Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1991 Oct 1;174(4):761–767. doi: 10.1084/jem.174.4.761

Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide

PMCID: PMC2118959  PMID: 1717630

Abstract

Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate- limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega- methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D- arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.

Full Text

The Full Text of this article is available as a PDF (750.8 KB).

Selected References

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

  1. Adamson R. H. Hydroxyguanidine--a new antitumour drug. Nature. 1972 Apr 21;236(5347):400–401. doi: 10.1038/236400a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Cory J. G., Carter G. L. Leukemia L1210 cell lines resistant to ribonucleotide reductase inhibitors. Cancer Res. 1988 Feb 15;48(4):839–843. [PubMed] [Google Scholar]
  4. Cory J. G., Mansell M. M., George C. B., Wilkinson D. S. Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid. Arch Biochem Biophys. 1974 Feb;160(2):495–503. doi: 10.1016/0003-9861(74)90426-3. [DOI] [PubMed] [Google Scholar]
  5. Crawford C. R., Ng C. Y., Noel L. D., Belt J. A. Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters. J Biol Chem. 1990 Jun 15;265(17):9732–9736. [PubMed] [Google Scholar]
  6. Ding A. H., Nathan C. F., Stuehr D. J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol. 1988 Oct 1;141(7):2407–2412. [PubMed] [Google Scholar]
  7. Drapier J. C., Hibbs J. B., Jr Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible. J Clin Invest. 1986 Sep;78(3):790–797. doi: 10.1172/JCI112642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fontecave M., Eliasson R., Reichard P. Enzymatic regulation of the radical content of the small subunit of Escherichia coli ribonucleotide reductase involving reduction of its redox centers. J Biol Chem. 1989 Jun 5;264(16):9164–9170. [PubMed] [Google Scholar]
  9. Fontecave M., Gerez C., Mansuy D., Reichard P. Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol. J Biol Chem. 1990 Jul 5;265(19):10919–10924. [PubMed] [Google Scholar]
  10. Granger D. L., Lehninger A. L. Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J Cell Biol. 1982 Nov;95(2 Pt 1):527–535. doi: 10.1083/jcb.95.2.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S., Tannenbaum S. R. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982 Oct;126(1):131–138. doi: 10.1016/0003-2697(82)90118-x. [DOI] [PubMed] [Google Scholar]
  12. Gross S. S., Stuehr D. J., Aisaka K., Jaffe E. A., Levi R., Griffith O. W. Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine. Biochem Biophys Res Commun. 1990 Jul 16;170(1):96–103. doi: 10.1016/0006-291x(90)91245-n. [DOI] [PubMed] [Google Scholar]
  13. Gräslund A., Ehrenberg A., Thelander L. Characterization of the free radical of mammalian ribonucleotide reductase. J Biol Chem. 1982 May 25;257(10):5711–5715. [PubMed] [Google Scholar]
  14. Hibbs J. B., Jr, Taintor R. R., Vavrin Z. Iron depletion: possible cause of tumor cell cytotoxicity induced by activated macrophages. Biochem Biophys Res Commun. 1984 Sep 17;123(2):716–723. doi: 10.1016/0006-291x(84)90288-2. [DOI] [PubMed] [Google Scholar]
  15. Hibbs J. B., Jr, Taintor R. R., Vavrin Z. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science. 1987 Jan 23;235(4787):473–476. doi: 10.1126/science.2432665. [DOI] [PubMed] [Google Scholar]
  16. Hibbs J. B., Jr, Taintor R. R., Vavrin Z., Rachlin E. M. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun. 1988 Nov 30;157(1):87–94. doi: 10.1016/s0006-291x(88)80015-9. [DOI] [PubMed] [Google Scholar]
  17. Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1989 Aug 25;264(24):13963–13966. [PubMed] [Google Scholar]
  18. Ignarro L. J., Gruetter C. A. Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite: possible involvement of S-nitrosothiols. Biochim Biophys Acta. 1980 Aug 13;631(2):221–231. doi: 10.1016/0304-4165(80)90297-4. [DOI] [PubMed] [Google Scholar]
  19. Iyengar R., Stuehr D. J., Marletta M. A. Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6369–6373. doi: 10.1073/pnas.84.18.6369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kosaka H., Wishnok J. S., Miwa M., Leaf C. D., Tannenbaum S. R. Nitrosation by stimulated macrophages. Inhibitors, enhancers and substrates. Carcinogenesis. 1989 Mar;10(3):563–566. doi: 10.1093/carcin/10.3.563. [DOI] [PubMed] [Google Scholar]
  21. Kwon N. S., Nathan C. F., Stuehr D. J. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J Biol Chem. 1989 Dec 5;264(34):20496–20501. [PubMed] [Google Scholar]
  22. Lagergren J., Reichard P. Purine deoxyribonucleosides counteract effects of hydroxyurea on deoxyribonucleoside triphosphate pools and DNA synthesis. Biochem Pharmacol. 1987 Sep 15;36(18):2985–2991. doi: 10.1016/0006-2952(87)90213-9. [DOI] [PubMed] [Google Scholar]
  23. Lancaster J. R., Jr, Hibbs J. B., Jr EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1223–1227. doi: 10.1073/pnas.87.3.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lepoivre M., Chenais B., Yapo A., Lemaire G., Thelander L., Tenu J. P. Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells. J Biol Chem. 1990 Aug 25;265(24):14143–14149. [PubMed] [Google Scholar]
  25. Marletta M. A., Yoon P. S., Iyengar R., Leaf C. D., Wishnok J. S. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry. 1988 Nov 29;27(24):8706–8711. doi: 10.1021/bi00424a003. [DOI] [PubMed] [Google Scholar]
  26. Nakagawara A., Nathan C. F. A simple method for counting adherent cells: application to cultured human monocytes, macrophages and multinucleated giant cells. J Immunol Methods. 1983 Jan 28;56(2):261–268. doi: 10.1016/0022-1759(83)90418-0. [DOI] [PubMed] [Google Scholar]
  27. Ochiai E., Mann G. J., Gräslund A., Thelander L. Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase. J Biol Chem. 1990 Sep 15;265(26):15758–15761. [PubMed] [Google Scholar]
  28. Pellat C., Henry Y., Drapier J. C. IFN-gamma-activated macrophages: detection by electron paramagnetic resonance of complexes between L-arginine-derived nitric oxide and non-heme iron proteins. Biochem Biophys Res Commun. 1990 Jan 15;166(1):119–125. doi: 10.1016/0006-291x(90)91919-j. [DOI] [PubMed] [Google Scholar]
  29. Reichard P., Ehrenberg A. Ribonucleotide reductase--a radical enzyme. Science. 1983 Aug 5;221(4610):514–519. doi: 10.1126/science.6306767. [DOI] [PubMed] [Google Scholar]
  30. Reichard P. Interactions between deoxyribonucleotide and DNA synthesis. Annu Rev Biochem. 1988;57:349–374. doi: 10.1146/annurev.bi.57.070188.002025. [DOI] [PubMed] [Google Scholar]
  31. Steeper J. R., Steuart C. D. A rapid assay for CDP reductase activity in mammalian cell extracts. Anal Biochem. 1970 Mar;34:123–130. doi: 10.1016/0003-2697(70)90092-8. [DOI] [PubMed] [Google Scholar]
  32. Stubbe J. Ribonucleotide reductases: amazing and confusing. J Biol Chem. 1990 Apr 5;265(10):5329–5332. [PubMed] [Google Scholar]
  33. Stuehr D. J., Gross S. S., Sakuma I., Levi R., Nathan C. F. Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide. J Exp Med. 1989 Mar 1;169(3):1011–1020. doi: 10.1084/jem.169.3.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stuehr D. J., Kwon N. S., Gross S. S., Thiel B. A., Levi R., Nathan C. F. Synthesis of nitrogen oxides from L-arginine by macrophage cytosol: requirement for inducible and constitutive components. Biochem Biophys Res Commun. 1989 Jun 15;161(2):420–426. doi: 10.1016/0006-291x(89)92615-6. [DOI] [PubMed] [Google Scholar]
  35. Stuehr D. J., Kwon N. S., Nathan C. F. FAD and GSH participate in macrophage synthesis of nitric oxide. Biochem Biophys Res Commun. 1990 Apr 30;168(2):558–565. doi: 10.1016/0006-291x(90)92357-6. [DOI] [PubMed] [Google Scholar]
  36. Stuehr D. J., Kwon N. S., Nathan C. F., Griffith O. W., Feldman P. L., Wiseman J. N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine. J Biol Chem. 1991 Apr 5;266(10):6259–6263. [PubMed] [Google Scholar]
  37. Stuehr D. J., Marletta M. A. Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma. J Immunol. 1987 Jul 15;139(2):518–525. [PubMed] [Google Scholar]
  38. Stuehr D. J., Marletta M. A. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7738–7742. doi: 10.1073/pnas.82.22.7738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stuehr D. J., Nathan C. F. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med. 1989 May 1;169(5):1543–1555. doi: 10.1084/jem.169.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tai A. W., Lien E. J., Moore E. C., Chun Y., Roberts J. D. Studies of N-hydroxy-N'-aminoguanidine derivatives by nitrogen-15 nuclear magnetic resonance spectroscopy and as ribonucleotide reductase inhibitors. J Med Chem. 1983 Sep;26(9):1326–1329. doi: 10.1021/jm00363a021. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES