The pCMV4 plasmid containing the cancer-promoting gene, c-erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the`neo' selectable marker. Several clones showing stable expression of c-erbB2/neu were established and characterized by determination of c-erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and a1,3-fucosyltransferases and the biological behavior of 7721 cells after c-erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLe x expression on the surface of mock cells was high, whereas expression of SDLe x , Le x and SLe a was absent or negligible. This is compatible with the abundant expression of a1,3-fucosyltransferase VII, very low expression of afucosyltransferase III/VI, and almost absent expression of a1,3-fucosyltransferase IV in the mock cells. After transfection of c-erbB2/neu, expression of SLe x and a1,3-fucosyltransferase VII were simultaneously elevated, but that of afucosyltransferase III/VI was not altered. The expression of both SLe x and a1,3-fucosyltransferase VII correlated positively with the expression of c-erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c-erbB2/neu-transfected cells.These increases also correlated positively with the expression intensities of c-erbB2/neu, SLe x and a1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLe x (KM93) and SDLe x (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLe x and SLe a did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of a1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c-erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of a1,3-fucosyltransferase VII and its specific product, SLe x , and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the cerbB2/neu gene is proposed to be a metastasis-promoting gene, and its effects are at least partially mediated by the increased expression of a1,3-fucosyltransferase VII and SLe x .Keywords: c-erbB2/ neu; hepatocarcinoma cells; metastasisassociated phenotype; sialyl Lewis X; a1, 3-fucosyltransferase.The c-erbB2 gene (also known as neu, HER-2 or NGL) encodes a transmembrane glycoprotein of molecular mass 185 kDa (p185), which shares extensive sequence homology with the receptor of epidermal growth factor (EGF-R) [1,2]. Amplification or overexpression of the c-erbB2/neu gene was first found in human breast cancer [3] ...
The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.
K1 killer strains of Saccharomyces cerevisiae secrete a polypeptide toxin to which they are themselves immune. The alpha and beta components of toxin comprise residues 45-147 and 234-316 of the 316-residue K1 preprotoxin. The intervening 86-residue segment is called gamma. A 26-residue signal peptide is removed on entry into the endoplasmic reticulum. The Kex2 protease excises the toxin components from the 290-residue glycosylated protoxin in a late Golgi compartment. Expression of a cDNA copy of the preprotoxin gene confers the complete K1 killer phenotype on sensitive cells. We now show that expression of immunity requires the alpha component and the N-terminal 31 residues of gamma. An additional C-terminal extension, either eight residues of gamma or three of four unrelated peptides, is also required. Expression of preprotoxin terminating at the alpha C-terminus, or lacking the gamma N-terminal half of gamma causes profound but reversible growth inhibition. Inhibition is suppressed in cis by the same 31 residues of gamma required for immunity to exocellular toxin in trans, but not by the presence of beta. Both immunity and growth inhibition are alleviated by insertions in alpha that inactivate toxin. Inhibition is not suppressed by kex2, chc1 or kre1 mutations, by growth at higher pH or temperature, or by normal K1 immunity. Inhibition, therefore, probably does not involve processing of the alpha toxin component at its N-terminus or release from the cell and binding to glucan receptors. Some insertion and substitution mutations in gamma severely reduce toxin secretion without affecting immunity. They are presumed to affect protoxin folding in the endoplasmic reticulum and translocation to the Golgi.
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