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Abstract

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

The process of cell division is highly ordered and regulated. Checkpoints exist to delay progression into the next cell cycle phase only when the previous step is fully completed. The ultimate goal is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints can become activated due to DNA damage, exogenous stress signals, defects during the replication of DNA, or failure of chromosomes to attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al., 2002). The tumor suppressor p53 plays a critical role in each of these cell cycle checkpoints and is reviewed here. J. Cell. Physiol. 209: 13–20, 2006. © 2006 Wiley-Liss, Inc.

p53 is a sequence-specific transcriptional regulator that is widely conserved in metazoans. p53 has presumably appeared in evolution as a response in multicellular organisms to the problem of uncontrolled cellular division and the accompanying increased probability of mutation. This notion is supported by the fact that the p53 protein or pathway is mutated or inactivated in most human cancers (Vogelstein et al., 2000). Consistent with this, p53-deficient mice are highly susceptible to spontaneous development of different types of tumors (Donehower et al., 1992; Harvey et al., 1993; Purdie et al., 1994).

Most of the tumor suppressor properties of p53 have been ascribed to its ability to function as a transcription factor. The human p53 gene encodes a 393-amino acid protein that is organized into several domains. The globular central domain of p53 (aa 102–292) has been shown to confer sequence-specific DNA binding activity to the protein (Wang et al., 1993). This domain contains the four conserved regions of p53 and the major mutation hot-spots found in tumor-derived mutants (Pavletich et al., 1993). An oligomerization domain has been mapped to residues 324–355 and p53 has been shown to bind DNA as a tetramer, more specifically a dimer of dimers (Friedman et al., 1993; Wang et al., 1994; McLure and Lee, 1998). The defined DNA consensus sequence for p53 binding consists of two repeats of the palindromic sequence 5′-RRRCA/TT/AGYYY-3′, separated by a 0–13-bp spacer (el-Deiry et al., 1992; Funk et al., 1992). With four pentamer repeats of 5′-RRRCW-3′, the organization of the p53 DNA binding site is consistent with p53 binding as a tetramer. The central domain of p53 also coordinates a zinc ion and metal chelators can inhibit DNA binding (Pavletich et al., 1993). Two independent acidic activation domains have been identified at the N-terminus of p53, between residues 1–42 and 43–73 (Fields and Jang, 1990; Venot et al., 1999). These have been shown to interact with factors of the basal transcription machinery such as TFIID (TBP) and TFIIH, as well as the Drosophila TAFII40 and TAFII60, and their human homologs TAFII31 and TAFII70 (Xiao et al., 1994; Lu and Levine, 1995; Thut et al., 1995; Farmer et al., 1996). The oncogenic protein Mdm2, the main negative regulator of p53 stability and activity, also binds to the N-terminal domain of p53 inhibiting its transcriptional activity (Oliner et al., 1993). Finally, a basic regulatory domain is located at the carboxyl terminus of p53, between residues 363 and 393. This domain has been shown to bind to DNA in a non-specific manner and to negatively regulate specific DNA binding by the central domain (Anderson et al., 1997). Together with the N-terminal domain, the C-terminus of p53 concentrates most of the many post-translational modification sites that contribute to regulate p53 function (Appella and Anderson, 2001) (Fig. 1A).

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Figure 1. p53 structure and pathways. A: p53 is organized in several domains including two N-terminal transactivation domains (TA1, TA2), a proline-rich domain (PRO), a central DNA binding domain (DBD), and a C-terminal regulatory domain (REG). Multiple residues clustered at the N- and C-terminus of p53 are post-translationally modified by phosphorylation (circles), acetylation and ubiquitination (squares), and sumoylation (triangle). B: p53 is stabilized and activated following oncogene activation and a variety of cellular stress. p14ARF induction and post-translational modification of p53 by multiple kinases ultimately lead to disruption of the interaction of p53 with its E3 ligase Mdm2. Activated p53 transcriptionally regulates the expression of a number of target genes that are involved in cell growth arrest, apoptosis, DNA repair, anti-angiogenesis, and feedback regulation of p53 itself.

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p53 can integrate signals from many different pathways that become activated as a result of a variety of stimuli such as DNA damage, hypoxia, and oncogene activation. In these conditions, p53 triggers various cellular responses that can lead to cell-cycle arrest, senescence, differentiation, DNA repair, apoptosis, and inhibition of angiogenesis. p53-dependent transcriptional regulation of p21, 14-3-3σ, Cdc25C, and GADD45 has been proposed to mediate cell growth arrest (el-Deiry et al., 1993; Hermeking et al., 1997). p53 can stimulate DNA repair by inducing the expression of p21, GADD45, and the p48 xeroderma pigmentosum protein (Smith et al., 1994; Li et al., 1994a; Pan et al., 1995; Hwang et al., 1999). Several p53 target genes including Bax, PUMA, Noxa, p53-AIP, PIG3, Fas/APO1, and KILLER/DR5 have been implicated in p53-induced apoptosis (Miyashita and Reed, 1995; Owen-Schaub et al., 1995; Polyak et al., 1997; Wu et al., 1997; Oda et al., 2000a,b; Nakano and Vousden, 2001). Transcriptional upregulation of thrombospondin-1, glioblastoma-derived angiogenesis inhibiting factor, and hypoxia-inducible factor-1α have been proposed to be involved in the inhibition of angiogenesis by p53 (Dameron et al., 1994; Van Meir et al., 1994; Fukushima et al., 1998; Ravi et al., 2000). Finally, several p53 target genes play a role in the regulation of the p53 through an auto-regulatory feedback loop: Mdm2, Pirh2, COP1, and cyclin G (Juven et al., 1993; Okamoto and Beach, 1994; Haupt et al., 1997; Okamoto et al., 2002; Leng et al., 2003; Dornan et al., 2004) (Fig. 1B).

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Figure 2. G1/S and G2/M checkpoints. Sequential activation of the G1/S cyclins D, E, and A result in hyperphosphorylation of pRb, which then releases E2F, allowing transcription of genes required for entry into S-phase. Cell growth arrest at the G1/S border is initiated by destruction of cyclin D1 and the cdk2 activator Cdc25A. p53 maintains this arrest by inducing the expression of the cdk inhibitor p21 that blocks this progression by inhibiting cdk2 complexes. The G2/M transition is driven by the cdc2/cyclin B complex. Following cellular stress, the G2/M arrest is initiated by phosphorylation of the cdc2/B-activating phosphatase, Cdc25C, which is then sequestrated in the cytoplasm by 14-3-3 proteins. A number of p53 target genes ensure the maintenance of this arrest: p21 inhibits the cdc2/B complex, as well as its activating kinase CAK; GADD45 promotes dissociation of the cdc2/B complex; 14-3-3 σ sequesters cdc2/B in the cytoplasm; Cdc25C expression is repressed by p53.

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The responses elicited by p53, mainly growth arrest and apoptosis, are frequently stimuli- and cell type-specific. The way p53 activates one pathway or the other, however, is poorly understood. It has been proposed that the decision may be driven by affinity (Vousden, 2000). According to this model, growth arrest target genes are regulated through high affinity p53 response elements, whereas the promoters of apoptotic target genes contain low affinity response elements or have additional requirements for transcriptional activation (Inga et al., 2002). In this setting, low levels of stress or DNA damage would induce levels of p53 that upregulate high-affinity growth arrest genes. In contrast, when levels of stress hit a cell leaving it in a situation beyond repair, the higher levels of induced p53 activate apoptotic genes. Alternatively, it has been proposed that specific post-translational modifications of p53 or the recruitment by p53 of specific co-activators could determine the different outcomes following cellular stress. Finally, although most of the studies on p53 function have focused on its transcriptional activity, p53 has been shown to play a transcriptional-independent role in the mitochondrial pathway of apoptosis (Mihara et al., 2003).

p53 AND THE G1/S CHECKPOINT

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

The G1/S checkpoint prevents initiation of DNA replication in cells that have damaged DNA. p53 plays a prominent role in the G1/S checkpoint. Expression of p53 ectopically or following DNA damage, arrests cells at the G1/S transition (Kastan et al., 1991; Lin et al., 1992). Cell cycle progression is driven by phosphorylation events mediated by cyclin/cdk complexes. Cyclin D/cdk4, cyclin E/cdk2, and cyclin A/cdk2 complexes sequentially phosphorylate the tumor suppressor pRb and its family members, resulting in release of the E2F family of transcription factors and transactivation of genes involved in DNA replication. These cyclin/cdk complexes are thus the main targets of the effectors of the G1/S checkpoint (Sherr, 1994). Following activation by DNA damage, p53 induces the expression of the cdk inhibitor p21. p21 is induced and localizes to the nucleus in p53 wild-type cells undergoing G1 arrest, but not in cells expressing mutant p53 (el-Deiry et al., 1993, 1994). p21 was originally thought to bind to and inhibit the activity of all G1 cyclin/cdk complexes (Harper et al., 1993). It was later established that although p21 does, in fact, inhibit cdk2 complexes, not only does it not inhibit cyclin D/cdk4, but it promotes the assembly of the complex (LaBaer et al., 1997). Expression of p21, even in the absence of p53, was sufficient to induce growth arrest at the G1 and G2 phases (Rousseau et al., 1999). Furthermore, p21 is required for efficient G1 arrest. Mouse embryo fibroblasts derived from p21-deficient mice have an impaired DNA damage induced-G1 arrest (Brugarolas et al., 1995; Deng et al., 1995). Finally, deletion of p21 by homologous recombination in the colon cancer cell line HCT116 completely abrogated the G1 arrest following DNA damage (Waldman et al., 1995).

Despite the clear role of p53 in the establishment of the G1 arrest, there is evidence that the first delay following DNA damage is p53-independent. This is consistent with the observation of a transient decrease in cyclin E/A-cdk2 activities after irradiation in the absence of p53 (D'Anna et al., 1997). Evidence has been reported that the phosphatase Cdc25A mediates this delay. Cdc25A regulates the activity of cdk2 by removing inhibitory phosphates near its amino terminus. A DNA damage-activated ATM/Chk2 pathway was shown to lead to phosphorylation of Cdc25A, resulting in its ubiquitin- and proteasome-dependent degradation, and consequently, inhibition of cdk2 (Mailand et al., 2000). Moreover, cyclin D1 has been shown to be degraded following genotoxic stress, and this was required for initiation of the G1 arrest. In addition, upon cyclin D1 degradation, p21 is released and redistributed to cdk2 complexes which are then inhibited by p21 (Agami and Bernards, 2000). The second and p53-dependent G1 delay would take place later after DNA damage, since it requires gene transcription, and it also involves the p53 activating kinases ATM, ATR, and Chk2 (Fig. 2). It is proposed that the p53-dependent arrest in G1 extends the delay initiated by Cdc25A, providing the cell with sufficient time to repair the damaged DNA (Nyberg et al., 2002). The reversibility of the p53-induced G1 arrest, however, appears to be cell-type specific (Di Leonardo et al., 1994; Agarwal et al., 1995; Carr, 2000).

p53 AND THE S-PHASE CHECKPOINT

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

The S-phase checkpoint actually involves three distinct modes that despite some differences, share components and are mutually coordinated. First is the replication checkpoint that is activated by stalling of replication forks due to nucleotide depletion or polymerase inhibition. The second is the intra-S-phase checkpoint that is activated when damaged DNA is detected during S-phase, outside of the replicons, and is therefore replication-independent. Finally, the S/M checkpoint prevents division before the cell has completely duplicated its genome (Bartek et al., 2004). In addition, stalled replication forks frequently collapse, resulting in DNA damage.

A role for p53 at the S-phase checkpoint has been argued in several reports. Cells differing in their p53 status were treated with inhibitors of DNA replication, aphidicolin (which blocks DNA polymerase), hydroxyurea (HU, which inhibits ribonucleotide synthase). In contrast to p53 expressing cells, a significant proportion of p53-null cells continued to enter mitosis, although after a much longer duration of the S phase. These mitotic cells, scored by high levels of phosphorylated H1b, contained less than a 4N DNA complement. Based on these results, p53 was proposed to ensure that cells do not enter mitosis with unreplicated DNA (Taylor et al., 1999).

Downregulation of Cdc7, a kinase that is involved in the firing of replication origins, arrested normal human fibroblasts in S-phase, although without strong activation of checkpoint pathways, measured by low levels of active ATM/ATR and Chk1/Chk2 kinases. Despite undetectable phosphorylation at Ser15, p53 expression was induced, followed by p21 upregulation. When the cells arrested in S-phase were subsequently transfected with p53 siRNA, DNA replication resumed in a fraction of cells with subsequent caspase activation and apoptotis. These results suggest that p53 is required for maintenance of S-phase arrest induced by Cdc7 downregulation (Montagnoli et al., 2004). Furthermore, inactivation of ATR (by expression of a catalytically inactive mutant) and p53 (by expression of Mdm2 or HPV E6) synergized at promoting premature chromatin condensation, a hallmark of mammalian cells that enter mitosis before completing DNA replication (Nghiem et al., 2002).

With the frequent occurrence of stalled replication forks and double strand breaks during DNA replication, it has been suggested that a transient delay in S-phase with the absence of a mechanism for maintenance could actually be beneficial to the cells. Non-error prone DNA repair by homologous recombination requires the presence of sister chromatids and therefore can only occur during S or G2 phases. Accordingly, a prolonged blockade in S phase would limit the possibility of efficient repair. In addition, it has been suggested that during a prolonged S-phase arrest, replication origins can regain competence, possibly leading to rereplication (Bartek and Lukas, 2001). Consistent with this idea, downregulation of cdk2 triggered S-phase arrest, accompanied by p53 and p21 induction and ATM/ATR activation. While some cells died of apoptosis, cdk2 inactivation lead to increased loading of MCM complexes onto chromatin, and cells acquired a >4N DNA content, indicating rereplication. However, arguing for a role of p53 at the S-phase checkpoint, this phenotype was enhanced when cdk2 inactivation was combined with downregulation of p53 (Zhu et al., 2004).

Finally, the combination of activated p53 and E2F-1 found during S phase, can result in apoptosis (Bartek and Lukas, 2001; Gottifredi et al., 2001). This possibility was raised by studies where p53 was shown to become stabilized after HU. However, in contrast to irradiation, HU-induced p53 appeared to be transcriptionally inactive, as it failed to upregulate a number of target genes that are usually induced after irradiation. Moreover, in contrast to irradiation, p53 activation and Ser15 phosphorylation induced by HU did not require ATM (Gottifredi et al., 2001). More recently, a new p53 isoform was described that results from alternative splicing at exons 7 to 9. Despite lacking 39 residues of the core domain, this Δp53 isoform was shown to bind to promoters and transactivate a subset of p53 target genes, in particular the p21 and 14-3-3σ growth arrest genes, but not the apoptotic PIG3 gene. Even more interesting is the fact that Δp53 is active only during S-phase in G1/S UV irradiated cells, when full-length p53 is inactive. The data presents a scenario where Δp53 and p53 work in a consecutive and almost mutually exclusive manner (Rohaly et al., 2005). The appearance of this apparently apoptosis-defective isoform of p53 could reconcile the idea that active p53 during S-phase could be deleterious to the cell due to induction of apoptosis, with the several reports that argue in favor of a role for p53 at the S-phase checkpoint. The novelty of the Δp53 isoform, combined with the unprecedented circumstances that surround it, although very promising and exciting, ask for more extensive and careful examination of the involvement of p53 at the S-phase checkpoint (Prives and Manfredi, 2005).

p53 AND THE G2/M CHECKPOINT

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

G2/M progression is driven by the maturation-promoting factor MPF, a complex of cyclin B1/cdc2. Cyclin B1 and cdc2 accumulate and associate in the cytoplasm during G2. The complex is kept inactive by phosphorylation of cdc2 at Thr14 and Tyr15. The entry into mitosis is triggered by translocation of the complex to the nucleus and activation by dephosphorylation by the Cdc25 phosphatases. Cdc2 is further activated by phosphorylation at Thr161 by the cdk-activating kinase CAK (Nurse, 1990).

DNA damage activates checkpoint mechanisms that arrest cells at G1/S, but also at G2/M. The critical cyclin B1/cdc2 complex is the main target of the G2 checkpoint pathways that involve activation of the ATM and ATR and their downstream substrates Chk1 and Chk2 (Nyberg et al., 2002). In addition to the G1/S arrest, p53 has been shown to participate in the G2/M checkpoint (Stewart et al., 1995). Similarly to what has been proposed for the G1 arrest, p53 is involved in the maintenance rather than the initiation of the G2 arrest. For example, wild-type p53 HCT116 cells were able to sustain a G2 arrest following IR, whereas the isogenic p53-null derivative arrested initially in G2 but then escaped and entered mitosis (Bunz et al., 1998). Several p53 target genes were shown to play a role in the p53-induced G2 arrest (Fig. 2).

Cdc25C, the phosphatase that promotes mitosis, is inhibited after DNA damage through phosphorylation at Ser216 by Chk1, Chk2, and other kinases. This modification creates a binding site for 14-3-3 proteins and this association sequesters Cdc25C in the cytoplasm and/or inhibits its phosphatase activity (Lin et al., 1992; Lopez-Girona et al., 2001). Cdc25C has been shown to be a target of repression by p53 following DNA damage (St Clair et al., 2004). In addition, a particular 14-3-3 isoform, 14-3-3σ, is also a p53 target gene and is upregulated following DNA damage (Hermeking et al., 1997). 14-3-3σ prevents proper nuclear localization of cyclin B1/cdc2 after DNA damage. Further, deletion of 14-3-3σ by homologous recombination in HCT116 cells, resulted in cell death in response to irradiation (Chan et al., 1999).

Although it had been suggested that p21 is a poor inhibitor of cdc2 in vitro, compared to other cyclin-dependent kinases (Harper et al., 1995), p21 was also implicated in the sustained G2 arrest, through inhibition of the cyclin B1/cdc2 complex activity (Bunz et al., 1998). Early studies had reported that p21 mRNA has a bimodal expression in cycling cells, peaking in G1, decreasing during S-phase, and accumulating again in G2 (Li et al., 1994b). Consistent with that, p21 was detected in the nucleus in late G2, and this correlated with a delayed entry into mitosis that was not observed in cells lacking p53 or p21. At this stage, p21 was found associated with inactive cyclin A/cdk2 complexes, and to a lesser extent those containing cyclin B1. It was proposed that p21, by inducing a pause before the onset of mitosis, could facilitate G2/M checkpoint implementation (Dulic et al., 1998). Using p53-null cells expressing wild-type p21, or a p21 mutant incapable of binding PCNA, it was shown that the ability of p21 to interact with PCNA is required for a sustained G2 arrest. p21 co-immunoprecipitated with the cyclin B1/cdc2 complex and PCNA in cells arrested in G2 after DNA damage. In contrast, in cells with no p21, the p21 mutant, or in the absence of DNA damage, PCNA was found associated with Cdc25C. Because the binding of PCNA to Cdc25C or p21 appeared to be mutually exclusive, the authors hypothesized that p21 may contribute to the G2 arrest by preventing the interaction of Cdc25C with the cyclin B1/cdc2 complex (Ando et al., 2001). Also, p21 was reported to retain cyclin B1/cdc2 complexes in the nucleus, preventing its activation by Cdc25C and recruitment to the centrosomes (Charrier-Savournin et al., 2004). Another study, however, showed that p21 inhibits the cyclin B1/complex not by preventing dephosphorylation at the inhibitory Thr14/Tyr15, but by preventing activation of cdc2 by CAK (Smits et al., 2000).

In contrast to these observations, a novel positive regulation of the cyclin B1/cdc2 complex by p21 has been described. A transient hyperphosphorylated form of p21 was detected in the nucleus in early G2, preceding the phosphorylation of Cdc25C, dephosphorylation of cdc2 at Thr14/Tyr15, and the nuclear accumulation of cyclin B1. The authors propose a model in which p21, following translocation to the nucleus during S-phase, becomes phosphorylated at Thr57, possibly by cdk2. The presence of p21 correlated with increased cyclin B1-associated kinase activity. Using cell extracts and working with mutants, examination of the formed complexes by immunoprecipitation revealed that hyperphosphorylation of p21 facilitates binding of p21 to Ser126-phosphorylated cyclin B1 and promotes the formation of the cyclin B1/cdc2 kinase complex (Dash and El-Deiry, 2005).

Another p53 target gene involved in the G2 arrest is GADD45. GADD45 has been shown to interact with PCNA (Smith et al., 1994; Kearsey et al., 1995). GADD45 also interacts with cdc2 and inhibits its kinase activity, presumably by causing dissociation of the cyclin B1/cdc2 complex (Zhan et al., 1999). Further, induction of GADD45 resulted in G2 arrest associated with increased cytoplasmic cyclin B1 (Jin et al., 2002), and overexpression of cyclin B1 and Cdc25C abrogated GADD45-induced G2. Of note, induction of G2 arrest by GADD45 requires the presence of wild-type p53 and depends on the type of DNA damage. Human cells with downregulated GADD45 expression or lymphocytes from GADD45 knock-out mice failed to arrest in G2 following UV or the radiomimetic MMS, but had an intact response after irradiation (Wang et al., 1999).

p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

The spindle assembly checkpoint ensures that cells will not enter anaphase, where chromosome segregation occurs, until all chromosomes are aligned at the equator and attached to the microtubules of the mitotic spindle. Through activation of Mad1/2 and other proteins, spindle defects or the presence of unattached kinetochores will result in inactivation of the anaphase promoting complex or cyclosome, APC/C. This complex regulates the degradation by the proteasome of a number of proteins that regulate sister chromatid cohesion, and spindle elongation, as well as cyclin B1. Degradation of these proteins is a prerequisite for progression of mitosis (Hardwick, 1998). However, even in the absence of a functional mitotic spindle and chromosome segregation, MPF can undergo spontaneous degradation, and cells can enter an apparent state of interphase, a process known as adaptation or mitotic slippage (Rieder and Maiato, 2004). Failure of the mitotic spindle checkpoint can lead to cells exiting mitosis and entering the next S phase with a 4N DNA content, resulting in endoreduplication.

p53 was initially believed to control the mitotic spindle checkpoint because p53-deficient fibroblasts failed to arrest after treatment with microtubule inhibitors, undergoing a second round of DNA synthesis in the absence of division and becoming octaploid (Cross et al., 1995; Di Leonardo et al., 1997). In addition, it was thought that spindle components themselves could be under regulation of p53, since p53 binds to centrosomes (Brown et al., 1994) and p53−/− mouse embryo fibroblasts contain an abnormal number of centrosomes (Fukasawa et al., 1996). More detailed examination showed that expression of p53 occurred after cells had exited mitosis and progressed to a G1-like state (Minn et al., 1996). The time spent in mitosis by cells treated with nocodazole was the same, regardless of their p53 status. In addition, there were strong similarities between the p53-dependent checkpoint following nocodazole and the p53-dependent G1 arrest following irradiation: requirement of p21, high cyclin E and low cyclin B1 protein levels, hypophosphorylated pRb, and flat morphology (Lanni and Jacks, 1998). The nature of the signal that causes G1 arrest due to spindle malfunction is unclear. A p53-dependent G1 arrest with a 4N DNA content was also observed when actin assembly was inhibited, preventing cytokinesis even when spindle formation and chromosome segregation could proceed normally (Andreassen et al., 2001). This suggests that it is tetraploidy itself, and not the activation of the mitotic checkpoint, which causes G1 arrest. However, another study argues that a prolonged mitotic arrest is required for tetraploid G1 arrest. HCT116 cells where Mad1/2 function was diminished by downregulation or by a dominant negative approach had a compromised spindle checkpoint following nocodazole treatment. They were able to escape mitosis and endoreduplicate despite expressing p53. However, when mitotic Mad1/2-deficient cells were forced into a prolonged mitotic arrest by treatment with a proteasome inhibitor, a reduced proportion of cells undergoing endoreduplication was observed. These results in turn suggest that not only functional p53 but also an intact spindle assembly checkpoint is required for the 4N G1 arrest (Vogel et al., 2004). These results show that p53 plays a critical role in preventing aneuploidy by blocking endoreduplication of tetraploid cells that result from mitotic failure.

ROLE OF p53 IN DNA REPAIR

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

In addition to growth arrest and apoptosis, the ability of p53 to regulate DNA repair after genotoxic stress represents another mechanism by which p53 helps maintaining genomic integrity. p53-deficient cells have impaired nucleotide-excision repair (NER). More specifically, the global genomic repair (GGR) rather than the transcription-coupled repair (TCR) appears to be affected by loss of p53 (Ford and Hanawalt, 1995).

The participation of p53 in DNA repair involves transcriptional regulation as well as direct interaction with mediators of DNA repair. For example, in addition to binding and inhibiting cyclin-dependent kinases, the p53 target p21 has been shown to bind to PCNA, the DNA polymerase δ loading, and processivity factor. Through this association, p21 was reported to block replication (Waga et al., 1994), but not PCNA-dependent NER (Li et al., 1994a). However, the role of p21 in DNA repair remains controversial (Adimoolam et al., 2001; Stivala et al., 2001; Wani et al., 2002; Bendjennat et al., 2003). As mentioned earlier, GADD45 is another p53-regulated gene that also binds to PCNA (Smith et al., 1994). GADD45 stimulated DNA excision repair in vitro, and downregulation of GADD45 affected DNA repair and sensitized cells to killing by UV and genotoxic drugs (Smith et al., 1996). Mouse embryo fibroblasts from p53 and GADD45 knock-out mice presented impaired GGR, whereas loss of p21 had only a marginal effect (Smith et al., 2000). The xeroderma pigmentosum p48 gene is transactivated by p53 and shown to be required for efficient GGR as well (Hwang et al., 1999).

Regulation of deoxynucleotide triphosphate pools appears to be characteristic of DNA repair, since their limited availability increases damage sensitivity (Nyberg et al., 2002). For instance, the ribonucleotide reductase-like protein p53R2 was reported to be induced by p53 after DNA damage by ionizing radiation, UV, and DNA damaging agents. Moreover, inhibition of p53R2 reduced DNA repair and cell survival after genotoxic stress. (Nakano et al., 2000; Tanaka et al., 2000).

p53 can directly interact with proteins involved in DNA repair. p53 binds to replication protein A (RPA), a single-stranded DNA binding protein involved in DNA replication, homologous recombination (HR), and NER (Janus et al., 1999). The association of p53 and RPA is disrupted after UV, and the released RPA can then participate in NER (Abramova et al., 1997). p53 can also bind to members of the TFIIH complex that couples transcription with NER. These include XPB, XPC, and the cdk7/cyclin H/p36 (CAK) complex. p53 is regulated by CAK through phosphorylation (Ko et al., 1997), linking the DNA repair machinery with p53 activation. The role of these interactions, however, is not clear. It is possible that they could help recruiting p53 to sites of DNA repair or transcription (Janus et al., 1999). Also, p53 inhibits the helicase activity of XPB, XPC and the repair protein Cockayne Syndrome B protein (CSB), but the mechanistic role of that has yet to be found.

In addition to binding to DNA repair proteins, p53 interacts directly with a variety of DNA structures. p53 binds to double-stranded and single-stranded DNA in a non-specific way, to ends of double-strand breaks, to Holliday junctions, and to DNA bulges caused by DNA mismatches. Furthermore, p53 has been reported to possess DNA-reannealing and DNA end-joining activities, the ability to promote DNA strand transfer, and a 3′ to 5′ exonuclease activity. In particular, studies on p53 exonuclease activity raised some interesting issues. The C-terminus of p53 appears to inhibit its exonuclease activity (Janus et al., 1999). Furthermore, p53 appears to provide proof-reading capacity to DNA polymerase α-primase complex and to HIV1 reverse transcriptase in vitro (Bakhanashvili, 2001; Melle and Nasheuer, 2002). Although still speculative, it is tempting to hypothesize that in its non-induced state in the absence of DNA damage p53 could participate in maintaining genome integrity, perhaps by enhancing the fidelity of DNA synthesis (Albrechtsen et al., 1999).

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

After so many years of viewing p53 almost exclusively as a stress-activated sequence-specific transcriptional regulator, more and more faces of this versatile protein are revealed: an increasingly complex regulation, a growing number of interacting proteins and target genes, some unexpected functions. For instance, the possibility of p53 performing a maintenance role in the absence of stress is still an open question. Technical advances and increased understanding of the mechanisms set in place by p53 will certainly be of great value in optimizing cancer therapies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED

A review examining an aspect of p53 biology is always difficult, given the extensive published literature. The authors apologize for selective citations which, by necessity, resulted in their failure to acknowledge all those who contributed to our current understandings. We thank Luis Carvajal, Dana Lukin, Anthony Mastropietro, Sejal Patel, Lois Resnick-Silverman, and Shohreh Varmeh-Ziaie for helpful discussions and their support. The research in the authors' laboratory was supported by grants from the National Cancer Institute (CA86001 and CA80058) and the Department of Defense Prostate Cancer Research Program (W81XWH-05-1-0109) and Breast Cancer Research Program (W81XWH-05-1-00305).

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. p53 AND THE G1/S CHECKPOINT
  4. p53 AND THE S-PHASE CHECKPOINT
  5. p53 AND THE G2/M CHECKPOINT
  6. p53 AND THE SPINDLE ASSEMBLY CHECKPOINT VERSUS A G1 TETRAPLOID CHECKPOINT
  7. ROLE OF p53 IN DNA REPAIR
  8. CONCLUSIONS
  9. Acknowledgements
  10. LITERATURE CITED