PROTEOLYTIC MODIFICATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS - COMPARISON OF CONDITIONED MEDIA FROM HUMAN CELL-LINES, CIRCULATING PROTEASES AND CHARACTERIZED ENZYMES

Proteolytic modification of circulating insulin-like growth factor bin ding protein-3 (IGFBP-3) has been described in a number of conditions. Using Western ligand blotting and SDS-PAGE analysis of fragmentation patterns of I-125-labelled IGFBP-3 and (125)-labelled IGFBP-1, we have examined conditioned media from cultured human cell lines for the pre sence of proteolytic activity and compared this with the action of cir cuiting proteases and with characterized enzymes including cathepsin D , kallikrein, plasmin and tissue plasminogen activator. I-125-Labelled IGFBP-3 was incubated with serum from pregnant women, patients follow ing heart surgery and patients with cancer of the breast, lung or head /neck. Following separation of the preincubated samples by SDS-PAGE, a distinct pattern of degradation fragments was observed which was simi lar in all cases. This proteolytic activity was inhibited in the prese nce of EDTA, phenanthroline and 4(-2-aminoethyl)-benzenesulphonylfluor ide,HCl, These proteases had no detectable effect no IGFBP-1. Serum-fr ee conditioned medium from a human dermal fibroblast cell line, a rabd omyosarcoma, a cervical, a bladder, a chorio- and two-tongue squamous cell carcinoma cell lines all contained proteolytic activity which fra gmented IGFBP-3. The pattern of fragments was similar in all cell line s but different from that produced by the circulating proteases. Six o ut of nine cell lines produced protease(s) which degraded IGFBP-1 in a ddition to IGFBP-3. Whilst all the characterized enzymes tested also f ragmented IGFBP-3 and plasmin cleaved IGFBP-1, none of these acted in the same way as either circulating or cell line-derived proteolytic ac tivity. The activity associated with the characterized enzymes and cel l lines was inhibited in the presence of serum from normal healthy sub jects. These results demonstrate that the serum of pregnant women, pos t-operative patients and patients with cancer contain circulating prot eases which cause fragmentation of IGFBP-3 but have little effect on I GFBP-1. Cell-derived proteases were shown to act on IGFBP-3 and IGFBP- 1 in a number of instances but are not active ill the presence of circ ulating inhibitors. These proteases may play an important role in regu lating the availability of IGFs to normal and neoplastic tissues.