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: 3169158
• 负责人: J. Ian Mason
• 金额: $ 15.03万
• 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 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3169158
• 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; nonblood lipoprotein; 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这一机制(S) 慢性(长期)内分泌、旁分泌和自分泌 性腺类固醇合成酶合成和活性的调节 单层培养的α肿瘤细胞。具体地说， 调节类固醇17α-羟基酶/C-17，20- 裂解酶(17α-羟基酶细胞色素P-450 P-450，17α) 大鼠间质肿瘤细胞将(不依赖于cAMP)药物(例如， 转化生长因子、表皮生长因子和雌激素)的合成 细胞色素P-45017α和可翻译的mRNA水平 在培养的大鼠间质肿瘤细胞中编码这种酶的人将 要下定决心。P-450 17α的合成速度将是 从细胞提取液中进行特异性免疫沉淀后的估测 在用(35S)蛋氨酸放射性标记细胞之后，还 形成一个由细胞RNA编程的体外翻译系统。 免疫后P-450 17α编码基因的总mRNA水平 不同的处理将通过Northern blotting使用 一种放射性标记的缺口翻译的特异性cDNAs探针。后 调控(未转录)5‘-侧翼序列的阐明 在大鼠P-450 17α基因中，对这一基因的“足迹”研究 该基因的区域将使用从 肿瘤细胞在cAMP处理前后，以确定 该基因表达的改变涉及一种组织特异性 与cAMP反应元件相互作用的反式作用因子。 将通过以下方式进一步描述调控序列 用基因重组技术分析调控基因序列的缺失 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活性和选择性中的作用 间质肿瘤微粒子17α-羟基酶/C-17，20-裂解酶和 调节细胞色素b5合成的因子的性质 在肿瘤细胞中的作用将被评估。大鼠间质瘤R2C细胞 除了胆固醇外，LINE还表达类固醇芳香酶 链裂解酶，但类固醇低表达 17α-羟基酶/C-17，20-裂解酶。我们将测定内分泌 旁分泌、自分泌和改变的肿瘤/组织特异性因子 17α-羟基酶和芳香化酶的表达水平 R2C细胞系。这些发现将为我们提供对 内分泌旁分泌的相互依赖作用和机制，以及 改变分化细胞功能的自分泌因子。

项目成果

The synthesis and metabolism of [6-3H]-25-hydroxycholesterol in rat adrenal tumor cells.

大鼠肾上腺肿瘤细胞中[6-3H]-25-羟基胆固醇的合成和代谢。

• DOI: 10.1016/0022-4731(83)90257-1
• 发表时间: 1983
• 期刊: Journal of steroid biochemistry
• 作者: Mason,JI;Murry,BA;Aberhart,DJ
• 通讯作者: Aberhart,DJ

In vitro studies of the adrenal metabolism of halogenated side-chain analogues of cholesterol.

胆固醇卤化侧链类似物的肾上腺代谢的体外研究。

• DOI: 10.1016/0005-2760(83)90122-4
• 发表时间: 1983
• 期刊: Biochimica et biophysica acta
• 作者: Mason,JI;Arunachalam,T;Caspi,E
• 通讯作者: Caspi,E

Immunohistochemical studies of steroidogenic enzymes (aromatase, 17 alpha-hydroxylase and cholesterol side-chain cleavage cytochromes P-450) in sex cord-stromal tumors of the ovary.

卵巢性索间质瘤中类固醇生成酶（芳香酶、17 α-羟化酶和胆固醇侧链裂解细胞色素 P-450）的免疫组织化学研究。

• DOI: 10.1016/0046-8177(89)90010-5
• 发表时间: 1989
• 期刊: Human pathology
• 影响因子: 3.3
• 作者: Sasano,H;Okamoto,M;Mason,JI;Simpson,ER;Mendelson,CR;Sasano,N;Silverberg,SG
• 通讯作者: Silverberg,SG

Immunolocalization of aromatase, 17 alpha-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary.

人类卵巢中芳香酶、17 α-羟化酶和侧链裂解细胞色素 P-450 的免疫定位。

• DOI: 10.1530/jrf.0.0850163
• 发表时间: 1989
• 期刊: Journal of reproduction and fertility
• 作者: Sasano,H;Okamoto,M;Mason,JI;Simpson,ER;Mendelson,CR;Sasano,N;Silverberg,SG
• 通讯作者: Silverberg,SG

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells.

牛肾上腺皮质细胞原代培养物中 ACTH 诱导 3-羟基-3-甲基戊二酰辅酶 A 还原酶、胆固醇生物合成和类固醇生成。

• 发表时间: 1986
• 期刊: The Journal of biological chemistry
• 作者: Rainey,WE;Shay,JW;Mason,JI
• 通讯作者: Mason,JI