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The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V
August 1992
Volume 308, number 1, 46,.49 FEBS 11394 O 1~.)2 Federation o£ European fliochemieal Societies 0014S793/92/$S.00
The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V Ellen W. Easton", Irene Blokland", Albert A. G e l d o P , B. R a m a n a t h Raot' and Dirk H, van den Eijnden" "Department o f Medical Chemistry, t/rij¢ Utdversit¢it, Van der Boecharstxtraat 7, 1081 BT Amsterdam° The N¢therlauds and ~Departmcnt o f Ettdocrinolagy. Acadcmi~ch Zickm~huis Vrije Unix'era'licit, PO Box 7057, tO0? MB Amsterdam. The Netherlatt~" Received 8 June 1992; ceviscd version received 22 lune 1992 Enzyme activities of N.acetyl~l'-,cosamit'~yltrunsl'cras¢ (GIcNAc-Tas¢) t-V involved in N.linked compk~x.typ¢ ¢arboltydrate synthesis were deter. mined in a non.metastatic honnonc.depend~nt rut p¢o~tat~ tumor (R332?.H) and a related, hormone-independent variant metastasizing to lymph nodes and lungs (R3327-MatL),Lu), tn the metastasi~in$ variant a significantly increased activity or both GIcNAc.Ta~ 11I and GIcNAc-'l'ase V was observed, wher~s the activities ofGleNAc-Ta~ [ and 11w~re essentially uncllanlied. The increase in activity of GIcNAc.Tas¢ Ill is particularly notcwortlw since it indicat~ that elevated expression of this enzyme cannot I:mconsidered its an exclusiv~ marker of hepatic maliilnaney, N-Acetyllllucosaminyltranffcras¢; Metasta,~is; Rat prostate tumor; R332?-MatLyLu
1, INTRODUCTION Prostate cancer is one if the major lethal malignancies in males, and death is always caused by metastasis to lungs, skeleton or lymph nodes [1]. The R3327 prostate tumor in the Copenhagen rat is often used to investigate the process of prostate tumor cell metastasis. Several R3327 tumor variants have become available, with differences in hormone sensitivity, metastatic potential and target organ specificity [2]. We have used two of these variants, the R3227-H variant (hormone-sensitive and non-metastasizing) and the R3327-MatLyLu variant (hormone-insensitive and metastasizing to lymph nodezi and lungs), to investigate the po~.~ibl¢ role of c¢11 surface carbohydrates in the metastatic process. Malignant tranfformation of cells has often been found to be associated with an increa~ in the average molecular weight of N-linked complex-type cell surface carbohydrates [3-10]. This increased molecular weight has been attributed to increased branching on the trimannosyl core caused by an elevated N-ac¢tylglucosaminyltransferase (GIcNAc-Ta~¢) V activity [11,12], to increased polyactosaminoglycan formation [8,10,I 3] or to increased ~ialylation [6,9], Furthermore, a correlation between (31cNAc-Tase V activity and metastatic behavior of cells was observed in marine MDAY-D2 cells [14] and in oncogene-transfected rat 2 fibroblasts [15]. In NIH 3T3 cells expressing the N.ras proto-oncoCorrespondenceaddress; D. H, van den Eijnden, D~partment of"Med. teal Cbezni~try. Vrije Universit¢it, Van der Boeehorststraat 7, NL1081 BT Amsterdam, The Netherlands, Fax: (31) (---0) 642 8555.
46
gen¢, an invasive potential was correlated with an increased GlcNAc-Tase III and V expression [10]. We have determined the activities of GIcNAc.Tase I-V, enzymes that are involved in the synthesis on Nlinked complex-type di-, tri- and tetra-anti,unary glycans. in the R3327-H and the R3327-MatLyLu prostate tumor variants to investigate whether there is a correlation between altered 81ycosylation as indicated by changed glyco~yltransferas¢ levels and metastatic potential. We found that in the m¢tastasizin~ variant the activity of two of the branching OlcNAc-Tases was significantly increased. 2. MATERIALS AND METHODS LI. Mtaerkd.¢ Oiillo~cchar[d¢ M a n ' . l ~ 3(Man~tl~ 3(Man=l~
6)M.ar;~1 ,
6)Man/JI~4GIcNAc.81.~4GIcNAc(Man~GIcNAc:) was :' la:ed from the urine or loco-intoxicatcdsh~p [16].which was kiu,~,;¢supplied by Dr, C.D. Warren (Ma~tchusctts General Hospital.Boston, MA). GlcNAcpi-.* 2Man¢l~ 3(Manal~ 6)Manp.O(CH.,hCOOCH~ (GA-3139A) was a kind gift of Dr. O. HinsBaul (University of All~rta, Edmonton, Alia.).Oligosaccharides GIcNAc~I~2Man~I~3(GIcNAc~I-.,2Man,',I ~6)- Man,al~ 4GIcNA¢ (di). GIeNAcflI~ 2(GIeNAcffl.-, 4)Mortal 3(GIcNA¢,~I ~ 2Murl¢l ~6)- Man,a I~4GIeNA¢ (tri),GIeNAc/J I..-+ 2Man~tl-.+3(GlcNAcpl.~4) (GIeNA~I-~2Man¢I...*6)- Man/~'l...~ 4GIcNAc (his)arld GIeNAepI-+ 2Man ~I...43(GIcNAc,~I-.+ 2(GIcNAc/II ~6)Man al .-46)- Man~tt-~4GIcNA¢ (tri') w~re obtained by •-galactosidase (jack bean) digestion [17] of the corrcspondinl| ff4-t,alactosylated compounds, which were kindly donated by Dr. G. Strecker (Universit¢! de Lille, Villen,,uve d'Ascq. France). Amberlite 1RA-743 wits obtained from Sillma (St, Louis, MO), UDP.[=~CIGIcNAc (302 Ci/mol) was obtained from Amersham internatiomtl (Amer~ham, UK), and UDP-[~HJGIcNAc (18,S Ci/mol)
Published b)' Elseoler Sci¢tw¢ Publishers B, V.
Volume 308, numl~r I
FEBS Lv"I'['ERS
(tom Dapont.New EnMland Nuclear (Boston, MA), Unlabeled UDPelk:NAt was purehasad from Sillma (St, Louis. MO). The unlabeled taEar nacl¢otide was used to dilute the radioactive compounds to the d=lired specific radioactivity. All other chemi~ls were of the ~ t quality available from commercial r.ourc¢~, 2,2. T.mors Col~nh~len r~t:. originally ohtained from the Mammalian Geneti~ and Animal Production .~a:tion, National Caner Institute. Bcthud.el, MD, were bred (brother x sister) and housed in our animal facilities according to the institutional animal w~farc regulations, The R3327.MatLyLu tumor (hormone.in=ndtiv¢ and metastmizin$ to lymph nod~ and lunMs)and R3327.H tumor (hormonc.f
2.4, Gtyem;yhra~er~¢ a.~xa,rs The meth0th for assaying the activity ofGIcNAc-Tas¢ I and II were modifigl from Harpaz und ~hachtcr [20], Incubation mixtures consisted or :50 nmot Man,GIcNA¢= (GIeNAc-Ta¢ l)or 100 nmol oliilotaccharidc GA-3139A (GIcNAc-Ta= 1I), 70 nmol UDP.[~HIGIcNA¢ (s~.ilic ra:lloaetivity I Ci/mol), S/Jmol 2.(A'.morpholina|ethanesuil'onato (MEb~ buffer, pH 6,3, I/Jmnl MnCI=, 0,1 #1 Triton X-100 and =11 homogematc (0,35-.O.9 ms of protein) in a total volume of S0 /at, Incubations were conducted for 1-2 h at 37"C, In the case of GIeNA¢-Tas¢ 1 the reaction was stopped by the addition of 450#1 of H;O and the rr,ixtur¢ was parsedthrouih a I ml column ofDowex [XtS 000-200 m~h), Cl'.form, The elaam and 3 washes of0.$ ml of H:O each were collected and radioactivity incorporated into the ao:eptor oligor.l¢¢harid¢ was counted by liquid scintillation. In the atsay of GIcNA¢-Tuse II the r~aetion was stopped by the addition of $ml of H:O. Separation of the labelled olilio~h:tride product from the radioactive sull~r-Aonor substrat¢was ¢~trricd out using Sop.PaRC. 1 rever~.pha~ cart'id.qes (Water, Milford. MA) according to Pal¢ic et al. [~ i} ~hc activit;es of GIeNAc.TASE 111.IV and V weredetermined .min!.u1:~t,e~,.~yusing oligosa=:haride di as a common a~:eptor sub. sir'ate. |neubations were cnnducted for 16 h at 37*C as deu:rihed by Koend¢l~an el al. [~7]. The reaction was stopped by adding 0.5 ml H:O and pasinil the mixture through a I ml column of Dowex I-Xg, Cl'.form, The column was washed twice with 0,? ml H=O.To the ¢ombined eluates 150#1 2,7 M NaBHd0,1 M NaOH was added and the mixture was left at room temperature for I h, Excessof NaBH= was destroyed by the addition of 4 M acetic acid, Cations were re. moved by passing the mixture throul~h a column of 0.$ ml Dow~ :50W.XI6, H" form and borate was removed by passageover a 2 ml column of Amberllte IRA.743 column. The columns were washed ~'ith 4,0 ml H;O. and the eluate was lyophiltzed and dissolved in 101:1 /al H~O. Separation of the different products was ao:omplished by HPLC I17]. In all assays, control incubations were carried oat without a~:cptor to ¢orr=:t for endogenous ace=pier activity. A ~ y s were done for each rat tumor homogenato at least in dupli~t=. In the HPLC assays radiola~led referenceswere added to allow correct product identifieslion,
August 1992
3. RESULTS A N D
DISCUSSION
In this study we investigated the po~ible correlation between aberrant N-glycosylntion and tumorcell metastasis. The activity of GIoNA~,-Tase I-V was assayed in two rat prostate tumor variants, and for each enzyme the activity in the g3327-MatLyLa tumor relative to that in the R332?-H tumor was taken as a measure for the change in expression in the metastasizing cells. GlcNAc-Tas¢ 1 and II each catalyze a committed step in the synthesis of/V-linked complex.type di-antennary glycans [20], while the other enzymes arc involved in the formation of bisecte.d (GIcNAc-Tas¢ II!) [22] and triand tetra-antcnnary (GIcNAc-Tas¢ IV and V) [23,24] structures. The R332?-H tumor is hormone-dependent and grows well in intact animals bat not in castrated rats, while the hormone-independent g3327-MatLyLu tumor grows equally well in intact and castrated animals [18]. Therefore the differences observed in the activities of the enzymes between the two variants arc most likely due to an acquired genetic disposition rather than to hormonal influences. No significant differcn~ I~twc=a the two variants was observed in the activity of GIcNAc-Ta~ I and GlcNAc-Tase iI (Table I). This indicates that th~ porch. tial to synthesize N-linked complex.type glycans is the same in both tumors. However, the activity of GlcNAcTasc III was highly increased in the metastasizing variant, while the activity of GIcNAc-Ta'~¢ V was elevated to a lesser extent (Table 1, Fig. 1). GlcNAc-Tase IV activity was below detection in either variant, although we cannot rule out the possibility that some GIcNAcTas¢ IV activity is present in the H.variant (Fig. 1). An increased activity of GIcNAo-Ta.~ V has boon correlated with malignant transformation in several cases [10.-12.14,15], In a number of these ~ s e s cl0vation in the expression of this enzyme was associated with metastasis or invasiv¢ potential. Increased activity of GIcNAc-Tas¢ I11. on the other hand, has been suggested to be a marker specific of hepatic malignancy, as high activities of this enzyme have b ~ n reported in rat [17,253 and haman [26] Table i N-Acctylglucosaminyltransfera~ (GIeNAe.Tas¢) activiti~ (noel.rag "s protcin.h':) in the non.metastatic, hormone.dependent R332?-H and the metastatic, hormone-independent R332?.MatLyLu rat prostate tumor variants GIcNA- Linka~ formed Tas¢
Activity in R~327.H
I I1
GIcNAcpl ~2Manofl-~3 4,7 GleN Ac/~1--42Man ocl-.46 10,8
III
GlcNAcfll--*#Man~l-*4
1V V
GIcNAcfll-.~Man¢i ~ 3 <0.00.~ GlcNAc~l~6Man,~l~6 0,O0.'i
0,02
Activity ratio (R3327R3327MatLyLu MatLyLu R3327-H) 5,0 14,2
1.1 1.3
0,13
6,S
<0.005 0,013
2.6
47
FEBS L E T T E R S
Volume 308. number I
|
II
!
.I
~ @OOO, >,
i~ q ~ '
soB,
7o
7s Etutlors
do ¢ime
as
9'o
Cmin3
Fig, I. Atgtl,, of GIcNAc-Ta~¢ Ill. 1V and V activid~ by HPLC. Transfera~ activi|ics were measured in homollcnatcs of the R332?.H (0) and the g332?-MatLyLu (S) prostate tame: variants usin~ olillosa~haride di as the accepter substrat¢.After incubation the products of illyeosyltransfera~ action were i~laled and reduced as de~ribed in z~lion 2. AnalDis of the r~altin~ materhtI was performed on a Liehrosorb-NH,. column. Elution was carried out ;u a flow rate of 2 ml.min"~ usinl a linear Ivadient of acetonitrildlS rnM potatsium phosphate, pH $,2,startln|at 4:1 (v/v)and decreasing acetonitril¢by 0,2~&.min "~ [l?l. The arros~ indicate the elation positon of the reduced forms of (I) the substrate(di). and the products of (2) GIcNAc,Tas¢ IV (tri),(3) GlcNAc.Tas¢ Ill (bls)and (4) Gk:NAc.Ta~ V (tri'},
hepatomas, prcneoplastic hepatic nodules [27]. and in human liver cirrhosis (a condition that may result in the development of a primary hepatoma) [26], In rat h e ~ t o m a s the increased GIcNAc-Tase lll activity was shown to correlate with the presence of bisected structures on glycoproteins of hepatoma origin while these structures were absent on glycoproteins produced by normal liver [28,29]. Recently we have reported an increased activity of OlcNAc-Tase llI to occur in mouse NIH 3T3 fibroblasts ¢xpre~ing the human N.ras protooncogen¢ JIG], This result correlated well with lectin binding studies of surface glycopeptides of N I H 3T3 cells transfected with eas-oncogenes, which showed an increased affinity for E-PHA [4], a lectin specific for bisected structures [30]. By analogy, based on the results obtained in the present study, we predict that an increased amount of bisected structures will also be prescnt on the R3327-MatLyLu cells, Elevated levels of GlcNAc-Tase Ill and bisected structures therefore are not an exclusive characteristic of neoplasia of Ihe liver, Moreover, it should be n o t ~ that the presen~ of bisected structures at the cell surface or on circulating glycoproteins,as such is not necessarilyindicativeof a malignant status. Apart from the cases mentioned above, bisected structureshav¢ also been found on a number of normal glycoproteins,including ovalbumin [31].ovotransf¢rrin[32],and ovomucoid [33] from hen oviduct: human red blood cellband 3 glycoprotein [34] and glycophorin [35]; and 7-glutamyllranspeptidase 48
August 1992
from bovine [36], rat [37] and human kidn¢y [38]. However, although the presence of bisected structures is a normal characteristic of some glycoproteins, an abetrantiy increased expression of GlcNAc-Tase III and its products may well be correlated with malignant transformation and a metaslatic potential, Although recently some lectin-binding studies have been performed with R-3327 tumor variants none of them have provided information about the amount of bisected and tri'-antennary structures [39-.41]. Howcvcr, based on our results we expect an increased amount of N-linked tri'-antennary structures to occur on the R3327.MatLyLu cells, in addition to the above mentioned increase in bisected structures, Since tumor cell adhesion is thought to play an important role in the establishment of site-specific tumor metastasis [42], and carbohydrate structures may act as specific ligands in cell-cell adhesion processes [43] it is tempting to speculate about a role of the increased amount of bisected and tri'.al~tennary structures in the R3327-MatLyLu vari. ant in its metastasis to lymph nodes and lungs. Arkno.'ledl~emems:We would like to thank Dr, D.H. Joxiass¢ for his helpful comments on this manuscripl.
REFERENCES [I] Geldof. A.A, and RuB, B,R, tiP90) Antican¢cr Rcs. i0, I3031306. [2] Is'sacs.,i.T,and ColTey. D.S. 0983) Clin,Oncol. 2. 479-,4glL [3] Yamashita. K,. Ohkura. T., Taehibana. Y., Tak:tsaki, S. =,rid Kobata, A. (lPtt4} ,i. Uiol, Cllem. 259. 101t34-101t,40.
[4] San|or. U.V., Gilbert. F, and Olick. M.C. (1984) Cancer R¢~, 44, 3730-3735, [5] Oolu:her. J.G.M,. Van der Bijl, M,M.W,. Nee0es. J.J., Hall, A,, Smc;s. L.A. ;tad Ploq~h, H.L, (191i8)EMI]O J. 7, 3361-3369. [6] Van Beck. W.P,, Smels. L,A. and Emmelot, P, (1973)Cancer Rcs, 33. 29l 3-2922, [?] Pierce. M. and Arango. J, (19K6) J, Biol, Chore, 261, 1077210777. [~] Hubbard. S,C, (19g?) J. Biol. Cnc.m. -"62. 16403.,16411, [9] W:trren. L., Fuhrer. J.P. and Buck. C,A. (1972) Prec. Natl. Acad. Sci, USA 69. 1~3g-l~42. [IO] "fasten,E.W., Bolschcr, J,G.M, and Vim den |.~ijlldcn.D.H. (1991) J, Biol. Chem. 266. 21674.-2i6t10. [I I] Yamashita. i¢... Ti,chibana. Y,. Ohkum. T, ;tad Kobat:t, A. { 19tL~) J. Biol.Chem, 260, 3963=3969. {12] Arango, ]. and Pierce. M, (1988)J, Cell, Biochcm, 3'], 22S.-23L [13] Van den Eijnden, D.H,, Kocndcrm:m. A,H,L. and Schiphorst. W.E.C.M. (Ig88)J. Biol. Chem. 263, 12461-124"]1. [14] Dermis, J.W.. Lafert~. S,. Watthornc. C,. Brcitman. M.L. and Kcrbel. R,S, (1987)Science 236. S82-StlS. [I 51 Dennis, J.W.. Kosh, K.. Bryce, D,-M, and Breitman. M,L. (lPtlP) Once.end' 4, 1~S3-860. [[6] $adch. S,,Warren. C.D,. Daniel.P,F,,Bagg¢, 13.,.fames,L,F.and Jeanloz, R.W, (1983) FEBS Left. 163. 104-109. [17] goenderma:.. A.H,L.. Koppcn. P,L,. Kocleman, C,A,M, and Van den Eij~den,D.H, (19~9) Ear..I,Biochem, 181. 651-655. [181 Geldof, A,A, and RuB, B,R, (lrJP0) Pros|ate 16, '3-79-290, [lg] Peterson. G.L. (1977) Anal. Biochem. i't3,346-356. [20l Harpaz, N. and Schachtcr, H, (19B0) J, Biol, Chem. 255. 4~854B93, [21l Palcic, M,M., Hccrzc, L,D,, Pierce. M, :rod Hindsgaul, O, (Igtltl) Glycocolqu~lte J. S, 49-63,
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{22] Nara~imhan. S. (1982) .L Biol. Chem. 2~t7. 10235-10242. [23] (.He¢.~on.P.A. and Schaehter. H. (1983) J. Biol. Chera. 25t~.61826173,
[24] Cummings. R.D.. Trowbrid~. [.S. and Korn."eld. S. (19t(2) J. Biol. Chem, 2~T. 13421-13427. [2~1] Nishikawa. A.. Fujii. S.. SuBiyama. T.. Hayashi. N, and Tuniguchi. N. (|grill) Biochera. Biophys. R~. Commun. 152. 10% IL2. (26] lshibashl. K.. Nishikawa. A.. Hayashi. N.. Ka~haru. A.. Solo. N.. Fujii. S.. Kamada. T. and Taniguehi. N. (1989) Clin. Chim. Acta It, S. 325-332. [2"/] Nara~imhan. S.. Sehaehzer. H. and Rajalakshmi. S. (19art) J. Biol. Chem, 2~3, 1273-1281, [2t~] Kob,~ta, A. and Yamashita, K. (19~4) Pure AppI. Chem. ~6, g21-t~32, [29] Y'arnashita. R.. Hitoi. A.. TanaBuehi. N.. Yoko~w=. N.. T~ukada. Y. and K.obata. A. ([983) Cancer R~. 17. 1010-1027. [30] Cumminlts. R.D. and Kornl'¢ld. $. (19tt2) J. Biol. Chem. 257. 11230-11234. [31) Yamashita. K.. Taehibana. Y. and Kobata, A. (197t{) J. Biol. Chem. 25~1.31~2u3t~9, {32] Dorhmd. L.. Havcrkamp. J.. VJi¢ilentharl. J.F.G.. Spik. G.. Fourael. B. and Montreuil. J. (1979) Eur. J. Biochem. 100. 569$74.
AuBust 1992
[;13] Yamushit,. K.. Kamerlin$. J.P. and Kobatz. A, (19it.~) J. DioL Chem, 2~?, 12~09-12814, [34] Fukudu. M.. Dell. A. and Fukuda. M.N. (1984) J. Biol. Chem. 2~9, 471.12...4791. [35] ~!ashima. H.. Funhmayr. H. and Kobata. A. (19a0) J. flio|. Chem. 255, 9713-9718. [3G) Yamashita. K.. Tachibana. Y.. Shiehi. Ft. and Kobata. A. (1983) J. Bib:hem. (Tok>.o)9:3. |35-|47. [:17] Yama~hha. K.. Hitoi. A.. Muts~a. T.. T~uji. A.. Katunuraa. N. and Kobata. A. CI9~3) J. Biol. Chem. 25tl. 1098-1107. []til Yamashi|a. K.. Hitoi. A . Matsuda. Y.. Miura. T.. Kailanuma. N. and Kobata. A, (19Zl6) J, Biochem. (Tokyo) 9g. 551.G2. [391 Abel. P.D.r Foster. C.S.. Tebbutt. S. and Williarral. G. (1990) J. Urol. 144. 7(~0-765. [4Q] Bashir. 1.. Sikora. K. and Foster. C.S. (1990) Bi~chem. $o¢. Trans. lt~, g6~-969, [41] Palmeira. E. Sikora. K. and Foster. C.S. (1990) Dioehem. So¢. Trans. 1~. 1295-12~?. [42] Flier. J.S.. Underhill. L.H. and Zctter. B.R. (1990) N. EnBI. J. Meal. 322. 605-612. [431 Springer. T.A. and Lasky. L.A. (1991) Nature 349. 196-107.
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Volume 308, number 1, 46,.49 FEBS 11394 O 1~.)2 Federation o£ European fliochemieal Societies 0014S793/92/$S.00
The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V Ellen W. Easton", Irene Blokland", Albert A. G e l d o P , B. R a m a n a t h Raot' and Dirk H, van den Eijnden" "Department o f Medical Chemistry, t/rij¢ Utdversit¢it, Van der Boecharstxtraat 7, 1081 BT Amsterdam° The N¢therlauds and ~Departmcnt o f Ettdocrinolagy. Acadcmi~ch Zickm~huis Vrije Unix'era'licit, PO Box 7057, tO0? MB Amsterdam. The Netherlatt~" Received 8 June 1992; ceviscd version received 22 lune 1992 Enzyme activities of N.acetyl~l'-,cosamit'~yltrunsl'cras¢ (GIcNAc-Tas¢) t-V involved in N.linked compk~x.typ¢ ¢arboltydrate synthesis were deter. mined in a non.metastatic honnonc.depend~nt rut p¢o~tat~ tumor (R332?.H) and a related, hormone-independent variant metastasizing to lymph nodes and lungs (R3327-MatL),Lu), tn the metastasi~in$ variant a significantly increased activity or both GIcNAc.Ta~ 11I and GIcNAc-'l'ase V was observed, wher~s the activities ofGleNAc-Ta~ [ and 11w~re essentially uncllanlied. The increase in activity of GIcNAc.Tas¢ Ill is particularly notcwortlw since it indicat~ that elevated expression of this enzyme cannot I:mconsidered its an exclusiv~ marker of hepatic maliilnaney, N-Acetyllllucosaminyltranffcras¢; Metasta,~is; Rat prostate tumor; R332?-MatLyLu
1, INTRODUCTION Prostate cancer is one if the major lethal malignancies in males, and death is always caused by metastasis to lungs, skeleton or lymph nodes [1]. The R3327 prostate tumor in the Copenhagen rat is often used to investigate the process of prostate tumor cell metastasis. Several R3327 tumor variants have become available, with differences in hormone sensitivity, metastatic potential and target organ specificity [2]. We have used two of these variants, the R3227-H variant (hormone-sensitive and non-metastasizing) and the R3327-MatLyLu variant (hormone-insensitive and metastasizing to lymph nodezi and lungs), to investigate the po~.~ibl¢ role of c¢11 surface carbohydrates in the metastatic process. Malignant tranfformation of cells has often been found to be associated with an increa~ in the average molecular weight of N-linked complex-type cell surface carbohydrates [3-10]. This increased molecular weight has been attributed to increased branching on the trimannosyl core caused by an elevated N-ac¢tylglucosaminyltransferase (GIcNAc-Ta~¢) V activity [11,12], to increased polyactosaminoglycan formation [8,10,I 3] or to increased ~ialylation [6,9], Furthermore, a correlation between (31cNAc-Tase V activity and metastatic behavior of cells was observed in marine MDAY-D2 cells [14] and in oncogene-transfected rat 2 fibroblasts [15]. In NIH 3T3 cells expressing the N.ras proto-oncoCorrespondenceaddress; D. H, van den Eijnden, D~partment of"Med. teal Cbezni~try. Vrije Universit¢it, Van der Boeehorststraat 7, NL1081 BT Amsterdam, The Netherlands, Fax: (31) (---0) 642 8555.
46
gen¢, an invasive potential was correlated with an increased GlcNAc-Tase III and V expression [10]. We have determined the activities of GIcNAc.Tase I-V, enzymes that are involved in the synthesis on Nlinked complex-type di-, tri- and tetra-anti,unary glycans. in the R3327-H and the R3327-MatLyLu prostate tumor variants to investigate whether there is a correlation between altered 81ycosylation as indicated by changed glyco~yltransferas¢ levels and metastatic potential. We found that in the m¢tastasizin~ variant the activity of two of the branching OlcNAc-Tases was significantly increased. 2. MATERIALS AND METHODS LI. Mtaerkd.¢ Oiillo~cchar[d¢ M a n ' . l ~ 3(Man~tl~ 3(Man=l~
6)M.ar;~1 ,
6)Man/JI~4GIcNAc.81.~4GIcNAc(Man~GIcNAc:) was :' la:ed from the urine or loco-intoxicatcdsh~p [16].which was kiu,~,;¢supplied by Dr, C.D. Warren (Ma~tchusctts General Hospital.Boston, MA). GlcNAcpi-.* 2Man¢l~ 3(Manal~ 6)Manp.O(CH.,hCOOCH~ (GA-3139A) was a kind gift of Dr. O. HinsBaul (University of All~rta, Edmonton, Alia.).Oligosaccharides GIcNAc~I~2Man~I~3(GIcNAc~I-.,2Man,',I ~6)- Man,al~ 4GIcNA¢ (di). GIeNAcflI~ 2(GIeNAcffl.-, 4)Mortal 3(GIcNA¢,~I ~ 2Murl¢l ~6)- Man,a I~4GIeNA¢ (tri),GIeNAc/J I..-+ 2Man~tl-.+3(GlcNAcpl.~4) (GIeNA~I-~2Man¢I...*6)- Man/~'l...~ 4GIcNAc (his)arld GIeNAepI-+ 2Man ~I...43(GIcNAc,~I-.+ 2(GIcNAc/II ~6)Man al .-46)- Man~tt-~4GIcNA¢ (tri') w~re obtained by •-galactosidase (jack bean) digestion [17] of the corrcspondinl| ff4-t,alactosylated compounds, which were kindly donated by Dr. G. Strecker (Universit¢! de Lille, Villen,,uve d'Ascq. France). Amberlite 1RA-743 wits obtained from Sillma (St, Louis, MO), UDP.[=~CIGIcNAc (302 Ci/mol) was obtained from Amersham internatiomtl (Amer~ham, UK), and UDP-[~HJGIcNAc (18,S Ci/mol)
Published b)' Elseoler Sci¢tw¢ Publishers B, V.
Volume 308, numl~r I
FEBS Lv"I'['ERS
(tom Dapont.New EnMland Nuclear (Boston, MA), Unlabeled UDPelk:NAt was purehasad from Sillma (St, Louis. MO). The unlabeled taEar nacl¢otide was used to dilute the radioactive compounds to the d=lired specific radioactivity. All other chemi~ls were of the ~ t quality available from commercial r.ourc¢~, 2,2. T.mors Col~nh~len r~t:. originally ohtained from the Mammalian Geneti~ and Animal Production .~a:tion, National Caner Institute. Bcthud.el, MD, were bred (brother x sister) and housed in our animal facilities according to the institutional animal w~farc regulations, The R3327.MatLyLu tumor (hormone.in=ndtiv¢ and metastmizin$ to lymph nod~ and lunMs)and R3327.H tumor (hormonc.f
2.4, Gtyem;yhra~er~¢ a.~xa,rs The meth0th for assaying the activity ofGIcNAc-Tas¢ I and II were modifigl from Harpaz und ~hachtcr [20], Incubation mixtures consisted or :50 nmot Man,GIcNA¢= (GIeNAc-Ta¢ l)or 100 nmol oliilotaccharidc GA-3139A (GIcNAc-Ta= 1I), 70 nmol UDP.[~HIGIcNA¢ (s~.ilic ra:lloaetivity I Ci/mol), S/Jmol 2.(A'.morpholina|ethanesuil'onato (MEb~ buffer, pH 6,3, I/Jmnl MnCI=, 0,1 #1 Triton X-100 and =11 homogematc (0,35-.O.9 ms of protein) in a total volume of S0 /at, Incubations were conducted for 1-2 h at 37"C, In the case of GIeNA¢-Tas¢ 1 the reaction was stopped by the addition of 450#1 of H;O and the rr,ixtur¢ was parsedthrouih a I ml column ofDowex [XtS 000-200 m~h), Cl'.form, The elaam and 3 washes of0.$ ml of H:O each were collected and radioactivity incorporated into the ao:eptor oligor.l¢¢harid¢ was counted by liquid scintillation. In the atsay of GIcNA¢-Tuse II the r~aetion was stopped by the addition of $ml of H:O. Separation of the labelled olilio~h:tride product from the radioactive sull~r-Aonor substrat¢was ¢~trricd out using Sop.PaRC. 1 rever~.pha~ cart'id.qes (Water, Milford. MA) according to Pal¢ic et al. [~ i} ~hc activit;es of GIeNAc.TASE 111.IV and V weredetermined .min!.u1:~t,e~,.~yusing oligosa=:haride di as a common a~:eptor sub. sir'ate. |neubations were cnnducted for 16 h at 37*C as deu:rihed by Koend¢l~an el al. [~7]. The reaction was stopped by adding 0.5 ml H:O and pasinil the mixture through a I ml column of Dowex I-Xg, Cl'.form, The column was washed twice with 0,? ml H=O.To the ¢ombined eluates 150#1 2,7 M NaBHd0,1 M NaOH was added and the mixture was left at room temperature for I h, Excessof NaBH= was destroyed by the addition of 4 M acetic acid, Cations were re. moved by passing the mixture throul~h a column of 0.$ ml Dow~ :50W.XI6, H" form and borate was removed by passageover a 2 ml column of Amberllte IRA.743 column. The columns were washed ~'ith 4,0 ml H;O. and the eluate was lyophiltzed and dissolved in 101:1 /al H~O. Separation of the different products was ao:omplished by HPLC I17]. In all assays, control incubations were carried oat without a~:cptor to ¢orr=:t for endogenous ace=pier activity. A ~ y s were done for each rat tumor homogenato at least in dupli~t=. In the HPLC assays radiola~led referenceswere added to allow correct product identifieslion,
August 1992
3. RESULTS A N D
DISCUSSION
In this study we investigated the po~ible correlation between aberrant N-glycosylntion and tumorcell metastasis. The activity of GIoNA~,-Tase I-V was assayed in two rat prostate tumor variants, and for each enzyme the activity in the g3327-MatLyLa tumor relative to that in the R332?-H tumor was taken as a measure for the change in expression in the metastasizing cells. GlcNAc-Tas¢ 1 and II each catalyze a committed step in the synthesis of/V-linked complex.type di-antennary glycans [20], while the other enzymes arc involved in the formation of bisecte.d (GIcNAc-Tas¢ II!) [22] and triand tetra-antcnnary (GIcNAc-Tas¢ IV and V) [23,24] structures. The R332?-H tumor is hormone-dependent and grows well in intact animals bat not in castrated rats, while the hormone-independent g3327-MatLyLu tumor grows equally well in intact and castrated animals [18]. Therefore the differences observed in the activities of the enzymes between the two variants arc most likely due to an acquired genetic disposition rather than to hormonal influences. No significant differcn~ I~twc=a the two variants was observed in the activity of GIcNAc-Ta~ I and GlcNAc-Tase iI (Table I). This indicates that th~ porch. tial to synthesize N-linked complex.type glycans is the same in both tumors. However, the activity of GlcNAcTasc III was highly increased in the metastasizing variant, while the activity of GIcNAc-Ta'~¢ V was elevated to a lesser extent (Table 1, Fig. 1). GlcNAc-Tase IV activity was below detection in either variant, although we cannot rule out the possibility that some GIcNAcTas¢ IV activity is present in the H.variant (Fig. 1). An increased activity of GIcNAo-Ta.~ V has boon correlated with malignant transformation in several cases [10.-12.14,15], In a number of these ~ s e s cl0vation in the expression of this enzyme was associated with metastasis or invasiv¢ potential. Increased activity of GIcNAc-Tas¢ I11. on the other hand, has been suggested to be a marker specific of hepatic malignancy, as high activities of this enzyme have b ~ n reported in rat [17,253 and haman [26] Table i N-Acctylglucosaminyltransfera~ (GIeNAe.Tas¢) activiti~ (noel.rag "s protcin.h':) in the non.metastatic, hormone.dependent R332?-H and the metastatic, hormone-independent R332?.MatLyLu rat prostate tumor variants GIcNA- Linka~ formed Tas¢
Activity in R~327.H
I I1
GIcNAcpl ~2Manofl-~3 4,7 GleN Ac/~1--42Man ocl-.46 10,8
III
GlcNAcfll--*#Man~l-*4
1V V
GIcNAcfll-.~Man¢i ~ 3 <0.00.~ GlcNAc~l~6Man,~l~6 0,O0.'i
0,02
Activity ratio (R3327R3327MatLyLu MatLyLu R3327-H) 5,0 14,2
1.1 1.3
0,13
6,S
<0.005 0,013
2.6
47
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Volume 308. number I
|
II
!
.I
~ @OOO, >,
i~ q ~ '
soB,
7o
7s Etutlors
do ¢ime
as
9'o
Cmin3
Fig, I. Atgtl,, of GIcNAc-Ta~¢ Ill. 1V and V activid~ by HPLC. Transfera~ activi|ics were measured in homollcnatcs of the R332?.H (0) and the g332?-MatLyLu (S) prostate tame: variants usin~ olillosa~haride di as the accepter substrat¢.After incubation the products of illyeosyltransfera~ action were i~laled and reduced as de~ribed in z~lion 2. AnalDis of the r~altin~ materhtI was performed on a Liehrosorb-NH,. column. Elution was carried out ;u a flow rate of 2 ml.min"~ usinl a linear Ivadient of acetonitrildlS rnM potatsium phosphate, pH $,2,startln|at 4:1 (v/v)and decreasing acetonitril¢by 0,2~&.min "~ [l?l. The arros~ indicate the elation positon of the reduced forms of (I) the substrate(di). and the products of (2) GIcNAc,Tas¢ IV (tri),(3) GlcNAc.Tas¢ Ill (bls)and (4) Gk:NAc.Ta~ V (tri'},
hepatomas, prcneoplastic hepatic nodules [27]. and in human liver cirrhosis (a condition that may result in the development of a primary hepatoma) [26], In rat h e ~ t o m a s the increased GIcNAc-Tase lll activity was shown to correlate with the presence of bisected structures on glycoproteins of hepatoma origin while these structures were absent on glycoproteins produced by normal liver [28,29]. Recently we have reported an increased activity of OlcNAc-Tase llI to occur in mouse NIH 3T3 fibroblasts ¢xpre~ing the human N.ras protooncogen¢ JIG], This result correlated well with lectin binding studies of surface glycopeptides of N I H 3T3 cells transfected with eas-oncogenes, which showed an increased affinity for E-PHA [4], a lectin specific for bisected structures [30]. By analogy, based on the results obtained in the present study, we predict that an increased amount of bisected structures will also be prescnt on the R3327-MatLyLu cells, Elevated levels of GlcNAc-Tase Ill and bisected structures therefore are not an exclusive characteristic of neoplasia of Ihe liver, Moreover, it should be n o t ~ that the presen~ of bisected structures at the cell surface or on circulating glycoproteins,as such is not necessarilyindicativeof a malignant status. Apart from the cases mentioned above, bisected structureshav¢ also been found on a number of normal glycoproteins,including ovalbumin [31].ovotransf¢rrin[32],and ovomucoid [33] from hen oviduct: human red blood cellband 3 glycoprotein [34] and glycophorin [35]; and 7-glutamyllranspeptidase 48
August 1992
from bovine [36], rat [37] and human kidn¢y [38]. However, although the presence of bisected structures is a normal characteristic of some glycoproteins, an abetrantiy increased expression of GlcNAc-Tase III and its products may well be correlated with malignant transformation and a metaslatic potential, Although recently some lectin-binding studies have been performed with R-3327 tumor variants none of them have provided information about the amount of bisected and tri'-antennary structures [39-.41]. Howcvcr, based on our results we expect an increased amount of N-linked tri'-antennary structures to occur on the R3327.MatLyLu cells, in addition to the above mentioned increase in bisected structures, Since tumor cell adhesion is thought to play an important role in the establishment of site-specific tumor metastasis [42], and carbohydrate structures may act as specific ligands in cell-cell adhesion processes [43] it is tempting to speculate about a role of the increased amount of bisected and tri'.al~tennary structures in the R3327-MatLyLu vari. ant in its metastasis to lymph nodes and lungs. Arkno.'ledl~emems:We would like to thank Dr, D.H. Joxiass¢ for his helpful comments on this manuscripl.
REFERENCES [I] Geldof. A.A, and RuB, B,R, tiP90) Antican¢cr Rcs. i0, I3031306. [2] Is'sacs.,i.T,and ColTey. D.S. 0983) Clin,Oncol. 2. 479-,4glL [3] Yamashita. K,. Ohkura. T., Taehibana. Y., Tak:tsaki, S. =,rid Kobata, A. (lPtt4} ,i. Uiol, Cllem. 259. 101t34-101t,40.
[4] San|or. U.V., Gilbert. F, and Olick. M.C. (1984) Cancer R¢~, 44, 3730-3735, [5] Oolu:her. J.G.M,. Van der Bijl, M,M.W,. Nee0es. J.J., Hall, A,, Smc;s. L.A. ;tad Ploq~h, H.L, (191i8)EMI]O J. 7, 3361-3369. [6] Van Beck. W.P,, Smels. L,A. and Emmelot, P, (1973)Cancer Rcs, 33. 29l 3-2922, [?] Pierce. M. and Arango. J, (19K6) J, Biol, Chore, 261, 1077210777. [~] Hubbard. S,C, (19g?) J. Biol. Cnc.m. -"62. 16403.,16411, [9] W:trren. L., Fuhrer. J.P. and Buck. C,A. (1972) Prec. Natl. Acad. Sci, USA 69. 1~3g-l~42. [IO] "fasten,E.W., Bolschcr, J,G.M, and Vim den |.~ijlldcn.D.H. (1991) J, Biol. Chem. 266. 21674.-2i6t10. [I I] Yamashita. i¢... Ti,chibana. Y,. Ohkum. T, ;tad Kobat:t, A. { 19tL~) J. Biol.Chem, 260, 3963=3969. {12] Arango, ]. and Pierce. M, (1988)J, Cell, Biochcm, 3'], 22S.-23L [13] Van den Eijnden, D.H,, Kocndcrm:m. A,H,L. and Schiphorst. W.E.C.M. (Ig88)J. Biol. Chem. 263, 12461-124"]1. [14] Dermis, J.W.. Lafert~. S,. Watthornc. C,. Brcitman. M.L. and Kcrbel. R,S, (1987)Science 236. S82-StlS. [I 51 Dennis, J.W.. Kosh, K.. Bryce, D,-M, and Breitman. M,L. (lPtlP) Once.end' 4, 1~S3-860. [[6] $adch. S,,Warren. C.D,. Daniel.P,F,,Bagg¢, 13.,.fames,L,F.and Jeanloz, R.W, (1983) FEBS Left. 163. 104-109. [17] goenderma:.. A.H,L.. Koppcn. P,L,. Kocleman, C,A,M, and Van den Eij~den,D.H, (19~9) Ear..I,Biochem, 181. 651-655. [181 Geldof, A,A, and RuB, B,R, (lrJP0) Pros|ate 16, '3-79-290, [lg] Peterson. G.L. (1977) Anal. Biochem. i't3,346-356. [20l Harpaz, N. and Schachtcr, H, (19B0) J, Biol, Chem. 255. 4~854B93, [21l Palcic, M,M., Hccrzc, L,D,, Pierce. M, :rod Hindsgaul, O, (Igtltl) Glycocolqu~lte J. S, 49-63,
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{22] Nara~imhan. S. (1982) .L Biol. Chem. 2~t7. 10235-10242. [23] (.He¢.~on.P.A. and Schaehter. H. (1983) J. Biol. Chera. 25t~.61826173,
[24] Cummings. R.D.. Trowbrid~. [.S. and Korn."eld. S. (19t(2) J. Biol. Chem, 2~T. 13421-13427. [2~1] Nishikawa. A.. Fujii. S.. SuBiyama. T.. Hayashi. N, and Tuniguchi. N. (|grill) Biochera. Biophys. R~. Commun. 152. 10% IL2. (26] lshibashl. K.. Nishikawa. A.. Hayashi. N.. Ka~haru. A.. Solo. N.. Fujii. S.. Kamada. T. and Taniguehi. N. (1989) Clin. Chim. Acta It, S. 325-332. [2"/] Nara~imhan. S.. Sehaehzer. H. and Rajalakshmi. S. (19art) J. Biol. Chem, 2~3, 1273-1281, [2t~] Kob,~ta, A. and Yamashita, K. (19~4) Pure AppI. Chem. ~6, g21-t~32, [29] Y'arnashita. R.. Hitoi. A.. TanaBuehi. N.. Yoko~w=. N.. T~ukada. Y. and K.obata. A. ([983) Cancer R~. 17. 1010-1027. [30] Cumminlts. R.D. and Kornl'¢ld. $. (19tt2) J. Biol. Chem. 257. 11230-11234. [31) Yamashita. K.. Taehibana. Y. and Kobata, A. (197t{) J. Biol. Chem. 25~1.31~2u3t~9, {32] Dorhmd. L.. Havcrkamp. J.. VJi¢ilentharl. J.F.G.. Spik. G.. Fourael. B. and Montreuil. J. (1979) Eur. J. Biochem. 100. 569$74.
AuBust 1992
[;13] Yamushit,. K.. Kamerlin$. J.P. and Kobatz. A, (19it.~) J. DioL Chem, 2~?, 12~09-12814, [34] Fukudu. M.. Dell. A. and Fukuda. M.N. (1984) J. Biol. Chem. 2~9, 471.12...4791. [35] ~!ashima. H.. Funhmayr. H. and Kobata. A. (19a0) J. flio|. Chem. 255, 9713-9718. [3G) Yamashita. K.. Tachibana. Y.. Shiehi. Ft. and Kobata. A. (1983) J. Bib:hem. (Tok>.o)9:3. |35-|47. [:17] Yama~hha. K.. Hitoi. A.. Muts~a. T.. T~uji. A.. Katunuraa. N. and Kobata. A. CI9~3) J. Biol. Chem. 25tl. 1098-1107. []til Yamashi|a. K.. Hitoi. A . Matsuda. Y.. Miura. T.. Kailanuma. N. and Kobata. A, (19Zl6) J, Biochem. (Tokyo) 9g. 551.G2. [391 Abel. P.D.r Foster. C.S.. Tebbutt. S. and Williarral. G. (1990) J. Urol. 144. 7(~0-765. [4Q] Bashir. 1.. Sikora. K. and Foster. C.S. (1990) Bi~chem. $o¢. Trans. lt~, g6~-969, [41] Palmeira. E. Sikora. K. and Foster. C.S. (1990) Dioehem. So¢. Trans. 1~. 1295-12~?. [42] Flier. J.S.. Underhill. L.H. and Zctter. B.R. (1990) N. EnBI. J. Meal. 322. 605-612. [431 Springer. T.A. and Lasky. L.A. (1991) Nature 349. 196-107.
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