Cyclin D1 gene polymorphism and susceptibility to lung cancer in a Chinese population
Shi Qiuling1,
Zheng Yuxin1,3,
Zheng Suhua2,
Xiao Cheng1,
Leng Shuguang1 and
He Fengsheng1
1 Institute of Occupational Health and Poison Control, Chinese Center for Disease Control, Beijing 100050, P. R. China
2 Beijing Institute of Tuberculosis and Chest Tumor, Beijing 100090, P. R. China
3 To whom correspondence should be addressed Email: yxzheng{at}163bj.com
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Abstract
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The objective was to study the relationship between cyclin D1 gene (CCND1) polymorphism and lung cancer in the Chinese population. Blood samples of 182 cases and 185 controls were collected from a hospital based case-control study. PCRSSCP was used to examine the G/A polymorphism in exon 4 of CCND1. The results showed that the frequencies of the CCND1 AA, GA and GG genotypes were 31.3, 46.7 and 22.0% respectively in cases, and 21.1, 53.0 and 25.9 respectively in controls. Adjusted by age (in years), sex and smoking status, multivariate logistic regression analysis showed that the AA genotype was associated with a significantly increased risk (OR = 1.87, 95% CI 1.013.45) for lung cancer. In the stratification analysis, the CCND1 AA variant genotype was associated with increased risk in individuals who were
50 years old (OR = 3.23, 95% CI 1.178.96) and males (OR = 2.46, 95% CI 1.185.10). According to histological types, there was significantly higher frequency of AA genotype in squamous cell lung cancer than that in controls (OR = 2.92, 95% CI 1.078.03). In conclusion, it is suggested that the CCND1 G/A polymorphism is associated with the early onset of lung cancer in men and contributes to susceptibility to lung cancer, especially squamous cell cancer, in this population.
Abbreviations: SSCP, single-strand conformation polymorphism
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Introduction
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Lung cancer is the leading cause of cancer death for men in China (1) and the incidence is increasing worldwide. Although cigarette smoking is accepted as the major cause of lung cancer, only 1015% of heavy smokers ultimately develop lung cancer (2). This implies that genetic variation in sensitivity to carcinogen exposure may play an important role in the etiology of lung cancer.
Recent molecular biological studies have clearly indicated that lung cancer is a disease caused by the accumulation of multiple genetic defects in both tumor suppressor genes and dominant oncogenes (3,4). In addition, cytogenetic studies have shown that lung cancer frequently contains chromosomal abnormalities, as also seen in most other cancers (5). A number of surveillance mechanisms exit in cells to ensure the maintenance of genomic stability against various damage to genome (6). Among them, the G1/S checkpoint arrests the cell cycle to prevent replication of damaged DNA and allow DNA damage to be repaired (7,8). Playing a role in the transition from G1 to S phase of the cell cycle, cyclin D1 has been considered as a key regulator essential for this progress, whose deregulation has been implicated in pathogenesis of several cancers, including lung cancer (9,10).
In 1995, Betticher and colleagues (11) identified a single base polymorphism (G870A) in exon 4 of cyclin D1 gene (CCND1), which increases alternative splicing. The variant nucleotide interferes with splicing from exon 4 to exon 5 because of its unique localization within a conserved splice donor region and the A allele can produce more altered transcripts due to the altered splicing. Although both the normal and the altered transcripts encode a protein that contains amino acids thought to be responsible for the cyclin D1 function, the protein encoded by the altered transcript may have a longer half-life. Therefore, it has been suggested that DNA damage in cells from subjects with the A allele may bypass the G1/S checkpoint more easily than damage in cells not carrying the polymorphism. Recently, studies have shown that subjects with the AA genotype had a higher risk of urinary bladder cancer (12) and squamous cell carcinoma of the head and neck than those with the other two genotypes (13).
Because cyclin D1 plays a critical role in cell cycle control and it is associated with risk of lung cancer, we conducted a hospital-based case-control study of 182 newly diagnosed lung cancer patients and 185 healthy controls to test the hypothesis that the G/A CCND1 polymorphism may modulate individual susceptibility to lung cancer.
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Materials and methods
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Study subjects
Eligible cases consisted of newly diagnosed patients with lung cancer who received treatment at the Beijing Institute of Chest Tumor from September 2001 to March 2002. All patients were diagnosed histologically with specimens obtained from biopsy or surgical resection. Demographic and epidemiological data, including tobacco use, were gathered by a questionnaire completed by trained interviewers and reviewed by a single reviewer during the hospitalization. The healthy controls were recruited from a community in Beijing during a similar time period. These control subjects were first surveyed by means of a short questionnaire to determine their willingness to participate in research studies. Using the same questionnaire as for the cases, the same interview group interviewed eligible subjects and collected the data including age, sex and smoking status.
After informed consent was obtained, a one-time 2 ml of blood sample was obtained from all participants and stored at -70°C for genotype analysis. The study was approved by our Institutional Review Committee.
Genotyping
Genomic DNA was obtained from blood by the routine high salt method (14). The DNA purity and concentration were determined by spectrophotometric measurement of absorbance at 260 and 280 nm.
PCR-based single-strand conformation polymorphism (SSCP) analysis was used to genotype the CCND1 polymorphism in exon 4 (13). For PCRs the primers were 5'-TACTACCGGCTCACACGCTTC-3'(sense), 5'-TTGGCACCAGCCTCGGCATTTC-3'(antisense), which generated a 138 bp fragment. These fragments were amplified in 25 µl of reaction mixture containing -50 ng genomic DNA, 1x PCR buffer (50 mM KCl, 10 mM TrisHCl, pH 9.0 at 25°C and 1% Triton X-100), 1.5 mM MgCl2, 0.2 mM each dATP, dTTP, dCTP and dGTP, 6.25 pmol each primer and 1 U Taq polymerase (Shanghai Sangon Company, China). PCR was performed by incubating the fragments and reaction mixture at 95°C for 5 min, subjecting them to 28 cycles of 94°C for 30 s and 65°C for 30 s and incubating them at 72°C for 1 min, with a final elongation at 72°C for 10 min.
For SSCP analysis, 4 µl PCR product was mixed with 4 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05% bromphenol blue). The mixture was denatured at 95°C for 10 min and then immediately put on ice. Five microliters of the mixture were loaded on a 5% polyacrylamide gel for electrophoresis at 1.5 W for 5 h. After electrophoresis the band shift patterns on gels were visualized by silver staining. Sequencing analysis was used to confirm the genotype. Based on these band shift patterns and sequencing results subjects were divided into three groups for CCND1 genotypes, GG, GA and AA (Figure 1). Furthermore, 10% of the samples genotypes were re-tested for quality control.

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Fig. 1. Representative screen for the CCND1 genotypes by silver-staining SSCP and DNA sequencing analysis. (A) PCRSSCP typing pattern of CCND1 genotypes. Lanes 2 and 6, GG homozygotes; lanes 1 and 4, GA heterozygotes; lanes 3 and 5, AA homozygotes. (B) Results of nucleotide sequence analysis by using anti-sense primer. Arrows indicate the location of the nucleotide at which the polymorphism occurs.
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Statistical analysis
Subjects who had smoked >100 cigarettes in their lifetime were defined as smokers. The observed genotype frequencies were compared with those calculated from HardyWeinberg equilibrium theory (p2 + 2pq + q2, where p is the frequency of the variant allele and q = 1 p). Pearson's
2 was used to test for the differences in the distributions between cases and controls. In this study, we hypothesized that the presence of the A allele might be associated with a higher risk of lung cancer. However, because it was unclear whether the A allele had a dominant, recessive or gene-dosage effect, statistical modeling was performed on the relative risk of the AA genotype or the GA genotype against the GG genotype, respectively. The ORs and 95% CI were calculated to estimate the relative risk. Adjusted ORs were obtained by fitting unconditional multivariate logistic regression models with adjustments for age, sex and smoking status. A P value of 0.05 for any test or model was considered to be statistically significant. All statistic tests were two-sided and performed with Statistical Package for Social Science V.10.0 (SPSS, Chicago, IL).
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Results
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There were 182 lung cancer cases and 185 controls included in this analysis. Table I summarizes selected characteristics of the subjects. The cases and controls were frequency matched by sex and smoking status. However, there were significantly more cases than controls in the >50 years' group (P = 0.041). The observed distribution of CCND1 genotypes in controls (GG:GA:AA, 48:98:39) was not statistically different from those (51:92:42) expected from the HardyWeinberg equilibrium equation (P = 0.822).
Distributions of the CCND1 genotype and allele frequency in lung cancer patients and controls are shown in Table II. The A allele frequency was higher in cases (0.546) than that in controls (0.479), but this difference was only borderline statistically significant (P = 0.054). Although the CCND1 AA genotype was more frequent in the cases (31.3%) than in the controls (21.1%), no statistically significant difference was found in univariate analysis (OR = 1.75; 95% CI 0.983.15). After adjustment for age, sex and smoking status, the AA genotype was associated with a significantly increased lung cancer risk (adjusted OR = 1.87; 95% CI 1.013.45) compared with the GG genotype. The adjusted OR for the CCND1 AG type was 1.02 (95% CI 0.601.74).
Table III shows the CCND1 genotype frequencies in subjects stratified by age, sex and smoking status. In the younger subgroup (age
50 years), the OR was 3.23 (95% CI 1.178.96) for the AA genotype after adjustment for sex and smoking status. Males exhibited an increased risk (adjusted OR = 2.46; 95% CI 1.185.10) associated with the AA genotype, but this was not found in females (adjusted OR = 0.96; 95% CI 0.303.15). No significant association between the AA genotype and smoking status was observed.
The associations between the CCND1 AA genotype and histological types of lung cancer are shown in Table IV. After adjustment with age, sex and smoking status, the CCND1 AA genotype was associated with a significantly higher risk in NSCLC (OR = 1.95; 95% CI 1.003.78), while there was no significant association in SCLC (OR = 1.51; 95%, CI 0.583.94). Among NSCLC, the OR was 2.92 (95% CI 1.078.03) for the AA genotype and squamous cell lung cancer patients, but there was no significant association between CCND1 gene polymorphism and lung adenocarcinoma (OR = 1.78; 95% CI 0.784.04).
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Discussion
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Cyclin D1 is one of the major cyclins involved in transition from G1 to S phase, being associated with CDK 4/6 in mid to late G1 phase. Both gene activation (due to amplification or chromosomal rearrangement) and/or protein overexpression of cyclin D1 have been described in a variety of tumors, including lung cancer (1517). Overexpression of cyclin D1 may lead to premature cell passage through the G1/S transition, resulting in propagation of unrepaired DNA damage, accumulation of genetic errors, and a selective growth advantage for the altered cells. Furthermore, in tumor tissues cyclin D1 overexpression or gene amplification is associated with a more rapid and frequent recurrence of cancer (1820). In lung cancer, a high frequency of CCND1 amplification/overexpression was reported as one of the important steps in carcinogenesis (21).
The G/A single base pair polymorphism in CCND1 could reportedly influence tumor susceptibility in several cancers (2225). The AA homozygotes express more altered transcript than the GG homozygotes and GA heterozygotes and the altered transcript fails to be spliced at the exon 4/intron 4 boundary, which does not contain exon 5 and terminates downstream of exon 4 (26). This results in an altered protein that lacks the destruction box encoded by exon 5, which is responsible for the increasing half-life of the cyclin D1 protein (11). Therefore, in individuals with the AA genotype, there are increasing steady-state levels of cyclin D1 protein, allowing cells to pass through the G1/S checkpoint more easily and promoting the transformation of cells. Zheng and colleagues (13) have shown that the frequency of the AA genotype was higher in SCCHN patients (23.6%) than in controls (16.5%) in non-Hispanic white population and that subjects with AA genotype tended to develop SCCHN at an earlier age than those with GG genotype. Recently the A allele of CCND1 was shown to be associated with an early age at onset of hereditary non-polypsis colorectal cancer, acting as a risk modifying gene (28). Furthermore, study in non-small cell lung cancer patients showed that the event-free survival was longer in patients with GG genotype (11), which indicated that the CCND1 G/A polymorphism was associated with clinical outcome of lung cancer. Similar results were found in ovarian cancer (29) and SCCHN (30). In agreement with those studies, we found that the CCND1 AA genotype was associated with a higher risk for lung cancer in a Chinese population and this risk is remarkably increased in younger subjects. This finding is consistent with Zheng's result in SCCHN. For an early age of onset of disease is a hallmark of genetic predisposition, these results imply that the CCND1 G/A polymorphism may play a role in the susceptibility to carcinogenesis of lung cancer.
An interesting result was obtained when we divided the cases into subgroups by histological types. There were no significant associations in the subgroups except for squamous cell lung cancer patients. This finding is consistent with previous reports about cyclin D1 overexpression in squamous cell lung carcinomas (31,32). According to epidemiological studies, which have shown that smoking was the major cause of squamous cell lung cancer in Chinese (33) and the PAH-adduct was one of the main DNA damage of smoking-caused lung cancer (34), there was indication that the CCND1 G/A polymorphism might be due to the individual susceptibility to some specific environmental carcinogens or DNA damage, although the specific relationship between cyclin D1 protein and PAH has not been reported. This result also provides an explanation for the result of sex stratification. A significant higher risk of subjects with the AA genotype was found in males, but not in females, because compared with 33.6% male squamous cell lung cancer cases, only 11.8% female patients in our study were squamous cell lung cancer, and smoking was not a risk factor in females. It seems that the CCND1 G/A polymorphism may not be a susceptibility marker for lung cancer of women in this population, but larger studies are needed to confirm this finding.
In conclusion, we found that the CCND1 G/A polymorphism may contribute to the susceptibility to lung cancer, especially in those who were young and male, in this Chinese population. In addition, the polymorphism may be a link in the smokingDNA damagelung cancer interaction. However, bias may arise from the unmatched age and limited numbers in the subgroups analyzed and possible selection bias inherent in a hospital-based case-control study. Therefore, these results remain preliminary and need to be validated in larger and well-designed studies.
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Acknowledgments
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The authors thank Dr Qingyi Wei (Department of Epidemiology, M.D. Anderson Cancer Center, TX) for editing the manuscript. This investigation was supported in part by the National Key Basic Research and Development Program (2002CB512903).
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Received July 18, 2002;
revised February 23, 2003;
accepted February 24, 2003.