Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1993 Dec;92(6):2761–2767. doi: 10.1172/JCI116894

Glutathione, cell proliferation, and 1,3-bis-(2-chloroethyl)-1-nitrosourea in K562 leukemia.

H Frischer 1, E J Kennedy 1, R Chigurupati 1, M Sivarajan 1
PMCID: PMC288475  PMID: 8254030

Abstract

We have pursued our findings of glutathione reductase (GSSG-R) deficiency and disturbed glutathione in cancer patients treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), by investigating how thiol metabolism, cell proliferation, and the nitrosourea interact in human K562 leukemia. Fasting cells arrested in G greatly increased their reduced glutathione (GSH) in response to growth factors. The rise in thiol began after several hours, peaked before DNA synthesis, and resulted from increased production. BCNU inactivated GSSG-R rapidly, and later retarded, doubled, and greatly prolonged GSH formation before stopping DNA synthesis. Pretreatment unlike post treatment with buthionine-S-R-sulfoximine (BSO) diminished BCNU's ability to block GSSG-R. Enzyme inhibition decreased with falling cellular GSH. In the leukemia system as in vivo, sequential BCNU-induced thiol alterations heralded delayed antiproliferative effects. Drug timing markedly affected both thiol and DNA syntheses. By destroying GSSG-R and delaying the upregulation of thiol synthesis while escalating GSH utilization and requirements, the nitrosourea created a striking and previously unrecognized window of vulnerability for GSH-dependent processes. During this period, altered GSH metabolism could contribute indirectly to BCNU's pleiotropic effects by interfering with DNA alkylation repair, glucose decarboxylation, deoxyribose formation, and possibly by influencing other aspects of proliferation. Acquired GSSG-R deficiency was also an early and sensitive marker for prodrug breakdown and activation.

Full text

PDF
2761

Selected References

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

  1. Ahmad T., Frischer H. Active site-specific inhibition by 1,3-bis(2-chloroethyl)-1-nitrosourea of two genetically homologous flavoenzymes: glutathione reductase and lipoamide dehydrogenase. J Lab Clin Med. 1985 Apr;105(4):464–471. [PubMed] [Google Scholar]
  2. Ali-Osman F., Caughlan J., Gray G. S. Decreased DNA interstrand cross-linking and cytotoxicity induced in human brain tumor cells by 1,3-bis(2-chloroethyl)-1-nitrosourea after in vitro reaction with glutathione. Cancer Res. 1989 Nov 1;49(21):5954–5958. [PubMed] [Google Scholar]
  3. Arrick B. A., Nathan C. F. Glutathione metabolism as a determinant of therapeutic efficacy: a review. Cancer Res. 1984 Oct;44(10):4224–4232. [PubMed] [Google Scholar]
  4. Bannai S. Induction of cystine and glutamate transport activity in human fibroblasts by diethyl maleate and other electrophilic agents. J Biol Chem. 1984 Feb 25;259(4):2435–2440. [PubMed] [Google Scholar]
  5. Bannai S., Tateishi N. Role of membrane transport in metabolism and function of glutathione in mammals. J Membr Biol. 1986;89(1):1–8. doi: 10.1007/BF01870891. [DOI] [PubMed] [Google Scholar]
  6. Brent T. P. Isolation and purification of O6-alkylguanine-DNA alkyltransferase from human leukemic cells. Prevention of chloroethylnitrosourea-induced cross-links by purified enzyme. Pharmacol Ther. 1985;31(1-2):121–140. doi: 10.1016/0163-7258(85)90040-3. [DOI] [PubMed] [Google Scholar]
  7. Chresta C. M., Crook T. R., Souhami R. L. Depletion of cellular glutathione by N,N'-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea as a determinant of sensitivity of K562 human leukemia cells to 4-hydroperoxycyclophosphamide. Cancer Res. 1990 Jul 1;50(13):4067–4071. [PubMed] [Google Scholar]
  8. D'Incalci M., Citti L., Taverna P., Catapano C. V. Importance of the DNA repair enzyme O6-alkyl guanine alkyltransferase (AT) in cancer chemotherapy. Cancer Treat Rev. 1988 Dec;15(4):279–292. doi: 10.1016/0305-7372(88)90026-6. [DOI] [PubMed] [Google Scholar]
  9. DeVita V. T., Denham C., Davidson J. D., Oliverio V. T. The physiological disposition of the carcinostatic 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in man and animals. Clin Pharmacol Ther. 1967 Jul-Aug;8(4):566–577. doi: 10.1002/cpt196784566. [DOI] [PubMed] [Google Scholar]
  10. Dumenco L. L., Allay E., Norton K., Gerson S. L. The prevention of thymic lymphomas in transgenic mice by human O6-alkylguanine-DNA alkyltransferase. Science. 1993 Jan 8;259(5092):219–222. doi: 10.1126/science.8421782. [DOI] [PubMed] [Google Scholar]
  11. Erickson L. C., Laurent G., Sharkey N. A., Kohn K. W. DNA cross-linking and monoadduct repair in nitrosourea-treated human tumour cells. Nature. 1980 Dec 25;288(5792):727–729. doi: 10.1038/288727a0. [DOI] [PubMed] [Google Scholar]
  12. Frischer H., Ahmad T. Consequences of erythrocytic glutathione reductase deficiency. J Lab Clin Med. 1987 May;109(5):583–588. [PubMed] [Google Scholar]
  13. Frischer H., Ahmad T., Nora M. V., Carson P. E., Sivarajan M., Mellovitz R., Ptak L., Parkhurst G. W., Chow H. S., Kaizer H. Biotransformation of primaquine in vitro with human K562 and bone marrow cells. J Lab Clin Med. 1987 Apr;109(4):414–421. [PubMed] [Google Scholar]
  14. Frischer H., Ahmad T. Severe generalized glutathione reductase deficiency after antitumor chemotherapy with BCNU" [1,3-bis(chloroethyl)-1-nitrosourea]. J Lab Clin Med. 1977 May;89(5):1080–1091. [PubMed] [Google Scholar]
  15. Frischer H., Bowman J. E., Carson P. E., Rieckmann K. H., Willerson D., Jr, Colwell E. J. Erythrocytic glutathione reductase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic dehydrogenase deficiencies in populations of the United States, South Vietnam, Iran, and Ethiopia. J Lab Clin Med. 1973 Apr;81(4):603–612. [PubMed] [Google Scholar]
  16. Frischer H., Carson P. E., Bowman J. E., Rieckmann K. H. Visual test for erythrocytic glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, and glutathione reductase deficiencies. J Lab Clin Med. 1973 Apr;81(4):613–624. [PubMed] [Google Scholar]
  17. Frischer H. Erythrocytic glutathione reductase deficiency in a hospital population in the United States. Am J Hematol. 1977;2(4):327–334. doi: 10.1002/ajh.2830020402. [DOI] [PubMed] [Google Scholar]
  18. Frischer H., Mellovitz R. L., Ahmad T., Nora M. V. The conversion of primaquine into primaquine-aldehyde, primaquine-alcohol, and carboxyprimaquine, a major plasma metabolite. J Lab Clin Med. 1991 Jun;117(6):468–476. [PubMed] [Google Scholar]
  19. Gerson S. L., Trey J. E., Miller K. Potentiation of nitrosourea cytotoxicity in human leukemic cells by inactivation of O6-alkylguanine-DNA alkyltransferase. Cancer Res. 1988 Mar 15;48(6):1521–1527. [PubMed] [Google Scholar]
  20. Griffith O. W., Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem. 1979 Aug 25;254(16):7558–7560. [PubMed] [Google Scholar]
  21. Hohl R. J., Kennedy E. J., Frischer H. Defenses against oxidation in human erythrocytes: role of glutathione reductase in the activation of glucose decarboxylation by hemolytic drugs. J Lab Clin Med. 1991 Apr;117(4):325–331. [PubMed] [Google Scholar]
  22. Hollwy R. W., Kiernan J. A. Control of the initiation of DNA synthesis in 3T3 cells: serum factors. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2908–2911. doi: 10.1073/pnas.71.7.2908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Holmgren A. Glutathione-dependent synthesis of deoxyribonucleotides. Purification and characterization of glutaredoxin from Escherichia coli. J Biol Chem. 1979 May 10;254(9):3664–3671. [PubMed] [Google Scholar]
  24. Holmgren A. Regulation of ribonucleotide reductase. Curr Top Cell Regul. 1981;19:47–76. doi: 10.1016/b978-0-12-152819-5.50019-1. [DOI] [PubMed] [Google Scholar]
  25. Jenkinson S. G., Jordan J. M., Lawrence R. A. BCNU-induced protection from hyperbaric hyperoxia: role of glutathione metabolism. J Appl Physiol (1985) 1988 Dec;65(6):2531–2536. doi: 10.1152/jappl.1988.65.6.2531. [DOI] [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Lee S. M., Crowther D., Scarffe J. H., Dougal M., Elder R. H., Rafferty J. A., Margison G. P. Cyclophosphamide decreases O6-alkylguanine-DNA alkyltransferase activity in peripheral lymphocytes of patients undergoing bone marrow transplantation. Br J Cancer. 1992 Aug;66(2):331–336. doi: 10.1038/bjc.1992.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mannervik B. The enzymes of glutathione metabolism: an overview. Biochem Soc Trans. 1987 Aug;15(4):717–718. doi: 10.1042/bst0150717. [DOI] [PubMed] [Google Scholar]
  29. McKenna R., Ahmad T., Ts'ao C. H., Frischer H. Glutathione reductase deficiency and platelet dysfunction induced by 1,3-bis(2-chloroethyl)-1-nitrosourea. J Lab Clin Med. 1983 Jul;102(1):102–115. [PubMed] [Google Scholar]
  30. Meister A., Anderson M. E. Glutathione. Annu Rev Biochem. 1983;52:711–760. doi: 10.1146/annurev.bi.52.070183.003431. [DOI] [PubMed] [Google Scholar]
  31. Meister A. Glutathione deficiency produced by inhibition of its synthesis, and its reversal; applications in research and therapy. Pharmacol Ther. 1991;51(2):155–194. doi: 10.1016/0163-7258(91)90076-x. [DOI] [PubMed] [Google Scholar]
  32. Nathan C. F., Arrick B. A., Murray H. W., DeSantis N. M., Cohn Z. A. Tumor cell anti-oxidant defenses. Inhibition of the glutathione redox cycle enhances macrophage-mediated cytolysis. J Exp Med. 1981 Apr 1;153(4):766–782. doi: 10.1084/jem.153.4.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. O'Driscoll B. R., Hasleton P. S., Taylor P. M., Poulter L. W., Gattameneni H. R., Woodcock A. A. Active lung fibrosis up to 17 years after chemotherapy with carmustine (BCNU) in childhood. N Engl J Med. 1990 Aug 9;323(6):378–382. doi: 10.1056/NEJM199008093230604. [DOI] [PubMed] [Google Scholar]
  34. Pegg A. E. Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 1990 Oct 1;50(19):6119–6129. [PubMed] [Google Scholar]
  35. Reed D. J. Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol. 1990;30:603–631. doi: 10.1146/annurev.pa.30.040190.003131. [DOI] [PubMed] [Google Scholar]
  36. Schallreuter K. U., Gleason F. K., Wood J. M. The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase. Biochim Biophys Acta. 1990 Aug 13;1054(1):14–20. doi: 10.1016/0167-4889(90)90199-n. [DOI] [PubMed] [Google Scholar]
  37. Smith A. C., Boyd M. R. Preferential effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on pulmonary glutathione reductase and glutathione/glutathione disulfide ratios: possible implications for lung toxicity. J Pharmacol Exp Ther. 1984 Jun;229(3):658–663. [PubMed] [Google Scholar]
  38. Suthanthiran M., Anderson M. E., Sharma V. K., Meister A. Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc Natl Acad Sci U S A. 1990 May;87(9):3343–3347. doi: 10.1073/pnas.87.9.3343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969 Mar;27(3):502–522. doi: 10.1016/0003-2697(69)90064-5. [DOI] [PubMed] [Google Scholar]
  40. Tong W. P., Kirk M. C., Ludlum D. B. Formation of the cross-link 1-[N3-deoxycytidyl),2-[N1-deoxyguanosinyl]ethane in DNA treated with N,N'-bis(2-chloroethyl)-N-nitrosourea. Cancer Res. 1982 Aug;42(8):3102–3105. [PubMed] [Google Scholar]
  41. Weiss R. B., Poster D. S., Penta J. S. The nitrosoureas and pulmonary toxicity. Cancer Treat Rev. 1981 Jun;8(2):111–125. doi: 10.1016/s0305-7372(81)80031-x. [DOI] [PubMed] [Google Scholar]
  42. Yarosh D. B., Hurst-Calderone S., Babich M. A., Day R. S., 3rd Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base. Cancer Res. 1986 Apr;46(4 Pt 1):1663–1668. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES