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.

Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

“…Senescence of human fibroblasts induced by the RASval12 oncogene is associated with a higher level of DNA damage than senescence induced by an activated RAF kinase192021. Consistent with this difference, we observed an accumulation of H2A.J in IMR90hTERT fibroblasts induced into senescence by RASval12 (Fig.…”

“…WI-38 human embryonic fibroblasts from the ATCC were immortalized with hTERT and grown in MEM (Invitrogen #21090)+10% Fetal Bovine Serum+1 mM Sodium Pyruvate+2 mM L -glutamine+0.1 mM non-essential amino acids1621. Cells were mycoplasma negative.…”

Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels

“…For example, contrary to the Raf effects observed in this study, Ras activation decreased SQSTM1 protein levels [44], while increasing ATG7 mRNA levels in human diploid fibroblast cells [45]. Indeed, although aberrant Ras or Raf activation triggers largely similar physiological effects, many of these effects are mediated via different mechanisms because Ras can activate additional pathways other than Raf/MEK/ERK, including the PI3-kinase/AKT and RalGDS pathways [46]. It is therefore conceivable that Raf/MEK/ERK alone may not be able to mediate balanced autophagy while Ras does so by activating additional pathways.…”

Physiological and Pathological Aging Affects Chromatin Dynamics, Structure and Function at the Nuclear Edge

“…SAHFs do not form in all senescent cells. Their production might depend on the senescence induction and are mainly associated with oncogenic senescence rather than replicative senescence (Narita et al, 2003; Zhang et al, 2005; Funayama et al, 2006; Jeanblanc et al, 2012). …”