HUMAN PROSTATIC 3A HYDROXYSTEROID DEHYDROGENASE

描述(改编自申请人的摘要)：在雄激素靶点中 组织3a-羟基类固醇脱氢酶(3a-HSD)可能调节 雄激素受体(AR)的相互转换占据 5a-双氢睾酮(5a-DHT，一种有效的雄激素) 5a-雄烷-3a，17b-二醇(3a-二醇，一种弱雄激素)。我们已经拿到了型号 2型和3型3a-HSD cDNAs在人前列腺中表达并过表达 大肠杆菌中的酶。对这些重组酶的动力学研究表明， 类型3 3a-HSD同时用作3a-和17b-HSD以停用活动 雄激素，而3型3a-HSD可使5a-DHT与3a-二醇相互转化。 3a-HSD基因在前列腺原代培养中的表达水平较高 上皮细胞多于基质细胞；3a-HSD mRNA水平升高 良性皮肤上皮细胞原代培养观察 前列腺增生症和前列腺癌组织。稳定的表达 表皮生长因子(EGF)上调3a-HSD mRNA水平 人前列腺细胞系LNCaP(雄激素敏感)和PC3(雄激素敏感) 不敏感)。 这项建议的重点是检查类型的生理功能 2和3a-HSD对雄激素代谢的调节及其活性 在调节前列腺细胞增殖方面，将进行研究。这将是 通过稳定地将2型和3型3a-HSD cDNA转染到 这些细胞。第二，内源性2型和3型3a-HSD水平 转录本将在从细胞系和正常细胞中提取的RNA中进行检测 用核糖核酸酶保护试验(RPA)检测前列腺癌。第三，EGF调节 将使用RPA检测2型和3型3a-HSD mRNA水平。中的更改 EGF介导的3A-HSD在细胞裂解物中的表达将通过 酶活性的免疫滴定。第四，要理解 原始性和EGF调节的2型和3型3a-HSD的表达 前列腺细胞，3a-HSD基因的5‘侧翼区将是 负责转录调控的测序和顺式作用元件 将确定两种亚型中的一种。

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.