Numerical analysis of etoposide induced DNA breaks - PubMed (original) (raw)
Numerical analysis of etoposide induced DNA breaks
Aida Muslimović et al. PLoS One. 2009.
Erratum in
- PLoS One. 2009;4(6). doi: 10.1371/annotation/290cebfd-d5dc-4bd2-99b4-f4cf0be6c838
Abstract
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Etoposide-induced DNA breaks detected by CFGE.
A homodimer of topoII binds and cleaves cellular DNA. Etoposide binds independently to each monomer to block religation and thereby locks the topoII-DNA complex. At low concentrations, only one of the topoII monomers will be bound by etoposide and unable to religate the break, resulting in a topoII-linked SSB (a). When both monomers are occupied by etoposide, a topoII-linked DSB will be generated (b). In CFGE cells are lysed and proteins removed from DNA by SDS and proteinase K, allowing detection of protein-linked SSBs and DSBs.
Figure 2. Strand breaks induced by etoposide and CLM.
SV40-transformed fibroblasts treated with (a) CLM (0–30 nM) or (b), etoposide (0–450 µM) for 40 min at 37°C. The induced levels of TSBs and DSBs were measured with neutral and alkaline CFGE. As a control, we also treated cells with the SSB-inducer H2O2 (200 µM) or DSB and SSB inducer CLM (15 nM) to demonstrate that neutral CFGE fails to detect SSBs (a, separate gel). Error bars represent variation in two separate experiments performed on two different days.
Figure 3. Induction of H2AX phosphorylation by etoposide- or CLM-induced DSBs.
SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM before analysis of H2AX phosphorylation and DSB-level by neutral CFGE. Error bars represent variation in two separate experiments performed on different days.
Figure 4. Effect on cell survival of etoposide- and CLM-induced DSBs and TSBs.
SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM for 40 minutes at 37°C before analysis of colony survival and levels of (a) TSBs or (b) DSBs by neutral and alkaline CFGEs calculated as described in materials and methods. Error bars represent variation in two separate experiments performed on two different days.
Figure 5. Cell survival and H2AX phosphorylation in response to etoposide or CLM.
SV40-transformed fibroblasts were treated with 0–150 µM etoposide or 0–5 nM CLM before analysis of colony survival and H2AX phosphorylation. Error bars represent variation in two separate experiments performed on two different days.
Figure 6. Induction of DSBs and H2AX phosphorylation at different cell-cycle stages.
G361 cells were treated with 3 or 10 nM CLM (a) or 75 µM or 250 µM Etoposide (b) for 40 minutes before DNA staining, FACS sorting of G1, S and G2 cells, and analysis of DSBs by neutral CFGE. G361 cells were untreated or treated with 0.1 nM CLM or 75 µM Etoposide (c) for 40 minutes before DNA staining and analysis of H2AX phosphorylation and DNA-content to examine H2AX phosphorylation in G1, S and G2 cells. G361 cells were treated with CLM (d) or Etoposide (e) for 40 minutes before DNA staining and analysis of H2AX phosphorylation. Error bars represent variation from two separate experiments performed on two different days.
Figure 7. Time-dependent induction of DSBs and H2AX phosphorylation.
Analysis of DSBs and H2AX phosphorylation in SV40-transformed fibroblasts treated with 3 nM CLM (a) or 250 µM etoposide (b) for 0, 20, 40, 80 or 160 minutes at 37°C before analysis of DSBs with neutral CFGE and H2AX phosphorylation. Error bars represent variation in two separate experiments performed on two different days.
Figure 8. Etoposide-induced DNA damage in cells.
A homodimer of topoII binds and cleaves cellular DNA, generating a topoII-linked DSB. Etoposide binds independently to each monomer to block religation, locking the topoII monomer to the DNA break. If only one of the topoII monomers is bound by etoposide and unable to religate the break, this results in a topoII-linked SSB (a). When both monomers are occupied by etoposide, a topoII-linked DSB will be stabilized (b). TopoII-linked DNA breaks that are encountered by RNA or DNA polymerases during etoposide exposure will be denatured and therefore unable to religate the breaks. Denatured topoII will be cleared from the breaks, resulting in free DSBs that can induce H2AX phosphorylation. The relative amounts of these breaks as a percentage of all etoposide-induced breaks are indicated.
References
- Nias AHW. An introduction to radiobiology, 2nd edition. John Wiley & Sons Ltd; 1998.
- Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer. 2003;3:155–168. - PubMed
- Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–5868. - PubMed
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