containing 10% fetal bovine serum (FBS) with 5 μg/mL ciprofloxacin ... LAPC4 cells were cultured in IMDM media (Life Technologies, Carlsbad, CA) containing.
Supplemental Material and Methods
Cell Culture and reagents Human prostate cancer cell lines LNCaP, LAPC4, PC3, and DU145 were obtained from American Tissue Type Culture Collection (Manassas, VA). C4-2, PrEC, BPH-1 and 973/hTERT were provided as a gift from Dr. William. B. Isaacs (Brady Urology Institute Johns Hopkins University). LNCaP-95 cell line was provided by Dr. Alan Meeker (Johns Hopkins University). All cells were cultured as previously described. (Litvinov et al., 2006) (Vander Griend et al., 2014). PrEcs were grown in PrEGM defined growth media containing Insulin, EGF, Bovine pituitary extract (BPE), triiodothyronine (T3), transferin, hydrocortisone (HC), epinephrine, and retinoic acid as supplied by manufacture (Lonza/Cambrex, Walkersville MD). The 957E/hTERT cells were cultured in keratinocyte serum free defined media supplemented with growth factors (GFs) as supplied by manufacture (Life Technologies, Carlsbad, CA). LNCaP, C4-2, BPH-1, DU145 and PC3 cells were cultured in RPMI-1640 media while (Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum (FBS) with 5 μg/mL ciprofloxacin hydrochloride (U.S. Biological, Swampscott, MA), and 50 μg/mL gentamicin (Quality Biological, Inc., Gaithersburg, MD). LAPC4 cells were cultured in IMDM media (Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum (FBS) with 5 μg/mL ciprofloxacin hydrochloride (U.S. Biological, Swampscott, MA), and 50 μg/mL gentamicin (Quality Biological, Inc., Gaithersburg, MD) along with 5nM R1881 (R1881, Perkin Elmer, Waltham, MA). WPMY-1 human prostate stromal cell line was a gift from Dr. Robert Getzenberg lab (Johns Hopkins University) were grown and maintained in DMEM supplemented with 10% FBS and 5 μg/mL ciprofloxacin hydrochloride (U.S. Biological, Swampscott, MA), and 50 μg/mL gentamicin (Quality Biological, Inc., Gaithersburg, MD). Cells were allowed to grow until 70% to 80% confluent and harvested with 0.05% trypsin/0.53 mmol/L EDTA (Cellgro) before each subsequent passage. All cells were maintained at 37 °C in an atmosphere containing 5% CO2. All restriction enzymes used are
obtained from New England Biolabs (Ipswich, MA, USA). Primary mouse monoclonal antibodies for AR (AR 441, dilution 1:1000) and polyclonal AR (N-20, dilution 1:1000) and mAb for XPO5 (dilution 1:1000) for western blot were from Santa Cruz Biotech, (Santa Cruz, CA) and from LSBio LifeSpan Bioscience Inc (Seattle, WA), IHC and TMAs staining were done using the prestige XPO5 polyclonal antibody (Sigma Aldrich, St Louis, MO and mAb-β Actin (1:25 000) and Anti-mouse IgG HRP-conjugated (1:20 000 Sigma Aldrich, St Louis, MO). All other chemical reagents and compounds were ordered from Sigma Aldrich, unless otherwise specified
Q-RT PCR and Western blot analysis mRNA (1 μg) from transiently transfected or stably selected prostate cancer cells with mock vector, shRNA XPO5 or Overexpressing wildtype (wt) XPO5 plasmid were reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Sybr green–based real-time qRT-PCR was performed using SYBR GreenER qPCR SuperMix (Invitrogen) according to the manufacturer’s instructions. All reactions were done in triplicate. Standard curves were generated by serial dilution of each sample, and the relative abundance of target gene mRNA was normalized to Actin mRNA (see Supplemental Table 1 for primers). For protein expression studies cells were washed with 1 × PBS and re-suspended with five volumes of cold lysis buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.5% NP-40) supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN). The cell lysate was incubated on ice for 30 min and then centrifuged for 10 min at 4 °C. Equal amounts of proteins were separated by SDS-PAGE, and the resolved proteins were then transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk in TBST overnight at 4 °C, the blot was incubated with primary antibody at 1 h at room temperature. The membrane was then probed with HRP-conjugated secondary antibody for 1 h and developed (ECL-Plus system, Amersham Pharmacia, Piscataway, NJ) using the manufacturer's protocol.
MTS assay The metabolic viability of the cells was monitored using 3-(4,5)-Dimethylthiazol-2-yl)-5-(3carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS assay kit CellTiter 96) from Promega (Madison, WI) as described by Hoti et al (1). Briefly cells were seeded onto 96-well plates and cultured in the presence of test agents for indicated time intervals. A mixture of MTS and phenazine methosulfate (an electron-coupling reagent; final concentrations, 333 μg ml–1 and 25 μM) was added, and cells were incubated for 30 min at 37 °C Formazan formed from the reduction of MTS was quantified by measurement of absorbance of the medium at 490 nm using a microplate reader (All data were normalized to the background signals) Luciferase assay Prostate cancer cells at a density of 1 × 104 cells per well were plated in a 96-well plate one day before transfection. Cells were co-transfected using 100 ng of pBk-PSE-PBN-Luc, 1 ng of pRLCMV and 100 ng of either mock pRFP-C-RS, or pRFP-C-RS shRNA-XPO5 or, overexpressing wild type XPO5 plasmid. The Luciferase assays (Dual-Glo Luciferase Assay System, Promega, WI) were performed at 24–48 h post transfection. All of the shRNA knock-down experiments were performed in quadruplets and normalized to the total Renilla Luciferase numbers per well. Luciferase activity is reported as percentage relative light-forming units.
BALB/C Nude Athymic mice and tumor implantations Four to six-week-old athymic BALB/c nude male mice, weighing approximately 20–24 g were obtained from Harlan (Indianapolis, IN). Mice were quarantined for a minimum of 5 days in the SPF Grade Animal House under 12 h light/dark cycles at 24–25 °C with a relative humidity of 50–55%. Institutional guidelines were followed in handling the animals. Tumors were established by subcutaneous (s.c) injection with DU145 cells (1 × 106) that were stably selected to express Vector control or shRNA against XPO5 or overexpressed XPO5. Cells were resuspended in 1 × PBS (PH 7.4; BioSource, Rockville, MD) mixed 1 × with Matrigel (BD
Biosciences, Palo Alto, CA), in both dorsal flanks of the animals. All tumors were measured twice a week for six weeks. Each group consisted of six animals.
Tissue Microarray and Staining Prostate cancer tissue microarray TMA (IMH-303) was obtained from IMGENEX (San Diego, CA) which contains 49 formalin fixed paraffin embedded (FFPE) 4.0 mm prostatectomy cores. Out of 49 cores 40 were adenocarcinomas of the prostate with Gleason scores between 6 and 9, the remaining nine cores were from the normal adjacent tissues. The mean age of the patients was 64.8 years (range 44-75 years). Using the primary antibodies to XPO5 (Sigma Aldrich polyclonal Prestige Antibodies, diluted 1:1,000) slides were stained using citrate buffer antigen retrieval protocol. Appropriate positive controls human prostate cancer cell line, LAPC4 cells overexpressing a wt XPO5 plasmid) were run concurrently. Similarly, cell lines were made by knocking down XPO5 with an shRNA again the XPO5 as negative control, beside controls, mock sections were also treated in an identical fashion except for replacing the primary antibody with non-immune rabbit IgG. All these stains were manually scored separately in the nucleus/nucleolus and cytoplasm of epithelial (cancer, non-neoplastic) cells by one pathologist (H.M.) who was blinded to patient identity. German Immunoreactive Score was calculated by multiplying the percentage of immunoreactive cells (0% = 0; 1-10% = 1; 11-50% = 2; 51-80% = 3; 81-100% = 4) by staining intensity (negative = 0; weak = 1; moderate = 2; strong = 3), as described (2). Cores with the immunoreactive score of 0 or 1 were considered negative (0), and those with the immunoreactive scores of 2-4, 6-8, and 9-12 were considered weakly positive (1+), moderately positive (2+), and strongly positive (3+), respectively. The Fisher’s exact test was used to evaluate the association between categorized variables as previously described (2).
1. 2.
Hoti N, Chowdhury W, Hsieh JT, Sachs MD, Lupold SE, Rodriguez R. Valproic acid, a histone deacetylase inhibitor, is an antagonist for oncolytic adenoviral gene therapy. Mol Ther 2006;14:768-78 Canacci AM, Izumi K, Zheng Y, Gordetsky J, Yao JL, Miyamoto H. Expression of semenogelins I and II and its prognostic significance in human prostate cancer. Prostate 2011;71:1108-14
