ABSTRACTS OF POSTER PRESENTATIONS Chairpersons: Masatbugu Ueda (Dept of Obstet & Gynecol, Osaka Med CoUege) Kazwhige Kfguchi (Dept of Obstet & Gynecoll, Unh.Hog., St Marianna Med Sch.) Hiroaki Kataoku (Dept of Pathologv, Miyazaki Med College) Hkashi Hashimto (Dept of Anatomy, Jikei Univ. Sch. Med) Hiroyuki Kwamto (Dept of Obstet & Gynecol, Sch. Med, Kitasato Univ.) Masato Nkhida (Kasumigaura Med Center) YoshihiroKikuchi (Dept of Obstet & Gynecol, National Defense Med College) Makoto Yasda (Dept of Obstet & GynecoL, Jihi Unv. Sch. Med) 39

P-3-01 Organogenesish m the embryoid bodies composed of human amnion

P-3-02 New Method for Forming Large Embryoid Bodies using the Wall of the CultumDishalongwithanAnalysiiof theirstruchual Characteristics

origin stem cellp and m o w EES cells

Megumi IGUCHI', Isamu I S W f l A ' , Tomoharu TAMAGAWA',chiek~ISHIWNA', ~aptshige KIGUCH12,Kahei ,"S S h i pS m 4 ,

Ipurpose]Weattemptedto induce* 'oninto variousoqpnsbyformingembryoidbodieshthe humanamnioticstem ~ellS(HAM-I)andmou~eEES cells (early emblyonic stem cells). w a l s a n d Methods] Onehundredcellsea& mixed an EGFPgene indudon human amnion origin stem cell line (HAM-]EGFP) and a ddY mouse EES7 cells vmt C U M in the hanging drop d o n tbrtwodaysand f m e d the embryoidbodies. The culane medium used wasaMEM supplemented with 1oo/oFBS and emtnyotqltii fsctols(ETFs. Fratfiermore,The ~blyOidbodKsWereCU~ats$sianaryconditionin thesamemedim.

[ResultsandConchrsion]?heeanbryoidbodies~and

formedthepimordialaqpnsofa~gemdisc origin(bloodisland,skin,digestive~ithelium, a l v d ,blood vessel, blood cell). lF was Fecogruzedinthesepimordialorgansweserecognized by .AHAM-] cell suggestsdud it is stem cell having muhipotency di-g to primordial organs of trilamigelmdiscorigin.

40

MotoyoshiYAMAMO'IO',Hisashi HASHIMOTO', ~oshiakiTACHIBANA', w o~~l,~asakazll AKAHORI~, TM YOKOSE', lsamu ISHWMA*, ~iroshi ISHIKAWA'

Abstract

Early embryonic stem (EES)cells, which were established h2 cell stage embryos obtained hddY mice, had similar charactensb ' 'csasembryonicstem(Es) cells.'ThesecellSvvt!?emainEained in an undifferentiated stageinpwthmediasupplmentedwithleukemia inhibitory tsctor&IF) and were capable of dilktmtiating into triploblastictissues under various growth fscbrs.It has been known that rxxmal sized embryoid bodies(EBs) form by moving LIE Inthisshrdy, largeEEisgladdly f d alongthe side wall ofa culture dish,particulariy at theboundary bemxntheairandthegrowth medim when cells weze cultllred foraconsiderableperiod oftime a n d ~ s u b w l t u *r m g.Wecallthismethodthe'tvall adhesion ~ p o c e c h u ememethod . itselfwas S i m p l e and did not needany insbum~exceptplashicdishes because only the side walls ofthe dishes we^ utilized.The mean thicknessofthe b e EBs wasabout 1.5 mm 3 monthsafkrestablishingthestaticcube.Theirs& was c o v e r e d with a monolam of cells and they contained an eosinoph~liccell matrix By electron miamcopy, some Charactensbc * ' struchnescouldbeobsened,suchas inlracistemal A particles which w m present inside the swellingofthe rougb endoplasnic reticulum. Since many tissuesderivedfrwn Escells areobtainedthmugh EBS, it is expected that efkient acquirement sufficient quantities of these structures using the wall adhesion culture procedurewill be a shortcut for using Es cells in regenerativemediiine.

P-3-03 Nuclear elimination and transplantation in the m o w OOCYfeS using chemical treatments

P-3-04 Generation of rabbit clones using transfer of feta) fibroblastp and liver cells

ohis.'.*and sato, K.'

INTRODUCTION: T&y, the studies in the mammalian oocytes,especially mucleationtdmques of oocytesor zygotes and nuclearbansphtath of various cellsare usinga miawnanipulator,elec.t~~fusiondevices, M v a t e d Virus or chemical h i o n techniques.Rapid reproduciblemicromanipulahonis often used as standard techuques for nuclear transfer in many hbomtaies. Electmhion is the same as miaomanipulation,and has easy protocols additionally. However, thesetechniques need expensivedevices and investigator's skills. Additionally, e h h i o n htmduce invariably activation in the human oocytes.This is a fatal problem in the study o n ~ m b t y oIniahedvirushasbeen ~ . hadyusedforfirsionbecauseofthattoxkeffedtothe embryos. The chemically induced fusion with polyethylene glycol need makinga lot of timecompared with the other methods.However, in this methods no expensivedevices are needed and, polyethylene glycol-indd h i m are able to reproQlce &iently the mmshaedembryosassameasmicromaniprlationor electrofUsiontechniquesInthisstudy,weaimedto establinewpotocolsthathavemanymeritsfornuclear eliminationand transplantation inthemouseoocytes METHODS: Activation and enucleation of m o u s e oocytes wereaccomplishedwith5%ethanoYmmedium supplementedwith demecolcine.Oocyte nucleus with cytoplasm was released into PVS as p & dpolar body including 2C.Enucleation was judged using Hoechst 33342. Nuclear donor was used muse zygotes. The mn&pellllcidaeofthe enucleated oocytesand zygotes were removed by acidtyrodetmtmentpairs ofa zygote and an mucleared oocyte were fused respectivelyusing poly&ylene glycol. RESULTS& DISSCUSSION: The rrmmm&d oocytes successfUlly developed to blastocyst embryos. We believe thatthosemethodsfornuclearbansfercoulddbute greatly in the embryologicalstudy using mammalian oocytes.

Intduction: Successrl developmentof clone embryos have been qorted in sheep, cattle, mice, jpts and &bits using a variety of somatic cell type as nuclear donors.The m e t h o Q l o g y ~ f o r e m b r y'onineachof 0~ these species is essmtdy similar.Drploid donor nuclei have been transplanted intoenucleated MII oocytesthat activated on,orakrbansfer.In rabbiits, nuclear transfer has been significantlyless a single o&ping was reportedby transferof a somatic cell nucleus. However, no live o@mng were obtahed in StUdKs using f d fibroblastsas nuclear donors.In thisstudy,we studied cloned &it genedon by usingtransfer of fetal fibroblast and liver cell nuclei, Methods:Japanesewhite~abbits~usedaseggQnors. They were supemby PMSG-hCG tmtmmts. Fetal fibmblastsand liver cellswere isolated hDay 16 of fetuses fiom a Dutch Beleted rabbits. Donor cells wae cuMO.5%fetalcalfserum(FCS)for3-4dayspior to use in nuclear tmder. Donor cells were t m n d d into enuclearedoocytes.Reconshuctedembryosmh in DMEM with 8 % FCS and20 embtyosderivedh m ~kwere~into41lecipii15emb1yos

derived f b m h e r cell nuclei we^ & dinto 3 recipients. Results: One mipient b a n s f 4 with embryos derived h m fibroblastnuclei delivered one male, and also two mipirmtstmnsfbd with embryos h m liver cells delivered 3 otlkpring. However, all bunnies died within several horn after bhth.

41

P-3-05 Establishment and cbacacterimtionof the brain stem cell line derived fmm the 14 days m gestation mouse embryo ~ ~ m o hTAMAGAWA’, ar~ ~sam ISHIWNA’, ~ MW IGUCHI’.Ciko ISHTWAXA’, Satoshi OH2,Kazushige KIGUC13,Kahei sATo4

Ipmpose] Apuposeofthisexpe&mtkthe

estabkhmentofamouse tnain skm cell starin [M~alsandMethods]TheddYmouseembryos(l4 days in gestation)obtained and their txain tissues wmrinsedtwice with thecuhm medium and minced with asharppairofscissorsand dissociatedwith 600 hnaseUnitD1spaseinthec~mediumfor3Ominat room t e m m , and centrifuged at900rpm for 10 min. Thesedimentswereresuspendedinthegrowth medium, placed in 3.5cm plastic dishes,and incubatedat 37’C in a humidified amospk containing5% C Q in air. l’hegrowthmediumusedwasaMEMsuplemented with 1O??FBS,50 pghnl of aqhxnycm, 10 ng/ml of nervegrowthfirctor(NGF)and IOn~rnlofleukania inhibitay filcta(LJF) at final pH 7.2-7.4. The medium waschangedtwiceaweek [Resultsand Conclusion] The bi-pohr cellsappeared in thepimaryculture,gmvtapidlyandformed cellclusters. The c e l l c h wen separated by the colony isolation technique and the bi-polar cells grew well without intemptjon for6monthsandthe serial pasageswzzsuccessivelycaniedout40times. The cells W e r e C M in aMEM supplemented with lo?? FEE. The cells prolifemted rapidly, and the population doubling time was about 24 hours. The dvomosomes show a mouse n o d kayotype.The cuhed cells we^ spindle in shape, but a n a s t o m d each other by a cytoplasmicprocesseswhengmwthmediumwerenot changed for 10 days. The c W cellswere composed ofthree kinds of cells. some cells (glial cells) contained Glial filnillaracid protein(GFAP)and SlOOpotein othercellS(neuron)contained inand peripherin. Thethirdcells(stem-likecells)w€?enot stainedwiththesedmand~alksline -activity.

42

P-HI6 In vitm daferentialion of mouse embryonic stem cells into neural cells Hayawa Etsuko’,T m w a Tomoham2,Tokieda yuk~, ~shiwata~samu’and sat0 Wi’.

Mouse mtnyonic stem cells (ES cells)are continuwsly pwing cek Mved 6wn the inner cell mass ofthe 3.5 day bl-. Escellscanbecubed in an undiifferentiatedsrateinvitroforextendedperiodsoftime, and I.etain the abilitytocontriito all cell lineages including the germline. Retinoic acid (RA) is a potent diffiitiationinducercomonlyusedtotrigger diffmtiation of ES cells specificallyinto endoderm-like and hblast-like cells. This study was canied out in order to understand digeraaiation of ES cells into neural cells.At day 0, ES cells was plated in cell c u b flasks COntainingDMEMsuppkmentedwith lo??FBSand 5 x 1O-’M 5 x 10% RA as a differentiation inducerin the absenceoffeedercellsandleukemiainhiiifsctor and cultured at 37’c in 5% cqin air.& day 2, ~cellswerecollectedandre!platedintocell cultwe flasks m DMEM suppkmentedwith 10% FBS, witbout Wand RA.Themediumwaschangedcncea

-

weekRe!jydlessoftheRAdensity,putativepmgedm of neurons and glral cells (nestin positive cells) W e E identified one day after the om& ofdiffitiation. The characters O f tfiese cells we^ confirmed with KT-ITR a n d h ’m ’ g.Someofthesecells~showed positive for astrocytespecificantigens(GFAP antigens), and other cells were showed positive for neumn-specitic antigens(cytoskelton poteins MAPZ).

P-347 Relationship between the ICM ratio and the developmental ability over the blastocyst stage

x

Junko Sunaga'), Ryoko Tomi m a i, ii), Hideyuki H. Motohashi i, and Kahei Sat0

P-3-08 Targeting Disruption of Hepatocyte Growth FactorActivator Inhibitor Qpe 1 (HAI-1)GemeResnlbt m Impaired Placental Development and Embryonic LetbalitymMice

i) Department of Applied Life Science. Nihon University Graduate School of Bioresoume Sciences, Fujisawa, h a g a w a , ii) Department of Obstetric and Gynecolow, Nippon Medical School, Sendagi, Tokyo Introduction: It become clear that the total cell number (TCN) and the ICM cell number depended on the environment during early development including the culture media. It is supposed that the TCN and the cell number of ICM affected the implantation and the subsequent development. In this study, we investigated the relationship between the ICM ratio and the developmental ability over the blastocyst stage in in v i m culture. Methods: ICR strain mice were used. After the PMSG-hCG injections, females were mated with males. Pronucleus (PN) and 8-cell stage embryos were flushed out from the oviducts at 22 and 64 hr post-hCG respectively. The embryos were cultured in KSOM-AA until the blastocyst stage. The produced blastocysts were co-cultured with mouse fetal fibroblasts as feeder cells in DMEM supplemented with 10% FBS. Some of them were stained with Hoechst and Propidiun iodide and counted the cell number of the ICM and trophectodenn. Results: At 98 hr after hCG injections, the ICM ratio of the blastocysts from PN stage (mean*SD : 29.1*6.3%) was significantly lower than that from 8-cell stage (43.7*6.1%). Over the 17 days culture, the embryos having a portion of autonomous beating were observed in both groups, though there was no significantdifference (PN; 8.3%, %cell; Thus, the ICM ratio was decreased by in 7.1 YO). vitro culture from PN to 8-cell stage. This result showed that the developmental ability in in vitro culture was not conditioned on the ICM ratio.

Hepatocytepwih fscbractivatorinhiiitortype1 (HAI-1) is a m e m b m n e a s m a serine p m e k inhibitor initially idenhifiedasapotentinhibitorof geowtfi tsdoractivator (HGFA). HAI-Iis expesed PreQrmnantlYin the epithelial cells in human body. Its mRNAisalsoabundantin the placenta, in which HAI-1is specdically expmsed by villous cytdmphoblasts. In order to adcbesthe precise pathophysiological rolesof HAI-I,we'mdvateditsgaKbytmngetedmutagew&m embryonic stem ~ek. Heterozygo~HAI- 1+/- mi= underwent Ixxmal organ development Compl& inactivationofthe HAI-1 gene resulted in embryonic lethality, which became evident at embryonic day 10.5 p o s t c o h(El 0.5). As early as E9.5,mutant eanbyos Itxaled growth~mwhichdidnot =fled impaired cell prolifkdon but d l e r lesultedfrom filed placed development Histological analysis indicated that HAI-1is essential for the formation of labyrinth layer of the placenta

43

P-3-09 Periosteum Cell plays a role in bone formation by recombinant human Growttddifferentiation factor-5 in murine calvaria and application to human cells

P-3-10 Establishment and characterization of a pmlactia secreting cell line derived from early Es cell of spontaneousdwarf rat

Yamamoto M I J , Yoshimoto T2, Sekiya H', Negishi Y4, Sakoda K2, Izumi Y2 and Taka0 S' I Research Centerfor Life Science Resources, Kagoshima University; 2Department of Periodontology, Kagoshima University Graduate School of Medical and Dental Sciences; 'First Department of Oral and Mmillofacial Surgery, School of Dental Medicine, Tsurumi University 'Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Teibo University

GrowtMdifferentiation factor-5 (GDF-5), a member of TGF-beta superfamily, is known to play a role in embryonic skeletal formation such as endochondral ossification, synovialjoint formation, and tendon and ligament development. The purpose of present study was to investigate the osteoinductive effect of recombinant human GDF-5 (rhGDF-5) on adult murine carvarial bone in vivo and carvalia-derived cells in vitro. Microcomputed tomography (pCT) image showed augmented bone formation on ddY mice calvariae surface injected with rhGDF-5. Histological staining on decalcified sections illustrated de novo bone formation on the injected surface. Typical cartilage tissue was confirmed by H.E. and A.B. staining. Cartilage-related molecules, such as type I1 collagen and amecan were positively stained on decalcified section of augmented bone. Carvalia-derived cells such as primary osteoblast(POB), periosteum cell(POS), and connective tissue fibroblast(CF) were isolated enzymatically from dissected neonatal murine calvaria. Consistent with in vivo observation, up-regulation of both molecules were also demonstrated in periosteum cell by RT-PCR analyses. Angiogenic growth factor VEGF and its receptor flk-I were also enhanced in cultured carvaliaderived cells in the presence of rhGDF-5, though osteogenic extracellular matrices were not increased. Similar effect was observed in human gingival fibroblast and periosteum cell culture. In conclusion, bone formation by rhGDF-5 was comparable to endochondral ossification, though the cells participating in it were different from cells in epiphyseal plate at long bones. This growth factor could be beneficial for bone regeneration in human.

44

[purpose]Thepurposeofthis~onistodescribe

the p.ocedurefor establishinga prolactin secretingcell lineh m ES cell and to give preliminary h f i d o n on themorphologdcharactensbc *sandfunctionofthecell line. [Materials and Methods]l'he early ES cellsof

spontaneousdwarfmt(SDR)werecuhredDMEM/F12 medium supplemented with 1O??FBS and 1ng of LIF/ml. The* Es cellsweFeCuWwith e m l x y w c ~ I s ~ w i t h oLIF u by t hanging drop method. During,the embryoid bodies have grown into small embryonicmonster by ETF-supplementedh, the pieces of primordialdiencephalenwere bansplanted into

them.'Iheprimodaloqpnswithcapillaryndswere appeaFed' g the g&s in the embryonicmonster. 'These primordial organs picked up by fine grass pipette,

and werecultuFed in the35 mm dishes.% di~immunoassayrevealedthatsomecelllineof primordial o w s e c d proktin. Then a mundish cell colony was isolated,and RES-PceU line was established [Results]"he RESP cell line seurted 170np)ml of proladin for 3 days. "he cells were rwnd or polygonal in shape and easily removed h m the bottom of the dish, and p w n as floatingclusters.Electron micrographs revealedthattheceushad~mimvill~desmosomes, small vesicles and lysosomes. Il>lscussion] l'he cell line is very h l for d i g on the paractine regulationof GH or GH receptors etc.

P-3-11 Establishment and characterizationof a human endometripl stem celi4ke cell strain

lsamu ISHIWATA', Tmoharu TAMAGAWA', Megumi IGUCHI', Chieko I S W M A ' , Kaplshige KIGUCM2, Kahei S A W

[pmpose] The stem cells may exist m the human endometrium.M m ,we would like to repoxthe establishmentandcharadenza$ * 'onofthehuman -stem CeMke sbain(HKUsc)and theresuh of OUT investigationon the biological properties of the cell sbain. -&and Methods]The endometrial tissues obtainedbycurettageundermagreemerrtofapa?jent weaelinsedtwicewith theculturemedium and minced with asharp pair ofscissors in the& medium

conCaining6OORonaseUnit/mlI)lspase fa30minat 37'candfinallycentnfu%edat300xgfa10min.The sedimentswereresuspendedmthegrowthmedi~ placed i n h plastic dishesand incubated at3732 in a humidified atmosphereContainig 5% C Q in air. The p w t h medium used was aMEM supplemented with 100/0 human umbilical blood smm, 50 pg strepeomycin /ml, I O n g / m l E G F a n d I ~ m l L I F , a t t p H 72-7.4. The smaU round cells proliferated rapidly and were separsded h m the cultures by the colonialcloning methods. -and Conclusion] We could p v e that the newly established cell shain (HKUSC)is identical tothe human edon-whalstem cell-like sbain with the followingfkts, a) the material culturedwas he n d o d u m . b) HKUSC cells have remainedviable in cuhure for over 6 months and over30 serial passages have been made, c) the CUM cells have an epithelial cell arrangementand alkaline phosphataseactivity, d) the chromosomesshow a human normalkaryotype,46,XX soon.

P-3-12 Periodontal Tissue and Regenerative -Induction of Medicine Differentiation of Human Periodontal Ligament Cells into OsteoblastsMotoyama S'), Hashimoto H2),Tachibana T 'I, Sat0 El),Kamoi K"

"Department of Periodontology, School of Dentistry at Tokyo, The Nippon Dental University; 21Departmentof anatomy(t), The Jikei University, School of Medicine Regenerative medicine, in particular that using somatic (tissue) stem cells, has received a considerable amount of attention in recent years, and in the field of periodontics, bone regeneration has been a major obstacle for some time. In this study, we attempted to induce differentiation of tissue stem cells in the periodontal ligament into osteoblasts. The placenta is an integral part of embryogenesis, and since odontogenesis occurs at weeks 7-10 of gestation in humans, we speculated that physiologically active substances that induce odontogenesis must be present in the early placenta during this time. Therefore, with patient consent, the early placenta (week 8 of gestation) was cultured in a DMEM/F 12 medium (1 0% FBS) as follows: the placenta was washed, pefised, cut into small pieces and then dissociated in 0.2% trypsin-0.02% EDTA/PBS(-) for primary culture. After confirming cellular proliferation, a conditioned medium was collected after three days of serum-free culturing, lyophilized and dialyzed to prepare placental embryotrophic factors (P-ETF). In addition, with patient consent, the periodontal ligament was collected during tooth extraction in orthodontic treatment. The periodontal ligament was minced and treated with the above-mentioned digestive enzyme solution for primary culture. The above-mentioned P-ETF was added to the cultured periodontal ligament cells for up to five generations to induce differentiation into osteoblasts. Osteoblasts were identified by anti-osteonectin, anti-osteopontin, anti-osteocalcin immunostaining and electron microscope. The results showed that P-ETF induced differentiation into osteoblasts in a dose-dependent manner, clarifying that tissue stem cells or the progenitor cells of osteoblasts are present in the cells that make up the periodontal ligament. Since P-ETF contains several growth factors and cytokines, we are planning to investigate which of these compounds or unidentified compounds induce differentiation. 45

P-3-13 The effect ofA M on ERKln phosphorylation m human periodontal tigament cellp

Iino MI),Okayasu K2),Osawa G2),Nakaya H2) and Kamoi K"

P-3-14 The Effects of Human Placental Embryonic Factor on Periodontal Wound Healing Keiko Enomoto*, Hisahiro Kamoi****,Soh %to* and Kyuichi Kamoi*

?he~ofpesentsadyevaluatedthe~of h~placencal~bryonic~~-~f~periodontal A c t i v i n , a m e m b e r o f T G F ~ s ~ , h a s s h o w n t o WOUdhealingiIlbeagledOgS.P-ETFwaS~in StoreinmKmalbOnemabix, promatebanefiacaoe culturedhuman pkcental cellsand includesVarious healingpocessand regukbone ~ l i i g aone s of biologicallyactiveproqeinssucjlasp~~~vedgrcrwth coupling &O .S I. Ihe-xewasa report hitActivin W r , transforing p w l h bctor-p, vasculm endothelial prwnotedcellpl~o~Alkalineph~hatase(ALp) p w t h fictor and hepatocyte p w t h fixtor, as well as activityandcollagenmetabolisninperiodontalli~ other less welldescribedangiogenesis and differentiate (PDL) cells.The purposeofthis study was to proteinFdctors,IheclassIIfivcationwasexperimentally theroleofactivinonALPdvity, mRNAexpression created in the mandibularpremolarsof six male beagle (RANKL,Opci COX-2 and Cbk-I)and levels of active dogs. Periodontal qpedvetnxbmnbwerethen MAPK (ERKIR)in PDLcells treated with Advin perfmedwithandwithtthetopicalapplicationof (5Ong/ml, IOOng/ml) a BMP-2 (SOnglml, positive P-ETF. It were assigned to be Watd in one of three ways control). randomly;(1) in the fxmbdm (flap surgery done), (2) PDL cells wete obtained h m healthy tissue of in the atelocoUagen group (atelocollagenvehicle), (3) in exbaated p o l a r teeth for orthodontic m.PDL thewgroup('onof P-ETF in collagen vehicle). cells \Mae i n c u w m D-MEM with 1oo/o ms and Four and eight weeksafkall therapies,a histobgml bated with Advin aBMP-2 fa 1,3 and 7daywUP evduationoftheeffedonnew?issueformationwasthen activitywasquantifiedinsupeana$ntculturedhuman performedby~periodantaltissue~on. PDL elk by U S A . W-PCR w a d ~ to identifL ~evaluatedparamecers~bone,cementumand mRNAlevekforRAMU,OPG, COX-2andCbtbl. conneaivehattachment regenaation, length of Td-ERKlO and @IO@KFERKI/~ prate'rnepitheli~~andankylosis.Histologically qwdfied by ELISA . evaluated,theamountofnewamentum and camedive ALP activity was signifhntiy inawsed (p4.05). tissueatkhment in the test group wem m m h in the Levels of RANKL, Cbfb-1 and COX-2 mRNA we^ control group (p4.01). The length of epithelium in the significantlyupregulated (p4.05). ERKlR w a more control group was more than in the test p u p 0 . 0 1 ) . activatedwithActivin.?hesr:resultsuggestthatActivin 'Iheamount ofnew bone in the test group was mle than promotesosteobksticchuader of PDL cells. RANKL in the contml group. We conclude that P-ETF promotes mRNA may ~gukdeosteaclastogenesisin PDL cells. new attachmenon the periodontal tissue regeneration PDLcells possess response toAdivin that may be alteml treatments. at the signal transduction level like BMP-2.

46

P-3-15 Towards elucidation of the molecular mechanism ofADPm

P-3-16 Dynamic behavior of paired claudin strands within apposing plasma membranes Hiroyuki Sasaki' and Shoichiro Tsukid

[purpose] Autosomal dominant plycystk kidneydisease (ADpKD) isoneofconunon l e d diseases,m h g in clonic renal hilure. Although mutated genes, polycystin-1 and plycystin-2 w m identified, the molecularmechanism of cystic dilatationof ducts was unknown. We identifieda novel gene, Makorin 1 which is a potentialtarget of plycystin genes. Here we proposes new shategy towads OfADPKD. * elucidationof the molecular mechamm

(M-1 We identified Makorinl ,which wasamutated gene hihe mouse exhibhg polycystic kidney and bone & f e . Developmental and biochemical analyss suggests that Makorinl is present in the Wnt signal transduction pathway. First, we Will elucidate how Makorin 1 reCeives and bansducessigrral. Particulary, we will focus on phosphorylationof Makorinl. Second, we will iden* interacting pFoteins OfMakorinl. Ihird,we will address thedevelOpnentalmleofMakorin1.

r-I We made several phospholylationspecificantibodiesof Makorinl totme phosphorylationsignals. C a l m d i n k, casei kinase specificallyphosphoylate Makorin on stimulation by seaum after its starvation.These signals propagate by an &Idependent Won.These signal hansdudion couples cell poliferation and gene expmsion profiles. Thus,we proposethat Makorinl nxeives signal fiwnoutside and mmducesinto nucleustochange cell adhesion and cell polarity. [conclusions] We examined Makorinl signal transductionpathway by tracing phosphorylation status on stimulation.Makorin 1 will receive sequential phosphqlation,and translocate into nucleus to adjust gene expression.

'Department of Molecular Cell Biology, Institute of DNA Medicine, The Jikei University School of Medicine, Nishi-Shinbashi, Minato-ku, Tokyo 105, Japan.; 'Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501,Japan. The tight junction (TJ) strand is a linear proteinaceous polymer within plasma membranes, and each TJ strand associates laterally with another TJ strand in the apposing membranes of adjacent cells to form "paired" TJ strands. Claudins have been identified as the major constituents of TJ strands, and when exogenously expressed in L fibroblasts, they polymerize into paired strands, which are morphologically indistinguishable from paired TJ strands. Here we show that GFP (green fluorescent protein) fusion protein with claudin-1 can also form similar paired strands in L fibroblasts, allowing us to directly observe individual paired claudin strands in live cells. These paired strands showed more dynamic behavior than expected: They were frequently broken and annealed, and dynamically associated with each other in both an end-to-side and side-to-side manner. Through this behavior of individual paired claudin strands, the network of strands was reorganized dynamically. Furthermore, FRAP (fluorescence recovery after photobleaching) analyses revealed that claudin molecules were not mobile within paired strands. Although these observations are not necessarily representative of TJ strandsper se in epithelial cells, they provide important information on the physicochemical properties of TJ strands and their possible implications in the physiological functions of TJs.

47

P-3-17 Establishment of a a4l line developed from human amnion to h a t hepatic f d w to sewe as an idealmaterial for artificial liver apparatus and cell transplantation. Tabei I', Ito R', Kyouda S',Ohkubo TI,Ishida Y', Tachibana? HashimatoH3, IshiwataI*, Ohi 9, !jatou K ' , K u b HI, Yamazaki Y',Yanaga K',W w a H2,

[Objeet]Althoughlierl~ansp~onhasbeoomethe ultimate therapy for he pat^^ Mure patients,the lack of absolute donor o w has opened the patb of development to adjwant andor alternative methods such as cell bansplantation and artificial liver apratus. The mearch to develop the mataial for these methodshas involved

~embpyonicstemcellsandtransfection,ofhepetocytetobecom ' e~ cells.Butmost trialsare stalled due to unansweredproblems involving fimctio~tumorigensis,immunogeneticand~ questions. The p h t a is usually disposedafter birth and amnion obired hmthiswasteexpresseslittle MHC class I and no class II. We focused on this material that also~ucesotherimmun~~lartolyfidws, hypathesipingtofind immatm CelkthatwoUldmatuIe intofunctionalhepadocytes.~]Amnioncells obhtinedfiompkrcenoidonatedforresearchstudywas used.The amnion cells WE cultured in gmwh medium supplemented with DMSO,sodium butyrate, HGF, FGF or Embryatrophic factorsto induce differentiation.Then they were CUM in collagen sponge to chamcmze * the dserartiated cells in 3dimensioncub. m&] Amnicellsare h i r l y d celkwilh illmame q t o p h a n d ~ d ~ ~ p o t e n After c y . sevd~the~plasnbecQmesrichwith various organelle, evolving the e l l s to differentiate. Within the collagen sponge 3dimention culturethe cells pmducedAlbumiiatypicalcharactenstr * 'cofan hepatocyte. Rate of differentationwas 7?!.[Coodlrsion] Analbumin producing cell line was sucoessfirlly induced anddifRmt&ed h t h e a m n i 0 n . ffan hepatic cell line issucus~Miyd~~fiomamnionceUs, immunological rejection is predicted mild due to h4HC inactiveness,regardlessofHLAmismatch.Thusmaking this Fesoufce an ideal material for artificialorgan and cell banspianlation.

48

P-3-18 Isolation of unditrerentiated cek Derived from rabbit Embryos Using Somatic Cell Nuclear Transfer

P 4 1 7 Biological characteristicsof cultured cells derived from various types of human brain tumors

Yorino Sato, Ai Kazami, Naoki Okamoto, Koji Hosaka,

lsamu ISHIWATA', Chieko ISHlWATA',.Megumi IGUCHI',~asayukiSOMA*,yoshiro SAW, mot0 SONOBE), Kaarshige KIGUCHI', Toshiaki TACHLBANA', k h i ISHKAWA5

Kahei Sat0 Department ofApplied L@ Skiewe, Gr-e Biomsme sCiences,Nihon Universiw

school of

Introduction: Embryonic stem cell (ES cell) will be useful in regenerathe medicine. Ln this study, we examined to produce reconstituted embryos derived from somatic cell nuclei. and to isolate undifferentiated cell lines h m the embryo in rabbit. Materials and methods: M I1 oocytes obtained f h m superovulatedJapanese White female rabbits and Dutch fibroblastcells were used as nuclear recipients and donors respectively. The oocytes were enuclated and injected donor cells usingthe micromanipulator.The reconstituted embryos were produced by two methods, electorofision and microinjection.The reconstituted embryos were activated with 7% ethanol for Sminutes, and they were exposed to 6diiethylaminopunne(DMAP) + Cycloheximide (CHX) in HTF medium for 3 hours to re& the chromosome condensation and the release of 2nd polar body. The embryos were cultured in modified HTF medium to develop 8cell stage. The embryos developed to blastmyst were cultud on the f d e r cells layer in Ham-F 12 medium. Results: The number of reconstituted embryos was 72. We observed that 38.8% of embryos developed to 8cell stage. The rate of development to morula and blastocyst were 26.3% and 15.6% respectively.Although it was a low rate, hatched embryos were confinned in some reconstituted embryos. Conclusion: The reconstituted embr?,osdeveloped to blastmyst and more stage in rdbbit. Father. there was not the difference attached cell populations comparison with control in morphology. However, developmentalrate was low than the control. It is necessary for effective developmentof reconstitutedembryosto improve culture condition.

1) Ishhvata Ob/GyHosp, 2) Ibarakikrn Health LQrvice Association,3) Mito National Hup,4) Lkpt of Oh/Gy,9 Mwiunm Univ, 5) &pt ofAnutotg Jikei Univ, Sch of Med

[Purpose] We attempted making culture the brain tumors, establishingthecell linesand clearingdifferences of their biological properties. [Materials and Methods] The 45 cases (7 cases of asmytoma, 2 cases of oligodendroglioma, 2 cases of glioblastoma,2 cases of ependimoma, 13 cases of meningioma, 6 cases of pituitary adenoma 5 cases of neurinoma, a malignant lymphoma, a choroid plexus papilloma.and 6 cases of metastatic tumor) were placed on culture. For culture, the tumors were minced with scissors, digested with 600 PU/ml Dispase, and finally centrih~dat 300% for 10 minutes. ' h e sediments were placed in 6cm plastic dishes, and incubated at 37°C in a humidified atmosphere 5% Ca in air. The growth medium used has been Ham's F-12containing 15% fetal calf sem. [Resultsand Conclusion] We succeeded in making a primary culture of 33 casg and maintained 1 7 cases vitro over considerableperiod oftime (over three month), and succeeded in establishing cell lines. In the early period of the primary culture, the astrocytoma cells were characteristics of contacting each other by their cytoplasmic processes and the oligodendrogliomacells were small spindle-shapedcells and were characterizedas posessing the cytoplasmic processes, the glioblastoma cells revearled neoplastic and pleopmorphic features and were characterizedas posessing the cytoplasmic pmcesses, the ependpoma cells were characterized as f m i n g the rosset-like cell anangemenf meningioma cells were! spindle-or round-shaped cells and characterized as forming psammoma bodies, pituhy adenoma cells were!round- or oval-shaped cells and producingGH, ACTH, prolactin,etc. the neurinoma cells were spindle- or fitxousshaped cells, and the m a l i w t lymphoma cells were round and formed cell aggregates floating in the culture medium.

P a 1 Establishment and charaderhationof a human breast cudnoma cell tine (Nab=) Ohi S', Kyoda S2,Tabei 12, Kubo H2,T a c h i T', Hashimot0HI,Yamazaki Y2,Yanaga K2,and Ishikawa

HI

A cell line was derived hbreast tumortissue of a humanfemaleanddefinedasNabcaThepathological

~iswasscintwxlscarc~HormOneFecepQIs weat negativeforeshngeqprogestenrneand HER2.The focusofthe lefi b r e a s t a u n o r w a s .sulgKAly ~ by method of modified radical mastecbmy (Patry).

s~lyremovedsampleswereimmedirbelycolleded for incubation. To isolate the cells h m the tumor tissue, the tissue was washed with Hanks solldionandwascut as small as possible using two razo~ blades m TrypsikEDTNPBS(-) solution. Dissectedti- and cellswwzthen placed into 50 ml centif&@ tube. Subsequently,the tube was stationed for about 30 sec, and t h e n t h e s u ~ c o n t a i n ignis0M cells was collected andtxandmd into anew 50 ml ofmtdigal tube and centdiged at 1,5OOrpm for5 minAfter m~gatjon,thesupematantwasmoved,and immediilythe pellet was suspended with fksh growth medium(GM,DMEM/FI2supplementedwith17.5% f d bovine smut&0.1% * aminoacids solution, 025clghnl fungizone,and 50U penicillin/ 50clghnl streptomycin). The cell suspensionwas p W into tissue cuituredishes, and medium renewal was done twiceaweek S u b c u m were performed at an appropiatetime. Welldeveloped smooth and mu& ER, lipid droplet, many mimfilaments,o h microvilli and desmomes in the attachment of cells weat o h e d using an eledm mimscopy.

P402 Establishment and characterizationof a human ovruian small cell cartinoma cell b e (023-1) SeCEefing PTH, PTHrP andACI'H Ohi S.',Niimi S?,Yamada IC2, Yasuda M?, TachibanaT', ~ashimotoH.], ana aka T2 and ~shikawaH.'

This is a novel cell line (OW) derived hm human ovariandcellcarcinomaseaeted PTH,PTH-rPand ACTH. 'Ihe OS1 cell line was e s t a b l i fiwn metastatiC focusOf utenrs.A patient 25-yea1~ld Japanesewoman. The first she meived left OvarieCtDmy on April 2002.The histopathologicaldngnosis was ovarian small cell carcinoma, p n , Nx, Mx. Then on June 2003,metestatic focusofuterus was edomied. Apart ofthe tumorwascut into small pieces with raux blades, and dissociatedwith 0.1YOtrypsin-0.02Y0 EDTN PBS(-)solution at room temperature.The singlecells and snallclusterswereseededinto6Omm dishesanddtmd in p w t h medium (GM:DMEM/F12supplemented with 20% fetalbovine senun and 0.1% nandammo acids solution)at 37T, 4.7% CQ in humidified air. Medium was exchanged twicea week. OS-1 cells grew asfloatingcdtunsmttredishes.Radioimm~of condihionedmediawasreyealedthatthe~~

hgeamomtofPTH,PT'HrPandACTH~ely. In OUT knows it is the first repottthat the cell l i semtingPTH,PTHrPandACTHwassvccessllly

established.WeexpectthatOS1u m b i i t o s h d y o n themechanismofed~pichoxmonesexxtion.

49

P

a Establishment and Characterization of JHUCS-1 Cell Line Derived from Carcinosarcoma of the Human Uterus.

Kyosuke Yamada’),Toshiaki Tachibana”, Yasushi lida”, Kazu Ueda’),Akihiko Misawa”, Nagazumi Suzuki”, Hiroyuki Takahashi3’, Hiroyuki Kato3’, Eizo Kimura”, Makoto Yasuda’), Tadao Tanaka” and Hiroshi Ishikawa2’

Department of Obstetrics and Gynecology’), Department of Anatom#), Department of Pathology3),The Jihi University School of Medicine Abstract The cell line designed JHUCS-1 was established from a carcinosarcoma (malignant mixed mesodermal tumor) of the uterus that was surgically removed from a 57-year-old Japanese woman. She underwent a hysterectomy, bilateral salpingo-oophorectomy, omentectomy and splenectomy for the uterine malignancy on October 18, 1998. She had received neither chemotherapy nor radiation therapy before surgery. The pathologic diagnosis was a carcinosarcoma (stage IVb, pTI bNOM I) and a metastatic sarcoma that was observed in the spleen. We carefi~llyexamined the histopathology of the original tumor after the cell line was established and noted differentiation into a neuroendocrine carcinoma within the tumor’s epithelial components. Immunohistochemical staining of the tumorous tissue that had been heterotransplanted was positive for Leu7. Additionally, secretary granules were observed in the grafted cells as determined by electron microscopy. These results support the existence of neuroendocrine cells within the JHUCS-I cell line. Since few reports have been published describing the establishment of cell lines in long-term culture of human uterine carcinosarcomas, and none have documented neuroendocrine differentiation, the JHUCS-I cell line we established will be used for future basic research studies on this tumor.

50

P-4-04 Establishment and chrvacterizationof human glioblastoma cell h e (HUB’I-n) Isamu I S W N A ’ , Chieko ISHIWATA’,M IGUCHI’, Masayuk~SOMA2,Yoshiro S Z o t o sONOBE3,KazLlshige KIGUCId, Toshiaki TACHIBANA~

Ipurpose] We would like to report the establkhment and Charactenzab ’ ‘onof a human glioblastomacell line (HUBTn)and the result of our investigation on the biologicalpopertiesof HUBTn [MaterialSandMehds] Theculturematerialswe~ obinedhm4crrsesofbraintumors. Thetumorswere minced* scissors,digested with 600 PU/ml Dispase, and finally mtrihgedat 3Ooxg for 10minutes. The sedimentswereplacedin 6cm plasticdishes, and incubatedat 37’C in a humidifiedatmoqhe~5% CQ in air. The pvdh medium used has been Ham’s F-12 m*g15%fdcalfsenrm.~and Conclusion] We succeeded in primary cultm of3 in4 c8ses. The long-tem passageculhaes wasnorgdfiom the pimaryculturesd i l d y , b u t g l i o b cell ~ line (HUBT~)wasestablishedbyc~g~nudemom tumorxenogdb. ’Ihislinegrewwellwithout intaruptionfor 4 years and was subcuhvated over 120 times. ‘The cells were spindle like and mnd m shape and neoplastic and pleomorphic featues containedGlial fibrillaracidpmtein(GFAP)andS-IOOproteinand multilayahgwithoutcorttadinhibition.A bougtEshapedlongpmjecticmwasnoted from a small cell. l’hecells p l i i rapidly, and the populadiondoublig timewasabout32hours.Thechmmosomenmber showed a wide distributionof aneuploidy. ’Ihe cultwe cells were easily bansplanted into the subcutis of nu& mice and producedthe tumor resemblingthe original tumor.

P405

Escabliphmentand characterization of

cell line derived from a human semus surface papillary carrinoma

MIZUHARAHIROSHI~, KOBAYASHIYOHICHI~, KANIsHl YOSUKE', s m souJLRolJcoNDo H A R ~ o ' , o H K u M A Y o s H w ' OHHAM , TAZURU],OKUDAYOSHIKO',W ~ A N A B EMARI], OKAMURAASAMI~, IIDATOMOHIRO~,KIGUCHI KAZUSHIGE],ISHIZUKABUNPEI', ISHTWHA ISM2

[object] We had d l y established a novel serous s u r f k e papillary carcinoma (SSPC)cell line HYKSSPC. [MaterialsandMettrodsl Thec.atcinomacellwas obtained h m 60 yeam old Japanese woman. After obtainingptientconsenttheasciticfluid was removed h t h e abdomii cavity and was used forcell culture.

Tumarcellweaeis0latedbym~gation(1OOO1pm5 minutes) fiwnthe ascitic fluid. Ihecells wese dispersed Ham's F12 medium supplemented with 15% of fetalcalf serum,and incubetedat337'cunder5% C Q in air. HYKSSPC was maintainedover 24 months, and successllly s m over33 times; k f m we designatedtfiis cell line (mu~ts]'Ihe population doublmgtimeofHYKSSPC was 5 1.4 hours.A phase cmtmt micrograph of HM(ssPcshowsthepavementstonelikeaIlangement witbout contactinhibition.'Ihechromosomenumber showed a wide disbiiutjon of aneuploid. Unfktunatelythe cells w a e could not xenotransplanted into the nude mice. [conc~usion]SSPC was first cIescrii as mesotheIioma resemblingpapillary ovarian adenocarinomaby Swerdlow in 1959.Nevertheless,the mechanisms of its pogress has not yet been clearedjust enough. So we believe fhlythat thiscell line will beamostuwhl material in investigation ofthe unusual disease.This cell line has been peserving somecharactasofdK~momawithgrowtfiin

.

V h .

P-4-06 A novel dline derived from a human small dlung carcinoma that secretes mPTH-*tedprOtein,and pmopiom~ocortin Mayumi ishikawa (I), KamhimKimm(2), Toshiaki Tachibana(3), Yrni Akishima (4), Masako Shimojo (1 ), Ha~imeUeshiba (I), Kumiko Tsuboi (l), Koichiro Nakata (2), YoshikjoAkasaka(4), Kapltoshi Shhuya (4), Gen Yoshin0 (5)

'Ihere ale few case reports describing snall cell lung minomas (SCLC), which secrete PTH-related pmtein (PTH-rP) and resuh in hyperralcemia We have establisheda novel cell line, derived fiom 37-year old , WOmenWitIl ~ , t h a t p N l d u c e d m - r Fand pFoopiosnelanocortin(POMC), and led to hypercalcemia The cell line, so called SLC-1, was gKiw as floting cell clusta in DMEM/F12 medium supplemented with 1oo/o FJ3S and had a populationdoublingtimeof 72 hours. ?he modal chromosome number was 47 (98%);marker c h m m m e s were not obsewed.The SLC-1 cell line

senetednotonlyYTH-rPbutalsoFTH,andbothwere dect.easedbyCaClzdmb&ah'on.Decteasingthe d o n of Ca" in the growth medium stimulated the SecFetionOfboth PTH-rPand rn since PTH-rPand PTHsecretionhtheSLc-1 cellswasrelatedtoCa++ u m d o n in the p w t h medium, the cell line might be usefill for the study of PTH-rP and PTH regulationas well as fot SCLC analysis. Onthe&hand,thecelkSeYXkdNterminal POMC, the precursor ofAClH, which was stimulated by

CRH. E~micrographs~vealedthatthereweretwo kinds of cells. However, it has not been know a single cell seaetes PTH,YTH-rPand POMC shultaneously or not. In summary, we established a novel cell line SLC-I, which derived SCLC,that pFoduced PTH-re PTH and POMC.

51

P408 Susceptibii test for chemotherapy usingendosapkbiopsy samples

Tabei I], Kyouda S',Ohi S2,Kubo H',Kashiwagi H', Yamazaki Y', Yarqy K', Tachibana ?,Hashimoto H2, lshikawa HI.

Lkp of S& & Anat.&, Jikei universrty school of Macine

A number of malignant cell lines derived fiom natural

killer oIJK>cell and n a t d killer-like T (NKT)-cell leukemias/lymphomashave been established over m t years. We undutookto furtherrefine the -C * 'onand classificationof the cell lines by analyzing their expression of T- and NKcell associated antigens and transcription factors (TFs). Cell lines used were: 7 N K ~ e l lines l (HANK-1, KHYG-1, NK-92, NK-Y S,Mu, SNK-6, YT), 5 NKTell lines (DERL2, DERL7, MOTN-I,MTA, SNT-8x5 T ~ e lllk derived from acute lymphoblasticleukemia (ALL) including3 gammddelta T a l l receptor (TCR) type (LOUCY, MOLT- 14, PEER) and 2 a l p h a h a TCR trpe (HPB-ALL, MOLT- 16)and 3 k l l v m (BCP) leukemia cell lines (HBL3, NALM-16, NALM- 19).Distinct azurophilic granules were seen in all NK- and NKT-cell lines. Epstein-Bam virus antigen was found to be positive for 5/7 N K e l l lines and 115 NKTcell line. NK activity was heterogeneousamong the cell lines. CDl %A, CD158B, CDl59 and CD212 were found to be specific for NKcell lines. All NKell lines showed positivity for CD2, CD7, CD56, CD94, cyCDl78 and HLA-DR However, expression of other antigens was heterogeneous. m i o n pfiles of TFs were also analyzed at mRNA and -in levels in parallel with immunoprofiling,morphological and finctional analyses. The following 13 TFs AML 1, CEBPA, E2A,

ETSl,GATAl,GATA2,GATA3,tKAROS,IRFI, PAX5 PUl, TBET and TCFl were analyzed.AMLI, E2A, ETSI, lKAROS and lRFl were found to be positive for all cell lines whereas GATAl turned out to be universally negative. CEBPA, PAX5 and PU 1 were negative for all cell lines except in three BCPell lines. GATA2 was positive for 315 T e l l lines but negative for the other cell lines. GATA3 was positive for 7/7 NK-, 415 NKT-, 515 T- and 2/3 BCP-ll lines. Most sbikingly,TBET was positive for all NK-and NKTcell lines and negative for all T- and BCPell lines. In conbast to the expmion of TBET, TCF 1 was negative for all NK- and NKTcell lines, being positive for 4/5 Tand 1/3 BCPell lines. Expression analysisof TFs revealed that NK- and NKTcell lines showed identical profiles, clearly distinct From those of the T-ALLor BCP-ALL leukemiaderived cell lines. The composite data on these NK-and NKTcell lines allow for the opedonat definition of typical neoplastic NK-and NKT-ll line profile^. NK- and NKTell leukemiallymphomacell lines will prove invaluable models for studies of normal and malignantNK- and NKTell biology. 52

Susceptibilitytest is no dwbt a very usehl and a reliile way to chooseand i m p v e efficacyand respanseratesof chemothempy. Meisummnts ofthe susceptibility device using the dissolved oxygen meter and the disposable oxygenelechodesensm, we have p v e d it to be rime convenient, economical, and time savingcomparedto conventional methods(M7T etc.). Results using only small amountsof biopsy samples were as precise as when using surgicallyobtained samplg without the need of complicatingand boublesome intenseculture incubation work [Methods] 5 Patients i n b u d d to our institdon foroperationongastrointestinalcarcinoma,had endoscopicexaminationwith mall biopsies of 5 to 10 pieces befm surgery. Suscepg'bilitestswere p e r f o r m e d on thesebiopsy samplesusing the dissolved oxygen meter. The mlts were compared to the susceptibilitytest mutts using surgically r e j d samplesthat were about 1crn3in size. Commerciallyavailable chemothaapyagents such aSCDDp, rmx+4DkCPT-I 1 etc.were used and effectiveslope of dissolved oxygen value was determined as -0.123 to -0.4 accordinglyh m preliminary experiments. Aithough less volume was neededin measuring using the biopsy samples, results of effective agentsmatchedusingregularsurgical Samples. [Results] The novel susoeptibility device was able to measure susceptibilityusing only small amountsof endoscopimllytakensamplesand m stwere made within 2 hours wilhout incubation or cell culturing 'IhiswillenablecancerpatientSwhowere inoperableand without s u r g i d samples for m v e n t i o d susceptibility test, who are the most likely to have chemotherapy, still have the chanceto have testsperfmed and receptive agentsdiagnosed.

P-44N

New approach to susceptibility test with metabolic anticancer drug

Kyouda Shigeya', Tabei Isao', Ohi Satoshi', Tachibana Toshiaki2,Kubo Hirotaka', Hashimoto Hisashi', Yanaga Katsuhiko', Yamazaki Yoji', lshikawa Hiroshi'

'

P410 PFOgnosticsignificance of PDZKl located on fkquentty amplified region lq21q22m drug resistant ovarian cancer Kudoh K, T h M.,Okamoto S.,Sasaki K., Fujii K, Hirata J., KitaT, Kikuchi Y.

Jikei Universir),School of Medicine Dept of Surgery; Jikei Universir),School of Medicine Dept. of Anatomy 11

Lkptmeni of Obsteb-icscndGynecdog,National D&me M & College,3-2NamiRz,T-a sairama 3.59-8.513,Jrpxn

Chemotherapy, an established agent or cocktail regimen, is usually chosen in relation to the pathogenesis of the cancer. We often experience dissimilar effect and receptivity rates of anticancer agents in different individuals with the same diagnosed histology. When conventional susceptibility tests are performed, a cell line from a surgically obtained specimen is established and an anticancer agent is selected through the MTT method etc., a susceptibility test that measures damaged cells. This method at times is known to takes time and is not always a realistic procedure. Especially, susceptibility test against metabolic anticancer drugs such as 5-FU has been very difficult and unreliable. 7 cell lines and dissociated tissues established and obtained from surgical specimens were used. Samples were from colon cancer, gastric cancer, hepatocellular carcinoma, cholangiocarcinoma, and pancreatic carcinoma. The sensitivity difference between chemotherapeuticagent in each cell line was observed with and without 5-FU. Disposable oxygen electrode sensor and multi-channel dissolved oxygen meter(D0X 10/96) was use to measure sensitivity. In all 7 cell lines 5-FU alone showed no susceptibility.When 5-FUwas added prior, some of the agents such as MMC ,ADM , CPA ,VP-I6 ,CPT-I 1 ,and ACNU in some of the individual samples demonstrated a positive change in susceptibility. The susceptibility test using the multi-channel dissolved oxygen meter can accurately measure multiple samples simultaneously and detect individuality of the neoplasm. Focusing on cell respiration as an indicator of cell metabolism and activity, this made it possible to measure susceptibility to chemotherapeuticagents faster, easier, and more economical than conventional methods. The novel susceptibility device was able to measure difference in susceptibility with metabolic drugs such as 5-FU and the influence to other agents in combination.

Ovariancanceristhemostlethal gvnecologic malignancy, whichfkpmtlyacmmpaniedwithdisseminatedintra abdominaltumors, and pgnosis is deeply affected by chemotkapy~af?ercytoreductivemqpy. Charactensbc . - genetic changes in chemotherapyresistant ovarian MIK'RIS, however, are not well outlined yet We a d p d whole chromosomal changes by compadve genomic hybridization (CGH) in 28 ovarian cancer s p e & e n S r n V e ! d a t f i I s t ~ b e f r n ~ . Amplificasion of 1q2 1q22 appears significantlyfrequent w . 0 1 8 3 , chi-sqw test) in chemmistant tumors (9/14) than chefnosensitivetumors (Z14).Those who hartxxalq21q22amplificationshoweddeteriorated5 year slwival rate (35%) than patientswithout the abenation(49%).Exprsion of PDZ domain containing I PDZKI), acandidategene on amplifiedchrwnosomal regionwasdeteckd by quantitative realtime PCRNo ~withPDZKIoverexpressedtumotreachedto2 years survival, while 33% patients with low expession achieved 5 year survival.In addition, two natural mistant ovarian cancer cell lines (MHand KK) also showed 1q2 1q22 amplification along with PDZK 1 over expression,but a drug Sensitive cell line (KF28) and its drugresiStantsublines(KF2s/Tx,KFrl3andKFrl3/Tx) didnotshowthechromosomeande~imchanges. PDZKl isknownto interad with MRFZ and reportedto have role m multidmg resistance. Our &indicate that acquired PDZK 1 overexpressiOn with 1q2 1422

chmmosomal amplification dhgcarcinogenesis have critical mle m formingdrug refktovovarian cancer.

53

P4ll

Identification of genes responsiblefor an increased mvasive ability of Fenalcell caminoma cells under hypoxk conditions

P 4 1 2 Gene and protein expression profiling between primary and metastatic human renal cell carcinoma cell lines derived from one patient

Kunihiko Yoshioka, Yoshio Ohno, Tatrmo Gondo,

YoshiiiroNakqpni,KazuhitoMatsushita,Noboru sakamoto,MayukoMasumoto,ShoujiKoga,~ Namiki, TekhirouAoyagi, Makoto Ohori,Tadashi Hatan0and Masaaki Tachibana

INTRODUCI'IONANDOBJECIlVES HypoXia is a ' 'c of solid tumon, which modulates common charadensb many gene expxions and thereby altering an mvasive ability4 a metadatic patentid.llis study wasdesigned toobtainbetterundastandigofmolecularev~ followinghypoxia in renalcell Carcinoma(RCC)cells. MATERIALSAND METHODS The DNAchip methods,using IntelliGene2 Human CHIP 1 (TAKARA BIO) were applied for two RCC cell lines, caki-1 and KU 19-20to obtain DNA display pmfiles. These cells have been demonsbatedto have quite different cell kineticsinresponsetohypoxkRES~~Hypoxia significantlyincFeased both cell motility and invasiveness and deaeasedthe ability ofcellstoa t h h to collagen 4 in Caki-l cells, while hypoxia did not affect cell W c s of KU 19-20cells.A significantchange in expression in responseto hypoxiawas shown by284genes(203 ~ell~and294 inaeased,481 -)inCaki-l genes (248 increased and 46 decreased) in KU19-20 cells. The observed change was poorjr concordantbetween these two cell lines. only 39 of 284 genes with increased and6withdec~expessiOn~conunonly in bothCaki-1 and KU 19-20 O ~ U Sd hypoxia h Caki-1 cells,expressionof gena related to apoptosisand matrix metallopteinases(MMPs), and cell adhesion molecules we~eincreased in response to hypoxia CONCLUSIONS RCC cell kinetics in responseto hypoxia correlated well with hypoxia-indud molecular events. The hypoxia-induced upregulationof genes related to apoptosis, h4MPs and cell adhesion molecules may pomote an inmad invasive abilityof RCC cells.

Yoshio Ohno', Takeshi Kawamura2,Kunihiko Yoshioka', Teiichiro Aoyagi', Makoto Ohori', Kazunori Namiki', Shoji Koga', Mayuko Matsumoto', Noboru Sakamoto', Kazuhiro Matsushita', Yoshihiro Nakagami', Tadashi Hatano', Toshihide Nishimura', Masaaki Tachibana' I

Department of urology, Tobo medical university; Clinical pmteome centec Tokyo medical university

2

Introduction: To characterize primary and metastatic cancer cells is essential to develop novel treatment strategy for advanced cancer. We examined the differences of gene and protein expression profiling between primary and metastatic renal cell carcinoma (RCC)cell lines originated from one patient. Materials and Methods: Both primary and metastatic renal cell carcinoma cell lines, which were simultaneouslyderived from surgically removed primary and metastatic lesions, were utilized. The DNA expression profile of the cells was assessed by the DNA chip method and the protein expression profiling of the serum free cell cultured media was screened by mass spectrometry. Results: The expression of hexabrachion (hypoxia related extracellular matrix glycoprotein) showed the most significant reduction in metastatic cells, followed by the expression of epidermal growth factor receptor, microsomal glutathione S transferase I and cadherin 6 (K-cadherin) by the DNA chip method. On the other hand, the expression of Laminin receptor 1 and TGF-beta markedly increased in the metastatic cells. Four proteins were over-expressed in cultured media from primary cell line and one protein was found in cultured media from metastatic cell line. Summary: These results indicate that metastatic cells may lose their biological properties of the epithelial cell origin and metastatic features may be related to the hypoxic events related to the expression levels of extracellular matrix protein.

P413 The analysis of Sonic Hedgehog Siinesopbgdeancercelllines

P414 Loss of mtederon gamma Feceptor expmsion m esophagealsquamouscell ma cells Michio Okabe,' Junichi Kaganoi,' Shim Na@ani,' Masato Kondo,'Yutaka Shimada,' Go W e ' Masayuki Imamura,12

Back gmmd Recentqorts have -that activation of Hedgehog (HH) signaling pathway, which is involved in the regulation of precursorcell proliferaton in normal development,also plays an important mle m tumorigenesisandhunormain~in v a r i o u s ~ o f cancer. Inthisstudy,wemVesligatedthearpessionand funCti0nofHl-l pathway m human esopha%eal cancer cell

lines. Mataials and methods: 34 human esophagealsquamow carcinoma cell lines established in our depment were used and the expnsion of HH pathway molecules such as SHH, FTCH, GLI 1, GLI 2, GLI 3 and SMOweae examined by RT-PCRThen the effect of CyClopamine, a specific inhiiiof HH signaling, on cell prolifedon was inV&gated. Results:SHHandGLI 1 we~eex~1~sedin31 of34cell lines (91%) ,while FKH, GLI 2,GLI 3 and SMO were expessed m all 34 cell lines.Cell prolikath was signikady inhibited by Cyclopamine in 5 cell l i examined that eqmsed all HH pathway molecules, while cell prolifdon was not affected in 3 cell lines lacking SHH and GLI 1 arp.ession. conClwion:HH signaling@way has an impomnt role in the regulationof prolifedan in human esophageal c81K;eT cell lines. It is suggested that inhibiton of HH signaling pathway could be a potentid candidate for novel therapies.

We pviously reportedthat epidermal p w t h factor (EGF>inducedadvationof signal t m s d m and activators ofbanmipion (STW 1 resulted in apoptosis in dvee cell l i i of esophageal squamouscell CalCiIloma (ESCC). Epidermal growth factor(EGF>induced apoptosishas been reported in a very limited number of CUM cell lines,such A43 1 cells and MDA-MB468. As compared with EGF, mterfemn (IFN)-garnma is one of the cytdcinesthat sbmgly activates STAT1 m various typesofcells.IFN-gammastimulationSmnglyinhiii growth in 10 of30 Escc cell lines, in the other20 cell lines however, inhibition of growth was less or minimal. undertying differencesin the sensitivity Themecharusm * of ESCC cells to IFN-gamma has not been well studied, and a better undembding ofthis phenomenonmay provide important cluesto the optimal clinical use of I F N - g m m a a s c y t o k m i h ~We . ~eporthaethatloss of IFN-gamma ~ e c e p t expmsion o~ was obsaved in 6 of the 20 cell lines in which growth was minimally inhibited by I F N - w Such IOSS involved FeoeptoT type I in ~ n e celllineandreceptortypeIIintheotherfie.Allfour independent immortali flonnalcell linesestablished h m the Rugol-stajned portions of msxted esophageal specimensh m four patients expressed both IFN-gamma receptor type I and type II. To our knowledge, this is the fiEtlVpttoSUggeStthatesophagealcancerS~~bein

ptbecategdasaFeceptor-relateddisease.

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P415 Establishment and characterization of angiogenetic factor (HGF) pmducing cell line de&ed fmm uudiffemntiated carciooma of human thymid KumiiTsuboi (l), S b Kitaharir (2), Kapltoshi shibuya(3x Gen Yoshin0(1)

P416 Characteristics of MCF-7-MT cells with high mobility and migratory properties, selected in Chemotaxcell chambers, to undergo apoptosis upon induction by camptothecin compared to the parental MCF-7 cells Yutaka Tagawa', Masamichi I d , Piotr Srn~lewski~'~, Dorota Halicka3,Zbigniew Darzynkiewid I Department

Hepatocytegrowth fktop(HGF) is nxogmbdas ~ p w t h a f K i a n ~ 'Iherealtfew c ~ . reportr that anaplastic (undiffantiated) thymid minoma sec~eatesHGF. We have establisheda novel cell line, derived hlarge anaplastic carcinomawith samm like giant cells. The cell line, so called ANN 10, was gown in DMEMFI2medium supplementedwjth lPhFE3S.A population doubling time of 42 hours at 2 1 passages.

?hekaryatypeanalysiishowshyperdiploid,andmodel number is 72.The cell secretedHGF ( 125 n&V3 days) in conditionedmedium, but did not seutted thpglobuli thymxi&andth~iin,whichwas~h diffamtiated thymid cminoma HGFhasbeen knownthatit is not only hepahc growth hctorbut is agiogenic ~ * A n a p l a S t i c CarcifKHna is highly malignant, rapidly invadingadjacent structures and metastasizingdvoughoutthebody. And also it has been known HGF stimulates othangiogenic b, such as W F . It might be caused by HGF

secretionhanaplasticthpid carcinoma,that metastasisoccursvery fkquently, and the growth is very fist than differentiatedthymid carcinoma, In summary, we establisheda novel cell line, which derived fiom the most aggmsiveand invasive neoplasm ofthpid, anaplastic h p i d carcinoma,ANA310. This is the first report ofanaplastic thymid carcinow poducing HGF.

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of Health Sciences, Nagasaki University School of Medicine, Japan; 2Department of Oral and Maxillofacial Surgery, Yamaguchi University School of Medicine, Japan: 'Brander Cancer Research Institute, New York Medical College, Vahalla, New York; 'Department of Hematology, Copernicus Hospital, Medical University of Lo&, Poland In order to select cells with high mobility and migratory properties, an in virro model was developed utilizing Chemotaxcell chambers (Kurashiki Bouseki, Japan). Namely, MCF-7 cells were cultured in the chambers and the cells that were able to migrate through 8 micrometer pores during 24 48 h and continue growth for a week, were selected. These cells were trypsinized and subjected to the selection through migration, as above, three additional times. The cells with high migratory properties were then denoted MCF-FMT, and were considered to be of more metastasis-prone phenotype than the parental MCF-7 cells. The clonogenic potential of MCF-7-MT cells was reduced from 56 to 45 % compared to parental cells, but the colonies, when analyzed by laser scanning cytometry, were larger and consisted of more cells. Following treatment with I50 nM CPT the induction of p53 (wt) was more pronounced in MCF-7 (from 4.1f0.5 to 38.7 f 2.1) than in MCF-7-MT (from 4.6 f 0.9 to 29.9 f 1.9) cells (p<0.05). The increase in Bax to Bcl-2 ratio in response to CPT was nearly twice higher in MCF-7-MT than in parental cells. Also, upon CPT treatment the frequency of apoptotic cells characterized by the presence of DNA strand breaks (TUNEL assay) was over three times higher in MCF-7-MT than in MCF-7 cultures. Conclusion: 1) The MCF-7-MT cells selected based on their increased motility and migratory properties through the 8 pm porous membranes had many phenotype properties similar to their parent MCF-7 cells. However, their colonies' circumference (but note area) increased which would indicate that following cell division the individual daughter cells had tendency to move out from each other. 2) The MCF-7-MT cells showed increased sensitivity to the topoisomerase I inhibitor CPT as reflected by the reduced clonogenicity and measured by the apoptotic assays (TUNEL, PAW cleavage). Compared with the parental MCF-7 cells they also had increased expression of Bax and higher Bax/Bcl-2 ratio. However, the induction of p53 upon CPT treatment was more pronounced in the case of MCF-7 cells compared to MCF-7-MT cells.

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