Journal
EXPERIMENTAL CELL RESEARCH
Volume 315, Issue 8, Pages 1505-1520Publisher
ELSEVIER INC
DOI: 10.1016/j.yexcr.2008.12.007
Keywords
Mouse embryonic stem cells; DNA repair foci; Gamma-H2AX; DNA single-strand breaks; Comet assay
Categories
Funding
- Canadian Cancer Society
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Pluripotent mouse embryonic stem cells (mES cells) exhibit similar to 100 large gamma H2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gamma H2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gamma H2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gamma H2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gamma H2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock. (C) 2008 Elsevier Inc. All rights reserved.
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