wihns' tumor 1-kts isoforms induce p53-independent ... · [cancerresearch57....

[CANCERRESEARCH57. 1353-1363.April1. 19971

ABSTRACT

The Wilma' tumor 1 gene (WTJ)encodes a transcription factor of thezinc-finger family. As a result ofalternative RNA splicIng, the gene can beexpressed as four polypeptides that differ in the presence or absence of astretch of 17 amino acids just NH2 terminal of the four zinc fingers and astretch of three amino adds (Â±KTS) between zinc fingers 3 and 4. In this

study, cDNA constructs encoding the four human Wilma' tumor 1 splicevariants were transiently transfected into the p53-negative Hep3B and thep53-positive HepG2 hepatoma cell lines. Morphological assessment of theWT1-expressfng cells showed that the WT1(â€”KTS)splice variants induced apoptosis in both cell lines, whereas the WT1(+KTS) isoforms didnot. The induction of apoptosis by the WT1(â€”KTS)isoforms appears tobe p53 Independent in the hepatoma cell lines. Furthermore, it was foundthat the WT1(â€”KTS)-induced apoptosis could not be suppressed by coexpression of either the Mr 21,000 E1B, the Bcl-2, or the BAG-i protein.Coexpression ofeither the epidernial growth factor receptor or the insulinreceptor, however, partially rescued the cells from apoptosis.

INTRODUCTION

The yÃ§j'j2gene is one of at least three genes involved in thedevelopment of Wilms' tumor, a pediatric malignancy of the kidney(1). The gene spans about 50 kb at the 11p13 locus, contains 10 exons,and specifies a 3-kb mRNA (2, 3). As a result of alternative RNAsplicing, the gene can be expressed as four multiple-zinc fingerpolypeptidesthat differ in the presence(+) or absence (â€”)of twostretches of aas, a stretch of 17 residues just NH2 terminal of the fourzinc fingers (Â±l7aa) and one of 3 residues (K, T, and 5) between zincfmgers 3 and 4 (Â±KTS; Ref. 4). The functions of these four different

WT1 isoforms remain to be elucidated, but several lines of evidenceindicate that each protein may have a separate function.

All four WT1 polypeptides contain four zinc fingers of the KruppelC2-H2 class in the COOH-terminal part and a proline/glutamine-richN'@2terminus (2), suggesting a role as a transcription factor. Transienttransfection assays indicated that each WT1 splice variant may havea different transcriptional activity on (partly) different target genes(5â€”10).

Some WT1 splice variants may also be involved in posttranscriptional events. It has been shown that the WT1(+KTS) proteins cobcalize and associate with proteins of the splicing machinery (1 1),suggesting the involvement of these WT1 isoforms in splicing.

Recently, the four splice variants were also found to have differentbiological effects. Stable expression of the WT1(â€”l7aaâ€”KTS) protein in an adenovirus-transformed baby rat kidney cell line resulted in

Received 1/27/97; accepted 5/31/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed. Phone: (31) 71-5276136; Fax;

SRa-Ins-R and EV-Ins-R, eukargotic expression vectors expressing the human insulinreceptor; WTAR, mulated WT1 gene derived from pateint AP. (31) 71-5276284.

2 â€˜p.@abbreviations used are: WTI, Wilma' tumor I; as, amino acid; EGF, epidermalgrowth factor; CMV, cytomegalovirus; DAPI, 2,4-diamidino-2-phenylindole; RSV, Roussarcoma virus; IGF, insulin-like growth factor; SRa-lns-R and EV-Ins-R, eukaryoticexpressionvectorsexpressingthe humaninsulinreceptor,WTAR,mutated1471genederived from patient AR.

increased tumorigenicity (12), whereas expression of theWT1(â€” l7aa+KTS) protein suppressed the tumorigenicity of these

cells (12, 13).How these diverse properties, ascribed to the WT1 splice variants,

reflect the possible physiological functions of the WTJ gene is stillunclear. On the basis of the expression pattern of the gene, it has beenhypothesized that WT1 plays an important role in the development ofthe kidney, the gonads, and the mesothelium (14â€”17).The tissuesexpressing WT1 during the development of these organs have incommon that they are mesodermal in origin and that they experiencea switch from mesenchymal to epithelial cells. During kidney development, for example, WT1 expression is first seen in the mesenchymal metanephric blastemal cells condensing and aggregating aroundthe uretic bud. Levels of WTI mRNA increase as the mesenchymalcells divide and differentiate into specialized epithelial nephrons (16,18). This process of proliferation and differentiation must be strictlyregulated both spatially and temporally, and the WT1 proteins may bekey regulators in this process.

Besides proliferation and differentiation, apoptosis (programmedcell death) also plays an important role in normal development (19).During development of the rat kidney, 3% of the cells within nephrogenic areas are apoptotic at any given time, implying that largescale apoptosis takes place during renal development (20). It is quitepossible that the WT1 proteins are also involved in this process.Recently, it has been shown that WT1 can induce apoptosis (21).Remarkably, WT1 was also found to be able to suppress p53-inducedapoptosis (22). This latter discovery is in agreement with the observation that a WT1-null mutation in mice resulted in failure of kidney,gonad, and mesothelium development (23). In these mice, the mesenchymal cells failed to differentiate but degenerated via apoptosis,indicating that WT1 expression is required for their survival.

To further investigate the possible apoptosis-inducing and apoptosis-suppressing potential of the WT1 proteins, we studied the effect ofexpression of each WT1 splice variant in Hep3B and HepG2 cells.Both cell lines have been established from liver tumor biopsies fromchildren (24) and are considered to be mesenchyme-derived epithelialcells (25). Recently, it has been shown that p53 overexpression caninduce apoptosis in Hep3B cells (26). In contrast to HepG2 cells,Hep3B cells do not express p53 (27), enabling us to investigate theeffect of p53 on WT1-induced apoptosis and of WTI on p53-inducedapoptosis. Our results show that none ofthe WT1 splice variants couldsuppress p53-induced apoptosis. In addition, the two WT1(â€”KTS)splice variants induced apoptosis in both hepatoma cell lines, whereasthe WT1(-l-KTS) isoforms did not. This indicates that the apoptoticpathway triggered by WT1(â€”KTS) is independent of p53. The observed apoptosis could be suppressed by the coexpression of either theEGF receptoror the insulinreceptor.

MATERIALS AND METhODS

Tissue Culture. For this study, we used two hepatoma cell lines, theHep3B and the HepG2 (24).The cells were cultured at 37Â°C,5% CO2. inDMEM supplemented with 10%FCS (Life Technologies, Inc.) and antibiotics.

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Wihns' Tumor 1-KTS Isoforms Induce p53-independent Apoptosis That Can Be

Partially Rescued by Expression of the Epidermal Growth Factor Receptor

or the Insulin Receptor

Aswin L. Menke, Avi Shvarts, Nicole Riteco, Reinier C. A. van Ham, Alex J. van der Eb, and Aart G. Jochemsen'

Laboratory ofMolecular Carcinogenesis. Leiden University, P. 0. Box 9503, 23t'XIRA Leiden, the Netherlands

Research. on January 6, 2020. © 1997 American Association for Cancercancerres.aacrjournals.org Downloaded from

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WTI(-KTS) ISOFORMS INDUCE APOFFOSIS IN HEPATOMA CELLS

monoclonal antibody raised against the first 193 an of the WT1 protein4; p53.PAb 122 (36), a mouse monoclonal antibody; E1B-2lkDa, 101 1 (37), a ratmonoclonal antibody; desmin, clone 33 (Monosan); EGF receptor, 1005 (SantaCruz Biotechnology), a polyclonal antibody raised against a peptide conesponding to aas 1005â€”1016 of the human EGF receptor; insulin receptor, Cl9

(Santa Cruz Biotechnology), a polyclonal antibody raised against ma 1365â€”1382 at the COOH terminus of the human insulin receptor @3chain; Bcl-2, 100(Santa Cruz Biotechnology), a mouse monoclonal IgG antibody derived byfusion of mouse myeloma cells with spleen cells from a mouse immunizedwith the human Bcl-2 protein; and BAG-l, a rabbit polyclonal antibody raisedagainst a peptide representing the COOH-terminal 16 ass of BAG-i (30).

FITC-labeled goat antirabbit, FITC-labeled goat antirat, or tetramethylrhodaminc isothiocyanate-labeled goat antimouse (GAM-TRITC) antibodies (JacksonImmunoresearch Laboratories, Inc., West Grove, PA) were used as second

antibodies. Nuclear DNA was stained by DAPI.Immunofluorescence. Cells were grown on glass slides, fixed with 80%

acetone for 5 mm at room temperature, and washed twice with PBS/0.05%Tween 20. Subsequently, they were preincubated for 1 h with PBS/0.05%Tween 20/5% normal goat serum, 1 h with the first antibody and 30 mm withthe second antibody coupled to a fluorescent dye (FITC or tetramethylrhodamine isothiocyanate) diluted in the preincubation mixture.

Western Analysis. Cells were grown on 9-cm dishes to 70% confluency,washed twice with ice-cold PBS, scraped in ice-cold PBS, centrifuged, andlysed in radioimmunoprecipitation assay buffer containing 20 mrvitetraethylammonium (pH 7.8), 0.14 M NaCI, 0.05% sodium deoxycholate, 0.05% SDS,

0.05% Triton X-lOO, 1 msi phenylmethylsulfonyl fluoride, and 0.1 ,xg/mltrypsin inhibitor. The protein concentration was determined by the Bradford

assay (Bio-Rad; Ref. 38). Proteins, 50 @gof each sample, were fractionated ona 10% SDS-polyacrylamide gel and subsequently transferred onto an Immobilon-P transfer membrane (Millipore) in ice-cold blotting buffer containing20% methanol, 20 m'vi Tris, and 150 miviglycine at 300 mA for 3 h. The blotswere blocked for I h in TBST [10 mMTris-HCI (pH 8.0), 150 mMNaC1,and0.2%Tween20]containing5%nonfatdrymilk(Nutricia),andsubsequentlyincubated for 1 h with the first antibody diluted in TBST and for 30 mm witha second antibody coupled to horseradish peroxidase (Amersham) diluted inthe blocking buffer. Protein bands were made visible with enhanced chemilu

minescence substrate buffers (Amersham Corp.) and autoradiography.Luciferase Assay. Cells were cultured on 3-cm Petri-dishes. Five hundred

p1 of precipitate contained 1 @xgof the luciferase reporter construct, I @xgofCMV-WTI, or 1 @xgof pCMV (control) and 8 @xgSalmon-sperm DNA as

carrier DNA. Two hundred pi of precipitate were added to the cells. In eachexperiment, the precipitates were made in triplicate. Exponentially growingcells were transfected overnight. The next morning, the cells were washedtwice with HEBS [Hepes-buffered saline (20 mM Hepes, pH 7.05, 150 mMNaCI, 5 mMKCI, 0.7 mMNa,HPO4 . 2H20)I and 24â€”28h later, lysates weremade in Cell Culture Lysis Reagent (Promega, Madison, WI). After 15 mm ofincubation at room temperature, the cell debris was removed by brief centrifugation. Protein concentrations were determined by the Bradford assay (BioRad). The luciferase activity was determined per 10 @xgof cellular lysate in a20-pi sample of the supernatant by adding 100 pAof Luciferase Assay Reagent(Promega). After 10 s of preincubation, the produced light was measured in aluminometer (Lumat LB9SO1; Berthold)

RESULTS

WT1 Expression Does Not Affect p53-induced Apoptosis inHep3B Cells. Recently,it has been shownthat p53 overexpressioncan induce apoptosis in Hep3B cells (26). Because the WT1 proteinsmay interact physically with p53 and influence its transcriptionalactivity (22), we wondered whether expression of the WTI isoformsmight affect apoptosis induced by p53. Hence, we transiently transfected a CMV-p53 plasmid into Hep3B cells with or without one ofthe four human cDNA constructs encoding the WT1 splice variants.The cells were screened for expression of p53 and WT1 by indirectimmunofluorescence. In general, 0.5â€”2%of the cells expressed the

A B

Fig. I . Morphological analysis of Hep3B cells expressing exogenous p53. Hep3B cellswere transfected with 2.5 @tgof CMV-p53. The cells were fixed 4 days after transfectionand stained with anti-p53 (PAb 122) and DAPI. A. a p53-positive apoptotic cell, characterized by the rounding up of the cell, detachment from the surface. and weak nuclearstaining (arrows). B. a p53-positive apoptotic cell. characterized by the fragmentation ofthe nucleus and the weak nuclear staining (arrows). B. middle. DAPI staining in combination with phase contrast. The p53-negative, nonapoptotic cells are characterized bynormal. DAPI-stained oval nuclei.

Plasmids and DNA Transfections. The CMV-p53, the CMV-Desmin, theCMV-Bcl-2, and the CMV-EIB-2lkDa vectors are based on the plasmidpCMV-neo-Bam containing the human CMV promoter (28). The CMV-p53

plasmid (28) encodes wild-type human p53. The CMV-Desmin plasmid3encodes human desmin, a component of type II intermediate filaments. CMVE1B-2lkDa (29) encodes the adenovirus 5E1B-2lkDa protein. The CMVBcl-2 plasmid encodes the Bcl-2 protein (26). The BAG-l cDNA was subcloned into the HindIll and XbaI sites of pRc/CMV (Invitrogen, Inc.; Ref. 30).The CMV-BAG-l encodes the mouse BAG-l protein. The SRa-Ins-R encodesthe human insulin receptor under control of the SRcxpromoter (31; a gift of Dr.R. A. Roth, StanfordUniversity, Stanford,CA). The EV-Ins-Rencodes thehuman insulin receptor cloned into an expression vector provided by Dr. A.

Ullrich (32) of the Max Planck Institute, Martinsried, Germany. The EVEGF-R encodes the human EGF receptor (33) cloned into the same expressionvector (32). The CMV-WTI vectors are based on the CMV vector pCB6+(34).TheCMV-WT1(â€”/â€”)encodestheWTI splicevariant,lackingboththel7aa and the KTS inserts. The CMV-WT1(+/â€”) encodes the WT1 splicevariant containing only the l7aa insert. The CMV-WT1(â€”/+) encodes theWTI splice variants containing only the KTS insert. The CMV-WTI(+/+)encodes the WT1 tumor 1 splice variants containing both inserts. The plasmidDNA was purified twice by centrifugation in a CsCl gradient. One day beforethe cells were transiently transfected by way of the calcium-phosphate precipitation method (35), they were passed at 30â€”40% confluence in 6-cm dishescontaining glass slides (indirect immunofluorescence), 9-cm dishes (Westernanalysis and DNA fragmentation analysis) or 3-cm dishes (luciferase assay).

Antibodies. Antibodies against the following antigens were used in thisstudy: WT1, Cl9 (Santa Cruz Biotechnology), a polyclonal antibody raisedagainst the COOH-terminal 19 an of the WTI protein, and H7, a mouse

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WT1(-KTS) ISOFORMS INDUCE APOFFOSIS IN HEPATOMA CELLS

Fig. 2. The effect of WT1 and E1B-2lkDa onthe induction of apoptosis by p53 in Hep3B cells.The cells were transfected with 2.5 pg of CMVp53 in the presence or absence of 7.5 @xgof CMVWT1or CMV-E1B-2lkDa.Threeindependentcxperiments were carried out, and per transfection, atleast 100 p53-positive cells were examined. Datashown are means; bars, SD.

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exogenous proteins. Both p53 and WT1 were localized in the nucleus.To investigate whether apoptosis was induced, the cells were stainedwith DAPI, which is known to stain intact nuclei strongly but apoptotic nuclei irregularly and weakly (26, 39, 40). In agreement withprevious studies (26), we found that 4 days after transfection, >90%of the p53-expressing Hep3B cells were apoptotic, as was evidentfrom the rounding up of the cells, the detachment from the surface ofthe culture dish (Fig. 1A), the weak or complete absence of nuclearstaining, and the fragmentation of the nucleus (Fig. 1B). Coexpressionof the gene for the adenovirus Mr 21,000 E1B protein resulted insuppression of the p53-induced apoptosis. Coexpression of any of theWT1 splice variants, however, had no effect (Fig. 2). This was not theresult of a lack of WT1 expression, because double immunofluorescence showed that both p53 and WT1 were expressed in the samecells (data not shown).

In agreement with previous studies on apoptosis in hepatoma celllines (41, 42), cellular DNA isolated from the transfected Hep3B cellsexhibited no â€œladderingâ€•(data not shown). As a positive control, wetransfected the VP3 gene of the chicken anemia virus that encodes theapoptin protein. This protein has been shown to induce apoptosis inboth Hep3B and Saos-2 cells (26). DNA laddering has been observedin the Saos-2 cells. However, we have not been able to detect DNAladdering in the Hep3B cells. These findings support previous observations that DNA fragmentation may not always occur during phys

iobogical cell death (43, 44).

WT1(â€”KTS) Expression Induces Apoptosis in Both Hep3B andHepG2 Cells. To investigate the potential of the four WT1 isoformsto induce apoptosis, we transiently transfected each of the four formsinto both Hep3B and HepG2 cells. As a negative control, a CMVDesmin plasmid was used. In Fig. 3, a few examples are shown ofapoptotic and nonapoptotic cells. The apoptotic cells are characterizedby the weak or complete absence of nuclear DAPI staining and/or thefragmentation of the nucleus, whereas the nonapoptotic cells shownormally stained oval nuclei. Expression of the WTI(â€”KTS) iso

forms induced apoptosis in a significant proportion of the transfected cells in both the Hep3B (Fig. 4A) and the HepG2 (Fig. 4B)cell lines, the WT1(â€”/â€”) splice variant being the most potentinducer. In contrast, the WTI(+KTS) isoforms did not seem toinduce apoptosis, because the percentage of apoptotic cells wascomparable to that obtained after transfection of a CMV-Desminconstruct. In further support of these findings, we found that the

percentage of apoptotic cells increases in time when either one ofthe WT1(â€”KTS) isoforms is expressed (Fig. 4C). We found nosuch increase when the WT1(+I+) isoform was expressed. Westem analysis showed that the observed differences in apoptosis arenot the result of differences in the expression level of the WTIisoforms (Fig. 4D). In agreement with a previous study (45), we

could not detect endogenous WT1 expression in either Hep3B orHepG2 cells.

Transfection of the RSV-WTAR construct, which encodes a mutant

Fig. 3. Morphological analysis of Hep3B andHepG2 cells expressing exogenous W1'l ordesmin. Hep3B and HepG2 cells were transfectedwith 2.5@ of CMV-WF1(-I-), CMVWT1(+/+) or CMV-Desmin. The cells were fixed4 days after transfection and stained with anti-WT1(Cl9) orantidesmin(clone33)andDAPI.Arrows,WT1- and desmin-positive cells. Expression of theWT1(â€”Iâ€”)isoform resulted in apoptosisin bothHep3B and HepG2 cells, characterized by the fragmentation of the nucleus and the weak nuclearstaining, whereas the expression ofthe WTI(+I+)isoform or desmin did not induce apoptosis. Thenonapoptotic cells have normal, DAPI-stained ovalnuclei.

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Fig. 4. The percentage of apoptotic Hep3B cells (A) and HepG2 cells (B) expressing the W'Fl isoforms. In three independent experiments, the cells were transfected with 2.5 ,xgCMV-WTI(â€”/â€”), CMV-WT1(+/â€”), CMV-WT1(â€”/+), CMV-WT1(+/+), RSV-WT1(â€”/â€”), and RSV-WTAR. Per transfection, at least 100 WT1-positive cells were examined 4days after transfection. As a negative control, CMV-Desmin was included in these experiments. C. percentage of apoptotic HepG2 cells 1, 2. 3, 4, 5, and 6 days after transfection. D,Western analysis of exogenous WT1 expression and endogenous p53 expression 1 day after transfection. A Western blot containing 50 ,xg of total cell extract was incubated withanti-WT1 (Cl9) or anti-p53 (PAb 122). E, percentage of apopotic Hep3B cells 4 days after transfection with RSV-WT1(â€”/â€”)and RSV-WTAR. Data shown are means; bars, SD.

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Fig. 5. A. morphological analysis of Hep3Bcells cotransfected with 2.5 @sgof CMVWT1(â€”/â€”)and 7.5 i@gofCMV- E1B-2lkDa. Thecells were fixed 4 days after transfection andstained with anti-WT1 (Cl9) or anti-E1B 2lkDa(101 1). A cell is shown expressing bothWTI(â€”/â€”)and E1B-2lkDa (A, arrows). The cellis apoptotic, characterized by condensed DNA,weak nuclear staining, and detachment from thecell surface. In addition, E1B-2lkDa protein islocalized in both the nucleus and the cytoplasm.Theothercellsarenotapoptoticandhavenormal,DAPI-stained oval nuclei. In these cells, EIB2lkDa is localized in the cytoplasm only. B. Westem analysisof the exogenousWT1andE1B-2lkDa expression 1 day after transfection. AWestern blot containing 50 ,xg of total cell extractwas incubated with anti-WT1 (Cl9) or anti-E1B-2lkDa (lGl 1).

form of WT1, lacking the third zinc finger (8, 46), did not induceapoptosis (Fig. 4E). These results indicate that an intact zinc fingerdomain is essential for the apoptosis-inducing potential of the WT1

protein. Transfection of the RSV-WT1(â€”/â€”)construct resulted in alower number of apoptotic cells compared to transfection with theCMV-WTI(â€”/--) 4 days after transfection. Because the RSV promoter is weaker than the CMV promoter, this may be the result of areduced expression level of WT1.

Expression of Adenovirus E1B-2lkDa Protein Does Not Suppress WT1-induced Apoptosis. The adenovirus E1B-2lkDa proteinhas been shown to inhibit apoptosis induced by a wide range ofapoptotic stimuli (47) and can repress both p53-dependent and p53-independent apoptotic pathways (48â€”50).To investigate whether thisprotein could also suppress WT1-induced apoptosis, we cotransfectedthe E1B-2lkDa-encoding plasmid together with the WT1 constructs.In nonapoptotic cells, the E1B-2lkDa protein was found to be locatedin the cytoplasm, whereas in apoptotic cells, it was detected in boththe cytoplasm and the nucleus (Fig. 5A). Western analysis suggestedthat the expression of the WT1 protein was somewhat down-regulatedby the expression of E1B-2lkDa (Fig. SB), but the immunofluorescence data indicated that this was due to a lower transfection efficiency (data not shown). These experiments indicate that expressionof the E1B-2lkDa protein does not suppress the WTI-induced apoptosis (Fig. 6).

Transcriptional Repressor Activity of the WT1 Isoforms Correlates with Apoptosis.inducing Potential. Induction of apoptosisby p53 appears to be independent of the transcriptional activationactivity of p53 (51) but may be linked to its transcriptional repressoractivity (52). To investigate whether a similar correlation exists for theWT1 splice variants, we performed transient transfection assays. As areporter, we used an IGF-ll promoter/luciferase construct consistingof the human IGF-II P3 promoter (53) in front of the luciferase gene.It has been shown that both the WT1(â€”KTS) and the WT1(+KTS)isoforms can bind to overlapping sequences in this promoter (6). Ascan be seen in Fig. 7, the WT1(â€”/â€”)and the WT1(+/â€”) splicevariants significantly suppressed the luciferase activity in both the

Hep3B and the HepG2 cells. In the HepG2 cells, expression of theWT1(+KTS) splice variants had no effect on the IGF-H P3 promoteractivity whereas in the Hep3B cells these forms seem to increase the

promoter activity about 2.5-fold. Thus, induction of apoptosis by theWT1(â€”KTS)isoforms correlated with the ability of these forms torepress the IGF-II P3 promoter activity in transient assays.

Target Genes. Because the observed apoptosis may be linked tothe transcription-repressing activity of the WT1(â€”KTS) splice van

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Fig. 6. The effect of E1B-2lkDa expression on the WT1-induced apoptosis in Hep3Bcells (A) and HepG2 cells(B). The cells were transfected with 2.5 @sgof CMV-WT1(â€”/â€”)and 7.5@ of CMV vector or 2.5 @xgof CMV-W'I'l(+/â€”) and 7.5 @gof CMV-E1B-2lkDa. As a negative control, the CMV-WT1(+/+) was included in these experiments.

Three independent experiments were carried out, and per transfection, at least 100 WT1-and E1B-2lkDa-positive cells were examined. Data shown are means; bars, SD.

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ants, it is possible that the induction of apoptosis occurs by thedown-regulation of intrinsic survival factors. We have tested the

involvement of two such factors, Bcl-2 and BAG-l. BAG-i has beenshown to prolong the survival of BALB/c3T3 cells after an apoptoticstimulus (30). In the Hep3B cells, however, the protein did notsuppress the WTi(â€”KTS)-induced apoptosis (Fig. 8, A and B). Actually, the opposite was found. Almost 80% of the cells that onlyexpressed the BAG-i protein were already apoptotic, indicating thatoverexpression of this protein induces apoptosis. This explains theobserved increase in the number of apoptotic cells in the cotransfection experiments (Fig. 9, Wfl(â€”/â€”) + BAG-i and WTJ(+/+) + BAG-i). As a control, we transfected mutant p53 and mutantWT1 (WTAR), which, like desmin, do not induce apoptosis in Hep3B

. cells (Fig. 8, C and D; Fig. 9).

Bcl-2, a putative target gene of WT1 (54), has been shown topromote cell survival and to inhibit apoptosis in certain cells (55â€”59).In the Hep3B cells, however, the protein did not suppress theWT1(â€”KTS)-induced apoptosis. Four days after cotransfectingWT1(â€”KTS)and Bcl, we could no longer detect any WT1-positive orBcl-2-positive cells suggesting that Bcl-2 may promote apoptosiseven stronger than BAG-i. This apoptosis-promoting effect couldwell be cell-type dependent since Bcl-2 expression could be detected

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Fig. 7. Transcriptional activity of the WTI isoforms inHep3B (A) and HepG2 (B) cells. Both cell lines weretransfected with 1 @gof reporter construct, containing thehuman IGF-ll P3 promoter in front of the luciferase gene,and 1 @gof the CMV vector, the CMV-WTi(â€”Iâ€”),theCMV-WTI(+/â€”), the CMV-WT1(â€”/+), or the CMVWT1(+/+) construct. In three independent experiments,the luciferase activity was measured 1 day after transfection. In each experiment, the transfections were carried outin triplicate. Data shown are means; bars, SD.

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Instead of inducing apoptosis by the down-regulation of intrinsicsurvival factors, the WTi(â€”KTS) isoforms may suppress the expression of factors that mediate the effects of external survival factors.External survival factors can be provided by the extracellular matrix(60), direct cell-cell contact (61), and growth factors (62). It isconceivable that the regulation of growth factor receptors by WT1may play an important role in the onset of apoptosis. It has beenshown that the down-regulation of, for instance, the EGF receptor byWT1 correlates with the induction of apoptosis in both U2OS and

Saos-2 cells (21). In addition, EGF may function as an exogenoussurvival factor (63). In agreement with these findings, we found thatcoexpression of the EGF receptor partially rescues the Hep3B cellsfrom WTi(â€”KTS)-induced apoptosis (Fig. 10, A and B; Fig. i 1A). Insupport of a role for the EGF receptor in preventing apoptosis, wefound that the addition of EGF to the medium reduces the numberapoptotic cells. The outcome of these experiments suggests that theWT1(â€”KTS) isoforms may induce apoptosis in Hep3B cells by the

down-regulation of the endogenous EGF receptor.Because the insulin receptor may also be a potential target gene of

WT1, we investigated the possible involvement of this receptor in the

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observed apoptosis. Using two different expression vectors, we foundthat coexpression of the insulin receptor partially rescued the cellsfrom apoptosis induced by WT1(â€”KTS) (Fig. 10, C and D; Fig. 11B).These results suggest that the WT1(â€”KTS) isoforms may also induceapoptosis in Hep3B cells by downregulating the insulin receptor. Incontrast to the findings with the EGF receptor, we found that theaddition of insulin did not result in an additional rescue of the Hep3Bcells. In contrast, we observed an increase in the number of apoptoticcells, especially in the Hep3B cells cotransfected with the SRa-Ins-Rconstruct. Transfection of this construct resulted in a higher expression level of the insulin receptor compared to transfection with theEV-Ins-R construct. The SRcx-Ins-R construct even increased thenumber of apoptotic cells when coexpressed with the WT1(+I+)protein (Fig. 1lB). These results suggest that overstimulation ofinsulin receptor signaling may be lethal to the cell as well.

DISCUSSION

WT1 Expression Does Not Affect p53-induced Apoptosis inHep3B Cells. In this study, we have investigated the potential of thefour WT1 splice variants to suppress or activate apoptosis. Our resultsshow that expression of any of these forms does not affect p53-induced apoptosis in Hep3B cells. This is in conflict with a recentpublication in which the expression of the WT1(â€”/--) isoform wasreported to suppress p53-induced apoptosis in both U2OS and E1A-

transformed baby rat kidney cells (64). Previous observations showingthat the effect of the WT1 isoforms is cell type dependent may explainthese discrepancies. For example, the WT1(â€”KTS) isoform functionsas a transcriptional repressor of the Egr-l promoter in NIH3T3 cells(64), whereas it behaves as a transcriptional activator of the samepromoter in both Saos-2 (64) and U2OS cells (21). Furthermore,expression of the WT1(â€”/â€”)isoform stimulated the tumorigenicpotential of adenovirus-transformed baby rat kidney cells (12),

whereas the same protein suppressed the tumorigenicity of ras-transformed NIH3T3 cells (65). Thus, the effect of each of the WT1 splicevariants may well be cell type dependent.

WT1(â€”KTS) Expression Induces Apoptosis in Both Hep3B andHepG2 Cells. Instead of suppressing p53-induced apoptosis, wefound that the WT1(â€”KTS) isoforms induce apoptosis in both thep53-negative Hep3B cells and the p53-positive HepG2 cells, indicating that the observed effect is p53 independent. This observation is inagreement with a previous report in which WT1-induced apoptosiswas observed in both p53-positive U2OS cells and p53-negative

WT1(-/-) BAG-i

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A

B

C

D

Fig. 8. Morphological analysis of Hep3B cells cotransfected with 2.5 @.tgof CMVWT1(â€”/â€”)and 7.5 @sgof CMV-BAG-1 (A and B), 2.5 @xgCMV-mtp53 (C). and 2.5 @gRSV-WTAR (D). The cells were fixed 4 days after transfection and stained with anti-WTI(H7 or Cl9), anti-BAG-I, or anti-p53 (PAb 122). A, a cell is shown expressing bothWTI(â€”Iâ€”)and BAG-i (arrows). The cell is not apoptotic and has a normal DAPI-stainednucleus. B, an apoptotic cell, characterized by condensed DNA, weak nuclear staining.and detachment from the cell surface. The apoptotic cell lies partially on top of anonapoptotic cell (arrows). In the apoptotic cell, BAG-l is localized in both the cytoplasmand the nucleus. C, nonapoptotic cells expressing mutant p53 (arrows). D, nonapoptoticcells expressing the mutant WTI protein WTAR (arrows).

â€œS

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Fig. 9. The effect of BAG-I expression on theWTI-induced apoptosis in Hep3B cells. The cellswere transfected with 2.5 @.&gof CMV-W1'l(â€”Iâ€”)and 7.5@ of CMV vector or 2.5 p@gof CMVWT1(+/â€”) and 7.5 @sgof CMV-BAG-l. As acontrol, the CMV-WT1(+/+) construct was cotransfected with the CMV vector or the BAG-lconstruct. As a negative control, mutant p53,WTAR, and desmin were also transfected. Twoindependent experiments were carried out, and pertransfection, at least 100 WT1- and BAG-l-positive cells were examined. Data shown are means;bars, SD.

1359

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VVTI(-KTS)ISOFORMSINDUCEAPOPTOSISIN HEPATOMACELLS

Targets of WT1. As a first step to determine the underlyingmechanism of the WT1-induced apoptosis, we have investigatedtranscription regulation by the four WT1 splice variants in both theHep3B and the HepG2 cells. The apoptosis-inducing WT1(â€”KTS)isoforms show transcriptional repressor activity in contrast to theWT1(+KTS) isoforms. Like p53-induced apoptosis (52), WT1-induced apoptosis may therefore be linked to its transcription-repressingactivity. This is in agreement with the finding that the WTAR protein,in which the DNA binding domain is disrupted (8, 46), did not induceapoptosis.

The WT1(â€”KTS) splice variants may down-regulate the expression of intrinsic survival factors. In this connection, it should benoted that both Hep3B and HepG2 cells express a basal level oftransforming growth factor /3 (66, 67), a factor that has been shownto induce apoptosis in both Hep3B cells (68, 69) and rat hepatocytes (42). Down-regulation of an apoptosis inhibitor could therefore be fatal. We have tested the effect of two such inhibitors:BAG-i and Bcl-2. In contrast to previous reports, we find thatexpression of either of these proteins results in cell death. In livercells, Bcl-2 may not function as an survival factor. Histologicalexamination of the liver in bcl2@ mice revealed no abnormalitiesof the liver, whereas almost every lymphocyte population wasdecreased (70). The exact biochemical mechanisms of action ofproteins belonging to the Bcl-2 gene family is largely unknown(71). The Bcl-2 gene product can bind to several homologousproteins including Bax, Bcl-xL, Bcl-xs, and Mcli (50, 72). TheBAG-i protein contains ubiquitin-like domains that may mediateprotein-protein recognition (73). Thus, both the BAG-i and theBcl-2 proteins may exert their effect via associating proteins. Wehypothesize that cell type-dependent expression of such proteinswill determine the ultimate effect of the Bcl-2 and the BAG-iprotein on the cell. In support of this, it has been shown that Bcl-2,Bax, or Bak can promote either cell death or cell survival, depending on the assay system used (74â€”76). Alternatively, the expression level of BAG-i and Bcl-2 may determine the effect of eachprotein, as has been suggested for the WTi protein (22).

The WT1(â€”KTS) isoforms may also induce apoptosis by thedown-regulation of factors that mediate the effect of externalsurvival factors that can be provided by the extracellular matrix(60), direct cell-cell contact (61), or growth factors (62). Theregulation of growth factor receptors by WT1 may play an important role in the onset of apoptosis. For instance, it has been shownthat the WT1(â€”/â€”)isoform can suppress the IGF-I receptor (77,78) and that the down-regulation of this receptor correlates withincreased apoptosis (79). The EGF receptor may also be involvedin the process of apoptosis. Down-regulation of this receptor byWT1 correlated with the induction of apoptosis in both p53-positive U2OS cells and p53-negative Saos-2 cells (21). In agreement with these results, we found that coexpression of the EGFreceptor rescued the Hep3B cells from WTi(â€”KTS)-induced apoptosis. In support of a role for the EGF receptor in survival of thehepatoma cells, we found that the addition of EGF to the mediumfurther reduced the number of apoptotic cells. These results mdicate that EGF receptor signaling plays an important role in therescue of WTI-induced apoptosis.

Expression of the insulin receptor also resulted in increased survival of the Hep3B cells. In support of these results, preliminary dataindicate that the WT1(â€”/â€”)isoform can also induce apoptosis inNIH3T3 cells (data not shown), whereas hardly any apoptotic cell canbe detected when the WT1(â€”Iâ€”)isoform is expressed in NIH3T3A14 cells in which the human insulin receptor is stably overexpressed(80). In contrast to the results with the EGF receptor, we do notobserve an additional rescue when we add the ligand to the medium.

DAPI

A

B

@Vi1T Ins-R __________

C @1 __D@ ii,' H â€˜@ 4,

Fig. 10. Morphological analysis of Hep3B cells cotransfected with 2.5 @sgof CMVWTI(â€”/â€”)and 7.5 ;.@gEV-EGF-R (A and B) or 2.5 @sgofCMV-WT1(â€”/â€”)and 7.5 @sgEV-Ins-R (C and D). The cells were fixed 4 days after transfection and stained withanti-WTI (H7). anti-EGF-R (1005), or anti-Ins-R (Cl9). A. a cell is shown expressingboth WTI(â€”/â€”)and the EGF receptor (arrows). The cell is not apoptotic and has anormal DAPI-stained nucleus. B, an apoptotic cell is shown, characterized by the fragmentationof the DNA,weak nuclearstaining,and detachmentfromthe cell surface(arrows). In the apoptotic cell, the EGF-R protein is localized in both the cytoplasm andthe nucleus. C. a nonapoptic cell is shown, expressing Vi@Tland the insulin receptor(arrows). Panel D shows an apoptotic cell, characterized by the apoptotic bodies and weaknuclearstaining(arrows).

Saos-2 cells (21). In the latter study, however, the authors foundthat all four WT1 forms could induce apoptosis, whereas weobserved this activity only for the WT1(â€”KTS) splice variants. Ithas been suggested that the expression level may determine theapoptotic effect of the WT1 protein (21). The transfection efficiency in each experiment (data not shown) and the expressionlevels of the four WT1 proteins were basically similar (Fig. 4D).We cannot exclude, however, that a higher expression level of theWT1(+KTS) isoforms will induce apoptosis. Alternatively, thedifferences may again be explained by the cell-type-dependenteffects of the WT1 splice variants. In this respect, we have foundthat stable (12) or transient expression5 of any of the four WT1isoforms in adenovirus-transformed baby rat kidney cells does not

induce apoptosis at all.The Hep3B cells appear to be more sensitive to WT1-induced

apoptosis compared to the HepG2 cells. Mock transfection with theCMV-Desmin construct shows that the background level of apoptosisis already higher in the Hep3B cells. Whether this is the result of theabsence of p53 remains to be elucidated.

5 A. L. Menke and A. G. Jochemsen. unpublished results.

1360



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Wl'l(-KTS) ISOFORMS INDUCE APOPTOSIS IN HEPATOMA CELLS

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0_0

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Fig. 11. The effect of EGF receptor expression or insulin receptor expression on the WTI-induced apoptosis in Hep3B cells. The cells were transfected with 2.5 @gofCMV-W'l'l(â€”/â€”) and 7.5 @.&gof EV vector, 2.5 @&gof CMV-WTI(â€”/â€”) and 7.5 @sgof EV-Ins-R, or 2.5 @gCMV-WT1(â€”/â€”) and 7.5 sg of SRa-Ins-R. As a control, the sametransfections were carried out with the CMV-WT1(+/+) construct. Two independent experiments were carried out, per transfection, at least 100 WTI-positive cells were examined.The cells were grown after transfection in the presence or absence of ectopic EGF (100 ng/ml) or insulin (1 ps@i).Data shown are means; bars. SD.

In this case, overstimuiation may be harmful to the cell and willneutralize the initial rescue by the insulin receptor. In conclusion, theWT1(â€”KTS) isoforms may induce apoptosis by the down-regulationof the endogenous insulin- and EGF receptor. Both genes have potential WT1-binding sites in their promoter (21, 8i), and it has beenshown in transient transfection assays that WT1 can down-regulatethe EGF receptor promoter activity (21).

The mechanism by which the insulin receptor and the EGF receptorcan rescue the WT1(â€”KTS)-induced apoptosis remains to be elucidated. Both receptors may exert their antiapoptotic effect via the raspathway (80, 82). Also, WT1 may be involved in this signaling route,given that it has been shown that WT1 can block the transformationof NIH3T3 cells via the ms pathway (65, 83). Other pathways mayalso be involved (84). Insulin can act anabolically on proteins predominantly by inhibiting proteolysis (85). Because apoptosis presumably occurs via the activation of proteases (86), this might be amechanism by which the insulin receptor rescues the Hep3B cells

ACKNOWLEDGMENTS

We thankDr. H. van Ormondt,Prof. Dr. N. D. Hastie,andProf. Dr. A. H.Wyllie for critically reading this manuscript, Dr. J. Morris and Dr. F. J.Rauscher III for their generous gift of the four CMV-WT1 constructs and theH7 monoclonal antibody, Dr. S. Takayama for his generous gift of theCMV-BAG-i expression vector and the polyclonal antibody raised againstBAG-i, and Drs. 0. C. M. van der Zon and D. Telting for the EGF receptorand insulin receptor expression vectors.

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1997;57:1353-1363. Cancer Res Aswin L. Menke, Avi Shvarts, Nicole Riteco, et al. Epidermal Growth Factor Receptor or the Insulin ReceptorApoptosis That Can Be Partially Rescued by Expression of the Wilms' Tumor 1-KTS Isoforms Induce p53-independent

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