Human prostate cancer cell lines PC-3, DU145 and LNCap, breast cancer cell lines MCF7 and MDA-MB-231, colon carcinoma cell lines SW480, SW620 and HCT116, and mouse embryonic fibroblast cell line NIH-3T3 were purchased from American Type Culture Collection. MCF10A cells (human mammary epithelial cell-line) were kindly gifted by Randolph C. Elble, Ph.D., the Department of Pharmacology at Southern Illinois University School of Medicine. Fetal bovine serum (FBS) and bovine serum (BS) were purchased from Invitrogen. GFP antibody was purchased from Clontech; Lamin A/C (sc-20681) and emerin antibodies were purchased from Santa Cruz biotechnology; anti-phosphorylated histone-H2AX polyclonal antibody was from Trevigen. Phospho-Chk2 (Thr68) was purchased from Cell Signaling Technology, Inc. Two normal tissues (Normal breast (A804144): 41 y; and Normal colon (A605057): 21 y and two tumor tissues (Breast tumor (A810179): 50 y and Colon tumor (A805131): 51 y) total RNA were purchased from BioChain Institute, Inc. GenePORTER® liposome was from Genlantis. The senescent cell staining kit was from Sigma-Aldrich. CometAssay Kit was from Trevigen. pEGFP-lamin A (denoted as LA-pEGFP), Δ50 pEGFP-lamin A (denoted as progerin-pEGFP) or a control empty vector were kindly given by Dr. Tom Misteli, NIH.
Cell culture and plasmids transfection
PC-3, DU145, LNCaP, MDA-MB-231, SW480, SW620 and HCT116 cells were grown in a RPMI 1640 medium supplemented with 10% FBS and antibiotics-antimycotics (100 units/ml penicillin, 100 μg/ml streptomycin and 250 μg/ml amphotericin B) in a humidified incubator under an atmosphere containing 5% CO2 at 37°C. MCF7 and A431 cells were cultured in a Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS and antibiotics-antimycotics. NIH-3T3 cells were grown in a DMEM with 10% BS and antibiotics-antimycotics. MCF10A cells were cultured in DMEM/F12 medium, supplemented with Horse Serum, EGF, Hydrocortizone, CholeraToxin, Insulin, and Pen/Strep. PC-3, MCF7 or NIH 3T3 growth to 70%-80% confluence were transfected with expressing plasmids using GenePORTER® liposome transfection reagent following manufacturer's instructions. Stable clones were obtained after fluorescence activated cell sorting (FACS) and G418 (0.4-0.8 mg/ml) selection.
RT-PCR and real-time PCR
The expression of progerin in established human cancer cell lines was evaluated by RT-PCR. Total RNA was extracted from cell cultures with an RNeasy Mini Kit (QIAGEN) and treated with RNase-free DNase. 2 ug of RNA were reverse transcribed using oligo-dT primers by Superscript TM III First-Strand synthesis system (Invitrogen). Primers for simultaneous detection of full length and truncated LMNA isoforms were located in exon 9 and exon 12 of LMNA (LMNA-RT9F: 5' GTGGAAGGCACAGAACACCT-3'; LMNA-RT12R: 5'-GTGAGGAGGACGCAGGAA-3') as described in . End-point PCR products were separated in 2% agarose gels and stained with ethidium bromide. Primers for specific detection of truncated LMNA (progerin) were located across the aberrant splice junction and in exon 12 (LMNA-RTspecM3F: 5' GCGTCAGGAGCCCTGAGC-3'. Three mismatches were introduced in the primer to avoid primer dimmers formation and cross hybridization with the full-length LMNA mRNA; LMNA-RTspec12R: 5'-GACGCAGGAAGCCTCCAC-3'). Primers used to amplify all LMNA transcripts were located across exon 8-exon 9 splice junction and in exon 9 (LMNA-RTnorm8F: 5'-GGTGGTGACGATCTGGGCT-3'; LMNA-Rtnorm9R: 5'-CCAGTGGAGTTGATGAGAGC-3'). Samples were analyzed in triplicate in three independent experiments. Use of the cryptic splice site in the different cell lines was calculated by normalizing Δ150 LMNA (progerin) RNA levels to the total LMNA RNA levels in each sample. Samples were analyzed in triplicate in three independent experiments. For the real-time quantitative PCR, the reactions were performed as described by Chen et al .
PC-3 cells with stable progerin-pEGFP, LA-pEGFP or pEGFP vector expression were subjected to different treatments. The cell lyses were collected with ice-cold RIPA buffer (Sigma, R0278) in the presence of a protease inhibitor cocktail (Sigma, S8830). Fifty micrograms of protein were fractionated by SDS-PAGE gels in a Bio-Rad Protean II system. After transferring proteins to a PVDF membrane, the membrane was blocked with Odyssey Blocking Buffer from LI-COR Biosciences for 60 min at room temperature and incubated with the primary antibody at appropriate dilutions in Odyssey Blocking Buffer at 4°C overnight. After overnight incubation with appropriate primary antibodies, the membrane was washed (3×) with TBS-T for a total of 15 min, probed with fluorescently-labeled secondary antibody (1:5000) for 50 min at room temperature and washed (3×) with TBS-T for a total of 15 min. The immunoblots were visualized by an Odyssey Infrared Imaging System (LI-COR).
Immunocytochemistry and Confocal Microscopy
PC-3 cells with stable progerin-pEGFP, LA-pEGFP or pEGFP vector expression were seeded at 0.3 × 106/well into 6-well plates with coverglasses to achieve 70% confluence. Twenty four hours later, the cells were fixed with 3% paraformaldehyde in PBS for 15 min, followed by permeabilization with 0.1% Triton X-100 for 1 min. After blocking in 1% BSA/1 × PBS containing 3% horse serum for 30 min, the slides were incubated with primary antibody against Lamin A/C (Santa cruz biotechnology, inc.) with 50 fold dilution for 1 h, washed three times with PBS and then incubated with Alexa Fluor® 568 goat anti-mouse IgG (H+L) secondary antibody for 1 h (1: 100 dilution each). The slides were then washed three times with PBS, counterstained in Prolong® Gold antifade reagent with DAPI and visualized with a BX41 system microscope (Olympus) or with an Olympus Fluoview confocal microscope using a 100× oil immersion objective lens (IX70 Olympus, Melville, NY, USA). Fluorescence was excited by the 488-nm line of an argon laser and the 568/647-nm line of a Krypton laser. Images were analyzed using Fluoview software.
Senescence-associated β-galactosidase (SA β-Gal) staining
PC-3 cells with stable progerin-pEGFP, LA-pEGFP or pEGFP vector expression were seeded in six-well plates. When reaching 70% confluence, the cells were washed twice with PBS and fixed in fixation buffer containing 2% formaldehyde/0.2% glutaraldehyde in PBS for 7 min at room temperature. SA-β-gal staining was performed in fresh senescence-associated X-Gal staining solution containing 1 mg/ml of 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-Gal), pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide and 2 mM MgCl2 in PBS at 37°C (no CO2). Incubation typically lasted for 16 h. Cells were rinsed in PBS and stored in PBS with 70% glycerol. Cells were then examined under a microscope at Χ200 magnification for a blue-green staining of the cytoplasm indicative of senescence.
Estimation of DNA damage by comet assay
We performed the comet assay on Comet Slide (Trevigen) following the manufacturer's instructions. Briefly, the cells were treated with camptothecin (0.1 μM) for 12 hours and the extent of DNA damage was measured 48 hours later. Dakin-frosted slides were covered with 100 ml of 0.6% normal melting point agarose, and the agarose was allowed to solidify under a cover slip on ice. The cover slips were then removed. Subsequently, aliquots (1 ml) of harvested cells containing 1 × 105 cells in culture medium were then centrifuged to pellets. The pellets were resuspended in 80 μl of 0.6% low melting point agarose, layered onto the normal melting point agarose and allowed to solidify under a fresh cover slip on ice. All the steps described were conducted under a reduced light level to prevent additional DNA damage. Slides were then placed immediately in a cold lysis buffer for 1 hour. After lysis, the slides were drained and placed in a horizontal gel electrophoresis tank, surrounded by ice and filled with fresh cold electrophoresis buffer to a level of ~0.25 cm above the slides. Slides were kept in the high pH buffer for 20 min, to allow DNA unwinding. Electrophoresis was then carried out for 20 min at 25 V and 300 mA. The slides were stained with SYBR Green and covered with a cover slip for immediate analysis. The slides were observed with a BX41 fluorescence microscope (Olympus, Center Valley, PA) and more than 100 cells were analyzed to give a representative result for the population of cells. The parameters of comet images such as the length of the tail moments were calculated using Comet Assay IV software (Perceptive Instruments Ltd).
Cell viability measurement
Cell viability was measured with Vi-CELL™ Series Cell Viability Analyzers (Beckman Coulter, Inc.), which is based on traditional cell viability method of trypan blue exclusion.
Animal model and histology
PC-3 (4 × 106) cells with stable progerin-pEGFP, LA-pEGFP or pEGFP vector expression were injected subcutaneously into nude mice. Five groups of nude mice were utilized. Tumor development was monitored 1 week after implantation. About 38 days after implantation, the experiment was terminated and the animals were sacrificed. Primary tumors were removed and fixed for hematoxylin & eosin staining and immunohistochemical analysis. Mitotic and apoptotic figures in tumor sections stained with H&E were counted through the microscope by trained technicians with a mechanical tabulator, in a double blind approach. Total background cells in each field were also counted and the percentage of positive cells [(X positive/Y total count) × 100] was calculated for each tumor.
The probability of statistically significant differences between two experimental groups was determined by Student's t-test. P < 0.05 was considered statistically significant in all calculations.