Although liver carcinogenesis in rats has been extensively investigated, the key molecular events which govern the various stages of the process have not been defined. Similarly, it has been difficult to precisely trace the cell lineages of the tumors which, in this system, emerge only after a very long induction period. The approach we have taken to study these problems centers on the isolation and characterization of epithelial cell populations which proliferate at the early stages of hepatocarcinogenesis and contain most markers of liver neoplasia. We now show that liver epithelial cells isolated from rats treated with carcinogens for 2-6 weeks, placed in culture and transfected with the EJ oncogene, can give rise to well differentiated hepatocellular carcinomas when injected into nude mice. Differentiation markers on cells of these tumors include the reactivity with a panel of monoclonal antibodies and presence of albumin mRNA. In this application we propose to: a) use this novel system to analyze in detail the cell lineages of hepatocellular carcinomas produced by EJ-transfected liver epithelial cells and search for clonal lines which may contain tumor stem cells; b) determine in vivo the developmental potential and tumorigenicity of liver epithelial cells by transplanting cells carrying appropriate marker genes into livers of syngeneic rats. The liver epithelial cell system we have developed also permits an analysis of the role of oncogenes and growth factors in the development of hepatocellular carcinomas. Although the EJ gene was used to transform these cells, it is unlikely that this gene plays a role in the development of most rat hepatocellular carcinomas in vivo. Instead, we propose to test the hypothesis that constitutive expression of EGF receptor signals coupled with overexpression of myc (or a member of the same gene family) may be sufficient to transform liver epithelial cells. Preliminary data indicate that liver epithelial cell transformation is associated with the blockage of an autocrine inhibitory circuit involving TGF-beta. The proposed experiments should lead to the identification of hepatocellular carcinoma cell lineages and provide new information on the developmental potential and mechanisms of tumorigenesis of liver epithelial cells.
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