PMID: 7544625Aug 17, 1995Paper

Purification of an angiogenesis inhibitor from culture medium conditioned by a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8

Biochimica Et Biophysica Acta
Y OhbaM Takigawa

Abstract

We previously reported that a novel human chondrosarcoma-derived chondrocytic 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 inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel filtration chromatography, ion-exchange chromtography, and reverse-phase HPLC, 200 micrograms of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte-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 conditions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a single band of 22K M(r) under nonreducing conditions. Its NH2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP, hCHIAMP at a dose of as little as 0.45 mi...Continue Reading

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Apr 3, 2001·Trends in Cardiovascular Medicine·H P Gerber, N Ferrara
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