Plasma Membrane Estrogen Receptors Signal to Antiapoptosis in Breast Cancer
Mahnaz Razandi,
Ali Pedram and
Ellis R. Levin
Department of Medicine The Long Beach Veterans Hospital
Long Beach, California 90822
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ABSTRACT
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Chemotherapy or irradiation treatment induces
breast cancer cell apoptosis, but this can be limited by estradiol
(E2) through unknown mechanisms. To investigate
this, we subjected estrogen receptor-expressing human breast cancer
cells (MCF-7 and ZR-751) to paclitaxel (taxol) or to UV irradiation.
Marked increases in cell apoptosis were induced, but these were
significantly reversed by incubation with E2.
Taxol or UV stimulated c-Jun N-terminal kinase (JNK) activity, which
was inhibited by E2. Expression of a
dominant-negative Jnk-1 protein strongly prevented taxol- or UV-induced
apoptosis, whereas E2 inhibition of apoptosis
was reversed by expression of constituitively active Jnk-1. As targets
for participation in apoptosis, Bcl-2 and Bcl-xl were phosphorylated in
response to JNK activation by taxol or UV; this was prevented by
E2. Taxol or UV activated caspase activity in a
JNK-dependent fashion and caused the cleavage of procaspase-9 to
caspase-9, each inhibited by E2. Independently,
the steroid also activated extracellular signal-regulated protein
kinase activity, which contributed to the anti-apoptotic
effects. We report novel and rapid mechanisms by which
E2 prevents chemotherapy or radiation-induced
apoptosis of breast cancer, probably mediated through the plasma
membrane estrogen receptor.
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INTRODUCTION
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Estrogen receptors are expressed in approximately 65% of human
breast cancer, implying that this sex steroid plays an important role
in the development and propagation of the disease (1, 2). Approximately
one third of women with breast cancer respond to ablative endocrine
therapy (3, 4), and anti-estrogens positively influence the course of
established breast cancer (5) or prevent the development of primary
disease (6). Based upon in vitro and in vivo
data, estrogen probably acts as both a growth factor and a survival
factor for breast cancer (1, 2).
The ability of estradiol (E2) to act as a
survival factor for breast cancer is not well understood, but a
substantial part of the effects probably occur through the prevention
of programmed cell death, apoptosis (7). Apoptosis is often initiated
when a cell is exposed to a stressful stimulus, which then triggers a
transmembrane signal to an intracellular protease cascade, primarily
composed of the caspase family (8). As a result, intracellular enzymes
are activated that cleave DNA and cause cell shrinkage, chromatin
condensation, and membrane blebbing. In the early course of
establishment of breast cancer, a cytokine response could induce
apoptosis of the cancer cells via the activation of cell surface
receptors for tumor necrosis factor, as an example (9). In established
breast cancer, treatment with chemotherapy or irradiation induces
apoptosis. In vitro, breast cancer treatment with
chemotherapy is markedly less effective in the setting of estrogen (10, 11). Thus, E2 may establish a survival advantage
in this setting, but the mechanisms of this effect are not well
understood.
The actions of E2 are traditionally thought to be
mediated by the nuclear estrogen receptor (ER), through the regulation
of target gene transcription (12). This occurs when ER either binds
estrogen response elements on the promoters of target genes, or acts
through protein-protein interactions involving a variety of
coactivators, corepressors, and the basal transcriptional machinery
protein complex. Emerging evidence, however, has implicated a second
distinct mechanism of E2 action, where this
steroid binds a putative plasma membrane ER and enacts signal
transduction (13, 14). Each mechanism could work cooperatively or
distinctly to effect cell biological actions. Conceivably, the ability
of E2 to prevent apoptosis in several target
cells (15, 16) could be initiated through signal transduction mediated
through the plasma membrane receptor.
As the anti-apoptotic effects of E2 are poorly
understood, we investigated the mechanism of action and whether the
membrane receptor mediates these effects of the sex steroid.
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RESULTS
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Apoptosis is Inhibited by Membrane ER Ligation
MCF-7 and ZR-751 cells were subjected either to a
brief (1-min) UV irradiation, followed by 4-h incubation, or to 20
µM taxol treatment for 4 h. As shown in Fig. 1A
, UV (panel b) induced 13% of the
MCF-7 breast cancer cells to undergo apoptosis, compared with 1% in
the control cells (panel a). Preincubation with 10 nM
E2 or 100 nM
E2-BSA (panels c and d), significantly prevented
the effect of UV, lowering cell death to 6% and 7%, respectively. The
protective effects of either estrogen compound were reversed by
ICI182,780, the specific ER antagonist (panels e and f). Similar
effects were seen for taxol-induced apoptosis (Fig. 1B
), although taxol
was not as potent in this regard as UV exposure. The data are summated
in the bar graphs below each composite figure. These short exposures
were chosen to support the idea that a rapid, nongenomic effect of
E2 might be involved. In contrast, neither 100
nM testosterone nor progesterone had any significant effect
on UV or taxol-induced apoptosis in MCF-7 cells (data not shown).

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Figure 1. A, Apoptosis of MCF-7 Cells Is Induced by UV
Irradiation Exposure for 1 Min (b), as Demonstrated by Terminal
Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling Staining
Compared with Control Cells (a)
This is inhibited by 10 nM E2 (c) or
100 nM E2-BSA (d), which is reversed
by 1 µM ICI182,780, an ER antagonist (e and f,
respectively). B, Taxol (20 µM) exposure for 4 h
induces apoptosis in MCF-7 cells (b), inhibited by
E2 or E2-BSA (c and d),
again reversed by ICI182,780 (e and f). The study shown here was
repeated three times. Bar graphs show the mean ±
SEM number of apoptotic cells in each condition,
based on combined data, which are shown below the
composites (C). Apoptotic cells are stained
yellow/green compared with viable cells (red)
stained with propidium iodide.
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These results indicate that E2 is rapidly acting
through a specific plasma membrane ER to inhibit apoptosis. This is
supported by the similar effects of E2-BSA, a
compound that has been shown by several laboratories to neither enter
the cell nor bind/activate the nuclear ER (17, 18, 19).
E2-BSA has previously been shown to be less
potent than E2, perhaps due to the steric
hindrance of E2 accessing its receptor, when
conjugated to BSA (17, 18, 19). We have previously shown that BSA by itself
does not affect signaling (18).
To further support the idea that E2 and
E2-BSA are acting through a membrane ER, we
transiently transfected the cells with an estrogen response element
(ERE)-luciferase reporter construct, as previously described (19). Over
8 h, E2 (10 nM) significantly
stimulated the reporter activity, but E2-BSA did
not (Fig. 2
). This indicates that
E2-BSA does not enter the cell to bind the
nuclear ER by 8 h, determined at several times later than the
inhibition of apoptosis shown here (4 h). These results also indicate
that E2 does not substantially dissociate from
the BSA, although this has recently been called into question (see
Discussion).

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Figure 2. Activation of an ERE-Luciferase Reporter Over Time
by E2 (10 nM), but Not by E2-BSA
(100 nM)
The results are the mean ± SEM luciferase activity,
determined from triplicate determinations per time point in each
experiment. All data were combined from three separate experiments. *,
P < 0.05, by ANOVA and Scheffes test for time
zero vs. 4 or 6 h.
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We also assessed apoptosis by labeling MCF-7 and another ER-positive
breast cancer cell, ZR-751, with bromodeoxyuridine and propidium
iodide; we then determined cell death by fluorescent-activated cell
sorting (FACS) analysis. As shown in Table 1
, UV and taxol induced at least, 6- and
4-fold respective increases in programmed death in either cell line.
E2 afforded between a 6680% protection against
apoptosis across both conditions and both cell lines. The effects of
E2-BSA were comparable to those of
E2, and all steroid actions were reversed
7090% by ICI182,780. In contrast, E2 had no
effect on UV- or taxol-induced apoptosis in an ER-negative cell line,
HCC1569 (data not shown).
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Table 1. Apoptosis in Breast Cancer Cells after
Exposure to Ultraviolet (UV) Irradiation or Taxol, in the Presence or
Absence of Estrogen
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To examine an early event in apoptosis, the cells were assessed for
cell membrane binding of annexin V, again determined by FACS. In the
cell undergoing programmed cell death, annexin V can bind
phosphatidlyserine, which is expressed in the outer plasma membrane
leaflet of dying cells. In both ER-positive breast cancer cell lines,
UV or taxol induced 16-fold (MCF-7) and 8.5-fold (ZR-75) increases in
annexin binding compared with control cells. E2
or E2-BSA significantly reversed this by 5286%
across all conditions and both cell lines (Table 2
). ICI182,780 substantially reversed the
effects of either E2 or
E2-BSA.
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Table 2. Annexin-V Binding to the Membrane of Breast
Cancer Cells Exposed to Ultraviolet (UV) Irradiation or Taxol, with or
without Estrogen
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Apoptosis Is Modulated through c-Jun N-terminal kinase (JNK)
Activity
We then determined whether the induction of apoptosis by UV or
taxol was dependent on JNK activity and could be modified by
E2. JNK is known to mediate the activation of
apoptosis in several cell types, in response to various stressors (20).
We first found that either UV or taxol could significantly activate JNK
activity by 7- and 2.5-fold, respectively, in MCF-7, and significantly
in ZR-751 cells (Fig. 3A
, upper and lower panels). We also found that
either E2 or E2-BSA caused
a 5067% reduction in the stimulated JNK activity seen in response to
either stressor in both cell lines. The comparable effects of the two
estrogen compounds were blocked by ICI182,780, and when considered
along with the rapidity of inhibition by E2
(determined after 15 min of cell exposure), these results support a
membrane ER as mediating this action. The effects of
E2 or E2-BSA were also
concentration related, with significant inhibition seen at 1 and 10 nm
E2 in both cell lines (Fig. 3B
). In contrast,
E2 did not reduce UV- or taxol stimulated-JNK
activity in HCC-1569 cells (data not shown).

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Figure 3. A, The c-Jun Kinase Activity in MCF-7 Cells
(upper) and ZR-751 Cells (lower) after
15-Min Exposure to UV (left) or Taxol
(right), in the Presence or Absence of E2,
E2-BSA, or ICI182,780
JNK was immunoprecipitated from the treated MCF-7 cells, as described
in Materials and Methods. A
representative experiment of JNK activity directed against
GST-c-Jun-(179) as substrate protein is shown, with the Jnk-1
immunoblots below each condition. The bar
graph represents mean results ± SE of three
experiments combined. *, P < 0.05 for control
vs. UV or taxol; +, P < 0.05 for UV
or taxol vs. UV or taxol plus E2 or
E2-BSA; ++, P < 0.05 for UV or taxol
plus E2 or E2-BSA vs. the former
plus ICI. B, Concentration-related inhibition of UV- or
taxol-stimulated JNK activity by E2 or E2-BSA
in MCF-7 (left) and ZR-751 (right). The
data reflect three separate experiments combined.
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A critical issue is whether JNK activation is necessary and
sufficient for the induction of apoptosis in this setting. As shown in
Fig. 4A
, MCF-7 cells transfected to
express a dominant-negative Jnk-1 showed a significant reduction in the
degree of apoptosis induced by UV [panel c vs. a
(control)] or by taxol [panel d vs. b (control)]. Control
MCF-7 cells were transfected with the empty plasmid vector and
subjected to either of the two apoptotic stimuli. The reversal induced
by dominant-negative Jnk-1 was substantial (6367% for UV or taxol;
Fig. 4B
), which must be considered in the context that transfection
efficiency does not allow complete inhibition of JNK activation. We had
previously determined our transfection efficiency using this construct
as approximately 75% (21). These results show the requirement of JNK
for apoptosis induced by these two agents. Because UV- or taxol-induced
JNK activation and apoptosis were blocked by this steroid, we
believe that this is a novel target for the anti-apoptotic effects of
estrogen.

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Figure 4. A, Apoptosis of pcDNA3-Transfected MCF-7 Cells in
Response to UV (a) or Taxol (b) Is Substantially Greater Than That in
Cells Transfected with Dominant-Negative Jnk-1 (pcDNA3 Flag-Jnk-1 APF),
Then Exposed to UV (c) or Taxol (d)
MCF-7 cells were transfected with dominant-negative (dom-neg) Jnk-1,
recovered, then subjected to UV or taxol. B, Quantitation of three
apoptosis experiments is shown in the bar graph. Control
apoptosis (pcDNA3 transfected, but not subjected to UV or taxol) was
2% and is not shown. *, P < 0.05 for UV or taxol
vs. UV or taxol plus dom-neg Jnk-1. C, Expression of
constituitively active Jnk-1 (Flag-Jnk-1) reverses the E2
or E2-BSA inhibition of UV (left)- or taxol
(right)-induced apoptosis. Data are from three
experiments combined. *, P < 0.05 for UV or taxol
vs. UV or taxol plus E2 or
E2-BSA; +, P <0.05 for UV or taxol plus
E2 or E2-BSA vs. the former in
the presence of active Jnk-1
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To further support this contention, we transfected MCF-7 cells
with a mildly constituitively active Jnk-1 expression plasmid, a vector
that we and others have previously characterized (20, 21). We then
exposed the cells to UV or taxol in the presence or absence of
E2 or E2-BSA. We found that
active Jnk-1 resulted in a nearly complete reversal of the ability of
E2 or E2-BSA to block UV-
or taxol-induced apoptosis (Fig. 4C
).
Bcl-2 and Bcl-xl Serve as Substrates for Jnk
What JNK targets are critical for the apoptosis-inducing action of
this enzyme? It has recently been shown that the anti-apoptotic protein
Bcl-2 can be phosphorylated. In several cell types, phosphorylation by,
for instance, protein kinase A or c-Jun kinase (JNK), down-regulates
Bcl-2 actions to prevent cell death (22, 23, 24), although concomitant
activation of phosphatases or phosphorylation of a different site may
lead to activation of the protein in some contexts (25). However,
recent data clearly indicate that JNK can inactivate Bcl-2 function by
directly phosphorylating the loop domain of this protein (24). We
determined that UV or taxol was capable of significantly stimulating
the phosphorylation of Bcl-2 (Fig. 5A
).
Further, the phosphorylation was mainly attributable to JNK activation,
as transfection of the MCF-7 cells with dominant-negative Jnk-1 almost
completely reversed this effect of UV or taxol.

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Figure 5. A, UV or Taxol Induces the Increased
Phosphorylation of Bcl-2 and Bcl-xl Proteins in a JNK-Dependent Fashion
after 15-Min Incubation
JNK1-APF, Dominant-negative (dom-neg) Jnk-1. A representative study is
shown, repeated three times to constitute the bar graph. *,
P < 0.05 for control vs. UV or
taxol; +, P < 0.05 for UV or taxol
vs. UV or taxol plus dom-neg Jnk-1. B, UV or taxol
induces the increased phosphorylation of Bcl-xl protein in a
JNK-dependent manner. A representative study from three experiments is
shown. C, E2 or E2-BSA inhibits UV- or
taxol-induced phosphorylation of Bcl-2 (left) and Bcl-xl
(right), which is reversed by expressing active Jnk-1
(Flag-JNK-1).
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Another important anti-apoptotic protein in this family is
Bcl-xl. We similarly found that UV and taxol induced JNK-dependent
phosphorylation of this protein in MCF-7 cells (Fig. 5B
). However, in
this situation, another kinase may additionally contribute. As
E2 or E2-BSA inhibits JNK
activation, we hypothesized that they would block UV- or taxol-induced
phosphorylation of Bcl-2 and Bcl-xl. We found this to be the case, in
that either estrogen compound significantly inhibited the
phosphorylation of these two proteins by 5364% (Fig. 5C
). To support
a JNK-related mechanism of action, the blocking of either Bcl-2 or
Bcl-xl phosphorylation by E2 was substantially
reversed when a constituitively active Jnk-1 was expressed in the MCF-7
cells (Fig. 5C
, lanes 8 and 9). This identifies a novel downstream
target for the anti-apoptotic effects of the membrane ER, and the
mechanism is probably mediated through the inhibition of JNK
activation.
Caspase Activation Is JNK Dependent and Is Inhibited by
E2 or E2-BSA
The inactivation of Bcl-2 or Bcl-xl induced by UV- or
taxol-induced phosphorylation might lead to activation of caspase
activity (22, 23). We determined this in whole cell lysates using a
fluorogenic substrate that assesses caspase-4, -5, and -9 activities
(26). UV- or taxol-treated cells showed 91% and 75% increases,
respectively, in caspase activity, compared with nontreated,
control MCF-7 cells (Table 3
). Adding
E2-BSA or E2 reduced the
enhanced caspase activity by 41%53% across both conditions. To
determine a role for JNK, MCF-7 cells were transfected with the empty
plasmid, pcDNA3, or the dominant negative Jnk-1 construct. In MCF-7
cells transfected with pcDNA3, the apoptosis was higher than that in
nontransfected cells. However, UV and taxol still significantly
enhanced caspase activity in this setting, by 68% and 51%,
respectively (Table 3
). Expression of Flag-Jnk-1 APF significantly
reduced this stimulated caspase activity by 65% and 51%,
respectively.
Active caspase-9 facilitates the cleavage and activation of the
death effector caspases, resulting in DNA fragmentation and apoptosis.
Caspase-9 activation mainly results from proteolytic processing of
procaspase-9. This processing is indirectly restrained by Bcl-2 or
Bcl-xl and is directly activated through association with the
cytochrome c-Apaf-1 complex. We therefore determined whether
UV and taxol cleaved procaspase-9 to caspase-9, yielding detectable
active fragments of the zymogen. In control MCF-7 cells, only the
46-kDa zymogen was detected. Both taxol and UV induced cleavage of the
procaspase, yielding smaller molecular mass bands at 34 kDa, which were
detected after stressor exposure (Fig. 6
). E2 or
E2-BSA substantially prevented the cleavage of
procaspase-9 in the setting of either stressor for the MCF-7 cells.

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Figure 6. Procaspase-9 Is Cleaved by UV (1-Min Exposure) or
Taxol (20 µM), Which Is Prevented by E2 or
E2-BSA
MCF-7 cells were exposed to UV or taxol in the presence or absence of
E2 or E2-BSA, as described, then incubated for
a total of 4 h. Cell lysates were then immunoprecipitated for
caspase-9, using a monoclonal antibody that recognizes unprocessed and
processed forms of the caspase-9 zymogen. After SDS-PAGE separation and
membrane transfer, Western blot for caspase-9 was carried out. A
representative study is shown, repeated three times.
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Extracellular Signal-Regulated Protein Kinase (ERK) Activation
Contributes to Antiapoptosis
Might there be a contribution of other signaling molecules to the
protective actions of E2? We determined the
possible role of the ERK member of the mitogen-activated protein kinase
family. Both E2 and E2-BSA
stimulated ERK activity by about 3-fold after 10-min exposure to the
MCF-7 cells, and this was prevented by ICI182,780 (Fig. 7A
). In the setting of UV or taxol,
incubation of MCF-7 cells with PD 98059, a mitogen-activated
extracellular protein kinase kinase (MEK) inhibitor, partially
reversed the anti-apoptotic effects of E2 or
E2-BSA; this reversal ranged from 3348% (Fig. 7B
). These results indicate that ERK activation by
E2 occurs through the plasma membrane ER, and
that this contributes to the anti-apoptotic effects of the sex steroid.

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Figure 7. A, E2 and E2-BSA Stimulate
ERK Activity
MCF-7 cells were incubated with the steroids for 10 min, ERK
activity was immunoprecipitated from cell lysate, and activity was
determined against myelin basic protein (MBP). A representative study
is shown, repeated two additional times. B, Bar graph of
three experiments combined delineating the effects of the MEK
inhibitor, PD 98059, to partially reverse the ability of E2
or E2-BSA to prevent apoptosis. Data are the mean ±
SE from three experiments. *, P < 0.05
for control vs. UV or taxol; +, P < 0.05 is UV or
taxol vs. either treatment plus E2 or
E2-BSA; ++, P < 0.05 is UV or taxol
plus E2 or E2-BSA vs. the former
plus PD 98059.
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DISCUSSION
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E2 is an acknowledged growth and/or survival
factor for several cell types (1, 2, 3, 27, 28). Prenatally,
E2 could limit the programmed cell death
mechanism of remodeling, which is used as part of developmental
plasticity in target organs (29). Postnatally, E2
protection of the ovarian follicle ensures fertility (30), whereas
antiapoptosis in vascular cells probably contributes to the
lowered incidence of arterial disease in estrogenreplaced
postmenopausal women (31). However, the effect of
E2 is cell and context specific. When
advantageous, E2 can also induce apoptosis to
protect against bone resorption by inducing the death of osteoclasts
(32). A very important effect of E2 in this
regard is the unfortunate ability of this sex steroid to prevent breast
cancer cells from undergoing apoptosis in response to chemotherapy or
radiation.
Here, we have shown that E2 prevents UV- or
taxol-induced apoptosis of ER-positive breast cancer cell lines and
have uncovered some novel mechanisms of action.
E2 partially, but significantly, prevents UV- or
taxol-induced JNK activation and separately stimulates the activation
of ERK. Each mechanism contributes to the anti-apoptotic effects of
this steroid (Fig. 8
).
E2 acts through a putative plasma membrane ER
(13, 14), a receptor that has not yet been physically isolated, but for
which strong functional evidence has now emerged (19, 33, 34, 35). We have
recently shown that a single cDNA for ER
(or ERß) can result in
the expression of both nuclear and plasma membrane binding proteins.
The membrane receptor is a G protein-linked receptor capable of
signaling through multiple pathways after G protein activation (19). In
the studies reported here, we show that E2 or
E2-BSA inhibits the UV- or taxol-induced rapid
activation of JNK. These effects are reversed by an ER antagonist.
Several laboratories have previously shown that
E2-BSA does not activate the nuclear ER (17, 19),
and that this compound can be used as a membrane ER-specific
ligand. However, this has recently been called into question. Stevis
et al. proposed that commercially prepared
E2-BSA substantially deconjugates to free
E2 and BSA, and that the conjugated
E2-BSA, but not free E2,
has nonspecific effects to activate signal transduction, at least in
neural cancer cells (36). They also did not find nuclear ER activation
by intact E2-BSA. In the studies presented here,
E2 and E2-BSA act similarly
to inhibit signaling to apoptosis, and their effects are always
significantly reversed by an ER antagonist. Additionally,
E2, but not E2-BSA,
activated an ERE-luciferase reporter gene; this indicates that
E2-BSA did not bind the nuclear ER and did not
dissociate substantially into free E2 and BSA.
Furthermore, the focus should be not whether
E2-BSA is a useful reagent to activate the
membrane ER, but, rather, what are the nongenomic, rapid actions of
E2? Importantly, the signal effects of
E2 rapidly occur (within 515 min) and are
unlikely to represent an unprecedented action of a nuclear
receptor.
The nongenomic actions of E2 probably result from
ER
ligation by the steroid. The latter conclusion derives from the
fact that MCF-7 cells express almost exclusively the ER
receptor
(37). Furthermore, we have previously shown in CHO cells transfected to
singly express either ER that E2 ligation of
ER
inhibits basal JNK activity (and stimulates ERK activation),
whereas in CHO-ERß cells, E2 activates JNK
(22). It has been established here and previously that JNK is essential
to apoptosis induced by UV or taxol in several cell types (24, 38, 39).
We have further defined downstream targets for
E2-related antiapoptosis in breast cancer. UV and
taxol were found to stimulate the phosphorylation, and hence the
inactivation, of Bcl-2. These agents also stimulated phosphorylation of
the Bcl-xl protein in MCF-7 cells. This occurs mainly through a
JNK-mediated mechanism, which is also inhibited through the membrane
ER. Recently, taxol-induced JNK activation has been shown to directly
phosphorylate/inactivate Bcl-2 (24). Active Bcl-2 (and Bcl-xl) is
proposed to prevent cytochrome c release from mitochondria
(40), and therefore inhibit the complexing of Apaf-1, cytochrome
c, and procaspase-9. In the presence of ATP, cytochrome
c induces the oligomerization of Apaf-1, which then cleaves
the procaspase-9 zymogen, yielding active caspase-9. We report here
that taxol or UV induces the activation of caspase activity in a
JNK-dependent fashion and also induces the cleavage of the procaspase-9
zymogen to caspase-9. These events are significantly prevented by
E2 or E2-BSA.
Caspase-9 cleaves/activates the death effector caspases, such as
procaspase-3 or -7, thereby effecting apoptosis (41, 42). Caspase-3 is
not functional in MCF-7 cells (43), but other effector caspases, such
as caspase-7, are present (confirmed by us). In addition to its actions
in preventing the release of cytochrome c, Bcl-xl has been
shown to form a ternary complex with Apaf-1 and caspase-9, perhaps
inhibiting the ability of Apaf-1 to activate caspase-9 (44). Active
Bcl-2 has many important functions in preventing cell death (40, 45),
and each, theoretically, could be affected by and contribute to the
effects of E2 shown here. It has previously been
demonstrated that E2 can stimulate the
transcription and protein synthesis of Bcl-2 (11, 46). Thus,
E2 can prevent cell death by acute and more
chronic modulation of both the activity and levels of this
anti-apoptotic protein. This may be an example of coordinated cellular
actions between the membrane and nuclear ERs, respectively.
The novel finding that UV and taxol induce Bcl-xl phosphorylation
through a JNK-dependent mechanism deserves comment. This important
anti-apo- ptotic protein can form homo- or heterodimers with other
family members, including Bcl-2, Bad, Bax, etc., and it is
believed that these interactions underlie the effects of Bcl-xl.
However, it is not clear what the effect of phosphorylation is on
Bcl-xl action. Analogous to Bcl-2, Bcl-xl contains a 60-amino acid loop
domain that partially suppresses the anti-apoptotic functions of this
protein (47). The identical domain in Bcl-2 can be phosphorylated by
JNK, and this phosphorylation disables Bcl-2 cell survival function
(24, 48). We would predict that this sequence is a target for Bcl-xl
phosphorylation via JNK, causing inactivation of anti-apoptotic
function. Very recently, Kufe and colleagues demonstrated that ionizing
radiation causes the translocation of JNK to the mitochondria of
leukemia cells, where JNK phosphorylates Bcl-xl (49). Expression of a
phosphorylation mutant Bcl-xl in these cells prevents ionizing
radiation-induced apoptosis, perhaps because the mutant protein cannot
be inactivated by phosphorylation. Collectively, the data suggest that
the ability of E2 to inhibit the JNK-induced,
inactivating phosphorylation of Bcl-2 provides a partial understanding
of the anti-apoptotic effects of this sex steroid and probably extends
to Bcl-xl function as well.
It is likely that E2 stimulates other signal
transduction mechanisms to inhibit apoptosis. In MCF-7,
E2 stimulates the cascade that activates the ERK
member of the mitogen-activated protein kinase family, and importantly
contributes to DNA synthesis in this cell (50, 51). This kinase is
recognized to mediate cell proliferation in response to a variety of
growth factors targeting a myriad of cell types (52, 53) and has also
been proposed to act as a survival protein (54, 55). We show here that
E2 or E2-BSA activates ERK,
and that inhibition of activated ERK with the soluble and specific
MEK-1 inhibitor, PD 98059, partially reverses
E2-induced antiapoptosis in MCF-7 cells. This
indicates that ERK contributes to E2 effects in
this regard. Singer et al. showed that
excitotoxicity-induced necrosis of neurons can be prevented by
E2 or nerve growth factor, mediated through ERK
signaling to unknown downstream targets (56). Several mechanisms of
ERK-induced protection against antiapoptosis have recently been
elucidated. These include the phosphorylation of the pro-apoptotic BAD
protein at serine 112 (57) and the activation of cAMP response
element-binding protein-mediated transcription (58). Thus, it is
likely that several pathways that originate from the plasma membrane ER
lead to the preservation of target cells, and that it is the overall
balance of pro- and anti-apoptotic signals that determines the fate of
a cell.
Exactly where JNK (or ERK) fits in the apoptotic pathway is not clear,
but we and others (48) have shown that JNK is upstream of Bcl-2. The
complete effector pathways that prevent apoptosis will need to be
defined in a situation-specific context (59). Furthermore, taxol is a
microtubule-stabilizing agent, which theoretically acts through several
mechanisms to induce apoptosis (60, 61). These conceivably could also
be influenced by E2 in a JNK-independent
fashion.
Based upon these studies, we speculate that the actions of the membrane
ER in breast cancer could underlie the ability of
E2 to effect cell survival in vivo
(62). Understanding the mechanisms by which E2
induces antiapoptosis in cancer provides theoretical targets to prevent
this undesirable action. Membrane ER-specific antagonists that are
targeted to cancer cells might be a future means to enhance the
response to therapy. This might also allow women to take hormone
replacement to preserve desirable genomic effects of the nuclear ER. To
support this strategy, further understanding of the discrete actions of
E2 at both the cell membrane and the nucleus must
occur.
 |
MATERIALS AND METHODS
|
---|
Cells and Apoptosis Determination
MCF-7 cells were grown on 18-mm coverslips in 12-well culture
dishes in DMEM/F-12 medium without phenol red, but with added
charcoal-stripped serum. For apoptosis studies, the cells were
subjected to 1 min of UV irradiation (20 J/cm2)
or paclitaxel (taxol; 20 µM) and incubated for 4 h
at 37 C in the presence or absence of 10 nM
E2 or 100 nM
E2-BSA. At the end of incubation, the cells were
washed with PBS and fixed with 1% freshly prepared paraformaldehyde in
PBS, pH 7.4, at 4 C overnight. Apoptosis was then determined by the
terminal deoxynucleotidyl transferase-stimulated incorporation of
nucleotides into the 3-OH end of damaged DNA in the cell, detected by
fluorescent antibodies to the nucleotides (terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling), using a kit from
Intergen (Purchase, NY). From each experimental condition,
400 cells were visually scored for apoptosis, viewed by fluorescence
microscopy using standard fluorescein excitation and emission filters.
In addition, FACS-based cell counting for apoptosis was carried out
after bromodeoxyuridine labeling, for MCF-7 and another ER-positive
cell line, ZR-751. Apoptosis in both of these cell lines was also
determined by FACS detection of annexin V binding using a kit
(Becton Dickinson and Co., Mountain View, CA). In early
apoptosis, the plasma membrane phospholipid, phosphatidylserine,
translocates from the inner to the outer membrane leaflet. In cells
undergoing apoptosis, phosphatidylserine is then available to bind
phospholipid-binding proteins, such as annexin V. HCC1569 cells served
as a control, ER-negative breast cancer cell line for these
experiments.
Transient Transfections
Breast cancer cell lines were grown to 6070% confluence in
DMEM/F-12 (MCF-7) medium or RPMI-1640 (ZR-751 and HCC1569), without
phenol red but with 10% FBS. The cells were then washed and
transiently transfected with 510 µg of fusion plasmids after
optimization depending on the plate size and the amount of cells. The
plasmids included c-Jun wild type or dominant-negative mutant (see
below), ERE-simian virus 40 luciferase (provided by Dr. B. Gehm), or
respective backbone vectors. DNA was amplified and isolated using the
QIAGEN maxi-prep kit (Chatsworth, CA), and care was taken
to minimize carryover of salts, alcohol, or other confounding reagents.
Transfections were performed with Lipofectamine reagent (Life Technologies, Inc., Grand Island, NY); cells were incubated with
liposome-DNA complexes at 37 C for approximately 5 h, followed by
overnight recovery in DMEM-F-12 medium containing 10% FBS. Then,
before experimental treatment, cells were synchronized in serum-free
DMEM-F-12 for 24 h and treated with 17ß-E2
and/or related compounds. Cotransfections with a green fluorescent
protein expression vector (Promega Corp., Madison, WI)
indicated approximately 7075% efficiency of transfection. Luciferase
activity was determined as previously described (19, 63). The
concentration of E2-BSA was calculated from the
number of E2 molecules attached to each BSA
molecule.
Kinase Assays
The c-Jun kinase activity was determined as previously described
(21). Briefly, MCF-7 cells were incubated under various conditions for
15 min, then the cells were lysed, and lysate was immunoprecipitated
for Jnk-1. JNK activity was determined against GST-c-Jun-(179)
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as
substrate. Laser densitometry of the phosphorylated bands was used for
quantitation, and three experiments were combined for statistical
analysis. Immunoblot of Jnk-1 protein assured equal amounts of protein
loaded in each condition. In some experiments, wild-type
(pcDNA3Flag-Jnk-1) or dominant-negative Jnk-1 (pcDNA3 Flag-Jnk-1 APF)
(58) was transfected into MCF-7 cells as previously described (21, 58).
ERK activity was determined as previously described (18). MCF-7 cells
were synchronized in serum- and growth factor-free medium for 24
h, then incubated with E2 or
E2-BSA for 10 min, and immunoprecipitated ERK
activity from cell lysates was determined against myelin basic protein
as the substrate. For apoptosis experiments, cells were subjected to UV
or taxol treatment with or without E2 or
E2-BSA in the absence or presence of the specific
ERK kinase (MEK-1) inhibitor, PD 98059 (10 µM).
Bcl-2 and Bcl-xl Phosphorylation
MCF-7 cells were incubated for 1 h at 37 C in
phosphate-free medium containing 5% dialyzed FBS. At the end of the
incubation, cells were washed and labeled with
32P (final concentration, 0.2 mCi/ml) for 2
h at 37 C in a CO2 incubator. Cells were further
incubated with or without taxol, UV, and E2 or
E2-BSA for 1 h. At the end of the labeling
period, cells were washed with ice-cold PBS, then lysed with buffer for
30 min on ice. The lysates were microcentrifuged, and Bcl-2 and Bcl-xl
immunoprecipitation from the supernatant was conducted using specific
antibodies (Santa Cruz Biotechnology, Inc.) at 4 C. After
centrifugation and washing, the immunoprecipitated Bcl-2 and Bcl-xl
were resolved by SDS-PAGE on a 12% gel, followed by
autoradiography.
Caspase Activity and Zymogen Proteolysis
Cultured MCF-7 cells in 100-mm dishes were exposed to UV or
taxol as described for the previous experiments. The cytosolic
extracts were repeatedly frozen in extraction buffer and thawed as
previously described (26). Cell lysates were then diluted and incubated
with 1 µM fluorescent substrate (caspase-4, -5, and -9
substrate) for 30 min at 30 C. At the end of the incubation, the
fluorescence of the cleaved substrates was determined using a
spectrofluorometer, set at an excitation wavelength of 400 nm and an
emission wavelength of 505 nm.
Cleavage of the zymogen, procaspase-9, was assessed. MCF-7 cells
were cultured as described, then treated with UV (1 min) or taxol (20
µM) for 4 h in the absence or presence of
E2 or E2-BSA. At the end of
the 4-h incubation, the cells were washed, then lysed in buffer for 30
min on ice. The lysates were microfuged, and the supernatants were
immunoprecipitated with anti-caspase-9 polyclonal antibody, raised
against a peptide corresponding to the unique amino acids 299318 of
human caspase-9 (Cayman Chemical, Ann Arbor, MI). The
immunoprecipitants were then analyzed by Western blotting using the ECL
kit from Amersham Pharmacia Biotech (Arlington Heights,
IL).
Statistical Analysis
Pooled data from multiple experiments were compared by ANOVA and
Scheffes test, using the StatView statistical program
(P < 0.05 as significant). Bar graphs represent the
mean ± SEM from at least three
experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank Roger Davis for the JNK plasmids.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ellis R Levin M.D., Long Beach Veterans Hospital, Medical Service (111-I), 5901 E 7th St., Long Beach, California 90822. E-mail: ellis.levin{at}med.va.gov
This work was supported by grants from the Research Service of the
Department of Veterans Affairs, and the NIH (HL-59890; to
E.R.L.).
Received for publication February 2, 2000.
Revision received May 2, 2000. Revision received June 7, 2000.
Accepted for publication June 12, 2000.
 |
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