DNA CROSSLINK REPAIR ENZYMES IN HUMAN CELLS

人体细胞中的 DNA 交联修复酶

• 批准号: 3174484
• 负责人: THOMAS P BRENT
• 金额: $ 12.44万
• 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 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3174484
• 关键词: DNA; alkylating agents; antineoplastics; bioassay; carmustine; cell bank /registry; chemical addition; chemical reaction; cis platinum compound; covalent bond; crosslink; cyclophosphamide; cytotoxicity; drug resistance; enzyme mechanism; enzyme substrate; enzyme substrate complex; fluorimetry; human tissue; hybridomas; laboratory mouse; mitomycin C; monoclonal antibody; neoplasm /cancer pharmacology; nitrosourea; nucleic acid probes; pest control; radiotracer

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.

有令人信服的证据表明，人类恶性肿瘤的反应 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率有关 链间交叉连接。 要检验的假设是 提出的研究是，交联的减少是由以下因素介导的： 酶DNA修复过程。 我们的目标是识别，隔离 并描述人类细胞中修复DNA的酶 交联剂或其前体 通过DNA中的06-烷基鸟嘌呤-DNA烷基转移酶（GATase）预防 用氯乙基亚硝基脲（CENUs）处理。 GATase，纯化 进一步来自培养的人淋巴母细胞或人肝脏， 用作探针以确定交联的分子机理 用CENUs和其他类型的药物处理的DNA中的形成， 在DNA中的06-鸟嘌呤处产生加合物（例如，clomesome， 米托唑胺，甲基苄肼，丝裂霉素C和顺铂）：我们将 确定GATase在多种人类肿瘤细胞系中的活性， 包括中枢神经系统肿瘤，以建立 细胞GATase水平与耐药性的关系。 GATase特异性抗体和cDNA探针的开发应 最终使GATase水平能够容易地在 临床活检标本。我们现有的体外方法学 为研究CENUs和相关药物而开发的 交联形成的研究 以及由其他双功能烷化剂诱导的酶修复 (e.g.,环磷酰胺、异环磷酰胺、丝裂霉素C和顺铂）。 必要时将使用活化衍生物。 DNA 链间交联将在体外通过测量 分离的DNA的复性与荧光，过滤- 结合或电泳方法。 纯化的人类DNA修复 酶，如烷基嘌呤-DNA糖基化酶或GATase，和粗酶 将测定来自人细胞的提取物的活性， 修复交联的单加合物前体和2）裂解存在的 交叉连接。这项研究的最终目的是建立 修复酶活性、交联形成和 交联持久性与细胞毒性和临床反应 化疗 这些信息应该能够预测治疗的效果。 基于修复酶的直接生化测定的致敏反应 肿瘤和正常组织中的水平。 干预这些修复 酶抑制过程可以提供一种策略， 克服癌症患者的耐药性。