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• W2619354234 abstract "A17 The ability to reliably and quantitatively detect DNA damage is critical to cancer research. Multi-parameter cytometric measurements of the levels of γH2AX and activated ATM expression allow estimation of the extent of DNA damage caused by multiple factors such as ionizing radiation, carcinogenic agents, anti-cancer drugs and correlation of this damage with the cell cycle phase. Using laser scanning cytometry (LSC) technology we measured globally per nucleus and by enumeration of immunofluorescence (IF) foci γH2AX expression and ATM activation (ATM-Ser1981 phosphorylation; pATM).
 In dual-label γH2AX/DNA and pATM/DNA content studies, we validated quantification of the number of foci representing individual double strand breaks (DSBs) per nucleus in relation to the cell cycle phase. At low levels of DSBs, both pH2AX and pATM presented as distinct foci, reporting the number of DSBs per nucleus. At higher levels, when foci were too dense for individual enumeration, the overall immunofluorescence integrated over the nucleus was successfully applied as a measure of the pH2AX activation. After establishing background (constitutive) levels of DNA damage in cell lines, we administered treatment with topoisomerase inhibitor camptothecin (CPT), which showed that cells in S-phase were all saturated with γH2AX, while individual foci could be enumerated in cells in G1 and G2. We also demonstrated that the number of foci was reduced after treatment with 2-deoxy-D-glucose, implicating metabolically generated oxidants as the cause of the DSBs. Dual-label pATM/DNA content studies showed similar results, with pATM aggregating in foci within nuclei in response to oxidative stress.
 Finally, we established a high-content DNA damage assay comprising simultaneous measurement of pH2AX, pATM (expression levels and individual foci counts for both) and DNA content. This novel multi-parameter biomarker assay allows simultaneous assessment of ATM activation, H2AX phosphorylation and correlation with the cell cycle phase. It is particularly well suited for detection of low levels of DNA damage, such as occurring constitutively. Our results suggest that activated ATM foci are fewer and more diffuse than those of γH2AX. However, since ATM activation is upstream of H2AX phosphorylation in the DNA DSB repair pathway, it could serve as a useful early marker for these lesions. We will present the results of several screens for compounds that induce or inhibit DNA damage, as well as an assessment of DNA protective properties of “antioxidants” available in general health stores." @default.
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• W2619354234 date "2007-10-01" @default.
• W2619354234 modified "2023-09-23" @default.
• W2619354234 title "High-content DNA damage assay: Simultaneous measurement of H2AX phosphorylation, ATM activation, and DNA content" @default.
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