David Goldschneider

ΔN-p73α Accumulates in Human Neuroblastic Tumors

Neuroblastic tumors (NTs), occurring in early childhood, display a wide spectrum of differentiation. Recurrent deletions involving the p73 locus are frequently observed in undifferentiated NTs. To address the question of the possible implication of p73 in neuroblastic differentiation, we investigated the status of the expression of this gene in a panel of differentiated and undifferentiated tumors. Although mutations were not found, p73 transcript profiles differed between undifferentiated and differentiated tumors. The frequency of the transcripts lacking exon 2 (species 1-3) appeared to be higher in undifferentiated than in differentiating and differentiated NTs. In contrast, products from using an alternate promoter (DeltaN-p73) were present in all NTs. In addition, only DeltaN-p73, but not full-length proteins, were detected by immunoblotting, suggesting a greater stability of N-truncated isoforms. Importantly, as in the adrenal medulla, most NTs showed p73-positive immunohistological staining with a cellular distribution and intensity varying according to the neuronal differentiation. Surprisingly, we observed redistribution of p73 from the nucleus to the cytoplasm during neuroblastic differentiation. Our data suggest that, in undifferentiated NTs, a link may exist between the accumulation of DeltaN-p73alpha variants and the nuclear exclusion of p53.

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 p53β 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 p53β 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.

MYCN enhances P-gp/MDR1 gene expression in the human metastatic neuroblastoma IGR-N-91 model.

Despite intensive high-dose chemotherapy and autologous hematopoietic stem cell transplantation, disseminated neuroblastoma (NB) frequently proves to be chemosensitive but not chemocurable, and more often so in NB-presenting MYCN amplification. To assess the direct relationship between the MYCN oncogene and chemoresistance acquisition during NB metastatic dissemination, we have studied MYCN and MDR1 genes using the human IGR-N-91 ectopic xenograft metastatic model. This characterized experimental in vitro model includes human neuroblasts derived from a subcutaneous primary tumor xenograft, disseminated blood cells, myocardium, and bone marrow (BM) metastatic cells. All IGR-N-91-derived neuroblasts harbor a consistent MYCN genomic content but, unlike primary tumor xenograft, BM, and myocardium, human neuroblasts elicit a concomitant increase in MYCN and MDR1 transcripts levels, consistent with chemoresistance phenotype and active P-gp. In contrast, no variation of MRP1 transcript level was associated with the metastatic process in this model. Using an MDR1 promoter-CAT construct, we have shown that the MycN protein activates MDR1 transcription both in exogenous transient MYCN-transfected SK-N-SH cells and in endogenous BM metastatic neuroblasts with an increase in the MYCN transcript level. Band-shift experiments indicate that IGR-N-91 cells enriched with the MycN transcription factor do bind to two E-box motifs localized within the MDR1 promoter. Overall, our data indicate that MYCN overexpression increment contributes to the acquired drug resistance that occurs throughout the NB metastatic process.

When p53 needs p73 to be functional: forced p73 expression induces nuclear accumulation of endogenous p53 protein

In human neuroblastoma (NB), wild type p53 protein does not elicit its archetypal human tumor suppressive activity so far described. To elucidate this alteration, substantial investigations using NB cell lines have underscored p53 protein nuclear localization defect and/or inappropriate conformation, but no definitive evidence has been provided so far. p73, the first homologue of the p53 gene, locates at the 1p36.3 locus, which is known to be deleted in various human tumors including NB. Unlike p53 mRNA, which specifies a single protein, p73alpha mRNAs encode two types of isoform (TAp73alpha and DeltaNp73alpha) resulting from the use of two different promoters, and eliciting or lacking NH(2)-terminal transactivation domain, respectively. DeltaNp73alpha inhibits p53 pro-apoptotic function in murine developing neurons and is abundantly expressed in human undifferentiated NB tumors. However, critical issues have been raised regarding p73alpha isoform roles, and their possible link to p53 are yet to be clarified in human NB using adenoviral infection approach.

Les récepteurs à dépendance DCC et UNC5H : rôle de l'apoptose dans le contrôle de la tumorigenèse

Des etudes recentes ont mis en relief un nouvel aspect des recepteurs cellulaires. Linteraction ligand-recepteur etait jusqua present consideree comme indispensable pour permettre lactivation du recepteur. Il a cependant ete suggere que certains de ces recepteurs, appeles « recepteurs a dependance », peuvent egalement etre actives en labsence de ligand et induire un signal specifique de mort cellulaire. Par consequent, lexpression dun ou plusieurs de ces recepteurs rend les cellules dependantes de la presence du ligand pour leur survie. Nous avons emis lhypothese que ce mecanisme permet dinhiber la croissance tumorale en conduisant a lapoptose les cellules «anormales» qui, en labsence de ligand, seraient normalement amenees a proliferer - croissance cellulaire locale ou proliferation a distance du site tumoral. Dans le meme ordre didee, au debut des annees 90, le groupe de Vogelstein a suggere quun gene, le gene DCC (pour deleted in colorectal cancer), pourrait jouer un role de suppresseur de tumeur car il etait retrouve delete dans plus de 70 % des cancers colorectaux et de nombreux autres cancers. Les donnees recueillies au cours des quinze dernieres annees nont pas permis dapaiser la controverse concernant limplication du gene DCC dans la suppression tumorale. Toutefois, notre equipe a pu montrer que DCC agit comme un recepteur a dependance qui induit la mort de la cellule lorsque son ligand, la netrine-1, est absent, et que des souris modifiees pour surexprimer la netrine-1 et bloquer ainsi lapoptose induite par le gene DCC etaient predisposees au developpement de cancers colorectaux. Ces donnees renforcent lhypothese selon laquelle les recepteurs a dependance auraient un role de suppresseurs de tumeurs. La presente revue decrit le role de linteraction netrine-1/recepteur comme nouveau mecanisme de controle du developpement tumoral.