The objective is to use rat hepatocytes to study regulation of differentiation in hepatocytes in vitro and to determine how the processes associated with transformation and malignancy alter the expression of differentiated functions in hepatocytes in vitro. These goals will be achieved using two in vitro hepatocyte systems recently developed and characterized in our laboratory (1) a long-term culture system for primary hepatocytes in which the cells are maintained in a chemically defined medium supplemented with dimethylsulfoxide (DMSO) and (2) a series of simian virus 40 (SV40) immortalized, transformed, and tumor cell lines derived from hepatocytes. We propose to (1) determine what transcriptional mechanisms are used by the cells in the two in vitro hepatocyte systems to achieve steady state levels of albumin mRNA comparable to those in liver; (2) measure mRNA levels and transcription of albumin and other liver-specific genes, common genes, oncogenes, and genes associated with hepatocarcinogenesis after exposure of DMSO-treated primary hepatocytes and hepatocyte cell lines to specific hormones and agents and in hepatocyte cell lines as they progress in their tumorigenic potential; (3) use SV40-immortalized hepatocyte cell lines to map regulatory elements for the albumin gene and (4) use the techniques of DNase I hypersensitivity and DNase I footprinting to identify protein binding sites on the regulatory regions of liver-specific genes and study the factors which interact with these sites. We will also continue to define what viral and cellular DNA sequences can convert a normal hepatocyte into an immortalized hepatocyte cell line, delineate what sequences will convert an immortalized hepatocyte cell line into a tumorigenic cell line and compare the effect of introducing these DNA sequences on the expression of differentiated functions.
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