Armstrong M J, Galloway S M
Merck Research Laboratories, Department of Genetic and Cellular Toxicology, West Point, PA 19486, USA.
Mutat Res. 1997 Feb 3;373(2):167-78. doi: 10.1016/s0027-5107(96)00234-5.
O6-Methylguanine (O6MeG) is important in induction of chromosome aberrations (abs), with the unusual property that new abs are produced in the second cycle after treatment; cells lacking repair by O6-alkylguanine DNA-alkyltransferase (AGT) have more abs at the second division (M2) than at the first (M1). These second-cycle abs are likely caused by attempted correction by mismatch repair (MMR) of O6MeG:T mispairs, since we previously showed that MMR-deficient human cells (MT1 lymphoblasts) treated with SN-1 methylating agents do not produce new abs at M2 and are resistant to killing. Here we used MMR-deficient rodent cells to examine ab induction by alkylators and by incorporated 6-thioguanine (6-tG) which produces mispairs. BrdUrd labeling was used to identify cells at first, second and third metaphase after treatment (M1, M2 and M3). MMR-deficient Chinese hamster Clone B cells were 10-fold more resistant to ab induction by methyl nitrosourea and 1-methyl-3-nitro-1-nitrosoguanidine compared to their MMR-proficient parent cells, CHO MT+. Both cell lines express AGT and can remove the methyl group from O6MeG. Clone B has twice the AGT activity of CHO MT+, but inhibition of AGT with O6-benzylguanine did not change ab induction, indicating that methylation tolerance of Clone B cells was due to defective MMR and not to increased repair of O6MeG. Confirming the importance of O6MeG in inducing abs, even when it is a minor component of the adducts induced, Clone B cells were 2-fold more resistant to ab induction by methyl methanesulfonate and dimethylsulfate, whereas they had normal sensitivity to ethyl nitrosourea and 1-ethyl-3-nitro-1-nitrosoguanidine. Clone B cells are also resistant to killing by 6-tG, and 6-tG induced few abs in MMR-deficient Clone B (6-fold lower than CHO MT+ cells). Since mispairs do not occur until the cell cycle following incorporation of 6-tG, new abs in MMR-proficient cells are expected one cell cycle later than with the methylators, i.e., at M3. As expected, in normal CHO MT+, high ab levels were seen at M3, but there was also ab induction at M2. Similarly, with methylating agents we saw higher levels of abs at M1 in the MMR-proficient CHO MT+ cells than in Clone B cells, suggesting that in the rodent cells, MMR is involved in ab formation from mispairs or modified base pairs induced in the first S-phase, such as O6MeG:C. These rodent cells thus differ from human MT1 lymphoblasts which had similar ab levels to their normal parent cells at the first metaphase after treatment with methylators.
O6-甲基鸟嘌呤（O6MeG）在诱导染色体畸变（abs）方面很重要，具有一种不寻常的特性，即处理后在第二个细胞周期会产生新的染色体畸变；缺乏O6-烷基鸟嘌呤DNA-烷基转移酶（AGT）修复功能的细胞在第二次分裂（M2）时比第一次分裂（M1）时有更多的染色体畸变。这些第二周期的染色体畸变可能是由于对O6MeG:T错配进行错配修复（MMR）的尝试性校正导致的，因为我们之前表明，用SN-1甲基化剂处理的MMR缺陷型人类细胞（MT1淋巴细胞）在M2时不会产生新的染色体畸变，并且对杀伤具有抗性。在这里，我们使用MMR缺陷型啮齿动物细胞来研究烷化剂和掺入产生错配的6-硫鸟嘌呤（6-tG）所诱导的染色体畸变。使用溴脱氧尿苷标记来识别处理后第一次、第二次和第三次中期（M1、M2和M3）的细胞。与它们具有MMR修复功能的亲代细胞CHO MT+相比，MMR缺陷型中国仓鼠克隆B细胞对甲基亚硝基脲和1-甲基-3-硝基-1-亚硝基胍诱导的染色体畸变具有1０倍的抗性。两种细胞系都表达AGT，并且能够从O6MeG上去除甲基。克隆B的AGT活性是CHO MT+的两倍，但用O6-苄基鸟嘌呤抑制AGT并没有改变染色体畸变的诱导，这表明克隆B细胞的甲基化耐受性是由于MMR缺陷，而不是由于O6MeG修复增加。即使O6MeG是诱导的加合物中的次要成分，克隆B细胞对甲磺酸甲酯和硫酸二甲酯诱导的染色体畸变也具有2倍的抗性，这证实了O6MeG在诱导染色体畸变中的重要性，而它们对乙基亚硝基脲和1-乙基-3-硝基-1-亚硝基胍具有正常的敏感性。克隆B细胞对6-tG杀伤也具有抗性，并且6-tG在MMR缺陷型克隆B中诱导的染色体畸变很少（比CHO MT+细胞低6倍）。由于错配直到6-tG掺入后的细胞周期才会出现，因此预计MMR修复功能正常的细胞中出现新的染色体畸变比甲基化剂诱导的要晚一个细胞周期，即在M3时。正如预期的那样，在正常的CHO MT+中，M3时观察到高染色体畸变水平，但在M2时也有染色体畸变的诱导。同样，对于甲基化剂，我们在MMR修复功能正常的CHO MT+细胞的M1时观察到的染色体畸变水平高于克隆B细胞，这表明在啮齿动物细胞中，MMR参与了第一次S期诱导的错配或修饰碱基对（如O6MeG:C）形成染色体畸变的过程。因此，这些啮齿动物细胞与人类MT1淋巴细胞不同，后者在用甲基化剂处理后的第一次中期时染色体畸变水平与其正常亲代细胞相似。
