INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEIN (IGFBP)-3 LEVELS IN CONDITIONED MEDIA OF HS578T HUMAN BREAST-CANCER CELLS ARE POSTTRANSCRIPTIONALLY REGULATED

Hs578T human breast cancer cells secrete insulin-like growth factor bi nding protein (IGFBP)-3 (41-kDa and 39-kDa) and IGFBP-4 (24-kDa) as ma jor BP species. In addition, cell surface-associated IGFBP-3 is demons trable by use of cell monolayer affinity cross-linking or by employing immunoperoxidase staining of the cell surface with specific polyclona l anti-human IGFBP-3 antibodies (alphaIGFBP-3gl and alphaIGFBP-3ngl). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translati onal by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media(CM) following addition of IGF-I and IGF-II (maximum 8-13-fold in crease at 100 ng/ml concentration), but not by insulin up to 1 mug/ml; 2) confirmation of IGF-induced increases in CM concentrations of IGFB P-3 by means of Western ligand blot, affinity cross-linking, and IGFBP -3-specific radioimmunoassay; 3) increase of IGFBP-3 in CM by addition of IGF analogs which retain full affinity for IGFBPs ([Leu27]IGF-II a nd [Tyr55,Gln56]IGF-I), but not by IGF analogs which have significantl y decreased affinity for IGFBPs ([Gln3,Ala4,Tyr15,Leu16]IGF-I, [Gln6,A la7,Tyr18,Leu19,Leu27]IGF-II and IGF-I/insulin hybrid); 4) no change i n IGFBP-3 mRNA following addition of IGFs; 5) existence of metal ion-d ependent IGFBP-3 specific protease in CM and protection of IGFBP-3 fro m protease by formation of [IGF:IGFBP-3] complexes; and 6) release of cell surface-associated IGFBP-3 into CM by addition of IGF peptides. T hese studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated events, including dissociatio n of cell surface-associated IGFBP-3 and protection of IGFBP-3 from pr otease activity.