Numerical analysis of etoposide induced DNA breaks.
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.


RESULTS
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
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.
Muslimović
Aida
A
Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.
Nyström
Susanne
S
Gao
Yue
Y
Hammarsten
Ola
O
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D045744:Cell Line, Tumor; D002469:Cell Separation; D002470:Cell Survival; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism; D006801:Humans; D008962:Models, Theoretical; D010766:Phosphorylation; D013997:Time Factors
D000972:Antineoplastic Agents, Phytogenic; C468783:H2AX protein, human; D006657:Histones; D005047:Etoposide