Han S, Hickey R J, Tom T D, Wills P W, Syväoja J E, Malkas L H
Department of Pharmacology and Experimental Therapeutics, School of Medicine, University of Maryland, Baltimore, MD, USA.
Biochem Pharmacol. 2000 Aug 1;60(3):403-11. doi: 10.1016/s0006-2952(00)00336-1.
The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.
抗代谢物1-β-D-阿拉伯呋喃糖基胞嘧啶（ara-C）已被用作治疗白血病的高效药物。活性代谢物1-β-D-阿拉伯呋喃糖基胞嘧啶三磷酸（ara-CTP）是DNA聚合酶α、δ和ε的强效抑制剂，负责抑制完整细胞的DNA合成。我们已经表明，一种具有许多人类细胞DNA复制装置预期特性的多蛋白复合物，可以很容易地从人类细胞和组织中分离出来，并且能够在体外支持依赖于起始点的DNA合成。DNA聚合酶α、δ和ε是这种称为DNA合成体的多蛋白复合物的组成部分，我们在此报告，这些与DNA合成体相关的DNA聚合酶的活性受到ara-CTP的不同抑制。与DNA合成体相关的DNA聚合酶α的抑制作用呈浓度依赖性增加，并且与猿猴病毒40（SV40）依赖起始点的体外DNA复制的抑制密切相关，而在100微摩尔浓度下，ara-CTP对与DNA合成体相关的DNA聚合酶δ的活性没有显著抑制作用。最近的研究表明，与合成体相关的DNA聚合酶ε在体外SV40 DNA复制中不起作用，这表明只有聚合酶α和δ驱动DNA复制叉。因此，我们的结果表明，ara-CTP对哺乳动物细胞DNA合成体活性的抑制主要是由于对与DNA合成体相关的DNA聚合酶α的抑制。这一观察结果意味着该药物可能靶向人类细胞中DNA合成过程的特定阶段。
