The human cervical cancer cell lines utilized were HeLa (HPV18 positive), Caski (HPV16 positive), and C33A (HPV negative). These and the osteosarcoma cell line SAOS2 as well as IMR-90 human fibroblasts were maintained in Dulbecco's modified Eagle medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. Primary keratinocytes were prepared from human foreskins as described previously (66
) and maintained in keratinocyte Epilife medium supplemented with human keratinocyte growth supplement (HKGS; Cascade Biologics, Portland, OR). Near-diploid immortalized keratinocytes that form skin (NIKS) as well as NIKS transfected with HPV16 (NIKS-HPV16) (12
) (kind gifts from Paul Lambert, University of Wisconsin—Madison) were cultured on irradiated J2-3T3 feeder cells as previously described (42
Viral and plasmid constructs.
Adenovirus (Ad) and Ad expressing E2ts (AdE2ts) have been described previously (62
), as well as the E2-TA and E2-TR expression plasmids (63
). Nontargeting and DEK-specific short hairpin (NTsh and DEKsh, respectively) lentiviral vectors were obtained through the Sigma Mission short hairpin RNA (shRNA) program (Sigma-Aldrich, St. Louis, MO). The relevant product numbers were SHC 002VC for the control nontargeting vector (NTsh) and TRCN0000013107 for the DEKsh targeting vector. The LXSN retroviral constructs were a generous gift from Denise Galloway (University of Washington, Seattle, WA). The cytomegalovirus (CMV)-driven DsRed lentiviral expression vector was a generous gift from the Malik laboratory (Cincinnati Children's Hospital, Cincinnati, OH). Specific lentiviral constructs targeting the HPV16 E6 open reading frame were custom designed through Sigma. The shRNA targeted the E6 coding region, nucleotides (nt) 196 to 216: 5′-GTACTGCAAGCAACAGTTACT-3′. Double-stranded RNAi oligonucleotides designed against the HPV18 E7 coding region (nt 694 to 712, 5′-GGAAGAAAACGATGAAATA-3′) as well as the siGenome nontargeting control were purchased from Dharmacon (Lafayette, CO).
Adenoviral infections using empty vector (Ad) and temperature-sensitive E2-expressing Ad (AdE2ts) were carried out as described previously (62
). Briefly, a total of 1 × 106
cells plated on 10-cm dishes were infected on two consecutive days with either 10 PFU or 10 infectious units (IU) of virus, as indicated in the figure legends, in 1 ml of phosphate-buffered saline (PBS) containing 4% fetal bovine serum (FBS) for 1 h. The virus was then aspirated, and cells were washed twice with PBS and then overlaid with medium. On the third day, cells were split and placed at 37°C for several hours to allow attachment and later shifted to the permissive temperature of 32°C for 3 days, after which they were shown to be irreversibly growth arrested (62
). For lentiviral infections, cells were infected with 4 ml of shRNA-expressing nontargeting (NTsh), DEK-targeting (DEKsh), or HPV16 E6-targeting (HPV16 E6/E7sh) virus for 8 h. The cells were then washed twice with PBS and overlaid with fresh medium. At 1 day postinfection, the cells were overlaid with medium containing 650 ng/ml puromycin for selection of a pure, virally transduced population. For retroviral infections, cells were incubated with 4 ml of viral supernatant of empty vector (LXSN) or HPV16 E7- or HPV16 E6/E7-expressing vectors (LXSN E7 or LXSN 16E6/E7, respectively) or with 2 ml of viral supernatant of HPV16 E6 (LXSN E6)-expressing vector using 8 μg/ml Polybrene for 4 h and then overlaid with fresh medium. The same was done using a pBabe empty vector or vector expressing oncogenic H-ras. Cells were then selected and maintained in 500 μg/ml G418-containing medium.
Transient transfections and viral infections.
Transient transfections were conducted as previously described (66
). Briefly, HeLa cells seeded on a 60-mm plate were transfected with a total of 4 μg of plasmid DNA using FuGENE transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. For cotransfections, cells were transfected with 400 ng of neomycin resistance plasmid and 3.6 μg of DNA. Cells were then selected in medium containing 900 μg/ml G418 for 8 days.
Migration and invasion assays.
Migration assays were conducted using 8.0-μm-ore-size polycarbonate transwell inserts (Corning Inc., Corning, NY) according to the manufacturer's instructions. Briefly, inserts were first rehydrated using 0.1 ml of medium in the top inserts and 0.6 ml in the wells of a 24-well dish for at least 1 h. A total of 1 × 105 cells were plated in the upper inserts and then allowed to migrate for at least 16 h. Nonmigrating cells were removed with a cotton swab while cells that had migrated were fixed in methanol for 10 min and stained with Giemsa dye (Sigma-Aldrich, St. Louis, MO) at a 1:20 dilution. Four quadrants of the membrane were then counted under a light microscope. For invasion assays, Matrigel-coated invasion inserts (BD Biosciences, Billerica, MA) were rehydrated with DMEM containing 10% FBS, for a final volume of 0.5 ml in the upper inserts and 0.75 ml in the wells of a 24-well dish. After incubation for a minimum of 2 h, the medium from the upper inserts was removed and replaced with 0.5 ml of serum-free medium containing 1 × 105 cells. Cells were allowed to migrate through the Matrigel for 20 to 22 h and then were stained and counted as described for migration assays.
Time-lapse video microscopy.
A total 100 μl of 1 × 105 cells were seeded as a colony in a four-chambered Lab-Tek coverglass slide (Nunc, Rochester, NY). The cells were allowed to attach for several hours and were gently washed with PBS and overlaid with Leibovitz's L-15 medium containing l-glutamine and 5% serum (Gibco, Invitrogen, Carlsbad, CA) in the absence of phenol red. Control and treated groups were imaged simultaneously using a Zeiss LSM510 confocal system attached to a Zeiss Axiovert 200 microscope and a heated stage. Images were captured at ×10 magnification every 5 min for at least 12 h using the LSM software with the MultiTime macro. Cell edges for at least 20 independent cells within the field of view were then tracked using ImageJ software with the MTrackJ plug-in, and average distance and/or velocity of each cell was determined. This was repeated for at least two different field views for each cell group.
Western blot analyses.
Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (1% Triton, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 0.16 M NaCl, 10 mM Tris [pH 7.4], 5 mM EDTA) supplemented with protease inhibitor cocktail (Pharmingen, San Diego, CA). Protein concentrations were determined with Bradford reagent (Bio-Rad, Hercules, CA). Aliquots containing equal amounts of total protein were boiled in SDS sample buffer and resolved by SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and probed overnight with the following antibodies used at the indicated dilutions: p53 monoclonal, 1:1,000; polyclonal cyclin A, 1:400; HPV18 E7, 1:200 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); monoclonal DEK, 1:1,000 (BD Biosciences, San Diego, CA); monoclonal Rac1, 1:1,000 (Upstream Biosciences, Calgary, Alberta, Canada); monoclonal actin, 1:20,000 (Seven Hills Bioreagents, Cincinnati, OH), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Fitzgerald, Acton, MA). Membranes were exposed to enhanced chemiluminescence reagents (Perkin Elmer, Boston, MA), and protein bands were detected by autoradiography. For HPV16 E7 detection, a mixture of 1:50 HPV16 E7 8C9 monoclonal antibody (Invitrogen, Carlsbad, CA) and 1:200 HPV16E7 ED17 monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted in 3% milk was used. Signal was then detected using Super Signal West Femto substrate (Thermo Scientific, Rockford, IL).
Preparation of ECM.
Ad- or AdE2ts-infected cells plated at a confluent density were washed twice with PBS and then once with water. Cells were then treated with 20 mM NH4OH at room temperature for 5 min and aspirated, and the underlying endogenous extracellular matrix (ECM) was rinsed twice with PBS. Fresh Ad-infected cells were then seeded onto the AdE2ts ECM and vice versa, and time-lapse migration assays were conducted as described above.
Cell cycle analyses.
Cell cycle analysis was conducted as previously described (67
). Briefly, HeLa cells were treated with medium containing the cell cycle inhibitors mimosine (0.5 mM) and thymidine (2.5 mM) for 16 h to induce cellular arrest in the G0
and S phases of the cell cycle, respectively. Cells were then washed and pelleted by centrifugation, and 1 × 106
cells were resuspended in 800 μl of solution containing 1% bovine serum albumin (BSA) in PBS, 100 μl of 500 μg/ml propidium iodide in 10 mM sodium citrate, pH 7.0, and 100 μl of boiled RNase A (10 mg/ml in 10 mM Tris-HCl, pH 7.5). Cells were then incubated at 37°C for 30 min, and cell cycle profiles were obtained using a flow cytometer (BD Biosciences, San Jose, CA).
Total RNA was harvested from cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription-PCR (RT-PCR) was performed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) or a QuantiTect reverse transcriptase kit (Qiagen, Valencia, CA) as described by the manufacturer. Quantitative RT-PCR (qRT-PCR) was carried out using an ABI 7500 system (Applied Biosystems, Foster City, CA) using SYBR green master mix (Applied Biosystems, Foster City, CA). The levels of mRNA assessed were all normalized to the c-abl, GAPDH, or EEFA1 reference gene. For verification of gene regulation identified from meta-analysis, RT-PCR was conducted using mRNA from three independent experiments, and all levels were normalized to the EEFA1 reference gene. A list of all primers used along with their respective sequences is given in .
Ad- and AdE2ts-infected cells were plated onto poly-d-lysine hydrobromide (Sigma, St. Louis, MO)-coated coverslips and fixed with 2% paraformaldehyde in PBS. Coverslips were incubated in 0.2% Triton X-100 in PBS for 2 min, blocked with 2% BSA in PBS for 2 h, washed twice with PBS, and incubated with primary antibody at 4°C overnight. The following antibody dilutions were used: E-cadherin, 1:50 (BD Biosciences, San Diego, CA); vimentin, 1:300 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); fibronectin, 1:300 (Sigma, St. Louis, MO); and Rac1, 1:200 (Upstream Biosciences, Calgary, Alberta, Canada). Cells were then washed and incubated in anti-mouse, anti-rabbit, or anti-goat Alexa-Fluor 568- or 555-conjugated secondary antibody (1:500; Invitrogen, Carlsbad, CA) at room temperature for 2 h. Coverslips were then mounted onto glass slides with DAPI (4′,6′-diamidino-2-phenylindole) Vector Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and images were taken using a Zeiss fluorescence microscope.
Rho GTPase pulldown assays.
HeLa cells infected with Ad or AdE2ts were washed with PBS and then lysed in buffer containing 20 mM Tris-HCl, pH 7.6, 100 mM NaCl, 10 mM MgCl, 1% Triton X-100, 0.2% SDS, and protease and phosphatase inhibitors. Cell lysates containing equal amounts of protein were then incubated with glutathione beads conjugated to glutathione S-transferase (GST)–p21-activated kinase 1 (PAK-1) for Rac1, and total levels were determined by immunoblotting using Rac1 (Upstream Biosciences, Calgary, Alberta, Canada) at a dilution of 1:500.
Senescence-associated β-galactosidase assay.
Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 15 min, washed with PBS, and incubated at 37°C for 16 to 36 h in SA-β-galactosidase staining solution as described previously (9
). Cells were analyzed under a light microscope.