ICANCER RESEARCH 55, 5386-5389, November 15, 19951 Regulation of the Proto- bcl-2 and c-myc by the Wilms' WTJ1

Stephen M. Hewitt, Shinshichi Hamada,2 Timothy J McDonnell, Frank J Rauscher III, and Grady K Saunders3

Departments of Biochemistry and Molecular Biology [S. M. H., S. H., G. F. S.], and Molecular Pathology fT. J. M.J. The University of Texas M. D. Anderson Center, Houston, Texas 77030: and Wistar Institute ofAnatomy and Biology, Philadelphia, Pennsylvania 19107 [F. J. R.]

ABSTRACT polycystic kidneys and a reduced number of nephrons (20—22).bcl-2 overexpression has been found in both renal cell carcinomas and The Wilms' tumor gene WTJ functions as a tumor suppressor gene, Wilms' tumors (23), suggesting that bcl-2 has a role in tumorigenesis. repressing transcription of several growth factors and receptors. The bcl-2 and c-myc proto-oncogenes are essential for regula c-myc, the cellular homologue of v-myc, is an immediate early tion of and with roles in development and growth-response gene that plays a role in proliferation and oncogen oncogenesis. We found that WTJ can repress transcription of both the esis (24). c-myc is capable of either driving cell proliferation or bcl-2 and c-myc promoters. This suggests that WT1 regulates bcl-2 and apoptosis, depending on other cellular signals (24). c-myc has been c-myc during renal development, and the loss offunctional WT1 results in demonstrated as essential for development, and homozygous null deregulation of bcl-2 and c-myc, contributing to tumor formation. c-myc embryos die in utero (25). Overexpression of c-myc in the developing kidney results in polycystic kidney disease and abnormal INTRODUCTION cellular proliferation (26). c-myc is expressed in some Wilms' tumors, although not at the levels at which N-myc is frequently observed (27). The Wilms' tumor gene WTJ encodes a 449-amino acid zinc-finger Both bcl-2 and c-myc are expressed at high levels in the developing (1, 2) that represses transcription from a number of metanephric blastema but are down-regulated as differentiation occurs target promoters (3—11).The WT1 protein has a proline-rich amino (23, 28). bcl-2 is expressed in the induced mesenchyme of the met terminus that mediates transcriptional repression (3) and four Cys-Cys His-His zinc fingers that bind DNA (12). WT1 binds a GC-rich motif, anephros, with highest bcl-2 expression occurring within the condens GNGNGGGNG (13), and mediates transcriptional repression (3). ing mesenchyme (23). bcl-2 expression is quickly reduced as cells WT1 is differentially spliced, resulting in four splice isoforms (14). differentiate to epithelia in the glomerulus (23, 29, 30). bcl-2 expres One alternative splice results in the removal of exon 5; this modifi sion is detectable in the tubules of the nephron (23, 29, 30). c-myc is cation has no known functional consequence. A second alternative expressed in the uninduced mesenchyme and continues to be ex splice occurs in exon 9 and involves removal of a 9-nucleotide pressed until condensation of the comma- and S-shaped bodies, when sequence encoding KTS,4 resulting in the of a spacer between epithelial differentiation occurs (28). c-myc is not detected in glomer zinc fingers three and four (14). The presence of the KTS sequences ular structures but is detectable in the tubules during elongation. The alters WT1 DNA binding (15) and may alter the WT1 targets of expression of these two proto-oncogenes is temporally related in the repression. WT1 is expressed at low levels in the induced mesen same cells. The increase in WT1 expression corresponds spatially and chyme (16, 17). Expression increases as differentiation to the epithelia temporally to the decrease in bcl-2 and c-myc expression during occurs in the comma- and S-shaped bodies (16, 17). WT1 expression epithelial differentiation in the developing kidney (16, 17, 23, 28—30). remains within the podocyte layer of the glomerulus, but is undetect able in the other structure of the nephron or collecting ducts in the adult (17). Analysis of homozygous null WT1 transgenic mice reveals MATERIALS AND METHODS that the metanephric blastema forms, but is unable to differentiate to epithelia, even in the presence of a strong inducer (18). We searched Cotransfections. Cotransfectionwasperformedbyelectroporationaccord the Eukaryotic Database (19) for potential WT1 targets that ing to a method described previously (31). Briefly, S X 106 HeLa cells were have multiple potential WT1-binding sites within their promoters, are electroporated with 2.5 p@gofreporter plasmid and with 0—10p@gofexpression expressed in the developing kidney, and may have a potential role in plasmid. The total amount expression vector in each transfection was adjusted oncogenesis and . The proto-oncogenes bcl-2 and c-myc to 10 @igwith an empty expression vector. A cytomegalovirus driven p3-ga lactosidase plasmid (pCMVJ3; 1.25 p@g)was used as an internal control in the were identified as potential targets of WT1 repression. transfections. The cells were suspended in 200 p1 of medium without serum, bcl-2 encodes a polypeptide involved in the suppression of apop electroporated at 260 and 960 g.tF, and cultured for 72 h. CAT assays were tosis and is essential for normal renal development (20—22), and performed with the Boehringer Mannheim CAT ELISA according to the bcl-2-deficient mice have prominent kidney lesions that resemble directions of the manufacturer. Results were normalized with f3-galactosidase activity. Results are the average of three independent transfections. Received 6/20/95; accepted 9/19/95. EMSAS. EMSAwere performedwith a protocolmodifiedfromCao et al. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with (32). The reaction volume of 10 p1 contained a final concentration of 20 m@i 18 U.S.C. Section 1734 solely to indicate this fact. HEPES (pH 7.5), 70 mM KCI, 5 mM MgCl2, 100 @MZnCl2,0.5 mM DTF, 1 This work was supported by grants from the NIH (CA34936 and CA16672). T. J. M. 0.05% NP4O, and 12% glycerol. Each reaction contained 10 @Mof32P-labeled and F. J. R. are Pew Scholars in the Biomedical Sciences. S. M. H. is an M.D/Ph.D. DNA probe, 500 ng of WT1 protein, I @gofpoly dIdC, and 0—150 @M Predoctoral Fellow at The University of Texas Graduate School of Biomedical Sciences and is supported by Grant T32 CA09299. competitor oligonucleotide. Reactions were carried out at room temperature 2 Present address: Department of Pathology, Shiga University of Medical Science, and electrophoresed on a 5% 0.5 X Tris-borate EDTA acrylamide gel at 4°C. Seta, Ohtsu, 520-21, Japan. The sequence of the double stranded oligonucleotides used in EMSA are EGR 3 To whom requests for reprints should be addressed, at Biochemistry and Molecular Biology, Box 1 17, The University of Texas M. D. Anderson Cancer Center, 1515 (GGCCCGGCGCGGGGGCGAGGQCG),BCL2(GcICCCTTCCFCCCGCGC Holcombe Boulevard, Houston, TX 77030; Fax: (713) 790—0329. CCGCCCGG),CMYC (GGCCFGCCCGCCCACrCTCCCGG),and their corn 4 The abbreviations used are: KTh, Lys-Thr-Ser; CAT, chloramphenicol acetyltrans plements. Oligonucleotideprobes were labeled with [a-32P]dCT'Pby using the ferase; EMSA, electrophoretic mobility shift assay; EGR1, early growth response gene 1; IGF-I1, insulin-like growth factor II; PDGF a-chain, platelet-derived growth factor a- Kienow fragment to fill in a G overhang. The WT1—KThand WF1+KTh chain. peptideshave been describedpreviouslyin Rauscheret a!. (12). 5386

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1995 American Association for Cancer Research. wTI REPRESSION OF bcl-2 AND c-myc A P2 start sites and contains three potential WT1-binding motifs (Fig. 3B). P1 XBI A P2 P @ 1@' II 1@introni r' i@——— The WT1 —KThand WT1 +KTS cDNA expression plasmids were Wx5 W W W WW W — each cotransfected with the murine c-myc P1 +P2 construct. Both 0.34 kb pro 100 bp WT1 —KTS and WT1 +KT5 repressed transcription from the c-myc promoter approximately 4-fold. An EMSA was used to confirm in B teraction of WT1 with the c-myc promoter. An oligonucleotide con Spi Spi AP2 GGGCCAGGGACGGGCGGAGG_GGCGGCTCGG_GCTGGCTCAGAGGAGGGCTC taming a potential WT1-binding site within the c-myc promoter (Fig. CREB 3B) bound both WT1 —KThand WT1 +KTS protein and competed TTTCTTTC'2T CTTTTTTTGA ATGAACGT@I @A@ITACGC ACCAGGAAAC C/EBP with both unlabeled EGR-1 and c-myc oligonucleotides (Fig. 4B). CGGTCGGCTG TGCACAGAAI GAAGTAAGAG GACCAGGCCA CCGACAGCCC Spi Spi AP2 Spi AP2 CGA@C@@ @CCTTCCTC C@GCGt@$@@ @CTCCGCGC CGCCTGCC@ DISCUSSION Spi AP2 Sp1AP2 -‘ -‘ - c@c@lt@c@GCC@CGC@1@CC@GC@CGCCGCTCTCCGTGGCCCCGCCGCGCTGC WT1 has been shown to repress transcription from IGF-II (4), CGCCGCCGCCGCTGCCAGCGAAGGTGCCGGGGCTCCGGGCCCTCCCTGCC insulin-like growth factor 1 (6), PDGF a chain (5), trans forming growth factor @31(11), colony-stimulating factor 1 (7), PAX2 GGCGGCCGTCAGCGCTCGGAGCGGGCTGCG (10), and a (9), as well as EGR1 (3) and WT1 Fig. 1. Panel A, human bcl-2 promoter region. Promoter activity maps to two regions, (8). IGF-II, PDGF a chain, and transforming growth factor @31are P1 and P2, separated by intron I (33). Bent arrows, start sites. W, WT1-binding motifs. The promoter region (0.34 kb pro) cloned into the CAT reporter plasmids is shown. A, growth factors expressed during renal development (4, 5, 11), and AccI; BI, Bglll; P, PszI; X, Xhol; orf, open reading frame. Panel B, sequence of the IGF-II and PDGF a chain are overexpressed in Wilms' tumor (40— promoter region (0.34 kb pro) contained in the CAT reporter vector. Start sites of transcription are marked in bold (—@*;Ref. 33). WT1 consensus motifs are shaded. Potential binding sites for Spi, AP2, CREB, and C/EBP are underlined. The oligonu cleotide used in the EMSA is italicized. A

1.2 RESULTS

Transcriptional Repression of the bcl-2 P1 Promoter by WT1. Transcription of human bcl-2 originates from two promoter regions 1.0 1.3-kb apart (33). Most transcription originates from promoter 1 (P1; Fig. 1A), a GC-rich region that lacks both TATA and CAT boxes (33). This region contains numerous potential binding motifs for the tran 0.8 WT1-KTS scription factors Spi and AP2 (Fig. 1B). Previous work in our 0 laboratory showed that transcription can be initiated from the 340-bp 0 P1 region, which includes the first three start sites of bcl-2 transcrip 0 0.6 tion.5 Sequence analysis of this region revealed five potential WT1- 0 WTI+KTS binding motifs with the consensus sequence GNGNGGGNG (Fig. 1B) that overlap with Spi- and AP2-binding sites. To examine the ability of WT1 to repress the bcl-2 promoter, we 0.4 transiently cotransfected WT1 cDNA expression plasmids with either the human WT1 —KTSor WT1 +KTS isoform driven by the cyto megalovirus promoter with CAT reporter constructs containing the 0.2 bcl-2 promoter P1. Cotransfection of either WT1 expression plasmid 0 1 2 3 4 5 repressed transcription from the bcl-2 promoter plasmid 2.5-fold at a Ratio of WT1 to bcl-2 Plasmid 4: 1 ratio of expression plasmid:reporter plasmid. To confirm that WT1 binds sequences within the bcl-2 promoter, we used EMSAS. A bcl-2 promoter oligonucleotide containing two overlapping WT1- binding motifs with the consensus sequence GNGNGGGNG (Ref. 13; B Fig. 1B) was used to bind WT1-KTS and could be competed with the WT1 -KTS WT1 + KTS addition of either unlabeled EGR1 consensus motif (GCGGGGGCG), a strong WT1-binding motif (12), or unlabeled bcl-2 oligonucleotide Protein: .4— DNA (Fig. 2B). By using WT1 + KTS in EMSA, we were able to demon Complex strate binding of WT1 +KTS protein to this oligonucleotide, as well as competition with both unlabeled oligonucleotides (Fig. 2B). Transcriptional Repression ofthe c-myc Promoter by WT1. We ‘ø—Free next assayed the ability of WT1 to repress the murine c-myc promoter. Probe The promoter region of murine c-myc has been well described by numerous investigators (34—38).Transcription of c-myc originates Fig. 2. A, relative CAT activity of the human bcl-2 promoter region cotransfected with from two major start sites, P1 and P2, 164-bp apart (Ref. 39; Fig. 3A). human WT1 cDNA expression plasmids WT1—KThand WT1+KTS into HeLa cells. The c-myc promoter is highly conserved between mouse and human WTI—KTSand WT1+KTS both repressed CAT activity from the bcl-2 promoter P1 by 2.5-fold. B, EMSA of WT1 binding motif present within bcl-2 promoter P1. DNA probe (78.8% nucleotide identity), and most transcription initiates from P2 is italicized in Fig. iB, Lane 1, free probe; Lane 2, WT1—KTSprotein; Lane 3, (39). We assayed a 667-bp region that encompasses both the P1 and WT1 —KTh protein, unlabeled probe competitor (X 15); Lane 4, WT1 —KTh protein, unlabeled EGR1 oligonucleotide (XiS); Lane 5, WT1+KTS protein; Lane 6, WT1+KTS protein, unlabeled probe competitor (X 15); Lane 7, W'I'l +KTS protein, unlabeled EGR1 5 5. Hamada, unpublished results. oligonucleotide (XiS). 5387

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1995 American Association for Cancer Research. wTl REPRES5ION OF bc!-2 AND c-myc 42). Unfortunately, the repression of growth factors does not provide A convincing evidence that WT1 functions as a tumor suppressor gene. The failure of the metanephric blastema to differentiate to epithelia in I .2 the presence of inducer in the homozygous null WT1 embryo (18), provides evidence that WT1 plays a role in epithelial differentiation. 1.0 WT1 repression of bcl-2 and c-myc may explain the role WT1 plays in epithelial differentiation. Defects in either bcl-2 and c-myc lead to defects in epithelial structures of the kidney (20—22,26). The repres sion of bcl-2 and c-myc by WT1 adds an additional class of transcrip tional targets of WT1 repression that implicates WT1 in both devel WT1-KTS opment and tumorigenesis. The ability of WT1 to repress bcl-2, c-myc, and growth factors is evidence that WT1 coordinately regulates both apoptosis and cell proliferation. Repression of bcl-2 and c-myc by WT1 during renal development corresponds to a reduction in mesenchymal proliferation IE and the onset of epithelial differentiation. The loss of functional WT1 0.2 results in the failure to repress bcl-2, and bcl-2 consequently sup presses apoptosis. Failure to repress c-myc because of WT1 0.0 or deletion results in apoptosis or cellular proliferation, depending on 0 1 2 3 4 5 the presence of growth factors. Interestingly, genetic cooperation of c-myc and bcl-2 has been demonstrated during multistep carcinogen Ratio of WT1 to c-myc Plasmid esis in transgenic strains of mice that overexpress these oncogenes (43, 44). This complementation is due to inhibition of c-myc-mediated apoptosis by bcl-2, without alterations in c-myc-enhanced prolifera tion (40). In the absence of wild-type WT1, growth factor expression B is no longer repressed, and growth signals in concert with c-myc expression result in cellular proliferation. It appears that loss of WT1 function deregulates multiple pathways that maintain cell growth in Protein: check, and the loss of this regulation can result in tumor formation. @4- DNA Complex

A ‘4- Free P1 5H@2 N XBI Probe 1@ @i1@ T T ii I 234567 WNTA TA W 50b Fig. 4. A, relative CAT activity of the murine c-myc promoter region cotransfected with 067 kb nm p human WT1 cDNA expression plasmids WT1—KTS and WT1+KTS in HeLa cells. WT1 —KTSand WT1 + KTh both repressed CAT activity from the c-myc promoter 4-fold. B, EMSA of WT1 binding motif present within c-myc promoters. The DNA probe is B 5'Mg2 italized in Fig. 3B. Lane 1, free probe; Lane 2, W'Fi—Kl'S protein; Lane 3, WT1—KTS CCGAGTTCCC AAAGCAGAGG GCGGGGAAAC GAGAGGAAGG AAAAAAATAG protein, unlabeled probe competitor (X 15); Lane 4, WTi —KThprotein, unlabeled EGR1 5 IMg3 oligonucleotide (X 15); Lane 5, WT1 +KTS protein; Lane 6, W'I'i + KTS protein, unla AGAGAGGTGG GGAAGGGAGA CTCTGG@IAA TCCC@c@c@A beled probe competitor (X 15); Lane 7, WT1+KTS protein, unlabeled EGRI oligonu TATA —P1 cleotide (XiS). =mcccT@1T AIATTCCGGG GGTCTGCGCGGCCGAGGACC

CTGCTCTCAG CTGCCGGGTC CGACTCGCCT CACTCAGCTC CCCTCCTGCC ME1a2 ME1a1 These results provide evidence that WT1 plays a critical role in the TCCTGAAGGG CAGCGTTCGC CGACGCTT@ @@AAAAAGAAGGGAGGGG TATA —P2 regulation of cell proliferation and differentiation in the developing A@GGATCCTGAGTCGCAGTh IAAAAGAAGC TTTTCGGGCG TTTTTTTCTG kidney and demonstrate that WT1 regulates pathways other than those

ACTCGCTGTA GTAATTCCAG CGAGAGACAG AGGGAGTGAG CGGACGGTTG of growth factors.

GAAGAGCCGT GTGTGCAGAG CCGCGCTCCG GGGCGACCTA AGAAGGCAGC ACKNOWLEDGMENTS TCTGGAGTGA GAGGGGCTTT GCCTCCGAGC [email protected]@t:@ME:?CTCCCCA We wish to thank Gail Fraizer for her advice and suggestions and Bruno ACCCTGCGAC TGACCCAACA TCAGCGGCCG CAACCCTCGC CGCCGCTGGG Calabretta for providing the rnurine c-myc P1+P2 CAT plasmid. We also AAAcGCC CATTGCAGCG GGCAGACACT TCTCACTGGA ACTTACAATC thank Angela Monarres, Lazar Kottical, and Ruby S. Desiderio for their assistance. TGCGAGCCAG GACAGGACTC CCCAGGCTCC GGGGAGGGAA GTCTA TTTGGGGACA GTGTTCTCTG CCTCTGCCCG CGATCAGCTCTCCTGAAAAG REFERENCES AGCTCCTC i. Call, K. M., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber, D. A., Rose, Fig. 3. Panel A, murine c-myc promoter region. Transcription initiates from the two E. A., Kral, A., Yeger, H., Lewis, W. H., Jones, C., and Housman, D. E. Isolation and start sites P1 and P2 (bent arrows). W, WT1-binding motifs of GNGNGGGNG. TA, characterization of a polypeptide gene at the human chromosome ii TATA boxes; T, attenuation signals in c-myc exon 1. The promoter region P1 +P2 Wilrns' tumor locus. Cell, 60: 509—520,1990. contained in the CAT reporter plasmid is shown (0.67 kb pro). A, AccI; B, BamHI; Bl, 2. Gessler, M., Poustka, A., Cavenee, W., Neve, R., Orkin, S., and Bruns, G. A. P. A BglII; H, HindIIl; n, NotI; P, PstI; and X, XhoI. Panel B, sequence of the murine c-myc zinc finer gene identified by chromosome jumping. Nature (Land.), 343: 774—778, promoter region contained in the CAT reporter vector. Start sites of transcription are 1990. marked in bold(—*,DJand —°P2).WT1 consensus motifs are shaded. The oligonucleotide 3. Madden, S. L., Cook, D. M., Morris, J. F., Gashler, A., Sukhatme, V. P., and used in the EMSA is italicized. Attenuation signals are double underlined. Binding sites Rauscher, F. J., III. Transcriptional repression mediated by the Wfl Wilms tumor described previously are underlined and identified (34—39). gene product. Science (Washington DC), 253: i550—i553,i99i. 5388

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Stephen M. Hewitt, Shinshichi Hamada, Timothy J. McDonnell, et al.

Cancer Res 1995;55:5386-5389.

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