GENETIC ANALYSIS OF PEPTIDE HORMONE ACTION

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We will investigate the molecular mechanisms by which peptide hormones regulate the transcription of specific genes in cultured mammalian cell lines. Biochemical studies in many laboratories have determined the first intracellular steps in these hormone response pathways, but the later steps that lead to regulation of transcription remain unknown. We will use a combination of molecular biology and somatic cell genetics to define the steps and components of these pathways. Promoters and regulatory sequences of hormonally responsive genes (prolactin and SV40 early) will be fused to the structural gene for Eco gpt, thus creating hormonally responsive selectable marker genes. These hybrid genes will be introduced into hormonally responsive cultured cell lines (GH3 and HepG2, respectively) where they can be regulated by a variety of hormones and inducers (TRH, EGF, phorbol esters, and calcium for prolactin; phorbol esters for SV40). Selective culture media can be used to select for or against the expression of the gpt gene either in the presence or the absence of inducers. We will select for cell variants that lack the normal hormone responses and then characterize these variants genetically and biochemically. These same response pathways are involved in regulation of cell proliferation; in fact most of the currently known oncogene products are believed to be components of these pathways. Thus, the biochemical details of these pathways are essential for understanding the nature of cancer.