**2.4.1 The role of glutathione in DNA synthesis**

Among the important roles that GSH plays in cellular physiology, and among the first to be described, was its role in DNA synthesis. The pentose phosphate pathway is a cellular source of NADPH that is involved in reductive biosynthesis. In this process, ribose-5 phosphate is formed and subsequently used for the synthesis of RNA, DNA and nucleotide coenzymes. Apart from the synthesis of nucleotides, NADPH is also required for the formation of amino acids, fatty acids, cholesterol, neurotransmitters and nitric oxide (NO). Furthermore, NADPH is the source of electrons in the process of reduction of ribonucleotides to deoxyribonucleotides catalyzed by the ribonucleotide reductase (Thelander & Reichard, 1979).

The route is initialized by two distinct but complemented systems, the thioredoxin system and the glutaredoxin system. Thioredoxin operates by transferring the electrons to ribonucleotide reductase, and they are supplied by the thioredoxin reductase and NADPH. The glutaredoxin system is initialized by the glutathione reductase, which reduces the GSSG to GSH using the NADPH as source of electrons. GSH is used by the glutaredoxin to provide the reducing power to the ribonucleotide reductase (Zahedi et al., 2009).

The crucial role of glutathione in DNA synthesis has been extensively documented (Thelander & Reichard, 1979; Holmgren, 1976). For instance, Dethlefsen and co-workers (Dethlefsen et al. 1988) showed that glutathione depletion inhibits DNA synthesis in mammary carcinoma cells. In addition, the vital importance of GSH in this process has also been demonstrated in human T lymphocytes (Suthanthiran et al., 1990). As mentioned above GSH is an indispensable requirement in eukaryotes. In contrast, it does not demonstrate the same importance in prokariotes. It has been shown that E. Coli lacking gshA, the rate limiting enzyme in the synthesis of GSH, can grow without GSH supplementation (Greenberg & Demple, 1986; Miranda-Vizuete et al., 1996). On the contrary, in yeast, the depletion of GSH does affect cell proliferation on the level different from DNA synthesis: mutants deficient in GCS after GSH withdrawal arrest cells in G1, whereas a strain with a defect in ribonucleotide reduction arrest cells in S phase (Wang et al., 1997). Since other possible explanations, such as protection against oxidative stress or protection against non-native protein disulfides, have been discarded (Spector el al., 2001), it appears that the essential function of GSH in yeast is related to the redox properties of its thiol group. Consequently, glutathione can be replaced by dithiothreitol, but not with a GSH analog where a thiol group has been substituted by a methyl group (Grant et al., 1996). Since GSH is the reductant for glutaredoxin as explained previously, and glutaredoxin is also

protection for protein-SH against irreversible modifications and protein damage in response to higher levels of oxidative stress (Dalle-Donne et al., 2007). Interestingly, it was demonstrated that glutathiolation as a posttranslational modification occurs not only during oxidative stress, but also under basal conditions and is involved in regulating distinct transcription factors, such as NF-kB (Pineda-Molina et al., 2001), its inhibitor factor IKK (Reynaert et al., 2006) and c-Jun (Canela et al. 2007). Apparently, the binding capacity of these proteins to DNA or other proteins is modulated by glutathiolation. This relatively recent focus on the implication of glutathiolation modulatory effects on protein function yielded important breakthrough in elucidation of the implication of this modification in various physiological and pathological situations (Giustarini et al. 2004) and raises

Among the important roles that GSH plays in cellular physiology, and among the first to be described, was its role in DNA synthesis. The pentose phosphate pathway is a cellular source of NADPH that is involved in reductive biosynthesis. In this process, ribose-5 phosphate is formed and subsequently used for the synthesis of RNA, DNA and nucleotide coenzymes. Apart from the synthesis of nucleotides, NADPH is also required for the formation of amino acids, fatty acids, cholesterol, neurotransmitters and nitric oxide (NO). Furthermore, NADPH is the source of electrons in the process of reduction of ribonucleotides to deoxyribonucleotides catalyzed by the ribonucleotide reductase

The route is initialized by two distinct but complemented systems, the thioredoxin system and the glutaredoxin system. Thioredoxin operates by transferring the electrons to ribonucleotide reductase, and they are supplied by the thioredoxin reductase and NADPH. The glutaredoxin system is initialized by the glutathione reductase, which reduces the GSSG to GSH using the NADPH as source of electrons. GSH is used by the glutaredoxin to

The crucial role of glutathione in DNA synthesis has been extensively documented (Thelander & Reichard, 1979; Holmgren, 1976). For instance, Dethlefsen and co-workers (Dethlefsen et al. 1988) showed that glutathione depletion inhibits DNA synthesis in mammary carcinoma cells. In addition, the vital importance of GSH in this process has also been demonstrated in human T lymphocytes (Suthanthiran et al., 1990). As mentioned above GSH is an indispensable requirement in eukaryotes. In contrast, it does not demonstrate the same importance in prokariotes. It has been shown that E. Coli lacking gshA, the rate limiting enzyme in the synthesis of GSH, can grow without GSH supplementation (Greenberg & Demple, 1986; Miranda-Vizuete et al., 1996). On the contrary, in yeast, the depletion of GSH does affect cell proliferation on the level different from DNA synthesis: mutants deficient in GCS after GSH withdrawal arrest cells in G1, whereas a strain with a defect in ribonucleotide reduction arrest cells in S phase (Wang et al., 1997). Since other possible explanations, such as protection against oxidative stress or protection against non-native protein disulfides, have been discarded (Spector el al., 2001), it appears that the essential function of GSH in yeast is related to the redox properties of its thiol group. Consequently, glutathione can be replaced by dithiothreitol, but not with a GSH analog where a thiol group has been substituted by a methyl group (Grant et al., 1996). Since GSH is the reductant for glutaredoxin as explained previously, and glutaredoxin is also

provide the reducing power to the ribonucleotide reductase (Zahedi et al., 2009).

interesting questions about its possible implications in cell proliferation.

**2.4.1 The role of glutathione in DNA synthesis** 

(Thelander & Reichard, 1979).

essential (Rodriguez-Manzaneque et al., 1999) presumably their vital importance may be interdependent. Then GSH seems to be important in S phase. During the process of DNA replication, errors, such as double-strand breaks (DSBs) that arise from stalled replication forks, require attention by the DNA damage response proteins. Thus, the correct control of DNA synthesis and probably essential molecules, such as GSH, are necessary for the correct DNA processing.

One of the most important proteins involved in DNA damage signaling pathway is the ataxia-telangiectasa mutated protein (ATM). This central signaling protein, mainly for DSBs, is involved in the repairing DNA process necessary after replication stress. Thus cells lacking ATM fail to execute many of the cellular responses to DNA damage (Zhou & Elledge, 2000). In addition, control of ATM responses after DNA replication may be necessary for the correct cell cycle control. In that way, ATM is a central component in the cell cycle regulation. Therefore, patients with ataxia telangiectasia have reduction in DNA synthesis (Painter & Young, 1980). Furthermore, a recent work published by Guo Z. and coworkers describes using a series of elegant experiments how ATM sense the redox changes to modulate their activity (Guo et al, 2010). Interestingly, these authors propose that ATM may regulate global cellular responses to oxidative stre*s*s, remarking the essential link between redox control and DNA interacting, remodeling or repairing proteins. In Fanconi anemia for instance, Castillo and coworkers have shown that ATM dependent phosphorylation of FANCD2, one of the main proteins in the Fanconi anemia pathway of DNA repair, is necessary for normal S-phase checkpoint activation after oxidative stress (Castillo et al, 2011).

#### **2.4.2 Regulation of telomerase activity by glutathione**

The eukaryotic chromosomes are capped by telomeres, which consist of TTAGGG DNA sequences repeated in tandem, associated with several proteins, which protect the final regions of chromosomes. These structures play an important role in the stability and the complete replication of the chromosomes. Conventional DNA polymerases cannot fully replicate the 3'-end of the lagging strand of linear molecules, and therefore in every cell division telomeric sequences are lost (Komberg, 1969). Telomerase is an important enzyme that ensures the maintenance of normal telomere length. This activity is high in human cancers (Kim et al., 1994), but virtually absent in normal human tissues, except germinal cells (Harley et al., 1990). Telomerase regulation is not completely understood, but its changes are related to both cancer and aging (Sharpless & Depinho, 2004). Studies carried out by Jady et al. show that human telomeres are more accessible during the S-phase (Jady et al., 2006) and that the telomerase assembly with telomeres takes place at this specific moment of the cell cycle (Jady et al., 2006; Tomlison et al., 2006). Telomerase plays a key role in cellular homeostasis, because it maintains the length of the telomeres. This especially important in germinal cells in which it is necessary to keep a normal telomeric length after many cellular divisions. Important contributions about the epigenetic control of telomeres have been reported recently (Koziel et al., 2011). In that way, Maria Blasco has suggested that telomeres are under epigenetic control (García-Cao et al., 2004). Mammalian telomeres and subtelomeric regions are enriched in epigenetic marks that are characteristic of heterochromatin. In addition, histone deacetylase enzymes, such as Sirt6, regulate the telomeric chromatin conformation in order to allow the interaction of WRN protein with these chromosomal regions (Michishita et al., 2008).

The Nuclear Compartmentation of Glutathione: Effect on Cell Cycle Progression 277

importance. This finding that telomere length, and, therefore, telomere structure, is tightly regulated in telomerase proficient cells invokes a connection between cell cycle, telomerase and telomere structure (Blasco, 2002). In other words, the mechanism that lies beneath telomerase regulation might be related with the mechanism that control cell proliferation. This opens a highly significant area for exploratory study and the diversity of processes and control mechanisms that could be involved in this phenomenon remain to be elucidated.

Pioneer work from Meister, (Meister & Anderson, 1983) correlated GSH synthesis and its degradation throughout the so-called -glutamyl cycle, and defined it as a cytosolic processes. The importance of cellular compartmentalization of GSH is two fold, first because it plays an important role in fighting against radical oxygen species (ROS). It is well known that these molecules have a very short half life and exert their action close to the place they were produced. Thus, the presence or absence of GSH could determine the development of localized oxidative damage for the cell structure or metabolic function developed in the vicinity. Secondly, GSH compartmentalization is of vital importance because of its role as a cellular detoxifying agent; it is known that tumours that have high glutathione levels are more resistant to chemotherapy, and the importance of nuclear (Voheringer et al., 1998) and mitochondrial (Benlloch et al. 2005) compartmentalization of GSH has been pointed out. The overview of compartmentalization of glutathione in mammal cells is a complicated matter. This is due to the presence in the literature of a number of contradictory reports. The reason for the controversy is mainly methodological. Until very recently most reports were mainly based on cell-fractionation techniques. Those techniques appear to be reliable for mitochondrial studies; however their usefulness in nuclear or even endoplasmic reticulum

Although the role of nuclear GSH in the synthesis of DNA (Thelander & Reichard, 1979) and in protection against oxidative damage or ionizing radiation (Biaglow et al., 1983) is well established, little is known about the concentration of GSH in the nucleus and its regulation. This is due to two main factors. The first is methodological: it is impossible to determine the nuclear concentration of GSH using standard cell fractionation and analytical approaches (for a review see Söderdahl et al., 2003). The second factor is that most, if not all, of the reports share the common view of nuclear GSH distribution in a static situation. Cells are usually studied under steady state conditions *i.e.* when they are confluent (G0/G1 phase of the cell cycle). The nuclear membrane dissolves during mitosis and is formed again around newly replicated DNA packed in chromosomes; this spectacular change involves a variety of regulatory mechanisms. Therefore, if the nuclear GSH distribution is studied, the cell

The role of GSH in cell cycle regulation has been addressed mainly from the point of view of its overall cellular content. This is surprising since it is in the nucleus where most cell cycle progression events take place. The nucleus changes dramatically during the different phases of cell cycle, and failing to consider the corresponding changes in its redox environment could confer an important disadvantage in elucidating the actual importance of glutathione

**3.1 The physiological importance of compartmentalization of glutathione** 

**3. Compartmentalization of glutathione** 

measurements is at least controversial.

cycle physiology should be carefully considered.

in the control of cell proliferation.

**3.1.1 Nucleus** 

There are evidence that point to a role of redox environment in a short term regulation of the activity of this important enzyme. Minamino et al., 2001, using vascular smooth muscle cells, reported that hypoxia up-regulates telomerase activity. Hypoxia is known to lower oxidative stress and thus to increase levels of glutathione. A specific inhibitor of telomerase, 2-[3-(trifluoromethyl) phenyl]isothiazolin-3-one, reacts with a key cysteine residue, which is essential for telomerase activity and must be kept reduced. Consequently, it has been reported that dithiothreitol reverses this inhibition (Hayakawa et al., 1999). Furthermore, antioxidants have been shown to inhibit nuclear export of telomerase reverse transcriptase and thus delay replicative senescence of endothelial cells (Haendeler et al., 2004). In conclusion, a critical cysteine residue must be kept reduced in order to maintain full telomerase activity. It is likely that the glutathione redox potential may be important in this process.

Previous findings of our group showed that telomerase is regulated by the shift in glutathione redox potential within values similar to those found *in* vivo and alterations in telomerase activity are coordinated with changes in critical cell cycle proteins, particularly Id2 and E2F4 (Borras et al., 2004).

Fig. 1. Reduced glutathione regulates telomerase activity in 3T3 fibroblasts.

Thus, physiological variations in glutathione level induce changes in telomerase activities that are in concordance with changes in cell cycle regulatory proteins. A number of reports have shown similar results. Brown et al., 2007 demonstrated for the first time *in vivo* that high hepatic glutathione levels correlate with increased telomerase activity. Also, the importance of glutathione regulation in telomerase activity has been proved in endothelial progenitor cells (EPC): impairment of antioxidant defences in EPC promoted oxidant mediated apoptosis and telomerase inactivation which subsequently lead to development and/or progression of atherothrombosis (Fujii et al., 2006).

Recent data suggest that telomerase activity is regulated and ordered by telomere structure and telomerase assembly. Experimental evidence suggests that the telomere structure may change in a cell cycle-dependent manner to restrict telomerase activity to S phase (Hug & Ligner, 2006). In addition, telomere structure and specially the telomeric G-overhangs generation are strictly regulated during S phase and prolonged to other cell cycle phases depending on whether are telomeres from the lagging or the leading telomere (Dai et al., 2010). For this to happen, the precise control of the changes not only in telomerase conformation, but in chromatin structure (i.e. in its compactation level) as well, is of vital importance. This finding that telomere length, and, therefore, telomere structure, is tightly regulated in telomerase proficient cells invokes a connection between cell cycle, telomerase and telomere structure (Blasco, 2002). In other words, the mechanism that lies beneath telomerase regulation might be related with the mechanism that control cell proliferation. This opens a highly significant area for exploratory study and the diversity of processes and control mechanisms that could be involved in this phenomenon remain to be elucidated.
