DNA CROSSLINK REPAIR ENZYMES IN HUMAN CELLS

这项研究基于令人信服的证据，即变量的敏感度 不同哺乳动物细胞类型对双功能抗肿瘤药物的影响 与DNA链间的形成程度或去除速度相关 交叉链接。这项提议的主要目的是识别、隔离和 表征可能参与修复的人类细胞酶 交联性损伤。具体地说，我们将测试假设 切除交联链的单加合物活性中间体，使细胞 通过抑制交联物的形成而相对抵抗。基材用于 体外酶分析将通过将DNA与一系列 双功能试剂，包括亚硝脲(例如，BCNU)；氮， 苯丙氨酸和磷酰胺芥末；丝裂霉素C；和顺铂。 将在体外通过测量复性能力来确定交联度 用荧光法测定DNA的含量。最初，已知的DNA修复酶如 因为DNA糖基酶或烷基转移酶的能力将被测试 以去除交联剂中间体。第二，新的活动 将在人类细胞提取液中寻求交联链前体切除。在……里面 此外，所有这些酶和提取物都将进行能力测试 割裂现有的交叉链接。 在进行体外酶研究的同时，人类细胞系具有不同的 对每种药物的敏感性将根据形成情况进行表征 以及体内交联链的去除，如DNA复性所确定的， 碱洗脱法和浮力密度离心法。 最终目标是将修复-酶活性、交联链 具有细胞毒性的形成和交联物持久性。这样的信息 将允许基于直接酶分析对药物反应的预测 正常组织或肿瘤组织。最后，对修复过程的干预 通过酶抑制可能为克服耐药性提供了一种手段。

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.