γH2AX responses in human buccal cells exposed to ionizing radiation

Abstract

DNA double strand breaks are induced by ionizing radiation (IR), leading to the phosphorylation of the core histone protein H2AX (termed cH2AX). The understanding of the cH2AX responses in irradiated human buccal cells is still very limited. We used visual scoring and laser scanning cytometry (LSC) methods to investigate cH2AX signaling following exposure of human buccal cells (from six individuals) to ionizing radiation at 0-4 Gy. The frequency of nuclei containing 15-30 cH2AX foci was significantly elevated 30 min post-IR exposure (by visual scoring). Concomitantly, there was a significant decrease in the frequency of cells without foci following exposure to IR. IR-induced cH2AX signal as determined by laser scanning cytometry (which included cH2AX integral and MaxPixel value) increased significantly in all individual's 2N nuclei 30 min post-IR and was similar for all three nuclear shapes identified. Individuals with the lowest baseline cH2AX integral (i.e., in nonirradiated cells) showed the greatest fold stimulation of cH2AX and significant dose-responses to IR doses of 1, 2, and 4 Gy. In 5 out of 6 individuals, the frequency of visually scored cH2AX in nuclei showed a strong correlation (up to r 5 0.999) with LSC scored cH2AX integrals. The cH2AX response and subsequent decline varied between individuals but remained elevated above baseline levels 24 h post IR exposure. cH2AX response in irradiated human buccal cells has potential to be used as an index of baseline DNA damage in population studies. The variable response to IR exposure between individuals should be taken into consideration when using the cH2AX assay for radiation biodosimetry. V C 2014 International Society for Advancement of Cytometry

Persistent γH2AX: A promising molecular marker of DNA damage and aging

“…In a recent study by our group, gH2AX levels remained elevated in g-irradiated human buccal cells compared with non-irradiated control cells for up to 24 h following exposure to 4 Gy of IR as measured by quantitative laser scanning cytometry [48]. These results suggest that radiation induced gH2AX levels in human buccal cells may remain elevated above the baseline gH2AX level for a relatively long time (up to 24 h).…”

“…area, integral, MaxPixel) after inducing DNA damage [48,53,54]. The frequency of visually scored gH2AX in human buccal cell nuclei showed a strong correlation with LSC measured gH2AX integral [48]. Taken together, both microscopy and cytometry-based methods are suitable to evaluate gH2AX formation and loss and the choice of the best gH2AX assay will depend on the purpose of the study.…”

“…More recently, the use of laser scanning cytometry has also been proposed as a useful tool to measure cellular DNA content for cell cycle stage evaluation in conjunction with multiple gH2AX parameters (e.g. area, integral, MaxPixel) after inducing DNA damage [48,53,54]. The frequency of visually scored gH2AX in human buccal cell nuclei showed a strong correlation with LSC measured gH2AX integral [48].…”

“…The use of fluorescence can be extended to the measurement of total gH2AX protein level, in particular, types of cells and tissues using western blot and flow cytometry techniques [42][43][44]. The gH2AX foci counting approach has been used in numerous studies to assess the relationship between gH2AX foci removal and the rate of DSBs repair [25,[45][46][47][48]. In radiation biology the number of DSBs positively correlates with gH2AX foci formation [6,23,24].…”

“…Buccal cells are an easily accessible source of tissue and have been investigated for radiation biodosimetry [48,104]. The kinetics of gH2AX induction and loss in buccal cells were investigated by counting gH2AX foci for up to 5 h after exposure to 2 Gy of IR [104].…”