Proc. Natl. Acad. Sci. USA Vol. 91, pp. 2703-2707, March 1994 Biochemistry A mouse B16 melanoma mutant deficient in glycolipids (glucosyltransferase/glucosylceramide/glucocerebroside) SHINICHI ICHIKAWA*t, NOBUSHIGE NAKAJO*, HISAKO SAKIYAMAt, AND YOSHIo HIRABAYASHI* *Laboratory for Glyco Cell Biology, Frontier Research Program, The Institute of Chemical and Physical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan; and *Division of Physiology and Pathology, National Institute of Radiological Sciences, 4-9-1 Anagawa, Chiba-shi, Chiba 260, Japan Communicated by Saul Roseman, December 21, 1993 ABSTRACT Mouse B16 melanoma cell line, GM-95 (for- cosyltransferase would provide an ideal tool. Although sev- merly designated as MEC-4), deficient in sialyllactosylceram- eral glycosylation mutant cells have been isolated by treat- ide was examined for its primary defect. Glycolipids from the ment with a mutagen followed by selection with lectins, mutant cells were analyzed by high-performance TLC. No defects in these mutants were involved in either glycoprotein glycolipid was detected in GM-95 cells, even when total lipid syntheses (for review, see ref. 3), nucleotide sugar syntheses, from 107 cells was analyzed. In contrast, the content of or nucleotide sugar transporters (4). Mutants defective in ceramide, a precursor lipid molecule of glycolipids, was nor- glycolipid-specific transferases have rarely been found. Re- mal. Thus, the deficiency of glycolipids was attributed to the cently, Tsuruoka et al. (5) isolated a mouse mammary car- first glucosylation step of ceramide. The ceramide glucosyl- cinoma mutant that gained GM3 expression by selection with transferase (EC 2.4.1.80) activity was not detected in GM-95 antilactosylceramide (anti-LacCer) monoclonal antibody cells. There was no significant difference of sialyllactosylcera- (mAb) in combination with complement (5). mide synthase activity, however, between GM-95 and the In this report, we demonstrate that a mutant B16 melanoma parental cells. The deficiency of glycolipids in GM-95 cells was cell line, GM-95 [formerly designated as MEC-4; ref 6] associated with changes of the cellular morphology and growth lacking in GM3 has a primary defect in ceramide glucosyl- rate. The parental cells showed irregular shapes and tended to transferase (UDP-glucose: N-acylsphingosine D-glucosyl- overlap each other. On the other hand, GM-95 cells exhibited transferase, EC 2.4.1.80; GlcT-1). GM-95 cells contained an elongated fibroblastic morphology and parallel arrange- virtually no glycolipid and exhibited altered morphology and ment. The population-doubling times of GM-95 and the pa- growth properties. rental cells in serum-free medium were 28 hr and 19 hr, respectively. MATERIALS AND METHODS Materials. mAb GMR-6 (anti-GM3; IgM) was provided by Glycolipids are a group ofmembrane components that embed T. Tai of the Tokyo Metropolitan Institute of Science (7). the lipid portion in the outer leaflet ofplasma membrane and Fluorescein isothiocyanate labeled anti-mouse goat IgM(p.) extend the sugar chain to the outer environment. In animal was purchased from Kirkegaard & Perry Laboratories. Tet- cells, most glycolipids exist as glycosphingolipids. Most ramethylrhodamine-conjugated myosin subfragment was ob- glycosphingolipids are derived from the simplest glycolipid, tained from Wako Pure Chemical, Osaka. CMP-sialic acid glucosylceramide (GlcCer), which serves as the base for [cytidine 5'-monophosphate, sialic acid (sialic-4,5,6,7,8,9,- >300 glycosphingolipids. GlcCer and its derived glycosphin- 14C monophosphate), specific activity 5.55 GBq/mmol] and golipids are present essentially in all animal cells. Another UDP-Glc {uridine diphosphate glucose [glucose-14C (U)], group of glycolipids is the galactosphingolipids, character- specific activity 11 GBq/mmol} were purchased from NEN. ized by the attachment of galactose to ceramide; they are Ceramide prepared from bovine brain sphingomyelin, unla- mainly expressed in brain and kidney. beled nucleotide sugars, and NBD-GlcCer (12-[N-methyl-N- It is postulated that glycolipids serve as signal molecules (7-nitrobenz-2-oxa-1, 3-diazoyl-4-yl)]-aminodecanoylsphin- because of the localization on the cell surface and structural gosyl ,B-D-glucoside) were obtained from Sigma. Lactosyl- diversity of the carbohydrate moieties attached to them. ceramide was prepared from buttermilk. The Hoechst stain Several lines of evidence suggest the importance ofglycolip- kit for mycoplasma test was obtained from Flow Laborato- ids in various cellular processes. For example, neurite ex- ries. Horseradish peroxidase-conjugated lectins were from tention of neuroblastoma cells by gangliosides, such as GM1 Seikagaku Kogyo, Tokyo. All other reagents used were of and GQ1b, differentiation of HL-60 leukemia cells to macro- analytical grade. Mouse B16 melanoma cells were from M. phage by sialyllactosylceramide (GM3), growth regulation of Taniguchi of Chiba University. cells by GM3 and GlcCer, and implications of various gly- Cell Lines and Culture Conditions. GM3-deficient GM-95 colipids to cell-cell recognition have been reported (for cells were isolated from MEB-4 cells. MEB-4 cells do not review, see refs. 1 and 2). However, most of these results express a melanoma-specific antigen recognized by mAbs were obtained under artificial conditions-i.e., by an addition M562 and M622 but express another melanoma antigen ofglycolipids in culture medium, an inhibition oftransferases recognized by M2590 mAb (anti-GM3, IgM) (8). To obtain by specific inhibitors, a removal of sugar chains from gly- clones lacking GM3, MEB-4 cells were treated with N-methyl- colipids by enzymatic digestion, or a blockade of glycolipids N'-nitro-N-nitrosoguanidine (1.5 ,ug/ml). GM3-negative cells with antibodies or toxins. Although these approaches are were selected by killing with M2590 mAb and rabbit com- very useful to study the functions of glycolipids, one cannot plement. After 10 times selection of the mutagenized cells exclude the intrinsic side effect. with M2590 mAb, 100 clones were isolated by limiting To study the biological functions of glycolipids under physiological conditions, mutant cells lacking a specific gly- Abbreviations: GlcCer, glucosylceramide or glucocerebroside; Lac- Cer, lactosylceramide; GM3, sialyllactosylceramide; GlcT-1, ceram- The publication costs of this article were defrayed in part by page charge ide glucosyltransferase; mAb, monoclonal antibody; HPTLC, high- payment. This article must therefore be hereby marked "advertisement" performance TLC. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2703 2704 Biochemistry: Ichikawa et al. Proc. Natl. Acad Sci. USA 91 (1994) dilution. The clones were then tested for reactivity toward dpm per reaction)/80 pg ofcell protein. The reaction mixture M2590 mAb by indirect immunostaining, and four clones with was incubated for 2 hr at 370C. After incubation, the reaction low or nonreactivity with the antibody were isolated. These was stopped with 500 p4 ofwater, and the sample was applied clone were designated as MEC-1, MEC-2, MEC-3, and to Bond Elute RP 18 column (Analytichem International, MEC-4 (6); they were renamed as GM-20, GM-70, GM-89, Harbor City, CA) equilibrated with water. The column was and GM-95, respectively, because they are GM3 mutants. washed with water, and the glycolipids were eluted with These cell lines were tumorigenic, and their metastatic prop- chloroform/methanol, 2:1. The eluent was dried and sub- erties have been characterized (6). In the present study, jected to TLC followed by autoradiography. GM-95 cells, which express no GM3, were examined to Glucocerebrosidase activity was measured according to determine the deficient point in GM3 synthesis. the method of Dinur et al. (18). The cells were routinely maintained in Dulbecco's modi- Cell Growth Assay. Cells were maintained in E-RDF me- fied Eagle's medium (DMEM) (GIBCO)/10%o fetal calfserum dium supplemented with RD-I 2 weeks before the growth under 5% C02/95% air. When serum-free conditions were assay. Cells (1 x 104) were plated in 96-well plates (Primaria; required, E-RDF medium supplemented with RD-I (Kyokuto Beckton Dickinson Labware). The cell number was deter- Pharmaceutical, Tokyo) was used (9). All cell lines were mined after staining with crystal violet by a colorimetric negative in mycoplasma contamination when tested with the assay described by Kung et al. (19). Hoechst 33258 staining kit (10). Protein Assay. Proteins were quantitated by using Micro Indict Immunfluorescent Stainin. Cells were allowed to BCA protein assay reagent kit (Pierce) (20). attach for 24 hr to glass coverslips. The cells were washed with phosphate-buffered saline (PBS), fixed with 10% (vol/ vol) formaldehyde, blocked for 1 hr with 10%6fetal calfserum, RESULTS and treated with GMR-6 mAb (1:1, culture supernatant). MorphdogWical Characterization of GO-Deficient GM-95 After being washed with PBS, the cells were poststained with Cells. B16 melanoma cells and its mutagenized subclone fluorescein isothiocyanate-labeled anti-mouse IgM. MEB-4 cells have been shown to express Gm3 on the cell Lpid Analyses. Cells grown in E-RDF medium were col- surface (6). We have isolated a GM3-deficient mutant, GM-95, lected by scraping at subconfluency, washed twice with PBS, from MEB-4 cells. Indirect immunofluorescent staining of and lyophilized. Total lipids were extracted with 20 vol of MEB-4 and GM-95 cells are shown in Fig. 1. MEB-4 cells chloroform/methanol, 2:1 (vol/vol), filtrated, and evapo- expressed GM3 strongly (Fig. 1A), whereas GM-95 cells did rated to dryness. The total lipids were then redissolved into not express GM3 (Fig. 1B). Actin filament was stained with a small volume of chloroform/methanol, 2:1 (vol/vol) and tetramethylrhodamine-conjugated myosin subfiagment to vi- chromatographed on a precoated silica-gel high-performance sualize all cells in the same field (Fig. 1 C andD). MEB-4 cells TLC (HPTLC) plate (E. Merck) in chloroform/methanol/ show irregular shapes and tended to overlap each other (Fig. water, 65:25:4 (vol/vol).