Chapter 18 Role of gangliosides in tumor progression

L Svennerholm. A.K Asbury. R A Reisfeld. K Sandhoff. K. Suzuki. G Tettamanti and G Toffano (Eds.) ProRress in Bruin Rerearch. V d 101 0 1994 Elsevier Science BV All nghts reserved.

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CHAPTER 18

Role of gangliosides in tumor progression Sen-itiroh Hakomori The Biomembrane Instinrre. 1701 Elliott Ave W, Seartle. WA 98i 19 and Deparrments of Pathobioiogy and Microbiology, Universiy of Washington. Seattle. WA 98195, U S A

Introduction Glycosphingolipids (GSLs), especially gangliosides and their breakdown products, play two important roles in functioning of normal cells: (i) they define specificity of cell-cell or cell-substratum interaction; and (ii) they control transmembrane signaling via modulation of receptor kinases, protein kinase C, or other kinases (Hakomori, 1990). Certain GSLs highly expressed in tumor cells or tissues have been defined by specific monoclonal antibodies (MAbs) and thereby identified as tumor-associated carbohydrate antigens (TACAs) resulting from aberrant GSL synthesis in tumors (Hakomori, 1985, 1989). Aberrant expression of GSLs in primary tumors has been correlated with metastatic potential and invasiveness of tumors (Hakomori, 1991b; Miyake et al., 1992). Our recent studies, therefore, have focused on the functional role of gangliosides in tumor progression.

Tumor-associated carbohydrate antigens as adhesion molecules Expression of such TACAs as H/Ley/Leb in primary lung carcinoma (particularly squamous cell carcinoma) (Miyake et al., 1992), sialosyl-Tn in colorectal carcinoma (Itzkowitz et al., 1990), sialosyl-Lex in colonic carcinoma (Irimura et al., unpublished), and GM3 in murine B16 melanoma (Kojima et al., 1992b) is closely correlated with invasiveness, metastatic potential, and degree of tumor progression. Typical examples illustrating the relationship

between expression of these antigens in primary tumor and patient survival are shown in Fig. 1. Metastatic properties of tumor cells depend closely on: (i) adhesiveness of tumor cells to specific areas of microvascular endothelial cells (ECs); and (ii) ability of tumor cells to activate platelets, leading to platelettumor cell adhesion and microembolism of tumor cell aggregates. Therefore, we investigated the possible role of TACAs as adhesion molecules. Some (if not all) of the TACAs shown in Fig. 1 have been identified as adhesion molecules, recognized by complementary carbohydrate (CHO) structures on target cells via CHO-CHO interaction (Hakomori, 199 la) or interaction between CHO and lectin (including selectin) (Phillips et al., 1990; Polley et al., 1991; Handa et al., 1991b; Berg et al., 1991). That is, tumor cells showing high surface expression of these TACAs have a higher probability of interacting with the complementary CHO or lectin on the target cell. An example is described in detail in the following section.

Adhesion of melanoma cells to endothelial cells through GM3-LucCer interaction: correlation with metastatic potential GM3 ganglioside, which is highly expressed at the surface of mouse B16 melanoma cells, is recognized as a melanoma-associated antigen defined by MAb M2590 (Hirabayashi et al., 1985; Nores et al., 1987). While GM3 is ubiquitous in normal cells and tissues, it occurs at a much higher density on B16 cells and

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Fig. 1. Survival of cancer patients with or without expression of specific TACAs in their tumors. (a) Expression of A antigen in human lung cancer (Lee et al., 1991). (b) Expression of IULey/Leb(precursor of A antigen) in human lung cancer (Miyake et al., 1992). (c) Expression of SLe” antigen in human colonic cancer (Irirnura, T. et al., unpublished). (d) Expression of sialosyl-Tn antigen in human colonic cancer (Itzkowitz et al., 1990).

human melanoma cells. MAb M2590 has a ‘density threshold’ for recognition of GM3. GM3 expression is high in highly-metastatic B16 variants BL6 and F10, low in less-metastatic variant B16F1, and minimal in non-metastatic variant B16/WA4 (Fig. 2 insert). Several lines of evidence show that B16-EC adhesion is mediated by GM3-lactosylceramide (LacCer) interaction: (i) The order of adhesion of B16 variants to LacCer-coated plates (Fig. 2A) or LacCerlfibronectin (FN) co-coated plates (Fig. 2B) is the same as the order of metastatic potential, whereas the variants were indistinguishable in terms of adhesion to FN-coated plates (Fig. 2C). (ii) LacCer, a simple GSL highly expressed on mouse and human microvascular ECs, can be surface-labeled by galactose oxidase/NaB3H, treatment (Gillard et al., 1990; Kojima et al., 1992b) (data not shown). Relative

degree of adhesion of the B 16 variants to non-activated ECs is in the same order as their metastatic potential, i.e. BL6> FlO> F1> WA4 (Fig. 3A). The differences among B16 variants in adhesion to activated ECs are much smaller than differences in adhesion to non-activated ECs (Fig. 3B). (iii) Adhesion of B161BL6 or B16F10 cells to ECs is inhibited by liposomes containing LacCer, GM3, or methyl-P-lactoside, but not methyl-P-N-acetyllactosaminide(Fig. 4). Adhesion of GM3-liposomes to LacCer-coated plates, or vice-versa, has been clearly observed, and its degree is correlated with density of both GSL species (Kojima and Hakomori, 1991). (iv) Involvement of other adhesion molecules in adhesion of non-activated ECs is minimal. For example, adhesion of B16 cells to plates coated with FN, integrin, or Ig family receptors did not vary significantly

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[FN] pg/ml Fig. 2. Degree of GM3 expression in B 16 melanoma variants with different metastatic potentials, and their adhesiveness to LacCer-coated, FN-coated. and FNLacCer co-coated plates. Insert (upper right): GM3 expression as revealed by cytofluorometry with anti-GM3 MAb DH2. A, B16/BL6 cells. B, B16ff10 cells. C, B16ff1 cells. D, A431 cells. E, 3T3 cells. F, B16/WA4 cells. Panel A: Adhesion of BL6 0, F10 A, F1 A,and WA4 0 cells to LacCer-coated plates. Panel B: Adhesion to FN/LacCer co-coated plates in the co-presence of 1 kglwell of LacCer. Panel C: Adhesion to FN-coated plates.

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Fig. 3. Adhesion of melanoma variants to non-activated and activated ECs. 0, BL6 cells. A, FIO. A F1. 0 , WA4. Adhesion to non-activated ECs (upper panel) was strongly inhibited when ECs were pre-treated with anti-LacCer MAb T5A7, or when B 16 cells were treated with MAb DH2 or sialidase (data not shown). Adhesion to actlvated ECs (lower panel) was unaffected by treatment with these reagents (data not shown).

among the four melanoma variants (Fig. 2C), and required at least 20-30 min incubation. In contrast, adhesion to LacCer-coated plates required <10 min (Fig. 5 ) . This time-dependent difference between melanoma cell adhesion to GSL-coated vs. FN-coated plates provides an explanation for observed differences in a dynamic flow adhesion system (see below). Unique characteristics of melanoma adhesion based on GM3-LacCer interaction, as compared with lectin- or integrin-based adhesion, in a dynamic flow system Adhesion of B 16 cells in a dynamic flow system is distinct in many respects from adhesion in a static system; importantly, in the dynamic system adhesion to LacCer- or Gg3-coated surfaces predominates over adhesion to integrin- or lectin-coated surfaces. We constructed a dynamic flow assembly similar to that desribed by Lawrence et al. (1990). In thls assembly, a tumor cell suspension is pumped through a parallelplate laminar flow chamber, and flows over a glass

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Fig. 4.Inhibitory effect of oligosaccharides and GSL-liposomes on adhesion of BL6 cells to LacCer-coated plates (A), human ECs (B), and mouse ECs (C). Significant inhibition was observed for LacCer-liposome A, Gg3-liposome A, and methyl-P-lactoside 0 . GM3-liposome 0 was moderately inhibitory. Methyl-P-N-acetyllactosaminide V and lactose V had no effect. Concentrations of liposomes are expressed in p M on the abscissa, while concentrations of oligosaccharides are in mM.

plate coated with the suspected adhesion molecule. Adhesion of tumor cells to the plate is recorded on videotape and quantified. These conditions are designed to mimic the microvascular environment in which metastatic deposition of tumor cells occurs. In contrast to static (non-flow) model adhesion systems, degree of adhesion in the dynamic system was greatly dependent on the time required for adhesion. That is, integrin-dependent adhesion (which requires a much longer incubation period than does GSL-GSL interaction in a static system) was minimal in the dynamic system (Fig. 6A). Even Con A- or Erythrina lectin-dependent adhesion were found to be less prominent than adhesion based on GSL-GSL interac-

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Fig. 6. Adhesion of BL6 cells to glass plates coat GSLs. lectins, and adhesive proteins in a dynam Panel A: BL6 cells suspended in phosphate-bufff 105 cells/ml) were introduced into the dynamic floi ious wall shear stress values (abscissa). GSL-lipr GSWml) were coated on a circular area (0.5 ern di plates. GSL absorbed on this area was calculated Number of adherent cells after 3 min flow was cc A,LacCer. 0 ,GM3. V. paragloboside. A, control ing GSL). Panel B: procedure as in Panel A bui instead of GSL-liposomes. V, Con A lectin, Elyrhn'na lectin. 200 pg/ml. 0,FN, 100 pg/ml. V A,Con A, 20 vg/ml. 0, Gg3-liposome (for compa! A). Panel C: inhibition of BL6 cell adhesion to plates. 0, untreated BL6 cells (control). A, cells M ethyl-p-lactoside. A,sialidase. 0,MAb DH2.

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inhibited by ethyl-P-lactoside, sialidase, and antiGM3 MAb (Fig. 6C). Adhesion of BL6, F10, F l , and WA4 cells to ECs in the dynamic system, and its inhibitability by various reagents, is shown in Fig. 7A,B. These findings suggest that B 16 melanoma cell adhesion to ECs in vivo is based on GM3-LacCer interaction, and initiates metastatic deposition. Thls process may in turn trigger a series of ‘cascade reactions’ leading to activation of ECs, expression of selectins and Ig family receptors, and enhanced tumor cell adhesion and migration (Kojima et al., 1992b). Assuming the above hypothesis is true, melanoma cell metastasis could be inhibited by oligosaccharides representing GM3 or LacCer. Indeed, methyl- or ethyl-P-lactoside co-injected or separately injected with tumor cells did inhibit melanoma cell metastasis to lung (Oguchi et al., 1990). More recently, we observed that BL6 cell adhesion to LacCer-coated plates or ECs was more strongly inhibited by 6deoxy-6-fluoro-Galp1 +4Glcpl-0-Me than by methyl-P-lactoside (Cai et al., 1992) (Fig. 8). Molecular modeling experiments indicate that the 6-

(dyneslcd)

Fig. 7. Inhibition of BL6 cell adhesion to mouse ECs by various reagents in a dynamic flow system. Panel A: B16 melanoma variants ( los cells/ml) were passed over a glass plate on which mouse ECs had been grown. 0, BL6. A,F10. 0 , F1. A, WA4. Panel B: Adhesion of BL6 cells treated with vanous reagents. A, MAb DH2. A, lactose, 100 mM. 0,ethyl-P-lactoside, 50 mh4. 0, control.

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hydroxymethyl group of the galactopyranosyl residue of LacCer is involved in hydrogen bond formation during GM3-LacCer interaction. Possible functional SigniJicance of GSL and CHO antigens in human cancer A mechanism similar to that described above for mouse B 16 melanoma metastasis may well operate in human cancers, since high GM3 expression has been reported for human melanoma and other cancers, and human ECs express LacCer. Similarly, expression of HLeY/Leb antigen, defined by MAb MIA-15-5, showed a correlation with human lung carcinoma malignancy (Miyake et al., 1992; see Fig. IB). This phenomenon could reflect interaction of H/LeY/Lebexpressing tumor cells with H-expressing ECs, based on H-H, H-Ley, and/or H-Leb interaction, as illustrated in Fig. 9. Expression of H antigen on microvascular ECs of various human organs has been well established on histochemical (Holthofer et al., 1982) and immunochemical (Handa, K, Tashiro, K and Hakomori, S, unpublished) bases. It is also possible that recognition of sialosyl-Lex(SLex)or sialosyl-Lea (SLe") by selectins, and activation of platelets by tumor cells inducing P-selectin expression, could facilitate tumor cell aggregation and deposition of these aggregates on ECs. The evidence for these hypotheses was recently reviewed (Hakomori, 199 1c).

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Fig. 9. Binding of H,-liposomes (left panel) and Ley-liposomes (right panel) containing ['4C]-cholesterolto plates coated with various GSLs. Strongest binding was observed for H-H and H-Ley interaction.

Recently, we identified the P-selectin epitope expressed upon platelet activation as SLea, in addition to SLex, Binding of P-selectin to these two epitopes is strongly inhibited by a low concentration of sulfated glycan (e.g., 1 kg/ml dextran sulfate or fucoidin). Binding of SLe" or SLe" to the lectin domain of P-selectin is conformationally regulated by non-specific sulfated glycan (Handa et al., 1991b). A similar trend is clearly observed for L-selectin, but less so for E-selectin. The proposed modulatory effect of sulfated glycan on selectin binding to SLe" and SLe" is illustrated in Fig. 10. The epitopes recognized by selectins are carried mainly by 0-linked CHO chains. We observed that adhesion of HL60, U937, or Col0205 tumor cells to ECs or platelets was abolished or greatly reduced if 0-glycosylation extension was inhibited by benzyl-aGalNAc (Kojima et al., 1992a). Expression of Pselectin is strongly down-regulated by N,N-dimethylor N,N,N-trimethyl-sphingosine,or calphostin-C (all of which inhibit protein kinase C), thus inhibiting selectin-mediated adhesion of tumor cells to platelets (Handa et al., 1991a). Although the mechanism by which tumor cell metastasis is mediated by selectins is not well understood, there are obviously a number of factors involved besides expression of selectin on ECs and platelets, and mode of presentation of SLe" or SLea at the tumor cell surface (density, 0-linked, N-linked, lipid-linked, molecular carrier species). Close access and contact between tumor cells and ECdplatelets is a prerequisite for activation of and selectin expression on ECs/platelets. Our findings clearly indicate that certain tumor cells showing high expression of GM3 or H/LeY/Leb adhere to non-activated ECs through GSL-GSL interaction, which may trigger activation of ECs. Tumor cells showing high expression of Le" may autoaggregate through Lex-Lexinteraction (Eggens et al, 1989), leading to microembolism and activation of ECs/platelets. This suggests the following possible sequence of events: (1) tumor cells adhere to non-activated ECs, or autoaggregate through CHO-CHO interaction; (2) subsequently, ECs or platelets are activated by tumor cells, which lead to surface expression of E- or P-selectin; (3)

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T

Sull. Glycon

Fig. 10. Proposed effect of sulfated glycans on P-selectin binding to SLe" or SLe" presented on glycoprotein or GSL. Gp, glycoprotein. Solid oval, SLe" or Sk".Sulfated glycans may interact with the EGF domain (E) or complement-regulatoy sequence repeat (C), thereby inducing confonnational changes of the lectin domain (L) of P-selectin. Sulfated glycans also have a clear inhibitory effect on L-selectin, but have less effect on E-selectin binding to SLe" or SLe" (Shiozawa, T., Handa, K., Nudelman, E.D. and Hakomori, S.. unpublised).

SLex or SLe" epitope is appropriately presented at the tumor cell surface via clustered, 0-linked membrane proteins; and (4) additional stronger adhesion and

enhancement of cell motility may take place through mechanisms involving integrin and Ig family receptors. GSLs are organized in clusters at the cell surface

Fig. 11. Proposed organization of GSLs at the cell surface. Upper left: clustering of globoside on the human erythrocyte surface (Tillack et al., 1983). Upper right: clustering of Forssman GSLs on Forssman-liposome. E. external surface. F, fractured internal surface. Lower panel: conceptual scheme of GSL patches expressed on plasma membrane. Gp, glycoprotein.

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Fig. 12. Proposed scheme for stepwise progression of adhesion between tumor cells and ECs. Adhesion of tumor cells to ECs in a dynamic flow system is initially mediated via GSL patches on both plasma membranes (GM3-LacCer, H-Ley, or H-H) (step l), followed by activation of ECs (step 2). leading to expression of selectin (SE) and binding of selectin to SLe” or SLe”on the tumor cell surface. This sends signal ‘Y’to activate cell motility (step 3). leading to reinforcement of adhesion by integrin receptor (Itgr) or immunoglobulin receptor (IgR) mechanisms.

(Tillack et al., 1983; Rock et al., 1990, 1991) (Fig. 11). Initial interaction between GSLs expressed on tumor cells and those on ECs may take place via these GSL clusters (Fig. 12). Thus, if initial interaction between GSL clusters were blocked, the whole adhesion process would be blocked. The process could also be blocked by inhibiting transmembrane signaling involved in selectin expression. During induction of P-selectin expression by tumor cells, the presence of sulfated proteoglycans may modulate Pselectin binding activity. The metastatic process is complex, involving many different factors and steps. We may therefore be able to block metastasis at various steps by application of

several different types of reagents: (i) oligosaccharides or their derivatives; (ii) anti-oligosaccharide MAbs; (iii) inhibitors of 0-glycosylation or O-glycosylation extension; and (iv) blockers of transmembrane signaling leading to selectin expression. In fact, we have successfully blocked metastasis using some of these reagents (Oguchi et al., 1990; Okoshi et al., 1991). Further studies along these lines are in progress.

Acknowledgment I thank Dr. Stephen Anderson for scientific editing and preparation of the manuscript.

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References Berg, E.L., Robinson, M.K, Mansson, 0.. Butcher, E.C. and Magnani, J.L. (1991) A carbohydrate domain common to both sialyl Lea and sialyl Le" is recognized by the endothelial cell leukocyte adhesion molecule ELAM-I. J. B i d . Chem., 266: 14869-14872. Cai, S., Hakomori, S. and Toyokuni, T. (1992) Application of protease-catalyzed regioselective esterification in synthesis of 6'deoxy-6'-fluoro- and 6-deoxy-6-fluorolactosides.J. Org. Chem., 57: 3431-3437. Eggens, I., Fenderson, B.A., Toyokuni, T., Dean, B., Stroud, M.R. and Hakomori, S. (1989) Specific interaction between Lex and Lex determinants: A possible basis for cell recognition in preimplantation embryos and in embryonal carcinoma cells. J. Biol. Chem., 2 6 4 94769484, Gillard, B.K. Jones, M.A.. Tumer, A.A., Lewis, D.E. and Marcus, D.M. ( 1990) Interferon-gamma alters expression of endothelial cell-surface glycosphingolipids. Arch. Biochem. Biophys., 279: 122-1 29. Hakomori, S. (1985) Aberrant glycosylation in cancer cell membranes as focused on glycolipids: Overview and perspectives. Cancer Res., 45: 2405-2414. Hakomori, S. (1989) Aberrant glycosylation in tumors and tumorassociated carbohydrate antigens. Adv. Cancer Res., 52: 257-33 I . Hakomori S, ( 1990) Bifunctional role of glycosphingolipids: Modulators for transmembrane signaling and mediators for cellular interactians. J. Biol. Chem.. 265: 18713-18716. Hakomori, S. (199la) Carbohydrate-carbohydrate interaction as an initial step in cell recognition. Pure Appl. Chem., 63: 473482. Hakomori, S. (1991b) Possible functions of tumor-associated carbohydrate antigens. Curr. Opin. fmmunol., 3: 64&653 Hakomori, S. ( 1 9 9 1 ~ )Possible new directions in cancer therapy based on aberrant expression of glycosphingolipids: Anti-adhesion and ortho-signaling therapy. Cancer Cells, 3: 461470. Handa, K, Igarashi, Y., Nisar, M. and Hakomori, S. (1991a) Downregulation of GMP- 140 expression on platelets by N,N-dimethyl and N,N,N-trimethyl derivatives of sphingosine. Biochernisrry, 30: 11682-11686. Handa, K, Nudelman, E.D., Stroud, M.R., Shiozawa, T. and Hakomon, S. (1991b) Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Lea and sialosyl-Lex, and sulfated glycans modulate this binding. Biochem. Biophys. Res. Commun.. 181: 1223-1 230. Hirabayashi, Y., Hanaoka, A., Matsumoto, M., Matsubara, T., Tagawa, M., Wakabayashi, S. and Taniguchi, M. (1985) Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma. J. B i d . Chem., 260: 13328-13333. Holthofer, H., Virtanen, I., Kariniemi, A.-L., Hormia, M., Linder, E. and Miettinen A. (1982) Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab. Invest.. 47: 60-67.

Itzkowitz, S.H., Bloom, E.J., Kokal, W.A., Modin, G., Hakomori, S. and Kim, Y.S. (1990) Sialosyl-Tn: A novel mucin antigen associated with prognosis in colorectal cancer patients. Cancer. 66: 196C-1966. Kojima, N., Handa, K, Newman, W. and Hakomori, S. (1992a) Inhibition of selectin-dependent tumor cell adhesion to endothelial cells and platelets by blocking 0-glycosylation of these cells. Biochem. Biophys. Res. Commun.. 182: 1288-1295. Kojima, N., Shiota, M., Sadahira, Y. and Hakomori, S. (1992b) Cell adhesion in a dynamic flow system as compared to static system: Glycosphingolipid-glycosphingolipidinteraction in the dynamic system predominates over lectin- or integrin-based mechanisms in adhesion of B 16 melanoma cells to non-activated endothelial cells. J. Biol. Chem.. 267: in press. Kojima N. and Hakomori S. (1991) Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction. J. Biol. Chem., 266: 17552-17558. Lawrence, M.B., Smith, C.W., Eskm, S.G. and McIntire, L.V. (1990) Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium. Blood. 75: 227-237. Lee, J.S., Ro, J.Y., Sahin, A.A.. Hong, W.K., Brown B.W., Mountain, C.F. and Hittelman, W.N. (1991) Expression of blood-group antigen A: A favorable prognostic factor in nonsmall-cell lung cancer. N . Engl. J. Med., 324: 1084-1090. Miyake, M., Taki, T., Hitomi, S. and Hakomori, S. (1992) Correlation of expression of WLeY/Leb antigens with survival in patients with carcinoma of the lung. N . Engl. J . Med.. 327: 14-18. Nores, G.A., Dohi, T., Taniguchi, M. and Hakomori, S. (1987) Density-dependent recognition of cell surface GM, by a certain anti-melanoma antibody, and GM, lactone as a possible immunogen: Requirements for tumor-associated antigen and immunogen. J. fmmunol., 139: 3171-3176. Oguchi, H., Toyokuni, T., Dean, B., Ito, H., Otsuji, E., Jones, V.L., Sadozai, K.K. and Hakomori, S. (1990) Effect of lactose derivatives on metastatic potential of B16 melanoma cells. Cancer Commun., 2: 31 1-316. Okoshi, H., Hakomori, S., Nisar, M., Zhou, Q., Kimura, S., Tashiro, K. and Igarashi, Y. (1991) Cell membrane signaling as target in cancer therapy 11: Inhibitory effect of N,N.N-trimethylsphingosine on metastatic potential of murine B16 melanoma cell line through blocking of tumor cell-dependent platelet aggregation. Cancer Res.. 5 1: 60194024. Phillips, M.L., Nudelman, E.D., Gaeta, F.C.A., Perez, M., Singhal, A.K. Hakomori, S. and Paulson, J.C. (1990) ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Le". Science. 250: 113C-1132. Polley. M.J., Phillips, M.L., Wayner, E.A., Nudelman, E.D., Singhal, A.K. Hakomori, S. and Paulson, J.C. (1991) CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM- 1) recognize the same carbohydrate ligand, sialyl-Lewis x. P roc. Narl. Acad. Sci. USA, 88: 6224-6228. Rock, P., Allietta, M., Young, W.W. Jr., Thompson, T.E. and Tillack, T.W. ( 1990) Organization of glycosphingolipids in

250 phosphatidylcholine bilayers: Use of antibody molecules and Fab fragments as morphologic markers. Biochemistry, 29: 84848490. Rock P., Allietta, M., Young, W.W. Jr., Thompson, T.E. and Tillack, T.W. (1991) Ganglioside GMl and asialo-G,, at low concentration are preferentially incorporated into the gel phase

in two-component, two-phase phosphatidylcholine bilayers. Biochemistn. 30: 19-25. Tillack, T.W., Allietta, M., Moran, R.E. and Young, W.W. Jr. (1983) Localization of globoside and Forssman glycolipids on erythrocyte membranes. Biochim. Biophvs. A m . 733: 15-24.