Single cell tracking reveals that Msh2 is a key component of an early-acting DNA damage-activated G2 checkpoint.
Dysfunction of cell-cycle checkpoints in DNA mismatch repair (MMR)-deficient cells in response to DNA damage has implications for anticancer therapy and genetic instability. We have studied the cell-cycle effects of MMR deficiency (Msh2(-/-)) in primary mouse embryonic fibroblasts (MEFs) exposed to cisplatin (10 microM x 1 h) using time-lapse microscopy. Kinetic responses of MEFs from different embryos and passage ages varied, but we report a consistent drug-induced inhibition of mitotic entry (approx. 50%). There was a loss of an early-acting (<5 h) delay in G2 to M transition in Msh2(-/-) cells, although a later-acting G2 arrest was apparently normal. This suggests that Msh2 primarily acts to delay mitotic entry of cells already in G2, that is, DNA damage incurred during G2 does not influence the cell once committed to mitotic traverse. Irrespective of Msh2 status, cisplatin treatment and the incurred DNA damage did not effect mitotic traverse or show any evidence for early (within 24 h) cell death. The results indicate that Msh2(-/-) status can result in the premature commitment to mitosis of a cell subpopulation, determined by the fraction residing in G2 at the time of damage induction. The findings suggest a new route to MMR-driven genetic instability that does not rely primarily on the integrity of the late-acting checkpoint.
Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
Nuria
N
Marquez
Sally C
SC
Chappell
Owen J
OJ
Sansom
Alan R
AR
Clarke
Jon
J
Court
Rachel J
RJ
Errington
Paul J
PJ
Smith
D000818:Animals; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D004268:DNA-Binding Proteins; D005347:Fibroblasts; D016195:G2 Phase / Q000187:drug effects; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D051718:MutS Homolog 2 Protein; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C497173:Msh2 protein, mouse; D051718:MutS Homolog 2 Protein; D002945:Cisplatin