PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein

BioMed Cen al Page 1 o 12 (page numbe no o ci a ion pu poses) Molecula Cance Open Access Resea ch PRIMA-1MET induces nucleola ansloca ion o Eps ein-Ba i us-encoded EBNA-5 p o ein Gyö gy S ube 1, Emilie Flabe g1,2,3, Gabo Pe anyi1,2,4, Ri a Ö ös1, Nina Rökaeus5, Elena Kashuba1,3, Klas G Wiman5, Geo ge Klein1 and Laszlo Szekely*1,2,3 Add ess: 1Depa men o Mic obiology, Tumo and Cell Biology (MTC), Ka olinska Ins i u e, S-171 77 S ockholm, Sweden, 2Ka olinska Ins i u e Visualiza ion Co e Facili y (KIVIF), Ka olinska Ins i u e, S-171 77 S ockholm, Sweden, 3Cen e o In eg a i e Recogni ion in he Immune Sys em (IRIS), Ka olinska Ins i u e, S-171 77 S ockholm, Sweden, 4Swedish Cen e o Disease Con ol (SMI), S-171 82 Solna, Sweden and 5Dep a men o Oncology-Pa hology, Cance Cen e Ka olinska (CCK), Ka olinska Ins i u e, S-171 76 S ockholm, Sweden Email: Gyö gy S ube - Gyo gy.S ube @ki.se; Emilie Flabe g - Emilie.Flab[email p o ec ed]; Gabo Pe anyi - Gab[email p o ec ed]; Ri a Ö ös - [email p o ec ed]; Nina Rökaeus - [email p o ec ed]; Elena Kashuba - [email p o ec ed]; Klas G Wiman - [email p o ec ed]; Geo ge Klein - Geo [email protected]; Laszlo Szekely* - [email p o ec ed] * Co esponding au ho Abs ac The low molecula weigh compound, PRIMA-1MET es o es he ansc ip ional ansac i a ion unc ion o ce ain p53 mu an s in umo cells. We ha e p e iously shown ha PRIMA-1MET induces nucleola ansloca ion o p53, PML, CBP and Hsp70. The Eps ein-Ba i us encoded, la ency associa ed an igen EBNA-5 (also known as EBNA-LP) is equi ed o he e icien ans o ma ion o human B lymphocy es by EBV. EBNA-5 associa es wi h p53-hMDM2-p14ARF complexes. EBNA- 5 is a nuclea p o ein ha ansloca es o he nucleolus upon hea shock o inhibi ion o p o easomes along wi h p53, hMDM2, Hsp70, PML and p o easome subuni s. He e we show ha PRIMA-1MET induces he nucleola ansloca ion o EBNA-5 in EBV ans o med B lymphoblas s and in ans ec ed umo cells. The PRIMA-1MET induced ansloca ion o EBNA-5 is no dependen on he p esence o mu an p53. I also occu s in p53 null cells o in cells ha exp ess wild ype p53. Bo h he na i e and he EGFP o DSRed conjuga ed EBNA-5 espond o PRIMA-1MET ea men in he same way. Image analysis o DSRed-EBNA-5 exp essing cells, using con ocal luo escence ime- lapse mic oscopy showed ha he nucleola ansloca ion equi es se e al hou s o comple e. FRAP ( luo escence eco e y a e pho obleaching) and FLIP ( luo escence loss in pho obleaching) measu emen s on li e cells showed ha he nucleola ansloca ion was accompanied by he o ma ion o EBNA-5 agg ega es. The p ocess is e e sible since he agg ega es a e dissol ed upon emo al o PRIMA-1MET. Ou esul s sugges ha mu an p53 is no he sole a ge o PRIMA-1MET. We p opose ha PRIMA-1MET may e e sibly inhibi cellula chape ons ha p e en he agg ega ion o mis olded p o eins, and ha EBNA-5 may se e as a su oga e d ug a ge o elucida ing he p ecise molecula ac ion o PRIMA-1MET. Published: 26 Ma ch 2009 Molecula Cance 2009, 8:23 doi:10.1186/1476-4598-8-23 Recei ed: 10 Ap il 2008 Accep ed: 26 Ma ch 2009 This a icle is a ailable om: h p://www.molecula -cance .com/con en /8/1/23 © 2009 S ube e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 2 o 12 (page numbe no o ci a ion pu poses) In oduc ion Eps ein-Ba i us (EBV) is he mos powe ul ans o m- ing agen o human cells. EBV in ec ed es ing B cells unde go blas ans o ma ion and de elop in o immo al lymphoblas oid cell lines (LCLs) in i o o in o immuno- blas ic lymphomas in immunocomp omised hos . Nine la en i al p o eins a e exp essed egula ly in he ans- o med cells: he nuclea an igens, EBNA1–6, and h ee memb ane p o eins, LMP-1, -2a and -2b. Six o hese p o- eins a e equi ed o immo aliza ion: EBNA-1, -2, -3 (3A), -5 (LP), -6 (3C) and LMP-1 [1,2] EBNA-5 and EBNA-2 a e he i s wo i al p o eins exp essed in newly in ec ed cells [3]. The co-exp ession o EBNA-2 and EBNA-5 plays an impo an ole al eady a he i s s eps o EBV induced ans o ma ion by d i ing es ing B cells in o he cell cycle [4]. The co-ope a ion be ween EBNA-2 and EBNA-5 is needed o he ac i a ion o LMP-1 and Cp i al p omo e s [5]. EBNA-5 p e e en ially accumula es in dis inc nuclea oci in EBV- ans o med lymphoblas oid cell lines. We ha e p e iously shown ha hese oci a e inside he PML bod- ies [6]. We ha e also shown ha he same oci con ained he e inoblas oma (Rb) p o ein and he hea shock p o- ein, Hsp70 [7]. A i icial sp eading o he ch oma in by exposu e o he o ces o luid su ace ension dis up ed he co-localiza ion g adually. I showed ha EBNA-5 is loca ed in he inne co e o he bodies whe eas PML o med he igid ou e shell [6]. Nuclea bodies wi h p ominen PML s aining a e seen in unin ec ed es ing B lymphocy es. This s aining pa e n does no change upon EBV in ec ion. In eshly in ec ed cells EBNA-5 is di usely dis ibu ed h oughou he nucleoplasm bu a e a ew days g adually eloca e o he PML bodies and emains he e in es ablished lymphoblas- oid cell lines [8]. The localiza ion o EBNA-5 o PML bod- ies is es ic ed o EBV- in ec ed human lymphoblas s. Exogenously exp essed EBNA-5 in any o he cell lines dis- ibu es homogeneously in he nucleoplasm [9]. Hea shock o high cell densi y induced me abolic s ess leads o he ansloca ion o EBNA-5 o he nucleoli bo h om he PML bodies o om he nucleoplasm [10]. Sub- sequen ly we ound ha p o easome inhibi o s induced also nucleola ansloca ion o EBNA-5 in bo h LCLs and ans ec ed cell lines. They also induced nucleola anslo- ca ion o Hsp70 p o ein and mu an p53. T ansloca ion o he la e was enhanced by he p esence o EBNA-5 [9]. In a sepa a e s udy, we ha e shown ha p o easome inhibi- o s can induce nucleola ansloca ion o a ious compo- nen s o he PML body and nuclea /nucleola accumula ion o p o easomes [11]. The p ecise ole o EBNA-5 in he i us induced ans o - ma ion has no ye been es ablished. I binds o p14ARF- hMDM2-p53 complexes. Fo ced o e exp ession o p14ARF leads o he o ma ion o ex anucleola inclu- sions wi h subsequen en apmen o hMDM2, p53 and EBNA-5 [6,12-14]. PRIMA-1 has been iden i ied h ough di e en ial sc een- ing o he s uc u al di e si y se o he NCI chemical lib a y, as a d ug ha selec i ely induces apop osis in mu an p53 bea ing human umo cells bu no in hei p53 -/- coun e pa s [15]. T ea men wi h PRIMA-1 sup- p esses he g ow h o mu an p53 exp essing human umo xenog a s in i o [16,17]. PRIMA-1 can es o e he ansc ip ional ansac i a ing unc ion o ce ain p53 mu an s and induce p53 dependen apop osis [15]. Recen ly, we ha e shown ha PRIMA-1MET, a me hyla ed de i a i e o he o iginal PRIMA wi h imp o ed p o-apop- o ic ac i i y, causes nucleola ansloca ion o mu an p53 and o PML, CBP and Hsp70. The le el o Hsp70 was signi ican ly inc eased by PRIMA-1MET ea men . The nucleola accumula ion o PML, CBP and Hsp70 was much mo e e icien in cells wi h mu an p53 as com- pa ed o p53-/- cells [18]. PRIMA-Dead, a compound s uc u ally ela ed o PRIMA-1MET bu unable o induce mu an p53-dependen apop osis, ailed o induce nucle- ola ansloca ion o mu an p53. These esul s sugges ed ha edis ibu ion o mu an p53 o nucleoli plays a ole in PRIMA-1MET induced apop osis. Conside ing he in ima e ela ionship be ween EBNA-5 and he p53 pa hway as well as he ob ious simila i ies be ween he p o easome inhibi o and PRIMA-1MET induced nucleola ansloca ion o p53, PML and Hsp70, we ha e now in es iga ed he e ec o PRIMA-1MET on he subcellula dis ibu ion o EBNA-5. He e we show ha PRIMA-1MET induces he nucleola ansloca ion o bo h i us encoded endogenous and ans ec ed exogenous EBNA5 and i s luo escen de i a i es GFP-EBNA5 and DSRed-EBNA-5. Me hods Cell cul u es All cell lines we e g own in Isco e's cell cul u e medium supplemen ed wi h 10% hea -inac i a ed FBS, 2 mM L- glu amine, 100 U/ml penicillin and 100 U/ml s ep omy- cin. The cells we e passaged e e y ou h day 1:5. Cul u es we e egula ly es ed o he absence o mycoplasma wi h Hoechs 33258 s aining. T ans ec ions we e done using Lipo ec amine Plus eagen (GibcoBRL) acco ding o he manu ac u e 's ins uc ions. In he p esen s udy he ol- lowing cell lines we e used: LSsp, EBV ans o med lym- phoblas oid B-cell line, H1299 lung adeno ca cinoma line (p53 -/-) and i s mu an p53 ans ec ed subline (wi h His Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 3 o 12 (page numbe no o ci a ion pu poses) 175), SW480, a colo ec al cance line wi h mu an p53 (A g o His 273 and P o o Se 309). MCF-7, a b eas ca - cinoma line bea ing wild- ype p53. Clones o MCF-7 and SW480 lines (S2 ca ying EBNA-5 and P2 ec o con ol), cons i u i ely exp essing EBNA-5 om a pBabe-EBNA-5 cons uc we e gene a ed using selec ion wi h 1 μg/ml pu omycin (Sigma). MCF7 cells cons i u i ely exp essing DsRed_EBNA-5 we e selec ed on 1 mg/ml G418 (Sigma). Cons uc s GFP-EBNA-5 was made by cloning an EBNA-5-encoding BamHI-EcoRI agmen om pBabe-EBNA-5, con aining ou W epea s and he unique C- e minal egion, in o BglII-EcoRI-clea ed pEGFP-N1 [13]. To p oduce ed usion p o ein EBNA-5 was ampli ied wi h p ime s con aining NheI-EcoRI 5'o e hangs. The clea ed PCR p oduc was cloned in o he co esponding si es o pDsRed1-N1 ec o (Clon ech). PRIMA-1MET- ea men and immuno luo escence s aining The monoclonal mouse an ibody JF186 was used agains EBNA-5 [19] and he MAb used agains B23/nucleophos- min was a gi om P. K. Chan, Baylo College o Medi- cine, Hous on, USA. The cells we e cul u ed on co e slips in six-well pla es un il hey ha e eached a densi y o 5 × 104 cells/cm2 and incuba ed o 24 h in he p esence o 50 μM PRIMA-1MET dissol ed in dime hylsulphoxide (DMSO). Cells ea ed wi h DMSO we e used as con ols. Cells we e ixed wi h me hanol-ace one (1:1) a -20°C and hen we e e-hyd a ed in phospha e-bu e ed saline (PBS) o 30 min. An ibodies we e dilu ed in blocking bu e (2% bo ine se um albumin, 0.2% Tween-20, 10% glyce ol in PBS). The cells we e s ained wi h p ima y MAbs o 1 h a oom empe a u e, ollowed by h ee washes in PBS, incuba ed wi h seconda y FITC o Texas ed-conjuga ed an ibodies, washed h ee imes and moun ed wi h 80% glyce ol solu ion in PBS con aining 2·5% 1,4-diazabicyclo-(2.2.2)oc ane (Sigma). Bisbenz- imide (Hoechs 33258) was added a a concen a ion o 0·4 μg/ml o he seconda y an ibody o DNA s aining. Li e cell mic oscopy Long e m li e cell luo escence imaging was ca ied ou in POC Mini chambe s. The DSRed-EBNA-5 exp essing cells we e g own on he ound co e glass inse o he POC Mini chambe (Ca l Zeiss), The chambe s we e loaded wi h 400 ul cell cul u e medium and we e used in ully closed mode. The imaging was ca ied ou on an Ul a iew LCI h ee line lase Nipkow spinning disc con- ocal mic oscope sys em wi h a CSU10 Yokogawa head (Pe kin Elme ) assembled on a Zeiss Axio e mo o ized mic oscope. Du ing he imaging he empe a u e was main ained a 37 C wi h he help o hea con olle block co e ed wi h a anspa en plexi shield. The hea loss, owa d he oil imme sion objec i e, was p e en ed by he use o a sepa a e objec i e hea he ing. Bo h he con ol- le block and he hea e ing we e con olled by a com- mon elec ic he mos a e. The ou dimensional image cap u e was ca ied ou using he cus om de eloped so - wa e "Fi eColo Mo ie" w i en by us, using he Openlab Au oma o isual p og amming en i onmen (Imp o i- sion). To ensu e he main enance o p ope ocal plane du ing he ex ended imaging pe iods we ha e de eloped an au o ocusing p og am module ha uses ecu si e min- imum/maximum in ensi y measu emen s a 100 sepa a e small squa es o a ma ix o e layed on he gaussian blu ed cen al egion o in e es (ROI) o he aw image. To ind he ocus, se ies o sho (50 mic osecond) expo- si ions a e made a di e en Z axis posi ions ha a e clus- e ed a ound he las used ocal plane, and ex ended in heigh ha is wice he leng h o he imaging s ack. The sec ion wi h he g ea es di e ence be ween he minimum and maximum in ensi y alues is selec ed as sha pes . The Z posi ion o he sha pes image hen se es as he cen al poin o he consequen cap u ing o 10 images a anged in a Z s ack wi h exposi ion imes in he ange o 500 o 1000 milliseconds. To con enien ly cap u e se e al hun- d eds o mul icolou images along he ime axis he indi- idual Z s acks we e collapsed "on- he- ly" using maximum in ensi y p ojec ion algo i hm. By sa ing he images a e e e y 200 ime poin s he p og am assu es ha he image cap u e p ocess is no limi ed by he RAM bu by he a ailable ha d disc space. Pho okine ic measu emen s o ecombinan EBNA-5 in li e cells To measu e he in acellula mobili y o EBNA-5 in di e - en in anuclea compa men s o ea ed and con ol cells we used an Ul a iew RS i e line lase Nipkow spinning disc con ocal sys em wi h a CSU22 Yokogawa head (Pe - kin Elme ) assembled on a Nikon in e ed luo escence mic oscope. To achie e pixel p ecise bleaching o selec ed a eas a gal anome ic Pho okinesis uni (Pe kin Elme ) wi h sepa a e lase inpu was ins alled be ween he Ul a iew uni and he pho opo o he mic oscope. The imelapse 4D imaging wi h single (FRAP) and epea ed (FLIP) bleach cycles was ca ied ou wi h he Ul a iew cap u e so wa e (Pe kin Elme ). The cap u ed images we e quan i ied using he analy ic ou ines o he Ul a iew p og am as well as he p og am ImageJ (Ras- band, W.S., ImageJ, U. S. Na ional Ins i u es o Heal h, Be hesda, Ma yland, USA, h p:// sb.in o.nih.go /ij/). Fo FRAP and FLIP s udies DSRed-EBNA-5 exp essing cells we e g own on he bo om glass o a POC Mini chambe . The selec ed a eas we e bleached using he 568 nm line o an A gon-K yp on lase . In a ypical FRAP eco ding wo p ebleach images we e ollowed by 100 bleach cycles ( o al 300–1000 ms) and he eco e y was measu ed by a se ies o 500 ms exposi ion o e 1 o 3 minu es (120–360 Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 4 o 12 (page numbe no o ci a ion pu poses) images). In he FLIP expe imen s he bleach cycles we e ollowed by he cap u e o en images and hen he bleach cycles we e epea ed se e al imes. Resul s Immuno luo escence s aining o EBNA-5 in ixed cells To s udy he e ec o PRIMA-1MET on he subcellula local- iza ion o EBNA-5, EBV ans o med lymphoblas oid cell lines and EBNA-5 ans ec ed umo cells we e ea ed wi h a ious concen a ions o he d ug o 24 hou s. Beside na i e EBNA-5 we ha e also used EBNA-5 conju- ga ed o he C- e minus o he luo escence p o ein EGFP o DSRed. Upon comple ion o he ea men he cells we e ixed wi h me hanol:ace on and s ained wi h he monoclonal an i-EBNA-5 an ibody JF186. We ound ha 12–24 hou s ea men wi h 50 uM PRIMA-1MET induced nucleola ansloca ion o EBNA-5 in all cell lines such as he lymphoblas oid cells LSsp exp essing i us encoded endogenous EBNA-5 (Figu e 1) and ans ec ed umo cell lines such as he colon ca ci- noma line SW480 (endogenous mu an p53; Figu e 2), he b eas ca cinoma line MCF7 (w p53) and he lung adeno-ca cinoma line H1299 (p53 null cells) as well as i s ans ec ed a ian exp essing mu an p53 (His175). The iden i y o he nucleolus was asce ained by B23 s aining and/o phasecon as imaging in pa allel. In un ea ed, ans ec ed cells EBNA-5 localized o he low DNA densi y a eas co esponding o he euch oma in (Figu e 3). I was ei he absen om he nucleolus o i p esen i s le el did no exceed he amoun in he nucleo- plasm (Addi ional ile 1 and addi ional ile 2). In he PRIMA-1MET-induced ansloca ion o i us encoded endog-eneous EBNA-5 in he Eps ein-Ba i us ans o med human lymphoblas oid cell line LSsp ha ca ies i us encoded EBNA-5 and ha bo s wild ype p53Figu e 1 PRIMA-1MET-induced ansloca ion o i us encoded endogeneous EBNA-5 in he Eps ein-Ba i us ans o med human lymphoblas oid cell line LSsp ha ca ies i us encoded EBNA-5 and ha bo s wild ype p53. 24 hou s ea men wi h 50 uM PRIMA-1MET leads o he disappea ance o EBNA-5 om he PML bodies and o eloca ion o he nucleolus. PRIMA-1MET-induced ansloca ion o exogenous EBNA-5 in mu an p53 ca ying SW480 colon ca cinoma cells a e 24 hou s ea men Figu e 2 PRIMA-1MET-induced ansloca ion o exogenous EBNA-5 in mu an p53 ca ying SW480 colon ca ci- noma cells a e 24 hou s ea men . Immuno luo es- cence s aining o EBNA-5 is g een, DNA s aining using Hoecsh 33258 is blue. Dis ibu ion o DSRed-EBNA-5 in he nucleus o a li ing MCF7 cell ha ca ies wild ype p53Figu e 3 Dis ibu ion o DSRed-EBNA-5 in he nucleus o a li - ing MCF7 cell ha ca ies wild ype p53. Single con ocal sec ion selec ed om he middle o a se ies o 21 images. Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 5 o 12 (page numbe no o ci a ion pu poses) ea ed cells he e was an o e all inc ease o EBNA-5 le els wi h a e y p ominen inc ease in he nucleolus. A e 20 hou s o ea men almos all di use nucleoplasmic EBNA-5 signal was concen a ed in nume ous dis inc oci e enly dis ibu ed h oughou he en i e nucleus. To es he possible in luence o EBNA-5 on he su i al a e o PRIMA-1MET ea ed cells we ha e compa ed wild ype and mu an p53 ca ying cells s ably ans ec ed wi h EBNA-5 and hei ec o con ols. PRIMA-1MET ea men had no e ec on he su i al a e o wild ype p53 ca ying MCF7 b eas ca cinoma cells wi h o wi hou EBNA-5 a any PRIMA-1MET concen a ions, al hough he cells showed p ominen nucleola ansloca ion o EBNA-5. Compa ing EBNA5 ans ec ed (S2) and ec o con ol ans ec ed (P2) SW480 colon ca cinoma cells ha exp ess mu an P53 we ound ha he p esence o EBNA-5 sligh ly sensi ized o he PRIMA-1MET e ec . (Addi ional ile 3). Kine ics o nucleola ansloca ion o DS- edEBNA5 cells in PRIMA-1MET ea ed cells To s udy he dynamics o PRIMA-1MET induced nuclea mo emen s o EBNA-5 we used an au oma ed con ocal mic oscopy me hod, de eloped by us, o li e cell imaging (see Ma e ials and Me hods). The echnique pe mi s con- inuous eco ding o li ing cells using a combina ion o luo escence and phase con as illumina ion o e se e al (6–24) hou s. We ound ha he cells ole a ed excellen ly he combina ion o 568 nm epi luo escence and 600 nm ansmi ed phase con as illumina ion. On he o he hand using sho e wa eleng hs (405 and 488 nm) always led o isible pho o oxic e ec s du ing p olonged expe i- men s. Imaging cells s ably ans ec ed wi h DSRed- EBNA-5 e ealed ha EBNA-5 ha was o iginally e enly dis ibu ed in he nucleoplasm successi ely accumula ed in he nucleoli be ween 6 and 10 hou s a e he PRIMA- 1MET ea men (Figu e 4 and Addi ional ile 4). EBNA-5 accumula ion was associa ed wi h he o ma ion o 15–20 ound o o oid pa icles o he size o 250–300 nm ha showed limi ed mo emen inside he nucleolus. The nucleola accumula ion has egula ly s a ed om a single ocus in a gi en nucleolus. Di e en nucleoli s a ed he p ocess a di e en ime. Some nucleoli showed up o ou hou s delay as compa ed o he ea lies accumula ing nucleoli in he same nucleus. A e 10 hou s ea men wi h PRIMA-1MET he nucleoli became sa u a ed wi h Time lapse se ies o PRIMA-1MET-induced ansloca ion o DSRed-EBNA-5 in s ably ans ec ed MCF7 cellsFigu e 4 Time lapse se ies o PRIMA-1MET-induced ansloca ion o DSRed-EBNA-5 in s ably ans ec ed MCF7 cells. Images a 1 hou in e al we e selec ed om a se ies o 720 images eco ded in e e y minu es o 12 hou s. Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 6 o 12 (page numbe no o ci a ion pu poses) EBNA-5. A his poin some o he b igh ly luo escen DSRed-EBNA-5 pa icles we e eleased om he nucleolus and mo ed a ound in he nucleoplasm by apid B ownian mo emen (Addi ional ile 5). The nucleola accumula- ion was also accompanied by an o e all inc ease o DSRed-EBNA-5 luo escence in ensi y (Figu e 5). E ec o PRIMA-1MET on he mobili y o DSRed-EBNA-5 We ca ied ou FRAP and FLIP analysis on un ea ed and PRIMA-1MET- ea ed, DSRed-EBNA-5 ans ec ed MCF7 cells o measu e he a e o mobili y o EBNA-5 in di e - en nuclea sub-compa men s. In he un ea ed con ol cells he bulk o DSRed-EBNA-5 was homogenously dis- ibu ed h oughou he nucleoplasm. This ac ion showed e y high mobili y. Using single bleaching FRAP wi h 2 um spo size he a e age hal eco e y ime (T 1/2) was 1.5 second ha co esponds o a di usion coe icien o 0.66 um2/s. In compa ison he calcula ed mobili y in ee solu ion would be 54.6 um2/s. Imaging he luo escence loss in he non-illumina ed a eas (FLIP) showed a apid deple ion o he homogene- ous signal om he en i e nucleoplasm. The mino popu- la ion o nucleola DSRed-EBNA-5 in he non- ea ed cells showed a highe esis ance o FLIP. The nucleola DSRed- EBNA-5 was localized o dis inc sepa a ed a eas inside he nucleolus. Using pixel p ecise bleaching o he indi- idual a eas in FRAP expe imen s we could iden i y wo le els o eco e y one in e media e (T 1/2 21 second) and one e y slow (T 1/2 >> 300 s). The p esence o he wo sepa a e nucleola subcompa men was also con i med by FLIP expe imen s. (Figu e 6 and Addi ional ile 6) T ea men wi h PRIMA-1MET led o he accumula ion o DSRed-EBNA-5 in well-de ined g anules in he nucleolus wi h e y low molecula mobili y. In o he wo ds, epea ed bleaching o adjacen nucleoplasmic a eas, sepa- a e DSRed-EBNA-5 posi i e oci in he same nucleolus o e en pa o he same g anules ailed o induce any signi - ican loss o luo escence in he non-bleached s uc u es (Figu e 7). A e 20 hou s o ea men DSRed-EBNA-5 in addi ion o he nucleola agg ega es o med igid bodies e enly sca - e ed in he nucleoplasm. FLIP showed ha hese bodies con ained DSRed-EBNA-5 wi h simila low exchange mobili y as he nucleola agg ega es. These bodies showed also ela i ely apid ansla ional B ownian mo emen simila ly o he ones eleased om o e illed nucleoli. An impo an dis inc ion howe e om he o me was ha he nucleoplasmic bodies we e somewha la ge (400 nm e sus 300 nm) and hei mo emen ajec o ies we e much mo e es ic ed (Figu e 7 and Addi ional ile 7). Whe eas he bodies eleased om he nucleolus could a el o any a ea o euch oma in he bodies ha we e p e- cipi a ed ou a he la e ime poin in he nucleoplasm mo ed wi hin a well-de ined sphe e o 800–1200 nm. A e 24 hou s o ea men he nucleoplasmic bodies inc eased in size bu no in numbe (Addi ional ile 8). Re e sibili y o PRIMA-1MET induced agg ega ion o DSRed-EBNA-5 PRIMA-1MET egula ly induces apop osis in mu an p53 exp essing cells. To explo e he possibili y whe he he p o ein agg ega ion phenomenon is a ea u e ad anced s age cellula agony we ha e ea ed DSRed-EBNA-5 exp essing, p53 -/-, H1299 cells wi h PRIMA-1MET o 12 hou s. These cells a e much less sensi i e o PRIMA-1MET induced apop osis han i s mu an p53 exp essing de i a- i es. The d ug ea men induced nucleola accumula ion o he p o ein in mos nucleoli. Remo ing PRIMA-1MET by epea ed washing wi h d ug ee medium led o he com- ple e dissolu ion o he agg ega es as i could be demon- s a ed by combined luo escen /phase con as ime lapse mic oscopy. No cy opa hic e ec s we e de ec ed a any ime du ing he expe imen (Figu e 8). Discussion The mos s aigh o wa d explana ion o he obse ed e ec s is ha PRIMA-1MET induces a g adual p ecipi a ion o EBNA-5. In un ea ed cells EBNA-5 mo es a ound wi h high mobili y in he nucleoplasm bu slows down in he nucleolus. We sugges ha his is due o he mechanical sie ing e ec o he densely a anged ch oma in ibe s in he zona ib illa is o he nucleolus (see Addi ional ile 2 o illus a ion o he densi y o ch oma in ibe s in he nucleoplasm and in he nucleolus in a li ing cell nucleus). In ou scena io he d ug ea men induces g adual agg e- PRIMA-1MET ea men leads o an o e all inc ease o DSRed-EBNA-5 le els as shown by plo ing he a e age luo escence in ensi y agains ime o he 720 ames shown in Figu e 4 a e backg ound sub ac ionFigu e 5 PRIMA-1MET ea men leads o an o e all inc ease o DSRed-EBNA-5 le els as shown by plo ing he a e - age luo escence in ensi y agains ime o he 720 ames shown in Figu e 4 a e backg ound sub ac- ion. Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 7 o 12 (page numbe no o ci a ion pu poses) Fluo escence loss in pho obleaching (FLIP) expe imen o un ea ed DSRed-EBNA-5 exp essing MCF7 cells show e y high mobili y o homogeneously dis ibu ed DSRed-EBNA5Figu e 6 Fluo escence loss in pho obleaching (FLIP) expe imen o un ea ed DSRed-EBNA-5 exp essing MCF7 cells show e y high mobili y o homogeneously dis ibu ed DSRed-EBNA5. Region o in e es 1 (ROI 1) was bleached o 300 ms ollowed by eco ding o 10 consecu i e images a 500 ms in e als. These cycles we e epea ed o 5 minu es. ROI 1 and 5, ep esen ing he o al bleached a ea and a selec ed bleached suba ea, showed apid eco e y o luo escence. ROI 2, ep esen ing a emo e non-bleached a ea in he nucleoplasm showed apid homogeneous dec ease o luo escence. ROI 6 in adjacen cell showed no changes. ROI 3 and 4 in he nucleolus show wo di e en ly equilib a ing compa men one as e (ROI 3) and one slowe (ROI 4). Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 8 o 12 (page numbe no o ci a ion pu poses) 20 hou s ea men wi h PRIMA-1MET leads o he loss o mobili y o DSRed-EBNA-5 in MCF7 cells as shown by FLIP expe i-men Figu e 7 20 hou s ea men wi h PRIMA-1MET leads o he loss o mobili y o DSRed-EBNA-5 in MCF7 cells as shown by FLIP expe imen . ROI 1 was bleached as abo e bu showed no signi ican eco e y. Adjacen ROI 2 and dis an ROI 3 showed no signi ican loss o luo escence. Molecula Cance 2009, 8:23 h p://www.molecula -cance .com/con en /8/1/23 Page 9 o 12 (page numbe no o ci a ion pu poses) ga ion o EBNA-5 and he agg ega es s a o clog he nucleola ch oma in ibe meshwo k. He e ogenei y in he sie ing e ec ("e ec i e po e size") o di e en subnu- cleola compa men s o e en be ween di e en nucleoli could explain he phenomena o : a.) ocal ini ia ion o nucleola accumula ion b.) nucleola subcompa men s wi h di e en mobili y iden i ied by FLIP c.) asynch o- nous accumula ion o EBNA-5 in di e en nucleoli. Along he same lines we also sugges ha upon p olonged (20+ hou s) ea men wi h PRIMA-1MET, he in si u all- ing ou o EBNA-5 p ecipi a es in he nucleoplasm is due o he con e gence be ween he size o he p ecipi a ed bodies and he sie ing dimensions o he 300 nm ch oma- in ib e meshwo k o he nucleoplasm. This is also con- sis en wi h he inding ha nucleoplasmic EBNA-5 agg ega es show spa ially es ic ed and uni o m andom walk ajec o ies (Addi ional ile 7 and Addi ional ile 8). Con o ma ional change due o mu a ion o he mal, acid- base o edox change ha leads o inc eased su ace expo- si ion o o he wise c yp ic hyd ophobic side chains is he mos usual cause o p o ein p ecipi a ion. Exposed hyd o- phobic su aces a e po en induce s o cellula hea shock esponses. Inc eased exposi ion o hyd ophobic su aces lead o inc eased exp ession o molecula chape ons ha ac i ely coun e ac he p ecipi a ion p ocess, by isola ing he mis olded p o eins om each o he and using he ene gy o hyd olyzed ATP o ac i ely "massaging" hei p o ein clien back o a na i e con o ma ion ha ep e- sen s he lowes le el o olding ene gy. Impo an ly mu an p53 was ecen ly ound in complex wi h Hsp90 in PRIMA-1MET ea ed cells [20]. Hsp90 is a majo cons i u- en o he p o eome, comp ising up o 5% o he o al mass o cellula p o eins. I emains o be elucida ed o wha ex en mu an p53 is associa ed wi h o he , less abundan hea shock p o eins in he p esence and absence o PRIMA-1MET. I is well known howe e ha mu an p53 o en com- plexes wi h Hsp70 [21]. Associa ion o Hsp70 and CHIP (ca boxy e minus o Hsp70-in e ac ing p o ein) is esponsible o ubiqui ina ion and deg ada ion o some o he p53 mu an s [22]. Ou p esen indings sugges ha PRIMA-1MET migh ac h ough e e sible inhibi ion o cellula chape on ac i i y. In his scena io he agg ega ion o EBNA-5 (o mu an p53) would be p e en ed by he cons i u i e ac i i y o Hsp70 ype cellula chape ons. Inhibi ion o chape on ac i i y by PRIMA-1MET would lead o he accumula ion o p o ein agg ega es as well as a eac- i e inc ease o Hsp70 exp ession. Di ec in e ac ion wi h he Hsp70 amily o chape ons is also a common denomina o o EBNA-5 and mu an p53. The Hsp70 amily membe s a e also among he mos po en an i-apop o ic agen s ha can block bo h he ex insic (memb ane signalling ini ia ed) and he in insic (mi ochond ial cy och ome C elease ini ia ed) p o-apop- o ic signal pa hways [23]. T ans o ma ion o p ima y B-cells by EBV is dependen on he EBNA-2 induced ac i a ion o nume ous i al and cel- lula genes [3,4,24,25]. EBNA-5 enhances his ansac i a- ion. Hsp70 is a majo complexing pa ne o EBNA-5 [26]. Ele a ed exp ession o Hsp70 inc eases, and sup- p essed exp ession o Hsp70 dec eases he e ec o EBNA- 5 on EBNA-2 media ed ansac i a ion. I has been sug- ges ed ha Hsp70 chape on ac i i y may help EBNA-5 in shu ling o ep esso s om EBNA2-enhanced p omo e s [27]. We ha e p e iously shown ha inhibi ion o p o easome ac i i y leads o synch onous nucleola ansloca ion o The PRIMA-1MET-induced nucleola ansloca ion o DSRed-EBNA-5 is e e sible in he p53 -/- H1299 cellsFigu e 8 The PRIMA-1MET-induced nucleola ansloca ion o DSRed-EBNA-5 is e e sible in he p53 -/- H1299 cells. The ansloca ion was induced by 12 hou s d ug ea - men ollowed by ex ensi e washing wi h d ug ee medium. Time-lapse mo ie was eco ded o 6 hou s, one ame pe minu e. The luo escence images a e combined wi h phase con as pic u es o demons a e he in ac cellula mo phol- ogy o he cells du ing he en i e leng h o he expe imen .