identification and characterization of specific changes ......fibronectin (m, 230,000). metabolic...

[CANCER RESEARCH 43, 303-312, January 1983]0008-5472/82/0042-0000$02.00

Identification and Characterization of Specific Changes Induced by

Retinoic Acid in Cell Surface Glycoconjugates of S91 MurineMelanoma Cells1

Reuben Lotan, George Neumann,2 and Varda Deutsch

Department of Biophysics, The Weizmann Institute of Science. Pehovot 76 i 00, Israel

ABSTRACT

The effect of retinole acid, a potent inhibitor of the proliferation of the murine S91 melanoma cells, on the biosynthesis ofglycoproteins and glycolipids and on their exposure on the cellsurface was investigated. The cells were cultured in the absence or presence of retinoic acid, and their proteins andglycoconjugates were labeled metabolically by incubation withradiolabeled amino acids or monosaccharides. Externally exposed cell membrane components were radiolabeled by: (a)NalO4:NaB3H4; (b) galactose oxidase:NaB3H4; (c) neuramini-dase and galactose oxidase:NaB3H4; or (d) lactoperoxidase-catalyzed 125Iiodination. The labeled molecules were solubi-

lized with detergents or extracted with organic solvents andthen analyzed by polyacrylamide gel electrophoresis or thin-layer chromatography followed by autoradiography or fluorog-raphy.

The most pronounced effect of retinoic acid on glycoproteinsynthesis was an enhancement of the incorporation of [3H]-glucosamine or [3H]mannose into a M, 160,000 glycoprotein

(gp160) without increasing the incorporation of amino acidsinto this molecule. Less prominent changes were induced byretinoic acid in the synthesis of glycosphingolipids. The onlyconsistent effect was a reduced incorporation of [14C]galactose

into GM3.The gp160 was accessible on the surface of intact retinoic

acid-treated cells and was susceptible to digestion by pro

teases or by neuraminidase.Direct cell surface-labeling Methods a, c, and d described

above revealed a remarkable increase in the labeling of gp160on the retinoic acid-treated cells. The labeling of cell surface

gangliosides was also modified by retinoic acid: Method alabeled glycolipids comigrating with GM3 and with GMi moreintensely, whereas Method c labeled GM] less intensely ontreated cells than on untreated cells.

The changes induced by retinoic acid in gp160 could bedetected after 12 hr of treatment, and the lowest effectiveconcentration was 0.1 JUM.

The results indicate that retinoic acid enhances the glyco-sylation of gp160, which is a component of the cell surfaceand contains W-acetylglucosamine, mannose, galactose (or N-acetylgalactosamine), and sialic acid. Because the changesinduced by retinoic acid in the melanoma cell surface precedeany changes in cell proliferation, they may be causally relatedto growth inhibition.

1Supported by the United States Israel Binational Science Foundation (Jeru

salem. Israel).2 Present address: Department of Botany. University of California, Los Ange

les, Calif. 90024.Received May 24, 1982; accepted August 17, 1982.

INTRODUCTION

The addition of certain retinoids to the growth medium ofvarious transformed and tumor cells has been shown to cause,in many different cell types, inhibition of anchorage-dependentand anchorage-independent growth and to modify cellular dif

ferentiation (for reviews, see Refs. 12 and 35). Although themechanism(s) by which retinoids modulate such fundamentalprocesses is not known, accumulating evidence indicating thatthese compounds can alter gene expression (7, 8, 12, 16, 32,35), cell membrane structure, and function (1, 9, 11, 12, 23,24, 26, 35, 37, 38, 46, 51) suggests that the cell nucleus andthe cell membrane are the 2 main targets for retinoid actions.

Retinoids may gain access to the cell nucleus, like steroidhormones, via specific cytoplasmic retinoid-binding proteins,

which have been found in various normal and malignant cells(8, 12, 35). Once in the nucleus, the retinoids may changegenomic expression (7, 8, 12, 35). A role for retinoids in thebiosynthesis of membrane glycoproteins has been indicated byvarious studies which demonstrated the direct involvement ofglycosylretinoid phosphates as sugar carriers in the transfer ofmonosaccharides to endogenous membrane acceptors (1, 3,11, 12, 24, 35, 48). However, at present, there is no strongevidence to indicate the extent to which the growth-inhibitoryeffects of retinoids depend on retinoid-binding proteins or on

modifications of cell membrane glycoconjugates.During the last few years, we have been studying the effects

of retinoids on a murine melanoma cell line (S91 C-2), which isvery sensitive to the growth-inhibitory action of retinoids (36,39, 40). In previous reports, we presented results which suggested that the cytoplasmic retinoid-binding protein found inthese cells plays a role in mediating the growth-inhibitory

effects of retinoids (39, 40). The present study was undertakento determine whether retinoic acid can modify the glycosylationand cell surface exposure of membrane glycoconjugates of theS91 melanoma cells, in an attempt to gain more insight into themechanism of retinoid action.

MATERIALS AND METHODS

Cell Culture. The murine S91 melanoma-cloned cell line C-2 used

in the present study has been described previously (36, 40). The cellswere cultured in Dulbecco's modified Eagle's minimum essential me

dium containing 10% fetal bovine serum and antibiotics (regular medium).

Treatment with Retinoic Acid. All-frans-ÃŸ-retinoic acid, the gift of

Dr. F. Frickel (BASF, Ludwigshafen, Federal Republic of Germany),was dissolved in ethyl alcohol or dimethyl sulfoxide and stored for upto 1 week at -70Â° under N2. Before each experiment, samples of the

stock solutions were diluted 1:1000 into the culture medium. Control

JANUARY 1983 303

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R. Lotan et al.

cultures received 0.1% of the appropriate solvent. Cells were refedwith fresh medium with or without retinoic acid every 3 days.

Metabolic Labeling of Glycoproteins and Proteins. Cells werecultured in 10-cm diameter dishes for 2 or 6 days in the absence or inthe presence of 10 UM retinoic acid in regular medium. Forty-eight hr

before the end of treatment, the medium was replaced with freshmedium (with or without retinoic acid) containing only 5% fetal calfserum and supplemented with 10-fiCi/ml amounts of either o-[6-3H]-

glucosamine (19 or 31 Ci/mmol; New England Nuclear, Boston, Mass.)or D-[2-3H]mannose (16 or 25 Ci/mmol; Amersham/Searle Corp.,

Arlington Heights, III.) to label glycoproteins.To label proteins, cells were cultured for 5 days in the absence or

presence of 10/IM retinoic acid in regular medium and then incubatedfor 2 hr in medium without methionine or without leucine. The cellswere then labeled for 24 hr either with L-[35S]methionine (50 /iCi/ml;1390 Ci/mmol; Amersham) or with i_-[4,5-3H]leucine (5 juCi/ml; 60 Ci/

mmol; New England Nuclear) in fresh medium (with or without retinoicacid) lacking methionine or leucine and containing only 5% fetal calfserum. At the end of incubation, the cells were washed with PBS3

detached with 2 mw EDTA in CMF-PBS and counted using a Model

ZBI Coulter Counter (Coulter Electronics, Hialeah, Fla.). The cells werethen solubilized and processed for PAGE in the presence of SDS andfluorography as described previously (38).

Radiolabeling of Cell Surface Proteins and Glycoproteins. Proteinsexposed on the surface of untreated and retinoic acid-treated cellswere radioiodinafed by lactoperoxidase-catalyzed 125l-iodination either

in situ, in monolayer cultures, as described previously (38), or in asuspension as follows. Cells were grown in 10-cm diameter dishes,washed with PBS, and detached with EDTA in CMF-PBS. The cells

were then washed with PBS and mixed with 0.5 ml of an iodinationsolution containing 40 mM D-glucose, 10 units lactoperoxidase (100units/mg; Calbiochem-Behring Corp., La Jolla, Calif.), 0.3 unit glucose

oxidase (1560 units per ml, type V from Aspergillus nicer; SigmaChemical Co., St. Louis, Mo.), and 0.2 mCi carrier-free I25I. After a 10-min incubation at 23Â°, 5 ml ice-cold serum-free medium were added;

the cells were pelleted, washed successively with medium and PBS,and then processed for SDS:PAGE as described (38).

Glycoproteins exposed on the cell surface were radiolabeled by 3methods: (a) the galactose oxidase;NaB3H4 (18); (b) the sialidase(neuraminidase) and galactose oxidase:NaB3H4 (18); and (c) theNalO4:NaB3H4 (17). These methods have been adapted to cultured

melanoma cells (34), and we used this procedure with a few modifications as follows. Subconfluent cultures, grown in 75-sq cm flasks orin 10-cm dishes, were washed with PBS and covered with 1.5 ml PBS

(adjusted to pH 6.9 with HCI) containing either: (a) 10 units of galactoseoxidase (AB Kabi, Stockholm, Sweden, or, when the latter becameunavailable, Sigma; 125 units/mg); or (b) 25 units of neuraminidase(Calbiochem-Behring Corp., La Jolla, Calif.) and 10 units of galactoseoxidase; or (c) PBS, pH 6.9 only and incubated at 37Â°for 1 hr. Other

cultures were placed on crushed ice and covered with 1.5 ml of 10 mMNalOi for 10 min. At the end of incubation, the enzymes or periodatesolutions were removed by aspiration, and the cell monolayers werewashed with PBS (pH 7.2) and once with 2 mM EDTA in CMF-PBS,

and the cells were detached with EDTA and washed in PBS (pH 7.2).After resuspension in PBS (pH 7.2) at 107 cells/ml in 0.5 ml, the cellswere radiolabeted by the addition of 1.25 mCi NaB3H4 (2.8 or 6.7 Ci/

mol; New England Nuclear or Amersham) and incubated for 15 min at23Â° in a ventilated hood. The labeled cells were washed in PBS and

processed for SDS:PAGE as described (38).Treatment of Cells with Exogenous Enzymes. Cells labeled meta-

3 The abbreviations used are: PBS, phosphate-buffered saline. pH 7.2 (containing, per liter of H2O: KH2PO, 0.25 g: Na2HPO4.7H2O, 2.16 g; KCI, 0.2 g,NaCI, 8.0 g; CaClz, 0.1 g, and MgCI2-6H2O. 0.1 g); CMF, calcium-free andmagnesium-free; PAGE, polyacrylamide gel electrophoresis; SDS, sodium do-decyl sulfate; TLC, thin-layer chromatography; gp160, glycoprotein migratingwith an apparent molecular weight of 160,000; WGA. wheat germ agglutinin.

bolically with [3H]glucosamine for 48 hr in 10-cm-diameter dishes were

washed with PBS, pH 6.9, and then incubated with 25 units of neuraminidase in 2 ml of the same PBS for 1 hr at 37Â°.The cells were then

washed with PBS and detached with EDTA. After solubilization, thecells were processed for SDS:PAGE as described (38). In some experiments, cells were treated with neuraminidase before they werelabeled by the NalO4:NaB3H4 method. Cells labeled metabolically with[3H]glucosamine were also treated, in monolayer, with a mixture of

trypsin and papain as described elsewhere (38).Electrophoretic Analysis of Labeled Glycoproteins and Proteins.

The labeled proteins and glycoproteins were analyzed by SDS-PAGE

followed by fluorography or autoradiography as described previously(38). Some fluorograms were scanned at 570 nm using a Gilford Model250B spectrophotometer. Relative increases in various glycoconju-

gates were estimated by calculations of the areas under certain peaks.Protein markers for molecular weight estimation included ovalbumin(M, 43,000), bovine serum albumin (M, 68,000), phosphorylase a (M,94,000), /?-galactosidase (M, 130,000), myosin (M, 210,000), and

fibronectin (M, 230,000).Metabolic Labeling, Extraction, and Chromatographie Analysis of

Glycolipids. Cells grown in the absence or in the presence of 10 JUMretinoic acid were labeled with 0.5 jiCi D-[1-14C]galactose per ml (56.5

mCi/mmol; New England Nuclear) during the last 24 hr of a 5-day

incubation. The cells were then washed with PBS, detached with EDTA,and washed with PBS. After pelleting by centrifugation, the cells (1 to2 x 107) were frozen at -70Â°. The cells were then thawed andhomogenized at 2 x 106/ml in chloroform:methanol (2:1) in a Dounce

homogenizer. Lipids were extracted overnight at room temperature in20 volumes of chloroform:methanol (2:1) by mixing the homogenatesunder a N2 atmosphere. The suspension was filtered over a sintered-glass funnel, and the filtrate was collected and stored at 4Â°under N2.

The insoluble residue was reextracted with chloroform methanol (1:2)for 5 hr. The suspension was filtered again, and the second filtrate wascombined with the first one. The combined extracts were evaporatedto dryness under a N2 stream and dissolved in chloroform:methanol(1:1). A small sample was used for analysis of total lipids by 2-dimensional high-performance TLC on TLC Silica Gel 60 F254-coated

aluminum sheets (5 x 7.5 cm) from Merck (Darmstadt, Federal Republic of Germany). Extracts from 1 x 106 cells were spotted on each

plate, and the plates were developed in the first and second dimensionswith chloroform:methanol:2.5 N NH4OH (60:30:4 and 60:35:8, respectively).

The remaining lipid extract was subjected to Folch partition (14) toseparate neutral glycolipids from gangliosides. Folch lower phase waswashed 5 times with theoretical upper phase before evaporating it todryness and dissolving it in chloroform:methanol (1:1). The Folch upperphases were combined, and their volume was reduced to 1 ml for acell extract of 107 cells. Nonlabeled brain gangliosides were added to

the upper phase to serve as a carrier before the material was dialyzedextensively to remove salt. The ganglioside preparation thus obtainedwas lyophilized.

The neutral glycolipids and the gangliosides were analyzed by TLCafter spotting extracts from 2 x 106 cells on precoated Silica Gel 60

glass plates (10 x 20 cm) from Merck using chloroform:methanol:2.5N NH4OH (60:30:4 and 60:35:8, respectively).

Standard glycolipid markers were chromatographed in parallel onthe same plates as were the analyzed samples. The markers included'"C-labeled CMH, ceramide dihexoside, and unlabeled ceramide trih-

exoside; all were obtained from Dr. D. Shapira (Department of OrganicChemistry, The Weizmann Institute, Rehovot, Israel. Purified bovinebrain gangliosides were obtained from Dr. B. Sela (Department ofBiophysics, The Weizmann Institute). '"C-Labeled GM3 was prepared

by metabolic labeling of B16-F1 cells. Gangliosides were visualized onplates by resorcinol-HCI (52). For autoradiography, the dried plateswere pressed against an X-ray film (Curix RP2 100 AFW, AGFA) and

kept in the dark at room temperature for 5 days.Radiolabeling of Cell Surface Glycolipids. Cells were labeled by 3

304 CANCER RESEARCH VOL. 43



Retinoic Acid-modified Cell Membrane Glycoproteins

methods, NalO4:NaB3H4, galactose oxidase:NaB3Hâ€ž,or neuraminidaseand galactose oxidase followed by NaB3H4. The cells were detached

with EDTA, suspended and washed in PBS, and resuspended in PBSat 1 x 107/ml. Treatments with NalO4 and enzymes were performed

as described above for glycoproteins except that the cells were treatedin suspension. The labeled cells were extracted with chloro-

forrrvmethanol (2:1) as described above, and samples of the lipidextract were spotted (extracts from 1 x 106 cells/spot) on high-

performance TLC Silica Gel 60 F2s4-coated aluminum sheets. The

plates were developed in chloroform:methanol:2.5 N NH4OH (60:35:8)and dried. For fluorography, the plates were immersed in 7% (w/v)PPO in diethyl ether (50), dried, placed against preflashed X-ray film(X-Omat AR; Eastman Kodak Co., Rochester, N. Y.), and kept in thedark for 2 weeks at -70Â°.

RESULTS

Alterations in Cellular Morphology Induced by RetinoicAcid. Cells exposed to 10 JUMretinole acid for longer than 72hr exhibited a modified morphology. The treated cells becamemore elongated and extended long, dendrite-like processes

(Fig. 1). Although the treated cells seemed smaller than theuntreated cells, their median volume [1313 Â±52 cujtim (S.E.)]measured after detachment with 2 rnw EDTA using a CoulterCounter, was similar to that of untreated cells (1125 Â± 37cujttm).

Removal of retinole acid from the growth medium resulted inthe reversal of the morphological changes within 48 hr (datanot shown).

Enhancement of Glycoprotein Glycosylation by RetinoicAcid. To determine whether retinole acid stimulates glycosyl-ation of cellular glycoproteins, we incubated untreated andretinole acid-treated cells with radioactive monosaccharides([3H]glucosamine or [3H]mannose) or amino acids ([3H]leucineor [35S]methionine). Analyses of the incorporation of these

precursors into trichloroacetic acid-insoluble material revealed20 to 40% reductions in their incorporation in retinoic acid-treated cells (Table 1). The incorporation of mannose was lessthan 10% of the incorporation of glucosamine.

Identification of the cellular components that were radiola-beled by the above precursors was achieved by SDS:PAGEand fluorography. Fig. 2 shows that retinoic acid enhanced atleast 4-fold the incorporation of both [3H]glucosamine and[3H]mannose into gp160. In addition to this major effect of

retinoic acid, there were a few minor changes in componentsindicated by arrowheads in Fig. 2.

In contrast with the changes induced by retinoic acid inmonosaccharide incorporation, there were no detectable effects on the incorporation of [36S]methionine into gp160 of

treated cells (Fig. 2, Lanes e and f). The same results wereobtained when the incorporation of [3H]leucine was similarly

analyzed and when Coomassie Brilliant Blue-stained slab gels

were observed (data not shown).Because metabolic labeling with radioactive monosaccharide

precursors may be influenced by differences in intracellularpool sizes of unlabeled monosaccharides in untreated andretinoic acid-treated cells, we used an additional method toidentify changes in the carbohydrate-containing macromole-cules of the cells. This method is based on the ability of sugar-

binding proteins (lectins) to bind glycoproteins in slab gels aftertheir separation by SDS:PAGE (5). Fig. 2 (Lanes g and h)shows that the pattern of components that can be identified by125I-WGA, a lectin that binds specifically both sialic acid (4, 21,

47) and A/-acetylglucosamine (2), is almost superimposable onthe pattern of [3H]glucosamine-labeled glycoproteins (Fig. 2;cf. Lanes g and h with Lanes a and b). Thus, more 125I-WGA

was bound to gp160 of retinoic acid-treated cells than to thecorresponding component of untreated cells (Fig. 2, Lanes gand h). In addition, 125I-WGA labeled more intensely a M,

210,000 component and M, 55,000 to 60,000 components oftreated cells.

Collectively, these results indicate that retinoic acid enhances the glycosylation of gp160 without increasing the synthesis of the polypeptide moiety. They also show that gp160oligosaccharide side chain(s) contains mannose and glucosamine (presumably as A/-acetylglucosamine).

Sensitivity of gp16O to Neuraminidase and Proteases. Theradlolabeling methods described above do not distinguish between cytoplasmic components and cell surface membraneconstituents. To gain some information on the location ofgp160 in retinoic acid-treated cells, we labeled such cells with[3H]glucosamine and then subjected the intact cells to enzymatic digestion of externally exposed proteins and sialoglyco-

proteins with the nonpenetrating enzymes neuraminidase or amixture of trypsin and papain. Analysis of the changes causedby these enzymes in the electrophoretic pattern of the labeledcomponents revealed that neuraminidase modified the mobilityof gp160, whereas the proteases caused the disappearance ofgp160 (Fig. 3). The neuraminidase-modified gp160 (asialo-

gp160) migrated further in the gels and had an apparentlylower molecular weight (M, 150,000). In addition, the amount

Table 1

Effect of retinoic acid on the incorporation of radiolabeled monosaccharides and amino acids intotrichloroacetic acid-insoluble material in melanoma cells

Radioactivity in trichloroacetic acid-insoluble material (dpm/105cells)8TreatmentNone

Retinoic acid[3H]Glucosamine25,530

Â±1.7206

19,780 Â±1,130[3H]Mannose2,280Â±290

1,710 Â±110[3H]Leucine86,050Â±6.620

52,040 Â±2,980[35S]Methionine674,790Â±51,920

506,800 Â±32.570

Cells were cultured in 10-cm diameter dishes in the absence or in the presence of 10 ^M retinoic acidfor 6 days, of which the last 48 hr were in medium containing only 5% fetal calf serum and 10-nCi/mlamounts of either o-{6-3H]glucosamine or D-{2-3H]mannose. Other cultures were labeled for the last 24 hrwith 5 /iCi L-f_4,5-3H]leucine per ml or with 50 /iCi/L-[35S]methionine per ml. The cells were washed withPBS. detached with 2 mM EDTA, and counted. All cultures contained 5 Â±0.2 x 106 cells/dish. Trichloroacetic acid (10%, w/v) was added to cell suspensions containing 106 cells each. After 3 hr at 4Â°, the

suspensions were filtered on glass fiber filters (Whatman GF/C), and the trichloroacetic acid-insolublematerial was collected on the filters. The filters were washed, dried, and counted in a scintillation counter.

6 Mean Â±S.E. of triplicate samples.

JANUARY 1983 305



R. Lotan et al.

of radioactivity in gp160 was reduced by 40 to 50% afterneuraminidase treatment.

These results suggested that gp160 is a component of thecell membrane, which is exposed on the surface of retinoicacid-treated cells and contains sialic acid.

Radiolabeling of gp160 on the Surface of Intact Cells. Tosubstantiate the indirect methods that indicated the surfacelocation of gp160, we used 3 complementary direct cell surface-labeling procedures: galactose oxidase:NaB3H4 (before

and after neuraminidase treatment), which is specific for galac-tosyl or /v-acetylgalactosaminyl residues (18); NalO4:NaB3H4,

which is specific for sialosyl residues (17); and lactoperoxi-dase-catalyzed iodination with 125I,which is specific for tyrosyl

and histidyl residues.Treatment with galactose oxidase followed by NaB3H4 re

sulted in a very weak radiolabeling (Fig. 4, Lanes c and d);however, a strong labeling was achieved when the cells weretreated with neuraminidase and galactose oxidase followed byNaB3H4 (Fig. 4, Lanes e and f). The latter method revealed that

a M, 150,000 glycoprotein (presumably asialo-gp160) waslabeled remarkably more intensely (at least 5-fold) on retinoicacid-treated cells than on control cells. Another band at M,130,000 showed moderately increased (about 2-fold) labelingon treated cells. The labeling with NalO4:NaB3H4 indicated a

striking increase (at least 5-fold) in the labeling intensity of

gp160 (Fig. 4, Lanes g and h). Neuraminidase digestion ofretinoic acid-treated cells before NalO4:NaB3H4 labeling abol

ished incorporation of tritium into gp160 (data not shown).These results demonstrate that gp160 is a cell membrane

glycoprotein which is exposed on the surface of retinoic acid-

treated cells and contains galactosyl.galactosaminyl, and sialosyl residues. The gp160 is the major sialo-galactoprotein on

the treated cells.Lactoperoxidase-catalyzed 126l-iodination of cells in suspen

sion revealed a decreased labeling of M, 210,000, 90,000,85,000, and 80,000 components and a relative increase in thelabeling of gp160 on the surface of retinoic acid-treated cellscompared to their labeling on untreated cells (Fig. 4, Lanes iand /). Since the metabolic labeling with amino acids failed toreveal increased synthesis of the gp160 polypeptide chain, theincreased labeling with lactoperoxidase suggests that gp160is more accessible to lactoperoxidase on treated than onuntreated cells, perhaps due to increased exposure of themolecule to the exterior. However, the possibility that there isan increase in the amount of gp160 in the membrane of treatedcells cannot be excluded.

Effect of Treatment Duration on Retinoic Acid-induced

Changes in gp160. Comparison of the glycosylation of gp160after 48 hr and after 6 days of treatment with retinoic acid (10fiM) revealed that the short treatment was sufficient to significantly enhance the incorporation of [3H]glucosamine into

gp160, but the effect of the longer treatment was considerablylarger (Fig. 5, cf. Lanes d, e, and f). Because of the lowincorporation of [3H]glucosamine during short pulses, we usedthe NalO4:NaB3H4 cell surface-labeling method to further ana

lyze the effect of treatment duration on glycosylation of gp160.As can be seen in Chart 1/4, the enhanced sialylation of gp160in retinoic acid-treated cells is a gradual process that can be

detected as soon as 12 hr after the addition of the compoundto the growth medium and that reaches a steady state after 3to 4 days.

0.61RE

0.4i02m

<n-

A1:â€¢Ai,\-â€¢ B1'A

i !-Iif

EXPOSURE TO RETINOIC ACID(DAYS)

RETINOIC ACIDUMI

Chart 1. Densitometer scans of gp160 labeled by NalO4-NaB3HÂ« on the

surface of untreated cells and on cells treated with 10 JIM retinoic acid for theindicated periods M) or treated with the indicated concentrations of retinoic acidfor 4 days (B). The labeled cells were solubilized, and the components wereseparated by SDSrPAGE and analyzed by fluorography. The developed filmswere scanned at 570 nm. Only the gp160 peak is shown.

Fig. 5 (Lanes a and c) shows the pattern of lactoperoxidase-catalyzed iodination of cells in monolayer (as opposed tolabeling cells after detachment and suspension shown in Fig.4, Lanes i and f). The labeling of substrate-attached control

cells is similar to the labeling in suspension except for anincreased labeling of a M, 240,000 protein (cf. Lane a in Fig.5 with Lane i in Fig. 4). Treatment with 10 /AMretinoic acid for48 hr was sufficient to reduce the extent of labeling of nearlyall iodinatable components except for the gp160. This effectbecame more pronounced after the longer treatment whengp160 became the major iodinatable component (Fig. 5, Lanec). Because there were no detectable changes in total cellproteins after treatment with retinoic acid (see Fig. 2, Lanes eand f), the iodination results suggest that retinoic acid changesthe accessibility of most membrane proteins to lactoperoxidase. Indeed, a quantitative analysis of trichloroacetic acidinsoluble radioactivity incorporated into control and into cellstreated with retinoic acid for 48 hr revealed a 50% reduction intotal cpm per mg protein.

Effect of Retinoic Acid Concentration on Enhanced Glycosylation of gp160. The physiological plasma concentrationof retinoic acid is 0.01 /IM (13), whereas the pharmacologicallyachievable concentration is 0.5 to 5 /Â¿M(15, 20). We thereforeexamined the enhancement of sialylation of gp160 after a 4-

day treatment with a range of retinoic acid concentrations.Chart 16 shows that treatment with 0.01 fiM retinoic acidcaused a small increase in labeling, whereas 0.1 fiM and higherconcentrations enhanced sialylation of gp160 by 3- to 5-fold.The effect of 0.01 /Â¿Mretinoic acid, although marginal, wasreproducible, but it required at least 4 days of treatment to bedetected.

Effect of Retinoic Acid on Glycolipid Biosynthesis. Sinceneutral glycosphingolipids and gangliosides are synthesizedfrom CMH by the sequential addition of monosaccharidesbeginning with o-galactose (10), we labeled cells metabolicallywith D-[14C]galactose during the last 24 hr of a 5-day incubation

in the absence or presence of retinoic acid and extracted thelipids. The incorporation of [14C]galactose into the lipid extractsof control and treated cells was 4.24 and 3.53 x 104 cpm/1x 106 cells, respectively. The glycosphingolipids were ana

lyzed by 2-dimensional TLC and autoradiography. Fig. 6, A

and B, shows that there are no significant qualitative differences in the glycolipid pattern between control (/A)and retinoicacid-treated (ÃŸ)cells. Since the gangliosides are not resolvedwell in this chromatography system, we separated them from




Retinole Acid-modified Cell Membrane Glycoproteins

neutral glycosphingolipids by Folch partition (14) and analyzedseparately the lower phase (neutral glycosphingolipids) andthe upper phase (gangliosides) by one-dimensional TLC (Fig.

6, C and F). These analyses showed no qualitative differencesbetween untreated and retinoic acid-treated cells. The pattern

of neutral glycosphingolipids is characterized by high proportions of glycolipids which migrate with a mobility similar to thatof CMH, ceramide dihexoside, ceramide trihexoside, and smallamounts of globoside (Fig. 6, C and D; Table 2). The ganglio-

side pattern is characterized by a high proportion of glycolipidswhich correspond in Chromatographie migration to GMs, GM,,and GDia and lower amounts of GM2and GDib (Fig. 6, E and Fand Table 2). It is noteworthy that variations in glycolipidbiosynthesis at different cell densities (22, 53) were also observed by us with S91 melanoma cells (data not shown).Therefore, the experiments were designed so that control andretinoic acid-treated cultures were analyzed at very similar

(Â±10%) final cell densities. On the basis of data obtained inseveral analyses, we concluded that the reduction in the synthesis of GM3in retinoic acid-treated cells is very reproducible,

whereas changes in GDia and GMi are variable. In some experiments, we used unlabeled cells (at least 107 cells) and ex

tracted the lipids and analyzed them by TLC. The plates weresprayed with resorcinol-HCI (52) and photographed, and trans

parencies of the photographs were scanned in a densitometer.The results of such experiments were usually in agreementwith the results of metabolic labeling, suggesting that theanalysis of "*C-labeled gangliosides represented the actual

amounts of total gangliosides in the cells.Radiolabeling of Glycolipids on the Surface of Intact Cells.

The exposure of cell surface glycolipids in untreated and inretinoic acid-treated cells was analyzed by labeling withNalO4:NaB3H4, with galactose oxidase:NaB3Hâ€ž,and with neur-aminidase and galactose oxidase followed by NaB3H4. The

labeled cells were extracted, and the lipids were analyzed byTLC and fluorography. Fig. 7 shows that the NalO4:NaB3H4

method labeled mainly a ganglioside (resorcinol positive) witha Chromatographie migration similar to that of GM3 and to alower extent a Gwi-like glycolipid (Fig. 7, Lanes b and f). The

Table 2Relative proportions of ["CJgalactose-labeled glycosphingolipids in untreated

and in retinoic acid-treated melanoma cells

% of [''CJgalactoseincorporated8GlycolipidGangliosidesGM3GÂ«GM,GOLGoibNeutral

glycolipidsCeramidemonohexosideCeramidedihexosideHematosideCeramide

trihexosideGlobosideControl55.56.010.919.18.533.830.525.68.21.9Retinoic

acidtreated34.38.222.323.711.540.534.418.04.52.6

a Cells were labeled with ["C]galactose as described in text. The lipids were

extracted and separated by Folch partition into a fraction containing mainlygangliosides and a fraction containing mainly neutral glycolipids. These werefurther separated by TLC. After autoradiography, zones on the plates thatcorresponded to radioactivity bands on the autoradiograms were scraped off theplates and collected into scintillation vials, and the radioactivity was determined.

labeling of these glycolipids on retinoic acid-treated cells wasabout 1.5-fold higher than on control cells. In some experi

ments, a glycolipid which migrated with a mobility similar toCMH was also labeled more intensely on treated than onuntreated cells.

Labeling with galactose oxidase:NaB3H4 was not efficient

(Fig. 7, Lanes c and g). However, treatment with neuraminidaseand galactose oxidase followed by NaB3H4 labeled a single

glycolipid with a migration similar to that of GMi. The labelingof this ganglioside on control cells was 3- to 4-fold more intensethan on retinoic acid-treated cells (Fig. 7, Lanes d and /?). It

should be emphasized that the cells were treated with NalO4 orthe enzymes after detachment and suspension; therefore, thedifferences in the exposure of the gangliosides are not due tothe differences in spreading of the cells on the substratum.

DISCUSSION

The results presented here demonstrate clearly that retinoicacid enhances the glycosylation of gp160, a cell surface membrane glycoprotein of S91 melanoma cells. In addition, retinoicacid modifies the exposure to the extracellular environment ofgp160 and of gangliosides that comigrate on thin-layer chro-

matograms with GM3and GMi- Although the accurate analysisof the oligosaccharide side chain(s) of gp160 must await purification of this molecule or of its glycopeptides, the results ofsugar incorporation and carbohydrate-directed cell surfacelabeling suggest that gp160 contains N-acetylglucosamine,mannose, galactose, and/or A/-acetylgalactosamine and sialicacid. They also show that retinoic acid increases the amount ofthese sugars on gp160. The increase in N-acetylglucosamine

and mannose in gp160 indicates that retinoic acid enhancesthe synthesis of asparagine-linked oligosaccharide side chains.These could be both the "high mannose" type and the"complex" type which contains also galactose and sialic acid

(25). However, our results do not exclude the possibility thatthe glycosylation of serine (or threonine)-linked oligosaccharide side chains, which contain galactose, N-acetylgalactosa-

mine, and sialic acid, is also stimulated by retinoic acid. Arecently reported analysis of cell surface glycopeptides of3T12-transformed fibroblasts showed that retinoic acid causesa shift from the "high-mannose" to the "complex"-type gly

copeptides (51 ). Such an effect is compatible with our findingson the intact gp160. The stimulation of the glycosylation of a"complex"-type (known also as serum glycoprotein-type) gly-

copeptide by retinol was demonstrated in corneas from vitaminA-deficient rats (28).

An additional characteristic of gp160 is its poor labeling withgalactose oxidase:NaB3H4 without concurrent neuraminidase

treatment. This suggests that most of the galactose and N-

acetylgalactosamine residues are substituted with sialic acid orthat their accessibility to galactose oxidase is stericallyhindered before removal of adjacent sialic acid residues. Similar observations were reported for human melanoma cells (34)and for a rat hepatocellular carcinoma (19).

The mechanism by which retinoic acid increases the incorporation of various monosaccharides into gp160 without adetectable increase in the incorporation of amino acids is notyet known. Others have also found that retinoids stimulate theincorporation of monosaccharides to a much greater extentthan they stimulated amino acid incorporation into glycopro-

JANUARY 1983 307



R. Lotan et al.

teins and mucins of normal or tumor cells (23, 30, 41). Aplausible explanation for such results is that the carbohydratemoieties of gp160 turn over at a faster rate than does theprotein moiety, as was reported for a M, 110,000 glycoproteinof rat liver plasma membrane (31). The half-lives of fucose,galactose, and sialic acid in the latter glycoprotein were 12.5,20, and 33 hr, respectively, whereas the half-lives of methio-

nine and arginine were 70 and 78 hr, respectively (31). Thismay mean that monosaccharides or oligosaccharides arecleaved off the protein by the action of glycosidases and arereattached to the protein by glycosyltransferases in a sequential manner several times in the life span of the protein. If weextrapolate these findings to gp160, we can explain our resultsby suggesting that retinole acid increases the amount of carbohydrate on gp160 either by inhibiting glycosidase or byenhancing glycosyltransferases. Whereas there is no experimental support for the inhibition of glycosidases, there areseveral reports on retinoid-enhanced glycoprotein galactosyl-

transferases in microsomes of trachÃ©alepithelium (48), in ratliver plasma membranes (9), and in human epithelial carcinoma(KB) cells (43). Preliminary results in our laboratory indicate anincreased galactosyltransferase activity in the retinole acid-

treated S91 melanoma cells. The most likely mechanism bywhich retinoids enhance glycosyltransferase activity is via theformation of glycosylretinoid phosphates, which can act asmonosaccharide carriers and donors for enzymatic glycosyltransfer reactions (11 ). Although most of the evidence on theformation of such lipid intermediates was obtained with retinol,a recent report has demonstrated the formation of a manno-sylphosphoryl derivative of retinole acid in 3T12-transformed

mouse fibroblasts (3).As there have been previous reports on the effects of reti

noids on cell surface components of cultured normal andtransformed cells, it seems appropriate to compare some ofthem with our results. Thus, retinol induced in NIL2K hamsterfibroblasts a decrease in the surface labeling of fibronectinwith a concomitant increase in the labeling of a M, 180,000galactose-containing glycoprotein (46). Radioiodination of cellsurface proteins on 3T6-transformed fibroblasts revealed that

retinole acid decreased the labeling of 2 M, 75,000 and M,90,000 proteins, while increasing the labeling of the M, 230,-

000 fibronectin (26). We have shown previously that retinoleacid enhances the glycosylation of a M, 230,000 cell membrane glycoprotein, which is not fibronectin, and the radioio-

dination of a M, 190,000 glycoprotein on the surface of thehuman cervical carcinoma cells HeLa (38). These results indicate that different cell types respond differently to treatmentwith retinoids by showing distinct cell surface changes. It isnoteworthy, however, that we have found recently that retinoicacid induces in murine B16 melanoma cells membranechanges that are very similar to those presented here for themurine S91 melanoma cells (37). This similar response suggests that, perhaps in closely related tumors, retinoic acid willinduce similar cell surface changes.

Few reports described the effects of retinoids on glycolipidbiosynthesis in cultured normal and transformed cells. Retinolincreased the synthesis of QMS in NIL2K and in their transformed counterpart NILpyT cells (46). In 3T3 fibroblasts, inwhich ganglioside synthesis depends strongly on the state ofcell contact (46, 53), retinol increased GM3synthesis in sparsecultures and stimulated GMI and GDia synthesis in touching

cells (46). There is no clear explanation for these changessince no effect of retinol on the activity of the glycosyltransferase involved in GM3 synthesis (CMP-sialic acid:

lactosylceramide sialyltransferase) could be detected (46). Incontrast, retinoic acid enhanced more than 14-fold the activity

of the above sialyltransferase in human epithelial carcinomacells (KB) and increased GM3 levels in these cells (43). Intransformed 3T12 mouse fibroblasts, retinoic acid caused anincrease in GMi and a decrease in GDib (42). Our results withthe S91 melanoma cells are different from those obtained withother cells, since we found that retinoic acid-treated cells

produced less GM3 than do untreated ones. The increases inGMI and GDia that we found in a few experiments are similar tothe results obtained with 3T3 cells (46).

The changes that retinoic acid induced in the accessibility ofGMSand GMi on the S91 melanoma cells could not be correlatedwith the level of these gangliosides in the cells and mayrepresent changes in the organization of membrane components (e.g., glycoproteins and glycolipids) that result in masking or unmasking of the gangliosides.

At present, we do not know what the function of gp160 is onthe S91 cells. The possibility that it is a degradation product offibronectin was excluded on the basis of lack of cross-reactivityof gp160 with antifibronectin antibodies. Such antibodies failedto bind to either untreated or retinoic acid-treated cells and didnot precipitate gp160 labeled by NalCvNaB3H4 from a 0.5%

Nonidet P-40 cell lysate (data not shown). Preliminary results

suggest that gp160 is not the receptor for epidermal growthfactor which has a similar molecular weight. Cell membraneglycoproteins of high molecular weight (M, >100,000) havebeen described that are implicated in growth control (33, 49),membrane transport and permeability (27, 44, 45), and adhesion to the substratum (6, 29, 51). Changes in such glycoproteins could result in modified cellular proliferation.

Retinoic acid inhibits the growth of S91 melanoma cells.Their growth rate is decreased, and their ability to form coloniesin agarose is reduced (40). The earliest changes induced byretinoic acid in the cell cycle can be detected 24 hr after itsaddition to the growth medium (40), whereas the increasedglycosylation of gp160 can be detected 12 hr after the additionof retinoic acid to the cells. It seems, therefore, that thechanges in the cell surface are not the result of reduced growthrate. It is tempting to speculate that the changes induced byretinoic acid in gp160 and in the organization of other cellmembrane components are causally related to growth inhibition. We have recently isolated mutants of S91 melanoma cellsthat are resistant to the growth-inhibitory effects of retinoicacid." Such mutants could be useful tools for the investigation

of the possible relationship between modifications of cell membrane components, cellular proliferation, and anchorage independence.

ACKNOWLEDGMENTS

We thank Dr. B. Sela for advice concerning glycolipid analyses.

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' R. Lotan and D. Lotan. unpublished observations.




Retinole Acid-modified Celt Membrane Glycoproteins

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16. Fuchs. E., and Green. H. Regulation of terminal differentiation of culturedhuman keratinocytes by vitamin A. Cell, 25: 617-625. 1981.

17. Gahmberg. C. G.. and Andersson, L. C. Selective radioactive labeling of cellsurface sialoglycoproteins by periodate-tritiated borohydride. J. Biol. Chem.,252. 5888-5894. 1977.

18. Gahmberg, C. G., and Hakomori. S. External labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythro-cytes. J. Biol. Chem., 248: 4311-4317, 1973.

19. Glenney, J. R.. Kaulfus, P. J., Mclntyre, B. W., and Walborg, E. F. Identification and partial characterization of the major galactoproteins present atthe surface of AS-30D hepatocellular carcinoma cells. Cancer Res., 40:2853-2859. 1980.

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21. Greenaway, P. J., and Le Vine. D. Binding of N-acetyl-neuraminic acid bywheat germ agglutinin. Nature (Lond.), 24): 191-192, 1973.

22. Hakomori, S. Structures and organization of cell surface glycolipid dependency on cell growth and malignant transformation. Biochim. Biophys. Acta..4/7 53-80. 1975.

23. Hassell. J. R., Newsome, D. A., and DeLuca, L. M. Increased biosynthesisof specific glycoconjugates in rat corneal epithelium following treatment withvitamin A. Invest. Ophthalmol. Vis. Sci.. 19: 642-647, 1980.

24. Helling, T., and Peterson, P. A. Galactosyltransfer in mouse mastocytoma:synthesis of a galactose-containing polar metabolite of retinol. Biochem.Biophys. Res. Commun., 46: 429-436, 1972.

25. Hubbard, S. C., and Ivatt, R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu. Rev. Biochem., 50: 555-583, 1981.

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27. Juliano. R. L.. and Ling. V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta., 455:152-162, 1976.

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31. Kreisel. W., Volk. B. A., Buchsei. R., and Reutter, W. Different half-lives ofthe carbohydrate and protein moieties of a 1,000-dalton glycoprotein isolated from plasma membranes of rat liver. Proc. Nati. Acad. Sei. U. S. A..77: 1828-1831, 1980.

32. Linder, S., Krondahl, U., Sennerstam, R., and Ringertz, N. R. Retinoic acid-induced differentiation of F9 embryonal carcinoma cells Exp. Cell Res..J32: 453-460, 1981.

33. Lipkin, G., Knecht, M. E., and Rosenberg. M. Glycoprotein-containing factorthat mediates contact inhibition of growth Ann. N.Y. Acad. Sci.. 3J2: 382-

391, 1978.34. Lloyd, K. O., Travessos, L. R., Takahashi. T., and Old, L. J. Cell surface

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35. Lotan, R. Effects of vitamin A and its analogs (retinoids) on normal andneoplastic cells. Biochim. Biophys. Acta, 605: 33-91. 1980.

36. Lotan, R.. Giotta, G.. Nork, E., and Nicolson, G. L. Characterization of theinhibitory effects of retinoids on the in vitro growth of two malignant murinemelanomas. J. Nati. Cancer Inst., 60: 1035-1041, 1978.

37. Lotan, R., Irimura, T., and Nicolson. G. L. Inhibition of experimental pulmonary metastasis by suppression or enhancement of the glycosylation ofmelanoma cell membrane glycoproteins. In: T. Galeotti, A. Cittadini, G. Neri,and S. Papa (eds.), Membranes In Tumor Growth, pp. 193-204. Amsterdam:Elsevier/North Holland Biomedicai Press. 1982.

38. Lotan, R., Kramer, R. H., Neumann, G.. Lotan, D., and Nicolson. G. L.Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line. Exp. Cell Res.. 130: 401-414,

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41. Marchok, A. C., Clark. J. N., and Klein-Szanto, A. Modulation of growth,differentiation, and mucous glycoprotein synthesis by retinyl acetate incloned carcinoma cell lines. J. Nati. Cancer Inst., 66: 1165-1174, 1981.

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46. Patt, L. M., Itaya, K., and Hakomori, S. Retinol induces density-dependentgrowth inhibition and changes in glycolipids and LETS. Nature (Lond.). 273:379-381. 1978.

47. Peters, B. P., Ebisu, S., Goldstein, J. Y., and Flashner. M. Interaction ofwheat germ agglutinin with sialic acid. Biochemistry. 78 5505-5511,1979.

48. Plotkin. G. M., and Wolf, G. Vitamin A and galactosyltransferase of trachÃ©alepithelium. Biochim. Biophys. Acta, 675: 94-102, 1980.

49. Raben, D., Lieberman, M. A., and Glaser, L. Growth inhibitory protein(s) inthe 3T3 cell plasma membrane. Partial purification and dissociation ofgrowth inhibitory events from inhibition of amino acid transport. J. Cell.Physiol., 108: 35-45. 1981.

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51. Sasak, W., DeLuca, L. M., Dion, L. D., and Silverman-Jones. C. S. Effect ofretinoic acid on cell surface glycopeptides of cultured spontaneously transformed mouse fibroblasts (BALB/c 3T12-3 cells). Cancer Res., 40: 1944-1949, 1980

52. Svennerholm, L. Quantitative estimation of sialic acids. M A colorimetrieresorcinol-hydrochloric acid method. Biochim. Biophys. Acta, 24. 604-611,1957.

53. Yogeeswaran. G., and Hakomori, S. Cell contact-dependent gangliosidechanges in mouse 3T3 fibroblasts and a suppressed sialidase activity oncell contact. Biochemistry, 74: 2151-2156, 1975.

JANUARY 1983 309



Fig. 1. Photomicrographs of S91 melanoma cells cultured for 4 days in the absence (/A) or presence (B) of 10processes extended by the treated cells. Phase-contrast, x 160.

retinole acid. Arrowheads, long-cellular

10~3x

molwt

230-

9P160+130-

94-

68-

43-

10~3x

molw t

-210

-130- 94

7- 'â€¢â€¢

- 68

- 43a b c d e f g h abc

Fig. 2. Fluorograms and autoradiograms of glycoproteins and proteins of cells grown for 6 days in the absence (Lanes a, c, e, and g) or presence (Lanes b, d, f,and n) of 10 JUMretinoic acid. The cells were radiolabeled with [3H]glucosamine (Lanes a and b), or [3H]mannose (Lanes c and cOduring the last 48 hr of incubationor with [35S]methionine (Lanes e and 0 during the last 24 hr of incubation. After membrane solubilization, similar amounts of trichloroacetic acid-insoluble radioactivitywere analyzed by SDSrPAGE in 6% acrylamide gels and fluorography. In Lanes g and h, cells were solubilized, extracts from a similar number of cells (5 x 105) wereseparated by SDS:PAGE, and the slab gels were incubated with I25l-labeled WGA and then processed for autoradiography as described previously (38). Numbers on

the left, electrophoretic migration of molecular weight markers. Arrows in Lanes a and h, gp160; arrowheads in Lanes a, c, d, and n, additional modified glycoproteins.Fig. 3. Fluorograms of glycoproteins of cells treated with 10 /IM retinoic acid for 5 days and labeled with [3H]glucosamine during the last 48 hr. The labeled cells

were further incubated with PBS (Lane a), with neuraminidase (Lane b), or with a mixture of trypsin and papain (Lane c) before their solubilization and analysis ofextracts from a similar number of cells by SDS:PAGE and fluorography. Arrow in Lane a, gp160; arrowhead in Lane b, asialo-gp160.

310



RetinÃ³te Acid-modified Celi Membrane Glycoproteins

10"3x

molwt

-210

-

Â«*â€¢

* Â« Â«Fig. 4. Fluorograms and autoradiograms of surface glycoproteins and proteins of cells cultured for 6 days in the absence (Lanes a. c, e. g, and /') or presence

(Lanes b, d. f, h, and Â»of 10 /IM retinole acid. The cells were suspended, and externally exposed carbohydrates were radiolabeled by reduction with NaBJt-U after

an incubation with: PBS (Lanes a and b); galactose oxidase (Lanes c and d); neuraminidase and galactose oxidase (Lanes e and 0; or NalO4 (Lanes g and h).Externally exposed proteins were labeled by lactoperoxidase-catalyzed 125l-iodination (Lanes / and Â».The labeled cells were solubilized, and extracts from a similarnumber of cells were subjected to SDS:PAGE in 5% acrylamide gels followed by fluorography or autoradiography. Arrowhead in Lane /, asialo-gp160, arrowheadsin Lane ;'. proteins that are labeled more intensely on untreated cells than on treated cells.

Fig. 5. Autoradiograms and fluorograms of cell surface proteins and glycoproteins of cells cultured in the absence of retinole acid for 6 days (Lanes a and d) orin the presence of 10 /IM retinoic acid for 2 days (Lanes b and e) or 6 days (Lanes c and 0. Intact adherent cells were labeled by lactoperoxidase-catalyzed iodination(Lanes a and c) or by a 48-hr incubation with pHJglucosamine (Lanes d and 0 The labeled cells were solubilized and subjected to SDS:PAGE in 5% acrylamide gelsfollowed by autoradiography or fluorography. There was no difference in the labeling of control Day 2 and Day 6 cells (not shown). Arrow, gp160; arrowheads in Lanea. high-molecular-weight components labeled more intensely on untreated than on treated cells.

JANUARY 1983 311



R. Lotan et al.

â€¢<*â€¢%

CMH â€”

CMH- "* '

/ 'M3

HematoCDH- Ãœ f::Wi t CTH

Globoâ€” t* â€¢

CTH

CDH-ff i "G

Â«l.

tOrigin- ' Originâ€” â€¢.^ Jti:

B CD E F

Fig. 6. Autoradiograms of glycolipids extracted from cells cultured in the absence of (Lanes A, C, and E) or presence of 10 JIM retinole acid (Lanes B. D, and F)for 5 days and then labeled with [14C]galactose for 24 hr. Total lipid extract was analyzed by 2-dimensional TLC (Lanes A and 8). Neutral glycolipids (Lanes C and

D) and gangliosides (Lanes Â£and F) were analyzed by one-dimensional TLC. Tentative assignment of bands was based on Chromatographie migration of standards.Hemato, hematoside; Globo, globoside; CDH, ceramide dihexoside; CTH, ceramide trihexoside.

B

GM3

GMI

f g hFig. 7. Fluorograms of glycolipids labeled on the cell surface. Cells were

cultured for 5 days in the absence (A) or presence (ÃŸ)of 10 fiM retinole acid andthen labeled by NaB3l-U alone (Lanes a and e), by NalO4:NaB3H4 (Lanes b and f),by galactose oxidase-NaB3H4 (Lanes c and g), or by neuraminidase and galactoseoxidase followed by NaB3H4 (Lane d and n). Lipid extracts of the labeled cellswere analyzed by TLC on high-performance TLC plates and fluorography. TheChromatographie migration of GM3and GMI on these plates is indicated on theleft.




1983;43:303-312. Cancer Res Reuben Lotan, George Neumann and Varda Deutsch Melanoma Cellsby Retinoic Acid in Cell Surface Glycoconjugates of S91 Murine Identification and Characterization of Specific Changes Induced

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