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INTRODUCTION |
Cell death is a physiological process that plays a major role in
the maintenance of tissue homeostasis. Deregulation of the delicate
balance of genes that encode proteins that either induce or inhibit
cell death, contributes to the development of human neoplasia.
The bcl-2 gene was first discovered because of its
involvement in the t(14;18) chromosomal translocations commonly found
in lymphomas (1). Bcl-2 expression can also be altered in a variety of
other cancers through various mechanisms, including loss of p53 tumor
suppressor, which down-regulates Bcl-2 expression in some tissues
(reviewed in Refs. 2 and 3). Bcl-2 functions as an inhibitor of cell
death, thus contributing to cell expansion by inhibiting physiological
cell turnover. Bcl-2 has also been shown to block cell death induced by
a number of cytotoxic drugs as well as
-irradiation and is therefore
believed to contribute to resistance to anticancer therapies (reviewed
in Refs. 2 and 3). In vertebrates, several Bcl-2 homologues have been identified, some of which function as inhibitors (Bcl-XL,
Mcl-1, and A1), and others as promoters of cell death
(Bcl-XS, Bax, Bak, and BAD). These Bcl-2 family members are
characterized by their ability to interact and form homo- and
heterodimers (4). The relative abundance of anti- and proapoptotic
Bcl-2 homologues determines the sensitivity of cells to apoptotic
signals (reviewed in Refs. 2 and 5), but the mechanisms by which Bcl-2
inhibits cell death remain to be fully elucidated.
It has been proposed that Bcl-2 may inhibit cell death by interfering
with the function of proapoptotic Bcl-2 homologues, by repressing the
release of cytochrome c from mitochondria, by the
sequestration of caspase activators, such as Apaf-1, by interfering with the production of free radicals by cytotoxic agents, or by regulating intracellular calcium homeostasis (for reviews, see Refs.
6-8). In addition, Bcl-2 has also been shown to bind and possibly
modulate the function of other mammalian proteins, including R-Ras (9),
Raf-1 (10), nuclear factor-
B (11), calcineurin (12), SMN (13), and
the p53-binding protein 53BP2 (14). Interestingly, the Bcl-2 protein
has a transmembrane C-terminal domain
(TM)1 that targets it to
intracellular membranes, most notably the outer membranes of
mitochondria and nuclei and the endoplasmic reticulum membranes
(15-17). Membrane anchorage appears to be required for optimal Bcl-2
function but may not be necessary for all of its antiapoptotic
functions (18, 19).
The tumor suppressor p53 functions primarily as a transcription factor
that controls genomic stability. The levels of p53 protein are normally
very low but rise dramatically when DNA damage has occurred (20, 21).
High levels of p53 protein cause cell cycle arrest, mostly in
G1, or apoptosis depending of the cellular context and the
gravity of the damage (22-24). The p53 gene is often deleted or
mutated in cancer cells (25, 26). This leads to increased
susceptibility to malignant transformation and blunted responses to DNA
damaging cytotoxic agents (reviewed in Ref. 27). The functional status
of p53 strongly correlates with its ability to bind specific DNA
sequences on target genes and to activate transcription. In addition,
p53 can repress transcription of some promoters lacking specific p53
binding sites, and it also possess some transcription-independent
functions that are probably relevant to DNA-repair responses (28, 29).
Among the genes that are activated by p53 are the
cyclin-dependent kinase (cdk) inhibitor waf-1/p21 (30), cyclin G (31), GADD45
(32), the p53 inhibitor mdm2 (33), and the
apoptosis-inducing genes bax (34) and KILLER/DR5 (35).
Interestingly, an inverse relationship has been found between mutated
p53 and Bcl-2 expression in some types of carcinomas (36-39). This
suggests that p53 and Bcl-2 may serve as inducer and repressor,
respectively, of the same apoptotic pathway. Studies with
bcl-2 transgenic mice and p53 knockout mice also
support this concept (40). In this regard, it is well known that Bcl-2 is a potent inhibitor of p53-induced apoptosis (41, 42). In addition,
in some cell systems, it has been demonstrated that Bcl-2 inhibits p53
translocation to the nucleus upon DNA damage (43) or when co-expressed
with particular proteins (44). Bcl-2 may also relieve p53-mediated
transcriptional repression (45, 46).
In this study, we report that high levels of Bcl-2 protein
significantly impair p53-dependent induction of the cdk
inhibitor p21CIP1/WAF1 after DNA damage in MCF7
breast cancer cells, without, however, blocking p53 nuclear import. In
agreement with these findings, we show that Bcl-2 strongly inhibits
p53-dependent transactivation of several promoters,
including bax, p21CIP1/waf-1,
mdm2, cyclin G, and GADD45 in
transient co-transfection assays, again without preventing p53 entry
into the nucleus. We also demonstrate that these inhibitory effects of
Bcl-2 require its anchorage in intracellular membranes. These findings
suggest that in some circumstances, Bcl-2 can block p53 function by
inhibiting its transcriptional activity. Thus, Bcl-2 may be capable of
interfering with p53 at several levels, probably depending on cell context.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The plasmid pGL3-bax contains the p53
response element and TATA box from the BAX promoter cloned
into BglII/HindIII sites of the pGL3enhancer
reporter plasmid (Promega, Inc). The plasmid pCMV-Bcl-2(
TM) was
generated by introducing a stop codon and XhoI site at
position 562 of Bcl-2 cDNA (47) and then subcloning the
HindIII/XhoI-digested fragment into
HindIII/XbaI sites of pRc-CMV (Invitrogen,
Carlsbad, CA). pCMV-Bcl-2(ActA) and pCMV-Bcl-2(Cb5) were generated by
subcloning a HindIII-digested fragment from pSPUTK-Bcl-2(ActA) and -Bcl-2(Cyt5) (48) into HindIII sites of pcDNA3 (Invitrogen, CA). All plasmids were sequenced to confirm identity and correct orientation. Other plasmids employed here were
previously described, including pCMV-Bcl-2 (49), pCMV-p53 (50), WWP-luc
containing a 2-kb fragment of the waf-1 promoter (51),
mdm2-luc containing 350 base pairs of the human mdm2
promoter (52), and pGL3-cyclinG-luc containing a 1.48-kb fragment of the cyclin G promoter (53). The control plasmid
pGL3promoter, carrying the SV-40 early region promoter, and
pGL3enhancer were purchased from Promega.
Cell Culture--
The human breast cancer cell line MCF7,
transformed human embryonic kidney 293 cells, 293T, containing the
large T antigen, and 293-EBNA cells, carrying the Epstain-Barr virus
EBNA-1, were obtained from the American Type Culture Collection
(Manassas, VA). Cells were maintained in a humidified atmosphere with
5% CO2 in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 1 mM glutamine,
100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technology,
Inc.). In some experiments, MCF7 cells were treated with 0.4 µM doxorubicin (Sigma).
Generation of MCF7-Bcl-2 Stable Transfectants--
MCF7 breast
cancer cells (3 × 105) were plated in 60-mm dishes
and grown overnight. For transfection, plasmid DNA (2 µg) was diluted
into 0.1 ml of Opti-MEM medium (Life Technologies) and combined with 5 µl of LipofectAMINE (Life Technologies) in 0.1 ml of Opti-MEM. After
incubation for 20 min, 0.8 ml of Opti-MEM was added, and the mixtures
were overlaid onto cells. After 4 h, 1 ml of Opti-MEM containing
20% fetal calf serum was added to the cultures. The next day, medium
was replaced with complete Dulbecco's modified Eagle's medium.
Transformants were selected by growth for 14 days in complete medium
containing 0.5 mg/ml G418 (Life Technologies). Polyclonal populations
were grown and assayed for stable transgene expression by immunoblotting.
Transfections and Enzyme Assays--
Cells at 60% confluency
were transfected by a standard calcium phosphate precipitate method.
The total amount of plasmid DNA used was normalized to 2 µg/well and
7 µg/plate for transfections in 12-well and 6-cm plates,
respectively, by the addition of empty plasmid. For reporter gene
assays, 0.2 µg of the
-galactosidase expression plasmid
pCMV-
-galactosidase was co-transfected with the luciferase reporter
gene to normalize for variations in transfection efficiency. Cells were
exposed to the precipitate for 5 h at 37 °C. For MCF7 cells, a
glycerol shock was applied. Cells were exposed to 15% glycerol in HBS
buffer (25 mM HEPES, pH 7.05, 0.75 mM
Na2HPO4, 140 mM NaCl) for 4 min.
The glycerol was removed by washing three times with PBS and
replacement with fresh medium. For 293 cells, the medium was replaced
without applying a shock. MCF7 stable cell lines were transfected using
the lipofection method described above. Cell extracts were prepared
48 h after transfection. For reporter gene experiments, cells
lysates were made as described by the manufacturer (Promega) and
assayed for luciferase and
-galactosidase activity. All transfection
experiments were carried out in triplicate, repeated at least three
times, and normalized for
-galactosidase activity.
Cell Extracts and Subcellular Fractionation--
For gene
expression experiments, cells were washed two times in PBS and lysed in
RIPA buffer containing protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 0.28 TIU/ml aprotinin, 50 µg/ml
leupeptin, 1 µM benzamidine, 0.7 µg/ml pepstatin). For
protein localization experiments, nuclear and nonnuclear
fractions were prepared according to the method of Schreiber
et al. (54). Briefly, cells were collected and washed two
times with ice-cold PBS. Cell pellets were resuspended in Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 2.5 mM dithiothreitol,
protease inhibitors) and left on ice for 15 min prior addition of
Nonidet P-40 to a final concentration of 0.6% (v/v). After
centrifugation, supernatants were collected, and the nuclear pellets
were washed two times in the same buffer. Pellets were finally
resuspended in Buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 25% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, 2.5 mM dithiothreitol, protease
inhibitors) and vigorously shaken for 10 min, and the postnuclear
supernatants were cleared by centrifugation. Fractions were normalized
for protein content based on the bicinchoninic acid method (Pierce) prior to the SDS-PAGE/immunoblot assay.
Immunoblotting--
Aliquots containing 25 or 35 µg of protein
were subjected to SDS-PAGE using 12% gels, followed by electrotransfer
to Immobilon-P transfer membranes (Millipore Corporation, Bedford, MA).
Immunodetection was accomplished using 1:1000 (v/v) of anti-p53
(monoclonal Ab-1 and Ab-2 1:1, Oncogene Research Products, Cambridge,
MA), anti-p21/WAF-1 (monoclonal Ab, PharMingen, La Jolla, CA) or
anti-Bcl-2 (rabbit antiserum 1701) antibodies (54), followed by
horseradish peroxidase-conjugated secondary antibody (Amersham
Pharmacia Biotech). Detection was performed using the ECL detection
method (Amersham Pharmacia Biotech).
Immunofluorescence Microscopy--
MCF7 stable transfectants
were seeded into polylysine-coated 8-well chambers (Nalge-Nunc
International) and grown overnight at 37 °C. Cells were then
co-cultured in 0.4 µM doxorubicin for 20 h further.
The cells were then washed, fixed with 3.7% formaldehyde in PBS for 10 min, rinsed with PBS, permeabilized with 0.15% Triton X-100 in PBS for
10 min, and blocked in a solution of 3% BSA in PBS/0.1% Triton X-100
for 1 h. The cells were then incubated with anti-p53 (1:50
monoclonal Ab-2, Oncogene Science Inc., San Diego CA), anti-p21 (1:50
monoclonal Ab-1, Oncogene Science Inc.) or anti-Bcl-2 (1:100
rabbit antiserum 1701) antibodies for 1 h. The slides were washed
three times for 5 min in PBS and incubated with a 1:50 dilution (v/v)
of fluorescein isothiocyanate-conjugated goat anti-mouse or
TRITC-conjugated swine anti-rabbit immunoglobulin (Dako, Inc.). The
washing step was repeated before applying coverslips using
Vectashield® mounting medium (Vector Laboratories, Inc.).
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RESULTS |
Bcl-2 Inhibits p53-dependent Transactivation of Target
Genes--
To investigate the influence of Bcl-2 on the
transactivation activity of p53, a co-transfection reporter gene assay
was carried out. For these experiments, MCF7 breast cancer and human
embryonic kidney 293T cells were transiently co-transfected with
plasmids encoding wild-type p53 and Bcl-2, together with a reporter
plasmid in which the luciferase gene is driven by a fragment of the
human bax gene promoter containing its p53-response element
and TATA box. As shown in Fig. 1,
A and B, expression of wild-type p53 induced
luciferase expression in both cell lines. Co-transfection of Bcl-2
strongly inhibited p53 transcriptional activity in a concentration-dependent manner in both cell lines, whereas
co-transfection of a control protein (BAG-1L) did not affect p53
transactivation. To preliminarily explore the mechanisms responsible
for the observed suppression of p53 transcriptional activity by Bcl-2,
nuclear and cytoplasmic extracts were prepared from 293T transfected
cells and tested for expression of p53 and Bcl-2 by immunoblotting. As
shown in Fig. 1C, Bcl-2 had no obvious effects on total p53 protein levels. In addition, Bcl-2 also did not inhibit p53
accumulation in the nucleus, as has been reported in some but not all
types of cells (41, 43-55). Tubulin staining was used to confirm the accuracy of the nuclear extraction procedure and to confirm loading of
equivalent amounts of the samples. Similar results were obtained in
MCF7 cells (data not shown). Taken together, these data indicate that
Bcl-2 not only inhibits downstream events in p53-mediated apoptosis
but, at least in certain cell lines, it also blocks p53 transcriptional
activity.

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Fig. 1.
Concentration-dependent
repression of p53 transcriptional activity by Bcl-2. 293T
embryonic kidney (A) and MCF7 breast cancer cells
(B) were transiently transfected in 12- and 6-well plates,
respectively, with a fixed amount of p53 (A, 0.5 µg;
B, 1.5 µg), -galactosidase (A, 0.3 µg;
B, 1.5 µg), and bax promoter luciferase
(A, 0.5 µg; B, 1.5 µg) expression plasmids
along with increasing amounts (A, 0-0.5 µg; B,
0.15-2 µg) of Bcl-2 or control plasmids. Two days after
transfection, reporter gene activities were measured and expressed as a
percentage of the average activity in the absence of Bcl-2 plasmid. In
contrast to the T-antigen expressing 293 cells, p53 strongly induced
apoptosis in the absence of Bcl-2 in MCF7 cells. The transcriptional
activity under these conditions could therefore not be determined in
these cells. C, Bcl-2 and p53 expression in transiently
transfected 293T cells. Two days after transfection with 0.5 µg of
p53, 0.4 µg of Bcl-2, and 1.6 µg of pRc-CMV, 293T cells were
collected, and cytosolic (C) and nuclear (N)
extracts were prepared. SDS-PAGE/immunoblot analysis was conducted
using 25 µg of total protein per lane and antibodies specific for
p53, Bcl-2 and tubulin.
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Bcl-2 Suppresses p53-mediated Transactivation of Multiple
p53-responsive Promoters--
To assess whether Bcl-2 affects the
ability of p53 to transactivate promoters other than the bax
gene promoter, similar experiments were performed using
luciferase-expressing reporter constructs driven by the p53-responsive
p21CIP1/WAF1, mdm2, or cyclin
G promoters. As shown in Fig. 2, as
with the bax promoter, Bcl-2 inhibited the
p53-dependent transcriptional activity of all promoters,
without, however, affecting their basal levels of activity. Under these
same conditions, Bcl-2 did not impair the transactivation of the
p53-independent SV40 early region promoter used as a control (Fig. 2).
Thus, Bcl-2 specifically inhibits p53-mediated transactivation of
target genes, apparently without significantly affecting
transactivation by other transcription factors.

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Fig. 2.
Bcl-2 inhibits p53-mediated transactivation
of multiple p53-responsive promoters. 293T cells were transiently
transfected with p53, Bcl-2, and -galactosidase expression plasmids
along with bax-luc, cyclinG-luc, wwp-luc, mdm2-luc, or pGL3-promoter
reporter genes. Luciferase activity was measured 2 days after
transfection. Results are expressed as luciferase relative to
-galactosidase enzymatic activity (n = 3).
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Inhibitory Effect of Bcl-2 Depends on Membrane Anchoring--
The
C-terminal domain of Bcl-2 contains a stretch of hydrophobic amino
acids that anchors the protein into cellular membranes (56, 57, 58).
Previously, Bcl-2 has been found in association with mitochondrial
membranes, the nuclear envelope and the endoplasmic reticulum (15-17).
To assess the importance of the subcellular localization of Bcl-2 for
the observed inhibitory effect on p53 transactivation, we compared p53
transcriptional activity in 293-EBNA cells transiently transfected with
p53 and either wild-type Bcl-2 or a Bcl-2 mutant, which lacks the
C-terminal TM domain. Interestingly, unlike Bcl-2, co-expression of
Bcl-2(
TM) with p53 did not interfere with p53-mediated
transactivation. In fact, this cytoplasmically distributed Bcl-2
deletion mutant slightly stimulated p53 activity, possibly by competing
with endogenous Bcl-2 protein. Immunoblot analysis confirmed that the
failure of Bcl-2(
TM) to suppress p53 was not attributable to lower
levels of expression of the truncated protein compared with wild-type
p53 (Fig. 3B).

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Fig. 3.
Membrane anchoring is required for
Bcl-2-mediated suppression of p53 transactivation. A,
293-EBNA cells were plated into 12-well plates and transiently
transfected with 0.5 µg of p53, 0.5 µg of bax-luc, and 0.2 µg of
-galactosidase expression plasmids, along with increasing amounts
(0.05-0.5 µg) of plasmids encoding wild-type Bcl-2, Bcl-2( TM),
Bcl-2(ActA), or Bcl-2(Cb5). Reporter gene activities were measured two
days after transfection and expressed as a percentage of the activity
in the absence of Bcl-2 proteins (mean ± SD; n = 4). B, wild-type Bcl-2 and Bcl-2-chimeric protein expression
in transiently transfected 293-EBNA cells was determined by
immunoblotting. Two days after transfection with 0.5 µg of p53, 1.6 µg of pRc-CMV, and 0.4 µg of Bcl-2 or Bcl-2-mutant expression
plasmids, cell lysates were prepared and SDS-PAGE/immunoblot analysis
was conducted using 25 µg of total protein per lane and antibodies
specific for Bcl-2.
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The inability of the Bcl-2(
TM) mutant to suppress p53
transcriptional activity could be due either to a specific requirement for the TM domain of Bcl-2 or because of a need for membrane anchoring. To distinguish between these two possibilities, we examined the effects
on p53 of Bcl-2 chimerical proteins in which the normal C-terminal TM
domain was substituted with membrane-anchoring domains from proteins
that reside in either the mitochondrial outer membrane (ActA) or the
endoplasmic reticulum (cytochrome b5 (Cb5)) (48). Similar to wild-type
Bcl-2, plasmids encoding either Bcl-2(ActA) or Bcl-2(Cb5) proteins
caused a concentration-dependent suppression of
p53-mediated gene transcription (Fig. 3A). The
mitochondria-targeting Bcl-2(ActA) protein was somewhat more potent
that the endoplasmic reticulum-targeting Bcl-2(Cb5) protein. Comparable
levels of expression of the wild-type and mutant proteins were
demonstrated by immunoblot analysis (Fig. 3B). Again, p53
protein production and nuclear localization were unaffected (data not
shown). These observations indicate that the inhibitory effect of Bcl-2
on p53 transactivation requires its insertion into membranes.
Bcl-2 Inhibits p53-dependent p21/WAF-1 Up-regulation
Following DNA Damage--
The role of Bcl-2 in p53-mediated
transactivation of target genes induced following DNA damage was
investigated using MCF7 breast cancer cells that had been stably
transfected with plasmids encoding Bcl-2, Bcl-2(
TM) or no inserted
cDNA (neo-control). Polyclonal cell populations expressing
comparable levels of ectopic proteins were chosen for experiments (Fig.
4). First the effects of DNA damage on
p53 protein levels were examined. Cells were exposed to 0.4 µM doxorubicin, and samples were harvested at different times. Nuclear and cytoplasmic extracts were prepared from each sample
and subjected to immunoblot analysis for p53 expression. No p53 protein
could be detected before cytotoxic treatment (data not shown). In
contrast, as shown in Fig. 4A, p53 nuclear expression was
strongly induced 24 h after doxorubicin treatment in MCF7 transfectants expressing either wild-type or mutant Bcl-2 protein, as
well as in MCF7 control transfectants expressing only the neomycin resistance gene. Thus, as previously observed in transient transfection experiments (see above), the presence of high levels of Bcl-2 proteins
did not interfere with p53 accumulation in the nucleus. Similar results
were obtained for cells exposed to UV radiation (data not shown).

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Fig. 4.
Bcl-2 represses p21/WAF-1 induction in MCF7
cells following exposure to DNA damaging agents. Human MCF7 breast
carcinoma cells were stably transfected with plasmids either encoding
wild-type Bcl-2 (MCF7-Bcl-2), encoding Bcl-2 lacking the TM domain
(MCF7-Bcl-2( TM)), or carrying the neo-resistance gene alone
(MCF7-Neo). Polyclonal populations expressing comparable levels of
Bcl-2 and Bcl-2( TM) were chosen for subsequent experiments.
A, cells were treated with 0.4 µM doxorubicin.
Cytosolic (Cyto) and nuclear (Nuclei) extracts
were prepared 24 h later for analysis by immunoblotting using
anti-p53 antibodies. B, cells were transiently cotransfected
with a p53-responsive WWP-luciferase plasmid containing a 2-kb fragment
of the p21/WAF-1 promoter and a -galactosidase expression
plasmid used to normalize for transfection efficiency. One day after
transfection, cells were treated with 0.4 µM doxorubicin
or 10 J/m2 UV radiation. Cell extracts were prepared
24 h later and assayed for luciferase and -galactosidase enzyme
activities. Results are expressed as a percentage of the maximal
promoter activity measured in neo-control cells (mean ± S.D.;
n = 3). C, Bcl-2 and p21 protein levels were
assessed using lysates from stably transfected MCF7 cells before and
after 24 h doxorubicin treatment. SDS-PAGE/immunoblot analysis was
conducted using 35 µg of total protein per lane and antibodies
specific for Bcl-2 and p21.
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Next, to assess whether Bcl-2 also inhibits p53 transcriptional
activity in the setting of DNA damage, MCF7 cells that stably expressed
Bcl-2 or Bcl-2(
TM) were co-transfected with a p53-responsive luciferase reporter gene plasmid (wwp-luc), which contains a 2-kb fragment of the p21/waf-1 promoter. A
-galactosidase
expression plasmid was co-transfected to normalize for transfection
efficiency. One day later, cells were treated with 0.4 µM
doxorubicin or 10 J/m2 UV radiation. Cell extracts were
prepared 24 h later and assayed for luciferase and
-galactosidase enzyme activities. As shown in Fig. 4B,
both doxorubicin and UV irradiation resulted in strong induction of the
p21/waf-1 promoter in MCF7-Neo control cells. In contrast,
transactivation of the p21/waf-1 promoter was reduced by
~70% in cells expressing high levels of Bcl-2. Unlike wild-type Bcl-2, MCF7 cells expressing Bcl-2(
TM) did not exhibit defects in
their p53-mediated transactivation of the p21/waf-1 gene
promoter after cytotoxic treatment (Fig. 4B). Cultures of
UV-irradiated MCF7-Bcl-2 cells also became confluent, suggesting that
cell proliferation was not inhibited despite treatment with DNA
damaging agents. In contrast, cell cycle arrest was observed for
Bcl-2(
TM) expressing cells (not shown).
To test whether the observed inhibition of the p53-mediated
transactivation of a plasmid-borne p21/waf-1 promoter also
applies to the endogenous p21/waf-1 gene, we examined the
relative levels of p21 protein induction by immunoblot analysis of
whole cell lysates prepared from MCF7 cells after exposure to
DNA-damaging agents. As shown (Fig. 4C), p21 protein
induction following treatment with doxorubicin was completely
suppressed in MCF7-Bcl-2 but not in MCF7-Bcl2(
TM) cells. Fig.
4C also presents immunoblot analysis of the Bcl-2 and
Bcl-2(
TM) proteins in stably transfected MCF7 cells, demonstrating
that these two proteins were produced at comparable levels, thus
excluding differences in their expression as a trivial explanation for
the failure of Bcl-2(
TM) to suppress p21 protein induction.
Finally, similar observations were made by immunofluorescence
microscopy, using antibodies specific for the Bcl-2, p53 or p21/WAF-1
proteins. As shown in Fig. 5A,
the wild-type Bcl-2 protein was located in the nuclear envelope and
perinuclear membranes, in a patter similar to that reported previously
(15-17). In contrast, Bcl-2(
TM) was present diffusely through the
cytosol and nucleus of MCF7 cells (Fig. 5B). Doxorubicin
treatment resulted in marked up-regulation of p53 protein
immunostaining in both MCF7-Bcl-2 and MCF7-Bcl-2(
TM) cells. In both
lines, most of the p53 immunoreactivity was found in the nuclei of
these cells (Fig. 5, C and D). Although p53
protein accumulated in the nuclei of doxorubicin-treated MCF7-Bcl-2 cells, expression of the p21/WAF-1 protein was not induced (Fig. 5E). In contrast, striking induction of nuclear p21/WAF-1
immunostaining was observed in MCF7-Bcl-2(
TM) cells (Fig.
5F). Thus, in agreement with the results obtained in
transient transfection reporter gene assays, overexpression of Bcl-2
strongly reduced the induction of the p53 target gene
p21/waf-1 after DNA damage, and this effect was again
strictly dependent on the presence of the C-terminal membrane-anchoring
domain of Bcl-2.

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Fig. 5.
Bcl-2 but not
Bcl-2( TM) inhibits p53-dependent
p21/WAF-1 protein induction following doxorubicin treatment.
MCF7-Bcl-2 (A, C, and E) and MCF7-Bcl-2( TM)
(B, D, and F) stable transfectants were treated
with 0.4 µM doxorubicin for 24 h. Cells were then
fixed, permeabilized, and immunostained with antibodies specific for
Bcl-2 (A and B), p53 (C and
D) and p21 (E and F). Antibodies were
detected by indirect immunofluorescence microscopy as described under
"Experimental Procedures." Immunostaining of MCF7-Neo cells is not
reported because of the high mortality of these cells when seeded into
polylysine-coated 8-well chambers.
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It is interesting to note that whereas MCF7-Neo cells died within 2 days after treatment, Bcl-2(
TM) and Bcl-2 transfectants survived for
about 4 and over 7 days, respectively (data not shown). Thus, despite
the inability of Bcl-2(
TM) to suppress p53-mediated transcriptional
activity, it can apparently interfere with some downstream steps in the
p53 pathway for apoptosis.
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DISCUSSION |
The Bcl-2 oncoprotein is known to arrest p53-mediated
apoptosis in many cell types by mechanisms that are still not fully elucidated. It has been demonstrated that Bcl-2 inhibits downstream apoptotic events, such as the release of cytochrome c from
the mitochondria and subsequent activation of caspases (59, 60). However, Bcl-2 can also interfere with the activation of some transcription factors such as NFAT (12) and NF-
B (11), promoting us
to explore whether Bcl-2 is also capable of regulating upstream events
in the p53 pathway. The data provided here indicate that overexpression
of Bcl-2 can suppress p53-mediated transactivation of p53 target genes.
This effect was observed both in transient co-transfection reporter
gene assays in which p53 was expressed from plasmids and in experiments
in which endogenous p53 protein was activated by DNA-damaging agents.
Thus, in addition to interfering with apoptotic events downstream of
p53 target genes, Bcl-2 appears to be capable of suppressing an
upstream step required for p53 function as a transcription factor.
We observed that anchoring of Bcl-2 to membranes is a requirement for
its inhibitory effect on p53 transcriptional activity. Targeting of
Bcl-2 to specific membrane compartments, such as endoplasmic reticulum
and mitochondria, did not significantly alter the ability of Bcl-2 to
inhibit p53. These observations are in agreement with the idea that at
least some of the functions of Bcl-2 can be attributed to its ability
to sequester cytosolic proteins at membranes (reviewed in Ref. 6). For
instance, Bcl-2 was shown to inhibit NFAT-4 transcription factor
signaling through sequestration of calcineurin, a phosphatase required
for its dephosphorylation and subsequent nuclear translocation (12). A
mutant of Bcl-2 lacking the membrane-anchoring domain still bound
calcineurin but did not sequester it at membranes and did not suppress
signaling through NFAT-4. Other reports have similarly described the
recruitment of cytosolic proteins to internal membranes by
overexpression of Bcl-2, including the protein kinase Raf1 (10) and the
chaperone regulator BAG-1 (61).
The presence of the C-terminal membrane-anchoring domain has been
reported to enhance the anti-apoptotic activity of Bcl-2 in most
cellular contexts, but it may not be absolutely required at least for
some of its functions (18, 19). In this regard, our observation that
Bcl-2(
TM) had no inhibitory effect on p53 transactivation of target
genes, but did partially suppress p53-induced apoptosis, indicates that
some of the cell death protective functions of Bcl-2 are independent of
its membrane insertion. These observations may be relevant to other
reports that have demonstrated that targeting of Bcl-2 to different
locations results in a preferential protection against distinct
apoptotic stimuli (48).
In contrast to previous reports that examined a Myc-transformed
erythroleukemia or prostate cancer cell lines (43, 44), in MCF7 and 293 cells, Bcl-2 did not inhibit the accumulation of p53 in the nucleus.
Similarly, nuclear translocation of p53 is reportedly normal in a
variety of other types of cell lines despite overexpression of Bcl-2
(40-42, 46, 57, 62). We therefore speculate that Bcl-2 reduces the
capacity of p53 to transactivate genes by regulating the activity of
p53 itself or by affecting transcriptional coactivators required for
p53 function. For instance, Bcl-2 might directly inactivate p53 by
regulating posttranslational modifications required for its function as
a transcription factor. In this regard, p53 has been shown to be
phosphorylated at multiple sites by different kinases, such as
DNA-activated kinases (63), the protein kinases CKI and CK II (64, 65),
cyclin-dependent kinases (66, 67), stress-activated kinases
(68), mitogen-activated protein kinases (69), Raf-1 (70), and PKC (71).
Phosphorylation status regulates p53 conformation and ability to
transactivate target genes (72). As mentioned above, Bcl-2 interacts
with some kinases and phosphatases, regulating their intracellular distribution. It is therefore conceivable that Bcl-2 indirectly regulates p53 function by sequestration of a kinase or phosphatase that
phosphorylates or dephosphorylates p53. Alternatively, Bcl-2 might
interact with transcriptional coactivators required by p53, inhibiting
their entry into the nucleus. Interestingly, Bcl-2 and p53 have been
reported to compete for binding to 53BP2 (14), a protein of unknown
function that was initially discovered in a two-hybrid based screen for
proteins that bind wild-type but not mutant p53 (73).
Recently, it has been demonstrated that p53 requires p300/CBP-family
transcriptional co-activators for its transcriptional function (74).
Proteins that disrupt this interaction, such as the E1a oncoprotein,
reduce p53-dependent transactivation of the bax
and p21/waf-1 promoters. Interestingly, Chawla et
al. (75) recently reported that CBP contains a signal-regulated transcription activation domain that is controlled by nuclear calcium
and calcium/calmodulin-dependent protein kinase IV. Calcium has also been shown to regulate p53 transcriptional activity under some
circumstances (76-78). Because Bcl-2 has been reported to regulate
calcium efflux from the endoplasmic reticulum under some circumstances
(79) and prevent nuclear accumulation of calcium (80), it is
conceivable that Bcl-2 might inhibit p53 transcriptional activity by
hindering Ca2+-dependent CBP activation.
Whatever the mechanism responsible for the suppression of p53-mediated
transcription by Bcl-2, comparison with other reports suggests that the
overall importance of this action of Bcl-2 may be dependent on cell
context. For instance, in hematopoietic cells, Bcl-2 overexpression has
been reported to inhibit apoptosis induced by a temperature sensitive
mutant allele of p53 without impairing its induction of G1
cell cycle arrest (62). Thus, in these cells, Bcl-2 apparently does not
abrogate p53-mediated transcription of genes involved in inhibiting
cell proliferation such as p21/waf-1. In contrast, Upadhyay
et al. (81) reported that Bcl-2 overexpression can interfere
with p53-mediated p21/WAF-1 induction in MCF10A breast cancer cells.
Similarly, in some cell lines, it has been reported that Bcl-2 can
inhibit p53-mediated repression of some promoters under circumstances
where p53-mediated transactivation was unaffected (45, 46). We
therefore speculate that Bcl-2 may have several potential mechanisms
for interfering with specific steps of p53 activation or function and
that the ultimate effect of overexpressing Bcl-2 may be dictated by the
relative levels or differential expression of various p53 cofactors or
regulators that Bcl-2 can perturb within cells. In summary, although
much remains to be learned about the specific mechanisms involved, the
observation that Bcl-2 inhibits p53-dependent
transactivation of target genes might have important consequences for
the response of cancer cells to cytotoxic treatments and thus
contribute to the ability of Bcl-2 to promote chemo- and radioresistance.