Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles

Cell Cycle. 2010 Jan 1;9(1):168-78. doi: 10.4161/cc.9.1.10363. Epub 2010 Jan 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Apoptosis / genetics
  • Apoptosis / physiology*
  • Blotting, Western
  • CHO Cells
  • Cell Cycle / drug effects
  • Cell Cycle / genetics
  • Cell Cycle / physiology*
  • Cricetinae
  • Cricetulus
  • DNA Breaks, Double-Stranded*
  • DNA Replication / drug effects
  • DNA Replication / genetics
  • DNA Replication / physiology*
  • Flow Cytometry
  • Fluoresceins / metabolism
  • Guanine / analogs & derivatives*
  • Guanine / metabolism
  • Methylnitronitrosoguanidine / pharmacology
  • Microscopy, Fluorescence
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism
  • Succinimides / metabolism


  • 5-(6)-carboxyfluorescein diacetate succinimidyl ester
  • Fluoresceins
  • Succinimides
  • Methylnitronitrosoguanidine
  • Guanine
  • O-(6)-methylguanine
  • O(6)-Methylguanine-DNA Methyltransferase