Biological response to 8-oxoguanine base released during DNA base excision repair

Aging and age-associated diseases have been associated with altered cellular signaling, chromatin perturbation, oxidative stress, and DNA damage, especially at the telomere. Despite having evolved a sophisticated surveillance system, oxidative DNA damage still accumulates in the cells of organismal tissues. 8-oxo-7,8-dihydroguanine (8-oxo-Gua) is a compound occurring in cells as a major oxidative lesion due to guanine’s susceptibility to oxidation. A base excision repair pathway initiated by 8-oxoguanine DNA glycosylase (OGG1) excises 8-oxo-Gua lesions from nuclear and mitochondrial DNA, releasing the free oxidized base. It is well-established that failure to repair 8-oxo-Gua can result in mutagenesis and carcinogenesis; however, the cellular response to free 8-oxo-Gua has never been investigated. Initial experiments show that 8-oxo-Gua induced premature senescence in a concentration-dependent manner when added to the medium of human diploid fibroblasts (HDFs), while other oxidized and unmodified bases had no effect. We address a potential cause-effect relationship between free 8-oxo-Gua and the premature senescence of HDFs as a model for aging. We hypothesize that free 8-oxo-Gua released during DNA base excision repair is an endogenous mediator of cellular senescence through oxidative stress and altered cellular signaling. Using cell culture models and advanced cellular and molecular biology methods such as microarray, ROS detection assays, immunoblot, qRT-PCR, shRNA and siRNA technology, microscopy, as well as LC/IDMS for quantification of 8-oxo-Gua to test this hypothesis, we: 1) established that 8-oxo-Gua-induced senescent cells exhibit the morphology, features, and gene expression profile of naturally senescent cells; 2) determined that 8-oxo-Gua activates Ras in an unscheduled, prolonged manner, and that its activation transduces through the Raf-MEK(1/2)-ERK(1/2) MAPK cascade; 3) identified a chemical transformation of 8-oxo-Gua to a hydroperoxide-like form under physiological pH and oxygen atmosphere; and 4) determined 8-oxo-Gua-induced oxidative stress, which arises from the Rac1-Ras-NOX-PLA2-LO pathways, does not have a significant role in the observed senescence.