Cell culture conditions, treatments with BA and irradiation
The human malignant glioma cell lines U251MG and U343MG (American Type Culture Collection) were grown in RPMI 1640 medium (Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (PAA, Cölbe, Germany), 1% sodium pyruvate (Invitrogen, Karlsruhe, Germany), 185 U/ml penicillin (Invitrogen) and 185 μg/ml streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 3% CO2. Hypoxia (< 1% O2) was achieved using a gas generator system as previously described . All experiments were performed with cells in their logarithmic growth phase. BA (Biosolution GmbH, Halle, Germany) was dissolved in dimethyl sulfoxide (DMSO) to achieve a 20 mM stock solution. Cells (3 × 105) were seeded in 25 cm2 flasks 24 h before treatment with 3 to 30 μM BA. Cells were treated with BA or DMSO for 24 h at 37°C under normoxic or hypoxic conditions. Additionally, cells were irradiated in tissue culture flasks (Greiner, Frickenhausen, Germany) with 2, 6 or 15 Gy 24 h after incubation with BA. Irradiation was accomplished with 6 MV photons and adequate bolus material on a SIEMENS ONCOR (Erlangen, Germany) linear accelerator at a dose rate of 2 Gy/min. At 1, 24 or 48 h after irradiation, cells were harvested for clonogenic assays, protein extraction and migration assays.
Clonogenic survival assays and radiosensitivity
The cytotoxicity of BA was evaluated using the clonogenic survival assay. The cells were trypsinized 1 h after irradiation. Based on the optimal plating efficacy (depending on the BA treatment and irradiation dose), 500-5,000 cells were seeded in 25 cm2 flasks. The cells were cultured in RPMI supplemented with 10% FCS in a humidified atmosphere of 3% CO2 at 37°C. The medium was changed after 5 days. Between 10 and 14 days after irradiation, the cells were fixed with paraformaldehyde (Sigma, Deisenhofen, Germany), and colony formation was visualized by staining with 10% Giemsa solution (Sigma, Deisenhofen, Germany). Only colonies with > 50 cells were scored to determine the surviving fraction (SF). The cytotoxicity of BA was defined as the ratio of colonies formed after treatment with different concentrations of BA to DMSO-treated control cells. The SF was defined as the ratio of colonies formed after irradiation with 0, 2, 6 or 15 Gy to the number of colonies formed in the unirradiated controls. The enhancement factor (EF) was defined as the ratio of the SF of BA-treated cells to DMSO-treated control cells dependent on the dose of irradiation. The data represent at least three independent experiments.
Migration assays and cell cycle analysis
Cell migration was assessed using modified Boyden chambers as previously described . Cells (2 × 104) were suspended in 300 μl of RPMI without FCS and were then added to the upper chamber (membrane filter with 8 μm pore size), while the bottom chamber was filled with 1 ml of RPMI supplemented with 20% FCS (as a chemoattractant). The assay was performed at 37°C in a humidified atmosphere containing 3% CO2 for at least 16 h. Non-migrating cells on the upper side of the transwell inserts were removed. The cells that had migrated to the bottom side of the membrane were trypsinized and counted with CASY DT (Schärfe System GmbH, Reutlingen, Germany). The data represent at least three independent experiments.
Furthermore, we used a wound scratch assay to determine the migration of cells after treatment with BA. Cells were grown in 6-well cell culture plates in RPMI medium containing 10% FCS and were cultured to 100% confluence. A uniform cell-free area was created by scratching the confluent monolayer with a 200 μl pipette tip. To determine the migration of glioma cells, the wound closure was observed at different time points. The wound scratch assay was performed three times in independent experiments.
Cells were analyzed for cell cycle distribution. About 5 × 105 cells were harvested and washed in PBS. Subsequently, 95% ethanol was added slowly until a final concentration of 80% was reached. The DNA content, which was indicated by the extent of staining of propidium iodide, was measured by flow cytometry in an FACSscan (Becton Dickinson, Heidelberg, Germany), using the CellFit software (Version 2.0).
Cells were washed, trypsinized and centrifuged. The supernatant of cells was washed with PBS and resuspended in 100 μl of lysis buffer (50 mM Tris at pH 8.0, 0.3 M NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 0.1% NP40 and protease inhibitors), followed by ultrasonic homogenization. After centrifugation at 14,000 g for 15 min, the supernatant was collected and the protein concentration was determined using the Bradford assay (BioRad, Munich, Germany). About 30 μg of total protein from each cell lysate was separated on a 10% NuPAGE Bis-Tris (Invitrogen) gel that was placed in an X-Cell SureLock Mini-Cell (Invitrogen). The membrane was blocked with 10% non-fat milk in TBST (50 mM NaCl, 30 mM Tris-HCl at pH 8.0 and 0.1% Tween) for 1 h and incubated with rabbit anti-human survivin antibody (1:1,000 dilution, clone AF886, R&D Systems, Wiesbaden, Germany), rabbit anti-human cleaved PARP (1:2,000, Cell Signaling, Danvers, MA, USA), mouse anti-human HIF1α antibody (1:1,000, BD Transduction Laboratories, Lexington, KY) and mouse anti-β-actin (1:5,000, Sigma, Deisenhofen, Germany) at 4°C overnight. After washing, the membranes were incubated with a horseradish peroxidase-labeled goat anti-rabbit or anti-mouse IgG (1:2,000, DAKO, Glostrup, Denmark) for 1 h at room temperature. For protein detection, membranes were incubated with ECL substrate or ECL Plus Blotting Detection System for 1 min (Amersham Pharmacia Biotech, Freiburg, Germany) and exposed to X-ray film (Biomax, Kodak, Braunschweig, Germany).
The experimental results were analyzed by paired Student's t-tests. A p-value of 0.05 was considered to be significant.