The Loss of γH2AX Signal is a Marker of DNA Double Strand Breaks Repair Only at Low Levels of DNA Damage

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Abstract
The induction of DNA double-strand breaks (DSBs) by genotoxic treatment leads to high toxicity and genetic instability. Various approaches have been undertaken to quantify the number of breaks and to follow the kinetic of DSB repair. Recently, the phosphorylation of the variant histone H2AX (named γH2AX), quantified by specific immunodetection approaches, has provided a valuable and highly sensitive method to monitor DSBs formation. Although it is admitted that the number of γH2AX foci reflected that of DSBs, contradictory reports leave open the question of a link between the disappearance of γH2AX signal and DSBs repair. We determined γH2AX expression (i) in cells either proficient or not in DSBs repair capacity, (ii) after exposure to ionizing radiation (IR) or calicheamicin γ1, a radiomimetic compound, (iii) and by three different immunodetection methods, foci numbering, flow cytometry or Western blotting. We showed here that γH2AX loss correlates with DSB repair activity only at low cytotoxic doses, when less than 100-150 DSBs breaks per genome are produced, independently of the method used. In addition, in DNA repair proficient cells, the early decrease in the number and intensity of γH2AX foci observed after a 2 Gy exposure was not associated with a significant change in the global γH2AX level as determined by Western blotting or flow cytometry. These results suggest that the dephosphorylation step of γH2AX may be limiting and that the loss of foci is mediated not only by γH2AX dephosphorylation but also through its redistribution towards the chromatin.

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