D5-Androstenediol is a natural hormone with androgenic activity in human prostate cancer cells (ARA70yhydroxyf lutamideycasodexytestosteroney7-oxo-DHEA)

It is known that androst-5-ene-3b,17b-diol (Adiol), a precursor of testosterone (T), can activate estrogen target genes. The androgenic activity of Adiol itself, however, is poorly understood. Using a transient transfection assay, we here demonstrate in human prostate cancer cells that Adiol can activate androgen receptor (AR) target genes in the presence of AR, and that AR coactivator ARA70 can further enhance this Adiol-induced AR transcriptional activity. In contrast to this finding, an active metabolite of dehydroepiandrosterone, 7-oxo-dehydroepiandrosterone, does not activate AR target gene in the absence or presence of ARA70. Thin layer chromatography analysis reveals that T, dihydrotestosterone, and 17b-estradiol are undetectable in human prostate cancer DU145 cells after treatment with Adiol. Additionally, a proteolysis assay shows that a distinct ligand-receptor conformational difference exists between T-AR and Adiol-AR. Together, the above findings and the fact that T, but not Adiol, can induce transcriptional activity in a mutant AR (mtAR708), suggest that, without being metabolized into T, Adiol itself may represent a natural hormone with androgenic activity in human prostate cancer cells. Because two potent antiandrogens, hydroxyf lutamide (Eulexin), and bicalutamide (casodex), that are widely used for the treatment of prostate cancer, fail to block Adiol-mediated induction of AR transcriptional activity in prostate cancer cells, the effectiveness of so-called ‘‘total androgen blockage,’’ a standard treatment for prostate cancer, may need to be reevaluated. Androst-5-ene-3b,17b-diol (D5-Androstenediol or Adiol), derived from dehydroepiandrosterone (DHEA) and convertible into testosterone (T) (for detail, see Fig. 1) (1), has been suggested to play a role in the regulation of immune responses (2), obesity (3), and the genesis of estrogen-sensitive carcinomas, such as breast cancer (4, 5). Since 1954 (6), Adiol has been known to have estrogenic activity at physiological concentrations (1, 7). Accordingly, the estrogenic effect of Adiol, which is mediated by the estrogen receptor (ER), has been proposed as an essential female hormone that can partially replace the loss of 17b-estradiol (E2) for postmenopausal women. Using transient transfection assay, Kokontis et al. (8) also reported that Adiol may convert to 5a-dihydrotestosterone (5a-DHT) and then induce the androgen target gene in human prostate cancer PC-3 cells. The androgenic activity of Adiol, despite its ultimate conversion to T, is poorly understood, and there is no reported evidence of androgen receptor (AR)-mediated effects of Adiol itself. Using the yeast growth assay, the mammalian two-hybrid system, and a transient transfection assay, we investigated the possible androgenic effect of Adiol in the presence of AR and ARA70. Our results suggest that Adiol itself can activate AR transcriptional activity in human prostate cancer cells, and that ARA70 can further enhance Adiolmediated activation of the AR. MATERIALS AND METHODS Chemicals and Plasmids. Adiol, DHEA, androst-5-ene7,17-dione (Adione), T, DHT, and E2 were purchased from Sigma, 7-oxo-DHEA was synthesized from DHEA as described (9), trilostane was provided by T.-M. Lin (University of Wisconsin, Madison), hydroxyflutamide (HF, Eulexin), and bicalutamide (casodex) were provided by G. Wilding (University of Wisconsin, Madison). pSG5-wild-type AR (wtAR) and pSG5-ARA70 were constructed as described (10), the two mutant ARs, mtAR877 [codon 877 mutation, threonine to serine, derived from a prostate cancer (11)] and mtAR708 [codon 708 mutation, glutamic acid to lysine, derived from a partial androgen insensitive syndrome patient (12)], were provided by S. P. Balk (Beth Israel Hospital, Boston) and H. Shima (Hyogo Medical College, Japan), respectively. pGAL0mtAR877 and pGAL0-mtAR708 were constructed by inserting fragments—mtAR877 and mtAR708, respectively—into the pGAL0 vector that contained the GAL4 DNA binding domain, as described (13). Similarly, pGAL4-VP16 that contained the GAL4 DNA binding domain-linked to activation domain (AD) of VP16 was used to construct the ARA70 fusion. Yeast Growth Assay. Yeast cells (gifts from A. J. Caplan, Mount Sinai Medical Center, New York, NY) (14) transformed with the reporter and expression plasmids were grown at 30°C overnight in -3SD medium (-histidine, -leucine, and -tryptophan) with 25 mM 3-aminotriazole (Sigma) and hormones. Yeast transformations were performed by using the modified lithium acetate transformation procedure (15). The cell density was determined from the OD660 value. Cell Culture, Transfections, and Reporter Gene Expression Assays. Human prostate cancer cell lines, DU145, PC-3, and LNCaP, were maintained in DMEM containing 5% fetal calf serum. Transfections and chloramphenicol acetyltransferase (CAT) assays were performed as described (13). Briefly, 4 3 105 cells were plated on 60-mm dishes 24 h before transfection, and the medium was changed to phenol red free DMEM with 5% charcoal-stripped fetal calf serum 1 h before transfection. The cells were transfected by using the calcium phosphate precipitation method. The total amount of DNA was adjusted The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9511083-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: Adiol or D5-androstenediol, androst-5-ene-3b,17bdiol; DHEA, dehydroepiandrosterone; T, testosterone; ER, estrogen receptor; AR, androgen receptor; E2, 17b-estradiol; Adione, androst5-ene-7,17-dione; DHT, dihydrotestosterone; HF, hydroxyflutamide; wtAR, wild-type AR; CAT, chloramphenicol acetyltransferase; TLC, thin layer chromatography, MMTV, mouse mammalian tumor virus. ‡To whom reprint requests should be addressed: e-mail: chang@ pathology.rochester.edu.