Biomedical subjects

Jean-Christophe Bourdon

Publications and source records attributed to Jean-Christophe Bourdon.

8 recordsLinked to original sources

PubMed2007

Expression of p53 isoforms in squamous cell carcinoma of the head and neck.

Recent data indicate that, similar to p63 and p73, several different p53 isoforms can be produced in humans through alternative initiation of translation, usage of an internal promoter and alternative splicing. These isoforms are reported to have varying functions and expressions. In squamous cell carcinoma of the head and neck (SCCHN), disruption of the p53 pathway is one of the most common genetic alterations. However, to our knowledge, no studies regarding the expression of different p53 isoforms in SCCHN have so far been performed. We screened for the expression of different p53 isoforms in SCCHN and clinically normal oral epithelia using nested RT-PCR. p53 mRNA was expressed in all tumours, all matched clinically normal tissue adjacent to the tumour and in buccal mucosa from healthy volunteers. Of the novel isoforms, p53beta was detected in the majority of samples analysed, and all of the recently described isoforms were also detected in at least some tumour and normal epithelium samples, with the exception of Deltap53 isoforms. We conclude that p53 variant mRNAs are expressed in both normal oral stratified epithelium and SCCHN. Improvements in methodologies and reagents to detect and quantify p53 isoform expression in clinical material will be required to correlate p53 status with clinical outcomes.

Carcinoma, Squamous Cell↗

PubMed2006

A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia.

PURPOSE: The antioncogene protein product p53 has not been studied previously in cancer patients during in vivo chemotherapy. This study examined the early p53 protein and gene expression during induction chemotherapy in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: Leukemic cells were collected from five AML patients during their first 18 hours of induction chemotherapy and examined for p53 protein and gene expression by one- and two-dimensional gel immunoblot and high-density gene expression arrays. RESULTS: Up-regulation of p53 protein expression was detected in AML patients posttreatment in vivo. One- and two-dimensional gel immunoblots showed two main forms of p53, denominated alpha p53 and delta p53, both recognized by various NH2-terminal directed antibodies. As a response to treatment, we detected rapid accumulation of alpha p53, with significantly altered protein expression levels already after 2 hours. The accumulation of alpha p53 was accompanied by increased transcription of putative p53 target genes and subsequent cytopenia in the patients. CONCLUSION: Up-regulation of the p53 protein and target genes seems to be a prominent feature in induction chemotherapy of AML. The rapid shift from a shorter p53 protein form (delta) toward the full-length protein (alpha) underscores the complexity of p53 protein modulation in patients undergoing chemotherapy.

Acute Disease↗

PubMed2006

Tumor susceptibility and apoptosis defect in a mouse strain expressing a human p53 transgene.

Activation of apoptosis is believed to be critical for the role of p53 as a tumor suppressor. Here, we report a new mouse strain carrying a human p53 transgene in the mouse p53-null background. Expression of human p53 in these mice was comparable with wild-type murine p53; however, transactivation, induction of apoptosis, and G(1)-S checkpoint, but not transrepression or regulation of a centrosomal checkpoint, were deregulated. Although multiple functions of p53 were abrogated, mice carrying the human p53 transgene did not show early onset of tumors as typically seen for p53-null mice. In contrast, human p53 in the p53-null background did not prevent accelerated tumor development after genotoxic or oncogenic stress. Such behavior of human p53 expressed at physiologic levels in transgenic cells could be explained by unexpectedly high binding with Mdm2. By using Nutlin-3a, an inhibitor of the interaction between Mdm2 and p53, we were able to partially reconstitute p53 transactivation and apoptosis in transgenic cells. Our findings indicate that the interaction between p53 and Mdm2 controls p53 transcriptional activity in homeostatic tissues and regulates DNA damage- and oncogene-induced, but not spontaneous, tumorigenesis.

Animals↗

PubMed2005

p53 isoforms can regulate p53 transcriptional activity.

The recently discovered p53-related genes, p73 and p63, express multiple splice variants and N-terminally truncated forms initiated from an alternative promoter in intron 3. To date, no alternative promoter and multiple splice variants have been described for the p53 gene. In this study, we show that p53 has a gene structure similar to the p73 and p63 genes. The human p53 gene contains an alternative promoter and transcribes multiple splice variants. We show that p53 variants are expressed in normal human tissue in a tissue-dependent manner. We determine that the alternative promoter is conserved through evolution from Drosophila to man, suggesting that the p53 family gene structure plays an essential role in the multiple activities of the p53 family members. Consistent with this hypothesis, p53 variants are differentially expressed in human breast tumors compared with normal breast tissue. We establish that p53beta can bind differentially to promoters and can enhance p53 target gene expression in a promoter-dependent manner, while Delta133p53 is dominant-negative toward full-length p53, inhibiting p53-mediated apoptosis. The differential expression of the p53 isoforms in human tumors may explain the difficulties in linking p53 status to the biological properties and drug sensitivity of human cancer.

Alternative Splicing↗

PubMed2005

The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor.

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters and that endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-kappaB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity.

Apoptosis↗

PubMed2004

Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo.

p53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNA-mediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity.

Cell Line, Tumor↗

PubMed2004

Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.

The only reported role for the conjugation of the NEDD8 ubiquitin-like molecule is control of the activity of SCF ubiquitin ligase complexes. Here, we show that the Mdm2 RING finger E3 ubiquitin ligase can also promote NEDD8 modification of the p53 tumor suppressor protein. Mdm2 is itself modified with NEDD8 with very similar characteristics to the autoubiquitination activity of Mdm2. By using a cell line (TS-41) with a temperature-sensitive mutation in the NEDD8 conjugation pathway and a p53 mutant that cannot be NEDDylated (3NKR), we demonstrate that Mdm2-dependent NEDD8 modification of p53 inhibits its transcriptional activity. These findings expand the role for Mdm2 as an E3 ligase, providing evidence that Mdm2 is a common component of the ubiquitin and NEDD8 conjugation pathway and indicating the diverse mechanisms by which E3 ligases can control the function of substrate proteins.

Animals↗

PubMed2003

Primary cultured neurons devoid of cellular prion display lower responsiveness to staurosporine through the control of p53 at both transcriptional and post-transcriptional levels.

We assessed the contribution of the cellular prion protein (PrPc) in the control of neuronal apoptosis by examining cell death in both human cells and murine primary cultured neurons. We first confirmed our previous finding that staurosporine-induced caspase activation is increased by PrPc overexpression in HEK293 cells. We show here that this phenotype is fully dependent on p53 and that the control of p53 activity by PrPc occurs at both transcriptional and post-transcriptional levels in human cells. Of most interest, we demonstrate that neuronal endogenous PrPc also controls a p53-dependent pro-apoptotic phenotype. Thus, DNA fragmentation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling)-positive cells were lower in primary cultured neurons derived from Zrch-1 mice embryos in which PrPc has been abrogated than in wild-type neurons. PrPc knock-out neurons also displayed drastically diminished caspase-3-like activity and immunoreactivity together with reduced p53 expression and transcriptional activity, a phenotype complemented in part by PrPc transfection. Interestingly, p53 expression was also reduced in the brain of adult Prnp-/- mice. Neuronal PrPc likely controls p53 at a post-transcriptional level because the deletion of cellular prion protein is accompanied by a higher Mdm2-like immunoreactivity and reduced phosphorylated p38 MAPK expression. We therefore propose that the physiological function of endogenous cellular prion could be to regulate p53-dependent caspase-3-mediated neuronal cell death. This phenotype likely occurs through up-regulation of p53 promoter transactivation as well as downstream by controlling p53 stability via Mdm2 expression.

Animals↗