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 由于明显缺乏O 6-烷基鸟嘌呤DNA而导致的烷化剂 烷基转移酶（O 6AT）;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，但不去除鸟嘌呤0-6位的烷基化损伤。 DNA中的其他位点。Dr. D. Yarosh与我们的单位合作， 能够从人肝中部分纯化该酶， 这种蛋白质的抗体。已经开始研究， 这种蛋白质的单克隆抗体。最近，一个人的cDNA克隆 O 6AT已被分离的基础上，它保护O 6AT缺乏 从某些烷基化剂中分离细菌（Tano，K，Shiota，S，Collier，J， Foote，RS和Mitra，S.一种新的抗肿瘤药物的分离和结构表征 编码O 6-烷基鸟嘌呤的人DNA修复蛋白的cDNA克隆。Proc. Natl. Acad. Sci. USA 1990;87：686-690）。使用基于此的寡核苷酸 序列，我们从人肝脏中分离出了全长O 6 AT cDNA克隆 我们建造的图书馆。利用这个克隆体作为探针， 发现O 6AT mRNA在所有肿瘤细胞系中均显著降低 (8)考察在mer+细胞系中，O 6AT mRNA水平变化10倍 并与特定细胞中已知的蛋白质水平相关。我们 这些发现可能对癌症治疗有重要意义， 例如BCNU。D.亚罗什有初步证据表明 肿瘤没有可检测的O 6AT活性。确定哪些肿瘤是合并的- 而那些mer+肿瘤具有低水平的O 6AT mRNA和蛋白质， 可能对化疗计划有用。正在努力 开发方法来测量体内O 6AT mRNA和蛋白水平。

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 O6-alkylguanine DNA alkyltransferase (O6AT); 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. Dr. D. Yarosh in collaboration with our unit has been able to partially purify the enzyme from human liver and raise polyclonal antibodies to this protein. Studies have been initiated to develop monoclonal antibodies to this protein. Recently, a cDNA clone for a human O6AT has been isolated on the basis that it protects O6AT-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 O6-alkylguanine. Proc. Natl. Acad. Sci. USA 1990;87:686-690). Using oligonucleotides based on this sequence, we have isolated a full-length O6AT cDNA clone from a human liver library which we had constructed. Using this clone as a probe, we have found that O6AT mRNA was markedly reduced in all the mer- tumor cells lines (8) examined. The level of O6AT 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 O6AT activity. Determining which tumors are mer- and which mer+ tumors have low levels of O6AT mRNA and protein would probably be useful in planning chemotherapy. Efforts are underway to develop approaches to measure O6AT mRNA and protein levels in vivo.