[Differential gene expression in nasopharyngeal carcinoma cell with reduced and normal expression of 6A8 alpha-mannosidase].

OBJECTIVE: To detect the differential display of mRNA expression between human nasopharyngeal carcinoma cell CNE-2L2 with reduced malignancy caused by transduction of a DNA antisense to 6A8 alpha-mannosidase cDNA (AS cell) and the wild type cell (W cell). METHODS: Differential display of mRNA expression was analyzed using DNA microarray analysis. The datasets were confirmed by Northern blotting and RT-PCR. RESULTS: Out of the 1069 genes analyzed, 34 genes were up-regulated in AS cells relative to W cells. Conversely, 42 genes were down-regulated. The genes, up-regulation of which might have suppressive effect on tumor malignant behaviors, were P130 mRNA for 130K protein, TGF-betaIIR alpha, GABBR1, TGFBR1, TNFAIP1, STANIN, E-CADHERIN, CTNNA1 and 2, RFX2, TMPO, etc. The genes, down-regulation of which might have suppressive effect on tumor malignant behaviors, were CD44, NDRG1, TGFB1, RPS5, LEGUMAIIN, CBS, CD59, SNRPA1, etc. The microarray datasets were confirmed by Northern blot and RT-PCR analysis. CONCLUSIONS: In comparison to the W cell, AS cell has up-regulation of 34 genes and down-regulation of 42 genes. Changes of the gene expression may play a role in the malignancy reduction of AS cell.

Suppression of 6A8 alpha-mannosidase gene expression reduced the potentiality of growth and metastasis of human nasopharyngeal carcinoma.

Suppression of alpha-mannosidases by chemicals has been shown to reduce the potentiality of growth and metastasis of various tumors. In our study, the effect of 6A8 alpha-mannosidase (MAN 6A8), recently discovered in our laboratory, on malignant behaviors of tumor cells was examined. Since the suppressive effect of chemicals on alpha-mannosidase is not specific, antisense technique was used to specifically inhibit expression of the MAN 6A8 in human nasopharyngeal carcinoma cells, CNE-2L2. Two cell clones, AS1 and AS2, with pronounced suppression of MAN 6A8 expression were developed. Wild-type (W), mock-transduced (M) and irrelevant DNA-transduced (IR) CNE-2L2 cells with normal expression of the enzyme were used as controls. Malignant behaviors of the cells were examined. Significant inhibition of growth of AS cells in vitro measured by MTT assay, colony formation and anchorage-independent colony formation was found. Pronounced inhibition of formation of tumors from AS cells inoculated into nude mice and metastasis was also observed. W, M and IR cells cultured in plate wells appeared dispersed with a fibroblastic or epithelial morphology, whereas AS cells were in compact sheets with an epithelioid organization. Since E-cadherin is the key factor in homophilic adhesion of epithelial cells, its expression on the surface of CNE-2L2 cells was determined. E-cadherin expression on AS cells was enhanced, whereas it was markedly diminished on W, M and IR cells. In addition, lamellipodia, which play an important role in cell spreading and mobility, almost disappeared on AS cells. The results demonstrate a significant suppressive effect of reduced expression of MAN 6A8 on malignant behaviors of CNE-2L2 cells.

[Inhibition of ER alpha-mannosidase expression causes reduction and shortening of microvilli on rat liver epithelial cell WB-F344].

OBJECTIVE: To study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture. METHODS: Recombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine. The transfected cells were selected in G418 medium and cloned by means of limiting dilution. Integration of the transfected DNA into host DNA was detected by neo PCR. Rat liver ER alpha-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-alpha-D-mannopyranoside as a substrate. Microvilli on cell surface were observed upon a scan electron microscope. The growth curves of the cells in culture were drawn. RESULTS: The cell clones transfected with antisense 6A8 showed reduction of ER alpha-mannosidase activity with various degrees. Clone AS1 and AS2 cell showed a pronounced reduction of the enzymatic activity. In the study on AS1 cells, Con A binding to the cells was found to be enhanced, cell growth in culture became slow from day 5. The microvilli on the cells were reduced and blunted. CONCLUSIONS: Transfection with antisense 6A8 resulted in reduction and blunting of microvilli on the surface of growing WB-F344 cells, which might be related to N-glycosylation modification.

[Inhibition of 6A8 alpha-manosidase expression induces decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2].

OBJECTIVE: To investigate the inhibitory effect of 6A8 alpha-manosidase expression on the adhesiveness of CNE-2L2 cells to laminin and the lamellipodia on cell surface. METHODS: 6A8 alpha-manosidase expression was detected by Western blotting. For assaying the adhesion of cells to laminin, cells were incubated in laminin-coated plate at 37 degrees C for 1 h, the adhered cells were stained with crystal purple dissolved in 0.1 mol/L Sodium Citrate/50% ethanol. Absorbance 540 nm was measured. Adhesion rate (R) was calculated according to formula R = AT/A100 x 100%. Here A100 represents 100% adhesion. lamellipodia on cell surface was observed upon a scanning electron microscopy. RESULTS: The adhesion rate of two clones (AS1 and AS2) with inhibition of 6A8 alpha-manosidase expression to laminin was 0.447 +/- 0.096 and 0.533 +/- 0.065 respectively. The adhesion rate of three controls with normal expression of 6A8 alpha-manosidase to laminin was 0.78 +/- 0.035, 0.7 +/- 0.05 and 0.80 +/- 0.04 respectively. The difference was significant (P < 0.01). CNE-2L2 cells with normal expression of 6A8 alpha-manosidase was rich in lamellipodia on their surface. Lamellipodia nearly disappeared on the cells with inhibition of 6A8 alpha-manosidase expression. CONCLUSIONS: Inhibition of 6A8 alpha-manosidase expression results in decrease of adhesion to laminin and reduction of lamellipodia of human nasopharyngeal carcinoma cell CNE-2L2.

[Molecular mechanism of metastasis inhibition of nasopharyngeal carcinoma cell CNE-2L2 induced by reduction of 6A8 alpha-mannosidase expression].

OBJECTIVE: To study the effect of inhibition of 6A8 alpha-mannosidase expression on adhesiveness among and E-cadherin expression on CNE-2L2 cells, and on metastasis of the tumors from the cells inoculated in nude mice. METHODS: Anchorage-independent adhesion among cells was examined in soft agar culture. E-cadherin expression was studied by immunofluorescence staining, immunohistological staining and RT-PCR. CNE-2L2 cells were subcutaneously inoculated into nude mice. Eight weeks later tumor metastasis was demonstrated by means of histological examination of lung sections. RESULTS: CNE-2L2 cells with suppression of 6A8 alpha-mannosidase expression (AS) became aggregated. E-cadherin expression on wild type cells was very weak. In contrast, it was greatly enhanced on AS cells. The enhancement was detected on both protein and mRNA levels. Lung metastasis of the tumor from inoculated AS cells were heavily inhibited in nude mice. CONCLUSION: Inhibition of 6A8 alpha-mannosidase expression results in enhancement of cell-cell adhesion and of E-cadherin expression on CNE-2L2 cells. Lung metastasis of the tumor grown from AS cell inoculate in nude mice is heavily suppressed.