Protocol

DNA Topoisomerases

Volume 582 of the series Methods in Molecular Biology™ pp 145-153

Date:

Cytometric Assessment of DNA Damage Induced by DNA Topoisomerase Inhibitors

  • Zbigniew DarzynkiewiczAffiliated withBrander Cancer Research Institute, New York Medical College
  • , Dorota H. HalickaAffiliated withBrander Cancer Research Institute, New York Medical College
  • , Toshiki TanakaAffiliated withBrander Cancer Research Institute, New York Medical CollegeFirst Department of Surgery, Yamaguchi University School of Medicine

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Abstract

Exposure of cells to inhibitors of DNA topoisomerase I (topo I) or topoisomerase II (topo II) leads to DNA damage that often involves formation of DNA double-strand breaks (DSBs). DNA damage, particularly induction of DSBs, manifests by phosphorylation of histone H2AX on Ser-139 which is mediated by one of the protein kinases of the phosphoinositide kinase family, namely ATM, ATR, and/or DNA-PK. The presence of Ser-139 phosphorylated H2AX (γH2AX) is thus a reporter of DNA damage. This protocol describes quantitative assessment of γH2AX detected immunocytochemically in individual cells combined with quantification of cellular DNA content by cytometry. The bivariate analysis of γH2AX expression versus DNA content allows one to correlate DNA damage with the cell cycle phase or DNA ploidy. The protocol can also be used to assess activation (Ser-1981 phosphorylation) of ATM; this event also revealing DNA damage induced by topo I or topo II inhibitors. Examples where DNA damage was induced by topotecan (topo I) and etoposide (topo II) inhibitors are provided.

Key words

Histone H2AX phosphorylation ataxia telangiectasia mutated ATM DNA double-strand breaks flow cytometry apoptosis cell cycle topotecan etoposide