title: Persistence and dynamics of DNA damage signal amplification determined by microcolony formation and live-cell imaging.
abstract: 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.
authors: Atomic Bomb Disease Institute, Graduate School of Biochemical Sciences, Nagasaki University, Nagasaki 852-8523, Japan.
authors: Yasuyoshi
authors: Y
authors: Oka
authors: Motohiro
authors: M
authors: Yamauchi
authors: Masatoshi
authors: M
authors: Suzuki
authors: Shunichi
authors: S
authors: Yamashita
authors: Keiji
authors: K
authors: Suzuki
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D059447:Cell Cycle Checkpoints / Q000235:genetics* / Q000528:radiation effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D003114:Colony-Forming Units Assay; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D055785:Gene Knockdown Techniques; D016158:Genes, p53; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000528:radiation effects; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D025521:Tumor Suppressor Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases