**Eidetic Analysis of the Premature Chromosome Condensation Process**

Dorota Rybaczek

*Department of Cytophysiology, University of Łódź Poland* 

*'Why does this written doe bound through these written woods? (…) Perched on four slim legs borrowed from the truth, She pricks up her ears beneath my fingertips (…)' (Szymborska, 1993)* 

#### **1. Introduction**

184 DNA Repair

Zhu, J.W., DeRyckere, D., Li, F.X., Wan, Y.Y., and DeGregori, J. (1999). A role for E2F1 in the

*Differ* 10, 12.(829-838).

induction of ARF, p53, and apoptosis during thymic negative selection. *Cell Growth* 

An exact transfer of genetic information depends on the accuracy of mechanisms duplicating DNA molecules in the S-phase and the precise division sister chromosomes during mitosis. The regulation systems of these processes (checkpoints) not only control the activation course of the factors imposing different metabolic specificity on each of the cell cycle phases, but first of all – supervising the proper chronology of events – they condition the behavior of the structural and functional genome integrity. Checkpoints receive signals of all abnormalities or structural damages to DNA and in response evoke reactions inhibiting successive transitions through the cell cycle to enable the expression of specific genes and activation of DNA repair factors. One of the easily perceptible effects of disorders in this signaling system is the induction of premature chromosome condensation (PCC). The present chapter is a review of the ways and mode of the induction of PCC. The term 'PCC' is inseparably associated with Johnson & Rao (1970) and their experiments on the premature mitosis induced by fusion of interphase and mitotic HeLa cells (G1/M, S/M and G2/M) which were originally carried out using Sendai virus. PCC process can be also induced by chemical signals. Drug-induced PCC provides the new knowledge that DNA replication is tightly coupled with the premature chromosome condensation and that the genome stability results first of all from the alternation of the S-phase and mitosis. The main objective of this review is to show that the PCC induction is possible from various subperiods of cell cycle. Moreover, it has been shown that there are cause-and-effect relationships between the chromosome structure defining 'PCC phenotype' and subperiods, e.g. of the S-phase, initiating the biosynthesis of 'early' or 'late' replicons. Attempts have been made to find answers to questions such as: How to force cells to break out of the rules being developed by Nature for billions of years? How – despite the interrupted, still unterminated process of genome replication – to force a cell to initiate its division? What mechanisms annihilate the subordination principle verified in the course of evolution: first create (DNA-duplicating Sphase) and then divide (mitosis – a stage of DNA condensation and formation of sister

Eidetic Analysis of the Premature Chromosome Condensation Process 187

Carefully designed experiments by Johnson & Rao (1970) have resulted in the first correct interpretation of the phenomenon of premature chromosome condensation (PCC). The term PCC appeared in the description of phenomena observed during the fusion of interphase and mitotic malignant HeLa cells. It has been shown that the PCC phenomenon is accompanied by disappearance of the nuclear envelope, chromatin condensation and the formation of mitotic spindle. Subsequent investigations have shown that the induction of PCC is inseparably connected with the activity of MPF complex. The experiments, involving fusion of interphase and mitotic cells have clearly shown that the cells in all the subperiods of interphase are characterized by capability to induce PCC. Thus: (i) if the fusion took place between a mitotic cell and an interphase cell in G1 phase, then PCC resulted in the formation of chromosomes consisting of a single chromatid only (univalent chromosomes); (ii) if the fusion took place between a mitotic cell and an interphase cell in S-phase, strongly fragmented chromosomes were formed in the PCC process, and their morphology constituted a specific reminder of the nucleoplasm organization in the period of the activity of the cell replication apparatus (a 'pulverized' appearance that consisted of univalent and bivalent chromosomes); (iii) if a mitotic cell and interphase cell in G2-phase participated in the combination, PCC resulted in the formation of chromosomes consisting of two chromatids apparently non-differentiated morphologically as compared to normal chromosomes, although probably less condensed (bivalent chromosomes). Despite the fact that the first observation of PCC was reported by Kato & Sandberg (1967) in virus mediated multinucleated fused cells of interphase and mitotic cells, it was Johnson & Rao who in 1970 properly defined the observed phenomenon as 'premature chromosome condensation' (PCC), and the condensed interphase chromatin as 'prematurely condensed chromosomes' (PCCs) (Gotoh & Durante, 2006). Generally, the PCC method facilitates the visualization of interphase chromatin as a condensed form of chromosome structure (Gotoh, 2007). Nowadays, the efficiency and scope of PCC induction

Premature chromosome condensation became a method used to: (1) distinguish a cell cycle stage (Cadwell et al., 2011) and the Rabl-orientation of interphase chromosomes, e.g. G1- PCCs and G2-PCCs (Cremer et al., 1982); (2) investigations in chromosome dynamics, also that in interphase chromatin, chromosome replication studies and DNA repair analysis (Gotoh & Durante, 2006, as cited in Cornforth & Bedford, 1983; Hittelman & Pollard, 1982; Hittelman & Rao, 1974; Mullinger & Johnson, 1983; Schor et al., 1975); (3) perform mutagenic assay (Gotoh, 2009, as cited in Cornforth & Bedford, 1983; Durante et al., 1996); (4) chromosome instability analysis (Bezrookove et al., 2003); (5) prenatal diagnosis (Gotoh & Durante, 2006, as cited in Srebniak et al., 2005); (6) karyotyping of chromosomes (Kowalska

Virus-mediated PCC was first reported more than 40 years ago. In 1983, cell fusion could be achieved by means of polyethylene glycol (PEG-induced or chemically-mediated; Pantelias & Maillie, 1983). This allowed the external MPF to migrate from the inducing mitotic cell to the interphase recipient. A few years later it was possible to obtain chemically-induced PCC (drug-induced PCC): initially from synchronized cells and later still from each phase of the cell cycle (Gotoh & Durante, 2006, as cited in Schlegel & Pardee, 1986, Schlegel et al., 1990; Yamashita et al., 1990). Detailed data concerning PCC induction reported so far in the world

et al., 2003); and (6) cytogenetic analysis of cancers (Darroudi et al., 2010).

**2.1 Induction of PCC in historical and methodological terms** 

have been proved by combining techniques.

literature are presented in Table 1.

descendant nuclei)? Interference in the regulatory systems of cell cycle is not a simple matter. The gene pool, whose products participate in the creation of these systems, is constantly changing with time to continually form new systems and new interactions. Huge difficulties in the development of effective and selective methods that would arrest the proliferation of cancer cells result from their multiplicity and complication degree, as well as from the possibilities of starting the mechanisms of substitutive and biochemical emergency systems. Studies on the mechanisms inhibiting cell divisions seem to be the shortest way to reach the desired end. This chapter shows the usefulness of attempts to force divisions in cells, simultaneously taking into account the strategy of anticancer therapy.

Therefore the PCC phenomenon constitutes in reality not only a significant fundamental problem in the biology of cell cycle, but it is also an issue of paramount importance in view of practical applications. The radio- and chemotherapy methods used in the treatment of malignant diseases lead to extensive damages to DNA, arresting the replication process of genetic material. Despite this fact, the inhibition of cancer cell proliferation most often is of temporary character or it comprises only part of their population. Drug-induced PCC gives a novel tool to characterize the role of the chromosome instability in cancer development. In this chapter, an attempt is also made to explain the molecular base of PCC induction, for which the starting-point is the biochemical organization of the S-phase checkpoints that block mitosis initiation and the mechanisms which make it possible to suppress their restrictive interactions.

#### **2. Discovery of the premature chromosome condensation (PCC)**

The process of separate mitotic chromosome formation from chromatin of an interphase cell nucleus is associated with the construction of giant complexes or macromolecules this consequence being a specific expression of molecular morphogenesis. Simultaneously, it results from action of a complicated regulatory system, causing long, replicated DNA molecules to assume a form adapted to the biomechanical processes of mitosis. Control over these processes is provided on many planes of molecular chromatin organization, e.g. by the association of their components with the nuclear matrix, by specific phosphorylations and dephosphorylations conditioned by changes in the activity of protein kinases and phosphatases or by translocations of some molecules along the length of the fibrils of condensing chromatin or along the arms of existing chromosomes. The degree of packing achieved by chromatin throughout its domain organization before the G2→M transition falls short of the culminant metaphase condensation, the concomitant structural changes always resulting from biochemical modifications that proceed in the protein scaffold of the chromosome under formation.

Attentiveness to the integrity of genome determines the fundamental principles governing the regulation of cell cycle: the replication of each DNA molecule during S-phase can take place only once. The second condition involves the initiation of mitosis: this cannot begin before the complete termination of DNA replication. Control over the course of successive phases of cell cycle is extraordinarily precise and rigorous since initiation of the S-phase is restricted exclusively to unreplicated post-mitotic chromatin. Meanwhile, it is known that competence to initiate mitosis is not always conditioned by the replicated state of chromatin (this does not mean however that control over this process is not precise; simply, it results from the closely specified timing involved in setting-up factors liberating the activity of MPF [i.e. Cdk1 kinase and cyclin B complex or maturation/mitosis promoting factor] and its cooperation with aspects of the activators' and inhibitors' character).

descendant nuclei)? Interference in the regulatory systems of cell cycle is not a simple matter. The gene pool, whose products participate in the creation of these systems, is constantly changing with time to continually form new systems and new interactions. Huge difficulties in the development of effective and selective methods that would arrest the proliferation of cancer cells result from their multiplicity and complication degree, as well as from the possibilities of starting the mechanisms of substitutive and biochemical emergency systems. Studies on the mechanisms inhibiting cell divisions seem to be the shortest way to reach the desired end. This chapter shows the usefulness of attempts to force divisions in cells,

Therefore the PCC phenomenon constitutes in reality not only a significant fundamental problem in the biology of cell cycle, but it is also an issue of paramount importance in view of practical applications. The radio- and chemotherapy methods used in the treatment of malignant diseases lead to extensive damages to DNA, arresting the replication process of genetic material. Despite this fact, the inhibition of cancer cell proliferation most often is of temporary character or it comprises only part of their population. Drug-induced PCC gives a novel tool to characterize the role of the chromosome instability in cancer development. In this chapter, an attempt is also made to explain the molecular base of PCC induction, for which the starting-point is the biochemical organization of the S-phase checkpoints that block mitosis initiation and the mechanisms which make it possible to suppress their restrictive interactions.

The process of separate mitotic chromosome formation from chromatin of an interphase cell nucleus is associated with the construction of giant complexes or macromolecules this consequence being a specific expression of molecular morphogenesis. Simultaneously, it results from action of a complicated regulatory system, causing long, replicated DNA molecules to assume a form adapted to the biomechanical processes of mitosis. Control over these processes is provided on many planes of molecular chromatin organization, e.g. by the association of their components with the nuclear matrix, by specific phosphorylations and dephosphorylations conditioned by changes in the activity of protein kinases and phosphatases or by translocations of some molecules along the length of the fibrils of condensing chromatin or along the arms of existing chromosomes. The degree of packing achieved by chromatin throughout its domain organization before the G2→M transition falls short of the culminant metaphase condensation, the concomitant structural changes always resulting from biochemical modifications that proceed in the protein scaffold of the

Attentiveness to the integrity of genome determines the fundamental principles governing the regulation of cell cycle: the replication of each DNA molecule during S-phase can take place only once. The second condition involves the initiation of mitosis: this cannot begin before the complete termination of DNA replication. Control over the course of successive phases of cell cycle is extraordinarily precise and rigorous since initiation of the S-phase is restricted exclusively to unreplicated post-mitotic chromatin. Meanwhile, it is known that competence to initiate mitosis is not always conditioned by the replicated state of chromatin (this does not mean however that control over this process is not precise; simply, it results from the closely specified timing involved in setting-up factors liberating the activity of MPF [i.e. Cdk1 kinase and cyclin B complex or maturation/mitosis promoting factor] and its co-

operation with aspects of the activators' and inhibitors' character).

simultaneously taking into account the strategy of anticancer therapy.

**2. Discovery of the premature chromosome condensation (PCC)** 

chromosome under formation.

Carefully designed experiments by Johnson & Rao (1970) have resulted in the first correct interpretation of the phenomenon of premature chromosome condensation (PCC). The term PCC appeared in the description of phenomena observed during the fusion of interphase and mitotic malignant HeLa cells. It has been shown that the PCC phenomenon is accompanied by disappearance of the nuclear envelope, chromatin condensation and the formation of mitotic spindle. Subsequent investigations have shown that the induction of PCC is inseparably connected with the activity of MPF complex. The experiments, involving fusion of interphase and mitotic cells have clearly shown that the cells in all the subperiods of interphase are characterized by capability to induce PCC. Thus: (i) if the fusion took place between a mitotic cell and an interphase cell in G1 phase, then PCC resulted in the formation of chromosomes consisting of a single chromatid only (univalent chromosomes); (ii) if the fusion took place between a mitotic cell and an interphase cell in S-phase, strongly fragmented chromosomes were formed in the PCC process, and their morphology constituted a specific reminder of the nucleoplasm organization in the period of the activity of the cell replication apparatus (a 'pulverized' appearance that consisted of univalent and bivalent chromosomes); (iii) if a mitotic cell and interphase cell in G2-phase participated in the combination, PCC resulted in the formation of chromosomes consisting of two chromatids apparently non-differentiated morphologically as compared to normal chromosomes, although probably less condensed (bivalent chromosomes). Despite the fact that the first observation of PCC was reported by Kato & Sandberg (1967) in virus mediated multinucleated fused cells of interphase and mitotic cells, it was Johnson & Rao who in 1970 properly defined the observed phenomenon as 'premature chromosome condensation' (PCC), and the condensed interphase chromatin as 'prematurely condensed chromosomes' (PCCs) (Gotoh & Durante, 2006). Generally, the PCC method facilitates the visualization of interphase chromatin as a condensed form of chromosome structure (Gotoh, 2007). Nowadays, the efficiency and scope of PCC induction have been proved by combining techniques.

Premature chromosome condensation became a method used to: (1) distinguish a cell cycle stage (Cadwell et al., 2011) and the Rabl-orientation of interphase chromosomes, e.g. G1- PCCs and G2-PCCs (Cremer et al., 1982); (2) investigations in chromosome dynamics, also that in interphase chromatin, chromosome replication studies and DNA repair analysis (Gotoh & Durante, 2006, as cited in Cornforth & Bedford, 1983; Hittelman & Pollard, 1982; Hittelman & Rao, 1974; Mullinger & Johnson, 1983; Schor et al., 1975); (3) perform mutagenic assay (Gotoh, 2009, as cited in Cornforth & Bedford, 1983; Durante et al., 1996); (4) chromosome instability analysis (Bezrookove et al., 2003); (5) prenatal diagnosis (Gotoh & Durante, 2006, as cited in Srebniak et al., 2005); (6) karyotyping of chromosomes (Kowalska et al., 2003); and (6) cytogenetic analysis of cancers (Darroudi et al., 2010).

#### **2.1 Induction of PCC in historical and methodological terms**

Virus-mediated PCC was first reported more than 40 years ago. In 1983, cell fusion could be achieved by means of polyethylene glycol (PEG-induced or chemically-mediated; Pantelias & Maillie, 1983). This allowed the external MPF to migrate from the inducing mitotic cell to the interphase recipient. A few years later it was possible to obtain chemically-induced PCC (drug-induced PCC): initially from synchronized cells and later still from each phase of the cell cycle (Gotoh & Durante, 2006, as cited in Schlegel & Pardee, 1986, Schlegel et al., 1990; Yamashita et al., 1990). Detailed data concerning PCC induction reported so far in the world literature are presented in Table 1.

Eidetic Analysis of the Premature Chromosome Condensation Process 189

A specific change occurred when the preliminary cell-free cytoplasmic extracts (obtained by the centrifugation of *Xenopus* egg cells) were used *in vitro* to study the assembly of 'synthetic nuclei' involving the conversion of chromatin sperms into mitotic chromosomes. The resulting expansion of knowledge concerning the action of inhibitors and/or activators used for PCC induction has created new opportunities and perspectives for researchers (Prokhorova et al., 2003). In cells with disturbed DNA structure or those blocked during DNA biosynthesis, PCC-type processes can be induced by various chemical compounds such as: (i) inhibitors of protein kinases, e.g. 2-aminopurine (Herbig et al., 2004), caffeine (Wang et al., 1999, Gabrielli et al., 2007), staurosporine, 7-hydroxystaurosporine (UCN-01), CEP-3891 (Kohn et al., 2002; Syljuåsen et al., 2005), wortmannin (WORT) (Liu et al., 2007) and Gö6976 (Jia et al., 2008) as well as (ii) inhibitors of protein phosphatases, e.g. calyculin A (CalA), okadaic acid (OA) and sodium metavanadate (Van) (Hosseini & Mozdarani, 2004; Rybaczek & Kowalewicz-Kulbat, 2011). In order to induce the PCC phenomenon, an incubation with a strong inhibitor, e.g. of a given kinase or phosphatase or alternating incubation in two different inhibitors, of which the former slows down the course of S phase (e.g. hydroxyurea or aphidicolin) and the latter specifically influences the activity of selected kinases or phosphatases is a frequently used approach. Such substances restore the activity of protein kinases and control the phosphorylation of subordinate proteins in the regulatory pathways of cell cycle simultaneously creating conditions necessary for the initiation of prophase chromosome condensation, thereby fulfilling the role of the inductors of Cdk1-

cyclin B complexes and realising mitotic phosphorylations (Sturgeon et al., 2008).

cells showing morphological features of premature mitosis (Figure 1A).

Disturbance of the efficiency of cell cycle checkpoints can result from the action of many factors followed by overriding or breakage of control over genome integrity and the course of various interphase subperiods and finally PCC induction. The premature mitosis induced in meristems of *V. faba* roots by prolonged incubation with a mixture of hydroxyurea and caffeine is characterized by strong differentiation between the morphological forms of chromosomes, which allows one to separate several different cell classes. The degree of chromosome fragmentation (number of sections lost in anaphase) probably determines the level of genetic material disintegration in cells blocked by hydroxyurea in S-phase. Such observations suggest that there is a relationship between the chromosome structure determining 'PCC phenotype' and S-phase subperiod (initiating the biosynthesis of 'early' or 'late' replicons), in which replication block has taken place. This assumes that the period of time elapsing between the beginning of PCC induction and the appearance of first cells with symptoms of premature mitosis depends on the number of replication units in which the biosynthesis processes of complementary DNA strands have been initiated but not terminated. The same mechanism may also explain the considerable differentiation between

Modern models assume that phosphorylation of the N-terminal fragments of H3 histones constitutes a preliminary molecular signal which makes initiation of chromosome condensation possible by creating conditions for the assembly of other proteins directly engaged in the structural metamorphoses of chromatin (e.g. condesin complexes). Therefore, the post-translation modifications of H3 molecules are only one of the symptoms reflecting the action of complicated regulatory system which leads to the increase in chromatin packing during prophase chromosome condensation. This view is consistent with observations pointing to considerable asynchrony of the period of intensified H3 histone phosphorylation and the initial stages of mitotic condensation among various organisms, as well as to the phosphorylation of H3 molecules in plant cells taking place in condensed


Table 1. Data concerning PCC induction reported in the word literature

*The first observation of PCC*  Johnson & Rao, 1970 *The first correct interpretation of PCC* 

Le Bourhis et al., 2010

*The first successfully applied polyethylene glycol (PEG) induced fusion just before PCC induction* 

*The first one successfully applied in chemically-induced PCC in S phase cells*  Ghosh et al., 1998; Schlegel et al., 1990; Yamashita et al., 1990

Rybaczek & Kowalewicz-Kulbat, 2011;

*The first one successfully applied in chemically-induced PCC in S phase cells*  Schlegel et al., 1990; Yamashita et al., 1990; Nghiem et al., 2001

> Sen & Ghosh, 1998; Rybaczek et al., 2008; Rybaczek & Kowalewicz-Kulbat, 2011; Steinmann et al., 1991

Bialojan & Takai, 1988; Cohen et al., 1990; Gotoh et al., 1995; Gotoh & Tanno, 2007; Ishihara et al., 1989; Prasanna et al., 2000

cited by Tam & Schlegel, 1995

**The way of induction of PCC References** 

(a) UV inactivated Sendai virus-induced PCC Kato & Sandberg, 1967

(a) (PEG)-induced fusion Pantelias & Maillie, 1983

(a) Okadaic acid Schlegel & Pardee, 1986

(b) Sodium metavanadate Ghosh et al., 1998;

(a) Caffeine Schlegel & Pardee, 1986

**B2.2. Adriamycin (Doxorubicin)** Hittelman & Rao, 1975

Table 1. Data concerning PCC induction reported in the word literature

(b) During heat exposure Mackey et al., 1988; Swanson et al., 1995; (c) In normal and transformed mammal cells Kovaleva et al., 2007 (d) After X-irradiation of HeLa cells Ianzini & Mackey, 1997

**A. Fusion-induced PCC** 

**B. Drug-induced PCC** 

(b) Caffeine 2-aminopurine Staurosporine 6-dimethylaminopurine

 (a) Okadaic acid Calyculin A

**B2. Without synchronization** 

(in any phase of cell cycle)

**C. Spontaneous PCC (SPCC)** 

development of endosperm

 **B1. With synchronization**  Cells had to be synchronized in G1- or S- or G2-phase before PCC induction  **B1.1. Induced by protein phosphatase inhibitors** 

 Exploits the action of external MPF **A1. Virus-induced fusion** 

**A2. Chemically-induced fusion** 

(b) PCC-type remodeling of the donor nucleus after somatic cell nuclear transfer (SCNT)

Exploits the activation of endogenous MPF

 **B1.2. Induced by protein kinase inhibitors** 

PCC induction occurs without synchronization

**B2.1. Induced by protein phosphatase inhibitors** 

(a) In the ontogenesis of generative cells and during the

A specific change occurred when the preliminary cell-free cytoplasmic extracts (obtained by the centrifugation of *Xenopus* egg cells) were used *in vitro* to study the assembly of 'synthetic nuclei' involving the conversion of chromatin sperms into mitotic chromosomes. The resulting expansion of knowledge concerning the action of inhibitors and/or activators used for PCC induction has created new opportunities and perspectives for researchers (Prokhorova et al., 2003). In cells with disturbed DNA structure or those blocked during DNA biosynthesis, PCC-type processes can be induced by various chemical compounds such as: (i) inhibitors of protein kinases, e.g. 2-aminopurine (Herbig et al., 2004), caffeine (Wang et al., 1999, Gabrielli et al., 2007), staurosporine, 7-hydroxystaurosporine (UCN-01), CEP-3891 (Kohn et al., 2002; Syljuåsen et al., 2005), wortmannin (WORT) (Liu et al., 2007) and Gö6976 (Jia et al., 2008) as well as (ii) inhibitors of protein phosphatases, e.g. calyculin A (CalA), okadaic acid (OA) and sodium metavanadate (Van) (Hosseini & Mozdarani, 2004; Rybaczek & Kowalewicz-Kulbat, 2011). In order to induce the PCC phenomenon, an incubation with a strong inhibitor, e.g. of a given kinase or phosphatase or alternating incubation in two different inhibitors, of which the former slows down the course of S phase (e.g. hydroxyurea or aphidicolin) and the latter specifically influences the activity of selected kinases or phosphatases is a frequently used approach. Such substances restore the activity of protein kinases and control the phosphorylation of subordinate proteins in the regulatory pathways of cell cycle simultaneously creating conditions necessary for the initiation of prophase chromosome condensation, thereby fulfilling the role of the inductors of Cdk1 cyclin B complexes and realising mitotic phosphorylations (Sturgeon et al., 2008).

Disturbance of the efficiency of cell cycle checkpoints can result from the action of many factors followed by overriding or breakage of control over genome integrity and the course of various interphase subperiods and finally PCC induction. The premature mitosis induced in meristems of *V. faba* roots by prolonged incubation with a mixture of hydroxyurea and caffeine is characterized by strong differentiation between the morphological forms of chromosomes, which allows one to separate several different cell classes. The degree of chromosome fragmentation (number of sections lost in anaphase) probably determines the level of genetic material disintegration in cells blocked by hydroxyurea in S-phase. Such observations suggest that there is a relationship between the chromosome structure determining 'PCC phenotype' and S-phase subperiod (initiating the biosynthesis of 'early' or 'late' replicons), in which replication block has taken place. This assumes that the period of time elapsing between the beginning of PCC induction and the appearance of first cells with symptoms of premature mitosis depends on the number of replication units in which the biosynthesis processes of complementary DNA strands have been initiated but not terminated. The same mechanism may also explain the considerable differentiation between cells showing morphological features of premature mitosis (Figure 1A).

Modern models assume that phosphorylation of the N-terminal fragments of H3 histones constitutes a preliminary molecular signal which makes initiation of chromosome condensation possible by creating conditions for the assembly of other proteins directly engaged in the structural metamorphoses of chromatin (e.g. condesin complexes). Therefore, the post-translation modifications of H3 molecules are only one of the symptoms reflecting the action of complicated regulatory system which leads to the increase in chromatin packing during prophase chromosome condensation. This view is consistent with observations pointing to considerable asynchrony of the period of intensified H3 histone phosphorylation and the initial stages of mitotic condensation among various organisms, as well as to the phosphorylation of H3 molecules in plant cells taking place in condensed

Eidetic Analysis of the Premature Chromosome Condensation Process 191

growing number of γ-tubulin molecules. Meanwhile, normal cells (and not the transformed ones) are subjected to the control mechanisms of G2 phase which, by the intervention of ATM/ATR kinases, monitor the condition of DNA structure and make it possible to arrest

**A B** Feulgen H3S10

PCC-like prophases

PCC-like metaphases

PCC-like anaphases

PCC-like telophases Fig. 1. A. Caffeine-induced PCC in Feulgen-stained root meristem cells of *Vicia faba*. The full array of aberrations included: chromosomal breaks and gaps, lost and lagging chromatids and chromosomes, acentric fragments and micronuclei. B. Immunofluorescence of phospho-H3 (S10; Cell Signaling) after caffeine-induced PCC in root meristem cells of *Vicia faba*. *Bar* 20 μm

the cell cycle just before the initiation of mitosis.

chromosomes. Meanwhile, S10 phosphorylation of H3 histone has turned out to be an excellent marker in phenotyping PCC from various subperiods of e.g. S-phase (Figure 1B).

The G2→M transition is a period of an increased sensitivity of cells also to the action of factors that directly do not lead DNA damage. For example, when cells entering the early stages of prophase are subjected to hypothermia, anoxia, osmotic shock or other stresses, their chromosomes are decondensed followed by a return to late G2 phase. This phenomenon usually is reversible following a period of stress adaptation or regression of mitosis (Mikhailov & Rieder, 2002). So, the question - what is the beginning of mitosis? – is not trivial, especially in the context of its irreversibility. It is known that almost always the very fact of initiation of S phase determines future mitosis. On the other hand, the fact of DNA replication involving a successful transition through the cell cycle checkpoints gives the process of genetic material segregation to two daughter cells the status of authority or even necessity. It helps when seeking the answer to the question 'what is the beginning of mitosis?' to recall the term 'antephase', historically associated with the paper by Furlough & Johnson published in 1951 (Pines & Rieder, 2001, as cited in Furlough & Johnson, 1951).

The term 'antephase' is used to describe the final stage of G2 phase directly preceding the first perceptible symptoms of prophase chromosome condensation. Cells in the middle stages of prophase (cf. neuroblasts of grasshopper, cells of newt or PtK1 cell line) subjected to ionizing radiation gradually decondense the chromosomes under formation, their proteins are dephosphorylated and the course of cell cycle is arrested. The same cells irradiated during late stages of prophase, despite strong chromosome fragmentation, initiate further mitosis stages. A similar process – gradual chromosome decondensation – is also observed when the disassembly of microtubules by nocodazole takes place before cell transition through the middle stage of prophase. The action of nocodazole in later periods of prophase induces colcemid-mitoses – the division of chromosomes deprived of communication with the microtubular spindle apparatus leading to the formation of polyploid nuclei. Thus there is a specified point, i.e. the final period of 'antephase', after which a cell is unable to return to the interphase condition. In the cells of many animal species (vertebrates), 'the point of the last chance to return to G2 phase' occurs during the final stages of prophase, the border here between interphase and mitosis being set considerably later than usual (Pines & Rieder, 2001).

Thus, there are cells in which advanced stages of chromosome condensation are an easily recognizable indicator of G2 phase termination ('antephase'), whence they become convenient subjects for studying the transient G2→M period at a molecular level. The activity of kinase Cdk1-cyclin A complexes increases during the G2 phase and reaches its maximum at the moment of nuclear envelope decomposition. The microinjection of these complexes into G2 cells induces a violent chromosome condensation but inhibitors of Cdk1 kinase (such as p21Waf1/Cip1) block the transition to mitosis and then the early-prophase cells return to the interphase state. This is a period during which inactive Cdk1-cyclin B complexes remain in the area of cytoplasm (Furuno et al., 1999). Thus, it seems that preparation for the entry into mitosis taking place in vertebrate cells during 'antephase' is mainly controlled by Cdk1-cyclin A complex and not by the association of Cdk1 with cyclin B. An important role is also played by Plk1 kinase, which controls organization of the cell centrosomal apparatus and, while phosphorylating Cdc25 phosphatase, indirectly activates Cdk1-cyclin B complexes (Kumagai & Dunphy, 1996). Another protein kinase, Aurora B (animals) or its homologue in yeast cells (Ipl1) induces chromosome condensation via phosphorylation of S10 in H3 histone (Hsu et al., 2000). In many types of cells, changes occur at the level of chromosome condensation causing centrosomes to interact with a 190 DNA Repair

chromosomes. Meanwhile, S10 phosphorylation of H3 histone has turned out to be an excellent marker in phenotyping PCC from various subperiods of e.g. S-phase (Figure 1B). The G2→M transition is a period of an increased sensitivity of cells also to the action of factors that directly do not lead DNA damage. For example, when cells entering the early stages of prophase are subjected to hypothermia, anoxia, osmotic shock or other stresses, their chromosomes are decondensed followed by a return to late G2 phase. This phenomenon usually is reversible following a period of stress adaptation or regression of mitosis (Mikhailov & Rieder, 2002). So, the question - what is the beginning of mitosis? – is not trivial, especially in the context of its irreversibility. It is known that almost always the very fact of initiation of S phase determines future mitosis. On the other hand, the fact of DNA replication involving a successful transition through the cell cycle checkpoints gives the process of genetic material segregation to two daughter cells the status of authority or even necessity. It helps when seeking the answer to the question 'what is the beginning of mitosis?' to recall the term 'antephase', historically associated with the paper by Furlough & Johnson published in 1951 (Pines & Rieder, 2001, as cited in Furlough & Johnson, 1951). The term 'antephase' is used to describe the final stage of G2 phase directly preceding the first perceptible symptoms of prophase chromosome condensation. Cells in the middle stages of prophase (cf. neuroblasts of grasshopper, cells of newt or PtK1 cell line) subjected to ionizing radiation gradually decondense the chromosomes under formation, their proteins are dephosphorylated and the course of cell cycle is arrested. The same cells irradiated during late stages of prophase, despite strong chromosome fragmentation, initiate further mitosis stages. A similar process – gradual chromosome decondensation – is also observed when the disassembly of microtubules by nocodazole takes place before cell transition through the middle stage of prophase. The action of nocodazole in later periods of prophase induces colcemid-mitoses – the division of chromosomes deprived of communication with the microtubular spindle apparatus leading to the formation of polyploid nuclei. Thus there is a specified point, i.e. the final period of 'antephase', after which a cell is unable to return to the interphase condition. In the cells of many animal species (vertebrates), 'the point of the last chance to return to G2 phase' occurs during the final stages of prophase, the border here between interphase and mitosis being set considerably later than usual (Pines & Rieder, 2001). Thus, there are cells in which advanced stages of chromosome condensation are an easily recognizable indicator of G2 phase termination ('antephase'), whence they become convenient subjects for studying the transient G2→M period at a molecular level. The activity of kinase Cdk1-cyclin A complexes increases during the G2 phase and reaches its maximum at the moment of nuclear envelope decomposition. The microinjection of these complexes into G2 cells induces a violent chromosome condensation but inhibitors of Cdk1 kinase (such as p21Waf1/Cip1) block the transition to mitosis and then the early-prophase cells return to the interphase state. This is a period during which inactive Cdk1-cyclin B complexes remain in the area of cytoplasm (Furuno et al., 1999). Thus, it seems that preparation for the entry into mitosis taking place in vertebrate cells during 'antephase' is mainly controlled by Cdk1-cyclin A complex and not by the association of Cdk1 with cyclin B. An important role is also played by Plk1 kinase, which controls organization of the cell centrosomal apparatus and, while phosphorylating Cdc25 phosphatase, indirectly activates Cdk1-cyclin B complexes (Kumagai & Dunphy, 1996). Another protein kinase, Aurora B (animals) or its homologue in yeast cells (Ipl1) induces chromosome condensation via phosphorylation of S10 in H3 histone (Hsu et al., 2000). In many types of cells, changes occur at the level of chromosome condensation causing centrosomes to interact with a growing number of γ-tubulin molecules. Meanwhile, normal cells (and not the transformed ones) are subjected to the control mechanisms of G2 phase which, by the intervention of ATM/ATR kinases, monitor the condition of DNA structure and make it possible to arrest the cell cycle just before the initiation of mitosis.

Fig. 1. A. Caffeine-induced PCC in Feulgen-stained root meristem cells of *Vicia faba*. The full array of aberrations included: chromosomal breaks and gaps, lost and lagging chromatids and chromosomes, acentric fragments and micronuclei. B. Immunofluorescence of phospho-H3 (S10; Cell Signaling) after caffeine-induced PCC in root meristem cells of *Vicia faba*. *Bar* 20 μm

cell cycle.

Eidetic Analysis of the Premature Chromosome Condensation Process 193

molecular construction must take into account the specificity of the successive phases of the

The mentioned analogies have probably contributed to the fact that the best known function of checkpoints in G2 phase consists in blocking the initiation of mitosis in case of DNA damage. Detection of structural anomalies liberates the action of two complementary and partly convergent molecular pathways centred on ATM/ATR kinases and a common purpose – maintenance the Cdk1 kinase complexes with cyclin B inactive (Mikhailov & Rieder, 2002; Paulsen & Cimprich, 2007). In one pathway, ATM directly (or indirectly) activates part of the regulatory system, with a key factor p53. This protein induces the synthesis of inhibitors of cyclin-dependent kinases, e.g. p21Waf1/Cip1. In the other pathway, independent of p53, ATM activates effector kinases Chk1 and Chk2 that, in turn, prevent the activation of Cdc25C phosphatase (sometimes by Plk1 kinase). Undoubtedly, we have here an excellent example of two coincident regulatory systems that mutually intensify the effectiveness of their individual interactions, while at the same time providing an example of 'economical' use of the same metabolic networks in three different cell cycle phases

Mitosis is the most dramatic and potentially most 'dangerous' cell cycle phase – involving the condensation of replicated chromosomes, their association with the kinetochores and finally the segregation of sister chromatids to opposite poles. Not only chromosomes replicated in S phase are divided (karyokinesis) but also all organelles (cytokinesis). Thus the checkpoints of mitosis monitor all the transient stages of this complicated process but primarily they control the condition of mitotic spindle by detecting abnormalities in its structural and functional organization (Cortez & Elledge, 2000). Chromatin condensation forming mitotic chromosomes under physiological conditions takes place only during mitosis. Condensins are the key elements in this process. Condensin I and II occur among vertebrates. Condensin I obtains access to chromosomes always after the nuclear envelope breakdown (NEBD) and then, in cooperation with condensin II responsible for the initial stages of condensation in prophase nuclei, promotes the assembly of metaphase chromosomes. Condensins are regulated by phosphorylation dependent on Cdk1, which was demonstrated in the studies utilizing *Xenopus* egg extract. Active MPF initiates the nuclear envelope breakdown to allow condensin I to acquire chromosomes. Hypothetically, it is assumed that Cdk1-cyclin A complex phosphorylates and activates condensin II to initiate the early stage of chromosome condensation inside the prophase nucleus. Next, directly after the nuclear envelope breakdown, Cdk1-cyclin B complex phosphorylates and

Cell cycle checkpoints act via the principle of establishing a cause-effect relationship between separate biochemical processes (Hartwell & Weinert, 1989) involving feedback loops (Elledge, 1996). The term 'checkpoint' refers to a definite subset of internal and external regulatory mechanisms that link further processes to the realization of earlier ones. At the same time, one has to remember to always take into account the existence of hysteresis in the molecular interaction of the control network in the cell cycle system. Hysteresis means that it takes more to push a system from point A to point B than it does to keep the system at stage B (Sha et al., 2003; Solomon, 2003). Generally, there are two classes of regulatory systems in the cell cycle: (i) intrinsic systems of a constitutive character sorting out the events directly connected with the cell cycle, and (ii) extrinsic systems that are revealed under the influence of inducing factors and are engaged only when DNA damage

(Brown & Baltimore, 2007; Matthews et al., 2007; Peddibhotla et al., 2009).

activates condensin I (Hirano, 2005).

is detected (Elledge, 1996).

### **3. Molecular origin of PCC induction**

Hypotheses describing the mechanism of PCC induction are based on the results of genetic and biochemical analyses of yeast cells and human and animal cells *in vitro*. Among plants, the induction of premature chromosome condensation sometimes spontaneously occurs in the ontogenesis of generative cells and during the development of endosperm (cited by Tam & Schlegel, 1995). The special nature of these phenomena, the role played in them by phytohormonal factors and conditioning connected with the specificity of plant cell mitotic divisions have not yet been recognized. Neither have the organization of checkpoints blocking the initiation of mitosis, nor the mechanism that could overcome their restrictive interactions, been explained.

During the cell cycle, there are mechanisms governing transition 'to' and 'from' proliferation and coordinating of the complexes responsible for the successive stages of cell cycle transition. The first regulatory system is connected with the transition from G0 phase to cell cycle, which results in the transcription of appropriate genes, this being realization of the program responsible for leaving the resting condition. This leads to the commencement of DNA synthesis and consequently the initiation of mitotic division. The action of the second regulatory system involves arresting the course of G1 phase – before the start of DNA replication – or G2 phase – before the initiation of mitosis.

In cell cycles of the G1-S-G2-M type, the function of two main checkpoints is directly associated with the coordination of transient stages determining the maintenance of genetic identity. The G1 phase checkpoint monitors the metabolic conditions of cells in the G1 phase, the integrity of the nuclear DNA structure prior to the beginning of the synthesis of the complementary chromosome set and the level of necessary substrates, enzymes and replication factors. In plant cytology, it is known as the 'Principal Control Point 1' (PCP1), being the counterpart of the START point in yeast and of the Restriction Point in animal cells. The second checkpoint, PCP2, of a more conservative character, plays a similar role in G2 phase. Its functions are connected with the assessment of internal and external conditions of the cell environment during mitosis and cytokinesis. PCP2 controls if the replication of DNA is properly terminated and also monitors the integrity of chromatin structure before its mitotic condensation (Del Campo et al., 2003). The checkpoint pathways functioning in G1 and G2 phases of cell cycle constitute however only part of the complex system formed by checkpoint mechanisms of the whole cell cycle (Bucher & Britten, 2008). For example, the mechanisms determining S-phase initiation and development are functionally connected with the system of three S-phase checkpoints: (i) the intra-S-phase checkpoint or DSB-induced replicationindependent intra-S-phase checkpoint which block mitosis initiation in the case of structural DNA damage; (ii) the replication checkpoint or replication-dependent intra-S-phase checkpoint which block mitosis initiation in the case of inhibition of DNA biosynthesis; and (iii) the replication-dependent S-M checkpoint which ensures that the G2 phase and mitosis can begin after complete genome replication (Bartek et al., 2004).

In many respects, the control mechanisms of G2 phase are similar to the regulatory systems that protect a cell against too rapid initiation of DNA replication. Still more analogies can be found by comparing both of them with the complex molecular systems of the S phase checkpoints. Admitting some simplification, it can be assumed that this continual repetition is not unusual: if monitoring of the DNA structure integrity is the basic aim of complicated biochemical mechanisms, the means serving that end do not have to be adapted for the realization of many targets, but just one – the most important. On the other hand, their

Hypotheses describing the mechanism of PCC induction are based on the results of genetic and biochemical analyses of yeast cells and human and animal cells *in vitro*. Among plants, the induction of premature chromosome condensation sometimes spontaneously occurs in the ontogenesis of generative cells and during the development of endosperm (cited by Tam & Schlegel, 1995). The special nature of these phenomena, the role played in them by phytohormonal factors and conditioning connected with the specificity of plant cell mitotic divisions have not yet been recognized. Neither have the organization of checkpoints blocking the initiation of mitosis, nor the mechanism that could overcome their restrictive

During the cell cycle, there are mechanisms governing transition 'to' and 'from' proliferation and coordinating of the complexes responsible for the successive stages of cell cycle transition. The first regulatory system is connected with the transition from G0 phase to cell cycle, which results in the transcription of appropriate genes, this being realization of the program responsible for leaving the resting condition. This leads to the commencement of DNA synthesis and consequently the initiation of mitotic division. The action of the second regulatory system involves arresting the course of G1 phase – before the start of DNA

In cell cycles of the G1-S-G2-M type, the function of two main checkpoints is directly associated with the coordination of transient stages determining the maintenance of genetic identity. The G1 phase checkpoint monitors the metabolic conditions of cells in the G1 phase, the integrity of the nuclear DNA structure prior to the beginning of the synthesis of the complementary chromosome set and the level of necessary substrates, enzymes and replication factors. In plant cytology, it is known as the 'Principal Control Point 1' (PCP1), being the counterpart of the START point in yeast and of the Restriction Point in animal cells. The second checkpoint, PCP2, of a more conservative character, plays a similar role in G2 phase. Its functions are connected with the assessment of internal and external conditions of the cell environment during mitosis and cytokinesis. PCP2 controls if the replication of DNA is properly terminated and also monitors the integrity of chromatin structure before its mitotic condensation (Del Campo et al., 2003). The checkpoint pathways functioning in G1 and G2 phases of cell cycle constitute however only part of the complex system formed by checkpoint mechanisms of the whole cell cycle (Bucher & Britten, 2008). For example, the mechanisms determining S-phase initiation and development are functionally connected with the system of three S-phase checkpoints: (i) the intra-S-phase checkpoint or DSB-induced replicationindependent intra-S-phase checkpoint which block mitosis initiation in the case of structural DNA damage; (ii) the replication checkpoint or replication-dependent intra-S-phase checkpoint which block mitosis initiation in the case of inhibition of DNA biosynthesis; and (iii) the replication-dependent S-M checkpoint which ensures that the G2 phase and mitosis

In many respects, the control mechanisms of G2 phase are similar to the regulatory systems that protect a cell against too rapid initiation of DNA replication. Still more analogies can be found by comparing both of them with the complex molecular systems of the S phase checkpoints. Admitting some simplification, it can be assumed that this continual repetition is not unusual: if monitoring of the DNA structure integrity is the basic aim of complicated biochemical mechanisms, the means serving that end do not have to be adapted for the realization of many targets, but just one – the most important. On the other hand, their

**3. Molecular origin of PCC induction** 

replication – or G2 phase – before the initiation of mitosis.

can begin after complete genome replication (Bartek et al., 2004).

interactions, been explained.

molecular construction must take into account the specificity of the successive phases of the cell cycle.

The mentioned analogies have probably contributed to the fact that the best known function of checkpoints in G2 phase consists in blocking the initiation of mitosis in case of DNA damage. Detection of structural anomalies liberates the action of two complementary and partly convergent molecular pathways centred on ATM/ATR kinases and a common purpose – maintenance the Cdk1 kinase complexes with cyclin B inactive (Mikhailov & Rieder, 2002; Paulsen & Cimprich, 2007). In one pathway, ATM directly (or indirectly) activates part of the regulatory system, with a key factor p53. This protein induces the synthesis of inhibitors of cyclin-dependent kinases, e.g. p21Waf1/Cip1. In the other pathway, independent of p53, ATM activates effector kinases Chk1 and Chk2 that, in turn, prevent the activation of Cdc25C phosphatase (sometimes by Plk1 kinase). Undoubtedly, we have here an excellent example of two coincident regulatory systems that mutually intensify the effectiveness of their individual interactions, while at the same time providing an example of 'economical' use of the same metabolic networks in three different cell cycle phases (Brown & Baltimore, 2007; Matthews et al., 2007; Peddibhotla et al., 2009).

Mitosis is the most dramatic and potentially most 'dangerous' cell cycle phase – involving the condensation of replicated chromosomes, their association with the kinetochores and finally the segregation of sister chromatids to opposite poles. Not only chromosomes replicated in S phase are divided (karyokinesis) but also all organelles (cytokinesis). Thus the checkpoints of mitosis monitor all the transient stages of this complicated process but primarily they control the condition of mitotic spindle by detecting abnormalities in its structural and functional organization (Cortez & Elledge, 2000). Chromatin condensation forming mitotic chromosomes under physiological conditions takes place only during mitosis. Condensins are the key elements in this process. Condensin I and II occur among vertebrates. Condensin I obtains access to chromosomes always after the nuclear envelope breakdown (NEBD) and then, in cooperation with condensin II responsible for the initial stages of condensation in prophase nuclei, promotes the assembly of metaphase chromosomes. Condensins are regulated by phosphorylation dependent on Cdk1, which was demonstrated in the studies utilizing *Xenopus* egg extract. Active MPF initiates the nuclear envelope breakdown to allow condensin I to acquire chromosomes. Hypothetically, it is assumed that Cdk1-cyclin A complex phosphorylates and activates condensin II to initiate the early stage of chromosome condensation inside the prophase nucleus. Next, directly after the nuclear envelope breakdown, Cdk1-cyclin B complex phosphorylates and activates condensin I (Hirano, 2005).

Cell cycle checkpoints act via the principle of establishing a cause-effect relationship between separate biochemical processes (Hartwell & Weinert, 1989) involving feedback loops (Elledge, 1996). The term 'checkpoint' refers to a definite subset of internal and external regulatory mechanisms that link further processes to the realization of earlier ones. At the same time, one has to remember to always take into account the existence of hysteresis in the molecular interaction of the control network in the cell cycle system. Hysteresis means that it takes more to push a system from point A to point B than it does to keep the system at stage B (Sha et al., 2003; Solomon, 2003). Generally, there are two classes of regulatory systems in the cell cycle: (i) intrinsic systems of a constitutive character sorting out the events directly connected with the cell cycle, and (ii) extrinsic systems that are revealed under the influence of inducing factors and are engaged only when DNA damage is detected (Elledge, 1996).

Eidetic Analysis of the Premature Chromosome Condensation Process 195

inhibition). In the embryonic evolution of *Drosophila melanogaster*, grapes (*grp*) – whose product is a homolog of Chk1 kinase – is one of the genes of checkpoints functioning in this period of morphogenesis. Grapes mutants show a shortened course of interphase, a defective chromosome condensation and delayed metaphase initiation (Yu et al., 2000). In this case, premature mitosis is caused firstly by overriding of function of S phase checkpoints (then chromosome condensation is not dependent on the termination of S phase) and secondly, by the moment of initiation of chromosome condensation (ICC) occurring with no delay. Thus, only the period between ICC and metaphase is shortened, which seems to be the direct cause of incomplete chromosome condensation. To sum up, in *D. melanogaster* the delay in *grp* embryo entry into metaphase is caused by chromatin

The facts presented above that barely outline extensive problems connected with the cellular control mechanisms show a huge complexity of both stimulating and inhibiting biochemical systems. The associated regulatory network comprises processes that activate the expression of genes at the moment desired for the cell and block the course of chemical reactions when their products could accumulate in excessive amount or prematurely encourage cell cycle transitions. It seems that all eukaryotic cells are equipped with sensory factors, signal transmitting systems and effector factors. The significance of these consists in the fact that they make DNA replication and chromosome division possible without endangering the whole information contained in them which is indispensable for the organism development

The successive phases of prematurely initiated mitosis follow an aberrated course. The loss of relatively large chromosome sections suggests that disturbances of post-replication repair processes in the G2 (G2-PCC) are responsible for this. On the other hand, much higher disintegration of genetic material in some chromosomes leads to the conclusion that this is symptomatic of mitosis initiated from cell subpopulations that have not yet finished the DNA replication process (S-PCC; Rybaczek et al., 2008). Some influence on the degree of DNA fragmentation can be exerted by chemical compounds used in studies of PCC induction, e.g. caffeine which additionally intensifies chromosome destruction during their individualization. It is certain that the losses or breaks in the chromosome continuity: (i) either illustrate the unreplicated areas of genome; (ii) or result from physical stresses created during the mitotic condensation and segregation of chromosomes; (iii) or originate from relative fragility of single-stranded DNA sections generated during retardation of replication forks under the conditions of nucleotide triphosphate deficiency (i.e. after the use

of replication inhibitors, e.g. hydroxyurea) (Cimprich, 2003; El Achkar et al., 2005).

The appearance of double strand breaks (DSBs) in DNA molecules is connected with the formation of immunofluorescence foci associated with the phosphorylated form of H2AX histones at S139 (DSB marker). The family of lysine-like H2A histones includes three subfamilies of proteins (H2A1-H2A2, H2AZ and H2AX). In mammalian cells, H2AX amounts to about 2 to 25% (probably almost 100% in *S. cerevisiae*) (Rogakou et al., 1998). The C-terminal motif of AQ(D/E)(I/L/Y) is the sequence distinguishing H2AX among H2A histones, while S139, ahead of it, is the site of γ-phosphorylation. Formation of γ-H2AX molecules resulting from the exposure of mammalian cells (or living mice) to the action of ionizing radiation (in sublethal or lethal doses) is an extremely violent process (Kurose et al.,

condensation defects rather than by partial DNA replication (Royou et al., 2005).

and maintenance of species continuity.

**4. PCC and DNA damages** 

Each of the cell cycle checkpoints comprises three essential parts: (i) capability of sensing that a cell cycle event is aberrant or incomplete, (ii) means by which this signal is transmitted, and finally, (iii) effectors that delay or block the cell cycle transitions until the problem is resolved. The position of arrest within the cell cycle varies depending on the phase during which the damage is sensed. Since the main role of all these checkpoints is to make a decision whether or not the cell division cycle can be continued, their particular elements deserve special attention as promising targets for pharmacological treatment of cancer (Deckert et al., 2009). The action of checkpoints of cell cycle crucially depends on the effectiveness of the system transmitting signals released by the cell sensory apparatus. The activation of mitotic protein kinases (M-Cdk) is then blocked which makes it possible to effect repairs or to terminate DNA replication or apoptosis induction (Khanna & Jackson, 2001; Zhou & Elledge, 2000).

The PCC phenomenon results from the overriding of S-M checkpoint. It blocks the ability of cells to make mitotic divisions after extensive DNA damage or under conditions of unfinished replication. DNA damage activates in the first place sensor kinases from the PIKK family, ATM and/or ATR which subsequently inhibit the formation of active MPFs by the phosphorylation of Chk2 and Chk1 kinases, which blocks the onset of mitosis. The initiated cascade of signals simultaneously activates repair factors including DNA-PK kinase which is essential for the repair of DNA suffering NHEJ-type damage. Blocking of the function of sensor ATM and/or ATR kinases can bring about avoidance of the restrictive interactions of S phase checkpoints causing premature mitosis (Block et al., 2004).

Knowledge of PCC mechanisms and S-M checkpoint action goes hand in hand with studies of malignant diseases and eagerness to maximize the beneficial effects of radio- and chemotherapy (Erenpreisa & Cragg, 2007). Despite evidence that overexpression of Cdk1 cyclin B complexes can promote PCC, it is not clear if these complexes initiate PCC in human cells. Studies of the import of cyclin B1 in human beings and in asteroid oocytes have shown that it is imported within the period of last several minutes of prophase, just after the initiation of chromosome condensation. These observations suggest that normal chromosome condensation is not initiated by Cdk1-cyclin B1 complexes. On the other hand, there is evidence indicating that the chromosome condensation and other phenomena occurring in the early prophase are initiated by Cdk2-cyclin A complexes present in this period within the cell nucleus. The initiation of cytoplasmic phenomena of mitotic character such as reorganization of Golgi apparatus and microtubular changes probably requires no import of Cdk1-cyclin B1 complexes into the nucleus (Takizawa & Morgan, 2000). On the other hand, the initiation of mitotic division occurs once Cdc25 phosphatase has dephosphorylated phosphate groups (both Y15 and T14) from the area of exposed pocket binding ATP within kinase p34cdc2. Thus Cdc25 phosphatase is the activator of mitosis.

Dysfunctions of S phase checkpoints also occur in mutated cells (Krause et al., 2001). Deletions of *wee1* and *mik1* genes cause disappearance of proteins transmitting signals about DNA structure damage or blocked replication. Such cells initiate mitosis, but underreplications result in broken chromosomes being lost in the central spindle zone. Similar results follow from changes in the activity balance of protein kinases and phosphatases caused by overexpression of *cdc25* genes. Disappearance of intra-S-phase checkpoint function, caused e.g. by the lack (or mutation) of *atr*, also brings about the initiation of mitosis by cells that contain partly replicated genetic material (as opposed to normal cells in which replication forks activate ATR if they meet a defect impeding the biosynthesis of DNA which leads to the activation of S phase checkpoints and cell cycle inhibition). In the embryonic evolution of *Drosophila melanogaster*, grapes (*grp*) – whose product is a homolog of Chk1 kinase – is one of the genes of checkpoints functioning in this period of morphogenesis. Grapes mutants show a shortened course of interphase, a defective chromosome condensation and delayed metaphase initiation (Yu et al., 2000). In this case, premature mitosis is caused firstly by overriding of function of S phase checkpoints (then chromosome condensation is not dependent on the termination of S phase) and secondly, by the moment of initiation of chromosome condensation (ICC) occurring with no delay. Thus, only the period between ICC and metaphase is shortened, which seems to be the direct cause of incomplete chromosome condensation. To sum up, in *D. melanogaster* the delay in *grp* embryo entry into metaphase is caused by chromatin condensation defects rather than by partial DNA replication (Royou et al., 2005).

The facts presented above that barely outline extensive problems connected with the cellular control mechanisms show a huge complexity of both stimulating and inhibiting biochemical systems. The associated regulatory network comprises processes that activate the expression of genes at the moment desired for the cell and block the course of chemical reactions when their products could accumulate in excessive amount or prematurely encourage cell cycle transitions. It seems that all eukaryotic cells are equipped with sensory factors, signal transmitting systems and effector factors. The significance of these consists in the fact that they make DNA replication and chromosome division possible without endangering the whole information contained in them which is indispensable for the organism development and maintenance of species continuity.

#### **4. PCC and DNA damages**

194 DNA Repair

Each of the cell cycle checkpoints comprises three essential parts: (i) capability of sensing that a cell cycle event is aberrant or incomplete, (ii) means by which this signal is transmitted, and finally, (iii) effectors that delay or block the cell cycle transitions until the problem is resolved. The position of arrest within the cell cycle varies depending on the phase during which the damage is sensed. Since the main role of all these checkpoints is to make a decision whether or not the cell division cycle can be continued, their particular elements deserve special attention as promising targets for pharmacological treatment of cancer (Deckert et al., 2009). The action of checkpoints of cell cycle crucially depends on the effectiveness of the system transmitting signals released by the cell sensory apparatus. The activation of mitotic protein kinases (M-Cdk) is then blocked which makes it possible to effect repairs or to terminate DNA replication or apoptosis induction (Khanna & Jackson,

The PCC phenomenon results from the overriding of S-M checkpoint. It blocks the ability of cells to make mitotic divisions after extensive DNA damage or under conditions of unfinished replication. DNA damage activates in the first place sensor kinases from the PIKK family, ATM and/or ATR which subsequently inhibit the formation of active MPFs by the phosphorylation of Chk2 and Chk1 kinases, which blocks the onset of mitosis. The initiated cascade of signals simultaneously activates repair factors including DNA-PK kinase which is essential for the repair of DNA suffering NHEJ-type damage. Blocking of the function of sensor ATM and/or ATR kinases can bring about avoidance of the restrictive

Knowledge of PCC mechanisms and S-M checkpoint action goes hand in hand with studies of malignant diseases and eagerness to maximize the beneficial effects of radio- and chemotherapy (Erenpreisa & Cragg, 2007). Despite evidence that overexpression of Cdk1 cyclin B complexes can promote PCC, it is not clear if these complexes initiate PCC in human cells. Studies of the import of cyclin B1 in human beings and in asteroid oocytes have shown that it is imported within the period of last several minutes of prophase, just after the initiation of chromosome condensation. These observations suggest that normal chromosome condensation is not initiated by Cdk1-cyclin B1 complexes. On the other hand, there is evidence indicating that the chromosome condensation and other phenomena occurring in the early prophase are initiated by Cdk2-cyclin A complexes present in this period within the cell nucleus. The initiation of cytoplasmic phenomena of mitotic character such as reorganization of Golgi apparatus and microtubular changes probably requires no import of Cdk1-cyclin B1 complexes into the nucleus (Takizawa & Morgan, 2000). On the other hand, the initiation of mitotic division occurs once Cdc25 phosphatase has dephosphorylated phosphate groups (both Y15 and T14) from the area of exposed pocket binding ATP within kinase p34cdc2. Thus Cdc25 phosphatase is the activator of mitosis. Dysfunctions of S phase checkpoints also occur in mutated cells (Krause et al., 2001). Deletions of *wee1* and *mik1* genes cause disappearance of proteins transmitting signals about DNA structure damage or blocked replication. Such cells initiate mitosis, but underreplications result in broken chromosomes being lost in the central spindle zone. Similar results follow from changes in the activity balance of protein kinases and phosphatases caused by overexpression of *cdc25* genes. Disappearance of intra-S-phase checkpoint function, caused e.g. by the lack (or mutation) of *atr*, also brings about the initiation of mitosis by cells that contain partly replicated genetic material (as opposed to normal cells in which replication forks activate ATR if they meet a defect impeding the biosynthesis of DNA which leads to the activation of S phase checkpoints and cell cycle

interactions of S phase checkpoints causing premature mitosis (Block et al., 2004).

2001; Zhou & Elledge, 2000).

The successive phases of prematurely initiated mitosis follow an aberrated course. The loss of relatively large chromosome sections suggests that disturbances of post-replication repair processes in the G2 (G2-PCC) are responsible for this. On the other hand, much higher disintegration of genetic material in some chromosomes leads to the conclusion that this is symptomatic of mitosis initiated from cell subpopulations that have not yet finished the DNA replication process (S-PCC; Rybaczek et al., 2008). Some influence on the degree of DNA fragmentation can be exerted by chemical compounds used in studies of PCC induction, e.g. caffeine which additionally intensifies chromosome destruction during their individualization. It is certain that the losses or breaks in the chromosome continuity: (i) either illustrate the unreplicated areas of genome; (ii) or result from physical stresses created during the mitotic condensation and segregation of chromosomes; (iii) or originate from relative fragility of single-stranded DNA sections generated during retardation of replication forks under the conditions of nucleotide triphosphate deficiency (i.e. after the use of replication inhibitors, e.g. hydroxyurea) (Cimprich, 2003; El Achkar et al., 2005).

The appearance of double strand breaks (DSBs) in DNA molecules is connected with the formation of immunofluorescence foci associated with the phosphorylated form of H2AX histones at S139 (DSB marker). The family of lysine-like H2A histones includes three subfamilies of proteins (H2A1-H2A2, H2AZ and H2AX). In mammalian cells, H2AX amounts to about 2 to 25% (probably almost 100% in *S. cerevisiae*) (Rogakou et al., 1998). The C-terminal motif of AQ(D/E)(I/L/Y) is the sequence distinguishing H2AX among H2A histones, while S139, ahead of it, is the site of γ-phosphorylation. Formation of γ-H2AX molecules resulting from the exposure of mammalian cells (or living mice) to the action of ionizing radiation (in sublethal or lethal doses) is an extremely violent process (Kurose et al.,

Eidetic Analysis of the Premature Chromosome Condensation Process 197

protein (ATRIP), there complexes being concentrated in those areas of cell nucleus showing DNA damage. The activator of ATR-ATRIP complexes in vertebrate cells consists of TopBP1 protein. Investigations utilizing cytoplasmic extracts of *Xenopus* egg cells have also shown that the association of ATR with chromatin takes place during the period of DNA replication, whereas it disappears after the termination of replication (Freire et al., 2006;

It is uncertain whether disturbances of DNA replication are detected by means of only one, universal, sensory mechanism. Maybe, regardless of the type of the factor blocking S phase, the DNA structure generated by replication forks is identical at the damage site. However, one cannot exclude the possibility that each type of the DNA structure disturbance exerts different, specific influence on replication forks and that the factors associated with

Literature shows that proper functioning of all multicellular organisms, including *Homo sapiens,* depends not only on their ability to produce new cells but also on the ability of each cell to annihilate itself when it becomes unwanted or damaged. This takes place also when the control mechanisms of the cell cycle have been overridden during a simultaneous strong and/or long-lasting action of stress stimulus. Thus, among cells induced to enter premature, unauthorized division, there are also those that choose the apoptosis pathway (Sahu et al., 2009). Therefore premature mitoses/PCC are described as a mitotic catastrophe, abortive or suicidal. During the induction of apoptosis – following PCC induction – chromatin undergoes drastic changes: previously usually dispersed, it suddenly begins to condense into one or more aggregates in the vicinity of a nuclear membrane (Figure 2A). Changes connected with the initial phase of apoptosis also involve formation of intranuclear membranous structures (sometimes strongly developed and multi-layered) adhering to the

 Fig. 2. Ultrastructure of an apoptotic cell of *V. faba* root meristem (after PCC induction caused by 5 mM caffeine). Selected fragments of the above micro-photography (marked

**D** 

 **A B C** 

Among unicellular organisms, irregularities in the organization of cell cycle control systems result in a decreased reproduction potential, while among multicellular mechanisms they

Harper & Elledge, 2007).

particular types of damage are also different.

**5. Consequences of PCC induction** 

nuclear envelope (Figure 2B-D).

with arrows) at magnification. *Bar* 1μm

2006). Half of the γ-H2AX histones appears after a 1-minute period of irradiation, while the maximal level is reached within 3 to 10 minutes of exposure (per 1 Gy of radiation about 1% of H2AX histone molecules undergoes γ-phosphorylation, which corresponds to about 2x106 bp DNA in the area of the double-strand break of its structure) (Paull et al., 2000; Rogakou et al., 1998). It is assumed that each group of these molecules indicates a single DSB region, therefore the formation of H2AX foci phosphorylated at S139 constitutes a sensitive test revealing the presence of structural genome damage (Friesner et al., 2005). Literature reports in recent years show the occurrence of fluorescence foci of H2AX molecules within the area of S139 during PCC induction (Huang et al., 2006; Rybaczek et al., 2007; Rybaczek & Kowalewicz-Kulbat, 2011). On the other hand, Stevens and his co-workers (2010) clearly indicate that PCC is γ-H2AX negative, and that γ-H2AX phosphorylation is only a hallmark of a chromosome fragmentation phenomenon. However, simultaneously they define PCC as occurring in interphase cells exposed to active MPF vs that occurring during mitosis chromosome fragmentation. According to other investigations, however, the actual definition of drug-induced PCC involves both the described phenomena, i.e. PCC and chromosome fragmentation (Riesterer et al., 2009; Rybaczek et al., 2008; Terzoudi et al., 2010).

To protect against disturbances during DNA biosynthesis, cells have developed a network of biochemical reactions known as DNA-replication-stress-response. The basic strategy of this response is retardation of processes, whose continuation would result, among other things, in the transfer of affected DNA molecules to a new cell generation. Therefore under the conditions of replication stress, the DNA biosynthesis rate is slowed down and onset of mitosis is - most frequently - completely blocked. This continues until the expression of specific genes and activation of repair factors (Deckert et al., 2009; Mosesso et al., 2010). Each structural disturbance (e.g. DSB) causes the rate of production of replication forks to slow down. Additionally, any limitation of the replication apparatus effectiveness (resulting, e.g. from the deficiency of nucleotide triphosphate or polymerase dysfunction) can facilitate DNA damage. In such a situation, the action of checkpoint sensory factors releases a cascade of signals supplied to various effector proteins through intermediary elements.

The detection of double-strand breaks in DNA molecules activates the biochemical pathway in which ATM kinase is the superior element. The processes proceeding with its contribution are triggered in all the phases of cell cycle, while the factors participating here may also assist the second pathway going in parallel. ATR is also activated by damage resulting in disruption of continuity in both DNA strands, but here the induction process occurs more slowly (Scott & Pandita, 2006). The pathway subordinated to ATR kinase is specialized first of all in reacting to disturbance of the replication fork function. These disturbances can results from endogenous interactions, chemotherapy or experimental procedures leading to the inhibition or disturbance of replication processes caused by hydroxyurea (HU) or ultraviolet radiation (UV). In both biochemical pathways, Cdc25 phosphatase is the target substrate.

Mec1 and Tel1 kinases in *S. cerevisiae* and Rad3 and Tel1 kinases in humans are homologues of both conservative signaling ATM and ATR kinases (Garber et al., 2005). Their substrates, kinases of the CHK family, are subordinate factors activated by ATM/ATR/Mec1/Rad3. Such groups jointly form the central pathway module of response to replication stress, which both records incoming information on the DNA condition and sends signals to replication forks. In human and *Xenopus* cells, ATR kinase is indispensable in the phosphorylation of Chk1 kinase and it occurs in stable combination with ATR-interacting protein (ATRIP), there complexes being concentrated in those areas of cell nucleus showing DNA damage. The activator of ATR-ATRIP complexes in vertebrate cells consists of TopBP1 protein. Investigations utilizing cytoplasmic extracts of *Xenopus* egg cells have also shown that the association of ATR with chromatin takes place during the period of DNA replication, whereas it disappears after the termination of replication (Freire et al., 2006; Harper & Elledge, 2007).

It is uncertain whether disturbances of DNA replication are detected by means of only one, universal, sensory mechanism. Maybe, regardless of the type of the factor blocking S phase, the DNA structure generated by replication forks is identical at the damage site. However, one cannot exclude the possibility that each type of the DNA structure disturbance exerts different, specific influence on replication forks and that the factors associated with particular types of damage are also different.
