p53 has a key role in the maintenance of the genetic stability and, thus, in preventing tumour development. The current model is that the p53-dependent responses are solely driven by p53α. The uncovering that the TP53 gene physiologically expresses several p53 isoforms, challenges this paradigm. According to the data gathered about p53 isoforms in the past ten years, we hypothesise that the so-called p53 is a multi-protein system composed by the twelve isoforms and that a p53-mediated cell response is, in fact, the sum of the intrinsic activities of the co-expressed p53 isoforms. Here, we report that the TP53 gene always co-expresses several p53 protein isoforms in cells, tumour tissues and also in normal human tissues. Physiologically, the HPV E6 protein differentially regulates the expression of all p53 isoforms to command replication. Differentially modulating isoforms expression using siRNAs, without changing extra-cellular signals, alters the cell-fate outcome. Furthermore, we establish that the p53 pathway is still active in cell lines devoid of canonical p53α expression, but retaining expression of other isoforms. Mechanistically, we demonstrate that, depending on post-translational modifications, the isoforms co-expressed in a given cell type, oligomerise together and regulate target-gene transactivation in a promoter-dependent manner. Altogether, this study shows that p53 functions as a multi-protein system that is functional in the absence of canonical p53α. The cellular content in p53 isoforms defines the cell response. The isoforms oligomerise together, depending on post-translational modifications, to finely tune target-gene expression in a promoter-dependent manner. These data indicate that the p53 isoforms would compose a cellular code. Cracking “the p53 isoforms’ code” will, therefore, enable to define cell responses and to develop new therapeutic strategies.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Thèses (Ph.D.)--Université de Sherbrooke (Canada), 2002. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Univ., Diss., 2009--Marburg. / Teilw. publ. in: The EMBO Journal, 2008,27, S. 1563 - 1574.

P53 empêche la réplication d'ADN cellulaire pathologique ainsi que viral en activant la transcription d'effecteurs en aval. L'augmentation d'expression de p53 et sa phosphorylation en réponse à des oncogènes ou à des dommages à l'ADN sont considérées comme déterminant l'activation transcriptionnelle de p53. Les mutations tumorales et les protéines virales convergent fonctionnellement en inactivant p53. Par exemple la protéine cellulaire MDM2 et la protéine adénovirale E1 B-55K ciblent toutes les deux p53 pour sa dégradation. Ceci représente la base du raisonnement de la création des molécules antagonistes de MDM2 et de l'adénovirus oncolytique où E1B-55K a été supprimé, ONYX-015 (Oncorine), comme thérapies anticancéreuses ciblant p53. Pourtant ici, nous montrons que E1B-55K est dispensable pour l'inactivation de p53 et nous révélons un mécanisme épigénétique nouveau et dominant qui inactive l'activité de p53, quel que soit le niveau et la phosphorylation de p53. En utilisant une approche génétique, nous dévoilons qu'E4-ORF3, une autre protéine adénovirale, est le point de nucléation de domaines d'hétérochromatine, menant à l'inactivation des promoteurs cibles de p53, en empêchant sa liaison et donc son activation transcriptionnelle. De plus, nous montrons qu'E4-ORF3 forme un échafaudage nucléaire qui dirige deux métyltransferases, SUV39H1 et SUV39H2, vers ces nouveaux domaines répressifs. Notre étude change la définition fondamentale du mécanisme d'inactivation de p53 dans les cellules infectées par un adénovirus, apportant une nouvelle lumière sur un mécanisme critique qui va maintenant permettre le développement de thérapies adénovirale vraiment sélectives du statut p53 des cellules. / P53 guards against pathological cellular and viral DNA replication by activating the transcription of downstream effectors. The induction of p53 levels and its phosphorylation in response to oncogenes and DNA damage is thought to determine p53 transcriptional activation. Tumor mutations and viral proteins functionally converge in inactivating p53. For example the cellular protein MDM2 and the adenoviral protein E1B-55k both target p53 for its degradation. This is the premise for MDM2 antagonists and the E1B-55k deleted oncolytic adenovirus, ONYX-015 (Oncorine) as p53-targeted cancer therapies. However, here we show that E1B-55k is dispensable for p53 inactivation and reveal a novel and dominant epigenetic mechanism that silences p53 activity, irrespective of p53 level and phosphorylation. Using a genetic approach, we reveal that E4-ORF3, another adenoviral protein, nucleates heterochromatin domain, leading to the silencing of p53 target promot er by preventing its binding and subsequent transcriptional activation. Moreover we show that E4-ORF3 forms a novel nuclear scaffold that directs two methyltransferases, SUV39H1 and SUV39H2, to this newly formed repressive domains. Our study changes the fundamental definition of how p53 is inactivated in adenovirus infected cells, and provides a critical mechanistic insight that could now enable the rational development of true p53 tumor selective adenoviral therapies.

L’altération génétique et/ou fonctionnelle de p53 est très répondue dans les cancers humains et est répertoriée dans plus d’un cas sur deux. Les traitements et molécules utilisés en chimiothérapie anticancéreuse induisent pour la plupart l’apoptose dépendante de p53 ce qui confère une résistance particulière aux tumeurs p53 déficientes. Nous avons développé des techniques se basant sur la vidéomicroscopie à haut débit et l’utilisation de cellules fluorescentesTP53+/+ et TP53-/- pour mettre en évidence des agents chimiques qui ciblent les cellules p53 déficientes. Nous avons identifié SP600125, un inhibiteur de kinases dont MPS1, Aurora A et Aurora B, et qui tue préférentiellement les cellules tumorales TP53-/- . Cette cytotoxicité sélective a été confirmée sur de nombreux modèles de cellules déficientes en p53 in vitro et in vivo sur des xénogreffes TP53+/+ et TP53-/- injectés à des souris nudes. Nous avons utilisé une autre molécule qui a un spectre d’inhibition semblable à SP600125, la reversine, et nous avons aussi trouvé qu’elle a une cytotoxicité sélective envers les cellules p53 déficientes.L’analyse vidéomicroscopique des cellules traitées nous révèle que la mort préférentielle des cellules P53 déficientes est intimement liée à un mécanisme de polyploïdisation. En effet, les cellules TP53-/- (contrairement à celles TP53+/+) traitées effectuent des mitoses aberrantes sans karyokinèse ni cytokinèse qui ne sont pas contrôlées par un arrêt du cycle cellulaire. Ces cellules succombent par catastrophe mitotique après activation de la voie mitochondriale de l’apoptose.Cette observation concorde avec le fait que l’inhibition des protéines anti-apoptotiques de la famille Bcl-2 sensibilise les cellules traitées alors que l’inhibition des protéines BAX, APAF-1 et les caspases protège les cellules TP53-/- de l’effet cytotoxique de SP600125 et la reversine.Ces résultats nous permettent d’envisager ces drogues (ou dérivées) dans la prévention des cas de tumeurs pré malignes et/ou dans le traitement des cas de cancers p53 déficients. / The genetic and/or functional alterations of p53 are highly prevalent in cancer and are reported for more than a half of all human cancers. Classic chemotherapy leads p53 mediated apoptosis conferring a drug resistance for p53 deficient cells. We developed in the laboratory a technique based on high-content videomicroscopy and fluorescent TP53+/+ and TP53-/- cells for the screening of molecules that targets p53 deficient cells. We discovered that SP600125, a kinase inhibitor, including MPS1, Aurora A and Aurora B, kills p53-deficient cells more efficiently than their p53-proficient counterparts. This selective cytotoxicity was confirmed in vivo in mice carrying p53-deficient and -proficient human xenografts. Than after we used an another inhibitor with a similar broad-spectrum kinase, reversine, and we found that this molecule have a selective toxicity for TP53-/- cells and this result was confirmed in vitro for both molecule.Videomicroscopy-based cell fate profiling revealed that the p53-deficient cell death is coupled to hyperploïdy mechanism. Indeed, TP53-/- (but not TP53+/+) undergo successive round of abortive mitosis and failed to arrest the cell cycle in response to treatment and cells became polyploidy and progressively succumbed to mitochondrial apoptosis. In line with this notion, the depletion of anti-apoptotic proteins of the BCL-2 family sensitized TP53-/- cells to the toxic effects of SP600125 and reversine. Moreover, the knockdown of BAX or APAF-1, as well as the chemical inhibition of caspases, limited the death of TP53-/- cells.Hence, SP600125 or reversine (and its analogues/derivatives) might be used for cancer chemoprevention (for eliminating pre-malignant cells that have inactivated p53) or chemotherapy of p53-deficient cancers.

O linfoma de Hodgkin à uma neoplasia com elevados Ãndices de sobrevida global e sobrevida livre de doenÃa e seu comportamento e bases moleculares tÃm sido estudados. Sabe-se que a p53 à uma proteÃna que controla a progressÃo do ciclo celular e mutaÃÃes do gene TP53 prolongam a meia-vida da proteÃna alterando os mecanismos de apoptose e o controle da proliferaÃÃo celular. Cerca de 50% dos tumores humanos tÃm mutaÃÃes no TP53 e acredita-se que nos outros restantes a via de sinalizaÃÃo da p53 esteja comprometida por outros mecanismos. No linfoma de Hodgkin tem sido encontrada expressÃo de p53 e a detecÃÃo de gene TP53 mutado à rara. Ainda nÃo està definido se a p53 serve como marcador prognÃstico no linfoma de Hodgkin. NÃo hà estudos sobre a expressÃo de p53 em linfoma de Hodgkin na populaÃÃo do estado do CearÃ. Os receptores de estrogÃnio(RE) jà foram detectados por imuno-histoquÃmica em cÃlulas de medula Ãssea normal, alÃm de outros tecidos nÃo hematopoiÃticos. Hà evidÃncia de que os estrogÃnios tÃm um papel na diferenciaÃÃo dos linfÃcitos B e regulaÃÃo da apoptose.Sendo o linfoma de Hodgkin uma neoplasia de origem na cÃlula B, onde a resposta imune parece contribuir na sua patogenia, torna-se de particular interesse avaliar o papel do estrogÃnio nesse processo. Ainda nÃo sabemos qual a expressÃo de receptores de estogÃnio nos linfomas e qual sua influÃncia no comportamento dessa neoplasia. Neste trabalho estudamos a expressÃo de p53 e receptor de estrogÃnio em linfoma de Hodgkin no CearÃ. Foram estudados 39 casos de linfoma de Hodgkin confirmado por exame histo-patolÃgico e por imuno-histoquÃmica e um grupo controle de 10 casos de linfonodos com hiperplasia reativa, nÃo relacionados a linfoma. Foi feito estudo imuno-histoquÃmico com marcadores para p53 e REα. A idade dos pacientes variou entre 5 e 63 anos, 21% foram do sexo masculino, a apresentaÃÃo inicial da doenÃa foi em linfonodo cervical em 59% dos casos e 56% dos indivÃduos apresentavam sintomas B. Quanto ao subtipo histolÃgico, 56% dos casos eram de linfoma de Hodgkin(LH) clÃssico esclerose nodular, 27% celularidade mista, 14% rico em linfÃcitos e 3% eram LH predominÃncia linfocitÃria. NÃo houve casos de depleÃÃo linfocitÃria na amostra estudada. A p53 foi positiva em 53% dos casos e no grupo controle em 10%. O RE foi negativo nos casos de LH, com marcaÃÃo persistente dos neutrÃfilos (citoplasma) e marcou em 80% dos casos, em cÃlulas histiocitÃrias, com marcaÃÃo para-nuclear (golgiana) e difusa citoplasmÃtica (fraca). ConcluÃmos que a expressÃo de p53 ocorre em mais de 50% dos casos de LH, conforme jà relatado na literatura e que hà expressÃo de REα citoplasmÃtica em tecido linfÃide, mas nÃo no LH. Hà expressiva marcaÃÃo no citoplasma dos neutrÃfilos. Outros estudos sÃo necessÃrios para avaliar a expressÃo do REβ e seu papel na patogÃnese do LH. / Hodgkinâs lymphoma is a entity where free disease and overal survive is prolonged by treatment and its molecular basis have been studied. In previus literature is described p53 as a protein that controls progress of celular cicle and TP 53 gene mutations prolong half-life of protein, modifing the mechanisms of apoptosis and regulation of celular proliferation. About of 50% of human cancers have TP53 mutations and the investigators believe that in the others the signaling mechanisms of p53 are altered by others ways. Hodgkinâs lymphoma cells stain for p53 frequently, but detection for TP53 gene mutation is rare. Itâs not be well stabilished if p53 is a prognostic factor in Hodgkinâs lymphoma. There is no trial about p53 expression on Hodgkinâs lymphoma in Cearà state. Estrogen receptor were detected by imuno-histochemical analysis in normal bone marrow cells and others non-hematopoietic tissues. There is evidence that estrogen have a role on differentiation of B-cells and on regulation of apoptosis. If is Hodgkinâs lymphoma(HL) a B-cell neoplasm, with immune response seems to contribute in its pathogeny, its interesting to study the role of estrogen on Hodgkinâs lymphoma and its influence on this neoplasm behavior. We studied p53 and estrogen receptor expression in Hodgkinâs lymphoma on Cearà state. Were studied 39 cases of Hodgkinâs lymphoma confirmed by histo-pathological studies and by immuno-histochemical analysis and a control group with 10 cases of benign linfonodal hyperplasia. The simples were stained with p53 and estrogen receptor(ER) α anti-bodies. The age of patitens varied between 5 and 63 years, 21% were males, the initial presentation of disease was cervical adenopathy on 59% of cases and B simptoms were present on 56% of patients. On classification by histological subtipes, 56% were classical HL nodular sclerosis, 27% mixed cellularity, 14% lymphocyte-rich and 3% were HL linfocyte predominance. In this sample, lymphocyte-depleted subtype was not found. Positive staining for p53 occurred on 53% of cases and 10% of control group. Estrogen receptor was negative in all cases of Hodgkinâs lymphoma, but showed positive stainning in neutrophilic cells. In control group was founded ER positivity in 80% of cases, in histiocytic cells, with para-nuclear stainning and cytoplasmic diffuse. In conclusion, p53 expression occurs in more than 50% of Hodgkinâs lymphoma cases, as described previously, and there is expression of ERα in lymphoid tissue, but not on HL. There is persistent positivity in neutrophil cytoplasma. Another investigations are necessary to avaliate ERα and ERβ on RS cells and their role on the pathogenesis of Hodgkinâs lymphoma.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Kinesiology and Health Science. / Typescript. Includes bibliographical references (leaves 71-78). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38823

The TP53 gene can express protein p53 and 11 another isoform proteins N- and/or C-terminally truncated by using two promoters and alternative splicing. The p53 isoforms are found in both healthy and tumorous tissues, and are intensively studied in relation to cancer diagnosis, prognosis and treatment. In this work, the p53 isoforms were subcloned into expression vectors by LR reaction adapted from Gateway cloning system. The expression vectors were designed for protein production by bacteria E. coli strain BL-21. The constructs containing p53 isoforms were encoded together with two fusion proteins, glutathione-S-transferase and polyhistidine tag under the control of the same promotor for the affinity chromatography protein isolation. All the clones underwent Sanger sequencing for verification after homologous recombination. Sequencing confirmed the accuracy of the subcloned isoforms p53, 133p53, 160p53, p53 and 160p53 into an expression vector pDEST15-N6xHis-GST-GW-DEST. Protein 160p53 was expressed in BL-21 and isolated using both HIS and GST tag interacion. Isolation using HIS tag yielded in a higher protein concentration then the isolation mediated by the interaction of the glutathione-S-transferase.

L’anémie de Blackfan-Diamond (ABD) est une érythroblastopénie congénitale rare, secondaire à un blocage de la maturation érythroïde entre les stades BFU-e et CFU-e. L’ABD est le plus souvent la conséquence d’une mutation germinale affectant un gène codant pour une protéine ribosomique (RP) de la petite ou de la grande sous-unité du ribosome. Quatorze gènes distincts ont été identifiés. Les gènes les plus fréquemment mutés sont les gènes RPL5, RPL11 et RPS19 (37% des patients). Plus rarement, l’ABD est la conséquence de mutations dans le gène TSR2 ou dans le gène GATA-1. Ce dernier code pour un facteur de transcription majeur de l’érythropoïèse. Chez les patients ABD, les mutations de GATA-1 induisent une perte quasi-totale de la forme longue de GATA-1 qui est nécessaire à la différenciation de la cellule érythroïde. Notre groupe a identifié deux phénotypes de l’ABD in vitro en fonction du gène muté. En cas d’haploinsuffisance RPS19, la prolifération érythroïde est moins réduite qu’en cas d’haploinsuffisance RPL5 ou de RPL11. Une haploinsuffisance RPS19 n’altère pas la différenciation érythroïde et n’induit pas d’apoptose contrairement à l’haploinsuffisance RPL5 ou RPL11 où il existe un retard de différenciation érythroïde et un excès net d’apoptose responsable au moins en partie de la diminution drastique de la prolifération érythroïde dans ces phénotypes.HSP70 est impliquée dans la survie cellulaire et la différenciation érythroïde en protégeant GATA-1 du clivage par la caspase-3, une protéase activée lors de la différenciation érythroïde terminale. Comme la différence entre les deux phénotypes d’ABD in vitro concernait la différenciation érythroïde et la survie cellulaire, nous avons émis l’hypothèse selon laquelle la mutation de certains gènes RP provoque un défaut d’expression d’HSP70 conduisant au blocage de la différenciation érythroïde et à l’excès d’apoptose retrouvés dans les phénotypes sévères d’ABD.Nous avons étudié différents patients atteints d’ABD, porteurs de mutations dans les gènes RPS19, RPL5 ou RPL11 et généré un modèle in vitro d’ABD en exprimant, dans des cellules CD34+ humaines issues de sang de cordon, des ARN interférents ciblant RPL5, RPL11 ou RPS19. Chez les patients comme dans le modèle reproduisant l’ABD, l’haploinsuffisance RPL5 ou RPL11 diminue drastiquement l’expression protéique de HSP70 et de GATA-1 (Western blot, microscopie confocale et en cytométrie couplée à des techniques d’imagerie, (technologie ImageStream) à la différence de 1’haploinsuffisance RPS19. Dans tous les cas, HSP70 est normalement transcrite et traduite. Les inhibiteurs du protéasome (MG132, lactacystine, bortezomib) restaurent l’expression10de HSP70. La diminution d’expression de HSP70 est donc liée à une dégradation protéasomale. L’invalidation de RPL11 induit une polyubiquitinylation importante de HSP70. La transduction lentivirale de l’ADN complémentaire d’HSP70 dans les cellules primitives invalidées pour RPL11 permet de restaurer l’expression de HSP70 et de GATA-1 à un niveau similaire aux contrôles et de rétablir la prolifération cellulaire et la différenciation érythroïde, confirmant le rôle clé de HSP70 dans le phénotype sévère RPL5+/Mut ou RPL11+/Mut. Les formes les plus sévères de l’ABD sont associées à la dégradation de HSP70 par le protéasome. La perte de la protéine chaperone de GATA-1 induit la perte de GATA-1, facteur de transcription majeur de la différenciation érythroïde. Une augmentation de l’expression de HSP70 pourrait ainsi constituer une nouvelle approche thérapeutique dans l’ABD. / Diamond-Blackfan anemia (DBA) is the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA-1 genes have been identified in a few DBA families. The GATA-1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the eytrhoid differentiation and the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA-1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA-1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation. As such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA-1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of12HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of depleted RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation, and reduced apoptosis, confirming a critical role for HSP70 in the erythroid defect in the RPL11+/Mut DBA phenotypes. The loss of HSP70 may explain the loss of GATA-1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder.