The long term goal of this research project is to elucidate the molecular and cellular developments of hepatocellular carcinoma (HCC) in rats by applying in vitro techniques of gene modification and clonal selection (cell selection/ clonal amplification) of cells with in vivo clonal analysis of lesion development after transplantation of modified cells into liver of host rats. In vivo studies are based on the classic experimental model of carcinogenesis in rats, and the cells used are WB-F344 rat liver epithelial cells which have stem-like properties and differentiate into hepatocytes when transplanted into livers of syngeneic host rats. Liver epithelial cells that defined express aberrations in the structure and/or expression of genes normally involved in the regulation of cell proliferation, death, and differentiation have been clonally isolated from tumorigenic populations transformed spontaneously or by exposure to a carcinogenic chemical in vitro, in work on this project. Additional clonal lines will be genetically engineered to contain mutations or deletions and/or constitutively express selected genes or gene combinations involved in regulating cell proliferation, death, and differentiation. Cells bearing known gene aberrations will be transplanted into syngeneic host rats in order to assess their ability to grow progressively to form specific preneoplastic lesions and/or HCC in the regulating environment of the liver. In work done on this project we have already shown that the microenvironment regulates liver epithelial cells unlike the subcutaneous (SC) and intraperitoneal (IP) microenvironments; some lines of aneuploid liver epithelial transformants that are highly tumorigenic in SC or IP sites differentiate into hepatocyte-like cells in the liver, in coordination with slowed proliferation, increased death, and delayed or suppressed tumorigenicity. Dr Grisham believes that by coupling clonal analysis of preneoplastic and neoplastic cellular lesions that develop in the liver from transplanted cells that contain specific aberrations in gene function, he can identify the specific genes whose abnormal functions are responsible for clonal emergence of specific preneoplastic lesions and HCC, and thereby delineate the molecular pathogenesis of HCC.