INSULIN-LIKE GROWTH-FACTOR (IGF)-INDEPENDENT ACTION OF IGF-BINDING PROTEIN-3 IN HS578T HUMAN BREAST-CANCER CELLS - CELL-SURFACE BINDING ANDGROWTH-INHIBITION

Estrogen receptor-negative Hs578T human breast cancer cells secrete in sulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 as majo r binding protein (BP) species. Our previous immunohistochemical studi es (Oh, Y., Muller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. (1992) Endocrinology 131, 3123-3125) have demonstrated the existence o f cell surface-associated IGFBP-3 and release of cell surface-associat ed IGFBP-3 into conditioned media by addition of IGF peptide in Hs578T cells. In this study, we have demonstrated that IGFBP-3 binding on th e cell surface is specific and receptor-mediated, by showing: 1) a dos e-dependent increase of IGFBP-3 binding by addition of divalent cation s (CaCl2 and MnCl2); 2) dose-dependent competition of I-125-IGFBP-3E. coli by unlabeled IGFBP-3E.coli (>80% competition at 100 nM), but not by IGFBP-1 or fibronectin. In addition, exogenous IGFBP-3 treatment re sulted in a significant inhibitory effect on monolayer growth of Hs578 T cells. This inhibitory effect of IGFBP-3 was shown to be specific an d IGF-independent by demonstrating: 1) dose-dependent inhibition on ce ll growth (60% inhibition at 20 nM) and inhibition on DNA synthesis (1 0 nM; p < 0.05, 20 nM; p < 0.005) by exogenous IGFBP-3E.coli, but not by IGFBP- 1; 2) absence of stimulatory effects on monolayer cell growt h by either native IGFs or IGF analogs which have significantly decrea sed affinity for IGFBPs, but retain full affinity for type 1 and 2 IGF receptors; 3) significant diminution of the IGFBP-3 inhibitory effect s on monolayer growth by coincubation with native IGFs, but not by coi ncubation with IGF analogs with decreased affinity for IGFBP-3. In con clusion, exogenous IGFBP-3 shows specific binding on the cell surface and can inhibit Hs578T cell monolayer growth by itself, suggesting the existence of specific membrane-associated proteins or receptors for I GFBP-3. Furthermore, IGF-I and -II can attenuate inhibitory effect of IGFBP-3 by forming IGF.IGFBP-3 complexes, thereby preventing cell surf ace binding of IGFBP-3.