Mutation Research 711 (2011) 167–173

Contents lists available at ScienceDirect

Mutation Research/Fundamental and Molecular
Mechanisms of Mutagenesis
journal homepage: www.elsevier.com/locate/molmut
Community address: www.elsevier.com/locate/mutres

Review

Reactive Oxygen Species (ROS)––Induced genetic and epigenetic alterations in

human carcinogenesis
Dominique Ziech a , Rodrigo Franco b , Aglaia Pappa c,∗ , Mihalis I. Panayiotidis d,e,∗∗
a
Department of Student Success Services, University of Nevada, Reno, NV 89557, USA
b
Redox Biology Center & School of Veterinary Medicine & Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA
c
Department of Molecular Biology & Genetics, Democritus University of Thrace, Dragana Campus, Alexandroupolis 68100, Greece
d
School of Community Health Sciences, University of Nevada, Reno, NV 89557, USA
e
Department of Pathology, Medical School, University of Ioannina, University Campus, Ioannina 45110, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Cancer is a multistage and complex process characterized by molecular alterations that underlie all three
Received 19 November 2010 phases of its development: (i) initiation, (ii) promotion and (iii) progression. Some of these molecular
Received in revised form 26 February 2011 events include alterations in gene expression that are regulated by both genetic and epigenetic mecha-
Accepted 28 February 2011
nisms. On the other hand, “oxidative stress” implies a cellular state where ROS production exceeds the
Available online 16 March 2011
cell’s ability to metabolize them resulting in excessive accumulation of ROS that overwhelms cellular
defenses. Such state has been shown to regulate both genetic and epigenetic cascades underlying altered
Keywords:
gene expression in human disease including cancer. Throughout this manuscript, we review the current
Reactive Oxygen Species (ROS)
Oxidative stress
state of knowledge on the role of ROS-induced oxidative stress in altering the genetic and epigenetic
Epigenetics involvement during human carcinogenesis.
DNA methylation © 2011 Elsevier B.V. All rights reserved.
Histone modifications
DNA damage
DNA repair
DNA methyltransferases
HATs
HDACs
Tumor suppressor genes
Oncogenes
Human carcinogenesis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2. Oxidative stress-induced genetic alterations in human carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3. ROS-induced oxidative stress based epigenetic alterations in human carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

∗ Corresponding author at: Department of Molecular Biology & Genetics, Democritus University of Thrace, Dragana Campus, Alexandroupolis 68100, Greece.
Tel.: +30 25510 30625.
∗∗ Corresponding author at: Department of Pathology, Medical School, University of Ioannina, University Campus, Ioannina 45110 Greece. Tel.: +30 25510 35689.
E-mail addresses: apappa@mbg.duth.gr (A. Pappa), mpanagiotidis@unr.edu (M.I. Panayiotidis).

0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2011.02.015
168 D. Ziech et al. / Mutation Research 711 (2011) 167–173
1. Introduction
The term “free radicals” refer to any molecular species with one
or more unpaired electron(s), a characteristic that provides them
with an “unstable” chemical nature and thus confers them with the
property of being very reactive. It is indeed this property that when
they react with DNA, RNA, proteins, and lipids, leads to cellular
toxicity. Free radicals include both reactive oxygen (ROS; such as,
superoxide (O2 − ), hydroxyl (OH− ), hydroperoxyl (HOO− ), peroxyl
(ROO− ) and alkoxyl (RO− ) radicals [4,7]) and nitrogen (RNS) species.
In particular, ROS production can be of endogenous (mitochondrial
and P450 metabolism, inflammatory cell activation, etc.) as well
as of exogenous (radiation, ozone, etc.) origin. In fact, the major-
ity of environmental, occupational and industrial pollutants are
chemicals able to generate free radical species (primarily through
their metabolic activation) that contribute to the pathophysiology
of human disease including cancer. Finally, when ROS generation
exceeds the cell’s ability to metabolize and detoxify them, a state
of “oxidative stress” emerges [45–47].
In general, cancer development is a multi-stage process char-
acterized by the cumulative action of a number of altered cellular
processes including those of replication, angiogenesis, apoptosis,
metastasis etc. However, although some of these altered cellu-
lar processes have been extensively studied, there is still much
to learn in regards to the molecular mechanism(s) involved. This
is of great importance in enhancing our understanding about the
three main stages of human carcinogenesis, namely initiation,
promotion and progression, and how they contribute to the com-
plexity of human cancer biology [45]. To this end, for example,
ROS-mediated DNA damage plays a role in the initiation of car-
cinogenesis as well as in malignant transformation [8,42] and
thus, it may represent a major contributor in the pathogenesis of
human carcinogenesis [7]. Finally, because human carcinogene-
sis is rather complex and multi-stage in origin, there are several
different molecular mechanisms that contribute to its pathogen-
esis that include, but are not limited to, those that regulate
gene expression and involve both genetic and epigenetic alter-
ations. It would certainly be of major interest to identify such
mechanism(s) in an attempt to elucidate their potential role as
biomarkers for better cancer diagnostics and therapeutic strategies
[30,48].
2. Oxidative stress-induced genetic alterations in human
carcinogenesis
The carcinogenicity of oxidative stress is primarily attributed to
the genotoxicity of ROS in diverse cellular processes [7]. For exam-
ple, hydroxyl radicals can react with pyrimidines and/or purines
as well as chromatin proteins, resulting in base modifications and
genomic instability respectively all of which can cause alterations in
gene expression [42]. In addition, data have implicated ROS accu-
mulation as being a common phenomenon in many cancer cells.
Such accumulation can cause direct DNA damage by increasing a
cell’s mutation rate and/or promoting and maintaining the onco-
genic phenotype by acting as a secondary messenger in intracellular
signaling cascades [2,3].
ROS-mediated DNA damage in addition to ineffective DNA
repair mechanisms are well established lesions common to human
cancers such as in the case of breast cancer cell lines and human
breast tumor tissue samples [37]. DNA alterations like strand
breaks, base modifications and DNA–protein cross linkages, are all
strongly implicated in the initiation stage of carcinogenesis [1].
For example, hydroxyl radicals are known to attack DNA caus-
ing single and/or double strand breaks as well as pyrimidine
and purine lesions both of which can affect the integrity of the
genome. Major oxidative DNA damage products include those of
8-oxo-7,8-dihydroadenine (8-oxoAde), 8-oxo-7,8-dihydroguanine
(8-oxoGua), 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG), and
5,6-dihydroxy-5,6-dihydrothymine as well as the ring-open
lesions of 4,6-diamino-5-formamido-pyrimidine and 2,6-diamino-
4-hydroxy-5-formamido-pyrimidine [4]. Elevated levels of such
DNA lesions have been recorded in many tumor types and are
strongly implicated in all stages of carcinogenesis [5]. Of these
oxidative lesions, 8-oxoGua has received most attention due to
its potential to serve as a reliable biomarker of oxidative stress
and thus as a promising biomarker of ROS-induced carcinogenesis
[2,4]. It is hypothesized to have strong mutagenic and carcinogenic
potential, and has been steadily observed in tissues containing low
concentrations of antioxidant enzymes and high concentrations of
ROS [4]. While 8-oxoGua adducts occur at approximately one in
105 guanidine residues, in normal human cells, its rate of forma-
tion increases by 35–50% in individuals using tobacco smoke – a
well known carcinogenic source of ROS [2]. In addition, almost 50%
higher rates of 8-oxoGua levels have been observed in lung, breast
or prostate cancer patients when compared to otherwise healthy
individuals [4]. Furthermore, recent investigations have showed
higher endogenous levels of 8-oxoGua in tumor tissues when com-
pared to controls, thus suggesting oxidative DNA damage as a
contributing factor in cancer development [12]. In addition, high
levels of 8-oxoGua and possibly other DNA lesions are suggested as
reliable risk factors associated with the transformation of benign to
malignant tumors [2]. Furthermore, 8-oxoGua lesions are known
to induce aberrant modifications in adjacent DNA a hypothesized
mechanism that significantly contributes to the genetic instability
and metastatic potential of tumor cells [4]. For example, forma-
tion of 8-oxoGua lesions have been shown to induce a cascade
of adjacent DNA base mutations, such as GC → TA transversions
in the RAS and the TP53 oncogene and tumor suppressor genes
respectively in both lung and liver cancer cells [4]. Under normal
conditions, DNA repair mechanisms including the DNA glycosy-
lases of 8-oxoguanine glycosylase (OGG1) and MutY glycosylase
homologue (MUTYH) (in the base excision repair pathway) excise
8-oxodG lesions when paired with cytosine and adenine respec-
tively [39]. However, in the presence of cumulative oxidative stress,
repair mechanisms often are ineffective. Mutations known to accu-
mulate in response to failed repair mechanisms are those found in
the Ras oncogene and the p53 tumor suppressor gene. Mutations
in the Ras oncogene (a membrane-bound G protein whose main
function is to regulate cell growth and oppose apoptotic effects)
have been well documented in about 30% of lung, skin, liver, blad-
der and colon cancers [2]. On the other hand, mutations in the p53
tumor suppressor gene have been established as common genetic
alterations whereby they lead to gene’s inactivation in more than
half of all human cancers. For instance, a recent study has observed
a direct relationship between oxidative stress, DNA damage and
elevated frequency of p53 mutations in human dysplastic colon
and in colorectal carcinoma [34]. In general, p53 is known to reg-
ulate cell cycle entry and also act as a redox-active transcription
factor whose function is linked in regulating ROS generation as
well as mediating ROS-induced apoptotic cell death. Several cyto-
sine residues in the central domain of the protein are critical for
p53 binding and it is in these regions where mutations most com-
monly occur [41]. ROS-mediated posttranslational modifications
have been shown to inhibit p53, pro-apoptotic enzymes’ (i.e. cas-
pases) function and also activate telomerases, metalloproteinases,
and DNA methyltransferases [34]. During normal cell proliferation,
p53 triggers mechanisms that eliminate oxidative DNA damage
in comparison to the presence of oxidative damage where p53
triggers apoptotic cell death. However, during the carcinogenic
process an imbalance between cell proliferation and cell death
exists that shifts towards uncontrolled cell proliferation [2]. Finally,
 D. Ziech et al. / Mutation Research 711 (2011) 167–173
p53-depleted cells exhibit significant disruption of cellular ROS
homeostasis characterized by reduced mitochondrial and cellu-
lar superoxide and increased cellular hydrogen peroxide levels
[40].
Exogenous sources of oxidative stress like tobacco smoking,
alcohol consumption, and ionizing radiation are all involved in cel-
lular toxicity induced by oxidative stress [49,50]. Ionizing radiation,
for example, alone, can induce genomic instability in cells, which is
hypothesized to enhance the rate by which mutations and other
genetic alterations occur. In fact, recent work has revealed that
after irradiation of prostate and cervical cancers, the risk for both
rectal and bladder tumors increase respectively [35]. Exogenous
exposure to ionizing radiation is also known to induce cumulative
oxidative stress, over time, and result in DNA damage, cell death,
altered proliferation states and ultimately carcinogenesis. More
specifically, ionizing radiation-induced oxidative stress has been
shown to induce PI3K, MAPK, JNK and p38 signaling pathways as
well as inhibit protein tyrosine phosphatase activity, both of which
play an important role in controlling cell proliferation and induc-
ing metastasis [35]. In addition, a recent study has suggested that
oxidative stress-induced cellular damage (via occupational expo-
sure to low doses of ionizing radiation) results in the generation
of a variant of the PTSG2 gene that may serve as a strong indi-
cator of increased breast cancer risk [36]. Furthermore, it is well
established that ionizing radiation induces a wide spectrum of
DNA damage lesions, including single strand breaks (SSBs), dam-
aged bases, abasic sites and high levels of complex DNA damage
such as double strand breaks (DSBs) and oxidatively induced non-
DSB clustered DNA lesions (OCDLs) lesions. DSBs are known to be
highly genotoxic DNA lesions because potentially they can lead to
chromosomal breakage and can arise as the result of exposure to
exogenous and endogenous sources of free radical production [43].
These lesions are generally repaired by the base excision repair
pathway however, data have implicated that unsuccessful lesions’
repair contributes to significantly increased rates of mutations
and genomic instability [44]. In a separate study, alcohol-induced
oxidative stress has been shown to generate excess hepatic DNA
damage leading to hepatocellular carcinoma [34]. Alcohol is known
to facilitate ROS production and the formation of an in vivo oxida-
tive microenvironment and thus enabling suitable conditions for
the development of cancer. While the underlying mechanism of the
relationship between oxidative stress, alcohol and cancer has yet to
be elucidated, it is hypothesized that excess ROS production results
from the oxidation of ethanol whereby a variety of by-products
cause antioxidant depletion and oxidative macromolecular dam-
age [35]. In addition, exposure to tobacco smoke has also been
strongly linked to oxidative stress-induced DNA damage and car-
cinogenesis [6]. Cigarette smoking is known to contain increased
levels of free radicals leading to the induction of DNA lesions and
various oxidized bases (i.e. 8-oxoGua). A vast number of data have
indicated the central role of tobacco smoke in the development
of a number of different human cancers like lung, oral, pharyn-
geal, laryngeal, esophageal, bladder, stomach, pancreatic, kidney,
uterine, cervical and myeloid leukemic ones [38]. Additionally, it
has been known that free radicals produced during auto-oxidation
of polyphenols in saliva of tobacco users are crucial to the ini-
tiation and promotion of oral, pharynx, larynx and esophageal
cancers. More specifically, cigarette smoke has been observed to:
(i) cause the oxidation of glutathione (GSH; a common antioxidant
known to protect against ROS damage), (ii) decrease blood antiox-
idant levels and (iii) increase superoxide radical release. Taken
together, the ability of genotoxic carcinogens to induce muta-
tions coupled with the failure of repairing this damage results in
“gain-of-function” activity of oncogenes (i.e. K-ras) and/or “loss-
of-function” activity of tumor suppressor genes (i.e. p53 and p16)
[35].
169
As discussed earlier, a significant effect of cumulative oxida-
tive DNA damage is the impairment of normal cellular repair
mechanisms. In fact, one of the main etiological hypotheses link-
ing genomic instability, mutagenesis and tumorigenesis is that
of deficient cellular repair mechanisms due to extensive oxida-
tive DNA damage and cellular injury. Complex DNA damage, such
as DSBs and OCDLs are considered the hallmarks of irradiation-
induced oxidative damage [51,52]. OCDLs are hypothesized to
challenge the normal cellular repair machinery via cumulative
cytotoxic and mutagenic effects. Additionally, endogenous forma-
tion of OCDLs are known to accumulate in X-irradiated mouse
skin tissues and human breast cancer cells, leading to induction
of genetic instability and mutagenesis [9]. In general, the base
excision pathway repairs DSBs and OCDLs efficiently. In addition,
the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)
is known to play an important role during the processing of DSBs.
Only recently DNA-PKcs interactions with traditional BER proteins,
like XRCC1, APE1 or Pol␤, have generated much interest in the
ability and involvement of DNK-PKcs to process oxidative DNA
lesions [43]. Nevertheless, it is well established that when left
unrepaired, these complex DNA lesions play critical roles in the
initiation, promotion and progression of carcinogenesis. In fact, to
date, such complex DNA lesions are thought to be able to serve as:
(i) biomarkers in human acute lymphoblastic leukemia cells [44],
(ii) human tumor tissues from patients with liver, ovary, kidney,
breast and colon cancer [53] as well as (iii) elevated breast cancer
risk [43].
Surmounting evidence has implicated the involvement of mito-
chondrial oxidative DNA damage in the carcinogenesis process.
In fact, ROS-mediated mutations in mitochondrial DNA (mtDNA)
have recently emerged as an important contributor to human
carcinogenesis [42]. Mutations in mitochondrial genes encoding
complexes I, III, IV and V, as well as within the hypervariable
region of mtDNA, have been identified in various human can-
cers. In general, mtDNA is more susceptible to oxidative damage
than nuclear DNA because (i) mitochondrial DNA is not protected
by histones, (ii) mitochondrial DNA repair capacity is limited,
and (iii) under physiological conditions, the mitochondria con-
verts roughly 3–5% of oxygen consumed into superoxide anion and
subsequently hydrogen peroxide. In addition, mtDNA is located
in close proximity to the respiratory chain and thus is conse-
quently readily exposed to ROS-induced oxidative damage. As a
result, mtDNA has more than two orders of magnitude higher
frequency of oxidative damage than that of nuclear DNA and signif-
icantly correlates with the development of cancer [41]. While the
underlying mechanism(s) by which mitochondrial DNA alterations
participate in the carcinogenic process has yet to be revealed, data
have suggested that aberrant mtDNA fragment insertion within
regions of nuclear DNA can contribute as a potential mecha-
nism for oncogenic activation [2]. On another note, exciting new
research has suggested that under conditions of excessive DNA
damage, p53 directly activates the mitochondrial death signaling
pathways in cancer cells [41]. This process is controlled by the
(i) MDM family of p53-binding regulatory proteins, (ii) associa-
tion between p53 and mitochondrial proteins of the BCL-2 family
and (iii) VDAC (known to influence permeability of the mitochon-
drial outer membrane). Taken together, these findings imply a
novel role for p53 (that is, its involvement in maintaining nor-
mal mtDNA copy number and cellular ROS homeostasis) and thus
proving how loss of p53 function may promote the carcinogenic
process [40]. Moreover, VDAC has been recently identified as a
critical target of ROS buildup (susceptible to superoxide injury)
leading to the subsequent stimulation of apoptosis. These find-
ings support the hypothesis that ROS-mediated cellular damage
induces a state of redox imbalance and pro-carcinogenic activity
[42].
170 D. Ziech et al. / Mutation Research 711 (2011) 167–173
3. ROS-induced oxidative stress based epigenetic
alterations in human carcinogenesis
In addition to causing genetic changes, ROS may lead to epige-
netic alterations that affect the genome and play a key role in the
development of human carcinogenesis [3]. More specifically, ROS
production is associated with alterations in DNA methylation pat-
terns [17,54]. In particular, hydroxyl radical-induced DNA lesions
(such as 8-hydroxyl-2-deoxyguanosine; 8-hydroxyguanine; 8-
OHdG [56–59], O6 -methylguanine [60,61] and single stranded DNA
[62]) have all been shown to contribute to decreased DNA methy-
lation by means of interfering with the ability of DNA to function
as a substrate for the DNA methyltransferases (DNMTs) and thus
resulting in global hypomethylation [13]. In addition, ROS-induced
oxidative stress can contribute to gene silencing by mechanisms
that involve aberrant hypermethylation of tumor suppressor gene
promoter regions and thus lead towards progression to a malig-
nant phenotype. For example, a recent study has shown that
exposure of hepatocellular carcinoma cells to hydrogen peroxide
(H2 O2 ) induced hypermethylation of the E-cadherin gene promoter
by increasing the expression of Snail (a transcription factor that
down-regulates the expression of E-cadherin) which then induced
methylation of the gene’s promoter by recruiting histone deacety-
lase 1 and DNA methyltransferase 1 as well [15]. Thus, we may
conclude that whether by means of DNA hypo- and/or hyperme-
thylation ROS-induced epigenetic alterations have been implicated
with malignant transformation and the progression of numer-
ous tumors [3]. More than 20 years ago, the impact of epigenetic
contribution to disease was first realized when various studies
distinguished human cancer from normal tissue based upon DNA
methylation levels [20]. Since then, a wide range of studies have
determined major epigenetic alterations in cancer cells, such as
silencing of tumor suppressor genes, activation of oncogenes and
defects in DNA repair [28]. DNA methylation, in part, is perhaps the
most extensively studied epigenetic modification, and is hypothe-
sized to play an important role in the regulation of gene expression
and chromatin architecture, in association with histone modifi-
cation(s) and other chromatin-associated proteins [17]. In fact,
aberrant DNA methylation is the most common molecular lesion
of cancer cells [25]. In general, during DNA replication, methy-
lation status is transmitted to corresponding daughter DNA by
maintenance of DNA methyltransferases, which recognize hemi-
methylated CpG sites at the replication fork [23]. Overall, CpG
islands are kept unmethylated, however, when methylated, are
known to repress the expression of the gene due to changes in chro-
matin structure and the resulting decreased affinity of transcription
factors [23]. Methylated cytosines account for approximately 1%
of total nucleotides and about 75% of all CpG dinucleotides in
the human genome. About 50–60% of gene promoters lie within
CpG islands, and it is estimated that the human genome contains
approximately 29,000 of such CpG sequences [17,28].
DNA methylation changes in cancer cells are characterized by (i)
regional CpG island hypermethylation and (ii) generalized genomic
hypomethylation [19]. Such hypomethylation is thought to induce
both genomic instability and activation of protooncogenes. Alterna-
tively, promoter CpG island hypermethylation is thought to induce
the silencing of tumor suppressor genes [13,24]. Such altered pat-
terns of methylation lead to aberrant gene expression, in part,
by affecting the ability of methylated DNA-binding proteins to
interact with nearby transcription factors [14]. For example, a
recent study has shown that various oxidized products (dimethyl
and methionine sulfoxide) may accumulate in the cytosol dur-
ing the initial stages of carcinogenesis and react with nearby
nucleotides, leading to aberrant methylation-induced gene silenc-
ing [29]. Exciting new research has suggested tumor suppressor
genes as major target genes of aberrant methylation in oxidative
stress-induced carcinogenesis [10]. In fact, many tumor suppres-
sor genes are inactivated via ROS-mediated aberrant methylation
of CpG island-rich promoter regions [16]. For example, studies
have showed that when exposed to oxidative stress, tumor sup-
pressor genes p15INK4B and p16INK4A accrued aberrant methylation
patterns and ultimately their expression was silenced [10]. E-
cadherin, another tumor suppressor gene, has also been shown to
undergo ROS-mediated hypermethylation leading to its silenced
expression. Such promoter hypermethylation is hypothesized to
occur via the recruitment of methyl-CpG binding proteins that
then recruit histone deacetylases, histone methyltransferases, het-
erochromatin proteins etc. [16]. These epigenetic events alter the
chromatin structure in ways that block the access of transcription
factor complexes to DNA and thus repressing transcription [16]. For
example, down-regulation of E-cadherin has been correlated with
epithelial-to-mesenchymal transitions, metastasis, and poor prog-
nosis in hepatocellular carcinoma [15]. Other tumor suppressor
genes, such as CDKN2A, RB, VHL and BRAC1, have also been identified
in cancer cells as being inactivated via oxidative-induced aber-
rant CpG island promoter methylation [21,55]. In addition, recent
studies have demonstrated that both MHL1 (a mismatch repair
gene) and MGMT (responsible for the removal of O6 -methylguanine
adducts) gene promoter regions are hypermethylation-induced
silenced thus leading to increased mutation rates in human colorec-
tal cancers [63–65]. Genome-wide screening of tumor DNA has led
to the identification of several tumor-suppressor genes displaying
oxidative stress-induced alterations in methylation patterns. Such
gene products include silencing of SOCS1 in hepatocellular carci-
noma, SLC5A8 in colon cancer and BMP3B in non-small-cell lung
cancer [21]. In a recent study, 68% of patients with non-small-cell
lung cancer, showed aberrant promoter methylation in at least one
of the following genes: p16, RASSF1A, FHIT, H-cadherin and RARˇ
[24]. Moreover, in patients with endometrial cancer aberrant DNA
hypermethylation was observed in three or more genes from a
panel of five analyzed (CDH13, HSPA2, MHL1, RASSF1A, SOCS2) [24].
Finally, in patients with cervical neoplasia, detection of a panel of
three genes (DAPK1, RARˇ, TWIST1) associated with improved diag-
nostics characterized by 95% specificity and sensitivity depending
primarily on tumor stage: 74% for invasive cancer and 52% for cervi-
cal intraepithelial neoplasia and carcinoma in situ, respectively [24].
Overall, aberrant methylation of RB, p16, VHL, hMLH1, E-cadherin
and BRAC1 have all been implicated in various cancers, including
stomach, colon, liver, breast, uterine, renal and those of hema-
tological origin [23]. However, it is also important to note that
while methylation of promoter CpG islands leads to gene silencing,
aberrant methylation of non-promoter CpG islands is also fre-
quently observed in cancer cells. For example, non-promoter CpG
islands overlapping exon 2 of the CDKN2A gene, are generally kept
unmethylated in normal cells while in cancer cells these regions
are susceptible to ROS-mediated aberrant methylation. Because
these non-promoter CpG regions are known to be highly suscepti-
ble to ROS-mediated aberrant methylation, much interest has been
invested in their use as diagnostic tools for tumor development
[21].
On the other hand, genome-wide hypomethylation has also
been detected in cancer cells. In fact, virtually all tumors exhibit
a global decrease in genomic DNA methylation compared to nor-
mal cells. So far, no relationship between local hypermethylation
and genomic hypomethylation has been found, thus suggesting
that these processes are independent [24]. However, only recently,
evidence has linked genomic hypomethylation with oncogene
overexpression in conjunction with chromosomal instability [25].
In turn, DNA hypomethylation-induced chromosomal instability
has also been linked to increased mutation rates and abnormal
gene expression [26]. Over the last two decades, research has fur-
ther solidified hypomethylation to be strongly linked with global
 D. Ziech et al. / Mutation Research 711 (2011) 167–173
genomic instability and tumor formation [21] and also causing the
activation of proto-oncogenes in many, if not almost all, human
tumors [17,23]. The majority of the large decreases in the methyl-
cytosine content of the genome are mainly due to hypomethylation
of repetitive sequences, as hypomethylation of single-copy genes
accounts for <5% of human DNA. Repetitive sequences (defined
as CpG-rich non-coding sequences of the genome) are known to
be involved in oxidative-stress induced aberrant hypomethylation
and include (i) satellites (SAT2 and SAT3) and (ii) interspersed
repeat sequences (LINEs, SINEs and LTRs) [22]. The CpG-rich SAT2
and SAT3 satellites are normally densely methylated and are
known to contribute to the establishment of heterochromatin. They
are located as tandem repeats in the pericentromeric and juxta-
centromeric heterochromatin of several chromosomes but with
chromosomes 1, 9 and 16 containing large arrays of these repeats
at the base of their long arms [22]. Diminished methylation at
these sites has been described in several human cancers and was
also hypothesized to include mechanisms involving ROS-induced
oxidative stress [22,24]. Alternatively, hypomethylation of LINE
sequences has been reported in many human germ cell cancers
and appears to generally parallel that of overall hypomethylation
[22]. Furthermore, hypomethylation of repetitive sequences leads
to chromosomal rearrangements and, indirectly, to changes in gene
expression patterns like in the case of hepatocellular carcinoma
where such hypomethylation was highly correlated with disease
recurrence [24]. While hypomethylation of repetitive sequences
seems to account for the greater part in cancer cells, decreased
methylation of single-copy genes has attracted most of the atten-
tion. It is believed that single-copy genes known to be silenced in
normal tissues become re-activated when aberrantly hypomethy-
lated. More specifically, reports have suggested the possibility of
major oncogenes like MYC and RAS, to be activated by hypomethy-
lation in cancer cells [22].
In addition, oxidative stress-induced damage has been shown
not only to interfere with the ability of DNA to function as
a substrate for DNA methyltransferases (DNMTs), but also to
reduce the methyl-accepting ability of DNA [13,17]. In mam-
malian cells, DNMTs consist of DNMT1, DNMT2, DNMT3a, DNMT3b
and DNMT3L. DNMTs 2 and 3L lack enzymatic function due to
the presence of an animo-terminal regulatory domain in DNMT2
and a catalytic domain in DNMT3L. On the other hand, DNMT1
is regarded as a maintenance methyltransferase and recognizes
hemi-methylated DNA. In addition, DNMTs 3a and 3b are classified
as de novo methyltransferases since they bind to both hemi-
methylated and unmethylated CpG sites and add methyl groups to
previously unmethylated cytosines. Finally, there are reports that
DNMT3L also participates in de novo methylation [20]. Together,
these enzymes catalyze the transfer of the methyl donor group from
S-adenosylmethionine (SAM) to the 5 -carbon of cytosines within
CpG islands in genomic DNA [13]. These enzymes are known to
contribute to the increase of promoter methylation status (via de
novo methylation) as well as ensuring inheritance of gene silenc-
ing (via maintenance methylation), both of which could account for
the acquisition of a malignant transformation phenotype [13,31].
Increased expression of DNMT1 has been found in early and late
stages of neoplasia suggesting its involvement in the generation
of abnormal methylation patterns in cancer cells [13]. In addi-
tion, it has been reported that patients with mutations in DNMT3b
have numerous chromosomal aberrations [25]. Data, from vari-
ous research groups, have also revealed an over-expression of all
DNMTs in several cancer types suggesting that methyltransferase
cooperativity (especially between DNMT1 and DNMT3b) maintains
DNA methylation and gene silencing in human cancer cells [30].
As it was mentioned earlier on, DNA methylation is hypothe-
sized to play an important role in the regulation of gene expression
and chromatin architecture, in association with histone modi-
171
fication and other chromatin-associated proteins [17]. Histone
modifications have been the subject of intense investigations for
many years and are currently defined as epigenetic modifiers
[26]. The importance of histone modifications lies in the fact
that they are essential during development and their deregulation
can lead to human malignancies. Thus, histones have emerged as
key carriers of epigenetic information, constituting a fundamen-
tal and critical regulatory system that extends beyond the genetic
system [28]. In general, histones have amino-terminal tails that
are targets of several posttranslational modifications, including:
methylation, acetylation, phosphorylation, sumolyation and ubiq-
uitination, among others [18,26]. Alterations in histone acetylation
patterns are present in many tumors [27]. In fact, a recent study
has reported that cigarette smoke exposure to normal bronchial
epithelial or pulmonary carcinoma cells triggers aberrant histone
H2AX phosphorylation on serine 139 and/or phosphorylation on
serine 198 [38]. Other studies have also observed the phosphory-
lation of histone H3T11 in prostate cancer as well as the global loss
of monoacetylation and trimethylation of histone H4, a common
hallmark in many human tumor cells [18]. Typically, acetylated his-
tones are associated with relaxed and transcriptionally competent
chromatin regions whereas hypoacetylated histones, on the other
hand, are associated with transcriptionally silent regions and are
characterized by condensed chromatin structure [26]. The acetyla-
tion of specific lysine residues by histone acetyltransferases (HATs),
for example, is associated with transcriptionally active regions
whereas their hypoacetylation is associated with a transcriptional
repressed chromatin. There are, however, instances whereby mod-
ifications on the histone tails are shown to be both activators and
inhibitors of transcription. The methylation of lysine residues, for
example, has been shown to lead to either transcriptional activation
or repression, depending on the site of methylation [18]. Overall,
HATs are enzymes that alter chromatin-based processes and thus
attributing to mutations in oncogenes, tumor suppressor genes
and/or DNA repair genes resulting in genomic instability, oncogenic
transformation and the development of cancer [28]. Modifications
of histone proteins are strongly linked to DNMTs as they recruit
histone deacetylases (HDACs) leading to histone deacteylation and
subsequent transcriptional repression. These synergistic effects
between DNMTs and HDACs have been shown to induce differenti-
ation, apoptosis and cell growth arrest in several cancer cell lines, as
well as induce the expression of specific cancer-related genes [18].
For example, re-expression of the hypermethylated genes MLH1,
TIMP3, CSKN2B and CDKN2A has been enhanced when exposed to
inhibitors of both DNMTs and HDACs. In particular, HDAC1 has been
over-expressed in a number of tumors, and is likely a contributor to
tumorigenic gene silencing [27]. Finally, recent studies have shown
that both HATs and HDACs are involved in the process of an effi-
cient DNA repair and that inhibition of enzymatic activity and/or
inhibition of the removal of histone acetylation respectively can
compromise the DNA repair machinery thus leading to mutation
fixation and genomic instability [66–70].
Because epigenetic alterations are potentially reversible there
is great potential for the development of effective strate-
gies in preventing against cancer formation [32]. Moreover,
because methylation status can be measured accurately, molec-
ular biomarkers of diagnosis, prognosis, prediction of treatment
and those related to risk assessment could be developed alongside
“state-of-the-art” molecular techniques [11,33]. DNA methylation
markers hold great promise as disease detectors, not only for
the early detection of undiagnosed malignancies but also for the
monitoring of therapeutic efficacy [33]. DNA methylation markers
have several advantages when compared to gene expression pro-
filing and proteomic approaches: (i) stability of the DNA molecule
across extraction and handling procedures, (ii) measurement of
DNA methylation can be compared with absolute reference points
172 D. Ziech et al. / Mutation Research 711 (2011) 167–173
(i.e. a baseline of unmethylated or completely methylated DNA),
(iii) abnormal methylation patterns in cancer cells differ qualita-
tively from normal cells, which allow for the development of assays
with high sensitivity and specificity, (iv) DNA methylation patterns
are fairly stable over time, and lastly, (v) once a gene is hypermethy-
lated in a developing tumor, the methylation signal tends to persist
as the tumor grows [32].
In summary, epigenetic therapies hold great promise for the
treatment of a wide variety of tumors. Strategies combining his-
tone deactylase and methyltransferase inhibitors appear to be
ideal in achieving maximal re-expression of silenced tumor sup-
pressor genes. Such inhibitors when combined with conventional
chemotherapies may represent a powerful strategy for the man-
agement of both early- and late-stage cancers [27].
4. Conclusions
Cancer is a rather complex, multifactorial and multistage dis-
ease with a number of molecular alterations involved in each stage
(namely initiation, promotion and progression) of its development.
Oxidative stress (a state of persistent generation of ROS over-
whelming cellular defenses) has long been known to contribute to
human carcinogenesis. Such contribution has been shown to reg-
ulate altered carcinogenic gene expression at the genetic as well
the epigenetic level by very distinct mechanisms. ROS-induced
genetic alterations involve modifications in DNA bases, strand
breaks, DNA–protein cross linkages etc. all of which increase muta-
tional rates in genes involved in major cellular pathways like
DNA repair etc. thereby making them ineffective. On the other
hand, ROS-induced epigenetic mechanisms involve changes in DNA
methylation profiles and/or histone modifications both of which
lead to silencing of tumor suppressor genes and/or oncogenic acti-
vation and thus contributing to malignant transformation. Finally,
given that epigenetic alterations could potentially be reversible,
there is great potential for the development of effective strategies
in preventing against human carcinogenesis.
Conflict of interest
None.
Acknowledgments
This work was supported, in part, by the National Institutes
of Health (Grant# P20RR17675), Centers of Biomedical Research
Excellence (COBRE) and Layman Award form the University of
Nebraska-Lincoln (Dr. Franco); a Marie Curie International Rein-
tegration Grant within the 6th European Community Framework
Program (MIRG-CT-2006-036585) (Dr. Pappa); and, in part, by the
School of Community Health Sciences, a Junior Faculty Research
Award from the University of Nevada-Reno and a Marie Curie Inter-
national Reintegration Grant within the 7th European Community
Framework Program (PIRG05-GA-2009-249315) (Dr. Panayiotidis).
References
[1] G.J. Kim, K. Chandrasekaran, W.F. Morgan, Mitochondrial dysfunction, per-
sistently elevated levels of reactive oxygen species and radiation-induced
genomic instability: a review, Mutagenesis 21 (2008) 361–368.
[2] M. Valko, C.J. Rhodes, J. Moncol, M. Izakovic, M. Mazur, Free radicals, metals
and antioxidants in oxidative stress-induced cancer, Chem. Biol. Interact. 160
(2006) 1–40.
[3] A.C.E. Campos, F. Molognoni, F.H.M. Melo, L.C. Galdier, C.R.W. Carneiro, V.
D’Almeida, M. Correa, M. Jasiulionis, Oxidative stress modulate DNA methy-
lation during melanocyte anchorage blockade associated with malignant
transformation, Neoplasia 9 (2007) 1111–1121.
[4] B. Tudek, A. Winczura, J. Janik, A. Siomek, M. Foksinski, R. Olinski, Involvement
of oxidatively damaged DNA and repair in cancer development and aging, Am.
J. Transl. Res. 2 (2010) 254–284.
[5] D. Trachootham, J. Alexander, P. Huang, Targeting cancer cells by ROS-mediated
mechanism: a radical therapeutic approach? Nat. Rev. 8 (2009) 579–591.
[6] B.P. Patel, U.M. Rawal, R. Rawal, A. Shukla, P.S. Patel, Tobacco, antioxidant
enzymes, oxidative stress, and genetic susceptibility in oral cancer, Am. J. Clin.
Oncol. 31 (2008) 454–459.
[7] K.W. Lee, H.J. Lee, Biphasic effects of dietary antioxidants on oxidative stress-
mediated carcinogenesis, Mech. Ageing Dev. 127 (2006) 424–431.
[8] N.I. Herath, B. Leggett, G. Macdonald, Review of genetic and epigenetic alter-
ations in hepatocarcinogenesis, J. Gastroenterol. Hepatol. 21 (2006) 15–21.
[9] E. Gollapalle, R. Wang, R. Adetolu, D. Tsao, D. Francisco, G. Sigounas, A. Georgak-
ilas, Detection of oxidative clustered DNA-lesions in X-irradiated mouse skin
tissues and human MCF-7 breast cancer cells, Radiat. Res. 167 (2007) 207–216.
[10] A. Toyokuni, Molecular mechanisms of oxidative stress-induced carcinogene-
sis: from epidemiology to oxygenomics, IUMBM Life 60 (2008) 441–447.
[11] A. Risch, C. Plass, Lung cancer epigenetics and genetics, Int. J. Cancer. 123 (2008)
1–7.
[12] H.I. Chen, S.H. Liou, S.F. Ho, K.Y. Wu, C.W. Sun, M.F. Chen, L.C.
Cheng, T.S. Shih, C.H. Loh, Oxidative DNA damage estimated by plasma
8-hydroxyxeoxyguanosine (8-OHdG): influence of 4,4 -methylenebis (2-
chloroaniline) exposure and smoking, J. Occup. Health. 49 (2007) 389–398.
[13] R. Franco, O. Schoneveld, A. Georgakilas, M. Panayiotidis, Oxidative stress, DNA
methylation and carcinogenesis, Cancer Lett. 266 (2008) 6–11.
[14] G. Egger, G. Liang, A. Aparicio, P.A. Jones, Epigenetics in human disease and
prospects for epigenetic therapy, Nature 429 (2004) 457–463.
[15] S.O. Lim, J.M. Gu, M.S. Kim, H.S. Kim, Y.N. Park, C.K. Park, J.W. Cho, Y.M. Park,
G. Jung, Epigenetic changes induced by reactive oxygen species in hepatocellu-
lar carcinoma: methylation of the E-cadherin promoter, Gastroenterology 135
(2008) 2128–2140.
[16] T. Ushijima, E.O. Takada, Aberrant methylations in cancer cells: where do they
come from? Cancer Sci. 96 (2005) 206–211.
[17] K.V. Donkena, C.Y. Young, D.J. Tindall, Oxidative stress and DNA methylation in
prostate cancer, Obstet. Gynecol. Int. 2010 (2010) 302051.
[18] A.T. Hauser, M. Jung, Targeting epigenetic mechanisms: potential of natural
products in cancer chemoprevention, Planta Med. 74 (13) (2008) 1593–1601.
[19] S.Y. Park, E.J. Yoo, N.Y. Cho, N. Kim, G.H. Kang, Comparison of CpG island hyper-
methylation and repetitive DNA hypomethylation in premalignant stages of
gastric cancer, stratified for Helicobacter pylori infection, J. Pathol. 201 (2009)
410–416.
[20] H. Kirk, W.T. Cefalu, D. Ribnicky, Z. Liu, K. Eilertsen, Botanicals as epigenetic
modulators for mechanisms contributing to development of metabolic syn-
drome, Metab. Clin. Exp. 57 (2008) S16–S23.
[21] T. Ushijima, Detection and interpretation of altered methylation patterns in
cancer cells, Nat. Rev. 5 (2005) 223–231.
[22] M. Hoffman, W. Schulz, Causes and consequences of DNA hypomethylation in
human cancer, Biochem. Cell Bio. 83 (2005) 296–321.
[23] K. Miyamoto, T. Ushijima, Diagnostic and therapeutic applications to epigenet-
ics, Jpn. J. Clin. Oncol. 35 (2005) 293–301.
[24] J. Paluszczak, W.B. Dubowska, Epigenetic diagnostics of cancer – the application
of DNA methylation markers, J. Appl. Genet. 47 (2006) 365–375.
[25] M. Esteller, Aberrant DNA methylation as a cancer-inducing mechanism, Annu.
Rev. Pharmacol. Toxicol. 45 (2005) 629–656.
[26] R.M. Brena, T.H.M. Huang, Quantitative assessment of DNA methylation: poten-
tial applications for disease diagnosis, classification, and prognosis in clinical
settings, J. Mol. Med. 84 (2006) 365–377.
[27] C. Balch, J. Montgomery, H.I. Paik, S. Kim, T.H.M. Huang, K.P. Nephew, New anti-
cancer strategies: epigenetic therapies and biomarkers, Front Biosci. 10 (2005)
1897–1931.
[28] Z. Herceg, Epigenetics and cancer: towards an evaluation of the impact of envi-
ronmental and dietary factors, Mutagenesis 22 (2007) 91–103.
[29] K. Kawai, Y.S. Li, M.F. Song, H. Kasai, DNA methylation by dimethyl sulfoxide
and methionine sulfoxide triggered by hydroxyl radical and implications for
epigenetic modifications, Bioorg. Med. Chem. Lett. 20 (2010) 260–265.
[30] D. Ziech, R. Franco, A. Pappa, V.N. Mitsi, S. Georgakila, A. Georgakilas, M.
Panayiotidis, The role of epigenetics in environmental and occupational car-
cinogenesis, Chem. Biol. Interact. 188 (2010) 340–349.
[31] M.J. Hitchler, F.E. Domann, An epigenetic perspective on the free radical theory
of development, Free Radic. Biol. Med. 43 (2007) 1023–1036.
[32] F.E. Ahmed, Colorectal cancer epigenetics: the role of environmental factors
and the search for molecular biomarkers, J. Environ. Sci. Health C 25 (2007)
101–154.
[33] P.W. Laird, Cancer epigenetics, Hum. Mol. Genet. 14 (2005) R65–R76.
[34] H. Bartsch, J. Nair, Chronic inflammation and oxidative stress in the genesis
and perpetuation of cancer: role of lipid peroxidation, DNA damage and repair,
Langenbecks Arch Surg. 391 (2006) 499–510.
[35] S. Mena, A. Ortega, J.M. Estrela, Oxidative stress in environmental-induced
carcinogenesis, Mutat. Res. 674 (2009) 36–44.
[36] S.J. Schonfeld, P. Bhatti, E.E. Brown, M.S. Linet, S.L. Simon, R.M. Weinstock,
A.A. Hutchinson, M. Stovall, D.L. Preston, B.H. Alexander, M.M. Doddy, A.J. Sig-
urdson, Polymorphisms in oxidative stress and inflammation pathway genes,
low-dose ionizing radiation, and the risk of breast cancer among US radiologic
technologists, Cancer Causes Control 21 (2010) 1857–1866.
[37] D.C. Francisco, P. Peddi, J.M. Hair, B.A. Flood, A.M. Cecil, P.T. Kalogerinis, G.
Sigounas, A.G. Georgakilas, Induction and processing of complex DNA dam-
 D. Ziech et al. / Mutation Research 711 (2011) 167–173
age in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells, Free
Radic. Biol. Med. 44 (2009) 558–569.
[38] H. Zhao, A.P. Albino, E. Jorgensen, F. Traganos, Z. Darzynkiewicz, DNA damage
response induced by tobacco smoke in normal human bronchial epithelial and
A549 pulmonary adenocarcinoma cells assessed by laser scanning cytometry,
Cytometry Part A 75A (2009) 840–847.
[39] A. Katafuchi, T. Nohmi, DNA polymerases involved in the incorporation of
oxidized nucleotides into DNA: their efficiency and template base prefer-
ence, Mutat. Res. (2010), doi10.1016/j.mrgentox.2010.06.004 [Epub ahead of
print].
[40] M.A. Lebedeva, J.S. Eaton, G.S. Shadel, Loss of p53 causes mitochondrial DNA
depletion and altered mitochondrial reactive oxygen species homeostasis,
Biochim. Biophys. Acta. 1787 (2009) 328–334.
[41] S.J. Ralph, S.R. Enríquez, J. Neuzil, E. Saavedra, R.M. Sánchez, The causes of cancer
revisited: “mitochondrial malignancy” and ROS-induced oncogenic transfor-
mation – Why mitochondria are targets for cancer therapy, Mol. Aspects Med.
31 (2010) 145–170.
[42] J.P. Fruehaug, F.L. Meyskens, Reactive oxygen species: a breath of life of death?
Clin. Cancer Res. 13 (2007) 789–794.
[43] P. Peddi, D.C. Francisco, A.M. Cecil, J.M. Hair, M.I. Panayiotidis, A.G. Georgakilas,
Processing of clustered DNA damage in human breast cancer cells MCF-7 with
partial DNA-PKcs deficiency, Cancer Lett. 269 (2008) 174–183.
[44] S.M. Holt, A.G. Georgakilas, Detection of complex DNA damage in ␥-irradiated
acute lymphoblastic leukemia pre-B NALM-6 cells, Radiat. Res. 168 (2007)
527–534.
[45] M. Panayiotidis, Reactive Oxygen Species (ROS) in multistage carcinogenesis,
Cancer Lett. 266 (2008) 3–5.
[46] A. Pappa, R. Franco, O. Schoneveld, A. Galanis, R. Sandaltzopoulos, M.I. Panayi-
otidis, Sulfur-containing compounds in protecting against oxidant-mediated
lung diseases, Curr. Med. Chem. 14 (2007) 2590–2596.
[47] R. Franco, M.I. Panayiotidis, Environmental toxicity, oxidative stress, human
disease and the “black box” of their synergism: how much have we revealed?
Mutat. Res. 674 (1–2) (2009) 1–2.
[48] D. Ziech, R. Franco, A.G. Georgakilas, S. Georgakila, V. Malamou-Mitsi, O. Schon-
eveld, A. Pappa, M.I. Panayiotidis, The role of reactive oxygen species and
oxidative stress in environmental carcinogenesis and biomarker development,
Chem. Biol. Interact. 188 (2) (2010) 334–339.
[49] R. Franco, R. Sanchez-Olea, E.M. Reyes-Reyes, M.I. Panayiotidis, Environmental
toxicity, oxidative stress and apoptosis: ménage a trios, Mutat. Res. 674 (1–2)
(2009) 3–22.
[50] R. Franco, M.I. Panayiotidis, Cell death or survival: the double-edged sword of
environmental and occupational toxicity, Chem. Biol. Interact. 188 (2) (2010)
265–266.
[51] M. Hada, A.G. Georgakilas, Formation of clustered DNA damage after high-LET
irradiation: a review, J. Radiat. Res. (Tokyo) 49 (3) (2008) 203–210.
[52] A.G. Georgakilas, Processing of DNA damage clusters in human cells: current
status of knowledge, Mol. Biosyst. 4 (1) (2008) 30–35.
[53] S. Nowsheen, R.L. Wukovich, K. Aziz, P.T. Kalogerinis, C.C. Richardson, M.I.
Panayiotidis, W.M. Bonner, O.A. Sedelnikova, A.G. Georgakilas, Accumulation
of oxidatively induced clustered DNA lesions in human tumor tissues, Mutat.
Res. 674 (1–2) (2009) 131–136.
[54] D. Ziech, R. Franco, A. Pappa, V. Malamou-Mitsi, S. Georgakila, A.G. Georgakilas,
M.I. Panayiotidis, The role of epigenetics in environmental and occupational
carcinogenesis, Chem. Biol. Interact. 188 (2) (2010) 340–349.
173
[55] A.G. Georgakilas, K. Aziz, D. Ziech, S. Georgakila, M.I. Panayiotidis, BRCA1
involvement in toxicological responses and human cancer etiology, Toxicol.
Lett. 188 (2) (2009) 77–83.
[56] S.A. Weitzman, P.W. Turk, D.H. Milkowski, K. Kozlowski, Free radical adducts
induce alterations in DNA cytosine methylation, Proc. Natl. Acad. Sci. U.S.A. 91
(1994) 1261–1264.
[57] P.W. Turk, A. Laayoun, S.S. Smith, S.A. Weitzman, DNA adduct 8-hydroxyl-
2-deoxyguanosine (8-hydroxyguanine) affects function of human DNA
methyltransferase, Carcinogenesis 16 (1995) 1253–1255.
[58] P.W. Turk, S.A. Weitzman, Free radical DNA adduct 8-OH-deoxyguanosine
affects activity of HPA II and MSP I restriction endonucleases, Free Radic. Res.
23 (1995) 255–258.
[59] Y. Kuchino, F. Mori, H. Kasai, H. Inoue, S. Iwai, K. Miura, E. Ohtsuka, S. Nishimura,
Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the
modified base and at adjacent residues, Nature 327 (1987) 77–79.
[60] P.A. Hepburn, G.P. Margison, M.J. Tisdale, Enzymatic methylation of cytosine in
DNA is prevented by adjacent O6 -methylguanine residues, J. Biol. Chem. 266
(1991) 7985–7987.
[61] N.W. Tan, B.F.L. Li, Interaction of oligonucleotides containing 6-O-
methylguanine with human DNA (cytosine-5-)-methyltransferase,
Biochemistry 29 (1990) 9234–9240.
[62] J.K. Christman, G. Sheikhnejad, C.J. Marasco, R.J. Sufrin, 5-Methyl-2-
deoxycytidine in single stranded DNA can act in cis to signal de novo DNA
methylation, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 7347–7351.
[63] M.F. Kane, M. Loda, G.M. Gaida, J. Lipman, R. Mishra, H. Goldman, J.M. Jessup, R.
Kolodner, Methylation of the hMLH1 promoter correlates with lack of expres-
sion of hMLH1 in sporadic colon tumors and mismatch-repair-defective human
tumor cell lines, Cancer Res. 60 (2000) 4366–4371.
[64] J.G. Herman, A. Umar, K. Polyak, J.R. Graff, N. Ahuja, J-P.J. Issa, S. Markowitz, J.K.V.
Wilson, S.R. Hamilton, K.W. Kinzler, M.F. Kane, R.D. Kolodner, B. Vogelstein, T.A.
Kunkel, S.B. Baylin, Incidence and functional consequences of hMLH1 promoter
hypermethylation in colorectal carcinoma, Proc. Natl. Acad. Sci. U.S.A. 95 (1998)
6870–6875.
[65] M. Esteller, R.A. Risques, M. Toyota, G. Capella, V. Moreno, M.A. Peinado, S.B.
Baylin, J.G. Herman, Promoter hypermethylation of the DNA repair gene O(6)-
methylguanine-DNA methyltransferase is associated with the presence of G:C
to A:T transition mutations in p53 in human colorectal tumorigenesis, Cancer
Res. 61 (2001) 4689–4692.
[66] C.L. Peterson, J. Cote, Cellular machineries for chromosomal DNA repair, Genes.
Dev. 18 (2004) 602–616.
[67] A. Jazayeri, A.D. McAinsh, S.P. Jackson, Saccharomyces cerevisiae Sin3p facili-
tates DNA double-strand break repair, Proc. Natl. Acad. Sci. U.S.A. 101 (2004)
1644–1649.
[68] R. Murr, J.I. Loizou, Y.G. Yang, C. Cuenin, H. Li, Z.Q. Wang, Z. Herceg, Histone
acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of
DNA double-strand breaks, Nat. Cell. Biol. 8 (2006) 91–99.
[69] J.A. Downs, S. Allard, O. Jobin-Robitaille, A. Javaheri, A. Auger, N. Bouchard,
S.J. Kron, S.P. Jackson, J. Cote, Binding of chromatin-modifying activities
to phosphorylated histone H2A at DNA damage sites, Mol. Cell. 16 (2004)
979–990.
[70] B.A. Tamburini, J.K. Tyler, Localized histone acetylation and deacetylation trig-
gered by the homologous recombination pathway of double-strand DNA repair,
Mol. Cell. Biol. 25 (2005) 4903–4913.