Transfection of the c‐erbB2/neugene upregulates the expression of sialyl Lewis X, α1,3‐fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell line

Abstract

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] ...

Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates

“…Increased expression of UDP-galactose transporter gene (UGT1) in cancers was also suggested to be involved in the induction of these selectin ligands, because the introduction of the gene for UDP-galactose transporter conferred the induction of sialyl Lewis a and sialyl Lewis x expression on cultured cancer cells (8). Other investigators have suggested a role for fucosyltransferase VII (9)(10)(11) and even the involvement of another sugar transporter, GLUT1, for induction of sialyl Lewis x in cancers (12). However, the exact mechanism underlying this transcriptional induction has remained uncharacterized.…”

?-1,3-Fucosyltransferase-VII stimulates the growth of hepatocarcinoma cells via the cyclin-dependent kinase inhibitor p27Kip1

“…Other reagents, including dNTP, oligo (dT)-18, enhanced chemiluminescence (ECL) reagent and g-32 P-ATP (5 mCi/pmol) were commercially available in China. The H7721 cell line transfected with a-1,3-FucT-VII was established in our laboratory as previously reported [10]. Two transfectants were selected: one expressed a high level of a-1,3-FucT-VII mRNA and was named as FucTVII-H, another expressed moderate mRNA of a-1,3-FucT-VII and was designated FucTVII-M. H7721 cells mock-transfected with the empty vector pcDNA3.1 were used as the control, which expressed a low level of a-1,3-FucT-VII mRNA.…”

“…Our group has demonstrated that surface SLe x is a metastasis-related sugar structure. It was increased on H7721 human hepatocarcinoma cells after transfection of the metastasis-promoting gene c-erbB2/neu [10], or treatment with epidermal growth factor [11], whereas it was decreased by the metastasis-suppressive gene nm23H1 [12] or all-trans retinoic acid [11]. The metastatic potential, including cell adhesion to laminin, cell migration through a transwell and cell invasion through a matrigel, was always positively correlated to SLe x expression on the cell surface [10 -12].…”

“…The metastatic potential, including cell adhesion to laminin, cell migration through a transwell and cell invasion through a matrigel, was always positively correlated to SLe x expression on the cell surface [10 -12]. In addition, transfection of a-1,3-FucT-VII cDNA into H7721 cells to increase the synthesis of SLe x antigen led to an enhancement of the metastatic potential [10]. Clinical studies indicated that cancer cells with strong SLe x expression had a high risk of developing hematogenous metastasis, and this factor statistically affected the overall prognosis of the patients [13 -15].…”

Expression of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase-12 in Gastric and Colonic Cancer Cell Lines and in Human Colorectal Cancer

“…Cancer cell lines were cultured at 37°C, 5% CO 2 in RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin and streptomycin, as described by our laboratory [25]. …”