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

[Abstract]
Research on aging or anti-aging is receiving increasing attention in the aging society. Since the accumulation of senescent cells is closely related to the aging of individuals and organs, in vitro studies of cellular senescence and the search for anti-aging substances are important. However, as shown by Hayflick & Moore's study, which was the beginning of cellular senescence research, senescent cells are characterized by the arrest of cell division, which requires a huge amount of equipment and expense to secure a sufficient number of cells, especially in the search for anti-aging substances. Therefore, we attempted to induce cellular senescence by introducing double-stranded DNA (dsDNA), which was reported as a novel senescent cell marker in mouse embryonic fibroblasts during development, into normal human epidermal keratinocytes (NHEK), which are capable of dividing. In normal NHEK cultures, significant expression of Senescence Associated-β-Galactosidase (SA-β-Gal), a senescent cell marker, was observed after passage 5 (P5), but its expression was limited in P3 and P4 NHEKs. dsDNA into the NHEK of P4 showed a significant upregulation of SA-β-Gal expression. In addition, the enhanced expression of inflammatory cytokines (IL-1α, IL-6, IL-8, TNF-α), the increase in the expression of senescent cell marker p16INK4A, the increase in the number of γ-H2AX positive cells expressed during gene damage and the decrease in the number of Ki67 positive cells expressed in dividing cells were observed. These results indicate that dsDNA transfection induces the induction of senescent cell markers. Furthermore, mRNA expression of ceramide synthase CerS3, which is reported to be decreased in senile xeroderma, was also decreased in dsDNA-transfected cells. These results suggest that the introduction of dsDNA into cells in NHEK induces cellular senescence and provides a means to create a senescent cell model.