Accumulation of heparan sulfate in the culture of human melanoma cells with different metastatic ability

Glycosaminoglycans were metabolically labeled in subconfluent cultures of highly metastatic 7Gp122 and poorly metastatic IC8 variants and of the low metastatic parental M4Be human melanoma cell line. Proteoglycans were separated by DEAE Trisacryl chromatography from the culture medium, from the heparin extract of the cell layer and from the heparin-extracted cell residue lyzed with detergents. Glycosaminoglycans were released from the proteoglycans by reductive alkaline hydrolysis and heparan sulfate (HS) was detected by deaminative cleavage with nitrous acid. Expressed on cell protein basis, the labeled HS content in the medium and in the cell layer decreased with increasing metastatic ability. The extraction of HS with heparin from the 7Gp122 cells indicated that this variant was enriched in (polypeptide bound) HS non inserted into the plasma membrane, compared with the low metastatic IC8 and M4Be cells. The HS fraction in heparin extract and in the heparin-extracted cell residue exhibited molecular mass heterogeneity on gel permeation chromatography and it contained HS fragments. Scission with nitrous acid followed by molecular sieve chromatography of the degradation products indicated that the tetra- and disaccharide repeats separated by the N-sulfated glucosamine residues were present in about equal amounts and constituted 60% of the HS chains in the IC8 and M4Be cells. HS from 7Gp122, IC8 and M4Be cells did not bind antithrombin III with high affinity but it was capable of binding bFGF in in vitro assay.