Abstract
ATM-p53 pathway plays a critical role in maintaining genomic stability through its participation in multiple cell cycle checkpoints. After irradiation, ATM protein kinase is activated and autophosphorylated at the sites of DNA double-strand breaks (DSBs). Phosphorylated ATM (pATM) forms the initial foci in the chromatin region flanking DSBs. Subsequently, the number of foci decreases concurrently with DSBs repair, while some fraction of foci remain persistently. Thus, it can be hypothesized that the persistent foci formation of DNA damage checkpoint factors is involved in ATM-p53-dependent permanent cell cycle arrest. In the present study, we established single-cell based assay to test the hypothesis. Normal human diploid cells were irradiated with ionizing radiation and plated onto cover slips at low density. After incubation for 3 days, pATM and 53BP1 foci were investigated by fluorescence microscopy. In this experimental setting, no micro-colony which all cells had foci was observed in rapidly growing colonies, but the frequency of cells with foci was sufficiently high in the fraction of non-dividing and slowly growing colonies. Foci size in non-dividing and slowly growing cells is larger than those of rapidly growing cells. These results suggest that large persistent foci of pATM and 53BP1 are sufficient for permanent cell cycle arrest of normal human diploid cells exposed to radiation.
