From The Breakthrough Toby Robins Breast Cancer
Research Centre, Institute of Cancer Research, 237 Fulham Road, London
SW3 6JB and the ¶ Prostate Cancer Research Group, Department of
Cancer Medicine, Division of Medicine, Imperial College, Du Cane Road,
London W12 0NN, United Kingdom
Received for publication, February 20, 2003
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ABSTRACT |
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Glucocorticoids influence many physiological
processes, and in particular apoptosis, often with opposite effects
depending on the cell type examined. We found that during fibrosarcoma
development there is a strong increase in apoptosis at the tumor stage,
which is repressed by dexamethasone to levels observed in normal
fibroblasts. The anti-apoptotic Bcl-2 family protein
Bcl-xL is induced by dexamethasone at the
transcriptional level at all stages of fibrosarcoma development. The
ligand-activated glucocorticoid receptor (GR) activates the Bcl-x
promoter in transient transfection experiments, and GR binds to
specific Bcl-x promoter sequences in vitro and in
vivo. Furthermore, a GR antagonist abolishes this effect,
indicating that Bcl-xL induction is mediated by GR.
Importantly, exogenous Bcl-xL inhibits apoptosis and
caspase-3 activity in fibrosarcoma cells to levels found in
dexamethasone-treated fibrosarcoma cells. We conclude that
Bcl-xL is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis.
Glucocorticoids exert different effects on apoptosis and cell
growth depending on the tissues examined. In some cell types, for
example thymocytes and some leukemia cell lines, treatment with
glucocorticoids induces apoptosis (1). This has led to their common use
as chemotherapeutic agents in lymphomas and leukemias (2). In contrast,
glucocorticoids have been reported to inhibit apoptosis in a number of
other cell types, including glioma and astrocytoma cell lines (3),
fibroblasts (4), hepatoma cells (5), gastric cancer cell lines (6), and
mammary epithelial cells (7, 8).
The glucocorticoid receptor
(GR)1 belongs to a
superfamily of transcription factors that includes receptors for
steroid and thyroid hormones, retinoic acid, and vitamin D3
(9). GR is normally localized in the cytoplasm in a non-active state in
a complex that includes Hsp90. Upon hormone binding, GR changes conformation and migrates to the nucleus, where it induces or represses
transcription by binding to specific DNA sequences on target genes
(10).
Apoptosis, triggered by a variety of intra- and extracellular signals,
is important for normal development, to maintain tissue homeostasis,
and as a defense strategy against the emergence of cancer (11, 12). The
apoptotic program is executed by a family of cysteine proteases called
caspases, which are activated by proteolytic cleavage (13, 14). Once
activated, effector caspases cleave a variety of cellular substrates
including structural components, regulatory proteins, and other
caspases, resulting in the orchestrated collapse of the cell
characteristic to apoptosis.
Bcl-2 family proteins play critical roles in the control of apoptosis.
Two major groups of Bcl-2 family proteins exist; the pro-survival
members, including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, etc., and
the pro-apoptotic members, including Bax, Bak, Bok, etc. (see Ref. 15
for review). The ratio between these two groups of family members
determines whether a cell will live or die. Downstream of this
checkpoint lie the caspase pathway and mitochondria dysfunction, major
execution events that lead to irreversible cell death (16). Alterations
in the expression of anti-apoptotic members such as Bcl-2 and
Bcl-xL have been implicated in tumorigenesis in both clinical cases and transgenic models (17). In addition, Bcl-2 members
are also important determinants of anticancer drug sensitivity (18).
The conversion of a normal cell to a neoplastic one occurs in multiple
steps (19), and one approach to studying this process has been to
employ transgenic mice (20). Mice carrying the bovine papillomavirus
type I genome develop dermal fibrosarcomas in a process that involves
distinct proliferative stages. These are the normal fibroblasts (NF),
and then two histological grades of hyperplasia, mild fibromatosis (MF)
and aggressive fibromatosis (AF). Finally, at lower frequency, dermal
fibrosarcomas (FS) develop (21). The first molecular distinction
between the AF and the FS cells to be identified was a dramatic
increase in ligand-dependent GR transcriptional activity in
FS cells (22). This increase does not result from changes in the
intracellular levels of GR, hormone-dependent nuclear
translocation, or specific DNA binding activity, all of which are
unaltered throughout the progression. Moreover, analysis of the tumors
formed in mice upon inoculation of AF or FS cells indicates a direct
correlation between GR transcriptional activity and tumorigenic
potential (22).
To understand cancer progression it is important to determine the
mechanisms by which signaling proteins influence proliferation and
apoptosis at different stages of the tumorigenic process. Here we have
examined the effects of dexamethasone on apoptosis in the
multistep tumorigenic pathway of fibrosarcoma development. Our
observations point to Bcl-xL as a key GR target mediating the inhibition of apoptosis during fibrosarcoma progression.
DNA Plasmid Constructions--
The plasmid containing 3.2 kb
(
Fragments containing the putative GREs from the Bcl-x promoter were
cloned into a TK109-luciferase reporter construct: RE1 contains sequences of the murine Bcl-x promoter from
The putative GREs from the Bcl-x promoter were mutated using the
following oligonucleotides (nucleotides that were changed are
underlined) and their reverse complements: P1
(5'-CTCTGTGGCCAACAGTCCATTCTGCGAAAGACGGGAAAGTTGC-3'), P2
(5'-GCTGTGCAGAAGACCAGCTTTTTCCTGAGGCCATGTTATCC-CACAGCCAGG-3'), P3
(5'-GCTACATAGATTGAGGCCAGACTCGGCTGAAAAACTG-3').
The QuikChange XL site-directed mutagenesis kit (Stratagene) was
used following the instructions of the manufacturer. All mutations were
confirmed by DNA sequencing.
The fragment of murine Bcl-xL cDNA encompassing the
coding region was amplified by PCR from the total cDNA of NF cells
using HiFi polymerase (Roche Applied Science). This fragment was
cloned into pIRES2-eGFP (Clontech). Absence of
mutations within the insert was confirmed by sequencing in all cases.
Cell Culture and Transient Transfections--
Cultures were
established from skin and tumor tissues and maintained as reported
previously (22). Several cultures were tested from each stage of the
tumorigenic process: normal fibroblasts (23784, 40950); mild
fibromatosis (14249, 39614, 27877); aggressive fibromatosis (BPV3,
BPV7, BPV21); fibrosarcomas (BPV1, BPV22, BPV2, BPV11). For
experiments involving steroid hormone treatments, cells were maintained
in medium containing charcoal-stripped serum (24). COS-7 cells were
also maintained as above. Cell viability was determined by trypan blue
dye exclusion (25).
Cells were transiently transfected using the DEAE-dextran method and
the RNA Isolation and Northern Blot Analysis--
Total RNA was
isolated using the SV Total RNA isolation system (Promega)
according to the instructions of the manufacturer. Northern blot
analysis was performed using conventional techniques. The probe
encompassing 725 bp of the Bcl-xL cDNA was labeled
using a Random Primed DNA labeling kit (Roche Applied Science)
following the instructions of the manufacturer. Finally, the membrane
was exposed to x-ray film (Kodak X-Omat AR).
Preparation and Analysis of Cell Extracts--
Cells were
harvested by centrifugation in 40 mM Tris-HCl, pH 7.8, 10 mM EDTA, and 150 mM NaCl, washed in ice-cold
phosphate-buffered saline, and re-pelleted and frozen in liquid
nitrogen. Cells were resuspended in lysis buffer (50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium
deoxycholate, 1% Nonidet P-40, 10 µg/ml phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin/leupeptin/pepstatin A), incubated on ice
for 25 min, and centrifuged for 20 min at 14,000 rpm. Equal amounts of
extract protein (30 µg) were analyzed by immunoblot analysis as
described previously (22). Anti-Bcl-2 (Oncogene),
anti-Bcl-xL/S (Transduction Laboratories), anti-Bax (Zymed Laboratories Inc.), anti- Gel Retardation Assays--
Analysis of DNA-protein interactions
was performed as described (22). The following oligonucleotides were
used: consensus GRE-TAT (22), P1
(5'-TGGCCAACAGTACATTCTGTGAAAGAC-3'), P2
(5'-TGTGCAGAAGAACAGCTTTTTCC-TGAGGCCATGTTGTCCCACAG-3'), and P3
(5'-ATAGATTGAGGACAGACTGGGCTGAAA-3'); nonspecific, N
(5'-AGGATAACGGAGGCTGGGTAGGTGCAC-3'). To assess specificity of DNA
binding, 50-fold molar excess of unlabeled oligonucleotide (consensus
GRE, specific (S) or same as labeled oligonucleotide) or 200-fold molar
excess of a nonspecific oligonucleotide (N) was added to the reaction
prior to addition of the labeled probe.
Caspase Enzyme Activity Assay--
Caspase assays were performed
using a protocol based on the QuantiZymeTM assay system (Biomol). Cells
were lysed in buffer Q (50 mM HEPES, pH 7.4, 0.1% CHAPS,
0.1 mM EDTA, 0.1 mM dithiothreitol) for 30 min
on ice followed by centrifugation at 14,000 rpm for 10 min at
4 °C. Equal amounts of protein (30 µg) were added in triplicate to
wells in a 96-well plate and equilibrated to 37 °C for 15 min after
the addition of assay buffer (50 mM HEPES pH 7.4, 0.1%
CHAPS, 1 mM EDTA, 10 mM dithiothreitol, 100 mM NaCl, 10% glycerol). Fluorogenic peptide substrates
selective for caspase-1 (YVAD) or caspase-3 (DEVD) (final concentration
1 mM) were added to each well and incubated at 37 °C for
6 h. For controls, the caspase inhibitors Ac-YVAD-CHO or
Ac-DEVD-CHO, respectively, were used at a final concentration of 0.5 µM. Absorbance of the samples at 405 nm was measured
using a colorimetric plate reader (MRX) immediately after addition of
the substrate and after 6 h. The change in absorbance of each
sample (minus the inhibitor control) over the 6-h period was taken as
the specific caspase activity.
Chromatin Immunoprecipitation (ChIP) Assay--
Cells were
maintained in 10-cm dishes in medium containing charcoal-stripped serum
for at least 16 h and treated with or without 100 nM
dexamethasone for 1 h. The ChIP assay was performed as described
previously (26) except that the final samples were resuspended in 30 µl of water, and 2-4 µl of each sample was used for PCR
amplification (30 cycles) with the rTaq DNA polymerase (TaKaRa). The antibody used against GR was sc-1002X, and sc-751 against
cyclin A was used as a negative control (both from Santa Cruz
Biotechnology). The primers for the PCR were
5'-CCCAAAAGGATAGAATGAACTCTG-3' and 5'-CTGGTTATGTAGCTGTGGGCTGCC-3',
which amplify a 247-bp fragment corresponding to murine Bcl-x sequence
Cell Death Is Reduced by Dexamethasone Treatment--
Cell
proliferation increases during fibrosarcoma development in
vivo (27) and in cultured cells (21), and it is inhibited by
dexamethasone, a GR agonist (53). We examined the effects of
glucocorticoids on cell death during fibrosarcoma progression. We used low-passage primary cell lines representative of the
four stages in this multistep pathway: NF, MF, AF, and FS cells (see Table I). Cells from each stage were
cultured in the absence and presence of dexamethasone, and cell growth
was observed for 7 days at 24-h intervals. In the absence of
hormone, cells from the earlier stages of the progression proliferated
at a lower rate, as reported previously, whereas FS cells increased
rapidly in number (21). Cells cultured in the presence of dexamethasone appeared flattened (data not shown) and were fewer in number. Trypan
blue exclusion was used as a marker for viability of the cells. The
number of dead cells, calculated as a percentage of total population at
each of the progression stages (Fig.
1A, dead cells at day 7),
indicated that the extent of cell death was higher in the tumor cells
than in the primary cells and that dexamethasone reduced the number of
dead cells, particularly among FS cells. Fig. 1B shows the
proportion of dead FS cells on each day of culture. Thus dexamethasone
exerts a protective effect on FS cells.
To assess whether the observed cell death reflected apoptosis, we
measured the caspase activity in cells from all stages of fibrosarcoma
development. Caspase-3 has been shown to be one of the key effectors of
apoptotic cell death (28), whereas caspase-1 is involved in
inflammatory responses and also in some forms of apoptosis (29). In the
absence of hormone there was an increase in both caspase-1 and
caspase-3 activity during tumor progression (p < 0.01), particularly at the fibrosarcoma stage (Fig. 1, C and
D). Upon dexamethasone treatment, caspase-3 activity was
~50% reduced in FS cells (p < 0.005), suggesting an
inhibition of apoptosis. In contrast, caspase-1 activity was not
affected by the presence of hormone. In addition, when we examined the
extent of cell death by DNA fragmentation analysis, we detected a high
degree of DNA degradation in the FS cells, which was abolished by
hormone (data not shown). These results confirm that there is a strong
increase in apoptosis at the tumor stage of fibrosarcoma development
and that this is reduced by dexamethasone.
Changes in the Expression of Bcl-2 Family Proteins during
Fibrosarcoma Development--
Bax appears to be one of the major
pro-apoptotic Bcl-2 proteins that act as death effectors in
fibroblasts, and Bcl-2 and Bcl-xL serve as inhibitors of
Bax (30). Furthermore, Bcl-xL has been found to be
regulated by dexamethasone in some cell types (8, 31). Therefore, to
investigate changes in the level of expression of Bcl-2 family proteins
that might contribute to the regulation of apoptosis during
fibrosarcoma development, we examined the expression levels of these
proteins in the absence and presence of dexamethasone by immunoblot
analysis (Fig. 2). NF and MF cells expressed similar levels of the Bcl-2 family members examined hence
only MF results are shown in this figure. Expression of the
pro-apoptotic protein Bax was elevated in FS cells, but its protein
levels were not affected by dexamethasone (Fig. 2A). The increased expression of Bax protein correlates with increased apoptosis
in FS cells. However, the hormone-dependent inhibition of
apoptosis we observed in FS cells does not correlate with Bax levels.
Analysis of the expression of anti-apoptotic proteins (Fig.
2B) showed that Bcl-2 migrated as a doublet and remained
constant in all cell types throughout tumor progression. In contrast,
Bcl-xL expression clearly decreased during fibrosarcoma
progression. However, in the presence of dexamethasone its expression
was strongly induced, correlating with the much reduced level of
apoptosis in dexamethasone-treated FS cells (Fig. 1).
The combination of changes in the expression levels of apoptotic
proteins in FS cells, together with elevated caspase-3 activity, correlates with the increase in apoptosis at the tumor stage, suggesting that Bcl-2 proteins play a role in regulating apoptosis during fibrosarcoma development. Furthermore, Bcl-xL
induction in response to hormone in FS cells makes Bcl-xL a
candidate for mediating the reduction in apoptosis at the tumor stage.
Bcl-xL Expression Is Induced by Dexamethasone--
A
more detailed analysis of expression of Bcl-xL protein
during fibrosarcoma progression showed a clear increase in the fold induction of the protein by dexamethasone as fibrosarcoma
progresses (Fig. 3A). This
pattern of hormone-dependent induction of expression correlates with the transition in GR transactivation activity observed
during fibrosarcoma progression (22) and suggests that GR might be
involved directly in Bcl-xL regulation. The analysis presented involved representative cell cultures from the four stages of
the pathway (NF 40950, MF 14249, AF BPV3, FS BPV1). However, we
assayed the expression of Bcl-xL in two or more independent cell lines from each stage (see "Experimental Procedures") and found that all lines derived from a particular stage displayed consistent results. To determine the time course of Bcl-xL
induction in FS cells, we examined protein levels at various times
after dexamethasone treatment (Fig. 3B). Induction of
Bcl-xL protein could be detected as early as 2-4 h
following exposure to the hormone and remained elevated for 24 h.
Bcl-x Transcription Is Induced by Dexamethasone--
The rapid
increase in the level of Bcl-xL protein by dexamethasone in
FS cells suggested that this induction occurred at the transcriptional
level. Therefore, we examined Bcl-x mRNA expression after exposure
to dexamethasone in all stages of fibrosarcoma development. Northern
blot analysis revealed a major band of ~3-kb mRNA in all cell
stages (Fig. 4A), as reported
previously (32). In the absence of hormone, the expression level of the
Bcl-x transcript was reduced during tumor development, similar to the
observed decrease in protein levels. Bcl-x mRNA was increased by
the presence of dexamethasone.
To investigate the regulation of Bcl-x promoter activity by
dexamethasone, we transiently transfected a luciferase reporter construct containing a 3.2-kb genomic fragment of the murine Bcl-x promoter, Bcl-x(3.2), together with a
To assess whether the induction of Bcl-x transcription by dexamethasone
is dependent on GR, we tested the effect of the GR antagonist RU 40555
on transcriptional regulation of the Bcl-x promoter. In FS cells,
dexamethasone-dependent Bcl-x promoter activity was
inhibited by RU 40555 in a dose-dependent manner (Fig.
4D), whereas it did not have any effect by itself,
demonstrating a requirement for transcriptionally active GR to induce
Bcl-x expression.
GRE-like Sequences in the Bcl-x Promoter Mediate GR
Activation--
To understand further the transcriptional regulation
of Bcl-x expression by dexamethasone, we searched for potential
sequences within the Bcl-x promoter that confer hormone responsiveness. A series of luciferase vectors were constructed containing 5' fragments
of the 3.2-kb region of the Bcl-x promoter (Fig.
5A) and were transiently
transfected into FS cells. Analysis of these fragments showed a clear
reduction in the ability of the promoter to respond to dexamethasone,
from around 8-10-fold induction using the 3.2-kb fragment to a small
but significant (p < 0.005) induction using the most
proximal region (Fig. 5B). The highest induction from the
latter was obtained using Bcl-x(0.1), suggesting that this region
(
To investigate further the glucocorticoid-responsive sequences within
the Bcl-x promoter, the murine genomic Bcl-x sequence (GenBankTM accession number AF 088904) was screened for
potential GR binding sites by computer analysis (MatInspector program,
San Diego Workbench). Three candidate sequences, P1-P3 (with P2
containing two adjacent sites), were identified with some homology to
the consensus GRE sequence GGTACANNNTGTTCT (35) in the sequence between
Finally, two point mutations in positions known to be important for
GR-dependent transcription in the context of a consensus GRE (35) were introduced in each of the three putative GREs, and their
effect on dexamethasone-dependent induction of the Bcl-x promoter was determined in transient transfection assays. Single and
double combinations of these mutations decreased Bcl-x inducibility by
dexamethasone in an additive fashion (data not shown), whereas introduction of two point mutations in each of the putative GREs (Fig.
5F) resulted in strong reduction of
dexamethasone-dependent transcriptional activity in FS
cells (Fig. 5G). In NF cells the response to dexamethasone
is much weaker, but the inhibitory effect of mutating the GREs was
still detectable. Although mutation of these binding sites does not
abolish dexamethasone-dependent transcriptional activation
of the Bcl-x promoter in FS cells totally, this was not surprising,
because we know that the Bcl-x promoter contains other weak proximal
putative GREs (Fig. 5B), and it is still conceivable that
there are further weak putative GREs that remain unidentified. However,
our results suggest that the integrity of the three GREs we have
identified is required for GR-dependent activation of transcription.
GR Binds to the Bcl-x Promoter in Vitro and in Vivo--
The
ability of GR to bind to the identified GRE sequences was examined by
gel mobility shift assays. We incubated nuclear extracts from
hormone-treated FS cells in the presence of labeled oligonucleotides
containing either one of the putative GREs (Fig. 5C,
P1-3). A retarded complex was observed using P1 (Fig.
6A, lane b), P2
(lane g), and P3 (lane j) that migrated at the
same position as that originated by binding of FS cell extracts to the
consensus GRE-TAT (lane m). This complex has been shown to represent GR protein specifically bound to the GRE sequences (22). Incubation with 50-fold excess of the corresponding unlabeled oligonucleotides abolished binding to these sequences (Fig.
6A, lanes c, f, k, and
n), whereas 200-fold excess of nonspecific unlabeled
oligonucleotide had no effect (lanes d, h,
l, and o), confirming the specificity of the binding
reactions. The same experiment was performed with extracts from all
four stages of the tumorigenesis pathway, and as expected from previous
findings (22), GR binds to these sequences at all cell stages with
similar efficiency (data not shown). Furthermore, binding of GR to the labeled consensus GRE (Fig. 6B, lane b) is
disrupted by 50-fold excess of the unlabeled putative P1 (lane
c), P2 (lane d), and P3 (lane e), which is
as efficient as using the same molar excess of unlabeled consensus GRE
(S) (lane f). In the reverse experiment, binding to all
labeled putative GREs, P1, P2, and P3 (Fig. 6C, lanes
b, e, and h), could be abolished by competition with
unlabeled consensus GRE (S) (lanes c, f, and i).
In summary, these experiments demonstrate that GR can specifically bind
in vitro to non-consensus GRE sequences in the Bcl-x
promoter.
To determine whether GR interacts directly with this region of the
Bcl-x promoter in vivo, we used the chromatin
immunoprecipitation assay. Using an antibody raised against GR to
immunoprecipitate sequences bound by GR, we observed increased
occupancy by GR at the Bcl-x promoter after addition of dexamethasone
(Fig. 7, compare lanes 7 and
8), whereas no signal could be amplified from a fragment corresponding to a control element from the Bak promoter, which lacks
functional GREs. These observations were dependent on the use of an
antibody raised against GR to immunoprecipitate efficiently the
sequences of the Bcl-x promoter containing putative GREs, whereas a
control antibody (also rabbit serum, against cyclin A) failed to
recruit either of the promoter sequences in a
ligand-dependent manner (lanes 5 and
6). Thus, GR binds to the functional GREs sequences
identified in the Bcl-x promoter in vivo.
Exogenous Expression of Bcl-xL Reduces Apoptosis in FS
Cells--
Apoptosis is reduced by dexamethasone in FS cells, and this
correlates with transcriptional activation of the Bcl-x promoter and
increased expression of the anti-apoptotic protein, Bcl-xL. To determine whether enhanced Bcl-xL expression is
sufficient to reduce apoptosis in FS cells, we transfected an
expression vector encoding Bcl-xL, driven by the
cytomegalovirus promoter, into FS cells in the absence or presence
of dexamethasone. Transfection of this construct generated a level of
Bcl-xL protein expression similar to or higher than that
obtained by hormone treatment in FS cells transfected with the
cytomegalovirus vector alone (Fig. 8A). Dexamethasone did not
increase the level of Bcl-xL further. Determination of cell
death upon transfection with the Bcl-xL expression vector
alone indicated that the number of dead cells (Fig. 7B) and
caspase-3 activity (Fig. 8C) were reduced significantly (p < 0.005) compared with that achieved by
dexamethasone treatment of FS cells transfected with vector alone (or
untransfected; data not shown). Importantly, Bcl-xL
overexpression in the absence of hormone had a similar effect in both
assays, demonstrating that Bcl-xL inhibits apoptosis in FS
cells. The number of dead cells (Fig. 8B) and caspase-3
activity (Fig. 8C) were further reduced upon dexamethasone
treatment (p < 0.005) despite apparently constant
levels of Bcl-xL protein.
Apoptosis has been found to be widespread in many tumors (36, 37)
and yet limited in others (38). GR can display either pro-apoptotic or
anti-apoptotic activity depending on cell context (39, 40). We show
here that in the absence of glucocorticoids apoptosis increases during
fibrosarcoma development, whereas in the presence of dexamethasone it
is reduced to those levels found in the earlier stages of the
tumorigenic pathway.
Few studies of caspase activity during tumor development have been
reported. Caspase-3-like activity has been observed to increase during
the development of colorectal carcinoma (41), thus representing a
situation similar to that found in the absence of glucocorticoids
during fibrosarcoma development. Although involved in some forms of
apoptosis, caspase-1 also promotes inflammatory responses by regulating
cytokine signaling (42). Increasing caspase-1 activity during
fibrosarcoma development may therefore have implications not only for
apoptosis but also for inflammation.
Our results demonstrate that decreasing Bcl-xL expression
results primarily from decreasing Bcl-x transcription. This
correlates inversely with increasing transcription of the BPV-1 genome
and E5, E6, and E7 oncogene expression in MF, AF, and FS cells (21, 53). Both BPV-1 E6 and E7 oncoproteins sensitize cells to apoptosis induced by tumor necrosis factor- Increased Bcl-xL mRNA expression following
glucocorticoid treatment has been observed in human gastric cancer
cells (6), where enhanced Bcl-xL mRNA stability was
found to represent a part of the mechanism underlying the protective
effect of dexamethasone. However, dexamethasone does not affect the
stability of Bcl-xL mRNA in fibrosarcoma cells (53).
The reported increased expression of Bcl-xL mRNA in
human myeloid leukemic cells required 24 h of hormone treatment
(48), whereas we clearly detected up-regulation of Bcl-xL
after 2 h of dexamethasone treatment. Subsequent studies by other
groups have also demonstrated the importance of increasing Bcl-xL expression for the inhibition of apoptosis by
glucocorticoids in a variety of cell types (3, 8, 31, 49). However, none of these reports identified the mechanism by which GR might control Bcl-x expression.
Further extending the initial observations of these studies, we have
demonstrated that the up-regulation of Bcl-xL expression by
dexamethasone occurs at the transcriptional level and is increased during fibrosarcoma development. The increased expression of
Bcl-xL is most likely achieved by specific binding of GR to
the identified GRE-like sequences within the Bcl-x promoter. In fact,
these sequences also confer hormone responsiveness to a heterologous
promoter in COS-7 cells, indicating that the presence of
ligand-activated GR is sufficient to induce Bcl-x transcription in
other cell types. Furthermore, specific activation of the Bcl-x
promoter by dexamethasone requires the cooperation of the various GREs
identified, because deletion or mutation of these elements results in a
reduction in responsiveness. This observation supports the prediction
made by Nordeen and colleagues (35) that most natural response elements are suboptimal elements and that cooperativity among these individually weak sites contributes to the inducibility of the promoter. Because Bcl-x expression is higher in untreated NF than in untreated FS cells,
one possibility is that the Bcl-x promoter is repressed in FS cells and
that dexamethasone treatment results in de-repression. Although this
may be the case, the reduced expression of Bcl-x in FS cells is
unlikely to involve GR binding to GREs in the Bcl-x promoter, because
their mutation does not affect transcription in the absence of hormone.
Importantly, Bcl-xL is responsible for the ability of
dexamethasone to inhibit apoptosis in fibrosarcoma cells, as shown
by ectopic expression of Bcl-xL. As expression of
Bcl-xL decreases during fibrosarcoma development in the
absence of glucocorticoids, increasing up-regulation of
Bcl-xL expression by dexamethasone serves to maintain
Bcl-xL levels during tumor development, and thus
apoptosis remains approximately constant. In effect, Bcl-xL expression becomes increasingly dependent on glucocorticoids during fibrosarcoma development. Increased Bcl-xL expression and
reduced apoptosis correlate in FS cells with decreased caspase-3, but not caspase-1, activity in response to glucocorticoids. Such a correlation has also been reported by Messmer et al. (49).
Furthermore, levels of apoptosis in hormone-treated FS cells are
comparable with those in earlier cell types, despite the presence of
higher caspase-1 activity. These data indicate that caspase-3 is likely to play a more important role than caspase-1 in determining apoptosis in FS cells.
It may be noteworthy that we also observed dexamethasone regulation of
Bcl-x expression in both normal and transformed human mammary
epithelial cells. Apoptosis of immortalized mouse mammary epithelial
cells can be decreased by dexamethasone treatment, associated with
increased Bcl-x mRNA expression, within 2 h of hormone
treatment (8). Intriguingly, an increase in Bcl-xL protein
expression following dexamethasone treatment was readily detectable in
transformed SK-BR-3 breast cancer cells but was lower in normal
epithelial cells (data not shown). This represents a parallel situation
to that observed during fibrosarcoma progression, where the ability of
hormone to induce Bcl-xL expression increases during tumor
development; it may reflect the ability of normal cells to limit, or of
tumor cells to enhance, regulation of gene expression by specific
steroid hormone receptors as has been suggested previously (22). Such a
phenomenon may be a result of increased expression of steroid hormone
receptor co-factors during tumor development, and indeed co-activators
such as E6-AP and AIB1 are overexpressed in certain tumor types (50,
51).
It is interesting to consider that the discovery of regulation of
Bcl-xL expression by glucocorticoids during tumorigenesis may have clinical relevance. Glucocorticoids such as dexamethasone are
used as anti-emetics in the treatment of several cancers. One study has
identified a strong negative correlation between Bcl-xL
expression and sensitivity to a wide variety of cytotoxic agents in 60 cancer cell lines (18). Our results suggest that Bcl-xL
expression (and thus cell survival) may be promoted by dexamethasone.
Therefore, glucocorticoid treatment of tumors may reduce the cytotoxic
effects of chemotherapy on tumor cells and may be contraindicated in
those tumors with highly inducible Bcl-xL expression. A
similar situation may exist in malignant gliomas, where
glucocorticoids have been reported to interfere with chemotherapy response (52). Importantly, indications that expression of
Bcl-xL may be more glucocorticoid-dependent in
tumor cells than in normal cells suggest that glucocorticoid therapy
could have a certain degree of tumor specificity.
The enhanced expression of Bcl-xL by GR during fibrosarcoma
development reflects the unexpected transition in the transcriptional activity of GR and reveals an alternative mode of regulation for steroid hormone receptors. In summary, increasing GR transcriptional activity is likely to exert a protective effect by enabling the reduction of apoptosis at the tumor stage of fibrosarcoma development and, therefore, prolonging tumor cell survival.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3292 to
94) of the 5'-region of the murine Bcl-x promoter linked
to the luciferase reporter gene (23) was a kind gift of Gabriel
Nuñez (Ann Arbor, MI), and was designated Bcl-x(3.2). Serial
promoter deletion fragments were cloned into pGL2-basic (Promega) as
follows: Bcl-x(2.8) (
2829 to
94) as a BglII and
HindIII fragment; Bcl-x(0.6) (
679 to
94) by
SmaI and partial HindIII digestion; Bcl-x(0.2)
(
299 to
94) as an XmnI and HindIII fragment,
and Bcl-x(0.1) (
199 to 094) as a KpnI and
HindIII 110-bp fragment.
2963 to
2268.
This fragment was amplified by PCR using Bcl-x(3.2) as template and the
oligonucleotides A (5'-GTTTCCCAAAAGGATCCAATG-3') and C
(5'-AAATGCGGATCCTGACTGACTG-3'). The product was digested with
BamHI and inserted into TK109-luciferase. RE2
contains sequences from
2963 to
2826, which were amplified by PCR
using the oligonucleotides A and D (5'-CTGGTTATGTAGCTGTGGGCTGCC-3').
The PCR product was digested with BamHI and
BglII. RE3 contains sequences from
2349 to
2268 of the
Bcl-x promoter, which were amplified by PCR using the oligonucleotides
B (5'-AAGTGGATCCTCCTATGCTAC-3') and C. The product was digested with
BamHI.
-galactosidase expression vector 6RZ as internal control for
efficiency of transfection as described previously (22). After exposure
to the DNA/DEAE-dextran mixture, the cells were incubated for 36 h
in medium containing charcoal-stripped serum with or without 100 nM dexamethasone (Sigma). The GR antagonist RU 40555 was
kindly provided by Roussel-Uclaf (Romainville, France). Luciferase activity was measured according to the instructions of the
manufacturer (Promega).
-actin (Sigma), and
anti-tubulin (Sigma) were used as primary antibodies, followed by
incubation with the corresponding secondary antibody (horseradish
peroxidase-conjugated; Bio-Rad). Protein-antibody complexes were
visualized by an enhanced chemiluminescence immunoblotting detection
system (Amersham Biosciences).
2976 to
2730 from the transcription start site
(GenBankTM accession number AF 088904). As negative
control the primers 5'-TGGGCTGGCTCCCTGGTCAG-3' and
5'-CCTCGGTCACGGATCTTAGGC-3', which amplify a 112-bp fragment
corresponding to murine Bak sequence
960 to
848
(GenBankTM accession number Y 13232), were included in the
same PCR reactions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Stages of murine fibrosarcoma development
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Fig. 1.
Effects of dexamethasone
(Dex) on cell death during fibrosarcoma
development. A, the number of dead cells as a
percentage of total population of NF, MF, AF, and FS cells. Cells from
each stage of fibrosarcoma progression were plated at 2 × 104 cells/well 24 h prior to treatment with
(solid bars) or without (shaded bars) 100 nM dexamethasone. Values represent the mean percentage of
total cells counted in triplicate, within one representative experiment
(repeated three times). Error bars represent standard
deviation in all figures. B, dead FS cells on each day of
culture. C and D, caspase-1 and caspase-3 enzyme
activities in NF, MF, AF, and FS cells. Duplicate nonconfluent dishes
of cells were untreated or treated with dexamethasone for 24 h.
Values represent the average of three independent experiments. Caspase
activity is in absorbance units (×10 2).
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Fig. 2.
Expression of endogenous Bcl-2 family
proteins in MF, AF, and FS cells. Immunoblot analysis of
pro-apoptotic (Bax) (A) and anti-apoptotic (Bcl-2,
Bcl-xL) (B) from MF, AF, and FS cells untreated
( ) or treated (+) for 24 h with 100 nM dexamethasone
(Dex). Cell extracts were probed with antibodies against
various members of the Bcl-2 family (for details see "Experimental
Procedures").
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Fig. 3.
Bcl-xL protein
expression during fibrosarcoma progression following dexamethasone
(Dex) treatment. A, immunoblot
analysis of Bcl-xL protein from NF, MF, AF, and FS cells
untreated ( ) or treated (+) with 100 nM dexamethasone for
24 h. Cell extracts from each stage of fibrosarcoma progression
were probed with a rabbit antibody against Bcl-xL.
B, time course of Bcl-xL protein induction in FS
cells in response to dexamethasone for the indicated amount of time
(0-24 h). All cells were harvested 48 h after plating and were
growing exponentially.
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Fig. 4.
Regulation of Bcl-x transcription by
dexamethasone (Dex). A, cells from the
various stages of fibrosarcoma progression were untreated ( ) or
treated (+) with 100 nM dexamethasone for 4 h.
Northern blot analysis of Bcl-x mRNA levels using a 725-bp fragment
from the Bcl-xL cDNA (IMAGE 1395857) as probe.
The membrane was also probed for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). B, expression of the
Bcl-x(3.2) luciferase reporter construct in NF, MF, AF, and FS cells.
The cells were either untreated (black bars) or treated for
36 h with 100 nM dexamethasone (shaded
bars). Transcriptional activity of endogenous GR was analyzed. In
all transfection experiments, luciferase activity was normalized to the
-galactosidase activity of a co-transfected reporter. The values
presented are the mean of at least five different experiments.
Normalized luciferase activity units are ×103 in
all experiments. C and D, expression of
the Bcl-x(3.2) reporter in FS cells in response to increasing
concentrations of dexamethasone (C) or to various
hormone treatments, 100 nM dexamethasone, and/or the GR
antagonist RU 40555 (RU) (D).
-galactosidase expression plasmid to normalize for transfection efficiency, into cells from each
stage of fibrosarcoma progression. In the absence of dexamethasone, the
activity of the promoter decreased from NF to FS cells (Fig. 4B) suggesting that Bcl-xL protein expression
decreases during tumor development in the absence of dexamethasone,
primarily as a result of reduced Bcl-x transcription. Upon
dexamethasone treatment, promoter activity was strongly increased in FS
cells, indicating that glucocorticoids induce Bcl-x transcription (Fig.
4B). Various concentrations of dexamethasone were tested,
and concentrations as low as 10
10 M
significantly increased Bcl-x promoter activity (p < 0.005) (Fig. 4C).
199 to
94) contains a proximal weak glucocorticoid response
element (GRE). Although this fragment does not contain classical
consensus GRE sequences, it does contain putative binding sites for
factors known to interact with GR, such as C/EBP (33) and Oct-1 (34).
However, it appears that the major hormone-inducible promoter
sequences are located within
3292 to
679.
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Fig. 5.
Bcl-x promoter analysis. A,
Bcl-x promoter fragments. Dex, dexamethasone. B,
activity of the Bcl-x luciferase constructs shown in A in FS
cells. The values presented are the mean of four different transfection
experiments done in duplicates. Cells were untreated (black
bars) or treated for 36 h with 100 nM
dexamethasone (shaded bars). C, putative
dexamethasone-responsive sequences within the Bcl-x promoter.
D and E, activity of the Bcl-x constructs shown
in C in FS cells (D) and in COS-7 cells
(E) with conditions as described in B. F, triple mutant (mut) reporter resulting from
the mutation of 2 bp in each of the three GREs (P1,
P2, and P3) in the Bcl-x(3.2) reporter construct.
G, transcriptional activity of the wild type (wt)
and triple mutant Bcl-x(3.2) reporters (mut) with
conditions as described in B.
2944 and
2316 (Fig. 5C). To test whether these putative
GREs (or GRE-like elements) are sufficient to mediate hormone
induction, three fragments containing these sequences (RE1,
2963 to
2268, RE2,
2963 to
2826 and RE3,
2349 to
2268, Fig.
5C) were cloned into a luciferase reporter plasmid driven by
a TK109 promoter (TK109-luciferase). All three
reporter plasmids displayed a clear induction of transcription in
response to dexamethasone treatment (Fig. 5D). The largest fragment, RE1, which encompasses all four putative GREs,
displayed the strongest transcriptional activity, followed by RE2 and
RE3. Importantly, these sequences also conferred hormone responsiveness when co-transfected with a GR expression vector into COS-7 cells (Fig.
5E) although to a lesser extent than a reporter construct driven by a consensus GRE (TAT1, one copy of the GRE from the tyrosine
aminotransferase gene promoter in front of the TK109 promoter). A construct containing an unrelated sequence (consensus thyroid response element, TK) upstream of the
TK109-luciferase did not respond to dexamethasone. These
results confirm that dexamethasone-responsive sequences of the
Bcl-x promoter lie within (
2944 to
2826) and (
2330 to
2268).
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Fig. 6.
Gel retardation assays. in
vitro DNA binding of GR from nuclear extracts of
dexamethasone-treated FS cells. A, the various
32P end-labeled oligonucleotides used as probes
(P1-P3) are indicated at the top. Competition
reactions were performed using 50-fold excess of the corresponding
unlabeled oligonucleotide (specific (same as labeled) probe
(S)) (lanes c, f, k, and
n) or 200-fold excess of a nonspecific oligonucleotide
(N) (lanes d, h, l, and
o). B, a 32P end-labeled consensus
GRE-TAT oligonucleotide used as probe. Competition reactions with
50-fold excess of unlabeled oligonucleotides (P1-3,
lanes c-e, respectively, as well as GRE,
S, lane f) or nonspecific oligonucleotides
(N, lanes g-i). C, labeled
probes are indicated at the top). Competition reactions were
performed with unlabeled GRE (S, lanes c,
f, and i) or nonspecific oligonucleotide
(N, lanes d, g, and j). The
asterisk indicates the position of a nonspecific complex.
Lane a (in A-C), and lanes
e and i (in A) represent incubation in the
absence of extract.
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Fig. 7.
ChIP assay. Cells were untreated or were
incubated for 1 h with dexamethasone (Dex). Binding of
GR to the Bcl-x promoter in FS cells was determined in vivo
with the ChIP assay (lanes 7 and 8). As controls,
sample lysates were also incubated with an antibody against cyclin A
(lanes 5 and 6) or without antibody (lane
2), and PCR amplifications included primers designed to detect a
control segment from the endogenous Bak promoter that lacks functional
GREs. Lane 1 is the molecular weight marker;
lanes 3 and 4 represent input signals obtained
from 0.5% input chromatin, whereas ~3% of the immunoprecipitated
material was amplified. IP Ab, immunoprecipitation
antibody.
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Fig. 8.
Over-expression of Bcl-xL
in FS cells. A, FS cells were transfected with a
Bcl-xL expression vector or control vector and treated with
100 nM dexamethasone (Dex) for 48 h. Whole
cell extracts were analyzed for Bcl-xL protein expression.
B, number of dead cells in Bcl-xL-transfected FS
cell cultures. Values represent the mean of 12 independent counts
(×104). C, caspase-3 enzyme activity in
Bcl-xL-transfected FS cells. Caspase activity is given in
absorbance units (×10 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(43); thus it can be speculated that these proteins may inhibit Bcl-x transcription. Notably, the
angiogenic growth factor bFGF increases Bcl-x expression in epithelial
cells (44). Because the expression of bFGF becomes increasingly
extracellular at the AF stage of fibrosarcoma development (45), it is
possible that decreasing levels of intracellular bFGF contribute to
decreasing Bcl-xL expression in AF and FS cells. In
addition, the transcription factor AP-1 has been proposed to inhibit
Bcl-xL expression (46), and therefore the increased expression and activity of the AP-1 component proteins c-Jun and JunB
in AF and FS cells (22, 47) may also reduce Bcl-xL expression.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Gabriel Nuñez (University of Michigan Medical School, Ann Arbor) for providing the mouse Bcl-x(3.2) reporter, to Leanne Wiedemann (Stowers Institute for Medical Research, Kansas City, MO) for the human Bcl-xL cDNA, and to Roussel-Uclaf for RU 40555. We also thank Ramon Roca (Institute of Cancer Research, London) for help with various aspects of this work and Francesca Buffa (Institute of Cancer Research, London) for help with the statistical analysis. We very much appreciate Alan Ashworth and Pascal Meier for their critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Leopold Muller Trust and the Institute of Cancer Research.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.
§ Present address: Medical Molecular Biology Unit, Institute of Child Health, 30 Guilford St., London WC1N 1EH, United Kingdom.
To whom correspondence should be addressed. Tel.:
44-207-970-6001; Fax: 44-207-352-5241; E-mail: mariav@icr.ac.uk.
Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M301812200
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ABBREVIATIONS |
---|
The abbreviations used are: GR, glucocorticoid receptor; ChIP, chromatin immunoprecipitation; NF, normal fibroblast; MF, mild fibromatosis; AF, aggressive fibromatosis; FS, fibrosarcoma; GRE, glucocorticoid response element; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bFGF, basic fibroblast growth factor; TK, thymidine kinase.
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