Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation

Abstract

Mutations in isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) are found in a subset of gliomas. Among the many phenotypic differences between mutant and wild-type IDH1/2 gliomas, the most salient is that IDH1/2 mutant glioma patients demonstrate markedly improved survival compared with IDH1/2 wild-type glioma patients. To address the mechanism underlying the superior clinical outcome of IDH1/2 mutant glioma patients, we investigated whether overexpression of the IDH1(R132H) protein could affect response to therapy in the context of an isogenic glioma cell background. Stable clonal U87MG and U373MG cell lines overexpressing IDH1(WT) and IDH1(R132H) were generated, as well as U87MG cell lines overexpressing IDH2(WT) and IDH2(R172K). In vitro experiments were conducted to characterize baseline growth and migration and response to radiation and temozolomide. In addition, reactive oxygen species (ROS) levels were measured under various conditions. U87MG-IDH1(R132H) cells, U373MG-IDH1(R132H) cells, and U87MG-IDH2(R172K) cells demonstrated increased sensitivity to radiation but not to temozolomide. Radiosensitization of U87MG-IDH1(R132H) cells was accompanied by increased apoptosis and accentuated ROS generation, and this effect was abrogated by the presence of the ROS scavenger N-acetyl-cysteine. Interestingly, U87MG-IDH1(R132H) cells also displayed decreased growth at higher cell density and in soft agar, as well as decreased migration. Overexpression of IDH1(R132H) and IDH2(R172K) mutant protein in glioblastoma cells resulted in increased radiation sensitivity and altered ROS metabolism and suppression of growth and migration in vitro. These findings provide insight into possible mechanisms contributing to the improved outcomes observed in patients with IDH1/2 mutant gliomas.

Fig. 1.

Establishment of glioma stable cell lines overexpressing IDH1^WT, IDH1^R132H, IDH2^WT, and IDH2^R172K. (A) Schematic diagram of the IDH1-FLAG and IDH2-FLAG fusion protein amino acid sequence. The FLAG tag was linked to the C-terminus of IDH1 and IDH2. Amino acid residue 132 for IDH1 and 172 for IDH2 are indicated with (). (B) Representative western blot for FLAG-tagged IDH1^WT and IDH1^R132H or IDH2^WT and IDH2^R172K in stably transfected U87MG cells (2 representative independent clones for each construct, C1 and C2, are shown) using anti-FLAG, anti- IDH1^WT, and anti-IDH1^R132H antibodies. (C) Immunofluorescence showed the diffuse cytoplasmic distribution of IDH1^WT and IDH1^R132H proteins stained by anti-FLAG antibody (green), anti-IDH1^WT antibody (red), and anti-IDH1^R132H (green). Nuclei were counterstained with DAPI (blue). 40×, bar = 20μm (D) Immunofluorescence shows the punctate cytoplasmic distribution of IDH2^WT and IDH2^R172K proteins stained by anti-FLAG antibody (green), and nuclei were counterstained with propidium iodide (PI) (red), 40×, bar = 20μm. (E) ∼70-fold increase in 2-HG level was found in the conditioned medium of U87MG-IDH1^R132H cells by HPLC-MS. Data were normalized by cell number and expressed as fold change relative to U87MG-control (mean ± SEM, n =3). **P < .01 compared with U87MG-control and U87MG-IDH1^WT cells. (F) ∼140-fold increase in 2-HG level was found in the conditioned medium of U87MG-IDH2^R172K cells by HPLC-MS. Data were normalized by cell number and expressed as mean fold change relative to U87MG-control (mean ± SEM, n =3). **P < .01 compared with U87MG-control and U87MG-IDH2^WT cells.

Fig. 2.

Overexpression of IDH1^R132H or IDH2^R172K increased the sensitivity of glioma cells to radiation treatment. (A) Cells were treated with various doses of radiation and incubated for 48 h for U87MG cells or 72 h for U373MG cells. Viability was quantitated with MTT assay and expressed as mean percentage of untreated control cells (mean ± SEM, n =3). *P < .05, **P < .01 compared with control, IDH1^WT or IDH2^WT cells. (B) Cells were treated with 25 Gy of radiation for different incubation times. Viability was quantitated with MTT assay and expressed as mean percentage of untreated control cells (mean ± SEM, n = 3). *P < .05, **P < .01 compared with control, IDH1^WT or IDH2^WT cells. (C) Equal number of cells was seeded into 60-mm dishes and treated with different doses of radiation 24 h later. After 14 days, the cells were fixed and stained with 0.25% crystal violet. Mean clonogenic survival of cells after radiation was plotted on a logarithmic scale (mean ± SEM, n =3). *P < .05, **P <.01 compared with control, IDH1^WT or IDH2^WT cells.

Fig. 3.

IDH1^R132H and IDH2^R172K overexpression did not affect TMZ sensitivity. (A) Cells were treated with various doses of TMZ for 72 h. Viability was quantitated with MTT assay and expressed as mean percentage of untreated control cells (mean ± SEM, n =3). (B) Cells were treated with 400 μM of TMZ for different periods of time. Viability was quantitated with MTT assay and expressed as mean percentage of untreated control cells (mean ± SEM, n =3). (C) Clonogenic survival of cells after TMZ treatment was plotted on a logarithmic scale (mean ± SEM, n =3). *P < .05, **P < .01 compared with control, IDH1^R132H or IDH2^R172K cells.

Fig. 4.

U87MG-IDH^R132H cells showed significant increase in apoptosis following radiation treatment. (A) Apoptosis was assessed by YO-PRO-1 and propidium iodide (PI) staining after 48 h radiation treatment (0 Gy or 25 Gy). Harvested cells were evaluated by flow cytometry. Column plot summarized 3 independent experiments, each performed with triplicates (mean ± SEM, n =3). **P < .01 compared with U87MG-control and U87MG-IDH1^WT cells. (B) Apoptosis % was normalized to U87MG-control cells treated with 0 Gy and expressed as mean apoptotic index (mean ± SEM, n =3). **P< .01 compared with U87MG-control and U87MG-IDH1^WT cells.

Fig. 5.

U87MG-IDH^R132H cells showed increased oxidative response to radiation. Cells were incubated with the oxidant sensitive fluorescent dye DCFH-DA for 30 min before exposure to radiation, and ROS level was measured 90 min later by the plate reader. (A) The baseline ROS levels were measured by DCF assay and expressed as mean fold change relative to U87MG-control cells (mean ± SEM, n = 3). *P < .05, **P< .01 compared with U87MG-control and U87MG-IDH1^WT cells. (B) The ROS level after radiation treatment was measured by DCF assay and expressed as mean fold change relative to untreated U87MG-control, U87MG-IDH1^WT, and U87MG-IDH1^R132H cells (mean ± SEM, n =3). *P < .05 compared with U87MG-control and U87MG-IDH1^WT cells.

Fig. 6.

ROS scavenger protected the U87MG-IDH^R132H cells from radiation. Cells were pretreated with NAC for 4 h before exposure to different doses of radiation. Viability was determined 48 h later by MTT assay and expressed as mean percentage of untreated U87MG-control cells (mean ± SEM, n= 3). *P < .05, **P < 0.01 compared with U87MG-control and U87MG-IDH1^WT cells.

Fig. 7.

U87MG-IDH^R132H protein overexpression decreased cell proliferation, growth in soft agar, and migration. (A) Phase contrast microscopy pictures of U87MG-control, U87MG-IDH1^WT cells, and U87MG-IDH1^R132H cells in culture. Clusters are found at higher densities U87MG-control, U87MG-IDH1^WT cells (indicated by white arrows). 4×, bar = 100 μm (B) Cell proliferation was determined by MTT assay and expressed as mean absorbance value at 535 nm with reference at 660 nm (mean ± SEM, n =3). ***P < .001 compared with U87MG-control and U87MG-IDH1^WT cells. (C) Soft agar colony formation assay was performed by counting individual colonies (>50 µm) in 5 random microscopic fields, and representative micrographs are shown. The quantification of colony numbers are shown as mean ± SEM (n =3). 4×, bar = 100 μm, **P < .01 compared with U87MG-control and U87MG-IDH1^WT cells. (D) and (E) Monolayer cell migration scratch assay. Representative phase micrographs of U87MG-control, U87MG-IDH1^WT, and U87MG-IDH^R132H cells at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h after monolayer wounding are shown in D. Monolayer scratch wound distances were quantitated in (E) and expressed as mean ± SEM (n = 3). 4×, bar = 100 μm, *P < .05 compared with U87MG-control and U87MG-IDH1^WT cells. (F) Cell migration quantitated with a transwell migration assay. U87MG-control, U87MG- IDH1^WT, and U87MG-IDH1^R132H cells were plated in the transwell chamber for 5 h before migrated cells were fixed, stained, and counted. Top: representative micrographs of migrated cells. Bottom: migrated cell number is quantitated and expressed as mean percentage of U87MG-control (mean ± SEM, n =3). 4×, bar = 100 μm, **P < .01 compared with U87MG-control and U87MG-IDH1^WT cells.