Reitsema, T. J., Banáth, J. P., MacPhail, S. H. and Olive,P. L. Hypertonic Saline Enhances Expression of Phosphorylated Histone H2AX after Irradiation. Radiat. Res. 161, 402– 408 (2004).
Phosphorylation of histone H2AX at serine 139 occurs at sites surrounding DNA double-strand breaks, producing discrete spots called “foci” that are visible with a microscope after antibody staining. This modification is believed to create changes in chromatin structure and assemble various repair proteins at sites of DNA damage. To examine the role of chromatin structure, human SiHa cells were exposed to hypertonic salt solutions that are known to condense chromatin and sensitize cells to chromosome damage and killing by ionizing radiation. Postirradiation incubation in 0.5 M Na increased γH2AX expression about fourfold as measured by flow cytometry and immunoblotting, and loss of γH2AX was inhibited in the presence of high salt. Focus size rather than the number of radiation-induced γH2AX foci was also increased about fourfold. When high-salt treatment was delayed for 1 h after irradiation, effects on focus size and retention were reduced. The increase in focus size was associated with a decrease in the rate of rejoining of double-strand breaks as measured using the neutral comet assay. We conclude that γH2AX expression after irradiation is sensitive to salt-induced changes in chromatin structure during focus formation, and that a large focus size may be an indication of a reduced ability to repair DNA damage.