Effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (zidovudine) in human mononuclear cells

Abstract

The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed [3H]AZT phosphorylation in the same concentration range (20 to 100 ,uM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.The antiviral activities of 3'-azido-3'-deoxythymidine (AZT; zidovudine) and other 2',3'-dideoxynucleoside drugs in human immunodeficiency virus (HIV)-infected cells is thought to depend on three main factors: (i) the efficiency of phosphorylation to the triphosphate form, (ii) the efficiency with which the triphosphates inhibit viral reverse transcriptase, and (iii) the influence of physiological nucleosides on the former two processes (8). Concerning the last effect, it has been shown in ATH8 cells (9) and peripheral blood mononuclear cells (PBMC) (11) that thymidine (dThd) counteracts the antiviral activity of AZT. Uridine (Urd), on the other hand, was found not to affect the antiviral activity of AZT, although it antagonized the cytotoxic effect of AZT on bone marrow progenitor cells in a granulocyte-macrophage CFU assay (11). On this basis, Urd was suggested to have potential use in the treatment of human immunodeficiency virus (HIV) infections as a rescue from the hematologic side effects of AZT (11).To help understand the molecular basis of the abovedescribed phenomena, we asked in the present study whether the different effects of dThd and Urd on the anti-HIV action of AZT in PBMC and on the cytotoxicity of AZT in bone marrow mononuclear cells (BMMC) could be correlated with the effects of these nucleosides on the phosphorylation of AZT in these cells.Since monocytes constitute 20 to 30% of PBMC and since nucleoside metabolism is thought to differ between lymphoid cells and monocyte-macrophage lineage cells, purified monocytes and monocyte-derived macrophages (M/M) were subjected to separate analyses as well.PBMC were prepared from blood freshly drawn from healthy volunteers, and BMMC were prepared from a bone marrow aspirate from a healthy donor by Ficoll-Paque gradient centrifugation (5). Washed mononuclear cells were suspended in Dulbecco's modified Eagle's medium supplemented with L-glutamine, 10% heat-inactivated fetal calf serum, 4.5 g of D-glucose per liter, and antibiotics (D-MEM). Monocytes were separated from PBMC by counterflow centrifugal elutriation (16,17) and suspended in D-MEM (-3 x 107/ml). To prepare M/M, we plated monocytes in six-well * Corresponding author.Costar plates (5 x 106 cells per well) and cultured them for 1 day before starting the experiment.[3H]AZT (20 Ci/mmol) was from Moravek Biochemicals (Brea, Calif.). To measure AZT phosphoryl...

In vitroanti-HIV and Cytotoxicological Evaluation of the Triple Combination: AZT and ddC with HIV Proteinase Inhibitor Saquinavir (Ro 31-8959)

“…Thus further mutation of RT may be required to produce resistance to both AZT and ddC. Furthermore, the nucleoside analogues do not compete for phosphorylation since different kinases are involved (Cooney et a/., 1986, Furman et a/., 1986Starnes and Cheng, 1987), but both may compete with their corresponding natural substrates for phosphorylation (Balzarini et a/., 1987;Szebeni et a/., 1991). Thus the AZT/ddC combination may provide a number of different interactions and effects which operate at the same, early stages of the virus replicative cycle.…”

Cellular Pharmacology and Potency of HIV-1 Nucleoside Analogs in Primary Human Macrophages

“…A previous report by Szebeni et al (13) measured concentrations of zidovudine monophosphate (AZT-MP), AZT diphosphate (DP), or AZT-TP in lymphocytes and macrophages; however, the data were reported as a sum of AZT-MP plus AZT-DP plus AZT-TP, limiting the ability to delineate concentrations of the active metabolite, AZT-TP. Additionally, antiviral potency was not reported.…”

Effects of dideoxyinosine and dideoxycytidine on the intracellular phosphorylation of zidovudine in human mononuclear cells.

“…Although ZDV is only one of an increasing number of similarly acting nucleoside reverse transcriptase inhibitors, it is still the first-line therapy for HIV sufferers worldwide [4][5][6]. ZDV and other 2',3'-dideoxynucleosides are inactive against the target viral reverse transcriptase enzyme, requiring intracellular phosphorylation to their putative active triphosphate forms [7][8][9][10][11][12][13]. ZDV enters target lymphocytes by passive diffusion [14] and is converted via ZDV monophosphate and ZDV diphosphate to the active triphosphate metabolite (ZDV triphosphate), the intracellular enzymes involved being thymidine kinase, thymidylate kinase and pyrimidine nucleoside diphosphate kinase respectively [7].…”