The goal of project 3 is to examine the relationship between hepatocyte proliferation and expression of liver specific genes. Alphafetoprotein production is a characteristic of proliferating hepatocytes while albumin expression is a product of the adult, quiescent liver. Conditions that provoke hepatocyte division (regeneration, tumorigenic states, and fetal development) are associated with AFP synthesis. An understanding of the molecular basis for the regulation AFP and albumin expression may provide insight into the complex events that alter the proliferative state of the hepatocyte.
Our specific aims i nclude, 1) defining the cis-acting regulatory regions of alphafetoprotein and albumin that are responsive to factors that induce or inhibit proliferation, such as the membrane associated inhibitors of DNA synthesis isolated from normal senescent fibroblasts and immortalized human cells and stimulatory factors obtained from livers and serum of mice following hepatectomy. These assays will employ gene transfer of regulatory regions of each gene coupled to the bacterial enzyme chloramphenicol actyltransferase into human hepatoma cells. 2) characterizing biochemically, the DNA binding proteins that interact with regions of the DNA that have functional activity defined in specific aim 1. These studies will utilize """"""""band competition"""""""" or gel retardation assays and footprinting to identify the specific sequences in the gene with which the proteins interact. 3) the third aim is to identify and clone the regulatory gene(s) that govern alphafetoprotein expression by a novel genetic scheme. This study will utilize gene transfer and mutant selection to obtain a """"""""trans-acting factor"""""""" mutant. Complementation of the mutant phenotype and selection for the complementing gene will enable direct cloning of the regulatory loci. The trans-factor gene would then be available for an assessment of its role in the complex phenotype of hepatocytic proliferation at the molecular level. 4) a final aim is to immortalize human primary hepatocytes in order to examine the cellular genes that are modified.
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