Corresponding Authors

**1. Introduction** 

*2Department of Pathology, University of Melbourne, Melbourne,* 

*4Institute of Natural Sciences, Massey University, Auckland,* 

## **The Nucleolus and Ribosomal Genes in Aging and Senescence**

Nadine Hein1,\*, Elaine Sanij1,2,\*, Jaclyn Quin1,3, Katherine M. Hannan1, Austen Ganley4,# and Ross D. Hannan1,3,5,6,# *1Division of Cancer Research, Peter MacCallum Cancer Centre, Melbourne, 2Department of Pathology, University of Melbourne, Melbourne, 3Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, 4Institute of Natural Sciences, Massey University, Auckland, 5Department of Biochemistry and Molecular Biology, Monash University, Melbourne, 6School of Biomedical Sciences, The University of Queensland, Melbourne, 1,2,3,5,6Australia 4New Zealand* 

## **1. Introduction**

The nucleolus forms around the tandem repeats of the ribosomal RNA (rRNA) genes (rDNA) that are transcribed by RNA polymerase I (Pol I), giving rise to the production of rRNAs. These represent the nucleic acid backbone of the functional ribosomes in the cytoplasm, and as such rDNA transcription dictates the cells' protein translational capacity. More recently it has become apparent that the epigenetic status of these rDNA repeats and the integrity of the nucleolus can modulate cellular homeostasis beyond ribosome biogenesis. Such roles include mediating the titration of tumor suppressors and oncogenes, modulating the heterochromatic state of many RNA Polymerase II (Pol II) transcribed genes, and importantly, regulating the process of aging and senescence. This chapter will focus on the molecular and cellular evidence that the nucleolus and the rDNA repeats play critical roles in the control of aging and cellular senescence in yeast and mammals.

## **2. Introduction to rDNA transcription and the nucleolus**

This section will provide a brief overview of the regulation of rDNA transcription, however, for more details refer to (Tschochner & Hurt, 2003; McStay & Grummt, 2008).

<sup>\*</sup> These authors contributed equally to this work.

<sup>#</sup> Corresponding Authors

The Nucleolus and Ribosomal Genes in Aging and Senescence 173

steps not only include Pol I transcription of the pre-RNA precursor but its subsequent processing and modifications, the nuclear import of RP's, and the final assembly of the large and small ribosomal subunits followed by their export to the cytoplasm (Grummt & Pikaard, 2003; Moss et al., 2007). The process of ribosome biogenesis is fundamental to cellular life and consequently is highly conserved, and this is illustrated by the numerous

In budding yeast (*Saccharomyces cerevisiae)* and mammals the rRNA gene is transcribed exclusively by Pol I in the nucleolus. While in yeast this generates a 35S rRNA precursor which is processed into the mature 18S, 5.8S and 26S rRNAs (Fig. 2a), in mammals a 47S rRNA precursor is generated and processed to give 18S, 5.8S and 28S rRNAs (Fig. 2b). There are also numerous similarities in the other component of the ribosome, the RPs, which in both cases are transcribed by Pol II in the nucleoplasm. In growing yeast it has been established that ~40 nascent ribosomes leave the nucleolus every second, 80% of the total RNA is rRNA, and ~50% of total protein consists of RPs (Tschochner & Hurt, 2003). Overall the process of ribosome biogenesis consumes between 60-80% of the cells total energy both in yeast and mammals (Moss & Stefanovsky, 2002). Thus for both systems, even minor perturbations to ribosome biogenesis are likely to have major repercussions

similarities between yeast and mammals.

Fig. 2a. Organization of ribosomal RNA genes in yeast.

for the cell.

Fig. 1. Overview of ribosome biogenesis in mammalian cells.

## **2.1 Ribosome biogenesis**

Ribosome biogenesis dictates the capacity of a cell to grow and proliferate, and is one of the most energy consuming processes in eukaryotic cells (Grummt & Pikaard, 2003). The synthesis of a ribosome is a highly complex, yet exquisitely coordinated process, which utilizes all three DNA-dependent RNA polymerases (Pol I, Pol II and Pol III) to produce approximately equimolar amounts of numerous ribosomal proteins (RP) and four rRNA (Fig. 1). Transcription of the Pol I-transcribed rRNA genes (the rDNA) has traditionally been considered the major rate-limiting step in ribosome biogenesis. Consistent with this dogma, any perturbations in the cellular environment, such as nutrient withdrawal, altered growth factor signaling, cell cycle cues and stress, are directly accompanied by modulation of the rate of rDNA transcription. However, in addition to the two key components of the ribosome (rRNAs and RPs) a multitude of non-ribosomal proteins and non-coding RNAs have been identified as essential for various steps in the generation of new ribosomes. These

Fig. 1. Overview of ribosome biogenesis in mammalian cells.

Ribosome biogenesis dictates the capacity of a cell to grow and proliferate, and is one of the most energy consuming processes in eukaryotic cells (Grummt & Pikaard, 2003). The synthesis of a ribosome is a highly complex, yet exquisitely coordinated process, which utilizes all three DNA-dependent RNA polymerases (Pol I, Pol II and Pol III) to produce approximately equimolar amounts of numerous ribosomal proteins (RP) and four rRNA (Fig. 1). Transcription of the Pol I-transcribed rRNA genes (the rDNA) has traditionally been considered the major rate-limiting step in ribosome biogenesis. Consistent with this dogma, any perturbations in the cellular environment, such as nutrient withdrawal, altered growth factor signaling, cell cycle cues and stress, are directly accompanied by modulation of the rate of rDNA transcription. However, in addition to the two key components of the ribosome (rRNAs and RPs) a multitude of non-ribosomal proteins and non-coding RNAs have been identified as essential for various steps in the generation of new ribosomes. These

**2.1 Ribosome biogenesis** 

steps not only include Pol I transcription of the pre-RNA precursor but its subsequent processing and modifications, the nuclear import of RP's, and the final assembly of the large and small ribosomal subunits followed by their export to the cytoplasm (Grummt & Pikaard, 2003; Moss et al., 2007). The process of ribosome biogenesis is fundamental to cellular life and consequently is highly conserved, and this is illustrated by the numerous similarities between yeast and mammals.

In budding yeast (*Saccharomyces cerevisiae)* and mammals the rRNA gene is transcribed exclusively by Pol I in the nucleolus. While in yeast this generates a 35S rRNA precursor which is processed into the mature 18S, 5.8S and 26S rRNAs (Fig. 2a), in mammals a 47S rRNA precursor is generated and processed to give 18S, 5.8S and 28S rRNAs (Fig. 2b). There are also numerous similarities in the other component of the ribosome, the RPs, which in both cases are transcribed by Pol II in the nucleoplasm. In growing yeast it has been established that ~40 nascent ribosomes leave the nucleolus every second, 80% of the total RNA is rRNA, and ~50% of total protein consists of RPs (Tschochner & Hurt, 2003). Overall the process of ribosome biogenesis consumes between 60-80% of the cells total energy both in yeast and mammals (Moss & Stefanovsky, 2002). Thus for both systems, even minor perturbations to ribosome biogenesis are likely to have major repercussions for the cell.

Fig. 2a. Organization of ribosomal RNA genes in yeast.

The Nucleolus and Ribosomal Genes in Aging and Senescence 175

In contrast to yeast, the rDNA repeats of higher eukaryotes are located in multiple NORs (Fig. 2b). For example, humans have five NORs located on the short arms of the acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22) that contain ~70 copies of the rRNA genes (Sakai et al., 1995). During interphase two or more NORs coalesce to form multiple

A single rDNA unit, containing a transcribed region followed by intergenic spacer (IGS) is referred to as a canonical rDNA unit. However, non-canonical units, which form a palindromic structure are present and arranged as a mosaic with the canonical units in human NORs (Caburet et al., 2005). These non-canonical units are likely to be nonfunctional and it is believed that they are silenced since their transcription would result in antisense transcripts that could use the RNAi machinery to degrade the 47S precursor

The rDNA repeats were not sequenced during the human genome project due to their repetitive nature and high copy number, thus the full sequence of only two repeats have been reported (Kuo et al., 1996). A canonical human rDNA repeat (43 kb) consists of a 13 kb coding region that encodes the 47S pre-rRNA, which is rapidly processed at the external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions and ~30 kb of IGS (Fig. 2b). The IGS harbors several DNA regulatory elements, including the 47S rDNA promoter. The rDNA promoter has a bipartite architecture composed of a CORE and an upstream control element (UCE) located 100 bp upstream (McStay & Grummt, 2008). One or more terminator elements are located at the 5` and 3` ends of each mammalian rDNA repeat. Intriguingly, the IGS also contains one or more regions that are almost identical to the 47S rDNA promoter, and these are termed spacer promoters. Recent studies demonstrated that non-coding IGS transcripts play a role in the epigenetic control of rRNA gene silencing through modulation of the activity of the nucleolar remodeling complex

(NoRC), the rDNA silencing complex (Mayer et al., 2006) (see below for more detail).

It is well established that even in exponentially growing cells only a subset of rRNA genes are active. In yeast, active and inactive rRNA genes are randomly distributed in the single NOR (French et al., 2003). However in higher eukaryotes it is thought that active and silent rRNA genes are clustered, thus generating active and silent NORs. Silent NORs are condensed and do not contribute to the formation of nucleoli during interphase. It is likely that active NORs may contain a mosaic of active and inactive units however this remains a

While the precise mechanism controlling the silencing of rRNA genes is yet to be fully elucidated, there appears to be a marked difference between yeast and higher organisms in this process. For example, *S. cerevisiae* lacks the two major repressive methylation marks (CpG dinucleotide and H3K9 methylation) that are associated with the silencing of higher eukaryotic genes. Indeed, little is known about what regulates the number of active/inactive repeat units in yeast, although the histone deacetylase, Rpd3, is thought to play a role (Sandmeier et al, 2003), and TOR signaling regulates Pol I transcription and alters nucleolar

nucleoli in exponentially growing cells (McStay & Grummt, 2008).

rRNA. However, this has not been formally tested.

**2.3 Epigenetic regulation of Pol I transcription** 

matter for discussion (McStay & Grummt, 2008).

**2.3.1 Epigenetic silencing of rDNA** 

#### **2.2 Organization of eukaryotic ribosomal RNA genes**

The number of rDNA units per cell varies among eukaryotes, from 40 to ~19,000 in animals and from 150 to 26,000 in plants and correlates positively with genome size (Richard et al., 2008). In human cells there are up to 200 hundred copies of the ribosomal genes per haploid genome which are arranged in a head to tail orientation in clusters of tandem repeats. In yeast a single cluster consisting of ~150 copies of rRNA genes termed the nucleolar organizer region (NOR) is located on chromosome XII (Fig. 2a) and comprises over 10% of the whole genome (Kobayashi, 2011). During interphase a single nucleolus forms around this cluster. Two transcriptional regulatory DNA elements have been identified (Kulkens et al., 1991; Musters et al., 1989): the upstream element (UE), which is the binding site for the upstream activating factor (UAF); and the core promoter (CORE), which recruits the core factor (CF) (Elion & Warner, 1984). In addition to these regulatory DNA elements several non-coding regions have been identified within the gene and the IGS, including an origin of replication, called the ribosomal autonomous replicating sequence (ARS), and an expansion sequence containing a replication fork barrier (RFB) plus a bidirectional promoter (E-pro) that is required for rDNA amplification (Fig. 2a) (Brewer et al., 1992; Kobayashi et al., 1992).

Fig. 2b. Organization of ribosomal RNA genes in mammalian cells.

The number of rDNA units per cell varies among eukaryotes, from 40 to ~19,000 in animals and from 150 to 26,000 in plants and correlates positively with genome size (Richard et al., 2008). In human cells there are up to 200 hundred copies of the ribosomal genes per haploid genome which are arranged in a head to tail orientation in clusters of tandem repeats. In yeast a single cluster consisting of ~150 copies of rRNA genes termed the nucleolar organizer region (NOR) is located on chromosome XII (Fig. 2a) and comprises over 10% of the whole genome (Kobayashi, 2011). During interphase a single nucleolus forms around this cluster. Two transcriptional regulatory DNA elements have been identified (Kulkens et al., 1991; Musters et al., 1989): the upstream element (UE), which is the binding site for the upstream activating factor (UAF); and the core promoter (CORE), which recruits the core factor (CF) (Elion & Warner, 1984). In addition to these regulatory DNA elements several non-coding regions have been identified within the gene and the IGS, including an origin of replication, called the ribosomal autonomous replicating sequence (ARS), and an expansion sequence containing a replication fork barrier (RFB) plus a bidirectional promoter (E-pro) that is required for rDNA

**2.2 Organization of eukaryotic ribosomal RNA genes** 

amplification (Fig. 2a) (Brewer et al., 1992; Kobayashi et al., 1992).

Fig. 2b. Organization of ribosomal RNA genes in mammalian cells.

In contrast to yeast, the rDNA repeats of higher eukaryotes are located in multiple NORs (Fig. 2b). For example, humans have five NORs located on the short arms of the acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22) that contain ~70 copies of the rRNA genes (Sakai et al., 1995). During interphase two or more NORs coalesce to form multiple nucleoli in exponentially growing cells (McStay & Grummt, 2008).

A single rDNA unit, containing a transcribed region followed by intergenic spacer (IGS) is referred to as a canonical rDNA unit. However, non-canonical units, which form a palindromic structure are present and arranged as a mosaic with the canonical units in human NORs (Caburet et al., 2005). These non-canonical units are likely to be nonfunctional and it is believed that they are silenced since their transcription would result in antisense transcripts that could use the RNAi machinery to degrade the 47S precursor rRNA. However, this has not been formally tested.

The rDNA repeats were not sequenced during the human genome project due to their repetitive nature and high copy number, thus the full sequence of only two repeats have been reported (Kuo et al., 1996). A canonical human rDNA repeat (43 kb) consists of a 13 kb coding region that encodes the 47S pre-rRNA, which is rapidly processed at the external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions and ~30 kb of IGS (Fig. 2b). The IGS harbors several DNA regulatory elements, including the 47S rDNA promoter. The rDNA promoter has a bipartite architecture composed of a CORE and an upstream control element (UCE) located 100 bp upstream (McStay & Grummt, 2008). One or more terminator elements are located at the 5` and 3` ends of each mammalian rDNA repeat. Intriguingly, the IGS also contains one or more regions that are almost identical to the 47S rDNA promoter, and these are termed spacer promoters. Recent studies demonstrated that non-coding IGS transcripts play a role in the epigenetic control of rRNA gene silencing through modulation of the activity of the nucleolar remodeling complex (NoRC), the rDNA silencing complex (Mayer et al., 2006) (see below for more detail).

## **2.3 Epigenetic regulation of Pol I transcription**

It is well established that even in exponentially growing cells only a subset of rRNA genes are active. In yeast, active and inactive rRNA genes are randomly distributed in the single NOR (French et al., 2003). However in higher eukaryotes it is thought that active and silent rRNA genes are clustered, thus generating active and silent NORs. Silent NORs are condensed and do not contribute to the formation of nucleoli during interphase. It is likely that active NORs may contain a mosaic of active and inactive units however this remains a matter for discussion (McStay & Grummt, 2008).

## **2.3.1 Epigenetic silencing of rDNA**

While the precise mechanism controlling the silencing of rRNA genes is yet to be fully elucidated, there appears to be a marked difference between yeast and higher organisms in this process. For example, *S. cerevisiae* lacks the two major repressive methylation marks (CpG dinucleotide and H3K9 methylation) that are associated with the silencing of higher eukaryotic genes. Indeed, little is known about what regulates the number of active/inactive repeat units in yeast, although the histone deacetylase, Rpd3, is thought to play a role (Sandmeier et al, 2003), and TOR signaling regulates Pol I transcription and alters nucleolar

The Nucleolus and Ribosomal Genes in Aging and Senescence 177

In mammals transcriptionally active rRNA genes lack repressive histone modifications such as H3K9, H3K20 and H3K27 methylation and CpG DNA methylation (Conconi et al., 1989). Furthermore, they are associated with markers for active genes including H3K4 methylation and acetylation of histone H3 and H4 (Fig. 3). Importantly, transcriptionally active mammalian rDNA are characterized by the presence of UBF, which is enriched at the promoter and the transcribed regions of the repeat, and to a lesser extent at the IGS (Fig. 3) (Sanij et al., 2008; Wright et al., 2006). UBF seems to play multiple roles at the rDNA including transcriptional initiation, promoter escape and elongation control (Stefanovsky et al., 2006). Most likely these functions relate to the essential role UBF plays in maintaining active genes in an open, uncondensed configuration, which is achieved, in part, through the ability of UBF to outcompete histone linker H1, thus preventing the formation of higher order chromatin (Sanij & Hannan, 2009; Sanij et al., 2008). Of note, active rRNA genes are around tenfold less condensed then adjacent DNA and remain uncondensed during mitosis (Heliot et al., 1997). This is undoubtedly due to the continual association of UBF and a subset of the Pol I transcription machinery with the rDNA repeats, which maintains them in an under-condensed configuration to allow the rapid resumption of rDNA transcription as cells re-enter the cell cycle (Prieto & McStay, 2007;

One complex that has been described to promote the formation of an active chromatin environment for Pol I transcription is the chromatin remodeling complex B-WICH, which is

Fig. 3. Regulation of Pol I transcription in mammalian cells.

Roussel et al., 1996).

compaction (Tsang et al, 2007). Instead, the majority of studies looking at rDNA silencing in yeast have looked at the silencing of Pol II genes in the rDNA, and it is this form of silencing that is most strongly linked to senescence and aging, particularly through the Pol IIdependent E-pro promoter in the rDNA (Kobayashi & Ganley, 2005).

Silencing of E-pro is regulated by Sir2p, a member of the Sirtuin family of NAD+ dependent protein deacetylases. Sir2p also regulates the silencing of the telomeres and the mating loci (Guarente, 1999). The Sir2p analog in mammals SIRT1 is a key component of the energydependent nucleolar silencing complex (eNoSC), which has been reported to repress rRNA gene transcription in response to altered intracellular energy status (Murayama et al., 2008). Sir2p together with Net1 and Cdc14 are part of a well-known epigenetic regulator of the yeast rDNA locus, the regulator of nucleolar silencing and telophase exit (RENT) complex (Huang & Moazed, 2003). In addition, Sir2p in a complex with accessory proteins such as condensin or cohesin can repress recombination events within the rDNA repeats (Huang et al., 2006; Machin et al., 2004). More recently it has become clear that the stability of the rDNA repeats and their accurate replication depends on the proportion of the epigenetically silenced rRNA genes (Kobayashi, 2011).

In higher eukaryotes methylation of CpG dinucleotides is a common modification associated with establishing stable transcriptional repression. This covalent modification is catalyzed by the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, and is maintained during cell division (Klose & Bird, 2006). Stanchev et al (1979) demonstrated that CpG dinucleotide methylation marks are predominantly present in the promoter and enhancer regions of inactive rRNA genes (Fig. 3). In murine cells, methylation of a single CpG dinucleotide within the UCE (position -133 relative to the start of transcription) impairs the association of the Pol I transcription factor, upstream binding factor (UBF), to the rDNA and thus inhibits the assembly of the preinitiation complex (PIC) at the promoter (McStay & Grummt, 2008). In contrast, the human rDNA promoter contains ~25 CpG islands none of which are completely methylated or non-methylated. This suggests that the overall level of methylation rather than a binary on/off switch, dictates the transcriptional status of the rDNA. NoRC is the major complex involved in CpG methylation silencing of rDNA repeats (Santoro et al., 2002) (Fig. 3). The evolutionary logic underlying the additional complexity of rDNA silencing in higher eukaryotes compared to yeast is not clear but it potentially relates to regulation of cell differentiation and multicellular development.

#### **2.3.2 Active rDNA repeats and activation of Pol I transcription**

Regulation of rDNA transcription can occur at multiple levels, through regulatory elements defined by the primary DNA sequence as described above and also via the structure of the chromatin, which determines the accessibility of the DNA. Similar to regulation of Pol II and Pol III transcription, post-translational modification of the histones, such as acetylation, methylation, phosphorylation and ubiquitination, represent a key mechanism for the regulation of transcription by Pol I of active rDNA (Fig. 3).

In yeast it has been shown that the chromatin of actively transcribed rRNA genes is largely devoid of histone molecules, and instead is associated with the high-mobility group protein Hmo1, which interacts with the Pol I subunit Rpa49, binds across the entire 35S rDNA sequence and stabilize open rRNA gene chromatin (Hall et al., 2006; Merz et al., 2008).

compaction (Tsang et al, 2007). Instead, the majority of studies looking at rDNA silencing in yeast have looked at the silencing of Pol II genes in the rDNA, and it is this form of silencing that is most strongly linked to senescence and aging, particularly through the Pol II-

Silencing of E-pro is regulated by Sir2p, a member of the Sirtuin family of NAD+ dependent protein deacetylases. Sir2p also regulates the silencing of the telomeres and the mating loci (Guarente, 1999). The Sir2p analog in mammals SIRT1 is a key component of the energydependent nucleolar silencing complex (eNoSC), which has been reported to repress rRNA gene transcription in response to altered intracellular energy status (Murayama et al., 2008). Sir2p together with Net1 and Cdc14 are part of a well-known epigenetic regulator of the yeast rDNA locus, the regulator of nucleolar silencing and telophase exit (RENT) complex (Huang & Moazed, 2003). In addition, Sir2p in a complex with accessory proteins such as condensin or cohesin can repress recombination events within the rDNA repeats (Huang et al., 2006; Machin et al., 2004). More recently it has become clear that the stability of the rDNA repeats and their accurate replication depends on the proportion of the epigenetically

In higher eukaryotes methylation of CpG dinucleotides is a common modification associated with establishing stable transcriptional repression. This covalent modification is catalyzed by the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, and is maintained during cell division (Klose & Bird, 2006). Stanchev et al (1979) demonstrated that CpG dinucleotide methylation marks are predominantly present in the promoter and enhancer regions of inactive rRNA genes (Fig. 3). In murine cells, methylation of a single CpG dinucleotide within the UCE (position -133 relative to the start of transcription) impairs the association of the Pol I transcription factor, upstream binding factor (UBF), to the rDNA and thus inhibits the assembly of the preinitiation complex (PIC) at the promoter (McStay & Grummt, 2008). In contrast, the human rDNA promoter contains ~25 CpG islands none of which are completely methylated or non-methylated. This suggests that the overall level of methylation rather than a binary on/off switch, dictates the transcriptional status of the rDNA. NoRC is the major complex involved in CpG methylation silencing of rDNA repeats (Santoro et al., 2002) (Fig. 3). The evolutionary logic underlying the additional complexity of rDNA silencing in higher eukaryotes compared to yeast is not clear but it potentially relates

Regulation of rDNA transcription can occur at multiple levels, through regulatory elements defined by the primary DNA sequence as described above and also via the structure of the chromatin, which determines the accessibility of the DNA. Similar to regulation of Pol II and Pol III transcription, post-translational modification of the histones, such as acetylation, methylation, phosphorylation and ubiquitination, represent a key mechanism for the

In yeast it has been shown that the chromatin of actively transcribed rRNA genes is largely devoid of histone molecules, and instead is associated with the high-mobility group protein Hmo1, which interacts with the Pol I subunit Rpa49, binds across the entire 35S rDNA sequence and stabilize open rRNA gene chromatin (Hall et al., 2006; Merz et al., 2008).

dependent E-pro promoter in the rDNA (Kobayashi & Ganley, 2005).

to regulation of cell differentiation and multicellular development.

**2.3.2 Active rDNA repeats and activation of Pol I transcription** 

regulation of transcription by Pol I of active rDNA (Fig. 3).

silenced rRNA genes (Kobayashi, 2011).

Fig. 3. Regulation of Pol I transcription in mammalian cells.

In mammals transcriptionally active rRNA genes lack repressive histone modifications such as H3K9, H3K20 and H3K27 methylation and CpG DNA methylation (Conconi et al., 1989). Furthermore, they are associated with markers for active genes including H3K4 methylation and acetylation of histone H3 and H4 (Fig. 3). Importantly, transcriptionally active mammalian rDNA are characterized by the presence of UBF, which is enriched at the promoter and the transcribed regions of the repeat, and to a lesser extent at the IGS (Fig. 3) (Sanij et al., 2008; Wright et al., 2006). UBF seems to play multiple roles at the rDNA including transcriptional initiation, promoter escape and elongation control (Stefanovsky et al., 2006). Most likely these functions relate to the essential role UBF plays in maintaining active genes in an open, uncondensed configuration, which is achieved, in part, through the ability of UBF to outcompete histone linker H1, thus preventing the formation of higher order chromatin (Sanij & Hannan, 2009; Sanij et al., 2008). Of note, active rRNA genes are around tenfold less condensed then adjacent DNA and remain uncondensed during mitosis (Heliot et al., 1997). This is undoubtedly due to the continual association of UBF and a subset of the Pol I transcription machinery with the rDNA repeats, which maintains them in an under-condensed configuration to allow the rapid resumption of rDNA transcription as cells re-enter the cell cycle (Prieto & McStay, 2007; Roussel et al., 1996).

One complex that has been described to promote the formation of an active chromatin environment for Pol I transcription is the chromatin remodeling complex B-WICH, which is

The Nucleolus and Ribosomal Genes in Aging and Senescence 179

specific interaction between the promoter and terminator elements of actively transcribed rDNA repeats (Sander & Grummt, 1997). Thus by creating an rDNA loop, TTF1 is thought to promote efficient re-initiation of the Pol I complex at the rDNA promoter (Grummt et

Interestingly, in mammals not all the transcriptionally active rRNA genes of interphasic nucleoli are transcribed at any one time (Sanij & Hannan, 2009). Transcriptionally competent genes can be subdivided into two categories; active genes and pseudo-silent genes (Fig. 3). Active genes are undermethylated, bound by the cytoarchitectural chromatin remodeling factor UBF and are highly transcribed, whereas pseudo-silent rRNA genes are undermethylated and bound by linker-histone H1, but not by UBF, and thus are not transcriptionally active. This pseudo-silenced conformation of rDNA repeats, when induced by RNA inference mediated knock down of UBF, is stably propagated throughout the cell cycle of many generations in the absence of changes in CpG methylation and can be reversed by restoration of UBF to wild-type level (Sanij et al., 2008). Importantly, pseudosilencing seems to be a physiologically relevant phenomenon. For example, terminal differentiation of various cell types is associated with decreased UBF expression and a concomitant increase in the number of pseudo-silent rRNA genes (Poortinga et al., 2004; Poortinga et al., 2011; Sanij & Hannan, 2009). Moreover, the transition from a pre-malignant to malignant state is also associated with a decrease in the proportion of pseudo-silenced

*S. cerevisiae* and its unique genetic and biochemical attributes have proven to be an outstanding model organism to analyze many aspects of eukaryotic ribosome biogenesis. This is evident as much of our current understanding of the link between the nucleolus/rDNA transcription with aging and senescence comes from studies utilizing the

The highly repetitive nature of eukaryotic rDNA makes it one of the most fragile and dynamic regions of the genome, as recombination events within these repeats can cause either loss or gain of rDNA copies. Typically, such recombination events are highly regulated and are essential for maintenance of the rDNA copy number and the evolutionary stability of the rDNA repeats (Hawley & Marcus, 1989). Two mechanisms have been shown to be utilized for the repair of DNA double strand breaks (DSB) in the rDNA: homologous recombination (HR) and the single strand annealing (SSA) (Fishman-Lobell et al., 1992). Both these repair pathways can cause loss of rDNA copies, however the HR pathway can also result in a gain of copies. Despite the fluctuation resulting from the loss or gain of rDNA copies, an rDNA maintenance system provides a mechanism for the cell to keep copy number at a uniform level and ensure genomic stability (Fig. 4) (Kobayashi, 2006). Part of this maintenance system utilizes amplification of the rDNA, which rectifies the loss of rDNA copies (Kobayashi et al., 2004). Interestingly two of the

al., 1985; Henderson & Sollner-Webb, 1986).

**3. rDNA stability and aging in yeast** 

experimental advantages of budding yeast.

**3.1 Maintenance of rDNA copy number in yeast** 

**2.3.3 Pseudo-silenced rDNA repeats in higher eukaryotes** 

rRNA genes (Hannan RD and Bywater M, unpublished observation).

composed of the William syndrome transcription factor (WSTF), SNF2h and nuclear myosin (NM1) has been described to promote the formation of an active chromatin environment for Pol I transcription (Vintermist et al., 2011) (Fig. 3). The remodeling activity of the B-WICH complex is restricted to a specific 200 bp region around the promoter, which includes the UCE, CORE and transcriptional start site (Vintermist et al., 2011). An ATP-dependent chromatin remodeling complex (CSB IP/150) also promotes transcription of active rRNA genes. CSB IP/150 consists of the Crockayne syndrome protein B (CSB), TFIIH and TIF1B (Bradsher et al., 2002) (Fig. 3).

A key similarity between yeast and mammals is that the rate of rRNA transcription is regulated in response to stress signals and the availability of nutrients as sensed by the TOR pathway. In mammals the Pol I transcription factors, UBF and Pol I-specific transcription initiation factor 1A (TIF-1A)/RRN3 (Hannan et al., 2003; Mayer et al., 2004) have been reported to be activated by TOR kinase. Similar finding have been made in yeast for Hmo1 and Rrn3p. Specifically binding of Hmo1 to the rDNA is TORC1 dependent, and nutrient starvation or rapamycin (inhibitor of mTOR) treatment prevents this association (Berger et al., 2007). In the absence of Hmo1 the histone H4 deacetylase, Rpd3, can associate with the rDNA, resulting in rDNA condensation and a reduction of nucleolar size (Tsang et al., 2003).

The basal Pol I transcription machinery in yeast involves two multiprotein complexes, the UAF consisting of Rrn5, Rrn9, and Rrn10 and UAF30 (Keys et al., 1996; Siddiqi et al., 2001) and the CF, consisting of Rrn7p, Rrn11p, and Rrn6p (Steffan et al., 1996). Both complexes interact with the TATA-box-binding protein TBP. Binding of UAF to the promoter is essential for the recruitment of CF, once the UAF-CF is established active Pol I is recruited to initiate transcription. Initiation in yeast and mammals requires the essential Pol I-associated factor Rrn3p. Rrn3p is only found associated with a small fraction of Pol I, and in yeast this association requires Pol I phosphorylation. Upon initiation of transcription Pol I enters into the elongation phase of transcription and Rrn3p is released.

As with yeast, in mammals the initiation of transcription of active rRNA genes requires the assembly of a PIC at the promoter, although some of the components are species specific (Grummt, 2003; Moss et al., 2007). In the mammalian system the PIC (Fig. 3) contains the selectivity factor I (SL1), a complex itself of 4 or more TATA association factors (TAFs) unique to Pol I transcription plus the TBP that is utilized by all three Pol's (Learned et al., 1985; Zomerdijk et al., 1994). Our current understanding is that SL1 is recruited to the promoter by UBF, and consequently stabilizes UBF interaction with the rDNA promoter. Upon formation of a stable UBF/SL1 complex, active Pol I (defined by its association with RRN3 (Hempel et al., 1996; Yuan et al., 2002) is then recruited to complete the PIC. Following Pol I transcription mediated initiation and promoter clearance, RRN3 is thought to be released to be recycled for another round of transcription. Various steps in transcription including initiation or elongation, are also regulated in response to extracellular signals such as nutrients, amino acids, ATP, stress, which is mediated by signaling pathways, including the PI3K/AKT/mTOR, RAS/RAF/ERK and JNK pathways (Chan et al., 2011; Hannan et al., 2003; Mayer et al., 2005; Stefanovsky et al., 2001). More recently the transcription termination factor (TTF1), which was originally identified for its role in the termination of Pol I transcription, has been implicated in modulating DNA looping at the rDNA repeat thus facilitating a

composed of the William syndrome transcription factor (WSTF), SNF2h and nuclear myosin (NM1) has been described to promote the formation of an active chromatin environment for Pol I transcription (Vintermist et al., 2011) (Fig. 3). The remodeling activity of the B-WICH complex is restricted to a specific 200 bp region around the promoter, which includes the UCE, CORE and transcriptional start site (Vintermist et al., 2011). An ATP-dependent chromatin remodeling complex (CSB IP/150) also promotes transcription of active rRNA genes. CSB IP/150 consists of the Crockayne syndrome protein B (CSB), TFIIH and TIF1B

A key similarity between yeast and mammals is that the rate of rRNA transcription is regulated in response to stress signals and the availability of nutrients as sensed by the TOR pathway. In mammals the Pol I transcription factors, UBF and Pol I-specific transcription initiation factor 1A (TIF-1A)/RRN3 (Hannan et al., 2003; Mayer et al., 2004) have been reported to be activated by TOR kinase. Similar finding have been made in yeast for Hmo1 and Rrn3p. Specifically binding of Hmo1 to the rDNA is TORC1 dependent, and nutrient starvation or rapamycin (inhibitor of mTOR) treatment prevents this association (Berger et al., 2007). In the absence of Hmo1 the histone H4 deacetylase, Rpd3, can associate with the rDNA, resulting in rDNA condensation and a reduction of

The basal Pol I transcription machinery in yeast involves two multiprotein complexes, the UAF consisting of Rrn5, Rrn9, and Rrn10 and UAF30 (Keys et al., 1996; Siddiqi et al., 2001) and the CF, consisting of Rrn7p, Rrn11p, and Rrn6p (Steffan et al., 1996). Both complexes interact with the TATA-box-binding protein TBP. Binding of UAF to the promoter is essential for the recruitment of CF, once the UAF-CF is established active Pol I is recruited to initiate transcription. Initiation in yeast and mammals requires the essential Pol I-associated factor Rrn3p. Rrn3p is only found associated with a small fraction of Pol I, and in yeast this association requires Pol I phosphorylation. Upon initiation of transcription Pol I enters into

As with yeast, in mammals the initiation of transcription of active rRNA genes requires the assembly of a PIC at the promoter, although some of the components are species specific (Grummt, 2003; Moss et al., 2007). In the mammalian system the PIC (Fig. 3) contains the selectivity factor I (SL1), a complex itself of 4 or more TATA association factors (TAFs) unique to Pol I transcription plus the TBP that is utilized by all three Pol's (Learned et al., 1985; Zomerdijk et al., 1994). Our current understanding is that SL1 is recruited to the promoter by UBF, and consequently stabilizes UBF interaction with the rDNA promoter. Upon formation of a stable UBF/SL1 complex, active Pol I (defined by its association with RRN3 (Hempel et al., 1996; Yuan et al., 2002) is then recruited to complete the PIC. Following Pol I transcription mediated initiation and promoter clearance, RRN3 is thought to be released to be recycled for another round of transcription. Various steps in transcription including initiation or elongation, are also regulated in response to extracellular signals such as nutrients, amino acids, ATP, stress, which is mediated by signaling pathways, including the PI3K/AKT/mTOR, RAS/RAF/ERK and JNK pathways (Chan et al., 2011; Hannan et al., 2003; Mayer et al., 2005; Stefanovsky et al., 2001). More recently the transcription termination factor (TTF1), which was originally identified for its role in the termination of Pol I transcription, has been implicated in modulating DNA looping at the rDNA repeat thus facilitating a

(Bradsher et al., 2002) (Fig. 3).

nucleolar size (Tsang et al., 2003).

the elongation phase of transcription and Rrn3p is released.

specific interaction between the promoter and terminator elements of actively transcribed rDNA repeats (Sander & Grummt, 1997). Thus by creating an rDNA loop, TTF1 is thought to promote efficient re-initiation of the Pol I complex at the rDNA promoter (Grummt et al., 1985; Henderson & Sollner-Webb, 1986).

## **2.3.3 Pseudo-silenced rDNA repeats in higher eukaryotes**

Interestingly, in mammals not all the transcriptionally active rRNA genes of interphasic nucleoli are transcribed at any one time (Sanij & Hannan, 2009). Transcriptionally competent genes can be subdivided into two categories; active genes and pseudo-silent genes (Fig. 3). Active genes are undermethylated, bound by the cytoarchitectural chromatin remodeling factor UBF and are highly transcribed, whereas pseudo-silent rRNA genes are undermethylated and bound by linker-histone H1, but not by UBF, and thus are not transcriptionally active. This pseudo-silenced conformation of rDNA repeats, when induced by RNA inference mediated knock down of UBF, is stably propagated throughout the cell cycle of many generations in the absence of changes in CpG methylation and can be reversed by restoration of UBF to wild-type level (Sanij et al., 2008). Importantly, pseudosilencing seems to be a physiologically relevant phenomenon. For example, terminal differentiation of various cell types is associated with decreased UBF expression and a concomitant increase in the number of pseudo-silent rRNA genes (Poortinga et al., 2004; Poortinga et al., 2011; Sanij & Hannan, 2009). Moreover, the transition from a pre-malignant to malignant state is also associated with a decrease in the proportion of pseudo-silenced rRNA genes (Hannan RD and Bywater M, unpublished observation).

## **3. rDNA stability and aging in yeast**

*S. cerevisiae* and its unique genetic and biochemical attributes have proven to be an outstanding model organism to analyze many aspects of eukaryotic ribosome biogenesis. This is evident as much of our current understanding of the link between the nucleolus/rDNA transcription with aging and senescence comes from studies utilizing the experimental advantages of budding yeast.

## **3.1 Maintenance of rDNA copy number in yeast**

The highly repetitive nature of eukaryotic rDNA makes it one of the most fragile and dynamic regions of the genome, as recombination events within these repeats can cause either loss or gain of rDNA copies. Typically, such recombination events are highly regulated and are essential for maintenance of the rDNA copy number and the evolutionary stability of the rDNA repeats (Hawley & Marcus, 1989). Two mechanisms have been shown to be utilized for the repair of DNA double strand breaks (DSB) in the rDNA: homologous recombination (HR) and the single strand annealing (SSA) (Fishman-Lobell et al., 1992). Both these repair pathways can cause loss of rDNA copies, however the HR pathway can also result in a gain of copies. Despite the fluctuation resulting from the loss or gain of rDNA copies, an rDNA maintenance system provides a mechanism for the cell to keep copy number at a uniform level and ensure genomic stability (Fig. 4) (Kobayashi, 2006). Part of this maintenance system utilizes amplification of the rDNA, which rectifies the loss of rDNA copies (Kobayashi et al., 2004). Interestingly two of the

The Nucleolus and Ribosomal Genes in Aging and Senescence 181

DSB can occur at these paused replication forks, and they are repaired using HR. The outcome of this repair is dependent on the copy number of the rDNA (Kobayashi & Ganley, 2005). In cells containing wild-type copy number, Sir2p represses noncoding Pol IIdependent transcription at the bidirectional promoter located in the IGS (Fig. 4: E-pro OFF), enabling the association of cohesin with the IGS. Cohesin is a chromosome-associated multisubunit complex that connects sister chromatids and plays an essential role in the correct segregation of chromosomes during cell division and post-replicative DNA repair (Merkenschlager, 2010). Cohesin complex association with the IGS prevents the broken end from using a non-cognate repeat as the template for HR, thereby ensuring repair through equal sister chromatid recombination, with no rDNA copy number change. If the number of rDNA copies is reduced, however, a transcription-dependent rDNA amplification mechanism is activated whereby Sir2p repression is lifted, thus activating bidirectional Epro transcription (Fig. 4: E-pro ON). This non-coding transcription promotes the dissociation of cohesin from the IGS, allowing the broken end to use an unequal repeat as the repair template, resulting in a change of copy number. Copy number can either increase or decrease depending on whether the repeat used as the template for repair is upstream or downstream of the broken repeat (Ganley et al., 2005; Kobayashi & Ganley, 2005; Santangelo et al., 1988). If the template for repair is the same sister chromatid, a circular pop-out molecule, called an extra-chromosomal ribosomal circle (ERC) is formed. When rDNA copy number reaches wild-type levels E-pro transcription is silenced by Sir2p again, and rDNA amplification is inhibited. Sir2p mutant yeast cells can accumulate up to 300 copies due to non-restricted rDNA amplification (Kobayashi et al., 2004). Fob1p also meditates recombination events that are important for sequence homogenization of rDNA repeats and thus maintenance of the rRNA genes with identical or similar sequences (Ganley & Kobayashi, 2007). Whilst the mechanism by which cells monitor their rDNA copy number remains to be determined, it is clear that the maintenance of numerous copies of the rDNA

A recent landmark study (Ide et al., 2010) demonstrated that the number of silenced rDNA copies determines the cells sensitivity to DNA damage inducing agents such as ultraviolet (UV) radiation and methyl methanesulfonate (MMS) (Fig. 5). By using low-rDNA copy number strains (20 copies), the ratio of actively transcribed rRNA genes increased, and these strains were deficient in their ability to repair DNA damage during S-phase. Low copy number strains mutated in the Pol I subunit Rpa135p or Rrn3p were not impaired in their DNA repair capacity and consequently do not exhibit a higher sensitivity to DNA damaging agents. These findings suggest that rDNA transcription determines the sensitivity to DNA damage by inhibiting DNA repair (Ide et al., 2010). The authors also reported that this transcription-dependent sensitivity resulted from the inability of the multi subunit complex condensin, that is important for establishing and maintaining chromosome condensation, to associate with actively-transcribing rDNA units. This results in premature sister-chromatid separation, which impairs accurate sister-chromatid recombination required for DNA repair. Strikingly the major site of condensin complex occupation in the genome is the NOR. Consistent with this, the binding of mitotic condensin to the rDNA was shown to be reduced when Pol I transcription was elevated, and this impaired proper DNA repair and

chromatid cohesion, thus resulting in increased rDNA instability (Wang et al., 2006).

is very important for genomic stability.

**3.2 Silenced rDNA copies and DNA damage** 

key players in the regulation of rDNA copy number are also known as "aging associated genes", Fob1p and Sir2p (Fig. 4).

Fig. 4. Maintenance of rDNA copy number in yeast**.** Following initiation of replication from an ARS, Fob1p binding at the RFB inhibits replication fork progression. The outcome of the repair of these DSBs is dependent on the copy number of the rDNA. **"**E-pro OFF**"** illustrates cells containing wild-type copy number with no change in rDNA copy number. **"**E-pro ON**"** illustrates cells where rDNA copy number is altered, resulting in the production of an ERC.

During S-phase, initiation of DNA replication occurs at the origins of a subset of rDNA repeats (Pasero et al., 2002). The protein Fob1p binds in a sequence-specific manner to the rDNA at the RFB site, stalling the replication fork in one direction (Kobayashi, 2003). DNA

key players in the regulation of rDNA copy number are also known as "aging associated

Fig. 4. Maintenance of rDNA copy number in yeast**.** Following initiation of replication from an ARS, Fob1p binding at the RFB inhibits replication fork progression. The outcome of the repair of these DSBs is dependent on the copy number of the rDNA. **"**E-pro OFF**"** illustrates cells containing wild-type copy number with no change in rDNA copy number.

During S-phase, initiation of DNA replication occurs at the origins of a subset of rDNA repeats (Pasero et al., 2002). The protein Fob1p binds in a sequence-specific manner to the rDNA at the RFB site, stalling the replication fork in one direction (Kobayashi, 2003). DNA

**"**E-pro ON**"** illustrates cells where rDNA copy number is altered, resulting in the

genes", Fob1p and Sir2p (Fig. 4).

production of an ERC.

DSB can occur at these paused replication forks, and they are repaired using HR. The outcome of this repair is dependent on the copy number of the rDNA (Kobayashi & Ganley, 2005). In cells containing wild-type copy number, Sir2p represses noncoding Pol IIdependent transcription at the bidirectional promoter located in the IGS (Fig. 4: E-pro OFF), enabling the association of cohesin with the IGS. Cohesin is a chromosome-associated multisubunit complex that connects sister chromatids and plays an essential role in the correct segregation of chromosomes during cell division and post-replicative DNA repair (Merkenschlager, 2010). Cohesin complex association with the IGS prevents the broken end from using a non-cognate repeat as the template for HR, thereby ensuring repair through equal sister chromatid recombination, with no rDNA copy number change. If the number of rDNA copies is reduced, however, a transcription-dependent rDNA amplification mechanism is activated whereby Sir2p repression is lifted, thus activating bidirectional Epro transcription (Fig. 4: E-pro ON). This non-coding transcription promotes the dissociation of cohesin from the IGS, allowing the broken end to use an unequal repeat as the repair template, resulting in a change of copy number. Copy number can either increase or decrease depending on whether the repeat used as the template for repair is upstream or downstream of the broken repeat (Ganley et al., 2005; Kobayashi & Ganley, 2005; Santangelo et al., 1988). If the template for repair is the same sister chromatid, a circular pop-out molecule, called an extra-chromosomal ribosomal circle (ERC) is formed. When rDNA copy number reaches wild-type levels E-pro transcription is silenced by Sir2p again, and rDNA amplification is inhibited. Sir2p mutant yeast cells can accumulate up to 300 copies due to non-restricted rDNA amplification (Kobayashi et al., 2004). Fob1p also meditates recombination events that are important for sequence homogenization of rDNA repeats and thus maintenance of the rRNA genes with identical or similar sequences (Ganley & Kobayashi, 2007). Whilst the mechanism by which cells monitor their rDNA copy number remains to be determined, it is clear that the maintenance of numerous copies of the rDNA is very important for genomic stability.

#### **3.2 Silenced rDNA copies and DNA damage**

A recent landmark study (Ide et al., 2010) demonstrated that the number of silenced rDNA copies determines the cells sensitivity to DNA damage inducing agents such as ultraviolet (UV) radiation and methyl methanesulfonate (MMS) (Fig. 5). By using low-rDNA copy number strains (20 copies), the ratio of actively transcribed rRNA genes increased, and these strains were deficient in their ability to repair DNA damage during S-phase. Low copy number strains mutated in the Pol I subunit Rpa135p or Rrn3p were not impaired in their DNA repair capacity and consequently do not exhibit a higher sensitivity to DNA damaging agents. These findings suggest that rDNA transcription determines the sensitivity to DNA damage by inhibiting DNA repair (Ide et al., 2010). The authors also reported that this transcription-dependent sensitivity resulted from the inability of the multi subunit complex condensin, that is important for establishing and maintaining chromosome condensation, to associate with actively-transcribing rDNA units. This results in premature sister-chromatid separation, which impairs accurate sister-chromatid recombination required for DNA repair. Strikingly the major site of condensin complex occupation in the genome is the NOR. Consistent with this, the binding of mitotic condensin to the rDNA was shown to be reduced when Pol I transcription was elevated, and this impaired proper DNA repair and chromatid cohesion, thus resulting in increased rDNA instability (Wang et al., 2006).

The Nucleolus and Ribosomal Genes in Aging and Senescence 183

mammalian Sir2p orthologue, SIRT1 is part of the eNoSC complex, which mediates epigenetic silencing of rDNA in response to varying intracellular energy status (Murayama et al., 2008). It has been proposed that the SIRT1-eNoSC complex and epigenetic regulation of rDNA may provide a novel regulatory pathway for mammalian aging, which is

There is a clear prediction for aging factors in *S. cerevisiae,* as cell division is asymmetrical and the daughter cell receives a full lifespan, thus any aging factor must be preferentially sequestered in the mother cell and not passed on to the daughter. Indeed, Sinclair & Guarente (1997) demonstrated that aging wild-type yeast accumulated ERC, and these accumulated exclusively in mother cells. ERC accumulated even more rapidly in mutants (*sgs1*) that exhibit premature aging. In addition, accumulation of other extra-chromosomal genetic elements (i.e. plasmids) in the mother were shown to induce senescence. It was proposed that the accumulation of extra-chromosomal elements, including ERC and episomes, in the mother titrates genomic factors important for the maintenance of a young phenotype. A recent study suggested that it is not the ERC themselves that are the aging factor, but instead the rDNA recombination process that produces the ERC (Ganley et al., 2009). This study used strains with altered rDNA replication efficiencies. ERC exist in the cell effectively as plasmids because they harbour a replication origin. In the absence of selection, plasmid stability correlates with replication origin strength. Thus by altering rDNA replication strength, ERC production could be separated from their maintenance, and a strain with very little ERC accumulation was shown to age quickly when rDNA recombination was high. This study also reported that other episomes can induce genomic instability (Ganley et al., 2009), reconciling their results with those of Guarente and colleagues. ERCs have also been identified in *Drosophila* and humans (Gagnon-Kugler et al., 2009; Peng & Karpen, 2007) however its origin and role in aging is yet to be

Other aging theories propose that senescence is caused by an accumulation of DNA damage or cytoplasmic senescence factors that remain within the mother cell due to asymmetrical segregation. These theories are supported by the observation showing that oxidized (damaged) proteins predominantly accumulate in mother rather than daughter cells (Erjavec et al., 2007). Nucleolar rDNA is proposed to be particularly sensitive to the presence of elevated levels of oxidized proteins, as this leads to an impaired protein turnover and defects in DNA repair. Furthermore, the asymmetrical segregation of oxidized proteins is Sir2p dependent (Erjavec et al., 2007), leads to rDNA instability and the accumulation of ERC in the mother cell, which then promotes cellular senescence

The role of rDNA in aging is most clearly demonstrated in yeast with rDNA instability and cellular aging strongly correlating with rDNA copy number (Burkhalter & Sogo, 2004; Kobayashi et al., 2004). A recent review proposed the "rDNA theory" for aging. Specifically that dysfunction of DNA repair and the replication proteins, predominantly within the nucleolus of the mother cell, is a cause of increased rDNA instability. Because the nucleolus is the most sensitive cellular component to age-related DNA damage, the stability of the rDNA will in turn dictate the stability of the whole genome (Kobayashi,

associated with lower metabolic rates (Salminen & Kaarniranta, 2009).

determined.

(Kobayashi, 2008).

**3.4 A specific role for rDNA in aging** 

Fig. 5. Silenced rDNA copies and DNA damage in yeast. In cells with normal rDNA copy number, a proportion of genes are transcriptionally silent. Following DNA damage, condensin binds to untranscribed rDNA copies and facilitates sister chromatid cohesion, allowing repair of the rDNA by HR between sister chromatids at the stalled replication fork. Loss of rDNA copy number results in increased rates of transcription from the remaining rDNA repeats. Pol I transcription hinders the association of condensin with the rDNA, resulting in premature sister chromatid separation and preventing repair of the rDNA.

## **3.3 Aging in budding yeast**

Aging can generically be defined as a progressive functional decline, or a gradual deterioration of physiological function and loss of viability (Partridge & Mangel, 1999). In *S. cerevisiae* an aged phenotype becomes apparent when the mother cell gets beyond ~10 asymmetric cell divisions, and this includes enlargement of the cell and vacuole, extension of the cell division cycle, and sterility. Furthermore aged wild-type cells display an extremely enlarged and often fragmented nucleolus (Shore, 1998). The average lifespan of wild-type yeast is ~ 20 buddings, after which time the mother cell dies (Jazwinski, 2001). In contrast, daughter cells are born with a full budding capacity independent of the age of the mother cell. Studies in the late 90`s implicated a role for the nucleolus in yeast aging (reviewed in Guarente, 1997). As mentioned above Sirtuins are a protein family of NAD+ dependent protein deacetylases, and they are linked to the process of yeast aging by acting as silencing factors at a site termed the *AGE* locus, prolonging life span. It has been shown that Sir3p relocalizes to the nucleolus with age and that deletion of Sir2, 3 and 4 can abbreviate yeast life span (Kennedy et al., 1997). Later studies revealed that Sir2p could repress recombination in the rDNA locus, maintain rDNA stability and promote yeast longevity (Kaeberlein et al., 1999), suggesting a role for rDNA stability in the regulation of life span. Intriguingly, extra copies of Sir2 orthologs are capable of extending the lifespan of both worms and flies (Bauer et al., 2009; Tissenbaum & Guarente, 2001), suggesting an evolutionary role for Sir2 in regulation of longevity. As discussed earlier in section 2, the

Fig. 5. Silenced rDNA copies and DNA damage in yeast. In cells with normal rDNA copy number, a proportion of genes are transcriptionally silent. Following DNA damage, condensin binds to untranscribed rDNA copies and facilitates sister chromatid cohesion, allowing repair of the rDNA by HR between sister chromatids at the stalled replication fork. Loss of rDNA copy number results in increased rates of transcription from the remaining rDNA repeats. Pol I transcription hinders the association of condensin with the rDNA, resulting in premature sister chromatid separation and preventing repair of the rDNA.

Aging can generically be defined as a progressive functional decline, or a gradual deterioration of physiological function and loss of viability (Partridge & Mangel, 1999). In *S. cerevisiae* an aged phenotype becomes apparent when the mother cell gets beyond ~10 asymmetric cell divisions, and this includes enlargement of the cell and vacuole, extension of the cell division cycle, and sterility. Furthermore aged wild-type cells display an extremely enlarged and often fragmented nucleolus (Shore, 1998). The average lifespan of wild-type yeast is ~ 20 buddings, after which time the mother cell dies (Jazwinski, 2001). In contrast, daughter cells are born with a full budding capacity independent of the age of the mother cell. Studies in the late 90`s implicated a role for the nucleolus in yeast aging (reviewed in Guarente, 1997). As mentioned above Sirtuins are a protein family of NAD+ dependent protein deacetylases, and they are linked to the process of yeast aging by acting as silencing factors at a site termed the *AGE* locus, prolonging life span. It has been shown that Sir3p relocalizes to the nucleolus with age and that deletion of Sir2, 3 and 4 can abbreviate yeast life span (Kennedy et al., 1997). Later studies revealed that Sir2p could repress recombination in the rDNA locus, maintain rDNA stability and promote yeast longevity (Kaeberlein et al., 1999), suggesting a role for rDNA stability in the regulation of life span. Intriguingly, extra copies of Sir2 orthologs are capable of extending the lifespan of both worms and flies (Bauer et al., 2009; Tissenbaum & Guarente, 2001), suggesting an evolutionary role for Sir2 in regulation of longevity. As discussed earlier in section 2, the

**3.3 Aging in budding yeast** 

mammalian Sir2p orthologue, SIRT1 is part of the eNoSC complex, which mediates epigenetic silencing of rDNA in response to varying intracellular energy status (Murayama et al., 2008). It has been proposed that the SIRT1-eNoSC complex and epigenetic regulation of rDNA may provide a novel regulatory pathway for mammalian aging, which is associated with lower metabolic rates (Salminen & Kaarniranta, 2009).

There is a clear prediction for aging factors in *S. cerevisiae,* as cell division is asymmetrical and the daughter cell receives a full lifespan, thus any aging factor must be preferentially sequestered in the mother cell and not passed on to the daughter. Indeed, Sinclair & Guarente (1997) demonstrated that aging wild-type yeast accumulated ERC, and these accumulated exclusively in mother cells. ERC accumulated even more rapidly in mutants (*sgs1*) that exhibit premature aging. In addition, accumulation of other extra-chromosomal genetic elements (i.e. plasmids) in the mother were shown to induce senescence. It was proposed that the accumulation of extra-chromosomal elements, including ERC and episomes, in the mother titrates genomic factors important for the maintenance of a young phenotype. A recent study suggested that it is not the ERC themselves that are the aging factor, but instead the rDNA recombination process that produces the ERC (Ganley et al., 2009). This study used strains with altered rDNA replication efficiencies. ERC exist in the cell effectively as plasmids because they harbour a replication origin. In the absence of selection, plasmid stability correlates with replication origin strength. Thus by altering rDNA replication strength, ERC production could be separated from their maintenance, and a strain with very little ERC accumulation was shown to age quickly when rDNA recombination was high. This study also reported that other episomes can induce genomic instability (Ganley et al., 2009), reconciling their results with those of Guarente and colleagues. ERCs have also been identified in *Drosophila* and humans (Gagnon-Kugler et al., 2009; Peng & Karpen, 2007) however its origin and role in aging is yet to be determined.

Other aging theories propose that senescence is caused by an accumulation of DNA damage or cytoplasmic senescence factors that remain within the mother cell due to asymmetrical segregation. These theories are supported by the observation showing that oxidized (damaged) proteins predominantly accumulate in mother rather than daughter cells (Erjavec et al., 2007). Nucleolar rDNA is proposed to be particularly sensitive to the presence of elevated levels of oxidized proteins, as this leads to an impaired protein turnover and defects in DNA repair. Furthermore, the asymmetrical segregation of oxidized proteins is Sir2p dependent (Erjavec et al., 2007), leads to rDNA instability and the accumulation of ERC in the mother cell, which then promotes cellular senescence (Kobayashi, 2008).

### **3.4 A specific role for rDNA in aging**

The role of rDNA in aging is most clearly demonstrated in yeast with rDNA instability and cellular aging strongly correlating with rDNA copy number (Burkhalter & Sogo, 2004; Kobayashi et al., 2004). A recent review proposed the "rDNA theory" for aging. Specifically that dysfunction of DNA repair and the replication proteins, predominantly within the nucleolus of the mother cell, is a cause of increased rDNA instability. Because the nucleolus is the most sensitive cellular component to age-related DNA damage, the stability of the rDNA will in turn dictate the stability of the whole genome (Kobayashi,

The Nucleolus and Ribosomal Genes in Aging and Senescence 185

inhibition of rRNA synthesis and ribosome biogenesis and subsequent changes in nucleolar structure and function induce cell cycle arrest, implicating the nucleolus in regulation of cell survival and proliferation (Boisvert et al., 2007; Boulon et al., 2010). Indeed, a number of proteins regulated by their localization to the nucleolus, including the tumor suppressor protein ARF (alternative reading frame; p19ARF in mouse, p14ARF in human) and nucleophosmin (NPM), are involved in cellular senescence by mediating p53 stability (Colombo et al., 2002; Daniely et al., 2002). Therefore, it is likely that the nucleolus plays an active role in establishing and maintaining the senescent phenotype. Senescent cells are growth arrested in the transition from G1 to S-phase of the cell cycle (Sherwood et al., 1988). The role of the G1-S and G2-M cell cycle checkpoints is to ensure that the cell accurately duplicates its genome and successfully divide into the two daughter cells. The tumor suppressor factors p53 and retinoblastoma protein (RB) regulate these cell cycle checkpoints, which upon activation induce cell cycle arrest, senescence or apoptosis. Correct cell division requires increased protein synthesis that, in turn, is achieved by upregulation of ribosome biogenesis, thus these processes are tightly linked and potentially regulated by common mechanisms (Montanaro, 2008). For example, rDNA transcription and subsequent assembly of the nucleoli during G1 have been shown to be prerequisites for G1-S progression (Pardee, 1989; Sirri et al., 2002). Conversely, decreases in rates of rDNA transcription and disassembly of the nucleoli are observed during mitosis (Grummt, 1999; Pyronnet et al., 2001; Sirri et al., 2002). In fact, the nucleolus can sense and respond to cellular stress by modifying its size and content throughout interphase (Nalabothula et al., 2010). Consequently, it is not surprising that the nucleolus has been recognized as a central regulatory link between ribosome biogenesis and cell cycle progression (Carmo-Fonseca et al., 2000). Certainly, an ever increasing number of nucleolar proteins have been reported to play multiple roles in regulating ribosome biogenesis and cell cycle progression (Boisvert et al., 2007). Although, the exact molecular mechanisms responsible for mediating this crosstalk remain largely unknown, the p53 pathway is prevailing as an important link between ribosome

Early evidence for p53 as a key mediator of the crosstalk between ribosome biogenesis and cell cycle progression came from studies showing that inhibition of Bop1 (block of proliferation), a factor involved in rRNA synthesis and assembly, led to a p53-dependent G1 checkpoint arrest (Pestov et al., 2001). This is consistent with the notion that ongoing ribosome synthesis acts as a checkpoint at the G1-S boundary. In support of this inhibition of rRNA synthesis by microinjecting antibodies to UBF, disruption of the *TIF-IA* gene by Cre-dependent HR, or low doses of actinomycin D (Act D), leads to perturbations in nucleolar structure and function, p53-dependent G1-S cell cycle arrest and apoptosis

We have previously shown that inhibition of Pol I transcription by CX-5461, a small molecule inhibitor of initiation of rRNA synthesis, induces senescence in solid tumor cell lines (Drygin et al., 2011). Moreover, CX-5461 treatment or low doses of Act D induces premature senescence in TERT immortalized primary human fibroblasts (BJ-TERT) (Fig. 6) (Hahn et al., 1999). Within 24 hours of treatment with Act D or CX-5461, the protein levels of p53 and its transcriptional target, p21, are upregulated and sustained for a further 24 hours (Fig. 6a). Inhibition of rRNA synthesis correlates with the appearance of

biogenesis and the cell cycle (Pestov et al., 2001).

(Montanaro et al., 2007; Rubbi & Milner, 2003; Yuan et al., 2005).

2008). In both yeast and humans mutations within DNA repair genes result in a reduced lifespan (Park et al., 1999). In humans mutations associated with a premature aging phenotype (Werner and Bloom syndrome) are prominently found in RecQ homolog helicases, which are involved in rDNA repair (Ellis et al., 1995; Yu et al., 1996). In yeast, mutations in genes involved in rDNA transcription and elongation have been identified as modulators of rDNA stability and longevity (Heo et al., 1999; Hoopes et al., 2002; Merker & Klein, 2002). In conclusion whilst the findings in yeast clearly link the stability of rDNA, to aging and cellular senescence, relatively few studies in mammals investigating this link have been reported, predominantly due to the difficulties in studying this complicated part of the genome.

The idea that the rDNA has other, extra-coding functions has received increasing attention over the last few years (Kobayashi, 2011). In yeast, due to the fact that the rDNA cluster comprises ~10% of the genome, rDNA copy number and stability can influence the effective concentration of proteins and protein complexes located within the nucleus through titration. For example, studies investigating Sirtuins revealed that Sir2p is released from the nucleolus upon loss of rDNA copies. Sir2p, together with Sir3p, Sir4p and RAP1 can mediate silencing of telomeres and the mating-type loci. Intriguingly, depletion of ~50% of the rDNA repeats caused an increase in telomeric and mating-type gene silencing, suggesting that the effective concentration of Sir2p at different genomic loci plays a critical role in epigenetic regulation. The fact that mammalian rDNA comprises only ~0.3% of the genome raises the question of whether the association of genomic factors with the rDNA is sufficient to titrate silencing complexes to a similar extent to that reported in yeast. However, the mammalian nucleolus has been reported to influence various cellular functions via sequestering or releasing factors important for various cellular processes that regulate senescence and aging and this is described in more detail in section 4.

#### **4. The nucleolus and senescence in mammals**

Senescence is considered a manifestation of organismal aging at a cellular level, although this remains mechanistically unproven (Guarente, 1997; McCormick & Campisi, 1991). However, a number of studies have shown that senescent cells accumulate within mammalian tissues with increasing chronological age (Dimri et al., 1995; Y. Li et al., 1997; Pawelec et al., 1999). As discussed above, in yeast the rDNA locus, and hence the nucleolus, has been implicated in the regulation of longevity and senescence (Kobayashi, 2008). Consistent with this, a nontraditional role for the mammalian nucleolus is also now emerging that involves sequestration and release of tumor suppressors or oncogenes, cell cycle regulators and factors involved in modulating telomerase function (Olson et al., 2002; Olson, 2004; Olson & Dundr, 2005). Thus, similar to yeast, the mammalian nucleolus has been proposed to play an active role in senescence (Mehta et al., 2007; Olson et al., 2000).

Changes in nucleolar morphology are detected in aging cells (Mehta et al., 2007). While pre-senescent cells show a higher number of smaller nucleoli (Bemiller & Lee, 1978), senescent cells have a single prominent nucleolus. Cellular senescence however, does not always correlate with a concomitant decrease in rRNA gene transcription (Halle et al., 1997; Machwe et al., 2000). Even so, studies from many laboratories indicate that

2008). In both yeast and humans mutations within DNA repair genes result in a reduced lifespan (Park et al., 1999). In humans mutations associated with a premature aging phenotype (Werner and Bloom syndrome) are prominently found in RecQ homolog helicases, which are involved in rDNA repair (Ellis et al., 1995; Yu et al., 1996). In yeast, mutations in genes involved in rDNA transcription and elongation have been identified as modulators of rDNA stability and longevity (Heo et al., 1999; Hoopes et al., 2002; Merker & Klein, 2002). In conclusion whilst the findings in yeast clearly link the stability of rDNA, to aging and cellular senescence, relatively few studies in mammals investigating this link have been reported, predominantly due to the difficulties in

The idea that the rDNA has other, extra-coding functions has received increasing attention over the last few years (Kobayashi, 2011). In yeast, due to the fact that the rDNA cluster comprises ~10% of the genome, rDNA copy number and stability can influence the effective concentration of proteins and protein complexes located within the nucleus through titration. For example, studies investigating Sirtuins revealed that Sir2p is released from the nucleolus upon loss of rDNA copies. Sir2p, together with Sir3p, Sir4p and RAP1 can mediate silencing of telomeres and the mating-type loci. Intriguingly, depletion of ~50% of the rDNA repeats caused an increase in telomeric and mating-type gene silencing, suggesting that the effective concentration of Sir2p at different genomic loci plays a critical role in epigenetic regulation. The fact that mammalian rDNA comprises only ~0.3% of the genome raises the question of whether the association of genomic factors with the rDNA is sufficient to titrate silencing complexes to a similar extent to that reported in yeast. However, the mammalian nucleolus has been reported to influence various cellular functions via sequestering or releasing factors important for various cellular processes that regulate senescence and aging and this is described in more

Senescence is considered a manifestation of organismal aging at a cellular level, although this remains mechanistically unproven (Guarente, 1997; McCormick & Campisi, 1991). However, a number of studies have shown that senescent cells accumulate within mammalian tissues with increasing chronological age (Dimri et al., 1995; Y. Li et al., 1997; Pawelec et al., 1999). As discussed above, in yeast the rDNA locus, and hence the nucleolus, has been implicated in the regulation of longevity and senescence (Kobayashi, 2008). Consistent with this, a nontraditional role for the mammalian nucleolus is also now emerging that involves sequestration and release of tumor suppressors or oncogenes, cell cycle regulators and factors involved in modulating telomerase function (Olson et al., 2002; Olson, 2004; Olson & Dundr, 2005). Thus, similar to yeast, the mammalian nucleolus has been proposed to play an active role in senescence (Mehta et al., 2007; Olson et al.,

Changes in nucleolar morphology are detected in aging cells (Mehta et al., 2007). While pre-senescent cells show a higher number of smaller nucleoli (Bemiller & Lee, 1978), senescent cells have a single prominent nucleolus. Cellular senescence however, does not always correlate with a concomitant decrease in rRNA gene transcription (Halle et al., 1997; Machwe et al., 2000). Even so, studies from many laboratories indicate that

studying this complicated part of the genome.

**4. The nucleolus and senescence in mammals** 

detail in section 4.

2000).

inhibition of rRNA synthesis and ribosome biogenesis and subsequent changes in nucleolar structure and function induce cell cycle arrest, implicating the nucleolus in regulation of cell survival and proliferation (Boisvert et al., 2007; Boulon et al., 2010). Indeed, a number of proteins regulated by their localization to the nucleolus, including the tumor suppressor protein ARF (alternative reading frame; p19ARF in mouse, p14ARF in human) and nucleophosmin (NPM), are involved in cellular senescence by mediating p53 stability (Colombo et al., 2002; Daniely et al., 2002). Therefore, it is likely that the nucleolus plays an active role in establishing and maintaining the senescent phenotype.

Senescent cells are growth arrested in the transition from G1 to S-phase of the cell cycle (Sherwood et al., 1988). The role of the G1-S and G2-M cell cycle checkpoints is to ensure that the cell accurately duplicates its genome and successfully divide into the two daughter cells. The tumor suppressor factors p53 and retinoblastoma protein (RB) regulate these cell cycle checkpoints, which upon activation induce cell cycle arrest, senescence or apoptosis. Correct cell division requires increased protein synthesis that, in turn, is achieved by upregulation of ribosome biogenesis, thus these processes are tightly linked and potentially regulated by common mechanisms (Montanaro, 2008). For example, rDNA transcription and subsequent assembly of the nucleoli during G1 have been shown to be prerequisites for G1-S progression (Pardee, 1989; Sirri et al., 2002). Conversely, decreases in rates of rDNA transcription and disassembly of the nucleoli are observed during mitosis (Grummt, 1999; Pyronnet et al., 2001; Sirri et al., 2002). In fact, the nucleolus can sense and respond to cellular stress by modifying its size and content throughout interphase (Nalabothula et al., 2010). Consequently, it is not surprising that the nucleolus has been recognized as a central regulatory link between ribosome biogenesis and cell cycle progression (Carmo-Fonseca et al., 2000). Certainly, an ever increasing number of nucleolar proteins have been reported to play multiple roles in regulating ribosome biogenesis and cell cycle progression (Boisvert et al., 2007). Although, the exact molecular mechanisms responsible for mediating this crosstalk remain largely unknown, the p53 pathway is prevailing as an important link between ribosome biogenesis and the cell cycle (Pestov et al., 2001).

Early evidence for p53 as a key mediator of the crosstalk between ribosome biogenesis and cell cycle progression came from studies showing that inhibition of Bop1 (block of proliferation), a factor involved in rRNA synthesis and assembly, led to a p53-dependent G1 checkpoint arrest (Pestov et al., 2001). This is consistent with the notion that ongoing ribosome synthesis acts as a checkpoint at the G1-S boundary. In support of this inhibition of rRNA synthesis by microinjecting antibodies to UBF, disruption of the *TIF-IA* gene by Cre-dependent HR, or low doses of actinomycin D (Act D), leads to perturbations in nucleolar structure and function, p53-dependent G1-S cell cycle arrest and apoptosis (Montanaro et al., 2007; Rubbi & Milner, 2003; Yuan et al., 2005).

We have previously shown that inhibition of Pol I transcription by CX-5461, a small molecule inhibitor of initiation of rRNA synthesis, induces senescence in solid tumor cell lines (Drygin et al., 2011). Moreover, CX-5461 treatment or low doses of Act D induces premature senescence in TERT immortalized primary human fibroblasts (BJ-TERT) (Fig. 6) (Hahn et al., 1999). Within 24 hours of treatment with Act D or CX-5461, the protein levels of p53 and its transcriptional target, p21, are upregulated and sustained for a further 24 hours (Fig. 6a). Inhibition of rRNA synthesis correlates with the appearance of

senescence.

et al., 2003).

involved in cell cycle progression.

The Nucleolus and Ribosomal Genes in Aging and Senescence 187

Recent studies exploring the nucleolar proteome have revealed the involvement of the nucleolus in multiple biological processes including senescence, regulation of telomerase function, cell cycle regulation, and stress signaling (Boisvert et al., 2010; Boisvert et al., 2007). Furthermore, nucleolar structure and function are intimately linked with the regulation of p53 stabilization and activation of the p53 pathway, which firmly places the integrity of rDNA transcription and ribosome biogenesis at the centre of control of the cell cycle progression. In addition, over the last few years, significant advances have been made in understanding the higher order organization of nuclear structures, including the nucleolus, and their importance for the regulation of nuclear functions (Nemeth & Langst, 2011). Here, we address the impact of these recent results and discuss the molecular mechanisms underlying nucleolar function in the regulation of cell cycle progression and

An increasing number of nucleolar proteins have been reported to play multiple roles in regulating ribosome biogenesis and cell cycle progression. For instance, components of the DNA replication initiation machinery, origin of replication complex (ORC) and minichromosome maintenance (MCM) proteins, have been purified from human nucleoli (Boisvert et al., 2007; Couté et al., 2006) and were shown, in yeast, to associate with several 60S ribosomal synthesis factors that are required for pre-rRNA processing and are also essential for initiation of DNA replication by mediating the association of the ORC and MCM proteins at replication origins (Zhang et al., 2002). However, the most direct role for nucleoli in regulation of the cell cycle is via the sequestration or release of proteins directly

The tumor suppressor protein RB is an important regulator of senescence (Campisi & di Fagagna, 2007). RB is generally active during senescence, indeed its enforced expression has been shown to induce senescence (Narita et al., 2003). RB was initially reported to accumulate in the nucleolus and to have a repressive role in Pol I transcription (Cavanaugh et al., 1995; Hannan et al., 2000). Subsequently, Nucleolin (NCL), a multifunctional nucleolar protein essential for rRNA processing (Mongelard & Bouvet, 2007) was reported to associate with hypophosphorylated (active) RB (pRB) during the G1 phase of the cell cycle (Grinstein et al., 2006). pRB mediates a cell cycle checkpoint between G1 and S phase (Bartek et al., 1996) by targeting members of the E2F family of transcriptional activators (Chellappan et al., 1991) that are essential for cellular proliferation. While pRB has been reported to reside in the nucleoli in a cell type dependent manner (Angus et al., 2003) hyperphosphorylated RB (ppRB) is eliminated from the nucleolus until late S or G2 phase. Import of ppRB into the nucleolus in late S or G2 phase is mediated by its interaction with nucleolar NPM (Takemura et al., 2002). Although the functional significance of nucleolar retention and release of RB is currently unresolved, it has been proposed that its retention could represent a negative regulatory mechanism to sequester RB to prevent checkpoint activation during the cell cycle (Angus

By far the most convincing studies linking the dynamic release of nucleolar proteins and cell cycle progression were performed in yeast. Cdc14p is a protein phosphatase that is crucial

**4.1 The nucleolus in control of cell cycle regulation and senescence** 

single nucleoli, as visualized by fluorescence *in situ* hybridization (FISH) of rDNA (Fig. 6b), while in interphasic control cells the rDNA repeats are present as multiple nucleoli. The induction of p53 and nucleolar disorganization correlates with the subsequent appearance of H2A.X foci indicative of DNA damage, which is associated with senescence (Gire et al., 2004). After 96 hours of Pol I transcription inhibition, BJ-TERT cells appear bigger in size with a flat cell morphology and display acidic -galactosidase activity, characteristic phenotypic markers of senescence (Fig. 6c) (Dimri et al., 1995). UBF depletion in BJ-TERT cells is also associated with nucleolar disorganization and premature senescence, consistent with its role in establishing and maintaining nucleolar structure. Intriguingly, UBF depletion does not lead to decreased rates of rRNA synthesis, suggesting that the premature senescence may be a consequence of nucleolar disruption (Sanij E and Hannan RD, manuscript in preparation).

Fig. 6. Inhibition of Pol I transcription induces senescence. Following inhibition of Pol I transcription by treatment with either 5nM Act D or 1M CX-5461, BJ-TERT fibroblasts display hallmarks of senescence. a) Western blot analysis of p53 and p21 after treatment with either Act D or CX-5461. b) Following 48hr CX-5461 treatment, from left to right: RNA FISH to the 5'ETS region of the 47S pre-rRNA (red); DNA FISH to the rDNA (green) combined with immunofluorescent analysis for H2A.X (red). gDNA is visualized by DAPI (blue) c) -galactosidase staining of BJ-TERT cells after 96hr treatment with Act D or CX-5461.

single nucleoli, as visualized by fluorescence *in situ* hybridization (FISH) of rDNA (Fig. 6b), while in interphasic control cells the rDNA repeats are present as multiple nucleoli. The induction of p53 and nucleolar disorganization correlates with the subsequent appearance of H2A.X foci indicative of DNA damage, which is associated with senescence (Gire et al., 2004). After 96 hours of Pol I transcription inhibition, BJ-TERT cells appear bigger in size with a flat cell morphology and display acidic -galactosidase activity, characteristic phenotypic markers of senescence (Fig. 6c) (Dimri et al., 1995). UBF depletion in BJ-TERT cells is also associated with nucleolar disorganization and premature senescence, consistent with its role in establishing and maintaining nucleolar structure. Intriguingly, UBF depletion does not lead to decreased rates of rRNA synthesis, suggesting that the premature senescence may be a consequence of nucleolar disruption

Fig. 6. Inhibition of Pol I transcription induces senescence. Following inhibition of Pol I

transcription by treatment with either 5nM Act D or 1M CX-5461, BJ-TERT fibroblasts display hallmarks of senescence. a) Western blot analysis of p53 and p21 after treatment with either Act D or CX-5461. b) Following 48hr CX-5461 treatment, from left to right: RNA FISH to the 5'ETS region of the 47S pre-rRNA (red); DNA FISH to the rDNA (green) combined with immunofluorescent analysis for H2A.X (red). gDNA is visualized by DAPI (blue) c) -galactosidase staining of BJ-TERT cells after 96hr treatment with Act D or CX-5461.

(Sanij E and Hannan RD, manuscript in preparation).

Recent studies exploring the nucleolar proteome have revealed the involvement of the nucleolus in multiple biological processes including senescence, regulation of telomerase function, cell cycle regulation, and stress signaling (Boisvert et al., 2010; Boisvert et al., 2007). Furthermore, nucleolar structure and function are intimately linked with the regulation of p53 stabilization and activation of the p53 pathway, which firmly places the integrity of rDNA transcription and ribosome biogenesis at the centre of control of the cell cycle progression. In addition, over the last few years, significant advances have been made in understanding the higher order organization of nuclear structures, including the nucleolus, and their importance for the regulation of nuclear functions (Nemeth & Langst, 2011). Here, we address the impact of these recent results and discuss the molecular mechanisms underlying nucleolar function in the regulation of cell cycle progression and senescence.

## **4.1 The nucleolus in control of cell cycle regulation and senescence**

An increasing number of nucleolar proteins have been reported to play multiple roles in regulating ribosome biogenesis and cell cycle progression. For instance, components of the DNA replication initiation machinery, origin of replication complex (ORC) and minichromosome maintenance (MCM) proteins, have been purified from human nucleoli (Boisvert et al., 2007; Couté et al., 2006) and were shown, in yeast, to associate with several 60S ribosomal synthesis factors that are required for pre-rRNA processing and are also essential for initiation of DNA replication by mediating the association of the ORC and MCM proteins at replication origins (Zhang et al., 2002). However, the most direct role for nucleoli in regulation of the cell cycle is via the sequestration or release of proteins directly involved in cell cycle progression.

The tumor suppressor protein RB is an important regulator of senescence (Campisi & di Fagagna, 2007). RB is generally active during senescence, indeed its enforced expression has been shown to induce senescence (Narita et al., 2003). RB was initially reported to accumulate in the nucleolus and to have a repressive role in Pol I transcription (Cavanaugh et al., 1995; Hannan et al., 2000). Subsequently, Nucleolin (NCL), a multifunctional nucleolar protein essential for rRNA processing (Mongelard & Bouvet, 2007) was reported to associate with hypophosphorylated (active) RB (pRB) during the G1 phase of the cell cycle (Grinstein et al., 2006). pRB mediates a cell cycle checkpoint between G1 and S phase (Bartek et al., 1996) by targeting members of the E2F family of transcriptional activators (Chellappan et al., 1991) that are essential for cellular proliferation. While pRB has been reported to reside in the nucleoli in a cell type dependent manner (Angus et al., 2003) hyperphosphorylated RB (ppRB) is eliminated from the nucleolus until late S or G2 phase. Import of ppRB into the nucleolus in late S or G2 phase is mediated by its interaction with nucleolar NPM (Takemura et al., 2002). Although the functional significance of nucleolar retention and release of RB is currently unresolved, it has been proposed that its retention could represent a negative regulatory mechanism to sequester RB to prevent checkpoint activation during the cell cycle (Angus et al., 2003).

By far the most convincing studies linking the dynamic release of nucleolar proteins and cell cycle progression were performed in yeast. Cdc14p is a protein phosphatase that is crucial

The Nucleolus and Ribosomal Genes in Aging and Senescence 189

Fig. 7. Nucleolar regulation of telomere stability. Telomerase and the shelterin complex are regulated by the nucleolus through sequestration and release. In early S-phase, TERT moves

nucleoli. NCL-telomerase complexes are then exported to the telomeres during mid-S phase. TRF1 is regulated by NS and GNL3L in opposing manners. TRF2 is sequestered in the nucleolus during G1 and S phase, released to the nucleoplasm in G2, and returned to the

In addition to telomerase, components of the shelterin telomere binding complex, including telomeric repeat binding factors 1 (TRF1) and TRF2 have been reported as regulated in part by localization to the nucleolus (Fig. 7) (Lin et al., 2008; Tsai, 2009; Wong et al., 2002; Zhang et al., 2004). TRF1, which is required for establishing 'closed' structures at the telomeres that are inaccessible to telomerase, has been shown to be regulated by a number of nucleolar proteins, including nucleostemin (NS) and guanine nucleotide binding protein-like 3-like (GNL3L). NS binding to TRF1 enhances its degradation, while GNL3L binding stabilizes TRF1 (Zhu et al., 2009). Since the majority of TRF1 resides in the nucleoplasm, nucleolar retention of NS and GNL3L renders them inactive in modulating TRF1 activity. However, their nucleoplasmic localization during mitosis or in response to nucleolar stress may allow modulation of TRF1 in regulating

to the nucleoli and Cajal bodies containing TERC accumulate at the periphery of the

nucleoli at cytokinesis.

telomere capping (Tsai, 2009).

for promoting exit from mitosis into the G1 phase (Jin et al., 2008; Visintin et al., 1998; Zachariae et al., 1998). Cdc14p activation is also a prerequisite for successful chromosome segregation as it is necessary for condensin enrichment at the rDNA, which triggers rDNA segregation and ensures the completion of chromosome segregation (D'Amours et al., 2004). In G1 or during S phase, Cdc14p is sequestered to the nucleolus by its inhibitor Net1/Cfi1p, a component of the multifunctional protein complex RENT, where it remains inactive until the onset of anaphase, thereby preventing the premature onset of mitotic exit (Shou et al., 1999). The release of Cdc14p from the nucleolus is mediated through the sequential action of two regulatory networks: FEAR (CDC Fourteen Early Anaphase Release) and MEN (Mitotic Exit Network) that lead to Net1/Cfi1p phosphorylation reducing its affinity for Cdc14p and thus disassociation of the complex (Shou et al., 2002; Yoshida & Toh-e, 2002). The roles of the two human orthologs of Cdc14p (CDC14A and CDC14B) are not yet established. CDC14B has been shown to translocate from the nucleolus to the nucleoplasm following genotoxic stress in G2, leading to the activation of the APC/CDH1 complex and the establishment of a DNA damage induced G2 checkpoint (Bassermann et al., 2008). Nevertheless, a wider role in promoting mammalian cell cycle progression has been proposed for CDC14B including the governing of cell cycle re-entry after G2 block (De Wulf & Visintin, 2008).

Another example of nucleolar sequestration-mediated regulation of specific cellular activity during the cell cycle is the regulation of telomerase, the enzyme that adds telomeric repeats sequences to the ends of chromosomes (Fig. 7). Telomeres are composed of TTAGGG repeats that form a 3' overhang of 100-400 nucleotides forming a T-loop structure that is stabilized by telomeric proteins (Griffith et al., 1999). Telomeres maintain chromosome integrity by protecting against end shortening and end-to-end fusions (de Lange, 2005; Sahin & Depinho, 2010). If telomere length is not maintained, the telomeres will reach a critically short length, triggering the cell to undergo replicative senescence (Harley et al., 1990; Stewart et al., 2003). Although the telomeres and the nucleolus are separate subnuclear domains, multiple telomeric components have been detected in the nucleolus suggesting an underling regulatory connection between the nucleolus and telomeres (Tsai, 2009).

The ribonucleoprotein (RNP) telomerase is composed of the telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT), which catalyzes *de novo* repeat addition by utilizing TERC as a template (Greider & Blackburn, 1989). Outside of S phase, TERC and TERT are localised within distinct nucleoplasmic foci separate from telomeres. In early S-phase, TERT moves to nucleoli while Cajal bodies containing TERC accumulate at the periphery of nucleoli (Fig. 7). Nucleolar transportation of TERC and TERT has been proposed as a prerequisite step in the process of telomerase RNP biogenesis (Etheridge et al., 2002; Narayanan et al., 1999; Yang et al., 2002) and/or the transport of active telomerase, which occurs during mid-S phase (Tomlinson et al., 2006). Nucleolar localization of telomerase has also been reported to be mediated by NCL, which interacts with the active telomerase complex and is involved either in the assembly or maturation of telomerase. Nucleolar NCL-telomerase complexes are exported and maintained in the nucleoplasm and delivered to the telomeres (Khurts et al., 2004). Cell-cycle dependent nucleolar localization of telomerase is lost in transformed cells or following DNA damage (Wong et al., 2002).

for promoting exit from mitosis into the G1 phase (Jin et al., 2008; Visintin et al., 1998; Zachariae et al., 1998). Cdc14p activation is also a prerequisite for successful chromosome segregation as it is necessary for condensin enrichment at the rDNA, which triggers rDNA segregation and ensures the completion of chromosome segregation (D'Amours et al., 2004). In G1 or during S phase, Cdc14p is sequestered to the nucleolus by its inhibitor Net1/Cfi1p, a component of the multifunctional protein complex RENT, where it remains inactive until the onset of anaphase, thereby preventing the premature onset of mitotic exit (Shou et al., 1999). The release of Cdc14p from the nucleolus is mediated through the sequential action of two regulatory networks: FEAR (CDC Fourteen Early Anaphase Release) and MEN (Mitotic Exit Network) that lead to Net1/Cfi1p phosphorylation reducing its affinity for Cdc14p and thus disassociation of the complex (Shou et al., 2002; Yoshida & Toh-e, 2002). The roles of the two human orthologs of Cdc14p (CDC14A and CDC14B) are not yet established. CDC14B has been shown to translocate from the nucleolus to the nucleoplasm following genotoxic stress in G2, leading to the activation of the APC/CDH1 complex and the establishment of a DNA damage induced G2 checkpoint (Bassermann et al., 2008). Nevertheless, a wider role in promoting mammalian cell cycle progression has been proposed for CDC14B including the governing of cell cycle re-entry after G2 block (De Wulf

Another example of nucleolar sequestration-mediated regulation of specific cellular activity during the cell cycle is the regulation of telomerase, the enzyme that adds telomeric repeats sequences to the ends of chromosomes (Fig. 7). Telomeres are composed of TTAGGG repeats that form a 3' overhang of 100-400 nucleotides forming a T-loop structure that is stabilized by telomeric proteins (Griffith et al., 1999). Telomeres maintain chromosome integrity by protecting against end shortening and end-to-end fusions (de Lange, 2005; Sahin & Depinho, 2010). If telomere length is not maintained, the telomeres will reach a critically short length, triggering the cell to undergo replicative senescence (Harley et al., 1990; Stewart et al., 2003). Although the telomeres and the nucleolus are separate subnuclear domains, multiple telomeric components have been detected in the nucleolus suggesting an underling regulatory connection between the nucleolus and

The ribonucleoprotein (RNP) telomerase is composed of the telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT), which catalyzes *de novo* repeat addition by utilizing TERC as a template (Greider & Blackburn, 1989). Outside of S phase, TERC and TERT are localised within distinct nucleoplasmic foci separate from telomeres. In early S-phase, TERT moves to nucleoli while Cajal bodies containing TERC accumulate at the periphery of nucleoli (Fig. 7). Nucleolar transportation of TERC and TERT has been proposed as a prerequisite step in the process of telomerase RNP biogenesis (Etheridge et al., 2002; Narayanan et al., 1999; Yang et al., 2002) and/or the transport of active telomerase, which occurs during mid-S phase (Tomlinson et al., 2006). Nucleolar localization of telomerase has also been reported to be mediated by NCL, which interacts with the active telomerase complex and is involved either in the assembly or maturation of telomerase. Nucleolar NCL-telomerase complexes are exported and maintained in the nucleoplasm and delivered to the telomeres (Khurts et al., 2004). Cell-cycle dependent nucleolar localization of telomerase is lost in transformed cells or following DNA damage

& Visintin, 2008).

telomeres (Tsai, 2009).

(Wong et al., 2002).

Fig. 7. Nucleolar regulation of telomere stability. Telomerase and the shelterin complex are regulated by the nucleolus through sequestration and release. In early S-phase, TERT moves to the nucleoli and Cajal bodies containing TERC accumulate at the periphery of the nucleoli. NCL-telomerase complexes are then exported to the telomeres during mid-S phase. TRF1 is regulated by NS and GNL3L in opposing manners. TRF2 is sequestered in the nucleolus during G1 and S phase, released to the nucleoplasm in G2, and returned to the nucleoli at cytokinesis.

In addition to telomerase, components of the shelterin telomere binding complex, including telomeric repeat binding factors 1 (TRF1) and TRF2 have been reported as regulated in part by localization to the nucleolus (Fig. 7) (Lin et al., 2008; Tsai, 2009; Wong et al., 2002; Zhang et al., 2004). TRF1, which is required for establishing 'closed' structures at the telomeres that are inaccessible to telomerase, has been shown to be regulated by a number of nucleolar proteins, including nucleostemin (NS) and guanine nucleotide binding protein-like 3-like (GNL3L). NS binding to TRF1 enhances its degradation, while GNL3L binding stabilizes TRF1 (Zhu et al., 2009). Since the majority of TRF1 resides in the nucleoplasm, nucleolar retention of NS and GNL3L renders them inactive in modulating TRF1 activity. However, their nucleoplasmic localization during mitosis or in response to nucleolar stress may allow modulation of TRF1 in regulating telomere capping (Tsai, 2009).

The Nucleolus and Ribosomal Genes in Aging and Senescence 191

binds to MDM2 and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by MDM2, leading to the activation of p53 in the nucleoplasm (Honda & Yasuda, 1999; Palmero et al., 1998; Zindy et al., 1998). Under normal conditions, ARF is expressed at very low levels and is sequestered into to the nucleolus, due to its association with the nucleolar protein NPM (Gjerset & Bandyopadhyay, 2006; Korgaonkar et al., 2005). This prevents its interaction with MDM2. In contrast during replicative senescence of MEFs or stress induced by activation of oncogenes such as c-MYC and H-RAS, ARF rapidly accumulates to sufficient quantities and is able to bind and sequester MDM2 leading to p53 activation (Palmero et al., 1998; Sharpless et al., 2001; Weber et al., 1999). In addition ARF directly suppresses rRNA synthesis and processing to modulate ribosome biogenesis (Ayrault et al., 2006; Lessard et al., 2010; Sugimoto et al., 2003). Although, the importance of the later for modulation of p53 is unclear, a ribosome biogenesis-dependent-ARF pathway

The protein content of the nucleolus has been shown to change dramatically under various stress conditions (Boulon et al., 2010). It is now clear that, the nucleolar proteome undergoes distinct spatial and temporal alterations in response to different stress insults, suggesting that the nucleolus responds to different stress stimuli in a unique and specific manner (Moore et al., 2011). The landmark study by Rubbi and Milner (2003) proposed that disruption of nucleolar structure and function and subsequent release of nucleolar components into the nucleoplasm as a common denominator in most or possibly all p53 inducing stresses (Reviewed in Olson, 2004). Consistent with this, it is now recognized that inactivation of rDNA transcription, RP synthesis, rRNA processing, and the assembly and nucleolar export of the 40S and 60S ribosomal subunits (Zhang & Lu, 2009) are established mechanisms for causing nucleolar disruption and activation of the p53 pathway. From these observations a model of nucleolar surveillance of ribosome biogenesis (also termed nucleolar stress) has been proposed to integrate a diverse array of metabolic irregularities and oncogenic stimuli whereby the rate or efficiency of ribosome production serves as a signal by which cells could regulate cell-cycle progression via controlling p53 levels (Fig. 8) (Boulon et al., 2010; Deisenroth & Zhang, 2010; Ruggero & Pandolfi, 2003; Shcherbik & Pestov, 2010). In addition, due to the repetitive nature of the rRNA genes as well as the high rate of transcription by Pol I complexes, the rDNA is considered unstable and has been proposed to act as a potential sensor for DNA damage (Boisvert & Lamond, 2010). Signals associated with stalled polymerases and/or reduced rRNA transcription could activate p53 and possibly other DNA damage response pathways (Boisvert & Lamond, 2010; Kobayashi, 2008) leading to cell cycle arrest or programmed cell death (Drygin et al., 2009). Under conditions of "nucleolar stress", p53 stabilization can be achieved via different mechanisms including posttranslational modifications, protein-protein interactions and increases in the translation rate of p53 mRNA. Of these mechanisms perhaps the best documented is the role of RPs which are able to interact directly with MDM2 leading to p53 stabilization in response to ribosomal stress. Interestingly, while the RPs are required for p53 response to ribosomal stress, ARF in this context is not required, suggesting that different cellular conditions, oncogenic stress or ribosomal stress modulate the binding of either ARF or RPs to MDM2 and

may complement ARF's function in modulating the p53 pathway.

subsequent activation of p53 (Pan et al., 2011).

**4.2.2 Nucleolar stress** 

In addition, TRF2, the component of shelterin considered responsible for the formation of the protective telomeric T-loop structure required for protecting the telomeres, localizes to the nucleolus at G0 and S but diffuses into the nucleoplasm in G2 and returns to the nucleolus at cytokinesis (Fig. 7). Low dose of Act D, which specifically inhibits Pol I transcription, causes a delay in TRF2 release from nucleoli in G2 and mitotic cells displaying end-to-end chromosomal fusions, suggesting that the timely nucleolar retention/release of TRF2 regulates its nucleoplasmic function (Zhang et al., 2004).

Telomere attrition is recognized as a hallmark of aging cells (Harley et al., 1990). The p53 and p16INK4a-RB pathways are critical for establishing senescence in human cells (Campisi & di Fagagna, 2007). p53 is presumed to sense dysfunctional telomeres as damaged DNA, upon which it elicits the senescence response in part by increasing expression of the p21 CDKI, which in turn prevents the phosphorylation and inactivation of RB (Sherr & Roberts, 1999). In several mouse models, inappropriate p53 activity, either through deregulated expression of p53 or in response to constitutive stress like DNA damage, leads to premature aging (Maier et al., 2004; Tyner et al., 2002). As discussed later in this chapter, the nucleolus has been proposed as a central hub for sensing major cellular stress and transmitting signals for regulation of p53 levels and activity (Olson, 2004). It is therefore tempting to suggest that nucleoli may sense the DNA damage signal induced by damaged telomeres and activate a p53 response to implement senescence. Taken together, the nucleolus has emerged as a highly complex and multifunctional regulatory compartment involved in diverse biological processes including the regulation of proliferation and the execution of anti-proliferative responses such as cell cycle arrest and senescence.

#### **4.2 The nucleolus as a sensor of cellular stress**

One of the most intriguing roles proposed for the nucleolus is as a sensor of cellular stress and a means to couple cellular stress to the p53 pathway (Rubbi & Milner, 2003), a key regulator of senescence and longevity (Fig. 8) (Vigneron & Vousden, 2010) In this paradigm, under normal conditions, the nucleolus contributes to the maintenance of low p53 levels, while in response to cellular stress p53 levels and activity are dramatically elevated through the actions of select nucleolar proteins. Key to the nucleolar control of p53 is the oncogene MDM2 (mouse double minute 2; or HDM2 in humans). In proliferating cells, p53 activity is kept under surveillance by MDM2, via two complimentary mechanisms: (i) MDM2 acts as an E3 ubiquitin ligase directly transfering ubiquitin onto p53 thereby targeting it for 26S proteosomal degradation (Haupt et al., 1997; Kubbutat et al., 1997); and (ii) the direct binding of MDM2 to the N-terminal domain of p53 inhibits its transcriptional activity by abrogating its interaction with the basal Pol II transcription machinery (Momand et al., 1992; Oliner et al., 1993). The two best characterized mechanisms by which cellular stress modulates MDM2/p53 pathway in a nucleolar specific manner are in response to oncogenes (oncogenic stress) and to perturbations that alter ribosome biogenesis (nucleolar stress).

#### **4.2.1 ARF and oncogenic / replicative stress**

The *Ink4*/*Arf* locus encodes two tumor-suppressor proteins, p16INK4a and p19ARF, that govern the antiproliferative functions of RB and p53 proteins, respectively (Fig. 8). ARF binds to MDM2 and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by MDM2, leading to the activation of p53 in the nucleoplasm (Honda & Yasuda, 1999; Palmero et al., 1998; Zindy et al., 1998). Under normal conditions, ARF is expressed at very low levels and is sequestered into to the nucleolus, due to its association with the nucleolar protein NPM (Gjerset & Bandyopadhyay, 2006; Korgaonkar et al., 2005). This prevents its interaction with MDM2. In contrast during replicative senescence of MEFs or stress induced by activation of oncogenes such as c-MYC and H-RAS, ARF rapidly accumulates to sufficient quantities and is able to bind and sequester MDM2 leading to p53 activation (Palmero et al., 1998; Sharpless et al., 2001; Weber et al., 1999). In addition ARF directly suppresses rRNA synthesis and processing to modulate ribosome biogenesis (Ayrault et al., 2006; Lessard et al., 2010; Sugimoto et al., 2003). Although, the importance of the later for modulation of p53 is unclear, a ribosome biogenesis-dependent-ARF pathway may complement ARF's function in modulating the p53 pathway.

#### **4.2.2 Nucleolar stress**

190 Senescence

In addition, TRF2, the component of shelterin considered responsible for the formation of the protective telomeric T-loop structure required for protecting the telomeres, localizes to the nucleolus at G0 and S but diffuses into the nucleoplasm in G2 and returns to the nucleolus at cytokinesis (Fig. 7). Low dose of Act D, which specifically inhibits Pol I transcription, causes a delay in TRF2 release from nucleoli in G2 and mitotic cells displaying end-to-end chromosomal fusions, suggesting that the timely nucleolar retention/release of

Telomere attrition is recognized as a hallmark of aging cells (Harley et al., 1990). The p53 and p16INK4a-RB pathways are critical for establishing senescence in human cells (Campisi & di Fagagna, 2007). p53 is presumed to sense dysfunctional telomeres as damaged DNA, upon which it elicits the senescence response in part by increasing expression of the p21 CDKI, which in turn prevents the phosphorylation and inactivation of RB (Sherr & Roberts, 1999). In several mouse models, inappropriate p53 activity, either through deregulated expression of p53 or in response to constitutive stress like DNA damage, leads to premature aging (Maier et al., 2004; Tyner et al., 2002). As discussed later in this chapter, the nucleolus has been proposed as a central hub for sensing major cellular stress and transmitting signals for regulation of p53 levels and activity (Olson, 2004). It is therefore tempting to suggest that nucleoli may sense the DNA damage signal induced by damaged telomeres and activate a p53 response to implement senescence. Taken together, the nucleolus has emerged as a highly complex and multifunctional regulatory compartment involved in diverse biological processes including the regulation of proliferation and the execution of anti-proliferative

One of the most intriguing roles proposed for the nucleolus is as a sensor of cellular stress and a means to couple cellular stress to the p53 pathway (Rubbi & Milner, 2003), a key regulator of senescence and longevity (Fig. 8) (Vigneron & Vousden, 2010) In this paradigm, under normal conditions, the nucleolus contributes to the maintenance of low p53 levels, while in response to cellular stress p53 levels and activity are dramatically elevated through the actions of select nucleolar proteins. Key to the nucleolar control of p53 is the oncogene MDM2 (mouse double minute 2; or HDM2 in humans). In proliferating cells, p53 activity is kept under surveillance by MDM2, via two complimentary mechanisms: (i) MDM2 acts as an E3 ubiquitin ligase directly transfering ubiquitin onto p53 thereby targeting it for 26S proteosomal degradation (Haupt et al., 1997; Kubbutat et al., 1997); and (ii) the direct binding of MDM2 to the N-terminal domain of p53 inhibits its transcriptional activity by abrogating its interaction with the basal Pol II transcription machinery (Momand et al., 1992; Oliner et al., 1993). The two best characterized mechanisms by which cellular stress modulates MDM2/p53 pathway in a nucleolar specific manner are in response to oncogenes (oncogenic stress) and to

The *Ink4*/*Arf* locus encodes two tumor-suppressor proteins, p16INK4a and p19ARF, that govern the antiproliferative functions of RB and p53 proteins, respectively (Fig. 8). ARF

TRF2 regulates its nucleoplasmic function (Zhang et al., 2004).

responses such as cell cycle arrest and senescence.

**4.2 The nucleolus as a sensor of cellular stress** 

perturbations that alter ribosome biogenesis (nucleolar stress).

**4.2.1 ARF and oncogenic / replicative stress** 

The protein content of the nucleolus has been shown to change dramatically under various stress conditions (Boulon et al., 2010). It is now clear that, the nucleolar proteome undergoes distinct spatial and temporal alterations in response to different stress insults, suggesting that the nucleolus responds to different stress stimuli in a unique and specific manner (Moore et al., 2011). The landmark study by Rubbi and Milner (2003) proposed that disruption of nucleolar structure and function and subsequent release of nucleolar components into the nucleoplasm as a common denominator in most or possibly all p53 inducing stresses (Reviewed in Olson, 2004). Consistent with this, it is now recognized that inactivation of rDNA transcription, RP synthesis, rRNA processing, and the assembly and nucleolar export of the 40S and 60S ribosomal subunits (Zhang & Lu, 2009) are established mechanisms for causing nucleolar disruption and activation of the p53 pathway. From these observations a model of nucleolar surveillance of ribosome biogenesis (also termed nucleolar stress) has been proposed to integrate a diverse array of metabolic irregularities and oncogenic stimuli whereby the rate or efficiency of ribosome production serves as a signal by which cells could regulate cell-cycle progression via controlling p53 levels (Fig. 8) (Boulon et al., 2010; Deisenroth & Zhang, 2010; Ruggero & Pandolfi, 2003; Shcherbik & Pestov, 2010). In addition, due to the repetitive nature of the rRNA genes as well as the high rate of transcription by Pol I complexes, the rDNA is considered unstable and has been proposed to act as a potential sensor for DNA damage (Boisvert & Lamond, 2010). Signals associated with stalled polymerases and/or reduced rRNA transcription could activate p53 and possibly other DNA damage response pathways (Boisvert & Lamond, 2010; Kobayashi, 2008) leading to cell cycle arrest or programmed cell death (Drygin et al., 2009). Under conditions of "nucleolar stress", p53 stabilization can be achieved via different mechanisms including posttranslational modifications, protein-protein interactions and increases in the translation rate of p53 mRNA. Of these mechanisms perhaps the best documented is the role of RPs which are able to interact directly with MDM2 leading to p53 stabilization in response to ribosomal stress. Interestingly, while the RPs are required for p53 response to ribosomal stress, ARF in this context is not required, suggesting that different cellular conditions, oncogenic stress or ribosomal stress modulate the binding of either ARF or RPs to MDM2 and subsequent activation of p53 (Pan et al., 2011).

The Nucleolus and Ribosomal Genes in Aging and Senescence 193

proposed for how these various interactions might regulate p53 (Fig. 8). Under normal growth conditions, RPs are synthesized in equimolar amounts with the rRNAs and assembled with large and small ribosomal subunits in the nucleolus and transported to the cytoplasm to form functional ribosomes. In one model, so called "riding the ribosome", the interaction of p53 and/or MDM2 with the ribosomal subunits may facilitate p53/MDM2 transport from the nucleolus to the cytoplasm thus preventing p53 from interacting with its target genes in the nucleoplasm and/or promoting its ubiquitin-mediated degradation in the cytoplasm (Boulon et al., 2010). Conversely, stress signaling that impairs production and export of ribosome subunits, would be predicted to decrease p53/MDM2 transport to the cytoplasm, thus allowing p53 to activate transcription of its target genes in the nucleoplasm (Boulon et al., 2010). In a second and perhaps better described model, conditions that inhibit rRNA transcription or stall ribosome synthesis and assembly in the nucleolus are postulated to create a pool of free RPs (such as RPL5, RPL11 and RPL23) that are directly interact and sequester MDM2 resulting in suppression of p53 ubiquitination (Daniely et al., 2002; Deisenroth & Zhang, 2010; Lindstrom & Nister, 2010; Pestov et al., 2001; Warner & McIntosh, 2009; Zhang & Lu, 2009). However, Horn and Vousden (2008) observed a synergistic suppression of MDM2 activity through cooperation of RPL11 and RPL5, suggesting they have distinct roles in inhibiting MDM2 function. In addition, binding sites for ARF, RPL5, and RPL11 on MDM2 do not appear to overlap (Lindstrom et al., 2007; Zhang et al., 2003) suggesting that ARF and RPL5/L11 may respond to different stimuli and

converge at the point of MDM2 inactivation (Shcherbik & Pestov, 2010).

appropriate responses such as cell cycle arrest and senescence.

**with senescence** 

RPL26 is so far unique in its ability to bind the 5`untranslated region of the p53 mRNA and enhance its translation. Its interaction with MDM2 triggers its own ubiquitination and degradation, which in turn causes downregulation of p53 mRNA translation (Ofir-Rosenfeld et al., 2008). The diverse roles of RPs in the regulation of the MDM2-p53 pathway is further supported by the finding that knockdown of RPS6 not only affects 40S ribosomal biogenesis but also enforces RPL11 mRNA translation. This leads to an enhanced interaction between RPL11 and MDM2 leading to the accumulation and activation of p53 (Fumagalli et al., 2009). Since multiple RPs have separate mechanisms for activating p53, it is plausible that they may have distinct roles in sensing different types of signals leading to activation of nucleolar stress response. In summary there is now a robust set of data demonstrating that the nucleolus and rDNA transcription indirectly play an important role in the regulation of tumor suppressors and oncogenes such as ARF, MDM2 and p53 and thus perturbation in the nucleolus are predicted to have profound effects on cellular functions that are controlled by these factors. In this manner the nucleolus can be considered as a sensitive cellular stress detector integrating various perturbations in homeostasis and converting them to

**4.3 Alterations in genome organization in and around the nucleolus are associated** 

The nucleus is compartmentalized into substructures that perform distinct nuclear activities (Lanctot et al., 2007). These nuclear structures include the nucleoli, nuclear envelope, nuclear bodies, nuclear matrix and chromosome territories (Cremer & Cremer, 2001). Organization and spatial location of chromosomes and their interactions with other nuclear substructures ensures that transcription is correctly regulated (Misteli, 2004). The periphery

Fig. 8. The nucleolus as a sensor of cellular stress. Under normal conditions, p53 activity is maintained at low levels by MDM2, via two mechanisms: First, MDM2 ubiquitinates p53 thereby promoting its degradation; second, the binding of MDM2 to p53 abrogates its interaction with Pol II transcription machinery. Following oncogenic stress, ARF binds MDM2 and sequesters it in the nucleolus. Under nucleolar stress, p53 can be activated by the following mechanisms: (i). The co-transport of p53 and/or MDM2 with the ribosomal subunits to the cytoplasm is impaired; (ii). RPs interact directly with MDM2; (iii). 5.8S and 5S rRNA interact directly with MDM2; (iv). RPL26 binds p53mRNA and enhances its translation; (v). Increased RPL11 mRNA translation results in enhanced interaction between RPL11 and MDM2.

An increasing number of RPs (RPS3, RPS5, RPS7, RPL5, RPL11, RPL23, RPL26) as well as the 5.8S and 5S rRNAs are capable of interacting with MDM2 leading to p53 stabilization (Deisenroth & Zhang, 2010; Fontoura et al., 1992; Fumagalli et al., 2009; Ofir-Rosenfeld et al., 2008; Riley & Maher, 2007; Zhang & Lu, 2009). A number of different models have been

Fig. 8. The nucleolus as a sensor of cellular stress. Under normal conditions, p53 activity is maintained at low levels by MDM2, via two mechanisms: First, MDM2 ubiquitinates p53 thereby promoting its degradation; second, the binding of MDM2 to p53 abrogates its

interaction with Pol II transcription machinery. Following oncogenic stress, ARF binds MDM2 and sequesters it in the nucleolus. Under nucleolar stress, p53 can be activated by the following mechanisms: (i). The co-transport of p53 and/or MDM2 with the ribosomal subunits to the cytoplasm is impaired; (ii). RPs interact directly with MDM2; (iii). 5.8S and 5S rRNA interact directly with MDM2; (iv). RPL26 binds p53mRNA and enhances its translation; (v). Increased RPL11 mRNA translation results in enhanced interaction between RPL11 and MDM2.

An increasing number of RPs (RPS3, RPS5, RPS7, RPL5, RPL11, RPL23, RPL26) as well as the 5.8S and 5S rRNAs are capable of interacting with MDM2 leading to p53 stabilization (Deisenroth & Zhang, 2010; Fontoura et al., 1992; Fumagalli et al., 2009; Ofir-Rosenfeld et al., 2008; Riley & Maher, 2007; Zhang & Lu, 2009). A number of different models have been proposed for how these various interactions might regulate p53 (Fig. 8). Under normal growth conditions, RPs are synthesized in equimolar amounts with the rRNAs and assembled with large and small ribosomal subunits in the nucleolus and transported to the cytoplasm to form functional ribosomes. In one model, so called "riding the ribosome", the interaction of p53 and/or MDM2 with the ribosomal subunits may facilitate p53/MDM2 transport from the nucleolus to the cytoplasm thus preventing p53 from interacting with its target genes in the nucleoplasm and/or promoting its ubiquitin-mediated degradation in the cytoplasm (Boulon et al., 2010). Conversely, stress signaling that impairs production and export of ribosome subunits, would be predicted to decrease p53/MDM2 transport to the cytoplasm, thus allowing p53 to activate transcription of its target genes in the nucleoplasm (Boulon et al., 2010). In a second and perhaps better described model, conditions that inhibit rRNA transcription or stall ribosome synthesis and assembly in the nucleolus are postulated to create a pool of free RPs (such as RPL5, RPL11 and RPL23) that are directly interact and sequester MDM2 resulting in suppression of p53 ubiquitination (Daniely et al., 2002; Deisenroth & Zhang, 2010; Lindstrom & Nister, 2010; Pestov et al., 2001; Warner & McIntosh, 2009; Zhang & Lu, 2009). However, Horn and Vousden (2008) observed a synergistic suppression of MDM2 activity through cooperation of RPL11 and RPL5, suggesting they have distinct roles in inhibiting MDM2 function. In addition, binding sites for ARF, RPL5, and RPL11 on MDM2 do not appear to overlap (Lindstrom et al., 2007; Zhang et al., 2003) suggesting that ARF and RPL5/L11 may respond to different stimuli and converge at the point of MDM2 inactivation (Shcherbik & Pestov, 2010).

RPL26 is so far unique in its ability to bind the 5`untranslated region of the p53 mRNA and enhance its translation. Its interaction with MDM2 triggers its own ubiquitination and degradation, which in turn causes downregulation of p53 mRNA translation (Ofir-Rosenfeld et al., 2008). The diverse roles of RPs in the regulation of the MDM2-p53 pathway is further supported by the finding that knockdown of RPS6 not only affects 40S ribosomal biogenesis but also enforces RPL11 mRNA translation. This leads to an enhanced interaction between RPL11 and MDM2 leading to the accumulation and activation of p53 (Fumagalli et al., 2009). Since multiple RPs have separate mechanisms for activating p53, it is plausible that they may have distinct roles in sensing different types of signals leading to activation of nucleolar stress response. In summary there is now a robust set of data demonstrating that the nucleolus and rDNA transcription indirectly play an important role in the regulation of tumor suppressors and oncogenes such as ARF, MDM2 and p53 and thus perturbation in the nucleolus are predicted to have profound effects on cellular functions that are controlled by these factors. In this manner the nucleolus can be considered as a sensitive cellular stress detector integrating various perturbations in homeostasis and converting them to appropriate responses such as cell cycle arrest and senescence.

#### **4.3 Alterations in genome organization in and around the nucleolus are associated with senescence**

The nucleus is compartmentalized into substructures that perform distinct nuclear activities (Lanctot et al., 2007). These nuclear structures include the nucleoli, nuclear envelope, nuclear bodies, nuclear matrix and chromosome territories (Cremer & Cremer, 2001). Organization and spatial location of chromosomes and their interactions with other nuclear substructures ensures that transcription is correctly regulated (Misteli, 2004). The periphery

The Nucleolus and Ribosomal Genes in Aging and Senescence 195

Angus, S.P., Solomon, D.A., Kuschel, L., Hennigan, R.F. & Knudsen, E.S. (2003).

Ayrault, O., Andrique, L., Fauvin, D., Eymin, B., Gazzeri, S. & Seite, P. (2006). Human tumor

Bartek, J., Bartkova, J. & Lukas, J. (1996). The retinoblastoma protein pathway and the

Bassermann, F., Frescas, D., Guardavaccaro, D., Busino, L., Peschiaroli, A. & Pagano, M.

Bauer, J.H., Morris, S.N., Chang, C., Flatt, T., Wood, J.G. & Helfand, S.L. (2009). dSir2 and

Bemiller, P.M. & Lee, L.H. (1978). Nucleolar changes in senescing WI-38 cells. Mech Ageing

Berger, A.B., Cabal, G.G., Fabre, E., Duong, T., Buc, H., Nehrbass, U., Olivo-Marin, J.C.,

Berger, A.B., Decourty, L., Badis, G., Nehrbass, U., Jacquier, A. & Gadal, O. (2007). Hmo1 is

Boisvert, F.M., Lam, Y.W., Lamont, D. & Lamond, A.I. (2010). A quantitative proteomics

Boisvert, F.M. & Lamond, A.I. (2010). p53-Dependent subcellular proteome localization

Boisvert, F.M., van Koningsbruggen, S., Navascues, J. & Lamond, A.I. (2007). The

Boulon, S., Westman, B.J., Hutten, S., Boisvert, F.M. & Lamond, A.I. (2010). The nucleolus

Bradsher, J., Auriol, J., Proietti de Santis, L., Iben, S., Vonesch, J.L., Grummt, I. & Egly, J.M. (2002). CSB is a component of RNA pol I transcription. Mol Cell *10*, 819-829. Brewer, B.J., Lockshon, D. & Fangman, W.L. (1992). The arrest of replication forks in the rDNA of yeast occurs independently of transcription. Cell *71*, 267-276. Bridger, J.M. & Bickmore, W.A. (1998). Putting the genome on the map. Trends Genet *14*,

Bridger, J.M., Boyle, S., Kill, I.R. & Bickmore, W.A. (2000). Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts. Curr Biol *10*, 149-152. Burkhalter, M.D. & Sogo, J.M. (2004). rDNA enhancer affects replication initiation and

mitotic recombination: Fob1 mediates nucleolytic processing independently of

multifunctional nucleolus. Nat Rev Mol Cell Biol *8*, 574-585.

transcription factor phosphorylation. Oncogene *25*, 7577-7586.

restriction point. Current Opinion in Cell Biology *8*, 805-814.

checkpoint. Cell *134*, 256-267.

Mol Cell Biol *27*, 8015-8026.

Dev *8*, 417-427.

403-409.

melanogaster. Aging (Albany NY) *1*, 38-48.

damage. Mol Cell Proteomics *9*, 457-470.

following DNA damage. Proteomics.

under stress. Mol Cell *40*, 216-227.

replication. Mol Cell *15*, 409-421.

territories in live yeast. Nat Methods *5*, 1031-1037.

Retinoblastoma tumor suppressor: Analyses of dynamic behavior in living cells reveal multiple modes of regulation. Molecular and Cellular Biology *23*, 8172-8188.

suppressor p14ARF negatively regulates rRNA transcription and inhibits UBF1

(2008). The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response

Dmp53 interact to mediate aspects of CR-dependent lifespan extension in D.

Gadal, O. & Zimmer, C. (2008). High-resolution statistical mapping reveals gene

required for TOR-dependent regulation of ribosomal protein gene transcription.

analysis of subcellular proteome localization and changes induced by DNA

**6. References** 

of the nucleolus consists of satellite DNA repeats, which are proposed to play a role in the formation of perinucleolar heterochromatin (Manuelidis, 1984), and its been suggested they serve as a distinct nuclear space with a primary function in maintaining repressive chromatin states (Nemeth & Langst, 2011; van Koningsbruggen et al., 2010). For example the inactive X chromosome (Xi) must continuously visit the perinucleolar compartment during S phase to maintain its epigenetic status (Zhang et al., 2007). Other conserved chromosomal regions have also been shown to interact with the nucleolus including a fraction of the human centromeres (Bridger & Bickmore, 1998; Leger et al., 1994; Park & De Boni, 1992). Furthermore, chromosome mobility studies demonstrated that nucleolar-associated chromatin is significantly less mobile then other genomic regions. Specifically disruption of the nucleolar structure enhanced chromatin mobility, thus implying the nucleoli plays an active role in constraining chromatin movement and maintaining the three-dimensional organization of the genome within the nucleus (Chubb et al., 2002). This is further supported by the identification of specific interactions between repetitive and non-repetitive loci within the yeast genome including specific repeated elements that interact with rRNA genes. Therefore, it has been proposed that genomic architecture is organized by restricting the mobility of these repeat elements relative to the nucleolar interaction point (O'Sullivan et al., 2009). Similar results have been obtained using live cell imaging, with frequent interactions observed between the nucleolar and non-nucleolar chromatin (Berger et al., 2008). Intriguingly, extensive disorganisation of nuclear architecture, at the level of whole chromosomes, is associated with the transition from proliferative to senescent states. The human non-rDNA bearing chromosome 18, for instance, exhibits altered spatial positioning, changing from the apical edge of the nucleus in proliferating cells to nucleoli in senescent cells (Bridger et al., 2000). It is plausible that the reorganisation of the genome as the cells enter senescence is responsible for extensive changes in the transcriptional status of the genome (Foster & Bridger, 2005). Consitent with this, we have recently found that reductions in UBF levels lead to disruptions in nucleolar structure and acrocentric chromosome organization and induces premature senescence in primary human fibroblasts (Huang S, Hannan RD, manuscript in preparation). Together, the data suggest a functional role for nucleoli in the organization of the genome and in the regulation of cellular senescence.

#### **5. Conclusion**

The nucleolus is a highly evolutionary conserved subnuclear compartment traditionally associated with rDNA transcription and ribosome-subunit production. However it is now apparent that the nucleolus is dynamic in nature and its organization, size and protein composition changes dramatically during the cell cycle and under different cellular conditions including stress. Consistent with this dynamic nature the nucleolus has now been implicated in regulating additional important cellular processes beyond ribosome-subunit synthesis, including cell-cycle control, stress responses, senescence and aging. The fundamental role the rDNA repeats play in aging of fission yeast is now overwhelming. Similarly, in higher eukaryotes, nucleolar function in coupling ribosome subunit biogenesis and cell-cycle progression, through the activity of the tumor suppressor protein p53, places the nucleolus at the centre of coordinating cellular stress response and determining cell fate such as survival and senescence. It is likely we have only begun to scratch the surface of the detail by which eukaryotes have evolved to utilise the unique subnuclear domain, the nucleolus, to control fundamental cellular process such as aging and senescence.

## **6. References**

194 Senescence

of the nucleolus consists of satellite DNA repeats, which are proposed to play a role in the formation of perinucleolar heterochromatin (Manuelidis, 1984), and its been suggested they serve as a distinct nuclear space with a primary function in maintaining repressive chromatin states (Nemeth & Langst, 2011; van Koningsbruggen et al., 2010). For example the inactive X chromosome (Xi) must continuously visit the perinucleolar compartment during S phase to maintain its epigenetic status (Zhang et al., 2007). Other conserved chromosomal regions have also been shown to interact with the nucleolus including a fraction of the human centromeres (Bridger & Bickmore, 1998; Leger et al., 1994; Park & De Boni, 1992). Furthermore, chromosome mobility studies demonstrated that nucleolar-associated chromatin is significantly less mobile then other genomic regions. Specifically disruption of the nucleolar structure enhanced chromatin mobility, thus implying the nucleoli plays an active role in constraining chromatin movement and maintaining the three-dimensional organization of the genome within the nucleus (Chubb et al., 2002). This is further supported by the identification of specific interactions between repetitive and non-repetitive loci within the yeast genome including specific repeated elements that interact with rRNA genes. Therefore, it has been proposed that genomic architecture is organized by restricting the mobility of these repeat elements relative to the nucleolar interaction point (O'Sullivan et al., 2009). Similar results have been obtained using live cell imaging, with frequent interactions observed between the nucleolar and non-nucleolar chromatin (Berger et al., 2008). Intriguingly, extensive disorganisation of nuclear architecture, at the level of whole chromosomes, is associated with the transition from proliferative to senescent states. The human non-rDNA bearing chromosome 18, for instance, exhibits altered spatial positioning, changing from the apical edge of the nucleus in proliferating cells to nucleoli in senescent cells (Bridger et al., 2000). It is plausible that the reorganisation of the genome as the cells enter senescence is responsible for extensive changes in the transcriptional status of the genome (Foster & Bridger, 2005). Consitent with this, we have recently found that reductions in UBF levels lead to disruptions in nucleolar structure and acrocentric chromosome organization and induces premature senescence in primary human fibroblasts (Huang S, Hannan RD, manuscript in preparation). Together, the data suggest a functional role for nucleoli in the

organization of the genome and in the regulation of cellular senescence.

The nucleolus is a highly evolutionary conserved subnuclear compartment traditionally associated with rDNA transcription and ribosome-subunit production. However it is now apparent that the nucleolus is dynamic in nature and its organization, size and protein composition changes dramatically during the cell cycle and under different cellular conditions including stress. Consistent with this dynamic nature the nucleolus has now been implicated in regulating additional important cellular processes beyond ribosome-subunit synthesis, including cell-cycle control, stress responses, senescence and aging. The fundamental role the rDNA repeats play in aging of fission yeast is now overwhelming. Similarly, in higher eukaryotes, nucleolar function in coupling ribosome subunit biogenesis and cell-cycle progression, through the activity of the tumor suppressor protein p53, places the nucleolus at the centre of coordinating cellular stress response and determining cell fate such as survival and senescence. It is likely we have only begun to scratch the surface of the detail by which eukaryotes have evolved to utilise the unique subnuclear domain, the

nucleolus, to control fundamental cellular process such as aging and senescence.

**5. Conclusion** 


The Nucleolus and Ribosomal Genes in Aging and Senescence 197

Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens,

Drygin, D., Lin, A., Bliesath, J., Ho, C.B., O'Brien, S.E., Proffitt, C., Omori, M., Haddach, M.,

Drygin, D., Siddiqui-Jain, A., O'Brien, S., Schwaebe, M., Lin, A., Bliesath, J., Ho, C.B., Proffitt,

Elion, E.A. & Warner, J.R. (1984). The major promoter element of rRNA transcription in

Ellis, N.A., Groden, J., Ye, T.Z., Straughen, J., Lennon, D.J., Ciocci, S., Proytcheva, M. &

Erjavec, N., Larsson, L., Grantham, J. & Nystrom, T. (2007). Accelerated aging and failure to

Fishman-Lobell, J., Rudin, N. & Haber, J.E. (1992). Two alternative pathways of double-

Fontoura, B.M., Sorokina, E.A., David, E. & Carroll, R.B. (1992). p53 is covalently linked to

Foster, H.A. & Bridger, J.M. (2005). The genome and the nucleus: a marriage made by

French, S.L., Osheim, Y.N., Cioci, F., Nomura, M. & Beyer, A.L. (2003). In exponentially

Fumagalli, S., Di Cara, A., Neb-Gulati, A., Natt, F., Schwemberger, S., Hall, J., Babcock, G.F.,

Gagnon-Kugler, T., Langlois, F., Stefanovsky, V., Lessard, F. & Moss, T. (2009). Loss of

transcription and disrupts ribosomal RNA processing. Mol Cell *35*, 414-425. Ganley, A.R., Hayashi, K., Horiuchi, T. & Kobayashi, T. (2005). Identifying gene-

phylogenetic footprinting. Proc Natl Acad Sci U S A *102*, 11787-11792.

dependent mechanism of p53 induction. Nat Cell Biol *11*, 501-508.

synthesis and solid tumor growth. Cancer Res *71*, 1418-1430.

yeast lies 2 kb upstream. Cell *39*, 663-673.

telomerase. J Biol Chem *277*, 24764-24770.

5.8S rRNA. Mol Cell Biol *12*, 5145-5151.

9367.

*69*, 7653-7661.

helicases. Cell *83*, 655-666.

Mol Cell Biol *12*, 1292-1303.

Mol Cell Biol *23*, 1558-1568.

M., Rubelj, I., Pereira-Smith, O. & et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A *92*, 9363-

Schwaebe, M.K., Siddiqui-Jain, A., Streiner, N., Quin, J.E., Sanij, E., Bywater, M.J., Hannan, R.D., Ryckman, D., Anderes, K. & Rice, W.G. (2011). Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA

C., Trent, K., Whitten, J.P., Lim, J.K., Von Hoff, D., Anderes, K. & Rice, W.G. (2009). Anticancer activity of CX-3543: a direct inhibitor of rRNA biogenesis. Cancer Res

German, J. (1995). The Bloom's syndrome gene product is homologous to RecQ

segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p. Genes Dev *21*, 2410-2421. Etheridge, K.T., Banik, S.S., Armbruster, B.N., Zhu, Y., Terns, R.M., Terns, M.P. & Counter,

C.M. (2002). The nucleolar localization domain of the catalytic subunit of human

strand break repair that are kinetically separable and independently modulated.

evolution. Genome organisation and nuclear architecture. Chromosoma *114*, 212-9.

growing Saccharomyces cerevisiae cells, rRNA synthesis is determined by the summed RNA polymerase I loading rate rather than by the number of active genes.

Bernardi, R., Pandolfi, P.P. & Thomas, G. (2009). Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-

human ribosomal gene CpG methylation enhances cryptic RNA polymerase II

independent noncoding functional elements in the yeast ribosomal DNA by


Bystricky, K., Laroche, T., van Houwe, G., Blaszczyk, M. & Gasser, S.M. (2005).

Campisi, J. & di Fagagna, F.D. (2007). Cellular senescence: when bad things happen to good

Carmo-Fonseca, M., Mendes-Soares, L. & Campos, I. (2000). To be or not to be in the

Cavanaugh, A.H., Hempel, W.M., Taylor, L.J., Rogalsky, V., Todorov, G. & Rothblum, L.I.

Chan, J.C., Hannan, K.M., Riddell, K., Ng, P.Y., Peck, A., Lee, R.S., Hung, S., Astle, M.V.,

Chellappan, S.P., Hiebert, S., Mudryj, M., Horowitz, J.M. & Nevins, J.R. (1991). The E2F transcription factor is a cellular target for the RB protein. Cell *65*, 1053-1061. Chubb, J.R., Boyle, S., Perry, P. & Bickmore, W.A. (2002). Chromatin motion is constrained by association with nuclear compartments in human cells. Curr Biol *12*, 439-445. Colombo, E., Marine, J.C., Danovi, D., Falini, B. & Pelicci, P.G. (2002). Nucleophosmin regulates the stability and transcriptional activity of p53. Nat Cell Biol *4*, 529-533. Conconi, A., Widmer, R.M., Koller, T. & Sogo, J.M. (1989). Two different chromatin

Couté, Y., Burgess, J.A., Diaz, J.-J., Chichester, C., Lisacek, F., Greco, A. & Sanchez, J.-C.

Cremer, T. & Cremer, C. (2001). Chromosome territories, nuclear architecture and gene

D'Amours, D., Stegmeier, F. & Amon, A. (2004). Cdc14 and condensin control the

Daniely, Y., Dimitrova, D.D. & Borowiec, J.A. (2002). Stress-dependent nucleolin

de Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human

De Wulf, P. & Visintin, R. (2008). Cdc14B and APC/C tackle DNA damage. Cell *134*, 210-

Deisenroth, C. & Zhang, Y. (2010). Ribosome biogenesis surveillance: probing the ribosomal

regulation in mammalian cells. Nat Rev Genet *2*, 292-301.

protein-Mdm2-p53 pathway. Oncogene *29*, 4253-4260.

anchoring efficiency and territorial organization. J Cell Biol *168*, 375-387. Caburet, S., Conti, C., Schurra, C., Lebofsky, R., Edelstein, S.J. & Bensimon, A. (2005).

structures. Genome Res *15*, 1079-1085.

cells. Nat Rev Mol Cell Biol *8*, 729-740.

Ribosome Biogenesis in Cancer. Sci Signal *4*, ra56.

nucleolus. Nat Cell Biol *2*, E107-112.

product. Nature *374*, 177-180.

761.

*25*, 215-234.

*117*, 455-469.

telomeres. Genes Dev *19*, 2100-2110.

6022.

212.

Chromosome looping in yeast: telomere pairing and coordinated movement reflect

Human ribosomal RNA gene arrays display a broad range of palindromic

(1995). Activity of RNA polymerase I transcription factor UBF blocked by Rb gene

Bywater, M., Wall, M., Poortinga, G., Jastrzebski, K., Sheppard, K.E., Hemmings, B.A., Hall, M.N., Johnstone, R.W., McArthur, G.A., Hannan, R.D. & Pearson, R.B. (2011). AKT Promotes rRNA Synthesis and Cooperates with c-MYC to Stimulate

structures coexist in ribosomal RNA genes throughout the cell cycle. Cell *57*, 753-

(2006). Deciphering the human nucleolar proteome. Mass Spectrometry Reviews

dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell

mobilization mediated by p53-nucleolin complex formation. Mol Cell Biol *22*, 6014-


The Nucleolus and Ribosomal Genes in Aging and Senescence 199

Haupt, Y., Maya, R., Kazaz, A. & Oren, M. (1997). Mdm2 promotes the rapid degradation of

Hawley, R.S. & Marcus, C.H. (1989). Recombinational controls of rDNA redundancy in

Heliot, L., Kaplan, H., Lucas, L., Klein, C., Beorchia, A., Doco-Fenzy, M., Menager, M., Thiry,

Hempel, W.M., Cavanaugh, A.H., Hannan, R.D., Taylor, L. & Rothblum, L.I. (1996). The

Henderson, S. & Sollner-Webb, B. (1986). A transcriptional terminator is a novel element of

Henras, A.K., Capeyrou, R., Henry, Y. & Caizergues-Ferrer, M. (2004). Cbf5p, the putative

Honda, R. & Yasuda, H. (1999). Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53. EMBO J *18*, 22-27. Hoopes, L.L., Budd, M., Choe, W., Weitao, T. & Campbell, J.L. (2002). Mutations in DNA replication genes reduce yeast life span. Mol Cell Biol *22*, 4136-4146. Horn, H.F. & Vousden, K.H. (2008). Cooperation between the ribosomal proteins L5 and L11

Huang, J., Brito, I.L., Villen, J., Gygi, S.P., Amon, A. & Moazed, D. (2006). Inhibition of

Huang, J. & Moazed, D. (2003). Association of the RENT complex with nontranscribed and

Ide, S., Miyazaki, T., Maki, H. & Kobayashi, T. (2010). Abundance of ribosomal RNA gene

Jin, F., Liu, H., Liang, F., Rizkallah, R., Hurt, M.M. & Wang, Y. (2008). Temporal control of

Kaeberlein, M., McVey, M. & Guarente, L. (1999). The SIR2/3/4 complex and SIR2 alone

Kennedy, B.K., Gotta, M., Sinclair, D.A., Mills, K., McNabb, D.S., Murthy, M., Pak, S.M.,

homologous recombination by a cohesin-associated clamp complex recruited to the

coding regions of rDNA and a regional requirement for the replication fork block

the dephosphorylation of Cdk substrates by mitotic exit pathways in budding

promote longevity in Saccharomyces cerevisiae by two different mechanisms.

Laroche, T., Gasser, S.M. & Guarente, L. (1997). Redistribution of silencing proteins from telomeres to the nucleolus is associated with extension of life span in S.

the promoter of the mouse ribosomal RNA gene. Cell *47*, 891-900.

M., O'Donohue, M.F. & Ploton, D. (1997). Electron tomography of metaphase nucleolar organizer regions: evidence for a twisted-loop organization. Mol Biol Cell

species-specific RNA polymerase I transcription factor SL-1 binds to upstream

pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs. RNA *10*, 1704-1712. Heo, S.J., Tatebayashi, K., Ohsugi, I., Shimamoto, A., Furuichi, Y. & Ikeda, H. (1999). Bloom's

syndrome gene suppresses premature ageing caused by Sgs1 deficiency in yeast.

p53. Nature *387*, 296-299.

Genes Cells *4*, 619-625.

Genes Dev *13*, 2570-2580.

cerevisiae. Cell *89*, 381-391.

*8*, 2199-2216.

Drosophila. Annu Rev Genet *23*, 87-120.

binding factor. Mol Cell Biol *16*, 557-563.

in the p53 pathway. Oncogene *27*, 5774-5784.

rDNA recombination enhancer. Genes Dev *20*, 2887-2901.

protein Fob1 in rDNA silencing. Genes Dev *17*, 2162-2176.

copies maintains genome integrity. Science *327*, 693-696. Jazwinski, S.M. (2001). New clues to old yeast. Mech Ageing Dev *122*, 865-882.

yeast. Proc Natl Acad Sci U S A *105*, 16177-16182.


Ganley, A.R., Ide, S., Saka, K. & Kobayashi, T. (2009). The effect of replication initiation on gene amplification in the rDNA and its relationship to aging. Mol Cell *35*, 683-693. Ganley, A.R. & Kobayashi, T. (2007). Highly efficient concerted evolution in the ribosomal

Gire, V., Roux, P., Wynford-Thomas, D., Brondello, J.M. & Dulic, V. (2004). DNA damage checkpoint kinase Chk2 triggers replicative senescence. EMBO J *23*, 2554-2563. Gjerset, R.A. & Bandyopadhyay, K. (2006). Regulation of p14ARF through subnuclear

Greider, C.W. & Blackburn, E.H. (1989). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature *337*, 331-337. Griffith, J.D., Comeau, L., Rosenfield, S., Stansel, R.M., Bianchi, A., Moss, H. & de Lange, T. (1999). Mammalian telomeres end in a large duplex loop. Cell *97*, 503-514. Grinstein, E., Shan, Y., Karawajew, L., Snijders, P.J., Meijer, C.J., Royer, H.D. & Wernet, P.

protein and cancerous cell transformation. J Biol Chem *281*, 22223-22235. Grummt, I. (1999). Regulation of mammalian ribosomal gene transcription by RNA

Grummt, I. (2003). Life on a planet of its own: regulation of RNA polymerase I transcription

Grummt, I., Maier, U., Ohrlein, A., Hassouna, N. & Bachellerie, J.P. (1985). Transcription of

Hahn, W.C., Counter, C.M., Lundberg, A.S., Beijersbergen, R.L., Brooks, M.W. & Weinberg,

Hall, D.B., Wade, J.T. & Struhl, K. (2006). An HMG protein, Hmo1, associates with

Halle, J.P., Muller, S., Simm, A. & Adam, G. (1997). Copy number, epigenetic state and

Hannan, K.M., Brandenburger, Y., Jenkins, A., Sharkey, K., Cavanaugh, A., Rothblum, L.,

Hannan, K.M., Hannan, R.D., Smith, S.D., Jefferson, L.S., Lun, M. & Rothblum, L.I. (2000).

Harley, C.B., Futcher, A.B. & Greider, C.W. (1990). Telomeres shorten during ageing of

Guarente, L. (1997). Link between aging and the nucleolus. Genes Dev *11*, 2449-2455.

in Saccharomyces cerevisiae. Mol Cell Biol *26*, 3672-3679.

nucleolar transcription factor UBF. Mol Cell Biol *23*, 8862-8877.

between UBF and SL-1. Oncogene *19*, 4988-4999.

human fibroblasts. Nature *345*, 458-460.

polymerase I. Prog Nucleic Acid Res Mol Biol *62*, 109-154.

sequence data. Genome Res *17*, 184-191.

compartmentalization. Cell Cycle *5*, 686-690.

in the nucleolus. Genes Dev *17*, 1691-1702.

Nat Rev Mol Cell Biol *4*, 641-649.

*400*, 464-468.

Cell Biol *74*, 281-288.

DNA repeats: total rDNA repeat variation revealed by whole-genome shotgun

(2006). Cell cycle-controlled interaction of nucleolin with the retinoblastoma

mouse rDNA terminates downstream of the 3' end of 28S RNA and involves interaction of factors with repeated sequences in the 3' spacer. Cell *43*, 801-810. Grummt, I. & Pikaard, C.S. (2003). Epigenetic silencing of RNA polymerase I transcription.

R.A. (1999). Creation of human tumour cells with defined genetic elements. Nature

promoters of many ribosomal protein genes and throughout the rRNA gene locus

expression of the rRNA genes in young and senescent rat embryo fibroblasts. Eur J

Moss, T., Poortinga, G., McArthur, G.A., Pearson, R.B. & Hannan, R.D. (2003). mTOR-dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the

Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction


The Nucleolus and Ribosomal Genes in Aging and Senescence 201

Learned, R.M., Cordes, S. & Tjian, R. (1985). Purification and characterization of a

Leger, I., Guillaud, M., Krief, B. & Brugal, G. (1994). Interactive computer-assisted analysis of chromosome 1 colocalization with nucleoli. Cytometry *16*, 313-323. Lessard, F., Morin, F., Ivanchuk, S., Langlois, F., Stefanovsky, V., Rutka, J. & Moss, T. (2010).

Li, Y., Yan, Q. & Wolf, N.S. (1997). Long-term caloric restriction delays age-related decline in

Lin, J., Jin, R., Zhang, B., Chen, H., Bai, Y.X., Yang, P.X., Han, S.W., Xie, Y.H., Huang, P.T.,

Lindstrom, M.S., Jin, A., Deisenroth, C., White Wolf, G. & Zhang, Y. (2007). Cancer-

Lindstrom, M.S. & Nister, M. (2010). Silencing of ribosomal protein S9 elicits a multitude of

Machin, F., Paschos, K., Jarmuz, A., Torres-Rosell, J., Pade, C. & Aragon, L. (2004).

Machwe, A., Orren, D.K. & Bohr, V.A. (2000). Accelerated methylation of ribosomal RNA

Maier, B., Gluba, W., Bernier, B., Turner, T., Mohammad, K., Guise, T., Sutherland, A.,

Manuelidis, L. (1984). Different central nervous system cell types display distinct and

Mayer, C., Bierhoff, H. & Grummt, I. (2005). The nucleolus as a stress sensor: JNK2

Mayer, C., Schmitz, K.M., Li, J., Grummt, I. & Santoro, R. (2006). Intergenic transcripts

Mayer, C., Zhao, J., Yuan, X. & Grummt, I. (2004). mTOR-dependent activation of the

McCormick, A. & Campisi, J. (1991). Cellular aging and senescence. Current Opinion in Cell

McStay, B. & Grummt, I. (2008). The epigenetics of rRNA genes: from molecular to

regulate the epigenetic state of rRNA genes. Mol Cell *22*, 351-361.

chromosome biology. Annu Rev Cell Dev Biol *24*, 131-157.

polymerase I transcription factor TTF-I. Mol Cell *38*, 539-550.

telomerase function in human cells. J Cell Sci *121*, 2169-2176.

Mol Cell Biol *5*, 1358-1369.

Ophthalmol Vis Sci *38*, 100-107.

activation. PLoS One *5*, e9578.

isoform of p53. Genes Dev *18*, 306-319.

1068.

125-130.

1715-1724.

3123-3127.

*18*, 423-434.

Biology *3*, 230-234.

Genes Dev *19*, 933-941.

transcription factor that confers promoter specificity to human RNA polymerase I.

The ARF tumor suppressor controls ribosome biogenesis by regulating the RNA

proliferation capacity of murine lens epithelial cells in vitro and in vivo. Invest

Huang, C. & Huang, J.J. (2008). Nucleolar localization of TERT is unrelated to

associated mutations in the MDM2 zinc finger domain disrupt ribosomal protein interaction and attenuate MDM2-induced p53 degradation. Mol Cell Biol *27*, 1056-

cellular responses inhibiting the growth of cancer cells subsequent to p53

Condensin regulates rDNA silencing by modulating nucleolar Sir2p. Curr Biol *14*,

genes during the cellular senescence of Werner syndrome fibroblasts. FASEB J *14*,

Thorner, M. & Scrable, H. (2004). Modulation of mammalian life span by the short

nonrandom arrangements of satellite DNA sequences. Proc Natl Acad Sci U S A *81*,

inactivates the transcription factor TIF-IA and down-regulates rRNA synthesis.

transcription factor TIF-IA links rRNA synthesis to nutrient availability. Genes Dev


Keys, D.A., Lee, B.S., Dodd, J.A., Nguyen, T.T., Vu, L., Fantino, E., Burson, L.M., Nogi, Y. &

Khurts, S., Masutomi, K., Delgermaa, L., Arai, K., Oishi, N., Mizuno, H., Hayashi, N., Hahn,

Klose, R.J. & Bird, A.P. (2006). Genomic DNA methylation: the mark and its mediators.

Kobayashi, T. (2003). The replication fork barrier site forms a unique structure with Fob1p

Kobayashi, T. (2006). Strategies to maintain the stability of the ribosomal RNA gene repeats-

Kobayashi, T. (2008). A new role of the rDNA and nucleolus in the nucleus--rDNA

Kobayashi, T. (2011). Regulation of ribosomal RNA gene copy number and its role in

Kobayashi, T. & Ganley, A.R. (2005). Recombination regulation by transcription-induced

Kobayashi, T., Hidaka, M., Nishizawa, M. & Horiuchi, T. (1992). Identification of a site

Kobayashi, T., Horiuchi, T., Tongaonkar, P., Vu, L. & Nomura, M. (2004). SIR2 regulates

Korgaonkar, C., Hagen, J., Tompkins, V., Frazier, A.A., Allamargot, C., Quelle, F.W. &

Kubbutat, M.H., Jones, S.N. & Vousden, K.H. (1997). Regulation of p53 stability by Mdm2.

Kulkens, T., Riggs, D.L., Heck, J.D., Planta, R.J. & Nomura, M. (1991). The yeast RNA

initiation and complex formation in vitro. Nucleic Acids Res *19*, 5363-5370. Kuo, B.A., Gonzalez, I.L., Gillespie, D.A. & Sylvester, J.E. (1996). Human ribosomal RNA

Lam, Y.W., Lamond, A.I., Mann, M. & Andersen, J.S. (2007). Analysis of nucleolar protein

Lanctot, C., Cheutin, T., Cremer, M., Cavalli, G. & Cremer, T. (2007). Dynamic genome

and inhibits the replication fork. Mol Cell Biol *23*, 9178-9188.

instability maintains genome integrity. Bioessays *30*, 267-272.

cohesin dissociation in rDNA repeats. Science *309*, 1581-1584.

Saccharomyces cerevisiae. Mol Gen Genet *233*, 355-362.

function. Molecular and Cellular Biology *25*, 1258-1271.

individual rRNA genes in yeast. Cell *117*, 441-453.

preinitiation complex. Genes Dev *10*, 887-903.

Biological Chemistry *279*, 51508-51515.

Trends Biochem Sci *31*, 89-97.

155-161.

Sci *68*, 1395-1403.

Nature *387*, 299-303.

Acids Res *24*, 4817-4824.

dimensions. Nat Rev Genet *8*, 104-115.

760.

Nomura, M. (1996). Multiprotein transcription factor UAF interacts with the upstream element of the yeast RNA polymerase I promoter and forms a stable

W.C. & Murakami, S. (2004). Nucleolin interacts with telomerase. Journal of


modulating genome integrity and evolutionary adaptability in yeast. Cell Mol Life

required for DNA replication fork blocking activity in the rRNA gene cluster in

recombination between different rDNA repeats, but not recombination within

Quelle, D.E. (2005). Nucleophosmin (B23) targets ARF to nucleoli and inhibits its

polymerase I promoter: ribosomal DNA sequences involved in transcription

variants from a single individual and their expression in different tissues. Nucleic

dynamics reveals the nuclear degradation of ribosomal proteins. Curr Biol *17*, 749-

architecture in the nuclear space: regulation of gene expression in three


The Nucleolus and Ribosomal Genes in Aging and Senescence 203

Nemeth, A. & Langst, G. (2011). Genome organization in and around the nucleolus. Trends

O'Sullivan, A.C., Sullivan, G.J. & McStay, B. (2002). UBF binding in vivo is not restricted to

O'Sullivan, J.M., Sontam, D.M., Grierson, R. & Jones, B. (2009). Repeated elements coordinate the spatial organization of the yeast genome. Yeast *26*, 125-138. Ofir-Rosenfeld, Y., Boggs, K., Michael, D., Kastan, M.B. & Oren, M. (2008). Mdm2 regulates

Oliner, J.D., Pietenpol, J.A., Thiagalingam, S., Gyuris, J., Kinzler, K.W. & Vogelstein, B.

Olson, M.O., Dundr, M. & Szebeni, A. (2000). The nucleolus: an old factory with unexpected

Olson, M.O., Hingorani, K. & Szebeni, A. (2002). Conventional and nonconventional roles of

Olson, M.O.J. (2004). Sensing Cellular Stress: Another New Function for the Nucleolus? Sci

Olson, M.O.J. & Dundr, M. (2005). The moving parts of the nucleolus. Histochem Cell Biol

Palmero, I., Pantoja, C. & Serrano, M. (1998). p19ARF links the tumour suppressor p53 to

Pan, W., Issaq, S. & Zhang, Y. (2011). The in vivo role of the RP-Mdm2-p53 pathway in

Park, P.U., Defossez, P.A. & Guarente, L. (1999). Effects of mutations in DNA repair genes

Partridge, L. & Mangel, M. (1999). Messages from mortality: the evolution of death rates in

Pasero, P., Bensimon, A. & Schwob, E. (2002). Single-molecule analysis reveals clustering

Pawelec, G., Wagner, W., Adibzadeh, M. & Engel, A. (1999). T cell immunosenescence in

Peng, J.C. & Karpen, G.H. (2007). H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol *9*, 25-35. Pestov, D.G., Strezoska, Z. & Lau, L.F. (2001). Evidence of p53-dependent cross-talk between

Pardee, A.B. (1989). G1 events and regulation of cell proliferation. Science *246*, 603-608. Park, P.C. & De Boni, U. (1992). Spatial rearrangement and enhanced clustering of

association with nucleolar fusion. Exp Cell Res *203*, 222-229.

signaling oncogenic stress induced by pRb inactivation and Ras overexpression.

kinetochores in interphase nuclei of dorsal root ganglion neurons in vitro:

on formation of ribosomal DNA circles and life span in Saccharomyces cerevisiae.

and epigenetic regulation of replication origins at the yeast rDNA locus. Genes Dev

ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S

regulatory sequences within the vertebrate ribosomal DNA repeat. Mol Cell Biol

p53 mRNA translation through inhibitory interactions with ribosomal protein L26.

(1993). Oncoprotein MDM2 conceals the activation domain of tumour suppressor

Genet *27*, 149-156.

Mol Cell *32*, 180-189.

STKE *2004*, pe10-.

*123*, 203-216.

p53. Nature *362*, 857-860.

Ras. Nature *395*, 125-126.

Mol Cell Biol *19*, 3848-3856.

*16*, 2479-2484.

the old. Trends Ecol Evol *14*, 438-442.

vitro and in vivo. Exp Gerontol *34*, 419-429.

transition. Mol Cell Biol *21*, 4246-4255.

PLoS One *6*, e21625.

capabilities. Trends Cell Biol *10*, 189-196.

the nucleolus. Int Rev Cytol *219*, 199-266.

*22*, 657-668.


Mehta, I.S., Figgitt, M., Clements, C.S., Kill, I.R. & Bridger, J.M. (2007). Alterations to nuclear

Merkenschlager, M. (2010). Cohesin: a global player in chromosome biology with local ties

Merker, R.J. & Klein, H.L. (2002). hpr1Delta affects ribosomal DNA recombination and cell

Merz, K., Hondele, M., Goetze, H., Gmelch, K., Stoeckl, U. & Griesenbeck, J. (2008). Actively

Misteli, T. (2004). Spatial positioning; a new dimension in genome function. Cell *119*, 153-

Momand, J., Zambetti, G.P., Olson, D.C., George, D. & Levine, A.J. (1992). The mdm-2

Mongelard, F. & Bouvet, P. (2007). Nucleolin: a multiFACeTed protein. Trends Cell Biol *17*,

Montanaro, L., Mazzini, G., Barbieri, S., Vici, M., Nardi-Pantoli, A., Govoni, M., Donati, G.,

Moore, H.M., Bai, B., Boisvert, F.M., Latonen, L., Rantanen, V., Simpson, J.C., Pepperkok, R.,

Moss, T., Langlois, F., Gagnon-Kugler, T. & Stefanovsky, V. (2007). A housekeeper with

Musters, W., Knol, J., Maas, P., Dekker, A.F., van Heerikhuizen, H. & Planta, R.J. (1989).

Nalabothula, N., Indig, F.E. & Carrier, F. (2010). The Nucleolus Takes Control of Protein

Narayanan, A., Lukowiak, A., Jady, B.E., Dragon, F., Kiss, T., Terns, R.M. & Terns, M.P.

Narita, M., Nunez, S., Heard, E., Lin, A.W., Hearn, S.A., Spector, D.L., Hannon, G.J. & Lowe,

Trafficking Under Cellular Stress. Mol Cell Pharmacol *2*, 203-212.

Moss, T. & Stefanovsky, V.Y. (2002). At the center of eukaryotic life. Cell *109*, 545-548. Murayama, A., Ohmori, K., Fujimura, A., Minami, H., Yasuzawa-Tanaka, K., Kuroda, T.,

life span in Saccharomyces cerevisiae. Mol Cell Biol *22*, 421-429.

Mechanisms and Interventions, pp. 250-263.

histone molecules. Genes Dev *22*, 1190-1204.

transactivation. Cell *69*, 1237-1245.

156.

80-86.

Proteomics.

9661-9678.

J *18*, 5120-5130.

49.

to gene regulation. Curr Opin Genet Dev *20*, 555-561.

human osteosarcoma cell lines. Cell Prolif *40*, 532-549.

intracellular energy status. Cell *133*, 627-639.

genes during cellular senescence. Cell *113*, 703-716.

architecture and genome behavior in senescent cells. In Biogerontology:

transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of

oncogene product forms a complex with the p53 protein and inhibits p53-mediated

Trere, D. & Derenzini, M. (2007). Different effects of ribosome biogenesis inhibition on cell proliferation in retinoblastoma protein- and p53-deficient and proficient

Lamond, A.I. & Laiho, M. (2011). Quantitative proteomics and dynamic imaging of the nucleolus reveals distinct responses to UV and ionizing radiation. Mol Cell

power of attorney: the rRNA genes in ribosome biogenesis. Cell Mol Life Sci *64*, 29-

Oie, S., Daitoku, H., Okuwaki, M., Nagata, K., Fukamizu, A., Kimura, K., Shimizu, T. & Yanagisawa, J. (2008). Epigenetic control of rDNA loci in response to

Linker scanning of the yeast RNA polymerase I promoter. Nucleic Acids Res *17*,

(1999). Nucleolar localization signals of box H/ACA small nucleolar RNAs. EMBO

S.W. (2003). Rb-mediated heterochromatin formation and silencing of E2F target


The Nucleolus and Ribosomal Genes in Aging and Senescence 205

Santangelo, G.M., Tornow, J., McLaughlin, C.S. & Moldave, K. (1988). Properties of

Santoro, R., Li, J. & Grummt, I. (2002). The nucleolar remodeling complex NoRC mediates

Sharpless, N.E., Bardeesy, N., Lee, K.H., Carrasco, D., Castrillon, D.H., Aguirre, A.J., Wu,

Shcherbik, N. & Pestov, D.G. (2010). Ubiquitin and ubiquitin-like proteins in the nucleolus:

Sherr, C.J. & Roberts, J.M. (1999). CDK inhibitors: positive and negative regulators of G1-

Sherwood, S.W., Rush, D., Ellsworth, J.L. & Schimke, R.T. (1988). Defining cellular

Shore, D. (1998). Cellular senescence: lessons from yeast for human aging? Curr Biol *8*, R192-

Shou, W., Azzam, R., Chen, S.L., Huddleston, M.J., Baskerville, C., Charbonneau, H., Annan,

Shou, W., Seol, J.H., Shevchenko, A., Baskerville, C., Moazed, D., Chen, Z.W., Jang, J.,

Siddiqi, I.N., Dodd, J.A., Vu, L., Eliason, K., Oakes, M.L., Keener, J., Moore, R., Young, M.K.

Sinclair, D.A. & Guarente, L. (1997). Extrachromosomal rDNA circles--a cause of aging in

Sirri, V., Hernandez-Verdun, D. & Roussel, P. (2002). Cyclin-dependent kinases govern formation and maintenance of the nucleolus. Journal of Cell Biology *156*, 969-981. Sirri, V., Urcuqui-Inchima, S., Roussel, P. & Hernandez-Verdun, D. (2008). Nucleolus: the

Stancheva, I., Lucchini, R., Koller, T. & Sogo, J.M. (1997). Chromatin structure and

Stefanovsky, V., Langlois, F., Gagnon-Kugler, T., Rothblum, L.I. & Moss, T. (2006). Growth

Stefanovsky, V.Y., Pelletier, G., Hannan, R., Gagnon-Kugler, T., Rothblum, L.I. & Moss, T.

methylation of rat rRNA genes studied by formaldehyde fixation and psoralen

factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling. Mol Cell *21*, 629-

(2001). An immediate response of ribosomal transcription to growth factor

p19Arf predisposes mice to tumorigenesis. Nature *413*, 86-91.

and disassembly of the RENT complex. BMC Mol Biol *3*, 3.

transcription factor UAF. EMBO J *20*, 4512-4521.

cross-linking. Nucleic Acids Res *25*, 1727-1735.

fascinating nuclear body. Histochem Cell Biol *129*, 13-31.

phase progression. Genes Dev *13*, 1501-1512.

multitasking tools for a ribosome factory. Genes Cancer *1*, 681-689.

Biol *8*, 4217-4224.

Genet *32*, 393-396.

9086-9090.

complex. Cell *97*, 233-244.

yeast. Cell *91*, 1033-1042.

195.

639.

promoters cloned randomly from the Saccharomyces cerevisiae genome. Mol Cell

heterochromatin formation and silencing of ribosomal gene transcription. Nat

E.A., Horner, J.W. & DePinho, R.A. (2001). Loss of p16Ink4a with retention of

senescence in IMR-90 cells: a flow cytometric analysis. Proc Natl Acad Sci U S A *85*,

R.S., Carr, S.A. & Deshaies, R.J. (2002). Cdc5 influences phosphorylation of Net1

Charbonneau, H. & Deshaies, R.J. (1999). Exit from mitosis is triggered by Tem1 dependent release of the protein phosphatase Cdc14 from nucleolar RENT

& Nomura, M. (2001). Transcription of chromosomal rRNA genes by both RNA polymerase I and II in yeast uaf30 mutants lacking the 30 kDa subunit of


Poortinga, G., Hannan, K.M., Snelling, H., Walkley, C.R., Jenkins, A., Sharkey, K., Wall, M.,

Poortinga, G., Wall, M., Sanij, E., Siwicki, K., Ellul, J., Brown, D., Holloway, T.P., Hannan,

Prieto, J.L. & McStay, B. (2007). Recruitment of factors linking transcription and processing

Prieto, J.L. & McStay, B. (2008). Pseudo-NORs: a novel model for studying nucleoli. Biochim

Pyronnet, S., Dostie, J. & Sonenberg, N. (2001). Suppression of cap-dependent translation in

Richard, G.F., Kerrest, A. & Dujon, B. (2008). Comparative genomics and molecular dynamics of DNA repeats in eukaryotes. Microbiol Mol Biol Rev *72*, 686-727. Riley, K.J. & Maher, L.J., 3rd (2007). p53 RNA interactions: new clues in an old mystery.

Roussel, P., Andre, C., Comai, L. & Hernandez-Verdun, D. (1996). The rDNA transcription

Rubbi, C.P. & Milner, J. (2003). Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. EMBO J *22*, 6068-6077. Ruggero, D. & Pandolfi, P.P. (2003). Does the ribosome translate cancer? Nat Rev Cancer *3*,

Sahin, E. & Depinho, R.A. (2010). Linking functional decline of telomeres, mitochondria and

Sakai, K., Ohta, T., Minoshima, S., Kudoh, J., Wang, Y., de Jong, P.J. & Shimizu, N. (1995).

Salminen, A. & Kaarniranta, K. (2009). SIRT1 regulates the ribosomal DNA locus: Epigenetic

Sander, E.E. & Grummt, I. (1997). Oligomerization of the transcription termination factor

Sanij, E. & Hannan, R.D. (2009). The role of UBF in regulating the structure and dynamics of

Sanij, E., Poortinga, G., Sharkey, K., Hung, S., Holloway, T.P., Quin, J., Robb, E., Wong, L.H.,

transcriptionally active rDNA chromatin. Epigenetics *4*, 374-382.

active ribosomal RNA genes in mammals. J Cell Biol *183*, 1259-1274.

Human ribosomal RNA gene cluster: identification of the proximal end containing

candles twinkle longevity in the Christmas tree. Biochemical and Biophysical

TTF-I: implications for the structural organization of ribosomal transcription units.

Thomas, W.G., Stefanovsky, V., Moss, T., Rothblum, L., Hannan, K.M., McArthur, G.A., Pearson, R.B. & Hannan, R.D. (2008). UBF levels determine the number of

machinery is assembled during mitosis in active NORs and absent in inactive

granulocyte differentiation. EMBO J *23*, 3325-3335.

transcription in human cells. Genes Dev *21*, 2041-2054.

Nucleic Acids Res *39*, 3267-3281.

Biophys Acta *1783*, 2116-2123.

NORs. J Cell Biol *133*, 235-246.

stem cells during ageing. Nature *464*, 520-528.

Research Communications *378*, 6-9.

Nucleic Acids Res *25*, 1142-1147.

a novel tandem repeat sequence. Genomics *26*, 521-526.

RNA *13*, 1825-1833.

179-192.

mitosis. Genes Dev *15*, 2083-2093.

Brandenburger, Y., Palatsides, M., Pearson, R.B., McArthur, G.A. & Hannan, R.D. (2004). MAD1 and c-MYC regulate UBF and rDNA transcription during

R.D. & McArthur, G.A. (2011). c-MYC coordinately regulates ribosomal gene chromatin remodeling and Pol I availability during granulocyte differentiation.

of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of


The Nucleolus and Ribosomal Genes in Aging and Senescence 207

Visintin, R., Craig, K., Hwang, E.S., Prinz, S., Tyers, M. & Amon, A. (1998). The phosphatase

Wang, B.D., Butylin, P. & Strunnikov, A. (2006). Condensin function in mitotic nucleolar segregation is regulated by rDNA transcription. Cell Cycle *5*, 2260-2267. Warner, J.R. & McIntosh, K.B. (2009). How Common Are Extraribosomal Functions of

Weber, J.D., Taylor, L.J., Roussel, M.F., Sherr, C.J. & Bar-Sagi, D. (1999). Nucleolar Arf

Wong, J.M.Y., Kusdra, L. & Collins, K. (2002). Subnuclear shuttling of human telomerase induced by transformation and DNA damage. Nat Cell Biol *4*, 731-U735. Wright, J.E., Mais, C., Prieto, J.L. & McStay, B. (2006). A role for upstream binding factor in

Yang, Y., Chen, Y., Zhang, C., Huang, H. & Weissman, S.M. (2002). Nucleolar localization of hTERT protein is associated with telomerase function. Exp Cell Res *277*, 201-209. Yoshida, S. & Toh-e, A. (2002). Budding yeast Cdc5 phosphorylates Net1 and assists Cdc14 release from the nucleolus. Biochem Biophys Res Commun *294*, 687-691. Yu, C.E., Oshima, J., Fu, Y.H., Wijsman, E.M., Hisama, F., Alisch, R., Matthews, S., Nakura,

Positional cloning of the Werner's syndrome gene. Science *272*, 258-262. Yuan, X., Zhao, J., Zentgraf, H., Hoffmann-Rohrer, U. & Grummt, I. (2002). Multiple

Yuan, X., Zhou, Y., Casanova, E., Chai, M., Kiss, E., Grone, H.J., Schutz, G. & Grummt, I.

disruption, cell cycle arrest, and p53-mediated apoptosis. Mol Cell *19*, 77-87. Zachariae, W., Schwab, M., Nasmyth, K. & Seufert, W. (1998). Control of cyclin

Zhang, L.F., Huynh, K.D. & Lee, J.T. (2007). Perinucleolar targeting of the inactive X during S phase: evidence for a role in the maintenance of silencing. Cell *129*, 693-706. Zhang, S., Hemmerich, P. & Grosse, F. (2004). Nucleolar localization of the human telomeric

Zhang, Y. & Lu, H. (2009). Signaling to p53: ribosomal proteins find their way. Cancer Cell

Zhang, Y., Yu, Z., Fu, X. & Liang, C. (2002). Noc3p, a bHLH protein, plays an integral role in the initiation of DNA replication in budding yeast. Cell *109*, 849-860. Zhang, Y.P., Wolf, G.W., Bhat, K., Jin, A., Allio, T., Burkhart, W.A. & Xiong, Y. (2003).

Zhu, Q., Meng, L., Hsu, J.K., Lin, T., Teishima, J. & Tsai, R.Y. (2009). GNL3L stabilizes the TRF1 complex and promotes mitotic transition. J Cell Biol *185*, 827-839.

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway. Molecular and Cellular

repeat binding factor 2 (TRF2). J Cell Sci *117*, 3935-3945.

J., Miki, T., Ouais, S., Martin, G.M., Mulligan, J. & Schellenberg, G.D. (1996).

interactions between RNA polymerase I, TIF-IA and TAF(I) subunits regulate preinitiation complex assembly at the ribosomal gene promoter. EMBO Rep *3*,

(2005). Genetic inactivation of the transcription factor TIF-IA leads to nucleolar

ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting

Ribosomal Proteins? Molecular Cell *34*, 3-11.

sequesters Mdm2 and activates p53. Nat Cell Biol *1*, 20-26.

organizing ribosomal gene chromatin. Biochem Soc Symp, 77-84.

Cell *2*, 709-718.

1082-1087.

*16*, 369-377.

Biology *23*, 8902-8912.

complex. Science *282*, 1721-1724.

Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol

stimulation in mammals is mediated by ERK phosphorylation of UBF. Mol Cell *8*, 1063-1073.


Steffan, J.S., Keys, D.A., Dodd, J.A. & Nomura, M. (1996). The role of TBP in rDNA

Stewart, S.A., Ben-Porath, I., Carey, V.J., O'Connor, B.F., Hahn, W.C. & Weinberg, R.A.

Sugimoto, M., Kuo, M.L., Roussel, M.F. & Sherr, C.J. (2003). Nucleolar Arf tumor suppressor

Takemura, M., Ohoka, F., Perpelescu, M., Ogawa, M., Matsushita, H., Takaba, T., Akiyama,

Tissenbaum, H.A. & Guarente, L. (2001). Increased dosage of a sir-2 gene extends lifespan in

Tomlinson, R.L., Ziegler, T.D., Supakorndej, T., Terns, R.M. & Terns, M.P. (2006). Cell cycle-

Trumtel, S., Leger-Silvestre, I., Gleizes, P.E., Teulieres, F. & Gas, N. (2000). Assembly and

Tsai, R.Y.L. (2009). Nucleolar modulation of TRF1 A dynamic way to regulate telomere and

Tsang, C.K., Bertram, P.G., Ai, W., Drenan, R. & Zheng, X.F. (2003). Chromatin-mediated

Tschochner, H. & Hurt, E. (2003). Pre-ribosomes on the road from the nucleolus to the

Tyner, S.D., Venkatachalam, S., Choi, J., Jones, S., Ghebranious, N., Igelmann, H., Lu, X.,

van Koningsbruggen, S., Gierlinski, M., Schofield, P., Martin, D., Barton, G.J., Ariyurek, Y.,

Vigneron, A. & Vousden, K.H. (2010). p53, ROS and senescence in the control of aging.

Vintermist, A., Bohm, S., Sadeghifar, F., Louvet, E., Mansen, A., Percipalle, P. & Ostlund

chromosomes associate with nucleoli. Mol Biol Cell *21*, 3735-3748.

cell cycle by nucleostemin and GNL3L. Cell Cycle *8*, 2912-2916.

inhibits ribosomal RNA processing. Mol Cell *11*, 415-424.

binding to nucleophosmin/B23. Exp Cell Res *276*, 233-241.

Caenorhabditis elegans. Nature *410*, 227-230.

cerevisiae mutants. Mol Biol Cell *11*, 2175-2189.

cytoplasm. Trends Cell Biol *13*, 255-263.

associated phenotypes. Nature *415*, 45-53.

Aging (Albany NY) *2*, 471-474.

PLoS One *6*, e19184.

1063-1073.

2551-2563.

Nat Genet *33*, 492-496.

Cell *17*, 955-965.

6045-6056.

stimulation in mammals is mediated by ERK phosphorylation of UBF. Mol Cell *8*,

transcription by RNA polymerase I in Saccharomyces cerevisiae: TBP is required for upstream activation factor-dependent recruitment of core factor. Genes Dev *10*,

(2003). Erosion of the telomeric single-strand overhang at replicative senescence.

T., Umekawa, H., Furuichi, Y., Cook, P.R. & Yoshida, S. (2002). Phosphorylationdependent migration of retinoblastoma protein into the nucleolus triggered by

regulated trafficking of human telomerase to telomeres. Molecular Biology of the

functional organization of the nucleolus: ultrastructural analysis of Saccharomyces

regulation of nucleolar structure and RNA Pol I localization by TOR. EMBO J *22*,

Soron, G., Cooper, B., Brayton, C., Hee Park, S., Thompson, T., Karsenty, G., Bradley, A. & Donehower, L.A. (2002). p53 mutant mice that display early ageing-

den Dunnen, J.T. & Lamond, A.I. (2010). High-resolution whole-genome sequencing reveals that specific chromatin domains from most human

Farrants, A.K. (2011). The chromatin remodelling complex B-WICH changes the chromatin structure and recruits histone acetyl-transferases to active rRNA genes.


**1. Introduction**

2006).

Senescence, considered from the individual viewpoint can be characterized as a "progressive loss of fertility and increasing probability of death with increasing age"(Kirkwood & Austad, 2000). This phenomenon can also be considered from the populational perspective: senescent populations present increasingly higher death rates with increasing age (Masoro & Austad,

*Department of Pathology, School of Medicine at the University of Sao Paulo, Sao Paulo* 

**10**

*Brazil* 

**Senescence in Animals:** 

**Why Evolutionary Theories Matter** 

Thiago Monaco, Daniel Silvestre and Paulo S.P. Silveira

This is a clearly deleterious process, which seems difficult to conciliate with natural selection, which predicts evolution towards increasing fitness. Historically, the first evolutionary explanation able to conciliate these two processes is known as the mutation accumulation theory (Medawar, 1952). According to this theory, in age-structured populations the force of selection decreases with increasing age, allowing the accumulation of deleterious genes with age-specific effects on mortality rate (Hamilton, 1966). Under population genetics mechanisms, senescence is not necessarily deleterious: the original Medawar's proposition implies that the postponement of age-specific effects of harmful genes is equivalent to their elimination in such a way that they become effectively neutral. Hence, such postponement is

Medawar was convinced that these genes could only account for senescent manifestations encountered in protected populations after they reached ages not achievable in the wild and, therefore, that further explanation involving pleiotropy and linkage would be required to account for a gradual process of organic degeneration, but he did not elaborate on them. This was noted by Williams, who explained the maintenance of beneficial and deleterious traits

Essentially, the antagonistic pleiotropy theory relies on the existence of genes of a special kind, which are capable of increasing and decreasing fitness depending on the somatic environment and/or age. It is not necessary that the beneficial effects precede the deleterious effect as commonly believed e. g. (Futuyma, 1998; Masoro & Austad, 2006). Instead, Williams' original proposition only required an influx of pleiotropic alleles that may fixate in the population due to their overall beneficial effect. In this scenario, the observed senescence is understood as the composition of deleterious components from all present pleiotropic genes (Williams, 1957). Instead of basing his arguments on genetics, a somewhat different view was offered by Kirkwood, elaborating on the error catastrophe of Orgel (Orgel, 1963). He approached senescence from an ecological argument in which energy resources may be allocated either

beneficial and senescence can be regarded as a side effect of the process.

together, giving rise to the antagonistic pleiotropy theory.


## **Senescence in Animals: Why Evolutionary Theories Matter**

Thiago Monaco, Daniel Silvestre and Paulo S.P. Silveira *Department of Pathology, School of Medicine at the University of Sao Paulo, Sao Paulo Brazil* 

## **1. Introduction**

208 Senescence

Zindy, F., Eischen, C.M., Randle, D.H., Kamijo, T., Cleveland, J.L., Sherr, C.J. & Roussel, M.F.

Zomerdijk, J.C., Beckmann, H., Comai, L. & Tjian, R. (1994). Assembly of transcriptionally

apoptosis and immortalization. Genes Dev *12*, 2424-2433.

*266*, 2015-2018.

(1998). Myc signaling via the ARF tumor suppressor regulates p53-dependent

active RNA polymerase I initiation factor SL1 from recombinant subunits. Science

Senescence, considered from the individual viewpoint can be characterized as a "progressive loss of fertility and increasing probability of death with increasing age"(Kirkwood & Austad, 2000). This phenomenon can also be considered from the populational perspective: senescent populations present increasingly higher death rates with increasing age (Masoro & Austad, 2006).

This is a clearly deleterious process, which seems difficult to conciliate with natural selection, which predicts evolution towards increasing fitness. Historically, the first evolutionary explanation able to conciliate these two processes is known as the mutation accumulation theory (Medawar, 1952). According to this theory, in age-structured populations the force of selection decreases with increasing age, allowing the accumulation of deleterious genes with age-specific effects on mortality rate (Hamilton, 1966). Under population genetics mechanisms, senescence is not necessarily deleterious: the original Medawar's proposition implies that the postponement of age-specific effects of harmful genes is equivalent to their elimination in such a way that they become effectively neutral. Hence, such postponement is beneficial and senescence can be regarded as a side effect of the process.

Medawar was convinced that these genes could only account for senescent manifestations encountered in protected populations after they reached ages not achievable in the wild and, therefore, that further explanation involving pleiotropy and linkage would be required to account for a gradual process of organic degeneration, but he did not elaborate on them. This was noted by Williams, who explained the maintenance of beneficial and deleterious traits together, giving rise to the antagonistic pleiotropy theory.

Essentially, the antagonistic pleiotropy theory relies on the existence of genes of a special kind, which are capable of increasing and decreasing fitness depending on the somatic environment and/or age. It is not necessary that the beneficial effects precede the deleterious effect as commonly believed e. g. (Futuyma, 1998; Masoro & Austad, 2006). Instead, Williams' original proposition only required an influx of pleiotropic alleles that may fixate in the population due to their overall beneficial effect. In this scenario, the observed senescence is understood as the composition of deleterious components from all present pleiotropic genes (Williams, 1957).

Instead of basing his arguments on genetics, a somewhat different view was offered by Kirkwood, elaborating on the error catastrophe of Orgel (Orgel, 1963). He approached senescence from an ecological argument in which energy resources may be allocated either

While it is not necessarily clear what the relationship between the physiological and demographic components of senescence is, most "aging genes" described in the literature are simply genes whose variations influence the longevity of the studied species regardless of their physiological effect, and few genes were shown to affect the Mortality Rate Doubling Time (MRDT) of populations of mutants for such genes, and, therefore, to affect the speed of senescence (de Magalhães et al., 2005). Additionally, when strains carrying alleles for many of the so called *longevity genes* are mixed with wild populations, generally the "beneficial" mutation is lost over a few generations, indicating that although such variants increase

Senescence in Animals: Why Evolutionary Theories Matter 211

For these reasons, the first decision before staring to seek for "aging genes" should be which model of senescence to assume. Otherwise, we might not know how to interpret the findings in a coherent way: suppose that human carriers of a given mutation have an increase of 5% in their annual mortality from 30 years of age – are them carriers of a genetic disease or of a

Fig. 1. Three different models for the relationship between physiological and demographic senescence on the genetic architecture of senescence. (a) Genes negatively influence

physiological processes, which, then, lead to increasing effects on age-specific mortality. (b) The same genes that lead to physiological senescence independently lead to increasing age-dependent death rates, which are demografically measurable. (c) Different genes operate over physiological and demographic processes that are linked with senescence. Extracted from *Promislow, D. E. L. et al. Evolutionary Biology of Aging: Future Directions. In: Masoro EJ, Austad SN (Ed.). Handbook of the Biology of Aging. 6th. ed. San Diego: Academic Press, 2006.*

If we suppose that senescence is a unique genetic phenomenon whose physiological effects lead to its demographic aspects (Figure 1 (a)), then "genes of senescence" should exert age-dependent deleterious effects in the physiology of organisms, and because more frail individuals are more prone to dye from a given insult, such genes would also increase

longevity, they may exert a deleterious effect for fitness (Promislow et al., 2006).

deleterious mutation in a senescence pathway?

*217-242.*

mortality from their ages of onset.

to somatic cell maintenance or to reproduction, thus generating some sort of soma-germ conflict. Called disposable soma theory, it ultimately relies on the existence of specific genes controlling the accuracy of the transcription/translation machinery in an age-dependent manner. Kirkwood himself regards his theory as a specialization of the antagonistic pleiotropy of Williams (Kirkwood, 1977; Kirkwood & Holliday, 1975a;b; 1979). The difference is that Williams invokes the existence of genes responsible for beneficial and deleterious effects, but Kirkwood's theory, while not denying the existence of these genes, does not require them. The conflicting destination of energy either to body or reproduction maintenance would suffice for the evolution of senescence.

Senescence is a process that causes animals to become progressively less fertile (Medawar, 1946) and more vulnerable (Comfort, 1956) with age. It has long been noticed that senescence-associated frailty causes population death rates to rise exponentially with age (Gompertz, 1825).

Although a number of evidences have since been collected in support of each of these theories, in the last decades some phenomena have challenged all of them. This includes the effect of caloric restriction on longevity, the late-life mortality deceleration and the longevity pathways controlled by either a single or a few genes, such as the insulin pathway and the effect of sirtuins on longevity.

#### **2. Measuring senescence**

Although generally considered together, it is useful to take some time to consider the effects of senescence on individuals' survival and fertility (physiological senescence) or on populational survival curves (demographic senescence) separetly. By not doing so, the researcher may unwittingly take the risk of assuming demographic senescence to stem directly from physiological senescence. Although it might well be the case, there is no theoretical reason why it must be so.

The fact is that the genetic architecture of senescence, i.e., which genes are related to which measurable effects that we call senescence and how they relate to each other, will dictate the relationship between physiological and demographic senescence(s).

#### **2.1 What is the genetic architecture of senescence?**

*Genetic architecture* refers to the genetic basis of a phenotypic trait. Beyond comprehending the map of the genes linked to a given trait, genetic architecture considers all phenomena through which such genetic map produces the phenotype Masoro & Austad (2006).

The most common definition of the senescent phenotype combines individual effects (decrease in functional and reproductive abilities) with an effect which is measurable only in a population (age-dependent increase in mortality). This often leads us to conclude that it is exactly the same phenomenon that makes us individually more fragile and at greater risk of dying as we age.

Figure 1 shows that this is only one of the possible relationships between physiological senescence (progressive fall on functional capacity and fertility) and demographic senescence (increased mortality accompaining chronological aging) (Promislow et al., 2006).

2 Will-be-set-by-IN-TECH

to somatic cell maintenance or to reproduction, thus generating some sort of soma-germ conflict. Called disposable soma theory, it ultimately relies on the existence of specific genes controlling the accuracy of the transcription/translation machinery in an age-dependent manner. Kirkwood himself regards his theory as a specialization of the antagonistic pleiotropy of Williams (Kirkwood, 1977; Kirkwood & Holliday, 1975a;b; 1979). The difference is that Williams invokes the existence of genes responsible for beneficial and deleterious effects, but Kirkwood's theory, while not denying the existence of these genes, does not require them. The conflicting destination of energy either to body or reproduction maintenance would suffice for

Senescence is a process that causes animals to become progressively less fertile (Medawar, 1946) and more vulnerable (Comfort, 1956) with age. It has long been noticed that senescence-associated frailty causes population death rates to rise exponentially with age

Although a number of evidences have since been collected in support of each of these theories, in the last decades some phenomena have challenged all of them. This includes the effect of caloric restriction on longevity, the late-life mortality deceleration and the longevity pathways controlled by either a single or a few genes, such as the insulin pathway and the effect of

Although generally considered together, it is useful to take some time to consider the effects of senescence on individuals' survival and fertility (physiological senescence) or on populational survival curves (demographic senescence) separetly. By not doing so, the researcher may unwittingly take the risk of assuming demographic senescence to stem directly from physiological senescence. Although it might well be the case, there is no

The fact is that the genetic architecture of senescence, i.e., which genes are related to which measurable effects that we call senescence and how they relate to each other, will dictate the

*Genetic architecture* refers to the genetic basis of a phenotypic trait. Beyond comprehending the map of the genes linked to a given trait, genetic architecture considers all phenomena through

The most common definition of the senescent phenotype combines individual effects (decrease in functional and reproductive abilities) with an effect which is measurable only in a population (age-dependent increase in mortality). This often leads us to conclude that it is exactly the same phenomenon that makes us individually more fragile and at greater risk

Figure 1 shows that this is only one of the possible relationships between physiological senescence (progressive fall on functional capacity and fertility) and demographic senescence

(increased mortality accompaining chronological aging) (Promislow et al., 2006).

relationship between physiological and demographic senescence(s).

which such genetic map produces the phenotype Masoro & Austad (2006).

**2.1 What is the genetic architecture of senescence?**

the evolution of senescence.

(Gompertz, 1825).

sirtuins on longevity.

of dying as we age.

**2. Measuring senescence**

theoretical reason why it must be so.

While it is not necessarily clear what the relationship between the physiological and demographic components of senescence is, most "aging genes" described in the literature are simply genes whose variations influence the longevity of the studied species regardless of their physiological effect, and few genes were shown to affect the Mortality Rate Doubling Time (MRDT) of populations of mutants for such genes, and, therefore, to affect the speed of senescence (de Magalhães et al., 2005). Additionally, when strains carrying alleles for many of the so called *longevity genes* are mixed with wild populations, generally the "beneficial" mutation is lost over a few generations, indicating that although such variants increase longevity, they may exert a deleterious effect for fitness (Promislow et al., 2006).

For these reasons, the first decision before staring to seek for "aging genes" should be which model of senescence to assume. Otherwise, we might not know how to interpret the findings in a coherent way: suppose that human carriers of a given mutation have an increase of 5% in their annual mortality from 30 years of age – are them carriers of a genetic disease or of a deleterious mutation in a senescence pathway?

Fig. 1. Three different models for the relationship between physiological and demographic senescence on the genetic architecture of senescence. (a) Genes negatively influence physiological processes, which, then, lead to increasing effects on age-specific mortality. (b) The same genes that lead to physiological senescence independently lead to increasing age-dependent death rates, which are demografically measurable. (c) Different genes operate over physiological and demographic processes that are linked with senescence. Extracted from *Promislow, D. E. L. et al. Evolutionary Biology of Aging: Future Directions. In: Masoro EJ, Austad SN (Ed.). Handbook of the Biology of Aging. 6th. ed. San Diego: Academic Press, 2006. 217-242.*

If we suppose that senescence is a unique genetic phenomenon whose physiological effects lead to its demographic aspects (Figure 1 (a)), then "genes of senescence" should exert age-dependent deleterious effects in the physiology of organisms, and because more frail individuals are more prone to dye from a given insult, such genes would also increase mortality from their ages of onset.

that the removal of older, weaker and less fertile individuals from a population would enhance the survival of younger individuals and overall reproduction of the species (Weismann, 1889). Realizing his argument was circular (since it depended on older individuals being weaker and less fertile for senescence to evolve) Weismann withdrew his theory (Weismann, 1892).

Senescence in Animals: Why Evolutionary Theories Matter 213

More than half a century later, Medawar proposed the mutation accumulation theory of senescence (Medawar, 1952). He realized that even in an imaginary non senescent population, older individuals would be very rare simply because the cumulative incidence of death is necessarily dependent of age. This means that late acting mutations will affect population fitness only in the proportion of surviving individuals after such late ages. In other words, the force of natural selection decreases with age and deleterious mutations with effects that are

For Medawar, this would explain the existence of deleterious mutations fixed in ages to which individuals of a given species are not expected to survive in nature. Senescence evolved through such a process would only be observable in protected species, such as laboratory animals or our own species. Medawar, nonetheless, believed that animals did senesce in nature and, therefore felt the need for an early benefit to explain how a not so late acting

This was developed into the antagonistic pleiotropy theory (Williams, 1957). In short, Williams proposed that the fitness associated to mutations with more than one effect is the average fitness. Therefore, a mutation with earlier beneficial effects and later deleterious ones could be fixed by natural selection if the overall fitness be positive. Deleterious effects early enough to impact mortality in nature could be compensated by beneficial effects. Williams' theory depended on the existence of such special, pleiotropic genes, in numbers sufficient to

In 1977, elaborating on the mechanistic error catastrophe theory of Orgel (1963), Kirkwood proposed an ecological argument for the evolution of senescence (Kirkwood, 1977). Since evolution is centered on reproduction and not directly in survival, the energetic and metabolic cost of maintenance and repair could affect reproduction negatively if taken to perfection. Therefore, the level of body maintenance and repair that can evolve is the minimum to assure reproduction. Any deleterious mutation that do not decrease reproduction can not only be neutral, but can enhance fitness if it results in more reproductive resources. This is the

These three theories are not mutually excluding, and can explain different aspects of the

According to Haldane, a deleterious mutation with effect only on later ages may escape natural selection, because either most individuals will be dead or will have reproduced at such ages. For Haldane, this implies in the fall of the force of natural selection with advancing ages (Haldane, 1941). Nevertheless, he failed to turn this observation into a theory of senescence. It was Medawar who would do so. The gap between Haldane's observation of the falling force of natural selection and an evolutionary theory of senescence relies on the requirement of an age structure on populations for the fell in the force of selection. It is natural to suppose an

late enough are in fact neutral mutations, which could randomly accumulate.

explain the observed progressive increase in the effects of senescence.

deleterious mutation could evolve to fixation.

disposable soma theory of senescence.

evolution of senescence (Kirkwood & Austad, 2000).

**3.2 The mutation accumulation theory of senescence**

On the other hand, genes that determine effects on demographic senescence may exert independent effects on the physiology of organisms. Such effects might not be linked to the demographic effects of the same genes (Figure 1 (b)).

Finally, physiological senescence could be genetically independent from demographic senescence, so that there would be a "genetic modularity" between the two phenomena, in which different groups of genes participate in each process (Figure 1 (c)).

This text assumes when necessary that genes linked to physiological senescence may impact probability of death ( 1 (a)). It does so relying on the fact that there is little evidence that there may be a genetic variability to the age-dependent physiological decline without its influencing on demographic senescence (Wessells et al., 2004).

Once delimited the senescent phenotype, we review some genetic phenomena that may have importance for the genetic architecture of senescence. Such phenomena include:


Epistasis could function similarly to what is predicted on antagonistic pleiotropy theory: assuming two genes with positive effects for fitness, in which the first gene exerts a negative effect on the expression of the second gene, the first gene would have positive and negative effects on fitness The effect under selection, however, would be the average effect.

It is believed, since the formulation of the theory of mutation accumulation by Medawar, that senescence is a polygenic phenotype (Medawar, 1952). Indeed, recent decades have seen the description of "hundreds of aging genes" (Promislow et al., 2006). Summed to the fact that senescence is an early onset and gradually progressive phenotype in almost all of the species that has been described, it points to a polygenic inheritance with almost-continuity in organic response to genes that determine senescence.

#### **3. The evolutionary theories of senescence**

#### **3.1 Introduction**

It has always been difficult to conciliate senescence with natural selection, a biological mechanism generally expected to increase population fitness. Although acknowledged by Darwin (1872), the first tentative explanation for the evolution of senescence was offered by August Weismann in 1881. For Weismann, senescence had evolved for the good of species, in 4 Will-be-set-by-IN-TECH

On the other hand, genes that determine effects on demographic senescence may exert independent effects on the physiology of organisms. Such effects might not be linked to the

Finally, physiological senescence could be genetically independent from demographic senescence, so that there would be a "genetic modularity" between the two phenomena, in

This text assumes when necessary that genes linked to physiological senescence may impact probability of death ( 1 (a)). It does so relying on the fact that there is little evidence that there may be a genetic variability to the age-dependent physiological decline without its influencing

Once delimited the senescent phenotype, we review some genetic phenomena that may have

• Epistasis, when the expression of a gene negatively influences the expression of one

• Plasticity, when a single genotype can produce more than one distinct phenotype, such phenotypic diversity may occur among individuals of the same genotype, by action of different environmental influences on the same individual or *in the same individual at*

• Evolvability, when genotypic variations of a phenotype exist in a population and can lead to different degrees of adaptability, so that environmental changes will lead to

Epistasis could function similarly to what is predicted on antagonistic pleiotropy theory: assuming two genes with positive effects for fitness, in which the first gene exerts a negative effect on the expression of the second gene, the first gene would have positive and negative

It is believed, since the formulation of the theory of mutation accumulation by Medawar, that senescence is a polygenic phenotype (Medawar, 1952). Indeed, recent decades have seen the description of "hundreds of aging genes" (Promislow et al., 2006). Summed to the fact that senescence is an early onset and gradually progressive phenotype in almost all of the species that has been described, it points to a polygenic inheritance with almost-continuity in organic

It has always been difficult to conciliate senescence with natural selection, a biological mechanism generally expected to increase population fitness. Although acknowledged by Darwin (1872), the first tentative explanation for the evolution of senescence was offered by August Weismann in 1881. For Weismann, senescence had evolved for the good of species, in

effects on fitness The effect under selection, however, would be the average effect.

• Pleiotropy, when multiple phenotypic characteristics are influenced by a single gene;

importance for the genetic architecture of senescence. Such phenomena include:

• Quasi-continuity, while a variation in a gene affects minimally a phenotype;

• Polygyny, where multiple genes contribute to a phenotypic trait;

which different groups of genes participate in each process (Figure 1 (c)).

demographic effects of the same genes (Figure 1 (b)).

on demographic senescence (Wessells et al., 2004).

response to genes that determine senescence.

**3. The evolutionary theories of senescence**

another;

*different ages*;

readaptations.

**3.1 Introduction**

that the removal of older, weaker and less fertile individuals from a population would enhance the survival of younger individuals and overall reproduction of the species (Weismann, 1889).

Realizing his argument was circular (since it depended on older individuals being weaker and less fertile for senescence to evolve) Weismann withdrew his theory (Weismann, 1892).

More than half a century later, Medawar proposed the mutation accumulation theory of senescence (Medawar, 1952). He realized that even in an imaginary non senescent population, older individuals would be very rare simply because the cumulative incidence of death is necessarily dependent of age. This means that late acting mutations will affect population fitness only in the proportion of surviving individuals after such late ages. In other words, the force of natural selection decreases with age and deleterious mutations with effects that are late enough are in fact neutral mutations, which could randomly accumulate.

For Medawar, this would explain the existence of deleterious mutations fixed in ages to which individuals of a given species are not expected to survive in nature. Senescence evolved through such a process would only be observable in protected species, such as laboratory animals or our own species. Medawar, nonetheless, believed that animals did senesce in nature and, therefore felt the need for an early benefit to explain how a not so late acting deleterious mutation could evolve to fixation.

This was developed into the antagonistic pleiotropy theory (Williams, 1957). In short, Williams proposed that the fitness associated to mutations with more than one effect is the average fitness. Therefore, a mutation with earlier beneficial effects and later deleterious ones could be fixed by natural selection if the overall fitness be positive. Deleterious effects early enough to impact mortality in nature could be compensated by beneficial effects. Williams' theory depended on the existence of such special, pleiotropic genes, in numbers sufficient to explain the observed progressive increase in the effects of senescence.

In 1977, elaborating on the mechanistic error catastrophe theory of Orgel (1963), Kirkwood proposed an ecological argument for the evolution of senescence (Kirkwood, 1977). Since evolution is centered on reproduction and not directly in survival, the energetic and metabolic cost of maintenance and repair could affect reproduction negatively if taken to perfection. Therefore, the level of body maintenance and repair that can evolve is the minimum to assure reproduction. Any deleterious mutation that do not decrease reproduction can not only be neutral, but can enhance fitness if it results in more reproductive resources. This is the disposable soma theory of senescence.

These three theories are not mutually excluding, and can explain different aspects of the evolution of senescence (Kirkwood & Austad, 2000).

#### **3.2 The mutation accumulation theory of senescence**

According to Haldane, a deleterious mutation with effect only on later ages may escape natural selection, because either most individuals will be dead or will have reproduced at such ages. For Haldane, this implies in the fall of the force of natural selection with advancing ages (Haldane, 1941). Nevertheless, he failed to turn this observation into a theory of senescence.

It was Medawar who would do so. The gap between Haldane's observation of the falling force of natural selection and an evolutionary theory of senescence relies on the requirement of an age structure on populations for the fell in the force of selection. It is natural to suppose an

It is to note that the very existence of pleiotropic genes with antagonistic effects was not postulated by Williams. In a previous article, Sewall Wright describes an equation for

Senescence in Animals: Why Evolutionary Theories Matter 215

where *W* is the fitness of a gene and *S*1, *S*<sup>2</sup> ... *Sn* are the separate selective coefficients for each

Williams' merit was to note the implication of Wright's equation for the evolution of senescence in age structured populations. He applied to the Equation 1 the same reasoning applied by Medawar in relation to the age structure of populations: the magnitude of the effect of *Sn* of a gene may be reduced if it only starts at advanced ages. In a gene capable of expressing different effects on different ages, later effects will less be subject to natural

By considering the effects of a given gene in distinct ages of expression, however, Williams proposed that the measure of the magnitude of each effect in question (advantages or

where *Sn* is the effect under consideration, *mn* its magnitude or impact on fitness and *pn* is the proportion of a population's reproductive probability that is *relevant* to the age of

This allowed him to rewrite the Equation 1 considering the effect of age structure in the final

From this equation we may extract the simplest case: the one of a pleiotropic gene with a late deleterious effect and a very early beneficial effect. To demonstrate the formula we need to

Let us imagine a population (structured for simplicity on a human age-scale) with constant birth and death. This is necessary not to create an *ad hoc* argument, by starting with a previously non-senescent population and therefore with no age differences in mortality.

Let's say that this population has a constant mortality of 0.25 each 4 years, ie, 0.0625 per year, and that each 4-years extract is composed of 1000 individuals. This population age distribution is represented in Figure 2. The familiarity of this age distribution with any wild population is noticiable, as it is with any high-mortality human population (as an example,

In this non-senescent population, organisms do not lose fertility with the progression of age and all individuals have the same reproductive probability. Therefore, *px*, i.e., the proportion of the reproductive probability associated with each age will be the proportion of remaining individuals with ages equal or superior to that in the population, since the effect of *S* remain

Williams could thus formulate an evolutionary hypothesis for the permanence (regardless of natural selection) of detrimental effects whose expression was sufficiently early to be influential in wild populations. It is important to notice that neither the equation nor any

the Figure 3 represents the age distribution of the population of Afghanistan in 2008).

*W* = (1 + *S*1)(1 + *S*2)...(1 + *Sn*) , (1)

*Sn* = *mn pn* , (2)

*W* = (1 + *m*<sup>1</sup> *p*1)(1 + *m*<sup>2</sup> *p*2)...(1 + *mn pn*) . (3)

calculating the impact of a pleiotropic gene on fitness:

age-specific effect of such gene on fitness (Wright, 1956).

selection than earlier effects.

disadvantages) is given by

manifestation of the effect *Sn*.

selective coefficient of a pleiotropic gene:

after activated, i.e., be constant from its manifestation.

know the values of *p* for each age.

age structure with many young individuals and rare older ones if senescence exists. In such a population, Haldane's explanation for Huntington's Disease works well, but such a model, which already pressuposes senescence, cannot acount for its evolutionary origins.

Medawar postulated that age-independent environmental hazards such as hunger, predation, accidents, etc. were a sufficient condition for the establishment of populational age structures. Older individuals, Medawar claimed, were rare because they have been exposed to such risks (termed extrinsic mortality) longer than young individuals. In other words, the existence of age structures in wild populations is a function of environmental hazards and not of senescence. Even a non senescent population would have an age structure (Medawar, 1952).

The importance of Medawar's reasoning is that older individuals in age structured populations, being necessarily rarer than younger ones, not only do not compete for environmental resources: they also don't contribute much offspring to newer generations. This is the key for Medawar's mutation accumulation theory of senescence – and also a basis for the next two hypotheses, antagonistic pleiotropy and disposable soma theories of senescence.

#### Figure 3 (age structured population)

Deleterious mutations, provided that it effects happen in sufficiently late ages, could accumulate in the genome According to mechanisms of population genetics, senescence is not necessarily harmful: Medawar's original proposition implies that the postponement of the effects of age-specific deleterious genes for late ages is equivalent to their elimination. Thus, these genes become effectively neutral (Medawar, 1952).

According to this theory, such evolutionary mechanism could only explain the manifestations found in senescent populations protected after individuals reach ages above those found in nature, since in nature, the age of accumulation would coincide with the maximum age of living individuals.

Medawar accepted that the effects of senescence also occurred at ages commonly found in nature. For this reason, he became convinced that another mechanism, involving either pleiotropy or linkage, would be necessary to explain the process of early and gradual degeneration which is charachteristic of senescence (Medawar, 1952). Medawar, however, didn't advance more details on this hypothesis.

#### **3.3 The antagonistic pleiotropy theory**

Although the accumulation of mutations justifies the existence of deleterious genes with late expression (and thus the already established senescent state), it didn't seem to explain the slow onset of senescence (Williams, 1957).

Seeking to understand how deleterious genetic effects expressed in relatively early ages could escape selection, Williams grounded his theory on four assumptions: the existence of a somatic cell line, i.e., non-transferable in whole or part by sexual or asexual reproduction, the natural selection of different alleles at a population, a decreasing probability of reproduction with increasing ages, the existence of pleiotropic genes with different effects on fitness at distinct ages (antagonistic pleiotropy). According to this idea, the evolutionary fundamental process to the establishment of senescence is a selective action on the inheritance of a gene with antagonistic effects on its carrier's fitness (Williams, 1957).

6 Will-be-set-by-IN-TECH

age structure with many young individuals and rare older ones if senescence exists. In such a population, Haldane's explanation for Huntington's Disease works well, but such a model,

Medawar postulated that age-independent environmental hazards such as hunger, predation, accidents, etc. were a sufficient condition for the establishment of populational age structures. Older individuals, Medawar claimed, were rare because they have been exposed to such risks (termed extrinsic mortality) longer than young individuals. In other words, the existence of age structures in wild populations is a function of environmental hazards and not of senescence. Even a non senescent population would have an age structure (Medawar, 1952). The importance of Medawar's reasoning is that older individuals in age structured populations, being necessarily rarer than younger ones, not only do not compete for environmental resources: they also don't contribute much offspring to newer generations. This is the key for Medawar's mutation accumulation theory of senescence – and also a basis for the next two hypotheses, antagonistic pleiotropy and disposable soma theories of

Deleterious mutations, provided that it effects happen in sufficiently late ages, could accumulate in the genome According to mechanisms of population genetics, senescence is not necessarily harmful: Medawar's original proposition implies that the postponement of the effects of age-specific deleterious genes for late ages is equivalent to their elimination.

According to this theory, such evolutionary mechanism could only explain the manifestations found in senescent populations protected after individuals reach ages above those found in nature, since in nature, the age of accumulation would coincide with the maximum age of

Medawar accepted that the effects of senescence also occurred at ages commonly found in nature. For this reason, he became convinced that another mechanism, involving either pleiotropy or linkage, would be necessary to explain the process of early and gradual degeneration which is charachteristic of senescence (Medawar, 1952). Medawar, however,

Although the accumulation of mutations justifies the existence of deleterious genes with late expression (and thus the already established senescent state), it didn't seem to explain the

Seeking to understand how deleterious genetic effects expressed in relatively early ages could escape selection, Williams grounded his theory on four assumptions: the existence of a somatic cell line, i.e., non-transferable in whole or part by sexual or asexual reproduction, the natural selection of different alleles at a population, a decreasing probability of reproduction with increasing ages, the existence of pleiotropic genes with different effects on fitness at distinct ages (antagonistic pleiotropy). According to this idea, the evolutionary fundamental process to the establishment of senescence is a selective action on the inheritance of a gene

which already pressuposes senescence, cannot acount for its evolutionary origins.

senescence.

living individuals.

Figure 3 (age structured population)

Thus, these genes become effectively neutral (Medawar, 1952).

with antagonistic effects on its carrier's fitness (Williams, 1957).

didn't advance more details on this hypothesis.

**3.3 The antagonistic pleiotropy theory**

slow onset of senescence (Williams, 1957).

It is to note that the very existence of pleiotropic genes with antagonistic effects was not postulated by Williams. In a previous article, Sewall Wright describes an equation for calculating the impact of a pleiotropic gene on fitness:

$$W = (1 + S\_1)(1 + S\_2)...(1 + S\_n) \, . \tag{1}$$

where *W* is the fitness of a gene and *S*1, *S*<sup>2</sup> ... *Sn* are the separate selective coefficients for each age-specific effect of such gene on fitness (Wright, 1956).

Williams' merit was to note the implication of Wright's equation for the evolution of senescence in age structured populations. He applied to the Equation 1 the same reasoning applied by Medawar in relation to the age structure of populations: the magnitude of the effect of *Sn* of a gene may be reduced if it only starts at advanced ages. In a gene capable of expressing different effects on different ages, later effects will less be subject to natural selection than earlier effects.

By considering the effects of a given gene in distinct ages of expression, however, Williams proposed that the measure of the magnitude of each effect in question (advantages or disadvantages) is given by

$$S\_n = m\_n p\_n \, \tag{2}$$

where *Sn* is the effect under consideration, *mn* its magnitude or impact on fitness and *pn* is the proportion of a population's reproductive probability that is *relevant* to the age of manifestation of the effect *Sn*.

This allowed him to rewrite the Equation 1 considering the effect of age structure in the final selective coefficient of a pleiotropic gene:

$$W = (1 + m\_1 p\_1)(1 + m\_2 p\_2)...(1 + m\_n p\_n) \,. \tag{3}$$

From this equation we may extract the simplest case: the one of a pleiotropic gene with a late deleterious effect and a very early beneficial effect. To demonstrate the formula we need to know the values of *p* for each age.

Let us imagine a population (structured for simplicity on a human age-scale) with constant birth and death. This is necessary not to create an *ad hoc* argument, by starting with a previously non-senescent population and therefore with no age differences in mortality.

Let's say that this population has a constant mortality of 0.25 each 4 years, ie, 0.0625 per year, and that each 4-years extract is composed of 1000 individuals. This population age distribution is represented in Figure 2. The familiarity of this age distribution with any wild population is noticiable, as it is with any high-mortality human population (as an example, the Figure 3 represents the age distribution of the population of Afghanistan in 2008).

In this non-senescent population, organisms do not lose fertility with the progression of age and all individuals have the same reproductive probability. Therefore, *px*, i.e., the proportion of the reproductive probability associated with each age will be the proportion of remaining individuals with ages equal or superior to that in the population, since the effect of *S* remain after activated, i.e., be constant from its manifestation.

Williams could thus formulate an evolutionary hypothesis for the permanence (regardless of natural selection) of detrimental effects whose expression was sufficiently early to be influential in wild populations. It is important to notice that neither the equation nor any

long life. "

genes.

selection.

females (Sgro & Partridge, 1999).

as much as we do (Kirkwood & Rose, 1991).

**3.4 The disposable soma theory**

the evolution of senescence.

allocation between reproduction and body maintenance.

effects of these genes will be evident only in subjects in protected environments that enable a

Senescence in Animals: Why Evolutionary Theories Matter 217

While Medawar discussed the age of onset of a characteristic deleterious and Williams called attention to the magnitude of genetic effects, no theory explicited discussed the influence of the magnitude of extrinsic mortality and random genetic drift on the selection of deleterious

From the 1970s, a new theory for the evolution of senescence was proposed by Thomas Kirkwood (Kirkwood & Holliday, 1975a;b), reasoning initially not about evolutionary mechanisms as maladaptive as Medawar or on pleiotropic genes as Williams, but on a strong ecological basis. Elaborating on the theory Orgell's "error catastrophe " (Orgel, 1963), Kirkwood gave us a somewhat different view from what had previously been formulated on

Kirkwood addressed the issue of senescence under an ecological constraint in which energy resources available to individuals could be allocated either for maintenance somatic cells or for reproduction, generating a soma-germ conflict. Called disposable soma theory, this ultimately depends on the existence of specific genes that either influence or control the precision of the genetic replication / transcription and translation machineries in an age-dependent fashion; Kirkwood himself considered his theory as a specialization of the antagonistic pleiotropy theory of Williams (Kirkwood, 1977; Kirkwood & Holliday, 1979). The difference is that Williams posits the existence of genes for beneficial and deleterious effects, but the theory of Kirkwood, despite not denying the existence of these genes, do not need them. If, under Williams theory, natural selection would act on the average effects of a selected gene's mutant alleles, under Kirkwood's assumption a single genetic effect, determining the distribution of resources between reproduction and body maintenance would be sufficient for the evolution of senescence. Senescence would be the inevitable result of selection for an "'ideal" energy

This is the first theory to propose that the evolution of organisms can optimize the allocation of metabolic resources between the maintenance of the somatic lineage (the individual itself) and the effort of reproduction (investment in next generation). Under it, the physiological mechanisms that postpone senescence consume metabolic resources, which become less available for reproduction, and vice versa. As reproduction of the species by natural selection is prioritized, the body is "disposable" after its reproductive function has been sufficiently fulfilled, and the aging process may appear, without sufficient opposition from natural

There is some experimental support for this theory. When fruit flies are selected for a longer life expectancy, there is decrease in fertility. Conversely, exposure of females to earlier reproduction was correlated with a decline in their lifetimes as compared to virgin

The disposable soma theory changes the fundamental question about the evolution of senescence: instead of questioning why we senesce, we could wonder why we, humans, live

Fig. 2. Age distribution of an alleged non-senescent population exposed to a mortality of 6.25% per year.

Fig. 3. Age distribution of the Afghan population in 2008 (data from U.S. Census, International Database, available at http://www.census.gov/).

comment on Williams' classic article (Williams, 1957) infer what the number of effects of a pleiotropic gene should be, nor which effect, beneficial or deleterious should happen earlier. While it is clear that the simplest case of antagonistic pleiotropy would be of a gene with an early beneficial effect and a late deleterious one, Equation 3 does not imply that this is the only possibility.

The literature, however, did not follow this conclusion. Williams' antagonistic pleiotropy came to be regarded precisely as the case in which a mutation with two actions, an early advantage and a later disadvantage, it is positively selected. It is worth quoting verbatim the concept of antagonistic pleiotropy found in the *Handbook of Biological Aging*, of 2006, in a chapter written by the editor of the book (Masoro, 2006):

"Another genetic mechanism, proposed by Williams (1957), is referred to as antagonistic pleiotropy. It proposes that those genes that increase evolutionary fitness in early life will be selected for, even if they have catastrophic deleterious effects in late life. Again, the deleterious effects of these genes will be evident only in subjects in protected environments that enable a long life. "

While Medawar discussed the age of onset of a characteristic deleterious and Williams called attention to the magnitude of genetic effects, no theory explicited discussed the influence of the magnitude of extrinsic mortality and random genetic drift on the selection of deleterious genes.

#### **3.4 The disposable soma theory**

8 Will-be-set-by-IN-TECH

Fig. 2. Age distribution of an alleged non-senescent population exposed to a mortality of

Fig. 3. Age distribution of the Afghan population in 2008 (data from U.S. Census,

comment on Williams' classic article (Williams, 1957) infer what the number of effects of a pleiotropic gene should be, nor which effect, beneficial or deleterious should happen earlier. While it is clear that the simplest case of antagonistic pleiotropy would be of a gene with an early beneficial effect and a late deleterious one, Equation 3 does not imply that this is the only

The literature, however, did not follow this conclusion. Williams' antagonistic pleiotropy came to be regarded precisely as the case in which a mutation with two actions, an early advantage and a later disadvantage, it is positively selected. It is worth quoting verbatim the concept of antagonistic pleiotropy found in the *Handbook of Biological Aging*, of 2006, in a

"Another genetic mechanism, proposed by Williams (1957), is referred to as antagonistic pleiotropy. It proposes that those genes that increase evolutionary fitness in early life will be selected for, even if they have catastrophic deleterious effects in late life. Again, the deleterious

International Database, available at http://www.census.gov/).

chapter written by the editor of the book (Masoro, 2006):

6.25% per year.

possibility.

From the 1970s, a new theory for the evolution of senescence was proposed by Thomas Kirkwood (Kirkwood & Holliday, 1975a;b), reasoning initially not about evolutionary mechanisms as maladaptive as Medawar or on pleiotropic genes as Williams, but on a strong ecological basis. Elaborating on the theory Orgell's "error catastrophe " (Orgel, 1963), Kirkwood gave us a somewhat different view from what had previously been formulated on the evolution of senescence.

Kirkwood addressed the issue of senescence under an ecological constraint in which energy resources available to individuals could be allocated either for maintenance somatic cells or for reproduction, generating a soma-germ conflict. Called disposable soma theory, this ultimately depends on the existence of specific genes that either influence or control the precision of the genetic replication / transcription and translation machineries in an age-dependent fashion; Kirkwood himself considered his theory as a specialization of the antagonistic pleiotropy theory of Williams (Kirkwood, 1977; Kirkwood & Holliday, 1979). The difference is that Williams posits the existence of genes for beneficial and deleterious effects, but the theory of Kirkwood, despite not denying the existence of these genes, do not need them. If, under Williams theory, natural selection would act on the average effects of a selected gene's mutant alleles, under Kirkwood's assumption a single genetic effect, determining the distribution of resources between reproduction and body maintenance would be sufficient for the evolution of senescence. Senescence would be the inevitable result of selection for an "'ideal" energy allocation between reproduction and body maintenance.

This is the first theory to propose that the evolution of organisms can optimize the allocation of metabolic resources between the maintenance of the somatic lineage (the individual itself) and the effort of reproduction (investment in next generation). Under it, the physiological mechanisms that postpone senescence consume metabolic resources, which become less available for reproduction, and vice versa. As reproduction of the species by natural selection is prioritized, the body is "disposable" after its reproductive function has been sufficiently fulfilled, and the aging process may appear, without sufficient opposition from natural selection.

There is some experimental support for this theory. When fruit flies are selected for a longer life expectancy, there is decrease in fertility. Conversely, exposure of females to earlier reproduction was correlated with a decline in their lifetimes as compared to virgin females (Sgro & Partridge, 1999).

The disposable soma theory changes the fundamental question about the evolution of senescence: instead of questioning why we senesce, we could wonder why we, humans, live as much as we do (Kirkwood & Rose, 1991).

Hughes and others have found evidence in favor of mutation accumulation in the experimental evolution of accelerated senescence (decrease in MRDT) in fruit flies as the predominant phenomenon (Hughes et al., 2002), argument which is sustained by Cortopassi in relation to human senescence (Cortopassi, 2002). Physiological senescence in our species, however, is easily noticeable between the fourth and fifth decades of life. This is not incompatible with Medawar's observation (Medawar, 1952) that the accumulation of mutations explain only senescence in artificially protected populations: modern man is an

Senescence in Animals: Why Evolutionary Theories Matter 219

Pleiotropic mechanisms have currently been described. The gene for the juvenile hormone (JH) found in specimens of wild Drosophila is expressed early in its life cycle, and takes to an increase in fertility, early sexual maturation and augmented vitellogenesis. On the other hand, it undermines resistance to stress factors, reduces immunity and the maximum life expectancy (Flatt et al., 2005). In geese, artificial selection for early sexual maturation causes, as adverse effects, a faster reproductive senescence; quantitative analysis revealed a genetic correlation between these two features (Charmantier et al., 2006). Finally, several programmed cell death mechanisms known in mammals also perform "important vital functions such as energy production, metabolism differentiation or the cell cycle" (Ameisen, 2004; 2005). It was recently suggested that Alzheimer's disease, which appears to be specific to humans, could

Holliday argues that the mammals' life cycle strongly illustrates the idea behind the disposable soma theory: there is in mammals an inverse relationship between maximum reproductive potential and maximum longevity; small mammals are very short-lived and fertile, the great mammals are less fertile and very long-lived (Holliday, 1997; 2005). This contrast between longevity and reproduction also appears in a historical cohort analysis of demographic data of the aristocracy of Britain in which with female longevity correlated with

It is noteworthy that the evolutionary theories of senescence (mutation accumulation, antagonistic pleiotropy, and disposable soma) are not mutually exclusive. Although the three currently accepted evolutionary processes for the evolution of senescence can coexist, a current problem of the evolutionary research on senescence is to know how much each of the processes have contributed to the emergence of this phenomenon (Gavrilov & Gavrilova,

**4.3 The evolutionary theories of senescence and the increasing knowledge on evolution** Charlesworth's cumulative effect model (Charlesworth, 2001). The potential effect of genetic drift and natural selection on deleterious and pleiotropic mutations: effective population size on age-structured populations(Charlesworth, 1980; Felsenstein, 1971), infinite sites model of molecular mutation (Kimura & CROW, 1964), infinite alleles model of polymorphism and the

A greater effect of random genetic drift is expected in populations with age structure (where the effective population size, *Ne* is smaller than the real population size, *N*), than in populations without age structure (and where *Ne* ≈ *N*). Suppose that a genetic effect in a population is expressed at an advanced age (arbitrarily defined as significantly higher than

excellent example of a protected population.

be an example of antagonistic pleiotropy (Bufill & Blesa, 2006).

a lower number of children (Westendorp & Kirkwood, 1998).

neutral theory of evolution (Kimura and Ohta).

**4.3.1 Randon genetic drift**

2006).

## **4. The evolutionary theories of senescence today**

## **4.1 Is there senescence in the wild indeed?**

Does anyone die of old age? The existence of senescence in wild populations in their habitats, which, as mentioned, led Medawar and Williams to think of antagonistic pleiotropy, was harshly questioned in the literature by influential researchers like Hayflick and Comfort (Comfort, 1956; Hayflick, 2000).

In a large study, however, Promislow described significant evidence of senescence in 26 species of mammals *in natura* (Promislow, 1991). In fact, in recent years, several studies have demonstrated demographic senescence (Austad, 1993; Bronikowski et al., 2002; Ericsson et al., 2001; Orell & Belda, 2002) and reproductive senescence in mammals and birds in their habitats (Austad, 1993; Broussard et al., 2003; Ericsson et al., 2001; Reid et al., 2003; Saino et al., 2003).

Finally, a strong indication that senescence does exist in wild populations comes from our species: if the data collected by Gompertz and those who followed him can be extrapolated to primitive humans, then our mortality progresses under a Gompertzian regime from around 12 years of age. Senescence starting in such an early age certainly would have impacted on mortality of early human populations (Gompertz, 1825).

Evidence is thus, that death due to senescence is actually happening in the wild. We must change the question from someone dies of old to " someone dies because of senescence '?"And the answer, backed by extensive literature is a resounding yes (Carey & Judge, 2000).

#### **4.2 Findings supporting each evolutionary theory**

Evidence for the genetic basis of senescence accumulate in the literature. With respect to the specific theories on the evolution of senescence, experimental evidence supports each of the three theories mentioned above (Hughes & Reynolds, 2005).

In Drosophila, it is possible to obtain two distinct lineages in relation the speed of installation of its senescence by systematically separating over generations, the first (beginning of reproductive life) or the last oviposition (immediately prior to reproductive senescence). The flies of the the second group senesce more slowly and live up to 50% longer than the first group flies (Baret & Lints, 1993; Fukui et al., 1995; Luckinbill & Clare, 1985; Rose & Charlesworth, 1980; 1981).

According to Rose, while the flies in the control group focus their greater efficiency in the early reproductive life, the group submitted to selective pressure for older reproduction requires the of the opposite strategy. In both groups, according to the evolutionary concept of senescence, mutations of late manifestation accumulate and propagate to the new generations. These changes will not affect the flies of the first group, but the second group will largely benefit if such manifestations are delayed. Mutations that make deleterious effects to occur later will improve fitness of individuals in the second group, but not in the first group. This mechanism, over generations, makes the senescent manifestations in the second group to become even later, so that the flies of this group evolve longevity. Interestingly, even with the suspension of the selective pressure, the difference persists, and the strains of flies arising from these experiments remain more long-lived than wild flies (Rose, 1991).

10 Will-be-set-by-IN-TECH

Does anyone die of old age? The existence of senescence in wild populations in their habitats, which, as mentioned, led Medawar and Williams to think of antagonistic pleiotropy, was harshly questioned in the literature by influential researchers like Hayflick and

In a large study, however, Promislow described significant evidence of senescence in 26 species of mammals *in natura* (Promislow, 1991). In fact, in recent years, several studies have demonstrated demographic senescence (Austad, 1993; Bronikowski et al., 2002; Ericsson et al., 2001; Orell & Belda, 2002) and reproductive senescence in mammals and birds in their habitats (Austad, 1993; Broussard et al., 2003; Ericsson et al., 2001; Reid et al., 2003; Saino et al.,

Finally, a strong indication that senescence does exist in wild populations comes from our species: if the data collected by Gompertz and those who followed him can be extrapolated to primitive humans, then our mortality progresses under a Gompertzian regime from around 12 years of age. Senescence starting in such an early age certainly would have impacted on

Evidence is thus, that death due to senescence is actually happening in the wild. We must change the question from someone dies of old to " someone dies because of senescence '?"And

Evidence for the genetic basis of senescence accumulate in the literature. With respect to the specific theories on the evolution of senescence, experimental evidence supports each of the

In Drosophila, it is possible to obtain two distinct lineages in relation the speed of installation of its senescence by systematically separating over generations, the first (beginning of reproductive life) or the last oviposition (immediately prior to reproductive senescence). The flies of the the second group senesce more slowly and live up to 50% longer than the first group flies (Baret & Lints, 1993; Fukui et al., 1995; Luckinbill & Clare, 1985; Rose & Charlesworth,

According to Rose, while the flies in the control group focus their greater efficiency in the early reproductive life, the group submitted to selective pressure for older reproduction requires the of the opposite strategy. In both groups, according to the evolutionary concept of senescence, mutations of late manifestation accumulate and propagate to the new generations. These changes will not affect the flies of the first group, but the second group will largely benefit if such manifestations are delayed. Mutations that make deleterious effects to occur later will improve fitness of individuals in the second group, but not in the first group. This mechanism, over generations, makes the senescent manifestations in the second group to become even later, so that the flies of this group evolve longevity. Interestingly, even with the suspension of the selective pressure, the difference persists, and the strains of flies arising from these

the answer, backed by extensive literature is a resounding yes (Carey & Judge, 2000).

**4. The evolutionary theories of senescence today**

mortality of early human populations (Gompertz, 1825).

**4.2 Findings supporting each evolutionary theory**

three theories mentioned above (Hughes & Reynolds, 2005).

experiments remain more long-lived than wild flies (Rose, 1991).

**4.1 Is there senescence in the wild indeed?**

Comfort (Comfort, 1956; Hayflick, 2000).

2003).

1980; 1981).

Hughes and others have found evidence in favor of mutation accumulation in the experimental evolution of accelerated senescence (decrease in MRDT) in fruit flies as the predominant phenomenon (Hughes et al., 2002), argument which is sustained by Cortopassi in relation to human senescence (Cortopassi, 2002). Physiological senescence in our species, however, is easily noticeable between the fourth and fifth decades of life. This is not incompatible with Medawar's observation (Medawar, 1952) that the accumulation of mutations explain only senescence in artificially protected populations: modern man is an excellent example of a protected population.

Pleiotropic mechanisms have currently been described. The gene for the juvenile hormone (JH) found in specimens of wild Drosophila is expressed early in its life cycle, and takes to an increase in fertility, early sexual maturation and augmented vitellogenesis. On the other hand, it undermines resistance to stress factors, reduces immunity and the maximum life expectancy (Flatt et al., 2005). In geese, artificial selection for early sexual maturation causes, as adverse effects, a faster reproductive senescence; quantitative analysis revealed a genetic correlation between these two features (Charmantier et al., 2006). Finally, several programmed cell death mechanisms known in mammals also perform "important vital functions such as energy production, metabolism differentiation or the cell cycle" (Ameisen, 2004; 2005). It was recently suggested that Alzheimer's disease, which appears to be specific to humans, could be an example of antagonistic pleiotropy (Bufill & Blesa, 2006).

Holliday argues that the mammals' life cycle strongly illustrates the idea behind the disposable soma theory: there is in mammals an inverse relationship between maximum reproductive potential and maximum longevity; small mammals are very short-lived and fertile, the great mammals are less fertile and very long-lived (Holliday, 1997; 2005). This contrast between longevity and reproduction also appears in a historical cohort analysis of demographic data of the aristocracy of Britain in which with female longevity correlated with a lower number of children (Westendorp & Kirkwood, 1998).

It is noteworthy that the evolutionary theories of senescence (mutation accumulation, antagonistic pleiotropy, and disposable soma) are not mutually exclusive. Although the three currently accepted evolutionary processes for the evolution of senescence can coexist, a current problem of the evolutionary research on senescence is to know how much each of the processes have contributed to the emergence of this phenomenon (Gavrilov & Gavrilova, 2006).

#### **4.3 The evolutionary theories of senescence and the increasing knowledge on evolution**

Charlesworth's cumulative effect model (Charlesworth, 2001). The potential effect of genetic drift and natural selection on deleterious and pleiotropic mutations: effective population size on age-structured populations(Charlesworth, 1980; Felsenstein, 1971), infinite sites model of molecular mutation (Kimura & CROW, 1964), infinite alleles model of polymorphism and the neutral theory of evolution (Kimura and Ohta).

#### **4.3.1 Randon genetic drift**

A greater effect of random genetic drift is expected in populations with age structure (where the effective population size, *Ne* is smaller than the real population size, *N*), than in populations without age structure (and where *Ne* ≈ *N*). Suppose that a genetic effect in a population is expressed at an advanced age (arbitrarily defined as significantly higher than

Austad, S. N. (1993). Retarded senescence in an insular population of virginia opossums

Senescence in Animals: Why Evolutionary Theories Matter 221

Baret, P. & Lints, F. A. (1993). Selection for increased longevity in *drosophila melanogaster*: a

Bronikowski, A. M., Alberts, S. C., Altmann, J., Packer, C., Carey, K. D. & Tatar, M. (2002). The

Broussard, D. R., Risch, T. S., Dobson, F. S. & Murie, J. O. (2003). Senescence and

Bufill, E. & Blesa, R. (2006). [Alzheimer's disease and brain evolution: is alzheimer's disease

Carey, J. & Judge, D. (eds) (2000). *Longevity records: life spans of mammals, birds, reptiles,*

Charlesworth, B. (1980). *Evolution in Age Structured Populations*, Cambridge University Press,

Charlesworth, B. (2001). Patterns of age-specific means and genetic variances of mortality

Charmantier, A., Perrins, C., McCleery, R. H. & Sheldon, B. C. (2006). Quantitative genetics

Cortopassi, G. A. (2002). Fixation of deleterious alleles, evolution and human aging., *Mech*

Darwin, C. (1872). The origin of species by means of natural selection, or the preservation of

de Magalhães, J. P., Cabral, J. A. S. & Magalhaes, D. (2005). The influence of genes on the

Dobzhansky, T. (1973). Nothing in biology makes sense except in the light of evolution,

Ericsson, G., Wallin, K., Ball, J. P. & Broberg, M. (2001). Age-related reproductive effort and senescence in free-ranging moose, alces alces, *Ecology* (82): 1613–1620. Felsenstein, J. (1971). Inbreeding and variance effective numbers in populations with

aging process of mice: a statistical assessment of the genetics of aging., *Genetics*

rates predicted by the mutation-accumulation theory of ageing., *J Theor Biol*

of age at reproduction in wild swans: support for antagonistic pleiotropy models of

an example of antagonistic pleiotropy?], *Rev Neurol* 42(1): 25–33.

aging baboon: comparative demography in a non-human primate., *Proc Natl Acad*

age-related reproduction of female columbian ground squirrels, *Journal of Animal*

(*didelphis virginiana*), *Journal of Zoology* 229(4): 695–708.

new interpretation., *Gerontology* 39(5): 252–9. URL: *http://www.ncbi.nlm.nih.gov/pubmed/8314091*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/12082185*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/16402323*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/11343430*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/12044933*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/15466429*

overlapping generations., *Genetics* 68(4): 581–97. URL: *http://www.ncbi.nlm.nih.gov/pubmed/5166069*

favoured races in the struggle for life. URL: *http://darwin-online.org.uk*

*American Biology Teacher* 35: 125–129.

senescence., *Proc Natl Acad SciUSA* 103(17): 6587–92. URL: *http://www.ncbi.nlm.nih.gov/pubmed/16618935*

Comfort, A. (1956). *The Biology of Senescence*, Rinehart & Company, Inc., New York.

*amphibians and fish.*, Odense University Press.

*Sci U S A* 99(14): 9591–5.

*Ecology* 72(2): 212–219.

Cambridge.

210(1): 47–65.

169(1): 265–74.

*Ageing Dev* 123(8): 851–5.

the age of reproductive maturation of a species). We can use this to divide this population age into two subsets: that of individuals who have not expressed the genetic trait and those who have already expressed.

Let us consider what happens with the genetic trait in question under natural selection on these two subpopulations. Obviously, on the first subpopulation, the gene with the deleterious effect, not having expressed itself, is effectively neutral and therefore can evolve only by drift. In the second subpopulation, there is evolution by selection. Clearly, the effect of selection on the population as a whole will be less than what it would be if the mentioned gene was expressed at younger ages. This is just another way of saying that the force of selection falls with age.

Now let us consider the effect of genetic drift on the second subpopulation. Being it a fraction of the total population, its *Ne* will be considerably smaller than the the initial population's *Ne*. Thus, besides being subject to progressively smaller selection forces as a function of the age of onset, late onset of deleterious genes should be subject to progressively more intense phenomena of genetic drift. This point, not addressed in the current theories about the evolution senescence, might prove to be crucial.

This last aspect makes it fundamental to understand the roles of mutation, selection and drift as a whole in the evolution of senescence, since, at least in part, the force of selection declines with advancing ages precisely due to the decrease on the effective sizes of the subpopulations.

#### **5. Conclusion**

For decades, researchers in the field of senescence were divided between the proponents of proximal or mechanistic theories and the proponents of the distal or evolutionary theories of senescence (Masoro & Austad, 2006). Fortunately, recent decades have seen an excellent understanding of the importance of a joint reasoning between "mechanistic" and "evolutionary" thoughts: more and more studies focused on the evolution of senescence seek to understand its physiological mechanisms of onset and progression and researchers focusing on the mechanisms of senescence are increasingly seeking to understand the theoretical evolutionary basis of senescence (Masoro & Austad, 2006, Preface).

The evolutionary study of senescence seeks to explain why this phenomenon exists, providing researchers with mechanistic insights into what could be the proximal causes of senescence and how genetics produces the senescent phenotype (Kirkwood & Austad, 2000). By pointing to non-adaptive origins for senescence, the evolutionary theory may drive mechanistic researchers away from adaptive programs such as apoptosis as a plausible basis of senescence. After all, "nothing in biology makes sense except in the light of evolution" (Dobzhansky, 1973).

#### **6. References**

Ameisen, J. C. (2004). Looking for death at the core of life in the light of evolution., *Cell Death Differ* 11(1): 4–10.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/14647240*

Ameisen, J.-C. (2005). [selective "death programs" or pleiotropic "life programs"? looking for programmed cell death in the light of evolution], *J Soc Biol* 199(3): 175–89. URL: *http://www.ncbi.nlm.nih.gov/pubmed/16471257*

12 Will-be-set-by-IN-TECH

the age of reproductive maturation of a species). We can use this to divide this population age into two subsets: that of individuals who have not expressed the genetic trait and those who

Let us consider what happens with the genetic trait in question under natural selection on these two subpopulations. Obviously, on the first subpopulation, the gene with the deleterious effect, not having expressed itself, is effectively neutral and therefore can evolve only by drift. In the second subpopulation, there is evolution by selection. Clearly, the effect of selection on the population as a whole will be less than what it would be if the mentioned gene was expressed at younger ages. This is just another way of saying that the force of selection

Now let us consider the effect of genetic drift on the second subpopulation. Being it a fraction of the total population, its *Ne* will be considerably smaller than the the initial population's *Ne*. Thus, besides being subject to progressively smaller selection forces as a function of the age of onset, late onset of deleterious genes should be subject to progressively more intense phenomena of genetic drift. This point, not addressed in the current theories about

This last aspect makes it fundamental to understand the roles of mutation, selection and drift as a whole in the evolution of senescence, since, at least in part, the force of selection declines with advancing ages precisely due to the decrease on the effective sizes of the subpopulations.

For decades, researchers in the field of senescence were divided between the proponents of proximal or mechanistic theories and the proponents of the distal or evolutionary theories of senescence (Masoro & Austad, 2006). Fortunately, recent decades have seen an excellent understanding of the importance of a joint reasoning between "mechanistic" and "evolutionary" thoughts: more and more studies focused on the evolution of senescence seek to understand its physiological mechanisms of onset and progression and researchers focusing on the mechanisms of senescence are increasingly seeking to understand the theoretical

The evolutionary study of senescence seeks to explain why this phenomenon exists, providing researchers with mechanistic insights into what could be the proximal causes of senescence and how genetics produces the senescent phenotype (Kirkwood & Austad, 2000). By pointing to non-adaptive origins for senescence, the evolutionary theory may drive mechanistic researchers away from adaptive programs such as apoptosis as a plausible basis of senescence. After all, "nothing in biology makes sense except in the light of evolution" (Dobzhansky,

Ameisen, J. C. (2004). Looking for death at the core of life in the light of evolution., *Cell Death*

Ameisen, J.-C. (2005). [selective "death programs" or pleiotropic "life programs"? looking for programmed cell death in the light of evolution], *J Soc Biol* 199(3): 175–89.

have already expressed.

the evolution senescence, might prove to be crucial.

evolutionary basis of senescence (Masoro & Austad, 2006, Preface).

URL: *http://www.ncbi.nlm.nih.gov/pubmed/14647240*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/16471257*

falls with age.

**5. Conclusion**

1973).

**6. References**

*Differ* 11(1): 4–10.


URL: *http://www.ncbi.nlm.nih.gov/pubmed/12082185*


URL: *http://www.ncbi.nlm.nih.gov/pubmed/11343430*


URL: *http://www.ncbi.nlm.nih.gov/pubmed/12044933*


URL: *http://www.ncbi.nlm.nih.gov/pubmed/15466429*


Kirkwood, T. B. & Rose, M. R. (1991). Evolution of senescence: late survival sacrificed for

Senescence in Animals: Why Evolutionary Theories Matter 223

Luckinbill, L. S. & Clare, M. J. (1985). Selection for life span in drosophila melanogaster.,

Masoro, E. J. (2006). Are age-associated diseases an integral part of aging?, *in* E. J. Masoro &

Masoro, E. J. & Austad, S. N. (2006). *Handbook of the Biology of Aging*, sixth edition edn, Oxford

Orgel, L. E. (1963). The maintenance of the accuracy of protein synthesis and its relevance to

Promislow, D. E. L. (1991). Senescence in natural populations of mammals: A comparative

Promislow, D. E. L., Fedorka, K. M. & Burger, J. M. S. (2006). Evolutionary biology of aging:

Reid, J. M., Bignal, E. M., Bignal, S., McCracken, D. I. & Monaghan, P. (2003). Age-specific

Rose, M. R. (1991). *Evolutionary biology of aging*, 1 edn, Oxford University Press, Oxford, UK. Rose, M. R. & Charlesworth, B. (1981). Genetics of life history in drosophila melanogaster. i.

Saino, N., Ferrari, R. P., Romano, M., Rubolini, D. & MÃÂÿller, A. P. (2003). Humoral immune

Sgro, C. M. & Partridge, L. (1999). A delayed wave of death from reproduction in drosophila.,

Weismann, A. (1889). *Essays upon heredity and kindred biological problems. I*, Vol. 1, 2n ed. edn,

Weismann, A. (1892). *Essays upon heredity and kindred biological problems. II*, Vol. II, 2n ed. edn,

Wessells, R. J., Fitzgerald, E., Cypser, J. R., Tatar, M. & Bodmer, R. (2004). Insulin regulation of

response in relation to senescence, sex and sexual ornamentation in the barn swallow

Future directions., *in* E. J. Masoro & S. N. Austad (eds), *Handbook of the Biology of*

reproductive performance in red-billed choughs pyrrhocorax pyrrhocorax: patterns and processes in a natural population, *Journal of Animal Ecology* 72(5): 765–776. Rose, M. & Charlesworth, B. (1980). A test of evolutionary theories of senescence., *Nature*

S. N. Austad (eds), *Handbook of the Biology of Aging.*, 6th edn, Academic Press, London,

reproduction., *Philos Trans R Soc Lond B Biol Sci* 332(1262): 15–24.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/1677205*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/3930429*

Medawar, P. B. (1946). Old age and natural death, *Modern Quarterly* 1: 30–56.

*Aging.*, 6th edn, Academic Press, London, UK, pp. 217–242.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/6776406*

sib analysis of adult females., *Genetics* 97(1): 173–86. URL: *http://www.ncbi.nlm.nih.gov/pubmed/6790340*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/10617470*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/15565107*

Oxford, Clarendon Press, Amen Corner, E. C.

Oxford University Press Warehouse, Amen Corner, E. C.

heart function in aging fruit flies., *Nat Genet* 36(12): 1275–81.

(hirundo rustica)., *J Evol Biol* 16(6): 1127–34. URL: *http://www.ncbi.nlm.nih.gov/pubmed/14640404*

parus montanus, *Journal of Animal Ecology* 71(1): 55–64.

ageing., *Proc Natl Acad SciUSA* 49: 517–21. URL: *http://www.ncbi.nlm.nih.gov/pubmed/13940312*

Medawar, P. B. (1952). *An unsolved problem of biology*, H.K. Lewis and Co., Oxford, UK. Orell, M. & Belda, E. J. (2002). Delayed cost of reproduction and senescence in the willow tit

*Heredity* 55 ( Pt 1): 9–18.

Academic Press, London, UK.

study, *Evolution* 45(8): 1869–1887.

UK, pp. 43–62.

287(5778): 141–2.

*Science* 286(5449): 2521–4.


Gavrilov, L. A. & Gavrilova, N. S. (2006). Reliability theory of aging and longevity., *in* E. J. Masoro & S. N. Austad (eds), *Handbook of the Biology of Aging.*, 6th edn, Academic Press, San Diego, pp. 3–40.

Gompertz, B. (1825). On the nature of the function expressive of the law of human mortality and on a new mode of determining life contingencies., *Philos Trans Roy Soc London* (115): 513–85.

Haldane, J. B. S. (1941). *New Paths in Genetics*, Allen and Unwin, London.

Hamilton, W. D. (1966). The moulding of senescence by natural selection., *J Theor Biol* 12(1): 12–45.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/6015424*


URL: *http://www.ncbi.nlm.nih.gov/pubmed/15355246*

Kimura, M. & CROW, J. F. (1964). The number of alleles that can be maintained in a finite population, *Genetics* 49: 725–38.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/14156929*


URL: *http://www.ncbi.nlm.nih.gov/pubmed/1195775*

Kirkwood, T. B. & Holliday, R. (1975b). The stability of the translation apparatus., *J Mol Biol* 97(2): 257–65.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/1177323*

Kirkwood, T. B. & Holliday, R. (1979). The evolution of ageing and longevity., *Proc R Soc Lond B Biol Sci* 205(1161): 531–46.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/42059*

14 Will-be-set-by-IN-TECH

Flatt, T., Tu, M.-P. & Tatar, M. (2005). Hormonal pleiotropy and the juvenile hormone regulation of drosophila development and life history., *Bioessays* 27(10): 999–1010.

Fukui, H. H., Pletcher, S. D. & Curtsinger, J. W. (1995). Selection for increased longevity in drosophila melanogaster: a response to baret and lints., *Gerontology* 41(2): 65–8.

Gavrilov, L. A. & Gavrilova, N. S. (2006). Reliability theory of aging and longevity., *in* E. J.

Gompertz, B. (1825). On the nature of the function expressive of the law of human mortality

Hamilton, W. D. (1966). The moulding of senescence by natural selection., *J Theor Biol*

Holliday, R. (1997). Understanding ageing., *Philos Trans R Soc Lond B Biol Sci* 352(1363): 1793–7.

Hughes, K. A., Alipaz, J. A., Drnevich, J. M. & Reynolds, R. M. (2002). A test of evolutionary

Hughes, K. A. & Reynolds, R. M. (2005). Evolutionary and mechanistic theories of aging.,

Kimura, M. & CROW, J. F. (1964). The number of alleles that can be maintained in a finite

Kirkwood, T. B. & Holliday, R. (1975a). Commitment to senescence: a model for the finite and

Kirkwood, T. B. & Holliday, R. (1975b). The stability of the translation apparatus., *J Mol Biol*

Kirkwood, T. B. & Holliday, R. (1979). The evolution of ageing and longevity., *Proc R Soc Lond*

infinite growth of diploid and transformed human fibroblasts in culture., *J Theor Biol*

Holliday, R. (2005). Ageing and the extinction of large animals., *Biogerontology* 6(2): 151–6.

Masoro & S. N. Austad (eds), *Handbook of the Biology of Aging.*, 6th edn, Academic

and on a new mode of determining life contingencies., *Philos Trans Roy Soc London*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/16163709*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/7744268*

Press, San Diego, pp. 3–40.

*Annu Rev Entomol* 50: 421–45.

population, *Genetics* 49: 725–38.

53(2): 481–96.

97(2): 257–65.

*B Biol Sci* 205(1161): 531–46.

(115): 513–85.

12(1): 12–45.

Futuyma, D. (1998). *Evolution*, Sinauer Associates, Sunderland, MA, U.S.A.

Haldane, J. B. S. (1941). *New Paths in Genetics*, Allen and Unwin, London.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/6015424* Hayflick, L. (2000). The future of ageing., *Nature* 408(6809): 267–9. URL: *http://www.ncbi.nlm.nih.gov/pubmed/11089985*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/9460062*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/16034683*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/12386342*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/15355246*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/14156929* Kirkwood, T. B. (1977). Evolution of ageing., *Nature* 270(5635): 301–4. URL: *http://www.ncbi.nlm.nih.gov/pubmed/593350*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/11089980*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/1195775*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/1177323*

URL: *http://www.ncbi.nlm.nih.gov/pubmed/42059*

theories of aging., *Proc Natl Acad SciUSA* 99(22): 14286–91.

Kirkwood, T. B. & Austad, S. N. (2000). Why do we age?, *Nature* 408(6809): 233–8.

	- URL: *http://www.ncbi.nlm.nih.gov/pubmed/3930429*
	- study, *Evolution* 45(8): 1869–1887.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/6776406*


URL: *http://www.ncbi.nlm.nih.gov/pubmed/14640404*

Sgro, C. M. & Partridge, L. (1999). A delayed wave of death from reproduction in drosophila., *Science* 286(5449): 2521–4.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/10617470*


**11** 

*1,2Argentina 3France* 

**The Quest for Immortality in Triatomines:** 

**Hemimetabolous Hematophagous Insects** 

Delmi Canale2, Raúl L. Stariolo2 and Frédéric Menu3

*2Vectors Reference Center, National Coordination of Vectors Control,* 

*3Laboratory of Biometry and Biology Evolutionary, CNRS, UMR 5558,* 

*1Parasitological and Vector Studies Center (CEPAVE),* 

*National University of La Plata, La Plata,* 

**A Meta-Analysis of the Senescence Process in** 

Paula Medone1, Jorge Rabinovich1, Eliana Nieves1, Soledad Ceccarelli1,

*Direction of Transmitted Vectors Diseases, National Ministry of Health, Córdoba,* 

*Lyon University F-69000 Lyon, Claude Bernard University, Lyon 1 F-69622, Villeurbanne,* 

There are different views on senescence as a process. In its most general conception it represents the change in the biology of an organism as it ages. However this process may be viewed either at the physiological or at the demographic level. In the former sense senescence deals with changes affecting cells and tissues of the organism and their function and its effect on the organism as a whole (somatic senescence). In the demographic sense (actuarial senescence) the emphasis is in the population's survival decrease as a function of age (Promislow 1991, Tatar et al., 1993); this very general definition does not necessary imply somatic senescence (a physiological deterioration) because the organism may suffer an increased age-specific mortality rate because of an increased reproductive effort (Roff, 2002); the decrease in the reproductive performance with age may be termed reproductive senescence. Williams (1957), based on evolutionary arguments, claims that natural selection will frequently maximize vigor in youth at the expense of vigor later on and thereby he identifies senescence as a declination in vigor during adult life, using the term vigor as associated with a reproductive probability distribution. Here we are interested in this second approach to senescence, and we adhere to the definition of Rose (1991, cited in Roff 2002): "a persistent decline in the age-specific fitness components of an organism due to

According to Charlesworth & Partridge (1997) there are two main theories trying to explain the senescence process: (1) natural selection is less effective at reducing the frequency of later-acting mutations in populations, and so ageing is expected to evolve, and this is known as the "mutation accumulation" theory of ageing; (2) mutations that increase fitness at younger ages (perhaps because they increase fertility) but at the expense of decreasing

**1. Introduction** 

internal physiological deterioration".

	- URL: *http://www.ncbi.nlm.nih.gov/pubmed/9874369*

## **The Quest for Immortality in Triatomines: A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects**

Paula Medone1, Jorge Rabinovich1, Eliana Nieves1, Soledad Ceccarelli1, Delmi Canale2, Raúl L. Stariolo2 and Frédéric Menu3 *1Parasitological and Vector Studies Center (CEPAVE), National University of La Plata, La Plata, 2Vectors Reference Center, National Coordination of Vectors Control, Direction of Transmitted Vectors Diseases, National Ministry of Health, Córdoba, 3Laboratory of Biometry and Biology Evolutionary, CNRS, UMR 5558, Lyon University F-69000 Lyon, Claude Bernard University, Lyon 1 F-69622, Villeurbanne, 1,2Argentina 3France* 

## **1. Introduction**

16 Will-be-set-by-IN-TECH

224 Senescence

Westendorp, R. G. & Kirkwood, T. B. (1998). Human longevity at the cost of reproductive

Williams, G. C. (1957). Pleiotropy, natural selection and the evolution of senescence, *Evolution*

success., *Nature* 396(6713): 743–6.

11: 398–411.

URL: *http://www.ncbi.nlm.nih.gov/pubmed/9874369*

Wright, S. (1956). Modes of selection, *The American Naturalist* 90(850): 5–24.

There are different views on senescence as a process. In its most general conception it represents the change in the biology of an organism as it ages. However this process may be viewed either at the physiological or at the demographic level. In the former sense senescence deals with changes affecting cells and tissues of the organism and their function and its effect on the organism as a whole (somatic senescence). In the demographic sense (actuarial senescence) the emphasis is in the population's survival decrease as a function of age (Promislow 1991, Tatar et al., 1993); this very general definition does not necessary imply somatic senescence (a physiological deterioration) because the organism may suffer an increased age-specific mortality rate because of an increased reproductive effort (Roff, 2002); the decrease in the reproductive performance with age may be termed reproductive senescence. Williams (1957), based on evolutionary arguments, claims that natural selection will frequently maximize vigor in youth at the expense of vigor later on and thereby he identifies senescence as a declination in vigor during adult life, using the term vigor as associated with a reproductive probability distribution. Here we are interested in this second approach to senescence, and we adhere to the definition of Rose (1991, cited in Roff 2002): "a persistent decline in the age-specific fitness components of an organism due to internal physiological deterioration".

According to Charlesworth & Partridge (1997) there are two main theories trying to explain the senescence process: (1) natural selection is less effective at reducing the frequency of later-acting mutations in populations, and so ageing is expected to evolve, and this is known as the "mutation accumulation" theory of ageing; (2) mutations that increase fitness at younger ages (perhaps because they increase fertility) but at the expense of decreasing

The Quest for Immortality in Triatomines:

America (see WHO, 2007).

(

**2. Materials and methods 2.1 Demographic parameters** 

trade-offs, then our conclusions become much more robust.

(see section 2.4). The final list of species is shown in Table 1.

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 227

the best of conditions to detect trade-offs between reproductive effort and mortality. However, we think (in agreement with Mueller et al., 2005) that the identification of which aspects of the environment matter in the evolution of trade-offs can only be obtained by performing experiments in which these environmental variables are carefully manipulated; additionally, if even under such stable and near optimal conditions we are able to detect

Although life history traits such as fecundity, juvenile and adult survival, fasting capacity, developmental time, mortality patterns, and life span have been estimated under controlled conditions in the laboratory for a variety of triatomine species (about 500 scientific articles have been written on these aspects since 1910), very few studies have considered recent evolutionary ecology concepts (although see Menu et al., 2010) to shed some light on the trade-off aspects of life history traits. Understanding the mortality pattern in this group of insects is important both for academic and human health reasons. In the former sense we will provide elements to contribute to the theory of senescence and we discuss our results within of this theoretical background; in particular we will analyze the senescence pattern looking into the relationship between reproductive effort and mortality. In the latter sense, our analyses will provide information about a group of insects that are the vectors of Chagas disease, and represent a health threat estimated in 28 million people, living mostly in Latin

The basic information for the demographic parameters was obtained from a database compiled by one of us (JER). The original dataset comprised information on 534 case studies of triatomines representing 71 species; however, many of those cases had only partial information on demographic parameters and were not adequate for the present study. From the ones with complete information (55 cases) we selected 29 species that had relatively homogenous rearing conditions in the laboratory, to render them adequate for metaanalysis. The 29 species were later reduced to 27 species because two of them were not in the phylogenetic tree used for the application of the Phylogenetic Independent Contrast method

For each species we calculated basic life table parameters following Carey (2001): (i) agespecific survival or fraction alive at age *x* (l*x* = N*<sup>x</sup>* N0) (where N*x*= number of individuals alive at age *x*), (ii) age-specific period survival or fraction alive at age *x* surviving to *x* + 1 (*px* = l*x+1*  l*x*), (iii) age-specific period mortality (probability of dying over the one-week interval used (*qx* = 1 – *px*), and (iv) the force of mortality or instantaneous mortality rate

*<sup>x</sup>* = -ln(*px*)). For more details on the definitions and formulae of the life table parameters see Rabinovich & Nieves (2011). The original values obtained from the laboratory (N*x*, or number of individuals alive at age *x*, and M*x* or total number of eggs laid by all females of the cohort aged *x*) were processed with a special program (called TriTV) developed by one of us (JER) that calculates the life table statistics and population growth rate parameters. As it is usual with laboratory cohorts, data of the last time-units of the cohorts are based on a very small number of individuals; thus the mortality rate estimated over the last living individual were excluded from the analysis, due to the unreliability of the mortality rate

fitness at later ages (perhaps because they increase the death rate) can be incorporated into a population because natural selection will act more strongly on the earlier, beneficial effect. This is the reasoning behind the "antagonistic pleiotropy" or "trade-off" theory of ageing (Williams, 1957).

Abrams & Ludwig (1995), based on an extension of the "disposable soma" model (Kirkwood & Holliday, 1979), provide an explicit realization of the trade-off idea which postulates a conflict between the allocation of resources to reproduction and to the repair of somatic damage. A reduction in damage repair at a given age is assumed to cause an elevated death rate at all subsequent ages. Given a functional relationship between repair allocation and reproductive rate at a given age, the age-specific pattern of allocation to repair versus reproduction that maximizes life-time reproductive success can be determined, yielding a prediction of the age-specific pattern of mortality for the optimal life history (Charlesworth & Partridge, 1997).

Longevity and senescence patterns in mammals and birds are very variable according to the life-history of these organisms (Gaillard et al., 2004), and variation within and between phyla can be expected. Despite phylogenetic similarity is reciprocal to taxonomic level of relatedness (Cheverud et al., 1985) it is also possible that species phylogenetically related show different senescence and/or longevity patterns. So a comparative approach focused on the frequency of senescence in closely related species may contribute to our knowledge of the senescence process. Comparative studies in insects are scarce as compared to mammals and birds; within the insects, demographic analyses of senescence have been carried out mainly for the Diptera (Styer et al., 2007; Curtsinger et al., 1992; Carey et al., 1992, 2005; Fukui et al., 1993), and the Coleoptera (Tatar et al., 1993), and very few studies have considered hemimetabolous species (Dingle, 1966, Chaves et al., 2004a,b, Rabinovich et al., 2010).

In this chapter we investigate the frequency of senescence in a closely related species group of insects: the Triatominae (Hemiptera: Reduviidae). We compiled from the bibliography and resorted to personal data to have phylogeny and life history traits of 27 species reared under laboratory controlled and comparable conditions, and analyzed mortality and fecundity through several death and reproductive parameters. In particular we investigated: (a) species patterns of mortality with respect to age (from cohort studies that followed all individuals from the egg stage until the death of the last individual), (b) the relationship between mortality and different life-history traits (size, reproductive allocation), and (c) the relationship between mortality and environmental factors. Being the 27 selected triatomine species close relatives (they belonged to only five different genera), for our comparative study we included in the analysis a correction for the possible effect of the degree of phylogenetic relatedness.

The advantage of working on laboratory data is that we can estimate intrinsic mortality and fecundity rates without confounding effects resulting from extrinsic factors acting on mortality and fecundity (predation, accidental deaths, starvation, etc.). Even if work with triatomines has the advantage that under natural conditions all life stages occur in a single type of environment and have similar biological requirements, there is always the disadvantage that laboratory data does not reflect natural condition: insects are fed *ad libitum*, and predators, parasites and pathogens are kept out, so that it does not constitute the best of conditions to detect trade-offs between reproductive effort and mortality. However, we think (in agreement with Mueller et al., 2005) that the identification of which aspects of the environment matter in the evolution of trade-offs can only be obtained by performing experiments in which these environmental variables are carefully manipulated; additionally, if even under such stable and near optimal conditions we are able to detect trade-offs, then our conclusions become much more robust.

Although life history traits such as fecundity, juvenile and adult survival, fasting capacity, developmental time, mortality patterns, and life span have been estimated under controlled conditions in the laboratory for a variety of triatomine species (about 500 scientific articles have been written on these aspects since 1910), very few studies have considered recent evolutionary ecology concepts (although see Menu et al., 2010) to shed some light on the trade-off aspects of life history traits. Understanding the mortality pattern in this group of insects is important both for academic and human health reasons. In the former sense we will provide elements to contribute to the theory of senescence and we discuss our results within of this theoretical background; in particular we will analyze the senescence pattern looking into the relationship between reproductive effort and mortality. In the latter sense, our analyses will provide information about a group of insects that are the vectors of Chagas disease, and represent a health threat estimated in 28 million people, living mostly in Latin America (see WHO, 2007).

## **2. Materials and methods**

226 Senescence

fitness at later ages (perhaps because they increase the death rate) can be incorporated into a population because natural selection will act more strongly on the earlier, beneficial effect. This is the reasoning behind the "antagonistic pleiotropy" or "trade-off" theory of ageing

Abrams & Ludwig (1995), based on an extension of the "disposable soma" model (Kirkwood & Holliday, 1979), provide an explicit realization of the trade-off idea which postulates a conflict between the allocation of resources to reproduction and to the repair of somatic damage. A reduction in damage repair at a given age is assumed to cause an elevated death rate at all subsequent ages. Given a functional relationship between repair allocation and reproductive rate at a given age, the age-specific pattern of allocation to repair versus reproduction that maximizes life-time reproductive success can be determined, yielding a prediction of the age-specific pattern of mortality for the optimal life history (Charlesworth

Longevity and senescence patterns in mammals and birds are very variable according to the life-history of these organisms (Gaillard et al., 2004), and variation within and between phyla can be expected. Despite phylogenetic similarity is reciprocal to taxonomic level of relatedness (Cheverud et al., 1985) it is also possible that species phylogenetically related show different senescence and/or longevity patterns. So a comparative approach focused on the frequency of senescence in closely related species may contribute to our knowledge of the senescence process. Comparative studies in insects are scarce as compared to mammals and birds; within the insects, demographic analyses of senescence have been carried out mainly for the Diptera (Styer et al., 2007; Curtsinger et al., 1992; Carey et al., 1992, 2005; Fukui et al., 1993), and the Coleoptera (Tatar et al., 1993), and very few studies have considered hemimetabolous species (Dingle, 1966, Chaves et al., 2004a,b, Rabinovich et

In this chapter we investigate the frequency of senescence in a closely related species group of insects: the Triatominae (Hemiptera: Reduviidae). We compiled from the bibliography and resorted to personal data to have phylogeny and life history traits of 27 species reared under laboratory controlled and comparable conditions, and analyzed mortality and fecundity through several death and reproductive parameters. In particular we investigated: (a) species patterns of mortality with respect to age (from cohort studies that followed all individuals from the egg stage until the death of the last individual), (b) the relationship between mortality and different life-history traits (size, reproductive allocation), and (c) the relationship between mortality and environmental factors. Being the 27 selected triatomine species close relatives (they belonged to only five different genera), for our comparative study we included in the analysis a correction for the possible effect of the degree of

The advantage of working on laboratory data is that we can estimate intrinsic mortality and fecundity rates without confounding effects resulting from extrinsic factors acting on mortality and fecundity (predation, accidental deaths, starvation, etc.). Even if work with triatomines has the advantage that under natural conditions all life stages occur in a single type of environment and have similar biological requirements, there is always the disadvantage that laboratory data does not reflect natural condition: insects are fed *ad libitum*, and predators, parasites and pathogens are kept out, so that it does not constitute

(Williams, 1957).

& Partridge, 1997).

al., 2010).

phylogenetic relatedness.

## **2.1 Demographic parameters**

The basic information for the demographic parameters was obtained from a database compiled by one of us (JER). The original dataset comprised information on 534 case studies of triatomines representing 71 species; however, many of those cases had only partial information on demographic parameters and were not adequate for the present study. From the ones with complete information (55 cases) we selected 29 species that had relatively homogenous rearing conditions in the laboratory, to render them adequate for metaanalysis. The 29 species were later reduced to 27 species because two of them were not in the phylogenetic tree used for the application of the Phylogenetic Independent Contrast method (see section 2.4). The final list of species is shown in Table 1.

For each species we calculated basic life table parameters following Carey (2001): (i) agespecific survival or fraction alive at age *x* (l*x* = N*<sup>x</sup>* N0) (where N*x*= number of individuals alive at age *x*), (ii) age-specific period survival or fraction alive at age *x* surviving to *x* + 1 (*px* = l*x+1*  l*x*), (iii) age-specific period mortality (probability of dying over the one-week interval used (*qx* = 1 – *px*), and (iv) the force of mortality or instantaneous mortality rate (*<sup>x</sup>* = -ln(*px*)). For more details on the definitions and formulae of the life table parameters see Rabinovich & Nieves (2011). The original values obtained from the laboratory (N*x*, or number of individuals alive at age *x*, and M*x* or total number of eggs laid by all females of the cohort aged *x*) were processed with a special program (called TriTV) developed by one of us (JER) that calculates the life table statistics and population growth rate parameters. As it is usual with laboratory cohorts, data of the last time-units of the cohorts are based on a very small number of individuals; thus the mortality rate estimated over the last living individual were excluded from the analysis, due to the unreliability of the mortality rate

The Quest for Immortality in Triatomines:

species, the sum of squares (SSQ= (

**2.4 Phylogenetic Independent Contrasts** 

of freedom of each model.

package "ape".

**3. Results** 

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 229

(iv) the "Online Curve Fitting and Surface Fitting Web Site" (accessible at http://ZunZun.com/) (Christopoulos & Lew, 2000), and (v) the Solver utility of the Excel 2007 program. The reason for the variety of procedures resorted to for fitting the data, is that -even after smoothing- the age-specific instantaneous mortality data was quite irregular, and frequently a given software was not able to succeed in fitting the data to the models while another did. This would happen even if the parameter starting values for fitting the data would have been successfully estimated with the Solver utility of the Excel 2007 program. All software products used, if successful in fitting the data, provided the standard deviation of each parameter and the probability for deciding on the significance of the parameters' estimates. When more than one model fitted the data of the same

smallest SSQ was selected. We did not use the Akaike model selection criterion (Akaike, 1974) because the three models tested had the same number of parameters, so the SSQ goodness of fit values would not be affected by any penalty due to the number of degrees

Comparative studies, either among life-history traits or between those traits and environmental variables, frequently imply resorting to statistical methods such as regressions, correlations and contingency tables, which assume that the data is drawn from a common and independent distribution. However, the comparison among species that are related among each other with different degrees of phylogenetic relatedness does not necessary comply with this assumption. Methods are available to correct for this violation of the assumption of independence. For such purpose we used the Phylogenetic Independent Contrast method (Paradis, 2006) that corrects this violation when derived from phylogeny. The application of this method requires the topology of the phylogenetic tree, and an estimate of the degree of relatedness, generally expressed by the length of the branches of the phylogenetic tree. Calculations were carried out in language R using

The phylogenetic tree used for this purpose was the one provided by Silva de Paula et al. (2005), who analyzed the Reduviidae phylogeny by aligning groups of sequences using Clustal-X under gap opening/gap extension penalties, and treating the gaps as missing. For Cladistic analysis Silva de Paula et al. (2005) used the programs PAUP and MacClade to derive trees based both on maximum parsimony (MP) and on maximum likelihood (ML); parsimony bootstrap values were conducted with PAUP employing heuristic search with 100 bootstrap replicates. Decay index for the strict consensus trees (Bremer support) were retained using the decay commands performed by the MacClade software with the heuristic search command activated, and executed with PAUP. This phylogenetic tree covered 57 species of Triatominae, and included 27 species of the 29 species we had selected from the demographic dataset (see section 2.1); thus *Triatoma breyeri* and *Panstrongylus lignarius* were

deleted from the analysis, which remained composed by 27 species (Table 1).

The following are the main results obtained from our analyses.

mod)2) was calculated and the model with the

obs - 

estimates (Carey et al., 1992). The TriTV software was programmed in Delphi language, and is available under request to the second author (the interested user should have an adequate command of the Spanish language).

We considered of interest the division of the female's adult life in two, three or four periods of equal length, to analyze possible lag effects between reproductive effort and mortality. For that purpose, the reproductive effort (*mx*= number of female eggs per female per unit time) and the instantaneous mortality rates (*<sup>x</sup>*) were averaged for each of the periods in which the female's adult life was divided. We also accumulated mortality from the egg to the last adult female (∑*<sup>x</sup>*) and the female's *per capita* fecundity (∑*mx*) to look for a relationship of one with respect to the other along the female´s adult life (after scaling both between 0 and 1).

## **2.2 Mortality pattern models**

For several reasons (see Rabinovich et al., 2010) we preferred the use of mortality analysis over survival analysis; in particular because we agree with Carey (2001) in that, despite mortality and survival are intimately related, death can be considered as an event whereas survival is a "non-event", that is, the absence of the mortality event. For the analysis of the age-specific mortality pattern of the 27 triatomine species selected for this study the following two mortality models were used with the formulation proposed by Carey (2001): the Gompertz model

 $\mu\_{\mathbb{R}} = a$   $e^{-b\infty}$ 

and the Logistic model

$$\mu\_{\mathfrak{x}} = (n \propto^{n-1}) / (\mathfrak{g}^n + \mathfrak{x}^n)$$

We also used a third model based on reliability theory as proposed by Gavrilov & Gavrilova (2001), but simplified to two parameters, and which, for simplicity, will be called hereafter "Gavrilovs". Its formulation is given as:

$$
\mu\_x = n \; k^n \; \mathcal{X}^{n-1}
$$

In all models *x* is the age (in our case in weekly time-units), and for the interpretation of the model's parameters, one of them usually represents the "base" mortality rate, and the other the shape of the function (more directly related to the rate of increase of mortality with age, also called the ageing parameter in the case of the Gompertz model). The reason we selected these three models among about a dozen available models for the analysis of mortality patterns, is that Rabinovich et al. (2010), fitting seven models to the instantaneous mortality rate to another triatomine species (*R. neglectus*), found that these three models offered the best fit to the data.

#### **2.3 Model fitting to the data**

We used several tools and procedures to fit the three models to the age-specific instantaneous mortality data (*<sup>x</sup>*): (i) the R language (R Development Core Team, 2007), (ii) the Kolmogorov-Zurbenko Adaptive smoothing package (kza) in R language (used with parameter *q*= 2) (Zurbenko et al., 1996), (iii) the Statistica software (StatSoft, 2009), (iv) the "Online Curve Fitting and Surface Fitting Web Site" (accessible at http://ZunZun.com/) (Christopoulos & Lew, 2000), and (v) the Solver utility of the Excel 2007 program. The reason for the variety of procedures resorted to for fitting the data, is that -even after smoothing- the age-specific instantaneous mortality data was quite irregular, and frequently a given software was not able to succeed in fitting the data to the models while another did. This would happen even if the parameter starting values for fitting the data would have been successfully estimated with the Solver utility of the Excel 2007 program. All software products used, if successful in fitting the data, provided the standard deviation of each parameter and the probability for deciding on the significance of the parameters' estimates. When more than one model fitted the data of the same species, the sum of squares (SSQ= (obs - mod)2) was calculated and the model with the smallest SSQ was selected. We did not use the Akaike model selection criterion (Akaike, 1974) because the three models tested had the same number of parameters, so the SSQ goodness of fit values would not be affected by any penalty due to the number of degrees of freedom of each model.

## **2.4 Phylogenetic Independent Contrasts**

228 Senescence

estimates (Carey et al., 1992). The TriTV software was programmed in Delphi language, and is available under request to the second author (the interested user should have an adequate

We considered of interest the division of the female's adult life in two, three or four periods of equal length, to analyze possible lag effects between reproductive effort and mortality. For that purpose, the reproductive effort (*mx*= number of female eggs per female per unit

which the female's adult life was divided. We also accumulated mortality from the egg to

relationship of one with respect to the other along the female´s adult life (after scaling both

For several reasons (see Rabinovich et al., 2010) we preferred the use of mortality analysis over survival analysis; in particular because we agree with Carey (2001) in that, despite mortality and survival are intimately related, death can be considered as an event whereas survival is a "non-event", that is, the absence of the mortality event. For the analysis of the age-specific mortality pattern of the 27 triatomine species selected for this study the following two mortality models were used with the formulation proposed by Carey (2001):

> *x= a e bx*

*x= (n xn-1)/(gn + xn)* We also used a third model based on reliability theory as proposed by Gavrilov & Gavrilova (2001), but simplified to two parameters, and which, for simplicity, will be called hereafter

*x= n kn xn-1*  In all models *x* is the age (in our case in weekly time-units), and for the interpretation of the model's parameters, one of them usually represents the "base" mortality rate, and the other the shape of the function (more directly related to the rate of increase of mortality with age, also called the ageing parameter in the case of the Gompertz model). The reason we selected these three models among about a dozen available models for the analysis of mortality patterns, is that Rabinovich et al. (2010), fitting seven models to the instantaneous mortality rate to another triatomine species (*R. neglectus*), found that these three models offered the

We used several tools and procedures to fit the three models to the age-specific

(ii) the Kolmogorov-Zurbenko Adaptive smoothing package (kza) in R language (used with parameter *q*= 2) (Zurbenko et al., 1996), (iii) the Statistica software (StatSoft, 2009),

*<sup>x</sup>*): (i) the R language (R Development Core Team, 2007),

*<sup>x</sup>*) were averaged for each of the periods in

*<sup>x</sup>*) and the female's *per capita* fecundity (∑*mx*) to look for a

command of the Spanish language).

the last adult female (∑

**2.2 Mortality pattern models** 

between 0 and 1).

the Gompertz model

and the Logistic model

best fit to the data.

**2.3 Model fitting to the data** 

instantaneous mortality data (

"Gavrilovs". Its formulation is given as:

time) and the instantaneous mortality rates (

Comparative studies, either among life-history traits or between those traits and environmental variables, frequently imply resorting to statistical methods such as regressions, correlations and contingency tables, which assume that the data is drawn from a common and independent distribution. However, the comparison among species that are related among each other with different degrees of phylogenetic relatedness does not necessary comply with this assumption. Methods are available to correct for this violation of the assumption of independence. For such purpose we used the Phylogenetic Independent Contrast method (Paradis, 2006) that corrects this violation when derived from phylogeny. The application of this method requires the topology of the phylogenetic tree, and an estimate of the degree of relatedness, generally expressed by the length of the branches of the phylogenetic tree. Calculations were carried out in language R using package "ape".

The phylogenetic tree used for this purpose was the one provided by Silva de Paula et al. (2005), who analyzed the Reduviidae phylogeny by aligning groups of sequences using Clustal-X under gap opening/gap extension penalties, and treating the gaps as missing. For Cladistic analysis Silva de Paula et al. (2005) used the programs PAUP and MacClade to derive trees based both on maximum parsimony (MP) and on maximum likelihood (ML); parsimony bootstrap values were conducted with PAUP employing heuristic search with 100 bootstrap replicates. Decay index for the strict consensus trees (Bremer support) were retained using the decay commands performed by the MacClade software with the heuristic search command activated, and executed with PAUP. This phylogenetic tree covered 57 species of Triatominae, and included 27 species of the 29 species we had selected from the demographic dataset (see section 2.1); thus *Triatoma breyeri* and *Panstrongylus lignarius* were deleted from the analysis, which remained composed by 27 species (Table 1).

## **3. Results**

The following are the main results obtained from our analyses.

The Quest for Immortality in Triatomines:

genera.

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 231

Fig. 1 shows the instantaneous mortality and the fecundity rates of the 27 triatomine species analyzed. The senescence pattern is clear: instantaneous mortality rates stay at extremely low levels during most of the juvenile stages (except in the egg stage for some species) and during the first part of the adult stage, increasing greatly in the old ages (i.e., for old females). Most triatomine species seem to invest more in reproduction during the earlier and intermediate periods (but in general with lower effort) of the female's life. Only three species (*R. nasutus, R. neivai* and *D. maximus*) seem to invest more in the intermediate ages. However, six species (*T. eratyrusiformis, T. mazzotti, T. platensis, T. sordida, E. mucronatus,* and *P. geniculatus*) invest in reproduction during all periods of the reproductive life of the female. The difference in the reproductive effort pattern seem to be related to particular

Fig. 1. Age-specific fecundity and instantaneous mortality rates for 27 triatomine species.

accumulated fecundity (∑*mx*), both scaled from 0 to 1, in the form of a scatterplot (∑

∑*mx*); to facilitate interpretation of the different patterns, the scatterplots for all species were drawn in the same scale and with a 45º line to be used as a frame of reference. From those

relationships differ among the species of the six groups, the species classed in the first five groups invest strongly in reproduction before a high mortality has been accumulated to a high degree (most of the curves stay below the 45° line) although with increased initial

in weeks, but scaled from 0 to 100 to have a common scale for all species.

**3.2 Reproductive effort and mortality relationship** 

graphs we defined six groups with respect of the ∑

Fig. 2 shows the female adult accumulated mortality (∑

Age-specific fecundity (blue lines) and instantaneous mortality rates (red lines) for the 27 species analyzed. All species have a common scale for fecundity and mortality. Fecundity is represented in a scale from 0 to 25 ♀ eggs/♀/unit-time (not shown for better clarity); mortality is represented in a scale from 0 to 1 per unit-time; the x-axis is the age, originally

*<sup>x</sup>*) with respect the female adult

*<sup>x</sup>* vs ∑*mx* observed patterns. Despite the

*<sup>x</sup>* vs

#### **3.1 Demographic parameters**

Table 1, provides a list of the species analyzed and some of their main environmental information (average annual temperature and precipitation) and provides information about rearing conditions of each species analyzed. In the last column the initial number of eggs (both sexes) with which each cohort was initiated is given; as the sex ratio at the egg stage cannot be established, it was assumed to be 50% for each sex (see Rabinovich et al., 2010 for a justification of this procedure). Initial cohort size was between 35 and 500 eggs (mean: 167 eggs/cohort). Because the senescence analysis was based on female mortality, the initial number for each species was assumed to be one half of original total number of eggs. Feeding frequency of all cohorts was once a week.


Table 1. Natural environmental and laboratory rearing conditions of triatomine species selected for the analysis. R. H.=relative humidity. \* Pool of several cohorts. Published sources: a Rabinovich & Nieves (2010), b Rabinovich (1972), c Feliciangeli & Rabinovich (1985); for *T. patagonica* original data from Dr. Elena Visciarelli; the rest of species: original data from this study.

Table 1, provides a list of the species analyzed and some of their main environmental information (average annual temperature and precipitation) and provides information about rearing conditions of each species analyzed. In the last column the initial number of eggs (both sexes) with which each cohort was initiated is given; as the sex ratio at the egg stage cannot be established, it was assumed to be 50% for each sex (see Rabinovich et al., 2010 for a justification of this procedure). Initial cohort size was between 35 and 500 eggs (mean: 167 eggs/cohort). Because the senescence analysis was based on female mortality, the initial number for each species was assumed to be one half of original total number of

> Average annual temp. (ºC)

*Dipetalogaster maximus* Mexico 36,074 22.2 198.42 28 70 pigeon 35 *Eratyrus mucronatus* Venezuela 6,257,724 25.69 2340.64 26 60 chicken 200\* *Panstrongylus geniculatus* Venezuela 12,040,606 24.45 1843.01 26 60 chicken 100 *Panstrongylus herreri* Peru 354,387 21.46 1764.42 28 70 pigeon 105 *Panstrongylus megistus* Brazil 3,739,358 22.55 1281.21 28 70 pigeon 96\* *Rhodnius nasutus* Brazil 403,797 25.49 989.55 26 60 chicken 500\* *Rhodnius neglectus* <sup>a</sup> Brazil 2,381,373 23.3 1318.39 26 60 chicken 500\* *Rhodnius neivai* Venezuela 155,568 24.73 1347.3 26 60 chicken 100 *Rhodnius prolixus* Colombia 5,281,236 25.46 2286.3 28 70 pigeon 102 *Rhodnius robustus* Venezuela 3,133,393 25.68 2132.36 26 60 chicken 500\* *Triatoma delpontei* Argentina 705,366 20.58 733.59 28 70 pigeon 108\* *Triatoma dimidiata* Ecuador 2,176,134 24.26 1807.86 28 70 pigeon 45 *Triatoma eratyrusiformis* Argentina 540,347 16.72 407.53 28 70 pigeon 112\* *Triatoma garciabesi* Argentina 930,966 19.11 668.67 28 70 pigeon 132\* *Triatoma guasayana* Argentina 1,412,079 19.38 747.88 28 70 pigeon 108\* *Triatoma infestans* <sup>b</sup> Chile 5,198,083 19.26 957.3 26 60 chicken 500\* *Triatoma maculata* <sup>c</sup> Venezuela 152,506 24.26 1421.51 26 60 chicken 50\* *Triatoma matogrossensis* Argentina 205,258 22.3 815.29 28 70 pigeon 108 *Triatoma mazzotti* Mexico 267,872 20.82 876.65 28 70 pigeon 56 *Triatoma pallidipennis* Mexico 1,631,377 16.29 621.44 28 70 pigeon 138\* *Triatoma patagonica* Argentina 53,047 20.5 915.06 28 65 pigeon 90 *Triatoma platensis* Argentina 1,653,623 18.22 734 28 70 pigeon 126\* *Triatoma protracta* USA 2,082,288 16.27 404.55 28 70 pigeon 68\* *Triatoma pseudomaculata* Brazil 2,650,721 23.7 1198.89 28 70 pigeon 225\* *Triatoma rubrovaria* Argentina 470,659 18.57 1472.36 28 70 pigeon 201\* *Triatoma sordida* Brazil 4,408,479 22.25 1170.12 28 70 pigeon 120\* *Triatoma vitticeps* Brazil 524,660 21.06 1352.76 28 70 pigeon 135\* Table 1. Natural environmental and laboratory rearing conditions of triatomine species selected for the analysis. R. H.=relative humidity. \* Pool of several cohorts. Published sources: a Rabinovich & Nieves (2010), b Rabinovich (1972), c Feliciangeli & Rabinovich (1985); for *T. patagonica* original data from Dr. Elena Visciarelli; the rest of species: original

Average precipitation (mm/year)

Temp. (ºC)

Laboratory rearing conditions

Feeding source

Initial Number of eggs

R.H (%)

**3.1 Demographic parameters** 

Species Country

data from this study.

eggs. Feeding frequency of all cohorts was once a week.

Area of distribution (km2)

of origin

Fig. 1 shows the instantaneous mortality and the fecundity rates of the 27 triatomine species analyzed. The senescence pattern is clear: instantaneous mortality rates stay at extremely low levels during most of the juvenile stages (except in the egg stage for some species) and during the first part of the adult stage, increasing greatly in the old ages (i.e., for old females). Most triatomine species seem to invest more in reproduction during the earlier and intermediate periods (but in general with lower effort) of the female's life. Only three species (*R. nasutus, R. neivai* and *D. maximus*) seem to invest more in the intermediate ages. However, six species (*T. eratyrusiformis, T. mazzotti, T. platensis, T. sordida, E. mucronatus,* and *P. geniculatus*) invest in reproduction during all periods of the reproductive life of the female. The difference in the reproductive effort pattern seem to be related to particular genera.

Fig. 1. Age-specific fecundity and instantaneous mortality rates for 27 triatomine species.

Age-specific fecundity (blue lines) and instantaneous mortality rates (red lines) for the 27 species analyzed. All species have a common scale for fecundity and mortality. Fecundity is represented in a scale from 0 to 25 ♀ eggs/♀/unit-time (not shown for better clarity); mortality is represented in a scale from 0 to 1 per unit-time; the x-axis is the age, originally in weeks, but scaled from 0 to 100 to have a common scale for all species.

## **3.2 Reproductive effort and mortality relationship**

Fig. 2 shows the female adult accumulated mortality (∑*<sup>x</sup>*) with respect the female adult accumulated fecundity (∑*mx*), both scaled from 0 to 1, in the form of a scatterplot (∑*<sup>x</sup>* vs ∑*mx*); to facilitate interpretation of the different patterns, the scatterplots for all species were drawn in the same scale and with a 45º line to be used as a frame of reference. From those graphs we defined six groups with respect of the ∑*<sup>x</sup>* vs ∑*mx* observed patterns. Despite the relationships differ among the species of the six groups, the species classed in the first five groups invest strongly in reproduction before a high mortality has been accumulated to a high degree (most of the curves stay below the 45° line) although with increased initial

The Quest for Immortality in Triatomines:

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 233

patterns. A first group (Group I) shows a mortality pattern that we called "Late-spiky senescence" and is defined by a very long period in which the mortality rates are low and stable, with a high increase in the mortality rate at the end of the adult female life. In a second group, called "Gradual-medium senescence" (Group II), the increase in the mortality rate with age is more progressive than in the Group I and the mortality rate values stay at an intermediate level. In these two groups, no stabilization of mortality rate occurs at old age.

Fig. 3. Age specific mortality patterns of 27 species of triatomines.

latter covers from the egg stage to the last female adult alive.

The abbreviations in parenthesis indicate the best model that fitted the age-specific instantaneous mortality rate (Go= Gompertz, Lo= Logistic, and Ga= Gavrilovs). Axes have a common scale: from 0 to 1 in the ordinates (not shown) and from 0 to 100 in the abscissa; the

female adult mortality from Group 1 to Group 5; while Group 6 (R. neivai) has a different pattern with a curve that stays above this line during a long period, indicating no significant reproductive accumulation effort in relation to the accumulated mortality rate

Fig. 2. Grouping of the accumulated mortality and accumulated fecundity relationship

Female adult accumulated instantaneous mortality rate (∑*<sup>x</sup>*) (y- axis) with respect to the female adult accumulated fecundity (∑*mx*) (x- axis) (blue line), both scaled from 0 to 1. The 45º line was drawn as a reference (red line). Only selected species representative of each group are shown. For the criteria for grouping and description of the groups see text.

The species classed in first group (n=11) are: *T. garciabesi, T. guasayana, T. pallidipennis, T. vitticep, T. pseudomaculata, T. sordida, R. neglectus, R. prolixus, E. mucronatus, D. maximus* and *P. herreri;* in the second group (n=8): *T. delpontei, T. platensis, T. rubrovaria, T. pallidipennis, T. protracta, T. matogrossensis*, *T. mazzotti* and *P. megistus;* in the third group (n=4): *T. infestans, T. maculata, R. nasutus* and *R. robustus;* in fourth group (n=2): *T. eratyrusiformis* and *P. geniculatus*; in fifth group (n=1): *T. dimidiata*, and in last group (n=1): *R. neivai.* 

#### **3.3 Mortality pattern models**

Table 2 shows the coefficients of the fit of the kza- smoothed instantaneous mortality rate (x) to the three mortality models tested (Gavrilovs, Gompertz, and Logistic). Asterisks indicate a statistically significant coefficient with p< 0.05. The squared residuals (SSQ) are also shown. Certain mortality models could not be fitted to the instantaneous mortality rate data of some species; additionally, no single model fitted the mortality pattern of all species (Fig. 3, Table 2).

The fit is statistically significant for 14 out of 15 successful fits, for 10 out of 18 successful fits, and for 20 out of 21 successful fits, for the Gavrilovs, Gompertz, and Logistic models, respectively. From the results of the model fitting, Fig. 3 shows three main types of mortality

female adult mortality from Group 1 to Group 5; while Group 6 (R. neivai) has a different pattern with a curve that stays above this line during a long period, indicating no significant

Fig. 2. Grouping of the accumulated mortality and accumulated fecundity relationship

group are shown. For the criteria for grouping and description of the groups see text.

*geniculatus*; in fifth group (n=1): *T. dimidiata*, and in last group (n=1): *R. neivai.* 

female adult accumulated fecundity (∑*mx*) (x- axis) (blue line), both scaled from 0 to 1. The 45º line was drawn as a reference (red line). Only selected species representative of each

The species classed in first group (n=11) are: *T. garciabesi, T. guasayana, T. pallidipennis, T. vitticep, T. pseudomaculata, T. sordida, R. neglectus, R. prolixus, E. mucronatus, D. maximus* and *P. herreri;* in the second group (n=8): *T. delpontei, T. platensis, T. rubrovaria, T. pallidipennis, T. protracta, T. matogrossensis*, *T. mazzotti* and *P. megistus;* in the third group (n=4): *T. infestans, T. maculata, R. nasutus* and *R. robustus;* in fourth group (n=2): *T. eratyrusiformis* and *P.* 

Table 2 shows the coefficients of the fit of the kza- smoothed instantaneous mortality rate

The fit is statistically significant for 14 out of 15 successful fits, for 10 out of 18 successful fits, and for 20 out of 21 successful fits, for the Gavrilovs, Gompertz, and Logistic models, respectively. From the results of the model fitting, Fig. 3 shows three main types of mortality

x) to the three mortality models tested (Gavrilovs, Gompertz, and Logistic). Asterisks indicate a statistically significant coefficient with p< 0.05. The squared residuals (SSQ) are also shown. Certain mortality models could not be fitted to the instantaneous mortality rate data of some species; additionally, no single model fitted the mortality pattern of all species

*<sup>x</sup>*) (y- axis) with respect to the

Female adult accumulated instantaneous mortality rate (∑

**3.3 Mortality pattern models** 

(

(Fig. 3, Table 2).

reproductive accumulation effort in relation to the accumulated mortality rate

patterns. A first group (Group I) shows a mortality pattern that we called "Late-spiky senescence" and is defined by a very long period in which the mortality rates are low and stable, with a high increase in the mortality rate at the end of the adult female life. In a second group, called "Gradual-medium senescence" (Group II), the increase in the mortality rate with age is more progressive than in the Group I and the mortality rate values stay at an intermediate level. In these two groups, no stabilization of mortality rate occurs at old age.

Fig. 3. Age specific mortality patterns of 27 species of triatomines.

The abbreviations in parenthesis indicate the best model that fitted the age-specific instantaneous mortality rate (Go= Gompertz, Lo= Logistic, and Ga= Gavrilovs). Axes have a common scale: from 0 to 1 in the ordinates (not shown) and from 0 to 100 in the abscissa; the latter covers from the egg stage to the last female adult alive.

The Quest for Immortality in Triatomines:

Female's reproductive period

Age of first reproduction (weeks) (

the reproductive effort is high.

in terms of lifespan) using 27 species of triatomines.

instantaneous mortality rate (called hereafter

Table 4. Mean instantaneous mortality rate (

mean instantaneous mortality rate (

senescence"; see Section 3.3 for definition of each group).

**Independent variables Coefficient Std** 

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 235

bifurcations identify each node of the phylogenetic tree. Numbers above the branches indicate the length of each branch, estimated by a branch length/decay index (Bremer support); it is proportional to the distance to ancestors. To the right of each species there is a label with a roman numeral corresponding to the Group into which each species has been classified as following a certain age-specific mortality pattern (Group I= "Late-spiky senescence"; Group II= "Gradual-medium senescence", and Group III= "Gradual-low

No significant relationship exists between the female's post reproductive period (FPRP) and the total length of adults or the age of first reproduction (non scaled, weeks) (Table 3). The relationship is statistically significant and positive between FPRP and the age of first reproduction scaled by total longevity. A statistically significant and negative correlation exists between FPRP and the female's reproductive period (scaled by longevity), the female reproductive life period (weeks, i.e., not scaled), and fecundity (expressed as ♀ eggs/♀/life).

(longevity scaled) -0.12288 0.05419 -2.26754 0.03226 Total length of adults (mm) -0.00362 0.00192 -1.88994 0.07042 Female reproductive life period (weeks) -0.00113 0.00043 -2.61769 0.01481 Fecundity (♀eggs/♀/life) -0.00007 0.00002 -2.84123 0.00881

scaled by total longevity 0.11125 0.04857 2.29050 0.03070

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several independent variables on the female's post-reproductive period (scaled

Therefore, the female's post reproductive period decreases with reproductive effort, which means that females live less after their last reproduction in those triatomine species in which

We show in Table 4 a statistically significant and negative correlation between the mean

since the egg stage, but no statistically significant relationship with the female's total length.

**Independent variable Coefficient Std. Error t value p**  Total average ♀ longevity (from the egg stage) -0.00070 0.00014 -4.86 5.0E-05 Total length (mm) -0.00076 0.00105 -0.73 0.4740

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of average female longevity and total body length as independent variables on the

> *<sup>x</sup>* ).

Table 3. Female's post-reproductive period regressed on several independent variables

**Dev t value p** 

*<sup>x</sup>* ) and the average total female longevity

*<sup>x</sup>* ) regressed on longevity and total length

) 0.00068 0.00107 0.64255 0.52636


Table 2. Fit to the Gavrilovs, Gompertz, and Logistic mortality models.

Parameter values of the mortality models fitted to the laboratory instantaneous mortality rates (x). A "\*" indicates a statistically significant coefficient of p< 0.05. SSQ= (obs - mod)2 . A "-" indicates that the model could not be fitted to the data.

The third group (Group III), which we called "Gradual-low senescence", is defined by a low and progressively increasing instantaneous mortality rate with age. In this group, particularly in *R. neivai*, the mortality rate seems to stabilize at old ages. The senescence pattern variation does not seem to be homogeneous for a given genus: all senescence pattern groups seem to occur in all genera, particularly in the dominant ones (*Triatoma*, *Rhodnius*).

#### **3.4 Phylogenetic Independent Contrasts**

The results of a series of phylogenetic independent contrasts among several dependent variables related to mortality and other variables related to the longevity and reproduction of the triatomines are shown in Tables 3-10. For a better illustration about the phylogenetic relationships we present (Fig. 4) the phylogenetic tree provided by Silva de Paula et al. (2005) but restricted to the 27 species used in our analysis. In Fig. 4 numbers close to the

Species K n SSQ a b SSQ n g SSQ *D. maximus* 6.7E-03\* 18.78\* 1.7E-01 1.5E-09\* 1.6E-01\* 1.7E-01 18.94\* 149.8\* 1.7E-01 *E. mucronatus* - - - 1.6E-06\* 1.4E-01\* 1.8E-01 11.76\* 115.3\* 1.7E-01 *P. geniculatus* 6.7E-03\* 18.78\* 5.1E-02 1.1E-02\* 9.3E+00\* 4.8E-02 0.57 3310.3 5.0E-02 *P. herreri* - - - 7.4E-03\* 1.6E-02\* 7.2E-02 - - - *P. megistus* 1.1E-02\* 8.18\* 3.1E-01 7.9E-05\* 1.4E-01\* 3.3E-01 8.52\* 85.7\* 3.0E-01 *R. nasutus* 4.9E-03\* 3.46\* 9.4E-02 3.6E-03\* 4.5E-02\* 8.7E-02 3.56\* 200.1\* 9.4E-02 *R. neglectus* - - - 5.0E-03\* 3.9E-02\* 7.6E-02 2.64\* 260.3\* 7.1E-02 *R. neivai* 2.0E-03 0.82 4.6E-01 - - - 0.93\* 374.1\* 4.6E-01 *R. prolixus* 8.5E-03\* 6.32\* 7.2E-02 2.6E-04 9.7E-02\* 1.7E-01 6.49\* 115.8\* 1.6E-01 *R. robustus* - - - 2.1E-02\* 1.6E-02\* 3.2E-01 - - - *T. delpontei* 6.1E-03\* 29.39\* 3.2E-01 - - - 29.59\* 162.7\* 3.2E-01 *T. dimidiata* 1.2E-02\* 28.09\* 6.1E-01 - - - - - - *T. eratyrusiformis* - - - 1.3E-03 5.1E-02\* 1.3E-01 3.65\* 225.2\* 1.4E-01 *T. garciabesi* 1.2E-02\* 8.49\* 7.8E-02 4.1E-05 1.5E-01\* 8.7E-02 8.72\* 85.6\* 7.6E-02 *T. guasayana* 8.9E-03\* 88.89\* 7.4E-02 - - - 89.28\* 111.7\* 7.4E-02 *T. infestans* 2.3E-02\* 11.85\* 5.8E-02 6.0E-06 3.3E-01\* 5.4E-02 - - - *T. maculata* 4.2E-03\* 2.10\* 1.3E-01 1.0E-02\* 4.0E-02\* 1.3E-01 2.18\* 222.8\* 1.3E-01 *T. matogrossensis* - - - 2.6E-03\* 4.1E-02\* 7.7E-02 3.07\* 270.4\* 7.9E-02 *T. mazzotti* - - - 2.8E-08 3.3E-01\* 7.9E-02 15.69\* 63.6\* 7.7E-02 *T. pallidipennis* - - - 5.8E-05 7.0E-02\* 4.3E-01 - - - *T. patagonica* 1.3E-02\* 137.98\* 2.0E-02 - - - - - - *T. platensis* 5.8E-03\* 7.71\* 7.9E-02 - - - 7.78\* 172.5\* 7.9E-02 *T. protracta* - - - - - - 2.96\* 192.7\* 1.5E-01 *T. pseudomaculata* - - - - - - 5.64\* 163.4\* 1.2E-01 *T. rubrovaria* 1.1E-02\* 10.33\* 1.8E-01 8.2E-06 1.7E-01\* 1.8E-01 10.59\* 86.6\* 1.8E-01 *T. sordida* - - - - - - 146.65\* 60.1\* 4.3E-02 *T. vitticeps* - - - 1.9E-03 2.8E-02\* 2.0E-01 3.29\* 406.0\* 1.9E-01

Table 2. Fit to the Gavrilovs, Gompertz, and Logistic mortality models.

indicates that the model could not be fitted to the data.

**3.4 Phylogenetic Independent Contrasts** 

x). A "\*" indicates a statistically significant coefficient of p< 0.05. SSQ= (

(

Parameter values of the mortality models fitted to the laboratory instantaneous mortality rates

The third group (Group III), which we called "Gradual-low senescence", is defined by a low and progressively increasing instantaneous mortality rate with age. In this group, particularly in *R. neivai*, the mortality rate seems to stabilize at old ages. The senescence pattern variation does not seem to be homogeneous for a given genus: all senescence pattern groups seem to occur in all genera, particularly in the dominant ones (*Triatoma*, *Rhodnius*).

The results of a series of phylogenetic independent contrasts among several dependent variables related to mortality and other variables related to the longevity and reproduction of the triatomines are shown in Tables 3-10. For a better illustration about the phylogenetic relationships we present (Fig. 4) the phylogenetic tree provided by Silva de Paula et al. (2005) but restricted to the 27 species used in our analysis. In Fig. 4 numbers close to the

obs - 

mod)2 . A "-"

Gavrilovs model Gompertz model Logistic model

bifurcations identify each node of the phylogenetic tree. Numbers above the branches indicate the length of each branch, estimated by a branch length/decay index (Bremer support); it is proportional to the distance to ancestors. To the right of each species there is a label with a roman numeral corresponding to the Group into which each species has been classified as following a certain age-specific mortality pattern (Group I= "Late-spiky senescence"; Group II= "Gradual-medium senescence", and Group III= "Gradual-low senescence"; see Section 3.3 for definition of each group).

No significant relationship exists between the female's post reproductive period (FPRP) and the total length of adults or the age of first reproduction (non scaled, weeks) (Table 3). The relationship is statistically significant and positive between FPRP and the age of first reproduction scaled by total longevity. A statistically significant and negative correlation exists between FPRP and the female's reproductive period (scaled by longevity), the female reproductive life period (weeks, i.e., not scaled), and fecundity (expressed as ♀ eggs/♀/life).


Table 3. Female's post-reproductive period regressed on several independent variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several independent variables on the female's post-reproductive period (scaled in terms of lifespan) using 27 species of triatomines.

Therefore, the female's post reproductive period decreases with reproductive effort, which means that females live less after their last reproduction in those triatomine species in which the reproductive effort is high.

We show in Table 4 a statistically significant and negative correlation between the mean instantaneous mortality rate (called hereafter *<sup>x</sup>* ) and the average total female longevity since the egg stage, but no statistically significant relationship with the female's total length.


Table 4. Mean instantaneous mortality rate ( *<sup>x</sup>* ) regressed on longevity and total length Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of average female longevity and total body length as independent variables on the mean instantaneous mortality rate ( *<sup>x</sup>* ).

Fig. 4. Phylogenetic tree of the 27 triatomine species analyzed left from the original phylogenetic tree based on 72 species of Reduviidae proposed by Silva de Paula (2005)

The Quest for Immortality in Triatomines:

effort.

(weeks).

the Gompertz model.

Age of first reproduction (weeks) (

mortality after the first reproduction (1/e

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 237

Two indicators of mortality, total mean female mortality (1/e0) and the mean female

effort indicators (total eggs per female per life and reproductive weeks of females), decreasing with higher reproductive effort (Table 5). The mortality indicator 1/e0 is statistically significant and negatively correlated with both reproductive effort indicators used. Therefore, the total mean female mortality seems to decrease with the reproductive

are significantly correlated to two reproductive

Dependent variables: total mean ♀ mortality (1/e0)

) -0.01470 0.01375 -1.06885 0.30100

*<sup>x</sup>* data that could be fitted to

**Independent variable Coeff. Std. Err. T value p** 

**Independent variable Coeff. Std. Err. T value p** 

Total eggs/♀/life -2.7E-05 2.5E-06 -11.05 4.1E-11

Reproductive weeks of ♀ -0.00092 0.00012 -7.96 2.6E-08

Total eggs/♀/life -3.0E-05 4.5E-06 -6.72 4.8E-07

Reproductive weeks of ♀ -0.00127 9.4E-05 -13.54 5.2E-13 Table 5. Two indicators of mortality rate regressed on two reproductive effort variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of reproductive effort indicators as independent variables on two measures of mortality using 27 species of triatomines. The parameter e0 is the expectation of life at birth

scaled by total longevity -0.67365 0.89178 -0.75539 0.46099

scaled by lifespan -0.98590 0.87855 -1.12219 0.27834

Total female longevity (weeks) 0.00478 0.00692 0.69122 0.49933 Total length (mm) -0.03625 0.02683 -1.35086 0.19554 Table 6. The parameter *b* of the Gompertz model regressed on several independent variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several independent variables on the parameter *b* of the Gompertz mortality model.

represents the female's age (weeks) of first reproduction.

**Independent variable Coefficient Std t value p**  Average ♀eggs/♀/week -0.01099 0.07154 -0.15368 0.87979 Average ♀eggs/♀/life 0.00076 0.00133 0.56869 0.57746

The phylogenetic tree used was reduced to the 18 species with

Dependent variable: mean ♀ mortality after first reproduction (1/e

Fig. 4. Phylogenetic tree of the 27 triatomine species analyzed left from the original phylogenetic tree based on 72 species of Reduviidae proposed by Silva de Paula (2005) Two indicators of mortality, total mean female mortality (1/e0) and the mean female mortality after the first reproduction (1/e are significantly correlated to two reproductive effort indicators (total eggs per female per life and reproductive weeks of females), decreasing with higher reproductive effort (Table 5). The mortality indicator 1/e0 is statistically significant and negatively correlated with both reproductive effort indicators used. Therefore, the total mean female mortality seems to decrease with the reproductive effort.


Table 5. Two indicators of mortality rate regressed on two reproductive effort variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of reproductive effort indicators as independent variables on two measures of mortality using 27 species of triatomines. The parameter e0 is the expectation of life at birth (weeks).


Table 6. The parameter *b* of the Gompertz model regressed on several independent variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several independent variables on the parameter *b* of the Gompertz mortality model. The phylogenetic tree used was reduced to the 18 species with *<sup>x</sup>* data that could be fitted to the Gompertz model. represents the female's age (weeks) of first reproduction.

The Quest for Immortality in Triatomines:

Colwell's rain index of

Temperature coefficient of

Minim temperature of coldest

Maximum temperature of the

Maximum rain of the rainiest

Number of super-humid

Minimum rain of the driest month

mortality (1/e0) using 27 species of triatomines.

equal length, some correlations between

when divided in two periods, the

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 239

**Independent variables Coefficient Std Err t value p**  Surface area (km2) -0.00140 0.00022 -6.43226 9.8E-07 Modal latitude (degrees) 0.00034 0.00022 1.54316 0.13536 Modal longitude (degrees) 0.00025 0.00021 1.20700 0.23900 Average altitude (m) 1.1E-05 1.1E-05 1.05245 0.30266 Average annual temperature (ºC) -0.00060 0.00180 -0.33135 0.74315 Minimum annual temperature (ºC) 0.00162 0.00137 1.18693 0.24641 Maximum annual temperature (ºC) 0.00225 0.00210 1.07133 0.29425 Average annual precipitation (mm) -1.5E-05 4.2E-06 -3.56725 0.00149 95% Lower precipitation (mm) -2.6E-05 7.7E-06 -3.33849 0.00264 95% Upper precipitation (mm) -9.3E-06 2.7E-06 -3.47074 0.00190

predictability -0.15913 0.04713 -3.37684 0.00240 Rain coefficient of variation ( %) 0.00114 0.00044 2.61404 0.01494 Temperature amplitude (ºC) 0.00047 0.00121 0.38703 0.70201

variation ( %) 0.00072 0.00055 1.30837 0.20265 Average annual NDVI -0.05010 0.03350 -1.49561 0.14727 NDVI coefficient of variation ( %) 0.00804 0.00263 3.06192 0.00520 Average AET (mm) -0.00023 0.00015 -1.47877 0.15169

month (ºC) 0.00088 0.00093 0.94976 0.35133

warmest month (ºC) -9.8E-05 0.00272 -0.03617 0.97144

month (mm) -0.00033 0.00008 -3.99956 0.00050

(mm) -0.00011 3.2E-05 -3.26415 0.00317 Number of dry months/year 0.00197 0.00108 1.83082 0.07907 Number of humid months/year 0.00214 0.00181 1.18209 0.24829

months/year -0.00200 0.00089 -2.25957 0.03282

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several climatic variables used as independent variables on the average

statistically non-significant with respect to the mean reproductive effort (first line in Table 9). However, if we divide the individual female adult life in two, three or four periods of

effort for the same period are statistically significant and positive (Table 9 except the first

given period and the reproductive effort in the previous period(s) (Table 10). For instance,

positively correlated to the reproductive effort during the first period (first line in Table

*<sup>x</sup>* were

*<sup>x</sup>* for a

*<sup>x</sup>* for a given period and the mean reproductive

*<sup>x</sup>* value in the second period is significantly and

Table 8. Average mortality (1/e0) regressed on various geographic/climatic variables

The results obtained from the regressed of mean instantaneous mortality rate

line). Furthermore, we observe several significant positive correlations between

No statistically significant relationship was observed between the parameter *b* of the Gompertz mortality model (which is related to the shape of the mortality pattern) and the life history traits indicated in Table 6. Additionally, no statistically significant correlation was observed between the parameter *b* of the Gompertz mortality model and the geographic and/or climatic variables indicated in the table (Table 7).


Table 7. The *b* Gompertz model parameter regressed on geographic/climatic variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several climatic variables used as independent variables on the parameter *b* of the Gompertz model. The phylogenetic tree was reduced to the 18 species with *<sup>x</sup>* that could be fitted to the Gompertz model. The NDVI is the Normalized Difference Vegetation Index.

Table 8 shows that from several geographic and/or climatic variables analyzed only the surface area and various precipitation indicators are correlated (negatively) with the mean mortality rate (defined here as 1/e0), while with the coefficient of variation (in %) of the Normalized Difference Vegetation Index (NDVI, a common indicator of live green vegetation obtained from satellite data) it was found to be positively correlated. In other words, species' geographical range size and some climatic factors, mainly the ones related to precipitation, seem to be related to mean mortality.

However, mortality (1/e0) is probably not a good estimator of mean mortality because in our data the mortality rate varies greatly with age and this violates the hypothesis underlying this parameter as estimator of mean mortality rate. In consequence, we also used a more reliable estimator of the mean mortality rate from our data: the average for different time periods of the female's adult life ( *<sup>x</sup>* ) (see Tables 9 and 10).

No statistically significant relationship was observed between the parameter *b* of the Gompertz mortality model (which is related to the shape of the mortality pattern) and the life history traits indicated in Table 6. Additionally, no statistically significant correlation was observed between the parameter *b* of the Gompertz mortality model and the geographic

**Independent variable Coefficient Std Dev t value p** 

Surface area -0.02572 0.02877 -0.89415 0.38449

Average annual precipitation (mm) 0.00027 0.00023 1.19358 0.25004

95% Lower precipitation (mm) 0.00029 0.00043 0.66386 0.51623

95% Upper precipitation (mm) 0.00021 0.00014 1.52282 0.14732

predictability 2.40489 2.66950 0.90088 0.38101 Precipitation coefficient of variation ( %) 0.03058 0.02010 1.52139 0.14768

NDVI coefficient of variation ( %) -0.11072 0.12554 -0.88195 0.39086

Maximum rain of the rainiest month (mm) 0.00405 0.00520 0.77925 0.44721

Minimum rain of the driest month (mm) 0.00204 0.00170 1.20070 0.24734

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several climatic variables used as independent variables on the parameter *b* of

could be fitted to the Gompertz model. The NDVI is the Normalized Difference

Table 8 shows that from several geographic and/or climatic variables analyzed only the surface area and various precipitation indicators are correlated (negatively) with the mean mortality rate (defined here as 1/e0), while with the coefficient of variation (in %) of the Normalized Difference Vegetation Index (NDVI, a common indicator of live green vegetation obtained from satellite data) it was found to be positively correlated. In other words, species' geographical range size and some climatic factors, mainly the ones related to

However, mortality (1/e0) is probably not a good estimator of mean mortality because in our data the mortality rate varies greatly with age and this violates the hypothesis underlying this parameter as estimator of mean mortality rate. In consequence, we also used a more reliable estimator of the mean mortality rate from our data: the average for different

*<sup>x</sup>* ) (see Tables 9 and 10).

*<sup>x</sup>* that

Table 7. The *b* Gompertz model parameter regressed on geographic/climatic variables

the Gompertz model. The phylogenetic tree was reduced to the 18 species with

and/or climatic variables indicated in the table (Table 7).

Colwell's precipitation index of

Vegetation Index.

precipitation, seem to be related to mean mortality.

time periods of the female's adult life (


Table 8. Average mortality (1/e0) regressed on various geographic/climatic variables

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of several climatic variables used as independent variables on the average mortality (1/e0) using 27 species of triatomines.

The results obtained from the regressed of mean instantaneous mortality rate *<sup>x</sup>* were statistically non-significant with respect to the mean reproductive effort (first line in Table 9). However, if we divide the individual female adult life in two, three or four periods of equal length, some correlations between *<sup>x</sup>* for a given period and the mean reproductive effort for the same period are statistically significant and positive (Table 9 except the first line). Furthermore, we observe several significant positive correlations between *<sup>x</sup>* for a given period and the reproductive effort in the previous period(s) (Table 10). For instance, when divided in two periods, the *<sup>x</sup>* value in the second period is significantly and positively correlated to the reproductive effort during the first period (first line in Table

The Quest for Immortality in Triatomines:

**4.1 Senescense in the triatomines** 

this group, the regression analyses by the phylogeny.

**4. Discussion** 

ecology.

comparisons.

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 241

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of accumulated average fecundity on the accumulated average instantaneous

The following are our concepts on triatomine senescence in the light of evolutionary

Since the first attempt to explain evolution of ageing was made by Weismann (1891, cited in Kirkwood & Holliday 1979) senescence has been a major topic of research in evolutionary ecology, both from an experimental and a theoretical approach. Being such a general area of investigation it is not surprising that it has relied strongly on the use of a comparative approach across a wide range of taxa. The phylogenetic variation in rates of senescence has been considered a consequence of a combination of factors that decrease the rate of decline in reproductive probability (and intensifying selection against senescence) and factors that increase this rate (relaxing selection against senescence). We applied a comparative metaanalyses approach within a single subfamily (Triatominae) correcting, for the first time in

Williams (1957) has argued that the rate of senescence shown by any species will reflect the balance between a direct adverse selection of senescence as an unfavorable character, and an indirect, favorable selection through the age-related bias in the selection of pleiotropy genes. Thus positive variations in fecundity increase adult mortality rate, and affect other lifehistory traits (e.g., the shape of the distribution of reproductive effort with age) and thereby influence the evolution of senescence and phylogenetic variation. This theory predicts that (a) rapid morphogenesis should be associated with rapid senescence, (b) that senescence should always be a generalized deterioration of many organs and systems, and (c) that postreproductive periods should be short and infrequent in any wild population. The latter prediction seems to have been confirmed in the case of triatomines (at least in the laboratory). This is another reason why, despite having selected for our study a relatively low taxonomic level (the species of only five genera, so we can expect a strong degree of phylogenetic relatedness), it is important to have the phylogeny included in these

Our study shows that the 27 triatomine species analyzed present a senescence pattern that does not decrease at older ages. Mortality rate stays very low during most of the juvenile stages (except in the egg stage for some species) and during the first part of adult stage, and then increases greatly in the old ages in most species. This senescence pattern observed in triatomine species are in contrast with patterns reported for various Diptera: *Drosophila melanogaster* (Curtsinger et al., 1992; Pletcher & Curtsinger, 1998), *Ceratitis capitata* (Carey et al., 1992, 1998), *Anastrepha ludens* (Carey et al., 2005), and *Aedes aegypti* (Styer et al., 2007).

The mortality pattern of triatomines (Chaves et al., 2004a, Rabinovich et al 2010, and this study) seems to be more similar to that reported in *Oncopeltus fasciatus* (Dingle, 1966), *Dysdercus fasciatus* (Dingle, 1966), and *Callosobruchus maculatus* (Tatar et al., 1993) and is

mortality rates by periods of equal length (see text) using 27 species of triatomines.

10) or during the first and second ones (second line in Table 10). Similarly, when divided in three periods, the *<sup>x</sup>* value of the third period is significantly and positively correlated to the reproductive effort during the all three periods (lines 4 and 6 in Table 10). Similarly for four periods.


Table 9. Average mortality and fecundity variables lagged by periods

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of average fecundity ( *mx* ) on the average instantaneous mortality rates ( *<sup>x</sup>* ) by periods of equal length (see text) using 27 species of triatomines.


Table 10. Accumulated average mortality and fecundity variables lagged by periods

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of the effect of accumulated average fecundity on the accumulated average instantaneous mortality rates by periods of equal length (see text) using 27 species of triatomines.

## **4. Discussion**

240 Senescence

10) or during the first and second ones (second line in Table 10). Similarly, when divided

to the reproductive effort during the all three periods (lines 4 and 6 in Table 10). Similarly

*m̅* x 1/1 *μ̅ <sup>x</sup>* 1/1 0.00021 0.001126 0.187 0.85354 *m̅* x 1/2 *μ̅ <sup>x</sup>* 1/2 -0.00556 0.00047 -11.96 7.7E-12 *m̅* x 2/2 *μ̅ <sup>x</sup>* 2/2 0.01840 0.00501 3.67 0.00115 *m̅* x 1/3 *μ̅ <sup>x</sup>* 1/3 -0.00266 0.00027 -9.75 5.3E-10 *m̅* x 2/3 *μ̅ <sup>x</sup>* 2/3 -0.00696 0.00212 -3.29 0.00300 *m̅* x 3/3 *μ̅ <sup>x</sup>* 3/3 0.02206 0.00813 2.71 0.01190 *m̅* x 1/3 *μ̅ <sup>x</sup>* 2/3 -0.00437 0.00062 -7.00 2.0E-07 *m̅* x 2/3 *μ̅ <sup>x</sup>* 3/3 0.01603 0.00327 4.91 4.7E-05 *m̅* x 1/4 *μ̅ <sup>x</sup>* 1/4 -0.00216 0.00022 -9.60 7.3E-10 *m̅* x 2/4 *μ̅ <sup>x</sup>* 2/4 -0.01076 0.00103 -10.41 1.4E-10 *m̅* x 3/4 *μ̅ <sup>x</sup>* 3/4 0.00910 0.00678 1.34 0.37520 *m̅* x 4/4 *μ̅ <sup>x</sup>* 4/4 0.02308 0.00877 2.63 0.01438 *m̅* x 1/4 *μ̅ <sup>x</sup>* 2/4 -0.00769 0.00066 -11.73 1.2E-11 *m̅* x 2/4 *μ̅ <sup>x</sup>* 3/4 0.01196 0.00148 8.07 2.0E-08 *m̅* x 3/4 *μ̅ <sup>x</sup>* 4/4 0.00822 0.00607 1.35 0.23480

Simple lineal regression results, using the Phylogenetic Independent Contrast method, of

*m̅* x 1/2 *μ̅ <sup>x</sup>* 2/2 0.00528 0.00121 4.38 0.00019 *m̅* x 1+2/2 *μ̅ <sup>x</sup>* 2/2 0.01071 0.00197 5.45 1.2E-05 *m̅* x 1+2/3 *μ̅ <sup>x</sup>* 2/3 -0.00583 0.00102 -5.73 5.7E-06 *m̅* x 1+2/3 *μ̅ <sup>x</sup>* 3/3 0.00960 0.00203 4.73 7.5E-05 *m̅* x 2+3/3 *μ̅ <sup>x</sup>* 3/3 0.02390 0.00489 4.89 4.9E-05 *m̅* x 1+2+3/3 *μ̅ <sup>x</sup>* 3/3 0.01430 0.00276 5.18 2.3E-05 *m̅* x 1+2/4 *μ̅ <sup>x</sup>* 2/4 -0.00903 0.00079 -11.37 2.3E-11 *m̅* x 1+2/4 *μ̅* x 3/4 0.00970 0.00128 7.56 6.5E-08 *m̅* x 2+3/4 *μ̅ <sup>x</sup>* 3/4 0.02099 0.00267 7.85 3.3E-08 *m̅* x 2+3/4 *μ̅ <sup>x</sup>* 4/4 0.00450 0.00436 1.03 0.21760 *m̅* x 1+2+3/4 *μ̅ <sup>x</sup>* 3/4 0.01455 0.00176 8.25 1.3E-08 *m̅* x 1+2+3/4 *μ̅ <sup>x</sup>* 4/4 0.00231 0.00301 0.77 0.47810 *m̅* x 1+2+3+4/4 *μ̅ <sup>x</sup>* 4/4 0.00374 0.00383 0.98 0.31270

**variable Coefficient Std Err t value p** 

*<sup>x</sup>* ) by

the effect of average fecundity ( *mx* ) on the average instantaneous mortality rates (

Table 10. Accumulated average mortality and fecundity variables lagged by periods

*<sup>x</sup>* value of the third period is significantly and positively correlated

**variable Coefficient Std Err t value p** 

in three periods, the

**Independent variable** 

**Independent variable** 

for four periods.

**Dependent** 

Table 9. Average mortality and fecundity variables lagged by periods

periods of equal length (see text) using 27 species of triatomines.

**Dependent** 

The following are our concepts on triatomine senescence in the light of evolutionary ecology.

## **4.1 Senescense in the triatomines**

Since the first attempt to explain evolution of ageing was made by Weismann (1891, cited in Kirkwood & Holliday 1979) senescence has been a major topic of research in evolutionary ecology, both from an experimental and a theoretical approach. Being such a general area of investigation it is not surprising that it has relied strongly on the use of a comparative approach across a wide range of taxa. The phylogenetic variation in rates of senescence has been considered a consequence of a combination of factors that decrease the rate of decline in reproductive probability (and intensifying selection against senescence) and factors that increase this rate (relaxing selection against senescence). We applied a comparative metaanalyses approach within a single subfamily (Triatominae) correcting, for the first time in this group, the regression analyses by the phylogeny.

Williams (1957) has argued that the rate of senescence shown by any species will reflect the balance between a direct adverse selection of senescence as an unfavorable character, and an indirect, favorable selection through the age-related bias in the selection of pleiotropy genes. Thus positive variations in fecundity increase adult mortality rate, and affect other lifehistory traits (e.g., the shape of the distribution of reproductive effort with age) and thereby influence the evolution of senescence and phylogenetic variation. This theory predicts that (a) rapid morphogenesis should be associated with rapid senescence, (b) that senescence should always be a generalized deterioration of many organs and systems, and (c) that postreproductive periods should be short and infrequent in any wild population. The latter prediction seems to have been confirmed in the case of triatomines (at least in the laboratory). This is another reason why, despite having selected for our study a relatively low taxonomic level (the species of only five genera, so we can expect a strong degree of phylogenetic relatedness), it is important to have the phylogeny included in these comparisons.

Our study shows that the 27 triatomine species analyzed present a senescence pattern that does not decrease at older ages. Mortality rate stays very low during most of the juvenile stages (except in the egg stage for some species) and during the first part of adult stage, and then increases greatly in the old ages in most species. This senescence pattern observed in triatomine species are in contrast with patterns reported for various Diptera: *Drosophila melanogaster* (Curtsinger et al., 1992; Pletcher & Curtsinger, 1998), *Ceratitis capitata* (Carey et al., 1992, 1998), *Anastrepha ludens* (Carey et al., 2005), and *Aedes aegypti* (Styer et al., 2007).

The mortality pattern of triatomines (Chaves et al., 2004a, Rabinovich et al 2010, and this study) seems to be more similar to that reported in *Oncopeltus fasciatus* (Dingle, 1966), *Dysdercus fasciatus* (Dingle, 1966), and *Callosobruchus maculatus* (Tatar et al., 1993) and is

The Quest for Immortality in Triatomines:

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 243

With rare exceptions the Triatominae are exclusively hematophagous, and they also show strict hematophagy across all developmental stages. This may be a potential key to the explanation of the observed mortality pattern: hematophagy from vertebrate blood leads to the digestion of vertebrate hemoglobin resulting in the production of large amounts of heme, a potentially cytotoxic molecule that can exert biological damage (Graca-Souza et al., 2006). The evolution of hematophagy has resulted in many adaptations developed by blood-feeding insects and ticks to counteract those deleterious effects. Antioxidant enzymes and urates are known to play a major role in the protection of cells against free radical damage, and massive amounts of urates have been found in the haemolymph of *R. prolixus* (Souza et al., 1997). Given the strict hematophagy of triatomines these mechanisms that reduce the accumulation of oxidative substances associated with aging (Graca-Souzaet al., 2006) may be one of the basic

mechanisms that may help explain the senescence patterns observed in the triatomines.

periods show a delay in the effect of the latter on the former, indicating an important reproductive investment before a high accumulated instantaneous mortality rate occurs. The

female longevity suggests that species with high longevity can invest in reproduction with a relatively low mean mortality and then a smaller senescence rate than the species with low longevity. This type of relationship between reproductive effort and the effects on age-specific mortality rate is similar to the one found in the beetle *C. maculatus* (Tatar et al., 1993) and

Additionally, the female's post reproductive period decreases with reproductive effort indicating that longevity after their last reproduction is shorter in species in which the reproductive effort is high. The above results suggest a trade-off between mortality rate and reproductive effort: a large investment in the reproduction during the first and/or intermediate part of the female adult life that seems to result in an increase in high mortality, and then a high senescence rate at the end of the adult life. Such trade-off could contribute to explain the variation between species in the mean mortality and in the postreproductive period duration but probably not the difference in the senescence pattern since no significant relationship exists between the parameter *b* of the Gompertz model and other

The balance between extrinsic and intrinsic mortality rates is an important factor underlying the evolution and the diversity of senescence patterns. Williams (1957) proposed that organisms living in environments with high extrinsic mortality rate may evolve towards high senescence rates. Physiological senescence results from an optimal equilibrium between energy allocation in somatic maintenance and other competitive traits as reproduction (Kirkwood, 2005). In habitats in which life expectancy is short resulting from extrinsic factors (as high predation, starvation, etc.), the maintenance of costly mechanisms

Conversely, species living in habitats with low extrinsic mortality may increase energy allocation in somatic maintenance until old age and increase longevity by natural selection.

to guarantee the reparation of metabolic deterioration is not evolutionary stable.

*<sup>x</sup>* and average fecundity by

*<sup>x</sup>* and average

The relationships between average instantaneous mortality rates

variables used as indicators of reproductive effort.

**4.3 Triatominae senescence in the light of evolutionary ecology** 

negative relationship between the average instantaneous mortality rate

confirms the observation of Sulbaran & Chaves (2006) in the kissing bug *R. prolixus*.

consistent with models of senescence based on the decline in physiological functions with age (known as the "disposable soma" theory for the evolution of senescence; Kirkwood & Holliday, 1979; Kirkwood & Rose, 1991; Kirkwood & Austad, 2000; Kirkwood, 2002), which can be considered a consequence of the equilibrium predictions of the antagonisticpleiotropy and mutation accumulation hypotheses (Abrams & Ludwig, 1995).

In our comparative analysis we have used several measures of mortality. Although the use of the Gompertz model to describe the acceleration of mortality with age has been a matter of debate (Nusbaum et al., 1996), for a comparative study this model seems more sound than, e.g., maximum lifespan, particularly because of the sensitivity of maximum lifespan to the initial numbers of a cohort. Nusbaum et al. (1996) have analyzed the evolutionary relationships among several measures of mortality (Gompertz parameters, and average and maximum longevity) in 50 related populations of *D. melanogaster*; they included populations that had been selected for postponed aging and in their conclusions they give credit to a redundancy among these measures of aging, and consider that both the maximum lifespan and the Gompertz equation as adequate indices of aging in evolutionary research. This gives support to their use in our study.

#### **4.2 High variation in senescence pattern**

Our results show that the mortality pattern with age varies greatly among triatomine species (showing late-spiky, gradual-medium, and gradual-low senescence) despite our study was based on cohorts reared under very homogeneous environmental conditions; the present challenge is to understand which are the ultimate factors underlying such diversity of mortality patterns in triatomines (Fig. 3). These different patterns are observed in all the species we studied, even in the more phylogenetic related species within any given genus (Fig. 4). We do not dismiss the possibility that the small initial number of eggs of some cohorts may have played a role in the estimation of demographic parameters (e.g. threshold mortality) (Carey, 2001), and thus in the high degree of mortality patterns variability; however, we still think that the variability found is genuine and could reflects multiple underlying causes.

Despite most of the studied species show "late-spiky" (n= 14) or "gradual-medium" senescence (n= 9), the species in the "gradual-low" group (n= 4), particularly *R. neivai*, seem to show a weak late-life mortality plateau. Some demographic findings point to the existence of a late-life mortality plateau in a few dipteran insects (Mueller & Rose, 1996), with both antagonistic pleiotropy and mutation accumulation as driving population genetic mechanisms; this late-life attribute is a switch from accelerating mortality to a relatively stable mortality (Rauser et al., 2006); such plateaus seem to depend on the collection of high numbers of late-life data (Carey et al., 1992). One plausible explanation of the "plateau" behavior was introduced by Vaupel et al. (1979) assuming a life-long heterogeneity in the mortality rates: more robust subgroups survive to later ages, slowing the rate of decline in average survival probabilities at late ages among large cohorts. The older remaining individuals from the cohort are expected to be much more robust so that the mortality rate becomes a very shallow function of age, resembling a plateau. However this demographic heterogeneity model does not seem to be a reasonable explanation of demographic patterns and it has only a weak biological basis (Mueller et al., 2003).

consistent with models of senescence based on the decline in physiological functions with age (known as the "disposable soma" theory for the evolution of senescence; Kirkwood & Holliday, 1979; Kirkwood & Rose, 1991; Kirkwood & Austad, 2000; Kirkwood, 2002), which can be considered a consequence of the equilibrium predictions of the antagonistic-

In our comparative analysis we have used several measures of mortality. Although the use of the Gompertz model to describe the acceleration of mortality with age has been a matter of debate (Nusbaum et al., 1996), for a comparative study this model seems more sound than, e.g., maximum lifespan, particularly because of the sensitivity of maximum lifespan to the initial numbers of a cohort. Nusbaum et al. (1996) have analyzed the evolutionary relationships among several measures of mortality (Gompertz parameters, and average and maximum longevity) in 50 related populations of *D. melanogaster*; they included populations that had been selected for postponed aging and in their conclusions they give credit to a redundancy among these measures of aging, and consider that both the maximum lifespan and the Gompertz equation as adequate indices of aging in evolutionary research. This gives

Our results show that the mortality pattern with age varies greatly among triatomine species (showing late-spiky, gradual-medium, and gradual-low senescence) despite our study was based on cohorts reared under very homogeneous environmental conditions; the present challenge is to understand which are the ultimate factors underlying such diversity of mortality patterns in triatomines (Fig. 3). These different patterns are observed in all the species we studied, even in the more phylogenetic related species within any given genus (Fig. 4). We do not dismiss the possibility that the small initial number of eggs of some cohorts may have played a role in the estimation of demographic parameters (e.g. threshold mortality) (Carey, 2001), and thus in the high degree of mortality patterns variability; however, we still think that the variability found is genuine and could reflects multiple

Despite most of the studied species show "late-spiky" (n= 14) or "gradual-medium" senescence (n= 9), the species in the "gradual-low" group (n= 4), particularly *R. neivai*, seem to show a weak late-life mortality plateau. Some demographic findings point to the existence of a late-life mortality plateau in a few dipteran insects (Mueller & Rose, 1996), with both antagonistic pleiotropy and mutation accumulation as driving population genetic mechanisms; this late-life attribute is a switch from accelerating mortality to a relatively stable mortality (Rauser et al., 2006); such plateaus seem to depend on the collection of high numbers of late-life data (Carey et al., 1992). One plausible explanation of the "plateau" behavior was introduced by Vaupel et al. (1979) assuming a life-long heterogeneity in the mortality rates: more robust subgroups survive to later ages, slowing the rate of decline in average survival probabilities at late ages among large cohorts. The older remaining individuals from the cohort are expected to be much more robust so that the mortality rate becomes a very shallow function of age, resembling a plateau. However this demographic heterogeneity model does not seem to be a reasonable explanation of demographic patterns

pleiotropy and mutation accumulation hypotheses (Abrams & Ludwig, 1995).

support to their use in our study.

underlying causes.

**4.2 High variation in senescence pattern** 

and it has only a weak biological basis (Mueller et al., 2003).

With rare exceptions the Triatominae are exclusively hematophagous, and they also show strict hematophagy across all developmental stages. This may be a potential key to the explanation of the observed mortality pattern: hematophagy from vertebrate blood leads to the digestion of vertebrate hemoglobin resulting in the production of large amounts of heme, a potentially cytotoxic molecule that can exert biological damage (Graca-Souza et al., 2006). The evolution of hematophagy has resulted in many adaptations developed by blood-feeding insects and ticks to counteract those deleterious effects. Antioxidant enzymes and urates are known to play a major role in the protection of cells against free radical damage, and massive amounts of urates have been found in the haemolymph of *R. prolixus* (Souza et al., 1997). Given the strict hematophagy of triatomines these mechanisms that reduce the accumulation of oxidative substances associated with aging (Graca-Souzaet al., 2006) may be one of the basic mechanisms that may help explain the senescence patterns observed in the triatomines.

The relationships between average instantaneous mortality rates *<sup>x</sup>* and average fecundity by periods show a delay in the effect of the latter on the former, indicating an important reproductive investment before a high accumulated instantaneous mortality rate occurs. The negative relationship between the average instantaneous mortality rate *<sup>x</sup>* and average female longevity suggests that species with high longevity can invest in reproduction with a relatively low mean mortality and then a smaller senescence rate than the species with low longevity. This type of relationship between reproductive effort and the effects on age-specific mortality rate is similar to the one found in the beetle *C. maculatus* (Tatar et al., 1993) and confirms the observation of Sulbaran & Chaves (2006) in the kissing bug *R. prolixus*.

Additionally, the female's post reproductive period decreases with reproductive effort indicating that longevity after their last reproduction is shorter in species in which the reproductive effort is high. The above results suggest a trade-off between mortality rate and reproductive effort: a large investment in the reproduction during the first and/or intermediate part of the female adult life that seems to result in an increase in high mortality, and then a high senescence rate at the end of the adult life. Such trade-off could contribute to explain the variation between species in the mean mortality and in the postreproductive period duration but probably not the difference in the senescence pattern since no significant relationship exists between the parameter *b* of the Gompertz model and other variables used as indicators of reproductive effort.

## **4.3 Triatominae senescence in the light of evolutionary ecology**

The balance between extrinsic and intrinsic mortality rates is an important factor underlying the evolution and the diversity of senescence patterns. Williams (1957) proposed that organisms living in environments with high extrinsic mortality rate may evolve towards high senescence rates. Physiological senescence results from an optimal equilibrium between energy allocation in somatic maintenance and other competitive traits as reproduction (Kirkwood, 2005). In habitats in which life expectancy is short resulting from extrinsic factors (as high predation, starvation, etc.), the maintenance of costly mechanisms to guarantee the reparation of metabolic deterioration is not evolutionary stable.

Conversely, species living in habitats with low extrinsic mortality may increase energy allocation in somatic maintenance until old age and increase longevity by natural selection.

The Quest for Immortality in Triatomines:

when their death may occur (Gadgil & Bossert, 1970).

senescence in insects.

communication).

account.

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 245

called the "hypothesis of terminal investment" (Ricklef, 2000; Coulson & Fairweather, 2001; Ricklefs, 2008). Thus senescent individuals may invest more in reproduction than the nonsenescent individuals. Our observations in the triatomines do not support these predictions. Indeed most of the reproductive effort (about 50 to 90%) occurs in the first and intermediate part of the adult life and before the high mortality that takes place at older ages. Furthermore, the duration of the post-reproductive period decreases with reproductive effort suggesting a trade-off between reproduction allocation and future survival. Recently William's (1966) prediction and the concept of terminal investment have been criticized because of scarce empirical support and for resorting to two hypotheses based on quite unrealistic assumptions: (i) organisms must have a fixed longevity, which implies that individual trajectories cannot influence longevity, and (ii) in order to increase their reproductive effort at the end of their life during their last reproductive occasion(s), individuals must be able to anticipate their future, i.e., they have to use cues indicating

Environmental heterogeneity, and particularly high environmental uncertainty (stochastic effects), affect circumstances that are very important in insects, such as encounter rates associated to suitable oviposition sites, food and refuge availability, physiological state (e.g., reserves for producing oocytes, egg maturation rate, and somatic maintenance costs), and expected reproductive success that can lead to different patterns of behavior and rates of mortality and reproduction (Partridge & Mangel, 1999). Environmental stochasticity and/or density-dependence processes can select bet-hedging dormancy including a development delay in insects (e.g., Menu et al., 2000; Gourbière & Menu, 2009; Rajon et al., 2009). Such a risk spreading strategy has been postulated to exist in triatomines (Menu et al., 2010) and we recommend that this approach be incorporated in future studies for understanding

Mortality rates of triatomine vectors have epidemiological importance through the demography of its populations (Chaves et al., 2004a). Particularly, it has been shown in these insects that important changes in life-history traits take place when reared under laboratory conditions; for example, in *T. infestans*, *T. pseudomaculata*, *T. brasiliensis*, and *P. megistus* after only four years in the laboratory, the reduction in the number of eggs within the first month of oviposition and the average female life span was in average 37.5 and 45.9%, respectively (Perlowagora-Szumlewicz, 1976). Similar changes, including a reduction in life history traits and population growth parameters, during four successive and separate generations of *T. infestans* of Argentina (reared in the laboratory from a first generation from a sylvatic individual) have also been found (G. Martí, personal

The adaptation to a domestic environment causes similar effects on triatomines as the adaptation to laboratory conditions (Forattini, 1980): indoor conditions of rural houses provide better chances of feeding, and are characterized by smaller predation risks, and smaller fluctuations of temperature and relative humidity that exclude potentially deleterious extreme values. In our study we did not analyze possible adaptations to the laboratory because the precise number of laboratory generations was known for only a few cohorts; however, it is recommended that, when estimating trade-offs, and in particular the senescence pattern, the influence of such changes should be taken into

In triatomines no quantitative data exists concerning extrinsic mortality factors in the field. The levels of extrinsic mortality in relation to the habitat (domestic, peri-domestic and sylvatic), is still little known in triatomines. However, our results show a negative relationship between mortality rate and female longevity supporting Williams' (1957) prediction, contrary to other empirical observations that do not support this prediction (Promislow, 1991; Ricklefs, 1998; Reznicket et al., 2004), which has also been criticized on conceptual grounds due to the difficulty in separating extrinsic and intrinsic mortality rates (Williams & Day, 2003).

In a recent comparative analysis of mammal and bird survival senescence Jones et al. (2008) arrived to generalizations such as that mammals senesce faster than similarly sized birds. Furthermore, McCoy & Gillooly (2008) developed a model of natural mortality (relating body size and temperature to biological rate processes) and tested it with extensive field data from plants, invertebrates, fish, birds and mammals; their results indicate that much of the heterogeneity in natural mortality rates can be predicted, explicitly and quantitatively, despite the high diversity of extrinsic sources of mortality in natural systems, something that suggests that mortality rates may be governed by common rules.

We show that the geographical range size of species and some climatic factors, mainly the ones related to precipitation, seem to be related to total mean mortality (1/e0). These results suggest that the influence of geographic and climatic factors on the senescence pattern deserves further investigation. However, can the potential senescence that is observed and measured in the laboratory be expressed in natural habitats where extrinsic mortality occurs? In the field, triatomines could die before intrinsic mortality and decreasing fecundity occurs, due to extrinsic factors such as predation, parasitism, extreme climatic conditions, etc. Recent studies in vertebrates (Adams, 1985; Gaillard et al., 1993; Reznick et al., 2002; Rebke et al., 2010) show that senescence is observed under natural conditions, but that kind of information is lacking for triatomines and needs to be investigated. Williams (1957) claimed that greater rates of extrinsic mortality (age- and condition-independent) favored more rapid senescence, but Abrams (1991) showed that the effects of the "extrinsic" mortality affect differentially the rate of senescence as a function of the degree of densitydependence. Abrams (1991) also showed that mortality patterns, contrary to Williams' (1957) predictions, are possible when density-dependence is present, and acts primarily on the survival or fertility of later ages, or when most of the variation in mortality rates is due to variation in non-extrinsic mortality.

There are few laboratory evaluations of the density-dependent processes in triatomines. Rodríguez & Rabinovich (1980) showed that in *R. prolixus* density had a significant effect on the development rate of second, third, and fourth instars, but not on the survivorship of either the first or fifth instars, or even of the adults, nor on the instantaneous population parameters, or the age-specific parameters. Influence of density-dependence on the senescence pattern in triatomines still needs more research.

How organisms distribute their "investment" in reproduction with respect to age is a major question in the senescence theory. It will be optimal for an organism to "invest" more in its reproduction when it becomes old-aged (Williams, 1966). An extension of William's (1966) theory is that old individuals may increase their reproductive effort during their last reproductive occasion(s) because it is their last chance to reproduce. This extension was

In triatomines no quantitative data exists concerning extrinsic mortality factors in the field. The levels of extrinsic mortality in relation to the habitat (domestic, peri-domestic and sylvatic), is still little known in triatomines. However, our results show a negative relationship between mortality rate and female longevity supporting Williams' (1957) prediction, contrary to other empirical observations that do not support this prediction (Promislow, 1991; Ricklefs, 1998; Reznicket et al., 2004), which has also been criticized on conceptual grounds due to the difficulty in separating extrinsic and intrinsic mortality rates

In a recent comparative analysis of mammal and bird survival senescence Jones et al. (2008) arrived to generalizations such as that mammals senesce faster than similarly sized birds. Furthermore, McCoy & Gillooly (2008) developed a model of natural mortality (relating body size and temperature to biological rate processes) and tested it with extensive field data from plants, invertebrates, fish, birds and mammals; their results indicate that much of the heterogeneity in natural mortality rates can be predicted, explicitly and quantitatively, despite the high diversity of extrinsic sources of mortality in natural systems, something

We show that the geographical range size of species and some climatic factors, mainly the ones related to precipitation, seem to be related to total mean mortality (1/e0). These results suggest that the influence of geographic and climatic factors on the senescence pattern deserves further investigation. However, can the potential senescence that is observed and measured in the laboratory be expressed in natural habitats where extrinsic mortality occurs? In the field, triatomines could die before intrinsic mortality and decreasing fecundity occurs, due to extrinsic factors such as predation, parasitism, extreme climatic conditions, etc. Recent studies in vertebrates (Adams, 1985; Gaillard et al., 1993; Reznick et al., 2002; Rebke et al., 2010) show that senescence is observed under natural conditions, but that kind of information is lacking for triatomines and needs to be investigated. Williams (1957) claimed that greater rates of extrinsic mortality (age- and condition-independent) favored more rapid senescence, but Abrams (1991) showed that the effects of the "extrinsic" mortality affect differentially the rate of senescence as a function of the degree of densitydependence. Abrams (1991) also showed that mortality patterns, contrary to Williams' (1957) predictions, are possible when density-dependence is present, and acts primarily on the survival or fertility of later ages, or when most of the variation in mortality rates is due

There are few laboratory evaluations of the density-dependent processes in triatomines. Rodríguez & Rabinovich (1980) showed that in *R. prolixus* density had a significant effect on the development rate of second, third, and fourth instars, but not on the survivorship of either the first or fifth instars, or even of the adults, nor on the instantaneous population parameters, or the age-specific parameters. Influence of density-dependence on the

How organisms distribute their "investment" in reproduction with respect to age is a major question in the senescence theory. It will be optimal for an organism to "invest" more in its reproduction when it becomes old-aged (Williams, 1966). An extension of William's (1966) theory is that old individuals may increase their reproductive effort during their last reproductive occasion(s) because it is their last chance to reproduce. This extension was

that suggests that mortality rates may be governed by common rules.

(Williams & Day, 2003).

to variation in non-extrinsic mortality.

senescence pattern in triatomines still needs more research.

called the "hypothesis of terminal investment" (Ricklef, 2000; Coulson & Fairweather, 2001; Ricklefs, 2008). Thus senescent individuals may invest more in reproduction than the nonsenescent individuals. Our observations in the triatomines do not support these predictions. Indeed most of the reproductive effort (about 50 to 90%) occurs in the first and intermediate part of the adult life and before the high mortality that takes place at older ages. Furthermore, the duration of the post-reproductive period decreases with reproductive effort suggesting a trade-off between reproduction allocation and future survival. Recently William's (1966) prediction and the concept of terminal investment have been criticized because of scarce empirical support and for resorting to two hypotheses based on quite unrealistic assumptions: (i) organisms must have a fixed longevity, which implies that individual trajectories cannot influence longevity, and (ii) in order to increase their reproductive effort at the end of their life during their last reproductive occasion(s), individuals must be able to anticipate their future, i.e., they have to use cues indicating when their death may occur (Gadgil & Bossert, 1970).

Environmental heterogeneity, and particularly high environmental uncertainty (stochastic effects), affect circumstances that are very important in insects, such as encounter rates associated to suitable oviposition sites, food and refuge availability, physiological state (e.g., reserves for producing oocytes, egg maturation rate, and somatic maintenance costs), and expected reproductive success that can lead to different patterns of behavior and rates of mortality and reproduction (Partridge & Mangel, 1999). Environmental stochasticity and/or density-dependence processes can select bet-hedging dormancy including a development delay in insects (e.g., Menu et al., 2000; Gourbière & Menu, 2009; Rajon et al., 2009). Such a risk spreading strategy has been postulated to exist in triatomines (Menu et al., 2010) and we recommend that this approach be incorporated in future studies for understanding senescence in insects.

Mortality rates of triatomine vectors have epidemiological importance through the demography of its populations (Chaves et al., 2004a). Particularly, it has been shown in these insects that important changes in life-history traits take place when reared under laboratory conditions; for example, in *T. infestans*, *T. pseudomaculata*, *T. brasiliensis*, and *P. megistus* after only four years in the laboratory, the reduction in the number of eggs within the first month of oviposition and the average female life span was in average 37.5 and 45.9%, respectively (Perlowagora-Szumlewicz, 1976). Similar changes, including a reduction in life history traits and population growth parameters, during four successive and separate generations of *T. infestans* of Argentina (reared in the laboratory from a first generation from a sylvatic individual) have also been found (G. Martí, personal communication).

The adaptation to a domestic environment causes similar effects on triatomines as the adaptation to laboratory conditions (Forattini, 1980): indoor conditions of rural houses provide better chances of feeding, and are characterized by smaller predation risks, and smaller fluctuations of temperature and relative humidity that exclude potentially deleterious extreme values. In our study we did not analyze possible adaptations to the laboratory because the precise number of laboratory generations was known for only a few cohorts; however, it is recommended that, when estimating trade-offs, and in particular the senescence pattern, the influence of such changes should be taken into account.

The Quest for Immortality in Triatomines:

Vol.40, pp. 793–800

*Biology,* Vol.7, pp. 440-442

*Biology*, Vol.32, pp. 146-152

*Science,* Vol.258, pp. 461–463

*Entomology,* Vol.22, pp. 43-48

*Nat.,* Vol.104, No.935, pp. 1-24.

Brazil. *Revista de Saúde Pública,* Vol.14, pp. 265-299.

Vol.100, pp. 465–470

Vol.67, pp. 176-183

disease. *Acta Tropica,* Vol.92, pp. 119–125

*Automatic Control,* Vol.19, pp. 716–723

*Negl Trop Dis,* Vol.5, No.5, pp. e1045

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 247

Akaike, H. (1974). A new look at statistical model identification. *IEEE Transactions on* 

Barbu, C.; Dumonteil, E. & Gourbière, S. (2011). Evaluation of Spatially Targeted Strategies

Carey, J. R. (2001). Insect Biodemography. *Annual Review of Entomology,* Vol.46, pp. 79-110 Carey, J. R.; Liedo, P.; Müller, H.-G.; Wang, J.-L. & Vaupel, J. W. (1998). Dual Modes of Aging in Mediterranean Fruit Fly Females. *Science, New Series,* Vol.281, No.5379, pp. 996-998 Carey, J. R.; Liedo, P.; Muller, H-G.; Wang, J-L.; Senturke, D. & Harshman, L. (2005).

Carey, J. R.; Liedo, P.; Orozco, D. & Vaupel, J. W. (1992). Slowing of mortality rates at older

Charlesworth, B. & Partridge, L. (1997). Ageing: Levelling of the Grim Reaper. *Current* 

Chaves, L. F.; Hernández, M. J.; Revilla, T. A.; Rodríguez, D. J. & Rabinovich, J. E. (2004a).

Chaves, L. F.; Zamora, E. & Aldana, E. (2004b). Mortality profile of female *Rhodnius robustus* (Heteroptera: Reduviidae). *Revista de Saúde Pública,* Vol.38, pp. 466–468 Cheverud, J. M.; Dow, M. M. & Leutenegger, W. (1985). The Quantitative Assessment of

Christopoulos, A. & Lew, M. J. (2000). Beyond Eyeballing: Fitting Models to Experimental Data. Critical Reviews in *Biochemistry and Molecular Biology,* Vol.35, No.5, pp. 359–391 Coulson, J. C. & Fairweather, J. A. (2001). Reduced reproductive performance prior to death

Curtsinger, J. W.; Fukui, H. H.; Townsend, D. R. & Vaupel, J. W. (1992). Demography of

Dingle, H. (1966). The effect of population density on mortality & sex ratio in the milkweed

Dumonteil, E.; Gourbière, S.; Barrera-Perez, M.; Rodriguez-Félix, E.; Ruiz-Piña, H.; Banos-

Feliciangeli, M. D. & Rabinovich, J. E. (1985). Vital Statistics of Triatominae (Hemiptera:

Forattini, O. P. (1980). Biogeography, origin, and distribution of triatominae domiciliarity in

Fukui, H. H.; Xiu, L. & Curtsinger, J. W. (1993). Slowing of age-specific mortality rates in *Drosophila melanogaster*. *Experimental Gerontology,* Vol.28, pp. 585–599 Gadgil, M. & Bossert, W. H. (1970). Life Historical Consequences of Natural Selection. *Am.* 

Weight Among Primates. *Evolution,* Vol.39, No.6, pp. 1335-1351

ages in large medfly cohorts. *Science,* Vol. 258, pp. 457–461

to Control Non-Domiciliated *Triatoma dimidiata* Vector of Chagas Disease. *PLoS* 

Biodemography of a long-lived tephritid: Reproduction & longevity in a large cohort of female Mexican fruit flies, *Anastrepha ludens*. *Experimental Gerontology,*

Mortality profiles of *Rhodnius prolixus* (Heteroptera: Reduviidae), vector of Chagas

Phylogenetic Constraints in Comparative Analyses: Sexual Dimorphism in Body

in the Black-legged Kittiwake: senescence or terminal illness? *Journal of Avian* 

genotypes: failure of the limited life-span paradigm in *Drosophila melanogaster*.

bug, *Oncopeltus*, & the cotton stainer, *Dysdercus* (Heteroptera). *American Naturalist*,

Lopez, O.; Ramirez-Siera, M. J.; Menu, F. & Rabinovich, J. E. (2002). Geographic distribution of *Triatoma dimidiata* and transmission dynamics of *Trypanosoma cruzi* in the Yucatan peninsula Mexico. *American Journal of Tropical Medicine and Hygiene,*

Reduviidae) under Laboratory Conditions. II. *Triatoma maculata*. *Journal of Medical* 

Furthermore, as the data for our comparative analyses did not indicate the mean body length of the individuals used in the experiments we used the estimates provided by Galíndez Girón et al. (1998) based on the types and paratypes of triatomine measurements in museum collections. As the individual's body size distribution can vary with respect to each population, it is recommended that future cohort studies in the laboratory measure the body length of the individuals used. In consequence, we must take with some reservation the lack of a statistically significant correlation between life history traits and total length as obtained in this study and based on 27 triatomine species.

## **5. Conclusions**

Most studies in triatomines have investigated physiology, population genetics, phylogeny (see Gourbière et al., 2011 for a review) and ecology of a given species (e.g., Dumonteil et al., 2002; Gourbière et al., 2008; Barbu et al., 2011) but very few studies have investigated the evolution of life history traits in the light of evolutionary ecology concepts (Menu et al., 2010). Our study is the first comparative analysis to the senescence pattern and its relationship with life history traits in triatomines.

Our results indicate that triatomines show both an actuarial and a reproductive senescence, with a high diversity of mortality patterns, even within a given genus. We believe that the relationship between life history traits, and particularly the trade-off between reproductive effort and future survival, is central to understand this diversity in mortality patterns. In order to identify ultimate and proximate factors underlying this diversity, we need longitudinal studies conducted in the field in order to estimate if the potential senescence observed in the laboratory can be expressed under natural (sylvatic and domestic) conditions. The analysis of senescence in relation to other life history traits in triatomines has not only academic value but also impinges in the areas of vector population management and epidemiology.

## **6. Acknowledgments**

We are extremely grateful to the Vectors Reference Center of the National Ministry of Health, Córdoba, Argentina and to Dr. Elena Visciarelli for allowing the use of original data from 21 species of triatomines and from *T. patagonica*, respectively. We thank to Mr. Waldo Hasperué for his collaboration in the data processing with the computer program TriTV. This work has been supported by the French National Research Agency (grant reference «ANR-08-MIE-007»), the CNRS and the *Agencia Nacional de Promoción Científica y Tecnológica* of Argentina (grant PICT2008-0035).

## **7. References**


Furthermore, as the data for our comparative analyses did not indicate the mean body length of the individuals used in the experiments we used the estimates provided by Galíndez Girón et al. (1998) based on the types and paratypes of triatomine measurements in museum collections. As the individual's body size distribution can vary with respect to each population, it is recommended that future cohort studies in the laboratory measure the body length of the individuals used. In consequence, we must take with some reservation the lack of a statistically significant correlation between life history traits and total length as

Most studies in triatomines have investigated physiology, population genetics, phylogeny (see Gourbière et al., 2011 for a review) and ecology of a given species (e.g., Dumonteil et al., 2002; Gourbière et al., 2008; Barbu et al., 2011) but very few studies have investigated the evolution of life history traits in the light of evolutionary ecology concepts (Menu et al., 2010). Our study is the first comparative analysis to the senescence pattern and its

Our results indicate that triatomines show both an actuarial and a reproductive senescence, with a high diversity of mortality patterns, even within a given genus. We believe that the relationship between life history traits, and particularly the trade-off between reproductive effort and future survival, is central to understand this diversity in mortality patterns. In order to identify ultimate and proximate factors underlying this diversity, we need longitudinal studies conducted in the field in order to estimate if the potential senescence observed in the laboratory can be expressed under natural (sylvatic and domestic) conditions. The analysis of senescence in relation to other life history traits in triatomines has not only academic value but also impinges in the areas of vector population

We are extremely grateful to the Vectors Reference Center of the National Ministry of Health, Córdoba, Argentina and to Dr. Elena Visciarelli for allowing the use of original data from 21 species of triatomines and from *T. patagonica*, respectively. We thank to Mr. Waldo Hasperué for his collaboration in the data processing with the computer program TriTV. This work has been supported by the French National Research Agency (grant reference «ANR-08-MIE-007»), the CNRS and the *Agencia Nacional de Promoción Científica y Tecnológica*

Abrams P. A. & Ludwig, D. (1995). Optimality theory, Gompertz' law, & the disposable

Abrams, P. A. (1991). Life History and the Relationship between Food Availability and

Adams, C. E. (1985). Reproductive senescence, In: *Reproduction in Mammals,* C. R. Austin &

soma theory of senescence. *Evolution,* Vol.49, pp. 1055-1066

Foraging Effort. *Ecology,* Vol.72, No.4, pp. 1242-1252

R. V. Short (Eds.), 210-233. Cambridge, Univ. Press

obtained in this study and based on 27 triatomine species.

relationship with life history traits in triatomines.

management and epidemiology.

of Argentina (grant PICT2008-0035).

**6. Acknowledgments** 

**7. References** 

**5. Conclusions** 


The Quest for Immortality in Triatomines:

ISBN 978-0-387-32914-7

March 18-21, 1975

No.4, pp. 351-370

<httpwww.R-project.org>

Vol.135, No.3, pp. 252-262

*Reviews,* Vol.5, pp. 14-32

Vol.107, pp. 7841-7846

Vol.31, pp. 103-111

*Nat.,* Vol.152, No.1, pp. 24-44

*Ecology,* Vol.22, pp. 379-392

*Entomology,* Vol.48, No.4, pp. 775-787

A Meta-Analysis of the Senescence Process in Hemimetabolous Hematophagous Insects 249

Mueller, L. D.; Drapeau, M. D.; Adams, C. S.; Hammerle, C. W.; Doyal, K. M.; Jazayeri, A. J.;

Nusbaum, T. J.; Mueller, L. D. & Rose, M. R. (1996). Evolutionary Patterns Among Measures

Paradis, E. (2006). *Analysis of Phylogenetics and Evolution with R*. Springer-Verlag, Heidelberg.

Partridge, L. & Mangel, M. (1999). Messages from Mortality: the Evolution of Death Rates in

Perlowagora-Szumlewicz, A. (1976). Laboratory Colonies of Triatominae, Biology, and

Pletcher, S. D. & Curtsinger, J. W. (1998). Mortality Plateaus and the Evolution of Senescence: Why are Old-Age Mortality Rates so Low? *Evolution,* Vol.52, No.2, pp. 454-464 Promislow, D. E. L. (1991). Senescence in Natural Populations of Mammals: A Comparative

R Development Core Team (2007) *R: A Language and Environment for Statistical Computing*. R

Rabinovich J. E. & Nieves, E. L. (2011). Vital Statistics of Triatominae (Hemiptera:

Rabinovich, J. E. (1972). Vital statistics of Triatominae (Hemiptera: Reduviidae) under

Rabinovich, J. E.; Nieves, E. L. & Chaves, L. F. (2010). Age-specific mortality analysis of the

Rajon, E.; Venner, S. & Menu, F. (2009). Spatially heterogeneous stochasticity and the adaptive diversification of dormancy. *Journal of Evolutionary Biology,* Vol.22, pp. 2094-2103 Rauser, C. L.; Mueller, L. D. & Rose, M. R. (2006). The Evolution of Late Life. *Ageing Research* 

Rebke, M.; Coulson, T.; Becker, P. H. & Vaupel, J. W. (2010). Reproductive improvement and

Reznick, D.; Bryant, M. J. & Bashey, F. (2002). r- and K- selection revisited: the role of population regulation in life history evolution. *Ecology,* Vol.83, No.6, pp. 1509-1520 Ricklefs, R. E. (1998). Evolutionary Theories of Aging: Confirmation of a Fundamental

Ricklefs, R. E. (2000). Intrinsic aging-related mortality in birds. *Journal of Avian Biology,*

Ricklefs, R. E. (2008) The evolution of senescence from a comparative perspective. *Functional* 

of Aging. *Experimental Gerontology,* Vol.31, No.4, pp. 507-516

Study. *Evolution,* Vol.45, No.8, pp. 1869-1887

the Old . *Trends in Ecology & Evolution,* Vol.14, No.11, pp. 438-442

Ly, T.; Beguwala, S. A.; Mamidi, A. R. & Rose, M. R. (2003). Statistical Tests of Demographic Heterogeneity Theories. *Experimental Gerontology* Vol.38, pp. 373-386 Mueller, L. D.; Rauser, C. L. & Rose, M. R. (2005). Population Dynamics, Life History, &

Demography: Lessons From *Drosophila*. *Advances in Ecological Research,* Vol.37, pp. 77-99

Population Dynamics. *Proceedings of an International Symposium, New Approaches in American Trypanosomiasis Research,* pp. 63-82. Belo Horizonte, Minas Gerais, Brazil,

Foundation for Statistical Computing, Vienna, Austria. Available from

Reduviidae) Under Laboratory Conditions III. *Rhodnius neglectus*. *Journal of Medical* 

laboratory conditions. I. *Triatoma infestans* Klug. *Journal of Medical Entomology,* Vol.9,

dry forest kissing bug, *Rhodnius neglectus*. *Entomologia Experimentalis et Applicata,*

senescence in a longlived bird. *Proceedings of the National Academy of Sciences,*

Prediction, with Implications for the Genetic Basis and Evolution of Life Span. *Am.* 


Gaillard, J.–M. ; Viallefont, A. ; Loison, A. & Festa–Bianchet, M. (2004). Assessing Senescence

Galíndez Girón, I.; Carcavallo, R. U.; Jurberg, J.; Galvao, C.; Lent, H.; Barata, J. M. S.; Pinto

Gavrilov, L. A & Gavrilova, N. S. (2001) The reliability theory of aging and longevity. *Journal* 

Ghalambor, C. K.; Reznick, D. N. & Walker, J. A. (2004). Constraints on Adaptive Evolution: the

Gourbière, S. ; Dumonteil, E. ; Rabinovich, J. E. ; Minkoue, R. & Menu, F. (2008).

Gourbière, S.; Dorn, P.; Tripet, F. & Dumonteil, E. (2011). Genetics and evolution of

Graca-Souza, A. V.; Maya-Monteiro, C.; Paiva-Silva, G. O.; Braz, G. R. C.; Paes, M. C.;

Jones, O. R.; Gaillard, J.-M.; Tuljapurkar, S.; Alho, J. S.; Armitage, K. B.; Becker, P. H.; Bize,

Kirkwood, T. B. L. & Holliday, R. (1979). The Evolution of Ageing & Longevity, *Proceedings of the Royal Society of London, Series B, Biological Sciences* Vol.205, No.1161, pp. 531-546 Kirkwood, T. B. L. & Rose, M. R. (1991). Evolution of senescence: late survival sacrificed for reproduction. *Transactions of the Royal Society, London B,* Vol.332, pp. 15–24 Kirkwood, T. B. L. (2002). Evolution of Ageing. *Mechanisms of Ageing & Development* Vol.123,

Kirkwood, T. B. L. (2005). Understanding the odd science of aging. *Cell* Vol.120, pp. 437-447 McCoy, M. W. & Gillooly, J. F. (2008). Predicting natural mortality rates of plants and

Menu F.; Roebuck, J. P. & Viala, M. (2000). Bet-hedging diapause strategies in stochastic

Menu, F.; Ginoux, M.; Rajon, E.; Lazzari, C. R. & Rabinovich, J. E. (2010). Adaptive

Mueller, L. D. & Rose, M. R. (1996). Evolutionary theory predicts late-life mortality plateaus. *Proceedings of the National Academy of Sciences* Vol.93, pp. 15249-15253

Developmental Delay in Chagas Disease Vectors: An Evolutionary Ecology

*Journal of Tropical Medicine and Hygiene,* Vol78, pp. 133-139

continuum. *Ecology Letters,* Vol.11, pp. 664-673

animals. *Ecology Letters,* Vol.11, pp. 710-716

environment. *The American Naturalist*, Vol.155, pp. 724-734

Approach. *PLoS Neglected Tropical Diseases,* Vol.4, No.5, pp. 1-10

triatomines: from phylogeny to vector control. *Heredity,* pp. 1–13

Kirkwood, T. B. L. & Austad, S. N. (2000) Why do we age? *Nature* Vol.408, pp. 233-238

the Trinidadian Guppy (*Poecilia reticulata*). *Am. Nat.,* Vol.164, No.1, pp. 38-50 Gourbière, S. & Menu, F. (2009). Adaptive dynamics of dormancy duration variability:

Jurberg & H. Lent (Comps.), 53-73, FioCruz, Rio de Janeiro, Brazil

*of Theoretical Biology,* Vol.213, No.4, pp. 527–545

Vol.27, No.1, pp. 47–58

Vol.63, No.7, pp. 1879-1892

Vol.36, pp. 322-335

pp. 737-745.

Patterns in Populations of Large Mammals. *Animal Biodiversity & Conservation,*

Serra, O. & Valderrama, A. (1998). Morfology and external anatomy. A: General, In: *Atlas of Chagas' Disease Vectors in the Americas,* R. U. Carcavallo, I. Galíndez Girón, J.

Functional Trade-Off between Reproduction and Fast-Start Swimming Performance in

evolutionary trade-off and priority effect lead to suboptimal adaptation. *Evolution,*

Demographic and Dispersal Constraints for Domestic Infestation by Non-Domicilated Chagas Disease Vectors in the Yucatan Peninsula Mexico. *American* 

Sorgine, M. H. F.; Oliveira, M. F. & Oliveira, P. L. (2006). Adaptations against heme toxicity in blood-feeding arthropods. *Insect Biochemistry and Molecular Biology,*

P.; Brommer, J.; Charmantier, A.; Charpentier, M.; Clutton-Brock, T.; Dobson, F. S.; Festa- Bianchet, M.; Gustafsson, L.; Jensen, H.; Jones, C. G. & Lillandt, B.-G. (2008). Senescence rates are determined by ranking on the fast-slow life-history


**12** 

*Georgia* 

**Programming and** 

*1Georgian Systemic Research Center, Tbilisi,* 

*4I. Javakhishvili Tbilisi State University, Tbilisi,* 

*3Life Science Research Center, Tbilisi,* 

**Implementation of Age-Related Changes** 

*2Grigol Robakidze University's Oncology Department, Tbilisi,* 

Jaba Tkemaladze1, Alexander Tavartkiladze2 and Konstantin Chichinadze3,4

From the songs of my childhood I can remember one such song: " The cranes are flying far and they will never come back, the rumour is spread ... . " The author of the song text assumes that the cranes can not come back , but he/she can't bear the thought of losing lovely birds and adds: " it's a rumour !" However ,I am saying once more he lets the thought that maybe the cranes won't be back any more .... I loved this song and while listening sorrow used to enter my soul quietly. Although I knew that birds return from warm countries ... And still there was something ominous in those " never come back." Then , after so long in Dinara Kasradze's monograph ("Quantum satis ", 2005) I read the following: "Each species has it's own quantity of wing waving , after which the bird dies ." ..yhat's where that subconscious sorrow came from ... mine or of that song text author, as we mentioned above... Yes, it's so, the organism tissues , cells "get worn" while acting and get restored too; The only bad thing is that with age , damage of cells exceeds their reparation i. e. the mentioned fact becomes expressed with aging! Every creature turned to have its own strictly defined potential, after which the life expires. Then where is immortality? Only in fairy tales ? "The truth used to be written in fairy tails , the truth , written in a creative and "fairy" way " ,-"In fairy tales , many secrets of nature are explained in "fairy" language ,many things that are unsolved and not clear at all! "... Then where's immortality? The water or

With the age, damage of molecules exceeds its reparation i. e. the mentioned fact gets more expressed with aging. The question is : What are molecular reasons for wearing cells out. Hayflick's experiments are important in this way: in 1961 Leonard Hayflick demonstrated ( at Wister Institute , Philadelphia ) that the cells of normal human fetus managed to divide 40-60 times. The scientist in vitro observed human, normal, diploid fibroblast cultures and he saw that these cells have absolutely defined life expectancy - they stop multiplication and get older after 50 times division (doubling). On the contrary, fibroblasts of the patients , ill

spring of immortality ? Maybe it is nearby , here next to us...

**1. Introduction** 

**2. Hayflick's limit** 


## **Programming and Implementation of Age-Related Changes**

Jaba Tkemaladze1, Alexander Tavartkiladze2 and Konstantin Chichinadze3,4

*1Georgian Systemic Research Center, Tbilisi,* 

*2Grigol Robakidze University's Oncology Department, Tbilisi,* 

*Georgia* 

## **1. Introduction**

250 Senescence

Rodríguez, D. & Rabinovich, J. E. (1980). The Effect of Density on some Population

Souza, A. V. G.; Petretski, J. H.; Demasi, M.; Bechara, E. J. H.; Oliveira, P. L. (1997). Urate

StatSoft (2009) STATISTICA (Data Analysis Software System), version 9.0. Statsoft, Tulsa,

Styer, L. M.; Carey, J. R.; Wang, J.-L. & Scott, T. W. (2007). Mosquitoes do senesce: departure

Sulbaran, J. E. & Chaves, L. F. (2006). Spatial complexity and the fitness of the kissing bug, *Rhodnius prolixus*. *Journal of Applied Entomology,* Vol.130, No.1, pp. 51-55 Tatar M.; Carey, J. R. & Vaupel, J. W. (1993). Long-term cost of reproduction with & without

Vaupel, J. W. Manton, K. G. & Stallard, E. (1979). The impact of heterogeneity in individual frailty on the dynamics of mortality. *Demography* Vol.16, No.3, pp. 439-454. WHO (World Health Organization). 2007. Reporte sobre la enfermedad de Chagas. Grupo de trabajo científico 17–20 de abril de 2005, Buenos Aires, Argentina.. Williams, G. C. (1957). Pleiotropy, Natural Selection, & the Evolution of Senescence.

Williams, G. C. (1966). Natural Selection, the Costs of Reproduction, and a Refinement of

Williams, P. D. & Day, T. (2003). Antagonistic Pleiotropy, Mortality Source Interactions, and the Evolutionary Theory of Senescence. *Evolution*, Vol. 57, No. 7 pp. 1478-1488 Zurbenko, I.; Porter, P. S.; Rao, S. T.; Ku, J. Y.; Gui, R.; Eskridge, R. E. (1996). Detecting

Discontinuities in Time Series of Upper-air Data: Development and Demonstration of an Adaptive Filter Technique. *Journal of Climate*, Vol.9, No.12, pp. 3548-3560

Lack's Principle. *Am. Nat.,* Vol.100, No.916, pp. 687-690

Conditions. *Journal of Medical Entomology,* Vol.17, No.2, pp. 165-171 Roff, D. A. (2002). *Life History Evolution. Sinauer Associates*, Inc. Sunderland, Massachusetts, USA. Silva de Paula, A.; Diotaiuti, L. & Schofield, C. J. (2005). Testing the sister-group relationship

*Molecular Phylogenetics and Evolution,* Vol.35, pp. 712-718

*Biol. Med.,* Vol.22, pp. 209–214

*Hygiene,* Vol.76, pp. 111–117

mortality. *Evolution,* Vol.47, pp. 1302-1312

*Evolution,* Vol.11, No.4, pp. 398-411

OK, USA.

Parameters of *Rhodnius prolixus* (Hemiptera: Reduviidae) under Laboratory

of the Rhodniini and Triatomini (Insecta: Hemiptera: Reduviidae: Triatominae).

protects a blood-sucking insect against hemin induced oxidative stress. *Free Rad.* 

from the paradigm of constant mortality. *American Journal of Tropical Medicine &* 

accelerated senescence in *Callosobruchus maculatus*: analysis of age-specific

From the songs of my childhood I can remember one such song: " The cranes are flying far and they will never come back, the rumour is spread ... . " The author of the song text assumes that the cranes can not come back , but he/she can't bear the thought of losing lovely birds and adds: " it's a rumour !" However ,I am saying once more he lets the thought that maybe the cranes won't be back any more .... I loved this song and while listening sorrow used to enter my soul quietly. Although I knew that birds return from warm countries ... And still there was something ominous in those " never come back." Then , after so long in Dinara Kasradze's monograph ("Quantum satis ", 2005) I read the following: "Each species has it's own quantity of wing waving , after which the bird dies ." ..yhat's where that subconscious sorrow came from ... mine or of that song text author, as we mentioned above... Yes, it's so, the organism tissues , cells "get worn" while acting and get restored too; The only bad thing is that with age , damage of cells exceeds their reparation i. e. the mentioned fact becomes expressed with aging! Every creature turned to have its own strictly defined potential, after which the life expires. Then where is immortality? Only in fairy tales ? "The truth used to be written in fairy tails , the truth , written in a creative and "fairy" way " ,-"In fairy tales , many secrets of nature are explained in "fairy" language ,many things that are unsolved and not clear at all! "... Then where's immortality? The water or spring of immortality ? Maybe it is nearby , here next to us...

## **2. Hayflick's limit**

With the age, damage of molecules exceeds its reparation i. e. the mentioned fact gets more expressed with aging. The question is : What are molecular reasons for wearing cells out. Hayflick's experiments are important in this way: in 1961 Leonard Hayflick demonstrated ( at Wister Institute , Philadelphia ) that the cells of normal human fetus managed to divide 40-60 times. The scientist in vitro observed human, normal, diploid fibroblast cultures and he saw that these cells have absolutely defined life expectancy - they stop multiplication and get older after 50 times division (doubling). On the contrary, fibroblasts of the patients , ill

*<sup>3</sup>Life Science Research Center, Tbilisi,* 

*<sup>4</sup>I. Javakhishvili Tbilisi State University, Tbilisi,* 

Programming and Implementation of Age-Related Changes 253

consideration , telomeres must shorten very slowly-- by 3-6 nucleotides after one cell cycle; According to the Hayflick's limit, after certain number of divisions telomeres shorten by 150-

Nowadays, epigenetic theory of aging is delivered by B.A. Galitski (2009),according to which telomere erosion is explained first of all by activation of cell recombinazas, which are activated in respond to the DNA lesion, which is mainly caused by age depression of genome mobile elements. When after certain number of divisions telomeres absolutely disappear, the cell remains at certain stage of the cell cycle or it turns apoptosis mechanism on. It is interesting that A.M. Olivnikov ( who was the first to suppose existence of telomeres (1973,1996) ) said: "Telomeres action is proved. However, it shouldn't have had direct link

In spite of theories or certain plurality of opinions, it is clear today that cell aging is caused by a number of factors. Not only endogenous molecular way of cell aging is important, but also harmful exogenous affects during the whole life, which is followed by, so called, cell "wearing". In the course of time lethal changes are gathered in the cell lesions, which lead

Certainly human cells division is not infinite, except embryonic, sex and cancer cells. The cells which have short telomeres, are often "not valid" as their chromosomes are no longer stable. The chromosomes become less protected against various damaging factors, as in

During the experiment researchers (group- "Geron Corporation") could change the route of the aging process: They installed genes in the DNA. The genes were responsible for relomeraza synthesis. In these cells Hayflick's limit was doubled (100 divisions ) and the life expectancy accordingly increased . According to these scientists' opinion, it is possible to change ordinary human cells that they will be able to divide infinitely. In January 1998 all the media sources informed the world that American scientists were able to force human normal cells to overcome Hayflick's limit: Instead of aging, the cells continued to multiply; and what is remarkable, they didn't transform into cancer cells. Brilliant! Bravo! If it was managed to overcome Hayflick's limit and increase life expectancy so, that cancer transformation didn't take place! And still we must point out that much care and attention is

Telomeraza was discovered in 1984 by Carol W. Greider. For opening of the protecting mechanisms of chromosomes ( terminal replication by the help of telomerazas) Elizabeth Blackburn , Carol Grider and Jack Szostak received Nobel Prizes. Human telomeraza's structure was explained by Scott Cohen and his group (Australia, Children's medicine Research Institute, 2007). Telomeraza consists of telomeraza reverse transcriptaza (TERT.), telomeraza RNM (hTR or TERC) and diskerin (two molecules of each substance). TERT, as we pointed out, is the ferment reverse-transcriptaza, which creates one-string DNA on the basis of template one-string RNA (i.e. telomeraza represents a reverse - transcriptaza and a special molecule of RNA is connected with it , which is used as a matrix for the reverse-transcriptaza during lengthening of the telomeres.) Two subunits of the ferment are encoded by two different genes. TERT has a glove shape, which allows it to fix at the chromosome and add to it one-string parts. It is considered that telomeraza is the key to the cell immortality. Thanks to this ferment, cells multiply fast and don't get older.

the cells to death, as ability to respond to lesions is gradually reduced.

norm , their defending telomeres i.e. their endings can't protect them.

300 nucleotides.

with aging".

necessary in handling this process!

with Progeria or Verner syndrome (early aging) , used to double 10-12 times - instead of 40- 60 . Adopted term , so acceptable nowadays, Hayflick's limit comes from the above . This limit i.e. border was fixed in the culture as for all differential (i.e. mature) cells, also for other multi cell organisms. Maximum number of divisions in one organism is different according to the type of the cell and difference is much more expressed in different organisms. For most of the cells Hayflick's Limit is 52 divisions. The history of Hayflick's experiments is also interesting. He worked together with Paul Moorhead . In one of the tests they mixed equal quantities of normal males' fibroblasts ( that had been divided 40 times ) and normal females' fibroblasts (that had been divided only 10 times ) i.e. men's older cells were mixed with women's younger cells and they received mixed culture."Not mixed" culture (males') was used for control. When division in the controlling culture of the males cells stopped , in that very moment mixed culture was studied and it turned out that only women's cells were left there. This meant: The older cells "remembered" that they were old, even when they were surrounded by young cells.

Hayflick differed the following phases of cell division in the culture: At the beginning of the experiment, he called the "firstly" culture "phase one"; then the period when cells were multiplying was the "phase two". Division (doubling) period after numerous months was called "the third phase phenomenon" - when cell growth was reduced and stopped. The reason for replication aging is uncertain. It is supposed that genes of aging are activated. (They are located in the first and fourth chromosomes), the growth regulating genes are changed or lost (which at last causes growth inhibition in aging cells too. The genes, speeding (accelerating) aging process are revealed. For example, reducing signal transmission by means of Factor 1 , similar to insulin, causes Drosophila, also Nematode c. Elegans and increasing of life expectancy in mice. The reason for such difference in replication intensity is unknown. It can be caused by activating specific genes at an old age. For example, during reduction of replication Kinaza inhibitor genes (p21) are revealed. Replication aging is also induced by increased expression of p 161NK4a - the cell cycle inhibitor and DNA lesion.

#### **3. The telomeres**

The essence of genes' chromosome telomere shortening is interesting. The limited replication ability of the cell can be explained by the following: During each division, the chromosomal endings go through the unfinished replication (telomere shortening). After each division telomeres shorten, that ultimately causes stopping the cell division. Telomeres are important in stabilization of terminal parts of the chromosomes and also their fixing at the nucleus matrix. Telomeres gradually get shorter in later passages of the culture and also in older people's cell cultures. Telomeres are the longest in spermatozoids, they are longer in fetus than in an adult. As it seems, DNA loss in the terminal parts of chromosomes and telomere shortening causes deletion of important genes.

De novo synthesis of telomeres is regulated by means of enzym telomeraza. Correlation between the telomere length and telomeraza consistence is found. Hayflick's limit depends on telomere size reduction. Telomeres are DNA short repeated successions (TTAGGG) which are located at the end of chromosomes. If the cell doesn't have active telomeraza (as, for example, most of somatic cells), after each division the telomere size is reduced as DNA polymeraza can't replicate the ends of DNA molecule. In spite of this, taking this event into

with Progeria or Verner syndrome (early aging) , used to double 10-12 times - instead of 40- 60 . Adopted term , so acceptable nowadays, Hayflick's limit comes from the above . This limit i.e. border was fixed in the culture as for all differential (i.e. mature) cells, also for other multi cell organisms. Maximum number of divisions in one organism is different according to the type of the cell and difference is much more expressed in different organisms. For most of the cells Hayflick's Limit is 52 divisions. The history of Hayflick's experiments is also interesting. He worked together with Paul Moorhead . In one of the tests they mixed equal quantities of normal males' fibroblasts ( that had been divided 40 times ) and normal females' fibroblasts (that had been divided only 10 times ) i.e. men's older cells were mixed with women's younger cells and they received mixed culture."Not mixed" culture (males') was used for control. When division in the controlling culture of the males cells stopped , in that very moment mixed culture was studied and it turned out that only women's cells were left there. This meant: The older cells "remembered" that they were old, even when they

Hayflick differed the following phases of cell division in the culture: At the beginning of the experiment, he called the "firstly" culture "phase one"; then the period when cells were multiplying was the "phase two". Division (doubling) period after numerous months was called "the third phase phenomenon" - when cell growth was reduced and stopped. The reason for replication aging is uncertain. It is supposed that genes of aging are activated. (They are located in the first and fourth chromosomes), the growth regulating genes are changed or lost (which at last causes growth inhibition in aging cells too. The genes, speeding (accelerating) aging process are revealed. For example, reducing signal transmission by means of Factor 1 , similar to insulin, causes Drosophila, also Nematode c. Elegans and increasing of life expectancy in mice. The reason for such difference in replication intensity is unknown. It can be caused by activating specific genes at an old age. For example, during reduction of replication Kinaza inhibitor genes (p21) are revealed. Replication aging is also induced by increased expression of p 161NK4a - the cell cycle

The essence of genes' chromosome telomere shortening is interesting. The limited replication ability of the cell can be explained by the following: During each division, the chromosomal endings go through the unfinished replication (telomere shortening). After each division telomeres shorten, that ultimately causes stopping the cell division. Telomeres are important in stabilization of terminal parts of the chromosomes and also their fixing at the nucleus matrix. Telomeres gradually get shorter in later passages of the culture and also in older people's cell cultures. Telomeres are the longest in spermatozoids, they are longer in fetus than in an adult. As it seems, DNA loss in the terminal parts of chromosomes and

De novo synthesis of telomeres is regulated by means of enzym telomeraza. Correlation between the telomere length and telomeraza consistence is found. Hayflick's limit depends on telomere size reduction. Telomeres are DNA short repeated successions (TTAGGG) which are located at the end of chromosomes. If the cell doesn't have active telomeraza (as, for example, most of somatic cells), after each division the telomere size is reduced as DNA polymeraza can't replicate the ends of DNA molecule. In spite of this, taking this event into

were surrounded by young cells.

inhibitor and DNA lesion.

telomere shortening causes deletion of important genes.

**3. The telomeres** 

consideration , telomeres must shorten very slowly-- by 3-6 nucleotides after one cell cycle; According to the Hayflick's limit, after certain number of divisions telomeres shorten by 150- 300 nucleotides.

Nowadays, epigenetic theory of aging is delivered by B.A. Galitski (2009),according to which telomere erosion is explained first of all by activation of cell recombinazas, which are activated in respond to the DNA lesion, which is mainly caused by age depression of genome mobile elements. When after certain number of divisions telomeres absolutely disappear, the cell remains at certain stage of the cell cycle or it turns apoptosis mechanism on. It is interesting that A.M. Olivnikov ( who was the first to suppose existence of telomeres (1973,1996) ) said: "Telomeres action is proved. However, it shouldn't have had direct link with aging".

In spite of theories or certain plurality of opinions, it is clear today that cell aging is caused by a number of factors. Not only endogenous molecular way of cell aging is important, but also harmful exogenous affects during the whole life, which is followed by, so called, cell "wearing". In the course of time lethal changes are gathered in the cell lesions, which lead the cells to death, as ability to respond to lesions is gradually reduced.

Certainly human cells division is not infinite, except embryonic, sex and cancer cells. The cells which have short telomeres, are often "not valid" as their chromosomes are no longer stable. The chromosomes become less protected against various damaging factors, as in norm , their defending telomeres i.e. their endings can't protect them.

During the experiment researchers (group- "Geron Corporation") could change the route of the aging process: They installed genes in the DNA. The genes were responsible for relomeraza synthesis. In these cells Hayflick's limit was doubled (100 divisions ) and the life expectancy accordingly increased . According to these scientists' opinion, it is possible to change ordinary human cells that they will be able to divide infinitely. In January 1998 all the media sources informed the world that American scientists were able to force human normal cells to overcome Hayflick's limit: Instead of aging, the cells continued to multiply; and what is remarkable, they didn't transform into cancer cells. Brilliant! Bravo! If it was managed to overcome Hayflick's limit and increase life expectancy so, that cancer transformation didn't take place! And still we must point out that much care and attention is necessary in handling this process!

Telomeraza was discovered in 1984 by Carol W. Greider. For opening of the protecting mechanisms of chromosomes ( terminal replication by the help of telomerazas) Elizabeth Blackburn , Carol Grider and Jack Szostak received Nobel Prizes. Human telomeraza's structure was explained by Scott Cohen and his group (Australia, Children's medicine Research Institute, 2007). Telomeraza consists of telomeraza reverse transcriptaza (TERT.), telomeraza RNM (hTR or TERC) and diskerin (two molecules of each substance). TERT, as we pointed out, is the ferment reverse-transcriptaza, which creates one-string DNA on the basis of template one-string RNA (i.e. telomeraza represents a reverse - transcriptaza and a special molecule of RNA is connected with it , which is used as a matrix for the reverse-transcriptaza during lengthening of the telomeres.) Two subunits of the ferment are encoded by two different genes. TERT has a glove shape, which allows it to fix at the chromosome and add to it one-string parts. It is considered that telomeraza is the key to the cell immortality. Thanks to this ferment, cells multiply fast and don't get older.

Programming and Implementation of Age-Related Changes 255

phosphataza formation. These changes provide "turning off " the death mechanism by means of damaging chromosome destruction or apoptosis process. With this endless

In cell cultures, this model of cancer clears up the telomeraza's role in cancer development.

In cancers without TERT activation in cells , they mainly used other mechanism of telomeres' protection , which is called ALT (alternative lengthening of telomeres). Details of

According to Elizabeth Blackburn's works (1985, 2001), telomeraza is also involved in regulation of 70 genes which participate (or probably participate ) in cancer origin and development. Furthermore, telomeraza activates glycolysis, which allows cancer cells to use sugar in order to maintain growth and division rate. This speed is equal to the same process

Fight with the cancer is difficult. If it was possible to receive such a medication, which would cause telomeraza's blockage in cancer cells, then telomere shortening would be

Working in this direction has started ( Koreans have just created the preparation "Telovak", which in the opinion of creators, activates immune system, that ultimately is directed to suppress telomeraza. After trying it on volunteers, ill with cancer "Telovak" prolonged patients' life by merely one year. It also gifted three months of life to the patients who were on the last stage of cancer. The question is : Maybe not only in cancer cells is telomeraza

Cancer cell is divided infinitely, telomeraza's gene is expressed , " inserted " in it i.e. malignant cell looks like a sex or embryonic cell (Probably, these are the only cells and its

Hereby , we take the liberty of a small comment: Yes ! - Today, immortal cells of cancer are widely discussed , which is said to be the fault of telomerazas . However, it shouldn't be absolutely right. Not all multiplied cancer cells are divided, i.e. among them there are some cells that divide ,so there is the limit too. As regards the culture, it is possible to initiate growth in all the ways . Besides , in vivo , cancer cells gradually cannot divide . Ultimately, they don't have "strength " of even it and die (These are famous facts , pathology -anatomists like to point it out. I remember the talk with Omar Khardzeishvili about this very question) i.e. part of cancer cells die by themselves in the lifetime of the ill organism. (It is an axiom today too); However, the person dies before all the cancer cells

In human fibroblasts, Kamozin can increase Hayflicks's Limit by means of reduction of telomeres shortening quality. However, the scientists say , that vertebrates' cells have certain potential of replication. More importantly, A.Melk and his group (2003) saw that in vivo

multiplication of the cell begins.

this mechanism are unknown.

rate in embryo.

**5. Senescence** 

die !

In 90 percent of cancers telomeraza's activation is observed.

renewed. Mutations would appear and cancer cells would die!

suppressed? Subsequently, will it ultimately make life shorter?

product restores normal length of the telomere).

aging is possible without telomere shortening.

Telomeraza is the most intensively revealed in the embryonic cells , then-in Germinational cells, very slightly-in adult organism labile cells (cells, that must divide frequently, for example, intestinal epithelial cells), but their detection is difficult in the most of the somatic cells. (More correctly, it doesn't happen at all).

Telomeraza is used in the cosmetic production (The product TA-65 was received from the plant Astragal, which activates telomeraza). Michael Fossel supposed in one of the interviews (2006, int. by D, J Brown), that treatment by telomeraza could be used not only for struggle against cancer , but also for fight against aging i.e. for increasing life expectancy.

When cells come closer to the Hayflick's limit, it is possible to stop aging if deactivation of those genes will happen , which are responsible for creation of the albumin (p53 and pR6) , suppressing cancer . Sooner or later such , changed cells get to the condition , which is called "crisis" i.e. when big part of the cells (in the culture) die (However, sometimes the cell doesn't stop multiplication during the crisis period). As a rule, telomeres are absolutely destroyed at the time and condition of the chromosomes worsens after each division.

## **4. Cancer**

The naked endings of the chromosomes mean splitting of the both DNA strings. Neutralization of such type of lesion happens by connecting split endings. Also endings of different chromosomes can make confluence as they are no longer protected by telomeres. This story as if temporarily relieves telomere absence. However, in the anaphase of the cell division, linked chromosomes come apart quite accidentally, which is followed by a lot of mutations and chromosomal anomaly development and with the process development the cell genome is more and more injured. Ultimately the moment comes when apoptosis is turned on (The gene substance is so much injured ) or the mutation is added to the injured genome which activates telomeraza.

After activation of telomeraza some types of cells obtain immortality. Their chromosomes don't become less stable for the number of divisions and the death process doesn't start. Many cancer cells are considered to be immortal as the telomeraza gene is activated in them which allows these cells to divide infinitely and this is the reason for cancer growth.

Hela's cells are good example for cancer cells eternity, which were received in 1951 from Henrietta Lack's cervix cancer tissue. The name of the culture comes from the above. This culture is still used in studies today. Hela's cells are really immortal: They are produced daily in tons and they are descendants of the removed cancer cells from H. Lack's organism.

In spite of the fact, that cancer modeling in the cell culture is effective and it has already been used for years, it is still not exact. At first, it was unknown which was the influence, that causes cell multiplication in this model. Finding the answer became gradually possible: Different mutations were caused in the model cells (Which we meet in different cancer cells in human) which allowed the scientists to reveal several confluent mutations, that was sufficient for creating cancer cells from different types.

Mutations are confluent in different ways in different types of cells. However, in most of these confluences the following is fixed: 1.Activation of telomeraza 2.Cycle damage of the albumin p 53. 3. Activation of Ras , Myc and other protooncogenes . 4.Violation of PP2A phosphataza formation. These changes provide "turning off " the death mechanism by means of damaging chromosome destruction or apoptosis process. With this endless multiplication of the cell begins.

In cell cultures, this model of cancer clears up the telomeraza's role in cancer development. In 90 percent of cancers telomeraza's activation is observed.

In cancers without TERT activation in cells , they mainly used other mechanism of telomeres' protection , which is called ALT (alternative lengthening of telomeres). Details of this mechanism are unknown.

According to Elizabeth Blackburn's works (1985, 2001), telomeraza is also involved in regulation of 70 genes which participate (or probably participate ) in cancer origin and development. Furthermore, telomeraza activates glycolysis, which allows cancer cells to use sugar in order to maintain growth and division rate. This speed is equal to the same process rate in embryo.

Fight with the cancer is difficult. If it was possible to receive such a medication, which would cause telomeraza's blockage in cancer cells, then telomere shortening would be renewed. Mutations would appear and cancer cells would die!

Working in this direction has started ( Koreans have just created the preparation "Telovak", which in the opinion of creators, activates immune system, that ultimately is directed to suppress telomeraza. After trying it on volunteers, ill with cancer "Telovak" prolonged patients' life by merely one year. It also gifted three months of life to the patients who were on the last stage of cancer. The question is : Maybe not only in cancer cells is telomeraza suppressed? Subsequently, will it ultimately make life shorter?

## **5. Senescence**

254 Senescence

Telomeraza is the most intensively revealed in the embryonic cells , then-in Germinational cells, very slightly-in adult organism labile cells (cells, that must divide frequently, for example, intestinal epithelial cells), but their detection is difficult in the most of the somatic

Telomeraza is used in the cosmetic production (The product TA-65 was received from the plant Astragal, which activates telomeraza). Michael Fossel supposed in one of the interviews (2006, int. by D, J Brown), that treatment by telomeraza could be used not only for struggle against cancer , but also for fight against aging i.e. for increasing life expectancy. When cells come closer to the Hayflick's limit, it is possible to stop aging if deactivation of those genes will happen , which are responsible for creation of the albumin (p53 and pR6) , suppressing cancer . Sooner or later such , changed cells get to the condition , which is called "crisis" i.e. when big part of the cells (in the culture) die (However, sometimes the cell doesn't stop multiplication during the crisis period). As a rule, telomeres are absolutely

destroyed at the time and condition of the chromosomes worsens after each division.

The naked endings of the chromosomes mean splitting of the both DNA strings. Neutralization of such type of lesion happens by connecting split endings. Also endings of different chromosomes can make confluence as they are no longer protected by telomeres. This story as if temporarily relieves telomere absence. However, in the anaphase of the cell division, linked chromosomes come apart quite accidentally, which is followed by a lot of mutations and chromosomal anomaly development and with the process development the cell genome is more and more injured. Ultimately the moment comes when apoptosis is turned on (The gene substance is so much injured ) or the mutation is added to the injured

After activation of telomeraza some types of cells obtain immortality. Their chromosomes don't become less stable for the number of divisions and the death process doesn't start. Many cancer cells are considered to be immortal as the telomeraza gene is activated in them

Hela's cells are good example for cancer cells eternity, which were received in 1951 from Henrietta Lack's cervix cancer tissue. The name of the culture comes from the above. This culture is still used in studies today. Hela's cells are really immortal: They are produced daily in tons and they are descendants of the removed cancer cells from H. Lack's

In spite of the fact, that cancer modeling in the cell culture is effective and it has already been used for years, it is still not exact. At first, it was unknown which was the influence, that causes cell multiplication in this model. Finding the answer became gradually possible: Different mutations were caused in the model cells (Which we meet in different cancer cells in human) which allowed the scientists to reveal several confluent mutations, that was

Mutations are confluent in different ways in different types of cells. However, in most of these confluences the following is fixed: 1.Activation of telomeraza 2.Cycle damage of the albumin p 53. 3. Activation of Ras , Myc and other protooncogenes . 4.Violation of PP2A

which allows these cells to divide infinitely and this is the reason for cancer growth.

cells. (More correctly, it doesn't happen at all).

**4. Cancer** 

organism.

genome which activates telomeraza.

sufficient for creating cancer cells from different types.

Cancer cell is divided infinitely, telomeraza's gene is expressed , " inserted " in it i.e. malignant cell looks like a sex or embryonic cell (Probably, these are the only cells and its product restores normal length of the telomere).

Hereby , we take the liberty of a small comment: Yes ! - Today, immortal cells of cancer are widely discussed , which is said to be the fault of telomerazas . However, it shouldn't be absolutely right. Not all multiplied cancer cells are divided, i.e. among them there are some cells that divide ,so there is the limit too. As regards the culture, it is possible to initiate growth in all the ways . Besides , in vivo , cancer cells gradually cannot divide . Ultimately, they don't have "strength " of even it and die (These are famous facts , pathology -anatomists like to point it out. I remember the talk with Omar Khardzeishvili about this very question) i.e. part of cancer cells die by themselves in the lifetime of the ill organism. (It is an axiom today too); However, the person dies before all the cancer cells die !

In human fibroblasts, Kamozin can increase Hayflicks's Limit by means of reduction of telomeres shortening quality. However, the scientists say , that vertebrates' cells have certain potential of replication. More importantly, A.Melk and his group (2003) saw that in vivo aging is possible without telomere shortening.

Programming and Implementation of Age-Related Changes 257

structure formation ( fights with denaturation), inhibits free radicals of oxygen. Sirtuins also rise sensitivity on insulin and strengthen glucose metabolism. They can also be used in diabetes treatment. It's important that red wine activates sirtuins and this prolongs life.

So, probably nothing is impossible, among them defeating cancer disease and increasing life

`In spite of the fact that telomeraza is the key to cell immortality i.e. " infinity" of cancer cell is caused by telomeraza, we know and pointed out above that cancer cells ( where expression: of this enzym is high and where exactly telomeraza is "accused " in persistent division of the cells), ultimately the condition is achieved when they don't divide any more (

We set an example of studies above where there is indicated that aging is possible without telomere shortening or disappearing i.e. nor immortality or "not aging" reverse-process- the death (which follows the aging ) entirely depends on telomeres..... Consequently, it is doubtful again that telomeraza is the key to the cell immortality! Then, where is the

In our opinion the key to immortality should be that "thing" which causes permanent renovation! Though, it's difficult to imagine it in our dimension , where everything has its beginning and the end , still I remember " immortality water" from fairy tales ! - Where is the

There are such albumens-stress albumens (which appear during different stresses in the cell and protect it from lesion spreading) - Chaperon , Chaperonins, sirtuin molecules are very important and we pointed out above their protecting and " rescuing " occupation ! Red wine turned to be sirtuins action stimulator! There is Resveratrol in the same red wine -plant estrogen, Melatonin's precursor ! Melatonin is a hormone which acts as an obstacle for lesion, "wearing out "; Melatonin has the most expressed regenerative capacity and

In the preamble of my first monograph ( "melatonini" 2007) Dinara Kasradze wrote : "And in this system too, the most mysterious turned to be the Epiphysis... Epiphysis with its melatonin! However, it is the main thing where the life takes its origin (metaphor) -i.e. it is the "source " of light and darkness, mist and dawn . It causes sleep and waking up, tiredness and relaxing, wearing out and restoration, spring and autumn, summer and winter too .... It gives the world rhythm, biorhythm which gives us a birth, brings us up, breeds, ages or takes us into other..... It causes health and disease... and probably it is not surprising that in a creative way the "third eye" is called Epiphysis, maybe not entirely creatively. It can be so that overwhelming strength of the Lord is incarnated by means of Melatonin in our dimension.... Biological-cosmic key for immortality and constant transformations may probably lay ...... in the very magic molecule of Melatonin.... Who

Death is inevitable for any of living organisms, viruses, plants or animals. However, only multicellular animals, including human beings, die from ageing. A multicellulary organism

more exactly -can't ), so their "infinity" is doubtful! Where is the immortality?

consequently the ability to restore/maintain living structures!

**6. Centriole, differentiation, senescence** 

limit... However...

immortality!

immortality?

knows...."

The surgeon, Nobeliant Alexsis Carrel said : "All the cells , that grow in the culture , are immortal ; But if they reduced , the number of cell replications fell, it means that the way of cultivation, itself , is to be recovered and improved. " We take again the liberty of adding a comment of our own : "Growing cells in culture are immortal "- The multiplication process itself can be continuous. However, the cells probably change ; In this way a human is also immortal- by means of his/her generation or descendants ( since Adam till today ). Yes, exactly the same process! And don't let Carrel to deceive us. However, as regards the opinion, that "if the number of cell divisions reduced, then the way of cultivation must be improved. " This is right: It's necessary to improve conditions to increase birth-rate in people!).

Carrel's hypothesis (1921) was strengthened by the fact that the scientist had been growing the chicken's fibroblasts in the culture for 34 years. The part of scientists thought this was possible infinitely in vertebrates. However, there was a certain mistake during the experiment (i.e. in accuracy ) and most scientists didn't agree with the results. The mistake was in the fact that Carrel daily added chicken's embryonic, axial cells to the culture; This allowed the new cells to multiply and it wasn't multiplication of the initiate, original cells any more; but in this way cells' multiplication was possible unlimitedly. Some researchers are convinced that Carrel knew about this mistake (in the experimental procedure ), but he didn't admit it.

Briefly, Alexsis Carrel's theory didn't succeed and his mistake was evident too. However, Alexei Olivnikov's theory is acceptable and corresponds to Hayflick's experiments: Telomerazas' inhibition is a good remedy in malignant cancer treatment.

Ultimately, in reduction of life expectancy the following is important : 1. Reduction of the cell replication i.e. what Hayflick's limit means ; 2. Telomeres' shortening ; 3. Accumulation of cells and reduction of axial cells number; 4. Changes , revealed in the cell with aging: accumulation of free radicals, influence of radiation, generation of oxygen free radical O2 -, modification of albumins, lipids, nuclein acids , reduction of antioxidant mechanisms ( vitamin E, glutation peroxidaza) , accumulation of dot mutations; 5. Lowering of the reparation systems, lowering of the DNA helikaza activity i.e. defect (enzyme is involved in DNA replication, reperation and other functions, that are necessary for DNA perfection. Besides Verner syndrome, defect of this enzym is found in Ataxia-Telangiektazia); 6. Strengthened expression of antioxidant enzyme - SOD and katalaza (it has been seen in Drisophila) ; 7.It is thought that proteasome function can be lowered that is called proteolyses mechanisms. Its responsibility is elimination of abnormal and unnecessary intracellular albumen (Lesion of the organels which is one of the reasons for cell aging).

Consequently, with aging, in all the organ systems, there are physiological and structural changes. The rate of aging process is associated with genetic factors, nutrition specialty, social condition , disease development , associated with age. For example, atherosclerosis, diabetes type 2, osteoarthritis.

The most effective way for increasing life expectancy is limitation of calories. It depends on sirtuins. They have hystondeacetilaza activity and probably they promote expression of different genes , products of which increase life expectancy. This products consist of proteins which increase metabolic activity, reduce apoptosis , stimulates albumens' third

The surgeon, Nobeliant Alexsis Carrel said : "All the cells , that grow in the culture , are immortal ; But if they reduced , the number of cell replications fell, it means that the way of cultivation, itself , is to be recovered and improved. " We take again the liberty of adding a comment of our own : "Growing cells in culture are immortal "- The multiplication process itself can be continuous. However, the cells probably change ; In this way a human is also immortal- by means of his/her generation or descendants ( since Adam till today ). Yes, exactly the same process! And don't let Carrel to deceive us. However, as regards the opinion, that "if the number of cell divisions reduced, then the way of cultivation must be improved. " This is right: It's necessary to improve conditions to increase birth-rate in

Carrel's hypothesis (1921) was strengthened by the fact that the scientist had been growing the chicken's fibroblasts in the culture for 34 years. The part of scientists thought this was possible infinitely in vertebrates. However, there was a certain mistake during the experiment (i.e. in accuracy ) and most scientists didn't agree with the results. The mistake was in the fact that Carrel daily added chicken's embryonic, axial cells to the culture; This allowed the new cells to multiply and it wasn't multiplication of the initiate, original cells any more; but in this way cells' multiplication was possible unlimitedly. Some researchers are convinced that Carrel knew about this mistake (in the experimental procedure ), but he

Briefly, Alexsis Carrel's theory didn't succeed and his mistake was evident too. However, Alexei Olivnikov's theory is acceptable and corresponds to Hayflick's experiments:

Ultimately, in reduction of life expectancy the following is important : 1. Reduction of the cell replication i.e. what Hayflick's limit means ; 2. Telomeres' shortening ; 3. Accumulation of cells and reduction of axial cells number; 4. Changes , revealed in the cell with aging: accumulation of free radicals, influence of radiation, generation of oxygen free radical O2 -, modification of albumins, lipids, nuclein acids , reduction of antioxidant mechanisms ( vitamin E, glutation peroxidaza) , accumulation of dot mutations; 5. Lowering of the reparation systems, lowering of the DNA helikaza activity i.e. defect (enzyme is involved in DNA replication, reperation and other functions, that are necessary for DNA perfection. Besides Verner syndrome, defect of this enzym is found in Ataxia-Telangiektazia); 6. Strengthened expression of antioxidant enzyme - SOD and katalaza (it has been seen in Drisophila) ; 7.It is thought that proteasome function can be lowered that is called proteolyses mechanisms. Its responsibility is elimination of abnormal and unnecessary intracellular albumen (Lesion of the organels which is one of

Consequently, with aging, in all the organ systems, there are physiological and structural changes. The rate of aging process is associated with genetic factors, nutrition specialty, social condition , disease development , associated with age. For example, atherosclerosis,

The most effective way for increasing life expectancy is limitation of calories. It depends on sirtuins. They have hystondeacetilaza activity and probably they promote expression of different genes , products of which increase life expectancy. This products consist of proteins which increase metabolic activity, reduce apoptosis , stimulates albumens' third

Telomerazas' inhibition is a good remedy in malignant cancer treatment.

people!).

didn't admit it.

the reasons for cell aging).

diabetes type 2, osteoarthritis.

structure formation ( fights with denaturation), inhibits free radicals of oxygen. Sirtuins also rise sensitivity on insulin and strengthen glucose metabolism. They can also be used in diabetes treatment. It's important that red wine activates sirtuins and this prolongs life.

So, probably nothing is impossible, among them defeating cancer disease and increasing life limit... However...

`In spite of the fact that telomeraza is the key to cell immortality i.e. " infinity" of cancer cell is caused by telomeraza, we know and pointed out above that cancer cells ( where expression: of this enzym is high and where exactly telomeraza is "accused " in persistent division of the cells), ultimately the condition is achieved when they don't divide any more ( more exactly -can't ), so their "infinity" is doubtful! Where is the immortality?

We set an example of studies above where there is indicated that aging is possible without telomere shortening or disappearing i.e. nor immortality or "not aging" reverse-process- the death (which follows the aging ) entirely depends on telomeres..... Consequently, it is doubtful again that telomeraza is the key to the cell immortality! Then, where is the immortality!

In our opinion the key to immortality should be that "thing" which causes permanent renovation! Though, it's difficult to imagine it in our dimension , where everything has its beginning and the end , still I remember " immortality water" from fairy tales ! - Where is the immortality?

There are such albumens-stress albumens (which appear during different stresses in the cell and protect it from lesion spreading) - Chaperon , Chaperonins, sirtuin molecules are very important and we pointed out above their protecting and " rescuing " occupation ! Red wine turned to be sirtuins action stimulator! There is Resveratrol in the same red wine -plant estrogen, Melatonin's precursor ! Melatonin is a hormone which acts as an obstacle for lesion, "wearing out "; Melatonin has the most expressed regenerative capacity and consequently the ability to restore/maintain living structures!

In the preamble of my first monograph ( "melatonini" 2007) Dinara Kasradze wrote : "And in this system too, the most mysterious turned to be the Epiphysis... Epiphysis with its melatonin! However, it is the main thing where the life takes its origin (metaphor) -i.e. it is the "source " of light and darkness, mist and dawn . It causes sleep and waking up, tiredness and relaxing, wearing out and restoration, spring and autumn, summer and winter too .... It gives the world rhythm, biorhythm which gives us a birth, brings us up, breeds, ages or takes us into other..... It causes health and disease... and probably it is not surprising that in a creative way the "third eye" is called Epiphysis, maybe not entirely creatively. It can be so that overwhelming strength of the Lord is incarnated by means of Melatonin in our dimension.... Biological-cosmic key for immortality and constant transformations may probably lay ...... in the very magic molecule of Melatonin.... Who knows...."

## **6. Centriole, differentiation, senescence**

Death is inevitable for any of living organisms, viruses, plants or animals. However, only multicellular animals, including human beings, die from ageing. A multicellulary organism

Programming and Implementation of Age-Related Changes 259

Recent data indicate that centriole and nuclear cycles are not nterdependent (Gorgidze and Vorobjev, 1994). It was shown that nucleus controls the synthesis of 'building material' (basically proteins) used by centrioles, thereby having control over the centriole cycle. When a cell has such 'building material' in excess (i.e. oocytes), cycles of centriole duplication may occur even if mitotic cycle has not been launched (Manandkhar et al., 1990). Phillips and Rattner (1976) also demonstrated that inhibition of RNA or RNA/protein synthesis

As a rule, somatic cells contain a diploid set of chromosomes and a pair of centrioles (diplosome). This ratio is maintained by the parallel reproduction of nuclear DNA and centrioles, which takes place while cells are being prepared for the next division. In the process of gametogenesis, the number of chromosomes is reduced and the cell becomes haploid. Studies of spermatogenesis performed in insects (Sciara coprphila, Chrysopa carnea, Bombyx mori) and mammals (Heterohyrax syriacus and Memetes berdmorei) discovered that at the first meiotic division spindle poles contain a pair of centrioles, while at the second division e they contain only one centriole; in mammalians spermatocytes get two centrioles after both first and second divisions (Kriouchkova and nishchenco, 1999). On the contrary, oocytes of mammalians (mouse, rat, and rabbit) and Xenopus laevis do not have visible centriolar structures during both the meiotic divisions (Kriouchkova and Onishchenco, 1999). During the process of fertilization, the chromosomal sets of a spermatozoon and an oocyte are mixed. The chromosomal sets/centrioles ratio, typical for somatic cells, may be restored either in a zygote or early blastomeres. Thus, in sea urchins, the spindle pole at first cell division contains a diplosome, while in mice they are centriolefree at both first and second divisions. Only at third division does a centrosome get a pair of centrioles. The literature reveals several hypotheses, which try to explain the formation of centrioles in embryonic cells. These structures may be obtained exclusively from the paternal cell, as in sea urchins. In most mammalians zygotes, the centrosome is of maternal origin, but the centrioles are formed de novo only in the third cell cycle. It should be noted, however, that human centrioles, as well as centrioles of other mammalian species (sheep, cow, and marsupials), belong to the paternal sex cell (Breed et al., 1994; Nijs et al., 1996;

Some studies demonstrate that centrioles act as the regulators that control the course of every phase of a cell cycle influencing the processes that take place at least two phases

Centrioles seem to play some role in inheritance of tumorigenic properties. Taking into account aforementioned facts, what molecular mechanism underlying the centriolar activity may control determination of morphogenetic status of a somatic cell? Centrioles may contain differently encoded RNA molecules stacked in a definite order. During mitosis these RNA molecules are probably released one by one into cytoplasm. This process presumably changes the status of repressed and potentially active genes and, subsequently, the

suppresses the duplication of centrioles in cultured cells.

earlier (Neverova et al., 1996; Maniotis and Schliva, 1991).

**6.2 The rule of cell division** 

Schatten et al., 1996).

**6.3 Centrioles and cancer** 

morphogenetic status of a cell.

(animal), containing a multitude of irreversibly differentiated cells, develops from only one cell e the totipotential zygote. The potency to differentiate into a certain tissue or tissues (set by the factor which controls the possibility of repression and activation of genes) determines the individual histological state, or morphogenetic "status'" of a cell. The final morphogenetic status means that such a cell is committed to programmed death (apoptosis). A 'zero' morphogenetic status means that the cell has not been committed to any irreversible pathway, i.e. it remains totipotent. Modification of morphogenetic status of a cell changes the whole spectrum of the tissues into which this cell can differentiate. In ontogenesis, this spectrum consequently narrows (totipotential / pluripotential/ multipotential/ unipotential/ non-potential) until the cell reaches the state of final differentiation (Malaitcev et al., 2002).

## **6.1 Need for a self-replicating controller**

It has been established that morphogenetic status of a cell might be changed only through its division. We consider that cell division, differentiation and apoptosis may be controlled by a single intracellular structure. Naturally, this structure has to be self-replicating. Moreover, it has to have some way of counting or recording cell divisions. In a somatic cell potential candidates for this replicable structure are chromosomes, mitochondria (both contain DNA), and centrioles. To date the structures which might count cell divisions e and, therefore, determine the morphogenetical status of a cell e are generally thought to be telomeres (Bodnar et al., 1998; Greider, 1998; Shay and Wright, 2001). However, some data do not confirm the hypothesis that assigns the role of replication 'clock' to these parts of a chromosome.

Thus, the cells of mice bred by Blasco et al. (1997) did not have telomerase activity. Such mice were viable and could produce up to 6 generations, though the chromosomes of each subsequent generation had increasingly shortened telomeres. Only the sixth generation developed abnormalities caused by the extreme shortening of telomeres. The authors emphasised that 'ageing' of cells took place long before the marginal shortening of telomeres. Rudolph et al. (1999) showed that mice with inactivated genes of telomerase had shorter lifetime and increased frequency of oncological diseases, but physiological and biochemical tests did not reveal any signs of early ageing. Animal cloning disproves telomere hypothesis as well as other hypotheses, which suggest that a morphogenetic factor is located in nuclear DNA. In outline, the main point of cloning procedure is injection of the nucleus of a finally differentiated somatic cell into an oocyte with previously removed or destroyed nucleus. It was undoubtedly proved that the pattern of genes expression is changed to comply with the cytoplasm of a 'host' cell (Hardeman et al., 1986; Dominko et al., 1999). Injection of the nucleus of a frog carcinoma cells into an enucleated egg resulted in, not tumor cells, but normal tadpole development.

The key element of gene network controlling the processes of cell differentiation is an external factor for nuclear genome signal, which activates these groups of interactively expressing genes (Kolchanov et al., 2000). We suggest that the processes of cell ageing, differentiation and division are regulated by cytoplasmic factors. The structure, which regulates processes of irreversible differentiation, determination and modification of morphogenetic status, is most likely to be a centriole (centrosome).

Recent data indicate that centriole and nuclear cycles are not nterdependent (Gorgidze and Vorobjev, 1994). It was shown that nucleus controls the synthesis of 'building material' (basically proteins) used by centrioles, thereby having control over the centriole cycle. When a cell has such 'building material' in excess (i.e. oocytes), cycles of centriole duplication may occur even if mitotic cycle has not been launched (Manandkhar et al., 1990). Phillips and Rattner (1976) also demonstrated that inhibition of RNA or RNA/protein synthesis suppresses the duplication of centrioles in cultured cells.

## **6.2 The rule of cell division**

258 Senescence

(animal), containing a multitude of irreversibly differentiated cells, develops from only one cell e the totipotential zygote. The potency to differentiate into a certain tissue or tissues (set by the factor which controls the possibility of repression and activation of genes) determines the individual histological state, or morphogenetic "status'" of a cell. The final morphogenetic status means that such a cell is committed to programmed death (apoptosis). A 'zero' morphogenetic status means that the cell has not been committed to any irreversible pathway, i.e. it remains totipotent. Modification of morphogenetic status of a cell changes the whole spectrum of the tissues into which this cell can differentiate. In ontogenesis, this spectrum consequently narrows (totipotential / pluripotential/ multipotential/ unipotential/ non-potential) until the cell reaches the state of final differentiation (Malaitcev

It has been established that morphogenetic status of a cell might be changed only through its division. We consider that cell division, differentiation and apoptosis may be controlled by a single intracellular structure. Naturally, this structure has to be self-replicating. Moreover, it has to have some way of counting or recording cell divisions. In a somatic cell potential candidates for this replicable structure are chromosomes, mitochondria (both contain DNA), and centrioles. To date the structures which might count cell divisions e and, therefore, determine the morphogenetical status of a cell e are generally thought to be telomeres (Bodnar et al., 1998; Greider, 1998; Shay and Wright, 2001). However, some data do not confirm the hypothesis that assigns the role of replication 'clock' to these parts of a

Thus, the cells of mice bred by Blasco et al. (1997) did not have telomerase activity. Such mice were viable and could produce up to 6 generations, though the chromosomes of each subsequent generation had increasingly shortened telomeres. Only the sixth generation developed abnormalities caused by the extreme shortening of telomeres. The authors emphasised that 'ageing' of cells took place long before the marginal shortening of telomeres. Rudolph et al. (1999) showed that mice with inactivated genes of telomerase had shorter lifetime and increased frequency of oncological diseases, but physiological and biochemical tests did not reveal any signs of early ageing. Animal cloning disproves telomere hypothesis as well as other hypotheses, which suggest that a morphogenetic factor is located in nuclear DNA. In outline, the main point of cloning procedure is injection of the nucleus of a finally differentiated somatic cell into an oocyte with previously removed or destroyed nucleus. It was undoubtedly proved that the pattern of genes expression is changed to comply with the cytoplasm of a 'host' cell (Hardeman et al., 1986; Dominko et al., 1999). Injection of the nucleus of a frog carcinoma cells into an enucleated egg resulted

The key element of gene network controlling the processes of cell differentiation is an external factor for nuclear genome signal, which activates these groups of interactively expressing genes (Kolchanov et al., 2000). We suggest that the processes of cell ageing, differentiation and division are regulated by cytoplasmic factors. The structure, which regulates processes of irreversible differentiation, determination and modification of

et al., 2002).

chromosome.

**6.1 Need for a self-replicating controller** 

in, not tumor cells, but normal tadpole development.

morphogenetic status, is most likely to be a centriole (centrosome).

As a rule, somatic cells contain a diploid set of chromosomes and a pair of centrioles (diplosome). This ratio is maintained by the parallel reproduction of nuclear DNA and centrioles, which takes place while cells are being prepared for the next division. In the process of gametogenesis, the number of chromosomes is reduced and the cell becomes haploid. Studies of spermatogenesis performed in insects (Sciara coprphila, Chrysopa carnea, Bombyx mori) and mammals (Heterohyrax syriacus and Memetes berdmorei) discovered that at the first meiotic division spindle poles contain a pair of centrioles, while at the second division e they contain only one centriole; in mammalians spermatocytes get two centrioles after both first and second divisions (Kriouchkova and nishchenco, 1999). On the contrary, oocytes of mammalians (mouse, rat, and rabbit) and Xenopus laevis do not have visible centriolar structures during both the meiotic divisions (Kriouchkova and Onishchenco, 1999). During the process of fertilization, the chromosomal sets of a spermatozoon and an oocyte are mixed. The chromosomal sets/centrioles ratio, typical for somatic cells, may be restored either in a zygote or early blastomeres. Thus, in sea urchins, the spindle pole at first cell division contains a diplosome, while in mice they are centriolefree at both first and second divisions. Only at third division does a centrosome get a pair of centrioles. The literature reveals several hypotheses, which try to explain the formation of centrioles in embryonic cells. These structures may be obtained exclusively from the paternal cell, as in sea urchins. In most mammalians zygotes, the centrosome is of maternal origin, but the centrioles are formed de novo only in the third cell cycle. It should be noted, however, that human centrioles, as well as centrioles of other mammalian species (sheep, cow, and marsupials), belong to the paternal sex cell (Breed et al., 1994; Nijs et al., 1996; Schatten et al., 1996).

Some studies demonstrate that centrioles act as the regulators that control the course of every phase of a cell cycle influencing the processes that take place at least two phases earlier (Neverova et al., 1996; Maniotis and Schliva, 1991).

## **6.3 Centrioles and cancer**

Centrioles seem to play some role in inheritance of tumorigenic properties. Taking into account aforementioned facts, what molecular mechanism underlying the centriolar activity may control determination of morphogenetic status of a somatic cell? Centrioles may contain differently encoded RNA molecules stacked in a definite order. During mitosis these RNA molecules are probably released one by one into cytoplasm. This process presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

Programming and Implementation of Age-Related Changes 261

sequence of DNA loci activation in the offspring cells. Absence of centrioles in a stage of preleptotenic chromosome condensation in mice may be good illustration of 'zero' morphogenetic status of cells (Hartung and Stahl, 1977). Intracellular 'morphogenetic clocks'

Should centrioles indeed control the processes of cell differentiation and programmed

1. When a cell initially does not have centrioles (this situation denotes the absence of

Fig. 1. Causing irrevocable differentiation – Protein or RNA-hypothetical molecular code must be encoded in DNA or mitochondrial or nuclear. It is expected that as many differentiation molecular codes should be encoded in DNA as the quantity of irrevocable differentiation stages, characteristic for each particular species. Molecular structure of the differentiation, complexing at centriol must have the features of matrix - while forming new

2. If a cell launches centriole synthesis de novo (the centrioles are considered to be formed de novo as long as the cell is not committed to irreversible differentiation), it will be

totipotential and immortal, retaining its initial 'zero' morphogenetic status;

seems to be wound up at that moment returning to the initial 'zero' state.

**6.6 Conclusions that can be drawn** 

death, the following may be concluded:

centriols, it must repeat similar structure.

morphogenetical status), it will be totipotential;

Centrioles may have such molecules controlling morphogenetic status contained in their internal chamber (the area of two electron-dense linear structures). It is also possible that they are packed in definite order. Some molecules may differ from the preceding ones and control different morphogenetic status. Other ones can be exact copies, thus determining the same morphogenetic status. In each mitotic division, one of these molecules is released and 'lost' in the cytoplasm, so the centrioles of daughter cells contain one molecule less than the centrioles of the maternal cell. The number of molecules contained in centrioles decreases after each mitotic division. The last molecule triggers the processes of programmed death (i.e. apoptosis). Thus, the number of hypothetic molecules must correspond to the number of possible mitotic divisions, counting down from the cell having the first morphogenetic status to the last offspring cell having the final morphogenetic status (the ''Hayflick limit'', Hayflick, 1997).

#### **6.4 Importance of small MW RNA (siRNA)**

The best candidate for the role of the carrier of information on morphogenetic status seems to be low molecular weight RNA. Indirect evidence for this hypothesis may be the discovery of new classes of small RNA e so-called interfering RNA (siRNA) and micro-RNA (miRNA) e having regulatory activity. The phenomenon of RNA interference occurs to inhibit selectively gene expression in animal cells (Fire et al., 1998; McManus and Sharp, 2002; Montgomery et al., 1998), including mammalian cells (Wianny and Zornicka-Goets, 2000). MicroRNA appeared to act as a regulator of cell differentiation and development in higher organisms. There direct data confirm the role of these molecules in the process of cell division (Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2001). In 2002, it was found that the influence of siRNA might not be limited to only temporary 'knockout' of genes on RNA level. Small RNA can modify the chromatin structure and make genes active or silent for quite a long period of time (Zilberman et al., 2003). In addition to posttranscriptional and transcriptional homology-dependent gene silencing links between DNA structure and RNA expose themselves in many other phenomena, like dose-dependent compensation in drosophila and mammalian X-chromosome inactivation (Stuckenholz et al., 1999).

The whole body of existing e though sometimes contradictory e data demonstrating that RNA may be located inside of centrosome or linked to this structure (Heath, 1980; Heidemann et al., 1977; Lambert and Nagy, 2000; Nadejdina et al., 1982; Peterson and Berns, 1980) makes it possible to suggest that small RNA (of 20e300 nucleotides) may be an ideal candidate for the role of the molecule if these are inside the centrioles.

#### **6.5 The centriole as the carrier of controlling siRNA**

Filling of centrioles with the molecules of RNA in various species apparently takes place in different cells: those, which develop at the first of second meiotic division of gametogenesis (spermatogenesis or oogenesis, depending on species), or in the first blastomeres at the time of centriole formation de novo. Those molecules are transcribed from nuclear DNA and stacked in a definite order within the centrioles. Following this process subsequent release of RNA molecules during mitosis determines the expression of nuclear DNA. Nuclear genome presumably 'chooses' and 'places' into centrioles the information about the sequence of DNA loci activation in the offspring cells. Absence of centrioles in a stage of preleptotenic chromosome condensation in mice may be good illustration of 'zero' morphogenetic status of cells (Hartung and Stahl, 1977). Intracellular 'morphogenetic clocks' seems to be wound up at that moment returning to the initial 'zero' state.

## **6.6 Conclusions that can be drawn**

260 Senescence

Centrioles may have such molecules controlling morphogenetic status contained in their internal chamber (the area of two electron-dense linear structures). It is also possible that they are packed in definite order. Some molecules may differ from the preceding ones and control different morphogenetic status. Other ones can be exact copies, thus determining the same morphogenetic status. In each mitotic division, one of these molecules is released and 'lost' in the cytoplasm, so the centrioles of daughter cells contain one molecule less than the centrioles of the maternal cell. The number of molecules contained in centrioles decreases after each mitotic division. The last molecule triggers the processes of programmed death (i.e. apoptosis). Thus, the number of hypothetic molecules must correspond to the number of possible mitotic divisions, counting down from the cell having the first morphogenetic status to the last offspring cell having the final morphogenetic status (the ''Hayflick limit'',

The best candidate for the role of the carrier of information on morphogenetic status seems to be low molecular weight RNA. Indirect evidence for this hypothesis may be the discovery of new classes of small RNA e so-called interfering RNA (siRNA) and micro-RNA (miRNA) e having regulatory activity. The phenomenon of RNA interference occurs to inhibit selectively gene expression in animal cells (Fire et al., 1998; McManus and Sharp, 2002; Montgomery et al., 1998), including mammalian cells (Wianny and Zornicka-Goets, 2000). MicroRNA appeared to act as a regulator of cell differentiation and development in higher organisms. There direct data confirm the role of these molecules in the process of cell division (Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2001). In 2002, it was found that the influence of siRNA might not be limited to only temporary 'knockout' of genes on RNA level. Small RNA can modify the chromatin structure and make genes active or silent for quite a long period of time (Zilberman et al., 2003). In addition to posttranscriptional and transcriptional homology-dependent gene silencing links between DNA structure and RNA expose themselves in many other phenomena, like dose-dependent compensation in drosophila and mammalian X-chromosome inactivation (Stuckenholz et

The whole body of existing e though sometimes contradictory e data demonstrating that RNA may be located inside of centrosome or linked to this structure (Heath, 1980; Heidemann et al., 1977; Lambert and Nagy, 2000; Nadejdina et al., 1982; Peterson and Berns, 1980) makes it possible to suggest that small RNA (of 20e300 nucleotides) may be an ideal

Filling of centrioles with the molecules of RNA in various species apparently takes place in different cells: those, which develop at the first of second meiotic division of gametogenesis (spermatogenesis or oogenesis, depending on species), or in the first blastomeres at the time of centriole formation de novo. Those molecules are transcribed from nuclear DNA and stacked in a definite order within the centrioles. Following this process subsequent release of RNA molecules during mitosis determines the expression of nuclear DNA. Nuclear genome presumably 'chooses' and 'places' into centrioles the information about the

candidate for the role of the molecule if these are inside the centrioles.

**6.5 The centriole as the carrier of controlling siRNA** 

Hayflick, 1997).

al., 1999).

**6.4 Importance of small MW RNA (siRNA)** 

Should centrioles indeed control the processes of cell differentiation and programmed death, the following may be concluded:

1. When a cell initially does not have centrioles (this situation denotes the absence of morphogenetical status), it will be totipotential;

Fig. 1. Causing irrevocable differentiation – Protein or RNA-hypothetical molecular code must be encoded in DNA or mitochondrial or nuclear. It is expected that as many differentiation molecular codes should be encoded in DNA as the quantity of irrevocable differentiation stages, characteristic for each particular species. Molecular structure of the differentiation, complexing at centriol must have the features of matrix - while forming new centriols, it must repeat similar structure.

2. If a cell launches centriole synthesis de novo (the centrioles are considered to be formed de novo as long as the cell is not committed to irreversible differentiation), it will be totipotential and immortal, retaining its initial 'zero' morphogenetic status;

Programming and Implementation of Age-Related Changes 263

Fig. 3. Maintaining the quantity of the stem cells is explained by the reason, that irrevocable differentiation divisions have asymmetric nature (asymmetric division). Existence of asymmetric division and differentiation structure can also explain the fact that different types of cells give not only different (various), but also similar descendant

Many existing data may be explained on the basis of the proposed hypothesis:

1983). According to our conception they are fully potential and immortal;

belong to the fully potential and immortal cells;

phenomenon affecting the whole organism.

cell becomes immortal, though it has 'non-zero' morphogenetic status.

1. Centrioles are initially absent in the cells of higher plants (Sluiman, 1985), zygote and the first blastomeres of some animals (Abumuslimov et al., 1994 Calarco-Gillam et al.,

2. Centrioles formed de novo in a zygote and the first blastomeres (Maro et al., 1991)

3. Tumor and transformed cells are immortal, but not totipotential. This state may be due to the presence of a certain 'non-zero' morphogenetical status at the moment of

It is possible that centriolar and nuclear cycles of tumor and transformed cells are irreversibly disengaged. The nucleus looses its ability to perceive some intracellular and extracellular signals, including those which control the morphogenetic status of a cell. The

Based upon the proposed hypothesis, we suggest that centrioles realize their function of 'counting' mitotic divisions. The number of divisions (generations) of a cell is limited; this means that the number of cells of various types is fixed too. Consequently, the possibility of cellular regeneration will be also limited. Sooner or later, regenerating issues experience a lack of cells: then the organism will not be able to further provide its 'homeostatic' support for long-living cells (for instance, neurons). Thus, death from ageing seems to be a

cells.

transformation.

Fig. 2. Cell division, during which release of the molecule, causing differentiation happens, will finish with the irrevocable differentiation of the descendant cells. Probably, the molecule the albumen or RNA differentiation plays the role of the matrix. Ultimately, the small DNA molecule is formed, which enters the nuclear chromosomes, is inserted in them and so changes their structure and function, but not irrevocably.

3. Should a cell not to die due to programmed death when appropriate changes in centrioles do occur, it will be immortal, but not totipotential

Fig. 2. Cell division, during which release of the molecule, causing differentiation happens,

3. Should a cell not to die due to programmed death when appropriate changes in

will finish with the irrevocable differentiation of the descendant cells. Probably, the molecule the albumen or RNA differentiation plays the role of the matrix. Ultimately, the small DNA molecule is formed, which enters the nuclear chromosomes, is inserted in them

and so changes their structure and function, but not irrevocably.

centrioles do occur, it will be immortal, but not totipotential

Fig. 3. Maintaining the quantity of the stem cells is explained by the reason, that irrevocable differentiation divisions have asymmetric nature (asymmetric division). Existence of asymmetric division and differentiation structure can also explain the fact that different types of cells give not only different (various), but also similar descendant cells.

Many existing data may be explained on the basis of the proposed hypothesis:


It is possible that centriolar and nuclear cycles of tumor and transformed cells are irreversibly disengaged. The nucleus looses its ability to perceive some intracellular and extracellular signals, including those which control the morphogenetic status of a cell. The cell becomes immortal, though it has 'non-zero' morphogenetic status.

Based upon the proposed hypothesis, we suggest that centrioles realize their function of 'counting' mitotic divisions. The number of divisions (generations) of a cell is limited; this means that the number of cells of various types is fixed too. Consequently, the possibility of cellular regeneration will be also limited. Sooner or later, regenerating issues experience a lack of cells: then the organism will not be able to further provide its 'homeostatic' support for long-living cells (for instance, neurons). Thus, death from ageing seems to be a phenomenon affecting the whole organism.

Programming and Implementation of Age-Related Changes 265

In our point of view, the "key" to immortality must be "something" which caused constant renovation of the "worn". It's impossible to stop aging, only, it's possible to turn the biological clock back (even for several times). However, it's difficult to imagine in our dimension, where everything has its beginning and end... and still I can remember "water of

The fact that it was possible to induce totipotential qualities in the stem cell, gives us some hope. If we can discover the damaged DNA and replace it with healthy code, it's already

If we return this cell to the organism, theoritically, it is expected that the "aged" stem cells

[1] Abumuslimov SS, Nadezhdina ES, Chentcov YuS. Morphogenesis of centrioles and

[2] Alliegro M.C., at all. Centrosome-associated RNA in surf clam oocytes. Proc Natl Acad

[3] Alliegro MC, Satir P., 2009. Origin of the cilium: novel approaches to examine a

[4] Bartel D., Chen C. Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat. Rev. Genet. 2004; 5: 396-400. [5] Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA, et al. Telomere

[6] Blower MD, Feric E, Weis K, Heald R. Genome-wide analysis demonstrates conserved

[7] Blower M.D., Nachury M., Heald R., Weis K. A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly. Cell. 2005; 121: 223-234. [8] Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, et al.

[9] Breed W, Simerky C, Navara C, Vande Berg J, Schatten G. Distribution of microtubules

[10] Calarco-Gillam PD, Siebert MC, Hubble R, Mitchison T, Kirschner M. Centrosome

[11] Chang S., Johnston R., Frekjaer-Jensen C. et al. MicroRNAs act sequentially and

[12] Chapman M.J., Dolan M.F., Margulis L. Centrioles and kinetosomes: form, function,

[13] Chapman, M.J. and Alliegro, M.A. A symbiotic origin for the centrosome? Symbiosis.

centriolar evolution hypothesis. Methods Cell Biol. 2009; 94: 53-64

centrosomes in early mouse development: the Electron microscopic study.

shortening and tumor formation by mouse cells lacking telomerase RNA. Cell

localization of messenger RNAs to mitotic microtubules. J Cell Biol. 2007; 179(7):

Extension of life-span by introduction of telomerase into normal human cells.

in eggs and early embtyos of marsupial Monodelphis domestica. Dev Biol

development in early mouse embryos as defined by an autoantibody against

asymmetrically to control chemosensory laterality in the nematode. Nature. 2004;

immortality" from my childhood - Where is the immortality?

Tsitologiia 1994;11:1054e60 [in Russian].

Sci U S A. 2006; 103(24): 9034-9038

Science 1998 Jan 16;279(5349):334e5.

pericentriolar material. Cell 1983;35:621e9.

and evolution. Q Rev Biol. 2000; 75(4): 409-429.

1997;91:25e34.

1994;164:230e40.

430:785-789.

2007; 44: 23-32

1365-1373.

possible to return totipotential features to this cell.

"are replaced" by the "rejuvenated" stem cells.

**9. References** 

Our proposed hypothesis is verifiable, but needs also be tested to destruction. It may be easily checked if the chemical composition of the internal chamber of centrioles is studied more closely. According to the hypothesis, there must be some difference in chemical composition, as well as in ultrastructure (morphology), of the inner chamber of centrioles in different types of somatic cells. Attention should be also paid to monocellular organisms having centrioles.

It should be emphasised that until now the changes of ultrastructure of the centrosome during the processes of maturation of sex cells and their fusion during insemination, as well as in the course of the first embryonic cell cycles, have not yet been studied in any detail. This kind of investigation should throw light upon the problem of centrioles (diplosome) 'inheritance' in embryonic blastomeres. It would be interesting to know how and to what extent the hetero- and homo-gametic states are related to the 'inheritance' of centrioles. It is also important to find out what kind of divisions produce changes in morphogenetic status and how great is the number of such divisions.

#### **6.7 Final remarks**

The final resolution of the centriole question, however, can be through their transplantation from differentiated cells of one type into the cells of another type. Today such transplantations are quite possible. To preserve the functional activity of centrioles, they should be extracted immediately after their maturation is finished, i.e. in the middle of metaphase, and injected at the moment the chromosomes are formed. Of course, the centrioles of the recipient cell have to be removed earlier from the spindle poles. Then one could observe whether such 'hybridization' provokes changes in the expression of nuclear genes. In our opinion, the expression of nuclear genes should be changed to resemble that of the donor cell. It would confirm the hypothesis, which states that centrioles (diplosome) are actually the structure controlling the morphogenetical status of a cell.

According to this hypothesis, there must a constant number of stem cells, which is sufficient for regeneration of tissues and organs. However, this doesn't conform with reality. With aging, number of stem cells is reduced. It has to be searched: What is the reason for it? - Influence of the outer factors (e.g.: Gathering mutation in generations, which makes the cell lose the viability) or inner factors, the programmed process (e.g.: Telomeres' shortening in generations, that ends up with transformation in cancer cell or cell death).

## **7. Centrosomal RNA**

In 2006 a group of researchers discovered a specific centrosome-associated RNA, which is called cnRNA. New developments and findings in this direction will clarify the issue whether the differentiation molecules have a protein or RNA nature. It is desirable as well to identify which section of the ovule genome carries the information about these molecules.

### **8. Conclusion**

In spite of the fact, that Telomerase is the key to the cell immortality or that cancer cell "eternity" is caused by Telomerase, we know and have pointed out above that cancer cells (where enzyme expression is high and where every Telomerase is "to be blamed" for constant division of cells), ultimately achieve the condition when they don't divide any more (can't divide is more correct) - i.e. their "eternity" is suspicious! - Where is the immortality?

In our point of view, the "key" to immortality must be "something" which caused constant renovation of the "worn". It's impossible to stop aging, only, it's possible to turn the biological clock back (even for several times). However, it's difficult to imagine in our dimension, where everything has its beginning and end... and still I can remember "water of immortality" from my childhood - Where is the immortality?

The fact that it was possible to induce totipotential qualities in the stem cell, gives us some hope. If we can discover the damaged DNA and replace it with healthy code, it's already possible to return totipotential features to this cell.

If we return this cell to the organism, theoritically, it is expected that the "aged" stem cells "are replaced" by the "rejuvenated" stem cells.

## **9. References**

264 Senescence

Our proposed hypothesis is verifiable, but needs also be tested to destruction. It may be easily checked if the chemical composition of the internal chamber of centrioles is studied more closely. According to the hypothesis, there must be some difference in chemical composition, as well as in ultrastructure (morphology), of the inner chamber of centrioles in different types of somatic cells. Attention should be also paid to monocellular organisms having centrioles. It should be emphasised that until now the changes of ultrastructure of the centrosome during the processes of maturation of sex cells and their fusion during insemination, as well as in the course of the first embryonic cell cycles, have not yet been studied in any detail. This kind of investigation should throw light upon the problem of centrioles (diplosome) 'inheritance' in embryonic blastomeres. It would be interesting to know how and to what extent the hetero- and homo-gametic states are related to the 'inheritance' of centrioles. It is also important to find out what kind of divisions produce changes in morphogenetic status

The final resolution of the centriole question, however, can be through their transplantation from differentiated cells of one type into the cells of another type. Today such transplantations are quite possible. To preserve the functional activity of centrioles, they should be extracted immediately after their maturation is finished, i.e. in the middle of metaphase, and injected at the moment the chromosomes are formed. Of course, the centrioles of the recipient cell have to be removed earlier from the spindle poles. Then one could observe whether such 'hybridization' provokes changes in the expression of nuclear genes. In our opinion, the expression of nuclear genes should be changed to resemble that of the donor cell. It would confirm the hypothesis, which states that centrioles (diplosome) are

According to this hypothesis, there must a constant number of stem cells, which is sufficient for regeneration of tissues and organs. However, this doesn't conform with reality. With aging, number of stem cells is reduced. It has to be searched: What is the reason for it? - Influence of the outer factors (e.g.: Gathering mutation in generations, which makes the cell lose the viability) or inner factors, the programmed process (e.g.: Telomeres' shortening in

In 2006 a group of researchers discovered a specific centrosome-associated RNA, which is called cnRNA. New developments and findings in this direction will clarify the issue whether the differentiation molecules have a protein or RNA nature. It is desirable as well to identify which section of the ovule genome carries the information about these molecules.

In spite of the fact, that Telomerase is the key to the cell immortality or that cancer cell "eternity" is caused by Telomerase, we know and have pointed out above that cancer cells (where enzyme expression is high and where every Telomerase is "to be blamed" for constant division of cells), ultimately achieve the condition when they don't divide any more (can't

divide is more correct) - i.e. their "eternity" is suspicious! - Where is the immortality?

actually the structure controlling the morphogenetical status of a cell.

generations, that ends up with transformation in cancer cell or cell death).

and how great is the number of such divisions.

**6.7 Final remarks** 

**7. Centrosomal RNA** 

**8. Conclusion** 


Programming and Implementation of Age-Related Changes 267

[35] Klotz, C., Dabauvalle, M.C., Paintrand, M., Weber, T., Bornens, M. and Karsenti, E.

[36] Kolchanov NA, Anan'ko EA, Kolpakov FA, Podkolodnaia OA, Ignat'eva EV,

[37] Kriouchkova MM, Onishchenco GE. Structural and functional characteristics of the

[38] Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes

[39] Lambert JD, Nagy LM. Asymmetric inheritance of centrosomally localized mRNAs

[40] Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001;294:858e62. [41] Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans.

[42] Lecuyer E, Yoshida H, Parthasarathy N, Alm C, Babak T, Cerovina T, Hughes TR,

[43] Lopez de Heredia M, Jansen RP. mRNA localization and the cytoskeleton. Curr Opin

[44] Malaitcev VV, Bogdanov IM, Sukhikh GT. Contemporary introduction of biology of

[45] Manandkhar G, Khodjakov AL, Onishenko GE. Modified PEGDMSO- Serum method

[46] Maniotis A, Schliva M. Microsurgical removal of centrosomes block cell reproduction

[47] Margulis L., Chapman M., Guerrero R., Hall J. The last eukaryotic common ancestor

[50] Miska E., Alvarez-Saavedra E., Townsend M. et al. Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol. 2004; 5: R68. [51] Montgomery MK, Xu S, Fire A. RNA as a target of double/stranded RNA/mediated

[52] Nadejdina ES, Fais D, Chentsov YuS. Some biomedical aspects of centriole and basal

[53] Neverova AL, Uzbekov RE, Votchal MS, Vorobjev IA. Effect of microirradiation of the

with an inactivated centrosome. Tsitologiia 1996;2:145e54 [in Russian].

Proterozoic Eon. Proc Natl Acad Sci U S A. 2006; 103(35): 13080-13085 [48] Maro B, Gueth-Hallonet C, Aghion J, Antony C. Cell polarity and microtubule organisation during mouse early embryogenesis. Dev Suppl 1991;1:17e25. [49] McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat

(LECA): acquisition of cytoskeletal motility from aerotolerant spirochetes in the

genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA

bodies. Macrosomal Funct Cell 2 Sov-Ital. Sympos, Pushchino, 1982, b2, Moscow, p.

centrosome on cell behaviour. IV. Synthetic activity, spreading and growth of cells

Tomancak P, Krause HM. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell. 2007; 131(1):

Goriachkovskaia TN, et al. Gene networks. Mol Biol 2000;4:533e44.

coding for small expressed RNAs. Science 2001; 294:853e8.

during embryonic cleavages. Nature 2002; 420:682e6.

stem cell. Ark Pathol 2002;4:7e11 [in Russian].

induces high rate of fusion. Cell Biol IntRep 1990;14:82e6.

and centriole generation in BSC-1 cells. Cell 1991;67:495e504.

110: 405-415.

Cytol 1999;185:107e56.

Science 2001;294:862e4.

Cell Biol. 2004; 16(1): 80-85.

Rev Genet 2002;3:737e47.

1998;95:15502e7.

163 [in Russian].

174-187.

Parthenogenesis in Xenopus eggs requires centrosomal integrity. J. Cell Biol. 1990;

centrosome in gametogenesis and early embryogenesisi of animals. Intern Rev


[14] Chen C., Li L., Lodish H., Bartel D. MicroRNAs modulate hematopoietic lineange

[15] Chu C.Y. and Rana T.M. Small RNAs: regulators and guardians of the genome. J. Cell

[16] Dominko T, Mitalipova M, Haley B, Beyhan Z, Memili E, McKusick B, et al. Bovine

[17] Doxsey S., McCollum D., Theurkauf W. Centrosomes in cellular regulation. Ann. Rev.

[18] Etienne-Manneville S. Actin and microtubules in cell motility: which one is in control?

[19] Filipowicz, W., Bhattacharyya, S.N. and Sonenberg, N. Mechanisms of post-

[20] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific

[23] Gourlay C.W., Ayscough K.R. The actin cytoskeleton: a key regulator of apoptosis and

[24] Greider CW. Telomerase activity, cell proliferation, and cancer. Proc Natl Acad Sci

[25] Hardeman EC, Chiu CP, Minty A, Blau HM. The pattern of actin expression in human

[26] Hiedemann, S.R., Sander, G. and Kirschner, M.W. Evidence for a functional role of

[27] Hartung M, Stahl A. Preleptotene chromosome condensation in mouse oogenesis.

[28] Hayflick L. Mortality and Immortality on the cell level. Biochemistry 1997;11:1380e93. [29] Heath JB. Variant mitosis in lower eucaryotes: indicator of the evolution of mitosis. Int

[30] Heidemann SR, Sander G, Kirschner MW. Evidence for a functional role of RNA in

[31] Kim V.N., Han J, Siomi M.C. Biogenesis of small RNAs in animals. Nat Rev Mol Cell

[32] Kingsley E.P., Chan X.Y., Duan Y., Lambert J.D. Widespread RNA segregation in a

[33] Kloc M., Zearfoss N.R. and Etkin L.D. Mechanisms of subcellular mRNA localization.

[34] Kloc M., Etkin L.D. RNA localization mechanisms in oocytes. J Cell Sci. 2005; 118(Pt 2):

fibroblast mouse muscle heterokaryons suggests that human muscle regulatory

[21] Galitskii V.A. Epigenetic nature of ageing. Tsitologiia. 2009; 51(5):388-397. Russian [22] Gorgidze LA, Vorobjev IA. Centrioles in tissue culture cells are able to replicate in the

absence of the nucleus. Tsitologiia 1994;8:837e43 [in Russian].

ageing? Nat Rev Mol Cell Biol. 2005; 6(7): 583-589.

factors are produces. Cell 1986;47:123e30.

RNA in centrioles. Cell. 1977; 10, 337-350.

spiralian embryo. Evol Dev. 2007; 9(6): 527-539.

Cytogenet Cell Genet 1977;2:309e19.

oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol Reprod

transcriptional regulation by microRNAs: are the answers in sight? Nature Rev.

genetic interference by double/stranded RNA in Caenorhabditis elegans. Nature

differentiation. Science. 2004; 303: 83-86.

Cell and Dev. Biol. 2005; 21: 411-434.

Physiol. 2007; 213: 412-419.

Traffic. 2004; 5(7): 470-477.

Genet. 2008; 9: 102-114.

USA 1998 Jan 6;95(1):90e2.

Rev Cytol 1980;64:1e80.

Biol. 2009; 10(2): 126-39.

Cell. 2002; 108: 533-544.

269-82.

centrioles. Cell 1977;10:337e50.

1998;391:806e11.

1999;6:1496e502.


**13** 

*1Sweden 2P. R. China* 

**Cellular Degradation Machineries in** 

*1Department of Neuroscience, Karolinska Institutet, Stockholm,* 

*2Department of Human Anatomy, School of Medicine, Inner Mongolia University for Nationalities, Tongliao City,* 

Mikael Altun1, Max Grönholdt-Klein1, Lingzhan Wang1,2 and Brun Ulfhake1

**Age-Related Loss of Muscle Mass (Sarcopenia)** 

One of the most characteristic features of the aged is a change in body composition and human cross-sectional and longitudinal studies consistently demonstrate gain in fat mass and decline in lean mass (Attaix et al., 2005). Skeletal muscle mass is gradually reduced through both atrophy and loss of myofibers, and along with this connective tissue and intramyocellular lipids increase (Fig. 1) (St-Onge, 2005). Although sarcopenia is widely recognized it remains poorly understood and has not received appropriate attention until quite recently. Sarcopenia is often assumed to have a multi-factorial background (Adamo and Farrar, 2006; Attaix et al., 2005; Paddon-Jones and Rasmussen, 2009; Roth et al., 2006; Solomon and Bouloux, 2006). A sedentary life-style combined with periods of prolonged bed-rest during illnesses has well-established detrimental effects on muscle mass and function (Janssen et al., 2002). Skeletal muscles do not only generate the power to let us move but collectively they are a highly significant component of the systemic metabolic homeostasis machinery. The progressive loss of muscle mass with advancing age is evident in both the 'healthy' aging population and specialist patient populations. The reduced functional muscle mass is associated with increased morbidity, frailty and reduced quality of life (Baumgartner et al., 1998a). The prevalence of sarcopenia increases with about 5% per year and typically begins in the fourth decade of life (Baumgartner et al., 1998b). After the age of 50 years 1-2 % of the muscle mass is lost annually. Individuals with clinically manifest sarcopenia have 4 times greater risk of disability, three times greater risk of balance impairment, and 3 times greater risk of falling (Baumgartner et al., 1998b). Sarcopenia is the

single most common etiology to falls and fall-associated fractures in elderly.

**1.1 Regulation of muscle mass and tentative mechanisms in sarcopenia** 

Myofibers are complex structures build-up by merger of aligned myocytes (myotubes as intermediates). Thus, the myofiber has a common plasma membrane confining multiple sub domains each supported by one myonucleus. According to morphometric analysis the ratio myofiber volume to a myonucleus remains fairly constant suggesting an optimal size of the

**1. Introduction** 


## **Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia)**

Mikael Altun1, Max Grönholdt-Klein1,

Lingzhan Wang1,2 and Brun Ulfhake1

*1Department of Neuroscience, Karolinska Institutet, Stockholm, 2Department of Human Anatomy, School of Medicine, Inner Mongolia University for Nationalities, Tongliao City, 1Sweden 2P. R. China* 

## **1. Introduction**

268 Senescence

[54] Nijs M, Vanderswalmen P, Vandamme B, Segal-Bertin G, Lejeune B, Segal L, et al.

[55] Palacios I.M. and St Johnston D. Getting the message across: the intracellular

[56] Palazzo R.E., Vogel J.M. Isolation of centrosomes from Spisula solidissima oocytes.

[61] Roskelley C.D., Srebrow A., Bissell M.J. A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression. Curr Opin Cell Biol. 1995; 7(5): 736-47. [62] Rudolph KL, Chang S, Lee H, Blasco M, Gottlieb GJ, Greider C, et al. Longevity, stress response, and cancer in aging telomerasedeficient mice. Cell 1999;96:701e12. [63] Schatten G. The centrosome and its mode of inheritance: the reduction of the

[64] Schatten H, Schatten G, Mazia D, Balezon R, Simerly C. Behavior of centrosomes

[65] Shay JW,Wright WE. Ageing and cancdr: the telomere and telomerase connection.

[66] Sluiman HJ. A cladistic evaluation of the lower and higher green plants (Viridiplantae).

[67] Stuckenholz C, Kageyama Y, Kuroda MI. Guilt by association: noncoding RNAs,

[68] Tekotte H., Davis I. Intracellular mRNA localization: motors move messages. Trends

[69] Tkemaladze D., Chichinadze K. Potential role of centrioles in determining the

[70] Chichinadze K.N., Tkemaladze D.V. Centrosomal hypothesis of cellular ageing and

[71] Tkemaladze J., Chichinadze K. Centriole, Differentiation and Senescence. Rejuven Res.

[72] Tkemaladze J.V., Chichinadze K.N. Centriolar Mechanisms of Differentiation and

[73] Wianny F, Zernickab-Goets M. Specific interference with gene function by doublestranded RNA in early mouse development. Nat Cell Biol 2000;2:70e5. [74] Zilberman D, Cao X, Jacobsen SE. ARGONAUTE4 control of locusspecific siRNA accumulation and DNA and histone methylation. Science 2003;299:716e9.

differentiation. Adv Geront. 2008; 21(3): 367-371. Russian.

centrosome during gametogenesis and its restoration during fertilization. Dev.

during fertilization and cell division in mouse oocytes and sea urchin eggs. Proc

chromosome-specific proteins and dosage compensation in Drosophila. Trends

morphogenetic status of animal somatic cells (hypothesis). Cell Biol Int. 2005; 5:

Replicative Aging of Higher Animal Cells. Biochemistry (Moscow). 2005; 70(11):

[57] Pederson T. The centrosome: built on an mRNA? Nat Cell Biol. 2006; 8(7):652-654 [58] Peterson SP, Berns MW. The centriolar complex. Intern Rev Cytol 1980;64:81e106. [59] Peterson, S.P., and Berns, M.W. Evidence for centriolar region RNA functioning in spindle formation in dividing PTK2 cells. J. Cell. Sci., 1978; 34; 289-301. [60] Phillips SG, Rattner JB. Dependence of centriole formation on protein synthesis. J Cell

Hum Reprod 1996;11:2180e5.

Methods Cell Biol. 1999; 61: 35-56

614.

Biol 1976;70:9e19.

Biol. 1994; 165: 299-335.

Genet 1999;15:454e8.

2010; 13(2-3): 339-342.

370-374.

1288-1303.

Natl Acad Sci U S A 1996;83:105e9.

Plant Syst Evol 1985;149:217e32.

Genet. 2002; 18(12): 636-642.

Novartis Found Symp 2001;235:116e25.

Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection.

localization of mRNAs in higher eukaryotes. Annu Rev Cell Dev Biol. 2001; 17: 569-

One of the most characteristic features of the aged is a change in body composition and human cross-sectional and longitudinal studies consistently demonstrate gain in fat mass and decline in lean mass (Attaix et al., 2005). Skeletal muscle mass is gradually reduced through both atrophy and loss of myofibers, and along with this connective tissue and intramyocellular lipids increase (Fig. 1) (St-Onge, 2005). Although sarcopenia is widely recognized it remains poorly understood and has not received appropriate attention until quite recently. Sarcopenia is often assumed to have a multi-factorial background (Adamo and Farrar, 2006; Attaix et al., 2005; Paddon-Jones and Rasmussen, 2009; Roth et al., 2006; Solomon and Bouloux, 2006). A sedentary life-style combined with periods of prolonged bed-rest during illnesses has well-established detrimental effects on muscle mass and function (Janssen et al., 2002). Skeletal muscles do not only generate the power to let us move but collectively they are a highly significant component of the systemic metabolic homeostasis machinery. The progressive loss of muscle mass with advancing age is evident in both the 'healthy' aging population and specialist patient populations. The reduced functional muscle mass is associated with increased morbidity, frailty and reduced quality of life (Baumgartner et al., 1998a). The prevalence of sarcopenia increases with about 5% per year and typically begins in the fourth decade of life (Baumgartner et al., 1998b). After the age of 50 years 1-2 % of the muscle mass is lost annually. Individuals with clinically manifest sarcopenia have 4 times greater risk of disability, three times greater risk of balance impairment, and 3 times greater risk of falling (Baumgartner et al., 1998b). Sarcopenia is the single most common etiology to falls and fall-associated fractures in elderly.

#### **1.1 Regulation of muscle mass and tentative mechanisms in sarcopenia**

Myofibers are complex structures build-up by merger of aligned myocytes (myotubes as intermediates). Thus, the myofiber has a common plasma membrane confining multiple sub domains each supported by one myonucleus. According to morphometric analysis the ratio myofiber volume to a myonucleus remains fairly constant suggesting an optimal size of the

Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 271

The signaling events leading to myofiber atrophy converge onto members of the FOXO family of transcription factors, which in an active state induce atrophy by increased proteasome degradation of myofibrillar proteins (Bodine et al., 2001; Sacheck et al., 2007; Sacheck et al., 2004; Sandri et al., 2004; Stitt et al., 2004). Recently, it was discovered that the signaling pathway that activates FOXO also induces an increased degradation through autophagy and lysosomal degradation (Sandri et al., 2006; Solomon and Bouloux, 2006). This "atrophy program" is activated in a range of conditions such as disuse, denervation and systemic diseases (Solomon and Bouloux, 2006). Conversely, myofiber contraction and growth signals (e.g. IGF-1) acting via protein kinase B, mTOR, and pgc-1 induce synthesis of myofibrillar proteins, deactivation of FOXO and, in parallel, adaptation of the energy producing machinery (*idem*). The Activin A/myostatin pathway, acting on Act receptor IIB upstream of Smad, can activate myofibrillar proteolysis (i.e. induce muscle fiber atrophy) and depress SC proliferation (Zhou et al., 2010). This creates a regulatory link between cellular anabolism & catabolism and SC activation & deactivation which should work in

A growing body of evidence suggests that loss of muscle mass in elderly occurs through mechanisms more complex than those involved in disuse and disease atrophy in younger individuals (Altun et al., 2010; Edstrom et al., 2007; Edstrom and Ulfhake, 2005). In humans and rodents alike, a number of mechanisms have been suggested to underpin sarcopenia: 1) loss of innervation 2) disuse 3) impaired maintenance and repair including decline in endo-, para- and autocrine signaling (e.g. IGF-1) 4) systemic inflammation 5) imbalance between protein synthesis and degradation and 6) poor nutrition (Adamo and Farrar, 2006; Attaix et al., 2005; Paddon-Jones and Rasmussen, 2009; Roth et al., 2006; Solomon and Bouloux, 2006)*.*  However, awaiting more definitive evidence it remains unclear if the mechanism behind loss of muscle mass in elderly is different from that operating in muscle atrophy in young individuals. It should be noted that the progress over the past decade in our understanding changes in muscle mass in health and disease stems mainly from work done in laboratory

1. *Impaired regeneration of myonuclei from SCs.* The satellite cell pool has been reported to be reduced in aged individuals and the frequency of myonuclear apoptosis to increase, however, these changes have not been directly associated with the loss of muscle mass in sarcopenia. A reduced SC pool could be consequence of an imbalance of SC recruitment/activation and the replenishment of the SC pool (see above). Furthermore, reports suggest also that age-induced changes in the microenvironment influence utilization of the SC through negative effects on proliferation and differentiation of the SCs and that in the aged the SCs are not activated on demand as in adults (Carlson et al., 2009). Importantly, inhibition of, or failure, to activate SC may augment proliferation of other progenitor cells known to co-exist in the skeletal muscle (Christov et al., 2007). An interplay exists between myogenic cells and fibro/adipogenic cells, and myogenic cells are known to inhibit fibro/adipogenic cells in the SC niche (Rodeheffer, 2010). In animal models this has demonstrated to influence muscle regeneration i.e. the scar tissue formation and fat storage in the skeletal muscle (Fuso et

2. *Loss of innervation*. Observations in aged human skeletal muscle have shown loss of myofibers, myofiber-atrophy, a selective vulnerability of type IIa fibers and fiber-type grouping suggesting that an underlying mechanism is a progressive age-dependent denervation (Larsson, 1995). Direct observations in animal models have shown both a progressive denervation (Valdez et al., 2010a) and a dramatic increase in fibers re-

al., 2010), i.e. well-established features of the sarcopenic muscle.

concert in conditions with atrophy and hypertrophy, respectively.

animals, in particular rodents, and highlights:

domain supported by the machinery of one myonucleus. The impressive increase in muscle mass during late fetal and postnatal development but also in conditions of repair in adult muscle across life-span is dependent on recruitment of myogenic progenitors from the resident satellite cells (SC) in the stem cell niche (Kadi et al., 2005). SCs remain quiescent until activation through trophic stimuli e.g. IGF-1 and Notch-related signaling (Carlson et al., 2008). The activity of the SC pool is under the influence of several signaling pathways, enhancing or inhibiting SC proliferation, such as Wnt-Catenin, DeltaL/Jagged1-Notch or Smad2/3- TGF-/Activin/Myostatin (Carlson et al., 2008; Zammit, 2008).

Fig. 1**. Changes in fiber size and occurrence of nuclei with a central location in aged rat soleus muscle.** Eosin-Htx staining of soleus cross sections (8m) showing (A) young adult muscle where central nuclei (arrow) and very small fibers (double arrow) are infrequent. In early aging (B) fiber size becomes more irregular and the frequency of very small fibers increases (arrows). At advanced age (C) fiber size is highly irregular and centrally located nuclei are frequently occurring. Original micrographs shot with a x20 dry objective.

domain supported by the machinery of one myonucleus. The impressive increase in muscle mass during late fetal and postnatal development but also in conditions of repair in adult muscle across life-span is dependent on recruitment of myogenic progenitors from the resident satellite cells (SC) in the stem cell niche (Kadi et al., 2005). SCs remain quiescent until activation through trophic stimuli e.g. IGF-1 and Notch-related signaling (Carlson et al., 2008). The activity of the SC pool is under the influence of several signaling pathways, enhancing or inhibiting SC proliferation, such as Wnt-Catenin, DeltaL/Jagged1-Notch or

Fig. 1**. Changes in fiber size and occurrence of nuclei with a central location in aged rat soleus muscle.** Eosin-Htx staining of soleus cross sections (8m) showing (A) young adult muscle where central nuclei (arrow) and very small fibers (double arrow) are infrequent. In early aging (B) fiber size becomes more irregular and the frequency of very small fibers increases (arrows). At advanced age (C) fiber size is highly irregular and centrally located nuclei are frequently occurring. Original micrographs shot with a x20 dry objective.

Smad2/3- TGF-/Activin/Myostatin (Carlson et al., 2008; Zammit, 2008).

**A B**

**C**

The signaling events leading to myofiber atrophy converge onto members of the FOXO family of transcription factors, which in an active state induce atrophy by increased proteasome degradation of myofibrillar proteins (Bodine et al., 2001; Sacheck et al., 2007; Sacheck et al., 2004; Sandri et al., 2004; Stitt et al., 2004). Recently, it was discovered that the signaling pathway that activates FOXO also induces an increased degradation through autophagy and lysosomal degradation (Sandri et al., 2006; Solomon and Bouloux, 2006). This "atrophy program" is activated in a range of conditions such as disuse, denervation and systemic diseases (Solomon and Bouloux, 2006). Conversely, myofiber contraction and growth signals (e.g. IGF-1) acting via protein kinase B, mTOR, and pgc-1 induce synthesis of myofibrillar proteins, deactivation of FOXO and, in parallel, adaptation of the energy producing machinery (*idem*). The Activin A/myostatin pathway, acting on Act receptor IIB upstream of Smad, can activate myofibrillar proteolysis (i.e. induce muscle fiber atrophy) and depress SC proliferation (Zhou et al., 2010). This creates a regulatory link between cellular anabolism & catabolism and SC activation & deactivation which should work in concert in conditions with atrophy and hypertrophy, respectively.

A growing body of evidence suggests that loss of muscle mass in elderly occurs through mechanisms more complex than those involved in disuse and disease atrophy in younger individuals (Altun et al., 2010; Edstrom et al., 2007; Edstrom and Ulfhake, 2005). In humans and rodents alike, a number of mechanisms have been suggested to underpin sarcopenia: 1) loss of innervation 2) disuse 3) impaired maintenance and repair including decline in endo-, para- and autocrine signaling (e.g. IGF-1) 4) systemic inflammation 5) imbalance between protein synthesis and degradation and 6) poor nutrition (Adamo and Farrar, 2006; Attaix et al., 2005; Paddon-Jones and Rasmussen, 2009; Roth et al., 2006; Solomon and Bouloux, 2006)*.*  However, awaiting more definitive evidence it remains unclear if the mechanism behind loss of muscle mass in elderly is different from that operating in muscle atrophy in young individuals. It should be noted that the progress over the past decade in our understanding changes in muscle mass in health and disease stems mainly from work done in laboratory animals, in particular rodents, and highlights:


Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 273

This chapter will focus on the main cellular degradation machineries and our current understanding of how age-related changes in these systems impact skeletal muscle integrity.

Regulated proteolysis is instrumental for such diverse cellular processes as signaling, cell cycle progression, and apoptosis. Protein and organelle degradation enables recycling of the building blocks and is also an important survival response to starvation, whereby proteins are degraded to supply the organism with fuel for energy production. The accumulation of aggregates of misfolded proteins is a hallmark of cells and tissues of aged organisms, and protein aggregation occurs when damaged or partially unfolded proteins are not efficiently degraded. Disposal of proteins is in most conditions a selective and coordinated process, handled mainly by two cellular proteolytic systems, the ubiquitin-proteasomal system (UPS)

Work over the last 25 years has established the importance of regulating protein ubiquitination in a wide range of cellular functions including cell cycle control, transcriptional regulation, and diverse aspects of cell signaling. Lysine48 polyubiquitination targets proteins for degradation by the proteasome, a highly selective mechanism by which multiple cellular processes are being modulated. Collectively, the proteasome and enzymes involved in ubiquitination are referred to as the ubiquitin proteasomal system (UPS) (Fig. 3).

Fig. 3. **The ubiquitin proteasomal system.** Cartoon of the different components of the

ubiquitin-proteasomal system described in the text.

**2. Regulated proteolysis** 

and autophagy-lysosomal system (ALS).

**2.1 The ubiquitin proteasomal system (UPS)** 

expressing the embryonic isoform of myosin (eMyHC; Fig. 2) (Edstrom and Ulfhake, 2005) while the expression levels of adult MyHC isoforms decline (Altun et al., 2007b). In related work using a denervation/re-innervation animal model we have obtained evidence that the re-expression of nicotinic acetylcholine receptor subunit gamma (nAChR-) is a reliable marker for muscle denervation (Grönholdt-Klein et al., in preparation) and in animal models on sarcopenia we consistently find a dramatic increase in nAChR- expression. Earlier work also showed that spinal motoneurons innervating aged atrophic skeletal muscles show a regenerative phenotype suggesting an impaired contact with the target myofibers (Johnson et al., 1995). Combined, these observations suggest denervation as a significant component of sarcopenia.

3. *Protein degradation and proteotoxicity***.** Accumulation of damaged proteins is a hallmark of aging that is believed to reflect increased imbalance between generation and scavenging of radicals, and/or decreased ability to degrade damaged proteins (Harriman et al., 1970). A number of studies have reported on an accumulation of oxidatively damaged proteins in aged rodent muscle (Cai et al., 2004; Clavel et al., 2006) as well as increased levels of chaperones that selectively bind unfolded proteins (Clavel et al., 2006; Ferrington et al., 2005). Several groups have published observations suggesting a decreased proteasomal proteolysis as a mechanism for the buildup of worn-out and otherwise damaged proteins in aged rodent skeletal muscle (Ferrington et al., 2005), however, it is hard to reconcile such a reduced rate of proteolysis with the general loss of skeletal muscle mass characteristic of aging (sarcopenia).

Fig. 2**. Re-expression of the embryonic myosin (eMyHC) in the soleus muscle of an aged mouse.** Immunofluorescence micrograph of an 8 m thick cross-section through the mid portion of a soleus muscle from a 28 month-old C57BL/6J mouse. Light profiles often with an irregular out-line and small size indicate fibers expressing embryonic MyHC probably caused by an age-related loss of innervation. Original magnification x20.

This chapter will focus on the main cellular degradation machineries and our current understanding of how age-related changes in these systems impact skeletal muscle integrity.

## **2. Regulated proteolysis**

272 Senescence

observations suggest denervation as a significant component of sarcopenia.

skeletal muscle mass characteristic of aging (sarcopenia).

3. *Protein degradation and proteotoxicity***.** Accumulation of damaged proteins is a hallmark of aging that is believed to reflect increased imbalance between generation and scavenging of radicals, and/or decreased ability to degrade damaged proteins (Harriman et al., 1970). A number of studies have reported on an accumulation of oxidatively damaged proteins in aged rodent muscle (Cai et al., 2004; Clavel et al., 2006) as well as increased levels of chaperones that selectively bind unfolded proteins (Clavel et al., 2006; Ferrington et al., 2005). Several groups have published observations suggesting a decreased proteasomal proteolysis as a mechanism for the buildup of worn-out and otherwise damaged proteins in aged rodent skeletal muscle (Ferrington et al., 2005), however, it is hard to reconcile such a reduced rate of proteolysis with the general loss of

Fig. 2**. Re-expression of the embryonic myosin (eMyHC) in the soleus muscle of an aged mouse.** Immunofluorescence micrograph of an 8 m thick cross-section through the mid portion of a soleus muscle from a 28 month-old C57BL/6J mouse. Light profiles often with an irregular out-line and small size indicate fibers expressing embryonic MyHC probably

caused by an age-related loss of innervation. Original magnification x20.

expressing the embryonic isoform of myosin (eMyHC; Fig. 2) (Edstrom and Ulfhake, 2005) while the expression levels of adult MyHC isoforms decline (Altun et al., 2007b). In related work using a denervation/re-innervation animal model we have obtained evidence that the re-expression of nicotinic acetylcholine receptor subunit gamma (nAChR-) is a reliable marker for muscle denervation (Grönholdt-Klein et al., in preparation) and in animal models on sarcopenia we consistently find a dramatic increase in nAChR- expression. Earlier work also showed that spinal motoneurons innervating aged atrophic skeletal muscles show a regenerative phenotype suggesting an impaired contact with the target myofibers (Johnson et al., 1995). Combined, these

Regulated proteolysis is instrumental for such diverse cellular processes as signaling, cell cycle progression, and apoptosis. Protein and organelle degradation enables recycling of the building blocks and is also an important survival response to starvation, whereby proteins are degraded to supply the organism with fuel for energy production. The accumulation of aggregates of misfolded proteins is a hallmark of cells and tissues of aged organisms, and protein aggregation occurs when damaged or partially unfolded proteins are not efficiently degraded. Disposal of proteins is in most conditions a selective and coordinated process, handled mainly by two cellular proteolytic systems, the ubiquitin-proteasomal system (UPS) and autophagy-lysosomal system (ALS).

## **2.1 The ubiquitin proteasomal system (UPS)**

Work over the last 25 years has established the importance of regulating protein ubiquitination in a wide range of cellular functions including cell cycle control, transcriptional regulation, and diverse aspects of cell signaling. Lysine48 polyubiquitination targets proteins for degradation by the proteasome, a highly selective mechanism by which multiple cellular processes are being modulated. Collectively, the proteasome and enzymes involved in ubiquitination are referred to as the ubiquitin proteasomal system (UPS) (Fig. 3).

Fig. 3. **The ubiquitin proteasomal system.** Cartoon of the different components of the ubiquitin-proteasomal system described in the text.

Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 275

protein breakdown. Together these findings suggest that the enhanced capacity of the UPS may be a response to the increased generation of damaged polypeptides. Strong support for this conclusion is our finding of elevated levels of the ubiquitin ligases, CHIP and E6AP, in muscle of aged animals. CHIP ubiquitinates misfolded or mutated proteins bound to Hsp70 or Hsp90 (Connell et al., 2001), and recently a similar function in cellular quality control has been reported for E6AP (Mishra et al., 2009). Moreover, most of these conclusions about a negative impact of aging on proteasomal degradation in muscle were based exclusively on measurements of the 20S core particle. Although some degradation of unfolded or denatured proteins may occur by the free 20S (Jariel-Encontre et al., 2008), the bulk of proteasome-mediated degradation, even of oxidatively damaged proteins (Medicherla and Goldberg, 2008), seems to require the 26S proteasome and ubiquitylation of the substrate.

Fig. 4. **Degradation capacity towards different substrates of proteasomes isolated from triceps surae muscle of adult rats (Ad-AL), aged (Ag-AL) and aged rats maintained on dietary restriction (Ag-DR)** (original data reproduced from Altun et al., 2010). The higher content per unit muscle mass of proteasomes in aged (Ag-AL) muscles is paralleled by a corresponding increase in degradation of synthetic peptides designed for the chymotryptic and caspase sites of the proteasome (A) as well as casein (B). In (C) degradation of a native ubiquitinylated protein is shown illustrating that we could not detect any impairment of the degradation capacity of 26S proteasomes isolated from aged rats muscle. Further details on

experimental design in Altun at al., 2010.

However, ubiquitination does not only associate with proteasomal degradation, other forms of ubiquitination play roles in such diverse processes as transcriptional regulation, endocytosis, DNA modifications and stabilization of protein complexes (Harper and Schulman, 2006). Ubiquitin (Ub) is transcribed as precursor proteins from 4 genes and after cleavage to monomeric Ub, Ub-conjugation occurs through an enzymatic cascade where the E3-ligases (>600 E3 ligases have been identified so far) provide for target specificity. Prior to cleavage in the proteasome of a lys48 poly-Ub conjugated protein, the Ub-recognition signal is removed (Fig. 3). This serves to enhance passage of the targeted protein into the catalytic chamber and will also free ubiquitin for reuse and hinder the proteasomes to be preoccupied with destroying Ub [itself] (Hanna et al., 2007; Koulich et al., 2008). Hydrolysis of bound Ub and processing of Ub precursor proteins is accomplished by members of the large group of deubiquitinating enzymes (DUBs) (>90 members identified so far); DUBs are cysteine proteases that can be divided into distinct subfamilies based on sequence similarities and likely mechanism of action (reviewed in Nijman et al., 2005). Since DUBs can reshuffle ubiquitin from poly- to mono- or multi-ubiquitin chains, they are not only important for protein half-life but probably all cellular processes that involve ubiquitination of proteins (Clague and Urbe, 2006).

#### **2.2 Activation of the UPS in sarcopenia**

Because available data on the integrity of the UPS in the elderly was controversial we examined in 30-month old Sprague-Dawley rats the effects of aging on the content and activity of 26S proteasomes, proteasome-associated regulatory proteins and various other components of the UPS, including multiple DUBs (Altun et al., 2010). Muscles of these aged animals undergo marked atrophy compared to muscles of young adult animals and this age-related atrophy could be impeded if the animals were maintained on a restricted diet (Altun et al., 2007a). Analysis of proteasome protein content and proteasome degradation capacity in these animals revealed a 2-3 fold increase in proteasomes in the aged muscle. The increase in 26S proteasomes was suppressed completely in aged animals maintained on a restricted diet (30% of the consumption recorded for rats having free access to food). Since muscle wasting was reduced in these animals, these findings suggest that the buildup of proteasomes contributes to the loss of muscle mass in aged animals. In support of this notion we found that the levels of proteasome subunits in aged skeletal muscle were inversely correlated with muscle weight (subunits 1: r=-0.71, p<0.05; 5: r=-0.69; p<0.05), and no such inverse relationship was found in muscles of adult rats or aged animals maintained on dietary restriction (Altun et al., 2010). However, the underlying mechanism of this accumulation remains unclear. In contrast to muscles atrophying due to disuse, fasting or various systemic diseases, the age-related accumulation of proteasomes occurred without any increase in corresponding mRNAs. Thus, the accumulation of proteasomes in aged muscle must be due to enhanced subunit translation, more efficient assembly of the 26S particles, or slower degradation of the 26S particles (see below under 2.4).

Previous reports suggested that age-related decrease in the proteasome's peptidase activities were due to oxidative modifications (Bulteau et al., 2000; Conconi et al., 1996; Ferrington et al., 2005; Grune et al., 2001; Hayashi and Goto, 1998; Keller et al., 2000). However, we could not observe such defect in degrading capacity towards a range of substrates including ubiquitinated native proteins (Fig. 4). Instead, our data indicates that proteasome content increases during sarcopenia, and that these particles retain their full ability to function in

However, ubiquitination does not only associate with proteasomal degradation, other forms of ubiquitination play roles in such diverse processes as transcriptional regulation, endocytosis, DNA modifications and stabilization of protein complexes (Harper and Schulman, 2006). Ubiquitin (Ub) is transcribed as precursor proteins from 4 genes and after cleavage to monomeric Ub, Ub-conjugation occurs through an enzymatic cascade where the E3-ligases (>600 E3 ligases have been identified so far) provide for target specificity. Prior to cleavage in the proteasome of a lys48 poly-Ub conjugated protein, the Ub-recognition signal is removed (Fig. 3). This serves to enhance passage of the targeted protein into the catalytic chamber and will also free ubiquitin for reuse and hinder the proteasomes to be preoccupied with destroying Ub [itself] (Hanna et al., 2007; Koulich et al., 2008). Hydrolysis of bound Ub and processing of Ub precursor proteins is accomplished by members of the large group of deubiquitinating enzymes (DUBs) (>90 members identified so far); DUBs are cysteine proteases that can be divided into distinct subfamilies based on sequence similarities and likely mechanism of action (reviewed in Nijman et al., 2005). Since DUBs can reshuffle ubiquitin from poly- to mono- or multi-ubiquitin chains, they are not only important for protein half-life but probably all cellular processes that involve ubiquitination

Because available data on the integrity of the UPS in the elderly was controversial we examined in 30-month old Sprague-Dawley rats the effects of aging on the content and activity of 26S proteasomes, proteasome-associated regulatory proteins and various other components of the UPS, including multiple DUBs (Altun et al., 2010). Muscles of these aged animals undergo marked atrophy compared to muscles of young adult animals and this age-related atrophy could be impeded if the animals were maintained on a restricted diet (Altun et al., 2007a). Analysis of proteasome protein content and proteasome degradation capacity in these animals revealed a 2-3 fold increase in proteasomes in the aged muscle. The increase in 26S proteasomes was suppressed completely in aged animals maintained on a restricted diet (30% of the consumption recorded for rats having free access to food). Since muscle wasting was reduced in these animals, these findings suggest that the buildup of proteasomes contributes to the loss of muscle mass in aged animals. In support of this notion we found that the levels of proteasome subunits in aged skeletal muscle were inversely correlated with muscle weight (subunits 1: r=-0.71, p<0.05; 5: r=-0.69; p<0.05), and no such inverse relationship was found in muscles of adult rats or aged animals maintained on dietary restriction (Altun et al., 2010). However, the underlying mechanism of this accumulation remains unclear. In contrast to muscles atrophying due to disuse, fasting or various systemic diseases, the age-related accumulation of proteasomes occurred without any increase in corresponding mRNAs. Thus, the accumulation of proteasomes in aged muscle must be due to enhanced subunit translation, more efficient assembly of the

26S particles, or slower degradation of the 26S particles (see below under 2.4).

Previous reports suggested that age-related decrease in the proteasome's peptidase activities were due to oxidative modifications (Bulteau et al., 2000; Conconi et al., 1996; Ferrington et al., 2005; Grune et al., 2001; Hayashi and Goto, 1998; Keller et al., 2000). However, we could not observe such defect in degrading capacity towards a range of substrates including ubiquitinated native proteins (Fig. 4). Instead, our data indicates that proteasome content increases during sarcopenia, and that these particles retain their full ability to function in

of proteins (Clague and Urbe, 2006).

**2.2 Activation of the UPS in sarcopenia** 

protein breakdown. Together these findings suggest that the enhanced capacity of the UPS may be a response to the increased generation of damaged polypeptides. Strong support for this conclusion is our finding of elevated levels of the ubiquitin ligases, CHIP and E6AP, in muscle of aged animals. CHIP ubiquitinates misfolded or mutated proteins bound to Hsp70 or Hsp90 (Connell et al., 2001), and recently a similar function in cellular quality control has been reported for E6AP (Mishra et al., 2009). Moreover, most of these conclusions about a negative impact of aging on proteasomal degradation in muscle were based exclusively on measurements of the 20S core particle. Although some degradation of unfolded or denatured proteins may occur by the free 20S (Jariel-Encontre et al., 2008), the bulk of proteasome-mediated degradation, even of oxidatively damaged proteins (Medicherla and Goldberg, 2008), seems to require the 26S proteasome and ubiquitylation of the substrate.

Fig. 4. **Degradation capacity towards different substrates of proteasomes isolated from triceps surae muscle of adult rats (Ad-AL), aged (Ag-AL) and aged rats maintained on dietary restriction (Ag-DR)** (original data reproduced from Altun et al., 2010). The higher content per unit muscle mass of proteasomes in aged (Ag-AL) muscles is paralleled by a corresponding increase in degradation of synthetic peptides designed for the chymotryptic and caspase sites of the proteasome (A) as well as casein (B). In (C) degradation of a native ubiquitinylated protein is shown illustrating that we could not detect any impairment of the degradation capacity of 26S proteasomes isolated from aged rats muscle. Further details on experimental design in Altun at al., 2010.

Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 277

During muscle atrophy in adult animals induced by fasting, disuse or disease, there is a marked increase in protein ubiquitination and proteolysis, especially of myofibrillar components (Lecker et al., 1999; Mitch and Goldberg, 1996). If the rate of ubiquitination exceeds the rate of Ub-dependent proteolysis, Ub-protein will accumulate; this will also occur if proteins are damaged or rearranged (multimeric aggregates) in a way that hinders normal proteasomal degradation. Immunoblotting of muscle extracts with an anti-ubiquitin antibody reveal that the muscles from aged rats contained greater amounts of ubiquitylated proteins than those from adult rats (Fig.5B; see also (Altun et al., 2010)). An increase in free ubiquitin and ubiquitin conjugates with aging was also reported by others in rat muscles (Cai et al., 2004; Clavel et al., 2006). Since the 26S proteasomes isolated from aged muscle are fully active in degrading ubiquitylated proteins, the elevated levels of ubiquitylated proteins strongly suggest higher overall rates of protein ubiquitylation that exceed the rates of conjugate degradation or the formation of nondegradable Ub-protein aggregates. Immunohistochemical analysis (using the same antibody towards ubiquitin) show that some

Fig. 5. **Amount of ubiquitylated protein is increased in aged skeletal muscle.** (A) shows micrograph of an aged soleus muscle cut along fiber length and processed with an antibody that recognizes ubiquitylated proteins using the ABC peroxidase technique. Ub+ protein deposits appear in black (arrows) and vary in size but are usually localized to the peripheral domain of the fiber (original micrograph was taken with an x20 dry objective). (B) shows a immunoblot of muscle extract from adult, aged and aged rats maintained on dietary restriction (DR) using the same antibody as in (A). The amount of Ub-proteins is clearly

Ubiquitination is also involved in processes leading to abrogation of signal transduction and targeting of the receptors for destruction by the autophagosome lysosomal system (ALS; Fig. 6). One example is the monoubiquitin-dependent endocytosis and degradation of epidermal growth factor receptor (Haglund et al., 2003; Mizuno et al., 2005). Degradation through the LS involves not only proteins but also damaged cell organelles and large complexes (Cuervo et al., 2005; Terman and Brunk, 2006; Terman et al., 2006). Multiple vesicles that constantly fuse

higher in aged fed ad libitum, while aged rats on DR are more similar to adults.

**2.4 The autophagy lysosomal system (ALS)** 

**2.3 Accumulation of ubiquitinated proteins in sarcopenic muscles** 

of the fibers in aged skeletal muscles contain Ub+ deposits (Fig. 5A).

A decline in the cell's pool of free ubiquitin can limit the rate of proteolysis by the proteasome (Hanna et al., 2003; Kimura et al., 2009) and free ubiquitin is in turn released during degradation of ubiquitylated proteins by the DUBs associated with the 26S proteasome. Thus, in order to achieve high degradation rates, the capacity for deubiquitylation probably needs to be increased too. Using mechanism-based probes to assess the state of DUB activity and expression (Altun et al., 2010), eleven DUB-enzymes were found to be strongly up-regulated in the muscles of aged rats; including USP14 and Uch37 which are known to be associated with the 26S proteasome. USP14 and UCH37 trim off ubiquitin from the polyubiquitin chain and release ubiquitin monomers for re-use. In addition, USP14 controls gate opening into the 20S proteasome and facilitates substrate degradation (Peth et al., 2009). On the other hand, the yeast homolog of USP14, Ubp6, regulates the overall rate of proteolysis and appears critical in replenishing the free ubiquitin pool in yeast (Hanna et al., 2003; Hanna et al., 2007). Similarly, USP5 hydrolyses anchorless ubiquitin chains (Reyes-Turcu et al., 2006; Reyes-Turcu et al., 2008) and appears to work downstream of Rpn11, an intrinsic DUB on the proteasome, to prevent the binding of free ubiquitin chains to the 19S, which would inhibit proteolysis. Together these data illustrate important roles for deubiquitylation in the aged muscle, probably ensuring a supply of ubiquitin for enhanced proteolysis but perhaps also serving additional regulatory functions.

Loss of muscle mass can occur through increased protein degradation but also decreased protein synthesis or through some combination of these responses. With aging, sarcopenia develops over months to years depending on the species, unlike the rapid loss of muscle weight induced by fasting, disuse, and in various catabolic diseases, where marked atrophy (20-50% loss) can occur in rodents in several days. In these latter types of rapid atrophy, as described above, there is a common program of changes in the transcription of a set of atrophy-related genes (Lecker et al., 2006; Lecker et al., 2004; Sacheck et al., 2007). Several of the biochemical changes observed in aged atrophic muscle clearly distinguish them from those undergoing rapid atrophy in adult animals. Upon denervation or fasting, the atrophyspecific ubiquitin ligases, Atrogin-1/MAFbx and MuRF1, are induced by members of the FOXO family of transcription factors, and this induction is essential for the rapid weight loss (Bodine et al., 2001; Lecker et al., 2006; Sandri et al., 2004). Inhibition of FOXOs prevents their induction and the loss of muscle mass upon denervation, fasting, or glucocorticoid treatment (Sandri et al., 2004). In contrast, in the aged muscles, mRNAs for Atrogin-1/MAFbx, MuRF1 and the Ub-conjugating enzyme, E2-14K, were unchanged or lower than adult levels (Edstrom et al., 2006). Also, treatment with the glucocorticoid dexamethasone failed to induce Atrogin-1/MAFbx or MuRF1 or to cause muscle wasting, as it does in adult animals (Bodine et al., 2001; Gomes et al., 2001). However, MuRF1 protein increased in aged muscle, while Atrogin-1/MAFbx protein decreased (in accord with the mRNA data). Another distinction between sarcopenia and rapid atrophy is that the ubiquitin ligases, CHIP and E6AP, increased markedly in the aged muscle, though they do not rise in rapidly atrophying muscles. Their induction may reflect adaptations in the aged muscle to eliminate more efficiently misfolded proteins.

In summary, the finding of increased content of proteasomes and other UPS components (e.g. MuRF1) argues that proteolysis also increases in these muscles and may contribute to muscle wasting in the aged rats. Dietary restriction decreased levels of proteasomes and several other UPS components toward the levels in adult animal and partially inhibited the development of sarcopenia.

## **2.3 Accumulation of ubiquitinated proteins in sarcopenic muscles**

276 Senescence

A decline in the cell's pool of free ubiquitin can limit the rate of proteolysis by the proteasome (Hanna et al., 2003; Kimura et al., 2009) and free ubiquitin is in turn released during degradation of ubiquitylated proteins by the DUBs associated with the 26S proteasome. Thus, in order to achieve high degradation rates, the capacity for deubiquitylation probably needs to be increased too. Using mechanism-based probes to assess the state of DUB activity and expression (Altun et al., 2010), eleven DUB-enzymes were found to be strongly up-regulated in the muscles of aged rats; including USP14 and Uch37 which are known to be associated with the 26S proteasome. USP14 and UCH37 trim off ubiquitin from the polyubiquitin chain and release ubiquitin monomers for re-use. In addition, USP14 controls gate opening into the 20S proteasome and facilitates substrate degradation (Peth et al., 2009). On the other hand, the yeast homolog of USP14, Ubp6, regulates the overall rate of proteolysis and appears critical in replenishing the free ubiquitin pool in yeast (Hanna et al., 2003; Hanna et al., 2007). Similarly, USP5 hydrolyses anchorless ubiquitin chains (Reyes-Turcu et al., 2006; Reyes-Turcu et al., 2008) and appears to work downstream of Rpn11, an intrinsic DUB on the proteasome, to prevent the binding of free ubiquitin chains to the 19S, which would inhibit proteolysis. Together these data illustrate important roles for deubiquitylation in the aged muscle, probably ensuring a supply of ubiquitin for enhanced proteolysis but perhaps also serving

Loss of muscle mass can occur through increased protein degradation but also decreased protein synthesis or through some combination of these responses. With aging, sarcopenia develops over months to years depending on the species, unlike the rapid loss of muscle weight induced by fasting, disuse, and in various catabolic diseases, where marked atrophy (20-50% loss) can occur in rodents in several days. In these latter types of rapid atrophy, as described above, there is a common program of changes in the transcription of a set of atrophy-related genes (Lecker et al., 2006; Lecker et al., 2004; Sacheck et al., 2007). Several of the biochemical changes observed in aged atrophic muscle clearly distinguish them from those undergoing rapid atrophy in adult animals. Upon denervation or fasting, the atrophyspecific ubiquitin ligases, Atrogin-1/MAFbx and MuRF1, are induced by members of the FOXO family of transcription factors, and this induction is essential for the rapid weight loss (Bodine et al., 2001; Lecker et al., 2006; Sandri et al., 2004). Inhibition of FOXOs prevents their induction and the loss of muscle mass upon denervation, fasting, or glucocorticoid treatment (Sandri et al., 2004). In contrast, in the aged muscles, mRNAs for Atrogin-1/MAFbx, MuRF1 and the Ub-conjugating enzyme, E2-14K, were unchanged or lower than adult levels (Edstrom et al., 2006). Also, treatment with the glucocorticoid dexamethasone failed to induce Atrogin-1/MAFbx or MuRF1 or to cause muscle wasting, as it does in adult animals (Bodine et al., 2001; Gomes et al., 2001). However, MuRF1 protein increased in aged muscle, while Atrogin-1/MAFbx protein decreased (in accord with the mRNA data). Another distinction between sarcopenia and rapid atrophy is that the ubiquitin ligases, CHIP and E6AP, increased markedly in the aged muscle, though they do not rise in rapidly atrophying muscles. Their induction may reflect adaptations in the aged muscle to eliminate

In summary, the finding of increased content of proteasomes and other UPS components (e.g. MuRF1) argues that proteolysis also increases in these muscles and may contribute to muscle wasting in the aged rats. Dietary restriction decreased levels of proteasomes and several other UPS components toward the levels in adult animal and partially inhibited the

additional regulatory functions.

more efficiently misfolded proteins.

development of sarcopenia.

During muscle atrophy in adult animals induced by fasting, disuse or disease, there is a marked increase in protein ubiquitination and proteolysis, especially of myofibrillar components (Lecker et al., 1999; Mitch and Goldberg, 1996). If the rate of ubiquitination exceeds the rate of Ub-dependent proteolysis, Ub-protein will accumulate; this will also occur if proteins are damaged or rearranged (multimeric aggregates) in a way that hinders normal proteasomal degradation. Immunoblotting of muscle extracts with an anti-ubiquitin antibody reveal that the muscles from aged rats contained greater amounts of ubiquitylated proteins than those from adult rats (Fig.5B; see also (Altun et al., 2010)). An increase in free ubiquitin and ubiquitin conjugates with aging was also reported by others in rat muscles (Cai et al., 2004; Clavel et al., 2006). Since the 26S proteasomes isolated from aged muscle are fully active in degrading ubiquitylated proteins, the elevated levels of ubiquitylated proteins strongly suggest higher overall rates of protein ubiquitylation that exceed the rates of conjugate degradation or the formation of nondegradable Ub-protein aggregates. Immunohistochemical analysis (using the same antibody towards ubiquitin) show that some of the fibers in aged skeletal muscles contain Ub+ deposits (Fig. 5A).

Fig. 5. **Amount of ubiquitylated protein is increased in aged skeletal muscle.** (A) shows micrograph of an aged soleus muscle cut along fiber length and processed with an antibody that recognizes ubiquitylated proteins using the ABC peroxidase technique. Ub+ protein deposits appear in black (arrows) and vary in size but are usually localized to the peripheral domain of the fiber (original micrograph was taken with an x20 dry objective). (B) shows a immunoblot of muscle extract from adult, aged and aged rats maintained on dietary restriction (DR) using the same antibody as in (A). The amount of Ub-proteins is clearly higher in aged fed ad libitum, while aged rats on DR are more similar to adults.

## **2.4 The autophagy lysosomal system (ALS)**

Ubiquitination is also involved in processes leading to abrogation of signal transduction and targeting of the receptors for destruction by the autophagosome lysosomal system (ALS; Fig. 6). One example is the monoubiquitin-dependent endocytosis and degradation of epidermal growth factor receptor (Haglund et al., 2003; Mizuno et al., 2005). Degradation through the LS involves not only proteins but also damaged cell organelles and large complexes (Cuervo et al., 2005; Terman and Brunk, 2006; Terman et al., 2006). Multiple vesicles that constantly fuse

Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 279

Subsequently, the autophagosome fuses with a lysosome for degradation under the formation of an autophagolysosome (Fig. 6). With the formation of the AV, the Atg conjugase complexes disassociate, but the Atg 8 (the mammalian homologue is MAP-LC3) remains in the ALS until degradation. While, Atg8 may be a marker for AVs and [by its elimination also] the completion of this degradation route, the autophagy conjugase complexes indicate ongoing ALS formation/autophagy. The role of the autophagy-lysosomal system was not widely recognized until it was demonstrated that cathepsins (D and B+L, respectively; (Felbor et al., 2002)) and autophagy (Hara et al., 2006) were non-redundant for a normal development and that cells rely on a basal level of autophagy to keep them free of worn-out organelles and

There is solid evidence for the engagement of autophagy in major human degenerative diseases and in normal aging. A decreased capacity of the lysosomes to degrade waste will cause a build-up of damaged organelles and proteins disturbing cell homeostasis; and e.g. accumulation of dysfunctional mitochondria in aging cardiomyocytes, myofibers and neurons has been reported. Recently, several studies have shown that the induction of autophagy, by intervention of the above discussed signaling pathways, increase cellular clearance of aberrant proteins also when the normal degradation route operates via the proteasome or through CMA. The load on the ALS in normal aging is evident from the dramatic accumulation of lipofuscin by time (Fig. 7 G-H) and the concomitant upregulation of markers of lysosomal proteolysis. Lipofuscin forms due to iron-catalyzed intralysosomal peroxidation and its accumulation in the lysosomal compartment seems to set down the capacity of autophagy (Brunk and Terman, 2002). Oxidative stress, even mild stress, accelerates the formation/buildup of lipofuscin (Kurz et al., 2008) and stress can cause lysosomes to become leaky due to intralysosomal reactive iron mediated Fenton- reactions with production of hydroxyl radicals and resulting labilization of the lysosomal membrane. Even though the pH is suboptimal, several of the lysosomal enzymes are apparently proteolytically active if released to the cytosol

Analysis of atrophic skeletal muscle in aged rats (Altun et al., 2007b) revealed increased levels of molecules involved in iron transport (transferrin), binding (ferritin), as well as iron response element binding protein-1 activity; combined these observations suggest increased load on the iron-handling machinery. Measurements of iron levels revealed a significant accumulation in the aged skeletal muscle, providing further support for iron loading in senescence. Iron-loading increases the risk for common hallmarks of aging such as DNA damage, protein oxidation and misfolding, and lipid peroxidation and may accelerate the accumulation of lipofuscin (see above). Iron-load is not exclusive to aging-related muscle wasting, but is also evident in experimental disuse atrophy (Kondo et al., 1992) and iron-restriction has been shown to be

Until recently the UPS and the ALS were considered as independent pathways for protein degradation. The UPS offers a fast and highly specific mechanism to remove selected proteins. However, the targeted protein must be a monomer and unfolded to be able to enter the proteolytic lumen of the proteasome. The ALS represents a partly overlapping,

aberrant proteins.

**2.5 Signs of distress of the ALS during aging** 

and may then trigger cell degeneration/death (Chu, 2006).

beneficiary in certain inherited myopathies (Bornman et al., 1998).

**3. Cross talk between the UPS and the ALS** 

and fission constitute the lysosomal system. Lysosomal degradation is initiated through several pathways based on the substrate delivering mechanism (Fig. 6): Endocytosis (clathrin mediated pinching-off of membrane patches with/without inclusion of extracellular material) occurs and the resulting particle is referred to as an endosomes (carrying rab5). Endosomes can then develop into late endosomes, which are equipped with enzymes delivered by secretory vesicles from the trans-Golgi-network and sorted by mannose-6-phosphate receptors, which are markers for late endosomes. Late endosomes may also fuse with lysosomes (then they lose the mannose-6-phosphate receptors).

A second route is chaperone mediated autophagy (CMA), which targets proteins carrying a pentapeptide (KFERQ) motif that is recognized by the chaperon Hsc73 and through binding to the LAMP-2A receptor (lysosome associated membrane protein 2A; a marker of the lysosome), the targeted protein is taken up and degraded in the lysosomal lumen (Fig. 6). The major route for organelles and proteins targeted for lysosomal degradation is via autophagy, a process controlled by the autophagy-conjugase complexes Atg12-Atg5 and Atg8-Atg3 (Atg, autophagy related genes) whereby in-bulk cytoplasm becomes entrapped in a double membrane forming an autophagosome (autophagic vacuole, AV) (Terman and Brunk, 2004).

Fig. 6**. Schematic drawing of the autophagy-lysosomal system.** The different cargo routes to lysosomal degradation are illustrated and described in the text.

Subsequently, the autophagosome fuses with a lysosome for degradation under the formation of an autophagolysosome (Fig. 6). With the formation of the AV, the Atg conjugase complexes disassociate, but the Atg 8 (the mammalian homologue is MAP-LC3) remains in the ALS until degradation. While, Atg8 may be a marker for AVs and [by its elimination also] the completion of this degradation route, the autophagy conjugase complexes indicate ongoing ALS formation/autophagy. The role of the autophagy-lysosomal system was not widely recognized until it was demonstrated that cathepsins (D and B+L, respectively; (Felbor et al., 2002)) and autophagy (Hara et al., 2006) were non-redundant for a normal development and that cells rely on a basal level of autophagy to keep them free of worn-out organelles and aberrant proteins.

## **2.5 Signs of distress of the ALS during aging**

278 Senescence

and fission constitute the lysosomal system. Lysosomal degradation is initiated through several pathways based on the substrate delivering mechanism (Fig. 6): Endocytosis (clathrin mediated pinching-off of membrane patches with/without inclusion of extracellular material) occurs and the resulting particle is referred to as an endosomes (carrying rab5). Endosomes can then develop into late endosomes, which are equipped with enzymes delivered by secretory vesicles from the trans-Golgi-network and sorted by mannose-6-phosphate receptors, which are markers for late endosomes. Late endosomes may also fuse with

A second route is chaperone mediated autophagy (CMA), which targets proteins carrying a pentapeptide (KFERQ) motif that is recognized by the chaperon Hsc73 and through binding to the LAMP-2A receptor (lysosome associated membrane protein 2A; a marker of the lysosome), the targeted protein is taken up and degraded in the lysosomal lumen (Fig. 6). The major route for organelles and proteins targeted for lysosomal degradation is via autophagy, a process controlled by the autophagy-conjugase complexes Atg12-Atg5 and Atg8-Atg3 (Atg, autophagy related genes) whereby in-bulk cytoplasm becomes entrapped in a double membrane forming an autophagosome (autophagic vacuole, AV) (Terman and Brunk, 2004).

Fig. 6**. Schematic drawing of the autophagy-lysosomal system.** The different cargo routes

to lysosomal degradation are illustrated and described in the text.

lysosomes (then they lose the mannose-6-phosphate receptors).

There is solid evidence for the engagement of autophagy in major human degenerative diseases and in normal aging. A decreased capacity of the lysosomes to degrade waste will cause a build-up of damaged organelles and proteins disturbing cell homeostasis; and e.g. accumulation of dysfunctional mitochondria in aging cardiomyocytes, myofibers and neurons has been reported. Recently, several studies have shown that the induction of autophagy, by intervention of the above discussed signaling pathways, increase cellular clearance of aberrant proteins also when the normal degradation route operates via the proteasome or through CMA. The load on the ALS in normal aging is evident from the dramatic accumulation of lipofuscin by time (Fig. 7 G-H) and the concomitant upregulation of markers of lysosomal proteolysis. Lipofuscin forms due to iron-catalyzed intralysosomal peroxidation and its accumulation in the lysosomal compartment seems to set down the capacity of autophagy (Brunk and Terman, 2002). Oxidative stress, even mild stress, accelerates the formation/buildup of lipofuscin (Kurz et al., 2008) and stress can cause lysosomes to become leaky due to intralysosomal reactive iron mediated Fenton- reactions with production of hydroxyl radicals and resulting labilization of the lysosomal membrane. Even though the pH is suboptimal, several of the lysosomal enzymes are apparently proteolytically active if released to the cytosol and may then trigger cell degeneration/death (Chu, 2006).

Analysis of atrophic skeletal muscle in aged rats (Altun et al., 2007b) revealed increased levels of molecules involved in iron transport (transferrin), binding (ferritin), as well as iron response element binding protein-1 activity; combined these observations suggest increased load on the iron-handling machinery. Measurements of iron levels revealed a significant accumulation in the aged skeletal muscle, providing further support for iron loading in senescence. Iron-loading increases the risk for common hallmarks of aging such as DNA damage, protein oxidation and misfolding, and lipid peroxidation and may accelerate the accumulation of lipofuscin (see above). Iron-load is not exclusive to aging-related muscle wasting, but is also evident in experimental disuse atrophy (Kondo et al., 1992) and iron-restriction has been shown to be beneficiary in certain inherited myopathies (Bornman et al., 1998).

## **3. Cross talk between the UPS and the ALS**

Until recently the UPS and the ALS were considered as independent pathways for protein degradation. The UPS offers a fast and highly specific mechanism to remove selected proteins. However, the targeted protein must be a monomer and unfolded to be able to enter the proteolytic lumen of the proteasome. The ALS represents a partly overlapping,

Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 281

As in the aged rat muscles, the accumulation of proteasomes following lysosomal inhibition was not induced by a transcriptional up-regulation (data not shown) and therefore results likely from a reduced clearance of proteasomes due to an impairment of lysosomal function. Consistent with this, we observed lipofuscin accumulation in the aged rat muscle tissue (Fig.7 G and H). In addition, Cuervo and colleagues reported that autophagy declines in liver with aging but this effect is reduced by dietary restriction (Hanna et al., 2003; Watts et al., 2004). Activation of autophagy in muscle by dietary restriction could explain our findings that this regime prevented the increase in proteasomes with age without any change in proteasome mRNA levels. Combined these observations suggest the ALS as a

candidate pathway for proteasomal degradation awaiting more definitive evidence.

myofibers may be at risk to enter a state of proteotoxicity.

Ulfhake, 2005)). Still, regeneration fails and tissue atrophy progresses.

Human cross-sectional and longitudinal studies have consistently demonstrated a gain in fat mass and decline in lean mass during aging. Although aging-related muscle wasting, or sarcopenia, is widely recognized it still remains poorly understood. The reduced functional muscle mass is associated with increased morbidity and reduced quality of life. To maintain integrity muscle myofibers rely on degradation pathways to keep clean from worn-out organelles and damaged proteins. Our current understanding is that the autophagylysosomal system is in distress possibly driven by an age-dependent accumulation of reactive iron, while contrary to the widespread view, the complementary pathway for degradation of proteins, the UPS, is enhanced. The age-dependent increase in the muscle specific ubiquitin-ligase MuRF and ubiquitin-dependent proteasomal proteolysis is expected to occur in the progression of sarcopenia since this route is nonredundant for degradation of myofibrillar proteins. In addition, the UPS in aged skeletal muscle shows adaptations to an increased demand on degradation of aberrant proteins and an accumulation of ubiquitinated proteins. Combined these stigmata suggest that aged

Normal skeletal muscles have a good capacity to regenerate following wasting conditions such as disuse. The muscle regenerative response relies on signaling that evokes satellite cell replication and asymmetric division generating offsprings that will differentiate to myocytes via the myoblast stage. Such cells are then incorporated into the myofiber allowing it to grow (for example in response to an exercise stimulus). Poor capacity to regenerate muscle tissue at advanced age may depend on impaired signaling, exhaustion of the SC pool or changes in the extracellular matrix or stem-cell niche impeding the regeneration and incorporation of myoblasts into existing/regenerating myofibers (for references see Introduction). Assessments of the regenerative drive in aged sarcopenic muscle have, however, shown that myogenic differentiation factors are upregulated and that there are overt signs of incorporation of new nuclei into existing fibers (Fig. 1B,C; (Edstrom and

The triggering mechanism for the age-dependent fiber atrophy and fiber loss remains enigmatic. However, several lines of evidence converge towards support of the "neurogenic" theory (Gutman and Hanzlikova, 1972), which stipulates that sarcopenia is driven by a successive drop-out of motoneurons. Early evidence in favor of this theory was the observation of fiber-type grouping and also histological examination revealing regressive changes at the neuro-muscular junctions. The strongest argument against this theory is the absence of unbiased evidence of a significant age-dependent loss of

**4. Concluding remarks** 

partly complementary degradation route, taking care of folded and aggregated proteins as well as large complexes and organelles with heterogenic building blocks (carbohydrates, lipids and proteins). However, several lines of evidence indicate intersections between these two pathways. In neurons treated with proteasome inhibitors, aggregate-prone proteins normally degraded by the UPS are degraded by autophagy (Cuervo et al., 2004). Two recent papers also provide evidence that induction of the UPS and LS may occur via a common pathway in skeletal muscle (Mammucari et al., 2007; Zhou et al., 2010).

Several studies report that with increasing age the capacity for lysosomal proteolysis is reduced in postmitotic cells including myofibers, while the capacity to degrade proteins through the UPS is enhanced (see above). Given that transcription of proteasome subunits and the expression of proteins involved in the assembly of the 20S were unaltered in in aged rodents, we hypothesized that proteasome particles accumulate in muscles during aging because they are not degraded at the same rate as in young adults. It is currently unknown which mechanism is responsible for the degradation of proteasomes, although the lysosomal pathway was suggested to accomplish this task in hepatocytes (Cuervo et al., 1995). To test whether this mechanism may be present in skeletal muscle cells, we treated adult rats (Fig.7A-C) and rat muscle derived cell line L6, respectively, with the lysosomal inhibitor chloroquine followed by quantitation of proteasome levels and activity (Fig.7A-F). In these experiments, we observed an accumulation of proteasomes accompanied by increased active site labeling in the muscle extracts from both chloroquine treated animals and L6 cell extracts.

Fig. 7**. Compromised lysosomal proteolysis increases proteasome content and activity.**  (**A-C**) Adult animals treated with chloroquine (50mg/kg) for 16 days (1 injection/day). (**A**) Immunoblot against proteasome subunit b5 to assay proteasome content (top) and proteasome labeling (bottom) using the active site directed probe DansylAhx3L3VS. (**B**) Individual bands were quantified for b5 content and (**C**) active site directed probe DansylAhx3L3VS labeling of the b1, b2 and b5 catalytic subunits. (**D-F**) Rat muscle derived cell line L6 was treated when confluence reached about 70% with 50mM NH4Cl or 100mM chloroquine for 24 hour before lysis. Immunoblot against proteasome subunit b5 to assay proteasome content (**D** top panel**, E**) and active site labeling of the b1, b2 and b5 catalytic subunits (**D** lower panel**, F**). b-actin was used as loading control for immunoblotting (data not shown). All error bars are standard deviations. Statistical significance: \* p<0.05, \*\* p<0.01 and \*\*\* p<0.001. (**G, H**) Lipofuscin accumulation in aged skeletal muscle. Unstained longitudinal sections of soleus muscle form adult (A) and aged (B) rats show increased content of lipofuscin autofluorescence in aged sarcopenic muscle. The bar represents 100m

As in the aged rat muscles, the accumulation of proteasomes following lysosomal inhibition was not induced by a transcriptional up-regulation (data not shown) and therefore results likely from a reduced clearance of proteasomes due to an impairment of lysosomal function. Consistent with this, we observed lipofuscin accumulation in the aged rat muscle tissue (Fig.7 G and H). In addition, Cuervo and colleagues reported that autophagy declines in liver with aging but this effect is reduced by dietary restriction (Hanna et al., 2003; Watts et al., 2004). Activation of autophagy in muscle by dietary restriction could explain our findings that this regime prevented the increase in proteasomes with age without any change in proteasome mRNA levels. Combined these observations suggest the ALS as a candidate pathway for proteasomal degradation awaiting more definitive evidence.

## **4. Concluding remarks**

280 Senescence

partly complementary degradation route, taking care of folded and aggregated proteins as well as large complexes and organelles with heterogenic building blocks (carbohydrates, lipids and proteins). However, several lines of evidence indicate intersections between these two pathways. In neurons treated with proteasome inhibitors, aggregate-prone proteins normally degraded by the UPS are degraded by autophagy (Cuervo et al., 2004). Two recent papers also provide evidence that induction of the UPS and LS may occur via a common

Several studies report that with increasing age the capacity for lysosomal proteolysis is reduced in postmitotic cells including myofibers, while the capacity to degrade proteins through the UPS is enhanced (see above). Given that transcription of proteasome subunits and the expression of proteins involved in the assembly of the 20S were unaltered in in aged rodents, we hypothesized that proteasome particles accumulate in muscles during aging because they are not degraded at the same rate as in young adults. It is currently unknown which mechanism is responsible for the degradation of proteasomes, although the lysosomal pathway was suggested to accomplish this task in hepatocytes (Cuervo et al., 1995). To test whether this mechanism may be present in skeletal muscle cells, we treated adult rats (Fig.7A-C) and rat muscle derived cell line L6, respectively, with the lysosomal inhibitor chloroquine followed by quantitation of proteasome levels and activity (Fig.7A-F). In these experiments, we observed an accumulation of proteasomes accompanied by increased active site labeling in

pathway in skeletal muscle (Mammucari et al., 2007; Zhou et al., 2010).

the muscle extracts from both chloroquine treated animals and L6 cell extracts.

Fig. 7**. Compromised lysosomal proteolysis increases proteasome content and activity.**  (**A-C**) Adult animals treated with chloroquine (50mg/kg) for 16 days (1 injection/day). (**A**) Immunoblot against proteasome subunit b5 to assay proteasome content (top) and proteasome labeling (bottom) using the active site directed probe DansylAhx3L3VS. (**B**) Individual bands were quantified for b5 content and (**C**) active site directed probe DansylAhx3L3VS labeling of the b1, b2 and b5 catalytic subunits. (**D-F**) Rat muscle derived cell line L6 was treated when confluence reached about 70% with 50mM NH4Cl or 100mM chloroquine for 24 hour before lysis. Immunoblot against proteasome subunit b5 to assay proteasome content (**D** top panel**, E**) and active site labeling of the b1, b2 and b5 catalytic subunits (**D** lower panel**, F**). b-actin was used as loading control for immunoblotting (data not shown). All error bars are standard deviations. Statistical significance: \* p<0.05, \*\* p<0.01

and \*\*\* p<0.001. (**G, H**) Lipofuscin accumulation in aged skeletal muscle. Unstained longitudinal sections of soleus muscle form adult (A) and aged (B) rats show increased content of lipofuscin autofluorescence in aged sarcopenic muscle. The bar represents 100m Human cross-sectional and longitudinal studies have consistently demonstrated a gain in fat mass and decline in lean mass during aging. Although aging-related muscle wasting, or sarcopenia, is widely recognized it still remains poorly understood. The reduced functional muscle mass is associated with increased morbidity and reduced quality of life. To maintain integrity muscle myofibers rely on degradation pathways to keep clean from worn-out organelles and damaged proteins. Our current understanding is that the autophagylysosomal system is in distress possibly driven by an age-dependent accumulation of reactive iron, while contrary to the widespread view, the complementary pathway for degradation of proteins, the UPS, is enhanced. The age-dependent increase in the muscle specific ubiquitin-ligase MuRF and ubiquitin-dependent proteasomal proteolysis is expected to occur in the progression of sarcopenia since this route is nonredundant for degradation of myofibrillar proteins. In addition, the UPS in aged skeletal muscle shows adaptations to an increased demand on degradation of aberrant proteins and an accumulation of ubiquitinated proteins. Combined these stigmata suggest that aged myofibers may be at risk to enter a state of proteotoxicity.

Normal skeletal muscles have a good capacity to regenerate following wasting conditions such as disuse. The muscle regenerative response relies on signaling that evokes satellite cell replication and asymmetric division generating offsprings that will differentiate to myocytes via the myoblast stage. Such cells are then incorporated into the myofiber allowing it to grow (for example in response to an exercise stimulus). Poor capacity to regenerate muscle tissue at advanced age may depend on impaired signaling, exhaustion of the SC pool or changes in the extracellular matrix or stem-cell niche impeding the regeneration and incorporation of myoblasts into existing/regenerating myofibers (for references see Introduction). Assessments of the regenerative drive in aged sarcopenic muscle have, however, shown that myogenic differentiation factors are upregulated and that there are overt signs of incorporation of new nuclei into existing fibers (Fig. 1B,C; (Edstrom and Ulfhake, 2005)). Still, regeneration fails and tissue atrophy progresses.

The triggering mechanism for the age-dependent fiber atrophy and fiber loss remains enigmatic. However, several lines of evidence converge towards support of the "neurogenic" theory (Gutman and Hanzlikova, 1972), which stipulates that sarcopenia is driven by a successive drop-out of motoneurons. Early evidence in favor of this theory was the observation of fiber-type grouping and also histological examination revealing regressive changes at the neuro-muscular junctions. The strongest argument against this theory is the absence of unbiased evidence of a significant age-dependent loss of

Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 283

Bornman, L., Rossouw, H., Gericke, G.S., and Polla, B.S. (1998). Effects of iron deprivation

Brunk, U.T., and Terman, A. (2002). Lipofuscin: mechanisms of age-related accumulation

Bulteau, A.L., Petropoulos, I., and Friguet, B. (2000). Age-related alterations of proteasome structure and function in aging epidermis. Exp Gerontol *35*, 767-777. Cai, D., Lee, K.K., Li, M., Tang, M.K., and Chan, K.M. (2004). Ubiquitin expression is up-

Carlson, M.E., Silva, H.S., and Conboy, I.M. (2008). Aging of signal transduction pathways,

Carlson, M.E., Suetta, C., Conboy, M.J., Aagaard, P., Mackey, A., Kjaer, M., and Conboy, I.

Christov, C., Chretien, F., Abou-Khalil, R., Bassez, G., Vallet, G., Authier, F.J., Bassaglia, Y.,

Chu, C.T. (2006). Autophagic stress in neuronal injury and disease. J Neuropathol Exp

Clague, M.J., and Urbe, S. (2006). Endocytosis: the DUB version. Trends Cell Biol *16*, 551-559. Clavel, S., Coldefy, A.S., Kurkdjian, E., Salles, J., Margaritis, I., and Derijard, B. (2006).

Conconi, M., Szweda, L.I., Levine, R.L., Stadtman, E.R., and Friguet, B. (1996). Age-related

inactivation by heat-shock protein 90. Arch Biochem Biophys *331*, 232-240. Connell, P., Ballinger, C.A., Jiang, J., Wu, Y., Thompson, L.J., Hohfeld, J., and Patterson, C.

Cuervo, A.M., Bergamini, E., Brunk, U.T., Droge, W., Ffrench, M., and Terman, A. (2005).

Cuervo, A.M., Palmer, A., Rivett, A.J., and Knecht, E. (1995). Degradation of proteasomes by

Cuervo, A.M., Stefanis, L., Fredenburg, R., Lansbury, P.T., and Sulzer, D. (2004). Impaired

Edstrom, E., Altun, M., Bergman, E., Johnson, H., Kullberg, S., Ramirez-Leon, V., and

Edstrom, E., Altun, M., Hagglund, M., and Ulfhake, B. (2006). Atrogin-1/MAFbx and

Edstrom, E., and Ulfhake, B. (2005). Sarcopenia is not due to lack of regenerative drive in

Felbor, U., Kessler, B., Mothes, W., Goebel, H.H., Ploegh, H.L., Bronson, R.T., and Olsen,

rat Tibialis Anterior muscle. Mech Ageing Dev *127*, 794-801.

and influence on cell function. Free Radic Biol Med *33*, 611-619.

dystrophy. Biochem Pharmacol *56*, 751-757.

and pathology. Exp Cell Res *314*, 1951-1961.

shock proteins. Nat Cell Biol *3*, 93-96.

lysosomes in rat liver. Eur J Biochem *227*, 792-800.

sarcopenia during aging. Physiol Behav *92*, 129-135.

senescent skeletal muscle. Aging Cell *4*, 65-77.

Proc Natl Acad Sci U S A *99*, 7883-7888.

*425*, 42-50.

Med *1*, 381-391.

Neurol *65*, 423-432.

131-140.

*305*, 1292-1295.

Biol Sci Med Sci *61*, 663-674.

on the pathology and stress protein expression in murine X-linked muscular

regulated in human and rat skeletal muscles during aging. Arch Biochem Biophys

(2009). Molecular aging and rejuvenation of human muscle stem cells. EMBO Mol

Shinin, V., Tajbakhsh, S., Chazaud, B.*, et al.* (2007). Muscle satellite cells and endothelial cells: close neighbors and privileged partners. Mol Biol Cell *18*, 1397-1409.

Atrophy-related ubiquitin ligases, atrogin-1 and MuRF1 are up-regulated in aged

decline of rat liver multicatalytic proteinase activity and protection from oxidative

(2001). The co-chaperone CHIP regulates protein triage decisions mediated by heat-

Autophagy and aging: the importance of maintaining "clean" cells. Autophagy *1*,

degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science

Ulfhake, B. (2007). Factors contributing to neuromuscular impairment and

MuRF1 are downregulated in aging-related loss of skeletal muscle. J Gerontol A

B.R. (2002). Neuronal loss and brain atrophy in mice lacking cathepsins B and L.

motoneurons. However, as discussed at length elsewhere (Johnson et al., 1995) the denervation may be a peripheral process primarily involving subdomains of the motor axon's terminal arborization within a motor unit (see also (Valdez et al., 2010b)). Failure to maintain the distant axon arbor could cause branches and terminals to degenerate leaving myofibers vacated from innervation. As this process progresses more and more fibers will be denervated and read-outs validating this process are the increase in expression of the nAChR- subunit (Gronholdt-Klein et al., in preparation) and embryonic myosin (Edstrom and Ulfhake, 2005); and it should be noted that these proteins are re-expressed by young adult myofibers upon denervation. As denervation becomes significant, it will trigger the atrophy program by which MuRF1 and other enzymes will accelerate proteasomal myofibrillar protein degradation (see above). Recently, further direct histological evidence for this process was obtained by Valdez and coworkers (Valdez et al., 2010b). These authors also showed that exercise and dietary restriction slow-down the progression of agedependent denervation, at least the latter observation is consistent with our biochemical data on the UPS (see above). It will be important to seek proof of principle for the neurogenic theory in experimental animal models followed by validation in humans since it will impact the development of strategies to impede sarcopenia in humans.

#### **5. Acknowledgements**

This work was supported by a grant from the Swedish research council (VR 10820, to B. Ulfhake), Swedish research council post doc grant to M. Altun and a guest researcher stipendship from the PR China government to L. Wang.

#### **6. References**


motoneurons. However, as discussed at length elsewhere (Johnson et al., 1995) the denervation may be a peripheral process primarily involving subdomains of the motor axon's terminal arborization within a motor unit (see also (Valdez et al., 2010b)). Failure to maintain the distant axon arbor could cause branches and terminals to degenerate leaving myofibers vacated from innervation. As this process progresses more and more fibers will be denervated and read-outs validating this process are the increase in expression of the nAChR- subunit (Gronholdt-Klein et al., in preparation) and embryonic myosin (Edstrom and Ulfhake, 2005); and it should be noted that these proteins are re-expressed by young adult myofibers upon denervation. As denervation becomes significant, it will trigger the atrophy program by which MuRF1 and other enzymes will accelerate proteasomal myofibrillar protein degradation (see above). Recently, further direct histological evidence for this process was obtained by Valdez and coworkers (Valdez et al., 2010b). These authors also showed that exercise and dietary restriction slow-down the progression of agedependent denervation, at least the latter observation is consistent with our biochemical data on the UPS (see above). It will be important to seek proof of principle for the neurogenic theory in experimental animal models followed by validation in humans since it

will impact the development of strategies to impede sarcopenia in humans.

impairments of the aging rat. Physiol Behav *92*, 911-923.

skeletal muscle of aged rats. Muscle Nerve *36*, 223-233.

catabolic periods. Int J Biochem Cell Biol *37*, 1962-1973.

elderly in New Mexico. Am J Epidemiol *147*, 755-763.

stipendship from the PR China government to L. Wang.

Chem *285*, 39597-39608.

This work was supported by a grant from the Swedish research council (VR 10820, to B. Ulfhake), Swedish research council post doc grant to M. Altun and a guest researcher

Adamo, M.L., and Farrar, R.P. (2006). Resistance training, and IGF involvement in the maintenance of muscle mass during the aging process. Ageing Res Rev *5*, 310-331. Altun, M., Bergman, E., Edstrom, E., Johnson, H., and Ulfhake, B. (2007a). Behavioral

Altun, M., Besche, H.C., Overkleeft, H.S., Piccirillo, R., Edelmann, M.J., Kessler, B.M.,

Altun, M., Edstrom, E., Spooner, E., Flores-Moralez, A., Bergman, E., Tollet-Egnell, P.,

Attaix, D., Mosoni, L., Dardevet, D., Combaret, L., Mirand, P.P., and Grizard, J. (2005).

Baumgartner, R.N., Koehler, K.M., Gallagher, D., Romero, L., Heymsfield, S.B., Ross, R.R.,

Bodine, S.C., Latres, E., Baumhueter, S., Lai, V.K., Nunez, L., Clarke, B.A., Poueymirou,

ligases required for skeletal muscle atrophy. Science *294*, 1704-1708.

elderly in New Mexico. American Journal of Epidemiology *147*, 755-763. Baumgartner, R.N., Koehler, K.M., Gallagher, D., Romero, L., Heymsfield, S.B., Ross, R.R.,

Goldberg, A.L., and Ulfhake, B. (2010). Muscle wasting in aged, sarcopenic rats is associated with enhanced activity of the ubiquitin proteasome pathway. J Biol

Norstedt, G., Kessler, B.M., and Ulfhake, B. (2007b). Iron load and redox stress in

Altered responses in skeletal muscle protein turnover during aging in anabolic and

Garry, P.J., and Lindeman, R.D. (1998a). Epidemiology of sarcopenia among the

Garry, P.J., and Lindeman, R.D. (1998b). Epidemiology of sarcopenia among the

W.T., Panaro, F.J., Na, E., Dharmarajan, K.*, et al.* (2001). Identification of ubiquitin

**5. Acknowledgements** 

**6. References** 


Cellular Degradation Machineries in Age-Related Loss of Muscle Mass (Sarcopenia) 285

Kondo, H., Miura, M., Kodama, J., Ahmed, S.M., and Itokawa, Y. (1992). Role of iron in

Koulich, E., Li, X., and DeMartino, G.N. (2008). Relative structural and functional roles of

Kurz, T., Terman, A., Gustafsson, B., and Brunk, U.T. (2008). Lysosomes in iron metabolism,

Larsson, L. (1995). Motor units: remodeling in aged animals. J Gerontol A Biol Sci Med Sci *50* 

Lecker, S.H., Goldberg, A.L., and Mitch, W.E. (2006). Protein degradation by the ubiquitin-

Lecker, S.H., Jagoe, R.T., Gilbert, A., Gomes, M., Baracos, V., Bailey, J., Price, S.R., Mitch,

Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P., Burden,

Medicherla, B., and Goldberg, A.L. (2008). Heat shock and oxygen radicals stimulate

Mishra, A., Godavarthi, S.K., Maheshwari, M., Goswami, A., and Jana, N.R. (2009). The

Mitch, W.E., and Goldberg, A.L. (1996). Mechanisms of muscle wasting. The role of the

Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and Komada, M. (2005).

Paddon-Jones, D., and Rasmussen, B.B. (2009). Dietary protein recommendations and the prevention of sarcopenia. Curr Opin Clin Nutr Metab Care *12*, 86-90. Peth, A., Besche, H.C., and Goldberg, A.L. (2009). Ubiquitinated proteins activate the

Reyes-Turcu, F.E., Horton, J.R., Mullally, J.E., Heroux, A., Cheng, X., and Wilkinson, K.D.

Reyes-Turcu, F.E., Shanks, J.R., Komander, D., and Wilkinson, K.D. (2008). Recognition of

Rodeheffer, M.S. (2010). Tipping the scale: muscle versus fat. Nat Cell Biol *12*, 102-104.

mediated deubiquitination at endosomes. Mol Biol Cell *16*, 5163-5174. Nijman, S.M., Luna-Vargas, M.P., Velds, A., Brummelkamp, T.R., Dirac, A.M., Sixma, T.K.,

a common program of changes in gene expression. Faseb J *18*, 39-51. Lecker, S.H., Solomon, V., Mitch, W.E., and Goldberg, A.L. (1999). Muscle protein

ageing and apoptosis. Histochem Cell Biol *129*, 389-406.

and disease states. J Nutr *129*, 227S-237S.

skeletal muscle in vivo. Cell Metab *6*, 458-471.

misfolded proteins. J Biol Chem *284*, 10537-10545.

motif of unanchored ubiquitin. Cell *124*, 1197-1208.

ubiquitin-proteasome pathway. N Engl J Med *335*, 1897-1905.

295-297.

*Spec No*, 91-95.

*182*, 663-673.

enzymes. Cell *123*, 773-786.

T. J Biol Chem *283*, 19581-19592.

cell *36*, 794-804.

Mol Biol Cell *19*, 1072-1082.

oxidative stress in skeletal muscle atrophied by immobilization. Pflugers Arch *421*,

multiple deubiquitylating proteins associated with mammalian 26S proteasome.

proteasome pathway in normal and disease states. J Am Soc Nephrol *17*, 1807-1819.

W.E., and Goldberg, A.L. (2004). Multiple types of skeletal muscle atrophy involve

breakdown and the critical role of the ubiquitin-proteasome pathway in normal

S.J., Di Lisi, R., Sandri, C., Zhao, J.*, et al.* (2007). FoxO3 controls autophagy in

ubiquitin-dependent degradation mainly of newly synthesized proteins. J Cell Biol

ubiquitin ligase E6-AP is induced and recruited to aggresomes in response to proteasome inhibition and may be involved in the ubiquitination of Hsp70-bound

Regulation of epidermal growth factor receptor down-regulation by UBPY-

and Bernards, R. (2005). A genomic and functional inventory of deubiquitinating

proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Molecular

(2006). The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine

polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase


Ferrington, D.A., Husom, A.D., and Thompson, L.V. (2005). Altered proteasome structure,

Fuso, A., Ferraguti, G., Grandoni, F., Ruggeri, R., Scarpa, S., Strom, R., and Lucarelli, M.

Gomes, M.D., Lecker, S.H., Jagoe, R.T., Navon, A., and Goldberg, A.L. (2001). Atrogin-1, a

Grune, T., Shringarpure, R., Sitte, N., and Davies, K. (2001). Age-related changes in protein

Gutman, B., and Hanzlikova, V. (1972). Age changes in the neuromuscular system.

Haglund, K., Sigismund, S., Polo, S., Szymkiewicz, I., Di Fiore, P.P., and Dikic, I. (2003).

Hanna, J., Leggett, D.S., and Finley, D. (2003). Ubiquitin depletion as a key mediator of

Hanna, J., Meides, A., Zhang, D.P., and Finley, D. (2007). A ubiquitin stress response

Hara, T., Nakamura, K., Matsui, M., Yamamoto, A., Nakahara, Y., Suzuki-Migishima, R.,

Harper, J.W., and Schulman, B.A. (2006). Structural complexity in ubiquitin recognition. Cell

Harriman, D.G., Taverner, D., and Woolf, A.L. (1970). Ekbom´s syndrome and burning

Hayashi, T., and Goto, S. (1998). Age-related changes in the 20S and 26S proteasome activities in the liver of male F344 rats. Mech Ageing Dev *102*, 55-66. Janssen, I., Heymsfield, S.B., and Ross, R. (2002). Low relative skeletal muscle mass

Jariel-Encontre, I., Bossis, G., and Piechaczyk, M. (2008). Ubiquitin-independent degradation of proteins by the proteasome. Biochim Biophys Acta *1786*, 153-177. Johnson, H., Mossberg, K., Arvidsson, U., Piehl, F., Hökfelt, T., and Ulfhake, B. (1995).

Kadi, F., Charifi, N., Denis, C., Lexell, J., Andersen, J.L., Schjerling, P., Olsen, S., and Kjaer,

Keller, J.N., Huang, F.F., and Markesbery, W.R. (2000). Decreased levels of proteasome

Kimura, Y., Yashiroda, H., Kudo, T., Koitabashi, S., Murata, S., Kakizuka, A., and Tanaka, K.

symptoms of hindlimb incapacities. J Comp Neurol *359*, 69-89.

learned from human studies? Pflugers Arch *451*, 319-327.

toxicity by translational inhibitors. Mol Cell Biol *23*, 9251-9261.

induces altered proteasome composition. Cell *129*, 747-759.

(2010). Early demethylation of non-CpG, CpC-rich, elements in the myogenin 5' flanking region: a priming effect on the spreading of active demethylation. Cell

muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl

oxidation and proteolysis in mammalian cells. J Gerontol A Biol Sci Med Sci *56*,

Multiple monoubiquitination of RTKs is sufficient for their endocytosis and

Yokoyama, M., Mishima, K., Saito, I., Okano, H.*, et al.* (2006). Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature *441*, 885-889.

paraesthesiae. A biopsy study by vital staining and electron microscopy of the intramuscular innervation with a note on age changes in motor nerve endings in

(sarcopenia) in older persons is associated with functional impairment and physical

Increase in alpha-CGRP and GAP-43 in aged motoneurons: A study of peptides, growth factors, and ChAT mRNA in the lumbar spinal cord of senescent rats with

M. (2005). The behaviour of satellite cells in response to exercise: what have we

activity and proteasome expression in aging spinal cord. Neuroscience *98*, 149-156.

(2009). An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis.

function, and oxidation in aged muscle. Faseb J *19*, 644-646.

Cycle *9*, 3965-3976.

B459-467.

*124*, 1133-1136.

Cell *137*, 549-559.

Acad Sci U S A *98*, 14440-14445.

Scientechnica Ltd, Bristol, 1-20.

degradation. Nat Cell Biol *5*, 461-466.

distal muscles. Brain *93*, 393-406.

disability. J Am Geriatr Soc *50*, 889-896.


**14** 

 *Japan* 

Tetsuji Nagata1,2

**Cell Senescence as Observed** 

*Shinshu University School of Medicine, Matsumoto,* 

*2Shinshu Institute of Alternative Medicine and Welfare, Nagano,* 

*1Department of Anatomy and Cell Biology,* 

**by Electron Microscopic Radioautography** 

The term "cell senescence" initially means how the cells change when they get old due to their aging. It contains 2 meanings, one how a cell changes when it is isolated from in vivo original animals or plants such as in vitro cells in cell culture, while the other means how all the cells of an animal or a plant change in vivo due to the aging of the individual animal or plant. In order to study the cell senescence, we have 2 research techniques to clarify how cells get old, i.e., morphological technique and functional technique. The former employs microscopy either light microscope or electron microscope to observe the structure of cells and tissues, while the latter employs functional techniques such as physiological or biochemical to observe either the electric activities or chemical components. Since I am anatomist and had learned morphological technique, I employed to observe cells by light and electron microscopy. I had first studied the meaning of cell senescence many years ago (more than 50 years) how a cell changed when it was isolated from original experimental animals such as mice and rats by cell culture (Nagata 1956, 1957a,b), and then moved to the study on the latter cell senescence, i.e., how all the cells of an experimental animal change in vivo due to the senescence of the individual animal bodies (Nagata 1959, 1962, Nagata and Momoze 1959, Nagata et al. 1960a,b). Recently, I have been studying the senescent changes from the viewpoint of the cell nutrients which were incorporated and synthesized into various cells in individual animals during their senescence (Nagata 2010c). Therefore, this article deals with the cell senescence of animal cells in vivo, how the metabolism, i.e., incorporations and syntheses of respective nutrients, the macromolecular precursors, in various kinds of cells change due to the senescence of individual experimental animals such as mice and rats by means of microscopic radioautography. The incorporations and syntheses of various nutrients such as DNA, RNA, proteins, glucides, lipids and others in various kinds of cells of various organ in respective organ systems such as skeletal, muscular, circulatory, digestive, respiratory, urinary, reproductive, endocrine, nervous and sensory systems should be reviewed

referring many original papers already published from our laboratory.

When I was first asked early this year (April 2011) from the publisher, named InTech, Open Access Publisher in Croatia, to contribute this article as well as to edit the articles submitted

**1. Introduction** 


## **Cell Senescence as Observed by Electron Microscopic Radioautography**

## Tetsuji Nagata1,2

*1Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto, 2Shinshu Institute of Alternative Medicine and Welfare, Nagano, Japan* 

## **1. Introduction**

286 Senescence

Roth, S.M., Metter, E.J., Ling, S., and Ferrucci, L. (2006). Inflammatory factors in age-related

Sacheck, J.M., Hyatt, J.P., Raffaello, A., Jagoe, R.T., Roy, R.R., Edgerton, V.R., Lecker, S.H.,

Sacheck, J.M., Ohtsuka, A., McLary, S.C., and Goldberg, A.L. (2004). IGF-I stimulates muscle

ligases, atrogin-1 and MuRF1. Am J Physiol Endocrinol Metab *287*, E591-601. Sandri, M., Lin, J., Handschin, C., Yang, W., Arany, Z.P., Lecker, S.H., Goldberg, A.L., and

Sandri, M., Sandri, C., Gilbert, A., Skurk, C., Calabria, E., Picard, A., Walsh, K., Schiaffino, S.,

Solomon, A.M., and Bouloux, P.M. (2006). Modifying muscle mass - the endocrine

St-Onge, M.P. (2005). Relationship between body composition changes and changes in

Stitt, T.N., Drujan, D., Clarke, B.A., Panaro, F., Timofeyva, Y., Kline, W.O., Gonzalez, M.,

Terman, A., and Brunk, U.T. (2004). Myocyte aging and mitochondrial turnover. Exp

Terman, A., and Brunk, U.T. (2006). Oxidative stress, accumulation of biological 'garbage',

Terman, A., Gustafsson, B., and Brunk, U.T. (2006). The lysosomal-mitochondrial axis theory

Valdez, G., Tapia, J.C., Kang, H., Clemenson, G.D., Jr., Gage, F.H., Lichtman, J.W., and Sanes,

caloric restriction and exercise. Proc Natl Acad Sci U S A *107*, 14863-14868. Valdez, G., Tapia, J.C., Kang, H., Clemenson, G.D., Jr., Gage, F.H., Lichtman, J.W., and Sanes,

caloric restriction and exercise. Proc Natl Acad Sci U S A *107*, 14863-14868. Watts, G.D., Wymer, J., Kovach, M.J., Mehta, S.G., Mumm, S., Darvish, D., Pestronk, A.,

Zammit, P.S. (2008). All muscle satellite cells are equal, but are some more equal than

Zhou, X., Wang, J.L., Lu, J., Song, Y., Kwak, K.S., Jiao, Q., Rosenfeld, R., Chen, Q., Boone, T.,

ActRIIB antagonism leads to prolonged survival. Cell *142*, 531-543.

J.R. (2010a). Attenuation of age-related changes in mouse neuromuscular synapses by

J.R. (2010b). Attenuation of age-related changes in mouse neuromuscular synapses by

Whyte, M.P., and Kimonis, V.E. (2004). Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-

Simonet, W.S.*, et al.* (2010). Reversal of cancer cachexia and muscle wasting by

of postmitotic aging and cell death. Chem Biol Interact *163*, 29-37.

and Goldberg, A.L. (2007). Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic

growth by suppressing protein breakdown and expression of atrophy-related ubiquitin

Spiegelman, B.M. (2006). PGC-1alpha protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription. Proc Natl Acad

Lecker, S.H., and Goldberg, A.L. (2004). Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell

physical function and metabolic risk factors in aging. Curr Opin Clin Nutr Metab

Yancopoulos, G.D., and Glass, D.J. (2004). The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO

muscle wasting. Curr Opin Rheumatol *18*, 625-630.

diseases. Faseb J *21*, 140-155.

Sci U S A *103*, 16260-16265.

perspective. J Endocrinol *191*, 349-360.

transcription factors. Mol Cell *14*, 395-403.

and aging. Antioxid Redox Signal *8*, 197-204.

containing protein. Nat Genet *36*, 377-381.

others? J Cell Sci *121*, 2975-2982.

*117*, 399-412.

Care *8*, 523-528.

Gerontol *39*, 701-705.

The term "cell senescence" initially means how the cells change when they get old due to their aging. It contains 2 meanings, one how a cell changes when it is isolated from in vivo original animals or plants such as in vitro cells in cell culture, while the other means how all the cells of an animal or a plant change in vivo due to the aging of the individual animal or plant. In order to study the cell senescence, we have 2 research techniques to clarify how cells get old, i.e., morphological technique and functional technique. The former employs microscopy either light microscope or electron microscope to observe the structure of cells and tissues, while the latter employs functional techniques such as physiological or biochemical to observe either the electric activities or chemical components. Since I am anatomist and had learned morphological technique, I employed to observe cells by light and electron microscopy. I had first studied the meaning of cell senescence many years ago (more than 50 years) how a cell changed when it was isolated from original experimental animals such as mice and rats by cell culture (Nagata 1956, 1957a,b), and then moved to the study on the latter cell senescence, i.e., how all the cells of an experimental animal change in vivo due to the senescence of the individual animal bodies (Nagata 1959, 1962, Nagata and Momoze 1959, Nagata et al. 1960a,b).

Recently, I have been studying the senescent changes from the viewpoint of the cell nutrients which were incorporated and synthesized into various cells in individual animals during their senescence (Nagata 2010c). Therefore, this article deals with the cell senescence of animal cells in vivo, how the metabolism, i.e., incorporations and syntheses of respective nutrients, the macromolecular precursors, in various kinds of cells change due to the senescence of individual experimental animals such as mice and rats by means of microscopic radioautography. The incorporations and syntheses of various nutrients such as DNA, RNA, proteins, glucides, lipids and others in various kinds of cells of various organ in respective organ systems such as skeletal, muscular, circulatory, digestive, respiratory, urinary, reproductive, endocrine, nervous and sensory systems should be reviewed referring many original papers already published from our laboratory.

When I was first asked early this year (April 2011) from the publisher, named InTech, Open Access Publisher in Croatia, to contribute this article as well as to edit the articles submitted

Cell Senescence as Observed by Electron Microscopic Radioautography 289

the systematic results obtained by radioautography should be designated as radioauographology which means the science of radioautography (Nagata 1998b, 1999e, 2000e). This article deals with the results dealing with the radioautographic changes of

Explanation of Figures. From Nagata, T.: Acta Microsc. Vol. 6: Suppl. B. p. 42, 1997a. Brazil. Soc.

Fig. 1. Photographs showing the standard procedure for preparing EMRAG (electron

Fig. 1-2. A large wire-loop is dipped into the melted radioautographic emulsion and a thin film

Fig. 1-3. The emulsion film with the wire-loop (Fig. 1-2) is applied horizontally to the glass

Fig. 1-4. Ten glass blocks carrying 6 grid meshes each (Fig. 1-3) are attached on a glass slide

Fig. 1-6. Several glass slides, each carrying 10 glass blocks with grid meshes, are stored in a

Fig. 1-7. All the grid meshes on glass blocks are developed, fixed and stained simultaneously.

microscopy before exposure. Note the monolayer arrangement of the silver bromide crystals in

Fig. 1-5. The emulsion film picked up at random is checked by transmission electron

microscopic radioautograms) by the wire-loop method (Nagata 1982, 1985). Fig. 1-1. Six grid meshes carrying sections are placed on a square glass block.

Electron Microsc., San Paulo, Brazil

of the emulsion is obtained.

with Scotch tape.

block on which 6 grid meshes were placed (Fig. 1-1).

light tight slide box kept in a refrigerator at 4C for exposure.

Konica NR-H2 emulsion in this figure. x6,000.

individual cell by aging that should be included in radioautographology.

from around 40 authors throughout the world, I initially intended to write only one chapter entitled "Senescence as Analyzed by Microscopic Radioautography." However, when I was almost finishing this article in one chapter which consisted of the text around 140 pages and around 30 figures which might become over 170 pages altogether, the publisher requested me to submit only one chapter within exactly 16, 18, 20, 22, 24 or 26 pages including both text and figures. Since I could not agree with them in submitting such a short chapter dealing with all the research results of myself and my associates, I insisted to submit the large article in one chapter including so much information as the results of my research on senescence for more 50 years since 1955 up to the present time 2011, they requested me to divide the original one chapter into 3 or 4 short chapters.

Thus, I tried to divide the original draft into 4 chapters, including the foundations of radioautography and the results of its application to all the organ systems, i.e., skeletal, muscular, circulatory, digestive, respiratory, urinary, reproductive, endocrine, nervous and sensory organs altogether. As the results of dividing the initial one chapter into 4, the final chapters were consisted of more than 26 pages as the publisher requested. However, I am going to submit the longer chapters to the publisher and insist to publish those chapters as they are, otherwise, I would rather prefer to withdraw them from this book and would like to contribute them to any other suitable publishers in the world.

This first chapter deals with the methodology of microscopic radioautography as well as the first parts of the applications of radioautography to the organ systems, i.e. the organ of movement (skeletal and muscular system) and the circulatory system.

#### **1.1 Method in microscopic radioautography**

For the purpose of observing the localizations of the incorporations and syntheses of various nutrients synthesizing macromolecules in the human and animal bodies such as DNA, RNA, proteins, glucides and lipids in various kinds of cells of various organ in respective organ systems such as skeletal, muscular, circulatory, digestive, respiratory, urinary, reproductive, endocrine, nervous and sensory systems, we employed the specific techniques developed in our laboratory during these 50 years (Nagata 2002). The technique is designated as radioautography using RI-labeled compounds. Some scientists use another term autoradiography which is used as the synonym to radioautogarphy. However, the author prefers the term radioautography because of the etymological reason (Nagata 1996b). To demonstrate the localizations of macromolecular synthesis by using such RI-labeled precursors as 3H-thymidine for DNA, 3H-uridine for RNA, 3H-leucine for proteins, 3Hglucosamine or 35SO4 for glucides and 3H-glycerol for lipids are divided into macroscopic radioautography and microscopic radioautography. The techniques employ both the physical techniques using RI-labeled compounds and the histochemical techniques treating tissue sections by coating sections containing RI-labeled precursors with photographic emulsions and processing for exposure and development. Such techniques can demonstrate both the soluble compounds diffusible in the cells and tissues and the insoluble compounds bound to the macromolecules (Nagata 1972b). As the results, specimens prepared for EM RAG (electron microscopic radioautography) are very thick than conventional EM specimens and should be observed with high voltage electron microscopes in order to obtain better transmittance and resolution (Nagata 2001a,b). Such radioautographic techniques in details should be referred to other literature (Nagata 2002). On the other hand,

from around 40 authors throughout the world, I initially intended to write only one chapter entitled "Senescence as Analyzed by Microscopic Radioautography." However, when I was almost finishing this article in one chapter which consisted of the text around 140 pages and around 30 figures which might become over 170 pages altogether, the publisher requested me to submit only one chapter within exactly 16, 18, 20, 22, 24 or 26 pages including both text and figures. Since I could not agree with them in submitting such a short chapter dealing with all the research results of myself and my associates, I insisted to submit the large article in one chapter including so much information as the results of my research on senescence for more 50 years since 1955 up to the present time 2011, they requested me to

Thus, I tried to divide the original draft into 4 chapters, including the foundations of radioautography and the results of its application to all the organ systems, i.e., skeletal, muscular, circulatory, digestive, respiratory, urinary, reproductive, endocrine, nervous and sensory organs altogether. As the results of dividing the initial one chapter into 4, the final chapters were consisted of more than 26 pages as the publisher requested. However, I am going to submit the longer chapters to the publisher and insist to publish those chapters as they are, otherwise, I would rather prefer to withdraw them from this book and would like

This first chapter deals with the methodology of microscopic radioautography as well as the first parts of the applications of radioautography to the organ systems, i.e. the organ of

For the purpose of observing the localizations of the incorporations and syntheses of various nutrients synthesizing macromolecules in the human and animal bodies such as DNA, RNA, proteins, glucides and lipids in various kinds of cells of various organ in respective organ systems such as skeletal, muscular, circulatory, digestive, respiratory, urinary, reproductive, endocrine, nervous and sensory systems, we employed the specific techniques developed in our laboratory during these 50 years (Nagata 2002). The technique is designated as radioautography using RI-labeled compounds. Some scientists use another term autoradiography which is used as the synonym to radioautogarphy. However, the author prefers the term radioautography because of the etymological reason (Nagata 1996b). To demonstrate the localizations of macromolecular synthesis by using such RI-labeled precursors as 3H-thymidine for DNA, 3H-uridine for RNA, 3H-leucine for proteins, 3Hglucosamine or 35SO4 for glucides and 3H-glycerol for lipids are divided into macroscopic radioautography and microscopic radioautography. The techniques employ both the physical techniques using RI-labeled compounds and the histochemical techniques treating tissue sections by coating sections containing RI-labeled precursors with photographic emulsions and processing for exposure and development. Such techniques can demonstrate both the soluble compounds diffusible in the cells and tissues and the insoluble compounds bound to the macromolecules (Nagata 1972b). As the results, specimens prepared for EM RAG (electron microscopic radioautography) are very thick than conventional EM specimens and should be observed with high voltage electron microscopes in order to obtain better transmittance and resolution (Nagata 2001a,b). Such radioautographic techniques in details should be referred to other literature (Nagata 2002). On the other hand,

divide the original one chapter into 3 or 4 short chapters.

to contribute them to any other suitable publishers in the world.

**1.1 Method in microscopic radioautography** 

movement (skeletal and muscular system) and the circulatory system.

the systematic results obtained by radioautography should be designated as radioauographology which means the science of radioautography (Nagata 1998b, 1999e, 2000e). This article deals with the results dealing with the radioautographic changes of individual cell by aging that should be included in radioautographology.

Explanation of Figures. From Nagata, T.: Acta Microsc. Vol. 6: Suppl. B. p. 42, 1997a. Brazil. Soc. Electron Microsc., San Paulo, Brazil

Fig. 1. Photographs showing the standard procedure for preparing EMRAG (electron microscopic radioautograms) by the wire-loop method (Nagata 1982, 1985).

Fig. 1-1. Six grid meshes carrying sections are placed on a square glass block.

Fig. 1-2. A large wire-loop is dipped into the melted radioautographic emulsion and a thin film of the emulsion is obtained.

Fig. 1-3. The emulsion film with the wire-loop (Fig. 1-2) is applied horizontally to the glass block on which 6 grid meshes were placed (Fig. 1-1).

Fig. 1-4. Ten glass blocks carrying 6 grid meshes each (Fig. 1-3) are attached on a glass slide with Scotch tape.

Fig. 1-5. The emulsion film picked up at random is checked by transmission electron microscopy before exposure. Note the monolayer arrangement of the silver bromide crystals in Konica NR-H2 emulsion in this figure. x6,000.

Fig. 1-6. Several glass slides, each carrying 10 glass blocks with grid meshes, are stored in a light tight slide box kept in a refrigerator at 4C for exposure.

Fig. 1-7. All the grid meshes on glass blocks are developed, fixed and stained simultaneously.

Cell Senescence as Observed by Electron Microscopic Radioautography 291

histology. In contrast to the results obtained from DNA synthesis of almost all the organs, we have studied only several parts of the organ systems with regards RNA. The skeletal

The proteins found in animal cells are composed of various amino-acids which initially form low molecular polypeptides and finally macromolecular compounds designated as proteins. They are chemically classified into two, simple proteins and conjugated proteins. Therefore, the proteins can be demonstrated by showing specific reactions to respective amino-acids composing any proteins. Thus, the proteins contained in cells can be demonstrated either by morphological histochemical techniques staining tissue sections such as Millon reaction (Millon 1849) or tetrazonium reaction or otherwise by biochemical techniques homogenizing tissues and cells. To the contrary, the newly synthesized proteins but not all the proteins in the cells can be detected as macromolecular synthesis together with other macromolecules such as DNA or RNA in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography (Nagata 1992, 1994b,c, 1996a,b, 1997a, 2002, 2010c). The results obtained from protein synthesis should be described according to the order of organ systems in anatomy or histology. In contrast to the results obtained from DNA synthesis of almost all the organs, we have

The glucides found in animal cells and tissues are composed of various low molecular sugars such as glucose or fructose called monosaccharides which form compounds of polysaccharides or complex mucopolysaccharides connecting to sulfated compounds. The former are called simple polysaccharides, while the latter mucopolysubstances. Thus, the glucides are chemically classified into 3 groups, monosaccharides such as glucose or fructose, disaccharides such as sucrose and polysaccharides such as mucosubstances. However, in most animal cells polysaccharides are much more found than monosaccharides or disaccharides. The polysaccharides can be classified into 2, i.e. simple polyscaccharides and mucosubstances. Anyway, they are composed of various low molecular sugars that can be demonstrated by either histochemical reactions or biochemical techniques. To the contrary, the newly synthesized glucides but not all the glucides in the cells and tissues can be detected as macromolecular synthesis together with other macromolecules such as DNA, RNA or proteins in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography (Nagata 1992, 1994a,b,c, 1996a, 1997a, 2002, 2010c). The results obtained from glucides synthesis are described according to the order of organ systems in anatomy or histology. In contrast to the results obtained from DNA synthesis of almost all the organs, we have studied only several parts of the organ systems. The skeletal system, the muscular system and the circulatory

system, the muscular system or the circulatory system were not so much studied.

**1.2.3 The protein synthesis** 

studied only several parts of the organ systems.

**1.2.4 The glucide synthesis** 

system were not yet studied.

#### **1.2 Macromolecular synthesis**

The human body as well as the bodies of any experimental animals such as mice and rats consist of various macromolecules. They are classified into nucleic acids (both DNA and RNA), proteins, glucides and lipids, according to their chemical structures. These macromolecules can be demonstrated by specific histochemical staining for respective molecules such as Feulgen reaction (Feulgen and Rossenbeck 1924) which stains all the DNA contained in the cells. Each compounds of macromolecules such as DNA, RNA, proteins, glucides, lipids can be demonstrated by respective specific histochemical stainings (Pearse 1991) and such reactions can be quantified by microscpectrophotometry using specific wave-lengths demonstrating the total amount of respective compounds (Nagata 1972a). To the contrary, radioautography can only demonstrate the newly synthesized macromolecules such as synthetic DNA or RNA or proteins depending upon the RI-labeled precursors incorporated specifically into these macromolecules such as 3H-thymidine into DNA or 3H-uridine into RNA or 3H-amino acid into proteins (Nagata 2002).

Concerning to the newly synthesized macromolecules, the results of recent studies in our laboratory by the present author and co-workers should be reviewed in this article according to the classification of macromolecules as follows.

#### **1.2.1 The DNA synthesis**

The DNA (deoxyribonucleic acid) contained in cells can be demonstrated either by morphological histochemical techniques staining tissue sections such as Feulgen reaction (Feulgen and Rossenbeck 1924) or by biochemical techniques homogenizing tissues and cells. To the contrary, the synthetic DNA or newly synthesized DNA but not all the DNA can be detected as macromolecular synthesis together with other macromolecules such as RNA or proteins in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography, one of the morphological methods (Nagata 1992, 1994b,c,d, 1996a,b,c,d, 1997a, 2002, 2010c). The results should be here described according to the order of organ systems in anatomy or histology.

#### **1.2.2 The RNA synthesis**

The RNA (ribonucleic acid) contained in cells can be demonstrated either by morphological histochemical techniques staining tissue sections such as methyl green-pyronin staining or by biochemical techniques homogenizing tissues and cells. To the contrary, the synthetic RNA or newly synthesized RNA but not all the RNA in the cells can be detected as macromolecular synthesis together with other macromolecules such as DNA or proteins in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography, one of the morphological methods (Nagata 1992, 1994b,c, 1996a,b,c, 1997a, 2002, 2010c). The results obtained from RNA synthesis should be here described according to the order of organ systems in anatomy or histology. In contrast to the results obtained from DNA synthesis of almost all the organs, we have studied only several parts of the organ systems with regards RNA. The skeletal system, the muscular system or the circulatory system were not so much studied.

## **1.2.3 The protein synthesis**

290 Senescence

The human body as well as the bodies of any experimental animals such as mice and rats consist of various macromolecules. They are classified into nucleic acids (both DNA and RNA), proteins, glucides and lipids, according to their chemical structures. These macromolecules can be demonstrated by specific histochemical staining for respective molecules such as Feulgen reaction (Feulgen and Rossenbeck 1924) which stains all the DNA contained in the cells. Each compounds of macromolecules such as DNA, RNA, proteins, glucides, lipids can be demonstrated by respective specific histochemical stainings (Pearse 1991) and such reactions can be quantified by microscpectrophotometry using specific wave-lengths demonstrating the total amount of respective compounds (Nagata 1972a). To the contrary, radioautography can only demonstrate the newly synthesized macromolecules such as synthetic DNA or RNA or proteins depending upon the RI-labeled precursors incorporated specifically into these macromolecules such as 3H-thymidine into DNA or 3H-uridine into RNA or 3H-amino acid into proteins (Nagata

Concerning to the newly synthesized macromolecules, the results of recent studies in our laboratory by the present author and co-workers should be reviewed in this article

The DNA (deoxyribonucleic acid) contained in cells can be demonstrated either by morphological histochemical techniques staining tissue sections such as Feulgen reaction (Feulgen and Rossenbeck 1924) or by biochemical techniques homogenizing tissues and cells. To the contrary, the synthetic DNA or newly synthesized DNA but not all the DNA can be detected as macromolecular synthesis together with other macromolecules such as RNA or proteins in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography, one of the morphological methods (Nagata 1992, 1994b,c,d, 1996a,b,c,d, 1997a, 2002, 2010c). The results should be here described according to the order of organ systems in anatomy or

The RNA (ribonucleic acid) contained in cells can be demonstrated either by morphological histochemical techniques staining tissue sections such as methyl green-pyronin staining or by biochemical techniques homogenizing tissues and cells. To the contrary, the synthetic RNA or newly synthesized RNA but not all the RNA in the cells can be detected as macromolecular synthesis together with other macromolecules such as DNA or proteins in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography, one of the morphological methods (Nagata 1992, 1994b,c, 1996a,b,c, 1997a, 2002, 2010c). The results obtained from RNA synthesis should be here described according to the order of organ systems in anatomy or

according to the classification of macromolecules as follows.

**1.2 Macromolecular synthesis** 

2002).

histology.

**1.2.1 The DNA synthesis** 

**1.2.2 The RNA synthesis** 

The proteins found in animal cells are composed of various amino-acids which initially form low molecular polypeptides and finally macromolecular compounds designated as proteins. They are chemically classified into two, simple proteins and conjugated proteins. Therefore, the proteins can be demonstrated by showing specific reactions to respective amino-acids composing any proteins. Thus, the proteins contained in cells can be demonstrated either by morphological histochemical techniques staining tissue sections such as Millon reaction (Millon 1849) or tetrazonium reaction or otherwise by biochemical techniques homogenizing tissues and cells. To the contrary, the newly synthesized proteins but not all the proteins in the cells can be detected as macromolecular synthesis together with other macromolecules such as DNA or RNA in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography (Nagata 1992, 1994b,c, 1996a,b, 1997a, 2002, 2010c). The results obtained from protein synthesis should be described according to the order of organ systems in anatomy or histology. In contrast to the results obtained from DNA synthesis of almost all the organs, we have studied only several parts of the organ systems.

#### **1.2.4 The glucide synthesis**

The glucides found in animal cells and tissues are composed of various low molecular sugars such as glucose or fructose called monosaccharides which form compounds of polysaccharides or complex mucopolysaccharides connecting to sulfated compounds. The former are called simple polysaccharides, while the latter mucopolysubstances. Thus, the glucides are chemically classified into 3 groups, monosaccharides such as glucose or fructose, disaccharides such as sucrose and polysaccharides such as mucosubstances. However, in most animal cells polysaccharides are much more found than monosaccharides or disaccharides. The polysaccharides can be classified into 2, i.e. simple polyscaccharides and mucosubstances. Anyway, they are composed of various low molecular sugars that can be demonstrated by either histochemical reactions or biochemical techniques. To the contrary, the newly synthesized glucides but not all the glucides in the cells and tissues can be detected as macromolecular synthesis together with other macromolecules such as DNA, RNA or proteins in various organs of experimental animals by either morphological or biochemical procedures employing RI-labeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography (Nagata 1992, 1994a,b,c, 1996a, 1997a, 2002, 2010c). The results obtained from glucides synthesis are described according to the order of organ systems in anatomy or histology. In contrast to the results obtained from DNA synthesis of almost all the organs, we have studied only several parts of the organ systems. The skeletal system, the muscular system and the circulatory system were not yet studied.

chapter 4.

Cell Senescence as Observed by Electron Microscopic Radioautography 293

cells should be described separately from the normal organ system at the end of this book in

The Organ of Movement or locomotive organ of men and experimental animals consists of both the skeletal system and the muscular system. The former consists of many bones, around 200 in case of men while the latter consists of many skeletal muscles around 600 in case of men. We studied the macromolecular synthesis in a part of these locomotive organs in the experimental animals, but not all of them. The results should be described in the

The skeletal system of men and experimental animals consists of bones, joints and ligaments. We studied the DNA synthetic activities of the bones and joints of experimental

We studied the DNA synthetic activities of the bones and joints of experimental animals in

We studied the ossifications of salamander skeletons from hatching to senescence (Nagata 1998c). The fore-limbs (Fig. 2A) and hind-limbs (Fig. 2B) of salamanders were composed of skeletons consisting of bones and cartilages which were covered with skeletal muscles, connective tissues and epidermis consisting of stratified squamous epithelial cells in the outermost layer. The bones of juvenile salamanders at 4 weeks consisted of the hyaline cartilage (Figs. 2A, 2B). The hyaline cartilage consisted of spherical or polygonal cartilage cells or chondrocytes at the center. They were surrounded by rich interstitial ground substance which stained deep blue with toluidine blue staining. The spherical cartilage cells at the center of the bone changed their shapes to flattened shape under the perichondrium or free joint surfaces. Some of the nuclei of the chondrocytes were covered with silver grains when labeled with 3H-thymidine (Figs. 2A, 2B). Mitotic figures were frequently seen in spherical cartilage cells in young animals. Examination of radioautograms at the young stages such as 4 weeks after hatching showed that many spherical cartilage cells and flattened cartilage cells were predominantly labeled. At 6 weeks after hatching, the size of bones enlarged and the number of cartilage cells increased. At this stage, however, the number of labeled cells in the cartilage cells in both fore-limbs (Fig. 2C) and hind-limbs (Fig. 2D) decreased as compared with the previous stage (Figs. 2A, 2B). The size of bones in juvenile animals at 8, 9, 10, and 11 weeks enlarged gradually (Fig. 2E, 2F). Radioautograms at these stages showed that the number of the labeled cells remarkably reduced as compared with those of 4 and 6 weeks. In the adult salamanders at 8 months up to 12 months, the bones showed complete mature structure and examination of radioautograms revealed that the number of labeled cells reached almost zero (Kobayashi and Nagata 1994, Nagata 2006c). No difference was found on the morphology and labeling between the fore-limbs and hind-

animals in development and aging (Kobayashi and Nagata 1994, Nagata 1998c).

development and aging to senescence (Kobayashi and Nagata 1994, Nagata 1998c).

**2. Macromolecular synthesis in the organ of movement** 

following 2 sections, the skeletal system and the muscular system.

**2.1 Macromolecular synthesis in the skeletal system** 

**2.1.1 The DNA synthesis in the skeletal system** 

**2.1.1.1 The DNA synthesis in the bone** 

limbs at any stage.

#### **1.2.5 The lipid synthesis**

The lipids found in animal cells are chemically composed of various low molecular fatty acids. They are esters of high fatty acids and glycerol that can biochemically be classified into simple lipids and compound lipids such as phospholipids, glycolipids or proteolipids. The simple lipids are composed of only fatty acids and glycerol, while the latter composed of lipids and other components such as phosphates, glucides or proteins. In order to demonstrate intracellular localization of total lipids, we can employ either histochemical reactions or biochemical techniques. To the contrary, the newly synthesized lipids but not all of the lipids in the cells can be detected as macromolecular synthesis similarly to the other macromolecules such as DNA, RNA, proteins or glucides in various organs of experimental animals by either morphological or biochemical procedures employing RIlabeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography (Nagata 1992, 1994a,b,c,d,e, 1996a, 1997a, 2002, 2010c). However, we have not studied the lipids synthesis so much as compared to other compounds. We have studied only a few organs of the digestive system.

#### **1.3 The intracellular localization of the other substances**

The other substances than macromolecules that can also be demonstrated by radioautography are target tracers not the precursors for the macromolecular synthesis. They are hormones such as 3H-methyl prednisolone (Nagata et al. 1978b), neurotransmitters and inhibitors such as 14C-bupranolol, a beta-blocking agent (Tsukahara et al. 1980) or 3Hbefunolol (Nagata and Yamabayashi 1983, Yamabayashi et al. 1981), vitamins, drugs such as synthetic anti-allergic agent 3H-tranilast (Nagata et al. 1986b, Nishigaki et al. 1987, 1990a.b, Momose et al. 1989), hypolipidemic agent bezafibrate (Momose et al. 1993a,b, 1995), calmodulin antagonist (Ohno et al. 1982, 1983) or anti-hypertensive agent 3H-benidipine hydrochloride (Suzuki et al. 1994), toxins, inorganic substances such as mercury (Nagata et al. 1977b) and others such as laser beam irradiation (Nagata 1984). The details are referred to the previous publication on the radioatuographology (Nagata 2002). However, their relationships to the cell aging and senescence were not studied.

#### **1.4 Macromolecular synthesis in the normal organ systems**

With regards to the macromolecular synthesis such as DNA, RNA, proteins, glucides, lipids etc in various cells and tissues, we have studied various cells and tissues in almost all the organ systems in the experimental animals such as mice and rats. Therefore, the results are classified into the organ systems in anatomy and histology, i.e. the organ of movement including the skeletal system and the muscular system, the circulatory organs, the digestive organs, the respiratory organs, the urinary organs, the reproductive organs, the endocrine organs, the nervous system, and the sensory organs. Thus, the results should be described according to this order in the following chapters divided into 4 chapters.

#### **1.5 Macromolecular synthesis in the tumor cells**

As for the tumor cells, on the other hand, which do not belong to any organ systems of the normal organs but grow in any organ systems, the macromolecular synthesis in the tumor

The lipids found in animal cells are chemically composed of various low molecular fatty acids. They are esters of high fatty acids and glycerol that can biochemically be classified into simple lipids and compound lipids such as phospholipids, glycolipids or proteolipids. The simple lipids are composed of only fatty acids and glycerol, while the latter composed of lipids and other components such as phosphates, glucides or proteins. In order to demonstrate intracellular localization of total lipids, we can employ either histochemical reactions or biochemical techniques. To the contrary, the newly synthesized lipids but not all of the lipids in the cells can be detected as macromolecular synthesis similarly to the other macromolecules such as DNA, RNA, proteins or glucides in various organs of experimental animals by either morphological or biochemical procedures employing RIlabeled precursors. We have studied the sites of macromolecular synthesis in almost all the organs of mice during their aging from prenatal to postnatal development to senescence by means of microscopic radioautography (Nagata 1992, 1994a,b,c,d,e, 1996a, 1997a, 2002, 2010c). However, we have not studied the lipids synthesis so much as compared to other

The other substances than macromolecules that can also be demonstrated by radioautography are target tracers not the precursors for the macromolecular synthesis. They are hormones such as 3H-methyl prednisolone (Nagata et al. 1978b), neurotransmitters and inhibitors such as 14C-bupranolol, a beta-blocking agent (Tsukahara et al. 1980) or 3Hbefunolol (Nagata and Yamabayashi 1983, Yamabayashi et al. 1981), vitamins, drugs such as synthetic anti-allergic agent 3H-tranilast (Nagata et al. 1986b, Nishigaki et al. 1987, 1990a.b, Momose et al. 1989), hypolipidemic agent bezafibrate (Momose et al. 1993a,b, 1995), calmodulin antagonist (Ohno et al. 1982, 1983) or anti-hypertensive agent 3H-benidipine hydrochloride (Suzuki et al. 1994), toxins, inorganic substances such as mercury (Nagata et al. 1977b) and others such as laser beam irradiation (Nagata 1984). The details are referred to the previous publication on the radioatuographology (Nagata 2002). However, their

With regards to the macromolecular synthesis such as DNA, RNA, proteins, glucides, lipids etc in various cells and tissues, we have studied various cells and tissues in almost all the organ systems in the experimental animals such as mice and rats. Therefore, the results are classified into the organ systems in anatomy and histology, i.e. the organ of movement including the skeletal system and the muscular system, the circulatory organs, the digestive organs, the respiratory organs, the urinary organs, the reproductive organs, the endocrine organs, the nervous system, and the sensory organs. Thus, the results should be described

As for the tumor cells, on the other hand, which do not belong to any organ systems of the normal organs but grow in any organ systems, the macromolecular synthesis in the tumor

compounds. We have studied only a few organs of the digestive system.

**1.3 The intracellular localization of the other substances** 

relationships to the cell aging and senescence were not studied.

**1.4 Macromolecular synthesis in the normal organ systems** 

according to this order in the following chapters divided into 4 chapters.

**1.5 Macromolecular synthesis in the tumor cells** 

**1.2.5 The lipid synthesis** 

cells should be described separately from the normal organ system at the end of this book in chapter 4.

## **2. Macromolecular synthesis in the organ of movement**

The Organ of Movement or locomotive organ of men and experimental animals consists of both the skeletal system and the muscular system. The former consists of many bones, around 200 in case of men while the latter consists of many skeletal muscles around 600 in case of men. We studied the macromolecular synthesis in a part of these locomotive organs in the experimental animals, but not all of them. The results should be described in the following 2 sections, the skeletal system and the muscular system.

## **2.1 Macromolecular synthesis in the skeletal system**

The skeletal system of men and experimental animals consists of bones, joints and ligaments. We studied the DNA synthetic activities of the bones and joints of experimental animals in development and aging (Kobayashi and Nagata 1994, Nagata 1998c).

## **2.1.1 The DNA synthesis in the skeletal system**

We studied the DNA synthetic activities of the bones and joints of experimental animals in development and aging to senescence (Kobayashi and Nagata 1994, Nagata 1998c).

## **2.1.1.1 The DNA synthesis in the bone**

We studied the ossifications of salamander skeletons from hatching to senescence (Nagata 1998c). The fore-limbs (Fig. 2A) and hind-limbs (Fig. 2B) of salamanders were composed of skeletons consisting of bones and cartilages which were covered with skeletal muscles, connective tissues and epidermis consisting of stratified squamous epithelial cells in the outermost layer. The bones of juvenile salamanders at 4 weeks consisted of the hyaline cartilage (Figs. 2A, 2B). The hyaline cartilage consisted of spherical or polygonal cartilage cells or chondrocytes at the center. They were surrounded by rich interstitial ground substance which stained deep blue with toluidine blue staining. The spherical cartilage cells at the center of the bone changed their shapes to flattened shape under the perichondrium or free joint surfaces. Some of the nuclei of the chondrocytes were covered with silver grains when labeled with 3H-thymidine (Figs. 2A, 2B). Mitotic figures were frequently seen in spherical cartilage cells in young animals. Examination of radioautograms at the young stages such as 4 weeks after hatching showed that many spherical cartilage cells and flattened cartilage cells were predominantly labeled. At 6 weeks after hatching, the size of bones enlarged and the number of cartilage cells increased. At this stage, however, the number of labeled cells in the cartilage cells in both fore-limbs (Fig. 2C) and hind-limbs (Fig. 2D) decreased as compared with the previous stage (Figs. 2A, 2B). The size of bones in juvenile animals at 8, 9, 10, and 11 weeks enlarged gradually (Fig. 2E, 2F). Radioautograms at these stages showed that the number of the labeled cells remarkably reduced as compared with those of 4 and 6 weeks. In the adult salamanders at 8 months up to 12 months, the bones showed complete mature structure and examination of radioautograms revealed that the number of labeled cells reached almost zero (Kobayashi and Nagata 1994, Nagata 2006c). No difference was found on the morphology and labeling between the fore-limbs and hindlimbs at any stage.

Cell Senescence as Observed by Electron Microscopic Radioautography 295

The labeling indices of respective cell types changed with aging as expressed by mean in each group. The labeling index of the cartilage cells was lower than the epithelial cells. The peak of the labeling index of the cartilage cells in both fore-limbs and hind-limbs was found about 15-18% at 4 weeks after hatching (Fig. 2). The labeling index of the cartilage cells in both limbs at 6 weeks rapidly decreased to about 4-6%, then increased at 8 weeks to about 7- 8% and finally decreased to 2-3% gradually from 8 weeks to 9 weeks with aging and fell down to 0-1% at 10 weeks. The labeling index of cartilage cells from 10 weeks to 12 months kept very low around 0-1% (Fig. 3). Thus, the cartilages and bones of fore-limbs and hindlimbs of salamanders are demonstrated to complete the development by 10 weeks after

Fig. 3. Transitional curves of the labeling indices of the cartilage cells in the bones of the fore-limbs and the hind-limbs of salamanders labeled with 3H-thymidine at various ages from 4 weeks to 60 months (5 years) after hatching. Mean ± S.D. From Nagata, T.: Bulletin

The joints of an experimental animal such as mouse or human being are consisted of either 2 or 3 bones and the synovial membranes covering the ends of the bones. The synovial membranes are composed of the collagenous fibers interspersed with the synovial cells which are fibroblasts and lining cells. We studied macromolecular synthesis, both DNA and

Shinshu Inst. Alternat. Med. Vol. 2, p. 54, 2006, Nagano, Japan,

**2.1.1.2 The DNA synthesis in the joint** 

hatching (Kobayashi and Nagata 1994, Nagata 2006c).

Fig. 2D. Light microscopic radioautogram of the bone of a hind-limb of a salamander at 6 weeks after hatching. Only a few cartilage cells (arrow) are labeled. Magnification x 1200. Fig. 2E. Light microscopic radioautogram of the bone of a fore-limb of a salamander at 8 weeks after hatching. Only a few cartilage cells (arrow) are labeled. Magnification x 1200. Fig. 2F. Light microscopic radioautogram of the bone of a hind-limb of a salamander at 8 weeks after hatching. Only a few cartilage cells (arrow) are labeled. Magnification x 1200.

Fig. 2. Light microscopic radioautograms of the bones of either fore-limbs or hind-limbs of salamanders at various ages from 4 weeks to 8 weeks after hatching, injected with 3Hthymidine, fixed and processed for radioautography. Some of the cartilage cells (arrows) are labeled with silver grains due to 3H-thymidine incorporation demonstrating DNA synthesis. From Nagata, T.: Bulletin Shinshu Inst. Alternat. Med. Vol. 2, p. 53, 2006, Nagano, Japan Fig. 2A. Light microscopic radioautogram of the bone of a fore-limb of a salamander at 4 weeks after hatching. Many cartilage cells (arrows) are labeled with silver grains due to 3H-thymidine. Magnification. x 1200.

Fig. 2B. Light microscopic radioautogram of the bone of a hind-limb of a salamander at 4 weeks after hatching. Many cartilage cells (arrows) are labeled with silver grains due to 3H-thymidine. Magnification. x 1200.

Fig. 2C. Light microscopic radioautogram of the bone of a fore-limb of a salamander at 6 weeks after hatching. Only a few cartilage cells (arrow) are labeled. The numbers of labeled cells are fewer than the bone of a younger salamander at 4 weeks after hatching (Fig. 1A). Magnification x 1200.

Fig. 2. Light microscopic radioautograms of the bones of either fore-limbs or hind-limbs of salamanders at various ages from 4 weeks to 8 weeks after hatching, injected with 3Hthymidine, fixed and processed for radioautography. Some of the cartilage cells (arrows) are labeled with silver grains due to 3H-thymidine incorporation demonstrating DNA synthesis. From Nagata, T.: Bulletin Shinshu Inst. Alternat. Med. Vol. 2, p. 53, 2006, Nagano, Japan Fig. 2A. Light microscopic radioautogram of the bone of a fore-limb of a salamander at 4 weeks after hatching. Many cartilage cells (arrows) are labeled with silver grains due to

Fig. 2B. Light microscopic radioautogram of the bone of a hind-limb of a salamander at 4 weeks after hatching. Many cartilage cells (arrows) are labeled with silver grains due to

Fig. 2C. Light microscopic radioautogram of the bone of a fore-limb of a salamander at 6 weeks after hatching. Only a few cartilage cells (arrow) are labeled. The numbers of labeled cells are fewer than the bone of a younger salamander at 4 weeks after hatching (Fig. 1A).

3H-thymidine. Magnification. x 1200.

3H-thymidine. Magnification. x 1200.

Magnification x 1200.

Fig. 2D. Light microscopic radioautogram of the bone of a hind-limb of a salamander at 6 weeks after hatching. Only a few cartilage cells (arrow) are labeled. Magnification x 1200. Fig. 2E. Light microscopic radioautogram of the bone of a fore-limb of a salamander at 8 weeks after hatching. Only a few cartilage cells (arrow) are labeled. Magnification x 1200. Fig. 2F. Light microscopic radioautogram of the bone of a hind-limb of a salamander at 8 weeks after hatching. Only a few cartilage cells (arrow) are labeled. Magnification x 1200.

The labeling indices of respective cell types changed with aging as expressed by mean in each group. The labeling index of the cartilage cells was lower than the epithelial cells. The peak of the labeling index of the cartilage cells in both fore-limbs and hind-limbs was found about 15-18% at 4 weeks after hatching (Fig. 2). The labeling index of the cartilage cells in both limbs at 6 weeks rapidly decreased to about 4-6%, then increased at 8 weeks to about 7- 8% and finally decreased to 2-3% gradually from 8 weeks to 9 weeks with aging and fell down to 0-1% at 10 weeks. The labeling index of cartilage cells from 10 weeks to 12 months kept very low around 0-1% (Fig. 3). Thus, the cartilages and bones of fore-limbs and hindlimbs of salamanders are demonstrated to complete the development by 10 weeks after hatching (Kobayashi and Nagata 1994, Nagata 2006c).

Fig. 3. Transitional curves of the labeling indices of the cartilage cells in the bones of the fore-limbs and the hind-limbs of salamanders labeled with 3H-thymidine at various ages from 4 weeks to 60 months (5 years) after hatching. Mean ± S.D. From Nagata, T.: Bulletin Shinshu Inst. Alternat. Med. Vol. 2, p. 54, 2006, Nagano, Japan,

#### **2.1.1.2 The DNA synthesis in the joint**

The joints of an experimental animal such as mouse or human being are consisted of either 2 or 3 bones and the synovial membranes covering the ends of the bones. The synovial membranes are composed of the collagenous fibers interspersed with the synovial cells which are fibroblasts and lining cells. We studied macromolecular synthesis, both DNA and

Cell Senescence as Observed by Electron Microscopic Radioautography 297

The labeling indices revealed chronological changes, showing a peak at embryonic day 13 and decreasing gradually to 0% at 3 months after birth to month 24 (Fig. 5). We classified the graduation of the embryonic muscle development into 5 stages. Among them, the labeling index (LI) at stage I was the highest, while the LI at stage II was significantly lower than stage I, the LI at stage IV was significantly higher than stage II, and the LI at stage V was significantly lower than stage IV (Fig. 5). These changes accorded well with the primary and secondary myotube formation during the embryonic muscle development. We also studied the DNA synthesis of rat thigh muscles during the muscle regeneration after injury in rats (Sakai et al. 1977). When the skeletal muscles, i.e., the diaphragma, the rectus abdominis muscles and the gastrocnemius femoris muscle of adult Wistar rats were mechanically injured and labeled with 3H-thymidine, satellite cells were labeled during their regeneration. The satellite cells in the muscles of dystrophy chickens and normal control chickens were also labeled with 3H-thymidine, demonstrating DNA synthesis (Oguchi and Nagata 1980, 1981), which was later described in details in the review (Nagata 2002). Briefly, 2 groups of chickens, 4 dystrophy chickens and normal control chickens of both sexes aged 1 day and 21 days after hatching were used. All the animals received every 6 hrs intraperitoneal injections of 3H-thymidine 4 times successively and sacrificed. The superficial pectoral muscles were taken out, fixed, embedded in Epoxy resin and processed for LM and EMRAG. The results demonstrated that many nuclei of the satellite cells in all the experimental groups were labeled but none of the nuclei in the muscle fibers were labeled. The labeling indices of normal chickens at 1 day and 21 days were 4.59 and 3.86%, respectively. These results

showed that the LI decreased after hatching due to aging.

Fig. 5. The labeling indices of the muscular cells labeled with 3H-thymidine revealed

chronological changes, showing a peak at embryonic day 13 and decreasing gradually to 0% at 3 months after birth. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 114, 2002, Urban & Fischer, Jena, Germany

RNA syntheses of the synovial cells of the joints surgically obtained from 15 elderly human patients of both sexes aged from 50 to 70, suffering from rheumatoid arthritis (Kobayashi and Nagata 1994). Both the normal and rheumatoid cells were cultured and labeled in vitro with media containing precursors such as 3H-thymidine or 3H-uridine, fixed and radioautographed. DNA synthetic cells labeled with silver grains were observed by LM RAG (light microscopic radioautography) in both normal and rheumatoid cells. As the results, some labeled synovial cells with 3H-thymidine were found. However, no significant difference was observed between the labeling indices of normal and rheumatoid cells labeled with 3H-thymidine. From the results, it was concluded that the synovial cells synthesized DNA in both normal and rheumatoid conditions. However, the quantities of these macromolecules synthesized in these synovial cells varied in respective individuals and no significant difference was found between the labeling indices and grain counts in both normal and rheumatoid cells (Kobayashi and Nagata 1994).

## **2.2 Macromolecular synthesis in the muscular system**

The muscular system consists of various skeletal muscles amounting to around 600 in number in men and less in experimental animals such as rats and mice. We studied the aging changes of DNA synthesis in the intercostal muscles of aging ddY mice from prenatal day 13 through postnatal 24 months by 3H-thymidine RAG (Hayashi et al. 1993).

## **2.2.1 The DNA synthesis in the muscular system**

We studied the aging changes of DNA synthesis in the intercostal muscles of aging ddY mice from prenatal day 13 through postnatal 24 months in senescence by 3H-thymidine RAG (Hayashi et al. 1993). Many nuclei were labeled in myotubes at embryonic day 13-17 (Fig. 4C) during development, then the number of labeled nuclei decreased to embryonic day 18-19 (Fig. 4D), and less due to aging after birth.

Fig. 4. Light microscopic radioautograms of the skeletal muscle cells in the myotubes labeled with 3H-thymidine at embryonic day 13-17 (Fig. 3C), then the number of labeled nuclei decreased to embryonic day 18-19 (Fig. 3D), and less after birth. x260. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 62, 2001, Academic Press, San Diego, USA, London, UK.

RNA syntheses of the synovial cells of the joints surgically obtained from 15 elderly human patients of both sexes aged from 50 to 70, suffering from rheumatoid arthritis (Kobayashi and Nagata 1994). Both the normal and rheumatoid cells were cultured and labeled in vitro with media containing precursors such as 3H-thymidine or 3H-uridine, fixed and radioautographed. DNA synthetic cells labeled with silver grains were observed by LM RAG (light microscopic radioautography) in both normal and rheumatoid cells. As the results, some labeled synovial cells with 3H-thymidine were found. However, no significant difference was observed between the labeling indices of normal and rheumatoid cells labeled with 3H-thymidine. From the results, it was concluded that the synovial cells synthesized DNA in both normal and rheumatoid conditions. However, the quantities of these macromolecules synthesized in these synovial cells varied in respective individuals and no significant difference was found between the labeling indices and grain counts in

The muscular system consists of various skeletal muscles amounting to around 600 in number in men and less in experimental animals such as rats and mice. We studied the aging changes of DNA synthesis in the intercostal muscles of aging ddY mice from prenatal

We studied the aging changes of DNA synthesis in the intercostal muscles of aging ddY mice from prenatal day 13 through postnatal 24 months in senescence by 3H-thymidine RAG (Hayashi et al. 1993). Many nuclei were labeled in myotubes at embryonic day 13-17 (Fig. 4C) during development, then the number of labeled nuclei decreased to embryonic

Fig. 4. Light microscopic radioautograms of the skeletal muscle cells in the myotubes labeled with 3H-thymidine at embryonic day 13-17 (Fig. 3C), then the number of labeled nuclei decreased to embryonic day 18-19 (Fig. 3D), and less after birth. x260. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 62, 2001,

day 13 through postnatal 24 months by 3H-thymidine RAG (Hayashi et al. 1993).

both normal and rheumatoid cells (Kobayashi and Nagata 1994).

**2.2 Macromolecular synthesis in the muscular system** 

**2.2.1 The DNA synthesis in the muscular system** 

day 18-19 (Fig. 4D), and less due to aging after birth.

Academic Press, San Diego, USA, London, UK.

The labeling indices revealed chronological changes, showing a peak at embryonic day 13 and decreasing gradually to 0% at 3 months after birth to month 24 (Fig. 5). We classified the graduation of the embryonic muscle development into 5 stages. Among them, the labeling index (LI) at stage I was the highest, while the LI at stage II was significantly lower than stage I, the LI at stage IV was significantly higher than stage II, and the LI at stage V was significantly lower than stage IV (Fig. 5). These changes accorded well with the primary and

secondary myotube formation during the embryonic muscle development. We also studied the DNA synthesis of rat thigh muscles during the muscle regeneration after injury in rats (Sakai et al. 1977). When the skeletal muscles, i.e., the diaphragma, the rectus abdominis muscles and the gastrocnemius femoris muscle of adult Wistar rats were mechanically injured and labeled with 3H-thymidine, satellite cells were labeled during their regeneration. The satellite cells in the muscles of dystrophy chickens and normal control chickens were also labeled with 3H-thymidine, demonstrating DNA synthesis (Oguchi and Nagata 1980, 1981), which was later described in details in the review (Nagata 2002). Briefly, 2 groups of chickens, 4 dystrophy chickens and normal control chickens of both sexes aged 1 day and 21 days after hatching were used. All the animals received every 6 hrs intraperitoneal injections of 3H-thymidine 4 times successively and sacrificed. The superficial pectoral muscles were taken out, fixed, embedded in Epoxy resin and processed for LM and EMRAG. The results demonstrated that many nuclei of the satellite cells in all the experimental groups were labeled but none of the nuclei in the muscle fibers were labeled. The labeling indices of normal chickens at 1 day and 21 days were 4.59 and 3.86%, respectively. These results showed that the LI decreased after hatching due to aging.

Fig. 5. The labeling indices of the muscular cells labeled with 3H-thymidine revealed chronological changes, showing a peak at embryonic day 13 and decreasing gradually to 0% at 3 months after birth. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 114, 2002, Urban & Fischer, Jena, Germany

Cell Senescence as Observed by Electron Microscopic Radioautography 299

The mature blood cells circulating in the blood vessels of mammals are classified into 3 types, the erythrocytes, the leukocytes and the blood platelets. Those mature cells are formed either in the lymphatic tissues in the lymphatic organs or the myeloid tissues in the bone marrow, where various immature cells, lymphoblasts, erythroblasts, myeloblasts, meylocytes, and megakaryocytes can be observed. Among these blood cells, we studied macromolecular synthesis and cytochemical localization in leukocytes, megakaryocytes and blood platelets. As for the granulocytes, normal rabbit granulocytes were shown by EM RAG and X-ray microanalysis to incorporate 35SO4 into the Golgi apparatus and to the

On the other hand, the DNA, RNA and mucosubstance synthesis of mast cells from Wistar strain rats were studied by 3H-thymidine, 3H-uridine and 35SO4 radioautography, demonstrating incorporation changes of those normal mast cells from abnormal mastocytoma cells (Murata et al. 1977a). Mast cells were widely found distributing in the loose connective tissues of most mammals, as well as in the serous exudate in the peritoneal cavity as one of the free cells. We studied the fine structure and nucleic acid and mucosubstance syntheses of normal mast cells and Dunn and Potter's mastocytoma cells in mice and rats by electron microscopic radioautography (Murata et al. 1977b, 1979). As the results, some of the normal mast cells and mastocytoma cells incorporated 3H-thymidine, 3H-uridine and 35SO4, demonstrating DNA, RNA and mucosubstance syntheses. The incorporation of 3H-thymidine was observed in the nuclei and mitochondria. The labeling index of 3H-thymidine incorporation in the nuclei and mitochondria of normal mast cells was very low (0.37%) while that of mastocytoma cells was high (2-5%). These results suggested that the macromolecular synthesis such as nucleic acids (DNA, RNA) and

The spleen is one of the blood cell forming organs and is composed of the lymphatic tissues. Among the macromolecular synthesis, both the DNA, RNA and protein syntheses in the

We studied 3H-thymidine incorporation into the splenic cells of aging mice from newborn to adult and senescence in connection with the lysosomal acid phosphatase activity (Olea 1991, Olea and Nagata 1991, 1992a). The acid phosphatase activity as demonstrated by means of cerium substrate method was observed in the splenic tissues at various ages from postnatal day 1, week 1 and 2, month 1, 2 and 10. Electron dense deposits were localized in the lysosomes of macrophages, reticular cells and littoral cells in all the aging groups. The intensity of the reaction products as visually observed increased from day 1 to week 1, reaching the peak at 1 week, and decreased from week 2 to month 10 due to aging. The incorporation of 3H-thymidine, on the other hand, demonstrating DNA synthesis, was mainly observed in the hematopoietic cells in the spleens from postnatal day 1 to month 10 animals (Olea and Nagata 1991, 1992a). The labeling index was the maximum at day 1 and decreased to week 1, 2, 4, 8 and 40. A correlation between DNA synthesis and AcPase activity was examined by comparing two cell populations in the cell cycle, the S-phase cells

granules demonstrating glucosaminoglycan synthesis (Murata et al. 1978, 1979).

mucosubstances were higher in tumor cells than normal blood cells.

**3.4 Macromolecular synthesis in the spleen** 

**3.4.1 The DNA synthesis in the spleen** 

spleen were studied.

**3.3 The DNA synthesis in the blood cells** 

#### **2.2.2 The protein synthesis in the muscular system**

We studied only 3H-taurin incorporation in the skeletal muscles of both normal and dystrophy adult mice incubated in Eagle's medium containing 3H-taurin in vitro at varying time intervals from 1 min to 5, 10, 30 and 60 min (1 hour). The silver grains were observed over the skeletal muscle cells as well as over the smooth muscle cells and the endothelial cells in the arteries, both the nuclei and cytoplalsm, by LM and EM RAG, showing taurin incorporation into the proteins (Terauch et al. 1988, Terauch and Nagata 1993, 1994). However, the aging changes were not studied yet.

## **3. Macromolecular synthesis in the circulatory system**

The circulatory system or cardiovascular organs consists of the heart, the arteries, the veins, the capillaries, the blood, the lymphatic organs and the spleen. Among these cardiovascular organs, we studied the heart, the artery, some blood cells and the spleen.

#### **3.1 The nucleic acid synthesis in the heart**

Among the macromolecular synthesis, the nucleic acid synthesis, both DNA and RNA, in cultured cells from the hearts of chick embryos was studied by LM RAG (Nagata and Nawa 1966a,b). The fibroblasts of chick hearts in culture proliferated extensively and produced many binucleate cells. We compared the nucleic acid synthesis in mononucleate cells and binucleate cells in the heart fibroblasts. The incorporation of 3H-thymidine into each nucleus of a binucleate cell was a little less than that of a mononucleate cell, but the total of the two nuclei of a binucleate cell was almost twice as that of a mononucleate cell. The incorporation of 3H-uridine in the two nuclei of a binucleate cell was almost twice as that of a mononucleate cell, while the incorporation of 3H-uridine in the cytoplasm of a binucleate cell was not so much as twice as a mononucleate cell. From these results, it was concluded that the nucleic acid synthesis both DNA and RNA increased in binucleate cells than mononucleate cells of chick embryo heart fibroblasts (Nagata and Nawa 1966a,b).

#### **3.2 Localization of drugs in the artery**

The structure of the blood vessels, both arteries and veins consist of 3 layers, from inside to outside, the tunica intima, the media and the adventitia. Those layers are formed with connective tissues and the smooth muscles. We studied the localization of antihypertensive drugs in the supramesenteric arteries of the spontaneous hypertensive rats (Suzuki et al. 1994). Two kinds of anti-hypertensive drugs, labeled with RI, 3H-benidipine hydrochloride (Kyowa Hakko Kogyo Co., Shizuoka, Japan) and 3H-nitrendipine (New England Nuclear, Boston, MA, USA) were used. Both intravenous administrations into rats and in vitro incubation for 10 to 30 min were employed. For light and electron microscopic radioautography, both the conventional wet-mounting radioautograms after chemical fixation for insoluble compounds and the dry-mounting radioautograms after cryo-fixation and freeze-substitution for soluble compounds were prepared. The silver grains due to the anti-hypertensive drugs were localized over the plasma membranes and the cytoplasm of the fibrocytes in the intima and the smooth muscle cells in the media, suggesting the pharmacological active sites. However, the localization of synthetic DNA was not studied.

## **3.3 The DNA synthesis in the blood cells**

298 Senescence

We studied only 3H-taurin incorporation in the skeletal muscles of both normal and dystrophy adult mice incubated in Eagle's medium containing 3H-taurin in vitro at varying time intervals from 1 min to 5, 10, 30 and 60 min (1 hour). The silver grains were observed over the skeletal muscle cells as well as over the smooth muscle cells and the endothelial cells in the arteries, both the nuclei and cytoplalsm, by LM and EM RAG, showing taurin incorporation into the proteins (Terauch et al. 1988, Terauch and Nagata 1993, 1994).

The circulatory system or cardiovascular organs consists of the heart, the arteries, the veins, the capillaries, the blood, the lymphatic organs and the spleen. Among these cardiovascular

Among the macromolecular synthesis, the nucleic acid synthesis, both DNA and RNA, in cultured cells from the hearts of chick embryos was studied by LM RAG (Nagata and Nawa 1966a,b). The fibroblasts of chick hearts in culture proliferated extensively and produced many binucleate cells. We compared the nucleic acid synthesis in mononucleate cells and binucleate cells in the heart fibroblasts. The incorporation of 3H-thymidine into each nucleus of a binucleate cell was a little less than that of a mononucleate cell, but the total of the two nuclei of a binucleate cell was almost twice as that of a mononucleate cell. The incorporation of 3H-uridine in the two nuclei of a binucleate cell was almost twice as that of a mononucleate cell, while the incorporation of 3H-uridine in the cytoplasm of a binucleate cell was not so much as twice as a mononucleate cell. From these results, it was concluded that the nucleic acid synthesis both DNA and RNA increased in binucleate cells than

mononucleate cells of chick embryo heart fibroblasts (Nagata and Nawa 1966a,b).

The structure of the blood vessels, both arteries and veins consist of 3 layers, from inside to outside, the tunica intima, the media and the adventitia. Those layers are formed with connective tissues and the smooth muscles. We studied the localization of antihypertensive drugs in the supramesenteric arteries of the spontaneous hypertensive rats (Suzuki et al. 1994). Two kinds of anti-hypertensive drugs, labeled with RI, 3H-benidipine hydrochloride (Kyowa Hakko Kogyo Co., Shizuoka, Japan) and 3H-nitrendipine (New England Nuclear, Boston, MA, USA) were used. Both intravenous administrations into rats and in vitro incubation for 10 to 30 min were employed. For light and electron microscopic radioautography, both the conventional wet-mounting radioautograms after chemical fixation for insoluble compounds and the dry-mounting radioautograms after cryo-fixation and freeze-substitution for soluble compounds were prepared. The silver grains due to the anti-hypertensive drugs were localized over the plasma membranes and the cytoplasm of the fibrocytes in the intima and the smooth muscle cells in the media, suggesting the pharmacological active sites. However, the localization of synthetic DNA

**2.2.2 The protein synthesis in the muscular system** 

However, the aging changes were not studied yet.

**3.1 The nucleic acid synthesis in the heart** 

**3.2 Localization of drugs in the artery** 

was not studied.

**3. Macromolecular synthesis in the circulatory system** 

organs, we studied the heart, the artery, some blood cells and the spleen.

The mature blood cells circulating in the blood vessels of mammals are classified into 3 types, the erythrocytes, the leukocytes and the blood platelets. Those mature cells are formed either in the lymphatic tissues in the lymphatic organs or the myeloid tissues in the bone marrow, where various immature cells, lymphoblasts, erythroblasts, myeloblasts, meylocytes, and megakaryocytes can be observed. Among these blood cells, we studied macromolecular synthesis and cytochemical localization in leukocytes, megakaryocytes and blood platelets. As for the granulocytes, normal rabbit granulocytes were shown by EM RAG and X-ray microanalysis to incorporate 35SO4 into the Golgi apparatus and to the granules demonstrating glucosaminoglycan synthesis (Murata et al. 1978, 1979).

On the other hand, the DNA, RNA and mucosubstance synthesis of mast cells from Wistar strain rats were studied by 3H-thymidine, 3H-uridine and 35SO4 radioautography, demonstrating incorporation changes of those normal mast cells from abnormal mastocytoma cells (Murata et al. 1977a). Mast cells were widely found distributing in the loose connective tissues of most mammals, as well as in the serous exudate in the peritoneal cavity as one of the free cells. We studied the fine structure and nucleic acid and mucosubstance syntheses of normal mast cells and Dunn and Potter's mastocytoma cells in mice and rats by electron microscopic radioautography (Murata et al. 1977b, 1979). As the results, some of the normal mast cells and mastocytoma cells incorporated 3H-thymidine, 3H-uridine and 35SO4, demonstrating DNA, RNA and mucosubstance syntheses. The incorporation of 3H-thymidine was observed in the nuclei and mitochondria. The labeling index of 3H-thymidine incorporation in the nuclei and mitochondria of normal mast cells was very low (0.37%) while that of mastocytoma cells was high (2-5%). These results suggested that the macromolecular synthesis such as nucleic acids (DNA, RNA) and mucosubstances were higher in tumor cells than normal blood cells.

### **3.4 Macromolecular synthesis in the spleen**

The spleen is one of the blood cell forming organs and is composed of the lymphatic tissues. Among the macromolecular synthesis, both the DNA, RNA and protein syntheses in the spleen were studied.

## **3.4.1 The DNA synthesis in the spleen**

We studied 3H-thymidine incorporation into the splenic cells of aging mice from newborn to adult and senescence in connection with the lysosomal acid phosphatase activity (Olea 1991, Olea and Nagata 1991, 1992a). The acid phosphatase activity as demonstrated by means of cerium substrate method was observed in the splenic tissues at various ages from postnatal day 1, week 1 and 2, month 1, 2 and 10. Electron dense deposits were localized in the lysosomes of macrophages, reticular cells and littoral cells in all the aging groups. The intensity of the reaction products as visually observed increased from day 1 to week 1, reaching the peak at 1 week, and decreased from week 2 to month 10 due to aging. The incorporation of 3H-thymidine, on the other hand, demonstrating DNA synthesis, was mainly observed in the hematopoietic cells in the spleens from postnatal day 1 to month 10 animals (Olea and Nagata 1991, 1992a). The labeling index was the maximum at day 1 and decreased to week 1, 2, 4, 8 and 40. A correlation between DNA synthesis and AcPase activity was examined by comparing two cell populations in the cell cycle, the S-phase cells

Cell Senescence as Observed by Electron Microscopic Radioautography 301

Clermont Y.: The contractime elements in the limiting membrane of the seminiferous

Cui, H.: Light microscopic radioautographic study on DNA synthesis of nerve cells in the

Cui, H., Gao, F., Nagata, T.: Light microscopic radioautographic study on protein synthesis in perinatal mice corneas. Acta Histochem. Cytochem. 33, 31-37, 2000. Duan, H., Gao, F., Li, S., Hayashi, K., Nagata, T.: Aging changes and fine structure and DNA

Duan, H., Gao, F., Li, S., Nagata, T.: Postnatal development of esophageal epithelium in

Duan, H., Gao, F., Oguchi, K., Nagata, T.: Light and electron microscopic radioautographic

Feulgen, R., Rossenbeck, H.: Mikroskopische-chemischer Nachweis einer Nukeinsaeure von Thymus der Thymonukeinsaeure Z. Physik. Chem. 135, 203-248, 1924. Fujii, Y., Ohno, S., Yamabayashi, S., Usuda, N., Saito, H., Furuta, S., Nagata, T.: Electron

Gao, F.: Study on the macromolecular synthesis in aging mouse seminiferous tubules by light and electron microscopic radioautography. Cell. Mol. Biol. 39, 659-672, 1993. Gao, F., Toriyama, K., Nagata, T.: Light microscopic radioautographic study on the DNA

Gao, F., Li, S., Duan, H., Ma, H., Nagata, T.: Electron microscopic radioautography on the

Gao, F., Toriyama, K., Ma, H., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in aging mice corneas. Cell. Mol. Biol. 39, 435-441, 1993. Gao, F., Ma, H., Sun, L., Jin, C., Nagata, T.: Electron microscopic radioautographic study on

Gao, F., Chen, S., Sun, L., Kang, W., Wang, Z., Nagata, T.: Radioautographic study of the

Gao, F., Jin, C., Ma, H., Sun, L., Nagata, T.: Ultrastructural and radioautographic studies on

Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick embryo. I. Light microscopic radioautography. Shinshu Med. J. 32, 231-240, 1984a. Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick

IgG myeloma patient. J. Clin. Electr. Microsc. 13, 582-583, 1980.

synthesis of esophageal epithelium in neonatal, adult and old mice. J. Clin. Electron

mouse: a light and electron microscopic radioautographic study. Cell. Mol. Biol. 39,

study on the incorporation of 3H-thymidine into the lung by means of a new

microscopic radioautography of DNA synthesis in primary cultured cells from an

synthesis of prenatal and postnatal aging mouse retina after labeled thymidine

DNA synthesis of prenatal and postnatal mice retina after labeled thymidine

the nucleic acids and protein synthesis in the aging mouse testis. Med. Electron

macromolecular synthesis of Leydig cells in aging mice testis. Cell. Mol. Biol. 41,

DNA synthesis in Leydig cells of aging mouse testis. Cell. Mol. Biol. 41, 151-160,

embryo. II. Electron microscopic radioautography. Shinshu Med. J. 32, 241-248,

Clermont, Y.: Renewal of spermatogonia in man. Amer. J. Anat. 112, 35-51, 1963.

cerebella of aging mice. Cell. Mol. Biol. 41, 1139-1154, 1995.

tubules of rats. Exp. Cell Res. 15, 438-342, 1958.

Microsc. 25, 452-453, 1992.

nebulizer. Drug Res. 44, 880-883, 1994.

injection. Cell. Mol. Biol. 38, 661-668, 1992a.

Microsc. 27, 360-362, 1994.

145-150, 1995a.

1995b.

1984b.

injection. J. Clin. Electron Microsc. 25, 721-722, 1992b.

309-316, 1993.

which were labeled with 3H-thymidine and the non-S-phase cells or the interphase cells which were not labeled. It was demonstrated that the former showed an increase and decrease of much more AcPase activity with the aging while the latter less activity and no change.

#### **3.4.2 The RNA synthesis in the spleen**

On the other hand, the number of labeled cells and the grain counts in the hematopoietic cells in the spleens labeled with 3H-uridine, demonstrating RNA synthesis, from postnatal day 1 increased to 1 and 2 weeks, reaching the maximum, and decreased to 4, 8 and 40 weeks, different from the DNA synthesis (Olea and Nagata 1992b). These results demonstrated that AcPase activity, DNA and RNA synthetic activity changed due to aging.

#### **3.4.3 The protein synthesis in the spleen**

Among the circulatory organs, we first studied the protein synthesis in the spleens of aging mice at various ages. Several groups of litter mates, each 3, from fetal day 19 to postnatal day 1, 14, and month 6 to 12 (year 1) were administered with 3H-leucine and sacrificed, the spleens were taken out and processed for LM and EM RAG (Nagata and Olea 1999). The results demonstrated that the sites of incorporations were hematopoietic cells, i.e., lymphoblasts, myeloblasts, erythroblasts and littoral cells in the splenic tissues at every aging stage. In most labeled cells silver grains were observed over the nuclei, nucleoli, endoplasmic reticulum, ribosomes, Golgi apparatus and mitochondria. Quantitative analysis revealed that grain counts in respective cells were higher in young animals than adult aged animals. The grain counts and the labeling index increased from prenatal to postnatal day 14, reaching the maximum, then decreased to month 12. These results showed the increase and decrease due to aging of animals.

#### **4. Conclusion**

This chapter deals with the introductory remarks describing the method and procedure of microscopic radioautography as well as the first parts of its application to the organ systems such as the skeletal, muscular and the circulatory organs. The method and the procedure of microscopic radioautography described the detailed technology which was developed by the present author and associates since 1955 in our laboratory. The application of radaioautography to the skeletal, muscular and circulatory systems demonstrated the sites of macromolecular syntheses such as DNA, RNA, proteins, glucides and lipids in various organs as well as the quantitative changes due to aging of the experimental animals. These results should be very important to understand the fundamental changes in the respective bones, the skeletal muscles, and the cardiovascular organs as well as the contributions to the experimental biology and medicine throughout the world.

#### **5. References**

Chen, S., Gao, F., Kotani, A., Nagata, T.: Age-related changes of male mouse submandibular gland: A morphometric and radioautographic study. Cell. Mol. Biol. 41, 117-124, 1995.

which were labeled with 3H-thymidine and the non-S-phase cells or the interphase cells which were not labeled. It was demonstrated that the former showed an increase and decrease of much more AcPase activity with the aging while the latter less activity and no

On the other hand, the number of labeled cells and the grain counts in the hematopoietic cells in the spleens labeled with 3H-uridine, demonstrating RNA synthesis, from postnatal day 1 increased to 1 and 2 weeks, reaching the maximum, and decreased to 4, 8 and 40 weeks, different from the DNA synthesis (Olea and Nagata 1992b). These results demonstrated that AcPase activity, DNA and RNA synthetic activity changed due to aging.

Among the circulatory organs, we first studied the protein synthesis in the spleens of aging mice at various ages. Several groups of litter mates, each 3, from fetal day 19 to postnatal day 1, 14, and month 6 to 12 (year 1) were administered with 3H-leucine and sacrificed, the spleens were taken out and processed for LM and EM RAG (Nagata and Olea 1999). The results demonstrated that the sites of incorporations were hematopoietic cells, i.e., lymphoblasts, myeloblasts, erythroblasts and littoral cells in the splenic tissues at every aging stage. In most labeled cells silver grains were observed over the nuclei, nucleoli, endoplasmic reticulum, ribosomes, Golgi apparatus and mitochondria. Quantitative analysis revealed that grain counts in respective cells were higher in young animals than adult aged animals. The grain counts and the labeling index increased from prenatal to postnatal day 14, reaching the maximum, then decreased to month 12. These results showed

This chapter deals with the introductory remarks describing the method and procedure of microscopic radioautography as well as the first parts of its application to the organ systems such as the skeletal, muscular and the circulatory organs. The method and the procedure of microscopic radioautography described the detailed technology which was developed by the present author and associates since 1955 in our laboratory. The application of radaioautography to the skeletal, muscular and circulatory systems demonstrated the sites of macromolecular syntheses such as DNA, RNA, proteins, glucides and lipids in various organs as well as the quantitative changes due to aging of the experimental animals. These results should be very important to understand the fundamental changes in the respective bones, the skeletal muscles, and the cardiovascular organs as well as the contributions to the

Chen, S., Gao, F., Kotani, A., Nagata, T.: Age-related changes of male mouse submandibular

gland: A morphometric and radioautographic study. Cell. Mol. Biol. 41, 117-124,

change.

**3.4.2 The RNA synthesis in the spleen** 

**3.4.3 The protein synthesis in the spleen** 

the increase and decrease due to aging of animals.

experimental biology and medicine throughout the world.

**4. Conclusion** 

**5. References** 

1995.


Cell Senescence as Observed by Electron Microscopic Radioautography 303

Kobayashi, K., Nagata, T.: Light microscopic radioautographic studies on DNA, RNA and

Komiyama, K., Iida, F., Furihara, R., Murata, F., Nagata, T.: Electron microscopic

Kong, Y.: Electron microscopic radioautographic study on DNA synthesis in perinatal

Kong, Y., Nagata, T.: Electron microscopic radioautographic study on nucleic acid synthesis

Kong, Y., Usuda, N., Nagata, T.: Radioautographic study on DNA synthesis of the retina

Kong, Y., Usuda, N., Morita, T., Hanai, T., Nagata, T.: Study on RNA synthesis in the retina

Leblond, C. P.: Localization of newly administered iodine in the thyroid gland as indicated

Leblond, C. P.: The life history of cells in renewing systems. Am. J. Anat. 160, 113-158, 1981. Leblond, C. P., Messier, B.: Renewal of chief cells and goblet cells in the small intestine as

Li, S.: Relationship between cellular DNA synthesis, PCNA expression and sex steroid

Li, S., Nagata, T.: Nucleic acid synthesis in the developing mouse ovary, uterus and oviduct

Li, S., Gao, F., Duan, H., Nagata, T.: Radioautographic study on the uptake of 35SO4 in mouse ovary during the estrus cycle. J. Clin. Electron Microsc. 25, 709-710, 1992. Liang, Y.: Light microscopic radioautographic study on RNA synthesis in the adrenal glands

Liang, Y., Ito, M., Nagata, T.: Light and electron microscopic radioautographic studies on

Ma, H.: Light microscopic radioautographic study on DNA synthesis of the livers in aging

Ma, H., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of the

Ma, H., Nagata, T.: Studies on DNA synthesis of aging mice by means of electron microscopic radioautography. J. Clin. Electron Microsc. 21, 715-716, 1988b. Ma, H., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis in the

Ma, H., Nagata, T.: Study on RNA synthesis in the livers of aging mice by means of electron

livers in aging mice. J. Clin. Electron Microsc. 21, 335-343, 1988a.

microscopic radioautography. Cell. Mol. Biol. 36, 589-600, 1990b.

of aging mice. Acta Histochem. Cytochem. 31, 203-210, 1998.

of perinatal mouse retina. Med. Electron Microsc. 27, 366-368, 1994.

Histochem. Cytochem. 42, 982-982, 1994.

mouse retina. Cell. Mol. Biol. 39, 55-64, 1993.

Electron Microsc. 11, 428-429, 1978.

263-272, 1992a.

132: 247-259. 1958.

195, 1995.

1999.

1990a.

Mol. Biol. 38, 669-678, 1992b.

by radioiodine. J. Anat. 77, 149-152, 1943.

Histochemistry 102, 405-413, 1994.

mice. Acta Anat. Nippon. 63, 137-147, 1988.

protein syntheses in human synovial membranes of rheumatoid arthritis patients. J.

radioautographic study on 125I-albumin in rat gastric mucosal epithelia. J. Clin.

and retinal pigment epithelium of developing mouse embryo. Cell. Mol. Biol. 38,

and retinal pigment epithelium of mice by light microscopic radioautography. Cell.

shown by radioautography after injection of thymidine-3H into mice. Anat. Rec.

hormone receptor status in the developing mouse ovary, uterus and oviduct.

studied by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 185-

RNA synthesis in aging mouse adrenal gland. Acta Anat. Nippon. 74, 291-300,

hepatocytes of aging mice as observed by image analysis. Cell. Mol. Biol. 36, 73-84,


Gunarso, W., Gao, F., Cui, H., Ma, H., Nagata, T.: A light and electron microscopic

Gunarso, W., Gao, F., Nagata, T.: Development and DNA synthesis in the retina of chick

Hanai, T.: Light microscopic radioautographic study of DNA synthesis in the kidneys of

Hanai, T., Nagata, T.: Electron microscopic radioautographic study on DNA and RNA

Hanai, T., Nagata, T.: Study on the nucleic acid synthesis in the aging mouse kidney by light

Nagata, T., Ed., pp. 209-214, Shinshu University Press, Matsumoto, 1994b. Hanai, T., Nagata, T.: Electron microscopic study on nucleic acid synthesis in perinatal

Hanai, T., Usuda, N., Morita, T., Shimizu, T., Nagata, T.: Proliferative activity in the kidneys

Hayashi, K., Gao, F., Nagata, T.: Radioautographic study on 3H-thymidine incorporation at

Ito, M.: The radioautographic studies on aging change of DNA synthesis and the

Ito, M., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis and

Izumiyama, K., Kogure, K., Kataoka, S., Nagata, T.: Quantitative analysis of glucose after

Jamieson, J. D., Palade, G. E.: Intracellular transport of secretory proteins in the pancreatic

Jin, C.: Study on DNA synthesis of aging mouse colon by light and electron microscopic

Jin, C., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. J. Histochem. Cytochem. 43, 1223-1228, 1995a. Jin, C., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. Med. Electron Microsc. 28, 71-75, 1995b. Joukura, K.: The aging changes of glycoconjugate synthesis in mouse kidney studied by 3Hglucosamine radioautography. Acta Histochem. Cytochem. 29, 57-63, 1996. Joukura, K., Nagata, T.: Aging changes of 3H-glucosamine incorporation into mouse kidney observed by radioautography. Acta Histochem. Cytochem. 28, 494-494, 1995. Joukura, K., Usuda, N., Nagata, T.: Quantitative study on the aging change of

radioautography. Brain Res. 416, 175-179, 1987.

radioautography. Cell. Mol. Biol. 42, 255-268, 1996.

exocrine cells. J. Cell Biol. 34, 577-615, 1967.

Ed., pp. 127-131, Shinshu University Press, Matsumoto, 1994a.

mouse kidney tissue. Med. Electron Microsc. 27, 355-357, 1994c.

Histochem. 98, 309-32, 1996.

aging mice. Cell. Mol. Biol. 39, 81-91, 1993.

Biol. 43, 189-201, 1997.

39, 181-191, 1993.

1993.

1996.

1996.

radioautographic study on RNA synthesis in the retina of chick embryo. Acta

embryo observed by light and electron microscopic radioautography. Cell. Mol.

synthesis in perinatal mouse kidney. In, Radioautography in Medicine, Nagata, T.,

and electron microscopic radioautography. In, Radioautography in Medicine,

of aging mice evaluated by PCNA/cyclin immunohistochemistry. Cell. Mol. Biol.

different stages of muscle development in aging mice. Cell. Mol. Biol. 39, 553-560,

ultrastructural development of mouse adrenal gland. Cell. Mol. Biol. 42, 279-292,

ultrastructure of aging mouse adrenal gland. Med. Electron Microsc. 29, 145-152,

transient ischemia in the gerbil hippocampus by light and electron microscope

glycoconjugates synthesis in aging mouse kidney. Proc. Xth Internat. Cong. Histochem. Cytochem., Acta Histochem. Cytochem. 29, Suppl. 507-508, 1996.


Cell Senescence as Observed by Electron Microscopic Radioautography 305

Nagata, T.: A quantitative study on the ganglion cells in the small intestine of the dog. Med.

Nagata, T.: A radioautographic study on the RNA synthesis in the hepatic and the intestinal

Nagata, T.: On the increase of binucleate cells in the ganglion cells of dog small intestine due

Nagata, T.: A radioautographic study on the protein synthesis in the hepatic and the

Nagata, T.: Chapter 3. Application of microspectrophotometry to various substances. In ,

Nagata, T.: Electron microscopic dry-mounting autoradiography. Proc. 4th Internat. Cong.

Nagata, T.: Electron microscopic radioautography of intramitochondrial RNA synthesis of

Nagata, T.: Quantitative electron microscope radioautography of intramitochondrial nucleic

Nagata, T.: Electron microscopic observation of target cells previously observed by phase-

Nagata, T.:. Electron microscopic radioautography and analytical electron microscopy. J.

Nagata, T.: Radiolabeling of soluble and insoluble compounds as demonstrated by light and

Nagata, T.: Quantitative analysis of histochemical reactions: Image analysis of light and

Nagata, T. Quantitative light and electron microscopic radioautographic studies on

Nagata, T.: Electron microscopic radioautography with cryo-fixation and dry-mounting

Nagata, T.: Application of electron microscopic radioautography to clinical electron

Nagata, T.: Radioautography in Medicine. Shinshu University Press, 268pp, Matsumoto,

Nagata, T.: Radioautography, general and special. In, Histo- and Cyto-chemistry 1994, Japan

Society of Histochemistry and Cytochemistry, ed, pp. 219-231, Gakusai Kikaku Co.,

irradiated cultured cells. J. Clin. Electron Microsc. 17, 589-590, 1984. Nagata, T.: Principles and techniques of radioautography. In, Histo- and Cyto-chemistry

to experimental ischemia. Med. J. Shinshu Univ. 12, 93-113, 1967a.

epithelial cells of mice after feeding with special reference to binuclearity. Med. J.

intestinal epithelial cells of mice, with special reference to binucleate cells. Med. J.

Introduction to Microspectrophotometry. Isaka, S., Nagata, T., Inui, N., Eds.,

contrast microscopy: Electron microscopic radioautography of laser beam

1985, Japan Society of Histochemistry and Cytochemistry, Ed., Gakusai Kikaku Co.,

electron microscopy. Recent Advances in Cellular and Molecular Biology, Wegmann, R. J., Wegmann, M. A., Eds. Peters Press, Leuven, Vol. 6, pp. 9-21, 1992.

electron microscopic radioautograms. Acta Histochem. Cytochem. 26, 281-291,

macromolecular synthesis in several organs of prenatal and postnatal aging mice.

J. Shinshu Univ. 10, 1-11, 1965.

Shinshu Univ. 11, 49-61, 1966.

Shinshu Univ. 12, 247-257, 1967b.

Tokyo, pp. 207-226, 1985.

1993a.

1994c.

Tokyo, 1994d.

Olympus Co., Tokyo, pp. 49-155, 1972a.

Clin. Electron Microsc. 24, 441-442, 1991.

Chinese J. Histochem. Cytochem. 2: 106-108, 1993b.

procedure. Acta Histochem. Cytochem. 27: 471-489, 1994a.

microscopy. Med. Electron Microsc. 27; 191-212, 1994b.

Histochem. Cytochem. Kyoto, pp. 43-44, 1972b.

HeLa cells in culture. Histochemie 32, 163-170, 1972c.

acid synthesis. Acta Histochem. Cytochem. 5, 201-203, 1972d.


Ma, H., Nagata, T.: Collagen and protein synthesis in the livers of aging mice as studied by

Ma, H., Gao, F., Olea, M. T., Nagata, T.: Protein synthesis in the livers of aging mice studied by electron microscopic radioautography. Cell. Mol. Biol. 37, 607-615, 1991. Matsumura, H., Kobayashi, Y., Kobayashi, K., Nagata, T.: Light microscopic

Momose, Y., Nagata, T.: Radioautographic study on the intracellular localization of a

Momose, Y., Naito, J., Nagata, T.: Radioautographic study on the localization of an antiallergic agent, tranilast, in the rat liver. Cell. Mol. Biol. 35, 347-355, 1989. Momose, Y., Shibata, N., Kiyosawa, I., Naito, J., Watanabe, T., Horie, S., Yamada, J., Suga, T.,

Momose, Y., Naito, J., Suzawa, H., Kanzawa, M., Nagata, T.: Radioautographic study on the

Morita, T.: Radioautographic study on the aging change of 3H-glucosamine uptake in mouse

Morita, T., Usuda, N. Hanai, T., Nagata, T.: Changes of colon epithelium proliferation due to

Murata, F., Momose,Y. , Yoshida, K., Nagata, T.: Incorporation of 3H-thymidine into the nucleus of mast cells in adult rat peritoneum. Shinshu Med. J. 25, 72-77, 1977a. Murata, F., Momose, Y., Yoshida, K., Ohno, S., Nagata, T.: Nucleic acid and mucosubstance

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Ultrastructural and electron microscopic

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Mucosubstances of rabbit granulocytes studied

Nagata, T.: On the relationship between cell division and cytochrome oxidase in the Yoshida

Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. I. Observation on the smear preparation under normal conditions. Med. J. Shinshu Univ. 2: 187-198, 1957a. Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. II. Phase-contrast

Nagata, T.: Cell divisions in the liver of the fetal and newborn dogs. Med. J. Shinshu Univ. 4:

Nagata, T.: A radioautographic study of the DNA synthesis in rat liver, with special

reference to binucleate cells. Med. J. Shinshu Univ. 7, 17-25, 1962.

thymidine radioautography. Histochemistry 101, 13-20, 1994.

radioautography. Acta Pharmacol. Toxicol. 41, 58-59, 1977b.

radioautographic study of DNA synthesis in the lung of aging salamander,

hypolipidemic agent, bezafibrate, a peroxisome proliferator, in cultured rat

Nagata, T.: Morphometric evaluation of species differences in the effects of bezafibrate, a hypolipidemic agent, on hepatic peroxisomes and mitochondria. J.

intracellular localization of bezafibrate in cultured rat hepatoctyes. Acta Histochem.

individual aging with PCNA/cyclin immunostaining comparing with 3H-

metabolism of mastocytoma cells by means of electron microscopic

radioautographic studies on the mastocytoma cells and mast cells. J. Clin. Electron

by means of electron microscopic radioautography and X-ray microanalysis.

microscopic observations under normal conditions. Med. J. Shinshu Univ. 2: 199-

electron microsopic radioautography. Ann. Microsc. 1, 13-22, 2000.

Hynobius nebulosus. J. Histochem. Cytochem. 42, 1004-1004, 1994.

hepatocytes. Cell. Mol. Biol. 39, 773-781, 1993a.

Toxicol. Pathol. 6, 33-45, 1993b.

ileum. Cell. Mol. Biol. 39, 875-884, 1993.

Cytochem. 28, 61-66, 1995.

Microsc. 11, 561-562, 1978.

207, 1957b.

65-73, 1959.

Histochemistry 61, 139-150, 1979.

sarcoma cells. Shinshu Med. J. 5: 383-386, 1956.


Cell Senescence as Observed by Electron Microscopic Radioautography 307

Nagata, T.: Radioautographology, general and special: a novel concept. Ital. J. Anat.

Nagata, T.: Three-dimensional observations on thick biological specimens by high voltage

Nagata, T.: Biological microanalysis of radiolabeled and unlabeled compounds by

Nagata, T.: Electron microscopic radioautographic study on protein synthesis in pancreatic cells of perinatal and aging mice. Bull. Nagano Women's Jr. College 8, 1-22, 2000c. Nagata, T.: Light microscopic radioautographic study on radiosulfate incorporation into the tracheal cartilage in aging mice. Acta Histochem. Cytochem. 32, 377-383, 2000d. Nagata, T.: Introductory remarks: Special radioautographology. Cell. Mol. Biol. 46 (Congress

Nagata, T.: Three-dimensional high voltage electron microscopy of thick biological

Nagata, T.: Three-dimensional and four-dimensional observation of histochemical and

Nagata, T. : Special cytochemistry in cell biology. In, Internat. Rev. Cytol. Jeon, K.W., ed.,

Nagata, T. : Radioautographology General and Special, In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 37 No. 2, Urban & Fischer, Jena, pp. 57-226, 2002. Nagata T.: Light and electron microscopic study on macromolecular synthesis in amitotic hepatocyte mitochondria of aging mice. Cell. Mol. Biol. 49, 591-611, 2003. Nagata, T.: X-ray microanalysis of biological specimens by high voltage electron

Nagata T.: Aging changes of macromolecular synthesis in the uro-genital organs as revealed by electron microscopic radioautography. Ann. Rev. Biomed. Sci. 6, 13-78, 2005. Nagata T.: Electron microscopic radioautographic study on protein synthesis in hepatocyte

Nagata T.: Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata, T.: Electron microscopic radioautographic study on macromolecular synthesis in hepatocyte mitochondria of aging mouse. J. Cell Tissue Res. 7, 1019-1029, 2007c.

hepatocyte mitochondria of developing mice. The Sci. World J. 6: 1583-1598, 2006b.

by electron microscopic radioautography. I: Nucleic acid synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. Eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 245-

by electron microscopic radioautography. II: Protein synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 259-271,

mitochondria of developing mice. Ann. Microsc. 6, 43-54, 2006a.

cytochemical specimens by high voltage electron microscopy. Acta Histochem.

microscopy. In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 39, No. 4,

radioautography and X-ray microanalysis. Scanning Microscopy International, 14,

electron microscopy. Image Analysis Stereolog. 19, 51-56, 2000a.

Nagata, T.: Special radioautographology: the eye. J. Kaken Eye Res. 18, 1-13, 2000f.

Vol. 211, Chapter 2, Academic Press, New York, pp. 33-154, 2001c.

Embryol. 104 (Suppl. 1), 487-487, 1999e.

on line, 2000b.

258, 2007a.

2007b.

Suppl.), 161-161, 2000e.

specimens. Micron 32, 387-404, 2001a.

Urban & Fischer Verlag, Jena, pp. 185-320, 2004.

Cytochem. 34, 153-169, 2001b.


Nagata, T., Application of electron microscopic radioautography to clinical electron

Nagata, T.: Light and electron microscopic radioautographic study on macromolecular synthesis in digestive organs of aging mice. Cell. Mol. Biol. 41, 21-38, 1995a. Nagata, T.: Histochemistry of the organs: Application of histochemistry to anatomy. Acta

Nagata, T.: Three-dimensional observation of whole mount cultured cells stained with

Nagata, T.: Morphometry in anatomy: image analysis on fine structure and histochemical

Nagata, T.: Technique and application of electron microscopic radioautography. J. Electron

Nagata, T.: Techniques of light and electron microscopic radioautography. In,

Nagata, T.: Remarks: Radioautographology, general and special. Cell. Mol. Biol. 42 (Suppl.),

Nagata, T.: On the terminology of radioautography vs. autoradiography. J. Histochem.

Nagata, T.: Techniques and applications of microscopic radioautography. Acta Microsc. Vol.

Nagata T.: Three-dimensional observation on whole mount cultured cells and thick sections

Nagata, T.: Radioautographic study on collagen synthesis in the ocular tissues. J. Kaken Eye

Nagata, T.: Techniques of radioautography for medical and biological research. Braz. J. Biol.

Nagata, T.: Radioautographology, the advocacy of a new concept. Braz. J. Biol. Med. Res. 31,

Nagata, T.: Radioautographic studies on DNA synthesis of the bone and skin of aging

Nagata, T.: 3D observation of cell organelles by high voltage electron microscopy.

Nagata, T.: Application of histochemistry to anatomy: Histochemistry of the organs, a novel

Nagata, T.: Aging changes of macromolecular synthesis in various organ systems as

concept. Proc. XV Congress of the International Federation of Associations of

observed by microscopic radioautography after incorporation of radiolabeled

salamander. Bull. Nagano Women's Jr. College 6, 1-14, 1998c.

Microscopy and Analysis, Asia Pacific Edition, 9, 29-32, 1999a.

Anatomists, Ital. J. Anat. Embryol. 104 (Suppl. 1), 486-486, 1999b.

precursors. Methods Find. Exp. Clin. Pharmacol. 21, 683-706, 1999c. Nagata, T.: Radioautographic study on protein synthesis in mouse cornea. J. Kaken Eye Res.

stained with histochemical reactions by high voltage electron microscopy. In, Recent Advances in Microscopy of Cells, Tissues and Organs, Motta, P., Ed.,

Cytochem. Acta Histochem. Cytochem. 29 (Suppl.), 343-344, 1996b.

histochemical reactions by ultrahigh voltage electron microscopy. Cell. Mol. Biol.

reactions with special reference to radioautography. Ital. J. Anat. 100 (Suppl. 1),

Histochemistry and Cytochemistry 1996. Proc. Xth Internat. Congr. Histochem.

microscopy. Med. Electron Microsc. 27, 191-212, 1994e.

Anat. Nippon. 70, 448-471, 1995b.

41, 783-792, 1995c.

591-605, 1995d.

11-12, 1996c.

Microsc. 45, 258-274, 1996a.

Cytochem. 44, 1209-1209, 1996d.

Antonio Delfino Editore, Roma, pp. 37-44, 1997b.

6: Suppl. B. p. 19-42, 1997a.

Med. Res. 31, 185-195, 1998a.

Res. 15, 1-9, 1997c.

201-241, 1998b.

8, 8-14, 1999d.


Cell Senescence as Observed by Electron Microscopic Radioautography 309

Nagata, T.: Macromolecular synthesis in the livers of aging mice as revealed by electron

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata, T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA, RNA and

Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 12, 1-29, 2010h. Nagata, T., Cui, H., Gao, F.: Radioautographic study on glycoprotein synthesis in the ocular

Nagata, T., Cui, H., Kong, Y.: The localization of TGF-b1 and its mRNA in the spinal cords

Nagata, T., Cui, H., Liang, Y.: Light microscopic radioautographic study on the protein

Nagata, T., Fujii, Y., Usuda, N.: Demonstration of extranuclear nucleic acid synthesis in

Nagata, T., Ito, M., Chen, S.: Aging changes of DNA synthesis in the submandibular glands

Nagata, T. Ito, M., Liang, Y.: Study of the effects of aging on macromolecular synthesis in

Nagata, T., Iwadare, I., Murata, F.: Electron microscopic radioautography of nucleic acid

Nagata, T., Kawahara, I.: Radioautographic study of the synthesis of sulfomucin in digestive

Nagata, T., Kawahara, I., Usuda, N., Maruyama, M., Ma, H.: Radioautographic studies on

organs of mice. J. Trace Microprobe Analysis 17, 339-355, 1999.

Diego, St. Louis, Vol. 45, No. 1, pp. 1-80, 2010c.

Vol. 3, Formatex, Badajoz, Spain, in press, 2010g.

tissues. J. Kaken Eye Res. 13, 11-18, 1995.

Alternat Med Welfare 5, 25-37, 2010d.

Med. Welfare 5, 38-52, 2010f.

232, 2010e.

41-60 (2001).

1966.

Microsc. 1, 4-12, 2000a.

Toxicol. 41, 64-65, 1977c.

Exp. Clin. Pharmacol. 22, 5-18, 2000b.

microscopic radioautography. In, Prog. Histochem. Cytochem., Sasse, D., Ed., Elsevier, Amsterdam, Boston, London, New York, Oxford, Paris, Philadelphia, San

mitochondria in adrenal medullary cells of aging mice. Bulletin Shinshu Inst

adrenal cortical and medullary cells of aging mice. J. Biomed. Sci. Enginer. 4, 219-

mitochondria in adrenal cortical cells of aging mice. Bulletin Shinshu Inst. Alternat.

protein synthesis in adrenal cells of aging mice. Formatex Microscopy Series No. 3,

of prenatal and postnatal aging mice demonstrated with immunohistochemical and in situ hybridization techniques. Bull. Nagano Women's Jr. College, 7, 75-88, 1999a.

synthesis in the cerebellum of aging mouse. Bull. Nagano Women's Jr. College, 9,

mammalian cells under experimental conditions by electron microscopic radioautography. Proc. 10th Internat. Congr. Electr. Microsc. 2, 305-306, 1982b. Nagata, T., Hirano, I., Shibata, O., Nagata, T.: A radioautographic study on the DNA

synthesis in the hepatic and the pancreatic acinar cells of mice during the postnatal growth, with special reference to binuclearity. Med. J. Shinshu Univ. 11, 35-42,

of mice as observed by light and electron microscopic radioautography. Ann.

mouse steroid secreting cells using microscopic radioautography. Methods Find.

synthesis in cultured cells treated with several carcinogens. Acta Pharmacol.

the glycoconjugate synthesis in the gastrointestinal mucosa of the mouse. In,


Nagata, T.; Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata, T.; Aging changes of macromolecular synthesis in the mitochondria of mouse

Nagata, T.: Sexual difference between the macromolecular synthesis of hepatocyte

Nagata, T.: Protein synthesis in hepatocytes of mice as revealed by electron microscopic

Nagata, T.: Electron microscopic radioatuographic studies on macromolecular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata, T.: Applications of high voltage electron microscopy to thick biological specimens.

Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of mitochondria in adrenal medullary cells of aging mice. Open Anat. J. 1, 14-24, 2009g. Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 11, 1-17, 2009h. Nagata, T.: Electron microscopic radioautographic studies on macromoleclular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata T. Electron microscopic radioautographic study on mitochondrial RNA synthesis in

adrenocortical cells of aging mice. Open Anat J. 2, 91-97, 2010a.

Eds., Nova Biomed. Books, New York, pp. 133-161, 2009b.

Radiopharmaceutics 2, 118-128, 2009d.

Ann. Microsc. 9, 4-40, 2009f.

Welfare 4, 15-38, 2009i.

Welfare 4, 51-66, 2009j.

2222, 2010b.

Nagata, T.: Radioautographology, Bull. Shinshu Institute Alternat. Med. 2, 3-32, 2007f. Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in adrenal cortical cells of developing mice. J. Cell. Tis. Res. 8, 1303-1312, 2008a. Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in

2007d.

2008b.

2009a

1802, 2009e.

30-36, 2007e.

hepatocyte mitochondria of developing mice. Trends Cell Molec. Biol. 2, 19-33,

hepatocytes as revealed by microscopic radioautography. Ann. Rev. Biomed. Sci. 9,

adrenal cortical cells of developing and aging mice. The Sci. World J. 8, 683-697.

mitochondria in male and female mice in aging as revealed by electron microscopic radioautography. Chapter 22. In, Women and Aging: New Research, H. T. Bennninghouse, A. D. Rosset, Eds. Nova Biomed. Books, New York, pp. 461-487,

radioautography. In, Protein Biosynthesis. Esterhouse, T. E. and Petrinos, L. B.,

mitochondria of various cells. 18EMSM Conference Proc. 9th Asia-Pacific Microscopy Conference (APMC9), Kuala Lumpur, Malaysia, pp. 48-50, 2009c. Nagata, T.: Recent studies on macromolecular synthesis labeled with 3H-thymidine in

various organs as revealed by electron microscopic radioautography. Current

in adrenal medullary cells of developing and aging mice. J. Cell Tissue Res. 9, 1793-

mitochondria of some organs in aging animals. Bull. Shinshu Inst. Alternat. Med.

in adreno-cortical cells of aging ddY mice. Bull. Shinshu Inst. Alternat. Med.

adrenal medullary cells of aging and senescent mice. J Cell Tissue Res. 10, 2213-


Cell Senescence as Observed by Electron Microscopic Radioautography 311

Nagata, T., Shimamura, K., Kondo, T., Onozawa, M., Momoze, S., Okubo, M.: Relationship

Nagata, T., Steggerda, F. R.: Histological study on the deganglionated small intestine of the

Nagata, T., Steggerda, F. R.: Observations on the increase of binucleate cells in the ganglion

Nagata, T., Toriyama, K., Kong, Y., Jin, C., Gao, F.: Radioautographic study on DNA synthesis in the ciliary bodies of aging mice. J. Kaken Eye Res.12, 1-11, 1994. Nagata, T., Usuda, N.: Image processing of electron microscopic radioautograms in clinical

Nagata, T., Usuda, N.: Studies on the nucleic acid synthesis in pancreatic acinar cells of

Nagata, T., Usuda, N.: Electron microscopic radioautography of protein synthesis in

Nagata, T., Usuda, N.: In situ hybridization by electron microscopy using radioactive

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of nucleic acid

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of lipid synthesis in pancreatic cells of aging mice. J. Clin. Electr. Microsc. 23, 841-842, 1990. Nagata, T., Usuda, N., Maruyama, M., Ma, H.: Electron microscopic radioautographic study

Nagata, T., Usuda, N., Suzawa, H., Kanzawa, M.: Incorporation of 3H-glucosamine into the

Nagata, T., Yamabayashi, S.: Intracellular localization of 3H-befunolol by means of electron

Nagata, T., Yoshida, K., Murata, F.: Demonstration of hot and cold mercury in the human

Nagata, T., Yoshida, K., Ohno, S., Murata, F.: Ultrastructural localization of soluble and

radioautography. J. Clin. Electron Microsc. 25, 646-647, 1992.

electron microscopy. J. Clin. Electron. Microsc. 18, 451-452, 1985.

probes. J. Histochem. Cytochem. 41, 1119-1119, 1993b.

Intern. Cong. Electr. Microsc. 3, 2281-2282, 1984.

1960a.

1964.

1993a.

1988b.

1978b.

153-158, 1960b.

dog. Physiologist 6, 242-242, 1963.

Microsc. 19, 486-487, 1986.

Microsc. 16, 737-738, 1983.

Toxicol. 41, 60-61, 1977b.

some organs of toads in summer and winter. Med. J. Shinshu Univ. 5, 147-152,

of binuclearity to cell function in some organs. II. Variation of frequencies of binucleate cells in some organs of dogs owing to aging. Med. J. Shinshu Univ. 5,

cells of the dog's intestine due to experimental ischemia. Anat. Rec. 148, 315-315,

aging mice by means of electron microscopic radioautography. J. Clin. Electron

pancreatic acinar cells of aging mice. Acta Histochem. Cytochem. 26, 481-481,

synthesis in pancreatic acinar cells of prenatal and postnatal aging mice. Proc. XIth

on lipid synthesis in perinatal mouse pancreas. J. Clin. Electr. Microsc. 21, 756-757,

pancreatic cells of aging mice as demonstrated by electron microscopic

microscopic radioautography of cryo-fixed ultrathin sections. J. Clin. Electron

thyroid tissues by means of radioautography and chemography. Acta Pharmacol.

insoluble 3H-methyl prednisolone as revealed by electron microscopic drymounting radioautography. Proc. 9th Internat. Congr. Electr. Microsc. 2, 40-41,

Glycoconjugate in Medicine, Ohyama, M., Muramatsu, T., Eds, pp. 251-256, Professional Postgrad. Service, Tokyo, 1988a.


Nagata, T., Kong, Y.: Distribution and localization of TGFb1 and bFGF, and their mRNAs in

Nagata, T., Ma, H., Electron microscopic radioautographic study on mitochondrial DNA synthesis in hepatocytes of aging mouse. Ann. Microsc. 5, 4-18, 2005a. Nagata, T., Ma, H., Electron microscopic radioautographic study on RNA synthesis in hepatocyte mitochondria of aging mouse. Microsc. Res. Tech. 67, 55-64, 2005b. Nagata, T., Momoze, S.: Aging changes of the amitotic and binucleate cells in dog livers.

Nagata, T., Morita, T., I. Kawahara, I.: Radioautographic studies on radiosulfate incorporation in the digestive organs of mice. Histol. Histopathol. 14, 1-8, 1999b. Nagata, T., Murata, F.: Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy. Histochemistry 54, 75-82, 1977. Nagata, T., Murata, F., Yoshida, K., Ohno, S., Iwadare, N.: Whole mount radioautography of

Nagata, T., Nawa, T.: A modification of dry-mounting technique for radioautography of

Nagata, T., Nawa, T.: A radioautographic study on the nucleic acids synthesis of binucleate

Nagata, T., Nawa, T., Yokota, S.: A new technique for electron microscopic dry-mounting radioautography of soluble compounds. Histochemie 18, 241-249, 1969. Nagata, T., Nishigaki, T., Momose, Y.: Localization of anti-allergic agent in rat mast cells

Nagata, T., Ohno, S., Kawahara, I., Yamabayashi, S., Fujii, Y., Murata, F.: Light and electron

Nagata, T., Ohno, S., Murata, F.: Electron microscopic dry-mounting radioautography for

Nagata, T., Ohno, S., Yoshida, K., Murata, F.: Nucleic acid synthesis in proliferating

Nagata, T., Olea, M. T.: Electron microscopic radioautographic study on the protein synthesis in aging mouse spleen. Bull. Nagano Women's Jr. College 7, 1-9, 1999. Nagata, T., Shibata, O., Omochi, S.: A new method for radioautographic osbservation on

Nagata, T., Shibata, O., Nawa, T.: Simplified methods for mass production of

Nagata, T., Shibata, O., Nawa, T.: Incorporation of tritiated thymidine into mitochondrial

Nagata, T., Shimamura, K., Onozawa, M., Kondo, T., Ohkubo, K., Momoze, S.: Relationship

DNA of the liver and kidney cells of chickens and mice in tissue culture.

of binuclearity to cell function in some organs. I. Frequencies of binucleate cells in

soluble compounds. Acta Phamacol. Toxicol. 41, 62-63, 1977a.

cultured cells as observed by high voltage electron microscopy. Proc. Fifth Internat.

cells in cultivated fibroblasts of chick embryos. Med. J. Shinshu Univ. 11, 1-5, 1966b.

demonstrated by light and electron microscopic radioautography. Acta Histochem.

microscopic radioautography of nucleic acid synthesis in mitochondria and peroxisomes of rat hepatic cells during and after DEHP administration. Acta

peroxisomes of rat liver as revealed by electron microscopical radioautography.

aging mice. Bull. Nagano Women's Jr. College 6, 87-105, 1998.

Professional Postgrad. Service, Tokyo, 1988a.

Acta Anat. Nipponica 34, 187-190, 1959.

Cytochem. 19, 669-683, 1986b.

Histochem. J. 14, 197-204, 1982a.

Histochemie 10, 305-308, 1967b.

Histochem. Cytochem. 16, 610-611, 1979.

isolated cells. Histochemie 2, 255-259, 1961

radioautograms. -Acta Anat. Nippon.42, 162-166, 1967a.

Conf. High Voltage Electron Microsc. 347-350, 1977d.

water-soluble compounds. Histochemie 7, 370-371, 1966a.

Glycoconjugate in Medicine, Ohyama, M., Muramatsu, T., Eds, pp. 251-256,

some organs of toads in summer and winter. Med. J. Shinshu Univ. 5, 147-152, 1960a.


Cell Senescence as Observed by Electron Microscopic Radioautography 313

Sato, A.: Quantitative electron microscopic studies on the kinetics of secretory granules in G-

Sato, A., Iida, F., Furihara, R., Nagata, T.: Electron microscopic raioautography of rat

Shimizu, T., Usuda, N., Yamanda, T., Sugenoya, A., Iida, F.: Proliferative activity of human

Sun, L.: Age related changes of RNA synthesis in the lungs of aging mice by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 1061-1072, 1995. Sun, L., Gao, F., Duan, H., Nagata, T.: Light microscopic radioautography of DNA synthesis

Sun, L., Gao, F., Nagata, T.: Study on the DNA synthesis of pulmonary cells in aging mice by light microscopic radioautography. Cell. Mol. Biol. 41, 851-859, 1995a. Sun, L., Gao, F., Jin, C., Duan, H., Nagata, T.: An electron microscopic radioautographic

Sun, L., Gao, F., Jin, C., Nagata, T.: DNA synthesis in the tracheae of aging mice by means of

Sun, L., Gao, F., Nagata, T.: A Light Microscopic radioautographic study on protein

Suzuki, K., Imada, T., Gao, F., Ma, H., Nagata, T.: Radioautographic study of benidipine

Terauchi, A., Mori, T., Kanda, H., Tsukada, M., Nagata, T.: Radioautographic study of 3H-

Terauchi, A., Nagata, T.: Observation on incorporation of 3H-taurine in mouse skeletal

Terauchi, A., Nagata, T.: In corporation of 3H-taurine into the blood capillary cells of mouse

Toriyama, K.: Study on the aging changes of DNA and protein synthesis of bipolar and

Tsukahara, S., Yoshida, K., Nagata, T.: A radioautographic study on the incorporation of

Usuda, N., Nagata, T.: Electron microscopic radioautography of acyl-CoA mRNA by in situ

Usuda, N., Nagata, T.: The immunohistochemical and in situ hybridization studies on hepatic peroxisomes. Acta Histochem. Cytochem. 28, 169-172, 1995.

radioautography. Cell. Mol. Biol. 41, 593-601, 1995.

hybridization. J. Clin. Electron Microsc. 25, 332-333, 1992.

immnohistochemical studies. Cancer 71, 2807-2812, 1993.

pp. 201-205, Shinshu University Press, Matsumoto, 1994.

stomach G-cells by means of 3H-amino acids. J. Clin. Electron Microsc. 10, 358-359,

thyroid tumors evaluated by proliferating cell nuclear antigen/cyclin

in pulmonary cells in aging mice. In, Radioautography in Medicine, Nagata, T. Ed.,

study on the DNA synthesis of pulmonary tissue cells in aging mice. Med. Electron.

light and electron microscopic radioautography. Acta Histochem. Cytochem. 30,

synthesis in pulmonary cells of aging mice. Acta Histochem. Cytochem. 30, 463-

hydrochloride: localization in the mesenteric artery of spontaneously hypertensive

taurine uptake in mouse skeletal muscle cells. J. Clin. Electron Microsc. 21, 627-628,

muscle cells by light and electron microscopic radioautography. Cell. Mol. Biol. 39,

skeletal muscle. Radioautography in Medicine, Nagata, T. ed., Shinshu University

photo-receptor cells of mouse retina by light and electron microscopic

14C-bupranolol (beta-blocking agent) into the rabbit eye. Histochemistry 68, 237-

cells. Cell Tissue Res. 187, 45-59, 1978.

Microsc. 28, 129-131, 1995b.

rat. Drug Res. 44, 129-133, 1994.

211-220, 1997a.

470, 1997b.

1988.

397-404, 1993.

244, 1980.

Press, Matsumoto, 1994.

1977.


Nishigaki, T., Momose, Y., Nagata, T.: Light microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Electron microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Localization of the anti-allergic agent tranilast in the

Oguchi, K., Nagata, T.: A radioautographic study of activated satellite cells in dystrophic

Oguchi, K., Nagata, T.: Electron microscopic radioautographic observation on activated

Ohno, S., Fujii, Y., Usuda, N., Endo, T., Hidaka, H., Nagata, T.: Demonstration of

Ohno, S., Fujii, Y., Usuda, N., Nagata, T., Endo, T., Tanaka, T., Hidaka, H.: Intracellular

Olea, M. T.: An ultrastructural localization of lysosomal acid phosphatase activity in aging

Olea, M. T., Nagata, T.: X-ray microanalysis of cerium in mouse spleen cells demonstrating

Olea, M. T., Nagata, T. : Simultaneous localization of 3H-thymidine incorporation and acid

Olea, M. T., Nagata, T.: A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro. Cell. Mol. Biol. 38, 399-405, 1992b. Oliveira, S. F., Nagata, T., Abrahamsohn, P. A., Zorn, T. M. T.: Electron microscopic

Oliveira, S. F., Abrahamsohn, P. A., Nagata, T., Zorn, T. M. T.: Incorporation of 3H-amino

Pearse, A. G. E.: Histochemistry, Theoretical and Applied. 4th Ed. Vol. 1. 439 pp., 1980, Vol.

Sakai, Y., Ikado, S., Nagata, T.: Electron microscopic radioautography of satellite cells in

radioautographical study. Cell. Mol. Biol. 41, 107-116, 1995.

regenerating muscles. J. Clin. Electr. Microsc. 10, 508-509, 1977.

536, 1987.

65-71, 1990a.

Tokyo, 1980.

Res. 40, 272-275, 1990b.

Welfare of Japan, Tokyo, 1981.

Co., New York, 1982.

37, 155-163, 1991.

Cytochem. 24, 201-208, 1991.

Cell. Mol. Biol. 38, 115-122, 1992a.

Cell. Mol. Biol. 37, 315-323, 1991.

Edinburgh, London and New York, 1991.

radioautography. J. Electron Microsc. 32, 1-12, 1983.

localization of anti-allergic agent, tranilast, in rat mast cells. Histochem. J. 19, 533-

localization of an anti-allergic agent, tranilast, in rat mast cells. Cell. Mol. Biol. 36,

urinary bladder of rat as demonstrated by light microscopic radioautography. Drug

chicken muscle. In, Current Research in Muscular Dystrophy Japan. The Proc. Ann. Meet. Muscular Dystrophy Res. 1980, pp. 16-17, Ministry of Welfare of Japan,

satellite cells in dystrophy chickens. In, Clinical Studies on the Etiology of Muscular Dystrophy. Annual Report on Neurological Diseases 1981, pp. 30-33, Ministry of

intracellular localization of calmodulin antagonist by wet-mounting

localization of calmodulin antagonists (W-7). In, Calmodulin and intracellular Ca2+ receptors. Kakiuchi, S., Hidaka, H, Means, A. R., Eds., pp. 39-48, Plenum Publishing

mouse spleen: a quantitative X-ray microanalytical study. Acta Histochem.

acid phosphatase activity using high voltage electron microscopy, Cell. Mol. Biol.

phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

radioautographic study on the incorporation of 3H-proline by mouse decidual cells.

acids by endometrial stromal cells during decidualization in the mouse. A

2. 1055 pp., 1985, Vol. 3. Ed. with P. Stoward, 728 pp. Churchill Livingstone,


**15** 

 *Japan* 

Tetsuji Nagata1,2

**Macromolecular Synthesis** 

*Shinshu University School of Medicine, Matsumoto* 

*2Shinshu Institute of Alternative Medicine and Welfare, Nagano* 

*1Department of Anatomy and Cell Biology,* 

**in the Digestive and Respiratory Systems** 

This second chapter deals with the second parts of the application of microscopic radioautography to some of the visceral organ systems. The visceral organs can be divided into 5 organ systems according to anatomy and histology, i.e., the digestive system, the respiratory system, the urinary system, the reproductive system and the endocrine system. Among of them the digestive system consists of 2 parts, i.e., the digestive tract and the digestive glands. The former consists of simple tube structures such as the oral cavity, the esophagus, the stomach and the intestines, while the latter consists of complicated glandular structures such as the large digestive glands , i.e., the liver and the pancreas, while the respiratory system consists of 2 parts, one the respiratory tract such as the nose, the pharynx, the trachea, the bronchus, and final essential part the lungs. This chapter deals

The digestive system consists of the digestive tract and the digestive glands. The digestive tract can be divided into several portions, from the upper part to the lower part, i.e., the oral cavity, the pharynx, the esophagus, the stomach, the small and large intestines and the anus, while the digestive glands consist of the large glands such as the salivary glands, the liver and the pancreas and the small glands affiliated to the digestive tracts in the gastrointestinal walls such as the gastric glands including the fundic gland and the pyloric gland, the intestinal glands of Lieberkühn and the duodenal glands of Brunner. We have published many papers from our laboratory dealing with the macromolecular synthesis in respective digestive organs from the oral cavity to the gastrointestinal tracts and the digestive glands (Nagata 1992, 1993a,b, 1994a,b,c,d,e, 1995a,b,c,d, 1996a,b,c, 1999a,b,c, 2002, Nagata et al. 1979, 1982a, 2000a, Chen et al. 1995). The outline of the results concerning to the macromolecular synthesis in the digestive organs should be here described in the order of

The oral cavity consists of the lips, tongue, teeth, and the salivary glands. The DNA synthesis of mucosal epithelia of the 2 upper and lower lips and the tongue as well as the 3

with the digestive organs and the respiratory organs, respectively.

**1.1 Macromolecular synthesis in the digestive system** 

systematic anatomy and special histology as follows.

**1.1.1 Macromolecular synthesis in the oral cavity** 

**1. Introduction** 


## **Macromolecular Synthesis in the Digestive and Respiratory Systems**

## Tetsuji Nagata1,2

*1Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 2Shinshu Institute of Alternative Medicine and Welfare, Nagano Japan* 

## **1. Introduction**

314 Senescence

Usuda, N., Hanai, T., Morita, T., Nagata, T.: Radioautographic demonstration of

Uwa, H., Nagata, T.: Cell population kinetics of the scleroblast during ethisterone-induced

Watanabe, I., Makiyama, M. C. K., Nagata, T.: Electron microscopic radioautographic

Yamabayashi, S., Gunarso, W., Tsukahara, S., Nagata, T.: Incorporation of 3H-befunolol

Yamada, A., Nagata, T.: Ribonucleic acid and protein synthesis in the uterus of pregnant

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of DNA synthesis in

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of RNA synthesis of

Yoshinaga, K.: Uterine receptivity for blastcyst implantation. Ann. N. Y. Acad. Sci. USA, 541,

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Study of ethionine

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Radioautographic

pp.181-184, Peeters Press, Leuven, 1992.

Microscopica 6. 130-131, 1997.

Cell. Mol. Biol. 39, 1-12, 1993.

window. Cell. Mol. Biol. 38, 763-774, 1992b.

implantation. Cell. Mol. Biol. 38, 211-233, 1993.

9, 693-694, 1976.

365, 1992a.

424-431, 1988.

Microsc. 7, 349-350, 1974.

Electron. Microsc. 10, 372-373, 1977.

peroxisomal acyl-CoA oxidase mRNA by in situ hybridization. In, Recent advances in cellular and molecular biology, Vol. 6. Molecular biology of nucleus, peroxisomes, organelles and cell movement. Wegmann, R. J., Wegmann, M., Eds,

anal-fin process formation in adult females of the Medaka. Dev. Growth Different.

observation of the submandibular salivary gland of aging mouse. Acta

(beta blocking agent) into melanin granules of ocular tissues in the pigmented rabbits. I. Light microscopic radioautography. Histochemistry 73, 371-375, 1981. Yamada, A. T.: Timely and topologically defined protein synthesis in the periimplanting

mouse endometrium revealed by light and electron microscopic radioautography.

mouse during activation of implantation window. Med. Electron Microsc. 27, 363-

the endometria of pregnant-ovariectomized mice during activation of implantation

peri-implanting pregnant mouse during activation of receptivity for blastocyst

pancreatitis by means of electron microscopic radioautography. J. Clin. Electron

study of protein synthesis in pancreatic exocrine cells of alcoholic rats. J. Clin.

This second chapter deals with the second parts of the application of microscopic radioautography to some of the visceral organ systems. The visceral organs can be divided into 5 organ systems according to anatomy and histology, i.e., the digestive system, the respiratory system, the urinary system, the reproductive system and the endocrine system. Among of them the digestive system consists of 2 parts, i.e., the digestive tract and the digestive glands. The former consists of simple tube structures such as the oral cavity, the esophagus, the stomach and the intestines, while the latter consists of complicated glandular structures such as the large digestive glands , i.e., the liver and the pancreas, while the respiratory system consists of 2 parts, one the respiratory tract such as the nose, the pharynx, the trachea, the bronchus, and final essential part the lungs. This chapter deals with the digestive organs and the respiratory organs, respectively.

## **1.1 Macromolecular synthesis in the digestive system**

The digestive system consists of the digestive tract and the digestive glands. The digestive tract can be divided into several portions, from the upper part to the lower part, i.e., the oral cavity, the pharynx, the esophagus, the stomach, the small and large intestines and the anus, while the digestive glands consist of the large glands such as the salivary glands, the liver and the pancreas and the small glands affiliated to the digestive tracts in the gastrointestinal walls such as the gastric glands including the fundic gland and the pyloric gland, the intestinal glands of Lieberkühn and the duodenal glands of Brunner. We have published many papers from our laboratory dealing with the macromolecular synthesis in respective digestive organs from the oral cavity to the gastrointestinal tracts and the digestive glands (Nagata 1992, 1993a,b, 1994a,b,c,d,e, 1995a,b,c,d, 1996a,b,c, 1999a,b,c, 2002, Nagata et al. 1979, 1982a, 2000a, Chen et al. 1995). The outline of the results concerning to the macromolecular synthesis in the digestive organs should be here described in the order of systematic anatomy and special histology as follows.

## **1.1.1 Macromolecular synthesis in the oral cavity**

The oral cavity consists of the lips, tongue, teeth, and the salivary glands. The DNA synthesis of mucosal epithelia of the 2 upper and lower lips and the tongue as well as the 3

Macromolecular Synthesis in the Digestive and Respiratory Systems 317

Fig. 6. LM and EM RAG of the digestive organs. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 118, 2002, Urban &

Fischer, Jena, Germany

large salivary glands and many small glands of aging mice from fetal day 19 to postnatal 2 years were studied by LM and EM RAG labeled with 3H-thymidine. The glucide and glycoprotein syntheses by 3H-glucosamine and radiosulfate incorporations of the submandibular and sublingual glands of aging mice were also studied.

#### **1.1.2 The DNA synthesis in the oral cavity**

We first studied the DNA synthesis of the submandibular glands in 10 groups of aging mice at various ages from embryo to postnatal 2 years (Chen et al. 1995, Nagata et al. 2000a). The submandibular gland of male mouse embryonic day 19 consisted of the glandular acini and duct system (Fig. 5A). The duct system was composed of the juxtaacinar cells (JA), the intercalated duct cells (ICD) and the striated duct cells (SD). Many labeled developing acinar cells (AC), JA and ICD cells were observed. At postnatal day 1 to 3 (Fig. 5B), there was more JA cells and secretory granules than those of former stage. JA cells were cuboidal cells in shape, characterized by small darkly stained granules in the supranuclear cytoplasm and by basophilic mitochondria mostly at the basal half of the cells. JA cells were present at the acinar-intercalated duct junction of the mouse submandibular gland. Many labeled AC, JA, ICD and SD cells were also observed by electron microscopy (Fig. 5C). At postnatal 2 weeks to 3 months, developing immature acinar cells gradually matured to acinar cells, and JA cells increased and granular convoluted duct cells (GCT) appeared.

At postnatal 6 months to 2 years, the GCT cells were very well developed and were composed of the taller cells packed with many granules and became highly convoluted, and only a few labeled cells were found. The aging changes of frequency of 5 main individual cell types in submandibular glands of male mouse from embryonic day 19 to postnatal 2 years of age were counted. On embryonic day 19 of age, the gland consisted of developing acinar cells (49%), intercalated duct cells (37%), juxta-acinar (JA) cells (3%), striated duct (SD) cells (11%). At birth, JA cells increased rapidly to 32%, thereafter decreased gradually. At 1 month of age, JA cells disappeared and granular convoluted tubule (GCT) cells appeared and increased rapidly in number with age. They reached a maximum at 6 months. Then they decreased gradually from 6-21 months. The quantity proportion of acini was relatively stable during these periods. The frequency of ICD cells (Fig. 5C) was the highest (37%) at 1 day after birth. Thereafter it gradually decreased month by month and reached 2.6% at 21 months, while the ratio of SD cells persisted in 7%-12% from embryonic day 19 to postnatal 2 weeks and it disappeared at 3 months after birth. The proliferative activity of the cell population is expressed by the labeling index which is defined as the percentage of labeled nuclei with 3H-thymidine in a given cell population. The labeling index of the entire gland cells increased from 13.6% at embryonic 19 to 18.3% at neonate, when it reached the first peak (Fig. 6A, B). Then it declined to 2.2% at 1 week of age. A second small peak (2.9%) occurred at 2 weeks. Thereafter, the labeling index decreased progressively to less than 1% at 4 weeks of age and then remained low. The analysis of the labeling indices of respective cell types revealed that the first peak at neonate was due to the increased labeling indices of AC, ICD and JA cells, and the second peak at 2 weeks was due to the increase of ICD and SD cells. Thereafter, the labeling index of ICD cells decreased steadily but remained higher than those of any other cell types. Since the labeling index of ICD cells was more than the other cell types and persisted for a long time, it was suggested that ICD cells concerned with the generation of other cell types (Nagata 2002).

large salivary glands and many small glands of aging mice from fetal day 19 to postnatal 2 years were studied by LM and EM RAG labeled with 3H-thymidine. The glucide and glycoprotein syntheses by 3H-glucosamine and radiosulfate incorporations of the

We first studied the DNA synthesis of the submandibular glands in 10 groups of aging mice at various ages from embryo to postnatal 2 years (Chen et al. 1995, Nagata et al. 2000a). The submandibular gland of male mouse embryonic day 19 consisted of the glandular acini and duct system (Fig. 5A). The duct system was composed of the juxtaacinar cells (JA), the intercalated duct cells (ICD) and the striated duct cells (SD). Many labeled developing acinar cells (AC), JA and ICD cells were observed. At postnatal day 1 to 3 (Fig. 5B), there was more JA cells and secretory granules than those of former stage. JA cells were cuboidal cells in shape, characterized by small darkly stained granules in the supranuclear cytoplasm and by basophilic mitochondria mostly at the basal half of the cells. JA cells were present at the acinar-intercalated duct junction of the mouse submandibular gland. Many labeled AC, JA, ICD and SD cells were also observed by electron microscopy (Fig. 5C). At postnatal 2 weeks to 3 months, developing immature acinar cells gradually matured to acinar cells, and JA

At postnatal 6 months to 2 years, the GCT cells were very well developed and were composed of the taller cells packed with many granules and became highly convoluted, and only a few labeled cells were found. The aging changes of frequency of 5 main individual cell types in submandibular glands of male mouse from embryonic day 19 to postnatal 2 years of age were counted. On embryonic day 19 of age, the gland consisted of developing acinar cells (49%), intercalated duct cells (37%), juxta-acinar (JA) cells (3%), striated duct (SD) cells (11%). At birth, JA cells increased rapidly to 32%, thereafter decreased gradually. At 1 month of age, JA cells disappeared and granular convoluted tubule (GCT) cells appeared and increased rapidly in number with age. They reached a maximum at 6 months. Then they decreased gradually from 6-21 months. The quantity proportion of acini was relatively stable during these periods. The frequency of ICD cells (Fig. 5C) was the highest (37%) at 1 day after birth. Thereafter it gradually decreased month by month and reached 2.6% at 21 months, while the ratio of SD cells persisted in 7%-12% from embryonic day 19 to postnatal 2 weeks and it disappeared at 3 months after birth. The proliferative activity of the cell population is expressed by the labeling index which is defined as the percentage of labeled nuclei with 3H-thymidine in a given cell population. The labeling index of the entire gland cells increased from 13.6% at embryonic 19 to 18.3% at neonate, when it reached the first peak (Fig. 6A, B). Then it declined to 2.2% at 1 week of age. A second small peak (2.9%) occurred at 2 weeks. Thereafter, the labeling index decreased progressively to less than 1% at 4 weeks of age and then remained low. The analysis of the labeling indices of respective cell types revealed that the first peak at neonate was due to the increased labeling indices of AC, ICD and JA cells, and the second peak at 2 weeks was due to the increase of ICD and SD cells. Thereafter, the labeling index of ICD cells decreased steadily but remained higher than those of any other cell types. Since the labeling index of ICD cells was more than the other cell types and persisted for a long time, it was suggested that ICD cells concerned with the

submandibular and sublingual glands of aging mice were also studied.

cells increased and granular convoluted duct cells (GCT) appeared.

**1.1.2 The DNA synthesis in the oral cavity** 

generation of other cell types (Nagata 2002).

Fig. 6. LM and EM RAG of the digestive organs. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 118, 2002, Urban & Fischer, Jena, Germany

Macromolecular Synthesis in the Digestive and Respiratory Systems 319

(10-20/cell). The numbers of silver grains at 30 min were less than those at 12 hr. From the results, it was concluded that glycoprotein synthesis was demonstrated in both the submandibular and sublingual glands by radiosulfate incorporation. In the salivary glands the silver grains were more observed in serous cells than mucous cells at 30 min, while in mucous cells more at 12 hr than 30 min after the injection. These results show the time difference of glycoprotein synthesis in the two salivary glands, showing inverse proportion

Fig. 7. Histogram showing the frequencies (A) and labeling indices (B) of the five individual cell types in the submandibular glands of male ddY mice at respective ages. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No.

2, p.118, 2002, Urban & Fischer, Jena, Germany

to DNA synthesis of these cells (Watanabe et al. 1997, Nagata 2002).

Fig. 6A. LMRAG of the submandibular gland of male mouse embryonic day 19 labeled with 3H-thymidine consisted with the glandular acini and duct system. The duct system was composed of juxtaacinar cells (JA), intercalated duct cells (ICD) and striated duct cells (ICD). Many labeled developing acinar cells (AC), JA and ICD cells were observed. x500.

Fig. 6B. LMRAG of the submandibular gland at postnatal day 3, labeled with 3H-thymidine. There were more JA cells and secretory granules than those of former stage (Fig. 5A). x500. Fig. 6C. EMRAG of an ICD cell of a mouse at postnatal day 3, labeled with 3H-thymidine observed by electron microscopy. Many silver grains are observed over the nucleus of an ICD. x10,000.

Fig. 6D. EMRAG of the esophageal epithelial cells of a newborn mouse at postnatal day 1, labeled with 3H-thymidine. Many silver grains are observed over one of the nuclei at left. x10,000.

Fig. 6E. LMRAG of the colonic epithelial cells of a mouse embryo at fetal day 19, labeled with 3H-thymidine. Many silver grains are observed over the nuclei of several epithelial cells in the bottom of the crypt. x800.

Fig. 6F. LMRAG of the ileum epithelial cells labeled with 3H-glucosamine of an old mouse at postnatal 6 months. Many silver gains are localized over the Golgi region of the 3 goblet cells as well as over the cytoplasm of several absorptive columnar epithelial cells. x 1,000. Fig. 6G. LMRAG of the colonic epithelial cells of a mouse at postnatal month 1, labeled with 35SO4 in vitro and radioautographed. x1,000.

Fig. 6H. EMRAG of a goblet cell in the deeper crypt of the colonic epithelial cells of an adult mouse after injection of 35SO4 and radioautographed. Many silver grains are observed over the Golgi region and mucous droplets of the goblet cell, demonstrating the incorporation of radiosulfate into sulfomucins. x4,800.

#### **1.1.3 The glucide synthesis in the oral cavity**

We studied the incorporations of 3H-glucosamine in the submandibular glands of 10 groups of litter mice at various ages. The animals from embryonic day 19, postnatal day 1, 3, 7, 14, and 1, 3, 6 months to 1 and 2 years were sacrificed after administration of 3H-glucosamine and the submandibular glands were processed for LM and EM RAG (Watanabe et al. 1997, Nagata 2002). The results showed that the silver grains appeared over the endoplasmic reticulum, Golgi apparatus and the secretory granules of the acinar cells, demonstrating the glycoprotein synthesis in these cells. Grain counting revealed that the counts increased from the fetal stage at embryonic day 19 to postnatal day 1 to 3, 7, 14, reaching the peak at day 14, then decreased to month 1, 3, 6, to year 1 and 2, showing the aging changes, inverse proportion to DNA synthesis of these cells.

On the other hand, the sulfate uptake and accumulation in sulfomucin in several digestive organs of mice were also studied by light microscopic radioautography (Nagata and Kawahara 1999, Nagata et al. 1999b). Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter of animals was sacrificed at 30 min after the intraperitoneal injections with phosphate buffered Na2 35SO4, and the other litter animals were sacrificed at 12 hr after the injections. Then the submandibular glands and the sublingual glands were taken out, fixed, embedded in epoxy resin, sectioned, radioautographed and analyzed by light microscopy. As the results, many silver grains were observed on serous cells of the salivary glands at 30 min and 12 hr after the injections

Fig. 6A. LMRAG of the submandibular gland of male mouse embryonic day 19 labeled with 3H-thymidine consisted with the glandular acini and duct system. The duct system was composed of juxtaacinar cells (JA), intercalated duct cells (ICD) and striated duct cells (ICD).

Fig. 6B. LMRAG of the submandibular gland at postnatal day 3, labeled with 3H-thymidine. There were more JA cells and secretory granules than those of former stage (Fig. 5A). x500. Fig. 6C. EMRAG of an ICD cell of a mouse at postnatal day 3, labeled with 3H-thymidine observed by electron microscopy. Many silver grains are observed over the nucleus of an

Fig. 6D. EMRAG of the esophageal epithelial cells of a newborn mouse at postnatal day 1, labeled with 3H-thymidine. Many silver grains are observed over one of the nuclei at left.

Fig. 6E. LMRAG of the colonic epithelial cells of a mouse embryo at fetal day 19, labeled with 3H-thymidine. Many silver grains are observed over the nuclei of several epithelial

Fig. 6F. LMRAG of the ileum epithelial cells labeled with 3H-glucosamine of an old mouse at postnatal 6 months. Many silver gains are localized over the Golgi region of the 3 goblet cells as well as over the cytoplasm of several absorptive columnar epithelial cells. x 1,000. Fig. 6G. LMRAG of the colonic epithelial cells of a mouse at postnatal month 1, labeled with

Fig. 6H. EMRAG of a goblet cell in the deeper crypt of the colonic epithelial cells of an adult mouse after injection of 35SO4 and radioautographed. Many silver grains are observed over the Golgi region and mucous droplets of the goblet cell, demonstrating the incorporation of

We studied the incorporations of 3H-glucosamine in the submandibular glands of 10 groups of litter mice at various ages. The animals from embryonic day 19, postnatal day 1, 3, 7, 14, and 1, 3, 6 months to 1 and 2 years were sacrificed after administration of 3H-glucosamine and the submandibular glands were processed for LM and EM RAG (Watanabe et al. 1997, Nagata 2002). The results showed that the silver grains appeared over the endoplasmic reticulum, Golgi apparatus and the secretory granules of the acinar cells, demonstrating the glycoprotein synthesis in these cells. Grain counting revealed that the counts increased from the fetal stage at embryonic day 19 to postnatal day 1 to 3, 7, 14, reaching the peak at day 14, then decreased to month 1, 3, 6, to year 1 and 2, showing the aging changes, inverse

On the other hand, the sulfate uptake and accumulation in sulfomucin in several digestive organs of mice were also studied by light microscopic radioautography (Nagata and Kawahara 1999, Nagata et al. 1999b). Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter of animals was sacrificed at 30 min after the intraperitoneal injections with phosphate buffered Na235SO4, and the other litter animals were sacrificed at 12 hr after the injections. Then the submandibular glands and the sublingual glands were taken out, fixed, embedded in epoxy resin, sectioned, radioautographed and analyzed by light microscopy. As the results, many silver grains were observed on serous cells of the salivary glands at 30 min and 12 hr after the injections

Many labeled developing acinar cells (AC), JA and ICD cells were observed. x500.

ICD. x10,000.

cells in the bottom of the crypt. x800.

radiosulfate into sulfomucins. x4,800.

35SO4 in vitro and radioautographed. x1,000.

**1.1.3 The glucide synthesis in the oral cavity** 

proportion to DNA synthesis of these cells.

x10,000.

(10-20/cell). The numbers of silver grains at 30 min were less than those at 12 hr. From the results, it was concluded that glycoprotein synthesis was demonstrated in both the submandibular and sublingual glands by radiosulfate incorporation. In the salivary glands the silver grains were more observed in serous cells than mucous cells at 30 min, while in mucous cells more at 12 hr than 30 min after the injection. These results show the time difference of glycoprotein synthesis in the two salivary glands, showing inverse proportion to DNA synthesis of these cells (Watanabe et al. 1997, Nagata 2002).

Fig. 7. Histogram showing the frequencies (A) and labeling indices (B) of the five individual cell types in the submandibular glands of male ddY mice at respective ages. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p.118, 2002, Urban & Fischer, Jena, Germany

Macromolecular Synthesis in the Digestive and Respiratory Systems 321

When incorporation of radiosulfate into sulfated complex carbohydrate in rat stomach was studied by labeling with 35SO4 in vivo, silver grains appeared over the glandular cells of the pyloric gland but not those of the fundic gland, demonstrating the mucous synthesis in the former glands (Nagata et al. 1988a, Nagata and Kawahara 1999). The radiosulfate uptake and accumulation in the stomach of mouse were also studied by light microscopic radioautography (Nagata et al. 1999b). Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter animals were sacrificed at 30 min after

were sacrificed 12 hr after the injections. Then the antrum and the fundus tissues of the stomachs were taken out. The tissues were fixed, dehydrated, embedded in epoxy resin, sectioned, radioautographed and analyzed. As the results, many silver grains were observed on the mucosa and submucosa of the stomach at 30 min after the injection. Then at 12 hr after the injection silver grains were observed on some of the fundic glands. The numbers of silver grains observed in the stomach especially over the pyloric glands at 30 min (a few per cell) were less than those (several per cell) at 12 hr. The results showed the time difference of glycoprotein synthesis in the stomach, showing inverse proportion to DNA synthesis

The intestines of mammals are divided into 2 portions, small and large intestines, which can be further divided into several portions, the small intestines into the duodenum, the jejunum and the ileum, while the large intestines into the caecum, the appendix vermiformis, the colon and the rectum. The intestinal tracts in any portions consist of the mucosa covered with columnar epithelial cells including absorptive and secretory cells, the submucosa, the smooth muscular layer and the serosa. We studied the macromolecular synthesis, both the DNA and the proteins in the intestines by LM and EMRAG mainly in the

We studied the DNA synthesis in the intestines by LM and EMRAG mainly in the epithelial cells (Nagata 2002). The DNA synthesis of small and large intestines of mice were studied by 3H-thymidine RAG (Fig. 6E). The cells labeled with 3H-thymidine were localized in the crypts of both small and large intestines, a region defined as the proliferative zone. In the colon of aging mice from fetal to postnatal 2 years, the labeled cells in the columnar epithelia were frequently found in the perinatal groups from embryo to postnatal day 1. However, the labeling indices became constant from the suckling period until senescence (Morita 1993, Morita et al. 1994). On the other hand, we examined the labeling indices of respective cell types in each layer of mouse colon such as columnar epithelial cells, lamina propria, lamina muscularis mucosae, tunica submucosa, inner circular muscle layer, outer longitudinal muscle layer, outer connective tissue and serous membrane of the colon and found that most labeling indices decreased after birth to 2 months except the epithelial cells which kept constant value to senescence (Jin and Nagata 1995a,b, Jin 1996) (Fig. 8). Similar results were also obtained from the cecal tissues of mouse by LM and EMRAG. We also studied immunostaining for PCNA/cyclin and compared to the results obtained from RAG (Morita

35SO4, and the other litter animals

**1.3.3 The glucide synthesis in the stomach** 

(Nagata and Kawahara 1999, Nagata 2002).

**1.4.1 The DNA synthesis in the intestines** 

epithelial cells (Nagata 2002).

**1.4 Macromolecular synthesis in the intestines** 

the intraperitoneal injections with phosphate buffered Na2

#### **1.2 Macromolecular synthesis in the esophagus**

The esophagus is the characteristic digestive tract including all the layers, the mucous membrane covered with the stratified squamous epithelia, the submucosa, the muscular layer and the serosa or adventitia. We studied the DNA synthesis of the esophagus of aging mice labeled with 3H-thymidine by LM and EM RAG (Duan et al. 1992, 1993). The labeled cells were mainly found in the basal layer of the esophageal epithelium (Fig. 6D). By electron microscopy the nuclei and nucleoli of labeled cells were larger than those of unlabeled cells, but contained fewer cell organelles (Duan et al. 1993). The labeling indices in respective aging groups showed a peak at postnatal day 1 and decreased with aging keeping a constant level around a few % from 6 months to 2 years after birth.

#### **1.3 Macromolecular synthesis in the stomach**

The stomach consists of the mucosa covered with the surface epithelia of the columnar epithelia, including the gastric glands, the submucosa, the muscular layer and the serosa.

#### **1.3.1 DNA synthesis in the stomach**

As for the turnover of fundic glandular cells shown by 3H-thymidine radioautography, it was extensively investigated with LM RAG by Leblond and co-workers (Leblond 1981, Leblond et al. 1958). They demonstrated that the DNA synthesis in the stomach increased at perinatal stages and decreased due to aging and senescence. However, the activity never reached zero but low activity continued until senescence. We studied the macromolecular synthesis including DNA, RNA, protein and glycoproteins in the gastric mucosa of both human and animal tissues by LM and EMRAG (Sato et al. 1977). As for the DNA synthesis, we obtained the same results as Leblond et al (1958, 1981). We have not carried out this study so much. Therefore, the minute details will be here omitted.

#### **1.3.2 Protein synthesis in the stomach**

We observed the secretion process in G-cells by EM RAG using 3H-amino acid (Sato 1978, Sato et al. 1977, Komiyama et al. 1978). When the stomach tissues were taken out from the adult Wistar rats at postnatal month 1 and were labeled with either 3H-glutamic acid or 3H-glycine in vitro at varying time intervals, silver grains in the EM radioautograms appeared first over the Golgi zones, then migrated to secretory granules and were stored in the cytoplasm, suggesting the secretory kinetics. We also studied the mechanism of serum albumin passing through the gastric epithelial cells into the gastric cells by EM RAG (Sato et al. 1977). When adult Wistar rat stomach tissues were labeled with 132I-albumin in vitro at varying time intervals, silver grains in the radioautograms appeared over rough endoplasmic reticulum within 3 min, then moved to the Golgi apparatus in 10 min, and on to secretory granules and into the lumen in 30 min, suggesting the pathway of serum albumin absorption from the blood vessels through the gastric mucous epithelial cells into the gastric lumen (Komiyama et al. 1978). These results demonstrated that the stomach cells of adult rats synthesized proteins and secreted. However, aging changes of these protein synthesis between the young and senescent animal were not yet completed.

## **1.3.3 The glucide synthesis in the stomach**

320 Senescence

The esophagus is the characteristic digestive tract including all the layers, the mucous membrane covered with the stratified squamous epithelia, the submucosa, the muscular layer and the serosa or adventitia. We studied the DNA synthesis of the esophagus of aging mice labeled with 3H-thymidine by LM and EM RAG (Duan et al. 1992, 1993). The labeled cells were mainly found in the basal layer of the esophageal epithelium (Fig. 6D). By electron microscopy the nuclei and nucleoli of labeled cells were larger than those of unlabeled cells, but contained fewer cell organelles (Duan et al. 1993). The labeling indices in respective aging groups showed a peak at postnatal day 1 and decreased with aging

The stomach consists of the mucosa covered with the surface epithelia of the columnar epithelia, including the gastric glands, the submucosa, the muscular layer and the serosa.

As for the turnover of fundic glandular cells shown by 3H-thymidine radioautography, it was extensively investigated with LM RAG by Leblond and co-workers (Leblond 1981, Leblond et al. 1958). They demonstrated that the DNA synthesis in the stomach increased at perinatal stages and decreased due to aging and senescence. However, the activity never reached zero but low activity continued until senescence. We studied the macromolecular synthesis including DNA, RNA, protein and glycoproteins in the gastric mucosa of both human and animal tissues by LM and EMRAG (Sato et al. 1977). As for the DNA synthesis, we obtained the same results as Leblond et al (1958, 1981). We have not carried out this

We observed the secretion process in G-cells by EM RAG using 3H-amino acid (Sato 1978, Sato et al. 1977, Komiyama et al. 1978). When the stomach tissues were taken out from the adult Wistar rats at postnatal month 1 and were labeled with either 3H-glutamic acid or 3H-glycine in vitro at varying time intervals, silver grains in the EM radioautograms appeared first over the Golgi zones, then migrated to secretory granules and were stored in the cytoplasm, suggesting the secretory kinetics. We also studied the mechanism of serum albumin passing through the gastric epithelial cells into the gastric cells by EM RAG (Sato et al. 1977). When adult Wistar rat stomach tissues were labeled with 132I-albumin in vitro at varying time intervals, silver grains in the radioautograms appeared over rough endoplasmic reticulum within 3 min, then moved to the Golgi apparatus in 10 min, and on to secretory granules and into the lumen in 30 min, suggesting the pathway of serum albumin absorption from the blood vessels through the gastric mucous epithelial cells into the gastric lumen (Komiyama et al. 1978). These results demonstrated that the stomach cells of adult rats synthesized proteins and secreted. However, aging changes of these protein synthesis between the young and senescent

keeping a constant level around a few % from 6 months to 2 years after birth.

study so much. Therefore, the minute details will be here omitted.

**1.2 Macromolecular synthesis in the esophagus** 

**1.3 Macromolecular synthesis in the stomach** 

**1.3.1 DNA synthesis in the stomach** 

**1.3.2 Protein synthesis in the stomach** 

animal were not yet completed.

When incorporation of radiosulfate into sulfated complex carbohydrate in rat stomach was studied by labeling with 35SO4 in vivo, silver grains appeared over the glandular cells of the pyloric gland but not those of the fundic gland, demonstrating the mucous synthesis in the former glands (Nagata et al. 1988a, Nagata and Kawahara 1999). The radiosulfate uptake and accumulation in the stomach of mouse were also studied by light microscopic radioautography (Nagata et al. 1999b). Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter animals were sacrificed at 30 min after the intraperitoneal injections with phosphate buffered Na2 35SO4, and the other litter animals were sacrificed 12 hr after the injections. Then the antrum and the fundus tissues of the stomachs were taken out. The tissues were fixed, dehydrated, embedded in epoxy resin, sectioned, radioautographed and analyzed. As the results, many silver grains were observed on the mucosa and submucosa of the stomach at 30 min after the injection. Then at 12 hr after the injection silver grains were observed on some of the fundic glands. The numbers of silver grains observed in the stomach especially over the pyloric glands at 30 min (a few per cell) were less than those (several per cell) at 12 hr. The results showed the time difference of glycoprotein synthesis in the stomach, showing inverse proportion to DNA synthesis (Nagata and Kawahara 1999, Nagata 2002).

## **1.4 Macromolecular synthesis in the intestines**

The intestines of mammals are divided into 2 portions, small and large intestines, which can be further divided into several portions, the small intestines into the duodenum, the jejunum and the ileum, while the large intestines into the caecum, the appendix vermiformis, the colon and the rectum. The intestinal tracts in any portions consist of the mucosa covered with columnar epithelial cells including absorptive and secretory cells, the submucosa, the smooth muscular layer and the serosa. We studied the macromolecular synthesis, both the DNA and the proteins in the intestines by LM and EMRAG mainly in the epithelial cells (Nagata 2002).

## **1.4.1 The DNA synthesis in the intestines**

We studied the DNA synthesis in the intestines by LM and EMRAG mainly in the epithelial cells (Nagata 2002). The DNA synthesis of small and large intestines of mice were studied by 3H-thymidine RAG (Fig. 6E). The cells labeled with 3H-thymidine were localized in the crypts of both small and large intestines, a region defined as the proliferative zone. In the colon of aging mice from fetal to postnatal 2 years, the labeled cells in the columnar epithelia were frequently found in the perinatal groups from embryo to postnatal day 1. However, the labeling indices became constant from the suckling period until senescence (Morita 1993, Morita et al. 1994). On the other hand, we examined the labeling indices of respective cell types in each layer of mouse colon such as columnar epithelial cells, lamina propria, lamina muscularis mucosae, tunica submucosa, inner circular muscle layer, outer longitudinal muscle layer, outer connective tissue and serous membrane of the colon and found that most labeling indices decreased after birth to 2 months except the epithelial cells which kept constant value to senescence (Jin and Nagata 1995a,b, Jin 1996) (Fig. 8). Similar results were also obtained from the cecal tissues of mouse by LM and EMRAG. We also studied immunostaining for PCNA/cyclin and compared to the results obtained from RAG (Morita

Macromolecular Synthesis in the Digestive and Respiratory Systems 323

different time intervals after feeding. The animals of the first group were injected with 3Huridine at 9 a.m. and fed at 10 a.m. for 30 min. and sacrificed at 11 a.m. 1 hour after the feeding and 2 hours after the injection, the 2nd group was sacrificed at 1 p.m. 3 hours after feeding and 4 hours after the injection, the 3rd group at 5 p.m., 7 and 8 hours later, the 4th group at 9 a.m. on the next day 23 and 24 hours later, and finally the 5th group at 1. p.m. on the next day 3 hours after refeeding and 28 hours after the injection. Then, the jejunums obtained from each animal were prepared for isolated cell radioautograms according to Nagata et al. (1961). The results demonstrated that the grain counts in mononucleate villus cells reached the maximum (20-30 grains per cell) 4 hours after injection and decreased (10- 20/cell) after 28 hours, while the counts in mononucleate villus cells only increased gradually from 4 hours (10/cell) to 28 hours (20/cell). In contrast to this, the grain counts of binucleate cells which appeared in villus cells increased parallely to the mononucleate villus cells (10-20/cell). It was concluded that the RNA synthesis in the jejunal epithelial cells was high in the following order: mononucleate crypt cells, binucleate cells and mononucleate villus cells. These results revealed that the feeding or refeeding affected the RNA synthesis

We first studied the incorporations of 3H-leucine and 3H-tryptophane in mouse small intestines in connection to the binuclearity before and after feeding (Nagata 1967b). The results showed that the incorporations of both amino acids were greater in binucleate intestinal epithelial columnar cells than mononucleate villus and crypt cells at both before and after feeding. However, the aging changes of these incorporations were not studied.

We also studied the aging changes of glucide synthesis by 3H-glucosamine uptake in the small intestines of mouse (Morita 1993), and found that the silver grains in the ileum columnar epithelial cells were mainly localized over the brush borders and the Golgi regions in these cells (Fig. 6F). The grain counting revealed that the numbers of silver grains over the brush borders and cytoplasm of the columnar epithelial cells increased in the villi (10-15/cell) than in the crypts (1-2/cell) from 6 months up to 2 years due to aging. The grain counting in other cell types also revealed that the number of silver grains in goblet cells, basal granulate cells, Paneth

The glycoprotein synthesis in goblet cells as well as in absorptive epithelial cells was also studied using 35SO4 incorporation in the duodenums, the jejunums and the colons of adult mice at varying time intervals at 30, 60, and 180 min after the administration (Nagata et al. 1988a, Nagata and Kawahara 1999, Nagata et al. 1999b). Silver grains were localized over the columnar absorptive cells and the goblet cells, especially over the Golgi regions and mucous granules of the goblet cells. By EM RAG the intracellular localization of silver grains in goblet cells was clearly shown in the Golgi apparatus. The results from grain counting revealed that the average grain counts were different in the upper and deeper regions of the crypts in the 4 portions and it was shown that silver grains over goblet cells in the lower region of the crypt transferred rapidly from 30 min to 180 min, while they transferred slowly in goblet cells in the upper region of the colonic crypt, leading to the conclusion that the rates of transport and secretion of mucous products of the goblet cells at these two levels in

of the intestinal epithelial cells (Nagata 1966).

**1.4.3 The protein synthesis in the intestines** 

**1.4.4 The glucide synthesis in the intestines** 

cells increased by aging, but did not in the undifferentiated cells.

et al. 1994). The colonic tissues of litter mice of six aging groups from the embryonic day 19, to newborn postnatal day 1, 5, 21, adult 2 months and senescent 12 months were fixed in methacarn solution, sectioned and immunostained for cyclin proliferating nuclear antigen (PCNA/cyclin) with the monoclonal antibody and the avidin-biotin peroxidase complex technique. The reaction appeared in the colonic epithelium from G1 to S phase of the cell cycle. The immunostaining positive cells were localized in the crypts of colons similarly to the labeled cells with 3H-thymidine by radioautography, a region defined as the proliferative zone. The positive cells in the columnar epithelia were frequently found in the perinatal groups from embryo to postnatal day 1, and became constant from postnatal day 5 until senescence. Comparing the results by immunostaining with the labeling index by radioautography, it was found that the former was higher in each aging group than the latter. The reason for the difference should be due to that PCNA/cyclin positive cells included not only S-phase cells but also the late G1 cells.

Fig. 8. Histogram showing aging changes of average labeling indices in respective tissue layers and cells of mouse colons at various ages from embryo to postnatal year 1, labeled with 3H-thyidine. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 123, 2002, Urban & Fischer, Jena, Germany

#### **1.4.2 The RNA synthesis in the intestines**

We studied the RNA synthesis of the small intestines of mice after feeding or refeeding under the restricted conditions (Nagata 1966). Five groups of ddY mice, each consisting of 5 individuals, total 25, were injected with 3H-uridine, an RNA precursor, and sacrificed at

et al. 1994). The colonic tissues of litter mice of six aging groups from the embryonic day 19, to newborn postnatal day 1, 5, 21, adult 2 months and senescent 12 months were fixed in methacarn solution, sectioned and immunostained for cyclin proliferating nuclear antigen (PCNA/cyclin) with the monoclonal antibody and the avidin-biotin peroxidase complex technique. The reaction appeared in the colonic epithelium from G1 to S phase of the cell cycle. The immunostaining positive cells were localized in the crypts of colons similarly to the labeled cells with 3H-thymidine by radioautography, a region defined as the proliferative zone. The positive cells in the columnar epithelia were frequently found in the perinatal groups from embryo to postnatal day 1, and became constant from postnatal day 5 until senescence. Comparing the results by immunostaining with the labeling index by radioautography, it was found that the former was higher in each aging group than the latter. The reason for the difference should be due to that PCNA/cyclin positive cells

Fig. 8. Histogram showing aging changes of average labeling indices in respective tissue layers and cells of mouse colons at various ages from embryo to postnatal year 1, labeled with 3H-thyidine. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 123, 2002, Urban & Fischer, Jena, Germany

We studied the RNA synthesis of the small intestines of mice after feeding or refeeding under the restricted conditions (Nagata 1966). Five groups of ddY mice, each consisting of 5 individuals, total 25, were injected with 3H-uridine, an RNA precursor, and sacrificed at

included not only S-phase cells but also the late G1 cells.

**1.4.2 The RNA synthesis in the intestines** 

different time intervals after feeding. The animals of the first group were injected with 3Huridine at 9 a.m. and fed at 10 a.m. for 30 min. and sacrificed at 11 a.m. 1 hour after the feeding and 2 hours after the injection, the 2nd group was sacrificed at 1 p.m. 3 hours after feeding and 4 hours after the injection, the 3rd group at 5 p.m., 7 and 8 hours later, the 4th group at 9 a.m. on the next day 23 and 24 hours later, and finally the 5th group at 1. p.m. on the next day 3 hours after refeeding and 28 hours after the injection. Then, the jejunums obtained from each animal were prepared for isolated cell radioautograms according to Nagata et al. (1961). The results demonstrated that the grain counts in mononucleate villus cells reached the maximum (20-30 grains per cell) 4 hours after injection and decreased (10- 20/cell) after 28 hours, while the counts in mononucleate villus cells only increased gradually from 4 hours (10/cell) to 28 hours (20/cell). In contrast to this, the grain counts of binucleate cells which appeared in villus cells increased parallely to the mononucleate villus cells (10-20/cell). It was concluded that the RNA synthesis in the jejunal epithelial cells was high in the following order: mononucleate crypt cells, binucleate cells and mononucleate villus cells. These results revealed that the feeding or refeeding affected the RNA synthesis of the intestinal epithelial cells (Nagata 1966).

## **1.4.3 The protein synthesis in the intestines**

We first studied the incorporations of 3H-leucine and 3H-tryptophane in mouse small intestines in connection to the binuclearity before and after feeding (Nagata 1967b). The results showed that the incorporations of both amino acids were greater in binucleate intestinal epithelial columnar cells than mononucleate villus and crypt cells at both before and after feeding. However, the aging changes of these incorporations were not studied.

## **1.4.4 The glucide synthesis in the intestines**

We also studied the aging changes of glucide synthesis by 3H-glucosamine uptake in the small intestines of mouse (Morita 1993), and found that the silver grains in the ileum columnar epithelial cells were mainly localized over the brush borders and the Golgi regions in these cells (Fig. 6F). The grain counting revealed that the numbers of silver grains over the brush borders and cytoplasm of the columnar epithelial cells increased in the villi (10-15/cell) than in the crypts (1-2/cell) from 6 months up to 2 years due to aging. The grain counting in other cell types also revealed that the number of silver grains in goblet cells, basal granulate cells, Paneth cells increased by aging, but did not in the undifferentiated cells.

The glycoprotein synthesis in goblet cells as well as in absorptive epithelial cells was also studied using 35SO4 incorporation in the duodenums, the jejunums and the colons of adult mice at varying time intervals at 30, 60, and 180 min after the administration (Nagata et al. 1988a, Nagata and Kawahara 1999, Nagata et al. 1999b). Silver grains were localized over the columnar absorptive cells and the goblet cells, especially over the Golgi regions and mucous granules of the goblet cells. By EM RAG the intracellular localization of silver grains in goblet cells was clearly shown in the Golgi apparatus. The results from grain counting revealed that the average grain counts were different in the upper and deeper regions of the crypts in the 4 portions and it was shown that silver grains over goblet cells in the lower region of the crypt transferred rapidly from 30 min to 180 min, while they transferred slowly in goblet cells in the upper region of the colonic crypt, leading to the conclusion that the rates of transport and secretion of mucous products of the goblet cells at these two levels in

article.

from each other.

were formed at day 9 after birth.

**1.5.1.1 The DNA synthesis in hepatocyte nuclei** 

Macromolecular Synthesis in the Digestive and Respiratory Systems 325

1999a,b,c, 2002, Nagata et al. 1977a, Nagata and Nawa 1966b). The results obtained from the tissues of 3 groups of animals injected respectively with 3 kinds of RI-labeled precursors, i.e. 3H-thymidine, 3H-uridine and 3H-leucine, were already reported as several original articles and reviews (Ma 1988, Ma and Nagata 1988a,b, 1990a,b, 2000, Ma et al. 1991, Nagata 1996a,b, 1997a, 1998a, 1999c, 2001c, 2002, 2003, 2006a,b, 2007a,b, 2009a,b,c,d) or as a monograph in the series of Prog. Histochem. Ccytochem (Nagata 2002, 2004, 2010c). Therefore, the results from the livers in aging mice should be briefly summarized in this

As for the nucleic acid synthesis in hepatocytes, we first studied the difference between the mononucleate and binucleate hepatocytes of adult rats, injected with 3H-thymidine and radioautographed (Nagata 1962, 1994d). The results showed that the frequency of labeled cells was greater in the mononucleate cells (Fig. 9A) than in the binucleate cells. The labeled binucleate cells were classified into two types, i.e., a hepatocyte whose one of the two nuclei was labeled and a hepatocyte whose two nuclei were labeled. The former was more frequently observed than the latter. Grain counts revealed that the amount of DNA synthesized in the binucleate cell whose one nucleus was labeled was the same as the mononucleate cell, while the total amount of DNA synthesized in the binucleate cell whose two nuclei were labeled was almost twice as that of the mononucleate cell. These results suggested that the two nuclei of binucleate hepatocytes synthesized DNA independently

On the other hand, LM and EMRAG of prenatal and postnatal normal mice at various ages labeled with 3H-thymidine revealed that many silver grains were localized over the nuclei of various cell types consisting the liver, i.e., hepatocytes (Fig. 9A), sinusoidal endothelial cells (Fig. 8B), Kupffer's cells, Ito's fat-storing cells, bile ductal epithelia cells, fibroblasts and hematopoietic cells (Ma 1988, Ma and Nagata 1988a,b, 1990, Nagata 1995a). In hematopoietic cells in the livers of perinatal animals, silver grains were observed over the nuclei of erythroblasts, myeloblasts, lymphoblasts and megekaryocytes. However, most hematopoietic cells disappeared on postnatal day 14. At fetal day 19, the liver tissues were chiefly consisted of hepatocytes and haematopoietic cells and no lobular orientation was observed. At postnatal day 1 and 3, lobular formation started and finally the hepatic lobules

During the perinatal period, almost all kinds of cells were labeled with 3H-thymidine. Percentage of labeled hepatocytes was the highest at fetal day 19, and rapidly decreased after birth to day 3. From day 9 to 14, percentage of labeled hepatocytes (labeling index) decreased gradually and finally to the lowest at 24 months (Fig. 10A). When the labeling indices of hepatocytes in 3 hepatic acinar zones were analyzed, the indices decreased in zone 2 (intermediate zone) and zone 3 (peripheral zone) on days 3 and 9 after birth, whereas they increased in zone 1 (central) on day 9, and then they altogether decreased from day 14 to 24 months (Fig. 10B). When the size and number of cell organelles in both labeled and unlabeled hepatocytes were estimated quantitatively by image analysis with an image analyzer, Digigramer G/A (Mutoh Kogyo Co. Ltd., Tokyo, Japan) on EMRAG, the area size of the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, and the number of mitochondria in the unlabeled hepatocytes were more than the labeled cells (Ma and Nagata 1988a,b, Nagata 1995a,d).

the crypts were different. By EM RAG silver grains first appeared over the Golgi zone at 30 min. and then moved to the secretory granules at 60 and 180 min. The incorporation of Na235SO4 into sulfated complex carbohydrate was investigated in the mouse small and large intestines by LM and EM RAG as well as in the submandibular glands and the stomachs. Quantitative differences have been observed in the relative uptake of radiosulfate in the various labeled cells of each organ. Incorporation by the colon in goblet cells exceeded that elsewhere in the deep goblet cells of the colonic crypts migration of label progressed during the time tested from the supranuclear Golgi region to the deep position of the goblet and then extended throughout the mucosubstance in the goblet in the superficial goblet cells of the colon. The radioautographic and cytochemical staining differences between secretory cells in the deeper region compared with the upper region of the colonic crypts are considered to reflect differences in the rate of transport of secretory products in the theca and the rate of secretion at the low levels in the crypt (Figs. 6G,H). These results showed the time differences of glycoprotein synthesis in respective organs. The sulfate uptake and accumulation in several mouse digestive organs were also studied by LM RAG. Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter of animals was sacrificed 30 min after the intraperitoneal injections with phosphate buffered Na235SO4, and the other litter animals were sacrificed 12 hr after the injections. Then several digestive organs, the parotid gland, the submandibular gland, the sublingual gland, antrum and fundus of the stomach, the duodenum, the jejunum, the ileum, the caecum, the ascending colon and the descending colon were taken out and radioautographed. As the results, many silver grains were observed on villous cells and crypt cells of the small intestines and whole mucosa of the large intestines at 30 min after the injection. Then at 12 hr after the injection silver grains were observed on mucigen granules of goblet cells in the small intestines and the large intestines. The numbers of silver grains observed in respective organs at 30 min were less than those at 12 hr. From the results, it was concluded that the time difference of the glycoprotein synthesis was demonstrated in several digestive organs by radiosulfate incorporation, in reverse proportion to DNA synthesis. The total S contents in colonic goblet cells in upper and deeper regions of colonic crypts in aging mice were also analyzed by X-ray microanalysis (Nagata et al. 2000b, Nagata 2004). The results accorded well with the results from RAG (Nagata 2002) showing increase and decrease of mucosubstances in these cells due to development and aging to senescence.

#### **1.5 Macromolecular synthesis in the liver**

The liver is the largest gland in the human and the mammalian body and consists of several types of cells (Nagata 2010c). The hepatocyte is the main component of the liver, composing the liver parenchyma which form the hepatic lobules, surrounded by other types of cells such as the connective tissue cells, sinusoidal endothelial cells, satellite cells of Kupffer, Ito's fatstoring cells and bile epithelial cells. In the livers of perinatal animals, the liver tissues include hematopoietic cells such as erythroblasts, myeloblasts and magakaryocytes. We studied macromolecular synthesis by LM and EMRAG mainly in hepatocytes of rats and mice (Nagata 1993b, 1994a,b,c,d, 1995a,b,c, 1996a, 1997a, 1999c, 2002, 2003, 2006b, 2007a, 2009a,d,h,i, 2010c,h).

#### **1.5.1 The DNA synthesis in the liver**

We first studied the DNA synthesis in the liver tissues at various ages from embryo to postnatal 2 years (Nagata 1993a,b, 1994a,b,c,d, 1995a,b,c,d, 1996a,b,c,d, 1997a,b,c, 1998a,b,c, 1999a,b,c, 2002, Nagata et al. 1977a, Nagata and Nawa 1966b). The results obtained from the tissues of 3 groups of animals injected respectively with 3 kinds of RI-labeled precursors, i.e. 3H-thymidine, 3H-uridine and 3H-leucine, were already reported as several original articles and reviews (Ma 1988, Ma and Nagata 1988a,b, 1990a,b, 2000, Ma et al. 1991, Nagata 1996a,b, 1997a, 1998a, 1999c, 2001c, 2002, 2003, 2006a,b, 2007a,b, 2009a,b,c,d) or as a monograph in the series of Prog. Histochem. Ccytochem (Nagata 2002, 2004, 2010c). Therefore, the results from the livers in aging mice should be briefly summarized in this article.

## **1.5.1.1 The DNA synthesis in hepatocyte nuclei**

324 Senescence

the crypts were different. By EM RAG silver grains first appeared over the Golgi zone at 30 min. and then moved to the secretory granules at 60 and 180 min. The incorporation of Na235SO4 into sulfated complex carbohydrate was investigated in the mouse small and large intestines by LM and EM RAG as well as in the submandibular glands and the stomachs. Quantitative differences have been observed in the relative uptake of radiosulfate in the various labeled cells of each organ. Incorporation by the colon in goblet cells exceeded that elsewhere in the deep goblet cells of the colonic crypts migration of label progressed during the time tested from the supranuclear Golgi region to the deep position of the goblet and then extended throughout the mucosubstance in the goblet in the superficial goblet cells of the colon. The radioautographic and cytochemical staining differences between secretory cells in the deeper region compared with the upper region of the colonic crypts are considered to reflect differences in the rate of transport of secretory products in the theca and the rate of secretion at the low levels in the crypt (Figs. 6G,H). These results showed the time differences of glycoprotein synthesis in respective organs. The sulfate uptake and accumulation in several mouse digestive organs were also studied by LM RAG. Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter of animals was sacrificed 30 min after the intraperitoneal injections with phosphate buffered Na235SO4, and the other litter animals were sacrificed 12 hr after the injections. Then several digestive organs, the parotid gland, the submandibular gland, the sublingual gland, antrum and fundus of the stomach, the duodenum, the jejunum, the ileum, the caecum, the ascending colon and the descending colon were taken out and radioautographed. As the results, many silver grains were observed on villous cells and crypt cells of the small intestines and whole mucosa of the large intestines at 30 min after the injection. Then at 12 hr after the injection silver grains were observed on mucigen granules of goblet cells in the small intestines and the large intestines. The numbers of silver grains observed in respective organs at 30 min were less than those at 12 hr. From the results, it was concluded that the time difference of the glycoprotein synthesis was demonstrated in several digestive organs by radiosulfate incorporation, in reverse proportion to DNA synthesis. The total S contents in colonic goblet cells in upper and deeper regions of colonic crypts in aging mice were also analyzed by X-ray microanalysis (Nagata et al. 2000b, Nagata 2004). The results accorded well with the results from RAG (Nagata 2002) showing increase and decrease of

mucosubstances in these cells due to development and aging to senescence.

The liver is the largest gland in the human and the mammalian body and consists of several types of cells (Nagata 2010c). The hepatocyte is the main component of the liver, composing the liver parenchyma which form the hepatic lobules, surrounded by other types of cells such as the connective tissue cells, sinusoidal endothelial cells, satellite cells of Kupffer, Ito's fatstoring cells and bile epithelial cells. In the livers of perinatal animals, the liver tissues include hematopoietic cells such as erythroblasts, myeloblasts and magakaryocytes. We studied macromolecular synthesis by LM and EMRAG mainly in hepatocytes of rats and mice (Nagata 1993b, 1994a,b,c,d, 1995a,b,c, 1996a, 1997a, 1999c, 2002, 2003, 2006b, 2007a, 2009a,d,h,i, 2010c,h).

We first studied the DNA synthesis in the liver tissues at various ages from embryo to postnatal 2 years (Nagata 1993a,b, 1994a,b,c,d, 1995a,b,c,d, 1996a,b,c,d, 1997a,b,c, 1998a,b,c,

**1.5 Macromolecular synthesis in the liver** 

**1.5.1 The DNA synthesis in the liver** 

As for the nucleic acid synthesis in hepatocytes, we first studied the difference between the mononucleate and binucleate hepatocytes of adult rats, injected with 3H-thymidine and radioautographed (Nagata 1962, 1994d). The results showed that the frequency of labeled cells was greater in the mononucleate cells (Fig. 9A) than in the binucleate cells. The labeled binucleate cells were classified into two types, i.e., a hepatocyte whose one of the two nuclei was labeled and a hepatocyte whose two nuclei were labeled. The former was more frequently observed than the latter. Grain counts revealed that the amount of DNA synthesized in the binucleate cell whose one nucleus was labeled was the same as the mononucleate cell, while the total amount of DNA synthesized in the binucleate cell whose two nuclei were labeled was almost twice as that of the mononucleate cell. These results suggested that the two nuclei of binucleate hepatocytes synthesized DNA independently from each other.

On the other hand, LM and EMRAG of prenatal and postnatal normal mice at various ages labeled with 3H-thymidine revealed that many silver grains were localized over the nuclei of various cell types consisting the liver, i.e., hepatocytes (Fig. 9A), sinusoidal endothelial cells (Fig. 8B), Kupffer's cells, Ito's fat-storing cells, bile ductal epithelia cells, fibroblasts and hematopoietic cells (Ma 1988, Ma and Nagata 1988a,b, 1990, Nagata 1995a). In hematopoietic cells in the livers of perinatal animals, silver grains were observed over the nuclei of erythroblasts, myeloblasts, lymphoblasts and megekaryocytes. However, most hematopoietic cells disappeared on postnatal day 14. At fetal day 19, the liver tissues were chiefly consisted of hepatocytes and haematopoietic cells and no lobular orientation was observed. At postnatal day 1 and 3, lobular formation started and finally the hepatic lobules were formed at day 9 after birth.

During the perinatal period, almost all kinds of cells were labeled with 3H-thymidine. Percentage of labeled hepatocytes was the highest at fetal day 19, and rapidly decreased after birth to day 3. From day 9 to 14, percentage of labeled hepatocytes (labeling index) decreased gradually and finally to the lowest at 24 months (Fig. 10A). When the labeling indices of hepatocytes in 3 hepatic acinar zones were analyzed, the indices decreased in zone 2 (intermediate zone) and zone 3 (peripheral zone) on days 3 and 9 after birth, whereas they increased in zone 1 (central) on day 9, and then they altogether decreased from day 14 to 24 months (Fig. 10B). When the size and number of cell organelles in both labeled and unlabeled hepatocytes were estimated quantitatively by image analysis with an image analyzer, Digigramer G/A (Mutoh Kogyo Co. Ltd., Tokyo, Japan) on EMRAG, the area size of the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, and the number of mitochondria in the unlabeled hepatocytes were more than the labeled cells (Ma and Nagata 1988a,b, Nagata 1995a,d).

Macromolecular Synthesis in the Digestive and Respiratory Systems 327

Fig. 9B. EM RAG of a sinusoidal endothelial cells of the liver of a 14 day old mouse labeled with 3H-thymidine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9C. EM RAG of a hepatocyte of the liver of a 14 day old mouse labeled with 3H-uridine.

Fig. 9D. EM RAG of an Ito's fat-storing cell of the liver of a newborn 14 day old mouse labeled with 3H-uridine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9E. EM RAG of a hepatocyte of the liver of a 1 month old mouse labeled with 3Hleucine. Many silver grains were observed over the nucleus and mitochondria.

Fig. 9F. EM RAG of a sinusoidal endothelial cells of the liver of a newborn 14 day old mouse labeled with 3H-leucine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9G. EMRAG of a hepatocyte of the liver of an adult 2 month old mouse labeled with 3H-

Fig. 9H. EMRAG of a Kupffer cell of the liver of a newborn 1 day old mouse labeled with 3H-proline. Many silver grains were observed over the nucleus and mitochondria.

These data demonstrated that the cell organelles of the hepatocytes which synthesized DNA were not well developed as compared to those not synthesizing DNA during the postnatal development. In some of unlabeled hepatocytes, several silver grains were occasionally observed localizing over mitochondria and peroxisomes as was formerly reported (Nagata et al. 1967a,b, 1982b). The mitochondrial DNA synthesis was first observed in cultured hepatocytes of chickens and mice in vitro (Nagata et al. 1967a,b). The percentages of labeled cells in other cell types in the liver of aging mice such as sinusoidal endothelial cells, Kupffer's cells, Ito's fat-storing cells, bile ductal epithelia cells and fibroblasts showed also

When we observed DNA synthesis in the nuclei of mononucleate and binucleate hepatocytes, we also observed DNA synthesis in hepatocyte mitochondria (Ma 1988, Ma and Nagata 1988a,b, 1990a,b, Nagata and Ma 2005a). The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 mononucleate hepatocytes of each animal labeled with 3H-thymidine demonstrating DNA synthesis in 8 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 3, 9 and 14, month 1, 6, 12 and 24, were obtained. The number of total mitochondria per cell increased from perinatal stage (35-50/cell) to postnatal month 6 (95-105/cell), reaching the maximum, decreased to month 24 (85-90/cell), while the number of labeled mitochondria per cell increased from perinatal stage to postnatal day 14, reaching the maximum, decreased to month 6, then increased again to month 12, reaching the second peak and decreased again to month 24. Thus, the labeling indices in respective aging stages were calculated from the number of labeled mitochondria which showed an increase from perinatal stage to postnatal day 14, reaching the maximum and decreased to month 24. The results showed that the numbers of labeled mitochondria with 3H-thymidine showing DNA synthesis increased from prenatal embryo day 19 (3.8/cell) to postnatal day 14 (6.2/cell), reaching the maximum, and then decreased to month 6 (3.7/cell) and again increased to year 1 (6.0/cell), while the labeling indices increased from prenatal day 19 (11.8%) to postnatal day 14 (16.9%), reaching the maximum, then decreased to month 6 (4.1%), year 1 (6.4%) and year 2 (2.3%). The increase of the total number of mitochondria in mononucleate hepatocytes was stochastically significant (P<0.01), while the changes of number of labeled mitochondria and

labeling index in mononucleate hepatocytes were not significant (P<0.01).

proline. Many silver grains were observed over the nucleus and mitochondria.

decreases from perinatal period to postnatal 24 months. **1.5.1.2 The DNA synthesis in hepatocyte mitochondria** 

Many silver grains were observed over the nucleus and mitochondria.

Fig. 9. EM RAG of the liver. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 130, 2002, Urban & Fischer, Jena, Germany Fig. 9A. EMRAG of a hepatocyte of the liver of a 14 day old mouse labeled with 3Hthymidine. Many silver grains were observed over the nucleus and mitochondria.

Fig. 9. EM RAG of the liver. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 130, 2002, Urban & Fischer, Jena, Germany Fig. 9A. EMRAG of a hepatocyte of the liver of a 14 day old mouse labeled with 3Hthymidine. Many silver grains were observed over the nucleus and mitochondria.

Fig. 9B. EM RAG of a sinusoidal endothelial cells of the liver of a 14 day old mouse labeled with 3H-thymidine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9C. EM RAG of a hepatocyte of the liver of a 14 day old mouse labeled with 3H-uridine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9D. EM RAG of an Ito's fat-storing cell of the liver of a newborn 14 day old mouse labeled with 3H-uridine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9E. EM RAG of a hepatocyte of the liver of a 1 month old mouse labeled with 3Hleucine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9F. EM RAG of a sinusoidal endothelial cells of the liver of a newborn 14 day old mouse labeled with 3H-leucine. Many silver grains were observed over the nucleus and mitochondria. Fig. 9G. EMRAG of a hepatocyte of the liver of an adult 2 month old mouse labeled with 3Hproline. Many silver grains were observed over the nucleus and mitochondria. Fig. 9H. EMRAG of a Kupffer cell of the liver of a newborn 1 day old mouse labeled with 3H-proline. Many silver grains were observed over the nucleus and mitochondria.

These data demonstrated that the cell organelles of the hepatocytes which synthesized DNA were not well developed as compared to those not synthesizing DNA during the postnatal development. In some of unlabeled hepatocytes, several silver grains were occasionally observed localizing over mitochondria and peroxisomes as was formerly reported (Nagata et al. 1967a,b, 1982b). The mitochondrial DNA synthesis was first observed in cultured hepatocytes of chickens and mice in vitro (Nagata et al. 1967a,b). The percentages of labeled cells in other cell types in the liver of aging mice such as sinusoidal endothelial cells, Kupffer's cells, Ito's fat-storing cells, bile ductal epithelia cells and fibroblasts showed also decreases from perinatal period to postnatal 24 months.

#### **1.5.1.2 The DNA synthesis in hepatocyte mitochondria**

When we observed DNA synthesis in the nuclei of mononucleate and binucleate hepatocytes, we also observed DNA synthesis in hepatocyte mitochondria (Ma 1988, Ma and Nagata 1988a,b, 1990a,b, Nagata and Ma 2005a). The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 mononucleate hepatocytes of each animal labeled with 3H-thymidine demonstrating DNA synthesis in 8 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 3, 9 and 14, month 1, 6, 12 and 24, were obtained. The number of total mitochondria per cell increased from perinatal stage (35-50/cell) to postnatal month 6 (95-105/cell), reaching the maximum, decreased to month 24 (85-90/cell), while the number of labeled mitochondria per cell increased from perinatal stage to postnatal day 14, reaching the maximum, decreased to month 6, then increased again to month 12, reaching the second peak and decreased again to month 24. Thus, the labeling indices in respective aging stages were calculated from the number of labeled mitochondria which showed an increase from perinatal stage to postnatal day 14, reaching the maximum and decreased to month 24. The results showed that the numbers of labeled mitochondria with 3H-thymidine showing DNA synthesis increased from prenatal embryo day 19 (3.8/cell) to postnatal day 14 (6.2/cell), reaching the maximum, and then decreased to month 6 (3.7/cell) and again increased to year 1 (6.0/cell), while the labeling indices increased from prenatal day 19 (11.8%) to postnatal day 14 (16.9%), reaching the maximum, then decreased to month 6 (4.1%), year 1 (6.4%) and year 2 (2.3%). The increase of the total number of mitochondria in mononucleate hepatocytes was stochastically significant (P<0.01), while the changes of number of labeled mitochondria and labeling index in mononucleate hepatocytes were not significant (P<0.01).

Macromolecular Synthesis in the Digestive and Respiratory Systems 329

As for the binucleate hepatocytes, on the other hand, because the appearances of binucleate hepatocytes showing silver grains in their nuclei demonstrating DNA synthesis were not so many in the adult and senescent stages from postnatal month 1 to 24, only binucleate cells at perinatal stages when reasonable numbers of labeled hepatocytes were found in respective groups were analyzed. The number of mitochondria in binucleate hepatocytes at postnatal day 1 to 14 kept around 80 (77-84/cell) which did not show such remarkable changes, neither increase nor decrease, as shown in mononucleate cells. Thus, the number of mitochondria per binucleate cell, the number of labeled mitochondria per binucleate cell and the labeling index of binucleate cell in 4 groups from postnatal day 1 to 14 were counted. The number of mitochondria and the number of labeled mitochondria were more in binucleate cells than

mononucleate cells (Nagata 2007a,b,c,d,e, Nagata and Ma 2005a,b, Nagata et al. 1977a).

synthesis appeared over the nuclei and cytoplasm of hepatocytes.

The RNA synthesis in the liver was studied by 3H-uridine RAG. Silver grains due to RNA

When the RI-labeled precursor 3H-uridine was administered to experimental animals, or cultured cells were incubated in a medium containing 3H-uridine in vitro and LM RAG was prepared, silver grains first appeared over the chromatin of the nucleus and nucleolus of all the cells within several minutes, then silver grains spread over the cytoplasm within 30 minutes showing messenger RNA and ribosomal RNA (Nagata 1966, Nagata and Nawa

We studied quantitative changes of RNA synthesis in the livers of adult mice before and after feeding by incorporations of 3H-uridine. Five groups of ddY mice, each consisting of 5 individuals, total 25, were injected with 3H-uridine and sacrificed at different time intervals. The animals of the first group were injected with 3H-uridine at 9 a.m. and fed at 10 a.m. for 30 min. and sacrificed at 11 a.m. 1 hour after the feeding and 2 hours after the injection, the 2nd group was sacrificed at 1 p.m. 3 hours after feeding and 4 hours after the injection, the 3rd group at 5 p.m., 7 and 8 hours later, the 4th group at 9 a.m. on the next day 23 and 24 hours later, and finally the 5th group at 1. p.m. on the next day 3 hours after refeeding and 28 hours after the injection. Then, the livers were taken out from each animal, prepared for isolated cell radioautograms according to Nagata et al. (1961). The results demonstrated that the grain counts in both mononucleate and binucleate hepatocytes before feeding (15-25 grains per cell) increased 4 hours after feeding (30-40 grains per cell), reached the maximum in 24 hours (40-50 grains per cell), then decreased on the next day (30-40 grains per cell). It was concluded that the RNA synthesis in the binucleate hepatocytes was a little higher than the mononucleate hepatocytes at the same stages and both increased and decreased after feeding. These results revealed that the feeding or refeeding affected the RNA synthesis of the livers (Nagata 1966). Then, we studied aging changes of 3H-uridine incorporation in the livers and pancreases of aging mice at various ages from prenatal embryos to postnatal aged mice to senescence at month 12 and 24 by LM and EMRAG (Ma and Nagata 1990b, Nagata 1999c). When aged mice were injected with 3H-uridine, LM and EM RAG showed that silver grains were localized over the nucleoli, nuclear chromatin (both euchromatin and heterochromatin), mitochondria and rough surfaced endoplasmic reticulum of hepatocytes (Fig. 9C) and other

**1.5.2 The RNA synthesis in the liver** 

1966a,b, Nagata et al.1969).

**1.5.2.1 The RNA synthesis in hepatocyte nuclei** 

Fig. 10. Transitional curves of the labeling indices in the livers of aging mice after injection of 3H-thymine. Mean ± Standard Deviation. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 92, 2001, Academic Press, San Diego, USA, London, UK.

Fig. 10A. Labeling indices of hepatocytes, sinusoidal endothelial cells and hematopoietic cells, respectively.

Fig. 10B. Labeling indices of hepatocytes, sinusoidal endothelial cells and hematopoietic cells, respectively.

As for the binucleate hepatocytes, on the other hand, because the appearances of binucleate hepatocytes showing silver grains in their nuclei demonstrating DNA synthesis were not so many in the adult and senescent stages from postnatal month 1 to 24, only binucleate cells at perinatal stages when reasonable numbers of labeled hepatocytes were found in respective groups were analyzed. The number of mitochondria in binucleate hepatocytes at postnatal day 1 to 14 kept around 80 (77-84/cell) which did not show such remarkable changes, neither increase nor decrease, as shown in mononucleate cells. Thus, the number of mitochondria per binucleate cell, the number of labeled mitochondria per binucleate cell and the labeling index of binucleate cell in 4 groups from postnatal day 1 to 14 were counted. The number of mitochondria and the number of labeled mitochondria were more in binucleate cells than mononucleate cells (Nagata 2007a,b,c,d,e, Nagata and Ma 2005a,b, Nagata et al. 1977a).

## **1.5.2 The RNA synthesis in the liver**

328 Senescence

Fig. 10. Transitional curves of the labeling indices in the livers of aging mice after injection of 3H-thymine. Mean ± Standard Deviation. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 92, 2001, Academic Press, San Diego,

Fig. 10A. Labeling indices of hepatocytes, sinusoidal endothelial cells and hematopoietic

Fig. 10B. Labeling indices of hepatocytes, sinusoidal endothelial cells and hematopoietic

USA, London, UK.

cells, respectively.

cells, respectively.

The RNA synthesis in the liver was studied by 3H-uridine RAG. Silver grains due to RNA synthesis appeared over the nuclei and cytoplasm of hepatocytes.

#### **1.5.2.1 The RNA synthesis in hepatocyte nuclei**

When the RI-labeled precursor 3H-uridine was administered to experimental animals, or cultured cells were incubated in a medium containing 3H-uridine in vitro and LM RAG was prepared, silver grains first appeared over the chromatin of the nucleus and nucleolus of all the cells within several minutes, then silver grains spread over the cytoplasm within 30 minutes showing messenger RNA and ribosomal RNA (Nagata 1966, Nagata and Nawa 1966a,b, Nagata et al.1969).

We studied quantitative changes of RNA synthesis in the livers of adult mice before and after feeding by incorporations of 3H-uridine. Five groups of ddY mice, each consisting of 5 individuals, total 25, were injected with 3H-uridine and sacrificed at different time intervals. The animals of the first group were injected with 3H-uridine at 9 a.m. and fed at 10 a.m. for 30 min. and sacrificed at 11 a.m. 1 hour after the feeding and 2 hours after the injection, the 2nd group was sacrificed at 1 p.m. 3 hours after feeding and 4 hours after the injection, the 3rd group at 5 p.m., 7 and 8 hours later, the 4th group at 9 a.m. on the next day 23 and 24 hours later, and finally the 5th group at 1. p.m. on the next day 3 hours after refeeding and 28 hours after the injection. Then, the livers were taken out from each animal, prepared for isolated cell radioautograms according to Nagata et al. (1961). The results demonstrated that the grain counts in both mononucleate and binucleate hepatocytes before feeding (15-25 grains per cell) increased 4 hours after feeding (30-40 grains per cell), reached the maximum in 24 hours (40-50 grains per cell), then decreased on the next day (30-40 grains per cell). It was concluded that the RNA synthesis in the binucleate hepatocytes was a little higher than the mononucleate hepatocytes at the same stages and both increased and decreased after feeding. These results revealed that the feeding or refeeding affected the RNA synthesis of the livers (Nagata 1966).

Then, we studied aging changes of 3H-uridine incorporation in the livers and pancreases of aging mice at various ages from prenatal embryos to postnatal aged mice to senescence at month 12 and 24 by LM and EMRAG (Ma and Nagata 1990b, Nagata 1999c). When aged mice were injected with 3H-uridine, LM and EM RAG showed that silver grains were localized over the nucleoli, nuclear chromatin (both euchromatin and heterochromatin), mitochondria and rough surfaced endoplasmic reticulum of hepatocytes (Fig. 9C) and other

Macromolecular Synthesis in the Digestive and Respiratory Systems 331

respective aging stages were calculated from the number of labeled mitochondria and the number of total mitochondria per cellular profile area, respectively. The results showed that the numbers of labeled mitochondria with 3H-uridine showing RNA synthesis increased from prenatal embryo day 19 (3.3/cell) to postnatal month 1 (9.2/cell), reaching the maximum, and then decreased to month 6 (3.5/cell) and again increased to year 1 (4.0/cell) and year 2 (4.3/cell), while the labeling indices increased from prenatal day 19 (12.4%) to postnatal month 1 (16.7%), reaching the maximum, then decreased to year 1 (4.8%) and year 2 (5.3%). Stochastical analysis revealed that the increases and decreases of the number of labeled mitochondria from the perinatal stage to the adult and senescent stage were significant in contrast that the increases and decreases of the labeling indices were not significant (P<0.01). As for the binucleate hepatocytes, on the other hand, because the appearances of binucleate hepatocytes were not so many in the embryonic stage, only several binucleate cells (5-8 at least) at respective stages when enough numbers of binucleate cells available from postnatal day 1 to year 2 were analyzed. The results of visual counts on the number of mitochondria labeled with silver grains obtained from several (5 to 8) binucleate hepatocytes labeled with 3H-uridine demonstrating RNA synthesis in 8 aging groups at perinatal stages, postnatal day 1, 9, 14, and month 1, 2, 6, and year 1 and 2, were counted and the labeling indices in respective aging stages were calculated from the number of labeled mitochondria and the number of total mitochondria per cellular profile area calculated, respectively. The results showed that the number of labeled mitochondria increased from postnatal day 1 (2.3/cell) to day 9 (5.2/cell) and remained almost constant around 4-5, but the labeling indices increased from postnatal day 1 (2.1%) to postnatal day 9 (13.6%), remained almost constant around 13% (12.5-13.6%) from postnatal day 9 to month 1, then decreased to month 2 (6.1%) to month 6 (3.9%), and slightly increased to year 1 (6.3%) and 2 (5.3%). The increases and decreases of the number of labeled mitochondria and the labeling indices in binucleate hepatocytes were

We also studied intracellular localization of mRNA in adult rat hepatocytes localizing over the peroxisomes by means of in situ hybridization technique (Usuda and Nagata 1992, 1995, Usuda et al. 1992). However, its relationship to the aging of animals was not yet studied.

As for the protein synthesis in the liver, we first studied the incorporations of 3H-leucine and 3H-tryptophane in mouse hepatocytes in connection to the binuclearity before and after feeding (Nagata 1967b, Nagata et al. 1967a, Ma et al. 1991). Then, we also studied mitochondrial protein synthesis in the liver later (Nagata 2006a,b, 2007b,c,e, 2009b, 2010c).

We first studied the incorporations of amino-acids, 3H-leucine and 3H-tryptophane, in mouse mononucleate and binucleate hepatocytes before and after feeding (Nagata 1967b, Nagata et al. 1967a, Ma et al. 1991). The results showed that the incorporations of both amino acids were greater in binucleate hepatocytes than mononucleate. When 3H-leucine was injected into several groups of mice at various ages and the liver tissues were processed for LM and EM RAG, silver grains were observed over all cell types of the liver, i.e., hepatocytes (Fig. 9E), sinusoidal endothelial cells (Fig. 9F), ductal epithelial cells, Kupffer's cells, Ito's fat storing cells, fibroblasts and haematopoietic cells. In hepatocytes, number of silver grains in cytoplasm and karyoplasm increased from perinatal animals to postnatal 1

stochastically not significant (P<0.01).

**1.5.3 The protein synthesis in the liver** 

**1.5.3.1 The protein synthesis in hepatocyte nuclei** 

types of cells such as sinusoidal endothelial cells, Kupffer's cells, Ito's fat-storing cells (Fig. 9D), ductal epithelial cells, fibroblasts and haematopoietic cells in the livers at various ages. By quantitative analysis, the total number of silver grains in nucleus, nucleolus and cytoplasm of each hepatocyte increased gradually from fetal day 19 to postnatal days, reached the maximum at postnatal day 14 (30%), then decreased to 24 months (5%). The number of silver grains in nucleolus, when classified into two compartments, grains over granular components and those over fibrillar components both increased paralelly after birth, reached the maxima on day 14 (granular 6-7, fibrillar 1-2/per cell), then decreased to 24 months with aging. However, when the ratio (%) of silver grains over euchromatin, heterochromatin of the nuclei and granular components and fibrillar components of the nucleoli are calculated, the ratio remained constant at each aging point.

#### **1.5.2.2 The RNA synthesis in hepatocyte mitochondria**

The intramitochondrial RNA synthesis was first found in the cultured HeLa cells and the cultured liver cells in vitro using EM RAG (Nagata 1972c, d). Then, it was also found in any other cells in either in vitro or vivo (Nagata et al. 1977c, Nagata 2002). Observing light microscopic radioautograms labeled with 3H-uridine, the silver grains were found over both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo day 19 to postnatal day 1, 3, 9, 14, but also at the adult and senescent stages from postnatal month 1 to 2, 6, 12 and 24 (Nagata 2007a, c, d, e, f, Nagata and Ma 2005b). By electron microscopic observation, silver grains were detected in most mononucleate hepatocytes in respective aging groups localizing not only over euchromatin and nucleoli in the nuclei but also over many cell organelles such as endoplasmic reticulum, ribosomes, and mitochondria as well as cytoplasmic matrices from perinatal stage at embryonic day 19, postnatal day 1, 3, 9, 14, to adult and senescent stages at postnatal month 1, 2, 12 and 24. The silver grains were also observed in binucleate hepatocytes at postnatal day 1, 3, 9, 14, month 1, 2, 6, 12 and 24. The localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices but a few over the mitochondrial membranes and cristae when observed by high power magnification.

As the results, it was found that almost all the hepatocytes were labeled with silver grains showing RNA synthesis in their nuclei and mitochondria. Preliminary quantitative analysis on the number of mitochondria in 10 mononucleate hepatocytes whose nuclei were intensely labeled with many silver grains (more than 10 per nucleus) and other 10 mononucleate hepatocytes whose nuclei were not so intensely labeled (number of silver grains less than 9) in each aging group revealed that there was no significant difference between the number of mitochondria, number of labeled mitochondria and the labeling indices in both types of hepatocytes (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated in 10 hepatocytes selected at random in each animal in respective aging stages regardless whether their nuclei were very intensely labeled or not. The results obtained from the number of mitochondria in mononucleate hepatocytes per cellular profile area showed an increase from the prenatal day (mean ± standard deviation 26.2± /cell) to postnatal day 1 to day 14 (38.4-51.7/cell), then to postnatal month 1-2 (53.7-89.2/cell), reaching the maximum, then decreased to year 1-2 (83.7-80.4/cell) and the increase was stochastically significant (P<0.01). The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 mononucleate hepatocytes of each animal labeled with 3H-uridine demonstrating RNA synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 6 and year 1 and 2, were counted. The labeling indices in

types of cells such as sinusoidal endothelial cells, Kupffer's cells, Ito's fat-storing cells (Fig. 9D), ductal epithelial cells, fibroblasts and haematopoietic cells in the livers at various ages. By quantitative analysis, the total number of silver grains in nucleus, nucleolus and cytoplasm of each hepatocyte increased gradually from fetal day 19 to postnatal days, reached the maximum at postnatal day 14 (30%), then decreased to 24 months (5%). The number of silver grains in nucleolus, when classified into two compartments, grains over granular components and those over fibrillar components both increased paralelly after birth, reached the maxima on day 14 (granular 6-7, fibrillar 1-2/per cell), then decreased to 24 months with aging. However, when the ratio (%) of silver grains over euchromatin, heterochromatin of the nuclei and granular components and fibrillar components of the

The intramitochondrial RNA synthesis was first found in the cultured HeLa cells and the cultured liver cells in vitro using EM RAG (Nagata 1972c, d). Then, it was also found in any other cells in either in vitro or vivo (Nagata et al. 1977c, Nagata 2002). Observing light microscopic radioautograms labeled with 3H-uridine, the silver grains were found over both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo day 19 to postnatal day 1, 3, 9, 14, but also at the adult and senescent stages from postnatal month 1 to 2, 6, 12 and 24 (Nagata 2007a, c, d, e, f, Nagata and Ma 2005b). By electron microscopic observation, silver grains were detected in most mononucleate hepatocytes in respective aging groups localizing not only over euchromatin and nucleoli in the nuclei but also over many cell organelles such as endoplasmic reticulum, ribosomes, and mitochondria as well as cytoplasmic matrices from perinatal stage at embryonic day 19, postnatal day 1, 3, 9, 14, to adult and senescent stages at postnatal month 1, 2, 12 and 24. The silver grains were also observed in binucleate hepatocytes at postnatal day 1, 3, 9, 14, month 1, 2, 6, 12 and 24. The localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices but a few over the mitochondrial membranes and cristae when

As the results, it was found that almost all the hepatocytes were labeled with silver grains showing RNA synthesis in their nuclei and mitochondria. Preliminary quantitative analysis on the number of mitochondria in 10 mononucleate hepatocytes whose nuclei were intensely labeled with many silver grains (more than 10 per nucleus) and other 10 mononucleate hepatocytes whose nuclei were not so intensely labeled (number of silver grains less than 9) in each aging group revealed that there was no significant difference between the number of mitochondria, number of labeled mitochondria and the labeling indices in both types of hepatocytes (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated in 10 hepatocytes selected at random in each animal in respective aging stages regardless whether their nuclei were very intensely labeled or not. The results obtained from the number of mitochondria in mononucleate hepatocytes per cellular profile area showed an increase from the prenatal day (mean ± standard deviation 26.2± /cell) to postnatal day 1 to day 14 (38.4-51.7/cell), then to postnatal month 1-2 (53.7-89.2/cell), reaching the maximum, then decreased to year 1-2 (83.7-80.4/cell) and the increase was stochastically significant (P<0.01). The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 mononucleate hepatocytes of each animal labeled with 3H-uridine demonstrating RNA synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 6 and year 1 and 2, were counted. The labeling indices in

nucleoli are calculated, the ratio remained constant at each aging point.

**1.5.2.2 The RNA synthesis in hepatocyte mitochondria** 

observed by high power magnification.

respective aging stages were calculated from the number of labeled mitochondria and the number of total mitochondria per cellular profile area, respectively. The results showed that the numbers of labeled mitochondria with 3H-uridine showing RNA synthesis increased from prenatal embryo day 19 (3.3/cell) to postnatal month 1 (9.2/cell), reaching the maximum, and then decreased to month 6 (3.5/cell) and again increased to year 1 (4.0/cell) and year 2 (4.3/cell), while the labeling indices increased from prenatal day 19 (12.4%) to postnatal month 1 (16.7%), reaching the maximum, then decreased to year 1 (4.8%) and year 2 (5.3%). Stochastical analysis revealed that the increases and decreases of the number of labeled mitochondria from the perinatal stage to the adult and senescent stage were significant in contrast that the increases and decreases of the labeling indices were not significant (P<0.01). As for the binucleate hepatocytes, on the other hand, because the appearances of binucleate hepatocytes were not so many in the embryonic stage, only several binucleate cells (5-8 at least) at respective stages when enough numbers of binucleate cells available from postnatal day 1 to year 2 were analyzed. The results of visual counts on the number of mitochondria labeled with silver grains obtained from several (5 to 8) binucleate hepatocytes labeled with 3H-uridine demonstrating RNA synthesis in 8 aging groups at perinatal stages, postnatal day 1, 9, 14, and month 1, 2, 6, and year 1 and 2, were counted and the labeling indices in respective aging stages were calculated from the number of labeled mitochondria and the number of total mitochondria per cellular profile area calculated, respectively. The results showed that the number of labeled mitochondria increased from postnatal day 1 (2.3/cell) to day 9 (5.2/cell) and remained almost constant around 4-5, but the labeling indices increased from postnatal day 1 (2.1%) to postnatal day 9 (13.6%), remained almost constant around 13% (12.5-13.6%) from postnatal day 9 to month 1, then decreased to month 2 (6.1%) to month 6 (3.9%), and slightly increased to year 1 (6.3%) and 2 (5.3%). The increases and decreases of the number of labeled mitochondria and the labeling indices in binucleate hepatocytes were stochastically not significant (P<0.01).

We also studied intracellular localization of mRNA in adult rat hepatocytes localizing over the peroxisomes by means of in situ hybridization technique (Usuda and Nagata 1992, 1995, Usuda et al. 1992). However, its relationship to the aging of animals was not yet studied.

## **1.5.3 The protein synthesis in the liver**

As for the protein synthesis in the liver, we first studied the incorporations of 3H-leucine and 3H-tryptophane in mouse hepatocytes in connection to the binuclearity before and after feeding (Nagata 1967b, Nagata et al. 1967a, Ma et al. 1991). Then, we also studied mitochondrial protein synthesis in the liver later (Nagata 2006a,b, 2007b,c,e, 2009b, 2010c).

## **1.5.3.1 The protein synthesis in hepatocyte nuclei**

We first studied the incorporations of amino-acids, 3H-leucine and 3H-tryptophane, in mouse mononucleate and binucleate hepatocytes before and after feeding (Nagata 1967b, Nagata et al. 1967a, Ma et al. 1991). The results showed that the incorporations of both amino acids were greater in binucleate hepatocytes than mononucleate. When 3H-leucine was injected into several groups of mice at various ages and the liver tissues were processed for LM and EM RAG, silver grains were observed over all cell types of the liver, i.e., hepatocytes (Fig. 9E), sinusoidal endothelial cells (Fig. 9F), ductal epithelial cells, Kupffer's cells, Ito's fat storing cells, fibroblasts and haematopoietic cells. In hepatocytes, number of silver grains in cytoplasm and karyoplasm increased from perinatal animals to postnatal 1

Macromolecular Synthesis in the Digestive and Respiratory Systems 333

numbers of mitochondria in mononucleate hepatocytes showed an increase from the prenatal day (34.5/cell) to postnatal days 1 (44.6/cell), 3 (45.8/cell), 9 (43.6/cell), 14 (48.5/cell), to postnatal months 1 (51.5/cell), 2 (52.3/cell), reaching the maximum at month 6 (60.7/cell), then decreased to years 1 (54.2/cell) and 2 (51.2/cell). The increase and decrease were stochastically significant (P<0.01). The results obtained from visual counting on the numbers of mitochondria labeled with silver grains from 20 mononucleate hepatocytes of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 2, 6 and year 1 and 2, were counted. The labeling indices in respective aging stages were calculated from the numbers of labeled mitochondria and the numbers of total mitochondria per cell. The results showed that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis increased from prenatal embryo day 19 (8.3/cell) to postnatal days 1 (9.6/cell), 3 (8.1/cell), 9 (8.9/cell), 14 (9.5/cell), and month 1 (11.2/cell), reaching the maximum, and then decreased to months 2 (9.1/cell), 6 (8.8/cell) to years 1 (6.7/cell) and 2 (2.2/cell), while the labeling indices increased from prenatal day 19 (20.1%) to postnatal days 1 (21.2%), 3 (21.6%), 9 (22.2%), 14 (23.1%), reaching the maximum, then decreased to month 1 (21.7%), 2 (17.4%), 6 (14.6%), and years 1 (12.4%) and 2 (4.4%). Stochastical analysis revealed that the increases and decreases of the numbers of labeled mitochondria as well as the labeling indices from the perinatal stage to the adult and senescent stages were significant (P<0.01). The results obtained from the numbers of mitochondria in binucleate hepatocytes showed an increase from the postnatal days 1 (66.2/cell), to 3 (66.4/cell), 14 (81.8/cell), to postnatal months 1 (89.9/cell), 2 (95.1/cell), and 6 (102.1), reaching the maximum at month 12 (128.0/cell), then decreased to years 2 (93.9/cell). The increase and decrease were stochastically significant (P<0.01). The results obtained from visual counting on the numbers of mitochondria labeled with silver grains from 10 binucleate hepatocytes of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 aging groups at postnatal day 1, 3, and 14, month 1, 2, 6 and year 1 and 2, were counted. The labeling indices in respective aging stages were calculated from the numbers of labeled mitochondria and the numbers of total mitochondria per cell which showed that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis increased from postnatal day 1 (7.3/cell) to day 3 (6.8/cell), 14 (10.2/cell), and month 1 (15.0/cell), 2 (15.9/cell), reaching the maximum at month 6 (19.6/cell), then decreased to year 1 (8.3/cell) and 2 (5.1/cell), while the labeling indices increased from postnatal day 1 (11.8%) to 3 (10.2%), 14 (12.5%), month 1 (18.3%) and 2 (18.7%), reaching the maximum at month 6 (19.2%), then decreased to year 1 (6.4%) and 2 (5.5%). Stochastical analysis revealed that the increases and decreases of the numbers of labeled mitochondria as well as the labeling indices from the newborn stage to the adult and senescent stages were significant (P<0.01). The silver grains due to 3H-leucine were also observed in sinusoidal endothelial cells (Fig. 8F), Kupfer's cells, Ito's fat-storing cells, ductal

epithelial cells, fibroblasts and hematopoietic cells at various ages.

When 3H-prolin was injected into aging mice at various ages, silver grains were localized over the nuclei, cytoplasmic matrix, endoplasmic reticulum, the Golgi apparatus, mitochondria and peroxisomes of all the cell such as hepatocytes (Fig. 9G), sinusoidal endothelial cells, Kupfer's cells (Fig. 9H), Ito's fat-storing cells, ductal epithelial cells, fibroblasts and hematopoietic cells at various ages. The number of silver grains in hepatocytes gradually increased from perinatal stage to postnatal month 1 to 6 due to aging, reaching the maximum and decreased to momth 24. The number of silver grains localized the extracellular collage fibrils and matrices was not so great in respective aging groups and

month young adult animals and decreased with aging to senescence at 24 months. Number of silver grains observed over respective cell organelles, the Golgi apparatus, mitochondria and endoplasmic reticulum, changed with aging, reaching the maxima at 1 month but the ratio remained constant at each point. When 3H-proline was injected into mice at various ages from prenatal embryos to postnatal senescence, quantitative changes of collagen and protein synthesis in the livers were studied by electron microscopic radioautography (Ma and Nagata 2000, Nagata 2006a). The silver grains due to 3H-proline showing collagen synthesis were localized over the nuclei, cytoplasmic matrix, endoplasmic reticulum, the Golgi apparatus, mitochondria and peroxisomes of almost all the cells such as hepatocytes (Fig. 9F), sinusoidal endothelial cells, Kupffer's cells (Fig. 9G), Ito's fat-storing cells, ductal epithelial cells, fibroblasts and haematopoietic cells at various ages. The number of silver grains in the cell bodies and nuclei, cytoplasmic matrix, endoplasmic reticulum, mitochondria, the Golgi apparatus and peroxisomes of hepatocytes gradually increased from embryo, reaching the maxima at postnatal month 1 and 6, and decreased with aging until 24 months. The grain counts of the cell bodies reached the maximum at month 6 and the nuclei at month 2, while that of endoplasmic reticulum at month 6 and mitochondria at month 1. The number of silver grains localized over the extracellular collagen fibrils and matrices was not so many in respective aging groups and did not show any remarkable changes with aging. From the results, it was concluded that 3H-proline was incorporated not only into collagen but also into the structural proteins of hepatocytes and increased and decreased due to aging under normal aging conditions.

#### **1.5.3.2 The protein synthesis in hepatocyte mitochondria**

When the aging mice at various ages from embryo to senescence were injected with 3Hleucine, it was found that almost all the hepatocytes, from embryonic day 19, postnatal day 1, 3, 9, 14, to adult and senescent stages at postnatal month 1, 2, 6, 12 and 24, incorporated silver grains (Fig. 9E). The silver grains were also observed in binucleate hepatocytes at postnatal day 1, 3, 9, 14, month 1, 2, 6, 12 and 24 (Nagata 2007a,b,c,d, 2006a,b, 2007b,c,e). The localizations of silver grains observed over the mitochondria were mainly on the mitochondrial matrices but a few over their nuclei, cytoplasmic matrix, endoplasmic reticulum, ribosomes, Golgi apparatus and mitochondria (Nagata 2006a, b, 2007b, c, e). In the mitochondria the silver grains were localized over the mitochondrial membranes and cristae when observed by high power magnification. Preliminary quantitative analysis on the number of mitochondria in 20 mononucleate hepatocytes whose nuclei were intensely labeled with many silver grains (more than 10 per nucleus) and other 20 mononucleate hepatocytes whose nuclei were not so intensely labeled (number of silver grains less than 9) in each aging group revealed that there was no significant difference between the number of mitochondria, number of labeled mitochondria and the labeling indices in both types of hepatocytes (P<0.01).

On the other hand, the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices were calculated in 10 binucleate hepatocytes selected at random in each animal in respective aging stages, regardless whether their nuclei were very intensely labeled or not, except the prenatal stage at embryonic day 19, because no binucleate cell was found at this stage, resulted in no significant difference between them. Thus, the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices were calculated in 20 hepatocytes selected at random in each animal in respective aging stages regardless whether their nuclei were very intensely labeled or not. The results obtained from the total

month young adult animals and decreased with aging to senescence at 24 months. Number of silver grains observed over respective cell organelles, the Golgi apparatus, mitochondria and endoplasmic reticulum, changed with aging, reaching the maxima at 1 month but the ratio remained constant at each point. When 3H-proline was injected into mice at various ages from prenatal embryos to postnatal senescence, quantitative changes of collagen and protein synthesis in the livers were studied by electron microscopic radioautography (Ma and Nagata 2000, Nagata 2006a). The silver grains due to 3H-proline showing collagen synthesis were localized over the nuclei, cytoplasmic matrix, endoplasmic reticulum, the Golgi apparatus, mitochondria and peroxisomes of almost all the cells such as hepatocytes (Fig. 9F), sinusoidal endothelial cells, Kupffer's cells (Fig. 9G), Ito's fat-storing cells, ductal epithelial cells, fibroblasts and haematopoietic cells at various ages. The number of silver grains in the cell bodies and nuclei, cytoplasmic matrix, endoplasmic reticulum, mitochondria, the Golgi apparatus and peroxisomes of hepatocytes gradually increased from embryo, reaching the maxima at postnatal month 1 and 6, and decreased with aging until 24 months. The grain counts of the cell bodies reached the maximum at month 6 and the nuclei at month 2, while that of endoplasmic reticulum at month 6 and mitochondria at month 1. The number of silver grains localized over the extracellular collagen fibrils and matrices was not so many in respective aging groups and did not show any remarkable changes with aging. From the results, it was concluded that 3H-proline was incorporated not only into collagen but also into the structural proteins of hepatocytes and increased and

When the aging mice at various ages from embryo to senescence were injected with 3Hleucine, it was found that almost all the hepatocytes, from embryonic day 19, postnatal day 1, 3, 9, 14, to adult and senescent stages at postnatal month 1, 2, 6, 12 and 24, incorporated silver grains (Fig. 9E). The silver grains were also observed in binucleate hepatocytes at postnatal day 1, 3, 9, 14, month 1, 2, 6, 12 and 24 (Nagata 2007a,b,c,d, 2006a,b, 2007b,c,e). The localizations of silver grains observed over the mitochondria were mainly on the mitochondrial matrices but a few over their nuclei, cytoplasmic matrix, endoplasmic reticulum, ribosomes, Golgi apparatus and mitochondria (Nagata 2006a, b, 2007b, c, e). In the mitochondria the silver grains were localized over the mitochondrial membranes and cristae when observed by high power magnification. Preliminary quantitative analysis on the number of mitochondria in 20 mononucleate hepatocytes whose nuclei were intensely labeled with many silver grains (more than 10 per nucleus) and other 20 mononucleate hepatocytes whose nuclei were not so intensely labeled (number of silver grains less than 9) in each aging group revealed that there was no significant difference between the number of mitochondria, number

of labeled mitochondria and the labeling indices in both types of hepatocytes (P<0.01).

On the other hand, the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices were calculated in 10 binucleate hepatocytes selected at random in each animal in respective aging stages, regardless whether their nuclei were very intensely labeled or not, except the prenatal stage at embryonic day 19, because no binucleate cell was found at this stage, resulted in no significant difference between them. Thus, the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices were calculated in 20 hepatocytes selected at random in each animal in respective aging stages regardless whether their nuclei were very intensely labeled or not. The results obtained from the total

decreased due to aging under normal aging conditions. **1.5.3.2 The protein synthesis in hepatocyte mitochondria**  numbers of mitochondria in mononucleate hepatocytes showed an increase from the prenatal day (34.5/cell) to postnatal days 1 (44.6/cell), 3 (45.8/cell), 9 (43.6/cell), 14 (48.5/cell), to postnatal months 1 (51.5/cell), 2 (52.3/cell), reaching the maximum at month 6 (60.7/cell), then decreased to years 1 (54.2/cell) and 2 (51.2/cell). The increase and decrease were stochastically significant (P<0.01). The results obtained from visual counting on the numbers of mitochondria labeled with silver grains from 20 mononucleate hepatocytes of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 2, 6 and year 1 and 2, were counted. The labeling indices in respective aging stages were calculated from the numbers of labeled mitochondria and the numbers of total mitochondria per cell. The results showed that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis increased from prenatal embryo day 19 (8.3/cell) to postnatal days 1 (9.6/cell), 3 (8.1/cell), 9 (8.9/cell), 14 (9.5/cell), and month 1 (11.2/cell), reaching the maximum, and then decreased to months 2 (9.1/cell), 6 (8.8/cell) to years 1 (6.7/cell) and 2 (2.2/cell), while the labeling indices increased from prenatal day 19 (20.1%) to postnatal days 1 (21.2%), 3 (21.6%), 9 (22.2%), 14 (23.1%), reaching the maximum, then decreased to month 1 (21.7%), 2 (17.4%), 6 (14.6%), and years 1 (12.4%) and 2 (4.4%). Stochastical analysis revealed that the increases and decreases of the numbers of labeled mitochondria as well as the labeling indices from the perinatal stage to the adult and senescent stages were significant (P<0.01).

The results obtained from the numbers of mitochondria in binucleate hepatocytes showed an increase from the postnatal days 1 (66.2/cell), to 3 (66.4/cell), 14 (81.8/cell), to postnatal months 1 (89.9/cell), 2 (95.1/cell), and 6 (102.1), reaching the maximum at month 12 (128.0/cell), then decreased to years 2 (93.9/cell). The increase and decrease were stochastically significant (P<0.01). The results obtained from visual counting on the numbers of mitochondria labeled with silver grains from 10 binucleate hepatocytes of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 aging groups at postnatal day 1, 3, and 14, month 1, 2, 6 and year 1 and 2, were counted. The labeling indices in respective aging stages were calculated from the numbers of labeled mitochondria and the numbers of total mitochondria per cell which showed that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis increased from postnatal day 1 (7.3/cell) to day 3 (6.8/cell), 14 (10.2/cell), and month 1 (15.0/cell), 2 (15.9/cell), reaching the maximum at month 6 (19.6/cell), then decreased to year 1 (8.3/cell) and 2 (5.1/cell), while the labeling indices increased from postnatal day 1 (11.8%) to 3 (10.2%), 14 (12.5%), month 1 (18.3%) and 2 (18.7%), reaching the maximum at month 6 (19.2%), then decreased to year 1 (6.4%) and 2 (5.5%). Stochastical analysis revealed that the increases and decreases of the numbers of labeled mitochondria as well as the labeling indices from the newborn stage to the adult and senescent stages were significant (P<0.01). The silver grains due to 3H-leucine were also observed in sinusoidal endothelial cells (Fig. 8F), Kupfer's cells, Ito's fat-storing cells, ductal epithelial cells, fibroblasts and hematopoietic cells at various ages.

When 3H-prolin was injected into aging mice at various ages, silver grains were localized over the nuclei, cytoplasmic matrix, endoplasmic reticulum, the Golgi apparatus, mitochondria and peroxisomes of all the cell such as hepatocytes (Fig. 9G), sinusoidal endothelial cells, Kupfer's cells (Fig. 9H), Ito's fat-storing cells, ductal epithelial cells, fibroblasts and hematopoietic cells at various ages. The number of silver grains in hepatocytes gradually increased from perinatal stage to postnatal month 1 to 6 due to aging, reaching the maximum and decreased to momth 24. The number of silver grains localized the extracellular collage fibrils and matrices was not so great in respective aging groups and

Macromolecular Synthesis in the Digestive and Respiratory Systems 335

Fig. 11. EM RAG of the pancreas. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 140, 2002, Urban & Fischer, Jena,

Germany

did not show any remarkable changes with aging. From the results, it was concluded that 3H-prolin was incorporated not only into collagen but also into the structural proteins of hepatocytes under normal aging conditions (Ma and Nagata 2000).

#### **1.5.4 The glucide synthesis in the liver**

We first studied 3H-glucose incorporation into glycogen in the livers of adult mice, in connection to soluble compounds (Nagata and Murata 1977, Nagata et al. 1977a,d). Soluble 3H-glucose, which was demonstrated by cryo-fixation (at -196C) in combination with drymounting radioautography, was localized over the nuclei, nucleoli, all the cell organelles and cytoplasmic ground substance of all the hepatocytes diffusely. On the other hand, by conventional chemical fixation and wet-mounting radioautography, silver grains were localized only over glycogen granules, endoplasmic reticulum and Golgi apparatus showing glycogen synthesis. However, the relationship of glycogen synthesis to aging has not yet been fully clarified.

#### **1.5.5 The lipids synthesis in the liver**

We observed lipids synthesis in the liver using 3H-glycerol in connection to soluble compounds (Nagata 1994a,d, Nagata and Murata 1977a, Nagata et al. 1977a,d). When adult mice were injected with 3H-glycerol and the livers were taken out, cryo-fixed in liquid nitrogen at -196C, then freeze-substituted, embedded in epoxy resin, dry-sectioned, and prepared for dry-mounting radioautography, many silver grains appeared over the nuclei and cytoplasm of hepatocytes diffusely. However, when the same liver tissues were fixed chemically in buffered glutaraldehyde and osmium tetroxide at 4C, dehydrated, embedded, wet-sectioned and radioautographed by conventional wet-mounting procedures, very few silver grains were observed only over the endoplasmic reticulum and the lipid droplets, which demonstrated insoluble macromolecular lipid synthesis accumulating into the lipid droplets. However, the aging change of the lipid synthesis in the liver has not yet been fully clarified.

#### **1.6 Macromolecular synthesis in the pancreas**

The pancreas is a large gland, next to the liver in men and animals, among the digestive glands connected to the intestines. It consists of exocrine and endocrine portions and takes the shape of a compound acinous gland. The exocrine portion is composed of ductal epithelial cells, centro-acinar cells, acinar cells and connective tissue cells, while the endocrine portion, the islet of Langerhans, is composed of 3 types of endocrine cells, A, B, C cells and connective tissue cells. Intracellular transport of secretory proteins in the pancreatic exocrine cells were formerly studied by Jamieson and Palade (1967) by EMRAG. We studied the macromolecular synthesis of the aging mouse pancreas at various ages.

#### **1.6.1 The DNA synthesis in the pancreas**

We first studied the DNA synthesis of mouse pancreas by LM and EMRAG using 3Hthymidine (Nagata and Usuda 1984, 1985, 1986, Nagata et al. 1986). Light and electron microscopic radioautograms of the pancreas revealed that the nuclei of pancreatic acinar cells (Fig. 11A), centro-acinar cells (Fig. 11B), ductal epithelial cells, and endocrine cells were labeled with 3H-thymidine. The labeling indices of these cells in 5 groups of litter mate mice, fetal day 15, postnatal day 1, 20, 60 (2 months) and 730 (2 years) were analyzed.

did not show any remarkable changes with aging. From the results, it was concluded that 3H-prolin was incorporated not only into collagen but also into the structural proteins of

We first studied 3H-glucose incorporation into glycogen in the livers of adult mice, in connection to soluble compounds (Nagata and Murata 1977, Nagata et al. 1977a,d). Soluble 3H-glucose, which was demonstrated by cryo-fixation (at -196C) in combination with drymounting radioautography, was localized over the nuclei, nucleoli, all the cell organelles and cytoplasmic ground substance of all the hepatocytes diffusely. On the other hand, by conventional chemical fixation and wet-mounting radioautography, silver grains were localized only over glycogen granules, endoplasmic reticulum and Golgi apparatus showing glycogen synthesis. However, the relationship of glycogen synthesis to aging has not yet

We observed lipids synthesis in the liver using 3H-glycerol in connection to soluble compounds (Nagata 1994a,d, Nagata and Murata 1977a, Nagata et al. 1977a,d). When adult mice were injected with 3H-glycerol and the livers were taken out, cryo-fixed in liquid nitrogen at -196C, then freeze-substituted, embedded in epoxy resin, dry-sectioned, and prepared for dry-mounting radioautography, many silver grains appeared over the nuclei and cytoplasm of hepatocytes diffusely. However, when the same liver tissues were fixed chemically in buffered glutaraldehyde and osmium tetroxide at 4C, dehydrated, embedded, wet-sectioned and radioautographed by conventional wet-mounting procedures, very few silver grains were observed only over the endoplasmic reticulum and the lipid droplets, which demonstrated insoluble macromolecular lipid synthesis accumulating into the lipid droplets. However, the

The pancreas is a large gland, next to the liver in men and animals, among the digestive glands connected to the intestines. It consists of exocrine and endocrine portions and takes the shape of a compound acinous gland. The exocrine portion is composed of ductal epithelial cells, centro-acinar cells, acinar cells and connective tissue cells, while the endocrine portion, the islet of Langerhans, is composed of 3 types of endocrine cells, A, B, C cells and connective tissue cells. Intracellular transport of secretory proteins in the pancreatic exocrine cells were formerly studied by Jamieson and Palade (1967) by EMRAG. We studied the macromolecular synthesis of the aging mouse pancreas at various ages.

We first studied the DNA synthesis of mouse pancreas by LM and EMRAG using 3Hthymidine (Nagata and Usuda 1984, 1985, 1986, Nagata et al. 1986). Light and electron microscopic radioautograms of the pancreas revealed that the nuclei of pancreatic acinar cells (Fig. 11A), centro-acinar cells (Fig. 11B), ductal epithelial cells, and endocrine cells were labeled with 3H-thymidine. The labeling indices of these cells in 5 groups of litter mate mice,

fetal day 15, postnatal day 1, 20, 60 (2 months) and 730 (2 years) were analyzed.

aging change of the lipid synthesis in the liver has not yet been fully clarified.

hepatocytes under normal aging conditions (Ma and Nagata 2000).

**1.5.4 The glucide synthesis in the liver** 

**1.5.5 The lipids synthesis in the liver** 

**1.6 Macromolecular synthesis in the pancreas** 

**1.6.1 The DNA synthesis in the pancreas** 

been fully clarified.

Fig. 11. EM RAG of the pancreas. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 140, 2002, Urban & Fischer, Jena, Germany

Macromolecular Synthesis in the Digestive and Respiratory Systems 337

On the other hand, LM and EMRAG of pancreas of mouse injected with 3H-uridine demonstrated its incorporation into exocrine and then in endocrine cells, and more in pancreatic acinar cells (Figs. 11C,D) than in ductal or centro-acinar cells (Nagata and Usuda 1986a,b, Nagata et al. 1986). Among the acinar cells, the number of silver grains increased after birth to day 14 and then decreased with aging. Quantification of silver grains in the nucleoli, chromatin, and cell body were carried out by X-ray microanalysis (Nagata 1991, 1993, 2004, Nagata and Usuda 1985), which verified the results obtained by visual grain counting. In EMRAG obtained from the pancreas of fetal day 19 embryos, newborn day 1 and newborn day 14 mice labeled with 3H-uridine, demonstrating RNA synthesis, the number of silver grains in the nucleoli, nuclear chromatin and cytoplasm increased (Nagata 1985, 1991, 1993a,b, 2004, Nagata and Usuda 1985, 1986). In order to quantify the silver contents of grains observed over the nucleoli, nuclei and cytoplasm, X-ray spectra were recorded by energy dispersive X-ray microanalysis (JEM-4000EX TN5400), demonstrating Ag-Ka peaks at higher energies. Thus, P/B ratios expressing relative silver contents were determined and compared between the two age groups. The results obtained by X-ray microanalysis in different cell compartments at postnatal day 1 and day 14 and he results obtained by visual grain counting in different cell compartments in day 1 day and day 14 animals were compared. The number of silver grains was calculated to express the counts per unit area to be compared with the XMA counts. These two results, the silver content analyzed by X-ray microanalysis and the results obtaining from visual grain counting were

As for the protein synthesis in the pancreas, 3H-leucine incorporation into endoplasmic reticulum, Golgi apparatus and to secretory granules of pancreatic acinar cells was first demonstrated by Jamieson and Palade (1967). We first studied 3H-glycine incorporation into these cell organelles of mouse pancreatic acinar cells in connection with soluble compounds by EM RAG (Nagata 2000c, 2007a). It was demonstrated that soluble 3H-glycine distributed not only in these cell organelles but also in the karyoplasm and cytoplasm diffusely. Then, the quantitative aspects of protein synthesis with regards the aging from fetal day 19, to postnatal day 1, 3, 7, 14 and 1, 2, 6 and 12 months were also clarified (Nagata and Usuda 1993a, Nagata 2000c). The results showed an increase of silver grain counts labeled with 3Hleucine after birth, reaching a peak from postnatal 2 weeks to 1 month (Fig. 10E), and

On the other hand, we also studied 3H-leucine incorporations into the pancreatic acinar cells of both normal adult rats and experimentally pancreatitis induced rats with either ethionine or alcohol (Yoshizawa et al. 1974, 1977). The results showed that the incorporations as indicated silver grain counts in the pancreatitis rats were less than normal control rats.

Concerning the glucide synthesis of the pancreas, we first studied the incorporation of 3Hglucose into the pancreatic acinar cells of mouse in connection with soluble compounds by

**1.6.2 The RNA synthesis in the pancreas** 

in good accordance with each other.

**1.6.3 The protein synthesis in the pancreas** 

decreasing from 2 months to 1 year (Fig. 11F).

**1.6.4 The glucide synthesis in the pancreas** 

However, its relation to the aging was not yet studied.

Fig. 11A. EM RAG of 2 pancreatic acinar cells of a 14 day old mouse labeled with 3Hthymidine, showing DNA synthesis. x10,000.

Fig. 11B. EM RAG of 2 centro-acinar cells of a 14 day old mouse labeled with 3H-thymidine, showing DNA synthesis. x10,000.

Fig. 11C. EM RAG of 3 pancreatic acinar cells of a 1 day old mouse labeled with 3H-uridine, showing RNA synthesis. x10,000.

Fig. 11D. EM RAG of 3 pancreatic acinar cells of a 14 day old mouse labeled with 3H-uridine, showing RNA synthesis. x10,000.

Fig. 11E. EM RAG of a pancreatic acinar cell of a 30 day old mouse labeled with 3H-leucine, showing protein synthesis. x10,000.

Fig. 11F. EM RAG of a pancreatic acinar cell of a 12 month old mouse labeled with 3Hleucine, showing protein synthesis. x10,000.

Fig. 11G. EM RAG of a pancreatic acinar cell of a 1 day old mouse labeled with 3Hglucosamine, showing glucide synthesis. x10,000.

Fig. 11H. EM RAG of a pancreatic acinar cell of a 14 day old mouse labeled with 3Hglucosamine, showing glucide synthesis. x10,000.

The labeling indices of these cells reached the maxima at day 1 after birth and decreased gradually to 2 years (Fig. 12). The maximum in the acinar cells proceeded to the ductal and centro-acinar cells, suggesting that the acinar cells completed their development earlier than the ductal and centro-acinar cells (Nagata et al. 1986).

Fig. 12. Transitional curves of the labeling indices of respective cell types of the pancreas of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 142, 2002, Urban & Fischer, Jena, Germany

## **1.6.2 The RNA synthesis in the pancreas**

336 Senescence

Fig. 11B. EM RAG of 2 centro-acinar cells of a 14 day old mouse labeled with 3H-thymidine,

Fig. 11C. EM RAG of 3 pancreatic acinar cells of a 1 day old mouse labeled with 3H-uridine,

Fig. 11D. EM RAG of 3 pancreatic acinar cells of a 14 day old mouse labeled with 3H-uridine,

Fig. 11E. EM RAG of a pancreatic acinar cell of a 30 day old mouse labeled with 3H-leucine,

The labeling indices of these cells reached the maxima at day 1 after birth and decreased gradually to 2 years (Fig. 12). The maximum in the acinar cells proceeded to the ductal and centro-acinar cells, suggesting that the acinar cells completed their development earlier than

Fig. 12. Transitional curves of the labeling indices of respective cell types of the pancreas of

aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 142, 2002, Urban & Fischer, Jena, Germany

Fig. 11F. EM RAG of a pancreatic acinar cell of a 12 month old mouse labeled with 3H-

Fig. 11G. EM RAG of a pancreatic acinar cell of a 1 day old mouse labeled with 3H-

Fig. 11H. EM RAG of a pancreatic acinar cell of a 14 day old mouse labeled with 3H-

Fig. 11A. EM RAG of 2 pancreatic acinar cells of a 14 day old mouse labeled with 3H-

thymidine, showing DNA synthesis. x10,000.

showing DNA synthesis. x10,000.

showing RNA synthesis. x10,000.

showing RNA synthesis. x10,000.

showing protein synthesis. x10,000.

leucine, showing protein synthesis. x10,000.

glucosamine, showing glucide synthesis. x10,000.

glucosamine, showing glucide synthesis. x10,000.

the ductal and centro-acinar cells (Nagata et al. 1986).

On the other hand, LM and EMRAG of pancreas of mouse injected with 3H-uridine demonstrated its incorporation into exocrine and then in endocrine cells, and more in pancreatic acinar cells (Figs. 11C,D) than in ductal or centro-acinar cells (Nagata and Usuda 1986a,b, Nagata et al. 1986). Among the acinar cells, the number of silver grains increased after birth to day 14 and then decreased with aging. Quantification of silver grains in the nucleoli, chromatin, and cell body were carried out by X-ray microanalysis (Nagata 1991, 1993, 2004, Nagata and Usuda 1985), which verified the results obtained by visual grain counting. In EMRAG obtained from the pancreas of fetal day 19 embryos, newborn day 1 and newborn day 14 mice labeled with 3H-uridine, demonstrating RNA synthesis, the number of silver grains in the nucleoli, nuclear chromatin and cytoplasm increased (Nagata 1985, 1991, 1993a,b, 2004, Nagata and Usuda 1985, 1986). In order to quantify the silver contents of grains observed over the nucleoli, nuclei and cytoplasm, X-ray spectra were recorded by energy dispersive X-ray microanalysis (JEM-4000EX TN5400), demonstrating Ag-Ka peaks at higher energies. Thus, P/B ratios expressing relative silver contents were determined and compared between the two age groups. The results obtained by X-ray microanalysis in different cell compartments at postnatal day 1 and day 14 and he results obtained by visual grain counting in different cell compartments in day 1 day and day 14 animals were compared. The number of silver grains was calculated to express the counts per unit area to be compared with the XMA counts. These two results, the silver content analyzed by X-ray microanalysis and the results obtaining from visual grain counting were in good accordance with each other.

## **1.6.3 The protein synthesis in the pancreas**

As for the protein synthesis in the pancreas, 3H-leucine incorporation into endoplasmic reticulum, Golgi apparatus and to secretory granules of pancreatic acinar cells was first demonstrated by Jamieson and Palade (1967). We first studied 3H-glycine incorporation into these cell organelles of mouse pancreatic acinar cells in connection with soluble compounds by EM RAG (Nagata 2000c, 2007a). It was demonstrated that soluble 3H-glycine distributed not only in these cell organelles but also in the karyoplasm and cytoplasm diffusely. Then, the quantitative aspects of protein synthesis with regards the aging from fetal day 19, to postnatal day 1, 3, 7, 14 and 1, 2, 6 and 12 months were also clarified (Nagata and Usuda 1993a, Nagata 2000c). The results showed an increase of silver grain counts labeled with 3Hleucine after birth, reaching a peak from postnatal 2 weeks to 1 month (Fig. 10E), and decreasing from 2 months to 1 year (Fig. 11F).

On the other hand, we also studied 3H-leucine incorporations into the pancreatic acinar cells of both normal adult rats and experimentally pancreatitis induced rats with either ethionine or alcohol (Yoshizawa et al. 1974, 1977). The results showed that the incorporations as indicated silver grain counts in the pancreatitis rats were less than normal control rats. However, its relation to the aging was not yet studied.

#### **1.6.4 The glucide synthesis in the pancreas**

Concerning the glucide synthesis of the pancreas, we first studied the incorporation of 3Hglucose into the pancreatic acinar cells of mouse in connection with soluble compounds by

Macromolecular Synthesis in the Digestive and Respiratory Systems 339

The tracheas of mammals are composed of ciliated pseudostratified columnar epithelia, connective tissues, smooth muscles and hyalin cartilages surrounding the epithelia. The changes of DNA synthesis of tracheal cells in aging mice were studied by LM and EMRAG (Sun et al. 1997a, Nagata 2000d). The tracheae of 8 groups of mice from fetal day 18 to 2 years after birth were examined. The results demonstrated that the DNA syntheses and morphology of tracheal cells in the mouse tracheae changed due to aging. The radioautograms revealed that the DNA synthesis in the nuclei of ciliated cells was observed only in the fetal animals (Fig. 13A). However, the DNA synthesis in nonciliated cells and basal cells was observed in both prenatal and postnatal animals (Fig. 13B). The labeling indices of respective cell types were analyzed (Sun et al. 1997a). As the results, the labeling indices of the epithelial cells showed their maxima on fetal day 18, then fell down from postnatal day 3 to 2 years (Fig. 14A). The ciliated cell could not synthesize DNA and proliferate in the postnatal stage. They are supposed to be derived by the division and transformation of the basal cells. On the other hand, the DNA synthesis of chondrocytes was the highest on embryonic day 18, and rapidly declined on postnatal day 3 (Fig. 13C). The chondrocytes lost the ability of synthesizing DNA at 2 months after birth (Fig. 14B). The labeling indices of other cells (including fibroblasts, smooth muscle and glandular cells) were the highest on fetal day 18 and fell down markedly

on the third day after birth and decreased progressively due to aging (Fig. 14C).

macromolecular precursors respectively (Figs. 13E, F, G, H).

**2.1.2.1 The DNA synthesis in the pulmonary cells** 

We studied the pulmonary tissues at various ages from embryo to postnatal 2 years of mice (Sun et al. 1995a,b). The pulmonary tissues obtained from ddY strain mice at various ages from embryo day 19 to adult postnatal day 30 and to year 2 consisted of several types of cells, i.e., the type I epithelial cells or the small alveolar epithelial cells, type II epithelial cells or large alveolar epithelial cells, interstitial cells and endothelial cells, which incorporated

The pulmonary tissues obtained from ddY strain mice at embryonic to early postnatal stages consisted of undifferentiated cells (Fig. 13E). However, they differentiated into several types of cells due to aging, the type I epithelial cells or the small alveolar epithelial cells (Fig. 13E), the type II epithelial cells or the large alveolar epithelial cells (Fig. 13F), the interstitial cells (Fig. 13G), the endothelial cells (Fig. 13H) and alveolar phagocytes or dust cells as we had formerly observed (Sun et al. 1995a,b). At embryonic day 16 and 18, the fetal lung tissues appeared as glandular organizations consisting of many alveoli bordering undifferentiated cuboidal cells and no squamous epithelial cells were seen (Figs. 13E, 13G). Mitotic figures were frequently observed in cuboidal epithelial cells. After birth, the structure of the alveoli was characterized by further development of the alveolar-capillary networks from postnatal day 1 to 3 and 7 (Fig. 13H). During the development, the cellular composition of the alveolar epithelium resembled that of the adult lung, with a mixed population of the type I and type II epithelial cells. Up to 1 and 2 weeks after birth, the lung tissues showed complete alveolar structure and single capillary system almost the same as the adult after 1 month (Fig. 13F) to 2 to 6 months, and further to senescent stage over 12 months to 22 months. On electron microscopic radioautograms of the pulmonary tissues labeled with 3H-thymidine, silver grains were observed over the nuclei of some pulmonary cells corresponding to the DNA synthesis in S-phase as observed by light microscopic radioautograpy (Sun et al. 1995a,b, 1997a).

**2.1.1 The DNA synthesis in the trachea** 

**2.1.2 The DNA synthesis in the lung** 

EM RAG (Nagata et al. 1977a). It was demonstrated that soluble 3H-glucose distributed not only in such cell organelles as endoplasmic reticulum, Golgi apparatus, mitochondria but also in the karyoplasm and cytoplasm diffusely. Then, the incorporation of 3H-glucosamine into the pancreases of aging mice at various ages was studied by LM and EM RAG (Nagata et al. 1992). When perinatal baby mice received 3H-glucosamine injections and the pancreatic tissues were radioautographed, silver grains were observed over exocrine and endocrine pancreatic cells. However, the number of silver grains was not so many (Fig. 11G). When juvenile mice at the age of 14 days after birth were examined, many silver grains appeared over the exocrine pancreatic acinar cells (Fig. 11H). Less silver grains were observed over endocrine pancreatic cells and ductal epithelial cells. The grains in the exocrine pancreatic acinar cells were localized over the nucleus, endoplasmic reticulum, Golgi apparatus and secretory granules, demonstrating glycoprotein synthesis. Adult mice at the ages of postnatal 1 month, 6 month or senile mice at the ages of 12 months or 24 months showed very few silver grains on radioautograms. Thus, the glucide synthesis in the pancreatic acinar cells of mice revealed quantitative changes, increase and decrease of 3Hglucosamine incorporation with aging (Nagata 1994a,b,c,d,e, Nagata et al. 1992).

#### **1.6.5 The lipids synthesis in the pancreas**

In order to demonstrate the lipids synthesis in the pancreas, several litters of ddY mice aged fetal day 19, postnatal day 1, 3, 7, 14, and 1, 2, 6 up to 12 months, were injected with 3Hglycerol and the pancreas tissues were prepared for LM and EM RAG. The silver grains were observed in both exocrine and endocrine cells of respective ages (Nagata 1995a,b, Nagata et al. 1988b, 1990). In perinatal animals from fetal day 19 to postnatal 1, 3, and 7 days, cell organelles were not well developed in exocrine and endocrine cells and number of silver grains was very few. In 14 day old juvenile animals, cell organelles such as endoplasmic reticulum, Golgi apparatus, mitochondria and secretory granules were well developed and many silver grains were observed over these organelles and nuclei in both exocrine and endocrine cells. The number of silver grains was more in exocrine cells than endocrine cells. In 1, 2, 6 month old adult animals, number of silver grains remained constant. In 12 month old senescent animals, silver grains were fewer than younger animals. It was demonstrated that the number of silver grains expressed the quantity of lipids synthesis, which increased from perinatal atages to adult and senescent stages and finally decreased to senescence.

#### **2. Macromolecular synthesis in the respiratory system**

The respiratory system consists of 2 parts, the air-conducting portion and the respiratory portion. The former are the nose, the pharynx, the larynx and the trachea, while the latter the lung. We studied the macromolecular synthesis in the pulmonary tissues as well as the tracheal tissues at various ages from embryo to postnatal 2 years.

#### **2.1 The DNA synthesis in the respiratory system**

Among the air-conducting portion and the respiratory portion we studied the macromolecular synthesis in the tracheal tissues as well as the pulmonary tissues at various ages from embryo to postnatal 2 years (Sun et al. 1994, 1995a,b, 1997a,b, Nagata 2000d).

## **2.1.1 The DNA synthesis in the trachea**

338 Senescence

EM RAG (Nagata et al. 1977a). It was demonstrated that soluble 3H-glucose distributed not only in such cell organelles as endoplasmic reticulum, Golgi apparatus, mitochondria but also in the karyoplasm and cytoplasm diffusely. Then, the incorporation of 3H-glucosamine into the pancreases of aging mice at various ages was studied by LM and EM RAG (Nagata et al. 1992). When perinatal baby mice received 3H-glucosamine injections and the pancreatic tissues were radioautographed, silver grains were observed over exocrine and endocrine pancreatic cells. However, the number of silver grains was not so many (Fig. 11G). When juvenile mice at the age of 14 days after birth were examined, many silver grains appeared over the exocrine pancreatic acinar cells (Fig. 11H). Less silver grains were observed over endocrine pancreatic cells and ductal epithelial cells. The grains in the exocrine pancreatic acinar cells were localized over the nucleus, endoplasmic reticulum, Golgi apparatus and secretory granules, demonstrating glycoprotein synthesis. Adult mice at the ages of postnatal 1 month, 6 month or senile mice at the ages of 12 months or 24 months showed very few silver grains on radioautograms. Thus, the glucide synthesis in the pancreatic acinar cells of mice revealed quantitative changes, increase and decrease of 3H-

glucosamine incorporation with aging (Nagata 1994a,b,c,d,e, Nagata et al. 1992).

**2. Macromolecular synthesis in the respiratory system** 

tracheal tissues at various ages from embryo to postnatal 2 years.

**2.1 The DNA synthesis in the respiratory system** 

In order to demonstrate the lipids synthesis in the pancreas, several litters of ddY mice aged fetal day 19, postnatal day 1, 3, 7, 14, and 1, 2, 6 up to 12 months, were injected with 3Hglycerol and the pancreas tissues were prepared for LM and EM RAG. The silver grains were observed in both exocrine and endocrine cells of respective ages (Nagata 1995a,b, Nagata et al. 1988b, 1990). In perinatal animals from fetal day 19 to postnatal 1, 3, and 7 days, cell organelles were not well developed in exocrine and endocrine cells and number of silver grains was very few. In 14 day old juvenile animals, cell organelles such as endoplasmic reticulum, Golgi apparatus, mitochondria and secretory granules were well developed and many silver grains were observed over these organelles and nuclei in both exocrine and endocrine cells. The number of silver grains was more in exocrine cells than endocrine cells. In 1, 2, 6 month old adult animals, number of silver grains remained constant. In 12 month old senescent animals, silver grains were fewer than younger animals. It was demonstrated that the number of silver grains expressed the quantity of lipids synthesis, which increased from perinatal atages to adult and senescent stages and finally

The respiratory system consists of 2 parts, the air-conducting portion and the respiratory portion. The former are the nose, the pharynx, the larynx and the trachea, while the latter the lung. We studied the macromolecular synthesis in the pulmonary tissues as well as the

Among the air-conducting portion and the respiratory portion we studied the macromolecular synthesis in the tracheal tissues as well as the pulmonary tissues at various ages from embryo to postnatal 2 years (Sun et al. 1994, 1995a,b, 1997a,b, Nagata 2000d).

**1.6.5 The lipids synthesis in the pancreas** 

decreased to senescence.

The tracheas of mammals are composed of ciliated pseudostratified columnar epithelia, connective tissues, smooth muscles and hyalin cartilages surrounding the epithelia. The changes of DNA synthesis of tracheal cells in aging mice were studied by LM and EMRAG (Sun et al. 1997a, Nagata 2000d). The tracheae of 8 groups of mice from fetal day 18 to 2 years after birth were examined. The results demonstrated that the DNA syntheses and morphology of tracheal cells in the mouse tracheae changed due to aging. The radioautograms revealed that the DNA synthesis in the nuclei of ciliated cells was observed only in the fetal animals (Fig. 13A). However, the DNA synthesis in nonciliated cells and basal cells was observed in both prenatal and postnatal animals (Fig. 13B). The labeling indices of respective cell types were analyzed (Sun et al. 1997a). As the results, the labeling indices of the epithelial cells showed their maxima on fetal day 18, then fell down from postnatal day 3 to 2 years (Fig. 14A). The ciliated cell could not synthesize DNA and proliferate in the postnatal stage. They are supposed to be derived by the division and transformation of the basal cells. On the other hand, the DNA synthesis of chondrocytes was the highest on embryonic day 18, and rapidly declined on postnatal day 3 (Fig. 13C). The chondrocytes lost the ability of synthesizing DNA at 2 months after birth (Fig. 14B). The labeling indices of other cells (including fibroblasts, smooth muscle and glandular cells) were the highest on fetal day 18 and fell down markedly on the third day after birth and decreased progressively due to aging (Fig. 14C).

#### **2.1.2 The DNA synthesis in the lung**

We studied the pulmonary tissues at various ages from embryo to postnatal 2 years of mice (Sun et al. 1995a,b). The pulmonary tissues obtained from ddY strain mice at various ages from embryo day 19 to adult postnatal day 30 and to year 2 consisted of several types of cells, i.e., the type I epithelial cells or the small alveolar epithelial cells, type II epithelial cells or large alveolar epithelial cells, interstitial cells and endothelial cells, which incorporated macromolecular precursors respectively (Figs. 13E, F, G, H).

#### **2.1.2.1 The DNA synthesis in the pulmonary cells**

The pulmonary tissues obtained from ddY strain mice at embryonic to early postnatal stages consisted of undifferentiated cells (Fig. 13E). However, they differentiated into several types of cells due to aging, the type I epithelial cells or the small alveolar epithelial cells (Fig. 13E), the type II epithelial cells or the large alveolar epithelial cells (Fig. 13F), the interstitial cells (Fig. 13G), the endothelial cells (Fig. 13H) and alveolar phagocytes or dust cells as we had formerly observed (Sun et al. 1995a,b). At embryonic day 16 and 18, the fetal lung tissues appeared as glandular organizations consisting of many alveoli bordering undifferentiated cuboidal cells and no squamous epithelial cells were seen (Figs. 13E, 13G). Mitotic figures were frequently observed in cuboidal epithelial cells. After birth, the structure of the alveoli was characterized by further development of the alveolar-capillary networks from postnatal day 1 to 3 and 7 (Fig. 13H). During the development, the cellular composition of the alveolar epithelium resembled that of the adult lung, with a mixed population of the type I and type II epithelial cells. Up to 1 and 2 weeks after birth, the lung tissues showed complete alveolar structure and single capillary system almost the same as the adult after 1 month (Fig. 13F) to 2 to 6 months, and further to senescent stage over 12 months to 22 months. On electron microscopic radioautograms of the pulmonary tissues labeled with 3H-thymidine, silver grains were observed over the nuclei of some pulmonary cells corresponding to the DNA synthesis in S-phase as observed by light microscopic radioautograpy (Sun et al. 1995a,b, 1997a).

Macromolecular Synthesis in the Digestive and Respiratory Systems 341

Fig. 13A. LM RAG of the tracheal epithelial cells of a prenatal day 18 mouse embryo labeled

Fig. 13B. LM RAG of the tracheal epithelial cells of a postnatal 1 month old mouse labeled

Fig. 13C. LM RAG of the tracheal cartilage cells of a prenatal day 19 mouse embryo labeled

Fig. 13D. LM RAG of the tracheal cartilage cells of a postnatal day 3 mouse labeled with

Fig. 13E. LM RAG of the lung of a 1 day old mouse labeled with 3H-thymidine, showing

Fig. 13F. LM RAG of the lung of a 1 month old mouse labeled with 3H-thymidine, showing

Fig. 13G. LM RAG of the lung of a prenatal day 16 mouse embryo labeled with 3H-uridine,

The DNA synthetic activity of respective pulmonary cells as expressed by labeling indices demonstrated increases from perinatal stage to developmental stage and decreased due to aging. We also studied inhalation of 3H-thymidine in air by means of a nebulizer into the lungs of 1 week old young mice as experimental studies and observed by LM and EM RAG (Duan et al. 1994). After 10 min. inhalation, the lung tissues were taken out and processed by either rapid freezing and freeze-substitution for dry-mounting radioautography or conventional chemical fixation for wet-mounting radioautography. By wet-mounting RAG silver grains were observed in the nuclei of a few alveolar type 2 cells and interstitial cells demonstrating DNA synthesis. By dry-mounting RAG, numerous silver grains were located diffusely over all the epithelial cells and interstitial cells demonstrating soluble compounds. The results showed that 3H-thymidine inhaled into the lung distributed over all the pulmonary cells but only some of the alveolar type 2 cells and interstitial cells did synthesize

On the other hand, we studied the aging changes of DNA synthesis in the lungs of salamanders, *Hynobius nebolosus*, from larvae at 2 month after fertilization, juvenile at 1 month, adults at 10 and 12 months after metamorphosis, and finally to senescence at 5 years by LM RAG after 3H-thymidine injections (Matsumura et at. 1994). The results showed that the labeling indices of in the ciliated cells and mucous cells in the superficial layer of young animals were higher than those of the basal cells and they decreased in adults,

On electron microscopic radioautograms of the pulmonary tissues labeled with 3Hthymidine, silver grains were observed over not only the nuclei of some pulmonary cells corresponding to the DNA synthesis in S-phase as observed by LM radioautograpy (Sun et al. 1995a,b) but also over the mitochondria by EMRAG (Sun et al. 1995b). Some mitochondria in both S-phase cells and interphase cells which did not show any silver grains over their nuclei were labeled with silver grains showing intramitochondrial DNA

Fig. 13H. LM RAG of the lung of a 7 day old mouse labeled with 3H-leucine, showing

with 3H-thymidine, showing DNA synthesis. x1,125.

with 3H-thymidine, showing DNA synthesis. x1,125.

with 35SO4 showing mucosubstance synthesis. x400.

35SO4 showing mucosubstance synthesis. x400.

demonstrating aging changes in salamanders.

**2.1.2.2 The DNA synthesis in mitochondria of mouse pulmonary cells** 

DNA synthesis. x750.

DNA synthesis. x750.

protein synthesis. x750.

DNA.

showing RNA synthesis. x750.

Fig. 13. LM RAG of the respiratory organs. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 102, 2001, Academic Press, San Diego, USA, London, UK.

Fig. 13. LM RAG of the respiratory organs. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 102, 2001, Academic Press, San Diego,

USA, London, UK.

Fig. 13A. LM RAG of the tracheal epithelial cells of a prenatal day 18 mouse embryo labeled with 3H-thymidine, showing DNA synthesis. x1,125.

Fig. 13B. LM RAG of the tracheal epithelial cells of a postnatal 1 month old mouse labeled with 3H-thymidine, showing DNA synthesis. x1,125.

Fig. 13C. LM RAG of the tracheal cartilage cells of a prenatal day 19 mouse embryo labeled with 35SO4 showing mucosubstance synthesis. x400.

Fig. 13D. LM RAG of the tracheal cartilage cells of a postnatal day 3 mouse labeled with 35SO4 showing mucosubstance synthesis. x400.

Fig. 13E. LM RAG of the lung of a 1 day old mouse labeled with 3H-thymidine, showing DNA synthesis. x750.

Fig. 13F. LM RAG of the lung of a 1 month old mouse labeled with 3H-thymidine, showing DNA synthesis. x750.

Fig. 13G. LM RAG of the lung of a prenatal day 16 mouse embryo labeled with 3H-uridine, showing RNA synthesis. x750.

Fig. 13H. LM RAG of the lung of a 7 day old mouse labeled with 3H-leucine, showing protein synthesis. x750.

The DNA synthetic activity of respective pulmonary cells as expressed by labeling indices demonstrated increases from perinatal stage to developmental stage and decreased due to aging. We also studied inhalation of 3H-thymidine in air by means of a nebulizer into the lungs of 1 week old young mice as experimental studies and observed by LM and EM RAG (Duan et al. 1994). After 10 min. inhalation, the lung tissues were taken out and processed by either rapid freezing and freeze-substitution for dry-mounting radioautography or conventional chemical fixation for wet-mounting radioautography. By wet-mounting RAG silver grains were observed in the nuclei of a few alveolar type 2 cells and interstitial cells demonstrating DNA synthesis. By dry-mounting RAG, numerous silver grains were located diffusely over all the epithelial cells and interstitial cells demonstrating soluble compounds. The results showed that 3H-thymidine inhaled into the lung distributed over all the pulmonary cells but only some of the alveolar type 2 cells and interstitial cells did synthesize DNA.

On the other hand, we studied the aging changes of DNA synthesis in the lungs of salamanders, *Hynobius nebolosus*, from larvae at 2 month after fertilization, juvenile at 1 month, adults at 10 and 12 months after metamorphosis, and finally to senescence at 5 years by LM RAG after 3H-thymidine injections (Matsumura et at. 1994). The results showed that the labeling indices of in the ciliated cells and mucous cells in the superficial layer of young animals were higher than those of the basal cells and they decreased in adults, demonstrating aging changes in salamanders.

## **2.1.2.2 The DNA synthesis in mitochondria of mouse pulmonary cells**

On electron microscopic radioautograms of the pulmonary tissues labeled with 3Hthymidine, silver grains were observed over not only the nuclei of some pulmonary cells corresponding to the DNA synthesis in S-phase as observed by LM radioautograpy (Sun et al. 1995a,b) but also over the mitochondria by EMRAG (Sun et al. 1995b). Some mitochondria in both S-phase cells and interphase cells which did not show any silver grains over their nuclei were labeled with silver grains showing intramitochondrial DNA

Macromolecular Synthesis in the Digestive and Respiratory Systems 343

Fig. 14. Histogram showing aging changes of average labeling indices in respective cell types of the trachea of aging mice labeled with 3H-thymidine. Mean ± Standard Deviation.

From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem.

Cytochem. Vol. 37, No. 2, p. 148, 2002, Urban & Fischer, Jena, Germany

Fig. 14A. Epithelial cells.

Fig. 14C. Other cells.

Fig. 14B. Chondrocytes in the cartilage.

synthesis. The intramitochondrial DNA synthesis was observed in all cell types, the type I epithelial cells, the type II epithelial cells (Figs. 13F,G), the interstitial cells (Fig. 13F) and the endothelial cells. Because enough numbers of electron photographs (more than 5) were not obtained from all the cell types in respective aging groups, only some cell types and some aging groups when enough numbers of electron photographs were available were used for quantitative analysis. The numbers of mitochondria per cell profile area, the numbers of labeled mitochondria per cell and the labeling indices of the type I epithelial cells in only a few aging groups were observed and counted. The labeling indices in respective aging stages were calculated from the number of labeled mitochondria and the number of total mitochondria per cellular profile area which were calculated, respectively. These results demonstrated that the labeling indices in these cell types decreased from prenatal stages at embryo day 16 to day 18 (20-25%), and further decreased to postnatal days up to senescent stages due to aging (Fig. 15).

#### **2.2 The RNA synthesis in the respiratory system**

We studied the lungs of aging mice among the respiratory organs after administration of 3H-uridine at various ages from prenatal embryonic day 16 to postnatal senescent month 22 as observed by LM and EM RAG (Fig. 13H).

#### **2.2.1 RNA synthesis of mouse pulmonary cells**

When the lung tissues of mice were labeled with 3H-uridine, RNA synthesis was observed in all cells of the lungs at various ages by RAG (Sun 1995, Sun et al. 1997b, Nagata 2002). Observing the light microscopic radioautograms labeled with 3H-uridine, the silver grains were found over both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo day 16, 18, to postnatal day 1, 3, 7, 14, but also at the adult and senescent stages from postnatal month 1 to 2, 6, 12 and 22. The number of silver grains, by electron micrography, changed with aging. The grain counts in type I epithelial cells increased from the 1st day after birth and reached a peak at 1 week and decreased gradually to month 22, while the counts in type II epithelial cells (Fig. 13H), interstitial cells and endothelial cells increased from embryo day 16 and reached peaks at 1 week after birth, then decreased to senescence.

#### **2.2.2 The RNA synthesis of mitochondria in pulmonary cells**

By electron microscopic radioautography, silver grains were observed in most pulmonary cells in respective aging groups localizing not only over euchromatin and nucleoli in the nuclei but also over many cell organelles such as endoplasmic reticulum, ribosomes, and mitochondria as well as cytoplasmic matrices from perinatal stage at embryonic day 16, 18, to postnatal day 1, 3, 7, 14, to adult and senescent stages at postnatal month 1, 2, 6, 12 and 22 (Sun 1995, Sun et al. 1997b, Nagata 2002). The localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices but a few over the mitochondrial membranes and cristae when observed by high power magnification. However, quantitative analyses on the number of mitochondria, the number of labeled mitochondria and the labeling index were not performed because enough number of EM RAG was not obtained.

synthesis. The intramitochondrial DNA synthesis was observed in all cell types, the type I epithelial cells, the type II epithelial cells (Figs. 13F,G), the interstitial cells (Fig. 13F) and the endothelial cells. Because enough numbers of electron photographs (more than 5) were not obtained from all the cell types in respective aging groups, only some cell types and some aging groups when enough numbers of electron photographs were available were used for quantitative analysis. The numbers of mitochondria per cell profile area, the numbers of labeled mitochondria per cell and the labeling indices of the type I epithelial cells in only a few aging groups were observed and counted. The labeling indices in respective aging stages were calculated from the number of labeled mitochondria and the number of total mitochondria per cellular profile area which were calculated, respectively. These results demonstrated that the labeling indices in these cell types decreased from prenatal stages at embryo day 16 to day 18 (20-25%), and further decreased to postnatal days up to senescent

We studied the lungs of aging mice among the respiratory organs after administration of 3H-uridine at various ages from prenatal embryonic day 16 to postnatal senescent month 22

When the lung tissues of mice were labeled with 3H-uridine, RNA synthesis was observed in all cells of the lungs at various ages by RAG (Sun 1995, Sun et al. 1997b, Nagata 2002). Observing the light microscopic radioautograms labeled with 3H-uridine, the silver grains were found over both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo day 16, 18, to postnatal day 1, 3, 7, 14, but also at the adult and senescent stages from postnatal month 1 to 2, 6, 12 and 22. The number of silver grains, by electron micrography, changed with aging. The grain counts in type I epithelial cells increased from the 1st day after birth and reached a peak at 1 week and decreased gradually to month 22, while the counts in type II epithelial cells (Fig. 13H), interstitial cells and endothelial cells increased from embryo day 16 and reached peaks at 1 week after birth, then

By electron microscopic radioautography, silver grains were observed in most pulmonary cells in respective aging groups localizing not only over euchromatin and nucleoli in the nuclei but also over many cell organelles such as endoplasmic reticulum, ribosomes, and mitochondria as well as cytoplasmic matrices from perinatal stage at embryonic day 16, 18, to postnatal day 1, 3, 7, 14, to adult and senescent stages at postnatal month 1, 2, 6, 12 and 22 (Sun 1995, Sun et al. 1997b, Nagata 2002). The localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices but a few over the mitochondrial membranes and cristae when observed by high power magnification. However, quantitative analyses on the number of mitochondria, the number of labeled mitochondria and the labeling index were not performed because enough number of EM

stages due to aging (Fig. 15).

decreased to senescence.

RAG was not obtained.

**2.2 The RNA synthesis in the respiratory system** 

**2.2.1 RNA synthesis of mouse pulmonary cells** 

**2.2.2 The RNA synthesis of mitochondria in pulmonary cells** 

as observed by LM and EM RAG (Fig. 13H).

Fig. 14. Histogram showing aging changes of average labeling indices in respective cell types of the trachea of aging mice labeled with 3H-thymidine. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 148, 2002, Urban & Fischer, Jena, Germany Fig. 14A. Epithelial cells.

Fig. 14B. Chondrocytes in the cartilage.

Fig. 14C. Other cells.

Macromolecular Synthesis in the Digestive and Respiratory Systems 345

then decreased gradually to month 22, while the counts in type II epithelial cells, interstitial cells and endothelial cells reached the highest levels on fetal day 16, declined progressively with aging from fetal day 18 to postnatal day 3, then increased again from postnatal to day 7, reached peaks at 1 week (day 7) after birth, then decreased to senescence. However, grain counting on cell organelles by electron microscopy was not performed because enough

The incorporation of 35SO4 in the trachea of aging mice was studied among the respiratory organs (Nagata 2000d). As the results, silver grains indicating the incorporations of radiosulfate were found over the cartilage matrices and the cartilage capsules in the hyaline cartilages of the tracheae of fetal (Fig. 13C) and postnatal newborn mice (Fig. 13D). The grain density as analyzed by grain densitometry was the maximum at the fetal day 19 (1200 grains per unit area). The grain density then decreased from the fetal day 19 to the postnatal day 1, 3, 7 (600/area), 14 (200/area) and reached 0 on day 30, and no silver grain was found in the animals aged from 1 to 12 months. The silver grains in the perinatal animals aged at postnatal day 1 and 3, disappearred from the internal layer to the external layer of the cartilage and from the interterritorial matrix to the territorial matrix and the cartilage capsule. In the juvenile animals aged at postnatal day 9 and 14, intense incorporations were observed disseminatedly over several groups of cartilage capsules in the external layer. The results indicated that the glycoproteins constituting the cartilage matrix were synthesized from prenatal to postnatal day 30. To the contrary, no incorporation of silver grains was observed in the aging animals from postnatal 1 to 12 months by both LM and EM RAG. These results demonstrated the aging changes of glycoprotein synthesis in the cartilage

Chen, S., Gao, F., Kotani, A., Nagata, T.: Age-related changes of male mouse submandibular

Clermont Y.: The contractime elements in the limiting membrane of the seminiferous

Cui, H.: Light microscopic radioautographic study on DNA synthesis of nerve cells in the

Cui, H., Gao, F., Nagata, T.: Light microscopic radioautographic study on protein synthesis in perinatal mice corneas. Acta Histochem. Cytochem. 33, 31-37, 2000. Duan, H., Gao, F., Li, S., Hayashi, K., Nagata, T.: Aging changes and fine structure and DNA

Duan, H., Gao, F., Li, S., Nagata, T.: Postnatal development of esophageal epithelium in

Duan, H., Gao, F., Oguchi, K., Nagata, T.: Light and electron microscopic radioautographic

Clermont, Y.: Renewal of spermatogonia in man. Amer. J. Anat. 112, 35-51, 1963.

cerebella of aging mice. Cell. Mol. Biol. 41, 1139-1154, 1995.

gland: A morphometric and radioautographic study. Cell. Mol. Biol. 41, 117-124,

synthesis of esophageal epithelium in neonatal, adult and old mice. J. Clin. Electron

mouse: a light and electron microscopic radioautographic study. Cell. Mol. Biol. 39,

study on the incorporation of 3H-thymidine into the lung by means of a new

numbers of EM RAG were not obtained at that time.

**2.4 The glucide synthesis in the respiratory system** 

matrix of mice at various ages during development and aging.

tubules of rats. Exp. Cell Res. 15, 438-342, 1958.

Microsc. 25, 452-453, 1992.

nebulizer. Drug Res. 44, 880-883, 1994.

309-316, 1993.

**3. References** 

1995.

Fig. 15. Histogram showing aging changes of average labeling indices in respective cell types of the tracheal epithelial cells of aging mice labeled with 3H-thymidine. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 149, 2002, Urban & Fischer, Jena, Germany

#### **2.3 The protein synthesis in the respiratory system**

We studied the protein synthesis only in the lungs of aging mice at various ages among the respiratory organs including the respiratory tract.

#### **2.3.1 Protein synthesis of aging mouse pulmonary cells**

When the lung tissues of mice at various ages were labeled with 3H-leucine, protein synthesis was observed in all types of cells, type I and type II epithelial cells, interstitial cells and endothelial cells, of the lungs at various ages from embryo to senescence (Sun et al. 1997b). Observing the light microscopic radioautograms, the silver grains were detected over both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo day 16, 18, to postnatal day 1 to 14 (Fig. 13I), but also at the adult and senescent stages from postnatal month 1 to 2, 6, 12 and 22. The number of silver grains, by light microscopic radioautography, changed with aging. The grain counts in type I epithelial cells were the highest on postnatal day 1, decreased on day 3 and increased again at 1 week, then decreased gradually to month 22, while the counts in type II epithelial cells, interstitial cells and endothelial cells reached the highest levels on fetal day 16, declined progressively with aging from fetal day 18 to postnatal day 3, then increased again from postnatal to day 7, reached peaks at 1 week (day 7) after birth, then decreased to senescence. However, grain counting on cell organelles by electron microscopy was not performed because enough numbers of EM RAG were not obtained at that time.

#### **2.4 The glucide synthesis in the respiratory system**

The incorporation of 35SO4 in the trachea of aging mice was studied among the respiratory organs (Nagata 2000d). As the results, silver grains indicating the incorporations of radiosulfate were found over the cartilage matrices and the cartilage capsules in the hyaline cartilages of the tracheae of fetal (Fig. 13C) and postnatal newborn mice (Fig. 13D). The grain density as analyzed by grain densitometry was the maximum at the fetal day 19 (1200 grains per unit area). The grain density then decreased from the fetal day 19 to the postnatal day 1, 3, 7 (600/area), 14 (200/area) and reached 0 on day 30, and no silver grain was found in the animals aged from 1 to 12 months. The silver grains in the perinatal animals aged at postnatal day 1 and 3, disappearred from the internal layer to the external layer of the cartilage and from the interterritorial matrix to the territorial matrix and the cartilage capsule. In the juvenile animals aged at postnatal day 9 and 14, intense incorporations were observed disseminatedly over several groups of cartilage capsules in the external layer. The results indicated that the glycoproteins constituting the cartilage matrix were synthesized from prenatal to postnatal day 30. To the contrary, no incorporation of silver grains was observed in the aging animals from postnatal 1 to 12 months by both LM and EM RAG. These results demonstrated the aging changes of glycoprotein synthesis in the cartilage matrix of mice at various ages during development and aging.

#### **3. References**

344 Senescence

Fig. 15. Histogram showing aging changes of average labeling indices in respective cell types of the tracheal epithelial cells of aging mice labeled with 3H-thymidine. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog.

We studied the protein synthesis only in the lungs of aging mice at various ages among the

When the lung tissues of mice at various ages were labeled with 3H-leucine, protein synthesis was observed in all types of cells, type I and type II epithelial cells, interstitial cells and endothelial cells, of the lungs at various ages from embryo to senescence (Sun et al. 1997b). Observing the light microscopic radioautograms, the silver grains were detected over both the karyoplasm and cytoplasm of almost all the cells not only at the perinatal stages from embryo day 16, 18, to postnatal day 1 to 14 (Fig. 13I), but also at the adult and senescent stages from postnatal month 1 to 2, 6, 12 and 22. The number of silver grains, by light microscopic radioautography, changed with aging. The grain counts in type I epithelial cells were the highest on postnatal day 1, decreased on day 3 and increased again at 1 week,

Histochem. Cytochem. Vol. 37, No. 2, p. 149, 2002, Urban & Fischer, Jena, Germany

**2.3 The protein synthesis in the respiratory system** 

**2.3.1 Protein synthesis of aging mouse pulmonary cells** 

respiratory organs including the respiratory tract.


Macromolecular Synthesis in the Digestive and Respiratory Systems 347

Hayashi, K., Gao, F., Nagata, T.: Radioautographic study on 3H-thymidine incorporation at

Ito, M.: The radioautographic studies on aging change of DNA synthesis and the

Ito, M., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis and

Izumiyama, K., Kogure, K., Kataoka, S., Nagata, T.: Quantitative analysis of glucose after

Jamieson, J. D., Palade, G. E.: Intracellular transport of secretory proteins in the pancreatic

Jin, C.: Study on DNA synthesis of aging mouse colon by light and electron microscopic

Jin, C., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. J. Histochem. Cytochem. 43, 1223-1228, 1995a. Jin, C., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. Med. Electron Microsc. 28, 71-75, 1995b. Joukura, K.: The aging changes of glycoconjugate synthesis in mouse kidney studied by 3Hglucosamine radioautography. Acta Histochem. Cytochem. 29, 57-63, 1996. Joukura, K., Nagata, T.: Aging changes of 3H-glucosamine incorporation into mouse kidney observed by radioautography. Acta Histochem. Cytochem. 28, 494-494, 1995. Joukura, K., Usuda, N., Nagata, T.: Quantitative study on the aging change of

radioautography. Brain Res. 416, 175-179, 1987.

radioautography. Cell. Mol. Biol. 42, 255-268, 1996.

exocrine cells. J. Cell Biol. 34, 577-615, 1967.

Histochem. Cytochem. 42, 982-982, 1994.

mouse retina. Cell. Mol. Biol. 39, 55-64, 1993.

by radioiodine. J. Anat. 77, 149-152, 1943.

Electron Microsc. 11, 428-429, 1978.

263-272, 1992a.

Mol. Biol. 38, 669-678, 1992b.

1993.

1996.

1996.

different stages of muscle development in aging mice. Cell. Mol. Biol. 39, 553-560,

ultrastructural development of mouse adrenal gland. Cell. Mol. Biol. 42, 279-292,

ultrastructure of aging mouse adrenal gland. Med. Electron Microsc. 29, 145-152,

transient ischemia in the gerbil hippocampus by light and electron microscope

glycoconjugates synthesis in aging mouse kidney. Proc. Xth Internat. Cong. Histochem. Cytochem., Acta Histochem. Cytochem. 29, Suppl. 507-508, 1996. Kobayashi, K., Nagata, T.: Light microscopic radioautographic studies on DNA, RNA and

protein syntheses in human synovial membranes of rheumatoid arthritis patients. J.

radioautographic study on 125I-albumin in rat gastric mucosal epithelia. J. Clin.

and retinal pigment epithelium of developing mouse embryo. Cell. Mol. Biol. 38,

and retinal pigment epithelium of mice by light microscopic radioautography. Cell.

Komiyama, K., Iida, F., Furihara, R., Murata, F., Nagata, T.: Electron microscopic

Kong, Y.: Electron microscopic radioautographic study on DNA synthesis in perinatal

Kong, Y., Nagata, T.: Electron microscopic radioautographic study on nucleic acid synthesis

Kong, Y., Usuda, N., Nagata, T.: Radioautographic study on DNA synthesis of the retina

Kong, Y., Usuda, N., Morita, T., Hanai, T., Nagata, T.: Study on RNA synthesis in the retina

Leblond, C. P.: Localization of newly administered iodine in the thyroid gland as indicated

Leblond, C. P.: The life history of cells in renewing systems. Am. J. Anat. 160, 113-158, 1981.

of perinatal mouse retina. Med. Electron Microsc. 27, 366-368, 1994.


Feulgen, R., Rossenbeck, H.: Mikroskopische-chemischer Nachweis einer Nukeinsaeure von Thymus der Thymonukeinsaeure Z. Physik. Chem. 135, 203-248, 1924. Fujii, Y., Ohno, S., Yamabayashi, S., Usuda, N., Saito, H., Furuta, S., Nagata, T.: Electron

Gao, F.: Study on the macromolecular synthesis in aging mouse seminiferous tubules by light and electron microscopic radioautography. Cell. Mol. Biol. 39, 659-672, 1993. Gao, F., Toriyama, K., Nagata, T.: Light microscopic radioautographic study on the DNA

Gao, F., Li, S., Duan, H., Ma, H., Nagata, T.: Electron microscopic radioautography on the

Gao, F., Toriyama, K., Ma, H., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in aging mice corneas. Cell. Mol. Biol. 39, 435-441, 1993. Gao, F., Ma, H., Sun, L., Jin, C., Nagata, T.: Electron microscopic radioautographic study on

Gao, F., Chen, S., Sun, L., Kang, W., Wang, Z., Nagata, T.: Radioautographic study of the

Gao, F., Jin, C., Ma, H., Sun, L., Nagata, T.: Ultrastructural and radioautographic studies on

Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick embryo. I. Light microscopic radioautography. Shinshu Med. J. 32, 231-240, 1984a. Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick

Gunarso, W., Gao, F., Cui, H., Ma, H., Nagata, T.: A light and electron microscopic

Gunarso, W., Gao, F., Nagata, T.: Development and DNA synthesis in the retina of chick

Hanai, T.: Light microscopic radioautographic study of DNA synthesis in the kidneys of

Hanai, T., Nagata, T.: Electron microscopic radioautographic study on DNA and RNA

Hanai, T., Nagata, T.: Study on the nucleic acid synthesis in the aging mouse kidney by light

Nagata, T., Ed., pp. 209-214, Shinshu University Press, Matsumoto, 1994b. Hanai, T., Nagata, T.: Electron microscopic study on nucleic acid synthesis in perinatal

Hanai, T., Usuda, N., Morita, T., Shimizu, T., Nagata, T.: Proliferative activity in the kidneys

Ed., pp. 127-131, Shinshu University Press, Matsumoto, 1994a.

mouse kidney tissue. Med. Electron Microsc. 27, 355-357, 1994c.

IgG myeloma patient. J. Clin. Electr. Microsc. 13, 582-583, 1980.

injection. Cell. Mol. Biol. 38, 661-668, 1992a.

Microsc. 27, 360-362, 1994.

Histochem. 98, 309-32, 1996.

aging mice. Cell. Mol. Biol. 39, 81-91, 1993.

Biol. 43, 189-201, 1997.

39, 181-191, 1993.

145-150, 1995a.

1995b.

1984b.

injection. J. Clin. Electron Microsc. 25, 721-722, 1992b.

microscopic radioautography of DNA synthesis in primary cultured cells from an

synthesis of prenatal and postnatal aging mouse retina after labeled thymidine

DNA synthesis of prenatal and postnatal mice retina after labeled thymidine

the nucleic acids and protein synthesis in the aging mouse testis. Med. Electron

macromolecular synthesis of Leydig cells in aging mice testis. Cell. Mol. Biol. 41,

DNA synthesis in Leydig cells of aging mouse testis. Cell. Mol. Biol. 41, 151-160,

embryo. II. Electron microscopic radioautography. Shinshu Med. J. 32, 241-248,

radioautographic study on RNA synthesis in the retina of chick embryo. Acta

embryo observed by light and electron microscopic radioautography. Cell. Mol.

synthesis in perinatal mouse kidney. In, Radioautography in Medicine, Nagata, T.,

and electron microscopic radioautography. In, Radioautography in Medicine,

of aging mice evaluated by PCNA/cyclin immunohistochemistry. Cell. Mol. Biol.


Macromolecular Synthesis in the Digestive and Respiratory Systems 349

Morita, T., Usuda, N. Hanai, T., Nagata, T.: Changes of colon epithelium proliferation due to

Murata, F., Momose,Y. , Yoshida, K., Nagata, T.: Incorporation of 3H-thymidine into the nucleus of mast cells in adult rat peritoneum. Shinshu Med. J. 25, 72-77, 1977a. Murata, F., Momose, Y., Yoshida, K., Ohno, S., Nagata, T.: Nucleic acid and mucosubstance

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Ultrastructural and electron microscopic

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Mucosubstances of rabbit granulocytes studied

Nagata, T.: On the relationship between cell division and cytochrome oxidase in the Yoshida

Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. I. Observation on the smear preparation under normal conditions. Med. J. Shinshu Univ. 2: 187-198, 1957a. Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. II. Phase-contrast

Nagata, T.: Cell divisions in the liver of the fetal and newborn dogs. Med. J. Shinshu Univ. 4:

Nagata, T.: A radioautographic study of the DNA synthesis in rat liver, with special

Nagata, T.: A quantitative study on the ganglion cells in the small intestine of the dog. Med.

Nagata, T.: A radioautographic study on the RNA synthesis in the hepatic and the intestinal

Nagata, T.: On the increase of binucleate cells in the ganglion cells of dog small intestine due

Nagata, T.: A radioautographic study on the protein synthesis in the hepatic and the

Nagata, T.: Chapter 3. Application of microspectrophotometry to various substances. In ,

Nagata, T.: Electron microscopic dry-mounting autoradiography. Proc. 4th Internat. Cong.

Nagata, T.: Electron microscopic radioautography of intramitochondrial RNA synthesis of

Nagata, T.: Quantitative electron microscope radioautography of intramitochondrial nucleic

Nagata, T.: Electron microscopic observation of target cells previously observed by phase-

irradiated cultured cells. J. Clin. Electron Microsc. 17, 589-590, 1984.

epithelial cells of mice after feeding with special reference to binuclearity. Med. J.

intestinal epithelial cells of mice, with special reference to binucleate cells. Med. J.

Introduction to Microspectrophotometry. Isaka, S., Nagata, T., Inui, N., Eds.,

contrast microscopy: Electron microscopic radioautography of laser beam

reference to binucleate cells. Med. J. Shinshu Univ. 7, 17-25, 1962.

to experimental ischemia. Med. J. Shinshu Univ. 12, 93-113, 1967a.

thymidine radioautography. Histochemistry 101, 13-20, 1994.

radioautography. Acta Pharmacol. Toxicol. 41, 58-59, 1977b.

Microsc. 11, 561-562, 1978.

207, 1957b.

65-73, 1959.

Histochemistry 61, 139-150, 1979.

J. Shinshu Univ. 10, 1-11, 1965.

Shinshu Univ. 11, 49-61, 1966.

Shinshu Univ. 12, 247-257, 1967b.

Olympus Co., Tokyo, pp. 49-155, 1972a.

Histochem. Cytochem. Kyoto, pp. 43-44, 1972b.

HeLa cells in culture. Histochemie 32, 163-170, 1972c.

acid synthesis. Acta Histochem. Cytochem. 5, 201-203, 1972d.

sarcoma cells. Shinshu Med. J. 5: 383-386, 1956.

individual aging with PCNA/cyclin immunostaining comparing with 3H-

metabolism of mastocytoma cells by means of electron microscopic

radioautographic studies on the mastocytoma cells and mast cells. J. Clin. Electron

by means of electron microscopic radioautography and X-ray microanalysis.

microscopic observations under normal conditions. Med. J. Shinshu Univ. 2: 199-


Leblond, C. P., Messier, B.: Renewal of chief cells and goblet cells in the small intestine as

Li, S.: Relationship between cellular DNA synthesis, PCNA expression and sex steroid

Li, S., Nagata, T.: Nucleic acid synthesis in the developing mouse ovary, uterus and oviduct

Li, S., Gao, F., Duan, H., Nagata, T.: Radioautographic study on the uptake of 35SO4 in mouse ovary during the estrus cycle. J. Clin. Electron Microsc. 25, 709-710, 1992. Liang, Y.: Light microscopic radioautographic study on RNA synthesis in the adrenal glands

Liang, Y., Ito, M., Nagata, T.: Light and electron microscopic radioautographic studies on

Ma, H.: Light microscopic radioautographic study on DNA synthesis of the livers in aging

Ma, H., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of the

Ma, H., Nagata, T.: Studies on DNA synthesis of aging mice by means of electron microscopic radioautography. J. Clin. Electron Microsc. 21, 715-716, 1988b. Ma, H., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis in the

Ma, H., Nagata, T.: Study on RNA synthesis in the livers of aging mice by means of electron

Ma, H., Nagata, T.: Collagen and protein synthesis in the livers of aging mice as studied by

Ma, H., Gao, F., Olea, M. T., Nagata, T.: Protein synthesis in the livers of aging mice studied by electron microscopic radioautography. Cell. Mol. Biol. 37, 607-615, 1991. Matsumura, H., Kobayashi, Y., Kobayashi, K., Nagata, T.: Light microscopic

Momose, Y., Nagata, T.: Radioautographic study on the intracellular localization of a

Momose, Y., Naito, J., Nagata, T.: Radioautographic study on the localization of an antiallergic agent, tranilast, in the rat liver. Cell. Mol. Biol. 35, 347-355, 1989. Momose, Y., Shibata, N., Kiyosawa, I., Naito, J., Watanabe, T., Horie, S., Yamada, J., Suga, T.,

Momose, Y., Naito, J., Suzawa, H., Kanzawa, M., Nagata, T.: Radioautographic study on the

Morita, T.: Radioautographic study on the aging change of 3H-glucosamine uptake in mouse

of aging mice. Acta Histochem. Cytochem. 31, 203-210, 1998.

livers in aging mice. J. Clin. Electron Microsc. 21, 335-343, 1988a.

microscopic radioautography. Cell. Mol. Biol. 36, 589-600, 1990b.

electron microsopic radioautography. Ann. Microsc. 1, 13-22, 2000.

Hynobius nebulosus. J. Histochem. Cytochem. 42, 1004-1004, 1994.

hepatocytes. Cell. Mol. Biol. 39, 773-781, 1993a.

Toxicol. Pathol. 6, 33-45, 1993b.

ileum. Cell. Mol. Biol. 39, 875-884, 1993.

Cytochem. 28, 61-66, 1995.

132: 247-259. 1958.

195, 1995.

1999.

1990a.

Histochemistry 102, 405-413, 1994.

mice. Acta Anat. Nippon. 63, 137-147, 1988.

shown by radioautography after injection of thymidine-3H into mice. Anat. Rec.

hormone receptor status in the developing mouse ovary, uterus and oviduct.

studied by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 185-

RNA synthesis in aging mouse adrenal gland. Acta Anat. Nippon. 74, 291-300,

hepatocytes of aging mice as observed by image analysis. Cell. Mol. Biol. 36, 73-84,

radioautographic study of DNA synthesis in the lung of aging salamander,

hypolipidemic agent, bezafibrate, a peroxisome proliferator, in cultured rat

Nagata, T.: Morphometric evaluation of species differences in the effects of bezafibrate, a hypolipidemic agent, on hepatic peroxisomes and mitochondria. J.

intracellular localization of bezafibrate in cultured rat hepatoctyes. Acta Histochem.


Macromolecular Synthesis in the Digestive and Respiratory Systems 351

Nagata, T.: Radioautographic study on collagen synthesis in the ocular tissues. J. Kaken Eye

Nagata, T.: Techniques of radioautography for medical and biological research. Braz. J. Biol.

Nagata, T.: Radioautographology, the advocacy of a new concept. Braz. J. Biol. Med. Res. 31,

Nagata, T.: Radioautographic studies on DNA synthesis of the bone and skin of aging

Nagata, T.: 3D observation of cell organelles by high voltage electron microscopy.

Nagata, T.: Application of histochemistry to anatomy: Histochemistry of the organs, a novel

Nagata, T.: Aging changes of macromolecular synthesis in various organ systems as

Nagata, T.: Radioautographology, general and special: a novel concept. Ital. J. Anat.

Nagata, T.: Three-dimensional observations on thick biological specimens by high voltage

Nagata, T.: Biological microanalysis of radiolabeled and unlabeled compounds by

Nagata, T.: Electron microscopic radioautographic study on protein synthesis in pancreatic cells of perinatal and aging mice. Bull. Nagano Women's Jr. College 8, 1-22, 2000c. Nagata, T.: Light microscopic radioautographic study on radiosulfate incorporation into the tracheal cartilage in aging mice. Acta Histochem. Cytochem. 32, 377-383, 2000d. Nagata, T.: Introductory remarks: Special radioautographology. Cell. Mol. Biol. 46 (Congress

Nagata, T.: Three-dimensional high voltage electron microscopy of thick biological

Nagata, T.: Three-dimensional and four-dimensional observation of histochemical and

Nagata, T. : Special cytochemistry in cell biology. In, Internat. Rev. Cytol. Jeon, K.W., ed.,

Nagata, T. : Radioautographology General and Special, In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 37 No. 2, Urban & Fischer, Jena, pp. 57-226, 2002. Nagata T.: Light and electron microscopic study on macromolecular synthesis in amitotic hepatocyte mitochondria of aging mice. Cell. Mol. Biol. 49, 591-611, 2003. Nagata, T.: X-ray microanalysis of biological specimens by high voltage electron

cytochemical specimens by high voltage electron microscopy. Acta Histochem.

microscopy. In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 39, No. 4,

concept. Proc. XV Congress of the International Federation of Associations of

observed by microscopic radioautography after incorporation of radiolabeled

radioautography and X-ray microanalysis. Scanning Microscopy International, 14,

salamander. Bull. Nagano Women's Jr. College 6, 1-14, 1998c.

Microscopy and Analysis, Asia Pacific Edition, 9, 29-32, 1999a.

Anatomists, Ital. J. Anat. Embryol. 104 (Suppl. 1), 486-486, 1999b.

precursors. Methods Find. Exp. Clin. Pharmacol. 21, 683-706, 1999c. Nagata, T.: Radioautographic study on protein synthesis in mouse cornea. J. Kaken Eye Res.

electron microscopy. Image Analysis Stereolog. 19, 51-56, 2000a.

Nagata, T.: Special radioautographology: the eye. J. Kaken Eye Res. 18, 1-13, 2000f.

Vol. 211, Chapter 2, Academic Press, New York, pp. 33-154, 2001c.

Antonio Delfino Editore, Roma, pp. 37-44, 1997b.

Res. 15, 1-9, 1997c.

201-241, 1998b.

8, 8-14, 1999d.

on line, 2000b.

Suppl.), 161-161, 2000e.

Embryol. 104 (Suppl. 1), 487-487, 1999e.

specimens. Micron 32, 387-404, 2001a.

Urban & Fischer Verlag, Jena, pp. 185-320, 2004.

Cytochem. 34, 153-169, 2001b.

Med. Res. 31, 185-195, 1998a.

Recent Advances in Microscopy of Cells, Tissues and Organs, Motta, P., Ed.,


Nagata, T.: Principles and techniques of radioautography. In, Histo- and Cyto-chemistry

Nagata, T.:. Electron microscopic radioautography and analytical electron microscopy. J.

Nagata, T.: Radiolabeling of soluble and insoluble compounds as demonstrated by light and

Nagata, T. Quantitative light and electron microscopic radioautographic studies on

Nagata, T.: Electron microscopic radioautography with cryo-fixation and dry-mounting

Nagata, T.: Application of electron microscopic radioautography to clinical electron

Nagata, T.: Radioautography in Medicine. Shinshu University Press, 268pp, Matsumoto,

Nagata, T.: Radioautography, general and special. In, Histo- and Cyto-chemistry 1994, Japan

Nagata, T., Application of electron microscopic radioautography to clinical electron

Nagata, T.: Light and electron microscopic radioautographic study on macromolecular synthesis in digestive organs of aging mice. Cell. Mol. Biol. 41, 21-38, 1995a. Nagata, T.: Histochemistry of the organs: Application of histochemistry to anatomy. Acta

Nagata, T.: Three-dimensional observation of whole mount cultured cells stained with

Nagata, T.: Morphometry in anatomy: image analysis on fine structure and histochemical

Nagata, T.: Technique and application of electron microscopic radioautography. J. Electron

Nagata, T.: Techniques of light and electron microscopic radioautography. In,

Nagata, T.: Remarks: Radioautographology, general and special. Cell. Mol. Biol. 42 (Suppl.),

Nagata, T.: On the terminology of radioautography vs. autoradiography. J. Histochem.

Nagata, T.: Techniques and applications of microscopic radioautography. Histol.

Nagata T.: Three-dimensional observation on whole mount cultured cells and thick sections

Cytochem. Acta Histochem. Cytochem. 29 (Suppl.), 343-344, 1996b.

Tokyo, pp. 207-226, 1985.

1993a.

1994c.

Tokyo, 1994d.

41, 783-792, 1995c.

591-605, 1995d.

11-12, 1996c.

Microsc. 45, 258-274, 1996a.

Cytochem. 44, 1209-1209, 1996d.

Histopathol. 12, 1091-1124, 1997a.

Clin. Electron Microsc. 24, 441-442, 1991.

Chinese J. Histochem. Cytochem. 2: 106-108, 1993b.

procedure. Acta Histochem. Cytochem. 27: 471-489, 1994a.

microscopy. Med. Electron Microsc. 27; 191-212, 1994b.

microscopy. Med. Electron Microsc. 27, 191-212, 1994e.

Anat. Nippon. 70, 448-471, 1995b.

1985, Japan Society of Histochemistry and Cytochemistry, Ed., Gakusai Kikaku Co.,

electron microscopy. Recent Advances in Cellular and Molecular Biology, Wegmann, R. J., Wegmann, M. A., Eds. Peters Press, Leuven, Vol. 6, pp. 9-21, 1992. Nagata, T.: Quantitative analysis of histochemical reactions: Image analysis of light and

electron microscopic radioautograms. Acta Histochem. Cytochem. 26, 281-291,

macromolecular synthesis in several organs of prenatal and postnatal aging mice.

Society of Histochemistry and Cytochemistry, ed, pp. 219-231, Gakusai Kikaku Co.,

histochemical reactions by ultrahigh voltage electron microscopy. Cell. Mol. Biol.

reactions with special reference to radioautography. Ital. J. Anat. 100 (Suppl. 1),

Histochemistry and Cytochemistry 1996. Proc. Xth Internat. Congr. Histochem.

stained with histochemical reactions by high voltage electron microscopy. In,

Recent Advances in Microscopy of Cells, Tissues and Organs, Motta, P., Ed., Antonio Delfino Editore, Roma, pp. 37-44, 1997b.


Macromolecular Synthesis in the Digestive and Respiratory Systems 353

Nagata, T.: Applications of high voltage electron microscopy to thick biological specimens.

Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of mitochondria in adrenal medullary cells of aging mice. Open Anat. J. 1, 14-24, 2009g. Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 11, 1-17, 2009h. Nagata, T.: Electron microscopic radioautographic studies on macromoleclular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata T. Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Macromolecular synthesis in the livers of aging mice as revealed by electron

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata, T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA, RNA and

Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 12, 1-29, 2010h. Nagata, T., Cui, H., Gao, F.: Radioautographic study on glycoprotein synthesis in the ocular

Nagata, T., Cui, H., Kong, Y.: The localization of TGF-b1 and its mRNA in the spinal cords

Nagata, T., Cui, H., Liang, Y.: Light microscopic radioautographic study on the protein

Nagata, T., Fujii, Y., Usuda, N.: Demonstration of extranuclear nucleic acid synthesis in

adrenocortical cells of aging mice. Open Anat J. 2, 91-97, 2010a.

Diego, St. Louis, Vol. 45, No. 1, pp. 1-80, 2010c.

Vol. 3, Formatex, Badajoz, Spain, in press, 2010g.

tissues. J. Kaken Eye Res. 13, 11-18, 1995.

Alternat Med Welfare 5, 25-37, 2010d.

Med. Welfare 5, 38-52, 2010f.

mitochondria of some organs in aging animals. Bull. Shinshu Inst. Alternat. Med.

in adreno-cortical cells of aging ddY mice. Bull. Shinshu Inst. Alternat. Med.

adrenal medullary cells of aging and senescent mice. J Cell Tissue Res. 10, 2213-

microscopic radioautography. In, Prog. Histochem. Cytochem., Sasse, D., Ed., Elsevier, Amsterdam, Boston, London, New York, Oxford, Paris, Philadelphia, San

mitochondria in adrenal medullary cells of aging mice. Bulletin Shinshu Inst

adrenal cortical and medullary cells of aging mice. J. Biomed. Sci. Enginer. 4, 219-

mitochondria in adrenal cortical cells of aging mice. Bulletin Shinshu Inst. Alternat.

protein synthesis in adrenal cells of aging mice. Formatex Microscopy Series No. 3,

of prenatal and postnatal aging mice demonstrated with immunohistochemical and in situ hybridization techniques. Bull. Nagano Women's Jr. College, 7, 75-88, 1999a.

synthesis in the cerebellum of aging mouse. Bull. Nagano Women's Jr. College, 9,

mammalian cells under experimental conditions by electron microscopic radioautography. Proc. 10th Internat. Congr. Electr. Microsc. 2, 305-306, 1982b. Nagata, T., Hirano, I., Shibata, O., Nagata, T.: A radioautographic study on the DNA

synthesis in the hepatic and the pancreatic acinar cells of mice during the postnatal

Ann. Microsc. 9, 4-40, 2009f.

Welfare 4, 15-38, 2009i.

Welfare 4, 51-66, 2009j.

2222, 2010b.

232, 2010e.

41-60 (2001).


Nagata T.: Aging changes of macromolecular synthesis in the uro-genital organs as revealed by electron microscopic radioautography. Ann. Rev. Biomed. Sci. 6, 13-78, 2005. Nagata T.: Electron microscopic radioautographic study on protein synthesis in hepatocyte

Nagata T.: Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata, T.: Electron microscopic radioautographic study on macromolecular synthesis in hepatocyte mitochondria of aging mouse. J. Cell Tissue Res. 7, 1019-1029, 2007c. Nagata, T.; Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata, T.; Aging changes of macromolecular synthesis in the mitochondria of mouse

Nagata, T.: Sexual difference between the macromolecular synthesis of hepatocyte

Nagata, T.: Protein synthesis in hepatocytes of mice as revealed by electron microscopic

Nagata, T.: Electron microscopic radioatuographic studies on macromolecular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Eds., Nova Biomed. Books, New York, pp. 133-161, 2009b.

Radiopharmaceutics 2, 118-128, 2009d.

Nagata, T.: Radioautographology, Bull. Shinshu Institute Alternat. Med. 2, 3-32, 2007f. Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in adrenal cortical cells of developing mice. J. Cell. Tis. Res. 8, 1303-1312, 2008a. Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in

hepatocyte mitochondria of developing mice. The Sci. World J. 6: 1583-1598, 2006b.

by electron microscopic radioautography. I: Nucleic acid synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. Eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 245-

by electron microscopic radioautography. II: Protein synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 259-271,

hepatocyte mitochondria of developing mice. Trends Cell Molec. Biol. 2, 19-33,

hepatocytes as revealed by microscopic radioautography. Ann. Rev. Biomed. Sci. 9,

adrenal cortical cells of developing and aging mice. The Sci. World J. 8, 683-697.

mitochondria in male and female mice in aging as revealed by electron microscopic radioautography. Chapter 22. In, Women and Aging: New Research, H. T. Bennninghouse, A. D. Rosset, Eds. Nova Biomed. Books, New York, pp. 461-487,

radioautography. In, Protein Biosynthesis. Esterhouse, T. E. and Petrinos, L. B.,

mitochondria of various cells. 18EMSM Conference Proc. 9th Asia-Pacific Microscopy Conference (APMC9), Kuala Lumpur, Malaysia, pp. 48-50, 2009c. Nagata, T.: Recent studies on macromolecular synthesis labeled with 3H-thymidine in

various organs as revealed by electron microscopic radioautography. Current

in adrenal medullary cells of developing and aging mice. J. Cell Tissue Res. 9, 1793-

mitochondria of developing mice. Ann. Microsc. 6, 43-54, 2006a.

258, 2007a.

2007b.

2007d.

2008b.

2009a

1802, 2009e.

30-36, 2007e.


Macromolecular Synthesis in the Digestive and Respiratory Systems 355

Nagata, T., Ohno, S., Yoshida, K., Murata, F.: Nucleic acid synthesis in proliferating

Nagata, T., Olea, M. T.: Electron microscopic radioautographic study on the protein synthesis in aging mouse spleen. Bull. Nagano Women's Jr. College 7, 1-9, 1999. Nagata, T., Shibata, O., Omochi, S.: A new method for radioautographic osbservation on

Nagata, T., Shibata, O., Nawa, T.: Simplified methods for mass production of

Nagata, T., Shibata, O., Nawa, T.: Incorporation of tritiated thymidine into mitochondrial

Nagata, T., Shimamura, K., Onozawa, M., Kondo, T., Ohkubo, K., Momoze, S.: Relationship

Nagata, T., Shimamura, K., Kondo, T., Onozawa, M., Momoze, S., Okubo, M.: Relationship

Nagata, T., Steggerda, F. R.: Histological study on the deganglionated small intestine of the

Nagata, T., Steggerda, F. R.: Observations on the increase of binucleate cells in the ganglion

Nagata, T., Toriyama, K., Kong, Y., Jin, C., Gao, F.: Radioautographic study on DNA synthesis in the ciliary bodies of aging mice. J. Kaken Eye Res.12, 1-11, 1994. Nagata, T., Usuda, N.: Image processing of electron microscopic radioautograms in clinical

Nagata, T., Usuda, N.: Studies on the nucleic acid synthesis in pancreatic acinar cells of

Nagata, T., Usuda, N.: Electron microscopic radioautography of protein synthesis in

Nagata, T., Usuda, N.: In situ hybridization by electron microscopy using radioactive

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of nucleic acid

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of lipid synthesis in pancreatic cells of aging mice. J. Clin. Electr. Microsc. 23, 841-842, 1990. Nagata, T., Usuda, N., Maruyama, M., Ma, H.: Electron microscopic radioautographic study

Nagata, T., Usuda, N., Suzawa, H., Kanzawa, M.: Incorporation of 3H-glucosamine into the

radioautography. J. Clin. Electron Microsc. 25, 646-647, 1992.

electron microscopy. J. Clin. Electron. Microsc. 18, 451-452, 1985.

probes. J. Histochem. Cytochem. 41, 1119-1119, 1993b.

Intern. Cong. Electr. Microsc. 3, 2281-2282, 1984.

Histochem. J. 14, 197-204, 1982a.

Histochemie 10, 305-308, 1967b.

dog. Physiologist 6, 242-242, 1963.

Microsc. 19, 486-487, 1986.

1960a.

1964.

1993a.

1988b.

153-158, 1960b.

isolated cells. Histochemie 2, 255-259, 1961

radioautograms. -Acta Anat. Nippon.42, 162-166, 1967a.

peroxisomes of rat liver as revealed by electron microscopical radioautography.

DNA of the liver and kidney cells of chickens and mice in tissue culture.

of binuclearity to cell function in some organs. I. Frequencies of binucleate cells in some organs of toads in summer and winter. Med. J. Shinshu Univ. 5, 147-152,

of binuclearity to cell function in some organs. II. Variation of frequencies of binucleate cells in some organs of dogs owing to aging. Med. J. Shinshu Univ. 5,

cells of the dog's intestine due to experimental ischemia. Anat. Rec. 148, 315-315,

aging mice by means of electron microscopic radioautography. J. Clin. Electron

pancreatic acinar cells of aging mice. Acta Histochem. Cytochem. 26, 481-481,

synthesis in pancreatic acinar cells of prenatal and postnatal aging mice. Proc. XIth

on lipid synthesis in perinatal mouse pancreas. J. Clin. Electr. Microsc. 21, 756-757,

pancreatic cells of aging mice as demonstrated by electron microscopic

growth, with special reference to binuclearity. Med. J. Shinshu Univ. 11, 35-42, 1966.


Nagata, T., Ito, M., Chen, S.: Aging changes of DNA synthesis in the submandibular glands

Nagata, T. Ito, M., Liang, Y.: Study of the effects of aging on macromolecular synthesis in

Nagata, T., Iwadare, I., Murata, F.: Electron microscopic radioautography of nucleic acid

Nagata, T., Kawahara, I.: Radioautographic study of the synthesis of sulfomucin in digestive

Nagata, T., Kawahara, I., Usuda, N., Maruyama, M., Ma, H.: Radioautographic studies on

Nagata, T., Kong, Y.: Distribution and localization of TGFb1 and bFGF, and their mRNAs in

Nagata, T., Ma, H., Electron microscopic radioautographic study on mitochondrial DNA synthesis in hepatocytes of aging mouse. Ann. Microsc. 5, 4-18, 2005a. Nagata, T., Ma, H., Electron microscopic radioautographic study on RNA synthesis in hepatocyte mitochondria of aging mouse. Microsc. Res. Tech. 67, 55-64, 2005b. Nagata, T., Momoze, S.: Aging changes of the amitotic and binucleate cells in dog livers.

Nagata, T., Morita, T., I. Kawahara, I.: Radioautographic studies on radiosulfate incorporation in the digestive organs of mice. Histol. Histopathol. 14, 1-8, 1999b. Nagata, T., Murata, F.: Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy. Histochemistry 54, 75-82, 1977. Nagata, T., Murata, F., Yoshida, K., Ohno, S., Iwadare, N.: Whole mount radioautography of

Nagata, T., Nawa, T.: A modification of dry-mounting technique for radioautography of

Nagata, T., Nawa, T.: A radioautographic study on the nucleic acids synthesis of binucleate

Nagata, T., Nawa, T., Yokota, S.: A new technique for electron microscopic dry-mounting radioautography of soluble compounds. Histochemie 18, 241-249, 1969. Nagata, T., Nishigaki, T., Momose, Y.: Localization of anti-allergic agent in rat mast cells

Nagata, T., Ohno, S., Kawahara, I., Yamabayashi, S., Fujii, Y., Murata, F.: Light and electron

Nagata, T., Ohno, S., Murata, F.: Electron microscopic dry-mounting radioautography for

soluble compounds. Acta Phamacol. Toxicol. 41, 62-63, 1977a.

cultured cells as observed by high voltage electron microscopy. Proc. Fifth Internat.

cells in cultivated fibroblasts of chick embryos. Med. J. Shinshu Univ. 11, 1-5, 1966b.

demonstrated by light and electron microscopic radioautography. Acta Histochem.

microscopic radioautography of nucleic acid synthesis in mitochondria and peroxisomes of rat hepatic cells during and after DEHP administration. Acta

organs of mice. J. Trace Microprobe Analysis 17, 339-355, 1999.

aging mice. Bull. Nagano Women's Jr. College 6, 87-105, 1998.

1966.

Microsc. 1, 4-12, 2000a.

Toxicol. 41, 64-65, 1977c.

Exp. Clin. Pharmacol. 22, 5-18, 2000b.

Professional Postgrad. Service, Tokyo, 1988a.

Acta Anat. Nipponica 34, 187-190, 1959.

Cytochem. 19, 669-683, 1986b.

Histochem. Cytochem. 16, 610-611, 1979.

Conf. High Voltage Electron Microsc. 347-350, 1977d.

water-soluble compounds. Histochemie 7, 370-371, 1966a.

growth, with special reference to binuclearity. Med. J. Shinshu Univ. 11, 35-42,

of mice as observed by light and electron microscopic radioautography. Ann.

mouse steroid secreting cells using microscopic radioautography. Methods Find.

synthesis in cultured cells treated with several carcinogens. Acta Pharmacol.

the glycoconjugate synthesis in the gastrointestinal mucosa of the mouse. In, Glycoconjugate in Medicine, Ohyama, M., Muramatsu, T., Eds, pp. 251-256,


Macromolecular Synthesis in the Digestive and Respiratory Systems 357

Oliveira, S. F., Abrahamsohn, P. A., Nagata, T., Zorn, T. M. T.: Incorporation of 3H-amino

Pearse, A. G. E.: Histochemistry, Theoretical and Applied. 4th Ed. Vol. 1. 439 pp., 1980, Vol.

Sakai, Y., Ikado, S., Nagata, T.: Electron microscopic radioautography of satellite cells in

Sato, A.: Quantitative electron microscopic studies on the kinetics of secretory granules in G-

Sato, A., Iida, F., Furihara, R., Nagata, T.: Electron microscopic raioautography of rat

Shimizu, T., Usuda, N., Yamanda, T., Sugenoya, A., Iida, F.: Proliferative activity of human

Sun, L.: Age related changes of RNA synthesis in the lungs of aging mice by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 1061-1072, 1995. Sun, L., Gao, F., Duan, H., Nagata, T.: Light microscopic radioautography of DNA synthesis

Sun, L., Gao, F., Nagata, T.: Study on the DNA synthesis of pulmonary cells in aging mice by light microscopic radioautography. Cell. Mol. Biol. 41, 851-859, 1995a. Sun, L., Gao, F., Jin, C., Duan, H., Nagata, T.: An electron microscopic radioautographic

Sun, L., Gao, F., Jin, C., Nagata, T.: DNA synthesis in the tracheae of aging mice by means of

Sun, L., Gao, F., Nagata, T.: A Light Microscopic radioautographic study on protein

Suzuki, K., Imada, T., Gao, F., Ma, H., Nagata, T.: Radioautographic study of benidipine

Terauchi, A., Mori, T., Kanda, H., Tsukada, M., Nagata, T.: Radioautographic study of 3H-

Terauchi, A., Nagata, T.: Observation on incorporation of 3H-taurine in mouse skeletal

Terauchi, A., Nagata, T.: In corporation of 3H-taurine into the blood capillary cells of mouse

Toriyama, K.: Study on the aging changes of DNA and protein synthesis of bipolar and

radioautography. Cell. Mol. Biol. 41, 593-601, 1995.

radioautographical study. Cell. Mol. Biol. 41, 107-116, 1995.

regenerating muscles. J. Clin. Electr. Microsc. 10, 508-509, 1977.

immnohistochemical studies. Cancer 71, 2807-2812, 1993.

pp. 201-205, Shinshu University Press, Matsumoto, 1994.

Edinburgh, London and New York, 1991.

cells. Cell Tissue Res. 187, 45-59, 1978.

Microsc. 28, 129-131, 1995b.

rat. Drug Res. 44, 129-133, 1994.

211-220, 1997a.

470, 1997b.

1988.

397-404, 1993.

Press, Matsumoto, 1994.

1977.

acids by endometrial stromal cells during decidualization in the mouse. A

2. 1055 pp., 1985, Vol. 3. Ed. with P. Stoward, 728 pp. Churchill Livingstone,

stomach G-cells by means of 3H-amino acids. J. Clin. Electron Microsc. 10, 358-359,

thyroid tumors evaluated by proliferating cell nuclear antigen/cyclin

in pulmonary cells in aging mice. In, Radioautography in Medicine, Nagata, T. Ed.,

study on the DNA synthesis of pulmonary tissue cells in aging mice. Med. Electron.

light and electron microscopic radioautography. Acta Histochem. Cytochem. 30,

synthesis in pulmonary cells of aging mice. Acta Histochem. Cytochem. 30, 463-

hydrochloride: localization in the mesenteric artery of spontaneously hypertensive

taurine uptake in mouse skeletal muscle cells. J. Clin. Electron Microsc. 21, 627-628,

muscle cells by light and electron microscopic radioautography. Cell. Mol. Biol. 39,

skeletal muscle. Radioautography in Medicine, Nagata, T. ed., Shinshu University

photo-receptor cells of mouse retina by light and electron microscopic


Nagata, T., Yamabayashi, S.: Intracellular localization of 3H-befunolol by means of electron

Nagata, T., Yoshida, K., Murata, F.: Demonstration of hot and cold mercury in the human

Nagata, T., Yoshida, K., Ohno, S., Murata, F.: Ultrastructural localization of soluble and

Nishigaki, T., Momose, Y., Nagata, T.: Light microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Electron microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Localization of the anti-allergic agent tranilast in the

Oguchi, K., Nagata, T.: A radioautographic study of activated satellite cells in dystrophic

Oguchi, K., Nagata, T.: Electron microscopic radioautographic observation on activated

Ohno, S., Fujii, Y., Usuda, N., Endo, T., Hidaka, H., Nagata, T.: Demonstration of

Ohno, S., Fujii, Y., Usuda, N., Nagata, T., Endo, T., Tanaka, T., Hidaka, H.: Intracellular

Olea, M. T.: An ultrastructural localization of lysosomal acid phosphatase activity in aging

Olea, M. T., Nagata, T.: X-ray microanalysis of cerium in mouse spleen cells demonstrating

Olea, M. T., Nagata, T. : Simultaneous localization of 3H-thymidine incorporation and acid

Olea, M. T., Nagata, T.: A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro. Cell. Mol. Biol. 38, 399-405, 1992b. Oliveira, S. F., Nagata, T., Abrahamsohn, P. A., Zorn, T. M. T.: Electron microscopic

Microsc. 16, 737-738, 1983.

Toxicol. 41, 60-61, 1977b.

1978b.

536, 1987.

65-71, 1990a.

Tokyo, 1980.

Res. 40, 272-275, 1990b.

Welfare of Japan, Tokyo, 1981.

Co., New York, 1982.

37, 155-163, 1991.

Cytochem. 24, 201-208, 1991.

Cell. Mol. Biol. 38, 115-122, 1992a.

Cell. Mol. Biol. 37, 315-323, 1991.

radioautography. J. Electron Microsc. 32, 1-12, 1983.

microscopic radioautography of cryo-fixed ultrathin sections. J. Clin. Electron

thyroid tissues by means of radioautography and chemography. Acta Pharmacol.

insoluble 3H-methyl prednisolone as revealed by electron microscopic drymounting radioautography. Proc. 9th Internat. Congr. Electr. Microsc. 2, 40-41,

localization of anti-allergic agent, tranilast, in rat mast cells. Histochem. J. 19, 533-

localization of an anti-allergic agent, tranilast, in rat mast cells. Cell. Mol. Biol. 36,

urinary bladder of rat as demonstrated by light microscopic radioautography. Drug

chicken muscle. In, Current Research in Muscular Dystrophy Japan. The Proc. Ann. Meet. Muscular Dystrophy Res. 1980, pp. 16-17, Ministry of Welfare of Japan,

satellite cells in dystrophy chickens. In, Clinical Studies on the Etiology of Muscular Dystrophy. Annual Report on Neurological Diseases 1981, pp. 30-33, Ministry of

intracellular localization of calmodulin antagonist by wet-mounting

localization of calmodulin antagonists (W-7). In, Calmodulin and intracellular Ca2+ receptors. Kakiuchi, S., Hidaka, H, Means, A. R., Eds., pp. 39-48, Plenum Publishing

mouse spleen: a quantitative X-ray microanalytical study. Acta Histochem.

acid phosphatase activity using high voltage electron microscopy, Cell. Mol. Biol.

phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

radioautographic study on the incorporation of 3H-proline by mouse decidual cells.


**16** 

*Japan* 

Tetsuji Nagata1,2

**Macromolecular Synthesis** 

*Shinshu University School of Medicine, Matsumoto* 

*2Shinshu Institute of Alternative Medicine and Welfare, Nagano* 

*1Department of Anatomy and Cell Biology,* 

**in the Urinary and Reproductive Systems** 

This chapter deals with the third parts of the application of microscopic radioautography to the organ systems, including the urinary organs, the reproductive organs and the endocrine

The urinary system consists of the kidney and the urinary tract. We studied only the macromolecular synthesis in the kidneys of several groups of aging mice by LM and EM RAG, while the localization of an anti-allergic agent was observed in the urinary bladders of

We studied only the DNA, RNA and glucides syntheses in the kidneys of several groups of

The kidneys of mammals microscopically consist of the nephrons, which can be divided into two portions, the renal corpuscles and the uriniferous tubules. The renal corpuscles are composed of the glomeruli which are covered with the Bowman's capsules. They are localized in the outer zone of the kidney, the renal cortex, while the uriniferous tubules are composed of two portions, the proximal portions and the distal portions which can further be divided into several portions which run from the outer zone of the kidney, the renal cortex, to the inner zone, the medulla. We studied the DNA synthesis by 3H-thymidine radioautography in 3 groups of ddY mouse embryos from prenatal day 13 (Fig. 16A), day 15 (Fig. 16B) to day 19 in vitro, as well as perinatal mice from embryonic day 19 to postnatal day 1, 8, 30, 60 and 365 (1 year) in vivo (Hanai 1993, Hani et al. 1993, Hanai and Nagata

**2. Macromolecular synthesis in the urinary system** 

**2.1 Macromolecular synthesis in the kidney** 

**1. Introduction** 

adult rats (Nagata 2005).

aging mice by LM and EM RAG.

**2.1.1 The DNA synthesis in the kidney** 

organs.

1994a,b).


## **Macromolecular Synthesis in the Urinary and Reproductive Systems**

Tetsuji Nagata1,2

*1Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 2Shinshu Institute of Alternative Medicine and Welfare, Nagano Japan* 

## **1. Introduction**

358 Senescence

Tsukahara, S., Yoshida, K., Nagata, T.: A radioautographic study on the incorporation of

Usuda, N., Nagata, T.: Electron microscopic radioautography of acyl-CoA mRNA by in situ

Usuda, N., Nagata, T.: The immunohistochemical and in situ hybridization studies on hepatic peroxisomes. Acta Histochem. Cytochem. 28, 169-172, 1995. Usuda, N., Hanai, T., Morita, T., Nagata, T.: Radioautographic demonstration of

Uwa, H., Nagata, T.: Cell population kinetics of the scleroblast during ethisterone-induced

Watanabe, I., Makiyama, M. C. K., Nagata, T.: Electron microscopic radioautographic

Yamabayashi, S., Gunarso, W., Tsukahara, S., Nagata, T.: Incorporation of 3H-befunolol

Yamada, A., Nagata, T.: Ribonucleic acid and protein synthesis in the uterus of pregnant

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of DNA synthesis in

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of RNA synthesis of

Yoshinaga, K.: Uterine receptivity for blastcyst implantation. Ann. N. Y. Acad. Sci. USA, 541,

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Study of ethionine

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Radioautographic

hybridization. J. Clin. Electron Microsc. 25, 332-333, 1992.

pp.181-184, Peeters Press, Leuven, 1992.

Microscopica 6. 130-131, 1997.

Cell. Mol. Biol. 39, 1-12, 1993.

window. Cell. Mol. Biol. 38, 763-774, 1992b.

implantation. Cell. Mol. Biol. 38, 211-233, 1993.

244, 1980.

9, 693-694, 1976.

365, 1992a.

424-431, 1988.

Microsc. 7, 349-350, 1974.

Electron. Microsc. 10, 372-373, 1977.

14C-bupranolol (beta-blocking agent) into the rabbit eye. Histochemistry 68, 237-

peroxisomal acyl-CoA oxidase mRNA by in situ hybridization. In, Recent advances in cellular and molecular biology, Vol. 6. Molecular biology of nucleus, peroxisomes, organelles and cell movement. Wegmann, R. J., Wegmann, M., Eds,

anal-fin process formation in adult females of the Medaka. Dev. Growth Different.

observation of the submandibular salivary gland of aging mouse. Acta

(beta blocking agent) into melanin granules of ocular tissues in the pigmented rabbits. I. Light microscopic radioautography. Histochemistry 73, 371-375, 1981. Yamada, A. T.: Timely and topologically defined protein synthesis in the periimplanting

mouse endometrium revealed by light and electron microscopic radioautography.

mouse during activation of implantation window. Med. Electron Microsc. 27, 363-

the endometria of pregnant-ovariectomized mice during activation of implantation

peri-implanting pregnant mouse during activation of receptivity for blastocyst

pancreatitis by means of electron microscopic radioautography. J. Clin. Electron

study of protein synthesis in pancreatic exocrine cells of alcoholic rats. J. Clin.

This chapter deals with the third parts of the application of microscopic radioautography to the organ systems, including the urinary organs, the reproductive organs and the endocrine organs.

## **2. Macromolecular synthesis in the urinary system**

The urinary system consists of the kidney and the urinary tract. We studied only the macromolecular synthesis in the kidneys of several groups of aging mice by LM and EM RAG, while the localization of an anti-allergic agent was observed in the urinary bladders of adult rats (Nagata 2005).

## **2.1 Macromolecular synthesis in the kidney**

We studied only the DNA, RNA and glucides syntheses in the kidneys of several groups of aging mice by LM and EM RAG.

## **2.1.1 The DNA synthesis in the kidney**

The kidneys of mammals microscopically consist of the nephrons, which can be divided into two portions, the renal corpuscles and the uriniferous tubules. The renal corpuscles are composed of the glomeruli which are covered with the Bowman's capsules. They are localized in the outer zone of the kidney, the renal cortex, while the uriniferous tubules are composed of two portions, the proximal portions and the distal portions which can further be divided into several portions which run from the outer zone of the kidney, the renal cortex, to the inner zone, the medulla. We studied the DNA synthesis by 3H-thymidine radioautography in 3 groups of ddY mouse embryos from prenatal day 13 (Fig. 16A), day 15 (Fig. 16B) to day 19 in vitro, as well as perinatal mice from embryonic day 19 to postnatal day 1, 8, 30, 60 and 365 (1 year) in vivo (Hanai 1993, Hani et al. 1993, Hanai and Nagata 1994a,b).

Macromolecular Synthesis in the Urinary and Reproductive Systems 361

Fig. 16A. LM RAG of the metanephros of a prenatal day 13.5 mouse embryo labeled with

Fig. 16B. LM RAG of the metanephros cortex of a prenatal day 15.5 mouse embryo labeled

Fig. 16C. LM RAG of the testis of a postnatal day 7 male mouse labeled with 3H-thymidine,

Fig. 16D. LM RAG of the testis of a postnatal year 1 male mouse labeled with 3H-thymidine,

Fig. 16E. LM RAG of the testis of a postnatal day 3 male mouse labeled with 3H-uridine in

Fig. 16F. LM RAG of the testis of a male mouse at postnatal day 1 labeled with 3H-leucine in

thymidine in vitro, showing DNA synthesis in granulosa cells (G) and theca cells (T). x400. Fig. 16H. LM RAG of the oviduct of a postnatal day 30 female mouse labeled with 3H-

The labeling indices by LM RAG in glomeruli (28 to 32%) and uriniferous tubules (31 to 33%) in the superficial layer were higher than those of labeling indices (10 to 12%) and (8 to 16%) in the deeper layer from the late fetal to the suckling period, then decreased with aging from weaning to senescence (Fig. 17). EM RAG revealed the same results (Hanai and Nagata 1994a,b,c). At the same time, immunocytochemical staining of PCNA/cyclin was carried out in the same animals in several aging groups as 3H-thymidine RAG (Hanai 1993, Hanai et al. 1993). The results from the PCNA/cyclin positive indices in respective aging groups were almost the same as the labeling indices with 3H-thymidine RAG. The incorporation of 3Hthymidine was formerly observed by EM RAG in mitochondrial matrix of cultured kidney cells from chickens and mice in vitro demonstrating mitochondrial DNA synthesis (Nagata

The RNA synthesis by incorporation of 3H-uridine into the kidneys of aging mice was studied by LM and EM RAG (Hanai and Nagata 1994a,b, Nagata 2002). When the kidneys of several groups of aging mice from embryo to postnatal 1 year were radioautographed with 3H-uridine either in vitro (embryonic day 15, 19 and postnatal day 1) and in vivo (embryonic day 19, postnatal day 1, 7, month 1, 2, 12), RNA synthesis was observed in all the cells of the kidney at various ages. The numbers of silver grains demonstrating the incorporation of 3Huridine in glomeruli (34.6 per cell) and uriniferous tubules (56.4 per cell) were higher in the superficial layer than those (15.6 and 18.6 per cell) in the deeper layer at embryonic day 15 and decreased gradually with aging. These results demonstrated the aging changes of RNA

The incorporations of 3H-glucosamine in the kidneys of aging mice were studies by LM RAG (Joukura 1996, Joukura and Nagata 1995) and EM RAG (Joukura et al. 1996). Silver grains were observed over all the cell type nephrons at embryonic day 19, i.e., glomerular epithelial cells, endothelial cells, mesangial cells, Bowman's capsular cells and tubule cells.

Fig. 16G. LM RAG of the ovary of a postnatal day 3 female mouse labeled with 3H-

thymidine in vitro, showing DNA synthesis in epithelial cells. x400.

3H-thymidine, showing DNA synthesis. x1,200.

showing DNA synthesis. x800.

showing DNA synthesis. x800.

et al. 1967b).

synthesis in the kidney.

vitro, showing RNA synthesis. x1,500.

vitro, showing protein synthesis. x1,125.

**2.1.2 The RNA synthesis in the kidney** 

**2.1.3 Glucide synthesis in the kidney** 

with 3H-thymidine, showing DNA synthesis. x1,200.

Fig. 16. LM RAG of the uro-genital organs. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 108, 2001, Academic Press, San Diego, USA, London, UK.

Fig. 16. LM RAG of the uro-genital organs. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 108, 2001, Academic Press, San Diego,

USA, London, UK.

Fig. 16A. LM RAG of the metanephros of a prenatal day 13.5 mouse embryo labeled with 3H-thymidine, showing DNA synthesis. x1,200.

Fig. 16B. LM RAG of the metanephros cortex of a prenatal day 15.5 mouse embryo labeled with 3H-thymidine, showing DNA synthesis. x1,200.

Fig. 16C. LM RAG of the testis of a postnatal day 7 male mouse labeled with 3H-thymidine, showing DNA synthesis. x800.

Fig. 16D. LM RAG of the testis of a postnatal year 1 male mouse labeled with 3H-thymidine, showing DNA synthesis. x800.

Fig. 16E. LM RAG of the testis of a postnatal day 3 male mouse labeled with 3H-uridine in vitro, showing RNA synthesis. x1,500.

Fig. 16F. LM RAG of the testis of a male mouse at postnatal day 1 labeled with 3H-leucine in vitro, showing protein synthesis. x1,125.

Fig. 16G. LM RAG of the ovary of a postnatal day 3 female mouse labeled with 3H-

thymidine in vitro, showing DNA synthesis in granulosa cells (G) and theca cells (T). x400. Fig. 16H. LM RAG of the oviduct of a postnatal day 30 female mouse labeled with 3H-

thymidine in vitro, showing DNA synthesis in epithelial cells. x400.

The labeling indices by LM RAG in glomeruli (28 to 32%) and uriniferous tubules (31 to 33%) in the superficial layer were higher than those of labeling indices (10 to 12%) and (8 to 16%) in the deeper layer from the late fetal to the suckling period, then decreased with aging from weaning to senescence (Fig. 17). EM RAG revealed the same results (Hanai and Nagata 1994a,b,c). At the same time, immunocytochemical staining of PCNA/cyclin was carried out in the same animals in several aging groups as 3H-thymidine RAG (Hanai 1993, Hanai et al. 1993). The results from the PCNA/cyclin positive indices in respective aging groups were almost the same as the labeling indices with 3H-thymidine RAG. The incorporation of 3Hthymidine was formerly observed by EM RAG in mitochondrial matrix of cultured kidney cells from chickens and mice in vitro demonstrating mitochondrial DNA synthesis (Nagata et al. 1967b).

## **2.1.2 The RNA synthesis in the kidney**

The RNA synthesis by incorporation of 3H-uridine into the kidneys of aging mice was studied by LM and EM RAG (Hanai and Nagata 1994a,b, Nagata 2002). When the kidneys of several groups of aging mice from embryo to postnatal 1 year were radioautographed with 3H-uridine either in vitro (embryonic day 15, 19 and postnatal day 1) and in vivo (embryonic day 19, postnatal day 1, 7, month 1, 2, 12), RNA synthesis was observed in all the cells of the kidney at various ages. The numbers of silver grains demonstrating the incorporation of 3Huridine in glomeruli (34.6 per cell) and uriniferous tubules (56.4 per cell) were higher in the superficial layer than those (15.6 and 18.6 per cell) in the deeper layer at embryonic day 15 and decreased gradually with aging. These results demonstrated the aging changes of RNA synthesis in the kidney.

## **2.1.3 Glucide synthesis in the kidney**

The incorporations of 3H-glucosamine in the kidneys of aging mice were studies by LM RAG (Joukura 1996, Joukura and Nagata 1995) and EM RAG (Joukura et al. 1996). Silver grains were observed over all the cell type nephrons at embryonic day 19, i.e., glomerular epithelial cells, endothelial cells, mesangial cells, Bowman's capsular cells and tubule cells.

Macromolecular Synthesis in the Urinary and Reproductive Systems 363

The urinary tract is composed of the ureter, the urinary bladder and the urethra. We studied the urinary bladder of adult rats by LM RAG after oral administration of 3H-tranilast, an antiallergic agent produced by Kissei Pharmaceutical Co. (Momose et al. 1989, Nishigaki et al. 1987, 1990a,b). It was found that this agent specifically localized over the transitional epithelium and the endothelium of the veins in the mucosa of normal adult rats. However, any study on the

The reproductive system or genital organs can be divided into two parts, the male genital organs and female genital organs. We studied the DNA and RNA syntheses and protein synthesis in several groups of aging mice, both male and female, by LM and EM RAG

The male genital organs consist of the testis and its excretory ducts such as ductuli efferentes, ductus epididymidis, ductus deferens, ejaculatory ducts, auxiliary glands and penis. We studied both DNA and RNA syntheses in these organs of several groups of ddY

Among the male genital organs, the testis was the main target of the scientific interests. Formerly, Clermont (1958, 1963) demonstrated using 3H-thymidine radioautography that several stages of development of the spermatogonia were found at different levels in the germinal epithelium of mature men and rodents, with the most primitive germ cells found at the base and the more differentiated cells located at higher levels. We studied the DNA

The structure of the testis of mammals is a compound tubular gland enclosed in tunica albuginea, a thick fibrous capsule. The parenchyma of the testis is composed of around 250 pyramidal compartments in men and animals, named lobules. Each lobule is made of convoluted seminiferous tubules, consisting of many spermatogenic cells differentiatiang to sperms among the supporting cells of Sertoli in the seminiferous epithelium, surrounded by the interstitial cells of Leydig. We first studied the macromolecular synthesis in the testis of aging male ddY mice at various ages (Gao 1993, Gao et al. 1994,1995a,b). When testicular tissues were labeled with 3H-thymidine and observed by LM and EM RAG, many spermatogonia and myoid cells as well as Leydig cells were labeled with 3H-thymidine at various ages from embryonic day 19 to postnatal day 1, 3, 7 (Fig. 16C), 14, month 1, 2, 6, 12 (Fig. 16D) and 24 (2 years). Silver grains were localized over the nuclei and several mitochondria of the spermatogonia showing DNA synthesis. Among of the aging groups, we counted the numbers of mitochondria per cell profile area, the numbers of labeled mitochondria per cell of the spermatogonia from 4 aging groups, prenatal embryonic day 19, postnatal day 3, and adults at month 1 and 6, and the labeling indices were calculated.

DNA synthesis in the ureter, the urinary bladder and the urethra was not carried out.

**3. Macromolecular synthesis in the reproductive system** 

**3.1 Macromolecular synthesis in the male genital organs** 

**3.1.1 The DNA synthesis in the male genital organs** 

synthesis in the testis of several groups of aging mice.

**3.1.1.1 The DNA synthesis in the testis** 

aging mice by LM and EMRAG using macromolecular precursors.

**2.2 Localization of drugs in the urinary tract** 

(Nagata 2002).

In newborn and suckling stages, from postnatal day 1, 3, 7 to 14, both the renal corpuscles and urinary tubules were well differentiated and the number of silver grains increased (Figs. 36 C, D, E F, G, H in Nagata 2002). The results from grain counting revealed that the numbers of silver grains in both the renal corpuscles and the uriniferous tubules were less in the embryonic stage, but increased postnatally and reached peaks at day 1 and 3, then decreased to senescence at 1 year. These results showed that glucide synthesis in the kidney cells also changed with aging of animals.

Fig. 17. Histogram showing aging changes of average labeling indices in respective cell types of the kidneys of aging mice labeled with 3H-thyidine. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 156, 2002, Urban & Fischer, Jena, Germany Fig. 17A. Labeling indices of the glomeruli and the uriniferous tubules of of mouse embryo from prenatal day 13 and 19.

Fig. 17B. Labeling indices of the glomeruli and the proximal and distal tubules of mouse embryo from prenatal day 19 to postnatal year 1. Abbreviations: GS=glomeruli of the superficial layer. US=uriniferous tubules of the superficial layer. GD=glomeruli of the deeper layer. UD=uriniferous tubules of the deeper layer. G=glomeruli. PT=proximal tubules. DT=distal tubules.

## **2.2 Localization of drugs in the urinary tract**

362 Senescence

In newborn and suckling stages, from postnatal day 1, 3, 7 to 14, both the renal corpuscles and urinary tubules were well differentiated and the number of silver grains increased (Figs. 36 C, D, E F, G, H in Nagata 2002). The results from grain counting revealed that the numbers of silver grains in both the renal corpuscles and the uriniferous tubules were less in the embryonic stage, but increased postnatally and reached peaks at day 1 and 3, then decreased to senescence at 1 year. These results showed that glucide synthesis in the kidney

Fig. 17. Histogram showing aging changes of average labeling indices in respective cell types of the kidneys of aging mice labeled with 3H-thyidine. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem.

Fig. 17A. Labeling indices of the glomeruli and the uriniferous tubules of of mouse embryo

Fig. 17B. Labeling indices of the glomeruli and the proximal and distal tubules of mouse embryo from prenatal day 19 to postnatal year 1. Abbreviations: GS=glomeruli of the superficial layer. US=uriniferous tubules of the superficial layer. GD=glomeruli of the deeper layer. UD=uriniferous tubules of the deeper layer. G=glomeruli. PT=proximal

Cytochem. Vol. 37, No. 2, p. 156, 2002, Urban & Fischer, Jena, Germany

from prenatal day 13 and 19.

tubules. DT=distal tubules.

cells also changed with aging of animals.

The urinary tract is composed of the ureter, the urinary bladder and the urethra. We studied the urinary bladder of adult rats by LM RAG after oral administration of 3H-tranilast, an antiallergic agent produced by Kissei Pharmaceutical Co. (Momose et al. 1989, Nishigaki et al. 1987, 1990a,b). It was found that this agent specifically localized over the transitional epithelium and the endothelium of the veins in the mucosa of normal adult rats. However, any study on the DNA synthesis in the ureter, the urinary bladder and the urethra was not carried out.

## **3. Macromolecular synthesis in the reproductive system**

The reproductive system or genital organs can be divided into two parts, the male genital organs and female genital organs. We studied the DNA and RNA syntheses and protein synthesis in several groups of aging mice, both male and female, by LM and EM RAG (Nagata 2002).

## **3.1 Macromolecular synthesis in the male genital organs**

The male genital organs consist of the testis and its excretory ducts such as ductuli efferentes, ductus epididymidis, ductus deferens, ejaculatory ducts, auxiliary glands and penis. We studied both DNA and RNA syntheses in these organs of several groups of ddY aging mice by LM and EMRAG using macromolecular precursors.

## **3.1.1 The DNA synthesis in the male genital organs**

Among the male genital organs, the testis was the main target of the scientific interests. Formerly, Clermont (1958, 1963) demonstrated using 3H-thymidine radioautography that several stages of development of the spermatogonia were found at different levels in the germinal epithelium of mature men and rodents, with the most primitive germ cells found at the base and the more differentiated cells located at higher levels. We studied the DNA synthesis in the testis of several groups of aging mice.

## **3.1.1.1 The DNA synthesis in the testis**

The structure of the testis of mammals is a compound tubular gland enclosed in tunica albuginea, a thick fibrous capsule. The parenchyma of the testis is composed of around 250 pyramidal compartments in men and animals, named lobules. Each lobule is made of convoluted seminiferous tubules, consisting of many spermatogenic cells differentiatiang to sperms among the supporting cells of Sertoli in the seminiferous epithelium, surrounded by the interstitial cells of Leydig. We first studied the macromolecular synthesis in the testis of aging male ddY mice at various ages (Gao 1993, Gao et al. 1994,1995a,b). When testicular tissues were labeled with 3H-thymidine and observed by LM and EM RAG, many spermatogonia and myoid cells as well as Leydig cells were labeled with 3H-thymidine at various ages from embryonic day 19 to postnatal day 1, 3, 7 (Fig. 16C), 14, month 1, 2, 6, 12 (Fig. 16D) and 24 (2 years). Silver grains were localized over the nuclei and several mitochondria of the spermatogonia showing DNA synthesis. Among of the aging groups, we counted the numbers of mitochondria per cell profile area, the numbers of labeled mitochondria per cell of the spermatogonia from 4 aging groups, prenatal embryonic day 19, postnatal day 3, and adults at month 1 and 6, and the labeling indices were calculated.

Macromolecular Synthesis in the Urinary and Reproductive Systems 365

At embryonic and neonatal stages, DNA synthesis of spermatocytes was weak and only a few labeled spermatogonia could be observed during the perinatal stages. The labeled spermatocytes were recognized at postnatal day 4 and 7 (Fig. 16C) and the number of labeled spermatogonia and spermatocytes increased from 2 and 3 weeks, keeping high level from month 1 to year 1 and 2 until senescence (Fig. 18A). However, the Sertoli's cells (Fig. 18B) and myoid cells (Fig. 18C) labeled with 3H-thymidine were frequently observed at perinatal stages from embryo to postnatal day 7, while the labeling indices of both cells decreased from young adulthood (postnatal 2 weeks) to senescence (Gao 1993, Gao et al. 1994,1995a). The interstitial cells of Leydig in the testis surrounding the seminiferous

tubules shall be described in the following section of the endocrine system in detail.

weeks together with DNA and protein syntheses (Fig. 16F) to senescence.

**3.1.3 The protein synthesis in the male genital organs** 

**3.1.3.1 The protein synthesis in the testis** 

Among of the male genital organs, we studied the RNA synthesis in the testis of several

We studied the RNA syntheses in aging mouse testis by LM and EM RAG, demonstrating the incorporations of 3H-uridine into various cells of the seminiferous tubules (Gao 1993, Gao et al. 1994, Nagata 2002). The RNA synthesis of various cells in the seminiferous tubules was studied using 3H-uridine. Silver grains due to 3H-uridine demonstrating RNA synthesis were observed over the nuclei and cytoplasm of all spermatogonia, spermatocytes, Sertoli's cells, myoid cells of immature mice at perinatal stages at day 1 and 3 (Fig. 16E), as well as in mature and senescent mice from month 1, 6 to year 1 and 2. The synthetic activities of spermatogonia, Sertoli's cells and myoid cells as shown by grain counting with 3H-uridine, as expressed by grain counting, were low (2-8 grain counts per 10 mm2) at the embryonic and neonatal stages but increased at adult stages and maintained high levels (10-20 grain counts per 10 mm2) until senescence. These results showed that DNA synthesis in myoid cells and Sertoli's cells increased at the perinatal stages and decreased from postnatal 2 weeks as described previously (Fig. 16A), while the RNA synthesis (Fig. 16E) in spermatogonia increased from postnatal 2

We studied the protein synthesis of the reproductive system in both the male and female

We studied the protein syntheses in aging mouse testis by LM and EM RAG, demonstrating the incorporations of 3H-leucine into various cells of the seminiferous tubules (Gao 1993, Gao et al. 1994, Nagata 2002). The protein synthesis of various cells in the seminiferous tubules was first studied after administration of 3H-leucine into aging male mice at various ages from perinatal to senescence at postnatal 2 years. Silver grains due to 3H-leucine incorporation demonstrating protein synthesis were observed over the nuclei and cytoplasm of all the cells, spermatogonia, spermatocytes, Sertoli's cells, myoid cells of all male mice at respective stages from perinatal to senescence. The synthetic activities of spermatogonia, Sertoli's cells and myoid cells as shown by the number of silver grains due to 3H-leucine, as expressed by grain

**3.1.2 The RNA synthesis in the male genital organs** 

groups of aging mice.

reproductive organs.

**3.1.2.1 The RNA synthesis in the testis** 

The results showed that the LI of the spermatogonia increased from embryonic day 19 (17%) to postnatal day 7 (25%) and month 1 (30%), reaching the maximum, then decreased to month 6 (20%) to year 2.

Fig. 18. Transitional curves of the labeling indices of respective cell types in the testis of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 160, 2002, Urban & Fischer, Jena, Germany Fig. 18A. The spermatogonia. Fig. 18B. The Sertoli cells.

Fig. 18C. The myoid cells.

At embryonic and neonatal stages, DNA synthesis of spermatocytes was weak and only a few labeled spermatogonia could be observed during the perinatal stages. The labeled spermatocytes were recognized at postnatal day 4 and 7 (Fig. 16C) and the number of labeled spermatogonia and spermatocytes increased from 2 and 3 weeks, keeping high level from month 1 to year 1 and 2 until senescence (Fig. 18A). However, the Sertoli's cells (Fig. 18B) and myoid cells (Fig. 18C) labeled with 3H-thymidine were frequently observed at perinatal stages from embryo to postnatal day 7, while the labeling indices of both cells decreased from young adulthood (postnatal 2 weeks) to senescence (Gao 1993, Gao et al. 1994,1995a). The interstitial cells of Leydig in the testis surrounding the seminiferous tubules shall be described in the following section of the endocrine system in detail.

## **3.1.2 The RNA synthesis in the male genital organs**

Among of the male genital organs, we studied the RNA synthesis in the testis of several groups of aging mice.

### **3.1.2.1 The RNA synthesis in the testis**

364 Senescence

The results showed that the LI of the spermatogonia increased from embryonic day 19 (17%) to postnatal day 7 (25%) and month 1 (30%), reaching the maximum, then decreased to

Fig. 18. Transitional curves of the labeling indices of respective cell types in the testis of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 160, 2002, Urban & Fischer, Jena, Germany

month 6 (20%) to year 2.

Fig. 18A. The spermatogonia. Fig. 18B. The Sertoli cells. Fig. 18C. The myoid cells.

We studied the RNA syntheses in aging mouse testis by LM and EM RAG, demonstrating the incorporations of 3H-uridine into various cells of the seminiferous tubules (Gao 1993, Gao et al. 1994, Nagata 2002). The RNA synthesis of various cells in the seminiferous tubules was studied using 3H-uridine. Silver grains due to 3H-uridine demonstrating RNA synthesis were observed over the nuclei and cytoplasm of all spermatogonia, spermatocytes, Sertoli's cells, myoid cells of immature mice at perinatal stages at day 1 and 3 (Fig. 16E), as well as in mature and senescent mice from month 1, 6 to year 1 and 2. The synthetic activities of spermatogonia, Sertoli's cells and myoid cells as shown by grain counting with 3H-uridine, as expressed by grain counting, were low (2-8 grain counts per 10 mm2) at the embryonic and neonatal stages but increased at adult stages and maintained high levels (10-20 grain counts per 10 mm2) until senescence. These results showed that DNA synthesis in myoid cells and Sertoli's cells increased at the perinatal stages and decreased from postnatal 2 weeks as described previously (Fig. 16A), while the RNA synthesis (Fig. 16E) in spermatogonia increased from postnatal 2 weeks together with DNA and protein syntheses (Fig. 16F) to senescence.

## **3.1.3 The protein synthesis in the male genital organs**

We studied the protein synthesis of the reproductive system in both the male and female reproductive organs.

#### **3.1.3.1 The protein synthesis in the testis**

We studied the protein syntheses in aging mouse testis by LM and EM RAG, demonstrating the incorporations of 3H-leucine into various cells of the seminiferous tubules (Gao 1993, Gao et al. 1994, Nagata 2002). The protein synthesis of various cells in the seminiferous tubules was first studied after administration of 3H-leucine into aging male mice at various ages from perinatal to senescence at postnatal 2 years. Silver grains due to 3H-leucine incorporation demonstrating protein synthesis were observed over the nuclei and cytoplasm of all the cells, spermatogonia, spermatocytes, Sertoli's cells, myoid cells of all male mice at respective stages from perinatal to senescence. The synthetic activities of spermatogonia, Sertoli's cells and myoid cells as shown by the number of silver grains due to 3H-leucine, as expressed by grain

Macromolecular Synthesis in the Urinary and Reproductive Systems 367

Fig. 19. Histogram showing aging changes of average labeling indices in respective cell types of female genital organs of aging mice labeled with 3H-thymidine. Mean ± Standard

The silver grains with 3H-thymidine showing DNA synthesis of the uterus was observed over some of the nuclei of all the cells in the epithelia, stroma and smooth muscles from postnatal day 1 to 60 (Li 1994, Li and Nagata 1995). The labeling indices with 3H-thymidine (Fig. 19B) were high (80-95%) at postnatal day 1 and decreased from day 3 to 60 (>10%). The silver grains showing RNA synthesis of the uterus were observed over all the nuclei and cytoplasm of all the cells in the uterine epithelia, stroma and smooth muscles from day 1 to 60. The number of silver grains in the uterine epithelium increased from postnatal day 1 to 7 and decreased from day 14 to 60, while they increased in the stroma from day 1 to 3 and

These results from the female genital organs showed that both DNA and RNA syntheses, as expressed by labeling indices and grain counting, were active in all kinds of cells, such as surface epithelial cells, stromal cells and follicular cells of the ovaries between postnatal

Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 166, 2002, Urban & Fischer, Jena, Germany

Fig. 19A. The ovary. Fig. 19B. The uterus. Fig. 19C. The oviduct.

decreased from day 7 to 60.

**3.2.1.3 The DNA synthesis in the uterus** 

counting, were low at the embryonic and neonatal stages but increased at adult stages and maintained high levels until senescence. These results showed that DNA synthesis in Sertoli's cells (Fig. 18B) and myoid cells (Fig. 18C) that increased at the perinatal stages and decreased from postnatal 2 weeks, while the DNA synthesis in spermatogonia increased from postnatal 2 weeks (Fig. 18A) together with RNA and protein syntheses to senescence.

## **3.2 Macromolecular synthesis in the female genital organs**

The female genital organs consist of the ovary, the oviduct, the uterus, the vagina and the external genitals. We studied the macromolecular synthesis in the ovary, oviduct and uterus of several litters of ddY mice in aging.

## **3.2.1 The DNA synthesis in the female genital organ**

Among the female genital organs, we studied the DNA synthesis in the ovary, oviduct and uterus of several litters of ddY mice in aging.

#### **3.2.1.1 The DNA synthesis in the ovary**

The ovary consists of the germinal epithelium covering the surface and the stroma containing many developing ovarian follicles depending upon the age of animals.

The nucleic acids, DNA and RNA, syntheses in the developing virgin mice ovaries of 6 litters, each 3 individuals, consisting of 36 female mice at various ages in respective precursors were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995, Li et al. 1992). The 3H-thymidine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovaries between postnatal days 1 to 7 and decreased from day 14 (Fig. 16G) and maintained a lower level to day 60, while 3H-uridine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovaries between postnatal days 1 to 7 and maintained medium levels from day 14 on.

The labeling indices with 3H-thymidine showing DNA synthetic activity were high in all the surface epithelial cells, follicular cells and stromal cells of mice at neonatal stage from postnatal day 1 to 7, but decreased from day 40 to day 60 at mature stage (Fig. 19A). The grain counts showing RNA synthetic activity were high at neonatal stage from day 1 to day 7, and maintained medium levels from day 14 to day 60 at mature stage.

### **3.2.1.2 The DNA synthesis in the oviduct**

The nucleic acids, DNA and RNA, syntheses in the oviducts of developing virgin mice at various ages were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995). The silver grains with 3H-thymidine showing the DNA synthesis were observed over many nuclei in all surface epithelial cells, stromal and smooth muscle cells at neonatal stage between postnatal day 1 to 3 and decreased from day 7 to 30 (Fig. 16H) and 60, while the silver grains showing the RNA synthesis with 3H-uridine were observed over the nuclei and cytoplasm of all the epithelial and stromal cells from postnatal day 1 to day 60. The labeling indices with 3H-thymidine were high at neonatal stage from postnatal day 1 to 3 but decreased from day 7 to day 60 (Fig. 19C). The grain counts with 3H-uridine were high at neonatal stage from postnatal day 1 to 3 and increased from day 7 to day 14 and decreased from day 30 to day 60. These results demonstrated an unparalleled alternation of DNA and RNA syntheses in the oviduct (Li and Nagata 1995).

Fig. 19. Histogram showing aging changes of average labeling indices in respective cell types of female genital organs of aging mice labeled with 3H-thymidine. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 166, 2002, Urban & Fischer, Jena, Germany Fig. 19A. The ovary.

Fig. 19B. The uterus.

366 Senescence

counting, were low at the embryonic and neonatal stages but increased at adult stages and maintained high levels until senescence. These results showed that DNA synthesis in Sertoli's cells (Fig. 18B) and myoid cells (Fig. 18C) that increased at the perinatal stages and decreased from postnatal 2 weeks, while the DNA synthesis in spermatogonia increased from postnatal 2

The female genital organs consist of the ovary, the oviduct, the uterus, the vagina and the external genitals. We studied the macromolecular synthesis in the ovary, oviduct and uterus

Among the female genital organs, we studied the DNA synthesis in the ovary, oviduct and

The ovary consists of the germinal epithelium covering the surface and the stroma

The nucleic acids, DNA and RNA, syntheses in the developing virgin mice ovaries of 6 litters, each 3 individuals, consisting of 36 female mice at various ages in respective precursors were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995, Li et al. 1992). The 3H-thymidine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovaries between postnatal days 1 to 7 and decreased from day 14 (Fig. 16G) and maintained a lower level to day 60, while 3H-uridine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovaries between postnatal days 1 to 7 and maintained medium levels from day 14 on.

The labeling indices with 3H-thymidine showing DNA synthetic activity were high in all the surface epithelial cells, follicular cells and stromal cells of mice at neonatal stage from postnatal day 1 to 7, but decreased from day 40 to day 60 at mature stage (Fig. 19A). The grain counts showing RNA synthetic activity were high at neonatal stage from day 1 to day

The nucleic acids, DNA and RNA, syntheses in the oviducts of developing virgin mice at various ages were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995). The silver grains with 3H-thymidine showing the DNA synthesis were observed over many nuclei in all surface epithelial cells, stromal and smooth muscle cells at neonatal stage between postnatal day 1 to 3 and decreased from day 7 to 30 (Fig. 16H) and 60, while the silver grains showing the RNA synthesis with 3H-uridine were observed over the nuclei and cytoplasm of all the epithelial and stromal cells from postnatal day 1 to day 60. The labeling indices with 3H-thymidine were high at neonatal stage from postnatal day 1 to 3 but decreased from day 7 to day 60 (Fig. 19C). The grain counts with 3H-uridine were high at neonatal stage from postnatal day 1 to 3 and increased from day 7 to day 14 and decreased from day 30 to day 60. These results demonstrated an unparalleled alternation of

7, and maintained medium levels from day 14 to day 60 at mature stage.

DNA and RNA syntheses in the oviduct (Li and Nagata 1995).

containing many developing ovarian follicles depending upon the age of animals.

weeks (Fig. 18A) together with RNA and protein syntheses to senescence.

**3.2 Macromolecular synthesis in the female genital organs** 

**3.2.1 The DNA synthesis in the female genital organ** 

of several litters of ddY mice in aging.

uterus of several litters of ddY mice in aging.

**3.2.1.1 The DNA synthesis in the ovary** 

**3.2.1.2 The DNA synthesis in the oviduct** 

Fig. 19C. The oviduct.

#### **3.2.1.3 The DNA synthesis in the uterus**

The silver grains with 3H-thymidine showing DNA synthesis of the uterus was observed over some of the nuclei of all the cells in the epithelia, stroma and smooth muscles from postnatal day 1 to 60 (Li 1994, Li and Nagata 1995). The labeling indices with 3H-thymidine (Fig. 19B) were high (80-95%) at postnatal day 1 and decreased from day 3 to 60 (>10%). The silver grains showing RNA synthesis of the uterus were observed over all the nuclei and cytoplasm of all the cells in the uterine epithelia, stroma and smooth muscles from day 1 to 60. The number of silver grains in the uterine epithelium increased from postnatal day 1 to 7 and decreased from day 14 to 60, while they increased in the stroma from day 1 to 3 and decreased from day 7 to 60.

These results from the female genital organs showed that both DNA and RNA syntheses, as expressed by labeling indices and grain counting, were active in all kinds of cells, such as surface epithelial cells, stromal cells and follicular cells of the ovaries between postnatal

Macromolecular Synthesis in the Urinary and Reproductive Systems 369

showed that the labeling index in the posterior margin of the joint plate rapidly increased and the scleroblast population in the central portion increased simultaneously from the 3rd to 5th day of ethisterone treatment. These results indicated that the scleroblasts and their precursor cells migrated from the peripheral portion to the central portion along the proxi-

We studied the RNA synthesis of female reproductive organs of aging mice after the

The ovary consists of the germinal epithelium covering the surface and the stroma

The nucleic acids, DNA and RNA, syntheses in the developing virgin mice ovaries of 6 litters, each 3 individuals, consisting of 36 female mice at various ages in 2 groups were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995, Li et al. 1992). The 3H-thymidine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovary between postnatal days 1 to 7 and decreased from day 14 (Fig. 16G) and maintained a lower level to day 60, while 3H-uridine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovary between

The labeling indices with 3H-thymidine showing DNA synthetic activity were high in all the surface epithelial cells, follicular cells and stromal cells of mice at neonatal stage from postnatal day 1 to 7, but decreased from day 40 to day 60 at mature stage. The grain counts showing RNA synthetic activity were high at neonatal stage from day 1 to day 7, and

The nucleic acids, DNA and RNA, syntheses in the oviducts of developing virgin mice at various ages were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995). The silver grains with 3H-thymidine showing the DNA synthesis were observed over many nuclei in all surface epithelial cells, stromal and smooth muscle cells at neonatal stage between postnatal day 1 to 3 and decreased from day 7 to 30 (Fig. 16H) and 60, while the silver grains showing the RNA synthesis with 3H-uridine were observed over the nuclei and cytoplasm of all the epithelial and stromal cells from postnatal day 1 to day 60. The labeling indices with 3H-thymidine were high at neonatal stage from postnatal day 1 to 3 but decreased from day 7 to day 60. The grain counts with 3H-uridine were high at neonatal stage from postnatal day 1 to 3 and increased from day 7 to day 14 and decreased from day 30 to day 60. These results demonstrated an unparalleled alternation of DNA and

The silver grains with 3H-uridine showing RNA synthesis of the uterus was observed over almost all the nuclei and cytoplasm of all the cells in the epithelia, stroma and smooth muscles from postnatal day 1 to 60 (Li 1994, Li and Nagata 1995). The labeling indices with

containing many developing ovarian follicles depending upon the age of animals.

postnatal days 1 to 7 and maintained medium levels from day 14 on.

maintained medium levels from day 14 to day 60 at mature stage.

distal axis of the joint plate.

**3.2.2 The RNA synthesis in the female genital organs** 

administration of 3H-uridine at various ages.

**3.2.2.1 The RNA synthesis in the ovary** 

**3.2.2.2 The RNA synthesis in the oviduct** 

RNA syntheses in the oviduct (Li and Nagata 1995).

**3.2.2.3 The RNA synthesis in the uterus** 

days 1 to 7, then they decreased from day 14 to 60. However, the DNA synthesis in the epithelial cells and the stromal cells of both the uteri and the oviducts was active at postnatal day 1 and 3 and decreased from day 7 to 60. The RNA synthesis in the uteri and oviducts was active at postnatal day 1, increased from day 1 to day 14, and decreased from day 30 to 60. The unparalleled alteration of the DNA and RNA syntheses was shown between the ovary and the uterus or oviduct (Li and Nagata 1995).

We also studied PCNA/cyclin immunostaining in the ovary, oviduct and uterus (Li 1994). It was demonstrated that PCNA/cyclin positive cells were observed in the ovarian follicular epithelium, ovarian interstitial cells, tubal epithelial cells, tubal interstitial cells, uterine epithelial cells and uterine interstitial cells. The positive cells increased from postnatal 1 day to 3 and 7 days, then decreased from 14 days to senescence. These results accorded well with the results obtained from the 3H-thymidine radioautography (Li 1994, Li and Nagata 1995). Moreover, the mucosubstance synthesis incorporating sulfuric acid was also carried out (Oliveira et al. 1991, Li et al. 1995).

### **3.2.1.4 The DNA synthesis in gametogenesis**

The gametogenesis consists of both spermatogenesis in male germ cells and the oogenesis in female germ cells, leading to the implantation and further development of blastcysts. The macromolecular synthesis, DNA, RNA and protein synthesis, in both the testis and the ovary were already described in the sections of male and female reproductive systems (3.7.1. and 3.7.2.) previously.

#### **3.2.1.5 The DNA synthesis in implantaion**

In order to detect the changes of DNA, RNA and protein synthesis of the developing blastcysts in mouse endometrium during activation of the implantation, ovulations of female BALB/C strain mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply 3Hthymidine was injected. The three regions of the endometrium, i. e. the interinplantation site, the antimesometrial and mesometrial sides of implantation site, were taken out and processed for LM and EM RAG. It was well known that the uterus of the rodent becomes receptive to blastcyst implantation only for a restricted period. This is called the implantation window which is intercalated between refractory states of the endometrium whose cycling is regulated by ovarian hormones (Yoshinaga 1988). We studied the changes of DNA synthesis by 3H-thymidine (Yamada and Nagata 1992a,b) incorporations in the endometrial cells of pregnant-ovariectomized mice after time-lapse effect of nidatory estradiol. As the results, the endometrial cells showed topographical and chronological differences in the nucleic acid synthesis. The cells labeled with 3H-thymidine increased after nidatory estradiol effects in the stromal cells around the blastocyst, but not in the epithelial cells. The results suggested that the presence of the blastocysts in the uterine lumen induced selective changes in the behavior of endometrial cells after nidatory estradiol effect showing the changes of DNA synthesis.

As for a lower vertebrate, cell proliferation and migration of scleroblasts and their precursor cells during ethisterone-induced anal-fin process formation of the medaka, orizias latipes, was studied by LM RAG labeled with 3H-thymidine (Uwa and Nagata 1976). The results showed that the labeling index in the posterior margin of the joint plate rapidly increased and the scleroblast population in the central portion increased simultaneously from the 3rd to 5th day of ethisterone treatment. These results indicated that the scleroblasts and their precursor cells migrated from the peripheral portion to the central portion along the proxidistal axis of the joint plate.

## **3.2.2 The RNA synthesis in the female genital organs**

We studied the RNA synthesis of female reproductive organs of aging mice after the administration of 3H-uridine at various ages.

## **3.2.2.1 The RNA synthesis in the ovary**

368 Senescence

days 1 to 7, then they decreased from day 14 to 60. However, the DNA synthesis in the epithelial cells and the stromal cells of both the uteri and the oviducts was active at postnatal day 1 and 3 and decreased from day 7 to 60. The RNA synthesis in the uteri and oviducts was active at postnatal day 1, increased from day 1 to day 14, and decreased from day 30 to 60. The unparalleled alteration of the DNA and RNA syntheses was shown

We also studied PCNA/cyclin immunostaining in the ovary, oviduct and uterus (Li 1994). It was demonstrated that PCNA/cyclin positive cells were observed in the ovarian follicular epithelium, ovarian interstitial cells, tubal epithelial cells, tubal interstitial cells, uterine epithelial cells and uterine interstitial cells. The positive cells increased from postnatal 1 day to 3 and 7 days, then decreased from 14 days to senescence. These results accorded well with the results obtained from the 3H-thymidine radioautography (Li 1994, Li and Nagata 1995). Moreover, the mucosubstance synthesis incorporating sulfuric acid was also carried

The gametogenesis consists of both spermatogenesis in male germ cells and the oogenesis in female germ cells, leading to the implantation and further development of blastcysts. The macromolecular synthesis, DNA, RNA and protein synthesis, in both the testis and the ovary were already described in the sections of male and female reproductive systems (3.7.1.

In order to detect the changes of DNA, RNA and protein synthesis of the developing blastcysts in mouse endometrium during activation of the implantation, ovulations of female BALB/C strain mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply 3Hthymidine was injected. The three regions of the endometrium, i. e. the interinplantation site, the antimesometrial and mesometrial sides of implantation site, were taken out and processed for LM and EM RAG. It was well known that the uterus of the rodent becomes receptive to blastcyst implantation only for a restricted period. This is called the implantation window which is intercalated between refractory states of the endometrium whose cycling is regulated by ovarian hormones (Yoshinaga 1988). We studied the changes of DNA synthesis by 3H-thymidine (Yamada and Nagata 1992a,b) incorporations in the endometrial cells of pregnant-ovariectomized mice after time-lapse effect of nidatory estradiol. As the results, the endometrial cells showed topographical and chronological differences in the nucleic acid synthesis. The cells labeled with 3H-thymidine increased after nidatory estradiol effects in the stromal cells around the blastocyst, but not in the epithelial cells. The results suggested that the presence of the blastocysts in the uterine lumen induced selective changes in the behavior of endometrial cells after nidatory estradiol effect showing

As for a lower vertebrate, cell proliferation and migration of scleroblasts and their precursor cells during ethisterone-induced anal-fin process formation of the medaka, orizias latipes, was studied by LM RAG labeled with 3H-thymidine (Uwa and Nagata 1976). The results

between the ovary and the uterus or oviduct (Li and Nagata 1995).

out (Oliveira et al. 1991, Li et al. 1995).

and 3.7.2.) previously.

the changes of DNA synthesis.

**3.2.1.4 The DNA synthesis in gametogenesis** 

**3.2.1.5 The DNA synthesis in implantaion** 

The ovary consists of the germinal epithelium covering the surface and the stroma containing many developing ovarian follicles depending upon the age of animals.

The nucleic acids, DNA and RNA, syntheses in the developing virgin mice ovaries of 6 litters, each 3 individuals, consisting of 36 female mice at various ages in 2 groups were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995, Li et al. 1992). The 3H-thymidine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovary between postnatal days 1 to 7 and decreased from day 14 (Fig. 16G) and maintained a lower level to day 60, while 3H-uridine incorporations were active in all surface epithelial cells, stromal and follicular cells of the ovary between postnatal days 1 to 7 and maintained medium levels from day 14 on.

The labeling indices with 3H-thymidine showing DNA synthetic activity were high in all the surface epithelial cells, follicular cells and stromal cells of mice at neonatal stage from postnatal day 1 to 7, but decreased from day 40 to day 60 at mature stage. The grain counts showing RNA synthetic activity were high at neonatal stage from day 1 to day 7, and maintained medium levels from day 14 to day 60 at mature stage.

## **3.2.2.2 The RNA synthesis in the oviduct**

The nucleic acids, DNA and RNA, syntheses in the oviducts of developing virgin mice at various ages were studied by 3H-thymidine and 3H-uridine radioautography (Li 1994, Li and Nagata 1995). The silver grains with 3H-thymidine showing the DNA synthesis were observed over many nuclei in all surface epithelial cells, stromal and smooth muscle cells at neonatal stage between postnatal day 1 to 3 and decreased from day 7 to 30 (Fig. 16H) and 60, while the silver grains showing the RNA synthesis with 3H-uridine were observed over the nuclei and cytoplasm of all the epithelial and stromal cells from postnatal day 1 to day 60. The labeling indices with 3H-thymidine were high at neonatal stage from postnatal day 1 to 3 but decreased from day 7 to day 60. The grain counts with 3H-uridine were high at neonatal stage from postnatal day 1 to 3 and increased from day 7 to day 14 and decreased from day 30 to day 60. These results demonstrated an unparalleled alternation of DNA and RNA syntheses in the oviduct (Li and Nagata 1995).

## **3.2.2.3 The RNA synthesis in the uterus**

The silver grains with 3H-uridine showing RNA synthesis of the uterus was observed over almost all the nuclei and cytoplasm of all the cells in the epithelia, stroma and smooth muscles from postnatal day 1 to 60 (Li 1994, Li and Nagata 1995). The labeling indices with

Macromolecular Synthesis in the Urinary and Reproductive Systems 371

estradiol. As the results, the endometrial cells showed topographical and chronological differences in the nucleic acid synthesis. The cells labeled with 3H-thymidine increased after nidatory estradiol effects in the stromal cells around the blastocyst, but not in the epithelial cells. The results suggested that the presence of the blastocysts in the uterine lumen induced selective changes in the behavior of endometrial cells after nidatory estradiol effect showing

As for a lower vertebrate, cell proliferation and migration of scleroblasts and their precursor cells during ethisterone-induced anal-fin process formation of the medaka, orizias latipes, was studied by LM RAG labeled with 3H-thymidine (Uwa and Nagata 1976). The results showed that the labeling index in the posterior margin of the joint plate rapidly increased and the scleroblast population in the central portion increased simultaneously from the 3rd to 5th day of ethisterone treatment. These results indicated that the scleroblasts and their precursor cells migrated from the peripheral portion to the central portion along the proxi-

We studied the protein synthesis of female reproductive organs of aging mice after the

We studied the protein synthesis of the developing blastocysts in female mouse endometrium during activation of the implantation. The ovulations of female BALB/C strain adult mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply. After the mice were injected with 3H-leucine, they were sacrificed and the uteri were processed for LM and EMRAG. We studied the changes of protein synthesis by 3H-leucine incorporations (Yamada 1993, Yamada and Nagata 1992a). As the results, the endometrial cells showed topographical and chronological differences in the protein synthesis. The cells labeled with 3H-leucine were observed in both epithelial cells and stromal cells. Quantitative analysis revealed that the number of silver grains increased from 0 hr to 3 and 6 hr, reaching the peak at 6 hr and decreased from 12 to 18 hr. The protein synthesis in the decidual cells of pregnant mice uteri was compared to the endometrial cells of virgin mice uteri using 3Hproline and 3H-tryptophane incorporations. The results demonstrated that silver grains were localized over the endoplasmic reticulum and the Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix suggesting that the decidual cells produced collagen in the matrix. The collagen synthesis in the mouse decidual cells by 3H-proline showed that silver grains were localized over the endoplasmic reticulum and Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix (Oliveira et al. 1991, 1995). However, analytical studies on protein synthesis in aging

In order to detect the changes of DNA, RNA and protein synthesis of the developing blastocysts in female mouse endometrium during activation of the implantation, ovulations

the changes of DNA synthesis.

distal axis of the joint plate.

**3.2.3 The protein synthesis of the female genital organs** 

administration of 3H-leucine at various ages. **3.2.3.1 The protein synthesis in the uterus** 

mice at various ages were not yet carried out.

**3.2.3.2 The protein synthesis in the implantation** 

3H-thymidine were high (80-95%) at postnatal day 1 and decreased from day 3 to 60 (>10%). The silver grains showing RNA synthesis of the uterus were observed over all the nuclei and cytoplasm of all the cells in the uterine epithelia, stroma and smooth muscles from day 1 to 60. The number of silver grains in the uterine epithelium increased from postnatal day 1 to 7 and decreased from day 14 to 60, while they increased in the stroma from day 1 to 3 and decreased from day 7 to 60.

These results from the female genital organs showed that both DNA and RNA syntheses, as expressed by labeling indices and grain counting, were active in all kinds of cells, such as surface epithelial cells, stromal cells and follicular cells of the ovaries between postnatal days 1 to 7, then they decreased from day 14 to 60. However, the DNA synthesis in the epithelial cells and the stromal cells of both the uteri and the oviducts was active at postnatal day 1 and 3 and decreased from day 7 to 60. The RNA synthesis in the uteri and oviducts was active at postnatal day 1, increased from day 1 to day 14, and decreased from day 30 to 60. The unparalleled alteration of the DNA and RNA syntheses was shown between the ovary and the uterus or oviduct (Li and Nagata 1995).

We also studied PCNA/cyclin immunostaining in the ovary, oviduct and uterus (Li 1994). It was demonstrated that PCNA/cyclin positive cells were observed in the ovarian follicular epithelium, ovarian interstitial cells, tubal epithelial cells, tubal interstitial cells, uterine epithelial cells and uterine interstitial cells. The positive cells increased from postnatal 1 day to 3 and 7 days, then decreased from 14 days to senescence. These results accorded well with the results obtained from the 3H-thymidine radioautography (Li 1994, Li and Nagata 1995). Moreover, the mucosubstance synthesis incorporating sulfuric acid was also carried out (Oliveira et al. 1991, 1995, Li et al. 1992).

### **3.2.2.4 The RNA synthesis in gametogenesis**

The gametogenesis consists of both spermatogenesis in male germ cells and the oogenesis in female germ cells, leading to the implantation and further development of blastcysts. The macromolecular synthesis, DNA, RNA and protein synthesis, in both the testis and the ovary were already described in the sections of male and female reproductive systems (8.1.1. and 8.1.2.) previously.

#### **3.2.2.5 The RNA synthesis in implantation**

In order to detect the changes of DNA, RNA and protein synthesis of the developing blastcysts in mouse endometrium during activation of the implantation, ovulations of female BALB/C strain mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply 3Hthymidine was injected. The three regions of the endometrium, i. e. the interinplantation site, the antimesometrial and mesometrial sides of implantation site, were taken out and processed for LM and EM RAG. It was well known that the uterus of the rodent becomes receptive to blastcyst implantation only for a restricted period. This is called the implantation window which is intercalated between refractory states of the endometrium whose cycling is regulated by ovarian hormones (Yoshinaga 1988). We studied the changes of DNA synthesis by 3H-thymidine (Yamada and Nagata 1992a,b) incorporations in the endometrial cells of pregnant-ovariectomized mice after time-lapse effect of nidatory

3H-thymidine were high (80-95%) at postnatal day 1 and decreased from day 3 to 60 (>10%). The silver grains showing RNA synthesis of the uterus were observed over all the nuclei and cytoplasm of all the cells in the uterine epithelia, stroma and smooth muscles from day 1 to 60. The number of silver grains in the uterine epithelium increased from postnatal day 1 to 7 and decreased from day 14 to 60, while they increased in the stroma from day 1 to 3 and

These results from the female genital organs showed that both DNA and RNA syntheses, as expressed by labeling indices and grain counting, were active in all kinds of cells, such as surface epithelial cells, stromal cells and follicular cells of the ovaries between postnatal days 1 to 7, then they decreased from day 14 to 60. However, the DNA synthesis in the epithelial cells and the stromal cells of both the uteri and the oviducts was active at postnatal day 1 and 3 and decreased from day 7 to 60. The RNA synthesis in the uteri and oviducts was active at postnatal day 1, increased from day 1 to day 14, and decreased from day 30 to 60. The unparalleled alteration of the DNA and RNA syntheses was shown

We also studied PCNA/cyclin immunostaining in the ovary, oviduct and uterus (Li 1994). It was demonstrated that PCNA/cyclin positive cells were observed in the ovarian follicular epithelium, ovarian interstitial cells, tubal epithelial cells, tubal interstitial cells, uterine epithelial cells and uterine interstitial cells. The positive cells increased from postnatal 1 day to 3 and 7 days, then decreased from 14 days to senescence. These results accorded well with the results obtained from the 3H-thymidine radioautography (Li 1994, Li and Nagata 1995). Moreover, the mucosubstance synthesis incorporating sulfuric acid was also carried

The gametogenesis consists of both spermatogenesis in male germ cells and the oogenesis in female germ cells, leading to the implantation and further development of blastcysts. The macromolecular synthesis, DNA, RNA and protein synthesis, in both the testis and the ovary were already described in the sections of male and female reproductive systems (8.1.1.

In order to detect the changes of DNA, RNA and protein synthesis of the developing blastcysts in mouse endometrium during activation of the implantation, ovulations of female BALB/C strain mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply 3Hthymidine was injected. The three regions of the endometrium, i. e. the interinplantation site, the antimesometrial and mesometrial sides of implantation site, were taken out and processed for LM and EM RAG. It was well known that the uterus of the rodent becomes receptive to blastcyst implantation only for a restricted period. This is called the implantation window which is intercalated between refractory states of the endometrium whose cycling is regulated by ovarian hormones (Yoshinaga 1988). We studied the changes of DNA synthesis by 3H-thymidine (Yamada and Nagata 1992a,b) incorporations in the endometrial cells of pregnant-ovariectomized mice after time-lapse effect of nidatory

between the ovary and the uterus or oviduct (Li and Nagata 1995).

out (Oliveira et al. 1991, 1995, Li et al. 1992). **3.2.2.4 The RNA synthesis in gametogenesis** 

**3.2.2.5 The RNA synthesis in implantation** 

and 8.1.2.) previously.

decreased from day 7 to 60.

estradiol. As the results, the endometrial cells showed topographical and chronological differences in the nucleic acid synthesis. The cells labeled with 3H-thymidine increased after nidatory estradiol effects in the stromal cells around the blastocyst, but not in the epithelial cells. The results suggested that the presence of the blastocysts in the uterine lumen induced selective changes in the behavior of endometrial cells after nidatory estradiol effect showing the changes of DNA synthesis.

As for a lower vertebrate, cell proliferation and migration of scleroblasts and their precursor cells during ethisterone-induced anal-fin process formation of the medaka, orizias latipes, was studied by LM RAG labeled with 3H-thymidine (Uwa and Nagata 1976). The results showed that the labeling index in the posterior margin of the joint plate rapidly increased and the scleroblast population in the central portion increased simultaneously from the 3rd to 5th day of ethisterone treatment. These results indicated that the scleroblasts and their precursor cells migrated from the peripheral portion to the central portion along the proxidistal axis of the joint plate.

## **3.2.3 The protein synthesis of the female genital organs**

We studied the protein synthesis of female reproductive organs of aging mice after the administration of 3H-leucine at various ages.

### **3.2.3.1 The protein synthesis in the uterus**

We studied the protein synthesis of the developing blastocysts in female mouse endometrium during activation of the implantation. The ovulations of female BALB/C strain adult mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply. After the mice were injected with 3H-leucine, they were sacrificed and the uteri were processed for LM and EMRAG. We studied the changes of protein synthesis by 3H-leucine incorporations (Yamada 1993, Yamada and Nagata 1992a). As the results, the endometrial cells showed topographical and chronological differences in the protein synthesis. The cells labeled with 3H-leucine were observed in both epithelial cells and stromal cells. Quantitative analysis revealed that the number of silver grains increased from 0 hr to 3 and 6 hr, reaching the peak at 6 hr and decreased from 12 to 18 hr. The protein synthesis in the decidual cells of pregnant mice uteri was compared to the endometrial cells of virgin mice uteri using 3Hproline and 3H-tryptophane incorporations. The results demonstrated that silver grains were localized over the endoplasmic reticulum and the Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix suggesting that the decidual cells produced collagen in the matrix. The collagen synthesis in the mouse decidual cells by 3H-proline showed that silver grains were localized over the endoplasmic reticulum and Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix (Oliveira et al. 1991, 1995). However, analytical studies on protein synthesis in aging mice at various ages were not yet carried out.

#### **3.2.3.2 The protein synthesis in the implantation**

In order to detect the changes of DNA, RNA and protein synthesis of the developing blastocysts in female mouse endometrium during activation of the implantation, ovulations

Macromolecular Synthesis in the Urinary and Reproductive Systems 373

Chen, S., Gao, F., Kotani, A., Nagata, T.: Age-related changes of male mouse submandibular

Clermont Y.: The contractime elements in the limiting membrane of the seminiferous

Cui, H.: Light microscopic radioautographic study on DNA synthesis of nerve cells in the

Cui, H., Gao, F., Nagata, T.: Light microscopic radioautographic study on protein synthesis in perinatal mice corneas. Acta Histochem. Cytochem. 33, 31-37, 2000. Duan, H., Gao, F., Li, S., Hayashi, K., Nagata, T.: Aging changes and fine structure and

Duan, H., Gao, F., Li, S., Nagata, T.: Postnatal development of esophageal epithelium in

Duan, H., Gao, F., Oguchi, K., Nagata, T.: Light and electron microscopic radioautographic

Feulgen, R., Rossenbeck, H.: Mikroskopische-chemischer Nachweis einer Nukeinsaeure von Thymus der Thymonukeinsaeure Z. Physik. Chem. 135, 203-248, 1924. Fujii, Y., Ohno, S., Yamabayashi, S., Usuda, N., Saito, H., Furuta, S., Nagata, T.: Electron

Gao, F.: Study on the macromolecular synthesis in aging mouse seminiferous tubules by light and electron microscopic radioautography. Cell. Mol. Biol. 39, 659-672, 1993. Gao, F., Toriyama, K., Nagata, T.: Light microscopic radioautographic study on the DNA

Gao, F., Li, S., Duan, H., Ma, H., Nagata, T.: Electron microscopic radioautography on the

Gao, F., Toriyama, K., Ma, H., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in aging mice corneas. Cell. Mol. Biol. 39, 435-441, 1993. Gao, F., Ma, H., Sun, L., Jin, C., Nagata, T.: Electron microscopic radioautographic study on

Gao, F., Chen, S., Sun, L., Kang, W., Wang, Z., Nagata, T.: Radioautographic study of the

Gao, F., Jin, C., Ma, H., Sun, L., Nagata, T.: Ultrastructural and radioautographic studies on

Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick

IgG myeloma patient. J. Clin. Electr. Microsc. 13, 582-583, 1980.

Clermont, Y.: Renewal of spermatogonia in man. Amer. J. Anat. 112, 35-51, 1963.

cerebella of aging mice. Cell. Mol. Biol. 41, 1139-1154, 1995.

tubules of rats. Exp. Cell Res. 15, 438-342, 1958.

Electron Microsc. 25, 452-453, 1992.

nebulizer. Drug Res. 44, 880-883, 1994.

injection. Cell. Mol. Biol. 38, 661-668, 1992a.

Microsc. 27, 360-362, 1994.

145-150, 1995a.

1995b.

injection. J. Clin. Electron Microsc. 25, 721-722, 1992b.

39, 309-316, 1993.

gland: A morphometric and radioautographic study. Cell. Mol. Biol. 41, 117-124,

DNA synthesis of esophageal epithelium in neonatal, adult and old mice. J. Clin.

mouse: a light and electron microscopic radioautographic study. Cell. Mol. Biol.

study on the incorporation of 3H-thymidine into the lung by means of a new

microscopic radioautography of DNA synthesis in primary cultured cells from an

synthesis of prenatal and postnatal aging mouse retina after labeled thymidine

DNA synthesis of prenatal and postnatal mice retina after labeled thymidine

the nucleic acids and protein synthesis in the aging mouse testis. Med. Electron

macromolecular synthesis of Leydig cells in aging mice testis. Cell. Mol. Biol. 41,

DNA synthesis in Leydig cells of aging mouse testis. Cell. Mol. Biol. 41, 151-160,

embryo. I. Light microscopic radioautography. Shinshu Med. J. 32, 231-240, 1984a.

**4. References** 

1995.

of female BALB/C strain mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply and 3Hleucine was injected. The three regions of the endometrium, i. e. the interinplantation site, the antimesometrial and mesometrial sides of implantation site, were taken out and processed for LM and EM RAG. It was well known that the uterus of the rodent becomes receptive to blastocyst implantation only for a restricted period. This is called the implantation window which is intercalated between refractory states of the endometrium whose cycling is regulated by ovarian hormones (Yoshinaga 1988). We studied the changes of protein synthesis by 3H-leucine (Yamada 1993, Yamada and Nagata 1992a) incorporations in the endometrial cells of pregnant-ovariectomized mice after time-lapse effect of nidatory estradiol. As the results, the endometrial cells showed topographical and chronological differences in the nucleic acid and protein synthesis. The cells labeled with 3H-leucine were observed in both epithelial cells and stromal cells. Quantitative analysis revealed that the number of silver grains as expressed by grain counting per mm2 in both the stromal and epithelial cells on the antimesometrial side with 3H-leucine increased from 0 hr to 3 and 6 hr, reaching the peak at 6 hr and decreased from 12 to 18 hr. These results suggested that the presence of the blastocysts in the uterine lumen induced selective changes in the behavior of endometrial cells after nidatory estradiol effect showing the changes of DNA, RNA and protein synthesis. The time coincident peak of RNA and protein synthesis detected in the endometrial cells at the anti-mesometrial side of the implantation site, probably reflected the activation moment of the implantation window. The protein synthesis in the decidual cells of pregnant mice uteri was compared to the endometrial cells of virgin mice uteri using 3Hproline and 3H-tryptophane incorporations. The results demonstrated that silver grains were localized over the endoplasmic reticulum and the Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix suggesting that the decidual cells produced collagen in the matrix. On the other hand, collagen synthesis in the mouse decidual cells was studied by LM and EM RAG using 3H-proline (Oliveira et al. 1991, 1995). Silver grains were localized over the endoplasmic reticulum and Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix. The results suggested that the decidual cells produced collagen into the matrix. The quantitative analysis showed that both incorporations in the decidual cells and the matrix increased in the pregnant mice than the endometrial cells in virgin mice.

#### **3.2.4 The glucide synthesis in the reproductive system**

Among the reproductive organs, only the mucosubstance synthesis with radiosulfate, 35SO4, was studied in the ovaries of mice during the estrus cycle.

#### **3.2.4.1 The glucide synthesis in the ovary**

Litter mate groups of female ddY mice, aged 8-10 weeks, were divided into 4 groups, diestrus, proestrus, estrus and metestrus according to the vaginal smears. The ovaries were taken out, labeled with 35SO4 in vitro and radioautographed. In all the animals, silver grains were localized over the granulosa and theca cells. Almost all compartments of the ovaries were labeled. The grain counts per cell changed according to cell cycle. From the results, it was concluded that all the cells of the ovary incorporated mucosubstances throughout the estrus cycle (Li et al. 1992).

## **4. References**

372 Senescence

of female BALB/C strain mice were controlled by pregnant mare serum gonadotropin and human chorionic gonadotropin, then pregnant female mice were ovariectomized on the 4th day of pregnancy (Yamada 1993, Yamada and Nagata 1992a,b, 1993). The delay implantation state was maintained for 48 hrs and after 0 to 18 hrs of estrogen supply and 3Hleucine was injected. The three regions of the endometrium, i. e. the interinplantation site, the antimesometrial and mesometrial sides of implantation site, were taken out and processed for LM and EM RAG. It was well known that the uterus of the rodent becomes receptive to blastocyst implantation only for a restricted period. This is called the implantation window which is intercalated between refractory states of the endometrium whose cycling is regulated by ovarian hormones (Yoshinaga 1988). We studied the changes of protein synthesis by 3H-leucine (Yamada 1993, Yamada and Nagata 1992a) incorporations in the endometrial cells of pregnant-ovariectomized mice after time-lapse effect of nidatory estradiol. As the results, the endometrial cells showed topographical and chronological differences in the nucleic acid and protein synthesis. The cells labeled with 3H-leucine were observed in both epithelial cells and stromal cells. Quantitative analysis revealed that the number of silver grains as expressed by grain counting per mm2 in both the stromal and epithelial cells on the antimesometrial side with 3H-leucine increased from 0 hr to 3 and 6 hr, reaching the peak at 6 hr and decreased from 12 to 18 hr. These results suggested that the presence of the blastocysts in the uterine lumen induced selective changes in the behavior of endometrial cells after nidatory estradiol effect showing the changes of DNA, RNA and protein synthesis. The time coincident peak of RNA and protein synthesis detected in the endometrial cells at the anti-mesometrial side of the implantation site, probably reflected the activation moment of the implantation window. The protein synthesis in the decidual cells of pregnant mice uteri was compared to the endometrial cells of virgin mice uteri using 3Hproline and 3H-tryptophane incorporations. The results demonstrated that silver grains were localized over the endoplasmic reticulum and the Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix suggesting that the decidual cells produced collagen in the matrix. On the other hand, collagen synthesis in the mouse decidual cells was studied by LM and EM RAG using 3H-proline (Oliveira et al. 1991, 1995). Silver grains were localized over the endoplasmic reticulum and Golgi apparatus of fibroblasts and accumulated over collagen fibrils in the extracellular matrix. The results suggested that the decidual cells produced collagen into the matrix. The quantitative analysis showed that both incorporations in the decidual cells and the matrix increased in

the pregnant mice than the endometrial cells in virgin mice.

**3.2.4 The glucide synthesis in the reproductive system** 

was studied in the ovaries of mice during the estrus cycle.

**3.2.4.1 The glucide synthesis in the ovary** 

estrus cycle (Li et al. 1992).

Among the reproductive organs, only the mucosubstance synthesis with radiosulfate, 35SO4,

Litter mate groups of female ddY mice, aged 8-10 weeks, were divided into 4 groups, diestrus, proestrus, estrus and metestrus according to the vaginal smears. The ovaries were taken out, labeled with 35SO4 in vitro and radioautographed. In all the animals, silver grains were localized over the granulosa and theca cells. Almost all compartments of the ovaries were labeled. The grain counts per cell changed according to cell cycle. From the results, it was concluded that all the cells of the ovary incorporated mucosubstances throughout the


Macromolecular Synthesis in the Urinary and Reproductive Systems 375

Joukura, K., Usuda, N., Nagata, T.: Quantitative study on the aging change of

Komiyama, K., Iida, F., Furihara, R., Murata, F., Nagata, T.: Electron microscopic

Kong, Y.: Electron microscopic radioautographic study on DNA synthesis in perinatal

Kong, Y., Nagata, T.: Electron microscopic radioautographic study on nucleic acid synthesis

Kong, Y., Usuda, N., Nagata, T.: Radioautographic study on DNA synthesis of the retina

Kong, Y., Usuda, N., Morita, T., Hanai, T., Nagata, T.: Study on RNA synthesis in the retina

Leblond, C. P.: Localization of newly administered iodine in the thyroid gland as indicated

Leblond, C. P.: The life history of cells in renewing systems. Am. J. Anat. 160, 113-158, 1981. Leblond, C. P., Messier, B.: Renewal of chief cells and goblet cells in the small intestine as

Li, S.: Relationship between cellular DNA synthesis, PCNA expression and sex steroid

Li, S., Nagata, T.: Nucleic acid synthesis in the developing mouse ovary, uterus and oviduct

Li, S., Gao, F., Duan, H., Nagata, T.: Radioautographic study on the uptake of 35SO4 in mouse ovary during the estrus cycle. J. Clin. Electron Microsc. 25, 709-710, 1992. Liang, Y.: Light microscopic radioautographic study on RNA synthesis in the adrenal glands

Liang, Y., Ito, M., Nagata, T.: Light and electron microscopic radioautographic studies on

Ma, H.: Light microscopic radioautographic study on DNA synthesis of the livers in aging

Ma, H., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of the

Ma, H., Nagata, T.: Studies on DNA synthesis of aging mice by means of electron microscopic radioautography. J. Clin. Electron Microsc. 21, 715-716, 1988b. Ma, H., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis in the

livers in aging mice. J. Clin. Electron Microsc. 21, 335-343, 1988a.

of aging mice. Acta Histochem. Cytochem. 31, 203-210, 1998.

of perinatal mouse retina. Med. Electron Microsc. 27, 366-368, 1994.

Histochem. Cytochem. 42, 982-982, 1994.

mouse retina. Cell. Mol. Biol. 39, 55-64, 1993.

Electron Microsc. 11, 428-429, 1978.

263-272, 1992a.

132: 247-259. 1958.

195, 1995.

1999.

1990a.

Mol. Biol. 38, 669-678, 1992b.

by radioiodine. J. Anat. 77, 149-152, 1943.

Histochemistry 102, 405-413, 1994.

mice. Acta Anat. Nippon. 63, 137-147, 1988.

glycoconjugates synthesis in aging mouse kidney. Proc. Xth Internat. Cong. Histochem. Cytochem., Acta Histochem. Cytochem. 29, Suppl. 507-508, 1996. Kobayashi, K., Nagata, T.: Light microscopic radioautographic studies on DNA, RNA and

protein syntheses in human synovial membranes of rheumatoid arthritis patients. J.

radioautographic study on 125I-albumin in rat gastric mucosal epithelia. J. Clin.

and retinal pigment epithelium of developing mouse embryo. Cell. Mol. Biol. 38,

and retinal pigment epithelium of mice by light microscopic radioautography. Cell.

shown by radioautography after injection of thymidine-3H into mice. Anat. Rec.

hormone receptor status in the developing mouse ovary, uterus and oviduct.

studied by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 185-

RNA synthesis in aging mouse adrenal gland. Acta Anat. Nippon. 74, 291-300,

hepatocytes of aging mice as observed by image analysis. Cell. Mol. Biol. 36, 73-84,


Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick

Gunarso, W., Gao, F., Cui, H., Ma, H., Nagata, T.: A light and electron microscopic

Gunarso, W., Gao, F., Nagata, T.: Development and DNA synthesis in the retina of chick

Hanai, T.: Light microscopic radioautographic study of DNA synthesis in the kidneys of

Hanai, T., Nagata, T.: Electron microscopic radioautographic study on DNA and RNA

Hanai, T., Nagata, T.: Study on the nucleic acid synthesis in the aging mouse kidney by

Nagata, T., Ed., pp. 209-214, Shinshu University Press, Matsumoto, 1994b. Hanai, T., Nagata, T.: Electron microscopic study on nucleic acid synthesis in perinatal

Hanai, T., Usuda, N., Morita, T., Shimizu, T., Nagata, T.: Proliferative activity in the kidneys

Hayashi, K., Gao, F., Nagata, T.: Radioautographic study on 3H-thymidine incorporation at

Ito, M.: The radioautographic studies on aging change of DNA synthesis and the

Ito, M., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis and

Izumiyama, K., Kogure, K., Kataoka, S., Nagata, T.: Quantitative analysis of glucose after

Jamieson, J. D., Palade, G. E.: Intracellular transport of secretory proteins in the pancreatic

Jin, C.: Study on DNA synthesis of aging mouse colon by light and electron microscopic

Jin, C., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. J. Histochem. Cytochem. 43, 1223-1228, 1995a. Jin, C., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. Med. Electron Microsc. 28, 71-75, 1995b. Joukura, K.: The aging changes of glycoconjugate synthesis in mouse kidney studied by 3Hglucosamine radioautography. Acta Histochem. Cytochem. 29, 57-63, 1996. Joukura, K., Nagata, T.: Aging changes of 3H-glucosamine incorporation into mouse kidney observed by radioautography. Acta Histochem. Cytochem. 28, 494-494, 1995.

radioautography. Brain Res. 416, 175-179, 1987.

radioautography. Cell. Mol. Biol. 42, 255-268, 1996.

exocrine cells. J. Cell Biol. 34, 577-615, 1967.

Ed., pp. 127-131, Shinshu University Press, Matsumoto, 1994a.

mouse kidney tissue. Med. Electron Microsc. 27, 355-357, 1994c.

1984b.

Histochem. 98, 309-32, 1996.

aging mice. Cell. Mol. Biol. 39, 81-91, 1993.

Biol. 43, 189-201, 1997.

39, 181-191, 1993.

1993.

1996.

1996.

embryo. II. Electron microscopic radioautography. Shinshu Med. J. 32, 241-248,

radioautographic study on RNA synthesis in the retina of chick embryo. Acta

embryo observed by light and electron microscopic radioautography. Cell. Mol.

synthesis in perinatal mouse kidney. In, Radioautography in Medicine, Nagata, T.,

light and electron microscopic radioautography. In, Radioautography in Medicine,

of aging mice evaluated by PCNA/cyclin immunohistochemistry. Cell. Mol. Biol.

different stages of muscle development in aging mice. Cell. Mol. Biol. 39, 553-560,

ultrastructural development of mouse adrenal gland. Cell. Mol. Biol. 42, 279-292,

ultrastructure of aging mouse adrenal gland. Med. Electron Microsc. 29, 145-152,

transient ischemia in the gerbil hippocampus by light and electron microscope


Macromolecular Synthesis in the Urinary and Reproductive Systems 377

Nagata, T.: A radioautographic study of the DNA synthesis in rat liver, with special

Nagata, T.: A quantitative study on the ganglion cells in the small intestine of the dog. Med.

Nagata, T.: A radioautographic study on the RNA synthesis in the hepatic and the intestinal

Nagata, T.: On the increase of binucleate cells in the ganglion cells of dog small intestine due to experimental ischemia. Med. J. Shinshu Univ. 12, 93-113, 1967a. Nagata, T.: A radioautographic study on the protein synthesis in the hepatic and the

Nagata, T.: Chapter 3. Application of microspectrophotometry to various substances. In ,

Nagata, T.: Electron microscopic dry-mounting autoradiography. Proc. 4th Internat. Cong.

Nagata, T.: Electron microscopic radioautography of intramitochondrial RNA synthesis of

Nagata, T.: Quantitative electron microscope radioautography of intramitochondrial nucleic

Nagata, T.: Electron microscopic observation of target cells previously observed by phase-

Nagata, T.:. Electron microscopic radioautography and analytical electron microscopy. J.

Nagata, T.: Radiolabeling of soluble and insoluble compounds as demonstrated by light and

Nagata, T.: Quantitative analysis of histochemical reactions: Image analysis of light and

Nagata, T. Quantitative light and electron microscopic radioautographic studies on

Nagata, T.: Electron microscopic radioautography with cryo-fixation and dry-mounting

Nagata, T.: Application of electron microscopic radioautography to clinical electron

Nagata, T.: Radioautography in Medicine. Shinshu University Press, 268pp, Matsumoto,

Nagata, T.: Radioautography, general and special. In, Histo- and Cyto-chemistry 1994,

Japan Society of Histochemistry and Cytochemistry, ed, pp. 219-231, Gakusai

irradiated cultured cells. J. Clin. Electron Microsc. 17, 589-590, 1984. Nagata, T.: Principles and techniques of radioautography. In, Histo- and Cyto-chemistry

epithelial cells of mice after feeding with special reference to binuclearity. Med. J.

intestinal epithelial cells of mice, with special reference to binucleate cells. Med. J.

Introduction to Microspectrophotometry. Isaka, S., Nagata, T., Inui, N., Eds.,

contrast microscopy: Electron microscopic radioautography of laser beam

1985, Japan Society of Histochemistry and Cytochemistry, Ed., Gakusai Kikaku

electron microscopy. Recent Advances in Cellular and Molecular Biology, Wegmann, R. J., Wegmann, M. A., Eds. Peters Press, Leuven, Vol. 6, pp. 9-21, 1992.

electron microscopic radioautograms. Acta Histochem. Cytochem. 26, 281-291,

macromolecular synthesis in several organs of prenatal and postnatal aging mice.

reference to binucleate cells. Med. J. Shinshu Univ. 7, 17-25, 1962.

J. Shinshu Univ. 10, 1-11, 1965.

Shinshu Univ. 11, 49-61, 1966.

Shinshu Univ. 12, 247-257, 1967b.

Co., Tokyo, pp. 207-226, 1985.

1993a.

1994c.

Kikaku Co., Tokyo, 1994d.

Clin. Electron Microsc. 24, 441-442, 1991.

Chinese J. Histochem. Cytochem. 2: 106-108, 1993b.

procedure. Acta Histochem. Cytochem. 27: 471-489, 1994a.

microscopy. Med. Electron Microsc. 27; 191-212, 1994b.

Olympus Co., Tokyo, pp. 49-155, 1972a.

Histochem. Cytochem. Kyoto, pp. 43-44, 1972b.

HeLa cells in culture. Histochemie 32, 163-170, 1972c.

acid synthesis. Acta Histochem. Cytochem. 5, 201-203, 1972d.


Ma, H., Nagata, T.: Study on RNA synthesis in the livers of aging mice by means of electron

Ma, H., Nagata, T.: Collagen and protein synthesis in the livers of aging mice as studied by

Ma, H., Gao, F., Olea, M. T., Nagata, T.: Protein synthesis in the livers of aging mice studied by electron microscopic radioautography. Cell. Mol. Biol. 37, 607-615, 1991. Matsumura, H., Kobayashi, Y., Kobayashi, K., Nagata, T.: Light microscopic

Momose, Y., Nagata, T.: Radioautographic study on the intracellular localization of a

Momose, Y., Naito, J., Nagata, T.: Radioautographic study on the localization of an antiallergic agent, tranilast, in the rat liver. Cell. Mol. Biol. 35, 347-355, 1989. Momose, Y., Shibata, N., Kiyosawa, I., Naito, J., Watanabe, T., Horie, S., Yamada, J., Suga,

Momose, Y., Naito, J., Suzawa, H., Kanzawa, M., Nagata, T.: Radioautographic study on the

Morita, T.: Radioautographic study on the aging change of 3H-glucosamine uptake in mouse

Morita, T., Usuda, N. Hanai, T., Nagata, T.: Changes of colon epithelium proliferation due to

Murata, F., Momose,Y. , Yoshida, K., Nagata, T.: Incorporation of 3H-thymidine into the nucleus of mast cells in adult rat peritoneum. Shinshu Med. J. 25, 72-77, 1977a. Murata, F., Momose, Y., Yoshida, K., Ohno, S., Nagata, T.: Nucleic acid and mucosubstance

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Ultrastructural and electron microscopic

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Mucosubstances of rabbit granulocytes studied

Nagata, T.: On the relationship between cell division and cytochrome oxidase in the

Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. I. Observation on the

Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. II. Phase-contrast

Nagata, T.: Cell divisions in the liver of the fetal and newborn dogs. Med. J. Shinshu Univ. 4:

thymidine radioautography. Histochemistry 101, 13-20, 1994.

radioautography. Acta Pharmacol. Toxicol. 41, 58-59, 1977b.

Yoshida sarcoma cells. Shinshu Med. J. 5: 383-386, 1956.

radioautographic study of DNA synthesis in the lung of aging salamander,

hypolipidemic agent, bezafibrate, a peroxisome proliferator, in cultured rat

T., Nagata, T.: Morphometric evaluation of species differences in the effects of bezafibrate, a hypolipidemic agent, on hepatic peroxisomes and mitochondria. J.

intracellular localization of bezafibrate in cultured rat hepatoctyes. Acta Histochem.

individual aging with PCNA/cyclin immunostaining comparing with 3H-

metabolism of mastocytoma cells by means of electron microscopic

radioautographic studies on the mastocytoma cells and mast cells. J. Clin. Electron

by means of electron microscopic radioautography and X-ray microanalysis.

smear preparation under normal conditions. Med. J. Shinshu Univ. 2: 187-198,

microscopic observations under normal conditions. Med. J. Shinshu Univ. 2: 199-

microscopic radioautography. Cell. Mol. Biol. 36, 589-600, 1990b.

electron microsopic radioautography. Ann. Microsc. 1, 13-22, 2000.

Hynobius nebulosus. J. Histochem. Cytochem. 42, 1004-1004, 1994.

hepatocytes. Cell. Mol. Biol. 39, 773-781, 1993a.

Toxicol. Pathol. 6, 33-45, 1993b.

ileum. Cell. Mol. Biol. 39, 875-884, 1993.

Cytochem. 28, 61-66, 1995.

Microsc. 11, 561-562, 1978.

1957a.

207, 1957b.

65-73, 1959.

Histochemistry 61, 139-150, 1979.


Macromolecular Synthesis in the Urinary and Reproductive Systems 379

Nagata, T.: Three-dimensional observations on thick biological specimens by high voltage

Nagata, T.: Biological microanalysis of radiolabeled and unlabeled compounds by

Nagata, T.: Electron microscopic radioautographic study on protein synthesis in pancreatic cells of perinatal and aging mice. Bull. Nagano Women's Jr. College 8, 1-22, 2000c. Nagata, T.: Light microscopic radioautographic study on radiosulfate incorporation into the tracheal cartilage in aging mice. Acta Histochem. Cytochem. 32, 377-383, 2000d. Nagata, T.: Introductory remarks: Special radioautographology. Cell. Mol. Biol. 46

Nagata, T.: Three-dimensional high voltage electron microscopy of thick biological

Nagata, T.: Three-dimensional and four-dimensional observation of histochemical and

Nagata, T. : Special cytochemistry in cell biology. In, Internat. Rev. Cytol. Jeon, K.W., ed.,

Nagata, T. : Radioautographology General and Special, In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 37 No. 2, Urban & Fischer, Jena, pp. 57-226, 2002. Nagata T.: Light and electron microscopic study on macromolecular synthesis in amitotic hepatocyte mitochondria of aging mice. Cell. Mol. Biol. 49, 591-611, 2003. Nagata, T.: X-ray microanalysis of biological specimens by high voltage electron

Nagata T.: Aging changes of macromolecular synthesis in the uro-genital organs as revealed by electron microscopic radioautography. Ann. Rev. Biomed. Sci. 6, 13-78, 2005. Nagata T.: Electron microscopic radioautographic study on protein synthesis in hepatocyte

Nagata T.: Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata, T.: Electron microscopic radioautographic study on macromolecular synthesis in hepatocyte mitochondria of aging mouse. J. Cell Tissue Res. 7, 1019-1029, 2007c. Nagata, T.; Electron microscopic radioautographic study on nucleic acids synthesis in

mitochondria of developing mice. Ann. Microsc. 6, 43-54, 2006a.

cytochemical specimens by high voltage electron microscopy. Acta Histochem.

microscopy. In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 39, No. 4,

hepatocyte mitochondria of developing mice. The Sci. World J. 6: 1583-1598, 2006b.

by electron microscopic radioautography. I: Nucleic acid synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. Eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 245-

by electron microscopic radioautography. II: Protein synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 259-271,

hepatocyte mitochondria of developing mice. Trends Cell Molec. Biol. 2, 19-33,

radioautography and X-ray microanalysis. Scanning Microscopy International, 14,

electron microscopy. Image Analysis Stereolog. 19, 51-56, 2000a.

Nagata, T.: Special radioautographology: the eye. J. Kaken Eye Res. 18, 1-13, 2000f.

Vol. 211, Chapter 2, Academic Press, New York, pp. 33-154, 2001c.

on line, 2000b.

258, 2007a.

2007b.

2007d.

(Congress Suppl.), 161-161, 2000e.

Cytochem. 34, 153-169, 2001b.

specimens. Micron 32, 387-404, 2001a.

Urban & Fischer Verlag, Jena, pp. 185-320, 2004.


Nagata, T., Application of electron microscopic radioautography to clinical electron

Nagata, T.: Light and electron microscopic radioautographic study on macromolecular synthesis in digestive organs of aging mice. Cell. Mol. Biol. 41, 21-38, 1995a. Nagata, T.: Histochemistry of the organs: Application of histochemistry to anatomy. Acta

Nagata, T.: Three-dimensional observation of whole mount cultured cells stained with

Nagata, T.: Morphometry in anatomy: image analysis on fine structure and histochemical

Nagata, T.: Technique and application of electron microscopic radioautography. J. Electron

Nagata, T.: Techniques of light and electron microscopic radioautography. In,

Nagata, T.: On the terminology of radioautography vs. autoradiography. J. Histochem.

Nagata, T.: Techniques and applications of microscopic radioautography. Histol.

Nagata T.: Three-dimensional observation on whole mount cultured cells and thick sections

Nagata, T.: Radioautographic study on collagen synthesis in the ocular tissues. J. Kaken Eye

Nagata, T.: Techniques of radioautography for medical and biological research. Braz. J. Biol.

Nagata, T.: Radioautographology, the advocacy of a new concept. Braz. J. Biol. Med. Res. 31,

Nagata, T.: Radioautographic studies on DNA synthesis of the bone and skin of aging

Nagata, T.: 3D observation of cell organelles by high voltage electron microscopy.

Nagata, T.: Application of histochemistry to anatomy: Histochemistry of the organs, a novel

Nagata, T.: Aging changes of macromolecular synthesis in various organ systems as

Nagata, T.: Radioautographology, general and special: a novel concept. Ital. J. Anat.

concept. Proc. XV Congress of the International Federation of Associations of

observed by microscopic radioautography after incorporation of radiolabeled

salamander. Bull. Nagano Women's Jr. College 6, 1-14, 1998c.

Microscopy and Analysis, Asia Pacific Edition, 9, 29-32, 1999a.

Anatomists, Ital. J. Anat. Embryol. 104 (Suppl. 1), 486-486, 1999b.

precursors. Methods Find. Exp. Clin. Pharmacol. 21, 683-706, 1999c. Nagata, T.: Radioautographic study on protein synthesis in mouse cornea. J. Kaken Eye Res.

stained with histochemical reactions by high voltage electron microscopy. In, Recent Advances in Microscopy of Cells, Tissues and Organs, Motta, P., Ed.,

Cytochem. Acta Histochem. Cytochem. 29 (Suppl.), 343-344, 1996b. Nagata, T.: Remarks: Radioautographology, general and special. Cell. Mol. Biol. 42 (Suppl.),

histochemical reactions by ultrahigh voltage electron microscopy. Cell. Mol. Biol.

reactions with special reference to radioautography. Ital. J. Anat. 100 (Suppl. 1),

Histochemistry and Cytochemistry 1996. Proc. Xth Internat. Congr. Histochem.

microscopy. Med. Electron Microsc. 27, 191-212, 1994e.

Anat. Nippon. 70, 448-471, 1995b.

41, 783-792, 1995c.

591-605, 1995d.

11-12, 1996c.

Res. 15, 1-9, 1997c.

201-241, 1998b.

8, 8-14, 1999d.

Embryol. 104 (Suppl. 1), 487-487, 1999e.

Med. Res. 31, 185-195, 1998a.

Microsc. 45, 258-274, 1996a.

Cytochem. 44, 1209-1209, 1996d.

Histopathol. 12, 1091-1124, 1997a.

Antonio Delfino Editore, Roma, pp. 37-44, 1997b.


Macromolecular Synthesis in the Urinary and Reproductive Systems 381

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata, T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA, RNA and

Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 12, 1-29, 2010h. Nagata, T., Cui, H., Gao, F.: Radioautographic study on glycoprotein synthesis in the ocular

Nagata, T., Cui, H., Kong, Y.: The localization of TGF-b1 and its mRNA in the spinal cords

Nagata, T., Cui, H., Liang, Y.: Light microscopic radioautographic study on the protein

Nagata, T., Fujii, Y., Usuda, N.: Demonstration of extranuclear nucleic acid synthesis in

Nagata, T., Ito, M., Chen, S.: Aging changes of DNA synthesis in the submandibular glands

Nagata, T. Ito, M., Liang, Y.: Study of the effects of aging on macromolecular synthesis in

Nagata, T., Iwadare, I., Murata, F.: Electron microscopic radioautography of nucleic acid

Nagata, T., Kawahara, I.: Radioautographic study of the synthesis of sulfomucin in digestive organs of mice. J. Trace Microprobe Analysis 17, 339-355, 1999. Nagata, T., Kawahara, I., Usuda, N., Maruyama, M., Ma, H.: Radioautographic studies on

Nagata, T., Kong, Y.: Distribution and localization of TGFb1 and bFGF, and their mRNAs in

aging mice. Bull. Nagano Women's Jr. College 6, 87-105, 1998.

Alternat Med Welfare 5, 25-37, 2010d.

Vol. 3, Formatex, Badajoz, Spain, in press, 2010g.

tissues. J. Kaken Eye Res. 13, 11-18, 1995.

Med. Welfare 5, 38-52, 2010f.

232, 2010e.

41-60 (2001).

1966.

Microsc. 1, 4-12, 2000a.

Toxicol. 41, 64-65, 1977c.

Exp. Clin. Pharmacol. 22, 5-18, 2000b.

Professional Postgrad. Service, Tokyo, 1988a.

mitochondria in adrenal medullary cells of aging mice. Bulletin Shinshu Inst

adrenal cortical and medullary cells of aging mice. J. Biomed. Sci. Enginer. 4, 219-

mitochondria in adrenal cortical cells of aging mice. Bulletin Shinshu Inst. Alternat.

protein synthesis in adrenal cells of aging mice. Formatex Microscopy Series No. 3,

of prenatal and postnatal aging mice demonstrated with immunohistochemical and in situ hybridization techniques. Bull. Nagano Women's Jr. College, 7, 75-88, 1999a.

synthesis in the cerebellum of aging mouse. Bull. Nagano Women's Jr. College, 9,

mammalian cells under experimental conditions by electron microscopic radioautography. Proc. 10th Internat. Congr. Electr. Microsc. 2, 305-306, 1982b. Nagata, T., Hirano, I., Shibata, O., Nagata, T.: A radioautographic study on the DNA

synthesis in the hepatic and the pancreatic acinar cells of mice during the postnatal growth, with special reference to binuclearity. Med. J. Shinshu Univ. 11, 35-42,

of mice as observed by light and electron microscopic radioautography. Ann.

mouse steroid secreting cells using microscopic radioautography. Methods Find.

synthesis in cultured cells treated with several carcinogens. Acta Pharmacol.

the glycoconjugate synthesis in the gastrointestinal mucosa of the mouse. In, Glycoconjugate in Medicine, Ohyama, M., Muramatsu, T., Eds, pp. 251-256,


Nagata, T.; Aging changes of macromolecular synthesis in the mitochondria of mouse

Nagata, T.: Sexual difference between the macromolecular synthesis of hepatocyte

Nagata, T.: Protein synthesis in hepatocytes of mice as revealed by electron microscopic

Nagata, T.: Electron microscopic radioatuographic studies on macromolecular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata, T.: Applications of high voltage electron microscopy to thick biological specimens.

Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of mitochondria in adrenal medullary cells of aging mice. Open Anat. J. 1, 14-24, 2009g. Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 11, 1-17, 2009h. Nagata, T.: Electron microscopic radioautographic studies on macromoleclular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata T. Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Macromolecular synthesis in the livers of aging mice as revealed by electron

adrenocortical cells of aging mice. Open Anat J. 2, 91-97, 2010a.

Diego, St. Louis, Vol. 45, No. 1, pp. 1-80, 2010c.

Eds., Nova Biomed. Books, New York, pp. 133-161, 2009b.

Radiopharmaceutics 2, 118-128, 2009d.

Ann. Microsc. 9, 4-40, 2009f.

Welfare 4, 15-38, 2009i.

Welfare 4, 51-66, 2009j.

2222, 2010b.

Nagata, T.: Radioautographology, Bull. Shinshu Institute Alternat. Med. 2, 3-32, 2007f. Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in adrenal cortical cells of developing mice. J. Cell. Tis. Res. 8, 1303-1312, 2008a. Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in

9, 30-36, 2007e.

2008b.

2009a

1802, 2009e.

hepatocytes as revealed by microscopic radioautography. Ann. Rev. Biomed. Sci.

adrenal cortical cells of developing and aging mice. The Sci. World J. 8, 683-697.

mitochondria in male and female mice in aging as revealed by electron microscopic radioautography. Chapter 22. In, Women and Aging: New Research, H. T. Bennninghouse, A. D. Rosset, Eds. Nova Biomed. Books, New York, pp. 461-487,

radioautography. In, Protein Biosynthesis. Esterhouse, T. E. and Petrinos, L. B.,

mitochondria of various cells. 18EMSM Conference Proc. 9th Asia-Pacific Microscopy Conference (APMC9), Kuala Lumpur, Malaysia, pp. 48-50, 2009c. Nagata, T.: Recent studies on macromolecular synthesis labeled with 3H-thymidine in

various organs as revealed by electron microscopic radioautography. Current

in adrenal medullary cells of developing and aging mice. J. Cell Tissue Res. 9, 1793-

mitochondria of some organs in aging animals. Bull. Shinshu Inst. Alternat. Med.

in adreno-cortical cells of aging ddY mice. Bull. Shinshu Inst. Alternat. Med.

adrenal medullary cells of aging and senescent mice. J Cell Tissue Res. 10, 2213-

microscopic radioautography. In, Prog. Histochem. Cytochem., Sasse, D., Ed., Elsevier, Amsterdam, Boston, London, New York, Oxford, Paris, Philadelphia, San


Macromolecular Synthesis in the Urinary and Reproductive Systems 383

Nagata, T., Steggerda, F. R.: Histological study on the deganglionated small intestine of the

Nagata, T., Steggerda, F. R.: Observations on the increase of binucleate cells in the ganglion

Nagata, T., Toriyama, K., Kong, Y., Jin, C., Gao, F.: Radioautographic study on DNA synthesis in the ciliary bodies of aging mice. J. Kaken Eye Res.12, 1-11, 1994. Nagata, T., Usuda, N.: Image processing of electron microscopic radioautograms in clinical

Nagata, T., Usuda, N.: Studies on the nucleic acid synthesis in pancreatic acinar cells of

Nagata, T., Usuda, N.: Electron microscopic radioautography of protein synthesis in

Nagata, T., Usuda, N.: In situ hybridization by electron microscopy using radioactive

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of nucleic acid

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of lipid synthesis in pancreatic cells of aging mice. J. Clin. Electr. Microsc. 23, 841-842, 1990. Nagata, T., Usuda, N., Maruyama, M., Ma, H.: Electron microscopic radioautographic study

Nagata, T., Usuda, N., Suzawa, H., Kanzawa, M.: Incorporation of 3H-glucosamine into the

Nagata, T., Yamabayashi, S.: Intracellular localization of 3H-befunolol by means of electron

Nagata, T., Yoshida, K., Murata, F.: Demonstration of hot and cold mercury in the human

Nagata, T., Yoshida, K., Ohno, S., Murata, F.: Ultrastructural localization of soluble and

Nishigaki, T., Momose, Y., Nagata, T.: Light microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Electron microscopic radioautographic study of the

radioautography. J. Clin. Electron Microsc. 25, 646-647, 1992.

electron microscopy. J. Clin. Electron. Microsc. 18, 451-452, 1985.

probes. J. Histochem. Cytochem. 41, 1119-1119, 1993b.

Intern. Cong. Electr. Microsc. 3, 2281-2282, 1984.

153-158, 1960b.

1964.

1993a.

1988b.

1978b.

536, 1987.

65-71, 1990a.

dog. Physiologist 6, 242-242, 1963.

Microsc. 19, 486-487, 1986.

Microsc. 16, 737-738, 1983.

Toxicol. 41, 60-61, 1977b.

binucleate cells in some organs of dogs owing to aging. Med. J. Shinshu Univ. 5,

cells of the dog's intestine due to experimental ischemia. Anat. Rec. 148, 315-315,

aging mice by means of electron microscopic radioautography. J. Clin. Electron

pancreatic acinar cells of aging mice. Acta Histochem. Cytochem. 26, 481-481,

synthesis in pancreatic acinar cells of prenatal and postnatal aging mice. Proc. XIth

on lipid synthesis in perinatal mouse pancreas. J. Clin. Electr. Microsc. 21, 756-757,

pancreatic cells of aging mice as demonstrated by electron microscopic

microscopic radioautography of cryo-fixed ultrathin sections. J. Clin. Electron

thyroid tissues by means of radioautography and chemography. Acta Pharmacol.

insoluble 3H-methyl prednisolone as revealed by electron microscopic drymounting radioautography. Proc. 9th Internat. Congr. Electr. Microsc. 2, 40-41,

localization of anti-allergic agent, tranilast, in rat mast cells. Histochem. J. 19, 533-

localization of an anti-allergic agent, tranilast, in rat mast cells. Cell. Mol. Biol. 36,


Nagata, T., Ma, H., Electron microscopic radioautographic study on mitochondrial DNA synthesis in hepatocytes of aging mouse. Ann. Microsc. 5, 4-18, 2005a. Nagata, T., Ma, H., Electron microscopic radioautographic study on RNA synthesis in hepatocyte mitochondria of aging mouse. Microsc. Res. Tech. 67, 55-64, 2005b. Nagata, T., Momoze, S.: Aging changes of the amitotic and binucleate cells in dog livers.

Nagata, T., Morita, T., I. Kawahara, I.: Radioautographic studies on radiosulfate incorporation in the digestive organs of mice. Histol. Histopathol. 14, 1-8, 1999b. Nagata, T., Murata, F.: Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy. Histochemistry 54, 75-82, 1977. Nagata, T., Murata, F., Yoshida, K., Ohno, S., Iwadare, N.: Whole mount radioautography of

Nagata, T., Nawa, T.: A modification of dry-mounting technique for radioautography of

Nagata, T., Nawa, T.: A radioautographic study on the nucleic acids synthesis of binucleate

Nagata, T., Nawa, T., Yokota, S.: A new technique for electron microscopic dry-mounting radioautography of soluble compounds. Histochemie 18, 241-249, 1969. Nagata, T., Nishigaki, T., Momose, Y.: Localization of anti-allergic agent in rat mast cells

Nagata, T., Ohno, S., Kawahara, I., Yamabayashi, S., Fujii, Y., Murata, F.: Light and electron

Nagata, T., Ohno, S., Murata, F.: Electron microscopic dry-mounting radioautography for

Nagata, T., Ohno, S., Yoshida, K., Murata, F.: Nucleic acid synthesis in proliferating

Nagata, T., Olea, M. T.: Electron microscopic radioautographic study on the protein synthesis in aging mouse spleen. Bull. Nagano Women's Jr. College 7, 1-9, 1999. Nagata, T., Shibata, O., Omochi, S.: A new method for radioautographic osbservation on

Nagata, T., Shibata, O., Nawa, T.: Simplified methods for mass production of

Nagata, T., Shibata, O., Nawa, T.: Incorporation of tritiated thymidine into mitochondrial

Nagata, T., Shimamura, K., Onozawa, M., Kondo, T., Ohkubo, K., Momoze, S.: Relationship

Nagata, T., Shimamura, K., Kondo, T., Onozawa, M., Momoze, S., Okubo, M.: Relationship

DNA of the liver and kidney cells of chickens and mice in tissue culture.

of binuclearity to cell function in some organs. I. Frequencies of binucleate cells in some organs of toads in summer and winter. Med. J. Shinshu Univ. 5, 147-152,

of binuclearity to cell function in some organs. II. Variation of frequencies of

soluble compounds. Acta Phamacol. Toxicol. 41, 62-63, 1977a.

cultured cells as observed by high voltage electron microscopy. Proc. Fifth Internat.

cells in cultivated fibroblasts of chick embryos. Med. J. Shinshu Univ. 11, 1-5, 1966b.

demonstrated by light and electron microscopic radioautography. Acta Histochem.

microscopic radioautography of nucleic acid synthesis in mitochondria and peroxisomes of rat hepatic cells during and after DEHP administration. Acta

peroxisomes of rat liver as revealed by electron microscopical radioautography.

Acta Anat. Nipponica 34, 187-190, 1959.

Cytochem. 19, 669-683, 1986b.

Histochem. J. 14, 197-204, 1982a.

Histochemie 10, 305-308, 1967b.

1960a.

Histochem. Cytochem. 16, 610-611, 1979.

isolated cells. Histochemie 2, 255-259, 1961

radioautograms. -Acta Anat. Nippon.42, 162-166, 1967a.

Conf. High Voltage Electron Microsc. 347-350, 1977d.

water-soluble compounds. Histochemie 7, 370-371, 1966a.

binucleate cells in some organs of dogs owing to aging. Med. J. Shinshu Univ. 5, 153-158, 1960b.


Macromolecular Synthesis in the Urinary and Reproductive Systems 385

Shimizu, T., Usuda, N., Yamanda, T., Sugenoya, A., Iida, F.: Proliferative activity of human

Sun, L.: Age related changes of RNA synthesis in the lungs of aging mice by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 1061-1072, 1995. Sun, L., Gao, F., Duan, H., Nagata, T.: Light microscopic radioautography of DNA synthesis

Sun, L., Gao, F., Nagata, T.: Study on the DNA synthesis of pulmonary cells in aging mice by light microscopic radioautography. Cell. Mol. Biol. 41, 851-859, 1995a. Sun, L., Gao, F., Jin, C., Duan, H., Nagata, T.: An electron microscopic radioautographic

Sun, L., Gao, F., Jin, C., Nagata, T.: DNA synthesis in the tracheae of aging mice by means of

Sun, L., Gao, F., Nagata, T.: A Light Microscopic radioautographic study on protein

Suzuki, K., Imada, T., Gao, F., Ma, H., Nagata, T.: Radioautographic study of benidipine

Terauchi, A., Mori, T., Kanda, H., Tsukada, M., Nagata, T.: Radioautographic study of 3H-

Terauchi, A., Nagata, T.: Observation on incorporation of 3H-taurine in mouse skeletal

Terauchi, A., Nagata, T.: In corporation of 3H-taurine into the blood capillary cells of mouse

Toriyama, K.: Study on the aging changes of DNA and protein synthesis of bipolar and

Tsukahara, S., Yoshida, K., Nagata, T.: A radioautographic study on the incorporation of

Usuda, N., Nagata, T.: Electron microscopic radioautography of acyl-CoA mRNA by in situ

Usuda, N., Nagata, T.: The immunohistochemical and in situ hybridization studies on hepatic peroxisomes. Acta Histochem. Cytochem. 28, 169-172, 1995. Usuda, N., Hanai, T., Morita, T., Nagata, T.: Radioautographic demonstration of

radioautography. Cell. Mol. Biol. 41, 593-601, 1995.

pp.181-184, Peeters Press, Leuven, 1992.

hybridization. J. Clin. Electron Microsc. 25, 332-333, 1992.

immnohistochemical studies. Cancer 71, 2807-2812, 1993.

pp. 201-205, Shinshu University Press, Matsumoto, 1994.

Microsc. 28, 129-131, 1995b.

rat. Drug Res. 44, 129-133, 1994.

211-220, 1997a.

470, 1997b.

1988.

397-404, 1993.

244, 1980.

Press, Matsumoto, 1994.

thyroid tumors evaluated by proliferating cell nuclear antigen/cyclin

in pulmonary cells in aging mice. In, Radioautography in Medicine, Nagata, T. Ed.,

study on the DNA synthesis of pulmonary tissue cells in aging mice. Med. Electron.

light and electron microscopic radioautography. Acta Histochem. Cytochem. 30,

synthesis in pulmonary cells of aging mice. Acta Histochem. Cytochem. 30, 463-

hydrochloride: localization in the mesenteric artery of spontaneously hypertensive

taurine uptake in mouse skeletal muscle cells. J. Clin. Electron Microsc. 21, 627-628,

muscle cells by light and electron microscopic radioautography. Cell. Mol. Biol. 39,

skeletal muscle. Radioautography in Medicine, Nagata, T. ed., Shinshu University

photo-receptor cells of mouse retina by light and electron microscopic

14C-bupranolol (beta-blocking agent) into the rabbit eye. Histochemistry 68, 237-

peroxisomal acyl-CoA oxidase mRNA by in situ hybridization. In, Recent advances in cellular and molecular biology, Vol. 6. Molecular biology of nucleus, peroxisomes, organelles and cell movement. Wegmann, R. J., Wegmann, M., Eds,


Nishigaki, T., Momose, Y., Nagata, T.: Localization of the anti-allergic agent tranilast in the

Oguchi, K., Nagata, T.: A radioautographic study of activated satellite cells in dystrophic

Oguchi, K., Nagata, T.: Electron microscopic radioautographic observation on activated

Ohno, S., Fujii, Y., Usuda, N., Endo, T., Hidaka, H., Nagata, T.: Demonstration of

Ohno, S., Fujii, Y., Usuda, N., Nagata, T., Endo, T., Tanaka, T., Hidaka, H.: Intracellular

Olea, M. T.: An ultrastructural localization of lysosomal acid phosphatase activity in aging

Olea, M. T., Nagata, T.: X-ray microanalysis of cerium in mouse spleen cells demonstrating

Olea, M. T., Nagata, T. : Simultaneous localization of 3H-thymidine incorporation and acid

Olea, M. T., Nagata, T.: A radioautographic study on RNA synthesis in aging mouse spleen

Oliveira, S. F., Nagata, T., Abrahamsohn, P. A., Zorn, T. M. T.: Electron microscopic

Oliveira, S. F., Abrahamsohn, P. A., Nagata, T., Zorn, T. M. T.: Incorporation of 3H-amino

Pearse, A. G. E.: Histochemistry, Theoretical and Applied. 4th Ed. Vol. 1. 439 pp., 1980, Vol.

Sakai, Y., Ikado, S., Nagata, T.: Electron microscopic radioautography of satellite cells in

Sato, A.: Quantitative electron microscopic studies on the kinetics of secretory granules in G-

Sato, A., Iida, F., Furihara, R., Nagata, T.: Electron microscopic raioautography of rat

after 3H-uridine labeling in vitro. Cell. Mol. Biol. 38, 399-405, 1992b.

radioautographical study. Cell. Mol. Biol. 41, 107-116, 1995.

regenerating muscles. J. Clin. Electr. Microsc. 10, 508-509, 1977.

Drug Res. 40, 272-275, 1990b.

Welfare of Japan, Tokyo, 1981.

Publishing Co., New York, 1982.

Cell. Mol. Biol. 38, 115-122, 1992a.

Cell. Mol. Biol. 37, 315-323, 1991.

Edinburgh, London and New York, 1991.

cells. Cell Tissue Res. 187, 45-59, 1978.

1977.

Cytochem. 24, 201-208, 1991.

37, 155-163, 1991.

radioautography. J. Electron Microsc. 32, 1-12, 1983.

Tokyo, 1980.

urinary bladder of rat as demonstrated by light microscopic radioautography.

chicken muscle. In, Current Research in Muscular Dystrophy Japan. The Proc. Ann. Meet. Muscular Dystrophy Res. 1980, pp. 16-17, Ministry of Welfare of Japan,

satellite cells in dystrophy chickens. In, Clinical Studies on the Etiology of Muscular Dystrophy. Annual Report on Neurological Diseases 1981, pp. 30-33, Ministry of

intracellular localization of calmodulin antagonist by wet-mounting

localization of calmodulin antagonists (W-7). In, Calmodulin and intracellular Ca2+ receptors. Kakiuchi, S., Hidaka, H, Means, A. R., Eds., pp. 39-48, Plenum

mouse spleen: a quantitative X-ray microanalytical study. Acta Histochem.

acid phosphatase activity using high voltage electron microscopy, Cell. Mol. Biol.

phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

radioautographic study on the incorporation of 3H-proline by mouse decidual cells.

acids by endometrial stromal cells during decidualization in the mouse. A

2. 1055 pp., 1985, Vol. 3. Ed. with P. Stoward, 728 pp. Churchill Livingstone,

stomach G-cells by means of 3H-amino acids. J. Clin. Electron Microsc. 10, 358-359,


**17** 

*Japan* 

Tetsuji Nagata1,2

**Macromolecular Synthesis in the Endocrine,** 

This chapter deals with the last forth part of the application of microscopic radioautography to the organ systems, including the endocrine system, the nervous system and the sensory organs. The endocrine system includes the hypophysis, the pineal body, the thyroid gland, the parathyroid gland, the thymus, the adrenal gland, the islet of Langerhans and the reproductive glands, i. e. the testis and the ovary. The nervous system includes the central nervous system, i.e. the brains and spinal cord and the peripheral nerves, i.e. the ganglion and nerves, while the sensory system includes, the skin, the visual, the stato-acoustic, the olfactory and the gustatory organs. We have studied some of these organs, not all of them

Among the endocrine organs, we studied macromolecular synthesis in the adrenal gland and the steroid secreting cells of both sexes, the Leydig cells of the testis and the ovarian follicular cells in mice. On the other hand, incorporation of mercury chloride into the human

Among the endocrine organs, we studied DNA synthesis in the adrenal glands and steroid secreting cells of both sexes, the Leydig cells of the testis and the ovarian follicular cells in

We studied the adrenal tissues of aging mice, both the adrenal cortex and the medulla, from embryo to postnatal 2 years in senescence. Some of the results were already published in several original articles (Ito 1996, Ito and Nagata 1996, Liang 1998, Liang et al. 1999, Nagata 1994, 1999c, 2000a,b, 2008a,b, 2009c,d,e,f,g,h,i,j, 2010a, Nagata et al. 2000b). The results shall

**2. Macromolecular synthesis in the endocrine system** 

thyroid tissues was also studied (Nagata 2002).

**2.1 The DNA synthesis in the endocrine system** 

**2.1.1 The DNA synthesis in the adrenal gland** 

be summarized in this review.

**1. Introduction** 

yet.

mice.

**Nervous and Sensory Systems** 

*2Shinshu Institute of Alternative Medicine and Welfare, Nagano* 

*1Department of Anatomy and Cell Biology,* 

*Shinshu University School of Medicine, Matsumoto* 


## **Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems**

## Tetsuji Nagata1,2

*1Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 2Shinshu Institute of Alternative Medicine and Welfare, Nagano Japan* 

## **1. Introduction**

386 Senescence

Uwa, H., Nagata, T.: Cell population kinetics of the scleroblast during ethisterone-induced

Watanabe, I., Makiyama, M. C. K., Nagata, T.: Electron microscopic radioautographic

Yamabayashi, S., Gunarso, W., Tsukahara, S., Nagata, T.: Incorporation of 3H-befunolol

Yamada, A., Nagata, T.: Ribonucleic acid and protein synthesis in the uterus of pregnant

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of DNA synthesis in

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of RNA synthesis of

Yoshinaga, K.: Uterine receptivity for blastcyst implantation. Ann. N. Y. Acad. Sci. USA,

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Study of ethionine

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Radioautographic

9, 693-694, 1976.

365, 1992a.

541, 424-431, 1988.

Microsc. 7, 349-350, 1974.

Electron. Microsc. 10, 372-373, 1977.

Microscopica 6. 130-131, 1997.

Cell. Mol. Biol. 39, 1-12, 1993.

window. Cell. Mol. Biol. 38, 763-774, 1992b.

implantation. Cell. Mol. Biol. 38, 211-233, 1993.

anal-fin process formation in adult females of the Medaka. Dev. Growth Different.

observation of the submandibular salivary gland of aging mouse. Acta

(beta blocking agent) into melanin granules of ocular tissues in the pigmented rabbits. I. Light microscopic radioautography. Histochemistry 73, 371-375, 1981. Yamada, A. T.: Timely and topologically defined protein synthesis in the periimplanting

mouse endometrium revealed by light and electron microscopic radioautography.

mouse during activation of implantation window. Med. Electron Microsc. 27, 363-

the endometria of pregnant-ovariectomized mice during activation of implantation

peri-implanting pregnant mouse during activation of receptivity for blastocyst

pancreatitis by means of electron microscopic radioautography. J. Clin. Electron

study of protein synthesis in pancreatic exocrine cells of alcoholic rats. J. Clin.

This chapter deals with the last forth part of the application of microscopic radioautography to the organ systems, including the endocrine system, the nervous system and the sensory organs. The endocrine system includes the hypophysis, the pineal body, the thyroid gland, the parathyroid gland, the thymus, the adrenal gland, the islet of Langerhans and the reproductive glands, i. e. the testis and the ovary. The nervous system includes the central nervous system, i.e. the brains and spinal cord and the peripheral nerves, i.e. the ganglion and nerves, while the sensory system includes, the skin, the visual, the stato-acoustic, the olfactory and the gustatory organs. We have studied some of these organs, not all of them yet.

## **2. Macromolecular synthesis in the endocrine system**

Among the endocrine organs, we studied macromolecular synthesis in the adrenal gland and the steroid secreting cells of both sexes, the Leydig cells of the testis and the ovarian follicular cells in mice. On the other hand, incorporation of mercury chloride into the human thyroid tissues was also studied (Nagata 2002).

## **2.1 The DNA synthesis in the endocrine system**

Among the endocrine organs, we studied DNA synthesis in the adrenal glands and steroid secreting cells of both sexes, the Leydig cells of the testis and the ovarian follicular cells in mice.

## **2.1.1 The DNA synthesis in the adrenal gland**

We studied the adrenal tissues of aging mice, both the adrenal cortex and the medulla, from embryo to postnatal 2 years in senescence. Some of the results were already published in several original articles (Ito 1996, Ito and Nagata 1996, Liang 1998, Liang et al. 1999, Nagata 1994, 1999c, 2000a,b, 2008a,b, 2009c,d,e,f,g,h,i,j, 2010a, Nagata et al. 2000b). The results shall be summarized in this review.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 389

established as observed by EMRAG. At postnatal day 3, orientation of 3 layers, especially the zona glomerulosa became evident. At postnatal day 9 and 14, the specific structure of the 3 layers was completely formed and the arrangements of the cells in respective layers became typical especially at day 14 (Fig. 21D) and month 1 (Fig. 21E) to 24. Observing the ultrastructure of the adreno-cortical cells by electron microscopy, cell organelles including mitochondria were not so well developed at perinatal and early postnatal stages from embryonic day 19 to postnatal day 9. However, these cell organelles, mitochondria, endoplasmic reticulum, Golgi apparatus, appeared well developed similarly to the adult stages at postnatal day 14. The zona glomerulosa is the thinnest layer found at the outer zone, covered by the capsule, consisted of closely packed groups of columnar or pyramidal cells forming arcades of cell columns. The cells contained many spherical mitochondria and well developed smooth surfaced endoplasmic reticulum but a compact Golgi apparatus in day 14 animals. The zona fasciculate was the thickest layer, consisted of polygonal cells that were larger than the glomerulosa cells, arranged in long cords disposed radially to the medulla containing many lipid droplets. The mitochondria were less numerous and were more variable in size and shape than those of the glomerulosa cells, while the smooth surfaced endoplasmic reticulum were more developed and the Golgi apparatus was larger than the glomerulosa. In the zona reticularis, the parallel arrangement of cell cords were anastomosed showing networks continued to the medullar cells. The mitochondria were less numerous and were more variable in size and shape than those of the glomerulosa cells like the fasciculata cells, as well as the smooth surfaced endoplasmic reticulum were developed and the Golgi apparatus was large like the fasciculata cells. However, the structure of the adrenal cortex tissues showed changes due to development and aging at

Observing both LM and EM RAG of the adrenal cortex labeled with 3H-thymidine, demonstrating DNA synthesis, the silver grains were found over the nuclei of some adrenocortical cells in S-phase of cell cycle mainly in perinatal stages at embryonic day 19, postnatal day 1 and day 3, while less at day 9 and day 14 to month 1-24 (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b,c,d, 2009c,d,e). Those labeled cells were found in all the 3 layers (Fig. 20), the zona glomerulosa (Fig. 20 left), the zona fasciculata (Fig. 20 middle) and the zona reticularis (Fig. 20 right), at respective aging stages. In labeled adreno-cortical cells in the 3 layers the silver grains were mainly localized over the euchromatin of the nuclei and only a few or several silver grains were found over the mitochondria of these cells as

To the contrary, most adreno-cortical cells were not labeled with any silver grains in their nuclei nor cytoplasm, showing no DNA synthesis after labeling with 3H-thymidine. The labeling indices in respective 3 zones in the cortex as well as the medulla showed the maxima at perinatal stages and decreased due to aging (Fig. 22A,B). Among many unlabeled adreno-cortical cells, however, most cells in the 3 layers were observed to be labeled with several silver grains over their mitochondria due to the incorporations of 3Hthymidine especially at the perinatal stages from embryonic day 19 to postnatal day 1, day 3, day 9 and 14 (Fig. 21D). The ultrastructural localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices and some over the cristae or

respective developmental stages.

observed by EM RAG (Fig. 21D,E).

membranes.

#### **2.1.1.1 The DNA synthesis in the adrenal cortex**

We studied the adrenal tissues of mice at various ages from embryo to postnatal 2 years (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e,f,g,h,i,j). The adrenal tissues obtained from ddY strain mice at various ages from embryo day 19 to postnatal day 30 of both sexes, consisted of the adrenal cortex and the adrenal medulla. The former consisted of 3 layers, zona glomerulosa (Fig. 20A), zona fasciculata (Fig. 20B) and zona reticularis (Fig. 20C), developing gradually with aging from perinatal stage at embryonic day 19 to postnatal stages as day 1, 3, 9, 14, month 1, 2, 6, 12, 24 as observed by light microscopy.

Fig. 20. LM RAG of a young mouse adrenal cortex, labeled with 3H-thymine, showing DNA synthesis (arrow) in 3 layers, zona glomerulosa (left), zona fasciculate (middle), zona reticularis (right). From Nagata, T.: Annals Microsc. Vol. 10, p. 61, 2010, Microsc. Soc. Singapore.

At embryonic day 19 and postnatal day 1, the 3 layers of the adreno-cortical cells, zona glomerulosa (Fig. 21D), zona fasciculate (Fig. 21E) and zona reticularis were composed mainly of polygonal cells, while the specific orientation of the 3 layers was not yet well

We studied the adrenal tissues of mice at various ages from embryo to postnatal 2 years (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e,f,g,h,i,j). The adrenal tissues obtained from ddY strain mice at various ages from embryo day 19 to postnatal day 30 of both sexes, consisted of the adrenal cortex and the adrenal medulla. The former consisted of 3 layers, zona glomerulosa (Fig. 20A), zona fasciculata (Fig. 20B) and zona reticularis (Fig. 20C), developing gradually with aging from perinatal stage at embryonic day 19 to postnatal

stages as day 1, 3, 9, 14, month 1, 2, 6, 12, 24 as observed by light microscopy.

Fig. 20. LM RAG of a young mouse adrenal cortex, labeled with 3H-thymine, showing DNA synthesis (arrow) in 3 layers, zona glomerulosa (left), zona fasciculate (middle), zona reticularis (right). From Nagata, T.: Annals Microsc. Vol. 10, p. 61, 2010, Microsc. Soc.

At embryonic day 19 and postnatal day 1, the 3 layers of the adreno-cortical cells, zona glomerulosa (Fig. 21D), zona fasciculate (Fig. 21E) and zona reticularis were composed mainly of polygonal cells, while the specific orientation of the 3 layers was not yet well

Singapore.

**2.1.1.1 The DNA synthesis in the adrenal cortex** 

established as observed by EMRAG. At postnatal day 3, orientation of 3 layers, especially the zona glomerulosa became evident. At postnatal day 9 and 14, the specific structure of the 3 layers was completely formed and the arrangements of the cells in respective layers became typical especially at day 14 (Fig. 21D) and month 1 (Fig. 21E) to 24. Observing the ultrastructure of the adreno-cortical cells by electron microscopy, cell organelles including mitochondria were not so well developed at perinatal and early postnatal stages from embryonic day 19 to postnatal day 9. However, these cell organelles, mitochondria, endoplasmic reticulum, Golgi apparatus, appeared well developed similarly to the adult stages at postnatal day 14. The zona glomerulosa is the thinnest layer found at the outer zone, covered by the capsule, consisted of closely packed groups of columnar or pyramidal cells forming arcades of cell columns. The cells contained many spherical mitochondria and well developed smooth surfaced endoplasmic reticulum but a compact Golgi apparatus in day 14 animals. The zona fasciculate was the thickest layer, consisted of polygonal cells that were larger than the glomerulosa cells, arranged in long cords disposed radially to the medulla containing many lipid droplets. The mitochondria were less numerous and were more variable in size and shape than those of the glomerulosa cells, while the smooth surfaced endoplasmic reticulum were more developed and the Golgi apparatus was larger than the glomerulosa. In the zona reticularis, the parallel arrangement of cell cords were anastomosed showing networks continued to the medullar cells. The mitochondria were less numerous and were more variable in size and shape than those of the glomerulosa cells like the fasciculata cells, as well as the smooth surfaced endoplasmic reticulum were developed and the Golgi apparatus was large like the fasciculata cells. However, the structure of the adrenal cortex tissues showed changes due to development and aging at respective developmental stages.

Observing both LM and EM RAG of the adrenal cortex labeled with 3H-thymidine, demonstrating DNA synthesis, the silver grains were found over the nuclei of some adrenocortical cells in S-phase of cell cycle mainly in perinatal stages at embryonic day 19, postnatal day 1 and day 3, while less at day 9 and day 14 to month 1-24 (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b,c,d, 2009c,d,e). Those labeled cells were found in all the 3 layers (Fig. 20), the zona glomerulosa (Fig. 20 left), the zona fasciculata (Fig. 20 middle) and the zona reticularis (Fig. 20 right), at respective aging stages. In labeled adreno-cortical cells in the 3 layers the silver grains were mainly localized over the euchromatin of the nuclei and only a few or several silver grains were found over the mitochondria of these cells as observed by EM RAG (Fig. 21D,E).

To the contrary, most adreno-cortical cells were not labeled with any silver grains in their nuclei nor cytoplasm, showing no DNA synthesis after labeling with 3H-thymidine. The labeling indices in respective 3 zones in the cortex as well as the medulla showed the maxima at perinatal stages and decreased due to aging (Fig. 22A,B). Among many unlabeled adreno-cortical cells, however, most cells in the 3 layers were observed to be labeled with several silver grains over their mitochondria due to the incorporations of 3Hthymidine especially at the perinatal stages from embryonic day 19 to postnatal day 1, day 3, day 9 and 14 (Fig. 21D). The ultrastructural localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices and some over the cristae or membranes.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 391

quick frozen, freeze-dried, embedded in Epoxy resin, dry-sectioned, and radioautographed by dry-mounting procedure for demonstrating soluble compounds, many silver grains

Fig. 21B. EM RAG of human thyroid cancer cells, labeled with 205HgCl2 in vitro, chemically fixed doubly in buffered glutaraldehyde and osmium tetroxide, dehydrated, embedded in Epoxy resin, wet-sectioned, and radioautographed by wet-mounting procedure for demonstrating insoluble compounds, less silver grains showing insoluble 205HgCl2

glutaraldehyde mixture, embedded in Lowicryl K4M, sectioned and immuno-stained with anti-keratin antibody by the protein-A gold technique, demonstrating keratin filaments.

Fig. 21E. LM RAG of the zona fasciculata of the adrenal cortex of a postnatal month 6 mouse

Fig. 21F. LM RAG of the zona gromeulosa of the adrenal cortex of a prenatal day 19 mouse

Fig. 21G. LM RAG of the interstitial tissues of a postnatal month 12 male mouse labeled with

Preliminary quantitative analysis on the number of mitochondria in 10 adreno-cortical cells whose nuclei were labeled with silver grains and other 10 cells whose nuclei were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were later calculated regardless whether their nuclei were labeled or not (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b,c,d, 2009c,d,e). The results obtained from the number of mitochondria in adreno-cortical cells in the 3 layers of respective animals in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, showed an gradual increase from the prenatal day 19 (glomerulosa 12.5, fasciculata 14.9, reticularis 15/2/cell) to postnatal day 14 and month 1, 2 (glomerulosa 62.2, fasciculata 64.0, reticularis 68.2/cell) to month 6 and 12. The increase from embryo day 19 to postnatal month 2 was stochastically significant (P <0.01). Then, they did not change

**2.1.1.1.2 The DNA synthesis in the mitochondria of mouse adreno-cortical cells** 

The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 adreno-cortical cells in the 3 layers of each animal labeled with 3Hthymidine demonstrating DNA synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, and month 1, 2, 6, 12 and 24 were reported previously (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e). The results demonstrated that the numbers of labeled mitochondria with 3H-thymidine showing DNA

Fig. 21H. LM RAG of the interstitial tissues of a postnatal day 3 male mouse labeled with

Fig. 21A. EM RAG of human thyroid cancer cells, labeled with 205HgCl2 in vitro,

Fig. 21C. EM photo of a human thyroid cancer cell, fixed in paraformaldehyde and

Fig. 21D. LM RAG of the zona glomelurosa of the adrenal cortex of a postnatal day 14

mouse labeled with 3H-thymidine, showing DNA synthesis. x900.

labeled with 3H-thymidine, showing DNA synthesis. x900.

labeled with 3H-uridine, showing RNA synthesis. x1,000.

3H-thymidine, showing DNA synthesis in Leydig cells. x1,000.

**2.1.1.1.1 The number of mitochondria of mouse adreno-corticl cells** 

3H-uridine, showing RNA synthesis in Leydig cells. x1,000.

significanctly from month 12 to 24 (Fig. 22A).

showing soluble 205HgCl2 incorporations. x15,000.

incorporations. x15,000.

x15,000.

Fig. 21. LM and EM RAG of the endocrine organs. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 115, 2001, Academic Press, San Diego, USA, London, UK.

Fig. 21. LM and EM RAG of the endocrine organs. From Nagata, T.: Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Vol. 211, No. 1, p. 115, 2001, Academic Press, San

Diego, USA, London, UK.

Fig. 21A. EM RAG of human thyroid cancer cells, labeled with 205HgCl2 in vitro, quick frozen, freeze-dried, embedded in Epoxy resin, dry-sectioned, and radioautographed by dry-mounting procedure for demonstrating soluble compounds, many silver grains showing soluble 205HgCl2 incorporations. x15,000.

Fig. 21B. EM RAG of human thyroid cancer cells, labeled with 205HgCl2 in vitro, chemically fixed doubly in buffered glutaraldehyde and osmium tetroxide, dehydrated, embedded in Epoxy resin, wet-sectioned, and radioautographed by wet-mounting procedure for demonstrating insoluble compounds, less silver grains showing insoluble 205HgCl2 incorporations. x15,000.

Fig. 21C. EM photo of a human thyroid cancer cell, fixed in paraformaldehyde and glutaraldehyde mixture, embedded in Lowicryl K4M, sectioned and immuno-stained with anti-keratin antibody by the protein-A gold technique, demonstrating keratin filaments. x15,000.

Fig. 21D. LM RAG of the zona glomelurosa of the adrenal cortex of a postnatal day 14 mouse labeled with 3H-thymidine, showing DNA synthesis. x900.

Fig. 21E. LM RAG of the zona fasciculata of the adrenal cortex of a postnatal month 6 mouse labeled with 3H-thymidine, showing DNA synthesis. x900.

Fig. 21F. LM RAG of the zona gromeulosa of the adrenal cortex of a prenatal day 19 mouse labeled with 3H-uridine, showing RNA synthesis. x1,000.

Fig. 21G. LM RAG of the interstitial tissues of a postnatal month 12 male mouse labeled with 3H-thymidine, showing DNA synthesis in Leydig cells. x1,000.

Fig. 21H. LM RAG of the interstitial tissues of a postnatal day 3 male mouse labeled with 3H-uridine, showing RNA synthesis in Leydig cells. x1,000.

## **2.1.1.1.1 The number of mitochondria of mouse adreno-corticl cells**

Preliminary quantitative analysis on the number of mitochondria in 10 adreno-cortical cells whose nuclei were labeled with silver grains and other 10 cells whose nuclei were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were later calculated regardless whether their nuclei were labeled or not (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b,c,d, 2009c,d,e). The results obtained from the number of mitochondria in adreno-cortical cells in the 3 layers of respective animals in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, showed an gradual increase from the prenatal day 19 (glomerulosa 12.5, fasciculata 14.9, reticularis 15/2/cell) to postnatal day 14 and month 1, 2 (glomerulosa 62.2, fasciculata 64.0, reticularis 68.2/cell) to month 6 and 12. The increase from embryo day 19 to postnatal month 2 was stochastically significant (P <0.01). Then, they did not change significanctly from month 12 to 24 (Fig. 22A).

## **2.1.1.1.2 The DNA synthesis in the mitochondria of mouse adreno-cortical cells**

The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 adreno-cortical cells in the 3 layers of each animal labeled with 3Hthymidine demonstrating DNA synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, and month 1, 2, 6, 12 and 24 were reported previously (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e). The results demonstrated that the numbers of labeled mitochondria with 3H-thymidine showing DNA

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 393

On the other hand, the labeling indices in respective aging stages were calculated from the number of labeled mitochondria, dividing by the number of total mitochondria per cell which were mentioned previously (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e,j, 2010e). The results showed that the labeling indices gradually increased from prenatal day 19 (glomerulosa 2.4, fasciculata 2.7, reticularis 2.6%) to postnatal day 14, month 1 and 2 (glomerulosa 8.5, fasciculata 7.8, reticularis 8.8%), reaching the maximum and decreased to month 6 (glomerulosa 4.1, fasciculata 4.2, reticularis 3.8%), 12 and 24 (Fig. 23C).

We studied the adrenal tissues of mice at various ages from embryo to postnatal 2 years. The adrenal tissues obtained from ddY strain mice at various ages from embryo day 19 to postnatal day 30 of both sexes, consisted of the adrenal cortex and the adrenal medulla (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e,g, 2010d,e,f,g). The former consited of 3 layers, zona glomerulosa, zona fasciculata and zona reticularis, developing gradually with aging as observed by light microscopy (Fig. 20), while the latter consisted of 2 cell types in one layer when observed by electron microscopy (Nagata 2009c,d,e, 2010d,e,f,g). At embryonic day 19 and postnatal day 1, the 3 layers of the adreno-cortical cells, zona glomerulosa, zona fasciculate and zona reticularis were composed mainly of polygonal cells, while the specific orientation of the 3 layers was not yet well established. However, the orientation of 3 layers became evident at day 3 and completely formed at day 14 (Fig. 20) and to month 1-24 (Fig. 21D,E,F). On the other hand, the medulla consisted of only one layer containing 2 types of cells, adrenalin cell and noradrenalin cell. The former contains adrenalin granlules with low electron density, while the latter contains noradrenalin

The adrenal medulla is the deepest layer in the adrenal glands, surrounded by the 3 layers of the adrenal cortex as observed by light and electron microscopy (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b,c,d, 2009c,d,e, 2010d,e,f,g), containing either adrenalin granules or noradrenalin granules. Quantitative analysis revealed that the numbers of mitochondria in both adrenalin and noradrenalin cells at various ages increased from fetal day 19 to postnatal month 1 due to aging of animals, respectively, but did not decrease to month 24, while the number of labeled mitochondria and the labeling indices of intramitochondrial DNA synthesis changed due to aging. When they were labeled with 3H-thymidine silver grains appeared over some nuclei of both cell types at perinatal stages, but they appeared almost all the cell bodies containing mitochondria. Quantitative analysis revealed that the numbers of mitochondria in both adrenalin and noradrenalin cells at various ages increased from fetal day 19 to postnatal month 1 due to aging of animals, respectively, while the number of labeled mitochondria and the labeling indices of intramitochondrial DNA synthesis incorporating 3H-thymidine increased from fetal day 19 to postnatal day 14 (2 weeks), reaching the maxima, and decreased to month 24. It was shown that the activity of intramitochnodrial DNA synthesis in the adrenal medullary cells in aging mice increased and decreased due to

**2.1.1.1.3 The labeling index of DNA synthesis in mouse adreno-cortical mitochondria** 

The increase and decrease were stochastically significant (P <0.01).

**2.1.1.2 The DNA synthesis in mouse adreno-medullary cells** 

granules with high electron density.

aging of animals.

synthesis gradually increased from prenatal embryo day 19 (glomerulosa 0.3, fasciculata 0.5, reticularis 0.4/cell) to postnatal day 14, month 1 and 2 (glomerulosa 5.3, fasciculata 5.0, reticularis 6.2/cell), reaching the maximum, then decreased to month 6, 12 and 24 (Fig. 22A). The increase and decrease were stochastically significant (P <0.01).

Fig. 22. Histogram showing aging changes of average labeling indices in respective cell types of the adrenal glands of aging mice labeled with 3H-thymidine showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 171, 2002, Urban & Fischer, Jena, Germany

Fig. 22A. The adrenal cortex.

Fig. 22B. The adrenal medulla.

synthesis gradually increased from prenatal embryo day 19 (glomerulosa 0.3, fasciculata 0.5, reticularis 0.4/cell) to postnatal day 14, month 1 and 2 (glomerulosa 5.3, fasciculata 5.0, reticularis 6.2/cell), reaching the maximum, then decreased to month 6, 12 and 24 (Fig. 22A).

Fig. 22. Histogram showing aging changes of average labeling indices in respective cell types of the adrenal glands of aging mice labeled with 3H-thymidine showing DNA

Germany

Fig. 22A. The adrenal cortex. Fig. 22B. The adrenal medulla.

synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 171, 2002, Urban & Fischer, Jena,

The increase and decrease were stochastically significant (P <0.01).

## **2.1.1.1.3 The labeling index of DNA synthesis in mouse adreno-cortical mitochondria**

On the other hand, the labeling indices in respective aging stages were calculated from the number of labeled mitochondria, dividing by the number of total mitochondria per cell which were mentioned previously (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e,j, 2010e). The results showed that the labeling indices gradually increased from prenatal day 19 (glomerulosa 2.4, fasciculata 2.7, reticularis 2.6%) to postnatal day 14, month 1 and 2 (glomerulosa 8.5, fasciculata 7.8, reticularis 8.8%), reaching the maximum and decreased to month 6 (glomerulosa 4.1, fasciculata 4.2, reticularis 3.8%), 12 and 24 (Fig. 23C). The increase and decrease were stochastically significant (P <0.01).

## **2.1.1.2 The DNA synthesis in mouse adreno-medullary cells**

We studied the adrenal tissues of mice at various ages from embryo to postnatal 2 years. The adrenal tissues obtained from ddY strain mice at various ages from embryo day 19 to postnatal day 30 of both sexes, consisted of the adrenal cortex and the adrenal medulla (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b, 2009c,d,e,g, 2010d,e,f,g). The former consited of 3 layers, zona glomerulosa, zona fasciculata and zona reticularis, developing gradually with aging as observed by light microscopy (Fig. 20), while the latter consisted of 2 cell types in one layer when observed by electron microscopy (Nagata 2009c,d,e, 2010d,e,f,g). At embryonic day 19 and postnatal day 1, the 3 layers of the adreno-cortical cells, zona glomerulosa, zona fasciculate and zona reticularis were composed mainly of polygonal cells, while the specific orientation of the 3 layers was not yet well established. However, the orientation of 3 layers became evident at day 3 and completely formed at day 14 (Fig. 20) and to month 1-24 (Fig. 21D,E,F). On the other hand, the medulla consisted of only one layer containing 2 types of cells, adrenalin cell and noradrenalin cell. The former contains adrenalin granlules with low electron density, while the latter contains noradrenalin granules with high electron density.

The adrenal medulla is the deepest layer in the adrenal glands, surrounded by the 3 layers of the adrenal cortex as observed by light and electron microscopy (Ito 1996, Ito and Nagata 1996, Nagata 2008a,b,c,d, 2009c,d,e, 2010d,e,f,g), containing either adrenalin granules or noradrenalin granules. Quantitative analysis revealed that the numbers of mitochondria in both adrenalin and noradrenalin cells at various ages increased from fetal day 19 to postnatal month 1 due to aging of animals, respectively, but did not decrease to month 24, while the number of labeled mitochondria and the labeling indices of intramitochondrial DNA synthesis changed due to aging. When they were labeled with 3H-thymidine silver grains appeared over some nuclei of both cell types at perinatal stages, but they appeared almost all the cell bodies containing mitochondria. Quantitative analysis revealed that the numbers of mitochondria in both adrenalin and noradrenalin cells at various ages increased from fetal day 19 to postnatal month 1 due to aging of animals, respectively, while the number of labeled mitochondria and the labeling indices of intramitochondrial DNA synthesis incorporating 3H-thymidine increased from fetal day 19 to postnatal day 14 (2 weeks), reaching the maxima, and decreased to month 24. It was shown that the activity of intramitochnodrial DNA synthesis in the adrenal medullary cells in aging mice increased and decreased due to aging of animals.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 395

When we studied macromolecular synthesis in the exocrine pancreatic cells of aging mice by LM and EMRAG we also studied the islet cells of Langerhans together with the exocrine cells, using RI labeled precursors such as 3H-thymidine for DNA (Nagata and Usuda 1985, 1986, Nagata et al. 1986a,b), 3H-uridine for RNA (Nagata and Usuda 1985, 1993b, Nagata et al. 1986a,b), 3H-leucine for proteins (Nagata 2000, Nagata and Usuda 1993a, 1995), 3Hglucosamine for glucides (Nagata et al. 1992), 3H-glycerol for lipids (Nagata et al. 1988b, 1990). The results showed that the islets cells, A, B and C cells, incorporated those precursors to synthesize DNA, RNA, proteins and glucides. The labeling index of DNA synthesis and the densities of silver grains showing RNA, proteins and glucides syntheses were high at prenatal and earlier postnatal stages from day 1 to day 14, then decreased from 1 month to 1 years due to aging. However, the labeling indices by 3H-thymidine and the grain counts by 3H-uridine and 3H-leucine in the endocrine cells were less than those in the

The cells of Leydig can be found in the interstitial tissues between the seminiferous tubules of the testis of mammals (Gao 1993, Gao et al. 1994, 1995a,b, Nagata et al. 2000b). They are identified as spherical, oval, or irregular in shape and their cytoplasms contain lipid droplets. We studied the macromolecular synthesis of the cells in the testis of several groups of litter ddY mice at various ages from fetal day 19 to postnatal aging stages up to 2 years senescence

The Leydig cells from embryonic stage to senescent stages were labeled with 3H-thymidine as observed by LMRAG (Fig. 21G). The changes of the numbers of labeled Leydig cells with the 3H-thymidine incorporation into the nuclei showing the DNA synthesis were found in these cells at different aging stages. Only a few cells were labeled after 3H-thymidine at embryonic day 19. At early postnatal stages, there was a slight increase of the number of labeled cells. The number of labeled cells from perinatal stage to postnatal 14 days and 1, 2, 6 months were similar to the values found at prenatal and early postnatal stages. The notable increases in the number of labeled cells of Leydig were found from 9 months to 2 years in senescence. The labeling indices with 3H-thymidine in perinatal stages to postnatal 6 months were low (5-10%) but increased at 9 months and maintained high level (50-60%) to 2 years (Gao 1993, Gao et al. 1994, 1995a, Nagata et al. 2000b). The labeling indices at senescent stages still maintained a relatively high level and they were obviously higher than those of young animals. By electron microscopy, typical Leydig cells contained abundant cell organelles such as smooth surfaced endoplasmic reticulum, Golgi apparatus and mitochondria with tubular cristae. The silver grains were mainly localized over the euchromatin of labeled nucleus. Some of the grains were also localized over some of the

The ovarian follicles in the ovaries of mature mice are one of the steroid secreting organs in female animals. We studied the DNA and RNA synthesis of the follicular cells in the developing ddY mice ovaries in several aging groups at postnatal day 1, 3, 7, 14, 30 and 60 by LM and EMRAG using 3H-thymidine and 3H-uridine (Li 1994, Li and Nagata 1995). From

by LM and EMRAG using 3H-thymidine, 3H-uridine and 3H-leucine incorporations.

**2.1.2 The DNA synthesis in the islets of Langerhans** 

**2.1.3 The DNA synthesis in the Leydig cells of the testis** 

mitochondria in both the nuclei labeled and unlabeled cells.

**2.1.4 The DNA synthesis in the ovarian follicles** 

exocrine cells at the same ages.

Fig. 23. Histogram showing aging changes of the respective cell types of the adrenal glands of aging mice labeled with 3H-thymidine showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Current Radiopharmaceutics, Vol. 2, p. 173, 2002. Fig. 23A(25). The number of mitochondria per cell.

Fig. 23B(26). The number of labeled mitochondria per cell.

Fig. 23C(27). The mitochondrial labeling index.

## **2.1.2 The DNA synthesis in the islets of Langerhans**

394 Senescence

Fig. 23. Histogram showing aging changes of the respective cell types of the adrenal glands of aging mice labeled with 3H-thymidine showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Current Radiopharmaceutics, Vol. 2, p. 173, 2002.

Fig. 23A(25). The number of mitochondria per cell. Fig. 23B(26). The number of labeled mitochondria per cell.

Fig. 23C(27). The mitochondrial labeling index.

When we studied macromolecular synthesis in the exocrine pancreatic cells of aging mice by LM and EMRAG we also studied the islet cells of Langerhans together with the exocrine cells, using RI labeled precursors such as 3H-thymidine for DNA (Nagata and Usuda 1985, 1986, Nagata et al. 1986a,b), 3H-uridine for RNA (Nagata and Usuda 1985, 1993b, Nagata et al. 1986a,b), 3H-leucine for proteins (Nagata 2000, Nagata and Usuda 1993a, 1995), 3Hglucosamine for glucides (Nagata et al. 1992), 3H-glycerol for lipids (Nagata et al. 1988b, 1990). The results showed that the islets cells, A, B and C cells, incorporated those precursors to synthesize DNA, RNA, proteins and glucides. The labeling index of DNA synthesis and the densities of silver grains showing RNA, proteins and glucides syntheses were high at prenatal and earlier postnatal stages from day 1 to day 14, then decreased from 1 month to 1 years due to aging. However, the labeling indices by 3H-thymidine and the grain counts by 3H-uridine and 3H-leucine in the endocrine cells were less than those in the exocrine cells at the same ages.

## **2.1.3 The DNA synthesis in the Leydig cells of the testis**

The cells of Leydig can be found in the interstitial tissues between the seminiferous tubules of the testis of mammals (Gao 1993, Gao et al. 1994, 1995a,b, Nagata et al. 2000b). They are identified as spherical, oval, or irregular in shape and their cytoplasms contain lipid droplets. We studied the macromolecular synthesis of the cells in the testis of several groups of litter ddY mice at various ages from fetal day 19 to postnatal aging stages up to 2 years senescence by LM and EMRAG using 3H-thymidine, 3H-uridine and 3H-leucine incorporations.

The Leydig cells from embryonic stage to senescent stages were labeled with 3H-thymidine as observed by LMRAG (Fig. 21G). The changes of the numbers of labeled Leydig cells with the 3H-thymidine incorporation into the nuclei showing the DNA synthesis were found in these cells at different aging stages. Only a few cells were labeled after 3H-thymidine at embryonic day 19. At early postnatal stages, there was a slight increase of the number of labeled cells. The number of labeled cells from perinatal stage to postnatal 14 days and 1, 2, 6 months were similar to the values found at prenatal and early postnatal stages. The notable increases in the number of labeled cells of Leydig were found from 9 months to 2 years in senescence. The labeling indices with 3H-thymidine in perinatal stages to postnatal 6 months were low (5-10%) but increased at 9 months and maintained high level (50-60%) to 2 years (Gao 1993, Gao et al. 1994, 1995a, Nagata et al. 2000b). The labeling indices at senescent stages still maintained a relatively high level and they were obviously higher than those of young animals. By electron microscopy, typical Leydig cells contained abundant cell organelles such as smooth surfaced endoplasmic reticulum, Golgi apparatus and mitochondria with tubular cristae. The silver grains were mainly localized over the euchromatin of labeled nucleus. Some of the grains were also localized over some of the mitochondria in both the nuclei labeled and unlabeled cells.

#### **2.1.4 The DNA synthesis in the ovarian follicles**

The ovarian follicles in the ovaries of mature mice are one of the steroid secreting organs in female animals. We studied the DNA and RNA synthesis of the follicular cells in the developing ddY mice ovaries in several aging groups at postnatal day 1, 3, 7, 14, 30 and 60 by LM and EMRAG using 3H-thymidine and 3H-uridine (Li 1994, Li and Nagata 1995). From

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 397

et al. 2000b, Nagata 2010a). The results showed that the labeling indices gradually increased from prenatal day 19 (glomerulosa 10.4, fasciculata 12.1, reticularis 13.1%) to postnatal day 1 (glomerulosa 12.6, fasciculata 11.4, reticularis 11.1%), 3, 9 (glomerulosa 16.6, fasciculata 18.0, reticularis 18.0%), reaching the maximum and decreased to day 14, month 1 (glomerulosa 11.4, fasciculata 11.0, reticularis 10.7%) 2 (glomerulosa 8.5, fasciculata 7.8, reticularis 8.8%), month 6 (glomerulosa 4.1, fasciculata 4.2, reticularis 3.8%), 12 and 24 (Fig. 24C). The increase

Fig. 24. Histogram showing aging changes of the respective cell types of the adrenal glands of aging mice labeled with 3H-uridine showing RNA synthesis. Mean ± Standard Deviation.

From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem.

Cytochem. Vol. 37, No. 2, p. 173, 2002, Urban & Fischer, Jena, Germany

and decrease were stochastically significant (P <0.01).

the results it was shown that both DNA and RNA synthesis in the ovarian follicular cells were observed (Fig. 16G). Quantitative analysis, as expressed with labeling indices and grain counts, revealed that both increased significantly from postnatal day 1 to 3, then decreased from day 7 to 60 (Fig. 19A). Comparing the results to other female genital cells, a paralleled alteration of both DNA and RNA synthesis was revealed between the ovarian follicular cells and other uterine or oviductal cells (Fig. 19B,C). On the other hand, the glycoconjugate synthesis as shown by the uptake of 35SO4 in mouse ovary during the estrus cycle was also demonstrated (Li et al. 1992).

## **2.2 The RNA synthesis in the endocrine system**

We studied the RNA synthesis of the adrenal glands and the cells of Leydig in the testis of aging mice among the endocrine organs after the administration of 3H-uridine at various ages.

## **2.2.1 The RNA synthesis in the adrenal glands**

The RNA synthesis in the adrenal glands in aging mice was studied in both the adrenal cortex and the adrenal medulla after administration of 3H-uridine in many groups of mice at various ages from perinatal stages to senescence at year 2.

#### **2.2.1.1 The RNA synthesis in aging mouse adreno-cortical cells**

Observing both LM and EM RAG labeled with 3H-uridine, demonstrating RNA synthesis, the silver grains were found over the nuclei and cytoplasm of almost all the adreno-cortical cells from perinatal stages to postnatal month 1-24 (Liang 1998, Liang et al. 1999, Nagata et al. 2000b, Nagata 2010a). Those labeled cells were found in all the 3 layers, the zona glomerulosa (Fig. 21F), the zona fasciculata and the zona reticularis, at respective aging stages. In labeled adreno-cortical cells in the 3 layers the silver grains were mainly localized over the euchromatin of the nuclei and several silver grains were found over the endoplasmic reticulum, ribosomes and mitochondria of these cells. The ultrastructural localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices and some over the cristae or membranes.

#### **2.2.1.2 The RNA synthesis in the mitochondria of mouse adreno-cortical cells**

The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 adreno-cortical cells in the 3 layers of each animal labeled with 3H-uridine demonstrating RNA synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, and month 1, 2, 6, 12 and 24 were reported previously (Liang 1998, Liang et al. 1999, Nagata et al. 2000b, Nagata 2010a). The results demonstrated that the numbers of labeled mitochondria with 3H-uridine showing RNA synthesis gradually increased from prenatal embryo day 19 (glomerulosa 0.3, fasciculata 0.5, reticularis 0.4/cell) to postnatal day 14, month 1 and 2 (glomerulosa 5.3, fasciculata 5.0, reticularis 6.2/cell), reaching the maximum, then decreased to month 6, 12 and 24 (Fig. 23B). The increase and decrease were stochastically significant (P <0.01).

#### **2.2.1.3 The labeling index of RNA synthesis in mouse adreno-cortical mitochondria**

On the other hand, the labeling indices in respective aging stages were calculated from the number of labeled mitochondria (Fig. 14B), dividing by the number of total mitochondria per cell (Fig. 24A) which were mentioned previously (Liang 1998, Liang et al. 1999, Nagata

the results it was shown that both DNA and RNA synthesis in the ovarian follicular cells were observed (Fig. 16G). Quantitative analysis, as expressed with labeling indices and grain counts, revealed that both increased significantly from postnatal day 1 to 3, then decreased from day 7 to 60 (Fig. 19A). Comparing the results to other female genital cells, a paralleled alteration of both DNA and RNA synthesis was revealed between the ovarian follicular cells and other uterine or oviductal cells (Fig. 19B,C). On the other hand, the glycoconjugate synthesis as shown by the uptake of 35SO4 in mouse ovary during the estrus

We studied the RNA synthesis of the adrenal glands and the cells of Leydig in the testis of aging mice among the endocrine organs after the administration of 3H-uridine at various ages.

The RNA synthesis in the adrenal glands in aging mice was studied in both the adrenal cortex and the adrenal medulla after administration of 3H-uridine in many groups of mice at

Observing both LM and EM RAG labeled with 3H-uridine, demonstrating RNA synthesis, the silver grains were found over the nuclei and cytoplasm of almost all the adreno-cortical cells from perinatal stages to postnatal month 1-24 (Liang 1998, Liang et al. 1999, Nagata et al. 2000b, Nagata 2010a). Those labeled cells were found in all the 3 layers, the zona glomerulosa (Fig. 21F), the zona fasciculata and the zona reticularis, at respective aging stages. In labeled adreno-cortical cells in the 3 layers the silver grains were mainly localized over the euchromatin of the nuclei and several silver grains were found over the endoplasmic reticulum, ribosomes and mitochondria of these cells. The ultrastructural localizations of silver grains over the mitochondria were mainly on the mitochondrial

The results of visual grain counts on the number of mitochondria labeled with silver grains obtained from 10 adreno-cortical cells in the 3 layers of each animal labeled with 3H-uridine demonstrating RNA synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, and month 1, 2, 6, 12 and 24 were reported previously (Liang 1998, Liang et al. 1999, Nagata et al. 2000b, Nagata 2010a). The results demonstrated that the numbers of labeled mitochondria with 3H-uridine showing RNA synthesis gradually increased from prenatal embryo day 19 (glomerulosa 0.3, fasciculata 0.5, reticularis 0.4/cell) to postnatal day 14, month 1 and 2 (glomerulosa 5.3, fasciculata 5.0, reticularis 6.2/cell), reaching the maximum, then decreased to month 6, 12 and 24 (Fig. 23B). The increase and

**2.2.1.3 The labeling index of RNA synthesis in mouse adreno-cortical mitochondria** 

On the other hand, the labeling indices in respective aging stages were calculated from the number of labeled mitochondria (Fig. 14B), dividing by the number of total mitochondria per cell (Fig. 24A) which were mentioned previously (Liang 1998, Liang et al. 1999, Nagata

**2.2.1.2 The RNA synthesis in the mitochondria of mouse adreno-cortical cells** 

cycle was also demonstrated (Li et al. 1992).

**2.2 The RNA synthesis in the endocrine system** 

**2.2.1 The RNA synthesis in the adrenal glands** 

matrices and some over the cristae or membranes.

decrease were stochastically significant (P <0.01).

various ages from perinatal stages to senescence at year 2.

**2.2.1.1 The RNA synthesis in aging mouse adreno-cortical cells** 

et al. 2000b, Nagata 2010a). The results showed that the labeling indices gradually increased from prenatal day 19 (glomerulosa 10.4, fasciculata 12.1, reticularis 13.1%) to postnatal day 1 (glomerulosa 12.6, fasciculata 11.4, reticularis 11.1%), 3, 9 (glomerulosa 16.6, fasciculata 18.0, reticularis 18.0%), reaching the maximum and decreased to day 14, month 1 (glomerulosa 11.4, fasciculata 11.0, reticularis 10.7%) 2 (glomerulosa 8.5, fasciculata 7.8, reticularis 8.8%), month 6 (glomerulosa 4.1, fasciculata 4.2, reticularis 3.8%), 12 and 24 (Fig. 24C). The increase and decrease were stochastically significant (P <0.01).

Fig. 24. Histogram showing aging changes of the respective cell types of the adrenal glands of aging mice labeled with 3H-uridine showing RNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 173, 2002, Urban & Fischer, Jena, Germany

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 399

Observing both LM and EM RAG labeled with 3H-leucine, demonstrating protein synthesis, the silver grains were found over the nuclei and cytoplasm of almost all the adreno-cortical and adreno-medullary cells from perinatal stages to postnatal month 1-24 (Nagata 2010c,d,e,f,g). Those labeled cells were found in all the 3 layers, the zona glomerulosa, the zona fasciculata and the zona reticularis, as well as in all the cells in the adreno-medullae at

In order to study the aging changes of intramitochondrial protein synthesis of mouse adrenal cells, 10 groups of developing and aging mice, each consisting of 3 individuals, total 30, from fetal day 19 to postnatal newborn at day 1, 3, 9, 14, adult at month 1, 2, 6 and senescent animals at month 12 (year 1) and 24 (year 2) were injected with 3H-leucine, an protein precursor, sacrificed 1 hr later and the adrenal tissues were fixed and processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, the number of mitochondria per cell, the number of labeled mitochondria with 3H-leucine showing protein synthesis per cell and the mitochondrial labeling index in each adreno-cortical cells, in 3 zones, as well as in each adreno-medullary cells, 2 types of cells in the medulla, the adrenalin cells and the noradrenalin cells, were calculated and the results in respective aging groups were compared with each others (Nagata 2010c,d,e,f,g). Preliminary quantitative analysis on the number of mitochondria in either 10 adreno-cortical cells or adreno-medullary cells whose nuclei and cytoplasm were labeled with silver grains and other 10 cells whose nuclei and cytoplasm were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated regardless whether their nuclei were labeled or not (Nagata 2010d,e,f,g). The results demonstrated that the number of mitochondria in adreno-cortical cells in 3 zones, the zona glomerulosa, fasciculata and reticularis of respective mice at various ages increased from fetal day 19 to postnatal month 1 reaching the plateau from month 1 to 24 due to development and aging of animals, respectively, while the number of labeled mitochondria per cell and the labeling index of intramitochondrial protein synthesis incorporating 3H-leucine increased from fetal day 19 to postnatal day 3 to month 2 and decreased to month 24. We carried out the quantitative analysis of these incorporations into nuclei and cell organelles of adrenal cells, both adrenal cortical cells and medullary cells, in aging mice from prenatal to postnatal newborn, juvenile, adult and senescent individuals

Observing EM radioautograms, the silver grains were found over the nuclei of some adrenocortical cells labeled with 3H-leucine demonstrating protein synthesis in all aging stages from perinatal stages at embryonic day 19, postnatal day 1 and day 3, day 9 and day 14 and adults at month 1, month 2, month 6, month 12 and month 24. Those labeled cells were found in all the 3 layers, the zona glomerulosa (Fig. 25A), the zona fasciculata and the zona reticularis (Fig. 25B, C), at respective aging stages. In the labeled adreno-cortical cells in 3 layers the silver grains were mainly localized over the euchromatin of the nuclei or a few or several silver grains were found over cytoplasmic organelles, especially over some of the mitochondria showing protein synthesis incorporating 3H-leucine. The localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices and some over the

mitochondrial membranes as observed by high power magnification (Fig. 25C).

**2.3.1 The protein synthesis in the adrenal gland** 

**2.3.1.1 Protein synthesis in mitochondria of mouse adrenal cells** 

respective aging stages.

(Nagata 2010a,b).

Fig. 24A. The number of mitochondria per cell. Fig. 24B. The number of labeled mitochondria per cell. Fig. 24C. The mitochondrial labeling index.

#### **2.2.2 The RNA synthesis in aging mouse adreno-medullary cells**

The adrenal medulla consists of 2 cell types, the adrenalin cells and noradrenalin cells. When they were labeled with 3H-uridine, an RNA precursor, silver grains appeared over almost all the cells, both nuclei and cytoplasm containing mitochondria (Liang et al. 1999, Nagata et al. 2000b, 2010b). Quantitative analysis revealed that the numbers of mitochondria in both adrenalin and noradrenalin cells at various ages increased from fetal day 19 to postnatal month 1 due to aging of animals, respectively, but did not decrease to month 24 (Fig. 24A), while the number of labeled mitochondria (Fig. 24B) and the labeling indices of intramitochondrial RNA synthesis incorporating 3H-uridine increased from fetal day 19 to postnatal month 1, reaching the maxima, but did not decrease to month 24 (Fig. 24C). It was shown that the activity of intramitochnodrial RNA synthesis in the adrenal medullary cells in aging mice increased but did not decrease due to aging of animals in contrast to DNA synthesis (Nagata 2010b).

#### **2.2.3 The RNA synthesis in the Leydig cells of the testis**

The cells of Leydig can be found in the interstitial tissues between the seminiferous tubules of the testis of mammals (Gao 1993, Gao et al. 1994, 1995a, Nagata et al. 2000b). They are identified as spherical, oval, or irregular in shape and their cytoplasms contain lipid droplets. We studied the macromolecular synthesis of the cells in the testis of several groups of litter ddY mice at various ages from fetal day 19 to postnatal aging stages up to 2 years in senescence by LM and EMRAG using 3H-thymidine, 3H-uridine and 3H-leucine incorporations.

The incorporation of 3H-uridine into RNA was observed in almost all the Leydig cells in the interstitial tissues of the testis from embryonic day 19 to 2 years after birth. A few silver grains over the nuclei and cytoplasm of the Leydig cells labeled with 3H-uridine were observed at embryonic day 19. The silver grains over those cells slightly decreased at postnatal day 1, 3 (Fig. 21H), 7 and 14. The number of the silver grains over the nuclei increased from postnatal 1 months onwards. The average number of silver grains over the cytoplasm increased gradually and reached the maximum at 12 months after birth. At each stage, the activity of RNA synthesis was specifically localized over the euchromatin in the nucleus and nucleolus as observed by EMRAG. From adult to senescent stages, the activity of RNA synthesis maintained a high level in their nuclei as compared to the cytoplasm. In the cytoplasm of Leydig cells in respective aging groups some of the mitochondria and endoplasmic reticulum were also labeled with silver grains. It is noteworthy that the average grain counts increased prominently in the senescent aging groups at 1 and 2 years after birth.

#### **2.3 The protein synthesis in the endocrine system**

We studied the protein synthesis of the adrenal glands and the cells of Leydig in the testis of aging mice among the endocrine organs after the administration of 3H-leucine at various ages.

## **2.3.1 The protein synthesis in the adrenal gland**

398 Senescence

The adrenal medulla consists of 2 cell types, the adrenalin cells and noradrenalin cells. When they were labeled with 3H-uridine, an RNA precursor, silver grains appeared over almost all the cells, both nuclei and cytoplasm containing mitochondria (Liang et al. 1999, Nagata et al. 2000b, 2010b). Quantitative analysis revealed that the numbers of mitochondria in both adrenalin and noradrenalin cells at various ages increased from fetal day 19 to postnatal month 1 due to aging of animals, respectively, but did not decrease to month 24 (Fig. 24A), while the number of labeled mitochondria (Fig. 24B) and the labeling indices of intramitochondrial RNA synthesis incorporating 3H-uridine increased from fetal day 19 to postnatal month 1, reaching the maxima, but did not decrease to month 24 (Fig. 24C). It was shown that the activity of intramitochnodrial RNA synthesis in the adrenal medullary cells in aging mice increased but did not decrease due to aging of animals in contrast to DNA

The cells of Leydig can be found in the interstitial tissues between the seminiferous tubules of the testis of mammals (Gao 1993, Gao et al. 1994, 1995a, Nagata et al. 2000b). They are identified as spherical, oval, or irregular in shape and their cytoplasms contain lipid droplets. We studied the macromolecular synthesis of the cells in the testis of several groups of litter ddY mice at various ages from fetal day 19 to postnatal aging stages up to 2 years in senescence by LM and EMRAG using 3H-thymidine, 3H-uridine and 3H-leucine

The incorporation of 3H-uridine into RNA was observed in almost all the Leydig cells in the interstitial tissues of the testis from embryonic day 19 to 2 years after birth. A few silver grains over the nuclei and cytoplasm of the Leydig cells labeled with 3H-uridine were observed at embryonic day 19. The silver grains over those cells slightly decreased at postnatal day 1, 3 (Fig. 21H), 7 and 14. The number of the silver grains over the nuclei increased from postnatal 1 months onwards. The average number of silver grains over the cytoplasm increased gradually and reached the maximum at 12 months after birth. At each stage, the activity of RNA synthesis was specifically localized over the euchromatin in the nucleus and nucleolus as observed by EMRAG. From adult to senescent stages, the activity of RNA synthesis maintained a high level in their nuclei as compared to the cytoplasm. In the cytoplasm of Leydig cells in respective aging groups some of the mitochondria and endoplasmic reticulum were also labeled with silver grains. It is noteworthy that the average grain counts increased prominently in the senescent aging groups at 1 and 2 years

We studied the protein synthesis of the adrenal glands and the cells of Leydig in the testis of aging mice among the endocrine organs after the administration of 3H-leucine at various

Fig. 24A. The number of mitochondria per cell. Fig. 24B. The number of labeled mitochondria per cell.

**2.2.2 The RNA synthesis in aging mouse adreno-medullary cells** 

**2.2.3 The RNA synthesis in the Leydig cells of the testis** 

**2.3 The protein synthesis in the endocrine system** 

Fig. 24C. The mitochondrial labeling index.

synthesis (Nagata 2010b).

incorporations.

after birth.

ages.

Observing both LM and EM RAG labeled with 3H-leucine, demonstrating protein synthesis, the silver grains were found over the nuclei and cytoplasm of almost all the adreno-cortical and adreno-medullary cells from perinatal stages to postnatal month 1-24 (Nagata 2010c,d,e,f,g). Those labeled cells were found in all the 3 layers, the zona glomerulosa, the zona fasciculata and the zona reticularis, as well as in all the cells in the adreno-medullae at respective aging stages.

#### **2.3.1.1 Protein synthesis in mitochondria of mouse adrenal cells**

In order to study the aging changes of intramitochondrial protein synthesis of mouse adrenal cells, 10 groups of developing and aging mice, each consisting of 3 individuals, total 30, from fetal day 19 to postnatal newborn at day 1, 3, 9, 14, adult at month 1, 2, 6 and senescent animals at month 12 (year 1) and 24 (year 2) were injected with 3H-leucine, an protein precursor, sacrificed 1 hr later and the adrenal tissues were fixed and processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, the number of mitochondria per cell, the number of labeled mitochondria with 3H-leucine showing protein synthesis per cell and the mitochondrial labeling index in each adreno-cortical cells, in 3 zones, as well as in each adreno-medullary cells, 2 types of cells in the medulla, the adrenalin cells and the noradrenalin cells, were calculated and the results in respective aging groups were compared with each others (Nagata 2010c,d,e,f,g). Preliminary quantitative analysis on the number of mitochondria in either 10 adreno-cortical cells or adreno-medullary cells whose nuclei and cytoplasm were labeled with silver grains and other 10 cells whose nuclei and cytoplasm were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated regardless whether their nuclei were labeled or not (Nagata 2010d,e,f,g). The results demonstrated that the number of mitochondria in adreno-cortical cells in 3 zones, the zona glomerulosa, fasciculata and reticularis of respective mice at various ages increased from fetal day 19 to postnatal month 1 reaching the plateau from month 1 to 24 due to development and aging of animals, respectively, while the number of labeled mitochondria per cell and the labeling index of intramitochondrial protein synthesis incorporating 3H-leucine increased from fetal day 19 to postnatal day 3 to month 2 and decreased to month 24. We carried out the quantitative analysis of these incorporations into nuclei and cell organelles of adrenal cells, both adrenal cortical cells and medullary cells, in aging mice from prenatal to postnatal newborn, juvenile, adult and senescent individuals (Nagata 2010a,b).

Observing EM radioautograms, the silver grains were found over the nuclei of some adrenocortical cells labeled with 3H-leucine demonstrating protein synthesis in all aging stages from perinatal stages at embryonic day 19, postnatal day 1 and day 3, day 9 and day 14 and adults at month 1, month 2, month 6, month 12 and month 24. Those labeled cells were found in all the 3 layers, the zona glomerulosa (Fig. 25A), the zona fasciculata and the zona reticularis (Fig. 25B, C), at respective aging stages. In the labeled adreno-cortical cells in 3 layers the silver grains were mainly localized over the euchromatin of the nuclei or a few or several silver grains were found over cytoplasmic organelles, especially over some of the mitochondria showing protein synthesis incorporating 3H-leucine. The localizations of silver grains over the mitochondria were mainly on the mitochondrial matrices and some over the mitochondrial membranes as observed by high power magnification (Fig. 25C).

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 401

25C Fig. 25. EMRAG of the adrenal cortical cells aging mice labeled with 3H-leucine showing protein synthesis in the nucleus as well as in a few mitochondria. From Nagata, T.: Annals

Fig. 25A. EMRAG of the zona glomerulosa of a juvenile mouse at postnatal day 14, labeled with 3H-leucine showing protein synthesis (several silver grains) in the nucleus as well as in

Fig. 25B. EMRAG of the zona reticularis of an old adult mouse aged at postnatal month 12, labeled with 3H-leucine showing protein synthesis in the nucleus and a few mitochondria. x

Fig. 25C. High power magnification EMRAG of Fig. 24B, the zona reticularis of an old adult mouse aged at postnatal month 12, showing protein synthesis in a few mitochondria at

Preliminary quantitative analysis on the number of mitochondria in either 10 adreno-cortical cells whose nuclei and cytoplasm were labeled with 3H-leucine showing silver grains and other 10 cells whose nuclei were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated regardless whether their nuclei were labeled or not (Fig. 26A). The results obtained from the number of mitochondria in adreno-cortical cells in the 3 layers of respective animals in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, showed an

**2.3.1.1.1 Number of mitochondria of adreno-cortical cells in aging mice labeled with** 

of Microscopy Vol. 4, p. 54-71, 2011.

a few mitochondria. x 3,000.

upper left corner. x15,000.

3,000

**3H-leucine** 

25A

25A

25B

25C

Fig. 25. EMRAG of the adrenal cortical cells aging mice labeled with 3H-leucine showing protein synthesis in the nucleus as well as in a few mitochondria. From Nagata, T.: Annals of Microscopy Vol. 4, p. 54-71, 2011.

Fig. 25A. EMRAG of the zona glomerulosa of a juvenile mouse at postnatal day 14, labeled with 3H-leucine showing protein synthesis (several silver grains) in the nucleus as well as in a few mitochondria. x 3,000.

Fig. 25B. EMRAG of the zona reticularis of an old adult mouse aged at postnatal month 12, labeled with 3H-leucine showing protein synthesis in the nucleus and a few mitochondria. x 3,000

Fig. 25C. High power magnification EMRAG of Fig. 24B, the zona reticularis of an old adult mouse aged at postnatal month 12, showing protein synthesis in a few mitochondria at upper left corner. x15,000.

#### **2.3.1.1.1 Number of mitochondria of adreno-cortical cells in aging mice labeled with 3H-leucine**

Preliminary quantitative analysis on the number of mitochondria in either 10 adreno-cortical cells whose nuclei and cytoplasm were labeled with 3H-leucine showing silver grains and other 10 cells whose nuclei were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated regardless whether their nuclei were labeled or not (Fig. 26A). The results obtained from the number of mitochondria in adreno-cortical cells in the 3 layers of respective animals in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, showed an

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 403

Fig. 26A.

Fig. 26B.

gradual increase from the prenatal day 19 (glomerulosa 13.5, fasciculata 14.9, reticularis 15.2/cell) to postnatal day 14 (glomerulosa 37.7, fasciculata 37.8, reticularis 39.8/cell), and to adult stages at postnatal month 1 (glomerulosa 41.5, fasciculata 42.3, reticularis 42.9/cell), then increased at month 2 (glomerulosa 64.2, fasciculata 65.1, reticularis 67.2/cell), but kept plateau from month 6 (glomerulosa 61.7, fasciculata 62.9, reticularis 62.1/cell), to month 12 (glomerulosa 59.4, fasciculata 70.5, reticularis 71.4/cell) and month 24 (glomerulosa 59.5, fasciculata 62.2, reticularis 63.3/cell). The increase from embryo day 19 to postnatal month 1 was stochastically significant (P <0.01).

#### **2.3.1.1.2 Number of labeled mitochondria of adreno-cortical cells in aging mice labeled with 3H-leucine**

The results of visual grain counting on the number of mitochondria labeled with silver grains obtained from 10 adreno-cortical cells in the 3 layers of each animal labeled with 3Hleucine demonstrating protein synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 3, 6, 12 and 24, showed that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis per cell gradually increased from prenatal embryo day 19 (glomerulosa 0.3, fasciculata 0.4, reticularis 0.4/cell) to postnatal day 1 (glomerulosa 0.5, fasciculata 0.6, reticularis 0.5/cell), day 3 (glomerulosa 1.2, fasciculata 0.8, reticularis 1.1/cell), day 9 (glomerulosa 0.8, fasciculata 1.1, reticularis 1.1/cell), day 14 (glomerulosa 1.5, fasciculata 1.5, reticularis 1.6/cell), and month 1 (glomerulosa 1.8, fasciculata 1.8, reticularis 2.2/cell) and month 2 (glomerulosa 5.4, fasciculata 5.3, reticularis 5.8/cell), reaching the maximum, then decreased to month 6 (glomerulosa 4.5, fasciculata 4.8, reticularis 5.1/cell), month 12 (glomerulosa 5.2, fasciculata 5.8, reticularis 6.0/cell) and 24 (glomerulosa 3.8, fasciculata 4.1, reticularis 4.3/cell), as demonstrated in Fig. 26B.

#### **2.3.1.1.3 Labeling index of mitochondria of adrenal cortical cells in aging mice labeled with 3H-leucine**

Finally, the labeling indices of adreno-cortical cells showing protein synthesis in respective aging stages were calculated from the number of labeled mitochondria (Fig. 26B) dividing by the number of total mitochondria per cell (Fig.26A), which were plotted in Fig. 26C, respectively.

The results showed that the labeling indices gradually increased from prenatal day 19 (glomerulosa 2.2, fasciculata 2.7, reticularis 2.6%) to postnatal newborn stage at postnatal day 1 (glomerulosa 2.2, fasciculate 2.4, reticularis 2.0%) and day 3 (glomerulosa 4.5 fasciculata 2.9, reticularis 3.9%), and to juvenile stage at postnatal day 9 (glomerulosa 2.8, fasciculate 3.7, reticularis 3.7%), day 14 (glomerulosa 3.9, fasciculate 3.9, reticularis 4.0%) and to the adult stage at month 1 (glomerulosa 4.3, fasciculate 4.2 reticularis 5.1%) and month 2 (glomerulosa 8.5, fasciculate 8.1, reticularis 8.6%), reaching the maximum, and decreased to month 6 (glomerulosa 7.3, fasciculate 7.6, reticularis 8.1%) to month 12 (glomerulosa 8.4, fasciculate 8.2, reticularis 8.4%) and finally to senescence at month 24 (glomerulosa 6.1, fasciculate 6.6 reticularis 6.8%), as is shown in the histogram (Fig. 26C).

From the results obtained it was shown that the labeling indices of the adreno-cortical cells in 3 layers of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 groups gradually increased from prenatal embryo day 19 to postnatal day 1, 3, 9, and 14, month 1, 2, reaching the maximum, and decreased to 6, 12 and 24, due to aging and senescence.

gradual increase from the prenatal day 19 (glomerulosa 13.5, fasciculata 14.9, reticularis 15.2/cell) to postnatal day 14 (glomerulosa 37.7, fasciculata 37.8, reticularis 39.8/cell), and to adult stages at postnatal month 1 (glomerulosa 41.5, fasciculata 42.3, reticularis 42.9/cell), then increased at month 2 (glomerulosa 64.2, fasciculata 65.1, reticularis 67.2/cell), but kept plateau from month 6 (glomerulosa 61.7, fasciculata 62.9, reticularis 62.1/cell), to month 12 (glomerulosa 59.4, fasciculata 70.5, reticularis 71.4/cell) and month 24 (glomerulosa 59.5, fasciculata 62.2, reticularis 63.3/cell). The increase from embryo day 19 to postnatal month 1

**2.3.1.1.2 Number of labeled mitochondria of adreno-cortical cells in aging mice labeled** 

**2.3.1.1.3 Labeling index of mitochondria of adrenal cortical cells in aging mice labeled** 

Finally, the labeling indices of adreno-cortical cells showing protein synthesis in respective aging stages were calculated from the number of labeled mitochondria (Fig. 26B) dividing by the number of total mitochondria per cell (Fig.26A), which were plotted in Fig. 26C,

The results showed that the labeling indices gradually increased from prenatal day 19 (glomerulosa 2.2, fasciculata 2.7, reticularis 2.6%) to postnatal newborn stage at postnatal day 1 (glomerulosa 2.2, fasciculate 2.4, reticularis 2.0%) and day 3 (glomerulosa 4.5 fasciculata 2.9, reticularis 3.9%), and to juvenile stage at postnatal day 9 (glomerulosa 2.8, fasciculate 3.7, reticularis 3.7%), day 14 (glomerulosa 3.9, fasciculate 3.9, reticularis 4.0%) and to the adult stage at month 1 (glomerulosa 4.3, fasciculate 4.2 reticularis 5.1%) and month 2 (glomerulosa 8.5, fasciculate 8.1, reticularis 8.6%), reaching the maximum, and decreased to month 6 (glomerulosa 7.3, fasciculate 7.6, reticularis 8.1%) to month 12 (glomerulosa 8.4, fasciculate 8.2, reticularis 8.4%) and finally to senescence at month 24 (glomerulosa 6.1,

From the results obtained it was shown that the labeling indices of the adreno-cortical cells in 3 layers of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 groups gradually increased from prenatal embryo day 19 to postnatal day 1, 3, 9, and 14, month 1, 2, reaching the maximum, and decreased to 6, 12 and 24, due to aging and

fasciculate 6.6 reticularis 6.8%), as is shown in the histogram (Fig. 26C).

The results of visual grain counting on the number of mitochondria labeled with silver grains obtained from 10 adreno-cortical cells in the 3 layers of each animal labeled with 3Hleucine demonstrating protein synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 3, 6, 12 and 24, showed that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis per cell gradually increased from prenatal embryo day 19 (glomerulosa 0.3, fasciculata 0.4, reticularis 0.4/cell) to postnatal day 1 (glomerulosa 0.5, fasciculata 0.6, reticularis 0.5/cell), day 3 (glomerulosa 1.2, fasciculata 0.8, reticularis 1.1/cell), day 9 (glomerulosa 0.8, fasciculata 1.1, reticularis 1.1/cell), day 14 (glomerulosa 1.5, fasciculata 1.5, reticularis 1.6/cell), and month 1 (glomerulosa 1.8, fasciculata 1.8, reticularis 2.2/cell) and month 2 (glomerulosa 5.4, fasciculata 5.3, reticularis 5.8/cell), reaching the maximum, then decreased to month 6 (glomerulosa 4.5, fasciculata 4.8, reticularis 5.1/cell), month 12 (glomerulosa 5.2, fasciculata 5.8, reticularis 6.0/cell) and 24 (glomerulosa 3.8, fasciculata 4.1, reticularis

was stochastically significant (P <0.01).

4.3/cell), as demonstrated in Fig. 26B.

**with 3H-leucine** 

**with 3H-leucine** 

respectively.

senescence.

Fig. 26B.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 405

2, then decreased at month 24, while the labeling indices increased from perinatal embryonic day to postnatal newborn and juvenile stages at day 9, then decreased from day 14 to senescence at month 24, then decreased to the adult stages at month 1 and 2, to month 6, 12 and 24. These changes demonstrate the chronological aging changes. The results obtained previously (Nagata 2008a,b,c,d, 2009a, Nagata & Ito 1996) indicated that mitochondria in the adreno-cortical cells proliferated from newborn to adult stages around month 1 and 2, showing mitochondrial DNA synthesis, while the mitochondrial RNA synthesis increased from newborn stage to postnatal day 9, then decreased from day 14, reaching the maximum, then decreased to month 24, but the RNA synthetic activity was kept plateau from day 14 to month 12, then decreased to senescent at month 24 due to aging

The adreno-medullary tissues obtained from ddY strain mice at various ages from embryo day 19 to postnatal day 30, consisted mainly of 2 cell types, as observed by electron microscopy, adrenalin cells and noradrenalin cells, developing gradually (Nagata 2010a,b). At embryonic day 19 and postnatal day 1, the adreno-medullary cells were composed mainly of polygonal epitheloid cells, surrounded by blood capillaries and fibroblasts. The medullary cells can be divided into 2 types by the ultrastructure of granules. Some of the medullary cells possessed many granules of medium electron density which were believed to correspond to the adrenalin granules, while some other cells possessed many granules of very high electron density which were believed to correspond to the noradrenalin granules. However, the numbers of mitochondria found in their cytoplasm were not so many. At postnatal day 1, day 3 and day 9, the 2 types of cells differentiated and the numbers of granules, both adrenalin and noradrenalin granules, increased respectively. Likewise, the numbers of mitochondria also increased from prenatal day to postnatal days. At postnatal day 14 to month 1, month 2, month 6, month 12 and month 24 the numbers of adrenalin and noradrenalin granules as well as mitochondria increased. At postnatal month 1 and 2, the ultrastructures of 2 cell types were completely developed and the arrangements of the cells in the medulla became typical as adult tissues. Thus, the ultrastructure of the adrenal medullary cells showed changes due to development and aging at respective developmental stages. The number of mitochondria per cell increased from perinatal stage to postnatal stages due to aging. The data were stochastically analyzed using variance and Student's t-test. The increases of mitochondrial numbers in both adrenalin and noradrenalin cells from embryonic day

**2.3.1.2 Protein synthesis in mitochondria of mouse adreno-medullary cells** 

19 to postnatal month 6 were considered to be significant at P value <0.01.

more day 14 and adults at month 1, month 2, month 6, month 12 and month 24.

Observing electron microscopic radioautograms, the silver grains were found over the nuclei of some adreno-medullary cells labeled with 3H-leucine, demonstrating protein synthesis less in perinatal stages at embryonic day 19, postnatal day 1, day 3, day 9, while

However, those labeled cells were found in all the 2 cell types, adrenalin cells and noradrenalin cells, at respective aging stages. In the labeled adreno-medullary cells the silver grains were mainly localized over the euchromatin of the nuclei and only a few or several silver grains were found over the mitochondria of adrenalin cells. Likewise, most noradrenalin cells were also labeled with several silver grains in their nuclei as well as in their cytoplasm not only over the mitochondria but also over ribosomes, showing protein

(Nagata 2010e).

Fig. 26C.

Fig. 26. Histogram showing aging changes of the mitochondria in each adreno-cortical cell in the 3 layers of respective animals in 10 aging groups. From Nagata, T.: Annals of Microscopy Vol. 4, p. 54-71, 2011.

Fig. 26A. Histogram showing aging changes of the average numbers of mitochondria per cell in each adreno-cortical cell in the 3 layers of respective animals in 10 aging groups Fig. 26B. Histogram showing aging changes of the average numbers of labeled mitochondria with 3H-leucine showing protein synthesis per cell in each adrenocortical cell in the 3 layers of respective animals in 10 aging groups.

Fig. 26C. Histogram showing aging changes of the average labeling index of mitochondria labeled with 3H-leucine showing protein synthesis per cell in each adreno-cortical cell in the 3 layers of respective animals in 10 aging groups.

As for the macromolecular synthesis in various cells in various organs of experimental animals observed by light and electron microscopic radioautography, it is well known that the silver grains due to radiolabeled 3H-thymidine demonstrated DNA synthesis, while the grains due to 3H-uridine demonstrated RNA synthesis. On the other hand, the radioautograms showing incorporations of 3H-leucine into mitochondria indicating mitochondrial protein synthesis (Nagata 2002, 2009c,d,e, 2010a,b,c). It was shown from the results that the silver grains localized over the mitochondria independently from the nuclei whether the nuclei were labeled with silver grains or not in almost all the cells in the 3 layers of the adreno-cortical cells from prenatal embryo day 19 to postnatal month 24, administered with 3H-leucine during the development and aging. The numbers of labeled mitochondria showing protein synthesis increased from perinatal day to postnatal adult stage at month 2, then kept plateau, while the labeled mitochondria with 3H-leucine showing protein synthesis increased from perinatal stage to postnatal adult stage at month

Fig. 26C. Fig. 26. Histogram showing aging changes of the mitochondria in each adreno-cortical cell in the 3 layers of respective animals in 10 aging groups. From Nagata, T.: Annals of

Fig. 26A. Histogram showing aging changes of the average numbers of mitochondria per cell in each adreno-cortical cell in the 3 layers of respective animals in 10 aging groups Fig. 26B. Histogram showing aging changes of the average numbers of labeled mitochondria with 3H-leucine showing protein synthesis per cell in each adrenocortical cell in the 3 layers

Fig. 26C. Histogram showing aging changes of the average labeling index of mitochondria labeled with 3H-leucine showing protein synthesis per cell in each adreno-cortical cell in the

As for the macromolecular synthesis in various cells in various organs of experimental animals observed by light and electron microscopic radioautography, it is well known that the silver grains due to radiolabeled 3H-thymidine demonstrated DNA synthesis, while the grains due to 3H-uridine demonstrated RNA synthesis. On the other hand, the radioautograms showing incorporations of 3H-leucine into mitochondria indicating mitochondrial protein synthesis (Nagata 2002, 2009c,d,e, 2010a,b,c). It was shown from the results that the silver grains localized over the mitochondria independently from the nuclei whether the nuclei were labeled with silver grains or not in almost all the cells in the 3 layers of the adreno-cortical cells from prenatal embryo day 19 to postnatal month 24, administered with 3H-leucine during the development and aging. The numbers of labeled mitochondria showing protein synthesis increased from perinatal day to postnatal adult stage at month 2, then kept plateau, while the labeled mitochondria with 3H-leucine showing protein synthesis increased from perinatal stage to postnatal adult stage at month

Microscopy Vol. 4, p. 54-71, 2011.

of respective animals in 10 aging groups.

3 layers of respective animals in 10 aging groups.

2, then decreased at month 24, while the labeling indices increased from perinatal embryonic day to postnatal newborn and juvenile stages at day 9, then decreased from day 14 to senescence at month 24, then decreased to the adult stages at month 1 and 2, to month 6, 12 and 24. These changes demonstrate the chronological aging changes. The results obtained previously (Nagata 2008a,b,c,d, 2009a, Nagata & Ito 1996) indicated that mitochondria in the adreno-cortical cells proliferated from newborn to adult stages around month 1 and 2, showing mitochondrial DNA synthesis, while the mitochondrial RNA synthesis increased from newborn stage to postnatal day 9, then decreased from day 14, reaching the maximum, then decreased to month 24, but the RNA synthetic activity was kept plateau from day 14 to month 12, then decreased to senescent at month 24 due to aging (Nagata 2010e).

#### **2.3.1.2 Protein synthesis in mitochondria of mouse adreno-medullary cells**

The adreno-medullary tissues obtained from ddY strain mice at various ages from embryo day 19 to postnatal day 30, consisted mainly of 2 cell types, as observed by electron microscopy, adrenalin cells and noradrenalin cells, developing gradually (Nagata 2010a,b). At embryonic day 19 and postnatal day 1, the adreno-medullary cells were composed mainly of polygonal epitheloid cells, surrounded by blood capillaries and fibroblasts. The medullary cells can be divided into 2 types by the ultrastructure of granules. Some of the medullary cells possessed many granules of medium electron density which were believed to correspond to the adrenalin granules, while some other cells possessed many granules of very high electron density which were believed to correspond to the noradrenalin granules. However, the numbers of mitochondria found in their cytoplasm were not so many. At postnatal day 1, day 3 and day 9, the 2 types of cells differentiated and the numbers of granules, both adrenalin and noradrenalin granules, increased respectively. Likewise, the numbers of mitochondria also increased from prenatal day to postnatal days. At postnatal day 14 to month 1, month 2, month 6, month 12 and month 24 the numbers of adrenalin and noradrenalin granules as well as mitochondria increased. At postnatal month 1 and 2, the ultrastructures of 2 cell types were completely developed and the arrangements of the cells in the medulla became typical as adult tissues. Thus, the ultrastructure of the adrenal medullary cells showed changes due to development and aging at respective developmental stages. The number of mitochondria per cell increased from perinatal stage to postnatal stages due to aging. The data were stochastically analyzed using variance and Student's t-test. The increases of mitochondrial numbers in both adrenalin and noradrenalin cells from embryonic day 19 to postnatal month 6 were considered to be significant at P value <0.01.

Observing electron microscopic radioautograms, the silver grains were found over the nuclei of some adreno-medullary cells labeled with 3H-leucine, demonstrating protein synthesis less in perinatal stages at embryonic day 19, postnatal day 1, day 3, day 9, while more day 14 and adults at month 1, month 2, month 6, month 12 and month 24.

However, those labeled cells were found in all the 2 cell types, adrenalin cells and noradrenalin cells, at respective aging stages. In the labeled adreno-medullary cells the silver grains were mainly localized over the euchromatin of the nuclei and only a few or several silver grains were found over the mitochondria of adrenalin cells. Likewise, most noradrenalin cells were also labeled with several silver grains in their nuclei as well as in their cytoplasm not only over the mitochondria but also over ribosomes, showing protein

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 407

**2.3.1.2.3 Labeling index of mitochondria of adreno-medullary cells in aging mice labeled** 

From the results, the labeling indices of mitochondrial protein synthesis in 2 cell types in respective aging stages were calculated from the number of labeled mitochondria dividing

The results showed that the labeling indices gradually increased from prenatal day 19 (adrenalin 2.8, noradrenalin 2.6%) to postnatal newborn day 1 (adrenalin 2.8, noradrenalin 2.4%), day 3 (adrenalin 3.3, noradrenalin 2.9%), day 9 (adrenalin 3.4, noradrenalin 3.3%) to juvenile stage at day 14 (adrenalin 3.6, noradrenalin 3.8%), reaching the maximum, and decreased to adult stages at month 1 (adrenalin 3.6, noradrenalin 3.5%), month 2 (adrenalin 3.1, noradrenalin 3.3%), month 6 (adrenalin 3.2, noradrenalin 3.3%), month 12 (adrenalin 2.8, noradrenalin 3.1%) and month 24 (adrenalin 2.9, noradrenalin 2.8%). From the results, the increases of the mitochondrial labeling indices in both the adrenalin and noradrenalin cells from embryo day 19 to postnatal day 14, as well as the decreases from day 14 to month 24

From the results obtained it was shown that intramitochondrial protein synthesis was observed in adreno-medullary cells, both the adrenalin cells and noradrenalin cells of developing and aging mice at various ages from prenatal embryos to postnatal newborn, young juvenile, adult and senescent stages and the number of mitochondria per cell showed increases due to aging, while the number of labeled mitochondria per cell and the labeling

As for the macromolecular synthesis in various cells in various organs of experimental animals observed by light and electron microscopic radioautography, it is well known that the silver grains due to radiolabeled 3H-thymidine demonstrate DNA synthesis, while the grains due to 3H-uridine demonstrate RNA synthesis and 3H-leucine protein synthesis (Nagata 1996, 1997, 2002, 2010). We formerly observed the intramitochondrial DNA synthesis as well as RNA synthesis in various cells in mice and rats (Nagata 1972a,b,c,d, 1974, 1984, 2001, 2001, Nagata et al. 1975, 1976, 1977). Lately we observed in the intramitochondrial DNA synthesis as well as RNA synthesis in the adrenal cortical and medullary cells, both the adrenalin cells and the noradrenalin cells at various ages from fetal day 19 to postnatal newborn day 1, 3, 9, juvenile day 14 and to adult month 1, 2, 6, 12 and 24 (Nagata 2008a,b, 2009a, 2010b,e), as well as the intramitochondrial protein synthesis during

These results demonstrated that the numbers of silver grains showing nuclear protein synthesis did not give any significant difference between the 2 cells types in the same aging groups from perinatal to senescent stages. The radioautograms showing incorporations of 3H-leucine into mitochondria indicating mitochondrial protein synthesis resulted in silver grain localization over the mitochondria independently from the nuclei whether the nuclei were labeled with silver grains or not in both cell types, adrenalin and noradrenalin cells, in the medullae from prenatal embryo day 19 to postnatal day 1, 3, 9 and 14, to postnatal month 1, 2, 6, 12 and 24, during the development and aging. The numbers of labeled mitochondria showing protein synthesis as well as the labeling indices increased from perinatal embryonic day to postnatal newborn and juvenile stages at day 14 to month 12, reaching the maxima, and then did not decrease to the senescent stages at month 24 (year 2).

**with 3H-leucine** 

by the number of total mitochondria per cell.

were stochastically significant (P <0.01).

indices showed increases and slight decreases due to aging.

the aging and senescence (Nagata 2010d,f,g,h).

synthesis due to the incorporations of 3H-leucine especially at the senescent stages from postnatal month 12 when observed at high power magnifications by high voltage electron microscopy. The localizations of silver grains over the mitochondria were not only on the mitochondrial matrices but also over mitochondrial membranes (Nagata 2010a,b).

#### **2.3.1.2.1 Number of mitochondria of adreno-medullary cells in aging mice labeled with 3H-leucine**

Preliminary quantitative analysis on the number of mitochondria in 10 adreno-medullary cells whose nuclei were labeled with silver grains and other 10 cells whose nuclei were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated in 10 adreno-medullary cells regardless whether their nuclei were labeled or not. The results obtained from the number of mitochondria in adreno-medullary cells of respective animals in 10 aging groups at perinatal stages, from prenatal embryo day 19 to postnatal day 1, 3, 9, 14, and month 1, 2, 6, 12, showed an gradual increase from the prenatal day 19 (adrenalin 17.8, noradrenalin 18.2/cell) to postnatal day 1 (adrenalin 21.5, noradrenalin 22.4/cell), day 3 (adrenalin 22.5, noradrenalin 22.9/cell), day 9 (adrenalin 23.5, noradrenalin 23.9/cell), day 14 (adrenalin 24.1, noradrenalin 24.4/cell), and to adult stages at postnatal month 1 (adrenalin 24.7, noradrenalin 23.9/cell), month 2 (adrenalin 25.1, noradrenalin 24.5/cell) and month 6 (adrenalin 24.8, noradrenalin 24.3/cell), month 12 (adrenalin 24.5, noradrenalin 24.1/cell), and month 24 (adrenalin 23.5, noradrenalin 23.3/cell). The increases of mitochondrial numbers in both adrenalin and noradrenalin cells from embryonic day 19 to postnatal month 2 were considered to be significant at P value <0.01. However, the slight decrease from month 6 to 24 was not significant (Nagata 2010a or b).

#### **2.3.1.2.2 Number of labeled mitochondria of adrenal medullary cells in aging mice labeled with 3H-leucine**

We counted the number of mitochondria labeled with silver grains obtained from 10 adreno-medullary cells in the 3 layers of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 3, 6, 12 and 24, as well as the number of all the mitochondria and calculated the labeling index.

The results demonstrated that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis per cell gradually increased from prenatal embryo day 19 (adrenalin 0.5, noradrenalin 0.5/cell), to postnatal day 1 (adrenalin 0.65, noradrenalin 0.6/cell), day 3 (adrenalin 0.7, noradrenalin 0.75/cell), day 9 (adrenalin 0.8, noradrenalin 0.8/cell), day 14 (adrenalin 0.8, noradrenalin 0.9/cell), reaching the maxima at month 1 (adrenalin 0.9, noradrenalin 0.85/cell), and decreased to month 2 (adrenalin 0.8, noradrenalin 0.82/cell), month 6 (adrenalin 0.81, noradrenalin 0.8/cell) month 12 (adrenalin 0.7, noradrenalin 0.75/cell) and month 24 (adrenalin 0.7, noradrenalin 0.65/cell). The data were stochastically analyzed using variance and Student's t-test. The increases of the numbers of labeled mitochondria in both adrenalin and noradrenalin cells from embryo day 19 to postnatal day 14 and month 1, as well as the decreases from month 1 to month 24 were stochastically significant (P <0.01).

synthesis due to the incorporations of 3H-leucine especially at the senescent stages from postnatal month 12 when observed at high power magnifications by high voltage electron microscopy. The localizations of silver grains over the mitochondria were not only on the

mitochondrial matrices but also over mitochondrial membranes (Nagata 2010a,b).

from month 6 to 24 was not significant (Nagata 2010a or b).

**labeled with 3H-leucine** 

calculated the labeling index.

stochastically significant (P <0.01).

**2.3.1.2.2 Number of labeled mitochondria of adrenal medullary cells in aging mice** 

We counted the number of mitochondria labeled with silver grains obtained from 10 adreno-medullary cells in the 3 layers of each animal labeled with 3H-leucine demonstrating protein synthesis in 10 aging groups at perinatal stages, prenatal embryo day 19, postnatal day 1, 3, 9 and 14, month 1, 3, 6, 12 and 24, as well as the number of all the mitochondria and

The results demonstrated that the numbers of labeled mitochondria with 3H-leucine showing protein synthesis per cell gradually increased from prenatal embryo day 19 (adrenalin 0.5, noradrenalin 0.5/cell), to postnatal day 1 (adrenalin 0.65, noradrenalin 0.6/cell), day 3 (adrenalin 0.7, noradrenalin 0.75/cell), day 9 (adrenalin 0.8, noradrenalin 0.8/cell), day 14 (adrenalin 0.8, noradrenalin 0.9/cell), reaching the maxima at month 1 (adrenalin 0.9, noradrenalin 0.85/cell), and decreased to month 2 (adrenalin 0.8, noradrenalin 0.82/cell), month 6 (adrenalin 0.81, noradrenalin 0.8/cell) month 12 (adrenalin 0.7, noradrenalin 0.75/cell) and month 24 (adrenalin 0.7, noradrenalin 0.65/cell). The data were stochastically analyzed using variance and Student's t-test. The increases of the numbers of labeled mitochondria in both adrenalin and noradrenalin cells from embryo day 19 to postnatal day 14 and month 1, as well as the decreases from month 1 to month 24 were

**3H-leucine** 

**2.3.1.2.1 Number of mitochondria of adreno-medullary cells in aging mice labeled with** 

Preliminary quantitative analysis on the number of mitochondria in 10 adreno-medullary cells whose nuclei were labeled with silver grains and other 10 cells whose nuclei were not labeled in each aging group revealed that there was no significant difference between the number of mitochondria and the labeling indices (P<0.01). Thus, the number of mitochondria and the labeling indices were calculated in 10 adreno-medullary cells regardless whether their nuclei were labeled or not. The results obtained from the number of mitochondria in adreno-medullary cells of respective animals in 10 aging groups at perinatal stages, from prenatal embryo day 19 to postnatal day 1, 3, 9, 14, and month 1, 2, 6, 12, showed an gradual increase from the prenatal day 19 (adrenalin 17.8, noradrenalin 18.2/cell) to postnatal day 1 (adrenalin 21.5, noradrenalin 22.4/cell), day 3 (adrenalin 22.5, noradrenalin 22.9/cell), day 9 (adrenalin 23.5, noradrenalin 23.9/cell), day 14 (adrenalin 24.1, noradrenalin 24.4/cell), and to adult stages at postnatal month 1 (adrenalin 24.7, noradrenalin 23.9/cell), month 2 (adrenalin 25.1, noradrenalin 24.5/cell) and month 6 (adrenalin 24.8, noradrenalin 24.3/cell), month 12 (adrenalin 24.5, noradrenalin 24.1/cell), and month 24 (adrenalin 23.5, noradrenalin 23.3/cell). The increases of mitochondrial numbers in both adrenalin and noradrenalin cells from embryonic day 19 to postnatal month 2 were considered to be significant at P value <0.01. However, the slight decrease

### **2.3.1.2.3 Labeling index of mitochondria of adreno-medullary cells in aging mice labeled with 3H-leucine**

From the results, the labeling indices of mitochondrial protein synthesis in 2 cell types in respective aging stages were calculated from the number of labeled mitochondria dividing by the number of total mitochondria per cell.

The results showed that the labeling indices gradually increased from prenatal day 19 (adrenalin 2.8, noradrenalin 2.6%) to postnatal newborn day 1 (adrenalin 2.8, noradrenalin 2.4%), day 3 (adrenalin 3.3, noradrenalin 2.9%), day 9 (adrenalin 3.4, noradrenalin 3.3%) to juvenile stage at day 14 (adrenalin 3.6, noradrenalin 3.8%), reaching the maximum, and decreased to adult stages at month 1 (adrenalin 3.6, noradrenalin 3.5%), month 2 (adrenalin 3.1, noradrenalin 3.3%), month 6 (adrenalin 3.2, noradrenalin 3.3%), month 12 (adrenalin 2.8, noradrenalin 3.1%) and month 24 (adrenalin 2.9, noradrenalin 2.8%). From the results, the increases of the mitochondrial labeling indices in both the adrenalin and noradrenalin cells from embryo day 19 to postnatal day 14, as well as the decreases from day 14 to month 24 were stochastically significant (P <0.01).

From the results obtained it was shown that intramitochondrial protein synthesis was observed in adreno-medullary cells, both the adrenalin cells and noradrenalin cells of developing and aging mice at various ages from prenatal embryos to postnatal newborn, young juvenile, adult and senescent stages and the number of mitochondria per cell showed increases due to aging, while the number of labeled mitochondria per cell and the labeling indices showed increases and slight decreases due to aging.

As for the macromolecular synthesis in various cells in various organs of experimental animals observed by light and electron microscopic radioautography, it is well known that the silver grains due to radiolabeled 3H-thymidine demonstrate DNA synthesis, while the grains due to 3H-uridine demonstrate RNA synthesis and 3H-leucine protein synthesis (Nagata 1996, 1997, 2002, 2010). We formerly observed the intramitochondrial DNA synthesis as well as RNA synthesis in various cells in mice and rats (Nagata 1972a,b,c,d, 1974, 1984, 2001, 2001, Nagata et al. 1975, 1976, 1977). Lately we observed in the intramitochondrial DNA synthesis as well as RNA synthesis in the adrenal cortical and medullary cells, both the adrenalin cells and the noradrenalin cells at various ages from fetal day 19 to postnatal newborn day 1, 3, 9, juvenile day 14 and to adult month 1, 2, 6, 12 and 24 (Nagata 2008a,b, 2009a, 2010b,e), as well as the intramitochondrial protein synthesis during the aging and senescence (Nagata 2010d,f,g,h).

These results demonstrated that the numbers of silver grains showing nuclear protein synthesis did not give any significant difference between the 2 cells types in the same aging groups from perinatal to senescent stages. The radioautograms showing incorporations of 3H-leucine into mitochondria indicating mitochondrial protein synthesis resulted in silver grain localization over the mitochondria independently from the nuclei whether the nuclei were labeled with silver grains or not in both cell types, adrenalin and noradrenalin cells, in the medullae from prenatal embryo day 19 to postnatal day 1, 3, 9 and 14, to postnatal month 1, 2, 6, 12 and 24, during the development and aging. The numbers of labeled mitochondria showing protein synthesis as well as the labeling indices increased from perinatal embryonic day to postnatal newborn and juvenile stages at day 14 to month 12, reaching the maxima, and then did not decrease to the senescent stages at month 24 (year 2).

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 409

1965) or chicken fibroblasts in tissue culture under abnormal conditions (Chévremont 1963). However, these authors used old-fashioned developers consisting of methol and hydroquinone (MQ-developer) which produced coarse spiral silver grains resulting in inaccurate localization over cell organelles when observed by electron microscopy. All of these authors showed photographs of electron radioautographs with large spiral-formed silver grains (2-3 µm in diameter) localizing not only over the mitochondria but also outside the mitochondria. In order to obtain smaller silver grains, we first used elon-ascorbic acid developer after gold latensification, which produced comma-shaped smaller silver grains (0.4-0.8 µm in diameter), then later we used phenidon developer after gold latensification, producing dot-like smaller silver grains (0.2-0.4 µm in diameter) localizing only inside the mitochondria showing ultrahigh resolution of radioautograms. These papers were the first which demonstrated intramitochondrial DNA synthesis incorporating 3H-thymidine with accurate intramitochondrial localization in avian and mammalian cells. With regards the resolution of electron microscopic radioautography, on the other hand, many authors discussed the sizes of silver grains under various conditions and calculated various values of resolutions (Salpeter et al. 1969, Nadler 1971, Uchida & Mizuhira 1971, Nagata 1972b,c). Those authors who used the M-Q developers maintained the resolution to be 100-160 nm (Salpeter et al. 1969, Nadler 1971), while those authors who used the elon-ascorbic acid developer (Nagata 1972b, , Uchida & Mizuhira 1971) calculated it to be 25-50 nm. When we used phenidon developer at 16C for 1 min after gold latensification, we could produce very fine dot-shaped silver grains and obtained the resolution around 25 nm (Nagata 1992, 1996, 1997, 2001, 2002, Murata et al. 1979). For the analysis of electron radioautographs, Salpeter et al. (1969) proposed to use the half-distance and very complicated calculations through which respective coarse spiral-shaped silver grains were judged to be attributable to the radioactive source in a certain territory within a resolution boundary circle. However, since we used phenidon developer after gold latensification to produce very fine dot-shaped silver grains, we judged only the silver grains which were located in the mitochondria which were dot-shaped very fine ones to be attributable to the mitochondria without any

problem as was formerly discussed (Nagata 1972a,b, c, 1996, 1997, 2001, 2002).

nuclei but also in their mitochondria.

Then we also demonstrated intramitochondrial DNA synthesis incorporating 3H-thymidine in some other established cell lines originated from human being such as HeLa cells (Nagata 1972a,b,c,d) or mitochondrial fractions prepared from in vivo mammalian cells such as rat and mouse (Nagata 1974, Nagata et al. 1975, 1976). It was later commonly found in various cells and tissues not only in vitro obtained from various organs in vivo such as the cultured human HeLa cells (Nagata et al. 1966, 1986, Nagata 1984), cultured rat sarcoma cells (Nagata et al. 1977), mouse liver and pancreas cells in vitro (Nagata & Murata 1977, Nagata et al. 1977, 1986), but also in vivo cells obtained from various organs such as the salivary glands (Nagata et al. 2000), the liver (Nagata 2003, 2006a,b, 2007a,b,c,d,e, Nagata & Ma 2005, Nagata et al. 1979, 1982a,b, Ma & Nagata 1988a,b, Ma et al. 1994), the pancreas (Nagata 1992, Nagata et al. 1986), the trachea (Sun et al. 1997), the lung (Sun et al. 1994, 1995a,b, Nagata 2007), the kidneys (Hanai & Nagata 1994, Nagata 2005), the testis (Gao et al. 1994, 1995), the uterus (Yamada et al. 1993, 1994), the adrenal glands (Ito 1996, Ito et al. 1996, Nagata 2008a,b, 2009g,j, 2010a,b), the brains (Cui et al. 1996), and the retina (Gunarso 1984, Gunarso et al. 1996, 1997, Kong & Nagata 1994, Nagata 1996) of mice, rats and chickens. Thus, it is clear that all the cells in various organs of various animals synthesize DNA not only in their

From the results obtained, it was shown that intramitochondrial protein synthesis was observed in adreno-medullary cells, both the adrenalin cells and noradrenalin cells of developing and aging mice at various ages from prenatal embryos to postnatal newborn, young juvenile, adult and senescent stages and the number of mitochondria per cell showed increases due to aging, while the number of labeled mitochondria per cell and the labeling indices showed increases and slight decreases due to aging (Nagata 2010a,b,c,d,e,f,g,h).

As for the macromolecular synthesis in various cells in various organs of experimental animals observed by light and electron microscopic radioautography, it is well known that the silver grains due to radiolabeled 3H-thymidine demonstrate DNA synthesis, while the grains due to 3H-uridine demonstrate RNA synthesis and 3H-leucine protein synthesis (Nagata et al. 1967a, b, Nagata 1972a, b, c, 2001, 2002). The previous results obtained from the studies on the adreno-cortical cells of aging mice by light microscopic radioautography revealed that silver grains indicating DNA synthesis incorporating 3H-thymidine were observed over the nuclei of some adreno-cortical cells at perinatal stages from postnatal day 1 to day 14 (Ito 1996, Ito & Nagata 1996). However, they did not observe the intramitochondrial RNA synthesis. We formerly observed the intramitochondrial DNA synthesis (Nagata et al. 1967a, b, Nagata 1972a, b, c, 2001, 2002) as well as RNA synthesis (Nagata 1972a,b,c,d, 1974, 1984, 2001, 2002, Nagata et al. 1975, 1976, 1977, Nagata & Murata 1977) in the adrenal medullary cells, both the adrenalin cells and the noradrenalin cells at various ages from fetal day 19 to postnatal newborn day 1, 3, 9, juvenile day 14 and to adult month 1, 2, 6, 12 and 24. In the present study, we also observed the intramitochondrial protein synthesis during the aging and senescence. The numbers of silver grains showing nuclear protein synthesis did not give any significant difference between the 2 cells types in the same aging groups from perinatal to senescent stages. The radioautograms showing incorporations of 3H-leucine into mitochondria indicating mitochondrial protein synthesis resulted in silver grain localization over the mitochondria independently from the nuclei whether the nuclei were labeled with silver grains or not in both cell types, adrenalin and noradrenalin cells, in the medullae from prenatal embryo day 19 to postnatal day 1, 3, 9 and 14, to postnatal month 1, 2, 6, 12 and 24, during the development and aging. The numbers of labeled mitochondria showing protein synthesis as well as the labeling indices increased from perinatal embryonic day to postnatal newborn and juvenile stages at day 14 to month 12, reaching the maxima, and then did not decrease to the senescent stages at month 24 (year 2).

With regards to DNA in mitochondria in animal cells or plastids in plant cells, many studies have been reported in various cells of various plants and animals since 1960s (Nass 1966, Nass and Nass 1963, Gibor and Granick 1964, Gahan and Chayen 1965). Most of these authors observed DNA fibrils in mitochondria which were histochemically extracted by DN'ase. Electron microscopic observation of the DNA molecules isolated from the mitochondria revealed that they were circular in shape, with a circumference of 5-6 µm. It was calculated that such a single molecule had a molecular weight of about 107 daltons (van Bruggen et al. 1966). Mitochondria of various cells also contained a DNA polymerase, which was supposed to function in the replication of the mitochondrial DNA. On the other hand, the incorporations of 3H-thymidine into mitochondria demonstrating DNA synthesis were observed by means of electron microscopic radioautography in lower organism such as slime mold (Guttes and Guttes 1964, Schuster 1965), tetrahymena (Stone and Miller Jr.

From the results obtained, it was shown that intramitochondrial protein synthesis was observed in adreno-medullary cells, both the adrenalin cells and noradrenalin cells of developing and aging mice at various ages from prenatal embryos to postnatal newborn, young juvenile, adult and senescent stages and the number of mitochondria per cell showed increases due to aging, while the number of labeled mitochondria per cell and the labeling indices showed increases and slight decreases due to aging (Nagata

As for the macromolecular synthesis in various cells in various organs of experimental animals observed by light and electron microscopic radioautography, it is well known that the silver grains due to radiolabeled 3H-thymidine demonstrate DNA synthesis, while the grains due to 3H-uridine demonstrate RNA synthesis and 3H-leucine protein synthesis (Nagata et al. 1967a, b, Nagata 1972a, b, c, 2001, 2002). The previous results obtained from the studies on the adreno-cortical cells of aging mice by light microscopic radioautography revealed that silver grains indicating DNA synthesis incorporating 3H-thymidine were observed over the nuclei of some adreno-cortical cells at perinatal stages from postnatal day 1 to day 14 (Ito 1996, Ito & Nagata 1996). However, they did not observe the intramitochondrial RNA synthesis. We formerly observed the intramitochondrial DNA synthesis (Nagata et al. 1967a, b, Nagata 1972a, b, c, 2001, 2002) as well as RNA synthesis (Nagata 1972a,b,c,d, 1974, 1984, 2001, 2002, Nagata et al. 1975, 1976, 1977, Nagata & Murata 1977) in the adrenal medullary cells, both the adrenalin cells and the noradrenalin cells at various ages from fetal day 19 to postnatal newborn day 1, 3, 9, juvenile day 14 and to adult month 1, 2, 6, 12 and 24. In the present study, we also observed the intramitochondrial protein synthesis during the aging and senescence. The numbers of silver grains showing nuclear protein synthesis did not give any significant difference between the 2 cells types in the same aging groups from perinatal to senescent stages. The radioautograms showing incorporations of 3H-leucine into mitochondria indicating mitochondrial protein synthesis resulted in silver grain localization over the mitochondria independently from the nuclei whether the nuclei were labeled with silver grains or not in both cell types, adrenalin and noradrenalin cells, in the medullae from prenatal embryo day 19 to postnatal day 1, 3, 9 and 14, to postnatal month 1, 2, 6, 12 and 24, during the development and aging. The numbers of labeled mitochondria showing protein synthesis as well as the labeling indices increased from perinatal embryonic day to postnatal newborn and juvenile stages at day 14 to month 12, reaching the maxima, and

then did not decrease to the senescent stages at month 24 (year 2).

With regards to DNA in mitochondria in animal cells or plastids in plant cells, many studies have been reported in various cells of various plants and animals since 1960s (Nass 1966, Nass and Nass 1963, Gibor and Granick 1964, Gahan and Chayen 1965). Most of these authors observed DNA fibrils in mitochondria which were histochemically extracted by DN'ase. Electron microscopic observation of the DNA molecules isolated from the mitochondria revealed that they were circular in shape, with a circumference of 5-6 µm. It was calculated that such a single molecule had a molecular weight of about 107 daltons (van Bruggen et al. 1966). Mitochondria of various cells also contained a DNA polymerase, which was supposed to function in the replication of the mitochondrial DNA. On the other hand, the incorporations of 3H-thymidine into mitochondria demonstrating DNA synthesis were observed by means of electron microscopic radioautography in lower organism such as slime mold (Guttes and Guttes 1964, Schuster 1965), tetrahymena (Stone and Miller Jr.

2010a,b,c,d,e,f,g,h).

1965) or chicken fibroblasts in tissue culture under abnormal conditions (Chévremont 1963). However, these authors used old-fashioned developers consisting of methol and hydroquinone (MQ-developer) which produced coarse spiral silver grains resulting in inaccurate localization over cell organelles when observed by electron microscopy. All of these authors showed photographs of electron radioautographs with large spiral-formed silver grains (2-3 µm in diameter) localizing not only over the mitochondria but also outside the mitochondria. In order to obtain smaller silver grains, we first used elon-ascorbic acid developer after gold latensification, which produced comma-shaped smaller silver grains (0.4-0.8 µm in diameter), then later we used phenidon developer after gold latensification, producing dot-like smaller silver grains (0.2-0.4 µm in diameter) localizing only inside the mitochondria showing ultrahigh resolution of radioautograms. These papers were the first which demonstrated intramitochondrial DNA synthesis incorporating 3H-thymidine with accurate intramitochondrial localization in avian and mammalian cells. With regards the resolution of electron microscopic radioautography, on the other hand, many authors discussed the sizes of silver grains under various conditions and calculated various values of resolutions (Salpeter et al. 1969, Nadler 1971, Uchida & Mizuhira 1971, Nagata 1972b,c). Those authors who used the M-Q developers maintained the resolution to be 100-160 nm (Salpeter et al. 1969, Nadler 1971), while those authors who used the elon-ascorbic acid developer (Nagata 1972b, , Uchida & Mizuhira 1971) calculated it to be 25-50 nm. When we used phenidon developer at 16C for 1 min after gold latensification, we could produce very fine dot-shaped silver grains and obtained the resolution around 25 nm (Nagata 1992, 1996, 1997, 2001, 2002, Murata et al. 1979). For the analysis of electron radioautographs, Salpeter et al. (1969) proposed to use the half-distance and very complicated calculations through which respective coarse spiral-shaped silver grains were judged to be attributable to the radioactive source in a certain territory within a resolution boundary circle. However, since we used phenidon developer after gold latensification to produce very fine dot-shaped silver grains, we judged only the silver grains which were located in the mitochondria which were dot-shaped very fine ones to be attributable to the mitochondria without any problem as was formerly discussed (Nagata 1972a,b, c, 1996, 1997, 2001, 2002).

Then we also demonstrated intramitochondrial DNA synthesis incorporating 3H-thymidine in some other established cell lines originated from human being such as HeLa cells (Nagata 1972a,b,c,d) or mitochondrial fractions prepared from in vivo mammalian cells such as rat and mouse (Nagata 1974, Nagata et al. 1975, 1976). It was later commonly found in various cells and tissues not only in vitro obtained from various organs in vivo such as the cultured human HeLa cells (Nagata et al. 1966, 1986, Nagata 1984), cultured rat sarcoma cells (Nagata et al. 1977), mouse liver and pancreas cells in vitro (Nagata & Murata 1977, Nagata et al. 1977, 1986), but also in vivo cells obtained from various organs such as the salivary glands (Nagata et al. 2000), the liver (Nagata 2003, 2006a,b, 2007a,b,c,d,e, Nagata & Ma 2005, Nagata et al. 1979, 1982a,b, Ma & Nagata 1988a,b, Ma et al. 1994), the pancreas (Nagata 1992, Nagata et al. 1986), the trachea (Sun et al. 1997), the lung (Sun et al. 1994, 1995a,b, Nagata 2007), the kidneys (Hanai & Nagata 1994, Nagata 2005), the testis (Gao et al. 1994, 1995), the uterus (Yamada et al. 1993, 1994), the adrenal glands (Ito 1996, Ito et al. 1996, Nagata 2008a,b, 2009g,j, 2010a,b), the brains (Cui et al. 1996), and the retina (Gunarso 1984, Gunarso et al. 1996, 1997, Kong & Nagata 1994, Nagata 1996) of mice, rats and chickens. Thus, it is clear that all the cells in various organs of various animals synthesize DNA not only in their nuclei but also in their mitochondria.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 411

We studied incorporations of mercury chloride into human thyroid tissues of both normal and cancer cells obtained from human patients (Nagata et al. 1977b, Nagata 1994a,b,c,d,e). The tissues were obtained surgically from human patients of both sexes in various ages suffering from the cancer of thyroids and the both normal and cancer cells were cut into small pieces (3x3 mm) aseptically which were incubated in a medium (Eagle's MEM) containing RI-mercury chloride (203HgCl2) and fixed either cryo-fixation at -196ºC and freeze-dried or chemically fixed with buffered glutaraldehyde and osmium tetroxide. The former tissue blocks were processed for dry-mounting radioautography, while the latter were processed for conventional wet-mounting radioautography. The results revealed that the silver grains appeared much more in the cancer cells processed for freeze-fixation and dry-mounting radioatuography (Fig. 21A) than the cancer cells processed for chemical fixation and wet-mounting radioatuography (Fig. 21B), as well as much more in the cancer cells than the normal cells under the same conditions. On the other hand, PCNA/cycline and both keratin kinase C and vimentin were immunostained in connection to DNA synthetic activity. It was found that PCNA/cycline, keratin kinase C and vimentin antibodeis were localized around the filaments in the thyroid cancer cells (Fig. 21C), demonstrating the relation between those antigens and DNA synthetic activity in cell cycle

The nervous system consists of the central nervous system and the peripheral nervous system. The former is divided into the brains and the spinal cord, while the latter into the cerebrospinal system and the autonomous system. We studied macromolecular synthesis of the brains, the spinal cord in the cerebrospinal system and the autonomic peripheral nerves

We studied the DNA synthesis of the brains, the spinal cord in the cerebrospinal system and the autonomic peripheral nerves in the autonomous system by LM and EM RAG (Cui 1995, Cui et al. 1996, Izumiyama et al. 1987, Nagata 1965, 1967a, Nagata and Stegerda 1963, 1964,

The brains of mammals consist of the cerebrum, the cerebellum and the brain stem. We studied on DNA synthesis and protein synthesis in the cerebellum of aging mouse (Cui 1995, Cui et al. 1996) as well as the glucose incorporation in the cerebrum of adult gerbils (Izumiyama et al. 1987). The DNA synthesis was examined in the cerebella of 9 groups of aging ddY strain mice from fetal day 19, to postnatal day 1, 3, 8, 14 and month 1, 2, 6, 12, each consisting of 3 litter animals, using 3H-thymidine, a DNA precursor, by LM and EM RAG (Cui 1995, Cui et al. 1996). The labeled nuclei, by the precursor, in both the neurons and glias, i.e., neuroblasts and glioblasts, were observed in the external granular layers of the cerebella of perinatal mice from embryonic day 19 (Fig. 27A) to postnatal day 1, 3, 7 and day 14 by LMRAG and EMRAG. The labeled nuclei disappeared at postnatal 1 month. The peak of labeling index was at postnatal day 3 in both neuroblasts and glioblasts (Fig. 28A, B). The

**2.4 The localization of mercury in the thyroid gland** 

(Shimizu et al. 1993, Nagata 1994b,c, Gao et al. 1994).

in the autonomous system by LM and EM RAG.

**3.1 The DNA synthesis in the nervous system** 

**3.1.1 The DNA synthesis in the brains** 

Nagata et al. 1999a).

**3. Macromolecular synthesis in the nervous system** 

The relationship between the intramitochondrial DNA synthesis and cell cycle was formerly studied in synchronized cells and it was clarified that the intramitochondrial DNA synthesis was performed without nuclear involvement (Nagata 1972b). However, the relationship between the DNA synthesis and the aging of individual animals and men has not yet been clarified except a few papers recently published by Korr and associates on mouse brain (Korr et al. 1997, 1998, Schmitz et al. 1999a,b,). They reported both nuclear DNA repair, measured as nuclear unscheduled DNA synthesis, and cytoplasmic DNA synthesis labeled with 3H-thymidine in several types of cells in brains such as pyramidal cells, Purkinje cells, granular cells, glial cells, endothelial cells, ependymal cells, epithelial cells as observed by light microscopic radioautography using paraffin sections observed by LMRAG. They observed silver grains over cytoplasm of these cells by light microscopy and maintained that it was reasonable to interpret these labeling as 3H-DNA outside the nuclei, which theoretically belonged to mitochondrial DNA without observing the mitochondria by electron microscopy. From the results, they concluded that distinct types of neuronal cells showed a decline of both unscheduled DNA and mitochondrial DNA syntheses with age in contrast that other cell types, glial and endothelial cells, did not show such age-related changes neither counting the number of mitochondria in respective cells nor counting the labeling indices at respective aging stages. Thus, their results from the statistics obtained from the cytoplasmic grain counting seems to be not accurate without observing mitochondria directly. To the contrary, we had studied DNA synthesis in the livers of aging mice (Nagata et al. 1979, Nagata 1982a,b, 2003, 2005, 2006a,b,c,d,e, 2010c, Ma et al. 1988, 1994, Ma & Nagata 1988) and clearly demonstrated that the number of mitochondria in each hepatocytes, especially mononucleate hepatocytes, increased with the ages of animals from the perinatal stages to adult and senescent stages, while the number of labeled mitochondria and the labeling indices increased from the perinatal stages, reaching a maximum at postnatal day 14, then decreased. We also demonstrated that the number of mitochondria and labeled mitochondria with 3H-thymidine, 3H-uridine and 3H-leucine in the adrenal glands in aging mice increased due to aging (Nagata 2009j, 2010a,b,d,e,f,g,h).

#### **2.3.2 The protein synthesis in the Leydig cells of the testis**

We studied the macromolecular synthesis of the cells in the testis of several groups of litter ddY mice at various ages from fetal day 19 to postnatal aging stages up to 2 years senescence by LM and EM RAG using 3H-thymidine, 3H-uridine and 3H-leucine incorporations (Gao 1993, Gao et al. 1994, 1995a, Nagata 2000b). The incorporation of 3H-leucine into proteins was observed in almost all the Leydig cells in the interstitial tissues of the testis. The silver grains were located over the nuclei and cytoplasm of respective Leydig cells. The aging change of protein synthesis of Leydig cells among different aging groups was also found (Nagata 2001c, 2002). At embryonic day 19, the silver grains of Leydig cells labeled with 3H-leucine was observed in both nucleus and cytoplasm and there was no obvious difference between the number of silver grains on the cytoplasm and the nucleus. The number of silver grains decreased at postnatal day 1 and then increased at day 3 and 7. However, the number of silver grains on both the nucleus and cytoplasm decreased from 1 month to 3 months and increased again from 6 months onwards maintaining a high level from adult to senescent stages. Some of the silver grains were also localized over some of the mitochondria in respective aging groups as observed by EM RAG. These results indicate that the DNA, RNA and protein syntheses in Leydig cells are maintained at rather high level even at senescent stages at postnatal 1 and 2 years when the animals survived for longer lives.

### **2.4 The localization of mercury in the thyroid gland**

410 Senescence

The relationship between the intramitochondrial DNA synthesis and cell cycle was formerly studied in synchronized cells and it was clarified that the intramitochondrial DNA synthesis was performed without nuclear involvement (Nagata 1972b). However, the relationship between the DNA synthesis and the aging of individual animals and men has not yet been clarified except a few papers recently published by Korr and associates on mouse brain (Korr et al. 1997, 1998, Schmitz et al. 1999a,b,). They reported both nuclear DNA repair, measured as nuclear unscheduled DNA synthesis, and cytoplasmic DNA synthesis labeled with 3H-thymidine in several types of cells in brains such as pyramidal cells, Purkinje cells, granular cells, glial cells, endothelial cells, ependymal cells, epithelial cells as observed by light microscopic radioautography using paraffin sections observed by LMRAG. They observed silver grains over cytoplasm of these cells by light microscopy and maintained that it was reasonable to interpret these labeling as 3H-DNA outside the nuclei, which theoretically belonged to mitochondrial DNA without observing the mitochondria by electron microscopy. From the results, they concluded that distinct types of neuronal cells showed a decline of both unscheduled DNA and mitochondrial DNA syntheses with age in contrast that other cell types, glial and endothelial cells, did not show such age-related changes neither counting the number of mitochondria in respective cells nor counting the labeling indices at respective aging stages. Thus, their results from the statistics obtained from the cytoplasmic grain counting seems to be not accurate without observing mitochondria directly. To the contrary, we had studied DNA synthesis in the livers of aging mice (Nagata et al. 1979, Nagata 1982a,b, 2003, 2005, 2006a,b,c,d,e, 2010c, Ma et al. 1988, 1994, Ma & Nagata 1988) and clearly demonstrated that the number of mitochondria in each hepatocytes, especially mononucleate hepatocytes, increased with the ages of animals from the perinatal stages to adult and senescent stages, while the number of labeled mitochondria and the labeling indices increased from the perinatal stages, reaching a maximum at postnatal day 14, then decreased. We also demonstrated that the number of mitochondria and labeled mitochondria with 3H-thymidine, 3H-uridine and 3H-leucine in the adrenal

glands in aging mice increased due to aging (Nagata 2009j, 2010a,b,d,e,f,g,h).

We studied the macromolecular synthesis of the cells in the testis of several groups of litter ddY mice at various ages from fetal day 19 to postnatal aging stages up to 2 years senescence by LM and EM RAG using 3H-thymidine, 3H-uridine and 3H-leucine incorporations (Gao 1993, Gao et al. 1994, 1995a, Nagata 2000b). The incorporation of 3H-leucine into proteins was observed in almost all the Leydig cells in the interstitial tissues of the testis. The silver grains were located over the nuclei and cytoplasm of respective Leydig cells. The aging change of protein synthesis of Leydig cells among different aging groups was also found (Nagata 2001c, 2002). At embryonic day 19, the silver grains of Leydig cells labeled with 3H-leucine was observed in both nucleus and cytoplasm and there was no obvious difference between the number of silver grains on the cytoplasm and the nucleus. The number of silver grains decreased at postnatal day 1 and then increased at day 3 and 7. However, the number of silver grains on both the nucleus and cytoplasm decreased from 1 month to 3 months and increased again from 6 months onwards maintaining a high level from adult to senescent stages. Some of the silver grains were also localized over some of the mitochondria in respective aging groups as observed by EM RAG. These results indicate that the DNA, RNA and protein syntheses in Leydig cells are maintained at rather high level even at senescent stages at postnatal 1 and 2

**2.3.2 The protein synthesis in the Leydig cells of the testis** 

years when the animals survived for longer lives.

We studied incorporations of mercury chloride into human thyroid tissues of both normal and cancer cells obtained from human patients (Nagata et al. 1977b, Nagata 1994a,b,c,d,e). The tissues were obtained surgically from human patients of both sexes in various ages suffering from the cancer of thyroids and the both normal and cancer cells were cut into small pieces (3x3 mm) aseptically which were incubated in a medium (Eagle's MEM) containing RI-mercury chloride (203HgCl2) and fixed either cryo-fixation at -196ºC and freeze-dried or chemically fixed with buffered glutaraldehyde and osmium tetroxide. The former tissue blocks were processed for dry-mounting radioautography, while the latter were processed for conventional wet-mounting radioautography. The results revealed that the silver grains appeared much more in the cancer cells processed for freeze-fixation and dry-mounting radioatuography (Fig. 21A) than the cancer cells processed for chemical fixation and wet-mounting radioatuography (Fig. 21B), as well as much more in the cancer cells than the normal cells under the same conditions. On the other hand, PCNA/cycline and both keratin kinase C and vimentin were immunostained in connection to DNA synthetic activity. It was found that PCNA/cycline, keratin kinase C and vimentin antibodeis were localized around the filaments in the thyroid cancer cells (Fig. 21C), demonstrating the relation between those antigens and DNA synthetic activity in cell cycle (Shimizu et al. 1993, Nagata 1994b,c, Gao et al. 1994).

## **3. Macromolecular synthesis in the nervous system**

The nervous system consists of the central nervous system and the peripheral nervous system. The former is divided into the brains and the spinal cord, while the latter into the cerebrospinal system and the autonomous system. We studied macromolecular synthesis of the brains, the spinal cord in the cerebrospinal system and the autonomic peripheral nerves in the autonomous system by LM and EM RAG.

## **3.1 The DNA synthesis in the nervous system**

We studied the DNA synthesis of the brains, the spinal cord in the cerebrospinal system and the autonomic peripheral nerves in the autonomous system by LM and EM RAG (Cui 1995, Cui et al. 1996, Izumiyama et al. 1987, Nagata 1965, 1967a, Nagata and Stegerda 1963, 1964, Nagata et al. 1999a).

## **3.1.1 The DNA synthesis in the brains**

The brains of mammals consist of the cerebrum, the cerebellum and the brain stem. We studied on DNA synthesis and protein synthesis in the cerebellum of aging mouse (Cui 1995, Cui et al. 1996) as well as the glucose incorporation in the cerebrum of adult gerbils (Izumiyama et al. 1987). The DNA synthesis was examined in the cerebella of 9 groups of aging ddY strain mice from fetal day 19, to postnatal day 1, 3, 8, 14 and month 1, 2, 6, 12, each consisting of 3 litter animals, using 3H-thymidine, a DNA precursor, by LM and EM RAG (Cui 1995, Cui et al. 1996). The labeled nuclei, by the precursor, in both the neurons and glias, i.e., neuroblasts and glioblasts, were observed in the external granular layers of the cerebella of perinatal mice from embryonic day 19 (Fig. 27A) to postnatal day 1, 3, 7 and day 14 by LMRAG and EMRAG. The labeled nuclei disappeared at postnatal 1 month. The peak of labeling index was at postnatal day 3 in both neuroblasts and glioblasts (Fig. 28A, B). The

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 413

from embryonic day 12, 14, 16, 19 and postnatal day 1, 3, 7, 14, 21, 28, 42 and 70, were used. For in situ hybridization, 35S-labeled oligonucleotide probes for TGF-1 were used to detect their messenger RNA. Cryosections were incubated under silicon cover slides with 100 µl of pre-incubation solution plus final concentration of 2.4 x 106 cpm/ml probes and 100 mM DTT for 16 hours. After washing with SSC and DTT, the slides were dried and processed for radioautography by dipping in Konica NR-M2 emulsion, which were exposed and developed. The results showed that TGF-1 mRNA was detectable in the meninges surrounding the spinal cord, but scarcely detected in spinal cord parenchyma (Fig. 27B). The localization of TGF-1 mRNA in the spinal cord suggested that TGF- 1 acted through paracrine mechanism in the morphogenesis of the spinal cord in mice. The localization of TGF-1 and its mRNA in the segments of the spinal cords of mice was also investigated with immunocytochemical techniques (Nagata et al. 1999). The tissues of lower cervical segments of the spinal cords of BALB/c mice, from embryonic day 12, 14, 16, 19 and postnatal day 1, 3, 7, 14, 21, 28, 42 and 70, the same as in situ hybridization were used. For immunocytochemistry, transverse cryosections of the spinal cords were cut and stained with rabbit anti-TGF-1 polyclonal antibody followed with ABC method. The results showed that positive immunoreactivities arose in the ventral horn motoneurons from the embryonic stage to postnatal neonates (Fig. 27B) up to the adults. The extracellular matrix of the white matter, however, showed positive immunocytochemical staining from postnatal day 14, and thereafter, and the immunoreactivity remained with aging. The whole white matter showed only background level of staining before postnatal day 14. The results indicated that TGF-1 regulates motoneuron growth and differentiation as well as they were probably correlated with formation, differentiation and regeneration of myelin of nerve tracts. The immunostaining with FGF antibody presented the same basal pattern as shown in TGF1 immunohistochemistry (Nagata and Kong 1988). The positive immunoreactivities were detected in ganglion cell layer, inner and outer plexiform layers, retinal pigment epithelial layer, choroidal and scleral layers. Since TGF-1 mRNA was detectable in the meninges surrounding the spinal cord by in situ hybridization but scarcely detected in spinal cord parenchyma, the disparate localization of TGF-1 polypeptide and TGF-1 mRNA in the spinal cord suggest that TGF-1 acts through paracrine mechanism in the morphogenesis of the spinal cord in mice. The negative control abolished virtually all reactivity when using the normal rabbit serum instead of primary antibody or using avidin-biotin-peroxidase complex

When 10 groups of aging ddY mice from fetal day 19, to postnatal day 1, 3, 7, 14 and month 1, 2, 6, 12 and 24, each consisting of 3 litter mates, using 3H-leucine, protein syntheses of both neuroblasts and glioblasts were observed by LM and EM RAG in the extragranular layers of perinatal animals (Cui 1995, Cui et al. 1996, 2000, Nagata et al. 2001). The silver grains due to 3H-leucine demonstrating protein synthesis were localized over the nuclei and cytoplasm of neuroblasts and glioblasts of embryos at fetal day 19 and the number of silver grains increased after birth from postnatal day 1, 3 to day 7 and onward. On day 3, some Purkinje cells were recognized incorporating silver grains. The number of silver grains in these cells increased from neonatal stages to mature adult stage at postnatal day 14 and 30, then decreased from month 1, 2, 6, 12 to 24. The increase and decrease of the silver grains were due to the aging changes of protein synthesis in the cerebella due to development and

solution only.

**3.3 The protein synthesis in the cerebellum** 

senescence of individual animals.

glioblasts of the external granular layer migrated inward, some of them formed the Bergmann glia cells located between Purkinje cells. Labeled nuclei of neuroblasts and glioblasts in the internal granular layers were observed at perinatal stages. The maximum of the labeling index in the internal granular layer was at postnatal day 3, similarly to the external granular layer. The endothelial cells of the cerebellar vessels were progressively labeled from embryos to neonates, reaching the peak at 1 week after birth and decreasing thereafter.

## **3.1.2 The DNA synthesis in the peripheral nerves**

We first studied the degeneration and regeneration of autonomous nerve cells in the plexuses of Auerbach and Meissner of the jejunums of 15 dogs which were operated upon to produce experimental ischemia of the jejunal loops by perfusing with Tyrode's solution via the mesenteric arteries for 1, 2, 3 and 4 hours (Nagata 1965, 1967, Nagata and Steggerda 1963, 1964). Tissue blocks were obtained from the deganglionated portions and the adjoining normal portions, which were fixed in Carnoy's fluid, embedded in paraffin, sectioned and stained with buffered thionine, methyl-green and pyronine and PAS. Some animals were injected with either 3H-thymidine or 3H-cytidine and the intestinal tissues obtained from ischemic portions and normal portions were processed for LM RAG. The results revealed that the ganglion cells in Auerbach's plexus showed various degenerative changes in accordance with the duration of ischemia. After 4 hours ischemia, most of the ganglion cells in Auerbach's plexus were completely destroyed. The degenerative changes in Auerbach's plexus after 4 hours ischemia were irreversible after 1 week recovery. The ganglion cells in the Meissner's plexus, on the other hand, were less sensitive to the ischemia. They recovered completely even after 4 hours ischemia. The PAS positive substances in degenerative ganglion cells in both plexuses decreased immediately after 4 hours ischemia. The DNA contents of ganglion cells in both Auerbach's and Meissner's plexus did not show any change before and after ischemia. The RNA contents decreased immediately after the ischemia (Nagata and Steggerda 1963, 1964). The number of binucleate cells in ganglion cells in both Auerbach's and Meissner's plexuses after 4 hours ischemia increased to 4.6% and 5.7% respectively. In contrast, in the non-ischemic normal control preparations, the binucleate cells occurred only 0.5% and 1.8% in Auerbach's and Meissner's plexus respectively. The high frequency of binucleate cells in the ganglion cells persisted for more than 100 days after the ischemia, indicating a possible regeneration of ganglion cells. The radioautographic study revealed that there was no evidence for DNA synthesis in both Auerbach's and Meissner's plexus from either ischemic or normal loops. The RNA synthesis was observed to be higher in gaglion cells in normal loop than ischemic loop and higher in Auerbach's than in Meissner's as expressed by grain counting. It was higher in binucleate ganglion cells than in mononucleate cells.

#### **3.2 The RNA synthesis in the nervous system**

We studied only messenger RNA in the spinal cords of aging mice from perinatal to postnatal adult stages by means of in situ hybridizaion.

#### **3.2.1 The RNA synthesis in the spinal cord**

The localization of TGF-1 mRNA in the segments of the spinal cords of mice was investigated by means of in situ hybridization techniques together with immunohistochemical staining (Nagata et al. 1999). The tissues of lower cervical segments of the spinal cords of BALB/c mice,

glioblasts of the external granular layer migrated inward, some of them formed the Bergmann glia cells located between Purkinje cells. Labeled nuclei of neuroblasts and glioblasts in the internal granular layers were observed at perinatal stages. The maximum of the labeling index in the internal granular layer was at postnatal day 3, similarly to the external granular layer. The endothelial cells of the cerebellar vessels were progressively labeled from embryos to

We first studied the degeneration and regeneration of autonomous nerve cells in the plexuses of Auerbach and Meissner of the jejunums of 15 dogs which were operated upon to produce experimental ischemia of the jejunal loops by perfusing with Tyrode's solution via the mesenteric arteries for 1, 2, 3 and 4 hours (Nagata 1965, 1967, Nagata and Steggerda 1963, 1964). Tissue blocks were obtained from the deganglionated portions and the adjoining normal portions, which were fixed in Carnoy's fluid, embedded in paraffin, sectioned and stained with buffered thionine, methyl-green and pyronine and PAS. Some animals were injected with either 3H-thymidine or 3H-cytidine and the intestinal tissues obtained from ischemic portions and normal portions were processed for LM RAG. The results revealed that the ganglion cells in Auerbach's plexus showed various degenerative changes in accordance with the duration of ischemia. After 4 hours ischemia, most of the ganglion cells in Auerbach's plexus were completely destroyed. The degenerative changes in Auerbach's plexus after 4 hours ischemia were irreversible after 1 week recovery. The ganglion cells in the Meissner's plexus, on the other hand, were less sensitive to the ischemia. They recovered completely even after 4 hours ischemia. The PAS positive substances in degenerative ganglion cells in both plexuses decreased immediately after 4 hours ischemia. The DNA contents of ganglion cells in both Auerbach's and Meissner's plexus did not show any change before and after ischemia. The RNA contents decreased immediately after the ischemia (Nagata and Steggerda 1963, 1964). The number of binucleate cells in ganglion cells in both Auerbach's and Meissner's plexuses after 4 hours ischemia increased to 4.6% and 5.7% respectively. In contrast, in the non-ischemic normal control preparations, the binucleate cells occurred only 0.5% and 1.8% in Auerbach's and Meissner's plexus respectively. The high frequency of binucleate cells in the ganglion cells persisted for more than 100 days after the ischemia, indicating a possible regeneration of ganglion cells. The radioautographic study revealed that there was no evidence for DNA synthesis in both Auerbach's and Meissner's plexus from either ischemic or normal loops. The RNA synthesis was observed to be higher in gaglion cells in normal loop than ischemic loop and higher in Auerbach's than in Meissner's as expressed by grain counting. It was

neonates, reaching the peak at 1 week after birth and decreasing thereafter.

**3.1.2 The DNA synthesis in the peripheral nerves** 

higher in binucleate ganglion cells than in mononucleate cells.

We studied only messenger RNA in the spinal cords of aging mice from perinatal to

The localization of TGF-1 mRNA in the segments of the spinal cords of mice was investigated by means of in situ hybridization techniques together with immunohistochemical staining (Nagata et al. 1999). The tissues of lower cervical segments of the spinal cords of BALB/c mice,

**3.2 The RNA synthesis in the nervous system** 

**3.2.1 The RNA synthesis in the spinal cord** 

postnatal adult stages by means of in situ hybridizaion.

from embryonic day 12, 14, 16, 19 and postnatal day 1, 3, 7, 14, 21, 28, 42 and 70, were used. For in situ hybridization, 35S-labeled oligonucleotide probes for TGF-1 were used to detect their messenger RNA. Cryosections were incubated under silicon cover slides with 100 µl of pre-incubation solution plus final concentration of 2.4 x 106 cpm/ml probes and 100 mM DTT for 16 hours. After washing with SSC and DTT, the slides were dried and processed for radioautography by dipping in Konica NR-M2 emulsion, which were exposed and developed. The results showed that TGF-1 mRNA was detectable in the meninges surrounding the spinal cord, but scarcely detected in spinal cord parenchyma (Fig. 27B). The localization of TGF-1 mRNA in the spinal cord suggested that TGF- 1 acted through paracrine mechanism in the morphogenesis of the spinal cord in mice. The localization of TGF-1 and its mRNA in the segments of the spinal cords of mice was also investigated with immunocytochemical techniques (Nagata et al. 1999). The tissues of lower cervical segments of the spinal cords of BALB/c mice, from embryonic day 12, 14, 16, 19 and postnatal day 1, 3, 7, 14, 21, 28, 42 and 70, the same as in situ hybridization were used. For immunocytochemistry, transverse cryosections of the spinal cords were cut and stained with rabbit anti-TGF-1 polyclonal antibody followed with ABC method. The results showed that positive immunoreactivities arose in the ventral horn motoneurons from the embryonic stage to postnatal neonates (Fig. 27B) up to the adults. The extracellular matrix of the white matter, however, showed positive immunocytochemical staining from postnatal day 14, and thereafter, and the immunoreactivity remained with aging. The whole white matter showed only background level of staining before postnatal day 14. The results indicated that TGF-1 regulates motoneuron growth and differentiation as well as they were probably correlated with formation, differentiation and regeneration of myelin of nerve tracts. The immunostaining with FGF antibody presented the same basal pattern as shown in TGF1 immunohistochemistry (Nagata and Kong 1988). The positive immunoreactivities were detected in ganglion cell layer, inner and outer plexiform layers, retinal pigment epithelial layer, choroidal and scleral layers. Since TGF-1 mRNA was detectable in the meninges surrounding the spinal cord by in situ hybridization but scarcely detected in spinal cord parenchyma, the disparate localization of TGF-1 polypeptide and TGF-1 mRNA in the spinal cord suggest that TGF-1 acts through paracrine mechanism in the morphogenesis of the spinal cord in mice. The negative control abolished virtually all reactivity when using the normal rabbit serum instead of primary antibody or using avidin-biotin-peroxidase complex solution only.

#### **3.3 The protein synthesis in the cerebellum**

When 10 groups of aging ddY mice from fetal day 19, to postnatal day 1, 3, 7, 14 and month 1, 2, 6, 12 and 24, each consisting of 3 litter mates, using 3H-leucine, protein syntheses of both neuroblasts and glioblasts were observed by LM and EM RAG in the extragranular layers of perinatal animals (Cui 1995, Cui et al. 1996, 2000, Nagata et al. 2001). The silver grains due to 3H-leucine demonstrating protein synthesis were localized over the nuclei and cytoplasm of neuroblasts and glioblasts of embryos at fetal day 19 and the number of silver grains increased after birth from postnatal day 1, 3 to day 7 and onward. On day 3, some Purkinje cells were recognized incorporating silver grains. The number of silver grains in these cells increased from neonatal stages to mature adult stage at postnatal day 14 and 30, then decreased from month 1, 2, 6, 12 to 24. The increase and decrease of the silver grains were due to the aging changes of protein synthesis in the cerebella due to development and senescence of individual animals.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 415

Fig. 27A. LM RAG of a prenatal day 19 mouse cerebellum labeled with 3H-thymidine,

Fig. 27B. LM RAG of the spinal cord of a postnatal day 14 mouse immunostained with rabbit anti-TGF-polyclonal IgG followed by ABC method, showing that the ventral horn

Fig. 27C. LM RAG of the optic vesicle of a day 2 chick embryo labeled with 3H-thymidine,

Fig. 27E. Dark-field LM photo of the scleral layer (top), choroid layer (middle) and pigment epithelium (bottom) of an adult 1 month old mouse demonstrating intense silver grains by

Fig. 27F. Bright-field LM photo of the scleral layer (top), choroid layer (middle) and pigment epithelium (bottom) of an adult 1 month old mouse demonstrating intense silver grains by

Fig. 27H. LM RAG of the skin of the fore-limb of a salamander at 6weeks after hatching

The incorporation of 3H-deoxyglucose was studied in the adult gerbil brains among the nervous system of experimental animals (Izumiyama et al. 1987). The changes of soluble deoxyglucose uptake in the hippocampus were studied after 3H-deoxyglucose injections by means of cryo-fixation, freeze-substitution and dry-mounting radioautography to demonstrate soluble compounds under normal and post-ischemic conditions. The results demonstrated that the neurons in the hippocampus subjected to ischemia revealed higher uptake of soluble glucose than normal control. The concentration of soluble 3Hdeoxyglucose was higher than the chemically fixed and wet-mounted radioautograms that demonstrated only insoluble compounds. However, the relation of glycogen synthesis to

The sensory system consists of five organs, i.e., the visual organ or the eye, the stato-acoustic organ or the ear, the gustatory organ or the tongue, the olfactory organ or the nose, and the dermis or the skin. Among these sensory organs, we mainly studied the visual organ and the skin (Gunarso 1983a,b, Gunarso et al. 1996, 1997, Gao et al. 1992a,b, 1993, Kong 1993, Kong and Nagata 1994, Kong et al. 1992a,b, Nagata 1998, 1999, 2000, Nagata et al. 1994,

Among the sensory organs, we mainly studied the DNA synthesis of the visual organ and

Fig. 27G. LM RAG of the cornea of a postnatal day 14 mouse labeled with 3H-thymidine, showing DNA synthesis in the epithelial nucleus (arrow) as well

labeled with 3H-thymidine, showing DNA synthesis. x900.

**4. Macromolecular synthesis in the sensory system** 

**4.1 The DNA synthesis in the sensory organs** 

the skin. They should be described separately.

Fig. 27D. LM RAG of the optic vesicle of a day 2 chick embryo labeled with 3H-uridine,

showing DNA synthesis. x900.

showing DNA synthesis. x750.

showing RNA synthesis. x750.

as in the stroma. x900.

motoneurons are strongly positive. x70.

in situ hybridization for TGF-mRNA. x450.

in situ hybridization for TGF-mRNA. x450.

**3.4 The glucide synthesis in the brains** 

aging has not yet been fully clarified.

Toriyama 1995).

Fig. 27. LM RAG of the neuro-sensory cells. From Nagata, T., Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Jeon, K. W. Ed., Academic Press, San Diego, USA, London, UK, Vol. 211, No. 1, p. 122, 2001.

Fig. 27. LM RAG of the neuro-sensory cells. From Nagata, T., Special Cytochemistry in Cell Biology, In, Internat. Rev. Cytol. Jeon, K. W. Ed., Academic Press, San Diego, USA, London,

UK, Vol. 211, No. 1, p. 122, 2001.

Fig. 27A. LM RAG of a prenatal day 19 mouse cerebellum labeled with 3H-thymidine, showing DNA synthesis. x900.

Fig. 27B. LM RAG of the spinal cord of a postnatal day 14 mouse immunostained with rabbit anti-TGF-polyclonal IgG followed by ABC method, showing that the ventral horn motoneurons are strongly positive. x70.

Fig. 27C. LM RAG of the optic vesicle of a day 2 chick embryo labeled with 3H-thymidine, showing DNA synthesis. x750.

Fig. 27D. LM RAG of the optic vesicle of a day 2 chick embryo labeled with 3H-uridine, showing RNA synthesis. x750.

Fig. 27E. Dark-field LM photo of the scleral layer (top), choroid layer (middle) and pigment epithelium (bottom) of an adult 1 month old mouse demonstrating intense silver grains by in situ hybridization for TGF-mRNA. x450.

Fig. 27F. Bright-field LM photo of the scleral layer (top), choroid layer (middle) and pigment epithelium (bottom) of an adult 1 month old mouse demonstrating intense silver grains by in situ hybridization for TGF-mRNA. x450.

Fig. 27G. LM RAG of the cornea of a postnatal day 14 mouse labeled with

3H-thymidine, showing DNA synthesis in the epithelial nucleus (arrow) as well as in the stroma. x900.

Fig. 27H. LM RAG of the skin of the fore-limb of a salamander at 6weeks after hatching labeled with 3H-thymidine, showing DNA synthesis. x900.

## **3.4 The glucide synthesis in the brains**

The incorporation of 3H-deoxyglucose was studied in the adult gerbil brains among the nervous system of experimental animals (Izumiyama et al. 1987). The changes of soluble deoxyglucose uptake in the hippocampus were studied after 3H-deoxyglucose injections by means of cryo-fixation, freeze-substitution and dry-mounting radioautography to demonstrate soluble compounds under normal and post-ischemic conditions. The results demonstrated that the neurons in the hippocampus subjected to ischemia revealed higher uptake of soluble glucose than normal control. The concentration of soluble 3Hdeoxyglucose was higher than the chemically fixed and wet-mounted radioautograms that demonstrated only insoluble compounds. However, the relation of glycogen synthesis to aging has not yet been fully clarified.

## **4. Macromolecular synthesis in the sensory system**

The sensory system consists of five organs, i.e., the visual organ or the eye, the stato-acoustic organ or the ear, the gustatory organ or the tongue, the olfactory organ or the nose, and the dermis or the skin. Among these sensory organs, we mainly studied the visual organ and the skin (Gunarso 1983a,b, Gunarso et al. 1996, 1997, Gao et al. 1992a,b, 1993, Kong 1993, Kong and Nagata 1994, Kong et al. 1992a,b, Nagata 1998, 1999, 2000, Nagata et al. 1994, Toriyama 1995).

#### **4.1 The DNA synthesis in the sensory organs**

Among the sensory organs, we mainly studied the DNA synthesis of the visual organ and the skin. They should be described separately.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 417

The visual organ consists of the eye and its accessory organs. The eye of mammals consists of the cornea, iris, cilliary body, lens, retina, choroid and sclera. We studied mainly the macromolecular synthesis in the retina of chickens and mice (Nagata 2000f). The nucleic acid syntheses, both DNA and RNA, were first studied in the ocular tissues of white Leghorn chick embryos from day 1 to day 14 incubations by LM and EMRAG (Gunarso 1984a,b, Gunarso et al. 1997, Gao et al. 1992a,b, 1993, Kong 1993, Kong and Nagata 1994, Kong et al. 1992a,b, Nagata et al. 1994). It was shown that the labeled cells with silver grains due to 3H-thymidine were most frequently observed in the nuclei of the retinal cells in the posterior region of the day 2 chick embryo optic vesicle (Fig. 27C) and the labeled cells moved from anterior to posterior regions due to aging by incubation in vitro. The number of labeled cells as expressed by labeling index (%), was more in the posterior regions than the anterior and the equatorial regions and more in the outer portions than in the inner portions at day 2, but the labeling index became more in the anterior regions than the equatorial and posterior regions at day 3, 4 and 7 and it became more in the inner portions than in the outer portions at day 7, decreasing from day 2 to 3, 4 and 7 in each regions (Fig. 28). On the other hand, the silver grains due to 3H-uridine were observed over the nuclei and cytoplasm of all retinal cells from day 2 to 7 (Fig. 27D) and the number of silver grains incorporating 3Huridine increased from day 1 to day 7 and it was more in the anterior regions than in the posterior regions at the same stage (Gunarso et al. 1996). On the other hand, DNA and RNA syntheses in the ocular tissues of aging ddY mice were also studied (Gao et al. 1993, Kong and Nagata 1994, Kong et al. 1992a,b). The ocular tissues taken out from several groups of litter ddY mice at ages varying from fetal day 9, 12, 14, 16, 19 to postnatal day 1, 3, 7, 14 were labeled with 3H-thymidine in vitro and radioautographed (Gao et al. 1992a,b, 1993, Kong 1993, Kong et al. 1992a,b, Toriyama 1995). Silver grains showing DNA synthesis were localized over the nuclei of retinal cells and pigment epithelial cells in the anterior, equatorial and posterior regions of perinatal animals (Fig. 27A). The labeling indices of the retina and pigment epithelium were higher in earlier stages than in later stages, during which they steadily declined (Fig. 28A,B). However, the retina and the pigment epithelium followed different courses in their changes of labeling indices during embryonic development. In the retina, the labeling indices in the vitreal portions were more than those in the scleral portions during the earlier stages. However, the indices of scleral portions were more than those in the vitreal portions in the later stages. Comparing the three regions of the retinae of mice, the anterior, equatorial and posterior regions, the labeling indices of the anterior region were generally higher than those of the equatorial and posterior regions (Fig. 28A). In the pigment epithelium (Fig. 28B), the labeling indices gradually increased in the anterior region, but decreased in the equatorial and the posterior regions through all developmental stages. These results suggest that the proliferation of both the retina and pigment epithelium in the central region occurred earlier than those of the peripheral regions (Nagata 1999a, Gao et al. 1992a,b, Kong 1993, Kong and Nagata 1994, Kong et al. 1992a, b). In the juvenile and adult stages, however, the labeled cells were localized at the middle of the bipolar-photoreceptor layer of the retina, where was supposed to be the

Fig. 28A. The neuroblasts in the extragranular layer of the cerebella. Fig. 28B. The glioblasts in the extragranular layer of the cerebella.

**4.1.1 The DNA synthesis in the visual organ** 

undifferentiated zone.

Fig. 28. Transitional curves of the labeling indices of respective cell types in the cerebella of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 186, 2002, Urban & Fischer, Jena, Germany

Fig. 28A. The neuroblasts in the extragranular layer of the cerebella. Fig. 28B. The glioblasts in the extragranular layer of the cerebella.

#### **4.1.1 The DNA synthesis in the visual organ**

416 Senescence

Fig. 28. Transitional curves of the labeling indices of respective cell types in the cerebella of

aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 186, 2002, Urban & Fischer, Jena, Germany The visual organ consists of the eye and its accessory organs. The eye of mammals consists of the cornea, iris, cilliary body, lens, retina, choroid and sclera. We studied mainly the macromolecular synthesis in the retina of chickens and mice (Nagata 2000f). The nucleic acid syntheses, both DNA and RNA, were first studied in the ocular tissues of white Leghorn chick embryos from day 1 to day 14 incubations by LM and EMRAG (Gunarso 1984a,b, Gunarso et al. 1997, Gao et al. 1992a,b, 1993, Kong 1993, Kong and Nagata 1994, Kong et al. 1992a,b, Nagata et al. 1994). It was shown that the labeled cells with silver grains due to 3H-thymidine were most frequently observed in the nuclei of the retinal cells in the posterior region of the day 2 chick embryo optic vesicle (Fig. 27C) and the labeled cells moved from anterior to posterior regions due to aging by incubation in vitro. The number of labeled cells as expressed by labeling index (%), was more in the posterior regions than the anterior and the equatorial regions and more in the outer portions than in the inner portions at day 2, but the labeling index became more in the anterior regions than the equatorial and posterior regions at day 3, 4 and 7 and it became more in the inner portions than in the outer portions at day 7, decreasing from day 2 to 3, 4 and 7 in each regions (Fig. 28). On the other hand, the silver grains due to 3H-uridine were observed over the nuclei and cytoplasm of all retinal cells from day 2 to 7 (Fig. 27D) and the number of silver grains incorporating 3Huridine increased from day 1 to day 7 and it was more in the anterior regions than in the posterior regions at the same stage (Gunarso et al. 1996). On the other hand, DNA and RNA syntheses in the ocular tissues of aging ddY mice were also studied (Gao et al. 1993, Kong and Nagata 1994, Kong et al. 1992a,b). The ocular tissues taken out from several groups of litter ddY mice at ages varying from fetal day 9, 12, 14, 16, 19 to postnatal day 1, 3, 7, 14 were labeled with 3H-thymidine in vitro and radioautographed (Gao et al. 1992a,b, 1993, Kong 1993, Kong et al. 1992a,b, Toriyama 1995). Silver grains showing DNA synthesis were localized over the nuclei of retinal cells and pigment epithelial cells in the anterior, equatorial and posterior regions of perinatal animals (Fig. 27A). The labeling indices of the retina and pigment epithelium were higher in earlier stages than in later stages, during which they steadily declined (Fig. 28A,B). However, the retina and the pigment epithelium followed different courses in their changes of labeling indices during embryonic development. In the retina, the labeling indices in the vitreal portions were more than those in the scleral portions during the earlier stages. However, the indices of scleral portions were more than those in the vitreal portions in the later stages. Comparing the three regions of the retinae of mice, the anterior, equatorial and posterior regions, the labeling indices of the anterior region were generally higher than those of the equatorial and posterior regions (Fig. 28A). In the pigment epithelium (Fig. 28B), the labeling indices gradually increased in the anterior region, but decreased in the equatorial and the posterior regions through all developmental stages. These results suggest that the proliferation of both the retina and pigment epithelium in the central region occurred earlier than those of the peripheral regions (Nagata 1999a, Gao et al. 1992a,b, Kong 1993, Kong and Nagata 1994, Kong et al. 1992a, b). In the juvenile and adult stages, however, the labeled cells were localized at the middle of the bipolar-photoreceptor layer of the retina, where was supposed to be the undifferentiated zone.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 419

Fig. 30. Transitional curves of the labeling indices in the three layers of the central area of the cornea of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard

Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 192, 2002, Urban & Fischer, Jena, Germany

Fig. 30A. The labeling index in the epithelium. Fig. 30B. The labeling index in the stroma. Fig. 30C. The labeling index in the endothelium.

Fig. 29. Transitional curves of the labeling indices in the three regions (A: anterior, E: eqauator, P: posterior) of the retina and the pigment epithelium of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 191, 2002, Urban & Fischer, Jena, Germany

Fig. 29A. The labeling index in the retina.

Fig. 29B. The labeling index in the pigment epithelium.

Fig. 29. Transitional curves of the labeling indices in the three regions (A: anterior, E: eqauator, P: posterior) of the retina and the pigment epithelium of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2,

p. 191, 2002, Urban & Fischer, Jena, Germany Fig. 29A. The labeling index in the retina.

Fig. 29B. The labeling index in the pigment epithelium.

Fig. 30. Transitional curves of the labeling indices in the three layers of the central area of the cornea of aging mice labeled with 3H-thymidine, showing DNA synthesis. Mean ± Standard Deviation. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p. 192, 2002, Urban & Fischer, Jena, Germany Fig. 30A. The labeling index in the epithelium. Fig. 30B. The labeling index in the stroma.

Fig. 30C. The labeling index in the endothelium.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 421

the labeling index of the epidermal cells was higher than the cartilage cells. The index of the dermal cells in the hind-limbs was at its maximum about 25% at 4 weeks, and fell down markedly with time from 6 weeks to 9 weeks. The labeling index of epidermal cells of the hind-limbs, on the other hand, had its maximum about 23% at 6 weeks, increasing from 20% at 4 weeks, and decreasing to about 18% at 8 weeks, then fell progressively with time, dropped to 5% at 9 weeks (Fig. 31). The labeling indices of the epidermal cells of both forelimbs and hind-limbs were almost the same from 9 weeks to 12 months, keeping low constant level about 4-5%, but never reaching 0. These results indicated that the cutaneous

Fig. 31. Transitional curves of the labeling indices of the epithelial cells in the epidermis of the fore-limbs (F) and the hind-limbs (H) of the salamanders at various ages from 4 weeks to

Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2,

We studied only the RNA synthesis in the chicken and mouse eyes among of the sensory

The RNA synthesis in the chicken eyes was studied with the ocular tissues of chicken embryos in incubation (Fig. 27D). Silver grains due to the incorporations of 3H-uridine were observed over all the nuclei, cell organelles, cytoplasm of all the cells in the optic cups in development showing the RNA synthesis (Gunarso 1984a,b, Gunarso et al. 1969). Grain counting revealed that the counts gradually increased from day 2 to 7 and the numbers of silver grains were the most in the nuclei, while the numbers between the 3 portions of the optic cups, the anterior, equator and the posterior portions decreased from the anterior to

12 months after hatching labeled with 3H-thymidine. From Nagata, T.:

the posterior at the same developmental stages (Gunarso 1984a).

p.197, 2002, Urban & Fischer, Jena, Germany

**4.3.1 The RNA synthesis in the eye** 

organs.

**4.3 The RNA synthesis in the sensory organs** 

cells belonged to the renewing cell population (Nagata 1998c).

In the corneas of aging mice, DNA synthesis was observed in all 3 layers, i.e., the epithelial, stromal and endothelial layers, at perinatal stages (Gao et al. 1993). The labeled cells with 3H-thymidine were localized in the epithelial cells at prenatal day 19, postnatal day 1, 14 (Fig. 27G) to 1 year, while the labeled cells in the stromal and endothelial layers were less. The labeling index of the corneal epithelial cells reached a peak at 1 month after birth and decreased to 1 year, (Fig. 30A), while the indices of the stromal (Fig. 30B) and endothelial (Fig. 30C) cells were low and reached a peak at 3 days after birth and disappeared completely from postnatal 1 month to 1 year (Nagata 1999c).

In the cilliary body, the labeled cells were located in the cilliary and pigment epithelial cells, stromal cells and smooth muscle cells from prenatal day 19 to postnatal 1 week, but no labeled cells were observed in any cell types from postnatal day 14 to 1 year (Nagata et al. 1994). The labeling indices of all the cell types in the cilliary body were at the maximum at prenatal day 19 and decreased gradually after birth reaching 0 at postnatal day 14. On the other hand, when the ocular tissues were labeled with 3H-uridine, silver grains appeared over all cell types at all stages of development and aging (Toriyama 1995, Nagata 2000f). The grain counts in the retina and the pigment epithelium increased from prenatal day 9 to postnatal day 1 in the retinal cells, while they increased from prenatal day 12 to postnatal day 7 in the pigment epithelial cells (Nagata 1999a,b, Nagata et al. 1994)

#### **4.2 The DNA synthesis in the skin**

The skin which covers the surface of the animal body can histologically be divided into 3 layers, the epidermis, the dermis and the hypodermis. We studied only the epidermal cells of young salamanders after hatching to 24 months during the aging by radioautography (Nagata 1998c). The fore-limbs and hind-limbs of salamanders were composed of skeletons consisting of bones and cartilages which were covered with skeletal muscles, connective tissues and epidermis consisting of stratified squamous epithelial cells in the outermost layer. We observed both the cartilage cells in the bone and the epithelial cells in the epidermis to compare the two cell populations. The skin of a salamander consisted of epidermis and dermis or corium which was lined with connective tissue layers designated as the subcutaneous layer. The former consisted of stratified squamous epithelium, while the latter consisted of dense connective tissues. The epithelial cells in the juvenile animals at 4 weeks after hatching were cuboidal in shape and not keratinized. Radioautograms labeled with 3H-thymidine at this stage showed that many cells were labeled demonstrating DNA synthesis at both the superficial and deeper layers (Fig. 27H), resulting very high labeling index. At 6 weeks after hatching, the superficial cells changed their shape from cuboidal to flattened squamous, while the deeper and basal cells remained cuboidal. The numbers of labeled cells were almost the same as the previous stage at 4 weeks, but they were localized at the basal layer. The shape of epithelial cells in juvenile animals at 8, 9, 10, and 11 weeks differentiated gradually forming the superficial corneum layer which appeared keratinized and the deeper basal layer. Radioautograms at these stages showed that the labeled cells remarkably reduced as compared with that of 4 and 6 weeks. In the adult salamanders at 8 months up to 12 months, the dermal and epidermal cells showed complete mature structure and examination of radioautograms revealed that the labeled cells were localized at only the basal cell layer and their number reached very low but at constant level. No difference was found on the morphology and labeling between the fore-limbs and hind-limbs at any stages. Comparing the labeling indices of both epidermal cells and the cartilage cells in the limbs,

In the corneas of aging mice, DNA synthesis was observed in all 3 layers, i.e., the epithelial, stromal and endothelial layers, at perinatal stages (Gao et al. 1993). The labeled cells with 3H-thymidine were localized in the epithelial cells at prenatal day 19, postnatal day 1, 14 (Fig. 27G) to 1 year, while the labeled cells in the stromal and endothelial layers were less. The labeling index of the corneal epithelial cells reached a peak at 1 month after birth and decreased to 1 year, (Fig. 30A), while the indices of the stromal (Fig. 30B) and endothelial (Fig. 30C) cells were low and reached a peak at 3 days after birth and disappeared

In the cilliary body, the labeled cells were located in the cilliary and pigment epithelial cells, stromal cells and smooth muscle cells from prenatal day 19 to postnatal 1 week, but no labeled cells were observed in any cell types from postnatal day 14 to 1 year (Nagata et al. 1994). The labeling indices of all the cell types in the cilliary body were at the maximum at prenatal day 19 and decreased gradually after birth reaching 0 at postnatal day 14. On the other hand, when the ocular tissues were labeled with 3H-uridine, silver grains appeared over all cell types at all stages of development and aging (Toriyama 1995, Nagata 2000f). The grain counts in the retina and the pigment epithelium increased from prenatal day 9 to postnatal day 1 in the retinal cells, while they increased from prenatal day 12 to postnatal

The skin which covers the surface of the animal body can histologically be divided into 3 layers, the epidermis, the dermis and the hypodermis. We studied only the epidermal cells of young salamanders after hatching to 24 months during the aging by radioautography (Nagata 1998c). The fore-limbs and hind-limbs of salamanders were composed of skeletons consisting of bones and cartilages which were covered with skeletal muscles, connective tissues and epidermis consisting of stratified squamous epithelial cells in the outermost layer. We observed both the cartilage cells in the bone and the epithelial cells in the epidermis to compare the two cell populations. The skin of a salamander consisted of epidermis and dermis or corium which was lined with connective tissue layers designated as the subcutaneous layer. The former consisted of stratified squamous epithelium, while the latter consisted of dense connective tissues. The epithelial cells in the juvenile animals at 4 weeks after hatching were cuboidal in shape and not keratinized. Radioautograms labeled with 3H-thymidine at this stage showed that many cells were labeled demonstrating DNA synthesis at both the superficial and deeper layers (Fig. 27H), resulting very high labeling index. At 6 weeks after hatching, the superficial cells changed their shape from cuboidal to flattened squamous, while the deeper and basal cells remained cuboidal. The numbers of labeled cells were almost the same as the previous stage at 4 weeks, but they were localized at the basal layer. The shape of epithelial cells in juvenile animals at 8, 9, 10, and 11 weeks differentiated gradually forming the superficial corneum layer which appeared keratinized and the deeper basal layer. Radioautograms at these stages showed that the labeled cells remarkably reduced as compared with that of 4 and 6 weeks. In the adult salamanders at 8 months up to 12 months, the dermal and epidermal cells showed complete mature structure and examination of radioautograms revealed that the labeled cells were localized at only the basal cell layer and their number reached very low but at constant level. No difference was found on the morphology and labeling between the fore-limbs and hind-limbs at any stages. Comparing the labeling indices of both epidermal cells and the cartilage cells in the limbs,

completely from postnatal 1 month to 1 year (Nagata 1999c).

day 7 in the pigment epithelial cells (Nagata 1999a,b, Nagata et al. 1994)

**4.2 The DNA synthesis in the skin** 

the labeling index of the epidermal cells was higher than the cartilage cells. The index of the dermal cells in the hind-limbs was at its maximum about 25% at 4 weeks, and fell down markedly with time from 6 weeks to 9 weeks. The labeling index of epidermal cells of the hind-limbs, on the other hand, had its maximum about 23% at 6 weeks, increasing from 20% at 4 weeks, and decreasing to about 18% at 8 weeks, then fell progressively with time, dropped to 5% at 9 weeks (Fig. 31). The labeling indices of the epidermal cells of both forelimbs and hind-limbs were almost the same from 9 weeks to 12 months, keeping low constant level about 4-5%, but never reaching 0. These results indicated that the cutaneous cells belonged to the renewing cell population (Nagata 1998c).

Fig. 31. Transitional curves of the labeling indices of the epithelial cells in the epidermis of the fore-limbs (F) and the hind-limbs (H) of the salamanders at various ages from 4 weeks to 12 months after hatching labeled with 3H-thymidine. From Nagata, T.: Radioautographology, General and Special. In, Prog. Histochem. Cytochem. Vol. 37, No. 2, p.197, 2002, Urban & Fischer, Jena, Germany

#### **4.3 The RNA synthesis in the sensory organs**

We studied only the RNA synthesis in the chicken and mouse eyes among of the sensory organs.

#### **4.3.1 The RNA synthesis in the eye**

The RNA synthesis in the chicken eyes was studied with the ocular tissues of chicken embryos in incubation (Fig. 27D). Silver grains due to the incorporations of 3H-uridine were observed over all the nuclei, cell organelles, cytoplasm of all the cells in the optic cups in development showing the RNA synthesis (Gunarso 1984a,b, Gunarso et al. 1969). Grain counting revealed that the counts gradually increased from day 2 to 7 and the numbers of silver grains were the most in the nuclei, while the numbers between the 3 portions of the optic cups, the anterior, equator and the posterior portions decreased from the anterior to the posterior at the same developmental stages (Gunarso 1984a).

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 423

postnatal 6 months. No silver grains were observed in the lamina limitans anterior (Bowman's membrane) and the lamina limitans posterior (Descemet's membrane). The grain densities by 3H-leucine incorporation in 3 layers, i. e., epithelial, stromal and endothelial layers, increased from embryonic stage to postnatal day 3 and 7, then decreased to 2 weeks and 1 year. The grain densities due to the glycoprotein synthesis with 3H-glucosamine were more observed in the endothelial cells of prenatal day 19 animals, but more in the epithelial cells of postnatal day 1, 3 and 7 animals. From the results, it was shown that the glycoprotein synthetic activity in respective cell types in the cornea of mouse changed with

The collagen synthesis in the ocular tissues was also demonstrated by the incorporation of 3H-proline in 4 groups of mice at various ages, from prenatal day 20, postnatal day 3, 7 and 30. The results showed that the sites of 3H-proline incorporation were located in the stromal fibroblasts in both cornea and the trabecular meshworks in the iridocorneal angle in prenatal and postnatal newborn mice. No silver grains were observed in the epithelial and endothelial cells. On EM RAG, silver grains were localized over the endoplasmic reticulum and Golgi apparatus of fibroblasts and over intercellular matrices consisting of collagen fibrils. From the quantitative analysis, the grain densities were more observed in the fibroblasts in postnatal day 7 animals than younger animals at fetal day 20 and postnatal day 3, 7 and 30. In the same aging groups, the grain densities were more in the cornea than the iridocorneal angle. It was concluded that the collagen synthetic activity was localized in the fibroblasts in the cornea and the trabecular meshworks in the iridocorneal angle and the

On the other hand, the distributions of some of the ophthalmological drugs used for the treatment of human glaucoma patients were examined in the ocular tissues by LM and EM

We studied the aging changes of glucide synthesis by 3H-glucosamine uptake in the ocular

The glycoprotein synthesis of the cornea in aging mouse as revealed by 3H-glucosamine incorporation was studied in several groups of aging mice at various ages from prenatal stages to senescence (Nagata et al. 1995). Silver grains were located in the epithelial cells, the stromal fibroblasts and the endothelial cells from prenatal day 19 to postnatal 6 months. No silver grains were observed in the lamina limitans anterior (Bowman's membrane) and the lamina limitans posterior (Descemet's membrane). On the other hand, the grain densities by 3H-leucine incorporation in 3 layers, i.e., epithelial, stromal and endothelial layers, increased from embryonic stage to postnatal day 3 and 7, then decreased to week 2 and year 1. The grain densities due to the glycoprotein synthesis with 3H-glucosamine were more observed in the endothelial cells of prenatal day 19 animals, but more in the epithelial cells of postnatal day 1, 3 and 7 animals. From the results, it was shown that the glycoprotein synthetic activity in respective cell types in the cornea of mouse changed with aging of the

activity changed with aging, reaching the maximum at postnatal day 7.

**4.5 The glucide synthesis in the sensory organs** 

**4.5.1 The glucide synthesis in the eye** 

RAG (Nagata 2000f). However, its relationship to the aging was not studied.

aging of the animals.

tissues of aging mice.

animals.

On the other hand, the ocular tissues of aging mice were also labeled with 3H-uridine. The silver grains demonstrating RNA synthesis appeared over all the cell types at all the stages of development and aging. The grain counts in the retina and the pigment epithelium increased from prenatal day 9 to postnatal day 1 in the retinal cells, while they increased from prenatal day 12 to postnatal day 7 in the pigment epithelial cells (Kong et al. 1992b).

On the other hand, the distribution and localization of TGF-1 and FGF and their mRNA in the ocular tissues of aging mice were also studied (Nagata and Kong 1998). The posterior segment of BALB/c mouse eyes from embryonic day 14, 16, 19 and postnatal 1, 3, 5, 7, 14, 28, 42 and 70 were used. For in situ hybridization, 35S-labeled oligonucleotide probes for TGF-1 and FGF were used to detect their mRNA. Cryo-sections were picked up on glass slides which were processed for in situ hybridization and for radioautography. As the results, silver grains mainly located in the scleral layers and some in the choroidal and pigment epithelial layers, but only background level of grains were found in the whole retina. In the radioautograms from embryonic day 14 to adult mice at week 10 (day 70), the significant distribution of silver grains representing TGF-1 mRNA was not detected in the whole retina. However, the significant silver grains were detected in scleral and chorioidal layers and mesenchymal cells at embryonic day 14, then the number of grains increased in these layers particularly in sclera from prenatal to postnatal neonate until adult (Fig. 27E). These results suggest that mRNA for TGF-1 and FGF were synthesized in scleral, choroidal and pigment epithelial layers, but their proteins were transferred to the target cells of the retina and elsewhere. Furthermore, it is suggested that TGF-1 and FGF may play important roles on retinal differentiation, development and aging, particularly during the late embryonic and newborn stages (Nagata and Kong 1998).

These results showed that RNA synthetic activities in the ocular cells changed due to the aging of individual animals.

#### **4.4 The protein synthesis in the sensory organs**

We studied only the protein synthesis in the mouse eyes among of the sensory organs.

#### **4.4.1 The protein synthesis of the eye**

The protein synthesis in the ocular tissues of aging mouse were studied in all the 3 layers of the eye, the tunica fibrosa, the tunica vasculosa and the tunica intima, or the cornea, cilliary bodies and the retina of the aging mouse at various stages after the administration of several precursors (Toriyama 1995, Nagata 1997c, 1999b,c,d, 2000e,f, 2001c, Nagata and Kong 1998, Cui et al. 2000).

The protein synthesis of the retina in aging mouse as revealed by 3H-leucine incorporation demonstrated that number of silver grains in bipolar cells and photoreceptor cells was the most intense at embryonic stage and early postnatal days. The peak was 1 day after birth and decreased from 14 days to 1 year after birth. (Toriyama 1995). The protein synthesis of the cornea as revealed by 3H-leucine incorporation (Nagata 1997c, 1999d, 2000f, 2001c, Nagata and Kong 1998, Cui et al. 2000) and the glycoprotein synthesis demonstrated by 3Hglucosamine (Nagata et al. 1995) were also studied in several groups of aging ddY mice. Silver grains of both 3H-leucine and 3H-glucosamine incorporations were located in the epithelial cells, the stromal fibroblasts and the endothelial cells from prenatal day 19 to

On the other hand, the ocular tissues of aging mice were also labeled with 3H-uridine. The silver grains demonstrating RNA synthesis appeared over all the cell types at all the stages of development and aging. The grain counts in the retina and the pigment epithelium increased from prenatal day 9 to postnatal day 1 in the retinal cells, while they increased from prenatal day 12 to postnatal day 7 in the pigment epithelial cells (Kong et al. 1992b).

On the other hand, the distribution and localization of TGF-1 and FGF and their mRNA in the ocular tissues of aging mice were also studied (Nagata and Kong 1998). The posterior segment of BALB/c mouse eyes from embryonic day 14, 16, 19 and postnatal 1, 3, 5, 7, 14, 28, 42 and 70 were used. For in situ hybridization, 35S-labeled oligonucleotide probes for TGF-1 and FGF were used to detect their mRNA. Cryo-sections were picked up on glass slides which were processed for in situ hybridization and for radioautography. As the results, silver grains mainly located in the scleral layers and some in the choroidal and pigment epithelial layers, but only background level of grains were found in the whole retina. In the radioautograms from embryonic day 14 to adult mice at week 10 (day 70), the significant distribution of silver grains representing TGF-1 mRNA was not detected in the whole retina. However, the significant silver grains were detected in scleral and chorioidal layers and mesenchymal cells at embryonic day 14, then the number of grains increased in these layers particularly in sclera from prenatal to postnatal neonate until adult (Fig. 27E). These results suggest that mRNA for TGF-1 and FGF were synthesized in scleral, choroidal and pigment epithelial layers, but their proteins were transferred to the target cells of the retina and elsewhere. Furthermore, it is suggested that TGF-1 and FGF may play important roles on retinal differentiation, development and aging, particularly during

These results showed that RNA synthetic activities in the ocular cells changed due to the

The protein synthesis in the ocular tissues of aging mouse were studied in all the 3 layers of the eye, the tunica fibrosa, the tunica vasculosa and the tunica intima, or the cornea, cilliary bodies and the retina of the aging mouse at various stages after the administration of several precursors (Toriyama 1995, Nagata 1997c, 1999b,c,d, 2000e,f, 2001c, Nagata and Kong 1998,

The protein synthesis of the retina in aging mouse as revealed by 3H-leucine incorporation demonstrated that number of silver grains in bipolar cells and photoreceptor cells was the most intense at embryonic stage and early postnatal days. The peak was 1 day after birth and decreased from 14 days to 1 year after birth. (Toriyama 1995). The protein synthesis of the cornea as revealed by 3H-leucine incorporation (Nagata 1997c, 1999d, 2000f, 2001c, Nagata and Kong 1998, Cui et al. 2000) and the glycoprotein synthesis demonstrated by 3Hglucosamine (Nagata et al. 1995) were also studied in several groups of aging ddY mice. Silver grains of both 3H-leucine and 3H-glucosamine incorporations were located in the epithelial cells, the stromal fibroblasts and the endothelial cells from prenatal day 19 to

We studied only the protein synthesis in the mouse eyes among of the sensory organs.

the late embryonic and newborn stages (Nagata and Kong 1998).

**4.4 The protein synthesis in the sensory organs** 

**4.4.1 The protein synthesis of the eye** 

aging of individual animals.

Cui et al. 2000).

postnatal 6 months. No silver grains were observed in the lamina limitans anterior (Bowman's membrane) and the lamina limitans posterior (Descemet's membrane). The grain densities by 3H-leucine incorporation in 3 layers, i. e., epithelial, stromal and endothelial layers, increased from embryonic stage to postnatal day 3 and 7, then decreased to 2 weeks and 1 year. The grain densities due to the glycoprotein synthesis with 3H-glucosamine were more observed in the endothelial cells of prenatal day 19 animals, but more in the epithelial cells of postnatal day 1, 3 and 7 animals. From the results, it was shown that the glycoprotein synthetic activity in respective cell types in the cornea of mouse changed with aging of the animals.

The collagen synthesis in the ocular tissues was also demonstrated by the incorporation of 3H-proline in 4 groups of mice at various ages, from prenatal day 20, postnatal day 3, 7 and 30. The results showed that the sites of 3H-proline incorporation were located in the stromal fibroblasts in both cornea and the trabecular meshworks in the iridocorneal angle in prenatal and postnatal newborn mice. No silver grains were observed in the epithelial and endothelial cells. On EM RAG, silver grains were localized over the endoplasmic reticulum and Golgi apparatus of fibroblasts and over intercellular matrices consisting of collagen fibrils. From the quantitative analysis, the grain densities were more observed in the fibroblasts in postnatal day 7 animals than younger animals at fetal day 20 and postnatal day 3, 7 and 30. In the same aging groups, the grain densities were more in the cornea than the iridocorneal angle. It was concluded that the collagen synthetic activity was localized in the fibroblasts in the cornea and the trabecular meshworks in the iridocorneal angle and the activity changed with aging, reaching the maximum at postnatal day 7.

On the other hand, the distributions of some of the ophthalmological drugs used for the treatment of human glaucoma patients were examined in the ocular tissues by LM and EM RAG (Nagata 2000f). However, its relationship to the aging was not studied.

## **4.5 The glucide synthesis in the sensory organs**

We studied the aging changes of glucide synthesis by 3H-glucosamine uptake in the ocular tissues of aging mice.

## **4.5.1 The glucide synthesis in the eye**

The glycoprotein synthesis of the cornea in aging mouse as revealed by 3H-glucosamine incorporation was studied in several groups of aging mice at various ages from prenatal stages to senescence (Nagata et al. 1995). Silver grains were located in the epithelial cells, the stromal fibroblasts and the endothelial cells from prenatal day 19 to postnatal 6 months. No silver grains were observed in the lamina limitans anterior (Bowman's membrane) and the lamina limitans posterior (Descemet's membrane). On the other hand, the grain densities by 3H-leucine incorporation in 3 layers, i.e., epithelial, stromal and endothelial layers, increased from embryonic stage to postnatal day 3 and 7, then decreased to week 2 and year 1. The grain densities due to the glycoprotein synthesis with 3H-glucosamine were more observed in the endothelial cells of prenatal day 19 animals, but more in the epithelial cells of postnatal day 1, 3 and 7 animals. From the results, it was shown that the glycoprotein synthetic activity in respective cell types in the cornea of mouse changed with aging of the animals.

Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 425

This study was supported in part by Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 02454564) while the author worked at Shinshu University School of Medicine by 1996 as well as Grants for Promotion of Characteristic Research and Education from the Japan Foundation for Promotion of Private Schools (No. 1997, 1998 1999, 2000) while the author worked at Nagano Women's Jr. College from 1996 to 2002. The author is also grateful to Grant-in-Aids for Scientific Research from the Japan Society for Promotion of Sciences (No. 18924034, 19924204, 20929003) while the author has been working at Shinshu Institute of Alternative Medicine and Welfare since 2005 up to the present time. The author thanks Dr. Kiyokazu Kametani, Technical Official, Department of Instrumental Analysis, Research Center for Human and Environmental Sciences, Shinshu University, for his technical assistance in electron microscopy during the

Chen, S., Gao, F., Kotani, A., Nagata, T.: Age-related changes of male mouse submandibular

Clermont Y.: The contractime elements in the limiting membrane of the seminiferous

Cui, H.: Light microscopic radioautographic study on DNA synthesis of nerve cells in the

Cui, H., Gao, F., Nagata, T.: Light microscopic radioautographic study on protein synthesis in perinatal mice corneas. Acta Histochem. Cytochem. 33, 31-37, 2000. Duan, H., Gao, F., Li, S., Hayashi, K., Nagata, T.: Aging changes and fine structure and

Duan, H., Gao, F., Li, S., Nagata, T.: Postnatal development of esophageal epithelium in

Duan, H., Gao, F., Oguchi, K., Nagata, T.: Light and electron microscopic radioautographic

Feulgen, R., Rossenbeck, H.: Mikroskopische-chemischer Nachweis einer Nukeinsaeure von Thymus der Thymonukeinsaeure Z. Physik. Chem. 135, 203-248, 1924. Fujii, Y., Ohno, S., Yamabayashi, S., Usuda, N., Saito, H., Furuta, S., Nagata, T.: Electron

Gao, F.: Study on the macromolecular synthesis in aging mouse seminiferous tubules by light and electron microscopic radioautography. Cell. Mol. Biol. 39, 659-672, 1993. Gao, F., Toriyama, K., Nagata, T.: Light microscopic radioautographic study on the DNA

IgG myeloma patient. J. Clin. Electr. Microsc. 13, 582-583, 1980.

Clermont, Y.: Renewal of spermatogonia in man. Amer. J. Anat. 112, 35-51, 1963.

cerebella of aging mice. Cell. Mol. Biol. 41, 1139-1154, 1995.

tubules of rats. Exp. Cell Res. 15, 438-342, 1958.

Electron Microsc. 25, 452-453, 1992.

nebulizer. Drug Res. 44, 880-883, 1994.

injection. Cell. Mol. Biol. 38, 661-668, 1992a.

39, 309-316, 1993.

gland: A morphometric and radioautographic study. Cell. Mol. Biol. 41, 117-124,

DNA synthesis of esophageal epithelium in neonatal, adult and old mice. J. Clin.

mouse: a light and electron microscopic radioautographic study. Cell. Mol. Biol.

study on the incorporation of 3H-thymidine into the lung by means of a new

microscopic radioautography of DNA synthesis in primary cultured cells from an

synthesis of prenatal and postnatal aging mouse retina after labeled thymidine

**7. Acknowledgments** 

course of this study.

1995.

**8. References** 

### **5. Macromolecular synthesis in the tumor cells**

We carried out several experiments dealing with the nucleic acid synthesis in some malignant tumor cells by means of LM and EM RAG.

#### **5.1 The DNA synthesis in the tumor cells**

The DNA synthesis in nuclei and mitochondria of cultured HeLa cells, an established cell line obtained from the carcinoma of the human uterus, or IgG myeloma cells from a human patient, labeled with 3H-thymidine were demonstrated (Nagata 1972b,c,d, Fujii et al. 1980). The incorporations of these precursors increased or decreased depending upon the aging of isolated cells in vitro, i.e., the days of incubation in vitro. The incorporations of 3Hthymidine demonstrating DNA synthesis increased immediately after the incubation and then gradually decreased due to incubation days, reaching zero within several days.

## **5.2 The RNA synthesis in the tumor cells**

The RNA synthesis in nuclei and mitochondria of cultured HeLa cells, or IgG myeloma cells from a human patient, labeled with 3H-uridine were demonstrated (Nagata 1972b,c,d, Fujii et al. 1980). The incorporations of these precursors increased or decreased depending upon the aging of isolated cells in vitro. The incorporations of 3H-uridine demonstrating RNA synthesis increased immediately after the incubation and then gradually decreased due to incubation days.

## **6. Conclusions**

From the results obtained, it was concluded that almost all the cells in various organs of all the organ systems of experimental animals at various ages from prenatal to postnatal development and senescence during the aging of cells and individual animals demonstrated to incorporate various macromolecular precursors such as 3H-thymidine, 3H-uridine, 3Hleucine, 3H-glucose or glucosamine, 3H-glycerol and others localizing in the nuclei, cytoplasmic cell organelles showing silver grains due to DNA, RNA, proteins, glucides, lipids and others those which the cells synthesized during the cell aging. Quantitative analysis carried out on the numbers of silver grains in respective cell organelles demonstrated quantitative changes, increases and decreases, of these macromolecular synthesis in connection to cell aging of respective organs. In general, DNA synthesis with 3H-thymidine incorporations in most organs showed maxima at perinatal stages and gradually decreased due to aging. To the contrary, the other syntheses such as RNA, proteins, glucides and lipids increased due to aging and did not remarkably decrease until senescence. Anyway, these results indicated that macromolecular synthetic activities of respective compounds in various cells were affected from the aging of the individual animals.

Thus, the results obtained from the various cells of various organs should form a part of special radioautographology that I had formerly proposed (Nagata 1999e, 2002), i.e., application of radioautography to the aging of cells, as well as a part of special cytochemistry (Nagata 2001), as was formerly reviewed. We expect that such special radioautographology and special cytochemistry should be further developed in all the organs in the future.

#### **7. Acknowledgments**

424 Senescence

We carried out several experiments dealing with the nucleic acid synthesis in some

The DNA synthesis in nuclei and mitochondria of cultured HeLa cells, an established cell line obtained from the carcinoma of the human uterus, or IgG myeloma cells from a human patient, labeled with 3H-thymidine were demonstrated (Nagata 1972b,c,d, Fujii et al. 1980). The incorporations of these precursors increased or decreased depending upon the aging of isolated cells in vitro, i.e., the days of incubation in vitro. The incorporations of 3Hthymidine demonstrating DNA synthesis increased immediately after the incubation and

The RNA synthesis in nuclei and mitochondria of cultured HeLa cells, or IgG myeloma cells from a human patient, labeled with 3H-uridine were demonstrated (Nagata 1972b,c,d, Fujii et al. 1980). The incorporations of these precursors increased or decreased depending upon the aging of isolated cells in vitro. The incorporations of 3H-uridine demonstrating RNA synthesis increased immediately after the incubation and then gradually decreased due to

From the results obtained, it was concluded that almost all the cells in various organs of all the organ systems of experimental animals at various ages from prenatal to postnatal development and senescence during the aging of cells and individual animals demonstrated to incorporate various macromolecular precursors such as 3H-thymidine, 3H-uridine, 3Hleucine, 3H-glucose or glucosamine, 3H-glycerol and others localizing in the nuclei, cytoplasmic cell organelles showing silver grains due to DNA, RNA, proteins, glucides, lipids and others those which the cells synthesized during the cell aging. Quantitative analysis carried out on the numbers of silver grains in respective cell organelles demonstrated quantitative changes, increases and decreases, of these macromolecular synthesis in connection to cell aging of respective organs. In general, DNA synthesis with 3H-thymidine incorporations in most organs showed maxima at perinatal stages and gradually decreased due to aging. To the contrary, the other syntheses such as RNA, proteins, glucides and lipids increased due to aging and did not remarkably decrease until senescence. Anyway, these results indicated that macromolecular synthetic activities of respective compounds in various cells were affected from the aging of the individual

Thus, the results obtained from the various cells of various organs should form a part of special radioautographology that I had formerly proposed (Nagata 1999e, 2002), i.e., application of radioautography to the aging of cells, as well as a part of special cytochemistry (Nagata 2001), as was formerly reviewed. We expect that such special radioautographology and special cytochemistry should be further developed in all the

then gradually decreased due to incubation days, reaching zero within several days.

**5. Macromolecular synthesis in the tumor cells** 

malignant tumor cells by means of LM and EM RAG.

**5.1 The DNA synthesis in the tumor cells** 

**5.2 The RNA synthesis in the tumor cells** 

incubation days.

**6. Conclusions** 

animals.

organs in the future.

This study was supported in part by Grant-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 02454564) while the author worked at Shinshu University School of Medicine by 1996 as well as Grants for Promotion of Characteristic Research and Education from the Japan Foundation for Promotion of Private Schools (No. 1997, 1998 1999, 2000) while the author worked at Nagano Women's Jr. College from 1996 to 2002. The author is also grateful to Grant-in-Aids for Scientific Research from the Japan Society for Promotion of Sciences (No. 18924034, 19924204, 20929003) while the author has been working at Shinshu Institute of Alternative Medicine and Welfare since 2005 up to the present time. The author thanks Dr. Kiyokazu Kametani, Technical Official, Department of Instrumental Analysis, Research Center for Human and Environmental Sciences, Shinshu University, for his technical assistance in electron microscopy during the course of this study.

### **8. References**


Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 427

Izumiyama, K., Kogure, K., Kataoka, S., Nagata, T.: Quantitative analysis of glucose after

Jamieson, J. D., Palade, G. E.: Intracellular transport of secretory proteins in the pancreatic

Jin, C.: Study on DNA synthesis of aging mouse colon by light and electron microscopic

Jin, C., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. J. Histochem. Cytochem. 43, 1223-1228, 1995a. Jin, C., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis in cecal epithelial cells of aging mice. Med. Electron Microsc. 28, 71-75, 1995b. Joukura, K.: The aging changes of glycoconjugate synthesis in mouse kidney studied by 3Hglucosamine radioautography. Acta Histochem. Cytochem. 29, 57-63, 1996. Joukura, K., Nagata, T.: Aging changes of 3H-glucosamine incorporation into mouse kidney observed by radioautography. Acta Histochem. Cytochem. 28, 494-494, 1995. Joukura, K., Usuda, N., Nagata, T.: Quantitative study on the aging change of

radioautography. Brain Res. 416, 175-179, 1987.

radioautography. Cell. Mol. Biol. 42, 255-268, 1996.

exocrine cells. J. Cell Biol. 34, 577-615, 1967.

Histochem. Cytochem. 42, 982-982, 1994.

mouse retina. Cell. Mol. Biol. 39, 55-64, 1993.

Electron Microsc. 11, 428-429, 1978.

263-272, 1992a.

132: 247-259. 1958.

195, 1995.

Mol. Biol. 38, 669-678, 1992b.

by radioiodine. J. Anat. 77, 149-152, 1943.

Histochemistry 102, 405-413, 1994.

transient ischemia in the gerbil hippocampus by light and electron microscope

glycoconjugates synthesis in aging mouse kidney. Proc. Xth Internat. Cong. Histochem. Cytochem., Acta Histochem. Cytochem. 29, Suppl. 507-508, 1996. Kobayashi, K., Nagata, T.: Light microscopic radioautographic studies on DNA, RNA and

protein syntheses in human synovial membranes of rheumatoid arthritis patients. J.

radioautographic study on 125I-albumin in rat gastric mucosal epithelia. J. Clin.

and retinal pigment epithelium of developing mouse embryo. Cell. Mol. Biol. 38,

and retinal pigment epithelium of mice by light microscopic radioautography. Cell.

shown by radioautography after injection of thymidine-3H into mice. Anat. Rec.

hormone receptor status in the developing mouse ovary, uterus and oviduct.

studied by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 185-

Komiyama, K., Iida, F., Furihara, R., Murata, F., Nagata, T.: Electron microscopic

Kong, Y.: Electron microscopic radioautographic study on DNA synthesis in perinatal

Kong, Y., Nagata, T.: Electron microscopic radioautographic study on nucleic acid synthesis

Kong, Y., Usuda, N., Nagata, T.: Radioautographic study on DNA synthesis of the retina

Kong, Y., Usuda, N., Morita, T., Hanai, T., Nagata, T.: Study on RNA synthesis in the retina

Leblond, C. P.: Localization of newly administered iodine in the thyroid gland as indicated

Leblond, C. P.: The life history of cells in renewing systems. Am. J. Anat. 160, 113-158, 1981. Leblond, C. P., Messier, B.: Renewal of chief cells and goblet cells in the small intestine as

Li, S.: Relationship between cellular DNA synthesis, PCNA expression and sex steroid

Li, S., Nagata, T.: Nucleic acid synthesis in the developing mouse ovary, uterus and oviduct

of perinatal mouse retina. Med. Electron Microsc. 27, 366-368, 1994.


Gao, F., Li, S., Duan, H., Ma, H., Nagata, T.: Electron microscopic radioautography on the

Gao, F., Toriyama, K., Ma, H., Nagata, T.: Light microscopic radioautographic study on DNA synthesis in aging mice corneas. Cell. Mol. Biol. 39, 435-441, 1993. Gao, F., Ma, H., Sun, L., Jin, C., Nagata, T.: Electron microscopic radioautographic study on

Gao, F., Chen, S., Sun, L., Kang, W., Wang, Z., Nagata, T.: Radioautographic study of the

Gao, F., Jin, C., Ma, H., Sun, L., Nagata, T.: Ultrastructural and radioautographic studies on

Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick

Gunarso, W.: Radioautographic studies on the nucleic acid synthesis of the retina of chick

Gunarso, W., Gao, F., Cui, H., Ma, H., Nagata, T.: A light and electron microscopic

Gunarso, W., Gao, F., Nagata, T.: Development and DNA synthesis in the retina of chick

Hanai, T.: Light microscopic radioautographic study of DNA synthesis in the kidneys of

Hanai, T., Nagata, T.: Electron microscopic radioautographic study on DNA and RNA

Hanai, T., Nagata, T.: Study on the nucleic acid synthesis in the aging mouse kidney by

Nagata, T., Ed., pp. 209-214, Shinshu University Press, Matsumoto, 1994b. Hanai, T., Nagata, T.: Electron microscopic study on nucleic acid synthesis in perinatal

Hanai, T., Usuda, N., Morita, T., Shimizu, T., Nagata, T.: Proliferative activity in the kidneys

Hayashi, K., Gao, F., Nagata, T.: Radioautographic study on 3H-thymidine incorporation at

Ito, M.: The radioautographic studies on aging change of DNA synthesis and the

Ito, M., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis and

Ed., pp. 127-131, Shinshu University Press, Matsumoto, 1994a.

mouse kidney tissue. Med. Electron Microsc. 27, 355-357, 1994c.

injection. J. Clin. Electron Microsc. 25, 721-722, 1992b.

Microsc. 27, 360-362, 1994.

Histochem. 98, 309-32, 1996.

aging mice. Cell. Mol. Biol. 39, 81-91, 1993.

Biol. 43, 189-201, 1997.

39, 181-191, 1993.

1993.

1996.

1996.

145-150, 1995a.

1995b.

1984b.

DNA synthesis of prenatal and postnatal mice retina after labeled thymidine

the nucleic acids and protein synthesis in the aging mouse testis. Med. Electron

macromolecular synthesis of Leydig cells in aging mice testis. Cell. Mol. Biol. 41,

DNA synthesis in Leydig cells of aging mouse testis. Cell. Mol. Biol. 41, 151-160,

embryo. I. Light microscopic radioautography. Shinshu Med. J. 32, 231-240, 1984a.

embryo. II. Electron microscopic radioautography. Shinshu Med. J. 32, 241-248,

radioautographic study on RNA synthesis in the retina of chick embryo. Acta

embryo observed by light and electron microscopic radioautography. Cell. Mol.

synthesis in perinatal mouse kidney. In, Radioautography in Medicine, Nagata, T.,

light and electron microscopic radioautography. In, Radioautography in Medicine,

of aging mice evaluated by PCNA/cyclin immunohistochemistry. Cell. Mol. Biol.

different stages of muscle development in aging mice. Cell. Mol. Biol. 39, 553-560,

ultrastructural development of mouse adrenal gland. Cell. Mol. Biol. 42, 279-292,

ultrastructure of aging mouse adrenal gland. Med. Electron Microsc. 29, 145-152,


Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 429

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Ultrastructural and electron microscopic

Murata, F., Yoshida, K., Ohno, S., Nagata, T.: Mucosubstances of rabbit granulocytes studied

Nagata, T.: On the relationship between cell division and cytochrome oxidase in the

Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. I. Observation on the

Nagata, T.: Studies on the amitosis in the Yoshida sarcoma cells. II. Phase-contrast

Nagata, T.: Cell divisions in the liver of the fetal and newborn dogs. Med. J. Shinshu Univ. 4:

Nagata, T.: A radioautographic study of the DNA synthesis in rat liver, with special

Nagata, T.: A quantitative study on the ganglion cells in the small intestine of the dog. Med.

Nagata, T.: A radioautographic study on the RNA synthesis in the hepatic and the intestinal

Nagata, T.: On the increase of binucleate cells in the ganglion cells of dog small intestine due to experimental ischemia. Med. J. Shinshu Univ. 12, 93-113, 1967a. Nagata, T.: A radioautographic study on the protein synthesis in the hepatic and the

Nagata, T.: Chapter 3. Application of microspectrophotometry to various substances. In ,

Nagata, T.: Electron microscopic dry-mounting autoradiography. Proc. 4th Internat. Cong.

Nagata, T.: Electron microscopic radioautography of intramitochondrial RNA synthesis of

Nagata, T.: Quantitative electron microscope radioautography of intramitochondrial nucleic

Nagata, T.: Electron microscopic observation of target cells previously observed by phase-

Nagata, T.:. Electron microscopic radioautography and analytical electron microscopy. J.

irradiated cultured cells. J. Clin. Electron Microsc. 17, 589-590, 1984. Nagata, T.: Principles and techniques of radioautography. In, Histo- and Cyto-chemistry

epithelial cells of mice after feeding with special reference to binuclearity. Med. J.

intestinal epithelial cells of mice, with special reference to binucleate cells. Med. J.

Introduction to Microspectrophotometry. Isaka, S., Nagata, T., Inui, N., Eds.,

contrast microscopy: Electron microscopic radioautography of laser beam

1985, Japan Society of Histochemistry and Cytochemistry, Ed., Gakusai Kikaku

reference to binucleate cells. Med. J. Shinshu Univ. 7, 17-25, 1962.

Yoshida sarcoma cells. Shinshu Med. J. 5: 383-386, 1956.

Microsc. 11, 561-562, 1978.

1957a.

207, 1957b.

65-73, 1959.

Histochemistry 61, 139-150, 1979.

J. Shinshu Univ. 10, 1-11, 1965.

Shinshu Univ. 11, 49-61, 1966.

Shinshu Univ. 12, 247-257, 1967b.

Co., Tokyo, pp. 207-226, 1985.

Clin. Electron Microsc. 24, 441-442, 1991.

Olympus Co., Tokyo, pp. 49-155, 1972a.

Histochem. Cytochem. Kyoto, pp. 43-44, 1972b.

HeLa cells in culture. Histochemie 32, 163-170, 1972c.

acid synthesis. Acta Histochem. Cytochem. 5, 201-203, 1972d.

radioautographic studies on the mastocytoma cells and mast cells. J. Clin. Electron

by means of electron microscopic radioautography and X-ray microanalysis.

smear preparation under normal conditions. Med. J. Shinshu Univ. 2: 187-198,

microscopic observations under normal conditions. Med. J. Shinshu Univ. 2: 199-


Li, S., Gao, F., Duan, H., Nagata, T.: Radioautographic study on the uptake of 35SO4 in mouse ovary during the estrus cycle. J. Clin. Electron Microsc. 25, 709-710, 1992. Liang, Y.: Light microscopic radioautographic study on RNA synthesis in the adrenal glands

Liang, Y., Ito, M., Nagata, T.: Light and electron microscopic radioautographic studies on

Ma, H.: Light microscopic radioautographic study on DNA synthesis of the livers in aging

Ma, H., Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of the

Ma, H., Nagata, T.: Studies on DNA synthesis of aging mice by means of electron microscopic radioautography. J. Clin. Electron Microsc. 21, 715-716, 1988b. Ma, H., Nagata, T.: Electron microscopic radioautographic studies on DNA synthesis in the

Ma, H., Nagata, T.: Study on RNA synthesis in the livers of aging mice by means of electron

Ma, H., Nagata, T.: Collagen and protein synthesis in the livers of aging mice as studied by

Ma, H., Gao, F., Olea, M. T., Nagata, T.: Protein synthesis in the livers of aging mice studied by electron microscopic radioautography. Cell. Mol. Biol. 37, 607-615, 1991. Matsumura, H., Kobayashi, Y., Kobayashi, K., Nagata, T.: Light microscopic

Momose, Y., Nagata, T.: Radioautographic study on the intracellular localization of a

Momose, Y., Naito, J., Nagata, T.: Radioautographic study on the localization of an antiallergic agent, tranilast, in the rat liver. Cell. Mol. Biol. 35, 347-355, 1989. Momose, Y., Shibata, N., Kiyosawa, I., Naito, J., Watanabe, T., Horie, S., Yamada, J., Suga,

Momose, Y., Naito, J., Suzawa, H., Kanzawa, M., Nagata, T.: Radioautographic study on the

Morita, T.: Radioautographic study on the aging change of 3H-glucosamine uptake in mouse

Morita, T., Usuda, N. Hanai, T., Nagata, T.: Changes of colon epithelium proliferation due to

Murata, F., Momose,Y. , Yoshida, K., Nagata, T.: Incorporation of 3H-thymidine into the nucleus of mast cells in adult rat peritoneum. Shinshu Med. J. 25, 72-77, 1977a. Murata, F., Momose, Y., Yoshida, K., Ohno, S., Nagata, T.: Nucleic acid and mucosubstance

thymidine radioautography. Histochemistry 101, 13-20, 1994.

radioautography. Acta Pharmacol. Toxicol. 41, 58-59, 1977b.

RNA synthesis in aging mouse adrenal gland. Acta Anat. Nippon. 74, 291-300,

hepatocytes of aging mice as observed by image analysis. Cell. Mol. Biol. 36, 73-84,

radioautographic study of DNA synthesis in the lung of aging salamander,

hypolipidemic agent, bezafibrate, a peroxisome proliferator, in cultured rat

T., Nagata, T.: Morphometric evaluation of species differences in the effects of bezafibrate, a hypolipidemic agent, on hepatic peroxisomes and mitochondria. J.

intracellular localization of bezafibrate in cultured rat hepatoctyes. Acta Histochem.

individual aging with PCNA/cyclin immunostaining comparing with 3H-

metabolism of mastocytoma cells by means of electron microscopic

of aging mice. Acta Histochem. Cytochem. 31, 203-210, 1998.

livers in aging mice. J. Clin. Electron Microsc. 21, 335-343, 1988a.

microscopic radioautography. Cell. Mol. Biol. 36, 589-600, 1990b.

electron microsopic radioautography. Ann. Microsc. 1, 13-22, 2000.

Hynobius nebulosus. J. Histochem. Cytochem. 42, 1004-1004, 1994.

hepatocytes. Cell. Mol. Biol. 39, 773-781, 1993a.

Toxicol. Pathol. 6, 33-45, 1993b.

ileum. Cell. Mol. Biol. 39, 875-884, 1993.

Cytochem. 28, 61-66, 1995.

mice. Acta Anat. Nippon. 63, 137-147, 1988.

1999.

1990a.


Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 431

Nagata, T.: Techniques of radioautography for medical and biological research. Braz. J. Biol.

Nagata, T.: Radioautographology, the advocacy of a new concept. Braz. J. Biol. Med. Res. 31,

Nagata, T.: Radioautographic studies on DNA synthesis of the bone and skin of aging

Nagata, T.: 3D observation of cell organelles by high voltage electron microscopy.

Nagata, T.: Application of histochemistry to anatomy: Histochemistry of the organs, a novel

Nagata, T.: Aging changes of macromolecular synthesis in various organ systems as

Nagata, T.: Radioautographology, general and special: a novel concept. Ital. J. Anat.

Nagata, T.: Three-dimensional observations on thick biological specimens by high voltage

Nagata, T.: Biological microanalysis of radiolabeled and unlabeled compounds by

Nagata, T.: Electron microscopic radioautographic study on protein synthesis in pancreatic cells of perinatal and aging mice. Bull. Nagano Women's Jr. College 8, 1-22, 2000c. Nagata, T.: Light microscopic radioautographic study on radiosulfate incorporation into the tracheal cartilage in aging mice. Acta Histochem. Cytochem. 32, 377-383, 2000d. Nagata, T.: Introductory remarks: Special radioautographology. Cell. Mol. Biol. 46

Nagata, T.: Three-dimensional high voltage electron microscopy of thick biological

Nagata, T.: Three-dimensional and four-dimensional observation of histochemical and

Nagata, T. : Special cytochemistry in cell biology. In, Internat. Rev. Cytol. Jeon, K.W., ed.,

Nagata, T. : Radioautographology General and Special, In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 37 No. 2, Urban & Fischer, Jena, pp. 57-226, 2002. Nagata T.: Light and electron microscopic study on macromolecular synthesis in amitotic hepatocyte mitochondria of aging mice. Cell. Mol. Biol. 49, 591-611, 2003. Nagata, T.: X-ray microanalysis of biological specimens by high voltage electron

Nagata T.: Aging changes of macromolecular synthesis in the uro-genital organs as revealed by electron microscopic radioautography. Ann. Rev. Biomed. Sci. 6, 13-78, 2005.

cytochemical specimens by high voltage electron microscopy. Acta Histochem.

microscopy. In, Prog. Histochem. Cytochem., Graumann, W., Ed., Vol. 39, No. 4,

radioautography and X-ray microanalysis. Scanning Microscopy International, 14,

concept. Proc. XV Congress of the International Federation of Associations of

observed by microscopic radioautography after incorporation of radiolabeled

salamander. Bull. Nagano Women's Jr. College 6, 1-14, 1998c.

Microscopy and Analysis, Asia Pacific Edition, 9, 29-32, 1999a.

Anatomists, Ital. J. Anat. Embryol. 104 (Suppl. 1), 486-486, 1999b.

precursors. Methods Find. Exp. Clin. Pharmacol. 21, 683-706, 1999c. Nagata, T.: Radioautographic study on protein synthesis in mouse cornea. J. Kaken Eye Res.

electron microscopy. Image Analysis Stereolog. 19, 51-56, 2000a.

Nagata, T.: Special radioautographology: the eye. J. Kaken Eye Res. 18, 1-13, 2000f.

Vol. 211, Chapter 2, Academic Press, New York, pp. 33-154, 2001c.

Med. Res. 31, 185-195, 1998a.

201-241, 1998b.

8, 8-14, 1999d.

on line, 2000b.

Embryol. 104 (Suppl. 1), 487-487, 1999e.

(Congress Suppl.), 161-161, 2000e.

Cytochem. 34, 153-169, 2001b.

specimens. Micron 32, 387-404, 2001a.

Urban & Fischer Verlag, Jena, pp. 185-320, 2004.


Nagata, T.: Radiolabeling of soluble and insoluble compounds as demonstrated by light and

Nagata, T.: Quantitative analysis of histochemical reactions: Image analysis of light and

Nagata, T. Quantitative light and electron microscopic radioautographic studies on

Nagata, T.: Electron microscopic radioautography with cryo-fixation and dry-mounting

Nagata, T.: Application of electron microscopic radioautography to clinical electron

Nagata, T.: Radioautography in Medicine. Shinshu University Press, 268pp, Matsumoto,

Nagata, T.: Radioautography, general and special. In, Histo- and Cyto-chemistry 1994,

Nagata, T., Application of electron microscopic radioautography to clinical electron

Nagata, T.: Light and electron microscopic radioautographic study on macromolecular synthesis in digestive organs of aging mice. Cell. Mol. Biol. 41, 21-38, 1995a. Nagata, T.: Histochemistry of the organs: Application of histochemistry to anatomy. Acta

Nagata, T.: Three-dimensional observation of whole mount cultured cells stained with

Nagata, T.: Morphometry in anatomy: image analysis on fine structure and histochemical

Nagata, T.: Technique and application of electron microscopic radioautography. J. Electron

Nagata, T.: Techniques of light and electron microscopic radioautography. In,

Nagata, T.: On the terminology of radioautography vs. autoradiography. J. Histochem.

Nagata, T.: Techniques and applications of microscopic radioautography. Histol.

Nagata T.: Three-dimensional observation on whole mount cultured cells and thick sections

Nagata, T.: Radioautographic study on collagen synthesis in the ocular tissues. J. Kaken Eye

stained with histochemical reactions by high voltage electron microscopy. In, Recent Advances in Microscopy of Cells, Tissues and Organs, Motta, P., Ed.,

Cytochem. Acta Histochem. Cytochem. 29 (Suppl.), 343-344, 1996b. Nagata, T.: Remarks: Radioautographology, general and special. Cell. Mol. Biol. 42 (Suppl.),

histochemical reactions by ultrahigh voltage electron microscopy. Cell. Mol. Biol.

reactions with special reference to radioautography. Ital. J. Anat. 100 (Suppl. 1),

Histochemistry and Cytochemistry 1996. Proc. Xth Internat. Congr. Histochem.

Japan Society of Histochemistry and Cytochemistry, ed, pp. 219-231, Gakusai

Chinese J. Histochem. Cytochem. 2: 106-108, 1993b.

procedure. Acta Histochem. Cytochem. 27: 471-489, 1994a.

microscopy. Med. Electron Microsc. 27; 191-212, 1994b.

microscopy. Med. Electron Microsc. 27, 191-212, 1994e.

1993a.

1994c.

Kikaku Co., Tokyo, 1994d.

41, 783-792, 1995c.

591-605, 1995d.

11-12, 1996c.

Res. 15, 1-9, 1997c.

Microsc. 45, 258-274, 1996a.

Cytochem. 44, 1209-1209, 1996d.

Histopathol. 12, 1091-1124, 1997a.

Antonio Delfino Editore, Roma, pp. 37-44, 1997b.

Anat. Nippon. 70, 448-471, 1995b.

electron microscopy. Recent Advances in Cellular and Molecular Biology, Wegmann, R. J., Wegmann, M. A., Eds. Peters Press, Leuven, Vol. 6, pp. 9-21, 1992.

electron microscopic radioautograms. Acta Histochem. Cytochem. 26, 281-291,

macromolecular synthesis in several organs of prenatal and postnatal aging mice.


Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 433

Nagata, T.: Electron microscopic radioautographic study on DNA synthesis of mitochondria in adrenal medullary cells of aging mice. Open Anat. J. 1, 14-24, 2009g. Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 11, 1-17, 2009h. Nagata, T.: Electron microscopic radioautographic studies on macromoleclular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata T. Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Macromolecular synthesis in the livers of aging mice as revealed by electron

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata, T.: Electron microscopic radioautographic study on mitochondrial RNA synthesis in

Nagata, T.: Electron microscopic radioautographic study on protein synthesis of

Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA, RNA and

Nagata, T.: Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging. Ann. Rev. Biomed. Sci. 12, 1-29, 2010h. Nagata, T., Cui, H., Gao, F.: Radioautographic study on glycoprotein synthesis in the ocular

Nagata, T., Cui, H., Kong, Y.: The localization of TGF-b1 and its mRNA in the spinal cords

Nagata, T., Cui, H., Liang, Y.: Light microscopic radioautographic study on the protein

Nagata, T., Fujii, Y., Usuda, N.: Demonstration of extranuclear nucleic acid synthesis in

adrenocortical cells of aging mice. Open Anat J. 2, 91-97, 2010a.

Diego, St. Louis, Vol. 45, No. 1, pp. 1-80, 2010c.

Vol. 3, Formatex, Badajoz, Spain, in press, 2010g.

tissues. J. Kaken Eye Res. 13, 11-18, 1995.

Alternat Med Welfare 5, 25-37, 2010d.

Med. Welfare 5, 38-52, 2010f.

Welfare 4, 15-38, 2009i.

Welfare 4, 51-66, 2009j.

2222, 2010b.

232, 2010e.

41-60 (2001).

mitochondria of some organs in aging animals. Bull. Shinshu Inst. Alternat. Med.

in adreno-cortical cells of aging ddY mice. Bull. Shinshu Inst. Alternat. Med.

adrenal medullary cells of aging and senescent mice. J Cell Tissue Res. 10, 2213-

microscopic radioautography. In, Prog. Histochem. Cytochem., Sasse, D., Ed., Elsevier, Amsterdam, Boston, London, New York, Oxford, Paris, Philadelphia, San

mitochondria in adrenal medullary cells of aging mice. Bulletin Shinshu Inst

adrenal cortical and medullary cells of aging mice. J. Biomed. Sci. Enginer. 4, 219-

mitochondria in adrenal cortical cells of aging mice. Bulletin Shinshu Inst. Alternat.

protein synthesis in adrenal cells of aging mice. Formatex Microscopy Series No. 3,

of prenatal and postnatal aging mice demonstrated with immunohistochemical and in situ hybridization techniques. Bull. Nagano Women's Jr. College, 7, 75-88, 1999a.

synthesis in the cerebellum of aging mouse. Bull. Nagano Women's Jr. College, 9,

mammalian cells under experimental conditions by electron microscopic radioautography. Proc. 10th Internat. Congr. Electr. Microsc. 2, 305-306, 1982b. Nagata, T., Hirano, I., Shibata, O., Nagata, T.: A radioautographic study on the DNA

synthesis in the hepatic and the pancreatic acinar cells of mice during the postnatal


Nagata T.: Electron microscopic radioautographic study on protein synthesis in hepatocyte

Nagata T.: Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata T.: Macromolecular synthesis in hepatocyte mitochondria of aging mice as revealed

Nagata, T.: Electron microscopic radioautographic study on macromolecular synthesis in hepatocyte mitochondria of aging mouse. J. Cell Tissue Res. 7, 1019-1029, 2007c. Nagata, T.; Electron microscopic radioautographic study on nucleic acids synthesis in

Nagata, T.; Aging changes of macromolecular synthesis in the mitochondria of mouse

Nagata, T.: Sexual difference between the macromolecular synthesis of hepatocyte

Nagata, T.: Protein synthesis in hepatocytes of mice as revealed by electron microscopic

Nagata, T.: Electron microscopic radioatuographic studies on macromolecular synthesis in

Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis

Nagata, T.: Applications of high voltage electron microscopy to thick biological specimens.

Eds., Nova Biomed. Books, New York, pp. 133-161, 2009b.

Radiopharmaceutics 2, 118-128, 2009d.

Ann. Microsc. 9, 4-40, 2009f.

Nagata, T.: Radioautographology, Bull. Shinshu Institute Alternat. Med. 2, 3-32, 2007f. Nagata, T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in adrenal cortical cells of developing mice. J. Cell. Tis. Res. 8, 1303-1312, 2008a. Nagata T.: Electron microscopic radioautographic study on mitochondrial DNA synthesis in

hepatocyte mitochondria of developing mice. The Sci. World J. 6: 1583-1598, 2006b.

by electron microscopic radioautography. I: Nucleic acid synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. Eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 245-

by electron microscopic radioautography. II: Protein synthesis. In, Modern Research and Educational Topics in Microscopy. Mendez-Vilas, A. and Diaz, J. eds., Formatex Micrscopy Series No. 3, Vol. 1, Formatex, Badajoz, Spain, pp. 259-271,

hepatocyte mitochondria of developing mice. Trends Cell Molec. Biol. 2, 19-33,

hepatocytes as revealed by microscopic radioautography. Ann. Rev. Biomed. Sci.

adrenal cortical cells of developing and aging mice. The Sci. World J. 8, 683-697.

mitochondria in male and female mice in aging as revealed by electron microscopic radioautography. Chapter 22. In, Women and Aging: New Research, H. T. Bennninghouse, A. D. Rosset, Eds. Nova Biomed. Books, New York, pp. 461-487,

radioautography. In, Protein Biosynthesis. Esterhouse, T. E. and Petrinos, L. B.,

mitochondria of various cells. 18EMSM Conference Proc. 9th Asia-Pacific Microscopy Conference (APMC9), Kuala Lumpur, Malaysia, pp. 48-50, 2009c. Nagata, T.: Recent studies on macromolecular synthesis labeled with 3H-thymidine in

various organs as revealed by electron microscopic radioautography. Current

in adrenal medullary cells of developing and aging mice. J. Cell Tissue Res. 9, 1793-

mitochondria of developing mice. Ann. Microsc. 6, 43-54, 2006a.

258, 2007a.

2007b.

2007d.

2008b.

2009a

1802, 2009e.

9, 30-36, 2007e.


Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 435

Nagata, T., Ohno, S., Yoshida, K., Murata, F.: Nucleic acid synthesis in proliferating

Nagata, T., Olea, M. T.: Electron microscopic radioautographic study on the protein synthesis in aging mouse spleen. Bull. Nagano Women's Jr. College 7, 1-9, 1999. Nagata, T., Shibata, O., Omochi, S.: A new method for radioautographic osbservation on

Nagata, T., Shibata, O., Nawa, T.: Simplified methods for mass production of

Nagata, T., Shibata, O., Nawa, T.: Incorporation of tritiated thymidine into mitochondrial

Nagata, T., Shimamura, K., Onozawa, M., Kondo, T., Ohkubo, K., Momoze, S.: Relationship

Nagata, T., Shimamura, K., Kondo, T., Onozawa, M., Momoze, S., Okubo, M.: Relationship

Nagata, T., Steggerda, F. R.: Histological study on the deganglionated small intestine of the

Nagata, T., Steggerda, F. R.: Observations on the increase of binucleate cells in the ganglion

Nagata, T., Toriyama, K., Kong, Y., Jin, C., Gao, F.: Radioautographic study on DNA synthesis in the ciliary bodies of aging mice. J. Kaken Eye Res.12, 1-11, 1994. Nagata, T., Usuda, N.: Image processing of electron microscopic radioautograms in clinical

Nagata, T., Usuda, N.: Studies on the nucleic acid synthesis in pancreatic acinar cells of

Nagata, T., Usuda, N.: Electron microscopic radioautography of protein synthesis in

Nagata, T., Usuda, N.: In situ hybridization by electron microscopy using radioactive

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of nucleic acid

Nagata, T., Usuda, N., Ma, H.: Electron microscopic radioautography of lipid synthesis in pancreatic cells of aging mice. J. Clin. Electr. Microsc. 23, 841-842, 1990. Nagata, T., Usuda, N., Maruyama, M., Ma, H.: Electron microscopic radioautographic study

electron microscopy. J. Clin. Electron. Microsc. 18, 451-452, 1985.

probes. J. Histochem. Cytochem. 41, 1119-1119, 1993b.

Intern. Cong. Electr. Microsc. 3, 2281-2282, 1984.

DNA of the liver and kidney cells of chickens and mice in tissue culture.

of binuclearity to cell function in some organs. I. Frequencies of binucleate cells in some organs of toads in summer and winter. Med. J. Shinshu Univ. 5, 147-152,

of binuclearity to cell function in some organs. II. Variation of frequencies of binucleate cells in some organs of dogs owing to aging. Med. J. Shinshu Univ. 5,

cells of the dog's intestine due to experimental ischemia. Anat. Rec. 148, 315-315,

aging mice by means of electron microscopic radioautography. J. Clin. Electron

pancreatic acinar cells of aging mice. Acta Histochem. Cytochem. 26, 481-481,

synthesis in pancreatic acinar cells of prenatal and postnatal aging mice. Proc. XIth

on lipid synthesis in perinatal mouse pancreas. J. Clin. Electr. Microsc. 21, 756-757,

Histochem. J. 14, 197-204, 1982a.

Histochemie 10, 305-308, 1967b.

dog. Physiologist 6, 242-242, 1963.

Microsc. 19, 486-487, 1986.

1960a.

1964.

1993a.

1988b.

153-158, 1960b.

isolated cells. Histochemie 2, 255-259, 1961

radioautograms. -Acta Anat. Nippon.42, 162-166, 1967a.

peroxisomes of rat liver as revealed by electron microscopical radioautography.

growth, with special reference to binuclearity. Med. J. Shinshu Univ. 11, 35-42, 1966.


Nagata, T., Ito, M., Chen, S.: Aging changes of DNA synthesis in the submandibular glands

Nagata, T. Ito, M., Liang, Y.: Study of the effects of aging on macromolecular synthesis in

Nagata, T., Iwadare, I., Murata, F.: Electron microscopic radioautography of nucleic acid

Nagata, T., Kawahara, I.: Radioautographic study of the synthesis of sulfomucin in digestive organs of mice. J. Trace Microprobe Analysis 17, 339-355, 1999. Nagata, T., Kawahara, I., Usuda, N., Maruyama, M., Ma, H.: Radioautographic studies on

Nagata, T., Kong, Y.: Distribution and localization of TGFb1 and bFGF, and their mRNAs in

Nagata, T., Ma, H., Electron microscopic radioautographic study on mitochondrial DNA synthesis in hepatocytes of aging mouse. Ann. Microsc. 5, 4-18, 2005a. Nagata, T., Ma, H., Electron microscopic radioautographic study on RNA synthesis in hepatocyte mitochondria of aging mouse. Microsc. Res. Tech. 67, 55-64, 2005b. Nagata, T., Momoze, S.: Aging changes of the amitotic and binucleate cells in dog livers.

Nagata, T., Morita, T., I. Kawahara, I.: Radioautographic studies on radiosulfate incorporation in the digestive organs of mice. Histol. Histopathol. 14, 1-8, 1999b. Nagata, T., Murata, F.: Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy. Histochemistry 54, 75-82, 1977. Nagata, T., Murata, F., Yoshida, K., Ohno, S., Iwadare, N.: Whole mount radioautography of

Nagata, T., Nawa, T.: A modification of dry-mounting technique for radioautography of

Nagata, T., Nawa, T.: A radioautographic study on the nucleic acids synthesis of binucleate

Nagata, T., Nawa, T., Yokota, S.: A new technique for electron microscopic dry-mounting radioautography of soluble compounds. Histochemie 18, 241-249, 1969. Nagata, T., Nishigaki, T., Momose, Y.: Localization of anti-allergic agent in rat mast cells

Nagata, T., Ohno, S., Kawahara, I., Yamabayashi, S., Fujii, Y., Murata, F.: Light and electron

Nagata, T., Ohno, S., Murata, F.: Electron microscopic dry-mounting radioautography for

soluble compounds. Acta Phamacol. Toxicol. 41, 62-63, 1977a.

cultured cells as observed by high voltage electron microscopy. Proc. Fifth Internat.

cells in cultivated fibroblasts of chick embryos. Med. J. Shinshu Univ. 11, 1-5, 1966b.

demonstrated by light and electron microscopic radioautography. Acta Histochem.

microscopic radioautography of nucleic acid synthesis in mitochondria and peroxisomes of rat hepatic cells during and after DEHP administration. Acta

aging mice. Bull. Nagano Women's Jr. College 6, 87-105, 1998.

1966.

Microsc. 1, 4-12, 2000a.

Toxicol. 41, 64-65, 1977c.

Exp. Clin. Pharmacol. 22, 5-18, 2000b.

Professional Postgrad. Service, Tokyo, 1988a.

Acta Anat. Nipponica 34, 187-190, 1959.

Cytochem. 19, 669-683, 1986b.

Histochem. Cytochem. 16, 610-611, 1979.

Conf. High Voltage Electron Microsc. 347-350, 1977d.

water-soluble compounds. Histochemie 7, 370-371, 1966a.

growth, with special reference to binuclearity. Med. J. Shinshu Univ. 11, 35-42,

of mice as observed by light and electron microscopic radioautography. Ann.

mouse steroid secreting cells using microscopic radioautography. Methods Find.

synthesis in cultured cells treated with several carcinogens. Acta Pharmacol.

the glycoconjugate synthesis in the gastrointestinal mucosa of the mouse. In, Glycoconjugate in Medicine, Ohyama, M., Muramatsu, T., Eds, pp. 251-256,


Macromolecular Synthesis in the Endocrine, Nervous and Sensory Systems 437

Olea, M. T., Nagata, T.: A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro. Cell. Mol. Biol. 38, 399-405, 1992b. Oliveira, S. F., Nagata, T., Abrahamsohn, P. A., Zorn, T. M. T.: Electron microscopic

Oliveira, S. F., Abrahamsohn, P. A., Nagata, T., Zorn, T. M. T.: Incorporation of 3H-amino

Pearse, A. G. E.: Histochemistry, Theoretical and Applied. 4th Ed. Vol. 1. 439 pp., 1980, Vol.

Sakai, Y., Ikado, S., Nagata, T.: Electron microscopic radioautography of satellite cells in

Sato, A.: Quantitative electron microscopic studies on the kinetics of secretory granules in G-

Sato, A., Iida, F., Furihara, R., Nagata, T.: Electron microscopic raioautography of rat

Shimizu, T., Usuda, N., Yamanda, T., Sugenoya, A., Iida, F.: Proliferative activity of human

Sun, L.: Age related changes of RNA synthesis in the lungs of aging mice by light and electron microscopic radioautography. Cell. Mol. Biol. 41, 1061-1072, 1995. Sun, L., Gao, F., Duan, H., Nagata, T.: Light microscopic radioautography of DNA synthesis

Sun, L., Gao, F., Nagata, T.: Study on the DNA synthesis of pulmonary cells in aging mice by light microscopic radioautography. Cell. Mol. Biol. 41, 851-859, 1995a. Sun, L., Gao, F., Jin, C., Duan, H., Nagata, T.: An electron microscopic radioautographic

Sun, L., Gao, F., Jin, C., Nagata, T.: DNA synthesis in the tracheae of aging mice by means of

Sun, L., Gao, F., Nagata, T.: A Light Microscopic radioautographic study on protein

Suzuki, K., Imada, T., Gao, F., Ma, H., Nagata, T.: Radioautographic study of benidipine

Terauchi, A., Mori, T., Kanda, H., Tsukada, M., Nagata, T.: Radioautographic study of 3H-

Terauchi, A., Nagata, T.: Observation on incorporation of 3H-taurine in mouse skeletal

radioautographical study. Cell. Mol. Biol. 41, 107-116, 1995.

regenerating muscles. J. Clin. Electr. Microsc. 10, 508-509, 1977.

immnohistochemical studies. Cancer 71, 2807-2812, 1993.

pp. 201-205, Shinshu University Press, Matsumoto, 1994.

Cell. Mol. Biol. 37, 315-323, 1991.

Edinburgh, London and New York, 1991.

cells. Cell Tissue Res. 187, 45-59, 1978.

Microsc. 28, 129-131, 1995b.

rat. Drug Res. 44, 129-133, 1994.

211-220, 1997a.

470, 1997b.

1988.

397-404, 1993.

1977.

radioautographic study on the incorporation of 3H-proline by mouse decidual cells.

acids by endometrial stromal cells during decidualization in the mouse. A

2. 1055 pp., 1985, Vol. 3. Ed. with P. Stoward, 728 pp. Churchill Livingstone,

stomach G-cells by means of 3H-amino acids. J. Clin. Electron Microsc. 10, 358-359,

thyroid tumors evaluated by proliferating cell nuclear antigen/cyclin

in pulmonary cells in aging mice. In, Radioautography in Medicine, Nagata, T. Ed.,

study on the DNA synthesis of pulmonary tissue cells in aging mice. Med. Electron.

light and electron microscopic radioautography. Acta Histochem. Cytochem. 30,

synthesis in pulmonary cells of aging mice. Acta Histochem. Cytochem. 30, 463-

hydrochloride: localization in the mesenteric artery of spontaneously hypertensive

taurine uptake in mouse skeletal muscle cells. J. Clin. Electron Microsc. 21, 627-628,

muscle cells by light and electron microscopic radioautography. Cell. Mol. Biol. 39,


Nagata, T., Usuda, N., Suzawa, H., Kanzawa, M.: Incorporation of 3H-glucosamine into the

Nagata, T., Yamabayashi, S.: Intracellular localization of 3H-befunolol by means of electron

Nagata, T., Yoshida, K., Murata, F.: Demonstration of hot and cold mercury in the human

Nagata, T., Yoshida, K., Ohno, S., Murata, F.: Ultrastructural localization of soluble and

Nishigaki, T., Momose, Y., Nagata, T.: Light microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Electron microscopic radioautographic study of the

Nishigaki, T., Momose, Y., Nagata, T.: Localization of the anti-allergic agent tranilast in the

Oguchi, K., Nagata, T.: A radioautographic study of activated satellite cells in dystrophic

Oguchi, K., Nagata, T.: Electron microscopic radioautographic observation on activated

Ohno, S., Fujii, Y., Usuda, N., Endo, T., Hidaka, H., Nagata, T.: Demonstration of

Ohno, S., Fujii, Y., Usuda, N., Nagata, T., Endo, T., Tanaka, T., Hidaka, H.: Intracellular

Olea, M. T.: An ultrastructural localization of lysosomal acid phosphatase activity in aging

Olea, M. T., Nagata, T.: X-ray microanalysis of cerium in mouse spleen cells demonstrating

Olea, M. T., Nagata, T. : Simultaneous localization of 3H-thymidine incorporation and acid

radioautography. J. Clin. Electron Microsc. 25, 646-647, 1992.

Microsc. 16, 737-738, 1983.

Toxicol. 41, 60-61, 1977b.

Drug Res. 40, 272-275, 1990b.

Welfare of Japan, Tokyo, 1981.

Publishing Co., New York, 1982.

Cell. Mol. Biol. 38, 115-122, 1992a.

Cytochem. 24, 201-208, 1991.

37, 155-163, 1991.

radioautography. J. Electron Microsc. 32, 1-12, 1983.

1978b.

536, 1987.

65-71, 1990a.

Tokyo, 1980.

pancreatic cells of aging mice as demonstrated by electron microscopic

microscopic radioautography of cryo-fixed ultrathin sections. J. Clin. Electron

thyroid tissues by means of radioautography and chemography. Acta Pharmacol.

insoluble 3H-methyl prednisolone as revealed by electron microscopic drymounting radioautography. Proc. 9th Internat. Congr. Electr. Microsc. 2, 40-41,

localization of anti-allergic agent, tranilast, in rat mast cells. Histochem. J. 19, 533-

localization of an anti-allergic agent, tranilast, in rat mast cells. Cell. Mol. Biol. 36,

urinary bladder of rat as demonstrated by light microscopic radioautography.

chicken muscle. In, Current Research in Muscular Dystrophy Japan. The Proc. Ann. Meet. Muscular Dystrophy Res. 1980, pp. 16-17, Ministry of Welfare of Japan,

satellite cells in dystrophy chickens. In, Clinical Studies on the Etiology of Muscular Dystrophy. Annual Report on Neurological Diseases 1981, pp. 30-33, Ministry of

intracellular localization of calmodulin antagonist by wet-mounting

localization of calmodulin antagonists (W-7). In, Calmodulin and intracellular Ca2+ receptors. Kakiuchi, S., Hidaka, H, Means, A. R., Eds., pp. 39-48, Plenum

mouse spleen: a quantitative X-ray microanalytical study. Acta Histochem.

acid phosphatase activity using high voltage electron microscopy, Cell. Mol. Biol.

phosphatase activity in mouse spleen: EM radioautography and cytochemistry.


**18** 

*Mexico* 

**Cellular Senescence and** 

Diego Julio Arenas-Aranda1,

*Mexican Institute of Social Insurance,* 

*Technical National Institute. Mexico City,* 

**Its Relation with Telomere** 

Elena Hernández-Caballero2 and Fabio Salamanca-Gómez1

*2Section of Posgrado's Studies and Investigation, High School of Medicine,* 

*1Unit of Medical Research in Human Genetics, Medical National Center 21st Century,* 

For years it was thought that cells under culture conditions were immortal; however, from the publication of the works of Leonard Hayflick, this concept changed. Hayflick demonstrated that somatic cells under culture conditions had a limited capacity to proliferate; they stop dividing and enter into permanent arrest in the cell cycle, known as senescence. A particular characteristic of this state is that the cell maintains its viability and metabolic activity, and despite the presence of nutrients and mitogens, it does not divide (Ouellette et al., 2000). There are reports of cells that have been maintained in this state during several decades (Michaloglou et al., 2005). Regarding the origin of this process in mammalian cells, two different and apparently contradictory hypotheses have been proposed: senescence as a mechanism that suppresses tumor development, and senescence as the loss of the cells' regenerative capacity *in vivo*. In terms of the first hypothesis, senescence possesses a beneficial effect for the organism because it would avoid the development of cancer; otherwise, the second hypothesis would exert a harmful effect on organisms in that it would favor aging. Commentary will appear later on that both hypotheses joined together in the antagonist pleiotropic hypothesis (Williams, 1957; Campisi

It was 50 years ago that Hayflick first reported on the condition of mortality that cells maintain (Hayflick & Moorhead, 1961), which the researcher himself cited in 1998 (Hayflick, 1998). To the present day, few have dared to enter into the study of Biogerontology. The generalized belief was that cells kept under culture conditions could replicate themselves indefinitely; if this were not possible, it would be due to lack of knowledge on the appropriate conditions for maintaining cells under culture (Hayflick, 2003). Carrel in 1921 stated that it was possible to keep chicken-heart fibroblasts indefinitely, maintaining these with embryonic tissue extract. However, as Hayflick noted, this experiment involved a great

**1. Introduction** 

& Adda di Fagagna, 2007; Campisi, 2011).

**2. Hayflick experiments** 


## **Cellular Senescence and Its Relation with Telomere**

Diego Julio Arenas-Aranda1,

Elena Hernández-Caballero2 and Fabio Salamanca-Gómez1

*1Unit of Medical Research in Human Genetics, Medical National Center 21st Century, Mexican Institute of Social Insurance, 2Section of Posgrado's Studies and Investigation, High School of Medicine, Technical National Institute. Mexico City, Mexico* 

## **1. Introduction**

438 Senescence

Terauchi, A., Nagata, T.: In corporation of 3H-taurine into the blood capillary cells of mouse

Toriyama, K.: Study on the aging changes of DNA and protein synthesis of bipolar and

Tsukahara, S., Yoshida, K., Nagata, T.: A radioautographic study on the incorporation of

Usuda, N., Nagata, T.: Electron microscopic radioautography of acyl-CoA mRNA by in situ

Usuda, N., Nagata, T.: The immunohistochemical and in situ hybridization studies on hepatic peroxisomes. Acta Histochem. Cytochem. 28, 169-172, 1995. Usuda, N., Hanai, T., Morita, T., Nagata, T.: Radioautographic demonstration of

Uwa, H., Nagata, T.: Cell population kinetics of the scleroblast during ethisterone-induced

Watanabe, I., Makiyama, M. C. K., Nagata, T.: Electron microscopic radioautographic

Yamabayashi, S., Gunarso, W., Tsukahara, S., Nagata, T.: Incorporation of 3H-befunolol

Yamada, A., Nagata, T.: Ribonucleic acid and protein synthesis in the uterus of pregnant

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of DNA synthesis in

Yamada, A., Nagata, T.: Light and electron microscopic raioautography of RNA synthesis of

Yoshinaga, K.: Uterine receptivity for blastcyst implantation. Ann. N. Y. Acad. Sci. USA,

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Study of ethionine

Yoshizawa, S., Nagata, A., Honma, T., Oda, M., Murata, F., Nagata, T.: Radioautographic

radioautography. Cell. Mol. Biol. 41, 593-601, 1995.

pp.181-184, Peeters Press, Leuven, 1992.

Microscopica 6. 130-131, 1997.

Cell. Mol. Biol. 39, 1-12, 1993.

window. Cell. Mol. Biol. 38, 763-774, 1992b.

implantation. Cell. Mol. Biol. 38, 211-233, 1993.

hybridization. J. Clin. Electron Microsc. 25, 332-333, 1992.

Press, Matsumoto, 1994.

244, 1980.

9, 693-694, 1976.

365, 1992a.

541, 424-431, 1988.

Microsc. 7, 349-350, 1974.

Electron. Microsc. 10, 372-373, 1977.

skeletal muscle. Radioautography in Medicine, Nagata, T. ed., Shinshu University

photo-receptor cells of mouse retina by light and electron microscopic

14C-bupranolol (beta-blocking agent) into the rabbit eye. Histochemistry 68, 237-

peroxisomal acyl-CoA oxidase mRNA by in situ hybridization. In, Recent advances in cellular and molecular biology, Vol. 6. Molecular biology of nucleus, peroxisomes, organelles and cell movement. Wegmann, R. J., Wegmann, M., Eds,

anal-fin process formation in adult females of the Medaka. Dev. Growth Different.

observation of the submandibular salivary gland of aging mouse. Acta

(beta blocking agent) into melanin granules of ocular tissues in the pigmented rabbits. I. Light microscopic radioautography. Histochemistry 73, 371-375, 1981. Yamada, A. T.: Timely and topologically defined protein synthesis in the periimplanting

mouse endometrium revealed by light and electron microscopic radioautography.

mouse during activation of implantation window. Med. Electron Microsc. 27, 363-

the endometria of pregnant-ovariectomized mice during activation of implantation

peri-implanting pregnant mouse during activation of receptivity for blastocyst

pancreatitis by means of electron microscopic radioautography. J. Clin. Electron

study of protein synthesis in pancreatic exocrine cells of alcoholic rats. J. Clin.

For years it was thought that cells under culture conditions were immortal; however, from the publication of the works of Leonard Hayflick, this concept changed. Hayflick demonstrated that somatic cells under culture conditions had a limited capacity to proliferate; they stop dividing and enter into permanent arrest in the cell cycle, known as senescence. A particular characteristic of this state is that the cell maintains its viability and metabolic activity, and despite the presence of nutrients and mitogens, it does not divide (Ouellette et al., 2000). There are reports of cells that have been maintained in this state during several decades (Michaloglou et al., 2005). Regarding the origin of this process in mammalian cells, two different and apparently contradictory hypotheses have been proposed: senescence as a mechanism that suppresses tumor development, and senescence as the loss of the cells' regenerative capacity *in vivo*. In terms of the first hypothesis, senescence possesses a beneficial effect for the organism because it would avoid the development of cancer; otherwise, the second hypothesis would exert a harmful effect on organisms in that it would favor aging. Commentary will appear later on that both hypotheses joined together in the antagonist pleiotropic hypothesis (Williams, 1957; Campisi & Adda di Fagagna, 2007; Campisi, 2011).

## **2. Hayflick experiments**

It was 50 years ago that Hayflick first reported on the condition of mortality that cells maintain (Hayflick & Moorhead, 1961), which the researcher himself cited in 1998 (Hayflick, 1998). To the present day, few have dared to enter into the study of Biogerontology. The generalized belief was that cells kept under culture conditions could replicate themselves indefinitely; if this were not possible, it would be due to lack of knowledge on the appropriate conditions for maintaining cells under culture (Hayflick, 2003). Carrel in 1921 stated that it was possible to keep chicken-heart fibroblasts indefinitely, maintaining these with embryonic tissue extract. However, as Hayflick noted, this experiment involved a great

Cellular Senescence and Its Relation with Telomere 441

while the combination of wild-type alleles increased the risk of lung cancer in individuals aged >60 years. The researchers concluded that p53 protects the organism against cancer at the beginning of life, but that it promotes the aging phenotype in older persons, including the appearance of cancer at the end of life. Another recent study suggests that allele 4 carriers enjoy a beneficial cognitive effect in youth, and that later at an advanced age present cognitive diminution, which could increase the risk of presenting Alzheimer disease, although it could be that this allele is not pleiotropically antagonistic, but rather that it interacts with other risk factors for Alzheimer disease (Tuminello & Han, 2011). Feltes et al. (2011) suggest that aging and age-associated diseases could be the result of a program of development that is activated from the embryo stage, that persists throughout life, and that is regulated by the interaction of protein networks that connect environmental with molecular signals. The protein networks of the immune system, the epigenetic network, and aerobic metabolism are subject to great selection pressure during embryogenesis. However, this pressure becomes more relaxed in the

After gametes are fused during the fertilization process, that primordial cell denominated the zygote begins a long journey in the formation of the individual. However, this journey begins with an accelerated expansion in the number of cells, which later decelerates. Once the organism has been formed, it will utilize cellular replication during its entire lifetime to

Somatic cells possess a limited number of possible cell divisions, after which these become refractory to mitogeneic stimuli and enter into replicative senescence. In 2001, Sin et al. cite that aging at the cellular level is the result of cell function alterations, such as the response to DNA structural changes that is reflected in the expression of genes. A senescent cell remains arrested at cell cycle stage G1, and although it does not divide again, it remains metabolically active for a long time. The accumulation of mutations and damage to the DNA, together with inefficient repair mechanisms, become critical with each cell division and cause genetic heterogeneity in aging cells (De, 2011). There are somatic cells that possess the capacity to renew themselves, such as epithelial and blood cells, while there are cells that once differentiated, do not divide again, such as neurons. The equilibrium in death maintains homeostasis in the individual; thus, excessive death can lead to tissue degeneration and the inability to die can lead to hyperplasia and finally, cancer. The accumulated errors in a senescent cell's DNA can alter this balance and cause diverse

To date, there is no senescence marker that is totally specific for this stage because not all senescent cells express the same markers. These cells can exhibit diverse changes that in their entirety can aid us in defining the senescent phenotype (Rodier & Campisi, 2011).

Change in cell volume is one of the most evident characteristics of a senescent cell, because cells may be observed that range from 1,000 µm2 in an early passage of human fetal

grow, regenerate its tissues, cure wounds, or during the immune response.

adult, which allows the initiation of aging-associated diseases.

**4. Somatic cells and senescence** 

diseases (Hotchkiss et al., 2009).

**5. Senescent phenotype** 

technical error because the extract with which the culture was nourished throughout 34 years was supplied with fresh cells throughout the entire time that the experiment lasted (Hayflick, 1998).

Hayflick identified three phases in his cellular proliferation experiments: phase I or that of primary culture, in which cells initiate their proliferation; phase II, that of rapid and continuous proliferation, and phase III, in which proliferation velocity diminishes and is finally detained (Hayflick & Moorhead, 1961). Hayflick concludes that cells possess some type of counting mechanism because they stop dividing after a certain number of duplications, between 40 and 60. This counting mechanism is conserved even after the cells are frozen and cultured anew (Hayflick & Moorhead, 1961; Hayflick, 1965).

Some years later, thanks to the work of McClintock on chromosomal ends (McClintock, 1941), Olovnikov formulated his theory concerning the problem of replication and the solution to this. Suggesting that the inability of polymerase to replicate chromosomal ends totally was what could lead to cellular senescence, he proposed that the ends could function as a buffer, avoiding the loss of important sequences, but that in turn, this buffer function could also could be lost with successive replications (Olovnikov, 1996). Later, with the discovery of the telomeric repeats sequence of and telomerase by Blackburn, the study of telomeres and their participation in the senescence process began.

Currently, the existence is accepted of a limit of normal somatic cell replication, denominated the Hayflick limit. However, once the importance is established of telomerase as the enzyme that synthesizes telomeres (Greider & Blackburn, 1985), it was discovered that this enzyme is found to be active in immortalized cell lines (Morin, 1989), tumor cells (Kim et al., 1994), stem cells (Chiu et al., 1996), and embryonic and germinal cell lines (Mantell & Greider, 1994; Wright et al., 1996).

## **3. The antagonistic pleiotropy hypothesis**

Antagonistic pleiotropy is a concept pertaining to Evolutionary Biology that proposes that some genes can have an impact on the physical state of the organism differentially throughout its lifetime (Williams, 1957; Tuminello & Han, 2011). It is suggested that senescence evolved as an example of antagonistic pleiotropy; thus, its characteristics are beneficial in a reproductively active organism; later in this organism's lifespan, these characteristics become deteriorating. That is, senescence is the result of the random accumulation, whether passive or active, of harmful mutations (Kirkwood, 1977), reducing vigor and longevity, after the individual has passed reproductive age (Walker, 2011), although there is no definitive evidence that supports the negative effect of senescence in old persons.

Diverse processes have been found that are considered examples of antagonistic pleiotropy. Inflammation is a vital process to fight against infections and for cicatrization at all ages, but is also a process that can have negative effects if it becomes chronic in old age (Hornsby, 2010). On analyzing the influence of TP53 polymorphisms on cancer with respect to age, Cherdyntseva et al. (2010) found a relationship between the presence of polymorphisms in both genes and the increase of the risk of lung cancer in young, but not in older, smokers,

technical error because the extract with which the culture was nourished throughout 34 years was supplied with fresh cells throughout the entire time that the experiment lasted

Hayflick identified three phases in his cellular proliferation experiments: phase I or that of primary culture, in which cells initiate their proliferation; phase II, that of rapid and continuous proliferation, and phase III, in which proliferation velocity diminishes and is finally detained (Hayflick & Moorhead, 1961). Hayflick concludes that cells possess some type of counting mechanism because they stop dividing after a certain number of duplications, between 40 and 60. This counting mechanism is conserved even after the cells

Some years later, thanks to the work of McClintock on chromosomal ends (McClintock, 1941), Olovnikov formulated his theory concerning the problem of replication and the solution to this. Suggesting that the inability of polymerase to replicate chromosomal ends totally was what could lead to cellular senescence, he proposed that the ends could function as a buffer, avoiding the loss of important sequences, but that in turn, this buffer function could also could be lost with successive replications (Olovnikov, 1996). Later, with the discovery of the telomeric repeats sequence of and telomerase by Blackburn, the study of

Currently, the existence is accepted of a limit of normal somatic cell replication, denominated the Hayflick limit. However, once the importance is established of telomerase as the enzyme that synthesizes telomeres (Greider & Blackburn, 1985), it was discovered that this enzyme is found to be active in immortalized cell lines (Morin, 1989), tumor cells (Kim et al., 1994), stem cells (Chiu et al., 1996), and embryonic and germinal cell lines

Antagonistic pleiotropy is a concept pertaining to Evolutionary Biology that proposes that some genes can have an impact on the physical state of the organism differentially throughout its lifetime (Williams, 1957; Tuminello & Han, 2011). It is suggested that senescence evolved as an example of antagonistic pleiotropy; thus, its characteristics are beneficial in a reproductively active organism; later in this organism's lifespan, these characteristics become deteriorating. That is, senescence is the result of the random accumulation, whether passive or active, of harmful mutations (Kirkwood, 1977), reducing vigor and longevity, after the individual has passed reproductive age (Walker, 2011), although there is no definitive evidence that supports the negative effect of senescence in

Diverse processes have been found that are considered examples of antagonistic pleiotropy. Inflammation is a vital process to fight against infections and for cicatrization at all ages, but is also a process that can have negative effects if it becomes chronic in old age (Hornsby, 2010). On analyzing the influence of TP53 polymorphisms on cancer with respect to age, Cherdyntseva et al. (2010) found a relationship between the presence of polymorphisms in both genes and the increase of the risk of lung cancer in young, but not in older, smokers,

are frozen and cultured anew (Hayflick & Moorhead, 1961; Hayflick, 1965).

telomeres and their participation in the senescence process began.

(Mantell & Greider, 1994; Wright et al., 1996).

**3. The antagonistic pleiotropy hypothesis** 

(Hayflick, 1998).

old persons.

while the combination of wild-type alleles increased the risk of lung cancer in individuals aged >60 years. The researchers concluded that p53 protects the organism against cancer at the beginning of life, but that it promotes the aging phenotype in older persons, including the appearance of cancer at the end of life. Another recent study suggests that allele 4 carriers enjoy a beneficial cognitive effect in youth, and that later at an advanced age present cognitive diminution, which could increase the risk of presenting Alzheimer disease, although it could be that this allele is not pleiotropically antagonistic, but rather that it interacts with other risk factors for Alzheimer disease (Tuminello & Han, 2011). Feltes et al. (2011) suggest that aging and age-associated diseases could be the result of a program of development that is activated from the embryo stage, that persists throughout life, and that is regulated by the interaction of protein networks that connect environmental with molecular signals. The protein networks of the immune system, the epigenetic network, and aerobic metabolism are subject to great selection pressure during embryogenesis. However, this pressure becomes more relaxed in the adult, which allows the initiation of aging-associated diseases.

## **4. Somatic cells and senescence**

After gametes are fused during the fertilization process, that primordial cell denominated the zygote begins a long journey in the formation of the individual. However, this journey begins with an accelerated expansion in the number of cells, which later decelerates. Once the organism has been formed, it will utilize cellular replication during its entire lifetime to grow, regenerate its tissues, cure wounds, or during the immune response.

Somatic cells possess a limited number of possible cell divisions, after which these become refractory to mitogeneic stimuli and enter into replicative senescence. In 2001, Sin et al. cite that aging at the cellular level is the result of cell function alterations, such as the response to DNA structural changes that is reflected in the expression of genes. A senescent cell remains arrested at cell cycle stage G1, and although it does not divide again, it remains metabolically active for a long time. The accumulation of mutations and damage to the DNA, together with inefficient repair mechanisms, become critical with each cell division and cause genetic heterogeneity in aging cells (De, 2011). There are somatic cells that possess the capacity to renew themselves, such as epithelial and blood cells, while there are cells that once differentiated, do not divide again, such as neurons. The equilibrium in death maintains homeostasis in the individual; thus, excessive death can lead to tissue degeneration and the inability to die can lead to hyperplasia and finally, cancer. The accumulated errors in a senescent cell's DNA can alter this balance and cause diverse diseases (Hotchkiss et al., 2009).

## **5. Senescent phenotype**

To date, there is no senescence marker that is totally specific for this stage because not all senescent cells express the same markers. These cells can exhibit diverse changes that in their entirety can aid us in defining the senescent phenotype (Rodier & Campisi, 2011).

Change in cell volume is one of the most evident characteristics of a senescent cell, because cells may be observed that range from 1,000 µm2 in an early passage of human fetal

Cellular Senescence and Its Relation with Telomere 443

Cell cycle arrest-associated replicative senescence is related with telomere shortening and, as previously noted, is a response for suppressing tumor formation and that can have aging of the organism as a side effect (Harley et al., 1990; Campisi, 2011). The cell population gradually stops dividing. (Thomas et al., 1997). Cell growth inhibition can be the result of cellular quiescence, whether due to lack of growth or nutrient factors or to other in- and extrinsic factors. This leads to the cell's exhibiting a low metabolic rate, low protein synthesis and cell functions, and the absence of growth (Blagosklonny, 2011). The senescent cell is defined by the permanent lack of replicative potential despite receiving a mitogenic stimulus (Rodier & Campisi, 2011). The cells remain arrested in G1, although on some occasions can be stopped in G2 (Harley et al., 1990). The central signaling pathways for senescence are represented by the p16-pRb Retinoblastoma (Rb) protein and the p53 tumor suppressor (Lowe & Sherr, 2003). The p14-p53-p21 pathway is partially telomere-dependent, while the p16-pRb pathway is independent of the presence of dysfunctional telomeres (Campisi & d'Adda di Fagagna, 2007). The product of *p53* gene accumulates in response to cellular stress, which activates a specific gene target program to restrict the growth of abnormal or damaged cells; the result can be apoptosis, transitory cell cycle arrest, or permanent arrest (Beausejour et al., 2003). Thus, the product of *p53 gene* possesses anti-cancerous as well as pro-aging effects depending upon the context of the individual's age (Campisi, 2005). Among p53 target genes are found the Cyclindependent kinase (CDK) inhibitor p21, the pro-apoptotic genes *BAX* and *APAF1*, and the E3 ubiquitin ligase, MDM2 (Vousden & Lu, 2002). *p53* expression is controlled by p19 (Arf). On the other hand, Rb expression is controlled by p16Ink4a, whose protein levels increase in senescent cells. p16Ink4a maintains pRb in a hypophosphorylated state, which inhibits cell proliferation and induces growth arrest through the pRB effect on E2F; this is necessary to activate the genes implicated in cell cycle progression (Campisi & d'Adda di Fagagna, 2007). Both signaling pathways interact and are reciprocally regulated. However, each can interrupt

As Blagosklonny (2011) cites, cell arrest is only one part of the senescence equation, because senescent cells also become resistant to apoptosis. There are mechanisms of defense that augment apoptosis resistance, increasing anti-apoptotic signaling and avoiding the death of damaged cells. The increase of apoptosis resistance is a cell safety mechanism, because if cells have an acute stress due to some damage, they possess the capacity of recovering their homeostasis. However, in aging, when stress becomes persistent apoptosis resistance can cause the survival of undesired cells (Hampel et al., 2004; Salminen et al., 2011). It has been observed that the equilibrium between apoptotic and pro-apoptotic proteins changes with age. Bcl-2 and Bcl-xL protein levels are higher in aging than in young fibroblasts, while proapoptotic Bax levels are higher in young cells (Rochette & Brash, 2008). Apoptosis markers such as FasL and cytochrome C decrease in serum and, on the other hand, levels of soluble Fas (an apoptosis inhibitor) increase (Kavathia et al., 2009). Salminen et al. (2011) suggest that apoptosis resistance can affect the host's defenses in age-related fashion, a situation that

There are some molecular senescence markers that are characteristic of damage to DNA, including the nuclear foci of phosphorylated histones H2AX and DNA damage response

meets promoted by the chronic inflammation that senescent cells develop.

**5.1 Changes in gene expression, growth arrest, and apoptosis resistance** 

the cell cycle independently.

**6. Senescence markers** 

fibroblasts up to 9,000 µm2 in terminal passages of the culture. Size increase has been correlated with changes in cytoskeletal organization (Wang & Gundersen, 1984), which leads to modification in cell shape. For example, fibroblasts lose their typical tapered form to acquire a flat appearance (Greenberg et al., 1977), apparently due to changes in the expression of diverse cytoskeletal proteins. Nishio et al. (2001) found that senescent fibroblasts contain three times the amount of the cytoskeletal protein vimentin as embryonic fibroblasts. Vimentin presents as dense filament bundles that are parallel to the longest cellbody axis in senescent cells, while in young cells vimentin formation is observed as a network of short and thin filaments. The authors also demonstrated that young fibroblasts acquire a senescent phenotype once they are transfected with a vector that over expresses the vimentin gene, while actin levels diminish in senescent fibroblasts (Nishio & Inoue, 2005). This diminution causes rigidity in old donor cells and increases in the kinesis of cell reorganization (Zahn et al., 2011). On the other hand, cellular adhesion of aging fibroblasts increases. It was found recently that the senescence of vascular epithelial cells induces an increase in cell adhesion proteins, which in turn increases the adhesion of monocytes to endothelial cells through their binding with Intracellular adhesion molecule 1 (ICAM1), contributing to the appearance of atherosclerosis (Yanaka et al., 2011).

Another characteristic of cellular senescence is change in diverse organelles; a very common occurrence comprises the increase in lysosome number and size. In lysosomes, granules of lipofuscin, the so-called aging pigment, accumulate (Brunk & Terman, 2002; Gutteridge, 1984). This material cannot be degraded by the cell's proteolytic machinery, is highly toxic, and inhibits the degradation of oxidized proteins (Bader et al., 2007; Höhn et al., 2011). Another lysosome-linked senescence biomarker is β-galactosidase, which is derived from the β-D-galactosidase and whose activity increases in senescent cells. In non-senescent cells, lysosomes possess a pH4-optimal function, while when the cell ages, the lysosomal compartment expands and β-galactosidase increases; thus, it is possible to detect a suboptimal pH of 6, a change known as senescence-associated β-galactosidase activity (Dimri et al., 1995; Lee et al., 2006). Senescence is also implicated in the deterioration of mitochondrial function and in the appearance of mutations in mitochondrial DNA, due to the lack of a repair system (Percy et al., 2008), and it is considered that aberrant production of Reactive oxygen species (ROS) can increase the mitochondrial mass (Hwang et al., 2009) and on the other hand accelerate telomere shortening and contribute to cellular aging (Liu et al., 2002) due to damage to the DNA. With respect to the nucleus, the increase in chromatin condensation is the most evident nuclear change. Regions are formed that are known as Senescence-associated heterochromatin foci (SAHF); these DNA regions are associated with heterochromatin proteins such as HP1 and H3K9m (Narita et al., 2003). SAHF are also evident in cells that become senescent because of oncogenic stress with H-Ras (Kosar et al., 2011).

Changes in senescent cells are also reflected in their functions; for example, fibroblasts under culture conditions adopt a matrix-degrading phenotype, while adrenal cortex epithelial cells produce an altered steroid-hormone profile (Campisi, 2000). Due to the increase in the secretion of pro-inflammatory proteins such as interleukins and chemokines, it is said that senescent cells are found in a pro-inflammatory state (Freund et al., 2010), which in an aging organism can favor tissue deterioration.

fibroblasts up to 9,000 µm2 in terminal passages of the culture. Size increase has been correlated with changes in cytoskeletal organization (Wang & Gundersen, 1984), which leads to modification in cell shape. For example, fibroblasts lose their typical tapered form to acquire a flat appearance (Greenberg et al., 1977), apparently due to changes in the expression of diverse cytoskeletal proteins. Nishio et al. (2001) found that senescent fibroblasts contain three times the amount of the cytoskeletal protein vimentin as embryonic fibroblasts. Vimentin presents as dense filament bundles that are parallel to the longest cellbody axis in senescent cells, while in young cells vimentin formation is observed as a network of short and thin filaments. The authors also demonstrated that young fibroblasts acquire a senescent phenotype once they are transfected with a vector that over expresses the vimentin gene, while actin levels diminish in senescent fibroblasts (Nishio & Inoue, 2005). This diminution causes rigidity in old donor cells and increases in the kinesis of cell reorganization (Zahn et al., 2011). On the other hand, cellular adhesion of aging fibroblasts increases. It was found recently that the senescence of vascular epithelial cells induces an increase in cell adhesion proteins, which in turn increases the adhesion of monocytes to endothelial cells through their binding with Intracellular adhesion molecule 1 (ICAM1),

Another characteristic of cellular senescence is change in diverse organelles; a very common occurrence comprises the increase in lysosome number and size. In lysosomes, granules of lipofuscin, the so-called aging pigment, accumulate (Brunk & Terman, 2002; Gutteridge, 1984). This material cannot be degraded by the cell's proteolytic machinery, is highly toxic, and inhibits the degradation of oxidized proteins (Bader et al., 2007; Höhn et al., 2011). Another lysosome-linked senescence biomarker is β-galactosidase, which is derived from the β-D-galactosidase and whose activity increases in senescent cells. In non-senescent cells, lysosomes possess a pH4-optimal function, while when the cell ages, the lysosomal compartment expands and β-galactosidase increases; thus, it is possible to detect a suboptimal pH of 6, a change known as senescence-associated β-galactosidase activity (Dimri et al., 1995; Lee et al., 2006). Senescence is also implicated in the deterioration of mitochondrial function and in the appearance of mutations in mitochondrial DNA, due to the lack of a repair system (Percy et al., 2008), and it is considered that aberrant production of Reactive oxygen species (ROS) can increase the mitochondrial mass (Hwang et al., 2009) and on the other hand accelerate telomere shortening and contribute to cellular aging (Liu et al., 2002) due to damage to the DNA. With respect to the nucleus, the increase in chromatin condensation is the most evident nuclear change. Regions are formed that are known as Senescence-associated heterochromatin foci (SAHF); these DNA regions are associated with heterochromatin proteins such as HP1 and H3K9m (Narita et al., 2003). SAHF are also evident in cells that become senescent because of oncogenic stress with H-Ras (Kosar et al.,

Changes in senescent cells are also reflected in their functions; for example, fibroblasts under culture conditions adopt a matrix-degrading phenotype, while adrenal cortex epithelial cells produce an altered steroid-hormone profile (Campisi, 2000). Due to the increase in the secretion of pro-inflammatory proteins such as interleukins and chemokines, it is said that senescent cells are found in a pro-inflammatory state (Freund et al., 2010),

which in an aging organism can favor tissue deterioration.

contributing to the appearance of atherosclerosis (Yanaka et al., 2011).

2011).

#### **5.1 Changes in gene expression, growth arrest, and apoptosis resistance**

Cell cycle arrest-associated replicative senescence is related with telomere shortening and, as previously noted, is a response for suppressing tumor formation and that can have aging of the organism as a side effect (Harley et al., 1990; Campisi, 2011). The cell population gradually stops dividing. (Thomas et al., 1997). Cell growth inhibition can be the result of cellular quiescence, whether due to lack of growth or nutrient factors or to other in- and extrinsic factors. This leads to the cell's exhibiting a low metabolic rate, low protein synthesis and cell functions, and the absence of growth (Blagosklonny, 2011). The senescent cell is defined by the permanent lack of replicative potential despite receiving a mitogenic stimulus (Rodier & Campisi, 2011). The cells remain arrested in G1, although on some occasions can be stopped in G2 (Harley et al., 1990). The central signaling pathways for senescence are represented by the p16-pRb Retinoblastoma (Rb) protein and the p53 tumor suppressor (Lowe & Sherr, 2003). The p14-p53-p21 pathway is partially telomere-dependent, while the p16-pRb pathway is independent of the presence of dysfunctional telomeres (Campisi & d'Adda di Fagagna, 2007). The product of *p53* gene accumulates in response to cellular stress, which activates a specific gene target program to restrict the growth of abnormal or damaged cells; the result can be apoptosis, transitory cell cycle arrest, or permanent arrest (Beausejour et al., 2003). Thus, the product of *p53 gene* possesses anti-cancerous as well as pro-aging effects depending upon the context of the individual's age (Campisi, 2005). Among p53 target genes are found the Cyclindependent kinase (CDK) inhibitor p21, the pro-apoptotic genes *BAX* and *APAF1*, and the E3 ubiquitin ligase, MDM2 (Vousden & Lu, 2002). *p53* expression is controlled by p19 (Arf). On the other hand, Rb expression is controlled by p16Ink4a, whose protein levels increase in senescent cells. p16Ink4a maintains pRb in a hypophosphorylated state, which inhibits cell proliferation and induces growth arrest through the pRB effect on E2F; this is necessary to activate the genes implicated in cell cycle progression (Campisi & d'Adda di Fagagna, 2007). Both signaling pathways interact and are reciprocally regulated. However, each can interrupt the cell cycle independently.

As Blagosklonny (2011) cites, cell arrest is only one part of the senescence equation, because senescent cells also become resistant to apoptosis. There are mechanisms of defense that augment apoptosis resistance, increasing anti-apoptotic signaling and avoiding the death of damaged cells. The increase of apoptosis resistance is a cell safety mechanism, because if cells have an acute stress due to some damage, they possess the capacity of recovering their homeostasis. However, in aging, when stress becomes persistent apoptosis resistance can cause the survival of undesired cells (Hampel et al., 2004; Salminen et al., 2011). It has been observed that the equilibrium between apoptotic and pro-apoptotic proteins changes with age. Bcl-2 and Bcl-xL protein levels are higher in aging than in young fibroblasts, while proapoptotic Bax levels are higher in young cells (Rochette & Brash, 2008). Apoptosis markers such as FasL and cytochrome C decrease in serum and, on the other hand, levels of soluble Fas (an apoptosis inhibitor) increase (Kavathia et al., 2009). Salminen et al. (2011) suggest that apoptosis resistance can affect the host's defenses in age-related fashion, a situation that meets promoted by the chronic inflammation that senescent cells develop.

#### **6. Senescence markers**

There are some molecular senescence markers that are characteristic of damage to DNA, including the nuclear foci of phosphorylated histones H2AX and DNA damage response

Cellular Senescence and Its Relation with Telomere 445

(SAHF) (Funayama, 2007). One characteristic of the senescence program is observed in cultured fibroblasts in chromatin reorganization through H3 methylation in the Lys9 residue and by protein recruitment in the heterochromatin. Some agents that interact with DNA, such as doxorubicin, cisplatin, taxol, vincristine, cytarabine, and etoposide, can

It has been suggested that the condensation of genes implicated in proliferation through SAHF formation can contribute directly to senescence-associated silencing. However, it was recently suggested that SAHF are the result of persistent damage to the DNA and that it is condensation of the genes that promote proliferation, to a greater extent than large-scale

Cellular senescence can additionally be induced prior to telomere shortening, which is also known as premature senescence or stress-induced premature senescence. One example are fibroblasts under culture, which are exposed to stress by abnormal concentrations of nutrients and growth factors, in addition to the absence of neighbor cells and extracellular matrix, which can lead to the senescent phenotype (Sherr & DePinho, 2000). It also results from exposure to mutagens, such as ionizing radiation, ROS, chemotherapeutic agents, or bacterial toxins (Campisi & d'Adda di Fagagna, 2007); thus, the molecular mechanisms of induction are nearly identical to replicative senescence, that is, it is mediated by ATM-p53 p21. Another type is oncogene-induced senescence (Vavrova & Rezacova, 2011). However, it has been observed that moderate stress leads to telomere shortening and that the main cause of shortening is due to the presence of damaged bases, which interferes with the replication fork in telomeres, increasing the extension of the non-replicated ends (von Zglinicki, 2002; Duan et al., 2005). Non-genotoxic stress induces senescence by means of a telomereindependent mechanism, which involves p16-pRB pathway activation by p16INK4a over

Matos et al. (2011) recently analyzed the role of copper in inducing senescence in WI-38 fibroblasts and found that on exposing these to subcytotoxic copper sulfate concentrations, the fibroblasts exhibited the appearance of the senescent phenotype and an increase in senescence-associated genes such as *p21*, *apoJ*, fibronectin, *TGF β1*, *IGFBP3*, and Hemo oxigenase-1 (*HO-1*). These results are interesting because of the participation that copper can have in the establishment and progression of diseases such as Alzheimer and that of Wilson. On the other hand, it is possible that oxidative stress produces telomere shortening, therefore senescence, as confirmed by Brandl et al. (2011), because on exposing articular chondrocytes to oxidative stress with a sublethal dose of H2O2, the authors observed accelerated telomere exhaustion with over regulation of p21 expression and sub expression of SIRT1 and XRCC5, once the cells had acquired the senescent phenotype. Cellular senescence also can be induced by stress in the cultured cells, such as continuous mitogenic stimulation (Serrano & Blasco, 2001). Sustained exposure of melanocytes to an aberrant mitotic stimulus causes senescence after an initial proliferative burst, such as that observed by Michaloglou et al. (2005) suggesting that oncogene-induced senescence represents a

produce the senescent phenotype in tumor cells (Chang et al., 1999).

**7.3 Senescence induced by stress and other factors** 

regulation (Ben-Porath & Weinberg, 2005).

factor of protection against cancer.

SAHF, which detains senescence-associated proliferation (Rai & Adams, 2011).

factors such as 53BP1, MDC1, and NBS1 (d'Adda di Fagagna, 2008), which explains why culture shock can trigger senescence without the participation of telomeres. p16 is used for identification of senescent cells due to that it is overexpressed in the majority of these cells (Krishnamurthy et al., 2004). Although it has been demonstrated that in *Caenorhabditis elegans* alterations in microRNA´s (miRNAs) expression are associated with aging, the role that these could play in mammals remains unknown. In *C. elegans*, lin-4 over expression leads to an extension in its life span, while loss of lin-4 reduces the life span (Boehm & Slack, 2005). Lin-4 acts on the lin-14 messenger, affecting not only the life span, but also the insulin signaling pathway (Hung et al., 2010).

## **7. Causes of cellular senescence**

In stem cells, it has been observed that premature differentiation and senescence are alternatives to DNA damage repair that can exert a beneficial effect on restricting the accumulation of defective stem cells. However, sensitivity to DNA damage and p53-related apoptosis induction differ widely among stem cells (Blanpain et al., 2011). The stem cells of melanocytes, for example, undergo premature differentiation, which reduces the stem cell pool and causes graying of the hair (Inomata et al., 2009). On the other hand, loss of TP63 (a member of the p53 family) in dermal precursors leads to skin ulceration and cicatrization defects due to genomic instability and the induction of senescence (Su et al., 2009).

Recently, a chromatin remodeling factor was found to be implicated in replicative senescence, the Jun 2 dimerization protein (JDP2), which binds to histones and inhibits the binding of Polycomb repressor complexes (PRC1 and PRC2) to p16 gene promotor (Huang et al., 2011).

#### **7.1 Telomere-induced senescense**

The loss of proliferative potential can be a genetically programmed process. The telomereassociated aging theory suggests that progressive telomere sequence loss triggers chronic p53 activation, which consequently leads the cell to halt its proliferation (Lee et al., 1998). Telomere shortening is a stochastic event; thus, telomere length varies greatly among individuals (Halaschek-Wiener et al., 2008).

Dysfunctional telomeres trigger the response to DNA damage, which includes activation of ATM, 53BP1, Mdc1, Chk2, and H2AX, in addition to over expression of cell cycle inhibitors p21Cip1/Waf1 and p16INK4a and under expression of different cell cycle proteins (Herbig et al., 2004).

#### **7.2 Senescence induced by non-telomeric chromatin alterations**

Non-genotoxic stress can cause perturbations in chromatin, that is, epigenetic changes that can alter the genetic schema of the cell. It has been demonstrated that global genome methylation diminishes with age. However, it has also been observed that the promoters of certain genes can be hypermethylated, thus silenced. Histones themselves undergo modifications during aging, whether because of methylation or hypoacetylation; all of these chromatin regions in senescent cells are observed as transcriptionally inactive domains

factors such as 53BP1, MDC1, and NBS1 (d'Adda di Fagagna, 2008), which explains why culture shock can trigger senescence without the participation of telomeres. p16 is used for identification of senescent cells due to that it is overexpressed in the majority of these cells (Krishnamurthy et al., 2004). Although it has been demonstrated that in *Caenorhabditis elegans* alterations in microRNA´s (miRNAs) expression are associated with aging, the role that these could play in mammals remains unknown. In *C. elegans*, lin-4 over expression leads to an extension in its life span, while loss of lin-4 reduces the life span (Boehm & Slack, 2005). Lin-4 acts on the lin-14 messenger, affecting not only the life span, but also the insulin

In stem cells, it has been observed that premature differentiation and senescence are alternatives to DNA damage repair that can exert a beneficial effect on restricting the accumulation of defective stem cells. However, sensitivity to DNA damage and p53-related apoptosis induction differ widely among stem cells (Blanpain et al., 2011). The stem cells of melanocytes, for example, undergo premature differentiation, which reduces the stem cell pool and causes graying of the hair (Inomata et al., 2009). On the other hand, loss of TP63 (a member of the p53 family) in dermal precursors leads to skin ulceration and cicatrization defects due to genomic instability and the induction of

Recently, a chromatin remodeling factor was found to be implicated in replicative senescence, the Jun 2 dimerization protein (JDP2), which binds to histones and inhibits the binding of Polycomb repressor complexes (PRC1 and PRC2) to p16 gene promotor (Huang

The loss of proliferative potential can be a genetically programmed process. The telomereassociated aging theory suggests that progressive telomere sequence loss triggers chronic p53 activation, which consequently leads the cell to halt its proliferation (Lee et al., 1998). Telomere shortening is a stochastic event; thus, telomere length varies greatly among

Dysfunctional telomeres trigger the response to DNA damage, which includes activation of ATM, 53BP1, Mdc1, Chk2, and H2AX, in addition to over expression of cell cycle inhibitors p21Cip1/Waf1 and p16INK4a and under expression of different cell cycle proteins (Herbig

Non-genotoxic stress can cause perturbations in chromatin, that is, epigenetic changes that can alter the genetic schema of the cell. It has been demonstrated that global genome methylation diminishes with age. However, it has also been observed that the promoters of certain genes can be hypermethylated, thus silenced. Histones themselves undergo modifications during aging, whether because of methylation or hypoacetylation; all of these chromatin regions in senescent cells are observed as transcriptionally inactive domains

**7.2 Senescence induced by non-telomeric chromatin alterations** 

signaling pathway (Hung et al., 2010).

**7. Causes of cellular senescence** 

senescence (Su et al., 2009).

**7.1 Telomere-induced senescense** 

individuals (Halaschek-Wiener et al., 2008).

et al., 2011).

et al., 2004).

(SAHF) (Funayama, 2007). One characteristic of the senescence program is observed in cultured fibroblasts in chromatin reorganization through H3 methylation in the Lys9 residue and by protein recruitment in the heterochromatin. Some agents that interact with DNA, such as doxorubicin, cisplatin, taxol, vincristine, cytarabine, and etoposide, can produce the senescent phenotype in tumor cells (Chang et al., 1999).

It has been suggested that the condensation of genes implicated in proliferation through SAHF formation can contribute directly to senescence-associated silencing. However, it was recently suggested that SAHF are the result of persistent damage to the DNA and that it is condensation of the genes that promote proliferation, to a greater extent than large-scale SAHF, which detains senescence-associated proliferation (Rai & Adams, 2011).

### **7.3 Senescence induced by stress and other factors**

Cellular senescence can additionally be induced prior to telomere shortening, which is also known as premature senescence or stress-induced premature senescence. One example are fibroblasts under culture, which are exposed to stress by abnormal concentrations of nutrients and growth factors, in addition to the absence of neighbor cells and extracellular matrix, which can lead to the senescent phenotype (Sherr & DePinho, 2000). It also results from exposure to mutagens, such as ionizing radiation, ROS, chemotherapeutic agents, or bacterial toxins (Campisi & d'Adda di Fagagna, 2007); thus, the molecular mechanisms of induction are nearly identical to replicative senescence, that is, it is mediated by ATM-p53 p21. Another type is oncogene-induced senescence (Vavrova & Rezacova, 2011). However, it has been observed that moderate stress leads to telomere shortening and that the main cause of shortening is due to the presence of damaged bases, which interferes with the replication fork in telomeres, increasing the extension of the non-replicated ends (von Zglinicki, 2002; Duan et al., 2005). Non-genotoxic stress induces senescence by means of a telomereindependent mechanism, which involves p16-pRB pathway activation by p16INK4a over regulation (Ben-Porath & Weinberg, 2005).

Matos et al. (2011) recently analyzed the role of copper in inducing senescence in WI-38 fibroblasts and found that on exposing these to subcytotoxic copper sulfate concentrations, the fibroblasts exhibited the appearance of the senescent phenotype and an increase in senescence-associated genes such as *p21*, *apoJ*, fibronectin, *TGF β1*, *IGFBP3*, and Hemo oxigenase-1 (*HO-1*). These results are interesting because of the participation that copper can have in the establishment and progression of diseases such as Alzheimer and that of Wilson. On the other hand, it is possible that oxidative stress produces telomere shortening, therefore senescence, as confirmed by Brandl et al. (2011), because on exposing articular chondrocytes to oxidative stress with a sublethal dose of H2O2, the authors observed accelerated telomere exhaustion with over regulation of p21 expression and sub expression of SIRT1 and XRCC5, once the cells had acquired the senescent phenotype. Cellular senescence also can be induced by stress in the cultured cells, such as continuous mitogenic stimulation (Serrano & Blasco, 2001). Sustained exposure of melanocytes to an aberrant mitotic stimulus causes senescence after an initial proliferative burst, such as that observed by Michaloglou et al. (2005) suggesting that oncogene-induced senescence represents a factor of protection against cancer.

Cellular Senescence and Its Relation with Telomere 447

not form heterodimers, which leads to the presence of two protein complexes on the telomeres: one formed through TRF1, and the other by means of a paralog, TRF2 (Karlseder, 2003). TRF1 forms homodimers in order to bind stably to the DNA thanks to its Myb domain and, by means of T-loop formation, its binding with Rap1 at telomere repeats induces superficial double-strand folding, which indicates that it participates in loop formation (Bianchi et al., 1999). In cis, TRF1 acts in as a negative telomere length regulator. Its over expression produces telomere shortening and a dominant negative allele produces inappropriate lengthening in such a way that the amount of protein affects telomere size (Smogorzewska & de Lange, 2002). TRF1 can control telomerase access through its interaction with proteins TIN2, PTOP/PIP1, and POT1 and regulates their activity on interacting with PINX1 (Zhou et al., 2001). It also binds to TANK1 and 2 (Smith et al., 1998). Elimination of TRF1 produces telomere lengthening, but the extension stabilizes eventually due to that now the TIN2/TINT1 complex associates with TRF2, blocking access to

On the other hand, the complex formed by TRF2 is particularly important for protecting single-strand of the degradation and DNA repair processes (van Steensel et al., 1998; Smogorzewska & De Lange, 2004). TRF2 couples in the binding between double- and singlestranded repeats to facilitate T-loop formation, thus protecting its ends. In this manner, it is responsible for linear telomeric folding for T-loop formation (Griffith et al., 1999) and is found in >100 copies per chromosome (de Lange, 2002); additionally, given that it is the stabilizer of this structure, a lesser amount of TRF2 leads to T-loop opening, an event that can lead to senescence (Karlseder, 2003). Additionally, loss of TRF2 activates the Ataxia-Telangiectasia protein (ATM) kinase pathway, because while this is present it impedes autophosphorylation. ATM activation leads to p53 over regulation and G1/S arrest by means of p21 (Karlseder et al., 1999). When this is displaced from the telomere employing a dominant negative allele, the cell loses its ability to recognize the difference between a natural DNA end and a broken end. On the other hand, its over expression accelerates telomere shortening, which can be the result of the increase in the activity of a nuclease, whose activity is mediated by TRF2 (van Steelsen et al., 1998); Karlseder et al. (2002) suggest that an increase in TRF2 can protect critically short telomeres, delaying induction of cellular senescence even when the telomeres have been reduced. It can protect the single chain

It can bind to proteins such as Rap1 and to others involved in DNA damage repair responses, such as the MRE11/RAD50/NBS1 complex, Ku86, and ERCC1/XPF. Among its activities in blocking DNA repair is found that of avoiding that the T-loop insertion site is treated as a Holliday structure. On the other hand, inhibiting the binding of Nonhomologous end-joining (NHEJ) and homologous recombination in telomeres and probably in non-telomeric breaks allows determination of which repair pathway the cell should use (Wright & Shay, 2005). On the other hand, TRF2 facilitates the degradation of telomeric DNA on interacting with the WRN exonuclease, whose loss-of-function is implicated in premature cellular senescence, increasing the frequency of cancer and genomic instability

There are diverse proteins that bind indirectly with telomeres; TIN2 has emerged as an important component of the telomere complex. It interacts with the telomere through the

telomerase (Houghtaling et al., 2004).

indirectly on recruiting Pot1.

(Machwe et al., 2004).

#### **8. The mammalian telomere**

Telomeres are restricted to chromosomal ends and present in eukaryotes as diverse protozoans, fungi, flagellates, plants, and animals. The greater part of telomere DNA is double-stranded; however, the terminal 3' end is single-stranded. Each telomere is composed of a great region of short repeats rich in G. The sequence comprising a telomere varies in length and complexity depending on the organism (Greider, 1996). In the case of humans and other mammals, the sequence is TTAGGG, while organisms such as yeasts possess irregular repeat sequences in which a T is followed by one, two, or three Gs (TG1-3), while other organisms lack A in their repeats, as occurs in the ciliate Tetrahymena, which presents the TTGGGG sequence, and the Paramecium, which is distinguished by the TTGGGG and TTTGGG alteration.

Telomere DNA consists of two regions: one is double-stranded, and the other is singlestranded at its terminal end. The G-rich chain is that which projects further than the C-rich chain in the 3' direction. This salient is essential for telomere formation, due to that linear chromosomes need to protect their ends from degradation. The 3'-OH salient invades double-stranded telomere repeats, forming a loop-like structure called the T-loop (Telomere loop), in such a way that the salient remains hidden in the double chain (Griffith et al., 1999). The T-loop avoids that the ends are considered as DNA breaks and preserves genome integrity. The exact structure of the T-loop's base is unknown, but there is a short, doublestranded DNA segment that forms a D-loop (Displaced loop) of TTAGGG repeats, which is displaced by the invasion of the 3' salient (De Lange, 2002). The D-loop region can include Holliday-type binding (the intermediate state in homolog recombination) or a quadruple G fold (Neidle & Parkinson, 2003). The 3' salient, which in humans is between 35 and 600 nucleotides long, is the result of the impossibility of replicating the last fragment of Okasaki and of post-replicative processing events (Stewart et al., 2003).

## **9. The shelterin**

Maintenance of telomeric structure and regulation of its functions are supplied by diverse proteins that stabilize it and that permit the cell to distinguish between a natural chromosomal end and a DNA break. Shelterin or Telosome is a six-protein complex whose function is to form and maintain the T-loop. TRF1 and TRF2 are the main shelterin proteins. TRF1 modules telomere length, while TRF2 stabilizes T-loop structure (Xin et al., 2008). Other proteins have been described that, in addition to associating with the telomere, possess other cellular functions, such as XRCC5, which participates in double-stranded DNA damage repair (Thacker & Zdzienicka, 2004). SIRT1 is a negative regulator of p53 and that which avoids growth arrest, senescence, and apoptosis (Guarente, 1999). Doublestranded telomere DNA is wrapped in protein complexes that specifically bind to doublestranded proteins and that participate in the regulation of their length, while the 3' salient is wrapped in one or more single-stranded binding proteins that protect it (McEachern et al., 2000). The TRF protein family has a similar architecture, defined by two characteristic sequences: both have a DNA-binding motif in their helix-turn-helix carboxyl-terminus (highly related with the Myb domain of cMyb), and both possess a centrally localized sequence motif known as the TRF homolog domain (TRFH), unique for this protein family, which allows it to form homodimers (Fairall et al., 2001). However, the TRFH domain does

Telomeres are restricted to chromosomal ends and present in eukaryotes as diverse protozoans, fungi, flagellates, plants, and animals. The greater part of telomere DNA is double-stranded; however, the terminal 3' end is single-stranded. Each telomere is composed of a great region of short repeats rich in G. The sequence comprising a telomere varies in length and complexity depending on the organism (Greider, 1996). In the case of humans and other mammals, the sequence is TTAGGG, while organisms such as yeasts possess irregular repeat sequences in which a T is followed by one, two, or three Gs (TG1-3), while other organisms lack A in their repeats, as occurs in the ciliate Tetrahymena, which presents the TTGGGG sequence, and the Paramecium, which is distinguished by the

Telomere DNA consists of two regions: one is double-stranded, and the other is singlestranded at its terminal end. The G-rich chain is that which projects further than the C-rich chain in the 3' direction. This salient is essential for telomere formation, due to that linear chromosomes need to protect their ends from degradation. The 3'-OH salient invades double-stranded telomere repeats, forming a loop-like structure called the T-loop (Telomere loop), in such a way that the salient remains hidden in the double chain (Griffith et al., 1999). The T-loop avoids that the ends are considered as DNA breaks and preserves genome integrity. The exact structure of the T-loop's base is unknown, but there is a short, doublestranded DNA segment that forms a D-loop (Displaced loop) of TTAGGG repeats, which is displaced by the invasion of the 3' salient (De Lange, 2002). The D-loop region can include Holliday-type binding (the intermediate state in homolog recombination) or a quadruple G fold (Neidle & Parkinson, 2003). The 3' salient, which in humans is between 35 and 600 nucleotides long, is the result of the impossibility of replicating the last fragment of Okasaki

Maintenance of telomeric structure and regulation of its functions are supplied by diverse proteins that stabilize it and that permit the cell to distinguish between a natural chromosomal end and a DNA break. Shelterin or Telosome is a six-protein complex whose function is to form and maintain the T-loop. TRF1 and TRF2 are the main shelterin proteins. TRF1 modules telomere length, while TRF2 stabilizes T-loop structure (Xin et al., 2008). Other proteins have been described that, in addition to associating with the telomere, possess other cellular functions, such as XRCC5, which participates in double-stranded DNA damage repair (Thacker & Zdzienicka, 2004). SIRT1 is a negative regulator of p53 and that which avoids growth arrest, senescence, and apoptosis (Guarente, 1999). Doublestranded telomere DNA is wrapped in protein complexes that specifically bind to doublestranded proteins and that participate in the regulation of their length, while the 3' salient is wrapped in one or more single-stranded binding proteins that protect it (McEachern et al., 2000). The TRF protein family has a similar architecture, defined by two characteristic sequences: both have a DNA-binding motif in their helix-turn-helix carboxyl-terminus (highly related with the Myb domain of cMyb), and both possess a centrally localized sequence motif known as the TRF homolog domain (TRFH), unique for this protein family, which allows it to form homodimers (Fairall et al., 2001). However, the TRFH domain does

**8. The mammalian telomere** 

TTGGGG and TTTGGG alteration.

**9. The shelterin** 

and of post-replicative processing events (Stewart et al., 2003).

not form heterodimers, which leads to the presence of two protein complexes on the telomeres: one formed through TRF1, and the other by means of a paralog, TRF2 (Karlseder, 2003). TRF1 forms homodimers in order to bind stably to the DNA thanks to its Myb domain and, by means of T-loop formation, its binding with Rap1 at telomere repeats induces superficial double-strand folding, which indicates that it participates in loop formation (Bianchi et al., 1999). In cis, TRF1 acts in as a negative telomere length regulator. Its over expression produces telomere shortening and a dominant negative allele produces inappropriate lengthening in such a way that the amount of protein affects telomere size (Smogorzewska & de Lange, 2002). TRF1 can control telomerase access through its interaction with proteins TIN2, PTOP/PIP1, and POT1 and regulates their activity on interacting with PINX1 (Zhou et al., 2001). It also binds to TANK1 and 2 (Smith et al., 1998). Elimination of TRF1 produces telomere lengthening, but the extension stabilizes eventually due to that now the TIN2/TINT1 complex associates with TRF2, blocking access to telomerase (Houghtaling et al., 2004).

On the other hand, the complex formed by TRF2 is particularly important for protecting single-strand of the degradation and DNA repair processes (van Steensel et al., 1998; Smogorzewska & De Lange, 2004). TRF2 couples in the binding between double- and singlestranded repeats to facilitate T-loop formation, thus protecting its ends. In this manner, it is responsible for linear telomeric folding for T-loop formation (Griffith et al., 1999) and is found in >100 copies per chromosome (de Lange, 2002); additionally, given that it is the stabilizer of this structure, a lesser amount of TRF2 leads to T-loop opening, an event that can lead to senescence (Karlseder, 2003). Additionally, loss of TRF2 activates the Ataxia-Telangiectasia protein (ATM) kinase pathway, because while this is present it impedes autophosphorylation. ATM activation leads to p53 over regulation and G1/S arrest by means of p21 (Karlseder et al., 1999). When this is displaced from the telomere employing a dominant negative allele, the cell loses its ability to recognize the difference between a natural DNA end and a broken end. On the other hand, its over expression accelerates telomere shortening, which can be the result of the increase in the activity of a nuclease, whose activity is mediated by TRF2 (van Steelsen et al., 1998); Karlseder et al. (2002) suggest that an increase in TRF2 can protect critically short telomeres, delaying induction of cellular senescence even when the telomeres have been reduced. It can protect the single chain indirectly on recruiting Pot1.

It can bind to proteins such as Rap1 and to others involved in DNA damage repair responses, such as the MRE11/RAD50/NBS1 complex, Ku86, and ERCC1/XPF. Among its activities in blocking DNA repair is found that of avoiding that the T-loop insertion site is treated as a Holliday structure. On the other hand, inhibiting the binding of Nonhomologous end-joining (NHEJ) and homologous recombination in telomeres and probably in non-telomeric breaks allows determination of which repair pathway the cell should use (Wright & Shay, 2005). On the other hand, TRF2 facilitates the degradation of telomeric DNA on interacting with the WRN exonuclease, whose loss-of-function is implicated in premature cellular senescence, increasing the frequency of cancer and genomic instability (Machwe et al., 2004).

There are diverse proteins that bind indirectly with telomeres; TIN2 has emerged as an important component of the telomere complex. It interacts with the telomere through the

Cellular Senescence and Its Relation with Telomere 449

Recently, it was discovered that Rap1 is an important factor for avoiding telomere

Telomerase is a Ribonucleoprotein (RNP) composed of two units: the catalytic subunit, TERT, and a RNA template, TERC. TERT is a member of the family of reverse transcriptases related with non-LTR retrotransposons and group II introns. Its reverse transcriptase domain is found at the middle of the carboxyl-terminus and it supplies the active site for catalysis (Cech, 2004). TERC, also called TR, is highly expressed in all tissues, it not being important whether they possess telomerase activity or not, and it contains a short sequence that acts as a template from which DNA repeats are copied (Cong et al., 2002). Telomerase carries its own template and is restricted to copying solely a small segment of its RNA. Thus, implicit in telomerase polymerization activity is its ability to specify the template region and its limits, and also a mechanism for maintaining itself as a stable RNP while carrying out synthesis, because it allows the template to move through the active site during the synthesis process of a repeat, to later translocate itself and initiate the synthesis of

When the telomere catalytic subunit in mouse mutates, the first generation that lacks telomerase activity is phenotypically normal, with long telomeres. After four to six generations, its telomeres become very short, and the mice suffer from infertility, proliferation defects, and the risk of apoptosis in organs that undergo constant turnover, which diminishes their life span, while a *TERC* gene mutation causes Dyskeratosis congenita (DKC), a disease in which, among other characteristics, the telomeres are

There are accessory factors that aid the telomerase in acting on the telomere, such as dyskerin and TP1. Dyskerin is important for ribosomal processing because it binds to many small nucleolar RNA, and it is implicit in TERC and even TERT processing or stability because its small nucleolar RNP domain reached maturation in the nucleolus and later binds to the dyskerin (Cech, 2004). TP1 can form one or more structures that mediate interactions with other telomerase- or telomere-binding proteins such as TRF. The TP1 pattern of expression is not restricted to tissues and cell lines that express telomerase activity;

Chai et al. (2006) found that it is possible that telomerase preferentially extends the leader chain of 20-30 nucleotides per replication round in order to produce a salient similar to that of the delayed chain, because the leader-chain salient is smaller, which otherwise would

Telomerase-to-telomere access regulation is carried out by telomere-associated proteins, for example, TRF1 and TRF2; on forming the T-loop, these inhibit telomerase binding, while POT1 binding to the salient does not permit coupling of the enzyme so that this would

Mutations in telomerase components produce premature dysfunctions in adult stem cells

recombination and fragility (Martínez & Blasco, 2011).

another repeat. (O'Reilly et al., 1999; Cech, 2004).

therefore, it is not an essential subunit (Harrington et al., 1997).

affect the conformation of telomere structure.

and reduce longevity (Mitchell et al., 1999).

extend it.

abnormally short (Blasco, 2005).

**10. The telomerase** 

TRFH domain of TRF1, negatively regulating telomere length. A truncated form of TIN2 produces abnormal telomerase-independent telomere lengthening; therefore, it is a TRF1 function mediator on potentiating the pairing of telomere repeats in a TRF1-dependent manner; in addition, it can lead to the telomerase-inhibiting telomere (Kanoh & Ishikawa, 2003). In addition to this telomere-size regulator function, another mutant form generates DNA damage response and senescence (Kim et al., 2004). Through this protein, TRF1 and TRF2 can interact, while TIN2 can stabilize TRF2-complex binding to the telomere on acting as a liaison between this and TRF1. On the other hand, thanks to its third domain, it binds to PIP1/PTOP/TINT1, which in turn serves to recruit POT1 (Liu et al., 2004; Ye & de Lang, 2004). On controlling the Poly (ADP-ribose) polymerase (PARP) activity of tankirase 1, TIN2 protects TRF1 on its removal from the telomere (Ye & de Lang, 2004).

Tankirase 1 interacts with the TRF1. It is a PARP telomere that adds poly ADP-ribose to TRF1, diminishing its affinity for telomeric DNA (Smith & de Lange, 2000). More than diminishing TRF1 affinity for telomere repeats, what TANK1 does on separating TRF1 from the DNA is to expose its myb domain, which is recognized and marked by ubiquitin, leading to degradation of the protein by the proteosome. If this were performed otherwise, TRF1 would bind again to the telomere and its separation would permit the telomerase to gain access to and extend it (Chang et al., 2003). TIN2 is what stabilizes TANK1 binding to the TRF1 complex, and although TANK1 is the lesser abundant of these two proteins, it is necessary for controlled dismantling of the telomere complex during S phase (Ye & de Lange, 2004).

TANK 2, recently identified as a Golgi-associated protein, shares 80% identity in the aminoacid sequence with TANK1, in addition to similar distribution; however, when this is over expressed, it induces rapid cell death with necrotic characteristics (Kaminker et al., 2001). In addition and similar to TANK1, it is a PARP modifier of TRF1 and possibly possesses little effect on telomerase activity (d'Adda di Fagagna et al., 2004).

There are a variety of other proteins that bind to telomeres, such as Ku, Rap1, PIP1/PTOP/TINT1, WRN, PINX1 (Stellwagen et al., 2003; Espejel et al., 2002; Kanoh & Ishikawa, 2003; Köning & Rhodes, 1997; Lei et al., 2000; Ye & de Lange, 2004; Crabbe et al., 2004), and ATM. On the other hand, repair-machinery proteins such as the Mre11/Rad50/ /Nsb1 complex (the MRN complex), which participates in double-stranded DNA repair, can play a role in telomere maintenance, although it does not directly bind with it but rather interacts through TRF2 and possibly participates in T-loop formation (Saldanha et al., 2003). Among these single-stranded binding proteins is found POT1; this binds to a telomeric salient with exceptionally high specificity. It adopts an oligosaccharide-oligonucleotide (OB) joining fold with two forks that overhang to form a clamp for binding to DNA (Lei et al., 2003). When the PTO1 binding domain is mutated, there is no telomeric end fusion, but rather an increase in telomere extension by the telomerase; thus, one of its functions is to block the access of this enzyme to the DNA. TRF1 interacts with POT1; thus, it is the length control terminal transductor for TRF1. The more TRF1, more POT1, which leads to an increase in telomerase inhibition (Mattern et al., 2004). POT1 is necessary to maintain structure in telomere salients, protecting the cell against apoptosis, avoiding chromosomal instability and senescence, and interacting with TRF2 at the T-loop formation point, with which it cooperates for maintaining telomere integrity (Yan et al., 2005).

Recently, it was discovered that Rap1 is an important factor for avoiding telomere recombination and fragility (Martínez & Blasco, 2011).

## **10. The telomerase**

448 Senescence

TRFH domain of TRF1, negatively regulating telomere length. A truncated form of TIN2 produces abnormal telomerase-independent telomere lengthening; therefore, it is a TRF1 function mediator on potentiating the pairing of telomere repeats in a TRF1-dependent manner; in addition, it can lead to the telomerase-inhibiting telomere (Kanoh & Ishikawa, 2003). In addition to this telomere-size regulator function, another mutant form generates DNA damage response and senescence (Kim et al., 2004). Through this protein, TRF1 and TRF2 can interact, while TIN2 can stabilize TRF2-complex binding to the telomere on acting as a liaison between this and TRF1. On the other hand, thanks to its third domain, it binds to PIP1/PTOP/TINT1, which in turn serves to recruit POT1 (Liu et al., 2004; Ye & de Lang, 2004). On controlling the Poly (ADP-ribose) polymerase (PARP) activity of tankirase 1, TIN2

Tankirase 1 interacts with the TRF1. It is a PARP telomere that adds poly ADP-ribose to TRF1, diminishing its affinity for telomeric DNA (Smith & de Lange, 2000). More than diminishing TRF1 affinity for telomere repeats, what TANK1 does on separating TRF1 from the DNA is to expose its myb domain, which is recognized and marked by ubiquitin, leading to degradation of the protein by the proteosome. If this were performed otherwise, TRF1 would bind again to the telomere and its separation would permit the telomerase to gain access to and extend it (Chang et al., 2003). TIN2 is what stabilizes TANK1 binding to the TRF1 complex, and although TANK1 is the lesser abundant of these two proteins, it is necessary for controlled dismantling of the telomere complex during S phase (Ye & de

TANK 2, recently identified as a Golgi-associated protein, shares 80% identity in the aminoacid sequence with TANK1, in addition to similar distribution; however, when this is over expressed, it induces rapid cell death with necrotic characteristics (Kaminker et al., 2001). In addition and similar to TANK1, it is a PARP modifier of TRF1 and possibly possesses little

There are a variety of other proteins that bind to telomeres, such as Ku, Rap1, PIP1/PTOP/TINT1, WRN, PINX1 (Stellwagen et al., 2003; Espejel et al., 2002; Kanoh & Ishikawa, 2003; Köning & Rhodes, 1997; Lei et al., 2000; Ye & de Lange, 2004; Crabbe et al., 2004), and ATM. On the other hand, repair-machinery proteins such as the Mre11/Rad50/ /Nsb1 complex (the MRN complex), which participates in double-stranded DNA repair, can play a role in telomere maintenance, although it does not directly bind with it but rather interacts through TRF2 and possibly participates in T-loop formation (Saldanha et al., 2003). Among these single-stranded binding proteins is found POT1; this binds to a telomeric salient with exceptionally high specificity. It adopts an oligosaccharide-oligonucleotide (OB) joining fold with two forks that overhang to form a clamp for binding to DNA (Lei et al., 2003). When the PTO1 binding domain is mutated, there is no telomeric end fusion, but rather an increase in telomere extension by the telomerase; thus, one of its functions is to block the access of this enzyme to the DNA. TRF1 interacts with POT1; thus, it is the length control terminal transductor for TRF1. The more TRF1, more POT1, which leads to an increase in telomerase inhibition (Mattern et al., 2004). POT1 is necessary to maintain structure in telomere salients, protecting the cell against apoptosis, avoiding chromosomal instability and senescence, and interacting with TRF2 at the T-loop formation point, with

protects TRF1 on its removal from the telomere (Ye & de Lang, 2004).

effect on telomerase activity (d'Adda di Fagagna et al., 2004).

which it cooperates for maintaining telomere integrity (Yan et al., 2005).

Lange, 2004).

Telomerase is a Ribonucleoprotein (RNP) composed of two units: the catalytic subunit, TERT, and a RNA template, TERC. TERT is a member of the family of reverse transcriptases related with non-LTR retrotransposons and group II introns. Its reverse transcriptase domain is found at the middle of the carboxyl-terminus and it supplies the active site for catalysis (Cech, 2004). TERC, also called TR, is highly expressed in all tissues, it not being important whether they possess telomerase activity or not, and it contains a short sequence that acts as a template from which DNA repeats are copied (Cong et al., 2002). Telomerase carries its own template and is restricted to copying solely a small segment of its RNA. Thus, implicit in telomerase polymerization activity is its ability to specify the template region and its limits, and also a mechanism for maintaining itself as a stable RNP while carrying out synthesis, because it allows the template to move through the active site during the synthesis process of a repeat, to later translocate itself and initiate the synthesis of another repeat. (O'Reilly et al., 1999; Cech, 2004).

When the telomere catalytic subunit in mouse mutates, the first generation that lacks telomerase activity is phenotypically normal, with long telomeres. After four to six generations, its telomeres become very short, and the mice suffer from infertility, proliferation defects, and the risk of apoptosis in organs that undergo constant turnover, which diminishes their life span, while a *TERC* gene mutation causes Dyskeratosis congenita (DKC), a disease in which, among other characteristics, the telomeres are abnormally short (Blasco, 2005).

There are accessory factors that aid the telomerase in acting on the telomere, such as dyskerin and TP1. Dyskerin is important for ribosomal processing because it binds to many small nucleolar RNA, and it is implicit in TERC and even TERT processing or stability because its small nucleolar RNP domain reached maturation in the nucleolus and later binds to the dyskerin (Cech, 2004). TP1 can form one or more structures that mediate interactions with other telomerase- or telomere-binding proteins such as TRF. The TP1 pattern of expression is not restricted to tissues and cell lines that express telomerase activity; therefore, it is not an essential subunit (Harrington et al., 1997).

Chai et al. (2006) found that it is possible that telomerase preferentially extends the leader chain of 20-30 nucleotides per replication round in order to produce a salient similar to that of the delayed chain, because the leader-chain salient is smaller, which otherwise would affect the conformation of telomere structure.

Telomerase-to-telomere access regulation is carried out by telomere-associated proteins, for example, TRF1 and TRF2; on forming the T-loop, these inhibit telomerase binding, while POT1 binding to the salient does not permit coupling of the enzyme so that this would extend it.

Mutations in telomerase components produce premature dysfunctions in adult stem cells and reduce longevity (Mitchell et al., 1999).

Cellular Senescence and Its Relation with Telomere 451

1996, Sprung et al. (1996) sought to study the telomere-length effect on HSV-tk promotor expression, utilizing the KB319 cell line (SV40-transformed fibroblasts); the authors integrated a plasmid with the neo-gene at the end of chromosome 13 and found no effect on neo-gene expression when telomere length ranged from 25-0.5 kb; this leads them to suggest that chromatin structural differences conferred by telomere length do not affect the expression of nearby genes. A 130-kb microdeletion in the end of chromosome 22q in cells from a child with mental retardation; the broken end had been repaired by telomere addition, and consequently a unique DNA region that was normally localized at a distance of >100 kb from the telomere was now adjacent to it. This was the model employed by Ofir et al. in 1999 to demonstrate that telomeric sequences influence the activation of nearby replication origins, delaying the synchronization of replication at mid-S phase. The latter suggests that if human telomere repeats silence genes adjacent to repaired ends, then it is also conceivable that nearby genes may be epigenetically inactivated. On the other hand, Kilburn et al. (2001) found that the presence of a telomere sequence in an *APRT* gene intron in hamster ovary cells only had a modest effect on its expression. Finally, it was Baur et al. in 2001 who, employing a luciferase reporter, supplied convincing evidence of transcriptional silencing near telomeres in humans. The authors found that on placing the reporter adjacent to telomere repeats, there was 10-times lower expression than when they placed it at non-telomeric sites. However, the authors also found that TPE in humans required a histone deacetylase, because on treating the telomeric clones with trichostatin A (a histone deacetylase inhibitor), luciferase expression was restored. Koering et al. (2002) obtained similar results utilizing the *EGFP* reporter gene in C33-A cells (undifferentiated cervical carcinoma), reverting the repressor effect on employing trichostatin A, and they suggested that the position effect depends on the organization of telomeric chromatin, due to that they encountered the release of heterochromatin proteins HP1 and HP1. Pedram et al. (2006) developed embryonic stem cell clones of mouse with unique-copy gene markers and found that telomeric transgenes were not silenced in cells obtained from 3-day-old embryos as a result of their demethylation during early development, which led the authors to suggest that TPE also plays a role in embryo development. On the other hand, Wright and Shay (1992) also propose that progressive changes in presenescent cells can be the result of the reorganization of telomere chromatin and of the corresponding silencing or desilencing of subtelomere genes. This hypothesis has been extended to include the possibility that silenced proteins can be released from telomeres when the latter are shortened, in order to affect the expression of genes at internal non-telomere sites (Wood and Sinclair, 2002). This idea was based on studies with *S. cerevisiae*, in which it was demonstrated that gene markers inserted <4 kb from telomere repeats are frequently repressed and replicated at the end of the S phase (Dubrana et al., 2001). Although the loss of TPE is not the senescence trigger, it can be responsible for progressive changes in gene

expression as a function of replicative age (Wood and Sinclair, 2002).

**relation to cellular senescence** 

**12. Role of telomere length in subtelomeric gene expression and its possible** 

To date, there is only one report to our knowledge that studies *in vivo* TPE in human subtelomere genes. The results obtained suggest that the expression of these genes can be influenced by alterations in local heterochromatin structure so that this obstructs access to transcriptional factors (Ning et al., 2003). Due to the fact that few evidence exist about of

#### **11. Telomere position effect (TPE)**

At present, it is accepted that telomere clipping can affect gene expression in subtelomeric regions, which can lead to modification of the Biology of the cell prior to initiation of replicative senescence (Baur et al., 2001).

The Position variegation effect (PVE) refers to inactivation of a gene, which occurs when it is removed from its normal context by means of a rearrangement or by transgene insertion. The best known example of this is the result of the expression of the euchromatic white gene in Drosophila, which is responsible for the red color of the eye. Provoked by a rearrangement in chromosome X that causes its relocation near the heterochromatin region, it eliminates function in some cells, which produces mottled pigmentation. The explanation for silencing of the white gene in some cells and not in others is that condensed and inactive conformation of pericentric heterochromatin is dispersed on the rearrangement break and randomly inactivates nearby genes (Henikoff, 1990). This chromosomal position effect affects up to a distance of approximately 1Mb and reflects a genetic inactivation gradient that is inversely correlated with distance (Wakimoto, 1998). The PVE phenomenon suggests that heterochromatin forms a transcriptionally repressor environment within which the presence of heterochromatically active resident genes is somewhat paradoxical, because these genes exhibit reciprocal heterochromatic PVE, that is, a heterochromatic gene will undergo variation if it is translocated to an euchromatic ambit. From this arises the suggestion that these genes have developed transcriptional dependence in factors that normally silence the expression of other genes (Schulze et al., 2005). Among proteins known to possess an important function in gene silencing in Drosophila is found HP1, which is a protein associated with pericentric heterochromatin. It has unequal distribution through the genome and is principally associated with the fourth chromosome. Specifically, it exerts an impact on structural organization and does not only cover the DNA or serves to direct all repetitive DNA sequences toward the repressive structure (Cryderman et al., 1999).

In the case of *Saccharomyces cerevisiae*, the heterochromatin is not cytologically visible; however, it presents position effects in three places: the telomeres; the rDNA locus, and the silent "mating-type" loci (HML and HMR). When a gene is found in one of these sites, its transcription is repressed. As in PVE, silencing depends on gene localization and not on its sequence (Chen & Widom, 2004). Several proteins are involved in the repressive chromatin, including H3 and H4 histones, their acetylases and associated deacetylases, the molecular regulators Sir1-Sir4, and the Origin recognition complex (ORC) (Pryde & Louis, 1999). The four Sir proteins are necessary for transcriptional silencing in HML and HMR, while only Sir2p, Sir3p, and Sir4p are required for telomere silencing and only Sir2p is required for rDNA silencing (Chen & Widom, 2004). TPE was described for the first time in 1990 through generation of a terminal deletion that caused the *URA3* gene (the gene necessary for uracil synthesis) to be localized 6 kb from a telomere. This new gene position provoked its transcriptional repression, which was lost when this was localized 20 kb from the telomere (Gottschling et al., 1990).

It was in 1992 that Wright and Shay set forth the possibility that TPE exists in humans. However, the first experimental proposal to identify TPE in humans, carried out by Bayne et al. (1994), did not yield positive results because changes were not found in the hygromycinresistant gene on generating deletions in the long arm of chromosome X. Again, this time in

At present, it is accepted that telomere clipping can affect gene expression in subtelomeric regions, which can lead to modification of the Biology of the cell prior to initiation of

The Position variegation effect (PVE) refers to inactivation of a gene, which occurs when it is removed from its normal context by means of a rearrangement or by transgene insertion. The best known example of this is the result of the expression of the euchromatic white gene in Drosophila, which is responsible for the red color of the eye. Provoked by a rearrangement in chromosome X that causes its relocation near the heterochromatin region, it eliminates function in some cells, which produces mottled pigmentation. The explanation for silencing of the white gene in some cells and not in others is that condensed and inactive conformation of pericentric heterochromatin is dispersed on the rearrangement break and randomly inactivates nearby genes (Henikoff, 1990). This chromosomal position effect affects up to a distance of approximately 1Mb and reflects a genetic inactivation gradient that is inversely correlated with distance (Wakimoto, 1998). The PVE phenomenon suggests that heterochromatin forms a transcriptionally repressor environment within which the presence of heterochromatically active resident genes is somewhat paradoxical, because these genes exhibit reciprocal heterochromatic PVE, that is, a heterochromatic gene will undergo variation if it is translocated to an euchromatic ambit. From this arises the suggestion that these genes have developed transcriptional dependence in factors that normally silence the expression of other genes (Schulze et al., 2005). Among proteins known to possess an important function in gene silencing in Drosophila is found HP1, which is a protein associated with pericentric heterochromatin. It has unequal distribution through the genome and is principally associated with the fourth chromosome. Specifically, it exerts an impact on structural organization and does not only cover the DNA or serves to direct all

repetitive DNA sequences toward the repressive structure (Cryderman et al., 1999).

In the case of *Saccharomyces cerevisiae*, the heterochromatin is not cytologically visible; however, it presents position effects in three places: the telomeres; the rDNA locus, and the silent "mating-type" loci (HML and HMR). When a gene is found in one of these sites, its transcription is repressed. As in PVE, silencing depends on gene localization and not on its sequence (Chen & Widom, 2004). Several proteins are involved in the repressive chromatin, including H3 and H4 histones, their acetylases and associated deacetylases, the molecular regulators Sir1-Sir4, and the Origin recognition complex (ORC) (Pryde & Louis, 1999). The four Sir proteins are necessary for transcriptional silencing in HML and HMR, while only Sir2p, Sir3p, and Sir4p are required for telomere silencing and only Sir2p is required for rDNA silencing (Chen & Widom, 2004). TPE was described for the first time in 1990 through generation of a terminal deletion that caused the *URA3* gene (the gene necessary for uracil synthesis) to be localized 6 kb from a telomere. This new gene position provoked its transcriptional repression, which was lost when this was localized 20 kb from the telomere

It was in 1992 that Wright and Shay set forth the possibility that TPE exists in humans. However, the first experimental proposal to identify TPE in humans, carried out by Bayne et al. (1994), did not yield positive results because changes were not found in the hygromycinresistant gene on generating deletions in the long arm of chromosome X. Again, this time in

**11. Telomere position effect (TPE)** 

replicative senescence (Baur et al., 2001).

(Gottschling et al., 1990).

1996, Sprung et al. (1996) sought to study the telomere-length effect on HSV-tk promotor expression, utilizing the KB319 cell line (SV40-transformed fibroblasts); the authors integrated a plasmid with the neo-gene at the end of chromosome 13 and found no effect on neo-gene expression when telomere length ranged from 25-0.5 kb; this leads them to suggest that chromatin structural differences conferred by telomere length do not affect the expression of nearby genes. A 130-kb microdeletion in the end of chromosome 22q in cells from a child with mental retardation; the broken end had been repaired by telomere addition, and consequently a unique DNA region that was normally localized at a distance of >100 kb from the telomere was now adjacent to it. This was the model employed by Ofir et al. in 1999 to demonstrate that telomeric sequences influence the activation of nearby replication origins, delaying the synchronization of replication at mid-S phase. The latter suggests that if human telomere repeats silence genes adjacent to repaired ends, then it is also conceivable that nearby genes may be epigenetically inactivated. On the other hand, Kilburn et al. (2001) found that the presence of a telomere sequence in an *APRT* gene intron in hamster ovary cells only had a modest effect on its expression. Finally, it was Baur et al. in 2001 who, employing a luciferase reporter, supplied convincing evidence of transcriptional silencing near telomeres in humans. The authors found that on placing the reporter adjacent to telomere repeats, there was 10-times lower expression than when they placed it at non-telomeric sites. However, the authors also found that TPE in humans required a histone deacetylase, because on treating the telomeric clones with trichostatin A (a histone deacetylase inhibitor), luciferase expression was restored. Koering et al. (2002) obtained similar results utilizing the *EGFP* reporter gene in C33-A cells (undifferentiated cervical carcinoma), reverting the repressor effect on employing trichostatin A, and they suggested that the position effect depends on the organization of telomeric chromatin, due to that they encountered the release of heterochromatin proteins HP1 and HP1. Pedram et al. (2006) developed embryonic stem cell clones of mouse with unique-copy gene markers and found that telomeric transgenes were not silenced in cells obtained from 3-day-old embryos as a result of their demethylation during early development, which led the authors to suggest that TPE also plays a role in embryo development. On the other hand, Wright and Shay (1992) also propose that progressive changes in presenescent cells can be the result of the reorganization of telomere chromatin and of the corresponding silencing or desilencing of subtelomere genes. This hypothesis has been extended to include the possibility that silenced proteins can be released from telomeres when the latter are shortened, in order to affect the expression of genes at internal non-telomere sites (Wood and Sinclair, 2002). This idea was based on studies with *S. cerevisiae*, in which it was demonstrated that gene markers inserted <4 kb from telomere repeats are frequently repressed and replicated at the end of the S phase (Dubrana et al., 2001). Although the loss of TPE is not the senescence trigger, it can be responsible for progressive changes in gene expression as a function of replicative age (Wood and Sinclair, 2002).

#### **12. Role of telomere length in subtelomeric gene expression and its possible relation to cellular senescence**

To date, there is only one report to our knowledge that studies *in vivo* TPE in human subtelomere genes. The results obtained suggest that the expression of these genes can be influenced by alterations in local heterochromatin structure so that this obstructs access to transcriptional factors (Ning et al., 2003). Due to the fact that few evidence exist about of

Cellular Senescence and Its Relation with Telomere 453

Beausejour, CM., Krtolica, A., Galimi, F., Narita, M., Lowe, SW., Yaswen, P. & Campisi, J.

Ben-Porath, I & Weinberg, RA. (2005). The signals and pathways activating cellular

Bianchi, A., Stansel, RM., Fairall, L., Griffith, JD., Rhodes, D. & de Lange, T. (1999). TRF1

Blagosklonny, M. (2011). Cell cycle arrest is not senescence. *Aging*, Vol.3, No.2, pp. 94-101,

Blanpain, C., Mohrin, M., Sotiropoulou, PA. & Passegue, E. (2011). DNA-damage response

Blasco, MA. (2005). Telomeres and human disease: ageing, cancer and beyond. *Nat Rev* 

Boehm, M. & Salck, F. (2005). A developmental timing microRNA and its target regulate life span in C. elegants. *Science*, Vol.310, No.5756, pp. 1954-1957, ISSN 0036-8075 Brandl, A., Hartmann, A., Bechmann, V., Graf, B., Nerlich, M. & Angele, P. (2011). Oxidative

Brunk, UT. & Terman, A. (2002). Lipofuscin: mechanisms of age-related accumulation and

Campisi, J. (2000). Cancer, aging and celular senescence. *In vivo*, Vol.14, No.1, (Jan-Feb), pp.

Campisi, J. (2005). Aging, tumor suppression and cancer: high wire-act! *Mech Ageing Dev*,

Campisi, J. & d'Adda di Fagagna, F. (2007). Cellular senescence: when bad things happen to good cells. *Nat Rev Mol Cell Biol*, Vol. 8, No.9, (Sep), pp. 729-740, ISSN 1471-0080 Campisi, J. (2011). Cellular senescence: Putting the paradoxes in perspective. *Curr opin Genet* 

Cech, TR. (2004). Beginning to understand the end of the chromosome. *Cell*, Vol.116, No.2,

Chai, W., Du, Q,, Shay, JW. & Wright, WE. (2006). Human telomeres have different

Chang, BD., Xuan, Y., Broude, EV., Zhu, H., Schott, B., Fang, J. & Roninson, IB. (1999). Role

Chang, W., Dynek, JN. & Smith, S. (2003). TRF1 is degraded by ubiquitina-mediated

overhang sizes al leading versus lagging strands. *Mol Cell*, Vol.21, No.3, (Feb 3), pp.

of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs. *Oncogene*, Vol.18, No.34, (Aug 28),

proteolysis after release from telomeres. *Genes Dev*, Vol.17, No.11, (Jun 1), pp. 1328-

*EMBO J*, Vol. 22, No.16, pp. 4212–4222, ISSN 1460-2075

No.20, pp. 5735-5744, ISSN 1460-2075

*Genet*, Vol. 6, No.8, pp. 611–622, ISSN 1471-0064

Vol.126, No.1, (Jan), pp. 51–58, ISSN 1872-6216

(Jan 23), pp. 273-279, ISSN 1097- 4172

*Dev*, Vol.21, No.1, (Feb), pp. 107- 112, ISSN 1879-0380

Jan 31), pp. 1114–1120, ISSN 1554-527X

ISSN 1878-5875

ISSN 1945-4589

ISSN 1873-4596

183-188, ISSN 1791-7549

427-435, ISSN 1097-4164

1333, ISSN 1549-5477

pp.4808-4818, ISSN 1476-5594

1875-9777

(2003). Reversal of human cellular senescence: roles of the p53 and p16 pathways.

senescence. *Int J Biochem Cell Biol*, Vol.37, No.5, (Epub2004 Dec 30), pp. 961-976,

binds a bipartite telomeric site with extreme spatial flexibility. *EMBO J*, Vol.18,

in tissue-specific and cancer stem cells. *Cell Stem Cell,* Vol.8, No.1, pp. 16–29, ISSN

Stress Induces Senescence in Chondrocytes. *J Orthop Res,* Vol.29, No.7, (Epub 2011

influence on cell function. *Free Radic Biol Med*, Vol.33, No.5, (Sep 1), pp. 611–619,

how age-related telomere length affects the expression of specific human subtelomeric genes, we analyzed the relationship between telomere length and gene expression levels in fibroblasts derived from human donors at ages ranging from 0-70 years. We studied three groups of genes localized 100-150 kb, 200-250 kb, and 300kb away from telomeres. We found that chromatin modifier-encoding genes *Eu-HMTase1*, *ZMYND11*, and *RASA3* were over expressed in adults and implicated in chromatin restructuring (Hernández-Caballero et al., 2009). These genes are interesting because can participate in cellular senescence through the p53-p21Cip1 pathway and can also participate in chromatin restructuring, interacting with remodeling factors including ATP-dependent helicases, histone deacetylases, and histone methyltransferases (Velasco et al., 2006; Zhang et al., 2007). On the other hand, *Eu-HMTase1* can regulate H3-K9 mono- and dimethylation in euchromatin (Tachibana et al., 2005).

## **13. Conclusions**

Undoubtedly the Antagonistic pleiotropy helped to understand the seemingly contradictory functions of the cellular senescence, nevertheless still it is not clear how does the senescence response balance tumour suppression, tissue regeneration and ageing phenotypes, for which it will be needed of a major number of studies.

Our results suggest that the expression of the subtelomeric genes modifies with the age, probably as result of decrease of the telomere length. How the changes on telomere length affected the expression of subtelomeric genes? Recently Martínez et al (2010) demonstrated that the RAP1 protein associated with the telomere, also it associates to the subtelomeric genes. Probably this protein might be involved in TPE.

The Senescence is a complex phenomenon where different factors are involved, the changes in expression of subtelomeric genes, as result of the age, is another variable that will help to the understanding cellular senescence.

#### **14. Acknowledgment**

The authors are grateful to Dra. Martha Ruíz and Maggie Brunner for the support in the preparation of the manuscript. This work was supported by grant Salud-2009-C01-115296, from Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico).

#### **15. References**


how age-related telomere length affects the expression of specific human subtelomeric genes, we analyzed the relationship between telomere length and gene expression levels in fibroblasts derived from human donors at ages ranging from 0-70 years. We studied three groups of genes localized 100-150 kb, 200-250 kb, and 300kb away from telomeres. We found that chromatin modifier-encoding genes *Eu-HMTase1*, *ZMYND11*, and *RASA3* were over expressed in adults and implicated in chromatin restructuring (Hernández-Caballero et al., 2009). These genes are interesting because can participate in cellular senescence through the p53-p21Cip1 pathway and can also participate in chromatin restructuring, interacting with remodeling factors including ATP-dependent helicases, histone deacetylases, and histone methyltransferases (Velasco et al., 2006; Zhang et al., 2007). On the other hand, *Eu-HMTase1* can regulate H3-K9 mono- and dimethylation in euchromatin (Tachibana et al.,

Undoubtedly the Antagonistic pleiotropy helped to understand the seemingly contradictory functions of the cellular senescence, nevertheless still it is not clear how does the senescence response balance tumour suppression, tissue regeneration and ageing phenotypes, for

Our results suggest that the expression of the subtelomeric genes modifies with the age, probably as result of decrease of the telomere length. How the changes on telomere length affected the expression of subtelomeric genes? Recently Martínez et al (2010) demonstrated that the RAP1 protein associated with the telomere, also it associates to the subtelomeric

The Senescence is a complex phenomenon where different factors are involved, the changes in expression of subtelomeric genes, as result of the age, is another variable that will help to

The authors are grateful to Dra. Martha Ruíz and Maggie Brunner for the support in the preparation of the manuscript. This work was supported by grant Salud-2009-C01-115296,

Bader, N., Jung, T. & Grune, T. (2007). The proteasome and its role in nuclear protein maintenance. *Exp Gerontol,* Vol.42, No.9, pp. 864-870, ISSN 1873-6815 Baur, JA., Zou, Y., Shay, JW. & Wright, WE. (2001). Telomere position effect in human cells.

Bayne, RA., Broccoli, D., Taggart, MH., Thomson, EJ., Farr, CJ. & Cooke, HJ. (1994).

Sandwiching of a gene within 12 kb of a functional telomere and alpha satellite does not result in silencing. *Hum Mol Genet*, Vol.3, No.4, pp. 539-546, ISSN 0964-

from Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico).

*Science*, Vol.292, No.5524, pp. 2075-2077, ISSN 1095-9203

2005).

**13. Conclusions** 

which it will be needed of a major number of studies.

genes. Probably this protein might be involved in TPE.

the understanding cellular senescence.

**14. Acknowledgment** 

**15. References** 

6906


Cellular Senescence and Its Relation with Telomere 455

Feltes, BC., de Faria Poloni, J. & Bonatto, D. (2011). The developmental aging and origins of

*Biogerontology*, Vol.12, No.4, (Epub 2011 Mar 5), pp. 293-308, ISSN 1389- 5729 Freund, A., Orjalo, AV., Desprez, PY. & Campisi, J. (2010). Inflammatory networks during

Funayama, R. & Ishikawa, F. (2007). Cellular senescence and chromatin structure. *Chromosoma*, Vol.116, No.5, (Epub 2007 Jun 20), pp. 431–440, ISSN 1432-0886 Gottschling, DE., Aparicio, OM., Billington, BL. & Zakian, VA. (1990). Position effect at S.

Greenberg, SB., Grove, GL. & Cristofalo, VJ. (1977). Cell size in aging monolayer cultures. *In* 

Greider, CW. & Blackburn, EH. (1985). Identification of a specific telomere terminal

Greider, CW. (1996). Telomere length regulation. *Annu Rev Biochem*, Vol.65, pp. 337-365,

Griffith, JD., Comeau, L., Rosenfield, S., Stansel, RM., Bianchi, A., Moss, H. & de Lange, T.

Guarente, L. (1999). Mutant mice live longer. *Nature*, Vol.402, No.6759, pp. 243-245, ISNN

Guarente, L. (1999). Diverse and dynamic functions of the Sir silencing complex. *Nat Genet*,

Gutteridge, JM. (1984). Age pigments: role of iron and copper salts in the formation of

Halaschek-Wiener, J., Vulto, I., Fornika, D., Collins, J., Connors, JM., Le, ND., Lansdorp, PM.

Hampel, B., Malisan, F., Niederegger, H., Testi, R. & Jansen-Durr, P. (2004). Differential

Harley, CB., Futcher, AB. & Greider, CW. (1990). Telomeres shorten during ageing of human

Harrington, L., McPhhail, T., Mar, V., Zhou, W., Oulton, R., Bass, MB., Arruda, I. &

Hayflick, L. & Moorhead, PS. (1961). The serial cultivation of human diploid cell strains. *Exp* 

Hayflick, L. (1998). A brief history of the mortality and immortality of cultured cells. *Keio J* 

fibroblasts. *Nature*, Vol.345, No.6274, pp. 458–460, ISSN 1476-4687

2010 May 3), pp. 238–246, ISSN 1471-499X

*Vitro*, Vol.13, No. 5, pp. 297–300, ISSN 0073-5655

Vol.23, No.3, pp. 281–285, ISSN 1546-1718

No.11-12, pp. 1713–1721, ISSN 1873-6815

No.5302, pp. 973-977, ISSN 1095-9203

*Cell Res* 1961; Vol.25, pp. 585- 621, ISSN 1090-2422

*Med*, Vol.47, No.3, pp. 174-182, ISSN 1880-1293

pp. 751-762, ISSN 1097-4172

413, ISSN 1097-4172

503-514, ISSN 1097-4172

ISSN 1545-4509

1476-4687

1872-6216

6216

health and disease hypotheses explained by different protein networks.

cellular senescence: causes and consequences. *Trends Mol Med*, Vol.16, No.5, (Epub

cerevisiae telomeres: Reversible repression of Pol II transcription. *Cell*, Vol.63, No.4,

transferase enzyme with two kinds of primer specificity. *Cell,* Vol.51, No.6, pp. 405–

(1999). Mammalian telomeres end in a large duplex loop. *Cell*, Vol. 97, No.4, pp.

fluorescent lipid complexes. *Mech Ageing Dev*, Vol.25, No.1-2, pp. 205–214, ISSN

& Brooks-Wilson, A. (2008). Reduced telomere length variation in healthy oldest old. *Mech Ageing Dev*, Vol.29, No.11, (Epub 2008 Aug 14), pp. 638–641, ISSN 1872-

regulation of apoptotic cell death in senescent human cells. *Exp Gerontol*, Vol.39,

Robinson, M. (1997). A mammalian telomerase-associated protein. *Science*, Vol.275,


Chen, L. & Widom, J. (2004). Molecular basis of transcriptional silencing in budding yeast.

Cherdyntseva, NV., Gervas, PA., Litvyakov, NV., Stakcheeva, MN., Ponomaryeva, AA.,

progression. *Exp Oncol*, Vol.32, No.3, (Sep), pp. 205-208, ISSN 1812-9269 Chiu, CP., Dragowska, W., Kim, NW., Vaziri, H., Yui, J., Thomas, TE., Harley, CB. &

Cong, Y., Wright, WE. & Shay, JW. (2002). Human telomerase and its regulation. *Microb Mol* 

Crabbe, L., Verdun, RE., Haggblom, CI. & Karlseder, J. (2004). Defective telomere lagging

Cryderman, DE., Morris, EJ., Biessmann, H., Elgin, S. & Wallrath, LL. (1999). Silencing at

d'Adda di Fagagna, F., Teo, S. & Jackson, SP. (2004). Functional links between telomeres and

d'Adda di Fagagna, F. (2008). Living on a break: cellular senescence as a DNA-damage

de Lange, T. (2002). Protection of mammalian telomeres. *Oncogene*, Vol.21, No.4, (Jan 21), pp.

De, S. (2011). Somatic mosaicism in healthy human tissues. *Trends Genet* Vol.27, No. 6, pp.

Dimri, GP., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, EE., Linskens,

Espejel, S., Franco, S., Rodríguez-Perales, S., Bouffler, SD., Cigudosa, JC. & Blasco, MA.

critically short telomeres. *EMBO J*, Vol.21, pp. 2207-2219, ISSN 1460-2075 Fairall, L., Chapman, L., Moss, H., de Lange, T. & Rhodes, D. (2001). Structure of the TRFH

*Acad Sci USA*, Vol.92, No.20, (Sep 26), pp. 9363–9367, ISSN 1091-6490 Duan, J., Zhang, Z. & Tong, T. (2005). Irreversible cellular senescence induced by prolonged

*Biol*, Vol.13, No.3, pp. 281-289, ISSN 1879-0410

Vol.8, No.2, pp. 351-361, ISSN 1097-4164

M., Rubelj, I., Pereira-Smith, O., Peacocke, M. & Campisi, J. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. *Proc Natl* 

exposure to H2O2 involves DNA-damage and- repair genes and telomere shortening. *Int J Biochem Cell Biol*, Vol.37, No. 7, pp. 1407–1420, ISSN 1878-5875 Dubrana, K., Perrod, S. & Gasser, SM. (2001). Turning telomeres on and off. *Curr Opin Cell* 

(2002). Mammalian Ku86 mediates chromosomal fusions and apoptosis caused by

dimerization domain of the human telomeric proteins TRF1 and TRF2. *Mol Cell*,

roles. *EMBO J*, Vol.18, No.13, (Jul 1), pp. 3724-3735, ISSN 1460- 2075

response. *Nat Rev Cancer*, Vol.8, No.7, pp. 512-522, ISSN 1474-1768

Dobrodeev, AY., Denisov, EV., Belyavskaya, VA. & Choinzonov, EL. (2010). Agerelated function of tumor suppressor gene tp53: contribution to cancer risk and

Lansdorp, PM. (1996). Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. *Stem Cells*, Vol.14,

strand synthesis in cells lacking WRN helicase activity. *Science*, Vol.306, No.5703,

Drosophila telomeres: Nuclear organization and chromatin structure play critical

proteins of the DNA-damage response. *Genes Dev*, Vol.18, No.15, (Aug 1), pp. 1781-

Biochem *Cell Biol*, Vol.82, No.4, (Aug), pp. 413-418, ISSN 1208-6002

No.2, pp. 239-248, ISSN 0250-6793

(Dec 10), pp. 1951-1953, ISSN 1095-9203

1799, ISSN 1549-5477

532-540, ISSN 1476-5594

217-223, ISSN 1476-5594

*Biol Rev*, Vol.66, No.3, pp. 407- 425, ISSN 1098-5557


Cellular Senescence and Its Relation with Telomere 457

Karlseder, J., Smogorzweska, A. & de Lange, T. (2002). Senescence induced by altered

Karlseder, J. (2003). Telomere repeat binding factors: Keeping the ends in check. *Cancer* 

Kavathia, N., Jain, A., Walston, J., Beamer, BA., Fedarko, NS. (2009). Serum markers of

Kilburn, AE., Shea, MJ., Sargent, G. & Wilson. JH. (2001). Insertion of a telomere repeat

Kim, NW., Piatyszek, MA., Prowse, KR., Harley, CB., West, MD., Ho, PL., Coviello. GM.,

Kim, S., Beausejour, C., Davalos, AR., Kaminker, P., Heo, S. & Campisi, J. (2004). TIN2

Kirkwood, TB. (1977). Evolution of ageing. *Nature*, Vol.170, No.5635, pp. 201–204, ISSN 1476-

Koering, CS., Pollice A., Zibella, MP., Bauwens, S. Puisieux, A., Brunori, M., Brun, C.,

König, P. & Rhodes, D. (1997). Recognition of telomeric DNA. *TIBS*, Vol.22, No.2, pp. 43-47,

Kosar, M., Bartkova, J., Hubackova, S., Hodny, Z., Lukas, J. & Bartek, J. (2011). Senescence-

Krishnamurthy, J., Torrice, C., Ramsey, MR., Kovalev, GI., Al-Regaiey, K., Su, L. &

Lee, BY., Han, JA., Im, JS., Morrone, A., Johung, K., Goodwin, EC., Kleijer, WJ., DiMaio, D. &

Lee, HW., Blasco, MA., Gottlieb, GJ., Horner, JW., Greider, CW. & DePinho, RA. (1998).

Lei, M., Podell, ER., Baumann, P. & Cech, TR. (2003). DNA self-recognition in the structure

galactosidase. *Aging Cell*, Vol.5, No.2, pp. 187–195, ISSN 1474-9726

*Letters*, vol.194, No.2, pp. 189-197, ISSN 1872-7980

(Epub 2004 Aug 3) pp. 43799–43804, ISSN 1083-351X

*EMBO Rep*, Vol.3, No. 11, pp. 1055-1061, ISSN 1469-221X

*Cycle*, Vol.10, No.3, pp. 457-468, ISSN 1551-4005

Vol.114, No.9, pp. 1299-1307, ISSN 1558-8238

No. 6676, pp. 569–574, ISSN 1476-4687

203, ISSN 1476-4687

Vol.21, No.1, pp. 126-135, ISSN 1097-4164

2011–2015, ISSN 1095-9203

1095-9203

1945-4589

4687

ISSN 0968-0004

telomere state, not telomere loss. *Science*, Vol.295, No.5564, pp. 2446-2449, ISSN

apoptosis decrease with age and cancer stage. *Aging* Vol.1, No.7, pp. 652–663, ISSN

sequence into a mammalian gene causes chromosome instability. *Mol Cell Biol*,

Wright, WE., Weinrich, SL. & Shay, JW. (1994). Specific association of human telomerase activity with immortal cells and cancer. *Science,* Vol.266, No.5193, pp.

mediates functions of TRF2 at Human Telomeres. *J Biol Chem*, Vol. 279, No.42,

Martins, L., Sabatier, L., Pulitzer, JF. & Gilson, E. (2002). Human telomeric position effect is determined by chromosomal context and telomeric chromatin integrity.

associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16 (ink4a). *Cell* 

Sharpless, NE. (2004). Ink4a/Arf expression is a biomarker of aging. *J Clin Invest*,

Hwang, ES. (2006). Senescence-associated b-galactosidase is lysosomal b-

Essential role of mouse telomerase in highly proliferative organs. *Nature*, Vol.392,

of Pot1 bound to telomeric single-stranded DNA. *Nature*, Vol.426, No.6963, pp. 198-


Hayflick, L. (2003). Living for ever and dying in the attempt. *Exp Gerontol*, Vol.38, No.11-12,

Henikoff, S. (1990). Position-effect variegation after 60 years. *Trends Genet*, Vol.6, No.12, pp.

Herbig, U., Jobling, WA., Chen, BP., Chen, DJ. & Sedivy, JM. (2004). Telomere shortening

Hernández-Caballero, E., Herrera-González, NE., Salamanca-Gómez, F. & Arenas-Aranda,

Höhn, A., Jung, T., Grimm, S., Catalgol, B., Weber, D. & Grune, T. (2011). Lipofuscin inhibits

Hornsby, PJ. (2011). Cellular aging and cancer. *Crit Rev Oncol Hematol*, Vol.79, No.2, pp. 189-

Hotchkiss, RS., Strasser, A., McDunn, JE. & Swanson, PE. (2009). Cell death. *N Engl J Med*,

Houghtaling, BR., Cuttonaro, L., Chang, W. & Smith, S. (2004). A dynamic molecular link

Hung, YC., Lee, JH., Chen, HM. & Huang, GS. (2010). Effects of static magnetic fields on the

Hwang, ES., Yoon, G. & Kang, HT. (2009). A comparative analysis of the cell biology of

Inomata, K., Aoto, T., Binh, NT., Okamoto, N., Tanimura, S., Wakayama, T., Iseki, S., Hara,

Kaminker, PG., Kim, S., Taylor, RD., Zebarjadian, Y., Funk, WD., Morin, GB., Yaswen. P. &

Kanoh, J. & Ishikawa, F. (2003). Composition and conservation of the telomeric complex. *Cell* 

Karlseder, J., Broccoli, D., Day, D., Hardy, S. & de Lange, T. (1999). p53- and ATM-

No.38, (Epub 2001 Jul 13), pp. 35891–35899, ISSN 1083-351X

*Mol Life Sci*, Vol.60, No.11, pp. 2295-2302, ISSN 1420-9071

TRF2. *Current Biology*, Vol.14, No.18, pp. 1621–1631, ISSN 1879- 0445 Huang, YC., Hasegawa, H., Wang, SW., Ku, CC., Lin, YC., Chiou, SS., Hou, MF., Wu, DC.,

modification. *J Biomed Biotechnol*, Epub 2010 Dec 12, ISSN 1110-7251

(Epub 2010 Dec 16), pp. 585-591, ISSN 1873-4596

Vol.361, No.16, pp. 1570-1583, ISSN 1533-4406

triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). *Mol Cell*, Vol.14, No.4, pp. 501-513, ISSN 1097-4164

DJ. (2009). Role of telomere length in subtelomeric gene expression and its possible relation to cellular senescence. *BMB Rep*, Vol.42, No.11, pp. 747- 751, ISSN 1976-

the proteasome by binding to surface motifs. *Free Radic Biol Med*, Vol.50, No.5,

between the telomere length regulator TRF1 and the chromosome end protector

Tsai, EM., Saito, S., Yamaguchi, N. & Yokoyama, KK. (2011). Jun dimerization protein 2 controls senescence and differentiation via regulating histone

development and aging of Caenorhabditis elegans. *J Exp Biol*, Vol.213, No.12, pp.

senescence and aging. *Cell Mol Life Sci*, Vol.66, No.15,( Epub 2009 May 7), pp. 2503-

E., Masunaga, T., Shimizu, H. & Nishimura, EK. (2009). Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation. *Cell*, Vol.137,

Campisi, J. (2001). TANK2, a new TRF1-associated Poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression. *J Biol Chem*, Vol.276,

dependent apoptosis induced by telomeres lacking TRF2. *Science*, Vol.283, No.5406,

pp. 1231-1241, ISSN 1873-6815

422–426, ISSN 0168-9525

195, ISSN 1040-8428

2079-2085, ISSN 1477-9145

No.6, pp. 1088–1099, ISSN 1097-4172

pp. 1321-1325, ISSN 1095-9203

2524, ISSN 1420-9071

670X


Cellular Senescence and Its Relation with Telomere 459

Ning, Y., Xu, JF., Li, Y., Chavez, L., Riethman, HC., Lansdorp, PM. & Weng, NP. (2003).

fibroblasts. *Hum Mol Genet*, Vol.12, No.11, pp. 1329-1336, ISSN 1460-2083 Nishio, K., Inoue, A., Qiao, S., Kondo, H. & Mimura, A. (2001). Senescence and cytoskeleton:

fibroblasts. *Histochem Cell Biol*, Vol.116, No.4, pp. 321–327, ISSN 1432-119X Nishio, K. & Inoue, A. (2005) Senescence-associated alterations of cytoskeleton:

O'Reilly, M., Teichmann, SA. & Rhodes, D. (1999). Telomerases. *Curr Opin Struc Biol*, Vol.9,

Ofir, R., Wong, AC., McDermind, HE., Skorecki, KL. & Selig, S. (1999). Position effect of

Olovnikov, AM. (1996). Telomeres, telomerase and aging: Origin of the Theory. *Exp Gerontol*,

Ouellette, MM., Liao, M., Herbert, BS., Johnson, M., Holt, SE., Liss, HS., Shay, JW. & Wright

Pedram, M., Sprung, CN., Gao, Q., Lo, AW., Reynolds, GE. & Murnane JP. (2006). Telomere

Percy, CJ., Power, D. & Gobe, GC. (2008). Renal ageing: changes in the cellular mechanism

Pryde, FE. & Louis, EJ. (1999). Limitations of silencing at native yeast telomeres. *EMBO J*,

Rai, TS. & Adams, PD. (2011). Lessons from senescence: Chromatin maintenance in non-

Riley, T., Sontag, E., Chen, P. & Levine, A. (2008). Transcriptional control of human p53 regulated genes. *Nat Rev Mol Cell Biol,* Vol. 9, No.5, pp. 402–412, ISSN 1097-4164 Rochette, PJ. & Brash, DE. (2008). Progressive apoptosis resistance prior to senescence and

Rodier, F. & Campisi, J. (2011). Four faces of cellular senescence. *J Cell Biol*, Vol.192, No.4,

Saldanha, SN., Andrews, LG. & Tollefsbol, TO. (2003). Assessment of telomere length and

Salminen, A., Ojala, J. & Kaarniranta, K. (2011). Apoptosis and aging: increased resistance to

proliferating cells. *Biochim Biophys Acta.* Aug 3, ISSN 0006-3002

No.1, pp. 56-65, ISSN 1879-033X

9258

ISNN 1440-1797

207–214, ISSN 1872-6216

1432-1033

No.20, pp. 11434-11439, ISSN 1091-6490

Vol.31, No.4, pp. 443-448, ISSN 1873-6815

*Biol*, Vol.26, No.5, pp. 1865-1878, ISSN 1098-5549

Vol.18, No.9, pp. 2538-2550, ISSN 1460-2075

(Epub 2011 Feb 14), pp. 547-556, ISSN 1540-8140

Nov 30), pp. 1021-1031, ISSN 1420-9071

Telomere length and the expression of natural telomeric genes in human

overproduction of vimentin induces senescent-like morphology in human

extraordinary production of vimentin that anchors cytoplasmic p53 in senescent human fibroblasts. *Histochem Cell Biol*, Vol.123, No.3, pp. 263–273, ISSN 1432-119X

human telomeric repeats on replication timing. *Proc Natl Acad Sci USA*, Vol.96,

WE. (2000). Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. *J Biol Chem*, Vol.275, No.14, pp. 10072-10076, ISSN 0021-

position effect and silencing of transgenes near telomeres in the mouse. *Mol Cell* 

of energy metabolism and oxidant handling. *Nephrology*, Vol.13, No.2, pp. 147–152,

control by the anti-apoptotic protein BCL-xL. *Mech Ageing Dev*, Vol.129, No.4, pp.

factors that contribute to its stability. *Eur J Biochem*, Vol.270, No.3, pp. 389-403, ISSN

apoptosis enhances the aging process. *Cell Mol Life Sci*, Vol.68, No.6, (Epub 2010


Liu, D., O'Connor, M., Qin, J. & Songyang, Z. (2004). Telosome. A mammalian telomere

Liu, L., Trimarchi, JR., Smith, PJ. & Keefe, DL. (2002). Mitochondrial dysfunction leads to

Lowe, SW. & Sherr, CJ. (2003). Tumor suppression by Ink4a-Arf: progress and puzzles. *Curr* 

Machwe, A., Xiao, L. & Orren, DK. (2004). TRF2 recruits the Werner syndrome (WRN)

Mantell, LL. & Greider, CW. (1994). Telomerase activity in germline and embryonic cells of

Martínez, P., Thanasoula, M., Caslos, AR., Gómez-López, G., Tejera, AM., Schoefther, S.,

Mattern, KA., Swiggers, JJ., Nigg, AL., Löwenberg, B., Houtsmuller, AB. & Zijlmans, JM.

McClintock, B. (1941). The stability of broken ends of chromosomes in Zea mays. *Genetics*,

McEachern, MJ., Krauskopf, A. & Blackburn, EH. (2000). Telomeres and their control. *Annu* 

Michaloglou, C., Vredeveld, LC., Soengas, MS., Denoyelle, C., Kuilman, T., van der Horst,

Mitchell, JR., Wood, E. & Collins, K. (1999). A telomerase component is defective in the

Morin, GB. (1989). The human telomere terminal transferase enzyme is a ribonucleoprotein

Narita, M., Nuñez, S., Heard, E., Narita, M., Lin, AW., Hearn, SA., Spector, DL., Hannon,

Neidle, S. & Parkinson, GN. (2003). The structure of telomeric DNA. *Curr Opin Struct Biol*,

CM., Majoor, DM., Shay, JW., Mooi, WJ. & Peeper, DS. (2005). BRAFE600 associated senescence-like cell cycle arrest of human naevi. *Nature*, Vol.436,

human disease dyskeratosis congenita. *Nature*, Vol.402, No.6761, pp. 551-555, ISSN

that synthesizes TTAGGG repeats. *Cell*, Vol.59, No.3, pp. 521–529, ISSN 1097-4172

GL. & Lowe, SW. (2003). Rb- mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. *Cell*, Vol.113, No.6, pp. 703–716, ISSN

Xenopus. *EMBO J*, Vol.13, No.13, pp. 3211-3217, ISSN 1460-2075

in human fibroblasts. *Age (Dordr),* Jun 22, ISSN 1574-1587

Vol.26, No.2, pp. 234-282, ISSN 1943-2631

No.7051, pp. 720-724, ISSN 1476-4687

Vol.13, No.3, pp. 275-283, ISSN 1879-033X

*Rev Genet*, Vol. 34, pp. 331-358, ISSN 1545-2948

No.49, (Epub 2004 Sep 20), pp. 51338-51342, ISSN 1083-351X

*Opin Genet Dev*, Vol.13, No.1, pp. 77–83, ISSN 1879-0380

1097-4172

ISSN 1476-5594

1098-5549

1476-4687

1097-4172

associated complex formed by multiple telomeric proteins. *J Biol Chem*, Vol.279,

telomere attrition and genomic instability. *Aging Cell*, Vol.1, No.1, pp. 40–46, ISSN

exonuclease for processing of telomeric DNA. *Oncogene*, Vol.23, No.1, pp. 149-156,

Dominguez, O., Pisano, DG., Tarsounas, M. & Blasco, MA. (2010). Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites. *Nat Cell Biol*, Vol.12, No.8, pp. 768-780, ISSN 1097-6256 Martínez, P. & Blasco MA. (2011). Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins. *Nat Rev Cancer*, Vol.11, pp. 161-176, ISSN 1474-175X Matos, L., Gouveia, A. & Almeida, H. (2011). Copper ability to induce premature senescence

(2004). Dynamics of protein binding to telomeres in living cells: Implications for telomere structure and function. *Mol Cell Biol*, Vol.24, No.12, pp. 5587-5594, ISNN


Cellular Senescence and Its Relation with Telomere 461

Vavrova, J. & Rezacova, M. (2011). The importance of senescence in ionizing radiation-

Velasco, G., Grkovic, S. & Ansieau, S. (2006). New insights into BS69 functions. *J Biol Chem*, Vol.281, No.24, (Epub 2006 Mar 24), pp. 16546-16550, ISSN 1083-351X von Zglinicki, T. (2002). Oxidative stress shortens telomeres. *Trends Biochem Sci*, Vol.27,

Vousden, KH. & Lu, L. (2002). Live or let die: the cell's response to p53. *Nat Rev Cancer*,

Wakimoto, BT. (1998). Beyond the nucleosome: Epigenetic aspects of position-effect variegation in Drosophila. *Cell*, Vol.93, No.3, pp. 321-324, ISSN 1097-4172 Walker, RF. (2011). Developmental theory of aging revisited: focus on causal and

Wang, E. & Gundersen, D. (1984). Increased organization of cytoskeleton accompanying the

Williams, G. (1957). Pleiotropy, natural selection, and the evolution of senescence. *Evolution*,

Wood, JG. & Sinclair, DA. (2002). TPE or not TPE? It's no longer a question. *TRENDS* 

Wright, WE. & Shay, JW. (1992). Telomere positional effects and the regulation of cellular

Wright, WE., Piatyszek, MA., Rainey, WE., Byrd, W. & Shay, JW. (1996). Telomerase activity

Wright, WE. & Shay, JW. (2005). Telomere-binding factors and general DNA repair. *Nat* 

Xin, H., Liu, D. & Songyang, Z. (2008). The telosome/shelterin complex and its functions. *Genome Biol*, Vol.9, No.9, (Epub 2008 Sep 18), pp. 232-236, ISSN 1465-6914 Yan, Q., Zheng, Y. & Harris. CC. (2005). POT1 and TRF2 cooperate to maintain telomeric

Yanaka, M., Honma, T., Sato, K., Shinohara, N., Ito, J., Tanaka, Y., Tsuduki, T. & Ikeda, I.

Ye, JZ. & de Lange, T. (2004). TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex. *Nat Genet*, Vol.36, No.6, pp. 618-623, ISSN 1546-1718 Zahn, JT., Louban, I., Jungbauer, S., Bissinger, M., Kaufmann, D., Kemkemer, R. & Spatz, JP.

cells. *Small*, Vol.7, No.10, (Epub 2011 May 2), pp. 1480-1487, ISSN 1613-6829 Zhang, W., Chan, HM., Gao, Y., Poon, R. & Wu, Z. (2007). BS69 is involved in cellular

(2011). Increased monocytic adhesion by senescence in human umbilical vein endothelial cells. *Biosci Biotechnol Biochem*, Vol.75, No.6, (Epub 2011 Jun 13), pp.

(2011). Age-dependent changes in microscale stiffness and mechanoresponses of

senescence through the p53-p21Cip1 pathway. *EMBO Rep*, Vol.8, No.10, (Epub 2007

in human germline and embryonic tissues and cells. *Dev Genet*, Vol.18, No.2, pp.

senescence. *Trends Genet*, Vol.8, No.6, pp. 193-197, ISSN 0168-9525

integrity. *Mol Cell Biol*, Vol.25, No.3, pp. 1070-1080, ISSN 1098-5549

mechanistic links between development and senescence. *Rejuvenation Res*, Vol.14,

aging of human fibroblasts in vitro. *Exp Cell Res*, Vol.154, No.1, pp. 191–202, ISSN

5500

1090-2422

No.7, pp.339–344, ISSN 0968-0004

Vol.11, pp. 398-411, ISSN 1558-5646

173-179, ISSN 0192-253X

1098-1103, ISSN 1347-6947

Aug 24), pp.952-958, ISSN 1469-3178

Vol.2, No.8, pp. 594-604, ISSN 1474-1768

No.4, (Epub 2011 Jul 18), pp. 429-36, ISSN 1557-8577

*Pharmacol Sci*, Vol.23, No.1, pp. 1-4, ISSN 1873-3735

*Genet*, Vol.37, No.2, pp. 116-118, ISSN 1546-1718

induced tumour suppression. *Folia Biologica*, Vol.57, No.2, pp. 41-46, ISSN 0015-


Schulze, SR., Sinclair, D., Fitzpatrick, KA. & Honda, BM. (2005). A Genetic and molecular

Sherr, CJ. & DePinho, RA. (2000). Cellular senescence: Mitotic clock or culture shock? *Cell*,

Sin, D., Kucia, M. & Ratajcsak, MZ. (2011). Nuclear and chromatin reorganization during

Smith, S., Giriat, I., Schmitt, A. & de Lange, T. (1998). Tankyrase, a poly (ADP-ribose)

Smith, S. & de Lange, T. (2000). Tankyrase promotes telomere elongation in human cells.

Smogorzewska, A. & de Lange, T. (2002). Different telomere damage signaling pathways in human and mouse cells. *EMBO J*, Vol.21, No.16, pp. 4338-4348, ISSN 1460-2075 Smogorzewska, A. & de Lange, T. (2004). Regulation of telomerase by telomeric proteins.

Sprung, CN., Sabatier, L. & Murnane, JP. (1996). Effect of telomere length on telomeric gene expression. Nucleic Acids Res, Vol.24, No.21, pp. 4336-4340, ISSN 1362-4962 Stellwagen, AE., Haimberger, ZW., Veatch, JR. & Gottschling, DE. (2003). Ku interacts with

*Genet*, Vol.33, No.4, (Epub 2003 Mar 24), pp. 492-496, ISSN 1546-1718 Su, X., Paris, M., Gi, YJ., Tsai, KY., Cho, MS., Lin, YL., Biernaskie, JA., Sinha, S., Prives, C.,

Tachibana, M., Ueda, J., Fukuda, M., Takeda, N., Ohta, T., Iwanari, H., Sakihama, T.,

mesothelial cells. *Exp Cell Res*, Vol.236, No.1, pp. 355-358, ISSN 1090-2422 Tuminello ER & Han SD. (2011). The apolipoprotein e antagonistic pleiotropy hypothesis:

Van Steensel, B., Smogorzewska, A. & de Lange, T. (1998). TRF2 protects human telomeres from end-to-end fusions. *Cell*, Vol.92, No.3, pp. 401-413, ISSN 1097-4172

telomerase RNA to promote telomere addition at native and broken chromosome ends. *Genes Dev*, Vol.17, No.19, (Epub 2003 Sep 15), pp. 2384-2395, ISSN 1549-5477 Stewart, SA., Ben-Porath, I., Carey, VJ., O'Connor, BF., Hahn, WC. & Weinberg, RA. (2003).

Erosion of the telomeric single-strand overhang at replicative senescence. *Nat* 

Pevny, LH., Miller. FD. & Flores ER. (2009). TAp63 prevents premature aging by promoting adult stem cell maintenance. *Cell Stem Cell*, Vol.5, No.1, pp. 64–75, ISSN

Kodama, T., Hamakubo, T. & Shinkai, Y. (2005). Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. *Genes & Dev*. Vol.19, No.7, pp. 815-826, ISSN 1549-5477 Thacker, J. & Zdzienicka, MZ. (2004). The XRCC genes: expanding roles in DNA doublestrand break repair. *DNA Repair*, Vol.3, No. 8-9, pp. 1081-1090, ISNN 1568-7856 Thomas, E., al-Baker, E., Dropcova, S., Denyer, S., Ostad, N., Lloyd, A., Kill, IR. & Faragher,

RG. (1997). Different kinetics of senescence in human fibroblasts and peritoneal

review and recommendations. *Int J Alzheimers Dis*, Vol.24, pp. 1-12, ISSN 2090-0252

Vol.13, No.6, pp. 748–753, ISSN 1879-0410

Vol.102, No.4, pp. 407–410, ISSN 1097-4172

*Curr Biol*, Vol.10, No.20, pp. 1299-1302, ISSN 1879-0445

*Annu Rev Biochem*, Vol.73, pp. 177-208, ISSN 0066-4154

4), pp. 76-84, ISSN 1423-0003

1095-9203

1875-9777

characterization of two Proximal heterochromatic genes on chromosome 3 of Drosophila melanogaster. *Genetics*, Vol.169, No.4, pp. 2165–2177, ISSN 1943-2631 Serrano, M. & Blasco, MA. (2001). Putting the stress on senescence. *Curr Opin Cell Biol*,

cell senescence and aging- A mini-review. *Gerontology*, Vol.57, No.1, (Epub 2010 Feb

polymerase at human telomeres. *Science*, Vol.282, No.5393, pp. 1484-1487, ISSN


The replicative limit of human fibroblasts has long provided a model to assess the molecular mechanisms which underlie cellular aging. In culture, fibroblasts which reach the end of their proliferative lifespan acquire profound molecular changes that limit their response to growth factors, and cause permanent exit from the cell cycle. Part of the senescence programme is due to a well established link between telomere attrition which occurs with each population doubling and the subsequent activation of the p53 tumour suppressor. Critical shortening of telomeres is thought to cause a form of DNA damage, that leads to the activation of caretaker proteins ATM, ATR or DNA-PK which activate p53, leading to the initiation of senescence through p53 effector genes. In addition, p53 mediates senescence by many other stimuli

Caveolins are the main scaffolding proteins driving the formation of caveolae (50-100 nm wide cave like invaginations at the plasma membrane) from lipid rafts and allows the organization of many signalling cascades. This compartmentalization concentrates receptors, proteins with lipid anchors, and the lipids from which second messengers are derived. In this capacity caveolin has also been shown to bind and inactivate many key components of mitogenic pathways through the caveolin scaffolding domain (CSD) and thus is often considered a tumour suppressor. About a decade ago, the original investigations into the relationship between caveolae and senescence showed elevated levels of the caveolin proteins during replicative senescence. Both the ectopic expression or endogenous upregulation of caveolin was shown to lead to p53 mediated senescent arrest. However, this upregulation has not been proven to lead to an increase of caveolae at the cell surface, but does lead to increased number of internalized structures. The mislocalized caveolar vesicles likely influence the regulation of mitogenic signals that normally are integrated through caveolae. In addition the caveolin protein has been shown to regulate many signalling cascades that have an impact on senescence, and on the activation of p53.

Primary human diploid fibroblasts (HDFs) have traditionally served as an experimental model to investigate cellular and molecular aspects of aging. Normal fibroblasts derived from human donors are neither immortal or transformed and are limited in the number of times they are capable of dividing *in vitro* and *in vivo*. This finite replicative lifespan terminates with the acquisition of a phenotype having distinct morphological and biochemical characteristics termed replicative senescence (Hayflick, 1965a; Hayflick and

including oxidative stress, DNA damaging agents and oncogenic activation.

**1. Introduction** 

**1.1 Fibroblast model of senescence** 

*Department of Biology, York University, Toronto* 

Keith Wheaton

*Canada* 

Zhou, XZ. & Lu, KP. (2001). The PIN2/TRF1 interacting protein PinX1 is a potent telomerase inhibitor. *Cell*, Vol.107, No.3, pp. 347-359, ISSN 1097-4172 **19** 

## Keith Wheaton

*Department of Biology, York University, Toronto Canada* 

## **1. Introduction**

462 Senescence

Zhou, XZ. & Lu, KP. (2001). The PIN2/TRF1 interacting protein PinX1 is a potent telomerase

The replicative limit of human fibroblasts has long provided a model to assess the molecular mechanisms which underlie cellular aging. In culture, fibroblasts which reach the end of their proliferative lifespan acquire profound molecular changes that limit their response to growth factors, and cause permanent exit from the cell cycle. Part of the senescence programme is due to a well established link between telomere attrition which occurs with each population doubling and the subsequent activation of the p53 tumour suppressor. Critical shortening of telomeres is thought to cause a form of DNA damage, that leads to the activation of caretaker proteins ATM, ATR or DNA-PK which activate p53, leading to the initiation of senescence through p53 effector genes. In addition, p53 mediates senescence by many other stimuli including oxidative stress, DNA damaging agents and oncogenic activation.

Caveolins are the main scaffolding proteins driving the formation of caveolae (50-100 nm wide cave like invaginations at the plasma membrane) from lipid rafts and allows the organization of many signalling cascades. This compartmentalization concentrates receptors, proteins with lipid anchors, and the lipids from which second messengers are derived. In this capacity caveolin has also been shown to bind and inactivate many key components of mitogenic pathways through the caveolin scaffolding domain (CSD) and thus is often considered a tumour suppressor. About a decade ago, the original investigations into the relationship between caveolae and senescence showed elevated levels of the caveolin proteins during replicative senescence. Both the ectopic expression or endogenous upregulation of caveolin was shown to lead to p53 mediated senescent arrest. However, this upregulation has not been proven to lead to an increase of caveolae at the cell surface, but does lead to increased number of internalized structures. The mislocalized caveolar vesicles likely influence the regulation of mitogenic signals that normally are integrated through caveolae. In addition the caveolin protein has been shown to regulate many signalling cascades that have an impact on senescence, and on the activation of p53.

#### **1.1 Fibroblast model of senescence**

Primary human diploid fibroblasts (HDFs) have traditionally served as an experimental model to investigate cellular and molecular aspects of aging. Normal fibroblasts derived from human donors are neither immortal or transformed and are limited in the number of times they are capable of dividing *in vitro* and *in vivo*. This finite replicative lifespan terminates with the acquisition of a phenotype having distinct morphological and biochemical characteristics termed replicative senescence (Hayflick, 1965a; Hayflick and

Additional phenotypic changes include increased actin stress fibers and an increase in cells containing irregular and multiple nuclei (Wheaton et al., 1996). These morphological changes were further shown to be dependent on the innate property of the number of mean population doublings of a particular cell strain, and were not influenced by proximity to lower passage cells, splitting ratio, viral contamination, media composition, or periods of storage in liquid nitrogen (Hayflick, 1965b; Hayflick and Moorhead, 1961). Senescent cells can be distinguished from low passage fibroblasts by the innate property to stain blue (Fig.

Although HDFs in culture are separated from their normal cellular environment, several observations support the use of HDFs grown *in vitro* as a valid model of biological aging. These include: 1) Fibroblasts isolated from young individuals are reproducibly able to undergo more mean population doublings (MPDs) then cells from old donors of the same species (Martin et al., 1970). 2) Fibroblasts from longer lived species undergo more population doublings than short lived species, indicating a relationship between the maximum lifespan of a species and the proliferative capacity of their fibroblasts grown in culture (Goldstein and Singal, 1974; Rohme, 1981). 3) Fibroblasts from individuals with premature aging syndromes such as progeria and Werner's syndrome undergo fewer population doublings in culture than normal cells (Brown, 1990). 4) Normal human fibroblasts in tissues stain with -gal, indicating senescence *in vivo* (Dimri et al., 1995). These and other observations regarding biochemical differences that accompany senescent HDFs suggest that the HDF model of cellular aging reflects the normal biological processes that occur during *in vivo* aging. In addition to this evidence it has become increasingly apparent that senescence is a natural barrier to oncogenic transformation in many different tumour

As a result of the refractory nature of senescent fibroblasts to mitogenic stimuli, many of the original studies exploring the molecular details of replicative senescence focused on various growth factor signalling pathways. The growth factors which have been reported to give optimal proliferative response in human fibroblasts are the epidermal growth factor (EGF), insulin like growth factor 1 (IGF-1) and dexamethasone (Phillips and Cristofalo, 1988). Although these tyrosine kinase receptors (RTKs) have many potential down stream mediators, the classical example is a cascade through the small GTPase Ras that leads to the mitogen-activated protein kinase (MAPK) cascade. Early studies investigating the loss of mitogenic response of senescent human diploid fibroblasts focused upon the growth factor receptor:ligand interactions (Gerhard et al., 1991; Phillips et al., 1983; Sell et al., 1993). In the cases of EGF, dexamethasone and IGF-1, it was found that the number of receptors remained the same per unit surface area in senescent cells and that the ligand affinity remained unchanged (Sell et al., 1993). Similarly, the binding kinetics of the glucocorticoid receptor and the insulin receptor have been shown to remain unaltered in senescent cells (Chua et al., 1986). The degradation process of EGF receptor has been shown to be largely unchanged with fibroblast age (Phillips et al., 1984). Studies have also looked at tyrosine autophosphorylation of EGF and PDGF and found no significant differences (Chua et al., 1986; Gerhard et al., 1991). Thus, the general consensus is that decrease in mitogen response is not due to alterations of receptor function or processing (Rattan and Derventzi, 1991).

1A) when processed with an acidic -galactosidase (-gal) assay (Dimri, 1995).

types (Collado and Serrano, 2010).

**2. The post-receptor block** 

Moorhead, 1961). This is a cell fate distinct from apoptosis or differentiation since the cells remain viable but are refractory to mitogenic signals. Senescing fibroblasts progressively acquire a flattened morphology with an increased cellular volume, an irregular shape, and increased accumulation of debris (Hayflick, 1965a; Hayflick and Moorhead, 1961).

**A)** Young and senescent primary fibroblasts prepared with an acidic -galactosidase assay, staining senescent cells blue. Note the enlarged and flattened morphology of senescent cells compared to young fibroblasts. **B)** A growth curve of AG08470 primary fibroblasts virally transfected with empty vector (Normal), human telomerase (hTERT) or p53 short hairpin RNA (p53 shRNA). This strain of fibroblasts undergo replicative senescence at a mean population doubling (MPD) of 34 (Normal). The knockdown of p53 protein levels using p53 shRNA extends the proliferative lifespan by 13 MPDs. The ectopic expression of hTERT immortalizes the fibroblast strain.

Moorhead, 1961). This is a cell fate distinct from apoptosis or differentiation since the cells remain viable but are refractory to mitogenic signals. Senescing fibroblasts progressively acquire a flattened morphology with an increased cellular volume, an irregular shape, and increased accumulation of debris (Hayflick, 1965a; Hayflick and Moorhead, 1961).

**A)** Young and senescent primary fibroblasts prepared with an acidic -galactosidase assay, staining senescent cells blue. Note the enlarged and flattened morphology of senescent cells compared to young fibroblasts. **B)** A growth curve of AG08470 primary fibroblasts virally transfected with empty vector (Normal), human telomerase (hTERT) or p53 short hairpin RNA (p53 shRNA). This strain of fibroblasts undergo replicative senescence at a mean population doubling (MPD) of 34 (Normal). The knockdown of p53 protein levels using p53 shRNA extends the proliferative lifespan by 13 MPDs. The ectopic expression of hTERT

Fig. 1. Cellular Senescence

immortalizes the fibroblast strain.

Additional phenotypic changes include increased actin stress fibers and an increase in cells containing irregular and multiple nuclei (Wheaton et al., 1996). These morphological changes were further shown to be dependent on the innate property of the number of mean population doublings of a particular cell strain, and were not influenced by proximity to lower passage cells, splitting ratio, viral contamination, media composition, or periods of storage in liquid nitrogen (Hayflick, 1965b; Hayflick and Moorhead, 1961). Senescent cells can be distinguished from low passage fibroblasts by the innate property to stain blue (Fig. 1A) when processed with an acidic -galactosidase (-gal) assay (Dimri, 1995).

Although HDFs in culture are separated from their normal cellular environment, several observations support the use of HDFs grown *in vitro* as a valid model of biological aging. These include: 1) Fibroblasts isolated from young individuals are reproducibly able to undergo more mean population doublings (MPDs) then cells from old donors of the same species (Martin et al., 1970). 2) Fibroblasts from longer lived species undergo more population doublings than short lived species, indicating a relationship between the maximum lifespan of a species and the proliferative capacity of their fibroblasts grown in culture (Goldstein and Singal, 1974; Rohme, 1981). 3) Fibroblasts from individuals with premature aging syndromes such as progeria and Werner's syndrome undergo fewer population doublings in culture than normal cells (Brown, 1990). 4) Normal human fibroblasts in tissues stain with -gal, indicating senescence *in vivo* (Dimri et al., 1995). These and other observations regarding biochemical differences that accompany senescent HDFs suggest that the HDF model of cellular aging reflects the normal biological processes that occur during *in vivo* aging. In addition to this evidence it has become increasingly apparent that senescence is a natural barrier to oncogenic transformation in many different tumour types (Collado and Serrano, 2010).

## **2. The post-receptor block**

As a result of the refractory nature of senescent fibroblasts to mitogenic stimuli, many of the original studies exploring the molecular details of replicative senescence focused on various growth factor signalling pathways. The growth factors which have been reported to give optimal proliferative response in human fibroblasts are the epidermal growth factor (EGF), insulin like growth factor 1 (IGF-1) and dexamethasone (Phillips and Cristofalo, 1988). Although these tyrosine kinase receptors (RTKs) have many potential down stream mediators, the classical example is a cascade through the small GTPase Ras that leads to the mitogen-activated protein kinase (MAPK) cascade. Early studies investigating the loss of mitogenic response of senescent human diploid fibroblasts focused upon the growth factor receptor:ligand interactions (Gerhard et al., 1991; Phillips et al., 1983; Sell et al., 1993). In the cases of EGF, dexamethasone and IGF-1, it was found that the number of receptors remained the same per unit surface area in senescent cells and that the ligand affinity remained unchanged (Sell et al., 1993). Similarly, the binding kinetics of the glucocorticoid receptor and the insulin receptor have been shown to remain unaltered in senescent cells (Chua et al., 1986). The degradation process of EGF receptor has been shown to be largely unchanged with fibroblast age (Phillips et al., 1984). Studies have also looked at tyrosine autophosphorylation of EGF and PDGF and found no significant differences (Chua et al., 1986; Gerhard et al., 1991). Thus, the general consensus is that decrease in mitogen response is not due to alterations of receptor function or processing (Rattan and Derventzi, 1991).

interaction of adapter molecule Shc at the plasma membrane, which likely compromises its downstream signalling. Interestingly, different splice forms of Shc may have a role in

Studies have also focused on the downstream effectors of EGFR in senescence, particularly on the MAPK (erk 1/2). MAPK is pivotal in transactivation of various transcription factors that drive the cell through proliferation. Although a general reduction in MAPK (Erk1/2) activation has not been seen in senescence (Kim et al., 2000; Tresini et al., 2001), there is a dramatic reduction in localization of activated Erk to the nucleus of senescent fibroblasts (Bose et al., 2004; Kim et al., 2000; Lim et al., 2000; Tresini et al., 2001). However, it has also been found that a phosphatase MKP-2 is stabilized and acts on Erk in senescent cells (Torres et al., 2003), explaining the reduction in active MAPK. This reduced MAPK activity leads to a lack of Elk-1 activation, which loses its ability to bind to the adjacent ets region of the serum response element in *c-fos* promoter. These studies also concede that the reduction in binding of elk-1 alone cannot explain the entire loss of binding at the *c-fos* promoter, or the repression of its transcription. Interestingly, there is an equal amount of nonphosphorylated MAPK (activated) in the nucleus of young and senescent cells, and although reduced, there is still some localization of phospho-MAPK to the senescent cell nucleus. Therefore, these studies raise the question of whether the reduction in MAPK and Elk-1 represents a significant threshold to suppress *c-fos* transcription and explain the post receptor block. This was confirmed when SRF, the primary transcription factor of c-fos and Egr-1, was shown to be hyper-phosphorylated in senescent cells by PKC, and suppress immediate early gene transcription (Atadja et al., 1994; Wheaton and Riabowol, 2004). It was shown that immediate early genes could be restored in senescent cells by blocking PKC activity independent of MAPK. However, even though immediate early genes could be restored, this was insufficient to allow senescent cells to re-enter the cell cycle (Wheaton and Riabowol, 2004). Not surprisingly, other factors are clearly involved in senescent arrest

Premature senescence can be induced by the introduction of oncogenic Ras (Serrano et al., 1997) or Raf (Zhu et al., 1998) or constitutively active MAPK (Lin et al., 1998) in primary fibroblasts. In every case, there is an upregulation of p21 and p16 and an induction of senescence which depends on p53 activity. Also the inducible levels of the *c-fos* transcript were found to be repressed in oncogenic Ras induced senescence (Serrano et al., 1997). This oncogenic Ras/Raf/MAPK pathway has been shown to impinge on the p38MAPK stress activated kinase, which is required for the induction of premature senescence (Debacq-Chainiaux et al., 2010). The p38 activity likely impinges directly on p53, or the Rb/p16 pathway to lead to this arrest. The induction of premature senescence likely represents a natural mechanism to block oncogenesis when there is an inappropriately sustained mitogenic signal. Thus, the perturbations of the cellular system as a whole induced by hyperactive Ras/Raf/MAPK are important to inducing premature senescence and only

Conventional PKCs (, , & isoforms) and novel PKCs (, , , & isoforms) are activated by the lipid second messenger 1,2 diacylglycerol (DAG) which is generated downstream of

cellular senescence and longevity (Migliaccio et al., 1999).

besides the refractory mitogenic response.

indirectly produce the post receptor block.

**2.2 Protein kinase C activity during senescence** 

However, it has been established that the downstream responses of growth factors, such as the induction of the immediate early genes *c-fos* and *egr-1*, are blunted in senescent cells (Meyyappan et al., 1999; Riabowol et al., 1992; Seshadri and Campisi, 1990). Thus, the senescence-specific growth block is thought not to be due to alterations in growth factorreceptor engagement or processing, but to a postreceptor block which leads to reduced immediate early gene responsiveness. Many studies have attempted to identify the points where signalling cascades are modified in senescent cells to account for this block.

#### **2.1 EGFR-Ras-MAPK cascade in senescence**

The signal transduction pathway through the epidermal growth factor receptor (EGFR) (and other RTKs) begins with ligand engagement, which initiates dimerization and autophosphorylation within the cytoplasmic domains of the receptors (Fantl et al., 1993). This, in turn, is followed by Src homology 2 domain (SH2) containing molecules interacting with the receptor and recruiting other factors including adaptors. The classic adaptor molecule is Grb2, which binds to the growth factor receptor directly or indirectly through Shc and is associated constitutively with the son of sevenless (SOS) protein. The SOS guanine nucleotide exchange factor catalyses the transformation of inactive GDP-ras to active GTP-ras (Boguski and McCormick, 1993). Activated Ras recruits Raf1 to the membrane allowing its activation (Moodie and Wolfman, 1994). Appropriately localized and activated Raf1 then initiates a kinase cascade through the MAPK cascade. Raf 1 phosphorylates MEK (MAP kinase kinase) which activates Erk1 and Erk2 (MAPK) and leads to MAPK nuclear localization. In the nucleus MAPK phosphorylates transcription factors such as Elk-1 & serum response factor (SRF), stimulating cell proliferation (reviewed in (Wheaton et al., 1996)).

Many studies have attempted to elucidate the role of signalling cascades in senescence, and investigations into the signalling pathways downstream of the EGFR are the most common. It is unclear whether these pathways are compromised in senescence, or whether they need to be intact to promote senescence. Considering that EGFR has been localized to caveolae it is of interest to evaluate studies that have seen a differential regulation in senescent cells. First, it has been found that the EGFR is cleaved to a 100 kD fragment in non-ionic detergent isolates from senescent but not from young primary fibroblasts. This activity appears to be confined to a specialized region of the plasma membrane in senescent cells and was independent of receptor turnover (Carlin et al., 1994). However, it is unclear whether this proteolytic processing is an artefact of the isolation procedure or is physiologically relevant. Second, several studies have reported an age dependent decline in mitogenic stimulation of rat hepatocytes which may be caused by a reduced association between Shc and the EGFR (Hutter et al., 2000; Palmer et al., 1999). Although total tyrosine phosphorylation levels on EGFR appear equivalent after serum stimulation in young and senescent cells, there is a specific reduction of phosphorylation on tyrosine 1173 (a known phosphotyrosine binding (PTB) domain that interacts with Shc (Okabayashi et al., 1994)) in senescent cells. The 1173 residue is known as an autophosphorylation site (Downward et al., 1985), but can also be targeted by Src kinase *in vitro* (Wright et al., 1996) and also is recognized by the SHP-1 phosphatase (Keilhack et al., 1998). This site was subsequently shown to be dephosphorylated by an upregulation of SHP-1 and other phosphatases in senescent human fibroblasts (Tran et al., 2003). Thus, the EGFR in senescence is regulated by differential

However, it has been established that the downstream responses of growth factors, such as the induction of the immediate early genes *c-fos* and *egr-1*, are blunted in senescent cells (Meyyappan et al., 1999; Riabowol et al., 1992; Seshadri and Campisi, 1990). Thus, the senescence-specific growth block is thought not to be due to alterations in growth factorreceptor engagement or processing, but to a postreceptor block which leads to reduced immediate early gene responsiveness. Many studies have attempted to identify the points

The signal transduction pathway through the epidermal growth factor receptor (EGFR) (and other RTKs) begins with ligand engagement, which initiates dimerization and autophosphorylation within the cytoplasmic domains of the receptors (Fantl et al., 1993). This, in turn, is followed by Src homology 2 domain (SH2) containing molecules interacting with the receptor and recruiting other factors including adaptors. The classic adaptor molecule is Grb2, which binds to the growth factor receptor directly or indirectly through Shc and is associated constitutively with the son of sevenless (SOS) protein. The SOS guanine nucleotide exchange factor catalyses the transformation of inactive GDP-ras to active GTP-ras (Boguski and McCormick, 1993). Activated Ras recruits Raf1 to the membrane allowing its activation (Moodie and Wolfman, 1994). Appropriately localized and activated Raf1 then initiates a kinase cascade through the MAPK cascade. Raf 1 phosphorylates MEK (MAP kinase kinase) which activates Erk1 and Erk2 (MAPK) and leads to MAPK nuclear localization. In the nucleus MAPK phosphorylates transcription factors such as Elk-1 & serum response factor (SRF), stimulating cell proliferation (reviewed in

Many studies have attempted to elucidate the role of signalling cascades in senescence, and investigations into the signalling pathways downstream of the EGFR are the most common. It is unclear whether these pathways are compromised in senescence, or whether they need to be intact to promote senescence. Considering that EGFR has been localized to caveolae it is of interest to evaluate studies that have seen a differential regulation in senescent cells. First, it has been found that the EGFR is cleaved to a 100 kD fragment in non-ionic detergent isolates from senescent but not from young primary fibroblasts. This activity appears to be confined to a specialized region of the plasma membrane in senescent cells and was independent of receptor turnover (Carlin et al., 1994). However, it is unclear whether this proteolytic processing is an artefact of the isolation procedure or is physiologically relevant. Second, several studies have reported an age dependent decline in mitogenic stimulation of rat hepatocytes which may be caused by a reduced association between Shc and the EGFR (Hutter et al., 2000; Palmer et al., 1999). Although total tyrosine phosphorylation levels on EGFR appear equivalent after serum stimulation in young and senescent cells, there is a specific reduction of phosphorylation on tyrosine 1173 (a known phosphotyrosine binding (PTB) domain that interacts with Shc (Okabayashi et al., 1994)) in senescent cells. The 1173 residue is known as an autophosphorylation site (Downward et al., 1985), but can also be targeted by Src kinase *in vitro* (Wright et al., 1996) and also is recognized by the SHP-1 phosphatase (Keilhack et al., 1998). This site was subsequently shown to be dephosphorylated by an upregulation of SHP-1 and other phosphatases in senescent human fibroblasts (Tran et al., 2003). Thus, the EGFR in senescence is regulated by differential

where signalling cascades are modified in senescent cells to account for this block.

**2.1 EGFR-Ras-MAPK cascade in senescence** 

(Wheaton et al., 1996)).

interaction of adapter molecule Shc at the plasma membrane, which likely compromises its downstream signalling. Interestingly, different splice forms of Shc may have a role in cellular senescence and longevity (Migliaccio et al., 1999).

Studies have also focused on the downstream effectors of EGFR in senescence, particularly on the MAPK (erk 1/2). MAPK is pivotal in transactivation of various transcription factors that drive the cell through proliferation. Although a general reduction in MAPK (Erk1/2) activation has not been seen in senescence (Kim et al., 2000; Tresini et al., 2001), there is a dramatic reduction in localization of activated Erk to the nucleus of senescent fibroblasts (Bose et al., 2004; Kim et al., 2000; Lim et al., 2000; Tresini et al., 2001). However, it has also been found that a phosphatase MKP-2 is stabilized and acts on Erk in senescent cells (Torres et al., 2003), explaining the reduction in active MAPK. This reduced MAPK activity leads to a lack of Elk-1 activation, which loses its ability to bind to the adjacent ets region of the serum response element in *c-fos* promoter. These studies also concede that the reduction in binding of elk-1 alone cannot explain the entire loss of binding at the *c-fos* promoter, or the repression of its transcription. Interestingly, there is an equal amount of nonphosphorylated MAPK (activated) in the nucleus of young and senescent cells, and although reduced, there is still some localization of phospho-MAPK to the senescent cell nucleus. Therefore, these studies raise the question of whether the reduction in MAPK and Elk-1 represents a significant threshold to suppress *c-fos* transcription and explain the post receptor block. This was confirmed when SRF, the primary transcription factor of c-fos and Egr-1, was shown to be hyper-phosphorylated in senescent cells by PKC, and suppress immediate early gene transcription (Atadja et al., 1994; Wheaton and Riabowol, 2004). It was shown that immediate early genes could be restored in senescent cells by blocking PKC activity independent of MAPK. However, even though immediate early genes could be restored, this was insufficient to allow senescent cells to re-enter the cell cycle (Wheaton and Riabowol, 2004). Not surprisingly, other factors are clearly involved in senescent arrest besides the refractory mitogenic response.

Premature senescence can be induced by the introduction of oncogenic Ras (Serrano et al., 1997) or Raf (Zhu et al., 1998) or constitutively active MAPK (Lin et al., 1998) in primary fibroblasts. In every case, there is an upregulation of p21 and p16 and an induction of senescence which depends on p53 activity. Also the inducible levels of the *c-fos* transcript were found to be repressed in oncogenic Ras induced senescence (Serrano et al., 1997). This oncogenic Ras/Raf/MAPK pathway has been shown to impinge on the p38MAPK stress activated kinase, which is required for the induction of premature senescence (Debacq-Chainiaux et al., 2010). The p38 activity likely impinges directly on p53, or the Rb/p16 pathway to lead to this arrest. The induction of premature senescence likely represents a natural mechanism to block oncogenesis when there is an inappropriately sustained mitogenic signal. Thus, the perturbations of the cellular system as a whole induced by hyperactive Ras/Raf/MAPK are important to inducing premature senescence and only indirectly produce the post receptor block.

#### **2.2 Protein kinase C activity during senescence**

Conventional PKCs (, , & isoforms) and novel PKCs (, , , & isoforms) are activated by the lipid second messenger 1,2 diacylglycerol (DAG) which is generated downstream of

al., 1999).

membranes.

**2.3 Phospholipase D activity during senescence** 

activity or inhibition by ceramide (Venable and Obeid, 1999).

**2.4 The phosphoinositide 3-kinase pathway during senescence** 

Due to the role of IGF-1 in the optimal proliferative response of primary fibroblasts (Phillips and Cristofalo, 1988), a common downstream effector of this pathway, phosphoinositide 3 kinase (PI3K) was assessed for its role during senescence. PI3K consists of a p85 regulatory subunit and a p110 catalytic subunit which selectively phosphorylates the 3-OH position of

Caveolar Vesicles in Cellular Senescence 469

et al., 2008). PKC has also been found in the caveolar membrane microdomains of human fibroblasts (Wheaton, 2002). The high activity of PKC in senescence may be due to the loss of Src activity in caveolae, which normally inactivates PKC through degradation (Blake et

Phospholipase D (PLD) activity is required for an integrated mitogenic response from a variety of receptors including RTKs (reviewed in (Exton, 1998)). PLD catalyses the hydrolysis of phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline. PA is an important second messenger in many biological processes (Exton, 1997) such as in a membrane localization of Raf-1 (Ghosh et al., 1996). PA can be modified further to the mitogenic lyso-PA by phospholipase A2 or to DAG by PA phosphohydrolase (McDermott et al., 2004). Activation of PLD is complex, involving phosphatidylinositol 4,5-bisphosphate (PIP2), PKC/, Rho family GTPases and ARF GTPase (Exton, 1999). PLD activity has been correlated with mitogenic activity (reviewed in (Foster and Xu, 2003)) and has been shown to be reduced in senescent cells (Venable et al., 1994), possibly due to the reduction of PKC

Ceramide acts as a second messenger in a variety of biological processes after being generated from membrane associated sphingomyelin by neutral sphingomyelinase (reviewed in (Foster and Xu, 2003)). The ceramide levels remain stable with fibroblast age, but rise 2-4 fold relative to young cells upon the onset of senescence and are accompanied by an increase in the neutral Mg2+ dependent sphingomyelinase activity. The addition of a synthetic, cell permeable analog of ceramide to young cells led to growth arrest and cellular senescence (Venable et al., 1995). These cells showed cellular arrest, lack of Rb phosphorylation, no AP-1 production (Venable et al., 1995) and stained positive with the gal test for senescence (Mouton and Venable, 2000). Ceramide acts to inhibit the activation of PLD, by possibly three mechanisms, inhibiting activation by PKC, translocation to the membrane and gene expression, but none of these has been clearly defined (Exton, 1999). Exogenous ceramide inhibits PLD activity in primary fibroblasts (Venable et al., 1994) and this inhibition was shown to depend on PKC interaction but not on phosphorylation or translocation in cell free systems (Venable et al., 1996). However, ceramide has been shown to disrupt the liquid order of lipid rafts, which leads to PLD inhibition (Gidwani et al., 2003). To this end, recent work has identified that the deficiency in PLD activity is specific to the senescent membrane fractions, and not to changes in the cytosol (Webb et al., 2010). The increase in ceramide in senescent membranes likely changes the properties of lipid rafts, which no longer supports an integrated PLD response. Thus, the elevated ceramide levels and PKC regulation are important factors in reduced PLD activity in senescent cell

many G-protein-coupled receptors and tyrosine-kinase receptors (Dempsey et al., 2000; Newton, 2001). PKC family members are activated in response to a large number of extracellular signals, and regulate a large number of effectors including receptors, kinases, cytoskeletal components and transcription factors. The outcomes of these signals include changes in proliferation, gene expression, differentiation, permeability, migration, hypertrophy, apoptosis and exocytosis. Each outcome depends on the cellular context, the originating signal and the particular isoform activated (Dempsey et al., 2000). Altered PKC activity has been observed in senescent fibroblasts where a change in serum-induced translocation of PKC from the cytosol to the plasma membrane was reported (De Tata et al., 1993). In contrast, exogenously added phorbol 12-myristate 13-acetate (PMA, a DAG analog) stimulated this translocation identically in young and old cells, implying that the difference in response to serum was due to changes in the production of DAG (De Tata et al., 1993). A similar differential response to serum vs. PMA was also reported for the induction of the *cfos* gene (Riabowol et al., 1992). Although old fibroblasts have a higher basal level of DAG (Vannini et al., 1994; Venable et al., 1994), the response due to serum induction was reported to be much lower in senescent cells (Chang and Huang, 1994). It should be noted that the specific isoforms were not noted in these studies, but are likely PKC or . Attenuated PKC response could contribute to the senescent phenotype in several ways. For example, decreased phosphorylation of substrates that are downstream of PKC could affect the expression of many growth-regulatory genes such as *c-fos* or other MAPK substrates. Alternatively, the higher basal amount of DAG in the resting state of senescent cells compared to young cells may also allow constitutive activation of some kinases. One such kinase, PKC was found to be required in oxidative stress induced senescence in human fibroblasts. PKC was shown to induce the senescent phenotype by acting on erk1/2 and Sp1, which converge on and up regulate transcription from the p21 promoter (Kim and Lim, 2009). Interestingly the knockdown of PKC restored proliferative capacity of senescent cells, implying that the increased basal levels in DAG in senescence constitutively activate PKC.

The PKC isoform has a multifunctional role in various processes including growth inhibition, differentiation, apoptosis and tumour suppression (Basu and Pal, 2010). Many groups have reported that PKC activation leads to growth inhibition, including CHO cells, smooth muscle, NIH 3T3 fibroblasts, human glioma cells, and capillary endothelial cells (reviewed in (Gschwendt, 1999)). Studies using ectopic PKC implicate p27 as part of the cellular arrest phenotype (Ashton et al., 1999). PKC also assists in the sustained upregulation of p21 message and protein levels in a p53-independent manner (Zezula et al., 1997). However, PKC is also capable of phosphorylating and activating p53 in response to genotoxic stress (Johnson et al., 2002; Yoshida et al., 2006). The activity of PKC is substantially higher in senescent cells, and has been shown to hyper-phosphorylate SRF, which prevents it from binding to DNA and transactivating the immediate early genes *egr-1* and *c-fos* (Wheaton and Riabowol, 2004). Similarly, increased PKC activity during senescence was shown to indirectly inactivate WARTS, a kinase required to exit cytokinesis, and arrest cells after nuclear fission but not before division (Takahashi et al., 2006). A subsequent report also established that constitutively active PKC can induce senescent phenotype when introduced to young fibroblasts (Katakura et al., 2009). Interestingly, the activation of PKC by PMA is known to be localized to caveolae in cardiomyocytes (Rybin

many G-protein-coupled receptors and tyrosine-kinase receptors (Dempsey et al., 2000; Newton, 2001). PKC family members are activated in response to a large number of extracellular signals, and regulate a large number of effectors including receptors, kinases, cytoskeletal components and transcription factors. The outcomes of these signals include changes in proliferation, gene expression, differentiation, permeability, migration, hypertrophy, apoptosis and exocytosis. Each outcome depends on the cellular context, the originating signal and the particular isoform activated (Dempsey et al., 2000). Altered PKC activity has been observed in senescent fibroblasts where a change in serum-induced translocation of PKC from the cytosol to the plasma membrane was reported (De Tata et al., 1993). In contrast, exogenously added phorbol 12-myristate 13-acetate (PMA, a DAG analog) stimulated this translocation identically in young and old cells, implying that the difference in response to serum was due to changes in the production of DAG (De Tata et al., 1993). A similar differential response to serum vs. PMA was also reported for the induction of the *cfos* gene (Riabowol et al., 1992). Although old fibroblasts have a higher basal level of DAG (Vannini et al., 1994; Venable et al., 1994), the response due to serum induction was reported to be much lower in senescent cells (Chang and Huang, 1994). It should be noted that the specific isoforms were not noted in these studies, but are likely PKC or . Attenuated PKC response could contribute to the senescent phenotype in several ways. For example, decreased phosphorylation of substrates that are downstream of PKC could affect the expression of many growth-regulatory genes such as *c-fos* or other MAPK substrates. Alternatively, the higher basal amount of DAG in the resting state of senescent cells compared to young cells may also allow constitutive activation of some kinases. One such kinase, PKC was found to be required in oxidative stress induced senescence in human fibroblasts. PKC was shown to induce the senescent phenotype by acting on erk1/2 and Sp1, which converge on and up regulate transcription from the p21 promoter (Kim and Lim, 2009). Interestingly the knockdown of PKC restored proliferative capacity of senescent cells, implying that the increased basal levels in DAG in senescence constitutively activate

The PKC isoform has a multifunctional role in various processes including growth inhibition, differentiation, apoptosis and tumour suppression (Basu and Pal, 2010). Many groups have reported that PKC activation leads to growth inhibition, including CHO cells, smooth muscle, NIH 3T3 fibroblasts, human glioma cells, and capillary endothelial cells (reviewed in (Gschwendt, 1999)). Studies using ectopic PKC implicate p27 as part of the cellular arrest phenotype (Ashton et al., 1999). PKC also assists in the sustained upregulation of p21 message and protein levels in a p53-independent manner (Zezula et al., 1997). However, PKC is also capable of phosphorylating and activating p53 in response to genotoxic stress (Johnson et al., 2002; Yoshida et al., 2006). The activity of PKC is substantially higher in senescent cells, and has been shown to hyper-phosphorylate SRF, which prevents it from binding to DNA and transactivating the immediate early genes *egr-1* and *c-fos* (Wheaton and Riabowol, 2004). Similarly, increased PKC activity during senescence was shown to indirectly inactivate WARTS, a kinase required to exit cytokinesis, and arrest cells after nuclear fission but not before division (Takahashi et al., 2006). A subsequent report also established that constitutively active PKC can induce senescent phenotype when introduced to young fibroblasts (Katakura et al., 2009). Interestingly, the activation of PKC by PMA is known to be localized to caveolae in cardiomyocytes (Rybin

PKC.

et al., 2008). PKC has also been found in the caveolar membrane microdomains of human fibroblasts (Wheaton, 2002). The high activity of PKC in senescence may be due to the loss of Src activity in caveolae, which normally inactivates PKC through degradation (Blake et al., 1999).

## **2.3 Phospholipase D activity during senescence**

Phospholipase D (PLD) activity is required for an integrated mitogenic response from a variety of receptors including RTKs (reviewed in (Exton, 1998)). PLD catalyses the hydrolysis of phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline. PA is an important second messenger in many biological processes (Exton, 1997) such as in a membrane localization of Raf-1 (Ghosh et al., 1996). PA can be modified further to the mitogenic lyso-PA by phospholipase A2 or to DAG by PA phosphohydrolase (McDermott et al., 2004). Activation of PLD is complex, involving phosphatidylinositol 4,5-bisphosphate (PIP2), PKC/, Rho family GTPases and ARF GTPase (Exton, 1999). PLD activity has been correlated with mitogenic activity (reviewed in (Foster and Xu, 2003)) and has been shown to be reduced in senescent cells (Venable et al., 1994), possibly due to the reduction of PKC activity or inhibition by ceramide (Venable and Obeid, 1999).

Ceramide acts as a second messenger in a variety of biological processes after being generated from membrane associated sphingomyelin by neutral sphingomyelinase (reviewed in (Foster and Xu, 2003)). The ceramide levels remain stable with fibroblast age, but rise 2-4 fold relative to young cells upon the onset of senescence and are accompanied by an increase in the neutral Mg2+ dependent sphingomyelinase activity. The addition of a synthetic, cell permeable analog of ceramide to young cells led to growth arrest and cellular senescence (Venable et al., 1995). These cells showed cellular arrest, lack of Rb phosphorylation, no AP-1 production (Venable et al., 1995) and stained positive with the gal test for senescence (Mouton and Venable, 2000). Ceramide acts to inhibit the activation of PLD, by possibly three mechanisms, inhibiting activation by PKC, translocation to the membrane and gene expression, but none of these has been clearly defined (Exton, 1999). Exogenous ceramide inhibits PLD activity in primary fibroblasts (Venable et al., 1994) and this inhibition was shown to depend on PKC interaction but not on phosphorylation or translocation in cell free systems (Venable et al., 1996). However, ceramide has been shown to disrupt the liquid order of lipid rafts, which leads to PLD inhibition (Gidwani et al., 2003). To this end, recent work has identified that the deficiency in PLD activity is specific to the senescent membrane fractions, and not to changes in the cytosol (Webb et al., 2010). The increase in ceramide in senescent membranes likely changes the properties of lipid rafts, which no longer supports an integrated PLD response. Thus, the elevated ceramide levels and PKC regulation are important factors in reduced PLD activity in senescent cell membranes.

#### **2.4 The phosphoinositide 3-kinase pathway during senescence**

Due to the role of IGF-1 in the optimal proliferative response of primary fibroblasts (Phillips and Cristofalo, 1988), a common downstream effector of this pathway, phosphoinositide 3 kinase (PI3K) was assessed for its role during senescence. PI3K consists of a p85 regulatory subunit and a p110 catalytic subunit which selectively phosphorylates the 3-OH position of

The p53 tumour suppressor protein is a transcription factor that under normal cellular conditions is unstable and inactive. In response to many forms of cellular stress, p53 becomes activated by post-translational modification and is able to transactivate target genes that regulate diverse cellular processes including, cell cycle progression, senescence, DNA repair, metabolism and cell survival (Appella and Anderson, 2001; Vousden and Lane, 2007). Active p53 protein promotes cell cycle arrest through transcriptional activation of many genes including: *p21WAF1* (el-Deiry et al., 1993), *GADD45*α (Kastan et al., 1992), *BTG2*  (Rouault et al., 1996), *REPRIMO* (Ohki et al., 2000), *14-3-3*σ (Hermeking et al., 1997), and Prl-3 (Basak et al., 2008). Different subsets of p53-responsive genes regulate DNA repair, metabolism, survival and apoptosis. The p53 protein has been implicated as one of the key mediators of both the onset (Hara et al., 1991; Shay et al., 1991) and maintenance (Beausejour et al., 2003; Gire and Wynford-Thomas, 1998) of cellular senescence. The transcriptional targets of p53 shown to initiate replicative senescence include *p21WAF1* (Brown et al., 1997; el-Deiry et al., 1993; Noda et al., 1994), BTG2 (Wheaton et al., 2010), GADD45α (Jackson and Pereira-Smith, 2006) and PAI (Kortlever et al., 2006). When p53 is knocked down by shRNA (or other method) in fibroblasts it extends the proliferative lifespan by 10-20 population

Various chemical agents and gamma irradiation can cause DNA double stranded breaks, which also lead to premature senescence. The biological process that occurs at a doubled stranded break involves hundreds of H2AX histones becoming phosphorylated (H2AX) as the signal propagates from the break site. This DNA damage can visualized in the nucleus of a cell as foci by immunofluorescence using a H2AX phospho-specific antibody (Rogakou et al., 1999; Rogakou et al., 1998). The kinase Ataxia telangiectasia mutated (ATM) acts as a sensor of DNA damage, becomes activated at the H2AX foci and phosphorylates p53 (Bakkenist and Kastan, 2003). This phosphorylation stabilizes and activates p53, and allows

Telomeres consist of a tandem repeat of 6 nucleotides (TTAGGG) that cap the ends of linear double stranded DNA in mammals. Human telomeres consist of 5-10 kilobase pairs at the end of every chromosome arm and have an overhang of single-stranded telomeric repeat of several hundred bases. If ends of chromosomes were not protected by telomeres the cell would detect the end of the DNA as a double stranded break and initiate a DNA damage response. Telomeres normally protect the double stranded chromosome ends by the formation of telomere-loops (t-loops) in which the telomeric single-stranded overhang hybridizes within the double-stranded telomeric region of a chromosome. T-loops are maintained and stabilized by a complex of proteins referred to as shelterin (de Lange, 1994). Telomeres are know to shorten with each round of DNA replication because of the "endreplication problem"; the inability of DNA polymerases to completely replicate the 3' end of linear DNA molecules (Harley et al., 1990). Telomeres will shorten to a critical length after a finite number of DNA replication cycles, and lose the ability to form the sheltern/t-loop cap at the ends of chromosomes. The DNA damage signal arising from eroded telomeres will activate ATM, leading in turn to p53 activation (Herbig et al., 2004). Thus, t-loop disruption as a consequence of telomere shortening exposes telomeric DNA that leads to p53 activation and the initiation of replicative senescence (Herbig et al., 2004; Karlseder et al., 2002; Li et al., 2003; Li et al., 2004; Stewart et al., 2003). This model is supported by studies that ectopically expressed telomerase (hTERT) allowing fibroblasts to bypass senescence (Figure 1B) and become immortalized by preventing telomere shortening (Bodnar et al., 1998; Vaziri and

doublings, by avoiding p53 induced senescent program (Fig. 1B).

it to transactivate its effector genes.

phophoinositides. The enzyme is implicated in both mitogenic and survival signals which are propagated through down stream effectors such as PKB/Akt, IRS-1 and p70 S6K (reviewed in (Vanhaesebroeck et al., 1997). Application of a specific PI3K inhibitor (LY294002) to young primary fibroblasts resulted in cell arrest and the senescent phenotype. Conversely, in the same study, inactivation of the Ras/Raf/MAPK pathway by PD98059 also arrested cell growth, but no induction of senescence was noted (Tresini et al., 1998). Another group using the same approach showed that p27 was highly up-regulated, but that the presumed mediators of senescence, p21, p16, and p53 were all down-regulated. They additionally showed that p27 was the only mediator of the response, since p27-/- mouse embryonic fibroblasts did not become senescent with application of the drug (Collado et al., 2000). Overall, the role of PI3K seems to be in maintaining continued growth, and its inhibition leads to senescence through a non-traditional mechanism of p27 up-regulation.

One of the main effectors of the PI3K pathway is the mammalian target of rapamycin (mTOR), a kinase that integrates signals from multiple mitogenic pathways. The mTOR protein is activated by PI3K through the pathway of PKB/Akt leading to TSC1/TSC2 inactivation, which normally acts as a guainine exchange protein for the small GTPase Rheb. Rheb-GDP holds mTOR in an inactive state, until it is activated through PI3K-Akt-TSC1/2, allowing GTP exchange (Li et al. 2004). The increased mTOR activity has a global effect on translation and cell growth, which is required for rapid cell proliferation (Guertin and Sabatini, 2007). However, it has been shown that serum mitogens are required for the full development of the senescent phenotype (Satyanarayana et al., 2004). The mitogen based induction of senescence has been shown to involve the activation of mTOR, because its inactivation by rapamycin prevents senescence, even in the presence of DNA damage (Leontieva and Blagosklonny, 2010). Recently, treatment with rapamycin has even been shown to extend the proliferative lifespan of human fibroblasts (Cao et al., 2011). These effects were shown to depend on increased autophagic degradation of proteins that enforce senescence, but may also involve a disconnection between mitogen induced translational control and promotion of the cell cycle. Thus, the roles of PI3K and mTOR in senescence would seem to oppose each other, even though they are in the same pathway. These divergent results are difficult to reconcile, but possibly can be explained by other downstream PI3K targets that promote proliferation over senescence. This is supported by the finding that transcriptional targets of the PI3K/PKB are not properly regulated in replicative senescence (Lorenzini et al., 2002). Conversely, in stress or oncogenic induced senescence the PI3K pathway has been shown to have a positive role in the onset of senescence (Binet et al., 2009; Matuoka et al., 2003), consistent with a role for mTOR.

#### **3. P53 and telomere attrition**

Replicative senescence is widely accepted to be triggered by critically short telomeres. Stress-induced senescence (also called premature senescence) occurs when cells are exposed to sub-lethal cellular stress such as DNA damage (Di Leonardo et al., 1994; Resnick-Silverman et al., 1998), oxidative stress (Chen and Ames, 1994), or oncogenic stress (Serrano et al., 1997). Both replicative and stress-induced senescence result in the accumulation of cells that are incapable of further divisions and arrest primarily in the G1 phase of the cell cycle. Thus, senescence is thought to represent an intrinsic barrier to oncogenesis by limiting proliferation.

phophoinositides. The enzyme is implicated in both mitogenic and survival signals which are propagated through down stream effectors such as PKB/Akt, IRS-1 and p70 S6K (reviewed in (Vanhaesebroeck et al., 1997). Application of a specific PI3K inhibitor (LY294002) to young primary fibroblasts resulted in cell arrest and the senescent phenotype. Conversely, in the same study, inactivation of the Ras/Raf/MAPK pathway by PD98059 also arrested cell growth, but no induction of senescence was noted (Tresini et al., 1998). Another group using the same approach showed that p27 was highly up-regulated, but that the presumed mediators of senescence, p21, p16, and p53 were all down-regulated. They additionally showed that p27 was the only mediator of the response, since p27-/- mouse embryonic fibroblasts did not become senescent with application of the drug (Collado et al., 2000). Overall, the role of PI3K seems to be in maintaining continued growth, and its inhibition leads to senescence through a non-traditional mechanism of p27 up-regulation. One of the main effectors of the PI3K pathway is the mammalian target of rapamycin (mTOR), a kinase that integrates signals from multiple mitogenic pathways. The mTOR protein is activated by PI3K through the pathway of PKB/Akt leading to TSC1/TSC2 inactivation, which normally acts as a guainine exchange protein for the small GTPase Rheb. Rheb-GDP holds mTOR in an inactive state, until it is activated through PI3K-Akt-TSC1/2, allowing GTP exchange (Li et al. 2004). The increased mTOR activity has a global effect on translation and cell growth, which is required for rapid cell proliferation (Guertin and Sabatini, 2007). However, it has been shown that serum mitogens are required for the full development of the senescent phenotype (Satyanarayana et al., 2004). The mitogen based induction of senescence has been shown to involve the activation of mTOR, because its inactivation by rapamycin prevents senescence, even in the presence of DNA damage (Leontieva and Blagosklonny, 2010). Recently, treatment with rapamycin has even been shown to extend the proliferative lifespan of human fibroblasts (Cao et al., 2011). These effects were shown to depend on increased autophagic degradation of proteins that enforce senescence, but may also involve a disconnection between mitogen induced translational control and promotion of the cell cycle. Thus, the roles of PI3K and mTOR in senescence would seem to oppose each other, even though they are in the same pathway. These divergent results are difficult to reconcile, but possibly can be explained by other downstream PI3K targets that promote proliferation over senescence. This is supported by the finding that transcriptional targets of the PI3K/PKB are not properly regulated in replicative senescence (Lorenzini et al., 2002). Conversely, in stress or oncogenic induced senescence the PI3K pathway has been shown to have a positive role in the onset of

senescence (Binet et al., 2009; Matuoka et al., 2003), consistent with a role for mTOR.

Replicative senescence is widely accepted to be triggered by critically short telomeres. Stress-induced senescence (also called premature senescence) occurs when cells are exposed to sub-lethal cellular stress such as DNA damage (Di Leonardo et al., 1994; Resnick-Silverman et al., 1998), oxidative stress (Chen and Ames, 1994), or oncogenic stress (Serrano et al., 1997). Both replicative and stress-induced senescence result in the accumulation of cells that are incapable of further divisions and arrest primarily in the G1 phase of the cell cycle. Thus, senescence is thought to represent an intrinsic barrier to oncogenesis by limiting

**3. P53 and telomere attrition** 

proliferation.

The p53 tumour suppressor protein is a transcription factor that under normal cellular conditions is unstable and inactive. In response to many forms of cellular stress, p53 becomes activated by post-translational modification and is able to transactivate target genes that regulate diverse cellular processes including, cell cycle progression, senescence, DNA repair, metabolism and cell survival (Appella and Anderson, 2001; Vousden and Lane, 2007). Active p53 protein promotes cell cycle arrest through transcriptional activation of many genes including: *p21WAF1* (el-Deiry et al., 1993), *GADD45*α (Kastan et al., 1992), *BTG2*  (Rouault et al., 1996), *REPRIMO* (Ohki et al., 2000), *14-3-3*σ (Hermeking et al., 1997), and Prl-3 (Basak et al., 2008). Different subsets of p53-responsive genes regulate DNA repair, metabolism, survival and apoptosis. The p53 protein has been implicated as one of the key mediators of both the onset (Hara et al., 1991; Shay et al., 1991) and maintenance (Beausejour et al., 2003; Gire and Wynford-Thomas, 1998) of cellular senescence. The transcriptional targets of p53 shown to initiate replicative senescence include *p21WAF1* (Brown et al., 1997; el-Deiry et al., 1993; Noda et al., 1994), BTG2 (Wheaton et al., 2010), GADD45α (Jackson and Pereira-Smith, 2006) and PAI (Kortlever et al., 2006). When p53 is knocked down by shRNA (or other method) in fibroblasts it extends the proliferative lifespan by 10-20 population doublings, by avoiding p53 induced senescent program (Fig. 1B).

Various chemical agents and gamma irradiation can cause DNA double stranded breaks, which also lead to premature senescence. The biological process that occurs at a doubled stranded break involves hundreds of H2AX histones becoming phosphorylated (H2AX) as the signal propagates from the break site. This DNA damage can visualized in the nucleus of a cell as foci by immunofluorescence using a H2AX phospho-specific antibody (Rogakou et al., 1999; Rogakou et al., 1998). The kinase Ataxia telangiectasia mutated (ATM) acts as a sensor of DNA damage, becomes activated at the H2AX foci and phosphorylates p53 (Bakkenist and Kastan, 2003). This phosphorylation stabilizes and activates p53, and allows it to transactivate its effector genes.

Telomeres consist of a tandem repeat of 6 nucleotides (TTAGGG) that cap the ends of linear double stranded DNA in mammals. Human telomeres consist of 5-10 kilobase pairs at the end of every chromosome arm and have an overhang of single-stranded telomeric repeat of several hundred bases. If ends of chromosomes were not protected by telomeres the cell would detect the end of the DNA as a double stranded break and initiate a DNA damage response. Telomeres normally protect the double stranded chromosome ends by the formation of telomere-loops (t-loops) in which the telomeric single-stranded overhang hybridizes within the double-stranded telomeric region of a chromosome. T-loops are maintained and stabilized by a complex of proteins referred to as shelterin (de Lange, 1994). Telomeres are know to shorten with each round of DNA replication because of the "endreplication problem"; the inability of DNA polymerases to completely replicate the 3' end of linear DNA molecules (Harley et al., 1990). Telomeres will shorten to a critical length after a finite number of DNA replication cycles, and lose the ability to form the sheltern/t-loop cap at the ends of chromosomes. The DNA damage signal arising from eroded telomeres will activate ATM, leading in turn to p53 activation (Herbig et al., 2004). Thus, t-loop disruption as a consequence of telomere shortening exposes telomeric DNA that leads to p53 activation and the initiation of replicative senescence (Herbig et al., 2004; Karlseder et al., 2002; Li et al., 2003; Li et al., 2004; Stewart et al., 2003). This model is supported by studies that ectopically expressed telomerase (hTERT) allowing fibroblasts to bypass senescence (Figure 1B) and become immortalized by preventing telomere shortening (Bodnar et al., 1998; Vaziri and

other lipid Caveolin cholesterol

phospholipid sphingolipid PtdIns(4,5)P2

EGFR

Shc

Shc

Grb2 SOS

RAS

Dynamin

EGF Cavins

to 4 bind to caveolin and regulate caveolae biogenesis, shape and trafficking.

**A)** Electron micrograph of a caveolae at the membrane surface of 3T3 L1 fibroblasts. **B)** Schematic diagram of the lipid and protein composition of caveolae. Phospholipids and PtdIns(4,5)P2 are concentrated on the inner leaflet, while sphingolipid is found in the outer leaflet of caveolae. Cholesterol is required for the caveolin proteins to bind and create a multimeric scaffold that forms the caveolar structure. Dynamin proteins surround the neck of the caveolae, and play a role in the fission of caveolar vesicles during endocytosis. Many receptor and signalling cascades are compartmentalized in caveolae, including the epidermal growth factor receptor (EGFR), the adaptors Shc, Grb & SOS, and Ras small GTPase. Cavins 1

Cytosol

Extracellular

A

B

Fig. 2. Caveolae Structure

Benchimol, 1998). Hence, critically short telomeres provide a physiological signal for activation of the p53-dependent replicative senescence program in human cells.

#### **4. Caveolae**

Caveolae are flask shaped invaginations which form at the plasma membrane from lateral assemblies of cholesterol and sphingolipids known as lipid rafts (reviewed in (Harder and Simons, 1997)). Lipid rafts are composed of sphingomyelin, glycosphingolipids and cholesterol which form microdomain rafts surrounded by the phosphatidylcholine rich plasma membrane. These rafts sequester a specific subset of transmembrane associated proteins (Simons and Ikonen, 1997). Thus, lipid rafts are organelle like environments which have been postulated to have specific functions in various cell types (Simons and Toomre, 2000). Lipid rafts are also found to form higher order structures, caveolae (little caves), when associated with the scaffolding transmembrane proteins (Figure 2A & B). Caveolae are distinct from other vesicle-like invaginations, such as clathrin coated pits, due to their small size and lipid raft composition (Kurzchalia and Parton, 1999). The catalyst of caveolar formation is the oligomerization of a class of sphingolipid and cholesterol binding proteins known as caveolins (Monier et al., 1996), which stabilize and induce the lipid rafts to coalesce (reviewed in (Harder and Simons, 1997)). Three caveolin genes have been identified encoding caveolin proteins 1, 2, and 3 (Glenney, 1992; Kurzchalia et al., 1992; Scherer et al., 1996; Tang et al., 1996 ; Way and Parton, 1996). Both the N- and C-termini of caveolins are exposed to the cytoplasm forming a hairpin structure in the membrane, and are palmitoylated (Dietzen et al., 1995). Caveolin monomers oligomerize into hetero- and homodimers and trimers as they are translated in the cytoplasm (Lisanti et al., 1993), traffic through the trans-Golgi network and associate at the plasma membrane (reviewed in (Rietveld and Simons, 1998)). Due to their unique lipid raft composition caveolae can be biochemically purified based on their insolubility in the detergent Triton X-100 as well as on their low buoyant density on sucrose gradients (reviewed in (Anderson, 1998)). The insolubility of caveolae is likely due to the enrichment in sphingolipids and cholesterol in contrast to the glycerophospholipid rich plasma membrane (Hanada et al., 1995).

Recent advances in the characterization of lipid rafts have identified a new class of proteins which are essential to the biogenesis and regulation of caveolae; cavins 1 to 4. Cavin 1 (or polymerase I and transcript release factor: PTRF) has been shown to be essential for the stability and organization of caveolar structures (Hill et al., 2008; Liu and Pilch, 2008). Cavins 1-4 are part of a heterologous complex which bind to mature caveolin proteins (Figure 2B) that have emerged as caveolae after being processed through the trans-golgi system (Hayer et al., 2010). This interaction is cavin 1 and caveolin dependent and is thought to be due to a cavin phosphatidyl-serine interaction domain in all cavin proteins; a lipid which is enriched in caveolae (Bastiani et al., 2009). Cavin-2 is thought to play a role in the curvature of caveolar structures (Hansen et al., 2009). Cavin-3 is known to be involved in caveolae endocytosis and coordinates internalization on microtubules (McMahon et al., 2009). Cavin-4 is specifically expressed in striated muscle and has been shown to have specific roles related to myogenesis (Tagawa et al., 2008) and contraction (Ogata et al., 2008). Caveolin and cavin expression are interdependent on one another, since the depletion of one leads to the suppression of the other. Thus, cavins have emerged as an essential component of caveolae regulation, stability, and biogenesis.

Benchimol, 1998). Hence, critically short telomeres provide a physiological signal for

Caveolae are flask shaped invaginations which form at the plasma membrane from lateral assemblies of cholesterol and sphingolipids known as lipid rafts (reviewed in (Harder and Simons, 1997)). Lipid rafts are composed of sphingomyelin, glycosphingolipids and cholesterol which form microdomain rafts surrounded by the phosphatidylcholine rich plasma membrane. These rafts sequester a specific subset of transmembrane associated proteins (Simons and Ikonen, 1997). Thus, lipid rafts are organelle like environments which have been postulated to have specific functions in various cell types (Simons and Toomre, 2000). Lipid rafts are also found to form higher order structures, caveolae (little caves), when associated with the scaffolding transmembrane proteins (Figure 2A & B). Caveolae are distinct from other vesicle-like invaginations, such as clathrin coated pits, due to their small size and lipid raft composition (Kurzchalia and Parton, 1999). The catalyst of caveolar formation is the oligomerization of a class of sphingolipid and cholesterol binding proteins known as caveolins (Monier et al., 1996), which stabilize and induce the lipid rafts to coalesce (reviewed in (Harder and Simons, 1997)). Three caveolin genes have been identified encoding caveolin proteins 1, 2, and 3 (Glenney, 1992; Kurzchalia et al., 1992; Scherer et al., 1996; Tang et al., 1996 ; Way and Parton, 1996). Both the N- and C-termini of caveolins are exposed to the cytoplasm forming a hairpin structure in the membrane, and are palmitoylated (Dietzen et al., 1995). Caveolin monomers oligomerize into hetero- and homodimers and trimers as they are translated in the cytoplasm (Lisanti et al., 1993), traffic through the trans-Golgi network and associate at the plasma membrane (reviewed in (Rietveld and Simons, 1998)). Due to their unique lipid raft composition caveolae can be biochemically purified based on their insolubility in the detergent Triton X-100 as well as on their low buoyant density on sucrose gradients (reviewed in (Anderson, 1998)). The insolubility of caveolae is likely due to the enrichment in sphingolipids and cholesterol in

activation of the p53-dependent replicative senescence program in human cells.

contrast to the glycerophospholipid rich plasma membrane (Hanada et al., 1995).

of caveolae regulation, stability, and biogenesis.

Recent advances in the characterization of lipid rafts have identified a new class of proteins which are essential to the biogenesis and regulation of caveolae; cavins 1 to 4. Cavin 1 (or polymerase I and transcript release factor: PTRF) has been shown to be essential for the stability and organization of caveolar structures (Hill et al., 2008; Liu and Pilch, 2008). Cavins 1-4 are part of a heterologous complex which bind to mature caveolin proteins (Figure 2B) that have emerged as caveolae after being processed through the trans-golgi system (Hayer et al., 2010). This interaction is cavin 1 and caveolin dependent and is thought to be due to a cavin phosphatidyl-serine interaction domain in all cavin proteins; a lipid which is enriched in caveolae (Bastiani et al., 2009). Cavin-2 is thought to play a role in the curvature of caveolar structures (Hansen et al., 2009). Cavin-3 is known to be involved in caveolae endocytosis and coordinates internalization on microtubules (McMahon et al., 2009). Cavin-4 is specifically expressed in striated muscle and has been shown to have specific roles related to myogenesis (Tagawa et al., 2008) and contraction (Ogata et al., 2008). Caveolin and cavin expression are interdependent on one another, since the depletion of one leads to the suppression of the other. Thus, cavins have emerged as an essential component

**4. Caveolae** 

#### Fig. 2. Caveolae Structure

**A)** Electron micrograph of a caveolae at the membrane surface of 3T3 L1 fibroblasts. **B)** Schematic diagram of the lipid and protein composition of caveolae. Phospholipids and PtdIns(4,5)P2 are concentrated on the inner leaflet, while sphingolipid is found in the outer leaflet of caveolae. Cholesterol is required for the caveolin proteins to bind and create a multimeric scaffold that forms the caveolar structure. Dynamin proteins surround the neck of the caveolae, and play a role in the fission of caveolar vesicles during endocytosis. Many receptor and signalling cascades are compartmentalized in caveolae, including the epidermal growth factor receptor (EGFR), the adaptors Shc, Grb & SOS, and Ras small GTPase. Cavins 1 to 4 bind to caveolin and regulate caveolae biogenesis, shape and trafficking.

B

Fig. 3. Caveolar Endocytosis

cytoskeleton and are important for potocytosis.

Type 2 Caveolae

Actin Filaments

Cavesome

Endosome Golgi

**A)** Electron micrograph of caveolar structures at the membrane surface of differentiated 3T3 L1 fibroblasts. **B)** A schematic diagram showing caveolar endocytosis. Type I caveolae invaginate in response to various stimulae and form caveolar vesicles (cavicles) that are transported on microtubules. Cavicles can fuse together in cavesomes, where various contents may be recycled, or are transported to endosomes or the Golgi. Type II caveolae invaginate, fuse, and transport back to the cell surface. They are confined by the actin

Type 1 Caveolae

Cavicle

Microtubule

A

Caveolae are a type of endocytic vesicle (much like clathrin coated pits) and can undergo internalization with particular stimuli. The process is dependent on dynamin (a GTPase involved in membrane fission) and cholesterol and leads to the production of enclosed vesicular caveolar structures with in the cytoplasm of the cell (Fig. 3A & B). For example, caveolae can be generated in transformed NIH 3T3 cells when a dominant negative dynamin is introduced, and is blocked in the presence of cholesterol sequestering agent (Li et al., 2001). This implies that even in cells that normally have no detectable caveolae, there is a steady state of caveolar invagination that is stabilized with dominant negative dynamin. Pinched off caveolae can be visualized as self contained vesicles (called cavicles) using electron microscopy (Mundy et al., 2002), and have been shown to fuse with a specialized endosome (that is caveolin positive) called caveosomes (Pelkmans et al., 2002). This pathway has been shown to be utilized by SV-40 virus, cholera toxin and albumin (reviewed in (Lajoie and Nabi, 2007)). Caveolae utilizing this pathway are known as type I (Fig. 3B) and this cavicle trafficking has been shown to depend upon transport along microtubles. These caveolar vesicles form into alternate morphologies in different cell types; in adipocytes, caveolar vesicles cluster into rosettes, and in endothelial cells, the vesicles elongate and fuse to form channels. A second population of caveolae (type 2) are involved in continuous rounds of fusion and invagination (Fig. 3B) but are restricted to remain near the cell membrane by the actin cytoskeleton (Pelkmans and Zerial, 2005). This cycle is highly regulated by various kinases (del Pozo et al., 2005) which can also shift these caveolae into long range type I transport. It is theorized that type 2 caveolae are largely responsible for the potocytosis of vitamins and ions (Anderson et al., 1992). Type 3 caveolae form elongated tubes from the cell surface in certain cell types and are not as well characterized. Several types of caveolae can be seen simultaneously in the same cell lineage (Fig. 3A & B), and often carry out specialized functions that depend on cell type (reviewed in (White and Anderson, 2005)).

In addition to the endocytic transport described above, it has recently been found that the EGFR has a functional role during trafficking through caveolar system. Under normal conditions, activated EGFR is recycled through clathrin coated pit mediated endocytosis and degraded or recycled through endosomes or lysomes respectively (reviewed in (Madshus and Stang, 2009)). However, in circumstances of sustained EGF signalling levels or the presence of oxidative stress or gamma irradiation, the caveolar system transports the EGFR through an endosomal endoplasmic route to the nucleus (reviewed in (Dittmann et al., 2009)). These functions have been shown to be dependent on active EGFR, which is capable of utilizing transport through the nuclear pore and becomes soluble in the nucleoplasm. This localization is thought to be very important in the resolution of DNA damage. To this end, nuclear EGFR has been shown to phosphorylate and activate DNA dependent protein kinase (DNA-PK), an important mediator of nonhomologous end joining (Dittmann et al., 2005). In addition, nuclear EGFR has been shown to regulate transcription of cyclin D1, iNOS and B-myb, and regulate PCNA by phosphorylation, all of which correlate with entry into the cell cycle (Dittmann et al., 2009). Many tumours have been reported to have a nuclear population of EGFR, and its presence is linked to continued proliferation. Thus, this cellular transport system is the target of chemotherapy agents that either inactivate EGF signalling by inhibition of src kinase or antibody therapies that promote the uptake of EGFR through caveolae and thus block its activation (Dittmann et al., 2009).

Caveolae are a type of endocytic vesicle (much like clathrin coated pits) and can undergo internalization with particular stimuli. The process is dependent on dynamin (a GTPase involved in membrane fission) and cholesterol and leads to the production of enclosed vesicular caveolar structures with in the cytoplasm of the cell (Fig. 3A & B). For example, caveolae can be generated in transformed NIH 3T3 cells when a dominant negative dynamin is introduced, and is blocked in the presence of cholesterol sequestering agent (Li et al., 2001). This implies that even in cells that normally have no detectable caveolae, there is a steady state of caveolar invagination that is stabilized with dominant negative dynamin. Pinched off caveolae can be visualized as self contained vesicles (called cavicles) using electron microscopy (Mundy et al., 2002), and have been shown to fuse with a specialized endosome (that is caveolin positive) called caveosomes (Pelkmans et al., 2002). This pathway has been shown to be utilized by SV-40 virus, cholera toxin and albumin (reviewed in (Lajoie and Nabi, 2007)). Caveolae utilizing this pathway are known as type I (Fig. 3B) and this cavicle trafficking has been shown to depend upon transport along microtubles. These caveolar vesicles form into alternate morphologies in different cell types; in adipocytes, caveolar vesicles cluster into rosettes, and in endothelial cells, the vesicles elongate and fuse to form channels. A second population of caveolae (type 2) are involved in continuous rounds of fusion and invagination (Fig. 3B) but are restricted to remain near the cell membrane by the actin cytoskeleton (Pelkmans and Zerial, 2005). This cycle is highly regulated by various kinases (del Pozo et al., 2005) which can also shift these caveolae into long range type I transport. It is theorized that type 2 caveolae are largely responsible for the potocytosis of vitamins and ions (Anderson et al., 1992). Type 3 caveolae form elongated tubes from the cell surface in certain cell types and are not as well characterized. Several types of caveolae can be seen simultaneously in the same cell lineage (Fig. 3A & B), and often carry out specialized functions that depend on cell type (reviewed in (White and

In addition to the endocytic transport described above, it has recently been found that the EGFR has a functional role during trafficking through caveolar system. Under normal conditions, activated EGFR is recycled through clathrin coated pit mediated endocytosis and degraded or recycled through endosomes or lysomes respectively (reviewed in (Madshus and Stang, 2009)). However, in circumstances of sustained EGF signalling levels or the presence of oxidative stress or gamma irradiation, the caveolar system transports the EGFR through an endosomal endoplasmic route to the nucleus (reviewed in (Dittmann et al., 2009)). These functions have been shown to be dependent on active EGFR, which is capable of utilizing transport through the nuclear pore and becomes soluble in the nucleoplasm. This localization is thought to be very important in the resolution of DNA damage. To this end, nuclear EGFR has been shown to phosphorylate and activate DNA dependent protein kinase (DNA-PK), an important mediator of nonhomologous end joining (Dittmann et al., 2005). In addition, nuclear EGFR has been shown to regulate transcription of cyclin D1, iNOS and B-myb, and regulate PCNA by phosphorylation, all of which correlate with entry into the cell cycle (Dittmann et al., 2009). Many tumours have been reported to have a nuclear population of EGFR, and its presence is linked to continued proliferation. Thus, this cellular transport system is the target of chemotherapy agents that either inactivate EGF signalling by inhibition of src kinase or antibody therapies that promote the uptake of EGFR through caveolae and thus

Anderson, 2005)).

block its activation (Dittmann et al., 2009).

**A)** Electron micrograph of caveolar structures at the membrane surface of differentiated 3T3 L1 fibroblasts. **B)** A schematic diagram showing caveolar endocytosis. Type I caveolae invaginate in response to various stimulae and form caveolar vesicles (cavicles) that are transported on microtubules. Cavicles can fuse together in cavesomes, where various contents may be recycled, or are transported to endosomes or the Golgi. Type II caveolae invaginate, fuse, and transport back to the cell surface. They are confined by the actin cytoskeleton and are important for potocytosis.

In addition to the MAPK pathway, EGFR has been shown to activate other downstream effectors such as phospholipase C (PLC) through PI3K (Jang et al., 2001) and PLD1 (Han et al., 2002)) in caveolae. PLC hydrolyses phosphatidylinositol- 4,5-bisphosphate (PIP2) to produce diacylglycerol and inositol 1,3,5,-trisphosphate (IP3), which activates PKC and mobilizes intracellular calcium respectively. PLD catalyses the hydrolysis of phosphatidylcholine to yield phosphatidic acid. These second messengers lead to the activation of Akt and mTOR which promote cellular proliferation (reviewed in (de

The first report examining caveolin protein in replicative senescence (Park et al., 2000) found that the protein was highly elevated as human fibroblasts reached high population doublings. This upregulation was correlated to an increased number of caveolar structures seen in the EM of senescent cells, which were thought to be caveolae. Additionally, the group found EGFR signalling was attenuated in senescence through the binding to caveolin. However, these studies did not address whether caveolin generated bona-fide functional caveolar structures or whether it represented a differential regulation of caveolar pathway. Unfortunately, the EM chosen to examine the senescent cells did not have a clear cross section of the outer membrane, and it was unclear whether the structures being scored were caveolae or cavicles. The paradigm then (as now) was that increased caveolin drives the formation of caveolae and the regulation of cavicle generation had not yet been fully elucidated. This lead to the interpretation that the number of caveolae increase in senescent cells. Additionally, the low numbers of caveolae counted in low passage fibroblasts was in direct contrast to what had been previously seen in human fibroblasts by EM (Rothberg et al., 1992) or by freeze fracture EM (Fujimoto et al., 2000). Since this initial report, other groups have visualized a similar elevation of caveolar structures, which appear to be vesicles, and not classical caveolae in senescent cells (Bai et al., 2011; Volonte and Galbiati, 2011). Figure 3A shows structures consistent with caveolae in differentiated 3T3 L1 fibroblasts. Note the flask like invaginations within the outer membrane and the opening to the extracellular space through a smaller pore like neck. Thus, classically defined caveolae do not increase during senescence, and potentially are lost, while cavicles consistent with increased endocytosis have been seen in all senescent cells examined so far (Figure 3A & B). Although our own work examining caveolae (Wheaton and Riabowol, 2004) originally appeared to radically differ from other published reports, it does support the idea of increased caveolar vesicles in senescent fibroblasts. A comparison of young and senescent cells indicated that senescent cells contained a higher total amount of caveolins 1 and 2, but had significantly less of both proteins in the caveolar fraction obtained by sucrose density centrifugation. Additionally, caveolar fractions from senescent cells completely lacked the tyrosine kinase activity associated with functional caveolae and the EGF response. Furthermore, old cells had little caveolar protein exposed to the outer plasma membrane as estimated by an *in vivo* biotinoylation assay and there was no caveolin 1 detected on the cell surface using immunofluorescence (Fig. 4A & B) and confocal microscopy. Together, these data suggest that a fundamental loss of signal integration at the plasma membrane of senescent cells is due to the loss of signalling competent caveolae. However, they do not rule out the possibility that increased caveolar vesicles are present within the cytoplasm, as has been conjectured above. In fact, the poor response of EGFR and tyrosine phosphorylation of

Laurentiis et al., 2007)).

**5. Caveolae and cavicles in senescence** 

#### **4.1 Cell signalling through caveolae**

There is significant evidence that many well-studied receptor signalling systems are localized and operate through caveolae. Caveolin protein has been shown to directly interact with many of these signalling molecules through a 20 amino acid domain known as the caveolin scaffolding domain (CSD: (Couet et al., 1997)). Interaction with this domain has been shown to hold the signalling molecules in an inactive conformation (Okamoto et al., 1998). The pepitide sequence is rich in aromatic amino acids and has been shown to bind to adenylyl cyclase (AC), heterotrimeric Gα and Gβγ, PI3K, endothelial nitric oxide synthase (eNOS), protein kinase A (PKA), PKC, ERK1/2 and Src family kinases. In each case, the CSD is associated with the inactivation of the particular pathway (reviewed in (Patel et al., 2008)). However, since the CSD also binds lipids, there is the possibility that there are two populations of caveolin, one integral with rafts and another associated with the membrane that is not part of caveolae (Parton and Simons, 2007). Alternatively, the CSD has been conjectured to release the signalling proteins it binds to upon conformational change with activation (Okamoto et al., 1998). Although the exact role of the CSD is controversial, it is clear that it is involved in regulating and possibly sequestering signalling molecules to the caveolae. The inhibitory signalling associated with caveolin proteins has been challenged by many studies which show a requirement for Caveolin 1 in activating signal cascades (Kurzchalia and Parton, 1999, White, 2005 #707). This theory is based on the caveolin driven generation of caveolae, which acts to compartmentalize many signalling cascades. Thus, the caveolar structures and caveolin protein act as a center of cross talk and integration of mitogenic responses in normal cycling cells.

Many proteins have been found to localize to caveolae either through direct interaction with caveolins or sequestered through the properties of the lipid microdomains (Zajchowski and Robbins, 2002). Localization of proteins to caveolae is also enhanced by palmitoylation. A significant number of these are membrane bound receptors and kinase cascades with their adaptor proteins. Lipid moieties in caveolae are often utilized as second messengers (figure 2B) or for docking to plasma membrane by signalling proteins. This allows caveolae to regulate the activation of signalling cascades by concentration or proximity of substrates and second messengers (Cohen et al., 2004). Many receptor signalling systems are localized to and operate through caveolae and include; Src family kinases, nitric oxide synthase, EGFR, PDGFR, PLC, PLD, PKC & , Ras, trimeric G-proteins, MEK and Erk2, among others. Some of these proteins can also be found to be concentrated in lipid rafts (reviewed in (de Laurentiis et al., 2007)).

The most thoroughly characterized RTK residing in caveolae is EGFR, which is also crucial to mitogenic reposnse in human fibroblasts. Whether the localization of EGFR in caveolae has a negative or positive role in EGFR signalling is controversial. There is strong evidence for both, and it may be difficult to reconcile the observations definitively. The first evidence of the MAPK pathway in caveolae found H-Ras, Grb2 and SOS in Rat-1 cells (Mineo et al., 1996). The EGFR was present in the caveolin fraction in unstimulated Rat-1 cells, but declined rapidly after stimulation by EGF. The same migration was found in human fibroblasts and depended on EGF engagement, active EGFR tyrosine kinase, and phosphorylation of the EGFR receptor (Mineo et al., 1999). These observations implied that the activation of EGFR occurred within the caveolae, and is required to migrate in order to attenuate the EGF response. This was supported by the finding that some common mutations in EGFR that are oncogenic are also incapable of migrating from caveolae when stimulated.

There is significant evidence that many well-studied receptor signalling systems are localized and operate through caveolae. Caveolin protein has been shown to directly interact with many of these signalling molecules through a 20 amino acid domain known as the caveolin scaffolding domain (CSD: (Couet et al., 1997)). Interaction with this domain has been shown to hold the signalling molecules in an inactive conformation (Okamoto et al., 1998). The pepitide sequence is rich in aromatic amino acids and has been shown to bind to adenylyl cyclase (AC), heterotrimeric Gα and Gβγ, PI3K, endothelial nitric oxide synthase (eNOS), protein kinase A (PKA), PKC, ERK1/2 and Src family kinases. In each case, the CSD is associated with the inactivation of the particular pathway (reviewed in (Patel et al., 2008)). However, since the CSD also binds lipids, there is the possibility that there are two populations of caveolin, one integral with rafts and another associated with the membrane that is not part of caveolae (Parton and Simons, 2007). Alternatively, the CSD has been conjectured to release the signalling proteins it binds to upon conformational change with activation (Okamoto et al., 1998). Although the exact role of the CSD is controversial, it is clear that it is involved in regulating and possibly sequestering signalling molecules to the caveolae. The inhibitory signalling associated with caveolin proteins has been challenged by many studies which show a requirement for Caveolin 1 in activating signal cascades (Kurzchalia and Parton, 1999, White, 2005 #707). This theory is based on the caveolin driven generation of caveolae, which acts to compartmentalize many signalling cascades. Thus, the caveolar structures and caveolin protein act as a center of cross talk and integration of

Many proteins have been found to localize to caveolae either through direct interaction with caveolins or sequestered through the properties of the lipid microdomains (Zajchowski and Robbins, 2002). Localization of proteins to caveolae is also enhanced by palmitoylation. A significant number of these are membrane bound receptors and kinase cascades with their adaptor proteins. Lipid moieties in caveolae are often utilized as second messengers (figure 2B) or for docking to plasma membrane by signalling proteins. This allows caveolae to regulate the activation of signalling cascades by concentration or proximity of substrates and second messengers (Cohen et al., 2004). Many receptor signalling systems are localized to and operate through caveolae and include; Src family kinases, nitric oxide synthase, EGFR, PDGFR, PLC, PLD, PKC & , Ras, trimeric G-proteins, MEK and Erk2, among others. Some of these proteins can also be found to be concentrated in lipid rafts (reviewed

The most thoroughly characterized RTK residing in caveolae is EGFR, which is also crucial to mitogenic reposnse in human fibroblasts. Whether the localization of EGFR in caveolae has a negative or positive role in EGFR signalling is controversial. There is strong evidence for both, and it may be difficult to reconcile the observations definitively. The first evidence of the MAPK pathway in caveolae found H-Ras, Grb2 and SOS in Rat-1 cells (Mineo et al., 1996). The EGFR was present in the caveolin fraction in unstimulated Rat-1 cells, but declined rapidly after stimulation by EGF. The same migration was found in human fibroblasts and depended on EGF engagement, active EGFR tyrosine kinase, and phosphorylation of the EGFR receptor (Mineo et al., 1999). These observations implied that the activation of EGFR occurred within the caveolae, and is required to migrate in order to attenuate the EGF response. This was supported by the finding that some common mutations in EGFR that are oncogenic are also

**4.1 Cell signalling through caveolae** 

mitogenic responses in normal cycling cells.

incapable of migrating from caveolae when stimulated.

in (de Laurentiis et al., 2007)).

In addition to the MAPK pathway, EGFR has been shown to activate other downstream effectors such as phospholipase C (PLC) through PI3K (Jang et al., 2001) and PLD1 (Han et al., 2002)) in caveolae. PLC hydrolyses phosphatidylinositol- 4,5-bisphosphate (PIP2) to produce diacylglycerol and inositol 1,3,5,-trisphosphate (IP3), which activates PKC and mobilizes intracellular calcium respectively. PLD catalyses the hydrolysis of phosphatidylcholine to yield phosphatidic acid. These second messengers lead to the activation of Akt and mTOR which promote cellular proliferation (reviewed in (de Laurentiis et al., 2007)).

## **5. Caveolae and cavicles in senescence**

The first report examining caveolin protein in replicative senescence (Park et al., 2000) found that the protein was highly elevated as human fibroblasts reached high population doublings. This upregulation was correlated to an increased number of caveolar structures seen in the EM of senescent cells, which were thought to be caveolae. Additionally, the group found EGFR signalling was attenuated in senescence through the binding to caveolin. However, these studies did not address whether caveolin generated bona-fide functional caveolar structures or whether it represented a differential regulation of caveolar pathway. Unfortunately, the EM chosen to examine the senescent cells did not have a clear cross section of the outer membrane, and it was unclear whether the structures being scored were caveolae or cavicles. The paradigm then (as now) was that increased caveolin drives the formation of caveolae and the regulation of cavicle generation had not yet been fully elucidated. This lead to the interpretation that the number of caveolae increase in senescent cells. Additionally, the low numbers of caveolae counted in low passage fibroblasts was in direct contrast to what had been previously seen in human fibroblasts by EM (Rothberg et al., 1992) or by freeze fracture EM (Fujimoto et al., 2000). Since this initial report, other groups have visualized a similar elevation of caveolar structures, which appear to be vesicles, and not classical caveolae in senescent cells (Bai et al., 2011; Volonte and Galbiati, 2011). Figure 3A shows structures consistent with caveolae in differentiated 3T3 L1 fibroblasts. Note the flask like invaginations within the outer membrane and the opening to the extracellular space through a smaller pore like neck. Thus, classically defined caveolae do not increase during senescence, and potentially are lost, while cavicles consistent with increased endocytosis have been seen in all senescent cells examined so far (Figure 3A & B). Although our own work examining caveolae (Wheaton and Riabowol, 2004) originally appeared to radically differ from other published reports, it does support the idea of increased caveolar vesicles in senescent fibroblasts. A comparison of young and senescent cells indicated that senescent cells contained a higher total amount of caveolins 1 and 2, but had significantly less of both proteins in the caveolar fraction obtained by sucrose density centrifugation. Additionally, caveolar fractions from senescent cells completely lacked the tyrosine kinase activity associated with functional caveolae and the EGF response. Furthermore, old cells had little caveolar protein exposed to the outer plasma membrane as estimated by an *in vivo* biotinoylation assay and there was no caveolin 1 detected on the cell surface using immunofluorescence (Fig. 4A & B) and confocal microscopy. Together, these data suggest that a fundamental loss of signal integration at the plasma membrane of senescent cells is due to the loss of signalling competent caveolae. However, they do not rule out the possibility that increased caveolar vesicles are present within the cytoplasm, as has been conjectured above. In fact, the poor response of EGFR and tyrosine phosphorylation of

These data suggest a model in which senescent cells are unable to organize and localize the components of functional caveolae to the plasma membrane. The pinched off caveolar vesicles observed in senescent cells may represent a unique misregulation of caveolae that could explain both the functional differences and the increase in caveolar structures seen previously (Wheaton, 2011). Thus, although increased expression of cavin-1 and caveolin proteins drive the biogenesis of caveolar structures, they unbalance the normal equilibrium between cavicles and cavaolae. This unbalance could promote invagination, but stall cavicle migration at some point in their migration along microtubles before they fuse with caveosomes or endosomes. Consistent with this idea, caveolin 1 staining is found at the cell periphery in young cells, but not at the membrane of senescent cells. However, there is very intense staining of caveolin within the interior of senescent cells (Fig. 4A & B). Using confocal microscopy with a deconvolution algorithm found intense caveolin-1 staining in senescent cells that was associated with fibre like structures (Fig. 5B). Confocal analysis of young cell caveolin -1 found it concentrated in regions of the cell periphery (Fig. 5A). This pattern of fibres is consistent with the caveolae associating with microtubules, and becoming stalled after internalization. The increased number of cavicles could represent a natural response to stress, in which cell arrest is maintained by sequestering key signalling receptors away from their ligands in the extracellular space. This would have a similar effect to EGFR antibody based chemotherapies which drive internalization of the EGFR through caveolae, but prevent its localization to the nucleus and activation (Dittmann et al., 2009). Considering internalization of EGFR through caveolae is a response to gentoxic agents, it is possible that DNA damage downstream of telomere attrition may do the same. Thus, the cell may respond to stress by down regulating mitogen activated cascades through internalization of caveolae. Recent studies in which caveolae are found to be redistributed by internalization during mitosis support this idea (Boucrot et al., 2011) . The caveolae in this case internalize through microtubules and could be a mechanism to block mitogenic

signals after the commitment to divide.

**5.1 Physical changes in senescent membranes and lipids** 

It is possible that some of the changes seen in caveolar structures can be explained by the unique properties and composition of the plasma membrane of old cells (Rutter et al., 1996; Schroeder et al., 1984). Rafts are comprised of a high concentration of sphingolipids and cholesterol, which have strong cohesive forces that counteract the entropic force inherent in a fluid mosaic membrane (reviewed in (Harder and Simons, 1997)). Examining senescent cells using proton magnetic resonance has shown that the ratio of cholesterol/ phosphatidylcholine increases as human fibroblasts age in culture, indicating an increased amount of mobile cholesterol (Rutter et al., 1996). Another early report sought to probe the lipid composition of these fibroblasts by using fluorescent probes. These labelled lipids can be used to determine the limiting anisotropy (fluidity) of membranes and showed that the microsomal fraction increased in fluidity with donor age (Schroeder et al., 1984). Lipid composition changes during fibroblast aging lead to higher lipid fluidity and may reflect the inability of significant raft domains to form with age, since rafts by nature are islands of less fluid lipid. This is in part is due to the levels of a species of phosphatidylcholine comprised of stearic acid and arachidonic acid becoming elevated in senescent fibroblasts (Naru et al., 2008). This dilutes the components of lipid rafts in the membrane of senescent cells.

its targets in caveolae could be due the separation of these structures from the extracellular space. Therefore, the increase in caveolar structures in senescence may represent a misregulation of the normal functional caveolae during the aging process. This implies that some part of the endocytic process is blocked, leading to accumulation and stabilization of cavicles during senescence. This is supported by the finding that endocytosis is reduced in senescent cells and is correlated with an upregulation of caveolin 1 (Park et al., 2002).

Fig. 4. Caveolin-1 Localization in Young and Senescent Fibroblasts Young (A) and senescent (B) Hs68 fibroblasts were immuno-stained with anti-caveolin-1 and Alexa 488 flourescent secondary antibodies. Caveolin-1 is concentrated at the cell periphery in young cells (A) and is largely internalized in senescent cells (B).

its targets in caveolae could be due the separation of these structures from the extracellular space. Therefore, the increase in caveolar structures in senescence may represent a misregulation of the normal functional caveolae during the aging process. This implies that some part of the endocytic process is blocked, leading to accumulation and stabilization of cavicles during senescence. This is supported by the finding that endocytosis is reduced in

senescent cells and is correlated with an upregulation of caveolin 1 (Park et al., 2002).

Fig. 4. Caveolin-1 Localization in Young and Senescent Fibroblasts

periphery in young cells (A) and is largely internalized in senescent cells (B).

Young (A) and senescent (B) Hs68 fibroblasts were immuno-stained with anti-caveolin-1 and Alexa 488 flourescent secondary antibodies. Caveolin-1 is concentrated at the cell

These data suggest a model in which senescent cells are unable to organize and localize the components of functional caveolae to the plasma membrane. The pinched off caveolar vesicles observed in senescent cells may represent a unique misregulation of caveolae that could explain both the functional differences and the increase in caveolar structures seen previously (Wheaton, 2011). Thus, although increased expression of cavin-1 and caveolin proteins drive the biogenesis of caveolar structures, they unbalance the normal equilibrium between cavicles and cavaolae. This unbalance could promote invagination, but stall cavicle migration at some point in their migration along microtubles before they fuse with caveosomes or endosomes. Consistent with this idea, caveolin 1 staining is found at the cell periphery in young cells, but not at the membrane of senescent cells. However, there is very intense staining of caveolin within the interior of senescent cells (Fig. 4A & B). Using confocal microscopy with a deconvolution algorithm found intense caveolin-1 staining in senescent cells that was associated with fibre like structures (Fig. 5B). Confocal analysis of young cell caveolin -1 found it concentrated in regions of the cell periphery (Fig. 5A). This pattern of fibres is consistent with the caveolae associating with microtubules, and becoming stalled after internalization. The increased number of cavicles could represent a natural response to stress, in which cell arrest is maintained by sequestering key signalling receptors away from their ligands in the extracellular space. This would have a similar effect to EGFR antibody based chemotherapies which drive internalization of the EGFR through caveolae, but prevent its localization to the nucleus and activation (Dittmann et al., 2009). Considering internalization of EGFR through caveolae is a response to gentoxic agents, it is possible that DNA damage downstream of telomere attrition may do the same. Thus, the cell may respond to stress by down regulating mitogen activated cascades through internalization of caveolae. Recent studies in which caveolae are found to be redistributed by internalization during mitosis support this idea (Boucrot et al., 2011) . The caveolae in this case internalize through microtubules and could be a mechanism to block mitogenic signals after the commitment to divide.

#### **5.1 Physical changes in senescent membranes and lipids**

It is possible that some of the changes seen in caveolar structures can be explained by the unique properties and composition of the plasma membrane of old cells (Rutter et al., 1996; Schroeder et al., 1984). Rafts are comprised of a high concentration of sphingolipids and cholesterol, which have strong cohesive forces that counteract the entropic force inherent in a fluid mosaic membrane (reviewed in (Harder and Simons, 1997)). Examining senescent cells using proton magnetic resonance has shown that the ratio of cholesterol/ phosphatidylcholine increases as human fibroblasts age in culture, indicating an increased amount of mobile cholesterol (Rutter et al., 1996). Another early report sought to probe the lipid composition of these fibroblasts by using fluorescent probes. These labelled lipids can be used to determine the limiting anisotropy (fluidity) of membranes and showed that the microsomal fraction increased in fluidity with donor age (Schroeder et al., 1984). Lipid composition changes during fibroblast aging lead to higher lipid fluidity and may reflect the inability of significant raft domains to form with age, since rafts by nature are islands of less fluid lipid. This is in part is due to the levels of a species of phosphatidylcholine comprised of stearic acid and arachidonic acid becoming elevated in senescent fibroblasts (Naru et al., 2008). This dilutes the components of lipid rafts in the membrane of senescent cells.

In addition, senescent cell membranes are characterized by the specific loss of cholesterol from the microsomal fraction (Nakamura et al., 2003), even if the overall cholesterol may rise with age (Park et al., 2000). As previously mentioned, cholerterol is essential for lipid raft and caveolae formation (Fig. 2B). The altered cholesterol in the membrane also likely affects various signal transduction pathways (Fielding and Fielding, 2004). Alteration of the raft composition or dynamics may interfere with raft coalescence or its ability to sequester caveolin proteins. The loss of caveolae at the surface of the plasma membrane in old cells supports this model, since it would indicate that the proteins are unable to associate with rafts due to altered dynamics or composition, and subsequently they are not transported to

Key signalling pathways that operate through caveolar structures which are inactivated or misregulated could lead to aspects of the senescent phenotype. Since many growth receptors have been found to be localized within, and to signal through caveolar structures, it is possible that one or more of these may modulate the mitogenic response in primary fibroblasts. Many signalling pathways shown to be altered in senescence, including MAPK, PI3K, PLD and PKC (reviewed in (Caino et al., 2009)) are localized to caveolae. Since a significant post receptor block of mitogenic signalling is associated with the development of senescence, the existence of microdomains where receptors and signalling cascades are believed to be linked offers a promising functional connection to these observations. Indeed, the absence of a membrane population of caveolae may prevent mitogenic signal propagation. Thus, the post receptor block could be the result of receptors being either uncoupled from internalized caveolae or sequestered from access to ligands in the extracellular space. These kinase cascades would subsequently be unable to maintain integrated downstream responses to ligand and contribute to senescent arrest. Alternatively, the increases in caveolin protein itself may lead directly to the attenuation of many kinase cascades by virtue of the tumour suppressive function of CSD binding to key components of the cascade. Thus, a mechanism linking the post receptor block to caveolae is either through misregulated caveolar internalization or the inhibitory CSD of caveolin, both of which are

The initial observation linking caveolae to repressed signal propagation in senescence noted that EGF signalling was attenuated (Park et al., 2000). These first studies observed an increase in caveolin-1 & 2 proteins, and therefore the CSD domain (Park et al., 2000; Wheaton et al., 2001). Consistent with this observation, ectopic caveolin 1 expression in fibroblasts was shown to induce premature senescence and thus lead directly to cellular arrest (Volonte et al., 2002). This was further supported by the suppression of caveolin-1 using small inferring RNA or antisense oligos in senescent fibroblasts. Knockdown of caveolin in fibroblasts restores the response to EGF and the cells are capable of entering the cell cycle (Cho et al., 2003). It is thought that this response works primarily through the interaction of the caveolin CSD with erk1/2, leading to attenuation of kinase activity (Engelman et al., 1998). Supporting this, the erk1/2 kinase has been previously reported to

Oxidative stress has been shown to cause irreversible growth arrest in NIH 3T3 cells and is dependent on the upregulation of caveolin-1 (Volonte et al., 2002). Such stress also leads to

**5.2 Upregulation of caveolin proteins and its effect on caveolar signalling** 

characterized by increased caveolin-1 protein expression.

be misregulated during senescence (see section 2.1).

the cell surface.

Fig. 5. Confocal Microscopy of Caveolin-1 Staining in Young and Senescent Fibroblasts. Young (A) and senescent (B) Hs68 fibroblasts were immuno-stained with anit-caveolin-1 and Alexa 488 fluorescent secondary antibodies. The images show a single confocal layer that was filtered through a de-convolution protocol to show the localization in sharper detail. The young cells have a high concentration of caveolin at the cell periphery, indicated by arrows. In senescent cells, caveolin is concentrated through out the cell on filaments consistent with microtubules.

Fig. 5. Confocal Microscopy of Caveolin-1 Staining in Young and Senescent Fibroblasts. Young (A) and senescent (B) Hs68 fibroblasts were immuno-stained with anit-caveolin-1 and Alexa 488 fluorescent secondary antibodies. The images show a single confocal layer that was filtered through a de-convolution protocol to show the localization in sharper detail. The young cells have a high concentration of caveolin at the cell periphery, indicated by arrows. In senescent cells, caveolin is concentrated through out the cell on filaments

consistent with microtubules.

In addition, senescent cell membranes are characterized by the specific loss of cholesterol from the microsomal fraction (Nakamura et al., 2003), even if the overall cholesterol may rise with age (Park et al., 2000). As previously mentioned, cholerterol is essential for lipid raft and caveolae formation (Fig. 2B). The altered cholesterol in the membrane also likely affects various signal transduction pathways (Fielding and Fielding, 2004). Alteration of the raft composition or dynamics may interfere with raft coalescence or its ability to sequester caveolin proteins. The loss of caveolae at the surface of the plasma membrane in old cells supports this model, since it would indicate that the proteins are unable to associate with rafts due to altered dynamics or composition, and subsequently they are not transported to the cell surface.

#### **5.2 Upregulation of caveolin proteins and its effect on caveolar signalling**

Key signalling pathways that operate through caveolar structures which are inactivated or misregulated could lead to aspects of the senescent phenotype. Since many growth receptors have been found to be localized within, and to signal through caveolar structures, it is possible that one or more of these may modulate the mitogenic response in primary fibroblasts. Many signalling pathways shown to be altered in senescence, including MAPK, PI3K, PLD and PKC (reviewed in (Caino et al., 2009)) are localized to caveolae. Since a significant post receptor block of mitogenic signalling is associated with the development of senescence, the existence of microdomains where receptors and signalling cascades are believed to be linked offers a promising functional connection to these observations. Indeed, the absence of a membrane population of caveolae may prevent mitogenic signal propagation. Thus, the post receptor block could be the result of receptors being either uncoupled from internalized caveolae or sequestered from access to ligands in the extracellular space. These kinase cascades would subsequently be unable to maintain integrated downstream responses to ligand and contribute to senescent arrest. Alternatively, the increases in caveolin protein itself may lead directly to the attenuation of many kinase cascades by virtue of the tumour suppressive function of CSD binding to key components of the cascade. Thus, a mechanism linking the post receptor block to caveolae is either through misregulated caveolar internalization or the inhibitory CSD of caveolin, both of which are characterized by increased caveolin-1 protein expression.

The initial observation linking caveolae to repressed signal propagation in senescence noted that EGF signalling was attenuated (Park et al., 2000). These first studies observed an increase in caveolin-1 & 2 proteins, and therefore the CSD domain (Park et al., 2000; Wheaton et al., 2001). Consistent with this observation, ectopic caveolin 1 expression in fibroblasts was shown to induce premature senescence and thus lead directly to cellular arrest (Volonte et al., 2002). This was further supported by the suppression of caveolin-1 using small inferring RNA or antisense oligos in senescent fibroblasts. Knockdown of caveolin in fibroblasts restores the response to EGF and the cells are capable of entering the cell cycle (Cho et al., 2003). It is thought that this response works primarily through the interaction of the caveolin CSD with erk1/2, leading to attenuation of kinase activity (Engelman et al., 1998). Supporting this, the erk1/2 kinase has been previously reported to be misregulated during senescence (see section 2.1).

Oxidative stress has been shown to cause irreversible growth arrest in NIH 3T3 cells and is dependent on the upregulation of caveolin-1 (Volonte et al., 2002). Such stress also leads to

leading to a increase in caveolar vesicles causes both the attenuation of the EGFR signalling (de Laurentiis et al., 2007) and an increase in the activity of the p53 tumour suppressor through elevated ROS (Volonte and Galbiati, 2009a). Both of these mechanisms may lead to DNA damage that is not localized to telomeres. This is also consistent with studies in senescent cells which demonstrate higher p53 activity and p21 expression (see section 3).

Overexpression of caveolin 1 protein has been shown to cause G1 arrest, which is dependent on p21 expression induced by increased p53 activity. Mouse embryonic fibroblasts expressing caveolin-1 have a reduced proliferative lifespan and a senescent morphology (Volonte et al., 2002). The aberrant caveolin-1 levels lead to G0/G1 cell cycle arrest, activation of p53 and upregulation of p21 (Galbiati et al., 2001). This shows that caveolin-1 expression mediates premature senescence through a p53/p21 dependent pathway. This pathway is further enhanced by caveolin 1 mediated inactivation of MDM2 and PP2A-C which act as negative regulators of p53 and ATM, respectively (Bartholomew et al., 2009; Volonte et al., 2009). The MDM2 protein is an ubiquitin ligase which targets p53 for degradation and keeps p53 at basal levels in unstressed conditions. Upon activation by stress p53 is phosphorylated at residues which prevent MDM2 association and in turn stabilizes p53. It has been shown during oxidative stress induced senescence that MDM2 is neutralized by caveolin and stabilizes p53 (Volonte et al., 2009). It is thought that the activation of p53 is usually achieved by the kinase ATM, which is sensor of DNA damage (see section 3). Auto-phosphorylation of ATM occurs when it binds to regions of DNA damage and dissociates as active monomers (Bakkenist and Kastan, 2003). ATM is turned off after repair is completed by the phosphatase PP2A-C. When modeling pulmonary emphysema in murine fibroblasts, it was found that oxidative stress caused the sequestration and neutralization of PP2A-C into caveolar enriched microdomains (Bartholomew et al., 2009). Thus, there appears to be a role for CSD in caveolin for the activation of p53 at many levels. To further demonstrate a role for the CSD in caveolin, it was shown that introduction of the peptide of this domain was able to cause premature

The family of cavin proteins have recently been investigated for their potential role during senescence. Cavin-1 (PTRF) and Cavin-3 (SRBC) both appear to be elevated through protein stabilization during replicative senescence, but not quiescence (Bai et al., 2011; Cong et al., 2006) Additionally, it has been shown that cavin-1 is upregulated during oxidative stress induced premature senescence (Volonte and Galbiati, 2011). The ectopic expression of cavin-1 leads to activation of p53, a decrease in MAPK activity, and induced premature senescence. Interestingly, the cavin-1 expression also induced DNA foci that were visualized by H2AX and did not colocalize with telomeres. The knockdown of cavin-1 extended proliferative lifespan, and reversed the H2AX in high passage cells (Bai et al., 2011). Similarly, the knockdown of PTRF prevented oxidative stress from inducing senescence and was shown to prevent the MDM2:caveolin 1 interaction leading to p53 activation (Volonte and Galbiati, 2011). Cavin-1 expression is known to have a direct affect on the levels of caveolin (Hansen and Nichols, 2010) and therefore the upregulation of cavin-1 in these systems likely explains the upregulation of caveolin observed during senescence. Although these two studies focused on PTRF, much of the data can be explained by the elevation of caveolin and caveolar vesicles. What is most significant is the generation of DNA damage

senescence by itself (Volonte et al., 2009).

seen downstream of caveolar vesicles (Wheaton, 2011).

tyrosine phosphorylation of caveolin-1 which depends on p38 and Src (Volonte et al., 2001) and drives the internalization of caveolae. The same stress (H2O2) was shown to cause premature senescence in human fibroblasts, with a relocalization of caveolin to the cytoplasm and nucleus that required p38 activity (Chretien et al., 2008). Thus stress induced senescence appears to upregulate caveolin and redistribute the protein (and likely caveolar structures). This is similar to what is seen in replicative senescence where caveolin protein is found in the cytoplasm (Wheaton et al., 2001) or when cavicles accumulate (Bai et al., 2011; Park et al., 2000; Volonte and Galbiati, 2011). Thus, p38 activated by stress may drive internalization of caveolae and causes receptor tyrosine receptor signalling to be attenuated.

Senescent cells have a flattened and enlarged morphology that is characterized by an increase in focal adhesions (Cho et al., 2004) and which lead to increased amount of actin stress fibres (Wheaton et al., 1996). Caveolin has also been reported to localize to focal adhesions through integrin and likely plays a role in mitogen signalling during cell adhesion. This interaction appears to be upregulated during senescence since caveolin-1 and paxillin (a focal adhesion marker) co-localize strongly in senescent cells (Cho et al., 2004). Furthermore, when caveolin-1 is knocked down in senescent cells, there is an inhibition of focal adhesion kinase, depolarization of anchored actin and reversion of the cells to a younger morphology (Cho et al., 2004). The origin of these morphological alterations is difficult to verify since caveolin repression affects other aspects of mitogenic signalling. However, it has been shown that breaking cell adhesion contacts influences the internalization of lipid rafts within transformed cells (del Pozo et al., 2004; del Pozo et al., 2005). In addition, the clustering of integrins is known to activate Fyn in a caveolin-1 dependent manner to allow anchorage dependent growth (Wary et al., 1998). Consistent with these studies it has been shown that localization of caveolin-1 to cholesterol enriched microdomains decreases in senescent fibroblasts when they are liberated from the substratum by scraping or trypsin (Inomata et al., 2006). This implies that internalization of lipid rafts occurs in senescent cells when there detached and reflects caveolae misregulation in senescent cells. The absence of caveolae when senescent cells are establishing new focal contacts could represent another way in which mitogenic signals are blocked.

In summary, inappropriate caveolin regulation in senescent cells may contribute to the senescence associated post receptor block in three ways. First, the CSD may bind and neutralize various signalling molecules, and prevent signal propagation. Second, caveolin drives internalization of caveolae which prevents receptors access to ligands. Third, caveolin may no longer be sequestered to lipid rafts preventing the development of caveolae to allow signal integration.

#### **5.3 P53, DNA damage and caveolae**

Although the most common interpretation in the literature is that telomere attrition is the origin of DNA damage in replicative senescence, a considerable amount of damage foci (H2AX) are not localized to telomeres in senescent cells (Sedelnikova et al., 2004). The H2AX-telomere foci are also dependent on whether these cells are cultured in normoxic (2% O2) conditions (Herbig et al., 2004). Thus, the possibility exists that other forms of stress cause DNA damage in parallel with telomere erosion, such as elevated reactive oxygen species (ROS) or replication fork collapse. These forms of stress may result from the misregulation of caveolae or the upregulation of caveolin. The upregulation of caveolin

tyrosine phosphorylation of caveolin-1 which depends on p38 and Src (Volonte et al., 2001) and drives the internalization of caveolae. The same stress (H2O2) was shown to cause premature senescence in human fibroblasts, with a relocalization of caveolin to the cytoplasm and nucleus that required p38 activity (Chretien et al., 2008). Thus stress induced senescence appears to upregulate caveolin and redistribute the protein (and likely caveolar structures). This is similar to what is seen in replicative senescence where caveolin protein is found in the cytoplasm (Wheaton et al., 2001) or when cavicles accumulate (Bai et al., 2011; Park et al., 2000; Volonte and Galbiati, 2011). Thus, p38 activated by stress may drive internalization of caveolae and causes receptor tyrosine receptor signalling to be attenuated. Senescent cells have a flattened and enlarged morphology that is characterized by an increase in focal adhesions (Cho et al., 2004) and which lead to increased amount of actin stress fibres (Wheaton et al., 1996). Caveolin has also been reported to localize to focal adhesions through integrin and likely plays a role in mitogen signalling during cell adhesion. This interaction appears to be upregulated during senescence since caveolin-1 and paxillin (a focal adhesion marker) co-localize strongly in senescent cells (Cho et al., 2004). Furthermore, when caveolin-1 is knocked down in senescent cells, there is an inhibition of focal adhesion kinase, depolarization of anchored actin and reversion of the cells to a younger morphology (Cho et al., 2004). The origin of these morphological alterations is difficult to verify since caveolin repression affects other aspects of mitogenic signalling. However, it has been shown that breaking cell adhesion contacts influences the internalization of lipid rafts within transformed cells (del Pozo et al., 2004; del Pozo et al., 2005). In addition, the clustering of integrins is known to activate Fyn in a caveolin-1 dependent manner to allow anchorage dependent growth (Wary et al., 1998). Consistent with these studies it has been shown that localization of caveolin-1 to cholesterol enriched microdomains decreases in senescent fibroblasts when they are liberated from the substratum by scraping or trypsin (Inomata et al., 2006). This implies that internalization of lipid rafts occurs in senescent cells when there detached and reflects caveolae misregulation in senescent cells. The absence of caveolae when senescent cells are establishing new focal

contacts could represent another way in which mitogenic signals are blocked.

signal integration.

**5.3 P53, DNA damage and caveolae** 

In summary, inappropriate caveolin regulation in senescent cells may contribute to the senescence associated post receptor block in three ways. First, the CSD may bind and neutralize various signalling molecules, and prevent signal propagation. Second, caveolin drives internalization of caveolae which prevents receptors access to ligands. Third, caveolin may no longer be sequestered to lipid rafts preventing the development of caveolae to allow

Although the most common interpretation in the literature is that telomere attrition is the origin of DNA damage in replicative senescence, a considerable amount of damage foci (H2AX) are not localized to telomeres in senescent cells (Sedelnikova et al., 2004). The H2AX-telomere foci are also dependent on whether these cells are cultured in normoxic (2% O2) conditions (Herbig et al., 2004). Thus, the possibility exists that other forms of stress cause DNA damage in parallel with telomere erosion, such as elevated reactive oxygen species (ROS) or replication fork collapse. These forms of stress may result from the misregulation of caveolae or the upregulation of caveolin. The upregulation of caveolin leading to a increase in caveolar vesicles causes both the attenuation of the EGFR signalling (de Laurentiis et al., 2007) and an increase in the activity of the p53 tumour suppressor through elevated ROS (Volonte and Galbiati, 2009a). Both of these mechanisms may lead to DNA damage that is not localized to telomeres. This is also consistent with studies in senescent cells which demonstrate higher p53 activity and p21 expression (see section 3).

Overexpression of caveolin 1 protein has been shown to cause G1 arrest, which is dependent on p21 expression induced by increased p53 activity. Mouse embryonic fibroblasts expressing caveolin-1 have a reduced proliferative lifespan and a senescent morphology (Volonte et al., 2002). The aberrant caveolin-1 levels lead to G0/G1 cell cycle arrest, activation of p53 and upregulation of p21 (Galbiati et al., 2001). This shows that caveolin-1 expression mediates premature senescence through a p53/p21 dependent pathway. This pathway is further enhanced by caveolin 1 mediated inactivation of MDM2 and PP2A-C which act as negative regulators of p53 and ATM, respectively (Bartholomew et al., 2009; Volonte et al., 2009). The MDM2 protein is an ubiquitin ligase which targets p53 for degradation and keeps p53 at basal levels in unstressed conditions. Upon activation by stress p53 is phosphorylated at residues which prevent MDM2 association and in turn stabilizes p53. It has been shown during oxidative stress induced senescence that MDM2 is neutralized by caveolin and stabilizes p53 (Volonte et al., 2009). It is thought that the activation of p53 is usually achieved by the kinase ATM, which is sensor of DNA damage (see section 3). Auto-phosphorylation of ATM occurs when it binds to regions of DNA damage and dissociates as active monomers (Bakkenist and Kastan, 2003). ATM is turned off after repair is completed by the phosphatase PP2A-C. When modeling pulmonary emphysema in murine fibroblasts, it was found that oxidative stress caused the sequestration and neutralization of PP2A-C into caveolar enriched microdomains (Bartholomew et al., 2009). Thus, there appears to be a role for CSD in caveolin for the activation of p53 at many levels. To further demonstrate a role for the CSD in caveolin, it was shown that introduction of the peptide of this domain was able to cause premature senescence by itself (Volonte et al., 2009).

The family of cavin proteins have recently been investigated for their potential role during senescence. Cavin-1 (PTRF) and Cavin-3 (SRBC) both appear to be elevated through protein stabilization during replicative senescence, but not quiescence (Bai et al., 2011; Cong et al., 2006) Additionally, it has been shown that cavin-1 is upregulated during oxidative stress induced premature senescence (Volonte and Galbiati, 2011). The ectopic expression of cavin-1 leads to activation of p53, a decrease in MAPK activity, and induced premature senescence. Interestingly, the cavin-1 expression also induced DNA foci that were visualized by H2AX and did not colocalize with telomeres. The knockdown of cavin-1 extended proliferative lifespan, and reversed the H2AX in high passage cells (Bai et al., 2011). Similarly, the knockdown of PTRF prevented oxidative stress from inducing senescence and was shown to prevent the MDM2:caveolin 1 interaction leading to p53 activation (Volonte and Galbiati, 2011). Cavin-1 expression is known to have a direct affect on the levels of caveolin (Hansen and Nichols, 2010) and therefore the upregulation of cavin-1 in these systems likely explains the upregulation of caveolin observed during senescence. Although these two studies focused on PTRF, much of the data can be explained by the elevation of caveolin and caveolar vesicles. What is most significant is the generation of DNA damage seen downstream of caveolar vesicles (Wheaton, 2011).

The upregulation of caveolin was shown to block thioredoxin reductase I activity, and thus raise the ROS levels within fibroblasts (Volonte and Galbiati, 2009b). Elevated ROS production is well known to damage DNA, activate p53 and lead to senescence (Chen et al., 1995). Thus, DNA damage could be caused by the presence of ROS being produced in cells that over express caveolin 1 or reach replicative senescence. Furthermore, the negative regulation by caveolin 1 of many key regulatory proteins involved in the p53 mediated DNA damage response could be ensuring that damage signals that lead to a senescent

The transfer of EGFR to the nucleus is well known to influence the resolution of H2AX damage foci. As previously described (section 4), EGFR is internalized in response to gamma radiation or oxidative stress through a caveolar mediated endocytotic pathway. This pathway leads to EGFR localization to the nucleus and phosphorylates targets such as DNA-PK which facilitate the completion of DNA repair (Dittmann et al., 2005). This function depends on EGFR kinase activity and is blocked by a class of radio-sensitizing agents that work by antagonizing EGFR such as Cetuximab or Gefitinib. Such drugs enhance DNA damage and lead to apoptosis of malignant cells because of unrepaired DNA damage. The EGF signalling pathway is also upstream of the survival kinases MAPK and Akt which assist in the resolution of DNA damage (Golding et al., 2009). Additionally, EGFR activity was found to modulate non-homologous end joining after gamma radiation (Kriegs et al., 2010). Taken together, EGFR signalling plays a major role in controlling cell cycle arrest in response to cellular stress and DNA damage. In support of the role of caveolae in this model, EGFR was found to transport to the nucleus in a caveolin and Src dependent mechanism after oxidative stress (Khan et al., 2006). Similarly, gamma radiation causes Src induced association between caveolin-1 and EGFR leading to internalization. This leads to nuclear localization and to control of DNA-PK activity (Dittmann et al., 2008). Caveolin-1 expression has been demonstrated to be up regulated by ionizing radiation and is required for both homologous recombination and non-homologous end joining (Zhu et al., 2010). In this case, caveolin-1 is pivotal in forming the caveolar vesicles that allow transport of the EGFR, and nuclear DNA-PK activation. Lastly, Gefitinib (an EGFR kinase inhibitor) can generate premature senescence in non-small lung cancer cells (Hotta et al., 2007). Thus, the current evidence strongly suggests mitogenic signals and EGFR internalization to the nucleus are required to resolve DNA damage. Furthermore, blocked

Although primary fibroblasts are genetically stable, they do undergo transient DNA damage foci as a result of mitogenic stimulation (Ichijima et al., 2005; McManus and Hendzel, 2005). These H2AX foci occur as a natural part of the synthesis of DNA or division, and are resolved by the time the cell returns to G0 of the cell cycle. Thus, it is possible that a signal from the EGFR could assist cycling cells to resolve this DNA damage. Therefore, EGFR would promote both the initiation of the cell cycle and its continued signalling would be required to resolve DNA damage arising from progress through the cell cycle. However, in senescent cells, the up regulation of caveolin antagonizes EGFR signalling (see section 5.2). Thus, the attenuation of the EGFR pathway during senescence could perpetuate the normally transient DNA damage foci in fibroblasts. The blocking of EGF signalling in this case would prevent the resolution of damage induced by replication stress during normal growth. This could explain the DNA damage seen downstream of

increased caveolar vesicles during aging as a form of replication stress (Fig. 6A & B).

outcome are reinforced.

EGFR activity leads to apoptosis or senescence.

Fig. 6 Model for Cavicle Induced Senescence.

**A)** In young fibroblasts mitogenic signalling is achieved through a caveolae localized EGFR cascade that activates transcription of growth promoting genes. Signalling competent caveolae are capable of internalizing and traffic to the endoplasmic reticulum (ER) in response to cellular stress. EGFR is transported via cavicles to the ER followed by localization to the nucleus where it can activate DNA protein kinase (DNA-PK ). DNA-PK is required to repair DNA damage that is generated by stress or during replication. **B)** In senescence fibroblasts mitogenic signalling occurs through EGFR, but is unable to initiate DNA replication. The majority of caveolae are internalized as cavicles, and the increased caveolin protein levels inactivate many signalling molecules or negative regulators of the p53 tumour suppressor (MDM2 and PP2A-C). In theory the EGFR is no longer able to localize to the nucleus and therefore DNA damage generated by reactive oxygen species (ROS), replication stress, and cellular aging cannot be repaired. The activation of p53 through DNA damage causes cellular senescence.

**Caveolae**

DNAPK

TrxR EGFR MDM2 PP2A

**Nucleus**

p53

**A)** In young fibroblasts mitogenic signalling is achieved through a caveolae localized EGFR cascade that activates transcription of growth promoting genes. Signalling competent caveolae are capable of internalizing and traffic to the endoplasmic reticulum (ER) in response to cellular stress. EGFR is transported via cavicles to the ER followed by localization to the nucleus where it can activate DNA protein kinase (DNA-PK ).

DNA-PK is required to repair DNA damage that is generated by stress or during replication. **B)** In senescence fibroblasts mitogenic signalling occurs through EGFR, but is unable to initiate DNA replication. The majority of caveolae are internalized as cavicles, and the increased caveolin protein levels inactivate many signalling molecules or negative regulators of the p53 tumour suppressor (MDM2 and PP2A-C). In theory the EGFR is no longer able to localize to the nucleus and therefore DNA damage generated by reactive oxygen species (ROS), replication stress, and cellular aging cannot be repaired. The

**ER**

**ER**

**Cavicles**

Senescent Fibroblast

**Cavicle**

Young Fibroblast

A EGF

EGFR

Mitogenic Signal

B

DNA Replication

H2AX

Signal DNAPK

ROS

Replication Stress

activation of p53 through DNA damage causes cellular senescence.

Mitogenic

Fig. 6 Model for Cavicle Induced Senescence.

The upregulation of caveolin was shown to block thioredoxin reductase I activity, and thus raise the ROS levels within fibroblasts (Volonte and Galbiati, 2009b). Elevated ROS production is well known to damage DNA, activate p53 and lead to senescence (Chen et al., 1995). Thus, DNA damage could be caused by the presence of ROS being produced in cells that over express caveolin 1 or reach replicative senescence. Furthermore, the negative regulation by caveolin 1 of many key regulatory proteins involved in the p53 mediated DNA damage response could be ensuring that damage signals that lead to a senescent outcome are reinforced.

The transfer of EGFR to the nucleus is well known to influence the resolution of H2AX damage foci. As previously described (section 4), EGFR is internalized in response to gamma radiation or oxidative stress through a caveolar mediated endocytotic pathway. This pathway leads to EGFR localization to the nucleus and phosphorylates targets such as DNA-PK which facilitate the completion of DNA repair (Dittmann et al., 2005). This function depends on EGFR kinase activity and is blocked by a class of radio-sensitizing agents that work by antagonizing EGFR such as Cetuximab or Gefitinib. Such drugs enhance DNA damage and lead to apoptosis of malignant cells because of unrepaired DNA damage. The EGF signalling pathway is also upstream of the survival kinases MAPK and Akt which assist in the resolution of DNA damage (Golding et al., 2009). Additionally, EGFR activity was found to modulate non-homologous end joining after gamma radiation (Kriegs et al., 2010). Taken together, EGFR signalling plays a major role in controlling cell cycle arrest in response to cellular stress and DNA damage. In support of the role of caveolae in this model, EGFR was found to transport to the nucleus in a caveolin and Src dependent mechanism after oxidative stress (Khan et al., 2006). Similarly, gamma radiation causes Src induced association between caveolin-1 and EGFR leading to internalization. This leads to nuclear localization and to control of DNA-PK activity (Dittmann et al., 2008). Caveolin-1 expression has been demonstrated to be up regulated by ionizing radiation and is required for both homologous recombination and non-homologous end joining (Zhu et al., 2010). In this case, caveolin-1 is pivotal in forming the caveolar vesicles that allow transport of the EGFR, and nuclear DNA-PK activation. Lastly, Gefitinib (an EGFR kinase inhibitor) can generate premature senescence in non-small lung cancer cells (Hotta et al., 2007). Thus, the current evidence strongly suggests mitogenic signals and EGFR internalization to the nucleus are required to resolve DNA damage. Furthermore, blocked EGFR activity leads to apoptosis or senescence.

Although primary fibroblasts are genetically stable, they do undergo transient DNA damage foci as a result of mitogenic stimulation (Ichijima et al., 2005; McManus and Hendzel, 2005). These H2AX foci occur as a natural part of the synthesis of DNA or division, and are resolved by the time the cell returns to G0 of the cell cycle. Thus, it is possible that a signal from the EGFR could assist cycling cells to resolve this DNA damage. Therefore, EGFR would promote both the initiation of the cell cycle and its continued signalling would be required to resolve DNA damage arising from progress through the cell cycle. However, in senescent cells, the up regulation of caveolin antagonizes EGFR signalling (see section 5.2). Thus, the attenuation of the EGFR pathway during senescence could perpetuate the normally transient DNA damage foci in fibroblasts. The blocking of EGF signalling in this case would prevent the resolution of damage induced by replication stress during normal growth. This could explain the DNA damage seen downstream of increased caveolar vesicles during aging as a form of replication stress (Fig. 6A & B).

Ashton, A.W., Watanabe, G., Albanese, C., Harrington, E.O., Ware, J.A., and Pestell, R.G.

Atadja, P.W., Stringer, K.F., and Riabowol, K.T. (1994). Loss of serum response element-

Bai, L., Deng, X., Li, J., Wang, M., Li, Q., An, W., A, D., and Cong, Y.S. (2011). Regulation of

Bakkenist, C.J., and Kastan, M.B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature *421*, 499-506. Bartholomew, J.N., Volonte, D., and Galbiati, F. (2009). Caveolin-1 regulates the antagonistic

Basak, S., Jacobs, S.B., Krieg, A.J., Pathak, N., Zeng, Q., Kaldis, P., Giaccia, A.J., and Attardi,

Bastiani, M., Liu, L., Hill, M.M., Jedrychowski, M.P., Nixon, S.J., Lo, H.P., Abankwa, D.,

Basu, A., and Pal, D. (2010). Two faces of protein kinase Cdelta: the contrasting roles of PKCdelta in cell survival and cell death. ScientificWorldJournal *10*, 2272-2284. Beausejour, C.M., Krtolica, A., Galimi, F., Narita, M., Lowe, S.W., Yaswen, P., and Campisi,

Binet, R., Ythier, D., Robles, A.I., Collado, M., Larrieu, D., Fonti, C., Brambilla, E., Brambilla,

Blake, R.A., Garcia-Paramio, P., Parker, P.J., and Courtneidge, S.A. (1999). Src promotes

Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B.,

Boguski, M.S., and McCormick, F. (1993). Proteins regulating Ras and its relatives. Nature

Bose, C., Bhuvaneswaran, C., and Udupa, K.B. (2004). Altered mitogen-activated protein

(1999). Protein kinase Cdelta inhibition of S-phase transition in capillary endothelial cells involves the cyclin-dependent kinase inhibitor p27(Kip1). J Biol

binding activity and hyperphosphorylation of serum response factor during

cellular senescence by the essential caveolar component PTRF/Cavin-1. Cell Res *21*,

pleiotropic properties of cellular senescence through a novel Mdm2/p53-mediated

L.D. (2008). The metastasis-associated gene Prl-3 is a p53 target involved in cell-

Luetterforst, R., Fernandez-Rojo, M., Breen, M.R.*, et al.* (2009). MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes. J Cell Biol *185*, 1259-

J. (2003). Reversal of human cellular senescence: roles of the p53 and p16 pathways.

C., Serrano, M., Harris, C.C.*, et al.* (2009). WNT16B is a new marker of cellular senescence that regulates p53 activity and the phosphoinositide 3-kinase/AKT

Shay, J.W., Lichtsteiner, S., and Wright, W.E. (1998). Extension of life-span by introduction of telomerase into normal human cells [see comments]. Science *279*,

kinase signal transduction in human skin fibroblasts during in vitro aging:

Anderson, R.G. (1998). The caveolae membrane system. Annu Rev Biochem *67*, 199-225. Anderson, R.G., Kamen, B.A., Rothberg, K.G., and Lacey, S.W. (1992). Potocytosis: sequestration and transport of small molecules by caveolae. Science *255*, 410-411. Appella, E., and Anderson, C.W. (2001). Post-translational modifications and activation of

p53 by genotoxic stresses. Eur J Biochem *268*, 2764-2772.

cellular aging. Mol Cell Biol *14*, 4991-4999.

pathway. Cancer Res *69*, 2878-2886.

cycle regulation. Mol Cell *30*, 303-314.

pathway. Cancer Res *69*, 9183-9191.

PKCdelta degradation. Cell Growth Differ *10*, 231-241.

**8. References** 

Chem *274*, 20805-20811.

1088-1101.

1273.

349-352.

*366*, 643-654.

Embo J *22*, 4212-4222.

#### **6. Conclusion**

The inhibition or reduction of components of mitogenic signaling cascades in senescent cell caveolar fractions is consistent with the loss of caveolae playing a causal role in the blunted growth response seen during cellular senescence. Collectively, these observations suggest that localization and integration of signalling cascades in caveolae are disrupted in senescent cells. The mitogenic pathways leading to MAPK, PLD, PKC isoforms and PI3K are all localized to caveolae and have been shown to have deficiencies in senescent cells. In the case of EGFR, the proper coordination with shc is disrupted, the CSD of caveolin blocks kinase activity, and inappropriate retention or internalization in caveolae occurs. It is unclear whether this difference influences the downstream localization of activated erk1/2 to the senescent nucleus, however several groups have noted a decreased erk stimulation by EGF with age. Evidence suggests that the composition of lipid rafts likely change in senescent cells and that this may influence how PI3K, PLD and PKC are regulated. Cholesterol levels are decreased in lipid raft fractions, PC levels increase in the plasma membrane, and ceramide levels increase with fibroblast age. These observations suggest an increase in fluidity of the membrane, which changes the properties of lipid rafts. The increases in ceramide disrupt PLD, and the increase in DAG constitutively activates PKC in caveolae. The decrease in src kinase activity in caveolae may prevent the inactivation of the PKC and induce senescence. The loss of PI3K signal integration through caveolae may be a part of the induction of senescence, explaining why inhibitors of the pathway induce premature senescence. These examples indicate that signal transduction pathways rely upon caveolae for signal integration and propagation that they become disorganized in senescent cells. Collectively, these signalling changes may explain their reduced or absent response to mitogens in senescent cells.

There has been a shift in the understanding of how the DNA damage occurring in senescence is generated. Remarkably, the increase of caveolar vesicles observed in the senescent state can itself lead to the generation of DNA damage foci in parallel with the well known DNA damage localized to eroded telomeres. The exact mechanism by which this is achieved is still unknown, but likely involves the strong inhibitory activities of the scaffolding protein caveolin 1. The proteins theoridoxin, MDM2 and PP2A-C are all inhibited or sequestered by increases in caveolin protein during senescence. Inhibition of theoridoxin leads to an increase in ROS, which leads to DNA damage and p53 activation. The activation of p53 in turn is augmented by inhibition of the negative regulators of p53, MDM2 and PP2A-C by caveolin. The DNA damage that leads to p53 mediated senescence may also be generated through failure to resolve H2AX foci that occur during normal replication. The increase of caveolin and cavin-1 proteins shift the balance of caveolar structures to internalized cavicles. These cavicles are no longer capable of integrating the EGFR cascade, which is required to transfer EGFR to the nucleus and resolve replication dependent DNA damage through proteins such as DNA-PK. The failure to repair DNA leads to replication stress, the activation of p53 and thus cellular senescence (Fig. 6A & B). Thus, the misregulation of caveolar vesicles interferes with the essential role of these structures in repairing DNA damage.

## **7. Acknowledgements**

I would like to thank Dr. S. Robbins and Dr. Kari Sampsel (University of Calgary) for use of the electron microscopy images. I would also like to thank Densie Campuzano for editing this manuscript.

#### **8. References**

486 Senescence

The inhibition or reduction of components of mitogenic signaling cascades in senescent cell caveolar fractions is consistent with the loss of caveolae playing a causal role in the blunted growth response seen during cellular senescence. Collectively, these observations suggest that localization and integration of signalling cascades in caveolae are disrupted in senescent cells. The mitogenic pathways leading to MAPK, PLD, PKC isoforms and PI3K are all localized to caveolae and have been shown to have deficiencies in senescent cells. In the case of EGFR, the proper coordination with shc is disrupted, the CSD of caveolin blocks kinase activity, and inappropriate retention or internalization in caveolae occurs. It is unclear whether this difference influences the downstream localization of activated erk1/2 to the senescent nucleus, however several groups have noted a decreased erk stimulation by EGF with age. Evidence suggests that the composition of lipid rafts likely change in senescent cells and that this may influence how PI3K, PLD and PKC are regulated. Cholesterol levels are decreased in lipid raft fractions, PC levels increase in the plasma membrane, and ceramide levels increase with fibroblast age. These observations suggest an increase in fluidity of the membrane, which changes the properties of lipid rafts. The increases in ceramide disrupt PLD, and the increase in DAG constitutively activates PKC in caveolae. The decrease in src kinase activity in caveolae may prevent the inactivation of the PKC and induce senescence. The loss of PI3K signal integration through caveolae may be a part of the induction of senescence, explaining why inhibitors of the pathway induce premature senescence. These examples indicate that signal transduction pathways rely upon caveolae for signal integration and propagation that they become disorganized in senescent cells. Collectively, these signalling changes may

explain their reduced or absent response to mitogens in senescent cells.

structures in repairing DNA damage.

**7. Acknowledgements** 

this manuscript.

There has been a shift in the understanding of how the DNA damage occurring in senescence is generated. Remarkably, the increase of caveolar vesicles observed in the senescent state can itself lead to the generation of DNA damage foci in parallel with the well known DNA damage localized to eroded telomeres. The exact mechanism by which this is achieved is still unknown, but likely involves the strong inhibitory activities of the scaffolding protein caveolin 1. The proteins theoridoxin, MDM2 and PP2A-C are all inhibited or sequestered by increases in caveolin protein during senescence. Inhibition of theoridoxin leads to an increase in ROS, which leads to DNA damage and p53 activation. The activation of p53 in turn is augmented by inhibition of the negative regulators of p53, MDM2 and PP2A-C by caveolin. The DNA damage that leads to p53 mediated senescence may also be generated through failure to resolve H2AX foci that occur during normal replication. The increase of caveolin and cavin-1 proteins shift the balance of caveolar structures to internalized cavicles. These cavicles are no longer capable of integrating the EGFR cascade, which is required to transfer EGFR to the nucleus and resolve replication dependent DNA damage through proteins such as DNA-PK. The failure to repair DNA leads to replication stress, the activation of p53 and thus cellular senescence (Fig. 6A & B). Thus, the misregulation of caveolar vesicles interferes with the essential role of these

I would like to thank Dr. S. Robbins and Dr. Kari Sampsel (University of Calgary) for use of the electron microscopy images. I would also like to thank Densie Campuzano for editing

**6. Conclusion** 

Anderson, R.G. (1998). The caveolae membrane system. Annu Rev Biochem *67*, 199-225.


Cong, Y.S., Fan, E., and Wang, E. (2006). Simultaneous proteomic profiling of four different

Couet, J., Li, S., Okamoto, T., Ikezu, T., and Lisanti, M.P. (1997). Identification of peptide and

of caveolin with caveolae-associated proteins. J Biol Chem *272*, 6525-6533. de Lange, T. (1994). Activation of telomerase in a human tumor. Proc Natl Acad Sci U S A

de Laurentiis, A., Donovan, L., and Arcaro, A. (2007). Lipid rafts and caveolae in signaling

De Tata, V., Ptasznik, A., and Cristofalo, V.J. (1993). Effect of the tumor promoter phorbol

Debacq-Chainiaux, F., Boilan, E., Dedessus Le Moutier, J., Weemaels, G., and Toussaint, O.

del Pozo, M.A., Alderson, N.B., Kiosses, W.B., Chiang, H.H., Anderson, R.G., and Schwartz,

del Pozo, M.A., Balasubramanian, N., Alderson, N.B., Kiosses, W.B., Grande-Garcia, A.,

Di Leonardo, A., Linke, S.P., Clarkin, K., and Wahl, G.M. (1994). DNA damage triggers a

Dietzen, D.J., Hastings, W.R., and Lublin, D.M. (1995). Caveolin is palmitoylated on multiple

Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens,

Dittmann, K., Mayer, C., Fehrenbacher, B., Schaller, M., Raju, U., Milas, L., Chen, D.J.,

Dittmann, K., Mayer, C., Kehlbach, R., and Rodemann, H.P. (2008). Radiation-induced

Dittmann, K., Mayer, C., and Rodemann, H.P. (2009). Nuclear EGFR as novel therapeutic

Downward, J., Waterfield, M.D., and Parker, P.J. (1985). Autophosphorylation and protein

regulated membrane domain internalization. Nat Cell Biol *7*, 901-908. Dempsey, E.C., Newton, A.C., Mochly-Rosen, D., Fields, A.P., Reyland, M.E., Insel, P.A.,

cell responses. Am J Physiol Lung Cell Mol Physiol *279*, L429-438.

and tandem mass spectrometry. Mech Ageing Dev *127*, 332-343.

by growth factor receptors. Open Biochem J *1*, 12-32.

human diploid fibroblasts. Exp Cell Res *205*, 261-269.

*91*, 2882-2885.

Med Biol *694*, 126-137.

domains. Science *303*, 839-842.

human fibroblasts. Genes Dev *8*, 2540-2551.

caveolae. J Biol Chem *270*, 6838-6842.

kinase. J Biol Chem *280*, 31182-31189.

and activation of DNA-PK. Mol Cancer *7*, 69.

9367.

growth states of human fibroblasts, using amine-reactive isobaric tagging reagents

protein ligands for the caveolin-scaffolding domain. Implications for the interaction

12-myristate 13-acetate (PMA) on proliferation of young and senescent WI-38

(2010). p38(MAPK) in the senescence of human and murine fibroblasts. Adv Exp

M.A. (2004). Integrins regulate Rac targeting by internalization of membrane

Anderson, R.G., and Schwartz, M.A. (2005). Phospho-caveolin-1 mediates integrin-

and Messing, R.O. (2000). Protein kinase C isozymes and the regulation of diverse

prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal

cysteine residues. Palmitoylation is not necessary for localization of caveolin to

M., Rubelj, I., Pereira-Smith, O.*, et al.* (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A *92*, 9363-

Kehlbach, R., and Rodemann, H.P. (2005). Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein

caveolin-1 associated EGFR internalization is linked with nuclear EGFR transport

target: insights into nuclear translocation and function. Strahlenther Onkol *186*, 1-6.

kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. J Biol Chem *260*, 14538-14546.

differential expression of low- density lipoprotein receptor. J Gerontol A Biol Sci Med Sci *59*, 126-135.


Boucrot, E., Howes, M.T., Kirchhausen, T., and Parton, R.G. (2011). Redistribution of

Brown, J.P., Wei, W., and Sedivy, J.M. (1997). Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science *277*, 831-834. Brown, W.T. (1990). Genetic diseases of premature aging as models of senescence. Annu Rev

Caino, M.C., Meshki, J., and Kazanietz, M.G. (2009). Hallmarks for senescence in

Cao, K., Graziotto, J.J., Blair, C.D., Mazzulli, J.R., Erdos, M.R., Krainc, D., and Collins, F.S.

Carlin, C., Phillips, P.D., Brooks-Frederich, K., Knowles, B.B., and Cristofalo, V.J. (1994).

Chang, Z.F., and Huang, D.Y. (1994). Decline of protein kinase C activation in response to

Chen, Q., and Ames, B.N. (1994). Senescence-like growth arrest induced by hydrogen

Chen, Q., Fischer, A., Reagan, J.D., Yan, L.J., and Ames, B.N. (1995). Oxidative DNA damage

Cho, K.A., Ryu, S.J., Oh, Y.S., Park, J.H., Lee, J.W., Kim, H.P., Kim, K.T., Jang, I.S., and Park,

Cho, K.A., Ryu, S.J., Park, J.S., Jang, I.S., Ahn, J.S., Kim, K.T., and Park, S.C. (2003). Senescent

Chretien, A., Piront, N., Delaive, E., Demazy, C., Ninane, N., and Toussaint, O. (2008).

Chua, C.C., Geiman, D.E., and Ladda, R.L. (1986). Receptor for epidermal growth factor retains normal structure and function in aging cells. Mech Ageing Dev *34*, 35-55. Cohen, A.W., Hnasko, R., Schubert, W., and Lisanti, M.P. (2004). Role of caveolae and

Collado, M., Medema, R.H., Garcia-Cao, I., Dubuisson, M.L., Barradas, M., Glassford, J.,

Collado, M., and Serrano, M. (2010). Senescence in tumours: evidence from mice and

(2011). Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells. Sci Transl Med *3*, 89ra58.

Cleavage of the epidermal growth factor receptor by a membrane-bound leupeptinsensitive protease active in nonionic detergent lysates of senescent but not young

growth stimulation during senescence of IMR-90 human diploid fibroblasts.

peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci U S A *91*, 4130-

and senescence of human diploid fibroblast cells. Proc Natl Acad Sci U S A *92*,

S.C. (2004). Morphological adjustment of senescent cells by modulating caveolin-1

phenotype can be reversed by reduction of caveolin status. J Biol Chem *278*, 27789-

Increased abundance of cytoplasmic and nuclear caveolin 1 in human diploid fibroblasts in H(2)O(2)- induced premature senescence and interplay with

Rivas, C., Burgering, B.M., Serrano, M., and Lam, E.W. (2000). Inhibition of the phosphoinositide 3- kinase pathway induces a senescence-like arrest mediated by

carcinogenesis: novel signaling players. Apoptosis *14*, 392-408.

human diploid fibroblasts. J Cell Physiol *160*, 427-434.

Biochem Biophys Res Commun *200*, 16-27.

status. J Biol Chem *279*, 42270-42278.

p38alpha(MAPK). FEBS Lett *582*, 1685- 1692.

p27Kip1. J Biol Chem *275*, 21960-21968.

humans. Nat Rev Cancer *10*, 51-57.

caveolins in health and disease. Physiol Rev *84*, 1341-1379.

caveolae during mitosis. J Cell Sci *124*, 1965-1972.

Med Sci *59*, 126-135.

Gerontol Geriatr *10*, 23-42.

4134.

4337-4341.

27795.

differential expression of low- density lipoprotein receptor. J Gerontol A Biol Sci


Hanada, K., Nishijima, M., Akamatsu, Y., and Pagano, R.E. (1995). Both sphingolipids and

Hansen, C.G., Bright, N.A., Howard, G., and Nichols, B.J. (2009). SDPR induces membrane curvature and functions in the formation of caveolae. Nat Cell Biol *11*, 807-814. Hansen, C.G., and Nichols, B.J. (2010). Exploring the caves: cavins, caveolins and caveolae.

Hara, E., Tsurui, H., Shinozaki, A., Nakada, S., and Oda, K. (1991). Cooperative effect of

Harley, C.B., Futcher, A.B., and Greider, C.W. (1990). Telomeres shorten during ageing of

Hayer, A., Stoeber, M., Bissig, C., and Helenius, A. (2010). Biogenesis of caveolae: stepwise assembly of large caveolin and cavin complexes. Traffic *11*, 361-382. Hayflick, L. (1965a). The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell

Hayflick, L. (1965b). The limited in vitro lifetime of human diploid cell strians. Exp Cell Res

Hayflick, L., and Moorhead, P.S. (1961). The serial cultivation of human diploid cel strains.

Herbig, U., Jobling, W.A., Chen, B.P., Chen, D.J., and Sedivy, J.M. (2004). Telomere

Hermeking, H., Lengauer, C., Polyak, K., He, T.C., Zhang, L., Thiagalingam, S., Kinzler,

Hill, M.M., Bastiani, M., Luetterforst, R., Kirkham, M., Kirkham, A., Nixon, S.J., Walser, P.,

Hotta, K., Tabata, M., Kiura, K., Kozuki, T., Hisamoto, A., Katayama, H., Takigawa, N.,

Hutter, D., Yo, Y., Chen, W., Liu, P., Holbrook, N.J., Roth, G.S., and Liu, Y. (2000). Age-

Ichijima, Y., Sakasai, R., Okita, N., Asahina, K., Mizutani, S., and Teraoka, H. (2005).

Inomata, M., Shimada, Y., Hayashi, M., Kondo, H., and Ohno-Iwashita, Y. (2006).

response. Biochem Biophys Res Commun *336*, 807-812.

Biochem Biophys Res Commun *343*, 489-495.

p53, and p21(CIP1), but not p16(INK4a). Mol Cell *14*, 501-513.

shortening triggers senescence of human cells through a pathway involving ATM,

K.W., and Vogelstein, B. (1997). 14-3-3 sigma is a p53-regulated inhibitor of G2/M

Abankwa, D., Oorschot, V.M., Martin, S.*, et al.* (2008). PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell *132*, 113-124.

Fujimoto, N., Fujiwara, K., Ueoka, H.*, et al.* (2007). Gefitinib induces premature senescence in non-small cell lung cancer cells with or without EGFR gene mutation.

related decline in Ras/ERK mitogen-activated protein kinase cascade is linked to a reduced association between Shc and EGF receptor. J Gerontol A Biol Sci Med Sci

Phosphorylation of histone H2AX at M phase in human cells without DNA damage

Detachment- associated changes in lipid rafts of senescent human fibroblasts.

diploid fibroblasts, TIG-1. Biochem Biophys Res Commun *179*, 528-534. Harder, T., and Simons, K. (1997). Caveolae, DIGs, and the dynamics of sphingolipid-

cholesterol microdomains. Curr Opin Cell Biol *9*, 534-542.

*13*, 3976-3988.

Chem *270*, 6254-6260.

Res *37*, 614- 636.

Exp Cell Res *25*, 585-621.

progression. Mol Cell *1*, 3-11.

Oncol Rep *17*, 313-317.

*55*, B125-134.

*37*, 614-636.

Trends Cell Biol *20*, 177-186.

human fibroblasts. Nature *345*, 458-460.

palmitoylation: implications for epidermal growth factor signaling. Mol Biol Cell

cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes. J Biol

antisense-Rb and antisense-p53 oligomers on the extension of life span in human


el-Deiry, W.S., Tokino, T., Velculescu, V.E., Levy, D.B., Parsons, R., Trent, J.M., Lin, D.,

Engelman, J.A., Chu, C., Lin, A., Jo, H., Ikezu, T., Okamoto, T., Kohtz, D.S., and Lisanti, M.P.

Exton, J.H. (1997). New developments in phospholipase D. J Biol Chem *272*, 15579-15582.

Exton, J.H. (1999). Regulation of phospholipase D. Biochim Biophys Acta *1439*, 121-133. Fantl, W.J., Johnson, D.E., and Williams, L.T. (1993). Signalling by receptor tyrosine kinases.

Fielding, C.J., and Fielding, P.E. (2004). Membrane cholesterol and the regulation of signal

Foster, D.A., and Xu, L. (2003). Phospholipase D in cell proliferation and cancer. Mol Cancer

Fujimoto, T., Kogo, H., Nomura, R., and Une, T. (2000). Isoforms of caveolin-1 and caveolar

Galbiati, F., Volonte, D., Liu, J., Capozza, F., Frank, P.G., Zhu, L., Pestell, R.G., and Lisanti,

Gerhard, G.S., Phillips, P.D., and Cristofalo, V.J. (1991). EGF- and PDGF-stimulated phosphorylation in young and senescent WI-38 cells. Exp Cell Res *193*, 87-92. Ghosh, S., Strum, J.C., Sciorra, V.A., Daniel, L., and Bell, R.M. (1996). Raf-1 kinase possesses

Gidwani, A., Brown, H.A., Holowka, D., and Baird, B. (2003). Disruption of lipid order by

Gire, V., and Wynford-Thomas, D. (1998). Reinitiation of DNA synthesis and cell division in

Glenney, J.R., Jr. (1992). The sequence of human caveolin reveals identity with VIP21, a

Golding, S.E., Morgan, R.N., Adams, B.R., Hawkins, A.J., Povirk, L.F., and Valerie, K. (2009).

Goldstein, S., and Singal, D.P. (1974). Senescence of cultured human fibroblasts: mitotic

Guertin, D.A., and Sabatini, D.M. (2007). Defining the role of mTOR in cancer. Cancer Cell

Han, J.M., Kim, Y., Lee, J.S., Lee, C.S., Lee, B.D., Ohba, M., Kuroki, T., Suh, P.G., and Ryu,

S.H. (2002). Localization of phospholipase D1 to caveolin-enriched membrane via

downstream signaling by FcepsilonRI. J Cell Sci *116*, 3177-3187.

component of transport vesicles. FEBS Lett *314*, 45-48.

versus metabolic time. Exp Cell Res *88*, 359-364.

Gschwendt, M. (1999). Protein kinase C delta. Eur J Biochem *259*, 555-564.

M.P. (2001). Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanism. Mol

distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-Otetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells. J

short-chain ceramides correlates with inhibition of phospholipase D and

senescent human fibroblasts by microinjection of anti-p53 antibodies. Mol Cell Biol

Pro- survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double- strand break repair in human glioma cells. Cancer Biol Ther *8*, 730-

Exton, J.H. (1998). Phospholipase D. Biochim Biophys Acta *1436*, 105-115.

of p53 tumor suppression. Cell *75*, 817-825.

transduction. Biochem Soc Trans *32*, 65-69.

structure. J Cell Sci *113 Pt 19*, 3509-3517.

Annu Rev Biochem *62*, 453-481.

Res *1*, 789- 800.

Biol Cell *12*, 2229-2244.

Biol Chem *271*, 8472-8480.

*18*, 1611-1621.

738.

*12*, 9-22.

Mercer, W.E., Kinzler, K.W., and Vogelstein, B. (1993). WAF1, a potential mediator

(1998). Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett *428*, 205-211.

palmitoylation: implications for epidermal growth factor signaling. Mol Biol Cell *13*, 3976-3988.


Li, G.Z., Eller, M.S., Firoozabadi, R., and Gilchrest, B.A. (2003). Evidence that exposure of the

Li, G.Z., Eller, M.S., Hanna, K., and Gilchrest, B.A. (2004). Signaling pathway requirements

Li, W.P., Liu, P., Pilcher, B.K., and Anderson, R.G. (2001). Cell-specific targeting of caveolin-

Lim, I.K., Won Hong, K., Kwak, I.H., Yoon, G., and Park, S.C. (2000). Cytoplasmic retention

Lin, A.W., Barradas, M., Stone, J.C., van Aelst, L., Serrano, M., and Lowe, S.W. (1998).

Liu, L., and Pilch, P.F. (2008). A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization. J Biol Chem *283*, 4314-4322. Lorenzini, A., Tresini, M., Mawal-Dewan, M., Frisoni, L., Zhang, H., Allen, R.G., Sell, C., and

Madshus, I.H., and Stang, E. (2009). Internalization and intracellular sorting of the EGF

Martin, G.M., Sprague, C.A., and Epstein, C.J. (1970). Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype. Lab Invest *23*, 86-92. Matuoka, K., Chen, K.Y., and Takenawa, T. (2003). A positive role of phosphatidylinositol 3-

McDermott, M., Wakelam, M.J., and Morris, A.J. (2004). Phospholipase D. Biochem Cell Biol

McMahon, K.A., Zajicek, H., Li, W.P., Peyton, M.J., Minna, J.D., Hernandez, V.J., Luby-

McManus, K.J., and Hendzel, M.J. (2005). ATM-dependent DNA damage-independent

Meyyappan, M., Wheaton, K., and Riabowol, K.T. (1999). Decreased expression and activity

Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P.P., Lanfrancone, L.,

Mineo, C., Gill, G.N., and Anderson, R.G. (1999). Regulated migration of epidermal growth

that regulates caveolae function. Embo J *28*, 1001-1015.

response and life span in mammals. Nature *402*, 309-313.

factor receptor from caveolae. J Biol Chem *274*, 30636-30643.

constitutive MEK/MAPK mitogenic signaling. Genes Dev *12*, 3008-3019. Lisanti, M.P., Tang, Z.L., and Sargiacomo, M. (1993). Caveolin forms a hetero-oligomeric

in human diploid fibroblasts. Mech Ageing Dev *119*, 113-130.

pathways in fibroblast senescence. Exp Gerontol *37*, 1149-1156.

the biogenesis of caveolae. J Cell Biol *123*, 595-604.

527-531.

1408.

*301*, 189-200.

Sci *122*, 3433-3439.

*82*, 225-253.

Cell *16*, 5013-5025.

Gerontol Geriatr *36*, 203-219.

senescence. J Cell Physiol *179*, 29-39.

telomere 3' overhang sequence induces senescence. Proc Natl Acad Sci U S A *100*,

for induction of senescence by telomere homolog oligonucleotides. Exp Cell Res

1 to caveolae, secretory vesicles, cytoplasm or mitochondria. J Cell Sci *114*, 1397-

of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence

Premature senescence involving p53 and p16 is activated in response to

protein complex that interacts with an apical GPI-linked protein: implications for

Cristofalo, V.J. (2002). Role of the Raf/MEK/ERK and the PI3K/Akt(PKB)

receptor: a model for understanding the mechanisms of receptor trafficking. J Cell

kinase in aging phenotype expression in cultured human diploid fibroblasts. Arch

Phelps, K., and Anderson, R.G. (2009). SRBC/cavin-3 is a caveolin adapter protein

mitotic phosphorylation of H2AX in normally growing mammalian cells. Mol Biol

of the immediate-early growth response (Egr-1) gene product during cellular

and Pelicci, P.G. (1999). The p66shc adaptor protein controls oxidative stress


Jackson, J.G., and Pereira-Smith, O.M. (2006). p53 is preferentially recruited to the promoters

Jang, I.H., Kim, J.H., Lee, B.D., Bae, S.S., Park, M.H., Suh, P.G., and Ryu, S.H. (2001).

Johnson, C.L., Lu, D., Huang, J., and Basu, A. (2002). Regulation of p53 stabilization by DNA

Karlseder, J., Smogorzewska, A., and de Lange, T. (2002). Senescence induced by altered

Kastan, M.B., Zhan, Q., el-Deiry, W.S., Carrier, F., Jacks, T., Walsh, W.V., Plunkett, B.S.,

Katakura, Y., Udono, M., Katsuki, K., Nishide, H., Tabira, Y., Ikei, T., Yamashita, M., Fujiki,

senescence programs of human normal diploid cells. J Biochem *146*, 87-93. Keilhack, H., Tenev, T., Nyakatura, E., Godovac-Zimmermann, J., Nielsen, L., Seedorf, K.,

Khan, E.M., Heidinger, J.M., Levy, M., Lisanti, M.P., Ravid, T., and Goldkorn, T. (2006).

caveolin-1-dependent perinuclear trafficking. J Biol Chem *281*, 14486-14493. Kim, H.S., and Lim, I.K. (2009). Phosphorylated extracellular signal-regulated protein

Kortlever, R.M., Higgins, P.J., and Bernards, R. (2006). Plasminogen activator inhibitor-1 is a

Kriegs, M., Kasten-Pisula, U., Rieckmann, T., Holst, K., Saker, J., Dahm-Daphi, J., and

Kurzchalia, T.V., Dupree, P., Parton, R.G., Kellner, R., Virta, H., Lehnert, M., and Simons, K.

Kurzchalia, T.V., and Parton, R.G. (1999). Membrane microdomains and caveolae. Curr

Lajoie, P., and Nabi, I.R. (2007). Regulation of raft-dependent endocytosis. J Cell Mol Med

Leontieva, O.V., and Blagosklonny, M.V. (2010). DNA damaging agents and p53 do not

associated with conversion to senescence. Aging (Albany NY) *2*, 924-935.

network-derived transport vesicles. J Cell Biol *118*, 1003-1014.

attenuation of receptor signaling. J Biol Chem *273*, 24839-24846.

transcription of p21Sdi1/Cip1/Waf1. J Biol Chem *284*, 15475-15486. Kim, K., Nose, K., and Shibanuma, M. (2000). Significance of nuclear relocalization of

fibroblasts. J Biol Chem *275*, 20685-20692.

Cell Biol *8*, 877-884.

(Amst) *9*, 889-897.

*11*, 644-653.

Opin Cell Biol *11*, 424-431.

damage and protein kinase C. Mol Cancer Ther *1*, 861-867.

telomere state, not telomere loss. Science *295*, 2446-2449.

human fibroblasts. Cancer Res *66*, 8356-8360.

FEBS Lett *491*, 4-8.

587-597.

of growth arrest genes p21 and GADD45 during replicative senescence of normal

Localization of phospholipase C-gamma1 signaling in caveolae: importance in EGF-induced phosphoinositide hydrolysis but not in tyrosine phosphorylation.

Vogelstein, B., and Fornace, A.J., Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell *71*,

T., and Shirahata, S. (2009). Protein kinase C delta plays a key role in cellular

and Bohmer, F.D. (1998). Phosphotyrosine 1173 mediates binding of the proteintyrosine phosphatase SHP- 1 to the epidermal growth factor receptor and

Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and

kinases 1 and 2 phosphorylate Sp1 on serine 59 and regulate cellular senescence via

ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent

critical downstream target of p53 in the induction of replicative senescence. Nat

Dikomey, E. (2010). The epidermal growth factor receptor modulates DNA doublestrand break repair by regulating non-homologous end-joining. DNA Repair

(1992). VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-

cause senescence in quiescent cells, while consecutive re-activation of mTOR is


Parton, R.G., and Simons, K. (2007). The multiple faces of caveolae. Nat Rev Mol Cell Biol *8*,

Patel, H.H., Murray, F., and Insel, P.A. (2008). Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annu Rev Pharmacol Toxicol *48*, 359-391. Pelkmans, L., Puntener, D., and Helenius, A. (2002). Local actin polymerization and

Pelkmans, L., and Zerial, M. (2005). Kinase-regulated quantal assemblies and kiss-and-run

Phillips, P.D., and Cristofalo, V.J. (1988). Classification system based on the functional

Phillips, P.D., Kaji, K., and Cristofalo, V.J. (1984). Progressive loss of the proliferative

growth factor, insulin, transferrin, and dexamethasone. J Gerontol *39*, 11-17. Phillips, P.D., Kuhnle, E., and Cristofalo, V.J. (1983). [125I]EGF binding ability is stable throughout the replicative life-span of WI-38 cells. J Cell Physiol *114*, 311-316. Rattan, S.I., and Derventzi, A. (1991). Altered cellular responsiveness during ageing.

Resnick-Silverman, L., St Clair, S., Maurer, M., Zhao, K., and Manfredi, J.J. (1998).

Riabowol, K., Schiff, J., and Gilman, M.Z. (1992). Transcription factor AP-1 activity is

Rietveld, A., and Simons, K. (1998). The differential miscibility of lipids as the basis for the formation of functional membrane rafts. Biochim Biophys Acta *1376*, 467-479. Rogakou, E.P., Boon, C., Redon, C., and Bonner, W.M. (1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol *146*, 905-916. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., and Bonner, W.M. (1998). DNA double-

Rohme, D. (1981). Ageing and the fusion sensitivity potential of human cells in culture:

Rothberg, K.G., Heuser, J.E., Donzell, W.C., Ying, Y.S., Glenney, J.R., and Anderson, R.G.

Rouault, J.P., Falette, N., Guehenneux, F., Guillot, C., Rimokh, R., Wang, Q., Berthet, C.,

Rutter, A., Mackinnon, W.B., Huschtscha, L.I., and Mountford, C.E. (1996). A proton

recycling of caveolae. Nature *436*, 128-133.

dynamin recruitment in SV40-induced internalization of caveolae. Science *296*, 535-

equivalency of mitogens that regulate WI-38 cell proliferation. Exp Cell Res *175*,

response of senescing WI-38 cells to platelet-derived growth factor, epidermal

Identification of a novel class of genomic DNA-binding sites suggests mechanism for selectivity in target gene activation by the tumor suppressor protein p53. Genes

required for initiation of DNA synthesis and is lost during cellular aging. Proc Natl

stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem

relation to tissue origin, donor age, and in vitro culture level and condition. Mech

(1992). Caveolin, a protein component of caveolae membrane coats. Cell *68*, 673-

Moyret-Lalle, C., Savatier, P., Pain, B.*, et al.* (1996). Identification of BTG2, an antiproliferative p53- dependent component the DNA damage cellular response

magnetic resonance spectroscopy study of aging and transformed human

185-194.

539.

396-403.

Bioessays *13*, 601- 606.

Dev *12*, 2102-2107.

*273*, 5858-5868.

682.

Ageing Dev *16*, 241-253.

pathway. Nat Genet *14*, 482-486.

fibroblasts. Exp Gerontol *31*, 669-686.

Acad Sci U S A *89*, 157-161.


Mineo, C., James, G.L., Smart, E.J., and Anderson, R.G. (1996). Localization of epidermal

Monier, S., Dietzen, D.J., Hastings, W.R., Lublin, D.M., and Kurzchalia, T.V. (1996).

Moodie, S.A., and Wolfman, A. (1994). The 3Rs of life: Ras, Raf and growth regulation.

Mouton, R.E., and Venable, M.E. (2000). Ceramide induces expression of the senescence

Mundy, D.I., Machleidt, T., Ying, Y.S., Anderson, R.G., and Bloom, G.S. (2002). Dual control

Nakamura, M., Kondo, H., Shimada, Y., Waheed, A.A., and Ohno-Iwashita, Y. (2003).

Naru, E., Takanezawa, Y., Kobayashi, M., Misaki, Y., Kaji, K., and Arakane, K. (2008).

Newton, A.C. (2001). Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev *101*, 2353-2364. Noda, A., Ning, Y., Venable, S.F., Pereira-Smith, O.M., and Smith, J.R. (1994). Cloning of

Ogata, T., Ueyama, T., Isodono, K., Tagawa, M., Takehara, N., Kawashima, T., Harada, K.,

Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994).

Okamoto, T., Schlegel, A., Scherer, P.E., and Lisanti, M.P. (1998). Caveolins, a family of

Palmer, H.J., Tuzon, C.T., and Paulson, K.E. (1999). Age-dependent decline in mitogenic

Park, S.C., Park, J.S., Park, W.Y., Cho, K.A., Ahn, J.S., and Jang, I.S. (2002). Down-regulation

Park, W.Y., Park, J.S., Cho, K.A., Kim, D.I., Ko, Y.G., Seo, J.S., and Park, S.C. (2000). Up-

dysfunction and conduction disturbance. Mol Cell Biol *28*, 3424-3436. Ohki, R., Nemoto, J., Murasawa, H., Oda, E., Inazawa, J., Tanaka, N., and Taniguchi, T.

binding sites of Shc in intact cells. J Biol Chem *269*, 18674-18678.

Chem *271*, 11930-11935.

Trends Genet *10*, 44-48.

*113*, 169-181.

*115*, 4327-4339.

Cell Res *211*, 90-98.

acylation or cholesterol. FEBS Lett *388*, 143-149.

human diploid fibroblasts. Exp Cell Res *290*, 381-390.

dermal fibroblasts in vitro. Hum Cell *21*, 70-78.

the G2 phase. J Biol Chem *275*, 22627-22630.

plasma membrane. J Biol Chem *273*, 5419-5422.

signal-regulated kinases. J Biol Chem *274*, 11424-11430.

hyporesponsiveness. Ann N Y Acad Sci *959*, 45-49.

Cells. J Biol Chem *275*, 20847-20852.

growth factor- stimulated Ras/Raf-1 interaction to caveolae membrane. J Biol

Oligomerization of VIP21-caveolin in vitro is stabilized by long chain fatty

histochemical marker, beta-galactosidase, in human fibroblasts. Mech Ageing Dev

of caveolar membrane traffic by microtubules and the actin cytoskeleton. J Cell Sci

Cellular aging- dependent decrease in cholesterol in membrane microdomains of

Increased levels of a particular phosphatidylcholine species in senescent human

senescent cell- derived inhibitors of DNA synthesis using an expression screen. Exp

Takahashi, T., Shioi, T., Matsubara, H.*, et al.* (2008). MURC, a muscle-restricted coiled-coil protein that modulates the Rho/ROCK pathway, induces cardiac

(2000). Reprimo, a new candidate mediator of the p53-mediated cell cycle arrest at

Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are

scaffolding proteins for organizing "preassembled signaling complexes" at the

stimulation of hepatocytes. Reduced association between Shc and the epidermal growth factor receptor is coupled to decreased activation of Raf and extracellular

of receptor- mediated endocytosis is responsible for senescence-associated

regulation of Caveolin Attenuates Epidermal Growth Factor Signaling in Senescent


Tran, K.T., Rusu, S.D., Satish, L., and Wells, A. (2003). Aging-related attenuation of EGF

Tresini, M., Lorenzini, A., Frisoni, L., Allen, R.G., and Cristofalo, V.J. (2001). Lack of Elk-1

Tresini, M., Mawal-Dewan, M., Cristofalo, V.J., and Sell, C. (1998). A phosphatidylinositol 3-

Vanhaesebroeck, B., Leevers, S.J., Panayotou, G., and Waterfield, M.D. (1997).

Vannini, F., Meacci, E., Vasta, V., Farnararo, M., and Bruni, P. (1994). Involvement of protein

Venable, M.E., Bielawska, A., and Obeid, L.M. (1996). Ceramide inhibits phospholipase D in

Venable, M.E., Blobe, G.C., and Obeid, L.M. (1994). Identification of a defect in the

Venable, M.E., Lee, J.Y., Smyth, M.J., Bielawska, A., and Obeid, L.M. (1995). Role of

Venable, M.E., and Obeid, L.M. (1999). Phospholipase D in cellular senescence. Biochim

Volonte, D., and Galbiati, F. (2009a). Caveolin-1, cellular senescence and pulmonary

Volonte, D., and Galbiati, F. (2009b). Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress- induced premature senescence. EMBO Rep *10*, 1334-1340. Volonte, D., and Galbiati, F. (2011). Polymerase I and Transcript Release Factor

Volonte, D., Galbiati, F., Pestell, R.G., and Lisanti, M.P. (2001). Cellular stress induces the

Volonte, D., Kahkonen, B., Shapiro, S., Di, Y., and Galbiati, F. (2009). Caveolin-1 expression

Volonte, D., Zhang, K., Lisanti, M.P., and Galbiati, F. (2002). Expression of caveolin-1

Vousden, K.H., and Lane, D.P. (2007). p53 in health and disease. Nat Rev Mol Cell Biol *8*,

the ATM-p53-p21 pathway. J Biol Chem *284*, 5462-5466.

(PTRF)/Cavin-1 Is a Novel Regulator of Stress-induced Premature Senescence. J

tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogenactivated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress. J Biol

is required for the development of pulmonary emphysema through activation of

induces premature cellular senescence in primary cultures of murine fibroblasts.

ceramide in cellular senescence. J Biol Chem *270*, 30701-30708.

pathway in senescent human fibroblast. Exp Cell Res *269*, 287-300.

activity. Exp Cell Res *289*, 359-367.

[see comments]. Cancer Res *58*, 1-4.

a cell-free system. J Biol Chem *271*, 24800-24805.

emphysema. Aging (Albany NY) *1*, 831-835.

Biochem Sci *22*, 267-272.

*8*, 279-282.

26040-26044.

Biophys Acta *1439*, 291-298.

Biol Chem *286*, 28657-28661.

Chem *276*, 8094-8103.

Mol Biol Cell *13*, 2502-2517.

275-283.

receptor signaling is mediated in part by increased protein tyrosine phosphatase

phosphorylation and dysregulation of the extracellular regulated kinase signaling

kinase inhibitor induces a senescent-like growth arrest in human diploid fibroblasts

Phosphoinositide 3- kinases: a conserved family of signal transducers. Trends

kinase C and arachidonate signaling pathways in the alteration of proliferative response of senescent IMR-90 human fibroblasts. Mech Ageing Dev *76*, 101-111. Vaziri, H., and Benchimol, S. (1998). Reconstitution of telomerase activity in normal human

cells leads to elongation of telomeres and extended replicative life span. Curr Biol

phospholipase D/diacylglycerol pathway in cellular senescence. J Biol Chem *269*,


Rybin, V.O., Guo, J., Gertsberg, Z., Feinmark, S.J., and Steinberg, S.F. (2008). Phorbol 12-

Satyanarayana, A., Greenberg, R.A., Schaetzlein, S., Buer, J., Masutomi, K., Hahn, W.C.,

Scherer, P.E., Okamoto, T., Chun, M., Nishimoto, I., Lodish, H.F., and Lisanti, M.P. (1996).

Schroeder, F., Goetz, I., and Roberts, E. (1984). Age-related alterations in cultured human fibroblast membrane structure and function. Mech Ageing Dev *25*, 365-389. Sedelnikova, O.A., Horikawa, I., Zimonjic, D.B., Popescu, N.C., Bonner, W.M., and Barrett,

Sell, C., Ptasznik, A., Chang, C.D., Swantek, J., Cristofalo, V.J., and Baserga, R. (1993). IGF-1

Serrano, M., Lin, A.W., McCurrach, M.E., Beach, D., and Lowe, S.W. (1997). Oncogenic ras

Seshadri, T., and Campisi, J. (1990). Repression of c-fos transcription and an altered genetic

Shay, J.W., Pereira-Smith, O.M., and Wright, W.E. (1991). A role for both RB and p53 in the

Stewart, S.A., Ben-Porath, I., Carey, V.J., O'Connor, B.F., Hahn, W.C., and Weinberg, R.A.

Tagawa, M., Ueyama, T., Ogata, T., Takehara, N., Nakajima, N., Isodono, K., Asada, S.,

Takahashi, A., Ohtani, N., Yamakoshi, K., Iida, S., Tahara, H., Nakayama, K., Nakayama,

Tang, Z., Scherer, P.E., Okamoto, T., Song, K., Chu, C., Kohtz, D.S., Nishimoto, I., Lodish,

Torres, C., Francis, M.K., Lorenzini, A., Tresini, M., and Cristofalo, V.J. (2003). Metabolic

(2003). Erosion of the telomeric single-strand overhang at replicative senescence.

Takahashi, T., Matsubara, H., and Oh, H. (2008). MURC, a muscle-restricted coiledcoil protein, is involved in the regulation of skeletal myogenesis. Am J Physiol Cell

K.I., Ide, T., Saya, H., and Hara, E. (2006). Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol *8*,

H.F., and Lisanti, M.P. (1996). Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J Biol Chem *271*,

stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts. Exp

cardiomyocyte caveolae. J Biol Chem *283*, 17777-17788.

family. Proc Natl Acad Sci U S A *93*, 131-135.

Biochem Biophys Res Commun *194*, 259-265.

p16INK4a. Cell *88*, 593- 602.

Biol *1*, 31-39.

1291-1297.

2255-2261.

Nat Genet *33*, 492- 496.

Physiol *295*, C490-498.

Cell Res *290*, 195-206.

responses and senescence signaling. Mol Cell Biol *24*, 5459-5474.

unrepairable double- strand breaks. Nat Cell Biol *6*, 168-170.

program in senescent human fibroblasts. Science *247*, 205-209.

regulation of human cellular senescence. Exp Cell Res *196*, 33-39. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature *387*, 569-572. Simons, K., and Toomre, D. (2000). Lipid rafts and signal transduction. Nat Rev Mol Cell

myristate 13- acetate-dependent protein kinase C delta-Tyr311 phosphorylation in

Zimmermann, S., Martens, U., Manns, M.P., and Rudolph, K.L. (2004). Mitogen stimulation cooperates with telomere shortening to activate DNA damage

Identification, sequence, and expression of caveolin-2 defines a caveolin gene

J.C. (2004). Senescing human cells and ageing mice accumulate DNA lesions with

receptor levels and the proliferation of young and senescent human fibroblasts.

provokes premature cell senescence associated with accumulation of p53 and


**20** 

*Spain* 

**Alternative Splicing in Endothelial Senescence:** 

The vascular endothelium is the thin monolayer of specialized cells that line the blood vessels of the cardiovascular system. This endothelium is more than a simple protective barrier since it possesses anticoagulatory properties, mediates the metabolites exchange and regulates the vascular tone and homeostasis maintenance. These functions are finely tuned by endothelial cells that, in the absence of any stimuli, remain in a quiescent stage (Conway & Carmeliet, 2004). In fact, endothelial cells occasionally divide in a normal vessel, displaying a very low turnover rate except for localized areas (Foteinos et al., 2008). Thus, the endothelium is quite sensitive to a variety of signals including shear stress and circulating factors that lead to endothelial activation. As a result of their own physiology along the lifespan, endothelial cells progressively accumulate reactive oxygen species and pro-oxidant metabolites due to an increased oxidative stress, damages in DNA and advanced cellular replication involving shortening of telomeres. Altogether, these alterations lead endothelial cells to reach senescence (Brandes et al., 2005; Foreman & Tang, 2003), which has been proposed to be at the cellular basis of most of the vascular pathologies associated with ageing, such as atherosclerosis or hypertension (Minamino & Komuro, 2008;

The major aspect of endothelial physiology implies the growth or formation of new blood vessels from pre-existing ones, process named angiogenesis which is mainly induced by metabolic requests (Fraisl et al., 2009). Angiogenesis plays a key role from the first steps during the embryonic development to the adult stage, and is involved in numerous physiological processes such as wound repair or the growth of the tissues (Carmeliet & Jain, 2011). However, angiogenesis and vascular remodelling decline with age and several lines of evidence indicate that ageing and endothelial dysfunction progress in parallel (Brandes et al., 2005; Ferrari et al., 2003; Minamino et al., 2004). In this sense, numerous efforts are

The angiogenesis process consists of two separate but balanced phases, activation and resolution, that are finely arranged by a suite of cytokines, among which the transforming

addressed to elucidate the molecular mechanisms that underlie vascular ageing.

**2. TGF-β in angiogenesis - Role of endoglin** 

**1. Introduction** 

Rodríguez-Mañas et al., 2009).

**Role of the TGF-β Co-Receptor Endoglin** 

*Centro de Investigación Biomédica en Red de Enfermedades Raras,* 

Francisco J. Blanco and Carmelo Bernabéu

*Centro de Investigaciones Biológicas, CSIC,* 


## **Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin**

Francisco J. Blanco and Carmelo Bernabéu *Centro de Investigaciones Biológicas, CSIC, Centro de Investigación Biomédica en Red de Enfermedades Raras, Spain* 

## **1. Introduction**

498 Senescence

Wary, K.K., Mariotti, A., Zurzolo, C., and Giancotti, F.G. (1998). A requirement for caveolin-

Way, M., and Parton, R.G. (1996). M-caveolin, a muscle-specific caveolin-related protein

Webb, L.M., Arnholt, A.T., and Venable, M.E. (2010). Phospholipase D modulation by

Wheaton, K. (2002). Regulation of Serum Response Factor during Cellular Senescence. In Biochemistry & Molecular Biology (Calgary, University of Calgary), pp. 208. Wheaton, K. (2011). Caveolar vesicles generate DNA damage and perpetuate cellular aging.

Wheaton, K., Atadja, P., and Riabowol, K. (1996). Regulation of transcription factor activity

Wheaton, K., Muir, J., Ma, W., and Benchimol, S. (2010). BTG2 antagonizes Pin1 in response

Wheaton, K., and Riabowol, K. (2004). Protein kinase C delta blocks immediate-early gene

Wheaton, K., Sampsel, K., Boisvert, F.M., Davy, A., Robbins, S., and Riabowol, K. (2001).

White, M.A., and Anderson, R.G. (2005). Signaling networks in living cells. Annu Rev

Wright, J.D., Reuter, C.W., and Weber, M.J. (1996). Identification of sites on epidermal

Yoshida, K., Liu, H., and Miki, Y. (2006). Protein kinase C delta regulates Ser46

Zajchowski, L.D., and Robbins, S.M. (2002). Lipid rafts and little caves. Compartmentalized signalling in membrane microdomains. Eur J Biochem *269*, 737-752. Zezula, J., Sexl, V., Hutter, C., Karel, A., Schutz, W., and Freissmuth, M. (1997). The cyclin-

Zhu, J., Woods, D., McMahon, M., and Bishop, J.M. (1998). Senescence of human fibroblasts

induced by oncogenic Raf. Genes Dev *12*, 2997-3007.

to mitogens and telomere disruption during replicative senescence. Aging Cell *9*,

expression in senescent cells by inactivating serum response factor. Mol Cell Biol

Loss of functional caveolae during senescence of human fibroblasts. J Cell Physiol

growth factor receptors which are phosphorylated by pp60src in vitro. Biochim

phosphorylation of p53 tumor suppressor in the apoptotic response to DNA

dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells. J Biol Chem *272*, 29967-29974. Zhu, H., Yue, J., Pan, Z., Wu, H., Cheng, Y., Lu, H., Ren, X., Yao, M., Shen, Z., and Yang, J.M.

(2010). Involvement of Caveolin-1 in repair of DNA damage through both homologous recombination and non-homologous end joining. PLoS One *5*, e12055.

Lett 1995 Nov 27;376(1- 2):108-12]. FEBS Lett *378*, 108-112.

ceramide in senescence. Mol Cell Biochem *337*, 153-158.

during cellular aging. Biochem Cell Biol *74*, 523-534.

growth. Cell *94*, 625-634.

Cell Res *21*, 993-994.

747-760.

*24*, 7298-7311.

*187*, 226-235.

Pharmacol Toxicol *45*, 587-603.

damage. J Biol Chem *281*, 5734-5740.

Biophys Acta *1312*, 85-93.

1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell

[corrected and republished with original paging, article originally printed in FEBS

The vascular endothelium is the thin monolayer of specialized cells that line the blood vessels of the cardiovascular system. This endothelium is more than a simple protective barrier since it possesses anticoagulatory properties, mediates the metabolites exchange and regulates the vascular tone and homeostasis maintenance. These functions are finely tuned by endothelial cells that, in the absence of any stimuli, remain in a quiescent stage (Conway & Carmeliet, 2004). In fact, endothelial cells occasionally divide in a normal vessel, displaying a very low turnover rate except for localized areas (Foteinos et al., 2008). Thus, the endothelium is quite sensitive to a variety of signals including shear stress and circulating factors that lead to endothelial activation. As a result of their own physiology along the lifespan, endothelial cells progressively accumulate reactive oxygen species and pro-oxidant metabolites due to an increased oxidative stress, damages in DNA and advanced cellular replication involving shortening of telomeres. Altogether, these alterations lead endothelial cells to reach senescence (Brandes et al., 2005; Foreman & Tang, 2003), which has been proposed to be at the cellular basis of most of the vascular pathologies associated with ageing, such as atherosclerosis or hypertension (Minamino & Komuro, 2008; Rodríguez-Mañas et al., 2009).

The major aspect of endothelial physiology implies the growth or formation of new blood vessels from pre-existing ones, process named angiogenesis which is mainly induced by metabolic requests (Fraisl et al., 2009). Angiogenesis plays a key role from the first steps during the embryonic development to the adult stage, and is involved in numerous physiological processes such as wound repair or the growth of the tissues (Carmeliet & Jain, 2011). However, angiogenesis and vascular remodelling decline with age and several lines of evidence indicate that ageing and endothelial dysfunction progress in parallel (Brandes et al., 2005; Ferrari et al., 2003; Minamino et al., 2004). In this sense, numerous efforts are addressed to elucidate the molecular mechanisms that underlie vascular ageing.

## **2. TGF-β in angiogenesis - Role of endoglin**

The angiogenesis process consists of two separate but balanced phases, activation and resolution, that are finely arranged by a suite of cytokines, among which the transforming

Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 501

**L-Endoglin** ...YIYSHTR**SPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA**

*ENG* **pre-mRNA Intron**

**Ex#12**

Fig. 1. The two endoglin isoforms. (A) The electron microscopy density map (grey) of the endoglin extracellular region shows the overall structure. The backbone of a theoretical atomic model of the endoglin monomer is fitted inside (adapted from Llorca et al., 2007). This structure is common to both endoglin variants. The transmembrane domain (red) and cytoplasmic tails (brown, L; blue, S) are schematized. (B) The amino acid sequence of the cytoplasmic domain is detailed for both isoforms. (C) The endoglin pre-mRNA is represented in the middle of the mature transcripts that originate each isoform. The

retention of the final intron by an alternative splicing process leads to S-endoglin expression.

S-endoglin arises as the result of an alternative splicing mechanism by which the last intron, between exons #13 and #14, is retained in the mature mRNA (Figure 1C). Consequently, an early stop codon appears in the open reading frame and truncates the mature protein in the cytoplasmic region. Although this mechanism of intron retention normally involves a rapid degradation by the nonsense-mediated decay machinery (Lareau et al., 2004; Nott et al., 2003), under certain conditions it may also lead to a biologically active isoform (Sakabe & de Souza, 2007); and this is the case of endoglin. Thus, when endothelial cells become senescent during the ageing process, they show an up-regulation of S-endoglin (Blanco et al., 2008). At this senescent stage, both endoglin isoforms are co-expressed likely forming heterodimers, as it occurs in mice (Perez-Gomez et al., 2005), and some of the cellular responses to TGF-β are oppositely regulated by each isoform. Indeed, the S-endoglin increase has an antiangiogenic role in the blood vessels and contributes to vascular pathology (Blanco et al.,

**L-Eng mRNA**

**S-Eng mRNA**

2008; Perez-Gomez et al., 2005; Velasco et al., 2008).

**L-Endoglin S-Endoglin**

**(136 nt)**

**Ex#13 Ex#14**

**Stop - S \***


**Stop - L**

**\***


**S-Endoglin**...YIYSHTR**EYPRPPQ**

Plasma membrane

**A**

**B**

**C**

Endoglin extracellular domain

growth factor (TGF)-β plays a dual role (Pardali et al., 2010). TGF-β is the prototypic member of a large family of multifunctional and evolutionarily conserved cytokines, including also activins and bone morphogenetic proteins (BMPs). Upon proteolytic activation, TGF-β circulates as a 25 kDa homodimer that elicits its cellular functions by binding to a membrane complex of type II (TβRII) and type I (TβRI or ALKs) receptors with cytoplasmic serine-threonine kinase activity (Kang et al., 2009). Endothelial cells express two different TβRIs, named ALK5 and ALK1, with distinct affinity for the ligand and different signalling pathways mediated mainly by Smad proteins (Smad2/3 and Smad1/5/8, respectively) (Massague et al., 2005). Moreover, endothelial cells also express endoglin, or CD105, an auxiliary TGF-β receptor that modulates the balance between ALK1 and ALK5 signalling. Endoglin is mainly expressed as a homodimeric protein of 180 kDa and is associated to the activation phase of angiogenesis, acting as a modulator between both phases. In this context, endoglin interacts with ALK1 and promotes the TGF-β/ALK1 signalling pathway (Blanco et al., 2005; Lebrin et al., 2004).

The TGF-β/endoglin pairing has been studied in different contexts such as differentiation (Tang et al., 2011), cancer (Bernabeu et al., 2009; Perez-Gomez et al., 2010) and other pathologies including liver fibrosis (Meurer et al., 2011) or preeclampsia (Venkatesha et al., 2006). However, endoglin plays a major role in angiogenesis as well as in vascular remodelling and homeostasis (Lopez-Novoa & Bernabeu, 2010; ten Dijke et al., 2008). Heterozygous mutations in the endoglin gene (*ENG*) are responsible for the vascular dysplasia named hereditary haemorrhagic telangiectasia (HHT) type 1 (McAllister et al., 1994; Shovlin, 2010), a rare genetic disease with autosomal dominant inheritance. These mutations lead to the development of abnormal vascular structures that are the basis of the characteristic HHT symptoms, including frequent and recurrent nosebleeds, telangiectases in the nasal and gastrointestinal tracts and large arteriovenous malformations in different organs such as lung, liver or brain (Mahmoud et al., 2010; Shovlin, 2010). Nonetheless, the HHT symptoms are not present at birth and normally appear during adolescence, getting worse with age. This is in line with the functional role of endoglin in angiogenesis and with previous observation that angiogenesis becomes impaired with ageing (Rivard et al., 1999).

### **2.1 Two alternatively spliced endoglin isoforms**

Most of published studies about endoglin are referred to L-endoglin (long endoglin) that is the predominantly expressed isoform. However, the expression of a short variant (Sendoglin) was described first in humans (Bellon et al., 1993) and later in mouse (Perez-Gomez et al., 2005). In humans, both isoforms share the identical large extracellular region and the transmembrane domain, so that the only difference resides in their cytoplasmic tails (Figure 1A). In the case of L-endoglin, this region is composed by 47 amino acids with a high frequency of serine and threonine residues susceptible to be phosphorylated. Also, the sequence serine-methionine-alanine, SMA, in the C-terminal end is a docking site for proteins with a PDZ domain and is involved in the cytoskeleton organization (Koleva et al., 2006). By contrast, the sequence of the S-endoglin cytoplasmic tail is 14 amino acids long and contains only one serine and threonine residues; also the last 7 residues are specific for this isoform (Figure 1B). These data suggest that L-endoglin and S-endoglin may elicit different functional effects on the endothelial cell.

growth factor (TGF)-β plays a dual role (Pardali et al., 2010). TGF-β is the prototypic member of a large family of multifunctional and evolutionarily conserved cytokines, including also activins and bone morphogenetic proteins (BMPs). Upon proteolytic activation, TGF-β circulates as a 25 kDa homodimer that elicits its cellular functions by binding to a membrane complex of type II (TβRII) and type I (TβRI or ALKs) receptors with cytoplasmic serine-threonine kinase activity (Kang et al., 2009). Endothelial cells express two different TβRIs, named ALK5 and ALK1, with distinct affinity for the ligand and different signalling pathways mediated mainly by Smad proteins (Smad2/3 and Smad1/5/8, respectively) (Massague et al., 2005). Moreover, endothelial cells also express endoglin, or CD105, an auxiliary TGF-β receptor that modulates the balance between ALK1 and ALK5 signalling. Endoglin is mainly expressed as a homodimeric protein of 180 kDa and is associated to the activation phase of angiogenesis, acting as a modulator between both phases. In this context, endoglin interacts with ALK1 and promotes the TGF-β/ALK1

The TGF-β/endoglin pairing has been studied in different contexts such as differentiation (Tang et al., 2011), cancer (Bernabeu et al., 2009; Perez-Gomez et al., 2010) and other pathologies including liver fibrosis (Meurer et al., 2011) or preeclampsia (Venkatesha et al., 2006). However, endoglin plays a major role in angiogenesis as well as in vascular remodelling and homeostasis (Lopez-Novoa & Bernabeu, 2010; ten Dijke et al., 2008). Heterozygous mutations in the endoglin gene (*ENG*) are responsible for the vascular dysplasia named hereditary haemorrhagic telangiectasia (HHT) type 1 (McAllister et al., 1994; Shovlin, 2010), a rare genetic disease with autosomal dominant inheritance. These mutations lead to the development of abnormal vascular structures that are the basis of the characteristic HHT symptoms, including frequent and recurrent nosebleeds, telangiectases in the nasal and gastrointestinal tracts and large arteriovenous malformations in different organs such as lung, liver or brain (Mahmoud et al., 2010; Shovlin, 2010). Nonetheless, the HHT symptoms are not present at birth and normally appear during adolescence, getting worse with age. This is in line with the functional role of endoglin in angiogenesis and with previous observation that angiogenesis becomes

Most of published studies about endoglin are referred to L-endoglin (long endoglin) that is the predominantly expressed isoform. However, the expression of a short variant (Sendoglin) was described first in humans (Bellon et al., 1993) and later in mouse (Perez-Gomez et al., 2005). In humans, both isoforms share the identical large extracellular region and the transmembrane domain, so that the only difference resides in their cytoplasmic tails (Figure 1A). In the case of L-endoglin, this region is composed by 47 amino acids with a high frequency of serine and threonine residues susceptible to be phosphorylated. Also, the sequence serine-methionine-alanine, SMA, in the C-terminal end is a docking site for proteins with a PDZ domain and is involved in the cytoskeleton organization (Koleva et al., 2006). By contrast, the sequence of the S-endoglin cytoplasmic tail is 14 amino acids long and contains only one serine and threonine residues; also the last 7 residues are specific for this isoform (Figure 1B). These data suggest that L-endoglin and S-endoglin may elicit

signalling pathway (Blanco et al., 2005; Lebrin et al., 2004).

impaired with ageing (Rivard et al., 1999).

**2.1 Two alternatively spliced endoglin isoforms** 

different functional effects on the endothelial cell.

Fig. 1. The two endoglin isoforms. (A) The electron microscopy density map (grey) of the endoglin extracellular region shows the overall structure. The backbone of a theoretical atomic model of the endoglin monomer is fitted inside (adapted from Llorca et al., 2007). This structure is common to both endoglin variants. The transmembrane domain (red) and cytoplasmic tails (brown, L; blue, S) are schematized. (B) The amino acid sequence of the cytoplasmic domain is detailed for both isoforms. (C) The endoglin pre-mRNA is represented in the middle of the mature transcripts that originate each isoform. The retention of the final intron by an alternative splicing process leads to S-endoglin expression.

S-endoglin arises as the result of an alternative splicing mechanism by which the last intron, between exons #13 and #14, is retained in the mature mRNA (Figure 1C). Consequently, an early stop codon appears in the open reading frame and truncates the mature protein in the cytoplasmic region. Although this mechanism of intron retention normally involves a rapid degradation by the nonsense-mediated decay machinery (Lareau et al., 2004; Nott et al., 2003), under certain conditions it may also lead to a biologically active isoform (Sakabe & de Souza, 2007); and this is the case of endoglin. Thus, when endothelial cells become senescent during the ageing process, they show an up-regulation of S-endoglin (Blanco et al., 2008). At this senescent stage, both endoglin isoforms are co-expressed likely forming heterodimers, as it occurs in mice (Perez-Gomez et al., 2005), and some of the cellular responses to TGF-β are oppositely regulated by each isoform. Indeed, the S-endoglin increase has an antiangiogenic role in the blood vessels and contributes to vascular pathology (Blanco et al., 2008; Perez-Gomez et al., 2005; Velasco et al., 2008).

Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 503

upon TGF-β treatment, Smad3 is able to interact with the transcription factor c-myc, so repressing the promoter of the hTERT gene, encoding the catalytic subunit of telomerase (Figure 2). Thus, the c-myc activity is blocked in the Smad3 complexes which negatively affects to the cell cycle (Li & Liu, 2007; Li et al., 2006). In addition, this repression of the hTERT promoter mediated by TGF-β can be alternatively reinforced by the activation of the TGF-β activated kinase (TAK)-1 pathway that abrogates the transcriptional activity of Sp1

> **Replicative senescence**

↑TGF-β

**Stress-induced senescence**

Fig. 2. The endothelial senescence. Endothelial cells extensively cultured *in vitro* enlarge their size and shape, showing a positive blue staining for the SA-β-gal activity. Endothelial senescence is reached by, at least, two different routes, including replicative or oxidative stress-induced. Both pathways involve the activation of p53 and are characterized by an

The characteristic and irreversible growth arrest observed in senescent cells occurs in the transition from phase G1 to phase S of the cell cycle and is dependent on the retinoblastoma family proteins, playing the tumour suppressor p53 a key role which senses the telomeric DNA damage (Wesierska-Gadek et al., 2005). In this transition, the abolition of p53 expression interferes with the senescence process that would be related to the low levels of PAI-1, one of the p53 target genes (Kortlever et al., 2008). Conversely, it is well known that the p53 overexpression or activation is able to arrest the cell cycle and launch the senescence program, suggesting that this process could be useful in cancer therapy (Chen & Goligorsky, 2006; Ewald et al., 2010; Rosso et al., 2006; Sugrue et al., 1997). Furthermore, it was demonstrated that the prolonged treatment with interferon (IFN)-γ induces cellular senescence in endothelial cells, involving cell cycle arrest and an up-regulation of p53 and

Another TGF-β target protein that is associated with endothelial senescence is the helixloop-helix (HLH) transcription factor Id1, or inhibitor of DNA binding 1. Id1 lacks a basic DNA-binding domain, but is able to form heterodimers with other HLH proteins, thereby inhibiting DNA binding, a process that is essential for cellular proliferation (Benezra et al., 1990). In epithelial cells, TGF-β induces the formation of a Smad3/ATF3 heteromeric complex that represses the Id1 expression and negatively regulates the cell cycle (Kang et al., 2003). Hence, the decrease in the Id1 expression is considered a biomarker of endothelial

↑ROS

**p53**

<sup>↓</sup>hTERT <sup>↑</sup>ROS Hayflick's limit

Senescent EC (SA-β-gal+) ↑PAI-1 ↓Id1

on the hTERT promoter (Fujiki et al., 2007).

Young EC (SA-β-gal-

p21 proteins cells (Kim et al., 2009).

senescent cells (Tang et al., 2002).

)

increase in PAI-1 expression and the repression of Id1.

## **3. Endothelial senescence and TGF-β**

It is well known that ageing *per se* is the major risk factor for the development of cardiovascular diseases. Thus, senescence has been widely and mainly analyzed in *in vitro* studies but there are also evidences that this process takes place *in vivo* (Erusalimsky & Kurz, 2005; Minamino & Komuro, 2007). The first evidence of cellular senescence in primary cultures *in vitro* is the deceleration in the proliferation, that is, an increase in the doubling time of the cell population. In parallel, cells experience morphological changes along theses passages that involve the augment of the cellular size and shape. However, these observations are usually complemented with a useful tool based on the abnormal behaviour associated with senescent cells of the lysosomal hydrolase β-galactosidase. Thus, the senescence-associated β-galactosidase (SA β-gal) activity at pH 6 is widely accepted as an easily detectable senescence histochemical marker (Dimri et al., 1995).

Endothelial senescence is a cellular process that is clearly linked to both ageing and the development of vascular pathologies as well (Brandes et al., 2005; Erusalimsky, 2009; Minamino & Komuro, 2007). Basically, senescence constitutes a stress and damage response phenomenon that involves a permanent growth arrest (Campisi & d'Adda di Fagagna, 2007). Consequently, senescent cells undergo diverse changes in gene and protein expression that lead to an impairment of cellular functions (Foreman & Tang, 2003; Young & Narita, 2009). Thus, these changes usually affect to the endothelial phenotype favouring a pro-inflammatory, pro-atherosclerotic, or a prothrombotic state (Erusalimsky, 2009).

Here, TGF-β plays an important role owing to its ability to prompt senescence in a variety of cell types (Cipriano et al., 2011; Kordon et al., 1995; Tremain et al., 2000; van der Kraan et al., 2011; Wu et al., 2009). In the vascular context, it has been reported, e. g., elevated levels of TGF-β in the aging varicose veins that likely favour the fibrous process and the consequent venous insufficiency (Pascual et al., 2007). In this sense, the profibrotic effect of TGF-β is mediated by the stimulation via Smad3 signalling of the plasminogen activator inhibitor (PAI)-1 expression, a key regulator of the synthesis and deposition of the extracellular matrix in the tissue homeostasis (Ghosh & Vaughan, 2011). Thus, the increase of TGF-β upregulates PAI-1 expression, which contributes to the accumulation of collagen and other extracellular matrix components. This PAI-1 increase is also in line with the decrease of the antithrombogenic properties of a senescent endothelium due to the inhibition of the urokinase- and tissue-type plasminogen activator (uPA and tPA, respectively)/plasmin axis (Comi et al., 1995; Schneiderman et al., 1992).

#### **3.1 Replicative senescence**

Senescence was initially considered to reflect the finite capacity for division that normal diploid cells exhibit when propagated in culture. This statement is based on the successive rounds of cell division that imply the progressively shortening and eventual dysfunction of telomeres, the physical ends of chromosomes, in a phenomenon known as Hayflick's limit (Hayflick, 2003; Shay & Wright, 2007). Thus, the down-regulation of telomerase, the enzyme responsible for maintaining the telomeres length, is clue for the senescence program. Besides, because telomerase is re-activated in the majority of neoplastic processes, it is postulated that inhibiting telomerase activity should result in senescence induction by telomere shortening which can cause the death of cancer cells (Folini et al., 2011). Interestingly, the senescence inducer TGF-β down-regulates the telomerase activity. Thus,

It is well known that ageing *per se* is the major risk factor for the development of cardiovascular diseases. Thus, senescence has been widely and mainly analyzed in *in vitro* studies but there are also evidences that this process takes place *in vivo* (Erusalimsky & Kurz, 2005; Minamino & Komuro, 2007). The first evidence of cellular senescence in primary cultures *in vitro* is the deceleration in the proliferation, that is, an increase in the doubling time of the cell population. In parallel, cells experience morphological changes along theses passages that involve the augment of the cellular size and shape. However, these observations are usually complemented with a useful tool based on the abnormal behaviour associated with senescent cells of the lysosomal hydrolase β-galactosidase. Thus, the senescence-associated β-galactosidase (SA β-gal) activity at pH 6 is widely accepted as an

Endothelial senescence is a cellular process that is clearly linked to both ageing and the development of vascular pathologies as well (Brandes et al., 2005; Erusalimsky, 2009; Minamino & Komuro, 2007). Basically, senescence constitutes a stress and damage response phenomenon that involves a permanent growth arrest (Campisi & d'Adda di Fagagna, 2007). Consequently, senescent cells undergo diverse changes in gene and protein expression that lead to an impairment of cellular functions (Foreman & Tang, 2003; Young & Narita, 2009). Thus, these changes usually affect to the endothelial phenotype favouring a

Here, TGF-β plays an important role owing to its ability to prompt senescence in a variety of cell types (Cipriano et al., 2011; Kordon et al., 1995; Tremain et al., 2000; van der Kraan et al., 2011; Wu et al., 2009). In the vascular context, it has been reported, e. g., elevated levels of TGF-β in the aging varicose veins that likely favour the fibrous process and the consequent venous insufficiency (Pascual et al., 2007). In this sense, the profibrotic effect of TGF-β is mediated by the stimulation via Smad3 signalling of the plasminogen activator inhibitor (PAI)-1 expression, a key regulator of the synthesis and deposition of the extracellular matrix in the tissue homeostasis (Ghosh & Vaughan, 2011). Thus, the increase of TGF-β upregulates PAI-1 expression, which contributes to the accumulation of collagen and other extracellular matrix components. This PAI-1 increase is also in line with the decrease of the antithrombogenic properties of a senescent endothelium due to the inhibition of the urokinase- and tissue-type plasminogen activator (uPA and tPA, respectively)/plasmin axis

Senescence was initially considered to reflect the finite capacity for division that normal diploid cells exhibit when propagated in culture. This statement is based on the successive rounds of cell division that imply the progressively shortening and eventual dysfunction of telomeres, the physical ends of chromosomes, in a phenomenon known as Hayflick's limit (Hayflick, 2003; Shay & Wright, 2007). Thus, the down-regulation of telomerase, the enzyme responsible for maintaining the telomeres length, is clue for the senescence program. Besides, because telomerase is re-activated in the majority of neoplastic processes, it is postulated that inhibiting telomerase activity should result in senescence induction by telomere shortening which can cause the death of cancer cells (Folini et al., 2011). Interestingly, the senescence inducer TGF-β down-regulates the telomerase activity. Thus,

pro-inflammatory, pro-atherosclerotic, or a prothrombotic state (Erusalimsky, 2009).

easily detectable senescence histochemical marker (Dimri et al., 1995).

**3. Endothelial senescence and TGF-β**

(Comi et al., 1995; Schneiderman et al., 1992).

**3.1 Replicative senescence** 

upon TGF-β treatment, Smad3 is able to interact with the transcription factor c-myc, so repressing the promoter of the hTERT gene, encoding the catalytic subunit of telomerase (Figure 2). Thus, the c-myc activity is blocked in the Smad3 complexes which negatively affects to the cell cycle (Li & Liu, 2007; Li et al., 2006). In addition, this repression of the hTERT promoter mediated by TGF-β can be alternatively reinforced by the activation of the TGF-β activated kinase (TAK)-1 pathway that abrogates the transcriptional activity of Sp1 on the hTERT promoter (Fujiki et al., 2007).

Fig. 2. The endothelial senescence. Endothelial cells extensively cultured *in vitro* enlarge their size and shape, showing a positive blue staining for the SA-β-gal activity. Endothelial senescence is reached by, at least, two different routes, including replicative or oxidative stress-induced. Both pathways involve the activation of p53 and are characterized by an increase in PAI-1 expression and the repression of Id1.

The characteristic and irreversible growth arrest observed in senescent cells occurs in the transition from phase G1 to phase S of the cell cycle and is dependent on the retinoblastoma family proteins, playing the tumour suppressor p53 a key role which senses the telomeric DNA damage (Wesierska-Gadek et al., 2005). In this transition, the abolition of p53 expression interferes with the senescence process that would be related to the low levels of PAI-1, one of the p53 target genes (Kortlever et al., 2008). Conversely, it is well known that the p53 overexpression or activation is able to arrest the cell cycle and launch the senescence program, suggesting that this process could be useful in cancer therapy (Chen & Goligorsky, 2006; Ewald et al., 2010; Rosso et al., 2006; Sugrue et al., 1997). Furthermore, it was demonstrated that the prolonged treatment with interferon (IFN)-γ induces cellular senescence in endothelial cells, involving cell cycle arrest and an up-regulation of p53 and p21 proteins cells (Kim et al., 2009).

Another TGF-β target protein that is associated with endothelial senescence is the helixloop-helix (HLH) transcription factor Id1, or inhibitor of DNA binding 1. Id1 lacks a basic DNA-binding domain, but is able to form heterodimers with other HLH proteins, thereby inhibiting DNA binding, a process that is essential for cellular proliferation (Benezra et al., 1990). In epithelial cells, TGF-β induces the formation of a Smad3/ATF3 heteromeric complex that represses the Id1 expression and negatively regulates the cell cycle (Kang et al., 2003). Hence, the decrease in the Id1 expression is considered a biomarker of endothelial senescent cells (Tang et al., 2002).

2010).

**A**

Control

S-endoglin

x25

x25 x80

(Figure adapted from Blanco et al., 2008).

x80

Fig. 3. S-endoglin expression in senescence. (A) The expression of S-endoglin in blood vessels can be revealed by *in situ* hybridization in the endothelium of human coronary artery (black arrow) and in some smooth muscle cells (red arrow). (B) The increase in the percentage of senescent endothelial cells *in vitro* (blue graph) is concomitant with the induction of S-endoglin (red graph). (C) Primary cultures of human umbilical vein endothelial cells (HUVECs) maintained *in vitro* along passages co-express both endoglin isoforms comparing young (Y) versus senescent (S) cells in RT-PCR assays. In parallel, PAI-1 is increased, while Id1 and telomerase (hTERT) are down-regulated in senescent cell. As a control, the expression levels of the TGF-β type I receptors ALK1 and ALK5 are not altered.

Nonetheless, little is known about the role of splicing in the vascular context during senescence. A recent study demonstrates that TGF-β induces the distal splice-site selection leading to an antiangiogenic variant of the vascular endothelial growth factor (VEGF) (Nowak et al., 2008), and this could be one of the reasons why there is a reduced capability

As described above, the role of TGF-β in senescence has been clearly established, modulating specific intracellular effectors and leading to the cell growth arrest. In a first

to form tubular-like structure by senescent endothelial *in vitro* (Chang et al., 2005).

Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 505

(AGEs) which have been implicated in age-related disease and aging itself; as well as the p53 acetylation in stress-induced senescence (Furukawa et al., 2007). In addition, a growing body of evidence supports the involvement of the post-transcriptional modifications that occur in senescence, i. e., the alternative splicing processes associated with senescence (Harries et al., 2011; Meshorer & Soreq, 2002). Thus, alterations in the splicing pattern have been described for several age-related diseases, such as the Hutchison Gilford progeria syndrome (Eriksson et al., 2003), or the Alzheimer's disease-related tauopathies (Chen et al.,

**B**

**% Senescent ECs**

> **C Y S S-endoglin L-endoglin**

> > **Id1 PAI-1 ALK1 ALK5**

2 3 4 5 6 7 8 9 10 11 12 13 14

**Number of passage**

**Ratio S-/L-Endo**

**hTERT**

**GAPDH**

#### **3.2 Oxidative stress-induced senescence**

Endothelial senescence can also be triggered by telomere-independent events that in general involve damages in the DNA. In this sense, the oxidative stress is a major stimulus for the induction of this type of senescence, which is due to the generation of reactive oxygen species (ROS, including oxygen ions and peroxides) in the mitochondria (Collins & Tzima, 2011; Erusalimsky & Skene, 2009). Thus, the cellular metabolism is the central source of ROS, but often they have an extracellular origin such as the one induced by radiation. In any case, ROS can either provoke or accelerate the development of senescence by damaging the DNA (Figure 2), which triggers multiple response mechanisms that usually act through the retinoblastoma protein family pathways, the final effectors of the senescence program (Campisi & d'Adda di Fagagna, 2007; Erusalimsky, 2009).

In cell culture, ROS induce an acute form of senescence termed stress-induced premature senescence, which does not require extensive cell culture but which resembles somehow the replicative one (Toussaint et al., 2000). This type of senescence is relatively easy to analyze in *in vitro* assays because the sole treatment with hydrogen peroxide (H2O2) for a short lapse of time is enough to prompt this type of senescence (Chen et al., 1998). By contrast, using antioxidant agents such as the grape stilbenoid resveratrol protect from the oxidative stressinduced premature senescence (Kao et al., 2010). Also, several lines of evidence show that ROS can interact and deplete the nitric oxide (NO) generated by the endothelium in the vasodilator responses, so contributing to the endothelial dysfunction associated to ageing (Grisham et al., 1998; Steiner et al., 2002). This is in line with the availability of NO-donors to inhibit endothelial cell senescence (Hayashi et al., 2006). In fact, comparing elderly with young adults one can find that the NO levels, or its bioavailability, are decreased in the first group but, interestingly, without any difference regarding to the expression levels or activation state of the endothelial nitric oxide synthase (eNOS), the enzyme responsible of the NO generation (Sun et al., 2004; Taddei et al., 2001). In parallel, this decrease in the NO levels attenuates the negative interference that it exerts on the TGF-β signalling pathway (Saura et al., 2005), which contributes to prompt the senescence program.

On the other hand, radiation is an exogenous trigger for ROS. In human skin fibroblasts, repeated exposure to ultraviolet-B light at subcytotoxic level is able to prompt premature senescence. Interestingly, this effect is mediated by the increase in the TGF-β expression and consequently by its downstream signalling pathway (Debacq-Chainiaux et al., 2005). In the vascular context, this source of ROS has been poorly studied beyond the methodological interest to induce premature senescence because endothelial cells enter rapidly in apoptosis due to their high sensitivity to radiation (Paris et al., 2001). In this regard, a recent study has demonstrated that ionizing radiation suppresses angiogenesis in mice and this effect is mediated through the TGF-β/ALK5-dependent inhibition of endothelial cell sprouting (Imaizumi et al., 2010).

## **4. Induction of S-endoglin and its role in endothelial senescence**

The molecular changes involved or associated to the senescent program not only concern to the induction or repression of a specific set of genes. Many of the changes described in the literature report post-translational modifications, e. g., the advanced glycation endproducts

Endothelial senescence can also be triggered by telomere-independent events that in general involve damages in the DNA. In this sense, the oxidative stress is a major stimulus for the induction of this type of senescence, which is due to the generation of reactive oxygen species (ROS, including oxygen ions and peroxides) in the mitochondria (Collins & Tzima, 2011; Erusalimsky & Skene, 2009). Thus, the cellular metabolism is the central source of ROS, but often they have an extracellular origin such as the one induced by radiation. In any case, ROS can either provoke or accelerate the development of senescence by damaging the DNA (Figure 2), which triggers multiple response mechanisms that usually act through the retinoblastoma protein family pathways, the final effectors of the senescence program

In cell culture, ROS induce an acute form of senescence termed stress-induced premature senescence, which does not require extensive cell culture but which resembles somehow the replicative one (Toussaint et al., 2000). This type of senescence is relatively easy to analyze in *in vitro* assays because the sole treatment with hydrogen peroxide (H2O2) for a short lapse of time is enough to prompt this type of senescence (Chen et al., 1998). By contrast, using antioxidant agents such as the grape stilbenoid resveratrol protect from the oxidative stressinduced premature senescence (Kao et al., 2010). Also, several lines of evidence show that ROS can interact and deplete the nitric oxide (NO) generated by the endothelium in the vasodilator responses, so contributing to the endothelial dysfunction associated to ageing (Grisham et al., 1998; Steiner et al., 2002). This is in line with the availability of NO-donors to inhibit endothelial cell senescence (Hayashi et al., 2006). In fact, comparing elderly with young adults one can find that the NO levels, or its bioavailability, are decreased in the first group but, interestingly, without any difference regarding to the expression levels or activation state of the endothelial nitric oxide synthase (eNOS), the enzyme responsible of the NO generation (Sun et al., 2004; Taddei et al., 2001). In parallel, this decrease in the NO levels attenuates the negative interference that it exerts on the TGF-β signalling pathway

On the other hand, radiation is an exogenous trigger for ROS. In human skin fibroblasts, repeated exposure to ultraviolet-B light at subcytotoxic level is able to prompt premature senescence. Interestingly, this effect is mediated by the increase in the TGF-β expression and consequently by its downstream signalling pathway (Debacq-Chainiaux et al., 2005). In the vascular context, this source of ROS has been poorly studied beyond the methodological interest to induce premature senescence because endothelial cells enter rapidly in apoptosis due to their high sensitivity to radiation (Paris et al., 2001). In this regard, a recent study has demonstrated that ionizing radiation suppresses angiogenesis in mice and this effect is mediated through the TGF-β/ALK5-dependent inhibition of endothelial cell sprouting

The molecular changes involved or associated to the senescent program not only concern to the induction or repression of a specific set of genes. Many of the changes described in the literature report post-translational modifications, e. g., the advanced glycation endproducts

**3.2 Oxidative stress-induced senescence** 

(Campisi & d'Adda di Fagagna, 2007; Erusalimsky, 2009).

(Saura et al., 2005), which contributes to prompt the senescence program.

**4. Induction of S-endoglin and its role in endothelial senescence** 

(Imaizumi et al., 2010).

(AGEs) which have been implicated in age-related disease and aging itself; as well as the p53 acetylation in stress-induced senescence (Furukawa et al., 2007). In addition, a growing body of evidence supports the involvement of the post-transcriptional modifications that occur in senescence, i. e., the alternative splicing processes associated with senescence (Harries et al., 2011; Meshorer & Soreq, 2002). Thus, alterations in the splicing pattern have been described for several age-related diseases, such as the Hutchison Gilford progeria syndrome (Eriksson et al., 2003), or the Alzheimer's disease-related tauopathies (Chen et al., 2010).

Fig. 3. S-endoglin expression in senescence. (A) The expression of S-endoglin in blood vessels can be revealed by *in situ* hybridization in the endothelium of human coronary artery (black arrow) and in some smooth muscle cells (red arrow). (B) The increase in the percentage of senescent endothelial cells *in vitro* (blue graph) is concomitant with the induction of S-endoglin (red graph). (C) Primary cultures of human umbilical vein endothelial cells (HUVECs) maintained *in vitro* along passages co-express both endoglin isoforms comparing young (Y) versus senescent (S) cells in RT-PCR assays. In parallel, PAI-1 is increased, while Id1 and telomerase (hTERT) are down-regulated in senescent cell. As a control, the expression levels of the TGF-β type I receptors ALK1 and ALK5 are not altered. (Figure adapted from Blanco et al., 2008).

Nonetheless, little is known about the role of splicing in the vascular context during senescence. A recent study demonstrates that TGF-β induces the distal splice-site selection leading to an antiangiogenic variant of the vascular endothelial growth factor (VEGF) (Nowak et al., 2008), and this could be one of the reasons why there is a reduced capability to form tubular-like structure by senescent endothelial *in vitro* (Chang et al., 2005).

As described above, the role of TGF-β in senescence has been clearly established, modulating specific intracellular effectors and leading to the cell growth arrest. In a first

Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 507

(Blanco et al., 2008; Jerkic et al., 2006). Taken together, the induction of S-endoglin during endothelial senescence might be at the basis of the development of cardiovascular pathologies associated with ageing, including atherosclerosis and hypertension (Figure 4).

Briefly, the alternative splicing is a molecular process by which organisms notably increase the diversity and functionality of their proteome from a finite number of genes. This process is carried out by the spliceosome, a huge ribonucleoprotein complex that works with amazing fidelity: i) skipping or shuffling exons; ii) selecting alternative splice sites; or iii) retaining introns (Graveley, 2001; Kwan et al., 2007). In humans, there are two distinct spliceosome complexes, named the major (M-Sp) and the minor (m-Sp) spliceosome. The M-Sp is involved in the vast majority of the splicing events and comprises five snRNPs named U1, U2, U4, U5, and U6 and a multitude of non-snRNP splicing factors (Jurica & Moore, 2003; Matlin et al., 2005; Zhou et al., 2002). Likewise, the m-Sp is composed by four unique snRNPs, U11, U12, U4atac, and U6atac, besides the U5 snRNP shared by both spliceosomes (Hall & Padgett, 1996; Tarn & Steitz, 1996). The m-Sp was first associated with the maturation of the so-called non-canonical introns but its role on standard splicing has been recently reported (Sheth et al., 2006; Will & Luhrmann, 2005). Interestingly, the difference between the major spliceosome and the minor spliceosome is their spatial segregation. While the M-Sp is in the nucleus, the m-Sp can be detected in the cytosol (Caceres & Misteli, 2007; Konig et al., 2007). In both cases, the spliceosome assembly is driven by a set of snRNPs that sequentially recognize the 5' and 3' splice sites, as well as the branch point element in between them (Burge et al., 1999). These snRNPs constitute the basal machinery of the spliceosome, besides a number of essential proteins that takes part in the spliceosome assembly. Moreover, there are several groups of auxiliary proteins that may regulate the alternative splicing. These splicing factors, or *trans*-elements, recognize binding sites, or *cis*elements, spatially distributed inside the introns or exons and act as silencers or enhancers (Moore & Silver, 2008; Singh & Valcarcel, 2005; Sperling et al., 2008; Wang et al., 2006). Unfortunately, the alternative splicing during endothelial senescence has been poorly studied so far, but its importance has been suggested by the lifespan extension provoked by

**4.1 Regulation of endoglin alternative splicing in senescence** 

the overexpression of the splicing factor SNEV (Voglauer et al., 2006).

(Nowak et al., 2010).

One of the best characterized groups of splicing factors is the serine/arginine (SR) protein family, from which the alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototypical member (Graveley, 2000). ASF/SF2 is involved in both constitutive and alternative splicing processes. Although ASF/SF2 is mainly found in the nuclear speckles, it continuously shuttles between the nucleus and the cytoplasm depending on the phosphorylation and/or methylation states, which in turn determines its activity (Sanford et al., 2008; Sanford et al., 2005; Sinha et al., 2010). In this context, it has been recently reported the role of ASF/SF2 in the regulation of the S-endoglin intron retention during endothelial senescence (Blanco & Bernabeu, 2011). In endothelial senescent cells, the subcellular pattern of ASF/SF2 is mainly cytoplasmic, where ASF/SF2 interferes with the minor spliceosome inhibiting the elimination of the last intron of endoglin mRNA. The role of cytoplasmic ASF/SF2 as a senescent inductor is supported by its antiangiogenic properties, because the inhibition of the ASF/SF2 phosphorylation promotes its cytoplasmic localization and this is associated with increased expression levels of the antiangiogenic isoform VEGF165b

step, TGF-β binds to the specific receptor complex at the endothelial cell surface. Then, the signal is transmitted into the cytoplasm by different pathways depending on the type I receptor present in the complex. Thus, ALK5 signals via Smad2 and Smad3, whereas ALK1 mainly activates Smad1 and Smad5. In the TGF-β receptor complex, the presence of the predominantly expressed isoform, L-endoglin, favours the ALK1/Smad1 pathway and is related to the activation phase of the angiogenesis (Blanco et al., 2005; Lebrin et al., 2004). However, a post-transcriptional change during endothelial senescence, such as the retention of the last and small intron in the endoglin mRNA, has important consequences. Thus, the up-regulation of S-endoglin *in vitro* and *in vivo* is clearly associated with the ageing (Figures 3A and 3B). The co-expression of S- and L-endoglin in the senescent endothelial cells is able to tilt the angiogenic balance toward the resolution phase (ALK5/Smad3 pathway) in detriment of the ALK1/Smad1 route (Blanco et al., 2008). Also, S-endoglin induces the upregulation of the PAI-1 and the repression of Id1, changes clearly associated to the cell cycle arrest in senescence (Figure 3C and 4).

Fig. 4. Functional effects of S-endoglin in endothelial senescence. The S-endoglin upregulation in aged endothelial cells promotes the ALK5/Smad3 signalling pathway. As a consequent, the vascular physiology is affected decreasing the angiogenesis, increasing the fibrosis and unbalancing the eNOS/COX-2 system which is related to hypertension. (Figure adapted from Blanco et al., 2008)

Furthermore, transgenic mice that overexpress the human S-endoglin isoform (*S-Eng*+) experience a significant increase in the mean arterial pressure and a failure in the control on the NO-dependent vascular homeostasis, similarly to what happens in the endoglin deficient mouse model (*Eng*+/-) that resembles the HHT disease (Blanco et al., 2008; Santibanez et al., 2007). Supporting this, a common compensatory mechanism takes place in *S-Eng*+ and *Eng*+/- mice involving the up-regulation of the cyclooxigenase (COX)-2 enzyme (Blanco et al., 2008; Jerkic et al., 2006). Taken together, the induction of S-endoglin during endothelial senescence might be at the basis of the development of cardiovascular pathologies associated with ageing, including atherosclerosis and hypertension (Figure 4).

## **4.1 Regulation of endoglin alternative splicing in senescence**

506 Senescence

step, TGF-β binds to the specific receptor complex at the endothelial cell surface. Then, the signal is transmitted into the cytoplasm by different pathways depending on the type I receptor present in the complex. Thus, ALK5 signals via Smad2 and Smad3, whereas ALK1 mainly activates Smad1 and Smad5. In the TGF-β receptor complex, the presence of the predominantly expressed isoform, L-endoglin, favours the ALK1/Smad1 pathway and is related to the activation phase of the angiogenesis (Blanco et al., 2005; Lebrin et al., 2004). However, a post-transcriptional change during endothelial senescence, such as the retention of the last and small intron in the endoglin mRNA, has important consequences. Thus, the up-regulation of S-endoglin *in vitro* and *in vivo* is clearly associated with the ageing (Figures 3A and 3B). The co-expression of S- and L-endoglin in the senescent endothelial cells is able to tilt the angiogenic balance toward the resolution phase (ALK5/Smad3 pathway) in detriment of the ALK1/Smad1 route (Blanco et al., 2008). Also, S-endoglin induces the upregulation of the PAI-1 and the repression of Id1, changes clearly associated to the cell cycle

> Angiogenesis Fibrosis Hypertension

Fig. 4. Functional effects of S-endoglin in endothelial senescence. The S-endoglin upregulation in aged endothelial cells promotes the ALK5/Smad3 signalling pathway. As a consequent, the vascular physiology is affected decreasing the angiogenesis, increasing the fibrosis and unbalancing the eNOS/COX-2 system which is related to hypertension. (Figure

Furthermore, transgenic mice that overexpress the human S-endoglin isoform (*S-Eng*+) experience a significant increase in the mean arterial pressure and a failure in the control on the NO-dependent vascular homeostasis, similarly to what happens in the endoglin deficient mouse model (*Eng*+/-) that resembles the HHT disease (Blanco et al., 2008; Santibanez et al., 2007). Supporting this, a common compensatory mechanism takes place in *S-Eng*+ and *Eng*+/- mice involving the up-regulation of the cyclooxigenase (COX)-2 enzyme

**Senescence**

**S-endoglin**

ALK5 ALK1

PAI-1/ECM synthesis eNOS COX-2

**Smad3** Smad1

arrest in senescence (Figure 3C and 4).

**L-endoglin**

ALK5 ALK1

Id1 eNOS COX-2

adapted from Blanco et al., 2008)

Smad3 **Smad1**

Briefly, the alternative splicing is a molecular process by which organisms notably increase the diversity and functionality of their proteome from a finite number of genes. This process is carried out by the spliceosome, a huge ribonucleoprotein complex that works with amazing fidelity: i) skipping or shuffling exons; ii) selecting alternative splice sites; or iii) retaining introns (Graveley, 2001; Kwan et al., 2007). In humans, there are two distinct spliceosome complexes, named the major (M-Sp) and the minor (m-Sp) spliceosome. The M-Sp is involved in the vast majority of the splicing events and comprises five snRNPs named U1, U2, U4, U5, and U6 and a multitude of non-snRNP splicing factors (Jurica & Moore, 2003; Matlin et al., 2005; Zhou et al., 2002). Likewise, the m-Sp is composed by four unique snRNPs, U11, U12, U4atac, and U6atac, besides the U5 snRNP shared by both spliceosomes (Hall & Padgett, 1996; Tarn & Steitz, 1996). The m-Sp was first associated with the maturation of the so-called non-canonical introns but its role on standard splicing has been recently reported (Sheth et al., 2006; Will & Luhrmann, 2005). Interestingly, the difference between the major spliceosome and the minor spliceosome is their spatial segregation. While the M-Sp is in the nucleus, the m-Sp can be detected in the cytosol (Caceres & Misteli, 2007; Konig et al., 2007). In both cases, the spliceosome assembly is driven by a set of snRNPs that sequentially recognize the 5' and 3' splice sites, as well as the branch point element in between them (Burge et al., 1999). These snRNPs constitute the basal machinery of the spliceosome, besides a number of essential proteins that takes part in the spliceosome assembly. Moreover, there are several groups of auxiliary proteins that may regulate the alternative splicing. These splicing factors, or *trans*-elements, recognize binding sites, or *cis*elements, spatially distributed inside the introns or exons and act as silencers or enhancers (Moore & Silver, 2008; Singh & Valcarcel, 2005; Sperling et al., 2008; Wang et al., 2006). Unfortunately, the alternative splicing during endothelial senescence has been poorly studied so far, but its importance has been suggested by the lifespan extension provoked by the overexpression of the splicing factor SNEV (Voglauer et al., 2006).

One of the best characterized groups of splicing factors is the serine/arginine (SR) protein family, from which the alternative splicing factor/splicing factor 2 (ASF/SF2) is the prototypical member (Graveley, 2000). ASF/SF2 is involved in both constitutive and alternative splicing processes. Although ASF/SF2 is mainly found in the nuclear speckles, it continuously shuttles between the nucleus and the cytoplasm depending on the phosphorylation and/or methylation states, which in turn determines its activity (Sanford et al., 2008; Sanford et al., 2005; Sinha et al., 2010). In this context, it has been recently reported the role of ASF/SF2 in the regulation of the S-endoglin intron retention during endothelial senescence (Blanco & Bernabeu, 2011). In endothelial senescent cells, the subcellular pattern of ASF/SF2 is mainly cytoplasmic, where ASF/SF2 interferes with the minor spliceosome inhibiting the elimination of the last intron of endoglin mRNA. The role of cytoplasmic ASF/SF2 as a senescent inductor is supported by its antiangiogenic properties, because the inhibition of the ASF/SF2 phosphorylation promotes its cytoplasmic localization and this is associated with increased expression levels of the antiangiogenic isoform VEGF165b (Nowak et al., 2010).

Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 509

#13 and #14. Thus, the up-regulated expression of S-endoglin is considered to be part of the endothelial senescence program. Moreover, *in vitro* and *in vivo* studies suggest that Sendoglin contributes to vascular pathology associated with ageing. In this regard, mutations in the human *ENG* gene are responsible for HHT-1, an autosomic dominant vascular disease whose symptoms increase and become worse with age. Currently, the haploinsufficiency of the predominantly expressed L-endoglin isoform is widely accepted as the pathogenic mechanism of the disease. Because S-endoglin is up-regulated in aged mice as well as during senescence of endothelial cells and S-endoglin counteracts the function of Lendoglin, the increased S-endoglin expression during ageing would increase the functional L-endoglin haploinsufficiency in HHT-1 and could explain why the symptoms become worse with ageing. Therefore, one could predict that the age-dependent penetrance of the

HHT-1 is due, at least in part, to the S-endoglin induction mediated by ASF SF2.

modulator of the vascular pathology associated with endothelial senescence.

professor in the *Centro de Investigaciones Biológicas*, CSIC, Spain.

1993), pp. 2340-2345, ISSN 0014-2980.

(April 1990), pp. 49-59, ISSN 0092-8674.

(July 2009), pp. 954-973, ISSN 0006-3002.

ISSN 1524-4571.

**6. Acknowledgments** 

**7. References** 

In summary, these data suggest an important role for the TGF-β co-receptor endoglin as a

This work was supported by grants of the Spanish Ministry of Science and Innovation to CB (SAF2010-19222) and *Genoma España* (MEICA). FJB is a post-doctoral researcher of the *Centro de Investigación Biomédica en Red* (CIBER) *de Enfermedades Raras*, ISCIII, Spain. CB is a research

Bellon, T.; Corbi, A.; Lastres, P.; Cales, C.; Cebrian, M.; Vera, S.; Cheifetz, S.; Massague, J.;

Benezra, R.; Davis, R.L.; Lockshon, D.; Turner, D.L. & Weintraub, H. (1990). The protein Id: a

Bernabeu, C.; Lopez-Novoa, J.M. & Quintanilla, M. (2009). The emerging role of TGF-beta

Blanco, F.J. & Bernabeu, C. (2011). Alternative splicing factor or splicing factor-2 plays a key

Blanco, F.J.; Grande, M.T.; Langa, C.; Oujo, B.; Velasco, S.; Rodriguez-Barbero, A.; Perez-

Blanco, F.J.; Santibanez, J.F.; Guerrero-Esteo, M.; Langa, C.; Vary, C.P. & Bernabeu, C. (2005).

*Cell*, Vol. 10, No. 5, (June 2011), pp. 896-907, ISSN 1474-9726.

Letarte, M. & Bernabeu, C. (1993). Identification and expression of two forms of the human transforming growth factor-beta-binding protein endoglin with distinct cytoplasmic regions. *European Journal of Immunology*, Vol. 23, No. 9, (September

negative regulator of helix-loop-helix DNA binding proteins. *Cell*, Vol. 61, No. 1,

superfamily coreceptors in cancer. *Biochimica et Biophysica Acta*, Vol. 1792, No. 10,

role in intron retention of the endoglin gene during endothelial senescence. *Aging* 

Gomez, E.; Quintanilla, M.; Lopez-Novoa, J.M. & Bernabeu, C. (2008). S-endoglin expression is induced in senescent endothelial cells and contributes to vascular pathology. *Circulation Research*, Vol. 103, No. 12, (November 2008), pp. 1383-1392,

Interaction and functional interplay between endoglin and ALK-1, two components

Fig. 5. Regulation of the alternative splicing of endoglin in senescent endothelial cells. In this hypothetical model, the last intron of the *ENG* gene is eliminated in the mature mRNA, so that L-endoglin is the predominantly expressed isoform. In this mRNA processing, both spliceosomes (nuclear M-Sp and cytoplasmic m-Sp) can be involved. However, in senescent endothelial cells, the splicing factor ASF/SF2 (green) is translocated to the cytoplasm, stabilizing the S-endoglin mRNA and interfering with the m-Sp activity. Consequently, ASF/SF2 promotes the intron retention, thus up-regulating the levels of S-endoglin mRNA (adapted from Blanco & Bernabeu, 2011).

## **5. Conclusions**

Vascular physiology progressively declines with age due to multiple factors including an increase in oxidative stress, DNA damage, and advanced cellular replication involving telomere attrition. All these events converge in the key molecule p53, which acts typically arresting the cell cycle and triggering the endothelial senescence. At this stage, the expression of many specific genes is modulated, regarding not only to their expression levels but also the post-translational modifications and alternative processing of their premature mRNA molecules, which give rise to interesting protein variants. Nowadays, it can be postulated that this phenomenon is at the cellular basis of several age-associated cardiovascular pathologies, such as hypertension or atherosclerosis.

TGF-β is able to induce endothelial senescence via a cell surface receptor complex that includes the type I (ALK1 and ALK5) and the type II signalling receptors as well as endoglin. Endoglin is a TGF-β co-receptor highly expressed as L-(long)-endoglin by endothelial cells which is associated with active angiogenesis foci and vascular remodelling processes. Conversely, an alternative spliced and shorter isoform (S-endoglin) with opposite effects to those of L-endoglin in the context of the TGF-β system has been described. Usually, S-endoglin is almost undetectable in endothelial cells, but is induced during senescence. In this up-regulation, the senescence-induced cytoplasmic localization of the splicing factor ASF/SF2 plays a key role favouring the retention of the intron between exons #13 and #14. Thus, the up-regulated expression of S-endoglin is considered to be part of the endothelial senescence program. Moreover, *in vitro* and *in vivo* studies suggest that Sendoglin contributes to vascular pathology associated with ageing. In this regard, mutations in the human *ENG* gene are responsible for HHT-1, an autosomic dominant vascular disease whose symptoms increase and become worse with age. Currently, the haploinsufficiency of the predominantly expressed L-endoglin isoform is widely accepted as the pathogenic mechanism of the disease. Because S-endoglin is up-regulated in aged mice as well as during senescence of endothelial cells and S-endoglin counteracts the function of Lendoglin, the increased S-endoglin expression during ageing would increase the functional L-endoglin haploinsufficiency in HHT-1 and could explain why the symptoms become worse with ageing. Therefore, one could predict that the age-dependent penetrance of the HHT-1 is due, at least in part, to the S-endoglin induction mediated by ASF SF2.

In summary, these data suggest an important role for the TGF-β co-receptor endoglin as a modulator of the vascular pathology associated with endothelial senescence.

## **6. Acknowledgments**

This work was supported by grants of the Spanish Ministry of Science and Innovation to CB (SAF2010-19222) and *Genoma España* (MEICA). FJB is a post-doctoral researcher of the *Centro de Investigación Biomédica en Red* (CIBER) *de Enfermedades Raras*, ISCIII, Spain. CB is a research professor in the *Centro de Investigaciones Biológicas*, CSIC, Spain.

## **7. References**

508 Senescence

**S-Eng**

Fig. 5. Regulation of the alternative splicing of endoglin in senescent endothelial cells. In this hypothetical model, the last intron of the *ENG* gene is eliminated in the mature mRNA, so that L-endoglin is the predominantly expressed isoform. In this mRNA processing, both spliceosomes (nuclear M-Sp and cytoplasmic m-Sp) can be involved. However, in senescent endothelial cells, the splicing factor ASF/SF2 (green) is translocated to the cytoplasm, stabilizing the S-endoglin mRNA and interfering with the m-Sp activity. Consequently, ASF/SF2 promotes the intron retention, thus up-regulating the levels of S-endoglin mRNA

Vascular physiology progressively declines with age due to multiple factors including an increase in oxidative stress, DNA damage, and advanced cellular replication involving telomere attrition. All these events converge in the key molecule p53, which acts typically arresting the cell cycle and triggering the endothelial senescence. At this stage, the expression of many specific genes is modulated, regarding not only to their expression levels but also the post-translational modifications and alternative processing of their premature mRNA molecules, which give rise to interesting protein variants. Nowadays, it can be postulated that this phenomenon is at the cellular basis of several age-associated

TGF-β is able to induce endothelial senescence via a cell surface receptor complex that includes the type I (ALK1 and ALK5) and the type II signalling receptors as well as endoglin. Endoglin is a TGF-β co-receptor highly expressed as L-(long)-endoglin by endothelial cells which is associated with active angiogenesis foci and vascular remodelling processes. Conversely, an alternative spliced and shorter isoform (S-endoglin) with opposite effects to those of L-endoglin in the context of the TGF-β system has been described. Usually, S-endoglin is almost undetectable in endothelial cells, but is induced during senescence. In this up-regulation, the senescence-induced cytoplasmic localization of the splicing factor ASF/SF2 plays a key role favouring the retention of the intron between exons

Senescence

**M-Sp**

**m-Sp**

L-Eng

**M-Sp**

**m-Sp**

cardiovascular pathologies, such as hypertension or atherosclerosis.

**m-Sp**

L-Eng

**5. Conclusions** 

(adapted from Blanco & Bernabeu, 2011).

L-Eng


Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 511

Eriksson, M.; Brown, W.T.; Gordon, L.B.; Glynn, M.W.; Singer, J.; Scott, L.; Erdos, M.R.;

Erusalimsky, J.D. (2009). Vascular endothelial senescence: from mechanisms to

Erusalimsky, J.D. & Kurz, D.J. (2005). Cellular senescence in vivo: its relevance in ageing and

Erusalimsky, J.D. & Skene, C. (2009). Mechanisms of endothelial senescence. *Experimental Physiology*, Vol. 94, No. 3, (October 2008), pp. 299-304, ISSN 1469-445X. Ewald, J.A.; Desotelle, J.A.; Wilding, G. & Jarrard, D.F. (2010). Therapy-induced senescence

Ferrari, A.U.; Radaelli, A. & Centola, M. (2003). Invited review: aging and the cardiovascular

Folini, M.; Venturini, L.; Cimino-Reale, G. & Zaffaroni, N. (2011). Telomeres as targets for

Foreman, K.E. & Tang, J. (2003). Molecular mechanisms of replicative senescence in

Foteinos, G.; Hu, Y.; Xiao, Q.; Metzler, B. & Xu, Q. (2008). Rapid endothelial turnover in

Fraisl, P.; Mazzone, M.; Schmidt, T. & Carmeliet, P. (2009). Regulation of angiogenesis by

Fujiki, T.; Miura, T.; Maura, M.; Shiraishi, H.; Nishimura, S.; Imada, Y.; Uehara, N.; Tashiro,

Furukawa, A.; Tada-Oikawa, S.; Kawanishi, S. & Oikawa, S. (2007). H2O2 accelerates

*Science*, Vol. 118, No. 4, (January 2005), pp. 743-758, ISSN 0021-9533. Dimri, G.P.; Lee, X.; Basile, G.; Acosta, M.; Scott, G.; Roskelley, C.; Medrano, E.E.; Linskens,

423, No. 6937, (April 2003), pp. 293-298, ISSN 0028-0836.

9367, ISSN 0027-8424.

pp. 326-332, ISSN 8750-7587.

pp. 1536-1546, ISSN 1460-2105.

2011), pp. 579-593, ISSN 1744-7631.

1251-1257, ISSN 0531-5565.

179, ISSN 1878-1551.

pp. 5258-5266, ISSN 0950-9232.

634-642, ISSN 0531-5565.

ISSN 8750-7587.

4539.

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway. *Journal of Cell* 

M.; Rubelj, I.; Pereira-Smith, O. & et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 92, No. 20, (September 1995), pp. 9363-

Robbins, C.M.; Moses, T.Y.; Berglund, P.; Dutra, A.; Pak, E.; Durkin, S.; Csoka, A.B.; Boehnke, M.; Glover, T.W. & Collins, F.S. (2003). Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. *Nature*, Vol.

pathophysiology. *Journal of Applied Physiology*, Vol. 106, No. 1, (November 2008),

cardiovascular disease. *Experimental Gerontology*, Vol. 40, No. 8-9, (June 2005), pp.

in cancer. *Journal of the National Cancer Institute*, Vol. 102, No. 20, (September 2010),

system. *Journal of Applied Physiology*, Vol. 95, No. 6, (December 2003), pp. 2591-2597,

anticancer therapies. *Expert Opinion on Therapeutic Targets*, Vol. 15, No. 5, (February

endothelial cells. *Experimental Gerontology*, Vol. 38, No. 11-12, (November 2003), pp.

atherosclerosis-prone areas coincides with stem cell repair in apolipoprotein Edeficient mice. *Circulation*, Vol. 117, No. 14, (April 2008), pp. 1856-1863, ISSN 1524-

oxygen and metabolism. *Developmental Cell*, Vol. 16, No. 2, (February 2009), pp. 167-

K.; Shirahata, S. & Katakura, Y. (2007). TAK1 represses transcription of the human telomerase reverse transcriptase gene. *Oncogene*, Vol. 26, No. 36, (February 2007),

cellular senescence by accumulation of acetylated p53 via decrease in the function

of the endothelial transforming growth factor-beta receptor complex. *Journal of Cellular Physiology*, Vol. 204, No. 2, (February 2005), pp. 574-584, ISSN 0021-9541.


Burge, C.; Tuschl, T. & Sharp, P. (1999). Splicing of precursors to mRNAs by the

Caceres, J.F. & Misteli, T. (2007). Division of labor: minor splicing in the cytoplasm. *Cell*, Vol.

Campisi, J. & d'Adda di Fagagna, F. (2007). Cellular senescence: when bad things happen to

Carmeliet, P. & Jain, R.K. (2011). Molecular mechanisms and clinical applications of angiogenesis. *Nature*, Vol. 473, No. 7347, (May 2011), pp. 298-307, ISSN 1476-4687. Cipriano, R.; Kan, C.E.; Graham, J.; Danielpour, D.; Stampfer, M. & Jackson, M.W. (2011).

Collins, C. & Tzima, E. (2011). Hemodynamic forces in endothelial dysfunction and vascular

Comi, P.; Chiaramonte, R. & Maier, J.A. (1995). Senescence-dependent regulation of type 1

Chang, M.W.; Grillari, J.; Mayrhofer, C.; Fortschegger, K.; Allmaier, G.; Marzban, G.;

Chen, J. & Goligorsky, M.S. (2006). Premature senescence of endothelial cells: Methusaleh's

Chen, Q.M.; Bartholomew, J.C.; Campisi, J.; Acosta, M.; Reagan, J.D. & Ames, B.N. (1998).

Chen, S.; Townsend, K.; Goldberg, T.E.; Davies, P. & Conejero-Goldberg, C. (2010). MAPT

Debacq-Chainiaux, F.; Borlon, C.; Pascal, T.; Royer, V.; Eliaers, F.; Ninane, N.; Carrard, G.;

*Cell Research*, Vol. 219, No. 1, (July 1995), pp. 304-308, ISSN 0014-4827. Conway, E.M. & Carmeliet, P. (2004). The diversity of endothelial cells: a challenge for

66, No. 2, (May 2005), pp. 286-294, ISSN 0008-6363.

131, No. 4, (November 2007), pp. 645-647, ISSN 0092-8674.

Vol. 108, No. 21, (May 2011), pp. 8668-8673, ISSN 1091-6490.

United States of America.

729-740, ISSN 1471-0080.

1873-6815.

ISSN 1465-6914.

1875-8908.

(June 2005), pp. 121-136, ISSN 0014-4827.

No. 5, (April 2006), pp. H1729-1739, ISSN 0363-6135.

Vol. 332 ( Pt 1), (May 1998), pp. 43-50, ISSN 0264-6021.

of the endothelial transforming growth factor-beta receptor complex. *Journal of Cellular Physiology*, Vol. 204, No. 2, (February 2005), pp. 574-584, ISSN 0021-9541. Brandes, R.P.; Fleming, I. & Busse, R. (2005). Endothelial aging. *Cardiovascular Research*, Vol.

spliceosoomes, In: *The RNA World*. R. Gesteland, T. Cech, and J. Atkins (eds.), pp. 525-560. Cold Spring Harbor Laboratory Press, ISBN 0-87969-589-7, Plainview, NY,

good cells. *Nature Reviews Molecular Cell Biology*, Vol. 8, No. 9, (August 2007), pp.

TGF-beta signaling engages an ATM-CHK2-p53-independent RAS-induced senescence and prevents malignant transformation in human mammary epithelial cells. *Proceedings of the National Academy of Sciences of the United States of America*,

aging. *Experimental Gerontology*, Vol. 46, No. 2-3, (October 2010), pp. 185-188, ISSN

plasminogen activator inhibitor in human vascular endothelial cells. *Experimental* 

therapeutic angiogenesis. *Genome Biology*, Vol. 5, No. 2, (January 2004), pp. 207,

Katinger, H. & Voglauer, R. (2005). Comparison of early passage, senescent and hTERT immortalized endothelial cells. *Experimental Cell Research*, Vol. 309, No. 1,

dilemma. *American Journal of Physiology. Heart and Circulatory Physiology*, Vol. 290,

Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrest but not cell replication. *Biochemical Journal*,

isoforms: differential transcriptional profiles related to 3R and 4R splice variants. *Journal of Alzheimer's Disease*, Vol. 22, No. 4, (October 2010), pp. 1313-1329, ISSN

Friguet, B.; de Longueville, F.; Boffe, S.; Remacle, J. & Toussaint, O. (2005).

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway. *Journal of Cell Science*, Vol. 118, No. 4, (January 2005), pp. 743-758, ISSN 0021-9533.


Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 513

Kao, C.L.; Chen, L.K.; Chang, Y.L.; Yung, M.C.; Hsu, C.C.; Chen, Y.C.; Lo, W.L.; Chen, S.J.;

Kim, K.S.; Kang, K.W.; Seu, Y.B.; Baek, S.H. & Kim, J.R. (2009). Interferon-gamma induces

Koleva, R.I.; Conley, B.A.; Romero, D.; Riley, K.S.; Marto, J.A.; Lux, A. & Vary, C.P. (2006).

Konig, H.; Matter, N.; Bader, R.; Thiele, W. & Muller, F. (2007). Splicing segregation: the

Kordon, E.C.; McKnight, R.A.; Jhappan, C.; Hennighausen, L.; Merlino, G. & Smith, G.H.

Kortlever, R.M.; Nijwening, J.H. & Bernards, R. (2008). Transforming growth factor-beta

Lareau, L.F.; Green, R.E.; Bhatnagar, R.S. & Brenner, S.E. (2004). The evolving roles of

Lebrin, F.; Goumans, M.J.; Jonker, L.; Carvalho, R.L.; Valdimarsdottir, G.; Thorikay, M.;

Li, H. & Liu, J.P. (2007). Mechanisms of action of TGF-beta in cancer: evidence for Smad3 as

Li, H.; Xu, D.; Li, J.; Berndt, M.C. & Liu, J.P. (2006). Transforming growth factor beta

Lopez-Novoa, J.M. & Bernabeu, C. (2010). The physiological role of endoglin in the

*Physiology*, Vol. 299, No. 4, (July 2010), pp. H959-974, ISSN 1522-1539. Llorca, O.; Trujillo, A.; Blanco, F.J. & Bernabeu, C. (2007). Structural model of human

3873.

2008), pp. 179-188, ISSN 0047-6374.

No. 35, (June 2006), pp. 25110-25123, ISSN 0021-9258.

No. 8, (August 2007), pp. 1210-1218, ISSN 1088-9051.

23, No. 20, (September 2004), pp. 4018-4028, ISSN 0261-4189.

pp. 273-282, ISSN 0959-440X.

(October 2007), pp. 56-68, ISSN 0077-8923.

(June 2006), pp. 25588-25600, ISSN 0021-9258.

131, No. 4, (November 2007), pp. 718-729, ISSN 0092-8674.

*Biology*, Vol. 168, No. 1, (March 1995), pp. 47-61, ISSN 0012-1606.

Ku, H.H. & Hwang, S.J. (2010). Resveratrol protects human endothelium from H(2)O(2)-induced oxidative stress and senescence via SirT1 activation. *Journal of Atherosclerosis and Thrombosis*, Vol. 17, No. 9, (July 2010), pp. 970-979, ISSN 1880-

cellular senescence through p53-dependent DNA damage signaling in human endothelial cells. *Mechanisms of Ageing and Development*, Vol. 130, No. 3, (December

Endoglin structure and function: Determinants of endoglin phosphorylation by transforming growth factor-beta receptors. *Journal of Biological Chemistry*, Vol. 281,

minor spliceosome acts outside the nucleus and controls cell proliferation. *Cell*, Vol.

(1995). Ectopic TGF beta 1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population. *Developmental* 

requires its target plasminogen activator inhibitor-1 for cytostatic activity. *Journal of Biological Chemistry*, Vol. 283, No. 36, (July 2008), pp. 24308-24313, ISSN 0021-9258. Kwan, T.; Benovoy, D.; Dias, C.; Gurd, S.; Serre, D.; Zuzan, H.; Clark, T.A.; Schweitzer, A.;

Staples, M.K.; Wang, H.; Blume, J.E.; Hudson, T.J.; Sladek, R. & Majewski, J. (2007). Heritability of alternative splicing in the human genome. *Genome Research*, Vol. 17,

alternative splicing. *Current Opinion in Structural Biology*, Vol. 14, No. 3, (June 2004),

Mummery, C.; Arthur, H.M. & ten Dijke, P. (2004). Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction. *The EMBO Journal*, Vol.

a repressor of the hTERT gene. *Annals of the New York Academy of Sciences*, Vol. 1114,

suppresses human telomerase reverse transcriptase (hTERT) by Smad3 interactions with c-Myc and the hTERT gene. *Journal of Biological Chemistry*, Vol. 281, No. 35,

cardiovascular system. *American Journal of Physiology. Heart and Circulatory* 

endoglin, a transmembrane receptor responsible for hereditary hemorrhagic

of SIRT1 by NAD+ depletion. *Cellular Physiology and Biochemistry*, Vol. 20, No. 1-4, (June 2007), pp. 45-54, ISSN 1015-8987.


Ghosh, A.K. & Vaughan, D.E. (2011). PAI-1 in Tissue Fibrosis. *Journal of Cellular Physiology*,

Graveley, B.R. (2000). Sorting out the complexity of SR protein functions. *RNA*, Vol. 6, No. 9,

Graveley, B.R. (2001). Alternative splicing: increasing diversity in the proteomic world. *Trends in Genetics*, Vol. 17, No. 2, (February 2001), pp. 100-107, ISSN 0168-9525. Grisham, M.B.; Granger, D.N. & Lefer, D.J. (1998). Modulation of leukocyte-endothelial

Hall, S.L. & Padgett, R.A. (1996). Requirement of U12 snRNA for in vivo splicing of a minor

Harries, L.W.; Hernandez, D.; Henley, W.; Wood, A.R.; Holly, A.C.; Bradley-Smith, R.M.;

Hayashi, T.; Matsui-Hirai, H.; Miyazaki-Akita, A.; Fukatsu, A.; Funami, J.; Ding, Q.F.;

Hayflick, L. (2003). Living forever and dying in the attempt. *Experimental Gerontology*, Vol.

Imaizumi, N.; Monnier, Y.; Hegi, M.; Mirimanoff, R.O. & Ruegg, C. (2010). Radiotherapy

Jerkic, M.; Rivas-Elena, J.V.; Santibanez, J.F.; Prieto, M.; Rodriguez-Barbero, A.; Perez-

Jurica, M.S. & Moore, M.J. (2003). Pre-mRNA splicing: awash in a sea of proteins. *Molecular* 

Kang, J.S.; Liu, C. & Derynck, R. (2009). New regulatory mechanisms of TGF-beta receptor

Kang, Y.; Chen, C.R. & Massague, J. (2003). A self-enabling TGFbeta response coupled to

*Cell*, Vol. 10, No. 5, (June 2011), pp. 868-878, ISSN 1474-9726.

38, No. 11-12, (December 2003), pp. 1231-1241, ISSN 0531-5565.

*Cell*, Vol. 12, No. 1, (July 2003), pp. 5-14, ISSN 1097-2765.

(June 2007), pp. 45-54, ISSN 1015-8987.

404-433, ISSN 0891-5849.

0027-8424.

1932-6203.

1879-3088.

2765.

1996), pp. 1716-1718, ISSN 0036-8075.

(April 2011), DOI: 10.1002/jcp.22783, ISSN 1097-4652.

(September 2000), pp. 1197-1211, ISSN 1355-8382.

of SIRT1 by NAD+ depletion. *Cellular Physiology and Biochemistry*, Vol. 20, No. 1-4,

interactions by reactive metabolites of oxygen and nitrogen: relevance to ischemic heart disease. *Free Radical Biology & Medicine*, Vol. 25, No. 4-5, (September 1998), pp.

class of eukaryotic nuclear pre-mRNA introns. *Science*, Vol. 271, No. 5256, (March

Yaghootkar, H.; Dutta, A.; Murray, A.; Frayling, T.M.; Guralnik, J.M.; Bandinelli, S.; Singleton, A.; Ferrucci, L. & Melzer, D. (2011). Human aging is characterized by focused changes in gene expression and deregulation of alternative splicing. *Aging* 

Kamalanathan, S.; Hattori, Y.; Ignarro, L.J. & Iguchi, A. (2006). Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 103, No. 45, (November 2006), pp. 17018-17023, ISSN

suppresses angiogenesis in mice through TGF-betaRI/ALK5-dependent inhibition of endothelial cell sprouting. *PLoS One*, Vol. 5, No. 6, (June 2010), pp. e11084, ISSN

Barriocanal, F.; Pericacho, M.; Arevalo, M.; Vary, C.P.; Letarte, M.; Bernabeu, C. & Lopez-Novoa, J.M. (2006). Endoglin regulates cyclooxygenase-2 expression and activity. *Circulation Research*, Vol. 99, No. 3, (July 2006), pp. 248-256, ISSN 1524-4571.

function. *Trends in Cell Biology*, Vol. 19, No. 8, (August 2009), pp. 385-394, ISSN

stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells. *Molecular Cell*, Vol. 11, No. 4, (April 2003), pp. 915-926, ISSN 1097-


Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 515

Pardali, E.; Goumans, M.J. & ten Dijke, P. (2010). Signaling by members of the TGF-beta

Paris, F.; Fuks, Z.; Kang, A.; Capodieci, P.; Juan, G.; Ehleiter, D.; Haimovitz-Friedman, A.;

Pascual, G.; Mendieta, C.; Garcia-Honduvilla, N.; Corrales, C.; Bellon, J.M. & Bujan, J. (2007).

Perez-Gomez, E.; Del Castillo, G.; Santibanez, J.F.; Lopez-Novoa, J.M.; Bernabeu, C. &

Rivard, A.; Fabre, J.E.; Silver, M.; Chen, D.; Murohara, T.; Kearney, M.; Magner, M.; Asahara,

Rodríguez-Mañas, L.; El-Assar, M.; Vallejo, S.; López-Dóriga, P.; Solís, J.; Petidier, R.;

Rosso, A.; Balsamo, A.; Gambino, R.; Dentelli, P.; Falcioni, R.; Cassader, M.; Pegoraro, L.;

Sakabe, N.J. & de Souza, S.J. (2007). Sequence features responsible for intron retention in human. *BMC Genomics*, Vol. 8, (February 2007), pp. 59, ISSN 1471-2164. Sanford, J.R.; Coutinho, P.; Hackett, J.A.; Wang, X.; Ranahan, W. & Caceres, J.F. (2008).

Santibanez, J.F.; Letamendia, A.; Perez-Barriocanal, F.; Silvestri, C.; Saura, M.; Vary, C.P.;

Saura, M.; Zaragoza, C.; Herranz, B.; Griera, M.; Diez-Marques, L.; Rodriguez-Puyol, D. &

(September 2010), pp. 556-567, ISSN 1879-3088.

44, No. 3, (March 2007), pp. 192-201, ISSN 1018-1172.

99, No. 1, (January 1999), pp. 111-120, ISSN 0009-7322.

No. 7, (December 2005), pp. 4339-4347, ISSN 0021-9258.

(July 2001), pp. 293-297, ISSN 0036-8075.

2005), pp. 4450-4461, ISSN 0950-9232.

pp. 226-238, ISSN 1474-9726.

0027-8424.

ISSN 0021-9541.

family in vascular morphogenesis and disease. *Trends in Cell Biology*, Vol. 20, No. 9,

Cordon-Cardo, C. & Kolesnick, R. (2001). Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. *Science*, Vol. 293, No. 5528,

TGF-beta1 upregulation in the aging varicose vein. *Journal of Vascular Research*, Vol.

Quintanilla, M. (2010). The role of the TGF-beta coreceptor endoglin in cancer. *The Scientific World Journal*, Vol. 10, (December 2010), pp. 2367-2384, ISSN 1537-744X. Perez-Gomez, E.; Eleno, N.; Lopez-Novoa, J.M.; Ramirez, J.R.; Velasco, B.; Letarte, M.;

Bernabeu, C. & Quintanilla, M. (2005). Characterization of murine S-endoglin isoform and its effects on tumor development. *Oncogene*, Vol. 24, No. 27, (April

T. & Isner, J.M. (1999). Age-dependent impairment of angiogenesis. *Circulation*, Vol.

Montes, M.; Nevado, J.; Castro, M.; Gómez-Guerrero, C.; Peiró, C. & Sánchez-Ferrer, C.F. (2009). Endothelial dysfunction in aged humans is related with oxidative stress and vascular inflammation. *Aging Cell*, Vol. 8, No. 3, (June 2009),

Pagano, G. & Brizzi, M.F. (2006). p53 Mediates the accelerated onset of senescence of endothelial progenitor cells in diabetes. *Journal of Biological Chemistry*, Vol. 281,

Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF. *PLoS One*, Vol. 3, No. 10, (October 2008), pp. e3369, ISSN 1932-6203. Sanford, J.R.; Ellis, J.D.; Cazalla, D. & Caceres, J.F. (2005). Reversible phosphorylation

differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative splicing factor. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 102, No. 42, (October 2005), pp. 15042-15047, ISSN

Lopez-Novoa, J.M.; Attisano, L. & Bernabeu, C. (2007). Endoglin increases eNOS expression by modulating Smad2 protein levels and Smad2-dependent TGF-beta signaling. *Journal of Cellular Physiology*, Vol. 210, No. 2, (October 2006), pp. 456-468,

Rodriguez-Puyol, M. (2005). Nitric oxide regulates transforming growth factor-beta

telangiectasia. *Journal of Molecular Biology*, Vol. 365, No. 3, (November 2006), pp. 694-705, ISSN 0022-2836.


Mahmoud, M.; Allinson, K.R.; Zhai, Z.; Oakenfull, R.; Ghandi, P.; Adams, R.H.; Fruttiger, M.

Massague, J.; Seoane, J. & Wotton, D. (2005). Smad transcription factors. *Genes & Development*, Vol. 19, No. 23, (December 2005), pp. 2783-2810, ISSN 0890-9369. Matlin, A.J.; Clark, F. & Smith, C.W. (2005). Understanding alternative splicing: towards a

McAllister, K.A.; Grogg, K.M.; Johnson, D.W.; Gallione, C.J.; Baldwin, M.A.; Jackson, C.E.;

Meshorer, E. & Soreq, H. (2002). Pre-mRNA splicing modulations in senescence. *Aging Cell*,

Meurer, S.K.; Tihaa, L.; Borkham-Kamphorst, E. & Weiskirchen, R. (2011). Expression and

Minamino, T. & Komuro, I. (2007). Vascular cell senescence: contribution to atherosclerosis. *Circulation Research*, Vol. 100, No. 1, (January 2007), pp. 15-26, ISSN 1524-4571. Minamino, T. & Komuro, I. (2008). Role of telomeres in vascular senescence. *Frontiers in* 

Minamino, T.; Miyauchi, H.; Yoshida, T.; Tateno, K.; Kunieda, T. & Komuro, I. (2004).

Nott, A.; Meislin, S.H. & Moore, M.J. (2003). A quantitative analysis of intron effects on

Nowak, D.G.; Amin, E.M.; Rennel, E.S.; Hoareau-Aveilla, C.; Gammons, M.; Damodoran, G.;

Nowak, D.G.; Woolard, J.; Amin, E.M.; Konopatskaya, O.; Saleem, M.A.; Churchill, A.J.;

*Cardiology*, Vol. 36, No. 2, (February 2004), pp. 175-183, ISSN 0022-2828. Moore, M.J. & Silver, P.A. (2008). Global analysis of mRNA splicing. *RNA*, Vol. 14, No. 2,

*Bioscience*, Vol. 13, (January 2008), pp. 2971-2979, ISSN 1093-4715.

Vol. 1, No. 1, (October 2002), pp. 10-16, ISSN 1474-9718.

(December 2007), pp. 197-203, ISSN 1469-9001.

694-705, ISSN 0022-2836.

386-398, ISSN 1471-0072.

pp. 345-351, ISSN 1061-4036.

683-699, ISSN 1873-3913.

5532-5540, ISSN 1083-351X.

ISSN 0021-9533.

1355-8382.

1524-4571.

telangiectasia. *Journal of Molecular Biology*, Vol. 365, No. 3, (November 2006), pp.

& Arthur, H.M. (2010). Pathogenesis of arteriovenous malformations in the absence of endoglin. *Circulation Research*, Vol. 106, No. 8, (March 2010), pp. 1425-1433, ISSN

cellular code. *Nature Reviews Molecular Cell Biology*, Vol. 6, No. 5, (June 2005), pp.

Helmbold, E.A.; Markel, D.S.; McKinnon, W.C.; Murrell, J. & et al. (1994). Endoglin, a TGF-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. *Nature Genetics*, Vol. 8, No. 4, (December 1994),

functional analysis of endoglin in isolated liver cells and its involvement in fibrogenic Smad signalling. *Cellular Signalling*, Vol. 23, No. 4, (December 2010), pp.

Vascular cell senescence and vascular aging. *Journal of Molecular and Cellular* 

mammalian gene expression. *RNA*, Vol. 9, No. 5, (April 2003), pp. 607-617, ISSN

Hagiwara, M.; Harper, S.J.; Woolard, J.; Ladomery, M.R. & Bates, D.O. (2010). Regulation of vascular endothelial growth factor (VEGF) splicing from proangiogenic to anti-angiogenic isoforms: a novel therapeutic strategy for angiogenesis. *Journal of Biological Chemistry*, Vol. 285, No. 8, (November 2009), pp.

Ladomery, M.R.; Harper, S.J. & Bates, D.O. (2008). Expression of pro- and antiangiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. *Journal of Cell Science*, Vol. 121, No. Pt 20, (October 2008), pp. 3487-3495,


Alternative Splicing in Endothelial Senescence: Role of the TGF-β Co-Receptor Endoglin 517

Tarn, W.Y. & Steitz, J.A. (1996). Highly diverged U4 and U6 small nuclear RNAs required

ten Dijke, P.; Goumans, M.J. & Pardali, E. (2008). Endoglin in angiogenesis and vascular diseases. *Angiogenesis*, Vol. 11, No. 1, (February 2008), pp. 79-89, ISSN 0969-6970. Toussaint, O.; Medrano, E.E. & von Zglinicki, T. (2000). Cellular and molecular mechanisms

Tremain, R.; Marko, M.; Kinnimulki, V.; Ueno, H.; Bottinger, E. & Glick, A. (2000). Defects in

ras. *Oncogene*, Vol. 19, No. 13, (April 2000), pp. 1698-1709, ISSN 0950-9232. van der Kraan, P.M.; Goumans, M.J.; Blaney Davidson, E. & Ten Dijke, P. (2011). Age-

*Research*, DOI: 10.1007/s00441-011-1194-6, (June 2011), ISSN 1432-0878. Velasco, S.; Alvarez-Munoz, P.; Pericacho, M.; Dijke, P.T.; Bernabeu, C.; Lopez-Novoa, J.M.

*Science*, Vol. 121, No. Pt 6, (February 2008), pp. 913-919, ISSN 0021-9533. Venkatesha, S.; Toporsian, M.; Lam, C.; Hanai, J.; Mammoto, T.; Kim, Y.M.; Bdolah, Y.; Lim,

*Nature Medicine*, Vol. 12, No. 6, (June 2006), pp. 642-649, ISSN 1078-8956. Voglauer, R.; Chang, M.W.; Dampier, B.; Wieser, M.; Baumann, K.; Sterovsky, T.; Schreiber,

Wang, Z.; Xiao, X.; Van Nostrand, E. & Burge, C.B. (2006). General and specific functions of

Wesierska-Gadek, J.; Wojciechowski, J.; Ranftler, C. & Schmid, G. (2005). Role of p53 tumor

Will, C.L. & Luhrmann, R. (2005). Splicing of a rare class of introns by the U12-dependent

Wu, S.; Hultquist, A.; Hydbring, P.; Cetinkaya, C.; Oberg, F. & Larsson, L.G. (2009). TGF-

Young, A.R. & Narita, M. (2009). SASP reflects senescence. *EMBO Reports*, Vol. 10, No. 3,

315, No. 18, (September 2009), pp. 3099-3111, ISSN 1090-2422.

(February 2009), pp. 228-230, ISSN 1469-3178.

1824-1832, ISSN 0036-8075.

pp. 746-759, ISSN 0014-4827.

15-28, ISSN 0867-5910.

1431-6730.

2006), pp. 61-70, ISSN 1097-2765.

ISSN 0531-5565.

for splicing rare AT-AC introns. *Science*, Vol. 273, No. 5283, (September 1996), pp.

of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. *Experimental Gerontology*, Vol. 35, No. 8, (December 2000), pp. 927-945,

TGF-beta signaling overcome senescence of mouse keratinocytes expressing v-Ha-

dependent alteration of TGF-beta signalling in osteoarthritis. *Cell and Tissue* 

& Rodriguez-Barbero, A. (2008). L- and S-endoglin differentially modulate TGFbeta1 signaling mediated by ALK1 and ALK5 in L6E9 myoblasts. *Journal of Cell* 

K.H.; Yuan, H.T.; Libermann, T.A.; Stillman, I.E.; Roberts, D.; D'Amore, P.A.; Epstein, F.H.; Sellke, F.W.; Romero, R.; Sukhatme, V.P.; Letarte, M. & Karumanchi, S.A. (2006). Soluble endoglin contributes to the pathogenesis of preeclampsia.

M.; Katinger, H. & Grillari, J. (2006). SNEV overexpression extends the life span of human endothelial cells. *Experimental Cell Research*, Vol. 312, No. 6, (January 2006),

exonic splicing silencers in splicing control. *Molecular Cell*, Vol. 23, No. 1, (June

suppressor in ageing: regulation of transient cell cycle arrest and terminal senescence. *Journal of Physiology and Pharmacology*, Vol. 56, No. 1, (March 2005), pp.

spliceosome. *Biological Chemistry*, Vol. 386, No. 8, (October 2005), pp. 713-724, ISSN

beta enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity. *Experimental Cell Research*, Vol.

signaling in endothelial cells. *Circulation Research*, Vol. 97, No. 11, (October 2005), pp. 1115-1123, ISSN 1524-4571.


Schneiderman, J.; Sawdey, M.S.; Keeton, M.R.; Bordin, G.M.; Bernstein, E.F.; Dilley, R.B. &

Shay, J.W. & Wright, W.E. (2007). Hallmarks of telomeres in ageing research. *Journal of Pathology*, Vol. 211, No. 2, (January 2007), pp. 114-123, ISSN 0022-3417. Sheth, N.; Roca, X.; Hastings, M.L.; Roeder, T.; Krainer, A.R. & Sachidanandam, R. (2006).

*Research*, Vol. 34, No. 14, (August 2006), pp. 3955-3967, ISSN 1362-4962. Shovlin, C.L. (2010). Hereditary haemorrhagic telangiectasia: pathophysiology, diagnosis

Singh, R. & Valcarcel, J. (2005). Building specificity with nonspecific RNA-binding proteins.

Sinha, R.; Allemand, E.; Zhang, Z.; Karni, R.; Myers, M.P. & Krainer, A.R. (2010). Arginine

Sperling, J.; Azubel, M. & Sperling, R. (2008). Structure and function of the Pre-mRNA

Steiner, D.R.; Gonzalez, N.C. & Wood, J.G. (2002). Interaction between reactive oxygen

Sugrue, M.M.; Shin, D.Y.; Lee, S.W. & Aaronson, S.A. (1997). Wild-type p53 triggers a rapid

Sun, D.; Huang, A.; Yan, E.H.; Wu, Z.; Yan, C.; Kaminski, P.M.; Oury, T.D.; Wolin, M.S. &

Taddei, S.; Virdis, A.; Ghiadoni, L.; Salvetti, G.; Bernini, G.; Magagna, A. & Salvetti, A.

*Hypertension*, Vol. 38, No. 2, (August 2001), pp. 274-279, ISSN 1524-4563. Tang, J.; Gordon, G.M.; Nickoloff, B.J. & Foreman, K.E. (2002). The helix-loop-helix protein

*Investigation*, Vol. 82, No. 8, (August 2002), pp. 1073-1079, ISSN 0023-6837. Tang, Y.; Yang, X.; Friesel, R.E.; Vary, C.P. & Liaw, L. (2011). Mechanisms of TGF-beta-

Vol. 286, No. 6, (January 2004), pp. H2249-2256, ISSN 0363-6135.

(September 1997), pp. 9648-9653, ISSN 0027-8424.

pp. 1115-1123, ISSN 1524-4571.

ISSN 0027-8424.

1532-1681.

1545-9993.

0969-2126.

7587.

2762-2774, ISSN 1098-5549.

signaling in endothelial cells. *Circulation Research*, Vol. 97, No. 11, (October 2005),

Loskutoff, D.J. (1992). Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 89, No. 15, (August 1992), pp. 6998-7002,

Comprehensive splice-site analysis using comparative genomics. *Nucleic Acids* 

and treatment. *Blood Reviews*, Vol. 24, No. 6, (September 2010), pp. 203-219, ISSN

*Nature Structure Molecular Biology*, Vol. 12, No. 8, (August 2005), pp. 645-653, ISSN

methylation controls the subcellular localization and functions of the oncoprotein splicing factor SF2/ASF. *Molecular Cell Biology*, Vol. 30, No. 11, (March 2010), pp.

splicing machine. *Structure*, Vol. 16, No. 11, (November 2008), pp. 1605-1615, ISSN

species and nitric oxide in the microvascular response to systemic hypoxia. *Journal of Applied Physiology*, Vol. 93, No. 4, (September 2002), pp. 1411-1418, ISSN 8750-

senescence program in human tumor cells lacking functional p53. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 94, No. 18,

Kaley, G. (2004). Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats. *American Journal of Physiology. Heart and Circulatory Physiology*,

(2001). Age-related reduction of NO availability and oxidative stress in humans.

id-1 delays onset of replicative senescence in human endothelial cells. *Laboratory* 

Induced Differentiation in Human Vascular Smooth Muscle Cells. *Journal of Vascular Research*, Vol. 48, No. 6, (August 2011), pp. 485-494, ISSN 1423-0135.


**21** 

*Bulgaria* 

**Quantification of Elastin,** 

**Collagen and Advanced Glycation End** 

Milena Atanasova1, Aneliya Dimitrova2, Boryana Ruseva3,

*2Department of Pathophysiology, Medical University of Pleven, Pleven, 3Department of Physiology, Medical University of Pleven, Pleven, 4Department of Chemistry, Medical University of Pleven, Pleven, 5Medical Center "Clinical Institute for Reproductive Medicine", Pleven, 6Center for Reproductive Health; Medical University of Pleven, Pleven,* 

*1Department of Biology, Medical University of Pleven, Pleven,* 

Angelina Stoyanova4, Miglena Georgieva5 and Emiliana Konova5,6

**Products as Functions of Age and Hypertension** 

Elastin and collagen are the major extracellular matrix (ECM) proteins that make up the framework of the elastic arteries structure. These two fibrous proteins are the main structural components of arterial walls, and they provide the strength and resilience needed by the aorta to accommodate the pressure and volume variations during each heartbeat (Coquand et al., 2011). Elastin is a polymer of linear polypeptide chains and constituting more than 50% of the dry weight of the proximal parts of the aorta. It is the main provider of tissue elasticity, while collagen acts to stiffen the wall and to limit its extensibility (Samila & Carter, 1981). During aging the aorta diameter and stiffness increases because of degenerative changes in elastin, resulting in a transfer of stress to less extensible collagenous components of the aorta

Elastin production takes place during late gestation and before the end of childhood, but expression in very low rate persists in adulthood. The turnover rate for collagen and elastin is low in healthy arteries, but vascular pathology upsets the regulatory pathways that maintain this balance. In response to hypertension, the overexpression of both proinflammatory and proteinase-inhibitory molecules dramatically increases arterial ECM synthesis (Arribas et al., 2006; Jacob et al., 2001; Jacob, 2003). Elastin and elastic fibers are progressively degraded by enzymatic processes involving an age-related imbalance between anti-proteases and proteases. In particular, elastin degrading enzymes, i.e. elastases, include several matrix metalloproteases (MMP), such as MMP-2 and MMP-9 (Jacob, 2003; McNulty et al., 2005). Imbalance in matrix metalloproteinase/tissue inhibitors of metalloproteinases may contribute to alteration in collagen turnover and extracellular matrix remodelling. However, the ECM proteins synthesized in response to hypertension have a threedimensional architecture that is functionally less optimal than those deposited during fetal development and may play an important role in determining the modulus of pathological

**1. Introduction** 

(Coquand et al., 2011).

Zhou, Z.; Licklider, L.J.; Gygi, S.P. & Reed, R. (2002). Comprehensive proteomic analysis of the human spliceosome. *Nature*, Vol. 419, No. 6903, (September 2002), pp. 182-185, ISSN 0028-0836.

## **Quantification of Elastin, Collagen and Advanced Glycation End Products as Functions of Age and Hypertension**

Milena Atanasova1, Aneliya Dimitrova2, Boryana Ruseva3, Angelina Stoyanova4, Miglena Georgieva5 and Emiliana Konova5,6 *1Department of Biology, Medical University of Pleven, Pleven, 2Department of Pathophysiology, Medical University of Pleven, Pleven, 3Department of Physiology, Medical University of Pleven, Pleven, 4Department of Chemistry, Medical University of Pleven, Pleven, 5Medical Center "Clinical Institute for Reproductive Medicine", Pleven, 6Center for Reproductive Health; Medical University of Pleven, Pleven, Bulgaria* 

## **1. Introduction**

518 Senescence

Zhou, Z.; Licklider, L.J.; Gygi, S.P. & Reed, R. (2002). Comprehensive proteomic analysis of

ISSN 0028-0836.

the human spliceosome. *Nature*, Vol. 419, No. 6903, (September 2002), pp. 182-185,

Elastin and collagen are the major extracellular matrix (ECM) proteins that make up the framework of the elastic arteries structure. These two fibrous proteins are the main structural components of arterial walls, and they provide the strength and resilience needed by the aorta to accommodate the pressure and volume variations during each heartbeat (Coquand et al., 2011). Elastin is a polymer of linear polypeptide chains and constituting more than 50% of the dry weight of the proximal parts of the aorta. It is the main provider of tissue elasticity, while collagen acts to stiffen the wall and to limit its extensibility (Samila & Carter, 1981). During aging the aorta diameter and stiffness increases because of degenerative changes in elastin, resulting in a transfer of stress to less extensible collagenous components of the aorta (Coquand et al., 2011).

Elastin production takes place during late gestation and before the end of childhood, but expression in very low rate persists in adulthood. The turnover rate for collagen and elastin is low in healthy arteries, but vascular pathology upsets the regulatory pathways that maintain this balance. In response to hypertension, the overexpression of both proinflammatory and proteinase-inhibitory molecules dramatically increases arterial ECM synthesis (Arribas et al., 2006; Jacob et al., 2001; Jacob, 2003). Elastin and elastic fibers are progressively degraded by enzymatic processes involving an age-related imbalance between anti-proteases and proteases. In particular, elastin degrading enzymes, i.e. elastases, include several matrix metalloproteases (MMP), such as MMP-2 and MMP-9 (Jacob, 2003; McNulty et al., 2005). Imbalance in matrix metalloproteinase/tissue inhibitors of metalloproteinases may contribute to alteration in collagen turnover and extracellular matrix remodelling. However, the ECM proteins synthesized in response to hypertension have a threedimensional architecture that is functionally less optimal than those deposited during fetal development and may play an important role in determining the modulus of pathological

Quantification of Elastin,

**2.2 Blood collection** 

of the aorta or mg/cm.

use.

length.

Laboratory Animals (Bayne, 1996).

**2.3 Quantification of elastin in the thoracic aorta** 

**2.4 Quantification of collagen in the thoracic aorta** 

collagen per cm aorta (Keeley et al., 1984).

Collagen and Advanced Glycation End Products as Functions of Age and Hypertension 521

the animal facility of the Medical University of Pleven and were allowed free access to tap water and a standard laboratory chow. Animals were housed and kept under a normal 12 h light/dark cycle at 22 º± 2ºC. They were divided into 6 groups: 2-month-old SHR (2mSHR, n=7, 4 female and 3 male); 4-month-old SHR (4m SHR, n=9, 5/4); 8-month-old SHR (8mSHR, n=14, 9/5); 2-month-old WKY (2mWKY, n=12, 5/7); 4-month-old SHR (4mWKY, n=9, 5/4); 8-month-old WKY (8mWKY, n=12, 5/7). Our experimental design was approved by the Animal Care and Use of Laboratory Animals group of the Ethical Committee of the University, based on the principles described in the Guide for the Care and Use of

At the end of the 2nd, 4th and 8th month, following overnight fasting, the abdominal cavity of rats was opened under pentobarbitone sodium anesthesia (26 mg/kg body weight, i.p.). Blood was collected from the bifurcation of the aorta and put at 37 C to clot. After separation 0.02% NaN3 was added to each serum sample and stored at – 20 C prior to

The quantity of elastin was measured after dissection of the descending thoracic aorta and cleaning of blood and surrounding adipose tissue. Length and width (both sides) of the vessels were recorded by using a grid in the eyepiece, after opening of the vessel in

Elastin was then quantified by using a protocol deriving from a previously described method (Wolinski, 1972) with small modifications. Briefly, after delipidation in acetone/diethyl ether (1:1, vol/vol) and drying, the dry weight was recorded by using a Kern ALS 120-4 balance (precision: 0.01 mg). Cell proteins were extracted by gentle agitation in 0.3% SDS for 24 h and then 3 times in 5 M guanidinium chloride with preservative 0.02% sodium azide for 2 h. After washing 3 times in distilled water the extracellular proteins, other than elastin remaining in the aortic segments, were solubilized by three 15-minute extractions in 1 ml of 0.1 M NaOH in a boiling water bath. Elastin was quantified by determining the dry weight of the residue as percent dry weight

The content of the aortic collagen was assayed by determination the hydroxyproline presented in NaOH solution. The solution was evaporated to dryness and hydrolyzed in 6N HCl under vacuum conditions for 24 hours at 110 °C. A colorimetric assay according Woessner (1961) was applied for determination of the hydroxyproline. Assuming that collagen contains 12.77% hydroxyproline by weight its quantity was presented as mg

**2.5 Direct determination of advanced glycation end-products (AGEs) formed** *in vivo* Soluble α-elastin was obtained from the descending thoracic aorta by the method of Partridge (1955). Insoluble elastin was hydrolyzed 5 times in 0.25 M oxalic acid in boiling

elastin tissue (Arribas et al., 2006; Jacob et al., 2001; Jacob, 2003). The increase of aortic stiffness is not only because of enzymatic degradation of elastin, but also is due to other mechanisms mainly including age-dependent increase in the collagen content and arterial wall thickening, non-enzymatic glycation of proteins (elastin and collagens), leading to the formation of deleterious advanced glycation end-products (AGEs) and related molecular cross-links which modify the tissue mechanical properties (Gibbons & Dzau, 1994; Corman et al., 1998; Lakatta, 2003; O'Routke, 2007).

Advanced glycation is a major pathway for the posttranslational modifications of tissue proteins and begins with non-enzymatic addition of sugars to the primary amino groups of proteins. These early glucose-derived Schiff bases and Amadori products undergo a series of inter- and intramolecular rearrangement, dehydration, and oxidation-reduction reactions and produce the late products termed advanced glycation end products (AGEs). Excessive accumulation of AGE on tissue proteins has been implicated in the pathogenesis of many of the sequels of diabetes and normal aging. Protein-linked AGEs act to crosslink connective tissue proteins and to chemically inactive nitric oxide activity and thus are associated with endothelial dysfunction. They also act as a recognition signals for AGE receptor systems that are present on diverse cell types (Yang et al.,1991; Vlassara, 1994). Accumulation of AGEs in vascular walls increases intimal medial thickening, collagen is impaired, more cross-linked and elastin fibrils are broken. Large arteries are stiffen and mechanical stress increases, leading to hypertension and all its deleterious consequences (Dart & Kingwell, 2001; Zieman & Kass, 2004)

Excessive accumulation of AGEs on tissue proteins changes their structure and respectively functions, reduces their susceptibility to degradation and none of the last place immunogenicity. Specifically, the interaction of AGEs with vessel wall components increases vascular permeability, the expression of procoagulant activity and the generation of ROS (Yan et al., 1994). Glycated proteins form common immunological epitopes which raise formation of population of anti-AGE autoantibodies (AGEAb). These antibodies recognize and react with AGE-epitopes regardless of the proteins they have been formed on. Assessment of the levels of these antibodies shows that they are present at low titres even in sera of healthy subjects, perhaps as a part of homeostatic mechanism which clears glycated structures via *in situ* destruction or via opsonization of the gflycated proteins and products of their degradation (Baydanoff et al., 1996). However, in the conditions of increased nonenzymatic glycation the homeostatic control is inefficient and the generation of these antibodies increases (Baydanoff et al., 1996).

The aim of our study was to investigate the effects of age and hypertension on quantity and quality of elastin and collagen in the aortic wall of the rats. In order to reach this aim we used the spontaneously hypertensive rats (SHR), that are appropriate genetical model for studying essential hypertension compared to normotensive Wistar-Kyoto rats **(**WKR) at 2-, 4- and 8-months of age.

## **2. Materials and methods**

#### **2.1 Animals**

Female and male Spontaneously Hypertensive Rats (SHR, n=30) and Wistar Kyoto Rats (WKY, n=33) were used. Animals were born and raised under conventional conditions in the animal facility of the Medical University of Pleven and were allowed free access to tap water and a standard laboratory chow. Animals were housed and kept under a normal 12 h light/dark cycle at 22 º± 2ºC. They were divided into 6 groups: 2-month-old SHR (2mSHR, n=7, 4 female and 3 male); 4-month-old SHR (4m SHR, n=9, 5/4); 8-month-old SHR (8mSHR, n=14, 9/5); 2-month-old WKY (2mWKY, n=12, 5/7); 4-month-old SHR (4mWKY, n=9, 5/4); 8-month-old WKY (8mWKY, n=12, 5/7). Our experimental design was approved by the Animal Care and Use of Laboratory Animals group of the Ethical Committee of the University, based on the principles described in the Guide for the Care and Use of Laboratory Animals (Bayne, 1996).

## **2.2 Blood collection**

520 Senescence

elastin tissue (Arribas et al., 2006; Jacob et al., 2001; Jacob, 2003). The increase of aortic stiffness is not only because of enzymatic degradation of elastin, but also is due to other mechanisms mainly including age-dependent increase in the collagen content and arterial wall thickening, non-enzymatic glycation of proteins (elastin and collagens), leading to the formation of deleterious advanced glycation end-products (AGEs) and related molecular cross-links which modify the tissue mechanical properties (Gibbons & Dzau, 1994; Corman

Advanced glycation is a major pathway for the posttranslational modifications of tissue proteins and begins with non-enzymatic addition of sugars to the primary amino groups of proteins. These early glucose-derived Schiff bases and Amadori products undergo a series of inter- and intramolecular rearrangement, dehydration, and oxidation-reduction reactions and produce the late products termed advanced glycation end products (AGEs). Excessive accumulation of AGE on tissue proteins has been implicated in the pathogenesis of many of the sequels of diabetes and normal aging. Protein-linked AGEs act to crosslink connective tissue proteins and to chemically inactive nitric oxide activity and thus are associated with endothelial dysfunction. They also act as a recognition signals for AGE receptor systems that are present on diverse cell types (Yang et al.,1991; Vlassara, 1994). Accumulation of AGEs in vascular walls increases intimal medial thickening, collagen is impaired, more cross-linked and elastin fibrils are broken. Large arteries are stiffen and mechanical stress increases, leading to hypertension and all its deleterious consequences (Dart & Kingwell, 2001; Zieman

Excessive accumulation of AGEs on tissue proteins changes their structure and respectively functions, reduces their susceptibility to degradation and none of the last place immunogenicity. Specifically, the interaction of AGEs with vessel wall components increases vascular permeability, the expression of procoagulant activity and the generation of ROS (Yan et al., 1994). Glycated proteins form common immunological epitopes which raise formation of population of anti-AGE autoantibodies (AGEAb). These antibodies recognize and react with AGE-epitopes regardless of the proteins they have been formed on. Assessment of the levels of these antibodies shows that they are present at low titres even in sera of healthy subjects, perhaps as a part of homeostatic mechanism which clears glycated structures via *in situ* destruction or via opsonization of the gflycated proteins and products of their degradation (Baydanoff et al., 1996). However, in the conditions of increased nonenzymatic glycation the homeostatic control is inefficient and the generation of these

The aim of our study was to investigate the effects of age and hypertension on quantity and quality of elastin and collagen in the aortic wall of the rats. In order to reach this aim we used the spontaneously hypertensive rats (SHR), that are appropriate genetical model for studying essential hypertension compared to normotensive Wistar-Kyoto rats **(**WKR) at 2-,

Female and male Spontaneously Hypertensive Rats (SHR, n=30) and Wistar Kyoto Rats (WKY, n=33) were used. Animals were born and raised under conventional conditions in

et al., 1998; Lakatta, 2003; O'Routke, 2007).

antibodies increases (Baydanoff et al., 1996).

4- and 8-months of age.

**2.1 Animals** 

**2. Materials and methods** 

& Kass, 2004)

At the end of the 2nd, 4th and 8th month, following overnight fasting, the abdominal cavity of rats was opened under pentobarbitone sodium anesthesia (26 mg/kg body weight, i.p.). Blood was collected from the bifurcation of the aorta and put at 37 C to clot. After separation 0.02% NaN3 was added to each serum sample and stored at – 20 C prior to use.

## **2.3 Quantification of elastin in the thoracic aorta**

The quantity of elastin was measured after dissection of the descending thoracic aorta and cleaning of blood and surrounding adipose tissue. Length and width (both sides) of the vessels were recorded by using a grid in the eyepiece, after opening of the vessel in length.

Elastin was then quantified by using a protocol deriving from a previously described method (Wolinski, 1972) with small modifications. Briefly, after delipidation in acetone/diethyl ether (1:1, vol/vol) and drying, the dry weight was recorded by using a Kern ALS 120-4 balance (precision: 0.01 mg). Cell proteins were extracted by gentle agitation in 0.3% SDS for 24 h and then 3 times in 5 M guanidinium chloride with preservative 0.02% sodium azide for 2 h. After washing 3 times in distilled water the extracellular proteins, other than elastin remaining in the aortic segments, were solubilized by three 15-minute extractions in 1 ml of 0.1 M NaOH in a boiling water bath. Elastin was quantified by determining the dry weight of the residue as percent dry weight of the aorta or mg/cm.

## **2.4 Quantification of collagen in the thoracic aorta**

The content of the aortic collagen was assayed by determination the hydroxyproline presented in NaOH solution. The solution was evaporated to dryness and hydrolyzed in 6N HCl under vacuum conditions for 24 hours at 110 °C. A colorimetric assay according Woessner (1961) was applied for determination of the hydroxyproline. Assuming that collagen contains 12.77% hydroxyproline by weight its quantity was presented as mg collagen per cm aorta (Keeley et al., 1984).

## **2.5 Direct determination of advanced glycation end-products (AGEs) formed** *in vivo*

Soluble α-elastin was obtained from the descending thoracic aorta by the method of Partridge (1955). Insoluble elastin was hydrolyzed 5 times in 0.25 M oxalic acid in boiling

Quantification of Elastin,

ages did not differ from age matched WKR.

32

0,0

0,5

1,0

**mg/cm**

**B**

**3.2 Collagen content in the thoracic aorta** 

1,5

2,0

2,5

36

40

 **%**

**A**

44

48

Collagen and Advanced Glycation End Products as Functions of Age and Hypertension 523

aorta (fig. 1A) SHR at different ages did not differ significantly, whereas in the WKR groups factor "age" was found to have a significant effect (p<0.05) – elastin quantity decreased with age. Elastin content was significantly larger (p<0.032) in 8-month-old hypertensive animals compared to age matched normotensive group. When the elastin quantity was presented as milligrams per centimeter of aorta (fig. 1B) the absolute amount of elastin in SHR at different

**Elastin content (%)**
