Abstract
We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.
Highlights
We have determined the relationship between the binding sites for acidic fibroblast growth factor and basic fibroblast growth factors (FGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and basic FGF (bFGF) in DNA synthesis assays
The ductal epithelial cell is represented by the benign human benign mammary (Huma) 123 epithelial cells, the myoepithelial cells correspond to the Huma 109 myoepithelial-like cells, and the Rama 27 fibroblasts are representative of a mammary stromal fibroblast
The kinetics and affinity of the slow/low binding site for bFGF in the mammary HS are similar to the binding site for bFGF characterized in heparin [19]
Summary
Materials and Cells—Human recombinant aFGF and bFGF were prepared as described previously [20, 21]. After the removal of the culture medium and washing the cell monolayers with 5 ml of PBS, 25 ml of fresh step-down medium (SDM; DMEM with 250 g/ml bovine serum albumin) was added to each culture dish. Near-confluent Rama 27 cells in 9-cm culture dishes (Nunc) were washed twice with PBS and fresh, low sulfur Routine Medium (DMEM without SO42Ϫ, containing 20 M methionine and 15 M cystine (both Sigma) and supplemented with 5% (v/v) dialyzed fetal calf serum, 50 ng/ml insulin, 50 ng/ml hydrocortisone, and 15 mM NaClO3 (Fluka, Glossop, UK)) was added. After 24 h, the cell monolayers were washed twice with 500 l of PBS, and 500 l of low sulfur SDM (DMEM without SO42Ϫ, containing 20 M methionine and 15 M cystine and supplemented with 250 g/ml bovine serum albumin and 15 mM NaClO3) was added. Control Rama 27 fibroblasts were used in DNA synthesis assays, in which case conventional DMEM was used throughout [30]
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