IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status

Radiotherapy and Oncology xxx (2015) xxx–xxx

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Original article

IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status Jacqueline Kessler a,⇑, Antje Güttler a, Henri Wichmann a, Swetlana Rot b, Matthias Kappler b, Matthias Bache a, Dirk Vordermark a a

Department of Radiotherapy; and b Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Germany

a r t i c l e

i n f o

Article history: Received 5 July 2015 Received in revised form 8 August 2015 Accepted 9 August 2015 Available online xxxx Keywords: Isocitrate dehydrogenase 1 IDH1 IDH1R132H Glioma Radiation Hypoxia

a b s t r a c t Background and purpose: In malignant glioma the presence of the IDH1 mutation (IDH1R132H) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy. Material and methods: We determined the influence of IDH1R132H mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (<1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1wt) and mutated IDH1 (IDH1R132H). Results: Overexpression of IDH1R132H in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1R132H-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p < 0.01 and 1.42, p < 0.01), U-343MG (DMF10: 1.78, p < 0.01 and 1.75, p < 0.01) and LN-229 (DMF10: 1.41, p < 0.05 and 1.68, p < 0.01) under normoxia and hypoxia, respectively. Conclusion: Our data indicate that IDH1R132H mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy. Ó 2015 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2015) xxx–xxx

In the last few years the understanding of glioma development has changed radically due to the discovery of mutations involving the gene encoding isocitrate dehydrogenase (IDH) enzymes. IDH1 mutations occur in a restricted spectrum of tumors, including gliomas [1] and acute myeloid leukemia [2]. In gliomas, IDH1 mutation has been found to be a frequent, likely early event in gliomagenesis, occurring in 70–80% of World Health Organization (WHO) grade II and III astrocytic and oligodendroglial tumors, as well as in the majority of WHO grade IV secondary glioblastomas that evolved from lower-grade tumors, but not in primary glioblastomas [3–5]. IDH1 mutation seems to dictate a particular path for oncogenic progression and its presence is associated with better clinical outcome in gliomas of all grades [1,3–5]. Mutation of ⇑ Corresponding author at: Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097 Halle(S), Germany. E-mail addresses: [email protected] (J. Kessler), antje.hahnel@ uk-halle.de (A. Güttler), [email protected] (H. Wichmann), swetlana. [email protected] (S. Rot), [email protected] (M. Kappler), matthias. [email protected] (M. Bache), [email protected] (D. Vordermark).

the IDH1 gene occurs in a mutually exclusive manner usually affecting the arginine residue at codon 132 in the enzyme active site resulting in a substitution of histidine for arginine [1,6]. IDH1 is localized in the cytoplasm and peroxisomes and catalyzes the oxidative decarboxylation of isocitrate into a-ketoglutarate; thereby it serves as a major source for cytosolic nicotinamide adenine dinucleotide phosphate (NADPH) [7]. NADPH is necessary for the regeneration of reduced glutathione which functions as the main antioxidant in mammalian cells. The mutated IDH1 enzyme IDH1R132H shows a strongly decreased enzyme activity to isocitrate and results in a new enzyme function catalyzing the NADPHdependent reduction of a-ketoglutarate to D-2-hydroxyglutarate (D-2HG) leading to D-2HG accumulation and lowering aketoglutarate as well as NADPH levels [6,8]. Low levels of NADPH have been linked to elevated oxidative stress which has been hypothesized to confer an increased sensitivity to radiotherapy and may account for the prolonged survival of patients harboring the mutation [8]. It is unclear by which mechanism IDH1R132H and the resulting alterations in cellular metabolism influence tumor behavior and whether IDH1 mutant gliomas have a less

http://dx.doi.org/10.1016/j.radonc.2015.08.007 0167-8140/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Kessler J et al. IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation R132H mutation in gliomas –>status. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.08.007

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aggressive phenotype or if they are more radiosensitive. Malignant gliomas, comprising anaplastic astrocytoma and glioblastoma multiforme respond poorly to modern multimodality treatment including surgery, radiotherapy and chemotherapy. With increasing malignancy, gliomas exhibit intratumoral hypoxia, which has been associated with poor responses to radio- or chemotherapy [9–13]. In this study we evaluate the influence of IDH1R132H mutant protein on cellular and radiobiological behavior in a panel of human malignant glioma cells (U-251MG, U-343MG, LN-229) under normoxic and hypoxic conditions. Material and methods Generation of constructs, cell culture conditions, proliferation and growth in 3D spheroid culture The establishment of stable cells overexpressing IDH1wt or IDH1R132H was performed as described in detail in the Supplements (Supplementary Method 1). The human glioma cells U-251MG (kindly provided by Dr. Ariane Söling, Department of Pediatrics, University Göttingen, Göttingen, Germany) and LN-229 (kindly provided by Annie-Claire Diserens, Laboratoire de Neurochirurgie, Lausanne, Switzerland) were both derived from grade IV glioblastoma, whereas U-343MG (CLS Cell Lines Service, Eppelheim, Germany) was derived from grade III glioma. For normoxic conditions (21% O2) cells were grown in RPMI 1640 medium (Lonza, Walkersville, USA), supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 185 U/ml penicillin and 185 lg/ml streptomycin in a humidified-air atmosphere incubator containing 5% CO2 at 37 °C. Monolayer (2D) cultures were grown in flasks, on cell culture plates (10 cm2), 24-well or 96-well plates depending on the experimental procedure. All experiments were performed with cells in logarithmic growth phase. The impact of IDH1R132H on viability and proliferation was determined by trypan blue exclusion method at different time points (24 h, 48 h, 72 h and 96 h) after seeding. Further, we studied cell growth in 3D spheroid culture using flat bottom 96-well plates. Wells were coated with 50 ll agarose (1.5% in RPMI; Sigma) before adding cell suspension. For spheroid generation, 200 ll/well of cell suspensions at optimized density (2  104 cells/ml) were dispensed into agarose coated 96well plates using a multichannel pipette. Plates were incubated for 7 days under normoxia or hypoxia and spheroid formation and growth were evaluated using an AxioVert 135 microscope (Zeiss, Jena, Germany). Hypoxia and irradiation Hypoxia (<1% O2) was achieved, 24 h after seeding, using a gas generator system as previously described [14]. The cells were cultured for 24 h under hypoxic conditions and irradiated inside the gas generator system (<1% O2). Irradiation at 0–10 Gy (0, 2, 5 Gy under normoxia and 0, 2, 5, 10 Gy under hypoxia) was accomplished in logarithmically growing cultures with 6 MV photons and adequate bolus material on a SIEMENS ONCOR (Erlangen, Germany) linear accelerator at a dose rate of 2 Gy/min. After irradiation, the cells were either kept in normoxia or hypoxia at 37 °C for 1 h or 72 h before harvesting for the clonogenic assay (1 h) or protein isolation (72 h). Quantitative real-time PCR, Western blot analysis, immunohistochemical and immunofluorescence staining Total RNA was isolated with TRIzol (Invitrogen, Karlsruhe, Germany) as supplied by manufacturer´s protocol. For cDNA synthesis we utilized 1 lg of RNA using RevertAid H-Minus first-strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) according

to the manufacturer’s instructions. The PCR reaction and Western blot were performed as described before [15]. Immunostaining was performed to examine the distribution of IDH1wt and IDH1R132H overexpression in glioma cells. Cells grown on coverslips in 24-well plates at a density of 150,000 cells/well were fixed with 4% paraformaldehyde. Immunohistochemical staining was performed by using Dako REALTM Detection System, Peroxidase/DAB+, Rabbit/Mouse (Dako, Hamburg, Germany) following the manufacturer’s instructions. For immunofluorescence analysis cells were permeabilized with 0.5% Triton X-100/PBS, blocked for 1 h in 1% BSA/PB and incubated overnight with primary antibodies and subsequently for 1 h with secondary antibody and counterstained with DAPI. Details of the utilized primer sequences and antibodies are described in the Supplementary Tables 1 and 2. Immunostaining was evaluated using an AxioVert 200 M microscope (Zeiss). Cell migration and colony formation assay To analyze the migration activity of IDH1 wt - or IDH1R132H-expressing cells we performed a wound-healing assay as previously described [16]. Images were taken just after wound creation at 0 h and again after 24 h and 48 h of incubation under normoxic or hypoxic conditions by using an AxioVert 135 microscope (Zeiss) and wound closure (lm2) was measured using AxioVision Rel 4.6. software. Cell survival after radiation treatment was assessed by colony formation assay. Therefore, cells were trypsinized 1 h after irradiation, seeded in 25 cm2 flasks at different densities ranging from 250–10,000 cells depending on treatment and irradiation dose, cultured for 14 days and then fixed with paraformaldehyde (Sigma– Aldrich). Colony formation (colonies of P50 cells) was visualized by staining with 10% Giemsa solution (Sigma–Aldrich). Survival fraction (SF) was determined as the ratio of number of colonies formed by irradiated cells to the number of colonies formed by nonirradiated cells. The dose-modifying factor at a 10% survival level (DMF10) was established to analyze the effects of IDH1wt or IDH1R132H on the radiosensitivity of glioma cells (DMF10 = radiation dose untreated cells/radiation dose treated cells). To fit the data a linear quadratic model ln S = (aD + bD2) was applied, using origin 8.0 (OriginLab Corporation, Massachusetts, USA). Statistical analysis Results are presented as mean ± SD of n = 3–4 independent experiments. Statistical analyses were performed by Student’s t-test. A p-value <0.05 was considered significant. Results Quantitative RT-PCR showed high IDH1 mRNA levels in transduced U-251MG (an average of 43-fold under normoxia; an average of 78-fold under hypoxia), U-343MG (an average of 38-fold under normoxia; an average of 69-fold under hypoxia) and LN-229 (an average of 43-fold under normoxia; an average of 70-fold under hypoxia) cells compared to cells stably transduced with the empty vector pLVX (Fig. 1A). Western blot analysis and immunostaining with anti-human IDH1 and mutant specific anti-IDH1R132H antibodies confirm the overexpression and the diffuse cytoplasmic distribution of IDH1wt- and IDH1R132H-protein in the different glioma cell lines under normoxia (21% O2) and hypoxia (<1% O2), suggesting that the oxygenation status did not influence the expression of IDH1R132H-protein (Fig. 1B, C and Supplementary Fig. 1). Overexpression of IDH1R132H resulted in an insubstantial reduction of the viability by 2.6% ± 1.9 to 6.0% ± 2.9 (not significant) and slightly decreased the proliferation of U-251MG-IDH1R132H, U-343MG-IDH1R132H and LN-229-IDH1R132H cells (by 8.1% ± 1.9 to 11.9% ± 3.9; (p < 0.05) compared to empty vector cells under

Please cite this article in press as: Kessler J et al. IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation R132H mutation in gliomas –>status. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.08.007

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Fig. 1. (A) mRNA expression level (qRT-PCR) of IDH1 in U-251MG, U-343MG and LN-229 cells under normoxic (21% O2) and hypoxic (<0.1% O2) conditions. (B) Representative Western blot for IDH1wt and IDH1R132H in stably transduced U-251MG, U-343MG and LN-229 cells under normoxia and hypoxia using anti-IDH1wt and anti-IDH1R132H. (C) Immunostaining shows the diffuse cytoplasmic distribution of IDH1wt- or IDH1R132H-protein using anti-IDH1wt and anti-IDH1R132H antibodies (immunofluorescence, bar = 100 lm; immunohistochemical staining, bar = 25 lm) in U-251MG cells under normoxic and hypoxic conditions (UT: untreated, pLVX: cells stably transduced with empty vector; *p < 0.05 and **p < 0.01).

normoxic and hypoxic conditions (Supplementary Fig. 2). However expression of IDH1R132H had little impact on cell proliferation in monolayer culture, IDH1R132H caused differences in growth properties in 3D cultures. In contrast to untreated cells, empty vector cells and IDH1wt-expressing cells which showed a compact 3D spheroid structure, IDH1R132H-expressing U-251MG, U-343MG and LN-229 cells formed more loose and uneven spheroids in normoxia and hypoxia (Fig. 2 and Supplementary Fig. 3). Migration activity in all cell lines was higher in normoxia than in hypoxia (Fig. 3 and Supplementary Fig. 4). Furthermore, 48 h after wounding the motility of U-251MG and LN-229 cells (glioblastoma grade IV) was higher compared to U-343MG cells (glioma grade III). Treatment with the empty vector or overexpression of IDH1wt had no influence on the cell migration as compared to the irrespective untreated U-251MG, U-343MG and LN-229 cells. In contrast, overexpression of IDH1R132H reduced the cell motility by 26.4% ± 3.8 (p < 0.01) in U-251MG, by 15.4% ± 2.1 (p < 0.05) in U-343MG and by 37.2% ± 4.6 (p < 0.01) in LN-229 cells compared to IDH1wt-cells under normoxia. In addition, under hypoxic conditions, expression of IDH1R132H also reduced the migration activity by 26.3% ± 3.9 (p < 0.01) in U-251MG, by 12.1% ± 1.2 (p < 0.01) in U-343MG and by 52.5% ± 1.0 (p < 0.01) in LN-229 cells as compared to the irrespective IDH1wt-cells. We next analyzed, whether expression of IDH1R132H sensitized glioma cells to radiation (Fig. 4). Treatment with the empty vector or overexpression of IDH1wt did not affect the clonogenic survival

compared to the respective untreated U-251MG, U-343MG and LN229 cells. In contrast, overexpression of IDH1R132H reduced the clonogenic survival under normoxia (by 9–12%) and hypoxia (by 29–36%) as compared to that of the IDH1wt-expressing cells. In U-251MG, U-343MG and LN-229 untreated, empty vector and IDH1wt-cells had similar clonogenic survival curves in normoxia and hypoxia, respectively (untreated and empty vector cells are not shown for clarity). Survival of U-251MG, U-343MG and LN229 increased under hypoxic conditions as compared to normoxia. Survival curves revealed that expression of IDH1R132H significantly enhanced the effect of radiation in grade III glioma cells under normoxia (U-343MG DMF10: 1.78, p < 0.01) and hypoxia (U-343MG DMF10: 1.75, p < 0.01). Further, expression of IDH1R132H significantly enhanced radiation sensitivity of the grade IV glioblastoma cells U-251MG and LN-229 under normoxic (U-251MG DMF10: 1.52, p < 0.01; LN-229 DMF10: 1.41, p < 0.05) and hypoxic (U251MG DMF10: 1.42, p < 0.01; LN-229 DMF10: 1.68, p < 0.01) conditions (Fig. 4). We next used the proteolytic cleavage of PARP via Western blot analysis to qualitatively assess induction of apoptosis (Fig. 5). Western blot analysis confirmed our findings, similar to that found using clonogenic survival assay whereas treatment with the empty vector or overexpression of IDH1wt did not induce a cleavage of PARP, expression of IDH1R132H slightly increased the cleavage of PARP in the non-irradiated glioma cells and effectively enhanced the radiation induced apoptosis of U-251MG, U-343MG and LN-229 glioma cells under normoxic and hypoxic conditions.

Please cite this article in press as: Kessler J et al. IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation R132H mutation in gliomas –>status. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.08.007

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Fig. 2. Effect of IDH1R132H on growth properties of U-251MG cells in 3D cultures under normoxic and hypoxic conditions. Bar = 200 lm (UT: untreated, pLVX: cells stably transduced with empty vector).

Discussion IDH1 mutation predicted an alternative pathway of oncogenic progression and its presence is associated, irrespective of grade, with better clinical outcome in gliomas [3–5]. To evaluate whether IDH1 mutation improved survival of patients with IDH1 mutant glioma, based on a divergent biological behavior or enhanced responses to therapy, we analyzed the cellular and radiobiological consequences of overexpressing the IDH1R132H mutant protein in a panel of human malignant glioma cells (U-251MG, U-343MG, LN229). We observed that IDH1R132H overexpression slightly reduced proliferation and caused an altered growth in 3D spheroid culture of the indicated cells under normoxic and hypoxic conditions. IDH1R132H-cells aggregated to more loose, uneven and irregular structures in 3D cultures; whereas untreated, empty vector and IDH1wt-cells form compact tumor spheroids. In normoxia, Li and colleagues have also shown a decreased proliferation and migration for U-87MG IDH1R132H-expressing cells [17]. Further, our data are consistent with observations achieved in U-87MG glioma cells, where IDH1R132H overexpression resulted in a decreased proliferation under normoxia and mice injected with U-87MG IDH1R132Hexpressing cells had a prolonged survival compared to mice injected with U-87MG IDH1wt-expressing cells [18]. In addition, using wound-healing assays we found that overexpression of IDH1R132H reduced the migratory activity compared to untreated, empty vector and IDH1wt-cells under normoxic and hypoxic conditions. A recent study by Qi and colleagues suggests that the prolonged survival of patients with IDH-mutated gliomas is primarily due to a less aggressive biological behavior according to tumor site and MRI features. They demonstrated that IDHmutated tumors were rarely located in the high-risk regions of the brain and significantly more likely to exhibit a unilateral pattern of growth, sharp tumor margins, homogeneous signal intensity and less contrast enhancement on MRI. This is consistent with our findings that overexpression of IDH1R132H slightly decreased proliferation, reduced the migration activity and caused an altered growth in 3D culture to more loose and uneven spheroids of U-251MG, U-343MG and LN-229 cells, indicating a less aggressive biological behavior of IDH1R132H-expressing cells. To further elucidate a possible influence of mutant IDH1 on response to radiation of U-251MG, U-343MG and LN-229 gliomas under normoxia and hypoxia, we applied clonogenic survival assay as well as Western blot analysis of PARP cleavage. We showed that in normoxia and hypoxia overexpression of IDH1R132H reduced the plating efficiency and slightly enhanced cleavage of PARP. In addition, survival curves revealed that expression of IDH1R132H signifi-

cantly enhanced the effect of radiation and effectively enhanced the radiation induced apoptosis of U-251MG, U-343MG and LN229 glioma cells independent of the oxygenation status. Similar to our results Wang and colleagues showed an increased sensitivity to radiation for U-87MG cells under mild hypoxic conditions [19]. Furthermore, our findings are in line with most recent clinical observations of improved response to radiochemotherapy, as detected by serial magnetic resonance imaging, in glioblastoma patients with IDH1 mutation as compared to wild-type patients [20]. In another study by the European Organisation for Research and Treatment of Cancer (EORTC), patients with tumors harboring IDH1 or IDH2 mutations showed similarly better outcomes independent of whether they received RT/PCV or RT alone. Due to the fact that all patients received RT, these data indicate that IDH1R132H predicts improved response to RT. Further, Li and colleagues demonstrated that IDH1R132H protein did not sensitize U-87MG cells to TMZ [17]. Additionally, we showed that grade III U343MG IDH1R132H-expressing cells are more sensitive to radiation as compared to grade IV U-251MG-IDH1R132H and LN-229IDH1R132H-expressing cells under normoxia and hypoxia, respectively. These results appear consistent with clinical studies, in which the median overall survival was 31 months for patients with an IDH mutant glioblastoma and 15 months for patients with wildtype IDH1, whereas the median overall survival was 65 months for patients with an anaplastic astrocytoma harboring an IDH mutation and 20 months for those without mutations [reviewed in Ref. 21] Our study and the clinical observations indicate that the survival benefit garnered by IDH mutation for patients with lowgrade or high-grade gliomas is indicative of the effect of IDH mutation on the response to radiotherapy. It is possible that slightly decreased proliferation, reduced migration and altered growth in 3D culture as well as the enhanced radiosensitivity is caused by IDH1R132H-dependently reduced cellular fitness. Under physiologic conditions, IDH1 regulates aspects of glutamine and glutamate metabolism, as well as the synthesis of N-acetylated amino acids. Under these settings isocitrate, a-KG and NADPH levels are balanced in a manner that reflects the cellular energy state [6]. The mutated IDH1 enzyme IDH1R132H shows a strongly decreased enzyme activity leading to D-2HG accumulation and lowering a-ketoglutarate as well as NADPH levels. NADPH is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger [6,8]. Thereby it has been suggested that glioma cells expressing mutant IDH1 have a diminished antioxidant capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress [8]. In the literature, it has been demonstrated that with increasing malignancy, gliomas

Please cite this article in press as: Kessler J et al. IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation R132H mutation in gliomas –>status. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.08.007

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Fig. 3. Influence of IDH1R132H on wound closure of U-251MG, U-343MG and LN-229 cells using wound-healing assay under normoxic and hypoxic conditions (UT: untreated, pLVX: cells stably transduced with empty vector; *p < 0.05 and **p < 0.01).

exhibit intratumoral hypoxia, which has been associated with poor response to radio- or chemotherapy [9–13]. In our study expression of IDH1R132H attenuated the hypoxia-induced radioresistance of malignant glioma cells U-251MG, U-343MG and LN-229. IDH1 catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate to a-KG and the NADPH/CO2-dependent reductive carboxylation of a-KG to isocitrate, which is essential for citrate synthesis under hypoxic conditions. Leonardi and colleagues showed for IDH1R132H-expressing cells an inactivation of the NADPH-dependent reductive carboxylation and suggested that these metabolic issues may contribute to the reduced aggressiveness of glioma cells expressing IDH1R132H [22]. In the literature early evidence suggests that targeting IDH1 appears to be an attractive approach in IDH-mutant gliomas. Zheng and colleagues developed a series of mutant IDH inhibitors which inactivate the mutant IDH1 enzyme via hydrogen bonds and elec-

trostatic interactions with a stronger affinity to the active site of the mutated IDH1 enzyme [23]. In addition, Rohle and colleagues have developed a small-molecule IDH1R132H mutation-specific inhibitor (AGI-5198). They observed a reduction in the 2-HG level and tumor volume in murine xenografts and a reversed differentiation block which is associated with IDH mutation in gliomas [24]. Further investigations are needed to clarify the effects of these IDH1 inhibitors on the vulnerability of the gliomas to established treatments such as radiotherapy. In conclusion this study suggests a reduced aggressiveness as well as a radiosensitizing effect of IDH1R132H under normoxic and hypoxic conditions in glioma cells with different degrees of malignancy. Our results demonstrate an important role of mutant IDH1 in response to radiation and they are consistent with the clinical observation of prolonged survival of patients harboring the IDH1 mutation.

Please cite this article in press as: Kessler J et al. IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation R132H mutation in gliomas –>status. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.08.007

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Fig. 5. PARP cleavage (Western blot) 72 h after irradiation with 5 Gy under normoxia and hypoxia (UT: untreated, pLVX: cells stably transduced with empty vector).

Thomas Greither, Jürgen Dittmer and Alexander Navarrete Santos for the helpful discussions and the use of their microscopes. This work was supported by the Wilhelm-Roux-Program (FKZ: VF 02/39).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.radonc.2015.08. 007.

References

Fig. 4. Clonogenic survival of U-251MG, U-343MG and LN-229 cells. IDH1R132Hexpressing cells have a reduced clonogenic survival after irradiation in normoxia (2–5 Gy) or hypoxia (2–10 Gy). Data represent the average values (±SD) of four independent experiments.

Conflict of interest The authors declare that they have no competing interests.

Acknowledgements We would like to thank our colleagues from the Department of Radiotherapy for their contribution to this study and their continuous support. We also would like to thank Kathrin Spröte, Gabriele Thomas, Angela Dittmer for their excellent technical assistance and

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Please cite this article in press as: Kessler J et al. IDH1R132H mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation R132H mutation in gliomas –>status. Radiother Oncol (2015), http://dx.doi.org/10.1016/j.radonc.2015.08.007