DNA damage response activation in mouse embryonic fibroblasts undergoing replicative senescence and following spontaneous immortalization.
Primary mouse embryonic fibroblasts (MEFs) are a popular tool for molecular and cell biology studies. However, when MEFs are grown in vitro under standard tissue culture conditions, they proliferate only for a limited number of population doublings (PD) and eventually undergo cellular senescence. Presently, the molecular mechanisms halting cell cycle progression and establishing cellular senescence under these conditions are unclear. Here, we show that a robust DNA damage response (DDR) is activated when MEFs undergo replicative cellular senescence. Senescent cells accumulate senescence-associated DDR foci (SDFs) containing the activated form of ATM, its phosphorylated substrates and gammaH2AX. In senescent MEFs, DDR markers do not preferentially accumulate at telomeres, the end of linear chromosomes. It has been observed that proliferation of MEFs is extended if they are cultured at low oxygen tension (3% O(2)). We observed that under these conditions, DDR is not observed and senescence is not established. Importantly, inactivation of ATM in senescent MEFs allows escape from senescence and progression through the S-phase. Therefore, MEFs undergoing cellular senescence arrest their proliferation due to the activation of a DNA damage checkpoint mediated by ATM kinase. Finally, we observed that spontaneously immortalized proliferating MEFs display markers of an activated DDR, indicating the presence of chromosomal DNA damage in these established cell lines.
Di Micco
Raffaella
R
IFOM Foundation-FIRC Institute of Molecular Oncology Foundation, Milan, Italy.
Cicalese
Angelo
A
Fumagalli
Marzia
M
Dobreva
Miryana
M
Verrecchia
Alessandro
A
Pelicci
Pier Giuseppe
PG
di Fagagna
Fabrizio d'Adda
Fd
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D016922:Cellular Senescence; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology*; D051379:Mice; D010100:Oxygen / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D010100:Oxygen