TOPOISOMERASE DRUG ACTIONS AT NUCLEAR MATRIX DNA DOMAIN

拓扑异构酶在核基质 DNA 域的药物作用

• 批准号: 6130427
• 负责人: DANIEL James FERNANDES
• 金额: $ 20.34万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6130427
• 关键词: DNA damage; DNA repair; DNA replication; DNA topoisomerases; camptothecin; enzyme activity; flow cytometry; globin; leukemia; neoplasm /cancer pharmacology; nuclear matrix; phosphorylation; podophyllin; protooncogene; tissue /cell culture

DESCRIPTION: (Applicant's Abstract) The nuclear matrix model of DNA replication and organization will be utilized in the proposed studies to address (a) important effects of topoisomerase-active drugs on primary and higher order chromatin structure and (b) the cellular responses to these drug effects (e.g., DNA repair, chromatin disorganization) that influences drug action. The central hypothesis to be tested is that topoisomerase I and II poisons irreversibly damage DNA by preferentially inducing double-strand DNA breaks in replication forks on the nuclear matrix of human CEM cells. The cleaved replication forks then detach from the nuclear matrix, which interferes with the ability of the DNA protein kinase (DNA-PK) and replication protein A (RPA) systems to signal DNA damage control and replication fork repair. Preferential cleavage of its c-myc and beta-globin genes within various nuclear matrix DNA loop domains will be quantitated using the PCR-stop assay (Specific Aim 1). The studies described in Specific Aim 2a will extend these observations by evaluating some potentially important biological consequences of drug-induced detachment of c-myc and beta-globin Okazaki fragments and replicating DNA loops from the nuclear matrix. Specific Aims 2b and 2c are designed to assess some of the cellular responses to VM-26 and camptothecin induced damage to matrix-attached DNA loops. To evaluate the repair of VM-26 and camptothecin induced cleavage, the rates of reversal of drug-induced cleavage in replicating and nonreplicating c-myc and beta-globin genes will be determined in CEM and VM-26 resistant VM-1 cells. Additional experiments are designed to quantitate the degrees of drug-induced RPA32 hyperphosphorylation and Ku70-DNA-Pkcs binding to replicating and nonreplicating DNA. These studies will determine whether the extent of activation of the DNA-PK-RPA system for replication fork arrest and double strand DNA break repair is related to the degree of reversal of drug-induced DNA cleavage in these cell lines. The proposed studies represent a novel approach for providing insights into important biological effects of the topoisomerase I and topoisomerase II drugs as well as the cellular responses to these effects that may influence drug cytotoxicity.

（申请人摘要）DNA复制的核基质模型