Phosphorylation of Histone H2AX on Ser 139 and Activation of ATM During Oxidative Burst in Phorbol Ester-Treated Human Leukocytes

Abstract

Oxidative burst is a defense mechanism used by specialized phagocytes such as granulocytes or monocytes to kill the invading microorganisms through generation of superoxide anions. Oxidative burst also results in DNA damage of the phagocytes. Phagocytes are terminally differentiated cells, some of very short life-span cells. We could find no reports whether oxidative burst-mediated DNA damage triggers in such cells histone H2AX-Ser139 phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM), the signals otherwise used to activate DNA repair and checkpoint pathways in proliferating cells. We now present the evidence that induction of oxidative stress in human peripheral blood leukocytes by phorbol myristate acetate (PMA) was associated with intense phosphorylation of histone H2AX and with ATM activation, seen already 60 min after exposure to PMA. The modifications of H2AX and ATM in individual granulocytes, monocytes and lymphocytes were detected prior to caspases activation and thus were unrelated to induction of apoptosis. A large intercellular variation in response was observed, and only a fraction of cells in these subpopulations showed H2AX and ATM modifications. The data are compatible with the earlier observations of DNA damage during oxidative burst and suggest that even in terminally differentiated cells that have a short life-span, DNA damage triggers recruitment of the DNA repair machinery. The observed H2AX phosphorylation in lymphocytes may reflect their DNA damage by the superoxide ions propagating from the neighboring granulocytes and/or monocytes.

Attenuation of the oxidative burst-induced DNA damage in human leukocytes by hyaluronan

“…Our present data confirm an earlier observation that the induction of oxidative burst in leukocytes by PMA leads to distinct DNA damage response as evidenced by phosphorylation of histone H2AX on Ser-139 and activation of ATM through its phosphorylation on Ser-1981 (16). The induction of ROS in cells undergoing the oxidative burst was detected using the permeant H2DCFDA probe which becomes fluorescent upon oxidation (27).…”

“…Similar to prior studies, the extent of DNA damage was assessed by the cytometric analysis of H2AX phosphorylation and ATM activation (16,(22)(23)(24)(25), the early events of the DNA damage response (14,15,26). The data show significant attenuation of both H2AX phosphorylation and ATM activation by HA, consistent with the earlier evidence of the protective effect of this constituent of intercellular matrix on oxidative DNA damage in other cell systems (17)(18)(19)(20)(21)(22).…”

“…It is likely therefore that the observed effects of HA, namely the attenuation of DNA damage response and reduction of ROS, are the consequences of HA internalization by these phagocytes and the intracellular neutralization of the reactive oxidants by this biopolymer. However, because the ROS, generated during the oxidative burst is mobile and has the ability to damage DNA of neighboring cells (13,16) it is also possible that the observed effect of HA was due to ROS neutralization outside the cells in the media in which the cells were suspended. We were unable however, to detect any differences in degradation of HA in tissue culture media samples in which the cells were suspended and treated with PMA versus the media from the untreated cells.…”

“…Activation of Ataxia Telangiectasia Mutated (ATM) through its phosphorylation on Ser1981 and phosphorylation of histone H2AX on Ser139 are early markers of a cell's response to DNA damage, particularly if the damage involves formation of DNA double-strand breaks (DSBs) (14,15). We recently reported that induction of oxidative burst in human peripheral blood leukocytes by PMA led to intense phosphorylation of H2AX and activation of ATM which was seen in monocytes, granulocytes as well as in lymphocytes (16). These data were consistent with earlier observations (13) that the reactive oxidants generated in phagocytes during oxidative burst were mobile and induced DNA damage in the neighboring lymphocytes.…”

Attenuation of acridine mutagen ICR-191 — DNA interactions and DNA damage by the mutagen interceptor chlorophyllin

“…The extent of DNA damagewas assessed by measurement the induction of H2AX phosphorylation, the marker considered to report genotoxic changes in DNA molecule in chromatin [32][33][34][35][36][37][38][39]. Indeed in numerous studies phosphorylation of H2AX was shown to strongly correlate with other markers of DNA damage, such as assessed by single cell gel electrophoresis (comet assay), micronucleus assay or clonogenicity [33][34][35][36][37]48]. Our data show for the first time that exposure of cells to ICR-191 mutagen for 1 h was adequate to induce phosphorylation of H2AX.…”