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Parallel pathways in RAF-induced senescence and conditions for its reversion

Abstract

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.

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Acknowledgements

We thank Chiara Castelli (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy) for pLXSN-hTERT, Marco Crescenzi (Istituto Superiore di Sanità, Rome, Italy) for GST-CKI plasmids, and Martin McMahon (University of California San Francisco, USA) for the RAF1-ER plasmids, the IMR90/GFP-RAF1-ER cells and for invaluable advice. JYT thanks Masashi Narita for fruitful discussions and we also thank Michel Toledano for comments on the manuscript. This work was supported by grants from the French National Research Agency (ANR-07-BLAN-0098-CSD8) and the French Association for Cancer Research (ARC 4917), and benefited from discussions within the CEA/Curie network on Replication, Chromosomal Instability, and Cancer. SR is the recipient of a postdoctoral fellowship from the French Association for Cancer Research, and KC received a CEA Irtelis doctoral fellowship through the CEA Plasticity and Instability of the Genome Intramural Program.

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Jeanblanc, M., Ragu, S., Gey, C. et al. Parallel pathways in RAF-induced senescence and conditions for its reversion. Oncogene 31, 3072–3085 (2012). https://doi.org/10.1038/onc.2011.481

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