Medvedeva, N. G., Panyutin, I. V., Panyutin, I. G. and Neumann, R. D. Phosphorylation of Histone H2AX in Radiation-Induced Micronuclei. Radiat. Res. 168, 493–498 (2007).
DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to γ radiation, the proportion of γ-H2AX-positive to γ-H2AX-negative micronuclei increases. The majority of the γ-H2AX-positive micronuclei are centromere-negative. The number of γ-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most γ-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of γ-H2AX-positive to γ-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are γ-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, γ-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with γ-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with γ-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with γ-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.