研究者詳細 - 時野　隆至

論文 - 時野　隆至

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Sequence motif discovery with computational genome-wide analysis

Akashi H, Aoki F, Toyota M, Maruyama R, Sasaki Y, Mita H, Tokura H, Imai K, Tatsumi H, Tokino T

Tumor Research ( Sapporo Medical University ) 41 59 - 69 2006年12月 [査読有り]

As a result of the human genome project and advancements in DNA sequencing technology, we can utilize a huge amount of nucleotide sequence data and can search DNA sequence motifs in whole human genome. However, searching motifs with the naked eye is an enormous task and searching throughout the whole genome is absolutely impossible. Therefore, we have developed a computational genome-wide analyzing system for detecting DNA sequence motifs with biological significance. We used a multi-parallel network computing system as a powerful computing engine. Furthermore, we improved the system to work as a background engine for web-based applications. The multi-parallel computing engine consists of a head processor, which issues control commands to data processing nodes for various kinds of jobs, such as retrieving arbitrary sequences, generating mapping images, and loading data sections from genome databases. We constructed the system to function as a flexible Client/Server structure connected over the network, and this system could be adapted to cope with increases in sequence data and to deal with algorithms for new investigation needs by slightly changing the control procedures and increasing the number of the processor node. We developed two additional tools to annotate the genome sequences. The first was the cDNA Reverse Splicing Tool, which divided cDNA sequences into exons and mapped them on the genomic sequence, and the second was DNA-Protein Translation Tool which showed open reading frames (ORFs) of whole genome. In order to examine the availability and efficiency of our system, we searched and identified p53RE (p53 response element) as a representative sequence motif on genomic sequences of chromosome 21 and 22. As a result, we detected 50,000 p53REs on fifty mega base genomic DNA sequences within 27 seconds.

DOI CiNii

短いDNA断片の塩基配列情報を用いた定量的ゲノム解析法の開発と癌研究への応用

見田裕章, 豊田実, 丸山玲緒, 明石浩史, 青木文夫, 佐々木泰史, 鹿島理沙, 鈴木拓, 井戸川雅史, 苗代康可, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 182 - 182 2006年09月

インシリコ解析による新規p53標的遺伝子群の網羅的な同定

丸山玲緒, 豊田実, 明石浩史, 佐々木泰史, 青木文夫, 鈴木拓, 見田裕章, 井戸川雅史, 今井浩三, 篠村泰久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 79 - 79 2006年09月

CHFR(checkpoint with FHA and RING domain)によるNF-kBシグナリング制御

鹿島理沙, 見田裕章, 豊田実, 佐々木泰史, 井戸川雅史, 鈴木拓, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 162 - 162 2006年09月

口腔扁平上皮癌におけるRAS関連遺伝子のジェネティックおよびエピジェネティックな異常の解析

今井崇, 秋野公臣, 豊田実, 鈴木拓, 佐々木泰史, 見田裕章, 井戸川雅史, 荻和弘, 鹿島理沙, 曽我部陽平, 今井浩三, 平塚博義, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 199 - 199 2006年09月

大腸癌におけるエピジェネティックな変化を指標とした診断への比較検討

渡邊嘉行, 豊田実, 鈴木拓, 岡本賢, 時野隆至, 伊東文生

日本癌学会総会記事 ( (一社)日本癌学会 ) 65回 52 - 52 2006年09月

Activation of the ribosomal protein L13 gene in human gastrointestinal cancer.

Kobayashi T, Sasaki Y, Oshima Y, Yamamoto H, Mita H, Suzuki H, Toyota M, Tokino T, Itoh F, Imai K, Shinomura Y

International journal of molecular medicine 18 ( 1 ) 161 - 70 2006年07月 [査読有り] [国際誌]

Although ribosomal proteins are major components of ribosomes, recent data have shown them to have extraribosomal functions apart from ribosome and protein biosynthesis. In our earlier study, we showed that ribosomal protein L13 mRNA was up-regulated in response to DNA damage in hamster cells. We report here that L13 expression is up-regulated in human gastrointestinal cancers. We also examined the biological role of L13 on human cancer cells. Knocking down L13 expression using small interfering RNA (siRNA) resulted in drastic attenuation of cancer cell growth with significant G1 and G2/M arrest of the cell cycle. Moreover, L13 siRNA significantly enhanced the cellular sensitivity to certain DNA damaging agents and, concordantly, L13-overexpressing cells demonstrated greater chemoresistance compared to parent cells, suggesting an inverse correlation between L13 expression and chemosensitivity. By using semiquantitative RT-PCR, we analyzed expression of L13 in freshly resected cancer tissue of the stomach, colorectum and liver. Up-regulation of L13 mRNA expression was observed in 10 (28%) of 36 gastric, 19 (41%) of 46 colorectal and 5 (20%) of 25 liver cancer tissue samples compared to adjacent normal tissue samples. We also found that increased expression of the L13 gene correlated with clinical staging in gastric cancers. The results of this study suggest that L13 plays an essential role in the progression of some gastrointestinal malignancies.

PubMed

DNA断片の塩基配列情報に基づいたゲノムコピー数解析法の開発

見田裕章, 豊田実, 丸山玲緒, 青木文夫, 明石浩史, 鹿島理沙, 佐々木泰史, 鈴木拓, 井戸川雅史, 藤井恭子, 篠村恭久, 今井浩三, 時野隆至

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 43回 63 - 63 2006年07月

がん抑制遺伝子p53により誘導される新規分子の網羅的同定

丸山玲緒, 豊田実, 青木文夫, 佐々木泰史, 明石浩史, 鈴木拓, 見田裕章, 時野隆至, 今井浩三, 篠村恭久

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 43回 64 - 64 2006年07月

p73によるIL-4シグナルの活性化と抗腫瘍活性

佐々木泰史, 見田裕章, 豊田実, 篠村恭久, 今井浩三, 時野隆至

日本臨床分子医学会学術総会プログラム・抄録集 ( 日本臨床分子医学会 ) 43回 85 - 85 2006年07月

Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation.

Maruyama R, Aoki F, Toyota M, Sasaki Y, Akashi H, Mita H, Suzuki H, Akino K, Ohe-Toyota M, Maruyama Y, Tatsumi H, Imai K, Shinomura Y, Tokino T

Cancer research 66 ( 9 ) 4574 - 83 2006年05月 [査読有り] [国際誌]

p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.

DOI PubMed

Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus.

Kusano M, Toyota M, Suzuki H, Akino K, Aoki F, Fujita M, Hosokawa M, Shinomura Y, Imai K, Tokino T

Cancer 106 ( 7 ) 1467 - 79 2006年04月 [査読有り] [国際誌]

BACKGROUND: The CpG island methylator phenotype (CIMP), which is characterized by simultaneous methylation of the CpG islands of multiple genes, has been recognized as one of the important mechanisms in gastrointestinal carcinogenesis. METHODS: Methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumor-related genes in 78 primary gastric carcinomas was examined using combined bisulfite-restriction analysis. Epstein-Barr virus (EBV)-associated gastric tumors were detected using real-time polymerase chain reaction analysis followed by an evaluation of the correlations between CIMP status, EBV-association, and genetic alteration of p53 and K-ras. The authors compared the clinicopathologic features of gastric carcinomas that had high CIMP methylation (CIMP-H) with tumors that had low CIMP methylation (CIMP-L) or negative CIMP methylation (CIMP-N). RESULTS: The methylation profiles of 12 genes showed nonrandom methylation, supporting the presence of CIMP in gastric carcinoma. No p53 mutations were detected among CIMP-H tumors, and no EBV association was detected in tumors that showed mutation of p53 and K-ras. In a multiple logistic regression model with CIMP-H as the dependent variable, proximal location (P = .011), diffuse type (P = .019), and less advanced pathologic TNM status (P = .043) contributed significantly to CIMP-H. Patients who had CIMP-N gastric tumors had a significantly worse survival than patients who had CIMP-H tumors (P = .004) or CIMP-L tumors (P = .012). EBV-associated tumors were associated strongly with CIMP-H, hypermethylation of tumor-related genes, and no p53 or K-ras mutation. CONCLUSIONS: CIMP status appeared to be associated with distinct genetic, epigenetic, and clinicopathologic features in gastric carcinomas. The finding that gastric carcinomas arose through different molecular pathways may affect not only tumor characteristics but also patient prognosis.

DOI PubMed

Epigenetic inactivation of the candidate tumor suppressor gene HOXB13 in human renal cell carcinoma

H. Okuda, M. Toyota, W. Ishida, M. Furihata, M. Tsuchiya, M. Kamada, T. Tokino, T. Shuin

Oncogene 25 ( 12 ) 1733 - 1742 2006年03月 [査読有り]

Epigenetic alterations like DNA methylation and the resulting inactivation of cancer-related genes often contribute to the development of various cancers. To identify the genes that are silenced by aberrant methylation in renal cell carcinoma (RCC), we subjected two RCC lines to methylated CpG island amplification/representational difference analysis. This identified 27 CpG islands. Combined bisulfite restriction analysis of these CpG islands in primary RCC cases revealed that four were methylated in a tumor-specific manner. One of these was identified as the human homeo-box gene B13 (HOXB13) gene, but the remaining three CpG islands were not associated with known genes. The methylation frequencies of HOXB13 in primary RCC samples and lines were 30 and 73%, respectively. The methylation status of HOXB13 correlated with the loss of its expression both in RCC lines and primary tumors, and methyltransferase inhibitor treatment induced the recovery of its expression. Exogenous expression of HOXB13 in RCC cells that lacked endogenous HOXB13 expression suppressed colony formation and induced apoptotic features. Furthermore, HOXB13 methylation correlated positively with tumor grade and microvessel invasion. These results suggest that HOXB13 is a novel candidate tumor suppressor gene in RCC and that its inactivation may play an important role in both RCC tumorigenesis and progression. © 2006 Nature Publishing Group All rights reserved.

DOI PubMed

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines

K Terasawa, M Toyota, S Sagae, K Ogi, H Suzuki, T Sonoda, K Akino, R Maruyama, N Nishikawa, K Imai, Y Shinomura, T Saito, T Tokino

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 94 ( 6 ) 914 - 921 2006年03月 [査読有り]

Transcription factor 2 gene ( TCF2) encodes hepatocyte nuclear factor 1 beta ( HNF1 beta), a transcription factor associated with development and metabolism. Mutation of TCF2 has been observed in renal cell cancer, and by screening aberrantly methylated genes, we have now identified TCF2 as a target for epigenetic inactivation in ovarian cancer. TCF2 was methylated in 53% of ovarian cancer cell lines and 26% of primary ovarian cancers, resulting in loss of the gene's expression. TCF2 expression was restored by treating cells with a methyltransferase inhibitor, 5- aza- 2'deoxycitidine ( 5- aza- dC). In addition, chromatin immunoprecipitation showed deacetylation of histone H3 in methylated cells and, when combined with 5- aza- dC, the histone deacetylase inhibitor trichostatin A synergistically induced TCF2 expression. Epigenetic inactivation of TCF2 was also seen in colorectal, gastric and pancreatic cell lines, suggesting general involvement of epigenetic inactivation of TCF2 in tumorigenesis. Restoration of TCF2 expression induced expression of HNF4 alpha, a transcriptional target of HNF1 beta, indicating that epigenetic silencing of TCF2 leads to alteration of the hepatocyte nuclear factor network in tumours. These results suggest that TCF2 is involved in the development of ovarian cancers and may represent a useful target for their detection and treatment.

DOI PubMed

Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis

KE King, RM Ponnamperuma, MJ Gerdes, T Tokino, T Yamashita, CC Baker, WC Weinberg

CARCINOGENESIS ( OXFORD UNIV PRESS ) 27 ( 1 ) 53 - 63 2006年01月 [査読有り]

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and Delta N. Delta Np63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, Delta N- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. Delta Np63 alpha declines with Ca2+-induced differentiation, while a smaller Delta N-form, Delta Np63(s), persists, suggesting unique functions of the two Delta N-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, Delta Np63 alpha, Delta Np63(p40), TAp63 alpha, TAp63 gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of Delta Np63(p40), which lacks the entire alpha-tail present in Delta Np63 alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length Delta Np63 alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of Delta Np63 alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the Delta N domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The Delta N-terminus is sufficient to maintain keratinocytes in a proliferative state, as both Delta N forms block Ca2+-mediated p21(WAF1) induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63 alpha or TAp63 gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

DOI

新規大腸癌抑制遺伝子RASSF2異常メチル化の腫瘍マーカーへの応用

豊田実, 秋野公臣, 鈴木拓, 時野隆至, 篠村恭久, 今井浩三

日本分子腫瘍マーカー研究会誌 ( 日本分子腫瘍マーカー研究会 ) 21 5 - 6 2006年

DOI CiNii

The role of T-fimbrin in the response to DNA damage: silencing of T-fimbrin by small interfering RNA sensitizes human liver cancer cells to DNA-damaging agents.

Ikeda H, Sasaki Y, Kobayashi T, Suzuki H, Mita H, Toyota M, Itoh F, Shinomura Y, Tokino T, Imai K

International journal of oncology 27 ( 4 ) 933 - 40 2005年10月 [査読有り] [国際誌]

Fimbrins (also known as plastins) are actin-binding proteins of the cortical cytoskeleton present in all cells and conserved from yeast to mammals. We previously reported that the up-regulation of T-fimbrin, a fimbrin isoform, in association with G2 arrest following DNA damage and that a lack of T-fimbrin expression shortens the radiation-induced G2 arrest in Chinese hamster ovarian cells. In this study, we further investigated the role of T-fimbrin in DNA-damage response using a panel of human liver cancer cell lines and small interfering RNA technology. T-fimbrin was differentially expressed in human liver cancer cell lines. Colony formation assays revealed that cell lines lacking T-fimbrin expression were highly sensitive to DNA damage compared to cell lines that express T-fimbrin. Using siRNA designed to target T-fimbrin, we demonstrated that silencing endogenous T-fimbrin causes a marked increase in the cellular sensitivity to VP-16 and UV irradiation. Moreover, T-fimbrin deletion abrogated UV-mediated cell cycle checkpoint, and consequently led to increased apoptotic cell death in resistant cells. These findings suggest that the status of T-fimbrin expression may be a useful molecular marker for predicting the responsiveness of cancer cells to treatment with chemotherapeutic drugs.

PubMed

分裂期チェックポイント分子CHFRの結合分子探索と機能解析

見田裕章, 豊田実, 鹿島理沙, 佐々木泰史, 秋野公臣, 鈴木拓, 井戸川雅史, 下重美紀, 山田哲司, 篠村恭久, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 64回 327 - 327 2005年09月

DNAの異常メチル化を指標とした大腸癌アベレージリスク群の設定と検診への応用

渡邊嘉行, 豊田実, 秋野公臣, 鈴木拓, 前畑忠照, 岡本賢, 奥瀬千晃, 今井浩三, 篠村恭久, 伊東文生, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 64回 540 - 540 2005年09月

RASエフェクター,RASSF2遺伝子は大腸がんにおける新規がん抑制遺伝子である(The Ras Effector RASSF2 is a Novel Tumor Suppressor Gene in Human Colorectal Cancer)

秋野公臣, 豊田実, 鈴木拓, 見田裕章, 佐々木泰史, 篠村恭久, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 64回 136 - 137 2005年09月

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