Parallel pathways in RAF-induced senescence and conditions for its reversion.
We developed a clonal WI-38hTERT/GFP-RAF1-ER immortal cell line to study RAF-induced senescence of human fibroblasts. Activation of the GFP-RAF1-ER kinase by addition of 4-hydroxy-tamoxifen led to a robust induction of senescence within one population doubling, accompanied by the assembly of heterochromatic foci. At least two pathways contribute in parallel to this senescence leading to the accumulation of p15, p16, p21 and p27 inhibitors of cyclin-dependent kinases (CKIs). Cells that traversed S phase after RAF1 kinase activation experienced a replicative stress manifested by phosphorylation of H2AX and Chk2 and synthesis of p21. However, about half the cells in the population were blocked without passing through S phase and did not show activation of DNA-damage checkpoints. When the cells were cultivated in 5% oxygen, RAF1 activation generated minimal reactive oxygen species, but RAF-induced senescence occurred efficiently in these conditions even in the presence of anti-oxidants or inhibitors of DNA checkpoint pathways. Despite the presence of heterochromatic foci, simultaneous knockdown of p16 and p21 with inactivation of the GFP-RAF1-ER kinase led to rapid reversion of the senescent state with the majority of cells becoming competent for long-term proliferation. These results demonstrate that replicative and oxidative stresses are not required for RAF-induced senescence, and this senescence is readily reversed upon loss of CKIs.
CEA, iBiTec-S, Service de Biologie Intégrative et Génétique Moléculaire-Bât, 142, CEA/Saclay, Gif-sur-Yvette, France.
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M
Jeanblanc
S
S
Ragu
C
C
Gey
K
K
Contrepois
R
R
Courbeyrette
J-Y
JY
Thuret
C
C
Mann
D016922:Cellular Senescence; D003260:Contact Inhibition; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004249:DNA Damage; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C493956:CDKN1A protein, human; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009363:Neoplasm Proteins; D017382:Reactive Oxygen Species; D019908:Proto-Oncogene Proteins c-raf; D048051:p38 Mitogen-Activated Protein Kinases; D010100:Oxygen