Cell culture and reagents
Two sub-cultured lines of PC-3 prostate cancer cells (PC-3-a and PC-3-b), as well as LNCaP, C4-2B, RWPE1, and HeLa cells were maintained as follows. PC-3-a cells were cultured in T-medium with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) [Huang et al., 2005
]. PC-3-b cells were grown in DMEM-F12 with 10% FBS. LNCaP cells were cultured in RPMI supplemented with 10 mM non-essential amino acids, 2 mM sodium pyruvate and 10% FBS. C4-2B cells, which promote formation of osteoblastic lesions [Wu et al., 1998
], were cultured in RPMI supplemented with 10% FBS, and HeLa cells were maintained in DMEM with 10% FBS. The above cell lines were also supplemented with 2 mM L-glutamine and penicillin-streptomycin. RWPE1 cells were maintained in reduced keratinocyte medium and supplements [Bello et al., 1997
]; these immortalized prostate cells are TGFβRII positive and express AR at low levels in standard culture conditions (without androgen supplementation) [Bello et al., 1997
]. Cell cultures were incubated at 37°C with 5% CO2
. All media and supplements were purchased from Gibco/Invitrogen (Grand Island, NY).
For treatment with DHT and TGFβ, normal growth medium was replaced with medium containing 2% charcoal stripped (CCS) FBS for 24 h, followed by incubation with DHT (10 or 20 nM) (Sigma, St. Louis, MO) or porcine TGFβ (10 or 50 ng/ml) or vehicle (ethanol for DHT and incomplete growth medium for TGFβ) for 24 h. Cells were harvested for protein and mRNA detection (see below).
Cell growth assays
For growth curves, PC-3-a and PC-3-b cells were plated at equal densities in 6-well plates (5×104 cells per well). Cells were harvested with trypsin/0.25% EDTA and counted in quadruplicate on days 1 to 6 after plating using Trypan Blue exclusion and a hemocytometer. For siRNA experiments PC-3-a cells were plated at ~30-40% confluence and transfected using Oligofectamine® (Invitrogen, Carlsbad, CA). Commercially available siRNA oligonucleotides for Runx2 and non-silencing oligonucleotides (Smartpool ON-TARGETplus) (Dharmacon, Lafayette, CO) were used, and treatment (50 nM) was carried out for 48 h, after which cells were re-plated in 10 cm plates at day 0 and counted in triplicate on days 1 to 4.
Western blot analysis
Cells were lysed in RIPA buffer supplemented with protease inhibitors (Complete, EDTA-free, Roche Diagnostics, Mannheim, Germany), and MG132 (25 μM). Equal amounts of protein lysates in SDS sample buffer were loaded on a 10% acrylamide gel and subjected to electrophoresis and immunoblotting (BioRad system).
Immunoblots were incubated for one hour with the following antibodies (purchased from Santa Cruz Biotechnology, Santa Cruz, CA): p57 (C-20), p27 (C-19), p21 (H-164), Cyclin D1 (DCS-6), Cyclin A (C-19), CDK4 (C-22), AR (441), and CDK2 (M-2). Antibodies against Cyclin E (HE-12) (Becton Dickinson, Franklin Lakes, NJ), Tubulin (DM1A) (Sigma), and Runx2 (mouse monoclonal clone 8G5) (MBL International, Woburn, MA) were purchased from the indicated vendors. Peroxidase labeled goat-anti-rabbit or goat-anti-mouse antibodies (Santa Cruz) were used as secondary antibodies and were visualized with enhanced chemiluminescence (ECL) chemistry (PerkinElmer, Waltham, MA) on BioMax film (Kodak, Rochester, NY).
PC-3 and LNCaP cells were plated in 6-well plates in normal growth medium. The next day, cells were incubated for 24 h in medium containing 2% charcoal stripped (CCS) FBS. Transient transfections were then performed in fresh 2% CCS-FBS containing medium using FuGENE6 (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Cells were transfected with one of three different reporters in which the luciferase gene is driven by promoters containing tandemly repeated regulatory elements: six binding sites for Runx2 (6×Runx2-luc, 500 ng), three TGFβ responsive elements each spanning a Smad and Runx2 binding site (3×TGFβRE-luc, 500 ng), or multiple androgen responsive elements (ARE-luc, 2 μg). Co-transfections were performed with plasmids expressing HA epitope-tagged Runx2 under control of the CMV promoter. A promoter-less luciferase vector construct (pGL2-Luc) was used as a negative control. The same total amount of DNA was used in every transfection. After 16 h of transfection, cells were treated with DHT (10 nM) or TGFβ (10 or 50 ng/ml) or vehicle (ethanol for DHT and incomplete growth medium for TGFβ) for 24 h. Cell lysates were analyzed for firefly luciferase activity with the dual-luciferase reporter assay system (Promega, Madison, WI). Firefly activity was normalized to Renilla (phRL-null, 25 ng per well) (Promega).
Total RNA purified using Trizol (Invitrogen, Carlsbad, CA) was subjected to DNase I digestion, and cDNA was prepared with the qScript cDNA synthesis kit (Quanta BioSciences, Gaithersburg, MD). Relative PCR quantitation was determined using a 7300 sequence detection system (Applied Biosystems, Foster City, CA) with SYBR Green chemistry (Bio-Rad, Hercules, CA). The relative mRNA expression was calculated with the ΔΔCT method. Real-time primer pairs were used to amplify human mRNA (in 5′-3′ direction): p57 forward AAG AGA TCA GCG CCT GAG AA and reverse TGG GCT CTA AAT TGG CTC AC; p27 forward AGA TGT CAA ACG TGC GAG TG and reverse TCT CTG CAG TGC TTC TCC AA; p21 forward GAC TCT AGG GTC GAA AAC G and reverse GGA TTA GGG CTT CCT CTT GG; RB forward CAC ATT CCT CGA AGC CCT TA and reverse TTT TTG TTG GTG TTG GCA GA; p107 forward ATG GAT GCT CCA CCA CTC TC and reverse GAG CGC TTC TTG GTG TAA GG; p130 forward ATT ACG CCG TCT CCA AGA TG and reverse ATG CAT TAA AGG CTG CTG CT; CDK4 forward GGA ACT CTG AAG CCG ACC AG and reverse ACA TCT CGA GGC CAG TCA TC; Cyclin D1 forward GAT CAA GTG TGA CCC GGA CT and reverse TCC TCC TCC TCT TCC TCC TC; Cyclin A2 forward CCT GCA AAC TGC AAA GTT GA and reverse AAA GGC AGC TCC AGC AAT AA; Cyclin B1 forward CAA GCC CAA TGG AAA CAT CT and reverse GGA TCA GCT CCA TCT TCT GC; Cyclin B2 forward ACT GCT CTG CTC TTG GCT TC and reverse TTT CTC GGA TTT GGG AAC TG; H4n/o forward AGC TGT CTA TCG GGC TCC AG and reverse CCT TTG CCT AAG CCT TTT CC; GAPDH forward ATG TTC GTC ATG GGT GTG AA and reverse TGT GGT CAT GAG TCC TTC CA; Runx2 forward CGG CCC TCC CTG AAC TCT and reverse TGC CTG CCT GGG GTC TGT A.
Immunological and histological analysis of tumors in xenografts
Animal studies were conducted in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols and the National Institutes of Health Guide for Care and Use of Laboratory Animals. PC-3-a and PC-3-b (1×105
cells) were injected into tibiae of SCID mice (3 mice per group) as described previously [Barnes et al., 2004
; Lee et al., 2003
]. Tumors were allowed to grow for a period of 6 weeks and images were acquired for tumor growth analysis. Tumors from injected tibiae were harvested and processed for immunohistochemistry as described [Rubin et al., 2000
]. Consecutive tissue sections were hydrated for antigen retrieval carried out in citrate buffer (pH 6.0) at 95°C. After peroxidase blocking, sections were incubated with Ki67 rabbit polyclonal antibodies (C-19, Santa Cruz) followed by HRP conjugated secondary antibodies (Dako Cytomation, Carpinteria, CA).
LNCaP cells were infected with an adenoviral vector expressing Runx2 and allowed to grow for 48 h. Cells were harvested and lysed in 800 μl of Nonidet P-40 buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Nonidet P-40, 1× Complete protease inhibitor (Roche) and 25 μM MG132 (Sigma). Following centrifugation of the lysate for 15 min, the supernatant was transferred to a clean microcentrifuge tube and precleared with 20 μl of protein A/G plus-agarose beads (Santa Cruz) at 4°C for 30 min. Beads were collected by centrifugation at 1,000 g for 5 min at 4°C. Runx2 antibody (PEBP2αA M-70; 3 μg) (Santa Cruz) was added to the pre-cleared cell lysate followed by incubation at 4°C for 1 h. To precipitate immunocomplexes, a suspension of protein A/G plus-agarose beads (50 μl) was added and further incubated at 4°C with agitation for 1 h. The beads were washed twice with 1 ml wash buffer (20 mM Tris, pH 8.3, 0.5% sodium deoxycholate, 0.5% Nonidet P-40, 50 mM NaCl, 2 mM EDTA, 1× Complete protease inhibitor and 25 μM MG132) and the bead pellet was subsequently suspended in an equal volume of 2×SDS sample buffer. AR proteins were analyzed by western blot analysis using mouse monoclonal antibodies (H-300, Santa Cruz) and secondary HRP conjugated goat anti-mouse antibodies.
Detection of Prostate Specific Antigen (PSA) in cell culture
Supernatants were collected from LNCaP cells stimulated with DHT and transfected with Runx2-HA expression plasmids (see luciferase assays). Supernatants from untransfected cells and vehicle treated cells were collected as negative controls. For measuring PSA protein levels, a PSA enzyme-linked immuno-sorbent assay (ELISA) kit was used (United Biotech Inc., Mountain View, CA). PSA levels were calculated using the standard curve provided in the kit.