Figure 7. The SRD5A3 mRNA level is lower in androgen- dependent xenografts than it is in androgen-independent AR-negative SCPC xenografts. PSA (top) and SRD5A3 (bottom) mRNA levels were analyzed by qRT-PCR for the androgen-dependent xenograft MDA PCa 183 and the androgen-independent AR-negative SCPC xenografts MDA PCa 144, MDA PCa 146, and MDA PCa 155. doi:10.1371/journal.pone.0028840.g007 ChIP assay. It is possible that AR indirectly regulates the transcription of SRD5A1 and SRD5A2, which is mediated by AR downstream transcription factors. It is also possible that AR can directly regulate the transcription of SRD5A1 and SRD5A2 but with AREs located in the distal promoter, enhancer, or introns of the genes, which were not uncovered by our study. Squelching may be another mechanistic possibility for AR- mediated transcription repression. AR has been reported to interact with the general transcription factor TFIIF [47], and AR can compete with T-cell factor (TCF) for binding with b-catenin, thus leading to the suppression of b-catenin/TCF-related transcription [48]. For negative regulation of SRD5A3 gene expression, we cannot rule out the possibility that AR competes for interaction with general transcription factors, thus interfering with the transcription of SRD5A3. However, given the direct binding of AR to the promoter of SRD5A3 as suggested by our ChIP and EMSA experiments, we believe that such a regulatory mechanism is unlikely. SRD5A3 is reportedly overexpressed in hormone-refractory samples from patients [10,11]. These reports suggest potential stage-dependent expression of 5a-reductase type in the prostate from SRD5A2 to SRD5A1 and SRD5A3, which is consistent with the fact that the use of 5a-reductase inhibitors is more relevant in early than in more advanced prostate cancers. It remains controversial whether SRD5A3 enzyme activity can be inhibited by finasteride or dutasteride. Although Yamana et al. reported that finasteride and dutasteride can block the activity of SRD5A3 [49], Titus and Mohler [11] stated in a book chapter that the 5a- Androgen Regulation of 5a-Reductase Isoenzymes reductase inhibitor dutasteride does not inhibit the activity of SRD5A3. The presence and expression level of SRD5A3, which is dependent on the presence of androgens (i.e., repression of SRD5A3 by androgens), could be an important contributor to resistance to therapy with a 5a-reductase inhibitor. Therefore, the role of SRD5A3 in both prostate cancer progression and prevention is worth further investigation. The differing expression of 5a-reductase isoenzymes may also contribute to response or resistance to 5a-reductase inhibitors. Because AR regulates 5a-reductase expression in a cell type– dependent manner, we would not expect to prevent prostate cancer in all men by administering finasteride (a specific SRD5A2 inhibitor) or dutasteride (an inhibitor of both SRD5A1 and SRD5A2); this concurs with the results of the PCPT and REDUCE trials. Additionally, whether the androgen regulation of 5a-reductase isoenzymes is testosterone- or DHT-driven needs further investigation. It also remains to be seen whether 5a- reductases are regulated by AR in a ligand-independent manner. This points to the fact that we may need combination therapy, possibly inhibiting 5a-reductase isoenzymes, the AR, and other factors involved in ligand-independent AR activation for efficient prevention of prostate cancer in all men. Supporting Information Figure S1 DHT regulates the mRNA level of 5a-reduc- tase in VCaP cells. In this experiment, VCaP cells were treated with ethanol (vehicle only) or with 1 nM, 10 nM, or 100 nM DHT for 24 hours. We quantified the mRNA levels of SRD5A1, SRD5A2, and SRD5A3 by using qRT-PCR. *p,0.05, ** p,0.01, *** p,0.001; 2-sided t test. (TIF) Figure S2 Testosterone regulates the mRNA level of 5a- reductase differently in different prostate cell lines. LNCaP (A), PWR-1E (B), BPH-1-AR (C), LAPC-4 (D), and C4- 2B4 (E) cells were treated with ethanol (vehicle only) or with 1 nM, 10 nM, or 100 nM testosterone for 24 hours. The mRNA levels of SRD5A1, SRD5A2, and SRD5A3 for all cells were quantified by using qRT-PCR. *p,0.05, **p,0.01, ***p,0.001; 2-sided t test. (TIF) Figure S3 The synthetic androgen R1881 regulates the mRNA level of 5a-reductase differently in different prostate cell lines. LNCaP (A), PWR-1E (B), BPH-1-AR (C), LAPC-4 (D), and C4-2B4 (E) cells were treated with ethanol (vehicle only) or 1 nM, 10 nM, or 100 nM R1881for 24 hours. The mRNA levels of SRD5A1, SRD5A2, and SRD5A3 for all cells were quantified by using qRT-PCR. *p,0.05, **p,0.01, ***p,0.001; 2-sided t test. (TIF) Figure S4 Regulation of 5a-reductase mRNA level by DHT is AR dependent in LAPC-4 cells. A, The AR protein level was analyzed by Western blotting with no siRNA, control siRNA, and four AR siRNA treatments. AR siRNA#1 and AR siRNA#2 had a stronger knockdown effect than the other two AR siRNAs did. B, LAPC-4 cells were treated with no siRNA, control siRNA or with AR siRNAs (siRNAs #1 and #2), followed by treatment with 2 nM DHT. The mRNA level of SRD5A1 was measured by using qRT-PCR and normalized to b-actin. The changes in mRNA levels resulting from DHT treatment are shown relative to the levels in cells treated with vehicle only. (TIF) Figure S5 Mutations in the nARE of SRD5A3 impair its binding with the AR. Mutations were made in the sequence of PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28840