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. 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 for diagnosis and treatment. The liver is composed of admixtures of numerous cell types and it is therefore essential to obtain enriched samples of cells of interest if we are to acquire meaningful data. Laser microdissection (LM) techniques procure 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 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. 2005, Oncogene). 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. Starting from groups of 2000 adjacent hepatocytes isolated by laser microdissection in the tumour zone (T) and in two cirrhotic nodules (NT1, NT2), full length HCV core sequences were obtained from 7 patients with HCC associated to HCV 1b infection. For each studied area around 10 clones were sequenced. The comparison of core protein sequences from each patient demonstrated core protein heterogeneity in 5/7 patients and confirmed the increased core protein heterogeneity in the tumorous area as compared to the non-tumorous part. Furthermore, we have also evidenced a genetic variability between the different cirrhotic nodules (NT1,NT2) of the non-tumorous area. Altogether, our results suggest the existence of independent viral compartments within the liver, and assume area-specific evolution of HCV variants. The phenotype effects (if any) of the mutations identified in vivo will be also examined for their effects on cell signalling involved in proliferation, transformation and also in fibrosis that leads to cirrhosis and HCC. The predominant HCV core sequences from T, NT1 and NT2 will be independently co-transfected with a reporter construct containing TGF-β responsive elements and fold stimulation after TGF-β_ treatment will be measured.
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, V. THIERS, F. DEMAUGRE U370)
This study will be using laser microdissection (LM) to isolate HCC and non-HCC hepatocytes from HCV infected patients, and combine LM 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) with the collaboration of U467. The liver samples studied in this project have been obtained from the "collection Nationale des CHC" (Inserm/DHOS)
In order to warrant the use of microdissection to perform our study, the protein profiles of liver sections obtained from tumour (T) and non-tumour (NT) 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. The comparative analysis of the differential expression profiles showed that the use of microdissection can expose deregulated proteins which are masked when experiments are performed using global hepatic homogenates. Indeed, 30 % of the proteins differentially expressed in LM extracts were not highlighted when analyzing global hepatic homogenates. Furthermore, higher expression ratios were observed between T and NT in the microdissected cells as compared to liver sections (p<0,001). Altered expression of some proteins we identified by mass spectrometry was further validated using Western blot analysis. However, the altered expression of the identified proteins has to be shown in a larger series of patients to demonstrate their possible interest in HCC.
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 Carcinoma (HCC), genetic variability, laser microdissection (LM), Proteome, 2D-PAGE electrophoresis