Dipyridamole, a commonly used coronary vasodilator and antithrombotic drug, was recently shown to potentiate the activity of 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine against the human immunodeficiency virus type 1 (HIV-1) in human monocyte-macrophages in vitro. We report in the present paper that in uninfected monocyte-macrophages dipyridamole significantly inhibits cellular salvage of [3H]deoxycytidine, whereas it does not affect the salvage of [3H]dideoxycytidine. Similar differential inhibition by dipyridamole of the salvage of thymidine, as opposed to 3'-azido-3'-deoxythymidine, was reported previously (G. V. Betageri, J. Szebeni, K. Hung, S. S. Patel, L. M. Wahl, M. Corcoran, and J. N. Weinstein, Biochem. Pharmacol. 40:867-870, 1990). Taken together, these observations suggest that inhibition of the salvage of competing physiological nucleosides may explain or contribute to the potentiating effect of dipyridamole on these antiviral dideoxynucleoside drugs.We recently reported that dipyridamole (DPM), a commonly used antiplatelet and coronary vasodilator drug (3), enhances the protection offered by 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) against human immunodeficiency virus type 1 (HIV-1) infection in vitro in human monocyte-derived macrophages (M/M) and T lymphocytes (15,16,22).In studying the mechanism of this phenomenon, we have demonstrated that DPM decreases the cellular uptake and phosphorylation of deoxythymidine (dThd) in MIM, whereas it has no effect on the uptake and phosphorylation of AZT (1). Because dThd antagonizes the antiviral activity of AZT (8, 14), we suggested that differential inhibition of the transport and/or phosphorylation of dThd, as opposed to AZT, may suppress this antagonism and thus promote AZT's antiviral action (1,16). ddC, like AZT, suppresses HIV-1 replication by inhibiting viral reverse transcriptase and/or by terminating viral DNA synthesis (7). ddC is about 10 times more potent than AZT in inhibiting HIV-1 in vitro, its activation pathway and toxicity profile are different from those of AZT (24), and AZTresistant HIV isolates do not show cross-resistance to ddC (13). For these reasons, ddC is being studied extensively, both as a single agent and in combination with AZT in clinical trials with HIV-infected patients (5, 24).As mentioned previously, the anti-HIV activity of ddC, like that of AZT, was potentiated by DPM in M/M (16). Here we wished to ask whether differential effects similar to those seen with AZT (1) Human peripheral blood monocytes were isolated by counterflow centrifugal elutriation, using pyrogen-free phosphate-buffered saline (B & P/Scott) in the elutriation step (19,20). Monocytes were suspended in Dulbecco modified Eagle medium (DMEM) supplemented with 2 mM glutamine and antibiotics and, to prepare M/M, they were allowed to adhere (106 cells per well) in six-well Costar plates. Following overnight incubation, the medium was replaced with DMEM containing 10% fetal calf serum (FCS), 2 mM glutamine, antibiotics, and 4...
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...
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