FRA-1 Expression Level Modulates Regulation of Activator Protein-1 Activity by Estradiol in Breast Cancer Cells
Alexandre Philips1,2,
Catherine Teyssier1,
Florence Galtier,
Corinne Rivier-Covas,
Jean-Marc Rey,
Henri Rochefort and
Dany Chalbos
Institut National de la Santé et de la Recherche
Médicale Hormones and Cancer (U 148), and
Université de Montpellier I 34090 Montpellier, France
 |
ABSTRACT
|
---|
We compared the effect of estradiol on activator
protein-1 (AP-1) activity in estrogen receptor positive (ER
+) and
estrogen receptor negative (ER
-) human breast cancer cell lines
transiently transfected with the AP-1-responsive reporter plasmid
AP-1-TK-CAT and an ER
expression vector. While estradiol increased
AP-1 activity in the ER
+ cell lines MCF7, ZR75.1, and T47D, it
decreased (MDA-MB231 and BT20 cells) or had no significant effect
(MDA-MB435 cells) on AP-1-mediated transcription in ER
- cells.
Estradiol also inhibited AP-1 activity in ER
-MDA-MB231 cells stably
transfected with ER
and in which ER
levels are close to those
found in MCF7. Use of ER
mutant expression vectors demonstrated that
the DNA-binding domain of ER
was needed for stimulation or
inhibition of AP-1 activity by estradiol but suggested that ER
binding to estrogen-responsive elements was not required for these
effects. Changes in regulation paralleled quantitative and qualitative
changes in protein binding to AP-1 sites, as demonstrated by gel shift
assay: protein binding was greater and DNA/protein complexes migrated
faster for ER
- than for ER
+ cells. In fact, by Northern blot, a
high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as
compared with ER
+ cells, and MDA-MB435 cells showed an intermediary
level of expression. The differential expression of Fra-1 in MCF7 and
MDA-MB231 cells was confirmed at the protein level by supershift
experiments. In addition, overexpression of Fra-1 in MCF7 cells
decreased the positive effect of estradiol while inhibition of Fra-1
expression in MDA-MB231 cells, by transient transfection of the Fra-1
antisense expression vector, abolished the negative effect of the
hormone. In conclusion, we demonstrated that ER
- breast cancer cell
lines differ from ER+ cells by a high level of AP-1 DNA-binding
activity due, at least in part, to high Fra-1 constitutive expression.
High Fra-1 concentration is crucial for the negative regulation of AP-1
activity by estradiol and thus may take part in estradiol-induced
inhibition of cell proliferation in ER
- breast cancer cells
transfected with ER
expression construct.
 |
INTRODUCTION
|
---|
Estrogens (1, 2), acting via nuclear steroid receptors, and growth
factors such as epidermal growth factor (EGF), insulin-like growth
factor I (IGF-I), and transforming growth factor-
, which bind to
tyrosine kinase-associated membrane receptors (3), are major mitogens
of human breast cancers. The two regulatory pathways are interactive.
Estrogens and growth factors have a synergic effect on the control of
cell growth (4). In addition, antiestrogens, widely used in the
treatment of breast cancer as inhibitors of estrogen-induced responses,
also inhibit (in the absence of estrogens) the effect of growth factors
on cell proliferation (5) and on regulation of specific genes
(6, 7, 8).
Regulation of eukaryotic gene expression generally occurs at the
transcription level due to the interaction of trans-acting
regulatory proteins with sequence elements located in promoter regions
of target genes. It is a complex phenomenon resulting from the
intervention of multiple extracellular stimuli acting via different
signal transduction pathways. One of these pathways involves steroid
hormone receptors that act as ligand-activated transcription factors.
These superfamily members are characterized by a highly conserved
DNA-binding domain (DBD), which forms two zinc finger structures, and a
less conserved COOH-terminal ligand-binding domain (LBD). Once
activated, they induce transcription of target genes after binding to
specific DNA sequences, called hormone-responsive elements, present in
their promoter region (reviewed in Refs. 9, 10). Conversely, binding
of growth factors to tyrosine kinase membrane receptors, through
generation of second messengers activating a cascade of kinases,
results in induction and/or activation of some transcription factors
(11, 12). Among them, activator protein-1 (AP-1) consists of dimers of
proteins encoded by fos and jun gene families,
which have been widely implicated in differentiation, cell
proliferation, and transformation (13). Jun (c-Jun, JunB, JunD) and Fos
(c-Fos, Fra-1, Fra-2, and FosB) proteins share a conserved region
containing the basic DBD and the leucine zipper dimerization motif. Jun
proteins can form homodimers or heterodimers with proteins of the Fos
family (14). These dimers regulate gene transcription through
interactions with a specific DNA sequence, the 12-O
tetra-decanoyl-phorbol-13 acetate (TPA) responsive element (TRE), also
referred to as the AP-1 site (11, 14).
Transcriptional interferences between estrogens and growth factor
pathways were recently observed. EGF and IGF-I were reported to
increase estrogen responsive element (ERE)-mediated responses (for a
review, see Ref. 15) by activating the estrogen receptor (ER
) by
phosphorylation through the mitogen-activated protein kinase pathway
(16, 17). We and others have previously shown that estradiol could
modulate AP-1 activity (18, 19, 20, 21). In transient transfection experiments
(19), estradiol increased basal AP-1-mediated transcription level in
the ER
positive (ER
+) human breast cancer cell line MCF7. A
positive effect of estrogens was also detected in the presence of IGF-I
or EGF in conditions whereby the hormone did not modify the synthesis
of c-Fos and c-Jun mRNA or after cotransfection with c-Fos and c-Jun
expression vectors. Antiestrogens, which inhibited growth
factor-induced cell proliferation, also inhibited growth factor-induced
AP-1 activity (19); therefore, regulation of AP-1 activity by
estradiol/ER
complexes was parallel to growth regulation.
While the role of estrogens in the promotion and development of breast
cancer has been well documented for many years, the mechanism by which
they stimulate the growth of hormone-responsive cancers is still poorly
understood. Estrogens can directly increase proliferation of ER
+
epithelial cancer cells, as demonstrated by the use of established
hormone-responsive human breast cancer cell lines (1, 2), and ER
is
necessary for their mitogenic effect. However, the presence of ER
alone does not seem to be sufficient to promote estrogenic regulation
of growth, as shown by studies on several types of ER
- cells (for a
review, see Ref. 22) including breast cancer (23, 24, 25) and normal
mammary cells (23). Surprisingly, when ER
was introduced by stable
transfection in ER
- cells to restore estrogen regulation, estradiol
inhibited cell proliferation, even when ER
was expressed at
physiological levels. Estradiol treatment of stably ER
-transfected
cells, which did not express endogenous ER
, was also shown to
decrease the number and volume of lung metastases when injected in nude
mice (25). The mechanisms involved in these opposite regulations are
presently unknown. In transfected ER
- cells, estradiol efficiently
induced transcription of an ERE-containing reporter construct and
triggered the expression of some (but not all) endogenous
estrogen-responsive genes (22, 23, 24).
Our hypothesis in the present study was that, in contrast to
ERE-mediated responses, AP-1-responsive genes could be regulated
differently by estradiol in ER
-transfected breast cancer cell lines
expressing or not endogenous ER
and whose growth is respectively
increased or decreased by the hormone. We therefore compared the effect
of estradiol on AP-1 activity in both cell types. Using transient
transfections, it was found that estradiol enhanced growth
factor-induced AP-1-mediated transcription in all ER
+ breast cancer
cell lines tested but inhibited AP-1 activity in ER
- cells
cotransfected with an ER
expression vector. We further investigated
the mechanism of this cell specificity. We presently report that ER
+
and ER
- cells express different concentrations of Fra-1, and that
high Fra-1 expression level is responsible for the negative regulation
of AP-1 activity by estradiol in ER
- breast cancer cell lines.
 |
RESULTS
|
---|
Reverse Regulation of AP-1 Activity by Estradiol in ER
+ and
ER
- Breast Cancer Cell Lines Transfected with ER
We first compared estradiol regulation of transcriptional activity
of AP-1 complexes (AP-1 activity) in MCF7 (ER
+) and MDA-MB231
(ER
-) human breast cancer cells. Cells were transfected with the
reporter construct (AP-1)4-TK-CAT containing four AP-1 binding sites
and the wild-type ER
expression vector HEGO. As shown in Fig. 1A
, basal AP-1-mediated chloramphenicol acetyltransferase (CAT) activity
was increased by estradiol in MCF7 cells but decreased by the hormone
in MDA-MB231 cells. Experiments were also conducted in the presence of
EGF or IGF-I, at concentrations that were maximally efficient for
inducing c-Fos and c-Jun (4, 19) (Fig. 1A
), or after cotransfection of
the c-Jun expression vector (Fig. 1B
). In MCF7 cells, 8 nM
EGF and 5 nM IGF-I, respectively, increased AP-1-dependent
transcription by 3.6- and 6.5-fold as compared with control cells. In
MDA-MB231 cells, 8 nM EGF increased AP-1 activity by only
50%, 5 nM IGF-I had no significant effect (Fig. 1A
), and
both growth factors were not more efficient when they were tested in a
wide range of concentrations between 0.1 nM and 1
µM (not shown). Induction of AP-1 activity by c-Jun
overexpression was also much lower in MDA-MB231 cells that in MCF7
cells (1.5-fold and 10-fold induction as compared with control cells,
respectively). Regardless of the experimental conditions, estradiol
treatment enhanced AP-1 activity in MCF7 cells but inhibited AP-1
activity in MDA-MB231 cells. Similar effects of estradiol were obtained
when the AP-1-responsive promoter was induced by cotransfection of
c-Jun expression vector (Fig. 1B
). In MDA-MB231
cells, estradiol inhibited AP1-mediated CAT activity in a
dose-dependent manner (Fig. 1C
). Doses of estradiol required to repress
AP-1 activity in MDA-MB231 cells (half-maximal inhibition at about 50
pM, maximal inhibition at 0.1 nM) were the same
as those previously reported to activate AP-1 activity in MCF7 cells
(19). Modulation of AP-1 activity by estradiol in both cell lines was
dependent on the integrity of AP-1 sites, since two mutations in the
AP-1 binding site totally abolished the hormonal effect (Fig. 1D
).

View larger version (43K):
[in this window]
[in a new window]
|
Figure 1. Effect of Estrogen on AP-1 Activity in MCF7 and
MDA-MB231 Breast Cancer Cell Lines Transfected with ER
In all experiments, steroid-stripped MCF7 and MDA-MB231 cells were
transfected for 16 h and then treated for 28 h with estradiol
(E2, 1 nM; black bars) or
vehicle (open bars), in combination with 8
nM EGF or 5 nM IGF-I when indicated. A, Cells
were transfected with 100 ng of wild-type-ER expression vector
(HEGO, 40) and 1 µg of (AP-1)4-TK-CAT. B, Cells were transfected with
HEGO (200 ng), (AP-1)4-TK-CAT (1 µg), and increasing concentrations
(0, 50, 200, 500 ng) of pCI-c-Jun. C, MDA-MB231 cells, transfected with
HEGO (200 ng) and 1 µg of (AP-1)4-TK-CAT reporter plasmid, were
incubated with increasing concentrations of estradiol as indicated. D,
Cells were transfected with HEGO (100 ng) and 1 µg mutated
(AP-1)4-TK-CAT reporter plasmid, which does not allow binding of AP-1
factors. CAT activity was measured in whole cell extracts after
normalization for ß-galactosidase activity, as described in the
Materials and Methods. The results are the mean
(±SD) of three independent experiments. They are expressed
in arbitrary units; CAT activity measured in control cells was assigned
a value of 1.
|
|
To determine whether estrogen stimulation of AP-1-dependent
transcription was restricted to MCF7 cells or could be extended to
other breast cancer cells, we then tested the effect of estradiol in
several cell lines transfected with the reporter plasmid (AP-1)4-TK-CAT
and the expression plasmid HEGO (Fig. 2
).
We have previously shown that, in MCF7 cells, estradiol, which induced
c-Fos (4, 19), had no additive effect on the accumulation of c-Fos mRNA
when it was induced by growth factors. The experiment was therefore
conducted in the presence of 8 nM EGF to eliminate an
effect of estradiol on c-Fos synthesis. As observed in MCF7 cells,
estradiol increased AP-1-dependent transcription in two other wild-type
ER
+ human breast cancer cell lines (T47D and ZR75.1). In contrast,
and as in the MDA-MB231 cell line, AP-1 activity was decreased by
estradiol in BT20 (
40% inhibition) and had no significant effect in
MDA-MB435 cells. Comparable effects of estradiol were obtained in the
absence of EGF (not shown).
ER
Concentration Is Not Responsible for the Reverse Regulation
of AP-1 Activity
The reverse effect of estradiol/ER
complexes on AP-1 activity
in MCF7 and MDA-MB231 cells could be the result of a different ER
expression level in the two cell lines. To eliminate this possibility,
increasing amounts of ER
expression vector were transfected in
cells together with the (AP-1)4-TK-CAT construct (Fig. 3A
).
In the absence of added estradiol, transfection with the ER
expression vector enhanced IGF-I-induced AP-1 activity in MCF7 cells
(2.7-fold induction for the higher HEGO concentration) and decreased
AP-1 activity by approximately 25% in MDA-MB231 cells. Moreover, in
MCF7 cells, the positive effect of estradiol, which was detected in the
presence of endogenous ER
alone, was further increased after ER
overexpression. In contrast, in MDA-MB231 cells, estradiol inhibited
AP-1-mediated CAT activity as soon as ER
expression vector was
cotransfected. However, no regulation was observed in the absence of
ER
expression, demonstrating that the receptor was necessary for the
estradiol-induced decrease in AP-1 activity observed in these ER
-
cells.
Regulation of AP-1 activity by estradiol was also analyzed in two
MDA-MB231 clones (HC1 and HE5) stably transfected with HEGO, and
expressing, respectively, 100 and 57 fmol of ER
per mg of cytosol
protein, concentrations roughly equivalent to ER
concentration in
MCF7 cells (25). As shown in Fig. 3B
, estradiol decreased AP-1 activity
in both sublines, while it had no effect in PB5 cells stably
transfected with the empty expression vector. We therefore concluded
that reverse regulation by estradiol in MCF7 and MDA-MB231 cells could
not be explained by ER
expression levels and that introduction of
ER
in wild-type ER
- cells was not sufficient to give them the
characteristics of wild-type ER
+ cells with respect to the AP-1
response.
Induction of ERE-Mediated Responses Is Not Required for Modulation
of AP-1 Activity by Estradiol
To determine whether induction of ERE-mediated responses was
required for regulation of AP-1 activity, we tested the effect of
estradiol in MCF7 and MDA-MB231 cells transfected with (AP-1)4-TK-CAT
and expression vectors of ER
mutated in the DBD (Fig. 4
).
Increasing concentrations of ER
mutant expression vectors were
transfected in MCF7 cells that express endogenous ER
(Fig. 4A
).
ER
mutants used in this study are derived from HEO, which differs
from the wild-type ER
of MCF7 cells by a point mutation in the LBD
of the protein, resulting in a lower affinity for estradiol (26).
Overexpression of the HEO mutant increased the effect of estradiol on
AP-1 activity; levels of induction by estradiol were higher than that
obtained with the wild-type HEGO construct, and no significant effect
was detected in the absence of hormonal treatment (compare Figs. 3
and 4
). In contrast to that observed with HEO, the HE11 mutant (27),
lacking DBD, was inefficient in increasing the effect of estradiol on
AP-1 activity. The results obtained with HE11 could be interpreted as a
requirement for the ERE-mediated induction of an intermediary factor.
Conversely, DBD could simply be required, for instance, as a structural
domain necessary in protein-protein interactions. The HE91 mutant,
which harbors two point mutations in the C-terminal side of the
N-terminal zinc finger of ER
, was then used in an attempt to
discriminate between these two possibilities. This mutant activates
transcription from a reporter gene containing a
glucocorticoid-responsive element, but not from a reporter gene
containing a consensus ERE (28). Its introduction in MCF7 cells,
increased the efficacy of estradiol in modulating AP-1 activity. In
contrast, dexamethasone inhibited AP-1-dependent transcription in cells
cotransfected with the wild-type glucocorticoid receptor (GR)
expression vector HGO. Transfection of HEO in MDA-MB231 cells had no
significant effect on AP-1 activity in the absence of added estradiol
but increased its inhibition by estradiol more efficiently than HEGO
transfection. In MDA-MB231cells, as in MCF7 cells, only HEO and HE91
allowed regulation of AP-1 activity, and the activated GR receptor had
a negative effect (Fig. 4B
).
These results therefore suggested that, in both cell lines, regulation
of AP-1 activity by estradiol required the DBD of ER
but not the
induction of ERE-controlled genes.
Differential Binding of trans-Acting Factors on AP-1
Sites in ER
+ and ER
- Cells
In an attempt to pinpoint the mechanisms involved in the reverse
regulation of AP-1 activity in ER
+ and ER
- cells, we compared
the ability of proteins from MCF7 and MDA-MB231 cells to bind the
polyoma virus TRE motif in electrophoretic mobility shift assays.
When equal amounts of cellular proteins were analyzed, the intensity of
the upper band was higher using MDA-MB231 cell extracts than with MCF7
cell extracts (Fig. 5A
). In addition, the retarded
complexes migrated differently for the two cell lines. This qualitative
difference was highlighted after correction for specific binding when
3-fold more proteins were used for MCF7 cells than for MDA-MB231 cells
(Fig. 5
, BD). In fact, DNA/proteins complexes migrated faster for
MDA-MB231 cells than for MCF7 cells.

View larger version (65K):
[in this window]
[in a new window]
|
Figure 5. In Vitro Binding Activity of MCF7 and
MDA-MB231 Proteins to AP-1 Sequences
Double-stranded oligonucleotides, corresponding to the polyoma virus or
collagenase TRE sequence, were 32P-labeled and incubated
for 20 min with MCF7 and MDA-MB231 nuclear extracts, prepared as
described in Materials and Methods, from cells incubated
for 4 h with IGF-I (5 nM). Complexes were separated on
a 5% nondenaturing acrylamide gel. The positions of specific
retardations are indicated by filled arrows. A, MCF7 and
MDA-MB231 nuclear extracts (10 µg proteins) were incubated with
labeled polyoma virus TRE. B, Fifteen micrograms of MCF7 proteins and 5
µg of MDA-MB231 proteins were incubated with polyoma virus TRE. Ten-
or 100-fold excess of nonradioactive collagenase TRE or vitellogenin
ERE competitors were added with the labeled probe when indicated.
C, Nuclear extracts (15 µg of MCF7 proteins and 5 µg of MDA-MB231
proteins) were incubated for 1 h at room temperature with 2 µg
of polyclonal antibodies in the binding reaction mixture before
addition of labeled polyoma virus TRE sequence. Jun and Fos
antibodies, respectively, react with all the members of Jun and Fos
families. Preimmune IgG (IgG, tracks 1 and 4) was used as control. The
large arrow shows the position of complexes retarded by
the presence of antibodies. D, Fifteen micrograms of MCF7 proteins and
5 µg of MDA-MB231 proteins were incubated with labeled collagenase
TRE.
|
|
Differences in retardation actually reflected differences in protein
binding to AP-1 sites. The upper band was efficiently blocked with an
excess of unlabeled oligonucleotides containing the collagenase
consensus TRE motif but not with oligonucleotides containing the
vitellogenin ERE motif. As shown in Fig. 5C
, it was supershifted after
incubation with antibodies directed against all the members of Jun or
Fos families. Anti-Fos antibodies almost completely supershifted the
band, suggesting that AP-1 complexes were primarily constituted of
heterodimers in cells treated for 4 h with the growth factor.
However, anti-Jun antibodies only supershifted a small fraction of the
DNA/protein complexes. This discrepancy is probably due to the weak
efficiency of anti-Jun antibodies since, in the same experimental
conditions, a larger fraction of the complexes was supershifted with
antibodies specifically directed against the c-Jun protein (not shown).
As shown in Fig. 5D
, the same differences in migration were obtained
when a labeled collagenase consensus TRE site was used in the
assay.
We then investigated AP-1 DNA-binding activity in several ER
+ and
ER
- breast cancer cell lines. Sp1 DNA-binding activity was tested
in parallel as a control of the efficiency of nuclear protein
extraction in the different cell lines. As shown in Fig. 6
, where the same amounts of protein cell extracts
from various cell lines were analyzed, higher protein binding to the
polyoma virus TRE site and faster migration of DNA/protein complexes
were detected for the three ER
- breast cancer cell lines tested
(MDA-MB231, MDA-MB435, and BT20), as compared with the three ER
+
cell lines MCF7, T47D, and ZR75.1. In the two ER
+ MDA-MB231 clones
(HE5 and HC1) the extent of retardation of TRE sequences was identical
to that obtained in wild-type MDA-MB231 cells, demonstrating that ER
expression was not sufficient to explain the slower migration with
ER
+ cells.
Differential Expression of Fos Family Members in ER
+ and ER
-
Breast Cancer Cell Lines
We then investigated whether the different results obtained with
ER
+ and ER
- cells in gel retardation assays were due to a
difference in the composition of AP-1 complexes in these cells.
We first compared by Northern blot experiments mRNA expression of Fos
and Jun family members in MCF7 and MDA-MB231 cells cultivated for
increasing periods of time in the presence of IGF-I (Fig. 7
). In MCF7 cells, IGF-I induced c-Fos, c-Jun, and
Fra-1 mRNAs. In contrast, in MDA-MB231 cells, mRNA levels were
increased by TPA but not changed by IGF-I (Fig. 7A
) or EGF (not shown)
treatment. These results were in agreement with those obtained in
transfection experiments where growth factors were only slightly
efficient (EGF) or inefficient (IGF-I) in inducing AP-1 activity,
contrary to what was observed in MCF7 cells (Fig. 1
). Fra-2, FosB,
JunB, and JunD mRNA expression were all at a very low level in both
MCF7 and MDA-MB231 cells (not shown). The most obvious difference
between the two cell lines was the high constitutive level of Fra-1
mRNA detected in MDA-MB231 cells. We therefore evaluated Fra-1 mRNA
expression in other ER
- and ER
+ breast cancer cell lines (Fig. 7B
). Fra-1 mRNA was, as in MDA-MB231 cells, highly expressed in ER
-
BT20 cells. It was undetectable in T47D and ZR75.1 ER
+ cell lines
under the same experimental conditions. An intermediary expression
level was found in ER
- MDA-MB435 cells.

View larger version (60K):
[in this window]
[in a new window]
|
Figure 7. Northern Blot Analysis of Members of AP-1 Complexes
in Breast Cancer Cell Lines
A, MCF7 and MDA-MB231 cells were grown in steroid-stripped medium, as
described in Materials and Methods. MCF7 cells were then
incubated with IGF-I (5 nM) and MDA-MB231 cells with IGF-I
(5 nM) or TPA (50 nM), for the indicated
periods of time. Forty micrograms of total RNA were analyzed on a 1%
agarose denaturing gel. After transfer onto nylon membrane, RNAs were
hybridized to 32P-labeled probes. B, Total RNA was
extracted from ER + (T47D, ZR75.1, MCF7) and ER - (MDA-MB231,
MDA-MB435, BT20) steroid-stripped cells and analyzed by Northern blot
as described in panel A. The 36B4 probe, which corresponds to a
constant RNA (48 ), was used as a loading control. The mRNA species
lengths (kb) are indicated on the left. Two bands were
detected with c-Jun (3.4 and 2.6 kb) and Fra-1 (3.3 and 1.7 kb) probes
in agreement with the literature (43 50 ).
|
|
The differential expression of Fra-1 was then confirmed at the protein
level by supershift experiments. Nuclear extracts from MCF7 and
MDA-MB231 cells were preincubated with antibodies specific for each Fos
protein before the addition of labeled oligonucleotides containing the
polyoma virus TRE site (Fig. 8
).
Anti-FosB antibodies had no effect on DNA/protein complexes obtained
from both cell lines. This absence of effect was not due to the
inability of these antibodies to form a supershift complex in the assay
since they were able to supershift in vitro translated
FosB/c-Jun complexes bound to DNA (not shown). A slight supershift of
DNA/protein complexes was observed with anti-c-Fos antibodies in MCF7
cells and with anti-Fra-2 antibodies in both cell lines. However, only
antibodies directed against Fra-1 substantially supershifted complexes
in MDA-MB231 cells. In contrast, they had no effect in MCF7 cells.

View larger version (121K):
[in this window]
[in a new window]
|
Figure 8. Immunological Detection of Fos Proteins in
Complexes Bound to AP-1 Sites in MCF7 and MDA-MB231 Cells
Extracts from steroid-withdrawn cells cultivated for 24 h with
IGF-I (5 nM) were incubated for 1 h at room
temperature with 2 µg of the indicated polyclonal antibodies in the
binding reaction mixture before addition of labeled oligonucleotides
containing polyoma virus TRE sequence. Two micrograms of preimmune IgG
(IgG, tracks 1 and 4) were used as control. Positions of specific
(filled arrow) and nonspecific (open
arrow) retardations are indicated. The largest filled
arrows mark antibody supershifts.
|
|
Fra-1 Concentration Modulates the Estradiol Effect on AP-1
Activity
Based on these results, we speculated that estradiol might
decrease AP-1 activity when large quantities of Jun/Fra-1 complexes are
present, as in ER
- cells. It would therefore be possible to reverse
the estradiol effect by modulating the Fra-1 expression level.
To test this hypothesis, we first overexpressed Fra-1 in MCF7 cells.
Cells were transfected with (AP-1)4-TK-CAT, HEGO, and increasing
amounts of pCI-Fra-1 expression vector. Experiments were performed in
cells cotransfected or not by pCI-c-Jun (Fig. 9A
). In
the absence of estradiol, Fra-1 overexpression increased the basal
level of AP-1 activity but had no significant effect when c-Jun
expression vector was cotransfected. In both cases, Fra-1
overexpression inhibited stimulation of AP-1-mediated transcription by
estradiol. With the highest amount of pCI-Fra-1 expression vector and
in the absence of c-Jun cotransfection, induction of CAT activity by
estradiol decreased by 50%. In addition, the efficacy of estradiol in
stimulating AP-1 activity, which was increased by c-Jun overexpression
alone, was decreased by 75% when Fra-1 expression vector was
cotransfected. To determine whether Fra-1 overexpression specifically
inhibited the estradiol-induced AP-1 activity, the effect of increasing
Fra-1 expression was tested in parallel in cells cotransfected by an
ERE-containing reporter plasmid. As shown in Fig. 9
, induction by
estradiol of the ERE-ß-Globine-luciferase construct was not
significantly altered by Fra-1 overexpression.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 9. Effect of Fra-1 Expression on Modulation of AP-1
Activity by Estradiol
A, Steroid-stripped MCF7 cells were transfected with HEGO (200 ng),
increasing concentrations of pCI-Fra-1 (0, 0.4, 1.2 µg), and either 1
µg (AP-1)4-TK-CAT (left panel) or 1 µg
ERE-ßGlobine-luciferase (right panel) reporter
plasmids. They were cotransfected with 100 ng pCI-c-Jun expression
plasmid when indicated. B, Steroid-stripped MDA-MB231 cells were
transfected with HEGO (200 ng), increasing concentrations of pCI-Fra-1
antisense expression plasmid (0, 0.6, 1.2 µg), and either 1 µg
(AP-1)4-TK-CAT (left panel) or 1 µg
ERE-ßGlobine-Luciferase (right panel) reporter
plasmids. Cells were then treated with or without 10 nM
estradiol (E2) for 28 h, and CAT activity was
evaluated as described in Fig. 1 . Results are expressed in arbitrary
units; CAT (left panels) or luciferase activity
(right panels) measured in mock-transfected cells
cultivated in the absence of estradiol was assigned a value of 1. They
all represent the mean (±SD) of three independent
experiments.
|
|
The crucial role of the Fra-1 concentration in controlling the sense of
regulation of AP-1 activity by estrogens was confirmed in MDA-MB231
cells in which the Fra-1 expression level was decreased. As shown in
Fig. 9B
, transfection of increasing amounts of Fra-1 antisense
expression vector lowered the basal AP-1 activity in a dose-dependent
manner. The inhibitory effect of estradiol, which was 65% in control
cells, was totally abolished with the highest concentration of
antisense construct. By contrast Fra-1 antisense had only a slight
effect on ERE-mediated transcription.
 |
DISCUSSION
|
---|
This study addressed the question of the cell specificity of
interferences between AP-1 complexes and activated ER
. The
estradiol-induced increase in AP-1 activity previously described in
MCF7 cells was also observed in other human breast cancer cell lines
expressing endogenous ER
. However, estradiol decreased this activity
in ER
- cell lines, after transfection of an expression vector
coding for ER
.
The difference in the regulation of AP-1 activity did not result from a
difference of ER
expression in the two cell types. First, estradiol
had an inhibitory effect in stably transfected MDA-MB231 cells that
expressed ER
at a concentration comparable to that of wild-type MCF7
cells. Second, constitutive overexpression of ER
in MCF7 cells did
not reverse the effect of estradiol but, conversely, enhanced the
stimulation of AP-1 activity by the hormone.
Differences in the regulation of AP-1 activity reflected variations in
protein binding to AP-1 sites. Protein binding was greater in ER
-
than in ER
+ cells, in agreement with results of Dumont et
al. (29) showing that AP-1 DNA-binding activity was increased in a
MCF7 variant expressing reduced ER
amount. Retardation of TRE
sequences was also qualitatively different in ER
+ and ER
- cells.
ER
, which alone did not bind to AP-1 sites (Ref. 18 and our
unpublished results), was however not responsible for the slower
migration of protein/DNA complexes obtained from ER
+ cells. First,
the same migration patterns were observed for wild-type MDA-MB231 cells
and clones stably transfected with ER
. Second, migration was not
modified by an excess of consensus vitellogenin ERE. Third, antibodies
directed against ER
were unable to supershift the complexes, and,
finally, the addition of baculovirus-produced mouse ER
did not
modify the migration pattern obtained with MDA-MB231 cells (not
shown).
The difference in migration thus most likely resulted from the
composition of AP-1 complexes, which varied in both kinds of cells. In
contrast to ER
+ cells, ER
- breast cancer cell lines expressed
high constitutive AP-1 binding activity due, at least in part, to high
Fra-1 levels. We do not know if high Fra-1 expression is related to the
adverse prognosis of ER
- breast cancer cells, which are more
aggressive and metastatic than the ER
+ forms. Fra-1 is significantly
expressed in cycling cells, in contrast to c-Fos, and injection of
neutralizing Fra-1 antibodies into Swiss 3T3 fibroblasts reduces DNA
synthesis, especially in exponentially growing cells (30). It is devoid
of the C-terminal transcriptional activation function (31), which is
present in c-Fos and seems to be required for transformation, as
measured by focus formation assay (31, 32), but not for tumor formation
in nude mice, which was reported for Fos family members lacking this
domain (32). In addition, although Fra-1 is, as c-Fos, capable of
heterodimerization with Jun proteins and subsequently increases binding
to the AP-1 site, it does not bind, unlike c-Fos, the TATA binding
protein (TBP) (33). In contrast to c-Fos, its overexpression was
reported to inhibit AP-1 activity controlled by c-Jun (34) that may
explain the low efficacy of c-Jun overexpression in inducing
AP-1-mediated transcription in MDA-MB231 cells in contrast to that
observed in MCF7 cells (Fig. 1B
). However, Fra-1 was also found to
enhance the transcriptional activity of JunD (34) and increased the
basal AP-1 activity in MCF7 cells (Fig. 9A
), suggesting that expression
of AP-1-controlled genes could differ in ER
+ and ER
- breast
cancer cells.
Estradiol regulation of AP-1 activity was correlated with the Fra-1
expression level. Fra-1 expression was high in MDA-MB231 and BT20
cells, and AP-1 activity was inhibited by estradiol, whereas it was low
in MCF7, ZR75.1, and T47D cells, and AP-1 activity was stimulated by
the hormone in this case. In MDA-MB435 cells showing an intermediary
level of expression, the estradiol effect was not significant. We
therefore tried to reverse the hormonal effect by modulating the
composition of AP-1 dimers. In MCF7 cells, increasing the Fra-1
concentration lowered the positive effect of estradiol. In addition,
decreasing the endogenous level of Fra-1 in MDA-MB231 cells completely
abolished the negative effect of the hormone. However, estradiol still
induced AP-1 in MCF7 cells, and no positive effect was observed in
MDA-MB231 cells. This could suggest that the Fra-1 level had only
reached an intermediary expression level in transfected cells (Fra-1
expression in MDA-MB231 cells transfected with the Fra-1 antisense
construct may, for instance, be lowered to a level comparable to that
detected in wild-type MDA-MB435 cells). Conversely, Fra-1 expression
level might be not sufficient to explain the opposite effect of
estradiol, and regulation may implicate another factor. This factor may
be the Fra-1 partner in AP-1 complexes whose expression (no differences
were however detected in the expression level of Jun family mRNAs, not
shown) or phosphorylation state may be different in ER
+ and ER
-
cells. Alternatively, it may be a transcription intermediary factor
such as CPB/p300 that has been implicated in mediating the
transcriptional effect of both nuclear receptors and AP-1 (35). In any
case, the Fra-1 concentration appeared crucial in the cell-specific
effect of activated ER
on AP-1 activity: increasing its
concentration lowered the positive effect, and a negative effect was
only observed in cells when many Jun/Fra-1 complexes were present.
AP-1 complex content was previously reported to be important in the
transcriptional interference between c-Jun and GR. Maroder et
al. (36) showed that c-Jun overexpression increased dexamethasone
stimulation of glucocorticoid-responsive element-dependent
transcription in the CEM leukemic T cell line, whereas c-Fos
overexpression had an inhibitory effect, suggesting an influence of the
intracellular c-Fos level in this regulation. Promotor regions of
proliferin (37, 38) and neurotensin/neuromedin N (NT/N) (39) genes,
which can be bound by both GR and AP-1 complexes, were also described
to be positively or negatively regulated by glucocorticoids, depending
on the relative amount of c-Jun and Fos protein families. In both
cases, glucocorticoids had a positive effect in the presence of c-Jun
homodimers. However, c-Fos overexpression reversed the regulation of
the proliferin gene (37), while it potentiated activation by the
hormone of the NT/N gene (39). Conversely, overexpression of Fra-1
negatively regulated NT/N (39) but positively regulated proliferin (38)
gene expression. In our study, using a single AP-1 site,
glucocorticoids inhibited AP-1 activity in both MCF7 and MDA-MB231
cells, although they expressed different endogenous Fra-1 levels (Fig. 4
).
The DBD was required for transcriptional interference between ER
and
AP-1 complexes. However, ER
did not bind to AP-1 sites (Ref. 18 and
our unpublished results) and the HE91 mutant (28), unable to activate
transcription from an ERE-containing reporter gene, produced the same
result as the wild-type receptor. These results therefore suggested
that regulation of AP-1 activity resulted from protein-protein
interactions. As for GR (39, 40), a physical in vitro
interaction between ER
and c-Jun was recently reported by Webb
et al. (21) and confirmed by us (C. Teyssier and
D. Chalbos, unpublished data). This interaction is not sufficient to
explain the cell-specific effect of estradiol (our results and Ref.
41). However, the presence of Fra-1 in AP-1 heterodimers might modify
physical interactions between c-Jun and activated ER
.
Stable expression of ER
in ER
- cells results in
estrogen-dependent inhibition of cell proliferation, whereas estradiol
increases the growth of cells expressing endogenous ER
. As
ERE-mediated transcription is increased by estrogens in both cell types
(22), it was tempting to speculate that the opposite effect of
estradiol on breast cancer cell proliferation, is the result of its
reverse effect on some AP-1-controlled responses. Positive regulation
of AP-1 activity, which was observed in all studied ER
+ breast
cancer cells, may be necessary for stimulation of cell proliferation.
Conversely, in ER
- breast cancer cells, high constitutive
expression of Fra-1 leading to negative regulation (or no regulation
for MDA-MB435) of AP-1 activity may explain the negative effect of the
hormone on cell growth or at least the absence of a positive effect.
However, ER
- MDA-MB453 breast cancer cells, in which estradiol was
reported to increase AP-1 activity (21), might be an exception if
activated ER
inhibits their proliferation. This could then suggest
that negative regulation of breast cancer cell growth might also
implicate alternative mechanism(s).
In conclusion, high constitutive expression of Fra-1 in ER
- breast
cancer cells is responsible for high AP-1 DNA-binding activity. The
Fra-1 concentration, which is low in ER
+ cells, is crucial in
directing to a positive or negative regulation of AP-1 activity by
estradiol and might play an important role on the opposite regulation
of ER
- and ER
+ breast cancer cell proliferation.
 |
MATERIALS AND METHODS
|
---|
Materials
Human recombinant EGF and IGF-I were purchased from Euromedex
(Souffelweyersheim, France) and Saxon Biochemicals (Hanover, Germany),
respectively. 17ß-Estradiol was obtained from Hoechst-Marion-Roussel
(Romainville, France) and prepared as 1000-fold concentrated stock
solutions in ethanol. Anti-Jun antibodies (raised against amino acids
247263 of mouse c-Jun), anti-Fos antibodies (amino acids 128152 of
human c-Fos), anti-c-Fos antibodies (amino acids 316 of human c-Fos),
anti-Fra-1 antibodies (amino acids 322 of human Fra-1), anti-Fra-2
antibodies (amino acids 322 of human Fra-2), and anti-FosB antibodies
(amino acids 102117 of mouse FosB) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
Cell Culture
MCF7 cells were maintained in Hams F-12/DMEM (1;1), and all
other cell lines were maintained in DMEM. All media were supplemented
with 10% FCS and 50 µg/ml gentamycin. For transient transfection
experiments, cells were stripped of endogenous steroids by successive
passages in phenol red free medium containing 10% (2 days), and then
3% (3 days) dextran-coated charcoal (DCC)-stripped FCS (DCC-FCS) (19).
They were then plated at about 80% confluence (106-2
x 106 cells per 35-mm diameter well) 24 h before
transfection. For gel retardation assays and RNA extraction, cells,
plated in T75 flasks (1:6 dilution), were stripped of endogenous
steroids by successive passages in phenol red free medium containing
10% (2 days), 3% (3 days), and finally 1% DCC-FCS (2 days).
Plasmids
(AP-1)4-TK-CAT and (mutated AP-1)4-TK-CAT plasmids were derived
from pG14XB and pG14XAB constructs, respectively (42). Four
head-to-tail copies of the wild-type AP-1-responsive element of the
polyoma virus enhancer were subcloned into the BamHI site of
the eukaryotic expression vector pBLCAT 8PN (19).
ERE-ßGlobine-Luciferase reporter plasmid was a gift of P. Balaguer
(Montpellier, France). Expression vectors for GR (glucocorticoid
receptor), ER
, and ER
mutants were kindly donated by P. Chambon
(Strasbourg, France). PCI-Fra-1 and pCI-c-Jun were constructed into pCI
vector (Promega, Madison, WI) by inserting whole cDNA sequences of
human c-Fra-1 (43) and mouse c-Jun (44) under the control of the human
cytomegalovirus immediate-early promoter. PCI-antisense Fra-1 construct
harbored whole cDNA sequence of c-Fra-1 in antisense orientation.
Transient Transfection and CAT and Luciferase Assays
Twenty four hours after plating, the medium was changed and
cells were transfected for 16 h using the calcium phosphate DNA
coprecipitation method as previously described (19). When cells were
transfected by an expression vector, the same amount of empty vector
was transfected in control cells. Two micrograms of CMV-ß
galactosidase expression plasmid were used for internal control of
transfection efficiency, and pSPT18 DNA was added up to 5 µg total
DNA per well. Cells were washed twice with phenol red free medium and
treated, as indicated, for 24 h in phenol red free medium
containing 1% DCC-FCS. CAT enzyme assays were performed in whole cell
extracts after normalization for ß-galactosidase activity (45).
Acetylated and nonacetylated forms of
[14C]chloramphenicol were separated by TLC.
Quantification was performed with a Fuji BAS1000 Bioimaging Analyzer
(Raytest, Paris, France). For luciferase assay, cells were lysed for 15
min in the cell culture lysis reagent from Promega. Luciferase activity
was measured, as described by Roux et al. (46), using an LKB
luminometer (LKB Instruments, Rockville, MD) and normalized for
ß-galactosidase activity (46).
Gel Retardation Assay
Cell extracts were prepared as described by Stein et
al. (47), aliquoted (10 µg/µl), frozen on dry ice, and stored
at -70 C. Protein concentrations were determined by a Bradford protein
assay (Bio-Rad SA, Yvry Sur Seine, France). Gel retardation assays were
performed using double-stranded oligonucleotides labeled by Klenow in
the presence of [32P]dCTP. The sequences of the
oligonucleotides were as follows. AP-1 binding sites:
5'-TCGACTGTGCTCAGTTAGTCACTTCC-3' and 5'-TCGAGGAAGTGACTAACTGAGCACAG-3'
(polyoma virus TRE sequence) and 5'-CTAGCTGTCTGAGTCATGCA-3' and
5'-AGCTTGCATGACTCAGACAG-3' (collagenase TRE sequence). Consensus Sp1
DNA-binding site: 5'-TCGATCGGGGCGGGGCGA-3' and
5'-GCTCGCCCCGCCC-CGAT-3'. In a typical binding reaction mixture (20
µl final volume), 515 µg of proteins were incubated with 3 µg
of poly(deoxyinosinic-deoxycytidylic)acid (Pharmacia Biotech, Saclay,
France) in binding buffer [50 mM Tris-HCl (pH 8), 12.5
mM MgCl2, 1 mM EDTA, 1
mM dithiothreitol, 10% glycerol] for 20 min at room
temperature. The 32P-labeled DNA (4 x 105
cpm, 0.5 pmol) was then added and the incubation continued for 20 min.
The resulting DNA-protein complexes were resolved from the free probes
by electrophoresis on a 5% nondenaturing polyacrylamide gel and
visualized by autoradiography. In competition experiments,
nonradioactive double-stranded competitor was added together with the
labeled probe. For super-shift experiments, cell extracts were
preincubated for 1 h, in binding buffer, with 2 µg antibodies
before the addition of the labeled double-stranded oligonucleotide. The
gels were dried and autoradiographed with intensifying screens at -80
C.
Northern Blotting
Total RNA (40 µg) was electrophoresed in 1% (wt/vol)
agarose gel containing formaldehyde and transferred to
Hybond-N+ membrane (Amersham Corp., Les Ulis, France). 36B4 (48),
c-Jun (43), c-Fos (49), and Fra-1 (43) cDNA probes were
32P-labeled by multiprime DNA synthesis using an Amersham
kit (SA, 109 cpm/µg). Hybridization in 50% formamide and
wash conditions were performed as previously described (8). Filters
were autoradiographed with intensifying screens at -80 C.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to P. Chambon for providing ER
and GR
expression vectors and 36B4 cDNA probe; B. Wasylyk for PB and PAB
constructs; M. Piechaczyk for c-Jun and Fra-1 cDNA plasmids; and P.
Balaguer for ERE-ßGlobine-Luciferase reporter construct. We also
thank J.-Y. Cance for photographs and colleagues in the laboratory for
critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dany Chalbos, Unité 148 INSERM, 60 Rue de Navacelles, 34090 Montpellier, France. E-mail:
chalbos{at}u148.montp.inserm.fr
This work was supported by the "Institut National de la Santé
et de la recherche Médicale", the Université of
Montpellier I, the "Association pour la Recherche sur le Cancer"
(Grants 1250 and 1411), the "Ligue Nationale contre le Cancer," and
the French "Ministère de la Recherche et de lEnseignement
Supérieur" (fellowship to C.T.).
1 The contributions of the two first authors of this manuscript should
be considered equivalent. 
2 Present address: UMR5535, IGGM, 1919 Route de Mende, 34293,
Montpellier Cedex 5, France. 
Received for publication October 17, 1997.
Revision received February 20, 1998.
Accepted for publication March 19, 1998.
 |
REFERENCES
|
---|
-
Allegra JC, Lippman ME 1978 Growth of a human breast
cancer cell line in serum-free hormone supplemented medium. Cancer Res 38:38233829[Abstract]
-
Chalbos D, Vignon F, Keydar I, Rochefort H 1982 Estrogens
stimulate cell proliferation and induce secretory proteins in a human
breast cancer cell line (T47D). J Clin Endocrinol Metab 55:276283[Abstract]
-
Lippman ME, Dickson RB, Bates S, Knabbe C, Huff K, Swain S,
McManaway M, Bronzert D, Kasid A, Gelman EP 1986 Autocrine and
paracrine growth regulation of human breast cancer. Breast Cancer Res
Treat 1:5970
-
Van der Burg B, de Groot RP, Isbrücker L, Kruijer W, de
Laat SW 1990 Stimulation of TPA-responsive element activity by a
cooperative action of insulin and estrogen in human breast cancer
cells. Mol Endocrinol 4:17201726[Abstract]
-
Vignon F, Bouton MM, Rochefort H 1987 Antiestrogens inhibit
the mitogenic effect of growth factors on breast cancer cells in total
absence of estrogens. Biochem Biophys Res Commun 159:15021508
-
Sumida C, Pasqualini JR 1989 Antiestrogens antagonize the
stimulatory effect of epidermal growth factor on the induction of
progesterone receptor in fetal uterine cells in culture. Endocrinology 124:591597[Abstract]
-
Katzenellenbogen BS, Norman MJ 1990 Multihormonal regulation
of the progesterone receptor in MCF7 human breast cancer cells:
interactionships among insulin/insulin-like growth factor-I, serum and
estrogen. Endocrinology 126:891898[Abstract]
-
Chalbos D, Philips A, Galtier F, Rochefort H 1993 Synthetic
antiestrogens modulate induction of pS2 and cathepsin-D messenger
ribonucleic acid by growth factors and adenosine 3',5'-monophosphate in
MCF7 cells. Endocrinology 133:571576[Abstract]
-
Beato M 1988 Gene regulation by steroid hormones. Cell 56:335344
-
Tsai M-J, OMalley B 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Hunter T, Karin M 1992 Control of transcription factor
activity by protein phosphorylation. Cell 70:375388[Medline]
-
Kerr LD, Inoue J-I, Verma I 1992 Signal transduction: the
nuclear target. Curr Opin Cell Biol 4:496501[CrossRef][Medline]
-
Angel P, Karin M 1991 The role of Jun, Fos and the AP-1
complex in cell proliferation and transformation. Biochim Biophys Acta 1072:129157[CrossRef][Medline]
-
Kouzarides T, Ziff E 1988 The role of leucine zipper in the
Fos-Jun interaction. Nature 336:646651[CrossRef][Medline]
-
Chalbos D, Philips A, Rochefort H 1994 Genomic cross-talk
between the estrogen receptor and growth factor regulatory pathways in
estrogen target tissues. Semin Cancer Biol 5:361368[Medline]
-
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 270:14911494[Abstract]
-
Bunone G, Briand P-A, Miksicek R, Picard D 1996 Activation of
the unliganded estrogen receptor by EGF involves the MAP kinase pathway
and direct phosphorylation. EMBO J 15:21742183[Abstract]
-
Gaub M-P, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P 1990 Activation of the ovalbumin gene by estrogen receptor involves the
Fos-Jun complex. Cell 63:12671276[Medline]
-
Philips A, Chalbos D, Rochefort H 1993 Estradiol increases and
anti-estrogens 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 19:1410314108
-
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 269:1643316442[Abstract/Free Full Text]
-
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 9:443456[Abstract]
-
Levenson AS, Jordan VC 1994 Transfection of human estrogen
receptor (ER) cDNA into ER-negative mammalian cell lines. J Steroid
Biochem Mol Biol 51:229239[CrossRef][Medline]
-
Zajchowski DA, Sager R 1991 Induction of estrogen regulated
genes differs in immortal and tumorigenic human mammary ephithelial
cells expressing a recombinant estrogen receptor. Mol Endocrinol 5:16131623[Abstract]
-
Jiang S-Y, Jordan VC 1992 Growth regulation of estrogen
receptor-negative breast cancer cells transfected with complementary
DNAs for estrogen receptor. J Natl Cancer Inst 84:580591[Abstract]
-
Garcia M, Derocq D, Freiss G, Rochefort H 1992 Activation of
estrogen receptor transfected into a receptor-negative breast cancer
cell line decreases the metastatic and invasive potential of the cells.
Proc Natl Acad Sci USA 89:1153811554[Abstract]
-
Tora L, Mullick A, Metzger D, Pouglikitmongkol M, Park I,
Chambon P 1989 The cloned human oestrogen receptor contains a mutation
which alters its hormone binding properties. EMBO J 8:19811986[Abstract]
-
Kumar V, Green S, Stack G, Berry M, Jiu J, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941951[Medline]
-
Mader S, Kumar V, de Verneuil H, Chambon P 1989 Three acids of
the oestrogen receptor are essential to its ability to distinguish an
oestrogen from a glucocorticoid-responsive element. Nature 338:271274[CrossRef][Medline]
-
Dumont JA, Bitonti AJ, Wallace CD, Baumann RJ, Cashman EA,
Croo-Doersen DE 1996 Progression of MCF-7 breast cancer cells to
antiestrogen-resistant phenotype is accompanied by elevated levels of
AP-1 DNA-binding activity. Cell Growth Differ 7:351359[Abstract]
-
Kovary K, Bravo R 1992 Existence of different Fos/Jun
complexes during the G0-to-G1 transition and during exponential growth
in mouse fibroblasts: differential role of Fos proteins. Mol Cell Biol 12:50155023[Abstract]
-
Wisdom R, Verma IM 1993 Transformation by Fos proteins
requires a C-terminal transactivation domain. Mol Cell Biol 13:74297438[Abstract]
-
Kovary K, Rizzo CA, Ryseck R-P, Noguchi T, Raynoschek C,
Pelosin J-M, Bravo R 1991 Constitutive expression of FosB and its short
form, FosB/SF, induces malignant cell transformation in Rat-1A cells.
New Biol 3:870879[Medline]
-
Metz R, Kouzarides T, Bravo R 1994 A C-terminal domain in
FosB, absent in Fos/SF and Fra-1, which is able to interact with the
TATA binding protein, is required for altered cell growth. EMBO J 13:38323842[Abstract]
-
Suzuki T, Okuno H, Yoshida T, Endo T, Nishina H, Iba H 1991 Difference in transcriptional regulatory function between c-Fos and
Fra-2. Nucleic Acids Res 19:55375542[Abstract]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R Gloss B, Lin
SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptor. Cell 85:403414[Medline]
-
Maroder M, Farina AR, Vacca A, Felli MP, Screpanti I, Frati L,
Gulino A 1993 Cell-specific bifunctional role of Jun oncogene family
members on glucocorticoid receptor-dependent transcription. Mol
Endocrinol 7:570584[Abstract]
-
Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: selectors of positive or negative
regulation from a single DNA element. Science 249:12661272[Medline]
-
Miner JN, Yamamoto KR 1992 The basic region of AP-1 specifies
glucocorticoid receptor activity at a composite response element. Genes
Dev 2:525530
-
Harrison RJ, McNeil GP, Dobner PR 1995 Synergistic activation
of neurotensin/neuromedin N gene expression by c-Jun and
glucocorticoids: novel effects of Fos family proteins. Mol Endocrinol 9:981993[Abstract]
-
Yang-Yen HF, Chambard J-C, Sun YL, 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 62:12051215[Medline]
-
Shemshedini L, Knauthe R, Sassone-Corsi P, Pornon A,
Gronemeyer H 1991 Cell-specific inhibitory and stimulatory effects of
Fos and Jun on transcription activation by nuclear receptors. EMBO J 10:38393849[Abstract]
-
Wasylyk B, Imler JL, Chatton B, Schatz C, Wasylyk C 1988 Negative and positive factors determine the activity of the polyoma
virus enhancer
domain in undifferentiated and differentiated cell
types. Proc Natl Acad Sci USA 85:79527956[Abstract]
-
Matsui M, Tokuhara M, Konuma Y, Nomura N, Ishizaki R 1990 Isolation of human fos-related genes and their expression
during monocyte-macrophage differentiation. Oncogene 5:249255[Medline]
-
Ryseck R-P, Hirai SI, Yaniv M, Bravo R 1988 Transcriptional
activation of c-jun during the G0/G1 transition
in mouse fibroblasts. Nature 334:535537[CrossRef][Medline]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning
Manual, ed 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Roux S, Térouanne B, Balaguer P, Jausons-Loffreda N,
Pons M, Chambon P, Gronemeyer H, Nicolas J-C 1996 Mutation of
isoleucine 747 by a threonine alters the ligand responsiveness of the
human glucocorticoid receptor. Mol Endocrinol 10:12141226[Abstract]
-
Stein B, Rahmsdorf HJ, Steffen A, Litfin M, Herrlich P 1989 UV-induced DNA damage is an intermediate step in UV-induced expression
of human immunodeficiency virus type 1, collagenase, c-fos, and
metallothionein. Mol Cell Biol 9:51695181[Medline]
-
Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A,
Chambon P 1982 Cloning of cDNA sequences of hormone-regulated genes
from the MCF-7 human breast cancer cell line. Nucleic Acids Res 10:78957903[Abstract]
-
Roux P, Verrier B, Klein B, Niccolino M, Marty L, Alexandre C,
Piechaczyk M 1991 Retrovirus-mediated gene transfer of a human
c-fos cDNA into mouse bone marrow stromal cells. Oncogene 6:21552160[Medline]
-
Davidson NE, Prestigiacomo LJ, Hahm HA 1993 Induction of
jun gene family members by transforming growth factor
but not 17ß-estradiol in human breast cancer cells. Cancer Res 53:291297[Abstract]