Retinoic Acid Disrupts the Golgi Apparatus and Increases the Cytosolic Routing of Specific Protein Toxins

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1 Retinic Acid Disrupts the Glgi Apparatus and Increases the Cytslic Ruting f Specific Prtein Txins YuNeng Wu,* Massim Gadina,* Jung-Hwa Ta-Cheng,* and Richard J. Yule* * Bichemistry Sectin, Surgical Neurlgy Branch, Natinal Institute f Neurlgical Disrders and Strke, Natinal Institutes f Health, Bethesda, Maryland 20892; and * Electrn Micrscpy Facility, Natinal Institute f Neurlgical Disrders and Strke, Natinal Institutes f Health, Bethesda, Maryland Abstract. All-trans retinic acid can specifically increase receptr mediated intxicatin f ricin A chain immuntxins mre than 10,000 times, whereas fluid phase endcytsis f ricin A chain alne r ricin A chain immuntxins was nt influenced by retinic acid. The immuntxin activatin by retinic acid des nt require RNA r prtein synthesis and is nt a cnsequence f increased receptr binding f the immuntxin. Vitamin D3 and thyrid hrmne T3, that activate retinic acid receptr (RAR) cgnates, frming heterdimers with mtinid X receptr (RXR), d nt affect the ptency f immuntxins. Amng ther retinids tested, 13-cis retinic acid, which binds neither RAR nr RXR, als increases the ptency f the ricin A chain immuntxin. Therefre, retinic acid receptr activatin des nt appear t be necessary fr immuntxin activity. Retinic acid ptentiatin f immtmtxins is prevented by brefeldin A (BFA) indicating that in the presence f retinic acid, the immuntxin is efficiently muted thrugh the Glgi apparatus en rute t the cytplasm. Directly examining cells with a mnclnal antibdy (Mab) against mannsidase II, a Glgi apparatus marker enzyme, demnstrates that the Glgi apparatus changes upn treatment with retinic acid frm a perinuclear netwrk t a diffuse aggregate. Within rain after remval f retinic acid the cell reassembles the perinuclear Glgi netwrk indistinguishable with that f nrmal cntrl cells. C6-NBD-ceramide, a vital stain fr the Glgi apparatus, shws that retinic acid prevents the flurescent staining f the Glgi apparatus and eliminates flurescence f C6-NBD-ceramide prestained Glgi apparatus. Electrn micrscpy f retinic acid-treated cells demnstrates the specific absence f any nrmal lking Glgi apparatus and a perinuclear vacular structure very similar t that seen in mnensin-treated cells. This vaculizatin disappears after remval f the retinic acid and a perinuclear Glgi stacking reappears. These results indicate that retinic acid alters intracellular muting, prbably thrugh the Glgi apparatus, ptentiating immuntxin activity independently f new gene expressin. Retinic acid appears t be a new reagent t manipulate the Glgi apparatus and intracellular traffic. As retinic acid and immuntxins are bth in clinical trials fr cancer therapy, their cmbined activity in viv wuld be interesting t examine. ETINOIC acid is a mrphgen that defines certain cell fates during develpment and has the ptential t treat cancer by inducing tumr cell differentiatin (34, 39). Retinic acid binds the retinic acid receptr (RAR) 1 causing it t frm heterdimers with the retinid X receptr (RXR) and induce gene transcriptin (3, 19, 45). In Dr. Massim Gadina was partially supprted by the Assciazine Italiana per la Ricerca sul Cancr. 1. Abbreviatins used in this paper: BFA, brefeldin A; C6-NBD-ceramide, N- [7-(4-nitrbenz-2-xa- 1,3-diazle)]-6-aminhexanyl-D-erythr-sphingsine; DT, diphtheria txin; PE, Pseudmnas extxin; RAR, refinic acid receptr; rra, recmbinant ricin A chain; RXR, retinid X receptr; Tfn, transferrin. additin t the well accepted rle f mtinids in transcriptin activatin, sme retinids may have direct effects n cell secnd messengers (9). Mnclnal antibdies cupled t prtein txins, called immuntxins, are being examined in numerus clinical trials fr treatment f cancer and autimmune diseases (36). Subsequent t cell surface binding by the mnclnal antibdy, the txic prtein subunit crsses the membrane surrunding the cytsl t reach the intracellular substrate. Ricin, fr example, enzymatically inactivates ribsmes inhibiting prtein synthesis and causing cell death (8, 30, 31). Hw the hydrphilic enzyme crsses int the cytsl is unknwn althugh endcytsis and intracellular muting t the prper cmpartment are required (17). The Glgi apparatus appears t be ne cmpartment thrugh which ricin must The Rckefeller University Press, /94/05/743/11 $2.00 The Jurnal f Cell Bilgy, Vlume 125, Number 4, May

2 IO t =" 0...,... -I...,......I...--I...-I i g, g pass en rute t the cytsl. Native ricin efficiently rutes thrugh the Glgi apparatus (12, 16, 38, 44) t the cytsl due t galactse-binding sites n the ricin B chain (17). When the ricin B chain is remved and enzymatically active A chain is linked t mnclnal antibdies reactive with cell surface mlecules such as the transferrin receptr, much less effi- cient entry int the cytsl ensues (43). Althugh the immuntxin is rapidly endcytsed via the transferrin receptr, it des nt traffic such that the enzymaticauy active A chain rapidly reaches the cytsl. In additin t ricin B chain, sme drugs that cause alteratins in the Glgi apparatus such as the inphre, mnensin, and lyssmtrpic amines cause a large increase in cell sensitivity t the immuntxins (2). Chlrquine (21), a lyssmtrpic agent, and the ricin B chain (14), have been tested in man fr their ability t imprve the anti-cancer activity f immuntxins. We find that retinic acid alters the Glgi apparatus mrphlgy and causes a tremendus ptentiatin f immuntxin txicity. This activity f retinic acid is nt the result f transcriptin activatin but appears t be a direct effect f retinids n the Glgi apparatus and intracellular traffic. Materials and Methds ffl e, D. A m 0 t$ m M 0 J >,, u) a ' O "1.... ' " "I " C 0 I... I....'!...ml... "11... i... I " I0" 10" I0" 10" I0" 1 Cncentratin Figure 1. Ptentiatin f 454A12-rRA, Tfn-rRA, and rra cyttxicity by all-trans rvtinic acid in U251, K562, and 9L cells. (A) U251 cells grwing in 96-well plates were incubated with increasing cncentratins f 454A12-rRA (circles) r rra (squares) in the presence (e, m) and in the absence (, n) f 10 #M retinic acid in leucine-free RPMI1640 medium; (B) K562 cells grwing in 96- well plates were incubated with increasing cncentratins f 454A12-rRA in the presence (e) r absence () f 10 #M retinic acid; (C) 9L cells were incubated with increasing cncentratins f 454A12-rRA (circles) r Tfn-rRA (squares) in the presence (e, m) r absence (, t3) f 10 #M retinic acid. After 3-h incubatin, ~4C-leucine was added fr anther h. Cells were harvested and cunted as described in Materials and Methds.,-8 Materials All-trans retinic acid was purchased frm Sigma Chem. C. (St. Luis, MO) and Calbichem Crp. (La Jlla, CA); brefeldin A (BFA), 13-cis retinic acid, all-trans retinl, 13-cis retinl, all-traus retinal, 9-cis retinal, and 13-cis retinal all frm Sigma Chem. C.; N-[7-(4-nitrbenz-2-xa-l,3- diazle)]-6-aminhexanyl-d-erythr-sphingsine (Ct-NBD-ceramide) and flurescein labeled gat anti-muse IgG cnjugate frm Mlecular Prbe, Inc. (Eugene, OR); 53FC3 Mab against mannsidase II was a generus gift frm Dr. Lippinctt-Schwartz (Natinal Institutes f Health); 1,25- dihydrxy vitamin D3 and L-3,3',5-trh'dthyrnin(T3) were frm Calbichem Crp.; 454A12-rRA was prepared as described (i1); diphtheria txin and Pseudmnas ex_txin were btained frm List Bilgical C.; transferrin-crm107 was prepared as described by Jhnsn et al. (18) and transferrin-pe was a generus gift frm Dr. Aslak Gdal (Hafslund Nycmed); 2F9-rRA (i1) and M6-rRA (13) were prepared as described; and transferrin-rra was a generus gift frm Dr. Jerry Fultn (Inland Labratries, Inc.). Cell Lines U251 (human glima) cells and MCF-7 (human breast cancer) cells, and 9L (rat glima) were grwn in DMEM cntaining 10% FCS, 2 mm giutamine, 1 mm sdium pyruvate, 0.1 mm nn-essential amin acids, and 10 #g/mi gentamycin. K562 (human erythrleukemia) cells were grwn in RPMI1640 cntaining 10% FCS, 2 mm glutamine, I mm sdium pyruvate, 0.1 mm nn-essential amin acids, and 10 #g/ml gentamycin; L2C cells, a spntaneus transplantable B cell leukemia, were maintained by serial passage in inbred strain 2 guinea pigs as reprted previusly (13). L2C cells were harvested frm the peripheral bld and purified in Lymphcyte Separatin Medium (Orgenn Teknika, Durham, NC), washed three times with HBSS, and resnspended in leucine-free RPMI1640 fr cyttxicity assay. Prtein Synthesis Assay Prtein synthesis inhibitin by DT, PE, ricin, and immuntxins was determined as described previusly (41). Briefly, cells were plated at cncentratins f cells/ml in 96-well micrtiter plates vernight in DMEM cmplete medium. Retinic acid (15 mm in DMSO) and BFA (10 mg/ml in ethanl) stck slutins were diluted int lcucine-free RPMI1640 medium withut FCS t the apprpriate cncentratins. The same amunt f DMSO and/r ethanl were added in the cntrl slutins. After remving the cmplete DMEM medium, cells were incubated in the abve leucine-free RPMI 1640 medium cntainln~ increasing cncentratins f prtein txins with r withut retinic acid r ther retinids and/r BFA The Jurnal f Cell Bilgy, Vlume 125,

3 i to /) /) G),z- >,, t,d -.$ [ A Retin0ic Acid ' ' ' " ' I '' ""q ' " " ' 1 " ' ' " ' I ' ' " " N ' ''"" 0"1310"1210"1110"1010" M6-rRA B Retinic Acid 0 I ' '""'1 ' "'"'1 ' " '"'1 ' '""'i " " ' ' 1 ' '... 10" j "7 2F9-rRA Figure 2. Ptentiatin f 2F9-rRA and M6-rRA cyttxicity by all-trans retinic acid in MCF-7 and L2C cells. MCF-7 cells (A) r L2C cells (B) grwing in 96-well plates were incubated with increasing cncentratins f 2F9- rra with r withut 10/~M retinic acid as indicated. After 3 h, the mediumwas remved and the cells were pulsed with ~4C-leucinc fr anther h. Cells were harvested and cunted as described in Materials and Methds. fr 3 h fllwed by a 1-h pulse with 0.1 ~tci 14C-leucine. Cells were harvested nt glass fiber filters using a PHD cell harvester, washed with water, dried with ethanl, and cunted. The results were expressed as the percentage f ~4C-leucine incrpratin in mck-treated cntrl cells. Vital Staining f the Glgi Apparatus C6-NBD-ceramide was used t stain the Glgi apparatus in living cells (28). Cells were treated with retinic acid either befre C6-NBD-ceramide staining r after stainin. In the case f C6-NBD-ceramide staining after retinic acid expsure, cells plated n cverslips were incubated in leucinefree RPMI1640 withut FCS cntaining 10/~M retinic acid r media cntaining an equivalent amunt f DMSO in cntrl cells. After 2 h, the abve medium was remved and fresh medium cntaining 5 ~tm C6-NBDceramide was added and incubated at 2 C fr 1 h fllwed by an additinal incubatin at 37 C fr 30 rain. After staining, cverslips with labeled cells were munted fr flurescence micmsc W. In the case f C6-NBDceramide staining befre rctinic acid expsure, cells wcm first incubated with 5 t~m C6-NBD-ceramide at 2 C fr rain, the medium was remved and washed twice fllwed by an additinal incubatin at 37 C fr 2 h in the presence r absence f 10 #M retinic acid. Cells thus treated were munted fr flurescence micrscpy. Immunstaining f the Glgi Apparatus fr Light Micrscpy 9L cells were cultured n cverslips in RPMII640 medium with r withut 10 #M retinic acid fr 3 h, and then fixed fr 10 min in 2% frmaldehyde in PBS at 25 C, washed in PBS cntaining 10% FCS. Cells were incubated with mnclnai antibdy t mannsidase II in PBS cntaining 10% FCS and 0.2 % sapnin fr I h, washed with PBS + I0 % serum. Cells were then incubated with flurescein-labcled gat anti-muse IgG in PBS cntaining A m ~ /I /') -,m I,. ft A I I I " 14 10" 13 10" 12 10" 11 10" 10, 1 454A12-rRA ) ,,,,,-,,...,.,,.-,,,,,-,...,,,,- 454A12-rRA Figure 3. The effect f cyclheximide and actinmycin D n all-trans retinic acid ptentiated cyttxicity f 454AI2-rRA t U251 cells. Cells were preincubated fr 30 min with (squares) r withut (circles) 1.2/~g/ml cyclhcximide (A), r 3 h with (squares) r withut (circles) 5.0 ~g/ml actinmycin D (B), and then further incubated with increasing cncentratins f 454AI2-rRA in the presence (e, a) r absence (, D) f 10 #M retinic acid. After 3 h, the medium was remved and the cells were washed with fresh medium three times befre pulsing with 14C-leucine. Prtein synthesis was measured as described in Fig. I. Wu et al. Retinic Acid Disrupts the Glgi Apparatus 745

4 A B 0 ~ t,,,- t J) t- "6 t. 0,, '... I I... I "... I ' " ' " ' t " ' A12-rRA Figure 4. Brefeldin A blcks all-trans retinic acid ptentiated cyttxicity f 454A12-rRA immuntxin. U251 ceils were incubated with increasing cncentratins f 454A12-rRA at 37"C with (e, m) r withut (, D) 10 #M retinic acid in the presence (squares) r absence (circles) f 10 ttg/ml brefeldin A. After 3 h, prtein synthesis was assayed as described fr Fig % serum and 0.2% sapnin fr 1 h, washed three times with PBS/serum, and then with PBS alne. The cverslips were munted in 75 % glycerl. Electrn Micrscpy Ceils were grwn in 4-well chamber slides vernight in DMEM cmplete medium, and then ceils were incubated in leucine-free RPMI1640 medium cntalnln~ 10 btm retinic acid r an equivalent amunt f DMSO in cntrl culture. After 3 h, cells were washed twice and fixed with 2.5 % glutaraldehyde in 0.1 M Na-cacdylate buffer, ph 7.2 fr min at rm temperature, cells then were further prcessed fr electrn micrscpy. Results All-trans Retinic Acid Ptentiates Receptr Mediated Cyttxicity f lmmuntxins 454AI2-rRA, an immtmtxin made by a disulfide linkage between a mnclnal antibdy against the human transferrin receptr (454A12) and recmbinant ricin A chain (rra), was incubated with the human glima cell line, U251. After 3 h, there was n inhibitin f prtein synthesis up t 10-9 M 454A12-rRA. In the presence f 10 #M retinic acid, cell prtein synthesis was inhibited 50% at M, a cncentratin mre than 10,000 times lwer than that which inhibited prtein synthesis in the absence f retinic acid (Fig. 1 A). AT 10 -I~ M immuntxin, prtein synthesis was nly 20% f cntrl after nly 3 h. Human erythrleukemia cells, K562, were als mre than 10,000 times mre sensitive t 454A12-rRA in the presence f retinic acid than in the absence f retinic acid (Fig. 1 B). Hwever, recmbinant ricin A chain by itself was nt detectably ptentiated by retinic acid (Fig. 1 A). 454A12-rRA was nt detectably txic t a nn-target cell line (9L glima) even in the presence f 10 I~M retinic acid (Fig. 1 C). Hwever, transferrin-rra, which can bind rat 9L cells, was ptentiated at least 1,000- fld by 10 ttm retinic acid (Fig. 1 C). Tw ther immuntxins, 2F9-rRA, against a human breast cancer antigen (11) and M6-rRA against a B cell surface iditype antigen (13), were examined fr ptentiatin f txicity by retinic acid. Assayed against their respective target cell lines, MCF-7 and L2C, bth immuntxins were ptentiated at least several rders f magnitude by 10 #M retinic acid (Fig. 2). Thus, f three cell surface receptrs examined, all deliver rra t the cytsl much mre efficiently in the presence f retinic acid than in the absence f retinic acid. In cntrast t the dramatic effect n receptr-mediated txicity f ricin immuntxins by retinic acid, n effect n fluid phase cyttxicity f rra r immuntxin was seen in the presence f retinic acid. Cmparisn f the Effect f AU-trans Retinic Acid, Other Retinids, Vitamin De and Triidthyrnine (Tj) n Immuntxin Ptency All-trans retinic acid binds the RAR causing it t heterdimerize with the RXR and activate gene transcriptin (3, 19, 29, 45). 9-cis retinic acid interacts with the RXR and als stimulates dirner frmatin and transcriptin activatin (1, 15, 23). All ther cis retinic acids d nt bind either RAR r RXR. We examined whether r nt 13-cis retinic acid, which binds t neither RAR nr RXR (1), wuld affect immuntxin activity. Our results indicate that 10 #M 13-cis retinic acid ptentiates immuntxins similarly t all-trans retinic acid (data nt shwn). Amng ther retinids tested, 10 ~M all-trans retinl shws ptentiatin similar t that f all-trans retinic acid, whereas 13-cis retinl, all trans-retinal, 13-cis retinal, and 9-cis retinal d nt seem t increase 454A12-rRA immuntxin ptency at 10 t~m cncentratins (data nt shwn). All-trans retinl has been recently demnstrated t be a ligand f RAR, whereas all-trans retinal des nt bind t RAR (35). Whether the cis-frms f retinl r retinal bind RAR r RXR receptr is nt knwn. Thus there is sme specificity amng different retinids in ptentiating the cyttxicity f immuntxins, hwever retinid receptr binding and the ptentiatin f the immuntxin d nt crrelate. The thyrid hrmne (%) receptr and the vitamin D3 receptr are hmlgus with RAR and als frm heterdimers with RXR t induce transcriptin activatin (19, 45). Up t 1 t~m thyrid hrmne (%) r 1 t~m 1,25-dihydrxy, vitamin D3 had n affect n the sensitivity f t 454A12-rRA immuntxin (data nt shwn). All-tmns Retinic Acid Ptentiatin f lmmuntxins Is Independent f Gene Expressin T test whether r nt new gene prducts induced by retinic acid result in immuntxin sensitizatin, cells were incubated with cyclheximide (Fig. 3 A) r actinmycin D (Fig. 3 B) befre expsure t retinic acid and 454A12-rRA. Fig. 3 shws that neither actinmycin D nr cyclheximide prevented the ptentiatin f 454A12-rRA cyttxicity by retinic acid. Thus the well established transcriptin activatin activity f retinic acid des nt appear t be the mechanism by which retinic acid increases cell sensitivity t ira- The Jurnal f Cell Bilgy, Vlume 125,

5 c to ~e 1 O0 80" " 40' 20' =... i... i Trf-CRM g 0 1 O0 DT -9 may affect muting f immuntxins thrugh the Glgi apparatus, the effect f BFA n the retinic acid ptentiatin f 454A12-rRA was examined. BFA, by inhibiting vesicular transprt frm the ER t the Glgi, results in cllapse f the cis-glg~ apparatus blcking the retrgrade vesicular transprt f vesicles frm the Glgi t the ER (6, 24). BFA was incubated with U251 cells in the presence f 454A12-rRA and retinic acid. Fig. 4 shws that BFA cmpletely blcks the ptentiatin f txicity by retinic acid. This indicates that 454A12-rRA rutes thrugh a BFA sensitive cmpartment, pssibly the Glgi apparatus r the ER, in the presence f retinic acid Trf PE )-9 1 Figure 5. Effects f all-tram retinic acid n the cyttxicity f DT, PE, tfn-crm107, and tfn-pe in U251 cells. U251 cells were incubated with increasing cncentratins f DT, PE, tfn-crm107, and tfn-pe as indicated in the figure with (e) r withut () 10 #M all-trans retinic acid fr 3 h. Prtein synthesis was assayed as described fr Fig. 1. muntxins. This cnclusin is als cnsistent with the rapid time curse f the activatin by retinic acid. After nly 3 h, the immuntxin is 10,000 times mre txic t cells whereas many f the effects f retinic acid n cellular differentiatin ccur days after expsure t retinic acid. Apparently retinic acid has a direct effect n cells that causes the increased sensitivity t immuntxins. AU-trans Retinic Acid Ptentiates Immuntxins at Steps Subsequent t Cell Surface Receptr Binding Immuntxins may be ptentiated by increasing the amunt f immuntxin bund t cell surface receptrs r by increasing the delivery f surface bund immuntxin t the cytsl cmpartment. T examine if retinic acid increases the binding f immuntxins t target cells, cells were incubated with 454A12-rRA fr 2 h at 4 C, and then washed t remve unbund immuntxin. The cells were divided in half and ne half was incubated in the presence and ne half was incubated in the absence f retinic acid fr 3 h at 37 C, and then the cells were pulsed with ~'C-leucine and harvested. The results shw that the ptentiatin f 454A12- rra ccurs even in cells washed befre adding the retinic acid (data nt shwn) indicating that the effect f retinic acid is nt a cnsequence f increased receptr binding. This leaves intracellular ruting and passage int the cytsl as the likely effect retinic acid has upn immuntxin ptency. Brefeidin A Blcks the AU-trans Retinic Acid Ptentiatin f lmmuntxin Txicity The Glgi apparatus has been implicated in the efficient ruting f native ricin t the cytsl thrugh functins f the rich B chain (17). T examine whether r nt retinic acid PE -8 The Effect f All-trans Retinic Acid n the Ptency f Other Prtein Txins and lmmuntxins In cntrast t rra chain immuntxins, which are ptentiated by inphres that disrupt the Glgi apparatus, diphtheria txin (DT) and Pseudmnas extxin A (PE) and their respective immuntxins, are blcked by mnensin, a carbxylic inphre. We examined the effect f retinic acid n the txicity f PE, DT, and transferrin cupled t PE and transferrin cupled t a diphtheria txin mutant, CRM107. Fig. 5 shws that, in cntrast t 454A12-rRA, DT, PE, and transferrin-crm107 (ffn-crm107) and transferrin- PE (tfn-pe) are nt ptentiated by retinic acid. DT and fin- CRM107 are actually inhibited t a small extent by retinic acid. These results are cnsistent with the mdel that retinic acid alters the muting f immuntxins thrugh the Glgi apparatus with sme degree f selectivity. The effect f retinic acid differs markedly frm that f mnensin, hwever. Retinic acid has little effect n DT and PE whereas mnensin blcks DT ver 1,000 times. AU-trans Retinic Acid Alters the Glgi Apparatus Mrphlgy Visualized by Immunstaining with an anti-mannsidase II Mnclnal Antibdy and by Vital Staining with Ct-NBD-Cemmide Immunstaining f the Glgi apparatus with a mnclnal antibdy against the Glgi marker, mannsidase II, shws that retinic acid causes a marked perturbatin in the Glgi apparatus (Fig. 6 b). In cntrl 9L cells the Glgi apparatus has a typical perinuclear netwrk appearance (Fig. 6 a). After treatment f 9L cells with 10/~M retinic acid the Glgi apparatus becmes clumped and diffuse with n perinuclear distributin. Upn remval f the retinic acid the typical perinuclear distributin f the Glgi apparatus reassembles by rain (Fig. 6 c). Thus retinic acid causes a reversible disslutin f the perinuclear Glgi netwrk when bserved with an anti-mannsidase H antibdy. C~-NBD-ceramide, a flurescent dye, is anther pwerful tl t study the structure and functin f the Glgi apparatus in living cells (28). We examined Ct-NBD-ceramide staining f the Glgi apparatus in U251 cells in the presence and absence f retinic acid (Fig. 7). The Glgi apparatus in cntrl cells (a) shws a perinuclear appearance as previusly reprted (28). In cells treated with 10 ~M retinic acid fr 2 h there is a dramatic inhibitin f Glgi flurescence (b). If the Glgi apparatus is stained first with C~-NBDceramide, and then incubated with retinic acid (Fig. 7 d) r withut (Fig. 7 c) fr 2 h, cells shw a dramatic decrease in flurescence labeling. Thus retinic acid disrupts the nr- Wu et al. Retinic Acid Disrupts the Glgi Apparatus 747

6 Figure 6. All-trans retinic acid treatment causes redistributin f Glgi apparatus stained with mab against mannsidase II. 9L cells grwn n cverslips in leucine-free RPMI1640 medium were treated with (b and c) r withut (a) 10/zM retinic acid. After 2 h, the medium was remved and the cells were washed twice. Cells were then either fixed in 2% frmaldehyde (a and b) r incubated further in DMEM cmplete medium fr min (c), and then fixed in 2% frmaldehyde. Cells were incubated with mab against mannsidase II in PBS cntaining 10% FCS and 0.2% sapnin fr rain, and washed. Cells were then incubated with flurescein-labeled gat anti-muse IgG in PBS cntaining 10% FCS and 0.2% sapnin fr anther rain. Cells were washed and munted in 75% glycerl. The Jurnal f Cell Bilgy, Vlume 125,

7 Figure 7. All-trans retinic acid treatment either prevents r disrupts the specific vital staining f the Glgi apparatus with C6-NBDceramide. U251 ceils grwn n glass cverslips in leucine-free RPMI1640 medium were treated with (b) r withut (a) 10 ~M retinic acid. After 2 h, the medium was remved and the cells were washed twice. Cells were then incubated in the same medium with 5 ttm tlurescently labeled C6-NBD-ceramide in the absence f au-trans retinic acid at 2 C fr 1 h, washed twice, and incubated fr 30 min at 37 C. Cells were carefully munted n glass slides and phtgraphed under a flurescent micrscpe. In c and d, cells were first stained with 5 ttm C6-NBD-ceramide, washed twice, and further incubated at 37 C in the presence (d) r absence (c) f 10/~M all-trans retinic acid fr 2 h withut C6-NBD-ceramide. Cells thus treated were munted and phtgraphed as described abve. mai Glgi apparatus when examined with the vital dye, C6- NBD-ceramide. AU-trans Retinic Acid Treatment Causes a Reversible Disappearance f the Glgi Apparatus Observed by Electrn Micrscpy T further examine the status f the Glgi apparatus in retinic acid-treated cells we used electrn micrscpy. Retinic acid treatment f U251 cells crrelated with a cmplete disappearance f nrmal Glgi cisterna and the appearance f large perinuclear vacules (Fig. 8). Retinic acid caused a similar disappearance f the Glgi apparatus and vaculizatin in 9L ceus (Fig. 9 B). Upn remval f the retinic acid, nrmal Glgi stacking reappeared and the swllen vacules disappeared within rain. (Fig. 9 C). These results indicate that the vaculized structures may at least partiaily be cmpsed f dilated Glgi apparatus. Mnensin causes massive dilatin f the Glgi apparatus (22) similar t the appearance f retinic acid-treated cells and als causes ptentiatin f rra immuntxins. The effect f retinic acid n the Glgi may relate t the mechanism f immuntxin ptentiatin. Discussin All-trans retinic acid selectively increases the ptency f certain immuntxins, rra cntaining immuntxins, via three different receptrs, n several different cell lines, are ptentiated by retinic acid whereas immuntxins with diphtheria txin and Pseudmnas txin are nt. Thus the Wu et al Retinic Acid Disrupts the Glgi Apparatus 749

8 Figure 8. All.tram retinic acid treatment causes disappearance f the Glgi apparatus and appearance f perinuclear vaculizatin. Human cells grwn in 4-well chamber slides were incubated in leucine-free RPMI1640 medium with r withut 10 ~M retinic acid, after 3 h, cells were washed twice and fixed with 2.5% glutaraldehyde in 0.1 M Na-cacdylate buffer, ph 7.2 fr min at rm temperature, cells then were further prcessed fr electrn micrscpy (M, mitchndrium; G, Glgi apparatus; N, nucleus; thick arrw, vaculized structures). A, cntrl cell (x 10,000); B, retinic acid-treated cells (x 10,000); C, cntrl cells (x 20,000); D, retinic acid-treated cells (x 20,000). effect seems t be independent f the cell surface receptr yet specific t the txin. Only receptr mediated pathways f intxicatin appear t be affected by retinic acid. rra alne, and nn-b'mding immtmtxins are nt detectably ptentiated by retinic acid. The sensitizatin f cells t immtmtxins by retinic acid is cmpletely blcked by BFA. BFA blcks the vesicular transprt frm the ER t the c/s-glgi apparatus causing a cllapse f the cis-glgi and a terminatin f the retrgrade vesicular transprt frm the cis-glgi back t the ER (24, The Jurnal f Cell Bilgy, Vlume 125,

9 Figure 9. All-trans retinic acid caused vaculisatin disappears after remving the drug. Rat 9L cells cultured in 4-well chamber slides were incubated with (B and C) r withut (A) 10 ~M retinic acid. After 3 h, cells were washed twice and either fixed (A and B) with glutaraldehyde as described in Fig. 8 r further incubated in cmplete DMEM medium fr rain (C) befre fixing. Cells were then prcessed fr electrn micrscpy (G, Glgi apparatus; M, mitchndrium; N, nucleus; thick arrw, vaculized structures; x 12,000). 25). The blck f the retinic acid ptentiatin by BFA suggests that retinic acid stimulates transprt f the immtmtxins thrugh the c/s-glgi, pssibly t the ER en rute t the cytsl. BFA has als been shwn t affect endsmes, lyssmes, and the trans-glgi netwrk (26, 33), and we cannt ascertain at this time which f the cellular effects f BFA cause the blckage f immuntxin ptentiatin by retinic acid. Retinic acid seems t be having an effect n cell sensitivity t txins smewhat like that f lyssmtrpic agents such as mnensin. As mnensin disrupts intraceilular traffic and causes mrphlgic alteratins in the Glgi apparatus (22), we examined retinic acid-treated cells by immunstaining with an anti-mannsidase II Mab, with the vital Glgi stain, Ct-NBD-ceramide, and by EM. Examinatin f the Glgi apparatus with a lipid that specifically stains the Glgi in living ceils (28), flurescent C6-NBD-ceramide, shws that retinic acid treatment caused a dramatic disappearance f the Glgi apparatus staining. This is nt because f decreased C6-NBD-ceramide uptake since treatment with retinic acid fllwing C6-NBD-ceramide staining als causes a dramatic decrease in flurescence intensity f the Glgi apparatus. Immunstaining f mannsidase II demnstrates the disruptin f the perinuclearly lcated Glgi apparatus by retinic acid treatment, and this disruptin is readily reversible upn remval f the retinic acid. Electrn micrscpy als shws a cmplete disappearance f the Glgi stacking, and the appearance f a perinuclear vaculizatin upn expsure f ceils t retinic acid. This vaculizatin is reversed and nrmal Glgi apparatus reappears after remval f retinic acid indicating that the vaculized structures are derived at least partially frm the swllen Glgi apparatus. The vaculizatin seen by EM caused by retinic acid in tw different cell lines (U251, 9L) resembles that caused by mnensin (22). Hwever, the results f Ct- NBD-ceramide staining f retinic acid-treated cells are ppsite t thse seen in mnensin-treated ceils. With mnensin, C6-NBD-ceramide staining f the Glgi is mre intense and punctate (27) whereas retinic acid eliminates C6-NBD-ceramide staining. BFA causes diminutin f the Glgi apparatus by EM and with NBD-ceramide in apprpriate cell types (5, 20). Thus, the effects f retinic acid n the Glgi presents a new pattern relative t that seen with previusly described drugs. The effects f retinic acid n ricin, diphtheria txin, and Pseudmnas extxin are cnsistent with this mdel that retinic acid alters the endcyttic ruting in cells. Native ricin, mdeccin, and abrin txicity are blcked a small amunt by 10 pm retinic acid in HeLa cells and Ver cells (37). Retinic acid als prtects U251 cells frm ricin txicity (data nt shwn). These results and the ptentiatin f rra immuntxins by retinic acid are similar t the effects seen with lyssmtrpic amines and inphres. Native ricin cntains a B chain that is thught t use a galactsebinding functin, intracellularly, t rute the txin thrugh the Glgi apparatus t reach the cytsl (17). The galactsebinding activity may allw ricin, in the trans-glgi, t bind t KDEL receptr-like glycprteins that cycle t and frm the cis-glgi and the ER (32, 40). Immuntxins that lack a B chain are much less ptent, apparently due t a deftciency in intraceilular ruting, rra immuntxins may recycle thrugh the trans-glgi back t the cell surface repeatedly in the absence f a B chain and disruptin f the Glgi with mnensin r retinic acid may allw trans-glgi t cis- Glgi mvement, sensitizing the ceil t the txin. Retinic acid, by disrupting the Glgi, may slightly disrupt the efficient B chain mechanism f Glgi transprt resulting in a small inhibitin f native ricin txicity while ptentiating the inefficient ricin A chain immuntxin ruting. Diphtheria txin requires a lw intravesicular ph fr the txin B chain t mediate membrane transprt t the cytsl (7). Endsmes r lyssmes may be the ptimal intraceilu- Wu et al. Retinic Acid Disrupts the Glgi Apparatus 751

10 lar site fr DT entry int the cytsl and, as BFA des nt inhibit DT, there is n evidence that ruting t r thrugh the Glgi is needed fr DT txicity. Mnensin and lyssmtrpic amines blck DT presumably simply by neutralizing lw ph in endsmes and lyssmes. Retinic acid des nt inhibit the intracellular ruting f DT t these cmpartments nr des it appear t alter the intracellular ph f these cmpartments frm which DT enters the cytsl. PE, like DT, is blcked by lyssmtrpic agents but appears t rute thrugh different intracellular cmpartments n its way t the cytsl (10). PE cntains a KDEL-Iike COOH-terminal sequence that can bind the KDEL receptr in the Glgi and is prpsed t rute PE ut f the Glgi t the ER fr efficient transprt t the cytsl (4). Cnsistent with this mdel BFA blcks PE txicity (42). The inhibitin f PE by mnensin may result frm its affect n the Glgi apparatus. Retinic acid had little affect n PE arguing that retinic acid is very specific in its ceuular perturbatin r that PE is already adept at Glgi t ER transfer and retinic acid des nt further facilitate this step. Even when different txins enter cells via the same receptr they are affected differently by retinic acid. Tfn-PE and tfn-crm107, tw immuntxins that traffic int cells via the transferrin receptr, are nt affected by retinic acid whereas tfn-rra and 454A12-rRA, rra immuntxins that use the transferrin receptr, are strngly ptentiated. The ptentiatin f immuntxins by retinic acid and mnensin, hwever, shws sme significant differences. Retinic acid has little affect n DT r PE whereas mnensin strngly blcks bth DT and PE, and BFA can blck retinic acid mediated immuntxin ptentiatin but nt mnensinmediated immuntxin ptentiatin (data nt shwn). Thus, similar t the results frm mrphlgy studies by EM, immunstalning and C6-NBD-ceramide staining, retinic acid shws sme characteristics f immuntxin ptentiatin similar t mnensin and sme very different frm mnensin. The effect f retinic acid n immuntxin activity is nt blcked by cyclheximide r actinmycin D indicating that the retinic acid directly affects cells and des nt functin via inductin f new gene expressin. Furthermre, examining a series f agents that bind r heterdimerize members f the retinic acid receptr, e.g., all-trans retinic acid, alltrans retinl, thyrxine (T3) and vitamin D3, and retinids that d nt bind the receptrs, e.g., all-trans retinal, 13-cis retinic acid, and ther cis-frms f retinl and retinal, shws a lack f crrelatin between immuntxin activatin and RAR r RXR receptr interactin. Thus the well established transcriptin activatin activity f retinic acid des nt appear t be the mechanism by which retinic acid increases cell sensitivity t immuntxins. The mrphlgical effects f retinic acid n the Glgi apparatus as shwn by vital staining and by EM study likely relate t the mechanism f immuntxin ptentiatin. The mlecular mechanisms f this new activity f certain retinids remains t be discvered. We thank Dr. Peter G. Pentchev and Dr. Katherine Wd fr many helpful discussins; and Ms. Patricia Jhnsn and Ms. Virginia Tanner fr their technical assistance. Received fr publicatin 22 December 1993 and iu revised frm 7 March R ef erell~.e$ 1. Allenby,G, M. T. Bcquel, M. Saunders, S. Kazmer, J. Speck, M. Rsenberger, A. Lvey, P. Kastner, L F. Gripp, P. Chambn, and A. A. Levin Retinic acid receptrs and retinid X receptrs: interactins with endgenus retinic acids. Prc. Natl. Acad. Sei. USA. 90: Casellas, P., B. J. P. Burrie, P. Grs, and F. K. Jansen Kinetics f cyttxicity induced by immuntxins: enhancement by lyssmtrpic amines and carbxyllc inphres.,l Bil. Chem. 259: Chambn, P., A. Zelent, M. Petkvich, C. Mendelshn, P. Lery, A. Krnst, P. Kastner, and N. Brand The family f retinic acid nuclear receptrs. In Retinids: 10 Years On. L-H. Sanrat, editr. Karger/Basel Chaudhary, V. K., Y. Jinn, D. L FitzGerald, and I. Pastan Pseudmngs extxln cntains a specific sequence at the carbxyl terminus that is required fr cyttxicity. Pre. ~Carl. Aead. Sd. USA. 87: Cben, C. H., Y. Kuwazuru, T. Yshida, M. Nambiar, and H. C. Wu Islatin and characterizatin f a brcfcldin A-resistant mutant f mnkey vet cells. Exp. Cell Res. 203: Dms, R. W., G. Russ, andj. W. Yewdell Brefeldin A redistributes resident and itinerant G154 prteins t the endplasmic reticulum. J. Cell Bil. 109: Draper, R., and M. Simn The entry f DT int the mammalian cell cytplasm: evidence fr lyssmal invlvement. 2". Cell Bil. 87: End, Y., K. Mitsul, M. Mtizuki, and K. Tsura The mechanism f actin f ricin and related txic lectins n enkaryti ribsmes..l Bil. Chem. 262: Evaln-Brin, D., F. Raynaud, S. Tumier, A. Plet, and W. B. Andersn Retinic acid and cellular signal transductin. In Rctinids: 10 Years Oll. J.-H. Saurat, editr. Karger/Basel Fitzgerald, D., and I. Pastan Pseudmnas extxin and derived cnjugates: interactins with mammalian cells. In Intracellular Trafficking f Prteins. C. J. Steer and L A. Hanver, editrs. Cambridge/ England Frankel, A., D. Ring, F. Tringale, and S. Hsieh-Ma Tissue distributin f breast cancer-assciated antigens defined by mnclnal antibdies..l Bil. Respnse Mdif. 4: Gnatas, N., A. Steiber, S. Kim, D. Graham, and S. Av~. 1975~ Internalizatin f neurnal plasma membrane ricin receptrs int Gl54 apparatus. Exp. Cell Res. 94: Gregg, E. N., S. H. Bridges, R. J. Yule, D. L. Lng, L. L. Hustn, M. J. Glenule, F. K. Stevensn, and I. Green Whle ricin and recmbinant ricin A chain iditype-specific immuntxins fr therapy f the guinea pig I.~C cell leukemia. J. Immunl. 138: Grssbard, M. L., A. S. Freedman; J. Ritz, F. Cral, V. S. Gldmacber, L. Elise, N. Spectr, K. Dear, J. M. Lambert, W. A. Blattler, L A. Taylr, and L. M. Nadler Sertherapy f B-cell neplasms with anti-b4-blcked ricin: a phase I trial f daily blus infusin. B/d. 79: Heyman, R. A., D. J. Mangelsdrf, J. A. Dyck, R. B. Stein, G. Eicbele, R. M. Evans, and C. Thaller Cis retinic acid is a high altiulty ligand fr the retinid X receptr. Cell. 68: Hudsn, T. H., and F. G. Grill Brefeldin A enhancement f ricin A chain immuntxins and blckade f intact ricin, mdeccin, and abrin..l. Bil. Chem. 266: Jhnsn, V. G., and R. Yule Iatra~llular muting and membrane translcatin f diphtheria txin and ricin. In lntracellular Trafficking f Prteins. C. J. Steer and J. A. Hanver, editrs. Karger/Basel Jhnsn, V. G., D. Wilsn, L. Greenfield, andr. J. Yule The rle f the diphtheria txin receptr in cytsl translcatin..l Bil. Chem. 263: Kliewer, S. A., K. Umesn, D. J. Mangelsdrf, and R. M. Evans Retinid X receptr interacts with nuclear receptrs in retinic acid, thyrid hrmne and vitamin D3 signalling. Nature (Lad.). 355: Ktistakis, N. T., M. G. Rth, and G. S. Blm ~1 cells cntain a nnditfnsible, dminant factr that makes the G154 apparatus resistant t brefeldin A..L Cell Bil. 113: Laurent, G., J. Pris, J.-P. Farcet, P. Carayn, H. Blythman, P. Casellas, P. Pncelet, and F. K. Jansen Effects f tberapy with TI01 ricin A-chain immantxin in tw leukemia patients. B/d. 67: Ledger, P. W., N. Uchida, and M. L. Tanzer Immuncytchemical lcalizatin f prcllagen and fibmnectin in human fibmblnsts: effects f the mnvalant inphre, mnensin. J. Cell Bil. 87: Levin, A. A., L. J. Sturzenbecker, S. Kazmer, T. Bnsakwski, C. Huseltn, G. Allenby, J. Speck, J. Kratzeisen, M. Rnsenberg, A. Lvey, and J. F. Gripp Nature (Lad.). 355: Lippinctt-Schwartz, J., L. C. Yuan, J. S. Bulfacin, and R. D. Klansner Rapid redistributin f 0154 prteins int the ER in cells treated with brefeldin A: evidence fr membrane cycling frm 0O154 t ER. Cell. 56: Lippinctt-Schwartz, J., J. Glickman, J. G. Dnaldsn, J. Rbbins, T. E. Kreis, K. B. Seamn, M. P. Shectz, and R. D. Klansner Frsklin The Jurnal f Cell Bilgy, Vlume 125,

11 inhibits and reverses the effects f brefeldin A n Glgi mrphlgy by a camp-independent mechanism. J. Cell Bil. 112: Lippinctt-Schwartz, J., L. Yuan, C. Tipper, M. Amherdt, L. Orci, and R. D. Klansner Brefeldin A's effects n endsmes, iysnsmes, and the TGN suggest a general mechanism fr regulating rganalle structure and membrane tralfic. Cell. 67: Lipsky, N. G., and R. E. Pagan Intracellular translcatin f flurescent sphinglipids in cultured flbrblasts: endgenusly synthesized sphingmyelin and gluccerebrside analgues pass thrugh the Glgi apparatus en rute t the plasma membrane. J. Cell Bil. 100: Lipsky, N. G., and R. E. Pagan A vital stain fr the Glgi apparatus. Science (Wash. DC). 228: Mangelsdff, D. J., E. S. Ong, J. A. Dyck, and R. M. Evans Nature (Lend.). 345: Olsnes, S., and A. Pihl Different bilgical prperties f the tw cnstituent peptide chaing f ricin, a txic prtein inhibiting prtein synthesis. Bichemistry. 12: Olsnes, S., K. Sandvig, K. Refsnes, and A. Phil Rates f different steps invlved in the inhibitin f prtein synthesis by the txic lectins abrin and ricin. J. Bil. Chem. 257: Pelham, H. R. B Cntrl f prtein exit frm the endplasmic reticulure. Annu. Rev. Cell Bil. 5: PeLham, H. R. B Multiple targets fr brefldin A. Cell. 67: Petkvich, M Regulatin f gene expressin by vitamin A: the rle f nuclear retinic acid receptrs. Ann. Rev. Nutr. 12: Repa, J. J., K. K. Hansn, and M. Clagett-Dame All-trans-retinl is a ligand fr the retinic acid receptrs. Prec. Natl. Acad. Scl. USA. 90: Rybak, S. M., and R. J. Yule Clinical use f immuntxins: mn- clnal antibdies cnjugated t prtein txins. Immunlgy and Allergy Clinics f N.A. 11: Sandvig, K., and S. Olsnes Effects f retinids and phrbl esters n the sensitivity f different cell lines t the plypeptide txins mdeccin, abrin, ricin and diphtheria. Bichem. J. 194: Sandvig, K., K. Prydz, S. H. Hansen, and B. Van Deurs Ricin transprt in brefldin A-treated cells: crrelatin between Glgi structure and txic effect. J. CeU Bil. 115: Thaller, C., S. M. Smith, and G. Eichele Retinids in vertebrate develpment: pattern frmatin in limbs and in the central nervus system. In Retinids: 10 Years On. J.-H. Saurat, editr. Karger/Basel Wales, R., L. M. Rberts, and J. M. Lrd Additin f an endplasmic reticulum retrieval sequence t ricin A chain significantly increases its cyttxicity t mammalian cells. J. Bil. Chem. 268: Wu, Y. N., S. M. Mikulski, W. Ardeh, S. M. Rybak, and R. J. Yule A cyttxic ribnuclense: study f the mechanism f ncnase cyttxicity. J. Bil. Chem. 268: Yshida, T., C. H. Chen, M. S. Zhang, and H. C. Wu Disruptin f the Glgi apparatus by brefeldin A inhibits the cyt~xicity f ricin, mdeccin, and Pseudmnas Txin. Exp. Cell Res. 192: Yule, R. J., and D. M. Neville Kinetics f prtein synthesis inactivatin by ricin anti-thy 1.1 mnclnal antibdy hybrids: rle f the ricin B chain demnstrated by recnstitutin. J. Bil. Chem. 257: Yule, R. J., and M. Clmbatti Hybridma cells cntaining intracellular anti-ricin antibdies shw ricin meets secretry antibdy befre entering the cytsl. Y. Bil. Chem. 262: Zhang, X. K., B. Hffmann, P. B.-V. Tran, G. Granpner, and M. Pfahl Retinid X receptr is an auxiliary prtein fr thyrid hrmne and retinic acid receptrs. Nature (Lend.). 355: Wu et al. Retinic Acid Disrupts the Glgi Apparatus 753

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