DNA CROSSLINK REPAIR ENZYMES IN HUMAN CELLS

人体细胞中的 DNA 交联修复酶

• 批准号: 3174488
• 负责人: THOMAS P BRENT
• 金额: $ 9.33万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1987-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3174488
• 关键词: DNA; alkyl group; alkyltransferase; antineoplastics; carmustine; cis platinum compound; crosslink; cytotoxicity; drug resistance; human subject; mitomycin C; neoplasm /cancer pharmacology; nitrosourea; phenylalanine; phosphamide

This research is based on compelling evidence that the variable sensitivity of different mammalian cell types to bifunctional antineoplastic drugs correlates with extent of formation or rate of removal of DNA interstrand cross-links. The major aim of this proposal is to identify, isolate and characterize human-cell enzymes potentially involved in repair of cross-link lesions. In particular, we will test the hypothesis that excision of monoadduct reactive intermediates of cross-links, renders cells relatively resistant by suppressing cross-link formation. Substrates for in vitro enzyme assay will be made by reacting DNA with a range of bifunctional agents, including the nitrosoureas (e.g., BCNU); nitrogen, phenylalanine and phosphoramide mustards; mitomycin-C; and cisplatin. Cross-linking will be determined in vitro by measuring the renaturability of DNA with fluorometric methods. Initially, known DNA repair enzymes such as DNA-glycosylases or alkyltransferase will be tested for their capacity to remove cross-link intermediates. Secondly, new activities for cross-link-precursor excision will be sought in human cell extracts. In addition, all these enzymes and extracts will be tested for their ability to cleave existing cross-links. In parallel with in vitro enzyme studies, human cell lines with varied sensitivity to each drug will be characterized with respect to formation and removal of cross-links in vivo, as determined by DNA renaturability, alkaline elution and buoyant density centrifugation methods. The ultimate goal is to correlate repair-enzyme activity, cross-link formation and cross-link persistence with cytotoxicity. Such information will allow predictions of drug response based on direct enzyme assay in normal or neoplastic tissue. Finally, intervention in the repair process by enzyme inhibition might provide a means to overcome drug resistance.

这项研究是基于令人信服的证据表明，变量的敏感性 不同的哺乳动物细胞类型的双功能抗肿瘤药物 与DNA链间的形成程度或去除速率相关 交叉链接。 该提案的主要目的是识别、分离和 表征可能参与修复的人类细胞酶 交叉连接病变。 特别是，我们将测试假设， 切除交联的单加合反应性中间体，使细胞 通过抑制交联形成而具有相对抗性。 的底物 体外酶测定将通过使DNA与一系列 双功能试剂，包括亚硝基脲（例如，BCNU）;氮， 苯丙氨酸和磷酰胺核苷;丝裂霉素-C;和顺铂。 将通过测量可复性在体外确定交联 DNA的荧光方法。 最初，已知的DNA修复酶， 因为DNA-糖基化酶或烷基转移酶的能力将被测试， 以除去交联中间体。 第二，新活动 将在人细胞提取物中寻求交联前体切除。 在 此外，将测试所有这些酶和提取物的能力， 来切割现有的交联。 在体外酶研究的同时，研究了具有不同 对每种药物的敏感性将根据形成 和体内交联的去除，如通过DNA复性性所确定的， 碱性洗脱和浮力密度离心法。 最终目标是将修复酶活性、交联 形成和交联持久性与细胞毒性。 此类信息 将允许基于直接酶试验预测药物反应， 正常或肿瘤组织。 最后，在修复过程中进行干预 通过酶抑制可能提供一种克服耐药性的方法。