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

Yasuyoshi Oka, Motohiro Yamauchi, Masatoshi Suzuki, Shunichi Yamashita, Keiji Suzuki

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)766-774
Number of pages9
JournalJournal of radiation research
Volume52
Issue number6
DOIs
Publication statusPublished - Nov 2011
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Radiation
  • Radiology Nuclear Medicine and imaging
  • Health, Toxicology and Mutagenesis

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