Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays

Abstract

Purpose: Exposure to ionizing radiation results in phosphorylation of histone H2AX (γH2AX) at sites of DNA double‐strand breaks. To determine the relationship between γH2AX formation and radiosensitivity, the rate of formation and loss of γH2AX were examined in several cultured cell lines following exposure to 250 kV X‐rays.

Materials and methods: Flow and image cytometry were both performed using a mouse monoclonal antibody against γH2AX. Immunoblotting was used to confirm cell line‐dependent differences in antibody staining. Cell lines examined included V79 and CHO‐K1 hamster cells, the human tumour cell lines SiHa, WiDr, DU145, WIL‐2NS, HT144, HCC1937 and U87, and the normal cell strain HFL1. Radiosensitivity was measured using a standard clonogenic assay.

Results: Using flow cytometry, γH2AX formation was detected 1 h after doses as low as 20 cGy. Peak levels of γH2AX were observed within 15–30 min after irradiation and both the rate of radiation‐induced γH2AX formation and loss were cell type dependent. Maximum levels of γH2AX formation were lower for HT144 cells mutant for the ataxia telangiectasia gene. Half‐times of loss after irradiation ranged from 1.6 to 7.2 h and were associated with a decrease in the total number of foci per cell. The half‐time of loss of γH2AX was correlated with clonogenic survival for 10 cell lines (r²=0.66).

Conclusions: γH2AX can be detected with excellent sensitivity using both flow and image analysis. The rate of γH2AX loss may be an important factor in the response of cells to ionizing radiation, with more rapid loss and less retention associated with more radioresistant cell lines.