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p53 in health and disease

Karen H. Vousden* and David P. Lane

Abstract | As a component of the response to acute stress, p53 has a well established role in protecting against cancer development. However, it is now becoming clear that p53 can have a much broader role and can contribute to the development, life expectancy and overall fitness of an organism. Although the function of p53 as a tumour suppressor ensures that we cant live without it, an integrated view of p53 suggests that not all of its functions are conducive to a long and healthy life.
ChIP analysis
A chromatin immunoprecipitation technique that identifies proteins that are bound to promoter sequences of genes in the context of endogenous chromatin.

Glycolysis
The metabolic pathway through which glucose is metabolized to provide energy.

Autophagy
A process in which parts of the cytoplasm, including the organelles it contains, are engulfed inside a membranous compartment and targeted to lysosomes for degradation.

BCL2 family
A family of proteins that share structural motifs and have an important role in positively and negatively regulating mitochondrial apoptotic pathways.

*Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK. Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673. Correspondence to K.H.V. e-mail: k.vousden@beatson.gla.ac.uk doi:10.1038/nrm2147

p53 is an intensively studied protein, its fame stemming mainly from its clear role as a tumour suppressor in humans and other mammals1. Loss or mutation of p53 is strongly associated with an increased susceptibility to cancer, and most functions of p53 have been considered in the light of how p53 might protect from malignant progression2. Some p53-null mice can develop normally3, an observation that has been taken to rule out major functions for p53 in normal physiology. But recent studies are questioning whether p53 is truly such a single-minded protein, and other functions of p53 that might be profoundly important during normal life are being uncovered. These include roles for p53 in regulating longevity and ageing, glycolytic pathways that might determine endurance and overall fitness, and apoptotic responses during ischaemic and other types of stress. Evidence for genetic variations in the activity of the p53 pathway in humans gives these ideas extra relevance4. The p53 protein has been extensively studied at both structural and functional levels (FIGS 1,2). One of the major mechanisms by which p53 functions is as a transcription factor that both positively and negatively regulates the expression of a large and disparate group of responsive genes5. Although some of these p53-responsive genes have an important role in mediating cell-cycle arrest, senescence and apoptosis (the best understood activities of p53), it is now evident that the ability of p53 to influence gene expression has wider reaching effects. Numerous studies, including the recently reported genomewide ChIP analyses6,7, have identified p53-regulated genes that could have a role in a number of different and sometimes unexpected responses. Although some of these still need to be fully validated, there is now clear evidence for a role of p53 in the regulation of glycolysis8,9 and autophagy10, the repair of genotoxic damage11, cell survival and regulation of oxidative stress12, invasion and motility13, cellular senescence14, angiogenesis15, differentiation16 and bone remodelling17.

In this review we outline how some of these more recently described activities of p53 might have a role not only in regulating cancer progression, but also in the control of other aspects of health and disease. We consider some of the signals that activate p53 and whether the p53 response is always advantageous to our well-being. We also discuss how some of the apparently opposing functions of p53 (for example, in driving both death and survival) might be integrated into an overall model of p53 activity.

p53: beyond regulating gene expression The ability of p53 to regulate gene expression is key for the activation of the responses shown in FIG. 1. However, transcriptionally independent activities of p53 that can potentiate the apoptotic response have also been described18. These functions involve a direct interaction of p53 with members of the BCL2 family of proteins, allowing p53 to function as a so-called BCL2-homology domain-3 (BH3)-only protein (BOX 1). Some discussion remains about the exact nature of this activity of p53, with evidence for functions both in the cytosol and directly at the mitochondria19. However, it seems clear that the transcriptionally independent apoptotic activity of p53 complements its ability to activate the expression of pro-apoptotic BH3-only proteins at the transcriptional level, as interfering with either of these functions can severely impair the death response. For example, mice that lack PUMA one of the key apoptotic transcriptional targets of p53 show profound defects in their apoptotic response following the induction of p53 in many tissues20. On the other hand, a small molecule that inhibits the interaction of p53 with anti-apoptotic BH3 proteins can also dramatically reduce the p53dependent apoptotic response without hindering the transcriptional functions of p53 (REF. 21). Interestingly, there is a direct link between the transcriptional and cytoplasmic functions of p53 in that PUMA the
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Hypoxia Telomere erosion Nutrient deprivation p53 Apoptosis Cell-cycle arrest Survival DNA repair Senescence Genomic stability DNA damage Ribosomal stress Oncogene activation

Figure 1 | Activation and functions of p53. p53 has a key role in integrating the cellular responses (pink boxes) to different types of stress (blue boxes). Activation of p53 can result in a number of cellular responses, and it is possible that different responses are induced by different stress signals. There is evidence that p53 can play a part in determining which response is induced through differential activation of target-gene expression. Although the importance of these responses to tumour suppression is clear, previously unanticipated contributions of these responses to other aspects of human health and disease are being uncovered. The role of p53 in tumour suppression, development and ageing is likely to depend on which cellular response is activated and on the context in which the activation occurs.

elevated expression of which results from the transcriptional activity of p53 can dislodge cytoplasmic p53 from an inhibitory interaction with the anti-apoptotic BH3 proteins, and so activate the transcriptionally independent apoptotic function of p53 (REF. 22) (BOX 1).

PUMA
(p53-upregulated mediator of apoptosis). A protein that belongs to the BCL2 family and that promotes mitochondrial outer membrane permeabilization and apoptosis.

Ubiquitin ligase
An enzyme that can transfer ubiquitin onto a protein, which can target the protein for degradation by the proteasome.

Control and release of p53 p53, through its cell-cycle-arrest and apoptotic activities, can have a strong inhibitory effect on cell growth, making it essential to hold p53 function in check during normal development. Multiple mechanisms exist to negatively control p53, including the regulation of protein activity, stability and subcellular localization through the action of numerous other proteins that work directly or indirectly to restrain p53. These p53-regulatory proteins include ubiquitin ligases that have a role in controlling p53 protein stability, enzymes involved in post-translational modification of p53 (such as kinases and acetylases), transcriptional coactivators that can modulate the transcriptional activity of p53, and many more (as described in a number of excellent recent reviews5,2326). Loss of these regulators, and subsequent failure to rein in p53 function, can have disastrous consequences to the survival of the organism. This is nicely illustrated by MDM2, one of the key ubiquitin ligases responsible for limiting the levels of p53. Deletion of Mdm2 in mice results in an extremely early embryonic lethality that is the direct result of a failure to restrain p53-mediated apoptosis27.
Signals to activate p53 are they all equal? Release of the tight control over p53 and activation of p53 is a well established response to stress. Analysis of the p53 orthologue in flies and worms shows that, as seen in mammalian systems, p53 is an integral part of the response to genotoxic stress2830. p53 is extremely sensitive to even low levels of DNA damage, a response that is thought to contribute to tumour suppression by either allowing for repair or by eliminating cells harbouring

potentially oncogenic alterations. However, many other signals can also activate p53, including inappropriate cell proliferation driven by oncogene activation, telomere erosion, nutrient deprivation and hypoxia 23 (FIG. 1). Importantly, these signals do not all engage p53 through the same pathways, but use different signalling molecules to stabilize and activate p53. For example, ARF, a small protein that binds and inhibits MDM2, has an important role in signalling to p53 in response to some oncogenes, but is not necessary for the activation of p53 in response to DNA damage31. Similarly, the ribosomal protein L11 has a role in activating p53 in response to ribosomal stress without a requirement for ARF32. So, different signals use different pathways to activate p53, leading to the interesting question: are all of these pathways equally important for the inhibition of tumour development? Although a response to genotoxic stress certainly seems to be the most ancient function of p53 in evolutionary terms, a recent study using a mouse model in which p53 can be switched on and off has indicated that the response of p53 to DNA damage might not be responsible for tumour suppression33. The studies show that p53 becomes important only after the bulk of the damage has been resolved, and conclude that the key tumour-suppressive function of p53 is to respond to oncogene activation that occurs as a consequence of the original genotoxic stress. Supporting this idea are studies showing that ARF which is necessary for oncogene-induced, but not DNA-damage-induced, activation of p53 is responsible for almost all the tumour-suppressor activity of p53 (REF. 34). These are startling and provocative suggestions because they make us reconsider the utility of the p53 response to DNA damage in regards to tumour suppression as well as the true role of p53 in DNA repair. Undoubtedly there will be modifications, complications and caveats to this story. Other equally compelling studies indicate that genotoxic stress is the key signal to activate p53 and tumour suppression in pre-cancerous lesions, and that DNA damage can be induced by the activation of several oncogenes in an ARF-independent manner35,36.

MDM2
A ubiquitin ligase that functions as an important negative regulator of p53. As the product of a p53-inducible gene, MDM2 functions in a negative-feedback loop with p53.

p53 activation by everyday stresses Despite the clear importance of the negative regulation of p53 during normal cell growth, a number of recent studies have led us to question one of the basic tenets of the field that p53 is held entirely inactive until induced by unusual, sporadic or severe stress, such as acute genotoxic stress or oncogene activation. It has become evident that despite the many levels of negative regulation that are in place to restrain p53, the everyday rigours of normal mammalian life can more systemically induce low levels of p53 activity. And, recent studies have revealed a hitherto unappreciated importance of p53 under conditions of apparently normal growth and development. Interestingly, induction of p53 through these mechanisms seems to have a role in responses beyond cell-cycle arrest and apoptosis, including an intriguing role of p53 in promoting survival37 as well as cell death.

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TransProline-rich activation domain p53 Phosphorylation Point mutations in tumours Phosphorylation Acetylation Methylation Ubiquitylation Neddylation Sumoylation Sequence-specific DNA-binding domain Oligomerization domain

p73 SAM domain p63

Figure 2 | Structure of p53 family members. The major functional domains of the p53 protein are shown, including the N-terminal transactivation domains, the central sequence-specific DNA-binding domain and the C-terminal regulatory domain. p53 is subject to numerous post-transcriptional modifications, including phosphorylation, acetylation, methylation and modification with ubiquitin-like proteins, that can affect the function and stability of p53. Phosphatases, de-acetylases and de-ubiquitylating enzymes have been identified that can reverse most of these modifications. Most of the point mutations found in naturally occurring cancers occur in the central DNA-binding domain, and the position of the hotspots for these mutations are indicated by the orange lightning bolts. The p53-related proteins p63 and p73 show a similar overall structure, although some isoforms of these p53 relatives also contain a C-terminal sterile -motif (SAM) domain. Multiple isoforms of each of these proteins have now been described16.

Warburg effect
An increase in aerobic glycolysis that is characteristic of cancer cells.

Regulation of glucose metabolism. Recent results have indicated a role for p53 in determining the response of cells to nutrient stress and in regulating pathways of glucose usage and energy metabolism (FIG. 3). Metabolic stress that results in low glucose levels has been shown to activate p53 through a pathway that involves AMP kinase (AMPK), and has been proposed to contribute to the short-term survival of cells suffering, hopefully temporary, starvation38. However, the loss of this response in tumours that lack functional p53 might also contribute to the capability of these cells to continue to proliferate in nutrient-poor conditions, and so provide a proliferative advantage to tumour cells that are attempting to
Box 1 | Apoptotic pathways and p53

grow abnormally. Such a starvation-induced activity of p53 is entirely consistent with the concept of p53 as a sentinel in the detection and response to potentially oncogenic stress, but could clearly have a much broader role in the response to metabolic stress. More surprisingly, roles for p53 in controlling different metabolic pathways under apparently normal growth conditions has also been recently described. For example, p53 has been shown to induce the expression of the copper transporter SCO2, which is required for the assembly of cytochrome c oxidase8. This allows p53 to enhance oxidative phosphorylation. Conversely, the loss of p53 activity in cells results in a reduction in oxygen consumption. The resultant defect in respiration would affect tumour cells (which are all defective in some way for p53 activity) particularly strongly, because the abnormal and deregulated proliferation that is characteristic of cancer cells makes them particularly energy demanding. In cancers, a dramatic increase in glycolysis called the Warburg effect might help solve this problem of sustained energy production. Interestingly, loss of p53 seems to be a root cause of the metabolic changes that characterize cancer cells, because restoration of SCO2 expression in p53deficient cancer cell lines can restore mitochondrial respiration8. Similarly, basal levels of p53 have been shown to have a role in regulating AIF expression, a mitochondrial protein that also contributes to efficient oxidative phosphorylation39. But what effect does this change in mitochondrial respiration have on the whole animal? Closer examination of p53-null mice led to the identification of an interesting, and hitherto undetected, defect in endurance. p53-null mice get exhausted very quickly during exercise presumably because they cannot efficiently generate energy through aerobic respiration8. This might be one of many cancer-independent phenotypes that results from the loss of p53 that have been overlooked owing to our extreme focus on tumour development.

One of the most dramatic responses to p53 is the induction of apoptosis, a type of programmed cell death. Apoptotic signals can engage two main pathways (which are also interconnected). These are the extrinsic pathway, which is induced through the activation of cell-surface receptors, and the intrinsic pathway, which responds to stress signals98. Although p53 has been implicated in both pathways, it predominantly seems to influence the intrinsic pathway. This apoptotic pathway leads to a perturbation of mitochondrial membrane potential, and so the release of apoptogenic factors from the mitochondrial intermembrane space into the cytoplasm. This triggers a cascade of events leading to caspase activation and cell death99. A family of proteins with structurally conserved domains, known as the BCL2homology (BH) domains, have a central role in the intrinsic apoptotic pathway. Two of these BH-domain proteins, BAX and BAK, function to promote apoptosis by regulating mitochondrial membrane potential. Anti-apoptotic BH2 proteins, such as BCL2 and BCLxL, negatively regulate BAX and BAK, whereas a further group of these proteins, the BCL2homology domain-3 (BH3)-only proteins, control these survival proteins100. One of the key contributions of p53 to apoptosis is the induction of the expression of genes that encode apoptotic proteins, functioning in both extrinsic and intrinsic pathways. Many potential apoptotic target genes of p53 have been described, including those that encode the BH3-domain proteins NOXA and PUMA. Deletion of many of the described apoptotic targets of p53 has little effect on the sensitivity of the cell to stress-induced apoptosis, possibly reflecting the multitude of other apoptotic signals that can be induced by p53. The dramatic effect that loss of PUMA has on the sensitivity of several different cell types to p53induced cell death is therefore particularly telling101,102, indicating that PUMA is a crucial mediator of apoptosis in response to p53. Interestingly, PUMA has been proposed to function to release cytoplasmic p53 from inhibitory interactions with anti-apoptotic BH3-domain proteins, allowing p53 to function in a transcriptionally independent manner as a BH3-only protein103.

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Glucose AMPK TIGAR p53 Sestrins Pentose phosphate pathway Lower levels of ROS Survival

p21

p53

Survival Pyruvate SCO2 p53

Lactate

Mitochondrial respiration

Figure 3 | p53 and metabolism. Some of the points at which p53 can affect metabolic pathways. This is a new and rapidly moving area of research, and the influence of p53 on metabolism is likely to be much broader than illustrated here. In response to nutrient stress, p53 can become activated by AMP kinase (AMPK), promoting cell survival through an activation of the cyclin-dependent kinase inhibitor p21. Other functions of p53 include regulating respiration, through the action of SCO2, or in decreasing the levels of reactive oxygen species (ROS), through the actions of TIGAR (Tp53-inducible glycolysis and apoptosis regulator) or sestrins.

TIGAR
A gene that encodes a protein that functions to lower intracellular reactive oxygen species levels by enhancing the pentose phosphate pathway.

Pentose phosphate pathway

An alternative pathway for glucose metabolism that generates NADPH, which is needed for the scavenging of reactive oxygen species by reduced glutathione.

Sestrins
A family of proteins that modulate peroxide signalling and regulate intracellular reactive oxygen species levels.

Antioxidant functions. Another recently described p53-inducible gene with a role in glycolysis is TIGAR (Tp53-inducible glycolysis and apoptosis regulator)9, which encodes a protein that shows some structural similarity to the bisphosphatase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6 bisphosphate (PFK2/FBPase-2), and can lower the intracellular levels of fructose-2,6-bisphosphate. An effect of TIGAR expression is therefore to decrease the activity of 6-phospho-1-kinase, a key glycolytic enzyme, thereby diverting the major glycolytic pathway into the pentose phosphate pathway. The resultant increase in nucleotide and NADPH production might have a number of interesting consequences, including an increase in glutathione levels to promote scavenging of reactive oxygen species (ROS). Although the apoptotic activity of p53 is mediated at least in part through increasing ROS levels40,41, a number of studies have shown a survival function for p53 in lowering intracellular ROS levels, involving the activity of p53-inducible genes such as TIGAR, sestrins42,43, aldehyde dehydrogenase-4 (ALDH4)44, and others45 (FIG. 3). Most interestingly, this antioxidant function of p53 is important in the absence of acute stress and acts to prevent the accumulation of DNA damage in response to day-to-day damage46. It is worth pointing out, however, that even among these low-stress p53-response genes, the sensitivity to p53 can differ. For example, SCO2 and sestrin levels are reduced by the removal of p53 even in unstressed cells, whereas basal TIGAR expression is not so clearly affected. It seems likely that rather than dividing into two discrete groups of genes that respond to high stress and low stress, p53-responsive genes will form a continuum that shows ever-increasing sensitivity to p53 and stress levels (FIG. 4). Based on current evidence, we can propose a model in which p53 can have two important, but fundamentally opposing, roles in response to stress (FIG. 4). The low levels

of DNA damage that are encountered during normal life are dealt with, through p53, by lowering ROS levels (and so reducing damage) and by promoting the survival of the slightly damaged cell to allow repair (a process to which p53 can also contribute). In response to more severe, sustained stress such as oncogene activation or exposure to high doses of radiation p53 switches from promoting survival and repair to the induction of apoptosis12. This dual role of p53 might explain an interesting paradox in the consequences of loss of PUMA, one of the major mediators of p53-induced apoptosis mentioned earlier. Whereas p53 is unable to induce apoptosis in many cell types in the absence of PUMA, PUMAdeficient mice do not show enhanced tumour development20 presumably because the tumour suppressor functions of p53 in response to constitutive stress remain intact (FIG. 4). However, knock-down of PUMA greatly accelerates tumorigenesis in cells that have acquired certain types of activated oncogene47 probably because the apoptotic response to eliminate damaged cells cannot be activated. Of course, both activities of p53 (survival and death) contribute to tumour suppression. The question that has not been fully explored yet is: are there other consequences of loss of p53, apart from enhancing cancer development?

Beyond cancer other roles for p53 Most current models of how p53 functions in tumour suppression evoke the stress-induced activation of p53, which results in a block to further outgrowth cells that have acquired some oncogenic potential. This reflects an importance of several of the responses that are induced by p53 (FIG. 1), including apoptosis, cell-cycle arrest, maintenance of genomic stability and senescence4850. However, it is now becoming clear that the response to p53 also influences several other aspects of life apart from cancer development.
p53 in development. In vertebrates, the two other p53 family members, p63 and p73, illustrate a requirement for at least some p53-like function during development16. These proteins are structurally similar to p53 (FIG. 2), especially in the core DNA-binding domain in the centre of these proteins, and function as transcription factors that regulate a similar set of genes to p53. Deletion of either p63 or p73 has severe effects on the normal development of mice, and a number of human developmental diseases have also been linked to mutations in p63 (REF. 51). Although loss of p63 and p73 can result in a predisposition to cancer development52, neither protein has the profound tumour-suppressive activity that is shown by p53. Simpler organisms, such as flies or worms, carry only one p53 family member, which has a role in stress-induced apoptosis in the germline of these animals53. On a structural basis, p63 and p73 have been suggested to be evolutionarily more ancient than p53, and the worm and fly p53 might be more accurately described as p63 or p73 (REF. 54). However, in these simple organisms the principal function of these p53 orthologues seems to be to protect the germline from the effects of genotoxic damage.

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Low/constitutive stress High/acute stress

p53 levels Cell-cycle arrest Anti-oxidant DNA repair Cell survival Prevention/repair of damaged cells Tumour suppression Development Longevity

p53 in lower organisms. It would be interesting to know whether birth defects are more common in patients with LiFraumeni syndrome, who carry a germline mutation in one p53 allele and have an extremely high incidence of cancer66. The darker side of p53. Although suppression of cancer or inhibition of teratogenesis are clearly desirable features of p53, the induction of p53 is not without cost. Most clearly, p53 is strongly activated in response to acute genotoxic stress such as is encountered following irradiation or chemotherapy, and the ensuing apoptosis is responsible for the classic symptoms of radiation sickness and side effects of cancer therapy. It is also clear that other forms of stress or trauma, not necessarily associated with potential malignant progression, can also lead to the activation of p53. For example, induction of p53 during ischaemia has been shown to contribute to damage through the activation of apoptosis, and a temporary inhibition of p53 function under these conditions might be highly beneficial in the prevention and management of injury to the liver, brain and kidneys6769, or in treatment following myocardial infarction70. A potential role for p53 in immunity or the response to viral infection has also been suggested23, although there is no clear indication yet as to the mechanistic basis for this activity. Equally tantalizing is the possibility that p53 plays a part in neurodegenerative syndromes such as Parkinsons disease, Alzheimers disease and Huntingtons disease71. In contrast to the positive role of p53 in protection from cancer, each of these examples reflects a more negative role for p53 in which induction of the p53 response causes, rather than solves, problems. The concept that p53 has a darker side becomes even more profound with the realization that despite its help in protecting from cancer, p53 might also contribute to many of the undesirable aspects of ageing (see below). Taken together, it seems possible that we pay a high price for the protection from tumour development that is provided by p53. The quest for eternal life p53 and ageing. One of the most interesting and provocative functions of p53 is in the regulation of lifespan, although whether p53 helps or hinders the ageing process is not yet clear. The observation that even a slight constitutive hyperactivation of p53 results in an alarming prematureageing phenotype in mice72,73 cast a dark cloud over the enthusiasm for systemic activation of p53 as a cancer therapeutic (BOX 2). In humans, a polymorphism in p53 that results in a slight reduction in its activity is associated with an enhanced cancer risk, but also with increased longevity74. How p53 might promote ageing is not yet clear, although a contribution of p53 to cellular senescence and the limitation of the proliferative capacity of stem cells has been proposed75. In Drosophila melanogaster, the extended lifespan that results from a reduction of p53 activity in neurons is not further enhanced by calorie restriction 76, which increases longevity in a number of organisms from yeast to mice.

Apoptosis

Cell death Elimination of damaged cells Tumour suppression Radiation sickness Ischaemia Development Ageing

Figure 4 | Regulation of life and death by p53. p53 functions in the response to both the constitutive stress that is encountered during normal growth and development, and to the acute stress signals that would be associated with oncogenic progression and other types of trauma. In this model, p53 responds to conditions of low or constitutive stress to play an important part in decreasing oxidative damage, and provides repair functions to mend low levels of DNA damage. These activities of p53 contribute to the survival and health of the cell as well as to the prevention of the acquisition of tumorigenic mutations, and might contribute to overall longevity and normal development. By contrast, acute stress that results in a more robust induction of p53 leads to the activation of apoptotic cell death and thereby the elimination of the damaged cell. This function removes cells that have acquired oncogenic alterations, and can contribute to neural tube closure during development, but carries accompanying detrimental effects of stress-induced toxicity, such as radiation sickness, neurodegenerative disease and premature ageing.

In light of the importance of p63 and p73 to normal development in mice, it is perhaps not so surprising that a closer analysis of the p53-null mice revealed that they also show developmental abnormalities55. Although many of these mice are normal at birth, females of some strains show neural tube-closure defects (or exencephaly) that reveals a role for p53 in normal development, at least under certain circumstances56,57. This phenotype is also seen in mice with defects in other components of mitochondrial death pathways, which indicates that the lack of p53 leads to a failure in progenitor cell apoptosis and so an overproduction of neural tissue. Furthermore, studies of developmental abnormalities induced by in utero exposure to ionizing radiation clearly show that p53 has a role in reducing the rate of birth defects56,58,59. Recent studies in zebrafish demonstrate a protective function of p53 against a broad range of early developmental defects that are associated with loss-of-function mutations in genes as diverse as those involved in ribosomal RNA synthesis60, DNA synthesis61, gut development62 and neuronal development63. These findings imply that the normal p53 response constantly monitors the early developmental process, presumably eliminating the odd aberrant cell or killing the embryo when the defects become too extreme58. Interestingly, p53 has a clear anti-teratogenic function64,65, presenting a much more tangible evolutionary advantage than the more frequently studied anti-tumour activity and illustrating an interesting parallel with the germline functions of

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Box 2 | p53 and therapy
The observation that p53 function is lost in most cancers makes it an attractive target for new therapies. Because p53 can induce tumour cell death, the most actively explored avenue is to identify small molecules that will allow the reactivation of p53 in cancers. For cancers that retain wild-type p53, but have suffered alterations that prevent the activation of p53, a number of compounds have been described that target MDM2, an important negative regulator of p53 (REFS 94 96,104106). These include compounds such as Nutlin-3, which block the interaction of p53 with MDM2, and HLI98, which directly target the ubiquitin-ligase activity of MDM2. Of course, these drugs are likely to activate p53 in both normal and tumour cells, but the observation that cancers are much more sensitive to apoptotic stimuli than normal cells raises the hope that these compounds will be sufficiently selective in their killing to be useful cancer therapeutics. The cancers that have mutant p53 require a different approach, and compounds have been described that help some of these mutant proteins refold to acquire at least some degree of wild-type function107,108. This is an attractive line of attack. In most cases, only tumour cells express the mutant protein, and so such drugs are likely to be highly selective with low toxicity to normal tissue. Another interesting approach it to try and selectively harness the fact that cancer cells lack p53, and identify compounds that will only kill in the absence of p53 (so-called synthetic lethality). Although activating p53 in cancer cells might be a good idea, there is also increasing interest in inhibiting p53 (REF. 109). The obvious application for this approach would be to protect normal cells from the side effects of chemotherapy, which are to a large extent a reflection of the apoptotic activity of p53 in the intestine and other proliferating tissues. More generally, however, inhibitors of p53 might also be useful in preventing other detrimental effects of p53.

These results indicate that p53 functions in the pathways that respond to caloric restriction such as those involving silent information regulator-2 (SIR2) and/or insulin signalling73,77. How these observations link to the function of p53 as a survival factor in response to glucose deprivation is not yet clear. Some of these results also hint at a possible role for the recently identified N- or C-terminally truncated isoforms of p53 (REF. 78) in controlling ageing72,73. Regulation of glycolysis by p53 can also affect cellular, and so potentially organismal, lifespan79. However, despite the evidence that p53 can induce premature ageing, it seems that this might not be a function of properly regulated p53. Mice containing an extra copy of the p53 gene, or with reduced expression of Mdm2, showed the expected protection from tumour development but no decrease in normal lifespan80,81. Although not fully resolved, one explanation for this apparent paradox is that the ageing phenotype observed in the earlier studies reflects an imbalance of p53 signalling, with the activation of some, but not all, p53 functions82,83. Although some activities of p53 would be predicted to contribute to accelerated ageing, it seems equally possible that lack of p53, and the associated enhanced oxidative stress, would also have the same effect. Indeed, a deficiency of the p53-related protein p63 has been shown to result in premature ageing that correlates with an induction of senescence34. A recent study in the fruitfly has also indicated that p53 is required for compensatory growth after tissue damage and might contribute to tissue repair, cell renewal and survival in other animals84. Indeed, mice expressing altered forms of p53 also show perturbed wound healing72. Taken together, is seems that perhaps there is just no escape from our steady decline to the Zimmer frame.

Cant live with it cant live without it The evidence that p53 has functions in addition to responding to severe stress to prevent tumour development raises an interesting issue: if we look beyond
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tumour suppression, is p53 good or bad for our health? Is it possible that activation of p53 can be useful or detrimental, depending on the extent or type of stress signal that is being responded to? These questions bring us back to the compelling evidence that p53activating signals function through different pathways and that the response to p53 can vary depending on cell type, environment and other contributing factors85. More specifically, the switchable-p53 mice described earlier33,86 provide a method to begin to explore some exciting avenues for cancer therapies (BOX 2). These studies show that delaying the induction of p53 until after the effects of the irradiation have largely been taken care of by other DNA-repair mechanisms protects the mice from the negative effects of p53-induced apoptosis that are normally observed in radiosensitive tissues. In other words, activation of p53 is responsible for not only the inhibition of cancer outgrowth but also for the debilitating toxic side effects of chemotherapeutic treatments in humans (FIG. 5). So perhaps drugs that turn off p53 function could be used to protect normal tissues from the effects of chemotherapy, allowing patients to tolerate much more aggressive (and so potentially more successful) treatment regimes. These mice might also allow us to delve more deeply into the contribution of p53 to other aspects of health and disease, if the premature death of these mice due to cancer can be prevented by periodic, brief pulses of p53 function. Given the large body of evidence that p53 can contribute to DNA repair11, it will be important to understand whether p53 is truly irrelevant for a full response to genotoxic damage or whether the antioxidant and repair functions of p53 can only cope with much lower levels of damage than have been investigated so far in these studies. Although it seems that the off switch in these mice is not absolutely complete86, therefore raising the possibility that the basal functions of p53 might still be present in these animals, this mouse model is perhaps the first of many that will allow us to address the question of whether a reduction in p53 activity results in accelerated or delayed ageing.

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evidence that they function by interfering with activities of p63 or p73 (REF. 90). Although wild-type p53 shows no obvious ability to bind p63 or p73, mutations in p53 can alter the proteins conformation. This allows p53 to bind its sibling proteins, resulting in an inhibition of p63 and/or p73 function. In addition, mutant forms of p53 acquire novel transcriptional activities that are also likely to contribute to the enhanced malignant potential of cells that express these proteins91,92. p53 mutants in normal cells. Although mutant p53 affects cancer-cell behaviour, it is also interesting to consider the consequences of mutant p53 expression in otherwise normal cells. Although most humans develop p53 mutations as somatic alterations in only a limited number of cells, unfortunate individuals that suffer from LiFraumeni syndrome carry a germline mutation in p53 and express both a mutant form and wild-type form of p53 in all tissues1. Patients with LiFraumeni syndrome show an extremely high incidence of many types of cancer, in which the wild-type p53 allele is often lost. However, the consequence of mutant-p53 expression in normal somatic tissue is not yet clear. Tissue-culture experiments have shown that mutant p53 can function as a dominant-negative inhibitor of wild-type p53. On the other hand, examination of mouse or human cells in which only one p53 allele is mutated indicate that under these conditions the mutant p53 does not accumulate to the high levels it does in cancers, and so does not efficiently block the activity of the wild-type p53. Although this might initially suggest that having one mutant p53 allele has no effect in normal cells, it is important to keep in mind that the p53 pathway is exquisitely sensitive to small changes in the levels or activity of p53, and that the patients with LiFraumeni syndrome express only half the normal amount of functional p53, regardless of any impact of the mutant protein. So, although mutant p53 cannot completely abolish wild-type p53 activity, there might still be a small, but nevertheless important, shortcoming in wild-type p53 activity in cells in which both wild-type and mutant proteins are expressed. The potential importance of what might seem to be only slight changes in p53 regulation is further illustrated by a polymorphism in the MDM2 promoter that results in modest changes in the levels of the MDM2 protein, one of the key regulators of p53 stability and function, that can have a profound effect on an individuals susceptibility to cancer93. Similarly in mice, even small adjustments to MDM2 activity have clear effects on p53 (REF. 81). Polymorphisms in p53 itself can also results in subtle, but potentially extremely important, differences in activity 4. It seems that the p53 pathway needs only to be slightly tweaked for an effect to be seen. The anticipated consequences of any decrease in p53 activity would be a reduced resistance to cancer development, as is clearly seen in the patients with LiFraumeni syndrome. But in the light of the newly discovered functions for p53, it is worth looking at these individuals more closely. Are they exhausted unusually quickly during exercise? If their cancer problems could be efficiently

Acute genotoxic damage Short-term response p53 off No short-term toxicity p53 on Radiation sickness

p53 off

p53 on

Long-term response

Tumour development

Tumour suppression

Figure 5 | Temporal regulation of p53 activity in response to DNA damage. A recent study from Evan and colleagues33 provides evidence that the immediate p53 response to DNA damage is detrimental and not necessary for tumour suppression. The absence of p53 immediately after DNA damage protects animals from radiation sickness, but does not prevent repair of the DNA damage. Persistent absence of p53 results in tumour development, as expected. However, even transient restoration of p53 activity after the resolution of the initial DNA damage can inhibit tumour development without the deleterious responses, such as widespread apoptosis in lymphoid organs and intestinal epithelium, that occur following the irradiation of mice with fully active p53. These results suggest that the induction of p53 in response to signals that persist beyond DNA damage, such as activated oncogenes, is the key to tumour suppression.

Functions of mutant p53 Just as wild-type p53 continues to surprise us by having roles in a much broader range of processes than previously thought, interesting new activities for mutant p53 are also being uncovered. Mutations in p53 are extremely common in tumours, and at some point during malignant progression about half of all cancers acquire a mutation in p53 (REF. 87). It is clear that loss of wild-type p53 function is vitally important for successful tumour progression, and that cancer-associated mutations result in the loss of most of the normal functions of p53, such as transcriptional activation, cell-cycle arrest and apoptosis.
p53 mutations in cancer. Mutations in p53 are different from those that inactivate other tumour-suppressor genes such as retinoblastoma (RB), in which only mutations resulting in a loss of the wild-type protein are selected. In the case of p53, many cancers retain expression of a p53 protein with only a single amino-acid change, which most often occurs in the core DNA-binding domain, leading to both the disruption of normal p53 function and the accumulation of high levels of mutant p53 (REF. 87). Many studies have indicated that the consequences of expressing these mutant forms of p53 are not equivalent to the simple loss of p53, and this is most clearly seen in mouse models in which expression of mutant p53 is strongly linked to a change in tumour spectrum and an increase in metastatic potential compared to p53-null mice88,89. How these mutant p53 proteins exert this influence on tumour spread is now being explored, with

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dealt with in some way, would they be slower to age or suffer less from neurodegenerative diseases? Might they be more able to survive a stroke? also available21,97, and judicious use of these drugs might allow us to switch p53 on and off in humans at will. When this might be advantageous will require further investigation in model systems. But, it seems that in the treatment of p53-null tumour cells with high doses of chemotherapy, temporary inhibition of p53 function in normal tissues might have a profound effect on reducing toxicity without further increasing the cancer risk. More speculatively, can we avoid the deleterious effects of p53 function in other pathologies, or even in ageing, by inhibiting p53 and maintaining tumour suppression through the periodic reactivation of p53? Although some of these questions seem to be approaching fantasy, p53 continues to surprise us.

Conclusions and perspectives The analysis of new mouse model systems might lead us to the conclusion that we are better off without p53 if cancers can be held at bay. Several small-molecule activators of p53 have been described, most of which function to protect p53 from the negative regulatory effects of MDM2 (REFS 9496) (BOX 2). Because these compounds are not genotoxic, they can induce p53 while avoiding many of the deleterious side effects of conventional chemotherapies. A number of p53 inhibitors are

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Competing interests statement

The authors declare no competing financial interests.

DATABASES
The following terms in this article are linked online to: OMIM: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=OMIM Alzheimers disease | Huntingtons disease | LiFraumeni syndrome | Parkinsons disease UniProtKB: http://ca.expasy.org/sprot p53 | p63 | p73 | PFK2/FBPase-2 | SCO2

FURTHER INFORMATION
Karen H. Vousdens homepage: http://www.beatson.gla.ac.uk/research/index.html?topic_ id=36 David P. Lanes homepage: http://www.imcb.a-star.edu.sg/ research/research_group/cell_cycle_control/6000000116_ article.html Access to this links box is available online.

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