Poly-L Lysine (PLL), laminin, heparin, human recombinant epidermal growth factor (EGF), gramicidin, nigericin, tributyltin, valinomcycin, propidium idodide (PI), TMZ, staurosporine (STS), and BMT were purchased from Sigma Chemicals (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F12 medium, B27 supplement (without vitamin A), goat anti-IgG secondary antibodies Alexa Fluor® 488, PBFI-AM, calcein-AM, MQAE, and pluronic acid were obtained from Invitrogen (Carlsbad, CA). Human recombinant basal fibroblast growth factor (bFGF), mouse/rat WNK1 affinity purified antibody, and human phospho-WNK1 (T60) affinity purified antibody were from R&D Systems (Minneapolis, MN). Accutase was from Millipore (Billerica, MA). Annexin V-FITC Apoptosis Kit was from Imgenex (San Diego, CA). NKCC (T4) antibody was from Developmental Studies Hybridoma Bank (Iowa City, IA). Caspase-3 antibody, caspase-8, and α-tubulin antibodies were from Cell Signaling (Beverly, MA). NKCC (R5) antibody against phosphorylated NKCC (p-NKCC) was a kind gift from Dr. Biff Forbush (Yale University).
Glioblastoma cells (GC)
All studies involving human tissue were performed with approval from the University of Wisconsin-Madison Institutional Review Board with informed consent obtained from patients. Primary glioma cell lines (GC #22, #99) were established as described before [14
]. Human glioblastoma multiforme cell line U87MG was obtained from the American Type Culture Collection (Manassas, VA). GC and U87MG were maintained in adherent cultures in plates coated with PLL and laminin in 90% DMEM supplemented with 10% FBS and PSA (100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B). Cultures were passaged approximately every 4 days with fresh medium at a density of 105
in a culture flask. Passages of 10–35 were used in this study.
GC cancer stem cells (GSC)
GSCs (#22 or #99) were isolated and characterized from the same GC line as previously reported [14
]. GSC exhibited high CD133 expression, stem cell multipotency, and tumor initiation capability. Upon EGF and bFGF removal, GSC maintained sphere-forming and tumor initiation ability [14
]. Cultures were passaged approximately every 7 days with 70% DMEM-high glucose and 30% Ham’s F12 supplemented with B27, 5 µg/ml heparin, PSA, and EGF and bFGF (20 ng/ml each). To seed cells in plates coated with PLL and laminin, GSC spheres were incubated in accutase for 5 min at 37°C and dissociated with a P200 pipette. Cells were seeded at 105
in culture flasks. Passages of 20–50 were used in this study.
Cell volume measurement
Cell volume change was determined using calcein as a marker of intracellular water volume, which was established previously [15
]. Briefly, cells on coverslips were incubated with 0.5 µM calcein-AM for 30 min at 37°C. The cells were placed in a heated (37°C) imaging chamber (Warner Instruments, Hamden, CT) on a Nikon Ti Eclipse inverted epifluorescence microscope equipped with perfect focus, a 40X Super Fluor oil immersion objective lens, and a Princeton Instruments MicroMax CCD camera. Calcein fluorescence was monitored using a FITC filter set (excitation 480 nm, emission 535 nm, Chroma Technology, Rockingham, VT). Images were collected every 60 sec with MetaFluor image-acquisition software (Molecular Devices, Sunnyvale, CA). Regions of interest (0.5 µm in diameter) were selected in the cytoplasm of ~20 cells. Baseline drift resulting from photobleaching and dye leakage was corrected as described before [15
]. The fluorescence change was plotted as a function of the reciprocal of the relative osmotic pressure and the resulting calibration curve applied to all subsequent experiments as described before [15
]. The HEPES buffered isotonic solution contained (in mM, pH 7.4): 100 NaCl, 5.4 KCl, 1.3 CaCl2
, 0.8 MgSO4
, 20 HEPES, 5.5 glucose, 0.4 NaHC03
, and 70 sucrose with 310 mOsm determined using an osmometer (Advanced Instruments, Norwood, MA). Anisosmotic solutions (240, 370, and 400 mOsm) were prepared by removal or addition of sucrose to the above solution.
Intracellular Cl− concentration ([Cl−]i) measurement
The fluorescent dye MQAE was used to determine [Cl−
as described by Rocha-Gonzalez [16
] with some modifications. Cells were incubated with 5 mM MQAE for 1–2 h (37°C) in a HEPES buffered isotonic solution. The coverslip was placed in the heated imaging chamber for 30 min before imaging. Using the Nikon Ti Eclipse inverted epifluorescence microscope and the 40X oil immersion objective lens, cells were excited every 60 sec at 340 nm and emission fluorescence at 460 nm recorded. Images were collected and analyzed with the MetaFluor image-processing software.
The fluorescence in MQAE-loaded cells drifted over time as a function of exposure to excitation light [16
] and the drift in MQAE florescence was corrected by a linear fit. At the end of each experiment, the MQAE florescence was calibrated under a steady state condition when [Cl−
were considered equal by exposing cells to a series of calibration solutions containing 10 µM tributylin and 5 µM nigericin [16
]. The series of Cl−
calibration solutions contained (in mM): 1.27 Ca(OH)2
, 0.8 MgSO4
, 5 HEPES, 5.5 glucose, 120 K+
, and variable Cl−
. In these solutions, Cl−
was varied from 0 to 60 mM keeping the sum of Cl−
equal to 120 mM. KSCN (150 mM) was used to quench the MQAE fluorescence, which was taken as background fluorescence. [Cl−
was determined from the MQAE fluorescence (drift-corrected, background-corrected) using the following equation: [Cl−
, where Fo
was the fluorescence in 0 mM [Cl−
was the fluorescence at any given time point, and Ksv
was the slope of the linear fit of MQAE fluorescence vs. the [Cl−
of the standards. A Ksv
of 13.4 ± 1.5 M−1
was calculated in our study, a value similar to that reported by others [16
Intracellular K+ ([K+]i) measurement
was determined by a modified method as described by Kiedrowski [17
]. Briefly, cells were incubated with 5 µM PBFI-AM plus 0.02 % pluronic acid at 37°C for 90 min. The coverslips were placed in the heated imaging chamber at 37°C. Cells were rinsed and images collected using the Nikon Ti Eclipse inverted epifluorescence microscope equipped with the 40X oil immersion objective lens. Cells were excited every 20 sec at 340 and 380 nm and the emission fluorescence at 510 nm recorded. Images were analyzed with the MetaFluor image-processing software. At the end of each experiment, a calibration was performed by exposing cells to standards of varying K+
concentrations plus gramicidin and valinomcycin (10 µM each). K+
standards contained 30 mM NaCl, 20 mM HEPES, and 1 mM MgCl2
was varied from 20 to 100 mM by substituting K+
-gluconate and LiCl such that the sum of K+
was 100 mM.
Quantification of apoptosis with live cell imaging
Cells were incubated either with TMZ (0 – 500 µM) or TMZ plus 10 µM BMT for 48 h. To determine apoptotic cell damage, cells were rinsed with ice-cold PBS and incubated for 30 min at RT with 100 µl binding buffer, 5 µl FITC Annexin-V, and 5 µl propidium iodide (PI) provided in the Annexin V-FITC Apoptosis Kit. Brightfield, FITC (Annexin-V), and Texas Red (PI) images were collected from 4 random fields in each well using the Nikon Ti Eclipse microscope and a 20X objective. The total number of cells in each field was counted in the brightfield image. Apoptotic cells were calculated as the percentage of the Annexin V+/PI− cells.
We found that 500 µM TMZ led to robust apoptotic volume decrease and early apoptosis in a very short time (1–4 hours). This raised concerns about the impact of cell volume change and cell membrane damage on dye retention in TMZ-treated cells in live cell imaging studies (cell volume, K+, and Cl−) and of detached cells on sample collection for immunoblotting assays. Therefore, for these studies, we reduced TMZ concentration to 100 µM.
Flow cytometry assay for apoptosis
Assays were carried out by using an Annexin V-FITC Apoptosis Kit. Cultures were treated with a control medium or a medium containing 10 µM BMT, 500 µM TMZ, or 500 µM TMZ plus 10 µM BMT for 4 h. STS (4 µM, 4 h) was used as a positive control. Cultures were rinsed with ice-cold phosphate-buffered saline (PBS; pH 7.4) and incubated with accutase (2 ml per 25 cm2 surface area) at 37°C for 5 min. The detached cells (from PBS wash as well as from accutase treatment) were collected by centrifugation (500×g, 10 min). The combined cell pellets were washed with PBS and resuspended in 100 µl binding buffer. Cells were incubated with 5 µl FITC Annexin-V and 5 µl PI in dark at RT for 20 min. After incubation, 400 µl binding buffer was added to each sample. Cells were analyzed using a Becton Dickinson Biosciences FACSCalibur (BD Biosciences, CA). Early apoptotic cells were determined as Annexin V-positive and PI-negative cells (Annexin V+/PI−).
Changes in cell size were also evaluated by forward light scattering vs. side light scattering 3D plots using Becton Dickinson Biosciences FACSCalibur and CellQuest software. Decrease in the forward light scattering values of the cells reflected a decrease in cell size.
Time-lapse assay for apoptotic volume decrease
Cultures in 35 mm glass bottom culture dishes were placed in a stage top incubator (Tokai Hit, Shizuoka-ken, Japan) at 37°C and 5% CO2. Cultures were treated with a control medium or a medium containing 500 µM TMZ, or 500 µM TMZ plus 10 µM BMT for 12 h. Time-lapse DIC images were collected every 15 min for 12 h using the Nikon Ti Eclipse microscope with perfect focus. The surface area of each cell was manually traced using MetaMorph (Molecular Devices, CA). 5 cells in each field were selected and their surface areas determined. Cell surface area values at each time were normalized to the corresponding values at time 0 h. Mean cell surface area (± SEM) for each time point was calculated by averaging the normalized areas of cells for each hour.
Cells were washed with ice-cold PBS that contained 2 mM EDTA and protease inhibitors as described before [18
]. Cells were lysed by sonication at 4°C. Protein content of the cellular lysate was determined with BCA Protein Assay Kit (Pierce, Rockford, IL). Samples and prestained molecular mass markers (Bio-Rad, Hercules, CA) were denatured in SDS reducing buffer (1:2 vol/vol) and heated at 90°C for 5 min. The samples were then electrophoretically separated on 10–15% SDS gels. After transferring to PVDF membranes, the blots were blocked in 7.5% nonfat dry milk in Tris buffered saline for 1 h at RT and then incubated with a primary antibody at 4°C overnight. The blots were probed with monoclonal T4 antibody against total NKCC1 (1:2000), polyclonal antibody (R5) against a diphosphopeptide containing T184 and T189 of shark NKCC1 (p
-NKCC; 1:1000, [19
]), polyclonal antibodies against total WNK1 (1:1000), polyclonal rabbit anti-phospho-WNK1 antibody (p
-WNK1; 1:2000), polyclonal anti-caspase-3 (full length and large fragment of cleaved caspase-3; 1:1000), monoclonal anti-caspase-8 (full length, cleaved intermediate p43/41 and active capspase-8 fragment p18; 1:500), or monoclonal alpha tubulin antibody. After rinsing, the blots were incubated with horseradish peroxidase-conjugated secondary IgG for 1 h at RT. Bound antibody was visualized with an enhanced chemiluminescence assay (Amersham, Piscataway, NJ).
Cell proliferation analysis
Cell proliferation analysis was performed using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit [MTS; 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, Madison, WI). Cells were treated with vehicle (DMSO), 10 µM BMT, 250 µM TMZ, or 250 µM TMZ + 10 µM BMT for 0, 24, and 48 h, respectively. 20 µl of CellTiter 96® AQueous One Solution Reagent was added into each well of a 96-well plate (5,000 cells/100 µl medium). After 1 h incubation, the absorbance of each well at 490 nm was determined with a microplate reader (Molecular Devices, Sunnyvale, CA). The background absorbance was subtracted from all readings. Data are expressed as relative MTS activity.
Cell cycle analysis
Monolayer cell cultures were rinsed with PBS and the medium collected (to harvest floating cells). After trypsinization, detached cells and floating cells were combined and fixed by slow addition of ice-cold 70% (v/v) ethanol and stored at 4°C for 1 h. The fixed cells were then pelleted (500×g, 20 min), and washed twice in PBS. The cells were incubated with 20 µg /ml PI in PBS containing 100 µg /ml ribonuclease A at 37°C for 30 min in the dark. Cell cycle distribution was analyzed by Becton Dickinson Biosciences FACSCalibur and ModFit LTTM software with 20, 000 events per determination. The amount of DNA is proportional to the amount of PI dye staining. DNA content in each phase of the cell cycle (G1, S and G2/M) was calculated with ModFit LTTM software.
Statistical significance was determined by student’s t-test or an ANOVA (Bonferroni post-hoc test) in the case of multiple comparisons. A P-value < 0.05 was considered statistically significant. N values represent the number of cultures used in each experiment.