Alterations in the glycolipid composition and cellular properties of ovarian carcinoma-derived RMG-1 cells on transfection of the alpha1,2-fucosyltransferase gene

Abstract

Transfection of the mouse Fut1 and Fut2, and human FUT1 genes into human ovarian carcinoma-derived RMG-1 cells resulted in 20-30-fold increases in cellular alpha1,2-fucosyltransferase activity, and in alteration of the glycolipid composition, including not only fucosylated products, but also precursor glycolipids. Although globo-series glycolipids were not significantly affected by the transfection, the major glycolipids belonging to the lacto-series type 1 chain family in RMG-1 cells and the transfectants were the Lc4Cer, Lewis a (Le)a and Leb, and H-1 glycolipids, respectively, suggesting that fucosylation of Lc4Cer to the H-1 glycolipid prevents the further modification of Lc4Cer to Lea and Leb in the transfectants. Also, the lacto-series type 2 chains in RMG-1 cells were LeX, NeuAc-nLc4Cer and NeuAc-LeX, and those in the transfectants were LeX and LeY, indicating that the sialylation of nLc4Cer and LeX is restricted by increased fucosylation of LeX. As a result, the amount of sialic acid released by sialidase from the transfectants decreased to 70% of that from RMG-1 cells, and several membrane-mediated phenomena, such as the cell-to-cell interaction between cancer cells and mesothelial cells, and the cell viability in the presence of an anticancer drug, 5-fluorouracil, for the transfectants was found to be increased in comparison to that for RMG-1 cells. These findings indicate that cell surface carbohydrates are involved in the biological properties, including cell-to-cell adhesion and drug resistance, of cancer cells.

Figure 1

Thin‐layer chromatography (TLC) of neutral glycolipids from RMG‐1 cells and the transfectant. Glycolipids, corresponding to 0.5 mg of dry cells, were chromatographed on a TLC plate with chloroform/methanol/water (65:35:8, v/v/v) and the spots were seen with orcinol‐H₂SO₄ reagent. 1, RMG‐1; 2, pcDNA3.1‐FUT1‐RMG‐1; 3, pcDNA3.1‐Fut2‐RMG‐1.

Figure 2

Thin‐layer chromatography (TLC)‐immunostaining of glycolipids from RMG‐1 cells and the transfectants. The antibodies used for detection were (A) anti‐Lc₄Cer, (B) anti‐H1, (C) anti‐Le^b, and (D) anti‐Le^Y. 1, pcDNA3.1‐Fut1‐RMG‐1; 2, pcDNA3.1‐Fut2‐RMG‐1; 3 and 4, pcDNA3.1‐FUT1‐RMG‐1.

Figure 3

Concentrations of (□) lacto‐series glycolipids in RMG‐1 cells and (▪) the transfectants with pcDNA3.1‐FUT1. The concentrations are expressed as micrograms of glycolipids per milligram of dry cells, and are the means for three experiments. tr, trace amount (<0.005 µg/mg of dry cells); *not detected.

Figure 4

Quantity of (□) sialic acid released from RMG‐1 cells and (▪) the transfectants with pcDNA3.1‐FUT1 on treatment with (A) Vibrio cholerae sialidase, and (B) adhesion of the cells to a mesothelial cell monolayer.