Comparison of Intracellular Cytokine Production With Extracellular CytoJrJ.

ne Levels Using Two Flow Cytometric Techniques

Background: We investigated the relation between intracellular cytokine production and extracellular cytokine levels by using two flow cytometric techniques. Methods: A two-color floe cytometric technique was used to measure interleukin (lL)-1, It-6, tumor necrosis factor a (TNF-a), IL-10, and IL-'2 production blocked intracellularly with brefeldin A in lipopolysaccharide (LPS)-stimulated CO 14 + monocytes and IL-2, IL-4, and IFN--y production in phorbol-12-mirystate-13-acetate (PMA)-stimulated C03+ T Iymphocytes in samples from patients with rheumatoid arthritis. A flow cytometric microsphere-based immunoassay was performed to detect cytokine secretion n plasma of PMA- and LPS-stimulated whole blood samples. Results: There was a strong linear correlation between extracellular quantitative (pg/ml) and intracellular semiquantitative detection of LPS-stimulated IL-' [3, IL-6, IL-lO, and IL-12 production (r > 0.9). For Iymphocytes, extracellularly detected IL-2 and IFN--y correlated well with percentages of cytokine-producing cells (r > 0.8). The percentages of IL-4-positive T cells were moderately correlated with the secreted amounts of IL-4 as detected with the microsphere-based immunoassay (r = 0.7). Conclusion: Overall, there was a good correlation between semiquantitative intracellular detection of cytokines and the secreted amounts of cytokines detected with the microsphere based immunoassay. Cytometry Part B (Clin. Cytometry) 55B:52-58, 2003. 2003 Wiley-Liss, Ine. Key terms: Cytokine; f10w cytometry; intracellular; extracellular In the past decacle, the anaysis of cytokine production became increasingly important in unranveling the curse of an immune response, in the evaluation of specific therapies, and in the searech of the pathophysiologie mechanisms at the base of allergic and autoimmune diseases, Several methocls have been developed to determine Cytokines in biological fluids and culture samples. The most popular method is enzyme-linked immunosorbent assay (ELISA), wich is applied to measure cytokine secretion in supenatants (1) At the mRNA level, polymerase chain reaction (2) and in situ hyhridization (3) are avaliable, whereas low citometry is used to determine intracellular cytokine production (4 -9) The intracellular flow cytometric method can identify the cytokine-producing cell type, but there is no absolute quantitative measurement of the produced cytokine. Recently, by using the same floe cytometric equipment, an extracellular microsphere-based method was development to quantify multiple secreted cytokines simultaneously in very small volume (10-14). We compared the results of both flow cytometric methods to detect cytokines interleukin (II)-2, interferon

tumor necrosis factor a (TNF-a). and IL-6 intracellularly and extracellularly. MATERIALS AND METHODS Study Population Twenty one samples were collected from seven patients with active rheumatoid arthritis (RA) according to the diagnostic criteria oof the American College of Rheumathology for the classification of RA (15). The RA patients were part of an RA population investigated in a study evaluating cytokine profiles at three different time points during anti TNF-a therapy (16). All patients gave informed consent for the study.

Culture Stimulated With Phorbol-12-Mirystate-13-Acetate One mililiter of peripheral whole blood (Vacuette heptarin tubes, Greiner Bio-one, Kremsmnster, Austria) was incubated for 6 h (previously reported as the optimal incuubation period for intracellular cytokine detection) (5-7) at 37 in a 5% CO2 humidified atmosphere with or without 50 ng/mL of phorbol-12-mirystate-1 3-acetate (PMA: Sigma, St. Louis, MO: previouslv considered the optimal concentration in titration experiments) (5-7)] 1ug/ml of ionomycin (Sigma), and ] 10Lg/ml of brefeldin A (Sigma) for intracellular dctection of cytokines or with 50 ng/mL of PMA and 1 ug/ml of ionomyc without brcfeldin A for extracellular production of cytokines. Lipopo Iysaccha ride-Sti mulated CuIture One milliiter of peripheral whole blood was simulated for 8h (previously described in kinetic studies as the optimal incubation period for intracellular cytokine detection (9) with or without 1 ug/ml of lipopolysaccharie!e Escherichia coli (LPS; seroltype 026:B6: Sigma)I and 10 ug/ml of brefeldin A (previously reported as an efficient cytokine secretion inhibitor in monocicles) (9) for imracellular detection of cytokines or with 1 ug/ml of LPS alone for extracellular detection of cytokines with the microsphere-based immunoassay.

Intracellular Cytokine Analysis in Lymphocytes Cytokine-producing T Ivmphocytes were determine as previously described (7), After stimulation,100 ul of stimulated blood was styned with CD3 and peridinin chlorophyll protein (PerCP: BD, EremboDegem, Belgium) for ) 1 5 min in the dark at 4C. Aftefteward, red blood cells were lysed, and white blood cells were fixed with FACSIysis (BD) for 20 min at room temperature (RT) After centrifugation, cell membranes were made permeable with 0.3% saponin (Sigma) in phosphate buffered saline (PBS), and cells were stained for 30 min at 4C with monocorinal anticnokine antibodies labeled With phycoerythryn (PE: ant-Hu IL-2-PE clone 5344.111, anti-Hu

IL-4PE clone 3010.211, and anti-Hu IFN-PE clone 25733. 11; BD). Cells were spun for 5 min at 1,000g and resuspended in PBS. Twenty thousand cells were measured on a FACScan (BD) flow cytometer and analyzed with WinMDI 2.8 software. Analysis gates were set on lymphocytes according to forward and side scatter propcnies CD3 T Iymphocytes were defined and gated on a density plot of CD3-PerCP versus side scatter (Fig 1.)wthin this gate, intracellular cytokineproducing cells were determined in a hislogram (PE emission: 585 nm: Fig 1). Markers were set on the 99th percentile by using isotype-matched irrelevant atibodies (mouse immunoglobulin [lg] G1 PE; RD) as a reference. Results were expressed as the percentage of cytokinc-producing CD3 cells. Specificity of antibodies was evalualed by evaluation of unpermeabilized cells: no staining With anticytokine PE-Iabeled antibodies was observed in unpemleabiJized cells for all enokines Cdala not shown) Staining of llI1stimulaled T lymphoC)1es \\'as also performed as a neg:niyc control (7) In addition, cr"stal

Iizahle fragmelll (Fe) blockmg with human 11'(; (Sigma) ()[ rat serum bcfore normal staining showed no dilferencc" for illlracelJular eytokille slaining. Funhcr. speciticitv \yas evalualcd by preincubation of the f1uoroel1rome (PFilabeled antibody with appropriate recombinant C)1okilltfor 60 mn at RT. For lE\-, and IL-2, a c1ose-dependclll inhibition up 10 ] 00"" of c\Lokinc-produeing cells \\a" reached when antic\Lokinc antibodies \Vere prcineubated with rceombinant enokine in a dose-dependem manne

up to J fJ.g/ml. Inhbition cxperimems for !lA production showed a paradoxical increase of IL-1 producng T cdls. which is in accordance \\'ith the findings of RosLling l"I al \';ho aIso did not conf1rm ami-Il.--l specif1citY \\ ilh the same ami- IL--l c!one (1-) Fluorcscence microscolw s!Jowcd high membrane positiYit\ for 11.-4 pr' ,ductio!l. suggesting that complexcs of recomhinanl IL-j Wilh antiIL-4 amibodies bind IL-1 receptors at the surface of T cdls. In an additional experimem. T cells WlTe not plTmeabilized and incubated with recombinam 11-1. Staining wirh ami-IL-4 -PE showed

recombinam IL-j hound to he cell surfact~ whether or nor Fc receptors were hlockul wilh heataggregated (65CC for ')0 min) human Igc' ISigma) lh, case, anricnokine-PEl its \IESF \-aluc for PE emissi()Jl is 1 (1.000. The standardi7aton Cllr;es \vere linear and had a immune complexes or r;ll senlm.
\':lrialion coeftieicnl ()f 2""

Intracellular Cytokine Analysis in Monocytes ImraceUular cnokine production b,- monocnes was derermined as preYiouslY described (<) After culture, 100 .l of whole blood was srained with CD 14 and fl!lorcscein isothiocvanate (FITC; BD) for 15 min in the elark at _j C( Afterward, reel blood ceUs were Iyseel. anel whte blood cdls were fixed Wilh FACSLysis (BD) for 20 min at RT. After centrifugation, ceU membranes were made pem1t'able with 0.3% saponin (Sigma) in PBS, anel ceils were staineel for 30 min at -lCC with PEIaheled anticvtokine amibodies against IL-l r-\, lL-6, and T\T-o (anti-Hu IL-I~-\ clone ASJO, ami-Hu IL- c!one A'd2. ami anti-!Iu T\I-n done (IOI.I J J 1. BD) (ells were spun for 'i min at I,OO(lp ami rcsuspended in PBS. Fortv thousand n'en's \\cn' measureu on a FACScan fiow C\1ometer and anal\'ed \\ith \Vin.\1DI softwarc:. Anal\'sis gates \\'Cre set on UJ 1I " eel" in a density plot according to FITe emission ('i0,(1 nm) am! si de scatter (Fig. 2). \X'ithin his gate. intraccllular enokine production (VE emisson: 585 nm) was evaluatcd in a histogram (Fig. 2). Results werc: expresscd as t he rnCIJl molccuies of equi,'alcnt soluble fJuorescein (.\Ibl) unirs lsotype-matched irre!cyant antihodies (mouse IgC; l-PL RD) were used as controls. Staining of unstmulatecl mono cItes was also performed as a neg;ni,'C conrml (l)) in acldition. Fc blocking wth human IgG (Sigma) or r:n SCnlm bcforc: normal staining showed no uiffcn:nces for intraceUular enokine staining Specificit\' 01' anribodies was preYiOusl~' e,-aluated hy anah'sis 01' unpermeabilin'u cells no staining with amicnokine PE-Iabeled antjl)odies was observeu in unpemleabilized cdIs for al! C\1okines un. For T:\TF-cx, J 00% inhihition of the initial .\1f5F signal for T:'\F- production in monoq1:es was reaehed in blocking experiments \v!Jcn J fl-g/ml of recomhinam T\"F-o: was added. Standardization of inrracellular enokinc cictection \\'as perfom1Cd by using microspheres \yih standardi7ed tIuorescence intensities. as prc,-iously reponed (l)) Semiqllan titati\e measuremcnrs Werl' standardized as ,'( >llo~ nI mean fluorescence imensities were recalculated as '\1ESF units b,using microspheres with diffcrenr staJlclarcliznl fluorescent imensities (code K OJ J O. Fluor()~'pIKTCS. Dak(). Glostnlp. Denmark) (18-20) In other w(,rels. if:1I1 ana!\zed monocne emits the same amount 01' tluorescen~e as J 0.000 'moleculcs nI' the same flunrochmme (iJl

Flow Cytometric Microsphere-Based Immunoassay l'\I\- and LPSstimul:ned whole hlood samples \I'Cre ccntrifugcd lor 20 mn al ] ,OOOg, and plasma was col Iccted and stured at - SWC umil assay. The microsphcre-baseci immunoassay (cnometric bead arL1\. ellA: BD Pharmingen. San Jose, CAl was performecl as described b, (,)ok ct al. (12). Briefiy, six microsphen' populations with distinct fluorescence imensities ~'cre clled "irh proprietan- d\'Cs (emission > 650 nm). These beads \\cre

coated with capture amibodies against cno I-:ine" and miXl-d w\th reeombinam standards or test sam pies (l'\1,\- and LPS-stimulated supernatams) amI he PF conjugatn! 1 emissiol1 5.\5 nm) detection cy10kine anlibodies tu form sandwich complexes consisting a c\' tokine antihod\-. a c\1okine, and a PE-labcled cytokine amibodY. In P.\lA-s'imuiated supemaraflts, [L2, 11.-4. IL-'i H_- J O. TI\F-(\ ami F:'\-"v were detecte(] with h~an 1"helper J and 2 eRA ikindly proYided hy BD); in LPS stimlllated supemar;ml'i. IL-12p""0, IL-6,IL- J O. T:'\F-o., I L-'\ and IL-I i3 werc measu-ed wth the human intlammati')!1 U\.\ (pllrCh;lsni from BD), The instrument setLIp was performd with CaliBRln heads. and rhe cnometer setup beads (RD) was done ;\cording lO the manlllacturer's instnlctions: 2.000 nCIlt' \\('re measured and anah'7ed. A monomeric microsphcrc P()j1u!; t ion ,yas gateu '>11 forward anel side scaners Dara were analyzed in t\\(H'olor Iluorescence dot plOh rcpresenting rhe different microsphere populations (emis sion ()'iO nm) and thc c,wkine concentratiol1 (accord inl..( !O PE emission 01' 'i.\,) nm). ,\kan fluorescence inren sir', \;tlues \\-cre collected. four-paramerric logisi( caiihralon cunTS were used. and results were expres'cd ;h picogram' plT milliliter. The detection limil ~-as 2 to 211 pg/ml.

Statistics \'aration coeniciems \\'erc calculated f()r st:lt1(brci clIrvcs. and corrclations hctween the differenT tcchniqul' \\Tre cvalllated wlth l'earson,S linear 'corrclation coclt cien!. P O,O'i 'ya s considered statisticaJI\ signitlcan1.

RESULTS Standard Curves lor Microsphere-Based Immunoassay Thc standard cunTS for 1L-2, [F1\-)', IL-'I IL-~ 11. I11 lL-] 2p-O, 1L-1[:\. IL-. IL-'\ and T:\F- were estahlished hl d'lemning the mean tluorescence associated wth lile diftcrcnt microspheres incubated in kno\\'n concenrr:l tio!1S 01' c\1okine (2(). -jO. SO, 156. ')12, 62'i. I..)')(), 2.".011 ami 'i.lll)() pg/ml) The shapes of the cunTS 1m IL} IL-~ IL-'i. ,IO. IL-12p-O. IIA), IL-8, and T\F-(\ wen: lIlear ,md S\mmurical. ()nly the cunTS for IE"-)' and IL- f-\ \Ven' ni 1\ s~'mme:ricaL AII 'Cllf\TS sh()wed a good reproducihil1l1 tilC yariation coefticiems of three cun'es oi each C\!ok!\W '...-ere :'.5':" f()r IL-2, 5:\"" for [L--l, 2-(':, for [L'i, 5 (," tm IL-l0. j()"o or 11.-12p-0. :,>.10(, f(lr 11.-6.2_5"" or HAi. 2.1'" lor T\f--. 9.1 or 11':\-" and 5.1 for ILI~), Comparison 01 Intracellular MESF With Extracellular

Detection 01 Cytokines in Monocytes ' I'or LPS-stimulated monocytes (C1)14 ~), intracdlulal' scmiquantitatin .. .\iESF detenion of IL-jf3, 11.-6. IL-l0. and IL-] 2 production was stl'onglY correLlted with cA1:racellular quantitatin' measurements OL-l(3: l' = 0.96. P < 0001. II.- r = 0.9'i. P < 0.001: IL-IO. r = 0.90. P < 0.01: JI.-] 2 r = 09:'>. P <: 0.001: Fig. :,>A-D). Comparison 01 Intacellular Percentage With Extracellular Detection of Cytokines in T Lymphocytes 1'01' h'mphocncs (CD:'> ~). percentages o intracelllllar [L-2 and IFN--y'-posit'\T T cells correlated well with extracclIlllal' detened anwunts of IL-2 and 11';\-, (lL-2: r ~- 0.:-;] . P = 0.01: IF:\-, r = O.RO. P <.: 0.001: Fig. 'lA.m For IL-I. the pcrcentages o 11.-4 -positiYC T ceIls corrdated moderately with the s(;'cr(;':cd amounts of IL-4 as detened "itb the microsphcr('-hascd immunoassay (r = 0.-0. P = (U)5: Fig 'le) In an additional exp(;'riment. a control hlood sampJc was stimulatt'd with diflcrent concentrations o P.\1A (50. 5. and O. 'i n~ml): t he rdation bctwecn perccntage 01 cnokinc-positiYc T cells and secreted amounts of c\'tokines \'.-as Cyalll;[n.!. lksults are shown in Figure 'i fol' 1 L--t. IL-2. and 11'\-,. DISCUSSION In the present stuch. \Ve compared intraceIlular ,,-ith cxtraccllular dctection of c\1.okines. Bccause he objt'clin: of this study ,,-as to compar(;' cnokine production in ;1 large rangc of yalues, R-\ paticnts wcre seIected: the hnud clnical and pathologic spectra of AA result in widc ranges 01 cnokinc production. which haye becn reponed in sneral studics including so me perfoffi1ed in our lahoraton C~9.16.2I-2:'>). First. a f]ow c\1.ometric tcchniqllc based on the single cell mcthod was lIsed to idcntify cytokineproducing P.\L\ stimlllated T Iymphocnes and LPS-stimulatcd monOC\LC' of IZA patients: cnhanced sensitiYity for detcction 01 cnokine-producing cells was achieyed by fixation, peffileabl lization of thc ceIls. and blocking of the cnokine produc tion in tht' Golgi apparatus with brefcldin A (t -9). Second. a microsphere-based tlow q1.ometric immllno ass;y was perforrncd in parallel to e\'alua[c c\1:okinc se crction Flow c)'1.ometric microspherc-based immunoa, sav' ,,-ere designed to pcm1it casy screening of secn:tnl

c\1:okincs in limited H)Jumes of sampl (onh ] (! ')() ~I) with the :1dyantage of simultaneoush' anal\7ing lllultiplc qtokines (10 -] 4) Valid,ltion reports of this c:xtr.I~- flo'.y C\1ometric tcchnique descrioed s<:nsiti\itics compa- rabie to those of ELlSA lechnolof,'T (range of sensitn'ities2- 8 pgJml) (10 -] 4). Jn our experiments. this IK\Y tech-

no]0F:' appearcd lO show highly reproducible scandard CUf\TS with \ariation cocfficients ranging from~' lO 9"" To accllratelv stain for intraceIlular c\'lo\;jnc pmduction. it is ol' critica] imporumce lO aiways includc :\Ppro priate control, (2). Therefore. n<:gati\T contn.\ expcriments. such as incuhating unstimula,cd ('('lis \Yith anticvwkine antibodics or staining llnpcnncabilizcd cclls for c\1okines and using fluorochromc-Iabclcd isol\'lx CO!l' tmls. ha\c hcen perfonned. Prehlocking of the <lnricvto kinc antibodies with a molar excess of rcCOmh1l1:lnt cno kine has bten proposed as the ultima!, tI )01 !" n'alu:ltl'

spcciliciry (2. '1.6) In this s!Ud\', addititmal inhibifion e'. periments showed complete inhibition ofT:\F-. H. 2. Ami staining. ()n :1 thcoretical basis. thcre might han' oe<:n sonw discrcpanci<:s hcrween intracdlular produClion ano extra ccllular secretion, especiaUy during the ven early phast \\'hcn lranscription begins. and in the late phasc, when s\nthesis is finished. Therefore, lime points in th~s stud\ e) h for Iymphocncs and 8 h for monoc.:1:es) wercchosc!l on the oasis of pr<:\iously reported kinetic studies [har ohs<:rn'd a decrease in c\1:okine prodUCllOf1 afler 1 ~ h "t incuhation \yith brcfeldin A. ('i.(,9.24,2'i) \1casurement ol' IL-2 and IE\-)' production. pcrfo1111 tll bv c!lumcrating c\1okine-producing T ~'mphoc\1es nI' hl qu:mtificatioll ol' qtokinc in culrurc plasma. Ppoduccd results that s!1o\Y<:d a good corrtlation. Vikingss(ill el al dcscrihcd a corrdation b<:Twccn quaptitatll'C mcasurc

Different Isoforms of HPV-16 E7 Protein are Present in Cytoplasm and Nucleus

lResearch Centa nfJnfccrinlls Discos!'.1 ,VdIU)/U! /SIiTlITe 01 f'lIh1ic cu!th. CIICl"I?iI\'uca, ,ljore/o,I, ;I.ft'xico :'Biol/1ed!ca! Rcscurch Jl1sr!wtc, \'urioI7U! A 1I0I7U!I!(il!1 [ "Facll!{\' o(\fedicinc, Aurol7ol/1ollS '171\crsin' .\lcxlco ut\fcxico, ,lf,',Yico Cm ,Ifnico

,\fore/m Srdlc, CUC/'IJ,I\'UUi, Jlorc!o,',

different recombina'lt E7 protein (\1S2-[7) [20J F urther characterization was performed by immunobLJ!. ilT1mlimmrCcipitation and immunofluorescence. The anti-E7 (C24 and C89) poiyclonal antihodles were prepared in our laboratory against Ihe eIl-E7 pm\ein puritied by eleetro-elution. BrieJly. rabbils \\ere immuri7cd 3 til';ll:S with 50 flg of

l'ecombmanr E7 pl'o\ein and rc:,ed fo;' [he presence of specific E7 :mlibodies ir. the same \\J\ as O1.\h, The anti-E7 Iclone ED]7, C20). the anti-c::lnexine (clon.: H-70) and the anti-p21 (('-29) were from Santa Cruz Biotechnology (Santa Cruz. CA.lS-\1 The anriG\1130 (clon.: 35)\\as from BD Biosci.:nces (San Jose. eA. lS\) In Vitro Cell Culture Conditions. CaSki eell line (human cenical epidermoid carcinoma. naturally llP\'- ] 6 transfonned). HaCaT cells (nonnal human keratmoeytes) ami Cos-7 (kidney monkey, SV 40 transfonned fihwhlasts) \\ere cultured in Dulbecco's \10dified Eaglc's meUnl!11 (0\!E\11 supplemented with lO% fetal bovme serum (FBS I and mainrained in a humidified atmosphere of 5"", CO2')5% ai!. ar noc In Vivo Cell Labeling. Celis \\ere lahcled with 50 pCi!ml (jSJ-methionine and [)\S]-cysleine (Pwmix. 100(1 (immol. GE Healthcare. Piscataway. \.U. l'S.\). in melhionine and cysteine free medium and 5"" of FBS in o\'ernight labeling cxperiments or \\ilh 250 L1Ciml [ methioninc-cysteine for l5 min in pulse-ehase: C'\rerimcnh. Cells labeled with (=P]-orthophosphate \\erc sraned I1 0.2(~o FBS for 48 h and then placed in rhosphalc Cree me dium for further 2 h, a:i descrihed pre\iouslv [2]]. Afcr tll]" time. cells were ine:ubated with 100 UCi mi orthophosphate (3000 Ci. mmol, GE flcalthcarc. Piscata\\ a\. !\J. eSA) for ] h (time: zcrnl. The re:mal11ing ,cll, \\ er'e: incubatcd with 1 0% FBS for d!fferent time:i and Uded fOl I h with orthophosphare. as above. hcfnre: eelb \\ere eollcc:tcd and Iy:ied for immunoprecipitation as de"cribecJ klcn\ Transient Transfection 01' HP\'-It E7 in Cos-7 Cells. COS-! eells were transfected using the DL\E-dc\tran mcthod as describcd pre\iously [22J. Cells were' :iceded on 8 well1vfultitest Slide (M? BiomedicJL Solon, OH l:SA) and grown up to 80~o conflllence Cells were trat:sf'ceted \Iith 5 lg of purified peD:-\A or pcONA-[7 plasmid rha cont1111' the nucleotides 562 to 858 from HPV-16 whteh encodes f,\i the [7 protein (GenBank access AF 47'7385) (a gift frmn Dr. l Bcrumen, Gcnomic Medicine Laboratory. Hospital Gcneral. !\1exieo D.F.). Cells were grown for different periods of time (O, 2, 4. 8. 1 ti, 24. 4:-\ and 72 h I aftcr \\ h id: the cell, \\cre fixed with p-fonnaldebvde and treated f()r imn;unolluorcscence. JS dcscribed hc!c)\\'

Immunoprecipitation and SDS-PACE. eells \\er,~ l\sed 1nd immunoprcc1llilaled as rkserihcd e ,e\\here [23 J. Briefl\. cell monolayers were Iysed in radtOim Tunopreeipi tJtion 'assa\' (RIPA) buffer in lhe preSene!2 of pr')Il~a,;e inh ihltors (Complete mini. Roehe Diagnosties. IndiJnapolis. r'J eSA). Cell hsates \\'ere clcared b\ ecntrifug:nion. and :iUpcrnatanh in~munopreeipitatc:d for '16

h at 4'C \\ilh 3 1g 01' purified TgG anti-E7 mAb:; or 30 ul 01' anli-::- ;'ahbil pohelonal antibodies. Tbe antigen-antihody com;-!:;\ \\as ii"1',unOpreeiDitctcd \\ith Prole;n A-Sepnaro,c ((iE fka!thcc:re. Piseala\\'ay, \.U. eSA) for 2 h at 4"C. Rahbit anli-llll1LISe (oald Protein :\-Sepharose \\as uscd for mAbs The immunopl'eeipitales were dlssoh'ed in Laemmli loading buffer eontaining ] 00 mM dilhiolhreitoi, folJo\\'ed by clcetrophoresis in a l5',,, SOS-polyacry1amide gel. Gels were treated for tluorography \\'ilh the Enlighting reagenl ('-:E1'\ Life Seienees Produets, Boston. \1A. CS.-\) and specifie h:1nds detcet<:d by exposing the gels to X-O\1A T film. Immunoblot. Pll1teins from different eelllines mere extraelcd in R1PA buffer. as oescribed pre\'iously [24J. in the presenee of proleasc inhihilOrs (RoC'he Oiagnostics. Inolanapo:!S,I'\ LS-\I. Total protein extraets 11 mg/ml) \\cre ineuhated with rhe C24 polyelonal antibody and proeessccl for immunopreeipitation as mentioncd bcfore. lmmunopreeipilates \\'ere separarcd on 15~o SDS-PAGE gels and transfcrred lO nitrncelluJose Protean membranes (0.45flm. Whatman International Ud, \.1iddlcsex. CK). as deseribed h~ To\\bin Jnd eo\\'orkers [25J. The membraryes \\'ere blocked with PBS-0.5" (J T\\een containing lOoo ski01 milk for 3(1 min at room tcmpcralur,c. Subscquently. lhe membranes \\'ere incuhaleo with a 1 :200 dilution of antiE7 polyclonal antibodies (l[ \\'ith a uoe dilurion of anti-E7 mAb, in PBS-Ci5" Twcen and 5"" skim m]k, o\ermght. BlDlS were \IJshed \\'ith PBS-O.5"" --\\c:n followed by ineubation for ] h under lhe same conditions \\ilh the secondary goat anri-rabbit or rahbit anli-mouse IgCi antibodies eonjugated with hOlseradlsh pcroxidase (OAKO. (arpinteria. CA. lSA). The :nembranes were cie\eloped according with the ehemiluminc,ecnce kit of Perkin Elmer (Waltham, Massachusells. t'S:\) manufacturer Instructions. \1embranes were exposed w X0\1,\ T film. Indirect Immunofluorescence. Cells al SO".(! eClnilucnce \\erc rinsed \\'ith PBS anJ fixed \\ith 4" u p-formakkhyde ir, PBS al rOOlll temperaturc fOl 20 min fol!(1\\ cd by trcarment \\itb penneabi!izatton buffer [] "o BSA In rBS c(\nt~:1I1ing eilher 3"" Triton X-]OO (for nuclear ,truclureSj ur (,2"" ,aponine (for cylosolie and il'lernal membranes)J rOl' 2i lll1l1 al 4"C. Subsequenlly, celis were incubated wirh anll-r- ~lntlbodics (serum diluted 1: 1 00 for anti-E7 pl)lyclonal Ah, and 2 ng ,ul OfPllrIficd 19G for anti-E7 m.Abs in permeabilizatiu!l buffer) for l6 h al 4"C. Slides \Vere rinsed wilb PBS and ineubated for 2 h al 4"( \\ith anti-rabbit IgG conugateG \\'itl1 AIc\a 488 Igreen) or ~!nti-mouse 19G eonJugated \\11h .\lcxa '94 (red) i dilution 1 :250 and 1 :8(JO. respeetl\ ely. \1olccular Probes. Carlsbad. CA. eSA). Specimens \Iere muuntcd rn 5(10 glyeerol in PBS and visualizecl under the Confoc:,~ \1teroseopc 5] O \1ETA (Car! Zeiss. Massaehusetts. l'S.!" 1 undel' the Plan-'\! e:oJluor 1 OOX /l.3 oil Ph3 Icn I 2-dimcnsional Polyacrylamide Gel Electrophoresis (2D Gels). lmmunoprccipitated E7 protnn \\as treared for isoelcclrofoeusing (lEF). as dcseribed prn iousl\ [2hJ \\:th :iOl1le modifieations. Brietly, E7 immunopreeipilate,; \\crc diss,\]vcd m lEF samplc buffer (9.5 \1 UJea. 2(h, ';P40. 2",. al1lpholincs 3-]0.100 m\1 DTT). JEF u:ied -"o ampbl,llI1cs (GE Healtheare. NJ. l:SA) pH 3-9 in 2 .. ' ml11 diameter 1'1\1\\aen limide gel mbes and fixed to the adaptor t'or ,:,cb eleelwphoresis apparatus (BRL Wrighrs\ill,:. PA lS.'\ I The IEF \\ a' carl'ied for 16 h ar 400 V and elcctrophoresi,

t'ur he seeond dil1lension \I'as run 1S dc'el'ibcd bcfore The geb \Vere lrea ted fOl !l uorography as desenhec pre\ iUl:'!\ lfPV-16 E7/soforms RESULTS Characterization of HPV-16 E7 Protein ,,th Different Antibodies. It has been reponed that E7 prot>:in shows diffcrent moJecular weights and thar the pht'sl'horyJation process is probably one of the causes for thest' diJTerence, Thus, we decided to identify the diffcrent fO!l]]s reponed using polyclonal and monoc lonal antibodies. produced in our labaratorv that can differentiate the \arious isc'f,)rms To th!s end. CaSki cells werc labelcd with "SJ-methlOnine-cy<;teJnc far ] 6 h and thc E7 protein immunoprecipi:Jted \\ith the differcnt antibodies a, describcd in \1aterials and \1ethodolagy. The result in Fig (1) shows an E7 protcn of ] 6 kD" hat was immunopreClpiuted v,ith the polyclona] antibodi,'S C24 and CSCJ. as well as with the mAhs CJ~. 84 and the eommercial EDJ 7. H(meve!'. a ] 7 kDa E7 pn 12111 \\as 0[. ser\ed only \vith thc polyelonal antihodics (e2:; anJ CSe I Fig. 1 A). In contras1. none of these two hal~ds \Vere ohserled in immunopreelpitates from HaCaT eells thm \\'el'C used as negative cantro],; Fig. lA). or with control rahhit (11' mousc scrum (data not shown). Fig. (1). Recogntion of HP\-16 E7 prolein b~ monoclonal and polydonal anti~odies. (A I J-L,CaT (JI I and CaSki le) ('e]!, \\crl' labcled with :"'S]-mcthion:1ccysteine ami imnwlc,'prcclrltacc! wih dffcrcnt antl-E7 pollclonal C:'4. C~l)) ;nd n;\b, (j~. 13..\. C2. DJ 1. C2U. A5, I-::DI-), The arro\\s sho\\ " J6 kIl! band rc(ognlzed b\ all antboc1ICS. al1d a 17 kDa ban dctcctcd ,dv h rnlyc10nal 3I1tlbodie,. m) Cel] extracts \\nc firsl IInmunoprecirtated \\ith he C24 polycloDal amhody. samples trcaled 'ro\'cstcm b10: and tested with polyclonal and mAbs. The :lTTC'" ,r1(1\\'s the ErrotCl!1 01' 16 kDa \,Ie also tcstcd thc c3pacity' of thc o.ntihodic:, :n rccogn:zc the E7 proIcln h\ \\.':~:IC:-:; ~;~11 :1 s: th., (:if~~C'r'_''! ~;l~l:: .. >~'tlL:r

The Oren I 'irolo;y ]ournul, 20011: olume 2 I ~ \\eights of the E7 protcin CQuld he trye re,ult of the differen! eehniqlle, 1I,ed Preliously, Vle have sh()wn th,at the ,ensitility of the direet Westem blot for E7 is 10\\ [27] and therefore. it is necessar~ 10 carry out an immllnoprec:pitation-Wc<;tem blot 10 lisu:.dize the E7 protein from eell extraets. In his way. eel!, were immunopreeipitated flrst with the polyclonal C24 antibody as this antibody' reeognized :2 forms of E7 protein. The precipitates \\'ere ,;eparated on a SDS-P/\GE gel. transferred to Prote:m memoranes and tested \\Jth the diffcrent mAbs as cleserihed pre,iously. The results III Fig lB! shO\vcd thar ~ ol' the 6 mA b:; tested reeognized the ] 6 J..:Da band of the FI protein. This hand \\3S also reeogn:zed ~\ he commereial m.\b ED] - acquired froll1 Santa Cruz. \\hich was 1I,ed as a posltive control. The reeogniti,'n 01' 10 kDa hand is speciflc. as this was notprescnt \\hen thc mAhs \lcrC te,ted \\ith the HaCaT immul1opreeipit;.tes u,ed a.;

negative cnntrob (Fig. lB). In the case of the poh'clonal :mtihodies (C:24 and C::<G), they on ly recognIzed the 16 kDa hand hut 110t the 1-' kDa hand m the Western hlo1. lt 15 possihlc that these antibodles only reeognize a eonformatlonal epitope in the E" protein that is los{ by denaturation during the Western hlot procss EIg. 1 Bl Proressing (lf HPY-16 E7 Protein. Proeessing of E7 protein \\as determined by pulse-ehase aheling of CaSki cells and immunoprceipitated with c,:S9 po]yelonal antihody (Fig. 2), The top panel of Fig 2! 5ho\\s the elcwophoretie analvsis of immunoprecipitates of [-;sS] lllethioninee\steine laheled [7 protein after 15 mn pulse. follo\\'ed hy eha,e of IIp to 6 h, It is elear that 'he E7 protein \Vas syn\hes:ed atter the 15 min pulse as :! 1 -, kDa protein ([7 a) lhat rcm:ined st:lhle for IIp to 1 h anci sub,equently disappeared (Fig. 2) \fter j h of chase .he E7a protein \\as processed to a ] 6 kDa banJ (E7b) and was :;tahle for up to 3 h of cbase Tig. 2) This pattern of bands \\'as not ob,erved 10 the ill1Inunoprecipitates 01' labelcd aCaT cells used as control F ig 2 The ] 5 kDa hand ohsencd in HaCaT cells al 6 h smallcr han the one obsened l'or r- in CaSki cellsl IS anon-spc'eIlc hand as it is not consistently ohsencd il1 e\periment rcpetitiOlb. The hands of the luurography \\'ere scanneJ te> caiculate lhe half-iifc "f thc E7 proteins according to Belle and :o\\'orkers [:2S]. The rc:sults sho\\'ed lhat he haIr ill,' (lf the [7a protein \\'as oniy ~O mm and -'0 min ior the faster !l10\mg band ol' E7h Fig :2. inwcr panel). The prcscnce Of:1 nOl1specific 2] kDa hand in pu15e-ehase eel b (HaCIT :lI1d CaSki) was due to the high lnels of lahelused in tlm kind of experiments. Recognition of 3 Different forros of HP\'.I6 E7 Protein by Differences in Charge. Thc pulse-chase e'periments showed that the E-'a protein was proc,:ssed 10 a faster Illoving form descrihed as E7h. At this pOll1L it ~\'as not c\car if there was an actu;! ehemical modificatioil nf h,' pre,tem. ,)r if the clifferenees \\Tre only due te a contc1mlat!onal ehange. For this reason. it was importan! to deter:l1inc if there was any difference in charge in addIlJOn to the dllfcrcnec in molecular \\cight between thcse .: isofonm el;' E'. ,oelcctrofoeusing of imnunopreeipttate, (Cf;9 antih"d\) oi olcrnight ["SJ methionine-cysteine labekd E7 pnl1ein' \\:1) performed followed by SDS-PAGE gels. j'hc separation 01 the E7 proteins n a pH gradient showed that E7:; and [7h ha\l' diffcrent net charge (Fig. 3. CaSkl. "pnts 2 ami i re,pccli\'c]yL HC)\\C\'CL uIlcxpcctcdl) arl ddditj(1na~ fC\fTi! ot

Fig. (2). Proces~ing and half-life 01' the HPV-lli E7 protein. ILlCaT ami CaSki eells \Vete pulse labekd Wllh (:; -methlol1llcecy~teine fr ] 5 mm anJ chascd t~)r ditT~rcnt TimeS (O. i. 3 and 6 h Uppc:r panel: Cell ex:raels were lmrnunopreclpltatc:c: \\ lth the a:1:iE7 polvclonal U9 antlbod". ,arnplcs separated by SDSP\(jl gel and band, \'lsua]ized by autu-radlography. lhe atr(J\\ s ShO"C0 a 1kDa IE"'a) and a ] 6 kDa (E/b) bands. lhe i 5 kDa banJ lJbserved :11 !laCaT eelb ;s a ~on-speC11ic band as lt is not ClJnSisle:ltly obsen L'li in expcrim~:1t rcpcririol1s. Lo\\cr panel: Sands \Vcr~ -;c:ll1nC'd ar:d p!oucd in a graph to ealcubte he half-life 01' the dl1'fcrerH t,)mb ')( E7 protelll

the [7 prorein in the cxtraet 01' CaSkl celIs lhat was not resolved in onc dimension gels was faund. The tlllrd band ohser\ed ([7al) showeJ a malecu]ar weight of 175 kDa (Fig. 3. C aSki. spot l). Tl1e lEP for caeh form 01' E7 protein "as eaiculated as 4.68 for [7a1 (SpOl ] ). 6.] 8 for the [7a fllrm (Spot 2) and 6.96 ror [7b (spot 3) This diffcrc:nec: in eharge among the 3 lsoi'orms 01' [protein. suggests th~1t thc proteins are !TIodifed in some wavs in rwo diffcrenl qc:ps. Fig. (3). Characterizalion 01' the HPV-16 E7 proteins by IEp. HaCaT and CaSkl eclh \\ere labcled with ["S]-mcthionlIle-cys:ell'lo ano.! immunoprccipia:ed \\ i:h :he C~9 polvelollal a:1tibouv. llce lTIrl1unoprccipitatcs \Vere :-icpar:lt~d in thc first Jirn(t}~lUn in a rI1 gradient from 3-9 for ]6 h. The ,eeona dimension \Ias run In a ] :'" PAGE gel and treated for tluorography. lhe nUlvrers O\'er tlce) CaSki panel show the ioeallzation of the 3 1501'011115 o:' IIP\, -16 1:proteins E7al. Ea and E/Di and these spo" \Vere C,)t obsef\ed rn the HaCaT clJnrol cdi:; Phosphorylation 01' the HP\'-16 E7 Proten. Pre\1. it has be en reportcli that pnsttranslation phosphorylaion oeellrs 1I1 E7 [2l)]. Aeeordingly, we dceidcd tu ldentify \\hich ofthc E7 forms W~b phosphorylatcd. To Ihis erd. eelb were starved for 4:-\ h and subscqucnlly labclcd with (P]orthophosphatc for I h at different times aftcr protein synthesis reactivation by addition of rBS as described in \lateriab and \lcthodology, and separatcd in 15% SDS-P.\GE gel. Thc starvation step m this systcm was introduced 10 avoid incorporation l)f (Pj-ort!wphosphate in Ihe peplide hackbone and to [001; for novel prorein phosphorylation. The result showcd a phosphorylated E7 protein wirh an apparent molccular wc:ight 01' 17.5 kOa (E7a 1.) thar accumulates over rhe time. ho\\e\er. the 16 kOa protein band IE7b) \\as not ohserved at any tllTIe. sllggesting that this E7 isoform IS not phosphorylated IFig. 4). Several reports have shown th~lt E7 cm be phosphorylateJ in ar least :2 differenr sites [21. 30. 3] l. and this eouk e'ulain the presence 01' a broad banJ thar cOllld cont~1in the 17.5 kDa (["'al) and the]7 kDa IE7al proteirE thar \"ere Jbser\eJ m the 20 gels. Fig. (4). phosphorylation o HpY-16 E7 protein. CaSki celh \Vere starved l'or 16 h. labeled with C:P]-onophospha:e anll h'1r\esled el! ditTere11l penod,; ol' lime. Cell extraets ,,\cre immulwpreclpilated with the anl:-[- polyclonal CS9 antlbody or wilh cl),ltrol rabbrr serum. lhe prceipl:ares \Vere separated 111 a 15 u aer\!amlde gel anu "lsuallzcd by autora':lography. The arrow ShOW5 :he phusphorviated E- protell1 of 1-.5 kDa. Cellular localizatan 01' the different forms 01' HP\~-16 E7 protcins. Cp to this point. lhe resulrs shuwcd thar HP\16 c- l'rom CaSki cells is proeessed in 3 ditlerent !TIolcclIlar \\clght fomls. one 01' whieh is phosphorylated (["'~ll). \Ve ,dso Jemonsrrared that potyelonal ano mAbs reeognizcJ diffcrc:nt l'orrns of rhe E7 protein. Taken together. this mforma[ion made us \\onJc:r abollt the eelluiar loealization 01' the 3 isol'omlS ol' E7. Ths. we used the differenr polyelonal and mAbs in cells fixed for immunotluoreseence anJ e()localized rhe E7 protein with antibodies against eellular markc:rs fllr nuelcus (p21). endoplasmie retieulum (ER; calnexin) and Golgi (G\1130). as previously reponed [-'2_'-t] The results 111 Fig. 15\ shov;ed that the po!yelonal anrihoJy CS9 recognized the E7 prorein in the nuelcus as \\ells as in cytoplasm. while the C24 polyc!onal main!y stained the eytplasm. When rnAbs were tested togcther with the eel[ular markers it

was e'. iJent that the ED 17 antibody reeogTI\zed lhe E7 protem in the nuclear compartmel1t as this antiblldy Cll-I'lCJ.iized only with the p21 nuclear marker (Fig. 5. [017. merged). The E7 prolcin rccognized by the 13:1 m\b Cl'localized ordy with he calnexin markcr. \\hich '<Iggc't-; th~ll he E~ form rccognizcd by hi, antibulh 1'; rrc<:1t in the ER (Fig 5. B4. mergcd) Ho\\c\cr. \\hcn lhe D m.\h \\'J' tcstcd a small par! of this 3ntihody c(\-I(){ . .'31l.7\_~d \\l:h thc calncxin markcr of ER and 3 strongcr el)- io, .. ~~;~:z:!!jr)n .;.,; \';3:' obscr\"cd \,"ith thc Golgi CJl\11 ~ rn~lkcr I ='. D]

I:1C~ge'~ 1 Thc~c res:...;'ts d'::.'j71'='I1~:r2~C~~ t}-:;.:: ~b'..' -- -'1",i:C''''' ;c.;

20 The Open 'jr%gv Journu/. :'008, J 'o/ume :' fc:ned wilh the peD\L".-[7 piasmid to invc:stl:,'ate dc l)(llt' expression and loealization 01' E7 protein al dlftcrcnt pc:riOlL of time and lcsted for immunofluorc:seenee, as descrihed n Materials and \'1ethodology, The rc:sults in fig 61 show thal the polyelonal C89 antibody idc:ntified .3 differen patterns of E"7 staining: at firq E7 is recognized b\ 1 () h alkr transkction in the cytoplasm af the cells, By 24 h the E7 protelD hecame concentrated in the periphen of thc lll'c!eus and by 4~ h the tluorescence was on1y ohsencd iniO th,: nuckus anc1 concentrated in the nucleoJus (Fig, 6, C~9) Wilh the Dl 1 antibody identifies E7 in ER and Colf'i) a \eJ'y light cywplasmic fluorcscence signal \\as ohsened hy l6 h after [7 transfection, that became \ery strong h\ 24 h (: g. 6, D 1 1 ) However. the fluorescence signal disarpeared totally hy 48 h after Cos-7 cells transfection, In contras!. the 84 mAb that rccognized onh the E7 protcin !TI the ER sho,\ed a strong fluorescence slgnal cmly at 24 h after E7 transfcction (Fig, 6. B4) Whi1c the ED17 mAb that recof'nlzed E7 protcin only J'aldOl'inos- Torres el u/_ in the nuclcus dentified the prolcin only aft::r 24 h 2nd the !luoresccnce signal was still pre'ient hy 48 h (Fig. 6. ED 7), In these exreriments the presence nf E7 expression \\as nOl detectcd. with any of the different antibodies tcsted. beforc tbe ] 6 h and aer 48 b (data not shov.n), The speeificit\ 01' each one of the antihodies was detemlined by im111urwtluorescence using Cos-7 transfected cells \\irh pcD"J.\ pl2sr1l1d alone as it was done for the pcD'JAE7 plasmid (data not ,ho\\'n). In Fig. (6), L.nly the 24 b time peD"JA control i, .;hown for eaeh one of the antihodies as this \\as the 11111e 01' be highest Ee expressil'n in lhe transfecled cells.

HPV-I6 E' Iwlorms (peAF acetyltransferase, TBP, AP-] f:m1 ti: fa('wrs) [4(1-n 1 and ""ith the seneseence-regulating pratein DEK [41J, aIllong others, All of these target proteins are present In the nUekll". howe\er the reports aoaut the distribution of lh,' 17 lJ1 he cel! have sho\\n that this protein is prescnt

11(11 nnly in llll' cieus but also m the eywplasm, Wc belie\'e that his ddTercn, tial distrioution of E7 in the eelIs is due to th" presenee nj different isoforrns of E7 that are produeed during ItS eelluL1r proeessing. T o test th IS hypothesis. monoe lona] and po]:elonal antibodics that reeoglllze difTerent ep11op"s in the ['7 protein \Vere developed in our ]aooratof\ The re,ults shO\\ed the presenee of 3 lsoforl11s r I IP\'-] (- E7 pwte1n that vary m molecular welght and r[p The,e E7 proleins were deserioed as E7a] (1'7,~ kDa and lE!' ()f .1(,:\). Eia (1kDa and IEP of 6,] 8) and E7b ( 16 kDa and IEP 6,%) The half-li\Ts of the E7 proteins \Vere eakulated lh 50 l11in for E7a and 70 min for [70, These results are eC)J1~;I,tent \Iith a previous repoI1 that sh()wed that the half-lifc uf [7 fWI11 CaSki eells was 55 mm and 70 min in SiHa ee]ls, alth()ugh these researehers only identified one E7 band protein in each eellline [30], Thc differenee oetween Smotkin :md Wettstelll results and ours was the use oi different an11-['7 antioodies and that these reeognized the different iSOrornF of the Eprotem, In the pulse-ehase experil11ents \Se identifled that E7 IS initially syntheslzed as a 17 kDa protein and :Jt.:r apprO\lmately j h, it is processed tn a faster mo\'mg oancl '.11' 1 {, }';Da \\ith a short halfl1fe, These results suggesl th:ll F7 unckrgnes some postnranslationa! modifications tha! generate thc shift in molccu]ar weigh" A kncl\\n modifcat,('11 1'or [- l' the phosphorylation ()f the serine resiclues :;] anJ ~2 [2C), 4~], alth()ugh sCine ..,] is a]so a potential phosph":'\]ati()n site' r 31, 46], Our resu]ts sho\\ed a oroad ph()sphoT\ lateo oand el" a ea:eulated In()lceular weight or ] 7,5 kDll th'l: c"luld he th(' ] 7 }';Da oano that wa' rceogni7ed m the im'11JllClprec'pll:ltlons, The ddTerence in moleeu]ar weight eou]d ['c dlle In thl' change in chliIgc that ma}.;es the pr()tcin tel he r,'t:rclccl 111 he SDS-PACiE gel. Another possibilit: :s that the :Inlhocli"s die! not reeognt7C e1'I'ieicntiy the non-phosphonlalc'd prolL'in l' has been repoI1ed for other mAbs suggestmg that the phosphorylated E7 protcm has el1stme antigen: rrelpenes (conformational changeS, H(me" CL thesc resullS (hd not account for the lower l11()lccular \leigh1 oal1d "I' 16 }';D:: ([70) that was identificd in the Westem hlot a:1d in thc imrnunopreeipitJtions, Ano1hcr as yet llnidcnti1'cd noclific'at](111 could cxist and he thc rcasan for thc prcscncc (11' this L~stcr nloying E7h pro!cin 4CC(lrding to the HP'I'-]h [7 protein seCjLJenee thc caJc~Iateo IEP of this protem is 4,05. howe\er. \\hen this eharaeteristie \\as measured with recomhinant E7 protcm prodlleee! in bactena the IEP ohtamed was 54 [4X], WhlT the IEP o! the I-IP\:-18 E: polcn f'Joduccd in \'itro (LlSln~: rahhit rctlculoeyte Iys:nes) ()r immunoprecipi1ated frc11~' HeLa ,el!s, there \\crc 3 hands orlhe same molcell]ar but d;tTer

en! rEP [49] The researeheis suggcstd that thl' \IJS II typ1cal pattCG1 for phosphc,rylatcd protc:ns c.nd d:l~ \\as siJni!ar to \\'hat we fo~md, tI "v> e\ eL in ()ur eX1eriI1len!' ~1,) IEP, but also he 1110kelll:".r \\'eights oI'the [7 11'7:: 1, E7a ancl E7h. \\ere c11fr2fe:1t Ifl (he:'D Th, l1g11Jr1 sug~:csts that th~ C~9 Jntihndy ;s rccc~g:1'7ng cLffcrcnt epJtopcs (\~' ~he [, isC'f~:Jrr:~ :h3t h2-,'C 7l,:'>t hccn fulj~ e tIara el C'~':ZC\..l the

The Opell ~ 'iroloKY Journal. 2008, ~ lumf' 2 21 ,\t least 2 distinct moieeular weights have been reported ror HPV-16 E7 by 2 dirferent grouP5 [50. 51], However, the ('Xc) antibody \Ias ahk te' rec()gnize isimultaneously 2 forms "f E; bv rad1Oimmun()preeipitation and 3 when 2D ge]s \Il're used, It has oeen shown that E7 s highlyhydrophoble and that thls charackris11c together with its conformational structure pmduce an anol11aious electrophoric hcha\ior, Purified E7 pr()tein IS prl'sent as oligol11ers, and teneis tn bc ,(\Iuhlc ami with a molceular \\eght close 10 the ealcuiatcd m the presenee nI' :\\1 ureJ [] 3, 52), It is possiblc tbt \\e \\ ere ablc to \'lsua]i1e the 3 fo,:n:; c)f E7 in the ,2D gel" not lmh' oecau:;e tlle dfferenee in c!1lrge. but oecause the 1rOteins were solubi]zed in ~y urca, When \\ c eXlcl11incd he localization of the different E'7 r()rl11S in the ee!lllar eornpartments by immunof1uorcscenee staining. it wa:; no:;erved that the E7 protein ""as firS1 ohscr\ed in the ER IS this \\as the fir:,t signa! obsef\'ed \vith the imtbodie:; (] 6 h), SuoseCjuent]y, the E7 pmtein was \ l,ualIzed in the CJolgi compllrtment (16 lO' 24 h) and fina]!: translocated t() the nl1elcus (-+8 h), whc[e i1 probably interaets \\Ith target proeil1s. ,;ueh llS pRb Fro1l1 this wO-k, ir is c Iear that 1he E7b I ] 6 KDli. reeognized by mAos) iso{orm IS local17ecl in the e:toplasm and nuclcus, blJlt the ()ealization of the 17 and ] 7,5 }';Da [-;sof<mm still needs to oe determ!ned as :here are not spccl'-e antioodies availah]e to 'diffcrentiate them, Aeeurding wlh (his proces:;ng 01' the E7 protein, it is po,;siole that the j1rutein eneounters some p()sttranslatl()nal modifieations dllnng the transi through c1ifferelll cel]ular eompartments that :11]0\\ he final local ization ()f the Studying the amn() aed seCjllenee 01' the HP\' -16 [7 pr,1leitL 1t cnuld be observeo hat it eontains a conscnsu:; .sequcnce ror ghcosylation of Asp 29, has 2 sulfation sites (Tyr 23 and 26) lInd :; ph()sphorylatiof1 sites hesidesthe aiready Krwwn Scr i], ,i2 ami 7 i r -+61 tI- today, only phosphorylation has t>een rC1urled for the E7 pr()tcin ami th1S posttranslaton:1i I1wdifi,':ltj()n only ace()unts fOi the differenec in eharge ident:fiecl in lhe ~ difierent forll1s of r-. hut other modifieatiom \\ il: sill !le::d to be studied e) lh:: differenees qn molceuiar \\cigilt identified bv our rc,Iyclonal and mAbs, During the proee"lI1g oI' the E7 proein 111 he eos,7 rran,fceted celk th:: i'iC1tein is locajized at the perll1uelein /one hv 2-+ h afier oeing 'ynthesizcd, This 10ealizatof1 i:; in 1lgieel1lent with 11 prc\ iOllS repon that showed that r- I'll1m 1 IP\' -16 was oosel\ eo lt1 FR, e: toplasm and the nu,' Iear ~11Cmhrane, \\here r- w:!' aoe lo actvate the o;-glueosdasc enz\'me in an allosterie \\ ay [] Suosequently. dur:rg tk e\nrCSSi()l1 01' the E- protein in the Cos- - r:msfceted eell' i 4~ h). (he protcin \Ias \isua!iLed in the nuelcus ano rcL1eal, i7ed in10 the ;ueleolus c,f the cells, These strtJctures la\e ,11S(l bcen reponed lO he present in CaSki cell:;, but onl: dllI lng he CJ2 \1 cell cycle phasc [53) The nue]ear 10ealiz:!1iol1 ,,1' he E7 pr()tein has becn reported in :;e\cral smdie, "1, 53. 541: hO\\-C-YCL this ~rotein does no! contain a COT~S(:n:,u-,; -;cquc-ncc re he trans]()catca nIo thc nuclcus. Thc tran~oc:l llon of the E'7 pro!cll1 UO this eompanment has oeen re 1(]neO lo oe through a nl,n-conventiona] Ran-OTP path\1 a\ ,lIld un intermediary camer protcin secms 10 be in\ Clll ed .5-.+ L :'\ltcrnati\"c!y. in \'j,:ro ch2.racteriz2tiof 01 ["'7

has dCl110nstL.1tcd tha! fOfillation of dinlcs 3nd.tetaj~jCr~ nI ['": dcrc-.nd . ...; nn pH changc~ \vhich aUov,,-- C\pCl~UrC cp(Jpc~ [l.~ l. 1t is also \\-e 11 cstab :ishcd that ce[; '"-'\~;~~j!~irlrncrt, ~~2 :~;;:.'r'J;2n\ir(\nr:le;:ts th(:;! t'~nctio:~ J:

21 Thc Opl'fl "rolo;;y Journal. ~(j()8, j 'o/ume ~ l'erent pHs [55.56], It is posslble then. that fmal ]clcaiintic)n of the dil'l'erent isol'orms of E7 in the ee!] eouid ""C dri\ ed b\ exposure ol' dil'ferent epllopes due 10 oli1'omC-Lation of Eindueed by ehanges In pH. durin1' their transi! [hrou1'h the difl'erent eellular eompartments. The low levels ol' E7 expression ol' the high risk HP\'s. together with the short half-lil'e or this protein. makes it difficult to carry on with bioehemieal experiments helpful to completely eharaeteri?e this protein \10st ol' the inform:ltlOn abtained in an el'fan to oioehemically eharaeteril'e E7 protein has be en generated from reeombinant E- plotein produced in bacteria or produced under in \'rli l'onditions, Howe\er. Sl)mC al' rhe posttranslatlcmal modifu!lions of rhl' prateins are prcdominantly oosened m ellkaryc'lic cclls, The lad: of these modil'ie3rions would ehange the molcelli3r weights as well as the nel ehar1'es of the protein. making it dlffieult 10 uetect the mtermediares or processec~ :'llrms ol' rhe H1'\" -] 6 E7 proteIn thar are reponed in rhls paper as \Ve used eukaryorie eel! culrure,;, In this wa\. 3 fcmns Cl I P\,-] (, EprateIns were identified \\ith polyelonai :lI1l, mAhs tlnt showed difl'ercnt molceular wei1'hts ano chr"crent IEP,; (E7al. E7a and E70i Due lo the raet thal the (Iffcrent aniioodies reeogntzed different epitopes. the :7 pntcin \\,j'; recognized In different eelluiar companmcn1s at dffcrent times during ilS processl11g, The [7 prolcin has hecn recognized a,; a m.l!ii'llnC1111n~1 prOleIn that inleracts \\'Ith 3 high vanef\ of tJr1'et proleins, lt wil] oe ol' great interesl to Identir\ the compktc molccular proecssing ol' lhe E7 pnlein (othcr posttransLJw1nai Jllodificarions) that \"tll a1ic'\\ ~ oener kno\\icdgc ahollr lhe srructure and the blological aeti\ity ol' thls oncogenle :,['(\1e1l1 and its roie in the transformation proee,

Enzyrne-Linked Irnrnunosorbent Assay for Screening of Canine Brucellosis L sing Recornbinant Cu-Zn Superoxide Dismutase

Dcpanmen -Jpp/ed rerlthlf~l' SC'i/:'!!C( and: FeS L!rc} ( enkr "Jr f"irfJ{O:O(lfl Dlseases, ()hill!r,'i nIHTSI(\ 1)' _'!grcu!rlll'i' Olhi 'Clennary _\fC'dIClne. OhlhU" }jof:kaldo 0\0\'555 '/,:;)(11/ {md "/,:J/oruron (:( }Juhhe J/cl1!rh. ('olh).:.'[ 'C!CrUld.!l '\/ea'lclIlc') C.l,'(!Ol1t!sang \'aT/ona/ ! "nn'crSlrL f)(j() GL{,11\ u-/)o/lg. Ji/,Ilt, (.;l'~'(jrl''':!/clm (j()/J -(J / '\;Olllh f,"orco ABSTRAer Hrl1.c(!!/a C(ll1S. a tJ~llltative mtraccllular !1~ilh()gcn_ 1" th:..'-.:all_";il1\ e agLnt nf ~anlflL hruLc]]o"l~ l'hc' d]aVI)()si~ (le canine bn ce]]osis is hascd on hactcrinlogical c'\anlination anc: \crtllngica] Pll'th(\d~ in;.']uding a~glutinaliofl and ~cJ diffusJun tests In 11'; ~tlld: fccombmanl B. cams (u-In surcroxldc clIsmlltasc (\1 11)) \\as lbt:c1 as an anl1g~n ror lhe cnz\'meImKed immunosorbenl assa\' (11.1\.\ 'Dle recombinant SO\) Shll\\cd a sreciic rcactlon \\Ith SCfUI1l mltctcd \\Ith !i cans m Weslem blot1im: and FLlS,\ These re'.uhs Sllg gesl !hal ELlSA using rccombinant SOD IS uscful m scrccning Clf camno' bEllcellclsls KE" \l.'ORDS brucellosis. Cu-ln surcroxldc dlsmlltasc Il.lS,\ Canine orlleellosis is \\idel: dislrioukd afClllnd Ihe \\ nrid and is an important disease due lo lhe tTnllor:lie Ins,es i: causes in animal produetinn. and the risk.s 10 h'.ll1J:lJl heallh 17] Reprodueti\ e disnrders. such as aO(lEtin!b and prem,ture hirths. are the cliniea] signals nllhis oactcria: disebe' in pregnant anima]s. Dj,gnl1Sis nflhe ,lise'ase i, h:l,ed nll h'I-'tcriological examination and stTnill:.:ical tc'sb Ij I "er(li(lg;' cal diagm)o;is is usual" perf(1rmed h\ the tuoe test. rapid slide agglutin:Itilln test. lmcl iCel imlllul",clifflhi,l[! test [-1-61. f1eme\er. agglutinati(l[l lesh SOllk"imes "i,,' false-pnsi:i\ e reactinn, due t() c!'(lss-rea;:liorJ' \\ ith nth;:l p:lthogens. allCi a general strateg: felr e]iminali'l',ueh LT,"Sreacli,'Ils is 10 use puri!iel all1i"CI, \1 ilh e[liw[lc,. \\c prC\ inu,;I: repclflcd lhat a ITlClhod in \1 hi,h LTuck hot salin extracled anti,:.:ens are' coa1ccl on lo ale\ hc~jds \I,111L1 be lIsc1ul in lhe seruln,:.:ieal dia,:.:nnsis llf eaninl' hrucell"si, 1:21. '\l11on,:.: the allligcns <:Xlractd in Ihis \1 l:. Cu-/n \upero'.;ide disrlllltase (SO!)) ShCl\\ed lhe Str(lnc,'S[ :lllli!2enic reac1on [1:2]. ]n the present pape~. 1\ e repnr1 , 'LTcenin:, melhod felr eanine hruee]jnsis lhing an en/lll1e-linkd jml11unosorhent assa\ (FUSA) \Iilh recomhinanl SO)) as antigcn. Th gen encodin;- SO!) \\as amptilicd i'nllll ;:!mllnllsoma! D''::\ isolated jrnl11 jJ conis h: Illcans oi' PCR II ith 5'-GTGATCi:\/\CiTCCTT:\TTT.\T -3' and '-1 I .\TJ CCJ .. \TCAC(jCCGC\(iCC-3 used ,1' Ihe pair l,t' primer,. Tne pwdllet \Ias clnned intn pCn]dn leTI< 11' , l1~ar:l Il i" lne. Shiga. Japan). The lrig!2cr faetm (IT) ,lIlI! 1 ]is-t:gge',l SOD \Ias c\pressed in :he E coli slrain j)1 Su. ;:d il' l'urific~ni()n \\"as pcrf(Jnncd ~h dl.~scrih(.d ~~ lhe n-;~lJllJ!~ll'lUr~'r ('\C1\ agn. Da~msladt. (ermall)). lhe '" utinn \1:1' scparakd lIsing ] 0 o SDS-PA(jj J.nd thcn tr;,l\klTC'd 1(1 --- * C()R.R.~>pu'<:)r""<cr \\\' !'\'1 }..:r:trtmcp! \ ~'tCT1il~t:n.

Public f-kJl1h. f acull:

\gricu]luc:. ~'am;lgu~';l ~ !11\i..'r,,1!\

1 (;-77-1 YoshiJa. YamaguchJ ~~-~~: 5 .aran c-maii\\'ataral ,"j ~arnagll-..:hl-U acjp IIl111lnhilnn-!' memhr,l1les 1 \lillipClrc, Bilicrica, \1.\. lS.A.I. The eftleiene:, lli' transfer \1<1' delamined (,'om,hsie hri]lianl hlue R-:~';[). and then the memhr:lne, \1 ne lested fe)r reacli\i;\ II ith anlibndics in eanine ser~,. lb;: tuhe agg]ulination te,;, \I,h perf(rllld as fe,IIe,,, s. !kaliILIlt]1 alL'd 11 canis QL-] _~ \1 hok-;:;:II antigcn,;, \1 ere' (lbtaine'd frnm ~iasat(l Lah,,;at(lries. [qual \o]ullles lO.' ll1'I"j'lhe Ilh(llc-cl'll dntigens (optieal densit: 01'(1)< at ~'I) ni, : ~llld ,;erum. \\hieh lIad he,'n serial" dilllled 2-illd Ilith 1)llS. \' ere inellh:ltd al )()e tlr 24 hE. .\giClutinatiCln iler, \1 ,'re ,klennined lrom lhe' tina] dilutilln nf scrum ShtHI in" ,(1"" a!.'glutinatinn. Sampks s)I(l\ling liter higher Ihan ](1(1 \\LTe Cilnsidered w he POSili\e .'. 10]. LUSA u,;ing rcenil1 hir::lI1t son \Ias perlrmed as illlo\\\. lo e(lal lhe reCl)nlhinall1 SO)) onw imllllll10plales felr FUSA. )O./iI of il ('11 ug mil Ilas added \O:. 96-\le]1 ]mmuno pIme ('\llne.Roc1l:st;:r. '\1'. l'.S .. \.) and ]di cl\ernight al I'C. Then. he \\ e'l]s \\ ere hj(l~'kl'd using 0.:'% oo\ine Se!'lll11 alollmin (1\S\: lr 30 l11i11. Sera dillllc:d 1200 v.ere adclcd lO lhe \\e'lls. lhe \Ielis \Iere ineubated al 370(' fl'r j hr; \Ine \\;hhe'd, cmd then horseradish pero'.idasc-iahelcd aqti-dllg gel (Santa Cruz BiotechnnJogy. Santa Cruz, C\. U .s.A. 1 \1 as addecL The \\lIs II cre incllhated al ;-e(' 1()f i hr. \1 ere' \1 ashc'J. and J slIbstrate'. 3.3' .).5' -t;rame'th) Ibenzidinc (1 \113 I liquid suostrale '\Skm leJi FLlSA (SIC\1A. "1 !\'ui,;. \lO. l :." .. \.1. \Ias JddeJ. The ahsorh:mel' II as measured a; -1O) nm o: cm :I IS.\ "eader IlllodL:'1 j';ll. 1\iQRlld. I Icrellks. CA. 1 .S.\.l. lel i,lcntif~ ,era infceteJ \\ih Il. canis. \Il' prfe1mled lh, llJh~' tes1 on car;inc StTUnl .,;;anlik;,; (11=22-+ \ ranJllnlly sclcctcd froT11 di_,-g~ , .. \)nsl.'cuti\'cly adrnllcd t() ani11I:!t in (orea h\ !1l1spiral slaff In 1.he' lest. anrih!ld-

i'e,; ;,) c<lnis \\ (Te IkkekJ in .,0 oflhe 224 \erlnll salllplc, : I ;lhlc 11. Rel\)rlbin,1l11 SUD \Ias ll' \\'esler"

10gl'lhl'r \1 ilh serurn lhat hacllc'qcd ,:L: ~-k'~:l:\,--' in lhe Iune ~leL:ll1tjna:jon t::st t0 :l'~! rr rcaclili!:, lhc recomninalll SOD Sh(lll ed Strc1il),.: "c':'::li\il: \\ ilh posiivc sera, hUI not Ilih negati\ e sera (Fig, : 1. lhesc' results indicated lha! the recollloinalll SOl) l'Cactd speci ical]: \Iilh Brucella jnkced sera, \\c hen ClclLTmincd \Ihether FUSA using recolllbinant SOD can be l;>plied W screening for canine bruc!]nsis using he dog serum sal1lpies from horea, Al] scrurn salllpks hal ing aOSllrha!llx 1:1.]ues 01' O\r 0.41 () or under 1i.40li I OD"",) \1 C"e: cithcr POSili\C or negati\ c in The tune agglutination te" rlig, :; j, Si" serum samples ha\ing an aosorhlnce \aluc k.'llll.'cn l1.410 and 0.401 (OD4:) producd a mixed resulL :nd lh llIbe ag:gluinationlest deeeted antibcldies tu H C(1!Ic in 4 01' lhem (Fig. 2), ImlllunOrcacile oands 1\ en: dek.::ted ir)r sera ilaving an aosorhan.::e la! uc 01' (1\ cr 04 I I ( ) D4!) i~ \\ c'ster: o!oning (Tabie 1), lhus, the aosoroance

mc:SUrcT',C:lts in FUSA \Iith rc'comoinant SUD an,J :he i:er, i:l ;11,: tuhe agglulinaion test sho\lt:J a similar ten(L'nc:, lha ELlSA "itll rcomoirialll SOD i" usdUI in e:mine hruceii(lSis, H()\\el 'cr, (Ir lhe' ,erUl1l k1\1 lh,nrhlnce \llucs of lrl1l1nd (1, 4(10 (OD4I1S), i \1 as djj~ ticll! 10 judge \1 heher he: wce posii\e nr negati\ e fm ~,minc hrllce\\os\s, fhc llIhe aggluinatio!l e, u,ing whole B. can!s amigens has oecn used lo diagno;e oruce! losis in dngs in .!apan 11 () 1, H(me\CL i has heen nn!cd hm nonspecitic reactions nccur in a uoe agglulinmion TSt using whole hactcria! ce'11 ami, gens as \Iell as inlhe rapid slide agglutination eS [2J. Mure re'cently, "ilh he' aim n' del elnping a se'rological diagnosis mehod lhat is e::lsicr tu :Krll1:1ll, Ile eOaled lale" oeads with antigens \'"lractcd 11: hOl sa!ine 1rlr USe in he agglll1ination tesl [121, IIU\IC\ er. he 2rude antig:ens e"traced in our lllelhe\d 1\ cre nOl specific ones and thercfre nnl useru] in l highl: sensiti\e serodiagllnsi, mehnd such !lS ELlSA []:21. Sinc,' SOD is a kml\1 n amigl'nie ;Jroein orB. ah()r!/Is 111, ib po(emi,J \alue as a \ accine fr,r kucellosis preveminn and as ) diagmls1ic reagen\ 1'or lh dise,he has oen iIl\S:igatd [S. C), 111, and he reslllTs ine)ur slud: sho\\cd :hat recolllhinalll SUD ,las lIsefullr lhe delCion "fcanine hrlleel!c)sis, FurhL sincl' ELIS .. \ can h 1I,ed fr \ el': ,mall Serunl ,ampic, and klllclk llIany ,alllples al a time, it Ilould be suilaoic j(1] lhe screening (lf \aluahk samples such as hose: fwrn II ild, lile or small animals, !3esides, comelllional serolc)gical and hacleril11c1gical tS1S \1 ould OC needed 10 diagnos ror canine oruce!losis. I:J J\:\ ahs()rh~Hl~ e Jlul'S o; "cr~j U'1lI8 ;t'l'ol11on:lJlt sur The har, nJI-

c~Ite samrle\ \\]lh JnS(I~tJ:Jncl' \'Jlu(':-' (lf 0\("- (i ..j.(( OD . , \'all:c:--; ar(' J\lTages 3nd standard dc\':al]()!i\ \,:'lflplcUk \\;,;:1::; frnll1 ti1l'C Idel111CJI c\rX.TllTlt'nt) i)r, !\'C :.md !\:\ mJc:ltt' a\ew~.:.' ::h'iorhancl' \:~ll:'" (I['en r(lsill\'e control (Infe'--'kdl "e~::L ten ne~at1\ l' clHHrol i ul1mf,\..,t-.:J Sl';-: ah~ :he tL:.' J.g~ IlJt1ll3tin!l test n(>23tl\:.. ,,:.:~, f ll)" SJmplcsl T:tcrs i"or :h( Une agglulln:ll~(\n te,,: (TATl \\"Ith R ;.-oms ;mtl~:~Tl 3f,-' md:.:ated J:,---( SO i. -1 ! () i. and --i : _~:2( 1) '-,-'\r,,-'cl!\'el~