Carcinogenesis vol.21 no.2 pp.167–172, 2000
The effect of hOGG1 and glutathione peroxidase I genotypes and
3p chromosomal loss on 8-hydroxydeoxyguanosine levels in lung
cancer
L.J.Hardie2, J.A.Briggs, L.A.Davidson1, J.M.Allan,
R.F.G.J.King, G.I.Williams and C.P.Wild
Molecular Epidemiology Unit, School of Medicine, University of Leeds and
1Institute of Pathology, Algernon Firth Building, Leeds LS2 9JT, UK
2To
whom correspondence should be addressed
Email: [email protected]
Polymorphic genes for the peroxide scavenger glutathione peroxidase I (GPX1) and 8-hydroxydeoxyguanosine
(8-OHdG) DNA glycosylase/apurinic (AP) lyase (hOGG1)
map to loci on chromosome 3p which are subject to frequent
loss of heterozygosity (LOH) in lung tumours. Levels of
the pro-mutagenic, oxidative DNA lesion 8-OHdG, were
measured in 37 paired normal and tumorous lung specimens using HPLC with electrochemical detection. Lung
tumours were also analysed for 3p LOH by fluorescent
PCR with Genescan analysis. No significant difference was
observed between 8-OHdG levels in tumour [7.7 ⍨ 6.7
(mean ⍨ SE) 8-OHdG/106 2⬘-deoxyguanosine (dG)] and
normal (8.1 ⍨ 8.8 8-OHdG/106 dG) lung tissue. Adduct
levels in normal lung tissue DNA were not associated with
constitutive hOGG1 genotype although there was a trend
towards lower 8-OHdG levels in individuals possessing the
ALA6 GPX1 polymorphism. Lung tumours exhibiting 3p
LOH (40%) contained higher levels of 8-OHdG adducts
(10.9 ⍨ 2.6 8-OHdG/106 dG) (P ⍧ 0.05) and lower GPX1
enzyme activity [45.5 nmol glutathione (GSH)/min/mg]
(P ⍧ 0.09) when compared with tumours without LOH at
these sites (5.55 ⍨ 0.87 8-OHdG/106 dG and 63.6 nmol
GSH/min/mg, respectively). In conclusion, tumours with
3p LOH at loci associated with hOGG1 and GPX1 appear
to have compromised oxidative defence mechanisms as
measured by reduced GPX1 enzyme activity and elevated
8-OHdG levels and this may affect the prognosis of lung
cancer patients.
Introduction
Lung cancer accounts for 18% of the global cancer burden in
men and over 1 000 000 new cases of this disease were
reported in 1990 (1). Cigarette smoke is implicated in the
development of 90% of these tumours (1), through exposure
to carcinogens such as benzo[a]pyrene, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone and reactive oxygen species (ROS)
generated by compounds including catechol and hydroquinone
(2). At least 35 different oxidative base modifications have
been reported when ROS react with DNA (3–7). One of the
most common lesions formed is the pro-mutagenic DNA
Abbreviations: AP, apurinic; dG, 2⬘-deoxyguanosine; GPX1, glutathione
peroxidase I; GSH, glutathione; HPLC-ECD, high pressure liquid chromatography with electrochemical detection; 8-OHdG, 8-hydroxydeoxyguanosine;
LOH, loss of heterozygosity; RFLP, restriction fragment length polymorphism;
ROS, reactive oxygen species.
© Oxford University Press
lesion, 8-hydroxydeoxyguanosine (8-OHdG) (6,8). Levels of
this adduct are reported to be raised in the peripheral leukocytes
and lung tissue of smokers compared with non-smokers (2,9).
Several in vivo models have demonstrated that 8-OHdG
mispairs preferentially with adenine during replication,
inducing GC→TA transversions (5,8,10).
In view of its abundance and mutagenicity, a number of
defence mechanisms operate to minimize 8-OHdG accumulation within the genome. Primary defence mechanisms include
antioxidants and enzymes such as glutathione peroxidase
(8,11). Glutathione peroxidases reduce organic peroxides and
hydrogen peroxides through the coupled oxidation of reduced
glutathione (GSH). Glutathione peroxidase I (GPX1) is the
major cytosolic form of this enzyme, but other isozymes are
found in the plasma and phospholipid membranes (12).
Once formed, 8-OHdG lesions are subject to DNA repair
primarily through the base excision repair pathway (13). A
key component of this pathway is a specific DNA glycosylase/
apurinic (AP) lyase which catalyses the release of 8-OHdG
and the cleavage of DNA at the AP site (14). Inactivation
of this 8-OHdG glycosylase generates a mutator phenotype
characterized by GC→TA transversions in Escherichia coli
and yeast (15,16). Recently the human homologue of this
gene, hOGG1 has been identified (17–19). The gene product
constitutes an 8-OHdG DNA glycosylase/AP lyase which
exhibits greatest specificity and activity for 8-OHdG:dC and is
completely inactive against 8-oxodeoxyguanosine:dA (17,19).
hOGG1 has also been shown to excise 2,6-diamino-4-hydroxy5-formamidopyrimidine residues in a similar manner to its
yeast homologue (20–22).
The level of 8-OHdG measured in a tissue at any one time
is an integration of a number of parameters including the level
of ROS, tissue redox status, cellular antioxidant defence
mechanisms and DNA repair systems. The effectiveness of
these latter systems may be subject to modulation by gene
polymorphisms and gene dosage effects. A number of hOGG1
polymorphisms have been described in Japanese populations,
and a Ser/Cys (0.57/0.43) substitution in exon 7 is highly
prevalent (23–25). Preliminary evidence from an E.coli complementation assay suggests that the hOGG1-Cys isoform
exhibits reduced 8-OHdG repair activity (23) and may play a
role in increasing susceptibility to squamous cell carcinoma
of the lung (25). Polymorphisms in GPX1 are characterized
by a variable polyalanine repeat and the six-alanine repeat
form (ALA6) also contains a proline→leucine substitution at
codon 198 towards the C-terminus (12). Thus, constitutive
genotype may play a significant role in determining 8-OHdG
levels within tissue DNA.
Finally, both GPX1 and hOGG1 locate to regions of chromosome 3p (3p21 and 3p25/26, respectively) which are subject
to frequent and early loss of heterozygosity (LOH) during
lung cancer development (12,18,26). Deletions at these sites
may compromise antioxidant and 8-OHdG repair mechanisms
via gene dosage effects, resulting in the accumulation of 8OHdG within dysplastic and neoplastic tissue.
167
L.J.Hardie et al.
The aims of this study were to examine the effect of GPX1
and hOGG1 genotype on lung 8-OHdG levels and to establish
whether 3p LOH events modulate 8-OHdG levels in lung
tumour tissue.
Materials and methods
Tissue collection
Resected lobectomy and pneumonectomy specimens were collected on ice at
surgery from untreated primary non-small cell lung cancer patients (n ⫽ 37)
at Leeds General Infirmary (LGI). Paired tumour and uninvolved peripheral
lung tissue (designated normal), which were surplus to diagnostic requirements,
were dissected by a consultant pathologist. Specimens were cut into pieces
(200–400 mg) and fixed either in formalin for histology or frozen immediately
at –80°C for molecular and biochemical analyses. Smoking status from
patients was limited to self-reported information, but all patients were either
current or former smokers. Most squamous cell carcinoma samples (82%)
collected were from male patients, as opposed to adenocarcinomas, which
were predominantly (75%) obtained from female patients.
Histology
Histopathological diagnosis was performed by hospital pathologists according
to the WHO lung carcinoma classification. To verify that tumorous specimens
contained ⬎50% tumour cells and that uninvolved tissue contained ⬍10%
contaminating tumour material, paraffin-embedded sections (5 µm) from lung
specimens were stained with haematoxylin and eosin and examined by light
microscopy. Normal and tumorous lung tissue was microdissected where
appropriate from sequential tissue sections (5 µm) and genomic DNA extracted
using proteinase K digestion followed by phenol/chloroform extraction as
described previously (27).
Genotyping and LOH analysis
DNA was subject to PCR using fluorescent primers directed against the
following chromosome 3p markers: GPX1, FAM 5⬘-GAAACTGCCTGTGCCACGTGACC-3⬘ and 5⬘-CGAGAAGGCATACACCGACTGGGC-3⬘;
hOGG1, FAM 5⬘-ACTAGTCTCACCAGCCGTGAC-3⬘ and 5⬘-TGGCCTTTGAGGTAGTCACAG-3⬘; D3S1259, FAM 5⬘-GCTGGACTATATTTGAAACTCATT-3⬘ and 5⬘-TTTCAGTGAGCCAAGATCGT-3⬘; D3S1351, TET
5⬘-ACAGAACCAGAACCAATAGG-3⬘ and 5⬘-AGCCTCCATAACTGCATGAA-3⬘.
Aliquots of 0.5 µl of template DNA were added to a PCR mix containing
0.5 U AmpliTaq Gold (Perkin-Elmer Cetus, Norwalk, CT), 10 pmol each
primer, 2.5 µl 10⫻ Taq buffer, 200 µM dNTPs, 1.5 mM MgCl2 and water to
a final volume of 25 µl. Cycling conditions for GPX1, D3S1259 and DS1351
were: 95°C for 15 min; 30 cycles of 1 min at 95°C, 1 min at 56°C, 1 min at
72°C; followed by an extension step for 10 min at 72°C. The annealing
temperature was modified to 50°C for the hOGG1 PCR. For every PCR
experiment, water served as a negative template control.
PCR products were separated on a 6% denaturing acrylamide gel and
analysed using a 373A automated DNA sequencer with Genescan analysis
(27). LOH was defined as a change in allele ratio of ⬍0.6 between normal
and matched tumour tissue DNA, which was reproduced on three independent
PCR occasions for each pair of DNA extractions.
hOGG1 genotyping analysis
Individuals were genotyped for the presence of the serine or cysteine
polymorphism at codon 326 of the hOGG1 gene by virtue of a Fnu4HI
restriction site. The hOGG1 PCR product (5 µl) (see above) was incubated
with 7 U of Fnu4HI overnight at 37°C. Fnu4HI cuts cysteine alleles generating
123/124 and 169/170 bp fragments from the primary 293 bp amplicon.
Products were visualized on a 2.0% agarose gel and compared with known
genotype standards (confirmed by direct sequencing). In addition to the lung
cancer cases, leukocytes from 47 healthy individuals were sampled from the
general population to estimate the prevalence of these polymorphisms within
Caucasians.
8-OHdG analysis
High pressure liquid chromatography with Coularray electrochemical detection
(HPLC-ECD) (ESA Inc., Chelmsford, MA) is considered a valid technique
for 8-OHdG adduct analysis, although considerable care must be exercised to
avoid artefactual 8-OHdG formation during sample collection and preparation.
Thus, a phenol-free method with ethanol precipitation was used to extract
tissue DNA in this study. Briefly, tissue (100 mg) from each specimen was
finely chopped on ice in 1 ml lysis buffer [800 mM guanidine–HCl, 30 mM
Tris–HCl (pH 8.0), 30 mM EDTA (pH 8.0), 5% Tween 20, 0.5% Triton
X-100]. Each sample was then homogenized on ice in 9.5 ml lysis buffer
using an Ultra-Turrax T25 (2⫻10 s bursts at 8000 r.p.m.). DNA was extracted
168
from the homogenized solution using G100 columns (Qiagen Ltd, Crawley,
UK) according to the manufacturer’s instructions except that DNA was
precipitated with absolute ethanol. DNA was washed twice with 70% ethanol
and resuspended in 100 µl 40 mM Tris–HCl (pH 8.5). Samples were hydrolysed
by the addition of DNase (7.5 U), phosphodiesterases I (0.004 U) and II
(0.003 U), alkaline phosphatase (1.5 U) and 20 mM MgCl2 for 2 h at 37°C
before analysis by HPLC-ECD. Nucleosides were separated on a 150⫻4.6 mm,
3 µm particle size C-18 reverse phase column (Chrompack, Millharbour, UK)
at 25°C using 100 mM sodium acetate buffer with 5% methanol (pH 5.2 with
H3PO4) at 1 ml/min. The electrochemical detector was set at oxidation
potentials of ⫹450 and ⫹850 mV for 8-OHdG and 2⬘-deoxyguanosine (dG)
analyses, respectively. Preceding electrodes were set at ⫹230 and ⫹800 mV,
respectively, to screen out other electrochemically active compounds. dG
eluted at 7.5 min and 8-OHdG at 11 min. 8-OHdG and dG levels were
quantitated from samples by comparing the peak areas obtained against
8-OHdG (ESA Inc.) and dG (Sigma, Poole, UK) standard curves generated
on the optimized electrode channels.
During the development of this technique, an association between homogenization time and 8-OHdG level was observed in our laboratory. Specifically,
when homogenization times were raised from 20 to 40 and 60 s (in 10 s
bursts), adduct levels rose by 34 and 71%, respectively. In view of this, tissue
homogenization times were standardized to exactly 2⫻10 s bursts throughout
this study. The coefficient of variation for replicate injections on the HPLCECD ranged between 1.91 and 5.96% and for duplicate independent tissue
extractions and analyses performed several weeks apart was 26%. Data
presented are from the primary extraction, where there was sufficient paired
normal and tumour tissue to permit parallel extractions and analyses.
GPX1 enzyme activity
Tumour tissue samples were washed thoroughly in ice-cold buffer [0.15 M
Tris–HCl (pH 7.5), 1 mM EDTA, 2 mM 2-mercaptoethanol] to remove
contaminating blood, before homogenization using an Ultra-Turrax T25
(2⫻10 s bursts at 13 500 r.p.m.). Homogenates were centrifuged initially at
10 000 g for 15 min at 4°C, before these supernatants were centrifuged
(105 000 g for 60 min at 4°C) and the resulting cytosolic fractions used for
enzyme and protein analyses. Glutathione peroxidase activity was measured
according to the method of Paglia and Valentine (28) where hydrogen peroxide
was used as substrate at a final concentration of 0.25 mM. The reaction mix
(1 ml) contained 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA,
1.5 mM sodium azide, 1 mM GSH, 0.16 mM NADPH, 0.33 U/ml glutathione
reductase and 200 µl of cytosolic fraction. After a 5 min preincubation, the
reaction was initiated by the addition of hydrogen peroxide. The rate of
reaction was determined at 20°C by measuring the decrease in absorbance at
340 nm. Cytosolic protein concentrations were measured using the Bio-Rad
protein assay (Bio-Rad, Hemel Hempstead, UK). Specific activity is given as
nmol GSH oxidized/min/mg cytosolic protein.
Statistics
Statistical analyses were performed using the Mann–Whitney U-test and
Kruskall–Wallis non-parametric ANOVA.
Results
The mean 8-OHdG levels measured in normal and tumorous
lung tissue from 20 adenocarcinoma and 16 squamous cell
carcinoma cases are shown in Figure 1. Adduct levels ranged
from 0.5 to 47 8-OHdG/106 dG in normal and 0.3 to 34
8-OHdG/106 dG in tumour DNA in this study. The majority
(80%) of normal and tumour samples contained ⬍10 8-OHdG/
106 dG. There were no significant differences observed in
8-OHdG levels either between normal and tumorous tissue or
across different tumour types (Figure 1). Furthermore, nodal
involvement was not related to tumour 8-OHdG levels. In
view of this, data from adenocarcinoma and squamous cell
carcinoma 8-OHdG levels were pooled for further analysis.
Only limited qualitative information was available on the
smoking status of these lung cancer cases, except that all cases
were known to be current or former smokers. Therefore adduct
levels could not be correlated quantitatively to smoking status.
The relationship between 8-OHdG level and possession of
the GPX1 ALA6 allele is shown in Figure 2. Although there
was a trend towards reduced 8-OHdG levels in the presence
Chromosome 3p and 8-OHdG levels in lung cancer
Fig. 1. The relationship between tumour histopathology and 8-OHdG levels
in normal and matched tumorous lung specimens. Data presented are mean
8-OHdG values/106 dG bases ⫾ SE.
Fig. 2. The relationship between possession of the GPX1 ALA6 allele and
8-OHdG levels in normal lung tissue. Data presented are mean 8-OHdG
values/106 dG bases ⫾ SE.
of the ALA6 allele this result was not statistically significant
(P ⫽ 0.421).
We also examined the association between hOGG1 genotype
and adduct level. A typical hOGG1 restriction fragment length
polymorphism (RFLP) result is shown in Figure 3A. It was
noted that the hOGG-Ser allele was more prevalent (0.76) in
this control Caucasian population compared with previous
reports on Japanese populations (0.59) (23,25). Furthermore,
hOGG1 allele frequencies (Ser/Ser:Cys/Ser:Cys/Cys) did
not vary significantly between the lung cancer cases
(0.53:0.41:0.06) and the general Caucasian population
(0.56:0.40:0.04) in this study, indicative that a strong hOGG1
allele selection pressure was not operating in the lung cancer
cases. This is consistent with the fact that levels of 8-OHdG
in normal lung DNA did not vary with hOGG1 genotype
(P ⫽ 0.472) (Figure 3B).
LOH at one or more markers on chromosome 3p was
observed in 40% of lung tumours. The mean 8-OHdG level
in these tumours was double that observed in tumours which
showed no evidence of LOH (P ⫽ 0.05) (Figure 4A). Furthermore, the GPX activity of these tumours was reduced by one
third when compared with tumours exhibiting no 3p LOH
(Figure 4B), although this was not significant (P ⫽ 0.09).
Fig. 3. (A) RFLP analysis of the codon 326 hOGG1 Cys/Ser polymorphism.
The primary amplification generates a 293 bp amplicon (lane 2). Fnu4H1
specifically cuts cysteine alleles generating 123/124 and 170/169 bp
products. Lane 1, 50 bp ladder; lane 2, primary amplification product;
lane 3, serine homozygote control digest; lane 4, cysteine/serine
heterozygote control digest; lane 5, cysteine homozygote control digest; lane
6, serine homozygote; lane 7, cysteine/serine heterozygote lung specimens.
(B) The relationship between hOGG1 genotype and 8-OHdG levels in
normal lung tissue. Data presented are mean 8-OHdG values/106 dG bases
⫾ SE.
Discussion
A role for specific gene polymorphisms in relation to susceptibility to lung cancer has been highlighted for a number of
genes, including myeloperoxidase (29) and glutathione
S-transferases (30,31). However, markers of biological effects
on the disease pathway are rarely used to demonstrate a
mechanistic link between genotype and disease. Such a link
would contribute to establishing a causal association between
the two. In this study, the significance of GPX1 and hOGG1
polymorphisms in protecting against oxidative stress and
potentially in the aetiopathogenesis of lung cancer was
addressed by examining their effect on levels of the promutagenic lesion 8-OHdG in lung tissue.
Since guanine is the most readily oxidized DNA base, the
use of 8-OHdG as a dosimeter for oxidative stress is the
subject of debate in the light of susceptibility to artefactual
formation during sample processing and measurement (32–
34). In particular, phenol, light, heat, methanol and silylation
steps in GC-EIMS have all been highlighted as potential
sources of artefactual 8-OHdG formation. Thus, rigorous
procedures were employed in our study to minimize and
169
L.J.Hardie et al.
Fig. 4. (A) The association between 3p chromosomal loss and 8-OHdG
levels in lung tumours. Data presented are mean 8-OHdG values/106 dG
bases ⫾ SE. (B) The effect of LOH on chromosome 3p on lung tumour
glutathione peroxidase activity. Data presented are mean nmol GSH
oxidized/min/mg cytosolic protein ⫾ SE.
control for sources of experimental error, which included: (i)
the transit time between surgery and sample storage was
minimized; (ii) tissue dissection was performed by a consultant
pathologist in order to optimize and standardize the quality of
tissue specimens collected; (iii) all tissue was processed using
a standardized laboratory protocol with carefully timed tissue
homogenizations, an additional factor which we found could
lead to an increase in adduct levels (see Materials and methods).
Levels of adduct which were measured in this study are
consistent with those reported by Inoue et al. (35), who
described a mean 8-OHdG level of 5.2 8-OHdG/106 dG in the
peripheral lung of lung cancer cases compared with 8.1
8-OHdG/106 dG in the current study. There was somewhat
more inter-individual variability observed in the present study,
but 80% of samples fell within the range (i.e. ⬍10 adducts/
106 dG) reported by Inoue et al. (35). Neither study found a
difference in 8-OHdG levels between non-tumorous lung tissue
from adenocarcinoma and squamous cell carcinoma patients.
These findings also concur with an earlier study (2) on the
central lung, where 8-OHdG levels did not differ between
normal and tumorous tissue nor across different lung tumour
types.
Preliminary findings presented here indicate a trend towards
lower 8-OHdG levels in normal lung tissue possessing the
ALA6 allele of the GPX1 gene, implying a possible functional
change in enzyme activity as a result of sequence characteristics. However, further studies are required to define the role
of this polymorphism in relation to the function of GPX1 and
its role in protection against oxidative DNA damage.
The hOGG1-Ser and -Cys polymorphisms were initially
identified by Kohno et al. (23). Based upon in vitro evidence
utilizing an E.coli complementation assay, the hOGG1-Cys
allele appeared to exhibit a poorer 8-OHdG repair activity
170
compared with the hOGG1-Ser isoform. In the current study,
we were unable to find evidence to support a role for the
hOGG1 genotype in determining 8-OHdG levels in nontumorous tissue from a series of 34 lung specimens. This
suggests that differences in 8-OHdG glycosylase activity within
hOGG1 polymorphic variants are insufficient to impact upon
tissue 8-OHdG levels. Alternatively, it may reflect a lack of
resolution regarding adduct analysis in that current methodologies are limited to measurements in total genomic DNA and
cannot be targeted to specific regions, e.g. transcriptionally
active sites. In addition, repair of 8-OHdG may not be wholly
effected by hOGG1. Monden et al. (36) confirmed that OGG1
(type 1a isoform) constitutes the major 8-OHdG glycosylase
activity detectable in human cells. However, other repair
enzymes also demonstrate some 8-OHdG glycosylase activity
in vitro and could contribute to the persistence of 8-OHdG
within tissues (37,38). Perhaps of most significance, Hazra
et al. (38) recently identified OGG2, which preferentially
excises 8-OHdG when paired with G or A. In light of the
substrate specificities outlined for OGG1 and OGG2, the
effects of hOGG1 polymorphisms may be more subtle and
perhaps require discrimination within a base pair context.
Furthermore, chromatin structure and sequence context are
likely to be important factors not only in determining the site
of 8-OHdG adduct formation, but may also affect efficiency
of DNA repair and adduct persistence. This information could
be particularly pertinent in relation to oxidative DNA damage
within target genes where mutation is key to disease initiation
and/or progression.
In principle, one of the limitations in the type of study we
describe here is that tissue specimens were collected from
people undergoing surgery and hence with prevalent disease.
Additionally, it is often a fact that there is incomplete quantitative data available on other environmental exposures, e.g.
smoking, as in this study. Thus, both the presence of disease
and these latter factors could act as confounders of the
relationship between adduct level and genotype. Therefore, for
a polymorphism to cause detectable change in 8-OHdG levels,
the association would have to be relatively strong.
Notwithstanding the caveats outlined above, a global elevation in 8-OHdG levels was observed in tumor tissue which
exhibited LOH within GPX1 or at loci close to hOGG1.
This appears to indicate that oxidative defences are indeed
compromised in lung tumours which exhibit 3p LOH at these
sites and is further supported by the finding that GPX1 enzyme
activity was reduced in these tumours. To date, most studies
on the lung have compared GPX1 activities across normal and
tumorous tissue with somewhat inconclusive results (39,40).
That GPX1 activity is reduced in some but not all tumours
relative to normal tissue may in part reflect 3p LOH events in
these tumours. In the current study, reduced GPX1 enzyme
activity does not appear to completely account for the elevated
8-OHdG levels observed in tumours with 3p LOH, because
the correlation between GPX1 enzyme activity and 8-OHdG
levels in tumours at the individual level was poor (data not
shown). Future studies will address whether hOGG1 activity
is also compromised in tumours exhibiting 3p LOH, although
the involvement of other genes which locate to these regions
of chromosome 3p cannot be precluded.
In conclusion, preliminary results indicate that endogenous
8-OHdG levels in the lung may be lower in individuals
possessing the GPX1 ALA6 polymorphism but are not affected
by the hOGG1 genotypes examined. 3p LOH at chromosomal
Chromosome 3p and 8-OHdG levels in lung cancer
sites associated with lung cancer development appear to
compromise antioxidant defences, including GPX1 enzyme
activity, leading to elevations in 8-OHdG levels within genomic
DNA. It is interesting to speculate that as a consequence of
elevated 8-OHdG levels, tumours with 3p LOH will have a
higher mutation frequency. This may result in heightened
tumour aggressiveness but may also affect responsiveness to
therapeutic agents when compared with tumours which have
not lost these sites on chromosome 3p, as many of these
agents, e.g. γ-irradiation, induce oxidative DNA damage.
Acknowledgements
The authors wish to thank the cardiothoracic surgical staff at Leeds General
Infirmary who were involved with the collection of lung tissue specimens and
Dr Martin Muers in the initiation of this study. Genomic DNA from the
control Caucasian population was kindly provided by Professor P.J.Grant and
Mr M.Stickland from the Molecular Vascular Medicine Unit at the University
of Leeds. This work was supported by a grant from the NHS Special Trustees
at Leeds General Infirmary to L.J.H., C.P.W. and M.M.
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Received August 24, 1999; revised October 11, 1999;
accepted October 12, 1999