HUMAN PROSTATIC 3A HYDROXYSTEROID DEHYDROGENASE

描述（改编自申请人的摘要）：雄激素目标 组织中的 3a-羟基类固醇脱氢酶 (3a-HSD) 可能调节 通过相互转化占据雄激素受体（AR） 5a-二氢睾酮（5a-DHT，一种强效雄激素） 5a-androstane-3a,17b-diol（3a-二醇，一种弱雄激素）。 我们已经获得了类型 2 型和 3 型 3a-HSD cDNA 在人前列腺中表达并过表达 大肠杆菌中的酶。 这些重组酶的动力学研究表明 3 型 3a-HSD 同时充当 3a-和 17b-HSD 来灭活活性 雄激素，而 3 型 3a-HSD 可将 5a-DHT 与 3a- 二醇相互转化。 前列腺原代培养物中 3a-HSD mRNA 水平较高 上皮细胞多于基质细胞； 3a-HSD mRNA 水平升高 在源自良性的上皮细胞的原代培养物中观察到 前列腺增生和前列腺癌组织。 稳定的表达 3a-HSD mRNA 的状态水平通过表皮生长 (EGF) 上调 人前列腺细胞系、LNCaP（雄激素敏感）和 PC3（雄激素 不敏感）。 该提案的重点是检查类型的生理功能 2型和3型3a-HSD调节雄激素代谢及其活性 将研究调节前列腺细胞增殖的作用。 这将 通过将 2 型和 3 型 3a-HSD cDNA 稳定转染至 这些细胞。 二、内源性2型和3型3a-HSD水平 将在从细胞系和正常细胞中提取的 RNA 中检查转录本 使用核糖核酸酶保护测定（RPA）检测前列腺。 三、EGF调节 将使用 RPA 检查 2 型和 3 型 3a-HSD mRNA 水平。 变化 通过 EGF 介导的 3a-HSD 表达将在细胞裂解物中进行检查 酶活性的免疫滴定。 四、要了解 组成型和 EGF 调节的 2 型和 3 型 3a-HSD 表达 前列腺细胞中，3a-HSD 基因的 5' 侧翼区域将是 负责转录调控的测序和顺式作用元件 将鉴定出两种亚型。

DESCRIPTION (Adapted from the Applicant's Abstract): In androgen target tissues 3a-hydroxysteroid dehydrogenases (3a-HSDs) may regulate the occupancy of androgen receptor (AR) by interconverting 5a-dihydrotestosterone (5a-DHT, a potent androgen) with 5a-androstane-3a,17b-diol (3a-diol, a weak androgen). We have obtained type 2 and type 3 3a-HSD cDNAs expressed in human prostate and overexpressed the enzymes in E. coli. Kinetic studies of these recombinant enzymes show that type 3 3a-HSD functions as both a 3a-and 17b-HSD to inactivate active androgens, whereas, type 3 3a-HSD interconverts 5a-DHT with 3a- diol. Levels of 3a-HSD mRNA were higher in primary cultures of prostatic epithelial cells than stromal cells; and elevated levels of 3a-HSD mRNA were observed in primary cultures of epithelial cells derived from benign prostatic hyperplasia and prostatic carcinoma tissues. Expression of steady state levels of 3a-HSD mRNA is up-regulated by epidermal growth (EGF) in human prostatic cell lines, LNCaP (androgen sensitive) and PC3 (androgen insensitive). The focus of this proposal is to examine the physiological functions of type 2 and type 3 3a-HSD in regulating androgen metabolism and their activities in modulating prostatic cell proliferation will be investigated. This will be accomplished by stably transfecting type 2 and type 3 3a-HSD cDNAs into these cells. Second, levels of endogenous type 2 and type 3 3a-HSD transcripts will be examined in RNA extracted from the cell lines and normal prostate using ribonuclease protection assay (RPA). Third, EGF-regulated type 2 and type 3 3a-HSD mRNA levels will be examined using RPA. Changes in 3a-HSD expression mediated by EGF will be examined in cell lysates by immunotitration of the enzyme activity. Fourth, to understand the constitutive and EGF- regulated type 2 and type 3 3a-HSD expression in prostatic cells, the 5'-flanking regions of the 3a-HSD genes will be sequenced and cis-acting elements responsible for transcription regulation of two isoforms will be identified.