An in vitro model system that can differentiate the stages of DNA replication affected by anticancer agents
An in vitro model system that can differentiate the stages of DNA replication affected by anticancer agents
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DOI：
10.1016/j.bcp.2004.03.021
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发表时间：
2004-07-01
影响因子：
5.8
通讯作者：
Malkas, LH
中科院分区：
文献类型：
--
作者：
Abdel-Aziz, W;Hickey, RJ;Malkas, LH
我们以前曾报道过一种新的体外人细胞衍生模型系统的潜在用途，以研究直接影响DNA复制过程的抗癌药物的作用机制。我们的无细胞系统使用多蛋白质DNA复制复合物（指定的DNA合成体），已被分离，表征，并广泛纯化，从各种各样的哺乳动物细胞和组织。DNA合成体能够在体外协调猴病毒40（SV 40）来源特异性和大T抗原依赖性的DNA复制。在这项研究中，基于合成体的无细胞系统进行了测试，以评估1-β-D-阿拉伯呋喃糖基胞嘧啶（ara-C），喜树碱（CPT）和阿霉素（DOX）的作用机制。使用一种新的合成体为基础的体外动力学测定，我们证明了DNA复制介导的合成体启动内的SV 40复制起点和进行双向的方式类似于发生在细胞内。已发现Ara-CTP、CPT和DOX影响由复合物介导的体外DNA复制过程的不同阶段。Ara-CTP抑制起始和延伸阶段，而CPT产生的大部分影响，通过抑制DNA复制的延伸阶段。DOX抑制由合成体介导的DNA合成的终止阶段。这里提出的数据支持我们的论点，DNA合成体是一个非常有效的体外模型系统，用于研究某些抗癌药物可以直接影响DNA复制过程的机制。(C)2004年爱思唯尔公司All rights reserved.
We have previously reported on the potential use of a novel in vitro human cell-derived model system to investigate the mechanism of action of anticancer agents that directly affect the process of DNA replication. Our cell-free system uses a multiprotein DNA replication complex (designated the DNA synthesome) that has been isolated, characterized, and extensively purified from a wide variety of mammalian cells and tissues. The DNA synthesome is competent to orchestrate simian virus 40 (SV40) origin-specific and large T antigen-dependent DNA replication in vitro. In this study, the synthesome-based cell-free system was tested to evaluate the mechanism of action of 1-beta-D-arabinofuranosylcytosine (ara-C), camptothecin (CPT), and doxorubicin (DOX). Using a novel synthesome-based in vitro kinetic assay, we demonstrated that DNA replication mediated by the synthesome is initiated within the SV40 replication origin and proceeds bidirectionally in a manner analogous to that occurring within the cell. Ara-CTP, CPT, and DOX have been found to affect different stages of the in vitro DNA replication process mediated by the complex. Ara-CTP inhibited both the initiation and elongation stages, whereas CPT produced most of its effects by inhibiting the elongation phase of DNA replication. DOX inhibited the termination stage of DNA synthesis mediated by the synthesome. The data presented here support our contention that the DNA synthesome represents a highly effective in vitro model system for investigating the mechanism by which some anticancer agents can directly affect the process of DNA replication. (C) 2004 Elsevier Inc. All rights reserved.