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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 recombinant 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. Moreover we study the relation between hepatitis B and C viruses and their host in order to develop antiviral agents. Finally we have established a protocol of therapeutic genetic vaccination against hepatitis B.



  report

cale

Hepatitis B Virus and Hepatocellular Carcinoma

Group leader: Marie Annick BUENDIA

The molecular basis of liver carcinogenesis in patients infected with hepatitis B virus remains incompletely understood. The viral regulatory protein HBX has been implicated in the tumoral process by its ability to activate transcription from a broad range of cellular promoters. We analyze the interactions of HBX with the co-activators CBP, p300 and PCAF in transcriptional activation of cellular and viral promoters, and the activity of wild-type and mutated HBX in cell cycle progression and apoptosis. Current studies are aimed at identifying cellular target genes of HBX by transcriptome analysis. To recapitulate the hepatic context in HBV-infected patients, most of our studies use primary human hepatocytes or mouse liver.

Molecular Mechanisms of Liver Oncogenesis

Group leader: Marie Annick BUENDIA

Our recent studies of the molecular steps involved in liver cell transformation 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. In recent studies of transgenic mice, we have found that activation of ß-catenin and loss of function of p53 represent alternative oncogenic pathways in liver cell transformation. Our project is to better characterize the network of cellular factors implicated in the abnormal reactivation of theWnt pathway in liver tumors. In a two-hybrid screen, we have recently identified new ß-catenin-interacting proteins. We are studying the functional interactions of ß-catenin with these partners in adherens junctions and Wnt signaling. The impact of ß-catenin activation on gene expression profiles and cell cycle progression is currently analyzed in primary human hepatocytes, and candidate target genes of ß-catenin in the liver context are under study.

 

Comprehensive allelotyping of human and murine hepatocarcinomas with microsatellite markers has revealed frequently altered chromosomal regions that contain candidate tumor suppressor genes. In human tumors, we have evaluated the implication of E-cadherin by genetic and expression studies combined with fine mapping of chromosome 16. In murine tumors induced by c-Myc, specific chromosomal alterations suggest disruption of the INK4/RB pathway as an important step in liver oncogenesis.

 

Genetics of primary liver cancer

Group Leader: Anne DEJEAN

One of our main research interest focuses on the genetic basis of hepatocellular carcinoma (HCC). Our first goal is to identify novel tumor suppressor genes involved in the development of HCC. To this aim, we searched for allelic deletions affecting tumor DNA through a genome-wide scanning for LOH. Such approach is 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 was found for a locus in 8p23, this particular region is now being studied in details. A second extensive allelotyping performed on chromosomes 8p has revealed the presence of three distinct deleted regions suggesting the existence of several tumor suppressor genes on 8p. To delineate smaller regions, further allelotyping on a large number of additional HCC tumors is ongoing in the lab. To this aim, we have also created a collection of 60 hepatoma-derived cell lines and are looking for double deletions. It should narrow down the regions of interest and allow the search for mutations in potentially transcribed regions. In parallel, we used comparative genomic hybridization (CGH) to detect amplified regions in the human genome susceptible to harbor oncogenes. We focus now on chromosome 1q which we found to be amplified in more than 60% of liver tumors. The powerful CGH-array technique will be applied to narrow down the regions of interest. Finally, a transcriptome study of HCC has been recently undertaken in collaboration with MA Buendia's group.

 

Functional compartmentalization of the nucleus and acute promyelocytic leukemia

Group 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 retinoic acid in this type of leukemia, implies a new type of nuclear 'organelle' in a human disease. The interest of our lab is focused on the role of these PML Nuclear Bodies both in 'normal' and 'pathological' cells. We will mainly concentrate on the relationship between these particular substructures and the chromatin compartment for which we recently established a link. In an attempt to investigate the signals that regulate the dynamics of the NBs, we recently found that both 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. An important effort will now be devoted to elucidate the role of this new type of post-translational modification in vivo.

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. The approaches with these viruses will have generic interest for the construction of therapeutic vaccines in human and veterinary medicine and for cancer immunotherapy. The only reservoir of HBV consists in more than 350 million chronically HBV-infected carriers worldwide. Chronic liver disease and hepatocellular carcinoma associated with chronic HBV infection are among the most important human health problems in highly endemic regions. Current therapeutic approaches to control chronic hepatitis such as interferon-alpha and lamivudine are not fully satisfactory. Patients with HBV-associated chronic hepatitis would greatly benefit from a specific immunotherapy with a therapeutic vaccine that could control this persistent infection.

The goal of therapeutic vaccination is to stimulate and sustain an antiviral immune response that will resolve a chronic infection in an already persistently infected individual. We are studying the immunogenicity of selected HBV envelope-based vaccine formulations for the induction or the broadening of T and B cell responses that are deficient in HBV chronic carriers. We proposed specific vaccine therapies using either currently available recombinant anti-hepatitis B vaccines or envelope-derived DNA vaccines. Based on our results in animal models, a phase I clinical trial with the DNA vaccine 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 will study the immune response in vaccinated patients before and during vaccine therapy.

In addition, we are studying the effect of immunostimulatory DNA motifs (CpG), cytokines or activators for dendritic cells as adjuvants for vaccination with recombinant or DNA-encoded hepatitis B surface antigen (HBsAg). These vaccines preparations are tested for their immunogenicity in various animal models : mice of different MHC haplotypes, mice transgenic for HBsAg as a model for HBV-chronic carriers, mice transgenic for a human MHC-class I molecule and primates used as preclinical models for vaccination in human.

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 will allow 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.

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/HBs VLP against a SHIV challenge (collaborative work with Y. Rivière , I.P. and R. LeGrand, CEA).

 

Relation between hepatitis B (HBV) and C (HCV) viruses and their host.

Both HBV and HCV are responsible for major public health problems, all the more worrying since the available anti-viral treatments show limited efficacy. Our studies aim at characterizing the molecular interactions taking place during infection by HBV and HCV in order to identify new therapeutic targets.

In the case of HBV, we focus on the regulatory X protein (HBx) because this protein is essential for productive infection although its precise function is still unknown. We have shown that interaction of X with the host protein UV-DDB or DDB1 is crucial not only for in vitro X-mediated activities (transactivation and apoptosis), but also for productive infection in vivo. Our recent results indicate that DDB1 modulates the stability of X by inhibiting its rapid proteasome-mediated degradation. In addition, we could narrow the DDB1-binding domain of X down to a 15-aminoacid region. These observations prompt us to search for anti-viral compounds targeting X-DDB1 interaction. In this line of research, we have established a cell line in which, X activity can be induced at the post-traductional level. Induction of X activity triggers a dramatic morphological change of the cells, followed by their apoptotic death. This system will be used for : (i) The evaluation of anti-viral candidates derived from the minimal DDB1-binding domain of X and the setting of functional screens of chemical compounds on their ability to rescue cells from X-induced apoptosis. (ii) The kinetic characterization of the molecular events triggered by induction of X activity.

The nature and the function of interactions arising between the mature viral proteins produced from the single HCV polyprotein precursor are largely unknown, due to the absence of an efficient system to propagate HCV in vitro. This also holds for interactions between the virus and the host proteomes. We have designed tools for expression in human cells of random sub-regions covering the viral polyprotein. We will use these expression systems to: (i) further study the interactions between viral proteins, which we previously explored using the yeast two-hybrid system. (ii) identify interactions between viral and host proteins, (iii) identify B and T viral epitopes.

Figure legend:

Phase contrast microscopy of primary human hepatocytes and expression of ß-catenin transduced with a recombinant lentiviral vector.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

DA Louise-Marie IP lmda@pasteur.fr

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

TRANSY Catherine DR2 INSERM ctransy@pasteur.fr

WEI Yu CR IP ywei@pasteur.fr

BIANCHI Julie INSERM

BISCHOF Oliver Post-doc

KIRSH Olivier Ph.D. Student

LABALETTE Charlotte DEA

LEVY Laurence Ph.D. Student

LOIRAT Delphine Ph.D. Student

NACERDDINE Karim Ph.D. Student

PUAUX Anne-Laure Ph.D. Student

SEELER Jacob Post-doc

WU Yuanfei Ph.D. Student

ZHANG Menghua Post-doc

BERGAMETTI Françoise Engineer, Paris 7

BOURGINE Maryline Engineer, IP

BRES Evelyne 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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