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Endocrinology Vol. 143, No. 5 1872-1879
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Requirement for IGF-I in Epidermal Growth Factor-Mediated Cell Cycle Progression of Mammary Epithelial Cells

Malinda A. Stull, Monica M. Richert1, Aimee V. Loladze and Teresa L. Wood

Department of Neuroscience & Anatomy, Penn State College of Medicine, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: Teresa L. Wood, Ph.D., Department of Neuroscience & Anatomy H109, Penn State College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: . twood{at}psu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Induction of cyclin proteins is required for progression of cells through the G1-S and G2-M cell cycle checkpoints and is a primary mechanism by which mitogens regulate cell cycle progression. IGF-I and the epidermal growth factor (EGF)-related ligands are mitogens for mammary epithelial cells in vitro and are essential for growth of the mammary epithelium during development. We report here that IGF-I in combination with EGF or TGF{alpha} is synergistic in promoting DNA synthesis in mammary epithelial cells in the intact mammary gland cultured in vitro. We further investigated the role of IGF-I and EGF in cyclin expression and cell cycle progression in the mammary gland and demonstrate that IGF-I and EGF induce expression of early G1 cyclins. However, we show that IGF-I, but not EGF, induces late G1 and G2 cyclins and is required for mammary epithelial cells to overcome the G1-S checkpoint. These data demonstrate that IGF-I is essential for cell cycle progression in mammary epithelial cells and that it is required for EGF-mediated progression past the G1-S checkpoint in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REGULATION OF CELL cycle progression is an essential mechanism by which growth factors and cytokines mediate cell proliferation. Early studies in fibroblasts demonstrated that recruitment of quiescent cells into the cell cycle required both a competence factor to promote entry from G0 into G1 as well as a progression factor to overcome the G1-S checkpoint (1, 2). It is now known that the mechanism by which growth factors promote entry into and progression through the cell cycle is by regulating levels and activation of cell cycle regulatory proteins. Cyclin proteins, the regulatory subunits of the cyclin-dependent protein kinases, are required for progression through the cell cycle and are primary targets for regulating progression through both the G1-S and G2-M cell cycle checkpoints. Cyclin expression increases in response to signaling by a wide variety of mitogens including growth factors, cytokines, and hormones. Not surprisingly, aberrant expression or overexpression of cyclins results in increased proliferation and, in many tissues, tumorigenesis (3, 4, 5, 6).

Regulation of cyclin expression has been of particular interest in developing mammary tissue. Genetic deletion of cyclin D1 selectively affects development of the mammary gland and retina (7, 8). Overexpression of cyclin D1 in mammary epithelial cells in vitro accelerates cell cycle progression by decreasing the length of G1 and reducing the requirement for growth factors (3). In addition, overexpression of either cyclin D1 or cyclin E in mammary glands of transgenic mice results in epithelial hyperplasia and carcinoma (4, 5). These results demonstrate that regulation of cyclin levels is a critical point in regulating cell cycle progression and proliferation of mammary epithelial cells in both normal and abnormal growth of this tissue.

Regulation of mammary epithelial growth depends on the interactions of several growth factors and hormones. The circulating ovarian hormones estrogen and progesterone have been well studied as essential mediators of mammary epithelial growth; however, locally produced growth factors also have critical roles in mammary development (for reviews, see Refs. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). The IGF type I receptor (IGF-IR) and the epidermal growth factor (EGF) receptor (EGFR) family (including the EGFR/ErbB1 and ErbB2–4) are tyrosine-kinase receptors with known essential functions in growth of mammary epithelium (18, 21, 22, 23, 24, 25). While ligands for these receptors, the IGFs and the EGF-related ligands, are mitogens for mammary epithelial cells (26, 27, 28, 29, 30), the cell cycle basis for how these factors mediate proliferation of these cells during normal mammary gland development is largely uncharacterized.

Limited data from studies on nonmammary epithelial cells suggest that IGF-I and the EGF-related ligands regulate cell cycle progression through a combination of mechanisms including induction of cyclin expression. Regulation of cyclin D1 expression has been the best studied of the cyclins because it is rate-limiting for progression of cells through the G1 phase of the cell cycle. IGF-I and EGF induce cyclin D1 in a number of tumor cell lines including breast cancer cells (31, 32, 33, 34, 35). That regulation of cyclin D1 is essential to IGF-I and EGF mitogenic actions is supported by studies in human pancreatic cells where expression of antisense cyclin D1 abolished the ability of either growth factor to stimulate proliferation of these cells (34). Growth factor and hormone induction of cyclin D1 expression can occur through transcriptional regulation (32, 33, 36, 37) and, as shown in recent studies, through stabilization of cyclin D1 mRNA (38). IGF-I also induces the late G1 cyclin, cyclin E, in human breast cancer cells, and IGF-I induction of both cyclin D1 and E in these cells can be enhanced by estrogen (35, 39). Fewer studies have addressed cyclin regulation in normal cells; however, EGF can induce cyclin D1 in fibroblast cell lines (40, 41). IGF-I induces cyclins D1 and E in ventricular myocytes in addition to cyclins A2 and B1, cyclins essential for progression through S and G2 phases of the cell cycle, respectively (42). IGF-I induction of cyclin A2 also was reported in skeletal muscle satellite cells (43).

The goal of this study was to determine how IGF-I and the EGF-related ligands coordinate to regulate proliferation and cell cycle progression in normal mammary epithelium during postnatal growth of mammary tissue. The focus of these experiments was specifically to test whether IGF-I and EGF regulate expression of the G1 and G2 cyclins that are essential for promoting cell cycle progression. Because normal growth of mammary epithelium requires stromal-epithelial interactions, we have used an organ culture system to study the mechanisms by which IGF-I and EGF regulate proliferation and cyclin expression during growth of mammary epithelium in an intact tissue environment. We demonstrate that IGF-I and EGF synergize to promote DNA synthesis in mammary epithelium. Moreover, we present data showing that IGF-I and EGF both induce early G1 cyclins but that IGF-I is required to induce cyclins essential for late G1, S and G2 progression. Finally, we demonstrate that IGF-I is essential for EGF-mediated progression of mammary epithelial cells into S phase in the intact mammary gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mammary gland organ culture
Abdominal mouse mammary glands were cultured in mammogenic hormones and growth factors as described previously (44). All animal experimentation protocols were approved by Penn State Hershey Medical Center animal care and use committees and follow National Institutes of Health guidelines. Abdominal mammary glands were isolated from 6 wk C57Bl6/J mice following 9 or 14 d of priming with estrogen and progesterone using either implantation with 9-d time release pellets of 3 mg progesterone and 3 µg estrogen (Innovative Research of America, Sarasota, FL) or daily injections of 50 µl of 20 mg/ml progesterone (Sigma, St. Louis, MO) and 20 µg/ml estrogen (Sigma) in sesame oil (Sigma). Each individual gland was placed in a 3.5-cm culture well and cut into 6 uniformly-sized pieces to facilitate growth factor access. Glands were cultured for 48 h in media consisting of Waymouth’s media (752/1, Life Technologies, Inc., Gaithersburg, MD) containing 100 U/ml penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life Technologies, Inc.), 5 µg/ml PRL (mouse recombinant or ovine PRL, provided by A. F. Parlow at NIDDK’s National Hormone & Peptide Program), 5 µg/ml hydrocortisone (Sigma), 1 µg/ml aldosterone (Sigma), and 50 ng/ml insulin (Sigma) with or without growth factors and with the addition of 3H-thymidine (NEN Life Science Products, Boston, MA; 5.0 µCi/ml) for the final 6 h (44). Growth factor treatments consisted of IGF-I (10 or 100 ng/ml; Upstate Biotechnology, Inc., Lake Placid, NY) and/or EGF (60 ng/ml; Becton Dickinson and Co., Bedford, MA), amphiregulin (60 ng/ml; Sigma) or TGF{alpha} (60 ng/ml; Sigma). Additional mammary glands were cultured in basal media or in IGF-I (100 ng/ml) with or without EGF (60 ng/ml) in the presence of the antiestrogen ICI 182,780 (300 nM to 1 µM; Tocris Cookson Ltd., Bristol, UK). Following specified times in culture, each gland was homogenized in tissue extraction buffer (20 mM Tris HCl, pH 7.4; 2% Triton-X-100) and triplicate aliquots were precipitated with 10% trichloroacetic acid (TCA), applied to Whatman (Fisher Scientific, Pittsburgh, PA) filters, and rinsed with 5% TCA and 70% EtOH. TCA precipitable counts were determined by liquid scintillation counting. Additional glands were analyzed directly following removal from animals without culture or were analyzed following 12 h of culture in either Waymouth’s media alone or Waymouth’s media containing insulin, aldosterone, hydrocortisone, and PRL as described above. For time-course studies, mammary glands were cultured as described above but pretreated for 12 h in basal media containing hormones to reduce DNA synthesis to baseline levels before incubation with growth factors. Glands were then analyzed 12, 24, and 48 h following growth factor addition.

For histological analysis of sites of 3H-thymidine incorporation, glands were cultured for 48 h as above and fixed in 4% paraformaldehyde, paraffin embedded, and sectioned. Five-micrometer sections were deparaffinized through xylenes and graded ethanols and exposed to NTB2 photographic emulsion (Kodak) for 6 wk. Sections were developed and counterstained with Contrast Red (Kirkegaard \|[amp ]\| Perry Laboratories, Gaithersburg, MD).

Additional experiments were performed to analyze changes in cyclin expression as described below. An ‘n’ of four glands per treatment group obtained from individual animals was used for all experiments, and all experiments were performed at least three times.

Ribonuclease protection assays
RNA and DNA were isolated from cultured mammary glands using the Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) as described by the manufacturer. Ribonuclease protection assays (RPAs; PharMingen, San Diego, CA) were performed as instructed by the manufacturer using 5–20 µg of RNA from each gland. Phosphor Imager analysis was used to quantify optical density of cyclin mRNA expression and expression of a control mRNA, glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Values obtained for cyclin mRNA levels were normalized according to levels of GAPDH mRNA in each sample. Statistical analyses were performed on adjusted values for four glands per treatment group in each experiment. 3H-thymidine incorporation into glands used for RNA analysis was performed by determining the average dpm of equivalent amounts of DNA analyzed in triplicate for each sample.

Immunohistochemistry
Glands were cultured, fixed, embedded in paraffin and sectioned as for histochemical analyses. Sections were deparaffinized in xylenes and graded ethanols, endogenous peroxidases blocked with 3% H2O2 in methanol, rinsed with PBS and incubated in PGB superblock (10% BSA; 0.05% NaN3; 10% normal goat serum in PBS, pH 7.4). Sections were incubated overnight with a rabbit polyclonal anticyclin B1 antibody (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; H-433). Sections were rinsed in PBS and detection of cyclin B1 performed using a goat antirabbit alkaline phosphatase conjugated secondary antibody (1:750; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).

Western blot analysis
Mammary tissue was removed from culture and rinsed in cold PBS. To enrich for epithelial cells, tissue was minced and digested with 5 mg of collagenase III (Worthington, Lakewood, NJ) in 1.5 ml of PBS containing phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium vanadate) for 1.5 h at 37 C with rotation. The digested tissue was then centrifuged at 6,000 x g for 5 min at 4 C, and the floating adipocytes and supernatant were discarded. The remaining epithelial-enriched cell pellet was washed thoroughly with PBS to remove collagenase and homogenized on ice in 100–200 µl of lysis buffer containing 50 mM HEPES pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.5% NP-40, 2.5 mM Na pyrophosphate, 1 mM ß-glycerophosphate, protease inhibitor cocktail (1:100; Sigma), and 1 mM NaVO4. Tissue lysates were sonicated on ice, clarified by centrifugation at 15,000 x g at 4 C for 10 min. The protein concentration was determined by the bicinchonic acid protein assay (Pierce Chemical Co., Rockford, IL) and 100 µg of lysate was used for Western blot analysis. The samples were boiled for 5 min, electrophoresed through a 10% SDS polyacrylamide gel, and transferred onto nitrocellulose. Membranes with transferred proteins were incubated for 1 h in blocking solution containing TBS with 0.05% Tween 20 and 5% nonfat powdered milk and then overnight with rabbit anticyclin B1 (1:500; Santa Cruz Biotechnology, Inc.; H-433) in blocking solution at 4 C. Membranes were washed with TBS containing 0.05% Tween 20 and then incubated for 1 h with goat antirabbit-HRP secondary antibody (1:5000, Jackson ImmunoResearch Laboratories, Inc.) at room temperature. Bands were visualized using enhanced chemiluminescence (Perkin-Elmer Corp., Boston, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IGF-I and EGF on DNA synthesis in cultured mammary glands
Initial studies were designed to determine the effect of IGF-I and EGF on promoting DNA synthesis in mammary tissue (Fig. 1AGo). Treatment of mammary glands with IGF-I alone at two concentrations (10 ng/ml or 100 ng/ml) significantly increased DNA synthesis when compared with control glands as assayed by incorporation of 3H-thymidine (Fig. 1AGo). In contrast, treatment of mammary glands with EGF at 60 ng/ml [a concentration previously used for mammary gland organ culture (45, 46)] had no effect on DNA synthesis (Fig. 1Go, A and B). A 2-fold increase in EGF concentration (120 ng/ml) also failed to induce DNA synthesis (data not shown). However, the combination of EGF and IGF-I was synergistic in promoting DNA synthesis when compared with glands cultured in IGF-I alone (Fig. 1Go, A and B). Similar to EGF, the EGF-related ligands TGF{alpha} (60 ng/ml) and amphiregulin (60 ng/ml) also failed to stimulate DNA synthesis (Fig. 1BGo). However, like the combination of IGF-I and EGF, IGF-I in combination with TGF{alpha} was synergistic in promoting DNA synthesis (Fig. 1BGo).



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  		Figure 1. IGF-I or IGF-I in combination with EGF increases DNA synthesis in cultured mammary glands. A and B, Analysis of 3H-thymidine incorporation into pubertal mammary glands cultured in basal media (Ctl) or in basal media containing IGF-I (10 ng/ml or 100 ng/ml; I) and/or EGF (60 ng/ml; E), amphiregulin (60 ng/ml; A) or TGF{alpha} (60 ng/ml; T). C, Analysis of 3H-thymidine incorporation into pubertal mammary glands cultured in basal media (Ctl) or in IGF-I (100 ng/ml) with or without EGF in the presence of the antiestrogen ICI 182,780 (300 nM; ICI). Values shown represent dpm in 25 ml of tissue homogenate (mean ± SE). n = 4 glands from individual animals for each treatment group. Data were analyzed by ANOVA followed by Fisher’s protected least significant difference (PLSD) post hoc test. Hatched bars (I and I/E): 10 ng/ml IGF-I. Solid bars (I and I/E), 100 ng/ml IGF-I. a, P <= 0.01 vs. Ctl; b, P < 0.05 vs. 10 ng/ml IGF-I; c, P < 0.01 vs. 100 ng/ml IGF-I; d, P < 0.01 vs. IGF-I.

 
Several lines of evidence support the hypothesis that IGF-mediated proliferation of breast cancer cells involves interactions with estrogen-mediated signaling (for review, see Ref. 47). For example, IGFs can enhance phosphorylation and transcriptional effects of ER{alpha} in breast cancer cells (48). Moreover, induction of the IGF-IR and of insulin receptor substrate-1, a primary intracellular target for the IGF-IR, results from estrogen activation of the ER{alpha} (49). To test whether IGF-I-induced DNA synthesis in the cultured glands required activation of the ER, glands were treated with IGF-I in the presence of the pure antiestrogen ICI 182,780 (Tocris Cookson Ltd.). Treatment with the antiestrogen had no significant effect on thymidine incorporation at either a standard dose (300 nM; Fig. 1CGo) or at doses up to 1 µM (not shown). The ability of the ICI 182,780 to block estrogen-mediated DNA synthesis was confirmed in assays on the MCF-7 breast cancer cell line. The addition of either 100 nM or 500 nM of the inhibitor reduced incorporation of 3H-thymidine by 90–95% in MCF-7 cells stimulated with estrogen (data not shown).

Analysis of DNA replication in mammary epithelial cells
Previous studies in our laboratory demonstrated that mammary glands cultured under conditions used in these studies are viable and show morphology of glands undergoing ductal growth with no evidence of alveolar differentiation following 5 d in growth factor conditions (Ref. 44 ; Fig. 2AGo). In addition, whole mount staining of mammary glands treated with IGF-I and EGF contained terminal end buds (TEBs), the highly proliferative structures at the leading edge of the growing ducts (Fig. 2AGo). To determine the cellular sites of DNA synthesis within the cultured mammary glands, additional glands were treated with growth factors in the presence of 3H-thymidine and analyzed by autoradiography (Fig. 2Go, B–D). Histological analysis of sections from glands treated in control media or with EGF alone failed to induce DNA synthesis as shown by the lack of incorporation of 3H-thymidine (Fig. 2BGo). In contrast, glands treated with IGF-I alone (data not shown) or the combination of IGF-I and EGF (Fig. 2CGo) showed numerous sites of thymidine incorporation predominantly in epithelial cells in ducts and TEBs. Moreover, the majority of labeled epithelial cells were in structures at the leading edge of the ducts as would be predicted for sites of maximal proliferation (Fig. 2DGo). DNA synthesis also was observed occasionally in the surrounding stroma and in fibroblast cells at the periphery of the fat pad in glands treated with either IGF-I or the combination of IGF-I/EGF.



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  		Figure 2. IGF-I and IGF-I/EGF induce DNA synthesis in TEBs and ducts of pubertal mammary glands. A, Whole mount iron-hematoxylin stained preparation of mammary gland after 5 d of culture on gel foam rafts in IGF-I (100 ng/ml) and EGF (60 ng/ml). B–D, Histological analyses of mammary glands cultured in EGF (B) or IGF-I/EGF (C, D) in the presence of 3H-thymidine as described in Materials and Methods. Arrows indicate TEBs at the leading edge of the ductal structures. Size bars in B and C, 10 µm.

 
IGF-I and EGF regulation of cyclin expression
To elucidate the mechanisms by which IGF-I and EGF-related ligands induce mammary epithelial proliferation, RPA analyses were used to measure changes in cyclin mRNA levels in mammary glands cultured in the presence of growth factors alone or in combination (Fig. 3Go). After 48 h in culture, either IGF-I or EGF was equally effective in inducing mRNA levels of cyclins D1 (Fig. 3AGo), D2 (Fig. 3BGo), and D3 (Fig. 3CGo). The D-type cyclins are induced in early G1 suggesting that IGF-I and EGF equally promote early G1 events. Moreover, the combination of IGF-I/EGF significantly increased expression of cyclin D1 over that seen with either growth factor alone (Fig. 3AGo). DNA isolated from the same glands confirmed previous results showing that, although EGF and IGF-I were equally effective in inducing mRNAs for the D-type cyclins, only IGF-I increased DNA synthesis (data not shown). Induction and activation of cyclin E occurs later in G1 subsequent to induction of the D-type cyclins and is required for cells to progress past the G1-S restriction point and to enter the DNA synthetic phase. In contrast to the results obtained for the D-type cyclins, IGF-I but not EGF induced cyclin E mRNA (Fig. 3DGo). Moreover, the combination of IGF-I/EGF had no additional effect on cyclin E expression over treatment with IGF-I alone (Fig. 3DGo).



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  		Figure 3. Expression of cyclin mRNA in cultured mammary glands after 48 h of growth factor treatment. A–F, Changes in mRNA levels for cyclin D1 (A), cyclin D2 (B), cyclin D3 (C), cyclin E, (D), cyclin A2 (E), and cyclin B1 (F). Values shown represent arbitrary optical density units (mean ± SE) after adjustment to values obtained from hybridization to GAPDH mRNA within each sample. n = 4 glands from individual animals for each treatment group. Statistical analyses were performed using ANOVA followed by Fisher’s PLSD post hoc test. a, P < 0.05 vs. Ctl; b, P < 0.005 vs. IGF-I or EGF.

 
Similar to the requirement for induction of G1 cyclins in the G1-S transition, induction of cyclin A2 and cyclin B1 is necessary for cells to traverse the G2-M checkpoint of the cell cycle. Cyclin B1, in particular, is tightly regulated during the cell cycle such that cyclin B1 mRNA and protein levels only accumulate at the G2-M transition. Moreover, loss of cyclin B1 during G2 contributes to G2 arrest (50, 51, 52). Similar to the G1 cyclins, IGF-I induced mRNAs for both cyclin A2 and cyclin B1 in the cultured mammary glands (Fig. 3Go, E and F).

To determine the time course of cyclin induction in response to IGF-I and EGF in our culture system, additional experiments were performed to analyze cyclin expression in glands following 12, 24, and 48 h of growth factor treatment. In addition, to reduce DNA synthesis and cyclin expression in the cells before growth factor treatments, glands were pretreated for 12 h in either control media (containing mammogenic hormones) or in basal media without hormones (see Materials and Methods). Glands in either condition demonstrated similar low levels of DNA synthesis following 12 h of pretreatment that were equivalent to the levels of DNA synthesis in glands cultured in control media for 48 h (data not shown). Moreover, these glands showed a significant and equal loss of expression of both cyclin A2 and cyclin B1 compared with glands analyzed immediately following removal from the animal (Fig. 4Go). Thus, while it is unlikely that the cells within the intact mammary tissue can be synchronized following the pretreatment paradigm such that they progress at a uniform rate through the cell cycle after stimulation, it is likely that the cells were predominantly in a noncycling state at the initiation of the growth factor treatments.



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  		Figure 4. G2 cyclin mRNA expression is reduced in mammary glands cultured for 12 h in the absence of growth factors. A and B, Changes in mRNA levels for cyclin A2 (A) and cyclin B1 (B) in glands at time 0 (T0) and in glands cultured for 12 h in Waymouth’s media with or without the addition of hormone supplements. Values shown represent arbitrary optical density units (mean ± SE) after adjustment to values obtained from hybridization to GAPDH mRNA within each sample. T0, Glands analyzed directly after removal from animals; 12(-), glands cultured for 12 h in Waymouth’s media alone; 12(+), glands cultured for 12 h in Waymouth’s media containing insulin (50 ng/ml), hydrocortisone (5 µg/ml), aldosterone (1 µg/ml), and PRL (5 µg/ml). n = 4 glands from individual animals for each treatment group. Statistical analyses were performed using ANOVA followed by Fisher’s PLSD post hoc test. a, P <= 0.005 vs. T0; b, P <= 0.01 vs. T0.

 
Because the previous studies demonstrated a similar loss of DNA synthesis and G2 cyclin expression in either pretreatment condition, glands in the time-course study were cultured following a 12-h pretreatment in media containing hormones. [Preliminary studies in our laboratory also indicated that insulin and PRL are essential for gland viability in the organ cultures (our unpublished data)]. After 12 h of growth factor treatment, there was minimal induction of either cyclin expression or DNA synthesis in response to EGF or IGF-I either alone or in combination (data not shown). After 24 h of treatment with growth factors, however, there was a significant increase in cyclin mRNA levels and DNA synthesis in glands treated with the combination of IGF-I and EGF (Fig. 5Go). While not always significant, the pattern of cyclin induction was similar to that observed at 48 h where EGF and IGF-I each induced the D-type cyclins. In general, the magnitude of early cyclin mRNA induction by the individual growth factors at 24 h was less than that seen at 48 h suggesting that more cells within the gland had responded to the growth factor treatments by 48 h. IGF-I treatment significantly induced cyclin D2 and cyclin D3 after 24 h (Fig. 5Go, A–C). IGF-I also significantly induced cyclin A2 and cyclin B1 mRNAs as well as DNA synthesis by 24 h when compared with control glands or to EGF-treated glands (Fig. 5Go, D–F). Similar to the results seen at 48 h, EGF alone had no effect on mRNA levels of cyclin A2 and cyclin B1 or on DNA synthesis (Fig. 5Go, D–F). Interestingly, treatment with EGF in combination with IGF-I resulted in a significant increase in cyclin A2 and cyclin B1 compared with IGF-I alone (Fig. 5Go, D–F).



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  		Figure 5. Expression of cyclin mRNA in cultured mammary glands after 24 h of growth factor treatment. A–E, Changes in mRNA levels for cyclin D1 (A), cyclin D2 (B), cyclin D3 (C), cyclin A2 (D), and cyclin B1 (E). F, Analysis of 3H-thymidine incorporation into DNA from the mammary glands used for RPAs. Values shown in A–E represent arbitrary optical density units (mean ± SE) after adjustment to values obtained from hybridization to GAPDH mRNA within each sample. Values shown in F represent average dpm (mean ± SE) calculated for each treatment group from 20 µg of DNA per gland. n = 4 glands from individual animals for each treatment group. Data were analyzed by ANOVA followed by Fisher’s PLSD post hoc test. a, P < 0.005 vs. I or E; b, P = 0.06 vs. Ctl; c, P <= 0.05 vs. Ctl; d, P < 0.05 vs. I or E.

 
To determine if the induction of cyclin mRNA levels corresponded to an increase in cyclin protein levels, additional glands were cultured for 48 h and used for protein extraction as well as for immunohistochemistry. Due to the minimal amount of protein obtained from pubertal-stage mammary glands, cyclin protein levels were undetectable for the D-type cyclins in the cultured glands. However, it was possible to determine protein levels for cyclin B1, which is essential for the progression of cells past the G2-M checkpoint. Results from immunoblot analyses confirmed that IGF-I induced cyclin B1 protein (Fig. 6AGo). Quantitation of cyclin B1 levels revealed that IGF-I induced a 5-fold increase in cyclin B1 protein (Fig. 6BGo). In contrast, EGF had no effect on levels of cyclin B1 protein (Fig. 6AGo), consistent with the lack of induction of cyclin B1 mRNA by EGF. Immunohistochemical analyses demonstrated increased cyclin B1 protein in IGF-I and IGF-I/EGF treated glands in the epithelium within terminal ductal regions where 3H-thymidine incorporation was observed (Fig. 6CGo), whereas no detectable cyclin B1-positive cells were seen in either control or EGF treated glands (Fig. 6DGo).



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  		Figure 6. IGF-I increases cyclin B1 protein levels. A, Western blot analysis of cyclin B1 protein. B, Changes in cyclin B1 protein levels across treatment groups after normalization to ß-actin. C and D, Immunohistochemistry for cyclin B1 in mammary glands cultured in the presence of IGF-I (C) or EGF (D). Size bars in C and D, 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that IGF-I is an essential mediator of cell cycle progression in epithelial cells in the intact mammary gland and is required for EGF to promote G1-S progression in these cells. Moreover, we have shown that IGF-I can mediate progression past G1-S in the absence of estrogen receptor stimulation. A primary mechanism by which IGF-I promotes cell cycle progression is through induction of G1 and G2 cyclins. During the G1 phase of the cell cycle, IGF-I induces levels of both the early D-type cyclins as well as cyclin E, which is required for progression past the G1-S checkpoint. It is also of interest that IGF-I induced expression of cyclins A2 and B1 that are induced during S phase and G2, respectively. These results support an important role for IGF-I in G2-M progression in addition to G1-S progression in mammary epithelial cells. A function for IGF-I in G2 progression was suggested previously from studies of uterine cell proliferation in the IGF-I null mutant mouse following E2 stimulation (53). However, it is interesting that IGF-I was not required for G1 progression in the uterine cells. In contrast, proliferation of mammary epithelium is completely inhibited in the IGF-I null mutant in response to ovarian hormone stimulation (25). Results presented here now demonstrate that these deficits may be due, at least in part, to an essential requirement for IGF-I in progression of mammary epithelial cells past the G1-S checkpoint.

The synergistic effect of IGF-I/EGF on DNA synthesis is reflected in their cooperative induction of cyclin D1. These results suggest that the two factors interact primarily to promote early G1 events, although a synergistic effect of IGF-I/EGF also was observed on induction of cyclins A2 and B1 after 24 h when the effect of IGF-I alone was less pronounced than after 48 h. The most surprising aspect of our results is that IGF-I was required for EGF to promote progression past the G1-S restriction point and to induce DNA synthesis in the epithelial cells. This result is not due to lack of availability of the EGFR because EGF induced expression of the D-type cyclins equivalent to levels induced by IGF-I. Thus, it is unlikely that the EGFR is limiting and that IGF-I is required for induction of the EGFR. Another possible mechanism for the observed IGF-I/EGF synergism is that signaling through the EGFR enhances signaling through the IGF-IR. Consistent with this hypothesis, a previously published report demonstrated that activation of the EGFR in prostate epithelial cells inhibited IGF-I-mediated degradation of insulin receptor substrate-1 (54). There is precedence for the idea that the IGF-IR may be essential for maximal actions of many other growth factors (55, 56, 57). Fibroblast cells generated from IGF-IR null mutant embryos have been used in a number of studies to show that the IGF-IR is required for these cells to grow in serum-free media supplemented with fibroblast growth factor, transforming growth factor-ß, TGF{alpha}, IGF-I, IGF-II, insulin, platelet-derived growth factor, and EGF, either alone or in combination (55). The loss of the IGF-IR in the null fibroblasts resulted, in part, in an inability of other growth factors to produce a prolonged activation of the MAPK, ERK2 (56). Interestingly, the IGF-IR null fibroblasts also demonstrated elongation of all phases of the cell cycle when grown in 10% serum-containing medium (55).

The results from the experiments presented here have important implications for interpreting previous studies on mitogenic actions of IGF-I and EGF on mammary epithelial cells. It is particularly noteworthy that previous in vitro studies showing effects of EGF on growth of mammary epithelium in mammary gland organ culture used serum-free media containing micromolar levels of insulin (45, 46, 58). Insulin at these superphysiological concentrations activates the IGF-IR in addition to the insulin receptor (59). Micromolar concentrations of insulin also were used in investigations of EGF-mediated growth in cultures of dissociated mammary epithelial cells (26, 29). Thus, previous studies reporting EGF-mediated proliferation of mammary epithelial cells have largely overlooked the effect of signaling through the IGF-IR.

The results presented here support an essential role for IGF signaling in EGF-mediated growth of mammary epithelium and support a model where IGF and EGF ligands cooperate to induce cyclin expression and promote cell cycle progression of mammary epithelial cells. It is clear, however, that both IGF-I and the EGF-related ligands are essential for normal mammary development because genetic deletion of IGF-I or of the combination of EGF, TGF{alpha}, and amphiregulin in mice results in severe deficits in ductal outgrowth (18, 24, 25). Based on our proposed model of IGF/EGF cooperation in proliferation of mammary epithelial cells, we predict that the levels of endogenously available IGF-I are insufficient to compensate for the defects produced by loss of the EGF-ligands in mammary glands in vivo. It is likely that the available concentrations of either growth factor family are lower than the levels used in the organ cultures; thus, the combined effect of the two ligand families may be necessary to stimulate proliferation of mammary epithelial cells. A critical test of this prediction for future experiments will be to determine whether excess exogenous IGF-I can compensate for the loss of EGF ligands in the mutant glands in vivo. A second prediction from our model is that excess EGF will not compensate for the loss of IGF-I in the mammary gland of IGF-I null mutant mice.

Finally, the use of an intact organ culture system in these experiments raises the possibility that the primary effects of either IGF-I or EGF might be on cells in either or both the epithelial or stromal compartments. Support for EGF actions on mammary epithelial growth through stromal cell signaling has been provided by previous studies demonstrating that wild-type mammary epithelium shows growth deficits in an EGFR-null stroma (60). The presence of EGF binding sites or of the EGFR has been reported on both stromal and epithelial cells (10, 28, 61, 62). Thus, it is possible that induction of proliferation in the epithelial cells with the combination of IGF-I and EGF in our experiments is due to an indirect effect of EGF via activating cells in the stromal compartment. However, as discussed previously, both IGF-I and EGF can promote proliferation of isolated mammary epithelial cells in culture (26, 27, 28, 29, 30), and mammary epithelial cells in vivo express the IGF-IR and the EGFR (10, 17, 44, 62, 63). Expression of a dominant-negative EGFR specifically in the mammary epithelium causes deficits in ductal growth further supporting a role for direct actions of EGF-related ligands on the epithelial cells (22). Similarly, recent results where IGF-IR null epithelium was rescued from embryonic mammary glands and transplanted into wild-type stroma demonstrated an essential role for epithelial IGF-IR in ductal growth (64). It is particularly relevant to the current study that the loss of ductal growth in the IGF-IR null transplants was correlated with a decreased percent of BrdU-labeled cells in the TEBs with no detectable difference in the percent of apoptotic cells. Taken together, these data support a model where the primary effect of IGF-I in promoting epithelial proliferation in the mammary organ cultures is through direct actions on the epithelial compartment while actions of EGF may occur through effects on both the stromal and epithelial compartments. An EGF effect on stromal cells implies production of a second, as yet unidentified, factor that cooperates with IGF-I to mediate epithelial growth. Ultimately, elucidation of the complex interactions between the epithelial and stromal compartments of the mammary gland during phases of growth and differentiation will require understanding the effects of hormones and growth factors on cellular components in both compartments.


   Acknowledgments
 
The PRL was provided by A. F. Parlow and The NIDDK National Hormone & Peptide Program. We thank Dr. Kang Li for technical assistance with tissue sectioning and Dr. Steve Levison and Maricarmen Planas-Silva for their helpful suggestions during manuscript preparation. We also thank Dr. Maricarmen Planas-Silva for assistance with experiments to test the ICI 182,780 antiestrogen on MCF-7 cell proliferation.


   Footnotes
 
This work was supported by the National Cancer Institute NRSA CA83174 (to M.A.S.), the American Cancer Society Research Planning Grant CNE-97721 (to T.L.W.), and the Department of Defense Breast Cancer Research Program Career Development Award DAMD17-99-1-9296 (to T.L.W.).

Abbreviations: EGF, Epidermal growth factor; EGFR, EGF receptor; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; IGF-IR, IGF type I receptor; PLSD, protected least significant difference; RPA, ribonuclease protection assay; TCA, trichloroacetic acid; TEBs, terminal end buds.

1 Current Address: Department of Pathology Box B-216, University of Colorado Health Sciences Center, 4200 East 9th Street, Denver, Colorado 80262. Back

Received August 29, 2001.

Accepted for publication January 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pledger W, Stiles C, Antoniades H, Scher C 1977 Induction of DNA synthesis in BALB/c3T3 cells by serum components: reevaluation of the commitment process. Proc Natl Acad Sci USA 74:4481–4485[Abstract/Free Full Text]
  2. Stiles C, Capone G, Scher C, Antoniades H, Van Wyk J, Pledger W 1979 Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci USA 76:1279–1283[Abstract/Free Full Text]
  3. Kamalati T, Davies D, Titley J, Crompton MR 1998 Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells. Clin Exp Metastasis 16:415–426[CrossRef][Medline]
  4. Wang T, Cardiff R, Zukerberg L, Lees E, Arnold A, Schmidt E 1994 Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature 369:669–671[CrossRef][Medline]
  5. Bortner D, Rosenberg M 1997 Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E. Mol Cell Biol 17:453–459[Abstract]
  6. Rodriguez-Puebla ML, LaCava M, Conti CJ 1999 Cyclin D1 overexpression in mouse epidermis increases cyclin-dependent kinase activity and cell proliferation in vivo but does not affect skin tumor development. Cell Growth Differ 10:467–472[Abstract/Free Full Text]
  7. Fantl V, Stamp G, Andrews A, Rosewell I, Dickson C 1995 Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev 9:2364–2372[Abstract/Free Full Text]
  8. Sicinski P, Donaher J, Parker S, Li T, Fazeli A, Gardner H, Haslam S, Bronson R, Elledge S, Weinberg R 1995 Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82:621–630[CrossRef][Medline]
  9. Imagawa W, Yang J, Guzman R, Nandi S 1994 Control of mammary gland development. In: Knobil E, Neill JD, eds. The physiology of reproduction, ed. 2. New York: Raven Press, Ltd.; 1033–1063
  10. DiAugustine R, Richards R, Sebastian J 1997 EGF-related peptides and their receptors in mammary gland development. J Mammary Gland Biol Neoplasia 2:109–117[CrossRef][Medline]
  11. Schroeder JA, Lee DC 1997 Transgenic mice reveal roles for TGF{alpha} and EGF receptor in mammary gland development and neoplasia. J Mammary Gland Biol Neoplasia 2:119–29[CrossRef][Medline]
  12. Bocchinfuso W, Korach K 1997 Mammary gland development and tumorigenesis in estrogen receptor knockout mice. J Mammary Gland Biol Neoplasia 2:323–334[CrossRef][Medline]
  13. Humphreys R, Lydon J, O’Malley B, Rosen J 1997 Use of PRKO mice to study the role of progesterone in mammary gland development. J Mammary Gland Biol Neoplasia 2:343–354[CrossRef][Medline]
  14. Cunha G, Young P, Hom Y, Cooke P, Taylor J, Lubahn D 1997 Elucidation of a role for stromal steroid hormone receptors in mammary gland growth and development using tissue recombinants. J Mammary Gland Biol Neoplasia 2:393–402[CrossRef][Medline]
  15. Fendrick J, Raafat A, Haslam S 1998 Mammary gland growth and development from the postnatal period to postmenopause: ovarian steroid receptor ontogeny and regulation in the mouse. J Mammary Gland Biol Neoplasia 3:7–22[CrossRef][Medline]
  16. Shyamala G 1999 Progesterone signaling and mammary gland morphogenesis. J Mammary Gland Biol Neoplasia 4:89–104[CrossRef][Medline]
  17. Wood T, Richert M, Stull M, Allar M 2000 The insulin-like growth factors (IGFs) and IGF binding proteins in postnatal development of murine mammary glands. J Mammary Gland Biol Neoplasia 5:31–42[CrossRef][Medline]
  18. Kleinberg D, Feldman M, Ruan W 2000 IGF-I: an essential factor in terminal end bud formation and ductal morphogenesis. J Mammary Gland Biol Neoplasia 5:7–17[CrossRef][Medline]
  19. Fantl V, Creer A, Dillon C, Bresnick J, Jackson D, Edwards P, Rosewell I, Dickson C 2000 Fibroblast growth factor signalling and cyclin D1 function are necesary for normal mammary gland development during pregnancy. A transgenic mouse approach. Adv Exp Med Biol 480:1–7[Medline]
  20. Silberstein G 2001 Postnatal mammary gland morphogenesis. Microsc Res Tech 52:155–162[CrossRef][Medline]
  21. Luetteke N, Phillips H, Qiu T, Copeland N, Earp H, Jenkins N, Lee D 1994 The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev 8:263–278
  22. Xie W, Paterson A, Chin E, Nabell L, Kudlow J 1997 Targeted expression of a dominant negative epidermal growth factor receptor in the mammary gland of transgenic mice inhibits pubertal mammary duct development. Mol Endocrinol 11:1766–1781[Abstract/Free Full Text]
  23. Sebastian J, Richards RG, Walker MP, Wiesen JF, Werb Z, Derynck R, Hom YK, Cunha GR, DiAugustine RP 1998 Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis. Cell Growth Differ 9:777–75[Abstract]
  24. Luetteke N, Qiu T, Fenton S, Troyer K, Riedel R, Chang A, Lee D 1999 Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development. Development 126:2739–2750[Abstract]
  25. Ruan W, Kleinberg D 1999 Insulin-like growth factor I is essential for terminal end bud formation and ductal morphogenesis during mammary development. Endocrinology 140:5075–5081[Abstract/Free Full Text]
  26. Imagawa W, Tomooka Y, Hamamoto S, Nandi S 1985 Stimulation of mammary epithelial cell growth in vitro: interaction of epidermal growth factor and mammogenic hormones. Endocrinology 116:1514–24[Abstract]
  27. Imagawa W, Spencer E, Larson L, Nandi S 1986 Somatomedin-C substitutes for insulin for the growth of mammary epithelial cells from normal virgin mice in serum-free collagen gel cell culture. Endocrinology 119:2695–2699[Abstract]
  28. Coleman S, Silberstein G, Daniel C 1988 Ductal morphogenesis in the mouse mammary gland: evidence supporting a role for epidermal growth factor. Dev Biol 127:304–315[CrossRef][Medline]
  29. Gabelman BM, Emerman JT 1992 Effects of estrogen, epidermal growth factor, and transforming growth factor-{alpha} on the growth of human breast epithelial cells in primary culture. Exp Cell Res 201:113–118[CrossRef][Medline]
  30. Fenton SE, Sheffield LG 1994 Control of mammary epithelial cell DNA synthesis by epidermal growth factor, cholera toxin, and IGF-1: specific inhibitory effect of prolactin on EGF-stimulated cell growth. Exp Cell Res 210:102–106[CrossRef][Medline]
  31. Yamamoto K, Hirai A, Ban T, Saito J, Tahara K, Terano T, Tamura Y, Saito Y, Kitagawa M 1996 Thyrotropin induces G1 cyclin expression and accelerates G1 phase after insulin-like growth factor I stimulation in FRTL-5 cells. Endocrinology 137:2036–2042[Abstract]
  32. Furlanetto RW, Harwell SE, Frick KK 1994 Insulin-like growth factor-I induces cyclin-D1 expression in MG63 human osteosarcoma cells in vitro. Mol Endocrinol 8:510–517[Abstract]
  33. Perry JE, Grossmann ME, Tindall DJ 1998 Epidermal growth factor induces cyclin D1 in a human prostate cancer cell line. Prostate 35:117–124[CrossRef][Medline]
  34. Kornmann M, Arber N, Korc M 1998 Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum. J Clin Invest 101:344–352[Medline]
  35. Dupont J, Karas M, LeRoith D 2000 The potentiation of estrogen on insulin-like growth factor I action in MCF-7 human breast cancer cells includes cell cycle components. J Biol Chem 275:35893–35901[Abstract/Free Full Text]
  36. Shambaugh 3rd GE, Lee RJ, Watanabe G, Erfurth F, Karnezis AN, Koch AE, Haines 3rd GK, Halloran M, Brody BA, Pestell RG 1996 Reduced cyclin D1 expression in the cerebella of nutritionally deprived rats correlates with developmental delay and decreased cellular DNA synthesis. J Neuropathol Exp Neurol 55:1009–1020[Medline]
  37. Altucci L, Addeo R, Cicatiello L, Dauvois S, Parker MG, Truss M, Beato M, Sica V, Bresciani F, Weisz A 1996 17ß-Estradiol induces cyclin D1 gene transcription, p36D1–p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells. Oncogene 12:2315–2324[Medline]
  38. Dufourny B, van Teeffelen HA, Hamelers IH, Sussenbach JS, Steenbergh PH 2000 Stabilization of cyclin D1 mRNA via the phosphatidylinositol 3-kinase pathway in MCF-7 human breast cancer cells. J Endocrinol 166:329–338[Abstract]
  39. Dupont J, Le Roith D 2001 Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects. Mol Pathol 54:149–154[Abstract/Free Full Text]
  40. Ravitz MJ, Yan S, Dolce C, Kinniburgh AJ, Wenner CE 1996 Differential regulation of p27 and cyclin D1 by TGF-ß and EGF in C3H 10T1/2 mouse fibroblasts. J Cell Physiol168:510–520
  41. Takuwa N, Fukui Y, Takuwa Y 1999 Cyclin D1 expression mediated by phosphatidylinositol 3-kinase through mTOR-p70(S6K)-independent signaling in growth factor-stimulated NIH 3T3 fibroblasts. Mol Cell Biol 19:1346–1358[Abstract/Free Full Text]
  42. Reiss K, Cheng W, Pierzchalski P, Kodali S, Li B, Wang S, Liu Y, Anversa P 1997 Insulin-like growth factor-1 receptor and its ligand regulate the reentry of adult ventricular myocytes into the cell cycle. Exp Cell Res 235:198–209[CrossRef][Medline]
  43. Chakravarthy MV, Abraha TW, Schwartz RJ, Fiorotto ML, Booth FW 2000 Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway. J Biol Chem 275:35942–35952[Abstract/Free Full Text]
  44. Richert M, Wood T 1999 The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions. Endocrinology 140:454–461[Abstract/Free Full Text]
  45. Barlow J, Casey T, Chiu J-F, Plaut K 1997 Estrogen affects development of alveolar structures in whole-organ culture of mouse mammary glands. Biochem Biophys Res Commun 232:340–344[CrossRef][Medline]
  46. Plaut K, Ikeda M, Vonderhaar B 1993 Role of growth hormone and insulin-like growth factor-I in mammary development. Endocrinology 133:1843–1848[Abstract]
  47. Yee D, Lee A 2000 Crosstalk between the insulin-like growth factors and estrogens in breast cancer. J Mamm Gland Biol Neoplasia 5:107–115[CrossRef][Medline]
  48. Katzenellenbogen BS 1996 Estrogen receptors: bioactivities and interactions with cell signaling pathways. Biol Reprod 54:287–293[Abstract]
  49. Lee A, Weng C-N, Jackson J, Yee D 1997 Activation of estrogen receptor-mediated gene transcription by IGF-I in humna breast cancer cells. J Endocrinol 152:39–47[Abstract/Free Full Text]
  50. Innocente S, Abrahamson J, Cogswell J, Lee J 1999 p53 regulates a G2 checkpoint through cyclin B1. Proc Natl Acad Sci USA 96:2147–2152[Abstract/Free Full Text]
  51. Taylor W, DePrimo S, Agarwal A, Agarwal M, Schonthal A, Katula K, Stark G 1999 Mechanisms of G2 arrest in response to overexpression of p53. Mol Biol Cell 10:3607–3622[Abstract/Free Full Text]
  52. Badie C, Bourhis J, Sobczak-Thepot J, Haddada H, Chiron M, Janicot M, Janot F, Tursz T, Vassal G 2000 p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line. Br J Cancer 82:642–650[CrossRef][Medline]
  53. Adesanya OO, Zhou J, Samathanam C, Powell-Braxton L, Bondy CA 1999 Insulin-like growth factor 1 is required for G2 progression in the estradiol-induced mitotic cycle. Proc Natl Acad Sci USA 96:3287–3291[Abstract/Free Full Text]
  54. Zhang H, Hoff H, Sell C 2000 Insulin-like growth factor I-mediated degradation of insulin receptor substrate-1 is inhibited by epidermal growth factor in prostate epithelial cells. J Biol Chem 275:22558–22562[Abstract/Free Full Text]
  55. Sell C, Dumenil G, Deveaud C, Miura M, Coppola D, DeAngelis T, Rubin R, Efstratiadis A, Baserga R 1994 Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts. Mol Cell Biol 14:3604–3612[Abstract/Free Full Text]
  56. Swantek J, Baserga R 1999 Prolonged activation of ERK2 by epidermal growth factor and other growth factors requires a functional insulin-like growth factor 1 receptor. Endocrinology 140:3163–3169[Abstract/Free Full Text]
  57. Jiang F, Frederick T, Wood T 2001 IGF-I synergizes with FGF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle. Dev Biol 232:414–423[CrossRef][Medline]
  58. Tonelli Q, Sorof S 1980 Epidermal growth factor requirement for development of cultured mammary gland. Nature 285:250–252[CrossRef][Medline]
  59. LeRoith D, Adamo M, Werner H, Roberts Jr CT 1991 Insulin-like growth factors, their binding proteins and receptors as growth regulators in normal physiology and pathological states. Trends Endocrinol Metab 2:134–139[CrossRef]
  60. Wiesen J, Young P, Werb Z, Cunha G 1999 Signaling through the stromal epidermal growth factor receptor is necessary for mammary ductal development. Development 126:335–344[Abstract]
  61. Coleman S, Daniel C 1990 Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor. Dev Biol 137:425–433[CrossRef][Medline]
  62. Haslam S, Counterman L, Nummy K 1992 EGF receptor regulation in normal mouse mammary gland. J Cell Physiol 152:553–557[CrossRef][Medline]
  63. Darcy KM, Wohlhueter AL, Zangani D, Vaughan MM, Russell JA, Masso-Welch PA, Varela LM, Shoemaker SF, Horn E, Lee PP, Huang RY, Ip MM 1999 Selective changes in EGF receptor expression and function during the proliferation, differentiation and apoptosis of mammary epithelial cells. Eur J Cell Biol 78:511–523[Medline]
  64. Bonnette SG, Hadsell DL 2001 Targeted disruption of the IGF-I receptor gene decreases cellular proliferation in mammary terminal end buds. Endocrinology 142:4937–4945[Abstract/Free Full Text]



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