and MluI sites, as were further deletion constructs. LNCaP cells were transfected with these constructs by using Fugene 6 reagent (Roche) or Lipofectamine 2000 (Invitrogen). To test the AR-dependent repression ability of SRD5A3, we also inserted its promoter (2191/272 bp) into the pGL3–4ARE– E4–luc construct at the PstI and XhoI sites. LNCaP cells were transfected with it and then grown in the absence and presence of DHT for 24 hours. A dual-luciferase assay was conducted according to Promega’s protocol. The luciferase ratio was derived by dividing the luciferase activity by the Renilla activity. Mutagenesis Mutations were made in the nARE region of the SRD5A3 promoter by using a QuickChange II XL site-directed mutagenesis kit (Strategene, Agilent Technologies, Inc., Santa Clara, CA) according to the manufacturer’s instructions. The sequence CTGTTTTGCGTCT was mutated to ATTTTTTTATTAT in the context of the pGL3–4ARE–E4–luc construct. Electrophoretic mobility-shift assay (EMSA) AR’s binding to double-stranded oligos was assessed in LNCaP cell nuclear extracts by performing EMSA with a LightShift kit (all kits for EMSA were from Thermo Scientific) according to the manufacturer’s instructions. Nuclear protein extracts were isolated from LNCaP cells, and protein concentrations were determined by using a BCA protein assay kit. Oligos were designed to cover the 2191/291 region on the SRD5A3 promoter and labeled by using a biotin 39-end DNA labeling kit according to the manufacturer’s instructions. AR binding to double-stranded oligos was assessed in LNCaP cell nuclear extracts by EMSA using a LightShift kit according to the manufacturer’s instructions. Unlabeled oligos were used in EMSA as one of the controls. Biotin-labeled DNA was detected on the nylon membrane by using a chemiluminescent nucleic acid detection module kit. Chromatin immunoprecipitation (ChIP) assay LNCaP cells were cultured in both the presence and absence of 100 nM DHT, and ChIP was performed by using a kit from Cell Signaling Technology, Inc. (Danvers, MA), according to their protocol. Briefly, cells were treated with 37% formaldehyde to cross-link proteins to DNA. Cross-linked chromatin was digested by micrococcal nuclease to a length of 150–900 bp, and the nuclear membranes were broken by sonication. AR-specific antibodies (Dako) were used to precipitate the short chromatin fragments, which were then washed and eluted. The protein–DNA cross-links were reversed and DNA further purified by using spin columns in each PCR reaction. The forward primer for the nARE was CTGTTTTGCGTCTTTGCTTCTG, and the reverse primer was GAGGTCCTTGGTCCTGGTC, which amplify a 120-bp product. Xenograft models RNA from the MDA PCa 183, MDA PCa 144, MDA PCa 146, and MDA PCa 155 xenograft models was kindly provided by Dr. Sankar N. Maity and Dr. Nora M. Navone. The MDA PCa 183 xenograft was derived from androgen-dependent prostate carci- noma, whereas the others were derived from AR-negative castrate-resistant prostate carcinomas with small-cell prostate carcinoma (SCPC) morphology. Quantification of the relative mRNA level of xenograft SRD5A3 and prostate-specific antigen (PSA) was done by using qRT-PCR with SYBR Green (Applied Biosystems, Inc.). qRT-PCR was Androgen Regulation of 5a-Reductase Isoenzymes performed as described in [36]. The primer sequences used were as follows: SRD5A3: forward, 59-TCCAAGCTGGCTT- CATGGTT-39 located on exon 2 and reverse, 59-CACTCGAA- GAGTCTTCGTAA-39 located on exon 3; PSA: forward, 59-GAGAGCTGTGTCACCATGTG-39 located on exon 1 and reverse, 59-CACAATCCGAGACAGGATGA-39 located on exon 2. Statistical analysis Data are presented as means 6 SD. Two-sided t tests were conducted for comparing means between androgen-treated samples and controls. Significance was set at a p value of 0.05. Results All three 5a-reductase isoenzymes are present in the tested human prostate cell lines, with varying expression patterns To identify good model systems for studying the functions of 5a- reductase isoenzymes, we evaluated the mRNA levels of the 5a- reductase isoenzymes in different prostate cell lines, including PWR-1E immortalized normal prostatic epithelial cells; BPH-1- AR benign prostatic hyperplasia (BPH) cells, which stably express AR; LAPC-4 and LNCaP androgen-sensitive prostate cancer cells; and C4-2B4 androgen-independent cells. PWR1-E, BPH-1-AR and LAPC-4 cells express wild-type AR, whereas LNCaP and C4- 2B4 cells express a mutant AR, T877A. (The characteristics of these cell lines, including their source, androgen sensitivity, and AR-expression status, are summarized in Table S1.) As Figure 1 illustrates, the mRNA of all three 5a-reductase isoenzymes was detected on qRT-PCR analysis of each cell line, indicating that all three are expressed in these cell lines. However, the mRNA level of each isoenzyme differed between cell lines, and each isoenzyme had a distinctive expression pattern. 5a-Reductase mRNA levels are regulated by androgens in a cell type–specific manner To test whether the mRNA level of 5a-reductase is regulated by androgens, we treated LNCaP cells with either ethanol (i.e., vehicle only) or with testosterone or DHT at different concentra- tions (1, 10, and 100 nM). The normal plasma concentration of testosterone in men ranges from 350 to 1050 ng/dL (12.1– 36.4 nM) [37], and the castrate level of testosterone is less than 50 ng/dL (1.73 nM) [38]. The plasma concentration of DHT is about 1/10 of the testosterone concentration [39,40]. Thus, we treated cells with concentrations of androgen that are close to the castrate, physiologic, and superphysiologic levels. Our qRT-PCR analysis showed that DHT treatment resulted in an increased level of SRD5A1 mRNA, whereas it led to decreased levels of SRD5A2 and SRD5A3 mRNA in LNCaP prostate cancer cells (Fig. 2A). We also tested the effect of DHT on other cell lines. DHT did not notably affect the mRNA level of 5a-reductase in PWR-1E cells (Fig. 2B), whereas in BPH-1-AR cells, DHT increased the mRNA levels of all three isoenzymes (Fig. 2C). In LAPC-4 cells, which express wild-type AR, DHT up-regulated SRD5A1 expression without affecting SRD5A2 and SRD5A3 expression (Fig. 2D). And in the androgen-independent C4-2B4 prostate cancer cells, DHT regulated 5a-reductase expression similarly to its regulation in LNCaP cells, but to a lesser degree (Fig. 2E). We found it interesting that DHT regulates the mRNA level of SRD5A3 in a cell type–specific manner, a finding we have not seen reported before. To evaluate whether this happens only in LNCaP or LNCaP-derived cell lines, we similarly treated VCaP cells, PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28840