Cell culture and transfection
Prostate cancer cells (PC3, CWR22Rv1 and LNCaP) were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) at 37°C in 5% CO2. Cells were plated into 12 well plates or 96 well plates 16 hours before transfection either in growth medium or, if not specified in the figure legend, in phenol-red-free RPMI (Invitrogen) with 5% charcoal stripped serum (Hyclone) and 1% penicillin/streptomycin. Transfections were performed with Fugene 6 (Roche) according to the manufacturer's protocol. For 12 well assays, each well received 0.02 μg of pCMV-LEF-1, 0.1 μg of LEF-luciferase reporter plasmid, and 0.01 μg pCMV-Renilla together with 0.5 μg each of the expression plasmids . For 96 well assays, each well received 2 ng of pCMV-Renilla, 10 ng each expression plasmids, 4 ng of pCMV-LEF-1 and either 20 ng of the LEF-luciferase reporter or 35 ng of the PSA-luciferase reporter plasmid.
Generation of AR Mutants
From a pCMV-Flag vector containing wild type AR, a 1507 bp fragment was excised utilizing the endogenous BstEII and SalI restriction sites. This fragment encompasses a region of the AR gene containing the three mutation sites. Using the primers listed below and the QuickChange II Site-Directed Mutagenesis kit (Stratagene), an appropriate single base pair substitution was generated for each of the mutants:
H874Y (CAT to TAT):
5' – TTGCGAGAGAGCTGTATCAGTTCACTTTTG – 3' and
5' – CAAAAGTGAACTGATACAGCTCTCTCGCAA – 3'
T877A (ACT TO GCT):
5' – AGCTGCATCAGTTCGCTTTTGACCTGCTAA – 3' and
5' – TTAGCAGGTCAAAAGCGAACTGATGCAGCT – 3'
W741L (TGG to TTG):
5' – ACCATGAGCCCCATCAAGGAGTACTGAAT – 3' and
5' – ATTCAGTACTCCTTGATGGGGCTCATGGT – 3'
W71C (TGG to TGT):
5' – ACCATGAGCCCCATACAGGAGTACTGAAT – 3' and
5' – ATTCAGTACTCCTGTATGGGGCTCATGGT – 3'
These mutant fragments were then cloned into the pCMV-Flag-AR vector lacking the corresponding 1507 bp wild type AR region. All of the mutant constructs were verified by DNA sequencing prior to these studies.
Luciferase assays were performed with the dual luciferase assay kit (Promega) according to the manufacturer's instructions. The luciferase activities were normalized with the Renilla luciferase activities. The activity of control GFP expression was set to 1, and the relative activities to GFP were then calculated for fold activation. Each experiment was carried out in triplicate with error bars representing standard deviation. All the experiments were repeated three times.
Cell growth assay
Guava Viacount: PC3 cells were plated into a 96 well plate at 1 × 104 cells per well in 100 μl phenol-red free RPMI with 5% charcoal stripped serum and 1% penicillin/streptomycin. After 24 hours, cells in each well were transfected with 100 ng of the indicated plasmids. To ensure transfection with equal amount of DNA, 100 ng of GFP was cotransfected into each well containing cells receiving only 100 ng of a single plasmid. Seven days after transfection, cells were trypsinized and stained with Guava PCA-96 Viacount reagent (Guava technologies) as indicated by the manufacturer. Cells were then transferred to a low-attachment 96 well plate and analyzed by Guava PCA-96 using the Guava ViaCount program. Each experiment was carried out in triplicate with error bars representing standard deviation.
[3H]-thymidine incorporation assay: LNCaP cells and LNCaP-Flag-AR cells were seeded at 6000/well in 96 well plates and maintained in phenol-red free RPMI medium supplemented with 5% charcoal-stripped serum. After 24 hours, cells were treated with either 100 ng/ml Wnt3A or control (PBS with 0.2% serum). Four days after the compound treatment, cells were labeled with [3H]-thymidine for four to five hours. Plates were then harvested and counted using TOPCOUNT. Each experiment was carried out in triplicate with error bars representing standard deviation and repeated three times.
Comparison of cell numbers in cells transfected with GFP, Wnt1 or AR alone, as well as Wnt1 and AR together was conducted using one-tail paired student's t test. P < 0.05 was considered statistically significant.
Soft Agar growth assay
For analysis of anchorage-independent growth of both treated and untreated parental LNCaP and LNCaP-Flag-AR cells, a soft agarose medium was employed. 1 × 104 trypsinized cells were resuspended in 1 ml of RPMI containing 10%FBS and 0.3% agarose (type I-A, Sigma) and layered onto a 0.5 ml cushion of 0.6% agarose in RPMI supplemented with 10%FBS in 24-well plates (Costar). The next day, cells were treated with either 10 nM DHT (Sigma) or 100 ng/mL Wnt3A (R&D Systems) in RPMI with 5% charcoal-stripped serum. The medium, with or without treatments, was then renewed by gentle aspiration every 5 days. Cultures were incubated at 37°C in a humidified incubator containing 5% CO2, and, after an approximate 4 week growth period, colonies were fixed in the presence of 10% formaldehyde in PBS. Colony growth was assessed by photography, noting size and frequency in a typical field of cells using bright-field microscopy (12.5×). Each experiment was carried out in duplicate. And the experiments have been repeated three times.
A tissue microarray was obtained from Asterand containing 4 normal prostate tissue and 9 prostate tumors. This microarray was stained with antibodies against AR (Santa Cruz at a 0.5 ug/ml concentration) and β-Catenin (BD Biosciences, diluted at 1:500), using consecutive slides for the two antibodies. For antigen retrieval, Citra Plus Solution (BioGenex) was used at 95°C for 15 minutes. Slides were stained using the BioGenex i6000 automated staining system. The DAKO Envision Plus kit was used for chromagen detection. As negative controls, isotype mouse IgG1 and rabbit IgG1 were used, and no staining was observed (data not shown).
Chromatin Immunoprecipitation Assay
LNCaP cells in RPMI with 5% charcoal-stripped serum and 1% Pen-Strep were treated with either 10 nM DHT (Sigma), 100 ng/mL Wnt3A (R&D Systems), or left untreated. After 16 hours the cells were fixed with 2% formaldehyde followed by lysis in 0.2 ml buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS, protease inhibitors) per 1 × 106 cells. After sonication and centrifugation, the supernatant was diluted 1:10 in IP Buffer (16.7 mM Tris-HCl pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton ×-100, protease inhibitors) for immunoprecipitation with either anti-AR polyclonal antibody (custom made) or anti-β Catenin mouse monoclonal antibody (Cell Signaling) as well as negative control mouse IgG or rabbit IgG antibodies. No signals were observed in negative controls (data not shown). After overnight incubation at 4°C, protein-DNA complex was then purified with 50 uL of Ultralink Immobilized Protein A/G beads (Pierce) followed by reverse crosslinking. DNA was then isolated using a Qiagen purification kit and eluted in 35 ul volumes. Typically, 2 uL of the purified DNA was used as template in the PCR reactions. The PCR products were analyzed using a Bioanalyzer 2100 (Agilent) and a DNA 1000 kit (Agilent). The amount of PCR product was compared with the input amount to calculate the percentages of input. This experiment was repeated three times and the average of percent input was plotted with error bars representing standard deviation.
The primers used were:
negative control β-Actin  (No signal was observed in AR IP and β-Catenin IP in Figure 6),
PSA Promoter and enhancer
human c-myc and cyclin D1 promoter regions ,