Tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), and transforming oncogenes disrupt normal controls of gene expression and cell proliferation by acting on intracellular signal transduction pathways. One of the earliest changes in gene expression elicited by TPA, and by several oncogenes, is induction of ornithine decarboxylase (ODC). The goal of this project is to understand how the regulation of ODC is altered during the progression of normal cells to neoplasia. Towards this aim, we will compare the mechanisms by which TPA and a physiological growth factor, insulin, regulate ODC mRNA levels in a series of normal and transformed rat liver cells. We hypothesize that insulin and TPA each activate protein kinase cascades which regulate ODC by mechanisms with both common and distinct features. In order to understand these pathways, we propose to characterize the molecular events leading to ODC induction through the following specific aims. (1.) What are the DNA sequence elements required to regulate ODC transcription? The cis-acting regulatory elements in the rat ODC gene will be identified using transient expression assays. Those elements required for basal expression, induction by TPA and insulin, and inhibition by retinoic acid will be mapped. Qualitative and/or quantitative changes in ODC expression and the activity of these cis-acting elements will be studied in a series of normal, v-raf and v-raf + v-myc- transformed rat liver epithelial cells (RLE), and in the H35 hepatoma cell. (2.) What are the DNA-binding proteins required for the regulation of ODC transcription? The factors which regulate ODC transcription will be identified and characterized by protein-DNA binding assays; novel factors will be cloned. (3.) How is the activity of transcription factors required for ODC expression regulated? We will determine if transcription factors involved in ODC regulation are phosphorylated in response to TPA or insulin, and if so, whether the phosphorylation has effects on either DNA binding or transcriptional activation. In addition, we will determine whether the product of the c-raf-1 oncogene acts as an intermediate in signal transduction pathways involved in the regulation of ODC transcription or translation. The relationship of these regulatory factors to tumorigenesis will be assessed in the normal and transformed RLE. The results of these studies will provide mechanistic insight into the regulation of proliferation and gene expression by insulin and TPA in normal and transformed liver epithelial and hepatoma cells, and will contribute to an understanding of alterations in gene expression during tumorigenesis.
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