Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium.

The carbohydrate antigen, sialyl Lex, is known to be a ligand for the cell adhesion molecule called ELAM-1 (E-selectin, endothelial cell leukocyte adhesion molecule-1), which is present on cytokine-activated human endothelial cells. Recently, we reported that another carbohydrate antigen, sialyl Lea, can also serve as a ligand for ELAM-1 (A. Takada, K. Ohmori, N. Takahashi, K. Tsuyuoka, K. Yago, K. Zenita, A. Hasegawa, and R. Kannagi, Biochem. Biophys. Res. Commun., 179: 713-719, 1991). Both sialyl Lex and sialyl Lea are expressed in many human malignant cells. In order to assess the contribution of these carbohydrate antigens to the adhesion of human malignant cells to vascular endothelium, we selected a panel of 12 cultured human epithelial cancer cell lines and a panel of 12 human leukemia cell lines which express sialyl Lex and/or sialyl Lea antigens. All 12 epithelial cancer cell lines exhibited a clearly ELAM-1-dependent adhesion to cytokine-activated human umbilical vein endothelial cells, while only 3 of the 12 leukemia cell lines exhibited significant participation of ELAM-1 in the adhesion. With regard to epithelial cancer cells, the adhesion of 6 cancer cell lines, mostly of colon and pancreas origin, was dependent almost exclusively on sialyl Lea. A significant contribution of the sialyl Lex antigen was noted in the adhesion of the other 6 cell lines, including cancers of lung and liver origin. These results imply that the sialyl Lea/ELAM-1 adhesion system, as well as the sialyl Lex/ELAM-1 adhesion system, plays an important role in the adhesion of human cancer cells to human umbilical vein endothelial cells. With regard to leukemia cells, on the other hand, adhesion of the 3 leukemia cell lines that showed ELAM-1-dependent adhesion was mediated by the sialyl Lex antigen, and none of these leukemia cell lines expressed sialyl Lea or exhibited sialyl Lea-dependent adhesion.

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies.

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.

Adhesion of human cancer cells to vascular endothelium mediated by a carbohydrate antigen, sialyl Lewis A.

Recently the lectin-like domain on ELAM-1 (endothelial leukocyte adhesion molecule-1) was shown to recognize a carbohydrate antigen, sialyl Lewis x. In this paper we demonstrate, by a series of inhibition experiments utilizing specific monoclonal antibodies and pure glycolipid preparations, that the sialyl Lewis a antigen serves as a specific ligand for ELAM-1 as well as sialyl Lewis x and plays a significant role in the ELAM-1-mediated binding of human cancer cells to activated endothelial cells.

Differentiation-dependent expression of sialyl stage-specific embryonic antigen-1 and I-antigens on human lymphoid cells and its implications for carbohydrate-mediated adhesion to vascular endothelium.

Expression of two developmentally regulated carbohydrate antigens, the sialyl stage-specific embryonic antigen-1 (SSEA-1) and I-antigens, in human lymphocytes and lymphocytic leukemia cells was investigated using specific monoclonal antibodies. Sialyl SSEA-1 was expressed only on natural killer (NK) cells, and was essentially absent on resting mature T and B cells among normal peripheral lymphocytes. On the other hand, the I-antigen was strongly expressed on virtually all mature B cells, moderately expressed on most mature T cells, but not expressed on NK cells in normal donors. Expression of the two antigens on normal T and B cells was reversible; in vitro stimulation of normal lymphocytes with concanavalin A (Con A) resulted in the loss of I-antigen and appearance of sialyl SSEA-1 on CD3+ T blasts, whereas stimulation with pokeweed mitogen led to loss of I-antigen expression and appearance of sialyl SSEA-1 antigen on CD19+ B blasts. Among lymphocytic leukemia cells, sialyl SSEA-1 was detected primarily on leukemia cells having immature properties such as most common acute lymphocytic leukemia (cALL) blasts, while the I-antigen was frequently expressed on malignant cells having relatively mature properties, such as those found in adult T-cell leukemia or chronic lymphocytic leukemia, and only occasionally on cALL blasts. Among normal peripheral lymphocytes, the sialyl SSEA-1+I-antigen- NK cells selectively underwent E-selectin (ELAM-1, endothelial-leukocyte adhesion molecule-1)-dependent adhesion to endothelial cells, while the I-antigen+sialyl SSEA-1- mature T and B cells did not, in line with the recent finding that sialyl SSEA-1 serves as a specific ligand for E-selectin. Con A blasts, which are sialyl SSEA-1+I-antigen-, also exhibited significant E-selectin-dependent adhesion to endothelial cells. These results indicate that expression of the sialyl SSEA-1 and I-antigens varies alternately depending on the differentiation/activation status of the lymphocytes, and that this at least partly regulates the behavior of lymphocytes at the vessel wall.

Characterization of a T cell line specific to an anti-Id antibody related to the carbohydrate antigen, sialyl SSEA-1, and the immundominant T cell antigenic site of the antibody.

The stage-specific embryonic Ag-1 (SSEA-1) is a carbohydrate Ag and regarded as an onco-developmental Ag. Sialyl SSEA-1 Ag, the sialylated form of SSEA-1, is frequently expressed in human cancer cells as well as in murine cancer cells. A mAb, FH-6, was shown to specifically recognize the Ag. We have generated five anti-Id Abs directed to the paratope-related idiotopes of the FH-6 Ab. One of these anti-Id Abs, Id-F2, increased the survival of host mice that were inoculated with Meth-A cells expressing the sialyl SSEA-1 Ag. To clarify the exact mechanism underlying the antitumor effect of the anti-Id Ab, we established a T cell line that recognized Id-F2 in association with MHC class II molecules. The T cell line was CD4+V beta 8+, and produced IL-2, exhibiting helper activity for B cells. The VH CDR2 region of the Id-F2 amino acid sequences turned out to be strongly immunogenic to T cells. When the immune complexes, consisting of the sialyl SSEA-1 Ag, FH-6, and Id-F2, were formed at the Meth-A cell-surface, the T cell line showed a strong proliferative response. The possible roles played by such T cell subsets in the anti-tumor effect are discussed.