 |
INTRODUCTION |
The BRCA1 tumor suppressor gene encodes a large
polypeptide of 1863 amino acids that contains at least two recognizable
protein motifs: a RING domain at the N terminus (1) and two tandem BRCT
repeats at the C terminus (2). To date, more than 200 different
germline mutations of BRCA1 have been implicated in familial
breast cancer (reviewed in Ref. 3). Although most of these are
frameshift or nonsense mutations that grossly truncate the BRCA1
reading frame, in some cases breast cancer susceptibility has been
attributed to more subtle defects that affect the BRCT coding
sequences. These include missense mutations that cause single amino
acid substitutions in either the first (A1708E) or second (M1775R) BRCT
domain and a nonsense mutation that truncates the second BRCT domain by
deleting 11 residues from the C terminus of BRCA1 (Y1853
). The fact
that these mutations confer susceptibility to breast cancer implies
that the BRCT domains play a crucial role in BRCA1-mediated tumor suppression.
Although its molecular functions remain obscure, recent studies have
implicated BRCA1 in several cellular processes, including cell growth
control, transcriptional regulation, and the maintenance of genomic
stability (reviewed in Ref. 4). The expression of BRCA1 varies with
cell cycle progression in most established cell lines (5-8). In
particular, the steady-state levels of BRCA1 products remain low or
undetectable in resting cells as well as during the early
G1 phase of the cell cycle. However, as cycling cells
traverse the G1/S boundary, the expression of BRCA1 is
induced such that the highest steady-state levels of BRCA1 gene
products occur during the S and G2/M phases. At the
G1/S transition, BRCA1 polypeptides also become
hyperphosphorylated and aggregate within distinct nuclear structures
(5, 9-13).
Consistent with these observations, other lines of evidence implicate
BRCA1 in one or more of the checkpoint pathways that control cell cycle
progression. Although Saccharomyces cerevisiae does not
possess a true ortholog of BRCA1, ectopic expression of human BRCA1
inhibits the growth of budding yeast (14). Interestingly, this growth
inhibition is abolished by tumor-associated lesions in the BRCT domains
of BRCA1, including the A1708E and M1775R missense mutations. It has
also been reported that overexpression of wild type BRCA1 inhibits S
phase progression (15) and that a C-terminal segment of BRCA1 (residues
1293-1863) can ablate the G2/M checkpoint of human mammary
epithelial cells, perhaps by dominant-negative inhibition of endogenous
BRCA1 (16).
Although sequence-specific DNA recognition by BRCA1 has not been
observed, experiments with GAL4p fusion proteins have shown that the
C-terminal sequences of BRCA1 affect RNA transcription both in
vivo and in vitro. Two groups have reported that a
hybrid polypeptide containing the DNA binding domain of GAL4p (residues 1-147) fused to the BRCT sequences of BRCA1 (residues 1528-1863) can
activate the transcription of GAL4p-responsive reporter genes and that
this transactivation potential is ablated by tumor-associated mutations
such as A1708E, M1775R, and Y1853 (17, 18). Recently, a similar
fusion protein that includes GAL4p residues 1-147 and BRCA1 residues
1560-1863 was shown to activate gene transcription in vitro
(19) and alter chromatin structure in vivo (20). It has also
been reported that BRCA1 co-purifies with the RNA polymerase II
holoenzyme complex (21, 22). These results as well as other data (15,
23, 24) suggest that BRCA1 may function as a regulator of RNA
transcription. In addition, a role for BRCA1 in processing nascent RNA
transcripts is suggested by the observation that BARD1, a protein that
associates with BRCA1 in vivo (25), forms a stable complex
with the RNA 3' cleavage factor CstF-50 (26).
We and others recently showed that BRCA1 interacts with the CtIP
polypeptide (27, 28). Significantly, the binding of CtIP is mediated by
the BRCT domains of BRCA1, and it is abolished by tumor-associated
lesions that affect these domains, such as the A1708E, M1775R, and
Y1853 mutations. Thus, the in vivo interaction of CtIP
and BRCA1 is likely to be important for BRCA1-mediated tumor
suppression. Although the function of CtIP is not known, it was
recently shown to bind several other key nuclear regulatory factors,
including CtBP1 and Rb1 (29-31). Given their common association with
CtIP, it is intriguing that CtBP1 and Rb1 each function as co-repressors of RNA transcription (32-42) and that CtBP, like Rb1 and
BRCA1, can also serve as a tumor suppressor in certain cellular
contexts (43, 44).
To explore the role of CtIP in BRCA1-mediated tumor suppression, we
have examined the basic biological properties of CtIP with respect to
those of BRCA1. Our results indicate that CtIP is a nuclear protein
expressed in a cell cycle-specific fashion similar to BRCA1. The
highest steady-state levels of CtIP occur during the S and
G2 stages of cell cycle progression, at a time when BRCA1
expression is also maximal. In addition, we found that a subset of
cellular CtIP polypeptides exist in a protein complex with BRCA1 and
its associated protein, BARD1, and that this complex remains stable in
cells subjected to genotoxic stress. These data support the notion that
CtIP interacts with and modulates the function of BRCA1.
| |
EXPERIMENTAL PROCEDURES |
Cells and Antibodies--
The HBL-100, T24, and HCT116 cell
lines were obtained from the American Type Tissue Culture Collection.
To generate CtIP-specific antibodies, glutathione
S-transferase
(GST)1 fusion proteins that
contain different segments of human CtIP were produced in
Escherichia coli. Each fusion protein was then purified as
described below and used to immunize rabbits or mice. The 14-1 mouse
monoclonal antibody was raised against a GST fusion protein containing
the C-terminal 278 amino acids of CtIP (residues 620-897). Rabbit
polyclonal antisera 210 and 211 were raised against a GST fusion
containing the C-terminal 208 amino acids of CtIP (residues 690-897).
Rabbit antisera 164 and 614 were generated by immunizing with a GST
fusion containing CtIP residues 58-369.
Immunoprecipitation and Co-immunoprecipitation Analysis--
The
CtIP/pSP6-FLAG expression plasmid was generated by inserting cDNA
sequences encoding full-length CtIP into the pSP6-FLAG vector (25).
CtIP/pSP6-FLAG was then used as template for in vitro
synthesis of radiolabeled CtIP in rabbit reticulocyte lysates (Promega)
containing [35S]methionine (ICN). To evaluate the
CtIP-specific antibodies, 5-µl aliquots of the programmed lysate were
diluted in 500 µl of radioimmune precipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet
P-40, 0.1% SDS, and 0.5% sodium deoxycholate), incubated with the
appropriate antibody reagent for 1 h at 4 °C, and
immunoprecipitated as described below. To prepare mammalian cell
lysates, HBL-100 and HCT116 cells were cultured in McCoy's 5A medium
supplemented with 10% fetal bovine serum. The cells were lyzed in low
salt Nonidet P-40 buffer (10 mM Hepes, pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA)
supplemented with 0.5 mM dithiothreitol, 0.05% SDS,
protease inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride)
and phosphatase inhibitors (10 mM 
-glycerophosphate 5 mM NaF and 0.1 mM vanadate). For
immunoprecipitation/co-immunoprecipitation analysis of mammalian cell
lysates, the appropriate amount of lysate was co-incubated with the
indicated antibodies at 4 °C for 1 h. After adding 50 µl of
protein A-Sepharose beads (20% slurry, Amersham Pharmacia Biotech),
the mixture was rocked at 4 °C for another 1-3 h. The beads were
then washed twice with low salt Nonidet P-40 buffer, twice with high
salt Nonidet P-40 buffer (1 M NaCl), and twice again with
low salt Nonidet P-40 buffer. Finally, the beads were boiled for 10 min
in 30 µl of 2× SDS loading buffer (0.1M Tris-HCl, pH
6.8, 4% SDS, 20% glycerol, 0.1% -mercaptoethanol, 0.004%
bromphenol blue), and the supernatant was fractionated by electrophoresis.
GST Pull-down Assays--
The BR-SZ/pSP6-FLAG expression
plasmid, which encodes the C-terminal 336 amino acids of BRCA1 (the
"SZ fragment," BRCA1 residues 1528-1863), was generated by
inserting BRCA1 cDNA sequences into the pSP6-FLAG vector.
BR-SZ/pSP6-FLAG was then used as a template for in vitro
synthesis of the radiolabeled SZ polypeptide in rabbit reticulocyte
lysates (Promega) containing [35S]methionine (ICN).
Plasmids that encode the various GST-CtIP fusion proteins were
generated by inserting appropriate CtIP cDNA sequences into the
pGEX-KG expression vector (45). Each GST-CtIP fusion protein was then
expressed in E. coli, purified by affinity chromatography on
glutathione-agarose beads, and retained as a 50% slurry in buffer C
(20 mM Hepes, pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol)
containing protease inhibitors. For each GST pull-down assay, a 6-µl
aliquot of the radiolabeled SZ fragment was mixed with 60 µl of
glutathione-agarose beads (loaded with 20 µg of the appropriate
GST-CtIP fusion protein) and 434 µl of radioimmune precipitation
buffer. Following a 1-h incubation at room temperature, the beads were
washed four times with radioimmune precipitation buffer. The bound SZ
polypeptides were eluted by boiling the beads for 10 min in 25 µl of
loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20%
glycerol, 0.1% -mercaptoethanol, and 0.004% bromphenol blue). The
beads were then pelleted by centrifugation, and the supernatant was
analyzed by SDS-PAGE.
Mammalian Two-hybrid Analysis--
Plasmids encoding the various
GAL4p and Vp16 hybrid polypeptides were constructed by inserting
cDNA sequences into the pCMV-GAL4 and pVP-FLAG5 expression vectors,
respectively (25). Mammalian expression plasmids encoding wild type or
mutant (C61G) versions of full-length BRCA1 were generated by inserting
the corresponding cDNA sequences into the pCB6 vector. The
mammalian two-hybrid assays were conducted in 293 cells as described
(46).
Cell Cycle Analysis--
T24 cells were synchronized and
analyzed as described (47). Western analyses were conducted using 120 µg of cell lysate for BRCA1 immunoblots and 50 µg for CDK2, cyclin
A, and CtIP immunoblots. In addition, total RNA was extracted from
cells harvested at each time point, and 20-µg aliquots of RNA were
evaluated by Northern filter hybridization. Each filter was hybridized
in succession with radiolabeled cDNA probes for CtIP, BRCA1, cyclin
A, or glyceraldehyde-3-phosphate dehydrogenase using ExpressHyb
solution (CLONTECH). The intensity of each
autoradiographic signal was then evaluated with Imagequant software
(Molecular Dynamics).
Cell Fractionation--
Whole cell lysates and the membranous,
cytoplasmic, and nuclear fractions were prepared from T24 cells as
described (47). Equivalent volumes of each fraction (corresponding to
30 µg of whole cell lysate) were evaluated by direct immunoblotting
with the CtIP-specific monoclonal antibody 14-1 or with monoclonal antibodies that recognize NuMA or 
-tubulin (Santa Cruz). For detection of BRCA1, equivalent volumes of each fraction (corresponding to 200 µg of whole cell lysates) were immunoprecipitated with a
BRCA1-specific antiserum (25), and the immunoprecipitates were
immunoblotted with the BRCA1-specific MS110 monoclonal antibody (Oncogene Research Products) (9).
Treatment of Cells with Genotoxic Agents--
Approximately
1 × 107 cells were seeded onto each 150-mm culture
dish. When the cultures reached 50-70% confluence (usually about
24 h after plating), the cells were subjected to genotoxic stress.
For UV irradiation, six dishes of cells were washed with warm
phosphate-buffered saline and exposed to 10 J/m2 UV light
in a Stratalinker (Stratagene). The irradiated cells were then supplied
with fresh tissue culture media and allowed to recover at 37 °C for
1 h before harvest. For adriamycin treatment, four dishes of cells
were provided with fresh tissue culture medium containing 0.2 µg/ml
adriamycin (Sigma) and incubated at 37 °C for 24 h before
harvest. For hydrogen peroxide treatment, six dishes of cells were
provided with fresh tissue culture medium containing 10 mM
hydrogen peroxide (48). After incubating at 37 °C for 15 min, the
cells were washed twice with warm phosphate-buffered saline, supplied
with fresh tissue culture medium without hydrogen peroxide, and
incubated at 37 °C for an additional 1 h before harvest. To
detect CtIP, 50-µg aliquots of the harvested cell lysate were
subjected to Western analysis with the CtIP-specific 14-1 monoclonal
antibody. To detect p53 and actin, 10-µg aliquots of the same lysates
were immunoblotted with the appropriate antibody reagent (Santa Cruz).
One-mg aliquots of the lysates were used to detect changes in the
mobility of the p220 BRCA1 polypeptide (11).
| |
RESULTS |
Characterization of CtIP-specific Antibodies--
We had
previously shown that exogenous CtIP molecules associate in
vivo with the endogenous BRCA1 polypeptides of mammalian cells
(27). To establish whether endogenous CtIP also interacts with
endogenous BRCA1, it was necessary to generate immunological reagents
that specifically recognize CtIP. Therefore, three distinct segments of
CtIP were expressed in E. coli as fusion proteins with GST
(see "Experimental Procedures"). Each fusion protein was purified
and used as an immunogen to generate either polyclonal or monoclonal
antibodies. The antibody reagents were then tested for
immunoprecipitation of full-length CtIP polypeptides synthesized by
in vitro translation in the presence of
[35S]methionine. Fig. 1
shows that radiolabeled CtIP, which migrates with an apparent molecular
weight of ~120 kilodaltons upon SDS-PAGE, was immunoprecipitated with
rabbit antisera raised against sequences from either the N-terminal
(antiserum 164; CtIP residues 58-369) or C-terminal (antiserum 210;
CtIP residues 690-897) halves of CtIP (lanes 2 and
7, respectively) but not with the corresponding pre-immune
sera (lanes 1 and 6). Radiolabeled CtIP was also
immunoprecipitated by a mouse monoclonal antibody raised against CtIP
residues 620-897 (antibody 14-1) (lane 12). As expected,
immunoprecipitation of 35S-labeled CtIP by antiserum 210 and monoclonal antibody 14-1, both of which recognize C-terminal
sequences of CtIP, was blocked by an excess of the GST-CtIP(620-897)
fusion protein (lanes 10 and 14) but not GST
alone (lanes 8 and 13) or the GST-CtIP(58-369) fusion protein (lane 9). Likewise, immunoprecipitation of
radiolabeled CtIP with antiserum 164, which was raised against an
N-terminal segment of CtIP, was blocked by an excess of the immunogen,
GST-CtIP(58-369) (lane 4) but not by GST alone (lane
3) or GST-CtIP(620-897) (lane 5). These results
indicate that the each of the three antibody reagents recognizes CtIP
in a highly specific manner.
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 1.
Characterization of CtIP-specific antibodies
using in vitro translated CtIP. CtIP polypeptides
were synthesized by in vitro translation in the presence of
[35S]methionine. Equivalent aliquots of radiolabeled CtIP
were then immunoprecipitated with CtIP-specific rabbit antiserum 164 (lanes 2-5), rabbit antiserum 210 (lanes 7-10),
or the corresponding pre-immune sera (lanes 1 and
6, respectively). Additional aliquots of CtIP were
immunoprecipitated with either the CtIP-specific mouse monoclonal
antibody 14-1 (lanes 12-14) or an isotype-matched control
antibody (lane 11). In some cases, immunoprecipitation was
conducted in the presence of a molar excess of the GST-CtIP(58-369)
fusion protein (lanes 4 and 9), the
GST-CtIP(620-897) fusion protein (lanes 5, 10,
and 14), or GST alone (lanes 3, 8, and
13). The immunoprecipitates were then fractionated by
SDS-PAGE, and the presence of radiolabeled CtIP in each
immunoprecipitate was detected by autoradiography. The mobilities of
the 220- and 97-kilodalton molecular mass markers are shown on the left
of the image.
|
|
The rabbit antisera (164 and 210) were then used to immunoprecipitate
endogenous CtIP from lysates of HBL-100 cells, an immortalized line of
normal human mammary epithelial cells (49). The immunoprecipitates were
fractionated by SDS-PAGE, and the presence of CtIP in each immunoprecipitate was determined by immunoblotting with the
CtIP-specific monoclonal antibody (14-1). As illustrated in Fig.
2, a single endogenous CtIP band of
~120 kilodaltons was detected in an untreated lysate of HBL-100 cells
(lane 1). A band with the same electrophoretic mobility was
also obtained by immunoprecipitation with CtIP-specific antisera 164 and 210 (lanes 3 and 7, respectively) but not
with the corresponding pre-immune sera (lanes 2 and
6, respectively). As expected, immunoprecipitation of
endogenous CtIP by antiserum 164 was blocked by an excess of the
immunogen, GST-CtIP(58-369) (lane 4), but not by GST alone
(lane 5).
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Immunoprecipitation of endogenous CtIP from
mammalian cell lysates. Lysates of HBL-100 cells (0.3 mg) were
immunoprecipitated with rabbit antisera raised against the N-terminal
(164) or C-terminal (210) sequences of CtIP (lanes 3 and
7, respectively) or with the corresponding pre-immune sera
(lanes 2 and 6, respectively). In some cases, the
GST-CtIP(58-369) fusion protein (lane 4) or GST alone
(lane 5) was included in the immunoprecipitation reaction.
The immunoprecipitates were then fractionated by SDS-PAGE, along with a
smaller aliquot (0.1 mg) of untreated HBL-100 cell lysate (lane
1). The presence of endogenous CtIP polypeptides was determined by
immunoblotting with the CtIP-specific monoclonal antibody (14-1). The
arrow indicates the endogenous form of CtIP, which has a
molecular mass of about 120 kilodaltons. The mobilities of the 220- and
97-kilodalton molecular mass markers are shown on the left of the
image.
|
|
The efficiency of immunoprecipitation with these antisera can be
estimated by comparing the intensities of the CtIP bands obtained by
immunoblotting the untreated HBL-100 lysate (lane 1) with
those obtained by immunoblotting the HBL-100 immunoprecipitates (lanes 3 and 7). As seen in Fig. 2, approximately
3-fold more CtIP is observed in the immunoprecipitates (lanes
3 and 7) than in 0.1 mg of the untreated HBL-100 cell
lysate (lane 1). Since the immunoprecipitates were each
derived from 0.3 mg of HBL-100 cell lysate, it could be determined that
under these conditions, both antisera immunoprecipitate endogenous CtIP
with an efficiency that approaches 100%.
In Vivo Association of Endogenous CtIP and BRCA1
Polypeptides--
The CtIP-specific antibodies were then used to test
whether endogenous CtIP and BRCA1 polypeptides interact in
vivo. Fig. 3 illustrates a
co-immunoprecipitation experiment using a lysate of HBL-100 cells. An
aliquot of the lysate (2.5 mg of total cellular protein) was
immunoprecipitated with CtIP-specific antiserum 210 (lane
3), and smaller aliquots of the same lysate (0.4 mg) were immunoprecipitated with a BRCA1-specific antiserum (lane 5)
or a BARD1-specific antiserum (lane 7). The
immunoprecipitates were then fractionated by SDS-PAGE, and the presence
of BRCA1 in each immunoprecipitate was determined by Western analysis
with a BRCA1-specific monoclonal antibody. As expected, BRCA1 was
immunoprecipitated with the BRCA1-specific antiserum (lane
5) and co-immunoprecipitated with the BARD1-specific antiserum
(lane 7). Significantly, BRCA1 was also
co-immunoprecipitated with the CtIP-specific antiserum (lane
3), but not with the corresponding pre-immune serum (lane 2), confirming that the endogenous BRCA1 and CtIP polypeptides are
associated in vivo.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
Co-immunoprecipitation of endogenous CtIP and
BRCA1 polypeptides. Lysates of HBL-100 cells were subjected to
immunoprecipitation analysis. A, the CtIP-specific antiserum
210 (lane 3) and the corresponding pre-immune serum
(lane 2) were used to immunoprecipitate equivalent aliquots
of cell lysate (2.5 mg). Smaller aliquots of the same lysate (0.4 mg)
were immunoprecipitated with BRCA1-specific (lane 5) or
BARD1-specific (lane 7) rabbit antisera or the corresponding
pre-immune sera (lanes 4 and 6). The
immunoprecipitates were then fractionated by SDS-PAGE along with an
aliquot (0.2 mg) of untreated HBL-100 cell lysate (lane 1).
The presence of endogenous BRCA1 polypeptides in each immunoprecipitate
was determined by immunoblotting with a BRCA1-specific monoclonal
antibody. The arrow indicates the full-length form of
endogenous BRCA1. The mobility of the 220-kilodalton molecular mass
marker is shown on the left of the image. B, The
CtIP-specific antiserum 210 (lane 3) and the corresponding
pre-immune serum (lane 2) were used to immunoprecipitate
equivalent aliquots of an HBL-100 cell lysate (0.36 mg). Larger
aliquots of the same lysate (1.2 mg) were immunoprecipitated with
BRCA1-specific (lane 5) or BARD1-specific (lane
7) rabbit antisera or the corresponding pre-immune sera
(lanes 4 and 6). The immunoprecipitates were then
fractionated by SDS-PAGE, along with an aliquot (0.1 mg) of the
untreated cell lysate (lane 1). The presence of endogenous
CtIP polypeptides in each immunoprecipitate was determined by
immunoblotting with the CtIP-specific monoclonal antibody 14-1. The
arrow indicates the full-length form of endogenous CtIP. The
mobilities of the 220- and 97-kilodalton molecular mass markers are
shown on the left of the image.
|
|
Given that the CtIP-specific antisera immunoprecipitate CtIP
polypeptides from these lysates quantitatively (Fig. 2), it is possible
to estimate the fraction of cellular BRCA1 that exists in a CtIP-bound
state. As seen in Fig. 3A, the intensity of the BRCA1 band
co-immunoprecipitated with the CtIP-specific antiserum (lane
3) is approximately 1.5-fold higher than that of the BRCA1 band
obtained from 0.2 mg of untreated cell lysate (lane 1).
Since the co-immunoprecipitated BRCA1 in lane 3 was derived
from 2.5 mg of cell lysate, it can be calculated that roughly
one-eighth of the cellular BRCA1 pool is bound to CtIP in
unsynchronized HBL-100 cells. In contrast, a comparison of the
intensities of the BRCA1 bands in lanes 1 and 7 suggests that most BRCA1 polypeptides are associated with BARD1 in
HBL-100 cells.
A reciprocal co-immunoprecipitation experiment is illustrated in Fig.
3B. In this case, HBL-100 lysates (1.2 mg of total cellular protein) were immunoprecipitated with the BRCA1-specific antiserum, and
smaller aliquots of the same lysate (0.36 mg) were immunoprecipitated with CtIP-specific antiserum 210. As shown, endogenous CtIP
polypeptides were present in immunoprecipitates obtained with the
BRCA1-specific antiserum (lane 5) but not with the
corresponding pre-immune serum (lane 4). Therefore, the
in vivo interaction of CtIP and BRCA1 was observed by
co-immunoprecipitation using either CtIP-specific (Fig. 3A)
or BRCA1-specific (Fig. 3B) antisera as the
immunoprecipitating agent. The in vivo association of
endogenous CtIP with BRCA1 was also demonstrated in other cell types,
including breast cancer lines (MCF7 and T47D), a bladder carcinoma line
(T24), and a T cell leukemia line (Jurkat) (data not shown).
The experiment presented in Fig. 3A indicates that the
BRCA1-specific antiserum immunoprecipitates BRCA1 polypeptides from HBL-100 lysates quantitatively (compare lanes 1 and
5). Given this information, it is possible to estimate the
fraction of cellular CtIP that exists in a BRCA1-bound state from the
results of Fig. 3B. As shown, the intensity of the CtIP band
immunoprecipitated with the BRCA1-specific antiserum (lane
5) is comparable with that of the CtIP band obtained from 0.1 mg
of untreated cell lysate (lane 1). Since the
immunoprecipitated CtIP in lane 5 was derived from 1.2 mg of
cell lysate, it appears that only a minor fraction (5-20%) of the
cellular CtIP pool is bound to BRCA1 in unsynchronized HBL-100 cells.
Similar estimates for the proportion of BRCA1-bound CtIP polypeptides
were obtained in other co-immunoprecipitation experiments using lysates
of HBL-100 epithelial cells or Jurkat lymphoblasts (data not shown).
CtIP Expression during Cell Cycle Progression--
Since the
expression of BRCA1 is cell cycle-dependent (5-8), we were
interested in knowing whether CtIP expression is also regulated with
respect to the cell cycle. Therefore, the steady-state levels of CtIP
mRNA and protein were measured in synchronized populations of
cultured cells. For this purpose, T24 bladder carcinoma cells were
arrested in G0 by contact inhibition (47). The arrested cells were then induced to grow by replating at low density, and equivalent cultures of the replated cells were harvested at various times after induction. For each time point, the cell cycle distribution profile was determined by fluorescence-activated cell sorter analysis (Fig. 4), and the steady-state levels of
CtIP mRNA and protein were evaluated by Northern hybridization
(Fig. 4) and Western analysis (Fig. 5),
respectively.
View larger version (55K):
[in this window]
[in a new window]
|
Fig. 4.
Northern blot analysis of mRNA expression
in synchronized T24 cells. T24 bladder carcinoma cells were
synchronized in a quiescent state by contact inhibition. At time 0, the
cells were replated at low density to induce cell cycle progression.
RNA obtained from cells harvested at various times after induction were
analyzed for levels of CtIP (panel A), BRCA1 (panel
B), cyclin A (panel C), and G3PDH (panel D)
transcripts by Northern hybridization. An asynchronous population of
T24 cells was also examined (lane 1). In addition, the cell
cycle distribution of the synchronized cultures at each time point was
determined by fluorescence-activated cell sorter analysis, and the cell
cycle distribution at each time point is listed beneath panel
D.
|
|
View larger version (55K):
[in this window]
[in a new window]
|
Fig. 5.
Expression of CtIP polypeptides during cell
cycle progression. T24 bladder carcinoma cells were synchronized
in a quiescent state by contact inhibition. At time 0, the cells were
replated at low density to induce cell cycle progression. Lysates of
cells harvested at various times after induction were immunoblotted
with antibodies specific for CtIP (panel A), CDK2
(panel B), BRCA1 (panel C), and cyclin A
(panel D). A lysate of asynchronous T24 cells was also
evaluated (lane 1). The cell cycle distribution of the
synchronized cultures at each time point is listed in Fig. 4.
|
|
As shown in Fig. 4, CtIP transcripts of approximately 3.6 kilobases
were detected by Northern analysis of RNA from unsynchronized T24 cells
(panel A, lane 1). Although the level of CtIP
transcripts was somewhat lower in G0/G1 cells
(panel A, lanes 2-4), this level increased
approximately 2-fold as cells began to traverse the G1/S
boundary at 16 h post-induction (panel A, lane
5). This level was then maintained until 28 h post-induction,
by which time most of the cycling cells had completed mitosis and
re-entered the G1 phase (panel A, lane
8). In contrast, BRCA1 and cyclin A transcripts were not detected
in resting cells or G1-cycling cells (panels B
and C, lanes 2-4). As expected, however,
transcription of these genes increased markedly at the G1/S
transition (panels B and C, lanes 5)
and remained at high levels throughout S and G2/M (panels B and C, lanes 6-8).
In contrast to the results obtained by Northern hybridization, Western
analysis revealed that the steady-state levels of CtIP protein
fluctuate significantly during cell cycle progression. As seen in Fig.
5, CtIP polypeptides were barely detectable in resting cells and
G1-cycling cells (panel A, lanes
2-5). However, CtIP expression increased markedly after the
G1/S transition (panel A, lane 7),
and high levels of CtIP protein were maintained throughout S and
G2/M (panel A, lanes 7-9). The
expression patterns of BRCA1 and other cell cycle regulatory proteins
were as expected (panels B-D). For example, the steady-state
levels of CDK2 remained relatively constant during cell cycle
progression (panel B). In contrast, BRCA1 and cyclin A were
undetectable in resting cells or G1-cycling cells
(panels C and D, lanes 2-5). Their
protein levels increased after the G1/S transition and
peaked in the S and G2/M phases (panels C and
D, lanes 7-9). Thus, although the expression
patterns of CtIP and BRCA1 mRNA transcripts are quite different
with respect to the cell cycle (Fig. 4), the steady-state levels of
their respective protein products are induced with the same kinetics
during cell cycle progression (Fig. 5).
The Subcellular Localization of CtIP--
To determine the
subcellular location of CtIP, we prepared nuclear, cytoplasmic, and
membrane fractions from unsynchronized cultures of T24 cells. The
quality of these fractions was assessed by Western analysis with
antibodies specific for either the nuclear matrix protein NuMA or the
cytoplasmic protein -tubulin. The same fractions were also examined
by immunoblotting with BRCA1- and CtIP-specific antibodies. As shown in
Fig. 6, CtIP was found in the nuclear
fraction of asynchronous T24 cells (panel B) along with
BRCA1 (panel A) and NuMA (panel C).
Significantly, -tubulin was observed exclusively in the cytoplasmic
fraction (panel D), indicating that cross-contamination of
the nuclear compartment with cytosolic proteins was minimal.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
CtIP resides in the nucleus. Whole cell
(W), nuclear (N), cytoplasmic (C), and
membrane (M) fractions were prepared from unsynchronized T24
cells (panels A-D). Equivalent aliquots of each fraction
were then subjected to Western analyses with antibodies specific for
BRCA1 (panel A), CtIP (panel B), NuMA
(panel C), or -tubulin (panel D).
|
|
Mapping the BRCA1 Interaction Domain of CtIP--
Having shown
that CtIP associates with BRCA1 in vivo, we wished to define
the specific sequences of CtIP that mediate its interaction with BRCA1.
Therefore, we constructed a series of bacterial expression vectors that
encode GST fusion proteins containing different segments of CtIP. Each
of the GST-CtIP fusion proteins was expressed in E. coli and
purified from bacterial extracts by affinity chromatography on
glutathione-agarose beads. The SZ fragment of BRCA1 was then produced
by in vitro translation in the presence of
[35S]methionine, and the in vitro interaction
between the radiolabeled SZ fragment and each of the different GST-CtIP
proteins was measured in a GST pull-down assay. As shown in Fig.
7A, the SZ fragment of BRCA1
bound the GST-CtIP (45-620), GST-CtIP (133-462), GST-CtIP(58-369), and GST-CtIP (133-369) fusion proteins strongly (lanes 3,
6, 7, and 8, respectively) and the
GST-CtIP (282-369) fusion protein weakly (lane 9). In
contrast, the SZ sequences did not bind either the parental GST
polypeptide (lane 2) or the GST-CtIP (621-897) and GST-CtIP
(45-132) fusion proteins (lanes 4 and 5,
respectively). Thus, amino acid residues 133-369 of CtIP are required
for efficient in vitro association with BRCA1.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 7.
Mapping the BRCA1-interacting sequences of
CtIP in vitro and in vivo.
A, the SZ fragment of BRCA1 (residues 1528-1863) was
synthesized by in vitro translation in the presence of
[35S]methionine. An aliquot of the radiolabeled SZ
fragment (2 µl) was fractionated by SDS-PAGE (lane 1).
Additional aliquots (6 µl) were incubated with glutathione-agarose
beads loaded with either the parental GST polypeptide (lane
2) or with a GST fusion protein containing the indicated segment
of CtIP (lanes 3-9). The beads were then washed and boiled
in sample buffer, and the eluants were fractionated by SDS-PAGE. The
presence of the radiolabeled SZ polypeptide in each eluant was then
detected by autoradiography. The arrow indicates the
mobility of the in vitro translated SZ polypeptide. The
mobilities of the molecular mass markers are shown on the left of the
image (in kilodaltons). B, individual cultures of 293 cells
were transfected with the G5LUC reporter plasmid, the -galactosidase
control plasmid, and the two indicated expression vectors. The GAL4p
expression vector encoded either the parental GAL4p DNA binding domain
(+) or the indicated GAL4-CtIP hybrid polypeptide; the CtIP amino acid
residues present within each GAL4-CtIP hybrid are shown. The VP16
expression vector encoded either the parental VP16 transactivation
domain (+) or the VP16-SZ hybrid polypeptide. Duplicate transfections
were conducted for each combination of expression plasmids, and the
normalized luciferase activities obtained from each transfection are
illustrated. RLU, relative light units.
|
|
The BRCA1-interacting sequences of CtIP were also evaluated in the
mammalian two-hybrid system (46, 50). For this purpose, we constructed
a series of mammalian expression vectors that encode the DNA binding
domain of GAL4p fused to different segments of CtIP. Each GAL4-CtIP
expression plasmid was co-transfected into 293 cells along with a
vector encoding either the VP16 transactivation domain alone or the
VP16-SZ hybrid protein. After 48 h, the cells were lyzed, and the
luciferase activity of each lysate was determined. As shown in Fig.
7B, the CtIP (45-897) and CtIP (133-369) segments of CtIP
interact with BRCA1 to a comparable degree in this assay (lanes
4 and 12). In contrast, the interaction of CtIP
(282-369) with BRCA1 is much lower (lane 16). Therefore,
amino acid residues 133-369 of CtIP are required for efficient
association with BRCA1 both in vitro and in
vivo.
The Existence of a Protein Complex Containing CtIP, BRCA1, and
BARD1--
Since BARD1 and CtIP interact with distinct regions of the
BRCA1 polypeptide (25, 27, 28), it is possible that both proteins can
associate with the same molecule of BRCA1, allowing the formation of a
multimeric complex that includes all three proteins. To examine this
possibility, a "bridge" two-hybrid experiment was conducted in
human 293 cells. The bridge experiment is a variation of the two-hybrid
assay that allows the detection of in vivo interactions involving three or more proteins (46). Therefore, 293 cells were
co-transfected with two expression vectors: one that encodes the DNA
binding domain of GAL4p fused to residues 26-142 of BARD1 (the
GAL4-BARD1 hybrid) and one encoding the transactivation domain of VP16
fused to residues 45-897 of CtIP (the VP16-CtIP hybrid). As shown in
Fig. 8, co-expression of GAL4-BARD1 and
VP16-CtIP did not induce a significant increase in luciferase activity
(lane 7), indicating that the BARD1 and CtIP moieties of
these hybrids do not interact in vivo. However, a marked
increase in luciferase activity occurred when the GAL4-BARD1 and
VP16-CtIP expression plasmids were co-transfected with a plasmid
encoding full-length BRCA1 (lane 8). This suggests that
BRCA1 can interact simultaneously with both the GAL4-BARD1 and
VP16-CtIP hybrids, allowing the formation of a trimeric protein complex
(GAL4-BARD1/BRCA1/VP16-CtIP) that bridges the GAL4-BARD1 and VP16-CtIP
hybrids and induces expression of the GAL4-responsive reporter gene. In
contrast, BRCA1 polypeptides that have the tumor-associated C61G
missense mutation did not induce reporter gene transcription
(lane 9), presumably because this mutation abolishes the
interaction between BRCA1 and the GAL4-BARD1 hybrid. These results
suggest that BRCA1, BARD1, and CtIP have the potential to form a
trimeric protein complex in mammalian cells.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 8.
Mammalian bridge two-hybrid analysis of a
protein complex involving CtIP, BRCA1, and BARD1. Each culture of
293 cells was co-transfected with the G5LUC reporter plasmid, the
-galactosidase control plasmid, and the three indicated expression
vectors. 1) The Gal4-X expression vector encoded either the parental
GAL4p DNA binding domain (+) or the GAL4-BARD1 hybrid
polypeptide. 2) The VP16-Y expression plasmid encoded either the
parental VP16 transactivation domain (+) or the VP16-CtIP hybrid
protein. 3) The BRCA1 expression plasmid encoded either wild type
(wt) BRCA1 or a derivative containing the tumor-associated
C61G missense mutation; in some cultures, the empty expression vector
(pCMV4) was used in lieu of the BRCA1 expression plasmid (lanes
1, 4, and 7). Duplicate transfections were
conducted for each combination of expression plasmids, and the
normalized luciferase activities obtained from each transfected culture
are illustrated. RLU, relative light units.
|
|
The formation of a protein complex involving the endogenous CtIP,
BRCA1, and BARD1 polypeptides was also evaluated. For this purpose,
lysates of HBL-100 cells were immunoprecipitated with a BARD1-specific
rabbit antiserum, and the presence of CtIP polypeptides in the
resulting immunoprecipitate were examined by Western analysis with the
CtIP-specific monoclonal antibody. As illustrated in Fig.
3B, CtIP polypeptides were co-immunoprecipitated with the BARD1-specific antiserum (lane 7) but not with the
corresponding pre-immune serum. This indicates that CtIP and BARD1 can
exist in the same protein complex in vivo, presumably by
virtue of their simultaneous interaction with BRCA1. In addition, the
intensities of the CtIP bands that were co-immunoprecipitated with the
BARD1-specific (lane 7) and BRCA1-specific (lane
5) antisera are comparable, suggesting that most, if not all,
CtIP-bound BRCA1 molecules are also associated with BARD1.
The Effect of Genotoxic Stress on the CtIP/BRCA1
Interaction--
BRCA1 and BARD1 are stable partners in the sense that
both proteins remain associated during the cellular response to DNA damage (11). In contrast, Li et al. (51) recently reported that the interaction between BRCA1 and CtIP is disrupted upon DNA
damage by treatment with UV light or adriamycin. To explore this
phenomenon, we tested the effects of genotoxic stress on the stability
of the BRCA1/CtIP interaction in HCT116, a line of human colon
carcinoma cells. Thus, cultures of HCT116 cells were subjected to
either UV-C irradiation (10 J/cm2), adriamycin (0.2 µg/ml), or hydrogen peroxide (10 mM), as described under
"Experimental Procedures." After treatment, cells were harvested, and lysates were prepared from the genotoxin- and mock-treated cultures. The protein composition of each lysate was then examined by
immunoblotting with monoclonal antibodies specific for BRCA1, CtIP,
p53, and actin. As shown in Fig.
9A, BRCA1 from mock-treated cultures migrates as a broad band with a molecular mass greater than
220 kilodaltons (lanes 1, 3, and 6).
However, BRCA1 polypeptides from UV- and adriamycin-treated cells
migrate more slowly (lanes 2 and 4), consistent
with the fact that BRCA1 becomes hyperphosphorylated in cells subjected
to these agents (11-13). A more modest reduction in the
electrophoretic mobility of BRCA1 was seen in cells treated with
hydrogen peroxide (lane 5). In addition, the steady-state levels of BRCA1 in HCT116 cells were diminished by genotoxic stress (lanes 2, 4, and 5), consistent with
previous observations (52).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
The effect of genotoxic stress on the
expression of BRCA1 and CtIP. Cultures of HCT116 colon carcinoma
cells were subjected to various forms of genotoxic stress, as described
under "Experimental Procedures." After treatment, the cultures were
harvested, and lysates were prepared from untreated and
genotoxin-treated cells. A, Western analysis of endogenous
BRCA1 in untreated cells (lanes 1, 3, and
6) and in cells exposed to UV irradiation (lane
2), adriamycin (ADR, lane 4), or hydrogen
peroxide (lane 5). B, Western analysis of the
endogenous CtIP, p53, and actin polypeptides of untreated cells
(lane 1) and cells exposed to UV irradiation (lane
2), adriamycin (lane 3), or hydrogen peroxide
(lane 4). The mobilities of the molecular mass markers are
shown on the left of the images (in kilodaltons).
|
|
The effect of genotoxic stress on the steady state levels of p53 and
CtIP polypeptides was also evaluated by Western analysis. As shown in
Fig. 9B, p53 levels were significantly higher in HCT116 cells exposed to UV irradiation (lane 2) or hydrogen
peroxide (lane 4), and they were dramatically higher in
cells treated with adriamycin (lane 3). In contrast, these
treatments did not appreciably alter the steady state levels of CtIP
(Fig. 9B).
The effect of genotoxic stress on the CtIP/BRCA1 interaction was
evaluated in the same cells. Thus, equivalent aliquots of each lysate
were co-immunoprecipitated with the BRCA1-specific antiserum, and the
presence of CtIP in each immunoprecipitate was determined by
immunoblotting with the CtIP-specific monoclonal antibody. As
illustrated in Fig. 10A,
CtIP was co-immunoprecipitated from untreated HCT116 cells with the
BRCA1-specific antiserum (lane 2). Likewise, CtIP was also
co-immunoprecipitated with this antiserum from cells treated with
either UV light (lanes 3), adriamycin (lane 4),
or hydrogen peroxide (lane 5). Thus, in contrast to a
previous report (51), our results indicate that BARD1 and CtIP remain
associated in cells subjected to a variety of genotoxic agents.
Furthermore, the experiment presented in Fig. 10B shows that
endogenous CtIP polypeptides were also immunoprecipitated from each of
these lysates with the BARD1-specific antiserum. Thus, the multimeric
protein complex containing CtIP, BRCA1, and BARD1 also appears to
remain stable during the cellular response to genotoxic stress.
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 10.
The effect of genotoxic stress on the
CtIP/BRCA1 interaction. A, lysates were prepared from
untreated (lanes 1 and 2) and UV (lane
3)-, adriamycin (lane 4)-, and hydrogen peroxide-
(lane 5)-treated HCT116 cells. Equivalent aliquots of each
lysate (1.0 mg) were immunoprecipitated with the BRCA1-specific
antiserum (lanes 2-5) or the corresponding pre-immune serum
(lane 1). The immunoprecipitates were then fractionated by
SDS-PAGE, and the presence of CtIP polypeptides in each
immunoprecipitate was examined by immunoblotting with the CtIP-specific
monoclonal antibody. B, lysates were prepared from untreated
(lanes 1 and 2), UV-treated (lane 3),
and adriamycin-treated (lane 4) HCT116 cells. Equivalent
aliquots of each cell lysate were immunoprecipitated with a
BARD1-specific antiserum (lanes 2-4) or the corresponding
pre-immune serum (lane 1). The presence of CtIP in each
immunoprecipitate was determined by Western analysis with the
CtIP-specific monoclonal antibody. The arrows denote the
electrophoretic mobility of endogenous CtIP; the mobilities of the
molecular mass markers are shown to the left of each image (in
kilodaltons).
|
|
| |
DISCUSSION |
Previous studies have shown that the expression and subcellular
localization of BRCA1 change dramatically during the cell cycle (5-8,
10-13). In particular, the steady-state levels of BRCA1 polypeptides
are low in resting cells, and they remain low during the early
G1 phase of cell cycle progression. However, due to an
induction of BRCA1 synthesis that occurs at the G1/S boundary, these levels are substantially higher in S and G2
phase cells. In addition, immunostaining experiments have shown that although BRCA1 polypeptides are diffusely distributed in the nuclei of
resting and G1 cycling cells, as proliferating cells cross the G1/S boundary, BRCA1 aggregates in distinct nuclear
structures (the "BRCA1 nuclear dots") together with the BRCA2,
Rad51, and BARD1 polypeptides (10, 11, 53). Furthermore, when S phase cells are subjected to genotoxic stress, these proteins reappear in
distinct nuclear structures that contain PCNA and incorporate bromodeoxyuracil (11, 53). It appears, therefore, that DNA damage
elicits the mobilization of BRCA1, BRCA2, Rad51, and BARD1, presumably
as a complex, from BRCA1 nuclear dots to sites of DNA replication.
Thus, although their precise functions have yet to be established, the
BRCA1 nuclear dots may serve as reservoirs for repair proteins that
preserve the integrity of replicating DNA in the face of genotoxic
stress (10, 11, 53). In any event, given these characteristic patterns
of BRCA1 expression and subcellular localization, it is important to
establish where and when CtIP polypeptides are available for
interaction with BRCA1.
CtIP was identified on the basis of its association with three distinct
nuclear proteins, CtBP1, BRCA1, and Rb1 (27-31). Using newly generated
antibody reagents to examine the subcellular distribution of CtIP, we
now show that endogenous CtIP polypeptides also reside primarily in the
nuclear fraction of mammalian cells. However, only a minor fraction
(5-20%) of the endogenous CtIP pool was immunoprecipitated with the
BRCA1-specific antiserum, despite the fact that almost all endogenous
BRCA1 polypeptides are recovered by direct immunoprecipitation with the
same antibody reagent under the same experimental conditions (Fig. 3).
It is possible that the BRCA1/CtIP interaction was not preserved
quantitatively during either cell lysis or the co-immunoprecipitation
procedure. However, a more likely explanation is that not all CtIP
polypeptides are associated with BRCA1 in vivo. For example,
formation of the CtIP/BRCA1 heterodimer may be restricted to a certain
subpopulation of cells (e.g. cells at a particular stage of
cell cycle progression), and/or only a fraction of the CtIP molecules
in a given cell may be bound to BRCA1.
A crude estimate of the proportion of endogenous BRCA1 polypeptides
that are associated with either BARD1 or CtIP can also be obtained from
the co-immunoprecipitation experiments. For example, more than 75% of
the endogenous BRCA1 polypeptides were co-immunoprecipitated with BARD1
(Fig. 3), suggesting that most, if not all, cellular BRCA1 is complexed
with BARD1. This result is consistent with previous immunostaining
data, which show that BARD1 co-localizes perfectly with BRCA1 in the
same nuclear dots of S phase cells (47). In contrast, however, only
10-20% of the endogenous pool of BRCA1 polypeptides was
immunoprecipitated with the CtIP-specific antisera (Fig. 3). Again,
formation of the CtIP/BRCA1 heterodimer may be restricted to a
particular subpopulation of cells, and/or only a subset of the
endogenous BRCA1 pool in a given cell may be bound to CtIP. In either
case, it would be intriguing to know whether the CtIP-bound and
CtIP-free forms of BRCA1 have distinct biochemical functions and
whether one or both of these forms is involved in tumor suppression.
To evaluate CtIP expression with respect to the cell cycle, the
abundance of CtIP gene products was examined in synchronized cells
representing various stages of cell cycle progression. With the
exception of a very modest (2-fold) increase at the G1/S
transition, the steady-state levels of CtIP transcripts remain
relatively constant throughout the cell cycle. In contrast, however,
CtIP protein expression is induced dramatically at the G1/S
transition, in parallel with that of BRCA1. Accordingly, high levels of
CtIP polypeptides were observed in S phase cells, whereas low levels were found in both resting cells and G1 cycling cells.
The steady-state levels of BRCA1 transcripts and polypeptides both
increase markedly at the G1/S boundary, implying that BRCA1 induction is mediated primarily by transcriptional regulation (5-8).
The 2-fold increase in CtIP transcripts that occurs at the
G1/S transition may contribute to the elevated levels of
CtIP protein observed in S phase cells. However, post-transcriptional effects need to be invoked to account for the full induction of CtIP
protein expression that occurs at G1/S. Possible mechanisms of post-transcriptional regulation include a differential efficiency of
CtIP mRNA translation at different stages of the cell cycle or
changes in the stability of CtIP transcripts or polypeptides with cell
cycle progression. Whatever the means by which CtIP expression is
controlled, our results show that the steady-state levels of BRCA1 and
CtIP polypeptides increase in parallel with the onset of DNA synthesis.
CtIP has now been reported to interact with three nuclear proteins
(BRCA1, CtBP1, and Rb1), each of which exhibits tumor suppression activity in some cellular settings and has also been implicated in some
aspect of transcriptional regulation. The interaction with CtBP1
requires a short amino acid motif (PLDLS) that lies in the central
region of CtIP (residues 490-494), whereas the Rb1 tumor suppressor
binds a LXCXE motif located within the N-terminal region (CtIP residues 153-157) (29-31). The data presented in Fig. 7
demonstrate that amino acid residues 133-369 of CtIP are required for
efficient association with BRCA1 both in vivo and in
vitro. Although this region of CtIP encompasses the
LXCXE motif responsible for Rb1 association, it
does not overlap with the CtBP1 binding PLDLS motif. Further studies
will be required to establish whether BRCA1 influences the proposed
interactions of CtIP with either CtBP1 or Rb1 and whether CtIP serves
as a regulatory link between the seemingly distinct pathways of tumor
suppression mediated by BRCA1, CtBP1, and Rb1. In this regard, it is
intriguing that the C-terminal sequences of BRCA1 were recently shown
to interact with RbAp46 and RbAp48, both of which have also been
identified as Rb1-binding proteins (54).
Li et al. (51) recently reported that the in vivo
association of BRCA1 and CtIP is disrupted in cells subjected to agents that induce DNA damage (e.g. UV light) and/or block DNA
replication (adriamycin). On the basis of this observation they
proposed that the BRCA1/CtIP interaction modulates BRCA1-mediated
transcriptional regulation of the p21 gene in response to
genotoxic stress. However, our results indicate that the interaction
between BRCA1 and CtIP remains stable in the face of genotoxic stress
induced by either UV light or adriamycin (Fig. 10). These results were
obtained using well characterized BRCA1- and CtIP-specific antibodies,
and they were observed in various cell types, including the same line
used by Li et al. (HCT116 human colon carcinoma cells) (51).
Although we cannot specify the cause of this discrepancy in the data,
our results indicate that these genotoxic agents do not abrogate the in vivo interaction of BRCA1 and CtIP.
and
TOP
ABSTRACT
INTRODUCTION
Current address: Laboratory of Biochemistry and Molecular Biology,
The Rockefeller University, 1230 York Ave., New York, NY 10021.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
