Akihiro Aioi
Development Laboratory, SEPTEM-SOKEN Co., Ltd.,
Tomozumi Imamichi
Frederick National Laboratory for Cancer Research, Applied and Developmental Research Directorate
Jun-ichi Kashiwakura
Department of Life Science, Faculty of Pharmaceutical Sciences, Hokkaido University of Sciences
Emiko Okuda-Ashitaka
Department of Biomedical Engineering, Osaka Institute of Technology

[Abstract]
Since the Hayflick-limit was reported in 1961, cellular senescence markers have been identified. Previously, we presented that the transfection of double-stranded DNA (dsDNA) into human keratinocytes induced cell senescence. Here we show upregulation of other cellular senescence markers and the putative mechanism under the dsDNA-induced cell senescence (DICS). The mRNA levels of IL-1α and TNF-α were significantly upregulated in dsDNA-transfected keratinocytes, concomitant with significant decrease in the ratio of Ki67 positive cells. These results strengthened the evidence for the induction of cellular senescence by dsDNA transfection. Next, to explore the underlying mechanism of DICS, the effects of AZ20, an inhibitor of ATR, on DICS were evaluated. The dsDNA-enhanced mRNA levels of IL-6 and IL-8 were abolished to the same levels of mock treatment by the addition of 10μM AZ20. Immunofluorescence staining indicated that the SA-β-Gal positive cells were significantly reduced in the AZ20-pretreated keratinocytes. Furthermore, the positive cell population of GATA4, which is a transcription factor involved in the downstream of ATR signaling pathway, was increased in dsDNA-transfected keratinocytes. These results suggest that ATR is involved in DICS. We believe that the dsDNA-induced senescent cell model contributes the development of anti-cell senescence products.