FORMATION OF ADDUCTS OF MITOMYCIN C WITH DNA

丝裂霉素 C 与 DNA 加合物的形成

• 批准号: 6240171
• 负责人: MARIA TOMASZ
• 金额: $ 2.6万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240171
• 关键词: DNA; DNA directed DNA polymerase; adduct; antineoplastic antibiotics; chemical models; chemical reaction; chemical structure function; chemical synthesis; crosslink; drug metabolism; gel electrophoresis; guanine; high performance liquid chromatography; mass spectrometry; mitomycin C; nuclear magnetic resonance spectroscopy; oligonucleotides

Mitomycin C (MC) is an antibiotic and antitumor agent widely used in clinical cancer chemotherapy. It is mode of action has been the subject of intensive study and speculation for almost 30 years, since the discovery that it induced covalent crosslinking of DNA. Recently we elucidated the basic chemistry of the activation of MC and its ultimate reaction products with DNA including the MC-DNA crosslink. Our objective is to relate each of the four major reaction products to the biological effects of Mc, i.e. to determine (a)which is more effective for antitumor and cytotoxic activities, (b) why is it more effective and (c) how to enhance the formation of such effective reactions in the cell. The broad and long-term objective is to understand the molecular basis of the antitumor activity of the mitomycin and make this knowledge applicable to new drug design and treatment protocols in cancer chemotherapy. The proposed work has the following specific aims: (1) Characterization of MC-DNA adducts on the structural level: (i) NMR study of mitomycin monoadduct an mitomycin intrastrand cross-link adduct. This involves chemical synthesis of oligonucleotide-MC (Columbia University). (ii) Gel electrophoresis of multimers of oligonucleotide-MC adducts to detect bending of DNA at the MC adduct sites. (iii) Determination of the structure of a newly discovered guanine-MC adduct by spectroscopic microscale methods (masspec, NMR). (2) Adduct structure-activity relationships: This involves chemical construction of oligonucleotides selectively modified by one of the four major adducts of MC at a single site. The constructs are ligated into longer sequences and applied to the following: (a) Effect of the different adducts on primer extension by DNA polymerases. (b) Effect of template modification on transcription by T7 RNA polymerase. (c) Action of urvABC excision endonuclease in vitro on the adducted constructs. (d) In vivo effects on survival and mutagenesis: The adducted oligonucleotides will be incorporated into a "shuttle vector" plasmid system and the resulting recombinant plasmid will be used to transfect COS cells or transform E. coli directly. Replicated plasmids will be isolated from mutant cells and analyzed for genomic sequence changes. (3) Analysis of MC-DNA adducts formed in intact cells. [3H]-labelled MC is used to treated mammalian cells in culture, followed by isolation of such analyses are to determine (a) the effect of hypoxia on adduct formation; (b) relative cytotoxicities of monofunctional and bifunctional adducts; (c) intracellular repair of MC- DNA adduct lesions; (d) different efficiency of repair of cross-link and monoadducts; (e) effect of hypoxia on repair; (f) effect of intracellular glutathione level on adduct patterns; (g) adduct patterns in MC-resistant mutant cell lines.

丝裂霉素C（MC）是一种广泛用于用于的抗生素和抗肿瘤剂 临床癌症化疗。 这是行动方式一直是主题 从近30年来的密集研究和投机活动中 发现它引起了DNA的共价交联。 最近我们 阐明了MC激活的基本化学及其最终的化学反应 与DNA的反应产物，包括MC-DNA交叉链接。 我们的目标 是将四种主要反应产物中的每一个与生物学联系起来 MC的效果，即确定（a）对抗肿瘤更有效的（a） 和细胞毒性活动，（b）为什么更有效，以及（c） 增强细胞中这种有效反应的形成。 宽阔 长期目标是了解 丝裂霉素的抗肿瘤活性，并使此知识适用 用于癌症化学疗法的新药物设计和治疗方案。 拟议的工作具有以下特定目的：（1）表征 结构水平上的MC-DNA加合物：（i）丝裂霉素的NMR研究 丝裂霉素单肌素内链交联加合物。 这涉及 寡核苷酸MC的化学合成（哥伦比亚大学）。 （ii）凝胶 寡核苷酸-MC加合物的多聚体的电泳以检测 DNA在MC加合站位置的弯曲。 （iii）确定 新发现的鸟嘌呤MC加合物的结构 微观方法（MassPec，NMR）。 （2）加合结构活性 关系：这涉及寡核苷酸的化学结构 由单个MC的四个主要加合物之一有选择地修改 地点。 将构建体连接成更长的序列，并应用于 以下内容：（a）不同加合物对引物扩展的影响 通过DNA聚合酶。 （b）模板修改对转录的影响 由T7 RNA聚合酶。 （c）URVABC切除核酸内切酶的作用 在加合构建体上的体外。 （d）体内对生存的影响和 诱变：加合的寡核苷酸将被掺入 “穿梭矢量”质粒系统和产生的重组质粒 将用于转染COS细胞或直接转化大肠杆菌。 复制的质粒将从突变细胞中分离出来，并分析 基因组序列变化。 （3）分析MC-DNA加合物形成的 完整的细胞。 [3H] - 标签的MC用于治疗哺乳动物细胞 培养，然后分离这种分析是为了确定（a） 缺氧对加合物形成的影响； （b）相对的细胞毒性 单功能和双功能加合物； （c）MC-的细胞内修复 DNA加合物病变； （d）交联的修复效率不同 单载; （e）缺氧对修复的影响； （f）细胞内的影响 加合物模式上的谷胱甘肽水平； （g）抗MC的加合图案 突变细胞系。