Cell lines and culture conditions
The prostate cancer cell lines LNCaP, DU145, PC3 and Tramp-C1 were obtained from the American Type Culture Collection (Manassas, VA). LNCaP cells were cultured in RPMI medium supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate (Invitrogen, Carlsbad, CA). Tramp-C1, PC3 and DU145 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12K (1:1). Both media contained 10% fetal bovine serum (GIBCO BRL, Lewisville, TX) and 5% penicillin/streptomycin and were maintained in a 37°C incubator in a 5% CO2 humidified atmosphere.
5-aza and trichostatin A (TSA) treatment
Cells were seeded at a density of 1×106 cells/100 mm dish and allowed to attach over 24 h. 5-aza (Sigma, St. Louis, MO) was added to the medium for final concentrations ranging from 0–10 μM for 72 h. At every 24 h interval, fresh medium containing the drug was added. For the synergistic study, cells were first incubated with 10 μM 5-aza for 72 h at 37°C and then 50 nM TSA (Sigma, St. Louis, MO) was added for an additional 16 h. The treated cells were washed once with phosphate buffered saline (PBS). Cells were allowed to recover for 24 h in drug-free medium in a 37 °C incubator in a humidified atmosphere with 5% CO2.
Transfection of siRNAs
To silence 14-3-3σ or MBD2 expression by RNA interference, 150,000 cells per well were seeded in a 6-well plate at least 20 h before transfection. siRNAs against 14-3-3σ, MBD2, or non-specific control (siControl) were transfected using siLentFect transfection reagent (Bio-Rad, Hercules, CA) as per the manufacturer's instructions. Two days post-transfection, the nearly confluent cells were trypsinized and the cells were used for cell proliferation assays, immunoblot analysis, RT-PCR analysis, and ChIP assay. Target sequences of the siRNAs for MBD2 knockdown (derived from the MBD2 gene at base pair 671 of sequence NM_003927) is 5′- aagaggatggattgcccggcc- 3′. The sequences of the siRNAs for 14-3-3σ gene knockdown are as follows: si14-3-3σ1, 5′ – catggcagccttcatgaaa – 3′; si14-3-3σ2, 5′ – cactcttcttgcagctgtt – 3′ and si14-3-3σ3, 5′ – ctctgatcgtaggaattga - 3′. “Smart pool” siRNAs that combined the above 14-3-3σ siRNAs 1-3 targeted against different regions of the 14-3-3σ mRNA sequence (NM_006142) were used for transfection to increase the knockdown effect.
Cells were washed and lysed in 1X cell lysis buffer (Cell Signaling Technology Inc., Beverly, MA). Protein concentrations were determined on diluted samples using a bicinchoninic acid procedure (Pierce Biochemical Company, Rockford, IL). Equal amounts of protein were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (BioRad, Hercules, CA). Membranes were blocked in a PBS solution with 5% non-fat dry milk and incubated with primary antibodies in blocking solution overnight at 4 °C. Membranes were then washed three times in PBS with 0.1% tween-20 (T-PBS) at 10 min intervals. Horseradish peroxidase conjugated secondary antibodies (Biomeda, Foster City, CA) were used for detection of immunoreactive proteins by chemiluminescence (Amersham Biosciences, Piscataway, NJ). The following antibodies were used: anti-MeCP2 (Upstate Biotechnology, Lake Placid, NY), anti-MBD2 (Upstate Biotechnology, Lake Placid, NY), anti-PCNA (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH (Abcam, Cambridge, MA). Antibodies against 14-3-3 isoforms, such as 14-3-3β, 14-3-3ξ, 14-3-3ε and 14-3-3σ, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Protein detection by Immunofluorescence
Cells were cultured on Lab-Tek II chamber slides (Nalge Nunc International, Naperville, IL) and fixed in 4% paraformaldehyde solution for 20 min, permeabilized in 0.2% Triton-X (Sigma) in PBS for 15 min, and blocked in PBS with 1% BSA for 1 h. Primary antibodies specific for 14-3-3σ (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS with 1% BSA and 0.05% Tween 20 were added for 1 h and detected with Texas redconjugated donkey anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were covered with mounting medium containing the nuclear counterstain DAPI. Fluorescent images were acquired using a charge-coupled device RT Slider Spot Camera (Diagnostic Instruments Inc, Burroughs Sterling Heights, MI) connected to a microscope (Olympus, Melville, NY) and managed by a computer equipped with the spot RT software v3.5 (Diagnostic instruments, Burroughs Sterling Heights, MI).
Bisulfite modification of genomic DNA
Genomic DNA was isolated from LNCaP, DU145, PC3 and Tramp-C1 prostate cancer cells using the DNeasy tissue kit (Qiagen, Germany). The bisulfite reaction was carried out on 5 μg of genomic DNA with the CpGenome DNA modification kit as recommended by the manufacturer (Chemicon, Temecula, CA). Briefly, genomic DNA was treated with 3.0 mM sodium bisulfite (pH 5.0) and 0.5 mM hydroquinone for 16 h at 50°C. With this treatment, all cytosine residues are converted to uracil in unmethylated DNA, but those that are already methylated (5-methylcytosine) are resistant to this treatment and remain as cytosine. The reaction mixture was then purified with the Promega Wizard Clean-UP Kit (Madison, WI) and desulphanated with 0.3 M sodium hydroxide for 20 min at 40°C. The converted DNA was then precipitated in cold ethanol, dissolved in H20, and stored at −20°C.
Methylation-specific PCR (MSP)
Methylation-specific PCR was performed with 25 μL using 3 units of platinum Taqpolymerase (Invitrogen, Carlsbad, CA) per reaction. We used the following MSP primers: (1) for unmethylated 14-3-3σ sequence: 5′-TTA TTA GAG GGT GGG GTG GAT TGT-3′ (sense) and 5′-CAA CCC CAA ACC ACA ACC ATA A-3′ (antisense); (2) for methylated 14-3-3σ sequence: 5′-GGT TTT TTC GGT TAG TTG CGC GGC G-3′ (sense) and 5′-CCA ACG AAA ACC TCG CGA CCT CCG-3′ (antisense). After denaturation at 95°C for 5 min, 40 PCR-cycles were completed with the bisulfite-treated genomic DNA as a template. 20 μL of the PCR-amplified fragments were loaded onto 2% agarose gels for analysis. Positive controls used for methylation-specific PCR included DNA from normal prostate tissues as unmethylated DNA control and CpGenome Universal methylated DNA as methylated DNA control (Chemicon International). Negative control MS-PCR reactions were performed using water only as template.
Cloning and bisulfite sequencing
To permit DNA sequencing of inclusive CpG sites within the CpG island of the 14-3-3σ gene, PCR products were purified by electrophoresis on 1% agarose gels using the Qiaquick™ gel extraction kit (Qiagen), ligated into pCR2.1pTOPO-TA vectors (Invitrogen), and then introduced into TOP10 One Shot competent bacteria (Invitrogen). Transformed cells were plated on LB agar containing 100 μg/mL ampicillin (Invitrogen) and incubated overnight at 37 °C. Ten individual colonies were selected and each inoculated into 3 mL LB broth containing 100 μg/mL ampicillin (Invitrogen) and grown overnight at 37 °C. The insert containing plasmid DNA was then extracted from the cells using the Eppendorf FastPlasmid mini-prep kit (Eppendorf, Westbury, NY) and resuspended in 30 μL of the included EB buffer. A minimum of six clones selected at random from each DNA was sequenced in automated DNA sequencer.
Promoter methylation analysis
The methylation status of the 14-3-3σ gene promoter was determined using HhaI-based promoter methylation analysis. Genomic DNA (1-2 μg) obtained from LNCaP, DU145, PC3 and Tramp-C1 prostate cancer cells were digested for 3 h with a methylation-sensitive enzyme, HhaI (New England Biolabs Inc., Beverly, MA), in a 20 μL reaction. 2 μL were used as template for 14-3-3σ promoter-specific PCR with the following primers: 5′-CAT GAA AGG CGC CGT GGA GAA-3′ (sense) and 5′-GCT GAT GTC CAT GGC CTC CTG G-3′ (antisense). PCR amplification indicates that the HhaI site within the 14-3-3σ promoter targeted sequence was methylated and protected from digestion.
Reverse transcription-PCR analysis
Cellular RNA was isolated from LNCaP, DU145, PC3 and Tramp-C1 prostate cancer cell lines using the Qiagen RNeasy kit. 1 μg of RNA was treated with DNase (10 units/μg of RNA for 1 h) and used as a template for the reverse transcription reaction (RT, 20 μL). The RT reaction mix (Invitrogen, Carlsbad, CA) contained 1 μL (10 pm) of primers. The resultant cDNA was then used in PCR reactions and analyzed by gel electrophoresis. The following primers were used: 5′-ACG ACA AGA AGC GCA TCA TTG-3′ (sense) and 5′-GGC ATC TCC TTC TTG CTG ATG T-3′ (antisense). PCR conditions were as follows: 95°C for 5 min, followed by 40 cycles at 95 °C for 1 min, 55°C for 1 min, and 72°C for 1 min. The final extension was at 72°C for 5 min.
Chromatin Immunoprecipitation (ChIP) and siRNA-coupled ChIP Assays
ChIP analysis was used to determine the binding activity of MeCP2 and MBD2 in the 14-3-3σ promoter in PC3, DU145, LNCaP and Tramp-C1 cells before and after 5-aza treatment. ChIP assays were performed per manufacturer's instructions (17-295, Upstate Biotechnology, Lake Placid, NY). Briefly, prostate cancer cells (~1×106 cells/100 mm dish) were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 min at 37°C. The cells were washed twice with ice-cold PBS containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A), harvested, and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths below 1000 bp (amplitude 60%, 4×10s, Fisher Sonic Dismembrator 60, Pittsburgh, PA). From each sonicated sample, 5% was used as the input control for immunoprecipitated fragments. The complexes were immunoprecipitated with antibodies specific for MeCP2 (no. 07-013) and MBD2 (no. 07-198) from Upstate Biotechnology. 10 μL of antibody were used for each immunoprecipitation according to the manufacturer's instructions. Antibody controls were also included for each ChIP assay; no precipitation was observed. The antibody/protein complexes were collected using salmon sperm DNA/protein A agarose slurry and washed several times per manufacturer's instructions. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3, and the cross-links were reversed by incubation at 65°C for 4 h in the presence of 200 nM NaCl. The samples were treated with proteinase K for 1 h, and the DNA was purified by phenol/chloroform extraction, ethanol precipitation, and resuspended in 30 μL of H2O. Initially, PCR was performed with different number of cycles or dilutions of input DNA to determine the linear range of the amplification; all results shown fall within this range. Following 30 cycles of amplification, PCR products were run on 2% agarose gels and analyzed by ethidium bromide staining. For the siRNA-coupled ChIP assay, LNCaP cells transfected with siRNA against MBD2 and shown knockdown effect were selected. The cells were harvested and the immunoprecipitated chromatin was analyzed by PCR with primers specific to the 14-3-3σ promoter.
MTT Cell proliferation assay
Proliferation of LNCaP, DU145, PC3 and Tramp-C1 cells was assessed with the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method using a cell proliferation kit (Chemicon, Temecula, CA). Cells were plated at density of 1×103 cells/well in 96-well plates and cultured under standard tissue culture conditions for 24 h. Cells were treated with 10 μM 5-aza for 72 h followed by 50 nM TSA for an additional 16 h. Treated cells were washed once with PBS. Cells were allowed to recover for 24 h in drug-free medium in a 37°C incubator in a humidified atmosphere with 5% CO2. Then, 10 μL of MTT reagent were added and the cells were incubated for another 4 h at 37°C. Acid-isopropanol (0.04 N HCl/isopropanol) was immediately added to all wells and mixed vigorously so that the dark blue crystals dissolved effectively. Absorbance was measured at 570 nm (Benchmark, BIORAD, Hercules, CA).