643 Altered Metabolism and Cell Surface Expression of Glycosphingolipids Caused by Vitamin E in Cultured Murine (K3T3) Reticulum Sarcoma Cells 1 Ganesa Yogeeswaran* and Innocent N. Mbawuike ~ Department of Microbiology and Biochemistry and the Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts Vitamin E caused a generalized reduction in the metabo- lism and cell surface expression of glycosphingolipids (GSL) in cultured Kirsten murine sarcoma virus- transformed nonproducer (K3T3) cells. Metabolism of gangliosides was decreased two- to fourfold in cells treated for 72 hr with I and 2/~g/ml but not with 12 ~g/ml vitamin E compared to control cultures. This was demon- strated by a quantitative reduction in precursor 3H- galactose label incorporated in ganglioside fraction and further substantiated by thin layer chromatography of colorimetrically and radiochemically detected GSL homc~ logues. The composition of neutral GSL homologues was only slightly changed. The cell surface expressions of sialoglycoconjugates, analyzed by selective periodate- borotritide labeling, were also diminished quantitatively. These results are discussed in light of a previously demon- strated increase in antigenicity of K3T3 cells treated with vitamin E and the reduced tumorigenicity of these cells when transplanted into mice fed vitamin E-supplemented diets. Lipids 21, 643-647 (1986). Balb/c mice fed a vitamin E-supplemented diet were significantly protected against transplantation of Kirsten murine sarcoma virus-transformed Balb/3T3 (K3T3) tumor cells (1). The extent of tumor inhibition was depen- dent on the degree of unsaturation of dietary fat. Anti- tumor activity of vitamin E was due to the stimulation of the host's immune response (1). An enhanced antibody synthesis in mice fed a vitamin E-supplemented diet was shown to require macrophages that exhibit increased Fc- and C3b-receptor mediated phagocytosis and the expres- sion of Ia antigen (2). The latter finding indicates that vitamin E can modulate the expression of membrane molecules and their functions. We hypothesize that vitamin E enhances the display of tumor cell surface antigens either by prevention of peroxidation of membrane polyunsaturated fatty acids (PUFA), thereby preserving the overall integrity and stability of the tumor cell (3), or by inducing changes in the expression of cell surface glycoconjugate antigens, suggested by the observation of an enhanced in vitro im- munization against vitamin E-pretreated K3T3 cells (1), which also showed increased binding of antitumor an- tibodies to K3T3 cells (4). Vitamin E has also been reported to reverse the morphological expression of the 1Glycosphingolipid and ganglioside nomenclature and abbreviations are as recommended by IUPAC/IUB (Lipids 12, 445-468, 1977). *To whom correspondence should be addressed at the USDA-Human Nutrition Research Center at Tufts University, 711 Washington St., Boston, MA 02111. 2Present address: Department of Microbiology, Georgetown Univer- sity Schools of Medicine and Dentistry, 3900 Reservoir Rd. NW, Washington, DC 20007. transformed phenotype in cultured K3T3 cells (5). In previous reports, the association of altered glycolipid pat- terns with antigenic activities in these murine reticulum sarcoma cells (6) and the importance of cell surface glycoconjugates in the tumorigenic and metastatic poten- tial of tumor cells have been well documented (7-9). Because K3T3 cells injected in vitamin E-fed mice tend to sequester the vitamin relative to other host tissues (10), the present report examines the effect of vitamin E on the expression and biosynthesis of cell surface glyco- sphingolipids (GSL) in cultured K3T3 cells. MATERIALS AND METHODS Cell culture. K3T3 cells (11) obtained from G. Todaro of the National Institutes of Health were cultured in Dulbec- co's Modified Eagle's Medium (DMEM, Grand Island Biological Co., Grand Island, New York}, supplemented with 10% heat-inactivated fetal calf serum (FCS, Sterile Systems, Logan, Connecticut), 2 mM glutamine, 100 units/ ml penicillin and 100 ~g/ml streptomycin {Grand Island Biological Co.). Cells were grown and maintained at 37 C in a 100% humidified incubator with 95% air and 5% CO2 and subcultured once every five days from confluent cultures following removal by trypsin. Preparation of vitamin E-supplemented medium and treatment of cells. DL-a-tocopherol oil (Hoffman-La Roche, Nutley, New Jersey) was dissolved in ethanol]pro pylene glycol {1:9, v/v) to make a final concentration of 10 mg/ml. Appropriate amounts of stock DI~a-tocopherol were added to FCS and incubated for 45 min at 37 C to allow vitamin E to bind serum lipoproteins. DMEM was then added to make 10% FCS and desired tocopherol con- centration. Control medium consisted of very low levels of vitamin E (30 ng/ml) present in 10% FCS and sup- plemented with traces of ethanoYpropylene glycol (1:9, v/v). Cells were cultured for 72 to 96 hr in vitamin E- supplemented or control medium. 3H-Galactose incorporation. Ca. 1 X 10 ~ control or vitamin E-treated K3T3 cells were cultured in duplicate 100-mm tissue culture plates for 72 hr. These cultured cells were terminally labeled for an additional 24 hr in the same medium with 1/~Ci/ml galactose-D[4,5-3H(N)] {51.7 Ci/mM, New England Nuclear Corp., Boston, Mas- sachusetts}. Labeled cells harvested by gentle scraping with a rubber policeman were precipitated in 10% tri- chloroacetic acid (TCA) overnight at 0-4 C. TCA-pre- cipitable glycoconjugate was washed three times with ic~ cold 10% TCA and finally with 0.5% TCA before lipid extraction. Selective periodate-borotritide labeling of cell surface sialoglycoconjugates. Cell surface sialyl components were labeled selectively at sialic acid residues by mild periodate oxidation at 0-4 C, followed by reduction with sodium borotritide according to the procedure of Gahmberg and LIPIDS, VoL 21, No. 10 (1986)
