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: 3169152
• 负责人: J. Ian Mason
• 金额: $ 14.94万
• 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 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3169152
• 关键词: DNA footprinting; NADPH cytochrome c2 reductase; adrenocorticotropic hormone; affinity chromatography; aldehyde lyase; autoradiography; cellular oncology; centrifugation; cholesterol; cyclic AMP; cytochrome P450; cytochrome b; enzyme biosynthesis; epidermal growth factor; gel electrophoresis; gel filtration chromatography; gene expression; high density lipoproteins; hormone binding protein; hormone biosynthesis; hormone regulation /control mechanism; hormone related neoplasm /cancer; laboratory mouse; laboratory rat; luteinizing hormone; messenger RNA; neoplasm /cancer genetics; neoplastic cell; platelet derived growth factor; pregnanolone; radioimmunoassay; radiotracer; scintillation spectrometry; spectrometry; steroid 17alpha monooxygenase; steroid biosynthesis; testis neoplasms; tissue /cell culture; transforming growth factors; tritium

The objectives of this research are to elucidate the mechanism(s) of chronic (long term) endocrine, paracrine and autocrine regulation of steroidogenic enzyme synthesis and activity in gonad alpha tumor cells in monolayer culture. Specifically, the processes that regulate the steroid 17 alpha-hydroxylase/C-17,20- lyase (17 alpha-hydroxylase cytochrome P-450 P-450, 17 alpha) of rat Leydig tumors cells will (cAMP-independent) agents (e.g., transforming growth factors, EGF and estrogens) on the synthesis of cytochrome P-450 17 alpha and the level of translatable mRNA that encodes this enzyme in cultured rat Leydig tumor cells will be determined. The rate of synthesis of P-450 17 alpha will be estimated after its specific immunoprecipitation from cell extracts following radiolabeling of cells with (35S)methionine, and also form an in vitro translation system programmed with cellular RNA. The levels of total mRNA that encodes P-450 17 alpha after the various treatments will be quantified by Northern blotting using a radiolabeled nick-translated specific cDNA probe. After the elucidation of the regulatory (untranscribed) 5'-flanking sequence of the rat P-450 17 alpha gene, "foot-printing" studies of this region of the gene will be conducted using nuclei isolated from tumor cells before and after cAMP treatment, to determine whether the altered expression of this gene involves a tissue-specific trans-acting factor that interacts with a cAMP-responsive element. Further delineation of regulatory sequences will be achieved with deletion analysis of regulatory gene sequence constructs with the bacterial chloramphenicol acetyl transferase reporter gene. Utilizing a polyclonal antibody directed against rat cytochrome b5' the role of cytochrome b5 to alter the activity and selectivity of 17 alpha-hydroxylase/C-17,20-lyase of Leydig tumor microsomes and the nature of factors that regulate the synthesis of cytochrome b5 in tumor cells will be evaluated. The rat Leydig tumor R2C cell line expresses steroid aromatase in addition to cholesterol side chain cleavage enzyme, but demonstrates low expression of steroid 17 alpha-hydroxylase/C-17,20-lyase. We will determine endocrine paracrine, autocrine and tumor/tissue specific factors that alter the levels of expression of 17 alpha-hydroxylase and aromatase in the R2C cell line. These findings will provide insight into the interdependent actions and mechanisms of endocrine paracrine, and autocrine factors to alter differentiated cell function.

本研究的目的是阐明机制（S） 慢性（长期）内分泌、旁分泌和自分泌 性腺中类固醇生成酶合成和活性的调节 单层培养的α肿瘤细胞。 具体而言是 调节类固醇17 α-羟化酶/C-17，20- 裂解酶（17 α-羟化酶细胞色素P-450 P-450，17 α） 大鼠Leydig肿瘤细胞将（cAMP非依赖性）药剂（例如， 转化生长因子，EGF和雌激素）的合成 细胞色素P-450 17 α和可翻译mRNA的水平 在培养的大鼠睾丸间质肿瘤细胞中编码这种酶的基因 被确定。 P-450 17 α的合成速率将为 从细胞提取物中特异性免疫沉淀后估计 在用（35 S）甲硫氨酸放射性标记细胞后， 形成了一个体外翻译系统，用细胞RNA编程。 在细胞凋亡后，编码P-450 17 α的总mRNA水平 各种处理将通过北方印迹定量， 放射性标记的缺口翻译特异性cDNA探针。 后 阐明调控（未转录）5 '-侧翼序列 大鼠P-450 17 α基因的“足迹”研究， 将使用分离的细胞核进行基因的区域， cAMP处理前后的肿瘤细胞，以确定是否 该基因表达的改变涉及组织特异性 与cAMP反应元件相互作用的反式作用因子。 调控序列的进一步描绘将通过 用该方法对调控基因序列构建体进行缺失分析 细菌氯霉素乙酰转移酶报告基因。 利用针对大鼠细胞色素b5'的多克隆抗体 细胞色素b5在改变细胞色素B5的活性和选择性方面的作用 Leydig肿瘤微粒体的17 α-羟化酶/C-17，20-裂解酶和 调节细胞色素b5合成的因素的性质 将对肿瘤细胞进行评估。 大鼠睾丸间质细胞瘤R2 C细胞 除胆固醇侧外， 链裂解酶，但显示类固醇低表达 17 α-羟化酶/C-17，20-裂解酶。 我们将确定内分泌 旁分泌、自分泌和肿瘤/组织特异性因子， 17 α-羟化酶和芳香化酶的表达水平， R2 C细胞系。 这些发现将提供深入了解 内分泌旁分泌的相互依赖作用和机制， 自分泌因子以改变分化的细胞功能。