Oncogene (2005) 24, 4789–4798
& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00
www.nature.com/onc
Opposite effects of estrogen receptors alpha and beta on MCF-7 sensitivity
to the cytotoxic action of TNF and p53 activity
Sebastian A Lewandowski1,4, Jerome Thiery2, Abdelali Jalil2, Guy Leclercq3, Cezary Szczylik1
and Salem Chouaib*,2
1
Department of Oncology, Military Institute of Medicine, Szaserow 128 Street, 00-909 Warsaw, Poland; 2INSERM U487, ‘Cytokines
et Immunologie des Tumeurs Humaines’ Institut Gustave Roussy, 94805 Villejuif Cedex, France; 3Laboratoire J-C Heuson de
Cancérologie Mammaire, Service de Médecine Interne, Institut Jules Bordet, Université Libre de Bruxelles, 1 rue Héger-Bordet,
B-1000, Brussels, Belgium; 4Postgraduate School of Molecular Medicine, 3 Pasteur Street, 02-093 Warsaw, Poland
We have investigated the effect of estrogen on p53 cellular
location and its influence on tumor cell susceptibility to
tumor necrosis factor (TNF)-mediated cytotoxic action.
For this purpose, we have used the TNF-sensitive human
breast adenocarcinoma MCF-7 and its derivative, the
TNF-resistant 1001 clone. Our data indicate that
although estrogen receptor (ER)a is present in both cell
lines, estrogen treatment (1 108 M) has an influence
only on the MCF-7 cells and protects these cells from the
TNF cytotoxicity. This protective effect is associated with
translocation of p53 from the nucleus to the cytoplasm in
p53 wild-type MCF-7 and not in p53-mutated 1001 cells.
The translocation of p53 in MCF-7 cells results in a
decrease in its transcriptional activity, as revealed by
diminished p21WAF1/CIP1 induction and an altered ratio of
Bax and Bcl-2 proteins. The estrogen-induced effects are
reversed by the selective estrogen inhibitor 182, 780
(1 106 M). Interestingly, transient transfection of
MCF-7 cells with ERb but not ERa cDNA encoding
plasmid results in retention of p53 in the nucleus, a
subsequent potentiation of its transcriptional activity, and
in an increased MCF-7 sensitivity to TNF. The estrogen
effects on p53 location and transcriptional activity may
involve the mdm2 protein since both events were reversed
following MCF-7 transfection with plasmid encoding the
ARF cDNA. These studies suggest that estrogen-induced
MCF-7 cell survival in the presence of TNF requires a
transcriptionally active p53 and, more importantly,
indicate that introduction of ERb can attenuate the
estrogen effects on the p53 protein location, its transcriptional activity and also results in a potentiation of cell
sensitivity to TNF-mediated cell death.
Oncogene (2005) 24, 4789–4798. doi:10.1038/sj.onc.1208595;
published online 2 May 2005
Keywords: estrogen receptor; p53; cell death
*Correspondence: S Chouaib; E-mail: [email protected]
Received 16 July 2004; revised 4 February 2005; accepted 8 February
2005; published online 2 May 2005
Introduction
It is well established that a better understanding of
the interplay between tumor-associated proapoptotic
and antiapoptotic pathways may offer novel modalities
of manipulating tumor cell growth and thereby improving the effectiveness of cancer treatment. Estrogen
(17b-estradiol), apart from its role in reproduction,
is also known to regulate the growth of hormoneresponsive tissues, eventually leading to carcinogenous
progression (Sommer and Fuqua, 2001). Clinical
and epidemiological studies correlate the prolonged
estrogen treatment of post-menopausal women with an
increase rate of breast cancer occurrences (Rossouw
et al., 2002). Besides growth stimulation, estrogen
can also exert protective effect on breast cancer
cells undergoing stress stimuli such as UV, taxol
(Razandi et al., 2000) oxidative stress (Sudoh et al.,
2001; Kanda and Watanabe, 2003) or tumor necrosis
factor (TNF) (Burow et al., 2001). There are two distinct
types of estrogen receptor (ER), the ERa and ERb
(Green et al., 1986; Mosselman et al., 1996; Nilsson
et al., 2001). Both share similar domain design,
estrogen-binding affinity (Kuiper et al., 1997) and
recognize the same DNA sequence, the estrogen
response element (ERE) (Klein-Hitpass et al., 1986) in
promoters of estrogen responsive genes (Pace et al.,
1997; Hyder et al., 1999; Hall and Korach, 2002). On the
other hand, the receptors have diverse expression
patterns and exert dissimilar transcriptional activities
in response to estrogen (Hall and McDonnell, 1999;
Kuiper et al., 1997). The knockout mouse models for
ERa and ERb further support the view on diverse
actions of both receptors. While the ductal growth of
mammary gland is reduced in ERa knockout mice
(Mueller et al., 2002), it remains unaffected in ERb
knockouts (Krege et al., 1998). Both receptors have also
been shown to have different roles in the proliferation of
the normal mammary gland (Cheng et al., 2004). The
precise roles of ERa and ERb on the proliferation and
protection are still unknown. They have been so far
shown to exhibit unique properties on the cell cycle
progression, activation of the mitogen-activated protein
kinase pathway and protein kinase C expression (Burow
Effects of ERa and ERb on p53 activity and TNF response
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4790
et al., 2000; Wade et al., 2001; Liu et al., 2002; Paruthiyil
et al., 2004; Strom et al., 2004).
The p53 protein is one of the most extensively studied
tumor suppressors that promotes DNA repair, cell cycle
arrest and apoptosis (Vousden and Lu, 2002). There are
two most prominent features of p53. Firstly, it arrests
the cell cycle by the induction of p21 (WAF1/CIP1)
gene. Secondly, it controls two types of the apoptotic
pathways: the extrinsic, based on cell surface receptors
(Owen-Schaub et al., 1995; Wu et al., 1997; Muller et al.,
1998), and the intrinsic, which centers on the mitochondrial proteins from the Bcl-2 family (Miyashita et al.,
1994b; Kroemer and Reed, 2000). Acquisition of
somatic p53 mutation results in a decreased sensitivity
to cytotoxic drugs and host immune responses. Recently, it has been reported that estrogen was able to
alter p53 location and modulate the p53 pathway in ERpositive cells (Molinari et al., 2000; Kato et al., 2002),
indicating that estrogen has an ability to modulate p53
action. However, the mechanisms by which ERs mediate
the protective effect remains unknown, yet is of great
importance for improving breast cancer cytotoxic
treatment.
These studies were designed to better understand the
functional interaction between estrogen, the tumor
suppressor p53 protein and the influence of such
interaction on the cytotoxic action of TNF. The latter
has a powerful direct tumor-killing capability (Chouaib
et al., 1991) and has been shown to play a role in tumor
regression mediated by killer cells. TNF is released by
NK cells, cytotoxic T lymphocytes (CTLs), and significantly contributes to the local immune response to
the tumor. Thus, when its secretion is confined to the
area of tumor growth, TNF may fulfill its promise as an
anticancer agent. Previously, we have provided evidence
for the association between the resistance of human
breast adenocarcinoma cell line MCF-7 to the cytotoxic
actions of TNF and the loss of p53 function (Cai et al.,
1997b). This insensitivity can be reversed by the
reintroduction of wild-type, transcriptionally active
p53 (Ameyar et al., 1998). Here we describe the
protective effect of estrogen on ERa-positive MCF-7
cells on TNF-mediated cytotoxicity and its association
with altered cellular localization and transcriptional
activity of p53. Interestingly, those properties can be
reversed not only with antiestrogen ICI 182, 780
treatment but also by the introduction of ERb cDNA.
Results
The differential response to estrogen in the presence of
TNF is not related to altered expression of ERa by
MCF-7 and 1001 cells
We have first investigated the effect of estrogen on the
TNF cytotoxic action in the TNF-sensitive MCF-7 and
TNF-resistant derivative 1001 cells. Data depicted in
Figure 1a indicate that estrogen-deprived MCF-7 cells
were sensitive to TNF, while estrogen treatment resulted
in resistance of MCF-7 cells to the cytotoxic action of
Oncogene
Figure 1 The differential response to estrogen in the presence of
TNF is not related to altered expression of ERa. (a) Estrogendeprived MCF-7 (’) and 1001 (&) cells were treated with DMSO
(0.01%), estrogen (E2) (1 108 M) and ICI 182, 780 (1 106 M)
for 24 h and exposed to 100 ng/ml of TNF. Cell viability was
measured using the viability assay as described in Materials and
methods. Data presented are the mean7s.d. of triplicate determinations. The comparator treatment for both MCF-7 and 1001 cells
was 0 ng/ml TNF without steroid treatment (DMSO). The level of
significance was Po0.004 (*) for compared DMSO vs E2 and E2 vs
E2 þ ICI groups in MCF-7 cells as determined by two-tailed
Student’s t-test. (b). Expression of ERa in MCF-7 and 1001 cells. In
all, 50 mg of cell lysate was analysed using Western blotting for ERa
expression as described in Materials and methods. Similar results
were obtained in three independent experiments
TNF, similar to the 1001 clone. The protective effect of
estrogen was reversed by co-treatment with specific
inhibitor ICI 182, 780. Results from cells treated only
with dimethylsulfoxide (DMSO) were used as point of
reference for each cell line. Estrogen stimulation of
starved cells in the absence of TNF has significantly
increased the viability of only MCF-7 cells (data not
shown). The level of significance for the MCF-7 TNF
sensitivity test of DMSO- vs E2- and E2- vs E2 þ ICItreated groups was Po0.004, as determined by twotailed Student’s t-test. We have next examined if the
differential responsiveness to estrogen was due to an
altered expression of ERa in MCF-7 and 1001 cells. As
shown in Figure 1b, Western blotting analysis indicates
a comparable ERa expression by both cell lines,
suggesting that the lack of response to estrogen in
TNF-treated 1001 cells is not associated with altered
ERa expression.
Exogenous estrogen stimulates the translocation of p53
protein in MCF-7 but not in 1001 cells, and results in a
decrease in its transcriptional activity
It has been previously reported that MCF-7 cells
stimulated with estrogen displayed an export of the
p53 protein from the nucleus to the cytoplasm. We have
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
4791
thus asked if this effect might occur in the TNF-resistant
clone 1001 carrying a mutated p53. When cultured in
FBS supplemented media, MCF-7 cells show p53 mostly
positioned in the cytoplasm (data not shown). However,
after estrogen deprivation p53 is mostly accumulated in
the nucleus. Following estrogen stimulation, MCF-7
cells exhibit a substantial change by promoting p53
export from the nucleus to the cytoplasm. The later
event was not observed in 1001 cells. Co-treatment with
both estrogen and estrogen inhibitor ICI 182, 780
diminished the p53 export in MCF-7 and showed no
effect in 1001 (Figure 2a). This indicates that estrogen
has an effect on the wt p53 protein location and is
unable to export it in the 1001 clone. To test whether the
p53 translocation in MCF-7 was associated with a
functional effect on its transcriptional activity, we have
examined the influence of estrogen on three p53regulated proteins: p21, the regulator of the cell cycle
and the ratio between Bax and Bcl-2 proteins, a
common marker for mitochondria contribution to
apoptosis. Data shown in Figure 2b indicate that
estrogen induced a significant decrease in p21 protein
expression following irradiation in MCF-7 but not in
1001 cells. Co-treatment with estrogen and ICI 182, 780
partially diminished estrogen-mediated effect. Since
some of the Bcl-2 family members are directly regulated
by p53, we have asked if the p53 shift from the nucleus
to cytoplasm affects the expression of Bax and Bcl-2
proteins. To determine the expression level of Bax and
Bcl-2, we have carried out Western blotting of estrogendeprived MCF-7 and 1001 cells treated with estrogen
and ICI 182, 780 for 24 h. Data shown in Figure 2c
indicate that estrogen treatment decreased the level of
Bax and increased the level of Bcl-2, changing the
overall ratio between the two proteins only in MCF-7
cells (Figure 2b). These data indicate that the estrogendependent cytoplasmic export of p53 results in a
subsequent inhibition of its transcriptional activity and
affects expression of p53-regulated proteins involved in
the control of cell cycle and apoptotic process.
Differential influence of ERa and ERb on p53
transcriptional activity
Since the balance between ERa and ERb may control
the estrogen effect, we have asked whether the
introduction of ERb protein affects the observed
estrogen response. Two expression plasmids pERa and
pERb were introduced to MCF-7 cells. The transfection
efficiency was determined by Western blotting
(Figure 3a). The functional properties of introduced
receptors were compared by examining their transcriptional activity on a construct with ERE and luciferase
gene (Figure 3b). The endogenous ERa in MCF-7 cells
induced transcription after estrogen treatment and this
activity was reversed with specific inhibitor. With
overexpression of ERa, the luciferase activity was
increased as compared to endogenous level and also
reversible by this inhibitor as shown in Figure 3b. The
introduction of ERb resulted in a decrease in the ERE
transcriptional activity level after estrogen stimulation.
To examine if the introduction of ERb has an effect on
estrogen-mediated p53 transcriptional activity, we have
analysed the p21 protein induction after gamma
irradiation. The data depicted in Figure 3c indicate that
overexpression of ERa induced a decrease in p21
expression while the introduction of ERb resulted in
almost twofold increase of p21 as compared with basal
induction level. These results further confirm the
differential properties of the two ERs on p53 transcriptional activity.
ERa and ERb have antagonistic effects on MCF-7 cell
sensitivity to TNF
To compare the effects of ERs on the sensitivity to TNF,
we have transiently transfected the cells grown in regular
FBS-supplemented media with pERa and pERb for 3 h.
After an additional 21 h, the cells were exposed to
various concentrations of TNF (Figure 4). Cells
transfected with pERb showed an increased sensitivity
to TNF at concentrations ranging from 5 to 100 ng/ml
than those transfected with pERa (Po0.001) or pcDNA
3.1 (Po0.05) as determined by Student’s t-Test. These
results demonstrate the differential influence of ERa and
ERb on cell viability in the presence of TNF and suggest
an antagonistic effect of ERb and ERa.
Introduction of ERb prevents p53 export in estrogentreated MCF-7 cells
To find out if ERb has an effect on estrogen-stimulated
p53 translocation, we have examined estrogen-deprived
MCF-7 cells transfected with pERa and pERb after
estrogen stimulation. Confocal microscopy analysis
using anti-p53 antibody (Figure 5) indicates that empty
vector transfected cells with endogenous ERa showed
estrogen-dependent, inhibitor-reversible translocation of
the p53 protein similarly to those transfected with
pERa. However, the pERb-transfected cells maintained
p53 in the nucleus after estrogen treatment. These data
suggest that the introduction of ERb can potently
reduce estrogen-stimulated p53 protein translocation.
Mdm2 accumulation is associated with estrogen-mediated
effects on p53 location and transcriptional activity
We have asked if estrogen effects on p53 involve the
mdm2 protein. For this purpose, we have transiently
transfected MCF-7 cells with a plasmid encoding the
ARF protein known to induce rapid degradation of
mdm2. Such transfection resulted in accumulation of
p53 in the nucleus and a lack of p53 export to the
cytoplasm after estrogen stimulation (Figure 6a) as
determined by immunofluorescent staining and confocal
microscopy. Additionally, MCF-7 cells transfected with
pARF were resistant to estrogen-mediated decrease of
gamma irradiation stimulated expression of p21 protein
(Figure 6b). These results suggest that estrogen effects
on p53 localization and activity are at least in part
mediated by mdm2. In order to track the cellular
localization of mdm2 after estrogen stimulation, we
Oncogene
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
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Figure 2 Effect of exogenous estrogen on the translocation of p53 protein and its transcriptional activity in MCF-7 and 1001 cells.
MCF-7 and 1001 cells were preincubated in hormone-free media for 6 days and then treated with DMSO (0.01%), estrogen (E2)
(1 108 M) and ICI 182, 780 (1 106 M) for 24 h. (a) p53 protein localization in MCF-7 and 1001 cells determined by confocal
microscope imaging and immunofluorescence. (b) Expression of p21 protein following estrogen treatment. MCF-7 and 1001 cells were
exposed to 5 Gy dose of gamma irradiation (’). Nonirradiated cells (&) were used for comparison. p21WAF1/CIP1 expression was
determined after subsequent 6 h by Western blotting. Basal expression of p21 in MCF-7 was used as control [C] for 1001 samples.
Relative band intensity in respect to actin was calculated using Bio-Profil software and is shown as a graph. (c) Expression of Bax and
Bcl-2 following estrogen treatment. In all, 50 mg of cell lysate was analysed by Western blotting for Bax and Bcl-2 expression as
described in Materials and methods. Relative band intensity was calculated in respect to actin using Bio-Profil software and is shown as
a graph presenting the ratio between expression level of Bax and Bcl-2
have transiently transfected estrogen-deprived MCF-7
cells with pERa and pERb plasmids followed by steroid
treatment. Confocal microscopy analysis using antimdm2 antibody indicates that MCF-7 cells exposed to
Oncogene
estrogen accumulate the mdm2 protein in the nucleus.
The transfection with pERb plasmid significantly
impaired such accumulating effects after estrogen
treatment (Figure 6c).
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
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Figure 4 ERa and ERb have opposing effects on MCF-7 cells
sensitivity to TNF. MCF-7 cells were cultured in FBS supplemented medium and transiently transfected with pERa (&), pERb (’)
encoding plasmids and pcDNA 3.1 ( ) as control. At 24 h after
transfection, the cells were exposed to a range of TNF concentrations. Cell viability was measured using the MTT assay as
described in Materials and methods. Results from nontreated cells
(0 ng/ml TNF) were used as point of reference within each
transfection. Data presented are the means7s.d. of triplicate
determinations. The level of significance was Po0.005 (*) for ERa
vs ERb transfected cells and Pp0.01 (**) for ERb vs pcDNA 3.1,
as determined by the Student’s t-test. Similar results were obtained
in three independent experiments
Discussion
Figure 3 Differential influence of ERa and ERbon p53 transcriptional activity. (a) MCF-7 cells were transiently transfected with
pERa and pERb plasmids and pcDNA 3.1 as control. After 24 h
50 mg of cell lysate was analysed by Western blotting for presence of
ERa and ERb as described in Materials and methods. (b)
Transcriptional activity of introduced receptors. MCF-7 cells were
preincubated in hormone-free media for 6 days. Then cells were
transfected with the respective ER plasmids together with two
luciferase reporter plasmids 3 Vit-ERE-Luc and pRL as described in Materials and methods, followed by treatment with
DMSO (’), estrogen (’) or estrogen and ICI 182, 780 ( )
containing media. After 24 h 1 104 cells were used for analysis of
luciferase activity. Result is shown in relative luciferase units
(3 Vit-ERE-Luc/pRL) of triplicate measurements. (c) Expression
of p21 protein after transfection with pERa and pERb. MCF-7
cells cultured in FBS supplemented medium were transiently
transfected with pERa, pERb encoding plasmids and pcDNA 3.1
as control. After 24 h MCF-7 cells were exposed to 5 Gy dose of
gamma irradiation (’) and nonirradiated cells (&) were used for
comparison. p21WAF1/CIP1 expression was determined after
subsequent 6 h by Western blotting. Relative band intensity with
respect to actin was calculated using Bio-Profil software and is
shown as a graph. Similar results were obtained in two independent
experiments
ER plays an important role in the development,
progression and treatment of breast cancer (Sommer
and Fuqua, 2001). It has been proven to be a valuable
predictive and prognostic factor in the clinical management of this disease. Consequently, its inhibition has
become one of the major strategies for the prevention
and treatment of breast cancer. However, there is
increasing evidence that this receptor interacts with coregulatory proteins and several signal transduction
pathways other than the so-called ‘classical’ ligandmediated stimulation of genes containing EREs (Driggers and Segars, 2002; Segars and Driggers, 2002).
Several reports have demonstrated that estrogen promotes cell survival in estrogen-responsive cells (Razandi
et al., 2000; Sudoh et al., 2001; Kanda and Watanabe,
2003). We have first examined, in the course of these
studies, the ability of estrogen to influence cell survival
in response to TNF taking advantage of our cell model
based on the sensitive MCF-7 breast carcinoma cell line
and its resistant derivative 1001 clone, displaying a
mutated p53, to the cytotoxic action of TNF. It is well
established that, among the players involved in the
regulation of cell survival and death, p53 has a major
function in transducing stress to apoptotic machinery of
the cell. In a previous report, we have shown that wildtype p53 is involved in the cytotoxic action of TNF and
that p53 function contributes to resistance of tumor cells
to TNF-induced killing (Cai et al., 1997b). This is
consistent with the importance of p53 status as a
determinant of cellular response to DNA-damaging
Oncogene
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
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Figure 5 The export of p53 after estrogen treatment in MCF-7 cells is altered following the transfection with pERb. Confocal
microscopy analysis of p53 protein localization. MCF-7 cells were preincubated in hormone-free media for 6 days and then transiently
transfected with pERa, pERb encoding plasmids and pcDNA 3.1 as control. At 6 h after transfection, the cells were exposed to
estrogen/inhibitor-containing media. After another 24 h, cells were fixed and analysed by immunofluorescence for p53 protein staining.
Transfection efficiency was determined using cotransfection of respective plasmids together with pGFP plasmid in the ratio 1 : 1. Cells
were analysed using FACS with an average of 68%75.8% positive fluorescent cells (data not shown). Similar results were obtained in
two independent experiments
factors (Brown and Wouters, 1999) and its key role in
the development or progression of many types of cancer.
We have therefore attempted to elucidate the cross-talk
between estrogen, p53 and TNF cytotoxic action.
Here we demonstrated, based on the p53 wild-type
parental MCF-7 cells and the p53-mutated 1001 clone,
that although both cell types express ERa they respond
differentially to the effect of estrogen under TNF
stimulation. This suggests a possible relationship between the effect of estrogen and p53 function. Accumulating evidence has been provided indicating that the
activity of p53, including post-translational modifications and the ability to interact with other proteins, can
be further controlled by regulation of the subcellular
localization of components of the p53-response pathways. Obviously, p53 is actively transported into and
out of the nucleus and can be localized to distinct
structures in both the nucleus and cytoplasm. Here we
show that the estrogen effect on MCF-7 is associated
with an export of the p53 protein from the nucleus to the
cytoplasm and a subsequent decrease in its transcriptional activity. Interestingly, no such distribution was
observed in 1001 cells displaying a mutated p53 that has
lost its transcriptional activity (Cai et al., 1997b). The
mutation was described in the DNA-binding domain
and does not explain the inability to migrate between the
Oncogene
nucleus and cytoplasm. Besides impaired transcription,
p53 protein in the 1001 cells is likely unable to respond
to the mechanisms of its turnover such as ubiquitination, phosphorylation or acetylation (Brooks and Gu,
2003).
It is clearly established that the ERa mediates breast
cancer-promoting effects of estrogen. However, the role
of ERb in breast cancer remains elusive. Several studies
show that ERb has a counteracting effect on the ERa
activity (Cowley et al., 1997; Pettersson et al., 1997; Hall
and McDonnell, 1999; Wade et al., 2001; Liu et al.,
2002). In this regard, we have shown that ERb can
antagonize the effect of ERa. In fact, the introduction of
ERb- to ERa- positive MCF-7 cells results in three
observed effects. (i) It allows retention of p53 in the
nucleus after estrogen stimulation. (ii) It increases p53
transcriptional activity. (iii) It consequently leads to an
increased sensitivity to TNF. These observations further
underline that estrogen-induced effects on MCF-7 need
a transcriptionally active p53. Hence, our model can
reflect the mammary cancer progression, which usually
advances from hormone-dependent, p53-wt to hormone-independent, p53-mutated phenotype (Moll
et al., 1992; Berns et al., 2000; Caleffi et al., 1994).
Our results confirm the previously observed effects of
estrogen under TNF treatment in MCF-7 cells (Burow
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
4795
Figure 6 Mdm2 accumulation is associated with estrogen-mediated effects on p53 location and transcriptional activity. (a) Confocal
microscopy analysis of p53 staining in MCF-7 cells following pARF transfection and estrogen stimulation. Cells were preincubated in
hormone-free media for 6 days and then plated on glass coverslips in a 24-well plate. Following overnight attachments, cells were
transiently transfected with pARF and pcDNA 3.1 plasmids for 12 h. The cells were assayed 24 h after estrogen stimulation. Cells
showing accumulated p53 are indicated with arrows. (b) Expression of p21 protein after transfection with pARF and pcDNA 3.1
plasmids. MCF-7 cells were cultured in estrogen-deprived medium and transfected with the respective plasmids for 12 h and stimulated
with estrogen for 24 h. Then, cells were exposed to 5 Gy dose of gamma irradiation and assayed by Western blotting. (c) Confocal
microscopy analysis of mdm2 protein localization. MCF-7 cells were preincubated in hormone-free media for 6 days and then
transiently transfected with pERa, pERb encoding plasmids and pcDNA 3.1 as control. After transfection the cells were exposed to
estrogen/inhibitor-containing media. After another 24 h, cells were fixed and analysed by immunofluorescence for mdm2 protein
staining as described in Materials and methods. Cells displaying accumulated mdm2 protein are indicated with arrows. Similar results
were obtained in two independent experiments
et al., 2001) and also point to p53 as the target of
estrogen action. Recently, it has been reported that the
induced expression of ERb in the breast cancer cell line
T47D reduced 17b-estradiol-stimulated proliferation
when expression of ERb equals that of ERa and that
such an effect involves a functional interaction with
components of the cell cycle machinery in these cells
(Strom et al., 2004). In addition, recently it has been
shown that ERb inhibits proliferation by repressing
c-myc, cyclin D and cyclin A gene transcription and
increases the expression of p21 and p27, which leads to a
G2 cell cycle arrest (Paruthiyil et al., 2004).
Our findings can also shed light on the paradoxical
regulation of Bcl-2 family proteins by estrogen (Leung
and Wang, 1999). As reported by other authors,
estrogen induces the expression of Bcl-2 protein
(Teixeira et al., 1995; Burow et al., 2001; Kanda and
Watanabe, 2003), changing the overall ratio between
Bcl-2 and Bax (Huang et al., 1997; Leung and Wang,
1999) – a marker for mitochondria contribution in
apoptosis (Oltvai et al., 1993). Since p53 is shown to be a
positive transcriptional regulator for Bax (Miyashita
et al., 1994b; Miyashita and Reed, 1995) and a negative
for Bcl-2 (Miyashita et al., 1994a, b), estrogen-mediated
Oncogene
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
4796
decrease in p53 transcriptional activity would explain
the contradictory regulation of these proteins by
estrogen. Nevertheless, Bcl-2 may only represent one
pathway by which estrogen and ER function to promote
cell survival. Indeed, the effects of estrogen on survival
signaling may also occur through activation of other
signaling cascades. The constitutive high Bcl-2 levels in
1001 cells are also in line with what other teams have
observed in drug-resistant derivatives of MCF-7 (Lilling
et al., 2000).
There are two plausible explanations on how ERs can
mediate their effects on p53 protein. Firstly, ERa and
p53 were shown to interact directly, which results in a
decreased ERE-dependent transcription of ERa (Yu
et al., 1997; Liu et al., 1999). However, this interaction
occurs regardless of estrogen or inhibitor presence and
results in a protective rather than deactivating effect on
p53 (Liu et al., 2000). Secondly, estrogen stimulation of
ERa-positive cells can increase the expression of mdm2,
the main regulator of p53 localization and stability
(Kato et al., 2002; Phelps et al., 2003). Nevertheless, the
ERa and ERb expression status has not been clearly
defined in these reports.
Our findings suggest that mdm2 protein may be
involved in the mediation of estrogen effects on p53.
Firstly, the estrogen stimulation accumulates mdm2
protein in the nuclei of ERa-positive cells. Secondly, the
exogenous expression of the ARF protein results in the
accumulation of the p53 protein in the nucleus and in an
increase in its transcriptional activity despite estrogen
stimulation. ERa and ERb did not display similar
effects on the mdm2 accumulation following estrogen
treatment. The antagonistic roles of ERa and ERa on
the mdm2 expression could be explained by previously
described opposing activities of both receptors on the
AP-1 sequence within several gene promoters (Paech
et al., 1997; Weatherman and Scanlan, 2001). Such
effect could also apply to the AP-1 site present in the
mdm2 gene promoter. Experiments will be designed to
address this issue in our experimental model.
There are several implications resulting from estrogen-mediated suppression of p53 protein. First, it can
allow efficient growth of mammary gland cells by
suppressing the p53 activity and consequently the
growth-inhibiting proteins under its control. The concept of constantly blocked wild-type p53 protein implies
low pressure for p53 gene mutations (Wynford-Thomas
and Blaydes, 1998) and could explain low (9%)
mutation frequency in mammary gland (Caleffi et al.,
1994) as compared with overall rate (50–55%) (Hollstein
et al., 1996) in all human cancers. Secondly, prolonged
exposure to estrogen can allow damaged cells to bypass
the p53-regulated mechanisms of DNA repair and cell
elimination by p53-dependent apoptosis (Vousden and
Lu, 2002). This can ultimately result in acquisition of the
mutation in p53 itself, a key step in tumor progression
resulting in an increased cytotoxic and chemotherapeutic resistance (Cai et al., 1997b; Berns et al., 2000;
Geisler et al., 2001; Thiery et al., 2003).
In summary, our data show a differential effect of
estrogen on TNF-sensitive cells and their resistant
Oncogene
counterparts with compromised p53 transcriptional
activity, and highlight the ERb dominant-negative
action on ERa. Our observations also argue that the
balance between ERa and ERb plays a role in the
control of cell survival by a mechanism involving at least
in part the status of p53.
Materials and methods
Plasmids and reagents
Plasmids pRST7 ERa, (pERa), pRST7 ERb (pERb) and
3 ERE vitellogenin luciferase (3 Vit-ERE-Luc) reporter
construct were kind gifts of Dr Kenneth S Korach (NIEHS
NIH NC). The pARF-Myc plasmid was a kind gift from Dr
Yue Xiong (University of North Carolina Chapel Hill, NC,
USA). The Renilla-Luciferase (pRL) reporter was purchased
from Promega (Madison, WI, USA) and pcDNA3.1 plasmid
was purchased from Invitrogen (Carlsbad, CA, USA). Estrogen (17b-estradiol) was purchased from Sigma (St Louis, MO,
USA) and ICI 182, 780 was purchased from Tocris (Northpoint, UK). TNF-a (Beromun) was purchased from Boehringer Ingelheim (Ingelheim, Germany). FuGENE 6 transfection
reagent was purchased from Roche Molecular Biochemicals
(Mannheim, Germany). 3-[4,5-Dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide (MTT) and DMSO were purchased
from Sigma.
Cell culture and hormone treatments
MCF-7 TNF-resistant clone 1001 was established as described
(Cai et al., 1997a). All cells were cultured in RPMI with
glutamax (GIBCO, Carlsbad, CA, USA) supplemented with
5% fetal bovine serum (FBS) and 100 U/ml penicillin–
streptomycin solution in a humidified atmosphere with 5%
CO2. For 1001 cells, geneticin (GIBCO) was used as selective
agent at a concentration of 250 mg/ml. Before estrogen
treatments, the cells were cultured for 6 days in RPMI phenol
red-free medium (GIBCO) supplemented with 5% charcoaldextran-treated serum (HyClone, Logan, UT, USA) and 100 U
penicillin-streptomycin. For hormonal treatments, estrogen
was used at concentration of 1 108 M, ICI 182, 780 was used
at concentration of 1 106 M. DMSO was used as diluent for
stock solutions of E2, ICI and also as mock control treatment
at 0.01% v/v. For gamma irradiation, MCF-7 cells (5 105)
were exposed to 5 Gy dose of radiation and cultured for an
additional 6 h before protein harvest.
Western blotting
Cells were harvested in protein extraction buffer (25 mM
Hepes, pH 7.5,. 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
1% Triton -100, 0.5 mM DTT, 0.5% sodium deoxycholate,
10% glycerol). Complete Mini proteinase inhibitors cocktail
from Roche was added as suggested by the manufacturer. The
protein concentration was determined using BCA Protein
Assay Kit from Pierce (Rockford, IL, USA). Protein extracts
(50 mg) were separated on a 12% sodoum dodecyl sulfate
(SDS)–polyacrylamide gel and electroblotted on nitrocellulose
Hybondt membranes (Amersham Pharmacia Biotech, Aylesbury, UK). The transfer efficiency and protein equivalence
were determined with Ponceau Red (Sigma) staining. After
blocking, the membranes were blotted with anti-ERa antibody
(Cell Signaling, Beverly, MA, USA), ERb antibody (Zymed,
San Francisco, CA, USA), anti-p21/WAF1 (Oncogene,
Merck-KgaA, Darmstadt, Germany), anti-Myc mouse
Effects of ERa and ERb on p53 activity and TNF response
SA Lewandowski et al
4797
monoclonal antibody developed in our laboratory and Bax,
Bcl-2 or alpha-actin antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA, USA). Secondary antibodies against rabbit,
mouse and donkey (Santa Cruz) conjugated with horseradish
peroxidase (HRP) were used in the second blotting step.
Complexes were detected using ECL chemiluminescence kit
(Amersham Pharmacia Biotech) and autoradiography. Subsequent results were analysed using BioProfil Bio1D Software
(Vilber Lourmat, Paris, France).
Confocal scanning immunofluorescence microscopy
3 104 cells grown on sterile slides were subjected to hormone
treatments as described above. Slides were washed once with
PBS and fixed for 10 min in 4% paraformaldehyde (PFA)
solution in PBS and washed three times with PBS. Membranes
were permeabilized for 10 min with 0.1% SDS in PBS and
washed three times with PBS. Nonspecific sites were blocked
for 20 min with 10% FBS in PBS and washed once with PBS.
The cells were incubated for 2 h with anti-p53 monoclonal
antibody (DO-1) (Santa Cruz) or anti-mdm2 antibody
(SMP14, Santa Cruz). Nuclei were counterstained using
SYTOX green reagent (Molecular Probes, Eugene, OR,
USA). After three washes with PBS, anti-mouse secondary
antibody conjugated with Alexa 546 (Molecular Probes,
Eugene, OR, USA) was used for 1 h. Coverslips were mounted
with antifading Vectashield from (Vector, Burlingame, CA,
USA). Confocal microscopy analysis was performed on Zeiss
LSM 510 microscope.
Cell viability
MCF-7 cells (5 103) or 1001 (7.5 103) were plated in flatbottom 96-well plates. For TNF treatments the estrogendeprived cells were preincubated with estrogen, ICI 182, 780 or
DMSO for 24 h and then the media was switched to mixed
steroid and TNF solutions. After 72 h, MTT solution (2.5 mg/
ml in PBS) was added to a final concentration of 0.8 mg/ml.
Cells were incubated for additional 2 h and lysed with MTT
lysis buffer (50% N-N0 dimethyl formamide, 20%, SDS pH
4.7). The OD was measured at 550 nm with 630 nm wavelength
as reference. The percentage of cell viability was calculated as
follows: percentage of viability ¼ (A1/A0) 100, where A1 and
A0 represent absorbances obtained, respectively, for treated
and untreated cells. The average value of a triplicate
measurement was used for data analysis.
Transient transfection and luciferase reporter assays
In all, 3–4 104 cells/cm2 were transfected 24 h after plating
with 0.5 mg/ml. of either pERa, pERb or pcDNA3.1 plasmid
using the FuGENE 6 transfection reagent according to the
manufacturer’s instructions. For viability assays TNF treatments were applied 24 h after transfection. For luciferase
reporter assays, 1 104 MCF-7 cells were transfected for 6
hours with 0.15 mg of either pERa or pERb plasmid together
with 0.15 mg of the 3 Vit-ERE-Luc and 0.01 mg of pRL using
the FuGENE 6 reagent according to the manufacturer’s
instructions. The total amount of DNA was brought to 0.5 mg
with pcDNA3.1 plasmid. After 6 h, the transfection solution
was switched to hormonal treatments and incubated for an
additional 20 h. Cells were assayed with Dual-Luciferases
Reporter Assay System kit from Promega (Madison, WI,
USA) and data were collected using MicroLumat LB96P
luminomiter counter (Berthold, Bad Wildbad, Germany).
Abbreviations
ERs, estrogen receptors; TNF, tumor necrosis factor a; ERb,
estrogen receptor beta; ERa, estrogen receptor alpha; ERE,
estrogen response element; DMSO, dimethylsulfoxide; E2,
17b-estradiol; ICI, ICI 182, 780.
Acknowledgements
This work was supported by grants from INSERM, the
Association pour la Recherche sur le Cancer (Grants 5253–
5129). SAL is a recipient of a fellowship from Marie Curie (5th
PCRD, contract QLGA-1999-50406). JT is supported by a
fellowship from the Ligue Nationale Francaise de Recherche
Contre Le Cancer.
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