Article Figures & Data
Figures
- Fig. 1.
Biochemical analysis of the CWR22Rv1 AR. A, detergent lysates of LNCaP, CWR22Rv1, DU-145, and PC-3 cells were separated by 8% SDS-PAGE followed by immunoblot analysis with the AR441 AR monoclonal antibody. Arrows indicate the positions of the LNCaP and CWR22Rv1 full-length AR proteins (top) and the smaller CWR22Rv1 isoform (bottom). Molecular mass standards are indicated on the right. B, antibody mapping of the truncated CWR22Rv1 AR was performed by immunoblot analysis (as described in A) with antibodies specific for the NH2-terminal 21 residues (PG-21), a central region of the transactivation domain (AR441), and the COOH-terminal LBD (C-19). LNCaP (L) lysates were examined adjacent to CWR22Rv1 (C) for comparison. Arrows indicate the full-length and truncated AR proteins.
- Fig. 2.
Cellular distribution, DNA binding, and interaction of the CWR22Rv1 AR isoforms. A, nuclear (N) and cytosolic (C) extracts were prepared from LNCaP and CWR22Rv1 cells grown for 3 days in medium supplemented with FBS or in AD medium supplemented with CDT FBS (CDT). AR immunoblot analysis was performed with the AR441 monoclonal antibody on 8% SDS-PAGE-separated proteins from 50 μg of cytosolic extract and the cell-equivalent amount of nuclear extract for each sample (top panel). The integrity of nuclear/cytosolic fractionation was confirmed by partitioning of NSE to the cytosolic extract (bottom panel). B, DNA binding ability of full-length and truncated AR in CWR22Rv1 cells. Nuclear extracts were prepared from CWR22Rv1 cells cultured in the absence of androgen for 3 days (−) and stimulated with DHT (1 nm) for 2 h (+). These were combined with biotinylated double-stranded oligonucleotide probes representing the ARE and ARR of the PSA promoter in DNA pull-down assays described in “Materials and Methods.” Proteins in the resulting DNA-protein complexes were separated by 8% SDS-PAGE and analyzed by immunoblot analysis for the AR with the AR441 monoclonal antibody. The full-length (AR) and truncated (ARΔLBD) receptors are indicated by arrows. As a negative control, the assay was performed without the addition of the probe (Lane 2). CWR22Rv1 cell lysate was included as a reference for the two AR forms (Lysate, Lane 1). C, interaction between the two CWR22Rv1 AR forms was investigated by immunoprecipitation with the COOH terminus domain-specific antibody (C-19), followed by immunoblot analysis with the AR441 antibody that recognizes both the full-length and truncated AR proteins. Immunoprecipitation using the AR441 antibody served as a positive control for ARΔLBD immunoprecipitation.
- Fig. 3.
Molecular analysis of the CWR22Rv1 AR mRNA. A, the AR mRNA structure was analyzed by a RT-PCR mapping strategy. Primer pairs (“Materials and Methods”) were designed to amplify overlapping regions (segments I–VI) spanning the entire AR cDNA sequence. These segments are depicted relative to schematic representations of the AR mRNA and polypeptide. Exons 1–8, major protein domains, and poly-amino acid stretches are indicated. B, RT-PCR reactions were performed on total RNA isolated from LNCaP (L) and CWR22Rv1 (C) cells using the primer pairs for the segments described in A. In addition, the 5′-most trinucleotide repeat region (CAG) encoding the first polyglutamine tract was analyzed. Reaction products were analyzed by agarose gel electrophoresis in 2% agarose-TAE gels containing ethidium bromide and photographed under UV illumination. The AR mRNA segments analyzed are indicated by Roman numerals (I–VI) at the top of the gel. The positions of the 100-bp DNA ladder size standards are annotated on the left. C, automated DNA sequencing was performed on purified segment V amplification products from both LNCaP and CWR22Rv1. Sequences were aligned using the ClustalW program, and the CWR22Rv1 insertion sequence was subsequently identified by BLAST analysis. Regions of homology between the CWR22Rv1, LNCaP, and reference human AR sequences are darkly shaded. The CWR22Rv1 insertion (Exon 3′) is highlighted with light shading. The resulting gaps in the aligned reference and LNCaP sequences are denoted by dashes above the inserted sequence.
- Fig. 4.
RT-PCR analysis of AR in CWR22 and CWR22Rv1 xenografts. Top panel, two separate passages of the original CWR22 xenograft and four relapsed tumor strains were analyzed by RT-PCR using the primer pair for AR segment V (Fig. 3A) <$REFLINK> . RNA isolated from LNCaP and CWR22Rv1 cell lines served as references. Arrows on the left indicate AR amplification products. Bottom panel, the same samples were analyzed for PSA expression.
- Fig. 5.
Effects of androgen and antiandrogens on the proliferation of CWR22Rv1 cells. A, LNCaP (▪ and □) and CWR22Rv1 (• and ○) cells were cultured in the presence (filled symbols) or absence (open symbols) of androgen as described in “Materials and Methods.” Cells were plated at a density of 2 × 105 cells/dish. At the indicated times, cells were enumerated with a hemocytometer. Results are presented as the total number of cells/dish and represent the mean ± SD of three separate determinations for each sample. B, CWR22Rv1 cells were seeded into 24-well plates at a density of 1 × 104 cells/well in AD medium. The cells were left untreated (open bars) or exposed to increasing concentrations of DHT (1, 10, and 100 nm), Casodex (1, 10, and 100 nm), and flutamide (0.2, 0.5, and 1.0 μm) as indicated by right-hatched, horizontally lined, and filled bars, respectively. After 5 days, the total number of cells/dish was determined with a Coulter counter. For each set of treatments, results are presented as the mean fractional change in growth ± SD as compared with the untreated controls. C, CWR22Rv1 cells were cultured in androgen-containing medium (FBS) or in AD medium for a duration of 0 (4 h) to 10 days. Cells were harvested daily for immunoblot analysis of AR and NSE expression as described in “Materials and Methods.” The AR full-length and ARΔLBD isoforms are indicated.
- Fig. 6.
Androgen responsiveness of the CWR22Rv1 cell line. A, LNCaP and CWR22Rv1 cells were cotransfected with the pGL3E-Probasin androgen-responsive reporter construct plus a Renilla luciferase construct (pRL-SV40). Twenty-four h later, the medium was replaced with AD medium, and the cells were incubated for an additional 24 h in the absence of androgen (open bars) or the presence of 0.1 (right-hatched bars), 1.0 (horizontally lined bars), and 10 nm (filled bars) DHT. RLU contained in the cell lysates were determined with a dual-luciferase reporter assay. Instrument background was corrected in all experimental readings by subtracting the RLU values obtained with lysates from nontransfected cells. Results are expressed as the fold induction in luciferase activity (relative to untreated control cultures) calculated from the ratios of firefly:Renilla luciferase activities in each sample. Data points represent the mean ± SD of three separate determinations for each sample. B, secretion of PSA by LNCaP and CWR22Rv1 cells was assessed by immunoassay analysis of culture supernatants from cells cultured in the absence (open bars) or presence of 0.1 (right-hatched bars) or 1 nm DHT (filled bars) for 48 and 72 h. Results are expressed as the concentration of PSA (ng/ml) in the culture medium and represent the mean ± SD of three separate assays for each sample. C, LNCaP and CWR22Rv1 cells were cultured in medium supplemented with FBS or in AD medium supplemented with 10-fold increasing concentrations of mibolerone for 3 days. Cells were harvested daily for immunoblot analysis of PSA and AR expression as described in “Materials and Methods.”
- Fig. 7.
Alteration of AR expression and transactivation by duplication of exon 3 duplication mutation into the wild-type AR. A, 293T cells were transiently transfected with AR expression constructs for the wild-type (pcDNA3.1-hAR-WT, hAR-WT) and for a modified wild-type receptor containing the CWR22Rv1 exon 3 duplication mutation (pcDNA3.1-hAR-E3DM, hAR-E3DM). As a control, cells were transfected with empty vector (Vector). Thirty-six h after transfection, detergent lysates were prepared and analyzed by immunoblot analyses as described in Fig. 1A <$REFLINK> using the AR441 monoclonal antibody. The full-length (AR) and truncated (ARΔLBD) AR forms are indicated. LNCaP and CWR22Rv1 (22Rv1) cell lysates serve as references. B, PC-3 cells were cotransfected with either the hAR-WT or hAR-E3DM expression construct in combination with pGL3E-Probasin and pRL-SV40. Twenty-four h later, the cells were either left untreated (□) or treated with 1 nm DHT (▪) for an additional day. Cell lysates were then prepared, and dual-luciferase reporter assays were performed as described in “Materials and Methods” and the legend for Fig. 6A <$REFLINK> . The data are represented as mean ± SD fold induction in firefly luciferase activity relative to the untreated control for each transfection.
Volume 62, Issue 22
