|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2003 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 2002 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Our previous study showed that reverting the expression of the β-1,4-galactosyltransferase (β-1,4-GalT) II or V gene in cancer cells results in the suppression of tumor growth, suggesting the importance of the surface carbohydrates in cell growth. Since normal cells cease to proliferate upon interaction of cells at higher cell density, we investigated whether or not cell surface glycosylation, particularly N-glycosylation, change by different cell density. Mouse NIH3T3 cells grown at 20%, 50% and 100% in density were harvested by scraper, and membrane preparations were subjected to lectin blot analysis. CBB-staining showed that the expression of most constitutional proteins unchanges during growth except for 50 K and 250 K proteins whose amounts increase. When the blot was incubated with RCA-I, that binds to β-1,4-galactosylated oligosaccharides, the reactivity of lectin increased in 80 K,110 K,120 K and 160 K bands and particularly in 230 K band in proportion to the cell density. A similar but much weaker reactivity towards these protein bands was observed using L-PHA. Northern blot analysis showed that the expression of the β-1,4-GalT II gene only increases significantly at 100% cell density, suggesting that the galactose residues coded by β-1,4-GalT II is important for growth inhibition of normal cells. By using an asialo-transferrin-Sepharose column, 31 K protein was obtained as galactose-binding protein from highly dense cells, suggesting the involvement of this protein in binding to galactose residues expressed on cells at higher density.