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J. Biol. Chem., Vol. 277, Issue 47, 45695-45703, November 22, 2002
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From the Department of Biochemistry, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61801
Received for publication, August 8, 2002, and in revised form, September 18, 2002
The therapeutic efficacy of tamoxifen (TAM) in
cancer therapy is thought to arise primarily from its ability to
compete with estrogens for binding to the estrogen receptor (ER). We
show that TAM and its active metabolite, 4-hydroxytamoxifen (OHT), can
actively induce programmed cell death through distinct
ER-dependent and ER-independent pathways. The
ER-independent pathway is activated by 10-20 µM
TAM and OHT and by 10-20 µM 17 Estrogens, acting through estrogen receptors
(ERs),1 regulate the growth
and differentiation of cells of the reproductive system. Binding of
17 Resolution of the role of ER in TAM- and OHT-induced apoptosis is
complicated by the fact that available ER-positive and ER-negative breast cancer cell lines are derived from independent tumors. These
cell lines therefore differ in many respects other than ER content. To
simplify analysis of the role of ER in TAM- and OHT-induced apoptosis,
we therefore used ER-negative HeLa, human cervical carcinoma cells, and
HeLa cells stably transfected to express hER Many compounds that are known to induce programmed cell death (PCD)
work via pathways that involve mitochondria. The presence of an
apoptotic stimulus triggers a rapid increase in mitochondrial permeability, leading to mitochondrial dysfunction. One of the causes
of the mitochondrial permeability transition is the translocation of
the proapoptotic Bax protein from the cytosol to the mitochondria, where it forms selective channels in the outer mitochondrial membrane and facilitates the release into the cytosol of cytochrome c
(21, 22). In the classic apoptotic pathway, this cytosolic cytochrome c forms a complex with procaspase 9 and Apaf-1 called the
apoptosome, which leads to the ATP-dependent cleavage and
activation of pro-caspase 9, the initiator caspase in mitochondrial
apoptosis. Activation of pro-caspase 9 results in activation of
downstream executioner caspases, such as caspase 3 (23-25).
We find that OHT is able to induce two independent pathways of PCD. An
ER-independent pathway kills ER-negative HeLa cells, requires 10-20
µM TAM or OHT, and is not TAM-specific as it is also
triggered by the SERM raloxifene (RAL) and by E2. In
contrast, submicromolar amounts of TAM and OHT trigger cell death only
in ER-positive HeLa cells. This effect is blocked by pre-treatment with
E2, RAL, and ICI 182,780, demonstrating that binding of TAM and OHT to the ER is required for this pathway of programmed cell death. The ER-dependent and ER-independent pathways both
trigger a mitochondrial permeability transition and share other
features of mitochondrial apoptosis, such as translocation of the
proapoptotic Bax protein from the cytosol into the mitochondria and the
release of cytochrome c into the cytosol. However, in
contrast to ER-independent PCD, which displays typical apoptotic
markers such as PARP cleavage, chromatin condensation, and DNA
laddering, a different cell morphology, as well as the absence of those
markers, indicates that the ER-dependent pathway does not
involve caspase activation. The ER-dependent pathway does
not result in the cleavage and activation of pro-caspase 9, the
initiator caspase in mitochondrial apoptosis. The OHT·ER-mediated PCD
pathway resembles the caspase-independent pathway referred to as
necrosis-like PCD. This study describes a novel and highly specific
pathway for TAM·ER- and OHT·ER-induced programmed cell death and
suggests an additional mechanism to account for the therapeutic
efficacy of TAM.
Materials--
Polyclonal antibodies to PARP and cleaved caspase
9 were obtained from Cell Signaling Technologies (Beverly, MA). COX4
and cytochrome c antibodies were obtained from
Clontech Laboratories (Palo Alto, CA), and actin
antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).
DiOC6(3) and propidium iodide were obtained from Molecular
Probes (Eugene, OR) and R & D Systems (Minneapolis, MN), respectively.
The p38 inhibitor SB203580 was obtained from Calbiochem.
Cell Culture--
All cell lines were grown in Dulbecco's
modified Eagle's medium supplemented with 10%
charcoal-dextran-treated fetal bovine serum. HeLaER cells were
maintained in medium supplemented with 200 µg/ml G418
(Invitrogen). HeLaER cells were plated in medium without G418
the day before experiments and maintained in G418-free medium
throughout the 2-4-day time period of the experiments.
Mitochondrial Membrane Potential (
Cells were seeded at 6 × 105 cells per 100-mm plate
and incubated for 24 h. After treatment with the indicated
ligands, the cells were washed once with PBS and harvested using
PBS-EDTA. The cells were pelleted by centrifugation at 600 rpm for 5 min and resuspended in PBS. 40 nM DiOC6(3) was
added to the resuspended pellet, and the samples were incubated at
37 °C for 15 min, after which propidium iodide (PI) was added to 5 µg/ml. Relative fluorescence intensities were measured using a
Coulter XL benchtop flow cytometer with excitation at 488 nm and
emission at 520 nm.
Whole Cell Extract Preparation and Western Blotting--
Cells
were seeded in six-well plates at a density of 1.5 × 105 cells/well. After treatments, the cells were washed
once with PBS, and harvested with lysis buffer containing 0.02 M Tris, 1% Triton X-100, 0.14 M NaCl, 2 mM EDTA, 10 µg/ml of the protease inhibitors leupeptin,
pepstatin, and aprotinin, 1 mM phenylmethylsulfonyl fluoride, and dithiothreitol. Protein content was measured using Coomassie Blue reagent (Bio-Rad, Richmond, CA). Extracts were run on
SDS-polyacrylamide gels, and the proteins were electroblotted into a
nitrocellulose membrane. Membranes were blocked with 5% non-fat milk
for 1 h at room temperature, probed with primary antibody
overnight at 4 °C, washed three times with TBS-Tween, and incubated
with horseradish peroxidase-conjugated secondary antibody at room
temperature for 1 h. Signals were detected using the
SuperSignal® West Pico chemiluminescent substrate kit (Pierce).
Transient Transfections--
Transient transfections of HeLaER6
cells were performed using LipofectAMINETM 2000 reagent
(Invitrogen), using the manufacturer's protocol. 2 × 105 cells per well were seeded in 12-well plates. After
24 h the cells were transfected with the indicated amounts of
ATL4 reporter (26), pRLSV40, and PTZ18U. The indicated
concentrations of hormone were added 4 h after transfection. After
24 h the cells were harvested, and dual luciferase assays
(Promega) were performed using the manufacturer's protocol.
Electron Microscopy--
After treatment, cells were harvested
by scraping and collected into a pellet by centrifugation at 600 rpm
for 5 min. At the time of embedding, cells were pipetted out of the
epoxy infiltration mixture and put in small BEEM capsules, which were
filled up the rest of the way with fresh epoxy, spun to collect cells
at the bottom, and placed in the oven overnight at 90 °C to harden.
Blocks were sectioned at 60-90 nm with a Diatome diamond knife using a
Richart Ultracut E ultramicrotome. The sections were picked up with
200-mesh copper and stained with saturated uranyl acetate, silver, and
lead citrate. Sections were viewed on an H600 Hitachi transmission
electron microscope (Tokyo, Japan). Images were shot with Eastman Kodak
Co. electron microscopy film.
Cell Fractionation--
Mitochondrial extraction was performed
using the ApoAlert cell fractionation kit
(Clontech). HeLa or HeLaER6 cells were seeded at
1 × 107 cells per sample, and various ligands were
added after 24 h. After treatment, cell pellets were collected by
centrifugation at 600 × g for 5 min at 4 °C, washed
once, and resuspended in ice-cold fractionation buffer containing
protease inhibitor mixture (Clontech) and
dithiothreitol. After incubation on ice for 10 min, cells were
homogenized using 2 ml of Kontes Dounce tissue grinders (Fisher).
Homogenates were centrifuged at 700 × g for 10 min at
4 °C. Supernatants were centrifuged at 10,000 × g
for 25 min at 4 °C. Supernatants were collected and designated as cytosolic fractions, and pellets were resuspended in fractionation buffer and designated as mitochondrial fractions. Protein
concentrations were determined using the Coomassie Blue assay.
DNA Fragmentation Analysis--
5 × 105 cells
were plated in 100-mm plates. After appropriate treatments, the cells
were harvested by centrifugation at 2000 rpm and 4 °C. Cell pellets
were resuspended in lysis buffer containing 20 mM EDTA, 100 mM Tris, pH 8.0, and 0.8% (w/v) sodium lauryl sarcosine.
Lysates were incubated with 10 µl of 1 mg/ml RNase A/RNase T1 mixture
at 37 °C for 2 h and 10 µl of 20 mg/ml proteinase K for
16 h. Samples were run in a 1% agarose gel.
To facilitate comparisons between cells that lack ER and cells
that contain ER, we used ER-negative, wild-type HeLa cells and our
HeLaER6 cells stably transfected to express hER Preliminary studies based on cell morphology suggested that 10-20
µM TAM and OHT were toxic to HeLa and HeLaER6 cells and that submicromolar concentrations of TAM and OHT were not visibly toxic
to HeLa cells. To further compare the toxicities of TAM and OHT in
HeLaER6 cells and in the parental HeLa cells, we performed dose-response experiments. Because the pathway(s) of programmed cell
death induced by TAM and OHT were unknown, we elected to use a
quantitative assay for cell death based on the ability of mitochondria
to sequester the strong cationic dye DiOC6(3). Damaged mitochondria of apoptotic or necrotic cells exhibit decreased retention
of DiOC6(3) and are visualized as a distinct subpopulation of cells in flow cytometry.
TAM and OHT Induce Programmed Cell Death in HeLaER6 Cells--
To
determine whether PCD induced by TAM and OHT was
ER-dependent, we treated ER-negative HeLa cells and HeLaER6
cells with a range of TAM or OHT concentrations (0.1 nM-1
µM). The percentage of wild-type HeLa cells that were in
early stage programmed cell death (low retention of
DiOC6(3) but still impermeable to the DNA-intercalating
propidium iodide dye) was unaffected by concentrations of TAM or OHT
from 0.1 nM to 1 µM (Fig.
1). The HeLaER6 cells exhibited an
increase in the percentage of cells with decreased retention of
DiOC6(3) from 0.1 to 1 µM TAM. Lower
concentrations of OHT were needed to produce mitochondrial dysfunction.
The percentage of cells with low retention of DiOC6(3)
reached a plateau at 10 nM OHT with an IC50 of
~1 nM OHT (Fig. 1). Much lower concentrations of OHT than
TAM were also required to induce cell death in a second cell line,
HeLaER5 cells (data not shown). Across a range of concentrations, ~100-fold lower amounts of OHT than TAM were required to elicit a
given percentage of cell death (Fig. 1, compare 10 The Estrogen Receptor Is Involved in OHT- and TAM-induced Cell
Death--
Although these data were consistent with a role for ER in
apoptosis induced by low concentrations of TAM and OHT, it was
important to test this more directly. Because nM
concentrations of OHT induce death of HeLaER6 cells, it was possible to
carry out competition experiments using a large molar excess of other
ER ligands over OHT and to evaluate the effect of these ligands on
OHT-mediated programmed cell death. We used three structurally
dissimilar compounds that are representative of the major classes of ER
ligands. RAL is a well studied SERM that exhibits agonist activity in
bone and antagonist activity in many other tissues (5, 6, 28). ICI
182,780 is a nearly pure antagonist (29, 30), and E2 is the
prototypical estrogen. In preliminary studies we examined the effects
of E2, RAL, and ICI 182,780 on the HeLaER6 cells. 10 nM OHT effectively induces early stage cell death in the
HeLaER6 cells. In contrast, E2, RAL, and ICI did not induce
cell death at concentrations from 10 nM to 1 µM (data not shown).
To saturate the ER with the ligands, the cells were maintained briefly
in medium containing a 100-fold molar excess (1 µM) of
ICI 182,780, RAL, or E2. OHT was then added to 10 nM, and the cells were maintained in medium containing the
various ER ligands for 3 days and analyzed by flow cytometry. A
discrete shoulder corresponding to a subpopulation of cells undergoing
early stage programmed cell death was seen with the cells maintained in
medium containing OHT alone (Fig.
2A, OHT). The
presence of a 100-fold molar excess of ICI 182,780, RAL, or
E2 blocked the appearance of this subpopulation of cells
(Fig. 2, A-C). The quantitative flow cytometry
results were reflected in morphological changes in the cells. After 4 days in OHT-containing medium, the HeLaER6 cells were
sparse, and their morphology was very different from that observed in
the control, vehicle-treated cells. Control cells and cells maintained
in OHT plus ICI 182,780 or OHT plus RAL were dividing actively and
exhibited similar morphology (Fig. 2B).
1 µM TAM is required to induce maximal programmed cell
death in HeLaER6 cells. Because two of the other ER ligands are toxic to the cells at extremely high concentrations (see Fig. 6), it was not
possible to carry out traditional competition experiments in which
these ER ligands were present at a 100-fold molar excess over TAM.
However, because TAM binds to ER with a much lower affinity than
E2, RAL, and ICI, we elected to attempt competition
experiments in the presence of equimolar concentrations of the
competitors and TAM. Although they were not present in excess over TAM,
1 µM ICI 182,780, 1 µM RAL, or 1 µM E2 were able to almost completely protect
the cells from TAM-induced programmed cell death (Fig. 2D).
These data provide compelling evidence for a role for ER- in
TAM-induced apoptosis of HeLaER6 cells. The most straightforward explanation for the ability of equimolar concentrations of
E2, ICI 182,780, and RAL to block TAM-induced apoptosis is
that E2, ICI 182,780, and RAL are known to bind to hER An Inhibitor of the p38 Pathway Protects against OHT-induced
Apoptosis--
The cell death response is sometimes triggered by the
activation of stress-activated mitogen-activated protein kinase-based signaling pathways. It was of interest to determine whether the p38
kinase, and perhaps the JNK kinase, were involved in OHT-induced programmed cell death. Low, 0.5-2 µM, concentrations of
SB203580 specifically inhibit the p38 pathway (32). At higher
concentrations (5-10 µM) SB203580 also inhibits some JNK
subtypes (33, 34). Pre-treatment of the HeLaER6 cells with 1 µM SB203580 reduced the percentage of cells in early
stage programmed cell death by almost 40%. When the cells were
pre-treated with 5 µM SB203580, this subpopulation was
reduced by almost 90% (Fig. 3.3). These data suggest that activation of the p38 and perhaps JNK kinases plays a
major role in OHT-induced programmed cell death.
In HeLaER6 Cells OHT Does Not Induce Transcription from the
Consensus Estrogen Response Element--
A potential mechanism for
ER-dependent OHT cytotoxicity is binding of OHT·ER to
estrogen response elements in the DNA inducing expression of
pro-apoptotic genes. To determine whether OHT exhibits agonist activity
in HeLaER6 cells, we performed transient transfections using a reporter
gene containing four copies of the consensus estrogen response element
(ERE). E2 elicited a robust activation of the reporter,
whereas 10 nM OHT, ICI 182,780, or RAL did not activate the
reporter significantly (Fig. 4). Although
OHT is not a classical strong agonist in these cells, these data do not exclude the possibility that OHT·ER either activates unusual EREs in
specific genes or is tethered to genes containing AP-1 or SP-1 sites
(35).
The ER-dependent Programmed Cell Death Pathway Is
Distinct from Classical Caspase-dependent
Apoptosis--
When visualized by light microscopy, cells maintained
in medium containing 10 nM OHT did not display typical
apoptotic morphology and became elongated instead of rounded (Fig.
2B). In contrast, cells treated with 10-20 µM
OHT displayed typical apoptotic morphology, including cell shrinkage
and membrane blebbing, and detached from the surface of the plate (data
not shown). These observations led us to consider the possibility that
treatment with 10 nM OHT induces non-classical programmed
cell death. To examine cell morphology in more detail, we performed
electron microscopy. Prior to analysis the cells were treated with 10 nM OHT for 4 days or with 20 µM OHT for 2 days. Control apoptotic cells were obtained by treating the cells with
etoposide, a topoisomerase inhibitor. Cells treated with etoposide or
20 µM OHT exhibited classical markers of apoptosis, most
notably cell shrinkage and chromatin condensation (Fig.
5, A, B, and
D). In contrast, cells treated with 10 nM OHT
were significantly more elongated compared with untreated,
etoposide-treated, and 20 µM OHT-treated cells (Fig.
5C). There was a significant reduction in the number of
cellular organelles present compared with untreated cells, but
chromatin condensation was absent, as was membrane blebbing.
One of the defining features of caspase-dependent apoptotic
cell death is chromatin fragmentation (36, 37) and DNA laddering. To
further clarify whether the OHT·ER death program is apoptotic and
caspase-dependent, we assayed for the appearance of a DNA ladder by agarose gel electrophoresis. A DNA ladder was observed with
DNA extracted from 10 µM OHT-treated cells but not with
DNA from cells treated with 10 nM OHT (Fig. 5E).
Another widely used marker for apoptosis is
caspase-dependent cleavage of PARP into 89- and 22-kDa
fragments. PARP cleavage was not detected in extracts from HeLaER6
cells maintained in medium containing 10 nM OHT (Fig. 5F). However, cells maintained in 20 µM OHT
exhibit PARP cleavage. These results indicate that 10-20
µM OHT induces a caspase-dependent death
pathway whereas 10 nM OHT induces a caspase-independent death pathway that shares some features with classic apoptosis, such as
mitochondrial dysfunction.
The ER-independent Cell Death Pathway Involves Caspase Activation
and Is Not Specific for TAM and OHT--
10 µM OHT and
TAM begin to initiate death of HeLa cells, whereas 20 µM
OHT or Tam produces robust activation of the cell death pathway (Fig.
6A). To determine whether high
concentrations of other ER ligands induce apoptosis, we tested the
ability of 10-20 µM E2 and RAL to induce
apoptosis. Although 1 µM E2 and RAL protect against apoptosis induced by low concentrations of OHT and TAM (Fig.
2), 2 days in 10-20 µM E2 induces apoptosis
in ER-negative HeLa cells, about as well as similar concentrations of
OHT and TAM (Fig. 6A). 20 µM RAL triggered
apoptosis even more rapidly, with significant cell death observed less
than a day after adding it to the culture medium (data not shown).
These data indicate that the ER-independent death pathway is not
specific for TAM and OHT. To analyze whether these diverse ER ligands
activate classical caspase-dependent apoptosis, we examined
the ability of 20 µM OHT, TAM, RAL, E2, and
ICI 182,780 to induce PARP cleavage and cleavage of the precursor of
the initiator caspase, caspase-9. In both HeLa cells and HeLaER6 cells
20 µM TAM, OHT, RAL, and E2 induced PARP
cleavage and formation of activated caspase 9 (Fig. 6, B and
C). ICI 182,780 did not induce significant PARP cleavage or
caspase 9 activation (Fig. 6, B and C) and does
not kill HeLa cells (data not shown).
10 nM OHT Induces a Cell Death Pathway That Is
Dependent on Mitochondria--
Depolarization of the mitochondrial
membrane is widely recognized as one of the early markers of programmed
cell death. Mitochondrial membrane depolarization therefore formed the
basis of our initial assay for cell death. Because 10 nM
OHT causes loss of mitochondrial membrane potential
( Two Independent Pathways for TAM- and OHT-mediated Programmed Cell
Death--
To help resolve the often-conflicting data on the role of
ER, TAM, and OHT in the induction of programmed cell death, we
developed a simple and experimentally tractable model for TAM- and
OHT-induced apoptosis. We compared effects in wild-type, ER-negative,
HeLa cells, and in HeLaER6 cells, which are stably transfected to
express ER. Although we describe only our studies in HeLaER6 cells, our findings with a different, independent clone of HeLa cells stably transfected to express ER, HeLaER5 cells, are basically similar (data
not shown).
High concentrations of TAM and/or OHT have been reported to activate
caspases and trigger apoptotic cell death (7, 9, 11, 12). OHT and TAM
are not the only ER ligands that induce apoptosis at high
concentrations. For example, our results show that 10-20
µM RAL or E2, but not ICI 182,780, also
induce apoptosis of HeLa cells. Because apoptosis of HeLa cells
requires extremely high pharmacologic concentrations of TAM, OHT, RAL,
and E2 and is not very structure-specific, we elected to
focus primarily on programmed cell death induced by much lower
concentrations of OHT and TAM. Most of our studies were carried out at
10 nM OHT, a concentration 500-1000-fold lower than the
OHT concentration that kills ER-negative, wild-type HeLa cells.
An ER-dependent Pathway for TAM- and OHT-induced
Programmed Cell Death--
10 nM OHT and 1 µM TAM do not induce PCD in ER-negative HeLa cells and
kill ER-positive HeLaER6 cells. The dose-response curve for PCD shows
that the ability of TAM and OHT to induce PCD in HeLaER6 cells is
roughly proportional to their affinity for hER Potential Pathways of TAM·ER- and OHT·ER-induced Programmed
Cell Death--
Activation of the p38 and JNK kinases by various
cellular stresses such as reactive oxygen species and agents that
damage DNA is often associated with proapoptotic events such as
phosphorylation of p53 and depolarization of mitochondrial membranes
(39-43). We previously used a reporter gene assay to demonstrate that
high OHT concentrations activate the p38 signal transduction pathway in
HeLaER cells (10). 0.5-2 µM SB203580 specifically
inhibits p38 kinases, whereas 5-10 µM SB203580 can also
partially block JNK kinase activity. Our finding that 1 µM SB203580 partially blocks PCD induced by 10 nM OHT and that 5 µM SB203580 almost completely blocks the onset of mitochondrial dysfunction suggests involvement of the p38 pathway and perhaps the JNK pathway in the
OHT·ER-induced signaling events upstream of mitochondrial dysfunction.
ER activates transcription by binding to specific DNA sequences termed
EREs or by being tethered to the DNA through proteins bound at AP-1 and
SP-1 sites (35). When transcription is activated by tethering of ER to
AP-1 sites, SERMS such as TAM, OHT, ICI 182,780, and RAL act as potent
agonists of ER (44). Because nM concentrations of these
ligands exhibit differential effects on HeLaER6 cell viability, there
is no reason to propose that tethering of ER to DNA through AP-1 or
SP-1 sites plays a key role in OHT-mediated apoptosis. TAM and OHT have
virtually no agonist activity on a reporter gene containing consensus
EREs. However, given the great diversity of AP-1 sites, SP-1 sites, and
imperfect EREs present in native genes, it remains quite possible that
TAM- or OHT-activated genes could induce cell death. Although a few
genes have been identified as being induced by OHT·ER, none of the
known OHT-induced genes is a plausible candidate for an inducer of
programmed cell death. Microarray studies that are ongoing in several
laboratories will likely greatly expand the repertoire of TAM- and
OHT-inducible genes.
The ER-dependent and ER-independent Pathways of
Programmed Cell Death Are Different--
Consistent with some earlier
work suggesting that high concentrations of TAM and/or OHT induce
caspase-dependent apoptosis (9, 11), we observed the
hallmarks of caspase-dependent apoptosis, including DNA
laddering, chromatin condensation, and PARP cleavage when HeLaER6 cells
were treated with 10-20 µM OHT. In contrast, 10 nM OHT did not produce these markers, although it did
elicit mitochondrial dysfunction resulting in cell death. To determine where the OHT·ER death pathway diverges from the apoptotic pathway, we examined the events occurring upstream and downstream of
mitochondrial dysfunction. Our results indicated that up to the point
of cytochrome c release, the mitochondrial death pathways
induced by high and low OHT concentrations are parallel. However,
caspase 9 activation was only triggered by 10-20 µM OHT
and was not seen with 10 nM OHT even after 4 days of
treatment (Fig. 7C) (data not shown). These data demonstrate
that the ER-dependent and ER-independent pathways diverge
at the level of pro-caspase 9 activation. Although dramatic reductions
in ATP levels (45-51) and induction of heat shock proteins (52-54)
reportedly interfere with the activation of caspase 9, the molecular
basis for the failure of the OHT·ER PCD pathway to activate caspase 9 remains to be established.
The OHT·ER-mediated Cell Death Pathway Resembles Necrosis-like
Programmed Cell Death--
Three classes of mitochondrial
death pathways can be triggered downstream of mitochondrial changes
(23, 45): 1) the caspase-dependent pathway initiated by
formation of the apoptosome, 2) a caspase-independent pathway leading
to necrosis-like programmed cell death, and 3) an apoptosis-like but
caspase-independent pathway that results when the apoptosis-inducing
factor is released from the mitochondria. The first and third pathways
share some common morphological characteristics, most notably chromatin
condensation and margination that are not seen in the PCD pathway
induced by 10 nM OHT. The second pathway, however, is
programmed cell death in the absence of chromatin condensation or at
best with chromatin clustering to speckles (50, 55-57). Fibroblasts
(58), neurons (59), and hepatocytes (60) can undergo PCD through this
pathway, in which a controlled series of events, involving signaling,
control, and execution, gives rise to cell morphology more
characteristic of necrosis more than apoptosis. This pathway is also
sometimes referred to as "aborted apoptosis," in which the
standard apoptotic program is initiated, blocked at the level of
caspase activation, and then terminated by caspase-independent means
(45). PCD induced by 10 nM OHT bound to ER best fits this
model for programmed cell death.
In this work we describe two distinct pathways of OHT-induced
programmed cell death: a caspase-dependent apoptotic cell
death program inducible in the absence of ER by very high
concentrations of TAM, OHT, RAL, or E2, and an
ER-dependent and ligand-specific pathway induced by
nanomolar concentrations of OHT and submicromolar concentrations of TAM
bound to ER (Fig. 8). This novel
ER-dependent cell death pathway is caspase-independent and
resembles necrosis-like programmed cell death. These studies describe
an activity of TAM and OHT that is distinct from their ability to
compete with estrogens for binding to the ER and suggests additional
potential mechanisms for the effectiveness of tamoxifen in cancer
therapy and chemoprevention. When ER-positive cancer cells are exposed
to extremely high concentrations of TAM or OHT, cell death likely
results from a combination of the ER-dependent programmed
cell death pathway we describe, the ER-independent pathway that leads
to caspase-dependent apoptosis, and the ability of TAM to
bind the ER and thereby block the growth-promoting and antiapoptotic
effects of estrogen.
We are grateful to Dr. J. Katzenellenbogen
for the gift of raloxifene and to Dr. J. Zhou for helpful comments on
the manuscript. We are also indebted to Lou Ann Miller for the
preparation of electron microscopy specimens and to Dr. Barbara Pilas
for help with flow cytometry protocols.
*
This work was supported by NCI, National Institutes of
Health Grant CA90374.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 19, 2002, DOI 10.1074/jbc.M208092200
2
C. Mao and D. J. Shapiro, manuscript in preparation.
The abbreviations used are:
ER, estrogen
receptor;
SERM, selective estrogen receptor modulator;
E2, 17
Estrogen Receptor-dependent and Estrogen
Receptor-independent Pathways for Tamoxifen and
4-Hydroxytamoxifen-induced Programmed Cell Death*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol and
raloxifene, and occurs in ER-negative cells. The ER dependence of a
second pathway, caused by submicromolar concentrations of TAM and OHT, was demonstrated by the ability of the ER ligands 17-estradiol, raloxifene, and ICI 182,780 to effectively block the cell
death-inducing effects of TAM and OHT. Because the p38-specific
inhibitor SB203580 blocks OHT·ER-induced cell death, stress
kinase pathways are likely involved. ER-independent cell death triggers
classic caspase-dependent apoptosis. However, although
OHT·ER triggers some hallmarks of apoptosis, including Bax
translocation and cytochrome c release, the absence of
poly(ADP-ribose) polymerase cleavage or DNA laddering indicates
that the death pathway involved is caspase-independent. The
OHT·ER-dependent cell death pathway appears to diverge
from classical apoptosis at the level of caspase 9 activation. The ability to promote ER-dependent programmed cell death
represents a novel activity of TAM and OHT.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol (E2) to the ER induces a
conformational change that enables the ER to recruit transcriptional
coactivators and to induce expression of estrogen-regulated genes.
Several estrogen-inducible genes, including c-myc, TGF-
,
and cathepsin D, are implicated in malignant transformation or tumor
metastases (1-4). Tamoxifen (TAM) and its active metabolite,
4-hydroxytamoxifen (OHT), are nonsteroidal selective estrogen receptor
modulators (SERMs) that compete with E2 and other estrogens
for binding to ER. Structural studies and chromatin
immunoprecipitations show that OHT·ER induces an ER conformation that
does not recruit coactivators to target genes and in many cell and
promoter contexts recruits corepressors (5, 6). The therapeutic
effectiveness of TAM in treatment of hormone-dependent
cancers and in preventing breast cancer in high risk women is thought
to arise primarily from its ability to compete with estrogens for
binding to the ER. It is thought that TAM·ER and OHT·ER are unable
to effectively activate transcription of genes important for the growth
and development of estrogen-dependent tumors. However,
several often-conflicting studies show that TAM and OHT can actively
induce programmed cell death of cancer cells (reviewed in Ref. 7). The
mechanism(s) by which TAM and OHT induce programmed cell death have
been quite controversial, with even the identity of the toxic agents in
dispute. One group reported that high concentrations of TAM, but not
OHT, induce cell death (8). Others indicated that both TAM and OHT induce cell death (9). Although our recent report was consistent with a
role for ER in OHT-induced apoptosis (10), other workers suggest a
number of different mechanisms for TAM-induced apoptosis. The effects
of TAM might be mediated through an ER-independent increase in reactive
oxygen species, resulting in caspase activation (9, 11), or through an
influx of extracellular calcium (12, 13). In addition, effects of TAM
on the levels of proteins important in cell growth including protein
kinase C (14, 15), TGF- (16, 17), and c-Myc (18, 19) have
been reported.
(HeLaER6 cells) (20).
These cell lines differ only in the presence or absence of ER (and the
neomycin phosphotransferase gene that encodes resistance to G418).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m)
Measurement--
To quantitate the percentage of cells in the early
stages of programmed cell death, we used a flow cytometry-based method that employs the strong cationic dye DiOC6(3). In healthy
cells, the presence of a mitochondrial membrane potential
(m) allows DiOC6(3) to be sequestered in
the mitochondria. Cells that are in the early stages of programmed cell
death exhibit decreased mitochondrial retention of
DiOC6(3).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(20). To minimize
potential cell stress due to maintaining HeLaER6 cell cells in medium
containing the selective agent G418, HeLaER6 cells were plated in
medium without G418 the day before experiments and maintained in
G418-free medium throughout the experiments.
9
M OHT with 107 M TAM). OHT, the
active metabolite of TAM, binds to the ER with at least a 100-fold
higher affinity than TAM (27). In intact cells, OHT exhibits some
agonist activity and activates transcription of a reporter gene at
concentrations ~100-fold lower than are required for TAM
activation.2 These data
suggested that binding to the ER was important in TAM and OHT-induced
death in the HeLaER6 cells.
View larger version (17K):
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Fig. 1.
TAM and OHT induce cell death in HeLaER cells
but not in parental wild-type HeLa cells. HeLa (open
symbols) and HeLaER6 (filled symbols) cells were
treated with increasing concentrations of TAM ( 
and 
) or
OHT (
and 
) for 3 days. After harvesting, the cells were
double-stained with DiOC6(3) and PI and analyzed by flow
cytometry as described under "Experimental Procedures." Data
analysis was performed using Summit V.3 software. Cells with decreased
DiOC6(3) retention were identified after gating for
PI-positive cell subpopulations and quantitated for each sample.
View larger version (46K):
[in a new window]
Fig. 2.
Raloxifene, ICI 182,780, and E2
block TAM- and OHT-induced programmed cell death of HeLaER6 cells.
HeLaER6 cells were pre-treated with 1 µM raloxifene, 1 µM ICI 182,780, or 1 µM E2 for
30 min before adding OHT to 10 8 M or TAM to
106 M. A, after 3 days of
treatment OHT-treated cells were harvested, double-stained with
DiOC6(3) and PI, and analyzed by flow cytometry, and the
percentage of cells in early stage PCD was determined by flow
cytometry. B, after 4 days of treatment cell images were
obtained using phase-contrast microscopy at ×40. C,
graphical representation of the data in panel A showing that
OHT-induced death is blocked by a 100-fold molar excess of RAL, ICI
182,780, or E2. D, TAM-induced programmed cell
death is blocked by equimolar concentrations of RAL, ICI 182,780, or
E2.
with much higher affinity than TAM (31).
View larger version (14K):
[in a new window]
Fig. 3.
The cell death pathway induced by
10 8 M OHT involves the p38 stress-activated
protein kinases. Cells were pre-treated with 1 or 5 µM SB203580 for 30 min and then exposed to
108 M OHT. After 3 days of treatment the
cells were harvested, and the percentage of cells in early stage PCD
was determined by flow cytometry as described under "Experimental
Procedures."
View larger version (9K):
[in a new window]
Fig. 4.
E2, but not OHT, transactivates
an ERE-containing reporter gene. HeLaER6 cells were transfected
using LipofectAMINE 2000 reagent with pATL4, which contains
four copies of the consensus ERE-driving expression of a luciferase
reporter gene, and with the internal control plasmid pRLSV40. Four h
post-transfection 10 8 M E2, OHT,
ICI 182,780, RAL, or ethanol vehicle was added to separate wells. Cells
were harvested 24 h later and assayed for luciferase activity. The
data are representative of three separate experiments.
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Fig. 5.
OHT·ER-induced programmed cell
death is caspase-independent. Cells were treated with EtOH vehicle
(A), 12.5 nM etoposide (etop;
B), 10 nM OHT (C), or 20 µM OHT (D) and harvested for electron
microscopy. Electron micrographs were obtained at ×6,000
magnification. E, to analyze DNA laddering, cells were
treated with EtOH, 10 5 M OHT, or
108 M OHT for 3 days or etoposide for 2 days.
Cell extracts were treated with RNase A/RNase T1 mixture for 2 h
at 37 °C and with proteinase K for 16 h at 50 °C. Samples
were run in a 1% agarose gel. F, Western blot analysis of
whole cell extracts prepared from EtOH, 108 M
OHT, or etoposide-treated cells. Full-length, uncleaved PARP appears as
a 116-kDa protein; caspases activated by apoptotic stimuli cleave PARP
into an 89-KDa fragment and a 22-kDa fragment.
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Fig. 6.
At high concentrations TAM, OHT, RAL, and
E2 induce caspase-dependent apoptosis in both
ER-negative HeLa cells and HeLaER cells. A, HeLaER6
cells were treated with 10 or 20 µM TAM, OHT, or
E2, or with 12.5 µM etoposide
(etop) as a positive control, and assayed for early stage
cell death by flow cytometry as described under "Experimental
Procedures." HeLa (B) and HeLa-ER6 (C) cells
were maintained for 2 days in medium containing 12.5 µM
etoposide or 20 µM OHT, TAM, RAL, ICI 182,780, or
E2. Whole cell extracts were then prepared and analyzed by
Western blotting for cleavage and activation of pro-caspase 9 and for
PARP cleavage.
m), it was clear that mitochondria were involved in
the death pathway. To determine the point at which this death pathway
deviates from the classic caspase-dependent apoptosis
pathway, we looked at apoptotic markers at the steps before and after
mitochondrial membrane depolarization. We examined the ability of
OHT·ER to induce translocation of the pro-apoptotic protein, Bax,
from the cytosol into the mitochondria, the release of cytochrome
c from the mitochondria into the cytosol, and the activation
of the most upstream caspase involved in mitochondrial apoptosis,
pro-caspase 9. Mitochondria were isolated from HeLaER6 cells (Fig.
7A) and HeLa cells (Fig.
7B) treated with 10 nM OHT or with 10 µM or 20 µM OHT. 10 nM OHT, 10 µM OHT, and 20 µM OHT elicited a large
increase in mitochondrial Bax. The level of the control mitochondrial
protein, COX4, remained unchanged (Fig. 7, A and
B). Cytosol extracts exhibited increased cytochrome
c, whereas levels of the housekeeping cytosolic protein
actin remained constant. However, when we assayed for pro-caspase 9 activation, we saw that 20 µM OHT, but not 10 nM OHT, produced active, cleaved caspase 9 (Fig.
7C). These data demonstrate that the
ER-dependent and ER-independent programmed cell death
pathways diverge at the level of caspase 9 activation.
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Fig. 7.
The OHT-activated, ER-dependent,
and ER-independent pathways of mitochondrial-based programmed cell
death diverge at the level of pro-caspase 9 activation.
A, HeLaER6 cells were treated with EtOH vehicle or 10 nM OHT for 3 days or with etoposide (etop) or 10 µM OHT for 2 days. cyt c, cytochrome
c. B, HeLa cells were treated with EtOH, 12.5 µM etoposide, 10 µM OHT, or 20 µM OHT for 2 days. Cells were collected by centrifugation
and homogenized using Kontes Dounce homogenizers. Mitochondria and
cytosol were prepared by differential centrifugation as described under
"Experimental Procedures." Mitochondrial extracts were assayed for
Bax translocation in response to OHT treatment. COX4 is an endogenous
mitochondrial protein that was assayed as a control for extract
cross-contamination. Cytosol extracts were assayed for an increase in
cytochrome c. Levels of the cytosolic housekeeping gene
actin did not change in response to any of the treatments.
C, in HeLaER6 cells only 20 µM OHT produces
strong caspase 9 cleavage. Whole cell extracts were prepared from cells
treated with EtOH or 10 nM OHT for 3 days or with etoposide
or 10 or 20 µM OHT for 2 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(27) and
transactivation potential.2 These data suggested
that binding of TAM and OHT to the ER was important for induction of
PCD (Fig. 1). However, these data did not exclude the possibility that
either traces of estrogen in the medium, or the OHT·ER complex,
induce a HeLa cell protein that binds TAM and OHT and induces PCD. A
recent report suggests that an estrogen-inducible G-protein, GPR30, and
not binding of ligands to ER, is responsible for the growth stimulatory
effects of ER ligands (38). Because all of the ER ligands these
researchers tested, including E2, ICI 182,780, and TAM,
produced the same effect and stimulated cell growth, and we find that
submicromolar concentrations of TAM and OHT, but not E2,
RAL, and ICI 182,780, stimulate PCD, it is clear that binding to GPR30
is not responsible for TAM- and OHT-induced apoptosis. It is also
improbable that direct binding of TAM and OHT to an as yet unidentified
TAM·ER and OHT·ER inducible protein is responsible for programmed
cell death of HeLaER cells. The ability of the five different ER
ligands we tested (TAM, OHT, E2, RAL, and ICI 182,780) to
induce or protect against programmed cell death data roughly correlates
with their binding affinities for hER (31). For example, TAM exhibits a much lower affinity for hER than E2, RAL, and ICI
182,780, and equimolar concentrations of these compounds were able to
protect against TAM-induced PCD (Fig. 2). The related possibility that E2, RAL, and ICI 182,780 block the ability of TAM and OHT
to induce apoptosis, not because they compete with TAM and OHT for
binding to the ER, but because they induce one or more
anti-apoptotic proteins, seems remote. Because the nearly
pure antagonist ICI 182,780, the potent agonist E2, and the
SERM RAL all block TAM- and OHT-induced programmed cell death, it seems
highly improbable that these three ER ligands, with their very
different abilities to activate transcription, would exhibit
essentially equal abilities to induce an anti-apoptotic protein.
View larger version (15K):
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Fig. 8.
Schematic of the ER-independent and
ER-dependent pathways of OHT-induced programmed cell
death. Likely steps in the ER-independent (A) and
ER-dependent (B) pathways of TAM- and
OHT-induced PCD are shown.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 217-333-1788;
Fax: 217-244-5858; E-mail: djshapir@uiuc.edu.
ABBREVIATIONS
-estradiol;
TAM, tamoxifen;
OHT, 4-hydroxytamoxifen;
RAL, raloxifene;
ICI, ICI 182,780;
PCD, programmed cell death;
DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide;
PI, propidium
iodide;
PARP, poly(ADP-ribose) polymerase;
PBS, phosphate-buffered
saline;
JNK, c-Jun NH2-terminal kinase;
ERE, estrogen
response element.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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J. Cheng, D. V. Yu, J.-H. Zhou, and D. J. Shapiro Tamoxifen Induction of CCAAT Enhancer-binding Protein {alpha} Is Required for Tamoxifen-induced Apoptosis J. Biol. Chem., October 19, 2007; 282(42): 30535 - 30543. [Abstract] [Full Text] [PDF]
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R. R. Nazarewicz, W. J. Zenebe, A. Parihar, S. K. Larson, E. Alidema, J. Choi, and P. Ghafourifar Tamoxifen Induces Oxidative Stress and Mitochondrial Apoptosis via Stimulating Mitochondrial Nitric Oxide Synthase Cancer Res., February 1, 2007; 67(3): 1282 - 1290. [Abstract] [Full Text] [PDF]
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M. I. Torres-Arzayus, J. Yuan, J. L. DellaGatta, H. Lane, A. L. Kung, and M. Brown Targeting the AIB1 Oncogene through Mammalian Target of Rapamycin Inhibition in the Mammary Gland Cancer Res., December 1, 2006; 66(23): 11381 - 11388. [Abstract] [Full Text] [PDF]
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 L. Wang, X. Gao, P. Gao, W. Deng, P. Yu, J. Ma, J. Guo, X. Wang, H. Cheng, C. Zhang, et al. Cell-Based Screening and Validation of Human Novel Genes Associated with Cell Viability J Biomol Screen, June 1, 2006; 11(4): 369 - 376. [Abstract] [PDF]
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 X. Jiang, B. A. Orr, D. M. Kranz, and D. J. Shapiro Estrogen Induction of the Granzyme B Inhibitor, Proteinase Inhibitor 9, Protects Cells against Apoptosis Mediated by Cytotoxic T Lymphocytes and Natural Killer Cells Endocrinology, March 1, 2006; 147(3): 1419 - 1426. [Abstract] [Full Text] [PDF]
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 P. Rouanet, G. Linares-Cruz, F. Dravet, S. Poujol, S. Gourgou, J. Simony-Lafontaine, J. Grenier, A. Kramar, J. Girault, E. Le Nestour, et al. Neoadjuvant Percutaneous 4-Hydroxytamoxifen Decreases Breast Tumoral Cell Proliferation: A Prospective Controlled Randomized Study Comparing Three Doses of 4-Hydroxytamoxifen Gel to Oral Tamoxifen J. Clin. Oncol., May 1, 2005; 23(13): 2980 - 2987. [Abstract] [Full Text] [PDF]
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Q. Wang, X. Li, L. Wang, Y.-H. Feng, R. Zeng, and G. Gorodeski Antiapoptotic Effects of Estrogen in Normal and Cancer Human Cervical Epithelial Cells Endocrinology, December 1, 2004; 145(12): 5568 - 5579. [Abstract] [Full Text] [PDF]
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 M. B. Buck, K. Pfizenmaier, and C. Knabbe Antiestrogens Induce Growth Inhibition by Sequential Activation of p38 Mitogen-Activated Protein Kinase and Transforming Growth Factor-{beta} Pathways in Human Breast Cancer Cells Mol. Endocrinol., July 1, 2004; 18(7): 1643 - 1657. [Abstract] [Full Text] [PDF]
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 Y. Wang, X. Li, L. Wang, P. Ding, Y. Zhang, W. Han, and D. Ma An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression J. Cell Sci., March 15, 2004; 117(8): 1525 - 1532. [Abstract] [Full Text] [PDF]
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A. J. Krieg, S. A. Krieg, B. S. Ahn, and D. J. Shapiro Interplay between Estrogen Response Element Sequence and Ligands Controls in Vivo Binding of Estrogen Receptor to Regulated Genes J. Biol. Chem., February 6, 2004; 279(6): 5025 - 5034. [Abstract] [Full Text] [PDF]
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K. Chaturvedi and D. K. Sarkar Involvement of Protein Kinase C-Dependent Mitogen-Activated Protein Kinase p44/42 Signaling Pathway for Cross-Talk between Estradiol and Transforming Growth Factor-{beta}3 in Increasing Basic Fibroblast Growth Factor in Folliculostellate Cells Endocrinology, February 1, 2004; 145(2): 706 - 715. [Abstract] [Full Text] [PDF]
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D. J. Waxman and P. S. Schwartz Harnessing Apoptosis for Improved Anticancer Gene Therapy Cancer Res., December 15, 2003; 63(24): 8563 - 8572. [Abstract] [Full Text] [PDF]
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