Characterization of an amino-terminal fragment of insulin like growth factor binding protein 3 and its effects in MCF-7 breast cancer cells

This study describes the purification, characterization and actions of a pe ptide derived from proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 br east cancer cells. One millilitre of cell-conditioned medium at pH 5.5 full y proteolysed 10 mug plasma-derived IGFBP-3, yielding an immunoreactive fra gment of apparent molecular mass 21 kDa by SDS-PAGE. After purification to homogeneity by IGF-I affinity chromatography and reverse-phase HPLC, sequen ce analysis revealed the amino-terminus of IGFBP-3, and mass spectrometry i ndicated a molecular mass of 12 295 Da. Analysis of the corresponding fragm ent generated by proteolysis or a non-glycosylated IGFBP-3 mutant indicated a molecular mass of 9855 Da, consistent with cleavage after Arg(97). This suggests that the fragment derived from glycosylated IGFBP-3 contains -2.5 kDa carbohydrate on Asn(89). IGFBP-3[1-97] formed binary complexes with IGF s, but with reduced efficiency compared with intact IGFBP-3. IGFBP-3[1-97] at 11 nM inhibited IGf-I-stimulated DNA synthesis by 50-60% in MCF-7 breast cancer cells, similar to the inhibition observed with the intact protein, in the absence of IGF-I, DNA synthesis was inhibited by IGFBP-3[1-97], but not intact IGFBP-3. This suggests that the IGFBP-3 protease in MCF-7 cell m edium can generate an inhibitor of IGF-dependent and independent breast can cer cell growth (C) 2000 Harcourt Publishers Ltd.