Preferential cleavage sites have been determined for Fe 2؉ /H 2 O 2 -mediated oxidations of DNA. In 50 mM H 2 O 2 , preferential cleavages occurred at the nucleoside 5 to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG) 81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H 2 O 2 , preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe 2؉ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe 2؉ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe 2؉ with the three purine N 7 residues. Fe 2؉ -dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A 5m CGA were consistent with this model.

Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21 Cip1 , p53, and p16 INK4A tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.8-oxoguanine ͉ DNA damage ͉ p53 ͉ reactive oxygen species (ROS) W hen propagated in culture, normal somatic cells achieve a limited number of divisions before undergoing the loss of proliferative capacity termed replicative senescence (1). Several studies have suggested that cell senescence plays a role in organismic aging (2, 3) and that activation of senescence programs in cancer cells block tumor progression (4, 5). Consequently, elucidating the biochemical mechanisms of cellular senescence is critical for understanding the physiologic basis of aging and the mechanisms of tumorigenesis.Several lines of evidence indicate that cumulative damage to cellular constituents sustained during culture in vitro eventually triggers senescence (6, 7). Such damage can be inflicted by reactive oxygen species (ROS), which are byproducts of incomplete mitochondrial electron transfer (8). Despite the actions of detoxifying enzymes, such as superoxide dismutases (SOD1 and SOD2) and catalase, and low molecular weight antioxidants, increasing oxidative stress due to age-related mitochondrial dysfunction may eventually exceed the capacity of cellular antioxidant defenses (9). Indeed, both ROS levels and oxidative damage levels are found to be higher in late-passage cells relative to early-passage cells (10). Additionally, increased oxidative stress in the form of hyperoxia (11), treatment with hydrogen peroxide (12), shRNA-mediated knockdown of SOD1 (13), or oncogenic Ras overexpression (14, 15), causes cells to enter senescence prematurely. Conversely, culturing...

Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence (OIS). In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but induces a reduced proliferative rate and an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors.