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Different Patterns of Lectin Binding and Cell Surface Sialylation Detected on Related High- and Low-Metastatic Tumor Lines

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Different Patterns of Lectin Binding and Cell Surface Sialylation Detected on Related High- and Low-Metastatic Tumor Lines (CANCER RESEARCH 43, 5138-5144, November 1983] Different Patterns of Lectin Binding and Cell Surface Sialylation Detected on Related High- and Low-Metastatic Tumor Lines Peter Altevogt,1 Mina Fogel, Rachanee Cheingsong-Popov, Jim Dennis, Peter Robinson, and Volker Schirrmacher2 Institut fur Immunologie und Genetik, Deutsches Krebsforschungszentrum, 6900 Heidelberg, Federal Republic of Germany [P. A., M. F., R. C-P. P. R., V. S.¡,and The Hospital for Sick Children, Toronto, Ontario, Canada [J. D.J ABSTRACT tation of metastatic tumor cells by heterotypic cell interactions; and (c) in the arrest in organs by specific interactions with the We have analyzed cell surface-bound carbohydrates in two target tissue. different model systems for metastasis composed of closely Such important involvements of carbohydrates in metastasis related tumor cell lines with differing metastatic potential. The formation would imply that tumor cells with different metastatic first system studied was that of the DBA/2-derived T-lymphoma potential possess distinct qualitative or quantitative differences lines (Eb/ESb) and some recently established sublines of ESb in their glycosylation of membrane glycoconjugates. A correlation with altered metastatic behavior (ESb-M and ESb-MR). The between the quantity of cell surface-exposed SA3 and metastatic second system consisted of the highly metastatic MDAY-D2 capacity was recently found by Yogeeswaran (36) for a variety cells, a wheat germ agglutinin-resistant low metastatic subline of different tumor cell lines. Of particular importance seemed to MDW40, and two metastatic revertants from the latter. The cells be SA residues in association with subterminal sugar moieties were stained with fluorescein isothiocyanate-conjugated lectins which could be oxidized by galactose oxidase and subsequently and analyzed by flow cytofluorography. All low-metastatic tumor reduced with NaBH4 (36). lines expressed receptor sites for the lectins soybean agglutinin We have further investigated this topic with cell lines of 2 (SBA) and Vicia villosa (VV). The metastatic lines had the re independent tumor model systems for metastasis. Differences in spective lectin binding sites blocked by sialic acid (SA). A good glycosylation and the role of SA were studied by using a variety correlation was found within the cell lineages Eb —»ESb—»ESb- of FITC-conjugated lectins with different sugar specificities and M —ESb-MR and MDAY-D2 -»MDW40 -»MDW40M1 between flow cytofluorographic analysis. The first tumor system was the reactivity of SBA and VV and metastatic potential. DBA/2 T-lymphoma Eb and its spontaneous high-metastatic The amount of neuraminidase-accessible SA was similar for variant ESb (30). It was found that acceptor sites of SBA and all cell types (except MDW40) indicating differences in the posi VV were covered by SA on all high-metastatic ESb sublines but tioning of SA. For high-metastatic ESb cells, the Sialylation of not on parental type Eb cells or on low-metastatic variants from SBA and VV receptor sites was paralleled by a relative decrease ESb cells. Similar differences were noted in a second tumor of SA associated with receptor sites for peanut agglutinin. Low- system (9, 11, 18, 19). From these results, we suggest that the metastatic Eb cells, in contrast, had their peanut agglutinin Sialylation of particular carbohydrate residues on tumor cells receptor sites sialylated but expressed asialylated SBA and VV which are recognized by certain lectins may facilitate their met receptor sites. Eb cells were also found to have 2-fold higher astatic spread possibly by masking specific cellular adhesion activities in galactose-specific sialyltransferases. It is proposed sites. that the differences in positioning of SA on the cell surface leading to masking or unmasking of terminal sugars could influ MATERIALS AND METHODS ence the metastatic potential of tumor cells. Cells. The tumor cell Eb (Heidelberg subline of L5178Y) and its metastatic variant ESb were obtained from Professor P. Alexander INTRODUCTION (London, England). The etiology of the cell lines has been described (30). Both cell lines were passaged as ascites in syngeneic DBA/2 mice Alterations in cell surface carbohydrates have been found to (Bomholtgaard, Denmark) or grown in suspension in Roswell Park Mem severely affect the metastatic potential of experimental tumors. orial Institute tissue culture Medium 1640 supplemented with penicillin For example, tumor cell variants selected in the presence of (100 units/ml), streptomycin (100 /jg/ml), glutamine (5 HIM), 4-(2-hydrox- toxic concentrations of certain plant lectins appeared to have yethyl)-1-piperazineethanesulfonic acid (50 mM), 2-mercaptoethanol (5 x 10~5 M), and 5 or 10% fetal bovine serum. ESb-M is a plastic-adherent significantly changed metastatic behavior (11, 28, 34). Such selection procedures can induce changes in specific surface variant of ESb with highly decreased metastatic capacity (14). MDAY- carbohydrates (11, 12), glycosyltransferase activities (12, 15, D2 is a metastatic tumor cell line of DBA/2 and was established by Kerbel ef al. (18,19). A WGAR cell line MDW40, with altered tumorigenic- 23), and glycoprotein content (22). One could theoretically envis age at least 3 steps in the metastatic cascade that involve cell ity and metastatic capacity (9), and 2 metastatic and wheat germ agglutinin-sensitive revertants thereof (MDW40M1 and MDW40M4) have surface carbohydrate interactions: (a) in the release of tumor been described previously (11 ). cells from the primary tumor mass due to altered homotypic Staining of Tumor Cells with FITC-conjugated Lectins and Cyto adhesion phenomena; (b) in the mechanism of blood transpor- fluorographic Analysis. FITC-conjugated lectins (1 mg/ml) were ob tained from Medac (Hamburg, Federal Republic of Germany). Fluoro- 1To whom requests for reprints should be addressed. Recipient of a grant from Meyenburg-Stiftung. 3The abbreviations used are: SA, sialic acid; FITC, fluorescein isothiocyanate; * Supported by the Deutsche Forschungsgemeinschaft through Sonderfor- SBA, soybean agglutinin; VV, Vicia vil/osa: WGA", Wheat germ agglutinin-resistant; schungsbereich 136. HP, Helix oomatia, GalNAc, N-acetylgalactosamine; PNA, peanut agglutinin; o-Gal. Received September 2, 1982; accepted August 8, 1983. D-galactose; HBP, hepatocyte-binding protein; GlcNAc, N-acetylglucosamine. 5138 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1983 American Association for Cancer Research. Cell Surface Glycosylation and Metastatic Potential chrome/protein ratios (495/280 nm) were between 1.3 and 1.8. Cells absent on ESb tumor cells but present on Eb cells (1). (106) were incubated with 50 n\ of 1/20 diluted FITC-lectins in the absence Staining of Eb cells with SBA, VV, and HP was only slightly or presence of 0.2 M competitive sugars on ice for 45 min. The reaction increased after neuraminidase treatment, indicating that these was carried out in 0.9% NaCI solution supplemented with 1 HIM calcium receptor sites were mostly unsialylated and accessible for the and magnesium salts. Cells were washed and analyzed by flow cytofluo- rography using an ORTHO 50-H cell sorter. The argon laser was operated lectins (Chart 1). at 488 nm, and 3 x 104 cells were analyzed using settings to exclude ESb cells showed weak reactivity with PNA, while Eb cells nonviable cells according to scatter characteristics. Gains were kept at were negative as judged by the histograms in Chart 1. After identical settings for each FITC-lectin analyzed. neuraminidase treatment, both cell lines were stained by PNA, Neuraminidase Treatment of Cells. Cells were incubated in Dulbec- but the ESb cells still showed stronger reactivity as seen by the co's phosphate-buffered saline at 20 to 40 x 106/ml for 45 min at 37°in percentage of stained cells and by the fluorescence intensity. In the presence of 2.5 units of neuraminidase of Vibrio cholerae (Behring- contrast, Ricinus communis agglutinin was able to bind to both werke, Marburg, Federal Republic of Germany) and were then washed Eb and ESb cells, possibly via unblocked terminal o-galactose 3 times and analyzed for lectin binding as described above. residues. This binding was enhanced after treatment with neur SA Determination. Fifteen to 20 x 106 cells/ml were treated with 2 units of neuraminidase for 45 to 60 min at 37°.Neuraminidase-releasable aminidase and most strongly for Eb cells, indicating a higher degree of o-galactose sialylation (Chart 1S). No major differences SA of the supernatants was determined by the thiobarbiturate method in lectin binding properties of Eb and ESb cells were noted for u- of Warren (35). fucose- or o-mannose-detecting lectins [see results in Chart 16 Determination of Glycosyltransferase Activities. Cell homogenates with Ulex europaeus (L-fucose) or concanavalin A (o-mannose)]. from Eb and ESb were prepared according to the method of Chatterjee ef al. (8). One g of packed cells was resuspended in 3 ml of 0.25 M The results taken together indicate obvious differences be sucrose and 0.1 mw CaCI2 adjusted to pH 8.0 with 1 M Tris buffer and tween the low- and high-metastatic cells, Eb and ESb, in the homogenized with a glass homogenizer. The homogenate was spun at expression of receptor sites for lectins with specificity for GalNAc 1500 rpm for 10 min. The supernatant was collected and stored at -70°. residues (VV, SBA, and HP), which are expressed on Eb but not Asialofetuin and asialoagalactofetuin
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