Abstract
Hypoxia induces apoptosis in primary and transformed cells and in various tumor cell lines in vitro. In contrast, there is little apoptosis and predominant necrosis despite extensive hypoxia in human glioblastomas in vivo. We here characterize ultrastructural and biochemical features of cell death in LN-229, LN-18 and U87MG malignant glioma cells in a paradigm of hypoxia with partial glucose deprivation in vitro. Electron microscopic analysis of hypoxia-challenged glioma cells demonstrated early stages of apoptosis but predominant necrosis. ATP levels declined during hypoxia, but recovered with re-exposure to normoxic conditions unless hypoxia exceeded 8 h. Longer hypoxic exposure resulted in irreversible ATP depletion and delayed cell death. Hypoxia induced mitochondrial release of cytochrome c, but there was no cleavage of caspases 3, 7, 8 or 9, and no DNA fragmentation. Ectopic expression of BCL-XL conferred protection from hypoxia-induced cell death, whereas the overexpression of the antiapoptotic proteins X-linked-inhibitor-of-apoptosis-protein and cytokine response modifier-A had no effect. These findings suggest that glioma cells resist adverse effects of hypoxia until energy stores are depleted and then undergo necrosis rather than apoptosis because of energy deprivation.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- APAF 1:
-
apoptotic protease activating factor 1
- CD95L:
-
CD95 ligand
- Crm-A:
-
cytokine response modifier-A
- ECL:
-
enhanced chemiluminescence
- GFP:
-
green fluorescent protein
- TUNEL:
-
terminal dUTP in situ nick end-labeling
- VEGF:
-
vascular endothelial growth factor
- XIAP:
-
X-linked-inhibitor-of-apoptosis-protein
References
Rampling R, Cruickshank G, Lewis AD, Fitzsimmons SA and Workman P (1994) Direct measurement of pO2 distribution and bioreductive enzymes in human malignant brain tumors. Int. J. Radiat. Oncol. Biol. Phys. 29: 427–431
Valk PE, Mathis CA, Prados MD, Gilbert JC and Budinger TF (1992) Hypoxia in human gliomas: demonstration by PET with fluorine-18- fluoromisonidazole. J. Nucl. Med. 33: 2133–2137
Collingridge DR, Piepmeier JM, Rockwell S and Knisely JP (1999) Polarographic measurements of oxygen tension in human glioma and surrounding peritumoural brain tissue. Radiother. Oncol. 53: 127–131
Zagzag D, Amirnovin R, Greco MA, Yee H, Holash J, Wiegand SJ, Zabski S, Yancopoulos GD and Grumet M (2000) Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis. Lab. Invest. 80: 837–849
Holash J, Maisonpierre PC, Compton D, Boland P, Alexander CR, Zagzag D, Yancopoulos GD and Wiegand SJ (1999) Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 284: 1994–1998
Royds JA, Dower SK, Qwarnstrom EE and Lewis CE (1998) Response of tumour cells to hypoxia: role of p53 and NFkB. Mol. Pathol. 51: 55–61
Kleihues P, Burger PC, Collins VP, Newcomb EW, Ohgaki H and Cavenee WK (2000) Glioblastoma. In Pathology and Genetics of Tumours of the Nervous System, Kleihues P and Cavenee WK (eds) (Lyon: IARC Press) pp. 29–39
Kimura H, Braun RD, Ong ET, Hsu R, Secomb TW, Papahadjopoulos D, Hong K and Dewhirst MW (1996) Fluctuations in red cell flux in tumor microvessels can lead to transient hypoxia and reoxygenation in tumor parenchyma. Cancer Res. 56: 5522–5528
Helmlinger G, Yuan F, Dellian M and Jain RK (1997) Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat. Med. 3: 177–182
Read TA, Sorensen DR, Mahesparan R, Enger PO, Timpl R, Olsen BR, Hjelstuen MH, Haraldseth O and Bjerkvig R (2001) Local endostatin treatment of gliomas administered by microencapsulated producer cells. Nat. Biotechnol. 19: 29–34
Machein MR, Risau W and Plate KH (1999) Antiangiogenic gene therapy in a rat glioma model using a dominant-negative vascular endothelial growth factor receptor 2. Hum. Gene Ther. 10: 1117–1128
Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW and Giaccia AJ (1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379: 88–91
Araya R, Uehara T and Nomura Y (1998) Hypoxia induces apoptosis in human neuroblastoma SK-N-MC cells by caspase activation accompanying cytochrome c release from mitochondria. FEBS Lett. 439: 168–172
Amellem O, Stokke T, Sandvik JA, Smedshammer L and Pettersen EO (1997) Hypoxia-induced apoptosis in human cells with normal p53 status and function, without any alteration in the nuclear protein level. Exp. Cell Res. 232: 361–370
Saikumar P, Dong Z, Weinberg JM and Venkatachalam MA (1998) Mechanisms of cell death in hypoxia/reoxygenation injury. Oncogene 17: 3341–3349
Tohma Y, Gratas C, Van Meir EG, Desbaillets I, Tenan M, Tachibana O, Kleihues P and Ohgaki H (1998) Necrogenesis and Fas/APO-1 (CD95) expression in primary (de novo) and secondary glioblastomas. J. Neuropathol. Exp. Neurol. 57: 239–245
Steinbach JP and Weller M (2002) Mechanisms of apoptosis in CNS tumors: application to theory. Curr. Neurol. Neurosci. Rep. 2: 246–253
Tachibana O, Lampe J, Kleihues P and Ohgaki H (1996) Preferential expression of Fas/APO1 (CD95) and apoptotic cell death in perinecrotic cells of glioblastoma multiforme. Acta Neuropathol. 92: 431–434
Schiffer D, Cavalla P, Migheli A, Chio A, Giordana MT, Marino S and Attanasio A (1995) Apoptosis and cell proliferation in human neuroepithelial tumors. Neurosci. Lett. 195: 81–84
Plate KH, Breier G, Weich HA and Risau W (1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359: 845–848
Dong Z, Venkatachalam MA, Wang J, Patel Y, Saikumar P, Semenza GL, Force T and Nishiyama J (2001) Up-regulation of apoptosis inhibitory protein IAP-2 by hypoxia. Hif-1-independent mechanisms. J. Biol. Chem. 276: 18702–18709
Kim CY, Tsai MH, Osmanian C, Graeber TG, Lee JE, Giffard RG, DiPaolo JA, Peehl DM and Giaccia AJ (1997) Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions. Cancer Res. 57: 4200–4204
Teicher BA (1994) Hypoxia and drug resistance. Cancer Metast Rev. 13: 139–168
Hockel M, Vorndran B, Schlenger K, Baussmann E and Knapstein PG (1993) Tumor oxygenation: a new predictive parameter in locally advanced cancer of the uterine cervix. Gynecol. Oncol. 51: 141–149
Korshunov A, Golanov A, Sycheva R and Pronin I (1999) Prognostic value of tumour associated antigen immunoreactivity and apoptosis in cerebral glioblastomas: an analysis of 168 cases. J. Clin. Pathol. 52: 574–580
Rhodes RH (1998) Biological evaluation of biopsies from adult cerebral astrocytomas: cell-growth/cell-suicide ratios and their relationship to patient survival. J. Neuropathol. Exp. Neurol. 57: 746–757
Hossmann KA, Mies G, Paschen W, Szabo L, Dolan E and Wechsler W (1986) Regional metabolism of experimental brain tumors. Acta Neuropathol 69: 139–147
Mies G, Paschen W, Ebhardt G and Hossmann KA (1990) Relationship between blood flow, glucose metabolism, protein synthesis, glucose and ATP content in experimentally induced glioma (RG1 2.2) of rat brain. J. Neurooncol. 9: 17–28
Vaupel P (1992) Physiological properties of malignant tumours. NMR Biomed 5: 220–225
Leist M, Single B, Castoldi AF, Kuhnle S and Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J. Exp. Med. 185: 1481–1486
Eguchi Y, Shimizu S and Tsujimoto Y (1997) Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57: 1835–1840
Weller M, Frei K, Groscurth P, Krammer PH, Yonekawa Y and Fontana A (1994) Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines. J Clin Invest 94: 954–964
Glaser T, Wagenknecht B, Groscurth P, Krammer PH and Weller M (1999) Death ligand/receptor-independent caspase activation mediates drug-induced cytotoxic cell death in human malignant glioma cells. Oncogene 18: 5044–5053
Hermisson M, Wagenknecht B, Wolburg H, Glaser T, Dichgans J and Weller M (2000) Sensitization to CD95 ligand-induced apoptosis in human glioma cells by hyperthermia involves enhanced cytochrome c release. Oncogene 19: 2338–2345
Gerweck LE, Seneviratne T and Gerweck KK (1993) Energy status and radiobiological hypoxia at specified oxygen concentrations. Radiat Res 135: 69–74
Glaser T, Wagenknecht B and Weller M (2001) Identification of p21 as a target of cycloheximide-mediated facilitation of CD95-mediated apoptosis in human malignant glioma cells. Oncogene 20: 4757–4767
Saikumar P, Dong Z, Patel Y, Hall K, Hopfer U, Weinberg JM and Venkatachalam MA (1998) Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury. Oncogene 17: 3401–3415
Callahan DJ, Engle MJ and Volpe JJ (1990) Hypoxic injury to developing glial cells: protective effect of high glucose. Pediatr. Res. 27: 186–190
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X (1997) cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479–489
Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR and Martin SJ (1999) Ordering the cytochrome c -initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9- dependent manner. J. Cell Biol. 144: 281–292
Eguchi Y, Srinivasan A, Tomaselli KJ, Shimizu S and Tsujimoto Y (1999) ATP-dependent steps in apoptotic signal transduction. Cancer Res. 59: 2174–2181
Wagenknecht B, Glaser T, Naumann U, Kugler S, Isenmann S, Bahr M, Korneluk R, Liston P and Weller M (1999) Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma. Cell Death Differ 6: 370–376
Weller M, Rieger J, Grimmel C, Van Meir EG, De Tribolet N, Krajewski S, Reed JC, von Deimling A and Dichgans J (1998) Predicting chemoresistance in human malignant glioma cells: the role of molecular genetic analyses. Int. J. Cancer 79: 640–644
Leist M and Jaattela M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2: 589–598
Wang KK (2000) Calpain and caspase: can you tell the difference? Trends Neurosci 23: 20–26
Waterhouse NJ, Finucane DM, Green DR, Elce JS, Kumar S, Alnemri ES, Litwack G, Khanna K, Lavin MF and Watters DJ (1998) Calpain activation is upstream of caspases in radiation-induced apoptosis. Cell Death Differ 5: 1051–1061
Haraguchi M, Torii S, Matsuzawa S, Xie Z, Kitada S, Krajewski S, Yoshida H, Mak TW and Reed JC (2000) Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2. J Exp Med 191: 1709–1720
Green DR and Reed JC (1998) Mitochondria and apoptosis. Science 281: 1309–1312
Susin SA, Daugas E, Ravagnan L, Samejima K, Zamzami N, Loeffler M, Costantini P, Ferri KF, Irinopoulou T, Prevost MC, Brothers G, Mak TW, Penninger J, Earnshaw WC and Kroemer G (2000) Two distinct pathways leading to nuclear apoptosis. J Exp Med 192: 571–580
Reed JC (1997) cytochrome c: can't live with it-can't live without it. Cell 91: 559–562
Roth W, Fontana A, Trepel M, Reed JC, Dichgans J and Weller M (1997) Immunochemotherapy of malignant glioma: synergistic activity of CD95 ligand and chemotherapeutics. Cancer Immunol. Immunother. 44: 55–63
Probst H, Schiffer H, Gekeler V, Kienzle Pfeilsticker H, Stropp U, Stotzer KE and Frenzel-Stotzer I (1988) Oxygen dependent regulation of DNA synthesis and growth of Ehrlich ascites tumor cells in vitro and in vivo. Cancer Res. 48: 2053–2060
Dreier T, Scheidtmann KH and Probst H (1993) Synchronous replication of SV 40 DNA in virus infected TC 7 cells induced by transient hypoxia. FEBS Lett. 336: 445–451
Kendler A and Dawson G (1990) Progressive hypoxia inhibits the de novo synthesis of galactosylceramide in cultured oligodendrocytes. J Biol Chem 265: 12259–12266
Acknowledgements
This study was supported by a grant from the German Cancer Council (10-1802-We 5) to MW and a grant from the fortüne program of the University of Tübingen to JS (724-0-0). We thank P Supra for excellent technical assistance, and H-J Riedinger and G Probst for help with the experimental setup for hypoxic incubation.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by JA Cidlowski
Rights and permissions
About this article
Cite this article
Steinbach, J., Wolburg, H., Klumpp, A. et al. Hypoxia-induced cell death in human malignant glioma cells: energy deprivation promotes decoupling of mitochondrial cytochrome c release from caspase processing and necrotic cell death. Cell Death Differ 10, 823–832 (2003). https://doi.org/10.1038/sj.cdd.4401252
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.cdd.4401252
Keywords
This article is cited by
-
Calreticulin: a quintessential multifaceted protein with therapeutic potential
Journal of Proteins and Proteomics (2023)
-
Blood-brain barrier disruption defines the extracellular metabolome of live human high-grade gliomas
Communications Biology (2023)
-
Activating transcription factor 4 mediates adaptation of human glioblastoma cells to hypoxia and temozolomide
Scientific Reports (2021)
-
Serine-dependent redox homeostasis regulates glioblastoma cell survival
British Journal of Cancer (2020)
-
18F-FDG PET metabolic-to-morphological volume ratio predicts PD-L1 tumour expression and response to PD-1 blockade in non-small-cell lung cancer
European Journal of Nuclear Medicine and Molecular Imaging (2019)
