Teiichiro Honda

Multiple tumor suppressor genes are increasingly methylated with age in non-neoplastic gastric epithelia

A number of tumor suppressor and tumor‐related genes are silenced by promoter hypermethylation in gastric cancer. Hypermethylation is not restricted to cancer cells, but is also present in non‐neoplastic cells during aging. Such age‐related methylation in non‐neoplastic gastric epithelia is postulated to constitute a field defect that increases the risk for development of gastric cancer. To quantitatively evaluate age‐related methylation in non‐neoplastic gastric epithelia, we used a fiber‐type DNA microarray on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the promoter CpG island of four tumor suppressor genes, lysyl oxidase (LOX), p16, RUNX3 and tazarotene‐induced gene 1 (TIG1), were amplified by PCR using Cy5 end labeled primers. Methylation rates (MRs) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Non‐neoplastic gastric mucosa was obtained from 24 non‐cancer‐bearing stomachs at autopsy. MRs ranged from 0.0% to 77.2% (mean, 15.8%) for LOX, 0.0% to 45.8% (mean, 10.0%) for p16, 0.0% to 83.8% (mean, 9.0%) for RUNX3, and 0.0% to 46.1% (mean, 6.6%) for TIG1, and significantly correlated with aging (P < 0.01). The regression curves were: y = 0.013x2 − 0.6184x + 4.0512, R2 = 0.5728 (P < 0.001) for LOX; y = 0.0107x2 − 0.6055x + 5.2943, R2 = 0.7891 (P < 0.00001) for p16; y = 0.0182x2 − 1.2234x + 11.566, R2 = 0.5595 (P < 0.001) for RUNX3; and y = 0.0068x2 − 0.3586x + 2.4306, R2 = 0.4670 (P < 0.01) for TIG1. Thus, our present results are consistent with the notion that age‐related methylation is associated with cancer susceptibility in the elderly. Quantitative analysis of DNA methylation using DNA microarrays is a promising method for risk assessment in the development of gastric cancer. (Cancer Sci 2006; 97: 1155–1158)

Promoter hypermethylation of DAP‐kinase is associated with poor survival in primary biliary tract carcinoma patients

To clarify the clinicopathological significance of promoter hypermethylation of tumor suppressor and tumor‐related genes in biliary tract carcinomas, we examined the promoter methylation status of multiple genes in primary biliary tract carcinomas. These consisted of carcinomas of the bile duct, gallbladder, and duodenal ampulla. Surgical specimens were obtained from a total of 37 patients with biliary tract carcinoma. The cohort consisted of 23 patients with bile duct carcinoma, 9 patients with gallbladder carcinoma, and 5 patients with ampullary carcinoma. The methylation status of CHFR, DAP‐kinase, E‐cadherin, hMLH1, p16, RASSF1A, and RUNX3 was examined by methylation‐specif ic polymerase chain reaction (MSP). The correlation between methylation status and clinicopathological characteristics was then assessed. The methylation frequencies of CHFR, DAP‐kinase, E‐cadherin, hMLH1, p16, RASSF1A, and RUNX3 genes were 16.2%, 21.4%, 27.0%, 8.1%, 24.3%, 27.0%, and 56.8%, respectively, in primary biliary tract carcinomas. The number of methylated genes per sample was 2.17±0.28 (average±SD) in bile duct carcinomas, 1.80±0.97 in ampullary carcinomas, and 0.89±0.35 in gallbladder carcinomas, with a statistically significant difference between bile duct carcinomas and gallbladder carcinomas (P=0.02). As for clinicopathological significance, patients with a methylated RUNX3 promoter were significantly older than those with unmethylated RUNX3 (P=0.01), and DAP‐kinase methylation was more frequent in poorly differentiated tumors than in well to moderately differentiated ones (P=0.04). The overall survival rate was significantly lower in patients with methylated DAP‐kinase (P=0.009) or RUNX3 (P=0.034) compared to those with unmethylated genes. Furthermore, DAP‐kinase methylation‐positive status was independently associated with poor survival in multivariate analyses (hazard ratio=8.71, P=0.024). A significant proportion of primary biliary tract carcinomas exhibited promoter hypermethylation of tumor suppressor and tumor‐related genes, although bile duct carcinomas are more prone to being affected by promoter methylation than are gallbladder carcinomas. Hypermethylation of DAP‐kinase appears to be a significant prognostic factor in primary biliary tract carcinomas.

Hypermethylation of the TSLC1 Gene Promoter in Primary Gastric Cancers and Gastric Cancer Cell Lines

The TSLC1 (tumor suppressor in lung cancer–1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non‐small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non‐cancerous gastric tissues, by bisulfite‐SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO‐III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non‐cancerous gastric tissues, suggesting that this hypermethylation is a cancer‐specific alteration. KATO‐III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi‐allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers.

Demethylation of the Synuclein γ Gene CpG Island in Primary Gastric Cancers and Gastric Cancer Cell Lines

Purpose: Whereas synuclein γ (SNCG) gene expression is usually highly tissue-specific and restricted to the nervous system, SNCG is expressed in advanced-stage breast and ovarian cancers. When overexpressed, SNCG stimulates cancer cell proliferation and metastasis. It is thought that the molecular mechanism of CpG island demethylation may underlie aberrant SNCG expression. To determine whether aberrant SNCG expression and demethylation play a role in gastric carcinogenesis, we examined the expression and methylation status of SNCG in primary gastric cancers, gastric cancer cell lines, and non-neoplastic gastric mucosal tissues. Experimental Design: Ten gastric cancer cell lines, 105 primary gastric cancers, and 10 non-neoplastic gastric mucosal tissues were examined. SNCG expression and methylation status were examined by reverse transcription-PCR and bisulfite-single-strand conformational polymorphism followed by direct sequencing, respectively. The relationship between SNCG methylation status and various clinicopathological factors of the primary gastric cancers was then analyzed. Results: SNCG mRNA expression was observed in 5 of 10 cell lines. Analysis of cell lines positive for SNCG expression revealed that most of the SNCG CpGs were demethylated. SNCG mRNA was not expressed in the 10 non-neoplastic gastric mucosal tissues, although several CpGs were demethylated. Of the 105 primary gastric cancers, 40 (38.1%) showed apparent SNCG demethylation, similar to the result obtained using cell lines. SNCG demethylation was more frequent in primary gastric cancers positive for lymph node metastasis (51%; 26 of 51) than in cancers without lymph node involvement (26%; 14 of 54; P < 0.05), and also more common in stage II-IV (48%; 27 of 56) than in stage I (27%; 13 of 49) cancers (P < 0.05). Conclusions: Aberrant SNCG gene expression can occur via CpG island demethylation, and tends to occur during the more progressive stages of gastric carcinogenesis.

Spreading of methylation within RUNX3 CpG island in gastric cancer

RUNX3 is a novel tumor suppressor gene that is frequently silenced by promoter hypermethylation in gastric cancer. The methylation status of multiple regions within the RUNX3 promoter CpG island (3478 bp) was examined in gastric cancer cell lines, primary gastric cancers and non‐neoplastic gastric mucosa to clarify how methylation spreads within the CpG island. The critical regions for RUNX3 silencing were evaluated by analysis of cell lines. The most 5′ region of the CpG island was methylated in 90% (9/10) of gastric cancer cell lines, 96% (43/45) of primary gastric cancers and in 96% (43/45) of non‐neoplastic gastric mucosa. The frequencies of methylation were less near the transcription start site and were 40% (4/10) in cell lines, 53% (24/45) in primary gastric cancers and 11% (5/45) in non‐neoplastic gastric mucosa, where methylation was proven to be critical for gene silencing. Thus, hypermethylation initially occurs at the most 5′ region of the RUNX3 CpG island and spreads to the transcription start site before ultimately shutting down RUNX3 mRNA expression. The detection of hypermethylation at multiple regions within the RUNX3 CpG island may be useful in the diagnosis and risk assessment of gastric cancer. (Cancer Sci 2005)

Quantitative assessment of RUNX3 methylation in neoplastic and non-neoplastic gastric epithelia using a DNA microarray.

Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber‐type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end‐labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non‐neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non‐neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non‐neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation‐positive by conventional methylation‐specific polymerase chain reaction (MSP). This microarray‐based methylation assay is a promising method for the quantitative assessment of gene methylation.

Pancreatic fat accumulation, fibrosis, and acinar cell injury in the Zucker diabetic fatty rat fed a chronic high-fat diet.

Objective The histological alteration of the exocrine pancreas in obesity has not been clarified. In the present study, we investigated biochemical and histological changes in the exocrine pancreas of obese model rats. Methods Zucker lean rats were fed a standard diet, and Zucker diabetic fatty (ZDF) rats were divided into 2 groups fed a standard diet and a high-fat diet, respectively. These experimental groups were fed each of the diets from 6 weeks until 12, 18, 24 weeks of age. We performed blood biochemical assays and histological analysis of the pancreas. Results In the ZDF rats fed a high-fat diet, the ratio of accumulated pancreatic fat area relative to exocrine gland area was increased significantly at 18 weeks of age in comparison with the other 2 groups (P < 0.05), and lipid droplets were observed in acinar cells. Subsequently, at 24 weeks of age in this group, pancreatic fibrosis and the serum exocrine pancreatic enzyme levels were increased significantly relative to the other 2 groups (P < 0.01). Conclusions In ZDF rats fed a chronic high-fat diet, fat accumulates in pancreatic acinar cells, and this fatty change seems to be related to subsequent pancreatic fibrosis and acinar cell injury.

Quantitative assessment of gene methylation in neoplastic and non-neoplastic gastric epithelia using methylation-specific DNA microarray

A fiber‐type DNA microarray was used to calculate methylation rates (MR) of four tumor suppressor genes, lysyl oxidase (LOX), p16, RUNX3, and tazarotene‐induced gene 1 (TIG1). MR were calculated in 26 primary gastric cancers and corresponding non‐neoplastic gastric epithelia, and the results were compared to those of conventional methylation‐specific polymerase chain reaction (MSP). MR ranged from 0.1% to 69.1% (mean, 18.3%) for LOX, 0.5–74.1% (mean, 15.7%) for p16, 0.2–76.5% (mean, 22.7%) for RUNX3, and 0.6–41.2% (mean, 5.8%) for TIG1 in primary gastric cancers, and from 0.1% to 25.8% (mean, 8.7%) for LOX, 1.0– 23.2% (mean, 10.3%) for p16, 0.7–25.1% (mean, 5.5%) for RUNX3, and 1.8–27.6% (mean, 11.4%) for TIG1 in corresponding non‐neoplastic gastric epithelia. Although MR varied significantly across different samples for both neoplastic and non‐neoplastic gastric epithelia, high‐level methylation (MR >40%) was cancer specific and was observed in 19.2%, 19.2%, 30.8%, and 3.8% of primary gastric cancers for LOX, p16, RUNX3, and TIG1, respectively. All samples with high‐level methylation, as well as some samples with low MR (particularly <10%) were judged to be methylation positive on conventional MSP. Quantitative analysis of gene methylation using methylation‐specific DNA microarray is a promising method for cancer diagnosis.

Molecular and Cellular Phenotypic Profiles of Gastric Noninvasive Neoplasia

According to the Padova international classification, 52 gastric noninvasive neoplasias (NIN) were classified as follows: 20 low-grade NIN (L-NIN); 9 high-grade NIN including suspicion for carcinoma without invasion (H-NIN); and 23 high-grade NIN including carcinoma without invasion (Ca-NIN). The molecular and cellular phenotypic profiles were investigated and compared. The APC gene was mutated in seven (35%) L-NIN, two (22%) H-NIN, and two (9%) Ca-NIN tumors; APC mutations were significantly more frequent in L-NIN compared with Ca-NIN tumors (p < 0.05). Mutations of the p53 gene were found in five (22%) Ca-NIN tumors but were not observed in L-NIN or H-NIN tumors (p < 0.05). Loss of heterozygosity involving at least one chromosomal locus was detected in 14 (61%) Ca-NIN tumors but was not detected in L-NIN or H-NIN tumors. High-frequency microsatellite instability (MSI-H) was detected in one (5%) L-NIN tumor and in six (26%) Ca-NIN tumors. The frequencies of loss of heterozygosity and MSI-H were significantly higher in Ca-NIN than in L-NIN or H-NIN tumors (p < 0.05). Nuclear accumulation of p53 protein was detected in no L-NIN tumors, 1 (11%) H-NIN tumor, and 10 (44%) Ca-NIN tumors (p < 0.01). All tumors with loss of hMLH1 expression exhibited MSI-H (p < 0.01). Cellular phenotypic analysis revealed that seven (35%) L-NIN tumors and one (4%) Ca-NIN tumor had complete-type intestinal metaplastic phenotype and that one (5%) L-NIN tumor and one (4%) Ca-NIN tumor had a gastric foveolar epithelial phenotype, whereas the remaining tumors exhibited an ordinary phenotype. Thus, the complete-type intestinal metaplastic phenotype was more characteristic of L-NIN tumors than of H-NIN or Ca-NIN tumors (p < 0.01). In summary, the Padova international classification correlated with both the molecular and cellular phenotypic profiles. In practice, p53 and hMLH1 immunohistochemistry discriminated Ca-NIN from L-NIN and H-NIN tumors.

Pancreatic hyperechogenicity associated with hypoadiponectinemia and insulin resistance: A Japanese population study

AIM To examine the relationship between pancreatic hyperechogenicity and risk factors for metabolic syndrome. METHODS A general population-based survey of lifestyle-related diseases was conducted from 2005 to 2006 in Japan. The study involved 551 participants older than 40 year of age. Data for 472 non-diabetic adults were included in the analysis. The measures included the demographic factors, blood parameters, results of a 75 g oral glucose tolerance test, and abdominal ultrasonography. The echogenicity of the pancreas and liver was compared, and then the subjects were separated into two groups: cases with pancreatic hyperechogenicity (n = 208) and cases without (controls, n = 264). The differences between both groups were compared using an unpaired t-test or Fisher’s exact test. Multiple logistic regression analysis was used to determine the relationship between the pancreatic hyperechogenicity and clinical and biochemical parameters. RESULTS Subjects with pancreatic hyperechogenicity had decreased serum adiponectin concentration compared to control subjects [8.9 (6.5, 12.8) vs 11.1 (7.8, 15.9), P < 0.001] and more frequently exhibited features of metabolic syndrome. Logistic regression analysis showed that the following variables were significantly and independently associated with pancreatic hyperechogenicity: Presence of hypoadiponectinemia, increased body mass index (BMI), higher homeostasis model assessment of insulin resistance (HOMA-IR) score, and presence of fatty liver. Similar associations were also observed in subjects with pancreatic hyperechogenicity without fatty liver. Multivariate association analysis of data from participants without fatty liver showed that hypoadiponectinemia was significantly associated with pancreatic hyperechogenicity (OR = 0.93, 95%CI: 0.90 - 0.97, P < 0.001). This association was independent of other confounding variables. Additionally, an increased BMI and higher HOMA-IR score were significantly associated with pancreatic hyperechogenicity. CONCLUSION Pancreatic hyperechogenicity is independently associated with increased BMI, insulin resistance, and hypoadiponectinemia in the general population.