Törnevik Y, Eriksson S
Department of Biochemistry I, Karolinska Institute, Stockholm, Sweden.
Mol Pharmacol. 1990 Aug;38(2):237-43.
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.
2',3'-二脱氧胞苷（ddCyd）是一种有效的人类免疫缺陷病毒DNA复制抑制剂，其抗病毒活性需要通过细胞核苷激酶进行磷酸化。脱氧胞苷激酶（NTP：脱氧胞苷5'-磷酸转移酶，EC 2.7.1.74）负责形成二脱氧胞苷单磷酸，并且该酶受天然终产物dCTP的反馈调节。我们使用培养的人T淋巴母细胞（CEM）研究了细胞内dCTP水平的降低是否会影响ddCyd引起的生长抑制以及积累二脱氧胞苷三磷酸（ddCTP）的能力。使用亚毒性浓度的胸苷来降低dCTP池。还研究了3'-叠氮基-3'-脱氧胸苷（AZT）单独或与ddCyd联合对细胞生长、DNA前体池和ddCTP积累的影响。ddCyd和胸苷的联合导致CEM细胞的生长抑制是相加预期的两倍，而AZT和ddCyd的联合显示出相加作用。CEM细胞有效地积累了ddCTP，因此在10μM ddCyd（对应于EC50值）和6小时孵育下，ddCTP池比dCTP池高3倍。同时添加胸苷（10μM）使dTTP池增加2倍，dCTP水平降低50%，但ddCTP积累仅增加10%。相反，存在AZT（300μM，对应于EC50值）导致dCTP升高，而其他DNA前体池没有显著变化。在这种高浓度的AZT下，ddCTP的积累减少了42%。还发现，除了二脱氧胞苷单磷酸、二磷酸和三磷酸外，ddCyd还代谢为另外两种化合物，即脂核苷酸二脱氧胞苷二磷酸乙醇胺和二脱氧胞苷二磷酸胆碱，分别占总磷酸化ddCyd代谢产物的45%和6%，而单磷酸、二磷酸和三磷酸分别占磷酸化二脱氧核苷酸的3%、21%和25%。这些结果表明，在人淋巴母细胞中，二脱氧胞苷单磷酸的形成在ddCTP的合成中不是限速步骤，这与早期在小鼠细胞中观察到的情况明显不同（《分子药理学》32：798 - 806，1988）。此外，ddCyd引起的生长抑制似乎与dCTP和ddCTP之间的比例有关。在临床相关浓度下，ddCyd和AZT代谢之间没有直接干扰，这可能会鼓励将这些化合物联合用于抗人类免疫缺陷病毒治疗。
