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이질아메바에의해유도되는 JurkatT 세포사멸에있어 calpain 과 ROS 의역할 연세대학교대학원 의과학과 김경아

이질아메바에의해유도되는 JurkatT 세포사멸에있어 calpain 과 ROS 의역할 지도신명헌교수 이논문을석사학위논문으로제출함 2005 년 12 월일 연세대학교대학원의과학과김경아

김경아의석사학위논문을인준함 심사위원 인 심사위원 인 심사위원 인 연세대학교대학원 2005 년 12 월일

감사의글 돌이켜보면늘아쉽고안타까운것이지나온시간들입니다. 지난 2 년동안최선을다해실험에몰두하였다고생각했었는데, 막상논문을준비하면서는자꾸만부끄러움이앞섰습니다. 조금만더, 조금만더시간이주어진다면하는후회와아쉬움을뒤로하고, 더나은미래를향해성큼발을내딛어봅니다. 지난 2년간의대학원생활은제인생에있어다시오지않을행복하고도소중한순간들이었습니다. 훌륭하신교수님들께많은것을배웠고, 무엇보다도인간적으로그리고학문적으로평생을두고갚아도다갚지못할사랑과가르침을베풀어주신신명헌지도교수님께짧은글로는다표현하지못할깊은감사와존경을드리고싶습니다. 또한바쁘신와중에도귀중한시간을내주시어논문에대한충고를해주신용태순교수님과서주영교수님께도감사드립니다. 또한학위과정동안하찮은고민도진지하게들어주고따뜻한격려를아까지않았던이영아선생님과심서보선생님에게감사의마음을전합니다. 또한미처고마움을표시하지못한제모든주위분들께도진심으로감사드리고싶습니다. 마지막으로,27 년의삶동안제게많은힘과용기를주시고, 애정과인내로지켜봐주신어머니께가슴에서우러나오는깊은사랑과존경을드리며, 늘어머니께부끄럽지않은모습으로정진하겠다고약속드립니다. 제게힘을주셨던모든분들께다시한번감사드리며, 앞으로의삶에있어서도사랑과겸손, 그리고최선의노력을잊지않고정진할것임을약속드립니다. 감사합니다. 2005 년 12 월 김경아드림

< 차례 > 국문요약 1 Ⅰ. 서론 3 Ⅱ. 재료및방법 7 1. 시약및항체 7 2. 이질아메바영양형의무균배양 7 3.JurkatT 세포의무균배양 8 4. 세포생존율측정 8 5.JurkatT 세포에다양한억제제전처치 9 6. 세포내 Ca 2+ 수준측정 9 7. 유세포분석 (Flow cytometry) 10 8.Westernblot 10 9.DNA 단편형성 (fragmentation) 11 10. 통계처리 11 Ⅲ. 결과 12 1. 이질아메바에의한 JurkatT 세포사멸 12 2. 이질아메바에의해유도된 JurkatT 세포사멸과 DNA 단편형성에있어서아메바 Gal/GalNAclectin 의역할 15 3. 이질아메바에의한 JurkatT 세포내칼슘 (Ca 2+ ) 농도증가와칼슘의존성시스테인단백분해효소인 calpain 의활성 17 4. 이질아메바에의한 JurkatT 세포내 caspase 의활성 19 5.Calpain 활성을통한 caspase 의활성 21

6. 이질아메바에의해유도된 Jurkat T 세포에서의 PS 외향화 (externalization) 와 DNA 단편형성에 pancaspase 억제제와 calpain 억제제가미치는영향 24 7. 이질아메바에의한 Jurkat T 세포내의타이로신탈인산화 (tyrosinedephosphorylation) 와 SHP2 단백질타이로신인산가수분해효소 (protein tyrosine phosphatase) 의단백분해 (proteolysis) 27 8. 이질아메바에의해유도되는 JurkatT 세포내 calpastatin 의절단에있어서 calpain 과 caspase 의역할 30 9. 이질아메바에의해유도되는 Jurkat T 세포의 PS 외향화 (externalization) 와 DNA 단편형성에있어서 ROS 억제제의효과 32 IV. 고찰 38 V. 결론 43 참고문헌 44 영문요약 53

그림차례 Fig.1A.Viability ofjurkatt cels incubated with orwithoute. histolytica. 13 Fig.1B.Phosphatidylserine exposure in JurkatT cels incubated withorwithoute.histolytica 13 Fig.1C.Cleavage ofparp in JurkatT cels stimulated with E. histolytica 14 Fig. 1D. DNA fragmentation in Jurkat T cels induced by E. histolytica 14 Fig. 2A. Efect of TDG (amebic lectin inhibitor) on the E. histolyticainducedceldeathinjurkatt cels 16 Fig.2B.EfectofDgalactose on the E.histolyticainduced DNA fragmentationinjurkatt cels 16 Fig.3A.IncreasedcytosolicCa 2+ levelin JurkatT celsincubated withe.histolytica 18 Fig.3B.Cleavage ofcalpain smalregulatory subunit(28 kda)in JurkatT celsstimulatedwithe.histolytica 18 Fig.4A.Cleavageofcaspasesin JurkatT celsstimulatedwith E. histolytica 20 Fig. 4B. Efects of bongkrekic acid and zlehdfmk on E. histolyticainduced cleavage of caspase3 in Jurkat T cels 20 Fig.5A.Efectofcalpain inhibitor on the E.histolyticainduced cleavage of calpain smal regulatory subunit (28 kda) and caspase3injurkatt cels 22

Fig.5B.Efectofcalpain inhibitor on the E.histolyticainduced cleavageofcaspase6andcaspase7injurkatt cels 22 Fig. 5C. Efect of pancaspase inhibitor on the E. histolyticainduced cleavageofcalpain smalregulatory subunit (28kDa)inJurkatT cels 23 Fig.6A.Efectofvarious concentrations ofcalpeptin on the E. histolyticainduced PS externalization on the surface ofjurkat T cels 25 Fig.6B.EfectofvariousconcentrationsofzVADfmk on thee. histolyticainduced PS externalization on the surface ofjurkat T cels 25 Fig. 6C. Efects of calpeptin and zvadfmk on the E. histolyticainduceddna fragmentationinjurkatt cels 26 Fig.7A.Time course analysis of E.histolyticainduced protein dephosphorylationinjurkatt cels 28 Fig.7B.Proteolysis ofshp2 tyrosine phosphatase in JurkatT celsstimulatedwithe.histolytica 28 Fig.7C.Efectofcalpain inhibitor on the E.histolyticainduced proteolysisofshp2phosphataseinjurkatt cels 29 Fig.8A.Cleavageofcalpastatin in JurkatT cels stimulated with E.histolytica 31 Fig. 8B. Efects of calpeptin and zvadfmk on the E. histolyticainduced cleavage of calpastatin in Jurkat T cels 31 Fig.9A.Inhibitory efectofvarious concentrations ofdpion the E. histolyticainduced PS externalization on the surface of JurkatT cels 33

Fig.9B.Inhibitory efectofvarious concentrations ofdpion the E.histolyticainduced DNA fragmentation in Jurkat T cels 33 Fig.10.Efect ofvarious concentrations ofrotenone on the E. histolyticainduced PS externalization on the surface ofjurkat T cels 34 Fig. 11. Efect of various concentrations of ETYA on the E. histolyticainduced PS externalization on the surface ofjurkat T cels 34 Fig.12.Efectofvarious concentrations oflnmma on the E. histolyticainduced PS externalization on the surface ofjurkat T cels 35 Fig. 13A. Efect of various concentrations of NAC on the E. histolyticainduced PS externalization on the surface ofjurkat T cels 36 Fig. 13B. Efect of various concentrations of GSH on the E. histolyticainduced PS externalization on the surface ofjurkat T cels 36 Fig.14.Proposed modelofsignaling pathways ofjurkatt cel deathtriggeredby Entamoebahistolytica 37 표차례 표 1.TYIS33 배지의조성 8

국문요약 이질아메바에의해유도되는 JurkatT 세포사멸에있어 calpain 과 ROS 의역할 인체에아메바성대장염과간농양등을일으키는기생원충인이질아메바 (Entamoebahistolytica) 는방어면역에관여하는 T 림프구와중성구와같은면역세포를직접사멸시킴으로써숙주의면역반응을회피할수있다. 그러나이질아메바에의한숙주세포의사멸에관여하는세포내신호전달기전은잘알려져있지않다. 본연구에서는살아있는이질아메바의접촉에의해유도되는숙주세포의사멸에있어세포내 calpain 과 reactiveoxygen species(ros) 의신호역할을알아보았다.JurkatT 세포를이질아메바와배양시배지에배양한것과비교해서세포의생존율이급격히감소하였으며, 또한 phosphatidylserine (PS) 의바깥세포막으로의노출, Poly[ADPribose]polymerase(PARP) 의절단및 DNA 의단편형성이매우증가되었다. 이질아메바에의해유도되는세포사멸효과는아메바의 Gal/GalNAclectin 과숙주세포표면에있는 galactose 잔기와의결합을방해하는 thiodigalactoside(tdg) 또는 Dgalactose 를첨가하였을때효과적으로차단되었다. 또한이질아메바에노출시킨 Jurkat T 세포내에서는칼슘 (Ca 2+ ) 의농도가증가되었으며, 이때칼슘의존성시스테인단백분해효소인 calpain 의활성도유도되었다. 이질아메바는 JurkatT 세포내 caspases3,6, 및 7 의활성을유도하였고, 이것은 calpain 억제제인 calpeptin 을 T 세포에전처치시억제되었다. 반면에 pancaspase 억제제인 zvadfmk 는이질아메바에의한 T 세포내 calpain 의활성을억제시키지못하였다.zVADfmk 와 calpeptin 은둘 1

다이질아메바에의해유도된 PS 의노출을억제시키지못하였지만, zvadfmk 는이질아메바에의한 DNA 단편형성을의미있게억제하였다. 그러나 calpeptin 은이질아메바에의해유도되는 DNA 단편형성을억제시키지못하였다. 이질아메바는 JurkatT 세포의탈인산화 (dephosphorylation) 와 SHP2 타이로신인산가수분해효소 (tyrosine phosphatase) 의활성을강력하게유도하였고, Jurkat T 세포에 calpeptin 을전처치시이질아메바에의해유도된 SHP2 타이로신인산가수분해효소의활성이억제되었다. 이질아메바는 JurkatT 세포내에존재하는 calpain 의내재성억제인자인 calpastatin 의절단을유도하였고, 이것은 calpeptin 에의해서는억제되었으나 zvadfmk 에의해서는전혀억제되지않았다. 이질아메바에의해유도되는세포사멸은 NADPH 산화효소 (oxidase) 억제제인 diphenyleneiodonium chloride(dpi) 를전처치함으로써의미있게차단되었다. 반면사립체 (mitochondria) 억제제인 rotenone 및 5lipooxygenase(LO) 억제제인 eicosatetraynoicacid(etya) 는아메바에의한 JurkatT 세포사멸을막지못했다. 이런결과들을종합하여보면이질아메바에의한세포사멸에는 calpain 을통한 caspase 및인산가수분해효소의활성경로와 NADPH 산화효소활성을통해유리되는 ROS 를매개로한신호경로가매우중요함을알수있다. 핵심되는말 : 이질아메바,JurkatT cel, 세포사멸,calpain,reactive oxygenspecies,caspases,calpastatin,tyrosinephosphatase 2

이질아메바에의해유도되는 JurkatT 세포사멸에있어 calpain 과 ROS 의역할 < 지도교수신명헌 > 연세대학교대학원의과학과 김경아 Ⅰ. 서론 조직침범 (tissueinvasive) 기생원충인이질아메바는전세계적으로약 5천만명의사람이감염되어있으며, 해마다약 4만명이상이이로인해사망하고있다. 13 이질아메바는감염시인체의대장조직을괴사시켜심한설사와복통을동반하는대장염과치사율이높은간농양등을일으킬수있어매우중요한수인성전염병원체로인식되어지고있다. 2 이질아메바는생활주기 (life cycle) 에있어포낭 (cyst) 과영양형 (trophozoite) 이관찰되며, 포낭으로오염된물과음식물의섭취로인해감염이성립된다. 장내로도입된포낭형의아메바는회장하부에서탈낭한후영양형으로변화되어대장에이행한후장점막에침입하여이분열법으로분열증식한다. 4 이질아메바는숙주의면역기전을피하기위해방어면역에관여하는 T 림프구와중성구와같은면역세포들을직접사멸시킬수있다. 또한이질아메바가분비하는시스테인단백분해효소는숙주의보체계를구성하는단백질들과항체들의기능을손상시켜면역기능을약화시킬수있다. 5 이외에이질아메바는숙주와접촉하였을때 poreforming 단백질인 3

amoebapore 를분비한후숙주의막을손상시켜세포의죽음을유도할수있다. 6 이질아메바에의한세포사멸은일반적인세포사멸기전과는달리일어나는것으로이해하고있는데, 그증거로세포사멸억제단백질인 Bcl2 를과발현시켰을때자외선조사에의한세포사멸은억제되었지만이질아메바에의한세포사멸은전혀억제되지않았다. 7 또한이질아메바는 caspase9 억제제로처리된 caspase8 결손세포도쉽게죽일수있었다. 더욱이이질아메바는 Fas/Fas 리간드 (ligand) 나종양괴사인자 (tumornecrosisfactor[tnf]) 수용체를통한세포사멸신호전달경로가결여되어있는마우스의간세포도죽일수있었다. 8 이러한결과를종합해보면이질아메바에의한숙주세포의사멸은현재가지알려진고전적인세포사멸기전과는다른경로로세포의죽음이일어나는것으로추측된다. 이질아메바가숙주세포에접촉시세포내칼슘의농도가급격히비가역적으로증가되는것으로잘알려져있다. 9 이러한세포내칼슘이온농도의급격한변화는세포죽음과직접적으로연관되어있다. 예를들면이질아메바에의한중국햄스터난소 (Chinese hamster ovary [CHO]) 세포의세포용해 (cytolysis) 가칼슘킬레이터 (chelators) 인 EDTA 와 EGTA 를사용하였을때억제되었다고한다. 10 더욱이 CHO 세포를지연성칼슘차단제인 verapamil 로처리하였을때아메바에의한세포사멸이잘억제되었다. 11 따라서이질아메바에의한세포사멸과정에있어칼슘의존성시스테인단백분해효소인 calpain 의활성이 12 매우중요한역할을할수있음을추측할수있다. 이질아메바를 JurkatT 세포에접촉시세포내단백질들의타이로신탈인산화 (tyrosinedephosphorylation) 가일어나며, 이때단백질타이로신인산가수분해효소 1B (protein tyrosine phosphatase 1B [PTP1B]) 의활성은 calpain 의활성과직접적인관련이있다. 13 Calpain 은거의모든포유류세포내에존재하고있는매우중요한단백분해효소이며 14 4

세포주기조절, 전사인자활성, 분화및사멸등에관여하고있다. Calpain 은 μcalpain 과 mcalpain 2개의이형체 (isoforms) 로존재하며각각촉매작용 (catalytic) subunit (80 kda) 와조절작용 (regulatory)subunit(28kda) 가결합된이질이량체 (heterodimer) 의형태로있다. 15 비활성형의전효소 (proenzymes) 가활성화되기위해서는단백분해라는자가공정 (autoprocessing) 을필요로한다. 16 이러한 calpain 의활성은생체및실험실내세포사멸모델에서잘관찰된다. 17,18 최근세포사멸과정에있어 calpain 의활성과 caspase 의활성간에는상호신호조절이있는것으로보고되고있다. 1921 예를들면 calpain 은 caspase3,7,8,9, 및 12 의절단을유도하였다. 2224 반대로 caspase 는 calpain 의세포내억제인자인 calpastatin 을절단함으로써 calpain 의활성을촉진하기도한다. 25,26 그러나이질아메바에의한세포사멸시 calpain 과 caspase 의활성간의신호조절체계에대해서는전혀알려진것이없다. Reactive oxygen species (ROS) 는과산화물음이온 (superoxide anion [O 2 ]), 과산화수소 (hydrogen peroxide [H 2O 2]) 들을지칭하며이러한물질들은세포사멸을유도하는중요한신호조절물질이다. 최근보고에따르면이질아메바에의한세포사멸에있어세포내 ROS 의축적이매우중요함을알수있다. 즉 ROS 억제제인 diphenyleneiodonium chloride(dpi) 로세포를전처치하였을때이질아메바에의한중성구사멸과세포내 ROS 의양이매우크게억제되었다. 27 그러나아직까지이질아메바에의한 JurkatT 세포사멸에세포내 ROS 가어떤역할을할수있는지에대해서는전혀알려진것이없다. 본연구에서는이질아메바에의해유도되는 JurkatT 세포사멸에있어 calpain 과 ROS 의신호역할을정밀히조사하고자하였다. 이러한연구는이질아메바와숙주간의상호신호관계성과병원체의숙주면역회피기전을보다더깊이이해할수있을것으로생각된다. 이러한 5

연구결과는이질아메바증의병인을규명할수있어질병의새로운치 료법을개발하는데크게기여할것으로기대된다. 6

Ⅱ. 재료및방법 1. 시약및항체 Pancaspase 억제제 zvadfmk, calpain 억제제 calpeptin, diphenyleneiodonium chloride(dpi),no synthase 억제제 LNMMA, 5LO 억제제 ETYA, bongkrekic acid, caspase9 억제제 zlehdfmk, rotenone, 항산화제인 NAcetylLcysteine (NAC), Glutathione(GSH) 은 Calbiochem (LaJola,CA,USA) 으로부터구입 하였다. Thiodigalactoside (TDG) 는 Sigma chemial company (St. Louis,MO,USA) 으로부터 구입하였다.Fluorescein isothiocyanate (FITC) labeled annexin V는 BD Pharmingen (San Diego,CA, USA) 으로부터구입하였다.Fluo3/AM 은 Molecularprobes 로부터구 입하였다.Caspase3,6,7,PARP 와 SHP2 타이로신인산가수분 해효소 (tyrosine phosphatase) 에 대한 rabbit 다클론 항체와, phosphotyrosine 에 대한 mouse 단클론 항체는 Cel signaling technology (Beverly,MA,USA) 로부터구입하였다.Calpain 조절작 용 subunit(regulatory subunit) 과 calpastatin 에대한 mouse 단클론 항체는 Calbiochem (LaJola,CA,USA) 으로부터구입하였다. 2. 이질아메바영양형의무균배양 TYIS33 배지 ( 표 1) 에서이질아메바 (Entamoeba histolytica) HM1:IMSS 주를 37 에서무균배양하였다. 28 4872 시간동안배양한 log phase 시기의이질아메바를모았으며 10 분동안얼음에넣어두었다가 5분동안 4 에서 1,000rpm 으로원심분리하여수확하였다. 이질아메바를 10% fetalbovine serum,25 mm HEPES,gentamicin sulfate(50 mg/l) 가함유된 RPMI1640 배지로실험에사용할이질아메바를세척한후다시 RPMI1640 배지에부유시켰다. 실험에사용된아메바의생존율은 trypanblue 염색상 98% 이상이었다. 7

표 1.TYIS33 배지의조성 성분 Trypticase(BBL) Yeastextract(BBL) Glucose NaCl K 2HPO 4 KH 2PO 4 LcysteineHCl Lascorbicacid Ferricammonium citrate 용량 20g/L 10g/L 10g/L 2g/L 1g/L 0.6g/L 1g/L 200mg/L 22.8mg/L DistiledWater 880mL (ph 6.8) Fetalcalfserum 10% Vitamin2% 100mL 20mL 3.JurkatT 세포의무균배양인체 leukemia T 세포주 JurkatE61 (American Type Culture Colection, USA) 를 10% fetal bovine serum, 25 mm HEPES, gentamicin sulfate (50 mg/l) 가포함된 RPMI 1640 배지 (GibcoBRL) 에부유시킨후 37,CO 2 배양기 (5% 이산화탄소,95% 공기 ) 에서무균배양하였다.JurkatT 세포를 5분동안 4 에서 1,000 rpm 으로원침시켜모은후 RPMI1640 배지에풀어주었다. 실험에사용된 T 세포주의생존율은 trypanblue 염색상 99% 이상이었다. 4. 세포생존율측정 세포생존율은 trypan blue 염색방법을이용하여측정하였다. 48weltissue culture plate 에이질아메바와 Jurkat 의비율을 1:5 로 8

넣고 37,5% CO 조건하에서 30 분과 60 분동안배양하였다. 배양후세포를모아 PBS 로세척한후 0.4% 의 trypan blue 용액을세포와섞어 hemacytometer 를이용하여 200 개의세포를검경하여세포생존율을계산하였다. 이질아메바의 Gal/GalNAclectin 을통한접촉이숙주세포의사멸에중요한역할을하는지를조사하기위해 lectin 과당과의결합을방해하는 TDG 를첨가한후이질아메바에의한 T 세포의생존율을대조군과비교하였다. 5.JurkatT 세포에다양한억제제전처치 JurkatT 세포를 30 분동안 37 에서 zvadfmk(50,100,200 μ M),DPI(5,10,20,50 μm),rotenone(10,20,50 μm),lnmma (1, 2, 5 mm), ETYA (35, 65, 100 μm), NAC (5, 10, 20 mm), Glutathione (5, 10, 20 mm), bongkrekic acid (50, 100 μm), zlehdfmk (50, 100 μm) 로전처치하였다. Calpain 억제제 calpeptin 을 0.5mM and 1mM 로 15 분동안 JurkatT 세포에전처치하고,10 μm,100 μm,250 μm 로 1시간동안전처치하였다. 전처치한 T 세포는이질아메바와반응시키기전에한번 RPMI1640 배지로세척하였다. 모든실험에서사용된매개체대조 (vehicle control) DMSO 는배지총량의 1% 를넘지않았다. 6. 세포내 Ca 2+ 수준측정 JurkatT 세포 (4 10 5 /wel) 와이질아메바 (4 10 4 /wel) 를 48wel tissue culture plate 에서 5분동안 37,CO 2 배양기에서배양한후모았다. 모은세포를혈청이없는 RPMI1640 배지 400 μl에부유시킨후칼슘에민감한형광색소인 Fluo3/AM (MolecularProbes, 미국 ) 을 2 μm 이되도록첨가하여 15 분동안 37 에서염색하였다. 유세포분석기 (flow cytometer) 를이용하여세포내칼슘이온의증가여부를형광강도의변화를통해분석하였다. 9

7. 유세포분석 (Flow cytometry) 고사 (apoptosis) 를통한세포사멸의특징인세포외막으로의 PS 의이동은 annexin V 염색을통해측정하였다. Jurkat T 세포 (4 10 5 /wel) 와이질아메바 (8 10 4 /wel) 를 48wel tissue culture plate 에서 1시간동안 37,CO 2 배양기에서배양한후모았다. 세척용액 (0.1% sodium azide 와 1% FBS 을포함하는 phosphatebufered saline[pbs]) 으로두번세척한세포에 FITClabeled annexinv (2 μl ) 가함유되어있는부착용액 (binding bufer[10 mm Hepes,140 mm NaCl,2.5mM CaCl 2,pH 7.4])200 μl를첨가하여 15 분동안상온에서방치하였다. 유세포분석기를이용하여 annexinv 에염색되어진세포수와형광강도를측정하였다. 8.Westernblot 여러가지약물학적억제제로전처치한 Jurkat T 세포 (1 10 6 /group) 에이질아메바 (1 10 5 /group) 를첨가하고시간별로배양하였다. 자극이끝난후원심분리기를이용하여세포를모아차가운 PBS 를넣어원침시켰다. 모아진세포침사에차가운용해용액 (20 mm TrisHClpH7.5,60 mm βglycerophosphate,10 mm EDTA, 10mM MgCl 2,10mM NaF,2mM dithiothreitol,1mm Na 2VO 4,1 mm APMSF,1% NP40,5 μg /mlleupeptin) 을첨가한후얼음에 30 분동안방치하였다.30 분이지난후시료용액과혼합하여 100 에서 5분동안열처리하였다. 열처리가끝난시료를얼음에놓아 5분간방치한후원심분리하여상층액만을따로모아새로운튜브로옮겨실험에사용될때까지 20 에보관하였다. 이와같은방법으로얻은시료를 10%,12% 또는 15% sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDSPAGE) 상에서전기영동시킨후젤을 immobilon P polyvinylidenefluoride 막 (Milipore, 독일 ) 으로 100 V 에서 1시간동안이적시켰다. 이적시킨막을 5% 탈지분유 (nonfat 10

dry milk) 를함유하고있는 Trisbufered saline (TBS [0.1% Tween20]) 에넣은후상온에서 1시간동안반응시켰다.TBST 로한번세척한후막을 caspase3,6 and 7, 또는 calpain 조절작용 subunit 에대한일차항체로 4 에서 18 시간동안반응시켰다. 반응시킨막을 TBST 로세번세척한후 HRPconjugatedantirabbitIgG 항체또는 HRPconjugated antimouse IgG 항체로상온에서 1시간동안반응시켰다. 최종적으로, 항원항체반응은 LumiGLO (Cel Signaling,Beverly,MA,USA) 를사용하여확인하였다. 9.DNA 단편형성 (fragmentation) 이질아메바에의한 JurkatT 세포내 DNA 의단편형성을측정하기위해 JurkatT 세포 (5 10 6 /group) 와이질아메바 (5 10 5 /group) 를 1시간동안 37 에서배양한후모았다. 이질아메바의 Gal/GalNAc lectin 을통한세포사멸효과를확인하기위해경쟁적인억제제인 Dgalactose 를 Jurkat 세포에처리한후 5분뒤이질아메바를넣었다. 모은시료를 PBS 로한번세척하고 TaKaRa kit(#mk600) 을사용하여 DNA 를추출하였다.DNA 를 2% agarose 젤에전기영동하여 DNA 의단편형성을확인하였다. 10. 통계처리결과는 3~ 5번의실험을각각독립적으로수행하였고, 평균 ± 표준오차로나타내었다. 실험군과대조군의통계처리는 Student'sttest 를이용하여 p < 0.05 이면두군간에유의한차이가있는것으로간주하였다. 11

Ⅲ. 결과 1. 이질아메바에의한 JurkatT 세포사멸 JurkatT 세포를이질아메바와배양하였을때유도되는다양한세포사멸현상에대하여알아보았다.Jurkat 세포를이질아메바와함께배양한후 trypan blue 로염색하였을때죽은세포가반응 1시간후에현저히증가되었다. 이질아메바를첨가하지않은정상군의세포생존율이 100% 인데반해, 이질아메바를첨가한후 30 분과 60 분에서각각 75.0 과 51.6% 로감소되었다 (Fig. 1A). 또한이질아메바와 Jurkat 세포를배양한후, 세포외막에 phosphatidylserine(ps) 의노출을 annexinv 염색을통해확인하였다. 이질아메바를첨가한후 AnnexinV 양성세포율은평균 44% 로배지만을첨가한대조군과비교할때약 8배정도증가하였다 (Fig. 1B).DNA 복구효소인 Poly(ADPribose)polymerase (PARP1) 는세포사멸시절단되어진다고알려져있다. 29 따라서본연구에서는이질아메바에의해 Jurkat 세포내 PARP 의절단이유도되는지를알아보았다. 이질아메바를첨가후 JurkatT 세포내의 PARP 의절단은반응시간과첨가한아메바수에따라여러개의단백분획으로절단된것을잘볼수있다 (Fig. 1C). 더욱이 JurkatT 세포에이질아메바를첨가하였을때대조군에비해현저한핵 DNA 의단편형성이유도되었다 (Fig.1D). 12

Fig 1A. Viability of Jurkat T cells incubated with or without E. histolytica. 120 Viability (%) 100 80 60 40 * * 20 0 medium 30min 60min Entamoeba Jurkat T cells (4 10 5 /well) were incubated for 30 or 60 min at 37 with or without E. histolytica (8 10 4 /well). After incubation, cells were stained with trypan blue for quantification of viability of Jurkat T cells. Data are presented as mean ± SEM from three independent experiments. Significant differences from the value obtained with cells incubated with medium alone are shown. *, p < 0.05 Fig 1B. Phosphatidylserine exposure in Jurkat T cells incubated with E. histolytica. AnnexinV positive cells, % 60 50 40 30 20 * 10 0 medium Entamoeba Count AnnexinV Jurkat T cells (4 10 5 /well) were incubated for 60 min at 37 with or without E. histolytica (8 10 4 /well). After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from three independent experiments. Significant differences from the value obtained with cells incubated with medium alone are shown. *, p < 0.05 13

Fig 1C. Cleavage of PARP in Jurkat T cells stimulated with E. histolytica. Time (min) 0 1 5 15 PARP 116 kda + + + + + + + + + + + + + + + + + + + + E. histolytica (10 : 1) E. histolytica (20 : 1) E. histolytica (50 : 1) Jurkat T cells Jurkat T cells (1 10 6 /sample) were incubated for 115 min at 37 with or without E. histolytica at a ratio of 10:1, 20:1, or 50:1 (Jurkat T cells to E. histolytica). After incubation, whole cell lysates were subjected to SDSPAGE and blotted with antiparp Ab. The figure is representative of three experiments showing similar results. Fig 1D. DNA fragmentation in Jurkat T cells induced by E. histolytica. 1 2 3 4 5 Lane 1 : 100 b.p Marker Lane 2 : Only Jurkat (1 hr) Lane 3 : Jurkat + Entamoeba (30 min) Lane 4 : Jurkat + Entamoeba (1 hr) Lane 5 : Entamoeba histolytica only Jurkat T cells (4 10 6 /sample) were incubated for 60 min at 37 with or without E. histolytica (4 10 5 /sample). DNA fragmentation was analyzed by 2% agarose gel electrophoresis. An equal number of amebae and Jurkat T cells were incubated in medium alone as negative controls. 14

2. 이질아메바에의해유도된 JurkatT 세포사멸과 DNA 단편형성에있어서아메바 Gal/GalNAclectin 의역할이질아메바가숙주세포를사멸시키는데있어서아메바와세포간의단단한접촉이필요하다는것은잘알려져있다. 30 이에따라, 아메바의 Gal/GalNAc lectin 과 Jurkat 세포표면에있는당잔기 (sugar residue) 사이의결합을방해할수있는 thiodigalactoside(tdg) 의효과를관찰하였다. 이질아메바에의해유도된세포의죽음은 TDG 첨가에의해현저히감소한것을볼수있었다 (Fig.2A). 특히 TDG 50 mm 에서는이질아메바에의한세포사멸이크게억제되어배지만을첨가한대조군의결과와비슷한수치를보였다. 또한 Dgalactose 를첨가하였을때에도이질아메바에의한세포의 DNA 단편형성이효과적으로억제된것을확인하였다 (Fig.2B). 15

Fig 2A. Effect of TDG (amebic lectin inhibitor) on the E. histolyticainduced cell death in Jurkat T cells. No Entamoeba Jurkat (control) TDG 5 mm TDG 10 mm TDG 20 mm TDG 50 mm 0 20 40 60 80 100 120 Viability (%) * * * Jurkat T cells (4 10 5 /well) were incubated for 60 min at 37 with or without E. histolytica (8 10 4 /well) in the absence or presence of TDG (550 mm). After incubation, cells were stained with trypan blue for quantification of viability of Jurkat T cells. Data are presented as mean ± SEM from three independent experiments. Significant differences from the value obtained with cells incubated with E. histolytica in the absence of sugars. *, p < 0.05 Fig 2B. Effect of Dgalactose on the E. histolyticainduced DNA fragmentation in Jurkat T cells. 1 2 3 4 5 Lane 1 : 100 b.p Marker Lane 2 : Only Jurkat (1hr) Lane 3 : Jurkat + Entamoeba Lane 4 : [Jurkat + Dgalactose 100 mm] + Entamoeba Lane 5 : Entamoeba histolytica only Jurkat T cells (4 10 6 /sample) were incubated for 60 min at 37 with or without E. histolytica (4 10 5 /sample) in the absence or presence of 100 mm Dgalactose. DNA fragmentation was analyzed by 2% agarose gel electrophoresis. An equal number of amebae and Jurkat T cells were incubated in medium alone as negative controls. 16

3. 이질아메바에의한 JurkatT 세포내칼슘 (Ca 2+ ) 농도증가와칼슘의존성시스테인단백분해효소인 calpain 의활성세포내칼슘이온농도증가가 calpain 의활성에반드시필요하다. 12 또한, 이질아메바가세포사멸을유도할때세포내칼슘이온의농도를증가시킨다. 10,11 본연구에서는 JurkatT 세포내칼슘이온의농도변화를세포투과칼슘지시약 (celpermeable calcium indicator) 인 Fluo3/AM 을사용하였다. 이질아메바를첨가하였을때 JurkatT 세포내의칼슘농도가대조군에비해약 2배정도증가한것을볼수있다 (Fig.3A). Calpain 의활성은 caspase 와유사하게자가절단 (autocleavage) 를통해유도된다. 31 다음으로이질아메바를 JurkatT 세포에첨가한후칼슘의존성단백분해효소인 calpain 활성을알아보았다. 이질아메바를첨가한후 1분에서부터 calpain 의 28kDa 크기의조절작용 subunit 가자가분해 (autolysis) 된것을확인하였다 (Fig.3B). 17

Fig 3A. Increased cytosolic Ca 2+ in Jurkat T cells incubated with E. histolytica. Intracellular calcium (fold of control) 3 2 1 * Count 0 medium Entamoeba Fluo3/AM Jurkat T cells (4 10 5 /well) were incubated for 5 min at 37 with or without E. histolytica (8 10 4 /well). After incubation, cells were stained with fluo3/am fluorescent dye for flow cytometric measurement. Data are presented as mean ± SEM from three independent experiments. Significant differences from the value obtained with cells incubated with medium alone are shown. *, p < 0.05 Fig 3B. Cleavage of calpain small regulatory subunit (28 kda) in Jurkat T cells stimulated with E. histolytica. Time (min) 0 1 2 5 10 calpain small subunit 28 kda + + + + + + + + + + + + + + + + + + E. histolytica (10 : 1) E. histolytica (20 : 1) Jurkat T cells Jurkat T cells (1 10 6 /sample) were incubated for 110 min at 37 with or without E. histolytica at a ratio of 10:1, or 20:1 (Jurkat T cells to E. histolytica). After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticalpain small subunit Ab. The figure is representative of three experiments showing similar results. 18

4. 이질아메바에의한 JurkatT 세포내 caspase 의활성 Western blot 을이용하여 caspase3,6,7 의활성을분석하였다.Fig.4A 에서보면이질아메바를첨가후 Jurkat T 세포내의 caspase3,6,7 모두배양시간및첨가한아메바의수에비례하여잘라진것을볼수있으며이러한결과들은반응초기인 1분내에서도관찰할수있었다. 특히이질아메바의첨가에의해 Jurkat 세포내 caspase3 이절단되어활성형의절편인 20kDa 과 17kDa 이잘관찰되었다. 이질아메바에의해유도된 caspase3 활성에사립체 (mitochondria) 가관여하는지를알아보기위해사립체 (mitochondria) 막안정제 (membrane stabilizer) 인 bongkrekic acid 와 caspase9 억제제인 zlehdfmk 를 JurkatT 세포에전처치한후이질아메바에의해유도되는 caspase3 의활성에미치는영향을알아보았다. Bongkrekicacid 와 zlehdfmk 둘다 caspase3 의활성을억제시키지못하였다 (Fig.4B). 19

Fig 4A. Cleavage of caspases in Jurkat T cells stimulated with E. histolytica. Time (min) 0 1 5 15 caspase 3 cleaved form 20 kda 17 kda caspase 6 20 kda caspase 7 15 kda + + + + + + + + + + + + + + + + + + + + E. histolytica (10 : 1) E. histolytica (20 : 1) E. histolytica (50 : 1) Jurkat T cells Jurkat T cells (1 10 6 /sample) were incubated for 115 min at 37 with or without E. histolytica at a ratio of 10:1, 20:1, or 50:1 (Jurkat T cells to E. histolytica). After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticaspase 3, 6, 7 Ab. The figure is representative of three experiments showing similar results. Fig 4B. Effects of bongkrekic acid and zlehdfmk on the E. histolyticainduced cleavage of caspase 3 in Jurkat T cells. 100 μm 100 μm 50 μm 100 μm 50 μm 100 μm caspase 3 + + + + + + + + + + + + + + + + + + + + + + + + + + + + 20 kda 17 kda Jurkat T cells E. histolytica (10 : 1) EtOH (vehicle) Bongkrekic acid ZLEHDfmk Calpeptin 1 mm Pretreated Jurkat T cells (1 10 6 /sample) with bongkrekic acid (50100 μm), zlehdfmk (50 100 μm), calpeptin (1 mm), EtOH (1%) were incubated for 5 min at 37 in the absence or presence of E. histolytica (1 10 5 /sample) in a CO 2 incubator. After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticaspase 3 Ab. The figure is representative of three experiments showing similar results. 20

5.Calpain 활성을통한 caspase 의활성세포사멸과정에있어 calpain 과 caspases 가서로독립적으로작용하거나 19,32,33, 서로협력적으로작용한다는연구결과가있다. 34 따라서본연구에서는 pancaspase 억제제인 zvadfmk 와 calpain 억제제인 calpeptin 을이용하여이질아메바에의해유도되는 calpain 과 caspase 활성간의신호전달체계를알아보았다.Calpeptin 을 JurkatT 세포에전처치하였을때이질아메바에의해유도되는세포내 calpain 및 caspase3,6,7 의활성이대조군과비교하였을때효과적으로억제되었다 (Fig.5A,5B). 특히 calpeptin 1 mm 에서억제효과가가장극적으로보였다. 이와는반대로 pancaspase 억제제인 zvadfmk 를고농도인 100 μm 과 200 μm 로 JurkatT 세포에전처치하였을때에도이질아메바에의해유도된 calpain 의활성은전혀억제되지않았다 (Fig.5C). 21

Fig 5A. Effect of calpain inhibitor on the E. histolyticainduced cleavage of calpain small regulatory subunit (28 kda) and caspase3 in Jurkat T cells. calpain small subunit 28 kda caspase 3 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 20 kda 17 kda Jurkat T cells E. histolytica (10 : 1) E. histolytica (20 : 1) E. histolytica (50 : 1) DMSO (vehicle) Calpeptin 1 mm Pretreated Jurkat T cells (1 10 6 /sample) with 1 mm calpeptin or 1% DMSO (v/v) as a control for 15 min at 37 were incubated with or without E. histolytica at a ratio of 10:1, 20:1, or 50:1 (Jurkat T cells to E. histolytica) for 1 min at 37 in a CO 2 incubator. After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticalpain small subunit and anticaspase 3 Ab. The figure is representative of three experiments showing similar results. Fig 5B. Effect of calpain inhibitor on the E. histolyticainduced cleavage of caspase 6 and caspase 7 in Jurkat T cells. 1 mm 0.01 mm 0.1 mm 0.5 mm 1 mm caspase 6 20 kda caspase 7 + + + + + + + + + + + + + + + + Jurkat T cells E. histolytica (10 : 1) + + DMSO (vehicle) Calpeptin + + + + + 15 kda Pretreated Jurkat T cells (1 10 6 /sample) with calpeptin (0.011 mm) or 1% DMSO (v/v) as a control for 15 min at 37 were incubated with or without E. histolytica (1 10 5 /sample) for 1 min at 37 in a CO 2 incubator. After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticaspase 6 and anticaspase 7 Ab. The figure is representative of three experiments showing similar results. 22

Fig 5C. Effect of pancaspase inhibitor on the E. histolyticainduced cleavage of calpain small regulatory subunit (28 kda) in Jurkat T cells. 200 μm 50 μm 100 μm 200 μm calpain small subunit 28 kda + + + + + + + + Jurkat T cells E. histolytica (10 : 1) + + + + + + + + DMSO (vehicle) + + + + zvadfmk Pretreated Jurkat T cells (1 10 6 /sample) with zvadfmk (5200 μm) or 1% DMSO (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (1 10 5 /sample) for 1 min at 37 in a CO 2 incubator. After incubation, whole cell lysates were subjected to SDS PAGE and blotted with anticalpain small subunit Ab. The figure is representative of three experiments showing similar results. 23

6. 이질아메바에의해유도된 Jurkat T 세포에서의 PS 외향화 (externalization) 와 DNA 단편형성에 pancaspase 억제제와 calpain 억제제가미치는영향이질아메바에의해유도된 JurkatT 세포의 PS 의노출과 DNA 단편형성에 calpain 과 caspase 활성이필요한지를확인하기위해 Jurkat T 세포를이질아메바첨가전 pancaspase 억제제또는 calpain 억제제로전처치하였다.ZVADfmk 와 calpeptin 모두이질아메바에의한 JurkatT 세포의 PS 외향화를감소시키지못했다 (Fig.6A,6B). 또한이질아메바에의해유도되는세포내 DNA 단편형성에 calpain 과 caspase 의역할을알아보았다.Pancaspase 억제제를 JurkatT 세포에전처치했을때에는아메바에의해유도된 DNA 단편형성이현저히감소된것을볼수있었지만,calpain 억제제인 calpeptin 을처리하였을때에는전혀억제되지않았다 (Fig.6C). 24

Fig 6A. Effect of various concentrations of calpeptin on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 60 50 40 30 20 10 0 medium DMSO (vehicle) Calpeptin 10 μm Calpeptin 100 μm medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with calpeptin ( 10100 μm) or 1% DMSO (v/v) as a control for 60 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. Fig 6B. Effect of various concentrations of zvadfmk on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 35 30 25 20 15 10 5 0 medium DMSO (vehicle) zvadfmk 100 μm zvadfmk 200 μm medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with zvadfmk ( 100200 μm) or 1% DMSO (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. 25

Fig 6C. Effects of calpeptin and zvadfmk on the E. histolyticainduced DNA fragmentation in Jurkat T cells. 1 2 3 4 5 6 7 8 Lane 1 : 100 b.p Marker Lane 2 : Only Jurkat (1 hr) Lane 3 : [Jurkat + DMSO 1%] + Entamoeba Lane 4 : [Jurkat + calpeptin 10 μm] + Entamoeba Lane 5 : [Jurkat + calpeptin 100 μm] + Entamoeba Lane 6 : [Jurkat + calpeptin 250 μm] + Entamoeba Lane 7 : [Jurkat + pancaspase inhibitor 100 μm] + Entamoeba Lane 8 : Entamoeba histolytica only Pretreated Jurkat T cells (4 10 6 /sample) with or without 10250 μm calpeptin, 100 μm zvadfmk for 60 min at 37 were incubated for 60 min at 37 in the absence or presence of E. histolytica (4 10 5 /sample) in a CO 2 incubator. DNA fragmentation was analyzed by 2% agarose gel electrophoresis. An equal number of amebae and Jurkat T cells were incubated in medium alone as negative controls. 26

7. 이질아메바에의한 Jurkat T 세포내의타이로신탈인산화 (tyrosine dephosphorylation) 와 SHP2 단백질타이로신인산가수분해효소 (proteintyrosinephosphatase) 의단백분해 (proteolysis) 숙주세포의방어기작을교란시키고세포생존을방해하기위해이질아메바를포함한여러가지병원체들은숙주세포내의단백질타이로신인산가수분해효소 (protein tyrosinephosphatases[ptpases]) 를활성화시켜세포내신호전달에중요한단백질들의타이로신인산화 (tyrosine phosphorylation) 을방해할수있는것으로보고되어있다. 35,36 따라서, 본연구에서는이질아메바에의해유도되는 Jurkat 세포의타이로신탈인산화현상을먼저확인하였다.Jurkat 세포와이질아메바를함께배양한군에서이질아메바를첨가하지않은대조군에비해단백질들의현저한탈인산화가일어난것을보여주고있다 (Fig. 7A). 다음으로, 타이로신탈인산화를유도할수능력을갖고있는 SHP2 단백질타이로신인산가수분해효소의활성을알아보았다. 이질아메바를 Jurkat 세포에첨가한후 1분에 SHP2 인산가수분해효소가단백분해된것을확인하였다. 그러나이질아메바의자극에의해서는 SHP2 인산가수분해효소의인산화는유도되지않았다 (Fig.7B). 이러한결과는이질아메바에의해유도되는 SHP2 인산가수분해효소 (phosphatase) 의활성은인산화가아닌단백분해라는과정을통해이루어짐을강력히암시하고있다. 이질아메바에의해유도된 JurkatT 세포내에있는 PTPase 중 PTPase1B 는 calpain 에의해활성화될수있다는기존의연구결과에따라 12 본실험에서도 calpain 억제제인 calpeptin 을 Jurkat 세포에전처리한후 SHP2 인산가수분해효소의활성변화를알아보았다.Calpeptin 을전처치하였을때이질아메바에의해유도되는 SHP2 인산가수분해효소의절단양상이억제된것을확인할수있었다 (Fig.7C). 이러한결과는이질아메바에의한 Jurkat 세포내단백질들의탈인산화에는 calpain 을통한 SHP2 인산가수분해효소의활성이중요함을알수있다. 27

Fig 7A. Time course analysis of E. histolyticainduced protein dephosphorylation in Jurkat T cells. Time (min) phosphotyrosine 0 1 2 5 10 + + + + + + + + + + + + + + + + + + E. histolytica (10 : 1) E. histolytica (20 : 1) Jurkat T cells Jurkat T cells (1 10 6 /sample) were incubated for 110 min at 37 with or without E. histolytica at a ratio of 10:1, or 20:1 (Jurkat T cells to E. histolytica). After incubation, whole cell lysates were subjected to SDSPAGE and blotted with antiphosphotyrosine Ab. The figure is representative of three experiments showing similar results. Fig 7B. Proteolysis of SHP2 tyrosine phosphatase in Jurkat T cells stimulated with E. histolytica. Time (min) 0 1 2 5 10 SHP2 72 kda + + + + + + + + + + + + + + + + + + + E. histolytica (10 : 1) E. histolytica (20 : 1) Jurkat T cells H 2 O 2 10 mm PhosphoSHP2 Jurkat T cells (1 10 6 /sample) were incubated for 110 min at 37 with or without E. histolytica at a ratio of 10:1, or 20:1 (Jurkat T cells to E. histolytica). After incubation, whole cell lysates were subjected to SDSPAGE and blotted with antishp2 or antiposphoshp2 Ab. The figure is representative of three experiments showing similar results. 28

Fig 7C. Effect of calpain inhibitor on the E. histolyticainduced proteolysis of SHP2 phosphatase in Jurkat T cells. SHP2 72 kda + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Jurkat T cells E. histolytica (10 : 1) E. histolytica (20 : 1) E. histolytica (50 : 1) DMSO (vehicle) Calpeptin 1 mm Pretreated Jurkat T cells (1 10 6 /sample) with 1 mm calpeptin or 1% DMSO (v/v) as a control for 15 min at 37 were incubated with or without E. histolytica at a ratio of 10:1, 20:1, or 50:1 (Jurkat T cells to E. histolytica) for 1 min at 37 in a CO 2 incubator. After incubation, whole cell lysates were subjected to SDSPAGE and blotted with antishp2 Ab. The figure is representative of three experiments showing similar results. 29

8. 이질아메바에의해유도되는 JurkatT 세포내 calpastatin 의절단에 있어서 calpain 과 caspase 의역할 세포내에 존재하는 calpain 의 활성 억제인자인 calpastatin 은 caspase 또는 calpain 에매우특이적인기질로작용하여 calpain 의활 성을 조절하는 것으로 잘 알려져 있다. 25 이질아메바 첨가에 의해 Jurkat 세포내 calpastatin 은빠른시간안에다수의분획으로절단되 었다 (Fig.8A). 다음으로 이질아메바에 의해 절단된 calpastatin 이 caspases 와 calpain 의 활성에 의해 조절을 받는지 알아보기 위해 JurkatT 세포를이질아메바첨가전 pancaspase 억제제와 calpain 억제제로각각전처치하였다.Calpain 억제제인 calpeptin 을전처치 했을경우이질아메바에의한 calpastatin 의단백분해가억제된반면, pancaspase 억제제를 처리하였을 때는 전혀 억제되지 못하였다 (Fig.8B). 30

Fig 8A. Cleavage of calpastatin in Jurkat T cells stimulated with E. histolytica. Time (min) calpastatin 0 1 5 15 120 kda + + + + + + + + + + + + + + + + + + + + E. histolytica (10 : 1) E. histolytica (20 : 1) E. histolytica (50 : 1) Jurkat T cells Jurkat T cells (1 10 6 /sample) were incubated for 115 min at 37 with or without E. histolytica at a ratio of 10:1, 20:1, or 50:1 (Jurkat T cells to E. histolytica). After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticalpastatin Ab. The figure is representative of three experiments showing similar results. Fig 8B. Effects of calpeptin and zvadfmk on the E. histolyticainduced cleavage of calpastatin in Jurkat T cells. calpastatin 120 kda + + + + + + + + Jurkat T cells + + + + + E. histolytica (10 : 1) + + DMSO (vehicle) zvadfmk 200 μm + + + + Calpeptin 1 mm Pretreated Jurkat T cells (1 10 6 /sample) with or without 1 mm calpeptin, 200 μm zvadfmk were incubated for 1 min at 37 in the absence or presence of E. histolytica (1 10 5 /sample) in a CO 2 incubator. After incubation, whole cell lysates were subjected to SDSPAGE and blotted with anticalpastatin Ab. The figure is representative of three experiments showing similar results. 31

9. 이질아메바에의해유도되는 Jurkat T 세포의 PS 외향화 (externalization) 와 DNA 단편형성에있어서 ROS 억제제의효과세포내축적되는 ROS 는세포사멸과정에서중요한역할을한다고알려져있다. 따라서본연구에서는 ROS 의주요생산장소인 NADPH 산화효소 (oxidase), 사립체 (mitochondria) 및 5lipooxygenase(LO) 의역할을조사하였다. NADPH 산화효소 (oxidase) 의플라빈단백 (flavoprotein) 억제제인 DPI, 사립체의전자전달계 (electrontransport chain) 억제제인 rotenone 및 5LO 억제제인 ETYA 를각각전처치한후이질아메바에의해유도되는 Jurkat 세포의사멸의억제효과를알아보았다. 이질아메바에의해유도된 PS 노출 (44.4 ± 7.0%) 은 DPI20 μm 을전처치하였을때 16.4 ± 6.3% 으로크게억제되었다 (Fig.9A). 또한, 이질아메바에의한 Jurkat 세포의 DNA 단편형성도 DPI 의농도에의존적으로억제되었다 (Fig.9B). 반면 rotenone 및 ETYA 에의해서는이질아메바에의해유도된 PS 외향화가전혀억제되지않았다 (Fig.10,11). 또한 NO synthase 억제제인 LNMMA 를 JurkatT 세포에전처치하였을때에도이질아메바에의한세포사멸을억제하지못하였다 (Fig.12). 또한사립체에서방출된 ROS 를제거할수있는항산화제들인 NAC 과 GSH 를 JurkatT 세포에전처치하였을때에도이질아메바에의한세포사멸이억제되지않았다 (Fig.13A,13B). 32

Fig 9A. Inhibitory effect of various concentrations of DPI on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 60 50 40 30 20 10 medium DMSO (vehicle) DPI 5 μm DPI 10 μm DPI 20 μm * 0 medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with DPI (520 μm) or 1% DMSO (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. Significant differences from the value obtained with cells incubated with DMSO are shown. *, p < 0.05 Fig 9B. Inhibitory effect of various concentrations of DPI on the E. histolyticainduced DNA fragmentation in Jurkat T cells. 1 2 3 4 5 6 7 Lane 1 : 100 b.p Marker Lane 2 : Only Jurkat (1 hr) Lane 3 : [Jurkat + DMSO 0.5%] + Entamoeba Lane 4 : [Jurkat + DPI 10 μm] + Entamoeba Lane 5 : [Jurkat + DPI 20 μm] + Entamoeba Lane 6 : [Jurkat + DPI 50 μm] + Entamoeba Lane 7 : Entamoeba histolytica only Pretreated Jurkat T cells (4 10 6 /sample) with DPI (1050 μm) or 0.5% DMSO (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (4 10 5 /sample) for 60 min at 37 in a CO 2 incubator. DNA fragmentation was analyzed by 2% agarose gel electrophoresis. An equal number of amebae and Jurkat T cells were incubated in medium alone as negative controls. 33

Fig 10. Effect of various concentrations of rotenone on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 70 60 50 40 30 20 10 medium DMSO (vehicle) Rotenone 10 μm Rotenone 20 μm Rotenone 50 μm 0 medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with rotenone (1050 μm) or 1% DMSO (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. Fig 11. Effect of various concentrations of ETYA on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 60 50 40 30 20 10 0 medium EtOH ETYA 35 μm ETYA 65 μm ETYA 100 μm medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with ETYA (35100 μm) 1% EtOH (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. 34

Fig 12. Effect of various concentrations of LNMMA on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 70 60 50 40 30 20 10 medium LNMMA 1 mm LNMMA 2 mm LNMMA 5 mm 0 medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with LNMMA (15 mm) for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. 35

Fig 13A. Effect of various concentrations of NAC on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 70 medium 60 NAC 5 mm NAC 10 mm 50 NAC 20 mm 40 30 20 10 0 medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with NAC (520 mm) for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. Fig 13B. Effect of various concentrations of GSH on the E. histolyticainduced PS externalization on the surface of Jurkat T cells. AnnexinV positive cells, % 70 60 50 40 30 20 10 medium DMSO Glutathione 5 mm Glutathione 10 mm Glutathione 20 mm 0 medium Entamoeba Pretreated Jurkat T cells (4 10 5 /well) with rotenone (520 mm) or 1% DMSO (v/v) as a control for 30 min at 37 were incubated with or without E. histolytica (8 10 4 /well) for 60 min at 37 in a CO 2 incubator. After incubation, cells were stained with FITCconjugated annexin V for flow cytometric measurement. Data are presented as mean ± SEM from five independent experiments. 36

Fig. 14. Proposed Model of Signaling Pathways of Jurkat T cell death triggered by Entamoeba histolytica Entamoeba histolytica Jurkat T cells lectin Galactose / GalNAc ROS calpastatin degradation Intracellular Ca 2+? Phosphatase (SHP2) activation Calpain activation Caspases activation PS exposure dephosphorylation DNA fragmentation 37

Ⅳ. 고찰 이질아메바에의한숙주세포의사멸에는 caspase3 의활성을통해이루어지는것으로잘알려져있으나 calpain 과 ROS 의구체적인신호역할에대해서는아직까지밝혀져있지않고있다. 이연구에서는 calpain 과 ROS 를매개로하는신호전달경로가이질아메바에의해유도되는 JurkatT 세포사멸에매우중요한역할을하고있음을처음으로밝혔다. 이와같은결론은다음과같은결과들로부터얻었다. 첫째, 이질아메바와함께배양한 JurkatT 세포는배양 30 분내에대조군에비해유의한수준의세포사멸이잘관찰되었다. 둘째, 이질아메바에노출된후 5분내에칼슘이온의증가와칼슘의존성단백분해효소인 calpain 의활성이 JurkatT 세포에서잘관찰되었다. 셋째, 이질아메바의첨가로인한세포내 caspase3,6,7 의활성은 calpain 특이억제제인 calpeptin 처치에의해크게억제되었다. 이때 calpeptin 의전처치는이질아메바에의해유도된세포내 calpain 의활성을억제시켰다. 넷째, 이질아메바의접촉으로인한 calpain 의활성은고농도의 pancaspase 억제제 (zvadfmk) 에의해서전혀영항을받지않았다. 다섯째, 이질아메바의첨가로인해유도된 SHP2 타이로신인산가수분해효소의활성이 calpeptin 처치에의해크게억제되었다. 여섯째, 이질아메바에의한세포사멸 (PS 외향화및 DNA 단편형성 ) 은 ROS 억제제인 DPI 를처치하였을때크게억제되었다. 이상의결과를요약하면, 이질아메바에의한 Jurkat T 세포사멸에는 calpain 을통한 caspase 및인산가수분해효소의활성경로, 그리고 ROS 를통한신호전달이중요한역할을하는것으로생각된다 (Fig. 14) 이연구에서보여준 calpain 을통한 caspase 의활성경로와유사한신호전달체계는 etoposide 에의해유도된생쥐 T 세포사멸시, 37 B 세포클론의삭제과정시, 38 그리고영양물질을제거했을때유도되는 38

망막유래세포죽음시에 31 잘보고되어있다. 그러나이와반대로 calpain 이활성형의 caspase9 또는 caspase3 를직접절단함으로써 caspase 의세포사멸기능을방해할수있다는보고도있다. 3941 이와는반대로 caspase 는 calpain 의내재성억제인자인 calpastatin 을가수분해함으로써 calpain 의활성을촉진시킬수있다고한다. 16,20,25,42,43 Calpastatin 은세포사멸시절단된형태를보일수있다. 고사 (apoptosis) 를통한 JurkatT 세포사멸시에는 110kDa 의 calpastatin 단백질이 75 kda 과 35 kda 의조각으로절단되어지고 caspase 특이억제제에의해그절단이억제된다. 25 그러나 calpain 의기질로 calpastatin 이작용하였을때에는, 44,45 calpain 의단백분해작용으로인해많은수의조각으로 calpastatin 이분해된다. 46 이연구에서관찰한이질아메바에의한세포사멸모델에서는 calpain 이직접 calpastatin 을가수분해함으로써 calpain 의활성을조절하였다. 즉이질아메바에자극은 JurkatT 세포내다수의절단된형태의 calpastatin 을잘유도하였으며이러한 calpastatin 의절단은 calpain 특이억제제인 calpeptin 처치에의해서는잘억제되었지만 pancaspase 억제제에의해서는전혀억제되지않았다. 따라서이질아메바에의해서유도되는 calpain 활성을통한 calpastatin 의단백분해는 calpain 의활성을더욱증가시켜세포의생존과관련된여러가지단백질들 (caspases 및 PARP) 을가수분해함으로써세포사멸을촉진시키는것으로추측된다. 다양한세포사멸모델에서 calpain 과 caspase 모두활성화될수있는것으로잘알려져있다. 20,32,4749 그러나 calpain 의역할은세포의종류와세포사멸자극인자의종류에따라다르게나타날수있다. 예를들면번역 (translation) 억제제인 cycloheximide 에의한중성구의 DNA 단편형성은 calpain 억제제로억제되었지만 Fas 수용체를통한세포사멸은 calpain 억제제로억제되지않았다. 50 이러한결과는새로운유전자의발현이요구되는 (induction model)dna 단편형성에는 calpain 이필수적이지만새로운유전자발현이필요없는 (transduction 39

model)dna 단편형성에는 calpain 이무관함을알수있다. 본연구에서, 이질아메바에의한 DNA 단편형성은 pancaspase 억제제로는억제되었지만 calpain 억제제에의해서는전혀억제되지않았다. 이러한결과를종합하면이질아메바에의한세포의 DNA 단편형성은 calpain 의활성과직접적인관련이없음을알수있었다. 또한본연구에서이질아메바에의한 Jurkat T 세포의 PS 외향화는 pancaspase 억제제및 calpain 억제제에의해전혀억제되지않았다. 고사를통한세포사멸의한특징인 PS 외향화는일반적으로 caspase 의활성과관련이있으나 51,52 노화된혈소판의세포사멸그리고 etoposide 또는 staurosporine 에의한세포사멸시유도되는 PS 의전치는 caspase 의활성과무관하게일어난다고보고되어있다. 53,54 따라서이질아메바에의해유도되는 Jurkat T 세포의 PS 외향화에는 calpain 과 caspase 의활성이아닌다른신호전달물질의활성에의해서유도됨을잘알수있다. UVB 로유도한 JurkatT 세포의죽음은 caspase 활성과무관하게 PS 의노출이유도된다고보고되어있으며이것은 UVB 의자극에의한세포내 ROS 의생성과관련이있다. 55 또한최근이질아메바또는결핵균 (Mycobacterium tuberculosis) 에의한중성구세포사멸에있어서 NADPH 산화효소의활성으로인해방출되는 ROS 가매우중요한역할을하고있음이알려져있다. 56 이러한 ROS 의생산장소인 NADPH 산화효소는중성구같은식세포뿐만아니라 T 세포와같은비식세포 (nonphagocytic cel) 에서도유사한 NADPH 산화효소가존재하고있음이최근알려져있다. 57 따라서이질아메바에의해유도되는 JurkatT 세포의세포사멸기전을밝히기위해서는다양한세포기능의조절에중요한제2의전달자 (second messengers) 인 ROS 의역할을밝히는것이매우중요하다고생각하였다. 이밖에 ROS 의생성장소로는사립체와 5lipooxygenase 등이있다. 본실험에서는 NADPH 산화효소, 사립체전자전달계및 5lipooxygenase(LO) 의특 40

이억제제인 DPI,rotenone,eicosatetraynoic acid (ETYA) 를 Jurkat 세포에각각전처치한후이질아메바에의한세포사멸을측정하였다. 이질아메바에의한 JurkatT 세포의 PS 외향화와 DNA 단편형성은 DPI 처치에의해억제되었지만 rotenone 및 ETYA 에의해서는전혀억제되지않음을관찰하였다. 이러한결과는세포내 NADPH 산화효소에서유리되는 ROS 의생성이이질아메바에의한세포사멸에매우중요한역할을하고있음을시사하고있다. 그러나이질아메바에의한 JurkatT 세포사멸에있어 NADPH 산화효소의세밀한역할에대하여보다더정밀한연구를수행하여야할것으로생각된다. 이질아메바감염에의한숙주조직의손상은숙주세포로의부착 (adherence), 부착의존성세포용해 (contactdependentcytolysis) 및포식 (phagocytosis) 의 3가지단계를거쳐이루어지게된다. 이러한세가지과정은이질아메바의병원성과직접적인관련이있다. 이질아메바의부착에의한숙주세포사멸은세포내에있는고사유도기기가활성화됨으로써이루어지는것으로이해하고있다. 최근보고에의하면많은병원체들은감염시숙주세포의생존과죽음을조절할수있다. 괴사 (necrosis) 와달리고사를통한세포사멸은조직염증반응을유도하지않아병원체의생존에유리하며그리고과격한염증반응으로인한조직손상을막을수있어병원체와숙주에모두다유익한면역반응이다. 이질아메바외에도다른병원체에의한숙주세포의고사기전이자세히연구되고있다. 즉, 소의생식기에기생하며트리코모나스증 (trichomoniasis) 을일으키는기생원충인트리코모나스페투스 (Trichomonas foetus) 는 caspase3 의활성을유도하여생식기의상피세포를사멸시킨다. 58 GroupB Streptococcus(GBS) 는쥐의대식세포 (macrophages) 에서세포사멸을유도한다.GBS 는 βhemolysin 을생성하여숙주세포내의막투과성을증가시켜칼슘이온농도를높인다. 이러한변화로인한대식세포의사멸은 βhemolysin 생성억제제와칼슘억제제에의해억제되었으나 caspase 억제제에의해서는억 41

제되지않았다. 59 또한인체의위점막에기생하는 Helicobacterpylori 도숙주세포의면역반응을피하기위해대식세포를사멸시킬수있는것으로보고되고있다.Helicobacterpylori 에감염에의해세포사멸은세포내다아민산화효소 (polyamine oxidase 1) 의활성으로인한 ROS 의생산과사립체막분극 (depolarization) 을통해이루어진다. 60 재향군인병 (legionnaires'disease) 을일으키는레지오넬라뉴모필라 (Legionelapneumophila) 는인체의폐포에감염되어폐포상피세포와대식세포를사멸시킨다. 레지오넬라균에의한 U937 대식세포의사멸은 caspase3 의활성으로인한 PARP 의절단을통해이루어지며,caspase3 특이억제제처리시 caspase3 의활성과세포사멸이억제된다고보고되어있다. 61 이와는반대로세포내기생하는어떤병원체는숙주세포의죽음을억제시킴으로써병원체의증식과생존을확보하려한다. 예를들면톡소포자충 (Toxoplasmagondi) 은숙주세포에감염시 NFκB 의활성을통한고사억제 (antapoptotic) 단백질들의생산이증가되어세포사멸이억제된다. 62 따라서병원체에의한숙주세포의생존과사멸의조절기전에대한연구는체내에서일어나는숙주와병원체간의상호관계를정확히이해할수있어매우중요하다. 42

Ⅴ. 결론 이질아메바에의한세포사멸에는 calpain 을통한 caspase 및인산가수분해효소의활성경로와 ROS 를매개로한신호경로가매우중요함을알수있었다. 이러한신호전달경로는이질아메바에의한세포사멸의기전을보다확실히이해할수있어이질아메바증의병인과정을밝히는데도움을줄수있을것으로기대한다. 43

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