The long-term objective of this research is to elucidate the mechanisms of low-dose radiation-induced carcinogenesis through studying its effects on a series of specific membrane-associated pathways known to be rapidly activated in whole or in part by the tumor promoting agents 12-0-tetradecanoyl-phorbol-13-acetate (TPA) and epidermal growth factor (EGF), as well as by a variety of growth-promoting hormones and other cell-stimulatory agents. We will examine the effects of radiation on levels of key components of these pathways: Phosphatidylinositol-4,5-biphosphate, inositol-1,4,5-triphosphate, diacylglycerols, and free cytoplasmic Ca2+, as well as on the activity of the phosphorylating enzyme protein kinase C. These levels/activities will be assayed: 1) at selected times post-irradiation, to determine whether any radiation-induced effects are rapidly produced (as expected from direct membrane perturbations) or are produced at later times (expected if such effects arise from radiation-produced alterations in gene expression); 2) during a radiation-initiated/promotor (TPA, EGF, or the hormone cortisone)-enhanced transformation regiment, to determine whether initiating doses of radiation alter inositol phospholipid-associated pathways known to be activated by selected tumor promoters; and 3) at various post-irradiation times in the presence of large, transformation-suppressing dietary protease inhibitors (the Bowman-Birk inhibitor and the chick-pea inhibitor), which are likely to be membrane-active and whose action in inhibiting specific steps in the series of inositol phospholipid-related membrane-associated pathways could support a role for such step(s) in carcinogenesis. Additionally, we will perform a series of radiation-induced transformation assays in the presence of modulators of levels/activities of specific components (cytoplasmic (Ca2+, protein kinase C) of the pathways studied, to ascertain whether altering the availabiligy of these components affects the ability of cells to undergo radiation-induced or radiation-initiated/promotor-enhanced transformation. Experiments will be performed in an in vitro system utilizing the mouse embryo-derived C3H10T-1/2 cell line used extensively for in vitro transformation research. These studies will provide information about whether there are direct or indirect effects of low-dose radiation on membrane-associated cellular activation processes known to be induced by tumor promotors, as well as elucidate the role of these activation processes in low-dose radiation induced cell transformation as it is promoted by hormonal and other promoting agents and inhibited by dietary protease inhibitors.
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