研究者詳細 - 鈴木　拓

Misc - 鈴木　拓

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癌メチル化研究が切り開く新たな癌早期診断および治療 (特集エピジェネティクス--生化学的機構と疾患への関与)

伊東文生, 鈴木拓, 今井浩三

モレキュラ-メディシン ( 中山書店 ) 39 ( 7 ) 784 - 791 2002年07月

DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers

A Satoh, M Toyota, F Itoh, T Kikuchi, T Obata, Y Sasaki, H Suzuki, A Yawata, M Kusano, M Fujita, M Hosokawa, K Yanagihara, T Tokino, K Imai

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 86 ( 11 ) 1817 - 1823 2002年06月

Death-associated protein kinase is a positive regulator of programmed cell death induced by interferon, To investigate the role of epigenetic inactivation of death-associated protein kinase in gastrointestinal cancer, we examined the methylation status of the 5' CpG island of the death-associated protein kinase gene. Methylation of the 5' CpG island was detected in 3 of 9 colorectal and 3 of 17 gastric cancer cell lines, while among primary tumours, it was detected in 4 of 28 (14%) colorectal and 4 of 27 (15%) gastric cancers. By contrast, methylation of the edge of the CpG island was detected in virtually every sample examined. Death-associated protein kinase expression was diminished in four cell lines that showed dense methylation of the 5' CpG island, and treatment with 5-aza-2'-deoxycitidine, a methyltransferase inhibitor, restored gene expression, Acetylation of histones H3 and H4 in the 5' region of the gene was assessed by chromatin immunoprecipitation and was found to correlate directly with gene expression and inversely with DNA methylation, Thus, aberrant DNA methylation and histone deacetylation of the 5' CpG island, but not the edge of the CpG island, appears to play a key role in silencing death-associated protein kinase expression in gastrointestinal malignancies.

DOI

A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer

H Suzuki, E Gabrielson, W Chen, R Anbazhagan, M van Engeland, MP Weijenberg, JG Herman, SB Baylin

NATURE GENETICS ( NATURE AMERICA INC ) 31 ( 2 ) 141 - 149 2002年06月

Aberrant hypermethylation of gene promoters is a major mechanism associated with inactivation of tumor-suppressor genes in cancer. We previously showed this transcriptional silencing to be mediated by both methylation and histone deacetylase activity, with methylation being dominant. Here, we have used cDNA microarray analysis to screen for genes that are epigenetically silenced in human colorectal cancer. By screening over 10,000 genes, we show that our approach can identify a substantial number of genes with promoter hypermethylation in a given cancer; these are distinct from genes with unmethylated promoters, for which increased expression is produced by histone deacetylase inhibition alone. Many of the hypermethylated genes we identified have high potential for roles in tumorigenesis by virtue of their predicted function and chromosome position. We also identified a group of genes that are preferentially hypermethylated in colorectal cancer and gastric cancer. One of these genes, SFRP1, belongs to a gene family; we show that hypermethylation of four genes in this family occurs very frequently in colorectal cancer, providing for (i) a unique potential mechanism for loss of tumor-suppressor gene function and (ii) construction of a molecular marker panel that could detect virtually all colorectal cancer.

DOI PubMed CiNii

Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors

T Kikuchi, M Toyota, F Itoh, H Suzuki, T Obata, H Yamamoto, H Kakiuchi, M Kusano, JPJ Issa, T Tokino, K Imai

ONCOGENE ( NATURE PUBLISHING GROUP ) 21 ( 17 ) 2741 - 2749 2002年04月

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5' CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in I out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of IS (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P < 0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (-300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.

DOI

Increased expression of T-fimbrin gene after DNA damage in CHO cells and inactivation of T-fimbrin by cpg methylation in human colorectal cancer cells

Y Sasaki, F Itoh, T Kobayashi, T Kikuchi, H Suzuki, M Toyota, K Imai

INTERNATIONAL JOURNAL OF CANCER ( WILEY-LISS ) 97 ( 2 ) 211 - 216 2002年01月

When DNA damage is induced by unprogrammed extrinsic events, activating-cell-cycle checkpoints delay cell-cycle progression in the G1 or G2 phases and allow repair of a damaged template. In this study, we evaluated changes in gene expression upon radiation-induced G2 cell-cycle arrest using Chinese hamster ovary (CHO) cells. T-fimbrin an actin-binding protein, was overexpressed in CHO cells which G2 arrest had been induced by X-radiation. Northern blot analysis revealed that T-fimbrin gene expression was induced not only by X-radiation but also by a topoisomerase II inhibitor, etoposide. Transfection of CHO cells with a vector encoding T-fimbrin antisense RNA demonstrated that reduced T-fimbrin expression induced alterations in cell-cycle control; radiation-induced G2 arrest was short and decreased in cells transfected with antisense T-fimbrin. Additionally, T-fimbrin gene expression was suppressed in a human colorectal cancer cell line, SW948, because of promoter-specific DNA methylation. These results suggest that downregulation of T-fimbrin may be involved in cancer development through G2/M cell-cycle control in mammalian cells. (C) 2002 Wiley-Liss, Inc.

DOI PubMed CiNii

Aberrant methylation and histone deacetylation of cyclooxygenase 2 in gastric cancer

T Kikuchi, F Itoh, M Toyota, H Suzuki, H Yamamoto, M Fujita, M Hosokawa, K Imai

INTERNATIONAL JOURNAL OF CANCER ( WILEY-LISS ) 97 ( 3 ) 272 - 277 2002年01月

Cyclooxygenase 2 plays a critical role in the development of gastrointestinal cancers in both human and animal models. About 80% of the gastric cancer showed a high level of expression of cyclooxygenase 2, but a subset of cases do not express without unknown reason. Aberrant methylation of CpG island of COX-2 was examined by using a series of gastric cancer cell lines and primary gastric cancers. Two out of 8 cell lines (25%) and 11 out of 93 (12%) primary cancers showed aberrant methylation of the 5' region of COX-2. Methylation of COX-2 was closely associated with loss of expression and treatment of methylation inhibitor, 5-deoxy-2'-azacytidine restored the expression of COX-2. A combined treatment of 5-deoxy-2'-azacytidine and a histone deacetylese inhibitor, trichostatin A, restored re-expression of the gene synergistically and chromatin immunoprecipitation analysis revealed that histone of methylated COX-2 promoter is deacetylated, indicating the role of cytosine methylation and histone deacetylation in the silencing of the gene. These results indicate that a subset of gastric cancer with COX-2 methylation evolves through the pathway that is independent of COX-2 expression and that COX-2 inhibitor may not be useful to induce apoptosis in these cases. (C) 2002 Wiley-Liss, Inc.

DOI PubMed CiNii

DNA methylation changes in gastrointestinal disease

M Toyota, F Itoh, T Kikuchi, A Satoh, T Obata, H Suzuki, S Ishii, T Endo, T Tokino, K Imai

JOURNAL OF GASTROENTEROLOGY ( SPRINGER-VERLAG TOKYO ) 37 97 - 101 2002年

DNA methylation of the 5' region of genes is often associated with gene silencing in X-chromosome inactivation and imprinting. Recent studies have indicated that altered DNA methylation plays a role in the inactivation of multiple tumor suppressor genes and DNA repair genes such as p16INK4A and hMLH1. Colorectal adenomas have a relatively high frequency of methylation, and aberrant methylation is an early event during tumorigenesis. In aging patients, even colon epithelium which appears to be normal showed a significant amount of methylation in a subset of the genes. Colon mucosa from patients with inflammatory bowel disease also showed a high level of methylation. DNA methylation can be a specific diagnostic marker in gastrointestinal cancer and inflammatory bowel disease, for which there is no,perfect marker for a noninvasive diagnosis.

消化器癌におけるアポトーシス関連遺伝子Hrkの異常メチル化

小畑俊郎, 豊田実, 佐々木泰史, 佐藤亜由美, 菊地剛史, 鈴木拓, 伊東文生, 時野隆至, 今井浩三

日本癌学会総会記事 61st 2002年

J-GLOBAL

DNAメチル化による遺伝子不活化の分子機構

豊田実, 菊地剛史, 佐藤亜由美, 佐々木泰史, 小畑俊郎, 秋野公臣, 鈴木拓, 時野隆至, 今井浩三

日本癌学会総会記事 61st 2002年

J-GLOBAL

ヒト腫ようにおけるM期チェックポイント遺伝子CHFRの異常メチル化と発現抑制

佐藤亜由美, 豊田実, 佐々木泰史, 荻和弘, 小畑俊郎, 菊地剛, 鈴木拓, 伊東文生, 今井浩三

日本癌学会総会記事 61st 2002年

J-GLOBAL

Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus

H Kaneto, S Sasaki, H Yamamoto, F Itoh, M Toyota, H Suzuki, Ozeki, I, N Iwata, T Ohmura, T Satoh, Y Karino, T Satoh, J Toyota, M Satoh, T Endo, M Omata, K Imai

GUT ( BRITISH MED JOURNAL PUBL GROUP ) 48 ( 3 ) 372 - 377 2001年03月

Background/aim-Inactivation of the p16(INK4A) (p16) tumour suppressor gene by promoter region hypermethylation has been demonstrated not only in many types of tumours, including hepatocellular carcinoma (HCC), but also in early preneoplastic lesions in the lung, colon, oesophagus, and pancreas. The aim of this study was to examine the methylation status of the p16 promoter in pre- and/or non-neoplastic liver diseases. Patients/subjects/methods-The methylation status of p16 was evaluated in 22 HCC, 17 cirrhosis, 17 chronic hepatitis, nine primary biliary cirrhosis (PBC), eight autoimmune hepatitis, seven drug induced liver disease, six fatty liver, and three normal Liver tissues using methylation specific polymerase chain reaction (MSP). p16 protein expression was also examined by immunohistochemical staining. Results-Methylation of the p16 promoter was detected in HCC (72.7%, 16/22) and also in cirrhosis (29.4%, 5/17) and chronic hepatitis (23.5%, 4/17), all of which were positive for hepatitis B or C virus infections. Methylation was not detected in any of the other samples. All methylation positive HCC, cirrhosis, and chronic hepatitis samples showed loss of p16 expression, and a significant correlation was found between methylation and loss of expression. Analysis of serial samples from individual patients with methylation positive HCC revealed that loss of p16 expression with promoter methylation occurred in 18 of 20 patients at the stage of chronic hepatitis without clinically detectable carcinoma. Conclusions-Our results suggest that methylation of the p16 promoter and the resulting loss of p16 protein expression are early events in a subset of hepatocarcinogenesis and that their detection is useful in the follow up of patients with a high risk of developing HCC, such as those with hepatitis B or C viral infections.

DOI

潰よう性大腸炎におけるメチル化の異常と発癌リスク予測への応用

豊田実, 伊東文生, 菊地剛史, 鈴木拓, 小畑俊郎, 佐藤亜由美, 佐々木泰史, 時野隆至, 今井浩三

日本癌学会総会記事 60th 2001年

J-GLOBAL

Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 sigma gene in human hepatocellular carcinoma

N Iwata, H Yamamoto, S Sasaki, F Itoh, H Suzuki, T Kikuchi, H Kaneto, S Iku, Ozeki, I, Y Karino, T Satoh, J Toyota, M Satoh, T Endo, K Imai

ONCOGENE ( NATURE PUBLISHING GROUP ) 19 ( 46 ) 5298 - 5302 2000年11月

The 14-3-3 sigma gene has been implicated in G2/M cell cycle arrest by p53, Frequent inactivation of the 14-3-3 sigma gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 sigma gene in hepatocellular carcinoma (HCC), The methylation status of the 14-3-3 sigma gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP, The 14-3-3 sigma protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 sigma gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 sigma expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 sigma expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2'-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 sigma gene in HCC, Hypermethylation and the resulting loss of expression of the 14-3-3 sigma gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.

DOI

大腸癌における CpG island methylator phenotype およびジェネティックな異常の解析

豊田実, 伊東文生, 鈴木拓, 菊地剛史, 小畑俊郎, 山本博幸, 垣内英樹, 有村佳昭, 今井浩三

生物物理化学 = Journal of Electrophoresis 44 36 - 36 2000年10月

CiNii

Inactivation of the 14-3-3 sigma gene is associated with 5 ' CpG island hypermethylation in human cancers

H Suzuki, F Itoh, M Toyota, T Kikuchi, H Kakiuchi, K Imai

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 60 ( 16 ) 4353 - 4357 2000年08月

The cell cycle checkpoint plays an important role in maintaining the integrity of cells. Recently, one of the 14-3-3 protein family members, 14-3-3 sigma, was shown to be regulated by p53 and to play a role in the G(2)-M-phase checkpoint. To determine whether 14-3-3 sigma is inactivated in human cancers, the methylation status of the 5' region of 14-3-3 sigma was investigated in a series of gastric, colorectal, and hepatocellular cancer cell lines. Of 22 cell lines examined, 6 showed aberrant methylation. The methylation status of 14-3-3 sigma was found to be correlated with loss of expression, which was restored by 5-aza-2'-deoxycytidine treatment. Furthermore, normal G(2) arrest after DNA damage was not demonstrated in the cell lines with methylation, In primary gastric cancers, 14-3-3 sigma hypermethylation was observed frequently in 26 of 60 (43%) cases and observed more frequently in poorly differentiated adenocarcinomas (P = 0.0017). Our findings suggest that 14-3-3 sigma is inactivated by aberrant methylation of the 5' region in various human canters and that it might play an important role in the development of undifferentiated gastric cancers.

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer

H Suzuki, F Itoh, M Toyota, T Kikuchi, H Kakiuchi, Y Hinoda, K Imai

ELECTROPHORESIS ( WILEY-V C H VERLAG GMBH ) 21 ( 5 ) 904 - 908 2000年03月

A novel DNA methylation assay technique, termed bisulfite single-strand conformation polymorphism (bisulfite-SSCP), is a combination of sodium-bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite-SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite-SSCP.

DOI PubMed CiNii

Identification of genes highly expressed in G2-arrested Chinese hamster ovary cells by differential display analysis

Y Sasaki, F Itoh, H Suzuki, T Kobayashi, H Kakiuchi, M Hareyama, K Imai

JOURNAL OF CLINICAL LABORATORY ANALYSIS ( WILEY-LISS ) 14 ( 6 ) 314 - 319 2000年

Abnormal cell cycle regulation is believed to be an important step in tumorigenesis. In mammalian cells, DNA damage commonly leads to cell cycle arrest in G2; however, little is known about the detailed biochemical mechanisms underlying the DNA damage-induced G2 arrest. In order to identify genes differentially expressed in association with G2 arrest, differential display analysis was performed between exponentially growing Chinese hamster ovary (CHO) cells and G2-arrested CHO cells induced by etoposide, SN-38, or X-radiation. We identified five cDNA clones whose expression was upregulated in G2-arrested CHO cells. Sequence analysis revealed that three clones were homologous to known genes: isogene I of translation initiation factor eIF-4A, ribosomal protein L13, and translation repressor NAT1. The remaining two clones showed no homology to known genes. These results indicate that DNA damage can alter the expression of multiple genes, including translational regulators. J. Clin. Lab. Anal. 14: 314-319, 2000. (C) 2000 Wiley-Liss, Inc.

DOI

Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype

M Toyota, N Ahuja, H Suzuki, F Itoh, M Ohe-Toyota, K Imai, SB Baylin, JPJ Issa

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 59 ( 21 ) 5438 - 5442 1999年11月

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in cancer. In colorectal cancer, a group of tumors is characterized by a hypermethylator phenotype termed CpG island methylator phenotype (CIMP), which includes methylation of such genes as p16 and hMLH1, To study whether CIMP is present in gastric cancer, the methylation status of five newly cloned CpG islands was examined in 56 gastric cancers using bisulfite-PCR, Simultaneous methylation of three loci or more was observed in 23 (41%) of 56 cancers, which suggests that these tumors have the hypermethylator phenotype CIMP, There was a significant concordance between CIMP and the methylation of known genes including p16, and hMLH1; methylation of p16 was detected in 16 (70%) of 23 CIMP+ tumors, 1 (8%) of 12 CIMP intermediate tumors, and 1 (5%) of 21 CIMP- tumors (P < 0.0001). Methylation of the hMLH1 gene was defected in three of five tumors that showed microsatellite instability, and all three of the cases were CIMP+. The CIMP phenotype is an early event in gastric cancer, being present in the normal tissue adjacent to cancer in 5 of 56 cases. These results suggest that CIMP may be one of the major pathways that contribute to tumorigenesis in gastric cancers.

Distinct methylation pattern and microsatellite instability in sporadic gastric cancer

H Suzuki, F Itoh, M Toyota, T Kikuchi, H Kakiuchi, Y Hinoda, K Imai

INTERNATIONAL JOURNAL OF CANCER ( WILEY-LISS ) 83 ( 3 ) 309 - 313 1999年10月

Aberrant 5' CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor-suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLHI in association of promoter hypermethylation, In the present study, we investigated the 5' CpG island hypermethylation of hMLHI, E-cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation-specific FCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLHI methylation, 16 (26.2%) and 25 (40.9%) showed E-cadherin and p16 methylation respectively, and 8 (13.1%) presented high-frequency MSI (MSI-H). Of the a MSI-H patients, 5 presented hMLHI methylation, whereas no low-frequency MSI (MSI-L) and microsatellite stable (MSS) cases exhibited hMLHI methylation (5/8 vs. 0/43, p<0.00001). Furthermore, these patients also presented E-cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLHI methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers. (C) 1999 Wiley-Liss, Inc.

DOI PubMed CiNii

G2/M期集積細胞において発現が亢進している遺伝子の単離と解析 : differential display 法を用いて

佐々木泰史, 伊東文生, 垣内英樹, 鈴木拓, 吉田幸成, 日野田裕治, 今井浩三, 谷内昭

生物物理化学 = Journal of Electrophoresis 42 29 - 29 1998年09月

CiNii

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