Androgen Regulation of 5a-Reductase Isoenzymes Figure 6. AR directly interacts with the nARE of the SRD5A3 gene. A, Agarose gel electrophoresis of the digested chromatin of LNCaP cells grown in the absence and presence of 100 nM DHT. DNA fragments generally range between 150 and 900 bp. B, Digested chromatin of LNCaP cells grown in the absence and presence of 100 nM DHT was used in the immunoprecipitation experiment with anti-AR antibody or normal mouse immunoglobulin G. Afterward, the protein–DNA crosslink was reversed, and purified DNA was used in the PCR reactions with primers flanking the nARE region. C, Three oligo probes were designed to cover the 2191/291 bp region in the promoter of SRD5A3. D, AR binds to the nARE of SRD5A3. The three (biotin-labeled) oligo probes were incubated with LNCaP cell nuclear extract, with LNCaP cell nuclear extract plus unlabeled oligo probes, or with LNCaP cell nuclear extract plus AR-specific antibody. doi:10.1371/journal.pone.0028840.g006 imply a transition in the regulation of SRD5A3 expression with prostate cancer pathogenesis and progression—a loss of positive regulation and gain of negative regulation—which is consistent with the report that a lower testosterone level was found among prostate cancer patients with higher Gleason scores than that in those with lower Gleason scores [46]. It is also consistent with the observation that SRD5A3 is overexpressed in hormone-refractory prostate cancers in which the androgen level is low [10,11]. Our identification of an nARE in the promoter of SRD5A3 allows us to further investigate the transition from positive to negative regulation of SRD5A3 expression that occurs with prostate cancer progression over time. The results of our evaluation of xenograft models provide additional supporting evidence of the androgen-negative regula- tion of SRD5A3. Because we did not use castrate xenograft models and thus could not directly compare the effect of androgen withdrawal on the AR-positive and -negative cells in vivo, we did observe SRD5A3 overexpression in the AR-negative prostate carcinoma xenografts MDA PCa 144 and MDA PCa 146, which is consistent with results from our cell line study and previously reported work [10,11]. Although morphologically similar, the original donor tumor and the MDA PCa 155 subline xenograft have slight differences from the MDA PCa 144 and MDA PCa 146 xenografts in terms of molecular characterization (person al communication with Dr. Sankar N. Maity; a separate manuscript under review elsewhere). It is possible that such differences have an effect on the expression of SRD5A3, resulting in the relatively low level of SRD5A3 in MDA PCa 155. In this study, we did not detect an ARE in the proximal promoter of SRD5A1 and SRD5A2, either by promoter analysis or PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28840