Abstract
Cellular aging is characterized by the loss of DNA replication capability and is mainly brought about by various changes in chromatin structure. Here, we examined changes in MCM2-7 proteins, which act as a replicative DNA helicase, during aging of human WI38 fibroblasts at the single-cell level. We used nuclear accumulation of p21 as a marker of senescent cells, and examined changes in MCM2-7 by western blot analysis. First, we found that senescent cells are enriched for cells with a DNA content higher than 4N. Second, the levels of MCM2, MCM3, MCM4 and MCM6 proteins decreased in senescent cells. Third, cytoplasmic localization of MCM2 and MCM7 was observed in senescent cells, from an analysis of MCM2-7 except for MCM5. Consistent with this finding, fragmented MCM2 was predominant in these cells. These age-dependent changes in MCM2-7, a protein complex that directly affects cellular DNA replication, may play a critical role in cellular senescence.
Highlights
Cellular aging is attributable to various genotoxic stimuli such as telomere shortening, DNA damage, reactive oxygen species and excess growth stimulation by oncogenes (Kuilman et al, 2010), and it is associated with changes in chromatin structure (Ishimi et al, 1987; O’Sullivan and Kariseder, 2012) and in epigenetic regulation (Sidler et al, 2017)
We examined changes in MCM2–7 during cellular aging of WI38 fibroblasts at the single-cell level
Cells with a DNA content of 4N had higher levels of p21 on average (Fig. 1D); this finding is consistent with the notion that cells arrested at G2 phase by various stimuli to induce cellular aging bypass M phase to enter G1 phase and become senescent (Johmura et al, 2014)
Summary
Cellular aging is attributable to various genotoxic stimuli such as telomere shortening, DNA damage, reactive oxygen species and excess growth stimulation by oncogenes (Kuilman et al, 2010), and it is associated with changes in chromatin structure (Ishimi et al, 1987; O’Sullivan and Kariseder, 2012) and in epigenetic regulation (Sidler et al, 2017). The DNA damage checkpoint system that is activated by shortened telomeres and double-stranded DNA breakage plays a role in preventing cell cycle progression into S phase (d’Adda di Fagagna et al, 2003). This system includes activation of checkpoint kinases such as ATM and thereby activation of transcription factor p53. It has been shown that senescent cells have a DNA content of 4N (Johmura et al, 2014) These data indicate that cells arrested at G2 phase by various stimuli to induce cellular aging bypass M phase to enter. Biochemical analysis suggested that fragmented MCM2 was predominant in the cells
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