ABSTRACTS OF

ORAL PRESENTATIONS

Chairpersons:

Isamu Ishiwata (Ishiwata Obstet & GynecoL Hosp.)

Nobuhiro Deguchi (IDepk of Urology, Saitama Med Sch)

Kegi Kwamoto (Depk of Nuemsurgery, Kansai Med UnzV.)

33

0-01 Establishment and characterization of the stem cell strain derived from human amnion

T ~ ~ I M I U TAMAGAWA', I S ~ ~ U ISW~WA', ship S A l T d

[purpose] Pluripotent Stan cellsare poposed to be used in qmeratktfierawandhavebeensuggestedtoaristin thehematopoieticsystem, ~ t r a d , s k e ~ m u s c k , theskinanfl~nervoussystemamltheirpresencein ~locationshasbeenclfirmed Plwipotent stem cells also may exist m the human amniot icmemh. We established a pluripotent stem cell line fiom human

~alsandMethods]Amnionmembnwofhuman fUll-tmnplacentaweremincedwithasharppairof scissors in the c u b medium wntamn ' * g 600 PU/ml of Dspase for 30 min. and mtdbged at900 rpm for 10

medium,pbced m&cmplasticdkhs,andincubatedat 37'Cin a humidified atmosphere Containing 5% C Q in air. ~p~mediumusedwasah4EMsupplemented with 1O?h FBS, 10 ndml ofLIF and 10 ndml of EGF. "he snall and round cells proliferated in multilayer vaeaspimtedwithmicnxaplary,lran&mdtoanew ChdiSh .

min. lllesedimentsmresuspendedinthepwth

~andGmcI~] 'Iheamniot ics temceU sh.ain (HAM-l)wasestabliShedandthe!HMA-l Cellsgtw rapjdlyandtheserialpasagesweresuocessivelycanied out5otimes.?heHAM-lcellswerechsn;pctenzed * asthe following biological properties; They (i) wem derived fiom the human amnion membrane, (2) have an a b i i i of self-duplication while maintaining an UndiffeFentiated state and normal karyotype, (iii) can difFknmthte into a 3 germ layer embryo with totipotemy, and (iv) have high specific enzyme activity (alkaline -).

0 4 2 In v i t ~ ~ di€femtiation of mouse embryonic stem cell into -myocYtes.

~ p v m e n t ~ ~ k d ~ S c ~ , Gmtuaeschoolof Bionmnme Science, Nihopl uitiversiry F@wwa KawPvaJm

hbpyonic stan (ES) cells are pluripcrttent cell isolated from the inner cell mas ofblastocysts and capable of s e ~ d a n d d i f f d o n i n t o c e u u l a r ~ ~ o f alllk!e gelm layers. EScellsaleusefirlmto shdy cardiomyocytedithentiation in Vitro. In this study, w e investigated e 6 c i i t induction of d m c m of mouse ES cell intocardiomyocytesushgreCinoic=id (RA) and 5 4 d i n e ( 5 d ) .

ES cells (1 29BV shain) wen? cum in suspension or in hanging drop culture system at acell density ofabout 1 X Id cells per drop, supplemented with 5 X 10% and 5

embryoid bodies (EBs) were formed m both culture systems. EBs formed in hanging drop system vae tnmfi i to suspension or adhesion cultme system. Each EBswe~embedded into padin and stained by hemabxylin and eosin (HE) at 10,20,30 days.

RA(5XI0%4)or5-azaCcWbmt&d ' intothemuscle

~El3swerecvlturedmorelhan30daysbutdidnot

x 10% RA, 3 JlM S d .After 5-6 days ofculture,

AsaI.esulfHEstainingshowedhtEI3sculturedwitfi

andvessel-likecells hall txlltmsystem. In the suspension

c o n t r a c t ~ . E B s ~ 6 u m l h e h a n g i n g dFoptotheadhesiculturewith5-azacshowedthemost

spo~usly,whentheyweFecuMwidl5-aracinthe hanging-suspensioncuhsystan,andwithRA.

eEientqontmxuiycontracbn ' g.LittleEBscorctracted

34

0 4 3 Factom iuftuencing the bbtocyst cell Metentiation and developmental competence m vitro and m vivo of m o w partbenogenetic embryos: amino acids supplemented simplex optimizes medium (KSOWAA) and insulin-like gtuwtb factor 2

'Ihe aim ofthis study wastodetamine optimal culture condition sf or mouse^ 'CelTlbryoandtO evaluate the developmed competence in vitm and in vivo.B6CBFl micewereusedasoocytedonors.Oocytes were collected at 14-1 5 h post hCG injection and

producedby lOmMshrmtiumcMdeand5c(g/ml cytochalasi B . T k e m b r y o s w e r e c ~ in M16, M1 aAA, M164GF2, WM, KSOWAA and WWAA/IGF2duringpreimphtationpaiod. D e t e r m i of cell numben of inner cell mass

a&ation)wasperformedbyaflwrescerrtdoubledye t e c h n i q u e . s o m e o f ~ blastocystsweae transf;erredtoCD.l femalesonday2.5of

10 post coitus, and the imphtation sites and f&uses m

KSOWAA, 95-98 % developed to bktocyds. In particular, devekpned rate in KSOWAA/IGF2 was IW! (89/89). Exogenous IGF2 pmmotes cell p l i f i i c m m blastocysts, inall cases. In the p p o f M 164GF2, hower, only ICM cells were increased, in cmmsttoinc~easeofcellnumberofbdhICMandTEin KsoM/AA + IGF2. Following ban& of patthenogenebc embryos to recipients, 70-90 % ofthe

incubated fot 1 h. Diploid perttrenogenebc - embryoswere

dtrophedoderm rn) in b W s a t g e ( l 0 2 h w

-. 'Iherecipii~sagificedonday

examined.ofpardKnogenebc - embryosculturedin

emblyos implanted in all CBSes, and the somite stage f~weceob lahedonday l O o f ~ . l . h i S s h d y

embryos is dramatically i n f l u e n c e d by clearly showsthat the development in vitro and in vivo of

KSOulAAdorIGF2suclplementation

04 Identification of the interacting protein with Makorinl

'Yoshihisa yano, *Noriyuki yoshida, 'Tomoka wachi, Munehisa ueno, hobuhiro deguchi, 'Shinji

hirotsune

3

Division of genetic disease research; 2Division of Chemical biology, Osaka city university graduate school of medicine; 'Saitama medical school department of urology

I

Purpose Autosomal dominant polycystic kidney disease (ADPKD) is one of most common inherited renal diseases, exhibiting cystic dilatation of renal ducts. Although the mutated genes, polycystin-1 and polycystin-2 were identified, how these mutations cause cystic dilatation was unknown. We identified a potential molecular target of polycystins, Makorinl . Here, we will propose a new therapeutic strategy for ADPKD using Makorin 1. Methods We made mutant mouse exhibiting polycystic kidney and bone deformity by insertional mutagenesis. A mutated gene, Makorinl carries RING finger motif, and supposed to be belong to Wnt signal pathway. First, we will identify an interacting protein with Makorin 1. Second, we will elucidate how Makorin 1 transduces signals. Finally, we will address the molecular mechanism of polycystic kidney associated with a mutation of Makorin 1. Results We examined subcellular localization of Makorinl , and identified that Makorinl is located in cell membrane and nucleus, suggesting that Makorinl transduces signals From outside into nucleus. We designed several strategy to hunt interacting proteins with Makorinl . First, we made several antibodies to generate affinity column. Second, we generated GST-tagged Makorin 1 for column purification. By these methods, we found several targets of Makorinl . We are determining amino acid sequencing using MS. Conclusion Biochemical and developmental study suggested that Makorinl is present in the Wnt signal transduction pathway. We determined subcellular localization of Makorin 1. Makorin 1 are located at cell membrane and nucleus. We also found several interacting proteins with Makorin 1.

35

05 A K T A ~ A ~ O N A N D B A D PHOSPHORYLATION IN RENAL CELLCARCINOMA

SATOSHI HARA, MOTOTSUGU OYA, RYUICHI MIZUNOyAKIOHORIGUCHl, KEN MARUMOAND MASARUMURAI

INTRODUcTlONAND0BJECWES:Akthasbeen implicated m the oncogene& andor pmgiession of human

effeaor molecules involved in both cell p M o n and malignanttumors~Aktphosphorylatesmanykey

slwival. Bad isoneofthefargefmoleCules OfAld ProapoptoticBadisinactivatedthroughmlation by Akt. We investigated Akt &vation and Bad m l a t j o n m d cell carcinoma (RCC) and evaluakdthee~ofAktinhiiitorasapofential thapeutic option forthe treabnent of RCC. MaterialsandMetfiods:Thee>rpressionsof PhosphoryM Akt (Ser4731, total A14 PhosphoryW Bad(ser13~ and total Bad wereanalyzed by westem

Aktinhibitor..gro~inhibitionandthe~onof

andtheflOWCybm&C - = ~ w b , i n

blottingPhosphmdyIinosito~etheranalogwasusedasan

qoptosk were investigated by theAlamar bluemethod

two RCC ell lines, Caki-1 and KU19-20. Results showed mote than a 200% haeased phospholyW Akt expnsion incumpaisontothat incorresponding- kidneytissues.Akt~vationwasobse!vedmclearCell type, but not in papillary and chnwnophobetype. Bad pho+olylation was observed only m clear cell type. Akt i n h i i i induced theapopQsis of KUl9-20 ceUswith constitutiveAktactivation andBadphosphorylation. Howevery in Caki- 1 cells with a low Akt activation and low Bad phosphorylati- apoptosis was nat induced. conclusiol ls:Akt~vationandBad~Monmay

of 41 RCC tissue samples 25 (63.4%)

play a role in the plifimtion of clear cell type of RCC. Akt inhibitor may therefibre be a potenWy d hapeutic option for a subset of RCC whh an elevated Aktactivation.

06 Targeting gene therapy for pmtate cancercellsby usingliposome conjugated with IgM type antcprostatespecihc membrane antigen (PSMA) monoclonal antibody

[Objectlwe conjugated the cationic Iiposome with I ~ M type anti-PSMA monoclonal a n t i i (MAb) and examinedwkhertheseliposamecouldbeusedfor selective gem delivery for PSMA positive cancer cells.

developed by us. Ihe a n t i i wastmted with 50mM cysteineand IgM monomer subunits weat isolated. ?he monomers were mixed with poty-LIysine and the conjugates were eluted by cation exchange chromatography. ?hen the conjugates were incubated with plasmids, and hxeafkr into cationic liposames @MAmj. Lip). We tested the specificity of these

[ ~ a n d a n d ] I g M t y p e a n t i - p s M A M A b ~

liposomestopostadecanoercekPSMA(+)LNCaPand PSAh4(-) PC-3 and DU145, and the PSMA(-) bladder cancer cell line, T24. 'Ihe binding ofthese liposornestothe cellswereexaminedfluo~esceinemicroscope,andthe

method using CMv-fHhl plasmid. We also exBmined the p w t h inhibm effects ofthese liposomes with gene

the lipoxms conjugated with nan-specifk IgM MAb wefeincubated. [ R e s u h s l P S ~ ~ m j . L i w a s f d t o b i s e ~

in the surfice of PSAM(+) WCaP cells. nK mnsktion efficiency of PSMAcOnj. L i was 13.2Y0 in LNW, as

T24. In contrasf when theconlml Abwas usedthe transfection efkiency remained at about 3% in all of the cell lines. F ~ ~ d ~ e i m q the PSMAconj. Lipo demonsbated aselectivegrowthinhibitoryeffectonLNCaPcellsinvitro, but could natexertsignifbntei€&son p w t h ofPSMA negativecelllines. [ c o n c l u s i o n l ~ m h suggested that PSMAconj. Lipo could deliver genes to PSMA positive cells selectively and this targekd llposome could be applied to suicide p thempy for PSMA positive prostate cancer Cells.

trasfectimefiiciencywasassessedbyx-galstaining

Using HSV-TK plus picloVir. AS c~ntrol,

c0mparedto3~%inpC-3,2.90/0inDU145,and3.1%in

36

0-07 Thehti-MetastaticRoleof ThrombomoduiinEsprossionInIslet Cell-Derived OR, and Its Diagnostic Value

ABSTRACT Isletceutumors(lcrs~enQcrine~harisingfiom p a n c t e a t i c i s ~ o f L a n ~ a l e h i s t o l ~ ~ to diagnose as benign or malignant. Because molecular markers associated with the clinical h m c & m h a * . that most of insulinoma are usually benign while other ICTs are malignant, have not been identified. In this context, we newly fwndthat an €xldohM antiaaguht hbomodulin (Th4) was# m the IK)rmal islet p

insulinomagrwpshowednometastasls *togetherwithTM

cells and insulinoma, but not of& islet components or non-insulinama IcTs. Clinically, all subjects ( ~ 1 5 ) of the

expmsion in the lesions, while the other ICT groups showed a high incidence of mecastasis (82%) and a low expression rate of TM (6%). To examine the fiaaional

of ICTs, we tested the e m ofexogemus TM

cell line. In c e w

. . role of% especially regarding the clinical charactensb cs

w ~ m c e l l ~ i ~ a n d p ~ m using MIN6 lnsylinoma '

~ T M o v e r a r p l e s s i o n r e d u c e d c e l l p ~ o n ¶ a n d ~ c a B - i i c e l l ~ p o s s i b l y throughdired~onwithneuralcelladhesion molecule(NCAM).Takentogdher,dKse&are suggesting that TM may act as anti-metastatic molecule of insulinomas. In addition, TM is a clinically useful molecular marker not only for identi@ng gCeU+rigin ICTs (i.e., insulinom) but also for pmhctmg disease prognosis of ICTS.

0-08 RaplA Is a Regulator of the MAP-lrinaseSignahgPathwayin Malipant Melanoma

Malignant melanoma is an m e l y w i v e neoplasm with high m d i . Because melanoma cells are resistant to most^ * agents available now, a novel beanent sbatqg based on the understanding of molecular basis of the disease is strongly demanded. FK228 is a mentlydeveloped anticancer drug, which induces cell cycle arrest and apoFtosis in varioushrmor~bymodulatinggenetranscripzionthrough inhibition ofhistonedeacetyks. In this study, we invedgatedthe & d o f FK228 on melanomaand its mechanisms using6 melanomacell lines. FK228 inhibited B d J incorporation of melanoma cells with an IC50 of 10 nM, which is loo-times lower than that of Cmsul Cell cycleadysiirevea)ed~FK228 induced GI mest and apoptosis after6 h ofcubt. Using a DNA chip contanun * . g3893can~ef-relatedgenes,wefod~ 142 genes were u p - q u k d more than 2.5-fold, and 75 genes were newly i n d u c e d by FK228 in melanoma cells. The latter included Rapl A, a small GTP-binding pmtem of the Ras h d y . E x p s i o n of Rapl A but not Rapl B inaeased in FK228-mated melanoma cells in dose and

phospImyMonofc-Raf, MEKIR, and ERKlnwas accompaniedbytheincreaseinRaplAptuteinkvels. Among 6 cell linestested, RaplAovereFion was observed in 2 ceU lines with aV599E mutation of EM. Mmver, Rapl Aand ERafwere physically associated in melanomacek. Finaily, forced expaessiOn of Rap1 Am a cell line without B-Rafmutation/RaplA ov&m

viability was incteased by sBNA-medW of Rapl Aoverexpression ma cell line with ERafmutatbn. 'Ihese data suggest that RaplA is an internal regulator of the MAP-kinase signaling and plays a defensive role against otlcogenc activation of B-Raf in melanoma cells. The cytobxic effedofFK228 is at least in part mediated dvoughupreguLdionofRaplAindignantmeh~a

timed-m.mdeaeasein

d i n k induc€ion ofapoptosis. In cell

37

Dtpartmenr of N e m w q a y h a i Medical Unbemity

ms~specimensofgllomawereanalyzedusingof Lscaadstainedby imm~toehemicalmethodfor

rnalipancy. [Materialsandmethods] 39gliomaspecimeos were ~ a t ~ e a y m w ~ ~ 1 3 ~ ~ . These includeed(5 Primary cases and 8 recurrentcases) 2 astrocytomas9anaplasticastrocytomasand2 ghoblastoms. All of specimens were analyzed by LSC andfixedH&Efb~methodduringtheOperatian.l'ben

cyclin B1, D1 and p53 protknt to assay the p q p O S i S and

theimmunohistocherm 'cal stains were done for cyclins, p53proteinafterheoperation [ResuhSlWe found that 39 spahens of tumor with the parts ofcenter and edge showed higher cych B1, cyclin D1 andp53 expessiOnthanthe part ofpeaiphery. On the other hand, the expression of recurrent cases and primary cases w a similar with cycb B1, D1. But tbere was very

mdif€~wasthat in thecaseof tumarh peiphery, edge to centre parts, the percentage of diploid pattemteadedtobelower,bowever,thepemmtageof aneuploid pattan tended to be higher, the pmatage of

difhent expression with p53 ptein.

imeuploidpattem in recllrreotoses was hi@erthan primarycases. conclusion^ expression ofcyclin B1, D1 may be related to the malignancy, prognosis of solid glioma. 'The rate of expressiOn of p53 h primary cases are higher than recurrentcases.

0-10 An essential amino acid, I-leucine induces p w t h arrest and persistent ERKdation in gliom cells

L-leucine isone ofhge neutral amino acids,audis

I-leucine added to the medium enhances gliom cells p l i f d a n However, we found higher amcentmtion of 1-leucine induces growth inhiiition of C6 rat glioma cells and T98G human glioblastoma cells in a dose dependent manner. Cell cycle analyses revealed cell accumulation in G1 and G2, and S phase depletim cell cycle p r q p ~ ~ ~ h h G 1 to S, d b y BdU inmpodon, was ckxmsed- *

following application of 1-leucine ~ v e a l e d immediate morphological change in the shape of cells. 'Ihe same concentration of other amino acids, such as valine did not

blotting for phosphorylated ERKs defnoastrated marked andplongedactjvation~for72hours.Incontmst toERKs,JNKandp38we~ndsi@cantlyactivated through the study period L-leucine si& or its metabolism might affect cell cycle pmgmsion in glioma cells.

nutritionallyessddformammahn * cells.onemM0f

y. ob6.erdiofl of living cells

~ & t h e ~ l l ~ g u I a t i o n s o ~ d y . westem

38