研究者詳細 - 大内田　守

論文 - 大内田　守

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Txn1 mutation is a monogenic cause of chronic kidney disease associated with mitochondrial dysfunction in rats

Iori Ohmori, Mamoru Ouchida, Yoshiko Hada, Haruhito A. Uchida, Shinya Toyokuni, Tomoji Mashimo

BioRxiv 2023年8月

"Input/output cytokines" in epidermal keratinocytes and the involvement in inflammatory skin diseases. 査読国際誌

Shin Morizane, Tomoyuki Mukai, Ko Sunagawa, Kota Tachibana, Yoshio Kawakami, Mamoru Ouchida

Frontiers in immunology 14 1239598 - 1239598 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

Considering the role of epidermal keratinocytes, they occupy more than 90% of the epidermis, form a physical barrier, and also function as innate immune barrier. For example, epidermal keratinocytes are capable of recognizing various cytokines and pathogen-associated molecular pattern, and producing a wide variety of inflammatory cytokines, chemokines, and antimicrobial peptides. Previous basic studies have shown that the immune response of epidermal keratinocytes has a significant impact on inflammatory skin diseases. The purpose of this review is to provide foundation of knowledge on the cytokines which are recognized or produced by epidermal keratinocytes. Since a number of biologics for skin diseases have appeared, it is necessary to fully understand the relationship between epidermal keratinocytes and the cytokines. In this review, the cytokines recognized by epidermal keratinocytes are specifically introduced as "input cytokines", and the produced cytokines as "output cytokines". Furthermore, we also refer to the existence of biologics against those input and output cytokines, and the target skin diseases. These use results demonstrate how important targeted cytokines are in real skin diseases, and enhance our understanding of the cytokines.

DOI： 10.3389/fimmu.2023.1239598

Thioredoxin deficiency increases oxidative stress and causes bilateral symmetrical degeneration in rat midbrain. 査読国際誌

Iori Ohmori, Mamoru Ouchida, Hirohiko Imai, Saeko Ishida, Shinya Toyokuni, Tomoji Mashimo

Neurobiology of disease 175 105921 - 105921 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Thioredoxin, encoded by Txn1, acts as a critical antioxidant in the defense against oxidative stress by regulating the dithiol/disulfide balance of interacting proteins. The role of thioredoxin in the central nervous system (CNS) is largely unknown. A phenotype-driven study of N-ethyl-N-nitrosourea-mutated rats with wild-running seizures revealed the importance of Txn1 mutations in CNS degeneration. Genetic mapping identified Txn1-F54L in the epileptic rats. The insulin-reducing activity of Txn1-F54L was approximately one-third of that of the wild-type (WT). Bilateral symmetrical vacuolar degeneration in the midbrain, mainly in the thalamus and the inferior colliculus, was observed in the Txn1-F54L rats. The lesions displayed neuronal and oligodendrocytic cell death. Neurons in Txn1-F54L rats showed morphological changes in the mitochondria. Vacuolar degeneration peaked at five weeks of age, and spontaneous repair began at seven weeks. The TUNEL assay showed that fibroblasts derived from homozygotes were susceptible to cell death under oxidative stress. In five-week-old WT rats, energy metabolism in the thalamus was significantly higher than that in the cerebral cortex. In conclusion, in juvenile rats, Txn1 seems to play an essential role in reducing oxidative stress in the midbrains with high energy metabolism.

DOI： 10.1016/j.nbd.2022.105921

Aberrant serine protease activities in atopic dermatitis 査読

Shin Morizane, Ko Sunagawa, Hayato Nomura, Mamoru Ouchida

JOURNAL OF DERMATOLOGICAL SCIENCE 107 ( 1 ) 2 - 7 2022年7月

記述言語：英語出版者・発行元：ELSEVIER IRELAND LTD

Atopic dermatitis (AD) is a chronic inflammatory skin disease; the three major factors responsible for AD, i.e., epidermal barrier dysfunction, allergic inflammation, and itching, interact with each other to form a pathological condition. Excessive protease activities are characteristic abnormalities that affect the epi-dermal barrier in patients with AD. In normal skin, epidermal serine protease activities are controlled by kallikrein-related peptidases (KLKs) and their inhibitors, including lympho-epithelial Kazal-type-related inhibitor (LEKTI). In AD lesions, KLKs are excessively expressed, which results in the enhancement of epi-dermal serine protease activities and facilitates the invasion by allergens and microorganisms. In addition, some KLKs can activate protease-activated receptor 2 (PAR2) in epidermal keratinocytes and peripheral nerves, resulting in the induction of inflammation and itching. Furthermore, in AD patients with single nucleotide polymorphism (SNP) such as E420K and D386N of SPINK5 which encodes LEKTI, LEKTI function is attenuated, resulting in the activation of KLKs and easy invasion by allergens and microorganisms. Further analysis is needed to elucidate the detailed mechanism underlying the control of serine protease activities, which may lead to the development of new therapeutic and prophylactic agents for AD.(c) 2022 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jdermsci.2022.06.004

Novel animal model of combined generalized and focal epilepsy. 査読国際誌

Iori Ohmori, Mamoru Ouchida, Masakazu Shinohara, Kiyoka Kobayashi, Saeko Ishida, Tomoji Mashimo

Epilepsia 63 ( 7 ) e80-e85 2022年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Thioredoxin, encoded by Txn1, is a critical antioxidant that protects against oxidative damage by regulating the dithiol/disulfide balance of interacting proteins. We recently discovered the Adem rat, an epileptic rat harboring the Txn1-F54L mutation, characterized by wild running and vacuolar degeneration in the midbrain. This study aimed to characterize the classification of epilepsy in Adem rats. We performed simultaneous video-electroencephalographic recordings, magnetic resonance imaging, neurotransmitter measurements using gas chromatography-mass spectrometry (GC-MS), and immunohistochemistry. Adem rats exhibited absence, tonic, and focal seizures. The type of epilepsy was classified as combined generalized and focal epilepsy. Neurotransmitters in the midbrain and cortex were measured at 3 weeks of age, when neuronal cell death occurs in the midbrain. The results of GC-MS ruled out the dominance of the excitatory system in the midbrain and cortex of Adem rats. Activation of astrocytes and microglia was more pronounced at 5 weeks of age, at which time epileptic seizures occurred frequently. The underlying pathology in Adem rats remains unknown. However, glial cell activation and inflammation may play a significant role in the occurrence of epilepsy.

DOI： 10.1111/epi.17295

Multifaceted Analysis of IL-23A- and/or EBI3-Including Cytokines Produced by Psoriatic Keratinocytes 査読

Tachibana K, Tang N, Urakami H, Kajita Ai, Kobashi M, Nomura H, Sasakura M, Sugihara S, FJiang F, Tomonobu N, Sakaguchi M, Ouchida M, Morizane S

International Journal of Molecular Sciences 22 ( 23 ) 10.3390/ijms222312659 2021年11月

Txn1 mutation causes epilepsy associated with vacuolar degeneration in the midbrain.

Iori Ohmori, Mamoru Ouchida, Hirohiko Imai, Saeko Ishida, Shinya Toyokuni, Tomoji Mashimo

bioRxiv 2021年10月

VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines. 査読国際誌

Nami Kurozumi, Takayuki Tsujioka, Mamoru Ouchida, Kanae Sakakibara, Takako Nakahara, Shin-Ichiro Suemori, Masaki Takeuchi, Akira Kitanaka, Misako Shibakura, Kaoru Tohyama

Cancer science 112 ( 8 ) 3302 - 3313 2021年8月

記述言語：英語掲載種別：研究論文（学術雑誌）

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.

DOI： 10.1111/cas.14982

Scn1a and Cacna1a mutations mutually alter their original phenotypes in rats. 査読国際誌

Iori Ohmori, Kiyoka Kobayashi, Mamoru Ouchida

Neurochemistry international 141 104859 - 104859 2020年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

This study aimed to examine the effects of Cacna1a mutation on the phenotype of Scn1a-associated epilepsy in rats. We used rats with an N1417H missense mutation in the Scn1a gene and others with an M251K mutation in the Cacna1a gene. Scn1a/Cacna1a double mutant rats were generated by mating both Scn1a and Cacna1a mutants. We investigated general health and the epileptic phenotype in all these genotypes. The onset threshold of hyperthermia-induced seizures was examined at 5 weeks and spontaneous seizures were monitored using video-EEG recordings from 6 to 12 weeks of age. Scn1a/Cacna1a double mutants showed significantly reduced threshold for hyperthermia-sensitive seizures onset compared with the Scn1a mutants and had absence seizures having 6-7 c/s spike-wave bursts with changes in the spike-wave pattern, whereas Cacna1a mutants had regular 6-7 c/s spike-wave bursts. In Scn1a/Cacna1a double mutants, 6-7 c/s spike-wave bursts were accompanied with eyelid myoclonia and continuously shifting generalized clonic seizures, which were not observed in either Scn1a or Cacna1a mutants. Although a curvature of the spine was observed in rats of all these genotypes, the degree of curvature was more pronounced in Scn1a/Cacna1a double mutants, followed by Cacna1a and Scn1a mutants. Our results indicate that Cacna1a and Scn1a mutations mutually alter their original phenotypes in rats. The phenotype of absence seizures with eyelid myoclonia, generalized clonic seizures, and of spine curvature in the Scn1a/Cacna1a double mutants were similar to that observed in patients with Dravet syndrome.

DOI： 10.1016/j.neuint.2020.104859

Development of a novel analgesic for neuropathic pain targeting brain-derived neurotrophic factor. 査読国際誌

Kae Ikeda, Keita Hazama, Yoshitaro Itano, Mamoru Ouchida, Hideki Nakatsuka

Biochemical and biophysical research communications 531 ( 3 ) 390 - 395 2020年10月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Effective treatment of neuropathic pain is challenging as its underlying mechanism remains largely unknown. Recently, the participation of brain-derived neurotrophic factor (BDNF) in neuropathic pain has been attracting increased attention. BDNF binds to a member of the tyrosine kinase receptor family, the TrkB receptor, that is specific for BDNF and is the transmembrane receptor on the posterior horn of spinal cord. In the present study, we purified two proteins that included the BDNF-binding domain of TrkB (eTrkB) and eTrkB coupled with a liposomal outer surface (liposomal eTrkB) in order to inhibit the BDNF-TrkB pathway in neuropathic pain. Results of the pull-down assay showed that eTrkB was bound to BDNF. We investigated the neuropathic pain suppression effect of this purified protein by its intrathecal administration in a rat neuropathic pain model. Mechanical and thermal hyperalgesia induced by L5 lumbar nerve ligation was markedly suppressed by treatment with eTrkB protein. Furthermore, we showed a prolonged algetic inhibition by liposomal eTrkB protein treatment. In conclusion, this study suggests that eTrkB, which sequesters endogenous BDNF and inhibits the BDNF-TrkB pathway, may prove to be a novel analgesic to treat neuropathic pain.

DOI： 10.1016/j.bbrc.2020.07.109

Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients. 査読

Shin Morizane, Mamoru Ouchida, Ko Sunagawa, Saeko Sugimoto, Mina Kobashi, Satoru Sugihara, Hayato Nomura, Kazuhide Tsuji, Atsushi Sato, Yoshihiro Miura, Hiroaki Hattori, Kotaro Tada, Wook-Kang Huh, Akemi Seno, Keiji Iwatsuki

Acta medica Okayama 72 ( 3 ) 275 - 282 2018年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients.

DOI： 10.18926/AMO/56073

Novel analgesics targeting brain-derived neurotrophic factor for neuropathic pain. 査読

Hazama K, Ikeda K, Tsuge M, Itano Y, Ouchida M, Nakatsuka H

Kawasaki Medical Journal. 44 ( 2 ) 41 - 52 2018年

Human RAD 17 Polymorphism at Codon 546 Is Associated with the Risk of Colorectal Cancer. 査読

Yukiko Yasuda, Akiko Sakai, Sachio Ito, Kaori Sasai, Hiromasa Yamamoto, Nagahide Matsubara, Mamoru Ouchida, Hiroshi Katayama, Kenji Shimizu

Acta medica Okayama 71 ( 1 ) 59 - 68 2017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：OKAYAMA UNIV MED SCHOOL

Human RAD17 acts as an activator of checkpoint signals in response to DNA damage. Here we evaluated the association of hRAD17 Leu546Arg (rs1045051), a missense single nucleotide polymorphism, with the risk of colorectal cancer (CRC) in relation to smoking and alcohol consumption habits in 212 CRC patients and 1,142 cancer-free controls in a case-control study conducted in Japan. The results showed that the hRAD17 Leu/Arg genotype compared to the Leu/Leu genotypes was significantly associated with the protective effect on CRC risk with the adjusted odds ratio (OR) of 0.68 [95% confidence interval (CI): 0.49-0.95, p=0.024], and the males with the Arg/Arg genotype had a greater risk of CRC compared to those with the Leu/Leu and Leu/Arg genotypes (OR=1.87, 95% CI 1.03-3.40, p=0.04). In stratified studies, the protective effect of the Leu/Arg genotype on CRC risk was markedly higher in the light smokers (< 20 pack years) (OR=0.61, 95% CI 0.40-0.94, p=0.024) and the rectal cancer patients (OR=0.49, 95% CI 0.31-0.78, p=0.003). The risk of the Arg/Arg genotype was associated with heavy smoking (>= 20 pack-years) (OR=2.24, 95% CI 1.09-4.61, p=0.03). These findings suggest that the genetic variant of hRAD17 Leu546Arg polymorphism has a significant effect on CRC susceptibility in Japanese.

DOI： 10.18926/AMO/54826

A case of Dravet syndrome with cortical myoclonus indicated by jerk-locked back-averaging of electroencephalogram data. 査読国際誌

Yoshinori Kobayashi, Yoshiyuki Hanaoka, Tomoyuki Akiayma, Iori Ohmori, Mamoru Ouchida, Toshiyuki Yamamoto, Makio Oka, Harumi Yoshinaga, Katsuhiro Kobayashi

Brain & development 39 ( 1 ) 75 - 79 2017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：ELSEVIER SCIENCE BV

We report a female patient with Dravet syndrome (DS) with erratic segmental myoclonus, the origin of which was first identified in the cerebral cortex by the detection of myoclonus-associated cortical discharges. The discharges were disclosed through jerk locked back-averaging of electroencephalogram (EEG) data using the muscle activity of myoclonus as triggers. The detected spikes on the contralateral parieto-central region preceded myoclonic muscle activity in the forearms by 28-46 ms. The patient was six months old at the time of examination, and was developing normally before seizure onset at two months of age. She suffered from recurrent afebrile or febrile generalized tonic clonic seizures that often developed into status epilepticus. Interictal EEG and brain magnetic resonance imaging (MRI) showed no significant findings. The amplitudes of the somatosensory-evoked potentials were not extremely large. She has a chromosomal microdeletion involving SCN1A and adjacent genes. (C) 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.braindev.2016.07.005

Development of a novel analgesic for cancer pain targeting brain-derived neurotrophic factor. 査読

Tsuge M, Hazama K, Kanzaki H, Itano Y, Maeshima K, Ouchida M, Nakatsuka H

Kawasaki Medical Journal. 43 107 - 120 2017年

Genetic alterations in lung adenocarcinoma with a micropapillary component. 査読国際誌

Masashi Furukawa, Shinichi Toyooka, Kouichi Ichimura, Hiromasa Yamamoto, Junichi Soh, Shinsuke Hashida, Mamoru Ouchida, Kazuhiko Shien, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi

Molecular and clinical oncology 4 ( 2 ) 195 - 200 2016年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Pulmonary adenocarcinoma (PA) with a micropapillary component (PA-MPC) is known as an aggressive subtype of PA. The molecular profiles of PA-MPC have not been well characterized. the pathological reports of patients who underwent surgical resection for lung cancer between April, 2004 and May, 2012 were reviewed. Of the 674 patients diagnosed with PA, 28 were found to have MPC. A total of 138 resected PAs without MPC were selected in the same period to serve as age-, gender- and smoking status-matched controls to the PA-MPC group. Mutational status was determined by the following two methods: SNaPshot assay based on multiplex polymerase chain reaction (PCR), primer extension and capillary electrophoresis that was designed to assess 38 somatic mutations in 8 genes [AKT1, BRAF, endothelial growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), mitogen-activated protein kinase kinase 1, neuroblastoma RAS viral oncogene homolog, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA) and phosphatase and tensin homolog]; and a PCR-based sizing assay that assesses EGFR exon 19 (deletions), EGFR exon 20 (insertions) and human epidermal growth factor receptor 2 exon 20 (insertions). echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene (EML4-ALK) was screened by ALK immunohistochemistry and confirmed using the reverse transcription PCR assay and the break-apart fluorescence in situ hybridization assay. Regarding genetic alterations, 13 (46.4%) of the 28 PA-MPCs harbored mutually exclusive mutations: 9 (32.1%) EGFR mutations, 1 (3.6%) KRAS mutation and 3 (10.7%) EML4-ALK fusion genes. PAs without MPC harbored 42 (30.4%) EGFR mutations, 17 (12.3%) KRAS mutations, 3 (2.2%) EML4-ALK fusion genes and 1 (0.7%) PIK3CA mutation. EML4-ALK fusion genes appeared to occur significantly more frequently in PA-MPCs compared with PAs without MPC (P=0.027). Although the sample size was small, our study suggests that the molecular pathogenesis of PA-MPC may be different from that of other adenocarcinomas.

DOI： 10.3892/mco.2015.690

Five-aza-2'-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells. 査読国際誌

Takayuki Tsujioka, Akira Yokoi, Yoshitaro Itano, Kentaro Takahashi, Mamoru Ouchida, Shuichiro Okamoto, Toshinori Kondo, Shin-ichiro Suemori, Yumi Tohyama, Kaoru Tohyama

Scientific reports 5 16709 - 16709 2015年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.

DOI： 10.1038/srep16709

Cyclin C interacts with steroid receptor coactivator 2 and upregulates cell cycle genes in MCF-7 cells. 査読国際誌

Olivera Bozickovic, Tuyen Hoang, Ingvild S Fenne, Thomas Helland, Linn Skartveit, Mamoru Ouchida, Gunnar Mellgren, Jørn V Sagen

Biochimica et biophysica acta 1853 ( 10 Pt A ) 2383 - 91 2015年10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

Steroid receptor coactivator 2 (SRC-2) is a coactivator that regulates nuclear receptor activity. We previously reported that SRC-2 protein is degraded through the action of cAMP-dependent protein kinase A (PKA) and CAMP response element binding protein (CREB). In the study presented here, we aimed to identify proteins that interact with and thereby regulate SRC-2. We isolated cyclin C (CCNC) as an interacting partner with the SRC-2 degradation domain aa 347-758 in a yeast two-hybrid assay and confirmed direct interaction in an in vitro assay. The protein level of SRC-2 was increased with CCNC overexpression in COS-1 cells and decreased with CCNC silencing in COS-1 and MCF-7 cells. In a pulse-chase assay, we further show that silencing of CCNC resulted in a different SRC-2 degradation pattern during the first 6 h after the pulse. Finally, we provide evidence that CCNC regulates expression of cell cycle genes upregulated by SRC-2. In conclusion, our results suggest that CCNC temporarily protects SRC-2 against degradation and this event is involved in the transcriptional regulation of SRC-2 cell cycle target genes. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamcr.2015.05.016

Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression. 査読国際誌

Kumiko Yamamoto, Sachio Ito, Hiroko Hanafusa, Kenji Shimizu, Mamoru Ouchida

PloS one 10 ( 9 ) e0137887 2015年9月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Micro RNAs (miRNAs) regulate the expression of target genes posttranscriptionally by pairing incompletely with mRNA in a sequence-specific manner. About 30% of human genes are regulated by miRNAs, and a single miRNA is capable of reducing the production of hundreds of proteins by means of incomplete pairing upon miRNA-mRNA binding. Lately, evidence implicating miRNAs in the development of lung cancers has been emerging. In particular, miR-19a, which is highly expressed in malignant lung cancer cells, is considered the key miRNA for tumorigenesis. However, its direct targets remain underreported. In the present study, we focused on six potential miR-19a target genes selected by miRNA target prediction software. To evaluate these genes as direct miR-19a target genes, we performed luciferase, pull-down, and western blot assays. The luciferase activity of plasmids with each miR-19a-binding site was observed to decrease, while increased luciferase activity was observed in the presence of anti-miR-19a locked nucleic acid (LNA). The pull-down assay showed biotinylated miR-19a to bind to AGO2 protein and to four of six potential target mRNAs. Western blot analysis showed that the expression levels of the four genes changed depending on treatment with miR-19a mimic or anti-miR-19a-LNA. Finally, FOXP1, TP53INP1, TNFAIP3, and TUSC2 were identified as miR-19a targets. To examine the function of these four target genes in lung cancer cells, LK79 (which has high miR-19a expression) and A549 (which has low miR-19a expression) were used. The expression of the four target proteins was higher in A549 than in LK79 cells. The four miR-19a target cDNA expression vectors suppressed cell viability, colony formation, migration, and invasion of A549 and LK79 cells, but LK79 cells transfected with FOXP1 and TP53INP1 cDNAs showed no difference compared to the control cells in the invasion assay.

DOI： 10.1371/journal.pone.0137887

MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors (vol 15, pg 1778, 2009) 査読

T. Kubo, H. Yamamoto, W. W. Lockwood, Valencia, I, J. Soh, M. Peyton, M. Jida, H. Otani, T. Fujii, M. Ouchida, N. Takigawa, K. Kiura, K. Shimizu, H. Date, J. D. Minna, M. Varella-Garcia, W. L. Lam, A. F. Gazdar, S. Toyooka

INTERNATIONAL JOURNAL OF CANCER 136 ( 2 ) E1 - E1 2015年1月

Methylphenidate improves learning impairments and hyperthermia-induced seizures caused by an Scn1a mutation. 査読国際誌

Iori Ohmori, Nozomi Kawakami, Sumei Liu, Haijiao Wang, Ikuko Miyazaki, Masato Asanuma, Hiroyuki Michiue, Hideki Matsui, Tomoji Mashimo, Mamoru Ouchida

Epilepsia 55 ( 10 ) 1558 - 67 2014年10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-BLACKWELL

ObjectiveDevelopmental disorders including cognitive deficit, hyperkinetic disorder, and autistic behaviors are frequently comorbid in epileptic patients with SCN1A mutations. However, the mechanisms underlying these developmental disorders are poorly understood and treatments are currently unavailable. Using a rodent model with an Scn1a mutation, we aimed to elucidate the pathophysiologic basis and potential therapeutic treatments for developmental disorders stemming from Scn1a mutations.
MethodsWe conducted behavioral analyses on rats with the N1417H-Scn1a mutation. With high-performance liquid chromatography, we measured dopamine and its metabolites in the frontal cortex, striatum, nucleus accumbens, and midbrain. Methylphenidate was administered intraperitoneally to examine its effects on developmental disorder-like behaviors and hyperthermia-induced seizures.
ResultsBehavioral studies revealed that Scn1a-mutant rats had repetitive behavior, hyperactivity, anxiety-like behavior, spatial learning impairments, and motor imbalance. Dopamine levels in the striatum and nucleus accumbens in Scn1a-mutant rats were significantly lower than those in wild-type rats. In Scn1a-mutant rats, methylphenidate, by increasing dopamine levels in the synaptic cleft, improved hyperactivity, anxiety-like behavior, and spatial learning impairments. Surprisingly, methylphenidate also strongly suppressed hyperthermia-induced seizures.
SignificanceDysfunction of the mesolimbic dopamine reward pathway may contribute to the hyperactivity and learning impairments in Scn1a-mutant rats. Methylphenidate was effective for treating hyperactivity, learning impairments, and hyperthermia-induced seizures. We propose that methylphenidate treatment may ameliorate not only developmental disorders but also epileptic seizures in patients with SCN1A mutations.

DOI： 10.1111/epi.12750

PROTEOMIC ANALYSIS OF INVASION- AND ANGIOGENESIS-RELATED PROTEINS IN MALIGNANT GLIOMAS 査読

Yu Takahashi, Tomotsugu Ichikawa, Tomoko Maruo, Kazuhiko Kurozumi, Manabu Onishi, Mamoru Ouchida, Kentaro Fuji, Yosuke Shimazu, Tetsuo Oka, E. Antonio Chiocca, Isao Date

NEURO-ONCOLOGY 15 9 - 10 2013年11月

Truncated SSX protein suppresses synovial sarcoma cell proliferation by inhibiting the localization of SS18-SSX fusion protein. 査読国際誌

Yasushi Yoneda, Sachio Ito, Toshiyuki Kunisada, Yuki Morimoto, Hirotaka Kanzaki, Aki Yoshida, Kenji Shimizu, Toshifumi Ozaki, Mamoru Ouchida

PloS one 8 ( 10 ) e77564 2013年10月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

Synovial sarcoma is a relatively rare high-grade soft tissue sarcoma that often develops in the limbs of young people and induces the lung and the lymph node metastasis resulting in poor prognosis. In patients with synovial sarcoma, specific chromosomal translocation of t(X; 18) (p11.2; q11.2) is observed, and SS18-SSX fusion protein expressed by this translocation is reported to be associated with pathogenesis. However, role of the fusion protein in the pathogenesis of synovial sarcoma has not yet been completely clarified. In this study, we focused on the localization patterns of SS18-SSX fusion protein. We constructed expression plasmids coding for the full length SS18-SSX, the truncated SS18 moiety (tSS18) and the truncated SSX moiety (tSSX) of SS18-SSX, tagged with fluorescent proteins. These plasmids were transfected in synovial sarcoma SYO-1 cells and we observed the expression of these proteins using a fluorescence microscope. The SS18-SSX fusion protein showed a characteristic speckle pattern in the nucleus. However, when SS18-SSX was co-expressed with tSSX, localization of SS18-SSX changed from speckle patterns to the diffused pattern similar to the localization pattern of tSSX and SSX. Furthermore, cell proliferation and colony formation of synovial sarcoma SYO-1 and YaFuSS cells were suppressed by exogenous tSSX expression. Our results suggest that the characteristic speckle localization pattern of SS18-SSX is strongly involved in the tumorigenesis through the SSX moiety of the SS18-SSX fusion protein. These findings could be applied to further understand the pathogenic mechanisms, and towards the development of molecular targeting approach for synovial sarcoma.

DOI： 10.1371/journal.pone.0077564

Inhalation of 10% carbon dioxide rapidly terminates Scn1a mutation-related hyperthermia-induced seizures 査読国際誌

Iori Ohmori, Keiichiro Hayashi, Haijiao Wang, Mamoru Ouchida, Naohiro Fujita, Takushi Inoue, Hiroyuki Michiue, Teiichi Nishiki, Hideki Matsui

EPILEPSY RESEARCH 105 ( 1-2 ) 220 - 224 2013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：ELSEVIER SCIENCE BV

The aim of this study was to assess the anticonvulsant effect of carbon dioxide (CO2) on Scn1a mutation-related febrile seizures. We examined physiological changes in the blood gas levels after the induction of hyperthermia-induced seizures (HISs), which were associated with the Scn1a missense mutation. We determined the efficacy of inhalation of 5% or 10% CO2 to treat HISs. HISs were evoked in Scn1a mutant and wild-type (WT) rats by hot water baths. To determine the anticonvulsant effect of CO2 inhalation, rats were placed in a chamber filled with air or mixed gas containing 5% CO2 or 10% CO2 for 3 min, immediately after the induction of HISs. We also analyzed the blood gas levels at the end of inhalation of CO2. Hot water bathing induced a significant reduction in the partial pressure of CO2 (pCO(2)) and respiratory alkalosis in the WT and Scn1a mutant rats. HISs were evoked in 100% of the Scn1a mutant rats within 5 min, but in none of the WT rats. The Scn1a mutant rats demonstrated a higher HISs susceptibility associated with respiratory alkalosis than the WT rats. Inhalation of 10% CO2 shortened the seizure duration from 62.6 +/- 12.1 s to 15.5 +/- 1.0 s. Blood gas analysis after the inhalation of 10% CO2 demonstrated an elevated pCO(2) level and respiratory acidosis. Inhalation of 10% CO2 demonstrated a potent and fast-acting anticonvulsant effect against HISs. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.eplepsyres.2013.01.003

CACNA1A VARIANTS AS A POTENTIAL GENETIC MODIFIER OF DRAVET SYNDROME 査読

Ohmori, I, M. Ouchida, K. Kobayashi, Y. Ohtsuka, H. Matsui

EPILEPSIA 54 195 - 196 2013年6月

Proteomics-based analysis of invasion-related proteins in malignant gliomas. 査読国際誌

Tomoko Maruo, Tomotsugu Ichikawa, Hirotaka Kanzaki, Satoshi Inoue, Kazuhiko Kurozumi, Manabu Onishi, Koichi Yoshida, Hirokazu Kambara, Mamoru Ouchida, Kenji Shimizu, Seiji Tamaru, E Antonio Chiocca, Isao Date

Neuropathology : official journal of the Japanese Society of Neuropathology 33 ( 3 ) 264 - 75 2013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：WILEY-BLACKWELL

One of the insidious biological features of gliomas is their potential to extensively invade normal brain tissue, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. To investigate the molecular basis of invasion by malignant gliomas, proteomic analysis was performed using a pair of canine glioma subclones - J3T-1 and J3T-2 - that show different invasion phenotypes in rat brains but have similar genetic backgrounds. Two-dimensional protein electrophoresis of whole-cell lysates of J3T-1 (angiogenesis-dependent invasion phenotype) and J3T-2 (angiogenesis-independent invasion phenotype) was performed. Twenty-two distinct spots were recognized when significant alteration was defined as more than 1.5-fold change in spot intensity between J3T-1 and J3T-2. Four proteins that demonstrated increased expression in J3T-1, and 14 proteins that demonstrated increased expression in J3T-2 were identified using liquid chromatography-mass spectrometry analysis. One of the proteins identified was annexin A2, which was expressed at higher levels in J3T-1 than in J3T-2. The higher expression of annexin A2 in J3T-1 was corroborated by quantitative RT-PCR of the cultured cells and immunohistochemical staining of the rat brain tumors. Moreover, immunohistochemical analysis of human glioblastoma specimens showed that annexin A2 was expressed at high levels in the tumor cells that formed clusters around dilated vessels. These results reveal differences in the proteomic profiles between these two cell lines that might correlate with their different invasion profiles. Thus, annexin A2 may be related to angiogenesis-dependent invasion.

DOI： 10.1111/j.1440-1789.2012.01361.x

INHALATION OF CO2 SHOWS A POTENT AND FAST-ACTING ANTICONVULSANT EFFECT AGAINST SCN1A MUTATION-RELATED HYPERTHERMIA-INDUCED SEIZURES 査読

Ohmori, I, K. Hayashi, H. Wang, M. Ouchida, H. Matsui

EPILEPSIA 54 49 - 49 2013年6月

CACNA1A variants may modify the epileptic phenotype of Dravet syndrome 査読国際誌

Iori Ohmori, Mamoru Ouchida, Katsuhiro Kobayashi, Yoshimi Jitsumori, Akiko Mori, Hiroyuki Michiue, Teiichi Nishiki, Yoko Ohtsuka, Hideki Matsui

NEUROBIOLOGY OF DISEASE 50 209 - 217 2013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：ACADEMIC PRESS INC ELSEVIER SCIENCE

Dravet syndrome is an intractable epileptic syndrome beginning in the first year of life. De novo mutations of SCN1A, which encode the Na(v)1.1 neuronal voltage-gated sodium channel, are considered the major cause of Dravet syndrome. In this study, we investigated genetic modifiers of this syndrome.
We performed a mutational analysis of all coding exons of CACNA1A in 48 subjects with Dravet syndrome. To assess the effects of CACNA1A variants on the epileptic phenotypes of Dravet syndrome, we compared clinical features in two genotype groups: 1) subjects harboring SCN1A mutations but no CACNA1A variants (n = 20) and 2) subjects with SCN1A mutations plus CACNA1A variants (n = 20). CACNA1A variants detected in patients were studied using heterologous expression of recombinant human Ca(v)2.1 in HEK 293 cells and whole-cell patch-clamp recording.
Nine CACNA1A variants, including six novel ones, were detected in 21 of the 48 subjects (43.8%). Based on the incidence of variants in healthy controls, most of the variants seemed to be common polymorphisms. However, the subjects harboring SCN1A mutations and CACNA1A variants had absence seizures more frequently than the patients with only SCN1A mutations (8/20 vs. 0/20, p = 0.002). Moreover, the former group of subjects exhibited earlier onset of seizures and more frequent prolonged seizures before one year of age, compared to the latter group of subjects. The electrophysiological properties of four of the five novel Ca(v)2.1 variants exhibited biophysical changes consistent with gain-of-function. We conclude that CACNA1A variants in some persons with Dravet syndrome may modify the epileptic phenotypes. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.nbd.2012.10.016

High-frequency EEG oscillations in hyperthermia-induced seizures of Scn1a mutant rats. 査読国際誌

Katsuhiro Kobayashi, Iori Ohmori, Keiichiro Hayashi, Yuichiro Kitagawa, Mamoru Ouchida, Takushi Inoue, Yoko Ohtsuka

Epilepsy research 103 ( 2-3 ) 161 - 6 2013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：ELSEVIER SCIENCE BV

We examined high-frequency oscillations (HFOs) in the ictal cortical EEGs of hyperthermia-induced seizures in a rat model of febrile seizures with an SCN1A mutation as a means of investigating the pathophysiological mechanisms underlying the generation of febrile seizures. We used 13 male homozygous Scn1a-N1417H mutant rats (F344/NSlc-Scn1a(Kyo811)) and 10 wild-type control rats.
Generalized tonic-clonic seizures were induced in all mutant rats, and HFOs with frequencies ranging from 200 to 400 Hz were found to precede spikes during the clonic phases of these seizures in the ictal EEGs. The proportion of all spikes in each seizure that were associated with HFOs increased with age. In time-frequency spectra of the EEG data, the HFOs had a mean peak frequency of 301.1 +/- 65.4 Hz (range: 156.3-468.8 Hz) and a mean peak power of 24.6 +/- 3.8 dB (range: 11.4-33.4 dB); the peak power increased with age. Regarding the wild-type rats, a brief seizure without unmistakable HFOs was exceptionally induced in only one rat.
The generation mechanism of febrile seizures is still an unanswered question. The detection of HFOs from the ictal EEGs of hyperthermia-induced seizures may provide a cue to answering this open question, although in this research we were unable to provide sufficient evidence to prove that the generation of HFOs depended on the mutation. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.eplepsyres.2012.07.020

Spinal nerve injury causes upregulation of ErbB2 and ErbB3 receptors in rat dorsal root ganglia. 査読国際誌

Satoshi Mizobuchi, Hirotaka Kanzaki, Hiroki Omiya, Yoshikazu Matsuoka, Norihiko Obata, Ryuji Kaku, Hirochika Nakajima, Mamoru Ouchida, Kiyoshi Morita

Journal of pain research 6 87 - 94 2013年

記述言語：英語掲載種別：研究論文（学術雑誌）

It is generally known that peripheral nerve injury causes changes in expression of some growth factors in the dorsal root ganglion. Altered expression of ErbB receptors, a well-known growth factor in somatic cells, reportedly follows peripheral nerve injury in the spinal dorsal horn; however, it remains unknown whether the expression of these receptors is altered in the dorsal root ganglion after nerve injury. Therefore, this study examined the gene expression profiles of ErbB receptors in bilateral lumbar (L)4/L5 dorsal root ganglia, using L5-selective spinal nerve ligation in model rats as a peripheral nerve injury model. The expression of ErbB2 and ErbB3 was observed in the dorsal root ganglia of the mature rat, despite ErbB1 and ErbB4 showing only subtle expression. We also demonstrated that peripheral nerve injury induced significant increases in ErbB2 and ErbB3 in the ipsilateral dorsal root ganglion as compared with uninjured nerve. Expression changes in ErbB receptors appear to play important roles in nerve injury and subsequent nerve regeneration.

DOI： 10.2147/JPR.S40967

MicroRNA-21 correlates with tumorigenesis in malignant peripheral nerve sheath tumor (MPNST) via programmed cell death protein 4 (PDCD4) 査読国際誌

Satoru Itani, Toshiyuki Kunisada, Yuki Morimoto, Aki Yoshida, Tsuyoshi Sasaki, Sachio Ito, Mamoru Ouchida, Shinsuke Sugihara, Kenji Shimizu, Toshifumi Ozaki

JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY 138 ( 9 ) 1501 - 1509 2012年9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：SPRINGER

We investigated the miRNA profile in peripheral nerve tumors and clarified the involvement of miRNA in the development and progression of MPNST in comparison with neurofibroma (NF). In addition, we attempted to seek associations between the miRNA and their potential targets in MPNST.
Global miRNA expression profiling was investigated for clinical samples of 6 MPNSTs and 6 NFs. As detected by profiling analysis, the expressions of miR-21 in clinical samples of 12 MPNSTs, 11 NFs, and 5 normal nerves, and 3 MPNST cell lines were compared using quantitative real-time reverse transcription PCR. MPNST cell line (YST-1) was transfected with miR-21 inhibitor to study its effects on cell proliferation, caspase activity, and the expression of miR-21 targets.
Analysis of miRNA expression profiles in MPNST and NF revealed significantly altered expression levels of nine miRNAs, one of those, miR-21, and its putative target, programmed cell death protein 4 (PDCD4), were selected for further studies. miR-21 expression level in MPNST was significantly higher than that in NF (P < 0.05). In MPNST cells, transfection of miR-21 inhibitor significantly increased caspase activity (P < 0.01), significantly suppressed cell growth (P < 0.05), and upregulated protein level of PDCD4, indicating that miR-21 inhibitor could induce cell apoptosis of MPNST cells.
These results suggest that miR-21 plays an important role in MPNST tumorigenesis and progression through its target, PDCD4. MiR-21 and PDCD4 may be candidate novel therapeutic targets against the development or progression of MPNSTs.

DOI： 10.1007/s00432-012-1223-1

Novel direct targets of miR-19a identified in breast cancer cells by a quantitative proteomic approach. 査読国際誌

Mamoru Ouchida, Hirotaka Kanzaki, Sachio Ito, Hiroko Hanafusa, Yoshimi Jitsumori, Seiji Tamaru, Kenji Shimizu

PloS one 7 ( 8 ) e44095 2012年8月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PUBLIC LIBRARY SCIENCE

The miR-17-92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.

DOI： 10.1371/journal.pone.0044095

Genetic risk of hepatocellular carcinoma in patients with hepatitis C virus: A case control study 査読国際誌

Takeshi Tomoda, Kazuhiro Nouso, Akiko Sakai, Mamoru Ouchida, Sayo Kobayashi, Koji Miyahara, Hideki Onishi, Shinichiro Nakamura, Kazuhide Yamamoto, Kenji Shimizu

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY 27 ( 4 ) 797 - 804 2012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：WILEY-BLACKWELL

Backgroud and Aim: Chronic hepatitis C virus (HCV) infection is a well known risk factor for hepatocellular carcinoma (HCC). The aim of this study is to elucidate the genetic risk of development and recurrence of HCC in patients with HCV.
Methods: A total of 468 patients with HCV, including 265 with HCC were enrolled. We genotyped 88 single nucleotide polymorphisms (SNPs) in 81 genes expected to influence hepatocarcinogenesis using the iPLEX assay. Risk of HCC was clarified by stratifying patients into risk groups based on the multiplied odds ratio (MOR) for SNPs associated with HCC, and the cumulative effects on the development and recurrence of HCC were analyzed.
Results: Six SNPs associated with risk of HCC were identified (OR range: 0.29-1.76). These included novel SNPs for hepatocarcinogenesis with HCV CCND2 rs1049606, RAD23B rs1805329, CEP164 rs573455, and GRP78rs430397 in addition to the known SNPs MDM2 rs2279744 and ALDH2 rs671. MOR analysis revealed that the highest risk group exerted about a 19-fold higher relative OR compared with the lowest risk group (P = 1.08 x 10(-5)). Predicted 10-year HCC risk ranged from 1.7% to 96% depending on the risk group and the extent of fibrosis. Recurrence-free survival of radiofrequency ablation-treated HCC in the high risk group (n = 53) was lower than that of low risk group (n = 58, P = 0.038).
Conclusion: Single nucleotide polymorphisms of CCND2, RAD23B, GRP78, CEP164, MDM2, and ALDH2 genes were significantly associated with development and recurrence of HCC in Japanese patients with HCV.

DOI： 10.1111/j.1440-1746.2011.06948.x

Different Responses to 5-fluoraouracil in Mutagenicity and Gene Expression between Two Human Lymphoblastoid Cell Lines with or without TP53 Mutation 査読

Hiroaki Oka, Mamoru Ouchida, Takuya Kondo, Fumio Morita, Kenji Shimizu

ACTA MEDICA OKAYAMA 66 ( 2 ) 119 - 129 2012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：OKAYAMA UNIV MED SCHOOL

Human lymphoblastoid TK6 and WTK-1 cells are widely used to detect mutagens in vitro. TK6 cells have wild-type TP53 alleles, while WTK-1 cells have one allele of mutated TP53. Both cells were treated with 5-fluorouracil (5-FU), and gene mutation assay and micronucleus assay were performed to clarify the differential response related to the TP53 gene status. The effects of 5-FU on gene expression were assessed by microarray and quantitative RT-PCR analyses. In WTK-1 cells, 5-FU increased the frequency of cells with micronucleus and mutation. In TK6 cells, frequency of cells with micronucleus was increased but the mutation frequency was not. The cytotoxicity induced by 5-FU was more prominent in TK6 cells than in WTK-1 cells. Analysis of gene expression showed that the genes involved in the TP53 pathway were up-regulated in TK6 cells but not in WTK-1 cells. The differential responses to 5-FU between these cell lines appeared to be due to the difference in the TP53 gene status, thus providing a molecular basis for the bioassays using these cell lines in the toxicology field. Our results indicate that the clinical efficacy of 5-FU chemotherapy may depend on the TP53 genotype.

Expression changes of the neuregulin 1 isoforms in neuropathic pain model rats. 査読国際誌

Kanzaki H, Mizobuchi S, Obata N, Itano Y, Kaku R, Tomotsuka N, Nakajima H, Ouchida M, Nakatsuka H, Maeshima K, Morita K

Neuroscience letters 508 ( 2 ) 78 - 83 2012年2月

Eradication of hepatitis C virus subgenomic replicon by interferon results in aberrant retinol-related protein expression. 査読

Kazuko Koike, Akinobu Takaki, Nobuyuki Kato, Mamoru Ouchida, Hirotaka Kanzaki, Tetsuya Yasunaka, Hidenori Shiraha, Yasuhiro Miyake, Kazuhide Yamamoto

Acta medica Okayama 66 ( 6 ) 461 - 8 2012年

記述言語：英語掲載種別：研究論文（学術雑誌）

Hepatitis C virus (HCV) infection induces several changes in hepatocytes, such as oxidative stress, steatosis, and hepatocarcinogenesis. Although considerable progress has been made during recent years, the mechanisms underlying these functions remain unclear. We employed proteomic techniques in HCV replicon-harboring cells to determine the effects of HCV replication on host-cell protein expression. We examined two-dimensional electrophoresis (2-DE) and mass spectrometry to compare and identify differentially expressed proteins between HCV subgenomic replicon-harboring cells and their "cured" cells. One of the identified proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Full-length HCV genome RNA replicating and cured cells were also assessed using ELISA. Replicon-harboring cells showed higher expression of retinal dehydrogenase 1 (RALDH-1), which converts retinol to retinoic acid, and the cured cells showed higher expression of retinol-binding protein (RBP), which transports retinol from the liver to target tissues. The alteration in RBP expression was also confirmed by ELISA and Western blot analysis. We conclude that protein expression profiling demonstrated that HCV replicon eradication affected retinol-related protein expression.

DOI： 10.18926/AMO/49042

Antinociceptive effects of intrathecal landiolol injection in a rat formalin pain model. 査読

Satoshi Mizobuchi, Yoshikazu Matsuoka, Norihiko Obata, Ryuji Kaku, Yoshitaro Itano, Naoto Tomotsuka, Arata Taniguchi, Hiroyuki Nishie, Hirotaka Kanzaki, Mamoru Ouchida, Kiyoshi Morita

Acta medica Okayama 66 ( 3 ) 285 - 9 2012年

記述言語：英語掲載種別：研究論文（学術雑誌）

Perioperative beta-blocker administration has recently been recommended for patients undergoing cardiac or other surgery due to the beneficial cardiovascular effects of these agents. In addition, some studies have reported that perioperatively administered beta-blockers also have analgesic effects. In this study, to investigate the antinociceptive effects and the analgesic profile of landiolol, we examined the effects of intrathecal landiolol administration on nociceptive pain behavior and c-fos mRNA expression (a neural marker of pain) in the spinal cord using a rat formalin model. We found that pain-related behavior was inhibited by intrathecal landiolol administration. Moreover, the increase in c-fos mRNA expression on the formalin-injected side was less pronounced in rats administered landiolol than in saline administered controls. Thus, intrathecal administration of landiolol exhibited antinociceptive effects. Further investigation of the antinociceptive mechanism of landiolol is required.

DOI： 10.18926/AMO/48569

DNA repair/replication gene polymorphism and gastroenterological cancer risk 査読

T. Sonoyama, H. Kato, A. Sakai, Y. Yasuda, M. Ouchida, K. Yamamoto, K. Shimizu

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY 26 225 - 225 2011年10月

Dravet syndrome with an exceptionally good seizure outcome in two adolescents 査読国際誌

Katsuhiro Kobayashi, Iori Ohmori, Mamoru Ouchida, Yoko Ohtsuka

EPILEPTIC DISORDERS 13 ( 3 ) 340 - 344 2011年9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：JOHN LIBBEY EUROTEXT LTD

We present two children who exhibited the characteristics of Dravet syndrome during infancy and young childhood, with SCN1A mutation, but nevertheless achieved seizure freedom for at least four years during adolescence. These patients had no episodes of convulsive status epilepticus with a duration of more than 30 minutes and their overall favourable seizure outcome may be related to the prevention of convulsive status epilepticus.

DOI： 10.1684/epd.2011.0453

Identification of direct targets for the miR-17-92 cluster by proteomic analysis 査読国際誌

Hirotaka Kanzaki, Sachio Ito, Hiroko Hanafusa, Yoshimi Jitsumori, Seiji Tamaru, Kenji Shimizu, Mamoru Ouchida

PROTEOMICS 11 ( 17 ) 3531 - 3539 2011年9月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally repress the expression of target genes. Many miRNAs have been implicated in a number of diseases, including cancers. The miR-17-92 miRNA cluster is known as a body of oncogenic miRNAs, and has been shown to be overexpressed in several cancers, including lung cancer. Although the overexpression of miR-17-92 is clearly implicated in the development of lung cancer, only a few direct targets for the miR-17-92 cluster have been identified thus far. In this study, we examined miR-17-92 target profiles in SBC-3 small-cell lung cancer cells using a quantitative proteomic strategy to identify direct targets of the miR-17-92 cluster. By knocking down the expression of endogenous miR-19a, miR-20a and miR-92-1, which are contained in the cluster, 112 up-regulated proteins were detected and also identified as potential targets of these miRNAs. Among these candidate targets, we validated one direct target, RAB14. In conclusion, these findings suggest that proteomic approaches are valuable for identifying direct miRNA targets, and we were able to identify a novel direct target for the miR-92-1 using our proteomic strategy.

DOI： 10.1002/pmic.201000501

The Effect of SYT-SSX and Extracellular Signal-Regulated Kinase (ERK) on Cell Proliferation in Synovial Sarcoma 国際誌

Wenjuan Cai, Yan Sun, Wei Wang, Chunrong Han, Mamoru Ouchida, Wenbin Xia, Xiulan Zhao, Baocun Sun

PATHOLOGY & ONCOLOGY RESEARCH 17 ( 2 ) 357 - 367 2011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：SPRINGER

The character of Synovial sarcoma is the chromosomal translocation t(X; 18)(p11.2;q11.2), which results in the fusion of the SYT gene with a SSX gene. There is little study that could fully elucidate the mechanism of pathogenesis of this fusion transcript. This study is designed to gain more insight into the function of this fusion gene. We evaluated the whole genome expression in SYO-1 cells inhibited as a result of specific small interfering RNA for SYT-SSX. Cell proliferation and apoptosis were analyzed by flow cytometer and MTT. The proteins correlated with proliferation were also detected using western blot. TUNEL and Immunohistochemical stain assessment were also carried out on TMA of SS tissues. The mRNA level reduced over 90% caused by SYT-SSX specific siRNA. Five pathways were employed, that ERK1/2 pathway was differential significantly (p = 0.043218). Meanwhile, down-regulation of SYT-SSX fusion gene expression would inhibit the proliferation of SS cell and the survival rate decreased (34.1%), while apoptotic rate increased (10.92%). After transfected with SYT-SSX-specific siRNA it caused a block in G1/G0 phase (31.99%) of SYO-1 cells compared with control cells. The protein level of ERK1/2, p-ERK, and cyclin D1 altered in same trend with expression of SYT-SSX. In TMA stain assessment, SYT-SSX positive group with high ki-67 LI expressed more cyclin D1and CDK4 than the SYT-SSX negative group. High ki-67 LI was detected in cases with p-ERK expression. Meanwhile, cyclin D1 and CDK4 were shown to be more expressed in tumor cells with p-ERK expression. Our results suggest that the fusion gene SYT-SSX should be considered to play important role on SS cell growth via ERK pathway. This study may be valuable for understanding the pathogenic role and molecular mechanism of the fusion gene SYT-SSX in synovial sarcoma through the proposed genome-wide approach. Furthermore, the research would open up the possibility of using SYT-SSX and ERK as a therapeutic target.

DOI： 10.1007/s12253-010-9334-y

TP53 codon 72 polymorphism is associated with pancreatic cancer risk in males, smokers and drinkers

Takayuki Sonoyama, Akiko Sakai, Yuichiro Mita, Yukiko Yasuda, Hirofumi Kawamoto, Takahito Yagi, Masao Yoshioka, Tetsushige Mimura, Kei Nakachi, Mamoru Ouchida, Kazuhide Yamamoto, Kenji Shimizu

MOLECULAR MEDICINE REPORTS 4 ( 3 ) 489 - 495 2011年5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

Tumor protein p53 (TP53) is the best-known tumor suppressor gene and plays a crucial role in carcinogenesis. The TP53 Arg 72 Pro polymorphism has been reported to be a risk factor for several types of cancer, but its association with pancreatic cancer has not been fully evaluated. Therefore, we investigated the effects of this polymorphism on pancreatic cancer in relation to smoking and drinking habits by examining the distribution of the SNP genotypes in 226 pancreatic cancer patients and 448 healthy controls. The frequencies of Arg/Arg, Arg/Pro and Pro/Pro were found to be 37, 49 and 15% in the pancreatic cancer cases and 44, 46 and 10% in the controls, respectively. Compared to the controls with the Arg/Arg genotype, cases with Pro/Pro homozygosity exhibited a significantly increased risk [adjusted odds ratio (OR)=1.70; 95% confidence interval (CI) 1.01-2.88]. In stratified studies, the association was particularly strong in males (OR=2.62; 95% CI 1.32-5.23), particularly in those smoking in excess of 20 pack-years and drinking in excess of 23 g ethanol/day (OR=5.02; 95% CI 1.12-22.51). We found that the. TP53 Pro/Pro genotype compared to the Arg/Arg genotype had a profound effect on pancreatic cancer risk among males, particularly among heavy smokers and excessive alcohol drinkers.

DOI： 10.3892/mmr.2011.449

Therapy for hyperthermia-induced seizures in Scn1a mutant rats 国際誌

Keiichiro Hayashi, Satoshi Ueshima, Mamoru Ouchida, Tomoji Mashimo, Teiichi Nishiki, Toshiaki Sendo, Tadao Serikawa, Hideki Matsui, Iori Ohmori

EPILEPSIA 52 ( 5 ) 1010 - 1017 2011年5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-BLACKWELL

Purpose: Mutations in the SCN1A gene, which encodes the alpha 1 subunit of voltage-gated sodium channels, cause generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). N1417H-Scn1a mutant rats are considered to be an animal model of human FS+ or GEFS+. To assess the pharmacologic validity of this model, we compared the efficacies of eight different antiepileptic drugs (AEDs) for the treatment of hyperthermia-induced seizures using N1417H-Scn1a mutant rats.
Methods: AEDs used in this study included valproate, carbamazepine (CBZ), phenobarbital, gabapentin, acetazolamide, diazepam (DZP), topiramate, and potassium bromide (KBr). The effects of these AEDs were evaluated using the hot water model, which is a model of experimental FS. Five-week-old rats were pretreated with each AED and immersed in water at 45 degrees C to induce hyperthermia-induced seizures. The seizure manifestations and video-electroencephalographic recordings were evaluated. Furthermore, the effects of each AED on motor coordination and balance were assessed using the balance-beam test.
Key Findings: KBr significantly reduced seizure durations, and its anticonvulsant effects were comparable to those of DZP. On the other hand, CBZ decreased the seizure threshold. In addition, DZP and not KBr showed significant impairment in motor coordination and balance.
Significance: DZP and KBr showed potent inhibitory effects against hyperthermia-induced seizures in the Scn1a mutant rats, whereas CBZ exhibited adverse effects. These responses to hyperthermia-induced seizures were similar to those in patients with GEFS+ and SMEI. N1417H-Scn1a mutant rats may, therefore, be useful for testing the efficacy of new AEDs against FS in GEFS+ and SMEI patients.

DOI： 10.1111/j.1528-1167.2011.03046.x

Proteomic Analysis Reveals Aberrant Retinol Metabolism in Hepatitis C Virus Subgenomic Replicon-Harboring Cells

Kazuko Koike, Akinobu Takaki, Nobuyuki Kato, Mamoru Ouchida, Tetsuya Yasunaka, Yasuhiro Miyake, Kazuhide Yamamoto

GASTROENTEROLOGY 140 ( 5 ) S972 - S972 2011年5月

Decoy strategy targeting the brain-derived neurotrophic factor exon I to attenuate tactile allodynia in the neuropathic pain model of rats 国際誌

Norihiko Obata, Satoshi Mizobuchi, Yoshitaro Itano, Yoshikazu Matsuoka, Ryuji Kaku, Naoto Tomotsuka, Kiyoshi Morita, Hirotaka Kanzaki, Mamoru Ouchida, Masataka Yokoyama

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 408 ( 1 ) 139 - 144 2011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：ACADEMIC PRESS INC ELSEVIER SCIENCE

The mechanism underlying neuropathic pain is still largely unclear. Recently, much attention has been focused on the role of brain-derived neurotrophic factor (BDNF) as a neuromodulator in the spinal cord. We previously reported that the expression of Bdnf exon I mRNA was remarkably up-regulated in the dorsal root ganglion (DRG) neurons with the rat L5 spinal nerve ligation (SNL) model. In the present study, we investigated whether neuropathic pain response would be reduced by the inhibition of the Bdnf exon I in the rat SNL model. We identified the promoter region of exon I and synthesized the decoy ODNs targeting the region. Reverse transcription-polymerase chain reaction analysis confirmed that the decoy ODN treatment reduced SNL-induced Bdnf exon I mRNA up-regulation in ipsilateral L4 and L5 DRGs. Furthermore, post-treatment with the decoy ODNs significantly attenuated SNL-induced tactile allodynia. This study suggested that decoy ODNs targeting the Bdnf exon I might provide a novel analgesic strategy for the treatment of neuropathic pain. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.03.137

Cumulative epigenetic abnormalities in host genes with viral and microbial infection during initiation and progression of malignant lymphoma/leukemia 国際誌

Takashi Oka, Hiaki Sato, Mamoru Ouchida, Atae Utsunomiya, Tadashi Yoshino

Cancers 3 ( 1 ) 568 - 581 2011年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Although cancers have been thought to be predominantly driven by acquired genetic changes, it is becoming clear that microenvironment-mediated epigenetic alterations play important roles. Aberrant promoter hypermethylation is a prevalent phenomenon in human cancers as well as malignant lymphoma/leukemia. Tumor suppressor genes become frequent targets of aberrant hypermethylation in the course of gene-silencing due to the increased and deregulated DNA methyltransferases (DNMTs). The purpose of this article is to review the current status of knowledge about the contribution of cumulative epigenetic abnormalities of the host genes after microbial and virus infection to the crisis and progression of malignant lymphoma/leukemia. In addition, the relevance of this knowledge to malignant lymphoma/leukemia assessment, prevention and early detection will be discussed. © 2011 by the authors
licensee MDPI, Basel, Switzerland.

DOI： 10.3390/cancers3010568

Elevated oxidative stress is associated with ventricular fibrillation episodes in patients with Brugada-type electrocardiogram without SCN5A mutation

Masamichi Tanaka, Kazufumi Nakamura, Kengo Fukushima Kusano, Hiroshi Morita, Keiko Ohta-Ogo, Daiji Miura, Aya Miura, Koji Nakagawa, Takeshi Tada, Masato Murakami, Nobuhiro Nishii, Satoshi Nagase, Yoshiki Hata, Kunihisa Kohno, Mamoru Ouchida, Kenji Shimizu, Chikao Yutani, Tohru Ohe, Hiroshi Ito

Cardiovascular Pathology 20 ( 1 ) e37 - e42 2011年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Background: Brugada syndrome is a disease known to cause ventricular fibrillation with a structurally normal heart and is linked to SCN5A gene mutation. However, the mechanism by which ventricular fibrillation develops in cases of Brugada-type electrocardiogram without SCN5A mutation has remained unclear. Recently, oxidative stress has been implicated in the pathophysiology of cardiac arrhythmia. We also investigated oxidative stress levels in the myocardia of patients with Brugada-type electrocardiogram. Methods: Endomyocardial biopsy samples were obtained from 68 patients with Brugada-type electrocardiogram (66 males and two females). We performed histological and immunohistochemical analyses for CD45, CD68, and 4-hydroxy-2-nonenal-modified protein, which is a major lipid peroxidation product. Results: SCN5A mutation was detected in 14 patients. Ventricular fibrillation was documented in three patients with SCN5A mutation and in 11 without SCN5A mutation. In patients with SCN5A mutation, 4-hydroxy-2-nonenal-modified protein-positive area was not significantly different between the documented ventricular fibrillation (VF) group (VF+ group) and the group without documented VF (VF- group). However, in patients without SCN5A, the area was significantly larger in the VF+ group than that in the VF- group (P&lt
.05). All other parameters (fibrosis area, CD45, and CD68) were not different between the VF+ and VF- group in both SCN5A+ and SCN5A- patients. Conclusion: Oxidative stress is elevated in the myocardium of patients with Brugada-type electrocardiogram who have VF episodes and do not have SCN5A gene mutations. Oxidative stress may be associated with the occurrence of VF in patients with Brugada-type electrocardiogram without SCN5A mutation. © 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.carpath.2010.02.002

Cumulative kinetics of epigenetic abnormalities during initiation and progression of Adult T-cell Leukemia/Lymphoma (ATLL).

Oka T, Sato H, A Al-Kader L, Shinnou Y, Washio K, Takata K, Murakami I, Utsunomiya A, Ouchida M, Takahashi K, Yoshino T

Retrovirology 8 ( Suppl 1 ) 195 - 195 2011年

Electrophysiological properties of variant voltage-gated calcium channels in patients with epilepsy

Iori Ohmori, Mamoru Ouchida, Haijiao Wang, Yuichiro Kitagawa, Hiroyuki Michiue, Teiichi Nishiki, Hideki Matsui

NEUROSCIENCE RESEARCH 71 E294 - E294 2011年

Genetic seizure susceptibility underlying acute encephalopathies in childhood 国際誌

Katsuhiro Kobayashi, Mamoru Ouchida, Akihisa Okumura, Yoshihiro Maegaki, Itsuko Nishiyama, Hideki Matsui, Yoko Ohtsuka, Iori Ohmori

EPILEPSY RESEARCH 91 ( 2-3 ) 143 - 152 2010年10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 herein investigated risk factors of pediatric acute encephalopathy (AE) regarding the hitherto uncharacterized genetic background of seizure susceptibility underlying the pathogenesis of AE. The study included 15 patients with a history of various types of AE in childhood. We undertook the mutational analysis of the neuronal sodium channel alpha 1 subunit (SCN1A) gene which is the most representative gene for hyperthermia-induced seizure susceptibility.
Six patients (40%) had a positive family history of seizures or AE, especially febrile seizures, in first- or second-degree relatives. The SCN1A-R1575C mutation was detected in a patient with a history of acute encephalitis with refractory, repetitive partial seizures (AERRPS) and also in the patient's apparently healthy father.
In the present study, dense familial seizure predisposition was present in the patients with AE. Although the presence of seizure susceptibility alone is insufficient to cause AE, it can exacerbate seizures and the subsequent development of inflammatory reactions in the brain when environmental factors are included. Genetic seizure susceptibility may contribute to some types of AE in childhood. Crown Copyright (C) 2010 Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.eplepsyres.2010.07.005

A Missense Mutation of the Gene Encoding Voltage-Dependent Sodium Channel (Na(v)1.1) Confers Susceptibility to Febrile Seizures in Rats 国際誌

Tomoji Mashimo, Iori Ohmori, Mamoru Ouchida, Yukihiro Ohno, Toshiko Tsurumi, Takafumi Miki, Minoru Wakamori, Shizuka Ishihara, Takashi Yoshida, Akiko Takizawa, Megumi Kato, Masumi Hirabayashi, Masashi Sasa, Yasuo Mori, Tadao Serikawa

JOURNAL OF NEUROSCIENCE 30 ( 16 ) 5744 - 5753 2010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：SOC NEUROSCIENCE

Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Na(v)1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFS+ (generalized epilepsy with febrile seizures plus). Here, we generated Scn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances, Scn1a mutant rats exhibited remarkably high susceptibility to hyperthermia-induced seizures, which involve generalized clonic and/or tonic-clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Na(v)1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

DOI： 10.1523/JNEUROSCI.3360-09.2010

Identification of novel direct targets of miR-19a in MCF-7 breast cancer cells

Hirotaka Kanzaki, Mamoru Ouchida, Sachio Ito, Seiji Tamaru, Hiroko Hanafusa, Yoshimi Jitsumori, Takashi Oka, Kenji Shimizu

CANCER RESEARCH 70 2010年4月

Missense polymorphisms of PTPRJ and PTPN13 genes affect susceptibility to a variety of human cancers 国際誌

Yuichiro Mita, Yukiko Yasuda, Akiko Sakai, Hiromasa Yamamoto, Shinichi Toyooka, Mehmet Gunduz, Shunsuke Tanabe, Yoshio Naomoto, Mamoru Ouchida, Kenji Shimizu

JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY 136 ( 2 ) 249 - 259 2010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：SPRINGER

We investigated the association between incidence of various cancers and four single nucleotide polymorphisms (SNPs), two each in two protein tyrosine phosphatase (PTP) genes, PTPRJ and PTPN13, by a case-control study conducted in Japan.
The study samples comprised 819 cancer-free controls and 569 cancer cases including lung, head and neck, colorectal, and esophageal cancers.
Compared with the major homozygotes at the Arg326Gln SNP in PTPRJ, a likely homologue of the mouse SCC1 (susceptible to colon cancer), Arg/Gln or Gln/Gln genotypes exhibited an increased colorectal cancer risk with adjusted odds ratios (aOR) of 1.71 (P = 0.021) and 3.74 (P = 4.14 x 10(-4)), respectively. Increased risks were observed with one or more of the combination genotypes of Gln276Pro and Arg326Gln in PTPRJ for most cancer types (aOR range 10.13-55.08, Bonferroni-corrected P = 0.0454-7.20 x 10(-9)). In the PTPN13, major homozygotes of Ile1522Met showed an increased risk for lung squamous cell carcinomas (aOR 1.86), compared to the heterozygotes. Increased risks were observed with at least one of the combination genotypes of the two SNPs, Ile1522Met and Tyr2081Asp, for all but esophageal cancer examined (aOR 3.36-13.75), compared with double heterozygotes. Moreover, these high risks were seen also when all cancer cases were combined (aOR 1.81-6.84).
PTPRJ and PTPN13 SNPs were found to influence susceptibility to a wide spectrum of cancers. Because allelic frequencies of these SNPs are relatively common in many ethnic groups, these findings are worthy of further study.

DOI： 10.1007/s00432-009-0656-7

Frequent methylation of RASSF1A in synovial sarcoma and the anti-tumor effects of 5-aza-2'-deoxycytidine against synovial sarcoma cell lines 国際誌

Kunihiko Numoto, Aki Yoshida, Shinsuke Sugihara, Toshiyuki Kunisada, Yuki Morimoto, Yasushi Yoneda, Yasuko Fujita, Keiichiro Nishida, Mamoru Ouchida, Toshifumi Ozaki

JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY 136 ( 1 ) 17 - 25 2010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：SPRINGER

In this study, the methylation status of RASSF1A in synovial sarcomas and the effect of de-methylation on synovial sarcoma were examined.
The methylation status in 74 soft tissue sarcomas (STSs) including 21 synovial sarcomas was determined by methylation specific PCR. The effect of the de-methylating agent 5-aza-2'-deoxycytidine (5-Aza-dC) on synovial sarcoma was examined using synovial sarcoma cell lines (SYO-1 and HS-SY-II).
RASSF1A methylation was observed in 10 (47.6%) of 21 synovial sarcomas and in 10 (18.9%) of 53 the other STSs (P = 0.0295). De-methylation of the cells by treatment with 5-Aza-dC induced re-expression of RASSF1A and growth suppression of the cells. The calculated IC50 of 5-Aza-dC against the SYO-1 and the HS-SY-II cells were 0.9 and 1.3 mu M (96 h), respectively. With twice weekly administration of 1 or 10 mg/kg 5-Aza-dC, the growth of the mouse xenograft tumors of SYO-1 was significantly suppressed in comparison to the controls (P < 0.01).
This is the first report showing the anti-tumor effect of 5-Aza-dC on synovial sarcoma. 5-Aza-dC is suggested to have a good therapeutic potential against synovial sarcoma.

DOI： 10.1007/s00432-009-0632-2

Multi-Step Aberrant CpG Island Hyper-Methylation Is Associated with the Progression of Adult T-Cell Leukemia/Lymphoma 国際誌

Hiaki Sato, Takashi Oka, Yoko Shinnou, Takami Kondo, Kana Washio, Masayuki Takano, Katsuyoshi Takata, Toshiaki Morito, Xingang Huang, Maiko Tamura, Yuta Kitamura, Nobuya Ohara, Mamoru Ouchida, Koichi Ohshima, Kenji Shimizu, Mitsune Tanimoto, Kiyoshi Takahashi, Masao Matsuoka, Atae Utsunomiya, Tadashi Yoshino

AMERICAN JOURNAL OF PATHOLOGY 176 ( 1 ) 402 - 415 2010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 SOC INVESTIGATIVE PATHOLOGY, INC

Aberrant CpG island methylation contributes to the pathogenesis of various malignancies. However, little is known about the association of epigenetic abnormalities with multistep tumorigenic events in adult T cell leukemia/lymphoma (ATLL). To determine whether epigenetic abnormalities induce the progression of ATLL, we analyzed the methylation profiles of the SHP1, p15, p16, p73, HCAD, DAPK, hMLH-1, and MGMT genes by methylation specific PCR assay in 65 cases with ATLL patients. The number of CpG island methylated genes increased with disease progression and aberrant hypermethylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival by Kaplan-Meyer analysis. The present findings strongly suggest that the multistep accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression, and prognosis of ATLL, as well as indicate the value of CpG methylation and CIMP for new diagnostic and prognostic biomarkers. (Am J Pathol 2010, 176:402-415; DOI: 10.2353/ajpath.2010.090236)

DOI： 10.2353/ajpath.2010.090236

Multi-step accumulation of aberrant CpG island hypermethylation during progression of lymphoma/leukemia

Takashi Oka, Takami Kondo, Hiaki Sato, Yoko Nakatani, Kana Washio, Ichiro Murakami, Nobuya Ohara, Kiyoshi Takahashi, Mamoru Ouchida, Mitsune Tanimoto, Tadashi Yoshino

International Journal of Molecular Medicine 26 ( Sup1 ) S44 - S44 2010年

A CACNB4 mutation shows altered Cav2.1 function in a patient with Dravet syndrome

大守伊織, 大内田守, 御牧信義, 西木禎一, 富澤一仁, 松井秀樹

岡山医学会雑誌 121 ( 3 ) 149 - 156 2010年

Helicobacter pylori infection induce accumulation of aberrant CpG hypermethylation to promote development and progression of gastric MALT lymphoma

Takashi Oka, Takami Kondo, Hiaki Sat, Yoko Shinnou, Kana Washio, Toshiaki Morit, Katsuyoshi Takat, Nobuya Ohara, Mamoru Ouchida, Kenji Shimizu, Tadashi Yoshin

Proceedings of The 3rd International Symposium for Future Technology Creating Better Human Health and Society Comprehensive Approach to Cancer and Infectious Diseases 1 ( 1 ) 105 - 105 2010年

REIC/Dkk-3 stable transfection reduces the malignant phenotype of mouse prostate cancer RM9 cells 国際誌

Jie Chen, Masami Watanabe, Peng Huang, Masakiyo Sakaguchi, Kazuhiko Ochiai, Yasutomo Nasu, Mamoru Ouchida, Nam-Ho Huh, Kenji Shimizu, Yuji Kashiwakura, Haruki Kaku, Hiromi Kumon

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 24 ( 6 ) 789 - 794 2009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：SPANDIDOS PUBL LTD

The reduced expression in immortalized cells (REIC)/Dickkopf (Dkk)-3. a member of the Dkk gene family, is a tumor suppressor in a broad range of cancers. REIC/Dkk-3 transfected stable clones of mouse prostate cancer RM9 cells (RM9-REIC) and the empty vector-transfected control clone cells (RM9-EV) were established. Clones were used to evaluate the anti-cancer effects and a proteomics analysis of REIC/Dkk-3 continuous expression was performed. The RM9-REIC cells show a feeble appearance and the cell membrane shows irregular buds known as blebs. In vitro cell proliferation was significantly suppressed in RM9-REIC clones in comparison to the control. The apoptosis assay was done under standard culture conditions and RM9-REIC showed a higher incidence of apoptosis. The RM9-EV and RM9-REIC cells were orthotopically implanted into a C57BL/6 mouse prostate. After 2 weeks, the tumor growth was significantly inhibited in RM9-REIC cells in comparison to the control. Two-dimensional gel electrophoresis was used to examine the modification of protein expression by the gene transfection. The analysis with mass spectrometry disclosed that expression of peroxiredoxin-l, GST-P1. transgelin-2, MRP-L12, ARD, GRP78 and Sorcin were increased and eEF1A-1 and cyclophilin-40 protein were decreased in RM9-REIC cells. Therefore, REIC/Dkk-3 stable transfectants show a reduction of malignancy in mouse prostate cancer RM9 cells in vitro and in vivo. The result of the proteomics analysis might provide important clues to clarify the anti-cancer molecular mechanism of REIC/Dkk-3 gene transfer.

DOI： 10.3892/ijmm_00000293

Dravet症候群患者に認められたカルシウムチャネル機能異常を引き起こすCACNB4遺伝子変異

大守伊織, 大内田守, 御牧信義, 西木禎一, 富澤一仁, 松井秀樹

岡山医学会雑誌 121 ( 3 ) 149 - 156 2009年12月

記述言語：日本語出版者・発行元：岡山医学会

Dravet症候群患者に認められたカルシウムチャネル機能異常を引き起こすCACNB4遺伝子変異について検討した。SCN1A遺伝子解析で変異を認めたDravet症候群患者38例を対象とした。健常者200例を対照とした。Dravet症候群患者で、SCN1B遺伝子とGABRG2遺伝子に変異を持つ症例はなかったが、CACNB4遺伝子変異を持つ症例を1例見出した。全細胞記録では、R568X変異からは背景電流異常のナトリウム電流は認めなかった。変異チャネルが野生型に比し有意にBa2+電流が増大した。ピーク電圧、細胞キャパシタンス、電流密度も変異型が野生型よりも有意に高値であった。

その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2009&ichushi_jid=J00175&link_issn=&doc_id=20091215200002&doc_link_id=10.4044%2Fjoma.121.149&url=https%3A%2F%2Fdoi.org%2F10.4044%2Fjoma.121.149&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Accumulation of aberrant CpG hypermethylation by Helicobacter pylori infection promotes development and progression of gastric MALT lymphoma 国際誌

Takami Kondo, Takashi Oka, Hiaki Sato, Yoko Shinnou, Kana Washio, Masayuki Takano, Toshiaki Morito, Katsuyoshi Takata, Nobuya Ohara, Mamoru Ouchida, Kenji Shimizu, Tadashi Yoshino

INTERNATIONAL JOURNAL OF ONCOLOGY 35 ( 3 ) 547 - 557 2009年9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

Aberrant DNA hypermethylation is an important mechanism for the inactivation of tumor-related genes in human tumors. Gastric mucosa-associated lymphoid tissue (MALT) lymphomas arise from Helicobacter pylori-associated chronic gastritis: most patients are H. pylori-positive and eradication therapy is highly effective. In the present study, we used methylation-specific PCR to analyze the DNA methylation status of 11 tumor-related genes (Kip2, p16, hMLH-1, p15, p73, MGMT, DAPK, MINT1, MINT2, MINT31 and HCAD) in 21 specimens of MALT lymphoma, 5 specimens of MALT lymphoma with large cell component (high-grade MALT lymphoma), 15 specimens of diffuse large B-cell lymphoma (DLBCL), 8 specimens of complete remission of MALT lymphoma after eradication therapy, 5 specimens with no evidence of malignancy and PBMCs from 10 healthy donors. The average number of methylated genes was significantly greater in gastric lymphomas as compared to normal controls (P<0.001). The CpG island methylator phenotype (CIMP) was observed in 93.3% (14/15) of DLBCLs, 100% (5/5) of high-grade MALT lymphomas and 61.9% (13/21) of MALT lymphomas; in contrast, CIMP was not found in the control group (0%). The average number of methylated genes and the CIMP incidence significantly increased with H. pylori infection. Furthermore, aberrant CpG methylation of specific genes, such as p16, MGMT and MINT31, was consistently associated with H. pylori infection. These findings strongly suggest that H. pylori infection causes the aberrant DNA hypermethylation of specific genes and induces CIMP, which is an important epigenetic mechanism for the development and progression of gastric MALT lymphoma; additionally, our findings provide new epigenetic markers.

DOI： 10.3892/ijo_00000366

Identification of novel target proteins for miR-17-92 cluster by proteomic analysis

Hirotaka Kanzaki, Mamoru Ouchida, Sachio Ito, Seiji Tamaru, Hiroko Hanafusa, Kenji Shimizu

CANCER RESEARCH 69 2009年5月

MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors 国際誌

Takafumi Kubo, Hiromasa Yamamoto, William W. Lockwood, Ilse Valencia, Junichi Sob, Michael Peyton, Masaru Jida, Hiroki Otani, Tetsuva Fujii, Mamoru Ouchida, Nagio Takigawa, Katsuyuki Kiura, Kenji Shimizu, Hiroshi Date, John D. Minna, Marileila Varella-Garcia, Wan L. Lam, Adi F. Gazdar, Shinichi Toyooka

INTERNATIONAL JOURNAL OF CANCER 124 ( 8 ) 1778 - 1784 2009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：WILEY-LISS

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phosphoMET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. (C) 2008 Wiley-Liss. Inc.

DOI： 10.1002/ijc.24150

Hyperthermia-induced seizure-susceptible rats: a novel model of febrile seizures

Tomoji Mashimo, Iori Ohmori, Mamoru Ouchida, Yukihiro Ohno, Toshiko Tsurumi, Takafumi Miki, Minoru Wakamori, Yasuo Mori, Tadao Serikawa

NEUROSCIENCE RESEARCH 65 S47 - S47 2009年

REIC/Dkk-3 in normal fibroblasts suppresses tumor growth via induction of interleukin-7.

Chen J, Watanabe M, Huang P, Sakaguchi M, Ochiai K, Nasu Y, Ouchida M, Huh NH, Shimizu K, Kashiwakura Y, Kaku H, Kumon H

Int J Mol Med 24 ( 6 ) 789 - 794 2009年

Ca(v)2.1 DYSFUNCTION MAY BE A GENETIC MODIFIER OF SEVERE MYOCLONIC EPILEPSY IN INFANCY

Iori Ohmori, Mamoru Ouchida, Takafumi Miki, Nobuyoshi Mimaki, Shigeki Kiyonaka, Teiichi Nishiki, Kazuhito Tomizawa, Yasuo Mori, Hideki Matsui

JOURNAL OF PHYSIOLOGICAL SCIENCES 59 515 - 515 2009年

A CACNB4 mutation shows that altered Ca(v)2.1 function may be a genetic modifier of severe myoclonic epilepsy in infancy 国際誌

Iori Ohmori, Mamoru Ouchida, Takafumi Miki, Nobuyoshi Mimaki, Shigeki Kiyonaka, Teiichi Nishiki, Kazuhito Tomizawa, Yasuo Mori, Hideki Matsui

NEUROBIOLOGY OF DISEASE 32 ( 3 ) 349 - 354 2008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：ACADEMIC PRESS INC ELSEVIER SCIENCE

Mutations of SCN1A, encoding the voltage-gated sodium channel alpha l subunit, represent the most frequent genetic cause of severe myoclonic epilepsy in infancy (SMEI). The purpose of this study was to determine if mutations in other seizure susceptibility genes are also present and could modify the disease severity. All coding exons of SCN1B, GABRG2, and CACNB4 genes were screened for mutations in 38 SCN1A-mutation-positive SMEI probands. We identified one proband who was heterozygous for a de novo SCN1A nonsense mutation (R568X) and another missense mutation (R468Q) of the CACNB4 gene. The latter mutation was inherited from his father who had a history of febrile seizures. An electrophysiological analysis of heterologous expression system exhibited that R468Q-CACNB4 showed greater Ba(2+) current density compared with the wild-type CACNB4. The greater Ca(v)2.1 currents caused by the R468Q-CACNB4 mutation may increase the neurotransmitter release in the excitatory neurons under the condition of insufficient inhibitory neurons caused primarily by the SCN1A mutation. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.nbd.2008.07.017

PROTEOMICS-BASED ANALYSIS OF INVASION-RELATED PROTEINS IN MALIGNANT GLIOMAS

T. Maruo, T. Ichikawa, H. Kanzaki, M. Onishi, S. Inoue, K. Yoshida, H. Kambara, M. Ouchida, K. Shimizu, I. Date

NEURO-ONCOLOGY 10 ( 6 ) 1143 - 1143 2008年12月

EGFR変異とUracil-Tegafurによる肺腺癌術後補助療法の関連性についての検討

末久弘, 豊岡伸一, 堀田勝幸, 内田亜希子, 宗淳一, 藤原義朗, 松尾恵太郎, 大内田守, 高田穣, 木浦勝行, 伊達洋至

岡山医学会雑誌 120 ( 3 ) 265 - 269 2008年12月

記述言語：日本語出版者・発行元：岡山医学会

完全切除された肺腺癌患者に補助療法としてUracil-Tegafur(UFT)を投与した場合における、EGFR変異の予後判定因子としての可能性を検討した。また、in vitro系のEGFR遺伝子導入細胞株における5-FUの効果も検討した。UFT投与群68例、手術のみの経過観察群119例であった。EGFR変異は187例中79例に認め、49例がエクソン19欠失変異、30例がエクソン21点突然変異であった。女性、非喫煙者に有意に変異が多かった。単変量解析では、UFT投与群が経過観察群よりも、EGFR変異群が野生群よりも予後がよい傾向であった。多変量解析では、術後補助療法が独立して有意に予後を延長していた。EGFR変異群では術後補助療法による予後延長の効果は認めなかった。EGFR変異型細胞株は5-FU低感受性を有することが示唆された。

その他リンク： https://search.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2008&ichushi_jid=J00175&link_issn=&doc_id=20081205250002&doc_link_id=10.4044%2Fjoma.120.265&url=https%3A%2F%2Fdoi.org%2F10.4044%2Fjoma.120.265&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

Novel SCN5A Mutations and SNP in Patients with Brugada Syndrome

Kazufumi Nakamura, Daiji Miura, Kaoru Kobayashi, Mamoru Ouchida, Kenji Shimizu, Iori Ohmori, Nobuhiro Nishii, Satoshi Nagase, Hiroshi Morita, Yoshiki Hata, Takefumi Oka, Kengo Kusano, Tohru Ohe

CIRCULATION 118 ( 18 ) S981 - S981 2008年10月

Novel SCN5A Mutations and SNP in Patients with Brugada Syndrome

Kazufumi Nakamura, Daiji Miura, Kaoru Kobayashi, Mamoru Ouchida, Kenji Shimizu, Lori Ohmorl, Nobuhiro Nishii, Satoshi Nagase, Hiroshi Morita, Yoshiki Hata, Takefumi Oka, Kengo Kusano, Tohru Ohe

CIRCULATION 118 ( 18 ) S1485 - S1485 2008年10月

The allelic distribution of a single nucleotide polymorphism in the PDCD5 gene locus of Japanese non-small cell lung cancer patients. 査読

Nanba K, Toyooka S, Soh J, Tsukuda K, Yamamoto H, Sakai A, Ouchida M, Kobayashi N, Matsuo K, Koide N, Kusaka K, Shimizu K, Date H

Molecular medicine reports 1 667 - 671 2008年9月

Single nucleotide polymorphism in reuced folate carrier-1 gene and methyleneterathydrofolate reductase gene in patients with osteosarcoma

Y. Morimoto, A. Yoshida, M. Ouchida, T. Kunisada, K. Shimizu, T. Ozaki

EJC SUPPLEMENTS 6 ( 9 ) 117 - 118 2008年7月

成人T細胞白血病/リンパ腫(ATLL)における特異的DNAメチル化と病態との関連

佐藤妃映, 岡剛史, 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 48 85 - 85 2008年5月

A screening test for the prediction of Dravet syndrome before one year of age 国際誌

Junri Hattori, Mamoru Ouchida, Junko Ono, Susumu Miyake, Satoshi Maniwa, Nobuyoshi Mimaki, Yoko Ohtsuka, Iori Ohmori

EPILEPSIA 49 ( 4 ) 626 - 633 2008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：WILEY

Purpose: Our aim was to develop a screening test to predict Dravet syndrome before the first birthday based on the clinical characteristics of infants and the SCN1A mutation analysis.
Methods: Ninety-six patients who experienced febrile seizures before the age of one were enrolled. The patients were divided into two groups-the Dravet syndrome group (n = 46) and the non-Dravet syndrome group (n = 50). We compared the clinical characteristics before one year of age of the two groups. We analyzed all coding exons of the SCN1A gene by the direct sequencing method. Scores from 0 to 3 were assigned to each risk factor based on the odds ratio and p-value.
Results: An age of onset of febrile seizure <= 7 months, a total number of seizures >= 5, and prolonged seizures lasting more than 10 min. were regarded as significant risk factors for Dravet syndrome. Other factors highly predictive of this syndrome were hemiconvulsions, partial seizures, myoclonic seizures, and hot water-induced seizures. A total clinical score of six or above was the cutoff value indicating a high risk of Dravet syndrome. SCN1A missense and truncated mutations were detected significantly more often in the Dravet syndrome group than in the non-Dravet syndrome group.
Discussion: This simple screening test was designed to be used by general pediatricians. It could help to predict Dravet syndrome before one year of age. If the sum of the clinical risk score is >= 6, then the performance of an SCN1A mutation analysis is recommended.

DOI： 10.1111/j.1528-1167.2007.01475.x

Rasmussen encephalitis associated with SCN1A mutation 国際誌

Iori Ohmori, Mamoru Ouchida, Katsuhiro Kobayashi, Yoshimi Jitsumori, Takushi Inoue, Kenji Shimizu, Hideki Matsui, Yoko Ohtsuka, Yoshihiro Maegaki

EPILEPSIA 49 ( 3 ) 521 - 526 2008年3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

Mutations in the SCN1A gene, encoding the neuronal voltage-gated sodium channel alpha 1 subunit, cause SMEI, GEFS+, and related epileptic syndromes. We herein report the R1575C-SCN1A mutation identified in a patient with Rasmussen encephalitis. R1575C were constructed in a recombinant human SCN1A and then heterologously expressed in HEK293 cells along with the human beta 1 and beta 2 sodium channel accessory subunits. Whole-cell patch-clamp recording was used to define biophysical properties. The R1575C channels exhibited increased channel availability and an increased persistent sodium current in comparison to the wild-type. These defects of electrophysiological properties can result in neuronal hyperexitability. The seizure susceptibility allele may influence the pathogenesis of Rasmussen encephalitis in this case.

DOI： 10.1111/j.1528-1167.2007.01411.x

The association between RAD18 Arg302Gln polymorphism and the risk of human non-small-cell lung cancer 国際誌

Hirotaka Kanzaki, Mamoru Ouchida, Hiroko Hanafusa, Hiromasa Yamamoto, Hiromitsu Suzuki, Masaaki Yano, Motoi Aoe, Kazue Imai, Hiroshi Date, Kei Nakachi, Kenji Shimizu

JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY 134 ( 2 ) 211 - 217 2008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：SPRINGER

Purpose The repair enzyme RAD18 plays a key role in the post-replication repair process in various organisms from yeast to human, and the molecular function of the RAD18 protein has been elucidated. Single nucleotide polymorphism (SNP) of arginine (Arg, CGA) or glutamine (Gln, CAA) at codon 302 is known on RAD18; however, the association between the SNP and the risk of any human cancers including non-small-cell lung cancer (NSCLC) has not been reported. We therefore investigated the relationship between the polymorphism and the development and progression of human NSCLC.
Methods The study population included 159 patients with NSCLC and 200 healthy controls. The SNP was genotyped by polymerase chain reaction with the confronting two-pair primer (PCR-CTPP) assay. Genotype frequencies were compared between patients and controls, and the association of genotypes with clinicopathological parameters was also studied.
Results The Gln/Gln genotype was significantly more frequent in NSCLC patients (20.7%) than in healthy controls (11.5%)(P = 0.003). The increased risk was detected in NSCLC patients with the Gln/Gln genotype [Odds ratio (OR) = 2.63, 95% confidence interval (CI)=1.38-4.98]. As to the relationship of the SNP with clinicopathological parameters of NSCLC, significantly higher risks were detected in lung squamous cell carcinoma (LSC) (OR = 4.40, 95% CI = 1.60-12.1).
Conclusions Our results suggested that Gln/Gln genotype of the RAD18 SNP has the increased risk of NSCLC, especially of LSC. This is the first report to provide evidence for an association between the RAD18 Arg302Gln polymorphism and human NSCLC risk.

DOI： 10.1007/s00432-007-0272-3

Decreased expression of the SIN3A gene, a candidate tumor suppressor located at the prevalent allelic loss region 15q23 in non-small cell lung cancer 国際誌

Hiromitsu Suzuki, Mamoru Ouchida, Hiromasa Yamamoto, Masaaki Yano, Shinichi Toyooka, Motoi Aoe, Nobuyoshi Shimizu, Hiroshi Date, Kenji Shimizu

LUNG CANCER 59 ( 1 ) 24 - 31 2008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：ELSEVIER IRELAND LTD

To identify the tumor suppressor genes (TSG) associated with non-small cell lung cancers (NSCLC), we performed the loss of heterozygosity (LOH) analysis in NSCLC samples from 66 patients. We focused on the novel hot spot region on 15q14-24 with eight polymorphic microsatellite markers. Frequent allelic toss was detected in 33 of 48 informative cases (69%) at D15S984 on 15q23. We defined the fine map on the region and identified the SlN3A gene as a candidate TSG. The SIN3A gene product is a component of the histone deacetylase (HDAC) complex and plays essential rotes in early embryonic development and the proliferation and survival. of a variety of cells through the repression of diverse signaling pathways. Our expression analysis revealed more frequent down-regulation of the SIN3A mRNA in 19 of 31 cases (61%) of NSCLCs in comparison to those of other flanking genes (16-42%), albeit the correlation of the decreased expression with the LOH did not attain statistic significance. These results suggest that the attenuated function of SIN3A due to a decreased level of expression may result in epigenetic de-regulation of growth-related genes through histone acetylation, which leads to the tumorigenesis of tung cancer cells. To our knowledge, this is the first evidence of the down-regulation of the SlN3A gene in human cancer. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.lungcan.2007.08.002

Increased DOI-induced wet-dog shakes in adrenocorticotropic hormone-treated rats are not affected by chronic imipramine treatment: Possible involvement of enhanced 5-HT2A-receptor expression in the frontal cortex

Yoshihisa Kitamura, Kazuhiko Shibata, Kozue Akiyama, Shizue Kirnoto, Yoshika Fujitani, Kouhei Kitagawa, Hirotaka Kanzaki, Mamoru Ouchida, Kenji Shimizu, Hiromu Kawasaki, Toshiaki Sendo, Yutaka Gomita

JOURNAL OF PHARMACOLOGICAL SCIENCES 106 ( 1 ) 100 - 106 2008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：JAPANESE PHARMACOLOGICAL SOC

We examined the influence of imipramine, a traditional tricyclic antidepressant, on the binding to serotonin (5-HT)(2) receptors and levels of 5-HT2A-receptor mRNA in the frontal cortex of rats treated with adrenocorticotropic hormone (ACTH). Chronic treatment with ACTH significantly increased the binding of [H-3]-ketanserin to 5-HT2 receptors and the expression of 5-HT2A-receptor mRNA in the frontal cortex. However, it did not alter the concentration of 5-HT or 5-hydroxyindole acetic acid. The effect of chronic ACTH treatment on 5-HT2 receptor and 5-HT2A-receptor mRNA levels was not altered by the chronic administration of imipramine. Also, imipramine did not affect the hyperfunction of 5-HT2A receptors caused by chronic ACTH treatment. These findings suggest that chronic treatment with ACTH acts to increase 5-HT2A-receptor synthesis through increased gene transcription, without modulating presynaptic serotonergic neurotransmission.

DOI： 10.1254/jphs.FP0071615

Single nucleotide polymorphism in the RAD18 gene and risk of colorectal cancer in the Japanese population 国際誌

Hirotaka Kanzaki, Mamoru Ouchida, Hiroko Hanafusa, Akiko Sakai, Hiromasa Yamamoto, Hiromitsu Suzuki, Masaaki Yano, Motoi Aoe, Kazue Imai, Hiroshi Date, Kei Nakachi, Kenji Shimizu

ONCOLOGY REPORTS 18 ( 5 ) 1171 - 1175 2007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

The RAD18 gene, located on the human chromosome 3p24-p25, plays a crucial role in post-replication repair (PRR) in various organisms from yeast to humans. In the human RAD18 gene, one coding single nucleotide polymorphism (SNP) at codon 302, encoding either arginine (Arg, CGA) or glutamine (Gln, CAA), was reported. Although the molecular function of the RAD18 protein came to be elucidated, the association between the RAD18 Arg302Gln polymorphism and the risk of human cancer development was not examined. Therefore, we investigated the relationship between the polymorphism and the development of human primary colorectal cancer (CRC). The Arg302Gln polymorphism in 100 patients with CRC and 200 healthy controls were genotyped by the polymerase chain reaction with confronting two-pair primer (PCR-CTPP) assay. The Gln/Gln genotype was significantly more frequent in CRC (18.0%) than in the healthy controls (11.5%) (p=0.046). The increased risk was detected in CRC patients with the Gln/Gln genotype (Odds ratio [OR], 2.10; 95% confidence interval [CI], 1.00 to 4.40). When the relationship of the SNP with clinicopathological parameters of CRC was investigated, particularly in the well-differentiated grade and in the lymph node metastasis (NI) CRC patients, significantly higher risks were detected (OR, 7.00; 95% CI, 1.19-41.1 and OR, 3.71; 95% CI, 1.30-10.6, respectively). These results suggested that the RAD18 Arg302Gln polymorphism is associated with the risk of CRC. This report provides evidence for an association between the RAD18 Arg302Gln polymorphism and human CRC risk.

EGFR変異とUFTによる肺腺癌術後補助療法の関連性についての検討

末久弘, 豊岡伸一, 堀田勝幸, 内田亜希子, 宗淳一, 藤原義朗, 松尾恵太郎, 大内田守, 高田穣, 木浦勝行, 伊達洋至

肺癌 47 ( 5 ) 513 - 513 2007年10月

Epidermal growth factor receptor mutation status and adjuvant chemotherapy with uracil-tegafur for adenocarcinoma of the lung 国際誌

Hiroshi Suehisa, Shinichi Toyooka, Katsuyuki Hotta, Akiko Uchida, Junichi Soh, Yoshiro Fujiwara, Keitaro Matsuo, Mamoru Ouchida, Minoru Takata, Katsuyuki Kiura, Hiroshi Date

JOURNAL OF CLINICAL ONCOLOGY 25 ( 25 ) 3952 - 3957 2007年9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 SOC CLINICAL ONCOLOGY

Purpose
Adjuvant chemotherapy with uracil- tegafur has been demonstrated to prolong survival among patients with resected lung adenocarcinomas. Epidermal growth factor receptor ( EGFR) mutations have been reported to be present in lung adenocarcinomas. The present study evaluated whether the EGFR status could be used as a biologic predictor of the outcome of adjuvant chemotherapy with uracil- tegafur.
Patients and Methods
The EGFR mutational status of 187 patients with resected lung adenocarcinomas was determined using a polymerase chain reaction - based assay for EGFR exons 19 and 21; the results were then correlated with the effect of adjuvant uracil- tegafur chemotherapy on survival. The antiproliferative effect of fluorouracil ( FU) on adenocarcinoma cell lines with EGFR wild-type or mutant type status was examined by measuring the inhibitory concentrations at 50% ( IC(50)s).
Results
Among the 187 patients, 68 received uracil- tegafur as adjuvant chemotherapy, and 119 were not treated with any chemotherapeutic agents. EGFR mutations were present in 79 patients ( 43%). Overall, the adjuvant chemotherapy with uracil- tegafur significantly prolonged survival compared with the control group ( hazard ratio = 0.38; P =.005). The survival benefit of adjuvant chemotherapy with uracil- tegafur was also examined after stratifying the patients according to EGFR mutation status. Adjuvant chemotherapy significantly prolonged survival among patients with EGFR wild- type tumors ( hazard ratio = 0.34; P =.013) but not among patients with EGFR mutant tumors. In an in vitro experiment, the IC(50)s of EGFR mutant cells to FU were higher than those of wild-type cells, indicating that EGFR wild- type cells are more sensitive to FU than mutant cells.
Conclusion
EGFR status influenced the effect of adjuvant chemotherapy with uracil- tegafur. Adjuvant chemotherapy could be customized based on EGFR status.

DOI： 10.1200/JCO.2007.11.8646

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

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

カルバマゼピン投与ラットにおける血中と肝臓中の産生CYPmRNA量の相関性の検討

林瑶子, 北村佳久, 大内田守, 清水憲二, 千堂年昭, 五味田裕

TDM研究 23 ( 3 ) S165 2006年7月

Single nucleotide polymorphism at codon 133 of the RASSF1 gene is preferentially associated with human lung adenocarcinoma risk 国際誌

Hirotaka Kanzaki, Hiroko Hanafusa, Hiromasa Yamamoto, Yukiko Yasuda, Kazue Imai, Masaaki Yano, Motoi Aoe, Nobuyoshi Shimizu, Kei Nakachi, Mamoru Ouchida, Kenji Shimizu

CANCER LETTERS 238 ( 1 ) 128 - 134 2006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：ELSEVIER IRELAND LTD

The RASSF1 gene, a putative tumor suppressor gene located on human chromosome 3p21, gamers much attention for the frequent allelic loss and gene silencing via promoter hypermethylation in a variety of human malignancies. An association between a single nucleotide polymorphism (SNP) at codon 133 of the RASSF1 gene, encoding either alanine (GCT) or serine (TCT), and human cancer risk remains undefined. We therefore, investigated the distribution of the Ala133Ser SNP in 101 patients with lung cancer, 63 with head and neck cancer, 72 with colorectal cancer, 56 with esophageal cancer and 110 healthy controls by polymerase chain reaction and restriction enzyme-digestion assay. The heterozygous Ala/Ser genotype was significantly more frequent in lung cancer patients than in healthy controls (P = 0.028). The adjusted odds ratio (OR) for the patients with heterozygous Ala/Ser genotype as compared with the controls with the Ala/Ala genotype was 2.59 (95% confidence interval (CI); 1.11-6.04). The increased risk of the Ala/Ser genotype was found in lung cancer patients but not in other cancer patients we examined. The association was particularly strong in those lung cancer patients of male (adjusted OR; 3.33, 95% CI; 1.37-8.12), with adenocarinoma (adjusted OR; 3.33, 95% CI; 1.36-8.15), early stages (adjusted OR; 3.42, 95% CI; 1.33-8.75) and with smoking habit (adjusted OR; 2.70, 95% CI; 1.06-6.83). These results suggest that the RASSF1 Ala133Ser SNP is associated with development of lung cancer, especially of lung adenocarcinoma. The increased risk of the heterozygous genotype is intriguing, implying a close relation with the dimerization feature of RASSF1 proteins. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.canlet.2005.07.006

Molecular pathological analysis of the protein tyrosine phosphatase: SHP1 gene in the pathogenesis of lymphoma and leukemias

Takashi Oka, Yoko Nakatani, Takami Kondo, Hiaki Sato, Kana Washio, Masayuki Takano, Ichirou Murakami, Yuriko Nishikawa, Mamoru Ouchida, Nobuya Ohara, Kiyoshi Takahashi, Kazuhiko Hayashi, Mitsune Tanimoto, Kenji Shimizu, Tadashi Yoshino

CANCER RESEARCH 66 ( 8 ) 2006年4月

Abberant DNA methylation in the progression of adult T cell leukemia/lymphoma (ATLL)

Hiaki Sato, Takashi Oka, Yoko Nakatani, Takami Kondo, Masayuki Takano, Nobuya Ohara, Mamoru Ouchida, Kouichi Oshima, Kenji Shimizu, Mitsune Tanimoto, Kiyoshi Tkahashi, Yoshino Tadashi

CANCER RESEARCH 66 ( 8 ) 2006年4月

Identification of a candidate tumor suppressor gene RHOBTB1 located at a novel allelic loss region 10q21 in head and neck cancer 国際誌

LB Beder, M Gunduz, M Ouchida, E Gunduz, A Sakai, K Fukushima, H Nagatsuka, S Ito, N Honjo, K Nishizaki, K Shimizu

JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY 132 ( 1 ) 19 - 27 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：SPRINGER

Purpose: Aims of the study are to narrow-down the hotspot region on 10q21 defined by previous genome-wide loss of heterozygosity (LOH) analysis in head and neck squamous cell carcinomas (HNSCC) and to define candidate tumor suppressor genes (TSG) concerned with 10q21. Materials and methods: LOH analysis was carried out with ten polymorphic microsatellite markers. Expression analysis was performed by semi-quantitative RT-PCR, and mutation analysis by PCR and direct sequencing. Results: LOH analysis on 10q21 in 52 HNSCC indicated distinctive and frequent allelic loss at D10S589 (42%). Among flanking genes, we found the RHOBTB1 gene as a candidate TSG, since an intragenic marker demonstrated the highest LOH (44%). Expression analysis revealed down-regulation of RHOBTB1 mRNA in 37% of tumors. Interestingly, all the five tumors that showed decreased expression of RHOBTB1 were accompanied with LOH, supporting the haploinsufficiency and class 2 TSG characteristics of RHOBTB1. No pathogenic mutation of RHOBTB1 was found. Furthermore, another gene within the region, EGR2, was also taken under scope. LOH frequencies around the EGR2 gene were relatively low (23 and 33%). Albeit semi-quantitative expression analysis of EGR2 demonstrated downregulation in 45% of tumor samples, no relation was found between the expression levels and LOH status. Conclusion: Frequent allelic loss and decreased expression of RHOBTB1 suggested that this gene has a role in tumorigenesis of a subset of HNSCC.

DOI： 10.1007/s00432-005-0033-0

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

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

Aberrant methylation of p57KIP2 gene in lung and breast cancers and malignant mesotheliomas 国際誌

T Kobatake, M Yano, S Toyooka, K Tsukuda, H Dote, T Kikuchi, M Toyota, M Ouchida, M Aoe, H Date, HI Pass, H Doihara, N Shimizu

ONCOLOGY REPORTS 12 ( 5 ) 1087 - 1092 2004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

The p57KIP2 gene belongs to the Cip/Kip family of CDK inhibitors and has been demonstrated to be a tumor suppressor gene, being inactivated in various types of human cancers. We analyzed the methylation and expression status of p57KIP2 in lung and breast cancers, and in malignant mesotheliomas (MMs). The promoter region of p57KIP2 was determined by methylation-specific PCR (MSP) in samples of lung and breast cancer, and of MM. The expression of the gene in the cell lines was determined by RT-PCR and correlated with the methylation status. Aberrant methylation was detected by MSP in 9 of 27 (33%) and 25 of 78 (32%) lung cancer cell lines and tumors, respectively, 11 of 18 (61%) and 17 of 38 (45%) breast cancer cell lines and tumors, respectively, and 1 of 25 (4%) MM tumors. DNA methylation was detected but rarely in the corresponding non-malignant tissues. In addition, the gene expression was restored in the methylated cell lines following 5-aza-2'-deoxycytidine treatment, confirming that the methylation was indeed responsible for the gene down-regulation. We also examined the relationship between the p57KIP2 methylation status and the clinicopathological features of the primary tumors, and found that there was no relationship between the p57KIP2 methylation status and any of the examined clinicopathological features. In summary, our results demonstrate that p57KIP2 methylation associated with the gene down-regulation is frequently present in lung and breast cancers and plays an important role at the molecular level in the pathogenesis of these cancers.

SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3A, a component of histone deacetylase complex 国際誌

T Ito, M Ouchida, S Ito, Y Jitsumori, Y Morimoto, T Ozaki, A Kawai, H Inoue, K Shimizu

LABORATORY INVESTIGATION 84 ( 11 ) 1484 - 1490 2004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：NATURE PUBLISHING GROUP

Synovial sarcomas are soft-tissue tumors predominantly affecting children and young adults. They are molecular-genetically characterized by the SYT-SSX fusion gene generated from chromosomal translocation t(X; 18) (p11.2; q11.2). When we screened new gene products that interact with SYT or SSX proteins by yeast two-hybrid assay, we found that mSin3A, a component of the histone deacetylase complex, interacts with SYT but not with SSX. These results were confirmed by mammalian two-hybrid and pull-down assays. Analyses with sequential truncated proteins revealed a main mSin3A-interaction region on the SYT amino-terminal 93 amino acids, and another one on the region between 187th amino acid and break point. In luciferase assay, mSin3A repressed the transcriptional activity of reporter promoter mediated by SYT and hBRM/BRG1. Our results suggest that the histone deacetylase complex containing mSin3A may regulate the transcriptional activation mediated by SYT.

DOI： 10.1038/labinvest.3700174

高齢にて発症したQT延長症候群の1例同一家系内で異なる発症様式を示したLQT1の1家系

三浦大志, 中村一文, 久松研一, 森田宏, 森田志保, 永瀬聡, 齋藤博則, 草野研吾, 江森哲郎, 大江透, 大内田守, 清水憲二, 西内崇将, 幡芳樹, 水尾浩三, 大杉保

Therapeutic Research 25 ( 11 ) 2137 - 2141 2004年11月

記述言語：日本語出版者・発行元：ライフサイエンス出版(株)

症例は81歳,男性.生来失神歴はなかった.80歳の時に意識レベルが低下し,CT上脳萎縮,多発性脳梗塞を指摘された.このとき心電図でQT間隔の延長を指摘された(QTc=0.54).その後も誤嚥性肺炎を繰り返し近医に入退院を繰り返していた.81歳の時,肺炎で入院中に失神発作をきたし,著明なQT間隔の延長(QTc=0.64),Torsades de Pointes(TdP)を認めた.発作の誘因としては低カリウム血症が増悪因子となったと考えられ,電解質補正で心室性不整脈は再発を認めていない.孫(12歳,男性)にKCNQ1遺伝子の変異(V254M)によるQT延長症候群患者がおり,遺伝子検査にて同じ変異が認められた.本症例は高齢になるまでTdPや失神を発症せず,LQT1(V254M)の同一家系内でも,発症には電解質や自律神経など後天的な因子も大きく関与していると考えられた(著者抄録)

Tumor-specific exon creation of the HELLS/SMARCA6 gene in non-small cell lung cancer 国際誌

M Yano, M Ouchida, H Shigematsu, N Tanaka, K Ichimura, K Kobayashi, Y Inaki, S Toyooka, K Tsukuda, N Shimizu, K Shimizu

INTERNATIONAL JOURNAL OF CANCER 112 ( 1 ) 8 - 13 2004年10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

To identify tumor-suppressor genes on chromosome 10 in non-small cell lung cancers, we isolated 10 types of splicing variant of the HELLS/SMARCA6 gene transcripts. HELLS/SMARCA6 is a novel member of SNF2 family, which is implicated in cellular functions like chromatin remodeling. Variant I was an alternatively spliced isoform containing an insertion of a 44 ntd intronic sequence between exons 3 and 4, giving rise to a premature termination of translation. Expression of variant I was detected exclusively in lung cancer specimens (I I of 43 cases, 26%) but was not detected in corresponding normal tissues. The D10S520 marker in the proximity of the HELLS/SMARCA6 gene showed prevalent allelic loss (41%) compared to flanking markers (25-31%). These results suggest that loss of function of HELLS/SMARCA6 by allelic loss and aberrant proteins by tumor-specific exon creation may result in epigenetic deregulation, leading lung cells to malignancy or its progression. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/ijc.20407

Severity dependent up-regulations of LOX-1 and MCP-1 in early sclerotic changes of common carotid arteries in spontaneously hypertensive rats

Y Hamakawa, N Omori, M Ouchida, M Nagase, K Sato, Nagano, I, M Shoji, T Fujita, K Abe

NEUROLOGICAL RESEARCH 26 ( 7 ) 767 - 773 2004年10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：MANEY PUBLISHING

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) and monocyte chemoattractant protein- I (MCP-1) are molecules involving in the initiation and progression of atherosclerosis. In order to examine a possible difference in LOX-1 and MCP-1 expressions depending on the severity of early stage of atherosclerosis, we investigated atherosclerotic changes by exposure to hypertension and hyperlipidemia in common carotid arteries (CCAs) of stroke-prone spontaneously hypertensive rat (SHR-SP). Three rat model groups such as control [Wistar Kyoto rat (WKY) group], hypertension (SHR-SP group) and hypertension + hyperlipidemia [SHR-SP+high fat and cholesterol (HFC) group] were used. Body weights, brain weights, systolic blood pressures and serum levels of total cholesterol, low-density lipoprotein and triglyceride were measured at 0, 5, 10 and 15 days after appropriate diet. Immunohistochemistry showed that the positive area and the strength of LOX-1 and MCP-1 were larger in the SHR-SP+ HFC group than in the SHR-SP group, while no immunoreactivities were found in the WKY group. Conventional RT-PCR and real-time PCR analyses showed that mRNAs of those in the SHR-SP group were higher with greater up-regulation in the SHR-SP+ HFC group. LOX-1 and MCP-1 expressions were coordinately up-regulated at mRNA and protein levels in an early stage of sclerosis depending on the severity of atherosclerotic stress. Activations of LOX-1 and MCP-1 are collectively involved in the early stage of atherosclerosis.

DOI： 10.1179/016164104225016074

Loss of heterozygosity on chromosome 10q associated with malignancy and prognosis in astrocytic tumors, and discovery of novel loss regions 国際誌

S Daido, S Takao, T Tamiya, Y Ono, K Terada, S Ito, M Ouchida, Date, I, T Ohmoto, K Shimizu

ONCOLOGY REPORTS 12 ( 4 ) 789 - 795 2004年10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

Certain tumor suppressor genes (TSG) residing on human chromosome 10q are implicated in astrocytic tumors. We thoroughly examined loss of heterozygosity (LOH) on chromosome 10q in astrocytic tumors to determine the extent of deletion and their relation to prognostic variables of patients. We analyzed 63 astrocytic tumors, including 9 diffuse astrocytomas, 36 anaplastic astrocytomas, and 18 glioblastomas. DNAs from tumors and leukocytes were analyzed for LOH at 18 microsatellite loci by polymerase chain reaction using fluorescence-labeled primers. Then correlation between LOH and clinicopathological variables was examined statistically. Twenty-four (66.7%) anaplastic astrocytomas and 15 (83.3%) glioblastomas had at least one LOH on chromosome 10q. However, diffuse astrocytomas exhibited no LOH. Nineteen tumors (10 anaplastic astrocytomas and 9 glioblastomas) were believed to have a total loss of one chromosome 10. Analyses on 20 tumors with interstitial LOH revealed that most of the high LOH regions matched the location of known TSGs, while some novel LOH regions were found preferentially in anaplastic astrocytoma. The median survivals of the total, partial, and no loss groups were 10.1, 14.8, and 46.8 months, respectively, indicating a significant difference in the survivals of these groups (P=0.0289). Thus, analyzing chromosome 10q loss is helpful for diagnosing malignancy in astrocytic tumors and for predicting patients' survival. Our data also suggested that there are novel TSGs for anaplastic astrocytoma at 10q24 and 10q26.

Prevalent hyper-methylation of the CDH13 gene promoter in malignant B cell lymphomas 国際誌

Y Ogama, M Ouchida, T Yoshino, S Ito, H Takimoto, Y Shiote, F Ishimaru, M Harada, M Tanimoto, K Shimizu

INTERNATIONAL JOURNAL OF ONCOLOGY 25 ( 3 ) 685 - 691 2004年9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

CDH13 (H-cadherin) is a member of the cadherin superfamily, which plays an important role in cell recognition and adhesion. We examined the expression and methylation status of the CDH13 gene in diffuse large B cell lymphomas (B-DLCLs). We found decreased expression of the CDH13 gene in all of 6 hematopoietic cell lines by reverse transcription-polymerase chain reaction (RT-PCR). Promoter hypermethylation of the gene was detected in all 6 cell lines and in 13 of 19 (68%) B-DLCL samples by methylation-specific PCR. Interestingly, the methylation frequency of the CDH13 gene was comparable to those of the tumor suppressor genes p15 (68%) and p16 (74%) detected in B-DLCLs. Sequencing of bisulfite-treated DNA revealed hyper-methylation of the CpG islands of the CDH13 promoter in B-DLCLs and the cell lines. Treatment with 5-aza-2'-deoxycytidine restored CDH13 gene expression in a cell line in which promoter hyper-methylation and impaired expression of the CDH13 gene were observed. Loss of heterozygosity (LOH) around the CDH13 genes on chromosome 16q24 was detected in 6 of 15 (40%) informative cases with microsatellite marker D16S507 and in 6 of 15 (40%) cases with D16S422 in B-DLCLs. In all of 4 B-DLCL cases which showed both promoter methylation and LOH at the two marker loci, expression of the CDH13 gene was significantly low. These results suggest that silencing of the CDH13 gene by aberrant promoter methylation and allelic deletion is associated with tumorigenesis in a subset of B-DLCL.

Positive correlation between allelic loss at chromosome 14q24-31 and poor prognosis of patients with renal cell carcinoma 国際誌

H Kaku, S Ito, S Ebara, M Ouchida, Y Nasu, T Tsushima, H Kumon, K Shimizu

UROLOGY 64 ( 1 ) 176 - 181 2004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：ELSEVIER SCIENCE INC

Objectives. To report our development of a new application of the inter-AN long polymerase chain reaction (PCR) for genomic scanning to screen for tumor-specific alterations in tumor DNA. Using this method, we detected a rearranged chromosomal region in renal cell carcinomas (RCCs). We then examined tumor-specific allelic loss in this region using microsatellite markers and determined whether a relationship was present between this allelic loss and the clinicopathologic features of the patients.
Methods. The inter-Alu long PCR genomic scan method was performed using RCC DNA samples and primers specific for a minor subset of the human repeat sequence Alu. We analyzed DNA samples from 42 pairs of matched normal and nonpapillary RCC tissues with seven microsatellite markers.
Results. The inter-Alu long PCR genomic scan method revealed an altered DNA region on chromosome 14q24-31, which is the location of several putative tumor suppressor genes. At least one of seven microsatellite markers on chromosome 14q24-31 showed loss of heterozygosity in 23 (54.8%) of 42 informative cases of RCC. The prevalent loss region was confined to a 2-Mb region around D14S67. We found a positive correlation between the presence of the loss of heterozygosity on 14q24-31 and tumor stage (P < 0.05). We also found that cases with allelic loss at 14q24-31 had a poor prognosis (P = 0.045).
Conclusions. Our inter-Alu long PCR genomic scan method is a powerful method for the screening of DNA alterations, and our data suggest that the chromosome 14q24-31 region contains likely tumor suppressor genes associated with the progression of RCC. (C) 2004 Elsevier Inc.

DOI： 10.1016/j.urology.2004.03.015

Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in breast cancer 国際誌

H Dote, S Toyooka, K Tsukuda, M Yano, M Ouchida, H Doihara, M Suzuki, H Chen, JT Hsieh, AF Gazdar, N Shimizu

CLINICAL CANCER RESEARCH 10 ( 6 ) 2082 - 2089 2004年3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: Human DOC-2/DAB2 interactive protein (hDAB21P) gene is a novel member of the Ras GTPaseactivating family and has been demonstrated to be a tumor suppressor gene inactivated by methylation in prostate cancer. We analyzed methylation and expression status of hDAB21P in breast cancer.
Experimental Design: The promoter region of hDAB21P was divided into two regions (m2a and m2b) following our previous report on prostate cancer, and methylation status was determined in breast cancer cell lines with bisulfited DNA sequencing. Expression was semiquantified with real-time reverse transcription-PCR to rind that aberrant methylation showed the inverse relationship with expression. On the basis of sequence data, we developed methylation-specific PCR for m2a and m2b regions and applied to samples.
Results: Aberrant methylation was detected in 11 of 25 breast cancer cell lines (44%) and 15 of 39 primary tumors (38%) at the m2a region and in 12 of 25 cell lines (48%) and 13 of 39 tumors (33%) at the m2b region. In addition, gene expression was restored in methylated cell lines with 5-aza2'-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined the relationship between hDAB21P methylation and clinicopathologic features in primary tumors and found that methylation in the m2b region was associated with progressive nodal status of tumors.
Conclusions: We developed methylation-specific PCR for hDAB21P and examined its methylation status in breast cancer. Our results demonstrate that hDAB2IP methylation frequently is present in breast cancer and plays a key role in hDAB21P inactivation, suggesting the relationship between hDAB2IP methylation and lymph node metastasis of breast cancer.

DOI： 10.1158/1078-0432.CCR-03-0236

Establishment and characterization of a biphasic synovial sarcoma cell line, SYO-1 国際誌

A Kawai, N Naito, A Yoshida, Y Morimoto, M Ouchida, K Shimizu, Y Beppu

CANCER LETTERS 204 ( 1 ) 105 - 113 2004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：ELSEVIER SCI IRELAND LTD

We describe here the establishment of a new synovial sarcoma cell line, SYO-1, derived from a biphasic synovial sarcoma that developed in the groin of a 19-year-old female. The cell line was maintained for more than 70 passages (more than 24 months) in vitro. The SYO-1 cells in monolayer culture exhibited a spindle shape without conspicuous pleomorphism. Immunohistochemically, the cells were positive for vimentin, type IV collagen, S-100, mdm2, bcl-2, c-Met and c-Kit. Tumors developed by their implantation in nude mice histologically showed biphasic features that were composed of areas of fascicles of spindle cells and areas of compact proliferation of polygonal to ovoid cells, which occasionally formed epithelial plaque and expressed cytokeratin and EMA. SYO-1 cells harbored the characteristic t(X;18)(p11.2;q11.2) translocation by chromosome analysis and SYT-SSX2 chimeric transcript by RT-PCR. The SYO-1 cells, the first characterized cell line derived from biphasic synovial sarcoma retaining the characteristic genetic and phenotypic features of the tumor, will be useful for various investigations on synovial sarcoma, especially for its epithelial differentiation. (C) 2003 Published by Elsevier Ireland Ltd.

DOI： 10.1016/j.canlet.2003.09.031

高齢にて発症したQT延長症候群の一例

三浦大志, 中村一文, 久松研一, 森田宏, 森田志保, 永瀬聡, 齋藤博則, 草野研吾, 江森哲朗, 大江透, 大内田守, 清水憲二, 西内崇将, 幡芳樹, 水尾浩三, 大杉保

Therapeutic Research 68 ( Suppl.III ) 946 - 946 2004年

Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene silencing by aberrant CpG methylation 国際誌

M Koyama, T Oka, M Ouchida, Y Nakatani, R Nishiuchi, T Yoshino, K Hayashi, T Akagi, Y Seino

LABORATORY INVESTIGATION 83 ( 12 ) 1849 - 1858 2003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：LIPPINCOTT WILLIAMS & WILKINS

Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.

DOI： 10.1097/01.LAB.0000106503.65258.2B

Frequent deletion and increased mRNA expression of a candidate tumor suppressor gene at 19p13 region, BRG1, in oral cancers(2nd ASIAN SCIENCE SEMINAR IN OKAYAMA 2003)

Gunduz Esra, Gunduz Mehmet, Nagatsuka Hitoshi, Tsujigiwa Hidetsugu, Ouchida Mamoru, Shinnou Yasuo, Cengiz Beyhan, Shimizu Kenji, Nagai Noriyuki

Journal of hard tissue biology 12 ( 2 ) 73 - 73 2003年12月

Single nucleotide polymorphism in fibroblast growth factor receptor 4 at codon 388 is associated with prognosis in high-grade soft tissue sarcoma 国際誌

Y Morimoto, T Ozaki, M Ouchida, N Umehara, N Ohata, A Yoshida, K Shimizu, H Inoue

CANCER 98 ( 10 ) 2245 - 2250 2003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：JOHN WILEY & SONS INC

BACKGROUND. A recent study revealed that single nucleotide polymorphism (SNP) at codon 388 (Gly or Arg) of fibroblast growth factor receptor 4 (FGFR4) was associated with prognosis in patients with carcinoma of the breast and colorectal carcinoma. The purpose of the current study was to investigate the correlation between codon 388 SNIP and clinical prognosis in patients with sarcoma of the bone and soft tissues.
METHODS. Tumor samples were obtained from 143 patients with high-grade bone and soft tissue sarcomas at Okayama University Hospital between 1986-2002, and from 102 healthy volunteers. SNP of codon 388 was detected by sequencing and fragment length of polymerase chain reaction products digested by restriction enzyme. The chi-square test was used to compare genotype distribution and the Kaplan-Meier method was used for survival analysis. 2
RESULTS. With regard to FGFR4 genotypes in the 143 patients studied, 54 (37.8%) were Gly/Gly, 72 (50.3%) were Gly/Arg, and 17 (11.9%) were Arg/Arg, findings that were not significantly different from those of controls (P = 0.97). With regard to cumulative overall and metastasis-free survival, patients with the Gly/Gly genotype were found to have a better prognosis (P = 0.085 and P = 0.27, respectively). FGFR4 SNIP was found to be correlated significantly with overall and metastasis-free survival in patients with soft tissue sarcomas (P = 0.029 and P = 0.045, respectively), but not in those patients with bone sarcomas (P = 0.88 and P = 0.75, respectively).
CONCLUSIONS. In the current study, the authors found a significant correlation between FGFR4 SNP and prognosis in patients with soft tissue sarcoma, although the samples were comprised of various histologic types. This SNP might be used to improve the prediction of clinical prognosis and lead to new treatment strategies in patients with soft tissue sarcomas. (C) 2003 American Cancer Society.

DOI： 10.1002/cncr.11778

現代医学の焦点(254)広範な悪性リンパ腫・白血病発症に関連するSHP1遺伝子に関する最新知見

岡剛史, 大内田守

日本臨床 61 ( 11 ) 2025 - 2032 2003年11月

[Recent progress in the protein-tyrosine phosphatase SHP1 gene: the significant correlation of the SHP1 gene silencing with the onset of lymphomas/leukemias].

Takashi Oka, Mamoru Ouchida

Nihon rinsho. Japanese journal of clinical medicine 61 ( 11 ) 2025 - 32 2003年11月

記述言語：日本語掲載種別：研究論文（学術雑誌）

Using cDNA expression array and tissue microarray, we comprehensively and systematically analyzed the expression profile to investigate the lymphomagenesis. We detected the significant decrease of hematopoietic cell specific protein-tyrosine phosphatase SHP1 gene expression in various malignant lymphomas and leukemias. High-frequent silencing of the SHP1 gene by promoter methylation was detected in various kinds of leukemias and lymphomas as well as in many hematopoietic cell lines. The promoter methylation of the SHP1 gene was significantly correlated with the clinical stage. Loss of heterozygosity with microsatellite markers around the SHP1 gene was detected in 79% of informative ALL cases. These results suggest that loss of functional SHP1 is closely associated with the pathogenesis of leukemias/lymphomas.

Is phenotype difference in severe myoclonic epilepsy in infancy related to SCN1A mutations? 国際誌

Ohmori, I, Y Ohtsukaa, M Ouchida, T Ogino, S Maniwa, K Shimizu, E Oka

BRAIN & DEVELOPMENT 25 ( 7 ) 488 - 493 2003年10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 classified 28 patients with severe myoclonic epilepsy in infancy (SME) according to the presence or absence of myoclonic seizures and/or atypical absences. Eleven of the patients had myoclonic seizures and/or atypical absences, and we refer to this condition as 'typical SME (TSME)'. Seventeen of the patients had only segmental myoclonias, and we refer to this condition as 'borderline SME (BSME)'. We then analyzed the electroclinical and genetic characteristics of these two groups. Ten of the 11 TSME patients had a photoparoxysmal response at some time during their clinical course, while none of the BSME patients showed this response. TSME and BSME showed a significant difference in regard to gender ratio: female dominance in TSME and male dominance in BSME (P = 0.008). The detection rate of the voltage-gated sodium channel alpha1-subunit (SCN1A) gene mutations was 72.7 and 88.2% in TSME and BSME, respectively. There was no difference in the type or rate of mutation between TSME and BSME. We conclude that TSME and BSME show distinct differences in photoparoxysmal response and gender, which might be caused by some genetic mechanism(s) other than the SCN1A gene mutation. (C) 2003 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0387-7604(03)00038-X

Paroxysmal movement disorders in severe myoclonic epilepsy in infancy 国際誌

Y Ohtsuka, Ohmori, I, T Ogino, M Ouchida, K Shimizu, E Oka

BRAIN & DEVELOPMENT 25 ( 6 ) 401 - 405 2003年9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 report on the electroclinical findings and the results of a molecular genetic study of a patient with typical severe myoclonic epilepsy ill infancy (TSME) and three with borderline SME (BSME) who showed paroxysmal movement disorders, such as choreoathetosis, dystonia and ballismus, during their clinical course. BSME was defined as a clinical entity that shares common characteristics with TSME but lacks myoclonic seizures associated with ictal EEG changes. When the paroxysmal movement disorders were first observed, all the patients in this study were being treated with polytherapy including phenytoin (PHT), and these abnormal movements disappeared when PHT was discontinued or reduced. However, on other occasions, two of our cases also showed the same abnormal movements even when not being treated with PHT. One patient with TSME and two of the three patients with BSME had SCN1A gene mutations that lead to truncation of the associated protein. We conclude that paroxysmal movement disorders seen in SME patients were closely related to their AED therapy, especially the use of PHT. It is thought that patients with both TSME and BSME have some predisposition toward paroxysmal movement disorders, and that this predisposition is partly related to sodium channel dysfunction, although some other factors might influence the occurrence of this phenomenon. (C) 2003 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0387-7604(03)00025-1

Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR

S Toyooka, K Tsukuda, M Ouchida, M Tanino, Y Inaki, K Kobayashi, M Yano, J Soh, T Kobatake, N Shimizu, K Shimizu

ONCOLOGY REPORTS 10 ( 5 ) 1455 - 1459 2003年9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

Mutation of the Kirsten ras (K-ras) gene is one of most common alterations in solid tumors including lung and colorectal cancers. We developed new enriched PCR-RFLP assay to detect mutations of K-ras codon 61 at the 1st and 2nd letters and non-enriched PCR-RFLP assay to detect the 3rd letter mutation. One mutant allele among 103 wild-type alleles was detected by enriched PCR-RFLP assay, while one mutant in 10 wild-type alleles was detected by non-enriched PCR-RFLP assay for codon 61 3rd letter. We then examined K-ras codon 12, 13 and 61 mutations in lung and colorectal cancers using these assays. K-ras codon 12 mutation was detected in 10 of 109 (9%) lung cancer and 19 of 83 (23%) colorectal cancer cases: K-ras codon 13 mutation was detected in 2 of 83 (2%) colorectal and 0 of 109 NSCLC cases, respectively. There was no K-ras codon 61 mutation in either type of cancer. Our results demonstrate that enriched PCR-RFLP is a sensitive assay to detect K-ras codon 61 mutation, however, it was extremely rare in lung and colorectal cancers, suggesting organ-specific pathways in mutagenesis of the ras gene family.

Splicing isoform of SYT-SSX fusion protein accelerates transcriptional activity and cell proliferation 国際誌

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

CANCER LETTERS 199 ( 1 ) 35 - 43 2003年9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 SCI IRELAND LTD

The human SYT-SSX gene has two splicing isoforms (type N and 1), the latter of which contains an additional insertion of 93 bases. In the present study, we found increased transcriptional activity of the SYT-SSX type I protein in luciferase assay. When the SYT-SSX cDNAs were transfected to NIH3T3 cells, the type I transformant grew faster than the type N transformant. Furthermore, we evaluated the isoform ratio of the SYT or SYT-SSX transcripts in various tissues. Our results suggest that the SYT-SSX type I protein plays a critical role in the tumorigenesis of synovial sarcomas through increased transcriptional activity. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3835(03)00314-8

Genome-wide analyses on loss of heterozygosity in head and neck squamous cell carcinomas 国際誌

LB Beder, M Gunduz, M Ouchida, K Fukushima, E Gunduz, S Ito, A Sakai, N Nagai, K Nishizaki, K Shimizu

LABORATORY INVESTIGATION 83 ( 1 ) 99 - 105 2003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：LIPPINCOTT WILLIAMS & WILKINS

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor survival rate. Identifying the tumor suppressor gene (TSG) loci by genomic studies is an important step to uncover the molecular mechanisms involved in HNSCC pathogenesis. We therefore performed comprehensive analyses on loss of heterozygosity (LOH) using a genome-wide panel of 191 microsatellite markers in 22 HNSCC samples. We found 53 markers with significantly high LOH (>30%) on 21 chromosomal arms; the highest values of those were observed on 3p, 9p, 13q, 15q, and 17p, corresponding to D3S2432 (67%), D9S921-D9S925 (67%) and GATA62F03 (86%), D13S1493 (60%), D15S211 (62%), and D17S1353 (88%), respectively. Fifteen hot spots of LOH were defined in 13 chromosomal arms: 2q22-23, 4p15.2, 4q24-25, 5q31, Sp23, 9p23-24, 9q31.3, 9q34.2, 10q21, 11q21-22.3, 14q11-13, 14q22.3, 17p13, 18q11, and 19q12 as loci reported previously in HNSCCs. Furthermore, we identified five novel hot spots of LOH on three chromosomal arms in HNSCC at 2q33 (D2S1384), 2q37 (D2S125), 8q12-13 (D8S1136), 8q24 (D8S1128), and 15q21 (D15S211). In conclusion, our comprehensive allelotype analyses have unveiled and confirmed a total of 20 possible TSG loci that could be involved in the development of HNSCC. These results provide useful clues for identification of putative TSGs involved in HNSCC by fine mapping of the suspected regions and subsequent analysis for functional genes.

DOI： 10.1097/01.LAB.0000047489.26246.E1

Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy.

Iori Ohmori, Mamoru Ouchida, Yoko Ohtsuka, Eiji Oka, Kenji Shimizu

Review Series Neurology, 2, 14-17 2003年

難治性リンパ腫発症関連遺伝子SHP1に関する最新知見

岡剛史, 大内田守

日本臨床 61(11): 2025-2032 2003年

Gene silencing of the tyrosine phosphatase SHP1 gene by aberrant methylation in leukemias/lymphomas. 国際誌

Takashi Oka, Mamoru Ouchida, Maho Koyama, Yoichiro Ogama, Shinichi Takada, Yoko Nakatani, Takehiro Tanaka, Tadashi Yoshino, Kazuhiko Hayashi, Nobuya Ohara, Eisaku Kondo, Kiyoshi Takahashi, Junjiro Tsuchiyama, Mitsune Tanimoto, Kenji Shimizu, Tadaatsu Akagi

Cancer research 62 ( 22 ) 6390 - 4 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

High-frequent silencing of hematopoietic cell-specific protein-tyrosine phosphatase SHP1 gene by promoter methylation was detected in various kinds of leukemias and lymphomas, as well as in many hematopoietic cell lines, which is supported by our previous observation of strong decrease of SHP1 mRNA and protein. The promoter methylation of the SHP1 gene was clearly correlated with the clinical stage. Loss of heterozygosity with microsatellite markers near the SHP1 gene was shown in 79% of informative acute lymphoblastic leukemia cases. These results suggest that functional loss of SHP1 is associated with the pathogenesis of leukemias/lymphomas.

Prognostic value of loss of heterozygosity around three candidate tumor suppressor genes on chromosome 10q in astrocytomas 国際誌

K Terada, T Tamiya, S Daido, H Kambara, H Tanaka, Y Ono, K Matsumoto, S Ito, M Ouchida, T Ohmoto, K Shimizu

JOURNAL OF NEURO-ONCOLOGY 58 ( 2 ) 107 - 114 2002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：KLUWER ACADEMIC PUBL

We thoroughly examined loss of heterozygosity (LOH) around three candidate tumor suppressor genes on chromosome 10q to determine whether LOH of each tumor suppressor gene is associated with the previously defined clinical prognostic indices. We also examined whether LOH can help predict prognostic variables in astrocytomas.
We selected samples from 40 astrocytomas (grades 2-4), performed Ki-67 immunostaining, and counted positive cells. Using DNA from aliquots of tumor blocks and leukocytes, we investigated LOH around the PTEN, NEURL, and DMBT1 genes (10q23.3-26.1) with the silver staining procedure. We then statistically evaluated the relationship among histological features, regional LOH on chromosome 10q, and survival. The mean survival period for patients with LOH around PTEN was 7.2 months after surgery, while that for patients without LOH around PTEN was 21.4 months. Thus, LOH around PTEN was closely associated with a reduced overall survival (p = 0.0020) but LOH at NEURL or DMBT1 was not (p > 0.05).
The combined features of an increase in histological grading and Ki-67-positive cells and the presence of LOH around PTEN significantly correlated with poor prognosis. These factors may be useful predictors of survival, and LOH analysis of tumor suppressor genes on chromosome 10q can contribute greatly to the treatment of patients with astrocytoma.

DOI： 10.1023/A:1016017711033

Allelic loss and reduced expression of the ING3, a candidate tumor suppressor gene at 7q31, in human head and neck cancers 国際誌

M Gunduz, M Ouchida, K Fukushima, S Ito, Y Jitsumori, T Nakashima, N Nagai, K Nishizaki, K Shimizu

ONCOGENE 21 ( 28 ) 4462 - 4470 2002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：NATURE PUBLISHING GROUP

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 tumor suppressor genes (TSG). We analysed LOH at 7q3I region in 49 HNSCC by using six polymorphic microsatellite markers and found allelic deletion in 48% (22/46) of the informative cases. We detected two preferentially deleted regions, one is around D7S643 and the other around D7S486. When we redefined the map of 7q31 region according to the contiguous sequences, a recently identified gene, ING3, was found in the proximity of D7S643. ING3 protein harbors the PHD 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 expression level. Reverse-transcription-PCR analysis demonstrated decreased or no expression of ING3 mRNA in 50% of primary tumors as compared with that of matched normal samples. Especially, about 63% of tongue and larynx tumors showed the decrease and a tendency of higher mortality was observed in cases with decreased ING3 expression. All these findings suggest a possibility that the ING3 gene functions as a TSG in a subset of HNSCC.

DOI： 10.1038/sj.onc.1205540

Astrocytomaにおける10番染色体長腕遺伝子異常と悪性度との関連

大同茂, 田宮隆, 小野恭裕, 大塚真司, 松下博和, 黒住和彦, 大本尭史, 伊藤佐智夫, 大内田守, 清水憲二

Brain Tumor Pathology 19 ( Suppl. ) 96 - 96 2002年5月

Reduced expression of the REIC/Dkk-3 gene by promoter-hypermethylation in human tumor cells 国際誌

K Kobayashi, M Ouchida, T Tsuji, H Hanafusa, M Miyazaki, M Namba, N Shimizu, K Shimizu

GENE 282 ( 1-2 ) 151 - 158 2002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：ELSEVIER SCIENCE BV

The human REIC gene is a recently found mortalization-related gene and a candidate tumor suppressor gene expression of which is largely attenuated in many immortalized and tumor-derived cell lines (Biochem. Biophys. Res. Commun. 268 (2000) 20-24). To gain insight into the mechanisms of the down-regulation, we investigated the genomic structure and promoter activity of the human REIC gene. The gene, identical with the DKK-3 gene, resides on chromosome 11pl5.1, consists of nine exons, and has two promoters. Methylation in the main promoter region was detected in I I out of 21 cell lines tested (52%) derived from a variety of human tumors, in which the expression of the REIC gene was decreased, In ten of these 11 cell lines the minor promoter was also methylated. Similarly, the REIC gene expression was decreased in 14 of 24 fresh non-small cell lung cancer specimens (58%) compared to that in corresponding non-cancerous tissue, though allelic loss and tumor-specific mutation were rare. Of these 14 tumors, at least five tumors exhibited heavy methylation of the REIC promoter region. These results indicate that the down-regulation of the REIC gene expression is ascribed to the aberrant promoter p hyper-methylation at least in a subset of human tumors. The expression was restored upon treatment of SQ5 cells with 5-aza-deoxycytidine, confirming DNA methylation as the mode of downregulation. A notable single nucleotide polymorphism in the coding region (cSNP) with an amino acid substitution of glycine (GGG) to arginine (AGG) was found at codon 335 of the REIC gene. However, the distribution of the cSNP showed no significant difference between lung cancer patients and healthy population. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(01)00838-1

Effect of naturally occurring E2F-4 alterations on transcriptional activation and proliferation in transfected cells

H Takashima, Y Matsumoto, N Matsubara, Y Shirakawa, R Kawashima, M Tanino, S Ito, H Isozaki, M Ouchida, SJ Meltzer, K Shimizu, N Tanaka

LABORATORY INVESTIGATION 81 ( 11 ) 1565 - 1573 2001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：LIPPINCOTT WILLIAMS & WILKINS

E2F is a family of transcription factors implicated in the regulation of gene expression required for progression through the G(1)-S transition. We have previously detected tumor-specific mutations at a trinucleotide repeat coding sequence of E2F-4 gene in a subset of human sporadic colorectal cancers. The purpose of this study was to investigate the potential functional consequences of these E2F-4 mutations. We transfected NIH3T3 fibroblasts with expression constructs containing wild-type as well as mutant E2F-4 cDNA, and the effect of the E2F-4 mutations on proliferation was examined. Alteration in transactivation of the E2F consensus promoter sequence was also examined by transient cotransfection of a E2F-4 with a DP-2 construct into cultured human cells. Transfected cell clones overexpressing mutant E2F-4 grew more rapidly and showed higher proliferative activity by increased immunohistochemical staining for proliferating cell nuclear antigen (PCNA). All three mutant forms of E2F-4 showed elevated transactivation of the E2F consensus promoter sequence. Thus, expression of mutant E2F-4s confers a growth advantage in vivo, and this effect may be related to the acquisition of a neoplastic phenotype.

A novel WD40 repeat protein, WDC146, highly expressed during spermatogenesis in a stage-specific manner

S Ito, A Sakai, T Nomura, Y Miki, M Ouchida, J Sasaki, K Shimizu

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 280 ( 3 ) 656 - 663 2001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：ACADEMIC PRESS INC ELSEVIER SCIENCE

We have cloned a novel cDNA encoding a protein with eight WD repeat motifs and a domain similar to collagen. As the predicted size of the protein was 146 kDa, the gene was named WDC146. Here, we characterized the genomic structure, gene products, and the expression profiles. The human WDC146 gene had 22 exons spanning over 105 kb, and these exons were distributed in three islands intervened by two long introns of around 40 kb. A minimum promoter region was identified within a 0.5 kb 5'-upstream region of exon 1. WDC146 mRNA was most highly expressed in human testis on Northern blot analysis. In mouse tissues, the highest expression was also observed in testis. By in situ hybridization on rat tissues, WDC146 mRNA was detected preferentially in the pachytene stage of spermatocytes in testis, and weakly in white pulp/marginal band of spleen and in cortex of thymus. WDC146 protein was found to be localized in nucleus. These data implied that WDC146 protein may play important roles in the mechanisms of cytodifferentiation and/or DNA recombination. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2000.4163

HD-PTP: A novel protein tyrosine phosphatase gene on human chromosome 3p21.3

S Toyooka, M Ouchida, Y Jitsumori, K Tsukuda, A Sakai, A Nakamura, N Shimizu, K Shimizu

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 278 ( 3 ) 671 - 678 2000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：ACADEMIC PRESS INC

A human cDNA encoding a novel protein tyrosine phosphatase has been isolated. The phosphatase has unique features in its domain structure: a "Zn-hand" domain containing several SH3-binding motifs, a tyrosine phosphatase domain, a C-terminal PEST motif, and an N-terminal domain similar to yeast BRO1, an apoptosis-related mammalian AIP1 and to a RHO-binding protein, Rhophilin. The gene is located at chromosome 3p21.3, an area frequently deleted in many types of cancer, especially within the functionally defined narrow region. The gene may be a human homolog of the rat PTP-TD14 gene reported by others, which can suppress H-ras-mediated transformation. We identified a hemizygous missense mutation in a lung cancer cell line. Thus, the phosphatase gene may be a candidate for one of the tumor suppressor genes located on 3P21.3. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3870

A reverse transcriptase-polymerase chain reaction assay in the diagnosis of soft tissue sarcomas

N Naito, A Kawai, M Ouchida, T Dan'ura, Y Morimoto, T Ozaki, K Shimizu, H Inoue

CANCER 89 ( 9 ) 1992 - 1998 2000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：WILEY-LISS

BACKGROUND. Many types of sarcomas are characterized by specific chromosomal translocations that result in the production of novel chimeric genes. Detection of these fusion genes could be a sensitive molecular diagnostic assay. However, to the authors' knowledge there have been few systemic comparisons between the current histopathologic diagnosis and the presence or absence of particular fusion genes in patients with adult soft tissue sarcomas (STSs).
METHODS. Total RNA was extracted from 75 cases of STS and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of a variety of fusion transcripts. The results of the molecular assay were compared with standard histopathologic diagnoses.
RESULTS. Of the 18 tumors diagnosed as synovial sarcoma, 17 (94%) expressed SYT-SSX chimeric transcripts. AU nine myxoid liposarcomas were positive for FUS-CHOP fusion transcripts. Of the four cases of Ewing sarcoma, two had an EWS-FLI1 fusion transcript and one had an EWS-ERG fusion transcript. A clear cell sarcoma had a EWS-ATF1 fusion transcript. None of 19 cases of malignant fibrous histiocytoma nor 3 leiomposarcomas contained a fusion transcript. Three cases with an initial diagnosis other than synovial sarcoma expressed a SYT-SSX fusion transcript. A review of the slides and additional examination showed that a diagnosis of synovial sarcoma was appropriate for these cases. There was a trend for biphasic synovial sarcoma to contain the SYT-SSX1 fusion.
CONCLUSIONS, The authors believe RT-PCR assay for the detection of a specific fusion gene provides a useful tool for confirmation of the diagnosis of adult STS in diagnostically difficult cases and in retrospective studies. (C) 2000 American Cancer Society.

DOI： 10.1002/1097-0142(20001101)89:9<1992::AID-CNCR16>3.0.CO;2-R

グリオーマの悪性度と10番染色体遺伝子異常との関連性に関する検討

大同茂, 寺田欣矢, 神原啓和, 小野恭裕, 田宮隆, 松本健五, 大本尭史, 伊藤佐智夫, 大内田守, 清水憲二

日本脳神経外科学会総会抄録集 59回 221 - 221 2000年10月

Genomic structure of the human ING1 gene and tumor-specific mutations detected in head and neck squamous cell carcinomas

M Gunduz, M Ouchida, K Fukushima, H Hanafusa, T Etani, S Nishioka, K Nishizaki, K Shimizu

CANCER RESEARCH 60 ( 12 ) 3143 - 3146 2000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：AMER ASSOC CANCER RESEARCH

We characterized the genomic structure of the human ING1 gene, a candidate tumor suppressor gene, and found that the gene has three exons, We also demonstrated that four mRNA variants Here transcribed from three different promoter regions. Of 34 informative cases of head and neck squamous cell carcinoma, 68% of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located, Here we present the first report that three missense mutations and three silent changes were detected in the ING1 gene in 6 of 23 tumors with allelic loss at the 13q33-34 region. These missense mutations were found within the PHD finger domain and nuclear localization motif in ING1 protein, probably abrogating the normal function.

Functional mutation of DNA polymerase β found in human gastric cancer - Inability of the base excision repair in vitro

Akinari Iwanaga, Mamoru Ouchida, Kohji Miyazaki, Katsuji Hori, Tsunehiro Mukai

Mutation Research - DNA Repair 435 ( 2 ) 121 - 128 1999年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

DNA polymerase β (polβ) is one of mammalian DNA polymerases and is known to be involved in a G:T/G:U mismatch repair. In order to investigate an involvement of this enzyme in a base excision repair, we searched a mutation of human polβ in human gastric cancer and studied a function of the mutation. We observed cancer-specific missense mutations in 6 of 20 samples. All of these mutations were, however, heterozygous. We further analyzed the base excision repair activity of these mutants to know whether these mutants cause an error of mismatch repair. One of these mutants, which resulted in an amino acid substitution of Glu for Lys at codon 295, showed an inhibitory effect by in vitro base excision repair assay, suggesting that this mutation might play some role in carcinogenesis of the gastric mucosa.

DOI： 10.1016/S0921-8777(99)00036-1

Osteoclast-derived zinc finger (OCZF) protein with POZ domain, a possible transcriptional repressor, is involved in osteoclastogenesis 査読

A Kukita, T Kukita, M Ouchida, H Maeda, H Yatsuki, O Kohashi

BLOOD 94 ( 6 ) 1987 - 1997 1999年9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 SOC HEMATOLOGY

The differentiation of osteoclasts is regulated by transcription factors expressed in cells of osteoclast lineage. We isolated here a potential transcription factor from a cDNA library of an enriched population of preosteoclasts and osteoclasts, The cDNA encodes a protein with hi-terminal POZ domain and C-terminal Kruppel-like zinc fingers, We designate this protein as osteoclast-derived zinc finger (OCZF), OCZF was found to be rat homologue of mouse leukemia/lymphoma-related factor (LRF), Northern blot and in situ hybridization analysis showed OCZF mRNA at a high level in osteoclasts and kidney cells. OCZF had a nuclear targeting sequence and was localized in the nucleus of transfected cells. In addition, OCZF specifically bound to the guanine-rich consensus sequences of Egr-1 and c-Krox. Transient transfection assays indicate that OCZF can repress transcription activity like other POZ domain proteins. Furthermore, antisense but not sense phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA suppressed the formation of osteoclast-like multinucleated cells (MNCs) in bone marrow culture, whereas the same ODNs did not significantly affect the formation of macrophage polykaryons and mononuclear preosteoclast-like cells (POCs), These results suggest that OCZF is a unique transcription factor that plays an important role in the late stage of osteoclastogenesis, (C) 1999 by The American Society of Hematology.

Osteoclast-derived zinc finger (OCZF) protein with POZ domain, a possible transcriptional regulator, is involved in osteoclastogenesis.

Kukita A, Kukita T, Maeda H, Ouchida M, Yatsuki H, Kohashi O

Blood 1999年

Transforming activity of the RL-akt gene, a c-akt gene activated by long terminal repeat insertion in murine leukemia RL♂1 cells

Motohiko Tanino, Mitsutoshi Matsuo, Akiko Uenaka, Kazunori Tsukuda, Mamoru Ouchida, Eiichi Nakayama, Kenji Shimizu

Molecular Carcinogenesis 26 ( 4 ) 286 - 297 1999年

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 Inc.

The unique antigen peptide pRL1 on BALB/c radiation-induced leukemia RL♂1 cells is derived from the normally untranslated 5' region of the mouse c-akt gene. Insertion of an endogenous long terminal repeat into the first coding exon of the gene resulted in the enhanced production of an altered akt protein, RL-akt, and creation of the tumor rejection antigen peptide pRL1. In this study, we constructed an RL-akt-expressing vector to investigate the transforming ability and anti-apoptotic activity of RK-akt in NIH/3T3 cells. RL-akt-expressing clones formed more colonies than did c-akt-expressing clones in soft agar and exhibited increased saturation density, a lower serum requirement for growth, and tumorigenicity on athymic nude mice. Immunoblot analysis of subcellular protein distribution showed that a considerable proportion of RL-akt was distributed in the membrane fraction. Thus, RL-akt expressed in NIH/3T3 cells appeared to behave like the v-akt oncoprotein. Furthermore, the RL-akt gene conferred resistance to the apoptosis induced by the calcium ionophore A23187 and by ultraviolet irradiation of NIH/3T3 cells. These findings indicate that the RL-akt gene is able to transform cells and exerts an anti-apoptotic effect on recipient cells, thereby implicating the gene in leukemogenesis of RL♂1 cells.

DOI： 10.1002/(SICI)1098-2744(199912)26:4<286::AID-MC7>3.0.CO;2-U

Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells

Hitomi Yatsuki, Mamoru Outida, Yasuo Atsuchi, Tunehiro Mukai, Koichiro Shiokawa, Katsuji Hori

Biochimica et Biophysica Acta - Gene Structure and Expression 1442 ( 2-3 ) 199 - 217 1998年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

A Xenopus aldolase C gene (XACλ3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA- like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.

DOI： 10.1016/S0167-4781(98)00165-1

Inhibition of apoptosis by normal and aberrant Fli-1 and erg proteins involved in human solid tumors and leukemias. 国際誌

H Yi, Y Fujimura, M Ouchida, D D Prasad, V N Rao, E S Reddy

Oncogene 14 ( 11 ) 1259 - 68 1997年3月

記述言語：英語掲載種別：研究論文（学術雑誌）

Two ets family members, namely erg and Fli-1 are fused with two EWS family members namely EWS and TLS/FUS as a result of chromosome translocation in human solid tumors and leukemias. EWS-erg and EWS-Fli-1, which are involved in greater than 95% of Ewing family of tumors, were shown to function as transcriptional activators. TLS/FUS-erg, which is involved in human myeloid leukemias also functions as a transcriptional activator. Expression of these fusion proteins (EWS-erg and EWS-Fli-1) are shown to be essential for maintaining the oncogenic and tumorigenic properties of tumor cells. Cancer is thought to be caused not only by uncontrolled cell proliferation but also by deregulation of programmed cell death. Therefore, we have studied the role of normal (Fli-1 and erg) and aberrant fusion proteins (EWS-erg, EWS-Fli-1 and TLS/FUS-erg) in apoptosis. We have found that expression of normal (Fli-1 and erg) and aberrant fusion proteins inhibit the apoptosis of NIH3T3 cells induced by either serum deprivation or by treatment with calcium ionophore. We have also observed similar suppression of apoptosis in Ewing's sarcoma cells expressing EWS-Fli-1 and EWS-erg proteins suggesting that these fusion proteins may be responsible for the decreased ability of these tumor cells to undergo apoptosis. Inhibition of the expression of these aberrant fusion proteins by antisense RNA technique resulted in increased susceptibility to apoptosis leading to the death of tumor cells. Therefore, our results suggest that one can use therapeutic agents which can down regulate the expression of fusion proteins in combination with chemotherapeutic agents as an effective treatment for these human solid tumors and leukemias.

DOI： 10.1038/sj.onc.1201099

Telomerase activity in gynecologic tumors

Peng-Sheng Zheng, Tsuyoshi Iwasaka, Fumio Yamasaki, Mamoru Ouchida, Masatoshi Yokoyama, Yoshifumi Nakao, Koichi Fukuda, Toshitake Matsuyama, Hajime Sugimori

Gynecologic Oncology 64 ( 1 ) 171 - 175 1997年

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 Inc.

The standard telomeric repeat assay protocol (TRAP) was used to examine telomerase activity in 16 ovarian tumors, 16 cervical carcinomas, 4 uterine tumors, and 3 vaginal tumors. Telomerase activity was detected in 95% of these tumors, 88% of ovarian malignancies, and 100% of cervical, endometrial, and vaginal malignancies. In contrast, telomerase activity was not evident in normal tissues or in benign proliferative lesions, such as leiomyomas, condyloma acuminata, and simple endometrial hyperplasia. These results suggest that telomerase activation is associated with immortalization or malignant transformation of gynecologic tumors.

DOI： 10.1006/gyno.1996.4523

Growth suppression of a cervical cancer cell line (TMCC-1) by the human wild-type p53 gene

Peng-Sheng Zheng, Tsuyoshi Iwasaka, Mamoru Ouchida, Koichi Fukuda, Masatoshi Yokoyama, Hajime Sugimori

Gynecologic Oncology 60 ( 2 ) 245 - 250 1996年

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 Inc.

To investigate the effects of human wild-type p53 expression on the proliferation of cervical carcinoma cells, a plasmid, pMO7-hp53, which contains a full-length cDNA of the human wild-type p53 (wt-p53) gene, was transfected into a cell line (TMCC-1) derived from an endocervical type, human papilloma virus-positive adenocarcinoma of the uterine cervix. The exogenous wt-p53 expression induced growth suppression, morphological changes, and loss of anchorage-independent growth of the tumor cells. As the wt-p53 gene apparently plays a negative role in growth regulation of cervical carcinoma cells, this gene may possibly be of some use for treating subjects with a cervical carcinoma.

DOI： 10.1006/gyno.1996.0033

Cytological Changes in Two Cervical carcinoma cell lines after Transfection of The Wild Type p53 Gene : 査読

Iwasaka T, Zheng PS, Ouchida M, Yamasaki H, Yokoyama M, Sugimori H

Acta Obset. Gynecol. Scand. 75 ( 9 ) 797 - 803 1996年

Cytologic changes in two cervical carcinoma cell lines after transfection of the wild-type p53 gene

Tsuyoshi Iwasaka, Peng-Sheng Zheng, Mamoru Ouchida, Humio Yamasaki, Masatoshi Yokoyama, Hajime Sugimori

Acta Obstetricia et Gynecologica Scandinavica 75 ( 9 ) 797 - 803 1996年

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-Blackwell Publishing Ltd

Background. To examine morphological changes in cervical carcinoma cells after induction of overexpression of wild-type (wt) p53. Method. A dexamethazone-inducible wt-p53 cDNA was introduced into two cervical carcinoma cell lines (TMCC-1 and ME180) and morphological changes were examined under a phase contrast microscope and following Papanicolaou staining. Results. TMCC-1 clones obtained by transfection with wt-p53 gene had an altered morphology especially after induction of p53 expression by treatment with dexamethazone. These changes were characterized by cell flattening, multinucleation, micronucleation, and huge giant cells, yet the parental TMCC-1 had none of these morphological changes even after treatment with dexamethazone. Similar cellular changes were observed in ME180 clones obtained by transfection of wt-p53 gene. Conclusion. On the basis of our observations, we propose that the growth inhibition induced by expression of wt-p53 in these cervical carcinoma cell lines carrying HPV DNA sequences is not the result of G1 arrest but rather relates to a blockade in the M phase of the cell cycle.

DOI： 10.3109/00016349609054706

Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. 国際誌

M Ouchida, T Ohno, Y Fujimura, V N Rao, E S Reddy

Oncogene 11 ( 6 ) 1049 - 54 1995年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Cytogenetic analysis of Ewing's sarcoma, primitive neuroectodermal tumors and Askin tumors revealed characteristic translocations t(11;22) or t(21;22). Molecular analysis of these translocations revealed 5'-region of EWS gene (from band 22q12) is fused to the 3'-region of either Fli-1 gene (from band 11q24) or erg gene (from band 21q22). Functional characterization of the EWS-Fli-1 and EWS-erg chimeric proteins suggested that they function as transcriptional activators. In order to develop therapeutic agents, it is essential to know whether expression of the EWS-fusion gene products is coupled to tumorigenicity of Ewing's sarcoma cells and if targeting the EWS-fusion products results in loss of tumorigenicity of Ewing's sarcoma cells. For this reason, we have made stable Ewing's sarcomas expressing antisense EWS-Fli-1 or EWS-erg expression plasmids. Expression of antisense EWS fusion transcripts resulted in a significant loss of endogenous EWS-Fli-1 and EWS-erg proteins in Ewing's sarcoma cells. These cells expressing antisense EWS fusion transcripts showed loss of anchorage independent growth and tumorigenicity in nude mice unlike the parental Ewing's sarcoma cells. These results demonstrate the necessity of a certain threshold level of expression of EWS-fusion products in the clonogenicity and tumorigenicity of Ewing's sarcoma cells and therefore emphasizes the importance of targeting the EWS-fusion products as a therapy for Ewing family of tumors.

TLS/FUS fusion domain of TLS/FUS-erg chimeric protein resulting from the t(16;21) chromosomal translocation in human myeloid leukemia functions as a transcriptional activation domain. 国際誌

D D Prasad, M Ouchida, L Lee, V N Rao, E S Reddy

Oncogene 9 ( 12 ) 3717 - 29 1994年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

EWS and TLS/FUS genes, which code for RNA binding proteins are involved in a wide variety of human solid tumors. The TLS/FUS gene is involved both in human myxoid liposarcomas which carry a characteristic chromosomal translocation, t(12;16)(q13;p11) and in human myeloid leukemias with recurrent chromosomal translocation, t(16;21)(p11:q22). The TLS/FUS gene is fused to a transcriptional repressor, CHOP (in human myxoid liposarcomas) or transcriptional activator, erg (in human myeloid leukemias). To understand better the functional role of TLS/FUS-erg in human myeloid leukemias, we have cloned the TLS/FUS and TLS/FUS-erg cDNAs and studied the functional properties of their gene products. TLS/FUS protein binds to RNA in vitro and shows preferential binding to poly G. Both the amino- and the carboxy- terminal regions of TLS/FUS containing the conserved RNA binding motifs are needed for poly G specific RNA binding activity. The TLS/FUS fusion domain (TFD) appears to regulate the DNA binding activity of TLS/FUS-erg chimeric protein which shows weaker transcriptional activation properties compared to normal erg proteins. Mutational analysis of the TLS/FUS-erg chimeric protein reveals TFD to function as a transcriptional activation domain thus replacing the amino terminal transcriptional activation domain of the erg protein. Therefore alterations in both DNA binding and transcriptional activation properties of aberrant erg proteins may be responsible for the genesis of t(16;21) chromosomal translocation-bearing human myeloid leukemias.

THE EWS GENE, INVOLVED IN EWING FAMILY OF TUMORS, MALIGNANT-MELANOMA OF SOFT PARTS AND DESMOPLASTIC SMALL ROUND-CELL TUMORS, CODES FOR AN RNA-BINDING PROTEIN WITH NOVEL REGULATORY DOMAINS

T OHNO, M OUCHIDA, L LEE, Z GATALICA, VN RAO, ESP REDDY

ONCOGENE 9 ( 10 ) 3087 - 3097 1994年10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：STOCKTON PRESS

The EWS gene, which maps to band q12 of human chromosome 22, is involved in a wide variety of human solid tumors including Ewing sarcoma, related primitive neuroectodermal tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors. In these tumors, the EWS is fused to genes encoding transcriptional activators/repressors, like Fli-1 or erg or ATF 1 or wt1. To better understand the function of the EWS protein, we cloned the EWS cDNA. Sequence analysis of this cDNA revealed differential splicing invoving two exons encoding 72 amino acids. Both alternatively spliced transcripts, EWS and EWS-b, are expressed in a variety of cells. Because EWS proteins contain putative conserved RNA binding motifs, we studied the RNA binding properties of the EWS protein. The EWS-b protein binds to RNA in vitro and, specifically, to poly G and poly U. The RNA binding activity was localized to the carboxy terminal 86 amino acids, which constitute RGG box. Thus the amino terminal domain of EWS (NTD-EWS), which is involved in chromosome translocation may regulate the specificity of RNA binding activity of EWS. An EWS-erg chimeric protein, which is found in Ewing's sarcoma cells, functions as a transcriptional activator. Mutational analysis of EWS-erg chimeric protein revealed that NTD-EWS functions as a regulatory domain for the transcriptional activation properties of EWS-erg chimeric protein.

PCR法によるＡＢＯ式血液型（糖転移酵素の遺伝子型判定).

袖崎賢一郎, 的場梁次, 大内田守

法医学の実際と研究 36 33 - 37 1993年

Correlation between HPV Positivity and state of the p53 gene in cervical Carcinoma cell lines

Gynecologic oncology 48, 104-109 1993年

Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-ras oncogenes in invasive cervical carcinomas

Tsuyoshi Iwasaka, Masatoshi Yokoyama, Mamoru Oh-uchida, Norito Matsuo, Kouichi Hara, Kouzou Fukuyama, Toru Hachisuga, Kouichi Fukuda, Hajime Sugimori

Gynecologic Oncology 46 ( 3 ) 298 - 303 1992年

記述言語：英語掲載種別：研究論文（学術雑誌）

Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression. © 1992.

DOI： 10.1016/0090-8258(92)90220-D

Altered Structure and Expression of the p 53 Gene in Human Neuroepithelial Tumors

Tabuchi Kazuo, Fukuyama Kouzou, Mineta Toshihiro, Mamoru Oh-Uchida, Hori Katsuji

neurologia medico-chirurgica 32 ( 10 ) 725 - 732 1992年

記述言語：英語掲載種別：研究論文（学術雑誌）

The p53 gene, located on chromosome 17p13.1, may be important in the pathogenesis of human neuroepithelial tumors, because it is a tumor suppressor gene and genetic alteration is essential for cer tain human cells to acquire the neoplastic phenotype. The structure and expression of the p53 gene were investigated in cultured human glioma cells and biopsied specimens of neuroepithelial tumors. Immunocytochemical examination of p53 gene expression revealed positive nuclear staining in six of seven glioma cell lines tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis demonstrated unequivocal heterogeneity of migration rate in p53 bands. Pulse-chase analysis clearly showed an increased half-life of p53 in cultured human glioma cells. These abnormalities are presumably due to genetic alterations in the p53 gene. Nucleotide substitutions in exon 5, 7, or 8 of the p53 gene could be detected by polymerase chain reaction-single strand conformational polymorphic analysis in four of seven (57%) human glioma cell lines, and nine of 29 (31%) biopsied specimens of neuroepithelial tumors examined. The present results indicate that genetic alterations in the p53 gene are responsible for the tumorigenesis of at least some human neuroepithelial tumors. © 1992, The Japan Neurosurgical Society. All rights reserved.

DOI： 10.2176/nmc.32.725

Suppression of anchorage-independent growth of human glioblastoma cell by major histocompativility complex class I gene-Transfection.

Momozaki N, Oh-uchida M, Tabuchi K, Ikezaki K, Hori K

journal of Neurosurgery 76 845 - 849 1992年

ヒト神経膠腫における癌抑制遺伝子の単離と遺伝子および遺伝子産物の分子生物学的解析

大内田守

佐川がん研究助成振興財団研究報告集 2 13 1991年

Transformation‐specific Decrease of Phosphorylation of 80K Protein, a Substrate of Protein Kinase C, in NIH3T3 Cells

Mamoru Oh‐uchida, Kazumi Yano, Shoko Kawamoto, Kenji Shimizu

Japanese Journal of Cancer Research 81 ( 8 ) 799 - 806 1990年

記述言語：英語掲載種別：研究論文（学術雑誌）

Phosphorylation in normal and transformed NIH3T3 cells of the 80K protein, a specific substrate for protein kinase C, was compared by means of two‐dimensional gel analysis. We obtained evidence that NIH3T3 cells transformed by the c‐raf or H‐ras oncogene maintained a decreased level of phosphorylation of the 80K protein, with or without phorbol ester (TPA)‐stimulation, at all concentrations of serum tested while normal NIH3T3 cells maintained an elevated level of phosphorylation of the 80K protein. Furthermore, NIH3T3 cells transformed by N‐ras, K‐ras, src, mos or polyoma middle T antigen exhibited a decreased level of phosphorylation of the 80K protein. These events were confirmed by an analysis of a hormone‐inducible H‐ras transformant. Thus, phosphorylation of the 80K protein is inversely correlated with cellular transformation. Copyright © 1990, Wiley Blackwell. All rights reserved

DOI： 10.1111/j.1349-7006.1990.tb02648.x

グリオーマにおける癌抑制遺伝子の単離及びその解析

大内田守

日本脳神経財団報告書 6 73 1990年

Changes in the patterns of RNA synthesis in early embryogenesis of Xenopus laevis. 査読

Shiokawa K, Misumi Y, Tashiro K, Nakakura N, Yamana K, Oh-uchida M

Cell Differ Dev. 28 ( 1 ) 17 - 25 1989年

Activated c-raf-1 gene from human stomach cancer

Shimizu K, Nakatsu Y, Oh-uchida M, Nomoto S, Sekiguchi M

Gan To Kagaku Ryoho. 14 ( 6 ) 2140 - 2146 1987年6月

We previously isolated a novel human transforming sequence from a primary stomach cancer and identified the gene as an activated version of the c-raf-1 gene which is the human homologue of v-raf, a viral oncogene encoding a serine/threonine-specific protein kinase. Of 57 kbp of the human sequence isolated, a region of 40 kbp was found to be the minimum functional unit for the transforming activity, because a cosmid clone harboring this region was capable of inducing foci upon transfection. The size of the transcript of the transforming c-raf-1 gene was estimated to be about 2.8kb. Analyses of cDNA clones of this gene revealed that the gene was generated by substitution of the 5'-sequence (exons 1-5) of the normal c-raf-1 gene with an unrelated human sequence. We identified a region in the genomic clone where the rearrangement had occurred. The rearranged EcoRI fragment was detected in all the primary transformants obtained from two independent transfections, suggesting that the recombination had occurred in the primary cancer. The substituted cDNA sequence is composed of an open reading frame, which joins to exon 6 of the c-raf-1 gene in an in-phase manner. The substituted open reading frame encodes an extremely hydrophobic polypeptide. Thus, the putative product of the transforming gene seems to have a hydrophobic stretch ahead of the ser/thr-protein kinase domain of the c-raf-1 gene product. These results suggest that the truncation or replacement of the amino-terminal domain of the c-raf-1 protein leads to constitutive activation of the protein kinase residing in the downstream domain.

Structure of the Activated C-raf-1 gene from Human Stomach Cancer

Nakatsu Y, Nomoto S, Oh-uchida M, Shimizu K, Sekiguchi M

Cold Spring Harbor Symposia on Quantitative Biology 51 1986年

S III-2. Detection of human transforming genes using DNA-transfection

K. Shimizu, Y. Nakatsu, M. Ohuchida, M. Sekiguchi

Japanese Journal of Human Genetics 30 ( 2 ) 93 - 94 1985年

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