Stable cellular senescence is associated with persistent DDR activation.

Kenneth B Marcu,Fabrizio D’Adda Di Fagagna,Chiara Mondello,Francesca Rossiello,Marzia Fumagalli

doi:10.1371/journal.pone.0110969

Kenneth B Marcu, Fabrizio D’Adda Di Fagagna + Show 3 more

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https://doi.org/10.1371/journal.pone.0110969

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Abstract

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.

Highlights

Upon generation of a DNA damage, cells activate a cascade of events known as DNA damage response (DDR) to coordinate the DNA repair and the transient arrest of cell-cycle progression until DNA damage has been removed in full [1]
Cellular senescence has been causally linked with organismal aging [28,29,30,31] and DDR activation has been demonstrated in vivo in tissues of aging mammals including primates [5,12,32,33,34,35,36,37,38] and in human skin naevi, which are composed of oncogene-induced senescent melanocytes [39]
These cells in cultures can be considered very deeply senescent. When we analyzed these cultures for the presence of DDR foci, we discovered that the vast majority (70–80%) of cells in both cultures still displayed markers of DDR activation containing ATM pS1981, co-localizing with 53BP1 and cH2AX; their morphology and number per cell was similar to that previously observed in senescent cultures (Fig. 2a)

Summary

Introduction

Upon generation of a DNA damage, cells activate a cascade of events known as DNA damage response (DDR) to coordinate the DNA repair and the transient arrest of cell-cycle progression until DNA damage has been removed in full [1]. It has been reported that while early in the senescence process DDR can be readily detected in the majority of the cells, after prolonged establishment of cellular senescence, detection of markers of DDR activation is reduced. This has led some investigators to conclude that markers of an activated DDR are detectable only in ‘‘senescing’’ cultures of human fibroblasts and that they are lost when cultures are ‘‘fully senescent’’ [42,43]

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