Unliganded and Liganded Estrogen Receptors Protect against Cancer Invasion via Different Mechanisms

Nadine Platet, Séverine Cunat, Dany Chalbos, Henri Rochefort and Marcel Garcia

Institut National de la Santé et de la Recherche Médicale Unité Hormones et Cancer (U148) and Université de Montpellier I Montpellier, France 34090


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
While estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor {alpha} (ER{alpha}) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ER{alpha} action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ER{alpha} expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ER{alpha} indicated that amino acids 179–215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ER{alpha} and ERß reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206–211 of ER{alpha}, reversed hormone-independent ER{alpha} inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ER{alpha} interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ER{alpha}-positive cell lines, where ER{alpha} content was inversely correlated with cell migration. We conclude that ER{alpha} protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor {alpha} (ER{alpha}) is a ligand-inducible transcription factor that belongs to the superfamily of nuclear receptors (reviewed in Refs. 1 2 ). The more recently isolated ERß isoform has structural similarities to ER{alpha}, but a different tissue distribution (3 4 ). Estrogen signal transduction involved high-affinity binding to ERs, conformational changes of ERs and recruitment of transcriptional auxiliary factors, binding to estrogen response elements (EREs) in gene promoters, and regulation of transcriptional activity in conjunction with other transcription factors bound to their cognate sites in the promoter. Molecular analysis has shown that ER{alpha}, like other nuclear receptors, consists of separable domains responsible for DNA binding [DNA-binding domain (DBD), hormone binding domain (HBD)], and transcriptional activation. The N-terminal activation function (AF-1) of the purified receptor is constitutively active, whereas the activation function located within the C-terminal part (AF-2) requires hormone for its activity. In addition to this well known activation of target promoters containing EREs, other liganded ER actions involving recruitment of ER{alpha} to gene promoters lacking EREs via protein-protein interactions with promoter-bound transcription factors (5 6 7 8 9 ), or activation of the mitogen-activated protein kinase pathway (10 ), have been proposed.

Estrogens and their receptors are implicated in the etiology of breast cancer (reviewed in Ref. 11 ). In large clinical studies, ER{alpha} has been associated with a more favorable prognostic outcome in breast cancer patients (12 ). While the mitogenic action of estrogens in breast cancer cells is well established (13 ), several studies have correlated ER{alpha} expression to lower Matrigel invasiveness and reduced metastatic potential of breast cancer cell lines (14 15 ). This paradox suggests that ER expression could be associated with or involved in pathways that hinder cancer progression. A working hypothesis is that ER{alpha} protects against invasion of the basement membrane, an important step of the metastatic process required for cancer dissemination. Estrogen effects on cell invasiveness have been studied in vitro using Matrigel, a reconstituted basement membrane as host. Initial studies indicated that invasiveness of MCF7 breast cancer cells was increased by estradiol and also, paradoxically, by antiestrogens (16 17 ). More recent studies have confirmed the stimulatory effect of antiestrogens, such as 4-OH-tamoxifen and ICI 164,384 but not the estradiol-induced stimulation (15 18 19 20 21 22 ). By contrast, estradiol appeared to significantly reduce invasiveness, and this inhibition was reversed by antiestrogens. This conclusion was noted in several ER{alpha}-positive cancer cell lines established from breast (15 ) or ovary (20 ), and in different ER{alpha}-negative cancer cells constitutively expressing ER{alpha} after stable transfection (18 19 22 ). Similar results were also obtained on the migration of normal cells from vascular smooth muscle (21 ).

In the present study, after deletion of different ER domains, we demonstrate that ER{alpha}, expressed in ER-negative MDA-MB-231 breast cancer cells, prevents invasion in vitro via two distinct mechanisms, according to its unliganded or liganded status. Estrogen-induced inhibition requires ER{alpha} domains normally involved in the transcriptional activation of ERE-containing promoters. In contrast, the unliganded receptor action appears to depend on a discrete ER{alpha} region that includes the N-terminal zinc finger of DBD and probably involves protein-protein interaction.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of Invasiveness of Breast Cancer Cells by Unliganded ER{alpha} and Estrogen/Antiestrogen Treatment
The effect of ER{alpha} on invasiveness was assayed using a new method based on transient cotransfection of the ER{alpha}-expression vector (HEGO), or the empty pSG1 vector, with the pGL3 vector constitutively expressing luciferase and used as a marker to assess only transfected cells, as described in Materials and Methods. The percentage of luciferase-positive cells migrating through Matrigel was compared in HEGO-transfected or control pSG1-transfected cells. The validity and reproducibility of this cotransfection method have been demonstrated in the highly invasive ER{alpha}-negative MDA-MB-231 cell line (23). As shown in Fig. 1AGo, estradiol or antiestrogen treatments did not significantly affect invasion of control pSG1-transfected cells. ER{alpha} expression induced a 3.3-fold decrease in the invasiveness of transfected cells in a ligand-independent manner. Estradiol treatment reinforced this effect by an additional 2-fold reduction in invasiveness. Among antiestrogens, the partial agonist/antagonist OH-tamoxifen did not significantly affect invasion whereas the pure antiestrogen ICI 164,384 almost totally reversed the strong inhibition due to the unliganded ER{alpha}. In the presence of estradiol, both antiestrogens reversed the hormone action, but ICI 164,384 also reversed the effect due to the unliganded receptor. Immunofluorescence analysis after transient HEGO transfection revealed that treatment by 100 nM ICI 164,384 decreased the number of ER-positive nuclei by 6-fold as compared with control or estradiol-treated cells (data not shown). This indicated that reversal of the unliganded receptor effect by ICI 164,384 was probably due to this drastic decrease in receptor content.



View larger version (31K):
[in this window]
[in a new window]
 
Figure 1. Effect of ER{alpha} Transient Transfection on MDA-MB-231 Cell Invasion and Chemotaxis

A, ER{alpha}-negative MDA-MB-231 cells were transiently cotransfected with ER{alpha}-expressing vector (HEGO) or control vector (pSG1), and the luciferase-expressing vector (pGL3) used as a marker of transfected cells, as described in Materials and Methods. The percentage of Matrigel-invading cells was estimated in triplicates 24 h after invasion in the presence of 20 nM estradiol (E2), 100 nM 4-hydroxy-tamoxifen (OHT), 100 nM ICI 164,384 (ICI), or ethanol alone (C). Values represent mean ± SD of three experiments. B, Comparison of the effects of ER{alpha}-expressing vector or control vector on cell invasion (with Matrigel) and motility (without Matrigel). Data represent mean ± SD of three separate experiments. *, P < 0.01 vs. pSG1 control; •, P < 0.05 vs. HEGO control.

 
We used the same assay in the absence of Matrigel to analyze the effect of ER{alpha} on one component of the invasion process, i.e. cell motility (Fig. 1BGo). In the absence of ligand, ER{alpha} expression reduced cell motility to 69% of the control value, indicating that decreased invasion (30% of control) could be partly due to a motility effect.

Different ER{alpha} Domains Are Implicated in the Inhibition of Invasion Mediated by Unliganded and Hormone-Activated Receptors
To determine the ER{alpha} domains involved in the two inhibitions obtained in the absence and presence of estradiol, ER{alpha} deletion constructs were transfected in MDA-MB-231 cells, and their effects on cell invasion were compared (Fig. 2Go). The expression and nuclear localization of all mutated proteins were also verified by immunofluorescence as described in Materials and Methods. In most experiments, transfection efficiency reached about 6 ± 2% of total cells (data not shown). In the absence of ligand, the G400V-mutated ER{alpha} (HEO, 46% of pSG1) was slightly less inhibitory than the native ER{alpha} (HEGO, 29% of pSG1). Mutant HE15 (1–282) lacking the hormone binding domain and AF-2 function or HE91 containing two amino acid changes in the DBD (E203G, A207V), which prevented transactivation via an ERE (24 ), was as effective as the wild-type ER{alpha}, indicating that these functions are not involved in ligand-independent inhibition. Inversely, mutants HE19 (179–595) and HE11 ({Delta}185–251) truncated in A/B and C domains, respectively, were ineffective, suggesting that these domains are important. On the other hand, E2-induced inhibition was totally abolished when mutants deleted in the HBD (HE15) or in the DBD (HE11) or mutated in the ERE binding domain (HE91) or in the core region of AF2 (HEmutAF2) were transfected (Fig. 2Go). In contrast, deletion of the A/B region (HE19) did not influence the hormonal effect.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 2. ER{alpha} Domains Involved in the E2-Independent and E2-Induced Inhibition of Cancer Cell Invasion

MDA-MB-231 cells were transiently cotransfected with vectors expressing wild-type (HEGO) or mutated forms of ER{alpha}, and a luciferase expressing vector, as described in Fig. 1Go. Matrigel invasiveness of transfected cells treated or not with 10 nM estradiol was estimated as in Fig. 1Go. Values represent mean ± SD of three experiments.

 
Taken together, these data indicated that inhibition due to unliganded ER{alpha} and to the E2-activated receptor required different receptor domains. The hormone action required ERE binding and AF2, two functions classically involved in transcriptional activation of target genes. In contrast, these functions were dispensable for the unliganded receptor action, which appeared to be due only to its 1–282 amino-terminal region.

Inhibition of Invasiveness by Unliganded ER Involved the First Zinc Finger of the C Region
To further specify which part of the 1–282 amino acid region is involved in unliganded receptor action, A/B deletion mutants were analyzed. Deletions of any different part of the A/B region did not influence the inhibitory effect of the unliganded receptor (Fig. 3AGo). Moreover, a point mutation of serine 118, previously described as essential for steroid-independent receptor activation (25 26 ), to a nonphosphorylatable alanine (HE457 or HE15/457) did not alter ER{alpha} and ER{alpha}1–282 (HE15) efficiencies for inhibiting invasion (data not shown). The finding that the ER100 ({Delta}3–178) mutant was active while HE19 ({Delta}1–178) was inefficient indicated that the two N-terminal amino acids are required (Fig. 3AGo).



View larger version (32K):
[in this window]
[in a new window]
 
Figure 3. ER{alpha} Domains Involved in the Ligand-Independent Inhibition of Cancer Cell Invasion

Vectors expressing wild-type (HEGO) or mutated ER{alpha} were transiently transfected in MDA-MB-231 cells in estradiol-withdrawn culture conditions, as described in Fig. 1Go. For each vector, Matrigel invasion was estimated after 24 h in triplicate wells. Values represent mean ± SD of three experiments. A, Deletion mutants of A/B domains. B, Deletion mutants of C/D domains. NLS and hemagglutinin epitope tag (HA) are described in Materials and Methods. C, Immunofluorescence detection of ER{alpha} mutants. Twenty four hours after transfection with the indicated vectors, the cells were fixed and immunostained with anti-ER{alpha} antibody directed against the A/B domain. The percentage of ER-positive cells was evaluated by counting immunofluorescent nuclei in five areas, as described in Materials and Methods. Typical staining after transfection with vectors HE15-NLS (a), ER108 (b), HE384 (c), and ER103 (d). Bar, 10 µm.

 
Since these results excluded involvement of a specific A/B subregion, we then constructed mutants deleted in the C and D regions. The SV40 large T antigen nuclear localization signal (NLS) was added at the C terminus of HE15 to maintain nuclear targeting of mutants deleted in the three nuclear localization sequences in the 256–303 region of ER{alpha} (Fig. 3BGo). Analysis of mutants ER106, ER107, and ER108 progressively deleted from amino acids 281 to 216 indicated that the D region and the second zinc finger of the C region are not necessary for inhibition. Taken together, these data indicated that the 3–178 (Fig. 3AGo) and 216–281 (Fig. 3BGo) regions were dispensable for the inhibitory effect, and that the 179–215 first (or N-terminal) zinc finger region was essential. Finally, this was demonstrated using mutant ER103 ({Delta}150–215) derived from HE384 by additional deletion of this region, which was totally inactive. The relevance of the 179–215 region in the inhibitory effect of the unliganded receptor was also demonstrated using a short receptor mutant (ER110) containing only amino acids 1 and 2 and 178–215 of the receptor followed by NLS and a hemagglutinin epitope to detect its expression. This minimal sequence significantly reduced invasion to 52 ± 14% of control. Immunofluorescence analysis demonstrated that the marked differences in the efficiency of these mutants were a direct consequence of the mutations and not due to differences in protein expression (Fig. 3CGo). These data clearly identify an ER{alpha} region between amino acids 179 and 215, containing the first zinc finger, which is essential for inhibition of cell invasiveness in the absence of hormone.

The reversibility of the inhibitory effect of unliganded ER by ICI 164,384 was tested using receptor mutants either mutated in the ERE binding region (HE91) or deleted in HBD (HE15 and ER108). As shown in Fig. 4Go, the effect of ICI 164,384 was not impaired when the ERE binding domain was mutated. By contrast, deletion of E/F domains totally prevented antiestrogen action. These data strongly suggest that ICI 164,384 inhibits receptor action after binding to the HBD, but independently of ERE-mediated transcription.



View larger version (32K):
[in this window]
[in a new window]
 
Figure 4. Reversal of ER{alpha} Inhibition of Invasion by ICI 164,384 Requires HBD but not ERE Binding

After transfection with the indicated vectors, Matrigel-invading cells were estimated in triplicate after 24 h in the presence of 100 nM ICI 164,384 (ICI) or ethanol alone (control) as described in Fig. 3Go. Values expressed as percentage of pSG control transfection are mean ± SD of three experiments. *, Significantly different from corresponding ethanol-treated cells, P < 0.05.

 
Unliganded Receptor Action Is Specific to ER{alpha} and ERß: Analysis of Common Amino Acids
Since the DBD is well conserved within the nuclear receptor superfamily, we analyzed the effects of several receptors on MDA-MB-231 cell invasiveness in steroid-deprived culture conditions. As shown in Fig. 5AGo, receptors specific to androgens or glucocorticoids were totally ineffective on invasion in the absence of hormone. Among the estrogen/thyroid/retinoic acid receptor subfamily, the thyroid hormone receptor {alpha}1 (TR{alpha}1), vitamin D receptor (VDR), and retinoid acid receptor {alpha} (RAR{alpha}) were also inactive. Invasion was specifically decreased 3-fold and 2-fold by the expression of ER{alpha} or ERß, respectively. The lower efficiency of the ERß could be due to its endogenous presence in these cells, as previously suggested by mRNA studies (27 ). These data indicated that inhibition of invasiveness in the absence of hormone was ER{alpha}- and ERß-specific and pointed to amino acids common to these receptors. The 179–215 amino-acid sequence of ER{alpha} was compared with the corresponding regions of the other receptors tested (TR{alpha}1, VDR, and RAR{alpha}) (Fig. 5BGo). ER{alpha} showed higher homology with ERß than with any other receptor in this region. Seven amino acids (A186, Y191, W200, S201, A207, K210, G215) were found to be common to only ER{alpha} and ERß and might be essential for the action of the unliganded ER.



View larger version (50K):
[in this window]
[in a new window]
 
Figure 5. Effects of Different Unliganded Nuclear Receptors on Cell Invasiveness

A, Nuclear receptor expression vectors were transfected in MDA-MB-231 cells as described in Materials and Methods, and the percentage of migrating cells was compared with that obtained with the corresponding control vector alone. The expression of each receptor was verified in parallel transfection experiments by hormonal induction of a reporter gene under the control of a specific hormone responsive element and cotransfected with the corresponding receptor. Human thyroid hormone {alpha}1 receptor (TR{alpha}1), vitamin D3 receptor (VDR), retinoic acid {alpha} receptor (RAR{alpha}), glucocorticoid receptor (GR), and androgen receptor (AR) were used. *, Significantly different from pSG control, P < 0.05. B, Comparison of the amino acid sequence of the N-terminal zinc finger region 179–215 of ER{alpha} to corresponding regions of other receptors tested in the same family. The conserved cysteine residues that may tetrahydrally coordinate zinc to form the zinc finger are underlined. Amino acids common to only ER{alpha} and ERß are shown with arrows.

 
To further characterize the active region, we used two mutants, i.e. HE 73 and HE74 derived from HEO, each containing six amino acid changes from the ER sequence to the GR sequence (Fig. 6Go). The HE73 plasmid containing mutations of four critical amino acids (A186, Y191, W200, and S201) common to only ER{alpha} and ERß inhibited invasion as efficiently as the wild-type receptor. By contrast, the HE74 plasmid mutated at six positions (203, 204, 207, 212, 213, 214) including only the ER common A207 had no significant effect on invasion (88 ± 11%) even by transfecting a 3-fold higher plasmid concentration (data not shown). This inactivation confirms the importance of the whole 203–215 region but is not only dependent upon mutation of the common A207 since this amino acid was also mutated in the active mutant HE91 (see Fig. 2Go).



View larger version (20K):
[in this window]
[in a new window]
 
Figure 6. Influence of Mutations in the 179–215 Region of the ER{alpha} on Cell Invasiveness

Vectors expressing ER{alpha} (HEO) or mutant receptors HE73 or HE74, each containing six mutations (underlined amino acids) in the 179–215 region, were transfected in MDA-MB-231 cells and their effects on cell invasiveness were determined as described in Fig. 3Go. Arrows indicate amino acids common to ER{alpha} and ERß but not to other receptors (see Fig. 5BGo).

 
Modulation of the Hormone-Independent Antiinvasive Effect of ER by Calreticulin Expression
Calreticulin, a Ca2+-binding protein, is known to bind integrins at the cell surface and to modulate gene expression by interacting with the consensus motif KxFF[K/R]R present in the DBD of all nuclear receptors (reviewed in Ref. 28 ). In ERs, the KAFFKR motif (amino acids 206–211) is located in the first zinc finger domain (28 ) and contains an ER-specific alanine that is essential for ERE binding specificity (29 ). Calreticulin thus appears as a potential ER-interacting protein possibly involved in hormone-independent ER-induced inhibition of invasion. When calreticulin was coexpressed with HE15 by transient transfection in MDA-MB-231 cells, the inhibitory effect of this HBD-deleted mutant was totally repressed, and invasion was unaltered as compared with control cells (Fig. 7Go). These data confirmed the implication of the first zinc finger domain in the antiinvasive effect. However, when tested alone, calreticulin expression reduced Matrigel invasion to 31%, i.e. as efficiently as ER{alpha} expression. Similar inhibition was observed after transfection of 1–10 µg of calreticulin expression vector (data not shown). It is not surprising that calreticulin was a negative modulator of invasion since this protein is known to increase integrin-mediated cell adhesion and spreading (28 ). Taken together, these data indicate that calreticulin prevents the hormone-independent inhibition of invasion by ER{alpha}, probably through interaction with its first zinc finger region, and suggests that calreticulin prevents ER{alpha} interaction with another unknown nuclear factor that activates invasion.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 7. Calreticulin Expression Reversed Ligand-Independent ER{alpha} Inhibition of Invasion

MDA-MB-231 cells were transfected with the indicated concentrations (µg) of HE15 or calreticulin (CRT) expression vectors as described in Materials and Methods. In the absence of these vectors, identical concentrations of the corresponding empty vectors (pSG1 for HE15, pcDNA3 for CRT) were used as controls. The percentages of migrating cells were compared with those obtained with the corresponding control vectors alone. HE15 and calreticulin expression was verified in parallel by immunocytochemistry. Values represent mean ± SD of three independent experiments. *, Significantly different from control, P < 0.05.

 
Inverse Correlation between ER{alpha} Expression and Cell Motility in ER-Positive Cell Lines
ER-positive breast cancer cell lines are less invasive in vitro and less metastatic in vivo than their ER-negative counterparts (14 15 ). In all breast cancer cell lines studied by immunocytochemistry, we observed heterogeneous expression of ER{alpha}, with a wide range of intense to negative nuclear staining (Fig. 8AGo). We used this heterogeneity to test whether the ER{alpha} content in individual cells was correlated with their motility capacities. A motility test was used instead of the invasion test because of the low invasiveness of ER{alpha}-positive cell lines and the low seeding cell density required for immunostaining of individual cells. The percentage of immunostained cells was determined in the upper and lower side of the filter containing nonmigrating and migrating cells, respectively (Fig. 8AGo). The percentage of ER{alpha}-positive cells was lower in the migrating cell population than in the nonmigrating one. This difference was significant in three different cell lines, MCF7, ZR75.1, and T47D (Fig. 8BGo). In control experiments, we verified that this decrease in ER{alpha} positivity was not due to differences in cell densities between the two sides of the filter by immunostaining of serial dilutions of MCF7 cells (data not shown).



View larger version (46K):
[in this window]
[in a new window]
 
Figure 8. ER Immunostaining of Three Cell Lines before and after Migration through Transwells

A, Immunostaining of MCF7 cells on the upper (nonmigrating cells) (left panel) and lower (migrating cells) (right panel) sides of the filter after 6 h migration in phenol red-free medium containing 10% DCC-treated FCS was performed using antihuman ER{alpha}, 1D5 monoclonal antibody. Photographs at x20 showed strong nuclear immunoperoxidase staining of ER-expressing cells, light nuclear counterstaining with hematoxylin of ER-negative cells, and colorless circles corresponding to filter pores. Bar, 30 µm. B, Cells were immunostained with antihuman ER{alpha} antibody as in panel A. The percentage of cells with nuclear staining was evaluated from 400–800 cells in five different areas on the upper and lower sides of filters. *, P < 0.05 vs. nonmigrating cells.

 
The fact that unliganded ER{alpha} expression was correlated with decreased motility of the three breast cancer cell lines was consistent with direct evidence obtained by transfection of ER{alpha}-negative cells. Taken together, these in vitro data indicated a protective role of ER{alpha} against invasion by breast cancer cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several studies indicated that estrogen decreased in vitro invasiveness and motility of breast (15 18 22 ) and ovarian (20 ) cancer cells. In addition, evidence of estrogen-induced inhibition of cancer aggressiveness was obtained in an experimental murine metastasis model using ER{alpha}-expressing MDA-MB-231 cells (18 ). Moreover, epidemiological studies of breast cancer risk in women using hormone replacement therapy (HRT) are in accordance with a decrease of tumor invasion mediated by estrogen (30 ). Among women using HRT (80% received preparations containing estrogen alone), the risk of breast cancer increased, but these tumors were stage 1 with more favorable prognosis. Compared with tumors in never-users, those in HRT-users are less invasive to axillary lymph nodes and to distant sites. These data suggest that, in addition to their initial promoter role in breast cancer, estrogens could prevent spreading of cancer cells.

In this study, we used an original method that combines transient gene expression with Matrigel invasion assay to specify the receptor domains involved in estradiol inhibition of invasion. The estradiol effect is probably due to the classical activation of target gene transcription since it required ERE binding and AF2 domain integrity, two transactivation prerequisites. This strongly suggests that some estrogen-regulated genes negatively controlled invasion. Among estrogen-regulated proteins, those increasing cell-cell adhesion, such as E-cadherin, or decreasing matrix degradation, such as {alpha}1-antichymotrypsin are possible candidates (reviewed in Ref. 31 ).

The major finding of this study is the strong inhibitory effect of unliganded ER{alpha} on cancer cell invasion. In previous experiments using stable transfection, analysis of two transfectants was not sufficient to render a conclusive decision on the inhibitory effect of unliganded ER{alpha} on invasion (18 ). Possible artefacts related to the cell cloning procedure were eliminated by this new transient transfection approach. We could reproducibly inhibit cell invasion by full-length ER{alpha} expression in the absence of hormone. Evidence that ER{alpha} acted through an E2-independent mechanism was demonstrated by the action of different mutants lacking the hormone binding domain. Moreover, the strong inhibitory effect of the unliganded receptor was reversed by addition of the pure antiestrogen ICI 164,384 but not by the partial agonist/antagonist OH-tamoxifen. We have also shown that ICI 164,384 activity requires the E/F region, but not ERE-mediated transcription, and is associated with a 6-fold decrease in the number of ER-stained nuclei. This suggests that ICI 164,384 prevents unliganded receptor action by ER degradation after its binding to the HBD. These data are in agreement with previous studies in native ER-positive cells showing that ER{alpha} stability was decreased in the presence of ICI 164,384 (32 ) but not in the presence of OH-tamoxifen (33 ).

Using ER{alpha} mutants, we demonstrated that the 1–215 region (ER108) of the receptor was as efficient as the full-length receptor in the absence of ligand. Moreover, ERE-mediated transactivation and the A/B region containing the AF1 transactivation function were dispensable for this action. This contrasts with the previously proposed mechanism for steroid-independent activation of ER{alpha} requiring the functional A/B region, serine 118 phosphorylation, and ERE binding (25 26 ).

Analysis of mutants in the C and D regions revealed that deletion of the first zinc finger of the C region totally prevented ligand-independent inhibition, whereas other deletions flanking this zinc finger sequence were ineffective. Moreover, the minimal 178–215 sequence preceded by two terminal amino acids appeared sufficient to inhibit invasion. The active conformation of the critical 179–215 region is probably favored by the presence of a short (two or more amino acids) terminal sequence. As inhibition of invasion is restricted to the two ER isoforms among different members of the nuclear receptor superfamily, we identified seven amino acids common to only ER{alpha} and ERß by sequence comparison of the 179–215 region. However, the analysis of HE73 and HE74 mutants pointed out the importance of the 203–214 sequence rather than common amino acids located in the 186–207 region.

On the basis of these data, we propose that unliganded ER decreases invasiveness via interaction of the first zinc finger region with an unknown nuclear factor. The possibility that unliganded ER reversed invasion via a direct genomic effect due to DNA interaction was excluded by the activity of HE91, ER108, and ER110 mutants. Previous published data have shown that 1) mutations in HE91 prevented transactivation of ERE-mediated responses (24 ); and 2) deletions of the C-terminal zinc finger or of the 222–226 dimerization domain, as found in ER108 and ER110 mutants, abolished DNA binding of ER (34 ) and DBD dimerization (35 ). Moreover, other examples of DBD regions that have a transcriptional role distinct from DNA binding have already been described (36 37 ).

Among the possible candidate proteins interacting with the DBD region of ER{alpha} (38 39 40 ), we tested calreticulin since this protein was known to specifically interact with the first zinc finger of different nuclear receptors (28 40 ). Calreticulin expression was able to reverse the inhibitory effect of HE15 but also inhibited invasion when tested alone. This indicates that calreticulin is not the ER{alpha} interacting factor that positively regulates invasion, but its interaction could prevent the binding of ER{alpha} to this unknown activator. The possibility that ER{alpha} could interfere with AP1-directed gene activity (6 8 9 41 ) via a protein-protein interaction with the c-Jun protein (9 ) was excluded since the ER{alpha} region interacting with c-Jun is probably not the first zinc finger (Ref. 9 and C. Teyssier and D. Chalbos, unpublished data), and we showed that c-Jun overexpression did not influence the ER{alpha} effect on cell invasiveness (our unpublished data).

In mammary carcinogenesis, the promoting role and mitogenic effect of estrogens are well demonstrated, whereas the presence of ER{alpha} paradoxically seems to be associated with more differentiated and less invasive tumors. Moreover, large clinical studies have shown that ER{alpha} is a favorable prognostic marker in primary breast tumors. This was also confirmed in breast cancer cell lines in which ER{alpha} positivity was associated with low Matrigel invasiveness and low metastatic potential in mice. In this study, we present the following evidence that ER{alpha} per se could have a protective role against cancer progression: 1) Transient expression of the unliganded ER{alpha} and several mutants deleted in the hormone binding domain drastically reduced MDA-MB-231 cell invasiveness in Matrigel tests. 2) Studies in three ER{alpha}-positive cell lines showed that in hormone-deprived conditions, the ER{alpha} content was inversely correlated with cell motility. These hormone-independent effects of ER{alpha} also suggest a function for ER variants deleted in exon 3 (second zinc finger) or exon 4 (part of HBD) that have been found to occur naturally in normal and neoplastic estrogen target tissues (42 43 ). In addition to the antitumor properties of the transfected ER{alpha} previously shown in the presence of estrogen (18 44 45 ), the strong antiinvasive activity associated with expression of the unliganded receptor also suggests potential practical applications in cancer gene therapy.

We conclude that unliganded and hormone-activated ER{alpha} decrease in vitro cancer cell invasiveness via distinct mechanisms. This is evidence of a protective role of ER{alpha} in cancer progression and supports hormone-independent ER{alpha} function. This finding could help to explain the favorable prognosis associated with the presence of ER{alpha} in primary breast cancers and lead to new therapeutic applications.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Plasmids derived from the pSG5 or pSG1 vector expressing full-length human ER{alpha} (HEGO) or mutated forms (HE series) of the receptor were generously donated by P. Chambon, and their constructions have been previously described (46 47 ) except for HEmut-AF2 containing four mutations in the core activating domain of AF2 that prevent its action (Ref. 48 and V. Vivat and P. Chambon, unpublished).

ER100 plasmid ({Delta}3–178), a derivative of the HE344/384 ({Delta}3–50 and {Delta}150–178), was created by XhoI excision of amino acids 50 to 150. The ER103 expression vector ({Delta}150–216) was constructed by replacement of the HE384 XhoI-XbaI fragment (amino acids 179 to 379) with a PCR-generated fragment containing residues 216–379 with a XhoI site preceding amino acid 216. The deletion mutants ER106, ER107, and ER108 were constructed as follows. The expression vector HE15-NLS was first constructed using two complementary oligonucleotides coding for the SV40 large T antigen NLS Pro-Lys-Lys-Lys-Arg-Lys-Val (49 ), which were inserted at the 3'-end of ER{alpha}1–282 (HE15 coding sequence) between XhoI and AocI sites. These oligonucleotides contained a new KpnI site, in front of the NLS. Mutants ER106 ({Delta}270–281), ER107 ({Delta}251–281), and ER108 ({Delta}216–281) were constructed by replacement of the HE15-NLS NotI-XhoI fragment (amino acids 65 to 282) with PCR-generated fragments containing residues 65–269 (plasmid ER106), 65–250 (plasmid ER107), and 65–215 (plasmid ER108) in front of a new XhoI site. Mutant ER110 was constructed from ER108 by replacement of EcoRI fragment by PCR-generated fragments containing human influenza hemagglutinin tag sequence YPYDVPDYA between the NLS and stop codon. Other human receptor cDNAs corresponding to ERß (provided by S. Mosselman) vitamin D (VDR), thyroid hormone (TR{alpha}1), retinoic acid (RAR{alpha}), androgen (AR), and glucocorticoid (GR) receptors (provided by P. Chambon) were used in pSG5 expression vector. Calreticulin cDNA inserted in pcDNA3 expression vector (Invitrogen, San Diego, CA) was provided by M. Michalak.

Cell Culture and Transfection
Human breast cancer cell lines MDA-MB-231, MCF7, T47D, and ZR 75.1 were maintained in monolayer cultures in DMEM supplemented with 10% FCS and 50 µg ml-1 gentamycin. In all experiments, steroids were withdrawn from cells by 6 days of culture in phenol red-free DMEM supplemented with 10% dextran-coated charcoal-treated FCS (FCS-DCC).

Transient transfections of MDA-MB-231 cells were performed using the calcium phosphate DNA coprecipitation method. Near confluent cells were cotransfected in a 75-cm2 flask with 3.75 µg of steroid receptor expression vector or pSG control vector and 33.75 µg of pGL3 vector (Promega Corp., Madison WI) coding for luciferase. Cells were exposed to the precipitate for 8 h, washed three times with phenol red-free medium, and incubated for an additional 16 h in fresh medium before the invasion assay.

Matrigel Invasion Assay, Recovery of Luciferase Activity, and Calculation of the Percentage of Matrigel-Invading Cells
For the invasion assay, a suspension of 3.105 transiently transfected cells was layered in the upper compartment of a Transwell (Costar, Cambridge, MA) on a polycarbonate filter (8 µm pore size) previously coated with 30 µg of Matrigel basement membrane (Becton Dickinson and Co., Le Pont de Claix, France), vs. 30 µg/ml fibronectin (Sigma, St. Louis, MO) as attractant in the lower compartment. Cells were incubated for 24 h at 37 C in DMEM + 10% FCS-DCC in the presence of the indicated hormone or antihormone concentration. In parallel, a suspension of 3.105 transfected cells was layered on a 24-well plate to determine the total luciferase activity. After 24 h, cells were rinsed and lysed for 30 min with 100 µl (for migrating cells on the lower side of the filter) or 300 µl (for cells plated on 24-well plate) of lysis buffer containing 10% glycerol and 1% Triton X100 (Promega Corp.). Luciferase activities were determined on 100 µl samples by measuring the luminescence (15-sec integration time) in a LKB luminometer (LKB, Rockville, MD) after the injection of 100 µl of luciferase assay reagent (Promega Corp.). The percentage of migrating cells is given by the ratio of luciferase activity of invasive cells to luciferase activity of total cells x 100. Values are means of three independent experiments performed in triplicate. The interexperiment coefficient of variation determined from 30 values was 4%. The validity and reproducibility of the transfection/invasion technique was described in greater detail by Platet and Garcia (23 ).

For chemotaxis studies, cells were seeded on the Transwell filter as described for the invasion assay, but in the absence of Matrigel.

Immunocytochemistry
ER{alpha} immunostaining was performed 24 h after transient transfection of vectors expressing full-length and mutated ER{alpha}. Cells were fixed and permeabilized with 3.7% formaldehyde for 12 min, cold methanol for 4 min, and cold acetone for 2 min and then saturated with 2.5% goat serum in phosphate saline buffer containing 4% BSA overnight at 4 C. Immunostaining was performed using mouse monoclonal ER{alpha} antibodies directed against either the amino-terminal A/B region (50 ) (2.5 µg ml-1 of 1D5 antibody, from DAKO Corp., Carpinteria, CA) or carboxy-terminal amino-acids 495–594 (1 µg ml-1 C311 antibody, from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), with goat antimouse rhodamine-conjugated antibody (Immunotech, Marseille, France) as second antibody. For chemotaxis studies in ER-positive cell lines, ER{alpha} was detected by immunoperoxidase staining using 1D5 antibody, antimouse biotinylated antibody, and the Vectastain kit (Vector Laboratories, Inc., Burlingame CA). Calreticulin and hemagglutinin expression was detected by double immunofluorescence using C-17 goat antihuman calreticulin antibody from Santa Cruz Biotechnology, Inc., and mouse monoclonal antihemagglutinin antibody from Roche Molecular Biochemicals (Mannheim, Germany).


    ACKNOWLEDGMENTS
 
We thank P. Chambon (IGMBC, Strasbourg, France) for provision of and assistance with many of the ER expression vectors used in this study; we also thank S. Mosselman (N. V. Organon, Oss, Netherlands) and M. Michalak (University of Alberta, Edmonton, Canada) for providing the ERß and calreticulin expression vectors, respectively. We thank D. Derocq for technical suggestions and J. Y. Cance for artwork.


    FOOTNOTES
 
Address requests for reprints to: Dr. Marcel Garcia, Unité 540 INSERM, 60, rue de Navacelles, 34090 Montpellier, France.

N.P. is a recipient of MRES fellowship. This work was supported by the Institut National de la Santé et de la Recherche Médicale and the Association pour la Recherche sur le Cancer.

Received for publication August 30, 1999. Revision received March 27, 2000. Accepted for publication April 3, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Beato M, Herrlich P, Schütz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83:851–857[Medline]
  2. Mangeldorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  3. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  4. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  5. Blobel GA, Sieff CA, Orkin SH 1995 Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor. Mol Cell Biol 15:3147–3153[Abstract]
  6. Philips A, Chalbos D, Rochefort H 1993 Estradiol increases and antiestrogens antagonize the growth factor-induced activator protein-1 activity in MCF7 breast cancer cells without affecting c-fos and c-jun synthesis. J Biol Chem 268:14103–14108[Abstract/Free Full Text]
  7. Stein B, Yang MX 1995 Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-{kappa}B and C/EBPß. Mol Cell Biol 15:4971–4979[Abstract]
  8. Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 269:16433–16442[Abstract/Free Full Text]
  9. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443–456[Abstract]
  10. Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo P, Nola E, Auricchio F 1996 Tyrosine kinase/p21 ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF7 cells. EMBO J 15:1292–1300[Abstract]
  11. Pike MC, Krailo MD, Henderson BE, Casagrande JT, Hoel DG 1983 ‘Hormonal’ risk factors, ‘breast tissue age’ and the age-incidence of breast cancer. Nature 303:767–770[Medline]
  12. McGuire WL 1978 Hormone receptors: their role in predicting prognosis and response to endocrine therapy. Semin Oncol 5:2428–2433
  13. Lippman ME, Bolan G, Huff K 1976 The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long term culture. Cancer Res 36:4595–4601[Abstract]
  14. Thompson EW, Paik S, Brunner N, Sommers CL, Zugmaier G, Clarke R, Shima TB, Torri J, Donahue S, Lippman ME, Martin GR, Dixon RB 1992 Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines. J Cell Physiol 150:534–544[Medline]
  15. Rochefort H, Platet N, Hayashido Y, Derocq D, Lucas A, Cunat S, Garcia M 1998 Estrogen receptor mediated inhibition of cancer cell invasion and motility: an overview. J Steroid Biochem Mol Biol 65:163–168[CrossRef][Medline]
  16. Albini A, Graf J, Kitten GT, Kleinman HK, Martin GR, Veillette A, Lippman ME 1986 17ß-Estradiol regulates and v-Ha-ras transfection constitutively enhances MCF7 breast cancer cell interactions with basement membrane. Proc Natl Acad Sci USA 83:8182–8186[Abstract]
  17. Thompson EW, Reich R, Shima TB, Albini A, Graf J, Martin GR, Dickson RB, Lippman ME 1988 Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens. Cancer Res 48:6764–6768[Abstract]
  18. Garcia M, Derocq D, Freiss G, Rochefort H 1992 Activation of estrogen receptor into a receptor-negative breast cancer cell line decreases the metastatic and invasive potential of the cells. Proc Natl Acad Sci USA 89:1538–11542
  19. Garcia M, Derocq D, Platet N, Bonnet S, Brouillet JP, Touitou I, Rochefort H 1997 Both estradiol and tamoxifen decrease proliferation and invasiveness of cancer cells transfected with a mutated estrogen receptor. J Steroid Biochem Mol Biol 61:11–17[CrossRef][Medline]
  20. Hayashido Y, Lucas A, Rougeot C, Godyna S, Argraves WS, Rochefort H 1998 Estradiol and fibulin-1 inhibit motility of human ovarian- and breast-cancer cells induced by fibronectin. Int J Cancer 75:654–658[CrossRef][Medline]
  21. Kolodgie FD, Jacob A, Wilson PS, Carlson GC, Farb A, Verma A, Virmani R 1996 Estradiol attenuates directed migration of vascular smooth muscle cells in vitro. Am J Pathol 148:969–976[Abstract]
  22. Long BJ, Rose DP 1996 Invasive capacity and regulation of urokinase-type plasminogen activator in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, and a transfectant (S30) stably expressing ER. Cancer Lett 99:209–215[CrossRef][Medline]
  23. Platet N, Garcia M 1999 A new bioassay using transient transfection for invasion-related gene analysis. Invest Metastasis 18:198–208
  24. Mader S, Kumar V, de Verneuil H, Chambon P 1989 Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element. Nature 338:271–274[CrossRef][Medline]
  25. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract]
  26. Bunone G, Briand P-A, Miksicek RJ, Picard D 1996 Activation of the unliganted estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174–2183[Abstract]
  27. Dotzlaw H, Leygue E, Watson PH, Murphy LC 1996 Expression of estrogen receptor-ß in human breast tumors. J Clin Endocrinol Metab 82:2371–2374[Abstract/Free Full Text]
  28. Dedhar S 1994 Novel functions for calreticulin: interaction with integrins and modulation of gene expression? Trends Biochem Sci 19:269–271[CrossRef][Medline]
  29. Green S, Kumar V, Theulaz I, Wahli W, Chambon P 1988 The N-terminal DNA-binding ‘zinc finger’ of the oestrogen and glucocorticoid receptors determines target gene specificity. EMBO J 7:3037–3044[Abstract]
  30. Marsden J, Backs NPM 1996 Hormone replacement therapy and breast cancer. Endocr Relat Cancer 3:81–97
  31. Sheikh, MS, Garcia M, Pujol P, Fontana JA, Rochefort H 1995 Why are estrogen-receptor-negative breast cancers more aggressive than the estrogen-receptor-positive breast cancers? Invest Met 14:329–336
  32. Dauvois S, Danielian PS, White R, Parker MG 1992 Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89:4037–4041[Abstract]
  33. Pink JJ, Jordan VC 1996 Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines. Cancer Res 56:2321–2330[Abstract]
  34. Chambraud B, Berry M, Redeuilh, Chambon P, Baulieu EE 1990 Several regions of human estrogen receptor are involved in the formation of receptor-heat shock protein 90 complexes. J Biol Chem 265:20686–20691[Abstract/Free Full Text]
  35. Mader S, Chambon P, White JH 1993 Defining a minimal estrogen receptor DNA binding domain. Nucleic Acids Res 21:1125–1132[Abstract]
  36. Schena M, Freedman LP, Yamamoto KR 1989 Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities. Genes Dev 3:1590–1601[Abstract]
  37. Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205–1215[Medline]
  38. Budhram-Mahadeo V, Parker M, Latchman DS 1998 POU transcription factors Brn-3a and Brn-3b interact with the estrogen receptor and differentially regulate transcriptional activity via an estrogen response element. Mol Cell Biol 18:1029–1041[Abstract/Free Full Text]
  39. Powers CA, Mathur M, Raaka BM, Ron D, Samuels HH 1998 TLS (translocated-in-liposarcoma) is a high affinity interactor for steroid, thyroid hormone, and retinoid receptors. Mol Endocrinol 12:4–18[Abstract/Free Full Text]
  40. Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M, Bleackley RC, Michalak M 1994 Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature 367:476–480[CrossRef][Medline]
  41. Gaub M-P, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P 1990 Activation of the ovalbumin gene by the estrogen receptor involves the Fos-Jun complex. Cell 63:1267–1276[Medline]
  42. Erenburg I, Schachter B, Mira y Lopez R, Ossowski L 1997 Loss of an estrogen receptor isoform (ER{alpha}{Delta}3) in breast cancer and consequences of its reexpression: interference with estrogen-stimulated properties of malignant transformation. Mol Endocrinol 11:2004–2015[Abstract/Free Full Text]
  43. Park W, Choi J-J, Hwang E-S, Lee J-H 1996 Identification of a variant estrogen receptor lacking exon 4 and its coexpression with wild-type estrogen receptor in ovarian carcinomas. Clin Cancer Res 2:2029–2035[Abstract]
  44. Jiang SY, Jordan VC 1992 Growth regulation of estrogen receptor negative breast cancer cells transfected with cDNA’s for estrogen receptor. J Natl Cancer Inst 84:580–591[Abstract]
  45. Levenson AS, Kwaan HC, Svoboda KM, Weiss IM, Sakurai S, Jordan VC 1998 Oestradiol regulation of the components of the plasminogen-plasmin system in MDA-MB-231 human breast cancer cells stably expressing the oestrogen receptor. Br J Cancer 78:88–95[Medline]
  46. Ali S, Metzger D, Bornert J-M, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160[Abstract]
  47. Metzger D, Ali S, Bornert J-M, Chambon P 1995 Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells. J Biol Chem 270:9535–9542[Abstract/Free Full Text]
  48. Vom Baur E, Zechel C, Heery D, Heine MJS, Garnier JM, Vivat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand dependent interactions between the AF-2 activating domain of nuclear recptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124[Abstract]
  49. Kalderon D, Roberts BL, Richardson WD, Smith AE 1984 A short amino acid sequence able to specify nuclear location. Cell 39:499–509[Medline]
  50. Al Saati T, Clamens S, Cohen-Knafo E, Faye J-C, Prats H, Coindre JM, Wafflart J, Caveriviere P, Bayard F, Delsol G 1993 Production of monoclonal antibodies to human estrogen-receptor protein (ER) using recombinant ER (RER). Int J Cancer 55:651–654[Medline]