Depar t ment of Bi ochemi s t r y

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Depar t ment of Bi ochemi s t r y

Kiyoshi Ohkawa,Professor Koji Takada,Associate Professor Tadashi Asakura,Associate Professor

Res ear ch Act i v i t i es Canc e r r e s e ar c h

1 . Ea r l i e r di a g nos i s a nd t he r a py a r e t he mos t e f f e c t i v e me a ns t o c ont r ol c a nc e r . To e s t a bl i s h a noni nv a s i v e a nd e f f e c t i v e i n v i v o s y s t e m f or de t e c t i ng s ma l l t umor s t hr oug h mol e c ul a r t a r g e t i ng / i ma g i ng wi t h ul t r a s ound or i nf r a r e d f l uor e s c e nc e ,bot h CD1 4 7 ( e x t r a c e l l ul a r ma t r i x me t a l l opr ot e a s e i nduc e r , ba s i g i n)a nd t e na s c i n- C( TN- C) , mol e c ul e s t ha t f unc t i on a s pr omot e r s of t umor - c e l l mot i l i t y a nd me t a s t a s i s a nd a s pr og nos t i c ma r ke r s , we r e s e l e c t e d a s t a r g e t i ng mol e c ul e s f or c a nc e r de t e c t i on. The f i na l g oa l of our s t udy wa s t o r e a l i z e a nt i body - di r e c t e d e a r l y c a nc e r di a g nos i s wi t h a noni nv a s i v e i n v i v o de t e c t i on me t hod. The us e f ul ne s s of a nt i bodi e s a g a i ns t huma n CD1 4 7 a nd TN- C wa s f i r s t de t e r mi ne d bi oc he mi c a l l y . CD1 4 7 i s e x pr e s s e d by a l mos t a l l ma l i g na nt t umor s wi t h t he e x c e pt i on of s e v e r a l of t hos e wi t h a ne ur og e ni c na t ur e ,a nd t he a bi l i t y of a nt i bodi e s t o r e c og ni z e CD1 4 7 wa s c ons i s t e nt a nd e f f e c t i v e bot h i n v i t r o a nd i n v i v o . I n c ont r a s t ,TN- C wa s de t e c t a bl e i n onl y a f e w huma n c a nc e r c e l l l i ne s . I mmunoc y t ol og i c s t udi e s wi t h Al e x a 4 8 8 ―l a be l e d r a t a nt i - huma n TN- C a nt i bodi e s r e v e a l e d t he di s t i nc t but f oc a l a nd pa t c hy di s t r i but i on of i nt r a c e l l ul a r TN- C f l uor e s c e nc e wi t h f i ne i nt e r c e l - l ul a r de pos i t s . Fur t he r s t udy c onf i r me d t ha t TN- C e x pr e s s i on wa s l oc a l i z e d i n pi l e d- up c e l l s r a t he r t ha n i n f l a t - g r owi ng c e l l s . Sphe r oi d c ul t ur e ,a c onv e nt i ona l 3 - di me ns i ona l ( 3 D)c ul t ur e me t hod,s howe d TN- C e x pr e s s i on wa s hi g he r i n A4 3 1 c e l l s t ha n i n monol a y e r c ul t ur e . I n s phe r oi d- c ul t ur e d c e l l s TN- C wa s ov e r e x pr e s s e d a t bot h t he me s s e ng e r( m)RNA a nd pr ot e i n l e v e l s wi t h s i mul t a ne ous downr e g ul a t i on of E- c a dhe r i n a nd upr e g ul a t i on of v i me nt i n,a s i n t he e pi t he l i a l - me s e nc hy ma l t r a ns i t i on. Ef f e c t s of mi c r oe nv i r onme nt a l f a c t or s on TN- C e x pr e s s i on we r e f ur t he r e x a mi ne d wi t h a r a di a l f l ow bi or e a c t or( RFB)f or a 3 D c ul t ur e s y s t e m,whi c h pr ov i de s t i s s ue a r c hi t e c t ur e a nd mol e c ul a r f unc t i ons mi mi c ki ng t he i n v i v o e nv i r onme nt . I t i s of i nt e r e s t t ha t A4 3 1 c e l l s g r owi ng i n a n RFB s e c r e t e d a nd a c c umul a t e d l a r g e r a mount s of t r a ns f or mi ng g r owt h f a c t or( TGF)be t a 1 i n c ondi t i one d me di a t ha n di d c e l l s i n a 2 - di me ns i ona l monol a y e r c ul t ur e . A4 3 1 c e l l s i n RFB s howe d i nc r e a s e d e x pr e s s i on of t he mRNAs of TGF be t a 1 a nd TGF be t a r e c e pt or s 1 a nd 2 . The A4 3 1 c e l l s a l s o ov e r e x pr e s s e d ma t r i x me t a l l o- pr ot e i na s e 7 ,a downs t r e a m mol e c ul e of t he TGF be t a 1 s i g na l i ng pa t hwa y ,a nd s howe d i nc r e a s e d r e l e a s e of s ol ubl e 8 0 - kDa f r a g me nt s of E- c a dhe r i n i n c ondi t i one d me di a t i me - de pe nde nt l y ,r e s ul t i ng i n a de c r e a s e i n t he E- c a dhe r i n mol e c ul e a t t he c e l l s ur f a c e wi t hout downr e g ul a t i on of i t s mRNA e x pr e s s i on. Thi s de c r e a s e i n E- c a dhe r i n r e l e a s e pr omot e d t he l i be r a t i on of be t a - c a t e ni n a nd i t s nuc l e a r pa r t ne r ,upr e g ul a t i on of l y mphoi d e nha nc e r f a c t or( LEF)1 ,a nd a c c e l e r a t e d s e c r e t i on of Wnt pr ot e i n. Addi - t i ona l upr e g ul a t i on of a t r a ns c r i pt i ona l f a c t or ,hi g h mobi l i t y g r oup A2( HMGA2 ) ,a nd t he downs t r e a m pr ot e i n Sl ug wa s not e d. The TGF be t a 1 - de pe nde nt ,ma t r i x me t a l l o-

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Research Activities 2008 The Jikei University School of Medicine

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pr ot e i na s e 7 - me di a t e d upr e g ul a t i on of be t a - c a t e ni n / LEF1 a nd t he TGF be t a 1 - a c t i v a t e d HMGA2 pa t hwa y s c onv e r g e d t o c a us e Sl ug ov e r e x pr e s s i on due t o t he di s a s s e mbl y a nd f ur t he r r e pr e s s i on of E- c a dhe r i n. Goos e c oi d,a t r a ns c r i pt i ona l r e pr e s s or of E- c a dhe r i n, wa s a l s o upr e g ul a t e d. Ta ke n a l l t he r e s ul t s de s c r i be d a bov e t og e t he r ,A4 3 1 t umor c e l l s c ul t ur e d i n RFB i nduc e d t he e pi t he l i a l - me s e nc hy ma l t r a ns i t i on v i a t he TGF be t a 1 a ut oc r i ne l oop a nd ov e r e x pr e s s e d TN- C bot h a t t he mRNA a nd pr ot e i n l e v e l s ,a s we r e mol e c ul e s r e l a t e d t o t he e pi t he l i a l - me s e nc hy ma l t r a ns i t i on. Ot he r t r a ns c r i pt i ona l f a c - t or s ,Not c h / HEY1 a nd nuc l e a r f a c t or ka ppa B,we r e a l s o upr e g ul a t e d. The s e s i g na l s r e c r ui t e d t he ECM- c e l l r e mode l i ng a nd a ng i og e ne t i c mol e c ul e s . 3 D c ul t ur e i n a n RFB i s a us e f ul t ool f or c a nc e r bi ol og y ,a s a r e nude mi c e .

2 . Gl uc os e me t a bol i s m i s a not he r t a r g e t f or c a nc e r c he mot he r a py . CD1 4 7 i s a n a c c e s s or y s ubuni t of a he t e r ome r i c l a c t a t e t r a ns por t e r ,monoc a r box y l a t e t r a ns por t e r ( MCT) ,a me mbe r of t he SLC1 6 f a mi l y of s ol ut e t r a ns por t e r s . The MCTs t r a ns por t l a c t a t e a c r os s t he pl a s ma me mbr a ne ,a nd CD1 4 7 - MCT i nt e r a c t i on i s r e qui r e d f or MCT a c t i v i t y ,a s we l l f or t r a f f i c ki ng t o t he pl a s ma me mbr a ne . Thr e e - br omopy r uv a t e( 3 - Br PA) ,a py r uv a t e / l a c t a t e a na l og ,i s a pot e nt g l y c ol y t i c i nhi bi t or a nd c a ndi da t e a nt i c a n- c e r a g e nt . To c l a r i f y whi c h t r a ns por t e r s a r e i nv ol v e d i n t he c e l l ul a r i nf l ux of 3 - Br PA, t he r ol e of MCTs wa s e x a mi ne d. The mRNAs of MCT1 ,MCT2 ,MCT4 ,MCT8 , MCT9 ,a nd MCT1 4 we r e e x pr e s s e d a t v a r i ous l e v e l v s i n a 3 - Br PA- s e ns i t i v e pr os t a t e c a nc e r c e l l l i ne ,PC- 3 . To de t e r mi ne whi c h MCT mol e c ul e wa s i nv ol v e d i n 3 - Br PA t r a ns por t ,t he e x pr e s s i on of MCTs wa s i nhi bi t e d by s ma l l i nt e r f e r i ng( s i )RNAs . Re s i s t a nc e t o 3 - Br PA wa s f ound onl y whe n t he PC- 3 c e l l s we r e t r a ns f e c t e d wi t h t he MCT1 s i RNA. PC- 3 c e l l s pr e t r e a t e d wi t h MCT1 i nhi bi t or s we r e a l s o r e s i s t a nt t o 3 - Br PA. Fur t he r mor e ,s hor t ha i r pi n RNA e x pr e s s i on v e c t or s s pe c i f i c f or CD1 4 7 r e duc e d t he s e ns i t i v i t y of PC- 3 c e l l s t o 3 - Br PA. The s e r e s ul t s s ug g e s t t ha t t he MCT1 - CD1 4 7 c ompl e x i s e s s e nt i a l f or 3 - Br PA upt a ke .

3 . Re s i s t a nc e of t umor c e l l s t o c he mot he r a pe ut i c a g e nt s i s a s e r i ous pr obl e m i n c a nc e r t he r a py . A c onj ug a t e of dox or ubi c i n a nd g l ut a t hi one v i a g l ut a r a l de hy de( GSH- DXR) s t r ong l y i nhi bi t e d g l ut a t hi one S - t r a ns f e r a s e( GST)a c t i v i t y i n r a t he pa t oma AH6 6 c e l l s . Tr e a t me nt of t he c e l l s wi t h GSH- DXR i nduc e d a popt os i s v i a a c t i v a t i on of c - J un N- t e r mi na l ki na s e( J NK)by t he bi ndi ng of GSH- DXR t o t he a c t i v e c e nt e r of t he GSTP1 - 1 e nz y me , i nc l udi ng c y t oc hr ome c r e l e a s e f r om mi t oc hondr i a t o c y t os ol , c a s pa s e - 3 a c t i v a t i on,a nd DNA f r a g me nt a t i on. Thr oug h t he t r e a t me nt of t he c e l l s wi t h GSH- DXR i n a r e c e nt s t udy ,a not he r pos s i bl e c y t ot ox i c me c ha ni s m a f t e r t he a c t i v a t i on of J NK wa s de mons t r a t e d:t he i nduc t i on of a popt os i s v i a de a mi da t i on of B- c e l l l y mphoma 2 ,e x t r a l a r g e ,f ol l owe d by t he t r a ns l oc a t i on of Ba x t o mi t oc hondr i a . 4 . Si x c e l l l i ne s wi t h r e s i s t a nc e t o e pox omi c i n we r e e s t a bl i s he d. The e pox omi c i n- r e s i s t a nt c e l l l i ne s a r e r e l i a bl e t ool s f or t he t he r a pe ut i c e v a l ua t i on of pr ot e a s ome i n- hi bi t or s i n pr e c l i ni c a l t r i a l s . Mor e ov e r ,t he s e c e l l l i ne s ma y a l s o be us e f ul f or c l a r i f y i ng me c ha ni s ms of r e s i s t a nc e t o pr ot e a s ome i nhi bi t or s a nd e x a mi ni ng a wi de v a r i e t y of pr ot e a s oma l f unc t i ons . Thi s y e a r ,t he r e l a t i on be t we e n E- c a dhe r i n e x pr e s s i on a nd pr ot e a s oma l i nhi bi t i on wa s a na l y z e d.

19 Research Activities 2008 The Jikei University School of Medicine

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Ot he r r e s e ar c h

1 . Re g ul a t or y me c ha ni s ms of t r a ns c r i pt i ona l c o- a c t i v a t or wi t h PDZ- bi ndi ng mot i f ( TAZ)pr ot e i n l i nke d t o t he f i br obl a s t g r owt h f a c t or( FGF) / r e c e pt or s i g na l i ng ,whi c h pl a y s a n e s s e nt i a l r ol e i n os s i f i c a t i on,we r e de t e r mi ne d wi t h os t e obl a s t - l i ke MC3 T3 - E1 c e l l s . I t wa s be e n f ound t ha t FGF- 2 , whi c h i nhi bi t s bone mi ne r a l i z a t i on a nd s t i mul a t e s c e l l pr ol i f e r a t i on,r e v e r s i bl y r e duc e d TAZ pr ot e i n e x pr e s s i on i n MC3 T3 - E1 c e l l s . Re c e nt s t udi e s ha v e s hown t ha t FGF- 2 a nd a di pog e ni c di f f e r e nt i a t i on a r e r e l a t e d a nd t ha t t he TAZ pr ot e i n a c t s a s a t r a ns c r i pt i ona l r e g ul a t or dur i ng t he di f f e r e nt i a t i on.

Whe n pr e a di poc y t e - l i ke c e l l s we r e c ul t ur e d wi t h FGF- 2 ,e x pr e s s i on of t he mRNA of a P2 ,a n a di poc y t i c di f f e r e nt i a t i on ma ke r t r a ns c r i be d by pe r ox i s oma l pr ol i f e r a t or - a c t i v a t e d r e c e pt or( PPAR)g a mma ,wa s i nc r e a s e d. I n c ont r a s t ,FGF- 2 s i g ni f i c a nt l y de c r e a s e d t he e x pr e s s i on of TAZ,whi c h i s a c or e pr e s s or of PPAR g a mma . The s e r e s ul t s s ug g e s t t ha t t he a di pog e ni c di f f e r e nt i a t i on i nv ol v e d i n FGF- 2 r e s ul t s f r om t he r e duc t i on of t he TAZ l e v e l .

2 . Wi t h me t hods t o pur i f y a nd i de nt i f y ubi qui t i na t e d pr ot e i ns i n bi ol og i c a l ma t e r i a l s , s e v e r a l ubi qui t i n- pr ot e i n c onj ug a t e s i n Tr i s - s a l i ne s ol ubl e a nd Tr i s - s a l i ne - i ns ol ubl e but 2 % s odi um dode c y l s ul f a t e ― s ol ubl e f r a c t i ons we r e a na l y z e d f r om c a dmi um- e x pos e d huma n pr ox i ma l t ubul a r HK- 2 c e l l s a nd br a i ns of Ni e ma nn- Pi c k t y pe C( NPC)di s e a s e ( l i pi d s t or a g e di s e a s e wi t h pr og r e s s i v e ne ur ona l de a t h)mode l mi c e . Some of pur i f i e d ubi qui t i na t e d pr ot e i ns we r e de t e r mi ne d wi t h a mi no a c i d s e que nc i ng a na l y s i s . The HK- 2 c e l l s e x pos e d t o c a dmi um a t a c onc e nt r a t i on of 0 . 0 7 mM ( me di a n l e t ha l dos e ) s howe d a ma r ke d i nc r e a s e i n l e v e l s of ubi qui t i na t e d s i g na l t r a ns duc e r a nd a c t i v a t or of t r a ns c r i pt i on 6 . Me a n l e v e l s of s odi um dode c y l s ul f a t e ―s ol ubl e ubi qui t i n- pr ot e i n c onj ug a t e s i n c e r e br ums of NPC ( ‑/‑ )mi c e( 4 a nd 9 we e ks ol d)we r e s i g ni f i c a nt l y hi g he r( up t o t wi c e a s hi g h)t ha n i n wi l d- t y pe or he t e r oz y g ous mi c e .

Publications

Shimada Y,Fukuda T,Aoki K,Yukawa T, Iwamuro S,Ohkawa K,Takada K. A protocol for immunoaffinity separation of the accumulated ubiquitin-protein conjugates solubilized with sodium dodecyl sulfate.Anal Biochem 2008;

377:77‑82.

Asakura T,Maeda K,Omi H,Matsudaira H, Ohkawa K.The associ ation of deamidation of Bcl-xL and translocation of Bax to the mitochon- dria through activation of JNK in the induction of apoptosis by treatment with GSH-conjugated DXR.Int J Oncol 2008;33:389‑95.

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Research Activities 2008 The Jikei University School of Medicine