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From the Pathology Division,* National Cancer Center Research Institute, Tokyo, Japan; the Gastric Surgery Division, National Cancer Center Hospital, Tokyo, Japan; and the Department of Surgery I, Oita Medical University, Oita, Japan
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Abstract |
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To date, three enzymes, DNA methyltransferase 1 (DNMT1),9 DNMT3a, and DNMT3b,10 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,11 and targets replication foci by binding to proliferating cell nuclear antigen (PCNA),12 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.13
Overexpression of DNMT1 has been detected in several human cancers.14-16 With regard to gastric cancer, we have reported that DNMT1 mRNA expression levels were significantly higher in cancer tissues than in normal gastric mucosae.16 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 manner17 ) in gastric and colorectal cancers.16 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 pylori18 and Epstein-Barr virus (EBV) infection.19
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Materials and Methods |
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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.
Immunohistochemistry
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;20
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.21
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:22 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.
Methylation-Specific Polymerase Chain Reaction (MSP) and Combined Bisulfite Restriction Enzyme Analysis (COBRA)
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.23 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 hMLH124 and E-cadherin23 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.25 Bisulfite-modified DNA was amplified by PCR using previously described primers that were designed to amplify methylated and unmethylated genomic regions equally.17 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).
In Situ Hybridization
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.
Statistics
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.
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Results |
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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.
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. DNMT1 protein overexpression was significantly associated with the degree of histological differentiation (P < 0.001).
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. 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.
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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.
Correlation between DNMT1 Protein Overexpression and the DNA Methylation Status of Multiple CpG Islands
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.17
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
).
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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.
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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 x400 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
).
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To understand the background behind DNMT1 protein overexpression, etiological factors considered to be involved in gastric carcinogenesis were examined. Although 44 (46%) of the 96 patients examined showed H. pylori infection in their noncancerous mucosae, there was no significant correlation between H. pylori infection and DNMT1 protein overexpression (P = 0.113). Similarly, there was no correlation between H. pylori infection and CIMP (P = 0.146). The incidence of EBV infection was examined by in situ hybridization in the same cohort (Figure 8)
. Four patients (4%) had EBV infection in their cancer cells, and all four of these cancers showed DNMT1 protein overexpression. Furthermore, EBV infection was significantly associated with DNA hypermethylation of five or more C-type CpG islands (P < 0.001).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Regional DNA hypermethylation of CpG islands was detected even in noncancerous mucosae, which can contain precursor cells for cancers and/or precancerous lesions, such as intestinal metaplasia. However, the incidence and degree of DNA hypermethylation was increased in the gastric cancers compared with the noncancerous mucosae. These data are consistent with previous findings in precancerous conditions and cancers of various organs.3-8 Twenty-four percent of the gastric cancers were CIMP-positive, confirming that aberrant DNA methylation is associated with the multistage development of certain subgroups of gastric cancers. Targeting of the substrate DNA by DNMT1 may be disrupted by mechanisms such as dysfunction of p21WAF1,26 which competes with DNMT1 for binding to PCNA, in cancer cells.12 Moreover, it has recently been suggested that DNMT1 is capable of de novo methylating activity as well as having a maintenance function.13 Therefore, it is feasible that, in cancers, DNMT1 participates in the DNA hypermethylation of CpG islands that are not methylated in normal cells. We have previously reported that DNMT1 mRNA overexpression correlates significantly with CIMP in gastric and colorectal cancers.16 In the present study, we demonstrated a significant correlation between DNMT1 expression and CIMP in gastric cancers, even at the protein level. Moreover, among the C-type CpG islands examined, those of the hMLH1 and THBS-1 genes may be particularly targeted by overexpressed DNMT1. This is compatible with previous reports that silencing of the hMLH1 gene results in frequent microsatellite instability in gastric cancers.27,28
E-cadherin is one of the most important molecules involved in intracellular adhesion and cancer morphogenesis.29 In addition to the development of mutations,30-32 it has become apparent that the E-cadherin gene can be silenced by DNA hypermethylation around its promoter regions in gastric cancers.33,34 Indeed, reduced E-cadherin protein expression associated with DNA hypermethylation around the promoter region of the E-cadherin gene was significantly correlated with poorer glandular differentiation of the cancers examined in this study. We showed a significant correlation between DNMT1 protein overexpression and reduced E-cadherin protein expression in gastric cancers, indicating that the E-cadherin gene may also be a target of overexpressed DNMT1.
Although DNMT1 is a major DNMT in humans, so far two other enzymes, DNMT3a and DNMT3b, have also been shown to possess DNMT activity.1 Genomic methylation patterns may be established through cooperation among these three DNMTs, even in cancer cells.13 Moreover, it may be that unknown co-factors potently target these DNMTs to unmethylated substrates in a sequence-specific manner in cancers. Thus, DNMT1 overexpression alone may not explain regional DNA hypermethylation in cancers. Further studies on the cooperation between DNMT1 and other components of the DNA methylation machinery in tissue specimens may further our understanding of the basis of regional DNA hypermethylation during gastric carcinogenesis. Although the degree of DNA hypermethylation in noncancerous mucosae seemed slight, DNA methylation of CpG islands actually occurred in a considerable number of noncancerous mucosae that can contain precursor cells for cancer. However, DNMT1 overexpression was not detected immunohistochemically in noncancerous mucosae except for proliferative zones, in which DNMT1 performs maintenance methylation at the replication foci. The protein expression levels of DNMT1 in noncancerous mucosae may not reach the threshold that can be detected by the present immunohistochemical method, even though it promotes slight DNA hypermethylation. Otherwise, slight DNA hypermethylation in noncancerous mucosae may be attributable to alterations in components of the DNA methylation machinery other than DNMT1. Although frequent regional DNA hypermethylation has been reported in gastric type tumors,35 DNMT1 protein expression was not different between gastric and intestinal phenotypes in the present study. We assume again that unknown components of the DNA methylation machinery may potently target DNMT1 to substrate DNA, or that DNMTs other than DNMT1 may also participate in regional DNA hypermethylation in cancers showing gastric phenotype.
On the other hand, the overall 5-methylcytosine level is lower in cancer tissues than in normal tissues.1 In common with the previously reported immunohistochemical findings for DNMT1 in colorectal cancers36 and HCCs,21 some cancer cells exhibited very weak or no DNMT1 immunoreactivity, even in a gastric cancer that was considered DNMT1-positive according to the criteria described in the Materials and Methods section. Cancer cells with low DNMT1 levels may be prone to overall DNA hypomethylation.
An initial increase in DNMT1 mRNA expression in colon cancers became far more modest when the expression level was normalized according to that of a cell proliferation marker.37 DNMT1 mRNA is expressed mainly during the S-phase and, because tumor tissue is presumed to contain a greater proportion of dividing cells than normal tissue, some debate has arisen as to whether increased DNMT1 expression is because of an increase in the proportion of dividing cells or to an acute increase in DNMT1 expression per individual cell. This continuing discussion prompted us to compare DMNT1 immunoreactivity and the PCNA-labeling index in gastric cancers. In the present study, DNMT1 protein overexpression was not significantly associated with increased proliferative activity of gastric cancer cells; we have previously observed a similar discrepancy between DNMT1 expression and cell proliferative activity in precancerous conditions for urinary bladder carcinomas38 and in certain subgroups of HCCs.21 These findings suggest that DNMT1 protein overexpression does not result entirely from increased numbers of dividing cells during carcinogenesis.
Finally, we focused on etiological factors believed to be involved in gastric carcinogenesis to clarify the background behind DNMT1 protein overexpression. Although DNMT1 protein overexpression was not significantly associated with the incidence of H. pylori infection in corresponding noncancerous mucosae, all four of the EBV-positive cancers showed DNMT1 protein overexpression. EBV infection was significantly associated with the DNA hypermethylation of five or more C-type CpG islands, in accordance with a previous report that the average number of methylated CpG islands was higher in EBV-positive gastric cancers than in EBV-negative ones.39 These observations indicate that DNMT1 may play an important role in EBV-related gastric carcinogenesis via aberrant DNA methylation. Although it has previously been reported that transfection of latent membrane protein-1 of EBV induces the expression and activity of DNMT1,40 latent membrane protein-1 is not typically expressed in EBV-associated gastric cancers.41 Although the molecular mechanism governing how EBV infection results in overexpression of DNMT1 protein requires further elucidation, and our present findings must be interpreted with caution because of the small number of EBV-positive gastric cancers studied, EBV infection and other etiological factors may be associated with DNMT1 overexpression and contribute toward gastric carcinogenesis by inducing frequent DNA hypermethylation of multiple CpG islands.
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Footnotes |
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Supported by a Grant-in-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control; a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare of Japan; and a research resident fellowship from the Foundation for Promotion of Cancer Research in Japan (to T.N.).
Accepted for publication October 8, 2003.
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