From the Department of Clinical
Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv
69978, Israel and the ¶ Department of Pediatrics, Oregon
Health and Science University, Portland, Oregon 97239
Received for publication, November 14, 2002
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ABSTRACT |
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The insulin-like growth factor-I receptor
(IGF-IR) plays a critical role in transformation. The expression of the
IGF-IR gene is negatively regulated by a number of transcription
factors, including the WT1 and p53 tumor suppressors. Previous studies have suggested both physical and functional interactions between the
WT1 and p53 proteins. The potential functional interactions between WT1
and p53 in control of IGF-IR promoter activity were addressed by
transient coexpression of vectors encoding different isoforms of WT1,
together with IGF-IR promoter-luciferase reporter constructs, in
p53-null osteosarcoma-derived Saos-2 cells, wild-type p53-expressing
kidney tumor-derived G401 cells, and mutant p53-expressing, rhabdomyosarcoma-derived RD cells. Similar studies were also
performed to compare p53-expressing Balb/c-3T3 and clonally derived
p53-null, (10)1 fibroblasts and the colorectal cancer cell line
HCT116 +/+, which expresses a wild-type p53 gene, and its HCT116 The insulin-like growth factor-I receptor
(IGF-IR)1 is a
transmembrane heterotetramer that mediates the effects of the IGFs, IGF-I and IGF-II, on growth and differentiation (1-3). The IGF-IR plays a central role in cell cycle regulation, as demonstrated by the
fact that overexpression of this receptor in fibroblasts abrogates all
requirements for exogenous growth factors (4). In addition to its
important role during development, there is evidence pointing to a
pivotal role for the IGF-IR in tumorigenesis (5, 6). The IGF-IR is
highly expressed by most tumors and cancer cell lines, whereas
fibroblasts derived from mouse embryos in which the IGF-IR was
disrupted by homologous recombination are resistant to
transformation by a number of oncogenes, indicating that IGF-IR
function is an important prerequisite for cellular transformation (7,
8). Furthermore, the IGF-IR exhibits potent antiapoptotic effects that
are consistent with the role of IGFs as cell survival factors (9,
10).
Structural analysis of the IGF-IR promoter revealed that it contains
multiple binding sites for the WT1 Wilms' tumor suppressor protein, a
transcription factor whose inactivation has been implicated in the
etiology of a subset of Wilms' tumors, a pediatric kidney malignancy
(11, 12). The WT1 gene product is a nuclear protein of 52-54 kDa that
contains N-terminal transcriptional regulatory and self-association
domains and a C-terminal DNA and RNA binding domain that comprises four
zinc fingers of the C2-H2 class (13, 14) (Fig.
1). This domain binds to target DNAs
containing versions of a 5'-GCGGGGGCG-3' consensus sequence.
Alternative splicing of exon 5 and the use of an alternative splice
site at the end of exon 9 produces mRNAs encoding multiple WT1
isoforms (15). Using transient transfection assays, we have previously
shown that WT1 proteins lacking the exon 9-encoded Lys-Thr-Ser (KTS) insert between zinc fingers 3 and 4 were more effective than the alternatively spliced +KTS variants in suppressing the activity of
co-transfected IGF-IR promoter constructs (11, 16). Furthermore, using
electrophoretic mobility shift assays (EMSA) and DNase I footprint
analyses, we demonstrated that this transcriptional effect was
associated with specific binding of the WT1-KTS isoform to sites
located both upstream and downstream of the IGF-IR gene transcription
initiation site (12). In addition, stable expression of the WT1-KTS
isoform in kidney tumor-derived G401 cells resulted in a decreased rate
of cellular proliferation, decreased levels of IGF-IR mRNA and
protein, and reduced activity of transfected IGF-IR promoter constructs
(17).
/
derivative, in which the p53 gene has been disrupted by homologous
recombination. WT1 splice variants lacking a KTS insert between zinc
fingers 3 and 4 suppressed IGF-IR promoter activity in the absence of p53 or in the presence of wild-type p53. WT1 variants that contain the
KTS insert are impaired in their ability to bind to the IGF-IR promoter
and are unable to suppress IGF-IR promoter. In the presence of mutant
p53, WT1 cannot repress the IGF-IR promoter. Coimmunoprecipitation experiments showed that p53 and WT1 physically interact, whereas electrophoretic mobility shift assay studies revealed that p53 modulates the ability of WT1 to bind to the IGF-IR promoter. In summary, the transcriptional activity of WT1 proteins and their ability
to function as tumor suppressors or oncogenes depends on the cellular
status of p53.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of wild-type WT1
isoforms. Alternative splicing of exons 5 and 9 of the WT1 gene
generates four different isoforms of the WT1 protein. The WT1 isoform
that includes both the exon 5-encoded sequence and the exon 9-encoded
Lys-Thr-Ser (KTS) tripeptide is designated WT1 +/+. The isoform that
includes exon 5 and lacks the KTS insert is designated WT1 +/ . The
protein that lacks exon 5 and includes the KTS insert is designated WT1
/+. The protein that lacks both exon 5 and the KTS insert is
designated WT1
/
. The N-terminal domain of WT1 contains the first
300 amino acids, including putative repression domains. The C-terminal
domain of the protein consists of 150 amino acids containing four zinc
finger DNA-binding domains. The arrows in the Denys-Drash
and WAGR proteins denote the location of missense point
mutations.
IGF-IR gene expression is also regulated by the p53 tumor suppressor (18, 19). Specifically, transcription of the IGF-IR gene is negatively regulated by wild-type p53, whereas a number of tumor-derived, mutant versions of p53 were shown to significantly stimulate the activity of the IGF-IR promoter. Unlike WT1, p53 does not exhibit specific binding in vitro to the regulatory region of the IGF-IR gene. However, the results of EMSA and co-immunoprecipitation experiments indicate that the mechanism of action of p53 involves potential interactions with TBP, the TATA-box binding subunit of the general initiation factor TFIID, and with the Sp1 transcription factor, an important trans-activator of the IGF-IR promoter (20, 21).
Several lines of evidence suggest that p53 and WT1 act in concert to control cellular proliferation. Using in vitro immunoprecipitation and Western blot analyses, p53 and WT1 proteins were shown to physically interact (22). Additionally, immunohistochemical analysis demonstrated aberrant expression of p53, an event usually associated with mutation of the p53 gene, in a significant proportion of Wilms' tumors (23). p53 mutations were usually restricted to anaplastic regions of the tumors, suggesting that progression to anaplasia is associated with clonal expansion of cells that have acquired a p53 mutation (24). WT1 protein was shown to stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis (25). Finally, we have previously reported that WT1 and mutant p53 are co-expressed in aggressive, estrogen receptor-negative human breast tumors (26).
In view of the central role of the IGF-IR in cell cycle progression and
tumorigenesis, and to extend our previous observations on regulation of
IGF-IR gene expression by WT1 and p53, we have addressed the potential
functional and physical interactions between these important tumor
suppressors in the transcriptional control of the IGF-IR gene.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Plasmids, and DNA Transfections--
Saos-2 is a
human osteogenic sarcoma-derived cell line in which both p53 alleles
are deleted. G401 is a human malignant rhabdoid tumor cell line that
expresses wild-type p53. Saos-2 and G401 cells were obtained from the
American Type Culture Collection (Manassas, VA). RD is a human
rhabdomyosarcoma cell line that expresses a mutant p53 gene (Arg to Trp
mutation at codon 248, in the DNA-binding domain). RD cells were kindly
provided by Dr. Lee Helman (NCI, National Institutes of Health,
Bethesda, MD). Saos-2 and RD cells were grown in Dulbecco's modified
Eagle's medium, and G401 cells were cultured in McCoy's 5A medium.
Media were supplemented with 10% fetal bovine serum, 2 mM
glutamine, and 50 µg/ml gentamicin sulfate. We have previously
demonstrated that each of these cell lines expresses significant
amounts of IGF-IR mRNA and supports transcription of IGF-IR
promoter-driven reporter constructs (17, 18, 21). Balb/c-3T3 and (10)1
murine fibroblasts that express, respectively, wild-type p53 or no p53 were kindly provided by Dr. Moshe Oren (Weizmann Institute of Science,
Rehovot, Israel) (27). Balb/c-3T3 and (10)1 cells were grown in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum. The human colorectal cancer cell lines HCT116 +/+, which
expresses wild-type p53, and HCT116 /
, in which the p53 gene has
been disrupted by targeted homologous recombination, were kindly
provided by Dr. Bert Vogelstein (Johns Hopkins University School of
Medicine, Baltimore, MD) (28). HCT116 cells were grown in McCoy's 5A
medium with 10% fetal bovine serum.
For transient co-transfection experiments, a genomic DNA fragment
extending from nucleotides 476 to +640 (nucleotide 1 corresponds to
the transcription start site of the rat IGF-IR gene) was subcloned upstream of a promoterless firefly luciferase reporter in the p0LUC
vector. The promoter activity of this fragment and the locations of WT1
binding sites and their relative affinities have been previously described (12). The construction of WT1 expression plasmids (in
pcDNA3) containing or lacking the 17-amino acid exon 5-encoded sequence and the 3-amino acid exon 9-encoded fragment (KTS) has been
previously reported (16). Likewise, the construction of WT1 expression
vectors containing mutations associated with Denys-Drash syndrome (DDS;
Arg to Trp at codon 394) and with the Wilms' tumor, aniridia,
genito-urinary abnormalities, and mental retardation syndrome (WAGR;
Gly to Asp at codon 201) has been described (16).
Cells were seeded in six-well plates the day before transfection.
Saos-2 cells were transfected with 5 µg of reporter plasmid and
0.8-2.5 µg of expression plasmid (or empty pcDNA3), along with
2.5 µg of a -galactosidase expression plasmid (pCMV
;
Clontech, Palo Alto, CA) using the calcium
phosphate method. The total amount of transfected DNA was kept constant
by using pcDNA3 DNA. G401 cells were transfected with 0.5 µg of
reporter plasmid, 0.5-4 µg of expression vector, and 0.25 µg of
pCMV
using the Fugene-6 reagent (Roche Molecular Biochemicals). RD
cells were transfected with 1 µg of reporter plasmid and 0.25-0.5
µg of expression plasmid, along with 2.5 µg of pCMV
using the
Polyfect reagent (Qiagen GmbH). Balb/c-3T3 and (10)1 cells were
transfected with 5 µg of the p(
476/+640)LUC reporter plasmid,
0-2.5 µg of the WT1
/
vector, and 2.5 µg of pCMV
using the
GenePORTER reagent (Gene Therapy Systems, San Diego, CA). HCT116 cells
were transfected with 1 µg of the p(
476/+640)LUC plasmid, 0-0.5
µg of WT1
/
, and 0.5 µg of pCMV
using the Polyfect reagent.
Cells were harvested 40 h after transfection, and luciferase and
-galactosidase activities were measured as previously described
(29). Promoter activities were expressed as luciferase values
normalized for
-galactosidase activity.
In Vitro Transcription and Translation Reactions-- Coupled in vitro transcription/translation of WT1 proteins was performed using the TNT® T7 Quick-Coupled Transcription/Translation System (Promega, Madison, WI). Briefly, T7 RNA polymerase-driven in vitro transcription reactions were followed by in vitro translations in the presence of [35S]methionine using rabbit reticulocyte lysates. In vitro translation products were electrophoresed through 10% SDS-PAGE and exposed to Eastman Kodak Co. X-Omat film.
Western Immunoblotting--
Transiently transfected cells were
harvested with ice-cold PBS containing 5 mM EDTA and lysed
in a buffer composed of 150 mM NaCl, 20 mM
Hepes, pH 7.5, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 mM leupeptin, 1 mM
pyrophosphate, 1 mM vanadate, and 1 mM
dithiothreitol. Protein content of the lysates was determined using the
Bradford reagent (Bio-Rad) using bovine serum albumin as a standard. In some experiments, nuclear extracts were prepared as described previously (29). Samples were subjected to 10% SDS-PAGE, followed by
electrophoretic transfer of the proteins to nitrocellulose membranes.
Membranes were blocked with 3% bovine serum albumin in T-TBS (20 mM Tris-HCl, pH 7.5, 135 mM NaCl, and 0.1%
Tween 20) and then incubated either with an anti-WT1 antibody (C19; 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with an
anti-human IGF-IR -subunit antibody (C20; 0.2 µg/ml; Santa Cruz
Biotechnology). Membranes were then washed extensively with T-TBS and
incubated with horseradish peroxidase-conjugated secondary antibody.
Bands corresponding to WT1 and IGF-IR proteins were detected using the
SuperSignal West Pico® Chemiluminescent Substrate (Pierce).
Co-immunoprecipitation Studies--
Saos-2 cells were
transiently transfected with 3 µg of a WT1 /
expression vector
and 3 µg of a HA-tagged p53 expression vector (or empty
pcDNA3-HA) (30) using the Fugene-6 reagent. The pcDNA3-HA-p53
plasmid was kindly provided by Dr. William G. Kaelin (Harvard Medical
School, Boston, MA). 24 and 48 h after transfection, cells were
lysed and immunoprecipitated with an anti-WT1 monoclonal antibody (F6;
200 µg/ml; Santa Cruz Biotechnology). Immunoprecipitates were
electrophoresed through 8% SDS-PAGE and immunoblotted with an anti-p53
monoclonal antibody (DO-1; dilution 1:1000; Santa Cruz Biotechnology)
as described above. Membranes were then washed and incubated with
horseradish peroxidase-conjugated Protein A (ICN Biomedicals, Aurora,
OH). In some co-immunoprecipitation experiments, the WT1 plasmid was
cotransfected with the pC53-248W vector that encodes a mutant p53 (Arg
to Trp at codon 248), provided by Dr. Edward Mercer (Thomas Jefferson
University, Philadelphia, PA).
Electrophoretic Mobility Shift Assays--
Saos-2 cells were
transiently transfected with 4 µg of a WT1 /
expression vector
(or pcDNA3) and 4 µg of a p53 expression vector (pC53-SN3; also
provided by Dr. Edward Mercer) or empty pCMV-Neo-Bam. After
48 h, cells were lysed, and nuclear extracts were prepared as
described (29). The probe used in EMSA was a double-stranded
synthetic oligonucleotide with the sequence GCGCAGTGCGGGTGGGGGCGGAAGCGTGGGCGCGCG,
which corresponds to nucleotides
273/
238 of the IGF-IR
promoter and includes two high affinity WT1 binding sites (underlined)
(12). The complementary oligonucleotides were annealed in the presence
of 100 mM NaCl at 95 °C, and the double-stranded probe
was end-labeled with [
-32P]ATP in the presence of T4
polynucleotide kinase. Binding reactions were performed for 30 min on
ice in a buffer containing 20 mM HEPES, pH 7.8, 100 mM KCl, 1 mM dithiothreitol, 1 µg of
poly(dI-dC), 1 mM ZnCl2, 104 dpm of
labeled probe, 10% glycerol, and 10 µg of crude nuclear extract.
Changes in mobility were assessed by electrophoresis through native 5%
PAGE gels that were run at 120 V for 2 h in 0.5× TBE.
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RESULTS |
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The proximal ~500 bp of 5'-flanking and ~700 bp of the 5'-untranslated region of the IGF-IR promoter have been previously shown to contain 12 bona fide binding sites for WT1 (12). These elements bind the WT1 protein lacking the KTS insert between zinc fingers 3 and 4 with medium to high affinity, whereas the binding capacity of the WT1 isoform including the KTS insert is significantly reduced. Consistent with the results of these binding experiments, we showed that transient expression of WT1-KTS isoforms in Chinese hamster ovary cells repressed the activity of a cotransfected IGF-IR promoter-luciferase reporter by 80%, whereas WT1+KTS variants produced ~40% repression (16). To examine the transcriptional effect of WT1 in different p53 backgrounds, we extended our analysis to the osteosarcoma-derived Saos-2, kidney rhabdoid tumor-derived G401, and rhabdomyosarcoma-derived RD cell lines.
To verify that the expression constructs to be utilized in functional
assays of transcriptional activity encoded proteins of the expected
size, synthetic RNAs were generated by in vitro transcription of pcDNA3 constructs and used to program rabbit reticulocyte lysates for in vitro translation in a coupled
system. As shown in Fig. 2, the apparent
molecular weight of the [35S]methionine-labeled WT1
isoforms containing exon 5-encoding sequences (WT1 +/ and +/+) was
~54 kDa, and the size of the exon 5-lacking WT1 variants (WT1
/+
and
/
) was ~52 kDa.
|
To evaluate the ability of the various naturally occurring WT1 isoforms
to regulate the activity of the IGF-IR promoter in a p53-independent
manner, p53-null Saos-2 cells were cotransfected with a series of
expression vectors containing or lacking alternatively spliced exon 5 and 9 sequences (WT1 +/+, +/,
/+, and
/
), together with the
reporter plasmid p(
476/+640)LUC, which contains most of the proximal
region of the rat IGF-IR promoter. The results of co-transfection
experiments in Saos-2 cells are presented in Fig.
3. The WT1
/
isoform induced a
significant dose-dependent decrease in promoter activity
(37 ± 3.9% inhibition with 1.5 µg of the expression plasmid
and 65 ± 2% inhibition with 2.5 µg). These results replicate
our previous data using pCB6-derived as well as pcDNA3-derived
constructs in Chinese hamster ovary cells (11, 16). Transfection of the
WT1 +/
isoform inhibited promoter activity by 34 ± 6% at 2.5 µg of input DNA, whereas the KTS-containing variants, WT1 +/+ and
/+, had a limited effect on IGF-IR promoter activity.
|
To examine whether the transcriptional repression effect of the WT1-KTS
variants was associated with corresponding changes in the levels of
endogenous IGF-IR protein, Western blot analysis was performed. Saos-2
cells were transiently transfected with increasing amounts of the WT1
/
expression vector, and after 72 h, cells were lysed as
described under "Experimental Procedures." Cell lysates (50 µg)
were electrophoresed through 10% SDS-PAGE gels, after which they were
blotted onto nitrocellulose membranes. The upper half of each membrane
was incubated with an anti-IGF-IR
-subunit antibody, and the lower
half was incubated with an anti-WT1 antibody. Results of Western blot
analysis of transfected cells showed that increasing amounts of WT1
induced a dose-dependent decrease in the endogenous levels
of IGF-IR (Fig. 4). Maximal suppression
(~90% inhibition) was seen with 6 µg of WT1 expression plasmid.
|
In G401 cells, which express wild-type p53, the WT1 /
and +/
variants both suppressed promoter activity in a
dose-dependent manner, with maximal inhibition (53 ± 3.6 and 36 ± 14%, respectively) seen with 2-4 µg of
expression vector (Fig. 5A).
Neither of the two KTS-containing isoforms (
/+ and +/+) inhibited the
IGF-IR promoter in G401 cells (Fig. 5B).
|
To more rigorously assess the potential involvement of p53 on WT1
action and to avoid the confounding effect of different cellular
backgrounds, co-transfections were performed in p53-expressing Balb/c-3T3 and p53-null (10)1 murine fibroblasts. The (10)1 cell line
was clonally derived from Balb/c-3T3 cells; thus, both lines share a
common genetic background (27). As shown in Fig.
6, 1.5 µg of WT1 /
suppressed
promoter activity in Balb/c-3T3 cells by 82%, whereas the same amount
of vector repressed activity in (10)1 fibroblasts only by 17%. With
2.5 µg of DNA, WT1
/
induced a further decrease in activity in
(10)1 cells to 27%. In addition, co-transfections were performed in
the human colorectal cancer cell lines HCT116 +/+, containing wild-type
p53, and HCT116
/
, in which the p53 gene has been disrupted by
targeted homologous recombination (28). In these cells, the inhibitory
effect of the KTS-lacking WT1 +/
isoform on IGF-IR promoter activity
was also significantly enhanced in p53-expressing compared with
p53-null cells, although the differences between p53-containing and
p53-lacking cells were less pronounced than those seen between
Balb/c-3T3 and (10)1 cells (51 ± 4 versus 21 ± 3% inhibition at 0.05 µg of expression vector and 58 ± 6 versus 38 ± 4% at 0.1 µg of DNA) (Fig.
7A). Interestingly, the
KTS-containing WT1
/+ isoform had a similar effect in HCT116
/
and +/+ cells (Fig. 7B).
|
|
To assess the effect of WT1 expression in the presence of a mutant p53,
cotransfections were performed in the rhabdomyosarcoma-derived RD cell
line that expresses a p53 molecule mutated at codon 248. In these
cells, neither WT1 /
nor WT1
/+ affected promoter activity to a
significant extent (Fig. 8A).
To more rigorously compare the differential effects of wild-type and
mutant p53, triple transfections were performed in Saos-2 cells using
the p(
476/+640)LUC reporter, together with the WT1
/
plasmid
(0.25 µg) and minimal amounts (50 ng) of wild-type or codon
248-mutated (pC53-248W) p53 vectors. The rationale for using such low
doses of WT1 and p53 was to minimize the individual effect of each
tumor suppressor (18). Furthermore, we chose the codon-248 mutant to
replicate the p53 status in RD cells. As shown in Fig. 8B, WT1
/
suppressed promoter activity by 24%, whereas in the presence of wild-type p53, the inhibitory effect increased to 39%. On the other
hand, WT1
/
had essentially no effect in the presence of a mutant
p53 (2% inhibition).
|
In addition to analyzing the wild-type splice variants of WT1, we
assessed the transcriptional activities of the naturally occurring DDS-
and WAGR-associated mutant versions of WT1. For this purpose, transient
co-transfections were performed in Saos-2 cells using the
p(476/+640)LUC IGF-IR promoter-luciferase reporter construct,
together with increasing amounts of expression vectors encoding either
+/+ or
/
variants of the DDS- and WAGR-associated proteins.
Interestingly, both +/+ and
/
isoforms of the DDS-associated mutant
(harboring a point mutation in the DNA binding domain) and of the WAGR
mutant (displaying a point mutation in the middle portion of the
molecule) suppressed the activity of the IGF-IR promoter in a
dose-dependent manner. Thus, 2.5 µg of DDS
/
induced a 64% decrease, whereas DDS +/+ induced a smaller, albeit significant, decrease (35%) (Fig. 9A). At
the same DNA concentration, WAGR +/+ and
/
mutants suppressed
promoter activity by 60 and 45%, respectively (Fig. 9B).
|
Although physical interactions between p53 and WT1 proteins have been
previously reported (22), we undertook a series of experiments aimed at
establishing whether the functional cooperation between p53 and WT1
isoforms in our cellular systems was similarly associated with specific
protein-protein interactions. For this purpose, Saos-2 cells were
transiently transfected with 3 µg each of expression vectors encoding
WT1 /
and pcDNA3-HA-p53 (or empty pcDNA3-HA vector). After
24 and 48 h, cells were harvested, lysates were immunoprecipitated
with an anti-WT1 monoclonal antibody (F6), and the precipitates were
loaded onto 8% SDS-PAGE gels. After electrophoresis, complexes were
transferred to nitrocellulose membranes and blotted with an anti-p53
monoclonal antibody (DO-1). As shown in Fig.
10A, immunoblotting with the
p53 antibody identified p53 in anti-WT1 immunoprecipitates of cells
transfected with WT1
/
and pcDNA3-HA-p53 but not in cells
transfected with WT1
/
and empty pcDNA3-HA. On the other hand,
co-transfection of mutant p53 (pC53-248W) did not result in WT1/p53
co-immunoprecipitation (Fig. 10B).
|
To examine whether p53 expression has an effect on WT1 levels, Saos-2
cells were co-transfected with expression vectors encoding WT1 /
or
/+, together with a p53 expression vector (or empty vector). After
48 h, nuclear extracts were prepared, and WT1 protein levels were
assessed by Western blotting. The results obtained showed that the
levels of WT1 were significantly increased in the presence of p53 (Fig.
11A). To assess whether a
similar effect can be seen in the presence of endogenous p53 and to
determine whether p53 improves WT1 stability or its nuclear
translocation, whole cell and nuclear extracts were prepared from
HCT116
/
and +/+ cells that were transfected with 0, 1, or 3 µg
of a WT1
/
vector. As shown in Fig. 11B, WT1 abundance
was increased in both whole cells and in the nuclei of p53-containing
cells (compare lane 2 versus
lane 5 and lane 3 versus lane 6). To examine the effect
of mutant p53, Saos-2 cells were co-transfected with a WT1
/
vector
(or empty vector), together with expression vectors encoding wild-type
or mutant (codon 248) p53 (or empty vector). Results of Western
blotting showed that cotransfection of wild-type p53 increased the
abundance of both endogenous WT1 (compare lane 2 versus lane 1) and exogenously added
WT1 (compare lane 5 versus lane
4). On the other hand, mutant p53 did not affect the levels of WT1
(compare lane 3 versus lane
1 and lane 6 versus
lane 4) (Fig. 11C).
|
To determine whether potential physical interactions between WT1 and
p53 can affect the ability of WT1 to bind to the IGF-IR promoter, EMSAs
were performed using crude nuclear extracts of Saos-2 cells that were
transfected with WT1 /
, wild-type p53, or both vectors. A labeled
double-stranded synthetic oligonucleotide that extends from nucleotide
273 to
238 and that includes two high affinity WT1 binding elements
was employed for this purpose (12). Incubation of the labeled fragment
with nuclear extracts of WT1
/
-transfected cells generated one
retarded band, whereas nuclear extracts of p53-transfected cells did
not exhibit any specific binding to this promoter region. Cells that
were transfected with both WT1 and p53, however, showed a significant
decrease in the intensity of the shifted band, suggesting that p53
induced a reduction in the amount of the WT1-IGF-IR promoter complex
(Fig. 12A). A similar
decrease in the intensity of the WT1-IGF-IR promoter complex was seen
when nuclear extracts of HCT116
/
cells transfected with WT1
/
and p53 were employed in EMSA (compared with cells transfected with WT1
/
alone). Mutant p53 had a very small effect on the intensity of
the DNA-protein complex (Fig. 12B).
|
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DISCUSSION |
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The participation of the IGF-IR in the etiology of Wilms' tumor, or nephroblastoma, was inferred from studies that showed that administration of a monoclonal antibody against the human IGF-IR to nude mice bearing Wilms' tumor heterotransplants prevented tumor growth and resulted in partial tumor remission (31). Furthermore, the IGF-IR gene was overexpressed in Wilms' tumor, with the levels of IGF-IR mRNA in individual tumors being inversely correlated with the levels of WT1 mRNA (11). Consistent with the postulated role of WT1 as a tumor suppressor, we showed in early studies that the IGF-IR promoter is negatively regulated by a particular isoform of WT1 (12, 17). The transcriptional regulation of the WT1 gene, however, is extremely complex, and multiple versions of the protein that differ as a result of the alternative splicing of exon 5 and an alternative splice site at the end of exon 9 are usually coexpressed in tissues. In addition, a number of missense point mutations in the WT1 gene have been identified in Wilms' tumor-associated disorders, including the Denys-Drash and WAGR syndromes.
In a more recent study, we addressed the regulation of IGF-IR promoter by wild-type and mutant versions of WT1 (16). The results obtained demonstrated that DNA binding is a major determinant of WT1 action. However, our data also suggested that WT1 could function through additional mechanisms such as RNA binding or protein-protein association. Since p53 has been shown to differentially regulate transcription of the IGF-IR gene, with wild-type p53 acting as a potent suppressor and mutant p53 as a strong transactivator (18), and in view of the fact that p53 can interact with WT1, we investigated in the present study the potential functional and physical interactions between WT1 and p53 in transcriptional regulation of the IGF-IR gene.
In the absence of p53, or in the presence of wild-type p53, the
critical determinant of wild-type WT1 action is its ability to bind DNA
via its zinc finger domain. Thus, in the absence of the KTS tripeptide
between zinc fingers 3 and 4, WT1 functions as a suppressor of IGF-IR
promoter activity. The presence of the KTS insert abolished the
repressing action of WT1, probably as a result of the inability of
KTS-containing isoforms to efficiently bind to the IGF-IR promoter
region (12). Interestingly, tumor-associated, mutant forms of WT1 were
able to suppress IGF-IR promoter even in the presence of the KTS
insert. Following binding of WT1 to the promoter region, the
extent of its repressing activity seems to depend on the p53
status of the cell. Thus, we observed that, in wild-type p53-expressing
Balb/c-3T3 cells, the suppressive effect of WT1 was significantly more
pronounced that in p53-null (10)1 cells (82 versus 27%
inhibition). Similarly, WT1 +/, but not WT1
/+, was more potent in
HCT116 +/+ than in HCT116
/
cells. Furthermore, the ability of the
KTS-containing WT1
/+ isoform to suppress IGF-IR promoter activity in
HCT116 cells may reflect the fact that the effect of the KTS insert is,
to a certain extent, cell type-specific. The increased potency of WT1
in the presence of p53 is probably explained by the results of
transfection experiments showing a greater abundance of exogenous WT1
in Saos-2 cells that were co-transfected with p53 in comparison with
cells that were transfected only with WT1, as well as in HCT116 +/+ in
comparison with HCT116
/
. The finding that augmented WT1 levels are
seen in both whole cell and nuclear extracts of HCT116 +/+ cells
suggests that p53 improves WT1 stability. The possibility that p53
increases, in addition, the translocation of WT1 to the nucleus cannot
be discounted.
The results of EMSA experiments showing that p53 is unable to bind to the IGF-IR promoter fragment are consistent with our previous data demonstrating that the inhibitory effect of p53 is mediated via interaction with the TATA-box binding component of TFIID (TBP) at the initiator element and does not seem to involve specific DNA binding (18). The fact that p53 interferes with the binding of WT1 to specific WT1 sites in the promoter region seems, however, paradoxical in view of the enhanced transcriptional activity of WT1-KTS seen in the presence of p53. These results, suggesting that WT1-KTS isoforms display an augmented activity despite reduced binding due to p53, can be potentially explained by the results of studies showing that the mechanism of action of p53 involves interaction with additional transcription factors, including Sp1, which positively affects IGF-IR promoter activity, as well as with members of the basal transcription machinery such as TBP. Alternatively, p53 may enhance WT1 binding to additional cis-elements in the IGF-IR promoter. Similar enhancement of WT1 activity by p53 toward the growth arrest-associated GADD45 gene has been reported (32). Co-expression of p53 and WT1 strongly induced a GADD45 reporter construct, and this effect was mediated by a WT1 element in the promoter region. Since p53 does not bind directly to this promoter, these results indicate that p53 contributes to the effect of WT1 via protein-protein interactions.
In RD cells harboring a mutant p53, WT1 was unable to repress IGF-IR promoter constructs, regardless of the presence or absence of the KTS insert. These findings can be interpreted to suggest that a potential mechanism by which loss-of-function mutations of p53 result in uncontrolled cell growth may be linked to the inability of WT1 to suppress target promoters, including that of the IGF-IR gene. Furthermore, it is relevant to analyze these findings in light of current controversies regarding the role of WT1. Inconsistent with the classical tumor suppressor function of WT1, an important body of work has unequivocally demonstrated that, in certain cellular contexts, WT1 is required to inhibit apoptosis in vitro and in vivo. This oncogenic role of WT1 has been found to be associated with its capacity to up-regulate antiapoptotic genes such as bcl-2 (33). Therefore, and in view of the diametrically opposed activities of WT1 in the presence of wild-type or mutant p53, it is important to take into consideration the cellular status of p53 when assessing the role of WT1. Analysis of anaplastic and nonanaplastic regions of seven Wilms' tumors revealed that, in five out of six tumors with p53 mutations, the mutations were restricted to the anaplastic region (24). These results indicate that progression to anaplasia is associated with clonal expansion of cells that have acquired a p53 mutation. Up-regulation of the IGF-IR gene as a result of expression of aberrant p53 has been shown to be important for the growth and survival of malignant cells (34).
The IGF-II gene, which encodes the main ligand of the IGF-IR in
malignant cells, is also transcriptionally regulated by WT1 isoforms.
Using the P3 promoter of the mouse IGF-II gene in primary cultures of
wild-type and knock-out p53 embryos, it was demonstrated that the
effect of WT1 was independent of p53 status (35). In addition, the
activities of KTS-containing or lacking WT1 isoforms toward the IGF-II
P3 promoter were in contrast to their activities toward the IGF-IR
promoter reported in the present paper. Thus, WT1-KTS activated the
IGF-II promoter, whereas WT1 + KTS repressed its activity. These
differences may reflect the relative simplicity of the IGF-II promoter,
which contains only two WT1 binding sites versus the
multiple sites characterized in the IGF-IR promoter. This possibility
is supported by the differential activities of WT1 variants toward the
insulin receptor promoter, which also contains multiple WT1 binding
sites (36). Specifically, WT1+KTS effectively repressed promoter
activity, whereas WT1-KTS was able to repress the insulin receptor
promoter only in the presence of co-transfected CCAAT/enhancer-binding
protein or a dominant-negative p53 mutation.
In summary, we have demonstrated that WT1 isoforms lacking the KTS
insert are capable of suppressing IGF-IR promoter activity in the
absence of p53 or in the presence of wild-type p53. Alternatively spliced variants of WT1 impaired in their ability to bind to the IGF-IR
promoter due to the presence of the KTS insert are unable to suppress
IGF-IR promoter activity. In the presence of mutant p53, WT1 cannot
repress the IGF-IR promoter. The transcriptional activity of WT1 gene
products and their ability to function as tumor suppressors or
oncogenes depend to a large extent on the cellular status of p53.
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ACKNOWLEDGEMENTS |
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We thank Drs. L. Helman, M. Oren, B. Vogelstein, W. G. Kaelin, and E. Mercer for providing cell lines and reagents.
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FOOTNOTES |
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* This research was supported by United States-Israel Binational Science Foundation (Jerusalem, Israel) Grant 97-00151 (to H. W. and C. T. R.), Fogarty International Research Collaboration Award TW00997-03 (to C. T. R. and H. W.), National Institutes of Health Grant DK50810 (to C. T. R.), a Project Grant from the Israel Cancer Research Fund, New York (to H. W.), and a grant from the Recanati Foundation, Israel (to H. W.).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.
§ This work was performed in partial fulfillment of the requirements for a Ph.D. degree in the Sackler Faculty of Medicine, Tel Aviv University.
To whom correspondence should be addressed. Tel.:
972-3-6408542; Fax: 972-3-6406087; E-mail:
hwerner@post.tau.ac.il.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M211606200
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ABBREVIATIONS |
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The abbreviations used are: IGF-IR, insulin-like growth factor-I receptor; IGF, insulin-like growth factor; EMSA, electrophoretic mobility shift assay; DDS, Denys-Drash syndrome; WAGR, Wilms' tumor, aniridia, genito-urinary abnormalities, and mental retardation syndrome; HA, hemagglutinin.
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