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INTRODUCTION |
The BRCA2 gene was identified in 1996 as a
breast and ovarian cancer susceptibility gene (1, 2). The
BRCA2 gene encodes a 3,418-amino acid, cell cycle-regulated,
nuclear phosphoprotein (3, 4) that has been implicated in the response
to DNA damage. The evidence for a role in DNA repair came initially
from the observation that BRCA2 binds directly with RAD51 through the
exon 11-encoded BRC repeats (5, 6) and through an additional C-terminal
binding site in the mouse (7). This association with a protein involved
in meiotic and mitotic recombination and DNA double-stranded break
repair suggests a similar role for BRCA2. Further support for a role in
DNA repair comes from the observation that cells expressing a wild type
BRCA2 BRC4 domain show hypersensitivity to
-irradiation, an
inability to form RAD51 radiation-induced foci, and a failure of
radiation-induced G2/M, but not G1/S,
checkpoint control (8). Moreover, cells expressing mutant BRCA2 are
more sensitive to methyl methanesulfonate-induced DNA damage than cells expressing wild type BRCA2 (9). Animal models have also been used to
demonstrate an association between BRCA2 and the DNA damage response.
Brca2-null mouse embryos that do not survive past day 8 of embryogenesis are highly sensitive to
-irradiation (7). Similarly, mouse embryo fibroblast cell lines derived from
viable brca2
/
animals are
highly sensitive to DNA damage induced by a number of agents (10, 11).
BRCA2 has also been directly implicated in homologous recombination and
gene conversion using CAPAN-1 BRCA2 mutant cell lines and
homozygous mutant brca2 embryonic stem cells (12, 13) and in
transcription-coupled repair in response to 8-oxoguanine treatment
(14). Most recently, the C terminus of BRCA2 has been shown to bind
directly to single-stranded DNA and to promote strand transfer and
RAD51 loading onto DNA during homologous recombination (15).
The finding that BRCA2 was involved in the response to DNA damage led
to the hypothesis that DNA damage might result in the induction of
BRCA2 expression. However, the opposite has proven true. Specifically,
BRCA2 mRNA levels were significantly down-regulated in breast and
ovarian cancer cell lines after exposure to various DNA-damaging agents
including adriamycin (ADR)1
and camptothecin (16, 17). In an effort to characterize this response
to DNA damage better we investigated whether ADR and other DNA-damaging
agents affected BRCA2 promoter activity and BRCA2 mRNA
and protein levels. Here we show that ADR down-regulates the
BRCA2 promoter in a p53-dependent manner by
inhibiting binding of a USF transcription factor complex to the minimal
BRCA2 promoter. In addition, we demonstrate that ADR alters
BRCA2 mRNA and protein stability in a p53-dependent
manner, resulting in a significant reduction in BRCA2 mRNA and
protein levels. This suggests that BRCA2 levels are regulated tightly
in response to DNA damage.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The preparation of a series of pGL3 luciferase
reporter constructs (Promega) containing partial fragments of the
BRCA2 promoter has been described previously (18). A
pcDNA3.1 plasmid containing the wild type p53 cDNA (wtp53) was
provided by Wilma Lingle. CMV-USF1 and USF2-VP16 expression constructs
were provided by Michele Sawadogo and Howard Towle, respectively. An
R273L dominant negative p53 mutant construct (dnp53) was generated by
site-directed mutagenesis of the wtp53 construct using the QuikChange
kit (Qiagen) according to the manufacturer's instructions. PCR
primers for the site-directed mutagenesis were
5'-GGAACAGCTTTGAGGTGCTTGTTTGTGCCTGTCCTGG-3' (forward) and
5'-CCAGGACAGGCACAAACAAGCACCTCAAAGCTGTTCC-3'
(reverse). Plasmid DNA was isolated from colonies, and the
presence of the mutation was confirmed by DNA sequencing.
Cell Culture--
HCT116/p53+/+ and
HCT116/p53
/
cells were provided by Junjie Chen.
HCT116/p21+/+ and HCT116/p21
/
cells
were provided by Wafik El-Deiry. MCF7/pCMV and MCF7/E6 cells were
provided by Scott H. Kaufmann. All other cell lines were obtained from
the American Type Culture Collection. Human breast adenocarcinoma MCF7
cells, human colon carcinoma SW480 cells, HCT116/p53+/+
cells, HCT116/p53
/
, cells and human osteosarcoma U2OS
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% bovine calf serum (Hyclone), 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Human osteosarcoma p53-null Saos2 cells were propagated
in McCoy's 5A medium supplemented with 15% bovine calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. T47D, MCF7/pCMV, and
MCF7/E6 breast adenocarcinoma cells were maintained in RPMI 1640 with
10% bovine calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. MCF-10A cells were
maintained in MEGM (Clonetics).
Transient Transfection and Luciferase Reporter
Assays--
Transient transfections were performed in six-well plates
using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) with
0.5-2.0 µg of BRCA2 promoter luciferase reporter
construct and 0.1 µg of pRL-TK-Renilla luciferase
control vector (Promega). For ADR (Sigma) treatment experiments, cells
were transfected, grown for 24 h, and exposed to 0, 2.5, 5.0, or
10 µM ADR for 1 h in standard medium. The cells were
washed with serum-free medium and incubated at 37 °C in fresh
culture medium for another 24 h or the indicated time. Because
less than 10% of any cell type exhibited a toxic response to 5 µM ADR, drug toxicity had no effect on the outcome of the
study. In cotransfection experiments, cells were also transfected with
0.5 µg of wtp53, dnp53, USF1, USF2-VP16, or pcDNA3.1 control.
Protein lysates were prepared from the cells, and luciferase activities
were measured as described previously (18). Renilla
luciferase activity from a cotransfected pRL-TK control vector was used
for normalization. In some lower dose experiments cells were exposed to
0.7 µM ADR for 24 h, or the indicated time, in
standard medium and processed immediately.
Electrophoretic Mobility Shift Assays
(EMSAs)--
Double-stranded oligonucleotides containing bp
10 to
35 of the BRCA2 promoter and either a wild type or mutated
USF binding site (
20 to
13) were labeled with
[
-32P]ATP and used in EMSAs (18). Double-stranded DNA
probes were purified from the reaction mixture using a Bio-Gel P-100
column (Bio-Rad), incubated with whole cell extract from MCF7 cells, and separated on 5% polyacrylamide gels as described previously (18).
Supershift assays using anti-USF1 (Santa Cruz), and anti-USF2 (Santa
Cruz) antibodies were also performed as described previously (18).
Northern Blotting--
Cells were transiently transfected with
pcDNA3.1, wtp53, or dnp53 expression constructs or exposed to 5 µM ADR for 1 h. After further incubation for 24 h, poly(A)+ RNA was isolated. RNA samples (1.5 µg/lane)
were used for Northern blotting as described previously (18).
Western Blotting--
Cells were collected, washed with cold
phosphate-buffered saline twice, and then lysed at 4 °C for 1 h
in EBC buffer (0.5% Nonidet P-40, 120 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0), 1 mM
-mercaptoethanol, and Complete protease inhibitor mixture (Roche Molecular Biochemicals)). Equal aliquots of cell lysate
(30-100 µg/lane) were electrophoresed through 7 or 12% SDS-polyacrylamide gels after incubating for 15 min at 65 °C in 1×
loading buffer (100 mM Tris-HCl (pH 6.8), 10% glycerol,
0.01% bromphenol blue, 2% SDS, 100 mM dithiothreitol).
Proteins were then transferred to nitrocellulose membranes (Schleicher
& Schüll) and blocked in TBST with 5% bovine albumin (fraction
V) (ICN Biomedicals). Membranes were blotted with anti-BRCA2 (Ab2,
Oncogene Research), anti-USF1 (Santa Cruz) or anti-p53 (Santa Cruz)
antibodies and then incubated with horseradish peroxidase-conjugated
secondary antibodies (Amersham Biosciences). Signals were visualized
using the SuperSignal chemiluminescent detection system (Pierce).
Flow Cytometry--
MCF7 cells treated with ADR were washed with
phosphate-buffered saline, collected by centrifugation, and fixed in
ice-cold 95% ethanol at
20 °C for 12 h. The fixed cells were
permeabilized and stained with 0.05% Triton X-100, 100 µg/ml RNase
A, and 40 µg/ml propidium iodide at 37 °C for 30 min and analyzed
by flow cytometry. The data were processed with VERITY ModFit software, version 5.2, for DNA distribution analysis.
mRNA Stability--
MCF7 cells were incubated in the
presence or absence of 10 µg/ml
-amanitin (Sigma) and 5 µM ADR for 1 h. Medium was removed, and cells were
incubated further in the presence or absence of
-amanitin. Total RNA
was extracted from the cells by standard methods after different
periods of incubation. Total cDNA was prepared by oligo(dT) priming
of 1 µg of RNA template from each time point using the cDNA
preamplification kit (Invitrogen). The amount of BRCA2 mRNA present
in the cells at each time point was measured by semiquantitative
reverse transcription PCR using oligonucleotide primer pairs from the
5'- and 3'-ends of the BRCA2 cDNA. GAPDH was used as an internal
control. The BRCA2 forward primer was 5'-CAAGCAGATGATGTTTCCTGTCC; BRCA2
reverse, 5'-AGAACTAAGGGTGGGTGGTGTAGC; BRCA2 forward,
3'-GCAGTGAAGAATGCAGCAGA; BRCA2 reverse, 3'-CAATACGCAACTTCCACACG; GAPDH forward, CAACTACATGGTTTACATGTTC; GAPDH reverse,
GCCA- GTGGACTCCACGAC. PCR products were separated on 10%
polyacrylamide gels, stained with Sybr Green, and scanned on a PhosphorImager.
Protein Stability--
MCF7 cells were incubated with 20 µg/ml
cycloheximide and 5 µM ADR for 1 h. Medium was
removed, and cells were incubated further in the presence of
cycloheximide. Total protein was extracted from the cells by standard
methods after different periods of incubation, and Western blotting
with the BRCA2 Ab-2 antibody was performed. The intensities of Western
blot signals were quantitated by densitometry using NIH Image software,
version 1.62.
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RESULTS |
The BRCA2 Promoter Is Repressed by ADR--
ADR has recently been
shown to down-regulate BRCA2 mRNA levels in human breast cancer
cells such as MCF7 (16). To address whether the reduction in BRCA2
mRNA levels by ADR is dependent on BRCA2 promoter
regulation, MCF7 cells were transiently transfected with a
BRCA2 promoter reporter gene construct (pGL3Prom) (18) and
exposed to various concentrations of ADR for 1 h. After removal of
the drug and further propagation for 24 h, cell lysates were harvested for luciferase reporter assays. As shown in Fig.
1A, ADR treatment reduced
BRCA2 promoter activity in a dose-dependent manner, with 5 µM ADR down-regulating the promoter
activity by 85%. Similar effects of ADR treatment on the
BRCA2 promoter were detected in both MCF10A normal breast
epithelial cells (Fig. 1B) and U2OS osteosarcoma cells (data
not shown). Continuous exposure of MCF7 cells to lower doses of ADR
(0.7 µM) for 24 h also reduced promoter activity by
60% (Fig. 1C). Furthermore, treatment of MCF7 cells with 5 µM ADR for 1 h resulted in a gradual reduction in
promoter activity over time (Fig. 1D). Thus, ADR appears to repress the BRCA2 promoter in a dose- and
time-dependent fashion in both tumor and normal cell lines.
To confirm that the ADR-dependent repression was specific
to the BRCA2 promoter and was not a result of a generalized
effect on gene transcription, several other reporter constructs were
evaluated using luciferase assays. MCF7 cells were transiently
transfected with reporter constructs containing the Rous sarcoma
virus, CMV, nuclear factor-
B, and
-galactosidase promoters
and treated with 5 µM ADR. No repression of
these promoters was observed (data not shown), suggesting that
ADR specifically represses the BRCA2 promoter.

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Fig. 1.
Dose- and time-dependent
repression of BRCA2 promoter activity by ADR.
A, ADR inhibition of BRCA2 promoter activity in
MCF7 cancer cells is dose-dependent. Luciferase activity
was measured in MCF7 cells that were transfected with a
BRCA2 promoter reporter construct (pGL3Prom), exposed to
different concentrations of ADR for 1 h, and incubated for a
further 24 h. B, ADR inhibition of BRCA2
promoter activity in normal MCF10A cells is dose-dependent.
Luciferase activity in MCF10A cells was measured as described in
A. C, low dose ADR inhibits the BRCA2
promoter. Luciferase activity was measured in pGL3Prom-transfected MCF7
cells after exposure to 0.7 µM ADR for 24 h.
D, ADR-associated repression of the BRCA2
promoter is time-dependent. Luciferase activity was
measured in MCF7 cells that were transfected with pGL3Prom, exposed to
5.0 µM ADR for 1 h, and incubated further for
different times.
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Repression of the BRCA2 Promoter by ADR Is Dependent on
p53--
ADR is a potent DNA-damaging agent that induces p53
accumulation and p53-dependent cell death in
wtp53-expressing cell lines (19). Given that ADR regulates the
BRCA2 promoter and induces p53, we investigated whether ADR
down-regulates the BRCA2 promoter in a
p53-dependent manner. Initially, BRCA2 reporter assays were performed in p53-positive U2OS and p53-null Saos2 osteosarcoma cells in
an effort to address the role of p53 in regulation of the promoter
while minimizing tissue-specific differences. Treatment with either 5 µM ADR for 1 h or 0.7 µM ADR for
24 h did not affect BRCA2 promoter activity in Saos2
cells, whereas inhibition of the promoter was observed in U2OS cells
(Fig. 2A). Similarly, ADR
treatment of matched p53-null (HCT116/p53
/
) and p53
wild type (HCT116/p53+/+) HCT116 cells resulted in
repression of the BRCA2 promoter in p53 wild type cells but
not in p53-null or mutant cells (Fig. 2B). In addition,
reporter assays were performed in MCF7 cells stably expressing either
the human papilloma virus type 16 (HPV-16) E6 gene (MCF7/E6) or a CMV
vector control (MCF7/CMV). The E6 protein stimulates degradation of p53
through a ubiquitin pathway (20, 21), resulting in very low levels of
p53 expression in these cells. Reduced levels of p53 in MCF7/E6 cells
was verified by Western blotting (data not shown). A decrease in
BRCA2 promoter activity in response to ADR treatment was
only detected in the MCF7/CMV cells (Fig. 2C). Taken
together, these data suggest that repression of BRCA2
promoter activity by ADR is dependent on the presence of wtp53.

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Fig. 2.
p53 represses the BRCA2
promoter. A, repression of the BRCA2
promoter in osteosarcoma cells is p53-dependent. Luciferase
activity from pGL3Prom was measured 24 h after treatment with 5 µM ADR or Me2SO (DMSO) for 1 h in matched U2OS (p53+/+) and Saos2 (p53 / )
osteosarcoma cell lines. B, ADR does not repress
BRCA2 promoter activity in p53-deficient HCT116 cells.
Luciferase activity from pGL3Prom was measured 24 h after
treatment with 5 µM ADR or Me2SO for 1 h
in matched HCT116/p53+/+ and HCT116/p53 /
cells. C, ADR does not repress the BRCA2 promoter
after E6-associated degradation of p53. Luciferase activity from
pGL3Prom was measured 24 h after treatment with 5 µM
ADR or Me2SO for 1 h in matched MCF7/CMV and MCF7/E6
cells. D, ectopic expression of p53 in MCF7
(p53+/+) cells inhibits BRCA2 promoter activity.
Luciferase activity from pGL3Prom was measured 24 h after
treatment with 5 µM ADR or Me2SO for 1 h
in MCF7 cells transiently transfected with wtp53, dnp53 (R273L), or
control vector. E, ectopic expression of p53 inhibits
BRCA2 promoter activity in Saos2 (p53 / )
cells. Luciferase activity from pGL3Prom was measured 24 h after
treatment with 5 µM ADR or Me2SO for 1 h
in Saos2 cells transfected with wtp53, dnp53 (R273L), or control
vector. F, MMC and actinomycin D (Act-D) inhibit
BRCA2 promoter activity in a p53-dependent
manner. Luciferase activity from pGL3Prom was measured after 24 h
of treatment with 10 ng/ml actinomycin D or 30 µg/ml MMC in MCF7
(wtp53), Saos2 (p53-null), and T47D (p53 mutant) cells.
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Given that ADR-associated repression of the BRCA2
promoter is dependent on p53, it seemed likely that p53 could regulate
the BRCA2 promoter independently of ADR treatment. To
address this hypothesis a series of BRCA2 promoter reporter
assays was performed in MCF7 and Saos2 cells transiently transfected
with wtp53, dnp53, or a pcDNA 3.1 vector control. Wtp53 expression
in MCF7 (Fig. 2D) and Saos2 cells (Fig. 2E)
reduced BRCA2 promoter activity 10-fold relative to a vector
control, and transfection of increasing concentrations of wtp53 into
these cells resulted in dose-dependent repression of the
promoter (data not shown). In contrast, ectopic expression of the dnp53
mutant markedly up-regulated the BRCA2 promoter (Fig.
2D). This apparent induction of promoter activity is likely
caused by dnp53-associated inactivation of endogenous p53 and
consequent relief of repression. Thus, wtp53 appears to have a specific
inhibitory effect on the BRCA2 promoter.
To evaluate further the relevance of p53 to the ADR response, we
repeated these reporter assays in the presence of ADR. As before, ADR
treatment caused significant down-regulation of BRCA2 promoter activity in vector-transfected MCF7 cells, but had no effect
in Saos2 cells (Fig. 2, D and E). However, when
wtp53-transfected MCF7 cells were treated with ADR a 40-fold
down-regulation of the promoter, or 2-fold greater repression than with
wtp53 expression alone, was observed (Fig. 2D). The lack of
a synergistic effect between ADR and wtp53 expression is further
support for the concept that wtp53 and ADR influence the
BRCA2 promoter through a common signaling pathway.
Repression of the BRCA2 Promoter by Other DNA-damaging
Agents--
We also evaluated whether other DNA-damaging agents
associated with induction or stabilization of wtp53 were capable of
repressing the BRCA2 promoter. MCF7 cells were treated with
30 µg/ml mitomycin C (MMC), 10 ng/ml actinomycin D, 5-10
µM camptothecin, 10 J/m2 UV irradiation, and
2-10 Grays of
-irradiation, and luciferase assays were performed.
MMC and actinomycin D repressed the BRCA2 promoter similarly
to ADR, whereas the other agents had no apparent effect. In contrast,
none of the agents had an effect on the BRCA2 promoter in
the p53-null Saos2 and p53 mutant T47D cell lines (Fig. 2F).
These data suggest that only certain DNA-damaging agents can repress
the BRCA2 promoter and that the effect is uniformly dependent on the presence of wtp53.
In Vivo Repression of BRCA2 Expression by ADR Requires
Wtp53--
To confirm an in vivo effect of ADR and wtp53 on
endogenous BRCA2 expression, BRCA2 mRNA levels were measured in
MCF7 and Saos2 cells treated with 5 µM ADR. BRCA2
expression was decreased substantially in MCF7 cells but was not
affected in Saos2 cells after ADR treatment (Fig.
3A). Similarly, ectopic
expression of wtp53 resulted in decreased BRCA2 expression in MCF7 and
Saos2 cells, and the combination of ADR and wtp53 expression did not appear to have a synergistic effect. In addition, BRCA2 mRNA levels were not substantially affected by ADR treatment of MCF7 cells ectopically expressing dnp53 (Fig. 3B). The combination of
these data and the results from the reporter assays described above strongly suggest that ADR and wtp53 can regulate BRCA2 expression in vivo through inhibition of BRCA2 promoter
activity.

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Fig. 3.
ADR and p53 repress BRCA2 expression in
vivo. A, ADR reduces BRCA2 mRNA levels
in wtp53 but not in p53-null cells. BRCA2 expression levels in Saos2
and MCF7 cells were measured by Northern blot of poly(A)+
RNA 24 h after treatment with 5 µM ADR or
Me2SO for 1 h. B, wtp53 down-regulates
BRCA2 mRNA levels. BRCA2 expression levels in MCF7 cells
transfected with wtp53, dnp53, or vector control were measured by
Northern blot of poly(A)+ RNA 24 h after treatment
with 5 µM ADR or Me2SO for 1 h. A GAPDH
control probe was used to demonstrate equal loading.
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In Vivo Reduction in BRCA2 Protein Levels by ADR and
Wtp53--
Although ADR appears to down-regulate BRCA2
promoter activity and BRCA2 expression levels, down-regulation of BRCA2
protein levels must also be evident to speculate that
ADR-dependent repression of BRCA2 transcription can affect
BRCA2 function. To evaluate the effect of ADR on in vivo
BRCA2 protein levels, MCF7, U2OS, and MCF10A cell lines were treated
with 5 µM ADR. As shown in Fig.
4A, BRCA2 levels were
decreased significantly in ADR-treated cells. In addition, the amount
of BRCA2 in MCF7 cells decreased in a dose-responsive manner after
treatment with 0.5, 2.0, and 5.0 µM ADR (Fig.
4B). The expected dose-dependent increase in p53
levels was also observed. To evaluate whether ADR treatment had a
time-dependent effect on BRCA2 protein expression, MCF7 cells were treated with 5 µM ADR for 1 h, and BRCA2
protein levels were measured over a 24-h period. A gradual decrease in
BRCA2 levels over time was observed with the most significant changes occurring between 5 and 11 h post-treatment (Fig. 4C).
Interestingly, the decrease in BRCA2 correlated inversely with changes
in p53 levels. Similar results were obtained in response to continuous treatment with 0.7 µM ADR over a 24-h period, although
the most significant changes in BRCA2 and p53 were delayed and occurred between 9 and 13 h after initiation of treatment (Fig.
4D).

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Fig. 4.
ADR and p53 down-regulate BRCA2 protein
levels. A, ADR treatment reduces BRCA2 protein levels
in several cell lines. Protein extracts from MCF7, U2OS, and MCF10A
cells incubated for 24 h after exposure to 5 µM ADR
or Me2SO for 1 h were used for BRCA2 Western blots.
BRCA2 from 293T cells transiently transfected with vector and a BRCA2
expression construct was used as a size control, and BRCA2 mutant
CAPAN1 cells were used as a negative control. B,
ADR-associated depletion of BRCA2 and induction of p53 proteins are
dose-dependent. Protein extracts from MCF7 cells incubated
for 24 h after exposure to different doses of ADR for 1 h
were used for BRCA2 and p53 Western blots. C, depletion of
BRCA2 and induction of p53 by ADR are time-dependent.
Protein extracts from MCF7 cells incubated for different times after
exposure to 5 µM ADR for 1 h were used for BRCA2 and
p53 Western blots. D, depletion of BRCA2 and induction of
p53 by low dose ADR are time-dependent. Protein extracts
from MCF7 cells continuously exposed to 0.7 µM ADR for
different amounts of time were used for BRCA2 and p53 Western blots.
E, ADR-associated depletion of BRCA2 protein is
p53-dependent. Protein extracts from matched
HCT116/p53+/+ and HCT116/p53 / cells,
matched MCF7/CMV and MCF7/E6 cells, Saos2 cells (p53-null), and SW480
(p53 mutant) cells incubated for 24 after exposure to 5 µM ADR for 1 h were used for BRCA2 Western blots.
F, ectopic expression of p53 depletes BRCA2 protein. Protein
extracts from Saos2 cells transfected with vector, wtp53, and dnp53 and
incubated for 24 h after exposure to 5 µM ADR or
Me2SO for 1 h were used for BRCA2 and p53 Western
blots. DNA-PK served as a loading control for BRCA2 Western
blots.
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|
Next we evaluated whether the effect of ADR on BRCA2 protein levels was
dependent on p53. Matched p53-null (HCT116/p53
/
) and
p53 wild type (HCT116/p53+/+) HCT116 cells, matched MCF7/E6
and MCF7/CMV cells, SW480 p53 mutant cells, and Saos2 p53-null cells
were treated with 5 µM ADR, and cell lysates were Western
blotted for BRCA2 with the Ab2 antibody. As shown in Fig.
4E, an ADR-dependent reduction in BRCA2 protein
levels was observed in cells expressing wtp53 but not in p53-null or
p53 mutant cells. Further analysis showed that ectopic expression of
wtp53 in Saos2 cells (Fig. 4F) and MCF7 cells (data not
shown) also caused a significant reduction in BRCA2 levels. When
combining all of these data, it is evident that there is a strong
correlation between the effects of ADR and p53 on BRCA2 protein levels
and on BRCA2 promoter activity.
The ADR- and p53-responsive Element Is Adjacent to the
Transcription Initiation Site--
Having determined that ADR and p53
repress BRCA2 promoter activity, we sought to understand the
mechanism of repression. We began by identifying the ADR-responsive
region within the BRCA2 promoter using a series of
luciferase reporter constructs containing deleted forms of an 8-kb form
of the BRCA2 promoter in reporter assays (18). MCF7 cells
were transfected with the various constructs, and luciferase activities
were measured before and after treatment with 5 µM ADR.
All promoter constructs that contained the minimal promoter, located
between nucleotides
58 and
1, were repressed equivalently by ADR
treatment (data not shown), suggesting that the ADR-responsive
cis-element is located in the minimal BRCA2 promoter. However, as deletion of the minimal promoter between
58 and
19 eliminated basal promoter activity (18), we could not directly
evaluate the role of this region in the ADR response. To overcome this
problem a series of promoter reporter constructs containing
substitution mutations in the putative ATF and MLTF binding sites, in
the known USF-binding element, and in the two repeats of the GCGTCACG
tandem repeat sequence within the
58 to
19 region of the
BRCA2 promoter was used (18). The reporter constructs
containing the mutated
13 to
20 USF transcription factor binding
site and the overlapping
17 to
24 repeat sequence were 3-fold less
repressed in the presence of ADR than any other reporter construct
(data not shown). Similar results were obtained after ectopic
expression of wtp53. These data suggest that this USF binding site that
has been implicated previously in regulation of basal activity of the
BRCA2 promoter (18) also regulates the response to ADR and
wtp53.
Wtp53 Inhibits Binding of USF to the BRCA2 Promoter--
To
determine whether the USF transcription factor is directly involved in
p53- and ADR-dependent regulation of BRCA2
promoter activity, the ability of USF to bind to the BRCA2
promoter was evaluated. Two 26-bp (
10 to
35) oligonucleotide probes
containing either a wild type or mutated USF binding site (18) were
used in gel shift assays with lysates from MCF7 cells that had been treated with Me2SO or 5 µM ADR. As shown in
Fig. 5A, a specific protein
complex bound to the wild type oligonucleotide but not to the mutant
oligonucleotide and the ability of this complex to bind DNA decreased
over time when cells were treated with ADR (Fig. 5A).

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Fig. 5.
ADR and p53 inhibit binding of USF to the
BRCA2 promoter. A, ADR-associated
disruption of a BRCA2 promoter USF binding site
protein·DNA complex is time-dependent. EMSAs were
performed using oligonucleotide probes containing a wild type
(W) or mutated (M) USF binding site from the
BRCA2 promoter, and whole cell protein extract from MCF7
cells was incubated for 0, 5, 13, and 24 h after treatment with 5 µM ADR for 1 h. B, disruption of the USF
protein·DNA complex by ADR in osteosarcoma cells is
p53-dependent. EMSAs of protein extracts from Saos2
(p53-null) and U2OS (p53 wild type) cells incubated for 24 h after
treatment with 5 µM ADR (+) or Me2SO ( ) for
1 h are shown. C, ADR does not disrupt protein·DNA
complex formation in HCT116/p53 / cells. EMSAs of
protein extracts from HCT116/p53 / and
HCT116/p53+/+ cells incubated for 24 h after treatment
with 5 µM ADR or Me2SO for 1 h are
shown. D, ADR does not disrupt protein·DNA complex
formation after E6-associated degradation of p53. EMSA were performed
of protein extracts from MCF7/CMV and MCF7/E6 cells incubated for
24 h after treatment with 5 µM ADR or
Me2SO for 1 h. E, ectopic expression of p53
disrupts protein·DNA complex formation. EMSAs were performed of
protein extracts from MCF7 cells that had been transfected with vector,
wtp53, and dnp53 and incubated for 24 h after treatment with 5 µM ADR or Me2SO for 1 h. F,
p53 disrupts binding of USF to the BRCA2 promoter.
Supershift assays of protein·DNA complexes formed between wild type
USF binding site oligonucleotides and protein extracts from MCF7 cells
transfected with vector or wtp53 expression constructs were performed
using anti-USF2 antibody.
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To determine whether wtp53 was needed for ADR-dependent
inhibition of complex formation, we repeated the gel shift assays using
lysates from matched Saos2 and U2OS cells, matched
HCT116/p53
/
and HCT116/p53+/+ cells, and
matched MCF7/CMV and MCF7/E6 cells (Fig. 5, B-D). A similar
protein-oligonucleotide complex was evident in all cells, but
the relative intensity of the complex was significantly greater in p53
mutant or p53-null cells than in matched cells with endogenous wtp53.
Moreover, exposure to ADR further reduced the intensity of the
protein·DNA complex in wtp53-expressing cells but had no effect in
p53-null or mutant cells (Fig. 5, B-D). In addition, complex formation was reduced substantially by ectopic expression of
wtp53, similarly to the ADR treatment, but was enhanced by expression
of dnp53. This suggests that dnp53 inactivates endogenous or induced
wtp53 and prevents p53-dependent inhibition of complex formation (Fig. 5E).
To verify that the protein complex binding to the wild type
oligonucleotide contained USF, it was demonstrated that anti-USF2 antibodies could supershift the protein·DNA complex (Fig.
5F). Furthermore, Western blotting with anti-USF1 and
anti-USF2 antibodies demonstrated that the levels of these proteins
were not altered in response to ADR or p53 expression (data not shown).
Together these data suggest that the repressive effect of ADR and p53
on the BRCA2 promoter does not involve reduction in USF1 and
USF2 levels but is dependent on altered binding of USF2 and/or USF1 to
the promoter.
p53 Inhibits USF-dependent Induction of the BRCA2
Promoter--
To establish further the relevance of USF to p53- and
ADR-dependent repression of the BRCA2 promoter,
we tested whether p53 could block induction of the BRCA2
promoter by USF. Initially, a USF2-VP16 fusion protein that binds to
the USF site was used to transactivate the BRCA2 promoter in
MCF7 cells (18), and then the effect of wtp53 and ADR on the activated
promoter was evaluated. As shown in Fig.
6A, wtp53 partially inhibited
USF2-VP16-associated activation of the BRCA2 promoter,
whereas dnp53 further activated the promoter. Subsequently, the ability
of wtp53 to inhibit activation of the promoter by USF1 alone was
evaluated. Ectopic expression of USF1 in SW480 (p53 mutant) cells
up-regulated the BRCA2 promoter 4.5-fold. However, ectopic
expression of wtp53 completely blocked this effect (Fig.
6B). Similar effects were observed in the Saos2 p53-null and
T47D p53 mutant cell lines (data not shown). Thus, p53 inhibits
USF-dependent promoter activation.

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Fig. 6.
p53 represses USF-dependent
induction of the BRCA2 promoter. A,
p53 inhibits USF2-VP16 induction of the BRCA2 promoter.
Luciferase activity from the BRCA2 promoter reporter
construct was measured 24 h after transfection of MCF7 cells with
combinations of USF-VP16, wtp53, dnp53, and vector. B, p53
inhibits USF1 induction of the BRCA2 promoter. Luciferase
activity from the BRCA2 promoter reporter construct was
measured 24 h after transfection of p53 mutant SW480 cells with
combinations of USF1, wtp53, and vector control expression
constructs.
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ADR Enhances BRCA2 mRNA and Protein Degradation--
The data
described above show that ADR, MMC, and actinomycin D repress the
BRCA2 promoter in a p53-dependent manner.
However, although the effect is significant it does not entirely
account for the rapid decrease in BRCA2 mRNA and protein levels. In
fact, a comparison among promoter activity, mRNA levels, and
protein levels at various time points after ADR exposure (Figs.
1D and 4C) indicates that BRCA2 mRNA and
protein levels decreased more rapidly than BRCA2 promoter
activity. This suggests that ADR can influence BRCA2 mRNA and
protein levels through additional mechanisms and provides support for
earlier observations that DNA-damaging agents can decrease BRCA2
mRNA levels (16, 17) and that certain damaging agents such as UV
irradiation can enhance BRCA2 protein degradation (22).
To determine whether ADR altered the stability of BRCA2 mRNA, MCF7
cells were treated with
-amanitin to block transcription, and the
rate of decrease in BRCA2 mRNA in cells treated with 5 µM ADR and Me2SO was compared by
semiquantitative reverse transcription PCR. As shown in Fig.
7, A and B, ADR
substantially enhanced the rate of BRCA2 mRNA degradation.
Quantitation by densitometry showed that the half-life of BRCA2
mRNA in MCF7 cells is normally 9 h, whereas in the presence of
ADR it is reduced to ~3 h. In addition, by comparing mRNA levels
from MCF7 cells treated with ADR alone or with ADR and
-amanitin at
various time points we determined that promoter inhibition accounted
for 50% of the reduction in BRCA2 mRNA levels.

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Fig. 7.
ADR enhances BRCA2 mRNA degradation.
A and B, ADR reduces BRCA2 mRNA levels.
Semiquantitative reverse transcription PCR of BRCA2 mRNA from MCF7
cells treated with 5 µM ADR, -amanitin, and ADR plus
-amanitin for 1 h and incubated further for the indicated times
in the presence or absence of -amanitin was performed.
Oligonucleotide pairs for PCR were located at the 3'-end (A)
and 5'-end (B) of the BRCA2 coding region.
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We also attempted to measure the rate of BRCA2 protein degradation in
the presence and absence of ADR. MCF7 cells were treated with
cycloheximide, and BRCA2 protein levels were measured by Western
blotting and densitometry. The half-life of the BRCA2 protein was
estimated to be 5 h (data not shown). MCF7 cells were subsequently
treated with cycloheximide and 5 µM ADR, and the half-life was estimated to be 4 h (data not shown). Although the rate of protein degradation in response to ADR treatment was enhanced, it was not sufficient to account for a 50% reduction in the BRCA2 protein level 5 h after ADR treatment as shown in Fig.
4C. Interestingly, a previous study of UV-associated BRCA2
protein degradation suggested that an inducible factor required for
active degradation of BRCA2 in response to UV treatment could not be
synthesized in the presence of cycloheximide and that the rate of
UV-dependent BRCA2 protein degradation could not be
measured accurately (22). It is likely that a similar effect was
encountered in our experiments and that the actual rate of BRCA2
protein degradation in response to ADR is more rapid than the rate
detected in our cycloheximide experiments. Our finding that BRCA2
protein levels are reduced by ~50% and that BRCA2 mRNA levels
are also reduced by ~50% in MCF7 cells treated for 5 h with ADR
supports this observation because it is unlikely that the reduction in
mRNA levels can account for the entire reduction in protein. As a
result it is not possible to determine the relative contribution of
promoter inhibition, mRNA degradation, and protein degradation to
the reduction in BRCA2 protein in response to ADR, but it is clear
that each mechanism makes a substantial contribution.
Repression of the BRCA2 Promoter by ADR and p53 Is Independent of
Cell Cycle Arrest--
It is not known whether
p53-dependent cell cycle arrest is required for repression
of the BRCA2 promoter or whether these events are
independent of each other and are simply concurrent responses to p53
induction by ADR. To address this question, cell cycle profiles of
cells were measured at various time points after treatment with ADR,
and the results were compared with time course studies of
BRCA2 promoter activity and BRCA2 mRNA and protein levels from cells that had been treated similarly. It was noted that
ADR-dependent repression of the BRCA2 promoter
preceded ADR-associated induction of an S and G2/M cell
cycle checkpoint arrest (Fig. 8,
A and B). This was
noted after exposure to a short pulse of 5 µM ADR and in
response to continuous exposure to 0.7 µM ADR. Down-regulation of BRCA2 mRNA and protein also appeared to precede the onset of cell cycle arrest.

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Fig. 8.
ADR and p53 repression of the
BRCA2 promoter is independent of
p21Waf1/CIP1 and cell cycle arrest. A,
continuous exposure to low dose ADR induces a G2/M cell
cycle arrest. Fluorescence-activated cell sorter analysis of the
percentage of PI-stained MCF7 cells in each phase of the cell cycle was
performed after various periods of exposure to 0.7 µM
ADR. B, high dose ADR induces cell cycle arrest at S and
G2/M checkpoints. Fluorescence-activated cell sorter
analysis of the percentage of PI-stained MCF7 cells in each phase of
the cell cycle was performed after different periods of incubation
after treatment with 5 µM ADR for 1 h. C,
p21Waf1/CIP1 is not required for ADR repression of the
BRCA2 promoter. Luciferase activity from the
BRCA2 promoter reporter construct was measured 24 h
after exposure of HCT116/p21+/+ and
HCT116/p21 / cells to 5 µM ADR or
Me2SO for 1 h. D, p21Waf1/CIP1
is not required for repression of the BRCA2 promoter by p53.
Luciferase activity from the BRCA2 promoter reporter
construct was measured 24 h after transfection of
HCT116/p21+/+ and HCT116/p21 / cells with
vector, wtp53, and dnp53 expression constructs.
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Given that induction of cell cycle arrest by p53 is associated with
induction of p21Waf1/Cip1, we also investigated whether
p21Waf1/Cip1 was required for the ADR and p53 effect on the
BRCA2 promoter. The BRCA2 luciferase reporter
construct was transiently transfected into HCT116/p21+/+
and matched p21
/
cells, and promoter activity was
measured after treatment with 5 µM ADR and ectopic
expression of wtp53. BRCA2 promoter activity was repressed
equivalently in the matched cell lines by ADR (Fig. 8C) and
p53 (Fig. 8D), indicating that the ADR and p53 effect is
independent of p21Waf1/Cip1 and suggesting that the
BRCA2 promoter is repressed independently of the induction
of G1/S checkpoint arrest.
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DISCUSSION |
The effect of cellular stress and DNA-damaging agents on
BRCA2 has been a topic of much interest. Initially, BRCA2
mRNA levels were shown to be down-regulated in cells in response to
treatment with DNA-damaging agents such as ADR, MMC, and UV irradiation (16). In this study, we show that as part of the cellular response to
ADR-associated DNA damage the BRCA2 promoter is
significantly repressed in a p53-dependent manner. We have
established that in response to ADR, MMC, and actinomycin D, p53
inhibits binding of the USF transcription factor to the
BRCA2 minimal promoter, resulting in repression of basal
promoter activity and substantial decreases in BRCA2 mRNA and
protein levels. The inability of ADR treatment to repress the promoter
and decrease mRNA and protein levels in p53 mutant or null cells or
in cells expressing the dominant negative R273Lp53 indicates that this
is a p53-dependent process. However, it is important to
note that only ADR, MMC, and actinomycin D repressed the promoter by
inhibiting USF binding in a p53-dependent manner, whereas
other DNA-damaging agents such as UV irradiation,
-irradiation, and
camptothecin, which are also associated with induction of p53, had no
effect. This raises the possibility that only certain forms of DNA
damage can induce other factors, in addition to p53, which are required
for inhibition of USF binding to the promoter.
Although UV irradiation,
-irradiation, and camptothecin fail to
inhibit USF binding, these agents all reduce BRCA2 mRNA and protein
levels. This suggests that these agents either regulate transcription
of the BRCA2 gene through cis-elements and
enhancer/repressor binding sites that are not present in our 8-kb
promoter construct or that these agents influence the stability of
BRCA2 mRNA and protein. In this study we determined that ADR and
other DNA-damaging agents enhance BRCA2 mRNA and protein turnover,
resulting in decreased BRCA2 mRNA and protein levels. Given that
BRCA2 mRNA and protein are only reduced by ADR in cells expressing
wtp53, the suggestion is that p53 mediates BRCA2 promoter
activity and BRCA2 mRNA and protein turnover in response to ADR,
MMC, and actinomycin D but only regulates mRNA and protein turnover
in response to other DNA-damaging agents. Thus, BRCA2 protein levels
may be regulated by various DNA-damaging agents at the level of the
promoter, mRNA stability, and protein stability.
In this work we determined that ADR and p53 repress the
BRCA2 promoter by inhibiting the ability of USF to bind to
the promoter. To our knowledge, this is the first report showing that
p53-mediated transcriptional repression is functionally associated with
USF binding. We have shown previously that binding of USF to the
BRCA2 minimal promoter is required for basal transcription
of BRCA2 and for induction of the promoter and increased
expression of BRCA2 during the S and G2 phases of the cell
cycle (18). Thus, by inhibiting binding of USF to the promoter it
appears that ADR treatment and p53 actually prevent activation of the
BRCA2 promoter during S and G2 phases of the
cell cycle. Although p53 appears to regulate USF binding in response to
ADR treatment, p53 does not seem to bind directly to USF, as evidenced
by an inability of anti-p53 antibodies to supershift the USF complex
and by failure of p53 to coimmunoprecipitate with USF1 or USF2 (data
not shown). Because we have also shown that p53 does not regulate USF
expression levels, it seems likely that ADR or p53 may regulate and p53
may interact with other protein(s) that in turn modulate the ability of
USF to bind to the promoter. However, it is also possible that p53 may
be a part of the USF protein complex, but its presence in the complex
may not be detected easily by certain antibodies. USF1 has recently
been identified as a phosphoprotein (23, 24) whose DNA binding activity
is dependent on cyclin-dependent phosphorylation and can be
inhibited by the p53-inducible cyclin-dependent kinase inhibitor, p21Waf1/Cip1, which blocks phosphorylation of
USF1 (24). This suggests that ADR and p53 regulate BRCA2 expression
through p21Waf1/Cip1. However, we found that p53 and
ADR repressed BRCA2 promoter activity in cells lacking
p21Waf1/Cip1, suggesting that p21Waf1/Cip1
plays no role in USF-dependent regulation of the
BRCA2 promoter.
Repression of transcription of several genes by p53 is thought to be
the consequence of p53-dependent inhibition of other transcriptional activators (25-27) or components of the basal
transcription machinery (28-30). One mechanism of p53-associated
repression utilizes histone deacetylases, mediated by interaction with
Sin3a, to regulate target genes such as map4 and
stathmin negatively (19) and to repress the CHK1
gene through the p21Waf1/Cip1 protein (31). Another
mechanism of p53-dependent repression involves binding of
p53 to p300/CBP and subsequent interference in coactivation of
p300/CBP-dependent factors, such as AP-1 (26), hypoxia-inducible factor 1 (32), and nuclear factor-
B (33, 34).
Interestingly, it has been reported that p300 interacts functionally
with USF to potentiate the activation of USF target genes (35). Whether
the USF-dependent regulation of the BRCA2 promoter by p53 is actually mediated by p300/CBP or histone
deacetylases or as yet unidentified factors remains to be determined.
There is substantial evidence that BRCA2 plays a role in DNA repair.
Therefore, it is surprising that BRCA2 is down-regulated by p53 in
response to ADR-associated DNA damage. One possible explanation is that
p53 must down-regulate BRCA2 to induce cell cycle arrest and DNA damage
repair because BRCA2 appears to interact with p53 in a
RAD51·p53·BRCA2 complex and may partially repress p53- dependent transactivation of target promoters such
as p21Waf1/CIP1 (36). Alternatively, BRCA2 may
interfere with induction of p53-dependent apoptosis in
response to DNA damage in a similar manner. However, because BRCA2 is
itself involved in DNA repair this seems unlikely. It is also possible
that down-regulation of BRCA2 by p53 allows cells to initiate apoptosis
in response to DNA damage more efficiently, while the presence of BRCA2
provides sufficient DNA repair to prolong cell viability. An
alternative possibility is that p53 may down-regulate BRCA2 after DNA
repair is complete to inactivate the DNA repair machinery and to
release the cell from checkpoint arrest. This could also be a mechanism by which p53 regulates the extent and timing of DNA damage repair. The
observation that p53 only significantly affects the BRCA2 promoter and BRCA2 protein levels 6 h after induction of DNA
damage supports this possibility.
Previous reports have indicated that BRCA2 mRNA and protein levels
change in a cell cycle-dependent manner, with low levels in
G1 and significantly elevated levels in S and
G2 phases of the cell cycle (3, 4). In the work described
above we have shown that repression of the BRCA2 promoter
and associated down-regulation of BRCA2 mRNA and protein levels
preceded p53-dependent S and G2/M phase cell
cycle arrest in response to ADR treatment. This indicates that BRCA2
down-regulation is dependent on p53 but is independent of the effect of
p53 induction on the cell cycle. Future experiments will determine
whether BRCA2 down-regulation is required or contributes to
p53-dependent cell cycle arrest in response to DNA damage.
In conclusion, we have demonstrated that the BRCA2 promoter
is down-regulated by ADR in a p53-dependent manner and that
repression of the promoter is mediated by altered binding of USF to the
minimal promoter. We have also shown that inhibition of promoter
activity results in decreased BRCA2 mRNA and protein levels over
time, suggesting a direct effect on BRCA2 function. Furthermore, we found that ADR and other DNA-damaging agents increase the rate of BRCA2
mRNA and protein turnover in a p53-dependent manner. Given that BRCA2 appears to regulate the transactivation activity of
p53 and that p53 inhibits BRCA2 expression, it appears that BRCA2 and
p53 share a complex regulatory loop that may be directly associated
with the cellular response to DNA damage. Further studies are needed to
identify the specific mechanism by which p53 inhibits USF binding to
the BRCA2 promoter and to }better understand why BRCA2 is
down-regulated in response to specific DNA-damaging agents.