The following mammalian expression plasmids were used: pAcGFP1-Mito (mito-green), a green fluorescent label for mitochondria (Clonetech Laboratories, Inc. Mountain View, CA); dominant-negative Drp1 (DN-Drp1, K38A) (a kind gift from Alexander van der Bliek, University of California, Los Angeles, CA). Antibodies used were: anti-Drp1 (BD Biosciences, San Jose, CA); anti-PSA and anti-AR (Santa Cruz Biotechnology Inc., Santa Cruz, CA); anti-Mfn1 (Novus Biologicals Littleton, CO); anti-phospho-Drp1 (Ser-616), anti-COXIV, anti-mouse and anti-rabbit IgG (Cell Signaling, Danvers, MA); anti-Mfn2, anti-β-actin and anti-chicken IgG (Sigma-Aldrich Corporation, St. Louis, MO); anti-GAPDH and anti-Hsp90α (Chemicon International, Temecula, CA). The chemicals used were: charcoal/dextran-stripped FBS (CSS) from Hyclone (Logan, UT); the synthetic androgen methyl trienolone (R1881) from Perkin-Elmer Life Sciences (Boston, MA); bicalutamide and cycloheximide from Sigma-Aldrich Corporation (St. Louis, MO); chloro-benzothiazepin CGP37157 (CGP) from Calbiochem (San Diego, CA); and G418 from Cellgro (Manassas, VA). Scrambled siRNA was from Qiagen (Valencia, CA) and siRNA for AR was from Dharmacon RNAi Technologies (part of Thermo Scientific, Lafayette, CO).
Cell Culture and Treatment
Prostate cancer cell lines LNCaP, DU145, and PC3 were purchased from ATCC (Manassas, VA). LNCaP derived C4-2 cells were a gift from Dr. Leland Chung, Cedars-Sinai Medical Center, Los Angeles, CA. Cells were maintained in RPMI 1640 (Hyclone, Logan, Utah) containing 9% FBS, 0.5% penicillin-streptomycin, and 0.1% fungizone. The CWR-R1 cells (provided by Dr. E. Wilson, University of North Carolina, Chapel Hill, NC) were grown in Richter’s MEM. Non-tumorigenic prostate epithelial cells (P69, a gift from Dr. Leland Chung) were maintained in T-medium (GIBCO-InVitrogen, Carlsbad, CA). VCaP cells (ATCC) were maintained in DMEM-GlutaMax medium (GIBCO-InVitrogen, Carlsbad, CA) containing 10% serum and treated in the same medium containing 5% CSS. Cells were maintained in CSS (5%) containing steroid free medium for 48h and treated with R1881 for 24h or the indicated periods. In combination experiments, bicalutamide was added 8h prior to R1881 treatment for 24h. Cycloheximide (50µg/ml) was added 1h before and during R1881 treatment. CGP (50µM) was added after 24h of R1881 treatment for the indicated periods. Transfection with siRNA was performed using HiPer-Fect reagent (Qiagen, Valencia, CA) for 48h. Stably transfected LNCaP cells expressing GFP in the mitochondria, pAcGFP1-Mito (mito-green), were generated by electroporation using the Electro Square Porator™ ECM-830 (BTX Inc, San Diego, CA), followed by G418 (500µg/ml) selection.
Protein Extraction and Western Blotting
Cell lysates were processed as described previously (27
). For phosphorylation studies, phosphatase inhibitor cocktail 1 and 2 were added (Sigma-Aldrich Corporation, St. Louis, MO) to the lysis buffer.
Quantitative Real-Time Reverse Transcription-PCR
Total RNA was extracted using TRIzol (InVitrogen, Carlsbad, CA). The cDNA was synthesized using a Reverse Transcription System (Promega Corporation, Madison, WI) and was amplified by qRT-PCR using Mastercycler ep-realplex2 (Eppendorf, Westbury, NY) with the IQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA). Primers used in this study were: Drp1: 5′-CCAAGGTGCCTGTAGGTGAT-3′ and 5′-CAGCAGTGACAGCGAGGATA-3′; PSA: 5′-CATCAGGAACAAAAGCGTGA-3′and 5′-AGCTGTGGCTGACCTGAAAT-3′; and GAPDH: 5′-ACAGTCAGCCGCATCTTCTT-3′ and 5′-ACGACCAAATCCGTTGACTC-3′. Cycle treshhold (Ct) was normalized using the known Ct from the housekeeping gene GAPDH. To compare the relative levels of gene expression of Drp1 or PSA, ddCt values were calculated using gene expression from the untreated control cells and ddCt values were expressed as the real-fold increase in gene expression.
Chromatin immunoprecipitation (ChIP) was performed as previously described (28
). ChIP enrichment was tested on one aliquot of the isolated DNA by real-time (quantitative) PCR and the remainder was used for single-end SOLEXA library preparation.
ChIP-seq SOLEXA Library Preparation
Single-end SOLEXA sequencing libraries were prepared as previously described (28
Illumina BeadArrays and Analysis
Illumina beadarrays were performed using standard Illumina protocols as described previously (28
). Autocorrelation analysis of Illumina gene expression data was performed as in (28
). The Illumina gene expression data (28
) have been deposited on GEO at http://www.ncbi.nlm.nih.gov/gds?term=GSE18684
Stably transfected mito-green LNCaP cells were maintained in androgen-depleted medium for 48h before R1881 treatment for 24h and/or CGP (50µM) for the last 1h. Cells were washed and suspended in HBSS (GIBCO InVitrogen, Grand Island, NY) and at least 200 cells were examined for mitochondrial fragmentation under a fluorescent microscope (Zeiss Axioskop, Carl Zeiss Imaging Inc., Thornwood, NY). Mito-green cells were also grown on cover slips and treated as above. Cells were fixed with 4% paraformaldehyde and then analyzed using LSM 510 confocal microscopy (Carl Zeiss). Using the Z-stack option, signals from different planes were obtained and combined to give a single picture (maximum intensity projection). We considered cells to be undergoing mitochondrial fission when more than 80% of mitochondria in the cell exhibited fragmentation.
Cell Proliferation Assays
Proliferation of LNCaP cells was measured using the Quick Cell Proliferation Assay Kit (BioVision, Mountain View, CA). LNCaP cells (5×103 per well) seeded in 96-well microtiter plate were treated with bicalutamide (50µM) and/or R1881 (1nM). CGP (50µM) was added during the last 4h of treatment. Reagent from the kit was added and samples read in a microplate reader. For S-phase analysis, cells were treated with 1nM R1881 and fixed overnight in 70% ethanol, stained with propidium iodide (20µg/ml) and cells in S-phase were measured using a flow cytometry.
Measurement of Apoptosis
Total cell lysates (5µg) were analyzed for apoptosis using an M30 Apoptosense kit (Peviva, DiaPharma Group, West Chester, OH) as described previously (27
Human Prostate Tissue
Deidentified coded tissue were obtained post-surgery from prostate cancer (n=7) and hormone refractory metastatic prostate cancer patients (n=6) from the University of Michigan SPORE tissue bank. Tissues were lysed in buffer containing 7M urea, 2M thiourea, 100mM DTT, 2% octyl glucoside and EDTA-free protease inhibitor cocktail (Roche) and the proteins were separated by SDS-PAGE, transferred to membranes and immunoblotted for Drp1, PSA (positive control) or GAPDH (loading control).
Data are presented as means ± SEM. We compared group mean values, as appropriate, by Student’s unpaired two-tailed t test or one-way analysis of variance with Tukey’s multiple comparison test (GraphPad Prism). Significant differences were defined at ***P≤0.001, **P≤0.01 and *P≤0.05.