Adducts of mitomycin C with nucleotides.

描述（申请人提供）：丝裂霉素C（MC）是一种天然抗肿瘤抗生素，用于临床癌症化疗。MC在肿瘤细胞中产生六种不同的DNA加合物。其中之一是DNA链间交联，这是MC对互补DNA链进行双功能烷基化的结果，而其他是药物的加合物，仅与一条DNA链连接（单加合物和链内交联）。本提案的广泛，长期目标是阐明每六个加合物的MC的细胞毒性/抗肿瘤活性的关系。此外，将使用Fanconi贫血（FA）细胞检查由MC的氧化还原循环产生的活性氧（ROS）的推定毒性作用。该提案的第一个目的是确定已知的FA细胞对MC的超敏反应是否主要是由于ROS的产生或药物形成的DNA加合物。这些研究将更好地定义Faneoni贫血细胞独特敏感性的生化基础，并提供对丝裂霉素活性的见解。其他具体的目的是检测和表征独特的MC的不同DNA加合物的生化反应。在这种情况下，我们将研究细胞培养中活EMT 6肿瘤细胞的加合物修复的差异率。在亚细胞系统中，通过构建91-mer寡核苷酸双链体，用各种MC加合物进行位点特异性修饰，并将其用作uvrABC核酸内切酶或哺乳动物全细胞提取物中存在的修复酶的底物，研究交联和单加合物的核苷酸切除修复的差异模式。将使用相同的底物在细胞提取物中搜索交联特异性的“加合物结合蛋白”。还将研究不同丝裂霉素单加合物对DNA聚合酶绕过病变的差异抑制，使用合成的加合物-模板-引物复合物作为底物。这些后面的实验旨在鉴定单加合物5和6的异常低的细胞毒性的基础。这项研究将导致更好地了解MC的抗肿瘤活性和毒理学的分子和生化基础，更普遍地说，其他DNA交联剂，目前用于癌症化疗的主要药物类别。

DESCRIPTION (provided by applicant): Mitomycin C (MC) is a natural antitumor antibiotic used in the clinic for cancer chemotherapy. MC generates six different DNA adducts in tumor cells. One of these is a DNA interstrand cross-link, as a result of bifunctional alkylation of the complementary DNA strands by MC, while the others are adducts of the drug, linked to only one strand of DNA (monoadducts and an intrastrand crosslink). The broad, long-range objective of the present proposal is to elucidate the relationship of each of the six adducts to the cytotoxicity/antitumor activity of MC. In addition, the putative toxic role of reactive oxygen species (ROS), generated by redox cycling of MC, will be examined using Fanconi's Anemia (FA) cells. The first aim of the proposal is to determine whether the known hypersensitivity of FA cells to MC is primarily due to ROS generation or to DNA adduct formation by the drug. These studies will better define the biochemical basis of the unique sensitivities of Faneoni's anemia cells, as well as providing insights into the activities of the mitomycins. The other specific aims serve to detect and characterize biochemical responses unique to the different DNA adducts of MC. In this context we shall investigate differential rates of repair of the adducts in living EMT6 tumor cells in cell culture. Differential modes of nucleotide excision repair of cross-link and monoadducts will be investigated in subcellular systems by constructing 91-mer oligonucleotide duplexes modified site-specifically with the various MC adducts, and using these as substrates for uvrABC excinuclease, or for the repair enzymes present in a mammalian whole cell extract. The same substrates will be used to search for "adduct binding proteins", specific for the cross-link, in cell extracts. Differential inhibition of lesion bypass by DNA polymerases by the different mitomycin monoadducts will also be investigated, using synthetic adduct-template-primer complexes as substrates. These latter experiments are aimed at identifying the basis for the unusually low cytotoxicity of the monoadducts 5 and 6. This research will lead to a better understanding of the molecular and biochemical basis of the antitumor activity and toxicology of MC and, more generally, of other DNA cross-linking agents, a major class of drugs currently used in cancer chemotherapy.