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Experimental CM1 Research 92 (1975) 259-270

EXOGENOUS GLYCOSPHINGOLIPIDS SUPPRESS GROWTH RATE OF TRANSFORMED AND UNTRANSFORMED 3T3 MOUSE CELLS T. W. KEENAN’,

ERIKA SCHMID, W. W. FRANKE

and H. WIEGANDTa

IDivision of Membrane Biology and Biochemistry, Institute for Experimental Pathology, German Cancer Research Center, D-69 Heidelberg, and *PhysioIogisch-Chemisches Znstitut, Philbps-Universitit, D-355 MarburglL., BRD

SUMMARY Gangliosides added to culture media reduced both the growth rate and saturation density of SV40-virus transformed and untransformed 3T3 cells. Monosialogangliosides were much more effective than disialogangliosides in inhibiting growth rate. These gangliosides caused little or no cell damage or significant morphological alteration of the individual cells. Trisialoganglioside markedly reduced growth rate but in some experiments also caused cell damage and lysis. The isolated carbohydrate moiety of the ganglioside G otetlr the sialo-oligosaccharide galactopyranosyl-N-acetyl-galactosaminyl-(N-acetylneuraminyl)-galactosyl-glucose, did not inhibit growth of SV40 3T3 cells in culture. Ceramide alone was also ineffective as a growth inhibitor. However, the tetrahexosyl ceramide derived from the above ganglioside was equally as effective as the parent compound in retarding growth of SV40 3T3 cells. Similarly, mono-, di- and trihexosyl ceramides were also effective in inhibiting growth of these cells. Gangliosides added to the culture media were rapidly accumulated by cells, apparently at the plasma membrane. The accumulated ganglioside was not degraded by the cells. However, the accumulated ganglioside could be distinguished from gangliosides synthesized in vivo by the lability of the former to neuraminidase.

Glycosphingolipids, which appear to be ubiquitous among mammalian tissues, occur in various intracellular endomembranes but Abbreviations: Cer, ceramide; Glc, glucose; Gal, galactose; GalNAc, N-acetylgalactosamine; NeuNAc, N-acetylneuraminic acid; Lac, lactose; Gtri, gangliotriose (GalNAQl-4Galal-4Glc); Gtet, gangliotetraose (Gal~1-3GalNAc~1-4Gal,8l-4Glc). Glycosphingolipids are abbreviated by linking the short notation of the carbohydrate moiety and ceramide, e.g., Lac-Cer, Gtri-Cer, Gtet-Cer. Gangliosides are designated according to principles outlined earlier (ref. [35]): G stands for ganglioside, the index, e.g. G otet, is an abbreviation for its neutral sugar moiety. To this is added the number of sialic acid residues, a, b to distinguish between positional sialoisomers: G,,l ; NeuNAc-Gal-Glc-Cer; G-2; NeuNAc-NeuNAcGal-Glc-Cer: G,+,, 1: GalNAc-(NeuNAc)-Gal-GlcCer; G otetf; Gii:‘GglNAc-(NeuNAc)-Gal-Glc-Cer; 2a; NeuNAc-Gal-GalNAc-(NeuNAc)-Gal-GlcZ?& o&B; Gal-GalNAc-(NeuNAc-NeuNAc)-GalGlclCer; G otet3; NeuNAc-Gal-GalNAc-(NeuNAcNeuNAc)-Gal-Glc-Cer. 18 - 751808

are concentrated in plasma membrane [l-6]. In biosynthesis the carbohydrate moieties of these glycolipids are added in a stepwise manner by glycosyltransferases which are concentrated in Golgi apparatus [7, 81.While glycolipids, and in particular the sialic acidcontaining gangliosides, have long been implicated to function in cellular recognition and adhesion [9, lo], there has been a recent upsurge in interest in these membrane constituents due to altered glycolipid patterns manifested in transformed cells. Relative to nontransformed controls, virally and chemically transformed cell lines [ll-211 and solid tumors [22-251 have been reported to have altered neutral glycolipid and/or ganglioside patterns. In nearly all casesthis alteration in Exptl Cell Res 9.2 (1975)

260 Keenan et al. transformed cells and tumors has involved an accumulation of lower glycolipids at the expense of higher homologs. This simplifica-

tion in ganglioside pattern was found to correlate with depression in the level of a glycosyltransferase activity [l 1, 12, 19-251. While different glycosyltransferases are depressed

in

activity

in different

transformed

cells or tumors, the glycolipid substrate for the depressedtransferase accumulates at the expenseof higher glycolipid homologs. Moreover, these glycolipid changes appear to be correlated with a loss in contact inhibition [26-301. The foregoing observations suggest that glycosphingolipids of plasma membrane may influence cell growth and morphology. It was thus deemed desirable to ascertain if exogenously supplied gangliosides could bind to cell surface membrane and alter growth behaviour of cells in culture. It is known that glycolipid Lewis antigens are accumulated by erythrocytes [3 l] and that cultured cells derive lipids from serum ~321.Since initiation of this study, Laine & Hakomori [33] reported that globoside (a trivial name for GalNAc,!?l+ 3Galal --f4Gal,%+ 4Glc-ceramide) added to culture medium was incorporated into the plasma membrane of NIL and transformed NIL hamster cells. These authors also noted a concomitant reduction in growth rate and reduced saturation densities. We report here that several gangliosides alter growth behaviour of cultured cells. MATERIALS AND METHODS Clone No. 10 of the Swiss mouse 3T3 cell line, which had been selected and subcloned for optimal contact inhibition and which was cultured under strict 3T3 schedule [34], was used along with a SV40 DNA tumor virus-transformed 3T3 cell line. Cells were grown in Eagle’s basal medium (BME) supplemented with 10% fetal calf serum contained in Falcon cell culture plastic Petri dishes. Cells were counted with a Coulter electronic particle counter after being released from the monolayer with trypsin under standard Exptl Cell Res 9.2 (1975)

conditions. Morphology of the growing cells was observed in phase contrast using a Leitz inverted stage microscope. Gangliosides GLac1 from dog erythrocytes, GGtril from human Tay-Sachs brain and GGtetl from bovine brain were isolated as in previous studies 1361.Gctet2a, Gotet26, and Gotet3 were from normal human brain [37]. Ceramide, glucosyl ceramide and lactosyl ceramide were prepared from bovine milk fat globule membranes [36]. The asialo derivatives of and ‘htetl (Gtri-Cer apd Gtet-Cy resPccttvely) were prepared by mild actd hydrolyses [36] and the desphingo derivative of Go& was obtained by ozonolysis and alkaline fragmentation [38]. C&J was made radioactive by oxidation of the terminal galactose with galactose oxidase (from Sigma Chemical Co., St Louis, MO) and reduction with tritiated sodium borohydride [39, 401. The resultant product had a specific activity of 1.3 x lo6 cpm/nmole. Gangliosides were evaporated from chloroform-methanol in sterile tubes under a stream of nitrogen. Tubes were heated at lOO”C, cooled, and culture medium was added. For all but the lowest ganglioside homolog solution was effected by vigorous mixing with a Vortex mixer. With GLacl and the neutral glycolipids it was necessary to treat with ultrasound, with cooling intervals, to yield transparent solutions. Alternatively, dry gangliosides were directly dissolved in the growth medium at the desired concentrations. This application, however, was somewhat less effective, perhaps due to the formation of micelles. Gangliosides were extracted from cells washed with phosphate-buffered saline and their quantity was measured by sialic acid assay as in previous studies 1361. yhen radio?ctive.%d wasused,retake was deterrrnned by dtssolvmg washed cells m Nuclear Chicago Solubilizer and determining radioactivity in a toluene-based scintillation fluid. Protein was determined according to Lowry et al. [41]. For neuraminidase treatment washed cells were suspended in 0.02 M acetate buffer, pH 5.2, containing 0.15 M NaCl. Incubation was for 3 h at 37°C with 0.1 unit of neuraminidase (from Clostridium perfringens, Boehringer, Mannheim, BRD) per 10 mg protein. For comparison, bovine milk fat globule membranes [42] were also treated with neuraminidase. Thin layer chromatographic separation of gangliosides was as in previous studies [36]. G~tr11

RESULTS The SV40 transformed 3T3 cell line has been found to have only one major ganglioside, namely G-1 [15, 171. Other gangliosides are present only in trace quantities, if at all One factor contributing to this simplified ganglioside pattern has been shown to be a greatly diminished activity in the enzyme transferring N-acetylgalactosamine from UDP-N-acetylgalactosamine to G,,l

261

Exogenous gangliosides and cell growth 12 10 8 6 h 2 0

Fig. 1. Abscissa: days in culture; ordinate: cell no. x 10-s. Effect of monosialogangliosides on growth rate of SV40 T33 cells. 60000 cells were added to 2 ml of media without (0) or with 140 nmoles/ml of Go,1 (A) or Go,,1 (A). At the intervals noted, cells were harvested and counted as described in the text.

[17, 211.In contrast, untransformed 3T3 cells have high levels of activity in this enzyme and a complete complement of mono- and disialogangliosides [15, 17, 211. It was thus of interest to determine if GOt,,l or Go& would retard growth of SV40 3T3 cells. Both gangliosides reduced the growth rate and saturation density of these cells (fig. 1). G,,il was the more effective ganglioside, reducing the number of cells at saturation to about 50% of the control level. GGtetl reduced the number of cells at saturation to 60-70 % of control levels. However, this growth retardation was not selective for the transformed cells; growth rate of 3T3 cells was also depressed by addition of these gangliosides to the culture medium (fig. 2). Moreover, GL,,l, which is present in high amount in SV40 3T3 cells [I 5, 171,was also effective in reducing growth rate of both transformed and untransformed cells (fig. 2). At all concentrations of the three gangliosides tested, this effect on growth rate was greater with SV40 3T3 cells. With all three gangliosides there was a proportional, but not linear, decrease in growth rate of 3T3 cells as ganglioside concentration increased. This was not true with SV40 3T3 cells, where 70 to 100

nmoles/ml of GLscl or Gctetl had little or no more effect than ‘20:&oles/ml of the respective ganglioside. In contrast, there was a proportional decrease in the number of SV40 3T3 cells with increasing concentrations of Gctril. At concentrations higher than about 70 nmoles/ml, GhC1 crystallized from solution, thus preventing determination of the effect of this ganglioside at higher concentrations. Monosialogangliosides were much more effective than disialogangliosides in retarding growth of both 3T3 and SV40 3T3 cells (table 1). With each of the three monosialogangliosides, growth retardation was greater with SV40 3T3 cells than with untransformed 3T3 cells. At both 2 and 3 days of incubation, G,,,,l was more effective in growth retardation than were G,,, 1 or G,,,l, which were similar in effect. While the two disialogangliosides were much less effective than any of the monosialogangliosides at the sameconcentration, the greater growth retardation was noted with 3T3 cells. A remarkably effective ganglioside was the trisialoganglioside Gcte,3. However, in some experiments this ganglioside revealed unexplained cytotoxic effects;

I

I

I

,

I

0.05

0.10

0.05

0.10

Fig. 2. Abscissa: ganglioside concentration in ,umoles/ ml; ordinate: % of control cell no. Concentration dependence of the growth retarding effects of gangliosides on SW0 3T3 cells (A) and untransformed 3T3 cells (B). Gangliosides GLBcl (o), Gatril (0) of Go& (A) were added to media at the indicated levels. The two ml cultures, seeded with 200000 3T3 cells or 100000 SV40 3T3 cells. were incubated for 3 days and the cells were recovered and counted. Results after 2 days in culture were similar to those shown. Exptl Cell Res 92 (197.5

262

Keenan et al.

Table 1. Effect of gangliosides on the growth

moiety alone is not responsible for growth rate of 3T3 and SV40 3T3 ceW retardation, but rather the lipid moiety is also required. Gangliotetraosyl-ceramide was Concenequally as effective as the parent compound in tration SV40 3T3 3T3 retarding SV40 3T3 cell growth, suggesting (nmoles/ Ganglioside 2 Day 3 Day 2 Day 3 Day ml) that sialic acid is not necessaryfor this effect. Thus, addition of one sialic acid residue to the 51 56 65 51 48 G,d tetrasaccharide unit has no effect on growth 65 45 45 66 51 %tril 65 12 56 80 83 retarding properties of the glycosphingolipid. 85 82 80 18 65 Addition of a second sialic acid residue, which 65 86 84 G c&b 69 54 65 iS present in GGt,,2a and &,t2b, decreases 10 98 83 growth retarding ability. However, addition ’ Cells were cultured in 2 ml of media containing the of a third sialic acid apparently tends to indicated amount of eandioside in 8 cma dishes. At restore this ability. While the lipophilic moiethe indicated inter& -cells were recovered and ty of the ganglioside was required, ceramide counted as described in the text. Results are percentage of cells relative to controls without exogenous alone had no effect on cell growth (table 2). gangliosides. Initial inocula consisted of 100 000 SV40 It thus appears that both carbohydrate and 3T3 cells or 200 000 3T3 cells per culture. lipid moieties must be present to impart none of the other gangliosides were cyto- growth-inhibiting properties to the sphingotoxic at the concentrations tested (cf a fol- lipid. The number of carbohydrate residues lowing section). At greatly reduced concen- did not appear to be critical, however, since tration, G,,,3 was seen to have neither such Glc-Cer, Lac-Cer, Gtri-Cer and Gtet-Cer all cytotoxic effectsnor a strong growth retarding depressedgrowth of SV40 3T3 cells to about effect. One interesting effect of GGtit3 was 38 to 50% of control levels (table 2). This is that at 65 nmoles/ml it was more effective the same range of growth retardation noted with SV40 3T3 cells whereas at 10 nmoles/ ml the greater effect was with the untrans- Table 2. Effect of glycosphingolipids on the formed 3T3 cells. These growth repressing growth rate of SV40 3T3 celW effects of gangliosides were readily reproducible. With new passagesat the same in% of control cell numbers cubation time and in the presence of the Sphingolipid 2 Day 3 Day same level of ganglioside, the percentage of growth inhibition was always similar. For GLJ 67 59 55 37 example, in five separate experiments with G Gtri 1 G Gtetl 66 51 G,,,l the number of SV40 3T3 cells ranged Gal-GalNAc-(NeuNAcb Gal-Glc 104 89 from 49 to 59 % of controls after three days of Gtet-Cer 73 incubation. Gtri-Cer 52 3: 69 36 Attempts were made to determine which of Lac-Cer Glc-Cer z38 38 the constituents of a ganglioside was respon- Ceramide 89 sible for growth inhibition. Compared to the Cells were cultured in 2 ml of media containing an ganglioside, the isolated oligosaccharide por- ainitial concentration of 65 mnoles/ml of the indicated tion of native GGtet1 had little or no growth sphingolipid in 8 cm* Petri dishes. At the indicated cells were recovered and counted as indiretarding effect with SV40 3T3 cells (table 2). intervals cated in the text. Initial inocula consisted of 100 000 It is thus obvious that the carbohydrate cells/culture. Exptl Cell Res 92 (1975)

Exogenous gangliosides and cell growth

with monosialogangliosides at the sameinitial concentrations. The effective concentration of the neutral glycosphingolipids in solution was lower than the initial concentration. While all lipids could be dispersed to yield transparent solutions, during culture crystals were seen to form, in phase contrast, in cultures containing Glc-Cer, Lac-Cer or Gtri-Cer. Thus the effective concentration of these compounds while not determined, was certainly lower than that of the gangliotetraosyl-ceramide or gangliosides, which were not observed to crystallize from solution at the same concentrations. Additionally, cells cultured in media containing Glc-Cer or Gtri-Cer were sometimes seen to dissociate from the monolayer between the second and third day of incubation. When this occurred, all cells appeared to be dissociated and floated free in the media. With the exception of the cell damages sometimesnoted with G,,,,3 and an increased number of rounded off cells in the presenceof G,J (fig. 4b), none of the gangliosides tested considerably altered the morphology of the cells (figs 3 to 5). The decreased saturation density attained in ganglioside-enriched media was characterized by holes in the cell layer. Spike-like extensions of normal morphology were more frequently recognized with the treated cells (e.g. fig. 3) but this may simply reflect the lower cell density. The impression was gained that cells grown in gangliosidecontaining media had somewhat increased adhesivenessfor each other. A marked reduction of piling up of cells grown in gangliosideenriched media was noted throughout (figs 4, 5), even at comparably high cell densities, and this seemedto correlate with a tendency of the cells to reveal a more flattened appearance and a sharper, more defined cell outline. These observations gave the impression that SV40 3T3 cells were behaving more as contact inhibited cells in such media. The cell damag-

263

ing effect that was occasionally noted in the presence of Gcte,3 (figs 3d, 5d) was characterized by large amounts of cellular debris, something not seen in the presence of other gangliosides. Such an action of the ganglioside G otet3 is possibly due to special amphiphatic properties resulting from its structure. The other gangliosides are also surfactants, but since they are not cytotoxic it is probable that a detergent effect was not responsible for their growth retarding properties. The morphology of cells growing in neutral glycosphingolipid containing media was similar to the morphology in monosialoganglioside containing media (not shown). With neutral glycolipids there was little evidence of cell damage and the decreasedsaturation density was characterized by holes in the cell sheet. That exogenously supplied gangliosides were taken up by SV40 3T3 cells was demonstrated with both monosialo- and disialogangliosides. Radioactive Gctetl added to growing cultures was rapidly accumulated by cells (fig. 6). This uptake increased in direct proportion to cell numbers. On a per cell basis, uptake was nearly maximal in the first 24 h in culture; with 100 nmoles/ml of added ganglioside, at this time point there was 1.09 nmoles of 3H-Gct,tl bound per lo6 cells. The amount of ganglioside bound increased somewhat between the fourth and fifth days, when the cells were in stationary phase (fig. 6). The level of increase was from 1.20 to 1.41 nmoles/106 cells over this time interval. The average amount of 3H-Gctetl per IO6cells for the five day period was 1.15 nmoles. Radioactive GOtetl was incorporated as such and was not further metabolized by the cells. For example, cells harvested after three days had an amount of radioactivity equivalent to 1.17 nmoles GGtetl/106 cells. A total of 7 % of this radioactivity was associated with the insoluble material after chloroformmethanol extraction. Of the radioactivity in Exptl Cell Res 92 (197.5)

264 Keenan et al.

Fig. 3. Morphology of 3T3 cells cultured for 3 days inI media containing exogenous gangliosides. (0) Control without added ganglioside; (b) G,,,J; (c) Gotet2a; (c1) GGtet3. All gangliosides were present at a level of 65 nmoles/ml. The morphology of cells in the presence 01r other gangliosides is similar to that shown in (b) and (c) and is not shown. x 230. Exprl Cell Res 92 (1975)

Exogenous gangliosides and cell growth

264

of SV40 3T3 cells cultured for 3 days in media containing exogenous gangliosides. (a) Control without added ganglioside; (6) GGL,,l; (c) G, ml; 68 Gctet 1. All gangliosides were present at a level of 65 nmoles/ml. x 115.

Fig. 4. Morphology

Exptl Cell Res 92 (1975)

266

Keenan et al.

Fig. 5. Morphology of SV40 3T3 cells cultured for 3 days in media containing exogenous gangliosides. (a) G Gt,t2a: (b) Gate&; (c) and (d) Gotet3. In (a), (b) arId (d), gangliosides were present in a concentration of 65 nmoles/ml and in (c) at 10 nmolesjml. The cell (damages in the presence of Got,%3 (d), however, were not consistently observed. x 230. Exptl Cd

Res 92 (1975)

of ganglioside sialic acid. However, neuraminidase treatment of SV40 3T3 cells grown in G,,,,2b-containing medium resulted in a decrease of ganglioside sialic acid from 3.74 nmoles/mg protein in untreated cells to 3.06 nmoles/mg protein in treated cells. Only one of the two sialic acid residues in Gctet2b is labile to neuraminidase. Thus with uptake of 0.7 nmoles G,,,,2b/mg protein (1.4 nmoles of sialic acid), the amount of ganglioside sialic acid released,0.68 nmole/mg protein, is close Fig. 6. Abscissa: days in culture; ordinate: (left) cpm x to the theoretical amount of 0.7 nmole which lo-$; (right) cell no. x 10m6. Accumulation of radioactive Goti+,1 by SV40 3T3 could be released were the incorporated cells. Cells were cultured in media containing 100 ganglioside completely accessibleto neuraminmoles/ml of 3H-Gotetl (3 170 cpm/nmole). At the indicated intervals cells were harvested and washed nidase. One of the two major gangliosides of 3 times with phosphate-buffered saline. Portions were SV40 3T3 cells, G,,,l, is freely labile to taken for counting and the remainder was used for neuraminidase in solution. It is thus obvious determination of radioactivity as described in the text. n, Cell count; 0, cpm. that the exogenously supplied ganglioside does not behave as gangliosides synthesized the extract, 87 % was recovered in the GOtet1 in vivo with respect to neuraminidase lability. region of thin layer chromatograms. The re- Two possible explanations for this non-accesmaining 13 :A was nearly totally accounted sibility of endogenous gangliosides can be for in the regions corresponding to other suggested. One is that they are covered or masked by other lipids or proteins which gangliosides. That gangliosides become associated with cells does not indicate that they are in- Table 3. Lability of endogenous and exogenous corporated into membranes in the same con- gangliosides to digestion by neuraminidasea figuration as are gangliosides synthesized in Ganglioside sialic acid, vivo. The observations that ganglioside sialic nmoles/mg protein acid in intact cells r431and isolated membrane UnNeuraminidasevesicles [44, 451is resistant to neuraminidase Fraction treated treated under conditions where glycoprotein sialic acid is readily released provides a possibility Control ~~40cells 2.34 2.20 3.06 for testing the orientation of exogenously GangiiosideSW) &IS 3.74 Milk fat globule memsupplied gangliosides. SV40 3T3 cells cultured brane 6.03 5.92 in media containing GctOt2bwerefound to have SV40 3T3 cells were cultured in media without or 60 % more ganglioside sialic acid per mg awith 10 nmoles/ml of ganglioside Gotet2b. After 3 days protein than the same cells cultured in media cells were harvested with a rubber policeman, washed 3 times with phosphate-buffered saline, and resuswithout added ganglioside (table 3). Assuming pended in 0.02 M acetate buffered saline, pH 5.2. that the added ganglioside was not degraded One portion served as a control and was extracted The other portion was incubated for 3 h and the sialic acid re-utilized, this amounted immediately. at 37°C w&h 0.1 unit of neuraminidase/lO mg protein to an uptake of 0.7 nmole G,,,,2b/mg protein. for ‘3 h before extraction. For comparison, bovine fat globule membranes were treated in parallel. Treatment of control cells with neuraminidase milk Gangliosides were recovered and measured by caused only a miniscule decreasein the level methods cited in the text. Exptl Cell Res 92 (1975)

268 Keenan et al.

would prevent interaction with neuraminidase.Another is that gangliosides in the plasma membrane are oriented on the interior surface. While this seemsunlikely since gangliosides appear to be synthesized in the same topological orientation as are glycoproteins [7, 81, results obtained on neuraminidase treatment of intact cells [43] or isolated membrane vesicles [43, 451 do not rule out this possibility. However, when this potential orientation of gangliosides was tested with neuraminidase in a membrane fraction such as the milk fat globule membrane which exposes natural cell surface derived from the apical plasma membrane of mammary secretory ceils but can also be isolated in the form of membrane sheets permitting access to both faces of the membrane [42, 461,there was no decrease in ganglioside sialic acid after treatment (table 3). The major ganglioside of this membrane, G,,2, is quantitatively converted to Lac-Cer by neuraminidase in solution [36]. Under these digestion conditions, 70 % of the total sialic acid of the membrane was released. While-this result does not rule out an orientation for gangliosides on the inner surface of plasma membranes, it does clearly demonstrate that surface membrane gangliosides are in any case cryptic with respect to neuraminidase lability.

the initial observation of Laine & Hakomori [33], who obtained similar results by adding globoside to culture medium, and extends the observation to a series of gangliosides. While both mono- and disialogangliosides exert this effect, the monosialogangliosides are markedly more effective. Of all the gangliosides tested only trisialoganglioside caused in some experiments cell damage or lysis. Other gangliosides caused little or no morphologically detectable ceil damage. They simply reduced saturation density of cells, possibly by increasing the adhesiveness of cells for each other. The free carbohydrate moiety of GGtetl alone was ineffective in growth retardation, suggesting that the ceramide portion of the molecule is essential for growth depression. This corresponds to findings that addition of glucose, galactose or N-acetylgalactosamine at concentrations of up to about 70 pmoles/ml to cultures of various mammalian cell lines, including mouse 3T3 cells, did not change growth rate and cell morphology [47]. The ceramide portion is probably necessary to anchor the molecule in lipophilic regions of the surface membrane. Ceramide itself was ineffective in growth repression of SV40 3T3 cells, suggesting that at least some carbohydrate residues must be attached to ceramide to impart this effect to the molecule. The number of neutral carbohydrates attached to ceramide appears DISCUSSION not to be critical, since Glc-Cer, Lac-Cer, Cellular contents of certain glycosphingo- Gtri-Cer and Gtet-Cer were all nearly equally lipids increase when cells become contact effective in depressing growth of SV40 3T3 inhibited [26-301 and it has been suggested cells. Moreover+ gangliotetraosyl-ceramide that this increase may prevent S phase initia- was equally as effective as the parent gangliotion (see ref. [33]). This suggestion is sup- side in retarding growth. Thus the addition ported by observations that tumor cells, of sialic acid to the Gtet-Cer molecule does which have altered glycosphingolipid patterns, the following: Addition of one sialic acid are not contact inhibited [26-301. Results residue has no effect on the growth retarding contained herein show that cells cultured in properties. Addition of a second sialic acid media with exogenous gangliosides have a residue either to the ultimate galactose greatly depressedgrowth rate. This confirms (yielding Gctet2a) or to the existing sialic acid ExptI Cell Res 92 (1975)

Exogenousgangliosides and cell growth 269 (yielding G,,,,%) decreases the growth retarding properties markedly. Addition of a third sialic acid residue, however, does not enhance this tendency. Addition of gangliosides to the growth medium also depressed the growth rate of untransformed 3T3 cells. Thus artificially increasing the cellular ganglioside content does not selectively retard growth of cells which have altered in vivo glycolipid composition. Instead, these results and those of Laine & Hakomori [33] suggest that at least certain glycolipids have a general effect on the growth of mammalian cells in culture. The mechanism by which glycosphingolipids retard growth remains unknown. While it would be tempting to speculate that artificial enforcement of the glycosphingolipid content increases susceptibility of cells to contact inhibition, recent observations of Dulbecco & Elkington [48] suggest that this growth retardation may well be due to lowered utilization of medium components by cells cultured in glycosphingolipid-enriched medium. Exogenous gangliosides are accumulated by the cells and appear to become constituents of the plasma membrane. However, the accumulated gangliosides can be distinguished from those synthesized by the cell by the lability of the former to neuraminidase, thus suggestingthat the artificially supplied ganglioside assumes a configuration or localization in the membrane different from that of the native gangliosides. Whether this difference of the exogenously supplied ganglioside is responsible for its growth-retarding properties remains to be determined. This work was supported by the Deutsche Forschungsgemeinschaft. T. W. K. is on sabbatical leave from the Department of Animal Sciences, Purdue University, West Lafayette, Ind., USA. He is supported by USPHS Cancer Development award GM 70596 and grant GM 18760 from the National Institute of General Medical Science, by grant CA 13145 from the NCI, and is a senior US scientist awardee of the Alexander von Humboldt Foundation.

Note added in proof Since submission of this paper we became aware of a studyof the effects of glycolipids from R mutants of Salmonella minnesota on growth behaviour of rat embryo fibroblasts (Brailovsky, C, Trudel, M, Lallier, R & Nigam, V N, J cell biol 57 (1973) 124). These authors found an inverse correlation between carbohydrate chain length of the bacterial glycolipids and growth retarding effects. These glycolipids’were ineffective with primary rat embryo fibroblasts, nominally effective with SV40-transformed fibroblasts and maximally effective with spontaneously transformed rat embryo fibroblasts. Brailovsky et al. further found that the bacterial glycolipid with maximal growth retarding activity was not cytotoxic to the cells.

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