Lung cancer
Gaetano Caramori,1 Ian M Adcock,2 Paolo Casolari,1 Kazuhiro Ito,2 Elen Jazrawi,2
Loukia Tsaprouni,2 Gino Villetti,3 Maurizio Civelli,3 Chiara Carnini,3 Kian Fan Chung,2
Peter J Barnes,2 Alberto Papi1
< An additional table is
published online only. To view
this file please visit the journal
online (http://thorax.bmj.com).
1
Centro di Ricerca su Asma e
BPCO University of Ferrara,
Ferrara, Italy
2
Airway Disease Section,
Imperial College London, UK
3
Department of Pharmacology,
Chiesi Farmaceutici, Parma, Italy
Correspondence to
Gaetano Caramori, Centro di
Ricerca su Asma e BPCO,
University of Ferrara, Via
Savonarola, 9, Ferrara 44100,
Italy; gaetano.caramori@unife.it
Gaetano Caramori, Ian M
Adcock, Peter J Barnes and
Alberto Papi have contributed
equally to this work.
Received 29 November 2010
Accepted 24 February 2011
Published Online First
2 April 2011
ABSTRACT
Background Chronic obstructive pulmonary disease
(COPD) is characterised by oxidative stress and
increased risk of lung carcinoma. Oxidative stress causes
DNA damage which can be repaired by DNA-dependent
protein kinase complex.
Objectives To investigate DNA damage/repair balance
and DNA-dependent protein kinase complex in COPD
lung and in an animal model of smoking-induced lung
damage and to evaluate the effects of oxidative stress
on Ku expression and function in human bronchial
epithelial cells.
Methods Protein expression was quantified using
immunohistochemistry and/or western blotting. DNA
damage/repair was measured using colorimetric assays.
Results 8-OH-dG, a marker of oxidant-induced DNA
damage, was statistically significantly increased in the
peripheral lung of smokers (with and without COPD)
compared with non-smokers, while the number of
apurinic/apyrimidinic (AP) sites (DNA damage and repair)
was increased in smokers compared with non-smokers
(p¼0.0012) and patients with COPD (p<0.0148).
Nuclear expression of Ku86, but not of DNA-PKcs,
phospho-DNA-PKcs, Ku70 or g-H2AFX, was reduced in
bronchiolar epithelial cells from patients with COPD
compared with normal smokers and non-smokers
(p<0.039). Loss of Ku86 expression was also observed
in a smoking mouse model (p<0.012) and prevented by
antioxidants. Oxidants reduced (p<0.0112) Ku86
expression in human bronchial epithelial cells and Ku86
knock down modified AP sites in response to oxidative
stress.
Conclusions Ineffective DNA repair rather than strand
breakage per se accounts for the reduced AP sites
observed in COPD and this is correlated with a selective
decrease of the expression of Ku86 in the bronchiolar
epithelium. DNA damage/repair imbalance may
contribute to increased risk of lung carcinoma in COPD.
INTRODUCTION
Lung cancer remains a major worldwide health
problem, accounting for more than a sixth of cancer
deaths. The incidence of lung cancer is highest in
the more developed countries of North America and
northern Europe. In these regions, more than 90%
of cases in men and 80% of cases in women are
related to smoking.1
Cigarette smoking is also the most important
risk factor for chronic obstructive pulmonary
disease (COPD).2 Thus, lung carcinoma and COPD
share a common risk factor, tobacco smoking,
Thorax 2011;66:521e527. doi:10.1136/thx.2010.156448
Key messages
What is the key question?
< Is the balance between DNA damage and repair
altered in COPD lung?
What is the bottom line?
< In COPD lung there is an unbalanced oxidant-
induced DNA damage and repair correlated with
a selective decrease of the expression of Ku86
in the bronchiolar epithelium.
Why read on?
< Because it provides a molecular mechanism to
explain the increased risk of lung cancer in
patients with COPD, in addition to their smoking
habit.
through which they also possibly share similar
pathogenetic mechanisms that are still largely
unknown at the molecular level.
Furthermore, COPD is an independent risk factor
for lung carcinoma among smokers; increasing the
risk of lung cancer twofold, with the risk increasing
with COPD severity.3e5
COPD is also associated with increased oxidative
stress in the lower airways which can cause DNA
damage and lung carcinogenesis.6 Damage to DNA
induces several cellular responses that enable cells
either to eliminate or cope with the damage or to
activate programmed cell death, thereby eliminating cells with potentially harmful mutations.7
DNA repair mechanisms include direct repair,
base excision repair, nucleotide excision repair,
double-strand break (DSB) repair, and cross-link
repair.7 Efficient repair of DNA DSBs is essential for
the maintenance of chromosomal integrity.8 9 DSBs
are repaired either by homologous recombination or
by non-homologous end-joining (NHEJ) with the
latter being the primary pathway involved.8 9 Six
distinct proteins function in the NHEJ pathway
(Ku70, Ku86, XRCC4, DNA ligase IV, Artemis,
DNA-PKcs).8 9
The heterotrimeric DNA-dependent protein
kinase (DNA-PK) complex is a serine-threonine
kinase activated by the presence of DSBs in
DNA and composed of a catalytic subunit (DNAPKcs) and the DNA-binding heterodimer of the
regulatory subunits Ku proteins (Ku70 and Ku86).
Autophosphorylation of DNA-PKcs (phosphoDNA-PKcs (pS2612)) correlates with loss of protein
521
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Unbalanced oxidant-induced DNA damage and repair
in COPD: a link towards lung cancer
Lung cancer
METHODS
Participants
We recruited a total of 69 patients (64 undergoing lung resection
for a solitary peripheral carcinoma (11 non-smokers, 28 smokers
with normal lung function and 25 with COPD) and five nonsmokers undergoing lung transplantation). All patients were
recruited from the Respiratory Diseases Clinic of the University
Hospital of Ferrara, Italy and from the Royal Brompton
Hospital, Imperial College London, UK.
Table 1
For the DNA damage/repair and DNA fragility studies, 11
were smokers with COPD, 15 were smokers with normal lung
function and seven were non-smokers (table 1). Samples from 14
smokers with COPD, 13 smokers with normal lung function
and nine non-smokers were used for immunohistochemical and
western blotting studies (table 2). COPD was defined according
to international guidelines.2 All patients were stable at the time
of the study and were free from acute exacerbations of symptoms and from upper respiratory tract infections in the
2 months preceding the study. None had received glucocorticoids, theophylline, antibiotics or antioxidants within the
month preceding surgery or bronchodilators within the previous
48 h. Patients had no history of asthma or other allergic diseases.
Former smokers had stopped smoking for more than 1 year. Each
patient underwent medical history, physical examination, chest
radiography, electrocardiogram, routine blood tests, and
pulmonary function tests during the week prior to surgery. The
study was approved by the ethics committees of the University
Hospital of Ferrara and the Royal Brompton Hospital.
Pulmonary function tests were performed as previously
described16 according to published guidelines.17 Predicted values
for the different measures were calculated from the regression
equations published by Quanjer and colleagues.17
Experimental methods
The cigarette smoke-exposed mouse model, the immunohistochemistry in human and mouse lungs for DNA-PKcs, DNA-PKcs
(pS2612), Ku70, Ku86 and g-H2AFX, the DNA damage/repair
quantification and DNA fragility assays, the western blot analysis for Ku70 and Ku86 in human lung parenchyma, the culture
of primary human bronchial epithelial cells and the Ku86 knockdown siRNA in human bronchial epithelial cells are all described
in full detail in the online data repository.
Statistical analysis
Group data were expressed as mean and SD or median (25e75
percentiles), as appropriate. Analysis of variance for clinical data
and the KruskaleWallis test for histological data were used to
determine differences between groups. The ManneWhitney
U test was performed after the KruskaleWallis test when
appropriate. Bonferroni adjustment was applied when indicated.
Categorical values were compared by means of the c2 test.
A probability value of <0.05 was considered statistically
significant.
RESULTS
Clinical data
Tables 1 and 2 show the characteristics of the patients recruited.
There was no significant difference in the smoking history
between patients with COPD and smokers with normal lung
function (p¼0.8279 and p¼0.5369 for pack-years of COPD vs
smokers in tables 1 and 2). The prevalence of chronic bronchitis
Characteristics of participants taking part in the DNA damage/repair and DNA fragility studies
Participants
N
Age
Sex
Non-smokers
Smokers
7
15
51.6 (17.5)
69.5 (5.5)
3M/4F
13M/2F
COPD
11
69 (6.7)
11M
Smoking
history
All never
6 ex,
5 current
5 ex,
4 current
Pack-years
Chronic
bronchitis
FEV1 % pred
FEV1/FVC%
0
48.9 (28.6)
0
9
ND
94.6 (19.9)
ND
75.4 (4)
43.6 (26.6)
4
77.7 (15.6)
64 (1.4)
For patients with chronic obstructive pulmonary disease (COPD) and smokers with normal lung function predicted forced expiratory
volume in 1 s (FEV1% pred) and FEV1/forced vital capacity (FVC)% are post-bronchodilator values.
ND, not done in five participants; two had a FEV1/FVC ratio of 77(5.6)% of predicted.
M, male; F, female. Data expressed as mean (SD).
522
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kinase activity and dissociation of the DNA-PKcs-Ku complex.
DNA-PK is localised in the nucleus and it is critical during NHEJ
because it initially recognises and binds to the damaged DNA
and then targets the other repair activities to the site of DNA
damage.8e10
In addition to the regulatory function of the Ku proteins in
DNA-PK, heterodimers of both Ku70 and Ku86 also have independent DNA repair functions.8e10 It has been documented in
vitro that oxidative stress can induce nuclear loss of Ku proteins
in pancreatic epithelial cells.11
Apurinic/apyrimidinic (AP) sites are common lesions in DNA
and are formed either spontaneously or as intermediates during
the course of base excision repair of oxidised, deaminated and
alkylated bases.12 It has been estimated that about 23105 base
lesions are generated per cell per day. The level of AP sites in cells
is thought to be a good indicator of both ongoing DNA damage
and repair induced by chemical agents and cell ageing.12
8-Hydroxy-2-deoxyguanosine (8-OH-dG) is produced by the
oxidative damage of DNA by reactive oxygen and nitrogen
species and serves as an established marker of oxidative stress.13
8-OH-dG is increased in the peripheral lung of patients with
pulmonary emphysema.14 After a DNA DSB a variant histone
H2A, termed H2A.x (which replaces conventional H2A in
a subset of nucleosomes), is rapidly phosphorylated at Serine
139 by ATM kinase. Phospho-S139-H2A.x is also known as
g-H2AFX.15 This phosphorylation step can extend up to several
thousand nucleosomes from the actual site of the DSB and may
mark the surrounding chromatin for recruitment of proteins
required for DNA damage signalling and repair.15 Both 8-OH-dG
and g-H2AFX are considered sensors of oxidative stress-induced
DNA damage.
The aim of this study was to investigate the degree of
oxidant-induced DNA damage and DNA repair and the localisation and expression of DNA-PKcs, DNA-PKcs (pS2612), Ku70,
Ku86 and g-H2AFX in lung parenchyma of patients with COPD
compared with smokers with normal lung function and nonsmokers (control groups). The study also analysed the effects of
oxidative stress on Ku protein expression in primary human
bronchial epithelial cells in vitro, the functional effect of Ku86
knockdown in bronchial epithelial cells and the effect of cigarette smoke exposure on bronchial epithelial cell Ku86 expression in an animal model in vivo.
Lung cancer
Table 2 Characteristics of participants taking part in the immunohistochemistry and western blotting
studies
n
Age
Sex
Non-smokers
Smokers
9
13
71.2 (9.7)
68.7 (7.5)
3M/6F
11M/2F
COPD
14
70.9 (4.1)
11M
Smoking
history
All never
6 ex,
5 current
5 ex,
4 current
Pack-years
Chronic
bronchitis
FEV1 % pred
FEV1/FVC%
0
33.7 (20)
0
5
111.1 (16.4)
96.7 (19.6)
84.6 (6.7)
77.6 (4.5)
37.7 (23.8)
9
70.2 (16.2)
61 (7.8)
For chronic obstructive pulmonary disease (COPD) and smokers with normal lung function predicted forced expiratory volume in 1 s
(FEV1% pred) and FEV1/forced vital capacity (FVC)% are post-bronchodilator values.
M, male; F, female. Data expressed as mean (SD).
was 52% in the COPD group and 50% in the smokers with
normal lung function.
DNA damage/repair in lung parenchyma
DNA samples from patients with COPD were more fragile
and liable to degradation than those obtained from non-smokers
and smokers with normal lung function (figure 1A). In order
to explain this we examined markers of specific DNA damage
and repair. The degree of oxidative stress-induced DNA
damage was similar in healthy smokers and smokers with
COPD as determined by the level of 8-OH-dG/mg DNA
(figure 1B) and statistically significantly greater than that
observed in normal controls (0.26 (0.17) and 0.29 (0.17) vs 0.06
(0.04) respectively; p¼0.0033). However, the number of AP sites,
a marker of DNA damage and repair, was elevated in smokers
with normal lung function (2.863 (1.31)) compared with
healthy non-smokers (1.195 (0.33); p¼0.0012) and patients
with COPD (1.607 (0.73); p¼0.0148). The number of AP sites
in patients with COPD, however, was not statistically
significantly different compared with non-smokers (p¼0.2048)
(figure 1C). The current smoking status had no effect on
AP sites.
Immunohistochemistry for DNA-PKcs, DNA-PKcs (pS2612),
Ku70, Ku86 and g-H2AFX in human lung parenchyma
The authors postulated that there may be decreased DNA repair
in COPD, therefore components of this process were analysed.
Figure 1 (A) Ethidium bromide staining of
freeze-thawed DNA extracted from nonsmokers (n¼6), smokers with normal lung
function (n¼6) and patients with mild to
moderate COPD (n¼10). (B) Graphical
representation of 8-hydroxy-2deoxyGuanosine (8-OH-dG) content, an index
of oxidative stress-induced DNA damage, in
human lung parenchyma obtained from nonsmokers (n¼7), healthy smokers with normal
lung function (n¼15) and patients with COPD
(n¼11). Histograms represent mean (SD). (C)
Graphical representation of AP sites, indexes
of DNA damage and repair, in human lung
parenchyma obtained from non-smokers
(n¼7), healthy smokers with normal lung
function (n¼15) and patients with COPD
(n¼11). Histograms represent mean (SD).
Thorax 2011;66:521e527. doi:10.1136/thx.2010.156448
A
Ku86 was mainly localised to the nucleus in both structural and
inflammatory cells of the lungs (figure 2). The number of
bronchiolar epithelial cells without nuclei positively stained for
Ku86 was statistically significantly higher in patients
with COPD (median (IQR) 3.54% (1.85e5.75), n¼14), but
not in healthy smokers (1.5% (0.3e5.4), n¼13), compared with
non-smokers (1% (0.6e1.8), n¼9) (p<0.039). In addition,
the amount of Ku86 protein (0.26 (0.04) vs 0.4 (0.03) Ku86/actin
ratio OD arbitrary units; p¼0.026), but not total Ku70 (0.49
(0.14) vs 0.4 5(0.1); p¼0.82) in lung parenchyma demonstrated
a statistically significantly decrease in COPD (n¼6) compared
with smokers with normal lung function (n¼6) (figure 2D).
DNA-PKcs, DNA-PKcs (pS2612), Ku70 and g-H2AFX were
localised exclusively in the nucleus in all cells, including bronchiolar epithelial cells (all bronchiolar epithelial cells were
stained in all three groups). There was no statistically significant
difference in their localisation and global expression among the
three groups for DNA-PKcs and Ku70 (figures 3 and 4 and online
table S1) and between smokers with normal lung function and
patients with COPD for DNA-PKcs (pS2612) and g-H2AFX
(figures 3 and 4 and online table S1).
Furthermore, no statistically significant differences in the
percentage of endoalveolar macrophages stained for DNA-PKcs,
Ku70, and Ku86 among the three groups and between smokers
with normal lung function and patients with COPD for
DNA-PKcs (pS2612) and g-H2AFX were seen (all endoalveolar
macrophages were stained in all three groups).
Normal
Smokers
COPD
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10
B
M
C
p=0.0033
523
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Participants
Lung cancer
D
Ku86
A
Actin
N1
N2
S1
S2
S3
C1
C2
C3
p=0.026
Stimulation of primary human bronchial epithelial cells with
hydrogen peroxide in vitro produced a statistically significant
reduction in Ku86 but not Ku70 expression (p<0.0112; figure 6A,B).
Additionally, knockdown of Ku86 protein in bronchial epithelial
BEAS-2B cells using siRNA resulted in a statistically significant
reduction of the ability of these cells to induce AP sites in
response to H2O2 exposure (figure 6CeE).
Ku86/a
actin ratio
0.5
B
Effect of H2O2 on Ku70/Ku86 expression in primary human
bronchial epithelial cells
0.4
0.3
0 .2
0.1
0.0
600x
Smokers
COPD
DISCUSSION
Figure 2 Photomicrographs showing the bronchiolar epithelium from
a non-smoker (A), a healthy smoker with normal lung function (B) and
a patient with mild to moderate chronic obstructive pulmonary disease
(COPD) (C) immunostained for identification of Ku86+cells (brown).
Results are representative of those from nine non-smokers, 13 smokers
with normal lung function, 14 patients with mild/moderate COPD. (D)
Western blotting analysis of Ku86 (with actin controls) in whole lung
parenchyma lysates from one non-smoker (N), three healthy smokers
with normal lung function (S) and three patients with mild to moderate
COPD (C). Histograms represent the mean (SD) of the Ku86/actin ratio in
healthy smokers (n¼6) and patients with COPD (n¼6).
Immunohistochemistry for Ku70 and Ku86 in mouse lung
parenchyma
Using a smoke-exposed mouse model the authors were also able to
demonstrate predominant nuclear Ku 70 and Ku86 localisation in
bronchial epithelial cells (figure 5). The expression of Ku86 in the
bronchial epithelial cells was decreased to a statistically significant
extent only in susceptible A/J mice (p¼0.012) but not in C57BL/6J
mice (p¼0.44) exposed to cigarette smoke. This decrease was
prevented by treatment of A/J mice with N-acetylcysteine (NAC),
as determined by immunohistochemistry (A/J mice: median
(interquartile range) 75% (71%e83%) for controls vs 66% (57%e
70%) for smoke-exposed mice vs 73% (67%e78%) for NAC
+smoke-exposed mice; p¼0.012 for controls vs smoke-exposed
Figure 3 Photomicrographs showing
the bronchiolar epithelium from a nonsmoker (A, D), a healthy smoker with
normal lung function (B, E) and
a patient with mild to moderate chronic
obstructive pulmonary disease (COPD)
(C, F) immunostained brown for
identification of Ku70+ (left panel) and
DNA-PKcs+cells (right panel). Results
are representative of those from nine
non-smokers, 13 smokers with normal
lung function and 14 patients with mild
to moderate COPD. (G) Western
blotting analysis of Ku70 (with actin
controls) in whole lung parenchyma
lysates from four healthy smokers with
normal lung function (S) and four
patients with mild to moderate COPD
(C). Histograms represent the mean
(SD) of the Ku70/actin ratio in healthy
smokers (n¼6) and COPD (n¼6).
Ku70
DNA-PKcs
G
Ku70
Actin
D
A
S1
S2
S3
S4
C1
C2
C3
C4
p=0.8182
0.7
E
B
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Smokers
600x
524
There is a selective decrease in the level of the DNA repair
protein Ku86 in the bronchiolar epithelium of patients with
COPD. This has also been confirmed in the cigarette-smoke
exposed murine strain susceptible to lung cancer. In vitro, the
exposure of bronchial epithelial cells to H2O2 reproduced the
selective pattern of Ku86 expression decrease observed in COPD.
In addition, the study demonstrated for the first time that the
number of AP sites is significantly increased in lung parenchyma
from smokers with normal lung function compared with nonsmokers, whereas the level of 8-OH-dG is significantly increased
in lung parenchyma from all smokers with or without COPD
compared with non-smokers.
AP levels reflect both ongoing DNA damage and DNA repair of
the damage. To the authors’ surprise, this combined marker of
DNA damage/repair was higher in smokers with normal lung
function compared with smokers with COPD. In contrast,
8-OH-dG and g-H2AFX, markers of DNA damage, were not
significantly different between smokers with normal lung function and current and former smokers with COPD, reflecting
similar levels of smoking exposure. The absence of increased AP
levels in patients with COPD might be a consequence of a loss of
DNA repair secondary to a lack of Ku86 in bronchiolar epithelial
cells.
Furthermore, short-term (11 days) cigarette smoke exposure
in mice decreased K86 expression in bronchial epithelial cells
Ku7
70/actin ratio
C
C
600x
COPD
F
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mice; C57BL/6J: 75% (68%e88%) for controls vs 71% (65%e83%)
for smoke-exposed mice; p¼0.44) (figure 5). In contrast, the
expression of Ku70 in the bronchial epithelial cells was not
statistically significantly different between controls and smokeexposed mice in either A/J or C57BL/6J strains (data not shown).
Lung cancer
Healthy smoker
COPD
A
B
-H2AFX
-H2AFX
D
C
Phospho-DNA-PKcs
only in the A/J strains, susceptible to tobacco-smoke induced
lung cancer, and this effect is prevented by the treatment with
the antioxidant NAC. Finally, in vitro studies demonstrated that
in bronchial epithelial cells H2O2 can decrease Ku86, but not
Ku70, expression, and Ku86 knockdown demonstrates a significant modulation of AP levels by preventing restoration of levels
to baseline.
A previous study demonstrated a reduction of DNA-PK
activity in peripheral blood mononuclear cells (PBMCs) obtained
from patients with non-small cell lung cancer (NSCLC)
compared with those without lung cancer.18 The same study
A
also demonstrated that the DNA-PK activity in PBMCs reflected
that seen in bronchial epithelial cells.18 This reduced DNA-PK
activity may be linked to the decreased Ku86 expression reported
here. Several studies have already shown that despite leucocytes
not being the direct target of tobacco carcinogens, the level of
DNA damage in PBMCs correlates with tobacco smokinginduced DNA damage in human lung tissues.19 20 This is in
keeping with the concept of COPD having a major systemic
component.21
The absence of significant differences in bronchiolar expression of phospho-DNA-PKcs in this study may suggest that
D
C57BL/6J control
B
C
Phospho-DNA-PKcs
C57BL/6J smoke
Ku86
A/J control
E
F
A/J smoke
Figure 5 Photomicrographs showing the bronchial epithelium from a control (A, D) and a cigarette-smoke exposed (B, E) mouse respectively of the
C57BL/6J (A, B) and A/J (D, E) strains immunostained for identification of Ku86+cells (brown). Histograms represent the median (IQR) of the percentage
of bronchial epithelial cells with nuclear immunostaining for Ku86 on total number of bronchial epithelial cells in controls (n¼6 and 8 for C57BL/6J and
A/J strains respectively) and cigarette-smoke exposed mice (n¼7 and 11 for C57BL/6J and A/J strains respectively) of C57BL/6J (C) and A/J (F) strains.
Thorax 2011;66:521e527. doi:10.1136/thx.2010.156448
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Figure 4 Photomicrographs showing
the bronchial epithelium from a healthy
smoker with normal lung function (A,
C) and a patient with mild to moderate
chronic obstructive pulmonary disease
(COPD) (B, D) immunostained for
identification of g-H2AFX+(A, B) and
phospho-DNA-PKcs+(C, D) cells
(brown). Results are representative of
those from 13 healthy smokers with
normal lung function and 14 patients
with mild to moderate COPD.
Lung cancer
A
B
0.3
0.4
p=0.0112
0.2
Ku70 binding
(OD 45
50nm)
Ku86 binding
(OD 45
50nm)
0.6
00
0.0
1.2
0.1
00
0.0
H2O2
Control
E
200
H2O2
Control cells
ku86 siRNA
*p=0.0286
1.0
0.8
% AP
C
Ku86/GAPDH mR
RNA
Control
p=0.10
0.2
0.6
0.4
*p=0.0286
150
§p=0.0286
*p=0.0286
100
p=0.0313
*p=0.0286
p
0.2
50
0.0
Wt
Ku86 KD
0
D
0
0
4
4
12
12
Time H2O2 (100 M) exposure (hrs)
Ku86
Wt
Ku86 KD
DNA-PKcs autophosphorylation, which in vitro correlates
with loss of protein kinase activity and dissociation of the
DNA-PKcs-Ku complex,9 may not be an important mechanism
in COPD lungs.
COPD is an independent risk factor for lung carcinoma,
particularly for squamous cell carcinoma, among smokers.4 It is
also associated with increased oxidative stress which can cause
DNA damage.6 Lung cancer is up to five times more likely to
occur in smokers with airflow obstruction than those with
normal lung function5 and lung cancer is one of the commonest
causes of death among patients with COPD.22 The authors
hypothesise that a defective DNA repair mechanism in smokers
with COPD in response to oxidative stress will contribute to the
increased incidence of lung carcinoma in these patients.
Recent evidence has determined a critical role for acetylation
of histone H3 on lysine 56 (AcH3K56) in DNA damage and
repair.23 24 Furthermore, deacetylation of AcH3K56 is controlled
by histone deacetylases (HDACs) 1 and 2 and loss of HDAC1
and/or 2 can increase susceptibility to UVA irradiation.23 Further
evidence for a role of HDAC-containing complexes in the regulation of DNA damage and repair is seen in Caenorhabditis elegans
in response to ionising radiation25 and there also appears to be
an important role for sirtuins in deacetylating H3K56 in
mammalian cells.24 26 This suggests that in diseases such as
COPD where there is a decrease in both HDAC227 and SIRT128
expression/activity in the lungs there will be less protection
against DNA strand break and repair induced by environmental
factors, increasing the potential for somatic mutations and
increasing the risk of lung cancer.
Ku86-deficient mice have early senescence and mortality but
do not show an increased incidence of lung cancer.29 However,
the effects of long-term tobacco smoke exposure on these
animals have not been investigated. Further studies using
a conditional Ku86 knockout chronically exposed to tobacco
smoke will aid the understanding of the role of decreased Ku86
in facilitating lung cancer.
In conclusion, these data suggest that a reduced expression of
the DNA repair protein Ku86 in the nucleus of the bronchiolar
526
epithelial cells from patients with COPD can contribute to their
increased risk of developing lung carcinoma.
Funding The study was supported by grants from Fondazione Carife (Ferrara, Italy;
http://www.fondazionecarife.it/) and Associazione per la Ricerca e la Cura dell’Asma
(ARCA, Padova, Italy) and the Wellcome Trust.
Competing interests None.
Ethics approval This study was conducted with the approval of the Ethics
committees of the University Hospital of Ferrara and the Royal Brompton Hospital.
Contributors GC, PJB, KFC and AP contributed to the recruitment of the human
subjects of the study. GV and CC prepared the animal model. GC, PC, KI, EJ, LT
performed the experiments of molecular biology. They were all blinded on the clinical
characterisation of the human subjects and on the experimental conditions of the
animal model. IMA analysed and interpreted the clinical and experimental data. GC
and AP obtained funding. GC and IAM drafted the manuscript which was critically
revised for important intellectual contents by all other authors.
Provenance and peer review Not commissioned; externally peer reviewed.
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Figure 6 Graphical representation of
Ku86 (A) and Ku70 (B) expression,
measured by immunoenzymatic
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primary human bronchial epithelial cells
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