PURIFICATION OF AN ANGIOGENESIS INHIBITOR FROM CULTURE-MEDIUM CONDITIONED BY A HUMAN CHONDROSARCOMA-DERIVED CHONDROCYTIC CELL-LINE, HCS-2 8/

We previously reported that a novel human chondrosarcoma-derived chond rocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311-316, 1990]. In the present study, we purified the inhibi tor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel fi ltration chromatography, ion-exchange chromatography, and reverse-phas e HPLC, 200 mu g of the inhibitor was obtained from 6 liters of CM wit h 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondr ocyte-derived inhibitor of angiogenesis and metalloproteinase activity ), showed a single protein band with a molecular mass (M(r)) of 24 000 (24K) under reducing conditions and M(r) 22K under nonreducing condit ions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a sin gle band of 22K M(r) under nonreducing conditions. Its NH2-terminal am ino acid sequence determined up to the 11th amino acid residue was ide ntical with that of the tissue inhibitor of metalloproteinases-2 (TIMP -2). On Western blotting, anti-human TIMP-2 antibody cross-reacted wit h hCHIAMP. hCHIAMP at a dose of as little as 0.45 mu g significantly i nhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mu mol of spermine. Because HCS-2/8 is a clonal cell line, these find ings clearly showed for the first time that chondrocytes themselves pr oduce a potent inhibitor of angiogenesis, which is also an inhibitor o f gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseas es including solid tumors.