Introduction

The p53 protein was first described in 1979 as a transformation-related protein (DeLeo et al., 1979) and as a protein associating with the SV40 DNA tumour virus large T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979). As these early studies were carried out in the oncogene era, research focused on the possibility that p53 functioned to promote cell growth and division. Some of these initial studies showed that p53 was overexpressed in mouse and human tumour cells (Dippold et al., 1981) and microinjection of anti-p53 antibody inhibited serum induced DNA synthesis suggesting that p53 possessed an oncogenic property. Therefore, p53 at first seemed to meet the criteria of being a proto-oncogene. However, controversial results kept appearing that ran contrary to p53 functioning simply as a proto-oncogene. These studies suggested that the overexpression of p53 could also repress transformation. Almost 10 years later, it was discovered that the oncogenic properties of p53 were in fact due to mutations in p53 (Baker et al., 1989; Finlay et al., 1989; Hinds et al., 1989), and subsequent research with wild-type p53 clearly demonstrated that p53 was in fact a tumour suppressor gene (Vogelstein et al., 2000).

Following more than 20 years of extensive studies, we now know that p53 is a member of a family of proteins that has three members: p53, p63 and p73. Both p63 and p73 share over 60% amino-acid identity within the DNA binding region of p53 and all three can induce apoptosis. However, there are many structural and functional differences between p53 and its family members. Firstly, p63 and p73 do not bind to SV40 large T antigen (Dobbelstein and Roth, 1998; Marin et al., 1998). This is particularly surprising as the DNA binding region of p53 – the domain that binds large T – is the region that possesses the most homology between the three proteins. Secondly, mutations in p63 and p73 are rare in human cancer. Furthermore, the p63 and p73 genes produce several alternatively spliced products, which is in contrast to the p53 gene which has just one promoter encoding a protein of 393 amino acids (Moll and Zaika, 2001). Some of the splice variants of p63 and p73 may act as oncogenes by inhibiting the ability of full-length p63 and p73 to suppress growth. Interestingly, studies with p53-, p63- and p73-deficient mice established that the expression of p63 and p73 is more important for mouse development than p53, whereas p63 and p73 mice do not acquire tumours to the same extent as mice lacking p53 (Donehower et al., 1992; Yang et al., 1999, 2000).

Damage to the p53 gene is known to be one of the most frequently observed genetic alterations during tumorigenesis, and it is likely that pathways that allow activation of the p53 protein are also disrupted in many tumours where p53 remains intact. The p53 protein modulates cellular functions such as gene transcription, DNA synthesis, DNA repair, cell cycle arrest, senescence and apoptosis. Mutations in the p53 gene can result in abrogated function of the protein and it is this dysfunction that is linked to tumour progression and genetic instability. Wild-type p53 is a nuclear phosphoprotein with five evolutionarily conserved domains designated boxes I–V, and four main functional domains: a transactivation domain (residues 1–42); a polyproline-rich region consisting of five PXXP repeats (residues 61–94); a central core domain (residues 102–292) required for sequence-specific DNA binding (the sequence being at least two repeats of consensus 5'PuPuPuC(A/T)-(T/A)GPyPyPy-3') (El-Deiry et al., 1992); and the oligomerization domain (residues 324–355). In addition, the C-terminus (residues 311–393) contains a nuclear localization sequence and possesses both RNA and nonspecific DNA binding and single-stranded DNA annealing activities. The C-terminus also functions as a negative regulatory domain (Vousden and Lu, 2002).

Levels of p53 within the cell increase dramatically following stresses such as ionizing radiation, UV radiation, hypoxia, heat shock, growth factor withdrawal, oncogene activation and the application of cytotoxic drugs (Vogelstein et al., 2000; Vousden and Lu, 2002). This increase in protein level is attributed mainly to an increase in p53 stability and can be achieved through post-translational modifications (e.g. phosphorylation) and reduced interaction with the Mdm2 protein that normally targets p53 for degradation via the ubiquitin-mediated proteasome pathway. As p53 is a transcription factor, p53 is able to both initiate and suppress gene expression following cellular stress. Since the functions of p53 target genes are diverse, this may be the foundation by which p53 acts as a multifunctional protein, and may begin to explain why loss of p53 activity has been found to result in the development of human tumours that are associated with a range of phenotypes such as a lack of differentiation, increased levels of angiogenesis and metastasis.

The transactivation of target genes results in either arrest of the cell cycle at G1 or apoptosis, depending on the cellular context and type of activating agent. Among the many p53 target genes identified, perhaps the best known is p21^waf1/cip1, a cyclin-dependent kinase inhibitor. Unlike p53 knockout mice, mice lacking p21 do not develop tumours (Donehower et al., 1992; Deng et al., 1995). This implies that the tumours seen in p53 null mice do not arise as a result of them having impaired cell cycle arrest mechanisms, but that apoptosis is the dominant mechanism by which p53 inhibits tumour development. Consistent with this, accelerated tumour development was achieved in E-myc mice through enforced expression of Bcl-2, which allows the tumour cells to tolerate p53-mediated apoptosis (Schmitt et al., 2002). Therefore, it would appear that it is the ability of p53 to induce apoptosis rather than growth arrest that is central to its role as a tumour suppressor. Support for this theory has also arisen from analysis of the behaviour of different p53 mutants. A group of p53 mutants have been shown to have selectively lost the ability to induce apoptosis yet are still able to bring about growth arrest (Friedlander et al., 1996; Rowan et al., 1996). In light of these discoveries and other observations that we shall discuss further, the focus of this review will be on the participation of p53 in the induction of cell death.

Induction of apoptosis by p53

Dependence of p53-mediated apoptosis on transcription

The role of p53 in the transactivation and transrepression of genes is well established, and consequently the mechanisms via which p53 regulates gene expression have been examined in depth by analysing the effect various mutations have on the actions of p53. Within the transactivation domain in the N-terminus of p53, residues Leu22/Trp23 (human) or Leu25/Trp26 (mouse) are indispensable for the transcriptional activity of p53 (Lin et al., 1994). When residues Leu22/Trp23 or Leu25/Trp26 were replaced with Gln22/Ser23 or Gln25/Ser26, the resulting mutants completely lost their ability to activate or repress the expression of p53 target genes (Chao et al., 2000; Jimenez et al., 2000). Therefore, this p53 mutant enabled the contribution of transcription towards p53-mediated apoptosis in vivo to be assessed. A p53 (Gln25/Ser26) knock in mouse was generated and as expected, the endogenous p53 target genes that were analysed were neither induced nor repressed. Like p53 null mice, the p53 (Gln25/Ser26) knock in mice have an impaired apoptotic response following DNA damage and they were also susceptible to developing tumours. Thymocytes and embryonic fibroblasts derived from p53 (Gln25/Trp26) knock in mice were as resistant to DNA damage-induced apoptosis as those derived from p53 null mice, despite the fact that p53 (Gln25/Ser26) was expressed and retained its ability to bind DNA (Chao et al., 2000; Jimenez et al., 2000). This suggests that the role of p53 as a transcription factor is vital for its ability to induce apoptosis and inhibit tumour development.

By analysing a panel of tumour-derived p53 mutants for their ability to transform cells and induce apoptosis, it was observed that the transrepression and apoptotic functions of p53 were tightly associated. Interestingly, the proline-rich domain of p53 (amino acids 61–94) is required for apoptosis (Walker and Levine, 1996), but not for the transactivation of many p53 target genes (exceptions include the p53 inducible genes, PIG). An artificial p53 mutant that lacks the proline-rich region can induce Bax expression to levels comparable with those of wild-type p53, but is unable to induce apoptosis. However, the ability of these polyproline deletion mutants to repress transcription is abolished, indicating that the suppression of gene expression also plays an important role in p53-induced apoptosis (Venot et al., 1998). The importance of transrepression was further illustrated by a report demonstrating that Rb can specifically enhance the ability of p53 to induce apoptosis by facilitating the action of p53 as a transcriptional suppressor. By forming a trimeric complex with p53 via Mdm2, Rb can overcome the inhibitory effect that Mdm2 has upon p53-mediated transcriptional repression while not affecting the inhibition that Mdm2 exerts on p53-dependent transactivation (Hsieh et al., 1999). Many gene array analysis studies have also shown that p53 specifically curbs the expression of a large number of genes including antiapoptotic genes such as Bcl-2, MAP4 and survivin (Haldar et al., 1994; Miyashita et al., 1994; Murphy et al., 1996; Hoffman et al., 2002). Although the promoters of genes like survivin contain a p53 binding site, many of the genes that are suppressed by p53 do not. Hence, it remains unclear how p53 specifically blocks the expression of these group of genes. Interaction with TATA binding protein may be one way through which wild-type p53 represses gene expression (Seto et al., 1992; Farmer et al., 1996). Alternatively, Mdm2 may be recruiting corepressor proteins like hCtBP2 to inhibit p53-mediated transactivation. Forming protein complexes with general repressor proteins such as mSin3A may provide another potential explanation of how p53 may repress gene expression (Murphy et al., 1999; Zilfou et al., 2001). It was reported recently that hypoxia increases the formation of a p53-mSin3A complex, and as a result, p53 was unable to elevate gene expression but retained the capacity to downregulate p53 target genes (Koumenis et al., 2001).

p53 is, of course, able to elevate the expression of genes whose promoters contain p53-binding sites. Proapoptotic genes in which a p53 responsive element has been reported include Bax, IGF-BP3, DR5/KILLER, Fas/Apo-1, the PIGs, PAG608, PERP, PUMA, Noxa, PIDD, DRAL, Apaf1, Scotin and p53AIP1 (Vousden and Lu, 2002). The products of these genes may subsequently precipitate apoptosis by a number of methods. For example, many of these proapoptotic gene products such as Bax, Puma, Noxa and p53AIP1 localize to the mitochondria and promote the loss of mitochondrial membrane potential and cytochrome c release, resulting in the formation of the apoptosome complex with Apaf-1 and caspase 9 (Oda et al., 2000a; Nakano and Vousden, 2001; Yu et al., 2001; Matsuda et al., 2002). The subsequent activation of the caspase family of cysteine proteases results in cleavage of cellular substrates and production of the apoptotic phenotype. Genes encoding redox-regulating enzymes such as ferrodoxin reductase and the PIGs (p53-induced genes) control membrane integrity (Figure 1). Reactive oxygen species produced by the PIGs subsequently cause damage to the mitochondria and initiate apoptosis (Polyak et al., 1997). The expression of ferrodoxin reductase sensitizes cells to apoptosis induced by reactive oxygen species and the chemotherapy drug 5-fluorouracil (Hwang et al., 2001; Liu and Chen, 2002). Another class of proapoptotic genes that can be regulated by p53 such as DR5, Fas and PIDD, are components of the death receptor-mediated cell death pathway (Takimoto and El-Deiry, 2000; Wu et al., 2000). In this instance, caspase activation occurs at the plasma membrane following the clustering of death receptors that occurs following their occupation by factors such as TRAIL or the Fas ligand. p53 may also induce apoptosis via an ER-dependent mechanism by transactivating the expression of Scotin, a protein located in the ER and nuclear membrane (Bourdon et al., 2002).

Figure 1.

Pathways through which p53 induces adoptosis

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With the use of gene array technology, increasing numbers of p53 target genes are being identified, and a significant number of them encode proteins that can regulate cell death. However, knockout mice studies have indicated that so far no single p53-induced product can solely explain p53-mediated transcriptionally dependent apoptosis. Bax was the first apoptotic effector identified (Miyashita and Reed, 1995), but studies on Bax and p53 knockout mice found that the contribution an elevation in Bax expression levels makes to p53-mediated cell death is cell type specific (Bouvard et al., 2000; Knudson et al., 2001). Hence, Bax alone cannot be essential for p53-induced apoptosis. Recent studies demonstrated that mouse embryo fibroblasts lacking Noxa or PUMA are resistant to DNA damage-induced apoptosis, a process known to be mediated by p53 (Jeffers et al., 2003; Shibue et al., 2003; Villunger et al., 2003). Also, in the absence of Noxa, irradiation-induced apoptosis was reduced in the epithelial cells of the small intestinal crypts (Shibue et al., 2003). PUMA null thymocytes were resistant to apoptosis induced by DNA damage, although thymocytes derived from Noxa knockout mice retained their susceptibility (Villunger et al., 2003). This is particularly interesting as Noxa is strongly induced by p53 in thymocytes in response to -irradiation (Fei et al., 2002). All these studies demonstrate once again that although Noxa and PUMA are critical mediators of p53-induced apoptosis in MEFs, neither is sufficient in isolation to mediate p53-induced apoptosis in all tissues. It is therefore likely that a combination of gene products is important and it is possible that different combinations of target genes will be responsible for p53-induced apoptosis in different tissues. This may largely depend on the induction kinetics and tissue specificity of these target genes in response to the expression of p53.

p53-induced apoptosis via a transcription-independent pathway

There is growing evidence to suggest that p53 can induce apoptosis independently of its ability to bind DNA and moderate transcription. The first indication came from the observation that actinomycin D, an inhibitor of transcription, not only elevated p53 levels but also enhanced p53-mediated apoptosis, thereby implying that the transcriptional activity of p53 can be uncoupled from its apoptotic function (Caelles et al., 1994). Since then, several groups have reported instances where p53 is inducing apoptosis independently of any effect upon gene expression. There have been a number of reports recently that have demonstrated the direct localization of p53 to the mitochondria following DNA damage or hypoxia, where p53 can interact directly with antiapoptotic proteins such as Bcl-2 and Bcl-X_L. A transcriptionally inactive tumour-derived p53 mutant, p53H175, when targeted to mitochondria, induced apoptosis as effectively as mitochondria-targeted wild-type p53 (Marchenko et al., 2000; Moll and Zaika, 2001; Sansome et al., 2001; Mihara et al., 2003). This suggests that the action of p53 as a transcription factor may not be a requirement for it to induce death. The ability of p53 to locate to mitochondria is also associated with one particular polymorphic form of p53. Using a temperature-sensitive p53, it was reported that p53Arg72 induces apoptosis more efficiently than p53Pro72, and part of the explanation for this derived from the observation that more p53Arg72 than p53Pro72 is located in the mitochondria (Dumont et al., 2003). Therefore, p53 may translocate to the mitochondria at the onset of p53-induced apoptosis and contribute directly to apoptotic signalling at the mitochondria.

Tissue specificity in p53-mediated apoptosis: some are more equal than others

The ability of stress-activated p53 to induce apoptosis in cells grown in culture is well known. However, this is not always reflected in vivo, where the response to -irradiation, for example, varies from tissue to tissue. Moreover, p53 does not always induce apoptosis when it is upregulated under these circumstances. On studies carried out in mice, -irradiation only caused p53 activation in tissues like the spleen, thymus, haematopoietic bone marrow, intestine, ependyma and kidney. Even within the same tissues, -irradiation did not induce p53 in all cell types. The heterogeneity of the p53 response is also reflected in its ability to instigate death. A second group of tissues, which includes the kidney, salivary gland, myocardium, adrenal gland, choroid plexus and osteocytes, showed elevated levels of p53 following -irradiation but this did not result in apoptosis. Finally, -irradiation induced very little p53 accumulation in liver, skeletal muscle and certain parts of the brain (MacCallum et al., 1996). This study raised many important questions. Why is p53 stabilized to differing degrees in different tissues following -irradiation? Why is p53 only able to trigger apoptosis in certain tissues? What determines this tissue-specific responsiveness to p53?

Some answers to these questions may lie in recent investigations into the tissue-specific expression patterns of p53 target genes such as Bax, DR5, Bid, PUMA, Noxa, p21^waf1/cip1 and Mdm2 (Bouvard et al., 2000; Burns et al., 2001; Fei et al., 2002). Cells in the jejunum and ileum, for example, are highly susceptible to -irradiation-induced apoptosis, and interestingly, this stimulus also induced the expression of proapoptotic p53 target genes such as PUMA, Noxa, Bid and DR5 in these tissues. In the spleen, however, only PUMA, Noxa and Bid were induced by -irradiation. Moreover, p53-induced PUMA expression was seen in the splenic white pulp while Noxa and Bid were only induced in the red pulp (Fei et al., 2002). Even though the thymus and the spleen are two tissues that are very sensitive to ionizing radiation-induced p53-dependent apoptosis, very few of the p53 target genes that are either induced or repressed in these two tissues overlap (Burns and El-Deiry, 2003). This all suggests that the ability of p53 to initiate death in a particular tissue is related to the subset of p53 target genes that are either up- or downregulated in a particular circumstance. In agreement with this, -irradiation failed to induce the expression of any proapoptotic p53 target genes in liver, a tissue known to be resistant to apoptosis when exposed to -irradiation. Here, the elevation in p53 levels resulted in an increase in the expression of p21^waf1/cip1, which causes cell cycle arrest at G1 as a consequence of its action as a cyclin-dependent kinases inhibitor and may even prevent cells from dying of apoptosis (Fei et al., 2002). The variation in the response to elevated p53 levels from one tissue to another is also seen in the development of mouse embryos. In the early stages of development, almost all tissues accumulated p53 following -irradiation. The variety of responses seen in the adult only arises during the later stages of embryogenesis (MacCallum et al., 1996). These studies in mice illustrate the complexity of the p53 response in vivo: a stimulus that causes an elevation in p53 levels does not do so in all instances, and furthermore, when p53 is elevated, death does not always ensue.

Factors that may influence tissue-specific p53-dependent apoptosis

The type and magnitude of the p53 response depends on many factors. A model has been proposed where the outcome of p53 activation correlates with the levels of p53 present in the cell. In circumstances where p53 levels are only slightly elevated, cell cycle arrest occurs through the high-affinity binding of p53 to promoters that closely match the p53 promoter consensus sequence. Death is induced when the cell attains high levels of p53, suggesting that promoters regulating apoptosis-inducing genes have a lower affinity for p53 (Chen et al., 1996; Kaeser and Iggo, 2002). A further layer of complexity was revealed in a recent analysis of the regulation of the PIG3 gene, which found that p53 binds to and activates this promoter via a polymorphic microsatellite that is repeated 10, 15, 16 or 17 times (Contente et al., 2002). A higher number of repeats resulted in a more efficient interaction between the promoter and p53, and it will be interesting to find out whether any relationship can be established between such polymorphisms and tumour susceptibility.

Alternatively, covalent modifications such as phosphorylation can also regulate conformation and promoter specificity. It appears that phosphorylation of serine 46 on p53 increases the transcriptional upregulation of p53AIP1 (p53-regulated apoptosis inducing protein) (Oda et al., 2000b). Additionally, the strength of the interaction of p53 with a particular promoter might also be regulated by interactions with other cellular factors. The p53 protein has been shown to interact with a number of cellular and viral proteins. Examples include SV40 large T, HPV E6 protein, Adenovirus E1b, Mdm2, E2F, p300/CBP, p33ING2 and the ASPP protein family. All p53 interacting proteins can be classified into two main groups, general regulators of p53 or apoptotic-specific regulators of p53. The examples of the general regulators of p53 include Mdm2, p300/CBP, E2F1, securin and p33ING2. Mdm2 binds to the N-terminal transactivation domain of p53 and it can either target p53 for degradation (Haupt et al., 1997; Kubbutat et al., 1997) or inhibit the transcriptional activity of p53 (Oliner et al., 1993). In contrast, p300/CBP binds to the N- and C-terminus of p53 and enhances the transcriptional activity through its ability to acetylate both p53 and chromatin (Avantggiati et al., 1997; Gu and Roeder, 1997; Gu et al., 1997). Similarly, E2F1 and p33ING2 are induced by DNA-damaging agents in a similar manner to p53 (Blattner et al., 1999; O'Connor and Lu, 2000; Nagashima et al., 2001). The induced E2F1 can complex with p53 and enhance p53-mediated apoptosis (Hsieh et al., 2002). On the other hand, human securin (a category of proteins involved in the regulation of sister chromatid separation) binds to the DNA binding domain of p53 and blocks the transcriptional activity of p53 (Bernal et al., 2002). Interestingly, p53 also represses the expression of human securin in response to DNA damage (Zhou et al., 2003).

In contrast to the long list of general regulators of p53, very few proteins that interact with p53 specifically regulate the ability of p53 to cause apoptosis. Perhaps the best-known examples of this group of proteins are the ASPP (ASPP: Apoptotic-Stimulating Proteins of P53, also Ankryin repeats, SH3 domain and proline-rich region contain Protein) family. The ASPP family of proteins bind to the evolutionarily conserved DNA binding domain of p53. ASPP1 is a novel protein, whereas ASPP2 is the full-length version of the previously characterized 53BP2 (Iwabuchi et al., 1994) and Bbp (Naumovski and Cleary, 1996). ASPP1 and 2 share 48% identity, and share the closest homology in the N and C termini. Cotransfection assays reveal that ASPP1 and 2 specifically increase the transactivation of proapoptotic p53-responsive genes such as Bax and PIG3, but have little effect on other target genes such as Mdm2, cyclin G and p21^WAF-1/CIP-1 that are involved in other functions such as cell cycle arrest. Concomitant with their effect on gene expression, ASPP1 and ASPP2 also increased the proportion of cells undergoing p53-dependent apoptosis. Intriguingly, chromatin immunoprecipitation demonstrated that the expression of ASPP2 caused an eightfold increase in the amount of p53 bound to the Bax promoter when compared with p53 alone. Thus, ASPP 1 and 2 seem to selectively enable p53 to regulate specific apoptotic target genes, providing a potential mechanism for discriminating between apoptosis and cell cycle arrest (Samuels-Lev et al., 2001). The third member of the ASPP family is iASPP, a smaller protein with homology to the C termini of ASPP1 and ASPP2. iASPP behaves as an inhibitor of its larger siblings. Whereas ASPP1 and ASPP2 are able to enhance p53-mediated transcription of proapoptotic genes, iASPP antagonises this elevation in gene expression, and by extension iASPP also suppresses p53-mediated death. If ASPP1 and ASPP2 are potentially novel tumour suppressor genes, then by virtue of its ability to oppose the actions of these two proteins and inhibit p53-dependent apoptosis one could surmise that iASPP is an oncogene. Evidence to support this prediction exists: iASPP is able to cooperate with the oncogenes Ras, adenovirus E1A and E7 from the human papilloma virus to transform primary rat embryo fibroblasts in vitro (Bergamaschi et al., 2003). Therefore, members of the ASPP family are important regulators of p53 function, assisting the cell in its decision to live or die (Figure 2).

Figure 2.

A classification of the cellular regulators of p53 based on their ability to regulate p53-mediated apoptosis

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Apart from the ASPP family of proteins, p53 family members, p63 and p73 were also shown recently to be required for p53-induced apoptosis (Flores et al., 2002). JMY is another protein that specifically regulates the apoptotic function of p53 (Figure 3). JMY is a cofactor of p300 and JMY increases p53-dependent apoptosis by enhancing the ability of p53 to activate the expression of the Bax gene (Shikama et al., 1999). Although we do not yet know the biochemical mechanisms through which the ASPP family members, p63/p73 and JMY are able to regulate the promoter specificity of p53, the identification of cellular regulators like the ASPP family are beginning to let us understand how the ability of p53 to bring about cell death can differ so dramatically from tissue to tissue.

Figure 3.

A diagram to illustrate how the apoptotic function of p53 can be regulated in vivo in a tissue-specific manner in response to various stress signals

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Apoptosis: the most conserved property of p53

The deletion of surplus cells by apoptosis is crucial to the proper development of multicellular organisms. The phenotype of an apoptotic cell is conserved from nematodes to mammals. Thus, it is perhaps not surprising that many of the genes that are known to be involved in apoptosis in mammals such as the caspases, APAF1 and the Bcl-2 family have counterparts in C. elegans and Drosophila. For many years it was thought that p53 was a comparatively recent evolutionary development, until the identification of p53 homologues in Drosophila (Dmp53) and C. elegans (Cep-1) revealed that p53 is in fact a gene with a long evolutionary history (Brodsky et al., 2000; Jin et al., 2000; Ollmann et al., 2000; Derry et al., 2001; Schumacher et al., 2001). Most interesting of all is the observation that unlike mammalian p53, Drosophila and C. elegans p53 can induce apoptosis but not cell cycle arrest in response to DNA damage. In C. elegans, Cep-1 (C. elegans p53) is essential for the maintenance of genome integrity as it is required for DNA damage-induced cell death in germ cells. Similarly, Dmp53 (Drosophila p53) null mutants only displayed mild defects in development, longevity and fertility. However, the absence of Dmp53 renders cells unable to die of apoptosis in response to irradiation, although following this stimulus the cells are still able to enter cell cycle arrest (Sogame et al., 2003). This mirrors the role for p53 in mammals: p53 null mice develop normally, but thymocytes derived from these animals are resistant to the induction of apoptosis in response to DNA-damaging agents including ionizing radiation (Clarke et al., 1993; Lowe et al., 1993). This evidence illustrates that the ability to kill unwanted cells is perhaps the most ancient role of p53.

It is also becoming apparent that some of the pathways in which p53 plays a part are also conserved. For example, Dmp53 is able to switch on the expression of the reaper and sickle genes that have long been known to be required for programmed cell death in Drosophila. The reaper gene product promotes death by inhibiting DIAP proteins, which themselves act as inhibitors of the caspase family whose function is to dismantle the cell during apoptosis. Recently, human p53 has been found to elevate the expression of the gene encoding Omi/HtrA2, a proapoptotic serine protease that cleaves and inactivates the mammalian DIAP homologue cIAP1 (Jin et al., 2003). However, as Reaper downregulates DIAP1 by promoting its ubiquitination and degradation via the proteasome, these two pathways are not exactly analogous.

Conservation of the regulation of p53-mediated apoptosis has also been identified in other nonmammalian organisms. In zebra fish, for example, Mdm2-deficient embryos suffer from a massive increase in apoptosis and this is rescued by knocking down the levels of p53, in much the same way that the embryonic lethal phenotype of Mdm2 null mice can be rescued by crossing with p53 knockout mice (de Oca Luna et al., 1995; Langheinrich et al., 2002). However, the regulation of p53 by Mdm2 may only exist in vertebrates, as so far researchers have failed to identify an Mdm2 homologue in either Drosophila or C. elegans. Analysis of the Fugu fish genome has revealed that although many of the p53 target genes such as PIG3 and PIDD, p53DINP1 are conserved, orthologues related to Fas, Bax, PUMA, Noxa and p53AIP were not found (Le Bras et al., 2003). This raises the interesting question as to which elements of the p53 response are conserved. The discovery of C. elegans iASPP provided us with the first example of an evolutionarily conserved means of p53 regulation. iASPP is the most conserved member of the ASPP family and it is the only member that exists in C. elegans. Increased germ cell death is observed in nematodes where the expression of C. elegans iASPP (Ce-iASPP) has been reduced by RNAi, and this is reversed where Ce-iASPP is downregulated alongside Cep-1. Ce-iASPP can substitute for human iASPP in its role in suppressing p53-mediated gene expression and death as well as its ability to transform primary rat fibroblasts. Moreover, apoptosis induced in human cells by C. elegans p53 was either inhibited by human iASPP or stimulated by human ASPP1 and ASPP2 in the same manner as human p53 (Bergamaschi et al., 2003). Therefore, not only is the ability of p53 to induce apoptosis conserved but the regulation of p53 by the ASPP family is also preserved from nematode to man.

Inactivation of p53 in human cancer

Mutation in p53

The p53 gene is located on the small arm of chromosome 17 and consists of 11 exons. Mutations in p53 have been found in 50% of human tumours. Additionally, patients with the rare Li–Fraumeni syndrome have an inherited germline mutation in p53, and the subsequent loss of the wild-type allele leads to tumours of the brain, breast, connective tissue, haematological system and adrenal gland. Unlike many other tumour suppressor genes, more than 85% of p53 mutations result in single amino-acid substitutions rather than deletions or frameshifts (for examples see the p53 mutation databases at http://p53.curie.fr and http://www.iarc.fr/p53/index.html). The vast majority of these mutations are within the central region of p53 (residues 102–292). This region of p53 is the most highly conserved region not only between p53 and its homologues from Drosophila and C. elegans, but also with its mammalian family members p63 and p73. By examining the mutation spectrum of p53, it becomes apparent that all the amino-acid residues that are essential for contact with DNA are mutated in human tumours, which reinforces the importance of this property of p53 to tumour suppression. Disruption of the p53–DNA interaction is also caused by the mutation of residues that do not contact DNA directly but are required for structural maintenance. The best examples of this class of p53 mutants are R175H and G245 (Bullock and Fersht, 2001). A potential avenue for novel cancer therapies is the use of small molecules that can act to stabilize the structure of mutant p53 and hence reactivate the ability of mutant p53 to regulate transcription and apoptosis. These molecules include peptides, such as CDB3, which is based upon the interaction between ASPP2 and p53, and compounds isolated from chemical library screens such as CP-31398 and PRIMA-1 (Foster et al., 1999; Bykov et al., 2002, 2003; Friedler et al., 2002). However, it is unclear exactly how these molecules act upon the mis-folded mutant p53 to restore its activity.

In addition to the well-defined class of mutations that entirely abolish the ability of p53 to bind DNA, there is a growing number of newly identified p53 mutants that are able to bind the promoters of some p53 target genes such as p21^waf1/cip1 and Mdm2, but not the promoters of proapoptotic genes like Bax and PIG3. Consequently, this new class of p53 mutants can induce cell cycle arrest as effectively as wild-type p53, but they are unable to induce apoptosis (Friedlander et al., 1996; Ludwig et al., 1996; Ryan and Vousden, 1998; Smith et al., 1999). Interestingly, some of these mutations, R181L or R181C for example, occur on the residues within the central domain of p53 that contact the ASPP2 C-terminus but not DNA. Furthermore, ASPP1 and ASPP2 failed to enhance the apoptotic function of these two mutants (Gorina and Pavletich, 1996; Smith et al., 1999; Samuels-Lev et al., 2001). Therefore, it is tempting to speculate that mutations that compromise the interaction of p53 with proteins like ASPP1 or ASPP2 would interfere with the ability of p53 to induce apoptosis, and hence be selected for during tumour development. Consistent with this, the residues that are most repeatedly mutated in human tumours, R248 and R273, contact both DNA and ASPP2 (Gorina and Pavletich, 1996) (Figure 4).

Figure 4.

A diagram to show the functional domains of p53 and some of the sites that are frequently mutated in human tumours. Evolutionarily conserved boxes are indicated as I–V. Residues 1–42 encompass the transcriptional domain (TD) and the proline-rich region of p53 is located within residues 64–92 (PR). The DNA and ASPP binding domains (102–292) are indicated. The oligomerization domain (324–355) is labelled as OD while the C-terminus basic region is labelled as BR (356–393). Residues K120, S241, C277, R280, R283 only contact DNA and whereas H178, R181, M243, N247 only contact ASPP2. All these residues are mutated in human tumours but with lower frequency. Residues R175, G245, R249 and R282 control the structural integrity of p53 and are mutated at high frequency. However, the most frequently mutated p53 sites in human tumours are R248 and R273, and they contact both DNA and ASPP2

Full figure and legend (97K)

A 229 amino-acid C-terminal fragment of ASPP2 is the only molecule, apart from DNA, whose three-dimensional interaction with p53 has been analysed (Gorina and Pavletich, 1996). As mutations within p53 that result in a modified protein conformation are unable to bind DNA, so they are also frequently unable to interact with ASPP2. In fact, the resemblance between the interaction of p53 with DNA and its interaction with the C-terminus of ASPP2 is striking. This poses the question as to whether the interaction between p53 and the ASPP family members will prove to be as important as the p53–DNA interaction with respect to the ability of p53 to operate as a tumour suppressor. However, it must be remembered that the fragment of ASPP2 used to study its interaction with p53 represents less than one-quarter of the entire ASPP2 protein (1128 amino acids) and only the DNA binding region of p53 is used. Whether the full-length ASPP2 protein interacts with the full-length p53 in the same way remains to be seen, as does the nature of p53's interaction with the other two members the ASPP family.

Abnormal expression of cellular regulators of p53

Having established that p53 is mutated in 50% of human tumours, the flipside of this must be that 50% of human tumours retain wild-type p53. Therefore, this poses the question as to why the wild-type p53 that persists in these tumours is unable to perform its duty as a tumour suppressor. Examination of the mutation frequency of p53 in over 10 000 tumours has shown that the probability of p53 having acquired a mutation varies dramatically depending on the tissue in which the tumour originated. In lung cancer, for example, the p53 mutation rate is as high as 75%, whereas only around 30% of breast tumours and as few as 5% of leukaemias have acquired mutations in p53. Of course, p53 is not the only tumour suppressor gene to have been identified, and these other tumour suppressors may well be involved in the development of the 25% of lung cancers and 70% of breast cancers whose p53 remains unaltered. However, the acquisition of a mutation in p53 provides a tumour with a clear selective advantage. So how do tumours tolerate wild-type p53, and what factors allow these tumours to overcome the tumour suppression that p53 provides?

Overexpression of oncogenes is one way to bypass the requirement for p53 mutation. For example, carcinomas derived from mouse prostate cells that have been transformed by coexpressing the oncogenes ras and myc retain wild-type p53 (Lu et al., 1992), as do cervical cancers that express the human papilloma virus (HPV) E6 oncoprotein. This is largely due to the fact that E6 can bind to the central region of wild-type p53 and target it for degradation. In a similar fashion, elevated expression of the proto-oncogene Mdm2, also overcomes the tumour suppression function of p53. Amplification of Mdm2 is found in many tumour types including soft-tissue sarcomas, osteosarcomas, lymphoblastic leukaemia and bronchogenic carcinoma (Oliner et al., 1992; Freedman et al., 1999). Overexpression of iASPP and securin has also been found in human breast carcinomas and hepatocarcinomas, respectively, that express wild-type p53 (Bernal et al., 2002; Bergamaschi et al., 2003).

Alternatively, p53 function can be nullified by inactivating mutations in genes that encode positive regulators or effectors of p53. One such example is Chk2, a kinase that is thought to phosphorylate p53 on threonine 18 and serine 20, which prevents p53 from interacting with Mdm2 resulting in an elevation in p53 levels. Chk2 is a tumour suppressor that has been found to be inactivated in tumours including carcinomas of the lung, breast, colon and ovary as well as sarcomas and lymphomas. Furthermore, a subset of Li–Fraumeni patients who possess wild-type p53 have been shown to have inactivating mutations in Chk2 (Bell et al., 1999; Bartek and Lukas, 2003). Downstream of p53 activation, the p53 responsive gene Apaf-1 has been shown to be inactivated in chemoresistant malignant melanomas, a tumour type that rarely exhibits p53 mutations. As a result, the death pathway initiated by p53 cannot be completed (Soengas et al., 2001). Also, expression of ASPP1 and ASPP2 is frequently downregulated in human breast tumours expressing wild-type p53 (Samuels-Lev et al., 2001), plus there is evidence to suggest that a reduction in ASPP2 mRNA levels in lymphomas may be linked to a poor prognosis (Lossos et al., 2002). Taken together, this suggests that downregulation of proteins that cooperate with p53 to induce death or the increased expression of p53 inhibitors are both mechanisms that can prevent wild-type p53 from working effectively. Moreover, the expression patterns of individual p53 regulators are likely to be tissue specific. Therefore, the abnormal expression of p53 activators and/or inhibitors during tumour development in different tissues may explain why there is such a variation in p53 mutation rates in different tumour types. A greater understanding of how cellular factors regulate the apoptotic function of p53 will allow us to develop better strategies to treat cancer.

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