The A172 glioma cells (American Type Culture Collection, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies, Carlsbad, CA, USA), 4.5 g/L glucose and 100 U/ml penicillin/streptomycin (both from Sangon Biotech Co., Ltd., Shanghai, China). The cells were dissociated using enzyme-free cell dissociation solution (EMD Millipore, Bedford, MA, USA) and cultured at 37°C in a humidified atmosphere of 5% CO₂. The YC-1 (Sigma-Aldrich, St. Louis, MO, USA) and 2-deoxyglucose (2-DG; Sigma-Aldrich) inhibitors were dissolved in dimethyl sulfoxide (DMSO; Sangon Biotech Co., Ltd.) and sterilized water, respectively, prior to use.

The full-length human wild-type (WT) IDH1 coding sequence was amplified from the HEK293T cells (Sangon Biotech Co., Ltd.). The following synthesized primers (Invitrogen Life Technologies) were used to fuse the cDNA in-frame with a FLAG tag at the N-terminus: Forward 5′-TTT CGT ACG ATG GAT TAC AAG GAC GAC GAT GAC AAG TCC AAA AAA AT-3′ containing the MluI site and reverse 5′-TTT ACG CGT GGT ATG AAC TTA AAG TTT GG-3′ containing the BsiWI site. This was inserted into the MluI- and BsiWI-linearized pHR-SIN vector (Open Biosystems, Huntsville, AL, USA). The IDH1^R132H mutation was generated in the pHR-SIN-IDH1^WT using a QuikChange Site-Directed Mutagenesis kit according to the manufacturer’s instructions (Stratagene, Santa Clara, CA, USA) with the following primer sequences: 5′-R132H, 5′-ACC TAT CAT CAT AGG TCA TCA TGC TTA TGG G-3′ and 3′-R132H, 5′-TGA CCT ATG ATG ATA GGT TTT ACC CAT CCA C-3′.

The HEK293T cells (4×10⁶) were seeded into 60 mm plates in DMEM cell culture medium with 100 U/ml penicillin/streptomycin 1 day prior to transfection. The cells were transfected with either 5.2 μg pHR-SIN-IDH1^WT or pHR-SIN-IDH1^R132H, 2.36 μg pSPAX2 and 0.8 μg pMD2G plasmids using Lipofectamine 2000 (Invitrogen Life Technologies) in DMEM. After 6 h, the transfection media was replaced with DMEM cell culture medium without penicillin/streptomycin and the lentiviral particles were harvested 72 h post-transfection. The A172 glioma cells (total number: 2×10⁵) were plated in 60 mm plates in DMEM with 10% phosphate-buffered saline (PBS) at a confluence of 20% 1 day prior to transduction. The cells were transduced by adding 3 ml media containing viral particles and 6 μg/ml polybrene (Sigma-Aldrich). After 16 h, the conditioned media was replaced with DMEM containing 15% FBS.

The cells (5×10⁵) were seeded onto glass coverslips for 24 h, fixed with 4% paraformaldehyde and permeabilized using 0.4% Triton X-100 in PBS (all from Sangon Biotech Co., Ltd.) for 10 min. Non-specific binding was blocked using PBS containing 5% FBS and the cells were incubated with primary antibodies against FLAG (mouse monoclonal anti-FLAG M2; 1:500; F3165; Sigma-Aldrich) or IDH1^R132H (mouse monoclonal anti-human; 1:500; DIA-H09; Dianova GmbH, Hamburg, Germany) at 4°C overnight. As a negative control, cells were treated with immunoglobulin G under the same conditions. The cells were washed three times in PBS prior to incubation with the respective Alexa Fluor 594-conjuated secondary antibody (goat anti-mouse; 1:1,000; 8890; Cell Signaling Technology, Inc., Danvers, MA, USA). Following staining, the cells were imaged using a DM2500 Leica microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA).

The transfected A172 cells were immediately placed on ice and washed with ice-cold PBS. The total protein was prepared using radioimmunoprecipitation lysis buffer containing a protease inhibitor cocktail (1:1,000; 04693124001; Roche Diagnostics GmbH, Mannheim, Germany) and phenylmethanesulfonylfluoride (Sigma-Aldrich). The proteins were resolved on 8–12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) by electroblotting. The membranes were blocked using Tris-buffered saline with 0.1% Tween-20 (TBST; Sangon Biotech Co., Ltd.) containing 5% non-fat dry milk for 1 h and incubated at 4°C overnight with their respective primary antibodies against IDH1 (rabbit monoclonal anti-human; 1:1,000; 8137; Cell Signaling Technology, Inc.), IDH1R^132H (mouse monoclonal anti-human; 1:1,000; DIA-H09; Dianova GmbH) or FLAG (mouse monoclonal anti-FLAG M2; 1:1,000; F3165; Sigma-Aldrich). Immunolabeling was detected using enhanced chemiluminescence reagents (Sigma-Aldrich) and visualized using an Amersham Imager 600 (GE Healthcare, Uppsala, Sweden). β-actin was used as a loading control.

The total RNA was isolated from the cells using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. Subsequently, 1 μg total RNA was used as a template for RT in a Moloney Murine Leukemia Virus Reverse Transcriptase reaction (Fermentas, Burlington, Ontario, Canada). RT-qPCR was performed using SYBR Green Master mix (Applied Biosystems Life Technologies, Carlsbad, CA, USA) and β-actin was used as an internal control. The following primers were used: Glucose transporter 1 (Glut1), forward 5′-CTTTGTGGCCTTCTTTGAAGT-3′ and reverse 5′-CCACACAGTTGCTCCACAT-3′; hexokinase 2 (HK2), forward 5′-GATTGTCCGTAACATTCTCATCGA-3′ and reverse 5′-TGTCTTGAGCCGCTCTGAGAT-3′ and β-actin, forward 5′-GGCGGCACCACCATGTACCCT-3′ and reverse 5′-AGGGGCCGGACTCGTCATACT-3′. The PCR thermal cycling conditions were as follows: Cycling began with 2 min at 50°C and 10 min at 95°C. Thermal cycling proceeded with 40cycles of 95°C for 0.5 min and 60°C for 2 min. All reactions were performed in the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The comparative Ct method was used to calculate the expression of mRNA relative to that of β-actin.

The cells (5×10³) were seeded into 96-well plates and cultured for 24, 48, 72 or 96 h, respectively. Following the incubation period, MTT was added to each well at a final concentration of 5 mg/ml and the cells were incubated at 37°C for a further 4 h. The reaction was terminated by adding 150 μL DMSO and the absorbance was measured using a BioTek ELISA reader (BioTek Instruments Inc., Winooski, VT, USA) at a wavelength of 490 nm. Each experiment was performed in triplicate.

Cells were trypsinized (trypsin from Sigma-Aldrich) to generate a single-cell suspension and seeded into three parallel 60-mm dishes with 500 cells in each dish. Fourteen days following seeding, the colonies were stained with 0.5% crystal violet (Sangon Biotech Co., Ltd.). The number of colonies containing ≥50 cells was determined, and the results were reported as a percentage of the number of colonies in untreated cultures of each corresponding clone.

The cells were subjected to serum starvation for 12 h prior to being cultured in DMEM containing 25 mM glucose. The cells were washed three times with PBS and incubated for 3 h in DMEM containing 1 mCi/ml 2-Deoxy-D-(1,2–3H) glucose (PerkinElmer, Inc., Boston, MA, USA). Following incubation, the cells were washed three times using ice-cold PBS and solubilized in 1% SDS. The radioactivity of each aliquot was determined in a scintillation counter (LS6500 Multipurpose Scintillation Counter; Beckman Coulter, Fullerton, CA, USA). Each assay was performed in triplicate.

The cells (5×10⁵) were seeded into 60 mm dishes and incubated in DMEM with 10% FBS at 37°C overnight. The media was replaced with DMEM without FBS and the cells were incubated for 1 or 2 h. The supernatant was collected and the lactate levels were quantified by colorimetric assay using a Lactate Assay kit (BioVision, Inc., Milpitas, CA, USA), according to the manufacturer’s instructions.

Statistical analysis was performed using GraphPad Prism version 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was determined using Student’s t-test. The data are expressed as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of overexpression of human glioma-associated IDH1 mutant proteins within the context of an isogenic glioma cell, N-terminus FLAG-tagged IDH1^WT, IDH1^R132H and control constructs were produced and stably expressed in the A172 glioma cells (Fig. 1A). Western blot analysis using anti-FLAG, anti-IDH1 and mutant-specific anti-IDH1^R132H antibodies confirmed the expression of their respective IDH1 proteins (Fig. 1B). Subsequent immunofluorescence analysis revealed that the steady-state cellular localization of the IDH1^R132H mutant protein was indistinguishable from that of the IDH1^WT protein, indicating that the mutation caused no significant alteration to the targeting of IDH1 (Fig. 1C and D). The high specificity of the IDH1^R132H antibody was also indicated.

The role of IDH1^R132H in glioma remains to be fully elucidated. To assess the importance of IDH1^R132H in A172 glioma cell growth, an MTT assay was used to compare the proliferation rates between the two types of IDH1-transfected cells and the control cells. The results revealed that following incubation for 4 days, the growth curve for the IDH1^R132H cells were significantly higher compared with those for the IDH1^WT and the control cells (Fig. 2A; P<0.05). This observation was supported by colony formation assays, which revealed that clonogenicity was significantly increased in the mutant IDH1^R132H cells compared with the IDH1^WT and the control cells (Fig. 2B; P<0.01).

Increased glucose uptake and lactate secretion are characteristics of proliferating cells undergoing glycolysis (2,16). As IDH1 is important in metabolism, the present study hypothesized that impaired IDH1 activity may alter cell metabolism. Therefore, the levels of glucose uptake and extracellular lactate were measured to investigate whether the IDH1^R132H mutation was responsible for a metabolic shift in the IDH1^R132H A172 cells. The results demonstrated that the levels of glucose uptake and extracellular lactate in the IDH1^R132H cells were significantly higher compared with the IDH1^WT and control cells (Fig. 3A and B, respectively; P<0.05).

Glut1 and HK2 are important genes involved in cell glycolysis (17–19), therefore their expression levels were assessed in IDH1-transfected cells using RT-qPCR and western blot analysis. The results revealed that the mRNA and protein expression levels of Glut1 and HK2 were increased significantly (Fig. 3C and D; P<0.01) in the IDH1^R132H cells compared with the IDH1^WT or control cells.

To elucidate whether the metabolic shift triggered by IDH1^R132H was involved in cell proliferation, the A172 cells overexpressing IDH1 were treated with 2-DG, an inhibitor of glycolysis, at a final concentration of 12 mM (20) and the effects on cell growth were analyzed using an MTT assay. The results revealed that 2-DG abrogated the IDH1^R132H-induced cell proliferation (Fig. 3E; P<0.05).

These findings suggested that IDH1^R132H-induced glycolysis promoted cell proliferation in the A172 glioma cells.

The overexpression of mutant IDH1 has been demonstrated to reduce the levels of α-KG and increase the levels of HIF-1α (11). Therefore, the present study hypothesized that the IDH1^R132H mutation may be involved in promoting or stabilizing HIF-1α, thereby enhancing aerobic glycolysis. Western blot analysis confirmed that the protein expression of HIF-1α was increased in the IDH1^R132H cells compared with the IDH1^WT and the control cells (Fig. 4A).

To investigate whether HIF-1α is required in IDH1^R132H-induced glycolysis, the activity of HIF-1α was inhibited in the IDH1-tranfected cells by treating the A172 cells with YC-1 at a final concentration of 5 μM (21). Western blot analysis revealed that the levels of Glut1 and HK2 were reduced in the IDH1^R132H cells following exposure to YC-1, whereas no effects were observed in the IDH1^WT or control cells (Fig. 4B). Consistent with these findings, YC-1 treatment significantly decreased the rate of glucose uptake and extracellular lactate secretion in the mutant IDH1^R132H cells compared with the IDH1^WT or control cells (Fig. 4C and D, respectively; P<0.05). These results demonstrated that HIF-1α was important in modulating aerobic glycolysis in the IDH1^R132H A172 glioma cells.