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.

Approximately 20% of tumors contain activating mutations in the RAS family of oncogenes. As tumors progress to higher grades of malignancy, the expression of oncogenic RAS has been reported to increase, leading to an oncogene-induced senescence (OIS) response. Evasion of this senescence barrier is a hallmark of advanced tumors indicating that OIS serves a critical tumorsuppressive function. Induction of OIS has been attributed to either RAS-mediated production of reactive oxygen species (ROS) or to induction of a DNA damage response (DDR). However, functional links between these two processes in triggering the senescent phenotype have not been explicitly described. Our previous work has shown that, in cultured untransformed cells, preventing elimination of oxidized guanine deoxyribonucleotides, which was achieved by suppressing expression of the cellular 8-oxodGTPase, human MutT homolog 1 (MTH1), sufficed to induce a DDR as well as premature senescence. Here, we demonstrate that overexpression of MTH1 can prevent the oncogenic H-RAS-induced DDR and attendant premature senescence, although it does not affect the observed elevation in ROS levels produced by RAS oncoprotein expression. Conversely, we find that loss of MTH1 preferentially induces an in vitro proliferation defect in tumorigenic cells overexpressing oncogenic RAS. These results indicate that the guanine nucleotide pool is a critical target for intracellular ROS produced by oncogenic RAS and that RAS-transformed cells require robust MTH1 expression to proliferate.

Expansion of the genetic alphabet has been a long time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semi-synthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogs, such as propinyl isocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogs inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogs that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluoro benzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self pair formed between two d3FB analogs is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analog (dBEN). In all, the data suggest that structure, electrostatics and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.

Major contributors to therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) include mutations, a dense desmoplastic stroma that prevents drug delivery to the tumor, and activation of redundant signaling pathways. We have previously identified a mechanistic rationale for targeting STAT3 signaling to overcome therapeutic resistance in PDAC. In this study, we investigate the molecular mechanisms underlying the heterogeneous response to STAT3 and RAS pathway inhibition in PDAC. Effects of JAK/STAT3 inhibition (STAT3i) or MEK inhibition (MEKi) were established in ; and (PKT) mice and patient-derived xenografts (PDX). Amphiregulin (AREG) levels were determined in serum from human patients with PDAC, LSL- (KPC), and PKT mice. MEKi/STAT3i-treated tumors were analyzed for integrity of the pancreas and the presence of cancer stem cells (CSC). We observed an inverse correlation between ERK and STAT3 phosphorylation. MEKi resulted in an immediate activation of STAT3, whereas STAT3i resulted in TACE-induced, AREG-dependent activation of EGFR and ERK. Combined MEKi/STAT3i sustained blockade of ERK, EGFR, and STAT3 signaling, overcoming resistance to individual MEKi or STAT3i. This combined inhibition attenuated tumor growth in PDX and increased survival of PKT mice while reducing serum AREG levels. Furthermore, MEKi/STAT3i altered the PDAC tumor microenvironment by depleting tumor fibrosis, maintaining pancreatic integrity, and downregulating CD44 and CD133 CSCs. These results demonstrate that resistance to MEKi is mediated through activation of STAT3, whereas TACE-AREG-EGFR-dependent activation of RAS pathway signaling confers resistance to STAT3 inhibition. Combined MEKi/STAT3i overcomes these resistances and provides a novel therapeutic strategy to target the RAS and STAT3 pathway in PDAC. This report describes an inverse correlation between MEK and STAT3 signaling as key mechanisms of resistance in PDAC and shows that combined inhibition of MEK and STAT3 overcomes this resistance and provides an improved therapeutic strategy to target the RAS pathway in PDAC. http://cancerres.aacrjournals.org/content/canres/78/21/6235/F1.large.jpg .

2-Aminopurine (2-AP), a fluorescent analog of adenine, has been widely used as a probe for local DNA conformation, since excitation and emission characteristics and fluorescence lifetimes of 2-AP vary in a sequence-dependent manner within DNA. Using steady-state and time-resolved fluorescence techniques, we report that 2-AP appears to be unusually stacked in the internal positions of ATAT and TATA in duplex DNA. The excitation wavelength maxima for 2-AP within these contexts were red shifted, indicating reduced solvent exposure for the fluorophore. Furthermore, in these contexts, 2-AP fluorescence was resistant to acrylamide-dependent collisional quenching, suggesting that the fluorophore is protected by its stacked position within the duplex. This conclusion was further reinforced by the presence of a secondary peak at 275 nm in the fluorescence excitation spectra that is indicative of efficient excitation energy transfer from nearby non-fluorescent DNA bases. Fluorescence anisotropy decay and internal angular 'wobbling' motion measurements of 2-AP within these alternating AT contexts were also consistent with the fluorophore being highly constrained and immobile within the base stack. When these fluorescence characteristics are compared with those of 2-AP within other duplex DNA sequence contexts, they are unique.

Androgen deprivation (AD) is an effective method for initially suppressing prostate cancer (PC) progression. However, androgen-refractory PC cells inevitably emerge from the androgen-responsive tumor, leading to incurable disease. Recent studies have shown AD induces cellular senescence, a phenomenon that is cell-autonomously tumor-suppressive but which confers tumor-promoting adaptations that can facilitate the advent of senescence-resistant malignant cell populations. Because androgen-refractory PC cells emerge clonally from the originally androgen-responsive tumor, we sought to investigate whether AD-induced senescence (ADIS) affects acquisition of androgen-refractory behavior in androgen-responsive LNCaP and LAPC4 prostate cancer cells. We find that repeated exposure of these androgen-responsive cells to senescence-inducing stimuli via cyclic AD leads to the rapid emergence of ADIS-resistant, androgen-refractory cells from the bulk senescent cell population. Our results show that the ADIS phenotype is associated with tumor-promoting traits, notably chemoresistance and enhanced pro-survival mechanisms such as inhibition of p53-mediated cell death, which encourage persistence of the senescent cells. We further find that pharmacologic enforcement of p53/Bax activation via Nutlin-3 prior to establishment of ADIS is required to overcome the associated pro-survival response and preferentially trigger pervasive cell death instead of senescence during AD. Thus our study demonstrates that ADIS promotes outgrowth of androgen-refractory PC cells and is consequently a suboptimal tumor-suppressor response to AD.