Chronic inflammation has long been recognized as a risk factor for many human cancers. One mechanistic link between inflammation and cancer involves the generation of nitric oxide, superoxide and other reactive oxygen and nitrogen species by macrophages and neutrophils that infiltrate sites of inflammation. Although pathologically high levels of these reactive species cause damage to biological molecules, including DNA, nitric oxide at lower levels plays important physiological roles in cell signaling and apoptosis. This raises the question of inflammation-induced imbalances in physiological and pathological pathways mediated by chemical mediators of inflammation. At pathological levels, the damage sustained by nucleic acids represents the full spectrum of chemistries and likely plays an important role in carcinogenesis. This suggests that DNA damage products could serve as biomarkers of inflammation and oxidative stress in clinically accessible compartments such as blood and urine. However, recent studies of the biotransformation of DNA damage products before excretion point to a weakness in our understanding of the biological fates of the DNA lesions and thus to a limitation in the use of DNA lesions as biomarkers. This review will address these and other issues surrounding inflammation-mediated DNA damage on the road to cancer. More Than an Association Between Chronic Inflammation and CancerStemming from the original observations by Virchow, 1 the link between chronic inflammation and cancer is now recognized as essentially a cause-and-effect relationship. 2-7 Epidemiological evidence suggests that more than 20% of all cancers are caused by chronic infection or other types of chronic inflammation, 8 with multiple lines of evidence from laboratory-and population-based studies pointing to a persistent local inflammatory state in organspecific carcinogenesis 9-15 even for tumors not epidemiologically linked to infection or inflammation. There are extremely strong correlations between chronic exposure to asbestos and mesotheloima, 16,17 and chronic infections and cancer for liver flukes (O. viverrini) and cholangiocarcinoma, 18,19 Helicobacter pylori and gastric cancer, 20-22 viral hepatitis and liver cancer 23 and Schistosoma haematobium and bladder cancer. 24,25 Although the epidemiological evidence is well established, the mechanisms underlying the link between chronic inflammation and cancer are not. These mechanisms can be arbitrarily divided into biological and chemical as illustrated in Figure 1 for infection-induced inflammation. The initial infection leads to cell death and changes in cell phenotype, with the release of cytokines and chemotactic factors that cause infiltration of macrophages, neutrophils, lymphocytes and other immune cells. The biological side of chronic inflammation entails the effects of cytokines and chemokines on host cell cycle and apoptosis, whereas the chemical side involves generation of a variety of reactive oxygen and nitrogen species by activated phagocytes with the goal of er...

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing betathalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent proteinbeta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells.beta-thalassemia ͉ gene correction ͉ triplex-forming oligonucleotides ͉ gene targeting M utations in the beta-globin gene that affect any stage in beta-globin biogenesis can cause beta-thalassemia. Identified mutations include single base pair changes that lead to frameshift mutations or changes in canonical sequences that affect mRNA stability and processing (1). As monogenic disorders, betathalassemia and sickle cell anemia have attracted substantial efforts directed at gene therapy by gene replacement, and there has been ongoing progress in this regard. In one approach specific to the thalassemias in which the genetic defect affects mRNA splicing, antisense oligonucleotides have been used to manipulate the splice site choice in beta-globin premRNA to prevent aberrant splicing. Restoration of proper beta-globin splicing has been demonstrated in human erythroid cells derived from beta-thalassemic patients, and in transgenic mouse models containing splicing mutations in the beta-globin gene (2, 3).In this study, we use an antigene approach to correct a thalassemia-causing splice-site mutation at the level of chromosomal DNA in cultured cells, generating heritable, site-specific modification of the beta-globin gene. We have used peptide nucleic acids (PNAs), a class of triplex-forming molecules shown to be effective at provoking recombination and repair at chromosomal sites near PNA binding sites (4). PNAs contain standard nucleobases linked to a peptide-like backbone, and their advantages include resistance to nucl...

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.

Background: In the event of a nuclear accident, people are exposed to elevated levels of continuous low dose-rate radiation. Nevertheless, most of the literature describes the biological effects of acute radiation.Objectives: DNA damage and mutations are well established for their carcinogenic effects. We assessed several key markers of DNA damage and DNA damage responses in mice exposed to low dose-rate radiation to reveal potential genotoxic effects associated with low dose-rate radiation.Methods: We studied low dose-rate radiation using a variable low dose-rate irradiator consisting of flood phantoms filled with 125Iodine-containing buffer. Mice were exposed to 0.0002 cGy/min (~ 400-fold background radiation) continuously over 5 weeks. We assessed base lesions, micronuclei, homologous recombination (HR; using fluorescent yellow direct repeat mice), and transcript levels for several radiation-sensitive genes.Results: We did not observe any changes in the levels of the DNA nucleobase damage products hypoxanthine, 8-oxo-7,8-dihydroguanine, 1,N6-ethenoadenine, or 3,N4-ethenocytosine above background levels under low dose-rate conditions. The micronucleus assay revealed no evidence that low dose-rate radiation induced DNA fragmentation, and there was no evidence of double strand break–induced HR. Furthermore, low dose-rate radiation did not induce Cdkn1a, Gadd45a, Mdm2, Atm, or Dbd2. Importantly, the same total dose, when delivered acutely, induced micronuclei and transcriptional responses.Conclusions: These results demonstrate in an in vivo animal model that lowering the dose-rate suppresses the potentially deleterious impact of radiation and calls attention to the need for a deeper understanding of the biological impact of low dose-rate radiation.

One possible mechanism linking inflammation with cancer involves the generation of reactive oxygen, nitrogen and halogen species by activated macrophages and neutrophils infiltrating sites of infection or tissue damage, with these chemical mediators causing damage that ultimately leads to cell death and mutation. To determine the most biologically deleterious chemistries of inflammation, we previously assessed products across the spectrum of DNA damage arising in inflamed tissues in the SJL mouse model nitric oxide over-production (Pang et al., Carcinogenesis 28: 1807–1813, 2007). Among the anticipated DNA damage chemistries, we observed significant changes only in lipid peroxidation-derived etheno adducts. We have now developed an isotope-dilution, liquid chromatography-coupled, tandem quadrupole mass spectrometric method to quantify representative species across the spectrum of RNA damage products predicted to arise at sites of inflammation, including nucleobase deamination (xanthosine, inosine), oxidation (8-oxoguanosine), and alkylation (1,N6-etheno-adenosine). Application of the method to liver, spleen, and kidney from the SJL mouse model revealed generally higher levels of oxidative background RNA damage than was observed in DNA in control mice. However, compared to control mice, RcsX treatment to induce nitric oxide overproduction resulted in significant increases only in inosine and only in the spleen. Further, the nitric oxide synthase inhibitor, N-methylarginine, did not significantly affect the levels of inosine in control and RcsX-treated mice. The differences between DNA and RNA damage in the same animal model of inflammation point to possible influences from DNA repair, RcsX-induced alterations in adenosine deaminase activity, and differential accessibility of DNA and RNA to reactive oxygen and nitrogen species as determinants of nucleic acid damage during inflammation.