Insulin-like Growth Factor Receptor Levels Are Regulated by Cell Density and by Long Term Estrogen Deprivation in MCF7 Human Breast Cancer Cells
2001; Elsevier BV; Volume: 276; Issue: 43 Linguagem: Inglês
10.1074/jbc.m105892200
ISSN1083-351X
AutoresRuth Stephen, Lesley E. Shaw, Camilla Larsen, David L. Corcoran, Philippa D. Darbre,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoThis work describes a reciprocal relationship between cell density and levels of insulin-like growth factor receptors (IGFR) in MCF7 human breast cancer cells, which adds a new dimension to the mechanism of cross-talk between estrogen and insulin-like growth factors in the regulation of breast cancer cell growth. The reduced binding of both 125I-IGF1 and αIR3 anti-IGFR antibody to whole cells showed that IGFR are lost from the surface of MCF7 cells as cell density increases, and this occurred irrespective of the presence or absence of estradiol. Western immunoblotting further confirmed loss of type I IGFR from MCF7 cells with increasing cell density. Long term estrogen deprivation was found to increase the levels of IGFR at all cell densities, such that after 96 weeks of estrogen deprivation, IGFR levels had become similar at the highest cell density in the absence of estradiol to the IGFR levels at the lowest cell density in the estrogen-maintained cells, and the levels of IGFR could be increased still further by estradiol. This overexpression of IGFR in the estrogen-deprived cells correlated with a reversal of response to exogenously added ligand, in that concentrations of insulin, IGFI, and IGFII that had stimulated growth of the estrogen-maintained cells became growth inhibitory to the estrogen-deprived cells. Blockade of the IGFIR with the αIR3 anti-IGFR antibody could partially inhibit the growth of the estrogen-deprived cells, suggesting that up-regulation of IGFR in these cells may contribute to the mechanism of adaptation to growth in steroid-deprived conditions which results in progression to estrogen independence of cell growth. This work describes a reciprocal relationship between cell density and levels of insulin-like growth factor receptors (IGFR) in MCF7 human breast cancer cells, which adds a new dimension to the mechanism of cross-talk between estrogen and insulin-like growth factors in the regulation of breast cancer cell growth. The reduced binding of both 125I-IGF1 and αIR3 anti-IGFR antibody to whole cells showed that IGFR are lost from the surface of MCF7 cells as cell density increases, and this occurred irrespective of the presence or absence of estradiol. Western immunoblotting further confirmed loss of type I IGFR from MCF7 cells with increasing cell density. Long term estrogen deprivation was found to increase the levels of IGFR at all cell densities, such that after 96 weeks of estrogen deprivation, IGFR levels had become similar at the highest cell density in the absence of estradiol to the IGFR levels at the lowest cell density in the estrogen-maintained cells, and the levels of IGFR could be increased still further by estradiol. This overexpression of IGFR in the estrogen-deprived cells correlated with a reversal of response to exogenously added ligand, in that concentrations of insulin, IGFI, and IGFII that had stimulated growth of the estrogen-maintained cells became growth inhibitory to the estrogen-deprived cells. Blockade of the IGFIR with the αIR3 anti-IGFR antibody could partially inhibit the growth of the estrogen-deprived cells, suggesting that up-regulation of IGFR in these cells may contribute to the mechanism of adaptation to growth in steroid-deprived conditions which results in progression to estrogen independence of cell growth. insulin-like growth factors insulin-like growth factor receptors estrogen receptor fetal calf serum dextran-charcoal stripped FCS phosphate-buffered saline Tris-buffered saline antibody epidermal growth factor Estrogen regulation of breast cancer cell growth can be modulated by complex interactions with a variety of growth factors, particularly insulin-like growth factors (IGF)1 (1Yee D. Breast Cancer Res. Treat. 1998; 47: 1-302Crossref PubMed Scopus (16) Google Scholar). The extensive clinical literature provides evidence that, when estrogen receptors (ER) are present, estrogen is a main stimulus for growth of breast cancer cells both in vitro (2Lippman M.E. Dickson R.B. Regulatory Mechanisms in Breast Cancer: Advances in Cellular and Molecular Biology of Breast Cancer. Kluwer Academic Publishers, Norwell, MA1990: 1-452Google Scholar) and in vivo (3Miller W.R. Estrogen and Breast Cancer. Chapman & Hall, New York1996: 1-203Google Scholar). IGFs have been implicated in growth regulation of breast cancer cells (4Rasmussen A.A. Cullen K.J. Breast Cancer Res. Treat. 1998; 47: 219-233Crossref PubMed Scopus (82) Google Scholar) since they are mitogenic to such cells (5Cullen K.J. Yee D. Sly W.S. Perdue J. Hampton B. Lippman M.E. Rosen N. Cancer Res. 1990; 50: 48-53PubMed Google Scholar) and also because blockade of the IGF receptor can reduce growth of the cells in the absence of estrogen (6Arteaga C.L. Osborne C.K. Cancer Res. 1989; 49: 6237-6241PubMed Google Scholar). Breast cancer cell lines possess IGFII (7Yee D. Cullen K.J. Paik S. Perdue J.F. Hampton B. Schwartz A. Lippman M.E. Rosen N. Cancer Res. 1988; 48: 6691-6696PubMed Google Scholar), make several of the IGFBPs (8Oh Y. Breast Cancer Res. Treat. 1998; 47: 283-293Crossref PubMed Scopus (92) Google Scholar), and have functional IGF receptors (5Cullen K.J. Yee D. Sly W.S. Perdue J. Hampton B. Lippman M.E. Rosen N. Cancer Res. 1990; 50: 48-53PubMed Google Scholar), both type I (IGFIR) (9Surmacz E. Guvakova M.A. Nolan M.K. Nicosia R.F. Sciacca L. Breast Cancer Res. Treat. 1998; 47: 255-267Crossref PubMed Scopus (84) Google Scholar) and type II (IGFIIR) (10Oates A.J. Schumaker L.M. Jenkins S.B. Pearce A.A. DaCosta S.A. Arun B. Ellis M.J.C. Breast Cancer Res. Treat. 1998; 47: 269-281Crossref PubMed Scopus (101) Google Scholar). At a molecular level, there appear to be many cross-talk pathways between estrogen and IGF (11Nicholson R.I. McClelland R.A. Robertson J.F.R. Gee J.M.W. Endocr. Relat. Cancer. 1999; 6: 373-387Crossref PubMed Scopus (144) Google Scholar, 12Yee D. Lee A.V. J. Mammary Gland Biol. Neoplasia. 2000; 5: 107-115Crossref PubMed Scopus (213) Google Scholar). Estrogen can regulate levels of IGFII mRNA (7Yee D. Cullen K.J. Paik S. Perdue J.F. Hampton B. Schwartz A. Lippman M.E. Rosen N. Cancer Res. 1988; 48: 6691-6696PubMed Google Scholar), IGF-binding protein (8Oh Y. Breast Cancer Res. Treat. 1998; 47: 283-293Crossref PubMed Scopus (92) Google Scholar, 13Perachiotti A. Darbre P. Exp. Cell Res. 1994; 213: 404-411Crossref PubMed Scopus (8) Google Scholar), IGFIR (14Stewart A.J. Johnson M.D. May F.E.B. Westley B.R. J. Biol. Chem. 1990; 265: 21172-21178Abstract Full Text PDF PubMed Google Scholar), and downstream signaling molecules insulin receptor substrates IRS-1 and IRS-2 (15Lee A.V. Jackson J.G. Gooch J.L. Hilsenbeck S.G. Coronado-Heinsohn E. Osborne C.K. Yee D. Mol. Endocrinol. 1999; 13: 787-796Crossref PubMed Scopus (0) Google Scholar, 16Molloy C.A. May F.E.B. Westley B.R. J. Biol. Chem. 2000; 275: 12565-12571Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). On the other hand, IGFs can influence expression of ER (17Clayton S.J. May F.E. Westley B.R. Mol. Cell. Endocrinol. 1997; 128: 57-68Crossref PubMed Scopus (30) Google Scholar) and of estrogen-regulated genes such as pS2 (18Chalbos D. Philips A. Galtier F. Rochefort H. Endocrinology. 1993; 133: 571-576Crossref PubMed Scopus (66) Google Scholar,19Abdul-Wahab K. Corcoran D. Perachiotti A. Darbre P.D. Cell Proliferation. 1999; 32: 271-287Crossref PubMed Scopus (5) Google Scholar). Overexpression studies have shown that increasing limiting components of the IGF pathway can negate estrogen regulation (19Abdul-Wahab K. Corcoran D. Perachiotti A. Darbre P.D. Cell Proliferation. 1999; 32: 271-287Crossref PubMed Scopus (5) Google Scholar, 20Daly R.J. Harris W.H. Wang D.Y. Darbre P.D. Cell Growth Differ. 1991; 2: 457-464PubMed Google Scholar, 21Cullen K.J. Lippman M.E. Chow D. Hill S. Rosen N. Zwiebel J.A. Mol. Endocrinol. 1992; 6: 91-100Crossref PubMed Scopus (81) Google Scholar). Overexpression of IGFII alone could override the need for estrogen in MCF7 human breast cancer cells (20Daly R.J. Harris W.H. Wang D.Y. Darbre P.D. Cell Growth Differ. 1991; 2: 457-464PubMed Google Scholar, 21Cullen K.J. Lippman M.E. Chow D. Hill S. Rosen N. Zwiebel J.A. Mol. Endocrinol. 1992; 6: 91-100Crossref PubMed Scopus (81) Google Scholar) where the limiting component was ligand (20Daly R.J. Harris W.H. Wang D.Y. Darbre P.D. Cell Growth Differ. 1991; 2: 457-464PubMed Google Scholar) and not receptor (22Daws M.R. Westley B.R. May F.E.B. Endocrinology. 1996; 137: 1177-1186Crossref PubMed Scopus (37) Google Scholar), but in ZR-75–1 human breast cancer cells overexpression of IGFII could only override the estrogen requirement when IGFIR (the limiting component) were simultaneously overexpressed (19Abdul-Wahab K. Corcoran D. Perachiotti A. Darbre P.D. Cell Proliferation. 1999; 32: 271-287Crossref PubMed Scopus (5) Google Scholar). The interrelationship between IGFR and ER signaling is shown also in vivo through the tendency for levels of IGFR to correlate positively with levels of ER in breast cancers (3Miller W.R. Estrogen and Breast Cancer. Chapman & Hall, New York1996: 1-203Google Scholar,9Surmacz E. Guvakova M.A. Nolan M.K. Nicosia R.F. Sciacca L. Breast Cancer Res. Treat. 1998; 47: 255-267Crossref PubMed Scopus (84) Google Scholar), and it is thought that abnormally high levels of IGFRs may contribute to increase in tumor mass and/or aid tumor recurrence (9Surmacz E. Guvakova M.A. Nolan M.K. Nicosia R.F. Sciacca L. Breast Cancer Res. Treat. 1998; 47: 255-267Crossref PubMed Scopus (84) Google Scholar). Part of the molecular basis for these cross-talk pathways may relate to an ability of ER to interact with the AP1 pathway (23Paech K. Webb P. Kuiper G.G.J.M. Nilsson S. Gustafsson J.A. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2061) Google Scholar), but the full picture remains far from clear. Although some studies have shown that estrogen can alter IGFR levels (22Daws M.R. Westley B.R. May F.E.B. Endocrinology. 1996; 137: 1177-1186Crossref PubMed Scopus (37) Google Scholar), it is unknown whether this is a mechanism or a consequence of estrogen action on cell growth. Work in other systems has shown that growth factor receptor levels are regulated by cell density (24Clemmons D.R. Elgin R.G. James P.E. J. Clin. Endocinol. & Metab. 1986; 63: 996-1001Crossref PubMed Scopus (17) Google Scholar, 25Scott C.D. Baxter R.C. J. Cell. Physiol. 1987; 133: 532-538Crossref PubMed Scopus (22) Google Scholar, 26Rizzino A. Kazakopp P. Ruff E. Kuszynski C. Nebelsick J. Cancer Res. 1988; 48: 4266-4271PubMed Google Scholar, 27Veomett G. Kuszynski C. Kazakoff P. Rizzino A. Biochem. Biophys. Res. Commun. 1989; 159: 694-700Crossref PubMed Scopus (37) Google Scholar, 28Lichtner R.B. Schirrmacher V. J. Cell. Physiol. 1990; 144: 303-312Crossref PubMed Scopus (48) Google Scholar, 29Hamburger A.W. Mehta D. Pinnamaneni G. Chen L.C. Reid Y. Pathobiology. 1991; 59: 329-334Crossref PubMed Scopus (13) Google Scholar, 30Kuszynski C.A. Miller K.A. Rizzino A. In Vitro Cell. Dev. Biol. Anim. 1993; 29: 708-713Crossref Scopus (7) Google Scholar, 31Mizuno K. Higuchi O. Tajima H. Yonemasu T. Nakamura T. J. Biochem. (Tokyo). 1993; 114: 96-102Crossref PubMed Scopus (32) Google Scholar, 32Pocsik E. Mihalik R. Ali-Osman F. Aggarwal B.B. J. Cell. Biochem. 1994; 54: 453-464Crossref PubMed Scopus (25) Google Scholar, 33Parnas D. Linial M. J. Mol. Neurosci. 1997; 8: 115-130Crossref PubMed Scopus (14) Google Scholar, 34Liu W. Ellis L.M. Pathobiology. 1998; 66: 247-252Crossref PubMed Scopus (13) Google Scholar), and estrogen acts to increase cell density including saturation density (35Darbre P.D. Daly R.J. Proc. R. Soc. Edinb. Sect. B. 1989; 95: 119-132Google Scholar). Since regulation of cell density and contact inhibition are fundamental parameters in cell growth control, we have studied here the effects of cell density on IGFR levels in breast cancer cells grown in the presence and absence of estrogen. A further issue in estrogen growth control of breast cancer cells is the ability of these cells to escape from regulation by estrogen. Loss of response to endocrine therapy is a major problem in the clinical management of breast cancer (3Miller W.R. Estrogen and Breast Cancer. Chapman & Hall, New York1996: 1-203Google Scholar), and such loss of response can be modeled in vitro (36Darbre P.D. Daly R.J. J. Steroid Biochem. Mol. Biol. 1990; 37: 753-763Crossref PubMed Scopus (28) Google Scholar). In cell culture systems, breast cancer cells possess a remarkable ability to adapt to prevailing growth conditions and to escape from any imposed growth inhibition. While maintained in the presence of estradiol, estrogen-sensitive breast cancer cell lines remain growth-regulated by estrogen, in that growth is severely reduced by removal of estrogen or addition of anti-estrogen (37Glover J.F. Irwin J.T. Darbre P.D. Cancer Res. 1988; 48: 3693-3697PubMed Google Scholar). However, upon long term removal of estrogen from the culture medium, the cells gradually adapt, developing an ability to grow without estrogen until they can eventually grow at the same rate as they had done previously only in the presence of estrogen (38Katzenellenbogen B.S. Kendra K.L. Norman M.J. Berthois Y. Cancer Res. 1987; 47: 4355-4360PubMed Google Scholar, 39Welshons W.V. Jordan V.C. Eur. J. Cancer Clin. Oncol. 1987; 23: 1935-1939Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 40Daly R.J. Darbre P.D. Cancer Res. 1990; 50: 5868-5875PubMed Google Scholar). Similar adaptive processes enable the cells to escape from growth inhibition imposed by the anti-estrogens tamoxifen (41Parisot J.P. Hu X.F. DeLuise M. Zalcberg J.R. Br. J. Cancer. 1999; 79: 693-700Crossref PubMed Scopus (76) Google Scholar) and ICI 182,780 (42Jensen B.L. Skouv J. Lundholt B.K. Lykkesfeldt A.E. Br. J. Cancer. 1999; 79: 386-392Crossref PubMed Scopus (41) Google Scholar) or by retinoic acid (43Stephen R. Darbre P.D. Br. J. Cancer. 2000; 83: 1183-1191Crossref PubMed Scopus (12) Google Scholar). The molecular basis for this growth adaptation remains unknown, but recent work has shown that development of estrogen hypersensitivity resulting from up-regulation of ER and increased estrogen-regulated gene expression may be involved (44Jeng M.H. Shupnik M.A. Bender T.P. Westin E.H. Bandyopadhyay D. Kumar R. Masamura S. Santen R.J. Endocrinology. 1998; 139: 4164-4174Crossref PubMed Google Scholar). In this work, we have investigated whether there is any involvement of an IGF cross-talk pathway through development of hypersensitivity to IGF and increased IGFR levels. Stock MCF7 McGrath human breast cancer cells were kindly provided by Dr. K. Osborne at passage number 390 (45Osborne C.K. Hobbs K. Trent J.M. Breast Cancer Res. 1987; 9: 111-121Crossref Scopus (222) Google Scholar) and were dependent on estrogen for growth in monolayer culture as described previously (35Darbre P.D. Daly R.J. Proc. R. Soc. Edinb. Sect. B. 1989; 95: 119-132Google Scholar). Cells were maintained routinely as monolayer cultures in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal calf serum (FCS) (Life Technologies, Inc.), 10−8m 17β-estradiol (Steraloids, Croydon, UK), and 10 µg/ml insulin (Sigma) in a humidified atmosphere of 10% carbon dioxide in air at 37 °C. Estradiol was dissolved in ethanol and diluted 1/10,000 in culture medium. All cell stocks were subcultured at weekly intervals by suspension with 0.06% trypsin, 0.02% EDTA, pH 7.3. A new vial of cells was thawed from liquid nitrogen at the start of each experiment, which ensured that control cells of the starting passage number were available for comparison at any time. Freshly thawed cells were grown for 2 weeks as stock cultures (see above) and then suspended with phenol red-free 0.06% trypsin, 0.02% EDTA, pH 7.3, washed with phenol red-free RPMI 1640 medium (Life Technologies, Inc.) and replated in phenol red-free RPMI 1640 medium containing 5% dextran-charcoal stripped FCS (DCFCS) (46Darbre P. Yates J. Curtis S. King R.J.B. Cancer Res. 1983; 43: 349-354PubMed Google Scholar). Cells were routinely maintained in phenol red-free RPMI 1640 medium with 5% DCFCS, subculturing every 2–3 weeks during the initial period of slow growth, and increasing to subculturing at weekly intervals as the growth rate increased. Cells were suspended from stock plates (either estrogen-maintained or estrogen-deprived) by treatment with phenol red-free 0.06% trypsin, 0.02% EDTA, pH 7.3, added to an equal volume of phenol red-free RPMI 1640 medium (Life Technologies, Inc.) containing 5% DCFCS (36Darbre P.D. Daly R.J. J. Steroid Biochem. Mol. Biol. 1990; 37: 753-763Crossref PubMed Scopus (28) Google Scholar), and counted on a hemocytometer. Cells were then added to the required volume of phenol red-free RPMI 1640 medium containing 5% DCFCS at a concentration of 0.2 × 105 cells/ml and plated in monolayer in 0.5-ml aliquots into 24-well plastic tissue culture dishes (Nunc). After 24 h, the medium was changed to phenol red-free RPMI 1640 medium supplemented with 5% DCFCS and the required concentration of 17β-estradiol, insulin, IGFI (Roche Molecular Biochemicals), IGFII (Bachem), αIR3 antibody (Oncogene Science), or mouse IgG (Sigma). Culture medium was changed routinely every 3–4 days in all experiments. Cell counts were performed by counting released nuclei on a model ZBI Coulter Counter, as described previously (46Darbre P. Yates J. Curtis S. King R.J.B. Cancer Res. 1983; 43: 349-354PubMed Google Scholar). Cells were plated onto 24-well plastic tissue culture dishes in 0.5-ml aliquots in RPMI 1640 medium supplemented with 5% DCFCS at densities from 0.05–0.5 × 105 cells per dish and grown for 5 days. Cells were then washed in RPMI 1640 medium, transferred into serum-free medium (RPMI 1640 medium supplemented with 15 mm HEPES buffer, 0.25% w/v bovine serum albumin (fraction V, Sigma)), and left overnight. After 24 h, three dishes of each treatment were used to estimate cell numbers by Coulter counting as above, and three dishes were used to assay IGFR by radioligand binding assay. For the binding assay, cells were washed twice with ice-cold phenol red-free RPMI 1640 medium and incubated with 80,000 cpm of 125I-IGF-I (specific activity 200–250 Ci/mmol made by the IODO-GEN method (47Salacinski P.R.P. McLean C. Sykes E.C. Clement-Jones V.V. Lowry P.J. Anal. Biochem. 1981; 117: 136-146Crossref PubMed Scopus (1030) Google Scholar)) with or without 30 µm insulin in 0.2 ml of phenol red-free RPMI 1640 medium containing 1 mg/ml bovine serum albumin for 3 h at 4 °C. After incubation, cells were washed 3 times with ice-cold isotonic saline; the cell layer was solubilized in ice-cold 0.5m sodium hydroxide, and incorporated radioactivity was measured in a gamma counter. Since insulin at 30 µmsuppresses only receptor binding and does not bind to binding proteins (48Conover C.A. Endocrinology. 1992; 130: 3191-3199Crossref PubMed Scopus (188) Google Scholar), specific receptor binding was assessed by subtraction of non-receptor binding in the presence of insulin from total binding in the absence of insulin. Results are expressed as fmol125I-IGFI bound per 106 cells. Scatchard analysis was carried out as above but by adding increasing amounts of 125I-IGFI and by using 125I-IGFI radiolabeled to a specific activity of 2000 Ci/mmol (Amersham Pharmacia Biotech). Results are expressed on the y axis as ratios of bound (specific) IGFI to free IGFI and on the x axis as pmol of IGFI-specific binding per 1,000,000 cells per liter (pm). Values for the dissociation constant (K d) were calculated from the slope of the best-fit line. Stock MCF7 cells were plated at varying cell densities onto 22-mm diameter glass coverslips inside 3.5-cm plastic tissue culture dishes in phenol red-free RPMI 1640 medium containing 5% DCFCS either with or without 10−8m 17β-estradiol for 3 days. Cells were fixed on each coverslip in methanol at room temperature for 10 min and then washed in PBS. Each coverslip was incubated for 10 min with 20 µl of normal rabbit serum/80 µl of 0.5 m Tris-buffered saline, pH 7.6 (TBS), and then washed in TBS. Cells were incubated in 100 µl of TBS containing αIR3 antibody at 1:1000 dilution overnight at 4 °C and then washed in 400 ml TBS for 10 min. Cells were incubated in 100 µl of TBS containing biotinylated anti-chicken antibody (Dako) at 1:500 dilution at room temperature for 4 h and then washed in 400 ml of TBS for 10 min. Cells were finally incubated with 100 ml of PBS with streptavidin-fluorescein isothiocyanate conjugate (Dako) at 1:100 dilution at room temperature for 1 h, washed in tap water for 10 min, and wet-mounted using vectoshield sealed with cosmetic nail varnish. Microscopy was performed at once using a Leitz confocal microscope. Stock MCF7 cells were plated at varying densities onto 24-well plastic tissue culture dishes in RPMI 1640 medium supplemented with 5% DCFCS and 10−8m 17β-estradiol. After 5–7 days, 3 wells were counted (see above) to provide the cell density at the time of harvest. Other wells were harvested directly into 26 mm Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 0.01% w/v bromphenol blue. Samples were sonicated and boiled before loading onto SDS-6% polyacrylamide gel electrophoresis. Protein concentrations were determined using the BCA reagent according to manufacturer's instructions (Pierce), and 150 µg of protein was loaded per gel track. Rainbow-colored high molecular weight protein markers in the 14–220-kDa range were loaded as standards to each gel (Amersham Pharmacia Biotech). Proteins were transferred onto Hybond-ECL membranes (Amersham Pharmacia Biotech) by semi-dry Western blotting in 48 mm Tris, 39 mmglycine, 1.3 mm SDS, 20% v/v methanol. The membrane was blocked with 0.1% v/v Tween 20, 5% w/v dried milk in PBS at 4 °C for 18 h, and immunoblotted with a 1:100 dilution of monoclonal mouse anti-human IGFIR antibody (Ab-3, Oncogene Research) in 0.1% v/v Tween 20, 1% w/v dried milk in PBS (PBS-TM) for 30 min at room temperature. The epitope for this antibody is the extracellular domain of the type I IGFR. After washing 6 times for 5 min with 0.1% v/v Tween 20 in PBS (PBS-T), the membrane was incubated with a 1:1000 dilution of biotinylated rabbit anti-mouse antibodies (Dako, Copenhagen, Denmark) in PBS-TM for 30 min at room temperature. The membrane was washed again 6 times for 5 min in PBS-T and then incubated with a 1:1500 dilution of streptavidin-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) in PBS-TM for 30 min at room temperature. Enhanced chemiluminescence was performed according to the manufacturer's instructions, and the signal was detected on ECL-Hyperfilm (Amersham Pharmacia Biotech). MCF7 human breast cancer cells are maintained routinely in the presence of 17β-estradiol in order to maintain sensitivity to estrogen (37Glover J.F. Irwin J.T. Darbre P.D. Cancer Res. 1988; 48: 3693-3697PubMed Google Scholar, 40Daly R.J. Darbre P.D. Cancer Res. 1990; 50: 5868-5875PubMed Google Scholar,43Stephen R. Darbre P.D. Br. J. Cancer. 2000; 83: 1183-1191Crossref PubMed Scopus (12) Google Scholar). Fig. 1shows that these estrogen-maintained MCF7 cells possess receptors capable of binding125I-IGF1. However, following a series of experiments in which the cells were plated at different cell densities, the levels of125I-IGF1 binding were found to drop substantially as the cell density increased (Fig. 1). Levels per cell dropped by around one-fifth as cell numbers increased 10-fold from low density (50,000 cells per well of a 24-well plate) to high density (500,000 cells per well of a 24-well plate). Short term growth of the cells (up to 14 days) in the absence of estradiol resulted in a markedly reduced growth rate (Fig. 2A), but again the levels of 125I-IGF1 binding were found to decrease as cell density increased (Fig. 1). When comparing between cells at equivalent densities, short term removal of estradiol did not appear to have any effect on levels of 125I-IGF1 binding (Fig. 1).Figure 2Effect of long term estrogen deprivation on estrogen-regulated growth of MCF7 human breast cancer cells in monolayer culture. Cells were maintained either as stock cultures in the presence of 10−8m 17β-estradiol (A) or under conditions of estrogen deprivation in phenol red-free RPMI 1640 medium with 5% DCFCS for 68 weeks (B). Short term growth was then assessed in phenol red-free RPMI 1640 medium with 5% DCFCS in the absence of estradiol (open circles) or in the presence of 10−8m 17β-estradiol (closed circles). Bars indicate the standard error of triplicate dishes, and where not seen, the error was too small for visual display.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunofluorescent confocal microscopy was used to confirm and to visualize these effects of cell density on IGFR levels. Fig.3shows the immunofluorescent staining of IGFR localized on the cell membrane. The fluorescent staining was much more intense for cells at low density in small clusters on a culture dish (Fig. 3 A) than for cells at high density (Fig.3 B). When the cells formed an even monolayer on a culture dish, almost no fluorescent staining could be seen anymore (Fig.3 B). When cells were in large monolayer groups but at subconfluence, staining tended to be more intense around the edges of those cells not in contact with adjacent cells. The effects of cell density on IGFR levels were further investigated using Western immunoblotting. Antibody Ab-3 was raised to the extracellular domain of the type I IGFR and accordingly detected a band on Western immunoblotting of 135 kDa in size corresponding to the α-subunits of the IGFIR in MCF7 cells (Fig.4). This band was more intense in cells at low density (Fig. 4, track 1) than at high density (Fig.4, track 3). When MCF7 cells are maintained in the continuous presence of estradiol, the cells remain dependent on estradiol for growth. Short term removal of estradiol results in a much reduced growth rate (Fig.2 A). However, following long term estrogen deprivation, short term growth of the cells is no longer influenced by estradiol (Fig. 2 B). This occurs by a gradual adaptive process in which the cells gradually develop an ability over time to increase their growth rate in the absence of estrogen. A representative time course is shown in Fig. 5. The increase in basal growth rate in the absence of estrogen occurred gradually over a period of 12 weeks such that at the end growth in the absence of estrogen had increased to the same rate as that at which the cells had originally grown only in the presence of estradiol (Fig. 5). This time course is reproducible since the results shown in Fig. 5 have been repeated 3 times for this cell line. Levels of IGFR were found to also increase with increasing periods of estrogen deprivation (Fig. 6). At all time points, the levels of IGFR continued to decrease as cell density increased, but the curves were shifted to higher values as length of estrogen deprivation increased (Fig. 6). When the effect of cell density was taken into account, short term readdition of estradiol now increased levels of IGFR (Fig. 6). Thus, although levels of IGFR had been unaffected in the short term by estradiol in the estrogen-maintained cells (Fig. 1), IGFR levels were now increased by estradiol following estrogen deprivation (Fig. 6). Scatchard analysis was performed in order to ascertain whether there were any alterations in binding affinity following long term steroid deprivation (Fig. 7). However,K d values were similar for estrogen-maintained cells (Fig. 7 A, K d 0.11 nm) after 1 week of estrogen deprivation (Fig. 7 B, K d0.11 nm), after 108 weeks of estrogen deprivation (Fig.7 C, K d 0.13 nm), and after 108 weeks of estrogen deprivation followed by 1 week of estrogen readdition (Fig. 7 D, K d 0.12 nm). The K d values are in line with previously published (49Guvakova M.A. Surmacz E. Exp. Cell Res. 1997; 231: 149-162Crossref PubMed Scopus (128) Google Scholar) values for MCF7 cells assayed using similar methodology. In order to determine whether the increased IGFR levels play any role in the increased basal growth rate of estrogen-deprived cells, we used the αIR3 antibody that has biological blocking activity on IGFIR activity (50Kull F.C. Jacobs S. Su Y.F. Svoboda M.E. vanWyk J.J. Cuatrecasas P. J. Biol. Chem. 1983; 258: 6561-6566Abstract Full Text PDF PubMed Google Scholar). Addition of the αIR3 antibody was able to reduce growth of the estrogen-deprived cells in both the absence of estrogen (Fig. 8p = 0.002 for −E+IgG (track 3)versus −E+αIR3 (track 4)) and the presence of estrogen (Fig. 8 p = 0.006 for+E+IgG (track 6) versus +E+αIR3 (track 7)), although only to a limited extent. This was reproducible over 3 separate experiments using two different batches of αIR3 antibody. Since receptor levels are important in determining cellular sensitivity to ligands, we studied the effects of exogenous insulin, IGFI, and IGFII on the estrogen-deprived cells that had raised IGFR levels. Interestingly, whereas all these ligands stimulated growth of estrogen-maintained cells, all ligands showed a dose-dependent inhibition of growth of the estrogen-deprived cells (Fig. 9). Although insulin increased growth of estrogen-maintained cells at 1 and 10 µg/ml (Fig. 9 A, tracks 3 and 4), growth of the estrogen-deprived cells was inhibited by 10 µg/ml insulin (Fig. 9 B, track 3 (p < 0.001)). Whereas 10 and 100 ng/ml IGFI stimulated growth of the estrogen-maintained cells (Fig. 9 A, tracks 5 and6), 100 ng/ml IGFI became growth-inhibitory to the estrogen-deprived cells (Fig. 9 B, track 5 (p< 0.001)). Whereas 10 and 100 ng/ml IGFII s
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