HUMAN PROSTATIC 3A HYDROXYSTEROID DEHYDROGENASE

描述（改编自申请人的摘要）：在雄激素靶向中 组织3 α-羟基类固醇脱氢酶（3a-HSDs）可以调节 雄激素受体（AR）通过相互转换占据 5 α-双氢睾酮（5 α-DHT，一种强效雄激素）， 5a-雄甾烷-3a，17 b-二醇（3a-二醇，弱雄激素）。 我们得到了 2型和3型3a-HSD cDNA在人前列腺中表达，并过表达 E.杆菌 这些重组酶的动力学研究表明， 3型3a-HSD作为3a-HSD和17 b-HSD发挥作用， 雄激素，而3型3 α-HSD将5 α-DHT与3 α-二醇相互转化。 3a-HSD mRNA在前列腺原代培养中表达水平较高， 上皮细胞比基质细胞; 3a-HSD mRNA水平升高， 在来源于良性肿瘤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。 稳态表达式 3a-HSD mRNA的状态水平被表皮生长（EGF）上调， 人前列腺细胞系，LNCaP（雄激素敏感）和PC 3（雄激素 不敏感）。 这个建议的重点是研究类型的生理功能 2型和3型3a-HSD调节雄激素代谢及其活性 调节前列腺细胞增殖。 这将 通过将2型和3型3a-HSD cDNA稳定地转染入 这些细胞。 第二，内源性2型和3型3a-HSD水平 将在从细胞系和正常细胞系中提取的RNA中检查转录物。 前列腺使用核糖核酸酶保护测定（RPA）。 第三，EGF调节 使用RPA检测2型和3型3a-HSD mRNA水平。 变化 将在细胞裂解物中通过以下方法检测EGF介导的3a-HSD表达： 酶活性的免疫滴定。 第四，了解 组成型和EGF调节的2型和3型3a-HSD表达， 在前列腺细胞中，3a-HSD基因的5 '-侧翼区将被 负责转录调控的序列顺式作用元件 两种异构体将被识别。

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.