2',3'-Dideoxycytidine toxicity in cultured human CEM T lymphoblasts: effects of combination with 3'-azido-3'-deoxythymidine and thymidine.
2',3'-Dideoxycytidine (ddCyd), a potent inhibitor of human immunodeficiency virus DNA replication, requires phosphorylation by cellular nucleoside kinases for antiviral activity. Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) is responsible for the formation of dideoxycytidine monophosphate and this enzyme is controlled by feedback regulation by the natural endproduct, dCTP. We have examined whether a decrease in intracellular dCTP levels affects the growth inhibition caused by ddCyd, as well as the capacity to accumulate dideoxycytidine triphosphate (ddCTP), using human T lymphoblast (CEM) cells in culture. Subtoxic concentrations of thymidine were used to decrease the dCTP pool. The effects of 3'-azido-3'-deoxythymidine (AZT), alone or in combination with ddCyd, on cell growth, DNA precursor pools, and accumulation of ddCTP were also studied. The combination of ddCyd and thymidine led to growth inhibition of CEM cells that was twice what would be expected from addition, whereas the combination of AZT and ddCyd showed an additive effect. CEM cells accumulated ddCTP efficiently, so that with 10 microM ddCyd (corresponding to the EC50 value) and a 6-hr incubation the ddCTP pool was 3-fold higher than the dCTP pool. Simultaneous addition of thymidine (10 microM) increased the dTTP pool 2-fold and gave a 50% reduction in the dCTP level but only a 10% increase in ddCTP accumulation. The presence of AZT (300 microM, corresponding to the EC50 value) led, in contrast, to an elevation of dCTP and no significant change in the other DNA precursor pools. With this high concentration of AZT, the accumulation of ddCTP decreased 42%. It was also found that ddCyd is metabolized into two additional compounds, besides the dideoxycytidine mono-, di-, and triphosphate, i.e., the liponucleotides dideoxycytidine diphosphate-ethanolamine and dideoxycytidine diphosphate-choline, constituting 45 and 6% of the total phosphorylated ddCyd metabolites, respectively, whereas the mono-, di-, and triphosphate corresponded to 3, 21, and 25% of the phosphorylated dideoxynucleotides. These results indicate that the formation of dideoxycytidine monophosphate is not rate limiting in the synthesis of ddCTP in human lymphoblasts, which clearly differs from what was observed earlier in mouse cells (Mol Pharmacol 32:798-806 1988). Furthermore, growth inhibition by ddCyd seems to be related to the ratio between dCTP and ddCTP. There was no direct interference between ddCyd and AZT metabolism in clinically relevant concentrations, which may encourage the use of combination of these compounds for anti-human immunodeficiency virus treatment.
Törnevik
Y
Y
Department of Biochemistry I, Karolinska Institute, Stockholm, Sweden.
Eriksson
S
S
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003842:Deoxycytidine Kinase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D054306:Dideoxynucleotides; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D006801:Humans; D010766:Phosphorylation; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D013936:Thymidine / Q000494:pharmacology; D016047:Zalcitabine / Q000494:pharmacology*; D015215:Zidovudine / Q000494:pharmacology
D003845:Deoxycytosine Nucleotides; D054306:Dideoxynucleotides; D015215:Zidovudine; C037406:2',3'-dideoxycytidine 5'-triphosphate; D016047:Zalcitabine; D003842:Deoxycytidine Kinase; D013936:Thymidine