研究者詳細 - 大内田　守

食道扁平上皮癌の発癌過程におけるDNAメチル化の役割

石井龍宏, 村上純, 能登原憲司, 笹本博美, 神原健, 白川靖博, 猶本良夫, 大内田守, 清水憲二, Jass Jeremy R., 松原長秀, 田中紀章

岡山医学会雑誌 119 ( 2 ) 113 - 117 2007年9月

記述言語：日本語出版者・発行元：岡山医学会

食道扁平上皮癌切除例の正常粘膜、異形成病変(IEN)、癌病変(ESCC)の変化を解析した。対象は56例で、対照として健常人42例の食道正常粘膜を用いた。14種類の癌関連遺伝子のプロモーター領域のメチル化を検索したところ、食道扁平上皮癌切除例の各段階でのメチル化数はCOX2、hMLH1を除いた殆どのマーカーで健常人よりも増加していた。癌背景粘膜正常部においてもメチル化数は健常人よりも増加しており、IENはESCCと同程度のメチル化の蓄積を示していた。p53遺伝子のエクソン5-9の遺伝子配列に対してシーケンスを行ったところ、p53遺伝子突然変異はESCCで62.5%(35/56例)、IENで57%(12/21例)にみられたが、癌背景粘膜正常部、健常人には殆ど存在しなかった。メチル化異常は健常人→癌背景正常粘膜→異形成病変→癌病変と段階的に増加し、メチル化異常が高度に蓄積する異形成病変ではp53変異が出現し、これを一因に癌化に至ると考えられた。

DOI： 10.4044/joma.119.113

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その他リンク： http://eprints.lib.okayama-u.ac.jp/13191

T75M-KCNJ2 mutation causing Andersen-Tawil syndrome enhances inward rectification by changing Mg2+ sensitivity 国際誌

Yoshinori Tani, Daiji Miura, Junko Kurokawa, Kazufumi Nakamura, Mamoru Ouchida, Kenji Shimizu, Tohru Ohe, Tetsushi Furukawa

JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 43 ( 2 ) 187 - 196 2007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：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

Andersen-Tawil syndrome (ATS) is a multisystem inherited disease exhibiting periodic paralysis, cardiac arrhythmias, and dysmorphic features. In this study, we characterized the KCNJ2 channels with an ATS mutation (T75M) which is associated with cardiac phenotypes of bidirectional ventricular tachycardia, syncope, and QT(c) prolongation. Confocal imaging of GFP-KCNJ2 fusion proteins showed that the T75M mutation impaired membrane localization of the channel protein, which was restored by co-expression of WT channels with T75M channels. Whole-cell patch-clamp experiments in CHO-K1 cells showed that the T75M mutation produced a loss-of-function of the channel. When both WT and the T75M were co-expressed, the T75M mutation showed dominant-negative effects on inward rectifier K+ current densities, with prominent suppression of outward currents at potentials between 0 mV and + 80 mV over the E-K. Inside-out patch experiments in HEK293T cells revealed that co-expression of WT and the T75M channels enhanced voltage-dependent block of the channels by internal Mg2+, resulting in enhanced inward rectification at potentials 50 mV more positive than the EK. We suggest that the T75M mutation causes dominant-negative suppression of the co-expressed WT KCNJ2 channels. In addition, the T75M mutation caused alteration of gating kinetics of the mutated KCNJ2 channels, i.e., increased sensitivity to intracellular Mg2+ and resultant enhancement of inward rectification. The data presented suggest that the mutation may influence clinical features, but it does not directly show this. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yjmcc.2007.05.005

Epigenetically distinct malignant cell population forms lymphoma-involving lesions in acute type adult T cell Leukemia/Lymphoma (ATLL)

Takashi Oka, Hiaki Sato, Yoko Nakatani, Takami Kondo, Kana Washio, Masayuki Takano, Ichiro Murakami, Nobuya Ohara, Mamoru Ouchida, Koichi Ohshima, Kenji Shimizu, Mitsune Tanimoto, Kiyoshi Takahashi, Atae Utsunomiya, Tadashi Yoshino

AIDS RESEARCH AND HUMAN RETROVIRUSES 23 ( 4 ) 627 - 628 2007年4月

The effect of gefitinib on B-RAF mutant non-small cell lung cancer and transfectants 国際誌

Shinichi Toyooka, Akiko Uchida, Hisayuki Shigematsu, Junichi Soh, Atsuko Ogino, Minoru Takata, Katsuyuki Kiura, Mamoru Ouchida, Takayuki Kosaka, Motoi Aoe, Tetsuya Mitsudomi, Hiroshi Date

JOURNAL OF THORACIC ONCOLOGY 2 ( 4 ) 321 - 324 2007年4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：LIPPINCOTT WILLIAMS & WILKINS

We previously reported one patient with squamous cell carcinoma of the lung that showed the long-term effect to gefitinib with complete response. In the present report, we examine the epidermal growth factor receptor (EGFR) and K-RAS, HER2, and B-RAF mutations in this patient to find a B-RAF exon 11 mutation, resulting in a substitution of valine by phenylalanine at codon 470 (V470F) as a novel type of B-RAF mutation in human cancers. In addition, the fluorescence in situ hybridization analysis for EGFR showed the high polysomy status. B-RAF is a nonreceptor serine/threonine kinase whose kinase domain has a structure similar to other protein kinases, including EGFR members. Of interest, the B-RAF V470F mutation corresponds to a position similar to the EGFR G719X mutation located on the phosphate binding (P)-loop of EGFR that clamps ATP into the catalytic cleft. This observation suggests that gefitinib may have an anti-cancer effect on B-RAF mutant tumors. Indeed, previous reports demonstrated that H1666 cells harboring B-RAF G465V mutations showed sensitivity to gefitinib, inhibiting phosphorylation of ERK1/2. We examined the effect of gefitinib on transient transfectants of the B-RAF mutant, but no drastic inhibition of ERKI/2 phosphorylation that was one of the downstream molecules of B-RAF was induced by gefitinib.
In summary, we found a novel B-RAF V470F mutation in lung squamous cell carcinoma that showed response to gefitinib. However. our in vitro investigation did not explain the response observed in this particular patient. Further investigation is necessary to elucidate the mechanism of tumor sensitivity to EGFR tyrosine kinase inhibitors.

DOI： 10.1097/01.JTO.0000263716.92191.be

Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa

T. Ishii, J. Murakami, K. Notohara, H. M. Cullings, H. Sasamoto, T. Kambara, Y. Shirakawa, Y. Naomoto, M. Ouchida, K. Shimizu, N. Tanaka, J. R. Jass, N. Matsubara

GUT 56 ( 1 ) 13 - 19 2007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：B M J PUBLISHING GROUP

Background: Oesophageal squamous cell carcinoma (OSCC) often arises from preceding dysplastic lesions in the oesophageal epithelium. However, the molecular changes occurring in premalignant lesions are not well understood. An epigenetic change is an example of OSCC that may occur within the epithelium.
Aim: To investigate the methylation status of multiple promoters in cancer-derived DNA, as well as in the background epithelium of OSCC, including dysplastic lesions and non-neoplastic mucosa. The normal epithelium from patients without cancer was also examined. The findings were correlated with the mutational status of p53.
Patients and methods: 56 patients with advanced OSCC, 21 patients with intraepithelial neoplasia (IEN), 56 patients with a background of non-neoplastic epithelium, adjacent to the OSCC, and 42 normal control epithelia from healthy volunteers were studied. The promoter methylation status of SFRP1, SFRP2, DCC, APC, p16(INK4a), p14(ARF), MINT1, MINT2, MINT31, CACNA1G, COX2, DAPK, hMLH1 and MGMT was examined by methylation-specific single polymerase chain reaction or combined bisulphite restriction analysis. The mutation of p53 by direct sequencing was assessed.
Results: DNA methylation was observed in OSCC and in its background epithelium. The frequency of CpG island methylation increased from a baseline level in the background non-neoplastic epithelium, through IEN, to advanced OSCC. However, mutations in p53 were almost exclusively observed in IEN and OSCC. More extensive DNA methylation was seen in the neoplastic lesions (OSCC or IEN) having a p53 mutation than in those with wild-type p53.
Conclusion: DNA methylation is present at low levels in the non-neoplastic oesophageal epithelium and appears to contribute to the progression of the dysplasia-carcinoma sequence in OSCC carcinogenesis.

DOI： 10.1136/gut.2005.089813

Anti-tumor effect of bisphosphonate (YM529) on non-small cell lung cancer cell lines 国際誌

Ryuichiro Koshimune, Motoi Aoe, Shinichi Toyooka, Fumikata Hara, Mamoru Ouchida, Masaki Tokumo, Yoshifumi Sano, Hiroshi Date, Nobuyoshi Shimizu

BMC CANCER 7 ( 8 ) 1 - 8 2007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：BIOMED CENTRAL LTD

Background: YM529 is a newly developed nitrogen-containing bisphosphonate ( BP) classified as a third-generation BP that shows a 100-fold greater potency against bone resorption than pamidronate, a second-generation BP. This agent is, therefore expected to be extremely useful clinically for the treatment of osteoporosis and hypercalcemia. Recently, YM529 as well as other third-generation BPs have also been shown to exert anti-tumor effects against various types of cancer cells both in vitro or/and in vivo. In this study, we investigate the anti-tumor effect of YM529 on non-small cell lung cancer (NSCLC).
Methods: Direct anti-tumor effect of YM529 against 8 NSCLC cell lines ( adenocarcinoma: H23, H1299, NCI-H1819, NCI-H2009, H44, A549, adenosquamous cell carcinoma: NCI-H125, squamous cell carcinoma: NCI-H157) were measured by MTS assay and calculated inhibition concentration 50% (IC50) values. YM529 induced apoptosis of NCI-H1819 was examined by DNA fragmentation of 2% agarose gel electrophoresis and flowcytometric analysis (sub-G(1) method). We examined where YM529 given effect to apoptosis of NSCLC cells in signaling pathway of the mevalonate pathway by western blotting analysis.
Results: We found that there was direct anti-tumor effect of YM529 on 8 NSCLC cell lines in a dose-dependent manner and their IC50 values were 2.1 to 7.9 mu M and YM529 induced apoptosis and G(1) arrest cell cycle with dose-dependent manner and YM529 caused down regulation of phospholyration of ERK1/2 in signaling pathways of NSCLC cell line (NCI-H1819).
Conclusion: Our study demonstrate that YM529 showed direct anti-tumor effect on NSCLC cell lines in vitro, which supports the possibility that third-generation BPs including YM529 can be one of therapeutic options for NSCLC.

DOI： 10.1186/1471-2407-7-8

Loss or down-regulation of HLA class I expression at the allelic level in freshly isolated leukemic blasts 国際誌

Kozo Masuda, Akio Hiraki, Nobuharu Fujii, Toshiyuki Watanabe, Motoyuki Tanaka, Kosei Matsue, Yoichiro Ogama, Mamoru Ouchida, Kenji Shimizu, Kazuma Ikeda, Mitsune Tanimoto

CANCER SCIENCE 98 ( 1 ) 102 - 108 2007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：BLACKWELL PUBLISHING

Loss or down-regulation of human leukocyte antigen (HLA) class I expression has been demonstrated in a variety of solid tumors. To date, such altered HLA expression has not been studied extensively in freshly isolated leukemic blasts. If it occurs, leukemic cells could escape T-cell surveillance as a consequence. Genotypes of nine leukemic cell lines were determined using a polymerase chain reaction for HLA classes I and II. Cells were also examined for HLA beta 2-microglobulin, and allele-specific HLA protein expression using flow cytometry. Next, 44 samples of freshly isolated leukemic blasts from 43 patients with malignant hematological diseases were examined for allele-specific HLA expression using flow cytometry. Microsatellite analysis was performed to determine heterozygosity in the HLA region on chromosome 6. Genotype analysis for HLA class I together with microsatellite analysis demonstrated loss of HLA haplotype in HL-60 cells. No loss of HLA haplotype was observed in 44 samples of freshly isolated leukemic blasts. As reported previously, flow cytometric analysis rarely demonstrated loss or down-regulation of HLA expression at initial diagnosis (3/39; 7.7%); however, this was evident in two of five cases in relapse (40.0%), which contrasts with previous reports. In one patient with acute leukemia, HLA-A2 cell surface expression was present at initial diagnosis, lost at relapse, and completely restored after 48 h of culture in the presence of interferon-gamma. These results suggest loss of allele-specific HLA expression may be involved in the pathogenesis of relapse in patients with leukemia. The findings should be valuable in designing new strategies for clinical immunotherapy.

DOI： 10.1111/j.1349-7006.2006.00356.x

SCN1A missense mutation associated with infantile partial epilepsy

Iori Ohmori, Mamoru Ouchida, Katsuhiro Kobayashi, Yoko Ohtsuka, Kenji Shimizu, Teiichi Nishiki, Kazuhito Tomizawa, Hideki Matsui

NEUROSCIENCE RESEARCH 58 S187 - S187 2007年

HIGH FREQUENT GENE SILENCING OF HEMETOPOIETIC CELL SPECIFIC PROTEIN TYROSINEPHOSPHATASE SHP1 IN HEMETOPOIETIC CELL MALIGNANCIES.

Oka T, Ouchida M, Tanimoto M, Shimizu K, Yoshino T

International Journal of Medical and Biological Frontiers 11 11 - 42 2007年

The role of angiotensin type 2 receptor (AT2R) on nerve growth factor (NGF) induced-reinnervation of perivascular nerves

Namika Yoshida, ayako Yokomizo, Mitsuhiro Goda, Mamoru Ouchida, Kenji Simizu, Fusako Takayama, Hiromu Kawasaki

JOURNAL OF PHARMACOLOGICAL SCIENCES 103 219P - 219P 2007年

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

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, 大島孝一, 宇都宮與, 高橋聖之, 高橋聖之, 吉野正

日本病理学会会誌 96 ( 1 ) 2007年

成人T細胞白血病/リンパ腫(ATLL)における特異的DNAメチル化と病態・臨床症状との相関

佐藤妃映, 佐藤妃映, 岡剛史, 近藤孝美, 高野正幸, 神農陽子, 鷲尾佳奈, 高田尚良, 守都敏晃, 大原信哉, 大内田守, 大島孝一, 谷本光音, 宇都宮與, 高橋聖之, 吉野正

日本リンパ網内系学会会誌 47 2007年

Highly frequent allelic loss of chromosome 6q16-23 in osteosarcoma: Involvement of cyclin C in osteosarcoma 国際誌

Norihide Ohata, Sachio Ito, Aki Yoshida, Toshiyuki Kunisada, Kunihiko Numoto, Yoshimi Jitsumori, Hirotaka Kanzaki, Toshifumi Ozaki, Kenji Shimizu, Mamoru Ouchida

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 18 ( 6 ) 1153 - 1158 2006年12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

The molecular pathogenesis of osteosarcoma is very complicated and associated with chaotic abnormalities on many chromosomal arms. We analyzed 12 cases of osteosarcomas with comparative genomic hybridization (CGH) to identify chromosomal imbalances, and detected highly frequent chromosomal alterations in chromosome 6q, 8p, 10p and 10q. To define the narrow rearranged region on chromosome 6 with higher resolution, loss of heterozygosity (LOH) analysis was performed with 21 microsatellite markers. Out of 31 cases, 23 cases (74%) showed allelic loss at least with one marker on chromosome 6q. We identified two distinct commonly deleted regions on chromosome 6 using markers D6S1565 located at 6q16 and 6q23MS1 at 6q23. The expression analysis of genes located at the deleted region was performed, and the decreased mRNA expression of the CCNC gene, one of the regulators of cell cycle, was detected. Growth of osteosarcoma cell line was significantly suppressed after the CCNC cDNA transfection. Fine mapping of the deleted region containing a possible tumor suppressor gene and the transfection assay suggest that the CCNC is a candidate tumor suppressor gene.

Multiple splicing variants of Naf1/ABIN-1 transcripts and their alterations in hematopoietic tumors 国際誌

Yasuhiro Shiote, Mamoru Ouchida, Yoshimi Jitsumori, Yoichiro Ogama, Yoshinobu Matsuo, Fumihiko Ishimaru, Mitsune Tanimoto, Kenji Shimizu

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 18 ( 5 ) 917 - 923 2006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：PROFESSOR D A SPANDIDOS

Naf1 (Nef-associated factor 1)/TNIP1/ABIN-1 (A20-binding inhibitor of NF-kappa B activation) is a cellular protein that interacts and cooperates with the NF kappa B inhibiting protein A20. It is reported that Naf1 attenuates epidermal growth factor (EGF)/extracellular-signal-regulated kinase2 (ERK2) nuclear signaling. Naf1 also binds to Nef, which plays a key role in acquired immunodeficiency syndrome pathogenesis and HIV-1 virus replication. Naf1 mRNA consists of 18 exons and multiple splice variants have been reported; two isoforms for exon 1, deletion of exon 2 and isoforms a and beta for exon 18. Using specimens from 29 acute myeloid leukemia (AML) patients, we detected a high frequency of allelic loss on DNA at STS marker D5S2014 near the Naf1 gene. We there :ore performed mutation and expression analyses using :leukemia-lymphoma lines and 6 pairs of clinical AML samples. There was no mutation in the Naf1 coding region of any sample. As a result of expression analysis, we identified novel splice variants of the Naf1 gene; deletion of exon 16 (Naf1 alpha 2, Naf1 beta 2), deletion of exon 16 with an insertion (Naf1 alpha 3, Naf1 beta 3) and deletion of exons 16 and 17 (Naf1 alpha 4). Naf1 alpha 3 and beta 3 showed premature termination. In peripheral blood mononucleocytes (PBMNCs) from healthy adults, almost no expression of full-length Naf1 (Naf1(FL)). Naf1 alpha 3 and beta 3 were observed. In contrast, their expression was wear in AML blasts and in the majority of leukemia-lymphoma lines investigated. Naf1 alpha 2 was widely expressed in PBMNCs from healthy adults, AML blasts and cell lines, suggesting it is the main transcript of the Naf1 gene. Luciferase assay revealed that Naf1 alpha 2 had equal NF-kappa B inhibitory effect to that of Naf1(FL), while Naf1 alpha 4 was less effective. In clinical AML patients, the expression of Naf1 alpha 3 was much higher at diagnosis than on remission after chemotherapy, suggesting the possible dominant negative effect of Naf1 alpha 3.

Single nucleotide polymorphism of the AXIN2 gene is preferentially associated with human lung cancer risk in a Japanese population 国際誌

Hirotaka Kanzaki, Mamoru Ouchida, Hiroko Hanafusa, Masaaki Yano, Hiromitsu Suzuki, Motoi Aoe, Kazue Imai, Nobuyoshi Shimizu, Kei Nakachi, Kenji Shimizu

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 18 ( 2 ) 279 - 284 2006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：SPANDIDOS PUBL LTD

The AXIN2 gene, a negative regulator gene of Wnt/beta-catenin signaling, is a putative tumor suppressor gene on human chromosome 17q24. In the genomic locus on which the AXIN2 gene is located, allelic loss and rearrangement were frequently detected in many cancers. An association between human cancer risk and a single nucleotide polymorphism (SNP) at codon 50 of the AXIN2 gene, encoding either proline (CCT) or serine (TCT), remains undefined. We, therefore, investigated the distribution of the SNP at codon 50 in 110 healthy controls and 160 patients with non-small-cell lung cancer, 113 patients with colorectal cancer, and 63 patients with head and neck cancer. We found that the frequency of the homozygous T/T (Ser/Ser) genotype was significantly less in lung cancer patients (5.0%) than in healthy controls (13.6%) (p=0.005). As compared with the C/C (Pro/Pro) genotype of the controls, lung cancer patients with the T/T genotype showed reduced risk of cancer; the adjusted odds ratio (OR) for patients with the homozygous T/T (Ser/Ser) genotype was 0.31 (95% confidence interval (0), 0.12-0.79). The association was particularly strong in lung cancer patients with lung adenocarcinoma (LAD) (adjusted OR, 0.24; 95% Cl, 0.07-0.81), with well-differentiated grade cancer (adjusted OR, 0.12; 95% Cl, 0.01-0.99) and with moderately-differentiated grade cancer (adjusted OR, 0.18; 95% CI, 0.04-0.85). These results suggest that the AXIN2 Pro50Ser SNP is associated with development of lung cancer as a protective SNP, while an association between the AXIN2 SNP and risk of colorectal cancer and of head and neck cancer was not observed. This is the first report to show an association between the AXIN2 SNP and lung cancer risk.

Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer 国際誌

Michio Inukai, Shinichi Toyooka, Sachio Ito, Hiroaki Asano, Shuji Ichihara, Junichi Soh, Hiroshi Suehisa, Mamoru Ouchida, Keisuke Aoe, Motoi Aoe, Katsuyuki Kiura, Nobuyoshi Shimizu, Hiroshi Date

CANCER RESEARCH 66 ( 16 ) 7854 - 7858 2006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：AMER ASSOC CANCER RESEARCH

The threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene has been reported in progressing lesions after gefitinib treatment in non-small cell lung cancer (NSCLC) that causes sensitive tumors to become resistant to gefitinib. Alternatively, the EGFR T790M mutation might be present in small fractions of tumor cells before drug treatment, and the tumor cells harboring the T790M mutation might be enriched during the proliferation after drug treatment. We developed a mutant-enriched PCR assay to detect small fractions of cells with T790M mutation and used this technique to detect mutations in 280 NSCLCs, including gefitinib-treated 95 cases. Although the direct sequencing detected only I T790M mutant case, the mutant-enriched PCR (confirmed to enrich one mutant out of 1 X 10(3) wild-type alleles) detected 9 additional cases among 280 cases. As linkage to clinicopathologic factors, the T790M mutation showed no bias for sex, smoking status, or histology but was significantly more frequent in advanced tumors (9 of III cases) than in early-stage tumors (I of 169 cases; P = 0.0013). Among gefitinib-treated cases, gefitinib-sensitive mutations were found in 30 cases. The T790M mutation was present in 3 of 7 no-responders with the gefitinib-sensitive mutation and was not present in 19 responders (P = 0.014). Our results indicate that the T790M mutation is sometimes present in a minor population of tumor cells during the development of NSCLC and suggest that the detection of small fractions of T790M mutant alleles may be useful for predicting gefitinib resistance of NSCLCs with sensitive EGFR mutations.

DOI： 10.1158/0008-5472.CAN-06-1951