The rat gene encoding the serum protein alphafetoprotein (AFP) is active in the fetal liver and yolk sac, but is repressed after birth. In the adult, the AFP gene can be re-expressed under certain abnormal conditions, such as during hepatocarcinogenesis and in regeneration of a partial hepatectomy. The AFP gene is also active in some transplantable hepatocellular carcinomas (hepatomas), and in cell lines derived from these. The expression of the AFP gene makes an excellent model system for the development regulation of genes, and how this regulation becomes changed in cancer. A genetic approach to this problem is available because hepatoma cell lines can be transfected with mutated and rearranged AFP genes. In addition, transplantable hepatomas, normal liver and premalignant liver can provide large amounts of material for the isolation of factors that might be involved in AFP gene regulation. This powerful combination of approaches will be exploited to answer specific questions about how the rat AFP gene is regulated in these pathological states. Previous work (by others) has defined three DNA sequence elements 5' of the AFP gene that are involved in tissue specific expression: (1) and enhancer between -7 and -4 kb of the 5' end that increases the expression of the AFP gene in cells that produce AFP; (2) a negative regulatory element (NRE) at about - 3.5 kb that reduces AFP gene expression in AFP positive cell lines; and (3) a tissue specific promoter that functions only in AFP positive cell lines. Genes transfer experiments will be done to further define the structures of these elements and to answer questions about how the elements function together to regulate gene transcription. Other studies will determine whether DNase I hypersensitive sites are important for AFP gene transcription and whether repetitive DNA sequences mapping in the 5' flaking region are involved with AFP gene expression. Biochemical studies will identify DNA sequences that bind nuclear proteins from liver and hepatomas and to relate these DNA sequences to those defined in genetic experiments. These studies will lead directly to the purification of specific nuclear proteins that bind AFP regulatory elements, and to the isolation of monoclonal antibodies against these nuclear proteins. This work will ultimately lead to a complete understanding of how gene expression is altered during carcinogenesis and in the neoplastic state.
