Complicating the complexity of p53
Karen S. Yee and
Karen H. Vousden *
The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK
* To whom correspondence should be addressed. Tel: +44 (0)141 330 2424; Fax: +44 (0)141 943 0372; Email: k.vousden{at}beatson.gla.ac.uk
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Abstract
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Recent studies have suggested that the straightforward role of p53 as a transcription factor that functions by inducing apoptotic target genes to eliminate developing tumor cells is only part of a much more complicated story. There is now a firm body of evidence supporting a transcriptionally independent activity of p53 as a functional, if not structural, homologue of the BH3-only proteins. Although this information adds another nuance to the mechanism by which p53 can induce apoptosis, further studies indicate that the apoptotic function of p53 represents only a part of its tumor suppressive activity. Although complicating our understanding of p53, these new insights may also provide some exciting new targets for the design of therapeutics that can reactivate p53 in cancers.
The p53 tumor suppressor gene encodes one of the most intensively studied proteins. This interest in p53 is engendered not by an essential role for p53 during normal growth and development, but by the contribution of p53 to tumor suppression. Loss or alterations in the function of p53 have been found in most human cancers, including the major epithelial malignancies that may soon be responsible for most deaths in the Western world (1,2). Understanding and treating these cancers has become a matter of some urgency, and the p53 pathway is an attractive candidate for the development of targeted cancer therapies. But can such therapies be successfully developed? Compared with the kinases, the most popular and successful cancer drug targets to date, p53 remains a more elusive quarry. Many functions have been ascribed to p53 that relate to activities as diverse as transcriptional activation, mitochondrial membrane permeabilization, exonuclease activity, DNA repair and regulation of angiogenesis (36). The ability of p53 to signal a variety of growth inhibitory responses, including the induction of cell-cycle arrest, senescence, differentiation and apoptosis (7,8), establish a central role for p53 as a master regulator of tumor suppression (9). However, despite the efforts of many researchers over many years, the relative contributions of these activities to tumor suppression remain hotly disputed. Over the past few years even the most unassailable truths about p53 have been called into question, leaving us with the uncomfortable feeling that the more we learn, the less we know. Two examples of reasonably well-established facts about p53 that have recently become more complicated are the importance of p53 as a transcription factor and the role of apoptosis in tumor suppression, each of which we will discuss briefly in this review.
Of all the activities of p53, none is more firmly accepted than the function of p53 as a transcription factor (1012). Assembling into a tetramer, p53 shows sequence specific DNA binding activity through its central domain (Figure 1), and activates the expression of genes that contain p53 binding sites in their promoters by virtue of interactions of the N-terminal domain of p53 with the transcriptional machinery. Almost all tumor derived p53 mutants contain a point mutation within the DNA binding domain that prevents, or in some cases alters, the recognition of p53 binding sites. In general, p53 mutants expressed in tumors lose the ability to activate expression of the target genes that are responsive to wild-type p53although they may acquire unique transcriptional activities not shared by the wild-type proteinand this by itself suggests that the transcriptional function of p53 is likely to be important for tumor suppression. Identification of target genes that are activated by p53 has further supported this concept; of the hundreds of p53-inducible target genes that have been identified so far there is compelling evidence that several of these play a role in mediating the various downstream responses to p53. The p21Waf1/Cip1 cyclin-dependent kinase inhibitor was amongst the first of the p53 targets to be identified, and deletion of p21Waf1/Cip1 in a number of cell systems strongly abrogates the ability of p53 to induce a G1 cell-cycle arrest (13,14). More recently, the products of other transcriptional targets of p53 have been shown to play a role in mediating various p53 responses, including a large group of genes encoding proteins with apoptotic activity (3). Analysis of a number of these genes indicated that knocking out any one individually failed to protect the cells completely from p53-induced death, suggesting that the response reflects the combined effect of a number of targets. However, more recently, the BH3 domain protein PUMA has emerged as a promising candidate for a key p53-inducible apoptotic target (15,16). PUMA was shown to be critical for p53-mediated cell death in a number of cells and tissues, with knock-out or knock-down of PUMA resulting in a severely impeded to apoptosis (17,18) and in some models, enhancing tumor development (19). However, given the all-or-nothing threshold nature of the apoptotic response, it seems unwise to conclude that PUMA is the be-all and end-all to p53-induced apoptosis. Indeed, it is clear that in some cells types inactivation of other p53 target genes, like NOXA, has an equally profound effect on the apoptotic response (18,20). Taken together, it seems most likely that the coordinated activity of many of the p53-inducible apoptotic target genes will play a key role in governing the life and death decision in response to stresses that activate p53.

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Fig. 1. Diagram showing the domain structure of the p53 protein. The p53 protein is a transcription factor that contains several well-defined domains. At the N-terminus are the transactivation domain and a proline-rich region, which is required for apoptotic function. Within the N-terminus are the interaction sites of p53 with components of the transcriptional machinery as well as ubiquitin ligase Mdm2. The central domain harbors the sequence specific DNA binding region, where most of the tumor associated mutations occur. This central region also contains binding sites for interaction with members of the Bcl2 protein family. The C-terminal region contains the oligomerization domain as well as nuclear localisation and export signals. Several sites within the N-terminal region have been shown to be phosphorylated and the C-terminal region contains numerous sites of modification which influence stability, localization and activity of p53.
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Despite the strong evidence supporting the role of p53 as a transcription factor, there has been consistent debate over whether p53 may also have transcriptionally independent activities. Certainly, p53 also shows transcriptional repressor functions that are strongly correlated with apoptosis (2123). However, a number of early studies suggested that p53 may have an apoptotic function that is completely separate from the regulation of gene expression (2427). An explanation for this activity has been provided very recently with the observation that following stress, a proportion of p53 appears to function outside the nucleus, in the cytoplasm or at the mitochondria, and that p53 can be found in association with several members of the Bcl2 family of proteins (28). The Bcl2 related proteins are the key components of the intrinsic apoptotic pathway and fall into three main groups; those that can function to perturb mitochondrial membrane potential directly (Bax and Bak), the BH3-only proteins that directly or indirectly drive the activation of Bax and Bak, and the anti-apoptotic proteins that sequester pro-apoptotic family members and hold them inactive (such as Bcl2, BclxL and Mcl1). The BH3-only proteins are further divided into two groups; those that bind Bax and Bak directly to activate them (the activators, such as Bim and Bid) and those that bind the anti-apoptotic family members to release the activators (the enablers like Bad and Bik) (29,30) (Figure 2). Unexpectedly, it would seem that p53 can function in a manner analogous to the BH3-only proteins, with evidence to support two broad, but not mutually exclusive, models. In the first, p53 functions like an enabler BH3 domain protein, interacting with the anti-apoptotic proteins and presumably releasing pro-apoptotic BH3 domain proteins to drive apoptosis (Figure 2A and B) (31). The second model is based on the recent observation that p53 can function in a manner more analogous to the activator BH3-only proteins, by directly activating the apoptotic function of Bax or Bak (Figure 2C and D) (32,33). In either case, the remarkable parallels in the function between p53 and the BH3-only proteins is not reflected by any obvious amino acid sequence similarity, although it is possible that parts of p53 adopt a similar structure to the BH3 domain. Interestingly, however, mutations in the DNA binding domain of p53 that are frequently found in tumors also prevent the interaction of p53 with BclxL (31), indicating that the loss of apoptotic activity of these mutants may derive from the concomitant failure to induce transcription and loss of this mitochondrial activity.

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Fig. 2. Models of p53 function as a BH3-only protein. On the left, two possible roles for p53 as an enabler type BH3-only protein. In these models p53 is able to disrupt the interaction between anti-apoptotic proteins (such as BclxL, Bcl2 and Mcl1) and pro-apoptotic proteins. This could directly relieve the inhibition of Bax and Bak (A), or free activator type BH3 only proteins (Bid and Bim), which can then activate Bax and Bak (B). On the right, models in which p53 functions as an activator type BH3-only protein. Activation of Bax and Bak might involve binding and releasing them from interaction with the anti-apoptotic proteins (C). Alternatively, p53 itself may be held inactive by interaction with the anti-apoptotic proteins. In this case it is possible that other enabler BH3-only proteins like PUMA might be able to displace p53 from the anti-apoptotic proteins, thus allowing it to activate Bax/Bak (D). Note that although there is evidence to support each of the models, not all the interactions indicated in the Figure have been confirmed.
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Although in experimental systems the mitochondrial activity of p53 alone can be sufficient for cell death, it seems likely that under physiological conditions this function will cooperate with the ability of p53 to activate transcription of genes like PUMA to drive a full apoptotic response. Although it is possible that PUMA and mitochondrial p53 activate independent apoptotic signals that combine to push the cell over an apoptotic threshold, it is interesting to consider a closer relationship between these two p53 activities. One possibility is that the accumulation of mitochondrial p53 is an immediate response to stress, sensitizing the cells to further apoptotic signals like the expression of PUMA. Recent studies have shown that in mice subjected to DNA damage treatment, a rapid p53 mitochondrial translocation (which precedes p53 target gene activation) triggers an early wave of apoptosis, which is followed later by a second wave that is transcription dependent (34). In this study, mitochondrial p53 was found preferentially in radiosensitive organs or in cultured cells that respond to p53 by undergoing apoptosis, rather than a cell-cycle arrest. These results suggest that the ability to accumulate mitochondrial p53 may be a distinguishing feature between radiosensitive and radioresistant organs and a determinant of whether or not a cell will die in response to p53. However, another model has recently been proposed which intertwines the activity of p53 at the mitochondria and the function of PUMA even more closely (35). As mentioned earlier, p53 can interact with the pro-survival BH3 proteins like BclxL, and it is possible that this interaction serves to inhibit the activator function of p53. PUMA has a very high affinity for the pro-survival proteins and appears to function like an enabler BH3-only protein (36,37). Therefore, when expressed at sufficiently high levels, PUMA may be able to dissociate p53 from the pro-survival proteins and thereby drive the activation of apoptosis (Figure 2D). Clearly, this cannot be the only route through which PUMA functions, since PUMA induces apoptosis in p53-null cells too (15). However, it is likely that the principal role for PUMA is to release the enabler proteins (which would now encompass Bid, Bim and p53) from the anti-apoptotic proteins to drive the activation of Bax and Bak. This model nicely ties together the transcriptionally independent activity of p53 with the transcriptionally dependent activation of proteins like PUMA and suggests how the final apoptotic response may require both of these events.
The ability of p53 to function at the mitochondria has also led to a reassessment of the consequences of how nuclear/cytoplasmic shuttling of p53 might be regulated. Rather intriguingly, this brings us back to considering one of the main negative regulators of p53 function, Mdm2. The role of Mdm2 in controlling p53 function is clear from numerous studies in cells and in mice, and stress-induced inhibition of Mdm2 function is key to the activation of p53 (38). Mdm2 binds to the N-terminal region of p53 that also contains the transcriptional activation domain (Figure 1), and this interaction of Mdm2 with p53 can block the binding of other components of the transcriptional machinery and inhibit the ability of p53 to activate transcription. Mdm2 can also directly repress transcription, in part through its ability to function as a ubiquitin ligase (E3) and ubiquitinate histones (39). This E3 activity of Mdm2 is also critically important for the ubiquitination of p53, an activity that leads to the degradation and maintenance of low levels of p53 in unstressed cells. The importance of the negative regulation of p53 by Mdm2 has been demonstrated in many systems, and it is generally accepted that inhibition of Mdm2 will result in the activation of p53. It is, therefore, somewhat heretical to suggest that Mdm2 may also be enabling some p53 functions, but the observation that Mdm2 is required for p53 to export from the nucleus (40,41) suggests that Mdm2 activity might contribute to the mitochondrial or cytoplasmic activities of p53. The mechanism through which Mdm2 drives nuclear export of p53 is not completely clear, although it is associated with ubiquitination of p53 (42,43) and many involve the unmasking of the nuclear export sequences that are present in the C-terminal region of p53 (Figure 1). Ubiquitination of p53 by Mdm2, therefore, appears to have two roles, targeting of p53 to the proteasome for degradation and nuclear export. Differentiation between these two responses depends on the extent of ubiquitinationwhereas mono-ubiquitination of p53 is sufficient for nuclear export, polyubiquitination is necessary for degradation (43). Interestingly, in support of this model the p53 found at the mitochondria has been shown to be ubiquitin modified (44).
The idea that Mdm2 may actually contribute to an apoptotic activity of p53 provides some rationale for the observed differences in apoptotic activity of the two most common polymorphic forms of p53, carrying either arginine or proline at amino acid residue 72. The Arg72 variant shows an enhanced ability to interact with Mdm2 and Crm1 and is more efficiently exported from the nucleus and localized to the mitochondria than the Pro72 form (44). Consistent with the importance for this localization of p53, the Arg72 variant shows significantly higher apoptotic activity. Interestingly, the apoptotic defect of p53 proteins mutated in the N-terminus that had been ascribed to loss of transcriptional function (4547) may also reflect a failure to bind Mdm2 and so relocalize to the mitochondria.
Taken together, it seems that p53-induced apoptosis represents the culmination of many activities, including the activation of expression of a number of target genes, repression of gene expression and an ability to activate Bax or Bak in a transcriptionally independent manner. Dissecting the relative importance of each of these functions to the overall apoptotic activity of p53 will be complex, and the identification of a mutant that could separate these functions would certainly be extremely helpful.
The clarity of our vision of how p53 functions is becoming even more clouded with a growing appreciation of the importance of p53-induced responses other than apoptosis in preventing tumor development. Certainly, the ability to induce cell death has been strongly linked to the function of p53 as a tumor suppressor (48,49). Compelling studies showing that transformed cells are more sensitive to p53-mediated apoptosis, and that under conditions of Myc or E1A driven tumorigenesis in mice, blocking apoptosis by overexpression of Bcl2 (50) or loss of Apaf1 (51) could substitute for loss of p53, strongly suggest that the ability to induce cell death might be key to the success of p53 as a tumor suppressor. In vivo model systems examining Myc-induced lymphomas also provide elegant support for a tumor suppressive role of PUMAin these studies loss of PUMA was as effective at accelerating tumor development as loss of p53 (19). However, there is now accumulating evidence that simply preventing the apoptotic response to p53 is not the same as losing p53 function completely. For example, the PUMA knock-out mice do not resemble p53-null animals with respect to tumor development, despite showing profound defects in their apoptotic response in many tissues (17,18). These observations therefore suggest that other activities of p53, such as the induction of cell-cycle arrest or senescence and the contribution to the maintenance of genomic stability, also play an important role in tumor suppression. As with apoptosis, pinning down the contribution of the cell-cycle arrest response to p53 tumor suppression has been complicated. Although the deletion of p21WAF1/CIP1, one of the key mediators of the proliferative block induced by p53, can enhance susceptibility to cancer development in some models (52), loss of p21WAF1/CIP1 is not equivalent to loss of p53. Indeed in some systems, loss of p21WAF1/CIP1 may even impede tumorigenesis, possibly as a reflection of tissue and system-dependent pro-apoptotic and anti-apoptotic activities of p21WAF1/CIP1 (53). However, notwithstanding this complexity in the contribution of p21WAF1/CIP1, it seems clear that the proliferative block induced by p53 in the shape of cell-cycle arrest or senescence may be as important in preventing cancer development as the induction of cell death. Some of this evidence has come from a reexamination of specific tumor derived p53 mutants that dissociate p53's apoptotic and cell-cycle responses. Initial tissue culture studies indicated that while these mutants remain competent in the induction of cell-cycle arrest, they fail to induce apoptosis (54). Interestingly, these p53 mutants function to enhance the transformation of cells in culture (55), adding further weight to the suggestion that apoptosis is the key tumor suppressor function of p53. More recently, however, a knock-in mouse expressing one such mutant p53 was created and interestingly, despite showing a complete loss of p53-mediated apoptosis and a reduced p53-induced cell-cycle arrest, these mice retained some ability to resist tumor development (56). Although clearly more susceptible to cancer than their wild-type littermates (suggesting apoptosis does indeed play some role), the incidence of tumor development was far less than that seen in p53-null mice, strongly indicating that the ability to induce a proliferative block, which was matched by a maintenance of genomic stability, is an important weapon in the tumor suppressive armoury of p53. These model systems will encourage us to think again about which p53 activities we should be trying to restore for therapyshould we concentrate on apoptosis, cell cycle progression or even senescence (57)? Even if the ability of p53 to induce a proliferative block can inhibit tumor development, will it also be true that reinstating such a p53-dependent cell-cycle arrest can help to cure a cancer? More importantly, will attempts to modulate the proliferative block in tumor cells have effects on the apoptotic response? For example, the identification of p21WAF1/CIP1 as a survival factor that might ideally be inhibited as a part of tumor therapy (58) suggests that reactivating this p53 response may have unanticipated and undesirable consequences for tumor cell survival.
Clearly, there are many questions that remain to be answered, but despite the obvious gaps in our understanding of exactly how p53 works, the role of p53 in tumor suppression is indisputable. This has encouraged the development of numerous approaches to harnessing the power of p53 for tumor therapy, including gene therapy to directly express wild-type p53 (59) as well as the development of small molecules that can restore function to mutant p53 (60) or reactivate wild-type p53 (61). The latter approach depends on the observation that many tumors that retain a wild-type p53 gene show defects in the pathways that allow the activation of p53 in response to stress, in most cases resulting in an inability to turn off Mdm2. In these tumors, targeting Mdm2 would be expected to stabilize and activate p53. One extremely promising approach has been to identify small molecule inhibitors of the Mdm2p53 interaction (62). Although inhibition of proteinprotein interactions is not usually a favourite target for drug development, in this case, the success of the Nutlin compounds may reflect the extremely tightly defined interaction of a small domain of p53 into a deep pocket in Mdm2 (63). Other approaches include the use of antisense oligonucleotides to inihibit Mdm2 expression (64) and the identification of small molecules that inhibit the E3 activity of Mdm2 (65). Interestingly, although the inhibition of Mdm2 activity should also hinder the mitochondrial function of p53 by preventing nuclear export, the results indicate that p53 activity can be restored with sufficient efficiency to induce an apoptotic response.
Although promising, these approaches have by no means exhausted the potential of p53 as a therapeutic target. As we discover more about p53, the list of possible drug targets that might allow for the reactivation of at least some facet of p53 tumor suppression also increases. We look forward to a continuing boom in our understanding of the basic biology of p53 advancing, hand-in-hand, with an increased ability to exploit this knowledge for cancer therapy.
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Acknowledgments
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We are grateful to Kevin Ryan for his helpful comments. We would also like to thank Cancer Research UK and the West of Scotland Women's Bowling Association for providing generous support to KSY.
Conflict of Interest Statement: None declared.
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Received April 30, 2005;
accepted May 3, 2005.