Predicting Radiosensitivity with Gamma-H2AX Foci Assay after Single High-Dose-Rate and Pulsed Dose-Rate Ionizing Irradiation

Abstract

Gamma-H2AX foci detection is the standard method to quantify DNA double-strand break (DSB) induction and repair. In this study, we investigated the induction and decay of γ-H2AX foci of different tumor cell lines and fibroblasts with known mutations in DNA damage repair genes, including ATM, LigIV, DNA-PKcs, Rad51 and Rad54. A radiation dose of 2.4 Gy was used for either an acute single high-dose-rate (sHDR) exposure or a pulsed dose-rate (pDR) exposure over 24 h. The number of γ-H2AX foci was determined at 30 min and 24 h after sHDR irradiation and directly after pDR irradiation. In a similar manner, γ-H2AX foci were also examined in lymphocytes of patients with differences in normal tissue toxicity after a total radiation dose of 1 Gy. In an initial count of the number of foci 30 min after sHDR irradiation, repair-proficient cell types could not be distinguished from repair-deficient cell types. However at 24 h postirradiation, while we observed a large decrease in foci numbers in NHEJ-proficient cells, the amount of γ-H2AX foci in cell types with mutated NHEJ repair remained at high levels. Except for IRS-1SF cells, HR-deficient cell types eventually did show a moderate decrease in foci number over time, albeit to a lesser extent than their corresponding parentals or repair-proficient control cells. In addition, analysis of γ-H2AX foci after sHDR exposure of patients with different sensitivity status clearly showed individual differences in radiation response. Radiosensitive patients could be distinguished from the more radioresistant patients with γ-H2AX foci decay ratios (initial number of foci divided by residual number of foci). Significantly higher decay ratios were observed in patients without toxicities, indicating more proficient repair compared to patients with radiation-induced side effects. After pDR irradiation, no consistent correlation could be found between foci number and radiosensitivity. In conclusion, γ-H2AX formation is a rapid and sensitive cellular response to DNA DSBs. Decay ratios after sHDR exposure elucidated large differences in γ-H2AX foci kinetics between the repair-proficient or -deficient cell types and patients. This assay may be useful for measuring cellular radiosensitivity and could serve as a clinically useful test for predicting radiosensitivity ex vivo before treatment.

Quantitative γ-H2AX immunofluorescence method for DNA double-strand break analysis in testis and liver after intravenous administration of 111InCl3

“…This will cause a lower foci-count directly after a protracted exposure than after a single high-dose rate exposure with the same total absorbed dose. However, the foci count after a 24 h protracted irradiation is expected to be higher than 24 h after an acute exposure, as shown by van Oorschot et al [50]. In addition, the magnitude of the dose-rate effect does vary between cell types, due to the inherent radiosensitivity, in our study represented by two contrasting tissues, related to the degree of differentiation and proliferation but also the repair time of sub-lethal DNA damage.…”

The impact of melatonin and carbon ion irradiation on cancer stem cells

“…Phosphorylation of H2AX is a sensitive marker for detection of radiation-induced DSB [162,163]. The number of radiation induced γH2AX foci is closely related to the number of DSB (164,165). Slowly repaired or unrepaired DSB may account for cell death [32,166].…”

Compromized DNA repair as a basis for identification of cancer radiotherapy patients with extreme radiosensitivity

“…We also considered alternative classifiers, namely the apparent repair rate in the 1–8-hr time interval, which can be calculated using a simpler linear regression algorithm we described previously 39 , and the ratio of foci number at 1/24 hrs, suggested by van Oorschot et al 32, 38 . The scatter plots for NOR and OR groups are presented in Supplementary Figure 2.…”

“…In 22 relevant studies published in 2008-2016, the post-irradiation γ-H2AX response has been compared in “over-responders” (OR), “not-over-responders” (NOR), and normal blood donors 18-38 , including our report of a RS clinical case 39 . Thirteen studies reported the ability of the assay to predict RT-associated normal tissue toxicity, while 9 studies reported that the biomarker is not predictive.…”