Abstract
Estrogen receptor-negative Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 as major binding protein (BP) species. Our previous immunohistochemical studies (Oh, Y., Müller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. (1992) Endocrinology 131, 3123-3125) have demonstrated the existence of cell surface-associated IGFBP-3 and release of cell surface-associated IGFBP-3 into conditioned media by addition of IGF peptide in Hs578T cells. In this study, we have demonstrated that IGFBP-3 binding on the cell surface is specific and receptor-mediated, by showing: 1) a dose-dependent increase of IGFBP-3 binding by addition of divalent cations (CaCl2 and MnCl2); 2) dose-dependent competition of 125I-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 resulted in a significant inhibitory effect on monolayer growth of Hs578T cells. This inhibitory effect of IGFBP-3 was shown to be specific and IGF-independent by demonstrating: 1) dose-dependent inhibition on cell growth (60% inhibition at 20 nM) and inhibition on DNA synthesis (10 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 growth by either native IGFs or IGF analogs which have significantly decreased affinity for IGFBPs, but retain full affinity for type 1 and 2 IGF receptors; 3) significant diminution of the IGFBP-3 inhibitory effects on monolayer growth by coincubation with native IGFs, but not by coincubation with IGF analogs with decreased affinity for IGFBP-3. In conclusion, 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 IGFBP-3. Furthermore, IGF-I and -II can attenuate inhibitory effect of IGFBP-3 by forming IGF.IGFBP-3 complexes, thereby preventing cell surface binding of IGFBP-3.
Highlights
Insulin-like growth factors, IGF-I andIGF-11,’ are peptide cancer cells secrete insulin-like growth factor binding mitogens for multiplecell lines, including human breast canprotein (1GFBP)-3and IGFBP-4as major binding pro- cer cells [1,2,3]
We have demonstrated that IGFBP-3 binding on the cell surfaceis specific and receptor-mediated, by showing: 1)a dose-dependent increase of IGFBP-3 binding to be mediated through the type 1 IGF receptor, which has high affinity for both IGF-I and-11and low affinity for insulin [5,6]
Because of the higher binding affinity of IGFBP-3 for all of the deglycosylated IGFBP-3 forms from human IGFs, the type 1 IGF receptor is normally "masked," but can serum, Hs578T CM, and solubilized membranes were immu- be unmasked by low concentrations of [Leuz7]IGF-11.This noprecipitated with theIGFBP-3 antibody, aIGFBP-3ngl competition by membrane-associated IGFBP-3 for receptor
Summary
Insulin-like growth factors, IGF-I andIGF-11,’ are peptide cancer cells secrete insulin-like growth factor binding mitogens for multiplecell lines, including human breast canprotein (1GFBP)-3and IGFBP-4as major binding pro- cer cells [1,2,3]. Exogenous IGFBP-3 treatment resulted tioned media (CM) of a wide variety of cell types, where they in a significant inhibitory effect on monolayer growth modulate IGF peptide activity ina complex manner [16,17,18,19,20]. We have previously reported the cell surface association and dissociation of IGFBP-3 in Hs578T human breast cancer cells.
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