Neurol Sci DOI 10.1007/s10072-013-1607-2

ORIGINAL ARTICLE

An IDH1 mutation inhibits growth of glioma cells via GSH depletion and ROS generation Jinlong Shi • Hao Zuo • Lanchun Ni • Liang Xia • Longxiang Zhao • Mingjie Gong • Dekang Nie • Peipei Gong • Daming Cui • Wei Shi • Jian Chen

Received: 7 October 2013 / Accepted: 10 December 2013 Ó Springer-Verlag Italia 2013

Abstract The isocitrate dehydrogenase 1 (IDH1) gene mutation occurs frequently in glioma. While some studies have demonstrated that IDH1 mutations are associated with prolonged survival, the mechanism remains unclear. In this study, we found that growth was significantly inhibited in glioma cells overexpressing the mutated IDH1 gene. Furthermore, these cells were characterized by decreased intracellular NADPH levels accompanied by glutathione (GSH) depletion and reactive oxygen species (ROS) generation. Moreover, the increased apoptosis and the decreased proliferation were found in the glioma cells overexpressing the mutant IDH1 gene. Accordingly, our study demonstrates that using H2O2-regulated mutant IDH1 glioma cells could obviously increase the inhibition of cell growth; nevertheless, GSH had the opposite result. Our study provides direct evidence that mutation of IDH1 profoundly inhibits the growth of glioma cells, and we speculate that this is the major factor behind its association with prolonged survival in glioma. Finally, our study indicates that depletion of GSH and generation of ROS are the primary cellular events associated with this mutation. J. Shi and H. Zuo contributed equally to the work. J. Shi  H. Zuo  L. Ni  L. Xia  L. Zhao  M. Gong  D. Nie  P. Gong  W. Shi  J. Chen (&) Department of Neurosurgery, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu, People’s Republic of China e-mail: [email protected] W. Shi e-mail: [email protected] D. Cui Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, 301Yanchang Road, Shanghai 200072, People’s Republic of China

Keywords

Glioma  IDH1  ROS  GSH

Introduction Gliomas are the most common primary brain tumors in adults and are grouped into four grades according to the histopathological and clinical criteria established by the World Health Organization (WHO) [1]. Despite recent advances in surgical resection, chemotherapy and radiotherapy, the prognosis of glioma continues to be very poor [2, 3]. Recent analysis has shown that isocitrate dehydrogenase 1 gene (IDH1) mutations are very common in grade II and III gliomas, and are also frequent in secondary GBM, but rare in primary GBM [4–7]. The most common of these IDH1 mutations affects the arginine residue at position 132 of the amino acid sequence [8]. Retrospective studies have demonstrated that IDH1 mutations are associated with prolonged survival, and that patients with different grades of glioma have improved survival [4–7]. Previous studies have suggested that elevated levels in tumor cells of reactive oxygen species (ROS) induce cell cycle arrest, inhibit proliferation and promote apoptosis, thereby inhibiting tumor growth [9, 10]. ROS produced by eukaryotic cells during normal oxidative metabolism are scavenged by the cellular antioxidant system to maintain the redox balance. However, an imbalance between the production of ROS and the ability of the cellular antioxidant system to readily detoxify ROS results in oxidative stress [11, 12]. Glutathione (GSH) is the most abundant intracellular antioxidant, and is involved in the protection of cells against oxidative damage and in various detoxification mechanisms [13]. Reduction of intracellular GSH levels leads to accumulation of ROS in cells, and elevated levels are associated with apoptosis resistance [14].

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NADPH is an essential cofactor for the biosynthesis of GSH, and the oxidized form of glutathione, GSSG, is reduced to GSH in an NADPH-dependent reaction catalyzed by glutathione reductase [15]. IDH1 catalyzes the oxidative carboxylation of isocitrate to a-ketoglutarate, yielding reduced nicotinamide adenine dinucleotide phosphate (NADPH) [6, 9]. We speculated therefore that elevated ROS levels and the depletion of GSH that arise from mutation of IDH1 contributed to inhibition of tumor growth. Here, we investigated ROS-mediated inhibition of the growth of U87 glioma cells as the mechanism underlying the association between mutation of IDH1 and improved prognosis in glioma.

PCNA at 4 °C overnight. After three washes with PBS, cells were incubated with fluorescence Cy3-labeled secondary antibodies. Fluorescent images were captured using fluorescent microscopy. TUNEL The TUNEL method was performed to label the 30 -end of fragmented DNA in apoptotic U87 cells. Cells were fixed with 4 % paraform phosphate buffer saline, and then permeabilized by 0.1 % Triton X-100 for FITC end-labeling of fragmented DNA using TUNEL cell apoptosis detection kit. The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope using 488-nm excitation and 530-nm emission.

Methods Cell viability assay Cell culture U87 cell lines were cultured in DMEM (Gibco BRL, Grand Island, NY, USA) with 10 % fetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin–streptomycin mixture (Gibco BRL, Grand Island, NY, USA) in an incubator at 37 °C and 5 % CO2. Plasmid constructs and lentivirus production Full-length human wild-type IDH1 cDNA was fused inframe using the following adapter primers for IDH: forward primer, 5 0 -GAGGATCCCCGGGTACCGGTCGCCAC CATGTCCAAAAAAATCAGTGGCGG-30 ; reverse primer, 50 -TCACCATGGTGGCGACCGGAAGTTTGGCC TGAGCTAGTTTG-30 . The IDH1R132H alteration was generated in PGC-FU-IDH1-WT using the QuikChange method (Agilent Technologies, USA) with the following primer sequence: 50 R132H(50 -ACCTATCATCATAGGTCATCA TGCTTATGGG-30 ) and 30 R132H (50 -TGACCTATGAT GATAGGTTTTACCCATCCAC-30 ). IDH1R132H were cloned into PGC-FU-vector, which contains a biotin tag 50 of the green fluorescent protein (GFP) tag, generating PGCFU-IDH1R132H-GFP. Lentiviral particles were added to U87 cells, and 48 h later, infection efficiency was determined by analyzing GFP expression using flow cytometry. Immunofluorescent staining Immunofluorescent staining was carried out according to the instructions provided by the manufacturer. U87 cells were first fixed with methanol, and then blocked in 1 % BSA for 2 h. Cultures were then treated with a fluoresceinlabeled goat anti-mouse IgG (1:80 dilution in 1 % BSA). Nuclei were stained with DAPI (1 lg/ml). Cells were incubated with a mouse monoclonal antibody against

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The viability of U87 cells was measured using the WST-1 cell proliferation and cytotoxicity assay kit (Beyotime, Haimen, China). Cells transfected with IDH1R132H, transfected with vector and control cells were seeded in 96-well plates and then mixed with 10 ll WST-1 reagent. GSH (1 mM) and H2O2 (1 mM) were added to the respective groups. After culturing for 24, 48, 72, 96, and 120 h under standard conditions, the proliferation rate was calculated by absorption at 450 nm. The percentage of viable cells was calculated by comparison with that of untreated control cells. Cellular NADPH and GSH levels Intracellular GSH was measured using a colorimetric microplate assay kit (Beyotime Ins, China). Absorbance was read at 450 nm using a microplate reader. NADPH was measured using an AmpliteTM Colorimetric NADP/ NADPH Assay Kit (Amyjet Scientific Inc, China) in a white/clear bottom 96-well plate using a NOVOStar microplate reader (BMG Labtech). The absorbance increase was monitored using an absorbance plate reader at 570 nm. Measurement of intracellular ROS For analysis of intracellular ROS, the redox-sensitive fluorescent probe DCFH-DA was used with a reactive oxygen species assay kit (Beyotime Ins, China). Cells were incubated with 5 lM DCFH-DA for 30 min at 37 °C. The harvested cells were immediately analyzed at 485 nm excitation and 535 nm emissions using a fluorescence spectrophotometer. Tumorigenicity assay Animal experiments were performed in strict accordance with the Institutional Animal Care guidelines. Transfected

Neurol Sci

Fig. 1 Analysis of cell proliferation using immunofluorescent staining. Cells were stained as described in ‘‘Methods’’. Images were acquired by fluorescent microscopy. Scale bar = 100 lm

IDH1R132H cells were mixed with H2O2 (1 mM) or GSH (1 mM) and cultured for 1 h. Then IDH1R132H cells with H2O2 and GSH, respectively, and control cells (1 9 105) were injected subcutaneously into left front near upper extremity of female nude mice (6 weeks old; Experimental Animal Laboratories, Shanghai, China). The resulting tumors were monitored weekly, and tumor volume (mm3) was calculated using the standard formula: length 9 width 9 height 9 0.5236. All mice were killed at the sixth week after implantation, and tumors were harvested and individually weighed. Data were presented as tumor volume and tumor weight (mean ± SD). Statistical analysis Statistical analysis was performed with GraphPad Prism 5 software. Statistical significance was determined using Student’s t test. The results are expressed as mean ± standard error mean (SEM). P \ 0.001 was considered significant. Each experiment consisted of at least three replicates per condition.

Results Mutation of IDH1 inhibits the growth of glioma cells and cell proliferation First, we defined whether mutation of IDH1 inhibited cell growth. For immunostaining, U87 cells transfected with

IDH1R132H-GFP were incubated with an antibody against PCNA for 7 days. Transfected IDH1R132H affected a significant decrease about 10 % in cellular proliferation rate by contrast with control cells (Fig. 1). IDH1 mutation induces apoptosis in glioma cells We next performed a TUNEL assay to label the 30 -end of fragmented DNA in the apoptotic U87 cells. We stained the cell nucleus with DAPI and acquired the images by fluorescent microscopy. Figure 2 shows that cells transfected with the mutant IDH1 mutation have fewer cell nuclei than the control group. Moreover, apoptosis rate was increased about 12 % significantly in cells transfected with IDH1R132H compared with the control group. Mutation of IDH1 reduces the viability of glioma cells We next measured the viability of mutant IDH1-transfected U87 cell lines and evaluated the impact of changes in H2O2 or GSH levels on cell viability. The viability of cells overexpressing IDH1R132H was significantly lower than control cells (Fig. 3). As shown in Fig. 3a, the viability of cells treated with GSH was higher than those treated without GSH. Moreover, the viability of cells treated with H2O2 was lower than those treated without H2O2 (Fig. 3b). These results suggest that neither overexpression of IDH1R132H nor downregulation of GSH was conducive to cell proliferation.

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Fig. 2 Analysis of apoptosis of U87 cells. Images were captured using fluorescent microscopy. U87 cells transfected with PGC-FUvector or PGC-FU-IDH1R132H. Blue spots are cell nuclei stained with

DAPI. FITC-labeled TUNEL-positive cells were imaged by fluorescent microscopy, as shown by red spots. Scale bar = 25 lm (color figure online)

Fig. 3 Viability of cells overexpressing mutant IDH1 or control cells in the presence or absence of GSH or H2O2. a U87-R132H compared with U87, U87-vector, U87-R132H ? GSH and U87-vector ? GSH. b U87-R132H compared with U87, U87-vector, U87-R132H ? H2O2

and U87-vector ? H2O2. Cell viability was measured by WST assay. Each value represents the mean SD from 3–5 independent experiments

Mutation of IDH1 reduces intracellular NADPH and GSH in glioma cells

other two groups, while the two groups had not changed significantly.

Intracellular GSH plays important roles in the maintenance of redox status and defense against oxidative stress. NADPH is an essential cofactor for the regeneration of GSH by glutathione reductase [15, 16]. We next investigated intracellular NADPH and GSH levels in U87 cells transfected with PGC-FU-IDH1R132H-GFP or PGC-FUvector. Figure 4a, b shows the levels of NADPH and GSH in cells overexpressing IDH1R132H were lower than the

Intracellular ROS level

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To investigate the effect of mutation of IDH1 on ROS levels, we measured the levels of intracellular ROS in the U87 cells transfected with PGC-FU-IDH1R132H-GFP using the oxidant-sensitive probe DCFH-DA under a laser confocal scanning microscope. As shown in Fig. 4c, levels of intracellular oxidants were

Neurol Sci

Fig. 4 Graphical representation of the enzymatic activity in the U87 cells overexpressing mutant IDH1 compared with the control cells. a Intracellular NADPH levels were measured as described in ‘‘Methods’’. The values are expressed as mM. *P \ 0. 001. b Intracellular GSH levels were determined according to the instructions

contained with the kit. The values are expressed as mM. *P \ 0. 001. c For analysis of intracellular ROS, cells were incubated with DCFHDA, according to instructions in the reactive oxygen species assay kit. The values are expressed as mM. *P \ 0.001

Fig. 5 The tumor volume curves and the bar chart of the weight. a, d The mice were killed at the sixth week after implantation, and tumors were individually weighed. b The curve shows the tumor growth of R132H-transfected U87 cell or vector-transfected, and the

R132H-transfected U87 cell group treated with H2O2 or GSH. c Tumor tissues were weighted at the sixth week. (*P \ 0.01, **P \ 0.001). Data are presented as tumor volume (mean ± SD) and tumor weight (mean ± SD)

significantly increased in U87-R132H compared with the control groups. These results further confirm that the rate of ROS generation is affected by mutation of IDH1.

IDH1 mutations reduce tumorigenicity in nude mice Our in vitro studies suggested that overexpression of mutant IDH1 reduced clonogenic proliferation and induced

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apoptosis in glioma cells. We next asked whether overexpression of mutant IDH1 in glioma cell lines affected their ability to initiate tumors in nude mice. Tumor size was greatly reduced in mice inoculated with R132H-transfected U87 cell treated with H2O2, compared with the control groups (Fig. 5a, b). As anticipated, the group treated with GSH developed tumors with a markedly larger size than the group inoculated with cells transfected with IDH1R132H only (Fig. 5a, b). Similarly, the tumor weights are consistent with the results of tumor volume (Fig. 5c, d).

Discussion Using a lentiviral system, we demonstrated that overexpression of IDH1R132H, the most common type of IDH1 mutation in glioma, decreased the intracellular GSH levels, enhanced the levels of intracellular ROS and inhibited the growth of glioma cells. These findings are consistent with improved survival in patients with tumors containing this IDH1 mutant [6]. The association between the mutant IDH1 and reduced proliferation in glioma cells sheds light on why the IDH1 mutation is an independent favorable prognostic marker in glioma patients [5, 7]. The current study demonstrates that IDH1R132H overexpression results in reduction of glioma cell clonogenic proliferation due to reduced cell cycle activity [17, 18]. In agreement with previous studies showing that cells expressing IDH1R132H had increased apoptosis and were more sensitive to radiation [19]. We observed an increased rate of apoptosis rate in IDH1R132H-expressing cell lines compared with control cell lines. It is well known that the redox status of the cell plays a central role in the regulation of various cell functions and is crucial to the optimal function of many enzymes [20]. Since cancer cells contain a higher level of ROS than normal cells, they are more susceptible to phytochemicals which target ROS metabolism [21, 22]. We found that overexpression of mutant IDH1 in glioma cells resulted in an increase in intracellular ROS levels, demonstrating that the mutant IDH1 affected growth inhibition by induction of cell apoptosis and inhibition of proliferation. We speculated that ROS generation might be the primary mechanism of action of IDH1. It is well known that oxidative stress is caused by an imbalance between production of ROS and the ability of the antioxidant defense system to readily detoxify the reactive intermediates. At the same time, the GSH redox system is one of the most important antioxidant defense systems, being involved in the protection of cells against oxidative damage as well as in various detoxification systems [14, 23]. Given that previous studies have reported that a reduction in intracellular GSH is necessary for the formation of ROS [13, 24], we sought to clarify the

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exact mechanism of ROS production and GSH depletion in glioma cells transfected with mutant IDH1. Our data confirm that overexpression of mutant IDH1 increased GSH depletion in U87 cells, implying that depletion of GSH might weaken antioxidant defense systems and enhance the levels of ROS. IDH1 catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate and NADPH. Previous studies have shown that IDH-mediated NADPH production and overall NADPH levels are reduced in IDH1R132H gliomas in situ [6, 25]. The cellular antioxidant system relies heavily on NADPH [26]. Interestingly, NADPH is an essential cofactor for the biosynthesis of GSH, and GSSG can be reduced to GSH in an NADPH-dependent reaction catalyzed by glutathione reductase [15]. IDH1 therefore catalyzes the oxidative carboxylation of isocitrate to aketoglutarate, yielding reduced nicotinamide adenine dinucleotide phosphate (NADPH) and resulting in reduced GSH and increased ROS levels [6, 9, 27]. It is possible that the observed increased oxidative stress in IDH1 mutant cells is due to decreased buffering of ROS due to lower available levels of NADPH, and our study supports the notion that decreased NADPH is important in GSH depletion and ROS generation in U87 cells expressing IDH1R132H. In conclusion, our study demonstrates that mutation of IDH1 profoundly inhibits the growth of glioma cells, and GSH depletion and ROS generation are the possible mechanisms. However, a limitation of this study has been the use of a unique cell line; thus, one needs to be cautious in generalizing the observed findings, due to the wellknown biologic heterogeneity of the different glioma cell lines. Acknowledgments This study was supported by the Youth Fund of the National Natural Science Foundation of China (81201975; 81201979), the Youth Fund of the Natural Science Foundation of Jiangsu Province (BK2012224), the Natural Science Foundation of China Ministry of Health (2010-2-025), the Natural Science Foundation of Jiangsu Department of Health (H201124), the Six Major Human Resources Project of Jiangsu Province (2011-WS-065; 2010WS-038), the Natural Science Foundation of Jiangsu Colleges and Universities Grant (11KJB320010). Conflict of interest All the authors report no disclosures relevant to the manuscript. Animal experiments were performed in strict accordance with the Institutional Animal Care guidelines.

References 1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114(2):97–109 2. Wen PY, Kesari S (2008) Malignant gliomas in adults. N Engl J Med 359(5):492–507

Neurol Sci 3. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996 4. Ichimura K, Pearson DM, Kocialkowski S, Backlund LM, Chan R, Jones DT, Collins VP (2009) IDH1 mutations are present in the majority of common adult gliomas but rare in primary glioblastomas. Neuro Oncol 11(4):341–347 5. Sanson M, Marie Y, Paris S, Idbaih A, Laffaire J, Ducray F, El Hallani S, Boisselier B, Mokhtari K, Hoang-Xuan K, Delattre JY (2009) Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. J Clin Oncol 27(25):4150–4154 6. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD (2009) IDH1 and IDH2 mutations in gliomas. N Engl J Med 360(8):765–773 7. Kloosterhof NK, Bralten LB, Dubbink HJ, French PJ, van den Bent MJ (2011) Isocitrate dehydrogenase-1 mutations: a fundamentally new understanding of diffuse glioma? Lancet Oncol 12(1):83–91 8. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, Olivi A, McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y, Busam DA, Tekleab H, Diaz LA Jr, Hartigan J, Smith DR, Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ, Bigner DD, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW (2008) An integrated genomic analysis of human glioblastoma multiforme. Science 321(5897):1807–1812 9. Menon SG, Goswami PC (2007) A redox cycle within the cell cycle: ring in the old with the new. Oncogene 26(8):1101–1109 10. Zhang Z, Leonard SS, Huang C, Vallyathan V, Castranova V, Shi X (2003) Role of reactive oxygen species and MAPKs in vanadate-induced G(2)/M phase arrest. Free Radic Biol Med 34(10):1333–1342 11. Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK, Sen E (2007) Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Mol Cancer Ther 6(9):2544–2553 12. Deng S, Yang Y, Han Y, Li X, Wang X, Li X, Zhang Z, Wang Y (2012) UCP2 inhibits ROS-mediated apoptosis in A549 under hypoxic conditions. PLoS One 7(1):e30714 13. Guha P, Dey A, Sen R, Chatterjee M, Chattopadhyay S, Bandyopadhyay SK (2011) Intracellular GSH depletion triggered mitochondrial Bax translocation to accomplish resveratrolinduced apoptosis in the U937 cell line. J Pharmacol Exp Ther 336(1):206–214 14. Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2(1):48–58

15. Izawa S, Maeda K, Miki T, Mano J, Inoue Y, Kimura A (1998) Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in Saccharomyces cerevisiae. Biochem J 330(Pt 2):811–817 16. Rhee SG (1999) Redox signaling: hydrogen peroxide as intracellular messenger. Exp Mol Med 31(2):53–59 17. Shackelford RE, Kaufmann WK, Paules RS (1999) Cell cycle control, checkpoint mechanisms, and genotoxic stress. Environ Health Perspect 107(Suppl 1):5–24 18. Bralten LB, Kloosterhof NK, Balvers R, Sacchetti A, Lapre L, Lamfers M, Leenstra S, de Jonge H, Kros JM, Jansen EE, Struys EA, Jakobs C, Salomons GS, Diks SH, Peppelenbosch M, Kremer A, Hoogenraad CC, Smitt PA, French PJ (2011) IDH1 R132H decreases proliferation of glioma cell lines in vitro and in vivo. Ann Neurol 69(3):455–463 19. Li S, Chou AP, Chen W, Chen R, Deng Y, Phillips HS, Selfridge J, Zurayk M, Lou JJ, Everson RG, Wu KC, Faull KF, Cloughesy T, Liau LM, Lai A (2013) Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation. Neuro Oncol 15(1):57–68 20. Chuang JI, Chang TY, Liu HS (2003) Glutathione depletioninduced apoptosis of Ha-ras-transformed NIH3T3 cells can be prevented by melatonin. Oncogene 22(9):1349–1357 21. Konishi T, Shimada Y, Nagao T, Okabe H, Konoshima T (2002) Antiproliferative sesquiterpene lactones from the roots of Inula helenium. Biol Pharm Bull 25(10):1370–1372 22. Lawrence NJ, McGown AT, Nduka J, Hadfield JA, Pritchard RG (2001) Cytotoxic Michael-type amine adducts of alpha-methylene lactones alantolactone and isoalantolactone. Bioorg Med Chem Lett 11(3):429–431 23. Ghantous A, Gali-Muhtasib H, Vuorela H, Saliba NA, Darwiche N (2010) What made sesquiterpene lactones reach cancer clinical trials? Drug Discov Today 15(15–16):668–678 24. Franco R, Panayiotidis MI, Cidlowski JA (2007) Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation. J Biol Chem 282(42):30452–30465 25. Bleeker FE, Atai NA, Lamba S, Jonker A, Rijkeboer D, Bosch KS, Tigchelaar W, Troost D, Vandertop WP, Bardelli A, Van Noorden CJ (2010) The prognostic IDH1(R132) mutation is associated with reduced NADP?-dependent IDH activity in glioblastoma. Acta Neuropathol 119(4):487–494 26. Koehler A, Van Noorden CJ (2003) Reduced nicotinamide adenine dinucleotide phosphate and the higher incidence of pollution-induced liver cancer in female flounder. Environ Toxicol Chem 22(11):2703–2710 27. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Fantin VR, Jang HG, Jin S, Keenan MC, Marks KM, Prins RM, Ward PS, Yen KE, Liau LM, Rabinowitz JD, Cantley LC, Thompson CB, Vander Heiden MG, Su SM (2010) Cancerassociated IDH1 mutations produce 2-hydroxyglutarate. Nature 465(7300):966

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