Cell lines
The OVCAR3 and SKOV3 human ovarian cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). OVCAR3 cells were grown in RPMI 1640 (Wisent, St-Bruno, QC, Canada) supplemented with 20% heat-inactivated FBS (Wisent), 2 mM L-glutamine (Wisent), 100 units/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml insulin, and maintained at 37°C in a humidified 5% CO
2 incubator. The SKOV3 cell line was maintained in DMEM/F12 (Wisent) supplemented with 10% heat-inactivated FBS, glutamine andantibiotics. OVCAR3 R350 cells are a TRAIL-resistant isogenic cell line that was obtained by exposing OVCAR3 cells to stepwise increases of TRAIL over 4 months. This cell line has been described previously [
28]. The construction of the anti-MUC16 scFv has been previously described in detail [
26,
27]. Two independent stable OVCAR3 clones expressing the anti-MUC16 scFv (1:9#7 scFv, 1:9#9 scFv), and the control scFv (ctrl scFv) were generated by transfection of these plasmids into OVCAR3 cells and their validation has been described previously [
26,
27]. Derivation of MUC16CTD-expressing SKOV3 cells has also been previously described [
26,
27]. In this construct, MUC16CTD is tagged at the C-terminal with an His and a c-myc tag to allow easy detection of MUC16CTD expression.
Reagents
Recombinant human TRAIL was purchased from PeproTech, inc. (Rocky Hill, NJ). The TRAIL-R2 agonist antibody (clone 71903), the tetrapeptide caspase inhibitor, z-IETD-fmk, anti-XIAP and anti-caspase-8 antibodies were obtained from R&D Systems (Minneapolis, MN). Anti-caspase-9, anti-caspase-3, anti-Bid, and anti-Bcl-XL, anti-mouse HRP and anti-rabbit HRP antibodies were purchased from Cell Signaling (Beverly, MA). TRAIL-Flag and anti-TRAIL-R1 to R4 receptor antibodies used for flow cytometry were from Alexis Biochemicals (San Diego, CA). Anti-FADD and anti-TRAIL R2 used to perform Western blotting were from EMD Millipore (Etobicoke, ON, Canada). Anti-c-FLIPL and anti-c-FLIPS antibodies were purchased from Calbiochem (LaJolla, CA). Anti-Bcl-2 and anti-CA125 M11 antibodies were obtained from Dako (Burlington, ON, Canada) and anti-CA125 OC125 antibody was from Zymed (South San Francisco, CA). XTT, phenazine methosulfate, propidium iodide, cycloheximide, anti-Flag M2 and anti-tubulin were from Sigma (Oakville, ON, Canada). Anti-Bax and anti-myc-789 used for immunoprecipitation experiments were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-myc 9E10 antibody used for Western blot detection, real-time PCR Taqman Gene Expression Assay Master Mix, Flip primers for RT-PCR assays and Flip siRNA were from Life Technologies Inc (Burlington, ON, Canada). Anti-His antibody used for immunoprecipitation experiments was from Bioshop Canada (Burlington, ON). RNAse was obtained from Roche (Laval, QC, Canada).
Cytotoxicity assays
Cell viability was determined by the XTT assay. Briefly, cells were plated at 20,000 cells/well in 96-well plates. The next day, cells (confluence 60-70%) were treated with human recombinant TRAIL or anti-TRAIL-R2 agonist antibody as indicated and incubated for 48 h. In some experiments, synthetic caspase inhibitor (25 μM z-IETD-fmk) was added 1 h before the addition of 25 ng/ml of TRAIL. At the termination of the experiment, the culture media was removed and a mixture of PBS and fresh media (without phenol red) containing phenazine methosulfate and XTT was added for 30 min. The absorbance of each well was determined using a microplate reader at 450 nm (TecanSunrise, Research Triangle Pack, NC). The percentage of cell survival was defined as the relative absorbance of treated versus untreated cells. All assays were performed in triplicate and repeated three times.
Apoptosis assays
Caspase-3 fluorogenic protease assay was performed according the manufacturer’s protocol (R&D Systems, Minneapolis, MN). In brief, 3 × 106 cells were lysed in 250 μl of cell lysis buffer, and total cell lysates were incubated with 50 μM of DEVD-AFC substrate for 1 h. Caspase-3 activity was measured on a Versa Fluor fluoremeter (BioRad, Hercules, CA). Protein concentration of the lysates was measured with Bio-Rad protein assay kit according to the manufacturer’s recommendations.
To determine the sub-G0 DNA content, floating and adherent cells were harvested, washed with PBS/2% FBS and fixed with cold ethanol for 2 h. Cell pellets were resuspended, washed with PBS, filtered on nylon mesh membrane (40 μm mesh) to remove cell aggregates. Cells were then incubated with propidium iodide (final concentration 20 μg/ml in PBS, RNase A (0.5 mg/ml) and 0.1% Triton X100 overnight at 4°C. Cells were analysed on a FACSCAN flow cytometer (Becton Dickinson, Mississauga, ON).
The release of nucleosomal DNA into the cytoplasm as a measure of apoptosis was determined using the Cell Death Detection ELISA kit according to the manufacturer’s instructions (Roche, Laval, QC, Canada). Briefly, cells were lysed and the extracted cytoplasmic nucleosomal DNA was captured in ELISA wells containing anti-histone antibodies. The nucleosomal DNA was detected with an anti-DNA-POD conjugated antibody. The absorbance of each well was determined using a microplate reader at 410 nm (TecanSunrise, Research Triangle Pack, NC). Each sample was assayed in duplicate. Data are from three independent experiments.
The mitochondrial membrane integrity in OVCAR3 controls and knockdown cells was assayed using the MitoLight™ Apoptosis Detection Kit (EMD Millipore). Cells were cultured in Labtek II chamber slides (Nalge Nunc International, Naperville, IL) in RPMI1640 supplemented with 20% FBS at 37°C to achieve 70-80% confluence. Fresh media containing 200 ng/ml (OVCAR3 cells) or 500 ng/ml (SKOV3 cells) TRAIL was then added and cells were incubated at 37°C in 5% CO2 for 45-60 min. After treatment with TRAIL, unfixed cells were incubated with the MitoLight™ reagent (Chemicon, Billerica, CA) (dilution 1:1,500) for 20 min at 37°C. The wells were washed and identical fluorescein and rhodamine fields were digitized at 400× magnification using an Olympus X170 fluorescence microscope. Dye uptake by non-apoptotic mitochondria concentrates in the mitochondrial membrane and is visualized as the accumulation of red fluorescence in the organelles. However, apoptotic mitochondria are incapable of accumulating the dye due to loss of membrane potential and therefore the monomeric dye in the cytoplasm appears green.
Immunoprecipitation and immunoblot analysis
Whole cell extracts were prepared in lysing buffer containing protease inhibitors and were separated by 12% SDS-PAGE gels. Proteins were transferred to PVDF membranes (Roche, Laval, Québec, Canada) by electroblotting, and immunoblot analysis was performed as previously described (18). All primary antibodies were incubated overnight at 4°C in 5% milk. Proteins were visualized by enhanced chemiluminescence (GE Healthcare, Baie d’Urfé, Québec, Canada). SKOV3 empty vector and SKOV3 MUC16CTD cell lines were lysed for an hour on ice and immunoprecipitates were obtained using anti-HIS, anti-myc-789 or isotypic anti-mouse IgG monoclonal antibodies conjugated to protein-G agarose (EMD Millipore, Billerica, CA). Immune complexes were separated by SDS-PAGE, immunoblotted and probed with anti-c-myc 9E10 antibodies and visualized by enhanced chemiluminescence. For TRAIL DISC analysis, 80% confluent cells were stimulated with 1 μg/ml (OVCAR3) or 2 μg/ml (SKOV3) of Flag-tagged TRAIL and 3 μg/ml of anti-Flag M2 (pre-incubated for 15 min) in RPMI medium for 30 or 60 min. Cells were then washed with ice-cold PBS and lysed with lysis buffer containing 30 mM Tris-Hcl, 150 mM NaCl, 1% Triton supplemented with protease inhibitors. Lysates were cleared, normalized for protein concentration and the DISC were immunoprecipitated with protein G agarose beads overnight at 4°C on a rotating rod. For Western blot analysis, the beads were washed 4 times with lysis buffer and heated in lysis buffer 4×SDS before the supernatants were separated by 12% SDS-PAGE. NIH Image J software was used to quantify the intensity of each band on Western blots. In some experiments, cycloheximide (200 μM) in DMSO was added for the indicated time.
RT-PCR analysis
Total RNAs were extracted from Ctrl scFv- and MUC16 scFv-expressing OVCAR3 cells using Trizol (Life Technologies) according to the manufacturer’s recommendations. RNA integrity was verified on gel by ethidium bromide staining and quantification was performed by determining absorbance at 260 nm. For each sample, total RNA (2 μg) and 1 μM of oligo dT (Promega) were incubated for 5 min at 70°C followed by the addition of 90 units of the reverse transcriptase MMULV (Promega, Madison, WI) and 2.5 μM dNTP for 1 h at 42°C. Amplification of cDNA was done in a PCR reaction using the Taqman Gene Expression Assay Master mix from Applied Biosystems as follow: 2 min at 50°C, 10 min at 95°C and then 40 cycles of 15 s at 94°C and 1 min at 60°C on a StepOne Plus real time PCR system (Life Technologies Inc). The primers for the amplification of cFLIPL (Gene expression assay Hs00153439_m1) and cFLIPS (Gene expression assay Hs00354474_m1) were from Life Technologies Inc.
Flow cytometry for TRAIL receptors and MUC16 expression
Cell monolayers were detached using EDTA, washed with PBS and fixed with paraformadehyde 4% in PBS for 20 min. Cells were incubated with the following unlabeled primary antibodies (10 μg/ml) for 1 h at room temperature with human anti-TRAIL-R1, -R2, -R3, and -R4 antibodies. The isotypic control antibody was a normal mouse IgG (BD Biosciences, Mississauga, ON). After three washes with PBS, cells were incubated with PE-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) for 45 min at room temperature. Cells were washed three times and analyzed immediately using a FACSCAN flow cytometer (Beckton Dickinson). After they had reached 80-90% confluence, MUC16 knockdown cells and ctrl scFv cells as well as the parental OVCAR3 cells were fixed with 4% paraformaldehyde. Cell surface MUC16 tumor antigen was detected by flow cytometry using the anti-CA125 M11 mouse monoclonal antibody and revealed using an anti-mouse PE-conjugated (Jackson ImmunoResarch).