We continue to test the hypothesis that the activation of PKC (a major TPA receptor) is an initial event in tumor promotion and ODC-gene transcription by the tumor promoter TPA. Thus, the continuing goals of this project are two-fold: 1. To determine the role of PKC in tumor promotion by TPA. Specifically, we plan: a) To determine the tumor promoting activity of diacylglycerols (DG) which activate PKC. We reported that, under the conditions where TPA is a complete tumor promoter, DG is a stage II tumor promoter. We will investigate the mechanism underlying differences between the tumor promoting activities of TPA and DG. b) To determine whether differences in tumor promoting susceptibility of B2S (sensitive) and B2R (resistant) strains of mice are due to differential translocation and down-regulation of PKC isozymes by TPA. c) To directly demonstrate the role of PKC in tumor promotion by TPA, we plan to use C3H10T1/2 cell line. We will transfect these cells with PKC gene expression vectors and determine whether different PKC isozyme overproducing cells are more sensitive to tumor promotion by TPA than their parental cells. 2. To determine the role of PKC in TPA-induced ODC gene transcription. We plan to: a) Transfect T24 cells with PKC gene expression vector and clone PKC overproducing transfectants to directly demonstrate the role of PKC in ODC gene expression. b) Analyze the mechanism of enhanced ODC gene transcription by TPA. In this study, we will use the 5'-flanking region of ODC gene to determine TPA-responsive enhancer elements, isolate trans- acting factors (proteins) and determine whether the trans-acting factors are substrates for PKC which may increase in vitro transcription of ODC gene. c) Analyze the relation of PKC down regulation to superinduction of ODC activity after repeated TPA applications to mouse skin. This study will permit an evaluation of the role of PKC in ODC gene transcription and tumor promotion by TPA.
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