Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5100-5104, June 1993

Physical and functional interaction between WT1 and SHYAMALA MAHESWARAN*, SEON PARK*, AMY BERNARD*, JENNIFER F. MoRRISt, FRANK J. RAUSCHER IIlt, DAVID E. HILLY, AND DANIEL A. HABER*§ *Massachusetts General Hospital Center and Harvard Medical School, Charlestown, MA 02129; tThe Wistar Institute, Philadelphia, PA 19104; and tOncogene Science, Inc., Cambridge, MA 02139 Communicated by Kurt J. Isselbacher, February 22, 1993

ABSTRACT WTI is a tumor-suppressor expressed in rabbit polyclonal antibodies directed against overlapping the developing , whose inactivation leads to the devel- N-terminal peptides, aa 1-173 and 85-173, respectively (9); opment of Wilms tumor, a pediatric kidney cancer. WTI DG10 is a monoclonal antibody generated against human encodes a which binds to the EGRI con- WT1 synthesized in Escherichia coli. For immunoprecipita- sensus sequence, mediating transcriptional repression. We now tions, cells were labeled with [35S]methionine and extracted demonstrate that p53, the product of a tumor-suppressor gene with either ELB buffer (50 mM Hepes, pH 7.0/250 mM with ubiquitous expression, physically associates with WT1 in NaCl/0.5 mM EDTA/0.1% Nonidet P-40) or RIPA buffer (10 transfected cells. The interaction between WT1 and p53 mod- mM Tris, pH 7.4/150 mM NaCl, 1% Triton X-100/1% sodium deoxycholate/0.1% SDS). For sequential immunoprecipita- ulates their ability to transactivate their respective targets. In tions, cellular lysates were extracted with ELB buffer and the absence of p53, WT1 acts as a potent transcriptional immunoprecipitated with the first antibody. The immune activator of the early growth response gene 1 (EGRI) site, complex was then dissociated in RIPA buffer and the re- rather than a transcriptional repressor. In contrast, WT1 leased proteins were immunoprecipitated with the second exerts a cooperative effect on p53, enhancing its ability to antibody. Peptide maps ofimmunoprecipitated WT1 proteins transactivate the muscle creatine kinase . were generated using various concentrations ofStaphylococ- cus aureus V8 protease (10). For immunoprecipitation/ Wilms tumor has been linked to the inactivation of the WTI Western analysis, anti-p53 antibodies were covalently tumor-suppressor gene at the 11p13 chromosomal crosslinked to A-Sepharose (10), and after immuno- (reviewed in ref. 1). WTI encodes a developmentally regu- precipitation, proteins were released from the antibody by lated transcription factor of 52-54 kDa. The C terminus incubation with 2% SDS/50 mM Tris, pH 6.8, at room contains four zinc fingers which confer binding specificity to temperature for 5 min. Western blot analysis was performed the EGRI DNA consensus (2), while the N terminus mediates according to the ECL protocol (Amersham), using antibody transcriptional repression in transient transfection assays (3, WT-6F1 (9) at a dilution of 1:2000. To crosslink proteins in 4). A complex pattern of leads to distinct cells, cultures were incubated with 3 mM dithiobis(succin- WTI gene products (5). Alternative splice II, which encodes imidyl propionate) (DSP, Pierce; 100-mg/ml stock in dimeth- three amino acids [Lys-Thr-Ser (KTS)], interrupts the spac- yl sulfoxide) for 30 min, DSP was inactivated with 30 mM ing between the third and fourth zinc fingers, altering the ammonium acetate (pH 7.0), and protein was extracted. For DNA-binding specificity of WT1 protein (2, 6). WTI muta- two-dimensional gel electrophoresis, proteins were cross- tions detected in Wilms tumor specimens have also provided linked with DSP, immunoprecipitated, and resolved by SDS/ reagents to dissect the functional properties of WT1 protein. 3-10% PAGE. The lanes were excised, the crosslinking was Of particular interest is a dominant negative , reversed by incubation in 5% 2-mercaptoethanol/10%o glyc- WTAR (7), which encodes an in-frame of the third erol/2.3% SDS, 62.5 mM Tris, pH 6.8, and the proteins were and demonstrates oncogenic potential in baby rat electrophoresed in the second dimension. For gel filtration, kidney (BRK) cell transformation assays (8). Disruption of ELB extracts from unlabeled ceils were resolved on a Su- the DNA-binding domain by the WTAR mutation suggests perose 12 FPLC column (Pharmacia); fractions were con- that its dominant effect may result from interactions with centrated by ethanol precipitation and analyzed by Western other cellular proteins. blotting. Using stable BRK cell lines immortalized by transfection Chloramphenicol Acetyltransferase (CAT) Assays. Cultures with the adenovirus EIA gene along with WTI, we demon- were transfected by calcium phosphate/DNA precipitation strate the presence of a complex containing WT1 and p53 with expression constructs under the control ofthe cytomeg- proteins. This complex is also observed in BRK cells ex- alovirus promoter, and the total amount of promoter se- pressing mutant WTAR and a mutated p53 gene (codon 248), quence transfected into each dish was equalized by the as well as in specimens of sporadic Wilms tumor. A potential addition ofvector DNA. Transfection efficiencies were stan- functional interaction between WT1 and p53 is suggested by dardized by cotransfection of a human growth hormone transactivation assays using their respective target se- reporter construct, and all experiments were repeated at least quences. While WT1 enhances transcriptional activation by three times. CAT activity was quantitated by excising the p53, wild-type p53 appears to convert WT1 from a transcrip- appropriate sections ofthe TLC plates for scintillation count- tional activator to a transcriptional repressor. ing.

MATERIALS AND METHODS RESULTS Coimmunoprecipitation of WT1 and p53 in Transfected Immunoprecipitations and Western Blot Analyses. Three BRK Cells. To study WT1-associated proteins in an appro- anti-WT1 antibodies were used: WT-6F1 and WT-91 are Abbreviations: BRK, baby rat kidney; DSP, dithiobis(succinimidyl The publication costs ofthis article were defrayed in part by page charge propionate); MCK, muscle creatine kinase; CAT, chloramphenicol payment. This article must therefore be hereby marked "advertisement" acetyltransferase. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 5100 Downloaded by guest on September 26, 2021 Genetics: Maheswaran et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5101

a 1st Ab Nor.specifir aWf-91 priate kidney-derived cell type, we made use of EIA- immortalized BRK cell lines, stably transfected with con- N c\ C\j structs encoding either wild-type WT1 (clone A6) or mutant WTAR (clone 6.3) (8). Immunoprecipitation of WT1 from 2nd Ab C L BRK cells under nonionic detergent conditions (ELB buffer) ~ Q resulted in the coprecipitation of a protein comigrating with M p53, while immunoprecipitation of p53 coprecipitated a band kDa that comigrated with WT1. Coprecipitation of these proteins was reduced by extraction under more stringent conditions 66- (RIPA buffer), consistent with an unstable protein associa- .- 5 tion. To determine whether WT1 and p53 were in fact coimmunoprecipitated, we performed sequential immuno- precipitations with antibodies directed against these two proteins. We first used 6.3 cells, expressing transfected 45 - WTAR and a mutated endogenous p53 (with an Arg His substitution in codon 246 of the rat p53 gene, analogous to human codon 248). Radiolabeled, ELB-extracted lysates were immunoprecipitated with anti-WT1 antibody WT-91,

a p53 a p53 and the immune complex was dissociated in RIPA buffer 1st Ab Nonspecific PAB 122 PAS 122 before immunoprecipitation with anti-p53 antibody PAb421 or PAb122. Fig. la shows the precipitation of a protein, to to .-Q released from the WT1 immune complex, that comigrated 9D- 2nd Ab a an C-1 with authentic p53. Substitution of nonspecific antibodies of a: 33 4 + the same isotype for either the anti-WT1 or the anti-p53 kDa antibody abolished precipitation of p53. In the reciprocal experiment, immunoprecipitation of extracts with anti-p53 antibody PAbl22, dissociation of the immune complex, and

66 - immunoprecipitation with either of two anti-WT1 antibodies (WT-91 and DG10) precipitated a protein that comigrated - WTAR --- with authentic WTAR (Fig. lb). Use of nonspecific antibod-

45 - ies or preincubation of DG10 with unlabeled vaccinia- produced WT1 protein abolished the immunoprecipitation of WTAR from the p53 immune complex (Fig. lb). Sequential- immunoprecipitation experiments also demonstrated the co- precipitation of p53 and wild-type WT1 from extracts of A6 cells. Partial V8 protease digestion of WT1 either directly C V8 (,u g) 1 5 50 1 5 immunoprecipitated from A6 cells or sequentially immuno- kDa precipitated with anti-p53 followed by anti-WT1 antibodies

45 - confirmed the presence of authentic WT1 within the p53 immune complex (Fig. lc). Immunoprecipitation/Western Blot Analysis of BRK Cells and Wilms Tumor Specimens. We used a second approach to 29 - demonstrate the association between WT1 and p53, consist- ing of immunoprecipitation with the first antibody, followed 21 - by Western blot analysis of the immunoprecipitate with the second antibody. Unlabeled ELB-extracted proteins were 14.3 -. - .: immunoprecipitated with anti-p53 antibody and analyzed by

translated p53 or WT1. RIPA buffer was sufficiently stringent to dissociate the WT1/p53 complex without affecting the primary antigen-antibody interaction. (b) Immunoprecipitation with anti-p53 antibody followed by anti-WT1 antibodies. Radiolabeled proteins extracted with ELB buffer from 6.3 cells were first immunoprecip- itated with either PAb122 (lst Ab, lanes 3 and 4) or a nonspecific antibody of the same isotype (lst Ab, lanes 1 and 2). Immune WTI direct ip WTI Seq ip complexes were dissociated and reprecipitated with anti-WT-91 (2nd aWT DGIO ap53 PAbl22 Ab, lanes 2 and 4) or a 10-fold excess of nonspecific rabbit antibody Il (2nd Ab, lanes 1 and 3). WTAR directly immunoprecipitated from 6.3 aWTI DGI10 cells served as a marker (lane M). In a separate experiment, proteins FIG. 1. Coimmunoprecipitation of WT1 and p53 from BRK cells. first immunoprecipitated with PAb122 were reimmunoprecipitated (a) Immunoprecipitation with anti-WT1 antibody followed by anti- with DG10 (2nd Ab, lane 6) or with DG10 that had been preincubated p53 antibody. Radiolabeled proteins from 6.3 cells were extracted with unlabeled vaccinia-produced WT1 (2nd Ab, lane 7). (c) Partial with ELB buffer and equal amounts of protein were first immuno- V8 protease digestion of WT1. Radiolabeled extracts from A6 cells precipitated with either anti-WT-91 (aWT-91, 1st Ab, lanes 4-6) or were either immunoprecipitated with anti-WT1 antibody (direct ip) a nonspecific rabbit polyclonal antibody (lst Ab, lanes 1-3). WT1- or sequentially with anti-p53 antibody followed by release of asso- associated proteins were released by incubation in RIPA buffer and ciated proteins and immunoprecipitation with anti-WT1 antibody reimmunoprecipitated with monoclonal anti-p53 antibody (Seq ip). The appropriate bands were excised from a polyacrylamide PAb421 (2nd Ab, lanes 1 and 4) or PAb122 (2nd Ab, lanes 2 and 5) gel and analyzed by SDS/PAGE after digestion with various con- or a nonspecific antibody of the same isotype (2nd Ab, lanes 3 and centrations of S. aureus V8 protease. Directly and sequentially 6). Lane M shows the migration position of p53 directly immuno- immunoprecipitated proteins were analyzed in the same gel, but precipitated from 6.3 cells. None of the anti-p53 and anti-WT1 different exposures are shown to allow comparison of digestion antibodies used demonstrated any cross-recognition of in vitro products. Downloaded by guest on September 26, 2021 5102 Genetics: Maheswaran et al. Proc. Natl. Acad. Sci. USA 90 (1993) a Western WTI (aWT 6Fi) from A6 and 6.3 cells (Fig. 2a). The immunoprecipitation/ Western technique allowed us to analyze specimens of pri- mary sporadic Wilms tumors, expressing wild-type endoge- nous WT1 (7), for WT1/p53 complex. WT1 was detected in the p53 immune complex from three Wilms tumor specimens, M X ~ < *Z~ ::4j '7 but not from a control osteosarcoma cell line, U20S, that sit* .: expresses wild-type p53 but not WT1 (Fig. 2 b and c). WTI - Size of the WT1/p53 Complex. Immunoprecipitation of labeled proteins from A6 and 6.3 cells followed by two- -45 kD dimensional gel analysis showed that p53 was present within two subpopulations: a monomeric form migrating at 50-55 Lysote ap53 ip kDa (Fig. 3 a and b, open arrow) and a complex of 100-150 kDa whose individual components migrated at 50-55 kDa b Western WTI (aWT 6Fi) C Western:p53(PAb 122) (arrowhead). The size range of the complex allowed for the presence of two or three components of 50-55 kDa. To confirm the identity of the components within the p53 com- plex, unlabeled 6.3 cell lysates were immunoprecipitated WTi- : s.dai: L _j**, ...... p53 with anti-p53 antibody, resolved by two-dimensional gel electrophoresis, and analyzed by Western blot with anti-WT1 ..JL___ antibody (Fig. 3c). WTAR protein was identified within the Lysote ap53 ip Lysole 100- to 150-kDa p53 complex (arrowhead), comigrating in the FIG. 2. WT1 Western blot analysis of p53 immunoprecipitates. second dimension with marker WTAR protein (M). In the (a) BRK cell lines. ELB-extracted lysates from A6 (E1A/WT1) or 6.3 reciprocal experiment (Fig. 3d), immunoprecipitation with (ElA/WTAR) cells were immunoprecipitated with anti-p53 antibody anti-WT1 antibody followed by Western blot analysis with PAb122 that had been covalently linked to protein A-Sepharose, and immunoprecipitates were analyzed by Western blot using anti-WT1 anti-p53 antibody identified p53 within the WTAR complex antibody WT-6F1 (ap53 ip, lanes 1 and 2). For lane 3: one-fifth the (arrowhead), comigrating with authentic p53 (M). amount of proteins from 6.3 cells was crosslinked in the cells with Gel filtration ofA6 cell lysates showed that p53 was present DSP and extracted with more stringent (RIPA) buffer. Lane 4 either in a monomeric form (Fig. 4a, fraction 13) or in a represents a mock immunoprecipitation. Lane M contained A6 cell complex ranging from 100 to >669 kDa (fractions 1-4). The lysate; position of WT1 is indicated. (b) Specimens from sporadic larger complexes, consistent with the multimerization ofp53 Wilms tumors. ELB-extracted lysates were prepared from three (13), were not evident by two-dimensional gel analysis, sporadic Wilms tumors that express high levels of wild-type WT1 presumably because large crosslinked complexes could not (VG, CS, and CJ) and an cell line (U20S) that does not enter the gel under the conditions used. Analysis of gel express WT1. The extracts were immunoprecipitated with anti-p53 antibody and analyzed by Western blot using anti-WT1 antibody filtration fractions for WT1 protein (Fig. 4b) showed that (apS3 ip). One-tenth ofthe cellular lysate was analyzed alongside and most of the cellular WT1 was present in fraction 4, which served as marker for WT1 (Lysate). (c) Lysates from the Wilms contained the smaller p53 complexes. However, WT1 was tumors and U20S cells were also analyzed by Western blot with also found in larger complexes (fractions 1-3) and in a smaller anti-p53 antibody, demonstrating the presence of p53 protein. complex which did not contain p53 (fraction 5). Low levels of monomeric WT1 were also detected in fractions 12-14. Western blot with a rabbit polyclonal anti-WT1 antibody, Modulation of Transcriptional Activation by Cotransfection WT-6F1. WT1 was identified in the p53 immune complex of WT1 and p53 . To assess the functional conse- FIG. 3. Two-dimensional gel analysis of a Cel/Lu e ElA/WT1 b E1A/WTAR WT1 (WTAR) and p53 complexes. (a and b) Zmmunor70,ec,p,o1a/on p53 (PAb 122) p53 (PAb 122) Two-dimensional gel analysis of p53 com- plexes. Radiolabeled proteins from A6 (ElA/WT1) (a) and 6.3 (ElA/WTAR) (b) : ON..U/A' cells were crosslinked with DSP and immu- 92 _ j noprecipitated with PAbl22. Immunoprecip- itates were analyzed by electrophoresis in a 69- nonreducing dimension (horizontal), fol- -t lowed by reversal ofthe protein crosslinking _ and electrophoresis in a second, reducing 46 dimension (vertical). Open arrows indicate monomeric p53 (50-55 kDa) and arrowheads indicate the p53 released from a 100- to C M d M e 150-kDa complex. The amount of p53 in 6.3 66 cells was higher than that in A6 cells, con- _-pWTAR, ___ -p53 sistent with a prolongation of p53 half-life in P_ these cells (data not shown). (c) Unlabeled 45- proteins from 6.3 cells were crosslinked, immunoprecipitated with a mixture of anti- p53 antibodies (PAb242, -246, and -248), and I 2 o analyzed by two-dimensional electrophore- Ce/ 97L20eElA/WTAR EA 2WTAR E1A/WTAR sis and Western blot with anti-WT1 antibody Imnuoprecepl/l/otIn p53 ( mixture) WTI (aWT91) WTI(AWT 91) WT-6F1. In vitro translated WTAR protein WesterGn WTI5 (aWT 6Fl) p53 (PAb 122) Nonspecific was electrophoresed in the second dimen- sion as a positive control for antibody detection and as a size marker (M). Arrowhead indicates the position of WTAR released from the p53 immune complex (100-150 kDa). (d) Crosslinked proteins from 6.3 cells were immunoprecipitated with antibody WT-91, resolved in a two-dimensional gel and analyzed by Western blot with PAbl22. Protein lysate from 6.3 cells served as size marker and positive control for antibody detection (M). Arrowhead indicates p53 protein released from the WTAR immune complex (100-150 kDa). (e) The Western filter shown in d was stripped of PAb122 and probed with a nonspecific mouse monoclonal antibody. Downloaded by guest on September 26, 2021 Genetics: Maheswaran et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5103 a Western p53 (PAb 122) the other. p53 has been shown to activate transcription from 1 2 3 4 5 6 7 8 9 10 11 12 133 12 13 14 the muscle creatine kinase (MCK) promoter (14). Transfec- tion of wild-type p53 into Saos-2 osteosarcoma cells, with SIt deleted p53 genes (15), resulted in 8.6-fold activation of poog:.e MCK-CAT (Fig. Sa). Cotransfection of p53 with wild-type WT1, but not mutant WTAR, consistently enhanced MCK- b Western WTI (a WT 6Fi) CAT activity (36.3-fold transactivation), suggesting a coop- l erative interaction. In contrast, p53 exerted a suppressive effect on WT1 transactivation. WT1 (lacking the KTS splice) has been shown to be a transcriptional repressor ofthe EGRI promoter in 3T3 cells (ref. 3 and Fig. Sb). However, trans- 669 443 150 66 prolonged exposure fection of WTJ into Saos-2 cells, lacking endogenous p53, FIG. 4. Gel filtration ofBRK lysates. ELB-extracted lysates from resulted in 13.6-fold activation of transcription from the A6 cells were fractionated on a Superose 12 FPLC column. Serial EGRI promoter (Fig. Sb). Transcriptional activation of the fractions were collected, and proteins were ethanol-precipitated and EGR1 promoter by WT1 was also seen in HeLa cells, in analyzed by Western blot using anti-p53 (a) or anti-WT1 (b) anti- which p53 is inactivated by E6 protein (data not shown). In bodies. Prolonged exposure offractions 12-14 was required to detect A1.5 cells, rat embryo fibroblasts stably transfected with a monomeric p53 or WT1. Gel-filtration size markers were thyroglob- temperature-sensitive p53 mutant (16), WT1 demonstrated ulin (669 kDa), apoferritin (443 kDa), alcohol dehydrogenase (150 10-fold transcriptional suppression of the EGRJ promoter kDa), and bovine serum albumin (66 kDa). under conditions favoring the wild-type p53 conformation, and 3-fold transcriptional activation in the presence of the quences of the WT1/p53 interaction, we analyzed the ability mutant p53 conformation (Fig. 5c). To analyze the effect of of each protein to modulate the transactivational activity of the WT1/p53 interaction on a more restricted promoter, we b EGR1-CAT a MCK-CAT (Saos-2) (3T3) (Soos-2) fold c EGRI-CAT (Al.5) induction 1.1 1.2 1.0 8.6 9.9 36.3 1 0.09 1 13.2 1 0.07 1 3

40 b.. S _re 40Jf: 4

_. 4 It t I .N.. S. I. * , f*.: ½ ¼J K 4 > )4 A> '4 A~' -A'

_ 320 c 39°C d p3XEBS-CAT (Saos-2) fold induction 13 15- 18 1 4844.89336440445250

. W,

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. . a FIG. 5. Modulation of transcriptional activation by cotransfection of WTJ and p53 genes. Saos-2, 4P 3T3, or A1.5 cells were transiently transfected by 9* .4p @, calcium phosphate DNA precipitation with reporter constructs: p53-responsive MCK-CAT (a) and WT1- responsive EGR1-CAT (b and c) or the minimal WT1-responsive p3XEBS-CAT (d), along with the following cytomegalovirus promoter-driven expres- sion constructs: vector alone (Vector), wild-type p53 (p53), mutant p53 at codon 143 (11) (mp53), wild- type RB (RB), mutant RB J82 (12) (mRB), and WT1 constructs lacking alternative splice 11 (9) (-KTS) or encoding alternative splice 11 (+KTS), or mutant WTAR (7). Downloaded by guest on September 26, 2021 5104 Genetics: Maheswaran et al. Proc. Natl. Acad Sci. USA 90 (1993) used the 3XEBS-CAT reporter, containing three tandem and WT1 may thus serve as a model for the functional EGRI consensus sequences upstream of the minimal fos interaction between p53 and other transcription factors. promoter (3). Transfection of Saos-2 cells with WT1 resulted The genetic implications of the WT1/p53 interaction re- in potent transcriptional activation (44.8-fold) of 3XEBS- main to be defined. In humans, a germ-line mutation inacti- CAT (Fig. 5d). Reintroduction of wild-type p53 suppressed vating one p53 allele, Li-Fraumeni syndrome, leads to an WT1-mediated transcriptional activation. The suppressive increased incidence of multiple , including Wilms effect ofp53 was observed following transfection of <1 ,zg of tumor (ref. 21; J. Garber and F. Li, personal communication). p53 DNA, which did not affect the level of baseline tran- Dominant negative WT1 mutants that contain disrupted scription from the 3XEBS reporter or that from a control DNA-binding domains, such as WTAR, may mediate their promoter, suggesting a specific functional interaction rather effect through a nonproductive interaction with p53, whereas than general suppression of the cellular transcriptional ma- other WT1 may specifically disrupt the interaction chinery. No effect on transactivation by WT1 was seen with between these two proteins. Genetic analysis ofWT1 and p53 a mutant p53 construct [codon 143 (11)] or by constructs mutants may thus serve to complement the biochemical encoding the gene product (RB), which is also characterization ofthe WT1/p53 interaction. The interaction deleted in Saos-2 cells. between p53, a protein with potentially universal function in cellular growth control, and WT1, a tissue-specific transcrip- tion factor, may provide a framework for understanding the DISCUSSION action of these two tumor-suppressor genes. By studying stably transfected BRK cell lines and Wilms We are grateful to Drs. D. Housman, E. Harlow, P. Sharp, D. tumor specimens, we have identified an association between Dobson, and N. Dyson for critically reviewing our manuscript; to two tumor-suppressor genes, WTI and the p53 gene, that may Drs. S. Friend, T. Frebourg, S. van den Heuvel, S. Pillai, J. Licht, play a role in normal development and in tumorigenesis. In and A. Bruskin for helpful discussions; and to Drs. A. Levine, S. this study, we used sequential immunoprecipitations with Hauschka, and V. Sukhatme for valuable reagents. This work was anti-p53 and anti-WT1 antibodies, as well as immunoprecip- supported by grants from the James McDonnell and Edward itations with one antibody followed by Western analysis with Mallinckrodt Foundations and the National Institutes of Health (CA58596) (to D.A.H.); the W. W. Smith Charitable Trust, Irving A. the second antibody to demonstrate the presence of a WT1/ Hansen, Mary Rumsey, and Pew Foundations, and the National p53 complex in transfected cells. The WT1/p53 association Institutes of Health (CA52009 and CA47983) (to F.J.R.); and the was evident in BRK cells expressing either wild-type WT1 or Cancer Research Institute (to J.F.M.). mutant WTAR, and either wild-type p53 or a p53 mutant (analogous to the human codon 248 mutation). However, a 1. Haber, D. & Housman, D. (1992) Adv. Cancer Res. 59, 41-68. functional interaction between WT1 and p53 was observed 2. Rauscher, F., Morris, J., Tournay, O., Cook, D. & Curran, T. only with the wild-type proteins. (1990) Science 250, 1259-1262. The association between WT1 and p53 could represent 3. Madden, S., Cook, D., Morris, J., Gashler, A., Sukhatme, V. either direct binding or an interaction through a third protein. & Rauscher, F., III (1991) Science 253, 1550-1553. The WT1/p53 complex migrated at 100-150 kDa and con- 4. Drummond, I., Badden, S., Rohwer-Nutter, P., Bell, G., at Sukhatme, V. & Rauscher, F., III (1992) Science 257, 674-678. tained individual components migrating 50 kDa, allowing 5. Haber, D., Sohn, R., Buckler, A., Pelletier, J., Call, K. & for either two or three such components. Two-dimensional Housman, D. (1991) Proc. Natl. Acad. Sci. USA 88,9618-9622. gel analysis of BRK cells did not demonstrate the presence of 6. Bickmore, W., Oghene, K., Little, M., Seawright, A., van a distinct complex between p53 and , a 90-kDa protein Heyningen, V. & Hastie, N. (1992) Science 257, 235-237. that binds to p53 (17). This may reflect the lower abundance 7. Haber, D., Buckler, A., Glaser, T., Call, K., Pelletier, J., Sohn, of mdm2 in these cells, compared with the transfected WT1, R., Douglass, E. & Housman, D. (1990) Cell 61, 1257-1269. or its relative shielding from protein crosslinking. Like WT1, 8. Haber, D., Timmers, H., Pelletier, J., Sharp, P. & Housman, mdm2 is a zinc-finger transcription factor, but its functional D. (1992) Proc. Natl. Acad. Sci. USA 89, 6010-6014. interaction with p53 contrasts with that of WT1. Whereas 9. Morris, J., Madden, S., Tournay, C., Cook, D., Sukhame, V. & Rauscher, F., III (1991) 6, 2339-2348. cotransfection of the mdm2 gene inhibits activation of the 10. Harlow, E. & Lane, D. (1988) in Antibodies: A Laboratory MCK promoter by p53 (17), WT1 exerts an enhancing effect Manual (Cold Spring Harbor Lab. Press, Plainview, NY). on p53-mediated transactivation. Cotransfection of mutant 11. Baker, S., Markowitz, S., Fearon, E., Willson, J. & Vogelstein, WTAR had no effect on transactivation ofthe MCK promoter B. (1990) Science 249, 912-915. by p53, suggesting that an intact WT1 DNA-binding domain 12. Horowitz, J. M., Yandell, D., Park, S., Canning, S., Whyte, P., is required for this cooperative effect. Buchkovich, K., Harlow, E., Weinberg, R. & Dryja, T. (1989) The suppressive effect of p53 on transactivation by WT1 is Science 243, 937-940. particularly interesting, given the characterization of WT1 as 13. Kraiss, S., Quaiser, A., Oren, M. & Montenarh, M. (1988) J. a transcriptional repressor of growth-related genes such as Virol. 62, 4737-4744. and The 14. Weintraub, H., Hauschka, S. & Tapscott, S. (1991) Proc. Natl. EGRI IGF2 (3, 4). potent transcriptional activation Acad. Sci. USA 88, 4570-4571. by WT1 of the EGRI site in cells lacking wild-type p53 15. Masuda, H., Miller, C., Koeffler, H., Battifora, H. & Cline, M. indicates that transcriptional repression is not an intrinsic (1987) Proc. Natl. Acad. Sci. USA 84, 7716-7719. property of WT1. Instead, transcriptional repression by WT1 16. Martinez, J., Georgoff, I., Martinez, J. & Levine, A. J. (1990) may result from its interaction with other cellular proteins, Genes Dev. 5, 151-159. including p53. Such a model would be analogous to the effect 17. Momand, J., Zambetti, G., Oson, D., George, D. & Levine, A. of RB in transforming the transcription factor from a (1992) Cell 69, 1237-1245. transcriptional activator to a repressor (18). p53 itself has 18. Weintraub, S., Prater, C. & Dean, D. (1992) Nature (London) been shown to be capable of suppressing transcription from 358, 259-261. a number of different promoter elements that do not contain 19. Seto, E., Usheva, A., Zambetti, G., Momand, J., Horikoshi, N., Weinmann, R., Levine, A. & Shenk, T. (1992) Proc. Natl. its own target sequence. The mechanism underlying this Acad. Sci. USA 89, 12028-12032. effect may involve an association with TATA-binding protein 20. Agoff, S. N., Hou, J., Linzer, D. I. H. & Wu, B. (1993) Science (19), as well as a direct interaction with specific transcription 259, 84-87. factors such as WT1 or with CCAAT-binding factor (20). The 21. Garber, J., Goldstein, A., Kantor, A., Dreyfus, M., Fraumeni, reciprocal modulation of transcriptional activation by p53 J., Jr., & Li, F. (1991) Cancer Res. 51, 6094-6097. Downloaded by guest on September 26, 2021