Hyperplastic foci that appear in livers of rats after treatments with hepatocarcinogens may represent progeny of clones of initiated hepatocytes. When we place liver cells from rats treated in this manner into cell culture soon after treatment, a number of hepatocytes proliferated under conditions in which normal hepatocytes failed to grow or senesced. We have suggested that hepatocytes with this enhanced- growth variant (EGV) phenotype (able to proliferate in cell culture) may be the initiated clonal progenitors for focus-forming hepatocytes in vivo. Since normal hepatocytes are programmed such that they do not to grow or grow for only a very limited duration in cell culture, presumably these EGV cells have undergone some alteration in their ability to negatively control cell proliferation. Negative control of cell proliferation is manifest at the level of initiation of DNA replication and mitosis. Whereas human cells normally delay entry into S and/or M phases when DNA is damaged, this capacity is lost in EGV's. We hypothesize that these two alterations of negative regulation of cell proliferation (EGV phenotype and checkpoint failure) many be manifestations of the same genetic defect. From this we predict that cells that develop the EGV phenotype also should have impaired ability to inhibit initiation of S and/or M phases (G1 and G2 checkpoint delays) and replicon initiation when their DNA is damaged. Hepatocyte EGV's have been generated after transfection of human fetal liver cells with selected oncogenes. To test our hypothesis we propose to induce several human hepatocyte EGV's by transfection of various growth promoting genes or by treatment with chemical oncogenes. To test our hypothesis we propose to induce several human hepatocyte EGV's by transfection of various growth promoting genes or by treatment with chemical carcinogens. Characteristics of cultured human hepatocytes will be evaluated including morphology, karyotype and expression of hepatocyte-specific genes. Hepatocytes will be transfected with various genes (ori-SV40, truncated human c-myc, HPV E6/E7, etc.) during the initial phase of active cell proliferation or treated with chemical carcinogens using a novel protocol involving conditional EGV's. EGV's will be isolated based on their abilities to grow under conditions in which normal cells fail to grow or senesce. We will determine if the hepatocyte EGV phenotype is consistently associated with a defect in negative regulation of cell cycle progression, regardless of how the EGV's are induced. The intactness of the G/D and G2/M checkpoints and the ability to inhibit replicon initiation will be evaluated. This should test whether alteration in control of cell proliferation and the EGV phenotype are characteristics of an early stage of neoplastic evolution.
