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, obtained from a heterogenous population of colon carcinoma CX-1 cells, was transfected with an expression vector containing a fragment of cDNA for alpha1,3/4-fucosyltransferase in antisense orientation. After transfection, the cell line was isolated which did not express sialosyl Le(a) antigen and lacked the alpha1,3/4-fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was found that the specific lack of expression of sialosyl Le(a) carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to E-selectin. To evaluate which cellular glycoconjugates carry sialosyl Le(a) antigen, glycoproteins as well as glycolipids of CX-1.1 cells were analysed for the expression of this structure. Anti-sialosyl Le(a) antibodies detected multiple glycoprotein bands with apparent molecular masses of 65-280 kDa on western blots, and an intense band representing sialosyl Le(a)-ganglioside on a thin-layer chromatogram. 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 glycoproteins. However, treatment of CX-1.1 cells with O-sialoglycoprotease did not decrease either their binding to E-selectin-expressing Chinese hamster ovary cells, or binding of anti-sialosyl Le(a) antibodies to the cell surface. These results suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a)-ganglioside, which was inaccessible for the antibody and E-selectin in untreated cells. This hypothesis was confirmed 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 as well as gangliosides carrying sialosyl Le(a) structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to E-selectin.
Kłopocki
A G
AG
Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw.
Laskowska
A
A
Antoniewicz-Papis
J
J
Duk
M
M
Lisowska
E
E
Ugorski
M
M
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D018395:CA-19-9 Antigen; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology*; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology* / Q000503:physiopathology*; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D019040:E-Selectin / Q000502:physiology; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D005732:Gangliosides / Q000235:genetics* / Q000502:physiology*; D006017:Glycolipids / Q000502:physiology; D006023:Glycoproteins / Q000502:physiology; D006801:Humans; D008563:Membrane Lipids / Q000502:physiology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D015295:Antigens, Tumor-Associated, Carbohydrate; D018395:CA-19-9 Antigen; D017931:DNA Primers; D019040:E-Selectin; D005732:Gangliosides; D006017:Glycolipids; D006023:Glycoproteins; D008563:Membrane Lipids; D016372:RNA, Antisense; C064564:sialyl Le(a) ganglioside; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase