The objectives are two-fold: 1) to determine the role of Ca2+-activated, phospholipid-dependent protein kinase (PKC, a receptor for TPA) in mouse skin tumor promotion; and 2) to analyze the mechanism by which PKC activation transduces signal(s) for ornithine decarboxylase (ODS) induction, a biochemical change associated with skin-tumor promotion by TPA. Recently, we found that diacylglycerol (DG), an activator of PKC, and phospholipase C (which releases DG from membrane phospholipids) induced ODC activity which correlated with the amount of ODC mRNA in cultured newborn mouse epidermal cells. Also, palmitoylcarnitine (PC), an inhibitor of PKC, inhibited TPA-induced ODC and ODC mRNA and tumor promotion by TPA in intact mouse skin. These preliminary results have led us to hypothesize that PKC activation is an initial event in ODC-gene transcription and skin tumor promotion by TPA and that TPA-increased ODC mRNA is mediated by PKC-dependent phosphorylation, directly or indirectly, of nuclear proteins. Specifically, we plan to seek answers to the following questions: 1) Is PKC activation involved in tumor promotion? In this we plan to a) determine the tumor promoting activity of agents (e.g., diacylglycerols) which activate PKC; b) determine whether the agents which inhibit PKC (e.g., PC) also proportionally inhibit skin tumor promotion by TPA; c) determine whether PKC-dependent phosphorylation of endogenous epidermal proteins correlates to the degree of tumor promotion susceptibility in B2S, SENCAR, and CD-1 mice. 2) Does PKC affect ODC-gene transcription in cultured newborn mouse epidermal cells? 3) What are the physiological protein substrates (targets) for PKC? 4) Does PKC-dependent phosphorylation of nuclear proteins affect ODC-gene transcription? Tumor induction experiments will be performed by the initiation-promotion protocol. The rate of ODC-gene transcription will be determined by measurement of incorporation of (3H)uridine into ODC mRNA by DNA-excess filter hybridization. PKC-dependent phosphorylated proteins will be separated by 2-D gel electrophoresis and then characterized by peptide mapping and phosphoamino acid analysis. The role of PKC-mediated phosphorylated chromosomal proteins in regulating ODC-gene transcription will be directly examined by determining template activity of reconstituted chromatin. This study will permit an evaluation of the role of PKC in ODC induction and tumor promotion by TPA.
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