Cell lines and hybridomas
EL-4 (mouse lymphoma), L1210 (mouse lymphoma), Jurkat (human lymphoma) cell lines were cultured in RPMI-1640; IMR-32 (human neuroblastoma) and Neuro-2A (mouse neuroblastoma) cell lines were cultured in EMEM medium; human melanomas mS and A375 were cultured in DMEM medium. All culture mediums were supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone), 2 mM L-glutamine and antibiotic/antimycotic solution (Sigma). Hybridoma cells HB9326 were maintained in Hybri-Max RPMI-1640 medium, supplemented with 10% FBS, 2 mM L-glutamine and antibiotic/antimycotic solution. All cell lines except mS were kindly provided by Dr. E.V. Svirshchevskaya (Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry), cell line mS was kindly provided by Dr. S.E. Dmitriev (Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University), HB9326 hybridoma cell line was originally purchased from the American Type Culture Collection (ATCC) and kindly provided by Dr. Telford (Experimental Transplantation and Immunology Branch, NCI, National Institutes of Health).
Antibodies and reagents
Mouse ME361 (S2A) antibody produced by HB9326 hybridoma cells were purified as described previously . GD2-specific antibodies ME361 were purified from mouse ascites by affinity chromatography. Other anti-GD2 14G2a mAbs were purchased from Millipore Inc. Anti-GM2/GD2 synthase and anti-ALCAM antibodies, siRNA and primers for GM2/GD2 and GD3 synthases were purchased from Santa Cruz Biotechnology Inc.
Staining of EL-4, L1210, Jurkat, IMR-32, Neuro-2A, mS, and A375 cells with two type of GD2-specific antibodies 14G2a and ME361 was performed as described previously . In brief, cells were detached from the culture plates (adherent cells were trypsinized and washed two times with PBS) and were incubated with AlexaFluor-488-labeled or unlabeled anti-GD2 mAbs (1 μg per 106 cells) for 1 h and then washed in PBS supplemented with 1% FBS and 0.02% sodium azide. After that, in the case of unlabeled anti-GD2 mAbs, the cells were incubated with FITC-labeled anti-mouse IgG (1:1000) for 40 min, and then twice washed in PBS. All procedures were performed at 4°C. The samples were immediately analyzed using EPICS Coulter XL-MCL flow cytometer. In each sample at least 5,000 events were collected. For all samples, the analysis was performed in triplicate. The data was analyzed using FlowJo and WinMDI software.
Microscopy and immunofluorescence
EL-4, IMR-32 and mS cell lines were grown on glass coverslips (Fisher Scientific) placed into 6-well tissue culture plates (Greiner). The cells that were grown to 80% confluence were subsequently washed with PBS and fixed with 2% paraformaldehyde (PF) for 30 min at room temperature (RT). After which, cells were washed twice with PBS and quenched with 50 mM NH4Cl for 10 min. After washing with PBS, the cells were blocked with PBS containing 10% FBS and incubated with 100 μl anti-GD2 mAbs (10 μg/ml) for 1 h at 4°C and then with FITC-labeled anti-mouse IgG (titer 1:1000) for 40 min at 4°C. Stained cells were fixed with 2% PF for 30 min at RT, and then sequentially washed in PBS and distilled water. Counterstaining was performed with Hoechst 33342 (0.5 μg/ml) for 10 min, and finally cell preparations were mounted in Mowiol (Calbiochem-Behring GmbH). Slides were analyzed using a confocal laser scanning microscope EZ-C1 Eclipse TE2000 (Nicon) equipped with a Plan Apo 40X and 60X objectives. Images were collected with EZ-C1 program and processed with EC1 Viewer (Nikon).
Ganglioside purification and quantitation
Total cellular gangliosides were extracted from GD2-positive (EL-4, mS, IMR-32) and GD2-negative (Jurkat, L1210, A375, Neuro-2A) cell lines. Total lipid extracts were obtained by multiple extractions of the lyophilized cell pellets (5 × 107 cells) with chloroform/methanol (2:1 and 1:2 (v/v) at 4°C. At each stage, the hydrophobic extracts were separated from the pellet by centrifugation (12000 g, 10 min). Total lipid extracts were washed with water five times to separate gangliosides as described by Folch et al. . Gangliosides in the aqueous phase were further purified on the cartridge Strata-X (33 μm, 60 mg/3 ml; Phenomenex) and their concentrations were assessed by the modified resorcinol method . High-performance thin layer chromatography (HPTLC) analysis of gangliosides was performed on silica gel using 60 HPTLC plates (Merck) in chloroform/methanol/0.2% aq. CaCl2 (60:40:9, v/v/v) system. Then plates were dried in the flow of cool air, incubated at 110°C for 15 s, and visualized by spraying with resorcinol-HCl reagent and further heating for 20 min at 110°C. Total cellular ganglioside content was determined as the sum of individual gangliosides measured by HPTLC densitometry (Shimadzu CS-920) using known concentrations of bovine liver GM1 (0.1 – 1 μg) as standard.
Viability and cell death assays
Propidium Iodide (PI) assay
Analyses of cell death as determined by DNA fragmentation were performed using propidium iodide (PI) staining in accordance to previously described method  with modifications . The tumor cells (5 × 105 cells per sample) were incubated with anti-GD2 mAbs at concentration of 5 μg/ml for 24 h under standard culture conditions. After incubation the cells were fixed and permeabilized with ice cold ethanol at 4°C for 60 min, and washed twice with PBS by centrifugation for 10 min at 300 g. The cell pellets were resuspended in DNA staining buffer (PBS, 20 μg/ml PI (Sigma), 20 μg/ml RNase A (Fermentas)), and further incubated for 30 min at RT. For all samples, cell death analysis was performed in triplicate. An EPICS Coulter XL-MCL flow cytometer was used to evaluate percent of cells with lower intensity of fluorescence in FL3 channel, which is characteristic of cells with fragmented DNA. In each sample at least 5,000 events were registered. Data processing was performed using FlowJo and WinMDI software.
Antibody-induced decrease in cell viability was analyzed by colorimetric MTT (3-[[4, 5]-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; purchased from Sigma) assay previously described by Denizot and Lang . Briefly, tumor cells were cultured in 96-well flat-bottomed tissue culture plates (104 cells/well, Greiner) with serial dilutions of mAbs ME361 and 14G2a (concentration range was from 0.031 to 10.000 μg/ml) for 72 h under standard culture conditions. After incubation, the MTT solution (250 μg/ml final concentration) was added to each sample for 4 h. The optical density (OD) was read in a Multiscan FC microplate reader (Thermo Scientific) at a test wavelength of 540 nm. Cell viability was measured as ratio of OD540 of cells treatment with anti-GD2 mAbs to OD540 of control cells. All MTT experiments for each cell line were reproduced at least three times.
Apoptotic volume decrease (AVD)
Apoptotic volume decrease of EL-4 cells was detected by flow cytometry. Intact untreated cells or cells treated with anti-GD2 antibodies were distinguished as normal and shrunken populations by the changes in forward and side light scatter (FCS/SSC) characteristics. Cells with apoptotic volume decrease had reduced mean of forward scatter and increased mean of side scatter as compared with normal cells. In each sample at least 5,000 events were registered. The data was analyzed using FlowJo and WinMDI software.
Caspase-3 activation assay
Evaluation of caspase-3 activation was performed in accordance with the method described earlier . 2 × 106 of untreated or treated with anti-GD2 mAbs EL-4 cells were washed once with PBS. Then, the cell lysate was prepared using RIPA-buffer. 20 μl of the lysate was placed in each well of a 96-well plate and the volume was adjusted to 200 μl buffer (100 mM HEPES, 20% glycerol, 5 mM DTT, 0.5 mM EDTA). The plate was incubated for 30 min at +37°C and then solution of fluorescently labeled caspase substrate Z-DEVD-AFC (10 μM) was added to each well. The fluorescence intensity was measured using Glomax spectrofluorometer (Promega, USA) at wavelengths on excitation and emission 400 nm and 505 nm, respectively.
Plasma membrane permeability assay
The loss of plasma membrane integrity was analyzed using of fluorescent DNA binding dye 7-AAD (7-aminoactinomycin D; purchased from Sigma). EL-4 cells were washed once in PBS and resuspended in 0.5 ml of staining solution (PBS with 2 μg/ml 7-AAD). 7-AAD fluorescence of cells was analyzed by flow cytometry using FL3-channel. In each sample at least 5,000 events were collected. The data was analyzed using FlowJo and WinMDI software.
Assessment of mitochondrial membrane potential in living cells
Mitochondrial membrane potential (ΔΨm) was measured using fluorescent dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) (Sigma). The cell suspension was adjusted to a density of 1 × 106 cell/ml and incubated in complete medium for 15 min at RT in the dark with 20 nM DiOC6(3) or with 2 μg/ml JC-1. After which, the cells were washed twice in cold PBS, suspended in a total volume of 500 μl and analyzed by flow cytometry (FL1-channel for DiOC6(3), or FL1 and FL2 channels for JC-1). In each sample at least 5,000events were collected. The data was analyzed using FlowJo and WinMDI software.
Polystyrene microtiter plates (Greiner) were coated with gangliosides GD2, GM2, GD1b and GD3 that were obtained according to the method applied in our previous work , or kindly provided by Dr. Mikhalyov (Institute of Bioorganic Chemistry, Russia Academy of Sciences) at concentration 0.25 μg in 100 μl of 70% methanol per well. Following air drying, all wells of the plate were blocked with 2% BSA in PBS-T (0.05% Tween 20 in PBS) in 100 μl per well for 2 h at RT. Antibodies (100 μl per well in PBS-T) were added in triplicates at different concentrations. Following incubation for 2 h at 37°C and washing with PBS-T, HRP-goat anti-mouse IgG (1:12000) were added. After incubation for 1 h at 37°C and further washing, TMB color reaction was performed and OD was read using Multiscan FC microplate reader (Thermo Scientific) at 450 nm. Percent of cross-reactivity was measured as ratio of OD450 of TMB substrate in GM2-, GD1b- or GD3-coated wells to OD450 of TMB substrate in GD2-coated wells.
The amount of gangliosides adsorbed to each well was determined by using fluorescent-labeled gangliosides BODIPY-FL-C5-GM1 and BODIPY-FL-C5-GD3 (kindly provided by Dr. Mikhalyov). Fluorescent probes were coated at the same concentration as unlabeled gangliosides (0.25 μg in 100 μl per well in 70% methanol), and the same operations were performed for fluorescent probes except adding of antibodies. At the last stage BODIPY-labeled gangliosides GM1 and GD3 that were adsorbed on surfaces of the wells were subsequently dissolved in methanol and fluorescence was measured using a Dynatech Micro FLUOR Reader (excitation 490 nm, emission 510–570 nm). The amount of gangliosides that were adsorbed on the wells was measured using proper calibration curve (linear regression: RFU BODIPY-FL-C5-GD3 = 20.726 + (271.329 × amount of ganglioside per well), RFU BODIPY-FL-C5-GM1 = 36.396 + (248.714 × amount of ganglioside per well, RFU – relative fluorescence units). All experiments were repeated three times.
Modulation of GD2 expression
Downregulation of GD2 expression using PDMP inhibitor
In the initial experiments we determined optimal concentration of PDMP inhibitor and time of incubation to downregulate GD2 expression in EL-4 cells. EL-4 cells were treated with different concentrations of PDMP (at rage of 5–50 μM) for 2–7 days. The expression of GD2, cell viability, and cell death were analyzed by flow cytometry using surface staining for GD2, PI-, and MTT-tests. In these experiments, the cells were treated with 2.5-100 μM PDMP and incubated for 72 h. After selection of optimal concentration, EL-4 cells were cultured for 6 days in the presence of 15 μM PDMP before the analysis of cytotoxicity induced by treatment with anti-GD2 antibodies.
Knockdown of GM2/GD2 and GD3 synthases by siRNA
siRNA for mouse GM2/GD2 or GD3 synthases were purchased from Santa Cruz Inc. The cells were transfected with these siRNAs using lipophilic agent Lipofectamine-2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested 48 h post-transfection and further incubated with anti-GD2 mAbs for 24 h followed by performing PI-test.
Western blot analysis
Protein lysates of EL-4 cells were prepared using RIPA buffer (Assay Design). The proteins from cell lysate were fractionated in SDS-PAGE, and were transferred onto nitrocellulose membranes using a semi-dry transfer device V10-SDB (Biostep). Membranes were further incubated in blocking buffer (0.05% Tween 20, 5% nonfat dried milk in PBS) for 1 h at RT, followed by incubation in primary anti-GM2/GD2 synthase antibody (10 μg/ml) for 1 h at RT in PBS supplemented with 0.05% Tween 20 (PBS-T). After washing several times with PBS-T, the membranes were incubated for 1 h in HRP-conjugated secondary antibody (diluted 1:2000) at RT, and then were washed four times with PBS-T. The immunoreactive proteins were visualized using the Metal Enchanced DAB Substrate Kit (Thermo Scientific) according to the manufacturer’s instructions.
RNA isolation and cDNA synthesis
Cells transfected with siRNA that target GM2/GD2 synthase or control cells were dissolved in 0.5 ml of Trizol reagent for isolation of the total RNA as described by the manufacturer (Invitrogen). All RNA extractions were carried out in a chemical hood using RNAse-free labware. RNA quality and quantity were evaluated by agarose gel electrophoresis and UV spectrometry (NanoVue, GE Helthscare). Samples were stored at −80°C until used. For reverse transcription reaction, 2 μg of total RNA was reversely transcribed using MMLV-RT kits according to the manufacturer’s protocol (Evrogen).
Real time RT-PCR
A ten-fold serial dilution of the cDNA derived from EL-4 cells was prepared in order to make standard curves and determination of PCR efficiency primers for the GM2/GD2 synthase gene (Santa Cruz Biotechnology) and GAPDH housekeeping gene (Evrogen). For performance of real-time RT-PCR we used a DT-96 PCR machine (DNA-Technology LLC), and each reaction was performed in a total volume of 20 μl containing 2 μl of cDNA of the test sample or control sample (standard curve) with 5xSybrGreen-mix prepared according to the manufacturer's protocol (Eurogen). Final concentrations of the primer sets and MgCl2 were 10 μM and 3 mM, respectively. After the denaturation step at 95°C for 5 min, the amplification program was set at 40 cycles each consisting of denaturation at 95°C for 15 s followed by annealing at 58°C for 10s, extension at 72°C for 3 min, followed by detection at the specified acquisition temperature. Melting curve analysis was used for amplicon`s size estimation. Negative controls, samples without reverse transcription or cDNA template were included with every PCR run and were always negative (not shown). Relative gene expression was determined as the ratio of the GM2/GD2 synthase gene to the internal reference gene expression (GAPDH) based on the Ct values using QGENE software.
Graphs were created using SigmaPlot and MS Excel software. These results were presented as Mean ± S.E. of at least three independent experiments, or one representative experiment of three was shown. Statistical analysis was performed using Student's t-test, Mann–Whitney Rank Sum Test, Analysis of Variance (ANOVA), whereas differences between means were inspected with Dunnett’s multiple comparison and Student-Newman-Keuls multiple comparison post-hoc tests. Significance levels of P < 0.05 were considered statistically reliable.