Ras Proteins Induce Senescence by Altering the Intracellular Levels of Reactive Oxygen Species

Abstract

Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using this in vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O 2 ) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H 2 O 2 represents a critical signal mediating replicative senescence.

Enhanced elimination of oxidized guanine nucleotides inhibits oncogenic RAS-induced DNA damage and premature senescence

“…The relative difference in superoxide levels between H-RAS-transduced and nontransduced cells is not as great as that observed for total ROS levels ( Figure 2a). This quantitative discrepancy presumably reflects both the highly efficient dismutation of superoxide into hydrogen peroxide in BJ cells (Serra et al, 2003) and the contribution from elevated mitochondrial hydrogen peroxide production (Lee et al, 1999;Moiseeva et al, 2009), which is detected by chloromethyl-dichlorofluorescein diacetate but not hydroethidine.…”

“…Oncogenic RAS-mediated production of ROS has been demonstrated to be essential to its ability to induce senescence (Lee et al, 1999). Chemically, the types of ROS generated by oncogenic RAS, that is, hydrogen peroxide and superoxide radicals, have very low oxidation potential and are unable to directly damage DNA.…”

“…The senescence response to the RAS oncoprotein has been ascribed to its production of reactive oxygen species (ROS) (Irani et al, 1997;Lee et al, 1999) and to the resulting induction of a DNA damage response (DDR) (Di Micco et al, 2006;Mallette et al, 2007). However, the connection between these two RAS-induced effects has been unclear.…”

Oxidation resistance 1 is a novel senolytic target

“…OXR1 is crucial for protecting cells against oxidative stress by regulating the expression of several enzymes that detoxify ROS (Jaramillo‐Gutierrez, Molina‐Cruz, Kumar & Barillas‐Mury, 2010; Oliver et al., 2011; Yang et al., 2015), and it may be particularly important as a senolytic target of PL because SCs are known to produce high levels of ROS but remain resistant to oxidative stress (Supporting Information Figure S2) (Chandrasekaran, Idelchik & Melendez, 2017; Davalli, Mitic, Caporali, Lauriola & D'Arca, 2016; Lee et al., 1999; Lu & Finkel, 2008). In addition, LMD‐3, homolog of OXR1 in Caenorhabditis elegans , has been shown to protect against oxidative stress and accelerated aging in the worm (Sanada et al., 2014).…”

“…SCs are known to produce high levels of ROS, but they are highly resistant to oxidative stress (Chandrasekaran et al., 2017; Davalli et al., 2016; Lee et al., 1999; Lu & Finkel, 2008). This resistance may be due to the increased expression of OXR1 and its downstream targets in SCs; therefore, OXR1 may be a genuine senolytic target.…”