ROLE OF SIALOSYL LEWIS(A) IN ADHESION OF COLON-CANCER CELLS - THE ANTISENSE RNA APPROACH

To study whether the adhesion of colon cancer cells to E-selectin can be directly affected by changes in the expression level of sialosyl Le (a) antigen we created a specific loss-of-function phenotype. A stable subclone (CX-1.1) with high expression of sialosyl Le(a) structure, o btained from a heterogenous population of colon carcinoma CX-1 cells, was transfected with an expression vector containing a fragment of cDN A for alpha 1,3/4-fucosyltransferase in antisense orientation. After t ransfection, the cell line was isolated which did not express sialosyl Le(a) antigen and lacked the alpha 1,3/4-fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was f ound that the specific lack of expression of sialosyl Le(a) carbohydra te structure on the surface of colon cancer cells completely abolished their adhesion to E-selectin. To evaluate which cellular glycoconjuga tes carry sialosyl Le(a) antigen, glycoproteins as well as glycolipids of CX1.1 cells were analysed for the expression of this structure. An ti-sialosyl Le(a) antibodies detected multiple glycoprotein bands with apparent molecular masses of 65-280 kDa on western blots, and an inte nse band representing sialosyl Le(a)-ganglioside on a thin-layer chrom atogram Using O-sialoglycoprotease from Pasteurella haemolytica and an alkaline beta-elimination procedure, it was shown that protein-linked sialosyl Le(a) structures are carried mostly by mucin-type glycoprote ins. However, treatment of CX-1.1 cells with O-sialoglycoprotease did not decrease either their binding to E-selectin-expressing Chinese ham ster ovary cells, or binding of anti-sialosyl Le(a) antibodies to the cell surface. These results suggested that cleavage of sialomucins unc overed cryptic sialosyl Le(a)-ganglioside, which was inaccessible for the antibody and E-selectin in untreated cells. This hypothesis was co nfirmed to some extent by the higher accessibility of gangliosides to galactose oxidase on the surface of O-sialoglycoprotease-treated CX-1. 1 cells, comparing to untreated cells. We propose that glycoproteins a s well as gangliosides carrying sialosyl Le(a) structures, when proper ly exposed and present in high density on surface of cancer cells: can effectively support the adhesion of cancer cells to E-selectin.