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- W1986543139 abstract "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 −/− 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. 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 −/− 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. insulin-like growth factor-I receptor insulin-like growth factor electrophoretic mobility shift assay Denys-Drash syndrome Wilms' tumor, aniridia, genito-urinary abnormalities, and mental retardation syndrome hemagglutinin 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 (1LeRoith D. Werner H. Beitner-Johnson D. Roberts C.T., Jr. Endocr. Rev. 1995; 16: 143-163Google Scholar, 2Werner H. Rosenfeld R.G. Roberts C.T., Jr. The IGF System: Molecular Biology, Physiology and Clinical Applications. Humana Press, Totowa, NJ1999: 63-88Google Scholar, 3Werner H. LeRoith D. Cell. Mol. Life Sci. 2000; 57: 932-942Google Scholar). 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 (4Pietrzkowski Z. Lammers R. Carpenter G. Soderquist A.M. Limardo M. Phillips P.D. Ullrich A. Baserga R. Cell Growth Differ. 1992; 3: 199-205Google Scholar). In addition to its important role during development, there is evidence pointing to a pivotal role for the IGF-IR in tumorigenesis (5Werner H. LeRoith D. Adv. Cancer Res. 1996; 68: 183-223Google Scholar, 6Werner H. LeRoith D. Crit. Rev. Oncog. 1997; 8: 71-92Google Scholar). 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 (7Sell C. Rubini M. Rubin R. Liu J.-P. Efstratiadis A. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11217-11221Google Scholar,8Baserga R. Cell. 1994; 79: 927-930Google Scholar). Furthermore, the IGF-IR exhibits potent antiapoptotic effects that are consistent with the role of IGFs as cell survival factors (9Resnicoff M. Abraham D. Yutanawiboonchai W. Rotman H.L. Kajstura J. Rubin R. Zoltick P. Baserga R. Cancer Res. 1995; 55: 2463-2469Google Scholar,10Sell C. Baserga R. Rubin R. Cancer Res. 1995; 55: 303-306Google Scholar).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 (11Werner H., Re, G.G. Drummond I.A. Sukhatme V.P. Rauscher III, F.J. Sens D.A. Garvin A.J. LeRoith D. Roberts C.T., Jr. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5828-5832Google Scholar, 12Werner H. Rauscher III, F. Sukhatme V.P. Drummond I.A. Roberts C.T., Jr. LeRoith D. J. Biol. Chem. 1994; 269: 12577-12582Google Scholar). 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 (13Call K.M. Glaser T. Ito C.Y. Buckler A.J. Pelletier J. Haber D.A. Rose E.A. Kral A. Yeger H. Lewis W.H. Jones C. Housman D.E. Cell. 1990; 60: 509-520Google Scholar, 14Morris J.F. Madden S.L. Tournay O.E. Cook D.M. Sukhatme V.P. Rauscher III, F.J. Oncogene. 1991; 6: 2339-2348Google Scholar) (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 (15Haber D.A. Sohn R.L. Buckler A.J. Pelletier J. Call K.M. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9618-9622Google Scholar). 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 (11Werner H., Re, G.G. Drummond I.A. Sukhatme V.P. Rauscher III, F.J. Sens D.A. Garvin A.J. LeRoith D. Roberts C.T., Jr. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5828-5832Google Scholar, 16Tajinda K. Carroll J. Roberts C.T., Jr. Endocrinology. 1999; 140: 4713-4724Google Scholar). 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 (12Werner H. Rauscher III, F. Sukhatme V.P. Drummond I.A. Roberts C.T., Jr. LeRoith D. J. Biol. Chem. 1994; 269: 12577-12582Google Scholar). 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 (17Werner H. Shen-Orr Z. Rauscher III, F. Morris J.F. Roberts C.T., Jr. LeRoith D. Mol. Cell. Biol. 1995; 15: 3516-3522Google Scholar).IGF-IR gene expression is also regulated by the p53 tumor suppressor (18Werner H. Karnieli E. Rauscher III, F.J. LeRoith D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8318-8323Google Scholar, 19Prisco M. Hongo A. Rizzo M.G. Sacchi A. Baserga R. Mol. Cell. Biol. 1997; 17: 1084-1092Google Scholar). 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 bindingin 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 (20Beitner-Johnson D. Werner H. Roberts C.T., Jr. LeRoith D. Mol. Endocrinol. 1995; 9: 1147-1156Google Scholar,21Ohlsson C. Kley N. Werner H. LeRoith D. Endocrinology. 1998; 139: 1101-1107Google Scholar).Several lines of evidence suggest that p53 and WT1 act in concert to control cellular proliferation. Using in vitroimmunoprecipitation and Western blot analyses, p53 and WT1 proteins were shown to physically interact (22Maheswaran S. Park S. Bernard A. Morris J. Rauscher III, F. Hill D. Haber D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5100-5104Google Scholar). 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 (23Lemoine N.R. Hughes C.M. Cowell J.K. J. Pathol. 1992; 168: 237-242Google Scholar). 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 (24Bardeesy N. Beckwith J.B. Pelletier J. Cancer Res. 1995; 55: 215-219Google Scholar). WT1 protein was shown to stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis (25Maheswaran S. Englert C. Bennett P. Heinrich G. Haber D. Genes Dev. 1995; 9: 2143-2156Google Scholar). Finally, we have previously reported that WT1 and mutant p53 are co-expressed in aggressive, estrogen receptor-negative human breast tumors (26Silberstein G.B. Van Horn K. Strickland P. Roberts C.T., Jr. Daniel C.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8132-8137Google Scholar).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.RESULTSThe 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 (12Werner H. Rauscher III, F. Sukhatme V.P. Drummond I.A. Roberts C.T., Jr. LeRoith D. J. Biol. Chem. 1994; 269: 12577-12582Google Scholar). 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 (16Tajinda K. Carroll J. Roberts C.T., Jr. Endocrinology. 1999; 140: 4713-4724Google Scholar). 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 vitrotranscription 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.Figure 2In vitro transcription/translation analysis of WT1 isoforms. T7 RNA polymerase-driven in vitro transcription reactions were performed using the TNT® quick-coupled transcription/translation system. Each reaction included 1 μg of each of the following expression vectors: WT1 +/−, WT1 −/+, WT1 +/+, WT1 −/−, and empty pcDNA3. In vitrotranslation reactions were performed in the presence of [35S]methionine using rabbit reticulocyte lysates. 1 μl of each translation reaction (out of a total volume of 50 μl) was electrophoresed through 10% SDS-PAGE gels and exposed for 20 h to Kodak X-Omat film.View Large Image Figure ViewerDownload (PPT)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 (11Werner H., Re, G.G. Drummond I.A. Sukhatme V.P. Rauscher III, F.J. Sens D.A. Garvin A.J. LeRoith D. Roberts C.T., Jr. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5828-5832Google Scholar, 16Tajinda K. Carroll J. Roberts C.T., Jr. Endocrinology. 1999; 140: 4713-4724Google Scholar). 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.Figure 3Regulation of IGF-IR promoter activity by WT1 isoforms in Saos-2 cells. 5 μg of the p(−476/+640)LUC reporter plasmid were co-transfected into Saos-2 cells, along with increasing amounts of WT1-KTS (+/− and −/−) (A) or WT1+KTS expression vectors (−/+ and +/+) (B) (or empty pcDNA3) and 2.5 μg of the pCMVβ plasmid using the calcium phosphate method. Luciferase and β-galactosidase activities were measured after 48 h. Luciferase data were normalized for β-galactosidase. 100% represents the IGF-IR promoter-driven luciferase activity in the absence of WT1. Experiments were performed between three and eight times, each time in duplicate. Error bars, S.E. Where not shown, the S.E. bars are smaller than the symbolsize. *, p < 0.05 versus control; **,p < 0.01 versus control.View Large Image Figure ViewerDownload (PPT)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 β-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.Figure 4Effect of WT1 expression on endogenous IGF-IR gene expression. Saos-2 cells were transfected with increasing amounts of a WT1 −/− expression vector, and after 72 h, cells were lysed in the presence of protease inhibitors. Equal amounts of protein (50 μg) were separated by 10% SDS-PAGE, transferred to nitrocellulose filters, and blotted with an anti-WT1 antibody (upper panel) or with an anti-IGF-IR antibody (lower panel). The positions of the 52–54-kDa WT1 and 97-kDa IGF-IR β-subunit proteins are denoted byarrows.View Large Image Figure ViewerDownload (PPT)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. 5 A). Neither of the two KTS-containing isoforms (−/+ and +/+) inhibited the IGF-IR promoter in G401 cells (Fig. 5 B).Figure 5Regulation of IGF-IR promoter activity by WT1 isoforms in G401 cells. G401 cells were co-transfected with 0.5 μg of the p(−476/+640)LUC IGF-IR reporter construct, together with 0–4 μg of WT1-KTS (−/− and +/−) (A) or WT1+KTS (−/+ and +/+) (B) and 0.25 μg of pCMVβ, using the Fugene-6 reagent. Experiments were performed between three and five times, each time in duplicate. For further details, see the legend to Fig. 3.View Large Image Figure ViewerDownload (PPT)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 (27Harvey D.M. Levine A.J. Genes Dev. 1991; 5: 2375-2385Google Scholar). 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 (28Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Google Scholar). 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 ± 6versus 38 ± 4% at 0.1 μg of DNA) (Fig. 7 A). Interestingly, the KTS-containing WT1 −/+ isoform had a similar effect in HCT116 −/− and +/+ cells (Fig. 7 B).Figure 6Effect of p53 background on WT1 action.Murine Balb/c-3T3 (open bars) and (10)1 (closed bars) fibroblasts were transfected with 5 μg of the p(−476/+640)LUC reporter plasmid, together with 0, 1.5, or 2.5 μg of a WT1 −/− expression vector, and 2.5 μg of pCMVβ, using the GenePORTER reagent. A value of 100% was given to the promoter activity in the absence of WT1. The figure shows the results of a typical experiment performed in duplicate dishes and repeated at least four times.View Large Image Figure ViewerDownload (PPT)Figure 7Effect of WT1 isoforms on IGF-IR promoter activity in p53-lacking and -expressing HCT116 human colorectal cancer cells. A, HCT116 −/− (closed bars) and HCT116 +/+ (open bars) cells were co-transfected with the p(−476/+640)LUC reporter and increasing amounts of the WT1 +/− expression plasmid. The figure shows the results of three experiments performed in duplicate dishes. *,p < 0.02 versus p53-lacking cells.B, HCT116 −/− (closed bars) and HCT116 +/+ (open bars) cells were cotransfected with the p(−476/+640)LUC reporter and increasing amounts of the WT1 −/+ expression plasmid. The figure shows the results of four experiments, each in duplicate.View Large Image Figure ViewerDownload (PPT)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. 8 A). 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 (18Werner H. Karnieli E. Rauscher III, F.J. LeRoith D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8318-8323Google Scholar). Furthermore, we chose the codon-248 mutant to replicate the p53 status in RD cells. As shown in Fig. 8 B, 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).Figure 8Regulation of IGF-IR promoter activity by WT1 in the presence of mutant p53. A, RD cells were transfected with 1 μg of the p(−476/+640)LUC construct, together with 0–2 μg of WT1 −/− or −/+ expression vectors, and 2.5 μg of pCMVβ, using the Polyfect reagent. Luciferase values, normalized for β-galactosidase, are expressed as a percentage of the luciferase activity of the empty pcDNA3 expression vector. Experiments were repeated between three and five times, each in duplicate. B, Saos-2 cells were cotransfected with the p(−476/+640)LUC reporter, along with 0.25 μg of WT1 −/− (or empty pcDNA3) and 50 ng of wild-type or codon 248-mutant p53 (or empty pCMV-Neo-Bam vector). Shown are the results of a typical experiment, performed at least six times, each in duplicate dishes.View Large Image Figure ViewerDownload (PPT)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. 9 A). At the same DNA concentration, WAGR +/+ and −/− mutants suppressed promoter activity by 60 and 45%, respectively (Fig. 9 B).Figure 9Regulation of IGF-IR promoter activity by DDS - and WAGR-associated mutant WT1 proteins. 5 μg of the p(−476/+640)LUC reporter plasmid were co-transfected into Saos-2 cells together with increasing amounts of expression vectors encoding DDS +/+ and −/− (A) or WAGR +/+ and −/− (B) and 2.5 μg of pCMVβ. Luciferase data were normalized for β-galactosidase activity. Experiments were repeated between two and five times, each in duplicate. For further details, see the legend to Fig. 3.View Large Image Figure ViewerDownload (PPT)Although physical interactions between p53 and WT1 proteins have been previously reported (22Maheswaran S. Park S. Bernard A. Morris J. Rauscher III, F. Hill D. Haber D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5100-5104Google Scholar), 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. 10 A, 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. 10 B).Figure 10Co-immunoprecipitation of p53 and WT1. A, Saos-2 cells were co-transfected with 3 μg of an expression vector encoding WT1 −/− and 3 μg of pcDNA3-HA-p53 (or empty pcDNA3-HA vector). 24 and 48 h after transfection, cells were harvested, and lysates were immunoprecipitated with antibody F6, a monoclonal antibody against WT1. Precipitates were loaded onto 8% SDS-PAGE gels, electrophoresed, and transferred to nitrocellulose membranes that were blotted with an anti-p53 antibody. Thearrow denotes the position of p53. B, Saos-2 cells were cotransfected with 3 μg of a WT1 −/− vector, together with 3 μg of expression vectors encoding wild-type or mutant (codon 248) p53. After 24 h, cells were harvested and processed as described above.View Large Image Figure ViewerDownload (PPT)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. 11 A). 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. 11 B, 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. 11 C).Figure 11Effect of p53 expression on WT1 levels. A, Saos-2 cells were co-transfected with expression vectors encoding WT1 −/− or −/+, together with a p53 vector (or empty vector). After 48 h, nuclear extracts were prepared, and equal amounts of protein were electrophoresed through 10% SDS-PAGE, transferred to nitrocellulose filters, and blotted with an anti-WT1 antibody. The position of the 52–54-kDa WT1 protein is indicated.B, HCT116 −/− and +/+ cells were transfected with 0, 1, and 3 μg of a WT1 −/− vector. After 48 h, whole-cell and nuclear extracts were prepared, and WT1 protein levels were assessed by Western blotting. C, Saos-2 cells were co-transfected with a WT1 −/− vector (lanes 4–6) or empty pcDNA3 (lanes 1–3), together with wild-type p53 (lanes 2 and 5) or mutant (codon 248) p53 (lanes 3 and 6)" @default.
- W1986543139 created "2016-06-24" @default.
- W1986543139 creator A5003904670 @default.
- W1986543139 creator A5009899871 @default.
- W1986543139 creator A5034875396 @default.
- W1986543139 creator A5051870613 @default.
- W1986543139 date "2003-01-01" @default.
- W1986543139 modified "2023-10-16" @default.
- W1986543139 title "WT1-p53 Interactions in Insulin-like Growth Factor-I Receptor Gene Regulation" @default.
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