Skip to main content
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1986 Nov;78(5):1261–1269. doi: 10.1172/JCI112710

Deoxyadenosine triphosphate as a mediator of deoxyguanosine toxicity in cultured T lymphoblasts.

G J Mann, R M Fox
PMCID: PMC423812  PMID: 3490493

Abstract

The mechanism by which 2'-deoxyguanosine is toxic for lymphoid cells is relevant both to the severe cellular immune defect of inherited purine nucleoside phosphorylase (PNP) deficiency and to attempts to exploit PNP inhibitors therapeutically. We have studied the cell cycle and biochemical effects of 2'-deoxyguanosine in human lymphoblasts using the PNP inhibitor 8-aminoguanosine. We show that cytostatic 2'-deoxyguanosine concentrations cause G1-phase arrest in PNP-inhibited T lymphoblasts, regardless of their hypoxanthine guanine phosphoribosyltransferase status. This effect is identical to that produced by 2'-deoxyadenosine in adenosine deaminase-inhibited T cells. 2'-Deoxyguanosine elevates both the 2'-deoxyguanosine-5'-triphosphate (dGTP) and 2'-deoxyadenosine-5'-triphosphate (dATP) pools; subsequently pyrimidine deoxyribonucleotide pools are depleted. The time course of these biochemical changes indicates that the onset of G1-phase arrest is related to increase of the dATP rather than the dGTP pool. When dGTP elevation is dissociated from dATP elevation by coincubation with 2'-deoxycytidine, dGTP does not by itself interrupt transit from the G1 to the S phase. It is proposed that dATP can mediate both 2'-deoxyguanosine and 2'-deoxyadenosine toxicity in T lymphoblasts.

Full text

PDF
1261

Selected References

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

  1. Carson D. A., Kaye J., Matsumoto S., Seegmiller J. E., Thompson L. Biochemical basis for the enhanced toxicity of deoxyribonucleosides toward malignant human T cell lines. Proc Natl Acad Sci U S A. 1979 May;76(5):2430–2433. doi: 10.1073/pnas.76.5.2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carson D. A., Wasson D. B., Lakow E., Kamatani N. Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3848–3852. doi: 10.1073/pnas.79.12.3848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chan T. S. Deoxyguanosine toxicity on lymphoid cells as a cause for immunosuppression in purine nucleoside phosphorylase deficiency. Cell. 1978 Jul;14(3):523–530. doi: 10.1016/0092-8674(78)90238-6. [DOI] [PubMed] [Google Scholar]
  4. Chang C. H., Cheng Y. C. Substrate specificity of human ribonucleotide reductase from Molt-4F cells. Cancer Res. 1979 Dec;39(12):5081–5086. [PubMed] [Google Scholar]
  5. Cohen A., Barankiewicz J., Lederman H. M., Gelfand E. W. Purine and pyrimidine metabolism in human T lymphocytes. Regulation of deoxyribonucleotide metabolism. J Biol Chem. 1983 Oct 25;258(20):12334–12340. [PubMed] [Google Scholar]
  6. Cohen A., Doyle D., Martin D. W., Jr, Ammann A. J. Abnormal purine metabolism and purine overproduction in a patient deficient in purine nucleoside phosphorylase. N Engl J Med. 1976 Dec 23;295(26):1449–1454. doi: 10.1056/NEJM197612232952603. [DOI] [PubMed] [Google Scholar]
  7. Cohen A., Gudas L. J., Ammann A. J., Staal G. E., Martin D. W., Jr Deoxyguanosine triphosphate as a possible toxic metabolite in the immunodeficiency associated with purine nucleoside phosphorylase deficiency. J Clin Invest. 1978 May;61(5):1405–1409. doi: 10.1172/JCI109058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen A., Hirschhorn R., Horowitz S. D., Rubinstein A., Polmar S. H., Hong R., Martin D. W., Jr Deoxyadenosine triphosphate as a potentially toxic metabolite in adenosine deaminase deficiency. Proc Natl Acad Sci U S A. 1978 Jan;75(1):472–476. doi: 10.1073/pnas.75.1.472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fox R. M., Kefford R. F., Tripp E. H., Taylor I. W. G1-phase arrest of cultured human leukemic T-cells induced by deoxyadenosine. Cancer Res. 1981 Dec;41(12 Pt 1):5141–5150. [PubMed] [Google Scholar]
  10. Fox R. M., Tripp E. H., Piddington S. K., Tattersall M. H. Sensitivity of leukemic human null lymphocytes to deoxynucleosides. Cancer Res. 1980 Sep;40(9):3383–3386. [PubMed] [Google Scholar]
  11. Fox R. M., Tripp E. H., Taylor I. W. Analytical DNA flow cytometric analysis of deoxyadenosine toxicity in cultured human leukemic lymphoblasts. Mol Pharmacol. 1984 Sep;26(2):388–394. [PubMed] [Google Scholar]
  12. Giblett E. R., Anderson J. E., Cohen F., Pollara B., Meuwissen H. J. Adenosine-deaminase deficiency in two patients with severely impaired cellular immunity. Lancet. 1972 Nov 18;2(7786):1067–1069. doi: 10.1016/s0140-6736(72)92345-8. [DOI] [PubMed] [Google Scholar]
  13. Grever M. R., Siaw M. F., Jacob W. F., Neidhart J. A., Miser J. S., Coleman M. S., Hutton J. J., Balcerzak S. P. The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy. Blood. 1981 Mar;57(3):406–417. [PubMed] [Google Scholar]
  14. Gudas L. J., Ullman B., Cohen A., Martin D. W., Jr Deoxyguanosine toxicity in a mouse T lymphoma: relationship to purine nucleoside phosphorylase-associated immune dysfunction. Cell. 1978 Jul;14(3):531–538. doi: 10.1016/0092-8674(78)90239-8. [DOI] [PubMed] [Google Scholar]
  15. Henderson J. F., Brox L., Zombor G., Hunting D., Lomax C. A. Specificity of adenosine deaminase inhibitors. Biochem Pharmacol. 1977 Nov 1;26(21):1967–1972. doi: 10.1016/0006-2952(77)90003-x. [DOI] [PubMed] [Google Scholar]
  16. Henderson J. F., Scott F. W., Lowe J. K. Toxicity of naturally occurring purine deoxyribonucleosides. Pharmacol Ther. 1980;8(3):573–604. doi: 10.1016/0163-7258(80)90077-7. [DOI] [PubMed] [Google Scholar]
  17. Kazmers I. S., Mitchell B. S., Dadonna P. E., Wotring L. L., Townsend L. B., Kelley W. N. Inhibition of purine nucleoside phosphorylase by 8-aminoguanosine: selective toxicity for T lymphoblasts. Science. 1981 Dec 4;214(4525):1137–1139. doi: 10.1126/science.6795718. [DOI] [PubMed] [Google Scholar]
  18. Kefford R. F., Fox R. M., McCairns E., Fahey D., Muscat G. E., Rowe P. B. Terminal incorporation of 2'-deoxyadenosine into polyadenylate segments of polyadenylated RNA in G1-phase-arrested human T-lymphoblasts. Cancer Res. 1983 May;43(5):2252–2257. [PubMed] [Google Scholar]
  19. Kefford R. F., Fox R. M. Purine deoxynucleoside toxicity in nondividing human lymphoid cells. Cancer Res. 1982 Jan;42(1):324–330. [PubMed] [Google Scholar]
  20. Matsumoto S. S., Yu J., Yu A. L. Inhibition of RNA synthesis by deoxyadenosine plus deoxycoformycin in resting lymphocytes. J Immunol. 1983 Dec;131(6):2762–2766. [PubMed] [Google Scholar]
  21. Meuth M., Aufreiter E., Reichard P. Deoxyribonucleotide pools in mouse-fibroblast cell lines with altered ribonucleotide reductase. Eur J Biochem. 1976 Dec;71(1):39–43. doi: 10.1111/j.1432-1033.1976.tb11087.x. [DOI] [PubMed] [Google Scholar]
  22. Milthorpe B. FMFPAK1: a program package for routine analysis of single parameter flow microfluorimetric data on a low cost mini-computer. Comput Biomed Res. 1980 Oct;13(5):417–429. doi: 10.1016/0010-4809(80)90039-7. [DOI] [PubMed] [Google Scholar]
  23. Mitchell B. S., Kelley W. N. Purinogenic immunodeficiency diseases: clinical features and molecular mechanisms. Ann Intern Med. 1980 Jun;92(6):826–831. doi: 10.7326/0003-4819-92-6-826. [DOI] [PubMed] [Google Scholar]
  24. Mitchell B. S., Mejias E., Daddona P. E., Kelley W. N. Purinogenic immunodeficiency diseases: selective toxicity of deoxyribonucleosides for T cells. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5011–5014. doi: 10.1073/pnas.75.10.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Prem veer Reddy G., Pardee A. B. Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3312–3316. doi: 10.1073/pnas.77.6.3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Prentice H. G., Russell N. H., Lee N., Ganeshaguru K., Blacklock H., Piga A., Smyth J. F., Hoffbrand A. V. Therapeutic selectivity of and predication of response to 2'-deoxycoformycin in acute leukaemia. Lancet. 1981 Dec 5;2(8258):1250–1254. doi: 10.1016/s0140-6736(81)91491-4. [DOI] [PubMed] [Google Scholar]
  27. Reichard P. From deoxynucleotides to DNA synthesis. Fed Proc. 1978 Jan;37(1):9–14. [PubMed] [Google Scholar]
  28. 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]
  29. Sidi Y., Hudson J. L., Mitchell B. S. Effects of guanine ribonucleotide accumulation on the metabolism and cell cycle of human lymphoid cells. Cancer Res. 1985 Oct;45(10):4940–4945. [PubMed] [Google Scholar]
  30. Sidi Y., Mitchell B. S. 2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation. J Clin Invest. 1984 Nov;74(5):1640–1648. doi: 10.1172/JCI111580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Simmonds H. A., Goday A., Morris G. S., Brolsma M. F. Metabolism of deoxynucleosides by lymphocytes in long-term culture deficient in different purine enzymes. Biochem Pharmacol. 1984 Mar 1;33(5):763–770. doi: 10.1016/0006-2952(84)90460-x. [DOI] [PubMed] [Google Scholar]
  32. Solter A. W., Handschumacher R. E. A rapid quantitative determination of deoxyribonucleoside triphosphates based on the enzymatic synthesis of DNA. Biochim Biophys Acta. 1969 Feb 18;174(2):585–590. doi: 10.1016/0005-2787(69)90288-3. [DOI] [PubMed] [Google Scholar]
  33. Spaapen L. J., Rijkers G. T., Staal G. E., Rijksen G., Wadman S. K., Stoop J. W., Zegers B. J. The effect of deoxyguanosine on human lymphocyte function. I. Analysis of the interference with lymphocyte proliferation in vitro. J Immunol. 1984 May;132(5):2311–2317. [PubMed] [Google Scholar]
  34. Stoeckler J. D., Cambor C., Kuhns V., Chu S. H., Parks R. E., Jr Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions. Biochem Pharmacol. 1982 Jan 15;31(2):163–171. doi: 10.1016/0006-2952(82)90206-4. [DOI] [PubMed] [Google Scholar]
  35. Stoop J. W., Zegers B. J., Hendrickx G. F., van Heukelom L. H., Staal G. E., de Bree P. K., Wadman S. K., Ballieux R. E. Purine nucleoside phosphorylase deficiency associated with selective cellular immunodeficiency. N Engl J Med. 1977 Mar 24;296(12):651–655. doi: 10.1056/NEJM197703242961203. [DOI] [PubMed] [Google Scholar]
  36. Suhadolnik R. J., Baur R., Lichtenwalner D. M., Uematsu T., Roberts J. H., Sudhakar S., Smulson M. ADP-ribosylation of isolated nuclei from HeLa cells, rat liver, fetal rat liver, and Novikoff hepatoma. Effect of nicotinamide adenine dinucleotide analogs on template activity for DNA synthesis, incorporation into nuclear proteins, and a new 1''-3' osidic linkage. J Biol Chem. 1977 Jun 25;252(12):4134–4144. [PubMed] [Google Scholar]
  37. Tattersall M. H., Lavoie A., Ganeshaguru K., Tripp E., Hoffbrand A. V. Deoxyribonucleoside triphosphates in human cells: changes in disease and following exposure to drugs. Eur J Clin Invest. 1975 Apr;5(2):191–202. doi: 10.1111/j.1365-2362.1975.tb00446.x. [DOI] [PubMed] [Google Scholar]
  38. Taylor I. W., Milthorpe B. K. An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations. J Histochem Cytochem. 1980 Nov;28(11):1224–1232. doi: 10.1177/28.11.6159392. [DOI] [PubMed] [Google Scholar]
  39. Theiss J. C., Morris N. R., Fischer G. A. Pyrimidine nucleotide metabolism in L5178Y murine leukemia cells: deoxycytidine protection from deoxyguanosine toxicity. Cancer Biochem Biophys. 1976 May;1(4):211–214. [PubMed] [Google Scholar]
  40. Thelander L., Eriksson S., Akerman M. Ribonucleotide reductase from calf thymus. Separation of the enzyme into two nonidentical subunits, proteins M1 and M2. J Biol Chem. 1980 Aug 10;255(15):7426–7432. [PubMed] [Google Scholar]
  41. Thelander L., Reichard P. Reduction of ribonucleotides. Annu Rev Biochem. 1979;48:133–158. doi: 10.1146/annurev.bi.48.070179.001025. [DOI] [PubMed] [Google Scholar]
  42. Ullman B., Clift S. M., Gudas L. J., Levinson B. B., Wormsted M. A., Martin D. W., Jr Alterations in deoxyribonucleotide metabolism in cultured cells with ribonucleotide reductase activities refractory to feedback inhibition by 2'-deoxyadenosine triphosphate. J Biol Chem. 1980 Sep 10;255(17):8308–8314. [PubMed] [Google Scholar]
  43. Ullman B., Gudas L. J., Caras I. W., Eriksson S., Weinberg G. L., Wormsted M. A., Martin D. W., Jr Demonstration of normal and mutant protein M1 subunits of deoxyGTP-resistant ribonucleotide reductase from mutant mouse lymphoma cells. J Biol Chem. 1981 Oct 10;256(19):10189–10192. [PubMed] [Google Scholar]
  44. Ullman B., Gudas L. J., Clift S. M., Martin D. W., Jr Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: genetic model for immunodeficiency disease. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1074–1078. doi: 10.1073/pnas.76.3.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Waddell D., Ullman B. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency. J Biol Chem. 1983 Apr 10;258(7):4226–4231. [PubMed] [Google Scholar]
  46. Wickremasinghe R. G., Yaxley J. C., Hoffbrand A. V. Solubilization and partial characterization of a multienzyme complex of DNA synthesis from human lymphoblastoid cells. Eur J Biochem. 1982 Sep 1;126(3):589–596. doi: 10.1111/j.1432-1033.1982.tb06821.x. [DOI] [PubMed] [Google Scholar]
  47. Wilson J. M., Mitchell B. S., Daddona P. E., Kelley W. N. Purinogenic immunodeficiency diseases. Differential effects of deoxyadenosine and deoxyguanosine on DNA synthesis in human T lymphoblasts. J Clin Invest. 1979 Nov;64(5):1475–1484. doi: 10.1172/JCI109606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zamecnik P. Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A): its role in cellular metabolism. Anal Biochem. 1983 Oct 1;134(1):1–10. doi: 10.1016/0003-2697(83)90255-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES