FORMATION OF ADDUCTS OF MITOMYCIN C WITH DNA

丝裂霉素C（MC）是一种广泛应用于临床的抗生素和抗肿瘤药物， 临床癌症化疗。 它的作用方式一直是 近30年来的深入研究和猜测， 发现它诱导DNA的共价交联。 最近我们 阐明了MC活化的基本化学过程及其最终的 与DNA的反应产物，包括MC-DNA交联。 我们的目标 是将四种主要反应产物中的每一种与生物 Mc的作用，即确定（a）哪一种对抗肿瘤更有效 和细胞毒活性，（B）为什么它更有效和（c）如何 增强细胞中这种有效反应的形成。 广大 长期目标是了解 丝裂霉素的抗肿瘤活性，并使这些知识适用 癌症化疗中的新药设计和治疗方案。 本文的工作主要有以下几个方面：（1）表征 MC-DNA加合物结构水平的研究：（i）丝裂霉素的NMR研究 单加合物是丝裂霉素链内交联加合物。 这涉及 化学合成法制备的阿替西肽-MC（哥伦比亚大学）。 (ii)凝胶 电泳多聚体的阿贝肽-MC加合物，以检测 DNA在MC加合位点的弯曲。 (iii)测定 一种新鸟嘌呤-MC加合物光谱结构 微型方法（质谱、NMR）。(2)加合物构效 关系：这涉及寡核苷酸的化学结构 选择性修饰的四个主要加合物之一的MC在一个单一的 绝佳的价钱 将构建体连接成更长的序列并应用于 （a）不同加合物对引物延伸的影响 DNA聚合酶。 (b)模板修饰对转录的影响 T7 RNA聚合酶 (c)urvABC切除核酸内切酶的作用 在体外对内收构建体。(d)对存活率的体内影响， 诱变：加合的寡核苷酸将被掺入到一个含有寡核苷酸的细胞中。 "穿梭载体"质粒系统和所得重组质粒 将用于转染COS细胞或转化E. coli直接进行。 将从突变细胞中分离复制的质粒，并分析 基因组序列改变。 (3)MC-DNA加合物的分析 完整的细胞 [3H]-标记的MC用于处理哺乳动物细胞， 培养，然后分离这些分析是为了确定（a） 缺氧对加合物形成影响;（B）相对细胞毒性 单功能和双功能加合物;（c）MC-1的细胞内修复 DNA加合物损伤;（d）交联和交联的不同修复效率， 单加合物;（e）缺氧对修复的影响;（f）细胞内 （g）抗MC-小鼠中的加合物模式; 突变细胞系

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.