Oxidatively damaged DNA and its repair in colon carcinogenesis
Barbara Tudek1,2, Elżbieta Speina1*
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106
Warsaw, Poland; 2 Institute of Genetics and Biotechnology, Faculty of Biology, Warsaw University,
Pawińskiego 5a, 02-106 Warsaw, Poland
*Corresponding author: Elżbieta Speina
Institute of Biochemistry and Biophysics
Polish Academy of Sciences
Pawińskiego 5a
02-106 Warsaw, Poland
tel: (+4822) 592-3334; fax: (+4822) 592-2190
e-mail: elasp@ibb.waw.pl
Running Title: DNA repair in colon cancer
Abbreviations:
8-oxodG–8-oxo-7,8-dihydro-2’-deoxyguanosine;
8-oxoGua–8-oxo-7,8-
dihydroguanine; AD–adenoma; CRC–colorectal cancer; APC–adenomatus polyposis 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,N4etheno-2’-deoxycytidine;
1,N2-εdG–1,N2-etheno-2’-deoxyguanosine;
MMR–mismatch repair;
1
LPO–lipid
peroxidation;
ABSTRACT
Inflammation, high fat, high red meat and low fiber consumption have for long been known
as the most important etiological factors of sporadic colorectal cancers (CRC). Colon cancer
originates from neoplastic transformation in a single layer of epithelial cells occupying colonic
crypts, in which migration and apoptosis 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 abundantly in the early phase of the process. This is followed by
mutations in RAS, TP53, and other genes. Progression 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, and in blood leukocytes and urine, already
at the earliest stages of disease development. Defence mechanisms are deregulated in CRC patients:
(i) antioxidative vitamins level in blood plasma declines with the development of disease; (ii)
mRNA level of base excision repair enzymes in blood leukocytes of CRC patients 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 rate of 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.
Keywords: Colorectal cancer, oxidative stress, lipid peroxidation, 8-oxo-7,8-dihydroguanine, etheno
DNA adducts, DNA repair
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1. Introduction
Colorectal cancer (CRC) is one of the most frequent causes of death in Western countries.
The cases are roughly equally distributed between the sexes. Two main factors are considered in
pathogenesis of this cancer: (i) inflammation and high fat diet [1], which increases the number of
lesions in DNA and induces mutations in critical genes; and (ii) genetic predispositions.
Inflammation is associated with the release of large amounts of reactive oxygen and nitrogen
species [2] leading to oxidation of nucleic acids, proteins and lipids, and induction of several promutagenic DNA lesions.
The genetic predisposition to CRC may be connected to the deficiency in mismatch repair
system (MMR), which causes mutator phenotype of intestine cells (manifesting itself as
microsatellites instability – MSI) and development of hereditary non-polyposis colon carcinoma –
HNPCC (or Lynch syndrome). They may also be associated with the deficiency of the APC
(adenomatus polyposis coli) gene product, which functions are related to Wnt signalling pathways
and migration of intestine epithelium. Lack of APC protein leads to familial adenomatus polyposis
(FAP), a well described syndrome characterized by multiple adenomatous lesions which usually
appear in the second and third decades of life. The probability of malignant transformation of one of
the polyps is close to 100% [3]. A subset of patients present an attenuated form (AFAP)
characterized by fewer lesions (<100), a later age of onset, and a predilection for the proximal
colon. About 10-15% of AFAP cases reveal mutations in APC gene. More frequently, in 20-30% of
cases [4,5], AFAP has been correlated with a biallelic mutation of the MutY human homologue gene
(MYH). MutY is a DNA glycosylase, which is involved in elimination from DNA of adenine
incorrectly paired with 8-oxo-7,8-dihydroguanine (8-oxoGua). Oxidative DNA damage has been
linked to various pathological conditions, such as aging, carcinogenesis, neurodegenerative and
cardiovascular disease (reviewed by [6-9]). This review focuses on the role of oxidatively induced
DNA lesions in colon carcinogenesis. We discuss the mechanisms of tumor initiation (generation of
DNA damage and mutations) and multiple molecular steps in the colon cancer progression with
3
special focus on the deregulation of defense mechanisms, such as anti-oxidant response and DNA
repair.
2. Histopathology of CRC and mutations in critical genes
A single layer of epithelial cells lines the crypts of the colon and rectum. Throughout the
digestive tract, these crypts substantially increase the surface occupied by the epithelium. Four to
six stem cells at the base of each crypt give rise to three epithelial cell types, absorptive cells,
mucus-secreting goblet cells and epithelial cells. The cells multiply in the lower third of the crypts
and differentiate in the upper two-thirds. Normally, the birth rate of the colonic epithelial cells
precisely equals the rate of cell loss from the crypts apex to the lumen of the colon. The birth/loss
ratio increases under neoplastic transformation of cells originating from a single progenitor cell.
Polyp, a mass of cells protruding from the bowel wall is often the first observed syndrome of the
development of colon cancer. However, according to official medical classifications colonic polyps
are not recognized as cancer. There are two main types of polyps, which can be distinguished
histologically but not by their external appearance [10]. The non-dysplastic or hyperplastic type
contains large number of cells that have a normal morphology. Cell epithelium is lined up in a
single row along the basement membrane, and these polyps apparently have little tendency to
become neoplastic. Adenomatous polyp type is dysplastic, has abnormal intercellular organization.
Several layers of epithelial cells are observed; the nuclei of the epithelial cells are larger than
normal, and their position within the cells is often aberrant. Crypts crowd together in kaleidoscopic
pattern. As adenomas grow in size, they become more dysplastic. They are also more likely to
contain “villous” components, i.e., fingerlike projections of dysplastic crypts that can be
distinguished from the smooth contour of the less advanced “tubular” adenomas. As adenomas
progress, they are likely to become malignant, defined as the ability for invading surrounding tissue
and travel to distant organs through direct spread or transport by blood and lymphatic vessels.
Malignant tumors are not necessarily larger or more dysplastic than benign tumors; their sole
4
determining feature is invasiveness.
Polyps are the earliest clinical manifestation of colorectal neoplasia. However, the earliest
pre-carcinogenic changes in the colon can be observed under microscope after methylene blue
staining or examination of the colonic mucosa cross sections (Fig.1). These changes are termed
aberrant crypt foci (ACF). Like their larger counterparts, can be either dysplastic or non-dysplastic.
In mice or rats ACF are found as soon as 2-3 weeks following administration of colon carcinogens
[11], they grow with time giving rise to malignant tumors 6 month after animal intoxication with
carcinogen [12].
Consecutive stages of colon carcinogenesis are accompanied by acquiring mutations in
critical genes, which regulate cell proliferation, and/or apoptosis. A relatively limited number of
oncogenes and tumor suppressor genes, most prominently the APC, KRAS, and TP53 genes are
mutated in CRCs, and a larger collection of genes that are mutated in subsets of CRC have begun to
be defined. On the basis of the frequency of mutations occurring in different genes at particular
stages of adenoma to carcinoma progression, the sequence of genetic changes was proposed [13].
We will briefly summarize the genetic events in order to shed light how these changes may affect
the extent of oxidative DNA damage, and modulate its repair rate. The earliest genetic change in
colon carcinogenesis is mutation in the APC gene. About 70–80 % of sporadic adenomas and
carcinomas have somatic mutation that inactivate the APC gene, and its frequency is similar in the
earliest lesions, like microscopic adenomas composed of few dysplastic glands, and in advanced
adenomas and carcinomas, independently of the efficiency of the MMR system. APC protein
functions in the Wnt signalling pathway, in which stimulates proteasomal degradation of β-catenin,
an activator of TCF/LET (T cell factor/lymphoid enhancer family) transcription factors family. In
consequence APC inhibits transcription of several genes, such as proto-oncogenes, e.g. c-MYC or
cyclin D1, genes encoding membrane factors - matrix metalloproteinase 7 (MMP-7)/Matrilysin,
membrane type 1 MMP, laminin-5 γ2 chain, CD44, growth factors, e.g. FGF20, FGF9, and Wnt
pathway feedback inhibitors, such as AXIN2, Naked-1/-2, Dickkopf-1, WNT inhibitory factor 1.
5
Transcriptional program induced by β-catenin resembles the transcriptional program in stem cells at
the base of the colon crypts [14]. Thus, activation of β-catenin caused by APC mutation will
stimulate proliferation of epithelial cells in colonic crypts. Next, mutations in genes functioning in
EGFR signalling pathway, such as K-RAS, BREF or PTEN will further enhance cell proliferation
and survival, and drive progression of early adenomas to intermediate lesions. K-RAS is mutated in
almost 40% of intermediate and late adenomas and carcinomas. Inactivation of TGF-β response,
e.g. DCC (deleted in colorectal cancer) or TGF-β receptor mutations cause loss of growth inhibitory
effect of TGF-β and suppress apoptosis. These genetic changes are frequently observed in late
adenoma lesions. Loss of TP53 function is the late genetic change, which further stimulates
proliferation and survival promoting the shift of adenoma to carcinoma. TP53 mutations are
observed in about 50% of colorectal cancers, but much less frequently in adenomas. At least two of
these genetic changes, loss of APC and TP53 functions may affect DNA repair in colonic crypts,
and in consequence the extent of DNA damage, and potential of gaining new mutations (see chapter
5).
3. Oxidative stress in the development of colon cancer
The majority of pathological factors favouring development of colon cancer involve
induction of oxidative stress in the colon, which is defined as overproduction of oxygen species
combined with insufficiency in protective mechanisms of anti-oxidative defence [15,16]. Oxidative
stress is involved in the production of DNA damage and mutations associated with the initiation or
progression of human cancers [17,18]. The main pathological factors for the development of CRC
are inflammation, fat metabolism, consumption of meat and alcohol and tobacco smoking. All these
stimulate the release of ROS and RNS as well as lipid peroxidation (LPO) [1,19]. These compounds
play a dual role in cells. At low or moderate concentrations they are mediators of specific
physiological processes, whereas high concentrations can be detrimental to all components of the
cell.
6
3.1. Inflammation
Epidemiological studies suggest that inflammatory processes are linked with CRC. This is
true particularly in the association of inflammatory bowel disease and colorectal cancer [20].
During inflammation activated macrophages release large amounts of ROS and RNS [2], which
oxidise nucleic acids, proteins and lipids. Oxidation of polyunsaturated fatty acids generates
reactive aldehydes unsaturated in α, β-positions which, in contrast to ROS and RNS, are relatively
stable and can diffuse throughout the cell [19]. Short chain 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, glyoxal, 4-oxo-2-nonenal - ONE) [21]. Multiplicity of LPO products is
tremendously augmented by the existence of stereoisomers of several of these compounds.
Reactive aldehydes either react directly with DNA and proteins [22], or undergo further
oxidation to more reactive epoxy derivatives, such as 2,3-epoxy-4-hydroxynonanal [23]. The
affinity of epoxidized LPO products to nucleic acids is much higher than to proteins. Epoxidation of
enals may occur in the presence of hydrogen peroxide, but is also catalysed by enzymes producing
inflammatory mediators, prostaglandins and leukotrienes, such as cyclooxygenase-2 (COX-2) and
lipooxygenases (LOX) [24]. It was observed that COX-2 is overexpressed in 80% of colorectal
cancer tissues [25,26]. Increased levels of COX-2 were observed in epithelial cells, inflammatory
cells, as well as in stromal cells, but not in adjacent normal tissues [27]. COX-2 overexpression in
Min (multiple intestinal neoplasia) mice was sufficient to induce mammary tumors [28]. The
mechanism of tumor induction by COX-2 overexpression is complex, and involves the activity of
its enzymatic end-products, prostanoids. A number of theories have been proposed to explain the
role of tumor-derived prostanoids in promotion of angiogenesis, induction of tumor cell
proliferation, suppression of immune response, and protection against apoptosis [29-31].
Additionally, it was shown that arachidonic acid (AA) metabolism mediated by COX-2 and 5-LOX
results in the formation of heptanone-etheno (Hε)-DNA adducts. The Hε-DNA adducts are highly
7
mutagenic in mammalian cells suggesting that these pathways could be (at least in part) responsible
for the somatic mutations observed in tumorigenesis [32]. That is why inhibition of COX-2 by nonsteroid anti-inflammatory drugs or RNA interference 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 that high intake of heme, present
in red meat, is associated with an increased risk of colon cancer. Recently elucidated mechanisms
suggest that heme induces DNA damage and cell proliferation of colonic epithelial cells via
hydrogen peroxide produced by heme oxygenase. In Caco-2 cells the presence of heme oxygenase
inhibitor, zinc protoporphyrin, reduced DNA damage (measured by a comet assay) triggered by
hemin as well as cell hyperproliferation [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 acid hydroperoxides (LAOOH) led to an increase in cell death,
malondialdehyde formation, and DNA fragmentation and these effects were related to the peroxide
group and the heme present in Hb. SW480 cells treated with Hb and LAOOH presented a higher
level of the modified DNA bases 8-oxoGua and 1,N2-etheno-2'-deoxyguanosine (1,N2-Gua)
compared to the control. Incubations with Hb led to an increase in intracellular iron levels, which
correlated with DNA oxidation. Thus, Hb from either red meat or bowel bleeding caused by
inflammatory processes could act as an enhancer of fatty acid hydroperoxide genotoxicity, and in
consequence contribute to the accumulation of DNA lesions in colon cells [36].
Another frequently suggested risk factor for CRC development is chronic alcohol
consumption. One of the factors which may contribute to the development of alcohol-associated
cancer is the action of acetaldehyde, the first and most toxic metabolite of alcohol metabolism [37].
Acetaldehyde is produced by the oxidation of ethanol by hepatic NAD-dependent alcohol
dehydrogenase and 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
8
also from lipid peroxidation [39,40]. In hepatic cancer model, lean and obese Zucker rats higher
levels of etheno DNA adducts were observed in the liver DNA of alcohol-fed than in control
animals. This increase was correlated with the induction of cytochrome P-450 isoform 2E1 [41].
Thus, the formation of exocyclic DNA adducts, like propanodG, 1,N2-dG, dA and dC may be the
important mechanism associated with acetaldehyde related cancer risk.
The results of epidemiological studies also link tobacco smoking with the risk for CRC
development. However, the obtained data are controversial, and the molecular mechanism remains
unclear. Although in the leukocytes of lung cancer patients tobacco smoking increased the level of
one of the major oxidatively damaged DNA base, 8-oxoGua [42], in CRC patients no effect of
tobacco smoking on 8-oxoGua level in the leukocytes and 8-oxoGua urinary excretion was
observed [43].
3.3. DNA damage
Oxidatively damaged DNA has been blamed for the physiological changes associated with
degenerative diseases and cancer [44,45]. DNA damage caused by free radicals is the most
heterogeneous group of DNA lesions due to the wide variety of deleterious molecules produced in
cells in the conditions of oxidative stress. A plethora of damaged DNA bases are formed upon ROS
attack on genetic material, several of them reveal strong promutagenic properties [46]. One of the
major and best studied is 8-oxoGua, a typical biomarker of oxidative stress, which may play a role
in carcinogenesis [47]. The presence of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) residues
in DNA leads to GCTA transversions [48]. 8-OxodG is formed in DNA either via direct oxidation
of nucleic acids or can be incorporated from the nucleotide pool by DNA polymerases, the latter
process being an important source of DNA oxidation and genome instability [49,50].
LPO products may contribute to DNA damage to nearly the same extent as directly oxidized
bases. There are distinct groups of linear and cyclic LPO specific DNA lesions. Malondialdehyde
reacts in DNA with guanine, adenine and cytosine to form cyclic pyrimido-[1,2α]purine-10(3H)one-2'-deoxyribose (M1dG) adduct and linear N6-(3-oxopropenyl)-2'-deoxyadenosine (M1dA) and
9
N4-(3-oxopropenyl)-2'-deoxycytosine (M1dC) adducts, respectively [51]. M1dG is the major MDADNA adduct and was detected in human and rodent tissues. In E. coli cells, it induces transversions
to T and transitions to A with a frequency comparable with that of 8-oxoGua [15].
Exocyclic propano DNA adducts are formed by the attachment of unsaturated reactive
aldehyde to ring and extra-ring nitrogen atoms in DNA bases, which generates additional saturated,
six-membered ring in a DNA base. These adducts are derived from the reaction of DNA with, e.g.
acrolein, crotonaldehyde and HNE. Acr-dG, Cro-dG and HNE-dG were found in rodent and human
tissues [52,53]. Etheno DNA adducts posses five-membered exocyclic rings attached to DNA bases.
The exact pathway of their formation in vivo is not known, although it is clear that they are
generated by the exposition of DNA to lipid peroxides. Ethenoadducts are also introduced into
DNA by certain environmental carcinogens like vinyl chloride or its metabolite, chloroacetaldehyde
(CAA) [54]. Propano- and etheno-type adducts that are found in native mammalian DNA are
unsubstituted and substituted with side chains of different length, depending on the structure of
reacting LPO product. All exocyclic DNA adducts have strong miscoding potential. Unsubstituted
exocyclic DNA adducts are more mutagenic than toxic. With the increase of the side chain the toxic
properties increase and are related to the inhibition of replication and transcription [55,56].
Elevated levels of dA and dC were found in pro-carcinogenic colon diseases, like
inflammatory bowel disease, Crohn's disease and ulcerative colitis, and in addition in chronic
pancreatitis [20,57]. This suggests that LPO-derived DNA lesions contribute to CRC etiology.
4. Repair of oxidative DNA damage as a driving force to colon carcinogenesis
The major pathway for repair of oxidatively derived DNA lesions is base excision repair
system (BER). Although several oxidatively damaged bases are also repaired by mismatch repair
system and AlkB family dioxygenases, this review will focus on BER activity. In a classical view
BER consists of five steps: (i) lesion recognition, (ii) excision of the damaged base, (iii) AP site
incision and removal of the chemical residues that could block further steps of the pathway, (iv)
10
incorporation of the proper nucleotide(s) by DNA polymerases and (v) ligation of the DNA strand
to restore the original DNA molecule [58-61].
Damaged bases are recognized by DNA glycosylases, which excise from DNA a wide range
of oxidised and alkylated bases. Presently, 13 human DNA glycosylases with different, but partially
overlapping substrate specificity have been identified. Mechanistically two types of DNA
glycosylases can be distinguished, monofunctional enzymes, that excise damaged base leaving
behind an abasic (AP) site, which is subsequently cleaved by AP endonuclease (APE1) 5' to the AP
site, and bifunctional DNA glycosylases, which have endowed AP lyase activity that incises
phosphodiester bonds 3' to the AP site created by the glycosylase activity of the enzyme. This
review will focus on repair of three oxidative DNA lesions, 8-oxoGua, Ade and Cyt.
8-oxoGua is repaired by a three-step system, which (i) limits incorporation of 8-oxodGTP
into DNA during replication, (ii) excises 8-oxoGua from DNA, and (iii) corrects the base opposite
to 8-oxoGua in DNA. Different DNA polymerases can incorporate 8-oxodGTP to the DNA, and
replicative DNA polymerases incorporate this modified nucleotide usually opposite dA, generating
A:T → C:G mutations. Approximately a half of 8-oxodG in DNA arises from the direct DNA
oxidation, and the other half is a result of incorporation of 8-oxodGTP during DNA synthesis.
Incorporation of 8-oxodGTP into DNA by DNA polymerases is limited by the activity of MTH1
phosphohydrolase, which hydrolyzes 8-oxodGTP to 8-oxodGMP [62]. 8-OxodGMP is subsequently
dephoshorylated by nucleotidase and removed from the cell [63]. Many observations indicate a
direct correlation between 8-oxodG formation and carcinogenesis in vivo [47,64-67]. The second
line of defence is glycosylase MutY (MYH) that removes adenine paired with 8-oxoGua. Activity
of this enzyme prevents mutations G:C→T:A, when dATP is incorporated opposite 8-oxoGua
during replication [68]. After removal of Ade by MYH glycosylase, repair DNA polymerase
incorporates dCMP opposite 8-oxodG. Then 8-oxoGua can be excised by the third component of the
system, OGG1 glycosylase [69]. OGG1 glycosylase/AP lyase is the major enzyme catalyzing
excision of 8-oxoGua from DNA, but only when paired with Cyt or Thy [70,71]. The AP lyase
11
activity of the OGG1 protein is about 10-fold lower than that of the glycosylase [72]. When 8oxodGTP is included into DNA during replication it is probably removed by NEIL1 or NEIL2
glycosylases, which excises 8-oxoGua paired with dC/G/T/A or dC/A, respectively [73]. This
complex three-tier system prevents mutations induced by the presence of 8-oxodG in DNA, both
when this lesion is induced directly in the DNA, and when it is incorporated from the pool of
nucleotides during replication. Polymorphisms of MYH and OGG1 glycosylases are related to
modulation of susceptibility to colon cancer. MYH variations significantly increase CRC risk, and
of several mutations described, two are the most prevalent: Y165C (exon 7) and G382D (exon 13).
Mutated enzyme excises Ade from dA:8-oxodG pair with a decreased rate, increasing mutation
frequency. The association of MYH with CRC was first reported in 2002 [74].
Several polymorphic changes in the OGG1 gene have been described, with the most common
being Ser326Cys [75]. Polymorphic Cys326 OGG1 protein was found to have a lower enzymatic
activity, both when 8-oxoGua was excised from oligodeoxynucleotides [76], and when 8-oxoGua
and FapyGua were liberated from γ-irradiated DNA by purified Cys326 or Ser326 OGG1 enzyme
[75,77]. It was suggested that the presence of two OGG1 326Cys alleles may confer an increased
risk of lung, prostate and nasopharyngeal cancer [78-80], but no association with the risk for colon
cancer was found [81,82]. Our and other functional studies report a decrease in 8-oxoGua excision
rate in lung [42,83,84] and head and neck cancer patients [85]. Such a decrease in excision rate and
simultaneous increase in 8-oxodG and FapydG levels in cellular DNA favors a pro-oxidant state
and may accelerate the acquisition of mutations in critical genes leading to cancer. Such an idea is
basically derived from the observation that a pro-oxidant environment is characteristic for advanced
stages of cancer.
Ade is excised by alkylpurine-DNA-N-glycosylase (ANPG) [86], whereas Cyt by
thymine-DNA glycosylase (TDG); the latter also excises thymine from G:T pairs, resulting from
deamination of 5-methylcytosine [87]. Ethenocytosine is also excised by single-stranded uracil
DNA glycosylase, SMUG1 [88] and by MBD4 glycosylase, although activity of the latter is
12
strongly limited to CpG islands [89,90].
ANPG glycosylase has a wide substrate specificity, and besides Ade excises from DNA
also 1,N2-ethenoguanine [91], hypoxanthine and the alkylated bases, 3-methyladenine and 7methylguanine [86]. TDG excises Cyt, Thy and Ura from pairs with guanine, and its activity is
strongly stimulated by the next enzyme in the BER pathway, AP endonuclease, APE1 [92]. TDG
and APE1 also play the role of transcription regulators [93-96].
Ethenocytosine repair by the BER system is dependent not only on the level and activity of
TDG, MBD4 or SMUG1 DNA glycosylases, but also on the presence and/or activity of ANPG
glycosylase, which primary substrate is εAde. ANPG binds strongly to DNA containing εCyt, and
although does not excise this modified base, does not allow its excision by the proper εCyt
glycosylase, like TDG [97]. This may explain observation that the level of εCyt measured in
different human tissues was constantly higher than that of εAde [19].
4.1. Repair of 8-oxo-7,8-dihydro-2’-deoxyguanosine in colon adenoma and carcinoma patients
In order to get an insight into the importance of oxidative stress and repair of oxidatively
damaged DNA bases in the pathology of colon cancer, we examined groups of individuals at
different stages of disease development, namely CRC patients, patients who developed benign
adenomas (AD) and healthy individuals for the markers of oxidative stress, repair capacity of
oxidatively damaged DNA, and polymorphism of DNA repair genes.
Increased oxidative stress was observed both in CRC patients and in AD individuals. This
manifested in an elevation of the 8-oxodG level in leukocyte DNA and in urine as well as depletion
of antioxidant vitamins (ascorbic acid, α-tocopherol, retinol) and uric acid in blood plasma of CRC
patients and AD individuals in comparison to healthy controls [43]. Depletion of antioxidant
vitamins in blood plasma was gradual, which suggests that oxidative stress is increasing gradually
with the disease progression. This is supported by the fact that urinary level of 8-oxodG was
augmented already in AD patients, but 8-oxoGua level was enhanced only in CRC, but not in AD
individuals [43]. Urinary 8-oxodG may reflect oxidation of nucleotide pool, while 8-oxoGua
13
reflects rather oxidation of DNA. Since nucleotide pool is much more susceptible to oxidation than
dsDNA, 8-oxodG in urine may be a more sensitive indicator of oxidative processes in the body, and
shows that even in AD individuals the oxidative processes are enhanced. The suggested
mechanisms responsible for the oxidative stress in cancer patients include [44]: i/ granulocyte
activation with release of ROS; ii/ stimulation of cytokines, of which some, e.g. tumor necrosis
factor, produce large amounts of ROS; iii/ production of hydrogen peroxide by malignant cells,
which in advanced stages of cancer may be released into the blood stream and penetrate into other
tissues [98].
Interestingly, we also observed an increase of mRNA levels of repair enzymes, OGG1,
APE1 and MTH1 in leukocytes of CRC and AD individuals (Table I) [43]. Expression of APE1 and
OGG1 genes was shown to be induced by ROS [99], and this could explain induction of repair
enzymes transcription in leukocytes of CRC and AD individuals. On the other hand induction of
repair enzymes may be a part of a carcinogenic program, which might decrease apoptosis in
aberrant cells and/or favor genomic instability. Our results corroborate the findings of
Winnepenninckx and coworkers [100] that induction of most of the DNA repair genes occurs very
early in the carcinogenic process, e.g., four years before clinical manifestation of malignant skin
melanoma.
Increase of mRNA level of OGG1 and APE1 repair enzymes was accompanied by the
augmentation of 8-oxoGua excision rate in leukocytes of CRC patients (Fig. 2A) [43]. The higher
activity observed in the cell extracts from the cancer patients is even more meaningful in view of
the finding that homozygous OGG1 326Cys variants, which are generally characterized by lower
OGG1 activity, were much more frequent among the cancer patients than in the control and
adenoma groups (Table II) [43]. In our study the effect of the OGG1 Ser326Cys polymorphism on
8-oxoGua excision rate was clearly seen in the CRC patient group, in which Cys homozygotes were
found to have a decreased 8-oxoGua excision rate in comparison with Ser homozygotes (Fig. 2B)
[43]. Thus, the observed increase in 8-oxoGua repair capacity in CRC patients in comparison to
14
healthy individuals might be due to increased transcription from OGG1 and APE1 genes, which was
observed in AD and CRC individuals (Table I) [43]. Interestingly, the OGG1 genotype exerted a
limited effect on 8-oxodG level in leukocytes and urinary excretion of 8-oxoGua [43]. In contrast to
our studies, Janssen et al [101] and Jensen et al [102] reported that 8-oxoGua repair incision in
human lymphocytes was unaffected by the polymorphic status at codon 326 of the OGG1 gene.
These studies were performed on the population of healthy subjects only. It was shown that OGG1
Cys326 variant forms dimers with lower enzymatic activity only under conditions of oxidative
stress probably due to OGG1 cysteine 326 oxidation. [103]. In parallel, Bravard and coworkers
[104] reported recently that extracts from lymphoblastoid cell lines established from individuals
carrying the Cys326Cys genotype have almost 2-fold lower basal 8-oxoGua DNA glycosylase
activity than the Ser326Ser variant and were more sensitive to inactivation by oxidizing agents than
that of the Ser/Ser cells. Analysis of redox status of the OGG1 protein in cells confirmed that the
lower activity of OGG1-Cys326 was associated with the oxidation of Cys326 to form a disulfide
bond. Thus different degrees of oxidative stress in different studies might partially explain the
controversies in the literature.
In the group of healthy subjects there was significant positive correlation between mRNA
levels of the main enzymes involved in 8-oxoGua removal (OGG1 and APE1), and the amount of 8oxoGua in urine [43]. These findings suggested that the amount of 8-oxoGua in urine might be
attributed to DNA repair. It is in parallel with previous results, which also showed that DNA repair
was responsible for the presence of oxidatively damaged DNA lesions in urine [105]. However, we
did not find such correlation in the groups of adenoma individuals and carcinoma patients. The
main reason for this inconsistency may be aberrant DNA oxidation in cancerous and precancerous
conditions which may overshadow the subtle relationship observed in healthy subjects.
Alternatively, the excretion of 8-oxoGua might reflect the level of oxidative stress, independent of
the activities of the repair enzymes [106].
Also, only a weak positive correlation was found between 8-oxoGua level in urine and the
15
stage of disease [43].
4.2. Repair of etheno DNA adducts in colon carcinogenesis
In contrast to cleavage of 8-oxoGua, leukocytes excision activity of Ade and Cyt was
significantly lower in CRC patients when compared to controls matched for age, sex and a smoking
habit (Fig. 3A) [107]. However, in colon tissues, excision activities for Ade and Cyt were
significantly higher in tumor than in adjacent, asymptomatic colon tissue (Fig. 3B) [107]. In
leukocytes (PBL) of CRC patients dA and 1,N2-dG level was unexpectedly lower than in controls,
and the level of dA and dC also showed a tendency to be lower in tumor than in normal colon
tissue [107]. This suggests that the regulation of etheno adduct level and repair may be
multifactorial and may differ in target (colon) and surrogate (PBL) tissues.
The lower excision rate for Ade and Cyt in leukocytes of CRC patients was not linked to
mutations in exons of ANPG and TDG genes nor to down regulation of mRNA expression of ANPG
and TDG glycosylases or its BER activator, APE1 endonuclease; in the contrary, mRNA levels of
these repair enzymes were significantly higher in PBL of CRC than in healthy controls (Table I)
[43,107]. Thus direct inactivation/inhibition of repair enzymes activities by inflammatory mediators
released in the blood stream is a more likely explanation. Indeed, several DNA repair enzymes were
shown to be directly inhibited by nitric oxide [108-110], hydrogen peroxide [111] and LPO product,
4-hydroxynonenal [56,112]. As shown for several human cancer sites and animal tumors, persistent
oxidative stress and LPO-induced DNA damage increased already during premalignant stages in a
time-dependent manner [113]. A number of studies demonstrated increase of mRNA level of several
repair enzymes upon oxidative stress [99,114,115]. The observed lower Ade and Cyt excision
(Fig. 3A) and simultaneous lower level of etheno adducts in PBL of cancer patients [107] may thus
suggest that other mechanisms are up-regulated in CRC patients and determine the level of etheno
adducts in DNA. Other repair systems engaged in etheno adducts elimination from DNA include
e.g. oxidative demethylation by AlkB family proteins or nucleotide excision repair [116,117].
Of interest are the findings in tumorous colon tissue. The Ade and Cyt excising activities
16
were much higher in tumor than in the surrounding normal colon (Fig. 3B) and was consistent with
a 2-3 times lower dA and dC adduct levels [107]. Also in several other studies lower level of dA
and dC was observed in colon tumors than in colon tissues of cancer prone patients suffering from
ulcerative colitis, Crohn's disease or familial adenomatous polyposis [57,118].In our study in
normal colon a negative correlation was found between dC level in DNA and its excision rate (ρ =
-0.64, p = 0.013) [107]. This supports a role of BER in Cyt-elimination; however, does not exclude
other etheno adduct control systems. In several types of non tumorous human tissues previously
analyzed, the dA level in DNA was found to be lower than that of dC [113], consistent with our
observation that excision activity was found to be higher for Ade than for Cyt (Fig. 3) [107].
In order to understand a huge increase of excision activity of εAde and εCyt in colon tumor
tissue in comparison to asymptomatic colon margin, we measured mRNA level of ANPG and TDG
glycosylases, as well as APE1 endonuclease in tumor and normal colon tissues. Higher mRNA level
in tumor tissue was observed only for APE1 [107]. APE1 stimulates Cyt excision [92], and most
probably also εAde. Another possibility are frequent mutations in the APC gene in colon tumors
[119]. APC tumor suppressor protein may inhibit BER engaged in repair of Ade and Cyt since it
was shown that it can directly interact with DNA polymerase β and FEN1 endonuclease [120]. BER
activity was inversely associated with APC expression in several breast cancer cell lines [121].
In conclusion, we have shown, for the first time, that excision activity of Ade and Cyt is
lower in PBL of CRC patients when compared to healthy individuals. However, our unexpected
findings, that the level of dA and 1,N2-dG was lower in DNA of PBL from CRC patients than in
healthy controls currently remains mechanistically difficult to explain. If such etheno adducts,
particularly Cyt excision deficiency also occurs during premalignant stages in the target organ, still
may favor acquisition of mutations in critical genes leading to neoplastic transformation. TDG
glycosylase removes Thy and Ura paired with guanine, and may prevent induction of mutations
caused by cytosine and 5-methylcytosine deamination [122].
17
5. Aberrant repair of oxidatively damaged DNA bases and mutations in critical genes
Activity and expression of BER proteins is modulated by two signaling molecules, which
are frequently mutated in colon cancer, the APC and TP53 gene products. Thus, mutations in these
genes affect repair rate of oxidatively damaged DNA, and favor pro-mutagenic events within colon
epithelial cells. On the other hand at least three BER proteins, namely, APE1, TDG and OGG1 take
part in the regulation of gene expression.
Mutations in the APC gene are early events in colon carcinogenesis, and are found in about
80% of colon adenomas and carcinomas. The most frequent mutation results in truncation of the Cterminal part of the APC protein. Wild type APC protein directly interacts with DNA polymerase β,
FEN1 endonuclease [120], and as recently suggested, APE1 endonuclease [123] inhibiting the
assembly of BER proteins on damaged DNA and the activity of short and long patch BER (Fig. 4).
In LoVo, colon cancer cells expressing truncated APC protein an accelerated assembly of BER
proteins, as well as higher activity of APE1, FEN1 and Pol β were observed [123]. Activation of
APE1 may also occur as a consequence of TP53 gene mutations, which are late, but frequent events
in colon carcinogenesis (in about 50% of cancers). TP53 protein participates in downregulation of
APE1 transcription in cells treated with DNA damaging agents [124]. On the other hand TP53
mediates ubiquitination of APE1 by TP53 inhibitor, MDM2, and in consequence APE1 degradation
[125]. Thus, mutations in TP53 gene should additionally increase APE1 amount in cancer cells.
Increased APE1 activity may result in unbalancing of BER pathway, and may favor genomic
instability and cancer progression: in inflamed non-cancerous colon tissues of ulcerative colitis
patients (at high risk for colon cancer, that accumulate high etheno DNA adducts [126]), ANPG and
APE1 activities were significantly increased, and microsatellite instability (MSI) was positively
correlated with their imbalanced activities of repair enzymes [127]. Similarly, in yeast and human
cells over-expression of ANPG and/or APE1 was associated with frameshift mutations and MSI
[127].
APE1, besides DNA repair function, also plays a role of redox regulatory protein for several
18
transcription factors. For example, participates in HIF-1α mediated angiogenesis [128]. Thus at late
stages of CRC development, mutations in TP53 gene will lead to accumulation of APE1 protein,
and in addition to increasing genome instability will also favor angiogenesis and in consequence
tumor growth (Fig. 4).
Increased APE1 activity may also accelerate the excision rate of DNA glycosylases. TDG
and OGG1 were shown to be particularly susceptible to induction by APE1, since they bind
strongly to AP sites, and APE1 increases turnover of these enzymes on damaged DNA [92,129].
Increased activity of TDG may favour demethylation of promoters of several genes and in
consequence contribute to hypomethylation of DNA observed in colon cancer [13] and induction of
transcription of several genes. TDG interacts with the deaminase AID and the damage response
protein GADD45a. 5-Methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to
thymine and 5-hydroxymethyluracil, respectively, and subsequently TDG removes thymine and 5hydroxymethyluracil from dT:dG or 5OHMedU:dG pairs, which initiates BER and enables
reconstitution of G:C base pairs [130]. TDG is also necessary for recruiting p300 to retinoic acid
(RA)-regulated promoters, and is engaged in transcriptional control of genes during embryonic
development. Its deficiency is embrionaly lethal [122]. TDG status may also determine sensitivity
of CRC patients to chemotherapy with 5-fluorouracil (5-FU), a chemotherapeutic routinely used in
CRC treatment. Inactivation of TDG significantly increases resistance of mouse and human cancer
cells towards 5-FU. Excision of 5-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 [131].
OGG1 and APE1 stimulate transcription of genes regulated by c-Myc transcription factor
[132], and thus may contribute to acceleration of cell proliferation (Fig. 4). The mechanism
involves demethylation of histone H3 by specific FAD-containing enzyme, LSD1 demethylase
which generates hydrogen peroxide during demethylation [133]. In consequence a local oxidation
of DNA around the E-boxes and TSS region of Myc target genes is triggered. 8-OxodG, OGG1 and
19
APE1 were found to be engaged on the same RNA polymerase II and Myc occupied chromatin
regions of Myc targets and stimulate transcription of these genes. Silencing of either OGG1 or
APE1 inhibits Myc mediated transcription. It is possible that regulatory regions in a gene interact
with each other forming loops that often bridge considerable distances on the genomic loci [134].
Following Myc binding local demethylation of lysine 4 in histone H3 (H3K4me2) triggers
recruitment of the OGG1 and the downstream BER enzymes thus producing nicks on Myc target
chromatin. This may relax the DNA strands and could favor chromatin bending necessary for the
formation of chromatin loops that bring together RNAPII and Myc. OGG1 is also engaged in the
regulation of transcription from estrogen promoters [135].
6. Conclusions
Oxidative stress is induced at the early, adenoma, stage of colon carcinogenesis, and is
increasing during disease progression. CRC and AD patients reveal a huge increase of mRNA level
of base excision repair enzymes in blood leukocytes. However, increased transcription is not always
correlated with repair capacity of tissues. Activities of BER enzymes remain under a complex
regulative network, which besides stimulation of transcription includes direct inactivation by
oxidation stress products, interaction with other BER proteins, and with signalling molecules, which
are frequently mutated in colon cancer, that is APC and TP53 proteins. Mutated proteins do not
inhibit transcription and/or activity of base excision repair enzymes. This increases the amount of
repair enzymes within cells and introduces imbalance in the repair of oxidative DNA damage. Such
imbalance causes increased genome instability and may be a driving force in the disease
progression.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
20
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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Figure legends
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 with methylene blue. The
mucosal side was observed under the light microscope (magnification 100x); B, C. Cross sections
of mouse epithelium stained with hematoxylin and eosin (magnification 1000x).
28
Fig. 2. The mean and standard deviation of 8-oxoGua excision activity in leukocytes of CRC
patients and control individuals, in the entire groups (A) and in the groups distinguished on the
basis of OGG1 Ser326Cys polymorphism (B). ***, p<0.001; **, p<0.005; *, p<0.05. (MannWhitney U test). Adapted from [43] with permission from Oxford University Press.
Fig. 3. A) The mean and standard deviation of Ade and Cyt excision rate in leukocyte 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 Cyt excision rate in colon tumor and normal surrounding
tissues of CRC patients. ***, p<0.001 (Wilcoxon rank sum test). Derived from [107] with
permission from Elsevier.
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 APE1 activity 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. TDG interacts with the deaminase AID and may favor
demethylation of promoters and induction of transcription of several genes. OGG1 and APE1
stimulate transcription of genes regulated 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 HIF1α mediated angiogenesis and erythropoesis and may favor tumor growth. + symbol in circle
denotes stimulation or increase; APC–adenomatus polyposis coli; Pol β–DNA polymerase β; FEN
1–flap endonuclease 1; APE1–apurinic site endonuclease 1; ANPG–alkyladenine DNA Nglycosylase; OGG1–8-oxoGua DNA N-glycosylase; TDG–thymine DNA N-glycosylase; MBD4–
29
methyl-CpG binding domain protein; polyposis coli; AID–activation-induced deaminase, HIF-1α–
hypoxia-inducible factor 1α subunit.
30