The objective is to use rat hepatocytes to study the effect of virus infection and transformation on expression of differentiated liver cell functions and the influence of a differentiated hepatocyte on virus replication and virus gene expression. These goals will be achieved using hepatocytes maintained in chemically defined medium with or without supplementation with dimethylsulfoxide (DMSO). Use of these culture conditions has enabled us to study simian virus 40 (SV40) induced stimulation of albumin production from time of infection until transformation, reproducibly generate SV40 transformed colonies which retain the ability to synthesize albumin, and develop a quantitative assay for transformation of nonproliferating differentiated epithelial cells. We propose to (1) examine regulation of levels of RNA for albumin, other liver-specific genes and common genes with time in culture and after SV40 infection and transformation of hepatocytes, (2) use flow cytometry to determine whether the DNA content of a hepatocyte at time of isolation is critical to transformation frequency and or the properties of the transformants and (3) use other virus genes and oncogenes to establish what oncogenic information is required to transform hepatocytes from normal, partially hepatectomized and diet-modified animals. We have shown that DMSO-treated hepatocytes retain the morphological and biochemical properties of freshly isolated hepatocytes for at least 48 days and that from 20-48 days after plating, the cells undergo little change. We propose to use this plateau period to investigate herpes simplex virus (HSV) replication, HSV-induced alterations of host gene expression HSV-induced stimulation of repair DNA synthesis and expression of specific HSV genes in a differentiated cell. Chemically defined medium, the inducing agent DMSO and appropriate plating surfaces provide biological conditions in which differentiation over extended periods of time can be maintained. By adding (1) techniques of molecular biology, (2) available recombinant DNA for liver-specific genes and (3) the tool of flow cytometry to the biological systems we have generated, we can measure regulation of expression of specific differentiated functions in normal cells and after infection and transformation.
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