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Department of Molecular and Integrative Physiology (W.R.H., B.S.K.), Department of Chemistry (S.H.K., J.A.K.), and Department of Cell and Developmental Biology (C.C.F., Z.M.-E., B.S.K.), University of Illinois, Urbana, Illinois 61801; and Department of Medicine (R.S.), Breast Center, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, University of Illinois, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu.
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INTRODUCTION |
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One challenge faced in studies of the nongenomic action of estrogens has been the separation of these actions from those of the genomic pathway. Some approaches have involved the use of specific inhibitors of RNA or protein synthesis that selectively block effects that are mediated by transcription or directly derived protein products, or temporal studies in which analysis of the earliest responses are presumed not to involve transcription. Another approach has been to use estrogen ligands that are capable of selective action on one or the other pathway (9, 15, 16). In this regard, frequent use has been made of estrogens conjugated to cell-impenetrant macromolecules, such as estradiol-BSA (E2-BSA) (13, 17, 18, 19) or E2-peroxidase (20, 21, 22). Such reagents are presumed to be capable of interacting only with ER that is membrane associated and thus to stimulate only the nongenomic pathway.
Despite the widespread use of these agents, however, concern has been raised about the behavior of E2-BSA and other estrogen-protein conjugates (23). The site selected to link the estrogen (C-6 or C-17) can interfere with the binding of the tethered ligand to ER and can affect the biological outcome (19). Also, the linkage between the steroid and the macromolecule is not always chemically durable, so that free ligand can leak from the conjugate. Although it is possible to remove free ligand from E2-BSA by charcoal treatment (23), macromolecules such as BSA, being a natural biopolymer, could be subject to proteolytic degradation by cells, with potential release of smaller fragments that might gain access to nuclear ER. Thus, there is room for the development of estrogen-macromolecule conjugates having improved stability, purity, and receptor binding characteristics (23).
In this report, we describe the development and characterization of one member of a new class of estrogen-macromolecule conjugates, EDCs. The EDC is constructed by the attachment of an estrogen through the 17
position, a site that preserves a good deal of ER binding affinity (24), to an abiological macromolecule, a poly(amido)amine (PAMAM) dendrimer (25), through a hydrolytically stable linkage. The use of a PAMAM dendrimer core affords flexibility in macromolecule size and surface charge, and produces conjugates that can be further functionalized with fluorophores to trace the cellular distribution of the EDCs. Furthermore, the EDC can be characterized spectroscopically and can be rigorously treated (e.g. by extraction with organic solvents) to remove free ligand. In cell-based assays reported here, we find that the EDC localizes at the membrane/cytoplasm of cells and is excluded from the nucleus. It is effective in stimulating extranuclear, nongenomic activities at low concentrations but is very ineffective in stimulating nuclear-initiated estrogen target gene expression. Thus, EDC molecules provide useful tools for studying nongenomic vs. genomic pathways regulated by estrogen.
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) (25). The EDC used in this study is based on a generation 6.0 (G-6) PAMAM dendrimer, which nominally has a molecular weight of 58,048 and 256 surface primary amine functional groups; thus, it is a polycation. PAMAM dendrimers are also available with different surface functional groups, either amines (cationic surface), alcohols (neutral surface), or carboxylic acids (anionic surface), and these surface functional groups can be used for the attachment of small molecules and fluorophore tags (25).
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-ethynylestradiol (EE2) in which the ethynyl substituent is further derivatized with a para-substituted phenyl group that incorporates an aldehyde function (see Fig. 1
, top), the latter for attachment to the primary amine groups on the G-6 PAMAM dendrimer. Coupling is achieved by mixing the estrogen aldehyde (EE2-Ph-CHO) and the dendrimer in methanol and then reducing the imine function that forms spontaneously with sodium borohydride to produce the EDC. These two steps in the chemical conversion can be followed by 1H nuclear magnetic resonance (NMR) (Kim, S. H., and J. A. Katzenellenbogen, unpublished). Because the amine functions are present in such large excess, the degree of dendrimer substitution by the estrogen can be controlled simply by adjusting the molar ratios of estrogen aldehyde to dendrimer, and the level and uniformity of estrogen substitution can be confirmed by matrix-assisted laser desorption ionization mass spectrometry. The EDC we have studied here was based on a G-6 PAMAM dendrimer derivatized with the estrogen in a 20:1 stoichiometry; matrix-assisted laser desorption ionization mass spectrometry analysis confirmed this average stoichiometry. In addition, after attachment of the estrogen derivatives, amine-reactive fluorescent reagents can be added to provide the EDC with an optional fluorophore tag, termed "EDC-TMR"; the EDC-TMR material we have studied here had an average of five TMR tags, in addition to the 20 estrogen tags. Because many free amine functions remain on the dendrimer surface, both EDCs retain a strong net positive charge.
Despite the fact that the reaction of the estrogen aldehyde with the G-6 PAMAM dendrimer appeared to be quantitative, we took great care to ensure that any remaining estrogen monomer aldehyde (or the corresponding alcohol reduction product) was removed from the EDC before it was used in biological experiments. Thus, after estrogen-dendrimer coupling, the EDC product was subject to five rounds of extraction purification by centrifugal ultrafiltration on a Centriplus column (Millipore, Bedford, MA) through which the free estrogen is repeatedly eluted with methanol. In the final product, no free estrogen was evident upon analysis by either 1H NMR, thin layer chromatography (TLC), or HPLC.
As an additional test, unlabeled E2 was added to the EDC solution at 10 equivalents excess (comparable to the concentration used for the EDC synthesis). This unlabeled steroid was spiked with 1% [3H]E2 so that the washout of free ligand could be followed radiometrically. More than 99% of the radioactivity was washed away with the first washing step. Furthermore, the trace of residual radioactivity that remained with the EDC after four methanol washes did not correspond to E2 and was most likely due to minor radiochemical impurities present in the tracer. Thus, we believe that there is less than, and likely considerably much less than, 0.1% of free estrogen in our EDC preparations, a fact confirmed by their very low potency in gene regulation assays (see below). Furthermore, as expected from the chemical stability of the amine linkage between the estrogen and PAMAM dendrimer, there was no evidence that free estrogen is released upon storage of the EDCs.
ER Binding of EDCs
Our aim in tethering the estrogen component to the PAMAM dendrimer through the 17 position with a spacer was to create a conjugate in which the estrogen unit would still retain significant binding affinity for the ER (24). To confirm this, we evaluated the ability of the EDC to compete with [3H]E2 for binding to soluble ER in a competitive radiometric binding assay (26, 27). In this assay, the estrogen unit in the EDC bound to ER with an affinity that was 3.8 ± 0.8% that of free E2, which corresponds to a dissociation constant (Kd) of 6 nM. (It is of note that in all of our studies, the concentration of the dendrimer-tethered estrogen is considered to be that of the estrogen equivalents present in the conjugate, i.e. 20-fold higher than the concentration of the dendrimer itself, because the estrogen is tethered to the dendrimer in a 20:1 ratio.) The amine corresponding to the 17-EE2 aldehyde-derivatizing agent, which is the monomer equivalent to the dendrimer-tethered estrogen, also binds to ER with an affinity 3.8% that of E2. Thus, the relative binding affinity of the bound estrogen in the EDC is comparable to that of the free estrogen derivative used in performing the conjugation, which indicates that conjugation of this ligand to the G-6 PAMAM does not reduce the ability of the ligand to interact with ER.
Cellular Localization of EDCs
We examined the cellular localization of the EDC in ER-positive MCF-7 cells and compared this with ER-negative MDA-MB-231 breast cancer cells. Cells were incubated with the TMR-tagged conjugate, EDC-TMR, and we examined cellular localization by confocal microscopy, which allowed optical sectioning throughout different levels of the cells. As shown in Fig. 2, MCF-7 cells showed bright fluorescent speckles at the membrane and in the cytoplasm, and no nuclear localization was observed (Fig. 2A). Treatment with E2 resulted in a marked suppression of cellular fluorescence (Fig. 2B), suggesting that EDC-TMR uptake in untreated cells is the result of ER binding. Notably, MCF-7 cells exposed to TMR-labeled empty dendrimer showed only very low fluorescence, comparable to that in the E2-treated cells (Fig. 2C). ER-negative MDA-MB-231 cells showed minimal uptake of EDC-TMR, with no difference being apparent in untreated (Fig. 2D) vs. E2-treated cells (Fig. 2E).
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, F and G, EDC showed a punctate pattern of extranuclear localization that was similar at both times. Hence, the EDC remains outside the nucleus for considerable periods of time. For the studies in panels F and G, we used EDC labeled with fluorescein, which confirms that the distribution is the same as observed with EDC-TMR.
Analysis of Estrogen Target Gene Expression by EDCs vs. E2
One would expect that an ER ligand that remains outside the nucleus would have attenuated capacity to activate nuclear ER-mediated transcriptional events. Therefore, we compared the relative potency of E2 and the EDC to regulate four known endogenous estrogen-responsive genes in ER-positive human breast cancer (MCF-7) cells (28). For these studies, we used two different MCF-7 cell lines: MCF-7 K1 cells, the MCF-7 cell line used in our laboratory for many years, which have high ER levels and very low levels of Her2/neu expression, and MCF-7/Her2 cells, which have high ER levels and high levels of Her2/neu due to stable overexpression (29).
Dose-response studies are shown in Fig. 3for E2 vs. EDC stimulation of pS2, WISP2, cdc6, and progesterone receptor gene expression. All four genes showed maximal stimulation by 10–11 to 10 –10 M E2, with high levels of stimulation observed throughout the concentration range studied, up to 10–7 M. By contrast, stimulation of expression of these genes by EDC was not observed until 10–7 or 10–6 M EDC (concentrations in estrogen equivalents). As expected, the empty (unconjugated) dendrimer (denoted Dend) gave no stimulation of these genes. Hence, EDCs are very ineffective in stimulating nuclear target gene expression, requiring concentrations that are approximately 10,000-fold higher than those for E2. Figure 3 presents findings from MCF-7/Her2 cells, but we observed essentially identical dose responses and magnitudes of stimulation in MCF-7 K1 cells.
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, the ER antagonist ICI 182,780 fully blocked the WISP2 gene induction by the high concentration of EDC, whereas the MEK inhibitor was ineffective. Thus, the genomic effects seen with high EDC concentrations could be the result of very small amounts (0.01–0.1%) of free estrogen ligand that remain in the EDC preparation or are liberated from the EDC during incubation with cells.
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, ERK phosphorylation was stimulated by EDC at concentrations of 10–10 M ligand and above (in E2 equivalents), and underivatized EDC (i.e. empty dendrimer) evoked no increase in ERK phosphorylation. The level of ERK phosphorylation achieved with the EDC was approximately 50% that seen with heregulin (10 ng/ml), a ligand very effective in inducing ERK phosphorylation.
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). Maximal phosphorylation was observed at 2 and 5 min, with a decline thereafter, returning to the control level at 60 min. As shown in Fig. 5C, stimulation of ERK phosphorylation by EDC or E2 was blocked by the MEK inhibitor, PD98059, as was the low level of basal ERK phosphorylation in the control, vehicle-treated cells. It is of note that at 10–8 M, a >2-fold higher level of ERK phosphorylation was achieved by the EDC than by E2.
Comparison of ERK phosphorylation by E2 and EDC over a concentration range of the two compounds (Fig. 5D) showed a more robust stimulation by the EDC compared with E2, with enhanced ERK phosphorylation over that of the control level in cells being observed even at 10–12 M EDC (in estrogen equivalents). The greater effectiveness of the EDC might reflect its multivalent character. By contrast to the ERK activation observed with the EDC or E2 in MCF-7 cells, we found that the ER-negative MDA-MB-231 cells showed no enhancement of phospho-ERK with either EDC or E2 treatment (Fig. 5E).
We also investigated activation of two factors upstream of MAPK, the tyrosine kinase c-Src and the scaffolding molecule Shc, both of which have been implicated in nongenomic signaling by E2 (30, 31, 32, 33). As seen in Fig. 6, both E2 and the EDC increased c-Src and Shc phosphorylation, EDC somewhat more robustly than observed with E2.
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, E2 evoked a concentration-dependent increase in cell proliferation that was blocked by cotreatment with the antiestrogen ICI 182,780. By contrast, the EDC, tested at several concentrations, was ineffective in increasing proliferation. As expected, the empty dendrimer (Dend) did not increase cell proliferation. The EDC or the empty dendrimer did not affect the stimulatory response to E2, indicating that their inability to elicit enhanced proliferation of MCF-7 cells was not due to any possible toxicity of these compounds. We also investigated the effect of EDC, E2, and empty dendrimer on ER-negative MDA-MB-231 cells (Fig. 7B) and found that these agents did not affect the proliferation of these cells, such that neither stimulation nor suppression was observed.
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0.01–0.1% free estrogen) than we have achieved here. It is of note that dendrimers have been used to create a variety of small molecule conjugates that have been used in different biological studies (25, 34, 35, 36, 37, 38, 39).
The EDC Shows Extranuclear Localization and Has High Potency in Stimulating Nongenomic Estrogen Action, but Is Ineffective in Stimulating Estrogen Genomic Action
By confocal microscopy, we find that EDC shows pronounced uptake in ER-positive breast cancer (MCF-7) cells that is displaced by E2, whereas very little uptake is observed in ER-negative cells. The EDC is observed in the membrane and cytoplasm but is effectively excluded from the nucleus. The E2 displacement in ER-positive cells and the very low uptake in ER-negative cells are consistent with a process that is mediated by ER. However, there are reports of an additional estrogen binding protein, GPR30, which has been proposed to mediate some of the actions of estrogens (40, 41). Although future studies are needed to determine whether it might play any role in the actions of EDC, its reported presence in both MCF-7 and MDA-MB-231 cells suggests that it may not be the mediator of EDC actions (40, 41).
The punctate pattern of the EDC observed in the membrane/cytoplasm might account for the high potency with which the EDC is able to stimulate ERK phosphorylation, an effective response being seen with as low as 0.1 nM EDC. The multivalency of the EDC could also contribute to its high potency in stimulating nongenomic signaling, because its avidity for clusters of membrane-associated ERs, localized in caveolae (8), might be considerably higher than the affinity we have measured with free ER in solution.
We have demonstrated that the EDC is very effective in stimulating three well-recognized rapid, nongenomic responses: ERK phosphorylation, the result of MAPK activation, and phosphorylation of the tyrosine kinase c-Src and the scaffolding protein Shc. In dose-response studies of ERK phosphorylation, the EDC had an effectiveness considerably greater than that of E2. ERK phosphorylation by both EDC and E2 was inhibited by the MEK inhibitor PD98059.
It was our aim to develop an agent that would be very specific in stimulating nongenomic responses. Therefore, we have been careful to establish that the EDC we studied has little to no capacity to activate genomic actions of estrogens by performing dose-response studies to assess its action on endogenous genes. Although we find that the EDC can induce transcription of estrogen-responsive genes, this response is found only at very high concentrations of EDC, 3–4 orders of magnitude greater than that needed for stimulation by E2. There is a similar differential in potency between the stimulation by EDC of ERK phosphorylation vs. its genomic activity. This behavior is consistent with the exclusion of the EDC from the nucleus, preventing its access to nuclear ER required to activate genomic functions.
Based on its sensitivity to antiestrogens but not MEK inhibitors, the residual genomic activity of the EDC, found only at very high concentrations, is likely dependent on activation of nuclear ER. This activity could thus result from exceedingly small amounts of free estrogen ligand that remain in the EDC, even after the extensive washing procedure, or small amounts of free ligand released from the EDC upon incubation with cells (despite the chemical stability of its link), or small amounts of intact EDC that might eventually find access to the nucleus. Nevertheless, because we have carefully quantified this residual genomic activity of the EDCs, it allows us to define conditions under which EDCs can be used to selectively activate only estrogen nongenomic pathways. And, as an important control, we have found that underivatized, positively charged PAMAM dendrimers have no capacity to stimulate either genomic or nongenomic actions.
To compare the effects of EDC and E2 on cell proliferation, we used two MCF-7 cell lines that both express high levels of ER, but very different levels of Her2/neu. Because we found that MCF-7/Her2 cells had high rates of cell proliferation that could be only minimally increased by E2, as reported previously (42), whereas cell proliferation was markedly enhanced by E2 in MCF-7 K1 cells that have very low Her2/neu levels, we compared the effects of EDC vs. E2 in these MCF-7 cells. Using the EDC, we found that this agent was unable to elicit any stimulation of the proliferation of MCF-7 K1 cells, whereas E2 gave very robust stimulation. These findings imply that estrogen stimulation of proliferation in these cells requires ER nuclear genomic actions. The actions of EDC vs. E2 on other biological endpoints known to be influenced by estrogen in breast cancer cells, such as cytoskeletal reorganization, cell motility, etc., still remain to be investigated.
EDCs Should Prove Useful as Tools to Study the Nongenomic Pathway of Estrogen Signaling
The EDC we have described in this report is capable of activating the nongenomic estrogen signaling pathway at subnanomolar concentrations and has minimal capacity to stimulate estrogen genomic signaling. Thus, this agent, as well as similar ones under development in our laboratories, should prove to be useful tools for studying the biological consequences of estrogen action through these two pathways. With these PAMAM dendrimer-based estrogen conjugates it should be possible to parse the nongenomic vs. genomic pathways of estrogens and their interrelationships to understand further the complexities and important biology of estrogen action in a variety of estrogen target cells.
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-EE2 (Steraloids, Newport, RI), PAMAM generation-6 dendrimer (Dendritech, Midland, MI), Centriplus YM-30 filter (Millipore), tetramethylrhodamine-6-carboxylic acid succinimide ester and fluorescein-6-carboxylic acid succinimide ester (Invitrogen/Molecular Probes, Eugene, OR), 17ß-E2 (Sigma, St. Louis, MO).
Preparation of the EDC and Fluorophore-Labeled EDCs
Chemical details of the synthesis of the EDC and the TMR-labeled conjugate (EDC-TMR) will be described elsewhere (Kim, S. H., and J. A. Katzenellenbogen, unpublished). Briefly, EE2 was converted to the benzaldehyde derivative (EE2-Ph-CHO; Fig. 1) by a palladium-mediated coupling with p-bromobenzaldehyde. Attachment of this derivative to the G-6 PAMAM dendrimer was achieved by a two-step reductive amination: EE2-Ph-CHO and the PAMAM dendrimer were first mixed in methanol at a 20:1 stoichiometry and were allowed to stir at room temperature until free EE2-Ph-CHO could no longer be detected by 1H NMR analysis; sodium borohydride was then added, and the reaction was continued until the 1H NMR peak for the imine proton could no longer be detected. The reaction mixture was transferred to a filter (Centriplus YM-30, cut-off molecular weight 30,000), diluted with methanol, and centrifuged at approximately 3000 x g until the entire solution had passed through the filter, a procedure that was repeated a total of five times. The concentrated residue was dried under vacuum to afford a yellowish solid, EDC, which was immediately dissolved in deionized water. For the preparation of TMR-labeled EDC (EDC-TMR), the following step was inserted before the purification: five equivalents of tetramethylrhodamine-6-carboxylic acid succinimide ester were added to the reaction solution, and the mixture was stirred for 5 h. An identical procedure, using fluorescein-6-carboxylic acid succinimide ester, was used to prepare the corresponding fluorescein-EDC derivative. Matrix-assisted laser desorption mass spectrometric analysis confirmed that the EDC preparation contains approximately 20 molecules of E2 ligand per PAMAM dendrimer; the EDC-TMR and the fluorescein-labeled EDC also contain two to three molecules of fluorophore. These EDCs were stored in methanol at –20 C.
Radiometric Assays to Follow Removal of Free Ligand
To a sample of purified EDC solution (0.17 µmol based on the PAMAM dendrimer concentration at the beginning of experiment) in methanol (10.7 ml) was added 10x E2 (1.7 µmol with 1% [3H]E2). This mixture was placed in the Amicon filter and centrifuged, as above. More than 99% of the activity was removed by the first washing, but the sample was washed three more times. Although the final EDC residue still retained 0.46% of the radioactivity, this material was not E2. A concentrated sample of the EDC was subjected to TLC in methanol using plastic-backed silica TLC plates with fluorescent indicator (Merck, Darmstadt, Germany). In this system, free E2 migrates with an Rf of 1 (solvent front) and the EDC remains at the origin. Most of the radioactivity in the washed sample remained with the EDC (Rf = 0); no radioactivity was detected with the free E2. The activity that remains with the EDC and resists extraction with organic solvents is believed to correspond to oxidized radiolytic products of E2 that react covalently with the free amines of the EDC.
ER Binding Affinity Assays
Relative binding affinities were determined by a competitive radiometric binding assay as previously described (27), using 10 nM [3H]E2 as tracer (Amersham Biosciences, Piscataway, NJ), and purified full-length human ER (PanVera/Invitrogen, Carlsbad, CA). The dendrimer conjugates were assayed from 10–4 to 10–9 M as equivalents of E2 (20-fold lower than the molar concentration of the dendrimer itself). Incubations were for 18–24 h at 0 C, and the receptor-dendrimer complexes were adsorbed onto hydroxyapatite (Bio-Rad Laboratories, Inc., Hercules, CA), and the unbound dendrimer was washed away. The binding affinities are expressed as relative binding affinity values, with the relative binding affinity of E2 being set at 100. The values given are the average ± range or SD for two or more independent determinations. E2 binds to ER with a Kd of 0.2 nM.
Fluorescence Microscopy
The cellular localization of TMR-conjugated EDC, fluorescein-labeled EDC, or TMR-conjugated empty dendrimer was assessed by confocal fluorescence microscopy. For these studies, cells were treated with fluorophore-labeled EDC (100 nM estrogen equivalents) in the absence or presence of a 30-fold excess of E2, or with fluorophore-labeled empty dendrimer, for 45 min or 15 h. Cells were then washed in PBS-5 mM MgCl2-0.1 mM EDTA solution with 0.1% Triton-X for 30 sec, fixed on glass coverslips in 1.6% paraformaldehyde for 60 min, washed three times for 5 min in PBS-5 mM MgCl2-0.1 mM EDTA solution with 2 mM glycine, and then washed three times for 5 min in PBS-5 mM MgCl2-0.1 mM EDTA solution, mounted, and stained using Vectashield HardSet Mounting Medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) to identify the nuclei. Samples were observed through a Nikon TE 2000-S inverted microscope and a CARV spinning disk confocal (Atto Bioscience, Rockville, MD). Fluorescence images (20 optical sections per cell) were collected using a CoolSnap HQ camera (Photometrics, Tucson, AZ) and Metamorph software v6.1.
Cell Culture, RNA Extraction, and Real-Time PCR Analysis of Gene Expression Regulation
Studies used two MCF-7 human breast cancer cell lines. MCF-7 K1 human breast cancer cells, which contain high levels of ER and low Her2/neu, were maintained in culture exactly as previously described (28). MCF-7/Her2 cells (29), which have high levels of ER and high levels of Her2/neu expression, were maintained as previously described (42), in DMEM (Cambrex Bioscience, Walkersville, MD) supplemented with 10% fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA) and 60 ng/ml bovine insulin (Sigma-Aldrich Corp., St. Louis, MO). At 6 d before E2 treatment, cells were switched to phenol red-free media containing charcoal-dextran-treated calf serum. Medium was changed on d 2 and d 4 of culture, and cells were then treated with compounds as indicated. After cell treatments, total RNA was isolated, reverse-transcribed, and analyzed by real-time PCR exactly as described previously (28). Primers for the estrogen-regulated genes cdc6, progesterone receptor, pS2, and WISP2 have been described previously (28, 43).
Assays for Determination of ERK, Shc, and Src Phosphorylation
MCF-7/Her 2 and MCF-7 K1 cells were plated in 10-cm2 plates (106 cells per plate). After plating and overnight incubation in phenol red-free media containing CD-treated serum, cells were grown for 2 d in phenol red-free media containing 0.5% CD-FCS and were subsequently grown in serum-free medium for 24 h before treatment. After treatment with the indicated compounds for various times, cells were rinsed with cold PBS and then lysed by incubation with 500 µl per plate of cell lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with phenylmethylsulfonylfluoride (10 µM). Cell lysates were collected by scraping and were then sonicated (three times for 10 sec on ice) and centrifuged at 14,000 rpm. Cell supernatants were aliquoted and stored at –80 C. Protein concentration was determined using BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL). Proteins (20 µg) were separated by SDS-PAGE using 10% polyacrylamide gels containing sodium dodecyl sulfate and transferred onto nitrocellulose membranes (Pall, Pensacola, FL). Primary antibodies used for Western blotting were phospho-p44/42 MAPK (Thr202/Tyr204), p44/p42 MAPK Antibody, phospho-Shc (Tyr317), phospho-Src (Tyr416) (Cell Signaling Technology) and ß-actin (Sigma-Aldrich Corp.). Western blotting was performed according to the manufacturer’s instructions.
Proliferation Assays
Before being plated for proliferation assays, MCF-7 K1 or MCF-7/Her 2 cells were grown for 1 wk in phenol red-free MEM containing 5% charcoal-dextran-treated calf serum (CD-CS) or Improved MEM containing 10% CD-FCS, respectively. Cells were then plated (1000 cells/well) in 96-well plates in either MEM containing 5% CD-CS (MCF-7 K1) or Improved MEM containing 0.5% or 5% CD-FCS (MCF-7/Her2). ER-negative MDA-MB-231 cells were grown and processed for proliferation assays as described in our studies with MDA-MB-231 cells stably expressing various ERs (44). After plating and overnight attachment, cells were treated with appropriate ligands or 0.1% control ethanol vehicle for 6 d, with ligand/media changed on d 2 and d 4. Cell proliferation was assessed over 6 d using the MTS tetrazolium colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI) according to the manufacturer’s protocol.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CD, Charcoal dextran; E2, Estradiol; EDC, estrogen dendrimer conjugate; EDC-TMR, tetramethylrhodamine-labeled EDC; EE2, ethynyl E2; ER, estrogen receptor; FCS, fetal calf serum; MEK, MAPK kinase; NMR, nuclear magnetic resonance; PAMAM, poly(amido)amine; TLC, thin layer chromatography; TMR, tetramethylrhodamine.
This work was supported by grants from the National Institutes of Health [NIH CA 18119 (to B.S.K.), DK 15556 (to J.A.K.); National Cancer Institute P50 CA 058183 (Breast Cancer Specialized Program of Research Excellence Grant, to R.S.)]; and a grant from the Breast Cancer Research Foundation (to B.S.K.). W.R.H. and C.C.F. received support from NIH Grants T32 HD 07028 and T32 ES 07326, respectively.
Received for publication May 9, 2005. Accepted for publication November 15, 2005.
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localized in caveolae. Biochem Biophys Res Commun 263:257–262[CrossRef][Medline] at the plasma membrane. Mol Cell Biol 23:1633–1646-Shc association and Shc pathway activation. Mol Endocrinol 16:116–127 to the plasma membrane. Proc Natl Acad Sci USA 101:2076–2081 or ERß in human osteosarcoma cells: distinct and common target genes for these receptors. Endocrinology 145:3473–3486NURSA Molecule Pages Link:
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H. Abe, K. L. Keen, and E. Terasawa Rapid Action of Estrogens on Intracellular Calcium Oscillations in Primate Luteinizing Hormone-Releasing Hormone-1 Neurons Endocrinology, March 1, 2008; 149(3): 1155 - 1162. [Abstract] [Full Text] [PDF]
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 S. R. Hammes and E. R. Levin Extranuclear Steroid Receptors: Nature and Actions Endocr. Rev., December 1, 2007; 28(7): 726 - 741. [Abstract] [Full Text] [PDF]
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M. Chang, K.-w. Peng, I. Kastrati, C. R. Overk, Z.-H. Qin, P. Yao, J. L. Bolton, and G. R. J. Thatcher Activation of Estrogen Receptor-Mediated Gene Transcription by the Equine Estrogen Metabolite, 4-Methoxyequilenin, in Human Breast Cancer Cells Endocrinology, October 1, 2007; 148(10): 4793 - 4802. [Abstract] [Full Text] [PDF]
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 N. Heldring, A. Pike, S. Andersson, J. Matthews, G. Cheng, J. Hartman, M. Tujague, A. Strom, E. Treuter, M. Warner, et al. Estrogen Receptors: How Do They Signal and What Are Their Targets Physiol Rev, July 1, 2007; 87(3): 905 - 931. [Abstract] [Full Text] [PDF]
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C. M. Silva and M. A. Shupnik Integration of Steroid and Growth Factor Pathways in Breast Cancer: Focus on Signal Transducers and Activators of Transcription and Their Potential Role in Resistance Mol. Endocrinol., July 1, 2007; 21(7): 1499 - 1512. [Abstract] [Full Text] [PDF]
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 S. Kousteni, M. Almeida, L. Han, T. Bellido, R. L. Jilka, and S. C. Manolagas Induction of Osteoblast Differentiation by Selective Activation of Kinase-Mediated Actions of the Estrogen Receptor Mol. Cell. Biol., February 15, 2007; 27(4): 1516 - 1530. [Abstract] [Full Text] [PDF]
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 S. Tang, Z. Zhang, S. L. Tan, M.-H. E. Tang, A. P. Kumar, S. K. Ramadoss, and V. B. Bajic KBERG: KnowledgeBase for Estrogen Responsive Genes Nucleic Acids Res., January 12, 2007; 35(suppl_1): D732 - D736. [Abstract] [Full Text] [PDF]
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V. S. Likhite, F. Stossi, K. Kim, B. S. Katzenellenbogen, and J. A. Katzenellenbogen Kinase-Specific Phosphorylation of the Estrogen Receptor Changes Receptor Interactions with Ligand, Deoxyribonucleic Acid, and Coregulators Associated with Alterations in Estrogen and Tamoxifen Activity Mol. Endocrinol., December 1, 2006; 20(12): 3120 - 3132. [Abstract] [Full Text] [PDF]
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 R. X.-D. Song and R. J. Santen Membrane Initiated Estrogen Signaling in Breast Cancer Biol Reprod, July 1, 2006; 75(1): 9 - 16. [Abstract] [Full Text] [PDF]
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