Abstract
BackgroundEtoposide 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 FindingsTo 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/SignificanceThese 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.
Highlights
Cancer is often treated with agents that induce DNA doublestrand breaks (DSBs) that preferentially kill dividing cells and, are slightly more toxic to fast-growing tumor cells
Etoposide induces mainly single-stranded DNA breaks To examine the relative amounts of single-strand breaks (SSBs) and double-strand breaks (DSBs) produced by etoposide (Fig. 1), we measured DNA strand breaks in cells treated with etoposide
We used a combination of methods to measure DSBs, SSBs, toxicity and H2AX phosphorylation to examine the relative amounts of strandbreaks, DNA damage signaling and cell survival
Summary
Cancer is often treated with agents that induce DNA doublestrand breaks (DSBs) that preferentially kill dividing cells and, are slightly more toxic to fast-growing tumor cells. H2AX differs from its homologue H2A in that it contains a distinct C-terminal extension, with a consensus target sequence at serine 139 for the DSB-activated kinases ATM, ATR, and DNA-PK [4,5]. Together, these kinases are responsible for the formation of several thousands of phosphorylated H2AX surrounding the DSB [5,6,7,8]. Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules.
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