A STUDY OF TROPIC HORMONE ACTION IN CARCINOMA CELLS

这项研究的目的是阐明机制 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 单层培养中的α肿瘤细胞。 具体来说， 调节类固醇 17 α-羟化酶/C-17,20- 的过程 裂解酶（17α-羟化酶细胞色素 P-450 P-450，17α） 大鼠 Leydig 肿瘤细胞会（cAMP 独立）药物（例如， 转化生长因子、EGF 和雌激素）的合成 细胞色素 P-450 17 α 的含量和可翻译 mRNA 的水平 在培养的大鼠 Leydig 肿瘤细胞中编码这种酶的 被确定。 P-450 17 α 的合成速率为 从细胞提取物中进行特异性免疫沉淀后估计 用 (35S) 蛋氨酸对细胞进行放射性标记后，以及 形成一个用细胞RNA编程的体外翻译系统。 编码 P-450 17 α 的总 mRNA 水平 各种治疗方法将通过 Northern blotting 进行量化 放射性标记的切口翻译的特异性 cDNA 探针。 之后 阐明调节（非转录）5'-侧翼序列 大鼠 P-450 17 α 基因的“足迹”研究 该基因的区域将使用从分离的细胞核进行 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 响应元件相互作用的反式作用因子。 进一步描述调控序列将通过 调控基因序列构建体的缺失分析 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改变活性和选择性的作用 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 将评估肿瘤细胞中的情况。 大鼠 Leydig 肿瘤 R2C 细胞 除胆固醇外，该系还表达类固醇芳香酶 链断裂酶，但显示类固醇的低表达 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/组织特异性因素改变 17α-羟化酶和芳香酶的表达水平 R2C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的作用和机制，以及 自分泌因子改变分化细胞功能。

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.