of the 5a-reductase isoenzymes could contribute to both sensitivity and resistance. Unlike androgen ablation, which decreases prostatic testosterone and DHT, inhibition of 5a-reductase activity decreases DHT but increases testosterone [24,25,26]. Since 5a- reductase inhibitors change the testosterone-to-DHT ratio, and given the critical role of 5a-reductase in AR signaling, the different 5a-reductase expression levels may provide clues about response and resistance to 5a-reductase inhibitors in prostate cancer prevention. Androgens can affect the expression of SRD5A1 and SRD5A2 in different tissues and cell types. In the rat ventral prostate, positive regulation of SRD5A2 by androgen has been reported [27], and in the rat testis, negative regulation of SRD5A1 [28]. Androgen ablation led to decreased immunostaining of 5a-reductase [29]. SRD5A1 and SRD5A2 are also regulated by testosterone and DHT in T and B lymphoid cells [30] and in rat liver and brain [31,32,33,34]. However, how 5a-reductase expression is regulated in human prostate cells has not been extensively investigated. Our primary purpose of this study was thus to evaluate androgen regulation of the 5a-reductase isoenzymes in human prostate cells. We further investigated whether the regulatory effects of androgens on the 5a-reductases are mediated by AR and whether a direct interaction exists between the cis-regulatory elements of 5a-reductase isoenzymes and AR. Our data demonstrated cell type–specific androgen regulation of the isoenzymes that is mediated by AR. To our knowledge, this is the first demonstration that AR can directly bind to the negative androgen response element (nARE) of the SRD5A3 promoter in LNCaP prostate cancer cells. Our findings may have clinical implications for identifying men whose disease may benefit from 5a-reductase inhibitors. Materials and Methods Cell lines and cultures PWR-1E, LNCaP, and VCaP cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA); BPH- 1-GFP, BPH-1-AR, and C4-2B4 cells were a gift from Dr. Sue- Hwa Lin (The University of Texas MD Anderson Cancer Center, Houston, TX); and LAPC-4 cells were kindly provided by Dr. Robert Reiter (University of California, Los Angeles, CA). PWR-1E cells were maintained in serum-free keratinocyte medium (Invitrogen, Life Technologies Corp., Carlsbad, CA) supplemented with 50 mg/mL bovine pituitary extract, 5% L- glutamine, and 5 ng/mL epidermal growth factor. LNCaP, C4- 2B4, BPH-1-GFP, and BPH-1-AR cells were maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S). LAPC-4 cells were maintained in Iscove’s modified Dulbecco’s medium (Invitrogen) supplement- ed with 5% FBS and 1% P/S. VCaP cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS and 1% P/S. All cultures were maintained at 37uC in humidified air with 5% CO2. Cell lines were validated at MD Anderson’s Characterized Cell Line Core by STR DNA fingerprinting using the AmpF,STR Identifiler kit (Applied Biosystems, Life Technologies Corp., Carlsbad, CA). The STR profiles were compared to the known ATCC fingerprints, to the Cell Line Integrated Molecular Authentication Database version 0.1.200808 (http://bioinformatics.istge.it/clima/) [35], and to MD Anderson’s fingerprint database. The STR profiles of PWR-1E, LNCaP, C4-2B4, and VCaP cells matched known DNA fingerprints; those of BPH-1-AR and LAPC-4 cells were unique. Androgen Regulation of 5a-Reductase Isoenzymes Quantitative reverse-transcription PCR (qRT-PCR) Total RNA was extracted from each cell line by using an RNeasy Plus mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s protocol. qRT-PCR was performed by using a TaqMan One-Step RT-PCR kit (Applied Biosystems, Life Technologies Corp.), according to the manufacturer’s instructions. Briefly, the qRT-PCR setting for each reaction was 48uC for 30 minutes, 95uC for 10 minutes, and 42 cycles of 95uC for 15 seconds and 60uC for 1 minute. Human b-actin was used as the endogenous control in each reaction. Primer and probes for SRD5A1, SRD5A2, and SRD5A3 genes were also from Applied Biosystems. Androgen treatment Testosterone and DHT were purchased from Sigma-Aldrich (St. Louis, MO). R1881, a synthetic androgen, was kindly provided by Dr. Sue-Hwa Lin. Cells were seeded in 12-well plates with their regular growth medium. After serum starvation overnight, cells were exposed to ethanol (control) or to 1 nM, 10 nM, or 100 nM androgen. After 24-hour and 48-hour incubation, cells were harvested and RNA extracted for qRT- PCR analysis. Actinomycin D treatment BPH-1-AR, LNCaP, and PWR-1E cells were treated with dimethyl sulfoxide (DMSO; control) or 1 mg/mL or 5 mg/mL of actinomycin D for 30 minutes, followed by ethanol (control) or 10 nM DHT. After 24 hours’ incubation, cells were harvested and total RNA extracted. Transfection with small interfering RNA (siRNA) To knock down AR expression, we seeded LNCaP cells in 12- well plates, serum starved them overnight, and then transfected them with 20 nM AR siRNA or control siRNA by using DharmaFECT 1 (Dhamarcon, Inc., Thermo Fisher Scientific, Lafayette, CO). After incubation overnight, cells were exposed to ethanol (control) or 2 nM DHT. The AR siRNA and control siRNA were obtained from Dhamarcon. Western blot analysis After 24 hours of incubation with siRNA, the cells were harvested and centrifuged at 5,000 rpm for 5 minutes. Cell pellets were resuspended in RIPA buffer (Boston Bioproducts, Inc., Ashland, MA) with protease inhibitor (Roche, Mannheim, Germany), incubated for 20 minutes with occasional vortex mixing, and then centrifuged at 14,000 rpm for 10 minutes. The supernatant was decanted and saved for Western blotting. The whole protein-extraction procedure was performed at 4uC, and the protein concentration was measured by using the BCA assay (Thermo Fisher Scientific, Inc., Waltham, MA). The supernatant was boiled for 5 minutes, loaded onto polyacrylamide gel, run, and transferred to a PVDF membrane, which was then blocked in TBST (TBS with 0.2% Tween 20) + 5% milk for 1 hour before being probed with anti-AR antibody (Dako North America, Inc., Carpinteria, CA) in blocking buffer overnight at 4uC, followed by incubation at room temperature for 1 hour with secondary horseradish peroxidase–conjugated anti-mouse antibody. Detec- tion was performed by using an electrochemiluminescence kit (Amersham, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Luciferase assay The SRD5A3 promoter (21027/+155 bp) was cloned into the pGL2 basic vector (Promega Corp., Madison, WI) at the XhoI PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28840