Activation of the S phase DNA damage checkpoint by mitomycin C

J Cell Physiol. 2007 May;211(2):468-76. doi: 10.1002/jcp.20957.

Abstract

We have studied the rate of DNA synthesis, cell cycle distribution, formation of gamma-H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S-phase and induced the appearance of gamma-H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage-induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S-phase arrest induced by MMC is ATR-dependent. These findings were confirmed by experiments with ATR-deficient and AT cells. They indicate that the DNA damage ATR-dependent S-phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstadienes / pharmacology
  • Antibiotics, Antineoplastic / pharmacology*
  • Ataxia Telangiectasia / genetics
  • Ataxia Telangiectasia / metabolism
  • Ataxia Telangiectasia / pathology
  • Ataxia Telangiectasia Mutated Proteins
  • Caffeine / pharmacology
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / metabolism*
  • Cell Nucleus / drug effects*
  • Cell Nucleus / metabolism
  • Chromatin / metabolism
  • Cross-Linking Reagents / pharmacology*
  • DNA Damage / drug effects*
  • DNA Replication / drug effects
  • DNA-Binding Proteins / antagonists & inhibitors
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Histones / metabolism
  • Humans
  • Mitomycin / pharmacology*
  • Nuclear Matrix / metabolism
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / deficiency
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rad51 Recombinase / metabolism
  • Recombination, Genetic / drug effects
  • S Phase / drug effects*
  • Time Factors
  • Tumor Suppressor Proteins / antagonists & inhibitors
  • Tumor Suppressor Proteins / metabolism*
  • Wortmannin

Substances

  • Androstadienes
  • Antibiotics, Antineoplastic
  • Cell Cycle Proteins
  • Chromatin
  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • Protein Kinase Inhibitors
  • Tumor Suppressor Proteins
  • Caffeine
  • Mitomycin
  • ATM protein, human
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases
  • RAD51 protein, human
  • Rad51 Recombinase
  • Wortmannin