Relationship between DNA double-strand break rejoining and cell survival after exposure to ionizing radiation in human fibroblast strains with differing ATM/p53 status: implications for evaluation of clinical radiosensitivity
To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early (< or =4 h) after exposure to 8 Gy of gamma-radiation and also exhibited high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX (gamma-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and gamma-H2AX assays. Our data suggest that the gamma-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.