Abstract
Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. Their activities were both lost by digestion with trypsin, but remained unchanged by oxidation with periodate, suggesting the role of the protein portions in their molecules. The potency of the unabsorbed factor was inhibited specifically by alpha-methyl-D-mannoside or D-mannose, while that of the absorbed factor was inhibited specifically by N-acetyl-D-glucosamine, suggesting that these carbohydrates may be concerned with the respective receptor structures at the tumour-cell surface. The unabsorbed factor induced not only cell aggregation (as shown in the form of simple apposition) but also cell adhesiveness characterized by development of intermediate junctions, desmosomes and tight junctions, while the absorbed factor produced only simple apposition, suggesting their functional difference.
Highlights
Summary.-Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was
Ovomucoid, which is known as an N-acetyl-D-glucosamine-rich glycoprotein in its oligosaccharide chain, resulted in the complete inhibition of cell aggregation by the absorbed aggregation-promoting factor (APF), when tested at a concentration of 100,ug/
5 ml of the absorbed APF (1-2 mg/ml), dialysed against 0-02M phosphate buffer for 12 h at 4°C and concentrated, were eluted on D-mannose starch gel as described above, because ovomucoid sample has been known to contain a small quantity of D-mannose in the oligosaccharide chain
Summary
In vitro assay for cell aggregation.-For carbohydrate inhibition experiments, equal volumes (1-0 ml) of the unabsorbed APF (100 ,ug/ml) or the absorbed APF (500 ,ug/ml) and of AH109A cell suspension (1-5 x 106/ml in HBS free of glucose) were mixed in a Falcon tube (1.5 x 9 5 cm) and incubated at 37°C in a roller tube culture apparatus, model Te-Her (Hirasawa Co., Tokyo, Japan) run at 1 rotation/8 min (Kudo et al, 1976). These APFs respectively induced cell aggregation graded+ at each concentration indicated; over 5000 of the originally suspended cells were aggregated after 30 min of incubation. After dialysis against HBS free of glucose at 4°C for 12 h, to remove free periodate, each reaction mixture was tested for cell aggregation
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