Insulin-like Growth Factor (1GF)-independent Action of IGF-binding Protein-3 in Hs578T Human Breast Cancer Cells CELL SURFACE BINDING AND GROWTH INHIBITION*

Estrogen receptor-negative Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (1GFBP)-3 and IGFBP-4 as major binding pro- tein (BP) species. Our previous immunohistochemical studies (Oh, Y., Muller, H. L., Pham, H., Lamson, G., and Rosenfeld, R. G. (1992) EndocrinoEogy 131, 3123-3125) have demonstrated the existence of cell surface-associated IGFBP-3 and release of cell sur-face-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 show- ing: 1) a dose-dependent increase of IGFBP-3 binding by of cations (CaCL and MnC12); 2) dose-dependent competition of ‘Z61-IGFBP-3E. by unlabeled IGFBP-3E. (>80% but not by growth of cells.


Insulin-like Growth Factor (1GF)-independent Action
In addition, exogenous IGFBP-3 treatment resulted in a significant inhibitory effect on monolayer growth of Hs678T 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 e 0.05,20 nM;p e 0.005) by exogenous IGFBP-3E.
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 -11 can attenuate inhibitory effect of IGFBP-3 by forming IGF.IGFBP-3 complexes, thereby preventing cell surface binding of IGFBP-3.
Insulin-like growth factors, IGF-I and IGF-11,' are peptide mitogens for multiple cell lines, including human breast cancer cells (1)(2)(3). They share structural similarity with insulin and have their own high affinity receptors on the cell membrane (4). The mitogenic actions of both IGF-I and -11 appear to be mediated through the type 1 IGF receptor, which has high affinity for both IGF-I and -11 and low affinity for insulin (5)(6). In contrast, the type 2 IGF receptor, with high affinity for IGF-I1 and significantly lower affinity for IGF-I, is identical to the mannose 6-phosphate receptor, which is involved in the transport of lysosomal enzymes; its role in mediating IGF action is controversial (7)(8)(9).
The IGFs, but not insulin, also have high affinity for a family of IGF-binding proteins (IGFBPs), six of which have been cloned and sequenced (10)(11)(12)(13)(14)(15). IGFBP-1 through IGFBP-6 are found in many body fluids and in the conditioned media (CM) of a wide variety of cell types, where they modulate IGF peptide activity in a complex manner (16)(17)(18)(19)(20). Although the IGFBPs are presumed to regulate access of IGFs to their receptors, their precise biological roles remain unclear, since both stimulatory and inhibitory actions have been reported under varying conditions (21)(22)(23)(24)(25).
IGFBP-3 is the major binding protein in human serum and serves as a storage depot for IGFs (26), resulting in an increase of the half-life of IGFs in the circulation, and control IGF access to extravascular spaces (26,27). A variety of extrahepatic cell types synthesize and secrete IGFBP-3 (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Thus, the presence of IGFBP-3 in the cellular microenvironment can directly modulate IGF actions. Interestingly, it has been reported that the patterns of IGFBP production in human breast cancer cells correlate with estrogen receptor (ER) status; ER-positive cell lines produce IGFBP-2 as major B P species, whereas ER-negative cells produce IGFBP-3 as the major IGFBP (39). Furthermore, recent reports have demonstrated that purified mouse IGFBP-3 can bind to the chick embryo fibroblast cell surface and inhibit cell growth (40). This IGFBP-3 effect was speculated to be an IGF receptor-independent action (41). In addition, several reports have demonstrated that cell growth inhibitors, such as TGF-P and trans-retinoic acid can increase IGFBP-3 production in human fetal fibroblasts (42) and human breast cancer cells (43). It was speculated that the inhibitory effects on cell growth of these factors were partly mediated through IGFBP-3 action, but whether the IGFBP-3 inhibitory effect is IGF-dependent The abbreviations used are: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; CM, conditioned media; ER, estrogen receptor; HPLC, high performance liquid chromatography; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; Endo-F, endoglycosidase F PBS, phosphate-buffered saline; GAP, GTPase-activating protein; EGF, epidermal growth factor. remains unclear. We have previously reported the cell surface association and dissociation of IGFBP-3 in Hs578T human breast cancer cells. To further characterize the mechanism of IGFBP-3 action in vitro, we have investigated the surface binding and subsequent biological effects of IGFBP-3 in Hs578T human breast cancer cells and have demonstrated that the cell surface binding of IGFBP-3 is specific and divalent cation-dependent and that IGFBP-3 is a cell growth inhibitor whose mechanism is, at least in part, IGF-independent. with Gln, Ala, Tyr, Leu, and Leu substituted for Glu, Thr, Gln, Phe, and Tyr at amino acid positions 6, 7, 18, 19, and 27, respectively, was synthesized as described previously (46). This peptide was designed to have reduced affinity for both the IGFBPs and the type 1 IGF receptor. The product was purified by gel filtration to remove polymeric by-products, followed by additional HPLC steps. Active [Gln6,Ala7,Tyr'8,Leu1y,Leu27]IGF-II was monitored during purification by a type 2 IGF receptor binding assay using rat placental membranes. HPLC purified hIGFBP-1 from human amniotic fluid was kindly provided by Dr. D. R. Powell (Baylor College of Medicine, Houston, TX) (47). Recombinant hIGFBP-3 was the generous gift of Celtrix, Inc. (Santa Clara, CA) (48). Two forms of IGFBP-3 were provided 1) hIGFBP-3'"', a fully glycosylated form expressed in Chinese hamster ovary (CHO) cells, and 2) hIGFBP-3E. a nonglycosylated 29K core protein expressed in Escherichia coli cells. Highly specific rabbit polyclonal antibodies, aIGFBP-3gl and aIGFBP-Bngl, were raised against glycosylated IGFBP-3 and nonglycosylated IGFBP-3, respectively (49, 50). Pooled human sera were prepared and fractionated by Sephadex G-50 acid chromatography to separate IGFBPs from IGF peptides, as described previously (51). Iodination was performed by a modification of the chloramine-T technique, to specific activities of 350-500 pCi/pg for IGF-I and -11 and to 100 pCi/ pg for IGFBP-3E. coli peptides. Human fibronectin was purchased from Calbiochem.

Peptides and
Cell Cultures-The human breast cancer cell line Hs578T was obtained from Dr. Adeline Hackett at Peralta Cancer Research Institute (Oakland, CA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose, 110 mg/liter sodium pyruvate, and 10% fetal bovine serum until 80% confluent and then switched for 12 h to serum-free DMEM. Medium was aspirated again, and cells were maintained in serum-free DMEM with or without study peptides for 72 h, unless specifically indicated in the text. CM were collected and centrifuged at 1000 X g for 10 min to remove cell debris. The harvested CM from triplicate wells within each experiment were pooled and stored at -70 "C until assay.
Membrane Preparation-Crude microsomal Hs578T membranes were prepared as described previously (52). For solubilized membranes, Triton X-100 was added to crude membranes to a final concentration of 1% (v/v). After end-over-end mixing for 18 h at 4 "C, the mixture was centrifuged at 100,000 X g for 1 h and the supernatant frozen at -70 "C until assay.
Western Ligand Blots-Proteins from CM samples and solubilized membranes were size-fractionated by SDS-PAGE under nonreducing conditions and electroblotted onto nitrocellulose filters (0.45 pm pore size) using a Bio Trans unit (Gelman Sciences). Filters were incubated overnight with 1.5 X lo6 cpm of Iz5I-IGF-I or -1GF-11, washed, dried, and exposed to film.
Glycosyhtion Studies-Proteins were deglycosylated with endoglycosidase F (Endo-F) (18). For each sample, 100 p1 of CM, solubilized membranes, or 2 p1 of human serum were heated at 95 "C for 2 min. After cooling to 22 "C, 300 milliunits of Endo-F were added and the pH adjusted to 5.0. Samples were incubated at 37 "C for 3 h and reactions terminated by the addition of electrophoresis buffer, followed by boiling for 2 min. Subsequent Western ligand blots were performed, as described above. For further immunoprecipitations, deglycosylated samples were treated as described above.
1251-IGFBP-3E-' ' I i Cell Binding Assay-Binding assays were performed directly on confluent cell monolayers (0.1 X lo6 cells/well in 24-multiwell plates) after 72-h incubation in CM. Cells were washed three times with cold binding buffer (Hanks' balanced salt solutions without CaCIz and MgClZ, containing 25 mM HEPES, 25 mM Na-HCO,, and 0.5% bovine serum albumin, pH 7.4) and incubated with indicated reagents and 1251-IGFBP-3E. cdi (50,000 cpm) at 15 "C for 3 h, unless otherwise stated. Cells were then washed three times with PBS, pH 7.4, solubilized in 0.6 N NaOH, and counted in a y counter.
Monolayer Cell Replication Assay-Cells were grown in 12-multiwell dishes until 60% confluent (0.2 X lo6 cells/well) and then changed to serum-free media with or without reagents for 72 h. Cells were then gently detached from plates by PBS-EDTA and cell number counted using a hematocytometer. PHlThymidine Incorporation Assay-Cells were grown in 12-multiwell dishes until 80% confluent and media then replaced by serumfree media. Twenty-four h later, variable concentrations of reagents were added. After 23 h, 0.2 pCi of I3H]thymidine (25 Ci/mM; Amersham Corp.) in a volume of 25 p1 were added to each well for a 1.5-h pulse. Cells were harvested, and the rate of DNA synthesis was estimated by measuring the trichloroacetic acid-precipitable radioactivity, as described previously (46).
Statistical Analysis-Data were analyzed with a two-tailed Student's t test, using the software program Statview (Abacus Concept, Inc.).

RESULTS
Characterization of Secreted IGFBP-3 and Membraneassociated IGFBP-3" Fig. 1 presents a Western ligand blot of the IGFBPs present in the CM and solubilized membranes of the human breast cancer cell line Hs578T. Hs578T cells secrete a 41-kilodalton (kDa), 39-kDa, and 24-kDa IGF-binding species as major IGFBPs and a 28-kDa IGF-binding species as a minor BP form. The 41-and 39-kDa IGF-binding species, which were found in both the CM (lane 4) and solubilized membranes (lane 7) are similar to human serum IGFBP-3 (lane 1 ) by migration pattern in ligand blots. Indeed, when CM or solubilized membranes were immunoprecipitated with polyclonal antibodies aIGFBP-3gl and aIGFBP-3ng1, raised against glycosylated recombinant human IGFBP-3 (IGFBP-3CHo) and nonglycosylated IGFBP-3 (IGFBP-3E. coli), respectively (lanes 5-6 and 8-9), only the 41-and 39-kDa species were immunoprecipitated, as was true for human serum IGFBP-3 (lanes 2 and 3 ) . Thus, both antibodies recognized identical IGFBP-3 species in cell membranes and in CM.

IGFBP-3 Is a Cell Growth Inhibitor
To further investigate membrane-associated IGFBP-3, crude microsomal membranes were prepared. Cells were grown in DMEM with 10% fetal bovine serum until 90% confluent. Cells were then washed, changed into new serumcontaining media, and again grown to full confluence. Cells were subsequently washed with ice-cold PBS three times and detached and sonicated. The lysate was centrifuged differentially at 12,000 X g and 40,000 X g, and the resulting membrane pellet was resuspended in 50 mM HEPES, pH 7.4,0.15 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM magnesium sulfate, in a final concentration of 1 mg of membrane protein/ml.  (lanes 6, 7, 9, and 10). Interestingly, a typical 135-kDa a-subunit of the type 1 IGF receptor was not detected under reducing conditions (lanes 1 and 8 ) , indicating that the radiolabeled IGF was preferentially bound to IGFBP-3. How-ever, when samples were incubated in the presence of [Leuz7] IGF-11, which competes for occupancy of the IGFBPs, but not for the type 1 IGF receptor, a typical 135-kDa a-subunit and 270-kDa a-a dimer were readily identified (lanes 11 and 12). Thus, because of the higher binding affinity of IGFBP-3 for IGFs, the type 1 IGF receptor is normally "masked," but can be unmasked by low concentrations of [Leuz7]IGF-11. This competition by membrane-associated IGFBP-3 for receptor binding has undoubtedly caused difficulties in previous attempts to interpret IGF receptor data from Hs578T cells (19) and other cells. Thus, IGF analogs which have high binding affinity only for IGFBPs may be useful in identifying masked IGF receptors. In Fig. 2B, 12sII-IGF-II also bound primarily to membrane associated IGFBP-3 (lanes 1 and 6); addition of excess unlabeled IGF-I revealed a 250-kDa type 2 IGF receptor, presumably by displacing IZ5I-IGF-II from IGFBP-3 (lanes 2 and 3 ) . As expected, incubation with unlabeled IGF-I1 and [LeuZ7]IGF-II displaced radioligand from both type 2 IGF receptors and membrane-associated IGFBP-3, at concentrations of 20 and 200 ng/ml (lanes 4,5, 7, and 8).
These data indicate that IGFBP-3 expressed in Hs578T cells exists as a secreted form in CM and as a membraneassociated form on the cell surface. IGFBP-3 can be identified by its deglycosylation pattern and by immunoprecipitation with IGFBP-3-specific antibodies, aIGFBP-3gl and aIGFBP-3ngl.
Mechanism for Cell Surface Association of IGFBP-3-Our previous immunohistochemical studies have demonstrated that Hs578T cell surface binding of IGFBP-3 was increased by CaC12 and decreased by EDTA and that cell surfaceassociated IGFBP-3 was released into CM following binding of IGF peptide (55,56). To further investigate the factors that regulate IGFBP-3 binding on the cell surface, we performed binding assays to Hs578T cell monolayers using Iz5-I-IGFBP-3". colr . to the cell surface was increased by CaC12 and MnCI2 in a dose-dependent manner, with an approximately 1.5-fold increase following coincubation with CaC12 at a concentration of 1 mM ( p < 0.001) and a 3.5-fold increase following coincubation with 10 mM MnC12 ( p < 0.001). These findings indicate that divalent cations facilitate IGFBP-3 binding to the cell surface and suggest that IGFBP-3 binding is a specific and receptor-mediated event, such as the interaction of fibronectin with its receptor (57). Further characterization of specific IGFBP-3 binding to the cell surface was performed in the presence of 1 mM CaC12, as demonstrated in Fig. 3B. Because of the limited supply of recombinant human IGFBP-3, we used partially purified human serum IGF binding protein fractions derived from G-50 acid column chromatography to determine nonspecific binding of IGFBP-3 (51). As can be seen in the inset, the competition by serum IGFBP fractions was dose-dependent; the maximum decrease in labeled IGFBP-3". 'O" bound to the cell surface was observed with 10% serum IGFBP fractions. Therefore, nonspecific binding was determined in the presence of 10% human serum IGFBP fractions from G-50 acid chromatography. Fig. 3R shows that 3, with an ICso of 12 nM and with >80% competition at 100 nM. When purified human IGFRP-1 and human fibronectin were used to compete with labeled IGFBP-3". '"'li, no competition was observed at concentrations up to 50 nM for IGFBP-1 and 20 nM for fibronectin.
Cell Growth Inhibition by IGFBP-3"Further studies were performed to investigate the biological significance of IGFRP-3 binding to the cell surface. We have demonstrated previously that insulin, IGF-I, and IGF-I1 did not stimulate proliferation of Hs578T cells, despite their potent mitogenic effects on other breast cancer cell lines (1)(2)(3) and speculated that the absence of IGF effects could reflect interference by endogenous IGFBPs. Therefore, we tested the effect of endogenously secreted IGFBP-3 and exogenously added IGFBP-3"-'"" on Hs578T cell proliferation by using the IGF-I analog, [Gln3,Ala4,Tyr15,Leu16]IGF-I. As shown in Fig. 4A, neither IGF-I nor [Gln3,Ala4,Tyr's,Leu'fi]IGF-I, which has significantly decreased affinity for IGFBPs, but retains full affinity for IGF receptors, stimulated cell growth at concentrations up to 20 nM. This implies that the absence of IGF effects on cell growth is not due to interference by endogenously secreted IGFBPs. Nevertheless, when exogenous IGFBP-3". ''I' was added, it showed a significant inhibitory effect on monolayer growth of Hs578T cells (10 and 20 nM; p < 0.005). This inhibitory effect of exogenous IGFBP-3". '"Ii was dose-dependent, with 60% inhibition at a concentration of 20 nM. Cell growth inhibition was specific for IGFBP-3; exogenous IGFBP-1 showed no significant inhibitory effect at concentrations up to 20 nM. In addition, the inhibitory effect of IGFBP-3 could be demonstrated on DNA synthesis, using a [3H]thymidine incorporation assay, as shown in Fig. 4R. As expected, IGFBP-3".
To identify the mechanism for this inhibitory effect of IGFBP-3 on monolayer growth, and determine whether it was IGF-dependent or -independent, native IGF-I or These data indicate that the IGFBP-3 inhibitory effect on cell growth does not result from blocking the mitogenic actions of IGF-I by preventing its binding to IGF receptors. Rather, the results suggest a cell surface-specific action of IGFBP-3, itself. Our data also demonstrate that this IGFBP-3 inhibitory effect on cell growth can be diminished by formation of an 1GF.IGFBP-3 complex. IGF analogs with decreased affinity for IGFBP-3 therefore cannot attenuate the inhibitory effect of IGFBP-3.
'"" binding to Hs578T monolayers. Cells were incubated for 3 h at 15 'C with 50, 000 cpm lZ5I-IGFBP-3". mf' in the presence of various concentrations of CaCI2 and MgCI2. Cells were then washed, solubilized, and cell-associated radioactivity determined. Results   As can be seen, in the absence of IGFBP-3, both native IGF-I1 and [Gln6,Ala7,Tyr'8,Leu'g,Leu*7]IGF-II, in concentrations up to 100 nM, have no effect on monolayer growth. The similar results seen with IGF-I1 and an IGF-I1 analog which does not bind to IGFBPs again indicates that the failure of IGF to stimulate Hs578T cells cannot be attributed to interference by endogenous IGFBPs. As shown previously, exogenous IGFBP-3 is inhibitory ( p < 0.005), and this effect can be partially blocked by coincubation of IGFBP-3 with an IGF peptide (IGF-11). However, when exogenous IGFBP-3 is coincubated with [Gln6,Ala7,Tyr'8,Leu'g,Leu27]IGF-II, which has markedly reduced affinity for the binding protein, there is no attenuation of the inhibitory effects of IGFBP-3.
Our data thus clearly demonstrate that IGFBP-3, itself, can^ inhibit Hs578T cell monolayer growth and that IGF-I and IGF-I1 can attenuate this IGFBP-3 inhibitory effect by formation of IGF. IGFBP-3 complexes, thereby preventing cell surface binding of IGFBP-3.

DISCUSSION
Our previous studies demonstrated cell surface-associated IGFBP-3 in Hs578T cells, by use of cell monolayer affinity cross-linking and by immunoperoxidase staining of the cell surface with anti-IGFBP-3 antibodies. Nonreceptor-mediated post-translational regulation of IGFBP-3 by IGF peptides resulted from the release of cell surface-associated IGFBP-3 into CM by the binding of IGFs to IGFBP-3 (55). These results additionally indicated that the cell surface binding of IGFBP-3 is specific and is facilitated by divalent cations in Hs578T human breast cancer cells. Our current studies further demonstrate that IGFBP-3, itself, is a cell growth inhibitor, whose mechanism is IGF-independent.
Several investigators have previously postulated IGFBP-3 binding on the cell surface of different cell lines, such as bovine dermal fibroblasts (28), human neonatal fibroblasts (58), chick embryo fibroblasts (59), rat Sertoli cells (60), and Hs578T human breast cancer cells (55). This cell surface binding of IGFBPs was also observed with IGFBP-1 in porcine aortic smooth muscle cells (61) and IGFBP-5 in human fetal dermal fibroblast cells (62). The mechanism of the cell surface binding for IGFBP-5 is unclear, but the association of IGFBP-1 was speculated to result from membrane receptors of the integrin protein family, which would recognize the arginine-glycine-aspartic acid (RGD) tripeptide sequence in show no competition for IGFBP-3 cell surface binding, indicating that the mechanism of IGFBP-3 cell surface binding is not mediated through fibronectin receptors and is different from that of IGFBP-1. Recently, Baxter and co-workers (58) reported that heparin, which releases proteins attached to cell surface proteoglycans, could displace IGFBP-3 binding on human neonatal fibroblast cell surfaces. They suggested that the association of IGFBP-3 with fibroblasts occurs as a result of interaction with a proteoglycan in the membrane or matrix of the cell, rather than interaction with a specific receptor for IGFBP-3 (58). It is likely that negatively charged heparin can bind to IGFBP-3, consequently preventing IGFBP-3 binding to the cell surface, mimicking the effect of IGFs on IGFBP-3 binding to the cell surface. It should be noted that even though fibronectin (64) and fibroblast growth factors (65) bind to cell surface proteoglycans through their heparin binding domain, they also interact with their own specific receptors on the cell surface, indicating the existence of multiple binding sites, which may also be true for IGFBP-3.
Recently, Hare1 and co-workers (40) reported that mouse IGFBP-3 (mIGFBP-3) inhibited DNA synthesis and growth of mouse 3T3 fibroblasts and chick embryo fibroblasts. Stimulation of DNA and RNA synthesis, not only by IGF-I, but also by serum and phorbol-12-myristate-13-acetate, was inhibited by IGFBP-3 (40). Further studies showed that mIGFBP-3 can bind to chick embryo fibroblasts membranes with low affinity. Binding of IGFBP-3 to the membrane was specific, and binding sites per cell were estimated at 60,000 (59). We have recently reported that the growth of human IGFBP-3 transfected mouse Balb/c fibroblast cells is significantly slower (2.5-fold) than in control cells transfected with vector alone (66). When transfected cells were grown in insulin-containing media ( 5 Fg/ml), growth rates of the IGFBP-3 transfected cells were not restored to control levels, Exogenous Type 2 Insulin FIG. 6. Schematic diagram of IGFBP-3 interactions with receptors and IGFs on Hs678T cells. The activated oncogenic ras protein in Hs578T cells overides the cell proliferation effects of insulin and IGFs by escaping from down-regulation by GAP, which is tightly regulated by insulin and type 1 IGF receptors. On the other hand, IGFBP-3 binds to membrane-associated proteins and exerts its cell growth inhibitory effects. These inhibitory effects can be attenuated by the binding of IGFs resulting in formation of IGF. IGFBP-3 and preventing the association of IGFBP-3 with cell surface. even though the expressed IGFBP-3 does not bind insulin. These results suggest that exogenous or endogenous IGFBP-3 has inhibitory effects on cell growth, which, as in our studies, may be IGF-independent. However, such experiments were performed in non-human models, using IGFBP-3 from a different species; additionally, fibroblasts have true IGF effects, mediated through IGF receptors. Therefore, it is difficult to determine whether the IGFBP-3 inhibitory effects derive entirely from the inhibition of IGF-dependent actions (e.g. by sequestration of the IGF peptides) or are independent events.
Accordingly, the Hs578T human breast cancer cell system is an excellent model for IGFBP action. These cells are not stimulated by IGF-I, IGF-11, or IGF analogs. It is of note that Hs578T cells contain the Harvey (H)-ras oncogene, possessing the genetic alteration of a substitution of aspartic acid for glycine at position 12 (67). Ras proteins are part of a large family of guanosine triphosphatases (GTPases) whose function is to act as a switch along essential cellular pathways (68). Recent reports have demonstrated that GTPase-activating protein (GAP) becomes physically associated with, and phosphorylated by, the activated tyrosine kinase-containing growth factor receptors, such as the insulin receptor (69), platelet-derived growth factor receptor (70), and EGF receptor (71). After stimulation with ligands, there is an inhibition of GAP activity, resulting in the accumulation of ras bound to GTP. The point mutation in Hs578T ras protein leads to an oncogenic protein, with a major biochemical change in the ras protein: impaired GTPase activity (72). Therefore, the oncogenic Hs578T ras protein constitutively remains in the active GTP-bound form, resulting from its escape from down-regulation by GAP.
As schematized in Fig. 6, it is tempting to speculate that the ras protein is a dominant downstream element in tyrosine kinase-containing receptor pathways. The activated oncogenic ras protein overides the cell proliferative effects of insulin (3), IGFs (3), and EGF (73) in Hs578T cells. The lack of responsiveness of these cells to IGF-I thus enables one to investigate the IGF-independent actions of IGFBP-3. The inhibition of Hs578T cell replication by IGFBPs is IGFBP-3-specific and appears to result from cell surface association of IGFBP-3. Indeed, we are faced with the interesting situation where rather than IGFBPs modulating IGF actions, IGF peptides attenuate the inhibitory effects of IGFBP-3, by forming an 1GF.IGFBP-3 complex and thereby preventing cell association of the binding protein (55).
It is of note that there is a strong correlation between IGFBP-3 production and estrogen receptor status in human breast cancer cells (39). In addition, TGF-P (74) and transretinoic acid (43,75) inhibit breast cancer cell growth. TGF-/ 3 production is inhibited by estrogen and insulin treatment, but growth-inhibitory anti-estrogens and glucocorticoids strongly stimulate its secretion (76). Interestingly, these Hs578T inhibitors stimulate IGFBP-3 production in human fetal fibroblasts (58) and MCF-7 ER-positive breast cancer cells (43). It is reasonable to speculate that these inhibitory effects are, in part, mediated through IGFBP-3-specific actions, in addition to the blocking of IGF actions. In this respect, the production and proteolysis of IGFBP-3 as an inhibitor is tightly regulated to balance net growth rate in vivo.