γ-H2AX foci and the measurement of variation in radiosensitivities of human and rodent cells

Abstract

DNA double strand breaks produced by radiation or other agents results in massive phosphorylation of the H2AX histone variant in chromatin around the site of each break. Immunocytochemical detection of this phosphorylated form (γ-H2AX) reveals fluorescent foci in irradiated cells and was used in a low dose rate irradiation assay to resolve even relatively small differences in cellular radiosensitivity. This assay revealed highly significant differences in the radiation response for cells derived from humans and mice that were heterozygous or normal with respect to the ATM gene. The assay also revealed differences for cells from unaffected parents of children with retinoblastoma and for cells from about 30% of clinically normal individuals. Variation in γ-H2AX induction and disappearance after irradiation were also measured in mutant and wild type CHO cells and in normal human fibroblasts. Disappearance of γ-H2AX foci after irradiation was much slower for mitotic than for G1 cells although no differences were seen for another assay measuring DNA breakage by gel electrophoresis. The expressed γ-H2AX foci in G0/G1 or G2 cells can persist into metaphase but the levels expressed in mitosis is dependent on the repair capacity for the different DNA damages. γ-H2AX foci can disappear with either rejoined or misrejoined DSBs. Go premature chromosome condensation analysis showed γ-H2AX foci disappeared after rejoining of DSBs and even after misrejoining. I also observed that high expression of γ-H2AX in S phase cells is related to DNA replication. After the process of premature chromosome condensation, S-phase PCC showed huge expression of γ-H2AX foci related to replication.