研究者詳細 - 大内田　守

Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay 国際誌

H Asano, S Toyooka, M Tokumo, K Ichimura, K Aoe, S Ito, K Tsukuda, M Ouchida, M Aoe, H Katayama, A Hiraki, K Sugi, K Kiura, H Date, N Shimizu

CLINICAL CANCER RESEARCH 12 ( 1 ) 43 - 48 2006年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

Purpose: Mutations in the epidermal growth factor receptor (EGFR) gene have been reported to be present in non-small cell lung cancer (NSCLC) and related to the responsiveness of tumors to EGFR tyrosine kinase inhibitors, suggesting its usefulness as a biomarker. Because clinical samples contain tumor and normal cells or genes, a highly sensitive assay for detecting mutation is critical for clinical applications.
Experimental Design: The mutant-enriched PCR is a rapid and sensitive assay with selective restriction enzyme digestion. We developed the mutant-enriched PCR assay targeting exons 19 and 21 of EGFR and applied the developed assay to detect mutations in 108 cases of surgically resected specimens of NSCLCs,18 samples of computed tomography (CT)-guided needle lung biopsies, and 20 samples of pleural fluid. In addition, results were then compared with those from direct sequencing and a nonenriched PCR assay.
Results: The mutant-enriched PCR that was proved to enrich one mutant of 2 x 103 normal genes detected mutations in 37 cases of 108 resected tumors, seven samples of CT-guided lung biopsies, and seven samples of pleural fluid. Among mutant cases, four resected tumors, two CT-guided lung biopsies, and two pleural fluid were identified as additional mutant cases by the mutant-enriched PCR, which were considered normal based on nonenriched assays.
Conclusions: Our results indicate that EGFR mutations are readily detectable by mutant-enriched PCR in various clinical samples. Thus, mutant-enriched PCR may provide a valuable method of potentially detecting a small fraction of mutant genes in heterogeneous specimens, indicating its possible use in clinical application for NSCLC.

DOI： 10.1158/1078-0432.CCR-05-0934

異常メチル化によるSHP1 gene silencingと悪性リンパ腫・白血病発症機構

岡剛史, 中谷陽子, 近藤孝美, 佐藤妃映, 佐藤妃映, 高野正幸, 鷲尾佳奈, 大内田守, 大原信哉, 清水憲二, 谷本光音, 高橋聖之, 吉野正

日本癌学会学術総会記事 65th 2006年

成人T細胞白血病/リンパ腫(ATLL)における病期の進行とDNAメチル化の関連

佐藤妃映, 佐藤妃映, 岡剛史, 中谷陽子, 近藤孝美, 鷲尾佳奈, 高野正幸, 大原信哉, 大内田守, 宇都宮こう, 大島孝一, 高橋聖之, 吉野正

日本病理学会会誌 95 ( 1 ) 2006年

成人T細胞白血病/リンパ腫(ATLL)におけるCpG island methylater phenotype(CIMP)

佐藤妃映, 佐藤妃映, 岡剛史, 中谷陽子, 近藤孝美, 鷲尾佳奈, 大原信哉, 大内田守, 大島孝一, 谷本光音, 高橋聖之, 吉野正

日本リンパ網内系学会会誌 46 2006年

SHP1遺伝子発現抑制と悪性リンパ腫・白血病発症機構の解析

岡剛史, 大内田守, 中谷陽子, 近藤孝美, 佐藤妃映, 佐藤妃映, 鷲尾佳奈, 高野正幸, 村上一郎, 大原信哉, 高橋聖之, 吉野正

日本病理学会会誌 95 ( 1 ) 2006年

Heparanase is involved in angiogenesis in esophageal cancer through induction of cyclooxygenase-2 国際誌

T Okawa, Y Naomoto, T Nobuhisa, M Takaoka, T Motoki, Y Shirakawa, T Yamatsuji, H Inoue, M Ouchida, M Gunduz, M Nakajima, N Tanaka

CLINICAL CANCER RESEARCH 11 ( 22 ) 7995 - 8005 2005年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

Purpose: Both heparanase and cyclooxygenase-2 (COX-2) are thought to play critical roles for tumor malignancy, including angiogenesis, although it is unknown about their relationship with each other in cancer progression. We hypothesized that they may link to each other on tumor angiogenesis.
Experimental Design: The expressions of heparanase and COX-2 in 77 primary human esophageal cancer tissues were assessed by immunohistochemistry to do statistical analysis for the correlation between their clinicopathologic features, microvessel density, and survival of those clinical cases. Human esophageal cancer cells were transduced with heparanase c DNA and used for reverse transcription-PCR and Western blot to determine the expression of heparanase and COX-2. COX-2 promoter vector and its deletion/mutation constructs were also used along with transduction of heparanase c DNA for luciferase assay.
Results: Heparanase and COX-2 protein expression exhibited a similar pattern in esophageal tumor tissues, and their expression correlated with tumor malignancy and poor survival. Their expression also revealed a significant correlation with high intratumoral microvessel density. Up-regulation of COX-2 mRNA and protein was observed in esophageal cancer cells transfected with heparanase cDNA. COX-2 promoter was activated after heparanase cDNA was transduced and the deletion/mutation of three transcription factor (cyclic AMP response element, nuclear factor-KB, and nuclear factor-interleukin-6) binding elements in COX-2 promoter strongly suppressed its activity.
Conclusion: Our results suggest that heparanase may play a novel role for COX-2-mediated tumor angiogenesis.

DOI： 10.1158/1078-0432.CCR-05-1103

Deletional Mapping of 2q21-37 Region in Oral Cancer(International Symposium of Maxillofacial and Oral Regenerative Biology in Okayama 2005)

Cengiz Beyhan, Gunduz Mehmet, Gunduz Esra, Ouchida Mamoru, Shimizu Kenji, Inoue Tomio, Nagai Noriyuki

Journal of hard tissue biology 14 ( 2 ) 227 - 228 2005年10月

記述言語：英語出版者・発行元：硬組織再生生物学会

Tumor suppressor genes are defined as genetic elements whose loss or mutational inactivation allows cells to display one or more phenotypes of neoplastic growth. Frequent deletion in a chromosomal region suggests existence of a candidate tumor suppressor gene. We analyzed Ch2q21-37.3 region by using 17 polymorphic microsatellite markers in 39 matched oral normal and cancer tissues. Loss of heterozygosity (LOH) was detected at least one location in 36 of 39 (92%) tumor tissues. High deletions were detected at microsatellite marker locations, D2S2304 (35%), D2S111 (40%), D2S155 (35%), D2S164 (29%), D2S125 (71%) and D2S140 (39%). Three frequently deleted regions at 2q22, 2q35-36 and 2q37.3 were observed. Chromosomal 2q22-37.3 region is highly populated with genes. Several candidate tumor suppressor genes in this region including such as ING5, CASP8, CASP10, PPP1R7 and BOK are located. We are currently analyzing inactivation mutations and mRNA expressions in oral squaomus cell carcinomas.

DOI： 10.2485/jhtb.14.227

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その他リンク： http://search.jamas.or.jp/link/ui/2007049536

Frequent deletion and down-regulation of ING4, a candidate tumor suppressor gene at 12p13, in head and neck squamous cell carcinomas 国際誌

M Gunduz, H Nagatsuka, K Demircan, E Gunduz, B Cengiz, M Ouchida, H Tsujigiwa, E Yamachika, K Fukushima, L Beder, S Hirohata, Y Ninomiya, K Nishizaki, K Shimizu, N Nagai

GENE 356 109 - 117 2005年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

We previously showed two members of the ING family, ING1 and ING3 as a tumor suppressor gene in head and neck cancer. Progress in human genome sequencing provided additional information of the new members of the ING family genes. ING4 is localized to chromosome 12p13.31 region and harbors the PHD domain highly homologous among ING family proteins. We analyzed loss of heterozygosity at 12p12-13 region in 50 head and neck squamous cell carcinomas by using six highly polymorphic microsatellite markers and found allelic loss in 66% (33/50) of the informative cases. To clarify the role of ING4 in head and neck carcinogenesis, we first checked mutation status in tumor samples. As mutation of the ING4 gene was not found in head and neck cancers, we examined the mRNA expression level. Quantitative real-time RT-PCR analysis demonstrated decreased expression of ING4 mRNA in 76% of primary tumors as compared with that of matched normal samples. Since p53 dependent pathways of other ING family members have been shown, we examined p53 mutation status and compared with ING4 mRNA expression in tumor samples. However, no such direct relationship has been detected. In conclusion, frequent deletion and decreased mRNA expression of ING4 suggested it as a class two tumor suppressor gene and may play an important role in head and neck cancer. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2005.02.014

Mutation screen of GABRA1, GABRB2 and GABRG2 genes in Japanese patients with absence seizures 国際誌

M Ito, Ohmori, I, T Nakahori, M Ouchida, Y Ohtsuka

NEUROSCIENCE LETTERS 383 ( 3 ) 220 - 224 2005年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER IRELAND LTD

Absence seizures are classified into typical and atypical absences according to clinical and EEG characteristics. Although missense mutations in the GABA(A) receptor gamma 2 subunits (GABRG2) gene have recently been detected in two families with typical absence seizures, no study has been carried out to clarify the relationship between atypical absence and GABA(A) receptors. We performed mutation analysis of all the coding exons of GABA(A) receptor alpha 1, beta 2 and gamma 2 subunit (GABRA1, GABRB2 and GABRG2) genes by direct sequencing to clarify whether there was common molecular biological mechanism underlying both typical and atypical absences. We recruited 52 unrelated Japanese patients, thirty-eight with typical absences and 14 with atypical absences. They consisted of 38 with childhood absence epilepsy, three with Lennox-Gastaut syndrome, two with epilepsy with myoclonic-astatic seizures and nine with epilepsy with continuous spike-waves during slow wave sleep. All of the subjects were idiopathic or cryptogenic cases without any organic brain lesions or underlying diseases. We detected five polymorphisms (T156C in GABRA1, C1194T in GABRB2, and C315T, T588C and C1230T in GABRG2), and they are silent mutations. In conclusion, mutations in GABRA1, GABRB2 and GABRG2 do not seem to be a major genetic cause of epilepsy with typical and atypical absences in Japanese subjects. (c) 2005 Elsevier Ireland Ltd. All fights reserved.

DOI： 10.1016/j.neulet.2005.04.017

Fine deletional mapping of chromosome 4q22-35 region in oral cancer 国際誌

Y Shinno, E Gunduz, M Gunduz, H Nagatsuka, H Tsujigiwa, B Cengiz, YJ Lee, R Tamamura, M Ouchida, K Fukushima, K Shimizu, N Nagai

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 16 ( 1 ) 93 - 98 2005年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PROFESSOR D A SPANDIDOS

We analyzed the loss of heterozygosity of the long arm of chromosome 4 in 40 oral cancers, using 16 microsatellite markers based on data from the human genome sequence, and defined the deletional mapping of the region with putative tumor suppressor genes. Our data revealed two distinct commonly deleted regions around the markers, D4S2623 and D4S1644, with an allelic deletion of 44 and 39%, respectively. Additional mapping and use of the markers near one of these hot spots narrowed down the minimally deleted region about 1.5 Mbp around the marker, D4S2623. Caspase 6 is localized 280 kb from the marker, D4S2623. Fine mapping of this region with possible tumor suppressor gene suggests caspasc 6 as a putative tumor suppressor gene. Further molecular analysis of caspase 6 should be performed to clarify its role in oral carcinogenesis.

Significant growth suppression of synovial sarcomas by the histone deacetylase inhibitor FK228 in vitro and in vivo 国際誌

T Ito, M Ouchida, Y Morimoto, A Yoshida, Y Jitsumori, T Ozaki, H Sonobe, H Inoue, K Shimizu

CANCER LETTERS 224 ( 2 ) 311 - 319 2005年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER IRELAND LTD

About 97% of synovial sarcomas harbor the SYT-SSX fusion gene by chromosomal translocation. We found that the histone deacetylase (HDAC) inhibitor FK228 significantly suppressed the growth of synovial sarcoma cells as compared with that of osteosarcoma. The 50% growth inhibition IC50 value we obtained for FK228 was 0.02-0.2 nM, and it indicates that its suppression effect on synovial sarcoma cells is the highest of any of the HDAC inhibitors yet reported. It was not likely that the growth suppression of FK228 depends on the doubling time of these cells. Introduction of SYT-SSX cDNA into HEK293 cells enhanced the sensitivity of the cells for FK228. Immunostaining of the FK228-treated cells using an anti-acetyl-hi stone H3 antibody showed that FK228 inhibits deacetylation of histone. In a mice assay, the growth of synovial sarcoma cells was markedly inhibited by FK228 treatment, and the invasion of tumors into surrounding tissues was suppressed. These results suggest that FK228 may be useful in developing therapeutic strategies to treat synovial sarcoma. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.canlet.2004.10.030

Single nucleotide polymorphisms in the EXO1 gene and risk of colorectal cancer in a Japanese population 国際誌

H Yamamoto, H Hanafusa, M Ouchida, M Yano, H Suzuki, M Murakami, M Aoe, N Shimizu, K Nakachi, K Shimizu

CARCINOGENESIS 26 ( 2 ) 411 - 416 2005年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

EXO1 is a member of the RAD2 nuclease family and functions in DNA replication, repair and recombination. We investigated the relationship of single nucleotide polymorphisms (SNPs) at exon 10 (T439M) and exon 13 (P757L) of the EXO1 gene with development, progression and metastasis of colorectal cancer. For T439M, the Thr/Met genotype [odds ratio (OR) = 2.03, 95% confidence interval (CI) 1.04-3.98] and Thr/Met and Met/Met genotypes combined (OR = 2.37, 95% CI 1.23-4.56) demonstrated significant association with the development of colorectal cancer after adjusting for age, gender and smoking status. For P757L, patients with the Leu/Leu genotype showed a reduced risk of colorectal cancer (adjusted OR = 0.398, 95% CI 0.183-0.866) when the Pro/Leu and Pro/Pro genotypes were combined and used as the reference. The Leu/Leu genotype also had a reduced risk (adjusted OR = 0.373, 95% CI 0.164-0.850) when the Pro/Leu genotype was used as the reference. Individuals who carried both putative risk genotypes (Thr/Met and Met/Met for T439M and Pro/Leu for P757L) showed an adjusted OR of 4.95 (95% CI 1.56-15.7) compared with those who carried both low risk genotypes. Analysis of microsatellite instability (MSI) revealed that tumors from individuals who carried both putative risk genotypes tended to have a higher frequency of MSI positives than those from patients who carried both low risk genotypes, although a significant correlation was not found between EXO1 genotype and MSI status. This is the first report to provide evidence for an association of EXO1 gene polymorphisms with colorectal cancer risk. The EXO1 genotypes were not associated with any clinicopathological characteristics in colorectal cancer patients.

DOI： 10.1093/carcin/bgh335

Aberrant promoter methylation of human DAB2 interactive protein (hDAB2IP) gene in lung cancers 国際誌

M Yano, S Toyooka, K Tsukuda, H Dote, M Ouchida, T Hanabata, M Aoe, H Date, AF Gazdar, N Shimizu

INTERNATIONAL JOURNAL OF CANCER 113 ( 1 ) 59 - 66 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-LISS

The human DOC-2/DAB2 interactive protein gene (hDAB2IP) is a novel member of the Ras GTPase-activating gene family that is known to act as a tumor suppressor gene and is inactivated by methylation in prostate and breast cancers. We established previously a methylation-specific PCR (MSP) for the promoter region (m2a and m2b regions) of hDAB2IP and examined hDAB2IP methylation status in breast cancers. We analyzed the methylation and expression status of hDAB2IP in lung cancers. The methylation status of hDAB2IP was examined in lung cancer cell lines using bisulfite sequencing and MSP. Expression was examined using conventional and real-time RT-PCR, and methylation was found to be inversely correlated with expression, confirming that the MSP can also be used to examine hDAB2IP methylation status in lung cancers. Aberrant methylation was detected at the m2a region in 19 of 47 lung cancer cell lines (40%) and 26 of 70 primary tumors (37%) and at the m2b in 16 lines (34%) and 25 of 70 tumors (36%). Gene expression was restored in methylated cell lines supplemented with 5-aza-2'-deoxycytidine, confirming that methylation was responsible for downregulation. We also examined the relationship between hDAB2IP methylation and clinico-pathological features of the lung cancers and found that hDAB2IP methylation was associated with advanced disease stage. Our results demonstrate that hDAB2IP methylation is frequently present in lung cancers and plays a key role in hDAB2IP silencing. hDAB2IP methylation could be used as a biomarker for disease stage, reflecting the degree of clinico-pathological malignancy of lung cancer. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/ijc.20531

Genetic and epigenetic alterations of BRG1 promote oral cancer development 国際誌

E Gunduz, M Gunduz, M Ouchida, H Nagatsuka, L Beder, H Tsujigiwa, K Fukushima, K Nishizaki, K Shimizu, N Nagai

INTERNATIONAL JOURNAL OF ONCOLOGY 26 ( 1 ) 201 - 210 2005年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PROFESSOR D A SPANDIDOS

Loss of heterozygosity (LOH) analysis is a sensitive method to detect deletions of specific chromosome regions which are considered to harbor of putative tumor suppressor gene (TSG)s. Previous allelotype analyses in various human cancers suggested the presence of at least one TSG in chromosome 19p13 region. Functional analysis of BRG1, a member of SWI/SNF complex proteins, located at 19p 13.2, suggested it to be a candidate TSG in different types of human cancers. We examined LOH at 19p13 region in 39 oral cancers by using six microsatellite markers and found allelic deletion in 25 of 39 (64%) samples. A microsatellite marker near BRG1 locus showed the highest LOH in tumor samples. To clarify the role of BRG1 gene in oral carcinogenesis, we checked its mutation status in tumor samples. As mutation of the BRG1 gene was not detected in oral cancers, we examined the mRNA expression level. Quantitative RT-PCR analysis surprisingly demonstrated increased expression of BRG1 mRNA in 62% of primary tumors as compared with that of matched normal samples. Since functions of BRG1 and BRM, another member of SWI/SNF highly homologous to BRG1, may be interrelated, we also examined BRM mRNA expression in the same samples. In most samples, the expressions of BRG1 and BRM changed in the same direction consistent with reported data on human cancer cell lines. An important finding is that expression of an alternative in frame splicing form of BRG1 which includes exon 26 is selectively decreased or lost in most tumor samples. This unique 33 amino-acid sequence of BRG1 protein shows very high homology with heterogeneous nuclear ribonucleoprotein E and may affect the function and level of BRG1 through modifications on post-transcriptional control. All these findings suggest that the genetic and epigenetic alterations of BRG1 may have a role in oral cancer development.

膜肉腫細胞株に対するHDAC抑制剤FK228のin vivo,in vitroにおける抗腫瘍効果の検討.

伊藤達男, 尾崎敏文, 大内田守

医学のあゆみ 213,12,1041-1046 2005年

Frequent Deletion and Down-Regulation of ING3 in Head and Neck Cancer

Beyhan Cengiz, Mehmet Gunduz, Esra Gunduz, Mamoru Ohuchida, Kenji Shimizu, Noriyuki Nagai, Deng Chunfu, Goichi Kinoshita

Journal of Hard Tissue Biology 14 ( 2 ) 298 - 299 2005年

記述言語：英語掲載種別：研究論文（学術雑誌）

Loss of heterozygosity (LOH) has been frequently detected at chromosome 7q31 region in human head and neck squamous cell carcinomas (HNSCC) and many other cancers, suggesting the existence of the tumor suppressor genes (TSG). However, the targeted gene was yet identified. We analyzed LOH at 7q31 region in 49 HNSCC by using 6 polymorphic microsatellite markers and found allelic deletion in 48% of the informative cases. We detected two preferentially deleted locations one around D7S643 and the other around D7S486. When we redefined the map of 7q31 region according to the contiguous sequences, an ING1 like gene, ING3, which we named it due to homology to ING1, was found in the proximity of D7S643. ING3 protein harbors a PHD zinc finger domain highly homologous among ING family proteins, in which we previously found mutations in a related gene, ING1. As only one missense mutation of the ING3 gene was found in HNSCC, we examined the mRNA expression levels. RT-PCR analysis demonstrated decreased or no expression of ING3 mRNA in 45 % of primary tumors as compared with that of mathced normal samples. All these findings suggest a possibility that ING3 functions as a TSG in a subset of HNSCC. © 2005, THE SOCIETY FOR HARD TISSUE REGENERATIVE BIOLOGY. All rights reserved.

DOI： 10.2485/jhtb.14.298

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