Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1989 Sep;84(3):863–875. doi: 10.1172/JCI114247

Inhibition of tumor cell growth by interferon-gamma is mediated by two distinct mechanisms dependent upon oxygen tension: induction of tryptophan degradation and depletion of intracellular nicotinamide adenine dinucleotide.

T M Aune 1, S L Pogue 1
PMCID: PMC329730  PMID: 2503544

Abstract

Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro. This mechanism is not well understood. The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma. Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h. Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma. The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM. By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan. Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide. Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma. ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD. All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h. Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD. Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT. Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma. The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity. Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma. Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro. Intracellular glutathione has been shown to protect cells against oxidative damage by various agents. Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively. These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth. Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD.

Full text

PDF
863

Selected References

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

  1. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. doi: 10.1016/s0076-6879(84)05016-3. [DOI] [PubMed] [Google Scholar]
  2. Althaus F. R., Lawrence S. D., He Y. Z., Sattler G. L., Tsukada Y., Pitot H. C. Effects of altered [ADP-ribose]n metabolism on expression of fetal functions by adult hepatocytes. Nature. 1982 Nov 25;300(5890):366–368. doi: 10.1038/300366a0. [DOI] [PubMed] [Google Scholar]
  3. Althaus F. R., Lawrence S. D., Sattler G. L., Pitot H. C. ADP-ribosyltransferase activity in cultured hepatocytes. Interactions with DNA repair. J Biol Chem. 1982 May 25;257(10):5528–5535. [PubMed] [Google Scholar]
  4. 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]
  5. Arrick B. A., Nathan C. F., Griffith O. W., Cohn Z. A. Glutathione depletion sensitizes tumor cells to oxidative cytolysis. J Biol Chem. 1982 Feb 10;257(3):1231–1237. [PubMed] [Google Scholar]
  6. Berger S. J., Sudar D. C., Berger N. A. Metabolic consequences of DNA damage: DNA damage induces alterations in glucose metabolism by activation of poly (ADP-ribose) polymerase. Biochem Biophys Res Commun. 1986 Jan 14;134(1):227–232. doi: 10.1016/0006-291x(86)90551-6. [DOI] [PubMed] [Google Scholar]
  7. Borden E. C., Rosenzweig I. B., Byrne G. I. Interferons: from virus inhibitor to modulator of amino acid and lipid metabolism. J Interferon Res. 1987 Oct;7(5):591–596. doi: 10.1089/jir.1987.7.591. [DOI] [PubMed] [Google Scholar]
  8. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  9. Byrne G. I., Lehmann L. K., Kirschbaum J. G., Borden E. C., Lee C. M., Brown R. R. Induction of tryptophan degradation in vitro and in vivo: a gamma-interferon-stimulated activity. J Interferon Res. 1986 Aug;6(4):389–396. doi: 10.1089/jir.1986.6.389. [DOI] [PubMed] [Google Scholar]
  10. Byrne G. I., Lehmann L. K., Landry G. J. Induction of tryptophan catabolism is the mechanism for gamma-interferon-mediated inhibition of intracellular Chlamydia psittaci replication in T24 cells. Infect Immun. 1986 Aug;53(2):347–351. doi: 10.1128/iai.53.2.347-351.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clifford D. P., Repine J. E. Measurement of oxidizing radicals by polymorphonuclear leukocytes. Methods Enzymol. 1984;105:393–398. doi: 10.1016/s0076-6879(84)05054-0. [DOI] [PubMed] [Google Scholar]
  12. De la Harpe J., Nathan C. F. A semi-automated micro-assay for H2O2 release by human blood monocytes and mouse peritoneal macrophages. J Immunol Methods. 1985 Apr 22;78(2):323–336. doi: 10.1016/0022-1759(85)90089-4. [DOI] [PubMed] [Google Scholar]
  13. Denz H., Lechleitner M., Marth C., Daxenbichler G., Gastl G., Braunsteiner H. Effect of human recombinant alpha-2- and gamma-interferon on the growth of human cell lines from solid tumors and hematologic malignancies. J Interferon Res. 1985 Winter;5(1):147–157. doi: 10.1089/jir.1985.5.147. [DOI] [PubMed] [Google Scholar]
  14. Familletti P. C., Rubinstein S., Pestka S. A convenient and rapid cytopathic effect inhibition assay for interferon. Methods Enzymol. 1981;78(Pt A):387–394. doi: 10.1016/0076-6879(81)78146-1. [DOI] [PubMed] [Google Scholar]
  15. Farzaneh F., Zalin R., Brill D., Shall S. DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation. Nature. 1982 Nov 25;300(5890):362–366. doi: 10.1038/300362a0. [DOI] [PubMed] [Google Scholar]
  16. Fiskum G., Craig S. W., Decker G. L., Lehninger A. L. The cytoskeleton of digitonin-treated rat hepatocytes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3430–3434. doi: 10.1073/pnas.77.6.3430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Flohé L., Otting F. Superoxide dismutase assays. Methods Enzymol. 1984;105:93–104. doi: 10.1016/s0076-6879(84)05013-8. [DOI] [PubMed] [Google Scholar]
  18. Folkman J. Tumor angiogenesis. Adv Cancer Res. 1974;19(0):331–358. doi: 10.1016/s0065-230x(08)60058-5. [DOI] [PubMed] [Google Scholar]
  19. Hirata F., Hayaishi O. Possible participation of superoxide anion in the intestinal tryptophan 2,3-dioxygenase reaction. J Biol Chem. 1971 Dec 25;246(24):7825–7826. [PubMed] [Google Scholar]
  20. Johnstone A. P., Williams G. T. Role of DNA breaks and ADP-ribosyl transferase activity in eukaryotic differentiation demonstrated in human lymphocytes. Nature. 1982 Nov 25;300(5890):368–370. doi: 10.1038/300368a0. [DOI] [PubMed] [Google Scholar]
  21. Jones B. N., Gilligan J. P. o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J Chromatogr. 1983 Aug 26;266:471–482. doi: 10.1016/s0021-9673(01)90918-5. [DOI] [PubMed] [Google Scholar]
  22. Jones D. P., Thor H., Andersson B., Orrenius S. Detoxification reactions in isolated hepatocytes. Role of glutathione peroxidase, catalase, and formaldehyde dehydrogenase in reactions relating to N-demethylation by the cytochrome P-450 system. J Biol Chem. 1978 Sep 10;253(17):6031–6037. [PubMed] [Google Scholar]
  23. Knighton D. R., Hunt T. K., Scheuenstuhl H., Halliday B. J., Werb Z., Banda M. J. Oxygen tension regulates the expression of angiogenesis factor by macrophages. Science. 1983 Sep 23;221(4617):1283–1285. doi: 10.1126/science.6612342. [DOI] [PubMed] [Google Scholar]
  24. Kohn K. W., Erickson L. C., Ewig R. A., Friedman C. A. Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry. 1976 Oct 19;15(21):4629–4637. doi: 10.1021/bi00666a013. [DOI] [PubMed] [Google Scholar]
  25. Kuthan H., Tsuji H., Graf H., Ullrich V. Generation of superoxide anion as a source of hydrogen peroxide in a reconstituted monooxygenase system. FEBS Lett. 1978 Jul 15;91(2):343–345. doi: 10.1016/0014-5793(78)81206-x. [DOI] [PubMed] [Google Scholar]
  26. Lengyel P. Enzymology of interferon action--a short survey. Methods Enzymol. 1981;79(Pt B):135–148. [PubMed] [Google Scholar]
  27. McCord J. M., Keele B. B., Jr, Fridovich I. An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc Natl Acad Sci U S A. 1971 May;68(5):1024–1027. doi: 10.1073/pnas.68.5.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meister A. Selective modification of glutathione metabolism. Science. 1983 Apr 29;220(4596):472–477. doi: 10.1126/science.6836290. [DOI] [PubMed] [Google Scholar]
  29. Ozaki Y., Edelstein M. P., Duch D. S. Induction of indoleamine 2,3-dioxygenase: a mechanism of the antitumor activity of interferon gamma. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1242–1246. doi: 10.1073/pnas.85.4.1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pfeffer L. M., Wang E., Landsberger F. R., Tamm I. Assays to measure plasma membrane and cytoskeletal changes in interferon-treated cells. Methods Enzymol. 1981;79(Pt B):461–473. doi: 10.1016/s0076-6879(81)79060-8. [DOI] [PubMed] [Google Scholar]
  31. Pfefferkorn E. R. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan. Proc Natl Acad Sci U S A. 1984 Feb;81(3):908–912. doi: 10.1073/pnas.81.3.908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rice G. C., Bump E. A., Shrieve D. C., Lee W., Kovacs M. Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo. Cancer Res. 1986 Dec;46(12 Pt 1):6105–6110. [PubMed] [Google Scholar]
  33. Rinehart J. J., Malspeis L., Young D., Neidhart J. A. Phase I/II trial of human recombinant interferon gamma in renal cell carcinoma. J Biol Response Mod. 1986 Aug;5(4):300–308. [PubMed] [Google Scholar]
  34. Schraufstatter I. U., Hinshaw D. B., Hyslop P. A., Spragg R. G., Cochrane C. G. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide. J Clin Invest. 1986 Apr;77(4):1312–1320. doi: 10.1172/JCI112436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seto S., Carrera C. J., Kubota M., Wasson D. B., Carson D. A. Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes. J Clin Invest. 1985 Feb;75(2):377–383. doi: 10.1172/JCI111710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Siedlik P. H., Marnett L. J. Oxidizing radical generation by prostaglandin H synthase. Methods Enzymol. 1984;105:412–416. doi: 10.1016/s0076-6879(84)05057-6. [DOI] [PubMed] [Google Scholar]
  37. Sims J. L., Sikorski G. W., Catino D. M., Berger S. J., Berger N. A. Poly(adenosinediphosphoribose) polymerase inhibitors stimulate unscheduled deoxyribonucleic acid synthesis in normal human lymphocytes. Biochemistry. 1982 Apr 13;21(8):1813–1821. doi: 10.1021/bi00537a017. [DOI] [PubMed] [Google Scholar]
  38. Talmadge J. E., Tribble H. R., Pennington R. W., Phillips H., Wiltrout R. H. Immunomodulatory and immunotherapeutic properties of recombinant gamma-interferon and recombinant tumor necrosis factor in mice. Cancer Res. 1987 May 15;47(10):2563–2570. [PubMed] [Google Scholar]
  39. Taniguchi T., Hirata F., Hayaishi O. Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes. J Biol Chem. 1977 Apr 25;252(8):2774–2776. [PubMed] [Google Scholar]
  40. Turco J., Winkler H. H. Gamma-interferon-induced inhibition of the growth of Rickettsia prowazekii in fibroblasts cannot be explained by the degradation of tryptophan or other amino acids. Infect Immun. 1986 Jul;53(1):38–46. doi: 10.1128/iai.53.1.38-46.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ueda K., Hayaishi O. ADP-ribosylation. Annu Rev Biochem. 1985;54:73–100. doi: 10.1146/annurev.bi.54.070185.000445. [DOI] [PubMed] [Google Scholar]
  42. Wong G. H., Goeddel D. V. Induction of manganous superoxide dismutase by tumor necrosis factor: possible protective mechanism. Science. 1988 Nov 11;242(4880):941–944. doi: 10.1126/science.3263703. [DOI] [PubMed] [Google Scholar]
  43. Yoshida R., Hayaishi O. Overview: superoxygenase. Methods Enzymol. 1984;105:61–70. doi: 10.1016/s0076-6879(84)05009-6. [DOI] [PubMed] [Google Scholar]
  44. Yoshida R., Imanishi J., Oku T., Kishida T., Hayaishi O. Induction of pulmonary indoleamine 2,3-dioxygenase by interferon. Proc Natl Acad Sci U S A. 1981 Jan;78(1):129–132. doi: 10.1073/pnas.78.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. de la Maza L. M., Peterson E. M. Dependence of the in vitro antiproliferative activity of recombinant human gamma-interferon on the concentration of tryptophan in culture media. Cancer Res. 1988 Jan 15;48(2):346–350. [PubMed] [Google Scholar]
  46. de la Maza L. M., Peterson E. M., Fennie C. W., Czarniecki C. W. The anti-chlamydial and anti-proliferative activities of recombinant murine interferon-gamma are not dependent on tryptophan concentrations. J Immunol. 1985 Dec;135(6):4198–4200. [PubMed] [Google Scholar]
  47. van der Burg M., Edelstein M., Gerlis L., Liang C. M., Hirschi M., Dawson A. Recombinant interferon-gamma (immuneron): results of a phase I trial in patients with cancer. J Biol Response Mod. 1985 Jun;4(3):264–272. [PubMed] [Google Scholar]

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

RESOURCES