Abstract
That irradiated cells affect their unirradiated ‘bystander’ neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named γ-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces γ-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple γ-H2AX foci. After 18 h coculture with cells irradiated with 20 α-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with γ-rays, and in cultures containing media conditioned on γ-irradiated cells. DNA DSB repair proteins accumulated at γ-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying γ-H2AX focus formation may be responsible for its downstream manifestations.
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Acknowledgements
We thank RARAF staff, Columbia University, for experimental support, R Neumann, NIH, and L Feinendegen, Heinrich-Heine-University Dusseldorf, for critical reading of the manuscript, W DeGraff, NIH, for help with γ-irradiation of the cells and I Kareva, NIH, for technical assistance. This work was supported by grants from the National Institutes of Health (NIH/NIBIB) (P41-EB002033), Department of Energy (DOE) (DE-FG02-03ER63629), and National Aeronautics and Space Administration (NASA) (NAG9-1519).
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Sokolov, M., Smilenov, L., Hall, E. et al. Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts. Oncogene 24, 7257–7265 (2005). https://doi.org/10.1038/sj.onc.1208886
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DOI: https://doi.org/10.1038/sj.onc.1208886
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