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. 1999 May 15;340(Pt 1):291–297.

Protection against hydrogen peroxide cytotoxicity in rat-1 fibroblasts provided by the oncoprotein Bcl-2: maintenance of calcium homoeostasis is secondary to the effect of Bcl-2 on cellular glutathione.

M M Rimpler 1, U Rauen 1, T Schmidt 1, T Möröy 1, H de Groot 1
PMCID: PMC1220248  PMID: 10229685

Abstract

The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by dl-buthionine-(S, R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baffy G., Miyashita T., Williamson J. R., Reed J. C. Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J Biol Chem. 1993 Mar 25;268(9):6511–6519. [PubMed] [Google Scholar]
  2. Bellomo G., Mirabelli F., Richelmi P., Orrenius S. Critical role of sulfhydryl group(s) in ATP-dependent Ca2+ sequestration by the plasma membrane fraction from rat liver. FEBS Lett. 1983 Oct 31;163(1):136–139. doi: 10.1016/0014-5793(83)81180-6. [DOI] [PubMed] [Google Scholar]
  3. Bellomo G., Orrenius S. Altered thiol and calcium homeostasis in oxidative hepatocellular injury. Hepatology. 1985 Sep-Oct;5(5):876–882. doi: 10.1002/hep.1840050529. [DOI] [PubMed] [Google Scholar]
  4. Bian X., Hughes F. M., Jr, Huang Y., Cidlowski J. A., Putney J. W., Jr Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells. Am J Physiol. 1997 Apr;272(4 Pt 1):C1241–C1249. doi: 10.1152/ajpcell.1997.272.4.C1241. [DOI] [PubMed] [Google Scholar]
  5. Björkman U., Ekholm R. Hydrogen peroxide degradation and glutathione peroxidase activity in cultures of thyroid cells. Mol Cell Endocrinol. 1995 Apr 28;111(1):99–107. doi: 10.1016/0303-7207(95)03552-i. [DOI] [PubMed] [Google Scholar]
  6. Bojes H. K., Datta K., Xu J., Chin A., Simonian P., Nuñez G., Kehrer J. P. Bcl-xL overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal. Biochem J. 1997 Jul 15;325(Pt 2):315–319. doi: 10.1042/bj3250315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Distelhorst C. W., Lam M., McCormick T. S. Bcl-2 inhibits hydrogen peroxide-induced ER Ca2+ pool depletion. Oncogene. 1996 May 16;12(10):2051–2055. [PubMed] [Google Scholar]
  9. Distelhorst C. W., McCormick T. S. Bcl-2 acts subsequent to and independent of Ca2+ fluxes to inhibit apoptosis in thapsigargin- and glucocorticoid-treated mouse lymphoma cells. Cell Calcium. 1996 Jun;19(6):473–483. doi: 10.1016/s0143-4160(96)90056-1. [DOI] [PubMed] [Google Scholar]
  10. Dringen R., Hamprecht B. Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells. Brain Res. 1997 Jun 6;759(1):67–75. doi: 10.1016/s0006-8993(97)00233-3. [DOI] [PubMed] [Google Scholar]
  11. Ellerby L. M., Ellerby H. M., Park S. M., Holleran A. L., Murphy A. N., Fiskum G., Kane D. J., Testa M. P., Kayalar C., Bredesen D. E. Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2. J Neurochem. 1996 Sep;67(3):1259–1267. doi: 10.1046/j.1471-4159.1996.67031259.x. [DOI] [PubMed] [Google Scholar]
  12. Fanidi A., Harrington E. A., Evan G. I. Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature. 1992 Oct 8;359(6395):554–556. doi: 10.1038/359554a0. [DOI] [PubMed] [Google Scholar]
  13. Farber J. L., Kyle M. E., Coleman J. B. Mechanisms of cell injury by activated oxygen species. Lab Invest. 1990 Jun;62(6):670–679. [PubMed] [Google Scholar]
  14. Fossati P., Prencipe L., Berti G. Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine. Clin Chem. 1980 Feb;26(2):227–231. [PubMed] [Google Scholar]
  15. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  16. Guo Q., Sopher B. L., Furukawa K., Pham D. G., Robinson N., Martin G. M., Mattson M. P. Alzheimer's presenilin mutation sensitizes neural cells to apoptosis induced by trophic factor withdrawal and amyloid beta-peptide: involvement of calcium and oxyradicals. J Neurosci. 1997 Jun 1;17(11):4212–4222. doi: 10.1523/JNEUROSCI.17-11-04212.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Halliwell B., Cross C. E. Oxygen-derived species: their relation to human disease and environmental stress. Environ Health Perspect. 1994 Dec;102 (Suppl 10):5–12. doi: 10.1289/ehp.94102s105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hockenbery D. M., Oltvai Z. N., Yin X. M., Milliman C. L., Korsmeyer S. J. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993 Oct 22;75(2):241–251. doi: 10.1016/0092-8674(93)80066-n. [DOI] [PubMed] [Google Scholar]
  19. Jurma O. P., Hom D. G., Andersen J. K. Decreased glutathione results in calcium-mediated cell death in PC12. Free Radic Biol Med. 1997;23(7):1055–1066. doi: 10.1016/s0891-5849(97)00134-2. [DOI] [PubMed] [Google Scholar]
  20. Kroemer G., Dallaporta B., Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol. 1998;60:619–642. doi: 10.1146/annurev.physiol.60.1.619. [DOI] [PubMed] [Google Scholar]
  21. Link E. M. Enzymic pathways involved in cell response to H2O2. Free Radic Res Commun. 1990;11(1-3):89–97. doi: 10.3109/10715769009109671. [DOI] [PubMed] [Google Scholar]
  22. Lomonosova E. E., Kirsch M., de Groot H. Calcium vs. iron-mediated processes in hydrogen peroxide toxicity to L929 cells: effects of glucose. Free Radic Biol Med. 1998 Sep;25(4-5):493–503. doi: 10.1016/s0891-5849(98)00080-x. [DOI] [PubMed] [Google Scholar]
  23. Macho A., Hirsch T., Marzo I., Marchetti P., Dallaporta B., Susin S. A., Zamzami N., Kroemer G. Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J Immunol. 1997 May 15;158(10):4612–4619. [PubMed] [Google Scholar]
  24. Makino N., Mochizuki Y., Bannai S., Sugita Y. Kinetic studies on the removal of extracellular hydrogen peroxide by cultured fibroblasts. J Biol Chem. 1994 Jan 14;269(2):1020–1025. [PubMed] [Google Scholar]
  25. Marin M. C., Fernandez A., Bick R. J., Brisbay S., Buja L. M., Snuggs M., McConkey D. J., von Eschenbach A. C., Keating M. J., McDonnell T. J. Apoptosis suppression by bcl-2 is correlated with the regulation of nuclear and cytosolic Ca2+. Oncogene. 1996 Jun 6;12(11):2259–2266. [PubMed] [Google Scholar]
  26. McConkey D. J., Orrenius S. The role of calcium in the regulation of apoptosis. J Leukoc Biol. 1996 Jun;59(6):775–783. [PubMed] [Google Scholar]
  27. Meredith M. J., Cusick C. L., Soltaninassab S., Sekhar K. S., Lu S., Freeman M. L. Expression of Bcl-2 increases intracellular glutathione by inhibiting methionine-dependent GSH efflux. Biochem Biophys Res Commun. 1998 Jul 30;248(3):458–463. doi: 10.1006/bbrc.1998.8998. [DOI] [PubMed] [Google Scholar]
  28. Merry D. E., Korsmeyer S. J. Bcl-2 gene family in the nervous system. Annu Rev Neurosci. 1997;20:245–267. doi: 10.1146/annurev.neuro.20.1.245. [DOI] [PubMed] [Google Scholar]
  29. Mirkovic N., Voehringer D. W., Story M. D., McConkey D. J., McDonnell T. J., Meyn R. E. Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols. Oncogene. 1997 Sep 18;15(12):1461–1470. doi: 10.1038/sj.onc.1201310. [DOI] [PubMed] [Google Scholar]
  30. Murphy A. N., Bredesen D. E., Cortopassi G., Wang E., Fiskum G. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9893–9898. doi: 10.1073/pnas.93.18.9893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nicotera P., Bellomo G., Orrenius S. The role of Ca2+ in cell killing. Chem Res Toxicol. 1990 Nov-Dec;3(6):484–494. doi: 10.1021/tx00018a001. [DOI] [PubMed] [Google Scholar]
  32. Nicotera P., McConkey D., Svensson S. A., Bellomo G., Orrenius S. Correlation between cytosolic Ca2+ concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicology. 1988 Nov 14;52(1-2):55–63. doi: 10.1016/0300-483x(88)90196-5. [DOI] [PubMed] [Google Scholar]
  33. Orrenius S., McConkey D. J., Bellomo G., Nicotera P. Role of Ca2+ in toxic cell killing. Trends Pharmacol Sci. 1989 Jul;10(7):281–285. doi: 10.1016/0165-6147(89)90029-1. [DOI] [PubMed] [Google Scholar]
  34. RACKER E. The mechanism of action of glyoxalase. J Biol Chem. 1951 Jun;190(2):685–696. [PubMed] [Google Scholar]
  35. RALL T. W., LEHNINGER A. L. Glutathione reductase of animal tissues. J Biol Chem. 1952 Jan;194(1):119–130. [PubMed] [Google Scholar]
  36. Rauen U., Reuters I., Fuchs A., de Groot H. Oxygen-free radical-mediated injury to cultured rat hepatocytes during cold incubation in preservation solutions. Hepatology. 1997 Aug;26(2):351–357. doi: 10.1002/hep.510260215. [DOI] [PubMed] [Google Scholar]
  37. Reed J. C. Double identity for proteins of the Bcl-2 family. Nature. 1997 Jun 19;387(6635):773–776. doi: 10.1038/42867. [DOI] [PubMed] [Google Scholar]
  38. Reynolds J. E., Eastman A. Intracellular calcium stores are not required for Bcl-2-mediated protection from apoptosis. J Biol Chem. 1996 Nov 1;271(44):27739–27743. doi: 10.1074/jbc.271.44.27739. [DOI] [PubMed] [Google Scholar]
  39. Tyurin V. A., Tyurina Y. Y., Quinn P. J., Schor N. F., Balachandran R., Day B. W., Kagan V. E. Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection. Brain Res Mol Brain Res. 1998 Oct 1;60(2):270–281. doi: 10.1016/s0169-328x(98)00181-8. [DOI] [PubMed] [Google Scholar]
  40. Vlachaki M. T., Meyn R. E. ASTRO research fellowship: the role of BCL-2 and glutathione in an antioxidant pathway to prevent radiation-induced apoptosis. Int J Radiat Oncol Biol Phys. 1998 Aug 1;42(1):185–190. doi: 10.1016/s0360-3016(98)00203-x. [DOI] [PubMed] [Google Scholar]
  41. Voehringer D. W., McConkey D. J., McDonnell T. J., Brisbay S., Meyn R. E. Bcl-2 expression causes redistribution of glutathione to the nucleus. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2956–2960. doi: 10.1073/pnas.95.6.2956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhong L. T., Sarafian T., Kane D. J., Charles A. C., Mah S. P., Edwards R. H., Bredesen D. E. bcl-2 inhibits death of central neural cells induced by multiple agents. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4533–4537. doi: 10.1073/pnas.90.10.4533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zörnig M., Busch G., Beneke R., Gulbins E., Lang F., Ma A., Korsmeyer S., Möröy T. Survival and death of prelymphomatous B-cells from N-myc/bcl-2 double transgenic mice correlates with the regulation of intracellular Ca2+ fluxes. Oncogene. 1995 Nov 16;11(10):2165–2174. [PubMed] [Google Scholar]

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