Chromosomal instability, reproductive cell death and apoptosis induced by O6-methylguanine in Mex-, Mex+ and methylation-tolerant mismatch repair compromised cells: facts and models.
O6-Methylguanine (O6-MeG) is induced in DNA by methylating environmental carcinogens and various cytostatic drugs. It is repaired by O6-methylguanine-DNA methyltransferase (MGMT). If not repaired prior to replication, the lesion generates gene mutations and leads to cell death, sister chromatid exchanges (SCEs), chromosomal aberrations and malignant transformation. To address the question of how O6-MeG is transformed into genotoxic effects, isogenic Chinese hamster cell lines either not expressing MGMT (phenotypically Mex-), expressing MGMT (Mex+) or exhibiting the tolerance phenotype (Mex-, methylation resistant) were compared as to their clastogenic response. Mex- cells were more sensitive than Mex+ cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced chromosomal breakage, with marked differences in sensitivity depending on recovery time. At early recovery time, when cells out of the first post-treatment mitosis were scored, aberration frequency was about 40% reduced in Mex+ as compared to Mex- cells. At later stages of recovery when cells out of the second post-treatment mitosis were analyzed, the frequency of aberrations increased strongly in Mex- cells whereas it dropped to nearly control level in Mex+ cells. From this we conclude that, in the first post-treatment replication cycle of Mex- cells, only a minor part of aberrations (< 40%) was due to O6-MeG whereas, in the second post-treatment replication cycle, the major part of aberrations (> 90%) was caused by the lesion. Thus, O6-MeG is a potent clastogenic DNA damage that needs two DNA replication cycles in order to be transformed with high efficiency into aberrations. The same holds true for sister chromatid exchanges (SCEs). MNNG is highly potent in inducing SCEs in Mex- cells in the second replication cycle after alkylation. Under these conditions, SCE induction is nearly completely prevented by the expression of MGMT. This is opposed to SCE induction in the first post-treatment replication cycle, where higher doses of MNNG were required to induce SCEs and no protective effect of MGMT was observed. This indicates that SCEs induced in the first replication cycle after alkylation are due to other lesions than O6-MeG. In methylation tolerant cells, which are characterized by impaired G-T mismatch binding and MSH2 expression, aberration frequency induced by MNNG was weakly reduced in the first and strongly reduced in the second post-treatment mitoses, as compared to CHO wild-type cells. The results indicate that mismatch repair of O6-MeG-T mispairs is decisively involved in O6-MeG born chromosomal instability and recombination. We also show that Mex+ and methylation tolerant cells are more resistant than Mex- cells with regard to induction of apoptosis, indicating O6-MeG to be also an apoptosis-inducing lesion. The data are discussed as to the mechanism of cytotoxicity, aberration and SCE formation in cells treated with a methylating agent.
Kaina
B
B
Division of Applied Toxicology, University of Mainz, Germany.
Ziouta
A
A
Ochs
K
K
Coquerelle
T
T
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D016466:CHO Cells / Q000187:drug effects*; D016923:Cell Death / Q000235:genetics; D002869:Chromosome Aberrations; D002875:Chromosomes / Q000187:drug effects* / Q000648:ultrastructure; D006224:Cricetinae; D003412:Cricetulus; D018736:DNA Adducts / Q000737:chemistry*; D019175:DNA Methylation; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D004351:Drug Resistance; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology / Q000633:toxicity*; D008957:Models, Genetic; D009152:Mutagenicity Tests; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D012854:Sister Chromatid Exchange / Q000187:drug effects
D018736:DNA Adducts; D008769:Methylnitronitrosoguanidine; D006147:Guanine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase