Department of Epidemiology, The University of Texas M.D.Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
1 To whom correspondence should be addressed. Tel: +1 713 792 3020; Fax: +1 713 792 0807; Email: qwei{at}mdanderson.org
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Abstract |
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Abbreviations: ALL, acute lymphocyte leukemia; CI, confidence interval; OR, odds ratio; TSER, thymidylate synthase enhancer region; TS3'UTR, thymidylate synthase 3'-untranslated region; TYMS, thymidylate synthase
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Introduction |
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Thymidylate synthase (TYMS), a key enzyme of folate metabolism, catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate and 5,10-methylenetetrahydrofolate to dihydrofolate (9). Deoxyuridine monophosphate is essential for the provision of thymidine, a nucleotide needed for DNA synthesis and repair (10). Not surprisingly, therefore, impairment of the TYMS enzyme is associated with chromosome damage and fragile site induction (11,12), suggesting that DNA repair mechanisms can be affected by nucleotide availability. In addition, TYMS is a target for chemotherapeutic drugs such as 5-fluorouracil and TYMS mRNA and protein expression levels are considered prognostic indicators for several cancers (1316). Therefore, genetic variation and in vivo regulation of TYMS are likely to be important in both cancer etiology and outcome.
In humans, a polymorphic tandem repeat element in the TYMS promoter enhancer region (TSER) has been identified near the initiation start site. This variant is most frequently present as either two (2R) or three (3R) repeats (17), although more rare alleles such as 4R, 5R and 9R also exist (18). TSER*3R confers a higher translational efficiency than does TSER*2R and, indeed, an in vitro study in HeLa S3 cells has demonstrated that the TSER*2R allele is associated with less than half the TYMS protein expression as the TSER*3R allele (17). In vivo TYMS mRNA levels in tumor tissue were 3-fold lower among subjects with 2R/2R homozygotes than among subjects with 3R/3R homozygotes (19). Furthermore, the 3R/3R homozygous genotype has been associated with increased levels of TYMS protein expression and higher absolute enzyme activity (20). In turn, lower expression levels of TYMS may reduce the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate, thereby increasing the chance of uracil misincorporation into DNA, which could increase the number of DNA double-strand breaks in rapidly proliferating tissues. Recently, a second TYMS polymorphism was identified (21): a 6 bp deletion/insertion at bp 1494 in the 3'-untranslated region of the TYMS gene (TS3'UTR or 1494del6, dbSNP accession no. rs16430). To date, several reported studies have investigated a possible association between the TS3'UTR 6 bp deletion/insertion and risk of colon cancer, but no consistent results were demonstrated (2123).
Although the protective effect of the TSER tandem repeat polymorphism in lung cancer chemotherapy has been well studied (24,25), the association between TYMS polymorphisms and risk for lung cancer has not been studied. Therefore, in this study, as part of an ongoing hospital-based casecontrol study of lung cancer, we tested our hypothesis that the TSER and TS3'UTR polymorphisms are associated with lung cancer risk and that these polymorphisms may interact with nutrient intake.
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Materials and methods |
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Genotyping
Using 1 ml of whole blood from each blood sample, a leukocyte pellet was obtained from the 200 µl buffy coat fraction by centrifugation. Genomic DNA was extracted using a Qiagen DNA Blood Mini Kit (Qiagen, Valencia, CA). DNA purity and concentrations were determined by spectrophotometric measurement of absorbance at 260 and 280 nm.
To analyze the TSER 28 bp repeat polymorphism, we amplified the fragment using the following primers: TSER forward primer 5'-GTGGCTCCTGCGTTTCCCCC-3'; reverse primer 5'-GGCTCCGAGCCGGCCACAGGCATGGCGCGG-3' (17). PCR was performed in a total volume of 20 µl of 1x PCR buffer (50 mM KCl, 10 mM TrisHCl and 0.1% Triton X-100), 1.5 mM MgCl2, 0.15 mM deoxyribonucleotide triphosphates, 100 nM each primer, 10% dimethylsulfoxide, 1.5 U Taq polymerase (Sigma-Aldrich, St Louis, MO) and 50 ng genomic DNA. The cycling conditions consisted of: one cycle of 95°C for 5 min; 35 cycles of 95°C for 30 s, 63°C for 45 s and 72°C for 45 s; a final extension at 72°C for 10 min. The amplified fragments were separated in a 3% Nusieve agarose gel (Biowhittaker Molecular Applications, Rockland, ME). The fragments containing 3R and 2R repeats were 243 bp and 215 bp, respectively.
The TS3'UTR polymorphism was analyzed on the basis of PCRrestriction fragment length polymorphism. A fragment containing the 6 bp deletion/insertion was amplified using the primers 5'-CAAATCTGAGGGAGCTGAGT-3' and 5'-CAGATAAGTGGCAGTACAGA-3' (21) in 20 µl of reaction mixture containing 1x PCR buffer (50 mM KCl, 10 mM TrisHCl and 0.1% Triton X-100), 1.5 mM MgCl2, 0.15 mM deoxyribonucleotide triphosphates, 100 nM each primer, 1.5 U Taq ploymerase (Sigma) and 50 ng genomic DNA. The cycling conditions consisted of: a 5 min denaturation period at 95°C; 35 cycles of 95°C for 30 s, 58°C for 45 s and 72°C for 60 s; a 10 min extension at 72°C. The amplified fragments were digested with DraI (New England BioLabs, Beverly, MA) and the products separated in a 3% Nusieve agarose gel. The expected fragment sizes were 70 and 88 bp for the 6 bp inserted allele and 152 bp for the 6 bp deleted allele. More than 10% of the samples was randomly selected for repeat assays and the results were in 100% concordance.
Statistical analyses
The mean values of body mass index, total energy intake, total folate intake, dietary intake of vitamins B6 and B12 and pack-years smoked in cases and controls were compared using Student's t-test. Differences in categorized demographic variables, smoking status, alcohol consumption and allele and genotype frequencies between the cases and controls were tested by using the 2 test. The associations between genotypes and risk of lung cancer were estimated by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) from both univariate and multivariate unconditional logistic regression analyses. The reference groups were subjects carrying TSER 2R2R or TS3'UTR 0bp/0bp. Stratification analysis was used to estimate risk for subgroups by age, sex, smoking status, pack-years smoked, alcohol consumption and dietary intake of folate, vitamin B6 and vitamin B12. The tertile values of continuous variables were used as cut-off points. P values for interaction were determined by the likelihood ratio test for the models with and without a multiplicative interaction term. All tests were two-sided and all statistical analyses were performed with Statistical Analysis System software version 8e (SAS Institute, Cary, NC).
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Results |
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The TSER and TS3'UTR allele and genotype distributions among the cases and controls are shown in Table II. The TSER 2R allele frequencies were 0.466 and 0.458 in the cases and controls, respectively, and the TS3'UTR 6bp insertion allele frequencies were 0.704 and 0.678 in the cases and controls, respectively. These differences between the cases and controls were not statistically significant (P = 0.444 for TSER and P = 0.058 for TS3'UTR 6bp), although the 2R and 6bp insertion allele frequencies were slightly higher in the cases than in the controls. The distributions of these two genotypes in the controls were in HardyWeinburg equilibrium (P = 0.062 for TSER; P = 0.071 for TS3'UTR). The distributions of the three major TSER genotypes (3R3R, 3R2R and 2R2R) did not differ between the cases and controls, whereas the distributions of the three TS3'UTR genotypes differed significantly between the cases and controls (P = 0.043): the frequencies of the 0bp/0bp, 6bp/0bp and 6bp/6bp genotypes were 8.4, 42.4 and 49.2%, respectively, for the cases and 11.6, 41.3 and 47.1%, respectively, for the controls, suggesting that the 6bp insertion allele may be a risk allele.
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Results of analyses stratified by age, sex, smoking and alcohol use, daily dietary intake of folate, vitamin B6 and vitamin B12 are also summarized in Table III. Although no significant main effect of the TSER variant genotypes was found in each stratum, subjects >55 years old with one or two TS3'UTR 6bp insertion alleles had a significantly higher risk of lung cancer than did subjects with the 0bp/0bp genotype (OR = 1.57, 95% CI = 1.102.23). Similar results were also found for men (OR = 1.88, 95% CI = 1.222.89), current smokers (OR = 2.04, 95% CI = 1.263.29) and heavy smokers (OR = 1.73, 95% CI = 1.102.70) and current alcohol users (OR = 3.17, 95% CI = 1.785.64) (Table III). For nutrient intake, the subjects were categorized into low, medium and high intake groups on the basis of tertiles of the continuous variable in controls used as the cut-off points. As shown in Table III, for individuals with low vitamin B12 intake the 2R allele was associated with a significantly increased lung cancer risk (OR = 1.43, 95% CI = 1.022.01), whereas the 2R allele was associated with decreased risk of lung cancer in individuals with medium and high vitamin B12 intake (OR = 0.83, 95% CI = 0.581.17 for high vitamin B12 intake; OR = 0.88, 95% CI = 0.631.24 for medium vitamin B12 intake). These data were consistent with a significant interaction between this polymorphism and vitamin B12 intake (Figure 1) that was not evident for other nutrient intakes. Furthermore, we also found a significant interaction between the TS3'UTR 6bp variant genotypes and alcohol intake (Figure 2). For never and former drinkers the 6bp variant genotypes were not associated with an increased risk for lung cancer (OR = 1.16, 95% CI = 0.681.98 for never drinkers; OR = 1.01, 95% CI = 0.591.75 for former drinkers), whereas a >3-fold higher risk was found in current drinkers with the 6bp variant genotypes (OR = 3.17, 95% CI = 1.785.64) (Table III). This interaction was not evident for intake of nutrients. For different histological cell types the TS3'UTR 6bp insertion allele was only associated with a significantly higher risk of squamous cell lung cancer (OR = 2.05, 95% CI = 1.093.87) (Table III).
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Discussion |
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Because of the critical role TYMS plays in folate metabolism and DNA repair, TYMS polymorphisms have recently drawn special interest from laboratory, epidemiological and clinical investigators. Several lines of evidence support a role of variation in TYMS and the risk of cancer. For example, TYMS enzymatic activity and the level of TYMS mRNA expression were found to be significantly higher in tumor tissue than in normal tissue, particularly for stomach and colorectal cancers (26). A functional analysis involving the use of a transient expression assay with cell lines showed that the TSER tandem repeat may contribute to the efficiency of expression of the TYMS gene (17). Individuals with the TYMS 2R2R genotype had lower plasma folate levels than did those with the TYMS 3R3R genotype (27). However, most of the published studies on the association of TYMS have been with regard to colorectal cancer, with the two largest studies being in Caucasian populations.
In a relatively large, hospital-based, casecontrol study of 510 patients with colorectal cancer and 604 polyp-free controls, Ulrich et al. (23) noted that the TYMS polymorphisms and dietary folate intake had an interactive effect on risk. Specifically, among individuals with the 3R3R genotype high levels of folate intake were associated with a reduced colorectal cancer risk, while among individuals with the 2R2R genotype high levels of folate intake were associated with an increased risk. No association was observed between the risk of colorectal cancer and the TYMS 6bp deletion/insertion polymorphism (23). These findings were not supported by a later prospective nested casecontrol study of 270 cases of colorectal cancer and 454 controls, in which the 2R/2R genotype was found to be associated with reduced risk (27).
To date, no casecontrol study of an association between the TYMS polymorphisms and lung cancer risk has been reported, although other related descriptive studies are available. For example, it was found that high TYMS activity, as measured by immunohistochemistry, was associated with higher proliferative activity in non-small cell lung tumors (28) and that lung cancer tissue also tended to have higher TYMS mRNA levels than did normal lung tissue (29). In addition, the TSER 3R3R genotype was associated with a significantly higher level of TYMS mRNA than were other genotypes in non-small cell lung cancer tissue (30).
The possible effect of the TYMS polymorphisms may be inferred from the published data on other cancers. However, in contrast to the findings for colorectal cancer, a recent study of 71 cases of acute lymphocyte leukemia (ALL) in Caucasians and 114 matched Caucasian controls found that compared with the TSER 3R3R genotype, the TSER 2R2R genotype was associated with an increased risk for adult ALL (31). Similarly, a casecontrol study of 180 lymphoma cases and 494 controls showed that individuals with at least one TSER 2R allele had an increased risk for malignant lymphoma, compared with those without the TSER 2R allele (32).
To the best of our knowledge, the present large casecontrol study is the first one to investigate an association between the TSER polymorphism and lung cancer risk. If TYMS enzyme activity mediates folate metabolism and high folate levels reduce DNA damage or facilitate DNA repair, then it is expected that the 2R2R genotype would be associated with an increased risk of lung cancer. However, studies of the association between the TSER polymorphism and serum folate levels in both healthy individuals and cancer patients have yielded mixed results, suggesting that the effect of the TSER polymorphism and folate pathway on cancer may be specific to certain cancers or ethnic groups (27,33,34). The absence of an association between the TSER tandem repeat polymorphism and risk of lung cancer suggests that this polymorphism does not modify the risk of lung cancer associated with low dietary folate intake as observed in the present study. Since vitamin B12 is also involved in DNA methylation, the unexpected finding of an increased risk of lung cancer associated with the TSER variant genotype in the presence of lower vitamin B12 intake suggests that vitamin B12 intake modifies the risk of lung cancer. However, it is unclear to what extent this effect may be biased by intake of other nutrients or supplemental vitamins.
Although the significance of mutations in the 3'-untranslated region is not as obvious as mutations resulting in amino acid changes, the mRNA turnover rate can be affected by this kind of sequence variation. Differences in mRNA turnover alter the stability of a given mRNA, which in turn determines protein expression levels (35). Therefore, it is possible that the TS3'UTR polymorphism affects mRNA stability or secondary mRNA structures, leading to altered protein levels or responses to up-regulation of this enzyme. In our study, because the TS3'UTR (0bp/6bp) polymorphism was found to be in linkage disequilibrium with the TSER (2R3R) polymorphism, the 6bp insertion allele might similarly decrease enzyme activity as does the 2R allele. This hypothesis can partly explain our finding of a higher risk of lung cancer in individuals with the 6bp insertion allele. In addition, this association was more pronounced among older individuals (>55 years), men and heavy smokers. Older males in this study were likely to be heavy smokers who may have incurred more smoking-induced DNA damage. Because a suboptimal repair capacity for removing smoking-related carcinogen-induced adducts has been shown to be associated with an increased risk of lung cancer (4), our data suggest that the TS3'UTR polymorphism may play a role in modifying the risk associated with smoking by decreasing thymidine supply for DNA synthesis and repair. Recently, one functional study on the biological significance of the TS3'UTR polymorphism showed that the 6bp deletion allele had an 50% lower mRNA expression than did the 6bp insertion allele (36), which is inconsistent with our results. This suggests that other mechanisms are involved in this oncogenic pathway that need to be further elucidated.
With regard to the interaction between the TS3'UTR polymorphism and diet, the 6bp insertion allele was associated with a higher risk of lung cancer only in current alcohol drinkers. Although the mechanisms by which alcohol consumption interacts with the effect of TYMS in lung carcinogenesis are not known, there are several causal pathways that can explain this. First, chronic alcohol exposure impairs folate absorption (37). However, we do not have data on folate status measured in plasma, serum or tissue to evaluate this hypothesis. Second, alcohol itself may be associated with a risk of lung cancer. In particular, elevated DNA adducts of acetaldehyde, the primary oxidative metabolite of ethanol, were found in peripheral blood cells among alcoholics (38) and impaired DNA repair was also observed among alcohol-fed rats with DNA damage induced by carcinogens (39). These findings suggest that the role of TYMS in DNA repair is implicated in alcohol-induced DNA damage. Our data also suggested that alcohol intake may interact with cigarette smoking, because these two exposures tended to be highly correlated and there were significantly more ever drinkers among ever smokers (72.3%) than among never smokers (40.9%; P < 0.0001) in our study population. Furthermore, alcohol may act as a solvent of tobacco carcinogens (40) and may change the oxidative capacity of liver microsomes, leading to a reduced ability to metabolize tobacco carcinogens (41), or it may affect cellular metabolism, resulting in an increased metabolic activation of procarcinogens (41,42). However, we did not find interactive effects between these two polymorphisms with folate intake. This may be due to possible misclassification of dietary folate intake assessed by the questionnaire. Therefore, further studies with additional assessment of serum or plasma folate levels are warranted to better evaluate these genenutrition interactions.
Possible limitations in our hospital-based study design could have introduced a selection bias. However, the genotype distributions of our study were similar to those seen in other studies. For example, the frequencies of the TSER 3R3R, 2R3R and 2R2R genotypes among our 1140 non-Hispanic white controls from Texas were 30.8, 46.3 and 22.6%, respectively, compared with 26, 52 and 21%, respectively, for 623 population-based Caucasian controls from Minnesota (21) and 29, 48 and 23% for 454 cohort controls (93% Caucasians) in the Physicians' Health Study (26). Also, the frequencies of TS3'UTR 0bp/0bp, 6bp/0bp and 6bp/6bp among our 1140 controls were 11.6, 41.3 and 47.1%, respectively, compared with 10, 40 and 50%, respectively, for 623 Caucasian controls from Minnesota (21) and 14, 42 and 44% for the 454 controls in the Physicians' Health Study (26). Because our TYMS genotype frequency estimates in the hospital-based controls are similar to those in population-based and cohort-based controls, any selection bias is unlikely to be substantial. However, in a recent study of a Chinese population the frequencies of TS3'UTR 0bp/0bp, 6bp/0bp and 6bp/6bp among 348 Chinese controls were 45.7, 44.5 and 9.8%, respectively (43), suggesting a substantial ethnicity-related difference in the distribution of the TS3'UTR polymorphism. However, the 6bp/6bp genotype was associated with an increased risk for esophageal cancer in the Chinese population (43), a finding similar to that in the present study of lung cancer.
There were also some limitations in the dietary data analysis. For instance, the data did not include information on the intake of specific supplemental vitamins and folate and, therefore, we could not adjust for other possible confounding effects, which could also explain the unexpected interactions between vitamin B12 or alcohol consumption and the TYMS variant genotypes. Like all casecontrol studies of diet and cancer risk, the diet information obtained was retrospective and was for the year preceding the lung cancer diagnosis. However, several studies have examined the reproducibility of food frequency questionnaires under a wide variety of conditions and have shown correlations generally ranging from 0.5 to 0.7 for nutrient intakes measured at periods of 110 years apart (4446). The average reduction in the intake of nutrients was 0.07 over a 5 year period, suggesting that dietary changes over time were not substantial and repeated administration of the questionnaire was not necessary (45). It is also possible that the low intake of dietary vitamins and folate among the cases may be due to the symptomatic effects of the cancer. However, in this study >80% of the cases were interviewed within 15 days after diagnosis, thus reducing potential measurement errors attributable to recall bias as well as recent dietary changes after diagnosis.
In conclusion, in a large non-Hispanic white population we have demonstrated that the TS3'UTR polymorphism may be associated with an increased risk of lung cancer and that this association may be modified by alcohol consumption, an interaction that was also observed for the TSER polymorphism and vitamin B12 intake. Once our findings are replicated, these polymorphisms may be used to identify individuals at risk of developing lung cancer. However, the underlying biological relevance of these TYMS polymorphisms and the mechanisms of genediet interactions warrant further study.
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Acknowledgments |
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References |
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