8-hydroxyguanine and Neoplasms

Cells of the aerobic metabolic organism are inevitably subjected to the damage from reactive oxygen species (ROS). ROS cause multiple forms of DNA damage, among which the oxidation product of guanine G 8-hydroxyguanine (8-oxoG) is the most frequent DNA oxidative damage, recognized by the specific glycosidase OGG1 that initiates the base excision repair pathway. If left unrepaired, 8-oxoG may pair with A instead of C, leading to a mutation of G: C to T: A during replication. Thus, the accumulation of 8-oxoG or the abnormal OGG1 repair is thought to affect gene function, which in turn leads to the development of tumor or aging-related diseases. However, a series of recent studies have shown that 8-oxoG tends to be produced in regulatory regions of the genome. 8-oxoG can be regarded as an epigenetic modification, while OGG1 is a specific reader of this information. Substrate recognition, binding or resection by OGG1 can cause DNA conformation changes or affect histone modifications, causing up-regulation or down-regulation of genes with different properties. Thus, in addition to the potential genotoxicity, the association of guanine oxidative damage with development of tumors is closely related to its aberrant initiation of gene expression through epigenetic mechanisms. In this review, we summarize the underlying mechanism of 8-oxoG and repair enzyme OGG1 in tumor development and progression, with aims to interpret the relationship between DNA oxidative damage and tumor from a new perspective, and provide new ideas and targets for tumor treatment.. 有氧代谢有机体细胞无法避免活性氧(reactive oxygen species, ROS)的伤害。ROS会造成多种形式的DNA损伤,其中鸟嘌呤G的氧化产物8-羟鸟嘌呤(8-oxoG)是频度最高的一种DNA氧化损伤,由特异性的糖苷酶OGG1识别并开启碱基切除修复通路完成修复。8-oxoG如果没有及时修复,可能会在复制的过程中引入G:C配对到T:A配对的碱基颠换突变。因此8-oxoG的积累或OGG1修复功能异常被认为会影响基因功能,进而导致肿瘤或衰老相关疾病的发生,然而直接实验证据却极为有限。近年来一系列研究表明,8-oxoG倾向于产生在基因的调控区,在这种情形下,8-oxoG可视为一种表观遗传学修饰,而OGG1则是这一信息的特异性读取者,OGG1对底物的识别、结合或切除会引发DNA构象或组蛋白修饰的改变,进而引起基因表达的上调或下调。因此,除了潜在的遗传毒性,鸟嘌呤氧化损伤与肿瘤的关联与其通过表观遗传学机制引发基因表达的异常密切相关。本文对8-oxoG及修复酶OGG1与肿瘤发生发展的关联机制进行了分析与总结,旨在提示研究人员从新的视角解读DNA氧化损伤与肿瘤的关系,并为肿瘤的治疗提供新的思路和靶点。.

Topics: DNA; DNA Damage; DNA Glycosylases; DNA Repair; Guanine; Humans; Neoplasms; Oxidative Stress; Reactive Oxygen Species

MUTYH is a base-excision repair glycosylase that removes adenine opposite 8-oxoguanine (OG). Variants of MUTYH defective in functional activity lead to MUTYH-associated polyposis (MAP), which progresses to cancer with very high penetrance. Whole genome and whole exome sequencing studies have found MUTYH deficiencies in an increasing number of cancer types. While the canonical OG:A repair activity of MUTYH is well characterized and similar to bacterial MutY, here we review more recent evidence that MUTYH has activities independent of OG:A repair and appear centered on the interdomain connector (IDC) region of MUTYH. We summarize evidence that MUTYH is involved in rapid DNA damage response (DDR) signaling, including PARP activation, 9-1-1 and ATR signaling, and SIRT6 activity. MUTYH alters survival and DDR to a wide variety of DNA damaging agents in a time course that is not consistent with the formation of OG:A mispairs. Studies that suggest MUTYH inhibits the repair of alkyl-DNA damage and cyclopyrimidine dimers (CPDs) is reviewed, and evidence of a synthetic lethal interaction with mismatch repair (MMR) is summarized. Based on these studies we suggest that MUTYH has evolved from an OG:A mispair glycosylase to a multifunctional scaffold for DNA damage response signaling.

Topics: Animals; DNA; DNA Damage; DNA Glycosylases; DNA Repair; Guanine; Humans; Neoplasms; Signal Transduction

The generation of DNA modifications in cells is in most cases accidental and associated with detrimental consequences such as increased mutation rates and an elevated risk of malignant transformation. Accordingly, repair enzymes involved in the removal of the modifications have primarily a protective function. Among the well-established exceptions of this rule are 5-methylcytosine and uracil, which are generated in DNA enzymatically under controlled conditions and fulfill important regulatory functions in DNA as epigenetic marks and in antibody diversification, respectively. More recently, considerable evidence has been obtained that also 8-oxo-7,8-dihydroguanine (8-oxoG), a frequent pro-mutagenic DNA modification generated by endogenous or exogenous reactive oxygen species (ROS), has distinct roles in the regulation of both transcription and signal transduction. Thus, the activation of transcription by the estrogen receptor, NF-κB, MYC and other transcription factors was shown to depend on the presence of 8-oxoG in the promoter regions and its recognition by the DNA repair glycosylase OGG1. The lysine-specific histone demethylase LSD1, which produces H

Topics: Animals; Carcinogenesis; DNA; DNA Glycosylases; DNA Repair; Epigenesis, Genetic; Guanine; Guanine Nucleotide Exchange Factors; Humans; Neoplasms; Oxidation-Reduction; Oxidative Stress; Risk Factors; Signal Transduction; Transcription, Genetic

The maintenance of genetic stability is of crucial importance for any form of life. Prior to cell division in each mammalian cell, the process of DNA replication must faithfully duplicate the three billion bases with an absolute minimum of mistakes. Various environmental and endogenous agents, such as reactive oxygen species (ROS), can modify the structural properties of DNA bases and thus damage the DNA. Upon exposure of cells to oxidative stress, an often generated and highly mutagenic DNA damage is 7,8-dihydro-8-oxo-guanine (8-oxo-G). The estimated steady-state level of 8-oxo-G lesions is about 10(3) per cell/per day in normal tissues and up to 10(5) lesions per cell/per day in cancer tissues. The presence of 8-oxo-G on the replicating strand leads to frequent (10-75%) misincorporations of adenine opposite the lesion (formation of A:8-oxo-G mispairs), subsequently resulting in C:G to A:T transversion mutations. These mutations are among the most predominant somatic mutations in lung, breast, ovarian, gastric and colorectal cancers. Thus, in order to reduce the mutational burden of ROS, human cells have evolved base excision repair (BER) pathways ensuring (i) the correct and efficient repair of A:8-oxo-G mispairs and (ii) the removal of 8-oxo-G lesions from the genome. Very recently it was shown that MutY glycosylase homologue (MUTYH) and DNA polymerase lambda play a crucial role in the accurate repair of A:8-oxo-G mispairs. Here we review the importance of accurate BER of 8-oxo-G damage and its regulation in prevention of cancer.

Topics: Animals; DNA Repair; DNA-Directed DNA Polymerase; Guanine; Humans; Mutation; Neoplasms; Oxidative Stress; Oxygen

Base excision repair (BER) is the major pathway responsible for averting the mutagenic and cytotoxic effects of spontaneous hydrolytic, oxidative, and non-enzymatic alkylation DNA damage. In particular, this pathway recognizes and repairs base modifications, such as uracil and 8-hydroxyguanine, as well as abasic sites and DNA single-strand breaks. In this review, we outline the basic mechanics of the BER process, and describe the potential association of this pathway with aging and age-related disease, namely cancer and neurodegeneration.

Topics: Aging; DNA Repair; Guanine; Humans; Mitochondria; Neoplasms; Neurodegenerative Diseases

The most commonly measured marker of oxidative DNA damage is 8-oxo-7,8-dihydroguanine (8-oxoGua) or its deoxyribonucleoside (8-oxodGuo). Published estimates of the concentration of 8-oxoGua/8-oxodGuo in DNA of normal human cells vary over a range of three orders of magnitude. Analysis by chromatographic methods (GC-MS, HPLC with electrochemical detection (ECD) or HPLC-MS/MS) is beset by the problem of adventitious oxidation of guanine during sample preparation. An alternative approach, based on the use of the DNA repair enzyme formamidopyrimidine DNA N-glycosylase (FPG) to make breaks in the DNA at sites of the oxidised base, gives much lower values. ESCODD, the European Standards Committee on Oxidative DNA Damage, has been testing the ability of different laboratories using a variety of methods to measure 8-oxoGua in standard samples of 8-oxodGuo, calf thymus DNA, pig liver, oligonucleotides, and HeLa cells, and in lymphocytes isolated from blood of volunteers. HPLC-ECD is capable of measuring 8-oxodGuo induced experimentally in calf thymus DNA or HeLa cells with high accuracy. However, there is no sign of consensus over the background level of this damage, suggesting that, even though standard extraction procedures were used, variable oxidation of Gua is still occurring. GC-MS failed to detect a dose response of induced 8-oxoGua and cannot be regarded as a reliable method for measuring low levels of damage. HPLC-MS/MS as yet has not proved capable of measuring low levels of oxidative DNA damage. FPG-based methods seem to be less prone to the artefact of additional oxidation. Although they can be used quantitatively, they require careful calibration and standardisation if they are to be used in human biomonitoring. The background level of DNA oxidation in normal human cells is likely to be around 0.3-4.2 8-oxoGua per 10(6) Gua. An effort should be made to develop alternative, validated methods for estimating oxidative DNA damage.

Topics: Animals; Cattle; DNA; DNA Damage; Guanine; HeLa Cells; Humans; Neoplasms; Oxidation-Reduction

Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER events which occur sequentially on the two strands. The removal of the mismatched adenine is followed by preferential insertion of a cytosine leading to the formation of 8oxoG/C pairs which are then corrected by OGG1-mediated BER. Both repair events are inhibited by aphidicolin, suggesting that a replicative DNA polymerase is involved in the repair synthesis step. We propose that Pol delta/epsilon-mediated BER (long-patch BER) is the mode of repair when lesions persist or are formed at replication. Finally, we address the issues of the relative contribution of the two BER pathways to oxidative damage repair in vivo and the possible role of BER gene variants as cancer susceptibility genes.

Topics: Base Sequence; DNA Damage; DNA Glycosylases; DNA Repair; Guanine; Humans; Models, Genetic; Neoplasms; Oxidative Stress; Reactive Oxygen Species

A wide variety of oxidative DNA lesions are present in living cells. One of the best known lesions of this type is 8-oxoguanine (8-oxoGua) which has been shown to have mutagenic properties. An influence of antioxidative vitamins and labile iron pool on the background level of 8-oxoGua in cellular DNA is discussed and oxidative damage to free nucleotide pool as a possible source of 8-oxo-2'-deoxyguanosine in DNA and urine is described. An involvement of 8-oxoGua in the origin and/or progression of cancer is reviewed. It is concluded that a severe oxidative stress manifested as a high level of 8-oxoGua in cellular DNA as well as in urine of cancer patients is a consequence of development of many types of cancer. Although at present it is impossible to answer directly the question concerning involvement of oxidative DNA damage in cancer etiology it is likely that oxidative DNA base modifications may serve as a source of mutations that initiate carcinogenesis (i.e. they may be causal factors responsible for the process).

Topics: Carcinogens; Deoxyguanosine; DNA Damage; Guanine; Humans; Iron; Neoplasms; Oxidative Stress

8-Hydroxyguanine (oh8G) is a major base lesion produced by reactive oxygen species. oh8G in DNA causes G:C to T:A transversions and, thus, could be responsible for mutations that lead to carcinogenesis. A human DNA glycosylase/AP lyase encoded by the OGG1 gene has an activity to remove directly oh8G from DNA, and suppresses the mutagenic effect of oh8G. OGG1 protein has a helix-hairpin-helix-GPD motif as a domain for both DNA binding and catalysis, a nuclear localization signal, and a mitochondria targeting signal. Among multiple OGG1 isoforms, OGG1-type la is expressed predominantly in human cells and repairs chromosomal DNA in the nucleus. Inactivation of the OGG1 gene in yeast and mice leads to elevated spontaneous mutation frequency in the cells. The human OGG1 gene maps to chromosome 3p26.2, and allelic deletions of this region occur frequently in a variety of human cancers. Moreover, the OGG1 gene is somatically mutated in some cancer cells and is highly polymorphic among human populations. Repair activities of some mutated and polymorphic OGG1 proteins are lower than those of wild-type OGG1-type la-Ser326 protein and, thus, could be involved in human carcinogenesis.

Topics: Amino Acid Sequence; Animals; Carbon-Oxygen Lyases; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 3; Conserved Sequence; DNA Damage; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Exons; Genes; Guanine; Humans; Kinetics; Mice; Molecular Sequence Data; Mutagenesis; N-Glycosyl Hydrolases; Neoplasms; Oxidation-Reduction; Oxidative Stress; Polymorphism, Genetic; Protein Isoforms; Rats; Reactive Oxygen Species; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity

A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as byproducts of cell metabolism. Among DNA damages induced by ROS, 8-hydroxyguanine (8-OH-G) is certainly the product that has retained most of the attention in the past few years. The biological relevance of 8-OH-G in DNA has been unveiled by the study of Escherichia coli and Saccharomyces cerevisiae genes involved in the neutralization of the mutagenic effects of 8-OH-G. These genes, fpg and mutY for E. coli and OGG1 for yeast, code for DNA glycosylases. Inactivation of any of those genes leads to a spontaneous mutator phenotype, characterized by the increase in GC to TA transversions. In yeast, the OGG1 gene encodes a DNA glycosylase/AP lyase that excises 8-OH-G from DNA. In human cells, the OGG1 gene is localized on chromosome 3p25 and encodes two forms of hOgg1 protein which result from an alternative splicing of a single messenger RNA. The alpha-hOgg1 protein has a nuclear localization whereas the beta-hOgg1 is targeted to the mitochondrion. Biochemical studies on the alpha-hOgg1 protein show that it is a DNA glycosylase/AP lyase that excises 8-OH-G and Fapy-G from gamma-irradiated DNA. Several approaches have been used to study the biological role of OGG1 in mammalian cells, ranging from its overexpression in cell lines to the generation of homozygous ogg1-/- null mice. Furthermore, to explore a possible role in the prevention of cancer, the cDNA coding for alpha-hOgg1 has been sequenced in human tumors. All these results point to 8-OH-G as an endogenous source of mutations in eukaryotes and to its likely involvement in the process of carcinogenesis. A review of the recent literature on the mammalian Ogg1 proteins, the main repair system involved in the elimination of this mutagenic lesion, is presented.

Topics: Aging; Amino Acid Sequence; Animals; Base Pair Mismatch; Base Sequence; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Guanine; Humans; Mice; Molecular Sequence Data; N-Glycosyl Hydrolases; Neoplasms

One of the most prevalent products of oxygen radical injury in DNA is 8-hydroxyguanosine. Cells must be able to withstand damage by oxygen radicals and possess specific repair mechanisms that correct this oxidative lesion. However, when these defenses are oversaturated, such as under conditions of high oxidative stress, or when repair is inefficient, the miscoding potential of this lesion can result in mutations in the mammalian genome. In addition to causing genetic changes, active oxygen species can lead to epigenetic alterations in DNA methylation, without changing the DNA base sequence. Such changes in DNA methylation patterns can strongly affect the regulation of expression of many genes. Although DNA methylation patterns have been found to be altered during carcinogenesis, little is known about the mechanism(s) that produce this loss of epigenetic controls of gene expression in tumors. Replacement of guanine with the oxygen radical adduct 8-hydroxyguanine profoundly alters methylation of adjacent cytosines, suggesting a role for oxidative injury in the formation of aberrant DNA methylation patterns during carcinogenesis. In this paper, we review both the genetic and epigenetic mechanisms of oxidative DNA damage and its association with the carcinogenic process, with special emphasis on the influence of free radical injury on DNA methylation.

Topics: Animals; DNA Damage; DNA Methylation; Guanine; Humans; Neoplasms; Oxidants; Oxidative Stress; Reactive Oxygen Species

Topics: Animals; DNA; DNA, Neoplasm; Guanine; Humans; Neoplasms; Neoplasms, Experimental

Oxygen radicals are produced by ionizing radiation and many other environmental carcinogens. They are also produced in cells endogenously by the oxygen metabolism. Therefore it appears to be important to study DNA damage by oxygen radicals and its relation with mutagenesis and carcinogenesis. During a study on DNA damage caused in vitro by heated carbohydrates, which were being used as a model of cooked foods, a new type of DNA modification was discovered, the formation 8-hydroxyguanine (8-OH-Gua). Various oxygen radical forming carcinogenic agents (radiation, cigarette smoke components, asbestos + H2O2 etc.) have been found to be effective in the formation of 8-OH-Gua in DNA in vitro. The formation of 8-OH-Gua was also observed in cellular DNA in vivo after mice or cultured cells were irradiated by ionizing radiation. The 8-OH-Gua residue in DNA may cause mutagenesis and carcinogenesis, since 8-OH-Gua in DNA induces misreading during DNA synthesis in vitro. Among the various types of DNA damage induced by oxygen radicals, 8-OH-Gua can be most readily measured at high sensitivity by high-pressure liquid chromatography coupled to an electrochemical detector. Recently, we found that oral administration of several chemical carcinogens, which are known to produce oxygen radicals, induce 8-OH-Gua in rat target organ DNA. Therefore, 8-OH-Gua can be used as a marker for monitoring oxidative DNA damage as a means of evaluating the carcinogenic potency of various oxygen radical forming agents. We are also measuring 8-OH-Gua levels in human lymphocyte DNA in order to investigate the relation between oxidative DNA damage and the incidence of cancer.

Topics: Animals; DNA; DNA Damage; DNA Mutational Analysis; Free Radicals; Guanine; Humans; Lymphocytes; Mice; Neoplasms; Neoplasms, Experimental; Oxygen; Rats

Guanine is the most susceptible to oxidation among all the DNA bases, and 8-oxo-7,8-dihydroguanine (OG) is one of main oxidation products that can occur in any part of chromosomal DNA. OG in the telomere sequence is associated with telomere shortening, cell aging, and dysfunction, and it may induce cancers. The accurate detection of OG in telomeres is important to early clinical diagnosis and molecular research. Herein, we develop a simple and rapid method to sensitively measure 8-oxo-7,8-dihydroguanine (OG) in telomeres of cancer cells by using Bsu polymerase-mediated fluorescence coding. This method is very simple without the requirement for any nucleic acid amplification or specific restriction enzyme recognition reaction, and Bsu polymerase can selectively incorporate Cy5-dATP into the opposite site of OG, endowing this method with good specificity. Moreover, the introduction of single-molecule detection significantly improves the sensitivity. This method can detect OG within 70 min with a limit of detection (LOD) of 2.45 × 10

Topics: DNA Damage; Fluorescence; Guanine; HeLa Cells; Humans; Hydrogen Peroxide; Neoplasms; Oxidation-Reduction; Telomere

Professor Barbara Tudek received the Frits Sobels Award in 2019 from the European Environmental Mutagenesis and Genomics Society (EEMGS). This article presents her outstanding character and most important lines of research. The focus of her studies covered alkylative and oxidative damage to DNA bases, in particular mutagenic and carcinogenic properties of purines with an open imidazole ring and 8-oxo-7,8-dihydroguanine (8-oxoGua). They also included analysis of mutagenic properties and pathways for the repair of DNA adducts of lipid peroxidation (LPO) products arising in large quantities during inflammation. Professor Tudek did all of this in the hope of deciphering the mechanisms of DNA damage removal, in particular by the base excision repair (BER) pathway. Some lines of research aimed at discovering factors that can modulate the activity of DNA damage repair in hope to enhance existing anti-cancer therapies. The group's ongoing research aims at deciphering the resistance mechanisms of cancer cell lines acquired following prolonged exposure to photodynamic therapy (PDT) and the possibility of re-sensitizing cells to PDT in order to increase the application of this minimally invasive therapeutic method.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Carcinogenesis; Cell Line, Tumor; DNA Adducts; DNA Damage; DNA Repair; Guanine; History, 20th Century; History, 21st Century; Humans; Lipid Peroxidation; Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents

A new chemiluminescence aptasensor for sensitive and efficient detection of 8-hydroxyguanine based on the synergistic interaction of Ni NPs@l-histidine@aptamer@MBs has been developed, and it has been applied in the real urine samples of cancer patients.

Topics: Aptamers, Nucleotide; Guanine; Histidine; Humans; Luminescent Measurements; Magnetics; Metal Nanoparticles; Neoplasms; Nickel

The determination of the concentrations of urinary biomarkers of oxidative damage to DNA and RNA is difficult due to the low content of targets and the complex matrix of urine. A method using polystyrene/polypyrrole (PS/PPY) electronspun nanofibers as the adsorbent was introduced to the routine urinary treatment and determination of 8-OHdG and 8-oxoG for the first time. And 2-aminoethyl diphenylborate (DPBA) solution was creatively used in the loading and rinsing steps in order to promote the retention of the analytes as well as remove impurities. Under optimal conditions, 8-OHdG, 8-oxoG and IS were separated very well and exhibited a good linearity in the range of 0.5-50 ng mL

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Biomarkers; Chromatography, High Pressure Liquid; DNA; DNA Damage; Guanine; Humans; Limit of Detection; Linear Models; Neoplasms; Oxidative Stress; Reproducibility of Results; RNA; Solid Phase Extraction

The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC

Topics: Antineoplastic Agents; Caco-2 Cells; DNA Glycosylases; Enzyme Inhibitors; Guanine; HEK293 Cells; HeLa Cells; Humans; Neoplasms; Small Molecule Libraries

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N

Topics: Adenine; Base Pair Mismatch; Biosensing Techniques; DNA; DNA Damage; DNA Repair; Epigenesis, Genetic; Guanine; Humans; Models, Molecular; Nanopores; Nanotechnology; Neoplasms; Uracil

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Topics: Adenine; Base Pairing; Catalytic Domain; Crystallography, X-Ray; Cytosine; Cytotoxins; Deoxyguanine Nucleotides; DNA; DNA Damage; DNA Polymerase beta; DNA Repair; DNA Replication; Guanine; Humans; Hydrogen Bonding; Kinetics; Models, Molecular; Molecular Conformation; Mutagenesis; Neoplasms; Oxidation-Reduction; Oxidative Stress; Static Electricity; Substrate Specificity; Time Factors

Because of the importance to identify prognostic indicator for radiotherapy, herein we decided to check whether the parameters which describe oxidative stress/DNA damage may be used as a marker of the therapy. The aim of this work was to investigate whether fractionated radiotherapy of patients with cancer (n = 99) is responsible for oxidative DNA damage on the level of the whole organism and whether the biomarkers of the damage such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and its modified base 8-oxo-7,8-dihydroguanine (8-oxo-Gua) in urine and DNA may be used as a predictor of radiotherapy success.. All the aforementioned modifications were analyzed using techniques which involve high-performance liquid chromatography/electrochemical detection (HPLC/EC) or HPLC/gas chromatography-mass spectroscopy (GC-MS).. Of all analyzed parameters only patients with significantly elevated urinary excretion of the 8-oxo-Gua with concomitant unchanged level of 8-oxo-dG in leukocytes DNA in the samples collected 24 hours after the first fraction in comparison to the initial level have significantly increased survival time (60 months after the treatment, survival of 50% of the patients who fulfill the above mentioned criteria, in comparison with 10% of the patients who did not).. Results of our work suggest that patients with higher urinary 8-oxo-Gua and concomitant stable level of 8-oxo-dG in leukocytes DNA, after 24 hours of the first dose should be regarded as better responder to radiotherapy as being at lower risk of mortality.. The above mentioned statement could make it possible to use these parameters as markers to predict the clinical success.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Damage; Female; Guanine; Humans; Leukocytes; Male; Middle Aged; Neoplasms; Oxidation-Reduction; Oxidative Stress; Prognosis; Radiation Oncology; Survival Rate

Synthetic lethal approaches to cancer treatment have the potential to deliver relatively large therapeutic windows and therefore significant patient benefit. To identify potential therapeutic approaches for cancers deficient in DNA mismatch repair (MMR), we have carried out parallel high-throughput RNA interference screens using tumor cell models of MSH2- and MLH1-related MMR deficiency. We show that silencing of the PTEN-induced putative kinase 1 (PINK1), is synthetically lethal with MMR deficiency in cells with MSH2, MLH1, or MSH6 dysfunction. Inhibition of PINK1 in an MMR-deficient background results in an elevation of reactive oxygen species and the accumulation of both nuclear and mitochondrial oxidative DNA lesions, which likely limit cell viability. Therefore, PINK1 represents a potential therapeutic target for the treatment of cancers characterized by MMR deficiency caused by a range of different gene deficiencies.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; DNA Mismatch Repair; DNA Repair-Deficiency Disorders; Guanine; Humans; Membrane Potential, Mitochondrial; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasms; Nuclear Proteins; Protein Kinases; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transfection

The broad spectrum of oxidative damage DNA biomarkers: urinary excretion of 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), 8-oxoGua (8-oxo-7,8-dihydroguanine) as well as the level of oxidative damage DNA in leukocytes, was analyzed in cancer patients and healthy subjects.. 222 cancer patients and 134 healthy volunteers were included in the analysis, using methodologies which involve HPLC (high-performance liquid chromatography) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and HPLC/EC.. For the whole patient population (n=222) the median values of 8-oxoGua and 8-oxodG in urine samples were 12.44 (interquartile range: 8.14-20.33) [nmol/24 hr] and 6.05 (3.12-15.38) [nmol/24 hr], respectively. The median values of 8-oxoGua and 8-oxodG in urine samples of the control group (n=85) were 7.7 (4.65-10.15) [nmol/24 hr] and 2.2 (1.7-2.8) [nmol/24 hr], respectively. The level of 8-oxodG in DNA isolated from leukocytes of the patient population (n=179) and of the control group (n=134) was 4.93 (3.46-9.27) per 10'6 dG and 4.46 (3.82-5.31) per 10'6 dG, respectively.. The results suggest that oxidative stress in cancer patients, demonstrated by augmented amounts of these modifications in urine, could be typical not only for affected tissue but also for other tissues and even the whole organism. An assay that enables the determination of levels of basic markers of oxidative stress might be applied in clinical practice as an additional, helpful marker to diagnose cancer.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Biomarkers, Tumor; Case-Control Studies; Deoxyguanosine; DNA Damage; DNA, Neoplasm; Guanine; Humans; Leukocytes; Neoplasms; Oxidative Stress

Supplementary oxygen during resuscitation of the asphyxiated newborn is associated with long-term detrimental effects including increased risk of childhood cancer. It is suspected that the resuscitation procedure results in accumulated DNA damage and mutagenesis. Base excision repair (BER) is the major pathway for repair of premutagenic oxidative DNA lesions. This study addresses DNA base damage and BER in brain, lung, and liver in neonatal mice (P7) after hyperoxic resuscitation. Mice were randomized to 8% oxygen or room air for 60 min in a closed chamber and subsequent reoxygenation with 100% oxygen for 0 to 90 min. During this treatment, 8-oxoguanine accumulated in liver but not in lung or cerebellum. We observed a linear relation between 8-oxoguanine and reoxygenation time in liver DNA from hypoxic animals (n = 28; B = 0.011 [0.001, 0.020]; p = 0.037). BER activity was not significantly changed during resuscitation. Our data suggest that after hypoxia, the capacity for immediate repair in liver tissue is inadequate to meet increasing amounts of DNA damage. The duration of supplementary oxygen use during resuscitation should be kept as short as justifiable to minimize the risk of genetic instability.

Topics: Animals; Animals, Newborn; Cerebellum; DNA Damage; DNA Repair; Guanine; Hyperoxia; Liver; Lung; Mice; Mice, Inbred C57BL; Mutagenesis; Neoplasms; Oxygen; Oxygen Inhalation Therapy; Resuscitation; Risk

Vitamins A, E and C, and uric acid, which can scavenge free radicals should also protect DNA from the damage. It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease subsequent cancer development.. A relationship between basal level of antioxidants (vitamins A, C and E and uric acid) and oxidative DNA damage was assessed. For the first time, the broad spectrum of oxidative DNA damage biomarkers: urinary excretion of 8-oxodG, 8-oxoGua and 5HMUra as well as the level of oxidative DNA damage in leukocytes was analyzed in healthy subjects (n = 158).. Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of oxidative DNA damage products excreted into urine and the amount of 8-oxodG in leukocytes' DNA (with HPLC/EC technique). The level of antioxidant vitamins and uric acid was estimated by HPLC technique with fluorimetric and UV detection.. Analyses of relationship between the most common antioxidants (vitamins A, C, E and uric acid) and oxidative DNA damage products reveal weak, statistically significant negative correlation between retinol and all the measured parameters except 5HMUra. Vitamin C negatively correlates with urinary excretion of 8-oxodG and 8-oxoGua. Uric acid revealed statistically significant negative correlation with 8-oxodG in cellular DNA and urinary excretion of 5HMUra, while alpha-tocopherol correlates negatively only with 8-oxodG in cellular DNA. Good, significant (P < 0.0001), positive correlation (r = 0.61) was noted between urinary levels of the base, 8-oxoGua and the deoxynucleoside, 8-oxodG.. Our results suggest that oxidative DNA damage shows limited but significant response to antioxidants analyzed in this study and is more affected by many other cellular functions like antioxidant enzymes or DNA repair enzymes as well as genetics.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Aged; Aged, 80 and over; Antioxidants; Biomarkers; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA Damage; Female; Free Radical Scavengers; Guanine; Humans; Leukocytes; Male; Middle Aged; Neoplasms; Oxidation-Reduction; Oxidative Stress; Uric Acid; Vitamins

In order to assess the effect of potassium bromate (KBrO3) on the induction of tumor formation, a 1-year carcinogenesis study was performed using Ogg1 knockout mice (Ogg1(-/-)) and wild-type mice (Ogg1(+/+)). The mice were chronically exposed to KBrO3 by putting it in the drinking water for 29 weeks, at 2 g/l for the first 18 weeks, and then at 1 g/l for another 11 weeks. After termination of treatment the mice were kept for an additional 23 weeks. The amount of 8-hydroxydeoxyguanosine (8-OH-dG) in kidney DNA after 29 weeks of KBrO3 exposure reached 500 8-OH-dG/10(6) dG, almost 250-fold that of untreated wild-type mice. During the course of study the mice appeared normal, although a decrease of body weight gain in both Ogg1(-/-) and Ogg1(+/+) mice exposed to KBrO3, and some kidney malfunction in KBrO3 treated Ogg1(-/-) mice was observed. Surprisingly, when Ogg1(-/-) and Ogg1(+/+) mice were sacrificed at 52 weeks, no tumor formation could be found in kidney or other organs such as lung, liver, spleen, thymus, stomach and intestine. Microscopic examination also showed the absence of precancerous foci in all tissues of both Ogg1(-/-) and Ogg1(+/+) mice. A possible explanation is presented to reconcile these results with those of others which showed an increased incidence of tumor formation in untreated Ogg1(-/-) mice.

Topics: Administration, Oral; Animals; Bromates; Carcinogenicity Tests; DNA Damage; DNA Glycosylases; Guanine; Kidney; Mice; Mice, Knockout; Neoplasms

Topics: DNA Damage; DNA Repair; Genetic Predisposition to Disease; Guanine; Humans; Mathematics; Models, Genetic; Molecular Epidemiology; Neoplasms; Polymorphism, Genetic

Epidemiologic studies have revealed a complex association between human genetic variance and cancer risk. Quantitative biological modeling based on experimental data can play a critical role in interpreting the effect of genetic variation on biochemical pathways relevant to cancer development and progression. Defects in human DNA base excision repair (BER) proteins can reduce cellular tolerance to oxidative DNA base damage caused by endogenous and exogenous sources, such as exposure to toxins and ionizing radiation. If not repaired, DNA base damage leads to cell dysfunction and mutagenesis, consequently leading to cancer, disease, and aging. Population screens have identified numerous single-nucleotide polymorphism variants in many BER proteins and some have been purified and found to exhibit mild kinetic defects. Epidemiologic studies have led to conflicting conclusions on the association between single-nucleotide polymorphism variants in BER proteins and cancer risk. Using experimental data for cellular concentration and the kinetics of normal and variant BER proteins, we apply a previously developed and tested human BER pathway model to (i) estimate the effect of mild variants on BER of abasic sites and 8-oxoguanine, a prominent oxidative DNA base modification, (ii) identify ranges of variation associated with substantial BER capacity loss, and (iii) reveal nonintuitive consequences of multiple simultaneous variants. Our findings support previous work suggesting that mild BER variants have a minimal effect on pathway capacity whereas more severe defects and simultaneous variation in several BER proteins can lead to inefficient repair and potentially deleterious consequences of cellular damage.

Topics: DNA Damage; DNA Glycosylases; DNA Ligases; DNA Repair; Genetic Predisposition to Disease; Guanine; Humans; Mathematics; Models, Genetic; Molecular Epidemiology; Neoplasms; Polymorphism, Genetic

There is growing evidence suggesting that cytotoxic activity of cisplatin is closely associated with increased generation of reactive oxygen species (ROS). Therefore, this study was undertaken to examine oxidative DNA damage, which arises as a result of chemotherapy with cisplatin. Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of 8-oxoGua and 8-oxodG excreted into urine in cancer patients (n = 66) who received chemotherapy with cisplatin. One day after the infusion of the drug, significant increase in the amount of 8-oxoGua and 8-oxodG in urine of the patients was observed, when compared to the initial value (78%, p < 0.0001 and 22%, p = 0.0051). In the "nadir days" (when the most distinct cell death based on hematological cell counts can be observed), the level of modified base and nucleoside decreased in comparison with the aforementioned time point. These results, for the first time, indicate that oxidatively damaged DNA may be, at least in part, responsible for cisplatin induced cytotoxicity. Our results also demonstrate that cell death does not contribute to urinary 8-oxoGua and 8-oxodG in humans.

Topics: Antineoplastic Agents; Cisplatin; DNA Damage; Female; Guanine; Humans; Male; Neoplasms; Reactive Oxygen Species

Topics: Biomarkers, Tumor; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Environmental Exposure; Free Radicals; Genetic Predisposition to Disease; Guanine; Humans; Lung Neoplasms; N-Glycosyl Hydrolases; Neoplasms; Oxidative Stress; Phenotype; Polymorphism, Single Nucleotide; Research Design; Risk Factors; Smoking; Treatment Outcome

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (alpha-hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutations Arg(46)-->Gln (alpha-hOgg1-Gln(46)) and Arg(154)-->His (alpha-hOgg1-His(154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containing a multiplicity of base lesions. Michaelis-Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alpha-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154). The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using (32)P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with gamma-irradiated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg(154), in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) found in human tumors are defective in their catalytic capacities.

Topics: DNA; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Guanine; Humans; Kinetics; Mutation; N-Glycosyl Hydrolases; Neoplasms; Pyrimidines; Substrate Specificity

Consumption of a single serving of tomatoes by healthy human volunteers was sufficient to alter levels of oxidative DNA base damage in white cell DNA within 24 h. Levels of the mutagenic oxidized purine base 8-hydroxyguanine decreased, especially in those subjects whose initial levels of this base were higher than the mean. However, total DNA base damage remained unchanged since levels of 8-hydroxyadenine rose. The ability of tomato consumption to modulate oxidative DNA damage in the short term may indicate why daily consumption of fruits and vegetables is beneficial in decreasing cancer incidence.

Topics: Diet; DNA; DNA Damage; Fruit; Guanine; Humans; Leukocytes; Neoplasms; Oxidation-Reduction; Solanum lycopersicum; Vegetables

Topics: Base Composition; DNA Damage; DNA Glycosylases; DNA Repair; DNA Replication; DNA, Bacterial; Escherichia coli; Guanine; Mutation; N-Glycosyl Hydrolases; Neoplasms; Point Mutation