Tumori, 96: 726-733, 2010 Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma Shi-Lian Hu1, Xiang-Yong Kong1, Zhao-Dong Cheng1, Yu-Bei Sun2, Gan Shen3, Wei-Ping Xu4, Lei Wu4, Xiu-Cai Xu5, Xiao-Dong Jiang1, and Da-Bing Huang1 1 Department of Geriatrics, Affiliated Anhui Province Hospital, Anhui Medical University, Hefei; Department of Oncology, Anhui Province Hospital, Hefei; 3Cadre’s Ward of Anhui PPC Hospital, Hefei; 4 Anhui Evidence-based Medicine Center, Hefei; 5Anhui Provincial Hospital Center Lab, Hefei, China 2 ABSTRACT Aims and background. Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including gastric cancer. A number of reports on methylation of various genes in gastric cancer have been published, but most of these studies focused on cancer tissues or only a single gene. In this study, we determined the promoter hypermethylation status and mRNA expression of 4 genes: p16, Runx3, DAPK and CHFR. Methods. Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of p16, Runx3, DAPK and CHFR gene promoters in cancer and adjacent normal gastric mucosa specimens from 70 patients with gastric cancer, as well as normal gastric biopsy samples from 30 people without cancer serving as controls. In addition, the mRNA expression of p16, Runx3, DAPK and CHFR was investigated in 34 gastric cancer patients by RT-PCR. Bisulfite DNA sequence analysis was applied to check the positive samples detected by MSP. Results. When carcinoma specimens were compared with adjacent normal gastric mucosa samples, a significant increase in promoter methylation of p16, Runx3, DAPK and CHFR was observed, while all 30 histologically normal gastric specimens were methylation free for all 4 genes. The methylation rate of the 4 genes increased from normal stomach tissue to tumor-adjacent gastric mucosa to gastric cancer tissue. Concurrent methylation in 2 or more genes was found in 22.9% of tumor-adjacent normal gastric mucosa and 75.7% of cancer tissues. No correlation was found between hypermethylation and other clinicopathological parameters such as sex, age, and tumor location. However, the frequency of DAPK and CHFR methylation in cancer tissues was significantly associated with the extent of differentiation and lymph node metastasis (P <0.05) and the frequency of Runx3 methylation was significantly associated with tumor size (P <0.05). Weak expression and loss of expression of the 4 genes was observed in cancer tissues and was significantly associated with promoter hypermethylation (P <0.05). Conclusions. Promoter hypermethylation of p16, Runx3, DAPK and CHFR is frequent in gastric cancer. DAPK and CHFR promoter hypermethylation may be an important help in evaluating the differentiation grade and lymph node status of gastric cancer. Weak gene expression and loss of gene expression due to promoter hypermethylation may be a cancer-specific event. Free full text available at www.tumorionline.it Introduction Gastric cancer is one of the most frequent tumor types worldwide and the major cause of cancer-related deaths in China1. Its development is associated with genetic changes in the host, such as loss of heterozygosity and several types of mutations in tumor suppressor genes2. Key words: gastric carcinoma, p16, Runx3, DAPK, CHFR, DNA methylation Acknowledgments: This work was supported by the National Natural Science Foundation of China, item number 30672383. Correspondence to: Professor Shi-lian Hu, Department of Geriatrics, Anhui Province Hospital, Anhui Medical University, Hefei 230001, China. Tel +86-0551-2283589; e-mail [email protected] Received November 19, 2009; accepted April 8, 2010. PROMOTER METHYLATION OF GENES AND GASTRIC CARCINOMA Epigenetic alterations are now also regarded as key mechanisms of the development and progression of gastric carcinoma3,4. Methylation of CpG islands is one of the crucial epigenetic mechanisms: it can lead to changes in chromosome structure, DNA constitution, and DNA stability5,6. CpG islands are DNA segments of at least 0.5 kb with a high G:C and CpG content which are often located in the promoter or 5’-exon sequences of genes7. Aberrant methylation of CpG islands is tumor specific, and may result in inactivation of tumor suppressor genes8-11. Recently, in addition to its potential role in gene inactivation in human cancers, CpG island hypermethylation has been gaining attention as a molecular marker for tumor detection and prediction of cancer development. Aberrant methylation of the promoters of tumor suppressor genes such as p16, Runx3, DAPK and CHFR has been reported in many kinds of cancer12,13. p16 is a cell cycle regulator involved in the inhibition of G1 phase progression14. Loss of function of p16 results in higher cyclin D-dependent protein kinase activity and thus leads to aberrant phosphorylation of retinoblastoma protein, which accelerates cell growth15. Inactivation of p16 by homozygous deletion, point mutation or hypermethylation is one of the most commonly observed aberrations in tumors16. CHFR (checkpoint with forkhead-associated [FHA] and RING finger) encodes a protein with FHA and RING finger domains that functions in the mitotic checkpoint pathway, which governs the transition from prophase to prometaphase. It is localized at chromosome 12q24.3317,18. Runt-related transcription factor 3 gene (Runx3) was originally cloned as AML2 and localized on human chromosome 1p36.119,20. Death-associated protein kinase (DAPK), an actin-associated calcium/calmodulin-dependent enzyme with serine/threonine kinase activity21, is involved in apoptosis induced by tumor necrosis factor-alpha, Fas, and IFN-γ22. Recent studies have shown that silencing of p16, Runx3, DAPK and CHFR by hypermethylation of the CpG-rich promoter region occurred frequently in non-small cell lung cancer23, colorectal carcinomas24, esophageal carcinomas, and gastric carcinomas25,26. However, most of these studies were focused on cancer tissues or only a single gene, while there have been few studies on the relationship between clinicopathological prognostic factors and the methylation status of multiple genes in gastric cancer. In the present study we examined the promoter hypermethylation status of the human p16, Runx3, DAPK and CHFR genes in the DNA of primary gastric tumor tissues and adjacent normal gastric mucosa. We analyzed the relationship between the methylation of these genes and clinicopathological parameters. In addition, we determined the mRNA expression of p16, Runx3, DAPK and CHFR in 34 primary gastric cancers to analyze the potential relationship between loss of mRNA expression and promoter hypermethylation. 727 Material and methods Patients and tissue samples Tumor specimens were obtained from 70 patients. All of them gave their informed consent before collection of the samples according to institutional guidelines. They underwent surgical resection of primary gastric cancer at the Department of General Surgery, Anhui Provincial Hospital and the Department of General Surgery, the Third Affiliated Hospital of Anhui Medical University, between September 2007 and March 2009. For all these tumors, adjacent normal gastric mucosa (located at least 5 cm from the primary tumor) was also obtained. In addition, 30 normal gastric biopsy specimens serving as controls were obtained from people without any evidence of cancer. These samples were immediately frozen after resection and stored at -80 . Of the 70 gastric cancer patients, 31 had clinical stage I and II while 39 had clinical stage III and IV; the mean age of the patients was 62.11 years (range, 32-85 years). DNA extraction and bisulfite modification Genomic DNA was obtained from samples using a QIAamp DNA Micro Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Extracted DNA was identified by agarose gel electrophoresis and then quantified by calculating the A260/280 ratio. Genomic DNA was modified with sodium bisulfite as described previously27. Briefly, 1 µg of genomic DNA was denatured with 2M NaOH for 10 minutes at 37 °C. 10 mM hydroquinone and 3 µM sodium bisulfite at PH 5.0 were added and mixed. Samples were incubated at 50 for 16 hours. DNA was purified using the Wizard DNA purification kit (Promega, Madison, WI, USA), treated again with NaOH, precipitated with ethanol, and resuspended in water. This procedure converts unmethylated cytosine residues to uracil that is recognized as thymine by Taq polymerase, whereas the methylated cytosine remains unchanged. The modified DNA was either used immediately as a template for PCR or stored at -20 °C. Methylation-specific PCR For detection of promoter methylation status, methylation-specific polymerase chain reaction (MSP) was performed as described by Herman et al.28. Briefly, modified DNA was subjected to MSP using specific primers (Table 1). The PCR was carried out in a volume of 25 µL with 2 µL of bisulfite-modified DNA, 10x PCR buffer, 25 mM MgCl2, 10 pmol of each primer, 200 µM dNTPs, and 1 U of Hot-Goldstar Taq polymerase (Takara Biochemical, Japan). PCR conditions were as follows: after initial denaturing for 10 minutes at 95 °C, 35 cycles at 95 °C for 30 seconds, 30 seconds at different annealing temperatures with the primers (Table 1), and 72 °C for 30 seconds were followed by a final extension step at 72 °C for 10 minutes. Plasma DNA from a healthy individual was 728 SL HU, XY KONG, ZD CHENG ET AL Table 1 - Primers and conditions used for MSP and BSP Primers p16 (MSP) p16 (BSP) Runx3 (MSP) Runx3 (BSP) DAPK (MSP) DAPK (BSP) CHFR (MSP) CHFR (BSP) M U M U M U M U Forward sequence (5’-3’) Reverse sequence (5’-3’) Base pairs Temperature (ºC) ATAATAGCGGTCGTTAGGGCGTCG TTATGAGGGGTGGTTGTATGTGGG TTAATATGAGAATTGGTTAAAATTTATATT ATAATAGCGGTCGTTAGGGCGTCG TTATGAGGGGTGGTTGTATGTGGG TAGGGTTTTTAGGAGATTTTTTTT GGATAGTCGGATCGAGTTAACGTC GGAGGATAGTTGGATTGAGTTAATGTT TTTTTATTTATTTTTTAGTTGTGTTTT TTTCGTGATTCGTAGGCGAC TTTTGTGATTTGTAGGTGAT TGTTTATTAAGAGYGGTAGTTAAAG GCTTCTACTTTCCCGCTTCTCGCG AAAACAACCAACACAAACACCTCC AAAAAAAACACCAAACAATATTTAC GCTTCTACTTTCCCGCTTCTCGCG AAAACAACCAACACAAACACCTCC AACTTCCCCAACTCTTACTACTTC CCCTCCCAAACGCCGA CAAATCCCTCCCAAACACCAA CCTTAACCTTCCCAATTACTC GCGATTAACTAACGACGACG ACAATTAACTAACAACAACA AAAATCCTTAAAACTTCCAATCC 234 234 207 115 234 295 98 106 237 155 155 197 65 60 56 55 61 60 55 55 53 58 53 55 MSP, methylation-specific polymerase chain reaction; BSP, bisulfite DNA sequencing PCR; M, methylated specific primers; U, unmethylated specific primers. used as the positive control for unmethylation, the same plasma DNA was methylated with excess Sss I methyltransferase (New England Biolabs Inc, Beverly, MA, USA) to generate completely methylated DNA at all CpG islands and used as the positive control for methylation; distilled water without DNA was used as negative control. The PCR products were separated in 1.5% agarose gels, stained with ethidium bromide, and visualized directly under ultraviolet illumination (BioSpectrum Imaging System; Ultra-Violet Products Inc, Upland, CA, USA). Bisulfite DNA sequencing PCR (BSP) We sequenced the bisulfite-PCR products of the methylation-positive gastric cancer tissue as detected by MSP. Genomic DNA was modified with sodium bisulfite as described previously23. The bisulfite-modified DNA was used for PCR amplification. The PCR was performed as mentioned previously. The primer sequences and PCR annealing temperatures used for bisulfite DNA sequence analysis are shown in Table 1. PCR products were purified by E.Z.N.A.Cycle-Pure Kit (Omega, USA) as described previously. The sequence of PCR products was analyzed by Takara Biotechnology (Dalian, China). Reverse transcription-PCR (RT-PCR) Germany), then amplified by primer sets specific for the p16 and DAPK genes (Table 2). PCR was performed in a total volume of 25 µL with 1 µL of Taq, 2.5 µL of PCR buffer, 2 µL of MgCl2, 0.5 µL of dNTP, 1 µL of primers, 4 µL of cDNA, and 13 µL DNase-free water. PCR amplification consisted of 1 cycle at 95 °C for 5 minutes, 35 cycles at 95 °C for 30 seconds, at different annealing temperatures with the primers (Table 2) for 30 seconds, and at 72 °C for 30 seconds, followed by a final extension at 72 °C for 10 minutes. The PCR products were separated in 2% agarose gels, stained with ethidium bromide, and visualized directly under ultraviolet transillumination. Statistical analysis The data were processed by the SPSS 16.0 statistical software. The associations between p16, Runx3, DAPK and CHFR methylation and clinicopathological parameters were statistically analyzed using the chi-square test. Correlations between p16, Runx3, DAPK and CHFR methylation status and mRNA expression were analyzed using Fisher’s exact test. Differences were considered statistically significant at P values <0.05. Results Expression of p16, Runx3, DAPK and CHFR mRNA was analyzed by RT-PCR. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was generated using a first-strand cDNA synthesis kit (Qiagen, Promoter hypermethylation of p16, Runx3, DAPK and CHFR in gastric cancer As shown in Figure 1, only methylated and unmethylated primers achieved the expected amplified Table 2 - Primers and conditions used for RT-PCR Primers Forward sequence (5’-3’) Reverse sequence (5’-3’) Size (bp) p16 Runx3 DAPK CHFR GAPDH AGCCTTCGGCTGACTGGCTGG GAGTTTCACCCTGACCATCACTGTG AACCCATCATCCATGCCATC TAA AGGAAGTGGTCCCTC TGTG CTGCACCACCAACTGCTTAG CTGCCCATCATCATGACCTGG GCCCATCACTGGTCTTGAAGGTTGT TCTCTCCTTCTCGGTTCTTGA GGTTTGGGCATTTCTACGC TGAAGTCAGAGGAGACCACC 150 198 200 205 407 Annealing temperature 60 55 51 58 60 °C °C °C °C °C PROMOTER METHYLATION OF GENES AND GASTRIC CARCINOMA p16 Runx3 DAPK CHFR H2O N2 T2 N1 T1 UP MP U M U M U M U M U M U M U M Ma H2O N2 T2 N1 T1 UP MP U M U M U M U M U M U M U M Ma H2O N2 T2 N1 T1 UP MP 200bp 100bp 200bp 100bp p16, Runx3, DAPK and CHFR were significantly different between gastric cancer tissues and adjacent normal gastric mucosa (P <0.05). All 30 histologically normal gastric biopsy specimens were methylation free for all 4 genes. The methylation rate of the 4 genes increased from gastric biopsy specimens to adjacent gastric mucosa to gastric cancer tissue. Concurrent methylation in 2 or more genes was found in 22.9% of adjacent normal gastric mucosa and 75.7% of cancer tissues (Figure 3). The results of bisulfite DNA sequence analysis H2O N2 T2 N1 T1 UP MP U M U M U M U M U M U M U M Ma 200bp 100bp 200bp 100bp Figure 1 - Methylation-specific PCR analysis of p16, Runx3, DAPK and CHFR methylation status. H2O: negative control; T1, T2: tumor tissues; N1, N2: adjacent normal tissues; Ma: 100-bp DNA ladder; MP: methylated positive control; UP: unmethylated positive control; M: methylated; U: unmethylated. fragment size in methylation-positive control and unmethylation-positive control samples, respectively, while the blank control sample did not amplify any fragment. This suggested that the experimental technique and the use of primers and reagents was correct, and the experimental result credible. As shown in Figure 2, the frequency of promoter methylation in tumor tissues was 68.6% (48 of 70) in p16, 60.0% (42 of 70) in Runx3, 60.0% (42 of 70) in DAPK, and 48.6% (34 of 70) in CHFR. In adjacent normal gastric mucosa, hypermethylation was found with the following frequency: 12.9% (9 of 70) in p16, 27.1% (19 of 70) in Runx3, 14.3% (10 of 70) in DAPK, and 22.9% (16 of 70) in CHFR. The promoter methylation frequencies for 50 45 40 35 30 25 20 15 10 5 0 729 Gastric cancer Adjacent gastric mucosa Normal contral *P <0.05 We carried out direct sequence analysis in all the positive samples for 4 genes as determined by MSP, and discovered that all positive samples showed different degrees of methylation. Representative results of bisulfite sequence analysis for gene promoters in gastric cancer samples are shown in Figure 4. Association between promoter methylation and clinicopathological parameters The relationships between the methylation status of p16, Runx3, DAPK and CHFR promoters and established clinicopathological parameters in gastric cancer are summarized in Table 3. No correlation was found between hypermethylation and other clinicopathological parameters such as age, sex, and tumor location (P >0.05). However, we observed a significant association between hypermethylation of Runx3 and tumor size (P <0.05). A statistically significant correlation was also observed between the methylation frequency of DAPK and CHFR and differentiation grade and lymph node metastasis in gastric cancer. Poorly differentiated cancers exhibited higher promoter methylation frequency for DAPK and CHFR than well differentiated cancers, while cancers metastatic to lymph nodes showed 70.2% (33/47) and 57.4% (27/47) promoter methylation for DAPK and CHFR, respectively, which was significantly higher than the 39.1% (9/23) and 30.4% (7/23) found in cancers without lymph node involvement (both P <0.05). In addition, a significant association was ob- g0 40 35 30 g2 20 p 16 Runx 3 DAPX CHFR Figure 2 - Methylation frequency of gastric cancer, adjacent normal gastric mucosa, and normal gastric biopsy samples. Methylation of p16, Runx3, DAPK and CHFR was more frequently detected in gastric cancer than in adjacent gastric mucosa and normal gastric samples (P <0.05). 15 10 0 gene 1 gene 2 gene 3 gene 4 gene g3 25 g4 g1 g2 g0 g1 g3 g4 5 0 Gastric cancer Adjacent gastric mucosa Figure 3 - Different levels and numbers of gene methylation in gastric cancer and adjacent normal gastric mucosa. g, gene. 730 SL HU, XY KONG, ZD CHENG ET AL T C C 20 G A C C C G 30 GT T A C G A T T C G served between hypermethylation of DAPK and clinical stage (P <0.05). We failed to find a significant correlation between hypermethylation of p16 and clinicopathological parameters including sex, age, clinical stage, differentiation grade, and lymph node involvement (P >0.05). Expression of p16, Runx3, DAPK and CHFR mRNA in gastric cancer 120 T C G G C G T p16 In the 34 gastric cancer tissues examined in this study, the expression of mRNA was 26.5% in p16, 20.6% in Runx3, 29.4% in DAPK, and 32.4% in CHFR, which was lower than in adjacent normal gastric mucosa with 64.7% in p16, 67.6% in Runx3, 76.5% in DAPK, and 58.8% in CHFR. The levels of p16, Runx3 and DAPK mRNA expression were significantly different between cancer and adjacent normal gastric mucosa (P <0.05) (Figure 5). Moreover, as shown in Table 4, among the 34 gastric carcinoma samples available for RT-PCR analysis, a significant association was observed between the mRNA expression of the 4 genes and their hypermethylation (P <0.05). In the samples with hypermethylation of the 4 genes, we found only 1 sample with DAPK expression, while the remaining 3 genes showed loss of expression. Representative results of RT-PCR in human gastric cancer samples are shown in Figure 6. Runx3 90 G C G G TC G G CG T T A DAPK CHFR Figure 4 - Representative results of bisulfite DNA sequence analysis of p16, Runx3, DAPK and CHFR genes. Sodium bisulfite treatment of DNA converted all unmethylated cytosines to uracils, but the level of methylated cytosines was unaffected. Table 3 - Relationship between promoter hypermethylation and patients’ clinicopathological parameters Parameters No. p16 gene Runx3 gene DAPK gene CHFR gene M (%) P value M (%) P value M (%) P value M (%) P value 0.408 Gender Male Female 53 17 37 (69.8) 11 (64.7) 0.767 32 (60.4) 10 (58.8) 1.000 33 (62.3) 9 (52.9) 0.574 24 (45.3) 10 (58.8) Age (years) ≥60 <60 43 27 30 (69.8) 18 (66.7) 0.797 26 (60.5) 16 (59.3) 1.000 24 (55.8) 18 (66.7) 0.455 19 (44.2) 15 (55.6) 0.462 Clinical stage I/II III/IV 31 39 18 (58.1) 30 (76.9) 0.122 16 (51.6) 26 (66.7) 0.228 14 (45.2) 28 (71.8) 0.029 11 (35.5) 23 (59.0) 0.059 Differentiation High/ intermediate Low 25 45 16 (64.0) 32 (71.1) 0.597 15 (60.0) 27 (60.0) 1.000 10 (40.0) 32 (71.1) 0.021 8 (32.0) 26 (57.8) 0.048 Tumor size ≥5 cm <5 cm 43 27 31 (72.1) 17 (63.0) 0.441 30 (69.8) 12 (44.4) 0.046 29 (67.4) 13 (48.1) 0.136 24 (55.8) 10 (37.0) 0.147 Tumor location Cardia Gastric body Antrum 29 20 21 22 (75.9) 14 (70.0) 12 (57.1) 0.359 20 (69.0) 10 (50.0) 12 (57.1) 0.239 17 (58.6) 14 (70.0) 11 (52.4) 0.755 10 (34.5) 11 (55.0) 13 (61.9) 0.152 Lymph node metastasis N0 N1/N2/N3 23 47 14 (60.9) 34 (72.3) 0.413 14 (60.9) 28 (59.6) 1.000 9 (39.1) 33 (70.2) 0.019 7 (30.4) 27 (57.4) 0.043 PROMOTER METHYLATION OF GENES AND GASTRIC CARCINOMA Gastric cancer Adjacent gastric mucosa 30 25 *P <0.05 20 15 10 5 0 p 16 Runx 3 DAPX CHFR Figure 5 - mRNA expression of 4 genes in gastric cancer and adjacent normal gastric mucosa. mRNA expression of p16, Runx3 and DAPK was more frequently detected in gastric cancer than adjacent normal gastric mucosa (P<0.05). Table 4 - Relationship between promoter hypermethylation and mRNA expression of 4 genes in gastric cancer mRNA expression p16 mRNA expression P value Runx3 positive negative M U 0 9 P value positive negative 25 0 0.000 M U 0 7 mRNA expression DAPK 27 0 0.000 mRNA expression P value CHFR positive negative M U 1 9 p16 GAPDH DAPK GAPDH 22 2 1 1 2 2 0.000 3 3 P value positive negative 4 4 M U Runx3 GAPDH CHFR GAPDH 0 11 20 3 1 2 1 0.000 3 2 4 3 4 Figure 6 - Representative results of RT-PCR in human gastric cancer tissue samples. GAPDH was used as an internal control. 1, 2: tumor tissues. 3, 4: adjacent normal gastric mucosa. Discussion Altered gene silencing due to DNA hypermethylation is common in human malignancies. This affects multiple genes in cancer cells and the affected genes vary in chemical and disease-dependent aspects29,30. It is therefore necessary to analyze the methylation status of a panel of representative genes in each cancer type. For this purpose CpG island methylator phenotype (CIMP) was introduced by Toyota et al.31 In gastric cancer, hy- 731 permethylation of CpG islands in promoter regions often occurs in important tumor suppressor genes such as hMLH1, p16, CDH1 and APC32-34. In particular, aberrant methylation of the normally unmethylated CpG islands of many tumor suppressor genes is associated with transcriptional inactivation and loss of expression. In this study we examined the methylation status and mRNA expression of the p16, Runx3, DAPK and CHFR genes in primary gastric cancer both using MSP and RT-PCR. In previous studies, the frequencies of the promoter methylation of p16, Runx3, DAPK and CHFR in gastric cancer were 32-43%35-38, 50-64%39-41, 30-90%42-47 and 3052%48-50, respectively. Our results were similar, and the methylation rate of the 4 genes increased from gastric biopsy specimens to adjacent gastric mucosa to gastric cancer tissue. Moreover, the hypermethylation rates for the p16, Runx3, DAPK and CHFR promoters were significantly higher in gastric cancer tissues than in adjacent normal gastric mucosa and control gastric specimens (P <0.05). The overall findings suggest that promoter hypermethylation of the p16, Runx3, DAPK and CHFR genes is frequent in gastric cancer and may be an early molecular event in gastric carcinogenesis. Concurrent methylation of multiple genes appears to be a tumorspecific phenomenon. We found that hypermethylation of Runx3 was significantly associated with tumor size (P <0.05), and that promoter hypermethylation of DAPK and CHFR was associated with differentiation grade and lymph node metastasis in gastric cancer (P <0.05) but not significantly associated with other clinicopathological factors such as sex, age, and tumor location. In contrast to previous reports45,49-51, our data indicated that the hypermethylation rate of DAPK and CHFR was significantly higher in poorly differentiated than well-differentiated gastric cancer (PDAPK = 0.021, PCHFR = 0.048). This suggests that hypermethylation of the DAPK and CHFR genes may play a role in the histological differentiation of gastric cancer and is associated with tumor behavior. The hypermethylation rate of DAPK and CHFR was significantly higher in gastric cancer metastatic to lymph nodes than gastric cancer without lymph node involvement (PDAPK = 0.019, PCHFR = 0.043). In addition, we observed a significant association between hypermethylation of DAPK and clinical stage (P <0.05). Our results suggest that hypermethylation of DAPK and CHFR promoters occurs more often in poorly differentiated tumors and tumors with lymph node metastasis, as the hypermethylation rate of DAPK and CHFR increased with the degree of malignancy of the tumor cells. Hypermethylation of DAPK and CHFR may therefore be an important indicator of molecular biology for evaluating the degree of malignancy and lymph node status of gastric cancer. We determined the expression of p16, Runx3, DAPK and CHFR mRNA by RT-PCR in tissues of 34 patients included in this study, and investigated the correlation be- 732 tween hypermethylation of the 4 genes and mRNA expression. We found a higher level of p16, Runx3, DAPK and CHFR mRNA expression in adjacent normal gastric mucosa than in cancer tissues. Moreover, the levels of p16, Runx3 and DAPK mRNA expression were significantly different between cancer tissues and adjacent normal gastric mucosa. According to Kim et al.40 and Kato et al.52, Runx3 and DAPK mRNA expression is absent in methylated cell lines but present in unmethylated cell lines, and loss of Runx3 and DAPK mRNA expression was related to the aberrant methylation of their CpG islands. Our data in the present study are similar. In gastric cancer tissues with hypermethylation of 4 genes, we found only 1 sample presenting DAPK expression, while the other 3 genes showed loss of expression. Furthermore, a significant association was observed between mRNA expression of the 4 genes and their hypermethylation status (P <0.05). This suggests that promoter hypermethylation of the 4 genes is possibly the main cause of downregulation or loss of mRNA expression. Weak expression and loss of expression due to promoter hypermethylation may play an important role in the pathogenesis of gastric cancer. These results support our initial hypothesis and further studies are warranted to determine whether promoter hypermethylation in precancerous gastric lesions is associated with a higher risk of subsequent cancer development and how to interrupt the malignant transition by developing genetargeting therapies that may reverse aberrant methylation. 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