Genetic variations in LC risk assessment 4

ATM
In human cells, ATM is required for the early re-
sponse to ionizing radiation. ATM senses genomic
damage and initiates DNA repair through interact-
ing with the MRN (MRE11, RAD50, and NBS1)
complex and subsequently activating a series of
downstream signaling mediators [153]. In a Korean
study, an SNP in intron 62 (IVS62 +50G>A) ex-
hibited a significantly increased LC risk with an OR
of 1.6 (1.1–2.1) [154], and higher risks for haplo-
types and diplotypes containing the variant allele
with ORs of 7.6 (1.7–33.5) and 13.2 (3.1–56.1), re-
spectively. The close proximity of this SNP to ATM
PI3K and FAT domains suggests a potential func-
tional impact on ATM kinase activity.
NBS1
In the HR pathway, the first event is the resec-
tion of the DNA to yield single-strand overhangs
[118]. NBS1 is part of an exonuclease complex that
takes part in this step. Zienolddiny et al. and Mat-
ullo et al. showed no association between the NBS1
Glu185Gln polymorphism and LC risk [125,155],
but Lan et al. reported that homozygotes for this al-
lele had an increased risk of LC with an OR of 2.53
(1.05–6.08) [156].
XRCC3
XRCC3 is an RAD51-related protein involved in cat-
alyzing the DNA strand exchange reaction during
HR [118]. Five epidemiological studies found no
association between the thr241Met polymorphism
with LC risk [125,143,148,155,157].
LIG4
In the NHEJ pathway, LIG4 has an important role in
linking the ends of a double-strand break together
[118]. Matullo et al. found no association between
two LIG4 polymorphisms (Ala3Val and Thr9Ile) and
LC risk [155]. Sakiyama et al. reported that the vari-
ant allele of the Ile658Val polymorphism of LIG4
was associated with a reduced risk of squamous cell
carcinoma with an OR of 0.4 (0.1–0.8) [158].
MMR
The MMR system maintains the stability of the
genome during repeated duplication, by repairing
base–base mismatches, caused not only by errors
of DNA polymerases that escape their proofreading
function, but also by insertion/deletion loops that
result from slippage during replication of repetitive
sequences or during recombination [118]. So far,
only a few studies have investigated the connection
between MMR and LC.
MLH1 –93G>A polymorphism was studied for its
association with risk of LC and no overall associa-
tionwas identified [159]; Jung et al. investigated the
association of MSH2 –118T>C, IVS1 +9G>C, IVS10
+12A>G, and IVS12 –6T>C genotypes with LC risk
[160] and found that the presence of at least one
IVS10 +12G allele was associated with a decreased
risk of adenocarcinoma as compared with the IVS10
+12AA genotype with OR of 0.59 (0.40–0.88), and
the presence of at least one IVS12 –6C allele was as-
sociated with an increased risk of adenocarcinoma
as compared with the IVS12 –6TT genotype with an
OR of 1.52 (1.02–2.27) [160].
DNA damage and repair phenotypic assays
The phenotypic assays for DNA damage and repair
include measuring: (a) DNA damage/repair after a
chemical or physicalmutagen challenge (such as the
mutagen sensitivity, comet, and induced adduct as-
says); (b) unscheduled DNA synthesis; (c) cellular
ability to remove DNA lesions from plasmid trans-
fected into lymphocyte cultures in vitro by expres-
sion of damaged reporter genes (the host–cell reac-
tivation assay); (d) activity of DNA repair enzyme
(repair activity assay for 8-OH-Guanine) [161,162].
Mutagen sensitivity
The mutagen sensitivity assay quantifies chromatid
breaks induced by mutagens in cultured lympho-
cytes in vitro as an indirect measure of DRC
[163,164]. Bleomycin is a clastogenic agent that
mimics the effects of radiation by generating free
oxygen radicals capable of producing DNA single-
and double-strand breaks that initiate BER and DSB
repair [165].Wu et al. showed that higher BPDE and
bleomycin sensitivities were independently signifi-
cantly associated with increased risks of LC, a find-
ing that has been confirmed by other studies [3–
5,166,167].
Comet assay
The comet assay is a single-cell gel electrophoresis
method used to measure DNA damage in individ-
ual cells. It is a sensitive and versatile method with
high throughout potential [168,169]. The alkaline
version (pH > 13) of the comet assay can detect
DNA damage such as single-strand breaks, double-
strand breaks, and alkaline labile sites [170]. Com-
mon mutagens used in this assay include BPDE,
bleomycin, and γ-radiation. Wu et al. found that
higher γ-radiation- and BPDE-induced olive tail
moments, one of the parameters for measuring
DNA damage, were significantly associated with
2.32- and 4.49-fold risks of LC, respectively [171].
Rajaee-Behbahani et al. reported lower repair rate of
bleomycin-induced DNA damage using the alkaline
comet assay in LC patients compared with controls
[172].
DNA adducts
Using 32P postlabeling techniques, two studies by
the same group indicated a significant association
between the level of in vitro BPDE-induced DNA
adducts and risk for LC [173,174], suggesting sub-
optimal ability to remove the BPDE-DNA adduct re-
sulted in increased susceptibility to tobacco carcino-
gen exposure [174].
Host cell reactivation assay
The host cell reactivation assaymeasures globalNER
as a biomarker for LC susceptibility [175–177], by
quantifying the activity of a reporter gene (CAT
or LUC gene) in undamaged lymphocytes trans-
fectedwith BPDE-treated plasmids. Because a single
unrepaired BPDE-induced DNA adduct can block
reporter gene transcription [178], the measured
reporter gene activity reflects the ability of the trans-
fected cells to remove the adducts fromthe plasmid.
Reduced capacity to repair adducts is observed in
cases compared to controls and is associated with
an increased risk of LC with evidence of a signifi-
cant dose–response association between decreased
DRC and risk of LC [175–177].
8-OGG assay
The enzyme 8-oxoguanine DNA N-glycosylase is
encoded by the OGG1 gene and initiates the BER
pathway. The OGG activity assay monitors the abil-
ity ofOGG to remove an 8-oxoguanine residue from
a radiolabeled synthetic DNA oligonucleotide, gen-
erating two DNA products that can be distinguished
on the basis of size [179]. Paz-Elizur et al. showed
that OGG activity was significantly lower in periph-
eral blood mononuclear cells from LC patients than
in those fromcontrols. Individuals in the lowest ter-
tile of OGG activity exhibited an increased risk of
NSCLC compared with those in the highest tertile
(OR=4.8; 95%CI, 1.5–15.9) [179].Gackowski et al.
also reported that the repair activity of OGG was
significantly higher in blood leukocytes of healthy
volunteers than in LC patients [180].
Cell cycle control
The intricate cell cycle regulatory network is essen-
tial for cells to undergo replication, division, prolif-
eration, and differentiation. Anomalies of cell cycle
regulation genes are frequently observed in a vari-
ety of human malignancies including LC, and are
considered to be one of the most critical early-stage
events in carcinogenesis [181–184].
Genetic polymorphisms in cell
cycle-related genes
p53
p53 is the most important tumor suppressor gene of
the genome defense system regulating pivotal cel-
lular activities such as DNA damage response, DNA
repair, cell cycle control, and apoptosis. Three poly-
morphisms of the p53 gene have been commonly
studied in cancer susceptibility.Weston et al. first re-
ported the association between the Arg72Pro nsSNP
in exon 4 and increased LC risk [185], which was
confirmed by a number of subsequent studies in
various populations [186–191]. Functional assays
corroborated this finding by demonstrating the as-
sociation between the variant allele and increased
p53 mutations in tumor tissues, as well as a reduced
rate of apoptosis in white blood cells of LC patients
[189,192,193]. Wu et al. reported an association
with the variant genotype of both the intron 3 16-bp
deletion/insertion and the intron 6 polymorphisms
[187]. Analyses of haplotypes reconstructed using
these three polymorphisms demonstrated an in-
creased LC risk for the variant-harboring haplotypes
compared to the haplotype with wild-type alleles at
all three loci. This result was supported by func-
tional studies showing that the variant-harboring
haplotypes exhibited a reduced apoptotic index and
reduced DNA repair capacity [187]. Moreover, the
association with the intron 3 polymorphism was
confirmed in a recent large-scale European study,
which reported a 2.98-fold [194] increased LC risk
for the homozygous variant genotype. Although
most studies suggest a positive association between
these p53 polymorphisms and LC risk, disagree-
ments exist including a recent meta-analysis of
13 LC studies showing no LC risk association for
any of these polymorphisms [195].
p73
p73 may activate p53 down-stream transcriptional
effectors such as p21 to control cell cycle progression
and apoptosis [196]. A dinucleotide polymorphism
in the 5
UTR of p73 is associated with an increased
risk of LC in a Caucasian population but a protec-
tive effect in a Chinese population [197,198], sug-
gesting the possible existence of ethnic-specific risk
differentiation. Furthermore, a gene–dosage effect
by combining both p53 and p73 variant alleles to-
gether was demonstrated [199].
MDM2
MDM2, a ubiquitin ligase, negatively regulates p53
activity either by binding to the transactivation do-
main of p53 protein and inhibiting its transcrip-
tional activation of p21, or by targeting p53 pro-
tein to ubiquitin-mediated proteasome degradation
[200].AT toGtransversion in the intronic promoter
region of MDM2 was associated with increased LC
risk in Chinese [190], Koreans [201], and Euro-
peans [202]. Other studies exhibited similarly el-
evated risk, although not reaching statistical sig-
nificance [203,204]. The agreement amongst these
studies recapitulates the in vivo observation that
the variant allele upregulates MDM2 expression and
thus reduces p53 protein level [205].
HRAD9
HRAD9 is a phosphorylation target of ATM kinase
that plays a crucial role in DNA repair and cell cycle
arrest in response to DNA damage [206]. A nsSNP