A STUDY OF TROPIC HORMONE ACTION IN CARCINOMA CELLS

癌细胞中热带激素作用的研究

• 批准号: 3169156
• 负责人: J. Ian Mason
• 金额: $ 15.04万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-01-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3169156
• 关键词: adrenocorticotropic hormone; affinity chromatography; autoradiography; centrifugation; cholesterol; cyclic AMP; gel electrophoresis; gel filtration chromatography; high density lipoproteins; hormone biosynthesis; hormone regulation /control mechanism; luteinizing hormone; neoplastic cell; pregnanolone; radioimmunoassay; radiotracer; scintillation spectrometry; spectrometry; steroid biosynthesis; tissue /cell culture; tritium

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)

本研究的目的是阐明慢性病的发病机制 （长期）类固醇酶合成的热带激素调节 以及利用大鼠和小鼠 Leydig 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 在 hCG 刺激这些肿瘤细胞后用于类固醇生成。 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成分，即细胞色素 P450scc、睾丸素和 HMGCoA 还原酶（限速酶） 胆固醇生物合成。 Leydig 肿瘤细胞的蛋白质是 用[ 35 S]甲硫氨酸放射性标记。 放射性标记的范围 胆固醇侧链裂解酶以及 HMGCoA 的蛋白质 还原酶是在不同时间的 hCG 治疗后通过使用 免疫吸附技术和 SDS 聚丙烯酰胺凝胶电泳 (SDS-PAGE)。 这些免疫分离依赖于 IgG 片段的能力 抗纯化牛肾上腺和大鼠肝脏蛋白的抗血清 与相应的大鼠 Leydig 肿瘤细胞蛋白发生交叉反应。 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，成为 Leydig 肿瘤细胞的趋向剂 类固醇生成酶和蛋白质。 细胞培养物将被处理 存在或不存在 hCG 的情况下的各种生长因子。 我们将使用 IgG 牛肾上腺细胞色素 P450scc 抗血清的级分， 肾上腺氧还蛋白和大鼠肝脏 HMGCoA 还原酶特异性免疫分离 Western 检测 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 来自在存在或不存在 hCG 的情况下培养不同时间的细胞 并将在无细胞翻译系统中进行翻译。 后 免疫分离、SDS-PAGE 和放射自显影我们将确定其作用 hCG 来改变 mRNA 的含量，从而指导体外合成 胆固醇侧链裂解酶蛋白和 HMGCoA 还原酶。 (四)