Hypertonic Saline Enhances Expression of Phosphorylated Histone H2AX after Irradiation

Abstract 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.

[1]  P. Olive,et al.  Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays , 2003, International journal of radiation biology.

[2]  W. Dewey,et al.  Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions. , 1979, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[3]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[4]  T. Kosaka,et al.  Correlation between non-repairable DNA lesions and fixation of cell damage by hypertonic solutions in Chinese hamster cells. , 1990, International journal of radiation biology.

[5]  E. Rogakou,et al.  Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo , 1999, The Journal of cell biology.

[6]  Ying-li Yu,et al.  Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G1-Phase Cells , 2003, Radiation research.

[7]  R. Bonner,et al.  Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks , 2003, Nature Cell Biology.

[8]  K. Weibezahn,et al.  Relationship between double strand break rejoining and G2 block formation in V79 cells , 1986, Radiation and environmental biophysics.

[9]  A. Fornace,et al.  Rapid activation of G2/M checkpoint after hypertonic stress in renal inner medullary epithelial (IME) cells is protective and requires p38 kinase , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[10]  M. Hayashi,et al.  Hypertonic Treatment Inhibits Radiation-Induced Nuclear Translocation of the Ku Proteins G22p1 (Ku70) and Xrcc5 (Ku80) in Rat Fibroblasts , 2001, Radiation research.

[11]  W. Dewey,et al.  Enhancement of x-ray damage in synchronous Chinese hamster cells by hypertonic treatments. , 1972, Radiation research.

[12]  W. Dewey,et al.  Alterations in the radiosensitivity of CHO cells by anisotonic treatments: correlations between cell lethality and chromosomal aberrations. , 1979, Radiation research.

[13]  J. Schwartz,et al.  Metaphase chromosome and nucleoid differences between CHO-K1 and its radiosensitive derivative xrs-5. , 1993, Mutagenesis.

[14]  G. Raaphorst,et al.  Effect of salt solutions on radiosensitivity of mammalian cells. I. Specific ion effects. , 1977, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[15]  Olive Pl,et al.  Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines , 1994 .

[16]  P. Olive,et al.  Higher-order chromatin structure-dependent repair of DNA double-strand breaks: factors affecting elution of DNA from nucleoids. , 1998, Radiation research.

[17]  M. Kastan,et al.  DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation , 2003, Nature.

[18]  J. Soranson,et al.  The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage. , 1990, International journal of radiation biology.

[19]  B. Trask,et al.  Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus , 1995, The Journal of cell biology.

[20]  P. Olive,et al.  Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines. , 1994, Cancer research.