Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Mar 1;354(Pt 2):397–406. doi: 10.1042/0264-6021:3540397

Nitric oxide-dependent pro-oxidant and pro-apoptotic effect of metallothioneins in HL-60 cells challenged with cupric nitrilotriacetate.

S Liu 1, K Kawai 1, V A Tyurin 1, Y Y Tyurina 1, G G Borisenko 1, J P Fabisiak 1, P J Quinn 1, B R Pitt 1, V E Kagan 1
PMCID: PMC1221668  PMID: 11171119

Abstract

Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl(2)-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl(2)-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.

Full Text

The Full Text of this article is available as a PDF (242.3 KB).

Selected References

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

  1. Aravindakumar C. T., Ceulemans J., De Ley M. Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiol. Biochem J. 1999 Nov 15;344(Pt 1):253–258. doi: 10.1042/0264-6021:3440253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aravindakumar C. T., Ceulemans J., De Ley M. Steric effect and effect of metal coordination on the reactivity of nitric oxide with cysteine-containing proteins under anaerobic conditions. Biophys Chem. 2000 May 31;85(1):1–6. doi: 10.1016/s0301-4622(00)00147-2. [DOI] [PubMed] [Google Scholar]
  3. Chalvardjian A., Rudnicki E. Determination of lipid phosphorus in the nanomolar range. Anal Biochem. 1970 Jul;36(1):225–226. doi: 10.1016/0003-2697(70)90352-0. [DOI] [PubMed] [Google Scholar]
  4. Chen P., Munoz A., Nettesheim D., Shaw C. F., 3rd, Petering D. H. Stoichiometry and cluster specificity of copper binding to metallothionein: homogeneous metal clusters. Biochem J. 1996 Jul 15;317(Pt 2):395–402. doi: 10.1042/bj3170395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen P., Onana P., Shaw C. F., 3rd, Petering D. H. Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometries. Biochem J. 1996 Jul 15;317(Pt 2):389–394. doi: 10.1042/bj3170389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cherian M. G., Apostolova M. D. Nuclear localization of metallothionein during cell proliferation and differentiation. Cell Mol Biol (Noisy-le-grand) 2000 Mar;46(2):347–356. [PubMed] [Google Scholar]
  7. Cook J. A., Kim S. Y., Teague D., Krishna M. C., Pacelli R., Mitchell J. B., Vodovotz Y., Nims R. W., Christodoulou D., Miles A. M. Convenient colorimetric and fluorometric assays for S-nitrosothiols. Anal Biochem. 1996 Jul 1;238(2):150–158. doi: 10.1006/abio.1996.0268. [DOI] [PubMed] [Google Scholar]
  8. Culotta V. C., Klomp L. W., Strain J., Casareno R. L., Krems B., Gitlin J. D. The copper chaperone for superoxide dismutase. J Biol Chem. 1997 Sep 19;272(38):23469–23472. doi: 10.1074/jbc.272.38.23469. [DOI] [PubMed] [Google Scholar]
  9. Dimmeler S., Haendeler J., Nehls M., Zeiher A. M. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J Exp Med. 1997 Feb 17;185(4):601–607. doi: 10.1084/jem.185.4.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  11. Fabisiak J. P., Kagan V. E., Ritov V. B., Johnson D. E., Lazo J. S. Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis. Am J Physiol. 1997 Feb;272(2 Pt 1):C675–C684. doi: 10.1152/ajpcell.1997.272.2.C675. [DOI] [PubMed] [Google Scholar]
  12. Fabisiak J. P., Tyurin V. A., Tyurina Y. Y., Borisenko G. G., Korotaeva A., Pitt B. R., Lazo J. S., Kagan V. E. Redox regulation of copper-metallothionein. Arch Biochem Biophys. 1999 Mar 1;363(1):171–181. doi: 10.1006/abbi.1998.1077. [DOI] [PubMed] [Google Scholar]
  13. Fabisiak J. P., Tyurin V. A., Tyurina Y. Y., Sedlov A., Lazo J. S., Kagan V. E. Nitric oxide dissociates lipid oxidation from apoptosis and phosphatidylserine externalization during oxidative stress. Biochemistry. 2000 Jan 11;39(1):127–138. doi: 10.1021/bi9912544. [DOI] [PubMed] [Google Scholar]
  14. Fabisiak J. P., Tyurina Y. Y., Tyurin V. A., Lazo J. S., Kagan V. E. Random versus selective membrane phospholipid oxidation in apoptosis: role of phosphatidylserine. Biochemistry. 1998 Sep 29;37(39):13781–13790. doi: 10.1021/bi9808262. [DOI] [PubMed] [Google Scholar]
  15. Fernando M. R., Nanri H., Yoshitake S., Nagata-Kuno K., Minakami S. Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells. Eur J Biochem. 1992 Nov 1;209(3):917–922. doi: 10.1111/j.1432-1033.1992.tb17363.x. [DOI] [PubMed] [Google Scholar]
  16. Garrett S. H., Somji S., Todd J. H., Sens D. A. Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA. Environ Health Perspect. 1998 Sep;106(9):587–595. doi: 10.1289/ehp.98106587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glerum D. M., Muroff I., Jin C., Tzagoloff A. COX15 codes for a mitochondrial protein essential for the assembly of yeast cytochrome oxidase. J Biol Chem. 1997 Jul 25;272(30):19088–19094. doi: 10.1074/jbc.272.30.19088. [DOI] [PubMed] [Google Scholar]
  18. Gordge M. P., Meyer D. J., Hothersall J., Neild G. H., Payne N. N., Noronha-Dutra A. Copper chelation-induced reduction of the biological activity of S-nitrosothiols. Br J Pharmacol. 1995 Mar;114(5):1083–1089. doi: 10.1111/j.1476-5381.1995.tb13317.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gravina S. A., Mieyal J. J. Thioltransferase is a specific glutathionyl mixed disulfide oxidoreductase. Biochemistry. 1993 Apr 6;32(13):3368–3376. doi: 10.1021/bi00064a021. [DOI] [PubMed] [Google Scholar]
  20. Gutteridge J. M., Stocks J. Caeruloplasmin: physiological and pathological perspectives. Crit Rev Clin Lab Sci. 1981;14(4):257–329. doi: 10.3109/10408368109105866. [DOI] [PubMed] [Google Scholar]
  21. Halliwell B. Albumin--an important extracellular antioxidant? Biochem Pharmacol. 1988 Feb 15;37(4):569–571. doi: 10.1016/0006-2952(88)90126-8. [DOI] [PubMed] [Google Scholar]
  22. Halliwell B., Gutteridge J. M. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1–85. doi: 10.1016/0076-6879(90)86093-b. [DOI] [PubMed] [Google Scholar]
  23. Jacob C., Maret W., Vallee B. L. Control of zinc transfer between thionein, metallothionein, and zinc proteins. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3489–3494. doi: 10.1073/pnas.95.7.3489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jiang L. J., Maret W., Vallee B. L. The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3483–3488. doi: 10.1073/pnas.95.7.3483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kagan V. E., Ritov V. B., Tyurina Y. Y., Tyurin V. A. Sensitive and specific fluorescent probing of oxidative stress in different classes of membrane phospholipids in live cells using metabolically integrated cis-parinaric acid. Methods Mol Biol. 1998;108:71–87. doi: 10.1385/0-89603-472-0:71. [DOI] [PubMed] [Google Scholar]
  26. Kawai K., Liu S. X., Tyurin V. A., Tyurina Y. Y., Borisenko G. G., Jiang J. F., St Croix C. M., Fabisiak J. P., Pitt B. R., Kagan V. E. Antioxidant and antiapoptotic function of metallothioneins in HL-60 cells challenged with copper nitrilotriacetate. Chem Res Toxicol. 2000 Dec;13(12):1275–1286. doi: 10.1021/tx000119l. [DOI] [PubMed] [Google Scholar]
  27. Kojima H., Nakatsubo N., Kikuchi K., Kawahara S., Kirino Y., Nagoshi H., Hirata Y., Nagano T. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem. 1998 Jul 1;70(13):2446–2453. doi: 10.1021/ac9801723. [DOI] [PubMed] [Google Scholar]
  28. Lazo J. S., Kuo S. M., Woo E. S., Pitt B. R. The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs. Chem Biol Interact. 1998 Apr 24;111-112:255–262. doi: 10.1016/s0009-2797(97)00165-8. [DOI] [PubMed] [Google Scholar]
  29. Lippard S. J. Free copper ions in the cell? Science. 1999 Apr 30;284(5415):748–749. doi: 10.1126/science.284.5415.748. [DOI] [PubMed] [Google Scholar]
  30. Liu S. X., Fabisiak J. P., Tyurin V. A., Borisenko G. G., Pitt B. R., Lazo J. S., Kagan V. E. Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins. Chem Res Toxicol. 2000 Sep;13(9):922–931. doi: 10.1021/tx0000623. [DOI] [PubMed] [Google Scholar]
  31. Ma Y., Cao L., Kawabata T., Yoshino T., Yang B. B., Okada S. Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells. Free Radic Biol Med. 1998 Sep;25(4-5):568–575. doi: 10.1016/s0891-5849(98)00088-4. [DOI] [PubMed] [Google Scholar]
  32. Mallis R. J., Thomas J. A. Effect of S-nitrosothiols on cellular glutathione and reactive protein sulfhydryls. Arch Biochem Biophys. 2000 Nov 1;383(1):60–69. doi: 10.1006/abbi.2000.2048. [DOI] [PubMed] [Google Scholar]
  33. Maret W., Vallee B. L. Thiolate ligands in metallothionein confer redox activity on zinc clusters. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3478–3482. doi: 10.1073/pnas.95.7.3478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Misra R. R., Hochadel J. F., Smith G. T., Cook J. C., Waalkes M. P., Wink D. A. Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein. Chem Res Toxicol. 1996 Jan-Feb;9(1):326–332. doi: 10.1021/tx950109y. [DOI] [PubMed] [Google Scholar]
  35. Nielson K. B., Atkin C. L., Winge D. R. Distinct metal-binding configurations in metallothionein. J Biol Chem. 1985 May 10;260(9):5342–5350. [PubMed] [Google Scholar]
  36. Palmiter R. D. The elusive function of metallothioneins. Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8428–8430. doi: 10.1073/pnas.95.15.8428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pearce L. L., Gandley R. E., Han W., Wasserloos K., Stitt M., Kanai A. J., McLaughlin M. K., Pitt B. R., Levitan E. S. Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):477–482. doi: 10.1073/pnas.97.1.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Percival M. D., Ouellet M., Campagnolo C., Claveau D., Li C. Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid. Biochemistry. 1999 Oct 12;38(41):13574–13583. doi: 10.1021/bi991028u. [DOI] [PubMed] [Google Scholar]
  39. Pufahl R. A., Singer C. P., Peariso K. L., Lin S. J., Schmidt P. J., Fahrni C. J., Culotta V. C., Penner-Hahn J. E., O'Halloran T. V. Metal ion chaperone function of the soluble Cu(I) receptor Atx1. Science. 1997 Oct 31;278(5339):853–856. doi: 10.1126/science.278.5339.853. [DOI] [PubMed] [Google Scholar]
  40. Quesada A. R., Byrnes R. W., Krezoski S. O., Petering D. H. Direct reaction of H2O2 with sulfhydryl groups in HL-60 cells: zinc-metallothionein and other sites. Arch Biochem Biophys. 1996 Oct 15;334(2):241–250. doi: 10.1006/abbi.1996.0452. [DOI] [PubMed] [Google Scholar]
  41. Reed J. C. Cytochrome c: can't live with it--can't live without it. Cell. 1997 Nov 28;91(5):559–562. doi: 10.1016/s0092-8674(00)80442-0. [DOI] [PubMed] [Google Scholar]
  42. Stillman M. J., Cai W., Zelazowski A. J. Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+. J Biol Chem. 1987 Apr 5;262(10):4538–4548. [PubMed] [Google Scholar]
  43. Stubauer G., Giuffrè A., Sarti P. Mechanism of S-nitrosothiol formation and degradation mediated by copper ions. J Biol Chem. 1999 Oct 1;274(40):28128–28133. doi: 10.1074/jbc.274.40.28128. [DOI] [PubMed] [Google Scholar]
  44. Thornberry N. A., Rano T. A., Peterson E. P., Rasper D. M., Timkey T., Garcia-Calvo M., Houtzager V. M., Nordstrom P. A., Roy S., Vaillancourt J. P. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997 Jul 18;272(29):17907–17911. doi: 10.1074/jbc.272.29.17907. [DOI] [PubMed] [Google Scholar]
  45. Toyokuni S., Tanaka T., Nishiyama Y., Okamoto K., Nakashima Y., Hamazaki S., Okada S., Hiai H. Induction of renal cell carcinoma in male Wistar rats treated with cupric nitrilotriacetate. Lab Invest. 1996 Aug;75(2):239–248. [PubMed] [Google Scholar]
  46. Villa P., Kaufmann S. H., Earnshaw W. C. Caspases and caspase inhibitors. Trends Biochem Sci. 1997 Oct;22(10):388–393. doi: 10.1016/s0968-0004(97)01107-9. [DOI] [PubMed] [Google Scholar]
  47. Woo E. S., Dellapiazza D., Wang A. S., Lazo J. S. Energy-dependent nuclear binding dictates metallothionein localization. J Cell Physiol. 2000 Jan;182(1):69–76. doi: 10.1002/(SICI)1097-4652(200001)182:1<69::AID-JCP8>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  48. Yang J., Liu X., Bhalla K., Kim C. N., Ibrado A. M., Cai J., Peng T. I., Jones D. P., Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997 Feb 21;275(5303):1129–1132. doi: 10.1126/science.275.5303.1129. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES