CELLULAR AND BIOCHEMICAL EFFECTS OF TUMOR PROMOTERS

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The purpose is to continue our studies on the biochemical and molecular mechanisms that underlie the process of tumor promotion. Because of the evidence that the enzyme protein kinase C (PKC) plays a central role in growth control and mediates the action of the phorbol ester tumor promoters, we will concentrate on this enzyme system. Studies from several laboratories, including our own, indicate that the mammalian genome encodes several isoforms of this enzyme. Our major strategy will be to use genetic techniques to develop derivatives of the Rat 6 fibroblast cell line that stably overexpress the beta 1, gamma, or epsilon isoforms of PKC. These cell lines will be used to determine possible biochemical differences between these isoforms, to assess their effects on growth control and synergy with oncogenes, and to develop more specific inhibitors of PKC. Using 32P-labelling of intact cells, coupled with two dimensional gel electrophoresis and immunoprecipitation methods, we will attempt to identify the major protein substrates phosphorylated in these cells and the possible roles of these phosphoproteins in mediating the effects of various isoforms of PKC. Region-specific mutations will be introduced into different domains of PKC beta 1 to assess the biochemical functions of these domains and their roles in growth control. A specific project will explore the role of PKC in activation of human T cells and in replication of the human immunodeficiency virus. We will also isolate a full length cDNA sequence for the gene TPA-R1, whose expression is inhibited by TPA, and determine whether overexpression of this sequence suppresses the transformed phenotype. A long term goal is to develop transgenic strains of mice that overexpress specific isoforms and mutant forms of PKC, to define the role of this enzyme system in the multistage carcinogenic process in the intact animal. We are hopeful that the insights obtained from these studies, and the model systems that are developed, will provide a more rational basis for detecting potential tumor promoters in our environment and lead to new strategies of cancer prevention.