TOPOISOMERASE II-ACTIVE AGENTS AND THE NUCLEAR MATRIX

一些临床上重要的抗癌药物，包括 表鬼臼毒素(Vp-16，Vm-26)、氨基吖啶(m-AMSA)、 蒽环类药物(阿霉素、柔红霉素)和蒽二酮 已知可干扰DNA拓扑异构酶II 活动。然而，在我们的知识中仍然存在着巨大的差距 拓扑异构酶II抑制后的关键生化事件 导致阻断肿瘤细胞增殖的活性。脱氧核糖核酸 拓扑异构酶II是核基质中主要的非组蛋白蛋白。 因此，拟议继续研究的总体目标是 提供对药物稳定的机制的见解 拓扑异构酶II-DNA复合体干扰核基质 DNA的组织和复制功能。拟议的研究将 扩展我们正在进行的工作，并将利用新技术进行调查 与核相关的DNA复制叉处的药物相互作用 矩阵。 初步研究的具体目标是确定a)是否 由VM-26或m-AMSA稳定的拓扑异构酶II-DNA复合体是 与散装DNA相比，富含核基质DNA，以及b)相对于什么 拓扑异构酶II的α和β亚型参与的程度 与核基质和块状DNA形成可切割的复合体。这些 研究将在亲代CCRF-CEM细胞和 耐多重拓扑异构酶II活性的CEM/VM-L亚系 探员们。所获得的结果将为我们深入了解 DNA复制和AS中的个别拓扑异构酶II亚型 拓扑异构酶II活性物质的靶标。进一步的研究将涉及 检验药物稳定的拓扑异构酶II-DNA的工作假设 复合体是DNA复制移动的物理屏障 模板DNA上的复合体。这将通过以下方式实现 监测VM-26和m-AMSA对DNA运动的影响 复制分叉通过c-myc原癌基因和二氢叶酸 吉恩。需要检验的第二个工作假说是可切割的复合体 VM-26或m-AMSA诱导DNA复制叉的形成 导致DNA复制叉从细胞核中分离 矩阵。与药物诱发的程度有直接关系 核基质DNA中可切割复合体的形成及其程度 冈崎碎片从核基质中解离将提供 复制叉处DNA链断裂是关键损伤的证据 由这些拓扑异构酶II-激活剂诱导。

A number of clinically important anticancer agents including the epipodophyllotoxins (VP-16, VM-26), aminoacridines (m-AMSA), anthracyclines (doxorubicin, daunorubicin), and anthracenediones (mitoxantrone), are known to interfere with DNA topoisomerase II activity. However, large gaps persist in our knowledge concerning the critical biochemical events following inhibition topoisomerase II activity that lead to blockade of tumor cell proliferation. DNA topoisomerase II is a major nonhistone protein of the nuclear matrix. Accordingly, the overall goal of the proposed continuation studies is to provide insights into the mechanisms by which drug-stabilized topoisomerase II-DNA complexes interfere with the nuclear matrix functions of DNA organization and replication. The proposed studies will extend our ongoing work and will utilize new techniques for investigating drug interactions at DNA replication forks associated with the nuclear matrix. The specific objectives of the initial studies are to determine a) if topoisomerase II-DNA complexes stabilized by either VM-26 or m-AMSA are enriched in nuclear matrix DNA compared to bulk DNA, and b) to what extent the alpha and beta isoforms of topoisomerase II are involved in cleavable complex formation with nuclear matrix and bulk DNA. These studies will be carried out with parental CCRF-CEM cells and with the CEM/VM-l subline that is resistant to multiple topoisomerase II-active agents. The results obtained will provide insights into the importance of the individual topoisomerase II isoforms in DNA replication and as targets for topoisomerase II-active agents. Further studies will involve testing the working hypothesis that drug-stabilized topoisomerase II-DNA complexes act as physical barriers to the movement of DNA replication complexes along the template DNA. This will be accomplished by monitoring the effects of VM-26 and m-AMSA on the movement of DNA replication forks through the c-myc protooncogene and the dihydrofolate gene. A second working hypothesis to be tested is that cleavable complex formation in DNA replication forks induced by either VM-26 or m-AMSA results in the detachment of the DNA replication forks from the nuclear matrix. A direct relationship between the extent of drug-induced cleavable complex formation in nuclear matrix DNA and the extent of dissociation of Okazaki fragments from the nuclear matrix would provide evidence that DNA strand breaks at replication forks are critical lesions induced by these topoisomerase II-active agents.