[CANCER RESEARCH 37, 1333-1339, May 1977] Association of the Glycolipid Pattern with Antigenic Alterations in Mouse Fibroblasts Transformed by Murine Sarcoma Virus1 Gunter Rosenfelder,2 William W. Young, Jr.,3 and Sen-itiroh Hakomori DIvisionof BiochemicalOncology,FredHutchinsonCancerResearchCenter,andDepartmentsofMicrobiologyandPathobiology,Universityof Washington, Seattle,Washington98104 SUMMARY ganglioside in various hepatoma cells as compared with normal liver (6, 8, 29, 31, 34). The level of the neutral glycolipid, GalNAc/31—@4Galf3i—* The biological implications of such alterations are not 4Glc—aCer(asialo GM2), in BALB/c 3T3 mouse fibrobiasts known. The primary objective of the present study was to transformed by Kirsten murine sarcoma virus (3T3KiMSV) determine whether such an accumulation of a precursor was greatly increased compared to the nontransformed pa glycolipid would result in a change in the immunogenicity rental cells (3T3). This elevated chemical quantity was of the affected tumor cells. A previous study in this labora found to be localized on the surface of intact cells and tory indicated that the accumulation of lacto-N-neotetrao accessible to external reagents, as detected by immunoflu sylceramide (paragloboside) in polyoma-transformed ham orescence and labeling with galactose oxidase:NaB3H4. ster fibroblasts (NiLpy cells) was accompanied by such a Furthermore, immunization of rabbits with 3T3KiMSV cells change of immunogenicity. Sera of hamsters bearing NILpy but not with 3T3 cells resulted in antibody production tumors contained detectable amounts of antibody-like ma against asialo GM2. These results demonstrate the potential terial reactive with paragloboside as determined by comple usefulness of glycolipids as tumor-associated cell surface ment fixation (33). markers. BALB/c 3T3 cells transformed by the nonproductive Kir sten strain of murine sarcoma virus (3T3KiMSV cells) (1) INTRODUCTION were reported previously to have a drastically altered glyco lipid pattern due to the blocked synthesis of GM, gangiio Alterations of cellular glycolipid patterns have been de side from GM, ganglioside (9, 10). We now report the strik scribed as being associated with the process of cancer. ing accumulation of asialo GM2in these cells, in addition to These changes have been observed as a common pheno the changes already described. Coupled with this chemical typic marker of spontaneous tumors as well as tumor cells change are immunological alterations that indicate that asi transformed by DNA viruses, RNA viruses, and chemical alo GM2 could function as a tumor-associated antigen in carcinogens (for reviews see Refs. 4 and 15). In many in 3T3KiMSV cells. stances the blocked synthesis of more complex glycolipids in tumor cells is often accompanied by an accumulation of MATERIALS AND METHODS high levels of precursor glycolipid(s). Typical examples of this phenomenon include increased levels of lactosylceram AnimalsandCells. NewZealandWhitemalerabbitswere ide in a clone of BHK cells transformed by polyoma virus purchased from Gunther Fur Products, Kent, Wash. BALB/ (16), lacto-N-neotetraosylceramide in hamster NIL cells c male mice were purchased from Simonsen Laboratories, transformed by polyoma virus (12, 33), and GMI4 and GD1a Inc., Gilroy, Calif. BALB 3T3 (clone A31) and 3T3KiMSV (clone K-234) cells were obtained from Dr. M. Hatanaka, I This investigation was supported by National Cancer Institute Contract Frederick Cancer Research Center, Frederick, Md. Cells NO1-CB-43920,NIHResearchGrantCA20026,andAmericanCancerSociety Grant BC-9F. were cultured in Dulbecco's modified Eagle's medium sup 2 Recipient of a Fellowship grant from the Deutsche Forschungsgemein plemented with 10% calf serum in a 5% CO2 atmosphere. schaft. Glycolipids were radioactively labeled by growing the cells a Recipient of NIH Postdoctoral Fellowship 1F32 GM05281. 4 The abbreviations used are: GM,, GaIpl—ø3GaINAcpl-.4(NAcNeura2 for 48 hr with 0.3 @Ciof[‘4C]galactose(uniformly labeled; —‘3)Gal@1--@4Glc--@Cer;GD1a, NAcNeura2—@3Galp1-.3GaINAcf31--øspecific activity, >200 mCi/mmoIe) per ml of medium. Cells 4(NAcNeura2—@3)GaI@1—.4GIc—øCer;3T3KIM5V,BALB/c 3T3 fibroblasts were harvested by mechanical scraping, and the washed transformed by the Kirsten strain of murine sarcoma virus; GM,, GaINAc@1—@ 4(NAcNeura2—'3)Galfll—'4Glc-.Cer; asialo GM,, GaINAcp1-.4Gal@1--ø cell pellet was stored at —80°. 4GIc-.Cer; TLC, thin-layer chromatography; CTH, ceramide trihexoside GlycolipidAnalysis. Detailedproceduresfor the isola (GaIal—―4GaIftl-.4Glc--'Cer); CIT. ceramide tetrasaccharlde (globoside; GaINAcft1—.3GaIa1-.4Galft1-.4Glc—@Cer);PBS,140 mu NaCI, 1 mM CaCI,; tion, characterization, and structural analysis of glycolipids 0.5 mt.i MgCI,, and 10 mM sodium phosphate, buffered to indicated PH; CDH, from various organs and cultured cells have been described ceramide dihexoside (GaIftl-.4GIc--'Cer); cytolipin R, GaINAc$1 recently (20). Briefly, frozen cell pellets and organs were -.3GaIa1-.3Gal@1—.4GIc--'Cer; asialo GM,, [email protected]@1—. 4GIc-@Cer. extracted with chloroform:methanol (2:1 , v/v). The ganglio Received November 23, 1976; accepted January 26, 1977. side fraction was partitioned according to the method of MAY 1977 1333 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1977 American Association for Cancer Research. G. Rosenfelder et a!. Folch et a!. (ii), followed by dialysis of the upper layer several sites on a male New Zealand White rabbit. After 4 using dialysis tubing with a small pore size (Spectrapor weeks, precipitating antibody against asialo GM2 was de 4000; Spectrum Medical Industries, Inc. , Los Angeles, tected. This antiserum was purified by ammonium sulfate Calif.) to minimize loss of ganglioside. The neutral glyco precipitation and passage through a Sepharose-bovine se lipid fraction was purified by the acetylation procedure pre rum albumin affinity chromatography column (7) to remove viously described (27). Purified neutral glycolipid and gan anti-albumin antibodies. Antibodies against human brain glioside fractions from radiolabeled cells were analyzed by GM1 were produced and purified in an identical fashion. separate TLC and autoradiography. Immunization of rabbits with 3T3 and 3T3KiMSV cells was Purified asialo GM2from 3T3KiMSV cells was obtained by carried out by a modification of the protocol used for pre preparative TLC. Maximum recovery of glycolipid was paring anti-Forssman antibodies by injection of sheep achieved by treating the silica gel suspension with Dowex erythrocyte membranes (17). Cell monolayers were har 50 H@as previously described (25). Structural characteriza vested with 0.02% EDTA, and the suspensions of intact cells tion of asialo GM2 was accomplished by: (a) carbohydrate were injected i.v. into male New Zealand White rabbits. A analysis through gas chromatography, (b) oxidation with series of ii injections, consisting of 106cells/injection, was galactose oxidase followed by reduction with completed within 2 weeks, and the rabbits were bled 5 days [3H]borohydride and examination of the 3H activity through after the final inoculation. radioactivity counting of sugar peaks obtained on gas chro The reactivity of the antisera with various glycolipids was matography, (c) methylation analysis (3, 21), (d) total mass tested using a modification of a microcomplement fixation spectrometry after permethylation and reduction (18), and test as previously described (5). Test glycolipids were pre (e) enzymatic degradation with f3-N-acetylhexosaminidase pared with auxiliary lipids using a weight ratio of egg leci and f3-galactosidase of jack bean (22). thin:cholesterol:glycolipidof25:12.5:1.Theantiserawere The chemical quantity of asiaio GM2 in normal BALB/c also tested in microtiter plates for hemagglutination of a organs was determined by TLC of free glycolipids in the suspension of 2% guinea pig erythrocytes. The ability of solvent chloroform:methanol:water (60:35:8) and of acety various glycolipid-liposomes to inhibit this hemagglutina lated glycolipids mnthe solvent 1,2-dichloroethane:meth tion was tested as well. anol (91:9). The latter system made possible the identifica Immunofluorescence. Monolayers of 3T3 and 3T3KiMSV tion of small amounts of asialo GM2 in the presence of cells were grown in 3-cm dishes. All of the following steps CTH and ceramide tetrasaccharide. were performed at 4°.Aftergentle washing of the monolayer Cell Surface Labeling. Cell surface labeling was carried with PBS, pH 7.4, the cells were incubated for 30 mm with 1 out as previously described (12, 13). Washed cell monolay ml of rabbit anti-asialo GM2 or anti-GM1 purified as de ers were incubated for 2 hr at room temperature with 10 scribed above and diluted 1:10 with PBS. After additional units of galactose oxidase (AB Kabi, Stockholm, Sweden) washing, the monolayers were incubated for 30 mm with 1 per 15-cm plate in 2 ml PBS, pH 7.0. The cells were harvested ml of fluorescein-labeled goat anti-rabbit immunoglobulin by mechanical scraping, washed by centrifugation, and sus (Behring Diagnostics, Somerville, N. J.) diluted 1:10 with pended in 0.5 ml PBS, pH 7.4, containing 2.5 mCi of PBS. The monolayers were thoroughly washed, fixed with [3Hjsodium borohydride. After 30 mm incubation at room glutaraldehyde, and examined with a Zeiss Photomicro temperature, the cells were washed extensively and stored scope II. at —80°.Eachfrozen cell pellet was extracted with chioro form:methanoi