Detroit-562, Cal27, 006/1, 005A, 013, HN3, HN4, HN5, HN6, 015B, SIHN-5B, 011A, SIHN-11B, (head and neck cancer) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). PJ41 and PJ34 (head and neck cancer) were cultured in Iscove’s Modified Eagle’s Medium (IMEM, Gibco, Invitrogen, Paisley, UK) and Jurkat (leukemia) were cultured in Roswell Park Memorial Institute media (RPMI). DMEM and IMEM were supplemented with 5% (v/v) FCS and RPMI with 10% (v/v) FCS (PAA, Pasching, Austria). All media contained 1% (v/v) L-glutamine and 0.5% (v/v) penicillin/streptomycin and cells were kept at 37°C in a humidified atmosphere containing 10% CO2. All cell lines were obtained from Dr S Eccles, ICR, UK, except for Jurkat, which was obtained from Prof. R. Marais, ICR, UK.
Reovirus (Dearing Type 3) was obtained from OncolyticsTM Biotech Inc. (Calgary, Canada) and stored at −80°C. Neat stocks were in phosphate-buffered Saline (PBS) and 1:10 working dilutions were stored in DMEM containing 2% (v/v) FCS, 1% (v/v) glutamine and 0.5% (v/v) penicillin/streptomycin (plating media). New stocks of working dilutions were made periodically and titred by standard Tissue Culture Infectious Dose 50 (TCID50) assay on L929 cells, as described previously
Recombinant human EGF (R&D Systems, Abingdon, UK), along with the EGFR inhibitors Iressa/Gefitinib (Biaffin, Kassel, Germany) Tyrphostin-AG99 (Calbiochem, Merck Chemicals, Nottingham, UK) and EGFR blocking antibody ICR62 (from Dr Sue Eccles, ICR, UK) were used in cell kill assays, western blot and one-step growth curve assays. MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, p38 MAPK inhibitor SB202190 MEK1/2 and MEK 5 inhibitor PD184352 (all from Calbiochem, Merck Chemicals, Nottingham, UK) and Wortmannin (Sigma-Aldrich, Gillingham, UK) were used in cell kill and western blot analyses. ZVAD (R&D Systems, Abingdon, UK), chymotrypsin (CHT, Roche Applied Science, Burgess Hill, UK) and 2-aminopurine (2AP, Sigma Aldrich, Gillingham, UK) were used in cell kill assays. Camptothecin, (Sigma-Aldrich, Gillingham, UK) was used as a positive control for the induction of apoptosis in western blots.
Cell survival experiments
Cells were seeded at 5x103 in 96-well plates and incubated at 37°C for 24hrs before experimental conditions were applied. Where used, cells were treated with antibodies, inhibitors and ligands for 1–2 hrs before infection. Additional plating media was added to the wells 2-24hrs after infection and cell survival was assessed 96 hrs post-infection by MTT assay as described previously
. Reovirus IC50 values were determined by interpolation from a sigmoidal dose response curve fit of the log transformed survival data, derived using GraphPad Prism version 4.0c for Mac OS X (GraphPad Software Inc. San Diego, USA).
ISVPs and cores
Reovirus stocks were treated with a final concentration of 10μg/ml sequencing grade CHT reconstituted in 1 mM HCl plus sequencing buffer, as per manufacturer’s instructions. Following digestion the CHT was neutralised with FCS and equal volumes of virus were analysed by western blot (see below). Proteins were detected using polyclonal anti-reovirus goat serum (Oncolytics Biotech Inc). Rabbit anti-goat HRP-conjugated antibody (Pierce, Perbio, Aalst, Belgium) was used for secondary detection. For cell kill analyses ISVP and core particles were created as above, diluted out in plating media and used to infect cells. Survival was analysed as described above.
Assessment of cell surface EGFR
Cells were cultured in T175 flasks, harvested and 1×106 cells stained with ICR62 for 1 hr at 4°C. Primary antibody binding was detected using F(ab’)2 rabbit anti-rat FITC conjugated IgG (Serotec, Oxford, UK). Staining was analysed using a FACSCalibur machine (Becton Dickinson, Oxford, UK).
Cells were incubated at 37°C for 24hrs before treatment with inhibitors. Monolayers were washed twice with PBS and scraped into 200 μl of lysis buffer (LB), supplemented with complete mini protease inhibitor cocktail tablets (Roche Applied Science, Gillingham, UK) for EGFR and ERK1/2 detection, phosphatase and protease inhibitors, as previously described for AKT analysis
, and 10 μg/ml TLCK, 1 mM PMSF and a 1:100 dilution of protease cocktail I (all Sigma-Aldrich, Gillingham, UK) for pro-caspase 3 assay. Lysates were loaded into pre-cast sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, either Precise Protein gels (Pierce, Perbio, Aalst, Belgium) or NuPage Novex Bis-Tris gels (Invitrogen, Paisley, UK). Following electrophoresis proteins were transferred to polyvinylidene fluoride (PVDF) membranes and probed with specific primary antibodies as follows: murine anti-EGFR (Sigma-Aldrich, Gillingham, UK), rabbit anti-pY1068 EGFR (Invitrogen, Paisley, UK) rabbit anti-p44/42 MAPK, rabbit anti-phospsho-p44/42 MAPK, rabbit anti-AKT, rabbit anti-phospho-AKT, rabbit anti-EIF2α, rabbit anti-caspase 3 (all Cell Signalling Technology, Danvers, USA). Incubations with primary antibodies were followed by secondary labelling using sheep anti-mouse HRP (Amersham Biosciences, GE Healthcare, Amersham, UK) or goat anti-rabbit (Santa Cruz Biotechnology, Santa-Cruz, USA). SuperSignal West Pico Chemiluminescent Substrate (Pierce, Perbio, Aalst, Belgium) was used according to the manufacturers instructions for detection. Membranes were stripped between antibody staining procedures in Restore Western Blot Stripping Buffer (Pierce, Perbio, Aalst, Belgium) for 15mins at 37°C. Murine anti-α tubulin or anti-α tubulin (Sigma-Aldrich, Gillingham, UK), murine anti-GAPDH (Novus Biologicals, Littleton, USA) or rabbit anti-β actin (Cell Signalling Technology, Danvers, USA) were used for loading controls.
Active Ras Pull-Down and Detection
Cells were grown so they were sub-confluent in T75 flasks prior to harvesting, processing and western blotting for Ras small GTPase activation using the Active Ras Pull-Down and Detection Kit (Thermo Scientific, Rockford, IL, USA). Experiments were performed per protocol according to the manufacturer’s instructions.
One-step viral growth assays
Cells were seeded at 1×105 in 24 well plates and treated o/n at 37°C with plating media alone, or plating media containing EGFR ligand/inhibitors. The following day cells were infected with reovirus for 2 hrs. Monolayers were washed once with PBS and the ligand/inhibitors replaced. Cells were scraped into the supernatant and harvested at time points post-infection, freeze-thawed three times and titred by TCID50 assay on L929 cells, as described previously
Cells were plated at 5 × 105 in 6 cm dishes. Cells were treated with SB202190 (10 μM) for 2 hours, harvested, and analysed for phospho-p38 (Surveyor IC: Human/Mouse/Rat Phospho-p38∝ (T180/Y182) immunoassay #SUV869, R&D Systems, Minneapolis, USA). Experiments were performed according to the protocol provided for the assay by the manufacturers.
Cal27, HN3, HN5 and SIHN-5B cells plated at 1 × 106 in 10 cm dishes were treated with reovirus at an MOI of 5, or left untreated. Cells were incubated for 24 hours and supernatants were collected and spun down to remove cell debris. Samples were stored at −20°C until analysis for alpha, beta and gamma interferon by ELISA. IFN-α was analysed using match-paired antibodies from Mabtech, IFN-γ with match-paired antibodies from BD Biosciences and IFN-β using a kit from PBL Interferon Source according to the manufacturer’s instructions. Data were read on a Multiskan EX plate reader (Thermo Scientific) at 405 nm using Ascent software.
JAM-1 FACS Analysis
Cells were harvested with trypsin, pelleted and resuspended in FACS buffer (1% FCS in PBS). 1 × 105 cells in 100 μL were stained with 2 μL of JAM-A antibody (1H2A9, Santa Cruz, USA) or isotype control (mouse IgG2b-PE, Santa Cruz) and incubated for 30 minutes at 4°C. One millilitre of FACS buffer was added and cells were pelleted. Pellets were either resuspended in 500 μL PBS and analysed within an hour using a FACSCalibur machine (Becton Dickinson, Oxford, UK), or fixed in 1% paraformaldehyde for analysis within 5 days.
The data on EGFR status and reovirus cell killing were not normally distributed. Therefore Spearman’s rank correlation was used to test the correlation between EGFR status and reovirus cytotoxicity. A non-parametric test (Wilcoxon signed rank test) was used for testing of significance when evaluating the effects of agonists and inhibitors of the RGFR/Ras pathway on reovirus-induced cytotoxicity.