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• W1963567995 abstract "We evaluated the significance of aberrant DNA methyltransferase 1 (DNMT1) protein expression during gastric carcinogenesis. The protein expression of DNMT1, Muc2, human gastric mucin, E-cadherin, and proliferating cell nuclear antigen was examined immunohistochemically in gastric cancers and corresponding noncancerous mucosae from 134 patients. The DNA methylation status of the CpG islands of the p16, human MutL homologue 1 (hMLH1), E-cadherin, and thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT)-1, -2, -12, and -31 clones was examined by methylation-specific polymerase chain reaction and combined bisulfite restriction enzyme analysis. Epstein-Barr virus (EBV) infection was detected by in situ hybridization. Nuclear immunoreactivity for DNMT1 was not detected in any of the noncancerous epithelia, except in proliferative zones (positive internal control), but was found in 97 (72%) of the gastric cancers. DNMT1 overexpression correlated significantly with poorer tumor differentiation (P < 0.001), but not with the phenotype (gastric type versus intestinal type) of the cancer cells. It also correlated significantly with DNA hypermethylation of the CpG islands of the hMLH1 (P = 0.024) and THBS-1 genes (P = 0.043), and with the CpG island methylator phenotype in the gastric cancers (P = 0.007). Reduced E-cadherin expression correlated significantly with poorer tumor differentiation (P = 0.002), DNA hypermethylation of the E-cadherin gene (P < 0.001) and DNMT1 overexpression (P = 0.014). DNMT1 overexpression was also associated with EBV infection (a potential etiological factor in gastric carcinogenesis) but not with the proliferative activity of the cancer cells as indicated by the proliferating cell nuclear antigen-labeling index. These results suggest that DNMT1 overexpression may not be just a secondary effect of increased cancer cell proliferative activity, but may be associated with EBV infection and other etiological factors during gastric carcinogenesis. Furthermore, DNMT1 may play a significant role in the development of poorly differentiated gastric cancers by inducing frequent DNA hypermethylation of multiple CpG islands. We evaluated the significance of aberrant DNA methyltransferase 1 (DNMT1) protein expression during gastric carcinogenesis. The protein expression of DNMT1, Muc2, human gastric mucin, E-cadherin, and proliferating cell nuclear antigen was examined immunohistochemically in gastric cancers and corresponding noncancerous mucosae from 134 patients. The DNA methylation status of the CpG islands of the p16, human MutL homologue 1 (hMLH1), E-cadherin, and thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT)-1, -2, -12, and -31 clones was examined by methylation-specific polymerase chain reaction and combined bisulfite restriction enzyme analysis. Epstein-Barr virus (EBV) infection was detected by in situ hybridization. Nuclear immunoreactivity for DNMT1 was not detected in any of the noncancerous epithelia, except in proliferative zones (positive internal control), but was found in 97 (72%) of the gastric cancers. DNMT1 overexpression correlated significantly with poorer tumor differentiation (P < 0.001), but not with the phenotype (gastric type versus intestinal type) of the cancer cells. It also correlated significantly with DNA hypermethylation of the CpG islands of the hMLH1 (P = 0.024) and THBS-1 genes (P = 0.043), and with the CpG island methylator phenotype in the gastric cancers (P = 0.007). Reduced E-cadherin expression correlated significantly with poorer tumor differentiation (P = 0.002), DNA hypermethylation of the E-cadherin gene (P < 0.001) and DNMT1 overexpression (P = 0.014). DNMT1 overexpression was also associated with EBV infection (a potential etiological factor in gastric carcinogenesis) but not with the proliferative activity of the cancer cells as indicated by the proliferating cell nuclear antigen-labeling index. These results suggest that DNMT1 overexpression may not be just a secondary effect of increased cancer cell proliferative activity, but may be associated with EBV infection and other etiological factors during gastric carcinogenesis. Furthermore, DNMT1 may play a significant role in the development of poorly differentiated gastric cancers by inducing frequent DNA hypermethylation of multiple CpG islands. DNA methylation plays an important role in transcriptional regulation and chromatin remodeling in mammalian cells.1Jones PA Baylin SB The fundamental role of epigenetic events in cancer.Nat Rev Genet. 2002; 3: 415-428Crossref PubMed Google Scholar Both overall DNA hypomethylation and more regional DNA hypermethylation have been well documented in various cancers.1Jones PA Baylin SB The fundamental role of epigenetic events in cancer.Nat Rev Genet. 2002; 3: 415-428Crossref PubMed Google Scholar, 2Vachtenheim J Horakova I Novotna H Hypomethylation of CCGG sites in the 3′ region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer.Cancer Res. 1994; 54: 1145-1148PubMed Google Scholar, 3Kanai Y Ushijima S Tsuda H Sakamoto M Sugimura T Hirohashi S Aberrant DNA methylation on chromosome 16 is an early event in hepatocarcinogenesis.Jpn J Cancer Res. 1996; 87: 1210-1217Crossref PubMed Scopus (73) Google Scholar, 4Kanai Y Ushijima S Hui AM Ochiai A Tsuda H Sakamoto M Hirohashi S The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas.Int J Cancer. 1997; 71: 355-359Crossref PubMed Scopus (243) Google Scholar, 5Eguchi K Kanai Y Kobayashi K Hirohashi S DNA hypermethylation at the D17S5 locus in non-small cell lung cancers: its association with smoking history.Cancer Res. 1997; 57: 4913-4915PubMed Google Scholar, 6Kanai Y Ushijima S Ochiai A Eguchi K Hui AM Hirohashi S DNA hypermethylation at the D17S5 locus is associated with gastric carcinogenesis.Cancer Lett. 1998; 122: 135-141Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 7Kondo Y Kanai Y Sakamoto M Mizokami M Ueda R Hirohashi S Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis—a comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma.Hepatology. 2000; 32: 970-979Crossref PubMed Scopus (242) Google Scholar, 8Kanai Y Ushijima S Tsuda H Sakamoto M Hirohashi S Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis.Cancer Lett. 2000; 148: 73-80Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar Aberrant DNA methylation may be involved in carcinogenesis as a result of 1) increased gene mutagenicity because of deamination of 5-methylcytosine to thymine; 2) a possible association of aberrant DNA methylation with allelic loss; and 3) repression of gene transcription through methylation of CpG islands in regulatory regions of specific genes, including tumor-suppressor genes.1Jones PA Baylin SB The fundamental role of epigenetic events in cancer.Nat Rev Genet. 2002; 3: 415-428Crossref PubMed Google Scholar To date, three enzymes, DNA methyltransferase 1 (DNMT1),9Bestor T Laudano A Mattaliano R Ingram V Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. The carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases.J Mol Biol. 1988; 203: 971-983Crossref PubMed Scopus (725) Google Scholar DNMT3a, and DNMT3b,10Okano M Xie S Li E Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases.Nat Genet. 1998; 19: 219-220Crossref PubMed Scopus (1305) Google Scholar have been confirmed to possess DNMT activity. Of these, DNMT1 is the major and best known. As DNMT1 shows a preference for hemimethylated rather than unmethylated substrates in vitro,11Bestor TH Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain.EMBO J. 1992; 11: 2611-2617Crossref PubMed Scopus (393) Google Scholar and targets replication foci by binding to proliferating cell nuclear antigen (PCNA),12Chuang LS Ian HI Koh TW Ng HH Xu G Li BF Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1.Science. 1997; 277: 1996-2000Crossref PubMed Scopus (792) Google Scholar it seems to be a maintenance form of DNMT that copies methylation patterns after DNA replication. However, some workers have proposed that DNMT1 possesses both maintenance and de novo DNA methylation activity in vivo, regardless of its in vitro substrate preference.13Rhee I Bachman KE Park BH Jair KW Yen RW Schuebel KE Cui H Feinberg AP Lengauer C Kinzler KW Baylin SB Vogelstein B DNMT1 and DNMT3b cooperate to silence genes in human cancer cells.Nature. 2002; 416: 552-556Crossref PubMed Scopus (1044) Google Scholar Overexpression of DNMT1 has been detected in several human cancers.14Sun L Hui AM Kanai Y Sakamoto M Hirohashi S Increased DNA methyltransferase expression is associated with an early stage of human hepatocarcinogenesis.Jpn J Cancer Res. 1997; 88: 1165-1170Crossref PubMed Scopus (87) Google Scholar, 15Saito Y Kanai Y Sakamoto M Saito H Ishii H Hirohashi S Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis.Hepatology. 2001; 33: 561-568Crossref PubMed Scopus (234) Google Scholar, 16Kanai Y Ushijima S Kondo Y Nakanishi Y Hirohashi S DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancers.Int J Cancer. 2001; 91: 205-212Crossref PubMed Scopus (193) Google Scholar With regard to gastric cancer, we have reported that DNMT1 mRNA expression levels were significantly higher in cancer tissues than in normal gastric mucosae.16Kanai Y Ushijima S Kondo Y Nakanishi Y Hirohashi S DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancers.Int J Cancer. 2001; 91: 205-212Crossref PubMed Scopus (193) Google Scholar Moreover, during this previous study, we found that increased DNMT1 mRNA expression correlated significantly with the CpG island methylator phenotype (defined as frequent DNA hypermethylation of C-type CpG islands that are methylated in a cancer-specific but not an age-dependent manner17Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa JP CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2166) Google Scholar) in gastric and colorectal cancers.16Kanai Y Ushijima S Kondo Y Nakanishi Y Hirohashi S DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancers.Int J Cancer. 2001; 91: 205-212Crossref PubMed Scopus (193) Google Scholar However, most previous analyses concerning DNMT1 expression in human cancers have been performed at the mRNA level. To our knowledge, DNMT1 protein expression in gastric cancers has never been reported. The aim of this study was therefore to evaluate the significance of aberrant DNMT1 protein expression during gastric carcinogenesis. Firstly, we searched for correlations between DNMT1 protein expression and the clinicopathological features of gastric cancers. Secondly, to determine the targets of aberrantly expressed DNMT1 during gastric carcinogenesis, we examined the correlations between DNMT1 protein expression on the one hand and the DNA methylation status of multiple C-type CpG islands and E-cadherin expression on the other. Thirdly, to clarify the background behind aberrant DNMT1 protein expression, we investigated correlations with the proliferative activity of cancer cells (as indicated by the PCNA-labeling index) and with etiological factors that are believed to be involved in gastric carcinogenesis, such as Helicobacter pylori18Sipponen P Helicobacter pylori: a cohort phenomenon.Am J Surg Pathol. 1995; 19: S30-S36PubMed Google Scholar and Epstein-Barr virus (EBV) infection.19Shibata D Weiss LM Epstein-Barr virus-associated gastric adenocarcinoma.Am J Pathol. 1992; 140: 769-774PubMed Google Scholar Cancerous tissues and corresponding noncancerous mucosae were obtained from 134 patients with primary gastric cancer. These patients underwent surgery at the National Cancer Center Hospital, Tokyo, Japan, between 1998 and 2002. They included 92 men and 42 women with a mean (± SD) age of 61 ± 7 years (range, 45 to 83 years). None of the patients received any preoperative treatment, such as radiation or chemotherapy. Based on histological examinations, the 134 tumors were classified as 23 well differentiated, 31 moderately differentiated, and 80 poorly differentiated (including signet ring cell and mucinous carcinomas) adenocarcinomas. Five-μm-thick sections of formalin-fixed, paraffin-embedded tissue specimens from all 134 patients were deparaffinized and dehydrated. For antigen retrieval, the sections were heated for 10 minutes at 120°C in an autoclave. Nonspecific reactions were blocked with 2% normal swine serum. All sections were incubated with specific primary antibodies that recognized DNMT1 (goat polyclonal antibody, sc-10219, dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), Muc2 (mouse monoclonal antibody, Ccp58, dilution 1:100; Novocastra, Newcastle-on-Tyne, UK), human gastric mucin (mouse monoclonal antibody, 45M1, dilution 1:50; Novocastra), E-cadherin (mouse monoclonal antibody, HECD-1;20Shimoyama Y Hirohashi S Hirano S Noguchi M Shimosato Y Takeichi M Abe O Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas.Cancer Res. 1989; 49: 2128-2133PubMed Google Scholar dilution 1:500), PCNA (mouse monoclonal antibody, p56720, dilution 1:200; Transduction Laboratories, Lexington, KY) and H. pylori (rabbit polyclonal antibody, B0471, dilution 1:20; DAKO, Glostrup, Denmark), respectively. We previously confirmed the specificity of the goat anti-human DNMT1 polyclonal antibody by Western blotting analysis: an immunoreactive band of ∼193.5 kd, corresponding to the molecular mass of DNMT1, was detected in human cancer cells, but no nonspecific bands were detected with this antibody.21Saito Y Kanai Y Nakagawa T Sakamoto M Saito H Ishii H Hirohashi S Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas.Int J Cancer. 2003; 105: 527-532Crossref PubMed Scopus (223) Google Scholar All primary antibody incubations were conducted at 4°C overnight and were followed by incubation with biotinylated secondary antibodies (anti-goat IgG, anti-mouse IgG, or anti-rabbit IgG, dilution 1:200; Vector Laboratories, Burlingame, CA) at room temperature for 30 minutes. The sections were then treated with Vectastain Elite ABC reagent (Vector Laboratories). 3.3′-Diaminobenzidine tetrahydrochloride was used as the chromogen. All sections were counterstained with hematoxylin. The gastric cancers were classified into three phenotypes according to previously described criteria:22Egashira Y Shimoda T Ikegami M Mucin histochemical analysis of minute gastric differentiated adenocarcinoma.Pathol Int. 1999; 49: 55-61Crossref PubMed Scopus (84) Google Scholar the gastric type (positive for human gastric mucin), the intestinal type (positive for Muc2), and the mixed type (positive for both human gastric mucin and Muc2). For the evaluations of DNMT1 and PCNA expression, nuclear immunoreactivity in the proliferative zones of noncancerous foveolar epithelia was used as a positive internal control for all sections. Similarly, immunoreactivity in the cell membranes of noncancerous foveolar epithelia was used as a positive internal control for all sections during the evaluation of E-cadherin expression. As a negative control, the primary antibodies were omitted from the reaction sequence. High-molecular-weight DNA was extracted from 105 fresh paired samples of cancerous tissues and their corresponding noncancerous mucosae by phenol-chloroform extraction and dialysis. Bisulfite conversion was performed using 1 μg of genomic DNA and the reagents provided in the CpGenome DNA modification kit (Intergen, Purchase, NY). This process converts unmethylated cytosine residues to uracil, whereas methylated cytosine residues remain unchanged. The DNA methylation status of the CpG islands of the p16, MutL homologue 1 (hMLH1), and E-cadherin genes was determined by MSP. This technique is based on the principle that the DNA sequences of methylated and unmethylated genomic regions differ after bisulfite conversion and can thus be distinguished by sequence-specific polymerase chain reaction (PCR) primers.23Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5260) Google Scholar The bisulfite-modified DNA of the p16 gene was amplified using the primer sets provided in the CpG WIZ amplification kit (Intergen) and that of the hMLH124Herman JG Umar A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1717) Google Scholar and E-cadherin23Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5260) Google Scholar genes was amplified using the previously described primers. The DNA methylation status of the thrombospondin-1 (THBS-1) gene and the methylated in tumor (MINT)-1, -2, -12, and -31 clones was determined by COBRA.25Xiong Z Laird PW COBRA: a sensitive and quantitative DNA methylation assay.Nucleic Acids Res. 1997; 25: 2532-2534Crossref PubMed Scopus (1047) Google Scholar Bisulfite-modified DNA was amplified by PCR using previously described primers that were designed to amplify methylated and unmethylated genomic regions equally.17Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa JP CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2166) Google Scholar The amplified fragments were digested with restriction enzymes that digest DNA only if the CpG sites in their recognition sequences are methylated: TaqI for the THBS-1 gene and the MINT-1 and -2 clones, MaeII for the MINT-12 clone and BstUI for the MINT-31 clone, respectively. The reaction products were separated electrophoretically on a 3% agarose gel and stained with ethidium bromide. Signal intensities were measured using an image analyzer (model FMBIO-2; Takara, Ohtsu, Japan). Five-μm-thick sections of formalin-fixed, paraffin-embedded tissue specimens from all 134 patients were deparaffinized, dehydrated, and predigested with proteinase K. The sections were then hybridized with a digoxigenin-labeled EBV encoding RNA (EBER) 1 oligonucleotide probe (EBV detection kit; Nichirei, Tokyo, Japan) for 2 hours at 37°C. Anti-digoxigenin antibody-alkaline phosphatase was used with a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate to detect the EBER1 signal. Positive control specimens were provided by the manufacture. As a negative control, the EBER1 probe was omitted from the reaction sequence. Correlations between the incidence of DNMT1 immunoreactivity and variables including clinicopathological parameters, the DNA methylation status of CpG islands, E-cadherin expression, and H. pylori and EBV infection were analyzed using the chi-square test. Correlations between the PCNA-labeling index and clinicopathological parameters or DNMT1 immunoreactivity were analyzed using the Mann-Whitney U-test or the Kruskal-Wallis test. Differences with P values <0.05 were considered significant. Immunoreactivity for DNMT1 was detected in the nuclei, but not in the cytoplasm or cell membranes, of cells in the proliferating zones of foveolar epithelia, lymphocytes, and cancer cells (Figure 1). To discriminate definitely positive cases from cases with leaky background level signal, if more than 30% of the cells in a tissue sample exhibited nuclear staining the sample was considered to show positive immunoreactivity. None (0%) of the 134 noncancerous epithelia exhibited DNMT1 immunoreactivity (except in the proliferative zones, which acted as the positive internal control for the analysis), whereas 97 (72%) of the 134 gastric cancers were DNMT1-positive. Correlations between the incidence of nuclear immunoreactivity for DNMT1 and the clinicopathological features of the gastric cancers are shown in Table 1. DNMT1 protein overexpression was significantly associated with the degree of histological differentiation (P < 0.001).Table 1DNMT1 Protein Expression and the PCNA-Labeling Index in the Gastric CancersVariablesAnalyzedDNMT-1 positive [number of cases (%)]P†Chi-square test,PCNA-labeling index [mean ± SD (%)]PTumor differentiation Well differentiated237 (30%)<.00162 ± 250.165‡Kruskal-Wallis test. Moderately differentiated3118 (58%)64 ± 17 Poorly differentiated8072 (90%)58 ± 27Phenotype*Cellular phenotypes are defined as described in the Materials and Methods section. Gastric type5038 (76%)0.13760 ± 290.793‡Kruskal-Wallis test. Intestinal type3421 (62%)57 ± 29 Mixed type5034 (68%)62 ± 23Depth of invasion Mucosa/submucosa3828 (74%)0.42356 ± 320.125‡Kruskal-Wallis test. Muscularis propria/subserosa116 (55%)70 ± 18 Serosa8563 (74%)60 ± 24Vascular involvement Negative5541 (75%)0.49456 ± 300.533§Mann-Whitney U-test. Positive7956 (71%)63 ± 23Lymphnode metastasis Negative8160 (74%)0.93159 ± 270.465§Mann-Whitney U-test. Positive5337 (70%)61 ± 26* Cellular phenotypes are defined as described in the Materials and Methods section.† Chi-square test,‡ Kruskal-Wallis test.§ Mann-Whitney U-test. Open table in a new tab Next, we evaluated cellular phenotypes based on immunohistochemistry for Muc2 and human gastric mucin, as shown in Figure 2. Fifty (37%) of the cancers showed a gastric phenotype, 34 (26%) showed an intestinal phenotype, and a further 50 (37%) showed a mixed cellular phenotype. There was no significant correlation between DNMT1 protein overexpression and the cellular phenotypes. We then focused on the histological features of the noncancerous mucosae, as intestinal metaplasia is considered to be a precancerous lesion for adenocarcinomas with an intestinal phenotype. There was no significant correlation between DNMT1 protein overexpression in the gastric cancers and the presence or absence (or, if present, the degree) of intestinal metaplasia in the corresponding noncancerous mucosae (data not shown). DNMT1 protein overexpression was not significantly associated with other parameters relating to cancer aggressiveness, such as the depth of invasion, vascular involvement, or lymph node metastasis. Figure 3 shows examples of the PCR products from MSP and COBRA. The incidence of DNA methylation of the C-type CpG islands of each of the genes and MINT clones tested in the noncancerous mucosae and gastric cancers is summarized in Table 2. DNA methylation of at least one C-type CpG island was seen in 50 (48%) of the 105 noncancerous mucosae and 83 (80%) of the 105 gastric cancers examined. For all patients showing DNA methylation of a certain CpG island in both their noncancerous mucosa and cancer, the signal intensity of the reaction products reflecting the presence of methylated DNA was increased in the cancer compared with the corresponding noncancerous mucosa (Figure 3A). The DNA methylation status of each C-type CpG island in the gastric cancers is shown in Figure 4. When DNA hypermethylation was seen on three or more C-type CpG islands, we regarded the patient as being CpG island methylator phenotype (CIMP)-positive, based on previously described criteria.17Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa JP CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2166) Google Scholar Twenty-five (24%) of the 105 gastric cancers were considered CIMP-positive. Furthermore, there were significant correlations between DNMT1 protein overexpression and DNA hypermethylation of each CpG island of the hMLH1 (P = 0.024) and THBS-1 (P = 0.043) genes in the gastric cancers (Table 3). There was also a significant correlation between DNMT1 protein overexpression and CIMP for the gastric cancer tissues (P = 0.007, Table 3).Table 2The DNA Methylation Status of the C-Type CpG Islands of Various Genes and Clones in Noncancerous Mucosae and Gastric CancersNumber of tissue samples (%)Noncancerous mucosaeGastric cancersCpG islandsAnalyzedDNA hypermethylation detectedAnalyzedDNA hypermethylation detectedp1610418 (17)10323 (22)hMLH110515 (14)10518 (17)THBS-11052 (2)10425 (24)MINT110523 (22)10539 (37)MINT21051 (1)10526 (25)MINT121046 (6)10418 (17)MINT311050 (0)10510 (10) Open table in a new tab Figure 4Protein expression of DNMT1 and DNA methylation profiles for seven C-type CpG islands in 105 gastric cancers. The DNA methylation status was examined using MSP or COBRA (see Figure 3). Patient numbers are indicated on the vertical columns and the seven CpG islands are indicated on the top row. +, DNMT1 protein overexpression-positive; −, DNMT1 protein overexpression-negative; solid box, methylated; open box, unmethylated; ND, not done. When DNA hypermethylation was seen in three or more CpG islands, the patient was regarded as being CIMP-positive.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 3Correlations between DNMT1 Protein Overexpression and the DNA Methylation Status of Each C-Type CpG Island, CIMP and E-Cadherin Protein Expression in Gastric CancersDNMT1 expression (number of tissue samples)P*Chi-square test. Reduced: over 50% of the cancer cells in a particular patient's sample lacked or showed only slight E-cadherin immunoreactivity.PositiveNegativeCpG islands p16 Methylated1330.361 Unmethylated5624 hMLH1 Methylated2020.024 Unmethylated4925 THBS-1 Methylated1820.043 Unmethylated5125 MINT1 Methylated2860.091 Unmethylated4121 MINT2 Methylated1840.237 Unmethylated5123 MINT12 Methylated1330.361 Unmethylated5624 MINT31 Methylated1000.503 Unmethylated5927CIMP Positive1010.007 Negative5926E-cadherin expression Maintained48270.014 Reduced4910* Chi-square test. Reduced: over 50% of the cancer cells in a particular patient's sample lacked or showed only slight E-cadherin immunoreactivity. Open table in a new tab E-cadherin was detected in the cell membranes of epithelia from all (100%) of the 134 noncancerous mucosae. However, E-cadherin protein expression was considered to be reduced when more than 50% of the gastric cancer cells in a particular patient's sample lacked or showed only slight membranous E-cadherin immunoreactivity. Reduced E-cadherin expression was observed in 59 (44%) of the 134 gastric cancers (Figure 5). The incidence of reduced E-cadherin expression was 22% in well, 23% in moderately, and 59% in poorly differentiated adenocarcinomas, respectively, and reduced E-cadherin expression was significantly associated with poorer tumor differentiation (P = 0.002). DNA methylation of CpG island of the E-cadherin gene was seen in 20 (19%) of the 105 gastric cancers examined (Figure 3C) and there was a significant correlation between DNA hypermethylation of CpG island of the E-cadherin gene and reduced E-cadherin expression in the gastric cancers (P < 0.001). Furthermore, there was a significant correlation between DNMT1 protein overexpression and reduced E-cadherin expression in gastric cancers (P = 0.014, Table 3). In fact, coincidence of nuclear immunoreactivity of DNMT1 and lack of membrane immunoreactivity of E-cadherin was frequently observed in individual cancer cells. Examples of the results of immunohistochemistry for PCNA are shown in Figure 6. To evaluate the PCNA-labeling index, ∼300 cells per specimen were examined at a magnification of ×400 under a microscope and the cells that did and did not show nuclear immunoreactivity for PCNA were counted. The PCNA-labeling index was expressed as the percentage of the total cells that showed nuclear immunoreactivity. The PCNA-labeling index was increased even in well-differentiated adenocarcinomas, in which the incidence of overexpression of DNMT1 protein was still low (Table 1), and coincidence of nuclear immunoreactivity of PCNA and lack of nuclear immunoreactivity of DNMT1 was frequently observed in individual well-differentiated cancer cells. Thus, DNMT1 protein overexpression was not significantly associated with the PCNA-labeling index in the gastric cancers (P = 0.309, Figure 7).Figure 7Average PCNA-labeling indices in DNMT1 protein overexpression-positive (n = 97) and -negative (n = 37) gastric cancers. Error bar, SD DNMT1 protein overexpression was not significantly associated with the PCNA-labeling index in the gastric cancers (P = 0.309, Mann-Whitney U-test).View Large" @default.
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• W1963567995 title "Increased DNA Methyltransferase 1 (DNMT1) Protein Expression Correlates Significantly with Poorer Tumor Differentiation and Frequent DNA Hypermethylation of Multiple CpG Islands in Gastric Cancers" @default.
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