Characterization of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) Binding to Human Breast Cancer Cells: Kinetics of IGFBP-3 Binding and Identification of Receptor Binding Domain on the IGFBP-3 Molecule* (original) (raw)

Journal Article

,

1Department of Pediatrics (Y.Y., E.M.W., R.G.R., Y.O.), School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201

Search for other works by this author on:

,

2Department of Pediatrics (J.L.F.), University of Kentucky, Lexington, Kentucky 40536

Search for other works by this author on:

,

1Department of Pediatrics (Y.Y., E.M.W., R.G.R., Y.O.), School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201

Search for other works by this author on:

,

1Department of Pediatrics (Y.Y., E.M.W., R.G.R., Y.O.), School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201

Search for other works by this author on:

1Department of Pediatrics (Y.Y., E.M.W., R.G.R., Y.O.), School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201

*Address all correspondence and requests for reprints to: Youngman Oh, Department of Pediatrics, NRC 5, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3042.

Search for other works by this author on:

• PDF
• Split View
• • Article contents
  • Figures & tables
  • Video
  • Audio
  • Supplementary Data
• Cite

Cite

Yoshitaka Yamanaka, John L. Fowlkes, Elizabeth M. Wilson, Ron G. Rosenfeld, Youngman Oh, Characterization of Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3) Binding to Human Breast Cancer Cells: Kinetics of IGFBP-3 Binding and Identification of Receptor Binding Domain on the IGFBP-3 Molecule, Endocrinology, Volume 140, Issue 3, 1 March 1999, Pages 1319–1328, https://doi.org/10.1210/endo.140.3.6566
Close

Navbar Search Filter Mobile Enter search term Search

Abstract

Insulin-like growth factor binding protein-3 (IGFBP-3) binds to specific membrane proteins located on human breast cancer cells, which may be responsible for mediating the IGF-independent growth inhibitory effects of IGFBP-3. In this study, we evaluated IGFBP-3 binding sites on breast cancer cell membranes by competitive binding studies with IGFBP-1 through -6 and various forms of IGFBP-3, including synthetic IGFBP-3 fragments. Scatchard analysis revealed the existence of high-affinity sites for IGFBP-3 in estrogen receptor-negative Hs578T human breast cancer cells (dissociation constant (Kd) = 8.19 ± 0.97 × 10−9m and 4.92 ± 1.51 × 105 binding sites/cell) and 30-fold fewer receptors in estrogen receptor-positive MCF-7 cells (Kd = 8.49 ± 0.78 × 10−9m and 1.72 ± 0.31 × 104 binding sites/cell), using a one-site model. These data demonstrate binding characteristics of typical receptor-ligand interactions, strongly suggesting an IGFBP-3:IGFBP-3 receptor interaction. Among IGFBPs, only IGFBP-5 showed weak competition, indicating that IGFBP-3 binding to breast cancer cell surfaces is specific and cannot be attributed to nonspecific interaction with glycosaminoglycans. This was confirmed by showing that synthetic IGFBP-3 peptides containing IGFBP-3 glycosaminoglycan-binding domains competed only weakly for IGFBP-3 binding to the cell surface. Rat IGFBP-3 was 20-fold less potent in its ability to compete with human IGFBP-3Escherichia coli, as well as 10- to 20-fold less potent for cell growth inhibition than human IGFBP-3, suggesting the existence of species specificity in the interaction between IGFBP-3 and the IGFBP-3 receptor. When various IGFBP-3 fragments were evaluated for affinity for the IGFBP-3 receptor, only those fragments that contain the midregion of the IGFBP-3 molecule were able to inhibit 125I-IGFBP-3Escherichia coli binding, indicating that the midregion of the IGFBP-3 molecule is responsible for binding to its receptor. These observations demonstrate that specific, high-affinity IGFBP-3 receptors are located on breast cancer cell membranes. These receptors have properties that support the notion that they may mediate the IGF-independent inhibitory actions of IGFBP-3 in breast cancer cells.

THE INSULIN-LIKE GROWTH factors (IGFs), IGF-I and IGF-II, have been recognized as major regulators of mammary epithelial cell and breast cancer cell growth (1–4). Both IGF-I and IGF-II serve as potent mitogens for a number of breast cancer cell lines in vitro (1, 2, 4–6), and IGF-I or IGF-II messenger RNAs are detectable in the majority of human breast tumor specimens (7, 8). Six distinct IGF binding proteins (IGFBPs), which can bind IGFs with high affinity, have been identified and have been designated IGFBP-1 through IGFBP-6 (9–14). IGFBPs, which are produced by breast cancer cells, can either potentiate or inhibit IGF-induced DNA synthesis in cultured fibroblasts (15–17) and can interfere with IGF effects in transformed cells (18–20).

Delbe et al. (21) have demonstrated that purified mouse IGFBP-3 can bind to the chick embryo fibroblast cell surface and inhibit cell growth. Our laboratory and others have demonstrated a significant inhibitory effect of exogenous IGFBP-3 on the growth of Hs578T estrogen receptor (ER)-negative human breast cancer cells (22), human IGFBP-3 transfected mouse Balb/c fibroblast cells (23), and human IGFBP-3 transfected fibroblast cells, which were derived from mouse embryos homozygous for a targeted disruption of the type I IGF receptor gene (24). Factors that are known to inhibit the growth of human breast cancer cells, such as transforming growth factor-β (TGF-β) (25), retinoic acid (26), and antiestrogens (27, 28), may do so through their effects on IGFBP-3. We have demonstrated that the antiproliferative effects of TGF-β and retinoic acid in human breast cancer cells are mediated, at least in part, through IGFBP-3 action (29, 30), whereas Huynh et al. have shown that antiestrogen-induced growth inhibition of MCF-7 human ER-positive breast cancer cells is mediated similarly through increased IGFBP-3 action (31). In addition, Hembree et al. (32) have reported that treatment of human ectocervical cells with a retinoic acid receptor-specific ligand increases IGFBP-3 levels and suppresses cell proliferation. Furthermore, Buckbinder et al. (33) have demonstrated that IGFBP-3 is induced by the p53 tumor suppressor gene and is probably a mediator in p53 signaling. Previous studies from our laboratory have demonstrated that IGFBP-3 binds to specific membrane proteins (20, 26, and 50 kDa) located on Hs578T cells (34), which may be responsible for mediating these growth inhibitory effects of IGFBP-3 (22). Those were further confirmed by studies from Rajah et al. (35), showing that IGFBP-3 binds to specific membrane proteins and induces apoptosis through a p53- and IGF-independent mechanism in human prostate cancer cells and IGF receptor-negative mouse fibroblasts. Thus, recent studies have revealed that IGFBP-3 may have specific biological effects in various cell systems, including human breast cancer cells, which are not mediated through their interactions with IGFs (IGF-independent actions).

In this study, we characterize IGFBP-3 binding to breast cancer cells by employing competitive binding assays, and demonstrate a single class of specific, high-affinity membrane receptors for IGFBP-3, and identify a receptor binding domain on the IGFBP-3 molecule.

Materials and Methods

Materials

HPLC-purified hIGFBP-1 from human amniotic fluid was kindly provided by Dr. D. R. Powell (Baylor College of Medicine, Houston, TX) (36). Recombinant human IGFBP-3 (rhIGFBP-3), a nonglycosylated 29K core protein expressed in Escherichia coli cells, was the generous gift of Celtrix, Inc. (Santa Clara, CA) (37). rhIGFBP-2, -4, -5, and -6 were purchased from Austral Biologicals (San Ramon, CA). Intact rat IGFBP-3, NH2-terminal rat IGFBP-3 (rat IGFBP-31–160), and COOH-terminal rat IGFBP-3 (rat IGFBP-3161–265), which were purified from normal rat serum, were gifts from Dr. Nicholas Ling (Neurocrine Biosciences, Inc., San Diego, CA). Pooled human serum IGFBPs were prepared as previously described (36). Briefly, human sera from healthy male adult volunteers were collected and pooled. This pooled sera were chromatographed over a Sephadex G-50 column in formic acid to separate IGF peptides from the IGFBPs. IGF-I was purchased from Bachem California, Inc. (Torrance, CA). IGF-II was kindly provided by Eli Lilly & Co. (Indianapolis, IN). Iodination was performed by a modification of the chloramine-T technique, to specific activities of 350–500 μCi/μg for IGF-I and -II and to 100μ Ci/μg for IGFBP-3Escherichia coli peptides. Reagents used for SDS-PAGE were purchased from Bio-Rad Laboratories, Inc. (Richmond, CA). Na125I was obtained from Amersham Corp. (Arlington Heights, IL).

Cell Cultures

Hs578T (ER-negative) and MCF-7 (ER-positive) human breast cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD). Hs578T and MCF-7 were maintained in DMEM supplemented with 4.5 g/liter glucose, 110 mg/liter sodium pyruvate, and 10% FBS. Stock cultures were subcultured every 3 days.

Preparation of human IGFBP-3 fragments produced by matrix metalloproteinase-3 (MMP-3)

IGFBP-3 fragments (fragments a–e) were produced by MMP-3, as previously described (38, 39). In brief, 60 μg rhIGFBP-3Escherichia coli were digested by 200 ng MMP-3 (kindly provided by Dr. Hideaki Nagase, University of Kansas Medical Center, KS) in a total vol of 60 μl of 50 mm Tris (pH 7.5), 0.15 m NaCl, 10 mm CaCl2, 0.02% NaN3, 0.05% Brij 35 for 8 h at 37 C. The digestion was stopped by the addition of EDTA (final concentration: 10 mm), and the digestion products were analyzed by SDS-PAGE under reducing conditions. Five rhIGFBP-3Escherichia coli fragments were described as either NH2-terminal IGFBP-3 fragments: a) IGFBP-31–109 and b) IGFBP-31–99, or COOH-terminal IGFBP-3 fragments: c) IGFBP-3100–264, d) IGFBP-3110–264, and e) IGFBP-3177–264 (38, 39). To separate rhIGFBP-3Escherichia coli fragments produced by MMP-3 digestion, the digestion mixture was passed through a heparin-Sepharose column (Sigma Chemical Co.). All fractions were analyzed by SDS-PAGE and stained with Coomassie Blue. The wash fractions contained only the smallest IGFBP-3 fragments (fragments a and b) produced by MMP-3, which correspond to the first 100–110 NH2-terminal amino acids of IGFBP-3. Fragments c-e bound to the heparin column, and all three fragments were eluted. The calculated protein concentrations were performed using a modified Bradford method obtained from Bio-Rad, and were read against a BSA standard.

Preparation of synthetic human IGFBP-3 peptides

Peptides containing heparin binding domains were produced by solid-phase peptide synthesis, using 9-fluorenylmethoxycarbonyl chemistry, as previously described (39). The sequences are as follows: 149CKKGHAKDSQRYKVDYESQS167 (peptide IV) and 213CDKKGFYKKKQ[C-Acm]RPSKGR230 (peptide VI). Peptides were purified on a Vydac C-8 HPLC column, using a Gilson automated HPLC system, and were shown to be more than 98% pure. Sequence verification was performed by electrospray mass spectrometry. Peptides were synthesized with an additional NH2-terminal cysteine for use in thiol-coupling reactions. The internal cysteine in peptide VI was acetylmethylated, because it is normally involved in disulfide bond formation.

Baculovirus expression of rhIGFBP-3 fragments representing NH2-terminal domain or midregion of IGFBP-3 molecule

Plasmid constructs for IGFBP-31–87, IGFBP-388–148, and IGFBP-388–183 were prepared to express proteins in a baculovirus expression system (Life Technologies), as previously described (40). In brief, PCR was employed to make all constructs, using human IGFBP-3 complementary DNA (cDNA) as template, and further to add FLAG epitope sequences (DYKDDDDK) and a new stop codon immediately following COOH-termini of IGFBP-3 fragments. The signal peptide sequences of IGFBP-3 cDNA were ligated to NH2-termini of IGFBP-3 fragments. After sequencing, the FLAG-tagged IGFBP-3 fragment cDNAs were subcloned into pFASTBAC1 baculovirus expression vector and transfected into Sf9 insect cells, and viral recombinants were identified by immunoblotting with the anti-FLAG M2 antibody (Eastman Kodak Co.). Recombinant proteins were purified with anti-FLAG M2 affinity column using the serum-free media of HIGH-5 cells after infecting for 3 days at 27 C. The purified proteins were subjected to SDS-PAGE in a 15% gel and were stained with Coomassie Blue or transferred to nitrocellulose for immunodetection. Protein fractions were pooled, concentrated, and quantified by comparison with known amounts of BSA, by silver-staining (Bio-Rad).

125I-IGFBP-3 cell binding assay

Monolayer binding assays were performed as previously described (22). Confluent monolayers of human breast cancer cells (0.2 × 106 cells/well in 24-multiwell plates) were incubated in serum-free medium overnight. The cells were washed once with cold washing buffer (HBSS without CaCl2 and MgCl2, containing 25 mm HEPES and 25 mm NaHCO3, pH 7.4). Cell surface-bound endogeneous IGFBP-3 was then removed by rinsing the cells once with cold washing buffer containing 1 mm EDTA. The cells were washed once with cold washing buffer and incubated in 250 μl of binding buffer (HBSS without MgCl2, containing 25 mm HEPES, 25 mm NaHCO3, 1 mm CaCl2, and 0.5% BSA, pH 7.4) for 3 h at 15 C with 125I-IGFBP-3 (50,000 cpm) in the absence or presence of various concentrations of unlabeled IGFBP-3. The cells were washed with PBS and solubilized with 0.6 n NaOH. Radioactivity of the cell lysates was determined, and specific binding was calculated by subtracting nonspecific binding (cpm in the presence of unlabeled 10% human serum IGFBP fractions or 100 nm unlabeled IGFBP-3) from total binding. Binding parameters were determined by the curve-fitting program LIGAND (41). Cells from parallel wells were gently detached from plates by Trypsin/EDTA, and cell numbers were counted with a Coulter Counter (Coulter, Ltd., Beds, UK).

Specificity of IGFBP-3 binding to cell surfaces

Cell binding assays were performed as described above, by incubation of Hs578T cells with rh-125I-IGFBP-3 in the absence or presence of various concentrations of: 1) HPLC-purified IGFBP-1 from human amniotic fluid; and 2) rhIGFBP-2, -4, -5, and -6, or 3) intact rat IGFBP-3. 100 nm unlabeled IGFBP-3 was used for determination of nonspecific binding. These materials were characterized by Western ligand blots, to verify that they were intact and biologically active.

Identification of domains of IGFBP-3 responsible for binding to the IGFBP-3 receptor and to heparin

Cell binding assays were performed, as described above, by incubation of Hs578T cells with 125I-IGFBP-3 in the absence or presence of various concentrations of: 1) human IGFBP-3 fragments produced by MMP-3 digestion; 2) intact rat IGFBP-3, rat IGFBP-31–160, or rat IGFBP-3161–265; 3) synthetic IGFBP-3 peptides IV and VI; and 4) baculovirus-expressed rhIGFBP-3 fragments IGFBP-31–87, IGFBP-388–148, and IGFBP-388–183.

Monolayer cell replication assay

Hs578T cells were grown in 24-multiwell dishes until 60% confluent (3 × 104 cells/well) and then changed to 1% FBS containing media with or without reagents for 94 h. Cells were then gently detached from plates by trypsin-EDTA, and cell number was counted using a Coulter counter (Coulter, Ltd.).

Statistical analysis

Data were analyzed with a two-tailed Student’s t test, using the software program Instat 2.01 (Graphpad Software, Inc., San Diego, CA). Values are expressed as means ± sd.

Results

Characterization of IGFBP-3 binding to the breast cancer cells

Previous studies have demonstrated that IGFBP-3 has growth-inhibitory effects in ER-negative Hs578T and MDA-231 cells and ER-positive MCF-7 cells (31). In all three cell lines, the inhibitory effects of IGFBP-3 have been shown to be IGF-independent. Further studies have demonstrated the existence of IGFBP-3 interacting proteins on the cell surface of Hs578T cells (22); these cell surface proteins seem to have the characteristics of a specific IGFBP-3 receptor. To determine the characteristics of IGFBP-3 binding to its receptor on Hs578T and MCF-7 cells, competitive binding data regarding receptor affinity, number, and specificity were gathered. Because our previous studies have demonstrated that the binding of 125I-IGFBP-3 to the cell surface of Hs578T cells was increased by divalent cations (CaCl2 or MnCl2) in a dose-dependent manner (22), characterization of specific IGFBP-3 binding to the cell surface was performed in the presence of 1 mm CaCl2. Figure 1 shows the competitive binding curve and Scatchard analysis for the binding of 125I-IGFBP-3 to Hs578T and MCF-7 monolayers. Calculated binding affinity and number of binding sites for IGFBP-3 in Hs578T and MCF-7 cells are shown in Table 1. In both Hs578T cells and MCF-7 cells, the specific binding of 125I-IGFBP-3 was inhibited by unlabeled IGFBP-3 in a dose-dependent manner, with approximately 50% nonspecific binding (Fig. 1A). Scatchard analysis revealed a best fit for a one-site model in both Hs578T and MCF-7 cell lines (Kd = 8.19 ± 0.97 × 10−9 and 8.49 ± 0.78 × 10−9m, respectively) (Fig. 1B). The dissociation constants, number of binding sites/cell, and the percentage of specific binding for both cell lines are shown in Table 1, with data obtained from three independent experiments.

Fig 1

A, Competitive binding of 125I-IGFBP-3 to ER-negative Hs578T and ER-positive MCF-7 monolayers by unlabeled IGFBP-3. Cells were grown to confluence in 24-multiwell plates and maintained in serum-free media for 16 h. Cells were incubated for 3 h at 15 C with 125I-IGFBP-3 (50,000 cpm/well) in the presence of the indicated amounts of unlabeled IGFBP-3, and then were washed and solubilized and the total cell-associated radioactivity determined. Each point of the curve represents the mean of three independent experiments carried out in triplicate. B, Scatchard plot of IGFBP-3 binding to Hs578T and MCF-7 cells, using 100 nm unlabeled IGFBP-3 for determination of nonspecific binding. The level of binding was determined and analyzed, using the LIGAND computer program to gain the best fit for one-site analysis, after subtraction of 125I-IGFBP-3 binding in the presence of 100 nm unlabeled IGFBP-3. The circles indicate experimental data, and the solid lines indicate the resolved high-affinity and nonspecific components of the fitted curves. B/F indicates the ratio of bound-to-free ligand. Results are representative of three independent experiments.

Table 1

Characteristics of IGFBP-3 binding to IGFBP-3 receptor in human breast cancer cells

Binding studies were carried out on Hs578T cells and MCF-7 cells as described in Materials and Methods. The Kd value and the number of IGFBP-3 receptors were determined using LIGAND computer program to gain the best fit for one-site analysis after subtraction of 125I-IGFBP-3 binding in the presence of 100 nm unlabeled IGFBP-3. Each result is the mean ± sd for three independent experiments.

a

P < 0.01

b

P < 0.05 (Hs578T cells vs. MCF-7 cells).

Table 1

Characteristics of IGFBP-3 binding to IGFBP-3 receptor in human breast cancer cells

Binding studies were carried out on Hs578T cells and MCF-7 cells as described in Materials and Methods. The Kd value and the number of IGFBP-3 receptors were determined using LIGAND computer program to gain the best fit for one-site analysis after subtraction of 125I-IGFBP-3 binding in the presence of 100 nm unlabeled IGFBP-3. Each result is the mean ± sd for three independent experiments.

a

P < 0.01

b

P < 0.05 (Hs578T cells vs. MCF-7 cells).

Because we have previously demonstrated that competition for 125I-IGFBP-3 binding to the cell surface is also observed with 10% human serum IGFBP fractions derived from G-50 acid chromatography (22), the competitive binding assay was also performed after nonspecific binding was determined in the presence of unlabeled 10% human serum IGFBP fractions. Scatchard analysis of specific binding data revealed curvilinear plots in both cell lines, and statistical analysis resulted in preference for a two-site binding model (data not shown). Analysis of the high-affinity binding site in Hs578T cells (Kd = 8.11 ± 0.88 × 10−9m; 5.79 ± 1.32 × 105 binding sites/cell) and in MCF-7 cells (Kd= 9.92 ± 1.12 × 10−9m; 3.52± 0.61 × 104 binding sites/cell) demonstrates similar binding affinities in the two cell lines but approximately 20-fold less binding sites/cell in MCF-7 cells. Low-affinity binding characteristics in Hs578T cells (Kd = 2.20 ± 0.34 × 10−5m; 3.08 ± 0.58× 107 binding sites/cell) and in MCF-7 cells (Kd = 2.10 ± 0.27 × 10−5m; 2.26 ± 0.45 × 107 binding sites/cell) show a similar pattern in the two cell lines. In both cell lines, the dissociation constants for high-affinity binding and the number of binding sites/cell are similar to those observed when 100 nm unlabeled IGFBP-3 was employed for nonspecific binding. Under both conditions, the high-affinity dissociation constant in Hs578T cells is similar to that of MCF-7 cells. These findings suggest that a binding site with a Kd of 8–9 × 10−9 represents the high-affinity site for IGFBP-3 in both cell lines and that quantitative differences in specific binding between the two cell lines can be entirely explained by differences in the numbers of high-affinity binding sites per cell.

Specificity of IGFBP-3 binding to Hs578T cell surface

To establish the specificity of IGFBP-3 binding to Hs578T cells, competitive binding assays were performed, where the complete family of IGFBPs was used to compete for binding of 125I-IGFBP-3 (Fig. 2, A and B). Neither IGFBP-1, 2, 4, nor 6 were able to compete for 125I-IGFBP-3 binding. In contrast, IGFBP-5 was able to weakly compete, with approximately 10% of the potency of IGFBP-3 (24% displacement of 125I-IGFBP-3 at IGFBP-5 concentrations of 30 nm).

Fig 2

Specificity of IGFBP-3 binding to the IGFBP-3 receptor in Hs578T cells. Binding studies were carried out on Hs578T monolayers, as described in Materials and Methods, in the absence or presence of indicated concentrations of unlabeled IGFBP-1, 2, 3, or 4 (A) or IGFBP-3, 5, or 6 (B). Cell-associated radioactivity was determined as described in Fig. 1.

To evaluate the specificity of human IGFBP-3 binding to Hs578T cells, compared with IGFBP-3 from other species, intact rat IGFBP-3 was used for competitive binding assays. Rat IGFBP-3 consists of 265 amino acids, one amino acid longer than human IGFBP-3, and shows 83% homology with human IGFBP-3 (42). Most of the amino acid sequence variations between the two IGFBP-3s occur in the midregion of the IGFBP-3 molecules. Although intact rat IGFBP-3 inhibited 125I-IGFBP-3Escherichia coli binding in a dose-dependent manner, rat IGFBP-3 was 20-fold less potent than human IGFBP-3Escherichia coli in competing for 125I-hIGFBP-3 binding, suggesting the existence of species specificity in the interaction between IGFBP-3 and the IGFBP-3 receptor (Fig. 3A). These differences are unlikely to be attributable to glycosylation of rat IGFBP-3, because data from our laboratory and others indicate no difference in the ability of glycosylated and nonglycosylated human IGFBP-3 to associate with cell surfaces (data not shown; Ref. 43). Assays for inhibition of Hs578T cell growth were consistent with the relative affinities of IGFBP-3 preparation for cell surface binding. When human IGFBP-3 was added, it showed a significant inhibitory effect on monolayer growth of Hs578T cells (10 nm, P < 0.005). This inhibitory effect of IGFBP-3 was dose dependent. On the other hand, rat IGFBP-3 showed a growth inhibitory effect with 10–20 times less potency, compared with human IGFBP-3 (Fig. 3B). These data suggest that the less-homologous midregion of the IGFBP-3 molecule contributes significantly to the binding site on IGFBP-3 for cell surface association, because the amino acid sequences from COOH-terminal residues 157–249 between human and rat IGFBP-3 are almost identical.

Fig 3

A, Competitive binding of intact rat IGFBP-3 with 125I-IGFBP-3 in Hs578T cells. Binding studies were carried out on Hs578T monolayers, as described in Materials and Methods, in the absence or presence of indicated concentrations of unlabeled intact rhIGFBP-3 and intact rat IGFBP-3. Cell-associated radioactivity was determined as described in Fig. 1. B, Effects of rhIGFBP-3 and rat IGFBP-3 on monolayer growth in Hs578T cells. Hs578T cells were grown in 24-multiwell dishes until 60% confluent (0.1 × 106 cells/well) and then changed to 1% FBS containing media with or without reagents for 94 h. Cells were then gently detached from plates by trypsin-EDTA, and cell number was counted using a Coulter counter. Statistical significance, in comparison with control values, is indicated by * (P < 0.005).

Characterization of the receptor binding domain on the IGFBP-3 molecule

To determine the binding domain on the IGFBP-3 molecule responsible for binding to its receptor in Hs578T cells, human IGFBP-3 fragments, produced by MMP-3 digestion and rat IGFBP-3 fragments (rat IGFBP-31–160 and rat IGFBP-3161–265), were compared for their binding affinities for Hs578T monolayers in competitive binding assays. Figure 4A demonstrates that COOH-terminal fragments of human IGFBP-3 (a mixture of IGFBP-3100–264, IGFBP-3110–264, and IGFBP-3177–264) inhibited 125I-IGFBP-3 binding in a dose-dependent manner, with 10-fold less potency than intact human IGFBP-3. In contrast, NH2-terminal fragments of human IGFBP-3 (a mixture of IGFBP-31–109 and IGFBP-31–99) did not inhibit 125I-IGFBP-3 binding. In addition, neither rat NH2- nor COOH-terminal IGFBP-3 fragments inhibited 125I-IGFBP-3 binding, as shown in Fig. 4B.

Fig 4

Competitive binding of 125I-IGFBP-3 to Hs578T monolayers by human IGFBP-3 fragments (A) or rat IGFBP-3 fragments (B). Binding studies were carried out on Hs578T monolayers, as described in Materials and Methods, in the absence or presence of indicated concentrations of unlabeled human IGFBP-3 fragments (A) or unlabeled rat IGFBP-3 fragments (B). Cell-associated radioactivity was determined as described in Fig. 1.

To further narrow down the receptor binding site on the IGFBP-3 molecule, and, especially, to evaluate the significance of the midregion (44) and the heparin-binding domains near the COOH-terminus of the IGFBP-3 molecule (45) for receptor binding, we have generated two synthetic peptides containing the heparin-binding domain, as well as baculovirus-expressed rhIGFBP-3 fragments corresponding to the midregion of the IGFBP-3 molecule (Fig. 5). Peptide IV contains the sequence 149KKGHA153, which resembles a short heparin-binding domain (BBXBX; B = basic amino acid and X = nonbasic amino acid) and peptide VI contains the sequence 219YKKKQCRP226, which resembles a long heparin-binding motif (XBBBXXBX) (45, 46). IGFBP-31–87 represents the highly conserved NH2-terminal region, whereas IGFBP-388–183 and IGFBP-388–148 correspond to the nonconserved midregion of IGFBP-3, with or without heparin binding domains, respectively. Competition binding assays revealed that the two peptides that contain the heparin binding domains (peptide IV and VI) weakly inhibited 125I-IGFBP-3 binding (Fig. 6A); 15% and 29% of 125I-IGFBP-3 were displaced by 10 μm of peptide IV or VI, respectively, confirming that IGFBP-3 binding to the Hs578T cell surface cannot be attributed to interaction with GAGs. Indeed, as shown in Fig. 6B, the Kd for peptide VI was significantly lower than that of the high-affinity component of IGFBP-3 binding in Hs578T cells (Fig. 1B). On the other hand, the low-affinity binding characteristics of IGFBP-3 are virtually identical to those of peptide VI (Kd = 2.03 × 10−5m, 3.88 × 107 binding sites/cell). As would be predicted, when peptide VI was added, it showed no significant inhibitory effect on monolayer growth of Hs578T cells, at concentrations up to 300 nm (data not shown).

Fig 5

Schematic diagram of the structure of human IGFBP-3 and baculovirus-expressed IGFBP-3 fragments. The conserved and nonconserved regions among the high-affinity IGFBPs are indicated. The vertical lines represent cysteine residues. The sequences of two putative heparin binding domains are indicated. Symbols (*) represent putative N-glycosylation sites. IGFBP-31–87 represents the conserved NH2-terminal region, whereas IGFBP-388–148 and IGFBP-388–183 correspond to the nonconserved midregion of IGFBP-3, with or without heparin binding domains, respectively.

Fig 6

A, Competitive binding of 125I-IGFBP-3 to Hs578T monolayers by human IGFBP-3 peptides IV and VI. Binding studies were carried out on Hs578T monolayers, as described in Materials and Methods, in the absence or presence of indicated concentrations of unlabeled human IGFBP-3 peptides IV or VI. Cell-associated radioactivity was determined as described in Fig. 1. B, Scatchard plot of IGFBP-3 to Hs578T cells using peptide VI as a competitor. Binding studies were carried out on Hs578T monolayers, as described in Materials and Methods, in the absence or presence of indicated concentrations of unlabeled human IGFBP-3 peptides VI. Cell-associated radioactivity was determined as described in Fig. 1.

On the other hand, IGFBP-3 fragments, representing the midregion of IGFBP-3, inhibited 125I-IGFBP-3 binding with equipotency to intact IGFBP-3 (40% and 60% inhibition at the concentrations of 10 and 30 nm, respectively), regardless of heparin binding domains. The NH2-terminal fragment, IGFBP-31–87, showed no inhibition of IGFBP-3 binding at concentrations up to 30 nm (Fig. 7). These data indicate that the midregion of the IGFBP-3 molecule, even without a putative heparin binding motif, is probably responsible for the binding of IGFBP-3 to Hs578T cell surfaces and the accompanying growth-inhibitory effect.

Fig 7

Competitive binding of 125I-IGFBP-3 to Hs578T monolayers by baculovirus-expressed rhIGFBP-3 fragments. Experiments were performed using Hs578T monolayers, as described in Fig. 6A. Statistical significance, in comparison with control values, is indicated by * (P < 0.01).

Discussion

Our laboratory and others have demonstrated a significant IGF-independent inhibitory effect of: 1) exogenous IGFBP-3 on the growth of Hs578T ER-negative human breast cancer cells (22); 2) human IGFBP-3 transfected mouse Balb/c fibroblast cells (23); and 3) human IGFBP-3 transfected fibroblast cells, which were derived from mouse embryos homozygous for a targeted disruption of the type I IGF receptor gene (24). We have recently demonstrated that the antiproliferative effects of TGF-β and retinoic acid in human breast cancer cells are mediated, at least in part, through transcriptional regulation of IGFBP-3 (29, 30) and that the growth inhibitory effects of IGFBP-3 in these cells are not mediated through interaction with IGFs (IGF-independent actions). The significance of IGFBP-3 as a mediator of the growth inhibitory effects of other hormones has been studied by others, as with antiestrogens and the p53 tumor suppressor gene (31, 33). Furthermore, we have recently demonstrated that IGFBP-3 binds to specific membrane proteins (20, 26, and 50 kDa) located on Hs578T cells (22), which may be responsible for mediating the growth inhibitory effects of IGFBP-3 (34). In the same context, previous studies have demonstrated that IGFBP-3 binds to specific membrane proteins and induces apoptosis through a p53- and IGF-independent mechanism in human prostate cancer cells, supporting our previous results indicating IGF-independent action of IGFBP-3 and IGFBP-3 receptors (35).

IGFBP production by breast cancer cells is heterogeneous; the predominant secreted IGFBPs seem to correlate with the ER status of the cells (47). ER-negative cells predominantly secrete IGFBP-3 and IGFBP-4 as major species, whereas ER-positive cells secrete predominantly IGFBP-2 and IGFBP-4 as major species (48–50). The differential expression of IGFBPs in these two classes of breast cancer cells suggests that the biological significance of IGFBPs and, especially, of IGFBP-3 and its own receptor, may depend upon the ER status of the cells. In this study, accordingly, we used two human breast cancer cell lines: Hs578T cells (ER-negative) and MCF-7 cells (ER-positive). High-affinity 125I-IGFBP-3 binding data in Hs578T cells and MCF-7 cells reveal similar binding affinities in the two cell lines, regardless of whether 10% human serum IGFBP fractions or 100 nm IGFBP-3 was employed for nonspecific binding. Receptor affinities were identical in the two cell lines, although 20- to 30-fold lower receptor number/cell was observed in MCF-7 cells, relative to Hs578T cells, entirely accounting for the differences in specific binding between the two cell lines. These data indicate that high-affinity binding can be attributed to a specific IGFBP-3 binding site, which seems to have all the characteristics of a peptide ligand-receptor interaction and which can be found, although at varying concentrations, in both Hs578T and MCF-7 cells. The presence of a single high-affinity binding site for IGFBP-3 on Hs578T cells is consistent with our previous identification of a single binding site (estimated at 23 kDa) by monolayer cross-linking (34). On the other hand, studies employing cross-linking to Hs578T cell lysates suggested the presence of as many as three specific IGFBP-3 interacting proteins. The additional two binding sites may reflect either: 1) intracellular proteins contained within the whole cell lysates; 2) components of an IGFBP-3 receptor complex; or 3) precursor or degradation forms of the IGFBP-3 receptor.

While Hs578T cells seem to have the same putative IGFBP-3 receptors as MCF-7 cells, the former has about 30-fold more IGFBP-3 receptors than the latter. From these results, it would be predicted that IGFBP-3 would have more growth inhibitory effects on Hs578T cells than on MCF-7 cells, because of the higher IGFBP-3 receptor concentrations in Hs578T cells. Thus, the differences in the cellular response to IGFBP-3 between ER-negative cell lines and ER-positive cell lines may depend, at least in part, on the concentration of IGFBP-3 receptors, although further studies will be necessary in additional cell lines. Nevertheless, in our previous studies (22), the threshold for IGFBP-3 growth inhibitory effects was in the range of 10−9–10−8m, which is consistent with the affinity of IGFBP-3 for the IGFBP-3 receptor (Kd = 8.19 ± 0.97 nm). These binding characteristics are also in the range of those estimated by Delbe et al. in chick embryo fibroblasts (Kd = 10−8m and 60,000 binding sites/cell) (21).

To establish the specificity of IGFBP-3 binding to Hs578T cells, competitive binding assays were performed in which the family of IGFBPs was used to compete for binding of 125I-IGFBP-3. IGFBP-1, 2, 4, and 6 were not able to compete for 125I-IGFBP-3 binding, although IGFBP-5 weakly competed at a concentration of 30 nm, indicating that IGFBP-3 has highly specific binding sites on the cell surface. The mechanism of the cell surface binding for IGFBP-1 has been shown to result from α5β1-integrin receptors, which would recognize the arginine-glycine-aspartic acid (RGD) tripeptide sequence in IGFBP-1 (51). The fact that IGFBP-3 lacks this RGD sequence and that no competition was seen with RGD sequence-containing IGFBPs, such as IGFBP-1 and IGFBP-2, indicate that the mechanism of IGFBP-3 cell surface binding is unlikely to be mediated through integrin receptors and is different from that of IGFBP-1. IGFBP-3, IGFBP-5, and IGFBP-6 have putative heparin binding amino acid sequences: residues 149K-K-G-H-A153 and 219Y-K-K-K-Q-C-R-P226 in IGFBP-3; residues 139P-K-H-T-R-I144 and 225Y-K-R-K-Q-C-K-P232 in IGFBP-5; residues 172Y-R-K-R-Q-C-R178 in IGFBP-6. Heparin-like molecules are present on cell surfaces and in extracellular matrix (ECM), suggesting the possibility that IGFBP-3, 5, and 6 could bind to cell surfaces or to ECM through heparin-like molecules. Others have reported that heparin, which releases proteins attached to cell surface proteoglycans, could displace IGFBP-3 binding on human neonatal fibroblast (52) and rat sertoli cell surfaces (53), and that heparin modulates the binding of IGFBP-5 to osteoblastic cell membranes (54). Synthetic peptides, containing the C-terminal heparin binding motifs from IGFBP-3, -5, and -6, inhibit IGFBP-3 and IGFBP-5 binding to endothelial cell monolayers (55–57). Similarly, recent studies reported that the ability of IGFBP-3 to associate with the cell surface was lost in IGFBP-3 variants lacking residues 185–264 and in the 228KGRKR232 → MDGEA mutant, suggesting that residues 228–232 of IGFBP-3 are essential for the cell-surface association in Chinese hamster ovary cells (58). However, it is of note that the 228KGRKR232 → MDGEA (IGFBP-1 sequence) mutations are adjacent to the putative long binding domain (219YKKKQCRP226) affecting IGFBP-3 binding to heparin. It is possible that mutation of the basic residues 228–232 to acidic residues results in disruption of heparin binding to the putative long binding domain of IGFBP-3, thereby affecting the ability of IGFBP-3 to associate with the cell surface.

On the other hand, when heparin and heparan sulfate linkages of glycosaminoglycans (GAGs) on the cell surface are enzymatically removed by pretreatment with heparinase or heparitinase, IGFBP-3 binding is only minimally affected in human breast cancer cells, despite exogenously added soluble heparin or heparan sulfate, which inhibits 125I-IGFBP-3 binding to the cell surface in a dose-dependent manner (59). This suggests that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting binding of IGFBP-3 to cell-surface proteins specific for IGFBP-3, rather than competing with cell-surface GAGs for binding of IGFBP-3. In our study, IGFBP-5 and IGFBP-6 showed little or no ability to compete for 125I-IGFBP-3 binding, suggesting that 125I-IGFBP-3 binding to breast cancer cell surfaces through heparin-like molecules is not a major factor. This was further confirmed by experiments employing synthetic peptides IV and VI, which contain the two heparin binding domains, 149K-K-G-H-A153 and 219Y-K-K-K-Q-C-R-P226, respectively, and have been shown to bind several different GAGs, such as heparin, heparan sulfate, and dermatan sulfate, but with different affinities (60). Even though peptides IV and VI competed with IGFBP-3 binding to the cell surface, the Kds for peptides IV and VI were significantly lower (Fig. 6) than that of the high-affinity component of IGFBP-3 binding in Hs578T cells (Fig. 1B). Furthermore, in the presence of peptides IV or VI, the IGFBP-3 cell binding assay showed the single high-affinity binding site (data not shown). More evidently, the low-affinity binding characteristics of IGFBP-3, as shown in the two cell lines, when 10% human serum IGFBP fractions were used for nonspecific binding, are identical to those of peptide VI (Kd = 2.03 × 10−5m, 3.88 × 107 binding sites/cell), suggesting that the low-affinity binding component may be the result of interactions between IGFBP-3 and GAG-like molecules on the cell surface or in the ECM. In that case, the curvilinear Scatchard plot observed in the presence of 10% human serum IGFBPs may be partially caused by competition by IGFBP-5 for the low-affinity site. We have shown that the C-terminal heparin binding motif from IGFBP-5 has an affinity for heparin similar to that of the homologous heparin binding motif from IGFBP-3, whereas the homologous IGFBP-6 consensus sequence binds heparin with much less affinity (60). These observations possibly explain why IGFBP-6 association with the cell surface has not been demonstrated.

Glycosylated rat IGFBP-3 inhibited rh-125I-IGFBP-3Escherichia coli binding in a dose-dependent manner, although glycosylated rat IGFBP-3 shows 20-fold less potency than human IGFBP-3Escherichia coli, differences which cannot be explained simply by glycosylation. These observations suggest that the differences in IGFBP-3 binding affinity to cell surfaces depend on the species of origin of IGFBP-3. Most of the amino acid sequence variations between human IGFBP-3 and rat IGFBP-3 occur in the midregion of the IGFBP-3 molecules (residues 86–184), suggesting that the binding site on IGFBP-3 for the IGFBP-3 receptor is located in the midregion of the IGFBP-3 molecule. This was supported by the different levels of competition for IGFBP-3 binding by human IGFBP-3 fragments or rat IGFBP-3 fragments. First, human N-terminal IGFBP-3 fragments, which contain residues 1–109, failed to inhibit 125I-IGFBP-3 binding, indicating that the N-terminal domain of IGFBP-3 is not responsible for receptor binding. Secondly, a human mid-C-terminal fragment (residues 100–264) inhibited 125I-IGFBP-3 binding, whereas a rat C-terminal fragment (161–265) did not compete for 125I-IGFBP-3 binding, even though it contains a heparin binding motif that is identical to that of human IGFBP-3, indicating that the difference between the human C-terminal IGFBP-3 and rat C-terminal IGFBP-3 is attributed to the midregion, where human and rat IGFBP-3 contain different amino acid sequences. Whereas the C-terminal fragments of human IGFBP-3 contain the midregion of the IGFBP-3 molecule, the other peptides employed do not contain or only partially contain this region, consistent with the hypothesis that the midregion of the IGFBP-3 molecule is essential for IGFBP-3 binding to its receptor.

To test our hypothesis more directly, we have generated three rhIGFBP-3 fragments in a baculovirus expression system: 1) IGFBP-31–87, an NH2-terminal fragment containing the highly conserved sequences among IGFBPs, including the 12-cysteine cluster; 2) IGFBP-388–148, a fragment containing the midregion of IGFBP-3, but without heparin binding domains; and 3) IGFBP-3888–183, a fragment corresponding to the entire midregion of IGFBP-3, including heparin binding motif. Competitive binding assays revealed that only IGFBP-3 fragments, representing the midregion of IGFBP-3, bind to the putative IGFBP-3 receptor with equipotency, compared with intact IGFBP-3, indicating that the receptor binding site resides within the midregion of the IGFBP-3 molecule (possibly within amino acids 88–148).

These observations have shown that high-affinity IGFBP-3 receptors are located on breast cancer cell membranes. These receptors have properties that support the notion that they may mediate the IGF-independent inhibitory actions of IGFBP-3 in breast cancer cells. Recent studies postulated that the type V TGF-β receptor is the putative IGFBP-3 receptor in mink lung epithelial cells (61). It seems that IGFBP-3 binds to the type V TGF-β receptor specifically and competes with TGF-β for receptor binding. The growth inhibitory effect of IGFBP-3 is aborted by a TGF-β1 peptide antagonist in these cells, suggesting that IGFBP-3 is a functional ligand for the type V TGF-β receptor. However, at the present time, it is unclear whether the type V TGF-β receptor represents the primary IGFBP-3 receptor, whether it is identical to the putative receptor observed in breast and prostate cancer cell systems, and whether it plays a role in the growth-inhibitory actions of IGFBP-3 in those cell systems. Further characterization and sequencing of the IGFBP-3 receptor will be important in determining the mechanisms for the IGF-independent actions of IGFBP-3 in directly inhibiting cell growth in human breast cancer cells. Current studies are in progress to identify the IGFBP-3 receptor and to characterize the IGFBP-3 receptor-mediated signal transduction pathway(s) in human breast cancer cells.

*

This work was supported, in part, by NIH Grants CA-58110 and DK-51513 (to R.G.R.), by a Sumitomo Pharmaceutical Co. Fellowship Grant (to Y.Y.), by NIH Grant DK-02276 (to J.L.F.), and by U.S. Army Grants DAMD-17–96-1–6204 and DAMD-17–97-1–7204 (to Y.O.).

References

1

Osborne

CK

,

Coronado

EB

,

Kitten

LJ

,

Arteaga

CI

,

Fuqua

SA

,

Ramasharma

K

,

Marshall

M

,

Li

CH

1989

Insulin-like growth factor-II (IGF-II): a potential autocrine/paracrine growth factor for human breast cancer acting via the IGF-I receptor.

Mol Endocrinol

3

:

1701

–

1709

2

De Leon

DD

,

Wilson

DM

,

Powers

M

,

Rosenfeld

RG

1992

Effects of insulin-like growth factors (IGFs) and IGF receptor antibodies on the proliferation of human breast cancer cells.

Growth Factors

6

:

327

–

336

3

Furlanetto

RW

,

DiCarlo

JN

1984

Somatomedin-C receptors and growth effects in human breast cells maintained in long-term tissue culture.

Cancer Res

44

:

2122

–

2128

4

Huff

KK

,

Kaufman

D

,

Gabbay

KH

,

Spencer

EM

,

Lippman

ME

,

Dickson

RB

1986

Secretion of an insulin-like growth factor-I-related protein by human breast cancer cells.

Cancer Res

46

:

4613

–

4619

5

Westley

B

,

May

FE

1991

Estrogen-regulated messenger RNAs in human breast cancer cells.

Cancer Treat Res

53

:

259

–

271

6

Baxter

RC

,

Maitland

JE

,

Raisur

RL

,

Reddel

R

,

Sutherland

RL

(ed)

1983

High molecular weight somatomedin-C (IGF-I) from T47D human mammary carcinoma cells: immunoreactivity and bioactivity. In:

Spencer

EM

(ed) Insulin-Like Growth Factors/Somatomedins.

De Gruyter

,

Berlin

, pp

615

–

618

7

Cullen

KJ

,

Smith

HS

,

Hill

S

,

Rosen

N

,

Lippman

ME

1991

Growth factor messenger RNA expression by human breast fibroblasts from benign and malignant lesions.

Cancer Res

51

:

4978

–

4985

8

Paik

S

1992

Expression of IGF-I and IGF-II mRNA in breast tissue.

Breast Cancer Res Treat

22

:

31

–

38

9

Puustinen

A

,

Finel

M

,

Virkki

M

,

Wikstrom

M

1989

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli.

FEBS Lett

249

:

163

–

167

10

Kita

K

,

Konishi

K

,

Anraku

Y

1984

Terminal oxidases of Escherichia coli aerobic respiratory chain. I. Purification and properties of cytochrome b562-o complex from cells in the early exponential phase of aerobic growth.

J Biol Chem

259

:

3368

–

3374

11

Minghetti

KC

,

Goswitz

VC

,

GabrielNE, Hill

JJ

,

Barassi

CA

,

Georgiou

CD

,

Chan

SI

,

Gennis

RB

1992

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity.

Biochemistry

31

:

6917

–

6924

12

Uno

T

,

Nishimura

Y

,

Tsuboi

M

,

Kita

K

,

Anraku

Y

1985

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states.

J Biol Chem

260

:

6755

–

6760

13

Hata

A

,

Kirino

Y

,

Matsuura

K

,

Itoh

S

,

Hiyama

T

,

Konishi

K

,

Kita

K

,

Anraku

Y

1985

Assignment of ESR signals of Escherichia coli terminal oxidase complexes.

Biochim Biophys Acta

810

:

62

–

72

14

Tsubaki

M

,

Mogi

T

,

Anraku

Y

,

Hori

H

1993

Structure of the heme-copper binuclear center of the cytochrome bo complex of Escherichia coli: EPR and Fourier transform infrared spectroscopic studies.

Biochemistry

32

:

6065

–

6072

15

De Mellow

JS

,

Baxter

RC

1988

Growth hormone-dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human skin fibroblasts.

Biochem Biophys Res Commun

156

:

199

–

204

16

Blum

WF

,

Jenne

EW

,

Reppin

F

,

Kietzmann

K

,

Ranke

MB

,

Bierich

JR

1989

Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I.

Endocrinology

125

:

766

–

772

17

Liu

L

,

Brinkman

A

,

Blat

C

,

Harel

L

1991

IGFBP-1, an insulin like growth factor binding protein, is a cell growth inhibitor.

Biochem Biophys Res Commun

174

:

673

–

679

18

Ritvos

O

,

Ranta

T

,

Jalkanen

J

,

Suikkari

AM

,

Voutilainen

R

,

Bohn

H

,

Rutanen

EM

1988

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells.

Endocrinology

122

:

2150

–

2157

19

Campbell

PG

,

Novak

JF

1991

Insulin-like growth factor binding protein (IGFBP) inhibits IGF action on human osteosarcoma cells.

J Cell Physiol

149

:

293

–

300

20

Culouscou

JM

,

Shoyab

M

1991

Purification of a colon cancer cell growth inhibitor and its identification as an insulin-like growth factor binding protein.

Cancer Res

51

:

2813

–

2819

21

Delbe

J

,

Blat

C

,

Desauty

G

,

Harel

L

1991

Presence of IDF45 (mlGFBP-3) binding sites on chick embryo fibroblasts.

Biochem Biophys Res Commun

179

:

495

–

501

22

Oh

Y

,

Muller

HL

,

Lamson

G

,

Rosenfeld

RG

1993

Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition.

J Biol Chem

268

:

14964

–

14971

23

Cohen

P

,

Lamson

G

,

Okajima

T

,

Rosenfeld

RG

1993

Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth.

Mol Endocrinol

7

:

380

–

386

24

Valentinis

B

,

Bhala

A

,

DeAngelis

T

,

Baserga

R

,

Cohen

P

1995

The human insulin-like growth factor (IGF) binding protein-3 inhibits the growth of fibroblasts with a targeted disruption of the IGF-I receptor gene.

Mol Endocrinol

9

:

361

–

367

25

Knabbe

C

,

Lippman

ME

,

Wakefield

LM

,

Flanders

KC

,

Kasid

A

,

Derynck

R

,

Dickson

RB

1987

Evidence that transforming growth factor-beta is a hormonally regulated negative growth factor in human breast cancer cells.

Cell

48

:

417

–

428

26

Fontana

JA

,

Burrows-Mezu

A

,

Clemmons

DR

,

LeRoith

D

1991

Retinoid modulation of insulin-like growth factor-binding proteins and inhibition of breast carcinoma proliferation.

Endocrinology

128

:

1115

–

1122

27

Rochefort

H

1991

Mechanism of action of high-affinity antiestrogens. An overview.

Am J Clin Oncol

14

:

1

–

4

28

Wosikowski

K

,

Kung

W

,

Hasmann

M

,

Loser

R

,

Eppenberger

U

1993

Inhibition of growth-factor-activated proliferation by anti-estrogens and effects on early gene expression of MCF-7 cells.

Int J Cancer

53

:

290

–

297

29

Oh

Y

,

Muller

HL

,

Ng

L

,

Rosenfeld

RG

1995

Transforming growth factor-beta-induced cell growth inhibition in human breast cancer cells is mediated through insulin-like growth factor-binding protein-3 action.

J Biol Chem

270

:

13589

–

13592

30

Gucev

ZS

,

Oh

Y

,

Kelley

KM

,

Rosenfeld

RG

1996

Insulin-like growth factor binding protein 3 mediates retinoic acid- and transforming growth factor beta2-induced growth inhibition in human breast cancer cells.

Cancer Res

56

:

1545

–

1550

31

Huynh

H

,

Yang

X

,

Pollak

M

1996

Estradiol and antiestrogens regulate a growth inhibitory insulin-like growth factor binding protein 3 autocrine loop in human breast cancer cells.

J Biol Chem

271

:

1016

–

1021

32

Hembree

JR

,

Agarwal

C

,

Beard

RL

,

Chandraratna

RA

,

Eckert

R

1996

Retinoid X receptor-specific retinoids inhibit the ability of retinoic acid receptor-specific retinoids to increase the level of insulin-like growth factor binding protein-3 in human ectocervical epithelial cells.

Cancer Res

56

:

1794

–

1799

33

Buckbinder

L

,

Talbott

R

,

Velasco-Miguel

S

,

Takenaka

I

,

Faha

B

,

Seizinger

BR

,

Kley

N

1995

Induction of the growth inhibitor IGF-binding protein 3 by p53.

Nature

377

:

646

–

649

34

Oh

Y

,

Muller

HL

,

Pham

H

,

Rosenfeld

RG

1993

Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells.

J Biol Chem

268

:

26045

–

26048

35

Rajah

R

,

Valentinis

B

,

Cohen

P

1997

Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53- and IGF-independent mechanism.

J Biol Chem

272

:

12181

–

12188

36

Liu

F

,

Powell

DR

,

Hintz

RL

1990

Characterization of insulin-like growth factor-binding proteins in human serum from patients with chronic renal failure.

J Clin Endocrinol Metab

70

:

6208

37

Spratt

SK

,

Tatsuno

GP

,

Yamanaka

MK

,

Ark

BC

,

Detmer

J

,

Mascarenhas

D

,

Flynn

J

,

Talkington-Verser

C

,

Spencer

EM

1990

Cloning and expression of human insulin-like growth factor binding protein 3.

Growth Factors

3

:

63

–

72

38

Fowlkes

JL

,

Enghild

JJ

,

Suzuki

K

,

Nagase

H

1994

Matrix metalloproteinases degrade insulin-like growth factor binding protein-3 in dermal fibroblast cultures.

J Biol Chem

269

:

25742

–

25746

39

Fowlkes

JL

,

Serra

DM

,

Rosenberg

CK

,

Thrailkill

KM

1995

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) functions as an IGF-reversible inhibitor of IGFBP-4 proteolysis.

J Biol Chem

270

:

27481

–

27488

40

Yamanaka

Y

,

Wilson

EM

,

Rosenfeld

RG

,

Oh

Y

1997

Inhibition of insulin receptor activation by insulin-like growth factor binding proteins.

J Biol Chem

272

:

30729

–

30734

41

Munson

PJ

,

Rodbard

D

1980

Ligand: a versatile computerized approach for characterization of ligand-binding systems.

Anal Biochem

107

:

220

–

239

42

Albiston

AL

,

Herington

AC

1990

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

Biochem Biophys Res Commun

166

:

892

–

897

43

Conover

CA

1992

Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism.

Endocrinology

130

:

3191

–

3199

44

Rechler

MM

1993

Insulin-like growth factor binding proteins.

Vitam Horm

47

:

1

–

114

45

Fowlkes

JL

,

Serra

DM

1996

Characterization of glycosaminoglycan-binding domains present in insulin-like growth factor-binding protein-3.

J Biol Chem

271

:

14676

–

14679

46

Arai

T

,

Parker

A

,

Busby Jr

W

,

Clemmons

DR

1994

Heparin, heparan sulfate, and dermatan sulfate regulate formation of the insulin-like growth factor-I and insulin-like growth factor-binding protein complexes.

J Biol Chem

269

:

20388

–

20393

47

Figueroa

JA

,

Jackson

JG

,

McGuire

WL

,

Krywicki

RF

,

Yee

D

1993

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status.

J Cell Biochem

52

:

196

–

205

48

Yee

D

,

Favoni

RE

,

Lippman

ME

,

Powell

DR

1991

Identification of insulin-like growth factor binding proteins in breast cancer cells.

Breast Cancer Res Treat

18

:

3

–

10

49

Clemmons

DR

,

Camacho-Hubner

C

,

Coronado

E

,

Osborne

CK

1990

Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status.

Endocrinology

127

:

2679

–

2686

50

Figueroa

JA

,

Yee

D

1992

The insulin-like growth factor binding proteins (IGFBPs) in human breast cancer.

Breast Cancer Res Treat

22

:

81

–

90

51

Jones

JI

,

Gockerman

A

,

Busby Jr

WH

,

Wright

G

,

Clemmons

DR

1993

Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence.

Proc Natl Acad Sci USA

90

:

10553

–

10557

52

Martin

JL

,

Ballesteros

M

,

Baxter

RC

1992

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Endocrinology

131

:

1703

–

1710

53

Smith

EP

,

Lu

L

,

Chernausek

SD

,

Klein

DJ

1994

Insulin-like growth factor-binding protein-3 (IGFBP-3) concentration in rat Sertoli cell-conditioned medium is regulated by a pathway involving association of IGFBP-3 with cell surface proteoglycans.

Endocrinology

135

:

359

–

364

54

Andress

DL

1995

Heparin modulates the binding of insulin-like growth factor (IGF) binding protein-5 to a membrane protein in osteoblastic cells.

J Biol Chem

270

:

28289

–

28296

55

Bar

RS

,

Boes

M

,

Booth

BA

,

Dake

BL

,

Moser

DR

,

Erondu

NE

1994

Vascular endothelium, IGFs, IGF binding proteins In:

Baxter

R

,

Gluckman

P

,

Rosenfeld

R

(eds) The Insulin-like Growth Factors and Their Regulatory Proteins.

Excerpta Medica

,

Amsterdam

, pp

237

–

244

56

Booth

BA

,

Boes

M

,

Dake

BL

,

Linhardt

RJ

,

Caldwell

EE

,

Weiler

JM

,

Bar

RS

1996

Structure-function relationships in the heparin-binding C-terminal region of insulin-like growth factor binding protein-3.

Growth Regul

6

:

206

–

213

57

Booth

BA

,

Boes

M

,

Andress

DL

,

Dake

BL

,

Kiefer

MC

,

Maack

C

,

Linhardt

RJ

,

Bar

K

,

Caldwell

EE

,

Weiler

J

,

Bar

RS

1995 IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin binding domain.

Growth Regul

5

:

1

–

17

58

Firth

SM

,

Ganeshprasad

U

,

Baxter

RC

1998

Structural determinants of ligand and cell surface binding of insulin-like growth factor-binding protein-3.

J Biol Chem

273

:

2631

–

2638

59

Oh

Y

,

Gucev

Z

,

Ng

L

,

Muller

HL

,

Rosenfeld

RG

1995

Antiproliferative actions of insulin-like growth factor binding protein (IGFBP)-3 in human breast cancer cells.

Prog Growth Factor Res

6

:

503

–

512

60

Fowlkes

JL

,

Serra

DM

1996

Characterization of glycosaminoglycan-binding domains present in insulin-like growth factor-binding protein-3.

J Biol Chem

271

:

14676

–

14679

61

Leal

SM

,

Liu

Q

,

Huang

SS

,

Huang

JS

1997

The type V transforming growth factor beta receptor is the putative insulin-like growth factor-binding protein 3 receptor.

J Biol Chem

272

:

20572

–

20576

Copyright © 1999 by The Endocrine Society

Citations

Views

Altmetric

Metrics

Total Views 700

481 Pageviews

219 PDF Downloads

Since 3/1/2017

×

Email alerts

More on this topic

Related articles in PubMed

Citing articles via

• Latest

• Most Read

• Most Cited

More from Oxford Academic