Persistence and dynamics of DNA damage signal amplification determined by microcolony formation and live-cell imaging

Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from dna damaging agents. in the present study,we developed a microcolony assay,in which normal human diploid fibroblast-like cells exposed to ionizing radiation,were plated onto coverslips at very low density (3 cells/cm 2). cells were grown for up to 3 days,and phosphorylated atm at ser1981 and 53bp1 foci were analyzed as the markers for an amplified dna damage signal. we observed a dose-dependent increase in the fraction of non-dividing cells,whose increase was compromised by knocking down p53 expression. while large persistent foci were predominantly formed in non-dividing cells,we observed some growing colonies that contained cells with large foci. as each microcolony was derived from a single cell,it appeared that some cells could proliferate with large foci. a live-imaging analysis using htert-immortalized normal human diploid cells transfected with the egfp-tagged 53bp1 gene revealed that the formation of persistent large foci was highly dynamic. delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after x-irradiation. thus,our results indicate that amplified dna damage signal could be ignored,which may be explained in part by the dynamic nature of the amplification process.