Oxidatively damaged DNA and its repair in colon carcinogenesis

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Mutation Research 736 (2012) 82– 92

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Review

Oxidatively damaged DNA and its repair in colon carcinogenesis

Barbara Tudeka,b, Elzbieta Speinaa,∗

a Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Polandb Institute of Genetics and Biotechnology, Faculty of Biology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw, Poland

a r t i c l e i n f o

Article history:Received 9 January 2012Received in revised form 2 April 2012Accepted 16 April 2012Available online 25 April 2012

Keywords:Colorectal cancerOxidative stressLipid peroxidation8-Oxo-7,8-dihydroguanineEtheno DNA adductsDNA repair

a b s t r a c t

Inflammation, high fat, high red meat and low fiber consumption have for long been known as the mostimportant etiological factors of sporadic colorectal cancers (CRC). Colon cancer originates from neoplas-tic transformation in a single layer of epithelial cells occupying colonic crypts, in which migration andapoptosis program becomes disrupted. This results in the formation of polyps and metastatic cancers.Mutational program in sporadic cancers involves APC gene, in which mutations occur most abundantlyin the early phase of the process. This is followed by mutations in RAS, TP53, and other genes. Pro-gression of carcinogenic process in the colon is accompanied by augmentation of the oxidative stress,which manifests in the increased level of oxidatively damaged DNA both in the colon epithelium, andin blood leukocytes and urine, already at the earliest stages of disease development. Defence mecha-nisms are deregulated in CRC patients: (i) antioxidative vitamins level in blood plasma declines withthe development of disease; (ii) mRNA level of base excision repair enzymes in blood leukocytes of CRCpatients is significantly increased; however, excision rate is regulated separately, being increased for 8-oxoGua, while decreased for lipid peroxidation derived ethenoadducts, �Ade and �Cyt; (iii) excision rateof �Ade and �Cyt in colon tumors is significantly increased in comparison to asymptomatic colon margin,and ethenoadducts level is decreased. This review highlights mechanisms underlying such deregulation,which is the driving force to colon carcinogenesis.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832. Histopathology of CRC and mutations in critical genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833. Oxidative stress in the development of colon cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

3.1. Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843.2. Consumption of red meat and alcohol and tobacco smoking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 843.3. DNA damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

4. Repair of oxidative DNA damage as a driving force to colon carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854.1. Repair of 8-oxo-7,8-dihydro-2′-deoxyguanosine in colon adenoma and carcinoma patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 864.2. Repair of etheno DNA adducts in colon carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

5. Aberrant repair of oxidatively damaged DNA bases and mutations in critical genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 886. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Abbreviations: 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxoGua, 8-oxo-7,8-dihydroguanine; AD, adenoma; CRC, colorectal cancer; APC, adenomatous poly-posis coli; FapyGua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; APE1, apurinic site endonuclease 1; ROS, reactive oxygen species; RNS, reactive nitrogen species;BER, base excision repair; �Ade, 1,N6-ethenoadenine; �dA, 1,N6-etheno-2′-deoxyadenosine; �Cyt, 3,N4-ethenocytosine; �dC, 3,N4-etheno-2′-deoxycytidine; 1N2-�dG, 1,N2-etheno-2′-deoxyguanosine; LPO, lipid peroxidation; MMR, mismatch repair.

∗ Corresponding author. Tel.: +48 22 592 3334; fax: +48 22 592 2190.E-mail address: [email protected] (E. Speina).

0027-5107/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.mrfmmm.2012.04.003


B. Tudek, E. Speina / Mutation Research 736 (2012) 82– 92 83

1. Introduction

Colorectal cancer (CRC) is one of the most frequent causes ofdeath in Western countries. The cases are roughly equally dis-tributed between the sexes. Two main factors are considered inpathogenesis of this cancer: (i) inflammation and high fat diet [1],which increases the number of lesions in DNA and induces muta-tions in critical genes; and (ii) genetic predispositions.

Inflammation is associated with the release of large amountsof reactive oxygen and nitrogen species [2] leading to oxidationof nucleic acids, proteins and lipids, and induction of several pro-mutagenic DNA lesions.

The genetic predisposition to CRC may be connected to the defi-ciency in mismatch repair system (MMR), which causes mutatorphenotype of intestine cells (manifesting itself as microsatellitesinstability – MSI) and development of hereditary non-polyposiscolon carcinoma – HNPCC (or Lynch syndrome). They may alsobe associated with the deficiency of the APC (adenomatous poly-posis coli) gene product, which functions are related to Wntsignaling pathways and migration of intestine epithelium. Lackof APC protein leads to familial adenomatous polyposis (FAP), awell described syndrome characterized by multiple adenomatouslesions which usually appear in the second and third decades oflife. The probability of malignant transformation of one of thepolyps is close to 100% [3]. A subset of patients present an atten-uated form (AFAP) characterized by fewer lesions (<100), a laterage of onset, and a predilection for the proximal colon. About10–15% of AFAP cases reveal mutations in APC gene. More fre-quently, in 20–30% of cases [4,5], AFAP has been correlated witha biallelic mutation of the MutY human homologue gene (MYH).MutY is a DNA glycosylase, which is involved in elimination fromDNA of adenine incorrectly paired with 8-oxo-7,8-dihydroguanine(8-oxoGua). Oxidative DNA damage has been linked to variouspathological conditions, such as aging, carcinogenesis, neurode-generative and cardiovascular disease (reviewed by [6–9]). Thisreview focuses on the role of oxidatively induced DNA lesions incolon carcinogenesis. We discuss the mechanisms of tumor ini-tiation (generation of DNA damage and mutations) and multiplemolecular steps in the colon cancer progression with special focuson the deregulation of defense mechanisms, such as anti-oxidantresponse and DNA repair.

2. Histopathology of CRC and mutations in critical genes

A single layer of epithelial cells lines the crypts of the colonand rectum. Throughout the digestive tract, these crypts substan-tially increase the surface occupied by the epithelium. Four to sixstem cells at the base of each crypt give rise to three epithelial celltypes, absorptive cells, mucus-secreting goblet cells and epithe-lial cells. The cells multiply in the lower third of the crypts anddifferentiate in the upper two-thirds. Normally, the birth rate ofthe colonic epithelial cells precisely equals the rate of cell lossfrom the crypts apex to the lumen of the colon. The birth/loss ratioincreases under neoplastic transformation of cells originating froma single progenitor cell. Polyp, a mass of cells protruding from thebowel wall is often the first observed syndrome of the developmentof colon cancer. However, according to official medical classifica-tions colonic polyps are not recognized as cancer. There are twomain types of polyps, which can be distinguished histologicallybut not by their external appearance [10]. The non-dysplastic orhyperplastic type contains large number of cells that have a nor-mal morphology. Cell epithelium is lined up in a single row alongthe basement membrane, and these polyps apparently have littletendency to become neoplastic. Adenomatous polyp type is dys-plastic, has abnormal intercellular organization. Several layers of

epithelial cells are observed; the nuclei of the epithelial cells arelarger than normal, and their position within the cells is often aber-rant. Crypts crowd together in kaleidoscopic pattern. As adenomasgrow in size, they become more dysplastic. They are also more likelyto contain “villous” components, i.e., fingerlike projections of dys-plastic crypts that can be distinguished from the smooth contour ofthe less advanced “tubular” adenomas. As adenomas progress, theyare likely to become malignant, defined as the ability for invad-ing surrounding tissue and travel to distant organs through directspread or transport by blood and lymphatic vessels. Malignanttumors are not necessarily larger or more dysplastic than benigntumors; their sole determining feature is invasiveness.

Polyps are the earliest clinical manifestation of colorectal neo-plasia. However, the earliest pre-carcinogenic changes in the coloncan be observed under microscope after methylene blue stainingor examination of the colonic mucosa cross sections (Fig. 1). Thesechanges are termed aberrant crypt foci (ACF). Like their larger coun-terparts, can be either dysplastic or non-dysplastic. In mice or ratsACF are found as soon as 2–3 weeks following administration ofcolon carcinogens [11], they grow with time giving rise to malig-nant tumors 6 months after animal intoxication with carcinogen[12].

Consecutive stages of colon carcinogenesis are accompanied byacquiring mutations in critical genes, which regulate cell prolifer-ation, and/or apoptosis. A relatively limited number of oncogenesand tumor suppressor genes, most prominently the APC, KRAS, andTP53 genes are mutated in CRCs, and a larger collection of genes thatare mutated in subsets of CRC have begun to be defined. On the basisof the frequency of mutations occurring in different genes at par-ticular stages of adenoma to carcinoma progression, the sequenceof genetic changes was proposed [13]. We will briefly summarizethe genetic events in order to shed light how these changes mayaffect the extent of oxidative DNA damage, and modulate its repairrate. The earliest genetic change in colon carcinogenesis is muta-tion in the APC gene. About 70–80% of sporadic adenomas andcarcinomas have somatic mutation that inactivate the APC gene,and its frequency is similar in the earliest lesions, like microscopicadenomas composed of few dysplastic glands, and in advancedadenomas and carcinomas, independently of the efficiency of theMMR system. APC protein functions in the Wnt signaling path-way, in which stimulates proteasomal degradation of �-catenin,an activator of TCF/LET (T cell factor/lymphoid enhancer family)transcription factors family. In consequence APC inhibits transcrip-tion of several genes, such as proto-oncogenes, e.g., c-MYC or cyclinD1, genes encoding membrane factors – matrix metalloproteinase7 (MMP-7)/Matrilysin, membrane type 1 MMP, laminin-5 �2 chain,CD44, growth factors, e.g., FGF20, FGF9, and Wnt pathway feedbackinhibitors, such as AXIN2, Naked-1/-2, Dickkopf-1, WNT inhibitoryfactor 1. Transcriptional program induced by �-catenin resemblesthe transcriptional program in stem cells at the base of the coloncrypts [14]. Thus, activation of �-catenin caused by APC mutationwill stimulate proliferation of epithelial cells in colonic crypts. Next,mutations in genes functioning in EGFR signaling pathway, suchas K-RAS, BREF or PTEN will further enhance cell proliferation andsurvival, and drive progression of early adenomas to intermediatelesions. K-RAS is mutated in almost 40% of intermediate and lateadenomas and carcinomas. Inactivation of TGF-� response, e.g., DCC(deleted in colorectal cancer) or TGF- receptor mutations causeloss of growth inhibitory effect of TGF-� and suppress apoptosis.These genetic changes are frequently observed in late adenomalesions. Loss of TP53 function is the late genetic change, whichfurther stimulates proliferation and survival promoting the shiftof adenoma to carcinoma. TP53 mutations are observed in about50% of colorectal cancers, but much less frequently in adenomas. Atleast two of these genetic changes, loss of APC and TP53 functionsmay affect DNA repair in colonic crypts, and in consequence the


84 B. Tudek, E. Speina / Mutation Research 736 (2012) 82– 92

Fig. 1. Aberrant crypt foci. (A) A view from the lumen site of intact mouse colon. The colon was cut open along the longitudinal median axis, formalin fixed and stained withmethylene blue. The mucosal side was observed under the light microscope (magnification 100×); (B and C) cross sections of mouse epithelium stained with hematoxylinand eosin (magnification 1000×).

extent of DNA damage, and potential of gaining new mutations (seeSection 5).

3. Oxidative stress in the development of colon cancer

The majority of pathological factors favoring development ofcolon cancer involve induction of oxidative stress in the colon,which is defined as overproduction of oxygen species combinedwith insufficiency in protective mechanisms of anti-oxidativedefence [15,16]. Oxidative stress is involved in the production ofDNA damage and mutations associated with the initiation or pro-gression of human cancers [17,18]. The main pathological factorsfor the development of CRC are inflammation, fat metabolism,consumption of meat and alcohol and tobacco smoking. All thesestimulate the release of ROS and RNS as well as lipid peroxidation(LPO) [1,19]. These compounds play a dual role in cells. At low ormoderate concentrations they are mediators of specific physiolog-ical processes, whereas high concentrations can be detrimental toall components of the cell.

3.1. Inflammation

Epidemiological studies suggest that inflammatory processesare linked with CRC. This is true particularly in the associationof inflammatory bowel disease and colorectal cancer [20]. Duringinflammation activated macrophages release large amounts of ROSand RNS [2], which oxidize nucleic acids, proteins and lipids. Oxi-dation of polyunsaturated fatty acids generates reactive aldehydesunsaturated in �, �-positions which, in contrast to ROS and RNS,are relatively stable and can diffuse throughout the cell [19]. Shortchain reactive aldehydes are classified into the following families:2-alkenals (e.g., acrolein, crotonaldehyde, 2-hexenal), 4-hydroxy-2-alkenals (e.g., 4-hydroxy-2-hexenal – HHE, 4-hydroxy-2-nonenal– HNE) and ketoaldehydes (e.g., malondialdehyde – MDA, gly-oxal, 4-oxo-2-nonenal – ONE) [21]. Multiplicity of LPO productsis tremendously augmented by the existence of stereoisomers ofseveral of these compounds.

Reactive aldehydes either react directly with DNA and pro-teins [22], or undergo further oxidation to more reactive epoxy

derivatives, such as 2,3-epoxy-4-hydroxynonanal [23]. The affin-ity of epoxidized LPO products to nucleic acids is much higherthan to proteins. Epoxidation of enals may occur in the presenceof hydrogen peroxide, but is also catalysed by enzymes produc-ing inflammatory mediators, prostaglandins and leukotrienes, suchas cyclooxygenase-2 (COX-2) and lipooxygenases (LOX) [24]. Itwas observed that COX-2 is overexpressed in 80% of colorectalcancer tissues [25,26]. Increased levels of COX-2 were observedin epithelial cells, inflammatory cells, as well as in stromal cells,but not in adjacent normal tissues [27]. COX-2 overexpression inMin (multiple intestinal neoplasia) mice was sufficient to inducemammary tumors [28]. The mechanism of tumor induction byCOX-2 overexpression is complex, and involves the activity of itsenzymatic end-products, prostanoids. A number of theories havebeen proposed to explain the role of tumor-derived prostanoids inpromotion of angiogenesis, induction of tumor cell proliferation,suppression of immune response, and protection against apopto-sis [29–31]. Additionally, it was shown that arachidonic acid (AA)metabolism mediated by COX-2 and 5-LOX results in the forma-tion of heptanone-etheno (H�)-DNA adducts. The H�-DNA adductsare highly mutagenic in mammalian cells suggesting that thesepathways could be (at least in part) responsible for the somaticmutations observed in tumorigenesis [32]. That is why inhibitionof COX-2 by non-steroid anti-inflammatory drugs or RNA interfer-ence appeared a successful approach in colon cancer prevention[33,34].

3.2. Consumption of red meat and alcohol and tobacco smoking

Epidemiological and animal model studies have suggested thathigh intake of heme, present in red meat, is associated with anincreased risk of colon cancer. Recently elucidated mechanismssuggest that heme induces DNA damage and cell proliferationof colonic epithelial cells via hydrogen peroxide produced byheme oxygenase. In Caco-2 cells the presence of heme oxygenaseinhibitor, zinc protoporphyrin, reduced DNA damage (measured bya comet assay) triggered by hemin as well as cell hyperprolifera-tion [35]. The effect of heme may be modified by LPO products.The preincubation of colon adenocarcinoma, SW480, cells with



hemoglobin (Hb) and its subsequent exposure to linoleic acidhydroperoxides (LAOOH) led to an increase in cell death, malon-dialdehyde formation, and DNA fragmentation and these effectswere related to the peroxide group and the heme present inHb. SW480 cells treated with Hb and LAOOH presented a higherlevel of the modified DNA bases 8-oxoGua and 1,N2-etheno-2′-deoxyguanosine (1,N2-�Gua) compared to the control. Incubationswith Hb led to an increase in intracellular iron levels, which cor-related with DNA oxidation. Thus, Hb from either red meat orbowel bleeding caused by inflammatory processes could act as anenhancer of fatty acid hydroperoxide genotoxicity, and in conse-quence contribute to the accumulation of DNA lesions in colon cells[36].

Another frequently suggested risk factor for CRC developmentis chronic alcohol consumption. One of the factors which maycontribute to the development of alcohol-associated cancer is theaction of acetaldehyde, the first and most toxic metabolite ofalcohol metabolism [37]. Acetaldehyde is produced by the oxida-tion of ethanol by hepatic NAD-dependent alcohol dehydrogenaseand during threonine catabolism. Acetaldehyde reacts with 2′-deoxyguanosine in DNA to form 1,N2-propanodG and 1,N2-�dG[38]. Both these DNA lesions were described to derive also fromlipid peroxidation [39,36]. In hepatic cancer model, lean and obeseZucker rats higher levels of etheno DNA adducts were observed inthe liver DNA of alcohol-fed than in control animals. This increasewas correlated with the induction of cytochrome P-450 isoform 2E1[40]. Thus, the formation of exocyclic DNA adducts, like propan-odG, 1,N2-�dG, �dA and �dC may be the important mechanismassociated with acetaldehyde related cancer risk.

The results of epidemiological studies also link tobacco smokingwith the risk for CRC development. However, the obtained dataare controversial, and the molecular mechanism remains unclear.Although in the leukocytes of lung cancer patients tobacco smokingincreased the level of one of the major oxidatively damaged DNAbase, 8-oxoGua [41], in CRC patients no effect of tobacco smoking on8-oxoGua level in the leukocytes and 8-oxoGua urinary excretionwas observed [42].

3.3. DNA damage

Oxidatively damaged DNA has been blamed for the physio-logical changes associated with degenerative diseases and cancer[43,44]. DNA damage caused by free radicals is the most heteroge-neous group of DNA lesions due to the wide variety of deleteriousmolecules produced in cells in the conditions of oxidative stress.A plethora of damaged DNA bases are formed upon ROS attack ongenetic material, several of them reveal strong promutagenic prop-erties [45]. One of the major and best studied is 8-oxoGua, a typicalbiomarker of oxidative stress, which may play a role in carcinogen-esis [46]. The presence of 8-oxo-7,8-dihydro-2′-deoxyguanosine(8-oxodG) residues in DNA leads to GC→TA transversions [47].8-OxodG is formed in DNA either via direct oxidation of nucleicacids or can be incorporated from the nucleotide pool by DNApolymerases, the latter process being an important source of DNAoxidation and genome instability [48,49].

LPO products may contribute to DNA damage to nearly the sameextent as directly oxidized bases. There are distinct groups of lin-ear and cyclic LPO specific DNA lesions. Malondialdehyde reacts inDNA with guanine, adenine and cytosine to form cyclic pyrimido-[1,2�]purine-10(3H)-one-2′-deoxyribose (M1dG) adduct and lin-ear N6-(3-oxopropenyl)-2′-deoxyadenosine (M1dA) and N4-(3-oxopropenyl)-2′-deoxycytosine (M1dC) adducts, respectively [50].M1dG is the major MDA-DNA adduct and was detected in humanand rodent tissues. In Escherichia coli cells, it induces transversionsto T and transitions to A with a frequency comparable with that of8-oxoGua [15].

Exocyclic propano DNA adducts are formed by the attachmentof unsaturated reactive aldehyde to ring and extra-ring nitrogenatoms in DNA bases, which generates additional saturated, six-membered ring in a DNA base. These adducts are derived from thereaction of DNA with, e.g., acrolein, crotonaldehyde and HNE. Acr-dG, Cro-dG and HNE-dG were found in rodent and human tissues[51,52]. Etheno DNA adducts posses five-membered exocyclic ringsattached to DNA bases. The exact pathway of their formation in vivois not known, although it is clear that they are generated by theexposition of DNA to lipid peroxides. Ethenoadducts are also intro-duced into DNA by certain environmental carcinogens like vinylchloride or its metabolite, chloroacetaldehyde (CAA) [53]. Propano-and etheno-type adducts that are found in native mammalian DNAare unsubstituted and substituted with side chains of differentlength, depending on the structure of reacting LPO product. Allexocyclic DNA adducts have strong miscoding potential. Unsub-stituted exocyclic DNA adducts are more mutagenic than toxic.With the increase of the side chain the toxic properties increaseand are related to the inhibition of replication and transcription[54,55].

Elevated levels of �dA and �dC were found in pro-carcinogeniccolon diseases, like inflammatory bowel disease, Crohn’s dis-ease and ulcerative colitis, and in addition in chronic pancreatitis[20,56]. This suggests that LPO-derived DNA lesions contribute toCRC etiology.

4. Repair of oxidative DNA damage as a driving force tocolon carcinogenesis

The major pathway for repair of oxidatively derived DNA lesionsis base excision repair system (BER). Although several oxidativelydamaged bases are also repaired by mismatch repair system andAlkB family dioxygenases, this review will focus on BER activity.In a classical view BER consists of five steps: (i) lesion recogni-tion, (ii) excision of the damaged base, (iii) AP site incision andremoval of the chemical residues that could block further stepsof the pathway, (iv) incorporation of the proper nucleotide(s) byDNA polymerases and (v) ligation of the DNA strand to restore theoriginal DNA molecule [57–60].

Damaged bases are recognized by DNA glycosylases, whichexcise from DNA a wide range of oxidized and alkylated bases.Presently, 13 human DNA glycosylases with different, but partiallyoverlapping substrate specificity have been identified. Mecha-nistically two types of DNA glycosylases can be distinguished,monofunctional enzymes, that excise damaged base leaving behindan abasic (AP) site, which is subsequently cleaved by AP endonu-clease (APE1) 5′ to the AP site, and bifunctional DNA glycosylases,which have endowed AP lyase activity that incises phosphodiesterbonds 3′ to the AP site created by the glycosylase activity of theenzyme. This review will focus on repair of three oxidative DNAlesions, 8-oxoGua, �Ade and �Cyt.

8-OxoGua is repaired by a three-step system, which (i) lim-its incorporation of 8-oxodGTP into DNA during replication, (ii)excises 8-oxoGua from DNA, and (iii) corrects the base opposite to8-oxoGua in DNA. Different DNA polymerases can incorporate 8-oxodGTP to the DNA, and replicative DNA polymerases incorporatethis modified nucleotide usually opposite dA, generating A:T → C:Gmutations. Approximately a half of 8-oxodG in DNA arises from thedirect DNA oxidation, and the other half is a result of incorporationof 8-oxodGTP during DNA synthesis. Incorporation of 8-oxodGTPinto DNA by DNA polymerases is limited by the activity of MTH1phosphohydrolase, which hydrolyzes 8-oxodGTP to 8-oxodGMP[61]. 8-OxodGMP is subsequently dephoshorylated by nucleoti-dase and removed from the cell [62]. Many observations indicate adirect correlation between 8-oxodG formation and carcinogenesis



in vivo [46,63–66]. The second line of defence is glycosylase MutY(MYH) that removes adenine paired with 8-oxoGua. Activity of thisenzyme prevents mutations G:C → T:A, when dATP is incorporatedopposite 8-oxoGua during replication [67]. After removal of Adeby MYH glycosylase, repair DNA polymerase incorporates dCMPopposite 8-oxodG. Then 8-oxoGua can be excised by the third com-ponent of the system, OGG1 glycosylase [68]. OGG1 glycosylase/APlyase is the major enzyme catalyzing excision of 8-oxoGua fromDNA, but only when paired with Cyt or Thy [69,70]. The AP lyaseactivity of the OGG1 protein is about 10-fold lower than that ofthe glycosylase [71]. When 8-oxodGTP is included into DNA duringreplication it is probably removed by NEIL1 or NEIL2 glycosylases,which excises 8-oxoGua paired with dC/G/T/A or dC/A, respec-tively [72]. This complex three-tier system prevents mutationsinduced by the presence of 8-oxodG in DNA, both when this lesionis induced directly in the DNA, and when it is incorporated fromthe pool of nucleotides during replication. Polymorphisms of MYHand OGG1 glycosylases are related to modulation of susceptibilityto colon cancer. MYH variations significantly increase CRC risk, andof several mutations described, two are the most prevalent: Y165C(exon 7) and G382D (exon 13). Mutated enzyme excises Ade fromdA:8-oxodG pair with a decreased rate, increasing mutation fre-quency. The association of MYH with CRC was first reported in 2002[73].

Several polymorphic changes in the OGG1 gene have beendescribed, with the most common being Ser326Cys [74]. Polymor-phic Cys326 OGG1 protein was found to have a lower enzymaticactivity, both when 8-oxoGua was excised from oligodeoxynu-cleotides [75], and when 8-oxoGua and FapyGua were liberatedfrom �-irradiated DNA by purified Cys326 or Ser326 OGG1 enzyme[74,76]. It was suggested that the presence of two OGG1 326Cysalleles may confer an increased risk of lung, prostate and nasopha-ryngeal cancer [77–79], but no association with the risk for coloncancer was found [80,81]. Our and other functional studies reporta decrease in 8-oxoGua excision rate in lung [41,82,83] andhead and neck cancer patients [84]. Such a decrease in excisionrate and simultaneous increase in 8-oxodG and FapydG levelsin cellular DNA favors a pro-oxidant state and may acceleratethe acquisition of mutations in critical genes leading to cancer.Such an idea is basically derived from the observation that apro-oxidant environment is characteristic for advanced stages ofcancer.

�Ade is excised by alkylpurine-DNA-N-glycosylase (ANPG) [85],whereas �Cyt by thymine-DNA glycosylase (TDG); the latter alsoexcises thymine from G:T pairs, resulting from deamination of5-methylcytosine [86]. Ethenocytosine is also excised by single-stranded uracil DNA glycosylase, SMUG1 [87] and by MBD4glycosylase, although activity of the latter is strongly limited to CpGislands [88,89].

ANPG glycosylase has a wide substrate specificity, andbesides �Ade excises from DNA also 1,N2-ethenoguanine [90],hypoxanthine and the alkylated bases, 3-methyladenine and 7-methylguanine [85]. TDG excises �Cyt, Thy and Ura from pairswith guanine, and its activity is strongly stimulated by thenext enzyme in the BER pathway, AP endonuclease, APE1 [91].TDG and APE1 also play the role of transcription regulators[92–95].

Ethenocytosine repair by the BER system is dependent not onlyon the level and activity of TDG, MBD4 or SMUG1 DNA glycosylases,but also on the presence and/or activity of ANPG glycosylase, whichprimary substrate is �Ade. ANPG binds strongly to DNA containing�Cyt, and although does not excise this modified base, does notallow its excision by the proper �Cyt glycosylase, like TDG [96].This may explain observation that the level of �Cyt measured indifferent human tissues was constantly higher than that of �Ade[19].

Table 1mRNA level of DNA glycosylases (OGG1, ANPG and TDG) and APE endonuclease(APE1), and MTH1 phosphohydrolase in relation to 18S rRNA (×10−6) in leukocytesof CRC and AD patients and healthy controls.

mRNA level (arbitrary units)a p

CRC patients (C) AD patients (A) Controls (H)

OGG1 1.28 0.99 0.19 <0.001 (C vs. H)b

(0.71–5.58) (0.45–4.91) (0.06–0.54) <0.001 (A vs. H)b

n = 44 n = 38 n = 40 0.46 (C vs. A)b

12.14 – 1.08 <0.001(C vs. H)ANPG (4.06–37.07) (0.31–4.75)

n = 44 n = 397.97 – 0.81 <0.001 (C vs. H)

TDG (1.87–21.03) (0.26–1.84)n = 44 n = 3988.25 113.83 13.87 <0.001 (C vs. H)b

APE1 (42.88–362.51) (48.74–292.37) (2.32–43.04) <0.001 (A vs. H)b

n = 44 n = 38 n = 40 0.5 (C vs. A)b

86.82 – 5.26MTH1 (5.19–728.53) (0.33–11.69) 0.0012 (C vs. H)

n = 37 n = 19

Adapted from [42,105] with permission from Oxford University Press and Elsevier,respectively.All subjects were Caucasians.

a Comparison of mRNA levels, expressed as median and interquartile range,between CRC and AD patients and controls (Mann–Whitney U test).

b p values are after Bonferroni correction.

4.1. Repair of 8-oxo-7,8-dihydro-2′-deoxyguanosine in colonadenoma and carcinoma patients

In order to get an insight into the importance of oxidative stressand repair of oxidatively damaged DNA bases in the pathologyof colon cancer, we examined groups of individuals at differentstages of disease development, namely CRC patients, patients whodeveloped benign adenomas (AD) and healthy individuals for themarkers of oxidative stress, repair capacity of oxidatively damagedDNA, and polymorphism of DNA repair genes.

Increased oxidative stress was observed both in CRC patientsand in AD individuals. This manifested in an elevation of the 8-oxodG level in leukocyte DNA and in urine as well as depletion ofantioxidant vitamins (ascorbic acid, �-tocopherol, retinol) and uricacid in blood plasma of CRC patients and AD individuals in com-parison to healthy controls [42]. Depletion of antioxidant vitaminsin blood plasma was gradual, which suggests that oxidative stressis increasing gradually with the disease progression. This is sup-ported by the fact that urinary level of 8-oxodG was augmentedalready in AD patients, but 8-oxoGua level was enhanced only inCRC, but not in AD individuals [42]. Urinary 8-oxodG may reflectoxidation of nucleotide pool, while 8-oxoGua reflects rather oxi-dation of DNA. Since nucleotide pool is much more susceptible tooxidation than dsDNA, 8-oxodG in urine may be a more sensitiveindicator of oxidative processes in the body, and shows that evenin AD individuals the oxidative processes are enhanced. The sug-gested mechanisms responsible for the oxidative stress in cancerpatients include [43]: (i) granulocyte activation with release of ROS;(ii) stimulation of cytokines, of which some, e.g., tumor necrosisfactor, produce large amounts of ROS; (iii) production of hydrogenperoxide by malignant cells, which in advanced stages of cancermay be released into the blood stream and penetrate into othertissues [2].

Interestingly, we also observed an increase of mRNA levels ofrepair enzymes, OGG1, APE1 and MTH1 in leukocytes of CRC andAD individuals (Table 1) [42]. Expression of APE1 and OGG1 geneswas shown to be induced by ROS [97], and this could explain induc-tion of repair enzymes transcription in leukocytes of CRC and ADindividuals. On the other hand induction of repair enzymes may bea part of a carcinogenic program, which might decrease apoptosis



A

50

60

70

80

90

100

* * *

CRC patients

Controls

n = 68

B

0

10

20

30

40 n = 90

8-o

xo

Gu

a e

xcis

ion

activity

(p

mo

ls/h

/mg

pro

tein

)

100

110

120

*

* * *

* *

CRC patients

Controls

0

10

20

30

40

50

60

70

80

90

n = 13

n = 32

n = 14

n = 53

8-o

xo

Gu

a e

xcis

ion

activity

(pm

ols

/h/m

g p

rote

in)

* *

OGG1 Ser326Cys polymorphism

Ser/ Ser Ser/Cys Cys/ Cys

n = 34

Fig. 2. The mean and standard deviation of 8-oxoGua excision activity in leukocytesof CRC patients and control individuals, in the entire groups (A) and in the groupsdistinguished on the basis of OGG1 Ser326Cys polymorphism (B). ***, p < 0.001; **,p < 0.005; *, p < 0.05 (Mann–Whitney U test).

Adapted from [42] with permission from Oxford University Press.

in aberrant cells and/or favor genomic instability. Our results cor-roborate the findings of Winnepenninckx and coworkers [98] thatinduction of most of the DNA repair genes occurs very early in thecarcinogenic process, e.g., four years before clinical manifestationof malignant skin melanoma.

Increase of mRNA level of OGG1 and APE1 repair enzymeswas accompanied by the augmentation of 8-oxoGua excision ratein leukocytes of CRC patients (Fig. 2A) [42]. The higher activityobserved in the cell extracts from the cancer patients is even moremeaningful in view of the finding that homozygous OGG1 326Cysvariants, which are generally characterized by lower OGG1 activity,

were much more frequent among the cancer patients than in thecontrol and adenoma groups (Table 2) [42]. In our study the effect ofthe OGG1 Ser326Cys polymorphism on 8-oxoGua excision rate wasclearly seen in the CRC patient group, in which Cys homozygoteswere found to have a decreased 8-oxoGua excision rate in com-parison with Ser homozygotes (Fig. 2B) [42]. Thus, the observedincrease in 8-oxoGua repair capacity in CRC patients in compari-son to healthy individuals might be due to increased transcriptionfrom OGG1 and APE1 genes, which was observed in AD and CRC indi-viduals (Table 1) [42]. Interestingly, the OGG1 genotype exerted alimited effect on 8-oxodG level in leukocytes and urinary excretionof 8-oxoGua [42]. In contrast to our studies, Janssen et al. [99] andJensen et al. [100] reported that 8-oxoGua repair incision in humanlymphocytes was unaffected by the polymorphic status at codon326 of the OGG1 gene. These studies were performed on the popu-lation of healthy subjects only. It was shown that OGG1 Cys326variant forms dimers with lower enzymatic activity only underconditions of oxidative stress probably due to OGG1 cysteine 326oxidation [101]. In parallel, Bravard et al. [102] reported recentlythat extracts from lymphoblastoid cell lines established from indi-viduals carrying the Cys326Cys genotype have almost 2-fold lowerbasal 8-oxoGua DNA glycosylase activity than the Ser326Ser variantand were more sensitive to inactivation by oxidizing agents thanthat of the Ser/Ser cells. Analysis of redox status of the OGG1 pro-tein in cells confirmed that the lower activity of OGG1-Cys326 wasassociated with the oxidation of Cys326 to form a disulfide bond.Thus different degrees of oxidative stress in different studies mightpartially explain the controversies in the literature.

In the group of healthy subjects there was significant positivecorrelation between mRNA levels of the main enzymes involved in8-oxoGua removal (OGG1 and APE1), and the amount of 8-oxoGuain urine [42]. These findings suggested that the amount of 8-oxoGuain urine might be attributed to DNA repair. It is in parallel withprevious results, which also showed that DNA repair was respon-sible for the presence of oxidatively damaged DNA lesions in urine[103]. However, we did not find such correlation in the groups ofadenoma individuals and carcinoma patients. The main reason forthis inconsistency may be aberrant DNA oxidation in cancerous andprecancerous conditions which may overshadow the subtle rela-tionship observed in healthy subjects. Alternatively, the excretionof 8-oxoGua might reflect the level of oxidative stress, independentof the activities of the repair enzymes [104].

Also, only a weak positive correlation was found between 8-oxoGua level in urine and the stage of disease [42].

4.2. Repair of etheno DNA adducts in colon carcinogenesis

In contrast to cleavage of 8-oxoGua, leukocytes excision activ-ity of �Ade and �Cyt was significantly lower in CRC patients when

Table 2Genotypes and allelic frequencies of the OGG1 Ser326Cys polymorphism among CRC patients, individuals with AD and healthy controls.

OGG1 polymorphism CRC patients (C)(n = 74)an (%) AD patients (A)(n = 76)an (%) Controls (H)(n = 97)an (%), (%)b p

Ser326CysSer/Ser 38 (51.3) 46 (60.5) 63 (65), (81)c >0.0000 (C vs. H)Ser/Cys 19 (25.7) 29 (38.2) 33 (34), (18)d >0.079 (A vs. H)Cys/Cys 17 (23) 1 (1.3) 1 (1), (1)e >0.0000 (C vs. A)

Allelic frequencySer (C) 0.64 0.8 0.82Cys (G) 0.36 0.2 0.18

Adapted from [42] with permission from Oxford University Press.All subjects were Caucasians.

a Comparison of OGG1 polymorphic frequency between CRC and AD patients and controls (Chi-square distribution analysis).b Results from Hardy–Weinberg equilibrium.c Homozygous OGG1 Ser326Ser individuals.d Heterozygous OGG1 Ser326Cys individuals.e Homozygous OGG1 Cys326Cys individuals.



25

30

35

40

45

50

n = 56

* * *

activity

μg p

rote

in)

CRC pati ents

C ontrols

54=

A

0

5

10

15

20

n = 56n = 54

*

εAde εCyt

Excis

ion

(pm

ols

/h/ μ n

14

16

n = 54

* * * Tumor

Normal colo n

B

0

2

4

6

8

10

12

n = 54

n = 54

n = 54

* * *

εAde εCyt

Excis

ion

activity

(pm

ols

/h/μ

g p

rote

in)

Fig. 3. (A) The mean and standard deviation of �Ade and �Cyt excision rate inleukocyte DNA of CRC patients and control individuals. ***, p < 0.001; *, p < 0.05(Mann–Whitney U test). (B) The mean and standard deviation of �Ade and �Cytexcision rate in colon tumor and normal surrounding tissues of CRC patients. ***,p < 0.001 (Wilcoxon rank sum test).

Derived from [105] with permission from Elsevier.

compared to controls matched for age, sex and a smoking habit(Fig. 3A) [105]. However, in colon tissues, excision activities for�Ade and �Cyt were significantly higher in tumor than in adjacent,asymptomatic colon tissue (Fig. 3B) [105]. In leukocytes (PBL) ofCRC patients �dA and 1,N2-�dG level was unexpectedly lower thanin controls, and the level of �dA and �dC also showed a tendencyto be lower in tumor than in normal colon tissue [105]. This sug-gests that the regulation of etheno adduct level and repair may bemultifactorial and may differ in target (colon) and surrogate (PBL)tissues.

The lower excision rate for �Ade and �Cyt in leukocytes of CRCpatients was not linked to mutations in exons of ANPG and TDGgenes nor to down regulation of mRNA expression of ANPG andTDG glycosylases or its BER activator, APE1 endonuclease; in thecontrary, mRNA levels of these repair enzymes were significantlyhigher in PBL of CRC than in healthy controls (Table 1) [42,105].Thus direct inactivation/inhibition of repair enzymes activitiesby inflammatory mediators released in the blood stream is amore likely explanation. Indeed, several DNA repair enzymes wereshown to be directly inhibited by nitric oxide [106–108], hydro-gen peroxide [109] and LPO product, 4-hydroxynonenal [55,110].As shown for several human cancer sites and animal tumors, per-sistent oxidative stress and LPO-induced DNA damage increasedalready during premalignant stages in a time-dependent manner[111]. A number of studies demonstrated increase of mRNA levelof several repair enzymes upon oxidative stress [97,112,113]. Theobserved lower �Ade and �Cyt excision (Fig. 3A) and simultane-ous lower level of etheno adducts in PBL of cancer patients [105]

may thus suggest that other mechanisms are up-regulated in CRCpatients and determine the level of etheno adducts in DNA. Otherrepair systems engaged in etheno adducts elimination from DNAinclude, e.g., oxidative demethylation by AlkB family proteins ornucleotide excision repair [114,115].

Of interest are the findings in tumorous colon tissue. The �Adeand �Cyt excising activities were much higher in tumor than inthe surrounding normal colon (Fig. 3B) and was consistent with a2–3 times lower �dA and �dC adduct levels [105]. Also in severalother studies lower level of �dA and �dC was observed in colontumors than in colon tissues of cancer prone patients suffering fromulcerative colitis, Crohn’s disease or familial adenomatous polypo-sis [56,116]. In our study in normal colon a negative correlation wasfound between �dC level in DNA and its excision rate (� = −0.64,p = 0.013) [105]. This supports a role of BER in �Cyt-elimination;however, does not exclude other etheno adduct control systems. Inseveral types of non tumorous human tissues previously analyzed,the �dA level in DNA was found to be lower than that of �dC [111],consistent with our observation that excision activity was found tobe higher for �Ade than for �Cyt (Fig. 3) [105].

In order to understand a huge increase of excision activity of�Ade and �Cyt in colon tumor tissue in comparison to asymp-tomatic colon margin, we measured mRNA level of ANPG and TDGglycosylases, as well as APE1 endonuclease in tumor and normalcolon tissues. Higher mRNA level in tumor tissue was observedonly for APE1 [105]. APE1 stimulates �Cyt excision [91], and mostprobably also �Ade. Another possibility are frequent mutations inthe APC gene in colon tumors [117]. APC tumor suppressor pro-tein may inhibit BER engaged in repair of �Ade and �Cyt since itwas shown that it can directly interact with DNA polymerase �and FEN1 endonuclease [118]. BER activity was inversely associatedwith APC expression in several breast cancer cell lines [119].

In conclusion, we have shown, for the first time, that excisionactivity of �Ade and �Cyt is lower in PBL of CRC patients whencompared to healthy individuals. However, our unexpected find-ings, that the level of �dA and 1,N2-�dG was lower in DNA ofPBL from CRC patients than in healthy controls currently remainsmechanistically difficult to explain. If such etheno adducts, par-ticularly �Cyt excision deficiency also occurs during premalignantstages in the target organ, still may favor acquisition of mutationsin critical genes leading to neoplastic transformation. TDG glyco-sylase removes Thy and Ura paired with guanine, and may preventinduction of mutations caused by cytosine and 5-methylcytosinedeamination [120].

5. Aberrant repair of oxidatively damaged DNA bases andmutations in critical genes

Activity and expression of BER proteins is modulated by two sig-naling molecules, which are frequently mutated in colon cancer, theAPC and TP53 gene products. Thus, mutations in these genes affectrepair rate of oxidatively damaged DNA, and favor pro-mutagenicevents within colon epithelial cells. On the other hand at least threeBER proteins, namely, APE1, TDG and OGG1 take part in the regu-lation of gene expression.

Mutations in the APC gene are early events in colon carcinogen-esis, and are found in about 80% of colon adenomas and carcinomas.The most frequent mutation results in truncation of the C-terminalpart of the APC protein. Wild type APC protein directly interactswith DNA polymerase �, FEN1 endonuclease [118], and as recentlysuggested, APE1 endonuclease [121] inhibiting the assembly of BERproteins on damaged DNA and the activity of short and long patchBER (Fig. 4). In LoVo, colon cancer cells expressing truncated APCprotein an accelerated assembly of BER proteins, as well as higheractivity of APE1, FEN1 and Pol � were observed [121]. Activation of



APE1/

ANPG

+

Genome

instability Progression

+

Genome

instabilityProgression

Pol βFEN1

APE1

+TP53

APC

mutated +

+

TDG/MBD4

AID-media ted

demethyla tion of

gene promoter s

Transcription of

several gene s

Transcriptio n of

Myc-regulated

genes

Prolifera tio n

ProgressionOGG1

mutated +

++

HIF-1αAngiogenesis

Erythropoesi s

Fig. 4. The influence of APC and TP53 genes mutations on the activity of BER enzymes and genome stability. Mutations in APC and TP53 proteins lead to increase in APE1activity and transcription, respectively, and up-regulate BER. Increased APE1, ANPG and unbalanced BER pathway may favor genomic instability and cancer progression.APE1 activity may also accelerate the excision rate of DNA glycosylases, OGG1 and TDG/MBD4. APE1, TDG and OGG1 take part in the regulation of gene expression. TDGinteracts with the deaminase AID and may favor demethylation of promoters and induction of transcription of several genes. OGG1 and APE1 stimulate transcription of genesregulated by c-Myc and estrogen receptors, and thus may contribute to acceleration of cell proliferation. Increased APE1 activity may result in unbalancing of BER pathway,and may favor genomic instability and cancer progression. APE1 also participates in HIF-1� mediated angiogenesis and erythropoesis and may favor tumor growth. + symbolin circle denotes stimulation or increase; APC, adenomatous polyposis coli; Pol �, DNA polymerase �; FEN 1, flap endonuclease 1; APE1, apurinic site endonuclease 1; ANPG,alkyladenine DNA N-glycosylase; OGG1, 8-oxoGua DNA N-glycosylase; TDG, thymine DNA N-glycosylase; MBD4, methyl-CpG binding domain protein; AID, activation-induceddeaminase; HIF-1�, hypoxia-inducible factor 1� subunit.

APE1 may also occur as a consequence of TP53 gene mutations,which are late, but frequent events in colon carcinogenesis (inabout 50% of cancers). TP53 protein participates in downregulationof APE1 transcription in cells treated with DNA damaging agents[122]. On the other hand TP53 mediates ubiquitination of APE1by TP53 inhibitor, MDM2, and in consequence APE1 degradation[123]. Thus, mutations in TP53 gene should additionally increaseAPE1 amount in cancer cells.

Increased APE1 activity may result in unbalancing of BERpathway, and may favor genomic instability and cancer progres-sion: in inflamed non-cancerous colon tissues of ulcerative colitispatients (at high risk for colon cancer, that accumulate high ethenoDNA adducts [124]), ANPG and APE1 activities were significantlyincreased, and microsatellite instability (MSI) was positively cor-related with their imbalanced activities of repair enzymes [125].Similarly, in yeast and human cells over-expression of ANPG and/orAPE1 was associated with frameshift mutations and MSI [125].

APE1, besides DNA repair function, also plays a role of redoxregulatory protein for several transcription factors. For example,participates in HIF-1� mediated angiogenesis [126]. Thus at latestages of CRC development, mutations in TP53 gene will lead toaccumulation of APE1 protein, and in addition to increasing genomeinstability will also favor angiogenesis and in consequence tumorgrowth (Fig. 4).

Increased APE1 activity may also accelerate the excision rate ofDNA glycosylases. TDG and OGG1 were shown to be particularlysusceptible to induction by APE1, since they bind strongly to APsites, and APE1 increases turnover of these enzymes on damagedDNA [91,127]. Increased activity of TDG may favor demethyla-tion of promoters of several genes and in consequence contributeto hypomethylation of DNA observed in colon cancer [13] andinduction of transcription of several genes. TDG interacts with thedeaminase AID and the damage response protein GADD45a. 5-Methylcytosine and 5-hydroxymethylcytosine are first deaminatedby AID to thymine and 5-hydroxymethyluracil, respectively, andsubsequently TDG removes thymine and 5-hydroxymethyluracilfrom dT:dG or 5OHMedU:dG pairs, which initiates BER and enablesreconstitution of G:C base pairs [128]. TDG is also necessary forrecruiting p300 to retinoic acid (RA)-regulated promoters, andis engaged in transcriptional control of genes during embryonic

development. Its deficiency is embrionaly lethal [120]. TDG statusmay also determine sensitivity of CRC patients to chemotherapywith 5-fluorouracil (5-FU), a chemotherapeutic routinely used inCRC treatment. Inactivation of TDG significantly increases resis-tance of mouse and human cancer cells toward 5-FU. Excision of5-FU from DNA by TDG generates persistent DNA strand breaks,delays S-phase progression, and activates DNA damage signaling.Hence, excision of 5-FU by TDG mediates DNA-directed cytotoxicity[129].

OGG1 and APE1 stimulate transcription of genes regulatedby c-Myc transcription factor [130], and thus may contribute toacceleration of cell proliferation (Fig. 4). The mechanism involvesdemethylation of histone H3 by specific FAD-containing enzyme,LSD1 demethylase which generates hydrogen peroxide duringdemethylation [131]. In consequence a local oxidation of DNAaround the E-boxes and TSS region of Myc target genes is triggered.8-OxodG, OGG1 and APE1 were found to be engaged on the sameRNA polymerase II and Myc occupied chromatin regions of Myc tar-gets and stimulate transcription of these genes. Silencing of eitherOGG1 or APE1 inhibits Myc mediated transcription. It is possiblethat regulatory regions in a gene interact with each other form-ing loops that often bridge considerable distances on the genomicloci [132]. Following Myc binding local demethylation of lysine 4in histone H3 (H3K4me2) triggers recruitment of the OGG1 andthe downstream BER enzymes thus producing nicks on Myc tar-get chromatin. This may relax the DNA strands and could favorchromatin bending necessary for the formation of chromatin loopsthat bring together RNAPII and Myc. OGG1 is also engaged in theregulation of transcription from estrogen promoters [133].

6. Conclusions

Oxidative stress is induced at the early, adenoma, stage of coloncarcinogenesis, and is increasing during disease progression. CRCand AD patients reveal a huge increase of mRNA level of baseexcision repair enzymes in blood leukocytes. However, increasedtranscription is not always correlated with repair capacity of tis-sues. Activities of BER enzymes remain under a complex regulativenetwork, which besides stimulation of transcription includes directinactivation by oxidation stress products, interaction with other



BER proteins, and with signaling molecules, which are frequentlymutated in colon cancer, that is APC and TP53 proteins. Mutatedproteins do not inhibit transcription and/or activity of base exci-sion repair enzymes. This increases the amount of repair enzymeswithin cells and introduces imbalance in the repair of oxidativeDNA damage. Such imbalance causes increased genome instabilityand may be a driving force in the disease progression.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Funding

Funded by the Polish-French grant project 346/N-INCA/2008/0.We thank all patients and volunteers who participated in this study.

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Oxidatively damaged DNA and its repair in colon carcinogenesis