231
Biochimico et Biophysics Acta, 388 (1975) 231-242 @ Elsevier Scientific Publishing Company, Amster&m
- Printed in The Netherlands
BBA 56591
GANGLIOSIDES OF CHEMICALLY RAT EMBRYO CELLS
ROBERT
AND VIRALLY
TRANSFORMED
LANGENBACH
The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebr. 68105 (U.S.A.) (Received November 4th, 1974).
Summary The gangliosides of control rat embryo cells, 3_methylcholanthrene, Rauscher leukemia virus, and combined 3-methylcholanthrene-Rauscher leukemia virus transformants were examined using [’ 4 C] glucosamine as a tracer. All four cell lines exhibited a complex pattern of gangliosides. While N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosyl-ceramide was the major ganglioside in the control cell line, N-acetylneuraminyl-galactosyl-glucosylceramide was the major ganglioside in the three transformants. The 3-methylcholanthrene transformant possessed a ganglioside pattern different from that of the Rauscher leukemia virus transformant. Hydrolysis of the gangliosides indicated that galactosamine, N-acetyl- and N-glycolylneuraminic acid were the labeled components in all cell lines.
Introduction The cell surface appears to function in the regulation of cell growth, and therefore alterations of surface membrane components may be a crucial change leading to malignancy [l] . Gangliosides are important constituents of cellular Abbreviations and nomenclature: The nomenclature used to describe the gangliosides is that of Svennerholm [28J : GMj, Nacetylneuraminylgalactosylglucosyl-ceramide; GM2, Nacetylgalactosaminyl-(N-acetylneuraminyl)%alactosyl-glucosyl-ceramide; GM 1. galactosyl-N-acetylgalactosaminyl-(N-acetylneuaminyl)-galactosyl-glucosyl-ceramide; GD~a,N-acetylneurarninyl-galactosyl-Nacetylgalactosaminyl-(N-acetylneuraminyl)-g~actosyl-~ucosyl-cer~de; GDlb, galactosyl-iv-acetylgalactosaminyl-di-(N-acetylneuraminyl)-g~actosyl-~ucosyl-ceramide;GT.N-acetylneuraminyl%~actosyl-N-acetylgalactosaminyl-di-(N-acety~eur~~yl)-g~actosyl-~ucosyl-ceramide. The nomenclature for the cell lines is: 3-methylcholantbrene transformant, rat embryo cells transformed with 3-methylcholanthrene; Rauscher leukemia virus transformant. rat embryo cells transformed with Rauscher leukemia virus; 3-methylcholanthrene-Rauscher leukemia virus transformant. rat embryo cells transformed by combined 3-methylcholanthrene and Rauscher leukemia virus treatment: AcNeu. N-acetylneuraminic acid: glycolylNeu, N-glycolylneuraminic acid.
232
membranes, and it has been speculated that they are located mainly in the surface membrane [2,3] . In addition to their role as structural components in membranes, gangliosides are potential antigenic receptors and mediators of contact recognition between cells [ 2,3] . Many reports have been published concerning alterations of gangliosides after viral transformation of cells in culture.‘Studies with hamster cells transformed by polyoma virus [4], mouse fibroblasts transformed by either polyoma or SV-40 viruses [5-71, and chick embryo fibroblasts transformed by Rous sarcoma virus [8] have indicated a general simplification of the ganglioside pattern in the transformed cells as compared to control cells. In the transformed cells there was a greater quantity of ganglioside GM3 and a smaller quantity of gangliosides whose oligosaccharide chain was larger than sialyllactose [9] . Deficiencies in certain enzymes required for ganglioside biosynthesis have been observed in the virus-transformed cells [9,10] and this probably accounted for the altered patterns. Chemical transformation of cells in culture has also been achieved in several laboratories [ll-151, and now it is possible to study the effect of chemical transformation on cellular processes. A cell transformation system, developed by Freeman and his collaborators, appears to produce reliable results when screening known carcinogenic and noncarcinogenic chemicals [ 14,151. In this system, rat embryo cells were treated with Rauscher leukemia virus and a chemical carcinogen to produce a malignant transformation. While cells at a lower passage in culture were transformed only by the combined chemical-virus treatment, control cells at a slightly higher passsage could be transformed by either chemical or virus alone [16]. The transformants were tumorigenic in neonatal rats, while the control cell line was not. Thus, this system is amenable to comparative biochemical studies on chemical and virus transformed cells. A study of ganglioside alterations caused by chemical or viral transformation may aid in elucidating differences or similarities in the mechanism(s) by which these agents transform cells. Therefore, the present work was directed toward examining possible differences in the gangliosides of control, 3-methylcholanthrene-transformed, Rauscher leukemia virus-transformed and combined 3-methylcholanthrene-Rauscher leukemia virus-transformed rat embryo cells. Materials and Methods Cells and cell culture
The cell lines were obtained from Drs P. Price and A. Freeman of Microbiological Associates. The derivation of the transformed lines and some of their characteristics have been reported by Freeman’s laboratory [14,15], and by Ryan and Curtis [16]. Stock rat embryo cell lines (some properties of the cells are shown in Table I) were maintained in plastic tissue culture flasks (25 or 75 cm* , Falcon Industries, Oxnard, Calif.). The complete media was Eagle’s basal media (Grand Island Biological Co., Grand Island, N.Y.) and 10% heat-inactivated fetal calf serum (Grand Island Biological Co.). Cells were seeded in Petri dishes (60 or 100 mm, Falcon Industries) in complete media and were incubated in humidified incubators at 37°C with an atmosphere of 5% CO* in air. For passage, cells were washed with phosphate buffered saline and trypsinized
233
with 2 ml of 0.1% trypsin (Difco, Detroit, Mich.) for 5-7 min until single cells remained. Two ml of complete media was then added and an aliquot removed for counting with a Model B Coulter Counter (Coulter Electronics, Hialeah, Fla.). The remaining cells were centrifuged at 600 rev./min for approx. 5 min and the supernatant was replaced with a known volume of complete media. All experiments were conducted with cells within five passages after thawing. Tests for mycoplasma were negative. Saturation densities were determined by seeding 3 X 10’ cells in lo-cm Petri dishes. On the third day after plating, microscopic examination indicated that all cell lines had reached confluency. The cells were then given a media change and grown for two more days, at which time they were trypsinized and counted. Plating efficiencies were determined by seeding 200 cells in 60-mm Petri dishes. Between the fifth and eighth day the cells were fixed with methanol and stained with Giemsa (Matheson, Coleman and Bell, Norwood, Ohio). Colonies were counted and the plating efficiency determined. The growth rates of the cells were determined by seeding lo4 cells in lo-cm Petri dishes. At 24 h intervals for 4 days, the cell number was determined in triplicate dishes. The cell number vs time was plotted on semilog paper and the generation time calculated. Labeling
of cells with [’ 4 C]glucosamine
Cells were labeled with D -glucosamine-[ 1-l 4 C] hydrochloride (55 mCi/mol, Amersham Searle, Arlington Heights, Ill.) by adding the substrate in phosphate-buffered saline (0.15 ml) to dishes. of exponentially growing cells. The cells were given a medium change 12 h prior to adding the [’ 4 C] glucosamine to a final concentration of 0.75 /&i/ml. After two generation times the radioactive media was removed, the cell layer washed twice with phosphatebuffered saline, and the cells harvested by scraping with a rubber policeman [8]. The cells were centrifuged and the pellets stored frozen at -20°C. Extraction
of gangliosides
Labeled gangliosides were isolated from the cell pellet by the procedure of Suzuki [17], with slight modification. Because of the small amount of cellular material, 2 mg of bovine gangliosides were added and the cells were broken by sonification in the chloroform/methanol (2 : 1, by vol.). A Sonifier Cell Disruptor (Heat Systems-Ultrasonic, Inc., Model W185) with a microtip was used for 15 s at an intensity of 30 W. The cell debris removed by centrifugation and the chloroform/methanol extract was partitioned against an aqueous phase to separate the gangliosides (upper aqueous phase) from the neutral glycolipids (lower phase). The combined upper phases were concentrated under vacuum at room temperature with a Rotovapor (Brinkman Instruments) and then dialyzed overnight against distilled water at 4°C [17]. After dialysis the solvent was removed under vacuum and the [’ 4 C] glucosamine-labeled gangliosides were dissolved in chloroform/methanol (2 : 1, by vol.) and separated by thin-layer chromatography on Silica Gel G-coated plates (E. Merck, Darmstadt, Germany) with the solvent system of chloroform/methanol/ammonia/water (60 : 35 : 1 : 7, by vol.). Ganglioside standards were isolated by the method of Suzuki [17] from human brain obtained at autopsy. Bovine gangliosides, standard ganglio-
234
side GM2, AcNeu, and glycolylNeu were purchased from Supelco (Supelco Inc., Bellefonte, Pa.). Standards were detected with the resorcinol reagent
Radioactivity
determinations
Radioactive samples were counted by pipetting aliquots into scintillation vials containing 10 ml Aquasol (New England Nuclear, Boston, Mass.). Autoradiography of the labeled gangliosides was performed by exposing the developed thin-layer plate to Blue Sensitive X-ray film (Eastman Kodak Co., Rochester, N.Y.) for lo-14 days. Quantitative analysis of the individual gangliosides involved scraping the absorbent containing the radioactive band into a vial, adding 1 ml of ethanol, shaking the vial vigorously and adding 10 ml Aquasol. Protosol (New England Nuclear) was used to solubilize cellular debris. Counting efficiencies were determined with an internal standard. Identification
of labeled components
of cellular gangliosides
Labeled gangliosides were isolated by thin-layer chromatography and subjected to mild or vigorous acid hydrolysis. Mild acid hydrolysis to liberate sialic acid was carried out according to Ledeen et al. [19]. The sialic acid was chromatographed on Silica Gel G-coated plates using the solvent system npropyl alcohol/l M ammonia/water (6 : 2 : 1, by vol.). Standard AcNeu and glycolylNeu were detected with the resorcinol spray [18] . Hexose and hexosamines were liberated by hydrolysis in 1 M Hz SO, at 100°C for 2 h according to Yogeeswaran et al. [6] and chromatographed on Whatman No. 1 filter paper with ethyl acetate/pyridine/water (10 : 4 : 3, by vol.) as the solvent. Standards of glucose, galactose, glucosamine and galactosamine were also chromatographed and were detected by alkaline silver nitrate reagent [ 201. Results Properties
of the cell lines
Some characteristics TABLE
of the cells are given in Table I. All cell lines were
I
PROPERTIES
OF THE CONTROL
AND TRANSFORMED
Passage
RAT
EMBRYO
CELL LINES
Plating* efficiency
Doubling time
(%)
(h)
109
54
24
6.8 f 0.6
virus transformant 3-Methylcholanthrene
118
94
17
15.0 + 1.5
tra.nsfonnmt 3_MethylcholanthreneRauscher leukemia virus
105
91
12
22.5 f 4.0
115
94
17
12.0 f 2.0
Control Rauscher
Saturation* * density (cells/l0 cm dish
leukemia
transformant
* The mean of 10 dishes. ** The mean of 3 experiments
with standard
deviation
given.
* 10e6)
235
passed in culture approximately the same number of times and therefore passage number should not be a factor in comparisons. The plating efficiencies of the three transformed lines were between 90 and 95%. All the transformed lines had a more rapid doubling time than the control cells. The saturation densities of the Rauscher leukemia virus and the combined 3-methylcholanthrene-Rauscher leukemia virus transformants were approximately twice that of the control cells, while the 3-methylcholanthrene transformant had approximately three times the saturation density of the control. Scanning electron microscopic appearance of the cell lines The results of electron microscopic studies of the control and transformed cells lines are shown in Fig. 1. All photographs depict colonies of cells in the log phase of growth. The control cells (Fig. 1A) were contact inhibited and possessed a flat, smooth surface with a fibroblastic appearance. By contrast, the Rauscher leukemia virus transformant and the 3-methylcholanthrene-Rauscher leukemia virus transformant (Fig. 1B and 1C) were elongated cells with rough surfaces that grew in a random pattern with some piling up. The 3-methylcholanthrene transformant (Fig. 1D) possessed a somewhat different morphology in that the cells were rounded with a rough surface and showed a great propensity for piling up. The surface features and morphologies of the control and virustransformed rat embryo cells were in general agreement with the results reported by Porter et al. [21] for Balb/3T3 cells and their virus transformants. Incorporation of [’ 4 C]glucosamine into control and transformed rat embryo cells Labeled glucosamine, which has been demonstrated to be a useful precursor for studying ganglioside biosynthesis in vivo [22] and in vitro [6,8,23], was used to label the gangliosides. To compensate for the fact that rapidly growing cells synthesize membrane components at a faster rate than slower growing cells, all cell lines were grown in the presence of the substrate for two generation times. When [’ 4 C] glucosamine was added to dishes containing approx. 1.5 X lo6 cells per dish and the cells harvested at approximately 6 X lo6 cells per dish, the control cell line took up 2.1% of the glucosamine, while the transformants took up between 3.1% and 4.2% of the glucosamine (Table II, Expt 1). Thus, although the transformants were in contact with the [’ 4 C] glucosamine for a shorter time, in the late exponential phase of growth they incorporated more radioactivity per lo6 cells than the control cells. The data in Table II show the results of two experiments in which individual cellular fractions were analyzed for glucosamine incorporation. In both experiments the control cell line incorporated more glucosamine into the gangliosides (upper phase) than did the transformants. Similar incorporation results and distribution results have been found by Yogeeswaran et al. [6] for 3T3 cells and their viral transformants. Chromatography of the [’ 4 Clgangliosides Fig. 2 shows an autoradiogram of the chromatographed ganglioside fractions after dialysis from the control and transformed rat embryo cells. By simultaneous chromatography of standard ganglioside GM, and human brain
236
237
Fig. 1. Electron micrographs of control and transformed rat embryo cells. A. co ‘ntrol cell line; B, Ra uscher leukemia virus transformant; C. combined 3-methylcholanthrene-Rauscher let &kern)ia virus tran lSf0 D, 3-methylcholanthrene transformant. X 1100.
238 TABLE
II
DISTRIBUTION TRANSFORMED
OF RADIOACTIVITY IN LIPID RAT EMBRYO CELLS
AND
NONLIPID
FRACTIONS
OF CONTROL
AND
The data in Expt 1 were obtained when glucosamine was added to dishes containing 1.5 X lo6 cells and harvested at approximately 6 X lo6 cells per dish. The data in Expt 2 were obtained when glucosamine wasadded to dishes containing 1 X lo6 cells and harvested at approximately 4 X lo6 cells per dish. % Radioactivity
Control
Nonlipid residue Lipid extract Upper phase Lower phase
Rauscher leukemia virus transformant
3-Methylcholanthrene transformant
3-Methylcholantbrene-Rauscher leukemia virus transformant
Expt 2
Expt 1
Expt 2
96.1 3.9 3.6 0.4
8’7.5 12.5 10.1 2.4
92.1 7.9 6.6 1.3
Expt 1
Expt 2
Expt 1
Expt 2
Expt
83.6 16.4 14.8 1.6
88.2 11.8 10.8 1.0
85.1 14.9 11.3 3.6
92.6 7.4 4.8 2.6
90.2 10.8 10.5 0.3
1
gangliosides,
the radioactive bands were identified as gangliosides GM, , GM, , and GT. Each of the gangliosides GM3, GM, and GM, G&t, consisted of two closely migrating bands. The cellular gangliosides depicted in Fig. 2 could also be detected by the resorcinol method [lS].However, autoradiography was a more sensitive technique because components not readily detectable by the resorcinol spray were easily visualized by autoradiography. The intensity of certain bands in Fig. 2 suggested that quantitative differences existed in certain gangliosides between the control and transformed lines. GM,,
(=I.,
I
2
3
4
Fig. 2. Autoradiogram of gangliosides from control and transformed rat embryo cells. Gang&sides from control cells (l), Rauscher leukemia virus-transformed cells (2), 3-methylcholanthrene-transformed cells (3). 3-methylcholanthrene-Rauscher leukemia virus-transformed cells (4). Approx. 2500 cpm of the ganglioside fraction after dialysis was applied to the thin-layer plate for each cell line.
239 TABLE
III
DISTRIBUTION
OF RADIOACTIVITY
IN THE INDIVIDUAL
GANGLIOSIDES
Percentage radioactivity values are the mean of two samples with standard 90% of the 2500 cpm applied to the chromatogmm. Ganglioside
deviations.
Recovery
was 85-
% Radioactivity Control
Rauscher leukemia ViXUS
3-Methylcholanthrene transformant
3-MethylcholanthreneRauscher leukemia virus transformant
25.3 6.8 5.2 1.0 3.5 7.4 16.7 28.2
25.2 9.3 5.0 9.4 3.7 6.7 22.5 17.8
transformant
GM3 GM2 GM1 GDla
GDlb
GT X Origin
9.2 31.0 4.8 4.5 10.0 1.0 22.1 16.6
+ f f f r ? * f
0.2 0.1 0.1 0.6 1.2 0.1 0.5 0.2
18.8 14.1 6.6 6.4 7.5 1.6 23.0 21.9
f f * + t f C *
0.1 1.2 0.1 1.0 0.6 0.0 0.1 1.2
* 5.0 f 3.6 f 0.8 * 0.6 r 0.3 f 0.2 + 2.6 It 7.0
f + f f f f + *
5.2 0.7 0.3 0.3 1.3 0.1 2.2 4.2
The most striking difference was that GM2 was the major ganglioside in the control cells, while GM, was the major ganglioside in the three transformants. In addition, Fig. 2 shows that a ganglioside with a chromatographic mobility of GD, b was decreased in the three transformants compared to control cells, and the concentration of a ganglioside resembling GT was increased in the 3methylcholanthrene and combined 3-methylcholanthrene-Rauscher leukemia virus transformants compared to the control cells and Rauscher leukemia virus transformant. Quantitative analyses of the individual gangliosides are shown in Table III. As suggested by the autoradiogram, ganglioside GM3 was 2- to 3-fold greater and GM, was 2- to 4-fold lower in transformed cells than control cells. Incorporation of [’ 4 C] glucosamine into the other individual gangliosides is also presented in Table III and the results are in agreement with the relative intensity of the bands in the autoradiogram. The labeled component immediately below ga:iglioside GT in the autoradiogram ha8 not been identified, but is not a ganglioside because it appeared yellow after spraying with the resorcinol reagent. Distribution of radioactivity in the gangliosides Because the above observations indicated considerable utilization of [’ 4 C] glucosamine for ganglioside biosynthesis, experiments were performed to determine which components of the gangliosides contained the label. The gangliosides GM, through GT for all cell lines were isolated by thin-layer chromatography and extracted from the silica gel with chloroform/methanol (2 : 1, by vol.). Aliquots were then subjected to strong acid hydrolysis followed by paper chromatography to determine which hexoses were labeled [6], or mild acid hydrolysis followed by thin-layer chromatography to determine the amount of label in the sialic acid [19]. The combined results of the two hydrolysis procedures indicated that the majority of the label was present in
240 TABLE
IV
DISTRIBUTION
OF RADIOACTIVITY
In determining % radioactivity, aliquots means of duplicate analysis with standard Cell line
Percentage
IN THE INDIVIDUAL used for hydrolysis deviation.
COMPONENTS contained
OF THE GANGLIOSIDES
3000-5000
Ratio of AcNeu glycolylNeu
of radioactivity
Galactosamine
Sialic acid
Control
61.0
f 5.5
25.2
* 4.5
1.8 * 0.1
Rauscher leukemia virus transformant
50.3
* 2.5
37.3
f 5.0
5.5 * 1.5
3-Methylcholanthrene transformant
42.8
+_3.0
54.0
f 2.1
6.7 lr 1.7
3-MethylcholanthreneRauscher leukemia virus transformant
61.7
f 4.5
26.8
t 4.2
5.3 f 1.1
the galactosamine Approx. 4% of gangliosides from radioactivity in between 5.3 and
cpm.
The data are
to
and sialic acid moieties of the gangliosides (Table IV). the label was in the glucose and galactose fractions of the all cell lines (not shown). As shown in Table IV, the ratio of AcNeu to glycolNeu was 1.8 in the control cells and was 6.7 in the transformants.
Discussion The most salient alteration in the ganglioside pattern after both in vitro chemical (3-methylcholanthrene) and viral (Rauscher leukemia virus) transformation of rat embryo cells was the decrease of ganglioside GM, and the increase in the ganglioside GM3 fraction. Similar results have been reported after DNA [4--71 or RNA [ 8,241 viral transformation of various cell lines in culture. While in vitro studies with in vivo chemically induced hepatoma cells have shown a simplification of ganglioside pattern compared to normal hepatocytes [25], the effect of in vitro chemical transformation on cellular gangliosides has not been previously reported. The results indicate that the ganglioside patterns of all cell lines are complex and that differences between the normal and transformed lines arequantitative rather than qualitative. In both the control and transformed cell lines, the gangliosides GM3, GM, and GM, were each resolved as double bands by thin-layer chromatography. The presence of doublets in certain gangliosides has been previously reported [5,6], and has been attributed to differences in the sialic acid, sphingosine or fatty acid moieties. It is interesting that while the ganglioside patterns of the three transformants differed from the control, they also differed from one another. The gangliosides of the Rauscher leukemia virus transformant were similar to those of the control cell line, except that GM3 was the major labeled component in the transformant. By contrast, the ganglioside pattern of the 3-methylcholanthrene transformant was dramatically different from that of the control cell line and the other transformants. This transformant contained the fastest
241
migrating band of the ganglioside GM, fraction and a ganglioside resembling GT, as the major labeled components. Only low levels of radioactivity were detectable in gangliosides GM,, GM, , GD, a and GD, b. The combined 3methylcholanthrene-Rauscher leukemia virus transformant possessed a complex ganglioside pattern with features of both the Rauscher leukemia virus transformant (the increase in both ganglioside GM3 bands) and the 3-methylcholanthrene transformant (the presence of a ganglioside resembling GT). The increases in simpler gangliosides were in agreement with several previous reports of ganglioside GM, accumulation after viral transformation of cells in culture. Yogeeswaran et al. [6] and Brady and Mora [ 51 have reported an accumulation of ganglioside GM3 after polyoma or SV-40 transformation of 3T3 or AL/N cells in culture. Diringer et al. [24] reported several-fold increases of ganglioside GM3 in 3T3 cells transformed with murine leukemia virus and noticed a decrease in ganglioside GM, in mouse 3T3 cells or STU cells transformed with SV-40. Other studies have also shown a decrease in ganglioside GM, after transformation and the reasons for the discrepancies are not yet clear [ 21 . The saturation densities in Table I and the colony morphologies in Fig. 1 indicated that the transformants were much less sensitive to contact inhibition than the control cells. While some previous studies [6,26] have not revealed a direct correlation between saturation density and cellular glycolipid content, the results reported here suggest a relationship between saturation density and accumulation of ganglioside GM, . Other evidence for a role of glycolipids in regulation of cell growth comes from two types of experiments. First, flat revertants of virally transformed cells exhibited normal growth properties in culture and a reversion in ganglioside pattern back to that of the parent Swiss 3T3 cell line [9] . And second, addition of bacterial glycolipids to the media in which normal or SV-40 transformed rat embryo cells were growing resulted in an inhibition of growth of transformed cells, but not of the normal cells [27]. The lower incorporation of ’ 4 C into the sialic acids compared to the galactosamine (Table IV) was probably accounted for by glucosamine being a more direct precursor of the latter. In the case of the 3-methylcholanthrene transformant, where ganglioside GM3 was the major component (GM, does not contain galactosamine), greater than 50% of the label was present in the sialic acid moieties. In the other two transformants and the control cell line, where ganglioside GM2 and larger gangliosides were present (GM, and more complex gangliosides contain galactosamine as well as sialic acid), galactosamine was the major labeled component. The significance of the change in the ratio of AcNeu to glycolylNeu after both chemical and viral transformation of the rat embryo cells is not clear, but such alterations could affect the antigenic behavior or contact sensitivity of the transformants. The results presented here indicate that there are alterations in the ganglioside pattern after both chemical and viral transformation of the rat embryo cells. Furthermore, the ganglioside patterns of chemically and virally transformed cells are different. However, because of possible clonal variation, further studies are in progress to determine if other chemically transformed cells have altered gangliosides and if such ganglioside differences can be used for distinguishing between chemical and viral transformation.
242
Acknowledgments I wish to thank Mrs Dolores Colantoni for her excellent technical assistance and I am grateful to Drs R. Wilson and L. Malick for the electron microscopy. This investigation was supported by Public Health Service Contract No. NO1 CP33278. References 1 Ho&y, R.W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2840-2841 2 Hakomori, S. (1973) in Advances in Cancer Research (Klein, G. and Weinhouse. S., eds), Vol. 18, pp. 265-313, Academic Press, New York 3 Nigam, V.N. and Cantero, A. (1973) in Advances in Cancer Research (Klein, G. and Weinbouse. S. eds), Vol. 17. p. 1, Academic Press, New York 4 Hakomori. S. and Murakami, W.T. (1968) Proc. Natl. Acad. Sci. U.S. 59. 254-261 5 Brady, R.O. and Mora, P.T. (1970) Biochim. Biophys. Acta 218. 308-319 6 Yogeeswaran, G., Sheinin. R.. Wherrett. J.R. and Murray, R.K. (1972) J. Biol. Chem. 247, 5146-5158 7 Mora. P.T., Brady, R.O., Bradley. R.M. and McFarland, V.W. (1969) Proc. Natl. Acad. Sci. U.S. 63, 129(t1296 8 Hakomori. S., Saito, T. and Vogt, P.K. (1971) Virology 44, 609-621 9 Brady, R.O., Fishman, P.H., Mora, P.T. (1973) Fed. Proc. 32.102-108 10 Cumar, F.A., Brady, R.O., Kolodny, E.H., McFarland, V.W. and Mom, P.T. (1970) Proc. Natl. Acad. Sci. U.S. 67, 757-764 11 Reznikoff, C.A., Bertram, J., Brankow. D.W. and Heidelberger. C. (1973) Cancer Res. 33. 3239-3247 12 BerwaId. Y. and Sachs, L. (1965) J. Natl. Cancer Inst. 35.641-661 13 DiPaolo, J.A., Nelson, R.L. and Donovan, P.J. (1972) Nature 235, 278-280 14 Freeman, A.E., Weisburger, E.K.. Weisburger, J.H.. Wolford, R.G.. Maryak. J.M. and Huebner. R.J. (1973) J. NatI. Cancer Inst. 51, 799-808 15 Freeman, A.E.. Price, P.J.. Bryan. R.J., Gordon, R.J., Gilden. R.V., KeIIoff, G.J. and Huebner, R.J. (1971) Proc. Natl. Acad. U.S. 68, 445-449 16 Ryan, W. and Curtis, G. (1973) in The Role of Cyclic Nucleotides in Carcinogenesis, (Schultz, J. and Gratzner, H.G., eds). Vol. 6, p. 1, Academic Press, New York 17 Suzuki, K. (1965) J. Neurochem. 12,629-638 18 Svennerholm. L. (1967) Biochim. Biophys. Acta 24. 604-611 19 Ledeen, R., Salsman. K. and Cabrera, M. (1968) Biochemistry 7. 2287-2295 20 Trevelyan, W.E.. Procter, D.P. and Harrison. J.S. (1950) Nature 166, 444445 21 Porter, K.R.. Todaro. G.J. and Fonte, V. (1973) J. CeII. Biol. 59,633-642 22 Burton, R.M., Garcia-Bunnel, L.. Golden, M. and Belfour, Y.M. (1963) Biochemistry 2, 580-585 S. and Murray, R.K. (1970) J. Biol. Chem. 245. 23 Yogeeswaran. G., Wherrett, J.R., Chatterjee. 6718-6725 24 Diringer, H.. Strobel, G. and Koch, M.A. (1972) Hoppe-Seyler’s Z. Physiol. Chem. 353, 1769-1774 25 Brady, R.O.. Boreck. C. and Bradley, R.M. (1969) J. Biol. Chem. 244,6552-6554 26 Sakiyama, H. and Robbins, P.W. (1973) Fed. Proc. 32, 86-90 27 BraiIovsky, C., Trudel. M.. LaIIier, R. and N&am. V.N. (1973) J. CeII Biol. 57.124-132 28 Svennerholm, L. (1964) J. Lipid Res. 5. 145-184
Biochimico et Biophysics Acta, 388 (1975) 231-242 @ Elsevier Scientific Publishing Company, Amster&m
- Printed in The Netherlands
BBA 56591
GANGLIOSIDES OF CHEMICALLY RAT EMBRYO CELLS
ROBERT
AND VIRALLY
TRANSFORMED
LANGENBACH
The Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebr. 68105 (U.S.A.) (Received November 4th, 1974).
Summary The gangliosides of control rat embryo cells, 3_methylcholanthrene, Rauscher leukemia virus, and combined 3-methylcholanthrene-Rauscher leukemia virus transformants were examined using [’ 4 C] glucosamine as a tracer. All four cell lines exhibited a complex pattern of gangliosides. While N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosyl-ceramide was the major ganglioside in the control cell line, N-acetylneuraminyl-galactosyl-glucosylceramide was the major ganglioside in the three transformants. The 3-methylcholanthrene transformant possessed a ganglioside pattern different from that of the Rauscher leukemia virus transformant. Hydrolysis of the gangliosides indicated that galactosamine, N-acetyl- and N-glycolylneuraminic acid were the labeled components in all cell lines.
Introduction The cell surface appears to function in the regulation of cell growth, and therefore alterations of surface membrane components may be a crucial change leading to malignancy [l] . Gangliosides are important constituents of cellular Abbreviations and nomenclature: The nomenclature used to describe the gangliosides is that of Svennerholm [28J : GMj, Nacetylneuraminylgalactosylglucosyl-ceramide; GM2, Nacetylgalactosaminyl-(N-acetylneuraminyl)%alactosyl-glucosyl-ceramide; GM 1. galactosyl-N-acetylgalactosaminyl-(N-acetylneuaminyl)-galactosyl-glucosyl-ceramide; GD~a,N-acetylneurarninyl-galactosyl-Nacetylgalactosaminyl-(N-acetylneuraminyl)-g~actosyl-~ucosyl-cer~de; GDlb, galactosyl-iv-acetylgalactosaminyl-di-(N-acetylneuraminyl)-g~actosyl-~ucosyl-ceramide;GT.N-acetylneuraminyl%~actosyl-N-acetylgalactosaminyl-di-(N-acety~eur~~yl)-g~actosyl-~ucosyl-ceramide. The nomenclature for the cell lines is: 3-methylcholantbrene transformant, rat embryo cells transformed with 3-methylcholanthrene; Rauscher leukemia virus transformant. rat embryo cells transformed with Rauscher leukemia virus; 3-methylcholanthrene-Rauscher leukemia virus transformant. rat embryo cells transformed by combined 3-methylcholanthrene and Rauscher leukemia virus treatment: AcNeu. N-acetylneuraminic acid: glycolylNeu, N-glycolylneuraminic acid.
232
membranes, and it has been speculated that they are located mainly in the surface membrane [2,3] . In addition to their role as structural components in membranes, gangliosides are potential antigenic receptors and mediators of contact recognition between cells [ 2,3] . Many reports have been published concerning alterations of gangliosides after viral transformation of cells in culture.‘Studies with hamster cells transformed by polyoma virus [4], mouse fibroblasts transformed by either polyoma or SV-40 viruses [5-71, and chick embryo fibroblasts transformed by Rous sarcoma virus [8] have indicated a general simplification of the ganglioside pattern in the transformed cells as compared to control cells. In the transformed cells there was a greater quantity of ganglioside GM3 and a smaller quantity of gangliosides whose oligosaccharide chain was larger than sialyllactose [9] . Deficiencies in certain enzymes required for ganglioside biosynthesis have been observed in the virus-transformed cells [9,10] and this probably accounted for the altered patterns. Chemical transformation of cells in culture has also been achieved in several laboratories [ll-151, and now it is possible to study the effect of chemical transformation on cellular processes. A cell transformation system, developed by Freeman and his collaborators, appears to produce reliable results when screening known carcinogenic and noncarcinogenic chemicals [ 14,151. In this system, rat embryo cells were treated with Rauscher leukemia virus and a chemical carcinogen to produce a malignant transformation. While cells at a lower passage in culture were transformed only by the combined chemical-virus treatment, control cells at a slightly higher passsage could be transformed by either chemical or virus alone [16]. The transformants were tumorigenic in neonatal rats, while the control cell line was not. Thus, this system is amenable to comparative biochemical studies on chemical and virus transformed cells. A study of ganglioside alterations caused by chemical or viral transformation may aid in elucidating differences or similarities in the mechanism(s) by which these agents transform cells. Therefore, the present work was directed toward examining possible differences in the gangliosides of control, 3-methylcholanthrene-transformed, Rauscher leukemia virus-transformed and combined 3-methylcholanthrene-Rauscher leukemia virus-transformed rat embryo cells. Materials and Methods Cells and cell culture
The cell lines were obtained from Drs P. Price and A. Freeman of Microbiological Associates. The derivation of the transformed lines and some of their characteristics have been reported by Freeman’s laboratory [14,15], and by Ryan and Curtis [16]. Stock rat embryo cell lines (some properties of the cells are shown in Table I) were maintained in plastic tissue culture flasks (25 or 75 cm* , Falcon Industries, Oxnard, Calif.). The complete media was Eagle’s basal media (Grand Island Biological Co., Grand Island, N.Y.) and 10% heat-inactivated fetal calf serum (Grand Island Biological Co.). Cells were seeded in Petri dishes (60 or 100 mm, Falcon Industries) in complete media and were incubated in humidified incubators at 37°C with an atmosphere of 5% CO* in air. For passage, cells were washed with phosphate buffered saline and trypsinized
233
with 2 ml of 0.1% trypsin (Difco, Detroit, Mich.) for 5-7 min until single cells remained. Two ml of complete media was then added and an aliquot removed for counting with a Model B Coulter Counter (Coulter Electronics, Hialeah, Fla.). The remaining cells were centrifuged at 600 rev./min for approx. 5 min and the supernatant was replaced with a known volume of complete media. All experiments were conducted with cells within five passages after thawing. Tests for mycoplasma were negative. Saturation densities were determined by seeding 3 X 10’ cells in lo-cm Petri dishes. On the third day after plating, microscopic examination indicated that all cell lines had reached confluency. The cells were then given a media change and grown for two more days, at which time they were trypsinized and counted. Plating efficiencies were determined by seeding 200 cells in 60-mm Petri dishes. Between the fifth and eighth day the cells were fixed with methanol and stained with Giemsa (Matheson, Coleman and Bell, Norwood, Ohio). Colonies were counted and the plating efficiency determined. The growth rates of the cells were determined by seeding lo4 cells in lo-cm Petri dishes. At 24 h intervals for 4 days, the cell number was determined in triplicate dishes. The cell number vs time was plotted on semilog paper and the generation time calculated. Labeling
of cells with [’ 4 C]glucosamine
Cells were labeled with D -glucosamine-[ 1-l 4 C] hydrochloride (55 mCi/mol, Amersham Searle, Arlington Heights, Ill.) by adding the substrate in phosphate-buffered saline (0.15 ml) to dishes. of exponentially growing cells. The cells were given a medium change 12 h prior to adding the [’ 4 C] glucosamine to a final concentration of 0.75 /&i/ml. After two generation times the radioactive media was removed, the cell layer washed twice with phosphatebuffered saline, and the cells harvested by scraping with a rubber policeman [8]. The cells were centrifuged and the pellets stored frozen at -20°C. Extraction
of gangliosides
Labeled gangliosides were isolated from the cell pellet by the procedure of Suzuki [17], with slight modification. Because of the small amount of cellular material, 2 mg of bovine gangliosides were added and the cells were broken by sonification in the chloroform/methanol (2 : 1, by vol.). A Sonifier Cell Disruptor (Heat Systems-Ultrasonic, Inc., Model W185) with a microtip was used for 15 s at an intensity of 30 W. The cell debris removed by centrifugation and the chloroform/methanol extract was partitioned against an aqueous phase to separate the gangliosides (upper aqueous phase) from the neutral glycolipids (lower phase). The combined upper phases were concentrated under vacuum at room temperature with a Rotovapor (Brinkman Instruments) and then dialyzed overnight against distilled water at 4°C [17]. After dialysis the solvent was removed under vacuum and the [’ 4 C] glucosamine-labeled gangliosides were dissolved in chloroform/methanol (2 : 1, by vol.) and separated by thin-layer chromatography on Silica Gel G-coated plates (E. Merck, Darmstadt, Germany) with the solvent system of chloroform/methanol/ammonia/water (60 : 35 : 1 : 7, by vol.). Ganglioside standards were isolated by the method of Suzuki [17] from human brain obtained at autopsy. Bovine gangliosides, standard ganglio-
234
side GM2, AcNeu, and glycolylNeu were purchased from Supelco (Supelco Inc., Bellefonte, Pa.). Standards were detected with the resorcinol reagent
Radioactivity
determinations
Radioactive samples were counted by pipetting aliquots into scintillation vials containing 10 ml Aquasol (New England Nuclear, Boston, Mass.). Autoradiography of the labeled gangliosides was performed by exposing the developed thin-layer plate to Blue Sensitive X-ray film (Eastman Kodak Co., Rochester, N.Y.) for lo-14 days. Quantitative analysis of the individual gangliosides involved scraping the absorbent containing the radioactive band into a vial, adding 1 ml of ethanol, shaking the vial vigorously and adding 10 ml Aquasol. Protosol (New England Nuclear) was used to solubilize cellular debris. Counting efficiencies were determined with an internal standard. Identification
of labeled components
of cellular gangliosides
Labeled gangliosides were isolated by thin-layer chromatography and subjected to mild or vigorous acid hydrolysis. Mild acid hydrolysis to liberate sialic acid was carried out according to Ledeen et al. [19]. The sialic acid was chromatographed on Silica Gel G-coated plates using the solvent system npropyl alcohol/l M ammonia/water (6 : 2 : 1, by vol.). Standard AcNeu and glycolylNeu were detected with the resorcinol spray [18] . Hexose and hexosamines were liberated by hydrolysis in 1 M Hz SO, at 100°C for 2 h according to Yogeeswaran et al. [6] and chromatographed on Whatman No. 1 filter paper with ethyl acetate/pyridine/water (10 : 4 : 3, by vol.) as the solvent. Standards of glucose, galactose, glucosamine and galactosamine were also chromatographed and were detected by alkaline silver nitrate reagent [ 201. Results Properties
of the cell lines
Some characteristics TABLE
of the cells are given in Table I. All cell lines were
I
PROPERTIES
OF THE CONTROL
AND TRANSFORMED
Passage
RAT
EMBRYO
CELL LINES
Plating* efficiency
Doubling time
(%)
(h)
109
54
24
6.8 f 0.6
virus transformant 3-Methylcholanthrene
118
94
17
15.0 + 1.5
tra.nsfonnmt 3_MethylcholanthreneRauscher leukemia virus
105
91
12
22.5 f 4.0
115
94
17
12.0 f 2.0
Control Rauscher
Saturation* * density (cells/l0 cm dish
leukemia
transformant
* The mean of 10 dishes. ** The mean of 3 experiments
with standard
deviation
given.
* 10e6)
235
passed in culture approximately the same number of times and therefore passage number should not be a factor in comparisons. The plating efficiencies of the three transformed lines were between 90 and 95%. All the transformed lines had a more rapid doubling time than the control cells. The saturation densities of the Rauscher leukemia virus and the combined 3-methylcholanthrene-Rauscher leukemia virus transformants were approximately twice that of the control cells, while the 3-methylcholanthrene transformant had approximately three times the saturation density of the control. Scanning electron microscopic appearance of the cell lines The results of electron microscopic studies of the control and transformed cells lines are shown in Fig. 1. All photographs depict colonies of cells in the log phase of growth. The control cells (Fig. 1A) were contact inhibited and possessed a flat, smooth surface with a fibroblastic appearance. By contrast, the Rauscher leukemia virus transformant and the 3-methylcholanthrene-Rauscher leukemia virus transformant (Fig. 1B and 1C) were elongated cells with rough surfaces that grew in a random pattern with some piling up. The 3-methylcholanthrene transformant (Fig. 1D) possessed a somewhat different morphology in that the cells were rounded with a rough surface and showed a great propensity for piling up. The surface features and morphologies of the control and virustransformed rat embryo cells were in general agreement with the results reported by Porter et al. [21] for Balb/3T3 cells and their virus transformants. Incorporation of [’ 4 C]glucosamine into control and transformed rat embryo cells Labeled glucosamine, which has been demonstrated to be a useful precursor for studying ganglioside biosynthesis in vivo [22] and in vitro [6,8,23], was used to label the gangliosides. To compensate for the fact that rapidly growing cells synthesize membrane components at a faster rate than slower growing cells, all cell lines were grown in the presence of the substrate for two generation times. When [’ 4 C] glucosamine was added to dishes containing approx. 1.5 X lo6 cells per dish and the cells harvested at approximately 6 X lo6 cells per dish, the control cell line took up 2.1% of the glucosamine, while the transformants took up between 3.1% and 4.2% of the glucosamine (Table II, Expt 1). Thus, although the transformants were in contact with the [’ 4 C] glucosamine for a shorter time, in the late exponential phase of growth they incorporated more radioactivity per lo6 cells than the control cells. The data in Table II show the results of two experiments in which individual cellular fractions were analyzed for glucosamine incorporation. In both experiments the control cell line incorporated more glucosamine into the gangliosides (upper phase) than did the transformants. Similar incorporation results and distribution results have been found by Yogeeswaran et al. [6] for 3T3 cells and their viral transformants. Chromatography of the [’ 4 Clgangliosides Fig. 2 shows an autoradiogram of the chromatographed ganglioside fractions after dialysis from the control and transformed rat embryo cells. By simultaneous chromatography of standard ganglioside GM, and human brain
236
237
Fig. 1. Electron micrographs of control and transformed rat embryo cells. A. co ‘ntrol cell line; B, Ra uscher leukemia virus transformant; C. combined 3-methylcholanthrene-Rauscher let &kern)ia virus tran lSf0 D, 3-methylcholanthrene transformant. X 1100.
238 TABLE
II
DISTRIBUTION TRANSFORMED
OF RADIOACTIVITY IN LIPID RAT EMBRYO CELLS
AND
NONLIPID
FRACTIONS
OF CONTROL
AND
The data in Expt 1 were obtained when glucosamine was added to dishes containing 1.5 X lo6 cells and harvested at approximately 6 X lo6 cells per dish. The data in Expt 2 were obtained when glucosamine wasadded to dishes containing 1 X lo6 cells and harvested at approximately 4 X lo6 cells per dish. % Radioactivity
Control
Nonlipid residue Lipid extract Upper phase Lower phase
Rauscher leukemia virus transformant
3-Methylcholanthrene transformant
3-Methylcholantbrene-Rauscher leukemia virus transformant
Expt 2
Expt 1
Expt 2
96.1 3.9 3.6 0.4
8’7.5 12.5 10.1 2.4
92.1 7.9 6.6 1.3
Expt 1
Expt 2
Expt 1
Expt 2
Expt
83.6 16.4 14.8 1.6
88.2 11.8 10.8 1.0
85.1 14.9 11.3 3.6
92.6 7.4 4.8 2.6
90.2 10.8 10.5 0.3
1
gangliosides,
the radioactive bands were identified as gangliosides GM, , GM, , and GT. Each of the gangliosides GM3, GM, and GM, G&t, consisted of two closely migrating bands. The cellular gangliosides depicted in Fig. 2 could also be detected by the resorcinol method [lS].However, autoradiography was a more sensitive technique because components not readily detectable by the resorcinol spray were easily visualized by autoradiography. The intensity of certain bands in Fig. 2 suggested that quantitative differences existed in certain gangliosides between the control and transformed lines. GM,,
(=I.,
I
2
3
4
Fig. 2. Autoradiogram of gangliosides from control and transformed rat embryo cells. Gang&sides from control cells (l), Rauscher leukemia virus-transformed cells (2), 3-methylcholanthrene-transformed cells (3). 3-methylcholanthrene-Rauscher leukemia virus-transformed cells (4). Approx. 2500 cpm of the ganglioside fraction after dialysis was applied to the thin-layer plate for each cell line.
239 TABLE
III
DISTRIBUTION
OF RADIOACTIVITY
IN THE INDIVIDUAL
GANGLIOSIDES
Percentage radioactivity values are the mean of two samples with standard 90% of the 2500 cpm applied to the chromatogmm. Ganglioside
deviations.
Recovery
was 85-
% Radioactivity Control
Rauscher leukemia ViXUS
3-Methylcholanthrene transformant
3-MethylcholanthreneRauscher leukemia virus transformant
25.3 6.8 5.2 1.0 3.5 7.4 16.7 28.2
25.2 9.3 5.0 9.4 3.7 6.7 22.5 17.8
transformant
GM3 GM2 GM1 GDla
GDlb
GT X Origin
9.2 31.0 4.8 4.5 10.0 1.0 22.1 16.6
+ f f f r ? * f
0.2 0.1 0.1 0.6 1.2 0.1 0.5 0.2
18.8 14.1 6.6 6.4 7.5 1.6 23.0 21.9
f f * + t f C *
0.1 1.2 0.1 1.0 0.6 0.0 0.1 1.2
* 5.0 f 3.6 f 0.8 * 0.6 r 0.3 f 0.2 + 2.6 It 7.0
f + f f f f + *
5.2 0.7 0.3 0.3 1.3 0.1 2.2 4.2
The most striking difference was that GM2 was the major ganglioside in the control cells, while GM, was the major ganglioside in the three transformants. In addition, Fig. 2 shows that a ganglioside with a chromatographic mobility of GD, b was decreased in the three transformants compared to control cells, and the concentration of a ganglioside resembling GT was increased in the 3methylcholanthrene and combined 3-methylcholanthrene-Rauscher leukemia virus transformants compared to the control cells and Rauscher leukemia virus transformant. Quantitative analyses of the individual gangliosides are shown in Table III. As suggested by the autoradiogram, ganglioside GM3 was 2- to 3-fold greater and GM, was 2- to 4-fold lower in transformed cells than control cells. Incorporation of [’ 4 C] glucosamine into the other individual gangliosides is also presented in Table III and the results are in agreement with the relative intensity of the bands in the autoradiogram. The labeled component immediately below ga:iglioside GT in the autoradiogram ha8 not been identified, but is not a ganglioside because it appeared yellow after spraying with the resorcinol reagent. Distribution of radioactivity in the gangliosides Because the above observations indicated considerable utilization of [’ 4 C] glucosamine for ganglioside biosynthesis, experiments were performed to determine which components of the gangliosides contained the label. The gangliosides GM, through GT for all cell lines were isolated by thin-layer chromatography and extracted from the silica gel with chloroform/methanol (2 : 1, by vol.). Aliquots were then subjected to strong acid hydrolysis followed by paper chromatography to determine which hexoses were labeled [6], or mild acid hydrolysis followed by thin-layer chromatography to determine the amount of label in the sialic acid [19]. The combined results of the two hydrolysis procedures indicated that the majority of the label was present in
240 TABLE
IV
DISTRIBUTION
OF RADIOACTIVITY
In determining % radioactivity, aliquots means of duplicate analysis with standard Cell line
Percentage
IN THE INDIVIDUAL used for hydrolysis deviation.
COMPONENTS contained
OF THE GANGLIOSIDES
3000-5000
Ratio of AcNeu glycolylNeu
of radioactivity
Galactosamine
Sialic acid
Control
61.0
f 5.5
25.2
* 4.5
1.8 * 0.1
Rauscher leukemia virus transformant
50.3
* 2.5
37.3
f 5.0
5.5 * 1.5
3-Methylcholanthrene transformant
42.8
+_3.0
54.0
f 2.1
6.7 lr 1.7
3-MethylcholanthreneRauscher leukemia virus transformant
61.7
f 4.5
26.8
t 4.2
5.3 f 1.1
the galactosamine Approx. 4% of gangliosides from radioactivity in between 5.3 and
cpm.
The data are
to
and sialic acid moieties of the gangliosides (Table IV). the label was in the glucose and galactose fractions of the all cell lines (not shown). As shown in Table IV, the ratio of AcNeu to glycolNeu was 1.8 in the control cells and was 6.7 in the transformants.
Discussion The most salient alteration in the ganglioside pattern after both in vitro chemical (3-methylcholanthrene) and viral (Rauscher leukemia virus) transformation of rat embryo cells was the decrease of ganglioside GM, and the increase in the ganglioside GM3 fraction. Similar results have been reported after DNA [4--71 or RNA [ 8,241 viral transformation of various cell lines in culture. While in vitro studies with in vivo chemically induced hepatoma cells have shown a simplification of ganglioside pattern compared to normal hepatocytes [25], the effect of in vitro chemical transformation on cellular gangliosides has not been previously reported. The results indicate that the ganglioside patterns of all cell lines are complex and that differences between the normal and transformed lines arequantitative rather than qualitative. In both the control and transformed cell lines, the gangliosides GM3, GM, and GM, were each resolved as double bands by thin-layer chromatography. The presence of doublets in certain gangliosides has been previously reported [5,6], and has been attributed to differences in the sialic acid, sphingosine or fatty acid moieties. It is interesting that while the ganglioside patterns of the three transformants differed from the control, they also differed from one another. The gangliosides of the Rauscher leukemia virus transformant were similar to those of the control cell line, except that GM3 was the major labeled component in the transformant. By contrast, the ganglioside pattern of the 3-methylcholanthrene transformant was dramatically different from that of the control cell line and the other transformants. This transformant contained the fastest
241
migrating band of the ganglioside GM, fraction and a ganglioside resembling GT, as the major labeled components. Only low levels of radioactivity were detectable in gangliosides GM,, GM, , GD, a and GD, b. The combined 3methylcholanthrene-Rauscher leukemia virus transformant possessed a complex ganglioside pattern with features of both the Rauscher leukemia virus transformant (the increase in both ganglioside GM3 bands) and the 3-methylcholanthrene transformant (the presence of a ganglioside resembling GT). The increases in simpler gangliosides were in agreement with several previous reports of ganglioside GM, accumulation after viral transformation of cells in culture. Yogeeswaran et al. [6] and Brady and Mora [ 51 have reported an accumulation of ganglioside GM3 after polyoma or SV-40 transformation of 3T3 or AL/N cells in culture. Diringer et al. [24] reported several-fold increases of ganglioside GM3 in 3T3 cells transformed with murine leukemia virus and noticed a decrease in ganglioside GM, in mouse 3T3 cells or STU cells transformed with SV-40. Other studies have also shown a decrease in ganglioside GM, after transformation and the reasons for the discrepancies are not yet clear [ 21 . The saturation densities in Table I and the colony morphologies in Fig. 1 indicated that the transformants were much less sensitive to contact inhibition than the control cells. While some previous studies [6,26] have not revealed a direct correlation between saturation density and cellular glycolipid content, the results reported here suggest a relationship between saturation density and accumulation of ganglioside GM, . Other evidence for a role of glycolipids in regulation of cell growth comes from two types of experiments. First, flat revertants of virally transformed cells exhibited normal growth properties in culture and a reversion in ganglioside pattern back to that of the parent Swiss 3T3 cell line [9] . And second, addition of bacterial glycolipids to the media in which normal or SV-40 transformed rat embryo cells were growing resulted in an inhibition of growth of transformed cells, but not of the normal cells [27]. The lower incorporation of ’ 4 C into the sialic acids compared to the galactosamine (Table IV) was probably accounted for by glucosamine being a more direct precursor of the latter. In the case of the 3-methylcholanthrene transformant, where ganglioside GM3 was the major component (GM, does not contain galactosamine), greater than 50% of the label was present in the sialic acid moieties. In the other two transformants and the control cell line, where ganglioside GM2 and larger gangliosides were present (GM, and more complex gangliosides contain galactosamine as well as sialic acid), galactosamine was the major labeled component. The significance of the change in the ratio of AcNeu to glycolylNeu after both chemical and viral transformation of the rat embryo cells is not clear, but such alterations could affect the antigenic behavior or contact sensitivity of the transformants. The results presented here indicate that there are alterations in the ganglioside pattern after both chemical and viral transformation of the rat embryo cells. Furthermore, the ganglioside patterns of chemically and virally transformed cells are different. However, because of possible clonal variation, further studies are in progress to determine if other chemically transformed cells have altered gangliosides and if such ganglioside differences can be used for distinguishing between chemical and viral transformation.
242
Acknowledgments I wish to thank Mrs Dolores Colantoni for her excellent technical assistance and I am grateful to Drs R. Wilson and L. Malick for the electron microscopy. This investigation was supported by Public Health Service Contract No. NO1 CP33278. References 1 Ho&y, R.W. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2840-2841 2 Hakomori, S. (1973) in Advances in Cancer Research (Klein, G. and Weinhouse. S., eds), Vol. 18, pp. 265-313, Academic Press, New York 3 Nigam, V.N. and Cantero, A. (1973) in Advances in Cancer Research (Klein, G. and Weinbouse. S. eds), Vol. 17. p. 1, Academic Press, New York 4 Hakomori. S. and Murakami, W.T. (1968) Proc. Natl. Acad. Sci. U.S. 59. 254-261 5 Brady, R.O. and Mora, P.T. (1970) Biochim. Biophys. Acta 218. 308-319 6 Yogeeswaran, G., Sheinin. R.. Wherrett. J.R. and Murray, R.K. (1972) J. Biol. Chem. 247, 5146-5158 7 Mora. P.T., Brady, R.O., Bradley. R.M. and McFarland, V.W. (1969) Proc. Natl. Acad. Sci. U.S. 63, 129(t1296 8 Hakomori. S., Saito, T. and Vogt, P.K. (1971) Virology 44, 609-621 9 Brady, R.O., Fishman, P.H., Mora, P.T. (1973) Fed. Proc. 32.102-108 10 Cumar, F.A., Brady, R.O., Kolodny, E.H., McFarland, V.W. and Mom, P.T. (1970) Proc. Natl. Acad. Sci. U.S. 67, 757-764 11 Reznikoff, C.A., Bertram, J., Brankow. D.W. and Heidelberger. C. (1973) Cancer Res. 33. 3239-3247 12 BerwaId. Y. and Sachs, L. (1965) J. Natl. Cancer Inst. 35.641-661 13 DiPaolo, J.A., Nelson, R.L. and Donovan, P.J. (1972) Nature 235, 278-280 14 Freeman, A.E., Weisburger, E.K.. Weisburger, J.H.. Wolford, R.G.. Maryak. J.M. and Huebner. R.J. (1973) J. NatI. Cancer Inst. 51, 799-808 15 Freeman, A.E.. Price, P.J.. Bryan. R.J., Gordon, R.J., Gilden. R.V., KeIIoff, G.J. and Huebner, R.J. (1971) Proc. Natl. Acad. U.S. 68, 445-449 16 Ryan, W. and Curtis, G. (1973) in The Role of Cyclic Nucleotides in Carcinogenesis, (Schultz, J. and Gratzner, H.G., eds). Vol. 6, p. 1, Academic Press, New York 17 Suzuki, K. (1965) J. Neurochem. 12,629-638 18 Svennerholm. L. (1967) Biochim. Biophys. Acta 24. 604-611 19 Ledeen, R., Salsman. K. and Cabrera, M. (1968) Biochemistry 7. 2287-2295 20 Trevelyan, W.E.. Procter, D.P. and Harrison. J.S. (1950) Nature 166, 444445 21 Porter, K.R.. Todaro. G.J. and Fonte, V. (1973) J. CeII. Biol. 59,633-642 22 Burton, R.M., Garcia-Bunnel, L.. Golden, M. and Belfour, Y.M. (1963) Biochemistry 2, 580-585 S. and Murray, R.K. (1970) J. Biol. Chem. 245. 23 Yogeeswaran. G., Wherrett, J.R., Chatterjee. 6718-6725 24 Diringer, H.. Strobel, G. and Koch, M.A. (1972) Hoppe-Seyler’s Z. Physiol. Chem. 353, 1769-1774 25 Brady, R.O.. Boreck. C. and Bradley, R.M. (1969) J. Biol. Chem. 244,6552-6554 26 Sakiyama, H. and Robbins, P.W. (1973) Fed. Proc. 32, 86-90 27 BraiIovsky, C., Trudel. M.. LaIIier, R. and N&am. V.N. (1973) J. CeII Biol. 57.124-132 28 Svennerholm, L. (1964) J. Lipid Res. 5. 145-184
