Early Increase of Radiation-induced γH2AX Foci in a Human Ku70/80 Knockdown Cell Line Characterized by an Enhanced Radiosensitivity

Abstract

A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

“…Obviously, residual foci are still present in both cell lines 24 h after irradiation. In contrast with this are the results of Vandersickel et al [8], who did not observe residual foci in MCF10A cells and MCF10A cells silenced for Ku70 and Ku80 24 h after treatment. It might be that the cells with residual foci had already died and therefore were not scored.…”

“…Maximal values are reached about 30 min after irradiation. The number of these initial foci is similar to the number of expected DNA DSBs and decreases with the repair of these DSBs [7][8][9]. Since γ-H2AX is associated with DSB repair, the kinetics of the formation and loss of γ-H2AX foci are related to the efficiency of repair [10][11][12].…”

Inhibitory effects of valproic acid in DNA double-strand break repair after irradiation in esophageal squamous carcinoma cells

“…This phosphorylation rapidly spreads to a region of chromatin surrounding the DSBs, resulting in the formation of γH2AX foci. The main role of H2AX phosphorylation in the DNA damage response was reported to be the recruitment of DNA repair proteins to DSB sites and the preparation of a platform for signaling and repair (38,40). In the present study, we analyzed H2AX phosphorylation levels by western blotting, and examined γH2AX foci by immunocytochemistry as a marker of DNA DSB after irradiation.…”

“…Previously, γH2AX has been described as a highly sensitive method to detect irradiation-induced DSBs (38,39). In response to this, serine 139 within the conserved C-terminal region of the H2AX protein becomes phosphorylated by one of the DNA damage signaling kinases of the phosphatidylinositol-3-OH- kinase-like family.…”

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB)

“…AlexaFluor 488 goat anti-mouse, Invitrogen, Paisley, UK). All participants scored foci manually ) in a total of 50 cells per sample and in addition three laboratories scored a total of 200 cells per sample automatically using Cellprofiler (www.cellprofiler.org) or MetaCyte (Metasystems, Altlussheim, Germany; Vandersicklel et al 2010). Also, participants used the 2 Gy reference samples to test the validity of their calibration curves and the unirradiated samples gave an indication of the level of background staining achieved on the day of the exercise, as suggested in Rothkamm et al (2013b).…”