The Potentiation of Estrogen on Insulin-like Growth Factor I Action in MCF-7 Human Breast Cancer Cells Includes Cell Cycle Components
2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês
10.1074/jbc.m006741200
ISSN1083-351X
AutoresJoëlle Dupont, Michael Karas, Derek LeRoith,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoTo gain insight into the mechanisms involved in the cross-talk between IGF-1 receptor (IGF-1R) and estrogen receptor signaling pathways, we used MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression. Growth of NEO cells (control MCF-7 cells) was stimulated by both IGF-1 and estradiol (E2), and the addition of both mitogens resulted in a synergistic response. Estrogen enhanced IGF-1R signaling in NEO cells, but this effect was markedly diminished in SX13 cells. Estrogen was also able to potentiate the IGF-1 effect on the expression of cyclin D1 and cyclin E and on the phosphorylation of retinoblastoma protein in control but not in SX13 cells. IGF-1 increased the protein level of p21 and the luciferase activity of the p21 promoter, whereas it only reduced the protein level of p27 without affecting p27 promoter activity. Estrogen did not affect the p21 inhibitor, but it decreased the protein level of p27 and the p27 promoter luciferase activity. These effects of both mitogens were also observed at the level of association of both cyclin-dependent kinase inhibitors with CDK2 suggesting that IGF-1 and E2 affect the activity of both p21 and p27. Taken together, these data suggest that in MCF-7 cells, estrogen potentiates the IGF-1 effect on IGF-1R signaling as well as on the cell cycle components. Moreover, IGF-1 and E2 regulate the expression of p21 and p27 and their association with CDK2 differently. To gain insight into the mechanisms involved in the cross-talk between IGF-1 receptor (IGF-1R) and estrogen receptor signaling pathways, we used MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression. Growth of NEO cells (control MCF-7 cells) was stimulated by both IGF-1 and estradiol (E2), and the addition of both mitogens resulted in a synergistic response. Estrogen enhanced IGF-1R signaling in NEO cells, but this effect was markedly diminished in SX13 cells. Estrogen was also able to potentiate the IGF-1 effect on the expression of cyclin D1 and cyclin E and on the phosphorylation of retinoblastoma protein in control but not in SX13 cells. IGF-1 increased the protein level of p21 and the luciferase activity of the p21 promoter, whereas it only reduced the protein level of p27 without affecting p27 promoter activity. Estrogen did not affect the p21 inhibitor, but it decreased the protein level of p27 and the p27 promoter luciferase activity. These effects of both mitogens were also observed at the level of association of both cyclin-dependent kinase inhibitors with CDK2 suggesting that IGF-1 and E2 affect the activity of both p21 and p27. Taken together, these data suggest that in MCF-7 cells, estrogen potentiates the IGF-1 effect on IGF-1R signaling as well as on the cell cycle components. Moreover, IGF-1 and E2 regulate the expression of p21 and p27 and their association with CDK2 differently. insulin-like growth factor I cyclin-dependent kinase insulin receptor substrate 1 phosphatidylinositol 3-kinase cyclin-dependent kinase inhibitors phosphate-buffered saline serum-free medium retinoblastoma protein estrogen receptor insulin-like growth factor I receptor Iscove's minimal essential medium fluorescence-activated cell sorter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetraolium bromide phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis mitogen-activated protein estradiol Insulin-like growth factor-1 (IGF-1)1 and estrogens are important mediators of cellular proliferation and are intimately linked to the progression of a number of human cancers, notably breast cancer (1Lee A.V. Yee D. Biomed. Pharmacother. 1995; 49: 415-421Crossref PubMed Scopus (90) Google Scholar). It has been shown that an inhibition of IGF-1 receptor (IGF-1R) signaling with anti-IGF-1R antibodies or antisense RNA to the IGF-1R restricts breast cancer cell growth both in vitro andin vivo (1Lee A.V. Yee D. Biomed. Pharmacother. 1995; 49: 415-421Crossref PubMed Scopus (90) Google Scholar). The IGF-1R is expressed in a high percentage of primary human breast tumors, and this expression is positively correlated with the level of estrogen receptor (ER) (2Peyrat J.P. Bonneterre J. Breast Cancer Res. Treat. 1992; 22: 59-67Crossref PubMed Scopus (107) Google Scholar). In several tissues and cell lines, including normal breast (3Ruan W. Catanese V. Wieczorek R. Feldman M. Kleinderg D.L. Endocrinology. 1995; 136: 1296-1302Crossref PubMed Google Scholar), endometrial cancer cells (4Kleinman D. Karas M. Roberts Jr., C.T. LeRoith D. Phillip M. Segev Y. Levy J. Sharoni Y. Endocrinology. 1995; 136: 2531-2537Crossref PubMed Google Scholar), and estrogen-responsive breast cancer cells (MCF-7, ZR-75, and T47D) (5Stewart A.J. Johnson M.D. May F.E. Westley B.R. J. Biol. Chem. 1990; 265: 21172-21178Abstract Full Text PDF PubMed Google Scholar, 6Guvakova M.A. Surmacz E. Exp. Cell Res. 1997; 231: 149-162Crossref PubMed Scopus (128) Google Scholar), estrogen sensitizes the cells to the mitogenic effect of IGF-1. Consequently, the combined effects of estradiol (E2) and IGF-1 might stimulate the proliferation in mammary epithelium, thereby increasing the risk of breast cancer. The mechanisms involved in this sensitization at the level of IGF-1R signaling and cell cycle components have not yet been established. The components of IGF-1R signaling pathways that transduce the mitogenic stimulus to the cell cycle machinery have been partially identified. IGF-1 initiates its growth-promoting effects through its cognate transmembrane tyrosine kinase receptor. Upon activation by ligand binding, the IGF-1R tyrosine kinase phosphorylates several intracellular substrates such as the insulin receptor substrate (IRS) proteins (IRS-1 through -4) and Shc (7White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar). In breast cancer cell lines that express the ER (MCF-7, ZR-75 or T47-D), the mitogenic effects of IGF-1 are primarily mediated by IRS-1 (8Jackson J.G. White M.F. Yee D. J. Biol. Chem. 1998; 273: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 9Surmacz E. Burgaud J.L. Clin. Cancer Res. 1995; 1: 1429-1436PubMed Google Scholar). Activated IRS-1 serves as a multisite docking protein for numerous Src homology 2 domain-containing proteins. These proteins include the p85 regulatory subunit of phosphatidylinositol 3′-kinase (PI3K) and the adapter protein, Grb2. Some of the downstream effectors of PI3K are the serine/threonine protein kinase Akt/PKB and p70/S6 kinase (10Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar). The binding of IRS and Shc proteins to Grb2 and the associated guanine nucleotide exchange protein, mSos, results in activation of the Ras-Raf-MAP kinase pathway. The specific pathway involved in cell proliferation (i.e. PI3K versus MAP kinase) depends on the particular cell type (11Petley T. Graff K. Jiang W. Yang H. Florini J. Horm. Metab. Res. 1999; 31: 70-76Crossref PubMed Scopus (103) Google Scholar). In myoblasts, adipocytes, and 3T3 fibroblasts, IGF-1-induced cellular proliferation is clearly mediated by the Ras-Raf-MAP kinase pathway (12Coolican S.A. Samuel D.S. Ewton D.Z. McWade F.J. Florini J.R. J. Biol. Chem. 1997; 272: 6653-6662Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 13Teruel T. Valverde A.M. Benito M. Lorenzo M. Biochem. J. 1996; 319: 627-632Crossref PubMed Scopus (72) Google Scholar, 14Valverde A.M. Lorenzo M. Navarro P. Benito M. Mol. Endocrinol. 1997; 11: 595-607Crossref PubMed Google Scholar). In contrast, in MCF-7 cells, the proliferative response to IGF-1 is mediated by PI3K (15Dufourny B. Alblas J. van Teeffelen H.A. van Schaik F.M. van der Burg B. Steenbergh P.H. Sussenbach J.S. J. Biol. Chem. 1997; 272: 31163-31171Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). More specifically, it has been shown that in MCF-7 cells the PI3K pathway is involved in cyclin D1 synthesis and the hyperphosphorylation of the retinoblastoma protein (Rb) (15Dufourny B. Alblas J. van Teeffelen H.A. van Schaik F.M. van der Burg B. Steenbergh P.H. Sussenbach J.S. J. Biol. Chem. 1997; 272: 31163-31171Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Further studies have shown that Akt affects cyclin D translation in response to serum in MCF-7 cells (16Muise-Helmericks R.C. Grimes H.L. Bellacosa A. Malstrom S.E. Tsichlis P.N. Rosen N. J. Biol. Chem. 1998; 273: 29864-29872Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The mechanisms by which E2 induces cellular proliferation have not been well established. E2 acts through nuclear hormone receptors (ERα and -β), which, upon activation, may induce the transcription of various genes, including growth factors, their receptors, and substrates (17Molloy C.A. May F.E. Westley B.R. J. Biol. Chem. 2000; 275: 12565-12571Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Recent studies have shown that these growth factors may, in turn, decrease ERα gene expression while increasing the activity of the receptor in MCF-7 cells (18Stoica A. Saceda M. Fakhro A. Joyner M. Martin M.B. J. Cell. Biochem. 2000; 76: 605-614Crossref PubMed Scopus (83) Google Scholar). The effect of E2 on cellular proliferation may be mediated by the up-regulation of IGF-1R expression (5Stewart A.J. Johnson M.D. May F.E. Westley B.R. J. Biol. Chem. 1990; 265: 21172-21178Abstract Full Text PDF PubMed Google Scholar), IRS-1, and IRS-2 (19Lee A.V. Jackson J.G. Gooch J.L. Hilsenbeck S.G. Coronado-Heinsohn F Osborne C.K. Yee D. Mol. Endocrinol. 1999; 13: 787-796Crossref PubMed Scopus (0) Google Scholar) or by down-regulating the expression of the inhibitory IGF-binding proteins (20Huynh H. Yang X. Pollak M. J. Biol. Chem. 1996; 271: 1016-1021Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). E2 also has direct effects on specific components of the cell cycle. For instance, some studies have shown that E2 stimulates cellular proliferation through early activation of CDK2 and CDK4, phosphorylation of Rb, and increased expression of certain cyclins (21Foster J.S. Wimalasena J. Mol. Endocrinol. 1996; 10: 488-498Crossref PubMed Scopus (206) Google Scholar, 22Planas-Silva M.D. Weinberg R.A. Mol. Cell. Biol. 1997; 17: 4059-4069Crossref PubMed Scopus (235) Google Scholar, 23Prall O.W.J. Sarcevic B. Musgrove E.A. Watts C.K.W. Sutherland R.L. J. Biol. Chem. 1997; 272: 10882-10894Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). In this study, we investigated the mechanisms involved in the potentiation of IGF-1s by E2 on cell proliferation of MCF-7 human breast cancer cells. We exposed MCF-7 cells to IGF-1 and E2 either separately or in combination, and then we analyzed various components of IGF-1R signaling (IGF-1R, IRS-1, PI3K, Akt, and Erk1/2) and certain cell cycle molecules (cyclin D1, Rb, cyclin E, and two cyclin-dependent kinase inhibitors (CDKIs), p21 and p27). In order to determine the role of the IGF-1R in estrogen signaling, we used MCF-7-derived cells that express a reduced level (50%) of IGF-1R (24Neuenschwander S. Roberts Jr., C.T. LeRoith D. Endocrinology. 1995; 136: 4298-4303Crossref PubMed Google Scholar). The results show a potentiation of action of IGF-1 by E2 not only on the immediate downstream targets of IGF-1R signaling but also on certain cell cycle components. Moreover, this potentiation is dependent on the level of IGF-1R expression. Our results also indicate that IGF-1 and E2 have differential actions on the CDKIs, p21 and p27. The radionuclide [γ-32P]ATP (6000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Recombinant human IGF-1 was obtained from Genentech (South San Francisco, CA). 17β-Estradiol, phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, protein A-agarose, and phosphatidylinositol were obtained from Sigma. ICI 182,780 was kindly supplied by Dr. Alan Wakeling at Zeneca Pharmaceuticals (Macclefields, UK). Silica TLC plates were obtained from Whatman. Rabbit polyclonal antibodies to cyclin D1 (HD11), cyclin E (C19), CDK2 (M2), p27KIP-1 (C19), Erk1 (C16), and the IGF-1 receptor β subunit (C20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibody to IRS-1 and mouse monoclonal antibodies to p21WAF1/CIP1 and p85 were from Upstate Biotechnology, Inc. (Lake Placid, NY). The mouse polyclonal anti- phosphotyrosine antibody (PY20) was from Transduction Laboratories (Lexington, KY). Monoclonal anti-actin (clone AC) was obtained from Sigma. Rabbit polyclonal antibodies to phospho-Akt (Ser473), Akt, phospho-Erk1/2 (Thr202/Tyr204), and phospho-Rb (Ser780) were from New England Biolabs (Beverly, MA). All these antibodies were used with a 1/1000 dilution in Western blotting. Full-length promoter constructs for p21 and p27 were kindly donated by Dr. F. Kashanchi (NCI, National Institutes of Health, Bethesda) and Dr. T. Sakai (Prefectural University of Medicine, Japan), respectively. MCF-7 cells from ATCC (Manassas, VA) were cultured in IMEM supplemented with 10% fetal bovine serum, glutamine (2 mm), penicillin (100 IU/ml), and streptomycin (100 μg/ml). MCF-7 cells stably transfected with an antisense IGF-1R cDNA (SX13) (29Johnson M.R. Valentine C. Basilico C. Mansukhani A. Oncogene. 1998; 16: 2647-2656Crossref PubMed Scopus (53) Google Scholar) and corresponding control cell lines transfected with the empty vector (NEO) were maintained in the same medium supplemented with 800 μg/ml G418 (Geneticin, Life Technologies, Inc.). For growth studies, cells were seeded in 96-well plates (8–10,000 cells per well) in IMEM phenol red-free medium containing 5% charcoal-stripped fetal bovine serum. One day later, the medium was switched to that containing the serum-free medium plus the anti-estrogen ICI 182,780, (10 nm) for 48 h to synchronize cells in the G0 phase. The medium was then changed to that containing phenol red-free medium without serum and the various stimuli as described in the figure legends. As an indirect measure of growth, the 3-[4,5-dimethylthiazol 2-yl] 2,5-diphenyltetraolium bromide (MTT) assay was used as described previously (26Kleinman D. Karas M. Danilenko M. Arbell A. Roberts C.T. LeRoith D. Levy J. Sharoni Y. Endocrinology. 1996; 137: 1089-1095Crossref PubMed Scopus (51) Google Scholar). Determination of the surface IGF-1Rs on NEO and SX13 cells was obtained by flow cytometry. Cells were trypsinized and washed once in PBS and once in FACS buffer (0.1% sodium azide, 2% bovine serum albumin in PBS). Next, cells (106 cells/sample) were incubated 30 min on ice with 5 μg/ml IGF-1R-PE-conjugated mouse IgG1 antibody (PharMingen, San Diego, CA) diluted in FACS buffer. Background staining was evaluated using a mouse IgG1 isotype control (5 μg/ml) (PharMingen, San Diego, CA). Cells were washed three times and resuspended in 0.5 ml with FACS buffer. Finally, cells were examined for fluorescence intensity on a FACSCalibur using CellQuest software (both from Becton Dickinson, Mountain View, CA). Cell lysates were prepared in lysis buffer A (10 mm Tris (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40) containing various protease inhibitors (2 mm PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin) and phosphatase inhibitors (100 mm sodium fluoride, 10 mm sodium pyrophosphate, 2 mm sodium orthovanadate). Lysates were centrifuged at 12,000 × gfor 20 min at 4 °C, and then the protein concentration in the supernatants was determined using the BCA protein assay. After normalization for protein concentration (250 μg) various proteins were immunoprecipitated from the supernatants using 5 μg of appropriate antibodies at 4 °C overnight. The immunocomplexes were precipitated with 40 μl of protein A-agarose for 1 h at 4 °C. After two sequential washes using buffer A with a 1/2 dilution, the resulting pellets were boiled for 4 min in reducing Laemmli buffer containing 80 mm dithiothreitol. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked and probed with the various antibodies as indicated in the figure legends. After extensive washings, immunoreactivity was detected with the appropriate horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence. Densitometry was performed by scanning the radiographs and then analyzing the bands with the software MacBas version 2.52 (Fuji PhotoFilm). In some experiments, Western blotting was performed on whole cell lysates, using 50 μg of protein. PI3′-kinase activity was determined as described previously (27Dupont J. Derouet M. Simon J. Taouis M. Biochem. J. 1998; 335: 293-300Crossref PubMed Scopus (29) Google Scholar). Cell lysates were prepared on ice in extraction buffer B composed of 20 mm Tris (pH 7.5), 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 150 mmNa3VO4, 1% Nonidet P-40, 10% glycerol (v/v), 2 mm PMSF, 10 μg/ml aprotinin in phosphate-buffered saline (PBS). Cell lysates were clarified by centrifugation for 35 min at 40,000 × g at 4 °C. IRS-1 was immunoprecipitated from aliquots of the resulting supernatants (each containing 250 μg of total protein) by incubating them overnight at 4 °C with the αIRS-1 antibody (1/1000). Immunoprecipitates were collected with protein A-agarose beads and washed successively as follows: once in PBS containing 1% Nonidet P-40 and 100 μmNa3VO4, twice in a buffer containing 100 mm Tris-HCl (pH 7.5), 500 mm LiCl2, 100 μm Na3VO4, and finally, once in a buffer containing 10 mm Tris-HCl (pH 7.5), 100 mm NaCl, 1 mm EDTA, and 100 μmNa3VO4. The pellet was resuspended in 40 μl of a buffer containing 10 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 1 mm EDTA. To each tube was added 10 μl of MnCl2 (100 mm) and 20 μg of phosphatidylinositol. The reaction was initiated by the addition of 10 μl of ATP (440 μm) containing 30 μCi of [γ-32P]ATP. Reactions were incubated for 10 min at room temperature and then were stopped by the addition of 20 μl of HCl (8n) and 160 μl of CHCl3:CH3OH (1/1). After centrifugation (3,000 × g for 4 min at 4 °C), the organic phase was extracted and applied to a silica gel thin layer chromatography (TLC) plate. TLC plates were developed in CHCl3/CH3OH/H2O/NH4OH (120/94/22.6/4) and dried, and the radioactivity was quantitated with a PhosphorImager apparatus (FujiFilm, Stamford, CT). Three days prior to experiments, MCF-7-derived cells were stripped of endogenous steroids by passage in IMEM without phenol red containing 3% fetal bovine serum (charcoal-stripped). Cells were transiently transfected using the Effectene reagent according to the manufacturer's recommendations. Cells were plated in 6-well plates 24 h prior to transfection; each well received 1 μg of p21 or p27 promoter-luciferase construct and 0.5 μg of the pTKRenilla vector to normalize for transfection efficiency. All transfections included a reference sample with p0luc (for p21) or pGL2 basic (for p27). 16 h after transfection, cells were switched to serum-free medium for 24 h, followed by the addition of various stimuli for 24 h. Firefly and Renilla luciferase activities were measured using the Dual-luciferase System (Promega, Madison, WI), according to the manufacturer's instructions. All reported values are the means ± S.D. Statistical comparisons were made by a two-sided Student'st test. Statistical significant was assumed if a null hypothesis could be rejected at the p < 0.05. To characterize potential cross-talk and synergism between the IGF-1R and ER signaling on cellular proliferation, we used MCF-7 cells expressing an IGF-1R antisense cDNA (SX13) and control cells (NEO). SX13 cells have a significantly reduced level of IGF-1R (50% reduction on Western blot analysis, Fig. 1 A and Ref.24Neuenschwander S. Roberts Jr., C.T. LeRoith D. Endocrinology. 1995; 136: 4298-4303Crossref PubMed Google Scholar). However, the surface receptors were undetectable by flow cytometry suggesting that most of IGF-1Rs in SX13 cells are internalized (Fig.1 B, left panel, curve 1 versus curve 2). NEO and SX13 cells were partially synchronized in G0 phase by serum deprivation and using the anti-estrogen ICI 182,780 (10 nm) for 48 h. Under these conditions, more than 75% of cells were synchronized in G0 phase (data not shown). Next, cells were incubated with E2 (10 nm), IGF-I (1 nm), or a combination of both stimuli for 3 days. MTT assays were performed on each day as an indirect measure of cell proliferation. In NEO cells, IGF-1 treatment resulted in a 1.7- (p < 0.05) and 1.8 (p < 0.05)-fold increase in cell number after 48 and 72 h, respectively, as compared with cells incubated with Serum-free Medium (SFM, Fig.2). Treatment with E2 (10 nm) resulted in a 1.3- (p < 0.05) and 1.9 (p < 0.05)-fold increase in cell number after 48 and 72 h, respectively. No significant effect was observed after 24 h of stimulation with E2 (data not shown). Coexposure to both mitogens resulted in a 3.8- (p < 0.05) and 4.7 (p < 0.05)-fold increase in cell number after 48 and 72 h stimulation, respectively. These results indicate that IGF-1 and E2 exert a greater effect when cells are simultaneously exposed to these growth factors than when exposed to E2 or IGF-1 individually. Thus, E2 sensitizes the MCF-7 cell line to the mitogenic effect of IGF-1, at least after 48 h. At 72 h, the effect of both IGF-I and E2 may have been maximal, and thus no further synergism was observed. In SX13 cells, the effect of IGF-1 on cellular proliferation was abrogated; only E2 treatment resulted in a significant increase in cell number after 72 h (2.1 (p < 0.05)-fold compared with cells incubated in SFM). These results were confirmed in a second clone expressing a decreased IGF-1R level.Figure 2Abrogation of the synergistic effect of E2 and IGF-1 on cell growth in SX13 cells. MCF-7 cells were maintained in SFM in the absence or presence of IGF-1 (1 nm), E2 (10 nm), or a combination of these both mitogens for 3 days, as described under "Experimental Procedures." Cell number was determined indirectly each day using the colorimetric MTT method. Results are expressed as the mean ± S.D. of percentage of cell number increase as compared with cells maintained in SFM. The results are obtained from three independent experiments with 5 measurements per experiment for each condition. *, p < 0.05 indicates a significant synergistic effect between IGF-1 and E2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next identified the specific signaling components of the IGF-1R cascade that are sensitized by E2 treatment. SX13 and NEO cells were again treated with either IGF-1 (5 min) or E2 (48 h) separately or sequentially (E2 for 48 h followed by IGF-1 for 5 min). The tyrosine phosphorylation state of the IGF-1R β subunit and IRS-1, PI3K activity, and the association of p85 with IRS-1, Akt, and Erk1/2 phosphorylation were then determined. Treatment with IGF-1 for 5 min resulted in tyrosine phosphorylation of the IGF-1 receptor β subunit and IRS-1 in NEO cells, whereas the effect of IGF-1 on phosphorylation of these proteins in SX13 cells was markedly reduced (Fig. 3, panels 1 and3). In NEO cells, treatment with IGF-1 induced the phosphorylation of Erk1/2, and this induction was reduced by 50% in SX13 cells (Fig. 3, panel 5). The phosphorylation state of IGF-1R, IRS-1, and Erk1/2 was not regulated by E2 treatment alone in either SX13 or NEO cells. However, in both cell lines, after exposure to E2, IGF-1 stimulation resulted in enhanced tyrosine phosphorylation of the IGF-1R and IRS-1 compared with cells stimulated with IGF-1 alone (Fig. 3, panels 1 and 3). In NEO cells, treatment with E2 also increased the IGF-1-stimulated Erk1/2 phosphorylation, whereas in SX13 cells no potentiation of E2 was observed. In NEO cells, the increased phosphorylation of IRS-1 was paralleled with an increase in the total amount of IRS-1 immunoreactivity (Fig. 3, panel 4). This effect was markedly attenuated in SX13 cells. In contrast, treatment with E2 did not increase the total level of IGF-1R or the protein expression of Erk1/2 in either cell line (Fig. 3,panels 2 and 6). In NEO cells, we also show by flow cytometry analysis that the level of surface IGF-1Rs is unchanged by the treatment with E2 (Fig. 1, right panel, curve 2 versus curve 3). However, by using the same technique, in SX13 cells, 38% of cells are IGF-1R positives after E2 treatment. Thus, E2 induced a redistribution of IGF-1Rs to the cellular surface (Fig. 1, left panel, curve 2 versus curve 3). These results indicate that in SX13 cells, most of the IGF-1Rs are internalized, and treatment with E2 facilitates the translocation of IGF-1Rs to the plasma membrane at the cellular surface. Thus, we show that the potentiation of the effects of IGF-I by E2 can be explained, at least in part, by the increased phosphorylation of the IGF-IR, the increased IRS-1 expression, and the increased phosphorylation of IRS-1 and Erk1/2 in NEO cells. In SX13 cells, this potentiation of E2 on IGF-1 action, while seen with IGF-1R and IRS-1 phosphorylation, is not apparent with Erk1/2 phosphorylation or with cellular proliferation. The regulatory subunit of PI3K (p85) is a signaling molecule that binds directly to IRS-1 and is important for the proliferative effects of IGF-1 on MCF-7 cells (8Jackson J.G. White M.F. Yee D. J. Biol. Chem. 1998; 273: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 15Dufourny B. Alblas J. van Teeffelen H.A. van Schaik F.M. van der Burg B. Steenbergh P.H. Sussenbach J.S. J. Biol. Chem. 1997; 272: 31163-31171Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). We therefore investigated the effects of E2 and IGF-I on the activation of PI3K and Akt, one of its downstream targets. Treating MCF-7 cells with IGF-1 for 5 min resulted in an increase in the association of p85 with IRS-1 in NEO cells (data not shown). This also increased PI3K enzyme activity associated with IRS-1 (Fig. 4 B). This effect was not observed in SX13 cells. Phosphorylation of Akt at Ser473was also induced by IGF-1 in NEO cells but not in SX13 cells (Fig.4 C, upper panel). The PI3K activity and Akt phosphorylation were unaffected by the presence of E2 in either cell line (Fig. 4,B and C). This is similar to the results observed on tyrosine phosphorylation of the IGF-1R and IRS-1. In NEO cells, estrogen enhancement of the IGF-1R and IRS-1 tyrosine phosphorylation (shown in Fig. 3, panels 1 and 3) was also associated with a significant increase in IGF-1-induced PI3K activity and an increase in the phosphorylation state of Akt (Fig. 4,B and C). Densitometric analysis revealed that IGF-1 induction of PI3K activity and phospho-Akt in the presence of E2 were 1.5-fold higher than that induced by IGF-1 alone. Interestingly, in SX13 cells, PI3K activity or phosphorylation of Akt induced by IGF-1 or E2 separately was undetectable, whereas these were both increased when E2 and IGF-I were given simultaneously (Fig. 4, B andC). This effect was associated with an increase in cell surface expression of IGF-1Rs following E2 treatment of SX13 (Fig.1 B, left panel). In NEO and SX13 cells, E2 treatment increased by 35% (p < 0.05) the total level of p85 immunoreactivity (Fig. 4 A) but did not alter the total level of Akt immunoreactivity (Fig. 4 C, lower panel). Thus, the synergistic effects of E2 and IGF-I are reflected at the level of downstream targets of the IGF-IR. We also investigated the mechanisms involved in the synergism of the mitogenic effects of IGF-1 and E2 at the level of various cell cycle components. The elements of the cell cycle we examined included cyclin D1, cyclin E, and phospho-Rb immunoreactivity levels in NEO and SX13 cells. In a time course experiment, when MCF-7 cells were treated separately with IGF-1 and E2, maximal cyclin D1 and cyclin E protein levels were induced after 3 and 24 h of stimulation, respectively (data not shown). Next, we tested the effect of simultaneous treatment with E2 and IGF-1 on these important cell cycle components. In NEO cells, treatment with IGF-1 and E2 separately for 3 h resulted in a 2.8- (p < 0.05) and 2.5 (p < 0.05)-fold increase in cyclin D1 immunoreactivity, respectively, as compared with cells maintained in SFM (Fig.5 A). Coexposure to both mitogens resulted in a 5.5-fold increase (p < 0.05) in cyclin D1 protein expression (Fig. 5 A). In SX13 cells, treatment with IGF-1 and E2 increased cyclin D1 protein expression by 1.3- and 2-fold (p < 0.05), respectively. Moreover, E2 did not enhance the effects of IGF-1 in these cells (Fig.5 A). In NEO cells, after 24 h of exposure to IGF-1 and E2 separately, cyclin E protein expression was increased by 1.5- (p < 0.05) and 1.8 (p < 0.05)-fold, respectively, as compared with cells maintained in SFM (Fig.5 B). Simultaneously exposing cells to both stimuli resulted in a 2.7-fold increase (p < 0.05) in cyclin E protein expression. In SX13 cells, the effect of IGF-1 alone on cyclin E levels was undetectable, whereas E2 alone or combined with IGF-1 increased cyclin E protein expression by 1.5-fold (p < 0.05) (Fig. 5 B). Finally, we determined the effects of IGF-1 and E2, alone or in combination, on the phosphorylation state of Rb using a phospho-specific Rb antibody. In NEO cells, IGF-1 or E2 treatment increased the phospho-Rb immunoreactivity by 3-fold (p< 0.05) as compared with cells grown in SFM (Fig. 5 C). Coincubating NEO cells with IGF-1 and E2 induced a 4.5-fold increase (p < 0.05) in phospho-Rb immunoreactivity. Similar to the effects on cyclin D1 and cyclin E protein expression, phosphorylation of Rb was only increased by E2 treatment alone or in combination with IGF-1 in SX13 cells (Fig. 5 C). Similar results wer
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