From the Derald H. Ruttenberg Cancer Center and the Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029
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ABSTRACT |
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The WT1 gene encodes a zinc finger
DNA binding transcription factor and is mutated in up to 15% of Wilms
tumor cases. The WT1 protein binds to the promoters of many genes
through GC- or TC-rich sequences and can function both as a
transcriptional repressor and an activator in co-transfection assays
depending on the cell type, the structure of the test promoter, and
even the expression vectors used. Engineered expression of WT1 can lead
to growth suppression by both cell cycle arrest and induction of
apoptosis. However, the transcriptional activity of WT1 that is
required for growth control was not defined. We found that three
N-terminal tumor-associated missense mutations of WT1 were defective
for activation of both a synthetic reporter containing WT1-binding sites as well as the promoter of a WT1 responsive gene, p21. These mutants failed to inhibit cell growth but still retain their ability to
repress several putative WT1 target promoters. These results indicate
that activation and not repression by WT1 is the critical transcriptional activity of the protein responsible for its growth suppressing properties.
Wilms tumor, an embryonic malignancy of the kidney, affects
approximately 1 in 10,000 live births making it the most common solid
tumor of children (1). Histologically, this tumor develops from
malignant transformation of renal stem cells, metanephric blastema,
that persist in the developing fetal kidney where they may appear as
undifferentiated metanephric mesenchyme cell, stromal cells, or
epithelial cells (2). The association of Wilms tumor with the WAGR
(Wilms Aniridia Genitourinary
malformation, and mental Retardation) syndrome with
concomitant constitutional deletions of chromosome 11p13 led to
positional cloning of the WT1 gene (3, 4). WT1 is critical
for normal renal development. It is expressed in the condensing
mesenchyme, renal vesicle, and glomerular epithelium of the developing
kidney. Furthermore, mice homozygous for disruption of wt1
die before birth with complete agenesis of the kidney (reviewed in
Refs. 5-8).
WT1 encodes a zinc finger transcription factor and undergoes
alternative splicing at two positions yielding four isoforms (9). WT1-A
refers to an isoform that excludes all alternative splices while WT1-B
is spliced to include exon 5, encoding a 17-amino acid sequence
immediately N-terminal to the zinc finger domain. WT1-C contains a
3-amino acid insertion (KTS) between zinc fingers three and four and
WT1-D contains both the 17- and the 3-amino acid insertions (10). Zinc
fingers 2-4 of WT1 are 67% identical to the
EGR-11 protein and both
proteins can bind to a GC-rich sequence and affect transcription (9,
11, 12). WT1 can also bind to alternate sequences including TC-rich
sequences (13-16) and was shown to activate or repress transcription
of many genes whose promoter contain these sequences. These include
early growth response 1 (EGR-1) (9, 12) insulin like-growth factor 2 (IGF-II) (17), platelet-derived growth factor A chain (18, 19),
insulin-like growth factor 1 receptor (IGF-IR) (20, 21), epidermal
growth factor receptor (EGFR) (22), WT1 (23, 24), and others
(25-34).
Mutations in WT1 occur in about 15% of Wilms tumor cases
(reviewed in Refs. 5 and 35-37). Most WT1 point mutations are
localized to the zinc finger region and are predicted to produce
proteins incapable of binding to DNA (38-44). These are null mutants
when present in the homozygous or hemizygous state. When present in the
heterozygous state, these mutations can function as dominant negative
alleles, inhibiting the function of the wild-type protein (5, 45-47).
Point mutations of WT1 can also occur in the N-terminal effector
regions of the protein. Two such mutations, F112Y and P129L, were
isolated from kidney tumors of newborn rats treated with
N-nitroso-N'-methylurea (NMU) (48). These animals
developed tumors that were histopathologically very similar to human
Wilms tumors suggesting the involvement of the wt1 gene. The
codon 112 mutation was found in four rat nephroblastomas where in each
case, the wild-type wt1 allele was lost. The codon 129 mutation was found in a mesenchymal rat kidney tumor although in this
case, the wild-type allele was still present (48). Park et
al. (49) also described a WT1 mutation outside of the zinc finger
domain. This mutation, F154S, was identified in nephrogenic rest from two patients with Wilms tumor and was not further characterized until now.
In this study, we used these three WT1 tumor-associated
mutations, WT1-A(F112Y), WT1-A(P129L), and WT1-A(F154S), as
genetic tools to further gain insight into the molecular action of WT1 and its involvement in Wilms tumor. We found that all three mutants were defective for WT1-mediated transcriptional activation and were
also unable to suppress growth in colony formation assays. Surprisingly, all these mutants were still competent for
transcriptional repression of reporter genes in co-transfection assays,
suggesting that the transcriptional activation function of WT1, and not
transcriptional repression, is most critical for its ability to
suppress growth.
Transfection Assay--
NIH-3T3 cells, grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and 1 × penicillin/streptomycin, plated at a density of 2 × 105 (6-well dishes) or 0.9 × 105 (12-well
dishes), were transfected utilizing SuperFect Transfection Reagent
(Qiagen) according to the manufacturer's directions. The amount of
reporter and WT1 expression vectors used in each transfection are
indicated in the figure legends. The cells were harvested 48 h
after transfection and assayed for CAT activity using an enzyme-linked
immunosorbent assay (Roche Molecular Biochemicals) according to the
manufacturer's directions. Luciferase activity was measured using the
Dual-Reporter Assay System (Promega).
Immunoblotting--
293T cells, plated at a density of 4 × 106 in 10-cm dishes, were transfected with 20 µg of WT1-A
expression vectors by calcium phosphate method as described previously
(50). Western blots were performed using 0.1 µg/ml rabbit polyclonal
anti-WT1 C19 antibody (Santa Cruz Biotechnology) as described (51).
Immunofluorescence--
To detect WT1-A proteins by
immunofluorescence, 2 × 105 NIH-3T3 cells plated in
35-mm dishes were transfected by SuperFect reagent as above. WT1
expressing cells were detected using 0.1 µg/ml anti-WT1 C19 antibody
diluted in a 3% bovine serum albumin/phosphate-buffered saline
solution as described previously (50).
Colony Suppression Assay--
G401 and Saos-2 cells, grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, were plated in 10-cm dishes at a density of 1 × 106/ml and transfected in duplicate with 20 µg of WT1-A
expression vectors or empty expression vector by calcium phosphate
precipitation. Cells were incubated with the DNA-calcium phosphate
complex for 16-18 h, washed twice with phosphate-buffered saline, and
refed with fresh media for an additional 12 h. 72 h after
transfection, duplicate plates of cells were washed with
phosphate-buffered saline, trypsinized, pooled and diluted 1:11 into
media containing 0.5 mg/ml G418. Five plates for each duplicate pair of
transfectant were cultured for 2-3 weeks under selection. The cells
were stained with Giemsa dye and the number of G418-resistant colonies
were counted.
Plasmid Construction--
The RSV-WT1-A expression vector (45),
murine p21-CAT (52) (gift of B. Vogelstein), human WT1-Luc (53) (gift
of M. Eccles), hIGF-II-(17) (gift of I. Drummond), mEGR1-CAT (12), and
the Zif3tk-CAT (45) constructs were previously described.
The G5tk-Luc reporter construct was generously provided by
Peter Traber, University of Pennsylvania. WT1-A mutants were
constructed by mutagenic PCR using wild-type and mutant WT1
oligonucleotides and RSV-WT1-A as a DNA template (54). The top strand
of the 112 mutant was made by PCR using the 5' wild-type primer (O195),
5'-CGCGGATCCGAGACAGTGCCTGAGCGCCTT-3' and the 3' mutant primer (O191),
5'-GGGAGGACCGTAGGGTCCGTA-3'. This generated a 90-base pair
fragment. The bottom strand was made with the 5' mutant primer (O190)
5'-TACGGACCCTACGGTCCTCCC-3' and the 3' wild-type primer
(O127) 5'-GTCGACGTCGACTTATCCACTCTCGTACCCTATAC-3'. This
generated a 474-base pair fragment that was combined with the 90-base
pair product of the first PCR reaction and re-amplified using wild-type
primers O195 and O127 to produce a DNA fragment encoding amino acids
86-256. The 129 mutant was made in a similar way. The top strand was
made using the 5' wild-type primer O195 and the 3' mutant primer (O282)
5'-TAGGGCGCATTGAGGAACATCCT-3' while the bottom strand was
made with the 5' mutant primer (O296) 5'-AGGATGTTCCTCAATGCGCCCTA-3' and the 3' wild-type primer
O127. The products of the PCR reactions containing both the 112 and 129 mutations were digested with AgeI/NcoI and
inserted into pSP64-WT1-A digested with
AgeI/NcoI. The top strand of the 154 mutation was made in a reaction with the wild-type 5' primer 5'-GGCCATTCACCGGACA-3' and a mutant 3' primer 5'-GCCCCGTCGGAAGTGACCGT-3'. The
second PCR reaction was performed with a mutant 5' primer
5'-ACGGTCACTTCCGACGGGGC-3' and a wild-type 3' primer
5'-GAATTCGAATTCTCAAAGCGCCACGTGGGAGTTT-3'. The products from these
reactions were then re-amplified with wild-type primers to produce a
DNA fragment encoding amino acids 100-429 of WT1-A containing the
codon 154 mutation. This fragment was digested with
AgeI/EcoRI and inserted into
AgeI/EcoRI digested pSP64-WT1-A to produce the
full-length WT1 coding region containing the 154 mutation. To construct
RSV-WT1-A mutants, the appropriate pSP64-WT1-A mutants were digested to
release the entire WT1-A coding region and inserted into an expression
vector containing the Rous sarcoma virus (RSV) long terminal repeat.
All WT1-A mutations were confirmed by sequencing with an internal
primer (O138) 5'-GGCCAGTTCACCGGTACA-3' using dideoxy sequencing method
and Sequenase version 2.0 (U. S. Biochemical Corp.).
Biological Activity of WT1-A Missense Mutations--
Previous
studies have shown that WT1 can suppress growth when expressed in RM1
cells (a Wilms tumor cell line) (55), Saos-2 cells (22), and
ras transformed NIH-3T3 cells (56). Therefore, to determine
the biological activity of the WT1-A mutants, we performed colony
suppression assays in both G401 (an embryonic kidney tumor cell line)
and Saos-2 cells (osteosarcoma cells), both of which do not express any
detectable WT1 protein. As expected, wild-type WT1-A inhibited growth
in both cell lines, suppressing colony formation up to 100-fold when
compared with the empty WT1 expression vector. In contrast, all three
WT1-A mutants only suppress growth 2-3-fold (Fig.
1). To confirm that the growth
suppressive properties of WT1 was specifically due to the effector
domain of the protein and not the zinc finger domain, we also performed colony suppression assay with a construct expressing only the zinc
finger region of WT1. Fig. 1 also shows that the zinc finger domain of
WT1-A was not sufficient for growth suppression. In fact, cells
transfected with an expression vector for the zinc finger domain
promoted growth especially in G401 cells where there were at least 10 times more colonies when compared with the empty expression vector
(Fig. 1). From this assay, we can conclude that these WT1-A
tumor-associated mutations are functionally deficient for at least one
WT1 biological activity. These results also confirm that the role of
WT1 in growth control is a critical point in tumorigenesis.
WT1-A Mutants Are Competent for Transcriptional
Repression--
Like other transcription factors, WT1 has been shown
to be a bifunctional transcription factor capable of both activating and repressing target genes. The initial characterization of WT1 showed
that it was a transcriptional repressor whose potential target genes
including many growth-related genes. To investigate the repressive
function of both wild-type and mutant WT1-A proteins, we determined
their effect on natural WT1 responsive promoters. We utilized a WT1
expression vector containing the CMV promoter as we and others showed
that it facilitates the ability of WT1 to act as a repressor (51). We
first examined the effect of WT1-A proteins on the human WT1 promoter.
Fig. 2A shows that CMV-WT1-A repressed transcription of this promoter by nearly 90% of control levels. The codon 112 and 129 CMV-WT1-A mutants repressed this promoter
to comparable levels while CMV-WT1-A(F154S) repressed it by as much as
98% being an even stronger repressor. We next examined the effect of
WT1 proteins on the EGR-1 promoter, the first gene whose expression was
shown to be down-regulated by WT1. As shown in Fig. 2B, all
three WT1-A mutant proteins repressed this promoter to similar levels
when compared with wild-type protein. Similarly, both wild-type and
mutant WT1-A proteins repressed the human IGF-II P3 promoter to equal
levels (Fig. 2C). Last, we determined the effect of WT1-A
proteins on the EGFR promoter, another gene whose transcription is
down-regulated by WT1 expression (22, 57). As with the other promoters
tested, the EGFR promoter was similarly repressed by all WT1-A proteins
(data not shown). As a further test of transcriptional repression, all
three mutations were engineered into the N-terminal portion of WT1-A
(amino acids 1-182), fused to the DNA-binding domain of Gal4, and
examined for their ability to affect transcription of a reporter gene
containing Gal4-binding sites. In this assay, all the fusion proteins
were able to repress transcription as well as the wild-type fusion protein (data not shown). Taken together, these data indicate that all
three WT1-A mutants are still competent for transcriptional repression.
They also suggest that the repression function of WT1 is not the
critical activity of the protein responsible for its growth suppressive
properties.
WT1-A Mutations Fail to Activate Transcription of an Artificial
Reporter Gene--
As noted, WT1 is a bifunctional transcription
factor capable of both repressing and activating gene transcription. We
previously showed that WT1-A can activate a reporter gene containing
upstream WT1/EGR-1-binding sites (45). We therefore tested the ability of the WT1 mutants to activate this reporter in 3T3 cells. As demonstrated in Fig. 3, wild-type WT1-A
activated this reporter 7-fold while all three mutants, (WT1-A(F112Y),
WT1-A(P129L), and WT1-A(F-154S)) activated this reporter at most
2-fold. These results were seen in multiple experiments as well as in
293T cells (data not shown).
Expression and Cellular Localization of Wild-type and Mutant WT1-A
Proteins--
To be assured that all of the mutant proteins were
produced to similar levels in the cells, immunoblot analysis on
extracts from transfected cells was performed. Fig.
4 shows that all three mutant proteins
were expressed to comparable levels. Interestingly, the wild-type
protein was expressed to a lesser level compared with all three mutant
proteins (Fig. 4). To examine the cellular localization of the WT1-A
proteins, we performed indirect immunofluorescence on transiently
transfected cells. Our data shows that the mutant WT1-A proteins were
correctly localized to the cell nucleus similar to the wild-type
protein (Fig. 5). These results thus
confirmed that the transcriptional difference seen between the
wild-type and mutant WT1-A proteins were in fact due to a difference in their innate transcriptional activity and not to a lack of expression or to incorrect cellular localization.
WT1-A Mutant Proteins Are Unable to Activate the Murine
p21WAF1/CIP1 Promoter--
Cell growth is regulated at
many different points within the cell cycle, one being regulation of
the cyclin-dependent kinase (CDK) inhibitor
p21WAF1/CIP1. Recently, Englert et al. (58) showed
that induction of WT1-A led to up-regulation of endogenous
p21WAF1/CIP1 in an inducible Saos-2 cell line (58) and
stimulated the p21 promoter in co-transfection assays. We therefore
speculated that these mutant WT1-A proteins may be defective for WT1
mediated activation of the p21 promoter. To investigate this
possibility, we co-transfected NIH-3T3 cells with a full-length murine
p21 promoter-CAT reporter construct along with either wild-type or mutant RSV-WT1-A expression vectors. As shown in Fig.
6, wild-type WT1-A was able to activate
this promoter by a factor of nearly 40 while all three mutants could
only activate it up to 2-fold. This result is consistent with a model
in which these tumor-derived WT1-A mutations may have led to tumor
formation due to their inability to up-regulate the CDK inhibitor,
p21WAF1/CIP1, and control cell cycle progression.
The ability of WT1 to both activate and repress transcription has
made it difficult to decipher the molecular mechanism by which it
functions as a tumor suppressor gene and to understand what are the WT1
target genes relevant to this process. Here, we described three
tumor-derived WT1 mutations and their use as a genetic tool to
understand the critical transcriptional function of WT1. We showed
that, unlike the wild-type protein: 1) mutant WT1-A proteins were
unable to activate transcription of WT1 responsive promoters. 2) Mutant
WT1-A proteins were unable to suppress growth in colony forming assay.
3) Mutant WT1-A proteins, like the wild-type protein, were still
competent for transcriptional repression of putative WT1 target promoters.
When initially identified, WT1 was shown to be a transcriptional
repressor. Since then, our group as well as others showed that WT1 can
also function as a transcriptional activator (19, 51). Interestingly,
almost all of the studies that showed WT1 to act as a repressor of
transcription used a CMV expression vector. We demonstrated that the
empty CMV expression vector alone was capable of strongly repressing
basal transcription of target promoters suggesting that this potent
promoter may be sequestering trans-activating factors from
the transcription machinery (51). This indicated that the choice of
expression vector used would determine whether WT1 would act as a
repressor or an activator in in vitro assays and called into
question the notion that WT1 acted as a pure repressor.
The use of an inducible or stable systems for WT1 expression have, in
some cases, suggested that down-regulation of gene expression by WT1 is
related to its function as a growth suppressor. Werner et
al. (59) showed that in G401 cells, expression of WT1-A led to
decreased levels of IGF-1R protein and reduced cellular proliferation. WT1 (expressed from the CMV promoter) was also capable of repressing a
reporter gene containing IGF-1R promoter sequences, suggesting that the
growth suppressive nature of WT1 may be due, at least in part, to the
repression of IGF-1R (59). Englert et al. (22) and Menke
et al. (57) showed that induction of WT1-A in Saos-2, Hep3B,
and HepG2 cells led to p53 independent apoptosis. Apoptosis was
associated with reduced expression of the EGFR protein and was
partially rescued by constitutive expression of EGFR but not IGF-1R. Of
note, EGFR mRNA levels were not examined in this study. However, a
recent study showed that, when expressed in an inducible manner in
HEK293 cells, WT1 did not affect the mRNA levels of a number of
putative WT1 target genes including the EGFR (60). Given the recent
observations that WT1 can bind RNA (61) and associate with splicing
factors (62, 63) it is possible that WT1 down-regulates the expression
of EGFR and some other genes at the post-transcriptional level.
Recent studies have highlighted the ability of WT1 to activate
transcription of target genes. One group found that induction of WT1
expression was associated with up-regulation of the cell cycle
inhibitor p21 (58). Furthermore, in transient transfection experiments,
WT1 activated the p21 promoter. Another group created a 3T3 cell line
which inducibly expressed WT1 and found that WT1 expression was
correlated with activation of the endogenous syndecan gene, a marker of
renal differentiation. Co-transfection experiments showed that WT1
activated the syndecan promoter (32). We found that stable expression
of WT1 in 3T3 cells leads to the acquisition of a partial epithelial
phenotype and up-regulation of a number of markers of renal
differentiation (64), some of which contain WT1-binding sites in their
promoters and are activated by co-transfected WT1.2
Previous analyses of tumor-derived WT1 mutants have indicated that
single amino acid changes can alter the transcriptional function of the
protein. Two such mutations (S273G and G201D) were described by Park
et al. (49, 65). Utilizing the CMV promoter in
co-transfection assays, this group found that these mutant WT1-A
proteins activated rather than repressed expression of the EGR-1
reporter gene. However, there was no correlation of transcription with
a functional growth suppression assay. It is possible that these
proteins, when expressed from the RSV promoter may also have altered
transcriptional activation function. Nevertheless, this information
suggests that an imbalance in WT1 transcriptional function may be
sufficient to contribute to tumor development. Our study confirms that
missense WT1 mutations in the effector region of the protein can alter
its transcriptional activity. Furthermore, we showed a close
correlation between the inability of three WT1 mutants to activate
transcription and their inability to suppress cell growth. This
information also suggests that WT1 functions in large part as an
activator of genes that in turn suppress cell growth.
The exact identity of the genes responsible for the ability of WT1 to
suppress cell growth is not completely known but the p21WAF1/CIP1 gene is an excellent candidate. Kudoh et
al. (66) showed that micro-injection of WT1 inhibited cell cycle
progression in serum-starved and released NIH 3T3 cells, an effect that
was mapped mid to late G1 phase. Cell cycle arrest was
associated with down-regulation of both CDK2 and CDK4 kinase activities
(66) and was completely abrogated by co-injection of CDK4 and cyclin D1
or CDK2 and cyclin E cDNAs. This is consistent with induction of
the p21 CDK inhibitor. Englert et al. (58) showed that
induction of WT1 led to up-regulation of p21 protein, and found that
WT1 could activate the p21 promoter. This group later found that an
N-terminal deletion of WT1 [WT1 The three WT1 mutations characterized here are located in what was
previously identified by Gal4 fusion assay as the repression domain of
WT1 (85-179) (68). However, we found that these mutants did not affect
the ability of WT1 to repress transcription either in its native
conformation or as a Gal4-fusion protein. This suggests that there may
be overlapping but functionally distinct domains in the N-terminal
domain of WT1 (amino acids 1-180) that interact with transcriptional
co-activators and co-repressors. All three mutations may significantly
alter the secondary or tertiary structure of WT1 and thus their
interaction with partner proteins. Indeed, the mutations F112Y and
F154S introduce a potential phosphorylation site into the protein while
P129L removes a potential helix breaking residue.
The identity of putative WT1 co-activators are not yet know. p53 (69),
Par-4 (70), and Ciao-1 (71) were all identified as WT1-binding proteins
that affect its transcriptional function through interaction with the
zinc finger domains of WT1. Hence these proteins are unlikely to be
co-factors for WT1-mediated activation whose interaction is disrupted
by the N-terminal mutations. The only identified proteins interacting
with the N terminus of WT1 to date are human ubiquitin conjugating
enzyme (hUBC9) (68, 72) and Hsp-70 (67). Hsp70 binds to the N-terminal
180 amino acids of WT1. WT1/Hsp-70 interaction is required for WT1
mediated growth inhibition and activation of the p21 promoter. We are
currently determining if the three WT1 missense mutations we
characterized are defective for this interaction.
In summary, the results presented here are consistent with a model in
which the mutant WT1 proteins are defective for transcriptional activation because of failure to interact with some critical
component(s) of the transcriptional machinery. Moreover, we found that
transcriptional activation by WT1 is most closely correlated with its
ability to function as a growth suppressor. This suggests that loss of WT1 function in Wilms tumors leads to tumor development due to insufficient activation of genes involved in cell cycle control, differentiation, and apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mutant WT1-A proteins are defective for
growth suppression. G401 and Saos-2 cells were transfected with 20 µg of the CMV-murine (m) WT1-A expression vectors and
selected in media containing 0.5 mg/ml G418 for 2 to 3 weeks. Shown are
the average number of G418-resistant colonies for each quadruplicate
set of plates (± S.D.) for each vector tested.
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Fig. 2.
All WT1-A mutant proteins can still
repress transcription. A, 3T3 cells were co-transfected
with a 0.2 µg of human WT1 promoter luciferase reporter, 5 ng of
tk-renilla, and 2.0 µg of empty CMV, WT1-A, and mutant WT1-A
expression vectors. B, 293T cells were co-transfected with
0.2 µg of murine EGR-I promoter-CAT construct. C,
0.4 µg of hIGF-II P3 promoter-CAT construct, 5 ng of tk-renilla, and
2.0 µg of empty CMV, WT1-A, and mutant WT1-A expression vectors.
Following luciferase assay, the averages (± S.D. of three independent
experiments are shown.
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Fig. 3.
WT1-A mutants are deficient in activating the
Zif3tk-CAT reporter. NIH-3T3 cells were transfected
with 0.4 µg of Zif3tk-CAT reporter vector, 0.1 µg of
tk-GH as an internal control, and 2.0 µg of wild-type of mutant
RSV-WT1-A expression vectors. At 48 h after transfection, the
cells were harvested and assayed for CAT activity. The data represent
the average (±S.D.) of eight independent experiments.
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Fig. 4.
Expression of mutant WT1-A proteins.
293T cells were transfected with 20 µg of RSV-WT1-A expression
vectors by the calcium phosphate method. Forty-eight hours after
transfection, cells were harvested and protein concentration was
determined using Bio-Rad protein reagent. Whole cell extracts (100 µg) were then separated on a 10% SDS-polyacrylamide electrophoresis
gel, transferred to nylon membrane, and probed with anti-WT1 C19
antibody.
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Fig. 5.
Cellular localization of WT1-A proteins.
3T3 cells were transfected with 2.0 µg of empty RSV expression
vector, wild-type, or mutant RSV-WT1-A expression vectors and
immunostained for WT1 using the C19 antibody.
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Fig. 6.
Mutant WT1-A proteins cannot activate the
murine p21 promoter. 3T3 cells were co-transfected with 50 ng of
murine p21 promoter-CAT reporter vector, 0.1 µg of tk-GH, and 2.0 µg of wild-type or mutant RSV-WT1-A expression vectors. CAT activity
was measured and the results of three independent experiments are
shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6-180] was unable to suppress
growth and could not activate the p21 promoter but was still competent
for transcriptional repression of both the EGFR and EGR promoter (67).
These results are further supported by our the observation that the
three tumor-associated missense WT1 mutants localized in the same
N-terminal region of WT1 we analyzed were unable to up-regulate the
expression of p21 promoter in co-transfection studies. Taken together,
these results indicate that activation of p21 by WT1 is important for
the growth suppressive properties of WT1. Our data are consistent with
the notion that it is the ability of WT1 to activate and not repress transcription that is most critical for the role of the protein in
growth control. However, it remains likely that other WT1 target genes
required for growth suppression have not yet been identified. The
mutant WT1 proteins may still be able to regulate the expression of
some of these genes to the same degree as the wild-type protein. Alternatively, the mutant WT1 proteins might also be defective for
transcriptional repression of WT1 genes important for growth suppression that were not examined in our study. Nevertheless, these
mutants represent important tools that can be used to further understand the transcriptional function of WT1 and its involvement in
Wilms tumor.
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ACKNOWLEDGEMENTS |
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We thank Isabelle Gross for critically reviewing this manuscript, Yariv Houvras for valuable technical support, and Josina Reddy for initial work on the codon 154 mutant.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO1 CA59998 (to J. D. L.).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.
Supported by a United Negro College Fund-Merck Graduate Science
Research Fellowship.
§ Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Derald. H. Ruttenberg Cancer Center, Box 1130, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-659-5487; Fax: 212-849-2523; E-mail: jlicht{at}smtplink.mssm.edu.
2 S. Hosono and J. D. Licht, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: EGR-1, early growth response-1; IGF, insulin growth factor; EGFR, epidermal growth factor receptor; PCR, polymerase chain reaction; RSV, Rous sarcoma virus; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; CDK, cyclin-dependent kinase; NMU, N-nitroso-N'-methylurea.
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