From the Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Received for publication, April 19, 2002, and in revised form, September 19, 2002
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ABSTRACT |
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Tumor suppressor p53 induces the cellular
response to DNA damage mainly by regulating expression of its
downstream target genes. The human securin is an anaphase inhibitor,
preventing premature chromosome separation through inhibition of
separase activity. It is also known as the product of the human
pituitary tumor-transforming gene, pttg, a
proto-oncogene. Here we report that the expression of human securin is
suppressed in cells treated with the DNA-damaging drugs doxorubicin and
bleomycin. This suppression requires functional p53. Analysis of the
human securin promoter reveals that DNA-binding sites for Sp1 and NF-Y
are both required for activation of securin expression; however, only
the NF-Y site is essential for the suppression by p53. Our study
indicates that securin is a p53 target gene and may play a role in
p53-mediated cellular response to DNA damage.
The securin proteins are a family of functional homologues,
including the Pimples protein in Drosophila, Cut2 in fission
yeast, Pds1 in budding yeast, and vSecurin in vertebrates (1-4).
Securins play a critical role in regulating the separation of sister
chromatids during mitosis. They prevent premature chromosome separation
through inhibition of separase activity by forming tight
securin/separase complexes. At anaphase securins are degraded,
releasing separases, which facilitate the dissociation of sister
chromatids by cleaving the cohesin complex (5). Deletion of securins
has been found to cause abnormal chromosome segregation in fission
yeast and human (6, 7). Interestingly, the budding yeast
Saccharomyces cerevisiae lacking Pds1 fails to arrest when
it is treated with ionizing radiation (8, 9). In addition, ionizing
radiation induces Pds1 phosphorylation at multiple locations, and this
phosphorylation is thought to stabilize Pds1 and is essential for
mitotic arrest (10, 11). These data indicate that Pds1 is involved in
DNA damage checkpoints in budding yeast; however, it is unknown whether its functional homologue, vSecurin, participates in cellular responses to DNA damage in human.
DNA damage checkpoints are mechanisms that arrest the cell cycle and
repair the damaged DNA by regulating expression of relevant genes (12).
They are critical in maintaining genomic stability, which is vital to
the health and survival of the cell. Genetic instability is believed to
be an essential factor in the evolution of cancer in humans (13). One
of the most important proteins controlling cellular responses to DNA
damage in mammals is the tumor suppressor p53 (14, 15). By regulating
expression of its downstream targets, p53 induces cell cycle arrest,
promotes apoptosis, and facilitates DNA repair (14, 16).
To investigate whether vSecurin plays a role in the cellular response
to DNA damage as its homologue Pds1 does in yeast, we examined its
expression in several human cancer cell lines after treatment with the
chemotherapeutic drugs doxorubicin
(Dox)1 and bleomycin (Blm),
both of which induce strand breaks in DNA (17, 18). Although the
treated cells were mainly arrested at G2 phase, we
found that the securin protein levels were dramatically reduced by Dox
and Blm treatment. This drug-induced suppression of securin was
strictly dependent on the presence of functional p53. Further studies
on the human securin promoter revealed that activation of p53 by DNA
damage reduced DNA binding of the transcription factor NF-Y to
this promoter, resulting in repression of transcription. Our data
clearly indicate that securin is a downstream target of p53 and suggest
that suppression of securin plays an important role in cellular
response to DNA damage.
Plasmid Constructs--
The wild-type p53
(pCMVp53wt) and dominant negative mutant p53
(pCMVp53mt135) expression vectors were obtained from
Clontech Laboratories (Palo Alto, CA). A DNA
fragment encoding the influenza hemagglutinin (HA) epitope was fused to
the 5'-end of the p53mt135 coding region using PCR to
generate an HA-tagged p53mt135 expression construct,
pCMVHAp53mt135. The control plasmid pCMV Cell Culture, Transfection, and Luciferase Assay--
HCT116,
U2OS, Saos2, and DLD-1 were obtained from American Type Culture
Collection (ATCC, Manassas, VA) and maintained according to the ATCC
instructions. For transfection, U2OS and HCT116 were seeded into
12-well tissue culture plates and transfected using TransIT-LT1 transfection reagent according to the
manufacturer's instruction (Panvera). For each well, a total of 1 µg
of plasmid DNA containing 0.2 µg of promoter construct, 0.2 µg of
pCMV Northern and Western Blotting--
Cells were grown in 100-mm
tissue culture dishes with or without treatment of Dox (0.2 µM for U2OS, 0.5 µM for HCT116, 0.1 µM for Saos2, and 0.25 µM for DLD-1) or Blm
(50 microunits/ml for U2OS and HCT116, 25 microunits/ml for Saos2 and
DLD-1). Twenty-hours later, nocodazole (Noc) was added to a final
concentration of 0.2 µg/ml for HCT116, Saos2, and DLD1 and 0.1 µg/ml for U2OS as indicated. After incubation for an additional
16-18 h, cells in one set of the dishes were lysed with TRIzol reagent
(Invitrogen, Carlsbad, CA) for total RNA isolation according to the
manufacturer's instruction. The cells in another set were washed once
with cold phosphate-buffered saline and lysed with radioimmune
precipitation assay buffer for total protein isolation as previously
described (19). For detection of securin RNA transcript, Northern
blotting was performed as previously described (20). For detection of securin protein, the whole cell lysate was resolved by 12% SDS-PAGE. After electrophoresis, the proteins were transferred onto a
polyvinylidene difluoride membrane. The membrane was probed with a
rabbit anti-human securin antibody (2.5 µg/ml) (catalogue No.
34-1500, Zymed Laboratories, South San Francisco, CA) and subsequently
detected using the ECL Plus system (Amersham Biosciences, Piscataway,
NJ). For detection of Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared using the Nuclear Extract Kit from Active Motif (Carlsbad,
CA). A double-stranded synthetic DNA oligomer containing the CCAAT
boxes encompassing the initiation site of the human securin promoter
( DNA-damaging Agents Induce Suppression of Securin
Expression--
To study the regulation of securin in response to DNA
damage, we treated the human osteosarcoma cell line U2OS and the human colorectal cancer cell line HCT116, both containing functional p53,
with Dox and Blm. The treated cells were arrested at both G1 and G2 phases of the cell cycle, with most
cells arrested at G2 (Fig.
1A), indicating that these
cells contain functional cell cycle checkpoints. It has been reported
that human securin expression is cell cycle-dependent. Its
level is low in G1 phase, increases as cells enter S, and
peaks in G2/M (21). In U2OS and HCT116, the securin protein
was readily detectable in the untreated cycling cells by Western
blotting (Fig. 1B). As a control, we treated cells with the
anti-microtubule drug nocodazole and found the majority of cells were
arrested at M phase (Fig. 1A and data not shown). In
agreement with the previous findings (21), a strong band with a higher
apparent molecular mass than that in the untreated cycling cells was
observed in cells treated with nocodazole (Fig. 1B). This
band is presumably the phosphorylated form of the securin protein (21).
Surprisingly, the securin level was significantly lower in the Dox- and
Blm-treated U2OS and HCT116 cells, despite that the majority of the
treated cells were arrested at G2 (Fig. 1B),
indicating that securin expression was suppressed in cells treated with
DNA-damaging drugs. Because p53 plays a critical role in regulating
cellular response to DNA damages, we next examined securin expression
in p53-deficient human osteosarcoma Saos2 and colorectal cancer DLD-1
cells. When these cells were treated with Dox and Blm, only
G2 arrest was observed (Fig. 1C), consistent with the previous finding that p53 is required for DNA damage-induced G1 arrest (22). Interestingly, no securin suppression was
observed in the drug-treated Saos2 and DLD-1 cells. Instead, a higher
level of securin protein was found in the drug-treated cells compared with the untreated cycling cells (Fig. 1D), indicating that
the DNA damage-induced suppression of securin is
p53-dependent.
We next examined the securin RNA transcripts in cells treated with Dox
and Blm. The mRNA was readily detected in all untreated cells (Fig.
2, A-C). However, the
mRNA levels were profoundly diminished in U2OS and HCT116 cells
treated with drugs (Fig. 2, A and B). In
contrast, Dox and Blm treatment did not change the mRNA level in
p53-deficient DLD-1 cells (Fig. 2C). These data indicate
that the DNA damage-induced decrease in securin protein is, at least in
part, attributed to the transcriptional repression of the securin gene,
and requires the presence of a functional p53.
DNA Damage-induced Repression of the Securin Promoter Is Mediated
by p53--
To investigate how p53 was involved in the DNA
damage-induced suppression of securin, we analyzed the activity of the
human securin promoter during DNA damage. A 4-kb DNA fragment
containing the human securin promoter has been cloned in our
laboratory. Deletion mutagenesis studies reveal that the majority of
promoter activity is retained within the region from nucleotide
position
In agreement with the previous findings that DNA-damaging agents induce
p53 accumulation (23), p53 levels were significantly elevated in both
U2OS and HCT116 cells after treated with Dox and Blm (Data not shown).
Considering the fact that Dox and Blm only reduced securin expression
in p53-proficient cells, but not in p53-deficeint cells (Fig. 1), we
reasoned that transcriptional repression of the securin promoter by
DNA-damaging agents may be attributed to the accumulation of p53. To
test this possibility, we examined the effects of p19ARF on
luciferase expression from p-710Luc in U2OS and Saos2 cells. The
wild-type (wt) p53 has a very short half-life due to its rapid degradation mediated by Mdm2 (24, 25). Expression of p19ARF
blocks Mdm2 function, thereby inducing p53 accumulation (26, 27). In
p53-proficient U2OS cells, co-transfection of a p19ARF
expression vector, pcDNAp19ARF, significantly
suppressed luciferase expression from p-710Luc in a
dose-dependent manner (Fig. 3B). In contrast,
transfection of pcDNAp19ARF had no effect on p-710Luc
in p53-deficient Saos2 cells (Fig. 3B), suggesting that
activation of p53 is required for suppressing transcriptional
activation of the securin promoter. We next tested whether the direct
introduction of wt p53 would suppress securin promoter activity. The
promoter construct p-710Luc was co-transfected with wt p53 or mutant
p53 (p53mt135) expression constructs (pCMVp53wt
and pCMVHAp53mt135, respectively) into U2OS and Saos2
cells. p53mt135 is a dominant negative mutant containing a
single amino acid mutation, changing Cys-135 to Tyr, within its
DNA binding domain (28). Expression of the exogenous wt p53 suppressed
luciferase expression in both cell lines, whereas expression of the
mutant p53 failed to do so (Fig. 3C), indicating that p53 is
directly involved in repression of the securin promoter.
To further confirm that suppression of the securin promoter during drug
treatment was caused by p53 activation, we co-transfected p-710Luc with
pCMVHAp53mt135 into U2OS cells and treated the cells with
Dox. In the absence of p53mt135, the expression of
luciferase was suppressed significantly by Dox. As the amount of
pCMVHAp53mt135 increased, the degree in Dox suppression of
the luciferase expression was gradually reduced (Fig. 3D).
When a sufficient amount of HAp53mt135 construct was
included, the securin promoter activity was completely restored from
the Dox suppression (Fig. 3D). Because the
p53mt135 alone did not activate p-710Luc in p53-deficient
Saos2 cells (Fig. 3C), restoration of the securin promoter
activity from Dox suppression by p53mt135 is, therefore,
due to the neutralization of the wt p53 activated by drug treatment.
Taken together, these data convincingly demonstrate that DNA damage
agent-induced suppression of securin expression is mediated by p53.
Suppression of Securin Expression by p53 Is Mediated via the CCAAT
Sites Overlapping the Major Start Site on the Securin
Promoter--
The promoter sequence of securin between
Next, we investigated whether CCAAT boxes are required for p53-mediated
repression of the securin promoter. We mutated the CCAAT box by
changing two or more nucleotides in the consensus core sequence of
CCAAT (see "Experimental Procedures"). The mutated promoters were
inserted into pGL3-Basic and tested for their transcription activities.
It is necessary to point out that two CCAAT boxes are found overlapping
the major transcription initiation site, the forward CCAAT-3 with the
consensus sequence
NF-Y Complexes Bound to CCAAT-3 Were Reduced by Dox
Treatment--
To elucidate the mechanism by which p53 represses the
securin promoter, we performed EMSA to identify the proteins binding to
the CCAAT-3 site. A 30-bp oligomer, equivalent to the DNA sequence from
position
Several transcription factors are capable of binding to DNA containing
the CCAAT sequence, such as C/EBP, CBT/NF-1, and CBF/NF-Y (34). A
careful comparison between the consensus binding sequences of each
CCAAT-binding protein with the sequences around the CCAAT-3 in the
human securin promoter revealed that the sequence of this region
matched the binding site for NF-Y (CCAAT) (34) as well as the half site
for NF-1 (GCCAA) (35). NF-Y is a complex of three subunits, NF-YA,
NF-YB and NF-YC (36). To identify which transcription factor forms
complexes with CCAAT-3, we incubated nuclear extracts with antibodies
specifically against NF-1, NF-YA, NF-YB, and NF-YC, respectively,
before the labeled CCAAT-3wt was added. The supershifted bands were
seen in lanes with the nuclear extracts preincubated with antibodies
against NF-YA, NF-YB, and NF-YC (Fig. 5C, lanes
3-5), implying that the complexes contain all three subunits of
the NF-Y transcription factor. In contrast, no supershifts were found
in lanes with nuclear extracts preincubated with NF-1 antibody as well
as with normal IgGs from rabbit and goat (Fig. 5C,
lanes 6-8). A similar pattern of the supershift by
anti-NF-Y antibodies was also observed in lanes with nuclear extracts
of the Dox-treated cells (Fig. 5C, lanes 10-12).
These data indicate that the nuclear proteins binding to CCAAT-3 are NF-Y transcription factors. More interestingly, we found again that the
band intensities on the gel representing NF-Y·DNA complexes were much
lower in lanes with nuclear extract of the Dox-treated cells compared
with those in lanes with untreated cell extracts (Fig. 5C,
comparing lanes 2-8 and lanes 9-15). To
investigate whether this reduction in DNA-binding of NF-Y during Dox
treatment was p53-dependent, we performed EMSA with nuclear
extracts from p53-deficient Saos2 cells. A specific high molecular
weight band was found in both lanes with nuclear extracts from
untreated and Dox-treated cells (Fig. 5D, lanes 2 and 5). The bands were supershifted by NF-YA antibody (Fig.
5D, lanes 3 and 6), indicating that it is also the transcription factor NF-Y that binds to CCAAT-3 in Saos2
cells. However, there was no significant difference in the intensities
of the band between lanes with nuclear extracts from the untreated and
Dox-treated cells (Fig. 5D), suggesting that Dox treatment
does not reduce NF-Y binding to CCAAT-3 in Saos2 cells. To quantify DNA
binding of NF-Y, we performed EMSA with nuclear extracts from both U2OS
and Saos2 cells (Fig. 6A,
upper panel). As a control, a duplicate reaction was set up
and the sample was resolved on a SDS-PAGE gel, which was then stained with Coomassie Blue (Fig. 6A, lower panel),
showing equal amount of nuclear extracts was used in each reaction. We
used a PhosphorImager to measure radiation signals emitted from the
NF-Y·DNA complexes on the gel. As shown in Fig. 6B,
formation of the NF-Y·DNA complexes was reduced by 70% in
Dox-treated U2OS cells compared with that in the untreated cells.
However, Dox treatment did not have any effect on the complex formation
in Saos2 cells (Fig. 6B). These data indicate that
repression of the securin promoter by the DNA-damaging agent Dox is
attributed to the decline in NF-Y binding to its DNA sites, which
requires the presence of functional p53.
One of the possible mechanisms leading to the reduction of NF-Y DNA
binding is that expression of one or more NF-Y subunits may be
decreased in Dox-treated U2OS cells. To explore this possibility, we
prepared whole cell lysate from U2OS cells with or without Dox
treatment and examined the expression of each NF-Y subunit by Western
blotting. We did not detect any changes in protein level for any of the
NF-Y subunits in cells with Dox treatment compared with those found in
cells without Dox treatment (data not shown), indicating that NF-Y
expression is not affected by the elevated level of p53. Another
possibility is that p53 may interact with NF-Y and prevent it from
binding to DNA, similar to the previously reported suppression of human
hsp70 promoter by p53 (31). To investigate this possibility,
we performed EMSA with U2OS nuclear extract preincubated with purified
recombinant wt p53 protein or the same amount of BSA (Fig.
7, upper panel). To
demonstrate that the equal amount of nuclear extracts was used in each
EMSA reaction, samples from duplicate reactions were resolved by
SDS-PAGE and visualized by Coomassie Blue staining (Fig. 7, lower
panel). The recombinant wt p53 protein was purchased from BD
Pharmingen, which was expressed in a Baculovirus expression system and
purified from Sf9 insect cell lysate. As shown in Fig. 7,
addition of recombinant wt p53 protein into nuclear extract resulted in
a significant reduction in formation of the NF-Y·CCAAT complex,
whereas the addition of BSA failed to do so. Because no apparent
supershifted bands were seen in the lane with nuclear extract
preincubated with p53 protein (Fig. 7), it is unlikely that p53 forms a
ternary complex with NF-Y·DNA. This is consistent with the finding
that no supershift bands were found in the EMSA with nuclear extract
from Dox-treated U2OS cells (Fig. 5A). Considering that p53
has been shown to interact with NF-Y in repression of human
hsp70 promoter (31), we conclude that p53 interacts with NF-Y and prevents it from binding to the CCAAT site, therefore, resulting in the decreased securin gene transcription.
DNA damage activates p53, which induces cell cycle arrest,
allowing for repair of the damage. If the damage is beyond repair, p53
promotes programmed cell death. p53 functions mainly through inducing
or inhibiting expression of its target genes (16, 37, 38). For example,
p53 causes cell cycle arrest at G1 by stimulating expression of the CDK inhibitor p21, while p53-induced G2
arrest is mediated by inducing expression of 14-3-36, Gadd45,
p21, and Reprimo, as well as inhibiting Cdc2 and cyclin B1 (39). In
this study, we showed that DNA damage induced by Dox and Blm suppressed expression of securin in the presence of functional p53. We further demonstrated that activation of p53 alone is sufficient to cause repression of the securin promoter. Finally, we provided evidence demonstrating that p53 suppresses securin expression by reducing the
binding of the transcription factor NF-Y to its promoter. Our data
indicate that human securin is a p53 target gene, which is suppressed
in response to DNA damage.
Transcription factor NF-Y has been shown to play an important role in
regulating expression and mediating p53-repression of several cell
cycle-regulated genes, such as cyclin A, cyclin
B1, cyclin B2, cdc2, and cdc25C
(32, 33, 40, 41). The promoter of human securin contains four CCAAT
boxes, which are the DNA-binding sites for NF-Y. Interestingly, our
data show that not all of these sites are involved in activation and
p53-mediated repression of the promoter. When CCAAT-1 and -2 are
mutated, neither the transcription activity, nor the p53-mediated
repression of the promoter is significantly affected. However, when the
overlapped CCAAT-3 and -4 (represented as CCAAT-3 in this study) are
mutated, both promoter activity and the p53-mediated transcription
repression are severely compromised. This selective utilization of
NF-Y-binding sites for the regulation of securin promoter may be
attributed to the location and the surrounding DNA sequences of each
individual site. For example, CCAAT-1 is located 320 bp away from the
major transcription start site, and there are no other transcription
factor-binding sites nearby. CCAAT-2 is in between two Sp1 sites. It
could be important in activating the securin promoter. However, CCAAT-3
overlaps the major start site and has three Sp1-binding sites
juxtaposed at its upstream. This unique setting may make CCAAT-3 much
more prominent in activating transcription from the promoter and
undermines the potential role of CCAAT-2 in regulating the securin
promoter. The promoter of human securin is TATA-less and contains no
apparent initiator element. The NF-Y has been shown to activate
transcription by recruiting the basal transcription factor TFIID (42)
and co-activators P/CAF and p300 (43, 44). In addition, functional interaction between NF-Y and Sp1 is necessary in regulating expression of many genes, such as human A-myb (45), p27kip
(46), type A natriuretic peptide receptor (47),
metalloproteinsase-2 inhibitor (48), and cystathionine The human securin expression is cell cycle-regulated, which is low in
G1 phase and starts to accumulate in S phase (21). The
chromosomes are duplicated during S phase of the cell cycle. To ensure
the correct distribution of the newly duplicated chromosomes to
daughter cells, the sister chromatids have to be held together until
the cell cycle reaches anaphase when they are separated. As the securin
binds to separase and inhibits its function, the accumulation of
securin during S phase may be necessary to prevent premature separation
of the newly synthesized sister chromatids. Thus, it is conceivable
that a high level of Pds1, a homologue of human securin, is essential
for mitotic arrest in the budding yeast in response to DNA damage (10,
11). However, our data demonstrate that DNA damage results in
suppression of securin in human cells containing functional p53.
Therefore, this suggests that in vertebrates other mechanisms must
exist to hold the sister chromatids together during G2
arrest. Several studies have indicated that securin binding is not the
only mechanism that regulates sister chromatid separation. For example,
deletion of securin does not affect the cell cycle progression of the
mouse embryonic stem cells and human HCT116 (6, 56). Furthermore, the
high activity of Cdc2 has been shown to inhibit anaphase independent of
securin (57).
Numerous studies indicate that the vSecurin is a multifunctional
protein. In addition to anaphase inhibition, it is also known as the
product of pttg, which is a proto-oncogene (4, 20, 58). It
stimulates expression of c-myc (59), promotes
angiogenesis (60), and transforms NIH3T3 cells (20, 58). Recently,
securin has been shown to form complexes with the Ku protein (61). The Ku protein is the regulatory subunit of the DNA-dependent
protein kinase (DNA-PK), an essential component in the repair of DNA
double-strand breaks (62, 63). In addition, Ku has also been indicated
in maintaining the stability of telomeres (64, 65). Therefore, the
suppression of securin by p53 may lead to the inhibition of its
functions related to the Ku protein. For example, Dox and Blm have been
known to cause DNA double-strand breaks (17, 18). The main mechanism
for DNA double-strand break repair in mammalian cells is non-homologues
recombination end-joining, in which Ku plays an essential role by
activating DNA-PK. It has been reported that the Ku protein
disassociates from securin in the presence of sonicated DNA (61),
suggesting that Ku is released from the securin binding in the presence
of double-strand breaks. Considering that the securin functions as an
inhibitory protein when it binds to separin, we postulate that securin
may act as an inhibitor of Ku, thereby to prevent formation of DNA-PK
holoenzyme in the absence of DNA double-strand breaks and/or inhibit
other Ku-dependent functions. High levels of securin
expression may lead to the depletion of the function of Ku proteins by
securin binding, therefore adversely affecting the repair process of
the damaged DNA. We hypothesized that DNA damages activate p53, which
in turn suppresses securin expression and decreases its cellular
concentration. Consequently, more Ku proteins free of securin are
available for DNA repair or other Ku-dependent functions,
such as the protection of the telomere. It will be important to clarify
the physiological significance of securin suppression by p53 to fully
appreciate the role of this multifunctional protein in cell cycle
control, DNA damage repair, and malignant transformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
expressing
-galactosidase was obtained from Clontech. A
murine p19ARF expression construct
(pcDNAp19ARF) was kindly provided by R. A. DePinho. For p-710Luc, a DNA fragment containing the human securin
promoter region from
710 to +45 was isolated from
p(
711/+201)-Luc2 and
subsequently subcloned into pGL3-basic vector (Promega, Madison, WI).
The site-specific mutations in Sp1-binding sites and CCAAT boxes were
generated by PCR-based site-directed mutagenesis. For each
site-specific mutation, a pair of primers was designed to carry the
desired mismatched nucleotides according to the promoter sequences (see
below), and the reverse primer was phosphorylated at its 5'-end. PCR
was performed with p-710Luc as the template using a Takara LA
TaqTM Polymerase kit (Panvera, Madison, WI) under the
condition of 94 °C for 1 min, 56 °C for 1 min, and 68 °C for 8 min for a total of 21 cycles. The PCR products were polished with
Pfu DNA polymerase (Promega), treated with DpnI,
purified by agarose gel electrophoresis, and self-ligated. The
authenticity of the generated plasmids was confirmed by DNA sequencing.
The promoters with desired mutations were then subcloned into
pGL3-basic. The PCR primers are as follows (the underlined nucleotides
were mismatched): CTGGCTGCTTAGGTCCTTTC (forward),
pGGCTCCGATCCCAAAAGCTCTTTCTTGC (reverse) for pSp1-1mt; ACATCCACGGCCCCGCCTCCTG (forward), pGGTCACCAAGTAGAACCAATGG
(reverse) for pSp1-2mt; ATCCTCCTGGGCGGAAGAGCCAATAGGGCC
(forward), pGGGCCGTGGGCGTGGTCACCAAGTAGAACC (reverse) for
pSp1-3mt; GGAAGAGCCAATTGGGCCGCGAGTTG (forward), pATCCAGGAGGCGGGGCCGTGGGCGTGGTCACC (reverse) for pSp1-4mt;
AGGTTGTTTCCCCTATTTCTTC (forward),
pAGGTTGGGTCTAAAGAATACG (reverse) for pCAT-1mt;
GGGTTCTACTTGGTGACCAC (forward),
pGGGAAAGGACCTAAGCAGCCAG (reverse) for pCAT-2mt;
ACGTGGGCCGCGAGTTGTGGT (forward), pGGCTCTTCCGCCCAGGAGG
(reverse) for pCAT-3mt. For pSp1-23mt with mutation of Sp1-2 and
Sp1-3, a PCR was performed using primers ATCCTCCTGGGCGGAAGAGCCAATAGGGCC (forward) and
pGGTCACCAAGTAGAACCAATGG (reverse); For pSp1-123mt, a PCR was done
using the primers for pSp1-23mt and pSp1-1mt as the template; For
pSp1-Allmt, a PCR was performed using forward primer for pSp1-4mt,
reverse primer for pSp1-23mt, and pSp1-1mt as the template; For
pSp1-All-CAT-3mt, a PCR was performed using primers
CGTGGGCCGCGAGTTGTGGT (forward) and pTGGCTCTGTCACCAAGTAGAACC
(reverse) with pSp1-Allmt as the template.
, and the others as indicated, plus 3 µl of
TransIT-LT1 reagent were used. Twenty hours after
transfection, the cells were treated with doxorubicin (0.1 µM for U2OS and 0.5 µM for HCT116) or
bleomycin (50 microunits/ml for both cell lines) for an additional
24 h and lysed for 30 min at 4 °C with 200 µl of lysis buffer
(25 mM Trizma (Tris base) phosphate, 2 mM
trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetic acid, 1% Triton X-100, and 10% glycerol). 10 µl of cell
lysate was used for luciferase assay with 150 µl of assay buffer (25 mM Gly-Gly, pH 7.8, 4 mM EGTA, 15 mM Mg2SO4, 2 mM ATP,
and 1 mM dithiothreitol in 15 mM potassium
phosphate buffer, pH 7.8) and 45 µl of luciferin using a luminometer.
Another 10 µl of the lysate was used to measure
-galactosidase
activity. The luciferase activity was finally normalized against the
-galactosidase activity from the same well.
-actin protein, Western blotting was performed
exactly as previously described (19). For detection of p53, the mouse
monoclonal antibody Do-1 (Santa Cruz Biotechnology, Santa Cruz, CA) was
used to probe the membrane. To determine expression of transiently transfected wt and mutant p53, cells were similarly transfected as
described above and lysed with 200 µl of radioimmune precipitation assay buffer. The 5 µg of total protein was then resolved on a 10%
SDS-PAGE. After transfer, the membrane was probed with Do-1 antibody
for wt p53 and HA.11 (Covance, Richmond, CA) for
HA-p53mt135.
17 to +13) was labeled with [
-33P]ATP using T4
polynucleotide kinase. Nuclear extract (4 µg) was preincubated on ice
for 30 min with 1 µg of poly(dI-dC) (Sigma-Aldrich, St. Louis, MO)
with or without unlabeled competitor DNA, in 1× binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 0.25 mM EDTA, 0.5 mM tetrasodium pyrophosphate, and
10% glycerol with freshly added 0.25 mM
phenylmethylsulfonyl fluoride and 0.25 mM dithiothreitol). The end-labeled probe (0.01 pmol, ~30,000 cpm) was added into the mix
and incubated for an additional 20 min at room temperature. For the
supershift assay with antibody, 2 µg of each antibody specific for
NF-YA, NF-YC (H0209 and C-19, Santa Cruz Biotechnology), NF-YB
(Rockland, Gilbertsville, PA), NF-I (Santa Cruz Biotechnology) was
included in the preincubation mixture and the preincubation time was
extended to 1 h on ice. For p53/NF-Y interaction, nuclear extract
(2 µg) was preincubated with 2 µg of the purified recombinant p53
(BD Pharmingen, San Diego, CA) or bovine serum album (BSA) for 2 h
on ice before the labeled probe was added. DNA·protein complexes were resolved on a 5% native polyacrylamide gel in
1× Tris-glycine buffer at 10 mA at 4 °C. The gel was then dried and exposed to Kodak BioMax film. To quantify the protein·DNA complex, the radiation emission from the dried gel was recorded with a Storm
PhosphorImager system (Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
DNA-damaging agents induce growth arrest and
suppress securin expression in cancer cells containing wt p53.
Human cancer cells were treated with Dox and Blm for 36 h. As
controls, cells were also treated with nocodazole (Noc)
alone or with Noc plus Dox and Blm as indicated. Cells without any
treatment are presented as cycling cells. Cell cycle arrest was
determined by FACS as previously described (19), and securin expression
was measured by Western blotting as described under "Experimental
Procedures." A, FACS data for the wt p53-containing U2OS
and HCT116 cells showing growth arrest, mainly at G2 phase.
B, securin protein levels in U2OS and HCT116. C,
FACS data for the p53-deficient Saos2 and DLD-1 cells. D,
securin protein levels in Saos2 and DLD-1.
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Fig. 2.
DNA-damaging agents suppress securin
transcription in cells containing wt p53 but not in cells lacking
functional p53. U2OS (A), HCT116 (B), and
DLD-1 (C) were treated with Dox and Blm as indicated, and
the RNA transcripts of securin were detected by Northern blotting. The
28 S rRNAs in agarose gels stained with ethidium bromide before
transfer are shown as the control for equal total RNA loading.
710 to +201, which contains several Sp1 sites and CCAAT
boxes.2 Further studies showed that DNA sequence from +46
to +210 was not required for the promoter activity (data not shown).
Therefore, we considered the reporter plasmid p-710Luc, containing the
5'-flanking sequence from
710 to +45, as the wild-type promoter
construct. When p-710Luc was transiently transfected into
p53-proficient U2OS and HCT116 cells, treatment with either Dox or Blm
significantly suppressed luciferase expression from this construct
(Fig. 3A). In contrast, no
significant drug-induced suppression of the luciferase activity was
observed in p53-deficient Saos2 and DLD-1 cells (Fig. 3A).
These data indicate that the p-710Luc responds to DNA damage agents in
a similar way as the endogenous securin gene does.
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Fig. 3.
DNA-damaging agent-induced suppression of the
securin promoter is mediated by p53. A, the human
securin promoter activity from the transfected p-710Luc was suppressed
by Dox and Blm in p53-proficient U2OS and HCT116, but not in
p53-defective Saos2 and DLD1. B, transfection of a construct
expressing p19ARF (amount indicated) resulted in a
dose-dependent repression of securin promoter activity from
the co-transfected p-710Luc (0.2 µg) in U2OS (p53-proficient) but not
in Saos2 (p53-deficient). C, co-transfection of a wt
p53-expressing construct (p53wt, amount indicated) with 0.2 µg of p-710Luc resulted in a dose-dependent repression of
securin promoter activity in both U2OS and Saos2 cells, whereas
co-transfection of a construct expressing a HA-tagged dominant negative
mutant p53 (p53mt135) failed to do so in either of the
cells. D, co-transfection of the construct expressing
p53mt135 rescued Dox-induced transcriptional repression of
p-710Luc in U2OS cells. The data was plotted as arbitrary units with
that from the control set as 100. The data was represented as mean ± S.D. for results from at least three independent experiments. The
induction of the p53 protein by Dox and Blm (A) and
transient expression of p53wt or p53mt135
(D and E) were detected by Western blotting to
assure proper protein expression.
710 and +45
consists of four Sp1-binding sites and four CCAAT boxes (Fig.
4A). It has been shown that
p53 inhibits transcription from the human telomerase reverse
transcriptase promoter and the SV40 promoter by repression of Sp1 DNA
binding (29, 30). In addition, p53 suppresses expression of several
genes through CCAAT sites on their promoters (31-33). Therefore, it is
possible that transcription factors interacting with Sp1 sites and
CCAAT boxes are involved in the p53-mediated repression of the securin
promoter. To explore this possibility, we mutated each of the
Sp1-binding sites by changing the consensus sequence GGGCGG
to GGATGG and tested how these mutations affected securin
promoter activity in U2OS cells. Mutation of each Sp1-binding site
individually did not significantly change activity of the promoter
(pSp1-1mt, pSp1-2mt, pSp1-3mt, and
pSp1-4mt in Fig. 4B); however, when two or more
Sp1 sites were mutated simultaneously, the securin promoter activity
was dramatically decreased (pSp1-23mt,
pSp1-123mt, and pSp1-Allmt in Fig.
4B). Then, we tested the activities of these mutant promoter constructs in response to Dox treatment. Interestingly, Dox treatment suppressed the activity of all these constructs by ~90%, very similar to the Dox-mediated suppression of the wt promoter construct p-710Luc (Fig. 4B). These results indicate that the
transcription factor Sp1 is essential for basal transcription activity
of the securin promoter but not required in p53-mediated repression of the promoter.
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Fig. 4.
p53 represses the securin promoter through
CCAAT boxes overlapping the major transcription start site. The
human securin promoter constructs carrying mutated Sp1-binding sites
and CCAAT boxes were generated and tested in U2OS cells as described
under "Experimental Procedures." A, the location for
Sp1-binding sites and CCAAT boxes in the human securin promoter with
their core sequence underlined. B, the left
panel is a schematic representation of human securin promoter
constructs with mutant Sp1-binding sites shown as filled
squares. The middle panel shows the effects of these
mutations on promoter activity in U2OS cells. The data were plotted in
an arbitrary unit, where the activity from cells transfected with the
promoter-less construct pGL3-Basic was set as 1. The
right panel shows the effects of Dox treatment on the
activities of the promoter containing these mutations. The luciferase
activities from the Dox-treated cells are represented as percentages of
those from the untreated cells. C, the left panel
is a schematic representation of human securin promoter constructs with
mutated CCAAT boxes shown as filled circles. The
middle panel shows the effects of these mutations on the
promoter activity in U2OS cells. The right panel shows the
effects of Dox treatment on the activities of the promoter containing
these mutations. D, the reporter constructs carrying the
mutated CCAAT-3 were co-transfected with or without wt p53 expression
construct into U2OS cells. The luciferase activities from cells with wt
p53 construct were represented as percentages of those from the
corresponding cells with no wt p53 construct. All data were represented
as mean ± S.D. for results from at least three independent
experiments.
10AAGAGCCAATTGGGCC+6 and the
reversed CCAAT-4 with the sequence
8GAGCCAATTGGGCCGC+8 (Fig.
4A). The CCAAT-3 and CCAAT-4 overlap each other; therefore, changing the two nucleotides shared by them in the core region mutates
both CCAAT boxes. Thus, they were considered as one CCAAT box
represented by CCAAT-3, and the promoter construct pCAT-3mt actually
contained both mutated CCAAT-3 and CCAAT-4. As shown in Fig.
4C, mutation of CCAAT-1 and -2 did not affect the securin promoter activity in U2OS cells (Fig. 4C,
pCAT-1mt and pCAT-2mt). However, mutation of
CCAAT-3 reduced the promoter activity by ~90% compared with that of
the wt p-710Luc in U2OS cells (Fig. 4C,
pCAT-3mt). More interestingly, Dox treatment only slightly reduced activity of pCAT-3mt, while it significantly suppressed activity of pCAT-1mt and pCAT-2mt (Fig. 4C). In addition,
when the CCAAT-3 site was mutated in pSp1-Allmt, the generated
pSp1-All-CAT-3mt had an activity similar to that of the promoter-less
vector pGL3-Basic, which was much lower than that of pSp1-Allmt (Fig.
4C). Furthermore, Dox treatment failed to suppress the
promoter activity significantly from pSp1-All-CAT-3mt. These data
clearly demonstrate that the CCAAT boxes overlapping the start site,
represented as CCAAT-3, are required not only for transcription
activation, but also for Dox-induced suppression of the securin
promoter, whereas CCAAT-1 and -2 sites are dispensable for both
activities. A similar result was also observed in HCT116 cells (data
not shown). To further examine whether CCAAT-3 was critical for
p53-mediated repression of the securin promoter, pCAT-3mt and
pSp1-All-CAT-3mt were co-transfected with the pCMVp53wt
expression construct into U2OS cells. As expected, the wt p53 only
slightly reduced luciferase expression from the promoters with CCAAT-3
mutations, whereas it significantly reduced expression of luciferase
from their corresponding control promoters, p-710Luc and pSp1-Allmt
(Fig. 4D), demonstrating that CCAAT-3 is indeed the site
through which p53 represses the securin promoter.
17 to +13 of the human securin promoter containing CCAAT-3,
was synthesized and named as CCAAT-3wt. As a control, a similar
oligomer, named as CCAAT-3mt, was also generated to contain a mutated
CCAAT-3 by changing sequences of CCAAT to CCACG (Fig. 5A). CCAAT-3wt was
labeled with 33P and incubated with nuclear extracts
isolated from untreated U2OS cells or cells treated with Dox. The
protein·DNA complexes were analyzed by non-denaturing polyacrylamide
gels. A high molecular weight band was detected in the lane with
nuclear extract from untreated U2OS cells (Fig. 5B,
lane 2). This band diminished when the nuclear extract was
preincubated with excessive unlabeled CCAAT-3wt (Fig. 5B,
lanes 3 and 4), whereas preincubation with the
same amount of the unlabeled CCAAT-3mt did not affect the band (Fig.
5B, lanes 5 and 6). This indicates
that the protein·DNA complex formed with this oligomer is CCAAT-3
sequence-specific. The complexes were also found with nuclear extracts
from the Dox-treated cells (Fig. 5B, lane 7);
however, the intensity of the band was much lower than that with
nuclear extract of the untreated cells (Fig. 5B, comparing
lanes 2 and 7). This suggests that Dox treatment reduces binding of the nuclear proteins to this site.
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Fig. 5.
DNA-damaging agents repress securin
expression through NF-Y. A, oligomers used in EMSA
experiments. CCAATwt contains securin promoter sequence from 17 to
+13. CCAATmt contains the same DNA fragment except that the
CCAAT has been changed to CCACG. B,
nuclear extracts from U2OS with or without Dox treatment were incubated
with the
-33P-labeled CCAATwt and the DNA·protein
complexes were resolved by 5% native polyacrylamide gel. For
competition, the 10- to 100-fold of molar excessive unlabeled CCAATwt
or CCAATmt was preincubated with the nuclear extract before the labeled
probe was added. C, for supershift assays, each of the
antibodies against NF-YA, NF-YB, NF-YC, and NF-1, as well as the
control IgGs from rabbit (R-IgG) and goat (G-IgG)
were preincubated with nuclear extracts from U2OS before the labeled
probe was added. D, EMSA assays were performed using nuclear
extracts from Saos2 cells with or without Dox treatment. NF-YA antibody
was used in the supershift assay.
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Fig. 6.
DNA-damaging drugs induce decline in NF-Y DNA
binding. A, EMSA reactions using nuclear extracts from
U2OS and Saos2 with or without Dox treatment were prepared in
duplicate. After incubated with the -33P-labeled
CCAATwt, the DNA·protein complexes in one set of the reactions were
resolved by 5% native polyacrylamide gel (upper panel). The
samples from the duplicate reactions were resolved by 10% SDS-PAGE,
which was later stained with Coomassie Blue to demonstrate that equal
amounts of nuclear extracts were used in each reaction (lower
panel). B, the NF-Y·DNA complexes from U2OS and Saos2
with or without Dox treatment were quantified by a PhosphorImager. The
reading from the Dox-treated nuclear extracts was compared with that
from its untreated counterpart as 100.
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Fig. 7.
Recombinant wt p53 inhibits NF-Y DNA binding
in vitro. EMSA reactions using U2OS nuclear
extracts were prepared in duplicate. The nuclear extract (2 µg) was
preincubated with recombinant wt p53 (2 µg) or BSA (2 µg) before
the labeled CCAATwt was added. The DNA·protein complexes in one set
of the reactions were resolved by 5% native polyacrylamide gel
(upper panel). The samples from the duplicate reactions were
resolved by 10% SDS-PAGE, which was later stained with Coomassie Blue
to demonstrate that equal amounts of nuclear extracts were used in each
reaction (lower panel). The arrows indicate the
positions of BSA and wt p53.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-synthase-1b
(49). Therefore, it is likely that the CCAAT-3-bound NF-Y plays a
critical role in transcription initiation of the securin promoter,
whereas the Sp1 functions as an activator when bound to the upstream
sequences near the CCAAT-3. The binding of Sp1 to the nearby sequence
may stabilize the DNA binding of NF-Y by direct physical interaction (50, 51) or through interaction with the same co-activator, such as
p300 (52, 53). This is consistent with our finding that both Sp1 and
NF-Y are required for the optimal activation of the securin promoter.
It has been shown that Sp1 interacts with TFIID and is involved in
transcription initiation of tumor necrosis factor
-responsive gene
(54, 55). Thus, Sp1 may become the major player activating
transcription initiation in the absence of CCAAT-3, which may be
responsible for activities of the promoter with mutated CCAAT-3. The
initiation site activated by Sp1 may or may not be the same location as
that activated by the CCAAT-3-bound NF-Y. Nevertheless, it is
interesting to notice that this transcription activation mediated by
Sp1 in the absence of CCAAT-3 is resistant to the p53-mediated
repression. This is not due to the low promoter activity, because the
promoter with all Sp1 sites mutated is still sensitive to p53
expression despite that the promoter activity is even lower than that
lacking CCAAT-3. These results suggest that p53 represses the human
securin promoter by affecting the transcription initiation through the
CCAAT-3-bound NF-Y. Taken together, we propose a mechanism by which DNA
damage induces the suppression of human securin expression. The p53
activated by DNA damages interacts with transcription factor NF-Y,
preventing it from binding to the DNA overlapping the transcription
start site on human securin gene promoter. This reduces transcription initiation from the promoter, thus leading to the expression
suppression of human securin.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. A. DePinho for providing the plasmid pcDNAp19ARF and Dr. A. Klibanski for support and critically reviewing of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Individual National Research Service Award 5 F32 CA88519-02 (to Y. Z.) and Grant IRG-87-007-13 from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Comprehensive Cancer Center, University of
California at San Francisco, San Francisco, CA 94143.
§ Present address: Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115.
¶ To whom correspondence should be addressed: Massachusetts General Hospital, Neuroendocrine Unit, 55 Fruit St., Bulfinch 457, Boston, MA 02114. Tel.: 617-724-7392; Fax: 617-726-5072; E-mail: xzhang5@partners.org.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M203793200
2 K. R. Mehta, Y. Zhou, S. R. Johnson, M. Katai, X. Li, and X. Zhang, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: Dox, doxorubicin; Blm, bleomycin; DNA-PK, DNA-dependent protein kinase; wt, wild-type; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; HA, hemagglutinin; Noc, nocodazole; BSA, bovine serum albumin; FACS, fluorescence-activated cell sorting.
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REFERENCES |
---|
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---|
1. | Cohen-Fix, O., Peters, J. M., Kirschner, M. W., and Koshland, D. (1996) Genes Dev. 10, 3081-3093[Abstract] |
2. | Funabiki, H., Yamano, H., Kumada, K., Nagao, K., Hunt, T., and Yanagida, M. (1996) Nature 381, 438-441[CrossRef][Medline] [Order article via Infotrieve] |
3. | Stratmann, R., and Lehner, C. F. (1996) Cell 84, 25-35[Medline] [Order article via Infotrieve] |
4. |
Zou, H.,
McGarry, T. J.,
Bernal, T.,
and Kirschner, M. W.
(1999)
Science
285,
418-422 |
5. | Nasmyth, K. (2001) Annu. Rev. Genet. 35, 673-745[CrossRef][Medline] [Order article via Infotrieve] |
6. | Jallepalli, P. V., Waizenegger, I. C., Bunz, F., Langer, S., Speicher, M. R., Peters, J. M., Kinzler, K. W., Vogelstein, B., and Lengauer, C. (2001) Cell 105, 445-457[CrossRef][Medline] [Order article via Infotrieve] |
7. | Funabiki, H., Kumada, K., and Yanagida, M. (1996) EMBO J. 15, 6617-6628[Abstract] |
8. |
Cohen-Fix, O.,
and Koshland, D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14361-14366 |
9. | Yamamoto, A., Guacci, V., and Koshland, D. (1996) J. Cell Biol. 133, 99-110[Abstract] |
10. |
Wang, H.,
Liu, D.,
Wang, Y.,
Qin, J.,
and Elledge, S. J.
(2001)
Genes Dev.
15,
1361-1372 |
11. |
Sanchez, Y.,
Bachant, J.,
Wang, H., Hu, F.,
Liu, D.,
Tetzlaff, M.,
and Elledge, S. J.
(1999)
Science
286,
1166-1171 |
12. | Zhou, B. B., and Elledge, S. J. (2000) Nature 408, 433-439[CrossRef][Medline] [Order article via Infotrieve] |
13. | Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1998) Nature 396, 643-649[CrossRef][Medline] [Order article via Infotrieve] |
14. | Lakin, N. D., and Jackson, S. P. (1999) Oncogene 18, 7644-7655[CrossRef][Medline] [Order article via Infotrieve] |
15. | Wahl, G. M., and Carr, A. M. (2001) Nat. Cell Biol. 3, E277-E286[CrossRef][Medline] [Order article via Infotrieve] |
16. | el-Deiry, W. S. (1998) Semin. Cancer Biol. 8, 345-357[CrossRef][Medline] [Order article via Infotrieve] |
17. | Gewirtz, D. A. (1999) Biochem. Pharmacol. 57, 727-741[CrossRef][Medline] [Order article via Infotrieve] |
18. | Povirk, L. F. (1996) Mutat. Res. 355, 71-89[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Zhou, Y.,
Sun, H.,
Danila, D. C.,
Johnson, S. R.,
Sigai, D. P.,
Zhang, X.,
and Klibanski, A.
(2000)
Mol. Endocrinol.
14,
2066-2075 |
20. |
Zhang, X.,
Horwitz, G. A.,
Prezant, T. R.,
Valentini, A.,
Nakashima, M.,
Bronstein, M. D.,
and Melmed, S.
(1999)
Mol. Endocrinol.
13,
156-166 |
21. | Ramos-Morales, F., Dominguez, A., Romero, F., Luna, R., Multon, M. C., Pintor-Toro, J. A., and Tortolero, M. (2000) Oncogene 19, 403-409[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Bunz, F.,
Hwang, P. M.,
Torrance, C.,
Waldman, T.,
Zhang, Y.,
Dillehay, L.,
Williams, J.,
Lengauer, C.,
Kinzler, K. W.,
and Vogelstein, B.
(1999)
J. Clin. Invest.
104,
263-269 |
23. | Nelson, W. G., and Kastan, M. B. (1994) Mol. Cell. Biol. 14, 1815-1823[Abstract] |
24. | Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997) Nature 387, 296-299[CrossRef][Medline] [Order article via Infotrieve] |
25. | Kubbutat, M. H., Jones, S. N., and Vousden, K. H. (1997) Nature 387, 299-303[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Honda, R.,
and Yasuda, H.
(1999)
EMBO J.
18,
22-27 |
27. | Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J., and Bar-Sagi, D. (1999) Nat. Cell Biol. 1, 20-26[CrossRef][Medline] [Order article via Infotrieve] |
28. | Scheffner, M., Takahashi, T., Huibregtse, J. M., Minna, J. D., and Howley, P. M. (1992) J. Virol. 66, 5100-5105[Abstract] |
29. | Xu, D., Wang, Q., Gruber, A., Bjorkholm, M., Chen, Z., Zaid, A., Selivanova, G., Peterson, C., Wiman, K. G., and Pisa, P. (2000) Oncogene 19, 5123-5133[CrossRef][Medline] [Order article via Infotrieve] |
30. | Perrem, K., Rayner, J., Voss, T., Sturzbecher, H., Jackson, P., and Braithwaite, A. (1995) Oncogene 11, 1299-1307[Medline] [Order article via Infotrieve] |
31. | Agoff, S. N., Hou, J., Linzer, D. I., and Wu, B. (1993) Science 259, 84-87[Medline] [Order article via Infotrieve] |
32. |
Yun, J.,
Chae, H. D.,
Choy, H. E.,
Chung, J.,
Yoo, H. S.,
Han, M. H.,
and Shin, D. Y.
(1999)
J. Biol. Chem.
274,
29677-29682 |
33. |
Manni, I.,
Mazzaro, G.,
Gurtner, A.,
Mantovani, R.,
Haugwitz, U.,
Krause, K.,
Engeland, K.,
Sacchi, A.,
Soddu, S.,
and Piaggio, G.
(2001)
J. Biol. Chem.
276,
5570-5576 |
34. |
Mantovani, R.
(1998)
Nucleic Acids Res.
26,
1135-1143 |
35. | Meisterernst, M., Gander, I., Rogge, L., and Winnacker, E. L. (1988) Nucleic Acids Res. 16, 4419-4435[Abstract] |
36. | Mantovani, R. (1999) Gene (Amst.) 239, 15-27[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Zhao, R.,
Gish, K.,
Murphy, M.,
Yin, Y.,
Notterman, D.,
Hoffman, W. H.,
Tom, E.,
Mack, D. H.,
and Levine, A. J.
(2000)
Genes Dev.
14,
981-993 |
38. | Vogelstein, B., Lane, D., and Levine, A. J. (2000) Nature 408, 307-310[CrossRef][Medline] [Order article via Infotrieve] |
39. | Taylor, W. R., and Stark, G. R. (2001) Oncogene 20, 1803-1815[CrossRef][Medline] [Order article via Infotrieve] |
40. | Huet, X., Rech, J., Plet, A., Vie, A., and Blanchard, J. M. (1996) Mol. Cell. Biol. 16, 3789-3798[Abstract] |
41. | Bolognese, F., Wasner, M., Dohna, C. L., Gurtner, A., Ronchi, A., Muller, H., Manni, I., Mossner, J., Piaggio, G., Mantovani, R., and Engeland, K. (1999) Oncogene 18, 1845-1853[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Frontini, M.,
Imbriano, C.,
diSilvio, A.,
Bell, B.,
Bogni, A.,
Romier, C.,
Moras, D.,
Tora, L.,
Davidson, I.,
and Mantovani, R.
(2002)
J. Biol. Chem.
277,
5841-5848 |
43. |
Li, Q.,
Herrler, M.,
Landsberger, N.,
Kaludov, N.,
Ogryzko, V. V.,
Nakatani, Y.,
and Wolffe, A. P.
(1998)
EMBO J.
17,
6300-6315 |
44. |
Currie, R. A.
(1998)
J. Biol. Chem.
273,
1430-1434 |
45. | Facchinetti, V., Lopa, R., Spreafico, F., Bolognese, F., Mantovani, R., Tavner, F., Watson, R., Introna, M., and Golay, J. (2000) Oncogene 19, 3931-3940[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Inoue, T.,
Kamiyama, J.,
and Sakai, T.
(1999)
J. Biol. Chem.
274,
32309-32317 |
47. |
Liang, F.,
Schaufele, F.,
and Gardner, D. G.
(2001)
J. Biol. Chem.
276,
1516-1522 |
48. |
Zhong, Z. D.,
Hammani, K.,
Bae, W. S.,
and DeClerck, Y. A.
(2000)
J. Biol. Chem.
275,
18602-18610 |
49. | Ge, Y., Konrad, M. A., Matherly, L. H., and Taub, J. W. (2001) Biochem. J. 357, 97-105[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Wright, K. L.,
Moore, T. L.,
Vilen, B. J.,
Brown, A. M.,
and Ting, J. P.
(1995)
J. Biol. Chem.
270,
20978-20986 |
51. |
Roder, K.,
Wolf, S. S.,
Beck, K. F.,
and Schweizer, M.
(1997)
J. Biol. Chem.
272,
21616-21624 |
52. |
Faniello, M. C.,
Bevilacqua, M. A.,
Condorelli, G.,
de Crombrugghe, B.,
Maity, S. N.,
Avvedimento, V. E.,
Cimino, F.,
and Costanzo, F.
(1999)
J. Biol. Chem.
274,
7623-7626 |
53. |
Xiao, H.,
Hasegawa, T.,
and Isobe, K.
(2000)
J. Biol. Chem.
275,
1371-1376 |
54. | Chen, J. L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994) Cell 79, 93-105[Medline] [Order article via Infotrieve] |
55. |
Ainbinder, E.,
Revach, M.,
Wolstein, O.,
Moshonov, S.,
Diamant, N.,
and Dikstein, R.
(2002)
Mol. Cell. Biol.
22,
6354-6362 |
56. | Mei, J., Huang, X., and Zhang, P. (2001) Curr. Biol. 11, 1197-1201[CrossRef][Medline] [Order article via Infotrieve] |
57. | Stemmann, O., Zou, H., Gerber, S. A., Gygi, S. P., and Kirschner, M. W. (2001) Cell 107, 715-726[Medline] [Order article via Infotrieve] |
58. |
Pei, L.,
and Melmed, S.
(1997)
Mol. Endocrinol.
11,
433-441 |
59. |
Pei, L.
(2001)
J. Biol. Chem.
276,
8484-8491 |
60. |
Ishikawa, H.,
Heaney, A. P., Yu, R.,
Horwitz, G. A.,
and Melmed, S.
(2001)
J. Clin. Endocrinol. Metab.
86,
867-874 |
61. |
Romero, F.,
Multon, M. C.,
Ramos-Morales, F.,
Dominguez, A.,
Bernal, J. A.,
Pintor-Toro, J. A.,
and Tortolero, M.
(2001)
Nucleic Acids Res.
29,
1300-1307 |
62. |
Smith, G. C.,
and Jackson, S. P.
(1999)
Genes Dev.
13,
916-934 |
63. | Kharbanda, S., Yuan, Z. M., Weichselbaum, R., and Kufe, D. (1998) Oncogene 17, 3309-3318[Medline] [Order article via Infotrieve] |
64. |
Hsu, H. L.,
Gilley, D.,
Galande, S. A.,
Hande, M. P.,
Allen, B.,
Kim, S. H., Li, G. C.,
Campisi, J.,
Kohwi-Shigematsu, T.,
and Chen, D. J.
(2000)
Genes Dev.
14,
2807-2812 |
65. | d'Adda di Fagagna, F., Hande, M. P., Tong, W. M., Roth, D., Lansdorp, P. M., Wang, Z. Q., and Jackson, S. P. (2001) Curr. Biol. 11, 1192-1196[CrossRef][Medline] [Order article via Infotrieve] |