Numerical Analysis of Etoposide Induced DNA Breaks

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.

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci

“…The detection of cleavage at precise sites also found in vitro and at translocation breakpoint hotspots, as well as local base preferences for cleavage mirroring those in vitro, gave validation to our assay. Also, the 1.3%-3.4% TOP2A DNA DSBs that we found are consistent with prior studies (Zechiedrich et al 1989;Muslimovic et al 2009). Overall, our methodology achieved the genome-wide identification of TOP2A cleavage sites not previously accomplished.…”

“…Etoposide occupancy at both scissile bonds is required for DSBs, and most breaks formed at concentrations relevant to chemotherapy are SSNs (Bromberg et al 2003). Consistent with reports in which the vast majority of detected TOP2 cleavage events were SSNs (Bromberg et al 2003;Muslimovic et al 2009), we found 1.3%-3.4% of TOP2A cleavage events were DSBs and the remainder were SSNs (Supplemental Table S1; Supplemental Fig. S2).…”

“…The use of CIP to release the DNA is simpler because it is carried out directly on the bead-bound TOP2 without protease digestion. It is also critical for the enzyme used to hydrolyze the phosphodiester bond to have high activity at TOP2 SSNs as well as DSBs because most cellular TOP2 cleavage sites are SSNs (Zechiedrich et al 1989;Muslimovic et al 2009; data from the present study in Supplemental Table S1 and Supplemental Fig. S2).…”

Measurement of H2AX Phosphorylation as a Marker of Ionizing Radiation Induced Cell Damage

“…Single-stranded DNA damage is therefore not very toxic. As an example, over 100 000 hydrogen peroxide-induced single-stranded DNA damage is required to affect the viability of a human cell (Muslimovic, Nystrom et al 2009). However, template-directed repair is not possible when both DNA strands within 10-20 bp are damaged, called clustered DNA damage (Fig.…”

“…Several reports indicate that each IR-induced gamma-H2AX focus represents a single DSB, at least in G1 cells (Sedelnikova, Rogakou et al 2002;Pilch, Sedelnikova et al 2003;Rothkamm and Lobrich 2003). The level of gamma-H2AX induced by different drugs and radiation is closely correlated to the level of cell death (Banath and Olive 2003;Muslimovic, Nystrom et al 2009). This indicates that measurement of gamma-H2AX in patient cells will be a biologically important marker for the induction of DSBs and toxicity following chemotherapy and radiotherapy.…”