Chemicals and cell-lines
Antibodies against p53 (DO-1 and FL-393), GFP, pCdk5, Cdk5, p35, Bcl-2, cytochrome-C, PARP, COX-IV, β-Tubulin, β-Actin, HRP-linked secondary antibodies, Control, p53 and PP2A siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-specific p53 antibodies were purchased from Cell Signalling Technology (Danvers, MA). FITC and rhodamine-conjugated secondary antibodies were purchased from KPL (Gaithersburg, MD). APO-Direct TUNEL kit, Matrigel and Bax antibody was purchased from BD (Franklin Lakes, NJ). Dox was purchased from Sigma (St. Louis, MO). Hygromycin B and Tet system approved serum was purchased from Clontech (Mountain View, CA). G418 and MTT were purchased from USB (Cleveland, OH). Okadaic acid (OA), mitotracker dye, DMEM, FBS and Lipofectamine2000 were purchased from Invitrogen (Carlsbad, CA). Cdk2/5 inhibitor, PFTα and U0126 were purchased from Calbiochem (Gibbstown, NJ). Cdk5 siRNA was purchased from Dharmacon Inc (Lafayette, CO). Development of cell-lines is described in additional file 1.
Plasmids and transfection
pC53-SN3 and pG13CAT were a kind gift from Dr. Bert Vogelstein, John Hopkins, Baltimore, MD. p53 fragment of pC53-SN3 was sub cloned in BamH1 site of pTRE and renamed as pTREp53. pG13CAT contains 13 repeats of p53 consensus binding site inserted in the 5' end to polyomavirus basal promoter linked to CAT reporter gene. Cells were co-transfected with 2 μg of pG13CAT and 0.5 μg of pEGFPC1 which serves as an internal control for transfection. Bcl-2 fragment from pRc/CMVBcl-2 (kind gift from Dr. S. Soddu, Regina Elena Cancer Institute, Italy) was excised by HindIII and cloned into pTRE2 to obtain pTRE2Bcl-2. Cells were transfected with either 2.0 μg (for 35 mm plate) or 0.5 μg (for 96 well plate) plasmid by Lipofectamine2000 transfection reagent as per manufacturer's instructions.
Clonogenic-survival assay
Cells (500) were treated with indicated concentrations of Dox, OA or Cdk2/5 inhibitor based on the experimental design and incubated for 48 h. Cells were further grown for 21 days and thereafter colonies on the plate were stained with crystal-voilet.
Electrophoretic mobility shift assay (EMSA)
To visualize the DNA-binding activity of p53 in nuclear extracts of HTet23p53, HTet26p53, HTet43GFP and HeLa cells, EMSA was performed. After treatment with Dox, cells were harvested for the preparation of cytoplasmic and nuclear fractions by using nuclear extraction kit as per manufacturer's instructions (Chemicon, Billerica, MA). Nuclear lysates were incubated for 45 min at 4°C and cleared by centrifugation at 15,000 ×g for 15 min at 4°C. Equal amount of nuclear proteins were used for the binding reaction. Complementary oligonucleotides containing the sequences corresponding to putative p53 binding site (forward, 5'-GAACATGTCTAAGCATGCTG-3'; reverse, 5'-CAGCATTCTTAGACATGTTC-3') were annealed and 5'-end-labeled with 2 micro curie (ΔCi) [γ-32P] ATP using 10 U of T4 polynucleotide kinase (Invitrogen) for 90 min. Binding reaction was carried out in a final volume of 20 μl consisting of 10 mM Tris.HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 2.5% glycerol, 1 μg deoxyinosinic deoxycytidylic acid [poly(dI-dC)], 300 ng BSA, 5 μg nuclear extract, and 2 μl of [γ-32P] labeled oligonucleotide probe. Reaction mixtures were incubated for 20 min at room temperature. Samples were resolved on a native polyacrylamide gel. Gel was dried under vacuum at 80°C for 45 min by gel dryer (Bio-Rad) and DNA-protein complex were visualized by autoradiography.
Chloramphenicol acetyl transferase assay
Cells were co-transfected with pG13CAT and pEGFPC1 expression vector using Lipofectamine2000 as described in transfection section. After 18 h post-transfection, p53 was induced with Dox for 48 h with or without PFTα pretreatment for 1 h. CAT assay was performed as described earlier [2] except that the reaction time was reduced to 30 min at 37°C. Spots were quantified by phosphoimager (Bio-Rad). GFP intensity was directly measured from the cell lysates to check or correct for equal transfection efficiency as well to normalize the reporter activity. The fluorescence intensity of GFP in equal amount of lysate was measured by fluorimeter (Fluoroskan Ascent FL, Fisher Scientific) with excitation at 485 nm and emission at 510 nm.
SiRNA transfections
Cells were transfected with 100 nM control or p53 siRNA using Lipofectamine2000 [21]. Eighteen hour post-transfection, Dox was added with or without OA and further incubated for 48 h. Thereafter, western blot or MTT assay was performed. To knock-down PP2A and Cdk5; Cdk5 siRNA was transfected 12 h prior to PP2A siRNA transfection and then incubated with Dox for 48 h.
Immunoprecipitation and Chromatin-immunoprecipitation (ChIP) assay
After indicated treatment cells were lysed in RIPA buffer. Equal amount of protein (400 μg) was taken and lysates were pre-cleared with 50 μl protein A/G-plus agarose for 30 min. Fifty microgram lysates were run as input. Agarose beads were pelleted and supernatant was incubated with p53 specific antibody overnight at 4°C. Fifty microliter protein A/G-plus agarose was added in antibody-antigen complex with gentle shaking for 4 h at 4°C. The protein A/G-plus was separated by centrifugation at 4,000 rpm. Target and its associated proteins were disrupted and resolved on SDS-PAGE. The expression of Cdk5 and p53 was detected by western blotting.
For chromatin-immunoprecipitation assay cells or homogenized tumors which were earlier fixed with 1% para-formaldehyde for 15 min, were lysed with 500 μl of lysis buffer [5 mM PIPES (pH 8.0), 85 mM KCl and 1% NP-40]. After centrifugation (5000 rpm), nuclear pellets were resuspended in 150 μl buffer [50 mM Tris-Cl (pH 8.0), 10 mM EDTA and 1% NP-40]. To fragment DNA to approximately 500 bps, samples were sonicated and centrifuged for 10 min. Samples were diluted 10-fold in IP buffer [16.7 mM Tris-Cl (pH 8.0), 167 mM NaCl, 1% NP-40 and 1 mM EDTA]. Samples (400 μg) were incubated with anti-p53 or anti-goat IgG overnight. Remaining solutions (10-times diluted) were used as input. Protein A/G-plus agarose beads pre-blocked with salmon-sperm DNA were added to antibody-antigen complexes and incubated for 4 h. Immune-complexes were centrifuged and washed with buffer [20 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.5% SDS, 1% Triton X-100 and 2 mM EDTA] twice and with buffer containing 250 mM NaCl. Immune-complexes were eluted by 50 μl of buffer [1% SDS and 0.1 M NaHCO3] twice. Then 20 μl of 5 M NaCl was added and incubated at 65°C overnight. DNA was precipitated with ethanol. RT-PCR was performed with promoter primer pairs for p21 (F) 5'-GGC TGG TGG CTA TTT TGT CC-3', (R) 5'-TCC CCT TCC TCC CTG AAA AC-3', and bax (F) 5'-AGC GTT CCC CTA GCC TCT TT-3' and (R) 5'-GCT GGG CCT GTA TCC TAC ATT CT-3' at annealing temperature 57°C and 59°C respectively.
Mitochondrial and cytosolic fractionation
HTet26p53 cells were swelled in ice-cold hypotonic HEPES buffer [10 mM HEPES (pH 7.4) 5 mM MgCl2, 40 mM KCl, 1 mM PMSF and protease inhibitor cocktail] for 30 min and centrifuged at 1500 rpm to pellet the nuclei. The resulting supernatant was centrifuged at 10,000 rpm to pellet mitochondrial fraction. Supernatant was used as cytosolic fraction and mitochondrial pellet was washed with PBS twice. This pellet was lysed in mitochondrial buffer [10 mM MOPS (pH 7.4), 1 mM EDTA, and 4 mM KH2PO4, 1% NP-40, protease inhibitor cocktail] and centrifuged at 12,000 rpm for 30 min.
Immunostaining
Cells grown on Labtek chamber slides were treated with Dox for 48 h and processed for immunofluorescene study as described earlier [21]. Primary antibody against p53 (1:50) was added and incubated for 2 h at room temperature. Following incubation, cells were washed 5 times. Fluorescein isothiocyanate (FITC) or Rhodamine conjugated secondary antibodies (1:100) were added and incubated for 1 h at room temperature. After five washes, vectashield mounting medium containing DAPI was added and slides were examined by a confocal microscope (LSM510, Carl Zeiss, Germany). For mitotracker deep red staining, after indicated treatments cells were incubated with 200 μM of mitotracker dye for 20 min. These were then fixed and processed for immunofluorescence study by incubating with a Bax specific primary antibody and FITC conjugated secondary antibody. Slides were mounted with DAPI containing medium and images were acquired in confocal microscope. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) staining was performed as per manufacturer's protocol (BD) except the reaction time was increased to 3 h at room temperature. Cells were washed twice with binding buffer and PI solution was added. Slides were washed, mounted and observed under confocal microscope (META, Carl Zeiss).
Tumor growth
HTet23p53 or HTet43GFP cells (5 × 106) in 100 μl PBS mixed with 100 μl matrigel were injected s.c. into 4-6 week-old female NOD/SCID mice (Jackson Laboratories). Total 12 mice were injected with HTet23p53 cells on the right flank and 4 mice were injected with HTet43GFP cells on both the flanks. Out of two groups, one was fed on 500 ng/ml Dox in drinking water. Tumor development was monitored. After tumor-size reached to 5-10 mm in diameter, OA (40 pg/mice) was administered at the tumor site. Tumor-sizes were measured weekly by digital Vernier Caliper (Sigma) and tumor volume was calculated by formula V = [1/2 × (large diameter) × (small diameter)2.
MTT assay, FACS analysis and western blotting
For methylthiazole tetrazolium (MTT) assay, 7,500 cells were treated with Dox, OA and/or Cdk2/5 inhibitor as per experimental requirement and assayed for cell survival. For western blotting following indicated treatments, cells were washed thrice with ice-cold phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer (50 mM Tris-Cl, pH 7.5, with 120 mM NaCl, 10 mM NaF, 10 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1% NP-40 and protease inhibitor cocktail (Roche Diagnostics, Penzberg, Germany). Equal amount of protein was resolved on a polyacrylamide gel. Where ever possible blots were stripped by incubating the membranes at 50°C for 30 min in stripping buffer (62.5 mM Tris-Cl pH 6.7, 100 mM mercaptoethanol, 2% SDS) with intermittent shaking. Membranes were washed thoroughly with TBS and reprobed with required antibodies. Otherwise gels run in duplicates were probed for the desired proteins by western blotting and then compiled.
For FACS analysis cells were plated at a density of 5 × 105 cells in 35 mm plates and allowed to adhere for 24 h. Cells treated as per experimental requirement were harvested by trypsinization and processed for flow cytometric analysis. The fluorescence of propidium iodide (PI) was measured through a 585 nm filter in a flowcytometer (FACS Calibur, BD) for 10,000 cells. Data were analyzed using cell quest software (BD). Details of these are as published earlier [21, 22].
TUNEL staining
To detect apoptotic cells APO-DIRECT TUNEL assay kit (BD) was used followed by flow cytometric analysis as per the manufacturer's instructions with some modifications. Cells were incubated in DNA-labeling solution for 2 h at 37°C and analyzed by FACS Calibur (BD). PI stains total DNA and FITC conjugated dUTP stains apoptotic cells.
Reverse-Transcription-PCR
Total RNA from the cells or tumor samples was extracted using TRIzol™ reagent and PCR was performed as described [21] with following primers; p53 (F) 5-CTG AGG TTG GCT CTG ACT GTA CCA CCA TCC-3', (R) 5'-CTC ATT CAG CTC TCG GAA CAT CTC GAA GC-3'; e6 (F) 5'-TGT GTA TGG AGA CAC ATT GG-3', (R) 5'-ATA GTG CCC AGC TAT GTT GT-3'; β-actin (F) 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3', (R) CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3', at annealing temperature of 55°C and p21 (F) 5'-GGC GTT TGG AGT GGT AGA AA-3' (R) 5'-GAC ACC ACT GGA GGG TGA CT-3' at annealing temperature of 59°C for 25-30 cycles.
Statistical analysis
Statistical comparisons are made using student's paired t-test using SPSS10.0 (SPSS Inc., IL) and P- value < 0.05 was considered significant.