This project will characterize the sequential acquisition of genetic alterations during spontaneous neoplastic transformation of the liver epithelial stem cell line WB-F344. This multipotent hepatic stem cell population represents a significant target for spontaneous or carcinogen-induced tumorigenesis: neoplastically transformed WB-F344 cells produce hepatocellular carcinomas, adenocarcinomas, hepatoblastomas and undifferentiated spindle cell tumors. Analysis of spontaneous transformation will allow identification of the minimal necessary genotypic alterations required for neoplastic transformation. Our hypothesis is that neoplastic transformation of WB-F344 cells results from progressive genomic instability entrained by a genetic alteration which produces a disregulation of nuclear DNA metabolism. The genomic instability can precipitate the pleiotropic alterations of gene expression and cellular phenotype associated with neoplastic transformation. The first goal of this project is to characterize in cohort cell lineages the sequence of appearance of: loss of regulation of cycle stage transitions; aneuploidy; alterations of structure or expression of tumor suppressor genes (Wilm's tumor-1, retinoblastoma, p53); de novo expression of TGF-alpha and coordinate overexpression of c-myc and c-ras; and tumorigenicity. Cohort cell lineages will be followed through sequential cycles of selection by maintenance at confluence until a single tumorigenic clonal population is established from each lineage. The second goal is to characterize the differential capacity of the cohort tumorigenic clonal cell lines to form tumors in adult syngeneic animals when transplanted into the liver parenchyma. Clonal lines will be infected with the CRE-BAG2 retrovirus which carries both neomycin resistance and bacterial beta-galactosidase gene activity. We anticipate that these populations which are all tumorigenic when transplanted subcutaneously will display a range of tumorigenic capacities in the liver, from non-tumorigenic (cells appear to differentiate into normal hepatocytes) to tumorigenic producing undifferentiated tumors. Transplanted cells will be recovered from the liver based upon the presence of the gene for neomycin resistance, reestablished in culture, and phenotypically evaluated. Clonal lines which differentiated into apparently normal hepatocytes will be transplanted into the subcutaneous compartment to determine if the suppression of the transformed phenotype by the liver microenvironment is due to a permanent genetic reprogramming, or due to transient suppression dependent upon the presence of a transacting factor produced by the normal liver parenchyma.
