title: 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.
authors.affiliation: Academic Medical Center, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.
authors.forename: Bregje
authors.initials: B
authors.lastname: van Oorschot
authors.affiliation: Academic Medical Center, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Hovingh
authors.affiliation: Academic Medical Center, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.
authors.forename: Annelot
authors.initials: A
authors.lastname: Dekker
authors.affiliation: Academic Medical Center, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.
authors.forename: Lukas J
authors.initials: LJ
authors.lastname: Stalpers
authors.affiliation: Academic Medical Center, Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands.
authors.forename: Nicolaas A P
authors.initials: NA
authors.lastname: Franken
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology* / Q000528:radiation effects; D019583:Dose Fractionation, Radiation; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000502:physiology / Q000528:radiation effects; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011836:Radiation Tolerance / Q000502:physiology* / Q000528:radiation effects*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: C468783:H2AX protein, human; D006657:Histones