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Institut National de la Santé et de la Recherche Médicale, Unité 482, 75012 Paris, France
Address all correspondence and requests for reprints to: Anne-Marie Gaben, Institut National de la Santé et de la Recherche Médicale, Unité 482, 184 rue du Faubourg Saint Antoine, 75012 Paris, France. E-mail: gaben{at}st-antoine.inserm.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ER isoforms 
and ß have been shown to act as ligand-regulated transcription factors. (In breast cancer, the isoform is responsible for the estrogen dependence of the tumor, and we shall further use the abbreviation ER to denote this isoform.) Additional factors named coactivators and corepressors participate in the regulation of transcription by the ER, allowing a fine modulation of the level of gene expression as a function of the cellular context as well as of the ligand (see Refs.1 and 2 for reviews). According to the cell type, a given ER ligand can induce or not the expression of a particular gene. In addition, several synthetic molecules that bind to the ER have been designed, but they do not allow its transcriptional activity and thus act as pure antiestrogens. Transcriptional regulation does not account for all of the effects of estrogens. For instance, estrogen-induced vasodilatation is rapid and relies on the activation of nitric oxide synthase by a nongenomic mechanism that appears to involve trimeric G proteins (3) and Akt (4, 5). As a matter of fact, direct interaction between the ER and phosphatidylinositol 3-kinase (PI-3K) has been demonstrated (6, 7, 8). Upon activation by products of PI-3K, Akt phosphorylates downstream targets that stimulate growth and inhibit apoptosis. The antiapoptotic action of estrogens (and androgens, too) in neurons (9) as well as osteoblasts and osteocytes (10) has also been reported to proceed via nongenomic mechanisms requiring the ligand-binding domain of the ER molecule and limited to extranuclear cell compartments.
As far as the induction of cell proliferation by estrogens is concerned, it has been shown that the expression of several genes with important functions in the cell cycle is induced directly (c-fos;c-myc) or indirectly (cyclin D1) by estradiol, through the interaction of the ligand-activated ER with specific sequences in the promoters (11, 12, 13). On the other hand, several groups reported that estradiol induces a rapid activation of MAPK/ERK in breast cancer cells (14, 15, 16), putting in doubt the initial consensus postulating that the mitogenic effects of estrogens rely on transcriptional mechanisms. Similarly, a rapid, nontranscriptional activation of PI-3K by estradiol in breast cancer cells has been reported (17, 18).
The induction of MAPK/ERK activity by estradiol in breast cancer cells has been questioned by Caristi et al. (19), who reported that the activation of this enzyme by estradiol is a poorly reproducible event and can be observed also in mock-stimulated cells. Lobenhofer et al. (20, 21) also failed to detect any activation of MAPK by estradiol in the MCF-7 cells. These authors have also addressed the question of the activation of the PI-3K in this experimental model, with a negative result: there was no phosphorylation of Akt after estradiol treatment.
The most intriguing data are those of Auricchio’s group (Ref.17 and references therein). An extensive series of studies of this group led to the conclusion that in fact the mitogenic activity of estrogens is exclusively nongenomic, resulting from the activation of the PI-3K/Akt pathway, both in breast cancer cells and in fibroblasts (NIH3T3) transiently transfected with ER. This radical conclusion is in contrast with the observations made in several stable transfection models that have shown that introduction of ER into nonestrogen target cells does not confer inducibility of the cell cycle by estrogens (22, 23, 24, 25) and renders irrelevant the transcriptional regulation of cell cycle-related genes by estrogens. In the experiments described here, we have reexamined the mechanisms of the mitogenic action of estrogens.
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RESULTS |
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). The activating phosphorylation of MAPK/ERK was evaluated by Western blotting with the antibody against the phosphoThr202/Tyr204 epitope. The results showed that the MAPK/ERK phosphorylation observed at early time points was not due to the action of estradiol per se but rather to the fact that cells were placed in a fresh culture medium; indeed, the same phospho-MAPK/ERK signal was detected at 5 min when cells were placed in fresh medium with or without estradiol or ICI 182780 (compare lanes 2, 4, and 8). In contrast, there was no rapid effect on the phosphorylation of the MAPK/ERK when estradiol in excess was added directly into the culture medium with the antiestrogen. The addition of phorbol 12-myristate 13-acetate used as a positive control caused a rapid and transient induction of the MAPK/ERK phosphorylation (lane 12).
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We have then verified the effects of estradiol in two other breast cancer cell lines positive for ER, ZR-75.1, and T47D (Fig. 1B
). Estradiol did not induce a rapid phosphorylation of MAPK/ERK in the T47D cells. In the ZR-75.1 cells, phosphorylation of MAPK/ERK was rapidly and transiently induced by manipulation of the dishes, irrespective of whether estradiol (lane 3) or vehicle was added (lanes 2 and 4). In both T47D and ZR-75.1 cell lines, phorbol myristrate acetate induced MAPK/ERK phosphorylation as early as after 5 min (lanes 8 and 16). Flow cytometry of propidium iodide-stained nuclei showed that the cell cycle of the T47D cells was less markedly inhibited by estrogen deprivation than the MCF-7 cells, and that there was no inhibition in the ZR-75.1 cell line used in our experiments despite the presence of a functional ER in both these cell lines (data not shown).
The absence of rapid induction of MAPK/ERK phosphorylation by estradiol was also confirmed in the MCF-7, ZR-75.1, and T47D cells preincubated in phenol red-free DMEM without antiestrogen (data not shown).
2. Estradiol Does Not Induce PI-3K
The same type of experiments as those described for MAPK were carried out to evaluate the activation of PI-3K by estradiol.
Akt and S6 kinases (p70 and p85), downstream targets of PI-3K, were used as indicators of the activity of PI-3K. When MCF-7 cells were treated with insulin (agonist ligand of the insulin receptor as well as IGF receptor type I, activators of PI-3K), a strong signal of phosphorylated Akt was visualized with the anti-phosphoSer473Akt antibody at 5 min (Fig. 2A, lane 5) as well as at long time intervals (Fig. 2A, lanes 8 and 12). In quiescent cells, this antibody revealed a doublet, probably nonspecific because it was not eliminated when cells were incubated with LY294002, a powerful inhibitor of PI-3K (Fig. 2B, lane 1 vs. 3). The S6 kinases (p70 and p85) in quiescent MCF-7 cells were predominantly in their unphosphorylated, fast-migrating form. This form was the only one detected after incubation with LY294002 (Fig. 2B). When cells were exposed to estradiol for 5 min, there was no effect on the phosphorylation of Akt or of S6 kinases (Fig. 2A, lanes 2 and 3). At longer time periods (1–6 h), estradiol induced phosphorylation of S6 kinases (Fig. 2A, lanes 6, 7, 9, and 10), whereas Akt remained unphosphorylated. As expected, insulin caused a rapid and lasting phosphorylation of S6 kinases. The mitogenic action of estradiol (Fig. 2A, lanes 9 and 10) and of insulin, to a lesser extent (lane 12), was confirmed by the accumulation of cyclin D1 at t = 6 h.
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In the two other ER-positive breast cancer cell lines, T47D and ZR-75.1, estradiol failed to induce the phosphorylation of Akt (Fig. 2C), indicating that the absence of effect of estradiol on the activity of PI-3K is not restricted to the MCF-7 cell line studied in this work. There was a strong cyclin D1 signal in the ZR-75.1 cell line, irrespective of the treatment; in the T47D cells exposed to ICI 182780, cyclin D1 level was low and weakly induced by estradiol.
The absence of rapid induction by estradiol of the phosphorylation of Akt was also confirmed when cells (MCF-7, ZR-75.1, T47D) were preincubated in phenol red-free DMEM without antiestrogen (data not shown).
3. Pharmacological Inhibitors of Kinase Cascades Interfere with the G1 Phase Progression
To determine whether the activities of protein kinases known for their cell cycle regulatory activities are involved in the estrogen-stimulated G1 phase progression in breast cancer cells, we used inhibitors of src (PP1), PI-3K (LY294002), and MAPK (U0126). When added simultaneously with estradiol to quiescent MCF-7 cells, PP1 and LY294002 strongly inhibited the phosphorylation of the retinoblastoma protein (Rb) as well as the induction of the expression of cyclin D1 in early- and mid-G1 phase. U0126 caused only a partial inhibition of Rb phosphorylation and had little or no effect on the induction of cyclin D1 (Fig. 3). As expected, U0126 blocked the phosphorylation of Erk1 and Erk2, whereas LY294002 as well as PP1 had little effect. In agreement with the inhibition of phosphorylation of Rb, LY294002 and PP1 blocked the entry of the cells into S-phase, whereas U0126 inhibited approximately 50% of the effect of estradiol (Fig. 4).
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). This effect was not observed within the shorter time interval (8 h), indicating that late G1-phase cells were not prevented from entering S-phase. It is important to note at this point that the evaluation of S-phase cells by [3H]thymidine pulses led to artifactual data, indicating that the pharmacological kinase inhibitors interfered with processes required for the incorporation of thymidine, and not only with the G1-phase progression. This conclusion was verified in experiments with synchronized cells (data not shown).
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), indicating that the activity of the respective protein kinases is required for the mitogenic signaling processes also in late G1 phase. However, under these conditions there was only a weak effect in terms of G1/S transition (Fig. 4). The replacement of estradiol by the antiestrogen ICI 182780 in the culture medium after 12 h of stimulation resulted in a similar partial dephosphorylation of Rb (Fig. 5), without any effect on the entry into S-phase (data not shown). In contrast, the MAPK kinase (MAPKK) antagonist U0126 had little or no effect at this stage, either on Rb phosphorylation or on the G1/S transition (Figs. 4 and 5).
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and ß, alone or combined, failed to alter the activity of the estrogen-induced activity of the cyclin A promoter (data not shown). However, the siRNA targeting Akt 1 and 2 had a powerful inhibitory action (
80%) (Fig. 6A). The estrogen-dependent transcription directed by the vitellogenin estrogen response element (ERE) element used as a control was not modified by the expression of this siRNA vector (Fig. 6B).
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65%) of the activation of the cyclin A promoter in the estradiol-stimulated MCF-7 cells (Fig. 6A). Remarkably, the ERE-directed expression of the indicator gene was also inhibited by the MAPK/ERK siRNA (by 70%; Fig. 6B). The activity of ß-galactosidase (used as indicator of transfection efficiency) was not altered by cotransfection of the siRNA vector (data not shown).
As expected, a decreased level of Akt and MAPK/ERK1,2 proteins was observed in cells electroporated with specific siRNA expression plasmids (Fig. 6C). This decrease was related to the fraction of ß-galactosidase-positive cells (40%).
Preincubation of MCF-7 cells with the antiestrogen ICI 182780 leads to a strong depletion of the ER (29). The experiment shown in Fig. 6B indicated that this depletion of the receptor did not preclude the ERE-dependent transcriptional activation by estradiol, manifestly because a sufficient level of ER remains present. This fact is further documented by our data obtained earlier with the MELN cell line derived from the MCF-7 cells by stable transfection with the ERE-luciferase expression vector. When the MELN cells were preincubated with ICI 182780 and subsequently stimulated by estradiol, the expression of the transgene was rapidly induced (30). We compared the preincubation of MELN cells in media containing ICI 182780, 4-OH-tamoxifen, another antiestrogen that does not deplete the ER but causes its subcellular redistribution (31) and inhibits a part of its transcriptional activities (32), or without any added drug (Table 2). The cells were then placed in the presence of the same drug as during preincubation, or in a medium containing estradiol (in the absence of antiestrogen). The results showed that 1) the presence of antiestrogens strongly reduced (4-OH-tamoxifen) or abolished (ICI 182780) the residual activity of the ERE promoter; 2) the expression of the indicator gene after stimulation with estradiol was reduced by approximately 30% in cells preincubated with ICI 182780, and practically unaltered by preincubation with 4-OH-tamoxifen. We conclude that, despite the depletion of the receptor by ICI 182780, estradiol induces a near-maximal transcriptional activation of estrogen regulated genes.
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). The presence of insulin in the low-serum culture medium led to a high basal cyclin A promoter activity, and in these conditions, estradiol caused a strong inhibition of luciferase expression (Fig. 7B). When cells were transfected with the ER mutant without a functional DNA binding domain, estradiol had no effect on the activity of the cyclin A promoter. The functional activity of the wild-type ER was confirmed by the induction of the ERE-directed luciferase expression (Fig. 7A, inset).
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) showed a mixed cytoplasmic/nuclear staining. In cells transfected with the wild-type receptor and stimulated with estradiol, the staining was exclusively nuclear (Fig. 8B), whereas in cells transfected with the DNA binding-deficient mutant the receptor remained distributed between the cytoplasm and nucleus also in the presence of estradiol (data not shown). In a sharp contrast with the data published by the group of Auricchio (17), we have failed to observe any induction of BrdU incorporation by estradiol in NIH3T3 cells transfected with the ER (Fig. 8, A vs. B). Cells deprived of serum in the presence of insulin showed a higher baseline incorporation of BrdU (20%; data not shown) than those kept in the absence of insulin (10%). This percentage was not increased in cells positive for the ER, wild-type or DNA binding-deficient, treated or not with estradiol. On the contrary, among cells positive for ER, less than 10% incorporated BrdU, irrespective of stimulation or not with estradiol. Positive control cells, transfected with empty vector and stimulated with 10% NBBS, entered massively into S-phase as detected by BrdU incorporation (40% positive cells during the 2-h pulse; Fig. 8C).
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DISCUSSION |
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A second mechanism proposed for the mitogenic effects of estrogens is nontranscriptional. A series of reports in the course of the last years has led to the affirmation that the mitogenic action of estrogens is in fact entirely independent of their transcription-promoting activity, and relies on the activation of MAPK/ERK and PI-3K/Akt. The demonstration of nontranscriptional actions of estrogens in vascular endothelial cells gives an incidental support to this hypothesis.
Our data do not support the nontranscriptional mechanisms of the mitogenic action of estradiol. In the breast cancer cells MCF-7, estradiol is a powerful mitogen but fails to rapidly induce the phosphorylation of MAPK/ERK. The induction observed at long time intervals (hours of treatment) is in accordance with a possible indirect mechanism (34, 35). Overall, our results suggest that kinases of the early cascade (MAPK/ERK, PI-3K/Akt, src) play a role in the G1 phase progression in hormone-dependent breast cancer cells, but are not induced by estradiol. Lobenhofer et al. (20, 21) reached similar conclusions. Our experiments show that a total block of the activating phosphorylation of MAPK/ERK by U0126, however, did not prevent but only partially inhibited the mitogenic actions of estradiol. We also confirm the report of Caristi et al. (19) who pointed out the sensitivity of MAPK/ERK to incidental manipulations such as changes of temperature. In addition, we note that during estrogen and serum starvation the cells deplete the culture medium of certain nutrients, and simple replacement of the medium can lead to MAPK/ERK phosphorylation, possibly through cellular sensors of essential amino acids. The activation of MAPK by refeeding the cells does not lead to G1 phase progression as detected for instance by expression of cyclin D1 or phosphorylation of Rb.
We also did not detect phosphorylation of Akt within minutes of stimulation with estradiol, in contrast to the powerful effect of insulin used as a positive control of direct PI3-K activation. It has been reported that, although ER forms complexes with PI-3K (7, 8), this binding is ligand independent and can lead to the activation of ER transcription-regulating function rather than the opposite, the activation of PI-3K by the ER. At longer times, estradiol did induce the phosphorylation of S6 kinases p70 and p85. This delayed effect of estradiol can be indirect, for instance transmitted by an estrogen-induced autocrine factor (35, 36, 37), or could result from alterations in the activities of the phosphatases responsible for the dephosphorylation of S6 kinases. The question is not definitively settled as inhibitors of translation (cycloheximide) and transcription (actinomycin D) themselves induce phosphorylation of S6 kinases.
In this context, it should be noted that paradoxical observations concerning enzymes such as MAPK/ERK and PI-3K are abounding. For instance, Tsai et al. (38) have reported Akt activation by estrogen in ER-negative breast cancer cells. Observations of this sort are clearly without physiological significance: estrogens and antiestrogens are (unfortunately) devoid of any activity on the proliferation of ER-negative breast cancer cells.
As the studies on the mitogenic effects of estradiol presented in this work were carried out with cells preincubated with the antiestrogen ICI 182780, known to down-regulate the ER, one may suspect that the low level of the receptor was responsible for the absence of observable effects on MAPK/ERK and Akt phosphorylation. Two points are to be noted in this context. First, we have verified that estradiol does not induce rapid MAPK/ERK and Akt phosphorylations in cells preincubated in the absence of ICI 182780. Second, despite the down-regulation of the receptor, estradiol induces rapidly and efficiently the transcription of its target genes (Fig. 6B; Table 2; and Ref.30). Incidentally, our experiments (Table 2) indicate that estrogen deprivation is not easily achieved by a simple incubation of cells in estrogen-free medium: estradiol tends to remain associated with cell structures (ER or other) and can only be efficiently dislodged by exchange for an antiestrogen molecule.
The fact that estrogens do not induce the enzymes of the early kinase cascade by a direct, nontranscriptional mechanism does not imply that these enzymes are dispensable for the progression through the G1 phase in estrogen-stimulated cells. Chemical inhibitors of src and PI-3K inhibited the phosphorylation of Rb and G1/S transition, even when added as late as 12 h after stimulation of quiescent MCF-7 cells with estradiol. Note that, at this time, arrest of estrogenic stimulation by replacement of estradiol by ICI 182780 did not prevent the subsequent entry into S-phase. Blocking the MAPK/ERK pathway by the MAPK kinase inhibitor U0126 in late G1 phase was, however, without effect on the G1/S transition. Moreover, U0126 had little effect in terms of Rb phosphorylation. These observations support the notion that the early cascade kinases may play a permissive role throughout the G1-phase progression of breast cancer cells. Our experiments with siRNA vectors targeting Akt and MAPK/ERK support this possibility. The inhibition of the estradiol-induced activation of the cyclin A promoter by MAPK/ERK siRNA can be related to the fact that a basal phosphorylation of MAPK/ERK is present in the quiescent cells (Figs. 1 and 3). This does not appear to be the case for Akt: no phosphorylation of the Ser 473 residue could be detected at quiescence (Fig. 2). One can speculate that either a very low level of phosphorylation escapes detection by Western blotting or that nonphosphorylated Akt can display activity, kinase, or other (biological activity of unphosphorylated Akt has been recently demonstrated in a different context (39, 40).
An unexpected result seen in these experiments was the inhibition by MAPK siRNA of the ERE-directed transcription in estradiol-stimulated cells. At the same time, the expression of unrelated genes [exogenous ß-galactosidase; endogenous heat shock protein (hsp) 90] was not altered by this siRNA vector, indicating that depletion of MAPK/ERK1,2 did not reduce gene expression in a nonspecific manner. It is possible that MAPK/ERK proteins are involved in ER-dependent transcription, and that inhibition of MAPK/ERK activity, by a pharmacological drug or by depletion of the MAPK/ERK proteins, interfere with the cell cycle progression in hormone-dependent breast cancer cells through the inhibition of estrogen-induced gene expression.
There remains the question of why the results of our experiments are diametrically opposed to those of Auricchio’s group (17). The work published by these authors is coherent and well reasoned, and the experiments are convincing. However, we were unable to confirm their essential results, namely, the rapid induction by estradiol of PI-3K activity in the MCF-7 breast cancer cells (as detected by Akt phosphorylation) and of BrdU incorporation in NIH3T3 fibroblasts transiently transfected with the ER. It is possible that these disagreements may be due to the use of different sublines of the cells. The divergence of cell lines in the course of culture in different laboratories is well known. For instance, among several NIH3T3 lines we tested, only a minority became quiescent by serum starvation and responded to serum by the resumption of the cell cycle. However, we feel that the conclusion proposed, according to which the mitogenic action of estrogens in breast cancer cells is exclusively nongenomic, is untenable, and could lead to neglecting important research on the relationship between the transcriptional effects of estrogens and hormone-dependent cancer.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The NIH3T3 cells were treated similarly except that NBBS had to be used instead of FBS, to render the cells quiescent in low serum conditions (0.5% NBBS), and able to reinitiate the cell cycle upon refeeding with high-serum containing medium. For some experiments, the serum was rendered estrogen-free by treatment with active charcoal (0.5%) during 30 min at 65 C, then centrifuged and filtered through a 0.22-µm membrane.
The distribution of cells among the phases of the cell cycle was evaluated by staining with propidium iodide and flow cytometry. In some experiments, we used the incorporation of [3H]thymidine (1 µCi/ml) to evaluate the proportion of S-phase cells. At the end of the incubation with labeled thymidine, the cells were fixed by acidification with ascorbic acid (1 M, 3 drops/ml) and washed twice with PBS and twice with 5% trichloroacetic acid. The incorporated radioactivity was determined after dissolving the residual material in 0.1 N NaOH. To identify individual cells that had entered S-phase, BrdU (10 µM) incorporation was evaluated during 2-h pulses.
Pharmacological Inhibitors of Signaling Kinases
The inhibitors used in this study, U0126 (inhibitor of MAPK kinase/MAPKK, enzyme required for the activating phosphorylation of MAPK/ERK; Promega, Madison, WI), LY294002 (inhibitor of the PI-3K; Calbiochem, San Diego, CA) and PP1 (inhibitor of the src family of kinases; Biomol Research Laboratories, Plymouth Meeting, PA), all compete for the ATP binding to their respective enzyme targets (41, 42, 43, 44).
Western Blotting
Cells were harvested at 4 C in a Tris (50 mM, pH 7.4) buffer containing EDTA (20 mM) Nonidet P-40 (0.5%), NaCl (150 mM), dithiothreitol (1 mM), aprotinin (1 µg/ml), leupeptine (1 µg/ml), phenylmethylsulfonyl fluoride (0.3 mM), NaF (1 mM), and sodium orthovanadate (1 mM). The lysates were clarified by centrifugation (10,000 x g for 5 min). The total protein concentration was determined by Bio-Rad assay (Bio-Rad, Hercules, CA) Sodium dodecyl sulfate (1% final concentration) and 2-mercaptoethanol (100 mM) were added and the solutions were boiled for 2 min before fractionation by electrophoresis in a polyacrylamide gel (8% for Rb and S6 kinase, 10% for cyclin D1). The proteins were then electrotransferred onto a Hybond membrane and incubated with the appropriate antibodies followed by the peroxidase-tagged secondary antibody. The primary antibodies used were from Cell Signaling Technology (Beverly, MA) for total and phospho(Ser 473) Akt and for total ERK1/2 as well as phospho(Thr 202/Tyr 204) ERK1, ERK2; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) for S6K and hsp90, CLONTECH (Palo Alto, CA) for cyclin D1, and Pharmingen (Le Pont de Claix, France) for Rb. The detection of the signal was carried out with the enhanced chemiluminescence kit (Amersham Biosciences, Saclay, France).
Expression Vectors
Expression vectors of the ER were: HEGO, wild-type ER; HE 251G, ER mutated in the DNA binding domain (45, 46).
The indicator plasmids used were: ERE-Tk-Luc (luciferase cDNA cloned downstream of two palindromes of the ERE from the Xenopus vitellogenin gene (47); pCA-Luc (luciferase cDNA cloned downstream of the cyclin A promoter (48). The pCH110 plasmid, ß-galactosidase cDNA driven by the cytomegalovirus promoter (Sigma-Aldrich, St. Louis, MO) was used to normalize the data for transfection efficiency.
Short inhibitory RNA oligonucleotide directed to a conserved sequence of MAPK/ERK 1 and 2 was designed using the Target Finder Program (Ambion, Austin, TX). The following sequence was introduced into siRNA pSilencer vector (Ambion): 5'-GATCCC aag caa tga cca tat ctg cta TTCAAGAGA tag cag ata tgg tca ttg ctt TTTTTTGGAAA-3', downstream of the U6 polymerase III promoter.
siRNA-Akt 1,2 as well as PI-3K and ß expression plasmids were a generous gift of Dr. F. Czauderna (AtuGen, Frankfurt, Germany). Control cells were transfected with the empty U6-containing vector.
Transfection
a) Lipofection.
Cells were seeded in 35-mm dishes at approximately 20.103/cm2 and allowed to attach overnight. The transfection was carried out by the Lipofectamine method (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturers’ protocol. After a 3-h incubation with the DNA-containing liposomes, the cells were placed for a minimum of 6 h in phenol red-free medium containing charcoal-treated serum. MCF-7 cells were then synchronized in G0/early G1 phase by a 24 h incubation in phenol red-free, serum-free DMEM containing 10 nM ICI 182780, before stimulations as described in Results. In the case of NIH3T3 cells, the synchronization was carried out in phenol red-free DMEM supplemented with 0.5% charcoal-treated NBBS, with or without insulin (1 µM).
b) Electroporation.
Cell suspensions (5 x 106 cells) in 500 µl of Cytomix buffer [120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2 (pH 7.6)] with DNA were transferred to a 0.4-cm electroporation cuvette (Bio-Rad) and pulsed with a gene pulser apparatus (960 µF, 350 V). The transfections were carried out with 1.5 µg of MAPK/ERK 1,2 or Akt 1,2-siRNA expression vectors. Electroporation of 1.5 µg of the vector without insert was used as a control in these experiments. The ß-galactosidase expression plasmid pCH110 was included to allow the determination of the fraction of cells expressing exogenous DNA. The total amount of DNA was adjusted with salmon sperm DNA to 40 µg per transfection. After overnight incubation in phenol-red-free medium containing 10% charcoal-treated serum, the cells were incubated in serum- and phenol red-free medium supplemented with 10 nM antiestrogen ICI182780 during 48 h, then harvested and cell lysates analyzed by Western blotting for total MAPK/ERK and Akt proteins as described above. Hsp90, a constitutively expressed cellular protein, was revealed as an indicator of loading. To determine the transfection efficiency, cells in separate dishes were stained for ß-galactosidase. Approximately 50% of cells were viable after electroporation, and the fraction of cells positive for ß-galactosidase was between 30 and 40%.
Immunofluorescence
Cells were washed twice with PBS and fixed in an ethanol buffered by glycine (pH 2) during at least 20 min at –80 C. Cells were then permeabilized with Triton X-100 (0.1%, 10 min) and incubated for 30 min in a blocking solution of 10% skimmed milk in PBS. To reveal the ER, the cells were incubated for 2 h at room temperature with the rabbit polyclonal anti-ER antibody (Santa Cruz, clone HC-20, 1 µg/ml) in the blocking solution, washed twice with PBS and then incubated with the Texas red-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) diluted 1:100 in the blocking solution. To detect cells that had incorporated BrdU, the labeling and detection kit (Roche, catalog no. 1296736) was used, following the manufacturer’s instructions. Cell nuclei were stained with 4',6-diamino-2'-phenylindol hydrochloride (Roche Molecular Biochemicals, Mannheim, Germany); 1 µg/ml).
The red (ER), green (BrdU), and blue fluorescence was observed under a Leica fluorescence microscope (Rueil-Malmaison, France). Positive cells were counted visually.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; hsp, heat shock protein; MAPKK, MAPK kinase; NBBS, newborn bovine serum; PI-3K, phosphatidylinositol 3-kinase; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; Rb, retinoblastoma protein; siRNA, small interfering RNA.
Received for publication April 10, 2003. Accepted for publication July 26, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(i). J Biol Chem 276:27071–27076: a new model for anti-estrogen resistance. J Biol Chem 276:9817–9824 (ER) via interaction between ER and PI3K. Cancer Res 61:5985–5991 (ER), differentially affect ER extractability, proteasome-mediated stability, and transcriptional activity in human breast cancer cells. Mol Endocrinol 17:2013–2027NURSA Molecule Pages Link:
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 H. J. Han, J. S. Heo, and Y. J. Lee Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes Am J Physiol Cell Physiol, April 1, 2006; 290(4): C1067 - C1075. [Abstract] [Full Text] [PDF]
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 S. E. Robertson, S. R. G. Setty, A. Sitaram, M. S. Marks, R. E. Lewis, and M. M. Chou Extracellular Signal-regulated Kinase Regulates Clathrin-independent Endosomal Trafficking Mol. Biol. Cell, February 1, 2006; 17(2): 645 - 657. [Abstract] [Full Text] [PDF]
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 Z. T. Ruiz-Cortes, S. Kimmins, L. Monaco, K. H. Burns, P. Sassone-Corsi, and B. D. Murphy Estrogen Mediates Phosphorylation of Histone H3 in Ovarian Follicle and Mammary Epithelial Tumor Cells via the Mitotic Kinase, Aurora B Mol. Endocrinol., December 1, 2005; 19(12): 2991 - 3000. [Abstract] [Full Text] [PDF]
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J. H. Gutzman, S. E. Nikolai, D. E. Rugowski, J. J. Watters, and L. A. Schuler Prolactin and Estrogen Enhance the Activity of Activating Protein 1 in Breast Cancer Cells: Role of Extracellularly Regulated Kinase 1/2-Mediated Signals to c-fos Mol. Endocrinol., July 1, 2005; 19(7): 1765 - 1778. [Abstract] [Full Text] [PDF]
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