Abstract
Background: Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Based on this hypothesis, the relationship between APE1 polymorphism, smoking and the risk of lung cancer was explored. Materials and Methods: The distribution of the APE1 Asp148Glu polymorphisms in 98 lung cancer patients and 67 healthy individuals were compared using PCR-RFLP analysis. Results: Individuals carrying the APE1 Asp148Glu heterozygous and homozygous variant genotype had a 3.23-fold increased risk of lung cancer compared with these carrying the wild-type (Asp/Asp) genotype (p<0.0001), and those carrying the 148Glu homozygous genotype had a 3.17-fold increased risk (p=0.023). When stratified by smoking status, carriers of the Glu allele of APE1 were at a statistically increased risk of lung cancer among smokers (p=0.001). Conclusion: A statistically significant interaction of current smoking status with APE1 Asp148Glu polymorphism was found. These results suggest that the presence of one or two APE1 Glu allele was associated with the risk of developing lung cancer.
Oxidative DNA damage and DNA repair mediate the development of several human pathologies, including cancer. Lung cancer is the most frequent cancer type and approximately 80% of cases are caused by tobacco smoking in the Caucasian population (1, 2). Tobacco smoke contains many carcinogens and reactive oxygen species that produce bulky adducts, cross-links, DNA damage and DNA strand breaks requiring repair through multiple pathways (3). The major pathway for oxidative DNA damage repair is base excision repair (BER). Functional assays performed in blood leukocytes of cancer patients and matched controls show that specific BER pathways are decreased in cancer patients, and may be risk factors (1). The apurinic/apyrimidinic (AP) endonuclease (APE1) plays a central role in the BER pathway, which operates on small lesions such as oxidized or reduced bases, fragmented or nonbulky adducts, or those produced by methylating agents (4, 5) Functional studies on Asp148Glu polymorphism of APE1 have shown that the Glu variant may have altered endonuclease and DNA-binding activity and reduced ability to communicate with other BER proteins (6). Some studies found a lack of association of this polymorphism with lung cancer risk (7, 8). Cigarette smoking may induce DNA damage (9) and individuals with a reduced capacity for DNA repair would be expected to have more carcinogen DNA adducts in their tissue (10). Lung cancer patients may have a lower capacity for DNA repair when compared with healthy individuals and this may modulate the risk of lung cancer associated with smoking (11-13). Polymorphisms of DNA repair genes that impair their function should theoretically predispose an individual to development of tobacco-related cancer such as that in the lung (13). Polymorphisms which alter DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk (4). Based on this hypothesis, we wished to explore the relationship between APE1 polymorphisms, smoking and the risk of lung cancer.
Materials and Methods
Study participants. Ninety-eight lung cancer patients and 67 healthy individuals took part in this study. Lung cancer patients were recruited from the Yedikule Teaching Hospital for Chest Diseases and Thoracic Surgery. They were all newly diagnosed with histopathologically confirmed primary lung cancer and surgically treated, before any radiotherapy and chemotherapy. Cases included 48 adenocarcinomas, 8 squamous cell carcinomas and 42 other tumors with a variety of different pathologies (including large cell and small cell carcinomas, carcinoids and mixed types).
Sixty-seven healthy persons without any malignancy were selected for the control group that comprised only individuals with a negative family history of cancer.
Tobacco exposure history. Smoking status at interview was classified into three categories: current smokers (individuals who either were currently smoking or had quit smoking within the previous 1 year); never smokers [those who had smoked 100 cigarettes in their lifetime (before diagnosis for cases)]; former smokers (those who had quit smoking 1 year and more previously).
Isolation of DNA. Blood specimens were collected in tubes containing EDTA and DNA was prepared from leukocyte pellet by SDS lysis, ammonium acetate extraction and ethanol precipitation (14).
Identification of Polymorphisms of APE Asp148Glu gene. For APE1 genotyping (15), the polymorphism in APE1, exon 5, T/G, 148 Asp/Glu, was determined using the following primers (Fermentas, Lithuania) forward, 5′-CTGTTTCATTTCTATAGGCTA-3′; reverse, 5′-AGGAACTTGCGAAAGGCTTC-3′. Approximately 100 ng genomic DNA in a total volume of 50 μl was amplified by PCR. The reaction mixture consisted of PCR buffer (150 mM Tris-HCl, pH 8.0, 500 mM KCl), 2.5 mM MgCl2, 0.2 mM each dNTP, 0.2 μM each primer, and 1 U Taq polymerase (Fermentas, Lithuania). PCR conditions were 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 52°C for 45 s, 72°C for 45 s, and a final elongation step at 72°C for 5 min. The 164-bp PCR product was digested with FspBI-MaeI restriction enzyme (Fermentas, Lithuania) at 37°C for 6 h. The restricted products of APE1 codon 148 Asp/Asp, Asp/Glu, and Glu/Glu genotypes are represented by band sizes of 164, 164/144/20, and 144/20 bp, respectively.
Statistical analysis. All statistical analyses were carried out using SPSS version 7.5 for Windows (SPSS Inc, Chicago, IL, USA). Numerical values were analysed by Student's t-test. Differences in characteristics between lung cancer patients and controls were assessed with the chi-square test, as well as disparities in genotype and allele frequencies. The frequencies of APE1 alleleles were estimated by gene counting methods. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated to estimate the risk for lung cancer. The threshold for significance was p<0.05.
Results
As shown in Table I, the analysis included 98 cases and 67 controls. There was no statistically significant difference in mean age and gender distribution between cases and controls. However, as expected, more smokers were represented in the cases as compared with the controls (78.6% versus 59.7%; p=0.009, OR 2.47, 95% CI=1.24-4.91).
Table II shows the APE1 genotype frequencies among cases and controls. The APE1 148Glu allele frequencies for controls and cases were 0.20 and 0.41 respectively and the genotype distribution of cases differed significantly from that of controls (p=0.001). Cases were more frequently homozygous for the 148Glu allele (Glu/Glu) of APE1 (p=0.023, χ2 test) and more were carriers of the 148Glu allele of APE1 (p<0.0001, χ2 test) (Table II).
Characteristics of cases and controls.
Individuals carrying the APE1 Asp148Glu heterozygote or homozygous variant genotype (Asp/Glu+ Glu/Glu) had a 3.23-fold increased risk of lung cancer compared with the wild-type genotype (Asp/Asp) (OR=3.23, 95% CI=1.68-6.19, p<0.0001) and those carrying the 148Glu homozygous genotype had a 3.17-fold increased risk (OR=3.17, 95% CI=1.12-8.95, p=0.023) (Table II).
When stratified by smoking status, carriers of the Glu allele of APE1 were at a non-statistically increased risk of lung cancer among never smokers, but at a statistically increased risk of lung cancer among smokers (Asp/Asp versus Glu/Glu + Asp/Glu, OR=3.86, 95% CI=1.70-8.75, p=0.001) (Table III).
Discussion
DNA is vulnerable to injury due to endogenous and/or exogenous exposure such as exposure to carcinogens in tobacco smoke (16). DNA repair systems play an important role in maintaining the integrity of the genome. Deficiencies in this defense system are thought to contribute to the development of cancer. Reduced DNA repair capacity was associated with increased risk of cancer and genetic variation in DNA repair genes may influence DNA repair capacity; consequently, DNA repair genetic polymorphisms may affect cancer susceptibility (17).
BER genes play a key role by removing DNA damage from oxidation, deamination, and ring fragmentation (18) and exposure to tobacco smoking induces oxidative damage by generation of reactive oxygen species (19). APE1 is the rate-limiting enzyme in the BER pathway (20). Although the APE1 Asp148Glu polymorphism does not result in reduced endonuclease activity (7), individuals with the Glu allele may have higher sensitivity to ionizing radiation (15). Several polymorphisms in DNA repair genes and their association with lung cancer and/or DNA repair capacity have been studied. In some studies, no associations between the APE1 Asp148Glu polymorphism and lung cancer risk have been found (7, 21, 22) but others found significant association (23).
Distribution of APE1 Asp148Glu genotypes in patients with lung cancer and controls.
We investigated the association of APE1 Asp148Glu polymorphism with lung cancer risk in a Turkish population.
In this study, the allele frequency of APE1 codon 148 Glu for controls and cases was 0.20 and 0.41 respectively. The estimated allele frequency in our study differs from reported allele frequencies in other populations (4, 6, 15). The differences in allele frequencies detected among these studies might be due to heterogeneity of study populations, ethnic variation and different sample sizes.
We observed a significant association for lung cancer risk with the Asp148Glu heterozygous and with the homozygous variant genotype as reported by De Ruyck et al. (23), where they showed an increased lung cancer risk with the Asp/Glu genotype in Caucasians.
The risk for lung cancer among smokers is thought to increase with cumulative tobacco exposure (4, 24) and genetic susceptibility to lung cancer may depend on the level of exposure to tobacco smoke (4, 25, 26). Ito et al. (4) reported a significant interaction between APE1 148Glu genotype and current smoking. They reported an increased lung risk (OR=2.67, 95% CI=1.00-7.68) associated with the Glu/Glu genotype among light smokers. Misra et al. (7) reported Glu allele frequency of 0.52 for APE1 codon 148 among male smokers. A Chinese study reported an association between the APE1 148 Glu allele and increased lung cancer risk among heavily smoking men (13). Therefore, we examined the relationships between polymorphisms of APE1 Asp148Glu, smoking and the risk of lung cancer. We chose to use smoking status as a measure of smoking history. We found a non-statistically increased risk of lung cancer associated with the GluGlu/AspGlu genotype of APE1 among never smokers, while smokers had about a 3.9-fold increased risk for lung cancer. The allele frequency of APE1 codon 148 Glu among ever smokers was 62.3% in our lung cancer patients. We suggest that APE1 Asp148Glu might modify the risk of lung cancer attributable to cigarette smoking exposure.
Genotypes of APE1 Asp148Glu and lung cancer risk, stratified by smoking status.
The exact mechanism of how cigarette smoking changes the DNA repair capacity by each genotype of these DNA repair polymorphisms is unknown. One possible mechanism is that at high levels of exposure, the DNA repair capacity is saturated, even in individuals having higher repair capacity (25, 26).
Although the present case-control study is relatively small in sample size, the results show clearly that the APE1 Asp148Glu polymorphism may contribute to the genetic susceptibility for lung cancer. Further studies with a larger sample and more complete measures of tobacco exposure are needed to clarify the gene-smoking interaction.
- Received December 19, 2008.
- Revision received March 17, 2009.
- Accepted April 4, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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