A STUDY OF TROPIC HORMONE ACTION IN CARCINOMA CELLS

本研究的目的是阐明慢性 类固醇生成酶合成的（长期）向性激素调节 在利用大鼠和小鼠Leydig肿瘤细胞的肿瘤中的活性， 单层培养 我们已经确定胆固醇是从头合成的， 在适当的条件下可能是胆固醇的主要来源 用于这些肿瘤细胞的hCG刺激后的类固醇生成。 我们正在测定hCG治疗后三种激素含量的变化， 胆固醇侧链裂解酶的组分，即，细胞色素 P450 SCC、睾丸氧还蛋白和HMGCoA还原酶， 胆固醇生物合成 Leydig肿瘤细胞的蛋白质是 放射性标记的[35S]蛋氨酸。 放射性标记的程度 胆固醇侧链裂解酶蛋白以及HMGCoA 在hCG处理的不同时间后，通过使用 免疫吸附技术和SDS聚丙烯酰胺凝胶电泳 （SDS-PAGE）。 这些免疫隔离依赖于免疫球蛋白的IgG级分的能力。 抗纯化的牛肾上腺和大鼠肝蛋白的抗血清， 与相应的大鼠睾丸间质肿瘤细胞蛋白交叉反应。 我们将 确定哪种血清或生长因子可以作为假定的第二个 通过促进细胞生长和合成的Leydig肿瘤细胞的趋化剂 类固醇生成酶和蛋白质。 细胞培养物将用 在存在或不存在hCG的情况下的各种生长因子。 我们将使用IgG 抗牛肾上腺细胞色素P450 SCC的抗血清级分， 肾上腺素还蛋白和大鼠肝HMGCoA还原酶特异性免疫隔离 通过Western印迹分析Leydig肿瘤细胞裂解物中的相应蛋白， 印迹 促性腺激素和生长因子对细胞的影响 将评估这些蛋白质的含量。 将分离总RNA 来自在hCG存在或不存在下培养不同时期的细胞 并且将在无细胞翻译系统中被翻译。 后 免疫分离，SDS-PAGE和放射自显影，我们将确定的作用 的hCG，以改变mRNA的含量，指导在体外合成 胆固醇侧链裂解酶蛋白和HMGCoA还原酶。 （丁）

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 [35S]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)