Retinoids regulate essentially all important biological processes including liver development, and these processes are largely regulated by complex transcription networks. In the liver, we have identified a gene, the alpha-fetoprotein (AFP) gene, that can be up- and down-regulated by retinoic acid (RA). AFL represents an important marker for hepatocyte differentiation, maturation, and carcinogenesis. One function of AFP is to regulate the concentration of retinoids. Thus, it is possible that RA and AFP might regulate each other in order to balance each other's effects. The goals of this project are to explore the molecular mechanisms involved in RA-mediated AFP gene regulation and to understand the actions of RA and the roles of RA receptors in differentiation and antiproliferation of hepatoma cells. The first specific aim is to characterize a complex DNA element containing one inverted and three direct repeats of GGGTCA-like sequence, which is responsible for the up-regulation of the AFP gene in McA-RH8994 hepatoma cells. Gel-shift assay, transient transfection CAT assay, and mutagenesis study will be employed to analyze the importance of individual repeats in trans-activation of the AFP gene. The possibility of other co-factors involved in up-regulating the AFP gene also will be examined. The roles of RA receptors in this positive regulatory pathway will be analyzed using retinoic acid receptor (RAR) and retinoid x receptor (RXR) selective ligands, transient co-transfection with RA receptor expression plasmids, and gel mobility shift assay with specific anti-RAR and -RXR antibodies. RA also appears to play a role in the post-natal down-regulation of AFP, and Hep3B cells can be used as a model system to study this down- regulation. The motif responsible for down-regulation of the AFP gene by RA will be identified and characterized in Hep3B cells. Since down- regulation of the AFP gene is associated with regulation of the RAR and RXR genes in Hep3B cells, the roles RA receptors and other trans-acting factors involved in the negative regulatory pathway will be examined as well. In addition, because the RARbeta gene has been suggested to be a tumor suppressor gene, the roles of RARbeta in differentiation, antiproliferation, and apoptosis of hepatoma cells will be studied by restoration of RARbeta in RARbeta negative hepatoma cells and by inactivation of RARbeta in RARbeta positive hepatoma cell lines. The results obtained from this proposal will enhance our understanding in the actions of RA on hepatocyte differentiation and antiproliferation.
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