Cancer Res 1998 Potter 3627 32

8/3/2019 Cancer Res 1998 Potter 3627 32

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1998;58:3627-3632.Cancer ResPhilip M. Potter, Judith S. Wolverton, Christopher L. Morton, et al.by the Rabbit EnzymeCarboxylesterase: Influence on Irinotecan (CPT-11) MetabolismCellular Localization Domains of a Rabbit and a Human

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[ CA NC ER R ES EA RC H 5 8. 3 62 7-3 632 , A ug ust 1 5, IW 8]

Cellular Localization Dom ains of a Rabbit and a Hum an Carboxylesterase:Influence on Irinotecan (CPT-11) M etabolism by the Rabbit Enzym e1Philip M . Potter, Judith S. W olverton, Christopher L. M orton, M onika W ierdl, and M ary K. Danks2D ep ar tm en t o f Mol ec ul ar P ha rm ac ol og y, S t. J ud e C hi ld re n's R es ea rc h H os pit al . M em ph is , T en ne ss ee 3 81 05

ABSTRACTEnzyme activation of prodrugs to improve the therapeutic index ofspecific anticancer agents is an attractive alternativ e to current chem oth era py r eg im en s. T his stu dy a dd ress es th e p ote ntia l fo r a ctiv atin g irin o-tecan (CPT-11) with recom binant carboxylesterases (CEs). C Es are a

u biq uito us cla ss o f e nzymes th ou gh t to b e in vo lv ed in th e d eto xifica tio n o fx en ob io tics. T heir p rim ary amin o a cid seq ue nc e in dica tes th at th ese p rotein s s ho uld b e lo ca lize d to th e e nd op la sm ic reticu lu m. B y P CR -m ed ia tedmutagenesis of a rabbit liver and a human alveolar macrophage CEcD NA , expression in C os? cells, and subsequent im munohistochem icallocalization, w e have determ ined that an 18-am ino acid N11,-term i nulhydrophobic signal peptide is responsible for the localization of theseproteins to the endoplasm ic reticulum . By sim ilar approaches, w e haved em on stra ted th at th e COOH-te rm in al a min o a cid s H ILp re ven t sec retio n o f th e p ro tein s fro m the cell. E nzyma tic a ctiv ity w as lo st b y rem ov in gthe N H2-term inal dom ain; however, active enzym e could be detected inthe culture m edia of cells expressing the C OO H-term inally truncatedproteins. Secretion of CEs lacking the six CO OH-term inal am ino acidscould be prevented with brefeldin A, confirm ing that these truncatedenzym es w ere processed and released from cells by endoplasm ic reticu-lum -m ed ia te d e xo cy to sis . D ou ble -tr un ca tio n mu ta nt e nz ym es la ck in g b othNH2 - a nd COOH-term in al seq ue nc es d em on stra ted imm un osta in ing p atterns sim ilar to those of the N H2-term inally truncated proteins and alsolacked C E activity. In all cases, m etabolism of the classic esterase subs tr at e o -n itr op he ny l a ce ta te p re dic te d th e s en si ti vit y o f c el ls e xp re ss in g t herabbit CE to the anticancer agent CPT-11. In addition, the secretedenzym e sensitized C os? cells to this drug, indica ting that protein association w ith a lipid bilayer is not required for substrate m etabolism .INTRODUCTION

CEs3 are a fam ily of ubiquitous enzymes that are thought to beinvolved in the detoxification of xenobiotics ( 1). T hey are present invirtually all organism s ranging from bacteria to m ammals (R efs. 2-4and references therein). B ecause m any of these enzym es are abundantly expressed, num erous proteins have been purified to hom ogeneity, and the cD NA s encoding these C Es have been isolated (5, 6).C haracteristic m otifs that have been identified in the prim ary am inoacid sequence include the B -l and B-2 dom ains, the form er of w hichcontains a serine that is labeled by the reaction of these proteins w ithradioactive inhibitor m olecules, suggesting that it is part of the cataly tic ac tiv e site (7 ). A dd itio nally, an 1 8-res id ue h ig hly h yd ro ph ob icsignal sequence is present at the N H2 term ini of m any of these C Es.It has been presum ed, by hom ology to other signal peptides, that thissequence is responsible for the localization of these enzym es w ithinthe ER of cells (8).

Re ce iv ed 4 /1 0/ 98 ; a cc ep te d 6 /1 7/ 98 .T he costs of publicatio n of th is article w ere defrayed in part by the paym ent o f pagech arg es . T his a rticle m us t th er ef ore b e h er eb y m ar ked a dv ertis em en t in a cc ord an ce w ith1 8 U .S .C . S ectio n 1 73 4 s olely to in dic ate th is fa ct.' Supported by N IH Grants C A-66124. C A-63512, CA -23099, and C A-76202, theC an cer C en te r C ore G ran t P 30 -C A-2 17 65 . a nd b y th e A mer ica n L eb an es e S yria n A ss oc ia t ed Cha ri ti es .2 T o w hom requests for reprints should b e addressed, at D epartm ent of M olecularP ha rm ac ol og y, S t. J ud e C hi ld re n's R es ea rc h H os pi ta l, 3 32 N or th L au de rd al e, M emp hi s,TN 3 81 0 5. P h on e: ( 90 1) 49 5- 34 40 ; F ax : ( 90 1 ) 5 21 -1 66 8: E -ma il : m a ry .d an ks@s tj ud e. or g.3 T h e a bb re vi at io ns u se d a re : CE . C ar bo xy le st er as e: C PT -1 1. i ri no te ca n. 7 -e th yl -1 0-[ 4- (l -p ip er id in o) -l -p ip er id in o] ca rb on yl ox yc am pto th ec in : ER , e nd op la sm ic r et ic ul um :HA , i nf lu en za h ema gl ut in in : HRP , h o rs er ad is h p er ox id as e; o -NPA , w - ni tr op he ny l a ce ta te :SN -3 8 , 7 -e th yl -IO -h y dr ox yc amp to th ec in ; R I PA , r ad io immuno p re ci pi ta ti on a ss ay .

T he generation of reagents for the identification of specific C Es inmammal ia n c el ls h as b ee n u ns uc ce ss fu l, l ar ge ly d ue to t he h ig h d eg re e o fhom ology am ong the fam ily m em bers (8, 9). C onsequently, the in situanalysis of particular C E m olecules has been com plicated. W e haverec en tly iso la te d a ra bbit liv er C E th at ca n a ctiv ate th e p rod ru g C PT -1 1t o g en er at e th e p ote nt to po is omer as e I i nh ib ito r SN- 38 (1 0) . CPT -1 1 h asd emo ns tr at ed s ig nif ic an t a nti tumo r a ct iv ity i n h uman tumor x en og ra ft sg row n in imm un e-d ep riv ed m ic e (11 ), an d th is a ge nt is cu rren tly u nde revaluation for the treatm ent of hum an tum ors (12, 13). To facilitateanalysis of the C E encoded by this rabbit liver cD NA as w ell as a hum anh om olo gu e o f th is g ene , w e p rev iou sly ep ito pe-tag ged a nd e xpre sse dthese proteins in m ammalian cells. W e localized the hum an and rabbitC Es predom inantly to the E R by both immunofluorescence im age cy-t om et ry a nd g ol d- en ha nc ed e le ctr on m ic ro sc op y ( 14 ).P re vio usly p ub lis he d re su lts h av e d em on strated th at e nz ym e/p ro -drug com binations, such as H erpes sim plex virus thym idine kinase/ganciclovir and E scherichia coli cytosine deam inase/5-fluorocy-tosine, suggest that the generation of a bystander effect may bethe rap eutically b en efic ia l (1 5, 1 6). W e re aso ned , th erefo re, th at a CEthat is secreted from the cell m ight result in localized extracellularactivation of C PT -11 and possibly in enhanced tum or cytotoxicity.H ow ev er, be ca use in fo rm atio n c on ce rn in g th e ce llu la r p ro cess in g o fC Es is lim ited, w e undertook a series of studies to determ ine w hetherthe coding sequences of the enzymes could be m odified to alter thesu bce llu la r lo ca liza tio n o f th e p ro te in s. W e th erefo re p erfo rm ed P CR -m edia te d m utag en esis o f p ote ntial en zyme lo ca liza tio n d om ain s o f th ecDNA s. T his study identifies the m otifs responsible for subcellularlocalization of C Es and their retention w ithin the E R and m onitors theeffect of these modifications on the metabolism of both a simplesu bstra te. o -N PA . a nd an tic an ce r pro dru g C PT -1 1.MATERIALS AND M ETHODS

C ell L ines and P lasm ids. C os7 m onkey kidney cells w ere obtained fromAmerican T yp e C ultu re C ollectio n, an d R h3 0 h um an rh ab do my osarco ma cellsw ere derived from a bone m arrow aspirate of a patient at St. Jud e C hildren'sR esearch H osp ital (1 7). B oth c ell lin es w ere g ro wn in 9 0% DMEM co ntain in g1 0% P CS an d 2 mM g lu tam in e u nd er an atm osp here o f 9 0% air: 1 0% CO, at 3 7 C .T he m ammalian ex pres sio n v ec to rs p CIn eo an d p IR ES neo w ere o btain ed fro mP rome ga (Ma di so n, W I) a nd C lo nt ec h (P al o A lto . CA) , r es pe ct iv ely . T he p la sm idco ns tru cts u sed in th is stu dy an d th e C Es e nco ded b y th es e cDNA s are d escrib edin T ab le 1 an d F ig . 1 . E pito pe-tag ged cDNA s en co din g a h um an alv eo lar m ac rop hag e C E an d a rab bit liv er C E h av e b een d escrib ed p rev io usly (1 4).I mm un oh isto ch em ica l R ea gen ts. A nti-in flu en za H A a nd a nti-H A a ntibody conjugated to HRP w ere purchased from Santa Cruz Biotechnology(S an ta C ru z. CA ) a nd B oe hri ng er M an nh eim ( In di an ap oli s, I N) , re sp ec ti ve ly .T he an ti- -tu bu lin an tib od y w as o btain ed fro m IC N (C osta M esa, C A). T ex asR ed an d F IT C-co nju gate d rat im mu no glo bu lin s w ere p urc hased fro m J ack so nIm mu no Res earch L ab ora to ries (W est G ro ve, P A), an d th e DNA stain H oech st33342 w as obtain ed from Sigm a (St. L ouis, M O).C onstruction of D eleted cD NA s. A ll deletions at the 5' and 3' ends of thecD NA s w ere perform ed by PCR using Pfu polym erase (Stratagene, La Jolla,C A). This enzym e w as chosen because it has a m uch low er error rate than T aqpolymerase (18). Briefly, oligonucleotides 24-30 residues in length w ered es ig ned th at created artificial m eth io nin e in itiatio n o r te rm in atio n co do ns .PC R w as perform ed using the tagged full-length cD NA as a tem plate under thefo llo win g co nd itio ns: d en atu ratio n at 9 4 C fo r 4 5 s; an nealin g at th e ap pro -

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M ETA BO LISM O F C PT-ll BY A RA BBIT CA RBO XY LESTER ASEpiiate temperature for 1 m in; and extension at 72 Cfor 1 m in and 30 s. Theinitial 4 cycles of annealing w ere perform ed at 50 C,an d the subsequen t 19cycles w ere perform ed at 56 C. The reduced annealing tem peratures in theearly am plification cycles allo wed the fo rm atio n of the m utation s at the endso f th e tem plate. A ll o lig on ucleo tid es crea ted E ro RI site s w ith in th e am plifiedseq uen ce to allo w su bse qu en t m an ip ulatio n o f th e DNA frag men t. F rag men tswere ligated into pUC9, and the termini were sequenced to confirm theg en era tio n o f th e m uta tio ns .A ssay for C E A ctivity. W hole-cell extracts w ere prepared by sonication ofthe cells in m inim al volum es of 50 m M H E PE S (pH 7.4) on ice, and C E activityw as determined by the conversion of o-N PA to o-nitrophenol as describedpreviously (10, 19). A minimum of four assays were performed for eachsam ple , an d p ro tein co ncen tratio ns w ere d eterm in ed w ith th e B io ra d p ro teinassay reagent (Biorad, Hercules, CA) using BSA as a standard. Enzymeac tiv ities in m ed iu m w ere ca lcu lated p er m illiliter o f sam ple .SD S-P AG E A nalysis and W estern A nalysis. C ell sonicates w ere separated in 8% denaturing acrylamide gels as described by Laemm li (20). G elsw ere either stained in C oom assie B lue R -250, or proteins w ere transferred bye le ct ro bl ott in g t o Imm ob il on -P (M ill ip or e, B ed fo rd , MA ) fo r W e ste rn a na ly sis(2 1). T o facilitate th e d etec tio n o f e pito pe-tag ged p ro tein s p res en t o n Im mob ilo n-P m em bra nes, th e filters w ere in cu bated w ith th e ap pro priate an tib od y,a nd s pe cif ic imm un ore ac tiv ity w as d ete cte d b y c hemi lumin es ce nc e (e nh an ce dc hemi lumin es ce nc e: Am er sh am . A rli ng to n H eig ht s, I L) . T o re mo ve a nti bo die sfrom filters to allow subsequent reprobing, m em branes w ere stripped in 62.5m M Tris (pH 6.7), 2% SD S, and 100 mM 0-m ercaptoethanol at 55Cfor 45m in . T o co nfirm ap pro xim ate e qu al lo ad in g o f g els, m em bran es c on tain in g ce lle xtra cts w ere in cu ba te d w ith a n a nti -/ 3-t ub uli n a nti bo dy .

Im munoprecipitations of Proteins from Cell C ulture M edium . To detect the presence of tagged secreted pro teins, culture m ediu m w as rem ovedfrom actively growing cells and centrifuged at 1,000 x g for 5 m in. Thesupernatant w as transferred to a new tube, and the procedure w as repeatedtw ice. After centrifugation at 14,000 X g for 5 min, 900 /il of medium weredilu ted to 1 m l with 100 dof IOx RIPA buffer [IX RIPA buffer = 50 mMTris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% NP40, and 0.25% sodiumd eo xy ch ola te], an d 2 5 / Io f p ro tein A /G -ag aro se b ead s (S an ta C ru z B io technology) were added. The sam ples were rocked for 4 h at 4C,and the beadsw ere rem oved by centrifugation at 14,000 x g for 5 m in. T wo tgof anti-H Am onoclonal antibody w ere added to the cleared supernatant, and the sam plesw ere in ve rte d re pe ate dly a t 4 Co ve rn ig ht. P ro te in A /G -a ga ro se b ea ds (2 5 / xl)w ere added to the sam ples and rocked at 4 Cfor 6 h. A fter centrifugation at14,000 X g for 10 s, the medium was rem oved, and the pellet w as w ashed w ith1 ml of 1X RIPA buffer. This procedure was repeated six times, the beadsw ere re su sp en ded in 4 0 p .] o f S DS -P AG E lo ad in g b uffer, a nd th e sam ples w ereheated at 95C for 3 m in. Proteins were applied to 8% SDS-PAGE andt ra ns fe rre d t o I mm ob il on -P m em br an es b y e le ct ro bl ott in g. HA -t ag ge d p ro te in sw ere detected w ith an anti-H A-H RP-conjugated antibody and visualized byenhanced chem ilum inescence. C ontrol sam ples consisted of m edium spiked

NameT ab le 1 P las mid a iles crib etl in th is s tu dy

DescriptionpCInco Mammalian expression vectorpIRESneo Mammalian expression vectorpCIHDM CAR pCInco containing human alveolar macrophage CE cDNApC'IHUM IIA pCInco containing tagged human alveolar macrophage CE

cDN A (Ref. 14)pC 'IHUM HAN pC lneo containing N Hylcrm inally truncated lagged hum ana lv eo la r m ac ro ph ag e CH c DN ApC IH UM HA C pC Inco containing C OOH -lcrm inally truncated lagged hum analv eo lar m acro ph ag e C E cD NApC IH UM HANC pC Inco containing both NH r and C OO H-term inally truncatedta gg ed h um an alv eo lar m ac ro ph ag e C E c DN ApCIRABFl. pCInco containing rahhil liver CE cDNApCIRABHA pClneo containing tagged rabbit liver CE cDNA (Ref. 14)pC IR ABH AN pC Inco containing N H,-lcrm inally truncated tagged rabbitliver C E cD NApC IR AB HA C pC 'In eo containing C (X )H -term inally truncated tagged rabbitliver C E cD NApC lR AB HA NC pC lneo containing both N H,- and C OOH-lerminally truncatedla gg ed ra bb it liv er C E cD NApIRESRABFL pIRESnco containing rabbit liver CE cDNApIRE SR ABH A pIR ESnco containing tagged rabbit liver C E cDNA

pC IR A B F L, p C IHUMCARp IRESRABFL

p C IR A B HA , p C IH UMHAp IRESRABHA

N B -2 B -1 TEHIELiosaHA

pC IR ABHAN , pC IHUMHAN E

p C IR A B HA C , p C IH UMHA C

pC lR ABHANC , pC IHUMHANC I KM JF ig . 1 . S ch em atic re pre sen ta tio n o f th e m od if ie d C Es u sed in th is s tu dy . B -1 a nd B -2 .C E m otifs; H . //in diII; H A. influenza H A ep itope tag; N . 18-residue N H,-lentiinalh yd ro ph ob ic s ig na l p ep ti de ; T EHI EL . s ix COOH -t er in in al a mi no a ci ds .

w ith cell extracts expressing the H A-tagged proteins and m edium from cellst ra ns fe ct ed w it h p la sm id s e nc od in g u nta gg ed p ro te in s.T ra ns fectio n o f M amm alia n C ells. C os7 cells (IO 7) w ere ele ctro po ra tedwith 20 igof plasmid DNA in a volume of 200 /Iof PBS using a Bioradelectro po ra to r a nd a c ap acitan ce ex ten de r (B io rad ). O ptim ized co nd itio ns fo relectroporation w ere achieved using 260 V and 960 /xF. A fter transfection,c ells w er e p la te d i nto 7 5-c m2 fl as ks in fr es h m ed ia a nd h arv es te d b y tr yp sin iz a-tio n after 4 8 h . F or stab le tran sfectio n o f R h3 0, c ells w ere electro po rated u nd ersimilar conditions, except that the voltage w as reduced to 180 V. A fter 48 h,transfectants w ere selected in m edia containing 500 /ig/m l G 418 for 10 daysb efo re an aly sis. G ro wth in hib itio n ass ay s w ere p erfo rm ed as d escrib ed p rev iously (10, 22, 23).F luorescence Im munohistochem ical L ocalization of P roteins. C ellsw ere p lated o n g las s slid es an d allo we d to attach o vern ig ht. A fter th e rem ov alo f m ed ia, cells w ere fix ed in 2% p arafo rm ald eh yd e. a nd th e n uclear m em bran ew as permeabilized by incubation in 0.25% Triton X -100. A fter extensivew ashing in PB S, antibody diluted to 100 ng/m l in PB S containing 1% B SA w asapplied to the cells for 1 h. A ntibody localization w as determ in ed by incu bation w ith a T exas R ed or a FIT C-conjugated im mu noglobulin. Cell D NA w asstain ed w ith H oe ch st 3 33 42 to d efin e th e n uclear a rea fo r eac h cell. C ells w ereanalyzed on a Zeiss Axiovert 135TV microscope connected to a SiliconG rap hics C rim so n w ork statio n as d es crib ed p rev io usly (2 4).

RESULTSD eletion of the NI I, i m inai Hydrophobic Signal Peptide ofthe Rabbit and H um an CE. To determ ine whether the residues atthe NH2 terminus of the CEs affected subcellulur localization, wedeleted nucleotides encoding the first 18 am ino acids and created anartificial m ethionine initiation codon in each cD NA by PCR . A fterD NA sequencing to confirm the integrity of the constructs, plasm idspCIHUMHAN and pCIRABHAN (see Table 1 and Fig. 1) weretransfected into Cos? cells. Cell sonicates were prepared for bothWestern a nalys is a nd CE ac tiv ity assa ys. T he Wes te rn a naly sis s ho wnin Fig. 2A indicates that the proteins can be expressed in cells and aresta ble. H ow ev er, ve ry little C E a ctivity w as o bserv ed in c ell so nic ates,indicating that this N H2-term inal dom ain is crucial for enzym e function (T able 2). Immunohistochem ical staining of these cells w ith thea nti-HA a ntib od y demo nstrated d iffu se c yto pla sm ic stain in g co nsistent w ith a failure to localize to a particular cellular organelle. T hisp attern is v ery d iffe re nt fro m th e ty pica l ER stainin g o bserv ed in ce llstransfected w ith the tagged full-length C E cD NA s in pC IH UMHAand pC IR AB HA (Figs. 3 and 4: R ef. 14). N o C E protein w as detected3628



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M ETA BO LISM O F CRT -11 BY A RA BB IT C ARB OX YLE ST ERA SE

HA Ab

Tubulin __ _ __.^

B20 s e cs

5 min

986460369864SO36

986450

36986450

36F ig. 2 . A . W estern analysis of truncated C Es expressed in C os7 cells. W hole-celle xt ra ct s d er iv ed f ro m Cos 7 c el ls a ft er t ra ns ie nt t ra ns fe ct io n w it h t he i nd ic at ed p la sm id sw er e s ub jec ted to im mu no blo ttin g u sin g an an ti- HA an tib od y ( HA ) o r an ti- -tu bu lina ntib od y. B , W es te rn an aly sis o f im mu no pre cip itatio ns o f ce ll c ultu re m ed ia f ro m C os 7c ells a fte r tr an sien t tra ns fe ctio n w ith th e s am e p las mid s. T he m em bran e w as p ro be d w ithan anti-H A-H RP -co nju gated antibo dy and exposed to film for 20 s or 5 m in.

in th e cu ltu re m ed ium a s d ete rm in ed b y Weste rn a nd immu no prec ip itation analyses (F ig. 2B ) or enzym e activity assays (T able 2).D el et ion of t he Six C OO H-t er m inal A mi no A cids of th e R abbi tan d H uman C E. T o d et er m in e w het her t he COOH -t er m in al am in oacids were responsible for sequestering the CEs within the ER andpreventing secretion from the cell, w e rem oved the nucleotides encoding these residues and added an artificial term ination codon to thetagged C E cDNA s by PCR. T his procedure truncated both proteins bysix am ino acids, deleting the T EH IE L residues. A fter confirm ation ofconstruct integrity by DNA sequencing, the cDNA s w ere ligated intopCIneo to create pCIH UM HAC and pCIRABHAC (see Table 1 andFig. 1). Each plasm id w as transfected into C os?, and after 48 h, cellsw ere subjected to W estern analysis and immunohistochem istry, andCE activities were determ ined. The cell culture m edium was alsoassayed for enzym e activity and analyzed by W estern analysis. T able2 indicates the CE activity of cell extracts and cell culture m edium ,and Fig. 2A dem onstrates the presence of the tagged C OO H-term inally truncated proteins in cell sonicates. E nzym e activity w as also

present in the culture m edium (Table 2), and truncated H A-taggedprotein w as detected in aliquots of the sam e m edium by immunopre-c ip it at io n ( Fi g. 2B) . Immun oh is to ch em is tr y o f t he COOH-t ermin al lytruncated proteins confirm ed their localization to the E R (Figs. 3 and4), dem onstrating a sim ilar fine netw ork-like staining pattern to thefu ll-len gth tag ge d CEs. T o c on firm th at th e p ro te in w as b ein g a ctive lysecreted from transfected Cos? cells, we rem oved the m edium andreplaced it w ith serum -free culture m edium . A fter 4 h, aliquots of thism edium w ere rem oved for C E activity assays. A pproxim ately 6- and9 -fo ld m ore CE ac tiv ity w as d etec te d in se rum-fre e m edium h arv este dfrom C os? cells expressing either pC IH UMHA C or pC IRA BH AC ,respectively (T able 2), than in m edium harvested from cells transfected w ith cDNA s encoding either full-length or N H-,-term inallytruncated proteins. Additionally, both the rabbit and human H A-tagged C OO H-term inally truncated proteins could be detected byimmunoprecipitation from the culture m edium (Fig. 2B ).B ref el din T r eat ment of C os? C ell s E xpr essing C OO H-t er m in al ly T r u ncat ed CEs. T o con fi r m t hat ex tr acel lu lar secr et ion of t heCOOH-te rm in all y t ru nc at ed r ab bi t a nd h uman CE s was o cc ur ri ng v ia th eER , w e e xp os ed tr an sie ntl y t ra ns fe ct ed Cos ? c el ls t o 1 0 ju .g /m l b re fe ld inin s er um -f re e m ed ium f or 4 h . B re fe ld in is a f un ga l- de riv ed a nt ib io ti c t ha tcauses reversible loss of the ER in m am malian cells (25, 26). A fterb re feld in tre atm en t, ce ll so nica tes c on ta in ed sig nifica ntly g rea ter C Eactivity (T ab le 2 ) tha n d id un trea te d C os? p CIRABHAC c ells , co nc omitant w ith reduced levels of C E activity in the culture m edia. Immuno-f lu or es ce nc e s ta in in g o f b re fe ld in -t re at ed c el ls d emons tr at ed a pun ct at es ta in in g p at te rn c on si st en t w i th a d is ru pt io n o f ER int eg ri ty (Fi g. 5 /4 ) , a n dimmun op re ci pi ta tio n a nd We st er n a na ly sis o f c ult ur e m ed ia c on fi rm edth e low er lev els of se cre te d CE (F ig. 5 fi).Delet ion of Both the NH 2-ter minal and COOH -ter minal Doma ins. cDNAs encoding CEs lack ing both the 18- residueNH2- term ina lsig nal pe ptid e a nd th e 6 COOH-term in al am in o ac id s w ere co nstruc te db y P CR an d lig ated in to p CIne o to c rea te p CIRABHANC an d pC IHUM -HANC (see T able 1 and Fig. 1). A fter transfection into C os? cells, cellsonicates w ere assayed for C E activity, and tagged proteins w ere analy ze d b y We ste rn a na ly sis . I n a dd it io n, th e p re se nc e o f CE e nz yme i n c ellc ul tu re m ed ium was d et ermin ed b y b ot h a ct iv ity a ss ay s a nd immuno pr ec ip it at io n. F ig . 2A demons tr at es t he e xp re ss io n o f p ro te in s o f t he e xp ec te dsiz e in C os? ce ll ex tra cts, in dica ting tha t th e p ro te in s w ere stab le. H owev er, n o CE en zyme a ctiv ity co uld b e d etec te d in id entica l ex tra cts o r inth e c el l c ult ure m ed ium (Ta ble 2 ). Immu no hi sto ch em is tr y o f c el ls t ra nsfe cted w ith p CIRABHANC o r p CIHUMHANC demo nstrated a d iffu ses ta in in g p at te rn s im il ar to t ha t o f c el ls t ra ns fe ct ed w it h pCIRABHAN andpC IH UMHAN (Figs. 3 and 4).

T ab le 2 C E a ctiv ity in c eil ex tr ac ts a nd c ultu re m ed ia o f tra ns ie ntly trtin .s fec ledCos? ce llsCE a ctiv ity w as d eterm in ed s pec tr op ho to me lr ica lly b y m on ito rin g th e co nv er sio n o f -NPAt o n it ro ph en ol a t 4 20 nm.

CE e nz ym ectivitySampleCell

extract(/mol/min/mg)Media+

serum (48 h)(/xmol/min/ml)Media-

serum (4 h)(fimol/min/ml)Cos?Cos?+Cos?+Cos?+Cos?+Cos?+Cos?+Cos?+Cos?+Cos?+Cos? +pCIneopCIHUMHApCIHUMHANpCIHUMHACpCIHUMHANCpCIHUMHAC

+refpClRABHApCIRABHANpCIRABHACpCIRABHANCpCIRABHAC

+ Bref6.8347.36.5453.55.7825.1785.37.7918.36.80.420.20.318.80.468.414.638.3

.736.2.933.7.8109.837.5N37.432.7188.13.6"1.3}1.8

1.5NDND"3.94.425.24.613.04.14.1.0"" ND . n ot d et er min ed .bP < 0.001 using ANOVA.' B ref, b refeldin treatm en t, 10 fig/m l for 4 h.

3629



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M ETA BO LISM O F CPT-II B Y A RA BB IT CA RB OX YLESTE RA SE

P h a s e DMA An ti - HA

Nega t i veContro l

HA

HAN

HAC

HANC

F ig . 3 . F lu or es cen ce d etec tio n o f tru nca te d ep ilo pe -ta gg ed ra bb it C Es ex pr es se d in C os 7 ce lls . P has e-c on tra st p ho to mic ro gr ap hs an d flu or es cen ce im ag es o f th e s am e ce ll s ta in edw ith th e an ti-H A a ntib od y (H A) to d etec t C Es a nd H oec hs t 3 33 42 ( DN A) a re s ho wn . N eg ativ e co ntro ls a re s im ila r ce lls s ta in ed w ith an is oty pe -m atch ed p rim ary a ntib od y.3630



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M ETA BO LISM O F CPT-ll B Y A R AB BIT C AR BO XY LESTERA SE

F ig . 4 . I mm un of lu or es ce nc e p ho tomi cr og ra ph o fC os 7 c el ls a ft er t ra ns fe ct io n w it h th e in dic at ed p la s-m id s e nc od in g e pi to pe -t ag ge d h um an CE s.

HAN MAC HANC

- b re fe ld in brefeldin

B

98646036

F ig . 5 . A , i mm u no ch em ic al l oc al iz at io n o f t he COOH -te rm in al ly t ru nc ate d h um an CE s i nC os ? ce lls af ter tre atm en t w ith 1 0 f ig /m l b ref eld in fo r 4 h . B , im mu no pr ec ip itatio n o f ce llc ul tu re m e di a h ar ve st ed f rom Co s? c el ls e xp re ss in g t he COOH- te rm i na ll y t ru nc at ed r ab bi t a ndh um an CE s ( pC lRABHAC a nd p CIHUMHAC ) a ft er t re at me nt w ith b re fe ld in ( +B ).

E ff ect of Pr ot ei n T r u ncat ion on CTP-11-med iat ed C yt ot ox ici ty .Because the human alveolar m acrophage CE does not m etabolizeCPT-ll,4 the effect of the NH2- and COOH-terminal deletions onC PT -11 -m ed ia te d c yto to xicity w as d eterm in ed by tran sfe ctin g C os?cells w ith the various rabbit m utant cD NA s. A fter transfection w ithplasm ids pCIRABHA, pCIRABHAN, pCIRABHAC, or pCIR-A BH AN C, H A-tagged proteins w ere detectable in cell extracts byW estern analysis (F ig. 2A ); additionally, C E activity w as present inpCIRABHA and pCIRABHAC cells (Table 2). The IC50 values ofCos7 cells transfected with the various plasmids to CPT-ll areindicated in Table 3. As can be seen, the IC5() v alues for Cos? cellstra nsfec te d w ith p CIRABFL (th e fu ll-len gth u nta gg ed ra bb it live r C E)or pC IRA BH A or pC IR AB HA C are approxim ately 3-20-fold low erthan those of cells electroporated with pCIneo, pCIRABHAN, orpCIRABHANC. These data corroborate the pattern of CE enzym eactivities in these cell extracts as determ ined by the m etabolism ofo-N PA (T able 2). N o sensitization w as seen in cells expressing theh um an alve ola r m acro ph ag e CE (p CIHUMCAR ).

4 M . K . D an ks a nd P . M . P otter . C om pa ris on o f th e e ff icien cy o f C PT -1 1 ac tiv atio n b ya ra bb it an d a h um an c ar bo xy les te ra se fo r u se in en zy me /p ro dru g th erap y, m an usc rip t inpreparation.

B ec au se th e effic ac y o f tran sfe ctio n an d h en ce p ro te in ex pressio ncan vary in transient assays with Cos? cells, we ligated the taggedrabbit cDNA into pIRESneo to generate pIRESRABH A and transfe cted R h3 0 c ells . A s ex pec ted , ce lls e xp re ssing eith er th e fu ll-len gth(pIR ESR AB FL ) or the tagged (pIR ESR ABH A) rabbit C E w ere sensitized to CPT-ll (Table 4). These data confirm that the taggedenzym e m etabolizes C PT-11 to SN -38 in situ.DISCUSSION

T his study presents the novel observations that the NH2-term inalam in o ac id s targ et a ra bb it liv er C E a nd a h um an a lv eolar m acro ph ag eC E to the E R, and that the COOH-term inal residues prevent secretionfrom the cell. R em oval of the 18 hydrophobic N H2-term inal residuesalso resulted in loss of enzym e activity as m onitored by the m etabolism of o-N PA and C PT-11-m ediated cytotoxicity, even though thepro tein w as d ete cted b y Weste rn an aly sis o f c ell e xtrac ts . B eca use, noCE activity was detected in cells expressing the NH2-term inallytru nca te d p ro te in s, w e p resume th at en zyme pro cessin g w ithin th e ERis essential for catalytic activity. A dditionally, because the COOH-te rm in ally tru nca te d se cre te d p ro teins w ere a ctiv e, ass oc ia tio n w ith alip id b ilay er is n ot a re qu irem en t fo r a fu nc tio nal e nz ym e, b ut p ossiblyp os tt ra ns la tio n mod ifi ca tio n, s uc h a s th e a dd iti on o f o lig os ac ch ar id esor the formation of disulfide bonds by isomerases, is required.W hereas the exact purpose of m odification is unclear, it is probablyin volv ed in pro tein fo ldin g. B eca use in hib ito rs of g ly co sy la tio n s uc has tunicam ycin do not interfere w ith transport to the G olgi apparatusor E R, it is unlikely that the addition of these carbohydrate m oietiesis re qu ired for p ro te in tran sp ort thro ug h th ese o rg an d e s (27 ). A dd iti on all y, p ro te in s c an s ti ll b e a cti ve ly s ec re te d fr om c el ls b y e xo cy to sisin the presence of these inhibitors (28). O ur data suggest that m odification by the ER is required to generate active C Es, regardless ofw hether the enzym e is retained in the cell or secreted.R emov al o f t he s ix COOH-t ermin al ami no a cid s, TEH L , r es ult ed insecretion of the protein from cells (Fig. 2B and T able 2). B ecause F CSc on ta in s d ete ct ab le amo un ts o f CE a ct iv ity , a s i nd ic at ed b y t he a ss ay s o fserum -containing m edium (Table 2), w e replaced the m edium w ith am inim al volum e of serum -free DMEM . A fter 4 h, C E activity assaysco nfirm ed th e p re sen ce o f a ctiv e e nz ym e in th e m ed ia, ap prox im ate ly6 -9 -f old g re ate r t ha n t ha t o f th e s ampl es a na ly ze d f rom c el ls t ra ns fe ct edT ab le 3 /C so v alu es o f tr an sie ntlv tr an sf ec ied C os 7 c ells af ter tr ea tm en t w ith C PT -1 1C ells were exposed to C PT -11 for 2 h, 48 h after transfection. A fter 3 days, a tim eequivalent to three cell doublings, the cell num ber w as determ ined. IC 5() values w ereca lc ulate d fr om s ig mo id al d os e re sp on se c urv es u sin g th e G ra ph pad P ris m p ro gra m.

PlasmidpCIneopCIRABFLpCIRABHApCIRABHANpCIRABHACpCIRABHANCpCIHUMCARIC5()

(MM)200.90.956.0282.42160

3631American Association for Cancer ResearchCopyright 1998

on November 8, 2011cancerres.aacrjournals.orgDownloaded from


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M ETA BO LISM O F CPT-ll B Y A RA BB IT C ARB OX YLE STERA SET ab le 4 I CW v alu es o f Ira ns fe cle il R h3 0 c ells a fter tr ea tm en t w ith C PT -1

C ells were exposed to C RT -11 for 2 h, and the cell num ber was determ ined 3 dayslater. T he IC SO v alues w ere calcu lated from sigm oidal dose response curves using theG ra ph pa d P ri sm p ro gr am .Plasm id 1C, >IM)

pIRESneopIRESRABFLplRESRABHA4 1.7 7 .11 .1 9 0. 551 .05 0 .22

w ith t he f ull -le ng th cDNAs. Thi s s ug ge st s t ha t t he se p ro te in s c on fo rm tothe em pirical rules suggested by M unro and Pelham (29), w ho hypothe si ze d t ha t t he e le ct ro st at ic c ha rg e and /o r s tr uc tu re o f t he COOH-t erm in ala mino a cid res id ues ca used p ro te in s to be re ta in ed in th e ER. A lth ou ghth e ex act m ech an ism o f th is re te ntio n is u nclea r, it is a ppa ren t tha t th eseam ino a ci ds p re vent s ec re ti on .B refe ld in tre atm en t of ce lls tra ns ie ntly tra ns fe cted w ith pC IRAB -HAC or pCIHUM HAC resulted in the loss of integrity of the ER andthe accum ulation of the tagged enzym es in discrete vesicles (Fig. 5).In te restin gly, lev els of CE a ctiv ity in th e e xtra cts o f ce lls tre ated w ithbrefeldin increased, presum ably due to the prevention of secretion ofthe enzym e. Sim ultaneously, levels of CE in the cell culture m ediaw ere reduced. T he prevention of secretion of the C Es by brefeldintreatm ent did not result in the inactivation of the protein in the cells,s ug ge st in g t ha t t he g ly co sy la ti on a nd /o r d is ul fi de b on d f orma ti on ( an dhence intracellular activation of the CE) can occur in the Golgiapparatus. Previous reports have indicated that the activity of CE sm ay be dependent on the glycosylation of the protein (30). O ur dataare in agreem ent w ith these studies, indicating that if the rabbit liverand hum an alveolar m acrophage C Es are not targeted to the ER by theNH2 -term in al sig na l p eptide , th en th e pro tein s are in activ e. B eca usethe addition of carbohydrate residues occurs w ithin the G olgi and E R,we presume that glycosylation and/or disulfide bond formation ise sse ntial for th e c orre ct p ro ce ssin g a nd fo ld in g o f th e m atu re p ro te in .In contrast, rem oval of the CO OH -term inal residues does not affectenzym e activity but allow s secretion of the proteins from the cell.In summ ary , re mo val o f th e NH,-term in al s ig nal p eptide resu lte d int he f orma ti on o f c at al yti ca ll y in ac tiv e p ro te in s, w he re as r emov al o f t hes ix COOH-t ermin al r es id ue s r es ul te d i n th e s ec re tio n o f a ct iv e r ab bi t a ndhum an C Es. B ecause the rabbit C E can convert C PT -11 to SN -38 (10),the potential application of the secreted enzym e for an enhanced bys ta nd er e ff ec t w it h CPT -1 1 is u nd er in ve sti ga tio n. F or c el ls e xp re ss in g a nin tr ac el lu la r CE , CPT -1 1 a cti va tio n i s d ep en de nt o n d if fu si on o f t he d ru gacross the cell m em brane for conversion to SN -38. H ow ever, w ith asec reted CE, C PT -1 1 a ctiv atio n sh ou ld o cc ur in the e xtra ce llu la r flu id ,a nd the SN-38 g en er at ed s hould r ea di ly d if fu se i nt o t he s ur ro unding c el ls ,r es ul ti ng i n c yt oto xi ci ty e ve n in c el ls t ha t c an no t a ct iv at e CPT -1 1. We a recu rren tly asse ssin g th e e ffic ac y o f b oth th e ER-loc aliz ed a nd sec retedr ab bi t li ve r CE t o p ro du ce a b ys ta nd er e ffe ct in c ombin ati on w it h CPT -1 1in b oth cells in cu ltu re an d x en og rafts. A dd itio na lly , w e a re g en eratingb ac ulo vir us e xp re ss in g COOH-t erm in all y tr un ca te d CE s t o a ll ow r ap idi so la ti on and pur if ic at io n o f l ar ge amount s o f t he se e nzyme s f or s tr uc tu ra lstudies.ACKNOWLEDGMENTS

W e thank Dr. Pat M cGovern (Pharmacia Upjohn, K alamazoo, M I) for thegifts of C PT -11 and SN -38 and D avid W hipp le for excellent technical assistance. W e also thank C layton N aeve and the C enter fo r B iotechn ology for D NAs eq ue nc in g a nd o lig on uc le ot id e s yn th es is .

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