Key words: p53, DNA damage, phosphorylation, non-genotoxic stress, hypoxia, p14^arf

Abstract
Since the initial concept of p53 as a sensor of DNA-damage, the picture of the role of p53 has widened to include the sensing of much more diverse forms of stress, including hypoxia and constitutive activation of growth-promoting cascades. The pathways by which these processes regulate p53 are partially overlapping, but imply different patterns of post-translational modifications. In this review, we summarise current knowledge on post-translational modifications of p53, and we discuss how hypoxia and oncogene activation stresses may induce p53 independently of DNA damage.

Introduction
In 1984, Maltzman and Czyzyk provided the first demonstration that p53 was activated in response to UV [1]. This discovery was not fully recognised until 1991, when Kastan and his colleagues demonstrated that p53 was essential in cell-cycle arrest in response to irradiation or DNA-damaging agents [2]. At the same time, it became evident that the main biological activity of p53 was to regulate gene expression as a sequence-specific transcription factor [3]. Further studies showed that p53 induction in response to DNA-damage consisted of two interconnected processes, stabilisation of the protein (by escape from constitutive, proteasome-dependent degradation), and activation (by conversion from «latent» into «active» form with high affinity for specific DNA sequences in the regulatory regions of target genes) (for review see [4;5]).

Over the past ten years, p53 has been shown to be activated by a wide range of DNA damaging agents (Table 1A). DNA strand breaks induce a rapid and specific induction of p53, and it has been proposed that strand break formation may be the common denominator in the effect of most p53 activating agents [6]. This conclusion was based in part on experiments showing that the introduction of nucleases into cells could rapidly trigger p53 activity [7]. However, another hypothesis proposes that the main signal for p53 induction is inhibition of mRNA synthesis, either directly by the poisoning of RNA polymerase II or indirectly by the induction of elongation-blocking DNA lesions [8].

Significant differences exist in the extent and kinetics of p53 activation by various types of agents. For example, gamma irradiation induces rapid (2-4h) and transient (up to 6-8h) accumulation of p53, whereas carcinogens such as benzo(a)pyrene induces much slower (12-24h) and long-lasting (up to 2 days) accumulation reflecting intracellular metabolisation [9]. However, these differences may also reflect the fact that these agents use distinct signalling pathways to activate p53.

In parallel with studies on DNA-damage, it emerged that p53 could also be activated by types of stress which are not primarily genotoxic (Table 1B). It is difficult to disprove that these stresses do not induce subtle forms of DNA damage as a by-product of their effects on cells. However, taken together, the diversity of these factors indicates that p53 is responsive to a much broader range of stresses than strict DNA-damage.

In this review, we briefly describe the molecular mechanisms by which p53 «senses» various forms of stress and converts itself into a fully active transcription factor. We summarise current data showing that patterns of post-translational modifications differ, depending on the nature of the inducing agent. Furthermore, we discuss how non-genotoxic stresses may activate p53 through signalling pathways independent of DNA damage. It is not our objective to provide detailed coverage of the network of effectors involved in the tumour suppressive effects of p53, which have been the main subjects of several, recent reviews [10-12].

Mdm2 and JNK: bodyguards of p53 activation
The p53 protein is essentially expressed as a single transcript encoding a 53 kDa, nuclear phosphoprotein with the typical anatomy of a transcription factor, including a N-terminal region containing major (residues 1-42) and minor (residues 45-56) transactivation domains, a sequence specific DNA-binding domain (residues 98-296) and a complex C-terminal region containing tetramerization motifs (residues 319-363) and a domain exerting a negative regulation on DNA-binding activity (residues 364-393). In the «latent» (inactive) form, the protein is constitutively unstable and adopts a conformation in which the extreme C-terminal domains hinder the interactions of the DNA-binding domain with its target [13]. Activation of the protein proceeds through co-ordinated steps, including escape from proteasome-dependent degradation, release of the negative inhibition carried by the C-terminus, and conversion of the conformation of the DNA-binding domain into a form with high affinity for target DNA (see review in [5]).

Two proteins, Mdm2 and JNK, are responsible for the constitutive instability of p53 [14-17](Figure 1). These proteins bind p53 at distinct sites in the N-terminus and act as E3-ubiquitin ligases targeting p53 for degradation by the 26S proteasome. Mdm2 binds at residues 17-22 [18] in p53 and is generally considered as the universal regulator of p53 in response to genotoxic stress [19]. Mdm2, a ring-finger protein, is the product of an oncogene amplified in several tumours, including in particular sarcomas [20]. By binding to p53, mdm2 not only earmarks the protein for degradation but also conceals transcription activation domain and mediates p53 export from the nucleus into the cytoplasm [21]. Furthermore, mdm2 expression is activated by p53, defining a feedback loop in which mdm2 controls the level, extent and duration of p53 protein activation [22]. MDM2 knock-out mouse embryos do not develop and undergo massive apoptosis at an early, post-implantation stage. This phenotype is rescued in double, TP53 and MDM2 knock-outs, pointing out the role of mdm2 protein in keeping p53 activity in check [23;24]. Aside from effects on p53, mdm2 also regulates its own stability, interacts with transcription factors such as E2F1 [25;26], and regulates the Cyclin A promoter by interacting with TAFII [27]. Moreover, mdm2 plays a p53-independent role in the differentiation of normal epidermis [28]. It remains to be established whether this role involves interactions with the p53 homologues p63 and p73, which are both expressed in these tissues. JNK, the c-Jun N-terminal Kinase, plays two distinct roles in the control of p53 activity. When inactive, this kinase binds to residues 97-116 in p53 and targets p53 for degradation by the proteasome. Expression of a constitutively activated JNK, or activation of the upstream kinase MEKK1, increases the level of p53 by allowing escape from degradation [29]. In contrast, active JNK phosphorylates p53 on threonine 81 [30] and participates in its activation. JNK is activated in response to many signals which trigger p53 accumulation, including UV, X-radiation and oxidative stress by hydrogen peroxide [31]. Activation of p53 by these agents is prevented by a peptide blocking p53-JNK interaction [16]. However, the contribution of the JNK pathway to p53 activation in response to genotoxic stress is still poorly understood. Complexes between p53 and JNK are preferentially found in G0/G1, in contrast with mdm2-p53 complexes, which are mostly detectable in S and G2/M phases. It has been proposed that JNK may essentially act as a regulator of basal levels of p53 protein in non-stressed cells [32;33].

The picture emerging from these studies is that JNK and Mdm2 are constitutive repressors of p53, acting to maintain the concentration of the protein at sub-active levels. Induction of p53 thus essentially consists in de-repression by release of the protein from these two bodyguards (Figure 1). However, additional factors may participate in p53 degradation. For example, the kinase associated with the COP9 signalosome (CSN) phosphorylates p53 at threonine 155 (located within the DNA-binding domain). The CSN has structural homologies with the 26S proteasome and mutation of threonine 155 to valine inhibits p53 degradation in vitro [34]. Further studies are needed to determine how the CSN kinase co-operates with mdm2 and JNK in regulating p53 levels.

Activation of p53 by genotoxic stress
The mechanisms of p53 activation in response to various forms of DNA damage was the subject of several recent reviews [11;35;36;37]. The dissection of these pathways has been greatly facilitated by obtaining antibodies against specific phosphorylation sites in p53 [38;39]. Overall, these studies have shown that induction of p53 implies concerted post-translational modifications in the N- and C-terminal regions (Figure 2).

Modifications in the N-terminus: sensing DNA-damage
The N-terminus is a specialised «landing pad» for kinases transducing DNA damage signals. These kinases include the phosphatidyl-Inositol-3-Kinase family members ATM, ATR, DNAPK, and the cell cycle regulatory kinases Chk1 and Chk2. Additional sites have been identified for variants of Caseine Kinase-I (CK-I), for cell-cycle activating kinase (CAK) and for stress kinases p38 and c-Jun-N-terminal kinase (JNK) (Figure 2, see figure legends for detailed references). There is evidence that the signalling of DNA strand breaks induced by IR primarily requires the ATM/Chk2 connection. In the case of UV-induced damage, the signalling to p53 preferentially involves ATR, Chk1, p38 and JNK. Although the role of DNA-PK is still controversial, its function does not appear to be necessary for p53 activation in response to DNA damage [40].

Co-ordinated changes in the phosphorylation state of several of these sites are thought to be necessary to stabilise p53 by dissociation of complexes with mdm2 and/or JNK. In addition to the release of the two bodyguards, phosphorylations also contribute to increase the affinity of the N-terminus for components of the transcriptional machinery and for co-activators of transcription such as members of the histone acetyl-transferase family (CBP/p300 and pCAF) [41;42]. Phosphorylation on specific sites may also determine how active p53 selects between subsets of target genes for transcription. Phosphorylation at Serine 46, for example, has been shown to occur only at high levels of DNA damage, and to correlate with activation of AIP1, a target gene specifically involved in apoptosis [43].

The C-terminus as an integrator of multiple regulatory signals
Activation of p53 requires post-translational modifications in the C-terminus. The latter contains several, complementary nuclear localisation signals (main signal: residues 316-322), a leucine-rich nuclear export signal (residues 340-351), the structural elements responsible for p53 dimerization and tetramerization (residues 319-363), the lysines acting as binding sites for ubiquitin, and a basic region that negatively regulates the DNA-binding activity (residues 364-392). The kinases phosphorylating p53 in these regions include the cell-cycle dependent kinases cdc2 and Cdk2, Protein Kinase C (PKC), and Casein Kinase-II (CK-II) (Figure 2; see figure legends for detailed references). The participation of these kinases to responses to DNA-damage is not well understood. CK-II is activated in response to UV but the consequences of this activation are unknown. There is no clear evidence of changes in the level of phosphorylation of ser 315 (cdc2/Cdk2) in response to DNA damage. The two PKC sites, serines 376 and 378, form a binding site for the regulatory protein 14-3-3sigma. Binding of the latter protein requires dephosphorylation of serine 376 by a phosphatase activated in an ATM-dependent manner [44]. This observation suggests that phosphorylation by PKC might essentially inhibit p53 activation, a hypothesis supported by the fact that PKC inhibitors maximize p53-dependent apoptosis in response to IR [45].

Several lysines in the C-terminus are covalently modified by acetylation, including lysine 320, 373 and 382.

In addition, lysine 386 is modified by binding of SUMO-1, an ubiquitin-related protein. In contrast with ubiquitination, sumoylation does not tag proteins for degradation but rather stabilises them [46;47]. Acetylation occurs in response to many forms of DNA-damage. However, the consequences of acetylation are still poorly understood. First, acetylation may contribute to stabilise p53 by concealing lysines used as target sites for ubiquitin, therefore inhibiting degradation [48;49]. Second, acetylation may induce conformational rearrangements of the C-terminus, increasing DNA binding capacity. Third, acetylation may play a role in the regulation of compartmentalisation of p53 between nucleus and cytoplasm. Interestingly, lysine 320, a substrate of PCAF, is located within the main nuclear localisation signal of p53. Acetylation may provide a ?cross-talk between the N-terminus (binding site of acetyl transferases) and the C-terminus (containing target residues for acetylation). These co-ordinated regulations are probably essential for the integration of DNA-damage responses.

A global picture of p53 post-translational activation should take into account that p53 partners are also affected by modifications in response to stress. In particular, mdm2 is phosphorylated by ATM [50;51] and is regulated by sumoylation [52]. Other factors may play a role as non-covalent modifiers of the extent of p53 activation. Mice deficient in the PARP-1 gene (encoding poly-ADP ribose polymerase) show low levels of p53 accumulation in response to IR [53]. PARP-1 is rapidly activated after IR and is involved in the recruitment of DNA repair enzymes at the site of the lesion [54]. It is therefore possible that PARP-1 may control the degree of p53 induction by modulating the very first steps of the signalling cascade initiated by DNA damage.

Activation of p53 by non-genotoxic stress
In the early nineties, it was generally considered that DNA damage was the exclusive signal that triggers p53 protein activation. The notion that p53 could also be activated in response to non-genotoxic stress slowly emerged from two lines of work. First, in 1993, Lowe and Ruley demonstrated that overexpression of the adenovirus 5 E1A protein, which binds and neutralises the activity of the retinoblastoma protein, induced the stabilisation and accumulation of p53 [55]. Second, in 1994, Giacca and his collaborators showed that p53 was activated in response to low-oxygen conditions [56]. There is now good evidence that these two processes do not require the direct formation of DNA-damage as a signalling intermediate. Since these initial studies, non-genotoxic activation of p53 has been reported in response to numerous physiological processes as well as pharmacological agents (see Table 1B).

P53 activation in response to oncogene expression
The observation that expression of E1A activates p53 is the cornerstone of a series of studies showing that p53 is stabilised and activated in response to an oncogenic challenge, with the apparent consequence of suppressing transformation. The signalling mechanism involves p14arf, the product of the alternative reading frame of the cell-cycle regulatory gene INK4a/CDKN2a [57;58]. This gene encodes two structurally unrelated tumour suppressors, p16 (a cyclin kinase inhibitor acting in G1/S) and p14arf (p19 in the mouse) [59]. Together with TP53, this gene is the most frequently altered locus in human cancer [60]. Both p16 and p14arf accumulate during the serial passage of cells in culture, and are rapidly induced in response to promitogenic signals (including expression of oncogenic ras, stimulation of the MAPK signalling cascade, or inactivation of Rb). ARF expression is regulated by transcription factors of the E2F family, which are essential for entry and progression into S phase.

The p14arf protein interacts with mdm2 and interferes with its capacity to trigger p53 degradation [61;62]. The mechanism of this interference involves p14arf binding to the C-terminal part of mdm2 [63-65]. As p14arf is essentially located in the nucleolus, this interaction may tether mdm2 into the nucleolus and prevent mdm2-mediated export of p53 in the cytoplasm for degradation [66;67]. However, there is also evidence that p14arf stabilises p53 without relocation of mdm2 to the nucleolus [68]. Recent results indicate that activation of the p14arf-p53 pathway can suppress the transformation of primary epithelial cells in vitro [69]. Together, mdm2 and p14arf thus define two interconnected feedback loops controlling p53 stability (Figure 3). Activation of the p14arf pathway during senescence as well as in response to promitogenic signals may represent a physiological pressure that selects for TP53 mutations in cancer cells. It is now emerging that this pathway is also activated in response to multiple stimuli that affect cell-cycle progression, as well as microtubule depletion [70].

P53 activation in response to hypoxia
Hypoxia (1.5% O2), anoxia and several hypoxy-mimetic drugs (e.g. cobalt chloride, desferroxamine, nickel compounds) have been shown to induce p53 accumulation. However, there is evidence that the time-course of accumulation, the physiological consequences of p53 activation, and perhaps the mechanisms of induction, differ between hypoxia, anoxia and hypoxy-mimetic agents. In severe hypoxia (0.02% O2, close to anoxic conditions) p53 accumulates rapidly (within 2 to 4 hrs) and transactivates the expression of genes preferentially involved in G1 arrest [56]. This accumulation appears to result from down-regulation of mdm2 [71] and is accompanied by phosphorylation of p53 on serine 15, but not by interaction with p300/CBP and acetylation of lysine 382 [72]. In contrast with response to DNA damage, p53 induced by severe hypoxia is resistant to E6-mediated degradation, suggesting that the pathway of stabilisation is at least partially different[56;71].

Oxygen homeostasis is an essential requirement for biosynthesis of ATP through oxidative phosphorylation cascades, while keeping in check oxidation damage to proteins, lipids and nucleic acids. All cells possess sophisticated oxygen sensing mechanisms, coupled to signal transduction pathways controlling multiple processes including glucose/energy metabolism, cell proliferation and viability, erythropoiesis and iron metabolism and vascular development and remodelling [73]. How changes in oxygen levels are transduced to p53 is not known. It should be noted that its is difficult to exclude any contribution of DNA strand-break damage in the process of p53 induction by hypoxia/anoxia. Indeed, anoxia has been shown to result in the activation of specific endonucleases in vitro [74].

Figure 4 proposes three hypotheses to account for p53 protein activation in response to hypoxia/anoxia. The first implies a signalling role for the alpha subunit of the hypoxia inducible factor Hif-1. Hif-1 is an heterodimeric transcription factor consisting of Hif-1, a DNA-binding protein, and Hif-beta (also termed Aryl Hydrocarbon Nuclear Translocator, ARNT) [75]. In normoxic conditions Hif-alpha is constitutively targeted to proteasome-mediated degradation by pVHL, the protein product of the Von Hippel Lindau gene [76]. The latter protein binds to an oxygen-dependent degradation domain of Hif-alpha, containing a an hydroxylated proline (residue 564). Suppression of hydroxylation in response to anoxia/hypoxia results in the release of pVHL binding and in the stabilisation of Hif-alpha [77;78]. There is evidence that Hif-1 a forms a complex with wild-type p53 [79]. However, it is not clear whether this interaction is direct or indirect through binding common co-activators such as p300/CBP [80]. Whether binding of Hif-a results in p53 induction is even less clear. Recent results indicate that p53 may, in turn, participate in the regulation of Hif-alpha by inducing its degradation by mdm2 [81]. Thus, the exact role of the p53/Hif-alpha complex remains to be elucidated. The second, hypothetical pathway of p53 induction by hypoxia/anoxia involves changes in mitochondrial permeability leading to release of free radicals [82]. These free radicals may, in turn, trigger p53 activation, but whether this activation requires oxidative damage to DNA is not known. This hypothesis is supported by the fact that cells deficient for mitochondrial functions fail to accumulate p53 in response to hypoxia, and that accumulation of p53 is prevented by the addition of anti-oxidants such as N-acetylcysteine [82]. The third hypothesis is that p53 may be a direct sensor of hypoxic stress by modulation of proline hydroxylation in a manner analogous to Hif-1 alpha. In this respect, a good candidate as a target for hydroxylation is proline 98, a conserved residue which is never targeted by missense mutations human cancer and is located within the binding domain for JNK, one of the regulators of p53 protein degradation.

Activation of p53 by ribonucleotide depletion
Studies by Linke et al. (1996) have shown that p53 is activated by ribonucleotide biosynthesis inhibitors, inducing a G0 or early G1 arrest in the absence of replicative DNA synthesis or detectable DNA damage in normal human fibroblasts [83]. CTP, GTP, or UTP depletion alone is sufficient to induce arrest. In contrast to the p53-dependent response to DNA damage, the antimetabolite-induced arrest is highly reversible. The pathway of p53 activation by ribonucleotide depletion is unknown but does not appear to involve activation of p14arf [70].

Activation of p53 functions by changes in extracellular or intracellular architecture
Activation of p53 occurs in response to many factors that deregulate cell adhesion, intercellular communications and microtubule architecture or assembly. In 1997, Nigro et al. have shown that loss of cell anchorage resulted in an approximately 5-fold decrease in p53 levels in keratinocytes, which was reversible upon reattachment of cells to a substratum [84]. Recently, several studies have shown that depolymerization of microtubules in quiescent fibroblasts resulted in accumulation of transcriptionally active p53 that caused cell-cycle arrest at the G1/S boundary [85]. This p53 activation is correlated with activation of Erk1/2 MAP kinases, and is alleviated by specific inhibitors of these kinases [86;87]. It is not known whether these processes correspond to independent pathways of p53 activation, nor whether their effects are direct or indirect.

Conclusions: p53 as a sensor of multiple forms of stress
To date, at least three distinct biological processes have been shown to alleviate p53 repression through specific mechanisms: genotoxic stress, hypoxia/anoxia and activation of growth signalling cascades. It is not clear whether there is a specific pattern of post-translational changes associated with each of these processes. It is interesting to note that activation of p53 by hypoxia seems to follow different time trends and to induce different effects than response to acute genotoxic stress. Therefore, the pattern of post-translational modifications may determine the selection of the subsets of target genes regulated in response to p53 activation, explaining how active p53 select between apparently contradictory cellular responses such as cell cycle arrest or apoptosis. Thus, non-genotoxic stress (such as in particular hypoxia) may induce a long lasting, moderate accumulation of p53, with preferential activation of genes involved in cell-cycle and in differentiation. In contrast, acute genotoxic stress may induce rapid and transient accumulation of very high levels of p53, with preferential activation of target genes involved in apoptosis (Figure 5). The two responses are not fundamentally different in terms of maintenance of genetic integrity: while cells with acute damage are deleted, cells subjected to non-genotoxic stress are permanently removed from the pool of cells undergoing DNA replication.

The fact that multiple signals can cooperate in its activation confer to p53 the properties of a «superpower molecule» acting as an emergency brake to prevent unscheduled cell proliferation. Although TP53 is a member of a gene family that includes at least two other members, it is unique in its post-translationally regulated responsiveness to such a broad range of signals. This central position explains why TP53 is so frequently altered in human cancer. In particular, hypoxia and/or activation of growth signalling cascades are very common phenomena during the early steps of cancer development. Therefore, these two processes may drive the selection of cell clones having lost wild-type TP53 function.

Many questions are still unanswered in the mechanisms of p53 activation. The first is to determine what is the exact contribution of DNA modifications. It is clear that acute DNA damage is a strong inducer of p53, but there is also evidence that very moderate levels of DNA strand breaks, which do not represent as such a genotoxic hazard, may also lead to an increase in p53 levels. For example, TNF-alpha induces p53 protein accumulation through intracellular production of reactive oxygen species and the formation of limited amounts of DNA strand breaks (Furukawa and Hainaut, unpublished observations). These observations raise the possibility that moderate and transient DNA modifications can act as part of a second messenger system in the pathway of p53 activation. According to this hypothesis, DNA itself would behave as a stress sensor, and low doses of DNA damage might, in themselves, play a positive role in cell physiology, as intermediates in signal transduction cascades used by many physiological processes.

A second, important question which is linked to the former, is the role of redox regulation in the control of the p53 pathway. It is quite remarkable that p53 lies at the centre of a network of redox regulatory signals (Hainaut and Mann, in press). ROS act upstream of p53 (as inducers of DNA damage or as intermediates in the signalling of hypoxia), on the p53 protein itself through a set of conserved cysteine residues located within the DNA-binding surface, and downstream of p53 (since several genes regulated by p53 control ROS production, such as NOS2, COX2, GST and several of the PIG genes). The intracellular redox potential may be described as the «barometer» of the cell, which controls a complex adaptive machinery allowing the cell to maintain a continuity in energy supply and in DNA integrity despite changes in its environment. Even relatively small redox changes may act as modulators of p53 protein activities and may contribute to shift the balance between various pathways activated in response to p53.

Finally, it is clear that the p53 protein does not act alone but forms complexes with many other cellular components and with nuclear structures. This protein has a very complex intracellular distribution, and both the mechanisms and consequences of its activation may largely depend on its local concentration, availability and on the nature of its partners. These spatio-temporal aspects may contribute to the extreme heterogeneity of p53-dependent responses observed within a specific tissue exposed to an activator of p53, as demonstrated in intestinal cells of mice exposed to whole body irradiation [88]. Further insights into these mechanisms may help to design drugs that will selectively use non-genotoxic pathways to activate p53 for therapeutic or chemo-preventive purposes.

Acknowledgement
O.P is supported by a fellowship of the French Ligue contre la Cancer (Comité de la Loire).

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Figure legends

Figure 1
Pathways for p53 protein degradation The proteins mdm2 and JNK bind p53 in the N-terminal region at distinct sites. In non stressed cells, these proteins act as a E3-ubiquitin ligases targeting p53 for degradation by the proteasome pathway. These two mechanisms act independently and in different phases of cell cycle. P14arf , a product of INK4a gene, binds mdm2 and neutralises its activity towards p53.

Figure 2
Schematic representation of p53 and sites of post-translational modifications.
p53 contains a N-terminal transactivation domain (N-Ter, residus 1-42), a DNA binding domain (DBD, residus 98-296), and a flexible C-terminal regulatory domain (C-Ter, residus 319-393) which is thought to interact with DBD to maintain p53 in a «latent» inactive form. Conversion to active form imply changes in the pattern of post-translational modifications. Sites of phosphorylation, of acetylation and of binding by SUMO-1 are shown. Sites preferentially modified in response to UV, IR, or both are colour-coded. A number of kinases have been implicated in phosphorylations of p53, including Caseine Kinase-I (CK-I) at ser6, ser9 and thr18 [89;90], DNA-Protein Kinase (DNA-PK) at ser15 and ser37 [39;91], ATM/ATR at ser15 and ser37 [92;93], Chk 1/2 at ser20 [94;95], CDK activating kinase (CAK) at ser33, ser371 and ser376 [96;97], ERKs 1/2 at ser15 [98], p38 at ser15, ser33 and ser46 [98;99], Jun N-Terminal kinase (JNK) at thr81 [30], Cdk2/cdc2 at ser315 [100;101], Protein kinase C (PKC) at ser371, ser376 and ser378 [44;102;103], Caseine kinase-II (CK-II) at ser392 [104], CSN kinase at thr155 [34], unknown kinase at thr55 [105]. Members of the histone acetylase family acetylate p53: PCAF at lys320 [106;107], p300/CBP at lys373 and lys382 [106;107]. SUMO-1 enzyme binds covalently the ubiquitin like peptide SUMO-1 at lys386 [46 ; 47].

Figure 3
Feedback loops regulating p53 protein stability. Stability of p53 is regulated by two feedback loops controlling its transactivation capacity. In loop A , p53 induces mdm2, which in turn inhibits p53 transactivation and targets p53 for degradation (see figure 1). In loop B, hyperproliferative signals (or activation of oncogenes) induce p53 through p14arf, which is activated by a pathway involving Rb, E2F and DAP kinase. This pathway is negatively regulated by p53 through the cyclin dependent kinase inhibitor p21waf-1 (for details see text).

Figure 4
Three hypotheses for the mechanisms of p53 induction in response to low oxygen pressure. First, under hypoxic conditions, the mitochondria generates reactive oxygen species at moderate levels, sufficient for the induction of p53 through a pathway that may involve DNA strand-break damage. Second, an unknown sensor of oxygen pressure directly induces p53. Third, hif-1a interacts with p53 under hypoxia and is required to induce p53 activation.

Figure 5
Activation of p53 by genotoxic or non-genotoxic stress : overlapping signalling pathways but distinct repertoires of biological responses.

While acute genotoxic stress preferentially induce apoptosis, non-genotoxic stress may preferentially induce cell-cycle arrest. The image of a hourglass illustrates the notion that p53 is at the point of convergence of many incoming and outgoing signalling pathways.

Table 1: Activation of p53 protein by genotoxic and non genotoxic treatments

1 A: DNA-damaging agents
Type	Agent
Irradiation	UV	References
	Gamma rays	[109]
	X-rays	[110-112]
	Alpha particles	[113]
Carcinogens	Polycyclic Aromatic Hydrocarbons	[114]
	Mycotoxins	[110]
	Heavy metals (Cadmium)	[115]
Oxidative/nitrosative stress	Hydrogen peroxyde	[116]
	Nitric Oxide donors	[117]
	Cytotoxic drugs Platinum compounds	[116;118]
	Alkylating agent	[119]
	Antimetabolites 5-FU PALA Methotrexate	[116] [120] [121]
	Tumour antibiotics Mitomycin C Actinomycin D Bleomycin	[116;118] [109] [120]
	Anthracyclins (doxorubicin)	[122]
Topoisomerase inhibitors	Camptothecin	[116]

1 B: Non-genotoxic agents
Type	Agent	References
Anti-microtubule agents*	Taxanes Nocodazole	[116] [123] [124]
Ribonucleotide depletion		[125]
Hypoxia/Anoxia		[126]
Cell adhesion		[84]
Oncogene activation	Overexpression of E1A	[55]
Cytokines	TNF-alpha	[127;128]
Hyperthermia		[129]
Proteasome inhibitors		[130]
Senescence/telomere erosion		[131]
Polyamine analogs		[132]

*: While these agents are cytotoxic, there is no evidence that they induce primary DNA-damage.

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