Numerical analysis of etoposide induced DNA breaks

PLoS One. 2009 Jun 10;4(6):e5859. doi: 10.1371/journal.pone.0005859.


Background: Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.

Methodology/principal findings: To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.

Conclusions/significance: These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Phytogenic / pharmacology
  • Cell Line, Tumor
  • Cell Separation
  • Cell Survival
  • DNA Breaks, Double-Stranded / drug effects*
  • DNA Damage / drug effects*
  • Dose-Response Relationship, Drug
  • Etoposide / pharmacology*
  • Flow Cytometry
  • Histones / metabolism
  • Humans
  • Models, Theoretical
  • Phosphorylation
  • Time Factors


  • Antineoplastic Agents, Phytogenic
  • H2AX protein, human
  • Histones
  • Etoposide