p53 is a tumour suppressor protein that is stabilised and activated by DNA damage. DNA damage-induced p53 is able to bring about either cell cycle arrest or apoptosis by the induction of p53-responsive genes such as mdm2 and p21 waf-I. Mdm2 regulates p53 function by blocking the transcriptional transactivation domain of p53 and also by targeting p53 for degradation via an ubiquitin-mediated pathway. Increases in the levels and activity of p53 are brought about by post-translational modifications. The most widely studied modification of p53 is phosphorylation, mediated by several DNA damageactivated kinases. Poly(ADP-Ribose) Polymerase-l (PARP-l) is also a DNA damageactivated enzyme which covalently modifies several target proteins by poly(ADPribosylation). It is well established that PARP-1 plays a key role in DNA base excision repair. More recently, several studies have implicated PARP-1 in the regulation of p53 function in response to DNA damage, although the nature of this relationship has been controversial. This study aimed to clarify and investigate further the role of PARP-1 in p53 regulation using PARP-1 proficient and PARP-1 deficient mouse embryonic fibroblasts (MEFs) as well as a novel potent PARP-1 inhibitor (AGI4361; Ki < 6nM). In this study, both primary and immortalised PARP-l MEFs were used. Initial experiments revealed a tendency for PARP-l +/+ MEFs to develop p53 mutations during immortalisation. Interestingly. PARP-1 -/- MEFs retained wild-type p53, suggesting that the absence of PARP-l bypasses the requirement for p53 to be mutated during the immortalisation of MEFs. As these cells could not be used to analyse p53 responses, experiments were perfonned on primary PARP-l MEFs. However. the primary PARP-l- - MEFs were found to grow very slowly compared to their PARP-1 proficient counterparts. Interestingly. treatment of primary PARP-1+1+ MEFs with AG14361 had a similar effect on cellular growth. This growth inhibition in the absence of PARP-1 was only evident in primary and not immortalised cells. It was therefore decided to stably transfect immortalised PARP-l-- MEFs, expressing wild-type p53, with a plasmid construct containing PARP-l to produce an isogenic cell line pair. These cells have been used, together with a human colorectal carcinoma cell line (HCT-116) and the potent PARP-1 inhibitor AG14361 to analyse the p53 response to different DNA damaging agents. In response to ionising radiation and ultra violet radiation, the absence of PARP-1 did not alter the induction or activity of p53. In response to the alkylating agent temozolomide, treatment of PARP-l proficient MEFs with AG14361 potentiated the increase in p53 protein levels without affecting the transcriptional transactivation activity of p53, possibly due to an impaired repair of the DNA damage and hence increased signalling to p53 due to the persistence of DNA strand breaks. However, similar results were not obtained in the absence of PARP-1 protein (P ARP-1-/- MEFs) or in HCT -116 cells treated with AG 14361 The data presented do not support the hypothesis that PARP-1 is directly involved in the DNA damage induced regulation of p53. There may, however, be an altered p53 response in the absence of PARP-l when cells are treated with particular DNA damaging agents, due to an impaired DNA repair pathway.

Upon responding to cellular stress, p53 protein becomes stabilised and acts as a transcription factor mainly resulting from phosphorylation and acetylation of the protein. Nitration of p53 protein is poorly characterised by comparison with phosphorylation and acetylation. The main aim of this work was to study the effects of nitration on p53 functional activities and on p53-MDM2 protein-protein interactions. Preliminary work was to characterise the nitration of p53 protein over-expressed in E. coli BL21(DE3) which was then purified by a series of column chromatography. GST-MDM2 protein along with control GST protein were also overexpressed in BL21 which were subsequently purified by a single step batch purification before subjected to nitration. Peroxynitrite, a nitrating agent used in this study, was generated in vitro. Preliminary nitration work was carried out using BSA as a model protein as it is easily nitrated owing to its high number of tyrosine residues (19 residues). The present results showed that p53 and GST-MDM2 proteins were hardly nitrated as no strong nitro-tyrosine signals were obtained. This might be due to these proteins, being overexpressed in E. coli, were not properly folded resulting in hidden/cryptic tyrosine residues of which making nitration difficult to achieve. Peroxynitrite was shown to have a degrading property, reducing protein levels of peroxynitrite-treated p53, GST-MDM2 and GST proteins. Immunoprecipitation studies of cancer cell lysates with different p53 status treated with peroxynitrite showed very weak signals of nitro-p53 protein in mutant p53 cells whereby no nitro-p53 protein signal in wild-type p53 MCF7 cells. In addition, NO donor GSNO-treated MCF7 cells showed weak nitro-p53 protein signals. / Ministry of Science, Technology and Innovation (MOSTI) of Malaysia

The p53 tumour suppressor protein lies at the hub of a very complex network of cellular pathways including apoptosis, cell cycle arrest, DNA repair and cellular senescence. However, the mechanism of why and how p53 switches between apoptosis and cell cycle arrest, thereby determining a cell’s fate, remains a mystery to us. To enable us to investigate this ability of p53 to switch between cell cycle arrest and apoptosis, we developed a model which demonstrates similar p53 expression patterns but different functional outcomes. Treating cells with Cisplatin (a common chemotherapeutic drug) and Nutlin-3 (an MDM-2 inhibitor) results in similar high levels of p53 accumulation but different cellular responses. Cisplatin-treated cells undergo apoptosis while Nutlin-treated cells enter cell cycle arrest. Using this model, we explored the localization of p53 and in particular a C-terminal Ser 392 moiety in an attempt to identify how p53 is able to preferentially activate cell cycle arrest or apoptotic pathway.

La protéine suppresseur de tumeur p53 est soumise à de complexes régulations transcriptionnelles et posttraductionnelles. La découverte d’isoformes de p53 a introduit un degré de complexité supplémentaire auxmécanismes de régulation des fonctions de p53. On dénombre à ce jour douze isoformes qui diffèrent de p53dans leurs domaines N- et C-terminal. Cependant, les modes d’expression et de fonction de ces isoformes restentà être clarifiés.Dans cette thèse, nous nous sommes intéressés aux deux isoformes Δ40p53 et Δ133p53, en analysant leurinteraction avec p53 et en mesurant leur expression dans les mélanomes, un type de cancer où p53 est trèsrarement mutée. Nous montrons que Δ40p53 peut moduler l’activité de p53 avec un effet bi-phasique, tantôtactivateur ou répresseur du niveau d’expression et des fonctions de p53. Δ133p53 est produite par un promoteurP2 localisé dans le gène TP53. Nous avons montré qu’en réponse à un stress génotoxique, l’expression de Δ133p53 est régulée par p53, qui se lie au promoteur P2. Ceci suggère une boucle d’auto-régulation par p53, quiest capable de contrôler l’expression d’une isoforme inhibant ses propres fonctions. Enfin, les isoformes Δ40p53 et Δ133p53 sont surexprimées dans les tumeurs métastatiques de mélanomes comparées aux tumeurs noninvasives,suggérant à ces isoformes un rôle dans l’inactivation de p53 dans les cancers.Ainsi, Δ40p53 et Δ133p53 interagissent avec p53 de façon complexe, avec des effets plus contrastés que lasimple inhibition de l’activité suppressive de p53. Les isoformes de p53 jouent ainsi un rôle majeur dans lesactivités basales de p53, ainsi que dans l’inactivation fonctionnelle de p53 dans les cancers. / The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.

The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.

Le gène TP53 est extrêmement polymorphique avec 85 polymorphismes décrits. Certains de ces polymorphismes sont associés à une augmentation du risque de cancer, par exemple rs10425222 peut moduler les fonctions de p53. Cependant, pour d’autres, comme le rs17878362 qui est le polymorphisme intronique le plus étudié, leur association avec une augmentation du riques au cancer est controversée.Pour analyser l’association entre le polymorphisme rs17878362 et la susceptibilité au cancer, nous avons analysé son rôle dans des contextes de cancers sporadiques et familiaux. Les résultats obtenus pour le polymorphisme rs17878362 sont paradoxaux avec une augmentation des cancers sporadiques associée avec le génotype A2A2 alors que l’allèle A2 est associé avec un effet « protectif » chez les patients atteints du syndrome de Li-Fraumeni porteurs d’une mutation germinale de TP53 situé sur l’haplotype A1. Ces observations suggèrent que des haplotypes spécifiques de TP53 pourraient moduler les capacités suppressives de p53. Une hypothèse possible est que les différents haplotypes de TP53 présenteraienrt des mutations somatiques à des fréquences différentes dans la population.De plus, le gène TP53 exprime différentes isoformes, comme le D40p53, inhibant l’activité suppressive de p53. Le D40p53 peut être produite par le maintien de l’intron 2 par épissage alternatif. Nous avons montré que les G-quadruplexes, des structures tridimensionnelles formées dans des régions riches en G, sont formés dans l’intron 3 et régulent la rétention de l’intron 2 et la formation du transcrit p53I2. Nous avons aussi observé que le polymorphisme rs1652785 (localisé dans l’intron 2) semble réguler la stabilité du p53I2. Ces résultats suggèrent que les polymorphismes de TP53 localisés dans une région de 412 pb située entre l’exon 2 et l’exon 4 régulent l’expression des isoformes de p53 dans une séquence temporelle d’évènements en modulant la formation des pré-ARNm (rs17878362), la stabilité des ARNm (rs1642785) et les fonctions protéiques (rs10425222).L’expression des isoformes de p53 est donc finement régulée par des mécanismes impliquant les polymorphismes de TP53 qui sont aussi associés avec une altération dans la susceptibilité au cancer. / The TP53 gene is a highly polymorphic gene with 85 polymorphisms described. Some of these have been associated with an increase of cancer susceptibility, for example rs10425222 that can modulate certain p53 activities. However for others such as rs17878362, the most studied intronic polymorphism, the association with cancer risk is more controversial. To investigate the influence of rs17878362 on cancer susceptibility, we analysed its role in sporadic and familial contexts. The results are paradoxical with an increase of sporadic cancer associated with the rs17878362 A2A2 genotype whereas the rs17878362 A2 allele is associated with a “protective” effect in the context of Li-Fraumeni patients carrying a TP53 germline mutation on an A1 haplotype. These observations suggest that specific TP53 haplotypes could modulate p53’s tumour suppression capacities. A possible hypothesis to explain this could be that somatic mutations are carried on different haplotypes of TP53 present at different allele frequencies in the population. In addition, TP53 is expressed as several protein isoforms, such as D40p53, which inhibits p53’s suppressive activity. D40p53 can be produced from an alternative spliced transcript that retains intron 2. We have shown that G-quadruplexes, tri-dimensional structures formed in G-rich sequences, are formed in intron 3 and regulate the retention of intron 2 and the formation of the p53I2 transcript. We also observed that rs1642785 (located in intron 2) could regulate p53I2’s stability. These results suggest that the TP53 polymorphisms located in a 412 bp region located between exon 2 and exon 4 regulate the expression of p53 isoforms in a temporal sequence of events by modulating the pre-mRNA formation (rs17878362), mRNA stability (rs1642785) and protein functions (rs1042522).p53 isoforms’ expression is thus finely regulated by mechanisms involving TP53 polymorphisms, which are also associated with altered cancer susceptibility.