Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.

E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O-sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L-selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E-selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin-dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin-dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression.

Interaction of tumor cells with vascular endothelia: role of platelet-activating factor.

We investigated whether tumor cell/endothelia interaction can be influenced by platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a lipid mediator that promotes adhesiveness and extravasation of leukocytes in the inflammatory reaction. We found that the PAF receptor antagonist WEB 2086 prevents adhesion of melanoma Hs294T and colon carcinoma LS180 lines to IL-1-stimulated endothelial cells. Moreover, PAF stimulated the adhesiveness of Hs294T and LS180 cells to VCAM-1 and E- selectin, respectively, in an artificial model consisting of recombinant adhesive proteins bound to protein A-coated substrata. Thus, tumoral and not endothelial cell surface seems to be involved in the PAF-mediated enhancement of tumor cell adhesiveness to IL-1-activated endothelia. This observation is supported by the finding that Hs294T and LS180 cells express high affinity and functionally active receptors for PAF. By using specific inhibitors, we found that PAF-induced enhancement of cell adhesiveness was mediated by G-protein activation and protein tyrosine phosphorylation. In addition, protein tyrosine phosphorylation was observed in Hs294T and LS180 cells stimulated by PAF. In conclusion, we demonstrated that PAF-mediated activation of tumor cells enhances their adhesiveness to IL-1-stimulated vascular endothelia.

Lipid characteristics in metastatic cells.

Interest in lipid characteristics of metastatic cells was aroused by the consideration that the various lipid components of cell membranes influence a broad spectrum of cell surface biological functions which are involved in different steps of the metastatic cascade. Correlation between invasive properties and characteristics of cell surface components has been appropriately studied in a limited number of metastatic cell systems isolated by in vivo and in vitro procedures. The major findings of this study have been reported in this review. Among membrane lipid components, glycolipids and phospholipids appeared particularly affected in tumor cells which acquired a metastatic phenotype. In fact, the reduction of complex gangliosides typical of transformed cell lines was even more evident in a highly metastatic variant selected from RSV-transformed murine fibroblasts. The reduction of complex gangliosides, mainly GD1a, particularly affected the adhesion sites of this variant. In a fibrosarcoma line, T3 cells, the metastatic properties appeared to be correlated with the content and cell surface expression of Gb3ose, a glycolipid characteristic of this line. Moreover, a particularly high level of ether-linked lipids was found in high metastatic variants isolated from murine melanoma and fibrosarcoma lines, as well as in human mammary carcinomas. The findings considered in this review are discussed for their possible relevance to the invasive properties of metastatic cells.

Lectin reactivity of murine fibrosarcoma lines with a different metastatic potential.

Lectins are suitable tools for investigating the glycoconjugate characteristics of metastatic cells. In the present study, we investigated whether there were differences between high metastatic T3 cells and a low metastatic isolate in their reactivities to several lectins specific for galactosyl and sialyl groups. Analysis of reactivity of the two cell lines to wheat germ agglutinin revealed a complex pattern. In fact, T3 cells had high-affinity, neuraminidase-resistant as well as low-affinity, neuraminidase-sensitive receptors. Instead, the low metastatic isolate showed only high-affinity receptors, both neuraminidase-resistant and neuraminidase-sensitive The two cell lines reacted similarly to galactose-specific lectins. These findings indicate that sialyl groups, rather than galactosyl groups, affect the metastatic behavior of our cell system.

Composition of ether-linked sub-classes of glycerophospholipids in clones with a different metastatic potential isolated from a murine fibrosarcoma line (T3 cells).

An increase of ether-linked sub-classes of choline and ethanolamine glycerophospholipids has been shown in different types of tumor cells, and correlated with some of their specific biological parameters. In the present study, we examined the composition of ether-linked lipids in a series of clones with a different lung-colonizing potential isolated in our laboratory from a highly metastatic fibrosarcoma line (T3 cells). We found good correlation between the metastatic potential of T3 isolates and increased proportions of both alkylacyl and alkenylacyl subclasses in choline glycerophospholipids (CGP). Moreover, propagation of a weakly metastatic T3 clone in tissue culture led to the emergence of a sub-clone which expressed high metastatic potential together with a high level of alkylacyl and alkenylacyl CGP. No differences were found in the alkylacyl and alkenylacyl-ethanolamine glycerophospholipids (EGP) between the strongly and weakly metastatic T3 clones. We discuss the accumulation of alkylacyl and alkenylacyl CGP in metastatic cells for its possible role in metastatic diffusion by generation of platelet-activating factor (PAF).

Role of glycolipids in the metastatic process: characteristics of neutral glycolipids in clones with different metastatic potentials isolated from a murine fibrosarcoma cell line.

We investigated whether metastatic phenotype is associated with a characteristic glycolipid pattern. For this study, we developed a system of variants with different metastatic potentials that we isolated from the highly metastatic T3 murine fibrosarcoma line by culture in 0.3% agar or on plastic. The glycolipid profiles of T3 cells and of their highly metastatic isolates were characterized by a high level of globotriaosylceramide (Gb3ose). On the other hand, Gb3ose was reduced in a weakly metastatic clone isolated from T3 cells. A reduced level of Gb3ose was also found in a weakly metastatic subclone isolated from a highly metastatic T3 clone. Propagation of this subclone led to the emergence of a series of variants which expressed a high metastatic potential together with a high Gb3ose level. We also observed that Gb3ose was 10 times more prevalent on the cell surface in T3 cells than in a weakly metastatic clone. On the whole, these findings indicate that, in our system of metastatic cells, a high Gb3ose level correlates with metastatic phenotype. It is possible that the highly exposed Gb3ose in metastatic cells is relevant to the metastatic process in view of the role played by the unique molecular structure of this glycolipid in other models of cell-to-cell interaction.

Inhibition of colon carcinoma cell lung colony formation by a soluble form of E-selectin.

During metastasis, tumor cells adhere to vascular endothelia. E-selectin is an adhesive protein expressed by cytokine-activated endothelium that can support adhesion of colon cancer cells through the recognition of specific carbohydrate ligands. Using a series of colon carcinoma cell lines that displayed E-selectin adhesiveness and an increased metastatic capacity in cytokine-treated mice, we examined possible inhibition of cytokine-dependent experimental lung metastasis by a soluble form of E-selectin, the recombinant fusion protein E-selectin-immunoglobulin. We found that E-selectin-immunoglobulin bound to the surfaces of HT-29 colon carcinoma cells and blocked the formation of cytokine-inducible experimental lung metastases; control L-selectin-immunoglobulin also bound to HT-29 cells but had no effect on tumor cell lung colonization. E-selectin-immunoglobulin was found to interfere with E-selectin-dependent adhesion of HT-29 cells to activated vascular endothelium and to block the retention of these cells in the lung, a process that implies tumor cell adhesive interactions with the host vasculature. Our results demonstrate that E-selectin-immunoglobulin inhibits adhesion and formation of lung metastases by colon carcinoma cells and suggest that impairment of tumor cell-endothelium adhesion might represent a therapeutic approach to the metastatic diffusion of tumors.

Endothelial-leukocyte adhesion molecules in human disease.

An effective host response to infection or tissue damage requires focal accumulation of leukocytes. Leukocyte adhesion to the vessel wall, a key step in this process, depends on the ordered expression of specific endothelial cell surface molecules. The endothelial molecules that support adhesion include selectins that recognize leukocyte cell surface glycoconjugates as well as members of the immunoglobulin superfamily that interact with leukocyte integrins. Although inflammation can occur with minimal damage to the vessel wall and surrounding tissues, control mechanisms sometimes appear to fail, and the inflammatory response itself becomes a significant clinical problem. In this review, we discuss endothelial-leukocyte adhesion molecules with particular emphasis on their expression and function in human disease. Pathophysiological processes presented include atherosclerosis, ischemia-reperfusion injury, acute lung injury, rheumatoid arthritis, and graft rejection. A more detailed description of the discovery and characterization of the key molecules appears in the antecedent article entitled "Endothelial-Leukocyte Adhesion Molecules".

Constitutive expression of E- and P-selectin cognate ligands in human endothelial cells.

On human endothelial cells from umbilical cord (HUVEC) are present, in addition to E- and P-selectins, their cognate ligands. Differently from selectins, the ligand expression is constitutive and not modulated by interleukin-1beta. Such ligands appear to be different from the ones present in promyelocytic cells in order to promote cell adhesion to immobilized selectins. The expression of selectin-ligands on HUVEC cells suggest that selectins can participate in endothelial signalling besides their role as adhesion molecules for circulating blood cells. However, despite their role in chemotaxis, selectins do not contribute to HUVEC tube formation in Matrigel.

Inositol polyanions. Noncarbohydrate inhibitors of L- and P-selectin that block inflammation.

Selectins are cell adhesion molecules known to support the initial attachment of leukocytes to inflamed vascular endothelium through their recognition of carbohydrate ligands such as the tetrasaccharide sialyl Lewisx (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-). In the present study, we describe the inhibition of L- and P-selectin function by inositol polyanions, simple 6-carbon ring structures that have multiple ester-linked phosphate or sulfate groups. In a purified component competition assay, binding of L- and P-selectin-Ig fusion proteins to immobilized bovine serum albumin-sialyl Lewisx neoglycoprotein was inhibited by inositol hexakisphosphate (InsP6, IC50 = 2.1 +/- 1.4 microM and 160 +/- 40 microM), by inositol pentakisphosphate (InsP5, IC50 = 1.4 +/- 0.2 and 260 +/- 40 microM), and by inositol hexakissulfate (InsS6, IC50 = 210 +/- 80 microM and 2.8 +/- 0.9 mM); E-selectin-Ig binding was unaffected. Inositol polyanions diminished the adhesion of LS180 colon carcinoma cells to plates coated with L- and P-selectin-Ig but not with E-selectin-Ig. Inositol polyanions blocked polymorphonuclear leukocyte (PMN) adhesion to COS cells expressing recombinant transmembrane P-selectin but not to those expressing E-selectin. In addition, inositol polyanions diminished PMN adhesion to activated endothelial cells under rotation-induced shear stress, a process known to require L-selectin function. In vivo, the effects of inositol polyanions were studied in two murine models of acute inflammation. Intravenously administered InsP6 (two doses of 40 mumol/kg) inhibited PMN accumulation in thioglycolate-induced inflammation (55 +/- 10% inhibition) and in zymosan-induced inflammation (61 +/- 4% inhibition). InsP5 and InsS6 also inhibited inflammation in these models, although higher doses were required for InsS6. In conclusion, inositol polyanions are noncarbohydrate small molecules that inhibit L- and P-selectin function in vitro and inflammation in vivo.