A STUDY OF TROPIC HORMONE ACTION IN CARCINOMA CELLS

本研究旨在阐明慢性阻塞性肺疾病的发病机制。 (长期)促性激素对类固醇合成酶合成的调节 并利用大鼠和小鼠间质肿瘤细胞在肿瘤中的活性 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hCG刺激这些肿瘤细胞后的类固醇合成。 我们正在测定hCG治疗后三者含量的变化。 胆固醇侧链裂解酶的组成，即细胞色素 P450scc、素和HMGCoA还原酶，这是 胆固醇的生物合成。间质肿瘤细胞的蛋白质是 放射性标记的[35S]蛋氨酸。放射性标记的范围 胆固醇侧链裂解酶和HMGCoA的蛋白质 人绒毛膜促性腺激素处理不同时间后，用 免疫吸附技术与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法 (SDS-PAGE)。这些免疫隔离依赖于免疫球蛋白组份的能力 纯化的牛肾上腺蛋白和大鼠肝脏蛋白的抗血清 与相应的大鼠间质肿瘤细胞蛋白发生交叉反应。我们会 确定哪一种血清或生长因子可能是第二种 促进细胞生长和合成的间质肿瘤细胞趋向剂 类固醇合成酶和蛋白质。细胞培养将会被用 各种生长因子在hCG存在或不存在的情况下。我们将使用免疫球蛋白 牛肾上腺细胞色素P450scc抗血清的组份 肾上腺素和大鼠肝脏HMGCoA还原酶特异性免疫隔离 免疫印迹法检测Leyig肿瘤细胞裂解产物中的相应蛋白 吸墨水。促性腺激素和生长因子对细胞的影响 将对这些蛋白质的含量进行评估。总RNA将被分离出来 在有或没有hCG的情况下培养不同时间的细胞 并将在无单元翻译系统中进行翻译。之后 免疫隔离，十二烷基硫酸钠-PAGE和放射自显影我们将确定其作用 改变指导人绒毛膜促性腺激素体外合成的mRNA含量 胆固醇侧链裂解酶蛋白和HMGCoA还原酶。 (D)

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)