HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 43, 40080-40086, October 26, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/43/40080    most recent M105892200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stephen, R. L. Articles by Darbre, P. D. Search for Related Content PubMed PubMed Citation Articles by Stephen, R. L. Articles by Darbre, P. D. Social Bookmarking                      What's this?

Insulin-like Growth Factor Receptor Levels Are Regulated by Cell Density and by Long Term Estrogen Deprivation in MCF7 Human Breast Cancer Cells^*

Ruth L. Stephen, Lesley E. Shaw, Camilla Larsen, David Corcoran, and Philippa D. Darbre

From the Division of Cell and Molecular Biology, School of Animal and Microbial Sciences, the University of Reading, Whiteknights, P. O. Box 228, Reading RG6 6AJ, United Kingdom

Received for publication, June 25, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 IR3 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 IR3 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (1). 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 (2) and in vivo (3). IGFs have been implicated in growth regulation of breast cancer cells (4) since they are mitogenic to such cells (5) and also because blockade of the IGF receptor can reduce growth of the cells in the absence of estrogen (6). Breast cancer cell lines possess IGFII (7), make several of the IGFBPs (8), and have functional IGF receptors (5), both type I (IGFIR) (9) and type II (IGFIIR) (10).

At a molecular level, there appear to be many cross-talk pathways between estrogen and IGF (11, 12). Estrogen can regulate levels of IGFII mRNA (7), IGF-binding protein (8, 13), IGFIR (14), and downstream signaling molecules insulin receptor substrates IRS-1 and IRS-2 (15, 16). On the other hand, IGFs can influence expression of ER (17) and of estrogen-regulated genes such as pS2 (18, 19). Overexpression studies have shown that increasing limiting components of the IGF pathway can negate estrogen regulation (19-21). Overexpression of IGFII alone could override the need for estrogen in MCF7 human breast cancer cells (20, 21) where the limiting component was ligand (20) and not receptor (22), 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 (19). 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 (3, 9), and it is thought that abnormally high levels of IGFRs may contribute to increase in tumor mass and/or aid tumor recurrence (9). Part of the molecular basis for these cross-talk pathways may relate to an ability of ER to interact with the AP1 pathway (23), but the full picture remains far from clear. Although some studies have shown that estrogen can alter IGFR levels (22), 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 (24-34), and estrogen acts to increase cell density including saturation density (35). 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 (3), and such loss of response can be modeled in vitro (36). 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 (37). 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 (38-40). Similar adaptive processes enable the cells to escape from growth inhibition imposed by the anti-estrogens tamoxifen (41) and ICI 182,780 (42) or by retinoic acid (43). 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 (44). 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stock Culture of MCF7 Human Breast Cancer Cells-- Stock MCF7 McGrath human breast cancer cells were kindly provided by Dr. K. Osborne at passage number 390 (45) and were dependent on estrogen for growth in monolayer culture as described previously (35). 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.), 10⁸ M 17-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.

Long Term Estrogen Deprivation of Cells-- 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) (46). 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.

Cell Growth Experiments-- 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 (36), 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 17-estradiol, insulin, IGFI (Roche Molecular Biochemicals), IGFII (Bachem), IR3 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 (46).

Measurement of IGF Receptors-- 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 (47)) 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 0.5 M sodium hydroxide, and incorporated radioactivity was measured in a gamma counter. Since insulin at 30 µM suppresses only receptor binding and does not bind to binding proteins (48), 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 fmol ¹²⁵I-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.

Immunocytochemistry-- 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 10⁸ M 17-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 IR3 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.

Western Immunoblotting-- Stock MCF7 cells were plated at varying densities onto 24-well plastic tissue culture dishes in RPMI 1640 medium supplemented with 5% DCFCS and 10⁸ M 17-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 mM glycine, 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of Cell Density on IGFR Levels-- MCF7 human breast cancer cells are maintained routinely in the presence of 17-estradiol in order to maintain sensitivity to estrogen (37, 40, 43). Fig. 1 shows that these estrogen-maintained MCF7 cells possess receptors capable of binding ¹²⁵I-IGF1. However, following a series of experiments in which the cells were plated at different cell densities, the levels of ¹²⁵I-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).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of cell density on IGFR levels in estrogen-responsive MCF7 human breast cancer cells in monolayer culture. Stock estrogen-maintained cells were plated at varying densities and grown for 5 days in phenol red-free RPMI 1640 medium containing 5% DCFCS either without estradiol (open circles) or with 108 M 17--estradiol (closed circles). IGFR levels are expressed as fmol of 125I-IGF1 bound per 106 cells.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Effect 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 108 M 17-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 108 M 17-estradiol (closed circles). Bars indicate the standard error of triplicate dishes, and where not seen, the error was too small for visual display.

Immunofluorescent confocal microscopy was used to confirm and to visualize these effects of cell density on IGFR levels. Fig. 3 shows 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. 3A) than for cells at high density (Fig. 3B). When the cells formed an even monolayer on a culture dish, almost no fluorescent staining could be seen anymore (Fig. 3B). 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.

View larger version (92K): [in this window] [in a new window]   Fig. 3.   Effect of cell density on immunofluorescent staining of IGFR in estrogen-responsive MCF7 human breast cancer cells in monolayer culture as viewed by confocal microscopy. Photographs of cells at low (A) and high (B) density to show immunofluorescent staining of IGFR using IR3 antibody overlaid onto a Nomarsky image of the same field of view.

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 -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).

View larger version (76K): [in this window] [in a new window]   Fig. 4.   Effect of cell density on IGFIR levels in estrogen-responsive MCF7 human breast cancer cells in monolayer culture as assayed by Western immunoblotting. Cells were grown in stock culture medium containing estradiol and insulin and were harvested at a density on 24-well dishes of 1.46 ± 0.01 × 105 cells/well (low density) (track 1), 4.21 ± 0.09 × 105 cells/well (track 2), and 8.54 ± 0.08 × 105 cells/well (high density) (track 3). 150 µg of protein was loaded per well and immunoblotted using the Ab-3 antibody for which the epitope is the extracellular domain of the type I IGFR. Position of the 97-kDa protein marker is indicated. The 220-kDa protein marker was also seen on the gel but was located above the top of this photograph.

Effect of Estrogen Deprivation on Cell Growth and IGFR Levels-- 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. 2A). However, following long term estrogen deprivation, short term growth of the cells is no longer influenced by estradiol (Fig. 2B). 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.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Time course of the changes in growth response to estradiol in MCF7 human breast cancer cells following long term estrogen deprivation. Cells were grown for increasing periods of time under conditions of estrogen deprivation (phenol red-free RPMI 1640 medium with 5% DCFCS), and at varying time points cells were assayed for short term growth over 14 days in the absence (open circles) or presence of 108 M 17-estradiol (solid circles). Results are expressed as the number of cell doublings in 14 days, and bars represent the standard error of all 9 possible combinations of triplicate counts at day 0 and day 14. Where error bars are not seen, the error was too small for visual display.

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).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of long term estrogen deprivation on cell density regulation of IGFR levels in MCF7 human breast cancer cells in monolayer culture. Cells were grown for increasing periods of time under conditions of estrogen deprivation (phenol red-free RPMI 1640 medium with 5% DCFCS) and assayed after 8 (circles), 52 (triangles), and 96 (squares) weeks for IGFR levels by plating at varying densities and growing for 5 days in phenol red-free RPMI 1640 medium containing 5% DCFCS either without estradiol (open symbols) or with 108 M 17-estradiol (closed symbols). IGFR levels are expressed as fmol 125I-IGF1 bound per 106 cells.

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. 7A, K_d 0.11 nM) after 1 week of estrogen deprivation (Fig. 7B, K_d 0.11 nM), after 108 weeks of estrogen deprivation (Fig. 7C, K_d 0.13 nM), and after 108 weeks of estrogen deprivation followed by 1 week of estrogen readdition (Fig. 7D, K_d 0.12 nM). The K_d values are in line with previously published (49) values for MCF7 cells assayed using similar methodology.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Scatchard analysis of the binding characteristics of 125I-IGFI to MCF7 human breast cancer cells in monolayer culture after increasing periods of estrogen deprivation. Estrogen-maintained cells were grown for 7 days in phenol red-free RPMI 1640 medium containing 5% DCFCS either with 108 M estradiol (A) or without estradiol (B). Cells were grown for 108 weeks under conditions of estrogen deprivation (phenol-red-free RPMI 1640 medium with 5% DCFCS) (C) or for 108 weeks under conditions of estrogen deprivation followed by 7 days in the same medium but with 108 M estradiol added back (D). Cells were assayed in 24-well dishes at a final density of 3.9 × 105 cells/well (A), 3.1 × 105 cells/well (B), 3.6 × 105 cells/well (C), and 3.1 × 105 cells/well (D). Values for the dissociation constant Kd were calculated as 0.11 (A), 0.11 (B), 0.13 (C), and 0.12 nM (D).

Effect of IGFR Blockade on Growth of Estrogen-deprived Cells-- 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 IR3 antibody that has biological blocking activity on IGFIR activity (50). Addition of the IR3 antibody was able to reduce growth of the estrogen-deprived cells in both the absence of estrogen (Fig. 8 p = 0.002 for E+IgG (track 3) versus E+IR3 (track 4)) and the presence of estrogen (Fig. 8 p = 0.006 for +E+IgG (track 6) versus +E+IR3 (track 7)), although only to a limited extent. This was reproducible over 3 separate experiments using two different batches of IR3 antibody.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of IR3 antibody on growth of estrogen-deprived MCF7 human breast cancer cells. Cells were grown for 21 weeks under conditions of estrogen deprivation (RPMI 1640 medium with 5% DCFCS) and were then assayed for growth over 7 days in RPMI 1640 medium, 5% DCFCS without estradiol (E) (tracks 2-4) or in the presence of 108 M 17-estradiol (+E) (tracks 5-7) and as indicated with 5 µg/ml IgG (tracks 3 and 6) or 5 µg/ml aIR3 antibody (tracks 4 and 7). The plating density of the experiment is indicated (PE) (track 1). Bars indicate the standard error of triplicate dishes, and where not seen, the error was too small for visual display. *, p = 0.002 track 4 versus track 3; **, p = 0.006 track 7 versus track 6.

Sensitivity of Estrogen-deprived Cells to Insulin and IGFII-- 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. 9A, tracks 3 and 4), growth of the estrogen-deprived cells was inhibited by 10 µg/ml insulin (Fig. 9B, track 3 (p < 0.001)). Whereas 10 and 100 ng/ml IGFI stimulated growth of the estrogen-maintained cells (Fig. 9A, tracks 5 and 6), 100 ng/ml IGFI became growth-inhibitory to the estrogen-deprived cells (Fig. 9B, track 5 (p < 0.001)). Whereas 10 and 100 ng/ml IGFII stimulated growth of the estrogen-maintained cells (Fig. 9A, tracks 7 and 8), 100 ng/ml IGFII became growth-inhibitory to the estrogen-deprived cells (Fig. 9B, track 7 (p < 0.001)). The inhibitory action of insulin has been reproducible over at least 4 separate experiments. The inhibitory action of IGFI and IGFII has been repeated twice with similar results.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Effect of long term estrogen deprivation on the insulin, IGFI, and IGFII sensitivity of growth in MCF7 human breast cancer cells. Cells were maintained either as stock cultures in the presence of 108 M 17-estradiol (A) or under conditions of estrogen deprivation in phenol red-free RPMI 1640 medium with 5% DCFCS for 61 weeks (B). Short term growth was then assessed over 12 (A) or 7 days (B) in phenol red-free RPMI 1640 medium, 5% DCFCS with no addition () (A, track 2, and B, track 2), with insulin (Ins) at 1.0 (A, track 3) or 10 µg/ml (A, track 4, and B, tracks 3 and 4), with IGFI at 10 (A, track 5) or 100 ng/ml (A, track 6, and B, tracks 5 and 6), with IGFII at 10 (A, track 7) or 100 ng/ml (A, track 8, and B, tracks 7 and 8), or with 108 M 17-estradiol (E) (A, track 9, and B, track 9). The effect of addition of IR3 antibody at 5 µg/ml was assayed in tracks B4, B6, and B8. The plating density of the experiments is indicated (PE) (A, track 1, and B, track 1). Bars indicate the standard error of triplicate dishes, and where not seen, the error was too small for visual display.

Studies using the IR3 antibody confirmed previously published work (5) that IR3 inhibits growth stimulation by IGFI and IGFII but not by insulin in the estrogen-maintained cells (data not shown). However, addition of IR3 could reverse the inhibitory action by all three ligands (10 µg/ml insulin, 100 ng/ml IGFI, and 100 ng/ml IGFII) in the estrogen-deprived cells (Fig. 9B, tracks 3-8). Addition of IR3 removed the inhibitory action of 10 µg/ml insulin (p = 0.55 for Fig. 9B, track 4 versus track 2) and of 100 ng/ml IGFII (p = 0.10 for Fig. 9B, track 8 versus track 2). Addition of IR3 reduced the inhibitory effect of IGFI significantly (p = 0.001) but did not abolish the effect (p < 0.001 Fig. 9B, track 6 versus track 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results presented here demonstrate a reciprocal relationship between levels of IGFR and cell density during the growth of MCF7 human breast cancer cells in monolayer culture. IGFR levels were found to decrease with increasing cell density irrespective of the presence or absence of estradiol and irrespective of the estrogen responsiveness of the cells. Regulation of cell density by contact inhibition and anchorage dependence is fundamental to cell growth control, and down-regulation of receptor for a growth stimulator with increasing cell density would provide a logical contributory mechanism. The reduced binding of both ¹²⁵I-IGF1 and IR3 anti-IGFR antibody to whole cells suggests that IGFR are indeed lost from the MCF7 cell surface at increasing density. Western immunoblotting showed that cellular levels of type I IGFR were reduced with increasing density.

Although alteration of IGFR levels with cell density has not been described previously in human breast cancer cells, the phenomenon has been well documented for other receptors in other cell lines. Levels of epidermal growth factor (EGF) receptors (26, 28-30), transforming growth factor- receptors (26), platelet-derived growth factor receptors (26), fibroblast growth factor receptors (26, 27), IGF type II receptor (25), hepatocyte growth factor receptor (31), tumor necrosis factor receptor (32), ciliary neurotrophic factor receptor (33), and vascular endothelial growth factor receptor (KDR) (34) have all been shown to be reduced as cell density increases. Furthermore, a similar trend has been shown for growth factor-binding proteins (IGF-binding protein (51); heparan sulfate proteoglycans (52)) and for downstream signaling components (from EGF receptors (53); from transforming growth factor- receptors (54); from interleukin 1 receptor (55)), suggesting that whole signal transduction pathways can be down-regulated with increasing cell density. Density-dependent effects on EGF receptors have been shown to operate through extensive cell-cell adhesion that is mediated by excess E-cadherin (56), and in this respect it is interesting that overexpression of IGFIR in MCF7 cells has been reported to induce E-cadherin-mediated cell-cell adhesion (49). It is also interesting to note that regulation of the ErbB-2 protein is different, showing increasing activity in confluent cultures (57), and overexpression of this EGF receptor family member has been correlated with a worse prognosis in breast cancer (58). This regulation by cell density may explain the differing levels of IGFR reported in breast cancer cells in the literature (59) since the IGFR measurements may simply have been made on cells at different densities. Cell density will now need to be taken into account in any future assessments of cellular IGFR levels and particularly in studies of regulation where the regulator might alter cell density through effects on proliferation or apoptosis.

When taking cell density into account, we could not find any short term (5 days) regulation of IGFR levels by estradiol in the stock estrogen-maintained cells (Fig. 1). It was only following long term estrogen deprivation when we observed any short term estrogen regulation of IGFR levels (Fig. 6). Although IGFR levels have been reported to be dependent on estrogen (9, 14), this has not been a universal finding in all reports (41). Although results presented here suggest that discrepancies may be explained by lack of consideration to cell density in assays, another explanation could lie with the different culture conditions used. Our MCF7 cells are maintained routinely in the presence of estradiol as well as insulin (see "Experimental Procedures"), whereas other studies use only insulin, and memory effects from exposure to estrogen are known to last in cells for up to 14 days (37). Such memory effects have been interpreted to relate to the time taken to lose patterns of estrogen-induced gene expression and specifically to the half-lives of estrogen-regulated mRNAs and proteins in the cells. While some mRNAs such as c-Myc can be up- and down-regulated in minutes by estradiol (60), other estrogen-regulated mRNAs such as pS2 can take several days to down-regulate after estrogen removal (61). These studies suggest that the cells need to be deprived of estrogen for several weeks in order to measure any dependence of IGFR on estrogen.

However, long term estrogen deprivation had the effect not only of revealing an estrogen regulation of IGFR but also of generally increasing IGFR levels in the cells. As length of estrogen deprivation increased from 8 to 52 to 96 weeks, the general levels of IGFR increased in the cells, and the short term estrogen regulation of the IGFR became more marked. It is interesting to note that in the 96-week deprived cells, the 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. This up-regulation of IGFR may be a further part of multiple processes of adaptation to growth without estrogen described by others (44) to involve up-regulation of ER and increased ER target gene activation. Such raised levels of ER in the cells have been suggested to be important in enabling the cells to respond to reduced environmental levels of estrogen (44). In an analogous way, raised IGFR levels may enable the cells to respond to the reduced IGF levels of the DC-stripped FCS or low levels of IGFII produced endogenously (20). Transfection experiments have already demonstrated the importance of adequate IGFR levels in the response of breast cancer cells to IGFII in cell culture (19) and that increased levels of transfected IGFR can alter sensitivity of breast cancer cells to exogenously added IGFs (22). Furthermore, overexpression of fibroblast growth factor receptor in breast cancer cells has been shown to reverse responses to added fibroblast growth factor from growth stimulatory to growth inhibitory (62). Interestingly, overexpression of IGFR in the estrogen-deprived cells here also correlated with a reversal of responses to exogenously added ligand, in that insulin, IGFI, and IGFII also became growth inhibitory rather than growth stimulatory, and this growth inhibition could be reduced with the IR3 antibody that has biological blocking activity on the IGFIR function (50).

The fact that blockade of the IGFIR with the IR3 antibody could, at least partially, inhibit the growth of the estrogen-deprived cells suggests that this up-regulation of IGFR must serve some function in the loss of dependence on estrogen for growth. However, it is clearly not the whole story since the IR3 did not block all the growth. Likewise, previous authors (63) have felt that increase in androgen receptor in LNCaP prostate cancer cells and increase in ER in MCF7 human breast cancer cells (44) could only partially explain increased androgenic and estrogenic activities, respectively. It is therefore possible that overall mechanisms of adaptation to growth in steroid-deprived conditions involve multiple cross-talk pathways rather than a single pathway. Specifically, up-regulation of ER (44) and IGFR (reported here) simultaneously in MCF7 cells may interact in the adaptive processes during estrogen deprivation, through the ability of ER to increase components of IGFR signaling (7, 8, 13-16) and to influence AP1-mediated pathways (23), and the ability of IGFR to influence levels of ER (17) and of estrogen-regulated gene expression (18, 19).

	ACKNOWLEDGEMENT

We thank E. Hales for technical assistance.

	FOOTNOTES

^* This work was supported by the Felix Foundation (to R. S.), the Association for International Cancer Research (to D. C.), and the Breast Cancer Campaign (to L. E. S. and P. D. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-118-9875123 (Ext. 7035/7025); Fax: 44-118-9310180; E-mail: p.d.darbre@reading.ac.uk.

Published, JBC Papers in Press, July 16, 2001, DOI 10.1074/jbc.M105892200

	ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factors; IGFR, insulin-like growth factor receptors; ER, estrogen receptor; FCS, fetal calf serum; DCFCS, dextran-charcoal stripped FCS; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; Ab, antibody; EGF, epidermal growth factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				D. Alvaro, G. Macarri, M. G. Mancino, M. Marzioni, M. Bragazzi, P. Onori, S. G. Corradini, P. Invernizzi, A. Franchitto, A. F. Attili, et al. Serum and Biliary Insulin-like Growth Factor I and Vascular Endothelial Growth Factor in Determining the Cause of Obstructive Cholestasis Ann Intern Med, October 2, 2007; 147(7): 451 - 459. [Abstract] [Full Text] [PDF]

				D. Alvaro, B. Barbaro, A. Franchitto, P. Onori, S. S. Glaser, G. Alpini, H. Francis, L. Marucci, P. Sterpetti, S. Ginanni-Corradini, et al. Estrogens and Insulin-Like Growth Factor 1 Modulate Neoplastic Cell Growth in Human Cholangiocarcinoma Am. J. Pathol., September 1, 2006; 169(3): 877 - 888. [Abstract] [Full Text] [PDF]

				S. Glaros, N. Atanaskova, C. Zhao, D. F. Skafar, and K. B. Reddy Activation Function-1 Domain of Estrogen Receptor Regulates the Agonistic and Antagonistic Actions of Tamoxifen Mol. Endocrinol., May 1, 2006; 20(5): 996 - 1008. [Abstract] [Full Text] [PDF]

				L-A Martin, S Pancholi, C M W Chan, I Farmer, C Kimberley, M Dowsett, and S R D Johnston The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling Endocr. Relat. Cancer, December 1, 2005; 12(4): 1017 - 1036. [Abstract] [Full Text] [PDF]

				R I Nicholson, I R Hutcheson, S E Hiscox, J M Knowlden, M Giles, D Barrow, and J M W Gee Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S29 - S36. [Abstract] [Full Text] [PDF]

				C. M Staka, R. I Nicholson, and J. M W Gee Acquired resistance to oestrogen deprivation: role for growth factor signalling kinases/oestrogen receptor cross-talk revealed in new MCF-7X model Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S85 - S97. [Abstract] [Full Text] [PDF]

				S. R. Johnston Combinations of Endocrine and Biological Agents: Present Status of Therapeutic and Presurgical Investigations Clin. Cancer Res., January 15, 2005; 11(2): 889s - 899s. [Abstract] [Full Text] [PDF]

				R. I. Nicholson, I. R. Hutcheson, J. M. Knowlden, H. E. Jones, M. E. Harper, N. Jordan, S. E. Hiscox, D. Barrow, and J. M. W. Gee Nonendocrine Pathways and Endocrine Resistance: Observations with Antiestrogens and Signal Transduction Inhibitors in Combination Clin. Cancer Res., January 1, 2004; 10(1): 346S - 354S. [Abstract] [Full Text] [PDF]

				L.-A. Martin, I. Farmer, S. R. D. Johnston, S. Ali, C. Marshall, and M. Dowsett Enhanced Estrogen Receptor (ER) {alpha}, ERBB2, and MAPK Signal Transduction Pathways Operate during the Adaptation of MCF-7 Cells to Long Term Estrogen Deprivation J. Biol. Chem., August 15, 2003; 278(33): 30458 - 30468. [Abstract] [Full Text] [PDF]

				J.-W. Chen, T. Ledet, H. Orskov, N. Jessen, S. Lund, J. Whittaker, P. De Meyts, M. B. Larsen, J. S. Christiansen, and J. Frystyk A highly sensitive and specific assay for determination of IGF-I bioactivity in human serum Am J Physiol Endocrinol Metab, June 1, 2003; 284(6): E1149 - E1155. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/43/40080    most recent M105892200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stephen, R. L. Articles by Darbre, P. D. Search for Related Content PubMed PubMed Citation Articles by Stephen, R. L. Articles by Darbre, P. D. Social Bookmarking                      What's this?