DNA CROSSLINK REPAIR ENZYMES IN HUMAN CELLS

有令人信服的证据表明，人类的恶性反应 用双功能烷化剂对肿瘤进行化疗 与DNA的形成程度或去除速度有关的 链间交联剂。这一假设将在 建议的研究是，减少交联度是通过 酶的DNA修复过程。我们的目标是识别、隔离 并对可能修复DNA的人类细胞酶进行了表征 交联剂或其前体近期目标集中在交联剂上 DNA中06-烷基鸟嘌呤-DNA烷基转移酶(GATase)的预防作用 用氯乙基亚硝脲(Cenus)处理。GATase，纯化 进一步从培养的人类淋巴母细胞或人类肝脏中分离出来 用作探针以确定交联剂的分子机理 Cenus和其他类药物处理的DNA中的形成 在DNA中的06-鸟嘌呤处产生加合物(例如， 丝裂霉素C和顺铂)：我们将 测定多种人类肿瘤细胞系的GATase活性。 包括中枢神经系统肿瘤，以建立 细胞GATase水平与耐药的关系 GATase特异性抗体和cDNAs探针的研制 最终允许GATase水平很容易在 临床活检标本。我们拥有的体外方法学 为研究cenus而开发的相关药物将被应用 关于交联剂形成的研究 以及其他双官能团烷基化试剂诱导的酶修复 (例如环磷酰胺、异环磷酰胺、丝裂霉素C和顺铂)。 在必要时，将使用激活的衍生品。脱氧核糖核酸 链间交联度将在体外通过测量确定 荧光分光光度法、滤波法测定提取DNA的复性 结合或电泳法。两种纯化的人类DNA修复 酶，如烷基嘌呤-DNA糖基酶或GAT酶，以及粗酶 从人类细胞中提取的物质将被检测为具有以下活性：1) 修复交联链的单加合物前体和2)裂解存在 交联剂。这项研究的最终目标是建立 修复酶活性、交联剂的形成及其相互关系 交联剂持久性与细胞毒性和临床反应 化疗。这样的信息应该能够预测温度- 基于修复酶直接生化测定法的临床反应 在肿瘤组织和正常组织中的水平。对这些修复的干预 通过酶抑制的过程可以提供一种策略 克服癌症患者的耐药性。

There is compelling evidence that the response of human malignant tumors to chemotherapy with bifunctional alkylating agents is related to the extent of formation or rate of removal of DNA interstrand crosslinks. The hypothesis to be tested in the proposed research is that reduction of crosslinking is mediated by enzymic DNA repair processes. The goal is to identify, isolate and characterize the human-cell enzymes that may repair DNA crosslinks or their precursors Immediate aims focus on crosslink prevention by 06-alkylguanine-DNA alkyltransferase (GATase) in DNA treated with the chloroethylnitrosoureas (CENUs). GATase, purified further from cultured human lymphoblasts or human liver, will be used as a probe to define the molecular mechanism for crosslink formation in DNA treated with CENUs and other classes of drug that produce adducts at 06-guanine in DNA (e.g., clomesome, mitozolomide, procarbazine, mitomycin C and cisplatin): We shall determine GATase activity in a variety of human tumor lines, in- cluding tumors of the central nervous system, to establish the relationship between cellular GATase levels and drug resistance. Development of GATase-specific antibodies and cDNA probes should eventually permit GATase levels to be readily determined in clinical biopsy specimens. The in vitro methodologies that we have developed for studying the CENUs and related drugs will be applied to investigation of crosslink formation and enzymic repair induced by other bifunctional alkylating agents (e.g., cyclophosphamide, ifosfamide, mitomycin C and cisplatin). Where necessary activated derivatives will be used. DNA interstrand crosslinking will be determined in vitro by measuring the renaturability of isolated DNA with fluorometric, filter- binding or electrophoretic methods. Both purified human DNA repair enzymes, such as alkylpurine-DNA glycosylase or GATase, and cruder extracts from human cells will be assayed for activities that 1) repair monoadduct precursors of crosslinks and 2) cleave existing crosslinks. The ultimate goal of this research is to establish correlations of repair enzyme activity, crosslink formation and crosslink persistence with cytotoxicity and clinical response to chemotherapy. Such information should enable prediction of thera- peutic response based on direct biochemical assay of repair enzyme levels in tumor and normal tissues. Intervention in these repair processes by enzyme inhibition could provide a strategy for overcoming drug resistance in cancer patients.