Unit: Necker-Institut Pasteur joint Hepatitis Laboratory
Director: Christian Bréchot, Valérie Thiers
The activities of our laboratory, which includes the National Reference Centre for Hepatitis B and C viruses, focus on implementing of new methodological approaches for the study of hepatitis B and C infections. HBV and HCV are the two principal agents implicated in the development of hepatocellular carcinoma (HCC), in Europe. Working closely with the Inserm 370/Pasteur Joint Research Unit, we are developing a project, aimed at analysing the proteome of microdissected hepatocytes so as to clarify the mechanisms of carcinogenesis in HCV-infected patients. In parallel, the Reference Centre on hepatitis B and C viruses ensures the molecular characterization of specific B and C viral isolates, and investigates the residual risks of transmission of these viruses.
The principal risk factor associated with the onset of HCC is chronic hepatitis resulting from infection with HBV or HCV, although the molecular mechanisms underlying hepatocarcinogenesis are still obscure. The reference techniques for the diagnosis of HCC include serological tumour markers and imaging modalities, but there is an urgent need to identify molecular markers to enable earlier diagnosis and improve the prognosis for HCC. The identification of differentially expressed proteins during the multi-step process of hepatocarcinogenesis would provide valuable insights into the mechanisms of HCC and the determination of potential markers and drug targets for diagnosis and treatment. The liver is composed of admixtures of numerous cell types and it is therefore essential to obtain pure samples of the cells of interest if we are to acquire meaningful data. Laser-assisted microdissection techniques procure pure populations of hepatocytes from frozen liver sections. Using this experimental approach we will analyse in matched pairs of tumorous and non tumorous groups of hepatocytes
The heterogeneity of HCV Core protein " In situ "
Protein fingerprinting of HCC tissues
1 Study of HCV core gene variants, a potential protein for virus-induced transformation. (R. SOBESKY, N. DERIAN V. THIERS).
The core protein of HCV has been reported to fulfil a pleiotropic function in the viral replication cycle, as well as being a component of viral nucleocapsids. Data suggest that the expression of certain HCV proteins, such as core protein, is involved in viral persistence, modulation of the antiviral effect of interferon, cell proliferation and cell viability. Moreover, studies performed in our laboratory, in a few cases of HCV-positive HCC, have shown distinct biological effects of HCV core proteins encoded by sequences isolated from HCC tumour tissues (Delhem et al, 2001, Oncogene ; Pavio et al. manuscript under revision). These results suggest the distribution of different quasi-species in tumour tissue, thus opening the possibility of a selection of variants with modified functional properties. Consequently, the demonstration, in other subjects with HCV-associated HCC, of quasi-species with similar properties would provide arguments in favour of a selection of viral variants during chronic infection.The aim of our project is thus to extend these observations to a larger number of cases. For this study, we have selected 12 HCC patients infected by HCV genotype 1b. Groups of approximately 2000 adjacent hepatocytes isolated by laser microdissection in the tumour zone (T) and in two cirrhotic nodules (NT1, NT2) were analysed in 7 patients. The early results have already shown the feasibility to isolate full-length core from a limited number of hepatocytes. The comparison of predominent core protein sequences from each patient demonstrated core protein heterogeneity in 5/7 patients, either between the different cirrhotic nodules (n=2) or between the nodules and the tumoral zone (n=3). The impact (if any) of amino acid variations on the secondary structure of the protein will be studied using bioinformatic tools. The phenotype effects of the mutations identified in vivo will be also examined.
2. Proteomic expression profiling of microdissected hepatocytes from tumour and non tumour liver tissues of subjects with HCV-associated HCC. (ARECA network) (A. DOS SANTOS, F. DEMAUGRE U370,& V. THIERS)
This study will be using laser assisted microdissection to isolate HCC and non-HCC hepatocytes from HCV infected patients, and combine microdissection with two-dimensional electrophoresis (2DE). Protein fingerprinting of matched pairs of HCC and non-HCC tissues will be compared and the differentially expressed proteins identified by mass spectrometry (MALDI-TOF-MS). The liver samples studied in this project have been obtained from the "collection Nationale des CHC" (Supported by l'Inserm/DHOS)
In order to warrant the use of microdissection to perform our study, the protein profiles of liver sections obtained from tumour and non-tumour zones in the patients, were compared with those of hepatocytes sampled by microdissection from the same subjects. Thanks to the bioinformatic analysis of gels realized in triplicates, we determined the differential protein expression profiles under the different conditions studied. Proteins retained as being differentially expressed (usually expression ratio ? 2 ; unpaired Student's t-test; p ?0.05), are under identification by mass spectrometry with the collaboration of the group led by A. Edelman (U467). We have examined the relative variations of expression of a group 30 differentially expressed proteins. Globally higher expression ratios were observed between T and NT in the microdissected cells as compared to liver sections. Furthermore, for 8 proteins a differential expression between T and NT was only observed in microdissected cells. This observation suggests that LCM circumvent the cellular of heterogenity of cirrhotic liver and has an enrichment effect on the proteins of interest. Thus these data validate the choice of microdissection for our study, and demonstrate its feasibility. (This work is supported by ARC-Inserm).
3. Activities of the National Reference Centre (CNR) for B and C viral hepatitis (F. RIMLINGER, V. THIERS)
The activities of the CNR are closely linked to the research projects being carried out by the laboratory. Molecular data, obtained by phylogenetic analysis, can provide an opportunity to identify the routes of HCV transmission (patient to patient, nosocomial). Detailed analysis of virus nucleotide sequences, in informative viral regions, can provide evidence for a common source of infection in outbreaks of infection or horizontal transmission between pair of individuals. Studies on the nosocomial aspects of hepatitis B and C virus transmission are ongoing, in collaboration with the French Health Surveillance Institute "Institut de Veille Sanitaire". Analyses of the genetic diversity of particular HCV types, in different geographical regions, are performed in collaboration with the HepMed network (EU project, coordinated by B. Larouze) and Pasteur Institute network (PTR project coordinated by P. Mavromara).
Keywords: Molecular epidemiology, Hepatitis C virus (HCV), Hepatocellular Cracinoma (HCC), genetic variability, laser assisted microdissection, HCC, Proteome, 2D-PAGE electrophoresis