Balzarini J, De Clercq E
Biochim Biophys Acta. 1984 Feb 28;785(1-2):36-45. doi: 10.1016/0167-4838(84)90231-0.
Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.
为监测胸苷（dThd）类似物对完整小鼠白血病L1210细胞中胸苷酸（dTMP）合酶（5,10-亚甲基四氢叶酸：dUMP C-甲基转移酶，EC 2.1.1.45）的抑制作用，人们采用了两种策略。第一种方法基于测定2'-脱氧[5-³H]尿苷[（5-³H]dUrd）或2'-脱氧[5-³H]胞苷[（5-³H]dCyd）的³H释放量；第二种方法基于估算作为dTMP掺入DNA中的dCyd量。通过评估一系列18种dThd类似物对小鼠白血病L1210细胞中胸苷酸合酶的抑制作用，对这些方法的有效性进行了评估。一方面，dThd类似物对L1210细胞增殖的抑制作用与（i）它们对[5-³H]dCyd的³H释放的抑制作用（r = 0.926）以及（ii）它们对dCyd掺入DNA dTMP的抑制作用（r = 0.921）之间存在很强的相关性。事实证明，评估[5-³H]dCyd的³H释放是迄今为止所描述的测量胸苷酸合酶活性以及追踪完整L1210细胞中胸苷酸合酶抑制剂抑制作用的最便捷方法，因为该方法快速且非常灵敏，并且由于它优于评估[5-³H]dUrd的³H释放，因为它避免了抑制剂与胸苷激酶活性可能存在的相互作用。
