The Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) acts as a co-activator of EBNA-2, a transcriptional activator essential for Epstein-Barr virus (EBV)-induced B-cell transformation. Burkitt's lymphoma (BL) cells harboring a mutant EBV strain that lacks both the EBNA-2 gene and 3 exons of EBNA-LP express Y1Y2-truncated isoforms of EBNA-LP (tEBNA-LP) and better resist apoptosis than if infected with the wild-type virus. In such BL cells, tEBNA-LP interacts with the protein phosphatase 2A (PP2A) catalytic subunit (PP2A C), and this interaction likely plays a role in resistance to apoptosis. Here, 28 cellular and four viral proteins have been identified by mass spectrometry as further possible interactors of tEBNA-LP. Three interactions were confirmed by immunoprecipitation and Western blotting, namely with the A structural subunit of PP2A (PP2A A), the structure-specific recognition protein 1 (SSRP1, a component of the facilitate chromatin transcription (FACT) complex), and a new form of the transcription factor EC (TFEC). Thus, tEBNA-LP appears to be involved not only in cell resistance to apoptosis through its interaction with two PP2A subunits, but also in other processes where its ability to co-activate transcriptional regulators could be important.

Objectives Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with white patients. In this study, we aimed to identify genetic factors that may account for this difference. Methods We analyzed immunoglobulin heavy chain (IGH) genes' mutational status by performing next-generation sequencing in 25 Senegalese and 50 Italian patients with CLL. Results We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series. Conclusions The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background.

The incidence of CLL in Africa is not known for several reasons, among them the difficulty to access the diagnosis since there is only clinical features and cytology which are available in several countries in francophone Africa, especially in Senegal. The WHO criteria which have been published since 2001 from the first revised WHO classification of tumours of haematopoitic and lymphoid tissues cannot be applied since there is no facility for immunophenotyping and cytogenetic. Moreover, no research in place can be set up without any precise diagnosis. In this study, we have developed the flow cytometry technique in the laboratory of Immunology in CHU Le Dantec in Dakar to obtain the immunophenotype looking at the expression of Kappa/lambda light chains, CD19, CD22, CD20, FMC7, CD5, CD10, CD23, CD38. Peripheral blood smears were fixed according to the technique for FISH to detect the following cytogenetic abnormalities: trisomy12, deletion 13q14, deletion 11q22-23, deletion 17p. RNA to test by qRT-PCR the expression of microRNA mir15a, mir16-1, mir181a, mir181b, and mir 34a and 34b was obtained from peripheral blood lymphocytes. Twelve cases of CCL aged ranging from 50 to 80 year old were characterized at clinical level according to the Binet’s system: 1 stage A, 5 stage B, 6 stages C. In most of cases (9 cases) hyperlymphocytosis of small lymphocytes with clumped chromatin and scanty cytoplasm was very high (122.000 to 336 000/mm3). The “Matutes” score obtained by immunophenotyping was 5 or 4 for 11 patients in favor of typical CLL. The expression of CD38 which is a marker of unfavorable prognosis is positive in 9 patients. Nine patients had cytogenetic abnormalities: 3 trisomy 12, 6 del 13q14, 2 del 11q22-23, 1 del17p. Four patients had two simultaneous cytogenetic abnormalities (tri 12, tri 13 ; del13q14, del11q22-23 ; del13q24, del17p ; del13q24, tri12). The analysis of mi-RNA showed high expression of mir15a and mir 16-1 and low value of mir181a and mir 181b which were described in aggressive CLL ; however the expression of mir 34a and mir34b was also high. This study shows the aggressiveness of CLL in Senegal, probably due to the delay of the diagnosis. We have also demonstrated the possibility to set up immunophenotype and the possibility of a precise diagnosis of lymphoproliferative disorder in place, in western Africa and the series will be extended. Moreover we have also demonstrated the possibility to construct translational research project from samples characterized in the University Hospital in Dakar gathering the cases from different sanitary structures in the country. Such experiment would be a model for epidemiologic, clinico-biologic and translational research studies in order to set up in the country the capacity building for diagnosis and research. Disclosures: No relevant conflicts of interest to declare.

Recent data showed that p53 stimulates the expression of genes encoding not only pro-but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

PURPOSE:Gamma-irradiation leads to activation of p53 tumour suppressor gene and to p53-dependant stimulation of a large panel of cellular genes including proapoptotic genes involved in intrinsic and extrinsic pathways. Most in vivo published data referred to high (lethal) irradiation doses. The present study was performed to analyse the p53-dependent response to more relevant low irradiation doses.MATERIALS AND METHODS:Mice were whole body exposed to irradiation doses decreasing from 5 - 0.05 Gy. Gene expression was estimated by real time reverse transcriptase polymerase chain reaction measurements on RNA extracted from thymus and spleen. Apoptosis was evaluated by the percentage of either annexin V positive or sub-G1 cells.RESULTS:A 0.1 Gy irradiation dose already gives a significant stimulation of Puma (p53 up-regulated modulator of apoptosis), and 0.2 Gy of Bax (Bcl-2-associated X protein) and Killer/DR5 (Death Receptor 5). The expression of genes involved in the two apoptotic pathways was induced as soon as 1 h post-irradiation and reached a maximum at 3 h, the induction level depending on both the gene and the organ. A significant increase in the number of apoptotic cells is already detectable at 0.5 Gy with a maximum of induction at 6 h.CONCLUSIONS:Our results reveal the high in vivo sensitivity of p53-dependent transcriptional activation of genes involved in the two main apoptotic pathways, their stimulation preceding the induction of apoptosis.

The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53 beta isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122-2137]. Our results show, for the first time, that the p53 beta isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

We investigated the role of tumor suppressor p53 and Fas (CD95/APO-1), a member of the tumor necrosis factor receptor family, in neural progenitors response to γ-irradiation exposure. Telencephalic cells were obtained from wild-type C57Bl/6, or p53−/− or fas−/−, 15-day-old mouse embryos. They were cultured in conditions allowing neural progenitors to form proliferating clusters (neurospheres). A 2 Gy γ-irradiation induced a G1 cell cycle arrest and triggered apoptosis in wild-type neural progenitor cultures in correlation with an enhanced expression of p53 and of its downstream target p21WAF1, both of them acquiring a nuclear localization. These effects did not occur in p53−/− neural progenitors demonstrating the central role played by p53 in their response to ionizing radiation. Furthermore, the monoclonal antibody Jo2 directed against Fas induced apoptosis of wild type but not of fas−/− neural progenitors, indicating the existence of a functional Fas signaling pathway in neural progenitors. Ionizing radiation induced an increase of Fas membrane expression related to a p53-dependent increase of fas mRNA expression in wild-type neural progenitors. Moreover, fas−/− neural progenitors exhibited delayed radiation-induced apoptosis compared to wild-type cells. Therefore, these findings establish a role for Fas/CD95 related to p53 in the response of neural progenitors to γ-radiation exposure. Similar mechanisms could be triggered in neural progenitors in case of different stresses during brain development or in the course of various diseases affecting the adult brain.

The p53 tumor suppressor gene belongs to a multigene family that includes two paralogues, p63 and p73. p73alpha has common activities with p53, such as DNA binding and transactivation, and can thus activate the transcription of p53-responsive genes. Using the adenoviral system, we report that an overexpression of either wt-p73alpha or one of the two transcriptional inactive mutants, deltaNp73alpha or p73alphaR292H, induces an accumulation of the endogenous wt-p53 expressed in the three transformed cell lines, SK-N-SH, MCF-7 and U-2OS, without stimulating the p53 gene transcription. p73-mediated accumulation of p53 protein coincides with an increase of p53-target gene expression in cells expressing either wt-p73alpha or the transcriptional inactive mutant p73alphaR292H, but not deltaNp73alpha that encodes a dominant-negative mutant of both p73 and p53. The fact that an ectopic expression of p73alphaR292H leads to both accumulation of p53 and stimulation of p53 target gene expression strongly suggests that p73alpha is able to induce activation of p53. This was confirmed by showing that p73alphaR292H no longer stimulated Waf1/p21 expression in MCF7/R-A1 cells that expressed a transcriptional inactive mutant of p53. We thus conclude that p73alpha protein was able to both stabilize and activate wt-p53 protein, independent of the p73alpha transcriptional activity.

Le gene suppresseur de tumeur p53 code pour une proteine dont la fonction principale est de proteger l'organisme contre la proliferation de cellules potentiellement tumorigenes. Dans des conditions normales (cellules non stressees), la proteine p53 est inactive ; elle est maintenue a un faible niveau par son association avec l'oncoproteine Mdm2, qui provoque son transport du noyau vers le cytoplasme et sa degradation par la voie ubiquitine/proteasome. En reponse a un stress, p53 subit une serie de modifications post-traductionnelles dont la nature depend du stress. Ces modifications conduisent a son accumulation dans le noyau et son activation comme facteur de transcription. Dans le cas des radiations ionisantes, l'activation de p53 implique les kinases ATM, ATR, Chk2 et Chk1. Ces kinases phosphorylent l'extremite N-terminale sur les serines 15 (ATM et/ou ATR) et 20 (Chk2 et/ou Chkl). Ces phosphorylations permettent la dissociation du complexe p53/Mdm2 et donc la stabilisation de p53 et le recrutement des histones acetyl-transferase CBP/p300 et P/CAF. La fixation de CBP/p300 et P/CAF a l'extremite N-terminale permet l'acetylation de l'extremite C-terminale. De plus, ATM induit la dephosphorylation de l'extremite C-terminale sur la Ser376, creant ainsi un site de fixation pour les proteines 14-3-3. L'ensemble de ces modifications, phosphorylation, acetylation et association avec des co-facteurs, stimule l'activite transcriptionnelle de p53 avec, pour consequence, l'induction de l'expression de toute une serie de genes impliques, en particulier, dans l'arret du cycle cellulaire et l'apoptose. C'est par ces effets biologiques que p53 protege l'organisme de l'action deletere des radiations ionisantes.

Tumorigenicity and radiosensitivity of related cell lines expressing distinct p53 mutants were analyzed in parallel with key components of the antioxidant metabolic pathway. Six sublines deriving from the same parental cell population and expressing either the mutant p53K130R or p53V270F were investigated. Both mutations abrogate the transcriptional activity of p53 as well as its ability to induce apoptosis. The cells expressing p53K130R showed a higher tumorigenicity and a higher radiosensitivity than those expressing p53V270F. An increase in tumorigenicity was associated with a decrease in manganese-containing superoxide dismutase activity, and with further decreases in the glutathione content and glutathione peroxidase (GPX) activity. A positive correlation was found between GPX activity, glutathione content and cell survival following ionizing irradiation. The fact that sister cell lines exhibit different tumorigenicity and radiosensitivity while expressing a mutant p53 further supports the notion that knowledge of p53 status is not sufficient to predict tumor outcome, especially the response to irradiation. A better understanding of antioxidant defenses might be more informative.

p53 regulates the transcription of a number of genes among which are different redox-related genes. It has been proposed that these genes can induce a cellular oxidative stress leading to p53-dependent apoptosis (Polyak et al., 1997). MnSOD, the product of superoxide dismutase 2 (SOD2) gene, is one of the major cellular defences against oxidative stress. We demonstrate here that p53 is able to repress SOD2 gene expression and that this repression takes place at promoter level. We show the importance of this regulation for the p53 function, by demonstrating that an overexpression of MnSOD decreases p53-mediated induction of apoptosis. Moreover, we demonstrate that MnSOD overexpression decreases p53-gene expression at the promoter level. These findings raise the hypothesis that p53 and SOD2 genes are mutually regulated leading to the modulation of various cellular processes including apoptosis.

The mechanisms by which the p53 tumour suppressor protein would, in vivo, co-ordinate the adaptive response to genotoxic stress is poorly understood. p53 has been shown to transactivate several genes that could be involved in two main cellular responses, growth arrest and apoptosis. To get further insight into the tissue-specific regulation of p53 transcriptional activity, we performed an extensive study looking at the expression of four well characterized p53-responsive genes, before and after γ-irradiation in p53 wild-type (p53+/+) and p53-deficient (p53−/−) mice. The waf1, bax, fas and mdm2 genes were chosen for their different potential roles in the cellular response to stress. Our data demonstrate the strict p53-dependence of mRNA up-regulation for bax, fas and mdm2 in irradiated tissues and confirm such findings for waf1. They further highlight complex levels of regulatory mechanisms that could lead, in vivo, to selective transcriptional activation of genes by p53. In addition, our results provide arguments for the involvement of p53 in the basal mRNA expression of the four genes in some organs. Finally, in situ expression of Bax and p21Waf-1 protein suggests, at least in lymphoid organs, a direct correlation between selective p53-target gene expression and a particular response of a cell to ionising radiation.

p53 protein is a sequence-specific transcriptional activator which induces the expression of a number of cellular genes involved in different metabolic pathways. We report that the computer-selected sequence in human and mouse C-Ha-Ras gene confers to a reporter gene the ability to be directly transactivated by wild-type p53 either overexpressed or activated in response to a cellular stress. By analysing human transformed cell lines, we showed, at both mRNA and protein level, that the endogenous c-Ha-Ras gene expression is positively regulated by wt p53 protein. The stimulation of c-Ha-Ras gene expression in Saos-2Ts cells by a temperature shift down to the permissive temperature for the p53-wt conformation is associated with a significant increase in the activated form of p21c-Ha-Ras protein. Furthermore, in human transformed cell lines, the transient expression of a dominant interfering mutant of c-Ha-Ras greatly reduced the ability of p53 to induce apoptosis and inhibited the p53-dependent transactivation. This is due, at least in part, to a decrease in the protein (but not mRNA) level of the transiently expressed p53, indicating that inactivation of p21c-Ha-Ras signalling pathways led to a specific degradation of p53 protein. We therefore suggest that, by inducing c-Ha-Ras, p53 activates a positive feedback loop that counteracts the negative feedback loop mediated by Mdm2.

While p53 is dispensable for development, an excess of p53 has dramatic consequences on the embryogenesis and on the cell differentiation. In an attempt to analyse in vivo the effects of p53 activity, we have generated transgenic mice expressing the wild-type p53 under the control of the metallothionein I promoter. In the three transgenic lines established, exogenous p53 is expressed constitutively in the postmeiotic cells of transgenic males and two lines are subfertile. Transgenic males expressing the upper level of p53 produce few spermatozoa since the majority of developing spermatids undergo apoptosis. In the subfertile males exhibiting an intermediate amount of p53, teratozoospermia is obvious suggesting an altered terminal differentiation of postmeiotic cells. In contrast lower level of p53 does not lead the third line to sterility. These results suggest that the activity of p53 is dependent in vivo on the amount of p53 present within cells, as it has been already demonstrated in vitro.

Le gène p53 est l'un des plus importants suppresseurs de tumeur puisqu'il est muté dans près de la moitié des cas de cancers humains. L'inactivation de p53 par le changement d'un seul acide aminé contribue au phénotype néoplasique et confère une résistance aux traitements thérapeutiques. p53 est une phosphoprotéine nucléaire capable de fixer une séquence spécifique d'ADN. La protéine p53 existe normalement sous forme de tétramère ou de complexe de tétramères. C'est un activateur transcriptionnel de gènes spécifiques et elle peut aussi exercer une répression transcriptionnelle produite probablement par interaction avec d'autres facteurs de transcription ou avec la machinerie générale de transcription. Quoique p53 ne soit pas essentielle à la croissance normale et au développement, l'un de ses principaux rôles est de contrôler la réponse cellulaire aux dommages causés à l'ADN et à différents types de stress. Les signaux de stress induisent l'accumulation et l'activation biochimique de p53. Il y a deux modes par lesquels p53 inactive ou réduit la prolifération après dommages causés à l'ADN ou après divers autres stress : arrêt du cycle cellulaire et apoptose. Diverses études ont suggéré que l'arrêt de croissance et l'apoptose représentent deux fonctions indépendantes de p53. Le choix entre l'arrêt en G1 et l'apoptose dépend d'un réseau complexe de signaux régulateurs. Les conséquences de l'inactivation fonctionnelle de p53 doivent être considérées dans le contexte du type cellulaire, de l'état de différenciation, de la constitution génétique de la cellule et de l'environnement cellulaire. En dépit de cette complexité, les connaissances de plus en plus approfondies des fonctions de p53 offrent une nouvelle vision sur le mécanisme du développement néoplasique dans des situations spécifiques. Son utilisation a dès à présent un impact sur le développement de stratégies plus efficaces pour traiter le cancer.

Les petits virus a ADN causant des tumeurs tels que le virus simien 40 (SV40), des papillomavirus humains (HPV) et les adenovirus (Ad) dependent de la cellule hote pour la production de virus. Leurs interactions avec le systeme de croissance cellulaire apportent des informations fondamentales, d'une part sur la facon dont les proteines transformantes (« oncoproteines ») de ces virus participent a la transformation cellulaire et d'autre part sur les fonctions des genes suppresseurs de tumeur p53 et RB1.