Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

Paulina Rybak,Tytus Bernas,Lukasz Bujnowicz,Jerzy Dobrucki,Mirosław Zarębski,Krzysztof Berniak,Zbigniew Darzynkiewicz,Agnieszka Hoang

doi:10.18632/oncotarget.10411

Abstract

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Highlights

Phosphorylation of histone H2AX on serine 139 is an early step in cellular response to DNA damage, when the damage results in formation of a double-strand break (DSB)
We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs
In order to study γH2AX foci of various size and brightness we exposed A549 cells to CPT, MTX or ETP according to a protocol described in Figure 1A, and imaged foci of γH2AX, 53BP1 as well as DNA replication factories in cells in various sub-stages of S-phase (Figure 1B, 1C)

Summary

Introduction

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to DNA damage, when the damage results in formation of a double-strand break (DSB). ΓH2AX foci are readily detected by immunofluorescence microscopy and are generally regarded as markers of DSBs [2, 3]. ΓH2AX foci exhibit a wide range of sizes and levels of the phosphorylated histone. This fact was generally assumed to reflect no more than an increasing or decreasing extent of H2AX phosphorylation at different time periods elapsing from the induction of a DSB, reflecting the kinetic progression or regression of individual γH2AX foci. We postulate that some small foci containing low levels of γH2AX may be formed in response to DNA lesions other than DSBs

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