Figure 1.
The expression pattern of 5α-reductase in prostate cell lines varies.
Graphs depict the relative mRNA levels of the SRD5A1, SRD5A2, and SRD5A3 isoenzymes in PWR-1E, BPH-1-AR, LAPC-4, LNCaP, and C4-2B4 cells as determined on qRT-PCR analysis.
Figure 2.
DHT regulates the mRNA level of 5α-reductase differently in different prostate cell lines.
A, LNCaP cells were treated with ethanol (vehicle only) or different concentrations of DHT (1 nM, 10 nM, 100 nM) for 24 and 48 hours. PWR-1E (B), BPH-1-AR (C), LAPC-4 (D), and C4-2B4 (E) cells were treated the same way but for only for 24 hours. The mRNA levels of SRD5A1, SRD5A2, and SRD5A3 for all cell lines were quantified by qRT-PCR. *p<0.05, **p<0.01, ***p<0.001; 2-sided t test.
Figure 3.
Androgen regulation of 5α-reductase mRNA level is sensitive to actinomycin D treatment.
BPH-1-AR (A) and LNCaP (B), and PWR-1E (C) cells were treated for 30 minutes with DMSO (vehicle-only control) or actinomycin D at 1 µg/mL and 5 µg/mL concentrations. Treatment with either ethanol (vehicle only) or 10 nM DHT or testosterone followed. We quantified the mRNA levels of SRD5A1, SRD5A2, and SRD5A3 by qRT-PCR.
Figure 4.
Regulation of 5α-reductase mRNA level by DHT is AR dependent.
A, The AR protein level of each prostate cell line was analyzed by Western blotting. BPH-1-GFP cells were used as a negative control for AR expression. B, The AR protein level was analyzed by Western blotting for LNCaP cells (left) with three control and two AR siRNA treatments. The AR mRNA level was analyzed by qRT-PCR for PWR-1E cells (right), also with three control and two AR siRNA treatments. C, LNCaP and PWR-1E cells were treated with control siRNAs and AR siRNAs and then treated with 2 nM DHT. We measured the mRNA levels of SRD5A1, SRD5A2, and SRD5A3 by using qRT-PCR and normalized the values to β-actin. The changes in mRNA levels with DHT treatment are shown relative to the levels in cells treated with vehicle only.
Figure 5.
The SRD5A3 promoter contains a functional nARE.
A, Deletion analysis of SRD5A3 promoter to narrow down the location of the nARE-containing region. LNCaP cells were transfected with luciferase constructs containing a deletion series of SRD5A3 promoter and then treated with ethanol (vehicle only; −DHT) or 100 nM DHT (+DHT) for 24 hours. Cells were then harvested, and their lysates were used for the luciferase assay. B, SRD5A3 has an nARE. LNCaP cells were transfected with pGL3–4ARE–E4 constructs with and without insertion of the SRD5A3 promoter (−191/−72 bp) sequence in both orientations or with insertion of an SRD5A1 promoter fragment (−1601/−1501 bp). Cells were treated with ethanol (vehicle only) or 100 nM DHT for 24 hours and then harvested for the luciferase assay. C, Mutations in the nARE abolished its suppressive effect. LNCaP cells were transfected with pGL3–4ARE–E4 constructs with and without insertion of SRD5A3 promoter (−191/−72 bp) or with insertion of the mutated SRD5A3 promoter (−191/−72 bp) and then subjected to DHT treatment and the luciferase assay.
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 −191/−91 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.
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.