Altered Growth Behavior of Malignant Cells Associated with Changes in Externally Labeled Glycoprotein and Glycolipid

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1 Proc. Nat. cad. Sc. US Vol. 7, No. 12, Part I, pp , December 1973 ltered Growth Behavor of Malgnant ells ssocated wth hanges n Externally Labeled Glycoproten and Glycolpd (polyoma vrus/sman vrus 4/galactose oxdase) RL GUSTV GHMBERG ND SEN-ITIROH HKOMORI Department of Pathobology, School of Publc Health and Department of Mcrobology, School of Medcne, Unversty of Washngton, Seattle, Wash ommuncated by Herman M. Kalckar, July 2, 1973 BSTRT By use of galactose oxdase (E ), followed by reducton wth trtated sodum borohydrde, the surface structures of transformed 3T3 and NIL cells, under ordnary growth condtons, were characterzed by () deleton of the normally exstng glycoproten label and () appearance or ncrease of a new glycoproten label. NIL cells had a galactoproten label wth molecular weght 2, that was deleted n NIL cells transformed by polyoma vrus. 3T3 cells had a glycoproten label wth molecular weght of 3, that was lost after transformaton. Glycoprotens of transformed 3T3 cells, wth molecular weght 15,, and those of transformed NIL cells, wth molecular weght 85,, were not labeled n normal confluent cells, but became labeled after trypsn treatment. The label n glycolpds was quanttatvely dfferent n normal and transformed cells. The labelng pattern n glycoproten and glycolpds of transformed NIL and 3T3 cells became smlar to that of nontransformed cells when contact responses of transformed cells became conspcuous when cells were cultured n the presence of dextran sulfate or dbutyryl cyclc adenosne monophosphate, or n medum n whch glucose was replaced wth galactose. There s ncreasng evdence that the plasma membrane of tumor cells dffers from that of normal cells. Increased reactvty to lectns (1) and to ant-glycolpd antbody (2), ncomplete synthess of carbohydrate chans n glycolpd (3) and glycoprotens (4), and enhanced synthess of a specfc salylfucopeptde n transformed cells (5) are partcularly notceable. No drect evdence has been provded, however, on whether the altered glycolpd or glycoproten components are located on the external surface of the cell membrane. ontact response* can be restored n tumor cells by growng them under sutable condtons, such as n medum contanng dextran sulfate (6) or cyclc MP (7) or n medum n whch glucose was replaced by galactose (8). Because t recently became possble to specfcally label surface glycoprotens and glycolpds of cells wth galactose oxdase (E ) followed by reducton wth trtated sodum borohydrde (9), a technque based on the labelng of ceruloplasmn (1) and cerebrosde (11), we decded to compare surface labels of cells wth normal growth and of transformed cells wth nduced changes n growth characterstcs. bbrevatons: 3T3sv cells, mouse fbroblast 3T3 cells transformed wth Sman vrus 4; 3T3svpy cells, 3T3 cells doubly transformed wth Sman vrus 4 and polyoma vrus; NILpy cells, hamster NIL cells transformed wth polyoma vrus; PBS, phosphate-buffered salne. * "ontact response" collectvely ncludes varous cell contact phenomena such as contact nhbton, toponhbton, contact orentaton, and contact promoton EXPERIMENTL PROEDURE ells and ell ulture. Hamster NIL cells transformed wth polyoma vrus (NILpy cells) were obtaned after nfecton wth polyoma vrus, and one clone was solated n November T3, 3T6, 3T3sv (cells transformed wth sman vrus 4), and 3T3svpy (cells transformed wth sman vrus 4 and polyoma vrus) cells were cultured n Eagle's medum, modfed by Dulbecco and supplemented wth 1% fetalcalf serum, n a 5-6% O2 atmosphere. NIL and NILpy cells were cultured n Eagle's medum contanng 1% calf serum n a 3% O2 atmosphere. Inducton of Reversed Growth Behavor. Transformed cells changed ther morphology and became senstve to contact nhbton of growth when cultured n orgnal Eagle's medum (1 X amno acds and vtamns) and 1% calf serum, contanng 1lg (for 3T6 cells) or 4 jg (for NILpy) of dextran sulfate per ml of medum (6). The dextran sulfate (molecular weght about 5,) was kndly provded by Dr. M. Goto, Tohoku Unversty, Senda, Japan. The saturaton denstes of 3T6 cells and NILpy cells wth nduced contact senstvty were X 15/cm2 and 15/cm2, respectvely. ells grown n regular medum reached denstes as hgh as 5-8 X 15/cm2. NILpy and 3T3svpy cells were grown n the presence of.1 mm dbutyryl cyclc MP and 1 mm theophylln (12). The morphologcal change appeared more remarkable n NILpy cells than n-3t3svpy cells. fter 2-3 days n culture, the cells appeared well contact-orented and the sze of the cells ncreased. Freshly transformed NILpy cells showed remarkable changes n morphology and contact orentaton when cultured n Eagle's medum (twce the standard concentraton of amno acds and vtamns), n whch glucose was replaced wth galactose and whch was supplemented wth 1% fetal-calf serum. The Procedure for Surface Labelng was a slght modfcaton of a descrbed method (9). The cells from one plate (Falcon, 14 cm n dameter) were enough for labelng and were obtaned ether by scrapng wth a rubber polceman or by usng a.2% EDT soluton or a.25% trypsn soluton. The specfc actvty of the NaB3H4 was 6 /mmol. For further detals, see the legend to Fg. 2. nalyss of Labeled Glycoprotens and Glycolpds: Electrophoress was done wth nternal [14]formaldehyde-labeled standard l)rotens (13). The radoactve gels were slced and counted as descrbed (9). Unless otherwse ndcated, the cells were harvested by treatment wth.2% EDT. Glycolpds were extracted and parttoned (14). The neutral glycolpd

2 333 Bochemstry: Gahmberg and Hakomor Proc. Nat. cad. Sc. US 7 (1973) TBLE 1. hange of glycolpd label n NIL cells dependng on growth behavor % Dstrbuton of Glycolpd label % of label Lower n glyco- eramde eramde glycolpd lpd/ penta- tetra- (ceramde Growth Morphology Harvest total saccharde saccharde d-, tr- ells condtons and growth condtons label* (Forssman) (Globosde) saccharde) NIL Regular Eagle's ontact scraped medum, nhbted, EDT confluent orented trypsn NILpy Regular Eagle's Not nhbted, scraped medum, dsorented EDT confluent trypsn NILpy Eagle's medum Not nhbted, scraped t 64.1 Glc replaced orented EDT t 62.9 wth Gal trypsn t 63. NILpy Eagle's medum ontact EDT wth dextran nhbted, sulfate orented NILpy Eagle's medum ontact EDT wth cyclc MP nhbted, and theophylln orented * The rest of the label s due to glycoproten or nonspecfc labellng. t bout 5-8% hgher label n globosde n Gal-orented NILpy cells was reproduced n three separate experments, and the mean values are shown. Others are the mean values of two separate experments..l.., P.,.. *4 4 :. b,: I: * q I. %#. ::,: fe..", k k f"',":.."f. I *e~~~ to.%ft.. FIG. 1. Lght mcroscopc pcture of an autoradogram of a thn secton of labeled neuramndase-treated NIL cells. Most of the label s located at the surface membrane (orgnal magnfcaton X 1. The pcture was prepared by Ruth Tyler, Department of Bologcal Structure, Unversty of Washngton. W fracton was purfed from the lower phase by acetylaton (15) and analyzed by thn-layer chromatography and gas chromatography as descrbed (9). RESULTS Surface Localzaton of Label and Nonspecfc Label n NIL and 3T3 ells. The label was nearly exclusvely located on cell surfaces, as shown by autoradography of fxed and dssected cells (Fg. 1); the actvty of the label was proportonal to cell number. Nonspecfc labels wthout galactose oxdase were found n some protens and n lpds of NIL and 3T3 cells. Peaks "c" of ntact NIL and NILpy cells (Fg. 2 and B), "f" of neuramndase-treated NIL and NILpy cells (Fg. 2-H), and "e" of neuramndase-treated transformed 3T3 cells (Fg. 3 and D) were due to nonspecfc label. These protens wth nonspecfc label had smlar molecular weghts (56,-59,); the label was enhanced n transformed cells. Specfc Labelng Pattern of Normal and Transformed ells. The specfc label n glycolpds was shown by the presence of radoactvty n l)urfed glycolpd fracton by acetylaton and by the presence of radoactvty n galactose and galactosamne fractons separated on gas chromatography (9). Peak "a" wth an apparent molecular weght of 2, was found only n normal NIL cells (Fg. 2), but was totally absent n transformed NIL cells (Fg. 2B). Wthout neuramndase treatment, no apprecable glycoproten peak was obtaned for labeled 3T3 cells and ther transformants. Labeled peaks characterstc for transformed NIL and 3T3 cells were clearly demonstrated when cells were treated wth neuramndase. NILpy cells contaned the enhanced peak "d" wth an apparent molecular weght of 85, (Fg. 2D), and 3T3sv and 3T3svlpy cells had the l)romnent peak "c" wth

3 Proc. Nat. cad. Sc. US 7 (1978) ltered Growth Behavor of Malgnant ells 3331 OV Hb. M - LLL V - - 4o 4- BS OV I D p] d BS OV yto 2- E B-g' 9 OV yto c I ~ 2- n Q. - - F B- gao BS OV t 4-] 2 4o 2 41 FIG. 2. Sodum dodecyl sulfate-polyacrylamde gel electrophoress of labeled NIL cells. ells were harvested wth.2% EDT soluton, then treated n dfferent ways. and B were run n 5% polyacrylamde gels; all others (-H) were run n 7.5% polyacrylamde eels. () confluent NIL cells; (B) NILpy cells, drectly labeled wthout neuramndase treatment; () confluent NIL cells; (D) NILpy cells, treated wth neuramndase then labeled; (E) NILpy cells grown n the presence of 4 Ig/ml of dextran sulfate, treated wth neuramndase, then labeled; (F) NILpy cells grown n galactose medum (8), treated wth neuramndase, then labeled; (G) confluent NIL cells treated wth trypsn, then wth neuramndase, then labeled; (H) NILpy cells grown n the presence of dbutyryl cyclc MP, treated wth neuramndase, then labeled. ells were washed twce by centrfugaton n PBS (ph 7.) and suspended n.5 ml of PBS contanng 2 mm phenylmethylsulfonylfluorde (Sgma) to nhbt proteases. 1, of galactose oxdase dssolved n PBS (ph 7.) contanng 1 unts/ml (Sgma Type III) was then added, and the cells were ncubated at room temperature for 2 hr wth gentle shakng. The galactose oxdase dd not contan any measurable proteolytc or neuramndase actvty. In some experments, galactose oxdase was purfed by affnty chromatography on Sepharose 4B column. fter ncubaton the cells were washed twce n PBS (ph 7.4) and suspended n.5 ml of PBS (ph 7.4) to whch was added 5 l of trtated sodum borohydrde soluton contanng.5-1 m of NaB3H4, specfc actvty 6 /mmol (New England Nuclear orp; stored n.1 N NaOH soluton at -7). The reacton mxture was allowed to stand for 3 mn at room temperature. In some experments the cells were frst ncubated wth 5 l of Vbro cholerae neuramndase (albochem, Type B) that contaned 5 unts/ml n.5 ml of.1 M phosphate buffer (ph 6.) n the presence of protease nhbtor. ells ncubated wth dbutyryl cyclc MP were treated wth V. cholerae neuramndase n the presence of 1 mm dbutyryl cyclc MP, followed by the labelng procedure. #-gal, f-galactosdase; BS, bovne-serum albumn; OV, ovalbumn; cyto c, cytochrome c (each labeled wth [14]- formaldehyde and used as nternal marker). BPB, peak poston of bromphenol blue trackng dye. Peaks "d" and "f," whch showed a remarkable change on transformaton and on reverted cell behavor, are shaded. Peaks "c" of and B and "f" of -H were nonspecfcally labeled.

4 3332 Bochemstry: Gahmberg and Hakomor Proc. Nat. cad. Sc. US 7 (1973) 75 - E L czs o 5- B-goa BS OV yto :z 5c al B- go BS OV Typo a- Ix o Bo- ga BP OV,.ytc cs D F 41 c a-gol BS OV yc D L -) a Se en -) FIG. 3. Sodum dodecyl sulfate-polyacrylamde gel electrophoress of labeled 3T3 cells and ther transformants. ells were harvested wth EDT and labeled after neuramndase treatment. ll samples were analyzed n 7.5% polyacrylamde gels. () onfluent 3T3 cells treated wth neuramndase, then labeled; (B) growng 3T3 cells treated wth neuramndase, then labeled; () 3T3sv cells treated wth neuramndase, then labeled; (D) 3T3svpy cells treated wth neuramndase, then labeled; (E), confluent 3T3 cells treated wth trypsn, then wth neuramndase, then labeled, (F) 3T3svpy cells grown n the presence of dbutyryl cyclc MP, treated wth neuramndase, then labeled. Markers and labelng procedures are the same as for Fg. 2. Peaks "c" and "e," whch showed a remarkable change on transformaton and on reverted cell behavor, are shaded. Peak "e" contaned nonspecfc label. In a- D I B P-3 an apparent molecular weght of 15, (Fg. 3 and D). ctvely growng 3T3 cells showed also a relatvely large peak "c" (Fg. 3B), whle confluent 3T3 cells had no detectable peak "c." Instead, the confluent 3T3 cells had more label n the "b" peak and "g" peak (Fg. 3). The "g" peak was very low or not detectable n transformed cells. In ether 3T3sv or 3T3svpy cells, the presence of a relatvely hgh label at the hghest molecular-weght range (peak "a" of Fg. 3 and D) was notced. Modfed Surface Label by Trypsn and Other Reagents that Induced ltered Growth Behavor. If confluent NIL or 3T3 cells were treated wth trypsn and then labeled, there appeared a greatly enlarged peak "d" for trypsnzed NIL cells (Fg. 2G) and a peak "c" for trypsnzed 3T3 cells (Fg. 3E), whch were not markedly labeled wthout trypsnzaton. In normal 3T3 cells from ether confluent or growng cultures, the label n lpd was much greater than n transformed 3T3 cells. The relatve actvty of peak "b" greatly ncreased when contact response of NILpy cells became normal n the presence of dextran sulfate (Fg. 2E) or dbutyryl cyclc MP (Fg. 2H, compare wth and D). 3T3svpy cells whose contact behavor was restored by cyclc MP showed a greatly enhanced peak between "c" and "d" (Fg. 3F). The proporton of peaks "a"-"g" s qute characterstc for the type and physologcal state of cells. The glycoproten label profle sgnfcantly changed n NILpy cells grown n medum contanng galactose (Fg. 2F). The glycoproten profle of 3T6 cells resembled that of 3T3sv or 3T3svpy cells. When contact response was nduced by dextran sulfate, the peaks characterstc of transformed cells dsappeared, and the lpd label became promnent. Relaton of carbohydrate structures to ther label and the effect of cell contact on degree of labelng were clearly seen n the labelng pattern of glycolpds. s expected, neutral glycolpds of NIL cells were strongly labeled; the label n hgher glycolpds such as globosde and Forssman glycolpd decreased; the proporton of label n smpler glycolpds greatly ncreased n NILpy cells, n agreement wth the results of chemcal analyss (Table 1). sgnfcant ncrease (5-8%) of globosde label and decrease of label n smpler glycolpds was observed n NILpy cells grown n galactose medum, whose contact orentaton became obvously restored. Smlarly, the label n Forssman glycolpd sgnfcantly ncreased n NILpy cells whose contact response was restored and whose contact orentaton became obvous by ether dextran sulfate or dbutyryl cyclc MP (Table 1). The label n ceramde trsaccharde ncreased n nontransformed NIL cells after EDT or trypsn treatment, but was

5 Proc. Nat. cad. Sc. US 7 (1978) unchanged n NILpy cells, ndcatng that smpler glycolpds were consstently exposed n NILpy cells. DISUSSION lthough several experments usng labelng wth radoactve precursors have been done to dstngush membrane glycolpds and glycoprotens of normal and transformed cells (3-5), these studes were not desgned to dstngush surface structural dfferences. Therefore, the observed dfferences were dffcult to correlate wth the functon of cell surfaces. 3T3sv cells, whose contact response was partly restored by dbutyryl cyclc MP, have a decreased level of sulfated acd mucopolysacchardes (16). Morphologcal change of NILpy or 3T3sv cells nduced by dbutyryl cyclc MP dd not correlate wth glycolpd composton (17). Wth the galactose oxdase labelng method, major structural dfferences can be seen n exposed heteroglycans n complance wth the change of growth behavor. Transformed 3T3 cells contan a labeled glycoproten of apparent molecular weght of 15, (peak "c", Fg. 3), and NILpy cells contan a glycoproten of apparent molecular weght 85, (peak "d," Fg. 2F); both glycoprotens were not labeled n the confluent normal cells but were labeled only after treatment wth trypsn. Transformed 3T3 cells also contan more label n the salylgalactoproten a (Fg. 3 and D) of apparent molecular weght of 14,. Normal NIL and 3T3 cells were characterzed by hgher label at peaks "a" and "b" (apparent molecular weghts 2, and 13,), and 3T3 cells had a peak "gy' (molecular weght 3,). These peaks were reduced or deleted n transformed cells. The presence of a galactoproten n ntact NIL cells and ts complete absence n NILpy cells were well demonstrated n 5% gels (Fg. 2 and B), but not n 7.5% gels. n ncrease or creaton of label by neuramndase agrees wth the general formula of saloglycosdes n whch salyl resdue s lnked to a penultmate Gal or GalNc resdue (2), detectable by Rcnus communs lectn (21). In contrast to normal confluent cells, transformed cells and trypsnzed normal cells are easly agglutnated by varous lectns (1). Ths ncreased agglutnablty was consdered to be due to ncreased reactve stes to the agglutnns, but later work showed no essental dfference n the amount of bndng of agglutnns (18). Ths dfference n agglutnablty was explaned by clusterng of agglutnn receptors by the altered "flud dynamc" state of the membrane of the transformed and trypsnzed cells (19). Our results show that although the total label n the glycoprotens and glycolpds of normal and transformed cells s not very dfferent, some glycoprotens and glycolpds are much more exposed n the transformed cells, and become exposed n normal confluent cells only after trypsnzaton. Therefore, some of these normally cryptc specfc glycoprotens could be responsble for the observed dfferences n agglutnablty and possbly be clustered n certan regons of the cell membrane. The label profle of transformed cells shfted towards normal when the contact response of these transformed cells was restored by dextran sulfate or by cyclc MP. The ncreased label n globosde of NILpy cells, grown n a medum n whch all glucose was replaced by galactose and whch showed altered contact orentaton, s of specal nterest. Prevously, t was not possble to demonstrate glycolpd changes of galactose-orented cells, compared to normally cultured cells (8). By the surface labelng technque, however, galactose-orented cells have about 5-8% hgher label n ltered Growth Behavor of Malgnant ells 3333 globosde, wth the structure GalNc1l 3Galal -- 3Galol - 3Glc - eramde (22). Forssman glycolpds and globosde characterstc of normal NIL cells were much more heavly labeled n cells cultured n the presence of cyclc MP. These data strongly suggest that restoraton of contact nhbton or the presence of contact responses observed n the cultures n the presence of dextran sulfate or dbutyryl cyclc MP, or n galactose-orented medum s probably due to surface structural changs. Malgnantly transformed cells have a characterstc surface structure whch must be the bass of ther abnormal behavor, as the characterstc glycoproten and glycolpd profle was markedly modfed when cell growth behavor changed towards normal. 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