The objective of this proposal are to analyze the molecular processes linked to the mechanism of mouse skin tumor promotion by the tumor promoting agent 12-0-tetra-decanoylphorbol-13-acetate (TPA). Recently, we have shown, using mouse skin explants in culture, that Ca2+ may play a role in TPA induction of ornithine decarboxylase (ODC), a phenotypic change associated with skin tumor promotion by TPA. Ca2+ regulates some of the cellular functions via phosphorylation of nuclear proteins (catalyzed by Ca2+ dependent protein kinases), which may influence gene transcription. Since increased ODC activity by TPA is dependent on new RNA and protein synthesis, we hypothesize that the induction of ODC by TPA requires an increase in mRNA which is mediated by Ca+ dependent phosphorylation of epidermal nuclear proteins. Specifically, we plan to investigate: 1) alterations in the phosphorylation of nuclear proteins as a result of the application of TPA to intact mouse skin in vivo, 2) effects of Ca2+ manipulations on TPA-induced phosphorylation of epidermal nuclear proteins and the levels of mRNA for ODC in mouse skin explants in culture, and 3) effects of application of TPA to mouse skin on epidermal Ca2+ dependent protein kinases (both calmodulin as well as phospholipid requiring). Experiments will be performed with CD-1 mice, which are widely used in the study of mouse skin tumor promotion. The nuclear fraction of epidermis will be prepared by the hexylene glycol and sucrose method. The phosphorylated histones and nonhistone proteins from epidermal nuclei will be purified (Bio-Rex 70) and then separated by slab gel electrophoresis. RNA will be isolated from epidermis by homogenization in urea-lysis buffer, followed by phenol extraction, and CsCl gradient centrifugation. The level of mRNA for ODC will be determined by its translation in vitro reticulocyte lysate system); the translation product ODC will be quantitated by its binding to its radioactive irreversible inhibitor alpha-difluoromethylornithine. Kinase assays will be performed on partially purified epidermal fractions using [gamma-32P]ATP as a phosphate donor, and histones, casein, and phosvitin as receptor proteins. Since phosphorylation of nuclear proteins has been correlated with enhanced gene expression in several systems, the proposed study will be a step toward understanding the role of gene transcription in tumor promotion by TPA.
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