title: Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles.
abstract: The DNA adduct O(6)-methylguanine (O(6)MeG) induced by environmental genotoxins and anticancer drugs is a highly mutagenic, genotoxic and apoptotic lesion. Apoptosis induced by O(6)MeG requires mismatch repair (MMR) and proliferation. Models of O(6)MeG-triggered cell death postulate that O(6)MeG/T mispairs activate MMR giving rise to either direct genotoxic signaling or secondary lesions that trigger apoptotic signaling in the 2(nd) replication cycle. To test these hypotheses, we used a highly synchronized cell system competent and deficient for the repair of O(6)MeG adducts, which were induced by the S(N)1 methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We show that DNA double-strand breaks (DSBs) are formed in response to O(6)MeG at high level in the 2nd S/G(2)-phase of the cell cycle. This is accompanied by ATR and Chk1 phosphorylation, G(2)/M arrest and late caspase activation. Although cells undergo apoptosis out of the 2nd G(2)/M-phase, the majority of them recovers and undergoes apoptosis after passing through additional replication cycles. The late apoptotic effects were completely abolished by O(6)-methylguanine-DNA methyltransferase, indicating that non-repaired O(6)MeG is carried over into subsequent generations, eliciting there a late apoptotic response. We also demonstrate that with a low, non-toxic dose of MNNG the passage of cells through the 1st and 2nd S-phase is not delayed, although the dose is able to induce excessive sister chromatid exchanges. This suggests that a significant amount of O(6)MeG can be tolerated by recombination, which is a fast and highly efficient process preventing from S-phase blockage, DSB formation and cell death.
authors.affiliation: Department of Toxicology, Medical Faculty of Johannes Gutenberg-University Mainz, Mainz, Germany.
authors.forename: Steve
authors.initials: S
authors.lastname: Quiros
authors.forename: Wynand P
authors.initials: WP
authors.lastname: Roos
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D016466:CHO Cells; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D013388:Succinimides; D008769:Methylnitronitrosoguanidine; D006147:Guanine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase