Assuntos
Falha de Equipamento , Radioterapia/instrumentação , Coleta de Dados , Raios gama , Raios XRESUMO
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.
Assuntos
Adesão Celular/fisiologia , Neoplasias do Colo/patologia , Neoplasias do Colo/fisiopatologia , Gangliosídeos/genética , Gangliosídeos/fisiologia , RNA Antissenso/genética , Animais , Antígenos Glicosídicos Associados a Tumores/genética , Antígenos Glicosídicos Associados a Tumores/fisiologia , Sequência de Bases , Antígeno CA-19-9 , Células CHO , Neoplasias do Colo/genética , Cricetinae , Primers do DNA/genética , Selectina E/fisiologia , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Glicolipídeos/fisiologia , Glicoproteínas/fisiologia , Humanos , Lipídeos de Membrana/fisiologia , Fenótipo , Reação em Cadeia da Polimerase , Transfecção , Células Tumorais CultivadasRESUMO
The activity of serum alpha-6-fucosyltransferase, a platelet derived enzyme, determined in sera of 22 normal individuals and 86 patients with various disorders was positively correlated with platelet counts. When the enzyme activity in 1 microliters serum was calculated per 1000 of platelets in blood (coefficient F/P) an inverse correlation became evident in that F/P was proportionally the higher the lower was platelet count in blood. The F/P values were in a good agreement with the results of direct assays of enzyme activities in isolated platelets. Neither granulocytes, lymphocytes nor red cells significantly contributed to serum enzyme activity though granulocytes enhanced the thrombin-induced enzyme release from platelets. In platelets separated by centrifugation in density gradients the enzyme was shown to be present in platelets of intermediate and high density but missing from the light ones. It is suggested that alpha-6-fucosyltransferase of platelets may be a marker of the ploidy level of megakaryocytes.
