The broad, long term objective of this proposal is to understand the potential role of retinoic acid (RA) in the treatment of hepatoma.
The specific aims of the proposed experiments are to: 1) study how RA regulates the phenotype of hepatoma cells, 2) determine the manner in which alpha-fetoprotein (AFP) and albumin (A1b) genes are regulated during the phenotypical change, 3) assess the regulatory mechanisms, and 4) elucidate the roles of retinoic acid receptor (RAR) in the regulatory process, and during hepatocarcinogenesis. Our preliminary data indicated that in Morris hepatoma McA-RH 8994 cell line, RA enhanced the expression of AFP and A1b genes which are characteristic of hepatocyte differentiation. To understand the involved regulatory mechanism, the AFP mRNA half life and transcriptional rate of AFP gene will be examined by pulse chase analysis of 3H uridine incorporation and nuclear run off assay in RA treated or untreated McA-RH 8994 cells. The functional elements of AFP gene will be examined by transient transfection assay using various kinds of AFP-chloramphenicol acetyltransferase (AFP-CAT) constructs. The 5'-flanking rat AFP genomic elements implicating RA responsiveness will be determined. Gel shift assay will be employed to examine the in vitro binding property of the element and the presence of binding proteins. RARbeta gene was inactivated and resistant to RA in Morris hepatoma McA-RH 8994 and 7777 cell lines. This aberrant expression of a normal gene may relate to carcinogenesis and provides a model system for studying the role of RARbeta in transformation. The cis-acting element within the promoter region of RARbeta gene will be isolated from genomic libraries of normal rat liver and hepatoma cells. The RARbeta gene sequence will be examined and the presence of mutation will be identified. The trans-acting factors which are putatively involved in control of RARbeta gene expression will also be studied by gel shift assay. The results of this study will help us in understanding the roles of RAR in regulating liver specific gene expression and in hepatocarcinogenesis.
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