TISSUE CULTURE RESEARCH COMMUNICATIONS 組織培養研究 The Journal of Experimental & Applied Cel Culture Research Vol.30 No.1 March 2011 The O ficialjournaloft

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2 TISSUE CULTURE RESEARCH COMMUNICATIONS 組織培養研究 The Journal of Experimental & Applied Cel Culture Research Vol.30 No.1 March 2011 The O ficialjournalofthe Japanese Tissue Culture Association Editor-in-Chief: TetsujiOkamoto,DepartmentofMolecularOralMedicineandMaxilofacialSurgery,DivisionofFrontierMedicalScience,GraduateSchoolof & ManagingEditor: BiomedicalSciences,HiroshimaUniversity,1 2 3,Kasumi,Minami-ku, Hiroshima ,Japan AsociateEditors: HiroyukiKuramoto(KanagawakenYobouigakukyoukai) TohruMasui(NIBIO) TakahikoSuzuki(TokyoUniv.Scho.Med.) ForeignReviewingEditors: R.I.Freshney(Univ.ofGlasgow,UK) L.Hayflick(Univ.ofCalifornia,SanFrancisco;USA) E.M.Levine(TheWistarInstitute;USA) J.P.Mather(RavenBiotech,Inc.) W.L.McKeehan(TexasA&M University,USA) ReviewingEditors: J.Enami(ZenyakuKogyoCo.Ltd.) N-H Huh(OkayamaUniversity) I.Ii(LonzaJapan) Y.Kitano(KitanoClinic) M.Kumegawa T.Matsumura(YokohamaBioIndustryCenter) Y.Mitsui(TokushimaBunriUniversity) S.Nagamori(KyorinUniv.) N.Nakamura(RERF) A.Niwa T.Ohno(Cel-Medicine,Inc.) T.Suzuki

3 NoticeforPhotocopying Ifyouwishtophotocopyanyworkofthispublication,youhave togetpermissionfrom thefolowingorganizationtowhichlicensing ofcopyrightclearanceisdelegatedbythecopyrightowner. <AlusersexceptthoseinUSA> JapanAcademicAssociationforCopyrightClearance,Inc. (JAACC) 6-41Akasaka9-chome,Minato-ku,Tokyo Japan Phone FAX <UsersinUSA> CopyrightClearanceCenter,Inc. 222RosewoodDrive,Danvers,MA01923USA Phone FAX 複写される方へ本会は下記協会に複写に関する権利委託をしていますので 本誌に掲載された著作物を複写したい方は 同協会より許諾を受けて複写して下さい 但し ( 社 ) 日本複写権センター ( 同協会より権利を再委託 ) と包括複写許諾契約を締結されている企業の社員による社内利用目的の複写はその必要はありません ( 社外頒布用の複写は許諾が必要です ) 権利委託先 :( 中法 ) 学術著作権協会 東京都港区赤坂 乃木坂ビル電話 (03) FAX(03) なお 著作物の転載 翻訳のような 複写以外の許諾は 学術著作権協会では扱っていませんので 直接発行団体へご連絡ください また アメリカ合衆国において本書を複写したい場合は 次の団体に連絡して下さい CopyrightClearanceCenter,Inc. 222RosewoodDrive,Danvers,MA01923USA Phone FAX Coverphoto 細胞アレイ上に形成されたヒト肝細胞スフェロイドの共焦点レーザー顕微鏡像 ( 左 ) 肝細胞がトランスポーター依存的に排出する蛍光物質 CDF をプローブとして胆管腔への排出を調べたところ 明らかに輸送が確認された ( 中 ) このプールは培地中の Ca 2+ を除去することにより境界が不明瞭になった ( 右 ) この胆汁プールは肝細胞間に形成された微小胆管腔であり 排出機能も有していると考えられた ( 演題 YIA-3 より )

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16 YIA-1 YIA-2 YIA-3 YIA-4 14

17 OG-1-1 OG-1-2 OG-1-3 OG-1-4 OG-2-1 OG-2-2 OG

18 OG-2-4 OG-2-5 OG-2-6 OG-3-1 OG-3-2 OG

19 SP-1 SP-2 SP-3 SP-4 SP-5 SP-6 SP-7 17

20 WS-1 WS-2 WS-3 Session in English Cell culture stress models in aging and cancer Chairpersons: Dr. ISHII NAOAKI (Tokai University School of Medicine) Dr. WADHA RENU (National Institute of Advanced Industrial Science & Technology) OE-1 TOPIC- The role of oxidative stress from mitochondria on aging and are-related diseases -- P.89 Tokai University School of Medicine, Basic Medical Science and Molecular Medicine, Department of Molecular Life Science NAOAKI ISHII, TAKAMASA ISHII, MASAKI MIYAZAWA OE-2 TOPIC- Biology of stress protein MORTALIN: focus on cancer and neurodegeneration -- P.90 National Institute of Advanced Industrial Science & Technology (AIST), RENU WADHWA, SUNIL KAUL 18

21 OE-3 TOPIC- Molecular insights into Ashwagandha leaf-derived factors for stress and aging -- P.91 National Institute of Advanced Industrial Science & Technology (AIST) SUNIL KAUL, DIDIK PRIYANDOKO, NAVJOT SHAH, RENU WADHWA OE-4 hnrnp-k promotes tumor metastasis by induction of genes involved in extracellular matrix, cell movement and angiogenesis -- P.92 1National Institute of Advanced Industrial Science & Technology (AIST), 2Graduate School of Life & Environmental Sciences, University of Tsukuba RAN GAO 1,2, TETSURO ISHII 2, SUNIL KAUL 1, RENU WADHWA 1 OE-5 PINK1 knockdown increases the apoptosis induced by adenovirus-mediated REIC/Dkk-3 overexpression on bladder cancer cells -- P.93 Dept. Cell Biol., Okayama Univ. Grad. Sch. Med., Dent. & Pharm. YU JIN, HITOSHI MURATA, KEN KATAOKA, MASAKIYO SAKAGUCHI, NAM-HO HUH OG-4-1 OG-4-2 OG-4-3 OG

22 OG-5-1 OG-5-2 OG-5-3 OG-5-4 OS-1 OS-2 OS-3 20

23 The Japanese Tissue Culture Association 84th Annual Meeting Work on and Overcome Diseases (Setagaya, Tokyo) 27th, 28th, May, 2011 National Center for Child Health and Development Ookura, Setagaya-ku, Tokyo Japan Pres: Shin Enosawa National Center for Child Health and Development Clinical Research Center, Division for Advanced Medical Sciences Tokyo Medical University, Third Department of Surgery TEL: FAX:

24 Greetings When appointed to set up 84th annual meeting by Scoiety President, Dr. Takahiko Suzuki, I was deeply honored and a little embarassed whether I could manage the meeting. However, as things are going, I realized the Japanese Tissue Culture Society (JTCA) has firm hard- and softwares to support the holding of annual meeting. On publishing the Book of Abstracts, first of all, I would like to express my deepest gratitude for persons who have devoted to give hands. The opportunity that I became a member of JTCA was given by Dr. Toshiharu Matsumura (current Honorary member). At that time, Dr. Matsumura was forwarding the establishment of public human tissue bank and I joined his project. I learned the significance of research use of human materials and ethical considerations from him. The experience influenced my later research life. The present theme of the meeting, Work on and Overcome Diseases is a message derived from our human tissue bank work. I hear that early JTCA members exchanged enthusiastically the information about culture techniques to establish cell lines from surgically resected tumor tissues. This original spirit is well taken over at present, shown as activities such as Education and Research Committee, etc. Also each annual meeting values the session of original papers. This time I do my best to keep the tradition. I wish you all have an invaluable time at the meeting. Best regards, Shin Enosawa Meeting president 22

25 Guidance for participants 1. Place Lecture Hall in Hospital, Seminar Hall in Research Institute National Center for Child Health and Development Ookura, Setagaya-ku, Tokyo Japan (Access) <Please note following items> Please be quiet in Hospital Lobby, Elevator, etc, where patients and families are using. All areas of the Center, including the garden and pavements are smoke-free. There is no smoking room inside the Center. Please use smoking rooms in the fast food shops or restraunts nearby. An annual public event is being held from 26th to 29th of May in Science and Research Lab of NHK, one block apart from the Center. Because the visitors use the same bus services from train stations, please have enough time to come. No discount of parking fee. Under 30min = free, from 31min to 60min = 600, additional 30min = 300!(#%)*+'&($,+(!"#$%&'& -&. /0, -&. 0780,9 :)59;)<9 -&.9 07C0,9 3&4+')4+,+*+5+ 6)*+ A)'2&9B($7($74)."#9:#$( 23

26 !"#$%&'( )*&+'*,-."/.!"#$%&'( 0-#-'+,1..2*#3&4&- )*&+'*,-."/. 0-#-'+,1.2*#3&4&- 5-&'6'7'.5&+--& 2. Schedule 27th, May 2011 At Seminar Hall in Research Institute 9:00- Registration 9:25 Opening remark by Dr.Takahiko Suzuki, Society President 9:30-10:30 Young investigator s awards nominees 10:30-10:45 Break 10:45-11:29 Original papers (I) Establishment and characterization of cells 11:29-12:35 Original papers (II) Methodology 12:35-13:08 Original papers (III) Research infrastructure 13:08-14:00 Break At Lecture Hall in Hospital 14:00-17:00 Joint Symposium of Japan Tissue Culture Association and Japan Society for Alternative to Animal Experiments Culture technology of ES and ips cells and prospectus for alternative to animal experiments 17:00-17:10 Break 17:10-18:00 Plenary Lecture Dr.Hidenori Akutsu Establishment of human embryonic stem cell with an aim of clinical research 18:10- Reception at Restaurant TSUBASA, 12th floor in Hospital 24

27 28th, May 2011 At Lecture Hall in Hospital 8:30- Registration 8:50-9:30 General assembly meeting 9:35-10:35 3rd Session of Training for Tissue Culture Instructor Course III (Trial) 10:40-12:00 Session in English Cell culture stress models in aging and cancer 12:00-13:00 Break 13:00-13:44 Original papers (IV) Cell intactness and transformation 13:44-14:28 Original papers (V) Cornea 14:28-14:45 Break 14:45-16:45 Public Symposium Work on and Overcome Diseases based on the Collaboration between Patients/Families and Researchers/Doctors Supported by National Center for Child Health and Development 16:45 Closing remark 3. To speakers This time, the head office does not receive submission of presentation files. Please note that speakers are responsible for own presentation. Of course, organizer does best to prepare presentation equipment; There are PCs for projection and checking (Windows and Macintosh). The PCs are installed PowerPoint 2007 (Windows) and 2008 (Mac). Files are acceptable only by USB flash memory. May use personal PC. Please note that the time to connect is included in the presentation time. When you have any trouble, feel free to consult staffs. Presentation time; 4. To nominees for Young Investigator s Awards The session is held from 9:30 to 10:30, 27th (Friday) of May, at Seminar Hall in Research Institute. The speakers are required both to make oral presentation and to display poster presentation (size; approx. A0 (W84cm x L119cm). Posters are put up on a given place by 9:20. Time for presentation 25

28 and discussion are 10 min and 5 min, respectively. Nominees are reviewed on not only the quality of research but also presentation skills such as construction of story, way of data presentation, adequateness of reply, etc. According to YIA Institution of the Japanese Tissue Culture Association, board members determine awardees. Awardees are announced at General assembly meeting held on Saturday, 28th of May (8:50am-9:30am). All nominees are required to attend the meeting. Awardees receive diploma and prize. Awardees are obliged 1) to write an essay to member correspondence, 2) submit an article about the work to Tissue Culture Research Communications. 5. General assembly meeting Being held from 8:50 to 9:30 on Saturday, 28th of May. Reports, deliberations, and resolutions are scheduled. Also, YIA awardees are announced and commended. 6. Session of Training for Tissue Culture Instructor (Trial) Being held from 9:35 to 10:35 on Saturday, 28th of May. The session is open for all participants. Since the session deals general contents about tissue culture, please join the session. 7. Fees Please pay at the entrance of the Hall. Member 6,000 (including Japanese Society for Alternative to Animal Experiments) Student member 3,000 (including as above) Non member 12,000 (with an Abstract Book) Non member, student 6,000 ( as above ) Honorary member Free (Participation Card sent in advance) Public symposium Free 8. Reception Being held from 18:10 on Friday, 27th of May at Restaurant TSUBASA (12th Floor in Hospital). Fee; 2, Booth At Seminar hall in Research Institute on Friday, 27th of May. 26

29 10. Board member meeting Being held on Thursday, 26th of May at Meeting Room 11 (The next room to Lecture Hall in Hospital). Details are mailed to the members. 27

30 10:30 10:45 Time Table 27th, Friday, May th, Saturday, May :30- Registration 9:00- Registration 8:50 9:25 Opening remark 9:30 General Assembly meeting 9:30 9:35 3rd Session of Training for Young investigator s awards Tissue Culture Instructor nominees 10:35 (Trial) 13:08 14:00 17:00 17:10 Original papers 10:40 I Establishment and characterization of cells II Methodology 12:00 III Research infrastructure Break 13:00 Joint Symposium of Japanese Tissue Culture Association and Japan Society for Alternative to Animal Experiments Culture technology of ES and ips cells and prospectus for alternative to animal experiments Plenary Lecture Establishment of human embryonic stem cell with an aim of clinical research 18:00 18:10- Reception 14:28 Session in English Cell culture stress models in aging and cancer Break Original papers IV Cell intactness and transformation V Cornea 14:45 Public Symposium Work on and Overcome Diseases Based on the Collaboration between Patients/Families and 16:45 Researchers/Doctors 16:45 Closing remark 28

31 <Acknowledgments> (Japanese alphabetical order) Planning Committee Isao Asaka, Kanako Eto, Takahiko Suzuki, Kenichi Manaka, Naoki Yamamoto Program planning Isao Asaka, Yasuyuki Sakai, Toshiaki Takezawa (Joint Symposium) Akihiro Umezawa, Nobuyuki Kamata (YIA, Original papers) Renu Wadhwa (Session in English) Minori Kokado, Tohru Masui (Public symposium) Toshihiro Sakano, Kenichi Manaka (Edit of Abstract Book) Supporting companies ABI CO., LTD. MEDICAL BIOLOGICAL LABORATORIES CO., LTD. INNOMEDICS Medical Instruments Inc. Research Institute for the Functional Peptides Corning International K.K. Life Sciences Division Cell Science & Technology Institute, Inc. Sigma Aldrich Japan K.K. SUMITOMO BAKELITE CO., LTD. Sekiuchi Pharmacy, Inc. TAKACHO CO., LTD. DS Pharma Biomedical Co., Ltd. Transparent Inc. Nippon Becton Dickinson Company, Ltd. NIPRO CORPORATION MEDINET Co., Ltd. MED SHIROTORI CO., LTD YAKUKENSHA CO., LTD. Life Technologies Japan Ltd. Roche Diagnostics K.K. Support for public symposium National Center for Child Health and Development Contribution for poster Ronald McDonald House Charities Japan HOICHOI PRODUCTIONS INC Head office Mikako Kobayashi, Masaharu Dozen 29

32 PROGRAM YIA-1 YIA-2 YIA-3 YIA-4 OG-1-1 OG-1-2 Molecular biology of the role of CARF in aging and cancer: basic and interventional approaches: RUMANI SINGH 1,2, SUNIL KAUL 2, RENU WADHWA 2 1 Graduate School of Life & Environmental Sciences, University of Tsukuba, 2 National Institute of Advanced Industrial Science & Technology (AIST) RHAMM may control cell proliferation and differentiation.: HIROKO HATANO 1,2, HIDEO SHIGEISHI 1, NOBUYUKI KAMATA 1 1 Department of Oral and Maxillofacial Surgery, Division of Cervico-Gnathostomatology, Programs for Applied Biomedicine, Graduate School of Biomedical Sciences, Hiroshima University, 2 Research Fellow of the Japan Society for the Promotion of Science Functional Evaluation of 3D-culture human hepatocytes on a novel multi-well plate microfabricated with highly hydrophilic polymer: YURIKO TAKAHASHI, TOMOKO JOMURA, EMIKO OZEKI,TAKESHI IKEYA Transparent Inc. Establishment of disease models of human genetic disorder using hepatocytes isolated from surgically resected samples.: HATSUNE MAKINO, AKIHIRO UMEZAWA Department of Reproductive Biology, National Research Institute for Child Health and Development Establishment of immortalized human erythroid progenitor cell lines: YUKIO NAKAMURA, RYO KURITA Cell Engineering Division, BioResource Center, RIKEN Establishment and characterization of transformed cell lines from human ips teratoma: YOUJI MITSUI, MIZUNA KAMADA, TUTOMU KUMAZAKI,MEGUMI TOUJOU, TAIRA MATSUO, TOMOKO TAKAHASHI Department of Physiological Chemistry, Faculty of Parmaceutical Sciences at Kagawa campus, Tokusima Bunri University 30

33 OG-1-3 OG-1-4 OG-2-1 OG-2-2 OG-2-3 Comparative analysis of characteristics among human ips, ES and MSC Cell lines: MITSUHI HIRATA 1, MIDORI HAYASHIDA 1, DAIKI TATEYAMA 1, YUTAKA OZAWA 1, HIROKO MATSUMURA 1, MASASHI IEMURA 1, SHANDAR AHMAD 4, TOMOKO SHOFUDA 2, YONEHIRO KANEMURA 2, ARIHIRO KOHARA 1, HIROYUKI MIZUGUCHI 3,5, MIHO FURUE K 1 1 JCRB Cell Bank, Laboratory of Cell Cultures, Department of Disease Bioresources National Institute of Biomedical Innovation, 2 Institute for Clinical Research, National Hospital Organization Osaka National Hospital, 3 Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, 4 Laboratory of Bioinfomatics, National Institute of Biomedical Innovation, 5 Department of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University Isolation and culture of hepatic progenitor cells from liver cirrhosis secondary to biliary atresia: TAISUKE YAMAZAKI 1,4, MUREO KASAHARA 2, AKINARI FUKUDA 2, SEISUKE SAKAMOTO 2, TAKANOBU SHIGETA 2, ATSUKO NAKAZAWA 3, SHIN ENOSAWA 4, TAKAYOSHI TOKIWA 1 1 Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, 2 Division of Surgery, National Center for Child Health and Development, 3 Department of Clinical Pathology, National Center for Child Health and Development, 4 Division for Advanced Medical Science, National Center for Child Health and Development Feasibility of hydrogel-based microencapsulation for propagation and differentiation of induced pluripotent stem cells in scalable suspension culture: IKKI HORIGUCHI, MOHAMMADMAHFUZ CHOWDHURY, YASUYUKI SAKAI Institute of Industrial Science,The University of Tokyo Culture of liver-derived cells using oxygen-permeable membranes: YASUYUKI SAKAI, FANNY EVENOU, MORGAN HAMON,TERUO FUJII Institute of Industrial Science, Univeristy of Tokyo Evaluation of drug toxicity with human hepatocytes cultured in a micro-space cell culture system: KAZUAKI NAKAMURA 1, YOKO EJIRI 2, AKITO TANOUE 1 1 Department of Pharmacology, National Research Institute for Child Health and Development, 2 Tsukuba Research Center, Kuraray Co., Ltd. 31

34 OG-2-4 Development of the Multi-Flask for an effective use of the culture space.: KANAKO ETO 1, SUSAN QIAN 2, ELIZABETH ABRAHAM 2 1 Nippon Becton Dickinson Ltd., BioSciences, 2 Becton, Dickinson and Company, BioSciences OG-2-5 Characteristics of tissue cryopreserved by cell alive system, CAS : OG-2-6 MASAKAZU IWASAKA 2, MANABU KURITA 1, NORIO OOWADA 1 1 ABI Inc., 2 Department of Biomedical Engineering, Graduate School of Technology, University of Chiba Fate of bone marrow mesenchymal stem cells following the allogeneic transplantation of cartilaginous aggregates into osteochondral defects of rabbits: TOMOKAZU YOSHIOKA 1,2, HISASHI SUGAYA 2, HAJIME MISHIMA 2, ZEENIA KAUL 3, TOSHIMASA UEMURA 3, SUNIL KAUL 3, RENU WADHWA 3 1 Department of Orthopaedics Surgery, Ibaraki Southwest Medical Center, 2 Department of Orthopaedics Surgery, University of Tsukuba, 3 National Institute of Advanced Industrial Science and Technology OG-3-1 OG-3-2 OG-3-3 SP-1 Research use of remnant human tissues obtained from surgeries: AYA ONISHI-KASAMATSU, TAKUO KOSAKA, MOTONOBU SATOH, TOUHO YOSHIDA Japan Health Sciences Foundation, Health Science Research Resources Bank, Human Tissue Bank Activities of intractable disease bioresource bank in National Institute of Biomedical Innovation: YOUSUKE KAMEOKA 1, RYUICHI SAKATE 1, ICHIRO TAKAHASHI 1, REIKO TANUMA 1, MAKOTO HIRATA 1, KIYOSHI TAKEMURA 2, KYOKO MATSUSHITA2, YUKI SAKAGUCHI 2, ATSUNORI HIGASHINO 1, EMIKO ITO 1 1 Laboratory of Rare Disease Biospecimen, National Institute of Biomedical Innovation, 2 Office of Policy and Ethics Research, National Institute of Biomedical Innovation About the revision of Ethics Guideline for Human Genome/Gene Analysis Research.: TOHRU MASUI National Institute of Biomedical Innovation What should be done to establish a in vitro testing for safety evaluation?: HAJIME KOJIMA National Institute of Health Sciences 32

35 SP-2 SP-3 SP-4 SP-5 SP-6 SP-7 SL-1 WS-1 Usefulness and Problem in Three-dimensional cultured human skin model to evaluate skin permeation and concentration of chemical compounds: KENJI SUGIBAYASHI Faculty of Pharmaceutical Sciences, Josai University Development of culture technologies utilizing a vitrigel chamber and a TOSHI (tissue/organ sections for histopathology)-substratum and their advantages for extrapolating ADMET of chemicals in human: TOSHIAKI TAKEZAWA National Institute of Agrobiological Sciences Differentiation of ES/iPS cells into the hepatic lineage: SHOEN KUME 1, NOBUAKI SHIRAKI 1, TAIJI YAMAZOE 1, QIN ZENG 2, KATSUMI MOCHITATE 2 1 Institute of Molecular Embryology and Genetics, Kumamoto University, 2 Environmental Health Sciences Division, National Institute for Environmental Studies Establishment of Disease-specific ips Cells and The Applications: ISAO ASAKA Dept. Regulatory Science, Center for ips Cell Research and Application, Kyoto University Expectation to utilization of human pluripotent stem cells for drug discovery and development: NORIMASA JINNO, HIROYUKI ARAI, KAZUICHI NAKAMURA Non-Clinical Evaluation Subcommittee, Drug Evaluation Committee, Japan Pharmaceutical Manufacturers Association Licensing activity under ips cells patents and distribution of ips cells: MITSUOMI SHIRAHASHI ips Academia Japan, Inc. Establishment of human embryonic stem cell with an aim of clinical research: HIDENORI AKUTSU Research Institute, National Center for Child Health and Development A search method of cell lines and usage of the data sheet: TADAYOSHI UEDA DS Pharma Biomedical Co.,Ltd. Research and Development Div. Research Group II 33

36 WS-2 WS-3 OE-1 Procedure for Growth Curve Analysis of Animal Cell Lines: ISAO ASAKA Dept. Regulatory Science, Center for ips Cell Research and Application, Kyoto University Designing and Compliance in Research with Mammalian Cell Culture: SHIN ENOSAWA National Center for Child Health and Development The role of oxidative stress from mitochondria on aging and are-related diseases: NAOAKI ISHII, TAKAMASA ISHII, MASAKI MIYAZAWA Tokai University School of Medicine, Basic Medical Science and Molecular Medicine, Department of Molecular Life Science OE-2 Biology of stress protein MORTALIN: focus on cancer and neurodegeneration: OE-3 OE-4 RENU WADHWA, SUNIL KAUL National Institute of Advanced Industrial Science & Technology (AIST) Molecular insights into Ashwagandha leaf-drived factors for stress and aging: SUNIL KAUL, DIDIK PRIYANDOKO, NAVJOT SHAH,RENU WADHWA National Institute of Advanced Industrial Science & Technology (AIST) hnrnp-k promotes tumor metastasis by induction of genes involved in extracellular matrix, cell movement and angiogenesis: RAN GAO 1,2, TETSURO ISHII 2, SUNIL KAUL1,RENU WADHWA 1 1 National Institute of Advanced Industrial Science & Technology (AIST), 2 Graduate School of Life & Environmental Sciences, University of Tsukuba OE-5 PINK1 knockdown increases the apoptosis induced by adenovirus-mediated REIC/Dkk-3 overexpression on bladder cancer cells: YU JIN, HITOSHI MURATA, KEN KATAOKA,MASAKIYO SAKAGUCHI, NAM-HO HUH Dept. Cell Biol., Okayama Univ. Grad. Sch. Med., Dent. & Pharm. 34

37 OG-4-1 OG-4-2 OG-4-3 OG-4-4 OG-5-1 Ashwagandha leaf-derived Withanone protects normal human cells against the toxicity of methoxyacetic acid, a major industrial metabolite: DIDIK PRIYANDOKO 1,2, TETSURO ISHII 2, SUNIL KAUL 1, RENU WADHWA 1 1 National Institute of Advanced Industrial Science & Technology (AIST), 2 Graduate School of Life & Environmental Sciences, University of Tsukuba Alcoholic extract of Ashwagandha leaf and its component withanone induce differentiation in brain derived cancer cells: NAVJOT SHAH 1,2, TETSURO ISHII 2, RENU WADHWA1,SUNIL KAUL 1 1 National Institute of Advanced Industrial Science & Technology (AIST), 2 Graduate School of Life & Environmental Sciences, University of Tsukuba Expression of REIC/Dkk-3 in squamous epithelia and screening of its regulating factors: KEN KATAOKA, MASAKIYO SAKAGUCHI, GANG DU,NATSUMI MAEHARA, HITOSHI MURATA, HUH NAM-HO Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The Role of CTGF in the Growth of Malignant Mesothelioma Cells: MAKIKO FUJII 1, TAKESHI TOYODA 2, HAYAO NAKANISHI 3, YASUSHI YATABE 4, HIDEMI ITO 1, EISAKU KONDO 3, YASUE MATSUDAIRA 1, TOHRU TUJIMURA 5, YOSHITAKA SEKIDO 1 1 Division of Molecular Oncology, Aichi Cancer Center Research Institute, 2 Division of Pathology, National Institute of Health Sciences, 3 Division of Oncological Pathology, Aichi Cancer Center Research Institute, 4 Division of Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital, 5 Department of pathology, Hyogo College of Medicine A novel culture technology utilizing a collagen vitrigel chamber and its application to eye irritation test: CHIKA OKAMOTO 1, HIROYUKI YAMAGUCHI 1,2, TOSHIAKI TAKEZAWA 1 1 National Institute of Agrobiological Sciences, 2 Kanto Chemical Co. Inc. 35

38 OG-5-2 OG-5-3 OG-5-4 OS-1 OS-2 OS-3 Making immortalized cells by normal human corneal epithelium: NAOKI YAMAMOTO 1, KOJI HIRANO 2, HAJIME KOJIMA 3, MARIKO SUMITOMO 1, HIROMI YAMASHITA 1, MASASHI NAKAMURA 4, KAZUHIRO HARA 4, ATSUHIRO TANIKAWA 2, KOKI TANIGUCHI 1, MASAYUKI HORIGUCHI 2 1 Laboratory of Molecular Biology & Histochemistry, Fujita Health University Joint Research Laboratory, 2 Department of Ophthalmology, Fujita Health University School of Medicine, 3 Japanese Center for the Validation of Alternative Methods (JaCVAM), Division of Pharmacology, National Institute of Health Sciences, 4 Hoyu Co., Ltd. In Vitro Studies Of Toxic Damage Of Benzalkonium Chloride and Antiglaucoma Drugs In Stratified Human Cultivated Corneal Epithelial Sheets: SUGURU NAKAGAWA 1, SEIICHI YOKOO 1, TOMOHIKO USUI 1, SACHIKO OMICHI1,2, YOSAI MORI 3, KAZUNORI MIYATA 3, MAKOTO AIHARA 1, SHIRO AMANO 1, MAKOTO ARAIE 1,4 1 The Department of Ophthalmology, University of Tokyo School of Medicine, 2 JR Tokyo General Hospital, 3 The Department of Ophthalmology, Miyata Eye Hospital, 4 Kanto Central Hospital The mucin secretion of corneal epithelial stem cell and the corneal epithelium sheet: SEIICHI YOKOO, SATORU YAMAGAMI, TOMOHIKO USUI,SIROU AMANO Department of Ophthalmology Fuculty of Medicine University Hospital The Uviversity of Tokyo Medical research based on mutual understanding between patients and doctors: MICHIYA NATORI Research Institute, National Center for Child Health and Development Human tissues and cells as the other selves committed to combat against diseases: What learned from my thirty five year interaction with them: TOSHIHARU MATSUMURA 1,2 1 Yokohama BioResearch and Supply Inc., 2 Honorary member, The Japanese Tissue Culture Association Collaboration between Patients/Families and Researchers/Doctors for the Development of New Drugs: JUNICHIRO FUJIMOTO Clinical Research Center, National Center for Child Health and Development 36

39 37

40 YIA-1 Molecular biology of the role of CARF in aging and cancer: basic and interventional approaches CARF (Collaborator of ARF) was first cloned as an ARF-interacting protein and shown to regulate the p53-p21waf1-hdm2 pathway, a central to tumor suppression via senescence and apoptosis. CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis. In order to determine the mechanism of CARF silencing-induced apoptosis, we examined various cell death and survival pathways including the mitochondrial stress, ATM/ATR, Ras/MAP kinase and retinoblastoma cascades in cultured CARF-compromised cancer cells. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, CARF-knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and gh2ax, leading to induction of mitotic arrest and eventual apoptosis. CARF was upregulated during replicative, oncogenic and stress-induced senescence in cultured cells. We demonstrate the upregulation of CARF beyond a certain threshold level causes shift of cellular phenotype from growth arrest to transformation and hence may provide a potential molecular bridge between aging and cancer. 39

41 YIA-2 RHAMM. X. SV-40 T antigen h-tert cdna RHAMMreceptor for hyaluronan-mediated motility RHAMM G2/M RHAMM ERK1/2 ERK1/2 RHAMM TPX2 AuroraA RHAMM TPX2 centrosome mitotic spindle RHAMM ERK1/2 TPX2 RHAMM RHAMM cdna RHAMM sirna RHAMM RHAMM MC3T3E1 U2 RHAMM RHAMM RHAMM 40

42 MC3T3E1 U2 RHAMM ERK1/2 TPX2 41

43 YIA-3 3 in vivo 3 3 No Xenotech 3 Cell-able JCRB 9019 ATCC CCL-163 Cell-able cells / well (96 well type)410 5 cells / well (12 well type) Williams E 1% RM Teststerone 100 micro-mol/l 4 6beta-hydroxy testosterone6ohts Testosterone glucuronide TSGln UPLC (Waters) Testosterone rifampicin25 micro-mol/l 3 2 (uptake) 3,7 [3H(G)]-Taurocholic acid (PerkinElmer ) 1 micro-mol/l 2, 5, 10 min 3 42

44 2,4,7 5(6)-carboxy-2,7-dichlorofluorescein Diacetate CDF-DA37, 10min CDF 1 1 3,7 6OHTS 3.9 pmol/10 6 cells/min, 4.0 pmol/10 6 cells/min TSGln 1.7 pmol/10 6 cells/min, 1.8 pmol/10 6 cells/min OHTS 180 pmol/10 6 cells/min195 pmol/10 6 cells/min150 pmol/10 6 cells/min rifampicin 54 5 (800 pmol/10 6 cells/hr) 2 (uptake) 24.1 nmol/10 6 cells nmol/10 6 cells, nmol/10 6 cells nmol/10 6 cells 3 2, 4, 7 Ca 2+ bile pool Cell-able 3 Cell-able Cell-able 43

45 YIA in vitro PCR in vivo 4 CK8/18PAS 44

46 45

47 OG-1-1 ES ES ES ES ES ips ips ES ips Tal1/SCL ips ips-tal1/scl ips-tal1/scl ips 47

48 OG-1-2 TIG-1 TIG-1 OCT4, KLF4, SOX2 A pcx-oks-2a pcxcmyc Nucleofection 18 ips RNA K4 1FBS DMEM TIG-1 65PD 120PD 48

49 OG-1-3 hes hips ES/iPS hes/ips hes/ips JCRB National Stem Cell Bank ES/iPS EC hmsc neuroblastoma PCR hes/ips/ec neuroblastoma hmsc hes/ips Embryoid bodies hesf9(1) (2) ES/iPS 1) MK. Furue, et al. Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. PNAS 105: ) Inamura M., et al. Efficient generation of hepatoblasts from human ES cells and ips cells by transient overexpression of homeobox gene HEX. Molecular Therapy Feb;19(2):

50 OG-1-4 BA / BA Non-BA / EpCAM Thy-1 BA Non-BA EpCAM Thy-1 1 EpCAM Thy-1 RT-PCR BA EpCAM (n=5)non-ba %(=3)BA p(0.01)thy-1 BA (n=5)Non-BA (n=3) EpCAM BA p0.05 EpCAM Thy-1 EpCAM Thy-1 EpCAM BA Non-BA EpCAM Thy-1 Thy-1 BA Thy-1 -fetoprotein EpCAM Thy-1 / EpCAM Thy-1 50

51 51

52 OG-2-1 ES ips ips ips 3 ips -L- EDTA Nanog EGFP ips ips-mef-ng-20d-17 : BRC [1] m ips -L- -L- 50 /160 / 160 /nanog-gfp 10 -L- ips [1] Okita, K. et al., Nature 448: (2007). 53

53 OG (poly-dimethylsiloxane, PDMS) PDMS Hep G cells/cm 2 21%5 5% 21% PDMS in vivo 54

54 OG-2-3 in vitro HepG P450 P450 55

55 OG-2-4 CO2 CO2 BD Falcon Cell Culture Multi-Flask 3 5 T- 3 5 BD Falcon Cell Culture Multi-Flask T-BD Falcon Cell Culture Multi-Flask HepG2 5 x 10 6 cells/layer 48 Multi-Flask T-Flask BHK-21 3 x 10 6 cells/layer 72 56

56 OG-2-5 CASCells Alive System CAS CAS CAS CAS CAS Cells Alive System 57

57 OG % (Qdot655 ; Invitrogen) (Qdot655 antibody conjugation kit; Invitrogen) mg/ml % TGF-3 (RCCSTM-4D system; Synthecon, Houston, TX, USA) mm 4mm O 8 26 in vitro in vivo 58

58 59

59 OG-3-1 HSRRB kasamatsu et al., Tissue Cult. Res. Commun HSRRB 61

60 OG ips 62

61 OG

62 65

63 SP-1 R R R REACHRegistration, Evaluation, Authorization and Restriction of Chemicals EU ITSIntegrated Testing Strategy ITS in vitro in vitro 21 in vitro toxicology in vitro hclat human Cell Line Activation Test 67

64 SP-2 Hill Fick 2 2 MTT in vitro 3 3 Episkin EpiDerm Fick 2 Km Vmax 3 EpiDerm false positive false-negative 68

65 3 69

66 SP-3 ADMET 2009 in vitro OECD 2004 in vitro Reduction Replacement Refinement (1) (2) 70

67 (3) ES ES ES (1). HAB Newsletter 17(2): 9-12, (2). Yakugaku Zasshi 130(4): , (3). Organ Biology 15(2): ,

68 SP-4 ES/iPS ES/iPS ES/iPS M15 (Stem Cells 2008, Genes Cells 2008, BBRC 2009) 72

69 SP-5 ips induced Pluripotent Stem Cells ES Embryonic Stem Cells Oct3/4Sox2Klf4c-Myc 4 c-myc 3 ES ips ips ips ips <BR> ips Oct3/4Sox2Klf4c-Myc 4 ips ips 73

70 SP-6 ipses % ips ES ips herg 74

71 75

72 SP-7 ips ips ips ips ips ips ips ips ips ips ips ips 76

73 77

74 SL-1 ES ES 1998 ES First in Man Trial 1 ES ES 79

75 81

76 WS HL

77 WS-2 (lag phase) logarithmic growth phase stationary phase (death phase) 84

78 WS-3 ES ES 85

79 COIConflict of Interest 86

80 87

81 OE-1 TOPIC- The role of oxidative stress from mitochondria on aging and are-related diseases NAOAKI ISHII, TAKAMASA ISHII, MASAKI MIYAZAWA (Tokai University School of Medicine, Basic Medical Science and Molecular Medicine, Department of Molecular Life Science) We have previously demonstrated that excessive mitochondrial O2- caused by a mutation (G71E in C. elegans, I71E in Drosophila or V69E in mouse) in SDHC subunit of Complex II led to premature death in C. elegans and Drosophila, tumors in mouse cells and infertility in transgenic mice. In humans, it has been reported that the mutation in SDHB, SDHC or SDHD often results in familial paragangliomas (PGLs). Recently, we have established a Tet-mev-1 conditional transgenic mouse with our uniquely developed a Tet-On/Off system equilibrating expression levels from the transgene with endogenous that. The Tet-mev-1 mouse with mitochondrial respiratory chain dysfunction overproduced O2-. The mitochondrial oxidative stress resulted in excessive apoptosis leading to low birth weight and growth retardation in the neonatal developmental phase. We also observed that the Tet-mev-1 mouse has low fertility, pregnancy and delivery rates, and occasionally result in habitual abortion or maternal death. It has also been confirmed that this mutation caused chronic inflammations in lung, lacrimal gland, liver and pancreas, and also caused decline of learning and memory ability in the senior adult mouse. As it is thought that intracellular oxidative stress leads to life-style related and age-related diseases such as diabetes, arteriosclerosis, cancer and neuronal degenerative diseases, the Tet-mev-1 mouse could clarify that these diseases are caused by excessive O2- produced from electron leakage in mitochondria. 89

82 OE-2 TOPIC- Biology of stress protein MORTALIN: focus on cancer and neurodegeneration RENU WADHWA, SUNIL KAUL (National Institute of Advanced Industrial Science & Technology (AIST)) Mortalin is a member of hsp70 family of stress proteins. It was first cloned in our laboratory in 1993 in a cybrid screening system. Over the years, it has emerged as a dynamic protein with several essential functions including chaperoning, intracellular trafficking, mitochondrial import of proteins, cell proliferation and control of ROS production. It is upregulated in a variety of cancers, showed correlation with tumor aggressiveness and contribute to human cancers by inactivation of tumor suppressor protein p53. Consistent with this, cancer cells compromised for mortalin enter growth arrest or apoptosis. In contrast to the cancers, loss of mortalin was associated with age-associated neurodegenerative diseases including Alzheimer's and Parkinson s. Furthermore, manipulations of mortalin level in dopaminergic neurons resulted in significant changes in their sensitivity to PD phenotypes via pathways involving mitochondrial and proteasomal function as well as oxidative stress. These data demonstrate the key role of mortalin in mitochondrial function and stress management that are tightly linked to aging and cancer. 90

83 OE-3 TOPIC- Molecular insights into Ashwagandha leaf-drived factors for stress and aging SUNIL KAUL, DIDIK PRIYANDOKO, NAVJOT SHAH, RENU WADHWA (National Institute of Advanced Industrial Science & Technology (AIST)) Many of the traditional herbal extracts are increasingly being appreciated with Western models of integrative health sciences and evidence-based approach both in research and practice. Ashwagandha (Withania somnifera; Solanaceae) is one of the most commonly used plants in Ayurveda (world s oldest system of home medicine system). Extracts from different parts of the plant including root, shoot, seed and berry have been in use for a variety of health promoting effects. The main active constituents of Ashwagandha leaves are alkaloids and steroidal lactones (commonly known as Withanolides). We had previously investigated the biological activity in the alcoholic extract of Ashwagandha leaf extract (i-extract) and demonstrated that it has a strong anti-cancer activity. By chemical fractionation, the activity was assigned to its constituent Withanone (i-factor). We have investigated the gene targets and pathways involved in cytotoxicity of i-extract by loss-of-function screening (abrogation of i-extract induced cancer cell killing) achieved by introduction of a shrna and randomized ribozyme library in cell based viability assays. Both of these revealed the involvement of tumor suppressor protein p53 in i-extract induced killing of cancer cells. Normal human fibroblasts, on the other hand, showed down-regulation of p53 function and delayed senescence. Bioinformatics analyses revealed the involvement of apoptosis and insulin/igf signaling pathways that are well linked to the ROS signaling, DNA and mitochondrial damage in cancer cells. Based on these data, we propose that in the present scenario of increase in aging society and cancer incidence, i-extract and Withanone are cheap, easily available potential reagents that could be recruited for stress, aging and cancer management. 91

84 OE-4 hnrnp-k promotes tumor metastasis by induction of genes involved in extracellular matrix, cell movement and angiogenesis RAN GAO 1,2, TETSURO ISHII 2, SUNIL KAUL 1, RENU WADHWA 1 ( 1 National Institute of Advanced Industrial Science & Technology (AIST), 2 Graduate School of Life & Environmental Sciences, University of Tsukuba) Cancer is a leading cause of death and still awaits effective therapies. Rapid growth of industrialization has contributed to increasing incidence of cancer globally. One of the reasons that most of the cancers fail therapy is due to the metastasis. Hence identification of the factors leading to metastasis is highly important in designing of effective and novel anti-cancer therapeutics. In our earlier study, we found that the heterogeneous nuclear ribonucleoprotein K (hnrnp-k) is involved in metastasis. Here, we studied the effect of hnrnp-k on proliferation and metastatic properties of cancer cells in vitro and in vivo. We have established stably overexpressing hnrnp-k derivative cell lines of NIH3T3 and U2OS. hnrnp-k was compromised in metastatic HT1080 cell line using intracellular antibody reported in our earlier study. We studied the effect of hnrnp-k overexpression and silencing using xenograft and tail vein tumor cell injections in nude mice model. Molecular basis of the hnrnp-k induced malignant and metastasis phenotype was dissected by DNA microarray and pathway analyses. We found that the hnrnp-k regulates extracellular matrix, cell motility and angiogenesis pathways. Involvement of the selected genes (CCK, MMP3, PTGS2 and CTGF) and the pathways was validated by gene specific expression analysis. Our results demonstrated that the hnrnp-k is a potential target of metastasis therapy. 92

85 OE-5 PINK1 knockdown increases the apoptosis induced by adenovirus-mediated REIC/Dkk-3 overexpression on bladder cancer cells YU JIN, HITOSHI MURATA, KEN KATAOKA, MASAKIYO SAKAGUCHI, NAM-HO HUH (Dept. Cell Biol., Okayama Univ. Grad. Sch. Med., Dent. & Pharm. ) PINK1 is a serine/threonine kinase with a mitochondrial localization signal and mutations in the gene are causatively linked to an autosomal recessive form of Parkinson's disease. In addition, the expression levels of PINK1 are increased in different cancers with high metastatic potential. We have found that overexpression of PINK1 can up-regulate the phosphorylation of Akt through the mtorc2 complex in human neuroblasts. And it was also reported that PINK1 may regulate mitochondrial proteins. REIC/Dkk-3 was first identified as a down-regulated gene in normal human fibroblasts immortalization and it is significantly downregulated in a broad range of cancer cell types. Adenovirus-mediated REIC/Dkk-3 (Ad-REIC) overexpression has showed a therapeutic effect on human prostate cancer, testicular cancer, renal cell carcinoma, pleural mesothelioma, and breast cancers. But some cancer cells showed the resistance to Ad-REIC in different degrees and overexpression of Ad-REIC in these cells only had partial apoptosis effects. For bladder cancer cells, they are resistant to Ad-REIC because of the high expression levels of Bcl-2 and/or Bcl-xL which can reduce the apoptosis at mitochondria. For these reasons, we planned to combine the PINK1 knockdown by sirna with Ad-REIC overexpression to increase the therapeutic effect on Ad-REIC resistant bladder cancer cells. We started with seven bladder cancer cell lines, and six of them showed the resistant effect to Ad-REIC overexpression. Next, we checked the adenovirus infection efficiency of these six cell lines with adenovirus LacZ. Two cell lines showed low infection efficiency, but the other four cell lines showed high infection efficiency and resistance to Ad-REIC at the same time. With these four Ad-REIC resistant bladder cancer cell lines, we checked the apoptosis effect induced by PINK1 knockdown and Ad-REIC overexpression. The results showed PINK1 knockdown can remarkably enhance the apoptosis induced by Ad-REIC overexpression. Western blotting revealed that a anti-apoptotic protein of Bcl-2 family, Bcl-xL, has been down-regulated by PINK1 knockdown. We conclude that PINK1 knockdown increases the apoptosis induced by Ad-REIC overexpression through the down-regulation of Bcl-xL. 93

86 95

87 OG-4-1 Ashwagandha leaf-derived withanone protects normal human cells against the toxicity of methoxyacetic acid, a major industrial metabolite The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Toxicity of the industrial chemicals is recognized as an alarm to human health and hence the need for alternative non-toxic natural products or adjuvants that may serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. Based on these findings, we suggest the use of withanone as health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates. 97

88 OG-4-2 Alcoholic extract of Ashwagandha leaf and its component withanone induce differentiation in brain derived cancer cells Alcoholic extract of Ashwagandha (Indian ginseng) leaves (i-extract) was recently shown to possess anticancer activities that operate through selective activation of p53 pathway and induction of oxidative stress in cancer cells. In a sharp contrast to cancer cells, normal cells showed inactivation of p53 activity and protection against oxidative stress. Furthermore, we found that the low doses of i-extract and withanone induce differentiation of glioma and neuroblastoma cells. We next undertook C6 glioma and IMR32 neuronal cell regeneration models to investigate whether these brain-derived cells could recover from oxidative stress induced by hydrogen peroxide. We demonstrate that in response to the treatment with i-extract and withanone, but not withaferin A, glial and neuronal cell undergo differentiation as seen by upregulation of GFAP in C6, and NF-H, MAP-2, PSD-95 and GAP43 in IMR32 cells. We propose that the use of in vitro cell culture and differentiation models may serve as an easy and economic way to identify new factors that could be employed for functional therapy of brain cancers and other pathologies associated with aging. 98

89 OG-4-3 REIC/Dkk-3 REIC/Dkk-3 DickkopfDkk Dkk Dkk-1 Wnt- Dkk-3 REIC/Dkk-3 REIC/Dkk-3 REIC/Dkk-3 REIC/Dkk-3 REIC/Dkk-3 REIC/Dkk-3 HaCaTA431BSCC-93HSC-5 Tumor Necrosis FactorTNF- REIC/Dkk-3 TNF- REIC/Dkk-3 99

90 OG-4-4 CTGF (connective tissue growth factor) p14ink4a/p14arf, neurofibromatosis type2 (NF2) TGF-TGF- YAP Smad3 CTGF YAP Smad3 Smad7MMP2fibronectin YAP CTGF Smad3YAPp300 CTGF CCN (Cyr61CTGFNov) CCN kda 38 kd CTGF TGF-/ CTGF NCI-H290 focus assay CTGF focus CTGF CTGF NCI-H290 CTGF NCI-H290 CTGF 100

91 101

92 OG mm HCE-T TEER 5 HCE-T TEER TEER in vitro 103

93 OG-5-2 / p75ntrcd271 / / p75ntr In Vitro Cell Dev Biol-Animal., 2010 ihce-ny HCEJCRB Gene Bank SV40 Large T Ag SV40 Proteomics HCE-TRCB2280 HCE-T CD HCE ihce-ny HCEHCE-T ihce-ny Proteomics SV40 HCE-T SV40 H H

94 OG BAC Latanoprost R,LatanoprostPF R, TravatanZ R, Tapros R Xalatan R %, 0.002%, 0.01%, 0.02%. 0.2% BAC 10 WST-1 assay 0.01%BAC, 0.02%BAC, 0.2%BAC Xalatan R (P0.05) % BAC Xalatan R Latanoprost R, LatanoprostPF R, TravatanZ R, Tapros R Xalatan R BAC BAC 105

95 OG-5-4 SHEM 3T3 Trans Well 6Well Type cells/well DMEM/F12 + B27 + EGF 20ng/ml 10% HE PAS EGF Control MAPK ERK FR PAS EGF PAS PAS ERK FR PAS Control PAS ERK12 MAPK ERK 106

96 107

97 NCI COE 109

98

99

100 OS-1 112

101 113

102 OS-2 HeLa HeLa Henrietta Lacks HeLa HeLa

103 (1) (2,3) 1) 19: , ) 40(2): , ) 115

104 OS-3 CML CML CML CML 116

105 119

106 121

107 122

108 123

109 124

110 125

111 INSTRUCTIONSTO AUTHORS TISSUE CULTURE RESEARCH COMMUNICATIONS is the oficialjournalofthe JapaneseTissueCultureAssociation. Originalarticlesthatdealwithtissues,organsor celsfrom multicelularorganismsinvitrowilbeconsidered. Manuscriptsmustbe writeninenglishorjapanese,preferentialyinenglish. Fourtypesofpapersare published;(a)regulararticles,(b)invitedreview articlesandreview articles,(c)rapid communications,(d)generalinformations thatinclude meeting reports and book reviews. Manuscriptsinregulararticlesandrapidcommunicationswilbereviewedby twotothreerefereesinthefield. HOW TO SUBMIT MANUSCRIPTS Sendthreecompletesetsofmunuscripts(oneoriginaltypescriptandtwocopies) withaltablesandoriginalphotographsandacopyrighttransferform tothe JapaneseTissueCultureAssociationto: TetsujiOkamoto,ManagingEditor EditorialoficeofTissueCultureResearchCommunications. Dept.ofOral&Maxilo-FacialSurgery1.HiroshimaUniversitySchoolofDentistry,1 2 3,Kasumi, HIROSHIMA ,Japan. Telephone: Facsimile: HOW TO PREPARE MANUSCRIPTS Style Nomenclature Manuscriptsshouldbeclearlytypedwith doublespacingona4orinternationalsize papers.marginsofnotlessthan2cm shouldbe leftoneachside.eachmunuscriptconsistsof title,authors,afiliations,abstract,keywords, runningtitle,footnotesincludingabbreviations, introduction,materialsandmethods,results, discussion, acknowledgements, references, tables,andfigurelegends. Thefirstpageshouldcarry:title,thename ofauthor s,afiliations,runningtitle(notto exceed40letersandspaces)andcorrespondingauthor snameandfuladdress(telephone numberandalsofacsimilenumberifany).at mostfivekeywordsshouldbegiven.the secondpageshouldcontainabstract,nottoexceed 200words.Thetextshould starton page3. Alpagesshouldbenumbered.Regulararticlesshouldbeuseddescriptivesubheading,e.g.,Introduction,Materialsand Methods,Results,Discussion,Acknowledgements,and References. Diferentwriting styleshouldbeacceptedinothertypesofarticles. Namesofchemicalcompoundsshouldconform totheinternationalunionforpureand AppliedChemistry(IUPAC)nomenclatureand biochemicaltermstothoseoftheinternational UnionofBiochemistry(IUB). Measurement,Signs,andSymbols Unitsofmeasurementsmustbethosein

112 INTRODUCTION TO AUTHORS internationalusage.anynewunitsorsymbols usedshouldbeexplainedinthetext.alunits ofmeasurementsshouldbesiunits. Abbreviations Useofabbreviationshouldbeminimized. Wherenecessary,spelouttheabbreviated term folowedbytheabbreviationinparentheseswhereitisfirstcited. References Authorsareresponsiblefortheaccuracyof references.authorsshouldconfirm alreferencesonthefinalmanuscriptwithoriginal publications.referencesshouldbenumbered consecutivelyintheorderinwhichtheyare citedinthetextbymeansofnumericalsuperscripts,suchas 1),2),3).Regardlessofcitation method,folow IndexMedicusJournaltitle abbreviations.cite manuscriptsinpreparation, unpublishedresults, personalcommunications,etc.,inthetext. Therecommendedcitationstyleforreferencestomanuals,booksandjournalsisshown inthefolowingexamples: 1.Hart,R.W.andSetlow,R.B.:Correlation between deoxyribonucleic acid excision repairandlifespaninanumberofmammalianspecies.proc.natl.acad.sci.usa, 71, , Hayflick,L.:Thecelularbasisforbiologicalaging:Finch.C.E.andSchneider.F.L. (Eds).InHandbookoftheBiologyofAging, VanNostrandReinhold,New York,NY,pp ,1977. Tables Tablesshouldbenumberedconsecutively withromannumerals,i.e.,tablei,table I,.Altablesshouldhaveatitle.Headingsto columnshouldbebrief.ifanyfootnotesare required,usealphabeticsuperscripts,suchas a), b). Figures Figuresshouldbenumberedconsecutively inarabicnumbers,i.e.,fig.1,fig.2,.al photographsshouldbeidentifiedonthereverse sidewithfigurenumber,thefirstauthor sname andorientation.photographsintriplicates shouldbeprintedonglossypaper.singlecolumnfiguresmustnotexceed82 220mm. Double-columnfiguresmustnotexceed mm.Materialsofinappropriatesizewilbe reducedbytheeditorialo fice.titlesand legendstofiguresshouldbetypedonaseparate sheetorsheets.magnificationsofphotographs shouldbeindicatedinthelegendsand/orby scalesatachedonthephotographs.thecost ofalcoloredphotographsmustbebornebythe author. ProofReading TheEditorialO ficeprovidesauthorswith galeyproofsfortheirexaminationonlyforone time.theauthorsshouldgoovertheproof carefuly,butnonewinsertionsshouldbemade inthetext. Pagecharge Inprinciple,thelengthofregulararticle, review article,andrapidcommunicationare limitedto8,10and4printedpages,respectively,includingfiguresandtables.ifthelengthof eacharticleexceeds,theauthorsarerequired topay 5,000perpageforextrapages. Reprints Reprintsorderformsaresenttoauthors alongwiththeirproof. Copyrights Thecopyrightforarticleswhichappearsin TissueCultureResearchCommunicationsis heldbythejapanesetissuecultureassociation.usethecopyrighttransferform atached tothisjournal.form wilbereturedwith manuscriptsnotaccepted.

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