Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC → AT transitions and GC → TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to asses the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

Cadmium: cellular effects, modifications of biomolecules, modulation of DNA repair and genotoxic consequences (a review)

Cadmium is an important toxic environmental heavy metal. Occupational and environmental pollution with cadmium results mainly from mining, metallurgy industry and manufactures of nickel-cadmium batteries, pigments and plastic stabilizers. Important sources of human intoxication are cigarette smoke as well as food, water and air contaminations. In humans, cadmium exposures have been associated with cancers of the prostate, lungs and testes. Acute exposures are responsible for damage to these organs. Chronic intoxication is associated with obstructive airway disease, emphysema, irreversible renal failure, bone disorders and immuno-suppression. At the cellular level, cadmium affects proliferation, differentiation and causes apoptosis. It has been classified as a carcinogen by the International Agency for Research on Cancer (IARC). However, it is weakly genotoxic. Indirect effects of cadmium provoke generation of reactive oxygen species (ROS) and DNA damage. Cadmium modulates also gene expression and signal transduction, reduces activities of proteins involved in antioxidant defenses. Several studies have shown that it interferes with DNA repair. The present review focuses on the effects of cadmium in mammalian cells with special emphasis on the induction of damage to DNA, membranes and proteins, the inhibition of different types of DNA repair and the induction of apoptosis. Current data and hypotheses on the mechanisms involved in cadmium genotoxicity and carcinogenesis are outlined.     

The crosstalk between Wnt/β-catenin signaling pathway with DNA damage response and oxidative stress: Implications in cancer therapy

DNA repair is essential for maintaining genomic integrity in cells. The dependence of cancer cell survival on proper DNA repair provides an opportunity to treat defective tumors by DNA damaging agents. Not only Wnt signaling has important functions in controlling gene expression, as well as cell polarity, adhesion and behavior, it also highly interacts with DNA damage response (DDR) in different levels. Furthermore, oxidative stress, which is responsible for majority of DNA lesions, affects Wnt signaling in different ways. A better understanding of the cross-talk between these pathways and events could provide strategies for treatment of cancer cells with deficient DNA repair capacity. As such, we will give a brief overview of the importance of the DNA repair machinery, signaling mechanisms of Wnt/β-catenin pathway, and DDR. We will further review the interactions between Wnt signaling and DDR, and the impact of oxidative stress on Wnt signaling. Finally, Wnt signaling is discussed as a potential treatment strategy for cancer.     

Effect of gliclazide on DNA damage in human peripheral blood lymphocytes and insulinoma mouse cells

Type 2 diabetes mellitus is associated with increased oxidative stress. Free radicals produced during this stress may damage various cellular components. Gliclazide, a second-generation sulfonylurea, is an oral hypoglycemic drug that possesses antioxidant properties. Therefore, gliclazide may diminish the harmful consequences of oxidative stress in diabetic patients. The aim of our study was to evaluate the action of gliclazide on DNA damage and repair in normal human peripheral blood lymphocytes and insulinoma mouse cells (beta-TC-6). DNA damage and repair were induced by hydrogen peroxide, gamma and ultraviolet radiation and MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) in the presence or absence of gliclazide and were analysed by the alkaline comet assay. DNA double-strand breaks were assayed by pulsed-field gel electrophoresis. Gliclazide protected DNA of both kinds of cells from DNA damage induced by chemicals and radiations. These results suggest that gliclazide may diminish the risk of free radical-related diseases associated with type 2 diabetes mellitus and possibly cancer.     

The oxidative induction of DNA lesions in cancer cells by 5-thio-d-glucose and 6-thio-d-fructopyranose and their genotoxic effects. Part 3

Thio-sugars have been described as potent inhibitors of cancer cell growth but the detailed mechanism of action remains unknown. Herein we investigated the mechanism of their anticancer action in the HeLa cell line. We investigated two thio-sugars: 5-thio-d-glucose (FCP1) and 6-thio-β-d-fructopyranose (FCP2). We have observed that FCP1 as well as FCP2 clearly induced oxidative DNA lesions in cancer cells and increased the level of cellular ROS. A spin trap and antioxidants have decreased the level of DNA lesions induced by FCPs. FCPs also induced significant changes in the oxidative-stress gene expression. Therefore, we assume that ROS generation is correlated with the increased NOX5 expression by FCPs. Higher cyto- and genotoxicity of FCPs for HeLa cells in a low glucose environment suggested their role in the glucose metabolism. The data indicates that thio-sugars may become drug alternatives for the cancer treatment but such undertaking needs further studies.     

A new perspective on oxidation of DNA repair proteins and cancer

Reactive oxygen and nitrogen species (RONS) are formed as byproducts of many endogenous cellular processes, in response to infections, and upon exposure to various environmental factors. An increase in RONS can saturate the antioxidation system and leads to oxidative stress. Consequently, macromolecules are targeted for oxidative modifications, including DNA and protein. The oxidation of DNA, which leads to base modification and formation of abasic sites along with single and double strand breaks, has been extensively investigated. Protein oxidation is often neglected and is only recently being recognized as an important regulatory mechanism of various DNA repair proteins. This is a review of the current state of research on the regulation of DNA repair by protein oxidation with emphasis on the correlation between inflammation and cancer.     

When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3′-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide (“long-patch”) BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5′-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15–30 min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60–70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5′-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.     

Cisplatin resistance: Preclinical findings and clinical implications

Cisplatin is used for the treatment of many types of solid cancers. While testicular cancers respond remarkably well to cisplatin, the therapeutic efficacy of cisplatin for other solid cancers is limited because of intrinsic or acquired drug resistance. Our understanding about the mechanisms underlying cisplatin resistance has largely arisen from studies carried out with cancer cell lines in vitro. The process of cisplatin resistance appears to be multifactorial and includes changes in drug transport leading to decreased drug accumulation, increased drug detoxification, changes in DNA repair and damage bypass and/or alterations in the apoptotic cell death pathways. Translation of these preclinical findings to the clinic is emerging, but still scarce. The present review describes and discusses the clinical relevance of in vitro models by comparing the preclinical findings to data obtained in clinical studies.     

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H₂O₂ than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H₂O₂ treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.     

DNA damage responses in cancer stem cells: Implications for cancer therapeutic strategies

The identification of cancer stem cells(CSCs) that are responsible for tumor initiation, growth, metastasis, and therapeutic resistance might lead to a new thinking on cancer treatments. Similar to stem cells,CSCs also display high resistance to radiotherapy and chemotherapy with genotoxic agents. Thus, conventional therapy may shrink the tumor volume but cannot eliminate cancer. Eradiation of CSCs represents a novel therapeutic strategy. CSCs possess a highly efficient DNA damage response(DDR) system, which is considered as a contributor to the resistance of these cells from exposures to DNA damaging agents. Targeting of enhanced DDR in CSCs is thus proposed to facilitate the eradication of CSCs by conventional therapeutics. To achieve this aim, a better understanding of the cellular responses to DNA damage in CSCs is needed. In addition to the protein kinases and enzymes that are involved in DDR, other processes that affect the DDR including chromatin remodeling should also be explored.     

Inflammation-induced DNA damage,mutations and cancer

The relationships between inflammation and cancer are varied and complex. An important connection linking inflammation to cancer development is DNA damage. During inflammation reactive oxygen and nitrogen species (RONS) are created to combat pathogens and to stimulate tissue repair and regeneration, but these chemicals can also damage DNA, which in turn can promote mutations that initiate and promote cancer. DNA repair pathways are essential for preventing DNA damage from causing mutations and cytotoxicity, but RONS can interfere with repair mechanisms, reducing their efficacy. Further, cellular responses to DNA damage, such as damage signaling and cytotoxicity, can promote inflammation, creating a positive feedback loop. Despite coordination of DNA repair and oxidative stress responses, there are nevertheless examples whereby inflammation has been shown to promote mutagenesis, tissue damage, and ultimately carcinogenesis. Here, we discuss the DNA damage-mediated associations between inflammation, mutagenesis and cancer.     

DNA repair of clustered uracils in HeLa cells

Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

Comparative study of DNA damage and repair in head and neck cancer after radiation treatment

We compared DNA damage and the efficacy of its repair after genotoxic treatment with γ-radiation of lymphocytes and tissue cells isolated from patients with squamous cell carcinoma of head and neck (HNSCC) and healthy donors. Thirty-seven subjects with HNSCC and 35 healthy donors were enrolled in the study. The extent of DNA damage including oxidative lesions and efficiency of the repair were examined by alkaline comet assay. HNSCC cancer cells were more sensitive to genotoxic treatment and displayed impaired DNA repair. In particular, lesions caused by γ-radiation were repaired less effectively in metastasis of HNSCC than in healthy controls. The differences in radiation sensitivity of cancer and control cells suggested that DNA repair might be critical for HNSCC treatment. We conclude that γ-radiation might be considered as an effective therapeutic strategy for head and neck cancers, including patients in advanced stage of the disease with clear evidence of metastasis.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

Diverse functions of DNA glycosylases processing oxidative base lesions in brain

Endogenous and exogenous oxidative agents continuously damage genomic DNA, with the brain being particularly vulnerable. Thus, preserving genomic integrity is key for brain health and neuronal function. Accumulation of DNA damage is one of the causative factors of ageing and increases the risk of a wide range of neurological disorders. Base excision repair is the major pathway for removal of oxidized bases in the genome and initiated by DNA glycosylases. Emerging evidence suggest that DNA glycosylases have non-canonical functions important for genome regulation. Understanding canonical and non-canonical functions of DNA glycosylases processing oxidative base lesions modulating brain function will be crucial for the development of novel therapeutic strategies.     

Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells

We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5 h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.     

Efficiency,specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells

The oxidation product of guanine, 8-oxoguanine, is a major lesion formed in DNA by intracellular metabolism, ionizing radiation, and tobacco smoke. Using a recently developed method for the quantitative analysis of translesion replication, we have studied the bypass of 8-oxoguanine in vivo by transfecting human cells with a gapped plasmid carrying a site-specific 8-oxoguanine in the ssDNA region. The efficiency of bypass in the human large-cell lung carcinoma cell line H1299 was 80%, and it was similar when assayed in the presence of aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon. A similar extent of bypass was observed also in XP-V cells, defective in pol eta, both in the absence and presence of aphidicolin. DNA sequence analysis indicated that the major nucleotide inserted opposite the 8-oxoguanine was the correct nucleotide C, both in H1299 cells (81%) and in XP-V cells (77%). The major mutagenic event was the insertion of an A, both in H1299 and XP-V cells, and it occurred at a frequency of 16-17%, significantly higher than previously reported. Interestingly, the misinsertion frequency of A opposite 8-oxoguanine was decreased in XP-V cells in the presence of aphidicolin, and misinsertion of G was observed. This modulation of the mutagenic specificity at 8-oxoguanine is consistent with the notion that while not essential for the bypass reaction, pol eta and pol delta, when present, are involved in bypass of 8-oxoguanine in vivo.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.