p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

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.

Expression of C-terminal deleted p53 isoforms in neuroblastoma.

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 p53beta 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 p53beta 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.

New Interactors of the Truncated EBNA-LP Protein Identified by Mass Spectrometry in P3HR1 Burkitt's Lymphoma Cells.

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.

Preferential Usage of Specific Immunoglobulin Heavy Chain Variable Region Genes With Unmutated Profile and Advanced Stage at Presentation Are Common Features in Patients With Chronic Lymphocytic Leukemia From Senegal.

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.

A comprehensive study of p53 transcriptional activity in thymus and spleen of gamma irradiated mouse: high sensitivity of genes involved in the two main apoptotic pathways.

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.

p73alpha expression induces both accumulation and activation of wt-p53 independent of the p73alpha transcriptional activity.

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.

Involvement of p53 and Fas/CD95 in murine neural progenitor cell response to ionizing irradiation.

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 gamma-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 gamma-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 p21(WAF1), 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 gamma-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 neurogene BTG2TIS21/PC3 is transactivated by DeltaNp73alpha via p53 specifically in neuroblastoma cells.

The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of DeltaNp73alpha, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, DeltaNp73alpha inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the DeltaNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by DeltaNp73alpha depends on both p53 status (as it is not observed in a p53-/- neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that DeltaNp73alpha may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete DeltaNp73alpha C-terminus sequence as it is not observed with DeltaNp73beta, which lacks most of the C-terminal domain. We have previously shown that DeltaNp73alpha is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that DeltaNp73alpha not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.

Correlation between antioxidant status, tumorigenicity and radiosensitivity in sister rat cell lines.

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.

Homogeneous time-resolved fluorescence assay for identifying p53 interactions with its protein partners, directly in a cellular extract.

The p53 protein is a tumor suppressor that protects the organism against malignant consequences of DNA damage. Interaction of p53 with numerous cellular or viral proteins regulates its functional activity either positively or negatively. An approach leading to identification of such protein interactions directly in a cell extract could be of help in the development of screening assays to search for drugs acting on p53 in its cellular environment, either by disrupting its association with inhibitory proteins or by increasing its affinity for activating proteins. We show that the homogeneous time-resolved fluorescence (HTRF) assay based on the time-resolved amplified cryptate emission (TRACE) technology allows identification of such an interaction by simply adding a mixture of two labeled monoclonal antibodies, directly in a cellular extract. We validate this assay by studying p53/SV40-LTAg interactions. The antibodies directed against genuine p53 and SV40-LTAg epitopes were labeled with europium cryptate (donor) and XL665, a crosslinked allophycocyanin (acceptor), respectively. We demonstrated that a nonradiative energy transfer occurs between labeled antibodies only when p53 interacts with SV40-LTag, which opens up the possibility of extending this approach to other p53 partners to search for drugs that restore p53 tumor-suppressor activity.