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Abstract

p53 IN CANCER

p53 plays a key role in mediating cell response to various stresses, mainly by inducing or repressing a number of genes involved in cell cycle arrest, senescence, apoptosis, DNA repair, and angiogenesis. According to this important function, p53 activity is controlled in a very complex manner, including several auto-regulatory loops, through the intervention of dozens of modulator proteins (the p53 interactome). p53 mutations are observed in a significant minority of breast tumours. In the remaining cases, alterations of interactome components or target genes could contribute, to some extent, to reduce the ability of p53 to efficiently manage stress events. While the prognostic and predictive value of p53 is still debated, there is an increasing interest for p53-based therapies. The present paper aims to provide updated information on p53 regulation and function, with specific interest on its role in breast cancer, p53 mutations, and recent strategies for improving the efficacy of cancer treatment by therapeutic manipulation of p53.

Introduction
p53 protein was first identified in 1979 as a transformation- related protein and a cellular protein which accumulates in the nuclei of cancer cells and binds tightly to the simian virus 40 (SV402) large T antigen . The gene encoding p53 was initially found to have weak oncogenic activity as the p53 protein was observed to be overexpressed in mouse and human tumor cells . However, almost 10 years later, researchers discovered that it was a missense mutant of p53 which had originally been considered as wild-type p53 (wt p53) in that previous study, and that the oncogenic properties of p53 actually re sulted from a p53 mutation which was later called gain of oncogenic function . By the early 1990s, data from the first p53 knockout mice provided inarguable evidence in support of the potent tumor suppressor action of wt p53 . In subsequent studies, p53 became widely recognized as a tumor suppressor, and the p53 gene became probably the most common site for genetic alterations in human cancers . Subsequent research with wt p53 clearly demonstrated that p53 was a major guardian of the genome . The biological consequences of p53 activity include cell-cycle regulation, induction of apoptosis, development, differentiation, gene amplification, DNA recombination, chromosomal segregation, and cellular senescence . Presently, p53 is known to play a key role in practically all types of human cancers, and the mutation or loss of the p53 gene can be identified in more than 50% of all human cancer cases worldwide. This significant involvement in oncogenesis extends far beyond the simple role in viral transformation p53 was suspected of playing in earlier investigation. p53 belongs to an unique protein family which includes three members: p53, p63 and p73 . Although these proteins are structurally and functionally related to each other, p53 seems to have evolved in higher organisms to prevent tumor development, whereas p63 and p73 have clear roles in normal developmental biology . Because p53 plays a pivotal role in regulation of the cell cycle and induction of apoptosis, there has been enthusiasm about its potential for therapeutic application. It is not surprising that the prominent position p53 plays in tumor development has spurred extensive research into both its basic biologic and clinical aspects. To better understand the relationship between p53 antineoplastic activities and its structure and function, this review focuses on describing biochemical modifications of p53, p53 mutations and its applicability in cancer treatment.

The structure of p53

Human p53 is a nuclear phosphoprotein of MW 53 kDa, encoded by a 20-Kb gene containing 11 exons and 10 introns , which is located on the small arm of chromosome 17 . This gene belongs to a highly conserved gene family containing at least two other members, p63 and p73. Wild-type p53 protein contains 393 amino acids and is composed of several structural and functional domains : a N-terminus containing an amino-terminal domain (residues 1-42) and a proline-rich region with multiple copies of the PXXP sequence (residues 61-94, where X is any amino acid), a central core domain (residues 102-292), and a Cterminal region (residues 301-393) containing an oligomerization domain (residues 324-355), a strongly basic carboxyl terminal regulatory domain (residues 363-393), a nuclear localization signal sequence and 3 nuclear export signal sequence . The amino-terminal domain is required for trans activation activity and interacts with various transcription factors including acetyltransferases and MDM2 (murine double minute 2, which in humans is identified as Hdm2). The proline-rich region plays a role in p53 stability regulated by MDM2, wherein p53 becomes more susceptible to degradation by MDM2 if this region is deleted . The central core of this protein is made up primarily of the DNA-binding domain required for sequence-specific DNA binding (the consensus sequence contains two copies of the 10-bp motif 5-PuPuPuC(A/T)(T/A)GPyPyPy-3, separated by 0-13 bp). The basic C-terminus of p53 also functions as a negative regulatory domain , and has also been implicated in induction of cell death . According to the allosteric model, in which C-terminal tail of p53 was considered as a negative regulator and may regulate the ability of its core DNA binding domain to lock the DNA binding domain as an latent conformation. If the interaction between the Cterminus and the core DNA binding domain is disrupted by posttranslational modification (such as phosphorylation and acetylation), the DNA binding domain will become active, thus induce an enhance transcriptional activity. The centralregion of p53 is its most highly conserved region, not only when p53 is compared with its homologues from Drosophila and Caenorhabditis elegans, but also as compared with its mammalian family members, p63 and p73 . Structural studies of p53 have revealed that the majority of p53 mutations found in cancers are missense mutations that are mostly located in the central DNA-binding domain, and more than 80% of p53 mutation studies have focused on residues between 126306 . In the p53 family, both p73 and p63 show considerable homology with p53 and have similar domain structures including an oligomerization domain, with over 60% amino acid identity within the DNA binding region , and all three of these proteins can induce apoptosis . However, at the same time there are many structural and functional differences between p53 and its other two family members.

The physiological functions of p53

As a tumor suppressor, p53 is essential for preventing inappropriate cell proliferation and maintaining genome integrity following genotoxic stress . Following various intracellular and extracellular stimuli, such as DNA damage (by means including ionizing radiation, UV radiation, application of cytotoxic drugs or chemotherapeutic agents, and infectious virus), heat shock, hypoxia, and oncogene overexpression, wt p53 is activated and emerges as a pivotal regulatory protein which triggers diverse biological responses,both at the level of a single cell as well as in the whole organism . p53 activation involves an increase in overall p53 protein level as well as qualitative changes in the protein through extensive posttranslational modification, thus resulting in activation of p53-targeted genes . For example, in response to DNA damage which leads to double strand breaks in DNA (DSBs),

ATM (ataxiatelangiectasia mutated) protein kinase is activated which in turn activates Chk2 kinase . ATM and Chk2 then both phosphorylate p53 at distinct sites leading to p53dependent cell cycle arrest or apoptosis [. In addition, DNA damage can also lead to replication blockage, thus activating the ATR (ATM and Rad3-related) kinase. Consequently, both activated ATR and subsequently activated Chk1 phosphorylate and activate p53 . Genes activated by wt p53 are functionally diverse and constitute downstream effectors of signaling pathways that elicit diverse responses such as cell-cycle checkpoints, cell survival, apoptosis, and senescence. Many of the multiple functions of p53 including the primary role of p53 in tumor suppression, can be attributed to its ability to act as a sequence-specific transcription factor which regulates expression of different cellular genes to modulate various cellular processes , although protein- protein interactions may also play a role. In response to various types of stress, p53 is accumulated in the nucleus and binds to specific sites in the regulatory regions of p53- responsive genes, and then strongly promotes the transcription of such genes . The p53 downstream targets are differentially activated depending on the cell type, extent of the damage which has influenced p53 activation, and various other as yet unidentified parameters Many approaches have been employed to identify the targets of p53 in various experimental systems. As a result of these efforts, hundreds of physiologically p53 responsive genes have been reported. These genes include genes involved in cell cycle arrest and DNA repair, as well as apoptosis and senescence-related genes , such as genes for p21Waf1/Cip1, Gadd45 (growth arrest and DNA-damageinducible protein 45) and genes of the Bcl-2 family . Besides leading to direct or indirect transcriptional activation, elevated levels of wt p53 also cause repression of gene expression . Genes which may be repressed by p53 include bcl-2, bcl-X, cyclin B1, MAP4 and survivin, some of them are negative regulators of apoptosis . Intriguingly, using ovarian cancer cells infected with p53- expressing adenovirus indicated that approximately 80% of the putative p53-responsive genes are in fact repressed by p53 . The functions of p53 target genes are diverse, corresponding to p53s activity as a multifunctional protein.

Cell-cycle regulation
Among various cellular responses induced by p53, most notable are the induction of cell cycle arrest and apoptosis. It appears that the ability of p53 to prevent cell growth is pivotal to its tumor suppressor functions. p53 can induce cell cycle arrest in the G1, G2 and S phases of the cell cycleThe induction of cell cycle arrest at G1 and G2 by p53 provides additional time for the cell to repair genomic damage before entering the critical stages of DNA synthesis and mitosis. The arrested cells can be released back into the proliferating pool through p53s biochemical functions that facilitate DNA repair including nucleotide excision repair and base excision repair A cyclin-dependent kinase (CDK) inhibitor, p21waf1/Cip1 is perhaps the best known downstream target of p53 among the various p53 target gene products identified. p21waf1/Cip1 is a primary mediator of p53-dependent G1 cell cycle arrest following DNA damage In response to cellular stresses, p53 upregulates endogenous p21waf1/Cip1 mRNA and protein levels p21waf1/Cip1 binds cyclin-CDK complexes through the ZRXLmotif Overexpression of p21waf1/Cip1 induces G1 arrest by blocking cyclin E/CDK2-mediated phosphorylation of Rb and release of E2F which functions to induce expression of genes required for S phase entry this response is also governed by other p53 target gene products, as seen for example, in the increased expressionof Gadd45 and 14-3-3 that participate in p53-driven G2 arrest Gadd45 binds to CDC2 (i.e. CDK1), preventing cyclin B/CDC2

complex formation and subsequently inhibiting the kinase activity A scaffold protein 14-3-3 removes cyclin B/CDC2 from the nucleus to physically separate cyclin B/CDC2 from its target proteins. Overexpression of 14-3-3 induces G2 arrest.

Figure 2: p53 locating at the crossroads of complex networks of stress response pathways. Various intercellular orextracellular stresses elicit cellular responses directly or indirectly through p53 activation. p53 activates its downstream targets to perform various functions including cellcycle arrest, DNA repair, apoptosis, and senescence.

Induction of apoptosis
As a cellular gatekeeper one of roles of p53 is to monitor cellular stress and to induce apoptosis as necessary [38]. In tissues where stressors generate severe and irrevocable damage, p53 can initiate apoptosis, thereby eliminating damaged cells. Studies with flies and nematodes have shown that induction of cell death following genotoxic challenges appears to be a function of p53, whereas in higher organisms p53 activity is acquired for cell growth arrest. Apoptotic gene products which are induced by p53 include Bax (Bcl-2-associated X protein)[ DR5/KILLER (death receptor 5DRAL [Fas/CD95 (cell-death signalingreceptorPIG3 (p53-inducible gene 3Puma (p53-upregulated modulator of apoptosis)[ Noxa (from the Latin word for harm or damage]PIDD (p53- induced protein with death domainPERP (p53 apoptosis effector related to PMP-22Apaf-1 (apoptotic protease- activating factor-1) p53AIP1 (p53- regulated apoptosis-inducing protein 1and others. The p53 associated apoptotic targets can be divided into several groups based on their functions and their executed pathways (Figure 3). The products of these genes may induce

apoptosis through either an extrinsic pathway or an intrinsic pathway, namely the death receptor pathway and the mitochondrial pathway, respectively. The intrinsic apoptotic pathway is engaged when cells are challenged by stress and is dominated by the Bcl-2 family proteins The Bcl-2 family proteins are composed of three classes: antiapoptotic proteins Bcl2 and Bcl-XL, pro-apoptotic proteins Bax, Bak and Bcl-Xl , and pro-apoptotic BH3-only proteins Bid (BH3-interacting death agonist), Bad, Noxa, and Puma In the regulation of the intrinsic pathway, pro-apoptotic gene products such as Bax, Bid, Puma, Noxa, and p53AIP1 localize to the mitochondria and promote the loss of mitochondrial membrane potential and release of cytochrome c, resulting in the formation of the apoptosome complex with Apaf-1 and caspase 9. These apoptosis-related gene products mentioned above are closely associated with p53 function. Bax was the first identified p53-regulated pro-apoptotic Bcl-2 family member and p53-responsive elements have been unequivocally identified in the bax gene Bax is specificlly required for Puma-mediated apoptosis, and it also participates in the death response as an indirect target of p53 through PumaThe requirement for Bax in p53-mediated apoptosis appears to be cell-type dependent. Loss of Bax is responsible for nearly half of the accelerated tumor growth in brain tumors that is related to loss of p53 functionBax also accounts for nearly half of p53-dependent apoptosis induced by 5-fluorouracil (5-FU) in colorectal cancer cells On the other hand, Bax is dispensable for the apoptosis induced by irradiation in thymocytes and intestinal epithelial cells The first evidence which suggested that mitochondria might be involved in p53-dependent apoptosis was the observation that Bcl-2 protected cells from p53- dependent apoptosis Several Bcl-2 family proteins and mitochondrial proteins such as Puma, Noxa, p53AIP1, and PIGs are implicated in p53dependent apoptosis. They are activated in a p53-dependent manner following DNA damage. Puma induces very rapid apoptosis, which occurs within hours following its expression p53AIP1 can cause mitochondrial membrane potential dissipation by interacting with Bcl2p53 also regulates the genes encoding Apaf- 1, a key component of the apoptosome and PIG3, which may cause mitochondrial depolarization Nevertheless, activated p53 can directly or indirectly modulate the expression of its targeted proteins and other proteins that control mitochondrial membrane permeability, and can therefore modulate the release of mitochondrial proteins which further carry out apoptosis. Another p53-related class of pro-apoptotic gene products is the components of the death receptor-mediated extrinsic pathway. In this cell death pathway, p53 can promote apoptosis through activation of the death receptors located at the plasma membrane, including Fas/CD95 DR4 [81] and DR5 and lead to inhibition of the production of IAPs (inhibitor of apoptosis proteinsBoth DR5 and DR4 can trigger or induce apoptosis by TRAIL (tumor necrosis factorrelated apoptosis-inducing ligand), Fas ligand and chemotherapeutic agentsand Fas is indispensable for p53-dependent apoptosis in most tissues p53 may also induce apoptosis via an endoplasmic reticulum-dependent mechanism by transactivating the expression of Scotin, a protein located in the endoplasmic reticulum and in the nuclear membrane It has been suggested that the intrinsic apoptotic pathway is primarily utilized in p53-mediated apoptosis, whereas the extrinsic pathway is used to augment the apoptotic response . p53 can also promote apoptosis through transcription independent mechanisms (including direct shuttling of p53 to the mitochondrial membrane p53 probably has direct apoptotic activity in the absence of transcription or protein synthesis under certain conditions and in certain cell types In mitochondria, p53 directly binds to Bcl-XL/Bcl-2 to displace Bax or BH3 domain-only pro-apoptotic proteins, and thus facilitates Bax-dependent mitochondrial apoptotic changes p53 can also bind to mitochondrial Bak and induce Bak oligomerization, which facilitates the release of cytochrome c after permeabilization of the mitochondrial

membrane This is a very rapid response (30 min), which precedes the transcriptional response (taking at least 2 hThis direct response is tissue-specific and appears to be limited to radiosensitive tissues. While cell cycle arrest can function to inhibit the growth of normal cells, it seems that cells which have attained oncogenic activation are less susceptible to such inhibition The ability of p53 to induce apoptosis appears to be well correlated with its ability to suppress malignant transformation. Loss of p53-dependent apoptosis accelerates mouse brain tumorigenesis Similarly, the observation that mice harboring the p53 R172P mutant develop many tumors may be due to lack of p53-induced apoptosis These results reveal that regulation of apoptosis is an important and evolutionarily conserved tumor suppressor function of p53. It appears that transcriptional factors such as c-Myc, JMY (junction-mediating and regulatory protein), ASPP (Apoptotic- Stimulating Protein of p53) family, p63, and p73 can influence the balance between cell cycle arrest and apoptosis A crucial balance between Puma and p21Waf1/Cip1 has been identified which determines the onset of arrest or death in response to exogenous p53 expression in human colorectal cancer cellsGrowth arrest through activation of p21Waf1/Cip1 is the normal response to p53 expression in these cells. If p21Waf1/Cip1 is disrupted, cells die through apoptosis. However, when Puma is disrupted, apoptosis is prevented. Cell cycle arrest is not only a positive element of the p53 response, but is also a negative element for p53-dependent apoptosis in some situations. Induction of apoptotic genes alone is sometimes not sufficient to induce apoptosis, as the high levels of cell cycle inhibitors may dominant and lead to cell cycle arrest Apoptotic response can be enhanced through abolition of cell cycle arrest (for instance by suppression of p21Waf1/Cip1 or 14-3-3). Following p53 expression or DNA damage in colorectal cancer cells, apoptosis is inhibited through cell cycle arrest mediated by p21Waf1/Cip1 and/or 14-3-3 If p21Waf1/Cip1 or 14-3-3 is removed from these cells, cell death rather than cell cycle arrest may result. When Puma is removed, these cells become resistant to apoptosis [nder certain conditions, cell cycle arrest protects cells from apoptosis. However, under other circumstances, cells undergo apoptosis.

The regulation of p53 level and activity

Under normal circumstances, wt p53 is maintained at very low concentrations within the cells and exists mainly in an inactive latent form During the cell cycle progression, the low basal level of wt p53 has to be precisely controlled In normally growing cells, the half-life of p53 is limited to minutes, whereas cellular stress or exposure to DNAdamaging agents prolongs it to hours Increased levels of the p53 protein are primarily regulated through lengthening of its half-life. The level of p53 and its activities in the cell depend on the cells situation and extracellular stimuli.

Figure 3: p53-associated genes and pathways involved in apoptotic cell death. p53 induces apoptosis mainly via two pathways: extrinsic and intrinsic pathways. The p53-associated extrinsic pathway is mainly executed by activating caspase 8 to induce apoptosis, whereas the p53-associated intrinsic pathway is almost executed by influencing mitochondrial proteins, by which activate caspase 9 to induce apoptosis. In addition,p53 may directly activate Apaf-1 to induce apoptosis.

Genes involved in regulating p53 level and activity

The regulation of p53 level and activity involves a complex network of a multitude of cellular proteins including HPV16 E6 WT-1 E1B/E4), SV40 T-antigen MDM2 JNK Pirh2 [and PARP-1 Moreover, wt p53 can switch between a latent and an active form in its function as a transcription factor. The binding of SV40 T antigen, WT1 or E1B/E4 with p53 increases its stability, whereas the association of E6 or MDM2 with p53 accelerates its degradation. MDM2 is an important related protein, which is the product of a p53 inducible gene. The importance of MDM2 in the regulation of p53 levels is demonstrated by the fact that disruption of the MDM2 gene is lethal in early embryos, whereas the concurrent inactivation of the p53 gene rescues the animal from a lethal consequence MDM2 inhibits p53 activity by blocking its transcriptional activity, favoring its nuclear export and stimulating its degradation. MDM2 protein has been found to play an additional role in blocking the interaction of p53 with the transcriptional apparatus by binding to and shielding the transactivation domain of p53 within its N-terminus MDM2 protein possesses intrinsic E3 ubiquitin-ligase activity and mediates both the ubiquitylation and proteasomedependent degradation of p53Ubiquitinated p53 is exported to the cytoplasm, thereby moving it away from its site of action and promoting its rapid degradation by the proteosome MDM2 can also recruit the histone deacetylase 1 (HDAC1) to deacetylate key lysine residues in the Cterminus of p53, thus making them available for ubiquitination. The MDM2 gene itself also contains a p53dependent promoter and is transcriptionally regulated by p53 following challenge of the cell by various stresses In this fashion, the p53 protein regulates the MDM2 gene at the level of transcription and the MDM2 protein regulates the p53 protein at the level of its activity, and

an autoregulatory feedback loop is established that regulates both the activity of the p53 protein and the expression of the MDM2 gene. In this sequence of events, it is wt p53 that is targeted by MDM2 for degradation, whereas mt p53 is outside of this negative feedback loop and accumulates to high levels in cancer cells (as will be discussed later). It is becoming evident that a number of mechanisms exist to abolish MDM2-mediated degradation of p53, thereby allowing the maintenance of a p53 response initiated by various genotoxic stimuli Under stress conditions, distinct signaling pathways can be activated to prevent p53 from ubiquitylation and degradation through posttranslational modifications and abolishment of MDM2 activity. Several regulators of p53 have been identified recently, such as the positive regulator PML (promyelocytic leukaemia protein)[ and the negative regulators YY1 (Yin Yang1)[ survivin [and PLD (phospholipase DAll these regulators appear to influence p53 through MDM2, and they all appear to affect the transcriptional activity of p53.

Genomic and non-genomic actions of p53

In normal cells not exposed to stress, the level and activity of p53 are very low. Upon stress, p53 is activated through a series of post-translational modifications and becomes able to bind to specific DNA sequences. The p53 recognition sequence is very loose and has been found in several hundred genes that are differentially modulated (induced or repressed) depending on the cell type, the nature of stress and the extent of damage. At low cellular levels, p53 modulates only a subset of the genes regulated at higher levels. The kinetics of target gene modulation may also vary. In a study with a micro-array carrying 6000 capture sequences, 107 genes were found to be induced and 54 genes were repressed by p53 (Zhao et al. 2000). This result extrapolates to at least 500 up-regulated and 260 down-regulated p53 target genes. Table 1, based on several papers (Yu et al. 1999, Vousden & Lu 2002, Liang & Pardee 2003, Nakamura 2004, Miled et al. 2005) lists a nonexhaustive series of p53-target genes that have been found to be altered by various stresses in many cell types. Modulation of cell cycle-related genes by activated p53 may mediate arrest of cells at one of two major cell-cycle checkpoints, in G1 near the border of S-phase (key role played by P21WAF1=CIP1) or in G2 before mitosis (important roles for GADD45 and 14-3-3s). The transcriptional program less clearly defined. However, the observation, for instance, that mice lacking the P21WAF1=CIP1 gene (CDKN1A), unlike p53-null mice, do not develop tumours indicates that it is this apoptotic program that plays an essential role in p53 tumour suppression. p53 may modulate the expression of genes associated with either the extrinsic or the intrinsic apoptotic pathways. The extrinsic pathway (in which genes such as TNFRSF10A, TNFRSF10B, FAS, PERP, LRDD are implied) involves engagement of particular death receptors. The intrinsic pathway is triggered in response to DNA damage and is associated with mitochondrial depolarization and release of cytochrome c from the mitochondrial inter-membrane space into the cytoplasm. Some genes associated with this pathway are APAF1, BAK1, BAX, BCL2 (repressed), FDXR, PMAIP1, and BBC3. Both pathways lead to a cascade of activation of caspases, ultimately causing apoptosis. p53 could promote the convergence of the extrinsic and intrinsic pathways through BID regulation. Besides the regulation of apoptosis-related genes, p53 also appears to be able to act directly at the mitochondria. It can interact with BCL2 family members, such as the antiapoptotic BCL2 itself and BCL-XL, and the pro-apoptotic BAK, thereby triggering mitochondrial outer membrane permeabilization and apoptosis (Schuler & Green 2005). The quantitative, or even qualitative contribution of the direct, transcription-independent action to the global apoptotic activity of p53 has been debated. Observations such as the

radio-resistant phenotype of the PUMA (BBC3)- and NOXA (PMAIP1)- knockout mice have been used as arguments against the general importance of transcription independent mechanisms in vivo (Yu & Zhang 2005). It has also been observed that, in various cell lines, DNA damage induced by either ionizing radiation (IR) or topoisomerase inhibitors triggered a robust translocation of a fraction of p53 to mitochondria to a similar extent. Nevertheless, the cells succumbed to apoptosis only in response to topoisomerase inhibitors, but remained resistant to apoptosis induced by IR, suggesting that mitochondrial translocation of p53 does not per se lead to cell death (Essmann et al. 2005). Other investigators, by examining 179 mutant p53s, found no significant correlation between their apoptotic property and their ability to activate transcription of six p53responsive genes (CDKN1A, MDM2, SFN, and the apoptosis-related BAX, p53AIP1, BBC3) (Kakudo et al. 2005). It is possible that rapid transactivation- independent events could modulate the extent of apoptosis, which would however depend on transactivation-dependent events. However, recent observations suggest that the inverse could be true. Indeed, it has been shown that after genotoxic stress, the major regulator of apoptosis, BCL-XL, sequestered cytoplasmic p53. Nuclear p53 caused expression of PUMA, which then displaced p53 from BCL-XL, allowing p53 to induce mitochondrial permeabilization. Mutant BCL-XL that bound p53, but not PUMA, rendered cells resistant to p53-induced apoptosis irrespective of PUMA expression. These observations thus identify PUMA as the protein coupling the nuclear and cytoplasmic pro-apoptotic functions of p53 (Chipuk et al. 2005). The central core region of p53 is of key importance in regulating apoptotic function, either transcription-dependent or -independent, as supported by the number of mutations affecting this region in apoptosis-deficient p53 cells. In addition to inducing genes that drive apoptosis, p53 can also activate the expression of genes that inhibit survival signalling (such as PTEN) or inhibit inhibitors of apoptosis (such as BIRC5) (Vousden & Lu 2002, Haupt et al. 2003, Meek 2004, Nakamura 2004, Lu 2005, Yu & Zhang 2005). Besides the central core, the proline-rich domain has been specifically associated with the apoptotic activity of p53 (Walker & Levine 1996). Deletion of this region leads to a complete loss of the apoptotic activity of p53. It could constitute an auxiliary proteinbinding site and could be necessary for cellular cofactors specifically involved in the apoptotic activity of p53. The p53-regulated genes that bring about senescence are less well characterized. However, CSPG2 has been strongly associated with senescence in prostate cancer cells (Schwarze et al. 2005).

Posttranslational modifications of p53

In response to stress, p53 activity and its stabilization are also highly governed through complex networks of posttranslational modifications], including phosphorylation, acetylation, ADP-ribosylation, ubiquitylation, sumoylation, neddylation, and cytoplasmic sequestration [Most of these modifications occur in the N- and C-terminal regions of p53. Posttranslational phosphorylation and acetylation are the main modifications enhancing the transcription activating ability of p53 because these modifications generally result in p53 stabilization and accumulation in the nucleus, where p53 interacts with sequence-specific sites of its target genes Posttranslational modifications also prevent p53 from being targeted for degradation as these modifications of p53 at distinct sites may abolish the interaction between MDM2 and p53 .Up to this point, phosphorylation has been regarded as the most commonly reported protein modification that occurs in mammalian cells, although it appears that the frequency and importance of acetylation may actually rival phosphorylation as a crucial posttranslational modification. Phosphorylation of p53 generally results in its stabilization and has also been shown to increase its sequence-specific DNA binding Twenty serine and threonine sites on p53 have been identified to be phosphorylated in huma cells following DNA damage induced by

ionizing radiation or UV irradiation Most of these sites are located in the N-terminal region at the Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Thr55, and Thr81 residues, but there are also other sites in the C-terminal domain at Ser315, Ser366, Ser371, Ser376, Ser378, Thr387, and Ser392, and also in the central core at Ser149, Ser150, Ser155 Phosphorylation at most of these sites is induced by DNA damage. However, there are some sites such as Thr55 and Ser376, which are repressed upon genotoxic stress Many protein kinases have been implicated in phosphorylating p53. Different protein kinases phosphorylate several sites on p53, and in some instances the same site can be phosphorylated by more than one protein kinase. For example, phosphorylation at Ser15 is mediated by ATM/ATR, either directly or through Chk1/Chk2, or at Ser20 by Chk1/Chk2 Expression of oncogenic ras in primary human cancer cells causes phosphorylation of p53 at Ser33 and Ser46 by p38MAPK. It is of interest that the activity of p38MAPK is negatively regulated by PPM1D phosphatase, whose expression is upregulated by p53This forms a negative feedback loop to modulate p53 activity in normal cells. It has been shown that inactivation of p38MAPK or overexpression of PPM1D significantly reduces p53-dependent transactivation. Phosphorylation of p53 at Ser33, Thr81 and Ser315 gives rise to binding sites for Pin1, a peptidylprolyl isomerase that recognizes the phosphorylated Ser/Thr-Pro motif Overexpression of Pin1 enhances p53-dependent transactivation, gene expression and apoptosis in an enzyme activitydependent manner. In a Pin1-inducible cell line, the expression of Pin1 does not change the level of p53, but nevertheless increases the expression of p53 targets genessuggesting that Pin1 directly affects the transactivation ability of p53 through inducing conformational change. Stress-induced N-terminal phosphorylation increases p53 stability by dissociating the negative regulator MDM2. For example, Ser15, Thr18, and Ser20, which are located in the MDM2-binding site, are phosphorylated in response to DNA damage. This phosphorylation alleviates the inhibition or degradation of p53 by MDM2, leading to p53 stabilization and activation Phosphorylation of Ser20 by Chk1 and Chk2 in response to ionizing radiation is important in abolishing the p53-MDM2 interaction In addition, phosphorylation at Ser33 and Ser46 by p38, and phosphorylation of Thr81 by JNK can lead to p53 stabilization Much less is known about the biological consequences of p53 dephosphorylation. It has been reported that Ser376 and Thr55 are dephosphorylated in cells exposed to ionizing radiation, indicating that dephosphorylation may also contribute to the activation of p53. By contrast, a recent study showed that dephosphorylation of p53 was associated with increased p21Waf1/Cip1 expression together with increased caspase 3 activity and induction of apoptosis, presumably because of p53 transcriptional activity However, this was not demonstrated by direct evidence.

Dephosphorylation
In vitro dephosphorylation of p53 by the phosphatases PP1, PP2A, PP5, PPM1D and CDC14 has been shown. These may have different specificities, as shown, for instance, by the fact that PP1, but not PP2A, can dephosphorylate phospho-Ser15 (Haneda et al. 2004). PPM1D is of high interest, as it is induced by p53 and may dephosphorylate both p53 (at Ser15) and CHK1 (which may phosphorylate p53 at various sites) (Lu et al. 2006). Amplification of the PPM1D gene has been observed in breast cancer and seems to be associated with high aggressiveness (Rauta et al. 2006). Dephosphorylation of Ser376 by an ATM-regulated phosphatase allows 14-3-3s binding to phosphorylated Ser378, thereby contributing to p53 stabilization with consequent effects on site-specific DNA binding.

Acetylation may also have a crucial role in stabilization ofp53 and p53 transcriptional activation
Almost every type of cellular stress increases acetylation levels of p53 in a range of cell types. Several lysines can be acetylated on p53 and all are located in the C-terminal region, including Lys305, Lys320, Lys372, Lys373, Lys381, Lys382, and Lys386. These acetylated residues are located in the regulatory domains adjacent to the tetramerization domain. Two histone acetyltransferases (HATs) are known to acetylate p53: p300 or/and CBP acetylates the C terminus of p53 at Lys305, Lys372, Lys373, Lys381, and Lys382, whereas PCAF (p300/CBP-associated factor) acetylates Lys320 The consensus is that the recruitment of coactivators CBP and p300 stabilizes p53 and augments sequencespecific DNA binding following DNA damage Moreover, the acetylation of p53 can dramatically stimulate its sequence-specific DNA binding activity, possibly as a result of an acetylation-induced conformational change It has been shown that acetylation by p300/CBP and PCAF enhances the transactivation activity of p53 in cells, whereas deacetylation of p53 suppresses such activity Our recent study has shown that depsipeptide, an inhibitor of HDAC, can induce p21Waf1/Cip1 expression through acetylation of p53 at Lys373/Lys382 Another study suggests that HDAC inhibitors TSA and butyrate both activate p53 by acetylation of Lys320 and Lys373, upregulate PIG3 and Noxa expression, and also induce apoptosis in cancer cells with both wild and pseudo-wild-type p53 genesAnother p53-binding protein SIRT1 (human Sir2) is a deacetylase that can specifically deacetylate p53 at Lys382 and attenuate p53 transcriptional activityDeacetylation of p53 compromises its ability to induce cell cycle arrest and apoptosisSignificantly, inhibition of SIRT1 activity can increase p53 acetylation but does not alter,cell survival following DNA damage in primary human mammary epithelial cells and certain cell lines3. Acetylation may also regulate the stability of p53 by inhibiting its ubiquitylation induced by MDM2 The ubiquitylation and acetylation of p53 occur at the same sites in the C terminus, suggesting that these modifications may compete for the same residues. The acetylation sites of p53 are essential for ubiquitylation and subsequent degradation of p53 by MDM2. That is to say, p53 acetylation is directly involved in the regulation of its ubiquitylation and subsequent proteolysis induced by MDM2 [nhibition of p53 deacetylation leads to a longer half-life of endogenous p53 suggesting that acetylation of p53 may also contribute to p53 stabilization. On the other hand, in response to a variety of stress-inducing agents, MDM2 can inhibit acetylation of p53 mediated by p300/CBP in vitro and in vivo

Deacetylation
It is likely that deacetylation provides a quick acting mechanism to stop p53 function once transcriptional activation of target genes is no longer needed. Deacetylation of p53 may be performed by multiple histone deacetylases (HDACs), at least by HDAC 1-3. The deacetylase sirtuin 1 (SIRT1) shows an in vitro activity on p53 peptides and it seems that cellular p53 is a major in vivo substrate of SIRT1 but not of the other six known SIRT proteins (SIRT 2-7) (Michishita et al. 2005). In fact, both HDAC1 and SIRT1 could be critical for p53- dependent stress response (Gu et al. 2004). MTA2 (metastasis-associated protein 2)/PID (p53 target protein in the deacetylase complexes) specifically interacts with p53 both in vitro and in vivo, and its expression reduces significantly the steady state levels of acetylated p53 by recruiting the HDAC1 complex.

MTA2/PID expression strongly represses p53-dependent transcriptional activation, and, notably, it modulates p53-mediated cell growth arrest and apoptosis (Luo et al. 2000). Numerous proteins modulating p53 activity have been shown to interfere with acetylation/deacetylation processes. Phosphorylation and acetylation of p53 are interrelated For example, in response to UV or ionizing irradiation, the N terminus of p53 first becomes phosphorylated at Ser33 and Ser37, and in turn phosphorylated p53 activates p300 and PCAF to induce p53 acetylation at Lys373/Lys382 and Lys320, respectively In some cases, phosphorylation might be required for subsequent acetylation of p53. Phosphorylation of p53 at certain sites such as Ser15 and Ser20 enhances its interaction with p300/CBP and potentiates p53 acetylation suggesting that acetylation may have an important role in activating p53responsive genes by DNA-damaging agents, which is induced by phosphorylation of p53 at multiple sites. One recent study has demonstrated that p53 C-terminal phosphorylation by Chk1 and Chk2 may also modulate the level of p53 C-terminal acetylation These data indicate that p53 modulation is a complex process, and the biological consequences of p53 activation induced by certain stimuli may be dependent on p53 posttranslational modifications at multiple sites. Another mechanism allowing p53 to overcome targeting by MDM2 is modificationindependent and involves the upregulation of the human p14ARF (mouse p19ARF) protein. The ARF protein binds directly to MDM2 in a region distinct from the p53 binding region and prevents degradation of p53.

Ubiquitination
In normal cells, degradation is the only mechanism that abrogates all functions of p53, and this appears to be accomplished, in part, by the ubiquitin- 26S proteasome system (the other way is ubiquitin- independent). The highly conserved protein, ubiquitin, targets substrate proteins for degradation by the 26S proteasome to peptides. Ubiquitin ligases realise the last step of ubiquitination. These enzymes exhibit a high level of target specificity. In normal cells, the RING domain MDM2 is considered as the main ubiquitin ligase regulating the amount of p53. MDM2 binds to the N-terminal region and represses p53 activity via twomechanisms: by promoting p53 export to the cytoplasm and its consequent degradation and by blocking p53 transcriptional activation. The export of p53 requires an intact p53 NES. Several lysine residues located at the C-terminus of p53 may be MDM2ubiquitinated: Lys370, Lys372, Lys373, Lys381, Lys382, Lys386 (Rodriguez et al. 2000). The ubiquitination of these lysine residues in the p53 C-terminus, including Lys305, is required to expose the NES even when p53 is bundled as a tetramer. MDM2 is up-regulated by activated p53 and this generates a p53-MDM2 auto regulatory loop. According to a current view, DNA damage leads to destabilization and accelerated degradation of MDM2. This limits MDM2 binding to p53 during the stress response and enables p53 to accumulate and remain active, even as p53 transcriptionally activates more MDM2. Thus, the induction of MDM2 RNA by activated p53 may create a reserve of MDM2 that can inactivate p53 once the DNA damage stimulus has abated and MDM2 is restabilized. The physiological relevance of the p53-MDM2 loop is supported by various observations: (1) MDM2-knockout mice have an embryonic lethal phenotype (which can be abolished by the simultaneous inactivation of p53; (2) disruption of the p53-MDM2 interaction with synthetic competitive inhibitors is sufficient to induce a p53 response in cultured cells; (3) blocking MDM2 degradation via proteasome inhibition prevents p53 transactivation in DNA-

damaged cells; (4) the activity of MDM2 is controlled by numerous factors and the p53MDM2 loop is the focal point of the many different stresses that activate the p53 pathway . As many tumours inactivate wild-type p53 through MDM2 over-expression, exploiting the pathways that trigger MDM2 auto-degradation may be an important new strategy for chemotherapeutic intervention (Stommel & Wahl 2005). COP1 (constitutive photomorphogenesis protein 1) is a RING domain ubiquitin ligase that inhibits p53-dependent transcription. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Over-expression of COP1 correlates with a striking decrease in steady state p53 protein levels and attenuation of the downstream target gene, CDKN1A, in cancers that retain a wild-type p53 gene status. Moreover, like MDM2, COP1 is a p53-inducible gene (Dornan et al. 2004). The cytosolic chaperone-associated U-box domain ubiquitin ligase CHIP (C-terminus of hsc70-interacting protein) may induce the proteasomal degradation of p53. CHIP is thought to act in the quality control of protein folding, specifically ubiquitinating unfolded proteins associated with the molecular chaperones. CHIP-induced degradation has been observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the protein. Thus, mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association (Esser et al. 2005). The cullin-domain ubiquitin ligase CUL4A (cullin 4a) associates with MDM2 and p53, and ubiquitinates p53. Depletion of CUL4A leads to an accumulation of p53. CUL4A fails to increase the decay of p53 in mouse embryonic fibroblasts lacking MDM2. In addition, the CUL4A-mediated rapid decay of p53 is blocked by the MDM2 negative regulator p19ARF(ARF for alternate reading frame). The results provide evidence for a cooperative role of CUL4A in the MDM2-mediated proteolysis of p53 (Nag et al. 2004). The E6 oncoprotein of human papilloma viruses (HPVs) that are associated with cervical cancer utilizes the HECT domain ubiquitin ligase E6AP (E6associated protein) to target p53 for degradation. In normal cells (i.e. in the absence of E6), p53 degradation is mediated by MDM2 rather than by E6AP. In HPV-positive cancer cells, the E6- dependent pathway of p53 degradation is not only active but, moreover, is required for degradation of p53, whereas the MDM2-dependent pathway is inactive. As the p53 pathway was reported to be functional in HPV-positive cancer cells, this finding indicates clearly that the ability of the E6 oncoprotein to target p53 for degradation is required for the growth of HPV-positive cancer cells (Hengstermann et al. 2001). Nuclear localization of p53 is essential for its tumour suppressor function. In contrast to most other ligases that act, or are believed to act in the nucleus, PARC (p53-associated parkin-like cytoplasmic protein), a RING domain ubiquitin ligase, directly interacts with p53 in the cytoplasm of unstressed cells. In the absence of stress, inactivation of PARC induces nuclear localization of endogenous p53 and activates p53-dependent apoptosis. Overexpression of PARC promotes cytoplasmic sequestration of ectopic p53. This suggests that PARC is a critical regulator in controlling p53 subcellular localization and subsequent function (Nikolaev et al. 2003). PIRH2 (p53-induced protein, RING-H2 domaincontaining) is a RING domain ubiquitin ligase that promotes p53 ubiquitination independently of MDM2. Expression of PIRH2 decreases the level of p53 protein and abrogation of endogenous PIRH2 expression increases the level of p53. Furthermore, PIRH2 represses p53 functions including p53-dependent trans-activation and growth inhibition. PIRH2, like MDM2 and COP1, participates in an auto-regulatory feedback loop that controls p53 function (Leng et al. 2003).

Using an osteosarcoma cell line, it was shown that TOPORS (topoisomerase I-binding arginine-serinerich protein) could act on p53 as a RING fingercontaining ubiquitin ligase. Over-expression of TOPORS was shown to result in a decrease in p53 protein expression (Rajendra et al. 2004). However, the exact role of TOPORS remains unclear, as it has also been shown to sumoylate p53, thereby abrogating its transcription activity TOPORS was shown to associate with and stabilize p53, and to enhance the p53-dependent transcriptional activities of CDKN1A, MDM2 and BAX promoters. Over-expression of TOPORS consequently resulted in the suppression of cell growth by cell cycle arrest and/or by the induction of apoptosis (Lin et al. 2005). Although P300 is known as an acetyltransferase, it has been suggested that it could cooperate with MDM2 to induce p53 polyubiquitination. In the presence of MDM2, P300 could poly-ubiquitinate the p53 residues mono-ubiquitinated by MDM2, thus contributing to p53 degradation; in the absence of MDM2, P300 might only act as a p53 acetyltransferase and therefore stimulates the transcriptional activity of p53 (Kohn & Pommier 2005). Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. How can one explain the apparent redundancy of ubiquitin ligases? A possibility is that ubiquitin ligases are expressed or act optimally in different cell or tissue types. It is also possible that one or more of these ubiquitin ligases are involved in the maintenance of p53 levels in the non-stressed or basal state, while others act only after a stressinduced p53 is produced. It appears likely that each of these ubiquitin ligases form protein complexes in the cell and the associated proteins may well differ for each of these ligases, connecting them to different regulatory circuits.

Deubiquitination
USP7 (ubiquitin-specific protease 7, also known as HAUSP) has been shown to interact with p53, which can lead to p53 deubiquitination and stabilization. Its activity and global effect on p53 activity is, however, complex .Ubiquitin-independent p53 degradation. The proteasomal degradation of p53 is regulated by both (poly) ubiquitination, targeting p53 for degradation by the 26S proteasome and by a MDM2- and ubiquitin-independent process. This appears to be mediated by the core 20S catalytic chamber of the 26S proteasome and is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). NQO1 physically interacts with p53 in an NADHdependent manner and protects it from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper for these 20S proteasomes. By competing with NADH, NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. The NQO1 pathway plays a role in p53 accumulation in response to IR, as co-expression of NQO1- specific siRNA with p53 prevented the accumulation of the latter following IR. Escaping MDM2- mediated degradation is probably not sufficient for efficient p53 stabilization following IR, because p53 is still susceptible to 20S proteasomal degradation. In order to achieve efficient p53 accumulation following irradiation, NQO1-p53 interaction could be increased to eliminate p53 degradation by the 20S proteasomes. NQO1 might notably play a role in p53 accumulation under oxidative stress. Reactive oxygen species (ROS) are known to induce NQO1, which, in turn, reduces ROS. The ability of NQO1 to support p53 accumulation following oxidative stress may contribute to cellular defence mechanisms against ROS. The core 20S proteasomes are abundant and ubiquitously present in the cells. They have been widely regarded as being incapable of degrading folded proteins and are therefore considered to be latent proteasomes. Degradation studies with natively unfolded proteins

suggest that unstructured proteins might have an intrinsic capacity to enter the pore of the 20S proteasome. Furthermore, the unstructured protein even when flanked with wellstructured regions is still susceptible to 20S proteasomal degradation. Therefore, a common feature of ubiquitin-independent and 20S proteasomal degraded proteins could be the presence of an unstructured protein region. Indeed, both the Nand the C-terminal regions of p53 have been identified as unstructured regions and could facilitate p53 degradation by the 20S proteasomes. p53 could be inherently unstable and degraded by default by the 20S proteasome, unless stabilized by a molecule like NQO1. p53, when engaged in a large functional complex could be protected from 20S proteasomal degradation as a consequence of the masking of its unstructured regions. (For a review on NQO1 in p53 degradation, see Asher & Shaul 2005.) The tumour suppressor p19ARF, which inhibits the ability of MDM2 to target p53 for degradation (see below), also inhibits dicoumarol-induced p53 degradation. Therefore, p19ARF exhibits a double lock activity that inhibits p53 degradation by both the MDM2dependent and the NQO1-regulated pathway, ensuring maximal p53 accumulation under certain physiological conditions.

Sumoylation
The p53 residue Lys386 may be sumoylated. SUMO (small ubiquitin-related modifier) is a ubiquitinrelated protein that covalently binds to other proteins using a mechanism analogous to, but distinct from, ubiquitin. Protein inhibitor of activated STAT (PIAS)-1, PIASxa, PIASxb, PIASy function as SUMO ligases for p53. In contrast to ubiquitination, sumoylation is not involved in protein degradation. Sumoylation affects target protein function by altering sub-cellular localization of the protein or by antagonizing other modifications (for example ubiquitination at the same acceptor site). Sumoylation most frequently correlates with decreased transcriptional activity and thus repression of target genes. PIAS proteins exert a strong repressive effect on p53-dependent transactivation (Schmidt & Muller 2002). It is thought that the physical association of MDM2 with p53 is important for the enhancement of SUMO conjugation to p53. However, mutant p53 that does not associate with MDM2 is still sumoylated, albeit at a reduced level.

Methylation
The p53 residue Lys372 may be methylated by the SET9 (SET domain-containing protein 9) methyltransferase. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. SET9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site (Chuikov et al. 2004).

Neddylation
Unexpectedly, MDM2 was recently assigned a new role as neddylation ligase for p53. NEDD8 (neuronal precursor cell-expressed developmentally down-regulated protein 8) is a small ubiquitin-like protein. MDM2-dependent NEDD8 modification of p53 was shown to inhibit its transcriptional activity (Xirodimas et al. 2004).

ADP-ribosylation
Poly(ADP-ribosyl)ation is a reversible post-translational protein modification implicated in the regulation of a number of biological functions. It is catalysed mainly by the enzyme poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is rapidly activated by DNA strand breaks, which finally leads to the modulation of multiple protein activities in DNA replication, DNA repair and checkpoint control. PARP-1 may be involved in homologous

recombination, and poly(ADP-ribosyl)ation of p53 represents one possible mechanism that activates p53 as a recombination surveillance factor (Wesierska-Gadek et al. 1996).

O-glycosylation
Addition of bulky residues such as sugar groups could disrupt p53 intramolecular interactions involving the basic region, thus activating DNA binding by p53 (Shaw et al. 1996).

Modulators of p53 activity

Besides those biochemical modificators of p53 mentioned above, a considerable number of other proteins have been shown to interact with p53, thus underlining its crucial role in controlling cell fate. An extensive description of all these proteins (more than 100 have been identified) cannot be envisaged here, yet some of them will be discussed. Indeed, they illustrate how the activity of p53 may be quantitatively or qualitatively regulated, closely or remotely, by mechanisms allowing the finely tuned integration of various signals.

The key role of MDM2

As MDM2 is a major regulator of p53 level, it is not surprising that numerous proteins can modulate its own activity. This allows the integration of various stress signals.

Ribosomes, MDM2 and p53

The ribosomal proteins L5, L11 and L23 (RPL5, RPL11, and RPL23) lower MDM2 activity, thus preventing p53 ubiquitination and increasing its transcriptional activity. This suggests an important link between ribosomal biogenesis and p53 activity, perhaps highlighting a pathway that integrates the p53 response with protein synthesis (Coutts & La Thangue 2005). This link is also supported by the recent observation that another ribosomal protein, RPL26, is able preferentially to bind to the 50 untranslated region of p53 mRNA after DNA damage and to enhance association of p53 mRNA with heavier polysomes, which increases the rate of p53 translation, induces G1 cell-cycle arrest, and augments irradiation-induced apoptosis.

Growth factors, MDM2 and p53

AKT (v-akt murine thymoma viral oncogene homologue, also known as PKB/protein kinase B) is a serine/threonine kinase, which in mammals comprisesthree highly homologous members (AKT1- AKT3). AKT is activated in cells exposed to diverse stimuli such as hormones, growth factors (epidermal growth factor (EGF), insulin-like growth factor-I (IGFI) . . .), and extracellular matrix components. The activation mechanism occurs downstream of phosphoinositide 3-kinase (PI-3K), which is itself activated by phosphatidyl inositol triphosphate (PIP3). AKT signalling is believed to promote proliferation and increase cell survival by inhibiting apoptosis, thereby contributing to cancer progression. In agreement with this, phosphorylation of MDM2 at Ser166 and Ser188 by activated AKT results in inhibition of MDM2 self-ubiquitination and in its translocation into the nucleus where it reduces p53 activity Milne et al. 2004). PTEN (phosphatase and tensin homologue), a dual specificity PIP3 phosphatase that antagonizes AKT signalling, is capable of blocking MDM2 nuclear translocation, thus preventing the negative effects of growth factors on p53 activity. PTEN may be viewed as a tumour suppressor. In addition, PTEN appears to modulate MDM2 transcription by negatively regulating its P1 promoter in a p53-independent manner (Chang et al. 2004). Indeed, the induction of MDM2 gene transcription by p53 requires the P2 promoter (Kohn & Pommier 2005). The induction of PTEN has been shown to be essential for p53-mediated

apoptosis in mouse cells, underscoring the importance of the AKT survival signalling in determining the final outcome of the p53 response.

Oncogenes, MDM2 and p53

In the frequency of its disruption in human cancer, the CDKN2A (also known as INK4A/ARF) gene, located at 9p21, is second only to Tp53 (Haber 1997). In fact, this locus encodes two proteins translated in alternate reading frames: P16INK4A, a tumour suppressor, is a cyclin-dependent kinase inhibitor that acts upstream of retinoblastoma (RB) protein to promote cell-cycle arrest; P19ARF is more related to p53 activity. P16INK4A and P19ARF are often co-deleted in tumour cells, as notably observed in the widely used, wild-type p53 MCF-7 breast cancer cell line (see Craig et al. 1998), but mice lacking P19ARF alone are highly susceptible to breast cancer (Haber 1997), thus underlining its importance. P19ARF activates p53 by sequestering MDM2 into the nucleolus, thus preventing it degrading p53. The P19ARF-p53 axis is critical for eliminating potential tumour cells containing deregulated oncogene expression. The adenoviral proteins E1A and MYC, when over-expressed, may promote apoptosis through p53 activation. By the same pathway, VHaras Harvey rat sarcoma viral oncogene homologue (HRAS) may induce cell senescence. It has been shown that P19ARF is strictly required to mediate these effects on p53 (Lowe 1999). P19ARF may also mediate the positive effects of betacatenin on p53 activity (Harris & Levine 2005). Interestingly, P19ARF also inhibits dicoumarolinduced p53 degradation. For instance, E1A, which stabilizes p53 by inducing P19ARF, also inhibits dicoumarolinduced p53 degradation, which is mediated by NQO1. Therefore P19ARF exhibits a double lock activity that inhibits p53 degradation by both the MDM2-dependent and the NQO1regulated pathway, ensuring maximal p53 accumulation under certain physiological conditions.

ABL, MDM2 and p53

ABL (v-abl Abelson murine leukemia viral oncogene homologue) is a ubiquitously expressed nonreceptor tyrosine kinase and a critical factor that under physiological conditions is required for the maximal and efficient accumulation of active p53 in response to DNA damage. Mice that lack both p53 and ABL are not viable. ABL protects p53 by antagonizing the inhibitory effect of MDM2, an action that requires a direct MDM2 phosphorylation at Tyr394 by ABL, observed in vivo as well as in vitro. In addition, ABL has been shown to directly interact with p53 and could protect the latter from ubiquitination by other inhibitors of p53, such as the E6/E6AP complex that inhibits and degrades p53 in HPV-infected cells (Levav- Cohen et al. 2005).

MDM4 (MDMX), MDM2 and p53

MDM4 (mouse double minute 4, also known as MDMX) is a structural homologue of MDM2 that can bind to p53 and inhibit its transcription function. Knockout of MDM4 in mice results in embryonic lethality due to hyper-activation of p53. Thus, MDM4 is an essential regulator of p53 during embryonic development, which is not the case for MDM2. The current thought is that MDM4 inhibits p53 activity both directly and indirectly by facilitating the p53-MDM2 feedback loop. MDM4 alone does not promote p53 ubiquitination or degradation in vivo. However, formation of the MDM2-MDM4 heterodimer stimulates the ubiquitin ligase activity of MDM2 for itself and for p53, suggesting that MDM4 may serve as a regulator or cofactor of MDM2. Although the role of MDM4 in DNA damage-mediated

control of p53 activity remains unclear, MDM2 is believed to target MDM4 for degradation after DNA damage, thereby increasing p53 activity (Coutts & La Thangue 2005, Pan & Chen 2005). MDM4 also possesses the ability to inhibit 53- dependent transcription in an MDM2independent manner. This could be a consequence of inhibition of P300/CBP-mediated acetylation of p53 (reviewed in Marine & Jochemsen 2005). MDM4 over-expression can lead to transformation in cell culture; MDM4 gene amplification and over-expression have been observed in 5% of primary breast tumours, all of which retained wildtype p53. MDM4 is notably amplified and highly expressed in the widely used MCF-7, a breast cancer cell line harbouring wild-type p53, and siRNA-mediated reduction of MDM4 markedly inhibits the growth potential of these cells in a p53- dependent manner. Together, these results make MDM4 a putative drug target for cancer therapy (Danovi et al. 2004). USP7 has been shown to interact with p53, which can lead to p53 deubiquitination and stabilization. However, it appears that total ablation of USP7 is indeed accompanied by an increase in p53 levels. In fact, USP7 may indirectly affect p53 activity and stability by associating with MDM2, leading to MDM2 stabilization. Furthermore, USP7 may also bind to MDM4, leading to its deubiquitination and stabilization. Of interest, the deubiquitination activity of USP7 towards MDM2 and MDM4 is impaired after DNA damage. Indeed, MDM2 and MDM4 phosphorylation by the DNA damageactivated ATM lowers their affinity for USP7, providing a possible mechanism for the instability of MDM2 and MDM4 after DNA damage. This example shows that USP7, MDM2, MDM4, and p53 entertain complex interactions (Meulmeester et al. 2005a,b) The adenoviral protein E1A may stabilize p53 tumour suppressor through the activation of P19ARF (see above). E1A may also bind to MDM4 and form a complex with p53 in the presence of MDM4, resulting in the stabilization of p53 in a P19ARF- independent manner. Although it has no effect on the p53-MDM2 interaction, E1A facilitates MDM4 binding to p53 and inhibits MDM2 binding to MDM4, resulting in decreased nuclear exportation of p53 (Li et al. 2004).

Gankyrin, MDM2 and p53

Gankyrin, also known as PSMD10 (proteasome 26S subunit, non-ATPase, 10), is an ankyrin repeat oncoprotein commonly over-expressed in certain carcinomas. Gankyrin has an antiapoptotic activity in cells exposed to DNA damaging agents. Downregulation of gankyrin induces apoptosis in cells with wild-type p53. Gankyrin binds to MDM2, facilitating p53MDM2 binding, and increases ubiquitination and degradation of p53. Gankyrin also enhances MDM2 auto-ubiquitination in the absence of p53. Down-regulation of gankyrin reduced amounts of MDM2 and p53 associated with the 26S proteasome. Thus, gankyrin is a cofactor that increases the activities of MDM2 on p53 (Higashitsuji et al. 2005).

KAP1, MDM2 and p53

By interacting withMDM2, the nuclear co-repressor KAP1 (KRAB-associated protein 1, also known as TRIM28/tripartite motif-containing protein 28) inhibits p53 acetylation and promotes p53 ubiquitination and degradation. P19ARF competes with KAP1 in MDM2 binding and oncogene induction of P19ARF expression reduces MDM2-KAP1 interaction (Wang et al. 2005a).

RB1, MDM2 and p53

The RB1 (retinoblastoma 1) protein can be found in cells in a complex with MDM2 and p53, resulting in high p53 activity and enhanced apoptotic activity. RB1 is generally associated

with the transcription factors E2Fs. By complexing to RB1, MDM2 allows the liberation of E2Fs. Both MDM2 and RB1 may be phosphorylated and inhibited by the cyclin E-cdk2 complex. Following DNA damage, activated p53 stimulates the synthesis of P21WAF1=CIP1, the product of the CDKN1A gene. P21WAF1=CIP1 inhibits the cyclin Ecdk2 complex, and this, in turn, acts positively upon the RB1- MDM2 complex that promotes p53 activity and apoptosis (apoptosis-selective auto-regulatory loop associated with RB1) (Yamasaki 2003, Harris & Levine 2005). Of note, E2Fs not bound to RB1 contribute to p53 stabilization, notably by increasing transcription of P19ARF, ATM and CHK2, and switches the p53 response from G1 arrest to apoptosis, notably by up-regulating the expression of ASPP1, ASPP2, JMY and Tp53INP1, four pro-apoptotic cofactors of p53 (see below) (Hershko et al. 2005).

Interactions between p53 and p63/p73

p63 and p73 are highly related to p53. In contrast to p53, their genes are rarely affected by inactivating mutations. On the other hand, their targeted deletion causes severe developmental defects, in contrast to a deletion of Tp53. Hence, p63 and p73 appear responsible for biological effects that cannot be elicited by p53 alone. It has been speculated that, during the course of evolution, p63 and p73 have first pursued a broader range of activities, whereas p53 later specialized on genome maintenance (Blandino & Dobbelstein 2004). A role of p73 in resistance to various drugs has been suggested (Melino et al. 2002). A complex network of interactions between p53, p63 and p73 has been demonstrated. p63 and p73 may exist as isoforms. Long isoforms (TAp63, TAp73) are able to transactivate the same target genes as p53, while short isoforms (DeltaN-p63, DeltaN-p73) have an opposite activity via dominant negative mechanisms. While common genes may be activated by p53 and p73, recent microarray analysis has, however, suggested that the cellular response induced by p73 during adriamycin treatment could involve specific genes, as suggested by microarray analysis (Vayssade et al. 2005) Of interest, p53 has been shown to induce the expression of DeltaN-p73, at both the mRNA and protein levels, through a specific p53-responsive promoter element. This induction of DeltaN-p73 expression establishes an auto-regulatory feedback loop that keeps the trigger of cell death under tight control (Kartasheva et al. 2002).

Mechanisms of p53 apoptosis vs growth arrest p53 apoptotic co-regulators

Apoptosis appears as the critical function of p53 in tumour suppression (Haupt et al. 2003, Yu & Zhang 2005). The choice between growth arrest and apoptosis likely involves the complex interplay of numerous factors. 1. According to a quantitative model, genes involved in growth arrest contain high-affinity p53 binding sites in their promoter, while low-affinity sites are present in the promoter of apoptosis-related genes (Chen et al. 1996). This is in line with observations that increased levels or activity of p53 can lead to the onset of apoptosis, presumably by achieving a certain threshold level. Moreover, p53 mutants with marginally altered conformations retain sufficient activity to induce growth arrest but not apoptosis, presumably because they can still interact only with high-affinity sites. However, despite the degenerative nature of p53 binding sequences, the apoptotic targets of p53 do not necessarily contain low-affinity promoters. For example, chromatin immunoprecipitation experiments have revealed that the apoptotic gene BBC3 contains highaffinity p53 binding sites (Kaeser & Iggo 2002). The quantitative model is thus not sufficient. 2. According to a qualitative model, the selective activation of the p53 apoptotic genes is

mediated through the interaction of p53 with certain transcription co-activators. Several proteins may interact with p53 and specifically modulate apoptosis. For instance, ASPP1 (apoptosis stimulating protein of p53-1, also known as PPP1R13B/protein phosphatase 1, regulatory subunit 13B) and ASPP2 can both favour the interaction of p53 with the promoters of apoptotic genes BAX and Tp53I3/PIG3, but not that of MDM2, CCNG1 or CDKN1A (Yu & Zhang 2005). The effects of ASPP1 and ASPP2 may be counteracted by iASPP (inhibitor of ASPP), the most conserved inhibitor of p53-mediated apoptosis. Both P63 and P73 are thought to favour selective binding of p53 to apoptotic promoters BAX, PMAIP1/NOXA and PERP (Yu & Zhang 2005), an effect that could be mediated through their interaction with ASPP1 and ASPP2 (Bergamaschi et al. 2004). DAXX (death-associated protein 6) is a transcriptional repressor of CDKN1A (involved in cell growth arrest), but it does not affect the activation of proapoptotic genes, and therefore acts by influencing the balance between cell cycle arrest and proapoptotic p53 targets (Gostissa et al. 2004). STAT1 (signal transducer and activator of transcription 1) can act as a co-activator of p53 to induce expression of BAX, PMAIP1/NOXA, and FAS (Yu & Zhang 2005). As mentioned above, phosphorylation of the p53 residue Ser46 plays an important role in permitting the apoptotic function of the protein. The interaction between p53DINP1 and Ser46 may allow this phosphorylation. Additional proteins do not interact directly with p53, but have been implied in its apoptotic function. JMY (junction-mediating and regulatory protein) interacts with P300 to enhance, selectively, the ability of p53 to induce expression of apoptotic genes such as BAX (Yu & Zhang 2005). STRAP (serine/threonine kinase receptor associated protein) was originally identified as a JMY-interacting protein. After DNA damage, its phosphorylation by activated ATM allows its localization to the nucleus. It is believed that this prompts p53 acetylation through recruitment of P300/JMY and the subsequent enhancement of p53 apoptosis (Coutts & La Thangue 2005). E2F transcription factors may contribute to p53 stabilization by regulating genes such as P19ARF, ATM and CHK2. In addition, E2F1 has been shown to up-regulate the expression of four proapoptotic cofactors of p53 ASPP1, ASPP2, JMY and Tp53INP1 through a direct transcriptional mechanism (Hershko et al. 2005).

Other interactors modulating the p53 transcriptional activity

Proteins that modulate p53 activity may exert their positive or negative effects through various ways that will not be discussed here. Among positive regulators of p53 are 14-3-3s (Yang et al. 2003), activating transcription factor 3 (ATF3, Yan et al. 2005), BRCA1associated RING domain 1 (BARD1, Wu et al. 2006), breast cancer 1, early-onset (BRCA1, Fabbro et al. 2004), CAAT-binding transcription factor 2 (CTF2, Uramoto et al. 2003), hypoxia-inducible factor 1 alpha (HIF1a, Fels & Koumenis 2005), highmobility group box 1 (HMGB1, Banerjee & Kundu 2003), members of the ING (inhibitor of growth family) (Gong et al. 2005), nuclear factor Y (NF-Y, Imbriano et al. 2005), prohibitin (PHB, Fusaro et al. 2003), and STAT1 (Townsend et al. 2005). Among negative regulators are bone marrow kinase, X-linked (BMX, Jiang et al. 2004), CCAAT/ enhancer binding protein beta (C/EBPb, Schneider- Merck et al. 2006), DNA methyltransferase-3a (DNMT3, Wang et al. 2005c), Kruppel-like factor 4 (KLF4, Rowland et al. 2005), SIN3 homologue A, transcription regulator (SIN3A, Zilfou et al. 2001), STAT3 (Niu et al. 2005), Y box-binding protein 1 (YB1, Homer et al. 2005), and YY1 transcription factor (YY1, Sui et al. 2004, Yakovleva et al. 2004). Some of them may restrict p53 activity to specific promoters, for instance those of genes related to apoptosis.

The promyelocytic leukaemia (PML) protein is of specific interest. This tumour suppressor can selectively and dynamically recruit a number of proteins including p53 to form a sub-nuclear multiprotein chamber named PML-NBs, of which it is an essential component. After DNA damage, p53 is recruited into PML-NBs and modified by phosphorylations and acetylations, which in turn potentiate its transcriptional and proapoptotic activities. By sequestering p53, PML-NBs may regulate in a complex way its subnuclear distribution upon stress, thus allowing coordinate temporal patterns of p53-associated transcription (Bao-Lei et al. 2005, Coutts & La Thangue 2006).

Auto-regulatory loops in p53 action

The p53 pathway is intimately linked to other signal transduction pathways that may play a significant role in cancer. Most often, these pathways regulate entry of cells into the cell cycle. The coordination between p53 activity and these pathways may be ensured through a series of auto-regulatory loops. Here are some examples, notably based on the work of Harris & Levine (2005). 1. MDM2 is induced by p53. MDM2 promotes p53 degradation. 2. P19ARF down-regulates the MDM2 ubiquitin ligase activity, thus increasing p53 levels. Activated p53 down-regulates P19ARF. 3. Activated P38 protein kinase increases p53. Activated p53 induces PPM1D which inactivates P38 by preventing its phosphorylation by the RAS pathway. 4. Activated p53 induces COP1 and PIRH2.These ubiquitin ligases contribute to p53 degradation. 5. Activated p53 induces DeltaN-P73. DeltaNP73 represses p53 transcriptional activation. 6. MDM2 activity may be inhibited by phosphorylation on Thr216 (by the cyclin A/cdk2 complex). Activated p53 induces cyclin G; cyclin G makes a complex with PP2A phosphatase, which removes the phosphate at Thr216 and increases MDM2 activity, thus reducing p53 level (Ohtsuka et al. 2004). 7. Activated p53 induces the ubiquitin ligase, seven in absentia homologue (SIAH)-1. SIAH-1 degrades BETA-CATENIN, which is known to up-regulate P19ARF and, subsequently, to increase p53 levels. 8. Growth factors may activate AKT, which, in turn, phosphorylates and activates MDM2; it results in a decrease in p53 (survival pathway). p53 increases PTEN and PTEN decreases AKT activity. 9. Activated p53 induces 14-3-3s. 14-3-3s interacts with p53 and stabilizes it. 10. Activated p53 induces PML. PML helps to potentiate p53 activity. 11. PCAF is induced by p53. It contributes to p53 stabilization. 12. PPM1D is induced by p53. It may dephosphorylate both p53 and CHK1 (which may phosphorylate p53 at various sites), thus inactivating it (Lu et al. 2005). 13. BRCA1, CHK1 and CHK2 contribute to p53 activation upon stress. All three are downregulated by activated p53 (Lohr et al. 2003, Matsui et al. 2004).

Mechanisms for loss of p53 activity in cancer

p53 is subject to tight regulation at multiple levels. In cancer cells, its function can be compromised by various mechanisms: mutations of Tp53, alteration of p53 regulators, alteration of p53 target genes.

Mutation of p53
The p53 gene is often found to be genetically altered in tumors, and is one of the most frequently inactivated genes in human cancers Aberrant stimulation of cell proliferation leads to DNA replication stress, DNA DSBs, genomic instability, activation of the DNA damage

checkpoint, and ultimately p53-dependent apoptosis. p53-dependent apoptosis suppresses expansion of pre-cancerous lesions (p53 tumor suppressor function) and provides selective pressure for p53 inactivation The function of p53 tumor suppressor in cancers can be lost by various mechanisms, including lesions that prevent activation of p53, mutations within the p53 gene itself or mutations of downstream mediators of p53s function Acquired mutations (more than 18,000 mutations have been identified) in the p53 gene are found in all major types of human cancers. Approximately half of all human tumors have a mutation or loss in the p53 gene leading to inactivation of its functionFor example, p53 mutation frequency is 70% in lung cancer, 60% in cancers of colon, head and neck, ovary, and bladder, and 45% in stomach cancer. In many of the other approximately 50% human tumors in which p53 is not functionally inactive, p53 function is impaired owing to mutations in proteins operating either upstream or downstream of p53 targets, such as MDM2 or the E6 protein of HPV, or deletion of key p53 co-activators such as the ARF gene Genes other than p53 are also frequently mutated in human the LiFraumeni syndrome, who have an inherited germline mutation in one of the two p53 alleles, are at very high risk of developing cancer throughout their lifetimes The subsequent loss of the wt p53 allele leads to tumors of the brain, breast, connective tissue, hematological system and adrenal gland. p53 inactivation also leads to cancer development in tumors without p53 mutation. For example, mice deficient in wt p53 are susceptible to spontaneous tumorigenesis In tumors without p53 mutation, the p53 pathway is frequently inactivated by oncoproteins or by defective upstream signals Mutations in the p53 gene can result in abolition of protein function and this loss of function may be linked to tumor progression and genetic instability. Clearly, inactivation of p53 is a key event in carcinogenesis. The presence of mt p53 protein, rather than the complete lack of wt p53 activity may confer a selective advantage to evolution of tumor cells Indeed, it has been demonstrated that mt p53 can be a causative factor in tumor progression, because expression of some mt p53 proteins in tumor cells with a p53-null background leads to an increase of their tumorigenic potential in vitro and in vivo The realization that some mt p53 proteins most likely play important roles as oncogenic factors in cancer progression led to the formulation of the gain-of-function by mt p53 supposition. According to this hypothesis, mt p53 proteins unable to prevent uncontrolled growth and protect cells from genomic alterations may acquire novel activities that actually promote cell growth and survival .For example, the role of wt p53 as a transcription factor is vital for its ability to induce apoptosis and inhibit tumor development whereas mt p53 suppresses the expression of CD95 (Fas/APO-1) gene, which encodes a death receptor implicated in a variety of apoptotic responses This activity of mt p53 may be contributed to its gain of function effect in oncogenesis. Mutant p53 proteins generally show significant phosphorylation and acetylation at sites that are well known to stabilize wt p53, thus potentially facilitating accumulation of dysfunctional mt p53 in the nucleus, where it can act as an oncogene. In tumor cells, phosphorylation of p53 at Ser46 and/or functional interaction with apoptotic cofactors (such as ASPP, JMY and p63/p73) allows for the activation of apoptotic target genes. These cofactors can bind p53 directly or indirectly, and assist p53 DNA binding by directly interacting with p53-responsive promoters. It is possible that phosphorylation alters the conformation of p53 to either enhance interaction with apoptotic cofactors, or allow binding to apoptotic target promoters Although mt p53 proteins are often referred to as being transcriptionally inactive, some mt p53 proteins that are unable to activate transcription of wt p53-inducible genes can potentially activate transcription of genes associated with growth or survival-promoting activities Some p53 mutants are able to bind the promoters of some p53 target genes such as

p21waf1/cip1 and MDM2, but are not able to bind promoters of proapoptotic genes like bax and PIG3 Consequently, this class of mt p53 can induce cell cycle arrest as effectively as wt p53, but are unable to induce apoptosis Inactivation of p53 allows cancer cells to escape apoptosis, while still retaining the ability through Chk1 and Chk2 to arrest in G2 until all DSBs are repaired. This indicates that the transactivation deficiency of such mt p53 proteins applies only to a specific set of genes. Moreover, most promoters activated by mt p53 do not contain sequences resembling the wt-p53 consensus, suggesting that mt p53 may regulate transcription via response elements that are distinct from wt-p53 response elements It is interesting that different promoters activated by mt p53 show no sequence homology, suggesting that sequence specific recognition is unlikely to be a parameter determining the specificity of mt-p53 DNA binding. Certain work suggests that the interaction between mt p53 and DNA could be related to the conformation and structure of the DNA One recent study has shown that some inhibition of transcription by mt p53 requires binding of the protein to a different promoter site, independent from the presence of canonical p53 binding sites Sequence- specific DNA binding (SSDB) of wt p53 is not only sequence-specific, but also DNA structure-dependent, and is regulated by the non-SSDB activity of the p53 C-terminus In contrast to wt-p53SSDB, the specificity of mt-p53 DNA binding is determined exclusively by DNA topology, due to the impairment of sequence-specific recognition affected by mutations in the core domain . A characteristic feature of the p53 mutational map is the frequency of missense point mutations. Unlike many other tumor suppressor genes, more than 80% of p53 mutations result in single amino-acid substitutions that lead to the synthesis of a stable full-length protein rather than deletions, frameshifts or nonsense mutations like those found in most other tumor suppressor genes, such as APC (adenomatosis polyposis coliThese missense mutations lead to the synthesis of a protein that lacks specific DNA binding function and accumulates in the nucleus of tumor cells. The maintenance of mt p53 in tumor cells is believed to be required for both a dominant negative activity to inhibit wt p53 expressed by the remaining allele, and for a gain of function that transforms mutant p53 into a dominant oncogene Structural studies have revealed that the amino acid residues in the mutation hot spots of p53 are within the central region (residues encoding the central DNA binding domain of the protein, whereas few p53 mutations are found in the regulatory domains (N terminus, residues 199; C terminus, residues 301393). Certain p53 codons show an unexpectedly high mutation frequency, with 28% of the mutations affecting only six residues of p53, namely Arg175, Gly245, Arg248, Arg249, Arg273, and Arg282 Figure 1). Among these six residues, the most frequently mutated sites are Arg248 and Arg273 that contact both DNA and ASPP2. The result of mutational inactivation of p53 by single amino acid substitutions is that many tumor cells retain the ability to express the mt p53 protein. These proteins are often more stable than wt p53, and are present at very high levels in tumor cell The higher proportion of missense mutations in p53 suggests that expression of mt p53 may confer some selective advantage to the cells expressing the mutant protein over cells null in p53 or expressing the wt form. In a previous study, we have demonstrated that the human lung cancer cell line H719 lacks p53-dependent p21Waf1/Cip1 expression due to a missense mutation at codon 242 of the p53 gene (CysTrp). In contrast, p21Waf1/Cip1 expression in response to treatment with irradiation or etoposide in wt p53-harboring A549 cells is correlated with an increase in wtp53 It has been demonstrated that residues Leu22/Trp23 (human) and Leu25/Trp26 (mouse) within the transactivation domain in the N-terminus of p53 are indispensable for the

transcriptional activity of p53 In p53 mutants in which residues Leu22/Trp23 or Leu25/Trp26 are replaced with Gln22/Ser23 or Gln25/Ser26, there was complete loss of ability to activate or repress the expression of p53 target genes examination of the mutation frequency of p53 in over 10,000 tumors has shown that the probability of p53 acquiring a mutation varies dramatically depending on the tissue in which the tumor originates. In lung cancer, for example, the p53 mutation frequency is as high as 75%, whereas only around 30% of breast tumors and as few as 5% of leukemias have acquired mutations in p53 Several models have been proposed to explain the high frequency of p53 inactivation in human cancer, such as the guardian of the genome hypothesis and the resistance to apoptosis hypothesis and the former has attracted a good deal of attention. According to this hypothesis, p53 can guard the genome against accumulation of oncogenic mutations. The strongest evidence in support of this hypothesis comes from analysis of p53 knockout mice Since these mice develop tumors with 100% penetrance, the absence of p53 function must be conducive to the accumulation of oncogenic mutations and, by inference, the normal function of p53 may be to preserve genomic integrity.

Hereditary breast cancer and p53

As mentioned above, Tp53 mutations may be observed in the rare familial autosomal Li Fraumeni syndrome. It is characterized by a high incidence of multiple early cancers, including breast tumours. Other hereditary breast cancers may be due to mutations in genes coding for p53modulator proteins. A significant proportion of these cancers have been associated with mutations of BRCA1. BRCA1 may interact with p53 and has been viewed as a scaffold for p53 response (Hohenstein & Giles 2003). Of interest, BRCA1 tumours often express Tp53 mutations, but it remains to be established if this reflects the need for p53 inactivation for the development of BRCA1 tumours to occur, or rather if the loss of BRCA1associated DNA repair properties may explain, at least partly, the high frequency of Tp53 mutations (Lacroix &Leclercq 2005). Other mutations leading to familial syndromes accompanied by a high occurrence of breast cancer may affect BRCA2, ATM (Ataxia-Telangiectasia), CHEK2 (Li-Fraumeni-like syndrome), STK11/LKB (Peutz-Jeghers syndrome), or PTEN (Cowden syndrome) (Lacroix & Leclercq 2005). The products of two of these genes, ATM and CHEK2, are involved in p53 activation, while the product of PTEN increases p53 activity by antagonizing the cell survival effects mediated by the AKT-MDM2 pathway (see above).

p53 alterations, breast tumour characteristics, and prognosis

The potential relationships between p53 alterations and the expression of other tumour markers or pathological characteristics (grade) have been widely investigated. Many teams have also examined the value of p53 as a prognostic marker. The interpretation of data has, however ,often been complicated by the fact that most initial studies used immune histochemistry (IHC) to detect the amount of p53, while analysis of p53 mutations was performed by other investigators. The correlation between p53 accumulation measured by IHC and p53 mutation detected by sequencing has been estimated to be less than 75% in breast carcinomas (Norberg et al. 1998). Indeed, not all mutations yield a stable protein and some mutations lead to a truncated protein not detected by IHC. On the other hand, wild-type p53 may accumulate in some tumours as a result of a response to DNA damage or by binding to other cellular proteins, giving a positive IHC result. Breast tumours expressing a high amount of p53 (as measured by IHC) are more frequently ER-negative and progesterone receptor (PgR)-negative. They are also associated

with a high proliferation rate, high histological and nuclear grades, aneuploidy, and poorer survival. A high p53 level is frequently observed in tumours over-expressing ERBB2 (also known as Her-2/neu) (Feki & Irminger-Finger 2004). The same relationships have been observed when p53 mutations were taken into account, instead of p53 accumulation. For instance, in a large (543 individuals) analysis of patients with node-negative breast cancer, p53 mutations were more frequent in breast carcinomas with amplification of the ERBB2 gene (leading to ERBB2 over-expression). Patients with both p53 mutation and ERBB2 amplification were associated with poor survival. The groups with p53 mutations (both with or without ERBB2 amplification) were more likely to be ER- and PgR-negative, more likely to be grade 3 for both histological and nuclear grade, and less likely to have lobular subtype (Bull et al. 2004). The particularly bad prognosis associated with the coexistence of high ERBB2 and p53 alterations is supported by other studies (Rahko et al. 2003, Yamashita et al. 2004). In a meta-analysis of more than 9000 patients, the prognostic and predictive value of high p53 expression in breast cancer, as evaluated by IHC, was found to be weak (Barbareschi 1996). On the other hand, more than 25 studies to date involving over 6000 patients have revealed the strong prognostic significance of p53 mutations (reviewed in Borresen-Dale 2003). A meta-analysis of 16 of these studies including over 3500 patients (Pharoah et al. 1999) confirmed that mutations in the Tp53 confer a worse overall and disease-free survival in breast cancer cases, an effect that is independent of other risk factors. In several of the studies the presence of a Tp53 mutation was the single most adverse prognostic indicator for both recurrence and death. It seems that the prognostic significance of all types of mutations is not the same. Studies have shown that patients with mutations effecting or disrupting the zinc binding domains L2 and L3 (codons 163195 and 236251) or affecting amino acids directly involved in DNA binding, many of these residing in the zinc binding domain, were related with the poorest prognosis (reviewed in Borresen- Dale 2003). These findings indicate that not just p53 mutation per se but the full spectrum (i.e. different types, locations, and numbers) of p53 mutations needs to be examined when it is used as a prognostic marker of survival in breast cancer patients (Lai et al. 2004). Recent technological advances have allowed the simultaneous evaluation of multiple RNAs (microarrays) or proteins (tissue arrays) in tumour samples or breast cancer cell lines. These studies have revealed that the breast tumours could be sorted into a very few classes characterized by the high level of expression of specific groups of genes/ proteins. Moreover, these classes are stable, as most individual lesions largely maintain their portrait when they evolve from in situ to the metastatic state (reviewed in Lacroix et al. 2004). The number of classes that have been defined in most micro-array-based or tissue array-based studies is three. About two-thirds of tumours express features characteristic of luminal cells. These lesions are often well differentiated, have a low grade and demonstrate relatively high levels of cytokeratins 8/18/19, ER, PgR, BCL2, CDH1 (Ecadherin), the three transcription factors GATA3, FOXA1, XBP1 (Lacroix & Leclercq 2004b), Treefoil factor (TFF)1 (pS2), TFF3, SLC39A6, P21WAF1=CIP1, P27KIP1, and cyclin D1. In contrast to the luminal-like lesions, about 20% of tumours have a low level of the above cited markers, whereas they express relatively high levels of cytokeratins 5/6 and 17, CDH3 (P-cadherin), EGF receptor (EGFR), cyclin E, MIB1, MCM2, and other proliferation markers. Most of these basal/ myoepithelial-like tumours are poorly differentiated and have a high grade. Finally, tumours over-expressing ERBB2 as a consequence of gene amplification constitute a third class. It appears that p53 mutation is much more frequent in the basal/myoepitheliallike and ERBB2 classes than in the luminal-like one (82, 71 and 31% respectively, according to Sorlie et al. 2001). Moreover, the most well differentiated tumours

have a very low level of p53 alteration (13% in Sorlie et al. 2001). Of note, up to 100% mutant p53 have been observed in medullary carcinoma, a specific subtype of breast cancer with a basal/myoepithelial-like phenotype (de Cremoux et al. 1999). The existence of breast tumour classes suggests that any tumour biology reflects to a large extent the biology of the cell of origin at the time of initiation. Tumours originating from more undifferentiated epithelial cells have a rapid growth pattern and more aggressive behaviour and outcome compared with those originating in more differentiated epithelial cells. Neoplastic progression might be p53-dependent in the tumours with a less-differentiated, basal/myoepithelial-like phenotype and those over-expressing ERBB2, while it might be p53-independent in those tumours with a more differentiated, pure luminal form.

Therapeutic applications of p53

The insights which have been provided by p53 research over the years have been for improvement of diagnostic techniques, accuracy of prognosis, and treatment of cancer. Since about half of all human tumors have an abnormal p53 which can occur early in carcinogenesis, and since posttranslational modifications of p53 can reflect the type and magnitude of cellular stress p53 can be a useful biomarker in carcinogenesis. Indeed, p53 has been used as a molecular signature to study both target tissues and surrogate fluids such as blood in high-risk cancer populations Mutated p53 protein accumulation and posttranslational modification endpoints could also prove useful in studying the efficacy of chemopreventive agents As p53 plays a key role in the cellular response to stress, it serves as a major barrier to tumorigenesis. This obstacle has to be removed in order for tumor development to proceed and restoration of wt p53 function is thus a potential key in anticancer therapies. Since MDM2 is an important negative regulator of p53, MDM2 hyperactivity may inhibit the function of p53 and lead to the development of a wide variety of cancers. For example, 30% of human sarcomas show no p53 mutations, but have an overexpressed MDM2 gene. It is believed that inhibiting the E3 activity of MDM2 and blocking the interaction of p53 with MDM2 are potential effective strategies for killing certain tumor cells selectively by restoring the function of wt p53 Therefore, many studies have focused on the p53-MDM2 interaction as the basis of a drug development strategy. A series of small molecule inhibitors have been developed, and some of these can bind to MDM2 and block its interaction with p53, including peptides that have been shown to elevate the levels of p53 protein and its transcriptional activity and trigger p53-dependent apoptosis in tumor cells A class of small molecules named nutlins have been identified to block p53/MDM2 interaction in vitro and in vivo. Treatment of tumor cells with nutlins results in induction of p53 and its target genes and triggering of apoptosis. Recently, a novel series of benzodiazepinedione antagonists of the p53/MDM2 interaction have been discovered which increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wt p53 One study suggests that antisense oligodeoxynucleotides targeted against MDM2 and p21Waf1/Cip1 could be employed in a potential therapeutic strategy sensitizing tumor cells to certain antineoplastic agents One of the major concerns about blocking the p53/MDM2 interaction for use in treatment of cancer was the idea that activation of p53 might be toxic to normal tissues. However, certain data suggest that the mechanisms governing p53 activity in tumor cells and normal cells are quite different, so the different effects of p53 in reactivating different molecules in tumor cells and normal cells might provide a molecular basis for a therapy without the need for tumor targeting Another factor in p53 inactivation is the presence of the human papilloma virus (HPV). In cervical carcinomas, p53 is targeted by HPV encoded E6 protein, which potentiates p53 degradation and inactivates its function in 90% of cervical cancers Drugs that inhibit E6

should promote p53 reactivation and thus have selective therapeutic effect. It has been reported that leptomycin B and actinomycin D inhibit E6 expression, stabilize p53 and induce apoptosis in a model system of cultured cells Interestingly, both drugs can inhibit MDM2mediated inactivation of p53, possibly via inhibition of p53 ubiquitylation (leptomycin B) or by decreasing MDM2 gene transcription (actinomycin D) Although these specific molecules per se have limited therapeutic effect, these data serve to encourage a search for other compounds with similar effects. Because the apoptotic function of p53 is critical for tumor suppression, induction of apoptotic pathways through p53- induced apoptotic targets may be an attractive strategy for anti-cancer treatment. Furthermore, the p53 apoptotic targets, unlike p53, are rarely mutated in human cancers Some of the p53 apoptotic targets, such as bax, Puma, p53AIP1, Noxa and others could potentially be used as targets for gene therapy For example, adenoviral gene transfer of bax can act synergistically with chemotherapy to induce apoptosis in tumors A recent study has demonstrated that siRNA targeting of survivin, a negative regulator of apoptosis which is downregulated by p53, could be potentially useful for increasing sensitivity to anticancer drugs, especially in drug-resistant cells with mutated p53 However, the effects of p53dependent apoptosis are not always favorable for clinical use, and so the inhibitors of p53-mediated apoptosis might be used to transiently decrease apoptosis in normal tissues when patients are receiving high doses of radiation or chemotherapy In addition to reactivation of wt p53 in tumors, introduction of the wt p53 gene into tumors is also an important therapy. This is based on several observations including the fact that p53null thymocytes and intestinal stem cells are more resistant to radiation-induced apoptosis than their normal counterparts, and p53-null mouse embryonic fibroblast cells are resistant to apoptosis induced by oncogene overexpression and chemotherapeutic agentsp53 null colorectal cancer cells are resistant to apoptosis induced by the anticancer agent 5-FU Expression of wt p53 was found to cause rapid loss of cell viability with morphological characteristics of apoptosis Delivery of the wt p53 gene by replication defective adenoviruses in p53- null tumors can directly induce apoptosis and restore sensitivity to chemotherapeutic drugs. Irreversible cell cycle arrest is sufficient to elicit tumor regression after transfer of the p53 gene in p53-deficient tumor cells Gene therapy based on the introduction of wt p53 has been undergoing clinical trials, and some of the results of the clinical trials have been promising, such as in non-small cell lung cancer and in ovarian cancer Introduction of p53, p73 and p63 into colorectal cancer cell lines via adenoviral vectors results in adenovirusmediated p73 and p63 transfer, suggesting a potential novel approach for the treatment of human cancers, particularly for tumors that are resistant to p53 gene therapy However, improvement of the efficiency of gene therapy treatment is required, including development of the new generation vectors, since application of gene therapy strategy is limited at the moment largely by the low efficiency of infection by existing viral vectors in vivo and the robust immune response. New forms of p53 that have increased DNA binding to promoters of apoptosisinducing genes, resistance to degradation, and enhanced thermodynamic stability will likely be more therapeutically active A strategy that improves the antitumor efficacy using an adenovirus expressing p53 fusion to VP22 protein of herpex simplex virus type 1 has already been developed. There is a clear consensus that restoration of the function of mt p53 in tumor cells would also be of potential therapeutic benefit. This strategy should be specific to cancer cells, as normal cells contain no mt p53. Mutant p53 can be thought of as a loaded gun, present in abundance in tumor cells but with a jammed trigger Tumor cells should be sensitive to the restoration of the p53 pathway, because of specific suppression of this pathway mutation in neoplasia. Since mt p53 is unable to perform its function due to the defect in its folding which is produced by any one of many single amino acid substitutions, several approaches aimed at

reversing this defect and restoring the function of mt p53 have been tried during the past few years. One such potential approach is the use of several peptides and small molecule compounds that can act to stabilize the structure of mt p53, and thus restore the specific DNA-binding, transcription and apoptosis functions to mt p53. They include synthetic peptides derived from the C-terminus of p53 as well as peptides such as CDB3, and compounds isolated from chemical library screening such as CP-31398 and PRIMA-1 (p53 reactivation and induction of massive apoptosis). CDB3 stabilizes the structure of mt p53 proteins and it binds mt p53 and efficiently induces the refolding of two hot spot p53 mutants, His273 and His175, in cancer cells. The transactivation activity of p53 can also be rescued by CDB3 [PRIMA-1 selectively inhibits the growth of tumor cells by provoking apoptosis in a transcriptiondependent fashion through conformational manipulation of p53 mutants to restore sequence-specific DNA binding CP-31398 is a small synthetic molecule with the capacity to restore wt p53 function to mutants It has been suggested that it triggers apoptosis of human cancer cells through the intrinsic Bax/mitochondrial/caspase-9 pathway and can stabilize wt p53 protein However, the exact molecular mechanism by which these molecules act upon the mis-folded mt p53 to restore its activity is as yet unclear. The biochemical study of p53 may help us clarify its mechanisms of action for use in anticancer therapy. Data has suggested that the mechanism by which some chemotherapeutic agents confer antineoplastic activity is through inducing DNA damage, thereby activating p53, which in turn increases downstream target expression, leading to the inhibition of cell proliferation. In one previous study, we found that anticancer agent 5-aza-2'-deoxycytidine induces inhibition of cell proliferation by increasing p53dependent p21Waf1/Cip1 expression following DNA damage, and the resultant data have provided useful clues for judging the therapeutic efficacy of 5-aza-2'-deoxycytidine in the treatment of human cancer cells [Another study also showed that cisplatin, widely used in cancer chemotherapy, can trigger cell cycle arrest and apoptosis following DNA damage through two distinct pathways, one involving p53, the other mediated by the p53-related protein p73. As such, a better understanding of the signaling networks involved in cisplatin toxicity would likely provide a rational basis for the development of new therapeutic requires.

General conclusion
p53 was originally viewed as an oncogene, but during the past several decades it has come to be understood to be a tumor suppressor gene. During this time, many p53 family transcriptional targets have been identified as having the capacity to modulate various cellular processes including growth arrest, apoptosis, senescence, differentiation, and DNA repair. In fact, it has become evident that this small 53- kDa tumor suppressor is a molecular node at the crossroads of an extensive and complex network of stress response pathways. Deregulation of p53 has enormous influence on carcinogenesis as mt p53 can induce an increased epigenetic instability of tumor cells, facilitating and accelerating the evolution of the tumor. Understanding the mechanisms of p53s function is currently a major challenge in p53 research, and such knowledge may ultimately provide novel targets and approaches to therapeutic manipulation of the p53 pathway in the treatment of cancer. The challenge in the future will be to use our knowledge of p53 to develop more highly effective strategies and novel drugs for cancer prevention and treatment with fewer side effects. The crucial role of p53 as a mediator of stress in various cell types is demonstrated; however, its contribution to breast cancer has been difficult to evaluate. Indeed, the number of functions that it controls, the diversity of its mutations, the multiplicity of the proteins constituting its interactome, and the genetic variability inherent to cancer cell progression may result in a tumour suppressor effect as well as an oncogenic action of p53. As an

illustration of this complexity, the link between p53 and prognosis and prediction remains largely unclear, despite numerous studies. Further investigations are needed to determine under which conditions a therapeutic approach targeting p53 could be of real benefit to breast cancer patients. The potential importance of this approach is, however, underlined by the number of compounds that are being developed to increase p53 level and/or to correct the mutant protein.

REFERENCES.. 1. DeLeo AB, Jay G, Appella E, Dubois GC, Law LW, Old LJ. Detection of atransformation-related antigen in chemically inducedsarcomas and other transformed cells of the mouse. Proc Natl Acad Sci USA 76: 2420-2424, 1979. 2. Lane DP, Crawford LV. T antigen is bound to a host protein I SV40-transformed cells. Nature 278: 261-263, 1979. 3. Linzer DI, Levine AJ. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell 17: 43-52, 1979. 4. Dippold WG, Jay G, DeLeo AB, Khoury G, Old LJ. p53 transformation- related protein: detection by monoclonal antibody in mouse and human cells. Proc Natl Acad Sci USA 78: 1695-1699, 1981. 5. Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, vanTuinen P, Ledbetter DH, Barker DF, Nakamura Y, White R, Vogelstein B. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 244: 217-221, 1989. 6. Finlay CA, Hinds PW, Levine AJ. The p53 proto-oncogene can act as a suppressor of transformation. Cell 57: 1083-1093, 1989. 7. Sigal A, Rotter V. Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome. Cancer Re 60: 6788-6793, 2000. 8. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, Butel JS, Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Natur 356: 215-221, 1992. 9. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 253: 49-53, 1991. 10. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 351: 453456, 1991. 11. Lane DP. p53, guardian of the genome. Nature 358: 15-16, 1992. 12. Oren M, Rotter V. Introduction: p53--the first twenty years. Cell Mol Life Sci 55: 911, 1999. 13. Schmale H, Bamberger C. A novel protein with strong homology to the tumor suppressor p53. Oncogene 15: 1363-1367, 1997. 14. Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, Minty A, Chalon P, Lelias JM, Dumont X, Ferrara P, McKeon F, Caput D. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 90: 809-819, 1997. 15. Irwin MS, Kaelin WG. p53 family update: p73 and p63 develop their own identities. Cell Growth Differ 12: 337-349, 2001. 16. Lamb P and Crawford L. Characterization of the human p53 gene. Mol Cell Biol 6: 1379-1385, 1986. 17. Isobe M, Emanuel BS, Givol D, Oren M, Croce CM. Localization of gene for human

p53 tumour antigen to band 17p13. Nature 320: 84-85, 1986. 18. Slee EA, O'Connor DJ, Lu X. To die or not to die: how does p53 decide? Oncogene 23: 2809-2818, 2004. 19. Bode AM, Dong Z. Post-translational modification of p53 in tumorigenesis. Nat Rev Cancer 4: 793-805, 2004. 20. Vousden KH, Lu X. Live or let die: the cell's response to p53. Nat Rev Cancer 2: 594604, 2002. 21. Fields S, Jang SK. Presence of a potent transcription activating sequence in the p53 protein. Science 249: 1046-1049, 1990. 22. Lin J, Chen J, Elenbaas B, Levine AJ. Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes Dev 8: 1235-1246, 1994. 23. Sakamuro D, Sabbatini P, White E, Prendergast GC. The polyproline region of p53 is required to activate apoptosis but not growth arrest. Oncogene 15: 887-898, 1997. 24. Kern SE, Kinzler KW, Bruskin A, Jarosz D, Friedman P, Prives C, Vogelstein B. Identification of p53 as a sequence-specific DNAbinding protein. Science 252: 17081711, 1991. 25. Chen X, Ko LJ, Jayaraman L, Prives C. p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev 10: 2438-2451, 1996. 26. Kaelin WG Jr. The p53 gene family. Oncogene 18: 7701-7705, 1999. 27. Cho Y, Gorina S, Jeffrey PD, Pavletich NP. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265: 346-355, 1994. 28. Moll UM, Zaika A. Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett 493: 65-69, 2001. 29. Benard J, Douc-Rasy S, Ahomadegbe JC. TP53 family member and human cancers. Hum Mutat 21: 182-191, 2003. 30. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 408: 307-310, 2000. 31. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 88: 323-331, 1997. 32. Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumorsuppressor protein p53 by DNA-damaging agents. Oncogene 8: 307-318, 1993. 33. Matsuoka S, Huang M, Elledge SJ. Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 282: 1893-1897, 1998. 34. Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, Ziv Y. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281: 1674-1677, 1998. 35. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguch 36. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY, Taya Y, Prives C, Abraham RT. A role for ATR I the DNA damage-induced phosphorylation of p53. Genes De 13: 152-157, 1999. 37. Shieh SY, Ahn J, Tamai K, Taya Y, Prives C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev 14: 289-300, 2000. 38. Hofseth LJ, Hussain SP, Harris CC. p53: 25 years after its discovery. Trends Pharmacol Sci 25: 177-181, 2004.

39. Farmer G, Bargonetti J, Zhu H, Friedman P, Prywes R, Prives C. Wild-type p53 activates transcription in vitro. Nature 358: 83-86,1992. 40. Oren M. Decision making by p53: life, death and cancer. Cell Death Differ 10: 431442, 2003. 41. Yang Y, Li CC, Weissman AM. Regulating the p53 system through ubiquitination. Oncogene 23: 2096-2106, 2004. 42. Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene 22: 9030-9040, 2003. 43. Mack DH, Vartikar J, Pipas JM, Laimins LA. Specific repression of TATA-mediated but not initiator-mediated transcription by wild-type p53. Nature 363: 281-283, 1993. 44. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 277: 3247-3257, 2002. 45. Mirza A, Wu Q, Wang L, McClanahan T, Bishop WR, Gheyas F, Ding W, Hutchins B, Hockenberry T, Kirschmeier P, Greene JR, Liu S. Global transcriptional program of p53 target genes during the process of apoptosis and cell cycle progression. Oncogene 22: 3645-3654, 2003. 46. Agarwal ML, Agarwal A, Taylor WR, Stark GR. p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proc Natl Acad Sci USA 92: 8493-8497, 1995. 47. Zhou J, Ahn J, Wilson SH, Prives C. A role for p53 in base excision repair. EMBO J 20: 914-923, 2001. 48. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclindependent kinases. Cell 75: 805-816, 1993. 49. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 75: 817-825, 1993. 50. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 366: 701704, 1993. 51. Adams PD, Sellers WR, Sharma SK, Wu AD, Nalin CM, Kaelin WG Jr. Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors. Mol Cell Biol 16: 6623-6633, 1996. 52. Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, Harris CC, Fornace AJ. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53regulated protein Gadd45. Oncogene 18: 2892-2900, 1999.