Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1993 May;91(5):2326–2333. doi: 10.1172/JCI116463

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

W Y Gao 1, T Shirasaka 1, D G Johns 1, S Broder 1, H Mitsuya 1
PMCID: PMC288239  PMID: 8387546

Abstract

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy.

Full text

PDF
2326

Selected References

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

  1. Ahluwalia G., Cooney D. A., Mitsuya H., Fridland A., Flora K. P., Hao Z., Dalal M., Broder S., Johns D. G. Initial studies on the cellular pharmacology of 2',3'-dideoxyinosine, an inhibitor of HIV infectivity. Biochem Pharmacol. 1987 Nov 15;36(22):3797–3800. doi: 10.1016/0006-2952(87)90440-0. [DOI] [PubMed] [Google Scholar]
  2. Aoki S., Yarchoan R., Thomas R. V., Pluda J. M., Marczyk K., Broder S., Mitsuya H. Quantitative analysis of HIV-1 proviral DNA in peripheral blood mononuclear cells from patients with AIDS or ARC: decrease of proviral DNA content following treatment with 2',3'-dideoxyinosine (ddI). AIDS Res Hum Retroviruses. 1990 Nov;6(11):1331–1339. doi: 10.1089/aid.1990.6.1331. [DOI] [PubMed] [Google Scholar]
  3. Brockman R. W., Cheng Y. C., Schabel F. M., Jr, Montgomery J. A. Metabolism and chemotherapeutic activity of 9-beta-D-arabinofuranosyl-2-fluoroadenine against murine leukemia L1210 and evidence for its phosphorylation by deoxycytidine kinase. Cancer Res. 1980 Oct;40(10):3610–3615. [PubMed] [Google Scholar]
  4. Bukrinsky M. I., Stanwick T. L., Dempsey M. P., Stevenson M. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science. 1991 Oct 18;254(5030):423–427. doi: 10.1126/science.1925601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cheng N., Traut T. W. Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis. J Cell Biochem. 1987 Nov;35(3):217–229. doi: 10.1002/jcb.240350305. [DOI] [PubMed] [Google Scholar]
  6. Cheng Y. C., Domin B., Lee L. S. Human deoxycytidine kinase. Purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients. Biochim Biophys Acta. 1977 Apr 12;481(2):481–492. doi: 10.1016/0005-2744(77)90281-9. [DOI] [PubMed] [Google Scholar]
  7. Cheng Y. C., Ostrander M. Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization. J Biol Chem. 1976 May 10;251(9):2605–2610. [PubMed] [Google Scholar]
  8. Clark A. G., Holodniy M., Schwartz D. H., Katzenstein D. A., Merigan T. C. Decrease in HIV provirus in peripheral blood mononuclear cells during zidovudine and human rIL-2 administration. J Acquir Immune Defic Syndr. 1992;5(1):52–59. [PubMed] [Google Scholar]
  9. Cooley T. P., Kunches L. M., Saunders C. A., Ritter J. K., Perkins C. J., McLaren C., McCaffrey R. P., Liebman H. A. Once-daily administration of 2',3'-dideoxyinosine (ddI) in patients with the acquired immunodeficiency syndrome or AIDS-related complex. Results of a Phase I trial. N Engl J Med. 1990 May 10;322(19):1340–1345. doi: 10.1056/NEJM199005103221902. [DOI] [PubMed] [Google Scholar]
  10. Cooney D. A., Dalal M., Mitsuya H., McMahon J. B., Nadkarni M., Balzarini J., Broder S., Johns D. G. Initial studies on the cellular pharmacology of 2',3-dideoxycytidine, an inhibitor of HTLV-III infectivity. Biochem Pharmacol. 1986 Jul 1;35(13):2065–2068. doi: 10.1016/0006-2952(86)90571-x. [DOI] [PubMed] [Google Scholar]
  11. De Clercq E. HIV inhibitors targeted at the reverse transcriptase. AIDS Res Hum Retroviruses. 1992 Feb;8(2):119–134. doi: 10.1089/aid.1992.8.119. [DOI] [PubMed] [Google Scholar]
  12. Dolin R., Lambert J. S., Morse G. D., Reichman R. C., Plank C. S., Reid J., Knupp C., McLaren C., Pettinelli C. 2',3'-Dideoxyinosine in patients with AIDS or AIDS-related complex. Rev Infect Dis. 1990 Jul-Aug;12 (Suppl 5):S540–S551. [PubMed] [Google Scholar]
  13. Fischl M. A., Parker C. B., Pettinelli C., Wulfsohn M., Hirsch M. S., Collier A. C., Antoniskis D., Ho M., Richman D. D., Fuchs E. A randomized controlled trial of a reduced daily dose of zidovudine in patients with the acquired immunodeficiency syndrome. The AIDS Clinical Trials Group. N Engl J Med. 1990 Oct 11;323(15):1009–1014. doi: 10.1056/NEJM199010113231501. [DOI] [PubMed] [Google Scholar]
  14. Furman P. A., Fyfe J. A., St Clair M. H., Weinhold K., Rideout J. L., Freeman G. A., Lehrman S. N., Bolognesi D. P., Broder S., Mitsuya H. Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8333–8337. doi: 10.1073/pnas.83.21.8333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harel J., Rassart E., Jolicoeur P. Cell cycle dependence of synthesis of unintegrated viral DNA in mouse cells newly infected with murine leukemia virus. Virology. 1981 Apr 15;110(1):202–207. doi: 10.1016/0042-6822(81)90022-2. [DOI] [PubMed] [Google Scholar]
  16. Hartman N. R., Ahluwalia G. S., Cooney D. A., Mitsuya H., Kageyama S., Fridland A., Broder S., Johns D. G. Inhibitors of IMP dehydrogenase stimulate the phosphorylation of the anti-human immunodeficiency virus nucleosides 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine. Mol Pharmacol. 1991 Jul;40(1):118–124. [PubMed] [Google Scholar]
  17. Huang P., Farquhar D., Plunkett W. Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases. J Biol Chem. 1990 Jul 15;265(20):11914–11918. [PubMed] [Google Scholar]
  18. Johnson M. A., Fridland A. Phosphorylation of 2',3'-dideoxyinosine by cytosolic 5'-nucleotidase of human lymphoid cells. Mol Pharmacol. 1989 Aug;36(2):291–295. [PubMed] [Google Scholar]
  19. Klecker R. W., Jr, Collins J. M., Yarchoan R., Thomas R., Jenkins J. F., Broder S., Myers C. E. Plasma and cerebrospinal fluid pharmacokinetics of 3'-azido-3'-deoxythymidine: a novel pyrimidine analog with potential application for the treatment of patients with AIDS and related diseases. Clin Pharmacol Ther. 1987 Apr;41(4):407–412. doi: 10.1038/clpt.1987.49. [DOI] [PubMed] [Google Scholar]
  20. Lambert J. S., Seidlin M., Reichman R. C., Plank C. S., Laverty M., Morse G. D., Knupp C., McLaren C., Pettinelli C., Valentine F. T. 2',3'-dideoxyinosine (ddI) in patients with the acquired immunodeficiency syndrome or AIDS-related complex. A phase I trial. N Engl J Med. 1990 May 10;322(19):1333–1340. doi: 10.1056/NEJM199005103221901. [DOI] [PubMed] [Google Scholar]
  21. Masood R., Ahluwalia G. S., Cooney D. A., Fridland A., Marquez V. E., Driscoll J. S., Hao Z., Mitsuya H., Perno C. F., Broder S. 2'-Fluoro-2',3'-dideoxyarabinosyladenine: a metabolically stable analogue of the antiretroviral agent 2',3'-dideoxyadenosine. Mol Pharmacol. 1990 Apr;37(4):590–596. [PubMed] [Google Scholar]
  22. Mayers D. L., McCutchan F. E., Sanders-Buell E. E., Merritt L. I., Dilworth S., Fowler A. K., Marks C. A., Ruiz N. M., Richman D. D., Roberts C. R. Characterization of HIV isolates arising after prolonged zidovudine therapy. J Acquir Immune Defic Syndr. 1992;5(8):749–759. [PubMed] [Google Scholar]
  23. McElrath M. J., Steinman R. M., Cohn Z. A. Latent HIV-1 infection in enriched populations of blood monocytes and T cells from seropositive patients. J Clin Invest. 1991 Jan;87(1):27–30. doi: 10.1172/JCI114981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Meng T. C., Fischl M. A., Boota A. M., Spector S. A., Bennett D., Bassiakos Y., Lai S. H., Wright B., Richman D. D. Combination therapy with zidovudine and dideoxycytidine in patients with advanced human immunodeficiency virus infection. A phase I/II study. Ann Intern Med. 1992 Jan 1;116(1):13–20. doi: 10.7326/0003-4819-116-1-13. [DOI] [PubMed] [Google Scholar]
  25. Merigan T. C., Skowron G., Bozzette S. A., Richman D., Uttamchandani R., Fischl M., Schooley R., Hirsch M., Soo W., Pettinelli C. Circulating p24 antigen levels and responses to dideoxycytidine in human immunodeficiency virus (HIV) infections. A phase I and II study. Ann Intern Med. 1989 Feb 1;110(3):189–194. doi: 10.7326/0003-4819-110-3-189. [DOI] [PubMed] [Google Scholar]
  26. Mitsuya H., Jarrett R. F., Matsukura M., Di Marzo Veronese F., DeVico A. L., Sarngadharan M. G., Johns D. G., Reitz M. S., Broder S. Long-term inhibition of human T-lymphotropic virus type III/lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2',3'-dideoxynucleosides in vitro. Proc Natl Acad Sci U S A. 1987 Apr;84(7):2033–2037. doi: 10.1073/pnas.84.7.2033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mitsuya H., Yarchoan R., Broder S. Molecular targets for AIDS therapy. Science. 1990 Sep 28;249(4976):1533–1544. doi: 10.1126/science.1699273. [DOI] [PubMed] [Google Scholar]
  28. Munch-Petersen B., Tyrsted G., Dupont B. The deoxyribonucleoside 5'-triphosphate (dATP and dTTP) pool in phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Exp Cell Res. 1973 Jun;79(2):249–256. doi: 10.1016/0014-4827(73)90442-4. [DOI] [PubMed] [Google Scholar]
  29. Perno C. F., Yarchoan R., Cooney D. A., Hartman N. R., Webb D. S., Hao Z., Mitsuya H., Johns D. G., Broder S. Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido-2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine. J Exp Med. 1989 Mar 1;169(3):933–951. doi: 10.1084/jem.169.3.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schinazi R. F., Sommadossi J. P., Saalmann V., Cannon D. L., Xie M. Y., Hart G. C., Smith G. A., Hahn E. F. Activities of 3'-azido-3'-deoxythymidine nucleotide dimers in primary lymphocytes infected with human immunodeficiency virus type 1. Antimicrob Agents Chemother. 1990 Jun;34(6):1061–1067. doi: 10.1128/aac.34.6.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sherman P. A., Fyfe J. A. Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers. Anal Biochem. 1989 Aug 1;180(2):222–226. doi: 10.1016/0003-2697(89)90420-x. [DOI] [PubMed] [Google Scholar]
  32. Shirasaka T., Yarchoan R., O'Brien M. C., Husson R. N., Anderson B. D., Kojima E., Shimada T., Broder S., Mitsuya H. Changes in drug sensitivity of human immunodeficiency virus type 1 during therapy with azidothymidine, dideoxycytidine, and dideoxyinosine: an in vitro comparative study. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):562–566. doi: 10.1073/pnas.90.2.562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Törnevik Y., Jacobsson B., Britton S., Eriksson S. Intracellular metabolism of 3'-azidothymidine in isolated human peripheral blood mononuclear cells. AIDS Res Hum Retroviruses. 1991 Sep;7(9):751–759. doi: 10.1089/aid.1991.7.751. [DOI] [PubMed] [Google Scholar]
  34. Varmus H. E., Padgett T., Heasley S., Simon G., Bishop J. M. Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell. 1977 Jun;11(2):307–319. doi: 10.1016/0092-8674(77)90047-2. [DOI] [PubMed] [Google Scholar]
  35. Yarchoan R., Mitsuya H., Myers C. E., Broder S. Clinical pharmacology of 3'-azido-2',3'-dideoxythymidine (zidovudine) and related dideoxynucleosides. N Engl J Med. 1989 Sep 14;321(11):726–738. doi: 10.1056/NEJM198909143211106. [DOI] [PubMed] [Google Scholar]
  36. Yarchoan R., Mitsuya H., Thomas R. V., Pluda J. M., Hartman N. R., Perno C. F., Marczyk K. S., Allain J. P., Johns D. G., Broder S. In vivo activity against HIV and favorable toxicity profile of 2',3'-dideoxyinosine. Science. 1989 Jul 28;245(4916):412–415. doi: 10.1126/science.2502840. [DOI] [PubMed] [Google Scholar]
  37. Yarchoan R., Perno C. F., Thomas R. V., Klecker R. W., Allain J. P., Wills R. J., McAtee N., Fischl M. A., Dubinsky R., McNeely M. C. Phase I studies of 2',3'-dideoxycytidine in severe human immunodeficiency virus infection as a single agent and alternating with zidovudine (AZT). Lancet. 1988 Jan 16;1(8577):76–81. doi: 10.1016/s0140-6736(88)90283-8. [DOI] [PubMed] [Google Scholar]
  38. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]
  39. Zack J. A., Haislip A. M., Krogstad P., Chen I. S. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. J Virol. 1992 Mar;66(3):1717–1725. doi: 10.1128/jvi.66.3.1717-1725.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES