DNA DAMAGE BY ALKYLATING AGENTS AND THEIR REPAIR IN HUMAN TUMOR CELLS

D.亚罗什河S.日，三，和其他人已经表明，大约20%， 的人肿瘤细胞系和病毒转化细胞系对 由于明显不存在06-烷基鸟嘌呤DNA， 烷基转移酶（06 AT）;这种表型已被指定为mer-。 这 酶去除鸟嘌呤0-6位的烷基化损伤，但不 DNA中的其他位点。 最近，已经克隆了人06 AT的cDNA克隆。 分离的基础上，它保护06 AT缺陷型细菌， 某些烷基化剂（Tano，K，Shiota，S，Collier，J，Foote，RS， 和Mitra，S.一个cDNA克隆的分离和结构鉴定 编码α 6-烷基鸟嘌呤的人DNA修复蛋白。 Proc. Natl. Acad. Sci. USA 1990;87：686-690）。使用基于此的寡核苷酸 序列，我们已经分离了全长06 AT cDNA克隆从人类 我们构建的肝脏库。 利用这个克隆体作为探针， 已经发现06 AT mRNA在所有的肿瘤中显著减少， 检查细胞系。 06 AT mRNA水平在mer+中变化10倍。 细胞系，并与已知水平的蛋白质，特别是 细胞 我们的发现可能对癌症治疗有重要意义 其中使用诸如BCNU的试剂。 D.亚罗什有初步证据 一些脑肿瘤没有检测到06 AT活性。 确定 哪些肿瘤是mer-和哪些mer+肿瘤具有低水平的06 AT mRNA 和蛋白质可能对化疗计划有用。 努力 正在开发测量06 AT mRNA和蛋白质的方法 体内水平。 这涉及重组06 AT的生产 蛋白和使用该重组体的抗体的开发 蛋白

D. Yarosh, R. S. Day, III, and others have shown that approximately 20% of human tumor lines and viral transformed lines are hypersensitive to alkylating agents due to an apparent absence of 06-alkylguanine DNA alkyltransferase (06AT); this phenotype has been designated mer-. This enzyme removes alkylation damage at the 0-6 position of guanine but not at other sites in DNA. Recently, a cDNA clone for a human 06AT has been isolated on the basis that it protects 06AT-deficient bacteria from certain alkylating agents (Tano, K, Shiota, S, Collier, J, Foote, RS, and Mitra, S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for 06-alkylguanine. Proc. Natl. Acad. Sci. USA 1990;87:686-690). Using oligonucleotides based on this sequence, we have isolated a full-length 06AT cDNA clone from a human liver library which we had constructed. Using this clone as a probe, we have found that 06AT mRNA was markedly reduced in all the mer- tumor cells lines examined. The level of 06AT mRNA varied by 10-fold in mer+ cell lines and correlated with known levels of the protein in particular cells. Our findings may have important implications in cancer therapy where agents such as BCNU are used. D. Yarosh has preliminary evidence that some brain tumors have no detectable 06AT activity. Determining which tumors are mer- and which mer+ tumors have low levels of 06AT mRNA and protein would probably be useful in planning chemotherapy. Efforts are underway to develop approaches to measure 06AT mRNA and protein levels in vivo. This has involved the production of recombinant 06AT protein and the development of antibodies using this recombinant protein.