Abstract
DNA methylation is one of the major events in the early process of gastric carcinogenesis and also occurs in non-neoplastic gastric mucosa. Catechol-O-methyltransferase (COMT) catalyzes the methylation of various endobiotic and xenobiotic substances, and protects DNA from oxidative damage. The association between a common functional polymorphism of COMT Val158Met and DNA methylation status in the stomach was investigated. Patients and Methods: One hundred and sixty-nine gastric mucosa samples from non-cancer patients were obtained by endoscopy. The promoter methylation status of p14 and p16 was determined by methylation-specific PCR (MSP). The COMT Val158Met polymorphism was detected by PCR-restriction fragment length polymorphism (RFLP). Results: CpG island methylation was observed in 32.5% of the p14, and 37.9% of the p16. The methylation status of both p14 and p16 was not associated with gender or age, while p16 methylation was strongly associated with Helicobacter pylori infection (OR=4.71, 95% CI=2.35-9.46, p<0.0001). The Val/Val genotype held a significantly higher risk of p16 methylation (OR=3.27, 95% CI=1.05-10.25, p=0.0418). Conclusion: The COMT polymorphism may influence the susceptibility to gene methylation in the gastric mucosa. The promoter CpG island of p16 gene, but not of p14 may be one of the specific regions whose methylation is closely influenced by the COMT polymorphism.
Abbreviations: COMT, Catechol-O-methyltransferase; MSP, methylation-specific polymerase chain reaction.
Aberrant DNA methylation is an important mechanism in gene silencing. In many kinds of cancer, some genes seem to acquire aberrant methylation in their CpG islands. Some genes are also methylated in non-neoplastic tissues with aging and this alteration is known as age-related methylation (1, 2). In addition, it has been shown that gene methylation may be present in non-neoplastic colorectal mucosa in patients with inflammatory bowel disease (3, 4), esophageal mucosa in patients with Barrett's esophagitis (5, 6) and liver tissues in chronic hepatitis (7).
In the non-neoplastic human gastric mucosa, frequencies or levels of CpG islands methylation of certain genes correlate with Helicobacter pylori infection (8, 9), histological or serological severity of gastritis and gastric cancer occurrence (10-13), suggesting that aberrant DNA methylation is one of the major events which occurs early in the process of tumorgenesis in the stomach. These epigenetic events in the gastric mucosa may lead to transcriptional inactivation in specific genes and increase DNA damage or mutation and chromosomal instability.
p16(INK4a) and p14(ARF) are involved in the negative cell cycle regulation via the pRb and p53 pathways, respectively. The genes for these two proteins have an independent first exon (exon 1a and 1β, respectively) but share exons2 and 3 (14, 15). Methylation of p16 and p14 has been shown to be present in gastric cancer as well as premalignant lesions (16, 17). Thus, both genes may play crucial roles in cell cycle control, apoptosis and DNA repair in the stomach and their disorder may be closely associated with gastric carcinogenesis.
Hypermethylation of promoter region of p14 in gastric mucosa in 3 cases. DNA from specimens of gastric mucosa in case 2 showed a positive methylated band. U; un-methylated band M; methylated band.
Catechol-O-methyltransferase (COMT) is expressed in various mammalian tissues including the stomach (18-20) and has been shown to catalyze the methylation of various endobiotic and xenobiotic substances preventing quinine formation and redox cycling, and therefore might protect DNA from oxidative damage (21, 22). A G to A transition, which results in an amino acid change from valine to methionine at codon 158, leads to COMT activity of the Met/Met genotype which is a quarter of that of the wild genotype, and heterozygous individuals exhibit intermediate enzyme activity (23).
Because of the important role that COMT plays with respect to preventing DNA damage, the polymorphism of COMT may influence the susceptibility to methylation in the human gastric mucosa.
In the present study, the methylation status of p14 and p16 in non-neoplastic gastric mucosa samples and its relation to the COMT Val158Met polymorphism was investigated.
Patients and Methods
Tissue samples and DNA extraction. The study population comprised 169 non-cancer patients, attending the Endoscopy Center of Fujita Health University Hospital from January 2005 to July 2007. All patients underwent upper gastroscopy as part of a health check, as a secondary procedure following barium X-ray examination for suspected stomach cancer, or for the investigation of abdominal discomfort. Patients who had severe systemic disease, or malignancy in the stomach or other organ were excluded from this study. Biopsy specimens were taken from the antrum along the greater curvature, from grossly non-pathological mucosa in all patients. The specimens were cut into two pieces. One of the pieces was fixed in 10% buffered formalin and embedded in paraffin for microscopic histological examination and the other part was immediately frozen and stored at -80°C until use. Histological analysis of all the selected biopsy samples also showed that these samples contained more than 70% of epithelial cells. Genomic DNA was extracted directly from the frozen specimens using a standard phenol/chloroform method. H. pylori infection status was assessed by serological, histological analysis or urea breath test. Patients were diagnosed as infected when at least one of the diagnostic tests was positive. The Ethics Committee of the Fujita Health University School of Medicine approved the protocol and prior, written informed consent was obtained from all participants.
Characteristics of the 169 patients.
Bisulufite modification and methylation-specifc PCR (MSP). For the examination of DNA methylation, the genomic DNA was treated with sodium bisulfite using a BislFast DNA Modification Kit for Methylated DNA Detection (Toyobo, Co. Ltd., Osaka, Japan). MSP was carried out with the following primers: p14 methylated forward (MF), 5′-gtgttaaagggcggcgtagc-3′ and p14 methylated reverse (MR), 5′-aaaaccctcactcgcgacga-3′ which amplify a 122-bp product; p14 unmethylated forward (UF), 5′-tttttggtgttaaagggtggtgtagt-3′ and p14 unmethylated reverse (UR), 5′-cacaaaaaccctcactcacaacaa-3′ which amplify a 132-bp product (24); p16 methylated forward (MF), 5′-ttattagagggtggggcggatcgc-3′ and p16 methylated reverse (MR), 5′-accccgaaccgcgaccgtaa-3′ which amplify a 149-bp product; p16 unmethylated forward (UF), 5′-ttattagagggtggggtggattgt-3′ and p16 unmethylated reverse (UR), 5′-caaccccaaaccacaaccataa-3′ which amplify a 151-bp product (25). The annealing temperature and times were determined using DNA from the peripheral blood of a young individual without H. pylori infection and DNA methylated with SssI methylase (New England BioLabs Inc., Beverly, MA, USA). The MSP was carried out in a volume of 21 μL containing 0.1 μg of bisulfite-modified DNA. The DNA was denatured at 95°C for 5 minutes, followed by 33~35 cycles at 95°C for 30 s and according to the primers for 1 minute, and 72°C for 1 minute with a final extension at 72°C for 5 minutes. The MSP reactions were conducted using EX Taq HS (Takara Bio Inc., Shiga, Japan). The bands of MSP were detected by electrophoresis in 2.5% agarose gels stained with ethidium bromide (Figure 1).
COMT Val158Met genotypes. Using genomic DNA, the polymorphism at codon 158 in the COMT gene was determined by PCR-restriction fragment length polymorphism (RFLP) assays. The PCR primers 5′-tcgtggacgccgtgattcagg-3′ and 5′-aggtctgacaacgggtcaggc-3′ were used to amplify a 217-bp fragment of COMT that contains the polymorphic NlaIII site as well as one other constant NlaIII site. PCR was performed in a reaction volume of 25 μl containing 200 ng of genomic DNA, 10 pmol of each primer, 200 ng of each dNTP and 0.6 units Taq DNA polymerase (Toyobo). The reaction mixture was first denatured at 95°C for 5 min and then amplified by PCR for 32 cycles at 95°C for 30 s, at 55°C for 30 s, and at 72°C for 1 min, followed by a 10 min extension at 72°C. Twelve μl of PCR product were incubated with 10 units of NlaIII (New England Biolabs) in a volume of 20 μl at 37°C overnight. NlaIII cuts only the low-activity variant (COMT158Met) in addition to a second constant cleavage site in the PCR product. Thus, homozygotes for COMT158Val generated fragments of 136 and 81bp, heterozygotes gave 136, 96, 81, and 40 bp fragments and homozygotes for COMT158Met generated 96, 81, and 40-bp fragments. The 40bp fragment ran off the gel during electrophoresis. The 3 genotypes were scored after running on a 3.5% agarose gel with ethidium bromide 10 μg/mL.
Association between p14 and p16 promoter methylation and age, gender, H. pylori infection and COMT Val158Met genotypes.
Statistical analysis. Statistical analysis was conducted with two-sided chi-squre for the comparison of promoter DNA methylation frequencies of p14 and p16 between two groups. The association between DNA methylation status of p14 and p16 and age was examined by the Mann-Whitney U-test. A probability value of less than 0.05 was considered statistical significant.
Results
Study population. The characteristics of the participants is shown in Table I. After gastroscopy, 13 patients (7.7%) were diagnosed as having active peptic ulcer disease.
Association between methylation of p14 and p16 and gender, age, H. pylori infection and COMT Val158Met polymorphism. All 169 gastric mucosa samples were available for MSP analysis and COMT genotyping. CpG island methylation of p14 was observed in 55 (32.5%) and of p16 in 64 subjects (37.9%). The methylation status of both p14 and p16 was not associated with gender or age, while p16 methylation was strongly associated with H. pylori infection (OR=4.71, 95% CI=2.35-9.46, p<0.0001). The COMT genotype distribution in the 169 participants was 80 Val/Val (47.3%), 75 Val/Met (44.4%) and 14 Met/Met (8.3%). Although no association was found between the COMT genotypes and p14 methylation, the Met/Met genotype was significantly associated with p16 methylation (Met/Met vs. Val/Val/ + Val/Met; OR=3.27, 95% CI=1.05-10.25, p=0.0418). While such an association was not found for the Met carriers (Val/Val vs. Met carriers; OR= 0.93, 95% CI=0.50-1.74, p=0.82). The prevalence of patients in whom either p14 or p16 or both p14 and p16 were methylated was also investigated. Although H. pylori infection was significantly associated with methylation of either, and both p14 and p16 (p14 or p16: OR=2.89, 95% CI=1.53-5.45, p=0.001, both p14 and p16: OR=5.19, 95% CI=1.45-18.57, p=0.006), age, gender and COMT genotype were not associated with methylation of either p14 or p16 or both p14 and p16 (Table II).
Discussion
Methylation of p16 was strongly associated with H. pylori infection. This observation is in line with previous studies (8-13). In regard to the CpG sites, the Met/Met genotype held a significantly higher risk of p16 rather than p14 promoter methylation.
The amino acid change at codon 158 (Val to Met) of COMT results in lower thermostability, with enzyme activity that is up to four times less than that from the wild-type allele (26). The presence of the Val/Val genotype has been considered to be favorable because it seems to lower the risk of developing non-Hodgkin lymphoma and estrogen-associated carcinomas in women (27-29), and because it is associated with a higher tendency to remain free from an increase in prostate-specific antigen in men with prostate cancer (30).
The mechanisms of gene methylation are unknown. Several factors may contribute to this methylation, such as exogenous carcinogens, generated reactive oxygen, and host genetic differences (31). One of the most important factors causing oxidative stress in the gastric mucosa is H. pylori infection, which induces chronic inflammation (32, 33). Indeed, the methylation of certain genes in non-neoplastic gastric mucosa correlates with H. pylori infection (8, 9), histological or serological severity of gastritis and gastric cancer occurrence (11-13). However, not all patients with H. pylori infection or gene methylation develop gastric cancer. This difference may be attributed to some genetic factors. The present results provided the first evidence that the genetic polymorphisms of COMT may also be involved in DNA methylation in human gastric mucosa. Although the activity of COMT in the serum or gastric mucosa was not investigated, it is possible that the COMT polymorphism influences the activity and modifies the risk of DNA methylation in the gastric mucosa, and thus, influences the susceptibility to methylation-related carcinogenesis.
Meanwhile, no significant association between COMT polymorphism and p14 methylation status was found. In addition, the COMT genotype was also not associated with methylation of either p14 or p16. The promoter CpG island of p16 gene, but not of p14 may be one of the specific regions whose methylation is closely influenced by COMT polymorphism. However, why the interplay between COMT polymorphism and aberrant hypermethylation is different in different genes is still unexplained. Only a more extensive understanding of the regulation of methylation in relation to gene expression and carcinogenesis will allow us to fully interpret our findings.
- Received January 28, 2009.
- Revision received May 7, 2009.
- Accepted May 15, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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