Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Dental Medicine　Division of Dental Medicine　Department of Pathobiological Science

Affiliation (Master)

• Faculty of Dental Medicine　Division of Dental Medicine　Department of Pathobiological Science

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Profile and Settings

Profile and Settings

• Name (Japanese)

  Yasuda

• Name (Kana)

  Motoaki

• Name

  200901082577304496

Achievement

Research Interests

• ウイルス学   分子生物学   molecular virology   molecular biology   

Research Areas

• Life sciences / Virology
• Life sciences / Molecular biology

Research Experience

• 2002 - 北海道大学大学院歯学研究科口腔分子微生物学教室 助教授
• 2002 - Associate Professor

Published Papers

• Adenovirus infection controls processing bodies to stabilize AU-rich element-containing mRNA.

  Takeshi Kuroshima, Aya Yanagawa Matsuda, Elora Hossain, Motoaki Yasuda, Tetsuya Kitamura, Yoshimasa Kitagawa, Fumihiro Higashino

  Virology 573 124 - 130 2022/08 

  In the adenovirus-infected cells, virus mRNAs are selectively exported to the cytoplasm by virus early gene products to facilitate virus replication. We previously showed AU-rich elements (AREs) containing mRNAs are exported to the cytoplasm and stabilized in infected cells. Here, we analyzed ribonucleoprotein (RNP) granules in the cytoplasm that are involved in mRNA degradation to elucidate the mechanism of ARE-mRNA stabilization in adenovirus infected cells. Our findings showed that processing bodies (PBs) aggregate, then almost all PBs are translocated to aggresomes formed by adenoviral gene products during the late phase of infection. Furthermore, E4orf3 was required for the PBs translocation, and the same domains of E4orf3-mutants required to change the form of promyelocytic leukemia bodies were also needed for PBs translocation. Luciferase activity showed that these domains were critical for miRNA- and ARE-mediated mRNA decay. These findings suggest that adenovirus changes the behavior of PBs to prevent ARE-mRNA downregulation.
• Mammaglobin 1 mediates progression of trastuzumab-resistant breast cancer cells through regulation of cyclins and NF-κB.

  Ratih Kusumastuti, Yuji Kumagai, Seiichiro Ishihara, Atsushi Enomoto, Takashi Murakami, Motoaki Yasuda, Hisashi Haga

  FEBS open bio 12 (10) 1797 - 1813 2022/07/29 

  Overexpression of human epidermal growth factor receptor 2 (HER2) in various cancers is correlated with poor patient survival. Trastuzumab, a recombinant humanized monoclonal antibody against HER2, has been considered to be a first-line therapy for HER2-positive breast cancer patients, but its usefulness is limited by the development of resistance. In this study, we established resistant cells by long-term treatment with trastuzumab. These cells showed higher proliferation, invasion, and migration abilities than the wild-type cells. Mammaglobin 1 (MGB1), cyclin D1, E1, A2, and phosphorylated NF-κB (p-p65) were upregulated in resistant cells. These proteins regulate cell proliferation, migration, and invasion of resistant cells. Depletion of MGB1 decreased cyclin and p-p65 expression. Cyclin D1 and A2, but not E1 expression, were affected by p-p65 downregulation. In summary, our results indicate that MGB1 expression is increased in breast cancer cells that have gained resistance to trastuzumab, and suggest that MGB1 promotes aggressiveness through cyclin and NF-κB regulation.
• Advantages of Using Paclitaxel in Combination with Oncolytic Adenovirus Utilizing RNA Destabilization Mechanism.

  Elora Hossain, Umma Habiba, Aya Yanagawa-Matsuda, Arefin Alam, Ishraque Ahmed, Mohammad Towfik Alam, Motoaki Yasuda, Fumihiro Higashino

  Cancers 12 (5) 2020/05/12 

  Oncolytic virotherapy is a novel approach to cancer therapy. Ad-fosARE is a conditionally replicative adenovirus engineered by inserting AU-rich elements (ARE) in the 3'-untranslated region of the E1A gene. In this study, we examined the oncolytic activity of Ad-fosARE and used it in a synergistic combination with the chemotherapeutic agent paclitaxel (PTX) for treating cancer cells. The expression of E1A was high in cancer cells due to stabilized E1A-ARE mRNA. As a result, the efficiency of its replication and cytolytic activity in cancer cells was higher than in normal cells. PTX treatment increased the cytoplasmic HuR relocalization in cancer cells, enhanced viral replication through elevated E1A expression, and upregulated CAR (Coxsackie-adenovirus receptor) required for viral uptake. Furthermore, PTX altered the instability of microtubules by acetylation and detyrosination, which is essential for viral internalization and trafficking to the nucleus. These results indicate that PTX can provide multiple advantages to the efficacy of Ad-fosARE both in vitro and in vivo, and provides a basis for designing novel clinical trials. Thus, this virus has a lot of benefits that are not found in other oncolytic viruses. The virus also has the potential for treating PXT-resistant cancers.
• Conditionally Replicative Adenovirus Controlled by the Stabilization System of AU-Rich Elements Containing mRNA.

  Yohei Mikawa, Mohammad Towfik Alam, Elora Hossain, Aya Yanagawa-Matsuda, Tetsuya Kitamura, Motoaki Yasuda, Umma Habiba, Ishraque Ahmed, Yoshimasa Kitagawa, Masanobu Shindoh, Fumihiro Higashino

  Cancers 12 (5) 2020/05/11 

  AU-rich elements (AREs) are RNA elements that enhance the rapid decay of mRNAs, including those of genes required for cell growth and proliferation. HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, is involved in the stabilization of ARE-mRNA. The level of HuR in the cytoplasm is up-regulated in most cancer cells, resulting in the stabilization of ARE-mRNA. We developed the adenoviruses AdARET and AdAREF, which include the ARE of TNF-α and c-fos genes in the 3'-untranslated regions of the E1A gene, respectively. The expression of the E1A protein was higher in cancer cells than in normal cells, and virus production and cytolytic activities were also higher in many types of cancer cells. The inhibition of ARE-mRNA stabilization resulted in a reduction in viral replication, demonstrating that the stabilization system was required for production of the virus. The growth of human tumors that formed in nude mice was inhibited by an intratumoral injection of AdARET and AdAREF. These results indicate that these viruses have potential as oncolytic adenoviruses in the vast majority of cancers in which ARE-mRNA is stabilized.
• The neural ELAVL protein HuB enhances endogenous proto-oncogene activation.

  Tomoyuki Hatanaka, Fumihiro Higashino, Kanchu Tei, Motoaki Yasuda

  Biochemical and biophysical research communications 517 (2) 330 - 337 2019/09/17 

  The cytoplasmic distribution of the HuR/ELAVL1 (embryonic lethal abnormal vision 1) protein is recognized as an important prognostic factor of malignant tumors. However, the previous study suggests that exogenous over-expression of HuR is not sufficient for nuclear export. Conversely, the predominantly cytosolic distribution of neuron-specific human ELAV members, including HuB/ELAVL2, HuC/ELAVL3, and HuD/ELAVL4, has been reported. In the present study, we demonstrated the expression of HuB in several types of cancer cells, but expression of HuC and HuD was not observed. In addition, our results indicated that HuR and HuB formed a complex in the cytosolic fraction of cancer cells via the RRM3 region. Ectopic expression of HuB was capable of initiating the cytosolic translocation of HuR from the nucleus to the cytosol. Furthermore, HuB-transduced cancer cells displayed significant nuclear export of HuR, with quantitative PCR experiments revealing the simultaneous upregulation of HIF-1α, c-Fos, c-MYC, and Ets2 basal mRNA expression. Phorbol 12-myristate 13-acetate (PMA)-stimulated HuB-transduced cells demonstrated significantly enhanced activation of endogenous c-Fos and CREB dependent cascades. Finally, co-transfection of HuB with the E1 region of type 5 human adenovirus significantly enhanced E1 transformation activities but that of HuR with the E1 region did not. Collectively, our findings suggest that the neural Hu family protein HuB plays a major role in the activation of memory-related proto-oncogenes.
• Oncolytic potential of an E4-deficient adenovirus that can recognize the stabilization of AU-rich element containing mRNA in cancer cells.

  Aya Yanagawa-Matsuda, Yohei Mikawa, Umma Habiba, Tetsuya Kitamura, Motoaki Yasuda, Mohammad Towfik-Alam, Yoshimasa Kitagawa, Kazuyuki Minowa, Masanobu Shindoh, Fumihiro Higashino

  Oncology reports 41 (2) 954 - 960 2019/02 

  AU-rich elements (AREs) are RNA elements that enhance the rapid decay of mRNA. The fate of ARE-mRNA is controlled by ARE-binding proteins. HuR, a member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, is involved in the export and stabilization of ARE-mRNA. In the vast majority of cancer cells, HuR constitutively relocates to the cytoplasm, resulting in the stabilization of ARE-mRNA. Previously, we described that the adenovirus gene product, E4orf6, which is necessary for virus replication, participates in ARE-mRNA export and stabilization. In the present study, we showed the oncolytic potential of E4orf6-deleted adenovirus dl355, which is expected to be replicated selectively in cancer cells. Virus production and cytolytic activity of dl355 were higher in cancer cells than in normal cells. HuR-depletion downregulated dl355 replication, demonstrating that ARE-mRNA stabilization is required for the production of this virus. Tumor growth was inhibited in nude mice by an intratumoral injection of dl355. Furthermore, dl355 had a stronger oncolytic effect than E1B55k-deleted adenovirus. These results indicate that dl355 has potential as an oncolytic adenovirus for a large number of cancers where ARE-mRNA is stabilized.
• Adenovirus infection induces HuR relocalization to facilitate virus replication.

  Jumond P Jehung, Tetsuya Kitamura, Aya Yanagawa-Matsuda, Takeshi Kuroshima, Alam Towfik, Motoaki Yasuda, Hidehiko Sano, Yoshimasa Kitagawa, Kazuyuki Minowa, Masanobu Shindoh, Fumihiro Higashino

  Biochemical and biophysical research communications 495 (2) 1795 - 1800 2018/01/08 

  HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3'-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.
• Role of ATF5 in the invasive potential of diverse human cancer cell lines

  Akihiro Nukuda, Hiroki Endoh, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 474 (3) 509 - 514 0006-291X 2016/06 [Refereed][Not invited]
   

  Activating transcription factor 5 (ATF5) is a member of the ATF/CAMP response element-binding protein family. Our research group recently revealed that ATF5 expression increases the invasiveness of human lung carcinoma cells. However, the effects of ATF5 on the invasive potential of other cancer cells lines remain unclear. Therefore, in this study, we investigated the role of ATF5 in the invasive activity of diverse human cancer cell lines. Invasiveness was assessed using Matrigel invasion assays. ATF5 knockdown resulted in decreased invasiveness in seven of eight cancer cell lines tested. These results suggest that ATF5 promotes invasiveness in several cancer cell lines. Furthermore, the roles of ATF5 in the invasiveness were evaluated in three-dimensional (3D) culture conditions. In 3D collagen gel, HT-1080 and MDA-MB-231 cells exhibited high invasiveness, with spindle morphology and high invasion speed. In both cell lines, knockdown of ATF5 resulted in rounded morphology and decreased invasion speed. Next, we showed that ATF5 induced integrin-alpha 2 and integrin-beta 1 expression and that the depletion of integrin-alpha 2 or integrin-beta 1 resulted in round morphology and decreased invasion speed. Our results suggest that ATF5 promotes invasion by inducing the expression of integrin-alpha 2 and integrin-beta 1 in several human cancer cell lines. (C) 2016 Elsevier Inc. All rights reserved.
• Substrate stiffness regulates temporary NF-κB activation via actomyosin contractions.

  Ishihara S, Yasuda M, Harada I, Mizutani T, Kawabata K, Haga H

  Experimental cell research 319 (19) 2916 - 2927 1090-2422 2013/11/15 [Refereed][Not invited]
   

  Physical properties of the extracellular matrix (ECM) can control cellular phenotypes via mechanotransduction, which is the process of translation of mechanical stresses into biochemical signals. While current research is clarifying the relationship between mechanotransduction and cytoskeleton or adhesion complexes, the contribution of transcription factors to mechanotransduction is not well understood. The results of this study revealed that the transcription factor NF-κB, a major regulator for immunoreaction and cancer progression, is responsive to substrate stiffness. NF-κB activation was temporarily induced in H1299 lung adenocarcinoma cells grown on a stiff substrate but not in cells grown on a soft substrate. Although the activation of NF-κB was independent of the activity of integrin β1, an ECM-binding protein, the activation was dependent on actomyosin contractions induced by phosphorylation of myosin regulatory light chain (MRLC). Additionally, the inhibition of MRLC phosphorylation by Rho kinase inhibitor Y27632 reduced the activity of NF-κB. We also observed substrate-specific morphology of the cells, with cells grown on the soft substrate appearing more rounded and cells grown on the stiff substrate appearing more spread out. Inhibiting NF-κB activation caused a reversal of these morphologies on both substrates. These results suggest that substrate stiffness regulates NF-κB activity via actomyosin contractions, resulting in morphological changes.
• High Prevalence of Multiple Human Papillomavirus Infection in Japanese Patients with Invasive Uterine Cervical Cancer

  Hidemichi Watari, Rie Michimata, Motoaki Yasuda, Akihiro Ishizu, Utano Tomaru, Ying Xiong, Mohamed K. Hassan, Noriaki Sakuragi

  PATHOBIOLOGY 78 (4) 220 - 226 1015-2008 2011 [Refereed][Not invited]
   

  Objective: Multiple human papillomavirus (HPV) infection of the uterine cervix has been suggested as a risk factor for persistent HPV infection, resulting in the development of invasive cervical cancer. The aim of this study was to reveal the actual state of multiple HPV infection in Japanese patients with invasive cervical cancer. Methods: Sixty fresh-frozen invasive cervical cancer tissues were examined for genotyping of HPV. The presence of HPV genotypes was determined with an HPV-DNA array, which can discriminate 25 different HPV genotypes with high sensitivity and specificity. Results: Among 60 samples, 59 (96.7%) were positive for HPV. The three common genotypes were HPV-16 (83.3%), HPV-18 (45.0%) and HPV-52 (28.3%). Multiple HPV infection was observed in 47 of 60 samples (78.3%), among which 42 were infected with more than one high-risk genotype (70.0%). Multiple high-risk HPV infection was significantly more prevalent in patients below 40 years old (14/15, 93.3%) than in patients 40 years of age and over (28/45, 62.2%). Conclusion: The HPV-DNA array is the preferred method to detect HPV genotypes. Multiple HPV infection in Japanese patients with invasive cervical cancer seemed to be more frequent than reported in the literature. Copyright (C) 2011 S. Karger AG, Basel
• Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6

  T Okusawa, M Fujita, JI Nakamura, T Into, M Yasuda, A Yoshimura, Y Hara, A Hasebe, DT Golenbock, M Morita, Y Kuroki, T Ogawa, KI Shibata

  INFECTION AND IMMUNITY 72 (3) 1657 - 1665 0019-9567 2004/03 [Not refereed][Not invited]
   

  The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CG DPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasmafermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.
• Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans.

  Mari Fujita, Takeshi Into, Motoaki Yasuda, Tsugumi Okusawa, Sumiko Hamahira, Yoshio Kuroki, Akiko Eto, Toshiki Nisizawa, Manabu Morita, Ken-ichiro Shibata

  Journal of immunology (Baltimore, Md. : 1950) 171 (7) 3675 - 83 0022-1767 2003/10/01 

  S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-kappaB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2(DeltaS40-I64) (a TLR2 mutant with a deletion of the region of Ser(40) to Ile(64)) failed to activate NF-kappaB in response to FSL-1. The deletion mutant TLR2(DeltaC30-S39) induced NF-kappaB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu(178) to Ala (TLR2(E178A)), TLR2(E180A), TLR2(E190A), and TLR2(L132E) induced NF-kappaB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2(L107E), TLR2(L112E) (a TLR2 point mutant with a substitution of Leu(112) to Glu), and TLR2(L115E) failed to induce NF-kappaB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2(L115E), TLR2(L112E), and TLR2(DeltaS40-I64) were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2(E190A) were. In addition, these mutants, except for TLR2(E180A), functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser(40)-Ile(64) and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.
• Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins

  M Aoyagi, F Higashino, M Yasuda, A Takahashi, Y Sawada, Y Totsuka, T Kohgo, H Sano, M Kobayashi, M Shindoh

  ONCOGENE 22 (44) 6919 - 6927 0950-9232 2003/10 [Refereed][Not invited]
   

  The adenovirus E4orf6 is a viral oncoprotein known to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventually leading to the inhibition of apoptotic activity.
• Adenovirus E1B19K inhibits apoptosis induced by cobalt chloride-mimicked hypoxia

  YAMAZAKI Asako, YASUDA Motoaki, SHINDOH Masanobu, KOHGO Takao, FUKUSHIMA Kazuaki

  北海道歯学雑誌 23 (2) 123 - 133 0914-7063 2002/12/15 [Not refereed][Not invited]
  
• Adenovirus E4orf6 Suppresses the Apoptotic Activity by Bcl-2 Family Protein, BNIP 3

  Aoyagi Mariko, Higashino Fumihiro, Yasuda Motoaki, Kohgo Takao, Sano Hidehiko, Shindoh Masanobu

  Japanese journal of oral biology 歯科基礎医学会 44 (1) 40 - 47 0385-0137 2002/02/20 [Not refereed][Not invited]
  
• Extracellular ATP regulates cell death of lymphocytes and monocytes induced by membrane-bound lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium.

  Takeshi Into, Kazutaka Okada, Nobuo Inoue, Motoaki Yasuda, Ken-ichiro Shibata

  Microbiology and immunology 46 (10) 667 - 75 0385-5600 2002 

  The cytotoxicities of lipoproteins of Mycoplasma fermentans and Mycoplasma salivarium to a lymphocytic cell line, MOLT-4, and a monocytic cell line, HL-60, was upregulated by ATP added extracellularly in a dose-dependent manner. These lipoproteins induced ATP release and plasma membrane permeability increase in these cell lines. In addition, periodate-oxidized ATP, an antagonist for P2X purinergic receptors, suppressed the cytotoxicity of the lipoproteins, suggesting the possibility that P2X receptors for ATP play crucial roles in the cytotoxicity. Activation of caspase-3 induced by the lipoproteins, which was assessed by the cleavage of the synthetic substrate DEVD-pNA and the endogenous substrate poly(ADP-ribose) polymerase, was also upregulated and downregulated by extracellular ATP and periodate-oxidized ATP, respectively. On the basis of these results, this study suggests that mycoplasmal lipoproteins induce the permeability increase in lymphocytes and monocytes, by which ATP is released, and the ATP regulates the cytotoxicities of the lipoproteins to the cells, possibly by interaction with ATP receptors such as P2X purinergic receptors.
• Involvement of Rb protein expression and phosphorylation in progression of oral carcinomas

  Yoshiaki Deyama, Toshiaki Ara, Motoaki Yasuda, Sadaaki Takeyama, Yoshitaka Yoshimura, Noriyuki Sakakibara, Kanchu Tei, Yasunori Totsuka, Kuniaki Suzuki

  Oral Therapeutics and Pharmacology 21 (2) 59 - 67 0288-1012 2002 [Refereed][Not invited]
  
• 悪性黒色腫におけるetsがん遺伝子群転写因子E1AFの発現

  秦 浩信, 大廣 洋一, 東野 史裕, 安田 元昭, 戸塚 靖則, 山下 利春, 吉田 幸一, 田口 和典, 高田 尚幸, 藤堂 省

  日本癌学会総会記事 日本癌学会 60回 472 - 472 0546-0476 2001/09
• Pretreatment apoptotic scores do not predict response to radiation therapy in oropharyngeal squamous cell carcinoma

  K Tsuchiya, H Shirato, T Nishioka, A Yamazaki, S Hashimoto, K Kagei, K Oomori, M Yasuda, M Shindo, K Miyasaka

  ORAL ONCOLOGY 37 (2) 159 - 163 0964-1955 2001/02 [Refereed][Not invited]
   

  The prognostic value of tumor apoptosis was studied in patients with oropharyngeal squamous cell carcinoma treated with radical radiotherapy. Forty-eight patients with oropharyngeal squamous cell carcinoma who received radical radiotherapy between 1990 and 1995 were enrolled in the study. The radiation treatment for all patients involved the administration of 65 Gy in 26 fractions over a 6.5-week period. The apoptotic index (AI; the apoptotic cell count per 1000 tumor cells) was distributed from 0 to 10 with a median at 2 and a mode of 1. There was a significant linear correlation between the AI and mitotic index (MI) (r = 0.393, 95% confidence interval: 0.129-0.605). The cause-specific 5-year survival for patients with AI greater than the median was 46% and for the counterpart was 41%. There was no difference in cause-specific survival between AI/MI greater than the median (50%) and AI/MI smaller than the median (36%). The number of patients was too small to draw definite conclusions, but the AI and the AI/MI before treatment were not shown to have a prognostic value for oropharyngeal squamous cell carcinoma in our study. The primary sites and treatment methods may influence the prognostic value of AI even for the same histological types. (C) 2001 Elsevier Science Ltd. All rights reserved.
• Functional Dissection of BH3 Only Protein BNIP3

  Yasuda Motoaki, Yamazaki Katsuhisa, Hamahira Sumiko, Kawasaki Takao, Nakamura Motoyasu, Shindoh Masanobu

  Tumor research : experimental and clinical 札幌医科大学 36 23 - 28 0041-4093 2001 

  BNIP3 is a pro-apoptotic protein, which contains BH3 and trans-membrane domains. Previous studies demonstrated an association between ceBNIP3, C. elegans homologue of BNIP3, and ced-3. It seemed likely that the interaction between ceBNIP3 and ced-3 was an alternative cascade of caspase activation in C. elegans, however the detailed mechanism of this interaction is yet to be elucidated. We constructed several deletion mutants of BNIP3 and investigated their biological functions. As we expected, the trans-membrane domain of BNIP3 was not required for the association between BNIP3 and ced-3, however trans-membrane deletion mutant could not initiate the apoptotic cascade. Immunofluorescence study demonstrated that wild type BNIP3 and ced-3 co-localized on mitochondria, but trans-membrane deletion mutant did not. These results suggest that the recruitment of ced-3/caspase onto the mitochondrial membrane is essential for BNIP3 initiated apoptosis.
• 口腔扁平上皮癌の遺伝子異常

  進藤正信, 東野史裕, 安田元昭, 向後隆男

  病理と臨床 19 252 - 259 2001 [Not refereed][Invited]
  
• BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma

  M Shindoh, M Adachi, F Higashino, M Yasuda, K Hida, T Nishioka, M Ono, S Takayama, JC Reed, K Imai, Y Totsuka, T Kohgo

  ORAL ONCOLOGY 36 (5) 444 - 449 0964-1955 2000/09 [Refereed][Not invited]
   

  BAG-1 is a Bcl-2-binding: protein that functions as an anti-apoptotic molecule. III this report We show a possible correlation between BAG-1 expression levels and the probability of oral squamous cell carcinoma (SCC) progression. We investigated BAG-1 expression levels in 22 patients diagnosed with early lesions (T1 and T2) of oral SCCs using immunohistochemistry and western blotting. High steady-state levels of BAG-1 were detected in 13 out of 29 cases (59%). High BAG-1 expression was observed more frequently in cases with nodal metastasis (89%) than in those without nodal metastasis (38%) (P < 0.03), suggesting that BAG-I expression levels may correlate with the pathological stage of oral SCCs. Furthermore, BAG-I expression levels correlated with the WHO grade, i.e. 45% in grade-I cases as opposed to 72% in grade-II cases (P < 0.02). These data suggest that an analysis of BAG-I expression may be useful in establishing a prognosis for patients with oral SCCs, and especially in predicting the metastatic potential of SSCs. (C) 2000 Elsevier Science Ltd. All rights reserved.
• [The cellular target proteins of adenovirus gene products].

  Higashino F, Yasuda M, Shindoh M

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 8 45 1350 - 1357 0039-9450 2000/06 [Not refereed][Invited]
  
• Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes

  M Hanzawa, M Shindoh, F Higashino, M Yasuda, N Inoue, K Hida, M Ono, T Kohgo, M Nakamura, K Notani, H Fukuda, Y Totsuka, K Yoshida, K Fujinaga

  CARCINOGENESIS 21 (6) 1079 - 1085 0143-3334 2000/06 [Refereed][Not invited]
   

  Hepatocyte growth factor (HGF) is thought to play a role in cell motility and invasion. Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of tumor cells. We have previously reported that the Ets-oncogene family transcription factor E1AF positively regulates transcription of MMP genes in transient expression assays and that overexpression of the E1AF gene confers an invasive phenotype on breast cancer cells. Here we examined the effect of HGF on E1AF and MMP gene expression in terms of the invasive potential of the oral squamous cell carcinoma cell line HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation, In contrast, no obvious upregulation of MMP-1 and -9 mRNA was observed in ASE1AFHSC3 cells transfected with the antisense E1AF expression vector into parental HSC3 cells. The wild-type MMP-9 gene promoter was activated by endogenous E1AF in HSC3 cells, and chloramphenicol acetyltransferase (CAT) activities increased when HGF was added to transfected cells. On the other hand, CAT activity was reduced to almost two-thirds of the wild-type activity when HSC3 cells were transfected with a CAT reporter plasmid driven by a mutant MMP-9 promoter lacking the Ets-binding site, and induction of CAT activity was not observed upon addition of HGF. Analysis of organotypic raft cultures revealed that HSC3 cells invaded and degraded collagen gel actively upon addition of HGF. These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.
• Functional identification of the apoptosis effector BH3 domain in cellular protein BNIP1.

  M Yasuda, G Chinnadurai

  Oncogene 19 (19) 2363 - 7 0950-9232 2000/05/04 

  BCL-2 family proteins play a central role in apoptosis regulation in mammals and in C. elegans. Mammalian cellular and viral anti-apoptosis proteins such as BCL-2 and E1B-19K interact with several cellular proteins. Some of these interacting proteins promote apoptosis and belong to the BCL-2 family. Certain BCL-2 family proapoptotic proteins such as BAX and BAK share extensive sequence homology with BCL-2. In contrast, certain pro-apoptotic proteins such as BIK and BID share a single death effector domain, BH3, with other BCL-2 family proteins. By mutational analysis, we show that one of the cellular proteins, BNIP1 (previously Nip-1), that interacts with BCL-2 family anti-apoptosis proteins is a 'BH3 alone' pro-apoptotic protein. Transient transfection of BNIP1 induces a moderate level of apoptosis. Deletions of the N-terminal 32 amino acid region and the C-terminal trans-membrane domain did not significantly affect pro-apoptotic activity. In contrast, deletions encompassing a region containing a motif similar to the BH3-domain abrogated the apoptotic activity. Substitution of BNIP1 BH3 domain for the corresponding sequence in BAX efficiently restored the apoptotic activity of BAX, establishing the functional identity of the BH3 domain of BNIP1. The N-terminal deletions of BNIP1 (that retain the BH3 domain) enhanced the level of interaction with BCL-XL. Mutants containing the BH3 deletions were still able to heterodimerize with BCL-XL while mutants lacking both the N-terminal region and the BH3 domain were unable to heterodimerize, suggesting that BNIP1 may bind to BCL-XL via two different binding motifs.
• Induction of transformation and p53-dependent apoptosis by adenovirus type 5 E4orf6/7 cDNA.

  S Yamano, T Tokino, M Yasuda, M Kaneuchi, M Takahashi, Y Niitsu, K Fujinaga, T Yamashita

  Journal of virology 73 (12) 10095 - 103 0022-538X 1999/12 

  Adenovirus (Ad) E4orf6/7, one of the early gene products of human Ads, forms a stable complex with the cellular transcription factor E2F to activate transcription from the Ad E2 promoter. E2F cDNAs have growth-promoting and apoptosis-inducing activities when overexpressed in cells. We cloned Ad5 E4orf6/7 cDNA in both simian virus 40- and human cytomegalovirus-based expression vectors to examine its transforming and apoptotic activities. The cloned E4orf6/7 collaborated with a retinoblastoma protein (RB)-nonbinding and therefore E2F-nonreleasing mutant of Ad5 E1A (dl922/947) to morphologically transform primary rat cells, suggesting that E2F is an important cellular protein functioning downstream of E1A for transformation. In a G418 colony formation assay, E4orf6/7 was shown to suppress growth of untransformed rat cells. Moreover, a recombinant Ad expressing Ad5 E4orf6/7 induced apoptosis in rat cells when coinfected with wild-type p53-expressing Ad. Mutational analysis of E4orf6/7 revealed that both of the domains required for growth inhibition and transformation by E4orf6/7 lay in the C-terminal region, which is essential for transactivation from the upstream sequence of an E2a promoter containing E2F-binding sites. However, the smallest mutant of E4orf6/7, encoding the C-terminal 59 amino acids, failed to complement the RB-nonbinding dl922/947 mutant despite showing growth inhibition and E2F transactivation activities. Thus, it is suggested that a subregion of E4orf6/7 which is required for growth inhibition and transformation in collaboration with dl922/947 overlaps the transactivation domain of E4orf6/7.
• BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3.

  M Yasuda, J W Han, C A Dionne, J M Boyd, G Chinnadurai

  Cancer research 59 (3) 533 - 7 0008-5472 1999/02/01 

  Apoptosis is regulated by interaction of viral and cellular BCL-2 family antiapoptotic proteins with various pro-apoptotic proteins, several of which are also members of the BCL-2 family. Cellular protein BNIP3 is a BCL-2 family proapoptotic protein that interacts with viral antiapoptosis proteins such as adenoviruses E1B-19K and EBV-BHRF1 and cellular antiapoptosis proteins such as BCL-2 and BCL-xL. Database searches indicate that the human genome encodes an open reading frame for a protein, BNIP3alpha, that shares substantial homology with BNIP3. The BNIP3alpha open reading frame encodes a protein of 219 amino acids that contains a conserved BH3 domain and a COOH-terminal trans-membrane domain, characteristic of several BCL-2 family proapoptotic proteins. BNIP3alpha interacts with viral antiapoptosis protein E1B-19K and cellular antiapoptosis proteins BCL-2 and BCL-xL. Overexpression of BNIP3alpha in transfected cells results in apoptosis and suppresses the antiapoptosis activity of E1B-19K and BCL-xL. Like BNIP3, BNIP3alpha seems to be predominantly localized in mitochondria. These results suggest that BNIP3alpha is a structural and functional homologue of BNIP3. BNIP3 and BNIP3alpha seem to be the first examples of homologues among the various human proapoptotic proteins. Northern blot analysis reveals that BNIP3alpha is expressed ubiquitously in most human tissues. In contrast, BNIP3 is expressed well in several human tissues and less abundantly in certain tissues such as placenta and lung. These results suggest that although BNIP3 and BNIP3alpha may promote apoptosis simultaneously in most human tissues, BNIP3alpha may play a more universal role.
• Regulation of apoptosis by a Caenorhabditis elegans BNIP3 homolog.

  M Yasuda, C D'Sa-Eipper, X L Gong, G Chinnadurai

  Oncogene 17 (19) 2525 - 30 0950-9232 1998/11/12 

  We have identified a C. elegans protein, ceBNIP3, homologous to the human BCL-2/EIB-19K interacting BCL-2 family pro-apoptotic protein BNIP3. In transiently transfected mammalian cells, ceBNIP3 complexes with CED-9, the worm homolog of BCL-2. CeBNIP3 also efficiently heterodimerizes with the cell death protease proCED-3 by direct binding via the prodomain. Transfection of ceBNIP3 and CED-3 results in enhanced proteolytic processing of the CED-3 zymogen and in cooperative induction of apoptosis. Coexpression of CED-9 suppresses the cooperative cell death induced by ceBNIP3 and CED-3. In cells coexpressing CED-9, ceBNIP3 and CED-3, all three proteins exist as a ternary complex suggesting that CED-9 may suppress cooperative apoptosis induced by CED-3 and ceBNIP3 by simultaneous complex formation with CED-3 and ceBNIP3. Our results suggest that ceBNIP3 may be a novel component of the C. elegans apoptosis paradigm and may initiate apoptosis by recruiting CED-3 to mitochondria and other cytoplasmic membranes.
• Adenovirus E1B-19K/BCL-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence.

  M Yasuda, P Theodorakis, T Subramanian, G Chinnadurai

  The Journal of biological chemistry 273 (20) 12415 - 21 0021-9258 1998/05/15 

  Adenovirus E1B-19K and BCL-2 anti-apoptosis proteins interact with certain BCL-2 family pro-apoptotic proteins. A conserved domain, BH3, present in these proteins is essential for their pro-apoptotic activity and for heterodimerization with anti-apoptosis proteins. Cellular protein BNIP3 (previously NIP3) interacts with E1B-19K, BCL-2, BCL-xL, and EBV-BHRF1. BNIP3 contains a motif similar to the BH3 domain. Deletion of the BH3-like motif in BNIP3 abrogates its ability to heterodimerize with E1B-19K and BCL-xL. Substitution of the BH3 domain of BNIP3 for the corresponding sequences of BAX functionally restores the pro-apoptotic and protein heterodimerization activities of BAX. BNIP3 exhibits a delayed cell death activity that is partially relieved by deletion of the BH3 domain. BNIP3 suppresses the anti-apoptosis activity of BCL-xL in a BH3-dependent manner. BNIP3 contains a C-terminal trans-membrane (TM) domain similar to other BCL-2 family proteins and BNIP1 (previously NIP1). The TM domains of BNIP3 and BNIP1 can functionally substitute for the TM domain of a BCL-2 family member EBV-BHRF1. The BNIP3 TM domain exclusively targets the heterologous green fluorescent protein (GFP) to mitochondria. These results suggest that BNIP3 is a member of the BH3-contaning BCL-2 family of pro-apoptotic proteins and functions in mitochondria.
• Antisense E1AF transfection restrains oral cancer invasion by reducing matrix metalloproteinase activities

  K Hida, M Shindoh, M Yasuda, M Hanzawa, K Funaoka, T Kohgo, A Amemiya, Y Totsuka, K Yoshida, K Fujinaga

  AMERICAN JOURNAL OF PATHOLOGY 150 (6) 2125 - 2132 0002-9440 1997/06 [Refereed][Not invited]
   

  E1AF is a newly identified human ets-family transcription factor We have reported that E1AF can up-regulate transcription of matrix metalloproteinase (MMP) genes and confers invasive phenotype on human cancer cells. HSC3 is an oral squamous-cell-carcinoma-derived cell line, and it manifests high levels of E1AF and MMP-1 and -9 gene expression that are associated with invasive potential, We reconstructed an E1AF antisense expression vector, transfected HSC3 cells with the vector, and obtained HSC3AS cells that express E1AF antisense RNA, HSC3AS showed decreasing mRNA and protein levels of MMP-1, -3, and -9, Moreover, HSC3AS showed lower invasive potential in vitro three-dimensional raft culture and in vivo implantation into nude mice. These results imply that transfection of antisense E1AF inhibits tumor invasion by down-regulating MMP genes.
• Analysis of Oral Cancer Treated by Preoperative Radiotherapy

  HOSOKAWA Yoichiro, KANEKO Masayuki, YASUDA Motoaki, KANEKO MASANORI, HANZAWA Motoaki, NAKAMURA Motoyasu, SHINDO Masanobu, KOHGO Takao, AMEMIYA Akira, NOTANI Ken-ichi, FUKUDA Hiroshi, TOTSUKA Yasunori

  日本口腔科学会雜誌 46 (1) 65 - 71 0029-0297 1997/01/10 [Not refereed][Not invited]
  
• Correlated expression of matrix metalloproteinases and ets-family transcription factor E1A-F in invasive oral-squamous-cell-carcinoma-derived cell lines

  Shindoh M, Higashino F, Kaya M, Yasuda M, Funaoka K, Hanzawa M, Hida K, Kohgo T, Amemiya A, Yoshida K, Fujinaga K

  Am. J. Pathol. 148 693 - 700 1996 [Refereed][Not invited]
  
• DETECTION OF HUMAN PAPILLOMAVIRUS DNA-SEQUENCES IN ORAL SQUAMOUS-CELL CARCINOMAS AND THEIR RELATION TO P53 AND PROLIFERATING CELL NUCLEAR ANTIGEN EXPRESSION

  M SHINDOH, CHIBA, I, M YASUDA, T SAITO, K FUNAOKA, T KOHGO, A AMEMIYA, Y SAWADA, K FUJINAGA

  CANCER 76 (9) 1513 - 1521 0008-543X 1995/11 [Refereed][Not invited]
   

  Background. The etiology of oral squamous cell carcinoma (SCC) is still obscure. Since human papillomavirus (HPV) DNAs are associated with carcinoma of the uterine cervix, carcinomas of the oral cavity were investigated to ascertain if these viruses are present in squamous carcinomas of this anatomic site. Methods. Seventy-seven oral mucosal SCCs were examined for the presence of HPV DNAs by polymerase chain reaction and dot blot hybridization. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and p53 was performed and single strand conformation polymorphism analysis for p53 was undertaken. In situ hybridization detection of HPV-16 DNA also was performed. Results. Human papillomavirus-16 DNA was detected in 23 cases of oral SCC and both HPV-16 and HPV-18 DNA were detected in one case of tongue SCC. Humin papillomavirus DNAs were detected of 11 of 33 tongue, 4 of 15 gingival, 2 of 4 palate, 2 of 5 buccal mucosa, 3 of 7 maxillary sinus, and 2 of 11 the floor of the mouth SCCs. None were detected in SCCs of the retromolar region (0/ 2). Immunohistochemical examination for p53 was performed in 26 cases of oral SCC and the accumulation of p53 protein was observed in 6 cases (i.e., in 4 of 17 HPV DNA-negative cases and in 2 of 9 HPV DNA-positive cases). Single strand conformation polymorphism analysis confirmed gene mutations in all 6 cases. Human papillomavirus-18 DNA was predominantly identified in cancer cells that showed a morphologic resemblance to basal cells and its hybridized signal in keratinized cells was reduced by in situ hybridization detection. Immunohistochemical detection of PCNA revealed its cooccurrence with HPV-16 DNA in cancer cells. Conclusions. These results suggest that HPV-16 DNA sequences may have the capability to maintain the proliferative state of epithelial cells, and may contribute to the production of malignant phenotypes.
• Computed tomograph system image for a radiation treatment planning using a shell and trial of the superposition of the MRI.

  細川 洋一郎, 箕輪 和行, 澤村 強, 阿部 悟, 安田 元昭, 金子 正範, 大森 桂一, 中村 太保, 渡辺 良晴, 鎌田 正

  Shika Hoshasen 特定非営利活動法人 日本歯科放射線学会 35 (3) 177 - 181 0389-9705 1995
• Detection of Human Papillomavirus (HPV) DNA Sequences in Normal Oral Scrapes Using the Nested PCR.

  Shindoh Masanobu, Fujinaga Yukako, Yasuda Motoaki, Ishikawa Makoto, Kohgo Takao, Amemiya Akira, Sawada Yukiharu, Fujinaga Kei

  Tumor Research Sapporo Medical University 29 39 - 47 0041-4093 1994 

  We investigated the prevalence rate of HPV DNAs in normal mucosa in the oral region. The nested PCR method was utilized to detect target DNA sequences using HPV E6/E7 consensus primer pairs. Of 56 patients examined, HPV 6 and HPV 16 DNA sequences were detected in a 46-year-old male and a 35-year-old female, respectively. These results suggest that HPVs are uncom-mon in normal oral epithelium, and that we should carry out careful follow-up in HPV DNA-positive cases.
• 口腔扁平上皮癌におけるヒトパピロ―マウイルス (HPV) DNAの関与．

  進藤正信, 安田元昭, 雨宮 璋, 澤田幸治, 藤永

  北海道頭頚部腫瘍 13 5 - 8 1992 [Refereed][Not invited]
  
• A case of malignant melanoma derived from oral melanosis.

  NOTANI Ken-ichi, YAMAZAKI Yutaka, MIZUKOSHI Takanori, TOTSUKA Yasunori, FUKUDA Hiroshi, SHINDO Masanobu, YASUDA Motoaki, IIZUKA Tadashi, AMEMIYA Akira

  Journal of Oral Surgery Society of Japan Japanese Society of Oral and Maxillofacial Surgeons 35 (9) 2335 - 2342 0021-5163 1989 

  Sixty-four-year-old male complained of the diffuse brown pigmentation of labial gingiva (3-3). The pathological diagnosis of biopsy specimen was melanosis.
  A black tuberous solid mass was found in the same site 20 months later during the follow up period. As malignant melanoma was suspected, the extirpation (partial maxillectomy) of the tumor and postoperative adjuvant immunochemotherapy (DAV, OK-432) were done.
  Six months later, metastasis was found in cervical lymph nodes. Radical neck dissection (RND) was performed and nodal involvements were pathologically recognized.
  Two years after RND, he died of pneumonia. Neither primary recurrence nor distant metastasis were observed.
• Marginal resection of the mandible for carcinoma of the floor of the mouth.

  TOTSUKA Yasunori, USUI Yasuhiro, TEI Kanchu, YASUDA Motoaki, UY Henry G., KANBARA Yoshinobu, NOTANI Ken-ichi, FUKUDA Hiroshi, SHINDO Masanobu, IIZUKA Tadashi, KO-Go Takao, AMEMIYA Akira

  Journal of Oral Surgery Society of Japan Japanese Society of Oral and Maxillofacial Surgeons 34 (5) 907 - 919 0021-5163 1988 [Refereed][Not invited]
   

  This study was undertaken to determine the indication and value of marginal resection of the mandible as a method of treatment for squamous cell carcinoma of the floor of the mouth. Eighteen patients whose primary tumors were treated by utilizing marginal resection at the Department of Oral Surgery of Hokkaido University from 1977 to 1986, were studied clinically and histopathologically. Another ten patients who had undergone resection of the primary tumor by other surgical methods during the same period, were also studied histopathologically.
  Local recurrence was observed in one case and the 5 year survival rate was 71. 3%. The histopathological study revealed that the cortical bone was eroded or destroyed by tumor tissues in more than half of the cases where the primary tumor had been diagnosed to have gingival invasion, mandibular adhesion or mandibular invasion. Tumor invasion into the periodontal ligament and bone marrow was also found in some cases. However, bone erosion and tumor invasion were limited within a small area, and tumor tissues did not invade beyound the extent which was anticipated before the surgery. These results suggest that some of the squamous cell carcinoma of the floor of the mouth can be removed by marginal resection of the mandible, even if the tumor invades the gingiva, the periosteum and mandible. On the other hand, in cases where the primary tumor had been diagnosed as not having gingival invasion nor mandibular adhesion, varied amounts of fibrous tissues were observed between the tumor tissues and the mandible. This suggests that squamous cell carcinoma of the floor of the mouth can be removed without resecting the mandible, if normal tissue is interposed the tumor and the mandible clinically. However, in some cases, particularly where the lesion was accompanied with infection and/or pain, it might be difficult to ascertain if the tumor had invaded the mandible or not. Therefore, it is recommended that squamous cell carcinoma of the floor of the mouth be removed by utilizing marginal resection of the mandible, if mandibular invasion is doubtful.

MISC

• Activating transcription factor 5 promotes cancer cell invasion through transcriptional activation of integrins

  A. Nukuda, H. Endoh, S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  26-  2015  [Not refereed][Not invited]
  
• Activating transcription factor 5 enhances radioresistance and malignancy in cancer cells

  Seiichiro Ishihara, Seiichiro Ishihara, Motoaki Yasuda, Akihiro Ishizu, Masayori Ishikawa, Hiroki Shirato, Hisashi Haga, Hisashi Haga  Oncotarget  6-  (7)  4602  -4614  2015/01/01  [Not refereed][Not invited]
   

  Radiotherapy is effective for treating various types of tumors. However, some cancer cells survive after irradiation and repopulate tumors with highly malignant phenotypes that correlate with poor prognosis. It is not known how cancer cells survive and generate malignant tumors after irradiation. Here, we show that activating transcription factor 5 (ATF5) promotes radioresistance and malignancy in cancer cells after irradiation. In the G1-S phase of the cell cycle, cancer cells express high levels of ATF5, which promotes cell cycle progression and thereby increases radioresistance. Furthermore, ATF5 increases malignant phenotypes, such as cell growth and invasiveness, in cancer cells in vitro and in vivo. We have identified a new mechanism for the regeneration of highly malignant tumors after irradiation and shown that ATF5 plays a key role in the process.
• Involvement of UTR-dependent gene expression in the maintenance of cancer stem cell like phenotypes

  Motoaki Yasuda, Tomoyuki Hatanaka, Hiroki Shirato, Takeshi Nishioka  Oncology Letters  10-  (5)  3171  -3176  2015/01/01  [Not refereed][Not invited]
   

  © Spandidos Publications 2015. All rights reserved. The present study demonstrated the acquisition of additional malignant characteristics in irradiated mouse fibrosarcoma cells compared with the parent cells. Several reporter assays indicated that hypoxia-inducible factor (HIF)-1α, activator protein-1 and Ets-dependent transcription were activated in irradiated cells. The cis-elements in the 5'-untranslated region (UTR) of these transcription factors plays a major role in their expression in surviving irradiated cancer cells. By contrast, there were no evident differences between the 3'-UTR-dependent repression demonstrated by parent cells and irradiated cells. A small population of parental fibrosarcoma cells was also found to exhibit the same enhanced 5'-UTR-dependent HIF-1α expression as that demonstrated by irradiated cells. These observations may indicate that high-dose X-ray irradiation affects the majority of proliferating cancer cells, but not the cancer stem cells (CSCs), and an increased CSC population may explain the progressive phenotypes of the irradiated cells. It appears likely that the transcription factors that maintain stemness are regulated by the same 5'-UTR-dependent mechanism.
• Lung cancer cells after irradiation indicate viability and malignancy dependent on activating transcription factor 5.

  S. Ishihara, M. Yasuda, A. Ishizu, H. Haga  MOLECULAR BIOLOGY OF THE CELL  25-  2014/12  [Not refereed][Not invited]
  
• X-Ray Irradiation Induces Mitophagy and Cancer Metabolic Reprogramming

  T. Nishioka, H. Shirato, M. Yasuda  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  90-  S778  -S778  2014/09  [Not refereed][Not invited]
  
• Cell type-specific reciprocal regulation of HIF1A gene expression is dependent on 5′- and 3′-UTRs

  Motoaki Yasuda, Tomoyuki Hatanaka, Hiroki Shirato, Takeshi Nishioka  Biochemical and Biophysical Research Communications  447-  (4)  638  -643  2014/05/16  [Not refereed][Not invited]
   

  In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5′-UTR is sufficient for significant translational up-regulation. On the other hand, the 3′-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3′-UTR-dependent down-regulatory machinery predominates over the 5′-UTR-dependent up-regulation of HIF1A. However, 5′-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5′-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A. © 2014 Elsevier Inc. All rights reserved.
• Lung Cancer Cells That Survive Ionizing Radiation Show Increased Integrin α2β1- and EGFR-Dependent Invasiveness

  Xue Li, Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Masayori Ishikawa, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga  PLoS ONE  8-  (8)  e70905  2013/08/08  [Not refereed][Not invited]
   

  Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as a contributor to the limited benefit of radiotherapy; however, its mechanism is still unclear. We previously showed that subcloned lung adenocarcinoma A549 cells (P cells), which survived 10 Gy IR (IR cells), acquired high invasiveness in vitro. Here, we tried to identify the mechanism by which IR cells increase their invasiveness by examining altered gene expression and signaling pathways in IR cells compared with those in P cells. To simulate the microenvironment in vivo, cells were embedded in a three-dimensional (3D) collagen type I gel, in which the IR cells were elongated, while the P cells were spherical. The integrin expression pattern was surveyed, and expression levels of the integrin α2 and β1 subunits were significantly elevated in IR cells. Knockdown of α2 expression or functional blockade of integrin α2β1 resulted in a round morphology of IR cells, and abrogated their invasion in the collagen matrix, suggesting the molecule's essential role in cell spread and invasion in 3D collagen. Epidermal growth factor receptor (EGFR) also presented enhanced expression and activation in IR cells. Treatment with EGFR tyrosine kinase inhibitor, PD168393, decreased the ratio of elongated cells and cell invasiveness. Signaling molecules, including extracellular signal-regulated kinase-1/2 (Erk1/2) and Akt, exhibited higher activation in IR cells. Inhibition of Akt activation by treating with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 decreased IR cell invasion, whereas inhibition of Erk1/2 activation by mitogen-activated protein kinase kinase (MEK) inhibitor U0126 did not. Our results show that integrin α2β1 and EGFR cooperatively promote higher invasiveness of IR-survived lung cancer cells, mediated in part by the PI3K/Akt signaling pathway, and might serve as alternative targets in combination with radiotherapy. © 2013 Li et al.
• Irradiation-tolerant lung cancer cells acquire invasive ability dependent on dephosphorylation of the myosin regulatory light chain

  Seiichiro Ishihara, Motoaki Yasuda, Takeshi Nishioka, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Hisashi Haga  FEBS Letters  587-  (6)  732  -736  2013/03/18  [Not refereed][Not invited]
   

  Radiotherapy is one of the major treatment modalities for malignancies. However, cells surviving irradiation often display high levels of invasiveness. This study shows that irradiation-tolerant lung adenocarcinoma demonstrates high invasive capability depending on dephosphorylation of the myosin regulatory light chain (MRLC). In a collagen gel overlay condition, low-invasive subclones of lung adenocarcinoma (A549P-3) showed a round morphology and diphosphorylation of MRLC. In contrast, irradiation-tolerant A549P-3 cells (A549P-3IR) displayed high invasiveness and a lower level of MRLC diphosphorylation. In addition, inhibition of MRLC phosphatase activity decreased the invasive activity. These findings suggest that A549P-3IR cells acquire high invasiveness through MRLC dephosphorylation. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• 放射線により誘発されるがん細胞の再増殖 : 移植可能なマウス肉腫細胞を用いた実験から類推されるメカニズム

  西岡 健, 安田 元昭, 武島 嗣英, 芳賀 永, 宮井 優介, 柴田 健一郎, 山崎 理衣, 白土 博樹, 手塚 正博, 伊達 広行  北海道醫學雜誌 = Acta medica Hokkaidonensia  87-  (4)  192  -192  2012/08/01
• EGFR and Integrin alpha 2 beta 1 Dependent Invasiveness of Lung Adenocarcinoma Cells that Survived 10 Gy Ionizing Radiation.

  X. Li, S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, T. Nishioka, H. Haga  MOLECULAR BIOLOGY OF THE CELL  23-  2012  [Not refereed][Not invited]
  
• Viral-mediated stabilization of AU-rich element containing mRNA contributes to cell transformation.

  T Kuroshima, M Aoyagi, M Yasuda, T Kitamura, J P Jehung, M Ishikawa, Y Kitagawa, Y Totsuka, M Shindoh, F Higashino  Oncogene  30-  (26)  2912  -20  2011/06/30  [Not refereed][Not invited]
   

  E4orf6 is one of the oncogene products of adenovirus, and it also has an important role for transportation of cellular and viral messenger RNA (mRNA) during the late phase of virus infection. We previously revealed that E4orf6 controls the fate of AU-rich element (ARE) containing mRNA by perturbing the chromosome maintenance region 1-dependent export mechanism. Here, we show that E4orf6 stabilizes ARE-mRNA through the region required for its oncogenic activity and ubiquitin E3 ligase assembly. Cells that failed to stabilize ARE-mRNA after HuR knockdown were unable to produce colonies in soft agar, even when E4orf6 was expressed. Furthermore, the stabilized ARE-mRNA induced the transformation of rodent immortalized cells. These findings indicate that stabilized ARE-mRNA is necessary, if not all, for the oncogenic activity of E4orf6 and has the potential to transform cells, at least under a certain condition. Oncogene (2011) 30, 2912-2920; doi:10.1038/onc.2011.14; published online 14 February 2011
• Substrate stiffness regulates NF-kB expression and activity in cancer cells

  S. Ishihara, M. Yasuda, T. Mizutani, K. Kawabata, H. Haga  MOLECULAR BIOLOGY OF THE CELL  22-  2011  [Not refereed][Not invited]
  
• Radiation-induced Cancer Cell Repopulation: A Possible Mechanism Implied by Experiments Using Transplantable Mouse-derived Sarcoma Cell Line

  Takeshi Nishioka, Motoaki Yasuda, Tsuguhide Takeshima, Hisashi Haga, Yusuke Miyai, Ken-ichiro Shibata, Rie Yamazaki, Hiroki Shirato, Masahiro Teduka, Hiroyuki Date  CELL STRUCTURE AND FUNCTION  36-  (1)  13  -20  2011  [Not refereed][Not invited]
   

  Purpose: Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation". To better clarify the mechanism, gene expression profiling and pathological experiments were performed. Materials and Methods: A mouse fibrosarcoma cell line, QRsP, was used. Cells were irradiated with 10 Gy. Colony assay and cloning were performed. Six clones were established. cDNA analysis was performed on the clone that showed the largest number of colonies on the 2nd 10 Gy irradiation. Mouse transplantation experiment was then carried out. Results: cDNA analysis showed that cyclin-dependent kinase inhibitors, p16 and p57 were down-regulated; 14.8- and 12.0-fold, respectively for the tolerant clone. Matrix metalloproteinase 3 and 13 were up-regulated; 22.5- and 25.8-fold, respectively. Transplantation ratio was 100% (5/5) for the tolerant clone whereas it was 40% (2/5) for the parent. Under light microscope, the mean mitotic cell number was 4.0+/-3.9 for the parent, and 12.8+/-3.4 for the tolerant clone (p < 0.01, Student's t-test). Conclusions: This study implies that repopulation is not a temporary reaction to irradiation. It is caused probably by "clonal" gene-expression changes, though it remains unknown whether the changes are attributable to tolerant cell selection or to gene mutation/modification.
• アデノウイルス感染は宿主細胞のStress granulesとP‐Bodiesに特異的な変化をもたらす

  黒嶋雄志, 安田元昭, 北村哲也, 石川誠, 北川善政, 進藤正信, 東野史裕  生化学  83回・33回-  ROMBUNNO.2P-0664  -0664  2010/12  [Not refereed][Not invited]
  
• Integrin beta 1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

  Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Takeomi Mizutani, Kazushige Kawabata, Hiroki Shirato, Takeshi Nishioka  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  396-  (3)  651  -655  2010/06  [Not refereed][Not invited]
   

  Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin beta 1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin beta 1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin beta 1-dependent phenotype, and integrin beta 1 might be a potentially effective therapeutic target in combination with radiotherapy. (C) 2010 Elsevier Inc. All rights reserved.
• Matrix Metalloproteinase's Are Up-regulated in Sub-clones That Survived 10Gy Irradiation: A Possible Role of Hypoxic-inducible Factor-alpha (HIF1a)

  T. Nishioka, K. Tsutsumi, T. Takeshima, H. Shirato, M. Yasuda  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  78-  (3)  S631  -S631  2010  [Not refereed][Not invited]
  
• Cell Motility and Invasion of Surviving Tumor Cells after 10 Gy Irradiation

  K. Tsutsumi, M. Tsuda, N. Yazawa, H. Nakamura, M. Yasuda, R. Yamazaki, H. Shirato, H. Kawaguchi, Y. Ohba, T. Nishioka  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  75-  (3)  S538  -S539  2009  [Not refereed][Not invited]
  
• Increased Invasiveness and Non-spheroid Morphology Change of Human Lung Adenocarcinoma Cells That Survived 10 Gy Irradiation

  T. Nishioka, S. Ishihara, Y. Miyai, T. Mizutani, K. Kawabata, H. Shirato, R. Yamazaki, M. Yasuda, H. Haga, K. Kawabata  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  75-  (3)  S540  -S540  2009  [Not refereed][Not invited]
  
• Value of fluorodeoxyglucose positron emission tomography before radiotherapy for head and neck cancer : dose the standardized uptake value predict treatment outcome?

  SUZUKI K.  Jpn J Radiol  27-  (6)  237  -242  2009  [Not refereed][Not invited]
  
• Novel function of transcription factor ATF5: blockade of p53-dependent apoptosis induced by ionizing irradiation.

  Takeshi Nishioka, Yusuke Miyai, Hisashi Haga, Kazushige Kawabata, Hiroki Shirato, Akihiro Homma, Kenichiro Shibata, Motoaki Yasuda  Cell structure and function  34-  (1)  17  -22  2009  [Not refereed][Not invited]
   

  Purpose: To find a new molecule that affects p53-dependent radiosensitivity.Methods and Materials: A mouse sarcoma cell line, QRsP(p53+/+), was used. From this cell line, we established a radiosensitive clone and a radioresistant one. Colony assay, p53 gene transfer, a luciferase assay for p53 and p21, animal transplantation experiment, and DNA array analyses were performed.Results: Microarray showed marked reduction of a transcription factor, ATF5, both in vitro and in vivo for the radiosensitive clone. Interestingly, flow cytometric analysis demonstrated marked apoptosis for the radiosensitive clone by p53 cloned adenovirus infection. Luciferase reporter assay revealed that ATF5 suppressed the transactivational activity of p53 and p63. By ATF5 gene transfer, the radiosensitive clone regained resistance to both ionizing-radiation and Ad-p53 infection-induced cell death. Surprisingly, time-lapse cell migration observation revealed greater cell motility for ATF5-transfected radiosensitive clone.Conclusions: It seems likely that ATF5 is a potent repressor of p53 and elevated expression of ATF5 in a tumor may relate to enhanced malignant phenotypes, such as radioresistance or greater cell motility.
• Increased Motility and Invasiveness in Tumor Cells That Survive 10 Gy Irradiation

  Kaori Tsutsumi, Masumi Tsuda, Natsuka Yazawa, Hirotaka Nakamura, Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Rie Yamazaki, Hiroki Shirato, Hideaki Kawaguchi, Takeshi Nishioka, Yusuke Ohba  CELL STRUCTURE AND FUNCTION  34-  (2)  89  -96  2009  [Not refereed][Not invited]
   

  Radiotherapy is an important noninvasive treatment for many types of cancer. However, it has been reported that the proliferative, invasive, and metastatic capacities of tumor cells can be increased in the repopulated tumors that survive radiotherapy. We have previously established a radiation-surviving cell model for the human non-small cell lung cancer cell line H1299 by harvesting relic cells 14 days after irradiation (IR cells). Here, we report that cell invasion, cell migration, and cell adhesion are enhanced in these surviving cancer cells. The mRNA expression levels of matrix metalloproteinases (MMPs), including mmp1, mmp2, and mmp9, were upregulated in IR cells compared with parental cells. A gelatin zymogram, wound healing assay, and invasion assay showed increased MMP activity, cell motility, and invasiveness in IR cells, respectively. Moreover, IR cells adhered more tightly to collagen-coated dishes than parental cells. Consistently, paxillin, phosphorylated FAK, integrin beta 1, and vinculin were strongly localized at focal adhesions in IR cells, as visualized by immunofluorescence. In this report, we identify molecules responsible for the malignant properties of tumor cells that survive irradiation. These molecules may be important therapeutic targets for the control of repopulated tumors after radiotherapy.
• Novel function of transcription factor ATF5: Blockade of p53-dependent apoptosis induced by irradiation

  T. Nishioka, M. Yasuda, H. Haga, R. Yamazaki, K. Tsutsumi, H. Shirato  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  72-  (1)  S43  -S43  2008  [Not refereed][Not invited]
  
• Resveratrol modulates phagocytosis of bacteria through an NF-kappa B-dependent gene program

  Mitsuhiro Iyori, Hideo Kataoka, Haque Mohammad Shamsul, Kazuto Kiura, Motoaki Yasuda, Takashi Nakata, Akira Hasebe, Ken-ichiro Shibata  ANTIMICROBIAL AGENTS AND CHEMOTHERAPY  52-  (1)  121  -127  2008/01  [Not refereed][Not invited]
   

  Many studies have shown that the pharmacological effects of resveratrol, a phytoalexin polyphenolic compound, include protective effects against cancer and inflammation as well as enhancement of stress resistance. In this study, we examined whether resveratrol affected the phagocytosis of bacteria by macrophages and the activation of the transcription factor NF-kappa B after stimulation with or without the ligand FSL-1 for Toll-like receptor 2 (TLR2). Phagocytosis of Escherichia coli and of Staphylococcus aureus by THP-1 cells and RAW264.7 cells was inhibited by resveratrol in a dose-dependent manner regardless of stimulation with FSL-1. The NF-kappa B activity in HEK293 cells stably expressing TLR2 was also inhibited by resveratrol after stimulation with FSL-1. Resveratrol also inhibited both the translocation of p65 of NF-kappa B into nuclei in the transfectant and tumor necrosis factor alpha production by THP-1 cells or RAW264.7 cells. It has recently been reported that TLR-mediated signaling pathways lead to the upregulation of mRNAs of phagocytic receptors, including scavenger receptors and C-type lectin receptors. This study also demonstrated that FSL-1 induced the upregulation of mRNAs of phagocytic receptors such as macrophage scavenger receptor-1, CD36, DC-SIGN, and Dectin-1 and that the FSL-1-induced upregulation of their mRNAs was inhibited by resveratrol. In addition, it was found that the expression of DC-SIGN in HEK293 cells stably expressing DC-SIGN was reduced by resveratrol and that the phagocytic activity was significantly inhibited by resveratrol. Thus, this study suggests that resveratrol inhibited bacterial phagocytosis by macrophages by downregulating the expression of phagocytic receptors and NF-kappa B activity.
• The diacylated lipopeptide FSL-1 enhances phagocytosis of bacteria by macrophages through a Toll-like receptor 2-mediated signalling pathway

  Masako Mae, Mitsuhiro Iyori, Motoaki Yasuda, Haque Mohammad Shamsul, Hideo Kataoka, Kazuto Kiura, Akira Hasebe, Yasunori Totsuka, Ken-ichiro Shibata  FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY  49-  (3)  398  -409  2007/04  [Not refereed][Not invited]
   

  A significant amount of evidence has been accumulated to show that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, little is known about how signalling triggered by TLRs leads to the phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 mainly with the lipopeptide FSL-1 plays a role in the phagocytosis of pathogens by macrophages. FSL-1 enhanced the phagocytosis of Escherichia coli to a markedly greater extent than it did that of Staphylococcus aureus, but did not enhance the phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2(+/+) mice but not by those from TLR2(-/-) mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors, including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partly by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signalling pathways, and that TLR2 by itself does not function as a phagocytic receptor.
• DC-SIGNは Toll-like receptor の補助レセプターとして機能する

  柴田 健一郎, 片岡 嗣雄, 伊従 光洋, 木浦 和人, 長谷部 晃, 安田 元昭, 中田 貴  日本細菌学雑誌  62-  (1)  159  -159  2007/02/25
• E2Fを介した自然免疫応答抑制機構

  安田 元昭, 伊従 光洋, 木浦 和人, 長谷部 晃, 片岡 嗣雄, 柴田 健一郎  日本細菌学雑誌  62-  (1)  159  -159  2007/02/25
• Three-dimensional tumor cell movement in collagen gel: mesenchymal-to-amoeboid transition in a sub-clone that survived 10 Gy irradiation

  T. Nishioka, H. Haga, Y. Miyai, M. Yasuda, K. Tsutsumi, R. Yamazaki, H. Shirato, K. Kawabata  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  69-  (3)  S148  -S148  2007  [Not refereed][Not invited]
  
• Matrixmetalloproteinases : up-regulated in subclones that survived 10-Gy irradiation

  NISHIOKA Takeshi, YASUDA Motoaki, TSUTSUMI Kaori, HAGA Hisashi, SHIRATO Hiroki  Radiat Med  25-  (8)  430  -431  2007  [Not refereed][Not invited]
  
• CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex

  Takashi Nakata, Motoaki Yasuda, Mari Fujita, Hideo Kataoka, Kazuto Kiura, Hidehiko Sano, Kenichiro Shibata  CELLULAR MICROBIOLOGY  8-  (12)  1899  -1909  2006/12  [Not refereed][Not invited]
   

  It has demonstrated that the recognition of triacylated lipopeptides by Toll-like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF-kappa B reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF-kappa B luciferase reporter gene, S-(2,3-bispalmitoyloxy-propyl)N-palmitoyl-Cys-Lys-Lys-Lys-Lys (Pam(3)CSK(4)) and Pam(3)CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14-induced enhancement did not occur without cotransfection of TLR1. Both CD14(dS39-A48), a mutant with deletion of the part of possible N-terminal ligand-binding pocket, and anti-CD14 monoclonal antibody reduced the CD14-induced enhancement. Transfection of a TIR domain-deficient mutant of TLR2 (TLR2(dE772-S784)) or TLR1 (TLR1(dQ636-K779)) completely abrogated the CD14-induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam(3)CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance-based assay demonstrated that CD14 binds to Pam(3)CSK(4) at a dissociation constant of 5.7 mu M. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.
• The diacylated lipopeptide FSL-1 induces TLR2-mediated Th2 responses

  Kazuto Kiura, Hideo Kataoka, Motoaki Yasuda, Nobuo Inoue, Ken-ichiro Shibata  FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY  48-  (1)  44  -55  2006/10  [Not refereed][Not invited]
   

  The diacylated lipopeptide FSL-1 enhanced the generation of IgG antibodies in TLR2(+/+) mice, but not in TLR2(-/-) mice, when administered together with hen egg lysozyme as an antigen. Escherichia coli lipopolysaccharide enhanced the generation of antigen-specific antibodies in both TLR2(-/-) and TLR2(+/+) mice. In TLR2(+/+) mice, the level of enhancement due to FSL-1 was similar to that caused by lipopolysaccharide. Analysis of the IgG antibodies subclass demonstrated that the level of Th2-type IgG1 antibodies was higher than that of Th1-type IgG2a antibodies. Both FSL-1 and lipopolysaccharide induced production of IL-10 and IL-6 by splenocytes from TLR2(+/+) mice. Lipopolysaccharide also induced production of these cytokines by splenocytes from TLR2(-/-) mice, but FSL-1 did not. Neither FSL-1 nor lipopolysaccharide induced IL-12p70 production by splenocytes from either type of mice. FSL-1 upregulated B7.2 expression in B220(+) cells from TLR2(+/+) mice but not those from TLR2(-/-) mice, whereas lipopolysaccharide upregulated B7.2 expression in B220(+) cells from both types of mice. FSL-1 and, to a lesser extent, lipopolysaccharide activated mitogen-activated protein kinases in splenocytes. FSL-1 and, to a lesser extent, lipopolysaccharide induced the expression of c-Fos, which is known to be involved in Th2-type responses, in splenocytes. Thus, this study demonstrated that FSL-1 possessed TLR2-mediated Th2-type responses in vivo.
• Roles of N-linked glycans in the recognition of microbial lipopeptides and lipoproteins by TLR2

  Hideo Kataoka, Motoaki Yasuda, Mitsuhiro Iyori, Kazuto Kiura, Mitsuo Narita, Takashi Nalkatal, Ken-ichiro Shibata  CELLULAR MICROBIOLOGY  8-  (7)  1199  -1209  2006/07  [Not refereed][Not invited]
   

  Details of roles of carbohydrates attached to Toll-like receptors (TLRs) in the recognition of pathogen-associated molecular patterns and in the formation of the functional receptor complex still remain unknown. This study was designed to determine whether the glycans linked at Asn114, Asn199, Asn414 and Asn442 residues of TLR2 ectodomain were involved in the recognition of diacylated lipopeptide and lipoprotein. Single and multiple mutants were transfected into human embryonic kidney (HEK) 293 cells together with a NF-kappa B luciferase reporter plasmid. All of these mutants were expressed on the surface. SDS-PAGE of the transfectants demonstrated that these mutants migrated lower than wild-type TLR2 and their molecular masses decreased as the number of mutated Asn residues increased. TLIZ12(N114A), TLR2(N199A) and TLR2(N414A) as well as wild-type TLR2 induced NF-kappa B activation when stimulated with these ligands, whereas TLR2(N442A) failed to induce NF-kappa B activation. All of triple and quadruple mutants failed to induce NF-kappa B activation, but were associated with both wildtype TLR2 and TLR6 in the transfectants. TLR2(N114A,N199A) TLR2(N114A,N414A) and, to a lesser extent, TLR2(N114A,N442A), in which two N-linked glycans are speculated to be exposed to the concave surface of TLR2 solenoid, not only induce NF-kappa B activation but also are associated with wild-type TLR2 and TLR6. These results suggest that the glycan at Asn442 and at least two N-linked glycans speculated to be exposed to the concave surface of TLR2 solenoid are involved in the recognition of ligands by TLR2 and/or in formation or maturation of a functional TLR2 receptor complex.
• The synthetic analogue of mycoplasmal lipoprotein FSL-1 induces dendritic cell maturation through Toll-like receptor 2

  K Kiura, H Kataoka, T Nakata, T Into, M Yasuda, S Akira, N Inoue, K Shibata  FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY  46-  (1)  78  -84  2006/02  [Not refereed][Not invited]
   

  Granulocyte-macrophage colony-stimulating factor-differentiated bone marrow-derived dendritic cells were stimulated with the synthetic lipopeptide S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) or the Escherichia coli lipopolysaccharide. FSL-1 induced the production of TNF-alpha and IL-12 by C57BL/6-derived bone marrow-derived dendritic cells but not by bone marrow-derived dendritic cells from Toll-like receptor 2-deficient (TLR2(-/-)) mice. Lipopolysaccharide induced the production of TNF-alpha and IL-12 by bone marrow-derived dendritic cells derived from either type of mice. FSL-1 did not induce production of IL-10 by bone marrow-derived dendritic cells from either type of mice, whereas lipopolysaccharide induced small amounts of IL-10 by bone marrow-derived dendritic cells from both types of mice. The upregulation by FSL-1 of the expression of CD80, CD86 and the MHC class II molecule IA(b) was dose- and time-dependent on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells but not on the surface of TLR2(-/-)-derived bone marrow-derived dendritic cells. Lipopolysaccharide upregulated the expression of these molecules on the surfaces of bone marrow-derived dendritic cells from both types of mice. The expression of CD11c on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells was upregulated by stimulation with both FSL-1 and lipopolysaccharide up to 12 h; thereafter, the expression was downregulated. The results suggest that FSL-1 can accelerate maturation of bone marrow-derived dendritic cells and this FSL-1 activity is mediated by TLR2.
• Reduced transactivation activity of p53 and repressed CDK inhibitor expression observed in subclones that survived 10Gy irradiation: Possible mechanisms of "radiation induced cancer cell repopulation"

  T. Nishioka, M. Yasuda, H. Shirato  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  66-  (3)  S586  -S587  2006  [Not refereed][Not invited]
  
• X-ray irradiation altered chemosensitivity of a p53-null non-small cell lung cancer cell line

  Kaori Tsutsumi, Motoaki Yasuda, Takeshi Nishioka  CELL STRUCTURE AND FUNCTION  31-  (2)  47  -52  2006  [Not refereed][Not invited]
   

  Radiotherapy is an effective approach to treating many types of cancer. Recent progress in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates. However, the emergence of tolerant cells during or after radiotherapy remains problematic. In the present study, we first established a cell population from H1299, the p53-null non-small cell lung cancer cell line, by 10 Gy irradiation using 6 MV X-rays. The radio- and chemosensitivity of this cell population (referred to as H1299-IR) was determined using colony formation analyses and MTS assays. Compared with the parental cell line, the radiosensitivity of H1299-IR was apparently the same. H1299 and H1299-IR were both more radio tolerant than the A549 cell line. However, H1299-IR became significantly more sensitive to cisplatin, an antitumor agent. After exposure to 25 mu g/ml cisplatin for 2 h, parental cells steadily grew during the MTS assay, whereas the sensitivity of H1299-IR cells doubled both at 24 and 48 h. Microarray analysis of over 30,000 H1299-IR genes (Agilent Technology) revealed that 12 and 15 genes were up- (> 2.0) and down- (< 2.0) regulated, respectively. Rad51d (homologous recombination repair protein) gene was down-regulated 2.8-fold, whereas matrix metalloproteinase 1 (collagenase-1) gene was up-regulated 4.4-fold. These results indicated that some p53-null non-small cell lung cancers could be successfully treated when X-ray radiotherapy was administered with subsequent or concurrent cisplatin chemotherapy.
• Activation of Human Monocytes and Mouse Splenocytes by Single-Walled Carbon Nanotubes

  Kazuto Kiura, Yoshinori Sato, Motoaki Yasuda, Bunshi Fugetsu, Fumio Watari, Kazuyuki Tohji, Ken-ichiro Shibata  JOURNAL OF BIOMEDICAL NANOTECHNOLOGY  1-  (3)  359  -364  2005/09  [Not refereed][Not invited]
   

  This study was designed to investigate biological toxicities of single-walled carbon nanotubes (SWCNTs) and hat-stacked carbon nanofibers (H-CNFs) that possess different specific surface areas and graphene structures. These materials activated both human monocytic and mouse spleen cells to produce TNF-alpha, although the activity was significantly lower than those of microbial lipopeptide and lipopolysaccharide. The activity of the nanotubes was found to be higher than that of the nanofibers. Thus, these materials are recognized as non-self antigens that can stimulate the immune response.
• Detection of human papillomavirus from oral mucosa

  YASUDA M., SHIBATA K.  北海道歯学雑誌  26-  (1)  49  -50  2005/06/15
• Hamahira S., Nakamura M., Khan M.H., Choudhury A.R., Shibata K., Yasuda M. "Comprehensive detection of human papilomavirus in buccal cancer collected in Bangladesh", Dentistry in Japan 41:19-24 (2005)*

  2005  [Not refereed][Not invited]
  
• Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation

  T Into, K Kiura, M Yasuda, H Kataoka, N Inoue, A Hasebe, K Takeda, S Akira, K Shibata  CELLULAR MICROBIOLOGY  6-  (2)  187  -199  2004/02  [Not refereed][Not invited]
   

  Mycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Toll-like receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF-kappaB, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid. The activity was synergically augmented by co-expression of TLR6, but not by co-expression of other TLRs. Under the condition of co-expression of TLR2 and TLR6, the lipoproteins could induce maximum NF-kappaB activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant-negative forms of MyD88 and FADD, but not IRAK-4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant-negative forms also downregulated the activation of both NF-kappaB and caspase-8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2- and TLR6-mediated sequential bifurcate responses: NF-kappaB activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.
• DNA array法による子宮頚部細胞診検体中ヒトパピローマウイルスの検出

  渡利英道, 山本律, 水上尚典, 安田元昭, 柴田健一郎, 白田勝利, 西村訓弘, 白川洋三, 藤田博正  日本産科婦人科学会東北連合地方部会誌  (51)  2004
• 1-33.DNA array法による子宮頚部擦過細胞診検体中ヒトパピローマウイルスの検出(第3群 子宮頸部悪性腫瘍3)(一般演題)

  渡利 英道, 安田 元昭, 白田 勝利, 西村 訓弘, 藤田 博正, 柴田 健一郎, 藤堂 幸治, 山本 律, 水上 尚典, 櫻木 範明  日本産科婦人科學會雜誌  56-  (2)  345  -345  2004
• Okusawa T., Fujita M., Nakamura JI., Into T., Yasuda M., Yoshimura A., Hara Y., Hasebe A., Golenbock D.T., Morita M., Kuroki Y., Ogawa T., and Shibata KI."Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides ・・・

  2004  [Not refereed][Not invited]
   

  Okusawa T., Fujita M., Nakamura JI., Into T., Yasuda M., Yoshimura A., Hara Y., Hasebe A., Golenbock D.T., Morita M., Kuroki Y., Ogawa T., and Shibata KI."Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6." Infect Immun, 72(3):1657-1665, 2004*
• Toll-like receptor 2の107,112ならびに115番目のロイシンはリポペプチドならびにペプチドグリカンの認識に関与する

  藤田 真理, 森田 学, 引頭 毅, 片岡 嗣雄, 安田 元昭, 柴田 健一郎  日本免疫学会総会・学術集会記録  33-  121  -121  2003/11  [Not refereed][Not invited]
  
• Mycoplasma fermentansの細胞膜リポタンパク質はToll-like receptor(TLR)2ならびにTLR6を介してMyD88とFADDに依存的なアポトーシスを誘導する

  引頭 毅, 木浦 和人, 安田 元昭, 藤田 真理, 片岡 嗣雄, 柴田 健一郎  日本免疫学会総会・学術集会記録  33-  123  -123  2003/11  [Not refereed][Not invited]
  
• マイコプラズマの細胞膜リポタンパク質がToll様受容体(TLR)2ならびにTLR6を介して誘導するアポトーシスの解析

  引頭 毅, 木浦 和人, 安田 元昭, 柴田 健一郎  日本マイコプラズマ学会雑誌  (30)  69  -71  2003/10  [Not refereed][Not invited]
   

  マイコプラズマの細胞膜リポタンパク質(LPfer)の誘導する細胞死において,ヒトToll-like receptor(TLR) 2ならびにTLR 6,更にその下流分子の関与について検討した.LPferはヒトTLR 2遺伝子を導入したHEK293細胞では濃度依存的に細胞死を誘導し,TLR 2とTLR 6を共に発現した細胞ではTLR 2単独の場合よりも強い細胞死誘導活性を示した.LpferがTLR 2/6を介して誘導する細胞死はドミナントネガティブ型myeloid differentiation factor 88(MyD88),ドミナントネガティブ型Fas-associated death domain protein(FADD)遺伝子導入により顕著に抑制された.Lpferの細胞死誘導活性にはTLR 2の存在が必須であり,TLR 2とTLR 6両者の存在下でより強いこと,LpferはTLR 2/6によって確認された後,MyD88やFADDを介したシグナル経路を経て細胞死を誘導することが示唆された
• Toll様受容体2におけるリポペプチドならびにペプチドグリカンの認識部位について

  藤田 真理, 森田 学, 引頭 毅, 片岡 嗣雄, 濱平 須美子, 安田 元昭, 柴田 健一郎  歯科基礎医学会雑誌  45-  (5)  273  -273  2003/09/01  [Not refereed][Not invited]
  
• 口腔マイコプラズマ由来のリポタンパク質のアジュバント活性について

  木浦 和人, 黒岩 理暢, 引頭 毅, 安田 元昭, 井上 農夫男, 柴田 健一郎  歯科基礎医学会雑誌  45-  (5)  273  -273  2003/09/01
• DNAアレイ法を用いたヒトパピローマウイルスの網羅的検出法

  安田元昭, 白田勝利, 西村訓弘, 岩沢晶彦, 渡利英道, 桜木範明, 柴田健一郎  日本癌学会総会記事  62nd-  290  -290  2003/08/25  [Not refereed][Not invited]
  
• Overexpression of osteopontin-enhanced adhesive activity of osteoblast-like cell.

  A. Yokoyama, M. Yasuda, K. Yamazaki, T. Kawasaki, F. Higashino, M. Shindoh, T. Kohgo, A. Yamamoto, T. Hanawa  JOURNAL OF DENTAL RESEARCH  82-  B140  -B140  2003/06  [Not refereed][Not invited]
  
• ジアシルリポペプチドとToll-like receptor 2との相互作用

  藤田 真理, 森田 学, 奥沢 亜美, 引頭 毅, 安田 元昭, 柴田 健一郎  日本細菌学雑誌  58-  (1)  294  -294  2003/02  [Not refereed][Not invited]
  
• Fujita M, Into T, Yasuda M, Okusawa T, Hamahira S, Kuroki Y, Eto A, Nisizawa T, Morita M, Shibata K.:"Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human toll-like receptor 2 in the recognition of diac・・・

  2003  [Not refereed][Not invited]
   

  Fujita M, Into T, Yasuda M, Okusawa T, Hamahira S, Kuroki Y, Eto A, Nisizawa T, Morita M, Shibata K.:"Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans." J Immunol,171(7):3675-83(2003)*
• Aoyagi M, Higashino F, Yasuda M, Takahashi A, Sawada Y, Totsuka Y, Kohgo T, Sano H, Kobayashi M, Shindoh M. :"Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins." Oncogene, 22(・・・

  2003  [Not refereed][Not invited]
   

  Aoyagi M, Higashino F, Yasuda M, Takahashi A, Sawada Y, Totsuka Y, Kohgo T, Sano H, Kobayashi M, Shindoh M. :"Nuclear export of adenovirus E4orf6 protein is necessary for its ability to antagonize apoptotic activity of BH3-only proteins." Oncogene, 22(44):6919-6927(2003)*
• Membrane lipoproteins derived from Mycoplasma fermentans induce extracellular ATP- and P2X_7 purinoceptor-mediated cell death in lymphocytes and monocytes

  INTO Takeshi, OKADA Kazutaka, HASEBE Akira, INOUE Nobuo, YASUDA Motoaki, SHIBATA Ken-ichiro  日本マイコプラズマ学会雑誌 = Japanese Journal of Mycoplasmology  29-  68  -69  2002/11/18
• マイコプラズマ由来リポタンパク質の細胞死誘導活性

  岡田 和隆, 濱平 須美子, 引頭 毅, 長谷部 晃, 安田 元昭, 井上 農夫男, 柴田 健一郎  歯科基礎医学会雑誌  44-  (5)  386  -386  2002/09/20
• アデノウイルスE1B19Kの抗アポトーシス活性

  濱平 須美子, 長谷部 晃, 安田 元昭, 柴田 健一郎  歯科基礎医学会雑誌  44-  (5)  385  -385  2002/09/20
• Bacteroides forsythus由来リポタンパク質の歯周病における病因的役割について

  長谷部 晃, 荒川 真一, 石倉 裕晃, 吉村 篤利, 土田 信夫, 安田 元昭, 柴田 健一郎  歯科基礎医学会雑誌  44-  (5)  463  -463  2002/09/20  [Not refereed][Not invited]
  
• Human adenovirus E1B19K inhibits the apoptosis induced by p51

  YASUDA Motoaki, SUGAWARA Yuichiro, HAMAHIRA Sumiko, KOHGO Takao, NAKAMURA Motoyasu  北海道歯学雑誌  23-  (1)  2  -9  2002/06/15
• Gamma-irradiation induces overexpression of hepatocyte growth factor in human diploid fibroblasts

  YASUDA Motoaki, SHINDOH Masanobu, KHAN Mahfuzul H., KOHGO Takao, NAKAMURA Motoyasu  北海道歯学雑誌  23-  (1)  10  -15  2002/06/15
• Yasuda M, Shindoh M, Khan M, Kohogo T, Nakamura M "Gamma-irradiation induced overexpression of hepatocyte growth factor in human diploid fibroblast."Hokkaido J. Dent. Sci 23(1):10-15 , 2002*

  2002  [Not refereed][Not invited]
  
• p53 and p51 induces apoptosis by utilizing an adenovirus vector system

  SUGAWARA Y, YASUDA M, KHAN MH, NAKAMURA M  歯科放射線  41-  34  -34  2001/09/30
• Amount of contrast media on sialography

  OHOMORI K, YASUDA M, KANEKO M, SAWAMURA T, NAKAMURA M, FUKUDA H, KURODA M  歯科放射線  41-  97  -97  2001/09/30
• アデノウィルスE40rf6によるBcl-2ファミリーBNIP3の機能抑制

  青柳 麻里, 東野 史裕, 安田 元昭, 佐野 英彦, 向後 隆男, 進藤 正信  歯科基礎医学会雑誌  43-  (5)  638  -638  2001/08/20
• Exogenous Expression of Osteopontin Enhanced Adhesive Activity of Osteoblast-like Cell

  YOKOYAMA A., YASUDA M., YAMAZAKI K., TANIGUCHI N., KAWASAKI T., HIGASHINO F., SHINDOH M., KOHGO T.  日本補綴歯科學會雜誌 = The journal of the Japan Prosthodontic Society  45-  147  -147  2001/06/01
• nm23-h1 suppresses invasion of oral squamous cell carcinoma-derived cell lines without modifying matrix metalloproteinase-2 and matrix metalloproteinase-9 expression

  MH Khan, M Yasuda, F Higashino, S Haque, T Kohgo, M Nakamura, M Shindoh  AMERICAN JOURNAL OF PATHOLOGY  158-  (5)  1785  -1791  2001/05  [Not refereed][Not invited]
   

  nm23-N1 is a candidate gene for the suppression of cancer metastasis. Several studies on human breast, hepatocellular, gastric, ovarian, and colon carcinomas and melanomas have shown that reduced nm23-H1 expression was closely related to metastatic progression with poor prognosis. However, the biochemical mechanism by which nm23-H1 suppresses the metastasis has yet to be elucidated. In this study, we analyzed the correlation between nm23 expression, cell motility, and the invasive abilities of six different oral squamous cell carcinoma cell lines (HSC2, HSC3, HSC4, KB, OSC19, and OSC20). Reduced mRNA/protein expression of the nm23-H1 was observed in three cell lines (HSC2, HSC3, and HSC4). These cell lines exhibited increased cell motility and an invasive character on organotypic raft culture. On the other hand, the cell lines (KB, OSC19, and OSC20) that showed a higher expression of nm23-H1 exhibited a threefold to fivefold reduced motility and also reflected fewer invasions compared to the former three cell lines. Because the HSCS cells demonstrated the lowest nm23-H1 expression with the highest cell motility and invasive character, we established nm23-H1-transfected NSCS cell lines to investigate whether exogenous nm23-H1 protein could inhibit cell migration and invasive activity. These transfectants showed a significant reduction in cell motility with exogenous nn23-H1 in a dose-dependent manner, and exhibited a noninvasive character. An immunofluorescence study demonstrated a distinct stress-fiber distribution at peripheral region of these transfectants, However, no significant difference of matrix metalloproteinase (MMP)-2 and MMP-9 expression was observed between mock transfectant and nm23-H1-transfected cells. These findings suggest that nm23-H1 inhibits the invasive activity of oral squamous cell carcinoma by suppression of cell motility without altering the MMP-2 and MMP-9 status.
• nm23-H1 Suppressed the Migration Activity of Oral Squamous Cell Carcinoma Cells Under Stimulation of Type-I and -IV Collagen

  MAHFUJUL HAQ Khan, YASUDA Motoaki, SEJUTY Haque, NAKAMURA Motoyasu  北海道歯学雑誌  22-  (2)  168  -177  2001  [Not refereed][Not invited]
  
• M. Yasuda, K. Yamazaki, S. Hamahira, T. Kawasaki、M. Nakamura, M. Shindoh "Functional Dissection of BH3 Only Protein BNIP3" Tumor Res. 36:23-28 (2001)*

  2001  [Not refereed][Not invited]
  
• 31:BNIP3 activates ced-3 induced apoptosis

  Yamazaki K, Yasuda M, Kohgo T, Shindoh M  Oral medicine & pathology  5-  (2)  123  -123  2000/12/20
• アデノウイルスと細胞のクロストーク--アデノウイルスの遺伝子産物と相互作用する細胞内蛋白質

  東野 史裕, 安田 元昭, 進藤 正信  蛋白質核酸酵素  45-  (8)  1350  -1357  2000/06
• Functional identification of the apoptosis effector BH3 domain in cellular protein BNIP1.

  M Yasuda, G Chinnadurai  Oncogene  19-  (19)  2363  -7  2000/05/04  [Not refereed][Not invited]
   

  BCL-2 family proteins play a central role in apoptosis regulation in mammals and in C. elegans. Mammalian cellular and viral anti-apoptosis proteins such as BCL-2 and E1B-19K interact with several cellular proteins. Some of these interacting proteins promote apoptosis and belong to the BCL-2 family. Certain BCL-2 family pro-apoptotic proteins such as BAX and BAK share extensive sequence homology with BCL-2. In contrast, certain pro-apoptotic proteins such as BIK and BID share a single death effector domain, BH3, with other BCL-2 family proteins. By mutational analysis, we show that one of the cellular proteins, BNIP1 (previously Nip-1), that interacts with BCL-2 family anti-apoptosis proteins is a 'BH3 alone' pro-apoptotic protein. Transient transfection of BNIP1 induces a moderate level of apoptosis. Deletions of the N-terminal 32 amino acid region and the C-terminal trans-membrane domain did not significantly affect pro-apoptotic activity. In contrast, deletions encompassing a region containing a motif similar to the BH3-domain abrogated the apoptotic activity. Substitution of BNIP1 BH3 domain for the corresponding sequence in BAX efficiently restored the apoptotic activity of BAX, establishing the functional identity of the BH3 domain of BNIP1. The N-terminal deletions of BNIP1 (that retain the BH3 domain) enhanced the level of interaction with BCL-X(L). Mutants containing the BH3 deletions were still able to heterodimerize with BCL-X(L) while mutants lacking both the N-terminal region and the BH3 domain were unable to heterodimerize, suggesting that BNIP1 may bind to BCL-X(L) via two different binding motifs.
• Hanzawa M, Shindoh M, Higashino F, Yasuda M, Inoue N, Hida K, Ono M, Kohgo T, Nakamura M, Notani K, Fukuda H, Totsuka Y, Yoshida K, Fujinaga K."Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cellinvasion by activati・・・

  2000  [Not refereed][Not invited]
   

  Hanzawa M, Shindoh M, Higashino F, Yasuda M, Inoue N, Hida K, Ono M, Kohgo T, Nakamura M, Notani K, Fukuda H, Totsuka Y, Yoshida K, Fujinaga K."Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cellinvasion by activating matrix metalloproteinase genes." Carcinogenesis. 21(6):1079-85.(2000)*
• Shindoh M, Adachi M, Higashino F, Yasuda M, Hida K, Nishioka T, Ono M, Takayama S, Reed JC, Imai K, Totsuka Y, Kohgo T."BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma."Oral O・・・

  2000  [Not refereed][Not invited]
   

  Shindoh M, Adachi M, Higashino F, Yasuda M, Hida K, Nishioka T, Ono M, Takayama S, Reed JC, Imai K, Totsuka Y, Kohgo T."BAG-1 expression correlates highly with the malignant potential in early lesions (T1 and T2) of oral squamous cell carcinoma."Oral Oncol. 36(5):444-449. (2000)*
• Clinical analysis of adenoid cystic carcinoma treated with radiotherapy

  OHMORI K, HOSOKAWA Y, OBINATA K, KANEKO M, YASUDA M, YAHATA H, NAKAMURA M, WATANABE Y  歯科放射線  39-  90  -90  1999/10/01
• BAG-1 can modify radio-sensitivity with p53 independent manner

  SUGAWARA Y, YASUDA M, KHAN MH, NAKAMURA M  歯科放射線  39-  88  -88  1999/10/01
• BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3.

  M Yasuda, J W Han, C A Dionne, J M Boyd, G Chinnadurai  Cancer research  59-  (3)  533  -7  1999/02/01  [Not refereed][Not invited]
   

  Apoptosis is regulated by interaction of viral and cellular BCL-2 family antiapoptotic proteins with various pro-apoptotic proteins, several of which are also members of the BCL-2 family. Cellular protein BNIP3 is a BCL-2 family proapoptotic protein that interacts with viral antiapoptosis proteins such as adenoviruses E1B-19K and EBV-BHRF1 and cellular antiapoptosis proteins such as BCL-2 and BCL-x(L). Database searches indicate that the human genome encodes an open reading frame for a protein, BNIP3 alpha, that shares substantial homology with BNIP3, The BNIP3 alpha open reading frame encodes a protein of 219 amino acids that contains a conserved BH3 domain and a COOH-terminal trans-membrane domain, characteristic of several BCL-2 family proapoptotic proteins. BNIP3a interacts with viral antiapoptosis protein E1B-19K and cellular antiapoptosis proteins BCL-2 and BCL-x(L). Overexpression of BNIP3 alpha in transfected cells results in apoptosis and suppresses the antiapoptosis activity of E1B-19K and BCL-x(L). Like BNIP3, BNIP3 alpha seems to be predominantly localized in mitochondria, These results suggest that BNIP3 alpha is a structural and functional homologue of BNIP3, BNIP3 and BNIP3 alpha seem to be the first examples of homologues among the various human proapoptotic proteins. Northern blot analysis reveals that BNIP3a is expressed ubiquitously in most human tissues. In contrast, BNIP3 is expressed well in several human tissues and less abundantly in certain tissues such as placenta and lung. These results suggest that although BNIP3 and BNIP3 alpha may promote apoptosis simultaneously in most human tissues, BNIP3 alpha may play a more universal role.
• Yamano S, Tokino T, Yasuda M, Kaneuchi M, Takahashi M, Niitsu Y, Fujinaga K, Yamashita T :"Induction of transformation and p53-dependent apoptosis by adenovirus type 5 E4orf6/7 cDNA. ", J Virol., 73(12):10095-103 (1999)*

  1999  [Not refereed][Not invited]
  
• Regulation of apoptosis by a Caenorhabditis elegans BNIP3 homolog.

  M Yasuda, C D'Sa-Eipper, X L Gong, G Chinnadurai  Oncogene  17-  (19)  2525  -30  1998/11/12  [Not refereed][Not invited]
   

  We have identified a C. elegans protein, ceBNIP3, homologous to the human BCL-2/EIB-19K interacting BCL-2 family pro-apoptotic protein BNIP3. In transiently transfected mammalian cells, ceBNIP3 complexes with CED-9, the worm homolog of BCL-2. CeBNIP3 also efficiently heterodimerizes with the cell death protease proCED-3 by direct binding via the prodomain. Transfection of ceBNIP3 and CED-3 results in enhanced proteolytic processing of the CED-3 zymogen and in cooperative induction of apoptosis. Coexpression of CED-9 suppresses the cooperative cell death induced by ceBNIP3 and CED-3. In cells coexpressing CED-9, ceBNIP3 and CED-3, all three proteins exist as a ternary complex suggesting that CED-9 may suppress cooperative apoptosis induced by CED-3 and ceBNIP3 by simultaneous complex formation with CED-3 and ceBNIP3. Our results suggest that ceBNIP3 may be a novel component of the C. elegans apoptosis paradigm and may initiate apoptosis by recruiting CED-3 to mitochondria and other cytoplasmic membranes.
• Adenovirus E1B-19K/BCL-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence.

  M Yasuda, P Theodorakis, T Subramanian, G Chinnadurai  The Journal of biological chemistry  273-  (20)  12415  -21  1998/05/15  [Not refereed][Not invited]
   

  Adenovirus E1B-19K and BCL-2 anti-apoptosis proteins interact with certain BCL-2 family pro-apoptotic proteins. A conserved domain, BH3, present in these proteins is essential for their pro-apoptotic activity and for heterodimerization with anti-apoptosis proteins. Cellular protein BNIP3 (previously NIP3) interacts with E1B-19K, BCL-2, BCL-x(L), and EBV-BHRF1. BNIP3 contains a motif similar to the BH3 domain. Deletion of the BH3-like motif in BNIP3 abrogates its ability to heterodimerize with E1B-19K and BCL-x(L). Substitution of the BH3 domain of BNIP3 for the corresponding sequences of BAX functionally restores the pro-apoptotic and protein heterodimerization activities of BAX. BNIP3 exhibits a delayed cell death activity that is partially relieved by deletion of the BH3 domain. BNIP3 suppresses the anti-apoptosis activity of BCL-x(L) in a BH3-dependent manner. BNIP3 contains a C-terminal trans-membrane (TM) domain similar to other BCL-8 family proteins and BNIP1 (previously NIP1). The TM domains of BNIP3 and BNIP1 can functionally substitute for the TM domain of a BCL-2 family member EBV-BHRF1. The BNIP3 TM domain exclusively targets the heterologous green fluorescent protein (GFP) to mitochondria. These results suggest that BNIP3 is a member of the BH3-contaning BCL-2 family of pro-apoptotic proteins and functions in mitochondria.
• Adenovirus E1B-19K/Bcl-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence

  YASUDA M.  J. Biol. Chem.  273-  (20)  12415  -12421  1998  [Not refereed][Not invited]
   

  Adenovirus E1B-19K and BCL-2 anti-apoptosis proteins interact with certain BCL-2 family pro-apoptotic proteins. A conserved domain, BH3, present in these proteins is essential for their pro-apoptotic activity and for heterodimerization with anti-apoptosis proteins. Cellular protein BNIP3 (previously NIP3) interacts with E1B-19K, BCL-2, BCL-xL, and EBV-BHRF1. BNIP3 contains a motif similar to the BH3 domain. Deletion of the BH3-like motif in BNIP3 abrogates its ability to heterodimerize with E1B-19K and BCL-xL. Substitution of the BH3 domain of BNIP3 for the corresponding sequences of BAX functionally restores the pro-apoptotic and protein heterodimerization activities of BAX. BNIP3 exhibits a delayed cell death activity that is partially relieved by deletion of the BH3 domain. BNIP3 suppresses the anti-apoptosis activity of BCL-xL in a BH3-dependent manner. BNIP3 contains a C-terminal trans-membrane (TM) domain similar to other BCL-2 family proteins and BNIP1 (previously NIP1). The TM domains of BNIP3 and BNIP1 can functionally substitute for the TM domain of a BCL-2 family member EBV-BHRF1. The BNIP3 TM domain exclusively targets the heterologous green fluorescent protein (GFP) to mitochondria. These results suggest that BNIP3 is a member of the BH3-contaning BCL-2 family of pro-apoptotic proteins and functions in mitochondria.
• Yasuda M, D'Sa-Eipper C, Gong XL, Chinnadurai G. :"Regulation of apoptosis by a Caenorhabditis elegans BNIP3 homolog." Oncogene. 17(19):2525-30.(1998)*

  1998  [Not refereed][Not invited]
  
• AUTOMATIC INJECTING SIALOGRAPHY

  OHMORI Keiichi, SAWAMURA Tsuyoshi, OBINATA Ken'ichi, KANEKO Masanori, YASUDA Motoaki, NAKAMURA Motoyasu, FUKUDA Hiroshi  歯科放射線  37-  74  -74  1997/09/10
• Reduction of radiation-induced xerostomia in nasopharyngeal carcinoma using CT simulation with laser patient marking and three-field irradiation technique

  T Nishioka, H Shirato, T Arimoto, M Kaneko, T Kitahara, K Oomori, M Yasuda, S Fukuda, Y Inuyama, K Miyasaka  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  38-  (4)  705  -712  1997/07  [Not refereed][Not invited]
   

  Purpose: Tumor control and reduction of postirradiation xerostomia in patients with nasopharyngeal carcinoma (NPC) using the three-field irradiation technique based on the CT-based simulation with laser patient marking was investigated. Methods and Materials: Seventy-eight patients with NPC were consecutively treated between 1983 and 1993. In 33 patients treated before 1987, target volume was determined using a conventional x-ray simulator with a reference of CT images, and the primary site was treated by the conventional parallel-opposed two-held technique (Group I). In 45 patients treated from 1987, target volume was determined using a CT simulator slice by slice, the treatment field was projected onto the patient's skin by a laser beam projector mounted on a C-arm, and the primary site was irradiated by a three-fields (anterior and bilateral) technique (Group II). In Group II, the shape of each field was determined using a beam's eye view to reduce the dose to the bilateral parotid glands. The three-field technique reduced the dose to the superficial lobe of parotid gland to about two-thirds of the dose given by the two-field technique. Radiation-induced xerostomia was evaluated by clinical symptoms and radioisotope sialography. Results: The 5-year survival rate and disease-free survival rate were 46.6 and 31.2% in Group I, and 46.8 and 46.5% in Group II, A large variation in the volume of parotid glands were demonstrated, ranging from 9 cm(3) to 61 cm(3) among patients treated with CT simulation. Forty percent of the patients in Group II showed no or mild xerostomia, whereas all of the patients in Group I showed moderate to severe xerostomia (p < 0.01). The radioisotope sialography study showed that the mean secretion ratio by acid stimulation was improved from 3.8% in the Group I to 15.2% in the Group II (p < 0.01). Conclusions: CT simulation was useful to determine the size and shape of each field to reduce the dose to the parotid gland, of which size varies largely among individual patients. The three-field technique based on CT simulation with laser patient markings is suggested to result in superior complication-free survival in terms of salivary dysfunction than did the conventional two-field technique with x-ray simulatior for NPC. (C) 1997 Elsevier Science Inc.
• Hida K, Shindoh M, Yasuda M, Hanzawa M, Funaoka K, Kohgo T, Amemiya A, Totsuka Y, Yoshida K, Fujinaga K. :"Antisense E1AF transfection restrains oral cancer invasion by reducing matrix metalloproteinase activities."Am J Pathol. 150(6):2125-21323.(1997)*

  1997  [Not refereed][Not invited]
  
• Nishioka T, Shirato H, Arimoto T, Kaneko M, Kitahara T, Oomori K, Yasuda M, Fukuda S, Inuyama Y, Miyasaka K. "Reduction of radiation-induced xerostomia in nasopharyngeal carcinoma using CT simulation with laser patient marking and three-field irradiati・・・

  1997  [Not refereed][Not invited]
   

  Nishioka T, Shirato H, Arimoto T, Kaneko M, Kitahara T, Oomori K, Yasuda M, Fukuda S, Inuyama Y, Miyasaka K. "Reduction of radiation-induced xerostomia in nasopharyngeal carcinoma using CT simulation with laser patient marking and three-field irradiation technique." Int J Radiat Oncol Biol Phys. 38(4):705-12. (1997)*
• Diagnosis of Cervical Lymphnodes by Ultrasonography : Definition of Short Axis of Lymphnode

  SATOH Takafumi, YASUDA Motoaki, NAKAMURA Motoyasu  歯科放射線  36-  55  -55  1996/09/01
• Analysis of chemosensitivity and oncogene expression in oral squamous-cell carcinoma

  YAMANO Shigeru, YASUDA Motoaki, NAKAMURA Motoyasu  歯科放射線  36-  31  -31  1996/09/01
• Correlation between invasion ability and matrix metalloproteinase expression in oral squamous cell carcinomas

  YASUDA Motoaki, YAMANO Shigeru, NAKAMURA Motoyasu  歯科放射線  36-  32  -32  1996/09/01
• Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity

  Chiba, I, M Shindoh, M Yasuda, Y Yamazaki, A Amemiya, Y Sato, K Fujinaga, K Notani, H Fukuda  ONCOGENE  12-  (8)  1663  -1668  1996/04  [Not refereed][Not invited]
   

  The p53 gene has been indicated to be a tumor is found in mutated form in common human cancers, Human papillomavirus (HPV) has oncogenic activity in cervical and oral squamous cell carcinomas (SCCs), The E6 protein of HPV is known to bind with p53 protein and inactivate the tumor suppressor activity by promoting p53 degradation, Because of this background, we examined 38 primary, resected specimens of oral SCCs for detection of p53 mutations and HPV DNAs, Exons 5 through 8 of the p53 gene were examined using the PCR-SSCP method, p53 Mutations were observed in nine cases (24%), HPV-DNA detection and typing were performed using PCR with 'high risk group' HPV-specific primers, HPV DNA sequences were detected in eight cases (21%), The AvaII digestion pattern of PCR-amplified HPV DNA showed that HPV-16 was present in all eight cases, Seven cases were p53 mutation-positive/HPV-negative, six cases were p53 mutation-negative/HPV-positive, and two intraosseus SCC cases were p53 mutation-positive/HPV-positive. Thus, 15/38 (40%) cases had inactivation of the p53 protein. Interestingly, p53 mutation-negative/HPV-negative cases had a poorer prognosis than p53 mutation positive or HPV-positive cases (P<0.01), We conclude that (1) mutation in the p53 gene and/or HPV infection are frequent (40%) in oral SCC; (2) inactivation of p53 function by mutation and HPV infection are important genetic events in the development of 40% similar to of oral SCCs; (3) p53 mutation and HPV infection are not mutually exclusive events and (4) other oncogenes or tumor suppresor genes may be crucial in the development of oral SCC if the prognosis is poor.
• Detection of dna fragmentation in γ -irradiated rat tongue epithelium

  Motoaki Yasuda, Takeshi Nishioka, Hiroki Shirato, Motoyasu Nakamura  Journal of JASTRO  8-  (1)  63  -66  1996/01/01  [Not refereed][Not invited]
   

  Apoptosis is the predominant form of cell death and occurs under variety of physiological and pathological conditions. In the present study, we detected the apoptotic cells and S phase cells in rat tongue epithelium in both normal and irradiated conditions. After 24 hours from irradiation, cell cycle arrest was obvious and the number of apoptotic cell reached the maximum level; however there was no detectable DNA fragmentation in basal cells. © 1996, Japanese Society for Therapeutic Radiology and Oncology. All rights reserved.
• Yasuda M., et al., : "Detection of DNA Fragmentation in gamma-irradiated Rat Tongue Epitherium" J. Jpn. Soc. Ther. Radiol. Oncol. 63-66(1996)*

  1996  [Not refereed][Not invited]
  
• MORPHOLOGICAL AND FUNCTIONAL PRESERVATION OF RADIATION TREATMENT

  SHIRATO Hiroki, HASHIMOTO Seiko, NISHIOKA Takeshi, YASUDA Motoaki  頭頚部腫瘍  21-  (3)  576  -580  1995/11/01
• Recognition thresholds of sour sensation for cases irradiated on tongue

  KANEKO Masanori, YASUDA Motoaki, OHMORI Keiichi, NAKAMURA Motoyasu, NISHIOKA Takeshi, KITAHARA Toshihiro, SHIRATO Hiroki, MIYASAKA Kazuo  日本放射線腫瘍学会誌学術大会報文集  17-  (1)  116  -116  1995/10/01
• 形態・機能温存と放射線治療

  白土 博樹, 橋本 井子, 安田 元昭  頭頚部腫瘍  21-  (2)  287  -287  1995/05/12
• Simultaneous carboplatin and radiotherapy for all stages of head and neck squamous cell carcinoma

  Y. Hosokawa, T. Kamada, H. Shirato, K. Ohmori, M. Yasuda, H. Yahata, T. Nishioka, T. Kitahara, T. Arimoto, Y. Inuyama  Clinical Oncology  7-  (3)  168  -172  1995  [Not refereed][Not invited]
   

  The study investigated the toxicity and efficiency of the concomitant administration of radiotherapy and carboplatin to patients with head and neck carcinomas. Sixty-three patients with head and neck squamous cell carcinomas, other than nasopharyngeal cancer and Stage I (UICC) larygneal cancers, were treated by external radiotherapy and four courses of carboplatin at a dose of 100 mg/m2 per week. In two patients, only three courses were possible due to renal toxicity. In the other 61 patients, toxicities were self-limiting and no patient required interruption of carboplatin administration. No patient required discontinuation of radiotherapy because of acute toxicity. Of 61 evaluable patients, a complete response (CR) was obtained in 11.5% and a partial response (PR) in 60.7% at 40 Gy. In 41 patients treated to 65 Gy (including two patients with maxillary sinus carcinoma, who were treated by debulking surgery), CR was obtained in 76.9% and CR + PR was 100% at the end of treatment. The actuarial survival rate of the 63 patients at 2 years was 69.2%, with a median follow-up period of 24.4 months. One of 12 patients who received salvage surgery after radical radiotherapy has died due to poor wound healing after the surgery. The schedule was safe, providing a weekly check of serum samples was possible. It is likely that the rate of local control and vocal cord preservation in laryngeal tumours might improve if concurrent carboplatin is used. Careful follow-up is required to determine the long-term effect of concomitant carboplatin administration. © 1995 The Royal College of Radiologists.
• Prognostic Significance of the PCNA Labeling Index in the Radiotherapy of Oropharyngeal Carcinoma

  Takuro Arimoto, Motoaki Yasuda, Takayuki Nojima, K. Ukraperuvaluthi-Pillai, Tadashi Kamada, Hiroki Shirato, Akio Takamura  The Journal of JASTRO  7-  (4)  321  -329  1995/01/01  [Not refereed][Not invited]
   

  Prognostic significance of the proliferating cell nuclear antigen (PCNA; cyclin) labeling index and the DNA ploidy pattern in the radiotherapeutic treatment of the oropharyngeal carcinoma were analyzed retrospectively. Paraffin-embedded specimen of 26 patients were utilized for analysis from 77 patients who were treated by radiotherapy (RT) at Hokkaido University Hospital between 1983 and 1991. Twenty-six specimens were selected mainly because of the availability of specimen and thorough clinical information about local tumor outcome. Significance of “classical” clinical parameters, such as the size of primary tumor, stage of disease, the sub-site of the primary tumor origin, pathological subclassification, and the age of patient, was analyzed first in all the 77 patients. The predictive value of PCNA labeling index and the DNA ploidy pattern in relation to these parameters were then investigated in 26 patients to clarify the potentials of both parameters as a new prognosticator. Only the primary tumor size (T1-3 vs. T4), and the site of primary disease (primaries of the ant. faucial pillar, tonsillar fossa and the soft palate did better than the other site; base of tongue, posterior wall, and the valecullar origin) were the “classical” factors significantly influencing local tumor control (p < 0.05). PCNA labeling index had no definite correlation with these “classical” prognostic factors, but it was found to influence three-year local tumor control with the statistically significant level. In those whose PCNA labeling index were below 30%, all (15/15) were controlled locally for more than three years, whereas 6 out of 11 tumors relapsed locally when the PCNA labeling index exceeded 30% (p < 0.05). The tendency did not change even after patients were stratified by tumor size. DNA diploidy was closely related to the PCNA labeling index of less than 30%, and there was a tendency to influence the local tumor control favorably (0.05 < p < 0.1), but less significantly. © 1995, Japanese Society for Therapeutic Radiology and Oncology. All rights reserved.
• Hosokawa Y, Kamada T, Shirato H, Ohmori K, Yasuda M, Yahata H, Nishioka T, Kitahara T, Arimoto T, Inuyama Y. : "Simultaneous carboplatin and radiotherapy for all stages of head and neck squamous cell carcinoma." Clin Oncol (R Coll Radiol). 7(3):168-72.・・・

  1995  [Not refereed][Not invited]
   

  Hosokawa Y, Kamada T, Shirato H, Ohmori K, Yasuda M, Yahata H, Nishioka T, Kitahara T, Arimoto T, Inuyama Y. : "Simultaneous carboplatin and radiotherapy for all stages of head and neck squamous cell carcinoma." Clin Oncol (R Coll Radiol). 7(3):168-72.(1995)*
• Human Papillomavirus Genome in Oral Carcinoma and Their Metastatic Cervical Lymph Node Tissues.

  Yasuda Motoaki, Shindoh Masanobu, Kohgo Takao, Amemiya Akira, Sawada Yukiharu, Fujinaga Kei  Tumor Research  29-  29  -37  1994  [Not refereed][Not invited]
   

  Twenty cases of oral squamous cell carcinoma (SCC) with cervical lymph node metastasis were investigated. Both primary lesions and metastatic lymph nodes were analyzed for the involvement of human papillomavirus (HPV) DNAs utilizing the polymerase chain reaction (PCR) method and dot blot hybridization. HPV DNAs were detected in five cases. Four primary lesions contained HPV-16 DNA, and one contained both HPV-16 and HPV-18 DNAs out of 20 cases examined. No HPV DNAs were detected in metastatic lymph node tissues in cases where HPV DNAs could not be detected in primary cancer tissues. The same types of HPV DNAs as those found in primary lesions were detected in metastatic lymph nodes including those with HPV-16 and HPV-18.
• Takinami S, Kaga M, Yahata H, Kure A, Oguchi H, Yasuda M. :"Radiation-induced hypoplasia of the teeth and mandible. A case report." Oral Surg Oral Med Oral Pathol. Sep;78(3):382-4. (1994 )*

  1994  [Not refereed][Not invited]
  

Association Memberships

• 北海道大学歯学会   日本分子生物学会   日本生化学会   日本細菌学会北海道支部   日本癌学会   日本ウイルス学会   

Research Projects

• Development of oncolytic virus that induces tumor immunity for oral cancers

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2022/04 -2025/03 

  Author : 東野 史裕, 安田 元昭
• 難治性口腔がんに有効な腫瘍溶解ウイルスの開発

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 安田 元昭, 東野 史裕

   

  令和3年度は主に2つの実験を行い以下の結果を得ることができた。①ヒト5型アデノウイルスのE4領域に任意の遺伝子配列を挿入することが可能か否かを判定するために、E4領域約3500塩基対の配列のうちorf6、orf6/7領域をルシフェラーゼ遺伝子に改変したプラスミドを構築し、その発現をルシフェラーゼアッセイにて解析した。この実験により、orf6の開始コドンを保存することにより、E4orf1-orf4の転写・翻訳を損なうことなく候補遺伝子を組み込むことが可能であることが確認された。またこの転写・翻訳反応は細胞内でE1Aの発現がなくても進行することが明らかとなった。②オートファジー関連タンパク質どうしの結合性をmammalian two hybrid法にて解析した結果、BNIP1およびBNIP3に存在するLIR( LC3 interacting region)を介したLC3AおよびLC3Bとの直接的な結合を確認できた。一方、BNIP1およびBNIP3とSQSTM1/p62の結合性に関しては、BNIP1-SQSTM1/p62の組み合わせにおいてのみ有意な結合性が認められ、この結合の一部にはBH3ドメインの関与の可能性が示唆された。仮にアデノウイルスの構造タンパク質の一部（Hexon、Fiberなど）がBNIP1、BNIP3およびSQSTM1/p62と結合することによって選択的にオートファジーによる排除を受けているのであれば、E1B19Kなどのタンパク質による抗オートファジー効果は、宿主細胞のウイルス排除機構の不活化に対して大きな役割を果たしていると考えられた。
• Oncolytic virus targeting oral cancer stem cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2018/04 -2021/03 

  Author : YASUDA MOTOAKI

   

  All transcribed mRNAs, a minimum of 20 late viral RNAs, contain the common 5’UTR, which is called as Tripartite Leader, and this sequence is required for preferential translation of late viral proteins during late infection period. In the present study, we have demonstrated a novel role for E4 orf4 protein in the late adenoviral mRNA translation. Tripartite Leader tagged construct (TrLdr-LUC) transfected cells showed more than 10-fold higher luciferase activity compared with control transfectants, whereas no significant increase of luciferase mRNA amount was detected in TrLdr-LUC transfected cells. At least 2 folds increase of luciferase activities were observed, and the mutational analysis indicated that E4orf4, but not E4 orf3 or E4 orf6, was required for the enhanced translation. It is likely that E4 orf4 or OMB1 containing complex is involved in so-called Ribosome Shunt system.
• Applied of RNA stabilization system-based oncolytic virus to oral cancer

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2017/04 -2020/03 

  Author : Higashino Fumihiro

   

  In this study, we investigated the effects of the newly developed oncolytic adenovirus Ad + AU. Ad + AU replicated more efficiently in cancer cells than in normal cells, and further induced cell death. Ad + AU was suggested to be an oncolytic virus that is more effective than the clinically applied virus ONYX-015. As a result of examining the effect of Ad + AU using animals, it was clarified that Ad + AU has an oncolytic effect even in a cancer-bearing model. Furthermore, the combined effect of the anticancer drug and Ad + AU was examined, and when used in combination with paclitaxel, the anticancer activity of Ad + AU was enhanced. These results indicate that Ad + AU is a useful oncolytic virus.
• Analysis of comprehensive gene regulation systems in oral cancer stem cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2015/04 -2018/03 

  Author : YASUDA MOTOAKI

   

  HuR has been shown to stabilize ARE mRNA. Clinicopathological reports have demonstrated that the higher expression of HuR is one of the important prognostic factors of malignant tumor cases. In the present study, we found the novel functions of HuB, a neuron specific ELAV member, in oral squamous-cell carcinoma cell lines. The lower differentiated quamous-cell carcinoma cell (SAS) expressed a higher amount of HuB mRNA whereas the highly differentiated squamous-cell carcinoma cell (HSC2) expressed only a small amount of HuB mRNA. On the other hand,HuB transfected HSC2 (HSC2-HuB) showed an obvious nuclear export of HuR in the HuB dependent manner. The higher expressions of ARE mRNAs were observed in HSC2-HuB cells which expressed exogenous HuB. We also demonstrated that HSC2-HuB showed a modest keratinization morphology compared with parental HSC2 cells.
• Development of cancer sensor by utilizing oncolytic virus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2013/04 -2015/03 

  Author : Higashino Fumihiro

   

  In this study, we developed a cancer sensor by applying oncolytic adenovirus which we exploited in the previous study. We didn’t succeed to produce a GFP including adenovirus, but noticed that the co-infection of two kinds of viruses, a GFP-expressing adenovirus and an our oncolytic adenovirus, is available for a cancer sensor. In cancer cell such as HeLa and H1299, GFP was activated by co-infection with our oncolytic adenovirus, whereas it was not activated in normal BJ cells. The data indicates that co-infection of these virus is suitable for a cancer sensor.
• がん細胞は自身の生存をかけ放射線域を逃れるべく上皮間葉移行をなしうるか

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  Date (from‐to) : 2012/04 -2015/03 

  Author : 西岡 健, 芳賀 永, 安田 元昭, 武島 嗣英

   

  今年度の実験において我々は5'UTRおよび3'UTR依存性発現調節の候補遺伝子の機能解析を行った。5'UTR結合タンパク質に関してはSRSFファミリーが有望であると考えられた。これらタンパク質はHIF-laあるいはcMYCの5'UTRを挿入したルシフェラーゼリポーターからのタンパク質発現を数倍に活性化することができた。これら候補遺伝子の発現を、マイクロアレイにて比較したところ、予想通り高転移性株では発現が高い傾向が認められた。これらの発現が遺伝子プロモーター領域の配列の違いによるものか否かを判断するため、アレイ解析に用いた細胞のゲノムDNAを出発材料にしてプロモーター領域のシークエンスを行ったところ顕著な塩基置換は認められず、メチル化などのエピジェネティックな変化の解析が今後の課題として提起された。3'UTR依存性発現調節の候補遺伝子としてELAVLファミリータンパク質の1つであるELAVL2の機能解析を行った。ELAVL1はほぼ全ての細胞において多量に発現し主に核局在を示すが、ELAVL2は主に細胞質に局在する。ELAVL2導入細胞株ではELAVL1を導入した細胞よりも、ARE mRNA由来のタンパク質発現レベルが有意に上昇していた。蛍光タンパク質タグを付与したHuRを単独で強制発現させてもELAVL1は核局在を示したが、ELAVL1とELAVL2を同時に発現させた細胞では細胞質局在するELAVL1が認められた。two-hybrid法にて、ELAVL1どうしの結合よりELAVL1-ELAVL2間のほうがより強い結合であることが示された。この結合にはELAVL2のN末端、C末端領域が必要であった。ELAVL1の核から細胞質への局在変化にはELAVL2が大きく関わっていること、ELAVL2の発現量はがん細胞の悪性度と相関することが示唆された。これまで3'UTR依存性の翻訳抑制機構を担うと報告されてきたELAVL1はむしろHIF-1αの翻訳効率を低下させることが示され、従来考えられてきた調節機構はより複雑なものであることが示唆された。
• Investigation of mechanism on obstruction of osseointegration caused by micro movement by occlusion

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2011/04 -2015/03 

  Author : ATSURO Yokoyama, YASUDA Motoaki, YAMAMOTO Satoru, AMIZUKA Norio

   

  The purpose of this study is to investigate effects of micro movement by occlusion on osseointegration. Left first molars of maxillae in rats were extracted, and then, titanium screws were immediately inserted into the sockets. After 1 week of the implantation, occlusal loading was provided for 1 or 2 weeks in the experimental groups. Histochemical and histomorphometric assessment and analysis of gene expression by microarray were carried out. Trabecular thickness of the experimental group was thicker than the control group. The index of sclerostin-positive osteocytes located close to the implants bearing 2 weeks’ loading was significantly attenuated compared with the control group, indicating undisturbed activities of osteoblasts. Microarray analysis showed that gene expression for bone formation and BMP were higher in the experimental group. It was suggested that the early occlusal loading for immediate implant accelerate osseointegration.
• Development of new oncolytic adenovirus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2011/04 -2014/03 

  Author : Higashino Fumihiro

   

  In this study, we attempted to develop a new oncolytic adenovirus, which is able to propagate in cancer cells relative to normal cells. The protein-deleted adenovirus (Ad-delta) replicated in cancer cells, whereas the virus proliferation was restricted in normal cells. Furthermore, Ad-delta induced cell death of cancer cells and it was effective for human cancer xenografts in nude mice. These results indicate that Ad-delta is available as an oncolytic virus.
• Reconstruction of Jaw Bone with 3 dimension scaffold treated by functionalized carbon nano-material

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2010 -2012 

  Author : YOKOYAMA Atsuro, AKASAKA Tsukasa, YAMAMOTO Satoru, TAKITA Hiroko, SATO Yoshinori, YASUDA Motoaki, SHIBA Kiyotaka, YUDASAKA Masako

   

  Functionalized carbon nanomaterials were developed by covalent bond of b-FGF to multiwalled carbon nanotubes and absorption of simvastatin to carbon nanohorns, and effects of them on bone formation were evaluated by animal experiments. Extensive bone formation was observed in the scaffolds with functionalized carbon nanomaterials in comparison with normal carbon nanomaterials. Also, biocompatibility carbon nanomaterials was observed by animal experiments for a long term.
• Cross-talk between innate-immunological responses and oral oncogenesis.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2010 -2012 

  Author : YASUDA Motoaki, HIGASHINO Fumihiro

   

  Approximately 50% decreased protein expression was observed for3’UTR of HIF-1 containing constructs compared to the control vector. In particular, a highly-aggressive breast cancer cell line MDA-MB-453 demonstrated a higher protein expression compared to a less-aggressive breast cancer cell line MCF7. It is plausible that 3’UTR dependent degradation of HIF-1a mRNA is repressed in several cancer cells which indicate malignant phenotypes. We also found the same phenotype in oral squamous cell carcinoma cells. Thus, 3’UTR dependent regulation of mRNA degradation might play an important role in the progression (vascular induction, invasion oreptherial-mesenchymal transition) of oral cancers.
• Immunological approaches to the mechanisms of bisphosphonate-related osteonecrosis of the jaw

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2009 -2011 

  Author : DEYAMA Yoshiaki, TOTSUKA Yasunori, YASUDA Motoaki, IIZUKA Tadashi, YOSHIMURA Yoshitaka

   

  In this study, we investigated the effects of bisphosphonate on osteoclasts and their precursor cells to clarify one of the mechanisms of bisphosphonate-related osteonecrosis of the jaw(BRONJ). BRONJ-related bisphosphonates strongly inhibited osteoclastogenesis and bone resorption activity. On the other hand, they showed cytotoxicity and enhanced microbial pathogens-induced cell death.
• The secret of radiation-surviving cell : multidisciplinary approach to eradicate cancer

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2008 -2010 

  Author : NISHIOKA Takeshi, YASUDA Motoaki, HAGA Hisahi, TUTSMI Kaori, SAKAI Masaharu, HOMMA Akiniro

   

  Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation". To better clarify the mechanism, gene expression profiling and pathological experiments were performed. A mouse fibrosarcoma cell line, QRsP, was used. Cells were irradiated with 10 Gy. Colony assay and cloning were performed. Six clones were established. cDNA analysis was performed on the clone that showed the largest number of colonies on the 2nd 10 Gy irradiation. Mouse transplantation experiment was then carried out. cDNA analysis showed that cyclin-dependent kinase inhibitors, p16 and p57 were down-regulated ; 14.8- and 12.0-fold, respectively for the tolerant clone. Matrix metalloproteinase 3 and 13 were up-regulated ; 22.5- and 25.8-fold, respectively. Transplantation ratio was 100% (5/5) for the tolerant clone whereas it was 40% (2/5) for the parent. Under light microscope, the mean mitotic cell number was 4.0+/-3.9 for the parent, and 12.8+/-3.4 for the tolerant clone (p<0.01, Student's t-test). This study implies that repopulation is not a temporary reaction to irradiation. It is caused probably by "clonal" gene-expression changes, though it remains unknown whether the changes are attributable to tolerant cell selection or to gene mutation/modification.
• Oral Tissue Reconstruction with 3D Scaffolds and Surface Modification Using Carbon Nanotubes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2007 -2009 

  Author : YOKOYAMA Atsuro, AKASAKA Tsukasa, SATO Yoshinori, YAMAMOTO Satoru, YASUDA Motoaki, HIGASHINO Fumihiro, SHINDO Masanobu

   

  In this study, we developed 3D collagen scaffolds coated with carbon nanotubes to reconstruct jaw bone. It was shown that initial adhesion and spreading of osteoblastic cells on 3D collagen scaffold was accelerated by CNTs coating, especially center part of scaffold. Also, it was suggested to apply CNTs for reconstruction of jaw bone, because excellent osteoconductivy was observed when 3D collagen scaffolds coated with CNTs were implanted in bone.
• Radiation-induced highly-malignant cancer cells: the mechanism and how to deal with it

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2006 -2007 

  Author : NISHIOKA Takeshi, YASUDA Motoaki, HAGA Hisashi, HOMMA Akihiro, TSUTSUMI Kaori

   

  Novel function of transcription factor ATF5: blockade of p53-dependent apoptosis induced by irradiation. Purpose: p53-dependent cell death is considered as a predominant mechanism of tumor cell apoptosis induced by ionizing irradiation, and a large number of studies have shown that mutant p53-harboring tumor cells with a p53 gene mutation exhibit radioresistance. However, even tumor cells that express wild-type p53 display various degrees of radiosensitivity to ionizing irradiation. This indicates that there are additional pathways that affect p53-dependent cell death mechanisms. Here we describe a novel molecule that represses radiation-induced apoptosis by inhibiting trans-activation activity of p53. Materials and Methods: We irradiated QRsP cells, a mouse transplantable malignant cell line, at 10Gy and from surviving colonies 24 sub-clones were established. These clonal cells were re-irradiated and the most readio-sensitive clone, QRsPIR-5, was used in the current experiment. All sub-cloned cells had the same morphological appearances as the parental QRsP cells and there were no p53 mutations of among any of the clones. Colony assay indicated that the survival fraction of QRsPIR-5 cells at a dose of 10 Gy was less than 20% of the survival fraction of the parental QRsP cells. Flow cytometer analysis also indicated a higher apoptotic index after infection with recombinant adenovirus containing wild-type p53 (Ad-p53) in QRsPIR-5 cells compared with the parent cell (26.5% vs. 7.1%). Interestingly, the parental and QRsPIR-5 cells had the same degree of tumorigenicity in a transplant experiment. Results: Comprehensive cDNA array analyses demonstrated differential gene expressions between the parental and QRsPIR-5 cells, both in vitro and in vivo. Among these, 23 genes were expressed differently both in vitro and in vivo between the parent and QRsP-5 cells with a high stringent threshold (less than 0.5 or more than 2.0). One such gene, bZIP transcription factor ATF5, which might explain difference in radio-sensitivity. Exogenous expression of ATF5 gave QRsPIR-5 cells a radioresistance level similar to that of the parental cells (colony assay). Moreover, QRsPIR-5 cells gained resistance to Ad-p53-induced apoptosis. A luciferase reporter assay demonstrated that over-expressed ATF5 repressed the transcriptional activity of wild-type p53 drastically. Interestingly, time lapse analysis indicated accelerated motility in ATF5-transfected QRsPIR-5 cells. Conclusion: It is likely that ATF5 is a potent repressor of p53. Elevated expression of ATF5 in a tumor may relate to enhanced malignant phenotypes, such as radio-resistance or greater cell motility.
• Cross-talk between viral. infection and bacterial infection in oral region

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2006 -2007 

  Author : YASUDA Motoaki, SHIBATA Ken-ichiro

   

  In the present study, we demonstrated the involvement of toll-like receptor 2 (TLR2) in the recognition of human adenovirus type 5 (Ad5) and the repressive effect of adenovirus E1A to the NF-κB activation via TLR2 pathways. Mutational analysis revealed that adenovirus E1A inhibited the NF-κB activation utilizing two independent pathways involving transcriptional co-activator p300 and the transcription factor E2Fs. Molecular mechanism of the NF-κB repression was explained by the competition between E1A and NF-κB for the limited amount of nuclear p300/CBP coactivator or the complex formation between RelA/ p65 and E2Fs which are displaced from Rb family proteins by competitive E1A binding to Rb proteins. The complex formation between RelA/ p65 and E2Fs also abolished the E2F dependent transcriptional activation. It is revealed that human adenovirus possess the transcriptional interference strategy involving NF-κB /Rel family and E2F family proteins and that human adenovirus utilize the strategy for the activation of host cell cycle and the repression of host innate immunological response.
• Roles of Toll-like receptors in bacterial phagocytosis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2005 -2006 

  Author : SHIBATA Ken-ichiro, KUROKI Yoshio, YASUDA Motoaki

   

  Enormous lines of evidence have been accumulated that Toll-like receptors (TLRs) function as sensors for microbial invasion before uptake and degradation of bacteria. However, less is known about how signaling triggered by TLRs leads to phagocytosis of pathogens by phagocytes. This study was designed to determine whether stimulation of TLR2 with mainly the lipopeptide FSL-1 plays a role in phagocytosis of pathogens by macrophages. Diacylated lipopeptide FSL-1 markedly enhanced phagocytosis of S. aureus more strongly than that of E. coli; but did not enhance phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2+/+ mice but not those from TLR2-/-mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partially by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signaling pathways and that TLR2 by itself does not function as a phagocytic receptor. This study also demonstrated that CD14 and the C-type lectin DC-SIGN enhances the recognition of lignads by TLR2 and FSL-1 induced Th2 response in vivo.
• Development of regenerative therapy for jaw bone using scaffold with carbon nanotubes.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2004 -2006 

  Author : YOKOYAMA Atsuro, YASUDA Motoaki, AKASAKA Tsukasa, SHINDOH Masanobu, HORIUCHI Rumi

   

  In this study, investigation of biocompatibility of carbon nanotubes (CNTs), cell culture on the scaffold using with CNTs and fabrication of bulk CNTs were carried out for the purpose of reconstruction of jaw bone using with CNTs. CNTs were implanted in the subcutaneous tissue in rats for 2 years, and histological and ultramicrostructural investigation to CNTs were done. Some CNTs were observed in lysosome in macrophages and only slight granulomatous inflammatory response was recognized around CNTs at 2 years, although granulation tissue with phagocytes was seen around CNTs at early stage after implant. The degree of aggregation of CNTs became to slight with time. Most of typical structure of CNTs like tubes was not changed after 2 years by observation using TEM. These results suggested that CNTs was not strong prophlogistic substance and structure of crystal of CNTs was stable in vivo. Scaffolds with CNTs were made by vacuum filtration of the dispersed CNTs slurry onto polycarbonate (PC) membrane. Saos-2 (human osteoblast-like cell) was cultured on the scaffolds. Proliferation and alkaline phosphatase activity of Saos-2 cultured on the CNTs scaffolds were higher than those of PC membrane. Application of the CNTs scaffold for guided bone regeneration membrane to bone defects made on rat parietal bone was effective in osteogenesis compared to PC membrane. Bulk CNTs materials were fabricated by spark plasma sintering (SPS). Physical and mechanical properties of bulk CNTs were close to those of bone, and biocompatibility was excellent. These results showed that bulk CNTs made by SPS method was suitable for bone substitute materials. From these results, it was suggested that regenerative therapy for jaw bone using with CNTs would be possible.
• Molecular Mechanisms and Visualization of Radiation-induced tumor cell repopulation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2004 -2005 

  Author : NISHIOKA Takeshi, YASUDA Motoaki, HOMMA Akihiro, NAKADA Kunihiro

   

  Purpose/Objective : Treatment with any cytotoxic agent can trigger surviving cells in a tumor to divide faster than before. This phenomenon is widely recognized as "repopulation" among radiobiologists. Many clinical studies have also indicated that repopulation is one of the major treatment failure factors in radiotherapy. However, little is known about its molecular mechanism. To better clarify the mechanism, gene expression profiling and pathological experiments were performed. Materials/Methods : A mouse fibrosarcoma cell line, QRSP (p53 wild type), was used in this study. Cells in culture medium were irradiated with 10 Gy using a 4MV linear accelerator at a dose rate of 1.8Gy/min. Ten thousand irradiated cells were seeded onto culture dishes for colony assay and cloning. After 10 days, colonies were stained and counted. At the same time, 6 clones were established and stored at -80C for future use. To observe radio-resistance in these clones, cells were irradiated again with 10Gy and colonies were counted as described above. Oligonucleotide microarray analysis (Agilent Technologies) of the expression of over 20,000 genes was performed on the clone that showed the largest number of colonies (hereafter referred to as the "tolerant clone"), using the parental QRSP cell line as a control. Twenty thousand cells of the tolerant clone and of QRSP were injected subcutaneously into 5 female mice (C57BL/6) each. The mice were sacrificed 28 days later and the transplantation ratio, tumor volume, and pathological status between the two groups were compared. For histologic analyses, tumors were fixed in 10% formalin, embedded in paraffin, and stained with H&E for light microscopy. Two pathologists examined the samples and mitotic cells were counted in 3 randomly selected high-power fields. Results : The QRSP control line produced 25 colonies after 10Gy irradiation. For the 6 clones, the number of colonies produced after the second 10Gy irradiation was 86,42,38,34,5 and 3, respectively. Gene expression was compared between QPSP and the most tolerant clone (which produced 86 colonies). A total of 133 genes were up-regulated (i.e., >2.0-fold increase) and a total of 239 genes were down-regulated (i.e., <2.0-fold decrease) in the tolerant clone. Among the up-regulated genes, the following were expressed at a particularly high level : IL6 (36.0-fold increase), matrix metalloproteinase (MMP) 13 (25.8), MMP3 (22.5), and GRO1 oncogene (12.8). Among the down-regulated genes, p16 and p57 were expressed at a particularly low level : 14.8- and 12.0-fold decrease, respectively. On sacrifice, tumors were observed in 2 of 5 mice (40%) for QRSP, and in all 5 mice (100%) for the tolerant clone (p<0.05, chi-square test). The mean tumor volume was greater for the tolerant clone : 1.92+/-1.68(SD) versus 0.82+/-1.04 g. Of the 5 tumors from the tolerant clone, one showed deep muscle invasion on palpation and under microscope examination. The mean mitotic cell number was 4.0+/-3.9 for QRSP, and 12.8+/-3.4 for the tolerant clone (p<0.01, Student's t-test). Conclusions : Colony assay showed that radio-resistant clones were established following 10Gy irradiation. The microarray data of the most tolerant clone was distinctive in terms of genes related to cell-cycle regulation (cyclin-dependent kinase inhibitors p16 and p57) and aggressive nature (GRO1 and MMPs). These in-vitro gene signatures correlated well with in-vivo tumor status. This study implies that repopulation is related to "clonal" gene expression changes caused by irradiation, though it is unknown whether the changes are attributed to tolerant cell selection or to gene mutation/modification (such as methylation).
• Studies on recognition mechanism of oral microbes-derived lipopeptides by Toll-like receptors

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2004 

  Author : SHIBATA Ken-ichiro, KUROKI Yoshio, YASUDA Motoaki

   

  Toll-like receptor 2 (TLR2)plays a key role in the recognition of microbial lipoproteins/lipoeppetides. However, details in molecular mechanisms of the recognition still remains unknown. This study was therefore carried out to determine the molecular mechanism by which TLR2 recognizes microbial lipoproteins/lipoeppetides. We have found the following findings. 1.The level of the activity of bacterial triacylated lipopeptides to stimulate macrophages was lower than that of mycoplasmal diacylated lipoepptides. 2.Both lipid and peptide portions of mycoplasmal diacylated lipopeptides are involved in the recognition by TLR2. 3.Leucine residues at positions 107,112 and 115 are involved in the recognition of mycoplasmal lipoproteins/lipopeptide and S.aureus peptidoglycans by TLR2 in combination with TLR6. 4.There is at least one leucine residue in all 8 leucine-rich repeats of TLR2 which is responsible for the the recognition of mycoplasmal lipoproteins/lipopeptide and S.aureus peptidoglycans by TLR2 in combination with TLR6. 5.Leucine residues in β-sheet structure of leucine-rich repeats are involved in the recognition of mycoplasmal lipopeptides by TLR2 in combination with TLR6. 6.Mycoplasmal lipoproteins possesses the activity to induce apoptosis of HEK293 cells through TLR2/TLR2 and the activity is mediated by FADD and MyD88 downstream of TLR2.
• 歯根膜再生・人工歯根膜開発を目的とした歯根膜幹細胞の分化における遺伝子発現の解明-DNAマイクロチップを用いた歯根膜幹細胞の遺伝子発現の検索-

  日本学術振興会：科学研究費助成事業 萌芽研究

  Date (from‐to) : 2003 -2004 

  Author : 横山 敦郎, 安田 元昭

   

  本研究においては,歯根膜幹細胞の種々の細胞への分化に関する成長因子の同定および分化した細胞の遺伝子発現様式の差異を明らかにすることを目的に,以下の研究を行った. WKAウィスター系5週齢雄性ラットから,下顎切歯を抜去し,15%FBSおよび抗生剤を含むα-MEM中に静置し,2週後まで初代培養し,アウトグロースした細胞を歯根膜細胞として回収した.回収した細胞を,デキサメサゾン(Dex),アスコルビン酸(Asc),βグリセロフォスフェイト(βGP)を含む培地とこれらを含まないコントロールの培地の2種の培地で2週間培養し,骨関連タンパクであるオステオカルシンと歯根膜特有のタンパクであるXII型コラーゲンについてRT-PCRを行いmRNAの発現を検索した.XII型コラーゲンの発現は,コントロールとDexを含む培地の両者に同様に認められたが,オステオカルシンはDexを含む培地で著しく強く発現していた.この結果から,Dexで骨芽細胞に誘導される幹細胞が,採取された歯根膜細胞には含まれることが明らかとなった.この結果をもとに,Dexを含む培地,b-FGFを含む培地およびこれらを含まないコントロール培地の3種の培地で歯根膜細胞を3日培養した後,RNAを回収し,DNAマイクロチップで網羅的にmRNAの発現を解析した.その結果,mRNAの発現は,b-FGFを含む培地とコントロールの培地では,ほとんど差異が認められなかったが,Dexを含む培地とでは差異が認められた.この結果から,b-FGFは歯根膜幹細胞を分化させることなく増殖させ,またDexは,歯根膜幹細胞を骨芽細胞へ分化させることが示唆された.
• 骨芽細胞への分化で発現する遺伝子の導入細胞を用いた顎骨再生療法の開発

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2002 -2004 

  Author : 川崎 貴生, 進藤 正信, 安田 元昭, 横山 敦郎, 山本 悟, 東野 史裕

   

  次年度である本年度は,ラット骨髄細胞のデキサメサゾンを用いた骨芽細胞への誘導およびDNAマイクロチップを用いてデキサメサゾン誘導の遺伝子発現に対する影響を経時的に検索するとともに,発現に差違が認められた遺伝子についてRT-PCRを用いて定量的に解析した. 生後8週齢のフィッシャー系雄性ラットを実験動物として用い,Maniatopolusの方法に準じて,大腿骨から骨髄細胞を採取し,初代培養後,ディッシュに付着した細胞を骨髄幹細胞として分離した.この細胞をデキサメサゾンを含む培地で誘導した場合と含まない培地を用いた場合に分け培養した.培養後2,5,14日後に細胞を回収し,RNAを抽出し,DNAマイクロチップにて遺伝子の発現を解析した.骨形成に関係するいくつかの遺伝子に発現の差違が認められ,これらについてさらにRT-PCRで経時的に,定量的に解析した.アルカリフォスファターゼについては,2,5日後については,差違が認められなかったが,7,14日後はデキサメサゾンを含まないほうが,有意に強い発現を示した.BMP2に付いては,同様に2,5日では差違が認められなかったが,7日後ではデキサメサゾンで誘導したほうが有意に強い発現を示し,14日後では同様の発現であった.同じファミリーであるBMP3は,2,5日後ではデキサメサゾンを含まないほうが強い発現を示したが,7,14日後ではデキサメサゾンて誘導したほうが,強い発現を示した.フォリスタチンは,デキサメサゾンで誘導した場合,2日後から5日後にかけて発現強度が1/2に減弱し,その後発現強度は増強した.以上の結果から,ラット骨髄細胞をデキサメサゾンで誘導した場合,5〜7日後に骨芽細胞への分化が生じている可能性が示唆された.
• The removal system at irradiated apoptotic cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2002 -2003 

  Author : YASUDA Motoaki, HIGASHINO Fumibiro, SHINDOH Masanobu

   

  The ionizing irradiation is a powerful toll for killing the cancer cell. The irradiated cancer tissue will be shrinked during the radiological treatment. However, it is sometimes difficult to find the apoptotic cells with conventional pathological examination especially in the cases of solid tumors. This phenomenon may be explained by the rapid pahogocytosis of dead cancer cells. Phagocytic cell will recognize the surface proteins of dying cell, so called "eat me signal". Phosphatidyl serine is the candidate of such molecule, however there is a lot of such kind of molecules relating phagocytosis of dying cells. We established the cell line named QRSP-IR which already irradiated at the dose of 10Gy. We transplanted these two cell lines on the back of c57B6 mice. Transplanted QRSP-IR made a significant tumor mass in all cases (5/5) whereas only 40% of mice which injected parental cells exhibited the tumor mass. The mean volume of tumor mass with QRSP-IR was significantly lager than the mass with parent cell line. Pathological examination revealed that irradiated cell exhibited the higher mitotic index, focal invasion tendencies and the survival character from the removal system of host animal. Then we performed the comprehensive detection of gene expression by DNA chip analysis. The expression level of 592 genes was higher and 480 were lower in QRSP-IR mass. It was interesting that these genes included the CD14, scavenger receptor or other membranous proteins. We will find the key molecules from present candidate genes in the future.
• Research on factors involved in prognosis of oral carcinoma.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2002 -2003 

  Author : SHINDOH Masanobu, OHIRO Yoichi, HIGASHINO Fumihiro, KOBAYASHI Masanobu, TOTSUKA Yasunori, YASUDA Motoaki

   

  EWS/ETS is a chimeric protein identified in most Ewing's sarcomas. We demonstrate that telomerase is a new target of EWS/ETS fusions TERT mRNA were up-regulated by EWS/Ets ; however, EWS/Ets function in an EBS-independent manner and EWS/ETS fusion proteins were shown to activate human telomerase activity as a transcriptional co-activator. The adenovirus E4orf6 is a viral oncoprotein and it has been shown to inhibit the apoptotic activities of p53 and p73. We demonstrate that the adenovirus E4ort6 protein directly inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family and that the export signal of E4orf6 from the nucleus to the cytoplasm was shown to be important for the inhibition of apoptotic activity. We investigated the activation mechanism of MMP-2 in oral squamous cell carcinomas. CAT assay revealed that MT1-MMP promoter was activated by E1AF and Real-time RT-PCR study in 25 patients with tongue squamous cell carcinoma showed synergistical increasing expression of E1AF and MT1-MMP. These results indicate that E1AF positively regulates transcription from MT1-MMP genes, which plays an important role in invasion and metastasis of squamous cell carcinoma of the tongue by converting pro-MMP-2. into active-MMP-2 We investigated expression levels of VEGF-C mRNA in 48 cases of 0SCC by real-time RT-PCR. High levels of VEGF-C expression were identified 20 of 48 cases examined Expression level of VEGF-C was significantly associated with cancer cell metastasis in cervical lymph node. These results suggest that high expression of VEGF-C would be a predictive parameter for cervical lymph node metastasis of 0SCC, especially in the early stage tumors.
• Molecular etiological study on buccal carcinogenesis in Bangladesh

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2003 

  Author : YASUDA Motoaki, HIGASHINO Fumibito, SHINDOH Masanobu

   

  The prevalence of human papillomavirus (HPV) in healthy and pathological oral tissue samples collected in Bangladesh and Japan was analyzed. PCR amplification with a consensus primer set (1) and a novel DNA array system detecting twenty-five different HPV genotypes were utilized. PCR amplification was able to detect one copy of HPV plasmid. The DNA array system used in this study could detect fifty copies of HPV plasmid without cross hybridization. HPV was found in 2 of the 28 Japanese samples but not in any of the 55 normal mucosa or 21 cancer samples from Bangladesh. These results together with results of a previous study on prevalence of HPV in Japanese oral cancers (detection of HPV genome in 25% of oral cancer samples ; (2)) suggest that a geometrical difference in HPV infection exists, and that HPV plays an insignificant role in the carcinogenesis of buccal mucosae in Bangladesh. It is possible that differences in life styles contribute to the discrepancy in HPV detection rate. However, the mechanism of HPV transmission to the oral region is still obscure.
• With the aim of expansion of the application of dental implants for the aged person and the patient who have systemic disease : the development of dental implants combined with the cell tranfected gene of bone related protein

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2003 

  Author : YOKOYAMA Atsuro, YASUDA Motoaki, SHINDOH Masanobu, HIGASHINO Humihiro, YAMAMOTO Akiko, HANAWA Takao

   

  The purpose of this study was expansion of the application of dental implants for sparse bone of the aged person and the patient who have systemic disease. Firstly, we investigate the adhesive potential of exogeneous osteopontin(OP) and osteonectin(ON) expressing osteoblastic cells that we have established. The expression of exogeneous OP and ON was confirmed by Western blotting. Parental Saos2, Saos2-puro, Saos2-pcDNA3, and Saos2-OP and Saos2-ON were seeded on the polystyrene dish at a density of 1.5 x 10^5 cells in serum-free DMEM. The cell adhesion rates(CAR) were counted after 6, 12 and 24 hours of incubation. Also, the cell adhesive shear force (F) and cell adhesive area (A) were measured in serum-free DMEM after 24 hours. The CAR of Saos2-OP was higher than those of parental cells (p<0.05), and that of Saos2-ON was lower than those of parental cells (p<0.05). F/A of Saos2-OP had higher than the parental cells, though no significance was observed in the difference between them. These results suggest that human OP is a potent enhancer of osteoblast-like cell adhesion. Secondary, we investigated the effect of diabetes on bone tissue around endosseous implants after osseointegration. Titanium implants were inserted in the femora of rats. At 8 weeks after implantation, diabetes was induced by intra peritoneal injection of streptozotocin. Histopathological and histomorphometric evaluations were carried out. There was no effect of diabetes on the ratio of bone-implant contact, while the amount of newly formed bone in the control group was greater than that of the diabetic group. Next, we developed dental implants combined with the bone marrow cells. Undifferentiated bone marrow cells were cultured on dental implants with dexamethasone for 2 weeks. Dental implants with cultured bone marrow cells were implanted into subcutaneous tissue in rats. Histopathogical investigation and analysis by RT-PCR were done. At 4 weeks after surgery, bone tissue was observed on the surface of dental implants and expression of mRNA of osteocalcin was detected by RT-PCR. These results suggested that the dental implants combined with the cells transfected gene of bone protein were effective on the sparse bone of the aged person and the patient who have systemic disease.
• Induction of apoptosis in oral squamous cell carcinoma by activating caspase

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2002 

  Author : TOTSUKA Yasunori, YASUDA Motoaki, HIGASHINO Fumihiro, SHINDOH Masanobu, TEI Kanchu, KOBAYASHI Masanobu

   

  Bnip3 is a pro-apoptotic pretein, which contains BH3 and transmembrane domains. We constructed several deletion mutants of Bnip3 and investigated their biological functions. The transmembrane domain of Bnip3 was not required fo the association of Bnip3 and ced-3 ; however, transmembrane domain deleted mutant could not initiate the apoptotic cascade. Immunofluorescence study demonstrated that wild type Bnip3 and ced-3 co-localized on mitochondria, and transmembrane domain deleted mutanat could not show the co-localization. These results suggest that recruitment of ced-3/caspase onto the mitochondrial membrane is essential for Bnip3 intiated apoptosis. The adenovirus E4orf6 is a viral oncoprotein know to cooperate with the E1A gene product in transforming primary murine cells. It has been shown to inhibit the apoptotic activities of p53 and p73 through direct binding to these proteins. Here, we demonstrate that the adenovirus E4orf6 protein inhibits apoptosis mediated by BNIP3 and Bik, which are BH3-only proteins of the Bcl-2 family. This activity was not mediated by p53 and p73 because E4orf6 had the same effect on the apoptosis in Saos-2 cells that do not express p53-related genes. It was also ascertained that E4orf6 could change the mitochondrial localization of BNIP3 and Bik. A mutant lacking the nuclear export signal of E4orf6 failed to inhibit apoptosis and to translocate BNIP3 protein from the mitochondria. Moreover, it was also established that E4orf6 was able to interact with BNIP3 and Bik in vitro. In BNIP3 protein, the region required for the interaction included the transmembrane domain, which is required for the localization of BNIP3 to the mitochondria. These results suggest that E4orf6 is exported from the nucleus to the cytoplasm, enabling it to interact with BH3-only proteins, eventualy leading to the inhibition of apoptotic activity.
• With the aim of early convalescence of the occlusal function in the immediate implant after tooth extraction :

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2002 

  Author : YOKOYAMA Atsuro, HIGASHINO Humihiro, YASUDA Motoaki, SHINDOH Masanobu, NAKASU Masanori, KAWASAKI Takao

   

  The purpose of this study was to densify the sparse cancellous bone to achieve the early convalescence of the occlusal function in the immediate implant after tooth extraction. Firstly, we developed the calcium phosphate cement with the sufficient plasticity for easy molding into the desired shape. This cement had the good handling property and biocompatibility. Furthermore, we developed the sandwich technique and suggested that it was effective for the jaw bone augmentation. Although it was possible to make the calcium phosphate cement with porous structure, it was impossible to develop the calcium phosphate cement with bio-absorbability. It is necessary to make the cement with bio-absorbability for densification the cancellous bone. Therefore, we developed β-TCP-CM-chitin composite material that was injectable from the # 14 needle. No inflammatory response was observed when this composite was implanted in soft tissue and bone tissue. It showed this material had good biocompatibility. Active bone formation was observed in subcutaneous tissue when it was implanted with BMP. The problems such as BSE on the collagen are reported, while collagen was used for the carrier for BMP usual. It was suggested that this composite material was suitable for the carrier for BMP. Histological investigation was carried out to evaluate the effectiveness of this composite material as a bone substitute. At I week after implant, active bone formation was observed on the surface of the material. Absorption of the material and replacement to the bone was seen with time. It was showed that the cancellous bone was densified. These results suggested that the possibility of the early convalescence of the occlusal function in the immediate implant after tooth extraction.
• Analyses of relation between cytokine production, apoptosis and clinicopathological findings in Sjogren's syndrom patients

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2002 

  Author : HIROSHI Fukkuda, MOTOAKI Yasuda, ICHIZO Kobayashi, MASANOBU Shindo, CHIHIRO Sugiura

   

  This study reviled that there were correlations between functional disorder of a salivary gland and distinctive punctate-shadowing on a sialograpy (grade III and grade IV) as well as decreased number of CD 4/ CD 8 ratio of peripheral blood cells in Sjogren's syndrome (SS) patients. It has been reported that there was a significant correlation between destruction of peripheral duct of parotid gland and appearances of distinctive punctate-shadowing on a sialogram (grade III and grade IV). So that we speculated that histopathological destruction or disorder of peripheral duct in salivary glands might be a explicable reason of functional disorder of a salivary gland in SS patients. Further more some researchers reported that decreased number of CD 4/ CD 8 ratio of peripheral blood cells in SS patients might have correlation with cytokine productivity of peripheral lymphocytes of SS patients. In this study, we reported the increase in Th1/Th2 ratio in salivary gland as well as systemic increase correlated with the severity of the underlying condition assessed by sialograpy. We have to investigate and clarify the details of shift of cytokine network, Th1/Th2 balance and cause of functional disorder in SS patients Further more, Vissink reported that functional disorder of a salivary gland by irradiation was followed with histopathological change of the organ in rat experiment system. This might allowed us to assume that functional disorder of a salivary gland was followed with histopathological change in SS patients as well. Recently it was reported that disorder of signaling pathway in salivary gland had correlation with malfunction of salivary gland in SS as well as other salivary gland diseases. We thought that it was necessary to clarify the correlation between signaling pathway and funcional disorder of salivary gland in SS patients.
• Gene therapy for oral squamous cell carcinoma targeting tumor specific promoter activation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : SHINDOH Masanobu, KOBAYASHI Masanobu, YASUDA Motoaki, HIGASHINO Fumihiro

   

  Oral squamous cell carcinoma is the mostcommon malignant tumor in oral and maxillofacial region, and has a property that oral carcinoma is able to be recognized macroscopy because of the superficial occurrence of tumors. This characteristics contribute to the gene targeting therapy. First, we examined E1AF expression in normal and tumor tissues. E1AF is anete-oncogene family transcription factor, and we have noted that E1AF can upregulate promoter activities of several matrix metalloproteinase (MMP) genes and showed that invasive potentials of oral squamous cell carcinoma-derived cell lines are correlated with expression of E1AF and MMPs. We analyzed the E1AF expression in cell lines and resected tumors of non-small-cell lung cancers (NSCLC). Fifteen out of 17 cell lines of NSCLC and 12 of 19 tumors expressed E1AF mRNA while normal lung tissue and concomitant normal cells within tumors did not express E1AF mRNA. Similar results were obtained in oral carcinoma and normal tissues. RT-PCR was utilizedto examine the expression of E1AF. Among the 27 patients, E1AF was detected in 15 cases and was not detected in normal tissues.Bnip3 is a pro-apoptotic molecule that contains BH3 and trans-membrane ? domains. BH3 only proteins are now thought to be the initiator of apoptotic signals. Our results suggest that BnipS induces apoptosis by recruiting caspases onto mitochondrial membrane through TM domain. Bik, a BH3 only molecule, expressing adenovirus vector driven by E1AF promoter was reconstructed (Ad-F-Bik). SiHa, a squamous cell carcinoma cell line was transplanted into nude mice. Ad-F-Bik was applied in vivo tumors twice a week. Tumor growth was inhibited by application of Ad-F-Bik, and apoptosis of tumor cells was induced. These results indicate that gene therapy targeting tumor specific expressing molecule is effective using adenovirus vector.
• Caspase cascades which activated by ionizing irradiation

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : YASUDA Motoaki, SHINDOH Masanobu, HIGASHINO Fumihiro

   

  1. Radiation resistance of human normal and tumor cell lines. An enzymatic activities of caspase-8 and caspase-9 of human normal (MRC5) and tumor (HL60, MGF7) were analyzed after 10Gy gamma-irradiation. HL60 expressed a significant amount of activated caspases, whereas MRC5 showed only a few amount of active caspase-9 (no active caspase-8 was detected). We concluded that radiation resistance and an amount of active caspase were well corresponding. 2. A conditioned medium from irradiated MRC5 contained elevated amount of chemoattractants. We found that irradiated MRC5 derived conditioned medium contained increased amount of chemoattractants. Northern blotting and competitive activity of anti-HGF antibodies indicate that one of these chemoattractants is HGF. 3. Ionizing radiation induced gene expression (DNA array analysis) We performed DNA analysis for irradiated MRC5. There were no significant changes in bcl-2 related genes. We found the up-regulation of caspase-2,-6 and 10. We also found the up-regulation of apoptosis suppressing genes. We concluded that these up-regulation of anti-apoptotic proteins may explain the radiation resistance of normal connective tissue.
• The role of E1AF, an ets-oncogene family tranascription factor in cell cycle regulation.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2000 -2001 

  Author : TOTSUKA Yasunori, YASUDA Motoaki, HIGASHINO Fumihiro, SHINDOH Masanobu, KOBAYASHI Masanobu

   

  E1AF is a member of the ets-oncogene family transcription factor, and E1AF was shown to bind to the promoter region of MMPs that induce transcription from multiple MMP genes. We demonstrated that Ad type 12 E1A is able to suppress the invasion ability of a cancer cell line by inactivating E1AF expression. Ad12E1A expressing oral squamous cell carcinoma cell line was established. Northern blotting indicated that the expression of E1AF mRNA decreased in transfected cells. Reduced expression of MMPs was identified both in mRNA levels and in protein levels. Reduced transcriptional activity of MMP gene was confirmed by CAT assay. These results suggest that Ad12E1A downregulates E1AF which, in turn, restrains invasion ability of human cancer cells by way of downregulaton of MMP expression. Ca9.22 cells that manifest low levels of E1AF and MMP-1 and -9 expression were transfected with metallothionein inducible E1AF expression vector. MMP-9 and p21_ protein were synergistically increased in ZnCl stimulated MT9.22 cells, and MMP-9 and p21 protein were expressed in the same cells when E1AF was induced in MT9.22 cells. These results imply that invasive growth of tumor cells occur in static state of cell cycle and E1AF play key role of this phenomenon. Luciferase assays, using different amounts of E1AF and p53 expression vectors along with p2 1 promoter luciferase reporter plasmids, demonstrate an increase in the activity of p21. Colony formation assay indicates that E1AF is capable of tumor suppression similar to p53. These results, therefore, suggest that E1AF is able to interact synergistically with p53 ; in consequence, this interaction can influence p21 leading to an enhancement of its activity and eventually, tumor suppression.
• Effects on gene expression of bone and cell-adhesive proteins by biomaterials

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : YOKOYAMA Atsuro, HIGASHINO Fumihiro, YASUDA Motoaki, SHINDOH Masanobu, KAWASAKI Takao

   

  The purpose of this study was to investigate the mechanism of adhesion of osteoblast to biomaterials. Firstly, the effect of the difference of the biomaterials on the adhesion of osteoblasts to them was investingated. Cell adhesion rate of a human osteosarcoma derived cell line (Saos2) to hydroxyapatite was same to that to titanium incubated without serum, whereas cell adhesion rate of Saos2 to hydroxyapatite was higher than that to titanium with serum. From these results, we speculated that the proteins within serum effected cell adhesion to biomaterials. We carried out the tranfection of osteopontin (OP) into Saos2. OP is a noncollagenous protein in bone matrix. It is reported that OP enhanced adhesive activity of osteoblast-like cells in vitro. We investigated the effect on adhesive activity of osteoblast-like cell to titanium by transfection of OP. Human OP cDNA given FLAG tag to C-terminus was subcloned to retrovirus vector (pBabe-puro). Selection was carried out with DMEM contained ouromyciiL Saos2 cell line that expressed OP-FLAG was cloned (Saos2-OP). Expression of OP was observed in only Saos2-OP by the RT-PCR and production of OP included FLAG tag was confirmed in only Saos2-OP. There was a significant difference between the numbers of Saos2-OP and parental cells attached to the titanium disc and polystyrene dish after 12 and 24 hours of incubation in serum-free DMEM, although there was no significance among the numbers of attached cells after 6 hours. Observation of SEM revealed the difference between the shapes of Saos2-OP and Saos2. The shape of Saos2-OP was smaller and thicker, compared with Saos2, and there were small global structure on the surface of Saos2-OP, but not on the surface of Saos2. These results suggested that human osteopontin is a potent enhancer of osteoblast-like cell adhesion to titanium, and that transfection of osteopontin influenced the shape of Saos2.
• Relationship between expression of E1AF and histological malignancies of oral squamous cell carcinoma

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : ONO Mitsunobu, YASUDA Motoaki, HIGASHINO Fumihiro, SHINDOH Masanobu

   

  E1AF is an ets-oncogene family transcription factor. Previous reports have noted that E1AF can up-regulate transcription from multiple matrix metalloprotease (MMP) genes that confers invasive phenotype on human cancer cells. We have reported that the close correlation of E1AF expression and cancer cell malignancy by in vitro studies. This Implies that E1AF expression may contribute to predict the prognosis of patients with oral squamous cell carcinoma. In the present study, we have investigated the expression level of E1AF, MT1-MMP as well as vascular endothelial growth factor (VEGF) and evaluated the correlation to the metastatic potential in oral cancer patients. Thirty-two cases of pathologically diagnosed as squamous cell carcinoma were investigated. All cases were T1 or T2 in the clinical staging. Lymph node metastasis was identified in 18 of the 32 cases examined. E1AF was positive 11 of the 32 cases, and lymph node metastasis was identified in 7 of the 11 E1AF positive cases (63.3 %). Five of the 32 cases were MT1-MMP positive, and all 5 cases were among the E1AF positive case. Four of the 5 MT1-MMP positive cases showed lymph node metastasis. VEGF was positive in 11 cases and lymph node metastasis was observed in 10 of the 11 VEGF positive cases (90.9%) that was statistically significant. There were 17 cases of which E1AF or VEGF was positive, and lymph node metastasis was identified in 12 of the 17 cases (70.6%). There were 15 cases in which E1AF and VEGF both were negative, and lymph node metastasis was identified in only 6 of these 15 cases (40%). These results indicate that the expression of E1AF and VEGF are important factors to predict the invasive and metastatic potential of oral squamous cell carcinomas.
• An atempt of gene therapy for oral squamous cell carcinoma

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : SHINDOH Masanobu, KOBAYASHI Masanobu, YASUDA Motoaki, HIGASHINO Fumihiro

   

  ElAF and Bax activation was investigated since ElAF was shown to activate promoter activity of p21waf1/cip1, a cell cycle inhibitory molecule. CAT (Chloramphenicol acetyl transferase) assay was carried out to determine the promoter activity of bax by transfectipn with bax reporter plasmid, ElAF, wild type (wt) or mutant (mt) p53 expression plasmids into a p53 diffident cell line. CAT activity increased with ElAF in a dose-dependent manner and synergistical increase of the promoter activity was observed with wt p53 and ElAF. Protein expression of Bax was confirmed in ElAF transfected cells by Western blotting. Growth suppression assay was performed and decreased colony formation was observed in ElAF transfected p53 diffident cells. These results suggest that ElAF associates with p53 to induce apoptosis through activating bax. E4orf6 is a newly identified viral oncoprotein of the adenovirus. E4orf6 markedly enhances the ability of transformed rat BRK and human 293 cells to form tumors in nude mice. The E4orf6 protein has been shown to interact with p53 and p73 gene products and to block the induction of apoptosis which is mediated by these antioncogenes. We investigated whether or not E4orf6 can influence molecules that induce apoptosis other than the p53 family, because p53-independent apoptosis pathways have also been proposed. We demonstrate that the adenovirus E4orf6 protein suppresses the apoptotic activity of BNIP3, a member of a group of an apoptosis initiators of the Bcl-2 family. Expression of E4orf6 inhibits BNIP3-mediated apoptosis and its mitochondrial translocation. E4orl6 interacts with BNIP3. In addition, the nuclear export signal of E4orf6 is important for these functions of E4orf6. Our data suggest that the pro-apoptotic protein of the Bcl-2 family is a target of the E4orf6 protein in transformed cells.
• 口腔偏平上皮がんの放射線感受性に関与する遺伝子の同定

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1998 -1999 

  Author : 安田 元昭

   

  口腔がん症例の放射線治療では、腫瘍組織のほか周囲正常細胞にも照射は行われ、正常組織にも照射による遺伝子発現の変化が生じているはずである。正常組織より放出されるタンパク質が腫瘍細胞に大きな影響を与えているのは予想に難くなく、この腫瘍一間葉間の相互作用が腫瘍細胞の放射線感受性に影響を及ぼすと考えられる。また腫瘍細胞自身の遺伝子発現、特に細胞死抑制的に働く遺伝子の発現は治療効果を左右する大きな因子である。 申請者は平成10年度の研究により、10Gyの照射を行ったヒト正常線維芽細胞において、HGF,ファイブロネクチンの発現が、照射24時間後より上昇することを確認した。HGFは多様な生物活性を持つタンパク質であり、肝細胞の再生、その他肺、腎細胞の再生に関与しこれら臓器の創傷治癒に正の働きがあることが示されている。これらのことから、ある放射条件においては、正常結合組織に照射線照射により腫瘍細胞を生存の方向に導く可能性のあるタンパク質の発現亢進が起こってる可能性が示唆された。 平成11年度は、腫瘍細胞に種々のアポトーシス抑制遺伝子を導入し、これらが放射線により引き起こされる細胞死を克服できるか否かを検討した。Bcl-xl,アデノウイルスE1B19K,Bag-l,サイトメガロウイルスUL37を導入したH1299細胞に照射を行いそのプレート効率の比較を行ったところ、Bcl-xl,アデノウイルスE1B19K、サイトメガロウイルスUL37は対照細胞に比較して、明らかな細胞死抑制能を持つことが示されたが、Bag-1はわずかな抑制を示すのみであった。この結果は、通常行われている放射線照射により起こる腫瘍治療効果の大きな部分が、近年注目を集めているミトコンドリア関連タンパク質により制御されている可能性をしめすものである。
• Identification of targetgenes of E1AF using two hybrid system

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -1999 

  Author : SHINDO Masanobu, YASUDA Motoaki, KOBAYASH Masandu, YOSHIDA Koichi, HIGASHINO Fumihiro

   

  E1AF is a member of the ets-oncogene family transcription factor, and E1AF was shown to bind to the promoter region of MMPs that induce transcription from multiple MMP genes. In the present study, we demonstrated that Ad type 12 E1A (Ad12E1A), a highly tumorgenic adenovirus product is able to suppress the invasion ability of a cancer cell line. 12-12S cell lines were established by transfection with Ad12E1A expression plasmid into HSC3, a highly invasive oral squamous cell carcinoma cell line. Expression of E1AF mRNA decreased in 12-12S cells compared to that of the parental HSC3 cells. Reduced expression of MMPs in 12-12S cells was identified both in mRNA levels and in protein levels. Furthermore, in vitro invasion assay using Matrigel revealed that the invasion ability of 12-12S cells was suppressed by half compared to that of HSC3 cells. These results suggest that Ad12E1A downregulates E1AF which, in turn, restrains invasion ability of human cancer cells by way of downregulaton of MMP expression. This may suggest that a negative-feedback circuit exists between Ad 12 E1A and E1AF. We examined the effect of HGF on the E1AF and MMP gene expression in terms of the invasive potential of the HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation. In contrast, no obvious upregulation of MMP-1 and MMP-9 mRNA was observed in ASE1AFHSC3 cells that was transfected with antisense E1AF expression vector into parental HSC3 cells. These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.
• ABNORMALITIES IN DNA REPAIR AND GENOMIC IMPRINTING IN ORAL PRECANCEROUS LESIONS.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1996 -1997 

  Author : CHIBA Itsuo, YASUDA Motoaki, SHINDOH Masanobu, MORIUCHI Tetsuya

   

  Oral squamous cell carcinoma (SCC) is the most common neoplasm in Sri Lanka, accounting for approximately 30% of all cancers in males. Epidemiologic evidence indicates that there is an unequivocal relationship between betel chewing oral carcinogenesis, suggesting there may be specific genetic targets of betel quid ingredients.The p53 gene has been indicated to be a tumor suppressor gene that is found in mutated from in common human cancers ; however, there are few reports about carcinogen-specificp 53 mutation. Because of this background, primary, resected specimens from 23 oral SCC_s,7 leukoplakias and 2 oral submucous fibrosis were collected from oral SCC patients in Sri Lanka and were used for the p53 mutation analysis. Exons 5 through 8 of p53 gene were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing. Mutations in the p53 gene were frequent (10/23,43%) in oral SCC specimens from Sri Lanka. Moreover, the mutations clustered significantly in exon 5 (7/10,70%) of the p53 gene and small deletions and inclusions other than point mutations were observed. These results indicate that 1) betel quid chewing may cause specific fenetic changes, including mutation in the p53 gene, 2) mutations in tthe p53 gene are not rare vvents in SCC patients who are betel quid chewers, which is in contrast to previous reports, 3) exon 5 of the p53 gene could be one of the specific targets for some betel quid ingredients, and 4) betel quid chewing may be one of the critical environmental factors in the development of oral SCC.
• ラフトカルチャーによる口腔がんの浸潤・転移活性の評価と転写因子E1A-Fとの関連

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1995 -1995 

  Author : 進藤 正信, 藤永 恵, 千葉 逸朗, 安田 元昭

   

  1 口腔扁平上皮がんの浸潤・転移活性を評価する目的で、2種類の異なったコラーゲン(1型コラーゲンのみ・1型+4型コラーゲン)を担体としたラフトカルチャーを行った。使用した細胞は口腔扁平上皮がん由来細胞株HSC3、Ca9.22を用いた。 2 Northern blottingではHSC3は、MMP(Matrix metalloproteinase)1(間質型コラゲナーゼ)およびMMP9(Gelatinase B)とets-oncogene familyに属する転写因子E1AFのmRNAの発現が顕著だったが、Ca9.22はそれらの発現はほとんど示さなかった。 3 両細胞とも、1型コラーゲンゲルのみでのラフトカルチャーでは浸潤する傾向は示さなかったが、HGF(Hepatocyte growth factor)を産生するMRC5 fibroblastをゲル中に混じると、HSC3細胞はゲル中に浸潤した。しかし、Ca9.22はMRC5を加えたラフトカルチャーにおいても浸潤する傾向は認められなかった。 4 さらに、HGFで刺激すると、HSC3はE1AFの転写の亢進が認められ、MMP1、MMP9の転写亢進も認められたが、Ca9.22は反応を示さなかった。 5 MRC5を加えないコラーゲンゲルを用いたラフトカルチャーにおいても、HGFを培地に加えることで、HSC3は浸湿性の増殖を示した。 6 Ca9.22をファイブロネクチン処理すると、Northern blottingでMMP2の転写が亢進した。しかしMMP1、MMP9には著名な変化は認められなかった。ラフトカルチャーにおいては、Ca9.22/ファイブロネクチン処理したものでは、1型コラーゲンには、浸潤傾向を示さなかったが、1+4型コラーゲンでは浸潤性に増殖した。 7 口腔扁平上皮がん手術摘出材料より初代培養したがん細胞がん細胞を、同様にラクトカルチャーを行い検索した。膨張性の発育を示した腫瘍細胞では、ほとんどゲル中への浸湿傾向は認められなかった。頚部リンパ節転移巣から分離した腫瘍細胞は著しい浸潤傾向を示し、とくに1+4型コラーゲンで顕著だった。
• ダウン症候群児の歯周疾患発症における細胞接着因子の役割

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1995 -1995 

  Author : 工藤 真幸, 安田 元昭

   

  ダウン症候群(DS)においては一般に歯周疾患の罹患率が高いといわれている。一方,細胞接着因子のひとつであるLFA-1(lymphocyte function associated antigen-1)のβ_2鎖は21番染色体上にコードされており,Talorらは21トリソミ-であるDSではその発現が増大していると報告している。また,LFA-1の欠損症である白血球粘着異常症においてはその臨床的特徴として小児期からの重篤な歯周疾患の発症が認められる。本研究ではこのLFA-1とDS児における歯周疾患との関連について検討した。 [試料および方法] 北海道大学歯学部付属病院小児歯科外来に通院中のDS児についてその末梢血を採取し,フローサイトメトリーにて解析した。抗体としてはLFA-1のβ鎖を認識するCD18およびα鎖を認識するCDlla,それぞれのモノクローナル抗体を用い,リンパ球,好中球のフローサイトメトリーのパターンについてCoulter社製EPIC ELITE Flocytometry Systemにより検討した。またそれぞれの患児について下顎前歯部のデンタルX線写真を撮影し,歯槽骨の状態について検討した。 [結果] DS児から採取したリンパ球のCD18についてフローサイトメトリーを実施した結果,健常児のものと比較してそのパターンに変異が認められた。つまり健常児ではa,b,2つの明らかなピークをもつ二峰性のパターンが認められたが,DS児ではaのピークの低下によりbのみの1ピークに近いパターンとなった。これらのピークの変化はT細胞のサブセット比の変化と相関するとの報告もあるが、本研究ではa/b比とTh/Ts比の明らかな相関は認められず,下顎前歯部のbone lossとの関連も明確ではなかった。一方,好中球についてはDS児,健常児ともに1つのピークでこのような変異は認められなかった。今後はこの2峰性のピークの違いとDS児における免疫機構との関連について検討する予定である。
• Molecular biology of morphological preservation of radiotherapy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1994 -1995 

  Author : SHIRATO Hiroki, YASUDA Motoaki

   

  To investigate the mechanism of morphological preservation of radiation therapy (RT), clinical, pathological, and molecular biological research was undertaken. By photographical and radilogical comparison between the pre-RT morphology and post-RT morphology, it was conclusive that morphological preservation is not merely ordinal tissue repair, but rather complex regeneration. Immunostaning of E-cadherin, which is one of the cell-cell adhesion molecule important in morphological developmement, showed that E-cadherin is stained even 5 years after the 65 Gy irradiation to the larynxgeal surface. Cell migration assay showed that more number of epitherial cells, HSC3 and CA9-22, were migrated by the conditioned medium of MRC5 which was irradiated 10 Gy than that of non-irradiated MRC5 Immunobiochemical quantitative analysis showed that the conditioned medium of MRC5 has more amount of HGF after 10 Gy irradiation than that of non-irradiated MRC5. These resultus suggested that morphological preservation after radiotherapy is depend on the molucular control of normal tissue, of which mechanism still await more investigation.
• HPV E6/E7遺伝子とapoptosisに関する分子生物学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1994 -1994 

  Author : 安田 元昭

   

  頭頚部におけるがんのほとんどは扁平上皮がんであり、これらの治療に際しては手術療法の他、放射線療法、化学療法、温熱療法が単独あるいは併用されている。しかしながら保存的療法といえる放射線等に対する腫瘍感受性のメカニズムについての分子生物学的研究はさほど進んでいないのが現状である。その中で、近年の腫瘍学におけるトピックスの一つとしてp21(waf1/cip1/sdi1)に関する諸研究があげられる。このタンパク質はp53により発現誘導され、Cdk2またはcyclin DとCdk4複合体に結合し、G1 cyclinとCdk複合体酵素の活性を抑制すること、また細胞の老化に伴い増加してくることがあきらかにされており、腫瘍感受性においても大きな役割を果たしていると思われる。我々は、頭頚部扁平上皮がん細胞株を用い、CBDCA(carboplatin)感受性とp53,p21の発現に関する検討を行い以下のことを明らかにした。 使用した細胞はHSC2,HSC3,HSC4,Ca922,KBでありCBDCAを様々な濃度で作用させた。 1.CBDCAは頭頚部扁平上皮がんに対して増殖を抑制し、細胞死を惹起する事ができた。細胞死に際して形態的あるいは分子生物学的にDNA fragmentationが認められた。 2.最も感受性の高かったHSC3ではCBDCAによりp21の転写が活性化されたがp53転写は増強されず従来の報告とは異なった活性化経路の存在が示唆された。 3.CBDCA処理によりCa9-22,KBにてp53の転写が増強した。両細胞では同時にp21転写も増強されていたがp53のmutationおよびHPV18の存在を考慮すると、ここにおいてもp53 independentなp21活性経路が予想された。 4.p16はKBにおいてのみCBDCA処理により転写が増強した。
• 口腔扁平上皮癌の放射線感受性に関する分子生物学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1993 -1993 

  Author : 安田 元昭

   

  今回の科学研究補助金(奨励研究(A))により以下の検索を行った。 1.口腔癌症例におけるHPV DNAの検出およびp53 mutationの解析 20例の口腔扁平上皮癌のDNA sampleに対して、HPVについてはconsensus primerを用いたdetection assay、p53についてはPCR SSCP法によるmutationの検出を試みた。これらのうち8例にHPV16型の存在を確認し、5例にp53のmutationを認めた。今後症例を重ねて予後との比較検討を行う予定である。 2.口腔癌細胞株に発現するnm23遺伝子の解析 口腔癌細胞株HSC2,HSC3,HSC4,Ca992,KB,OSC20のmRNAを出発材料としてRT-PCR法によりnm23遺伝子の増幅を行い半定量的解析および制限酵素切断パターンの解析を行った。HAC4,Ca922,KB,OSC20にてnm23H1,H2遺伝子の発現に差異は認めなかったが、HSC2ではnm23H1の発現量が低く、HSC3ではnm23H2遺伝子のPCR productのsizeが小さいことが見いだされた。今後はこの事実が細胞浸潤能、移転能とどのような関わりをもつかを明らかにしていく予定である。方法論的には核MMP nRNAの発現量の解析、マトリゲルを用いた浸潤能の解析を行う計画である。 3.舌癌におけるPCNA陽性率と頚部リンパ節転移の相関について 初診時T1N0あるいはT2N0であった口腔舌癌24症例に対して抗PCNAモノクローナル抗体による免疫染色を施しその陽性率と後発頚部リンパ節転移との関係を検索した。これらのうち後発転移は6例に認められ、これらの症例ではPCNA 陽性率が30%以上であった。これによりぜつ扁平上皮癌細胞の増殖活性と転移能にはかなりの相関性があることが示唆された。
• DNA腫瘍ウイルス

  Date (from‐to) : 1986
• Adenovirus,Human Papillomavirus

  Date (from‐to) : 1986

Industrial Property Rights

• 特開2007-228818:細胞培養容器、その製造方法および細胞培養方法  2007/09/13

  西 泰治, 田崎 剛, 福田 始弘, 柴田 健一郎, 安田 元昭, 菊池 裕子  株式会社クラレ  200903080352157588
• 特許第3600616号:ヒトパピローマウイルスを検出するためのプライマーセット、検出方法および検出用DNAアレイ  

  柴田 健一郎, 安田 元昭, 道又 理恵, 西村 訓弘  株式会社ジェネティックラボ  201103066882580568