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

D.Yarosh，R.S.Day，III和其他人已经表明，大约20%的 人肿瘤细胞株和病毒转化株对 由于明显缺乏O6-甲基鸟嘌呤DNA而导致的烷基化试剂 甲基转移酶(O6MT)；这种表型被指定为mer-。这 酶可消除鸟嘌呤0-6位的烷基化损伤，但不能消除鸟嘌呤的烷基化损伤 DNA中的其他位置。我们和其他人已经分离到了O6MT的cDNA克隆。我们的 克隆已被用作测量mRNA水平的探针，并已被 用于合成抗体开发用的重组蛋白。我们有 既往研究发现，O6MT基因在所有肿瘤中的表达均显著降低 对细胞系进行检测。在MER+中，O6MT mRNA水平变化了10倍 并与已知的蛋白质水平特别相关 细胞。与D.Yarosh合作。O6MT基因表达水平与O6MT 现已在患者的活组织标本中检测到活性 肺部和脑部肿瘤。其活性在不同来源的肿瘤中差异很大。 患者，但在肿瘤和邻近的正常组织中相似 同一个病人。总体而言，O6MT的相对mRNA水平与 活性，但在几个肺活检中活性相当低，而 MRNA水平未见明显变化。这表明O6MT的表达也可能是 在翻译和/或翻译后级别进行控制。我们的 这一发现可能对癌症治疗有重要意义， 例如BCNU。还启动了研究，以检查 O6MT在NCI专家小组人肿瘤细胞系中的表达 药物筛选计划。将研究O6MT基因的反应 与ZO1 CM 07184-04中的其他应激反应基因相结合，并将 并入了这个项目。

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-methylguanine DNA methyltransferase (O6MT); 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. We and others have isolated O6MT cDNA clones. Our clone has been used as a probe to measure mRNA levels and has also been used to synthesize recombinant protein for antibody development. We have found previously that O6MT mRNA was markedly reduced in all the mer- tumor cells lines examined. The level of O6MT mRNA varied by 10-fold in mer+ cell lines and correlated with known levels of the protein in particular cells. In collaboration with D. Yarosh. O6MT mRNA levels and O6MT activity have now been determined in biopsy specimens of patients with lung and brain tumors. The activity varied widely in tumors from different patients, but was similar in tumor and adjacent normal tissue from the same patient. In general, relative O6MT mRNA levels correlated with activity, but in several lung biopsies the activity was quite low while mRNA levels were not. This indicates that O6MT expression may also be controlled at the translational and/or post-translational levels. Our findings may have important implications in cancer therapy where agents such as BCNU are used. Studies have also been initiated to examine the expression of O6MT in human tumor lines from the panel employed in the NCI drug screening program. The response of the O6MT gene will be studied with other stress-responsive genes in ZO1 CM 07184-04 and will be incorporated into this project.