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  Director : Pierre TIOLLAIS (tiollais@pasteur.fr)


  abstract

 

The research projects of our Unit cover the fields of oncogenesis, molecular virology and hepatitis B therapeutic vaccine. We are attempting to identify tumor suppressor genes involved in liver cancer, and to better understand the role of the viral protein HBX and of the ß-catenin oncogene in liver tumorigenesis. Our laboratory also studies the relationships between the functional compartmentalization of the nucleus and acute promyelocytic leukemia. Finally we have established a protocol of therapeutic genetic vaccination against hepatitis B.



  report

cale

Hepatitis B Virus and Hepatocellular CarcinomaGroup leader: Marie Annick BUENDIA

Chronic infection with hepatitis B virus (HBV) remains a major risk factor for the development of liver cancer. However, the molecular basis of viral-induced liver carcinogenesis remains incompletely understood. The viral regulatory protein HBx has been implicated as a co-factor in the tumoral process by its transcriptional and cytotoxic activities. To recapitulate the hepatic context in HBV-infected patients, most of our studies used primary human hepatocytes or mouse liver. We have shown that HBx expression sensitizes liver cells to apoptotic killing by the Fas pathway, and that HBx activates the expression of different apoptotic genes. We are currently analyzing the functional role of wild-type and mutant HBx isolated from human tumors in cell cycle progression, apoptosis and senescence of primary human cells. To better understand the mechanisms of transcriptional activation by HBx, we are studying the interactions of the viral protein with histone acetyl-transferases et deacetylases. Our recent data indicating that the HBx protein is modified by acetylation prompted us to search for the functional significance of this post-translational modification in known HBx activities.

Molecular Mechanisms of Liver OncogenesisGroup leader: Marie Annick BUENDIA

Primary liver cancer is a leading cause of death worldwide. Our recent studies of the molecular steps involved in the tumoral process have shown that activation of Wnt signaling by oncogenic mutations of ß-catenin plays an important role in the development of hepatocellular carcinoma and hepatoblastoma. Our ongoing project is to better characterize the network of cellular factors implicated in abnormal reactivation of the Wnt pathway in liver tumors. To this aim, we have searched for new binding partners of ß-catenin and for Wnt target genes that are involved in liver cell transformation. Several new ß-catenin-interacting proteins have been identified, including the LIM domain protein FHL2, which acts as a co-activator of the ß-catenin/Tcf complex. Furthermore, we have shown that binding of CBP/p300 with ß-catenin results in acetylation and enforced transcriptional activity of ß-catenin. The impact of ß-catenin activation on gene expression profiles has been analyzed in primary human hepatocytes and in murine hepatomas, leading to the identification of candidate target genes of the Wnt pathway in the liver context. Notably, we found that the IL-8 chemokine is a direct target of ß-catenin and might convey proliferative and angiogenic signals in tumoral cells. In parallel, we are studying the role of these diferent factors, as well as that of the ß-catenin-E-cadherin complex, in the human liver and in primary liver tumors. Detailed analysis of new oncogenic pathways will undoubtly provide tools for diagnostic and therapeutic purposes.

Photo: Hépatocytes humains primaires en contraste de phase et expression de la ß-caténine transduite par un vecteur lentiviral.

Genetics of primary liver cancerGroup Leader: Anne DEJEAN

One of our main research interest focuses on the genetic basis of hepatocellular carcinoma. Our first goal is to identify novel tumor suppressor genes involved in the development of primary liver cancer. To this aim, we searched for allelic deletions affecting tumor DNA through a genome-wide scanning for LOH coupled to a comparative genomic hybridization (CGH) study. Such approaches are of great value for locating candidate genes implicated in cancer development. The chromosomes most frequently affected by deletions were 1p, 4q, 6q, 8p, 13q and 16q. Since the highest percentage of deletions were found at chromosomes 8p and 4q ( >50%), these two regions have been studied in details. The study of tumors and tumor derived cell lines using LOH and reduction to homozygosity has pointed to three regions on 8p that likely harbor hepatic tumor suppressor genes. The role of these candidate genes in liver tumorigenesis will be studied by mutational and expression analysis, including ‘tissue arrays'. Putative suppressor genes at 4q will be identified by positional cloning using a novel approach, the CGH array. Moreover, a search for homozygous deletions in a collection of a hundred tumor cell lines, including sixty of hepatobiliary origin, revealed five candidate tumor suppressor genes not previously associated with liver cancer. We will carry out mutational analysis of these genes in different etiological contexts (infection with HBV and HCV, alcohol use, metabolic disorders). The mutational analysis will be complemented by functional studies. Finally, an extensive transcriptome analysis of hepatocellular carcinoma was undertook with the aim of defining the major expression profiles of this heterogenous class of tumors. This should hopefully lead to the establishment of a tumor classification system of significant prognostic and therapeutic value for primary liver cancer.

Functional compartmentalization of the nucleus and acute promyelocytic leukemiaGroup Leader: Anne DEJEAN

A major aspect of Acute Promyelocytic Leukemia (APL) pathogenesis involves (i) a chromosomal translocation affecting the gene encoding the retinoic acid receptor a (RARa) and the PML gene (giving rise to the fusion PML-RARa oncoprotein) as well as (ii) a retinoic acid- reversible disruption of a particular subnuclear structure, the PML Nuclear Body (NB). This latter observation, that provides a striking parallelel to the therapeutic effect of retinoids in this type of leukemia, implies a new type of nuclear 'organelle' in a human disease. While it is generally accepted that the eukaryotic cell nucleus is highly organized, its functional compartmentalization still remains poorly understood. Our work seeks to identify and characterize the signals that regulate the dynamics, and hence function, of the PML NBs both in normal and pathological contexts.

We will mainly concentrate on the relationship between these particular substructures and the chromatin compartment for which we recently established a link. Our goal is to clarify the role of the PML NBs in regulating chromatin dynamics by biochemical and localization studies with the aim of integrating transcriptional regulatory mechanisms into the framework of subnuclear architecture. Moreover we have shown that PML plays a major role in inducing premature cellular senescence by activation of the two p53 and RB pathways. Molecular and cellular mechanisms involved in this process will be analyzed through the identification of interacting proteins and target genes whose expression is regulated by PML during senescence. Finally, we showed that PML and SP100, the two major components of the NBs, were modified by covalent linkage with the ubiquitin-related SUMO-1 modifier. By contrast to ubiquitination, ‘sumoylation' does not target protein for degradation but is rather involved in subcellular localization. Notably, sumoylation of PML is necessary for its proper targeting to the NBs and for the maintenance of the structural integrity of these structures. Given the tight link between sumoylation and NB dynamics, a significant effort will be dedicated to the study of this new type of post-translational modification and its role in the general organization of the nucleus both in normal and leukemic cells.

The PML Nuclear Bodies

Preventive and therapeutic vaccines against chronic viral infections Group Leader: Marie-Louise MICHEL

The aims of our studies are to develop preventive and therapeutic vaccines against persisting hepatitis B virus (HBV) or human immunodeficiency virus (HIV) infections. Chronic liver disease and hepatocellular carcinoma associated with chronic HBV infections are among the most important human health problems in highly endemic regions. Current therapeutic approaches to control chronic hepatitis are not fully satisfactory. Patients with HBV-associated chronic hepatitis would greatly benefit from a specific immunotherapy, which could control this persistent infection. We are studying the immunogenicity of selected HBV envelope-based vaccine formulations for the induction and/or the broadening of T and B cell responses that are deficient in HBV chronic carriers. The effect of immunostimulatory DNA motifs (CpG) as adjuvant for vaccination with recombinant or DNA-encoded hepatitis B surface antigen (HBsAg) was studied in mice transgenic for HBsAg. To study the mechanisms involved in tolerance to HBsAg and underlying viral persistence during chronic infection we have developed HLA-A2/HBsAg double transgenic mice. This humanized animal model allows us to design new strategies aimed at restoring in vivo effector functions of peripherally tolerized T cells, that could be directly transferred to patients chronically infected with HBV. Based on our results in animal models, a phase I clinical trial with a DNA vaccine encoding HBsAg is ongoing for HBV-chronically infected patients. This trial conducted by clinicians from Necker Hospital (Pr. Brechot, Pr. Pol and Dr. H. Fontaine) will mainly serve to assess safety. But, we are studying the immune response in vaccinated patients before and during vaccine therapy.

We have used HBsAg as virus like particle (VLP) for the presentation of foreign epitopes derived from SIV/HIV to specifically activate the immune system. This approach is combined to genetic vaccination in order to provide T cell help and to enhance cytotoxic response to these viruses. In a pilot experiment in macaques, we are assessing the immunogenicity and protective efficacy of our DNA-based vectors encoding HIV/HBsAg VLP against a SHIV challenge (collaborative work with Y. Rivière , I.P. and R. LeGrand, CEA).

Keywords: hepatocellular carcinoma, b-catenin, genetic alterations, hepatitis B virus, antiviral inhibitors, genetic vaccine, immunotherapy, transgenic mice, PML nuclear bodies, SUMO, cellular senescence, chromatin, leukemia



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  BUENDIA Marie-Annick DR1 CNRS (mbuendia@pasteur.fr)

DEJEAN Anne DR1 INSERM (adejean@pasteur.fr)

MICHEL Marie-Louise DR2 INSERM (maloum@pasteur.fr)

NEUVEUT Christine CR2 INSERM (cneuveut@pasteur.fr)

PINEAU Pascal CR IP (ppineau@pasteur.fr)

SEELER Jacob CR IP (seeler@pasteur.fr)

TIOLLAIS Pierre Prof. IP/Paris 7 (tiollais@pasteur.fr)

TRANSY Catherine DR2 INSERM (ctransy@pasteur.fr)

WEI Yu CR IP (ywei@pasteur.fr)

BISCHOF Oliver Post-doc

CARAMEL Julie DEA Student

CORDINA Emilie DESS Student

DA SILVA Lucie Student ESTBA

KIRSH Olivier Ph.D. Student

LABALETTE Charlotte Ph.D. Student

LEVY Laurence Ph.D. Student

MALMASSARI Silvina Post-doc

MARTIN Nadine DEA Student

NACERDDINE Karim Ph.D. Student

PUAUX Anne-Laure Ph.D. Student

WU Yuanfei Ph.D. Student

BERGAMETTI Françoise Engineer, Paris 7

BOURGINE Maryline Engineer, IP

MARCHIO Agnès Engineer, IP

RENARD Claire-Angélique Technician, IP



GEORGES Monique IP

LEGOUT Claudine IP

LEGUEULT Catherine IP

QUEROL Chantal IP


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