The objectives of this research are to elucidate the mechanism of chronic (long-term) tropic hormonal regulation of steroidogenic enzyme synthesis and activity in tumors utilizing rat and mouse Leydig tumor cells in monolayer culture. We have determined that cholesterol synthesized de novo under appropriate conditions may be the major source of cholesterol utilized for steroidogenesis after hCG stimulation of these tumor cells. We are determining changes after hCG treatment in the content of the three components of the cholesterol side-chain cleavage enzyme, i.e., cytochrome P450scc, testodoxin, and HMGCoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. The proteins of the Leydig tumor cells are radiolabeled with [?35?S]methionine. The extent of radiolabeling of the proteins of the cholesterol side-chain cleavage enzyme as well as HMGCoA reductase is determined after varying times of hCG treatment by use of immunoabsorbent techniques and SDS polyacrylamide gel electrophoresis (SDS-PAGE). These immunoisolations rely on the ability of IgG fractions of antisera against purified bovine adrenal and rat liver proteins to crossreact with the corresponding rat Leydig tumor cell proteins. We shall determine which serum or growth factor may serve as a putative second tropic agent for Leydig tumor cells by promoting cell growth and synthesis of steroidogenic enzymes and proteins. Cell cultures will be treated with various growth factors in the presence or absence of hCG. We shall use IgG fractions of antisera against bovine adrenal cytochrome P450scc, adrenodoxin, and rat liver HMGCoA reductase to immunoisolate specifically the corresponding proteins from Leydig tumor cell lysates by Western blotting. The influence of gonadotropin and growth factors on the cell content of these proteins will be assessed. Total RNA will be isolated from cells cultured in the presence or absence of hCG for varying periods and will be translated in a cell-free translation system. After immunoisolation, SDS-PAGE, and autoradiography we shall determine the role of hCG to alter the content of mRNA that directs the in vitro synthesis of the cholesterol side-chain cleavage enzyme proteins and HMGCoA reductase. (D)
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