Abstract
Glutamate has been shown to function as a toxic agent in neuronal and glial cells, as well as an excitatory neurotransmitter throughout the central nervous system. In the present study, we examined the effect of increasing glutamate concentration on the induction of apoptosis in the two human glioblastoma cell lines GB-4 and GB-12. Glutamate exposure caused cell death of GB-4 and GB-12 in a dose-dependent manner. The cells were found to die via apoptosis in response to glutamate based on the following criteria: propidium iodide (PI) staining, H–E staining, electron microscopic analysis, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. The glutamate-induced apoptosis appears to involve the modulation of Bcl-2 family gene products such as Bcl-2, Bcl-xL, and Bax-α. Both Bcl-2 and Bcl-xL were down-regulated by glutamate at 24 h and further at 48 h. The apoptosis-promoting product p21 Bax-α was also down-regulated in GB-12 but slightly up-regulated in GB-4, accompanied by generation of variant form of p18 Bax-α in both cell lines. These findings suggest that glutamate toxicity results in cellular death via an apoptotic mechanism which appears to involve the Bcl-2/Bax-α molecular complex.
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References
Monaghan DT, Bridges RJ, Cotman CW: The excitatory amino acid receptors; their classes, pharmacology, and distinct properties in the function of the central nervous system. Annu Rev Pharmacol Toxicol 29: 365-402, 1989
Watkins JC, Krogsgaard-Larsen P, Honore T: Structure- activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists. Trends Pharmacol Sci 11: 25-33, 1990
Choi DW: Excitotoxic cell death. J Neurobiol 23: 1261-1276, 1992
Goldberg MP, Weiss JH, Pharm PC, Choi DW: N-methyl-D-aspartate receptors mediate hypoxic neuronal injury in cortical culture. J Pharmacol Exp Ther 243: 784-791, 1987
Novelli A, Reilly JA, Lysko PG, Henneberry RC: Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res 451: 205-212, 1988
Bannai S, Kitamura E: Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture. J Biol Chem 255: 2372-2376, 1980
Bannai S: Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem 261: 2256-2263, 1986
Deneke SM, Fanburg BL: Regulation of cellular glutathione. Am J Physiol 257: L163-L173, 1989
Froissard P, Monrocq H, Duval D: Role of glutathione metabolism in the glutamate-induced programmed cell death of neuronal-like PC12 cells. Eur J Pharmacol 326: 93-99, 1997
Kato S, Higashida H, Higuchi Y, Hatakenaka S, Negishi K: Sensitive and insensitive states of cultured glioma cells to glutamate damage. Brain Res 303: 365-373, 1984
Murphy TH, Miyamoto M, Sastre A, Schnaar RL, Coyle JT: Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2: 1547-1558,1989
Piani D, Fontana A: Involvement of the cystine transport system Xc– in the macrophage-induced glutamate-dependent cytotoxicity to neurons. J Immunol 152: 3578-3585, 1994
Kato S, Negishi K, Mawatari K, Kuo C-H: A mechanism for glutamate toxicity in the C6 glioma cells involving inhibition of cystine uptake leading to glutathione depletion. Neuroscience 48: 903-914, 1992
Pereira CMF, Oliveira CR: Glutamate toxicity on a PC12 cell line involves glutathione (GSH) depletion and oxidative stress. Free Radic Biol Med 23: 637-647,1997
Raff MC: Social controls on cell survival and cell death. Nature 356: 397-400, 1992
Williams GT, Smith CA: Molecular regulation of apoptosis: genetic controls on cell death. Cell 74: 777-779, 1993
Cory S: Regulation of lymphocyte survival by the bcl-2 gene family. Annu Rev Immunol 13: 513-543, 1995
Oltvai ZN, Milliman CL, Korsmeyer SJ: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74: 609-619, 1993
Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ: Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death. Cell 80: 285-291, 1995
Lawrence MS, Ho DY, Sun GH, Steinberg GK, Sapolsky RM: Overexpression of Bcl-2 with herpes simplex virus vectors protects CNS neurons against neurological insults in vitro and in vivo. J Neurosci 16: 486-496, 1996
Vekrellis K, McCarthy MJ, Watson A, Whitfield J, Rubin LL, Ham J: Bax promotes neuronal cell death and is downregulated during the development of the nervous system. Development 124: 1239-1249, 1997
Sakakura C, Sweeney EA, Shirahama T, Igarashi Y, Hakomori S, Tsujimoto H, Imanishi T, Ohgaki M, Yamazaki J, Hagiwara A, Sawai K, Yamaguchi T, Takahasi T: Overexpression of bax enhances the radiation sensitivity in human breast cancer cells. Surg Today 27: 90-93, 1997
Furukawa K, Estus S, Fu W, Mark RJ, Mattson MP: Neuroprotective action of cycloheximide involves induction of bcl-2 and antioxidant pathways. J Cell Biol 136: 1137-1149, 1997
Takahashi M: Generation of cytotoxic TNK cells following in vitro stimulation with class I-positive glioma cells and IL-2. J Tokyo Med Univ 55: 40-48, 1997
Del Bino G, Skierski JS, Darzynkiewicz Z: The concentration-dependent diversity of effects of DNA topoisomerase I and II inhibitors on the cell cycle of HL-60 cells. Exp Cell Res 195: 485-491, 1991
Mizuguchi J, Yamanashi Y, Ehara K, Tamura T, Nariuchi H, Gyotoku Y, Fukazawa H, Uehara Y, Yamamoto T: Tyrosine protein kinase is involved in anti-IgM-mediated signaling in BAL17 B lymphoma cells. J Immunol 148: 689-694, 1992
Hartley DM, Kurth MC, Bjerkness L, Weiss JH, Choi DW: Glutamate receptor-induced 45Ca2+ accumulation in cortical cell culture correlates with subsequent neuronal degeneration. J Neurosci 13: 1993-2000, 1993
MacDermott AB, Mayer ML, Westbrook GL, Smith SJ, Barker JL: NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal neurons. Nature 321: 519-522, 1986
Cho Y, Bannai S: Uptake of glutamate and cysteine in C-6 glioma cells and in cultured astrocytes. J Neurochem 55: 2091-2097, 1990
Haldar S, Jena N, Croce CM: Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci USA 92: 4507-4511, 1995
Cheng EH-Y, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM: Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 278: 1966-1968, 1997
Clem RJ, Cheng EH-Y, Karp CL, Kirsch DG, Ueno K, Takahashi A, Kastan MB, Griffin DE, Earnshaw WC, Veliuona MA, Hardwick JM: Modulation of cell death by Bcl-xL through caspase interaction. Proc Natl Acad SciUSA 95: 554-559, 1998
Kobayashi T, Ruan S, Clodi K, Kliche K-O, Shiku H, Andreeff M, Zhang W: Overexpression of Bax gene sensitizes K562 erythroleukemia cells to apoptosis induced by selective chemotherapeutic agents. Oncogene 16: 1587-1591, 1998
Nagai T, Yanase N, Mizuguchi J: Anti-CD4O-mediated prevention of apoptosis of human germinal center B cells. J Tokyo Med Univ 56: 88-98, 1998
Yanase N, Takada E, Yoshihama I, Ikegami H, Mizuguchi J: Participation of Bax-α in interferon α-mediated apoptosis in Daudi B lymphoma cells. J Interf Cytok Res 18: 855-861, 1998
Schlesinger PH, Gross A, Yin X-M, Yamamoto K, Saito M, Waksman G, Korsmeyer SJ: Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci USA 94: 11357-11362, 1997
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Nishi, T., Takahashi, M., Ito, H. et al. Participation of Bcl-2/Bax-α in Glutamate-induced Apoptosis of Human Glioblastoma Cells. J Neurooncol 44, 109–117 (1999). https://doi.org/10.1023/A:1006310815374
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DOI: https://doi.org/10.1023/A:1006310815374