disruption of wt1 gene expression and exon 5 splicing ... · the kts insert have separate and vital...

Disruption of WT1 gene expression and exon 5 splicing followingcytotoxic drug treatment: Antisense down-regulation of exon 5alters target gene expression and inhibits cell survival

Jane Renshaw,1 Rosanne M. Orr,2

Michael I. Walton,2 Robert te Poele,2

Richard D. Williams,1 Edward V. Wancewicz,3

Brett P. Monia,3 Paul Workman,2 andKathryn Pritchard-Jones1

1Section of Paediatrics, and 2Cancer Research UK Centreof Cancer Therapeutics, Institute of Cancer Research, Sutton,Surrey, United Kingdom and 3Isis Pharmaceuticals, Inc.,Carlsbad, California

AbstractDeregulated expression of the Wilms’ tumor gene (WT1)has been implicated in the maintenance of a malignantphenotype in leukemias and a wide range of solid tumorsthrough interference with normal signaling in differentia-tion and apoptotic pathways. Expression of high levels ofWT1 is associated with poor prognosis in leukemias andbreast cancer. Using real-time (Taqman) reverse transcrip-tion-PCR and RNase protection assay, we have shownup-regulation of WT1 expression following cytotoxic treat-ment of cells exhibiting drug resistance, a phenomenonnot seen in sensitive cells. WT1 is subject to alternativesplicing involving exon 5 and three amino acids (KTS) atthe end of exon 9, producing four major isoforms. Exon 5splicing was disrupted in all cell lines studied following acytotoxic insult probably due to increased exon 5 skipping.Disruption of exon 5 splicing may be a proapoptotic signalbecause specific targeting of WT1 exon 5–containingtranscripts using a nuclease-resistant antisense oligonu-cleotide (ASO) killed HL60 leukemia cells, which wereresistant to an ASO targeting all four alternatively splicedtranscripts simultaneously. K562 cells were sensitive toboth target-specific ASOs. Gene expression profilingfollowing treatment with WT1 exon 5–targeted antisenseshowed up-regulation of the known WT1 target gene,thrombospondin 1, in HL60 cells, which correlated with

cell death. In addition, novel potential WT1 target geneswere identified in each cell line. These studies highlight anew layer of complexity in the regulation and function ofthe WT1 gene product and suggest that antisense directedto WT1 exon 5 might have therapeutic potential. [MolCancer Ther 2004;3(11):1467–83]

IntroductionThe Wilms’ tumor gene (WT1) is located at the humanchromosome region 11p13 and encodes a developmentallyregulated transcription factor that is essential for normalgenitourinary development (reviewed in refs. 1, 2). In theadult, WT1 expression is restricted to specific cell types inkidney, gonads, hematopoietic and nervous system, andmesothelium (1). However, inappropriate and/or over-expression of WT1 has been reported in leukemias and awide range of solid tumors including prostate, breast, andlung as well as thyroid, testicular and ovarian carcinomas,melanoma, and mesothelioma (2). Although an oncogenic rolein these tumors has not been proven, experimental evi-dence suggests that expression of WT1 may contribute tothe maintenance of a malignant phenotype through a va-riety of mechanisms including inhibition of differentiationand apoptosis and increased proliferation (reviewed in ref. 2).

The WT1 protein consists of 10 exons with an activator/repressor domain near the NH2 terminus and four zincfingers of the Cys2-His2 type at the COOH terminus.Alternative translation initiation site usage (3, 4) andalternative splicing of WT1 mRNA (5) produces multipleWT1 protein isoforms. The two alternative splice regionscorrespond to the 17 amino acids of exon 5 (present only inmammals) and the last three amino acids (KTS) of exon 9(conserved in all vertebrates; ref. 6). Insertion of KTS altersthe spacing between zinc fingers 3 and 4, disruptingsequence-specific DNA binding and, in transient cotrans-fection assays, transcriptional regulation (reviewed inref. 2). In addition, +KTS isoforms have been shown tocolocalize with splicing factors in the nucleus, suggesting arole in RNA processing (7). In these assays, the presence orabsence of exon 5 has little impact.

The four major isoforms of WT1, designated WT1 (+/+),WT1 (+/�), WT1 (�/+), and WT1 (�/�) to indicate thepresence or absence of exon 5/KTS, respectively (see Fig. 1),are in general quoted as being coexpressed in a fixed ratioin normal tissues (5) and this is true for the presence orabsence of KTS (the WT1 KTS ratio). Studies of transgenicmice have shown that WT1 isoforms with and withoutthe KTS insert have separate and vital functions in sexdetermination and gonadal development, consistent with afixed expression ratio (8, 9). However, we and others haveshown that WT1 exon 5 ratios differ according to tissue and

Received 3/10/04; revised 8/6/04; accepted 9/15/04.

Grant support: Children’s Cancer Unit Fund, Royal Marsden Hospital NHSTrust, Sutton, UK (J. Renshaw, R.D. Williams) and Cancer Research UK(R.M. Orr, M.I. Walton, R. te Poele, P. Workman, and K. Pritchard-Jones).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Requests for reprints: Jane Renshaw, Section of Paediatrics, Institute ofCancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG,United Kingdom. Phone: 44-208-7224327; Fax: 44-208-7224321.E-mail: [email protected]

Copyright C 2004 American Association for Cancer Research.

Molecular Cancer Therapeutics 1467

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differentiation stage as well as between species (6, 10).Differential splicing of exon 5 has been confirmed inhuman kidney and Wilms’ tumor samples through analysisof native WT1 protein (11). Mice lacking the WT1 exon 5insert develop normally and are not compromised withrespect to fertility, embryo viability, or the capacity tolactate, suggesting that exon 5 is redundant in genitouri-nary development and function at least in mice (12).Nevertheless, the presence of the exon 5 insert and themaintenance of the correct balance between WT1 + exon5 and WT1 � exon 5 isoforms has been suggested to beessential for the regulation of critical cellular functions suchas proliferation, differentiation, and resistance to chemo-therapeutic drugs (13–16). Furthermore, disruption of exon5 splicing has been suggested to affect the regulation ofgenes downstream in the WT1 pathway, as has been shownin Wilms’ tumors that express reduced levels of WT1 exon5 variants relative to those lacking exon 5 (17).

In leukemias, expression of very high levels of WT1 hasbeen correlated with poor response to treatment (18–21),whereas high expression of WT1 mRNA has been shownto predict poor prognosis in breast cancer patients (22).Numerous studies using ectopic overexpression of indi-vidual WT1 isoforms have indicated that WT1 may in-terfere with apoptotic pathways, but the results are diverse,often conflicting, and cell type specific. For example,regulation of the antiapoptotic gene Bcl-2 by exon 5–containing isoforms has been suggested to mediate inducedresistance to apoptosis, because cells expressing the WT1(+/�) isoform and Bcl-2 are resistant to staurosporine-,vincristine-, and doxorubicin-induced apoptosis (16). How-ever, K562 cells stably expressing the various WT1 isoformsdid not exhibit increased resistance to doxorubicin orcisplatin, although induction of differentiation by 12-O-tetradecanoylphorbol 13-acetate was partially inhibited(23). By contrast, high levels of WT1 (+/�) expressionhave been shown to suppress epidermal growth factorreceptor (EGFR) and induce late-onset, p53-independentapoptosis in some cell types (24). It should be noted thatenforced expression of individual isoforms simultaneouslyalters both exon 5 and KTS ratios, making interpretation oftheir individual functions difficult. Definitive evidence thatWT1 overexpression contributes to a resistant phenotype isstill lacking in studies using this approach.

In our previous study, exon 5–containing WT1 tran-scripts were shown to be in excess in leukemias, whereas

in gonadal tumors they were nearer equivalence ormarginally underrepresented. These findings were in-triguing because the individual isoforms of WT1 seem tomediate different downstream cell type–specific biologi-cal effects that may involve isoform-specific interactionswith coregulatory binding proteins such as Par4 andcyclic AMP response element binding protein bindingprotein (reviewed in ref. 2). For example, Par4 interactswith both exon 5 and the zinc fingers of WT1 resulting inopposing effects on transcription and, in exon 5 interaction,the rescue of 293 cells from lethal UV light treatment (25,26). It is therefore probable that Par4 may mediate bothpositive and negative interactions with WT1 dictated, atleast in part, by the relative ratio of WT1 + to � exon 5isoforms.

Numerous reports have described cell type–specificregulation of WT1 expression following induction ofdifferentiation in leukemic, embryonal stem cell, andcarcinoma cell lines accompanied by growth arrest andapoptosis in some cell types (27–30). We initiated thepresent study to determine whether similar alterations inthe regulation of WT1 mRNA levels and/or alternativesplicing were induced following an apoptotic stimulussuch as cytotoxic drug treatment. The possibility ofdifferential regulation of WT1 expression in cell linessensitive to or with acquired resistance to a cytotoxic agentwas investigated using two paired cell lines: the pairedovarian papillary cystadenocarcinoma cell lines, CH1-Sand CH1-R (sensitive to and with acquired resistance tocisplatin, respectively), and the corresponding testiculargerm cell tumor lines, GCT27-S and GCT27-R. The levelsof total WT1 and individual alternatively spliced WT1variants were measured in these cell lines followingcisplatin treatment and, in the erythroleukemia cell lineK562, following treatment with doxorubicin. A combina-tion of radioactive reverse transcription-PCR (RT-PCR),quantitative real-time (Taqman) RT-PCR, and a RNaseprotection assay (RPA) was used.

We report here the novel observation that both WT1 exon5 splicing and total WT1 mRNA transcript expression weredynamically regulated following cytotoxic treatment. Dis-ruption of exon 5 splicing resulted from the down-regulation of WT1 exon 5–containing transcripts relativeto those lacking exon 5, probably by a mechanism ofinduced exon 5 skipping. Induction of total WT1 expressionwas seen only in those cell lines displaying a resistantphenotype. The downstream sequelae of disrupted exon 5splicing were investigated further using the leukemic celllines K562 and HL60 and a combination of antisense andgene expression profiling technology. Both cell linesexpress high levels of WT1, and HL60 cells have beenshown previously to be resistant to WT1-directed antisenseoligonucleotides (ASOs), which kill K562 cells (31). Theseresults provide novel insights into the role of WT1 exon 5splicing and total WT1 expression in the regulation of cellsurvival signaling pathways. Antisense targeted to WT1exon 5 may have broader therapeutic potential thanpreviously described WT1-directed ASOs.

Figure 1. Schematic representation of WT1 alternatively splicedvariants. Four main splice variants are represented schematically to showthe presence or absence of exon 5 and KTS along with their designations:WT1 (+/+), (+/�), (�/+), and (�/�) and WT1 + and � exon 5.

Disruption of WT1 Isoform Ratios1468




Materials andMethodsOligonucleotidesThe 20-mer, 2V-O-methoxyethyl chimeric oligonucleoti-

des consisting of a central window of eight 2V-deoxyunmodified sugar residues with flanking 2V-O-methox-yethyl regions and a fully thioated backbone weresynthesized by Isis Pharmaceuticals Inc. (Carlsbad, CA)as described previously (32). The two active compoundsselected for this study were ISIS 16609, sequence5V-GCCCTTCTGTCCATTTCACT-3V, targeting exon 5 (ASW-T1exon 5) and ISIS 16601, sequence 5V-CACATACA-CATGCCCTGGCC-3V, targeting the 3V untranslated region(UTR) of WT1 (ASWT13VUTR). Two oligonucleotides of thesame chemistry were used as controls: ISIS 15770, sequence5V-ATGCATTCTGCCCCCAAGGA-3V, a 5-10-5 gapmer tar-geting murine c-raf kinase (ASmc-raf) and lacking homol-ogy to any known human sequence, and ISIS 105730,sequence 5V-CCATCGACCTGCACCGATCA-3V, a scrambledsequence of ASWT13VUTR (ASWT1scram).

Cell Culture and DrugTreatmentK562 cl.6 cells, a subclone of the parent erythroleukemia

(33), were kindly provided by Prof. Adrian Newland andDr. Xu-Rong Jiang (London Hospital Medical College,London, United Kingdom). HL60 promyelocytic leukemiacells were obtained from the American Type CultureCollection (Manassas, VA). K562 cl.6 and HL60 cells weregrown in RPMI 1640 (HEPES buffered) supplemented with10% FCS (Biowest, Ringmer, East Sussex, United Kingdom)and penicillin (100 units/mL). The in-house paired ovariancell lines CH1-S and CH1-R (34) and testicular germ celltumor cell lines GCT27-S and GCT27-R (35) were culturedin DMEM (bicarbonate buffered) supplemented with 10%FCS. Doxorubicin (Sigma-Aldrich Co. Ltd., Gillingham,Dorset, United Kingdom) and cisplatin ( Johnson MattheyTechnology Centre, Reading, Berkshire, United Kingdom)were dissolved in sterile saline and cells were treated(100 AL drug in saline to 10 mL cultures) either continu-ously (doxorubicin) or for 2 hours (cisplatin) at aconcentration determined previously as the IC99 in clono-genic assays: K562, 0.1 Amol/L doxorubicin; CH1-S,3.8 Amol/L; CH1-R, 18.5 Amol/L; GCT27-S, 9.3 Amol/L;and GCT27-R, 40 Amol/L cisplatin. Antisense or controloligonucleotides were dissolved in PBS and introduced intoK562 or HL60 cells by low-voltage electroporation. Briefly,appropriately diluted ASOs (40 AL) were combined withcell suspension (360 AL) at 2 � 107 cells/mL and cells wereelectroporated (Bio-Rad Gene Pulser II electroporationsystem with Pulse Controller Plus capacitance extenderaccessory module, Bio-Rad Laboratories Ltd., HemelHempstead, Hertfordshire, United Kingdom) using 300 Vand a capacitor value of 1,000 AF, diluted to 10 mL, andincubated at 37jC for various times.

Clonogenic Cell Survival AssaysFollowing drug treatment, appropriate aliquots of cells

were serially diluted in complete medium. Aliquots (2 mL)of diluted cells were added to polystyrene tubes (ElkayProducts Ltd., Basingstoke, Hampshire, United Kingdom)containing medium (3 mL) supplemented with 20%

FCS and 0.2% Agar Noble (Difco Laboratories, Detroit,MI) and incubated at 37jC and colonies were counted after14 days. In several separate experiments, plating efficien-cies ranged from 22% to 38%, with 800 to 1,600 cells initiallyplated.

RNAExtraction and Radioactive RT-PCRAt the appropriate times following drug treatment, cells

were harvested and RNA was extracted using Trizol (LifeTechnologies Ltd., Paisley, United Kingdom). RNA (1 Ag)was reverse transcribed with SuperScript II and randomhexamer primers (100 pmol, Invitrogen Ltd., Paisley,United Kingdom) in a final volume of 20 AL according tothe manufacturer’s instructions. RT-PCR of all four WT1alternatively spliced mRNA transcripts was carried outusing 1 AL cDNA, [a-32P]dCTP, reduced cold dCTP, andprimer pair 297 and 298 spanning WT1 exons 4 to 10. RT-PCR methodology and method validation have beendescribed in detail previously (10). Four individual reactionmixes for each sample were set up in parallel and amplifiedfor 26, 29, 32, and 35 cycles, respectively, and PCR products482 (�/�), 491 (�/+), 533 (+/�), and 542 (+/+) bp longwere separated on standard denaturing polyacrylamidegels. Levels of [32P]dCTP incorporated into all fourtranscripts were visualized and analyzed using a Storm860 PhosphorImager and ImageQuant software (Amer-sham Pharmacia Biotech UK Ltd., Little Chalfont, Buck-inghamshire, United Kingdom) followed by adjustment forthe number of possible sites of incorporation of 32P in thealternatively spliced PCR products.

RNase Protection AssayA cDNA sequence spanning the whole of WT1 exon 5

and 70 bp of exon 4 was amplified by RT-PCR from asample of normal human testis. PCR products weresubcloned by standard methods and sequenced (automat-ed fluorescent sequencing) using an ABI PRISM 310Genetic Analyzer (Applied Biosystems, Applera UnitedKingdom, Warrington, Cheshire, United Kingdom). Highspecific activity WT1 antisense riboprobes were generatedusing 1 Ag of purified linearized vector, [a-32P]UTP, and aMaxiscript labeling kit (Ambion Europe Ltd., Huntindon,Cambridgeshire, United Kingdom) according to themanufacturer’s instructions but with the addition ofsingle-stranded binding protein (1 AL, Amersham Phar-macia Biotech UK). RPA analyses were carried outaccording to the manufacturer’s instructions using totalRNA (f10 Ag) and the RPA III kit (Ambion Europe). WT1RNase protected fragments, 121 (+ exon 5) and 70 (� exon5) bp, along with control actin (Ambion Europe) protectedfragments, were separated in 1 hour using 8% standarddenaturing polyacrylamide gels. Levels of [32P]UTP in-corporated into the protected fragments were visualizedand analyzed using a Storm 860 PhosphorImager asabove.

Real-time (Taqman) PCRThe primer pairs and probes for the quantitation of WT1 ,

thrombospondin 1 (THBS1), and glypican 5 (GPC5) levelswere designed using the Primer Express program (AppliedBiosystems) according to the recommended guidelines:





WT1 forward primer 5V-TACCCAGGCTGCAATAAGAG-ATATTTTAAG-3V, reverse primer 5V-CCTTTGGTGTCTT-TTGAGCTGGTC-3V, and probe 5V-CACTGGTGAGAAAC-CATACCAGTGTGACTTCAAGGACT-3V; THBS1 forwardprimer 5V-GACAGCATCCGCAAAGTGACT-3V, reverseprimer 5V-GACAGTGACACTCAGTGCAGCTATC-3V, andprobe 5V-TGAGCTGAGGCGGCCTCCCCTA-3V; and GPC5forward primer 5V-GGGCTGCCGGATTCG-3V, reverse prim-er 5V-CTGGTGCAACATGTAGGCTTTT-3V, and probe 5V-CGCGGGCAGGACCTGATCTTCA-3V. The primers weredesigned to amplify across exon/exon boundaries andwere confirmed to be lacking significant homology withany known DNA sequences by a search of the HGMPdatabase. All other genes were analyzed using Assays-on-Demand Gene Expression Products (Applied Biosystems).Taqman analysis was carried out according to themanufacturer’s instructions using an Applied Biosystems7700 Sequence Detector. Each assay sample was analyzedin triplicate and multiplexed to facilitate the measurementof gene expression levels relative to 18s rRNA expression(rRNA control reagents, Applied Biosystems) using thestandard curve method.

Protein Extraction and Estimation Using WesternImmunoblotting

At the appropriate times following drug treatment, f5 �106 cells were harvested, washed in PBS, and lysed using2% SDS containing 10% v/v protease inhibitor cocktail(Sigma-Aldrich). Sample protein concentrations were esti-mated using the Bio-Rad detergent-compatible microtiterplate protein assay according to the manufacturer’sinstructions. Prior to electrophoresis, an aliquot of eachsample was treated with RNase-free DNase (2 units,Ambion Europe) in the presence of Mg2+ (5 mmol/L), totalvolume 20 AL, at 37jC for 30 minutes followed by 75jC for5 minutes to minimize protein band distortion. Electro-phoretic separation of the + and � exon 5 WT1 isoforms(20 Ag protein loaded) was achieved using NuPAGE 10%Bis-Tris precast gels (Invitrogen, Groningen, Netherlands)run with NuPAGE MOPS and SDS denaturing runningbuffer. WT1 proteins were analyzed using a 1:1,000 dilutionof the rabbit polyclonal WT(C-19) (Santa Cruz Biotechnol-ogy, Santa Cruz, CA). Horseradish peroxidase–conjugatedanti-rabbit F(abV)2 fragment secondary antibodies (Amer-sham Bioscience UK Ltd.) were used at 1:2,000 dilution,detected using the Enhanced Chemiluminescence Plussystem (Amersham Bioscience UK Ltd.), and visualizedand analyzed using a STORM PhosphorImager 860 andImageQuant software. For WT1 estimation, a nonspecificband, running slightly faster than WT1 and shownpreviously to be minimally affected by antisense treatment,was used to adjust for differences in loading and sampleprotein concentration.

PolyA+mRNAIsolationandcDNAMicroarrayAnalysisMultiple (10�) aliquots of K562 and HL60 cells were

treated with ASO (10 Amol/L) as described above. After24 hours, total RNA was extracted and the samples werepooled. PolyA+ mRNA was isolated and purified fromtotal RNA using an Oligotex kit (Qiagen Ltd., Crawley,

West Sussex, United Kingdom.) and concentrated usingMicrocon YM-30 centrifugal filter devices (Millipore,Watford, Hertfordshire, United Kingdom). cDNA micro-array analyses of each sample were done in duplicate. Thepreparation of the cDNA microarray slides, and fluorescentlabeling of polyA+ mRNA samples using Superscript IIand Cy5-labeled or Cy3-labeled dCTP, was carried out asdescribed previously (36). The microarray hybridizationwas carried out according to a published protocol (37). Theso-called Institute of Cancer Research gene set, consistingof 5,603 IMAGE cDNA clones, is also described in detail inthis latter publication along with the image collection anddata analysis procedures. Briefly, array images wereacquired with an Axon GenePix 4000 scanner (AxonInstruments, Foster City, CA) and analyzed with the AxonGenePix Pro 3 software package (Axon Instruments). Datawere filtered for quality by automated spot flagging andmanual inspection. Fluorescence intensity ratios (I = Cy5/Cy3) were calculated after background subtraction andnormalized to the median expression ratio of all highquality spots. Intensity ratios were transferred to a Micro-soft Access database for IMAGE clone to gene assignments,data filtering, and group queries. Gene assignments werechecked and updated using National Center for Biotech-nology Information UniGene, formatted for local databaseimport by a Perl script.4

ResultsDynamic Alteration ofWT1 exon 5 Alternative Splic-

ing and Total WT1 Expression followingTreatment ofGonadal Cell Lines with Cisplatin

The paired human ovarian cell lines CH1-S and CH1-R aswell as the corresponding testicular germ cell tumor celllines GCT27-S and GCT27-R have been shown previouslyto undergo apoptosis following equitoxic concentrations ofcisplatin (38, 39). Following treatment with cisplatin at IC99

concentrations (concentration of drug reducing cell surviv-al by 99% of control levels in clonogenic assays), therelative levels of all four WT1 alternatively spliced tran-scripts were analyzed using radioactive RT-PCR (Fig. 2).WT1 exon 5 ratios were rapidly reduced in all four celllines, reaching a nadir at 8 hours post-treatment, whereasthe WT1 KTS ratios remained relatively constant through-out (data not shown). Control 0-hour exon 5 ratios andratios in samples processed at various times during the24-hour period were not significantly different to those ofuntreated, logarithmically growing cells. In both paired celllines, the extent of disruption of WT1 exon 5 ratios wasgreater in the resistant line than in the parent sensitive line,the mean F range of WT1 exon 5 ratios when expressed asa percentage of control ratios being 65.7 F 10% versus 48.5F 11.5% for the CH1-S and CH1-R cell lines, respectively,and 76.0 F 14.6% versus 32.4 F 8% for the GCT27-S andGCT27-R cell lines, respectively, at 8 hours post-treatment.

4 http://www.hgmp.mrc.ac.uk/~rdwillia/unigene.html.





Although revealing a common pattern of cisplatin-induced disruption of exon 5 splicing in all four cell lines,expression of the data as a ratio gives no indication ofabsolute alterations in the levels of the + and � exon 5transcripts. We therefore examined the levels of WT1amplification products with and without exon 5 generatedin these samples during the logarithmic phase of polymer-ase amplification (26 cycles; see Fig. 3 for ovarian cell linesand Fig. 4 for germ cell tumor cell lines). In the CH1-S andGCT27-S parent sensitive cell lines, exon 5–containingtranscripts were reduced to 63% and 69% of control levels,respectively, at 8 hours after cisplatin treatment, whereasthe levels of transcripts lacking exon 5 remained essentiallyunchanged at 92% and 104% of control levels, respectively(Figs. 3Aa and Ab and 4Aa and Ab). By 24 hours post-treatment, the relative ratios of all four alternatively splicedtranscripts had recovered to near control levels, althoughoverall levels were slightly reduced. By contrast, total WT1transcript levels were up-regulated in the resistant CH1-Rand GCT27-R lines following treatment, although theinduction kinetics differed (Figs. 3Ba and Bb and 4Ba andBb). In CH1-R cells, disruption of exon 5 splicing and up-regulation of alternatively spliced transcript levels occurredsimultaneously, and the excess production of WT1 � exon5 transcripts (459%) as compared with WT1 + exon 5

transcripts (285%) by 8 hours post-treatment was consistentwith increased exon 5 skipping during post-transcriptionalsplicing. In GCT27-R cells, disruption of exon 5 splicingand induction of WT1 expression occurred sequentially,suggesting independent regulation of the two processes.Down-regulation of WT1 + exon 5 transcript levels inGCT27-R cells followed a time course similar to that seen inthe sensitive cell lines and was maximal (41% of control

Figure 2. Dynamic regulation of WT1 exon 5 transcript ratios followingtreatment with cisplatin. WT1 exon 5 ratios (see Fig. 1) were determinedusing radioactive RT-PCR (as illustrated in Fig. 3) at various time pointsover a 24-hour period following treatment of (A) CH1-S (*, P < 0.04), (B)CH1-R (**, P = 0.009), (C) GCT27-S (*, P = 0.08), and (D) GCT27-R(**, P = 0.002) cells with IC99 concentration of cisplatin. WT1 exon 5transcript ratios are expressed as a percentage of ratios at 0 hour.

Figure 3. Radioactive RT-PCR analysis of WT1 + and � exon 5transcript levels following IC99 cisplatin treatment of CH1-S and CH1-Rcells. Levels of WT1 amplification products with and without exon 5generated in (A) CH1-S and (B) CH1-R cells were determined usingradioactive RT-PCR following 26 cycles (logarithmic phase) of amplifica-tion. Aa and Ba, typical gel images (inset ) of all four alternatively splicedRT-PCR products from selected time points along with superimposed linegraphs generated from these images. Ab and Bb, normalized (adjustedfor length) levels of + exon 5 (open columns ) and � exon 5 (closedcolumns) WT1 transcripts over the 24-hour time course. C and D,confirmation of total WT1 transcript levels in CH1-S (C) and CH1-R (D)cells using real-time (Taqman) analysis.





levels) at 8 hours post-treatment. Up-regulation of totalWT1 expression was apparent by 16 hours and maximal by24 hours post-treatment in this cell line, accompanied by arecovery and an overshoot of the WT1 exon 5 ratios.Confirmation of the differential regulation of total WT1expression in the resistant as compared with the sensitivecell lines was achieved using real-time (Taqman) RT-PCRanalysis of the same samples (Figs. 3C and D and 4C and D).

DynamicAlterationofWT1exon5AlternativeSplicingand Total WT1 Expression followingTreatment of aLeukemicCell LinewithDoxorubicin

K562 cells, in common with most leukemic cell lines,express high levels of WT1 with an excess of exon 5–containing transcripts (10, 20). Significantly, K562 cells havebeen shown to be relatively resistant to induction ofapoptosis, which does not occur until 48 to 72 hoursfollowing a variety of apoptotic stimuli including treatmentwith the clinically used anthracycline, doxorubicin (40). Todetermine whether the response to a cytotoxic insult wasdifferent in a leukemic cell line as opposed to a gonadal cellline, WT1 exon 5 ratios and total WT1 levels weredetermined in K562 cells following an IC99 treatment withdoxorubicin using RPA analysis (Fig. 5). As seen in theovarian and germ cell tumor lines following cisplatintreatment, there was a rapid reduction of WT1 exon 5 ratiosreaching a nadir of 57% at 8 hours and partially recovering24 hours following doxorubicin treatment (Fig. 5A). TotalWT1 levels are shown in Fig. 5B. After an initial reduction,both WT1 + and � exon 5 transcripts were up-regulated inconcert in a manner similar to that seen in CH1-R cells butachieving maximum levels 16 hours following treatment

Figure 4. Radioactive RT-PCR analysis of WT1 + and � exon 5transcript levels following IC99 cisplatin treatment of GCT27-S andGCT27-R cells. Levels of WT1 amplification products with and withoutexon 5 generated in (A) GCT27-S and (B) GCT27-R cells were determinedusing radioactive RT-PCR following 26 cycles (logarithmic phase) ofamplification. Aa and Ba, typical gel images (inset ) of all four alternativelyspliced RT-PCR products from selected time points along with super-imposed line graphs generated from these images. Ab and Bb, normalized(adjusted for length) levels of + exon 5 (open columns ) and � exon 5(closed columns ) WT1 transcripts over the 24-hour time course. C andD,confirmation of total WT1 transcript levels in (C) GCT27-S and (D)GCT27-R cells using real-time (Taqman) analysis.

Figure 5. RPA analysis of WT1 + and � exon 5 transcript levels andratios following IC99 doxorubicin treatment of K562 cells. Levels of WT1+ and � exon 5 transcripts in K562 cells were determined using RPAanalysis at various time points over a 24-hour time course followingtreatment with IC99 doxorubicin. A, WT1 exon 5 transcript ratios in K562cells following doxorubicin treatment expressed as a percentage of ratiosat 0 hour. B, total WT1 mRNA levels normalized to actin. C, typical gelimages of RNase protected fragments (inset ) along with line graphsgenerated from these images. D, normalized (adjusted for length) levels of+ exon 5 (open columns ) and � exon 5 (closed columns ) WT1transcripts over 24 hours following doxorubicin treatment.





(Fig. 5C and D). Again, there was an excess production ofWT1 � exon 5 transcripts (217%) as compared with WT1 +exon 5 transcripts (125%), consistent with increased exon 5skipping during post-transcriptional splicing.

Down-Regulation ofWT1exon 5^ Containing Tran-scriptsUsingaNuclease-ResistantASO

To further analyze the impact that disruption of exon 5splicing might have on cell viability and downstream sig-naling, we used an antisense approach. WT1 exon 5 tran-scripts were targeted specifically in an attempt to mimic thedrug-induced down-regulation of exon 5 transcript expres-sion seen in this study. An ASO targeting all four WT1 alter-natively spliced transcripts simultaneously was deemed anessential comparator to control for those effects due solelyto a balanced down-regulation of WT1 expression. Froma primary screen of 25 2V-O-methoxyethyl ASOs targetedthrough the 5V UTR to 3V UTR sequences of WT1 (datanot shown), two active oligonucleotides were selected forfurther evaluation: ISIS 16609, targeting exon 5 (ASW-T1exon 5), and ISIS 16601, targeting the 3V UTR regionof WT1 (ASWT13VUTR). The control ASO used was ISIS15770 (ASmc-raf), directed to mouse c-raf and with no se-quence homology to human DNA. K562 cells were used asthis cell line had been shown previously to be sensitive toan ASO directed to the translational start site of WT1 (31).

Validation of AntisenseActivityInitial experiments using RPA analysis examined total

WT1 transcript levels 5 hours following electroporation ofK562 cells with 10 Amol/L ASWT13VUTR and ASWT1exon5 as compared with electroporation in the presence of PBSalone or the control ASO, ASmc-raf (Fig. 6A). Total WT1transcript levels were equivalent in PBS-treated and ASmc-raf-treated cells and reduced to 55% and 30% of PBS controllevels following treatment with ASWT13VUTR and ASW-T1exon 5, respectively. The WT1 exon 5 ratios did not alteras total WT1 levels were reduced following treatment withASWT13VUTR. Following treatment with ASWT1exon 5,WT1 exon 5–containing transcripts were reduced to <10%of control levels, whereas those lacking exon 5 were onlyminimally affected (Fig. 6B). These data show thatASWT1exon 5 targets WT1 exon 5–containing transcriptsselectively at early time points and also confirm postsplicedexon 5–containing transcripts as the primary target. At 24hours following treatment, WT1 levels were reduced to26% and 22% of control levels by ASWT13VUTR andASWT1exon 5, respectively (Fig. 6C). At this time point,both WT1 + and � exon 5 transcripts were reduced byASWT1exon 5. However, selective targeting of WT1 + exon5 transcripts was still apparent, the WT1 exon 5 ratios being0.7 as compared with 1.8 following ASWT13VUTR (Fig. 6D).

WT1ASOActivity Is BothTime and Dose DependentThe concentration-dependent and time-dependent na-

ture of WT1 transcript reduction induced by both activeASOs during the first 4 hours of treatment was confirmedusing radioactive RT-PCR, RPA, and real-time PCR.Examples of typical images of WT1 splice variantsfollowing treatment are shown in Fig. 6E (top) using RT-PCR and Fig. 6E (bottom) using RPA. Using RT-PCR, the

Figure 6. Efficient targeting WT1 mRNA transcripts by ASWT13VUTRand ASWT1exon 5. RPA analysis of total WT1 levels in K562 cells 5 (A)and 24 (C) hours following treatment with PBS, ASmc-raf , ASWT13VUTR(AS-3 VUTR ), and ASWT1exon 5 (AS-exon 5) expressed as a percentage ofPBS levels. B and D, same samples expressed as counts (PhosphorImagerunits) and plotted individually as WT1 + exon 5 transcript levels (opencolumns) and WT1 � exon 5 transcript levels (closed columns ). E, typicalgel images of WT1 fragments generated by radioactive RT-PCR analysis(top ) and RPA analysis (bottom ) of ASO (10 Amol/L)– treated K562 cells.F, radioactive RT-PCR analysis of WT1 exon 5 ratios in K562 cells 4 hoursfollowing treatment with 0.5–10 Amol/L ASWT13VUTR (open columns)and ASWT1exon 5 (closed columns ) expressed as a percentage of ASmc-raf (10 Amol/L)– treated levels. G, time course of WT1 exon 5 ratios inK562 cells over the first 4 hours following treatment with 10 Amol/LASWT13VUTR (open columns ) and ASWT1exon 5 (closed columns)expressed as a percentage of ASmc-raf (10 Amol/L)– treated levels anddetermined by radioactive RT-PCR.H, RPA analysis of total WT1 transcriptlevels 4 hours following treatment with 0.5–10 Amol/L ASWT13VUTR(open columns ) and ASWT1exon 5 (closed columns ) expressed as apercentage of ASmc-raf (10 Amol/L)– treated levels.





WT1 + exon 5 transcripts were shown to be selectively andlinearly reduced by ASWT1exon 5 over a concentrationrange of 0.5 to 10 Amol/L at 4 hours (Fig. 6F) and selectiveactivity could be seen as early as 30 minutes followingtreatment at 10 Amol/L concentration (Fig. 6G). By contrast,the WT1 exon 5 ratios were not reduced by treatmentwith ASWT13VUTR. Total WT1 transcripts were reducedat 4 hours following treatment with both ASOs in aconcentration-dependent manner (Fig. 6H using RPA).

Because we had clearly shown efficient targeting of WT1expression in K562 cells, we used a simple nonradioactiveRT-PCR screen to compare the activity of 10 Amol/LASWT1exon 5 and ASWT13VUTR in K562 and HL60 cells 24hours following treatment. Equivalent activity was shownin both cell lines using both ASOs (data not shown).

Antisense activity was subsequently confirmed in the HL60cells used for cell survival studies using real-time (Taqman)RT-PCR (see below). Basal WT1 transcript levels in K562cells were shown to be f2-fold higher than in HL60 cells.

Efficient Targeting of WT1 Protein Expression inBoth K562 and HL60 Cells by ASWT1 exon 5 andASWT13VUTR

A reduction in WT1 isoform levels was shown at 24hours following WT1-directed ASO treatment of both K562and HL60 cells lines but not at 4 hours (data not shown).Although adequate separation of the WT1 + and � exon 5isoforms was achieved using NuPAGE gels, the splitting ofthe WT1 signal reduced the sensitivity of the Western blots,making visual interpretation of the protein bands difficult.Therefore, for clarity, typical Western blot images areshown in Fig. 7 (insets) along with the line graphsgenerated from these bands using the ImageQuant soft-ware. ASWT1scram was used as control following confir-mation that both WT1 isoform levels and exon 5 ratios wereequivalent following PBS, ASWT1mc-raf , and ASWT1-scram treatment at 10 Amol/L concentration (data notshown). WT1 exon 5–containing isoforms were overrepre-sented in both leukemia cell lines, suggesting a correlationbetween alternatively spliced mRNA transcript levels andWT1 protein isoform expression. In these experiments,there was a slight reduction of nonspecific band signal inantisense-treated samples compared with control samplesbut no dose response effect between 5 and 10 Amol/Lantisense concentrations. Concentration-dependent reduc-tion of total WT1 isoform levels with maintenance ofisoform ratios was seen in both K562 and HL60 cell linesfollowing ASWT13VUTR treatment. Differential targeting ofWT1 + exon 5 isoforms by ASWT1exon 5 was also seen inboth cell lines as shown by the concentration-dependentreduction in the WT1 exon 5 isoform ratios (see Table 1).However, targeting of exon 5 isoforms was associated with

Figure 7. Efficient targeting of WT1 protein expression in both K562and HL60 cells by ASWT13VUTR and ASWT1exon 5. Western immuno-blot analysis of WT1 protein 24 hours following ASWT13VUTR (5 and10 Amol/L) and ASWT1scram (10 Amol/L; Con ) treatment of K562 (A) andHL60 (C) cells and ASWT1exon 5 (5 and 10 Amol/L) and ASWT1scram(10 Amol/L) treatment of K562 (B) and HL60 (D) cells. Typical images ofWT1 protein bands (inset ) are shown along with the superimposedImageQuant line graphs generated from these bands using the ImageQuantsoftware.

Table 1. WT1 exon 5 ratios and total WT1 protein levels in K562and HL60 cells following ASWT1 exon 5 and ASWT13VUTRtreatment (mean F range of two experiments)

Treatment(24 h)

K562 HL60

WT1 exon5 ratios

% TotalWT1

WT1 exon5 ratios

% TotalWT1

ASWT1scram(10 Amol/L)

3.25 F 0.75 100.00 3.39 F 0.39 100.00

ASWT13VUTR(5 Amol/L)

4.45 F 0.40 37.2 F 7.6 3.70 F 0.70 81.8 F 8.2

ASWT13VUTR(10 Amol/L)

4.75 F 0.15 20.7 F 7.6 3.90 F 0.40 45.3 F 12.6

ASWT1scram(10 Amol/L)

3.24 F 0.27 100.00 4.28 F 0.18 100.00

ASWT1exon 5(5 Amol/L)

2.20 F 0.27 40.2 F 16.6 2.41 F 0.12 69.6 F 5.0

ASWT1exon 5(10 Amol/L)

1.59 F 0.11 23.7 F 12.7 1.82 F 0.03 27.5 F 3.1





reduced overall levels of WT1 protein as was seen at themRNA level at 24 hours following treatment (cf. Fig. 6D).A recent report showed that WT1 exon 5 isoforms mediatespecifically the transactivation of an antisense WT1promoter in the first intron of the WT1 gene probably byinteraction with an accessory protein (41). The regulatoryantisense WT1 mRNA product has been shown previouslyto positively regulate WT1 protein levels (42), providing apotential mechanism by which selective targeting of WT1exon 5–containing variants results in the subsequentdown-regulation of WT1 protein expression.

Cell Survival StudiesTwo publications have shown growth inhibition and

induction of apoptosis using ASOs targeted to thetranslation initiation site of WT1 in K562 cells, MM6myelomonocytic cells, and samples of fresh leukemic cells,whereas granulocyte-macrophage colony-forming unitsand HL60 cells were unaffected (31, 43). To determinewhether this cell type–specific cytotoxicity was WT1ASOtarget sequence specific, the survival of K562 and HL60cells following treatment with both WT1-targeted ASOswas determined using a soft agar clonogenic assay. In K562cells (Fig. 8A), the control ASO used in the validationexperiments, ASmc-raf , was nontoxic at concentrations upto 10 Amol/L, whereas the control ASO, ASWT1scram, wasnontoxic at 5 Amol/L but showed slight nonspecific WT1unrelated toxicity (f20% loss of cell survival at 10 Amol/L;data not shown). The IC50 concentrations of both WT1ASOs were comparable at 7 and 8 Amol/L, respectively,confirming the ability of WT1-targeted ASOs to reduce cellsurvival in K562 cells regardless of the targeted sequence.

In HL60 cells (Fig. 8B), however, ASWT13VUTR and thecontrol ASO, ASWT1scram, did not reduce clonogenicsurvival, in keeping with the previous report (31). Analysisof the residual HL60 cells from the clonogenic assays usingreal-time PCR showed that the absence of cytotoxicity wasnot due to the failure of ASWT13VUTR to target WT1mRNA in these experiments (Fig. 8C). By contrast,ASWT1exon 5 reduced cell survival to 41% of controllevels at 10 Amol/L. Thus, in HL60 cells, down-regulationof WT1 expression in itself was not sufficient to reduce cellsurvival, but when down-regulation of WT1 was associatedwith disruption of WT1 exon 5 ratios, significant cell killwas achieved.

cDNAMicroarray Screen ofWT1Target Gene Expres-sion in HL60 and K562 CellsTreatedwith ASWT13VUTRand ASWT1exon 5

cDNA microarray analysis was used to examine which, ifany, of the known WT1 target genes were differentiallyregulated by ASWT13VUTR and ASWT1exon 5 in theleukemic cell lines. The differential cytotoxicity profiles ofASWT13VUTR and ASWT1exon 5 in HL60 and K562 cellsalso provided an opportunity to investigate whether any ofthese genes were regulated in a manner that correlatedwith disruption of isoform ratios and also with toxicity.

Of 42 known putative WT1 target genes, 36 (listed inTable 2) were represented in our in-house gene expressionarray of 5,603 cDNA clones. Eleven genes were represented

by more than one clone. Gene expression profiles comparedfirst-strand cDNA populations from cells treated with 10Amol/L ASWT13VUTR or ASWT1exon 5 and cells treatedwith 10 Amol/L ASWT1scram as the reference. In K562cells, no alteration in the expression of any of the putativeWT1 target genes was detected following treatment witheither active ASO (data not shown). These genes seem,therefore, not to contribute to the cytotoxic activity of eitherASO in this cell line. In HL60 cells, which were resistant toASWT13VUTR treatment, again no alteration in the expression

Figure 8. Inhibition of cell survival by ASWT13VUTR and ASWT1 exon5 in K562 and HL60 cells. Soft agar clonogenic assays of cell survivalfollowing ASO treatment of K562 (A) and HL60 (B) cells. Control ASO(K562 cells: ASmc-raf and HL60 cells: ASWT1scram; n); ASWT13VUTR(E); ASWT1exon 5 (.). Points, mean of quadruplicate determinations;bars, SD. C, confirmation of efficient targeting of WT1 transcripts at24 hours posttreatment in the residual HL60 cells from the experiment inB using real-time PCR analysis.





Table 2. Microarray analysis of the expression of putative WT1 target genes 24 hours following treatment of HL60 cells with 10 Amol/LASWT1exon 5 or ASWT13VUTR using 10 Amol/L ASWT1scram as the reference

Symbol Gene name IMAGE clone ASWT13VUTRvs. ASWT1scram

ASWT1exon 5vs. ASWT1scram

ABCB1 ATP-binding cassette, subfamily B (MDR/TAP) 1 813256 f 0.96*

AR Androgen receptor 954356 0.68* 1.23*

AREG Amphiregulin (Schwannoma-derived GF) 1410444 f 0.86 F 0.11

AREG Amphiregulin (Schwannoma-derived GF) 1926453 0.87 F 0.01 1.01 F 0.13

BAX BCL2-associated X protein 2569476 0.85 F 0.12 0.87 F 0.13

BCL2 B-cell chronic lymphocytic leukemia/lymphoma 2 342181 0.83* 0.87 F 0.23

BCL2 B-cell chronic lymphocytic leukemia/lymphoma 2 2147728 1.11 F 0.09 0.90 F 0.10

CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 155 1.21 F 0.05 1.01 F 0.18

CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 251019 f 0.96*

CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) 270710 f f

CSF1 Colony-stimulating factor 1 (macrophage) 73527 f 1.05*

CSTA Cystatin A (stefin A) 345957 1.60* 1.27*

CTGF Connective tissue growth factor 898092 f 0.98*

CTGF Connective tissue growth factor 267256 0.73* 1.03*

EGFR EGFR 324861 0.80 F 0.07 1.00*

EGFR EGFR 669485 f 0.73*

EGFR EGFR 137017 f f

EGR1 Early growth response 1 840944 0.77 F 0.01 0.96 F 0.02

EGR1 Early growth response 1 753104 0.77 F 0.10 f

FGF1 Fibroblast growth factor 1 (acidic) 360478 f 0.94*

FOXD1 Forkhead box D1 382564 f 0.93*

GNAI2 G protein, a inhibiting activity polypeptide 2 530139 0.99 F 0.17 1.01 F 0.16

HSPA1A Heat shock 70-kDa protein 1A 155287 f 0.94*

IGF1R Insulin-like growth factor 1 receptor 682555 0.80 F 0.25 1.05 F 0.11

IGF1R Insulin-like growth factor 1 receptor 1709032 f f

IGF1R Insulin-like growth factor 1 receptor 148379 0.72 F 0.04 0.94 F 0.02

IGF2 Insulin-like growth factor 2 (somatomedin A) 245330 1.33 F 0.07 1.19 F 0.16

IGF2 Insulin-like growth factor 2 (somatomedin A) 207274 f 1.02*

IGF2 Insulin-like growth factor 2 (somatomedin A) 245330 1.31 F 0.07 1.09 F 0.16

IGF2 Insulin-like growth factor 2 (somatomedin A) 296448 0.98* 1.07*

IL-11 Interleukin-11 324183 f f

INHA Inhibin a 1758908 f 1.03*

INSR Insulin receptor 427812 1.12 F 0.04 1.02 F 0.01

MYB v-myb avian myeloblastosis virus

(AMV) oncogene homologue

243549 1.27 F 0.10 0.96 F 0.02

MYC v-myc AMV oncogene homologue 417226 0.85 FF 0.00 0.49 FF 0.02

MYC v-myc AMV oncogene homologue 812965 1.04 FF 0.13 0.51 FF 0.00

MYCN v-myc AMV related oncogene, neuroblastoma 41565 f 1.09*

NOV Nephroblastoma overexpressed gene 1113071 0.82* 0.91*

NR0B1 Nuclear receptor subfamily 0, group B, member 1 2338923 1.04* 1.07*

ODC1 Ornithine decarboxylase 1 796646 0.87 F 0.07 0.58 F 0.03

PAX2 Paired box gene 2 800137 f f

PDGFA Platelet-derived growth factor a polypeptide 435470 1.11* 1.15 F 0.09

PDGFA Platelet-derived growth factor a polypeptide 435470 1.25* 1.13*

RARA Retinoic acid receptor (2) 2356574 0.84* 1.02*

RARA Retinoic acid receptor a 461516 1.16 F 0.07 1.21 F 0.07

SALL2 Sal (Drosophila)-like 2 52430 1.04 F 0.32 1.06 F 0.01

SDC1 Syndecan 1 525926 f 0.89*

SOD1 Superoxide dismutase 1 950489 1.16 F 0.09 0.87 F 0.07

TGFB1 Transforming growth factor h1 136821 f 0.85*

THBS1 Thrombospondin 1 810512 0.85 FF 0.11 5.70 F 0.19

VDR Vitamin D (1,25-dihydroxyvitamin D3) receptor 815816 0.89 F 0.02 0.90*

WT1 Wilms’ tumor 1 503338 0.44* f

NOTE: Mean F range of two separate experiments. f, both spots flagged; *, replicate spot flagged (unacceptable quality).





of any of these genes by ASWT13VUTR was noted. How-ever, treatment with ASWT1exon 5, which decreased cellsurvival by 60%, resulted in the differential expression ofMYC , ornithine decarboxylase (ODC1), and THBS1 (Table2). The proto-oncogene MYC plays a key role in cellproliferation, differentiation, and apoptosis and is reportedto be amplified in HL60 cells (44), whereas THBS1 exhibitsnumerous biological activities including effects on celladhesion, migration, proliferation, and angiogenesis (45).

Using real-time Taqman RT-PCR, the differential regu-lation of both MYC and THBS1 were confirmed in the samesamples used for the microarray analysis (data not shown)and also in an independent experiment that included anadditional 48-hour time point to assess the duration ofaltered gene expression. This latter experiment confirmeddown-regulation of WT1 expression in HL60 cells by bothASOs over 48 hours (Fig. 9A). Down-regulation of MYCfollowing ASWT1exon 5 was seen at 24 hours (Fig. 9B) butnot to the degree seen in the microarray samples (Table 2).In addition, at 48 hours, MYC expression was reduced byboth ASOs revealing a lack of correlation between down-regulation of MYC and cell survival. Basal levels of MYCwere 10 times higher in HL60 cells than in K562 cells,supporting amplification in this cell line, and lack of down-regulation of MYC in K562 cells was confirmed.

The differential up-regulation of THBS1 by ASWT1exon5 but not ASWT13VUTR was confirmed at both 24 and 48hours following treatment (Fig. 9C) and was shown to beASWT1exon 5 dose dependent (Fig. 9D). These dataimplicate the THBS1 gene as a bona fide downstreamtarget of WT1. In these studies, THBS1 was only responsivein HL60 cells when down-regulation of WT1 was accom-panied by disruption of exon 5 isoform ratios. THBS1expression levels were f10 fold lower in K562 cells than inHL60 cells and were not disrupted by WT1ASO treatment.

Identification of Novel Putative WT1-ResponsiveGenes

In addition to the known WT1 regulated genes, weexamined the expression profiles of the remaining genes inthe expression array. A further 10 genes were found to bemodestly up-regulated (1.6- to 2.6-fold) specifically in HL60cells following ASWT1exon 5 treatment, mirroring thepattern of differential regulation displayed by THBS1(Table 3A). Similarly, a further five genes showed a patternof down-regulation concordant with MYC and ODC1 . Ourgene profile database was therefore searched for ASW-T1exon 5–induced disruption of gene expression specifi-cally in K562 cells. Eight genes were found to be modestlyup-regulated in this category, whereas only two werefound to be down-regulated (Table 3B). Significantly, theserepresent a different set of genes to those disrupted inHL60 cells by ASWT1exon 5. A further search for genes,common to both cell lines and disrupted by ASWT1exon 5,revealed seven that were commonly down-regulated butnone that were differentially up-regulated convincingly inboth cell lines (Table 3C).

This exercise was repeated to identify genes specificallyup-regulated or down-regulated by ASWT13VUTR in K562

cells and genes commonly disrupted in this cell line by bothASOs (Table 4A and B, respectively). From this lattercategory, the down-regulation of the genes encodingtetraspan 5 (TM4SF9) and GPC5 by both ASOs wasconfirmed using real-time PCR (Fig. 9E and F, respective-ly). Both genes may potentially contribute to WT1-directedantisense activity in this cell line. Transmembrane proteinsof the tetraspanin superfamily are implicated in a diverserange of biological phenomena, including cell motility,metastasis, cell proliferation, and differentiation (reviewedin refs. 46, 47). TM4SF9 was not expressed in HL60 cells.GPC5 is one of the membrane-bound heparan sulfateproteoglycan family of genes, members of which have been

Figure 9. Confirmation of disruption of gene expression following WT1ASO treatment of HL60 and K562 cells using real-time (Taqman) RT-PCRanalysis. WT1 (A), MYC (B), and THBS1 (C) transcript levels in HL60 cells24 and 48 hours following treatment with 10 Amol/L ASWT1scram (graycolumns), ASWT13VUTR (open columns ), and ASWT1exon 5 (blackcolumns), expressed as a percentage of ASWT1scram levels. D, dose-dependent increase of THBS1 in HL60 cells 24 hours followingASWT1exon 5 treatment (closed columns) and confirmation of lack ofdisruption of THBS1 following ASWT13VUTR (open columns). TM4SF9 (E)(E) and GPC5 (F) transcript levels in K562 cells following WT1 ASOtreatment as in HL60 cells above.





proposed to function in cellular growth control andmorphogenesis (48). GPC5 has been mapped to chromo-some 13q31-2 (49) and 13q32 (50). Interestingly, K562 cellshave been shown to harbor an amplicon spanning this

region (51), raising the possibility that GPC5 might beoverexpressed in K562 cells. Real-time PCR analysisconfirmed that expression of GPC5 was in excess of 40-fold higher in K562 cells compared with HL60 cells.

Table 3. Microarray expression profiling of genes disrupted specifically by ASWT1exon 5 in HL60 (A), K562 (B), and both HL60 andK562 (C) cells

Symbol Gene name IMAGEclone

HL60 K562

ASWT13VUTRvs. ASWT1scram




A. HL60 Cells

THBS1 Thrombospondin 1 810512 0.85 F 0.11 5.70 F 0.19 f 0.83*

IL-1h Interleukin-1h 324655 0.89* 2.64 F 0.21 0.89 F 0.07 1.14 F 0.09IL-1h Interleukin-1h 491763 0.63* 2.14 F 0.04 f 0.78 F 0.02BTG2 BTG family, member 2 213136 1.04 F 0.13 2.49 F 0.62 0.99 F 0.01 0.96 F 0.02NR4A2 Nuclear receptor subfamily 4, A, member 2 898221 0.91* 2.23 F 0.29 0.91* 1.13 F 0.17NDRG1 N-myc downstream regulated 842863 1.28 F 0.77 2.11 F 0.04 0.97 F 0.04 1.25 F 0.03SLC2A3 Solute carrier family 2, member 3 753467 1.17 F 0.02 1.96 F 0.05 1.08 F 0.07 1.19 F 0.06DTR Diphtheria toxin receptor 35828 1.09 F 0.19 1.94 F 0.12 f 1.02*

TYROBP TYRO protein tyrosine kinasebinding protein

148469 0.84 F 0.06 1.82 F 0.01 0.65* 0.84 F 0.11

CCL2 Chemokine (C-C motif) ligand 2 768561 0.94 F 0.06 1.69 F 0.15 0.83 F 0.10 1.06 F 0.01PHLDA1 Pleckstrin homology-like domain A1 667883 0.80 F 0.16 1.67 F 0.14 0.67 F 0.06 0.87 F 0.03

Hs cDNA FLJ33407 fis, cl. BRACE2010535 768638 0.92 F 0.05 1.66 F 0.08 0.80 F 0.13 1.09*

MYC v-myc AMV oncogene homologue 812965 1.04 F 0.13 0.51* 1.05 F 0.11 0.88 F 0.03MYC v-myc AMV oncogene homologue 417226 0.85* 0.49 F 0.02 0.87 F 0.16 0.75 F 0.01ODC1 Ornithine decarboxylase 1 796646 0.87 F 0.07 0.58 F 0.03 1.22 F 0.11 0.82 F 0.04ICSBP1 IFN consensus sequence binding protein 1 290230 0.82 F 0.10 0.51 F 0.05 f 0.83*

IFRD2 IFN-related developmental regulator 2 809946 0.79 F 0.06 0.56 F 0.08 1.07 F 0.17 0.81 F 0.13NME1 Nonmetastatic cells 1, protein(NM23A) 845363 0.92 F 0.08 0.57 F 0.07 1.20 F 0.31 0.81 F 0.12TRAP1 Heat shock protein 75 897570 1.07 F 0.15 0.66 F 0.06 1.45 F 0.14 1.20 F 0.02RPL19 Ribosomal protein L19 549101 1.41 F 0.19 0.68 F 0.10 1.30 F 0.14 1.00 F 0.18

B. K562 Cells

SIAT1 Sialyltransferase 1 897906 1.01* 0.89* 0.92* 2.21*

ETFA Electron transfer flavoprotein,a polypeptide

71672 0.95 F 0.12 1.07 F 0.09 0.91* 1.88 F 0.23

PDE8A Phosphodiesterase 8A 289972 f 1.02* 0.74 F 0.03 1.76 F 0.07PIR Pirin 234237 0.96* 0.96 F 0.06 1.20 F 0.19 1.71 F 0.06MAPK8 Mitogen-activated protein kinase 8 119133 f 1.16 F 0.04 1.13* 1.70 F 0.01PPIB Peptidylprolyl isomerase B (cyclophilin B) 756600 1.03 F 0.34 1.14 F 0.05 1.08 F 0.36 1.69 F 0.07APRT Adenine phosphoribosyltransferase 897774 0.98 F 0.32 1.08 F 0.03 1.09 F 0.21 1.66 F 0.24NQO2 NAD(P)H dehydrogenase, quinone 2 824024 0.91 F 0.22 1.07 F 0.06 1.34 F 0.51 1.61 F 0.05MPP1 Membrane protein, palmitoylated 1 (55 kDa) 296880 1.12 F 0.29 0.88 F 0.06 1.75 F 0.35 0.61 F 0.03BTK Bruton agammaglobulinemia

tyrosine kinase2014424 1.56 F 0.30 0.95 F 0.04 1.43 F 0.10 0.53*

C. HL60 and K562 Cells

NSEP1 Nuclease-sensitive element bindingprotein 1

949932 1.17 F 0.03 0.45 F 0.05 1.49 F 0.23 0.37 F 0.06

AHCY S-adenosylhomocysteine hydrolase 840364 0.86 F 0.07 0.50* 0.86 F 0.11 0.44 F 0.01CDC45L CDC45 (Saccharomyces cerevisiae ,

homologue) like453107 0.95 F 0.24 0.51 F 0.06 1.61 F 0.33 0.41 F 0.04

ANXA11 Annexin A11 810117 0.91* 0.55 F 0.04 1.25 F 0.22 0.48 F 0.08APLP2 Amyloid h (A4) precursor-like protein 2 240249 1.00 F 0.08 0.61 F 0.04 0.93 F 0.11 0.35 F 0.04UMPK UMP kinase 344243 0.88* 0.64 F 0.08 1.19 F 0.24 0.56 F 0.07CUL4A Cullin 4A 2310644 1.16 F 0.05 0.66 F 0.02 1.70 F 0.14 0.61*






In addition to the genes listed in Table 4B, several geneswere found to be differentially regulated by both WT1-directed ASOs in both cell lines. For example, the geneencoding adaptor-related protein complex 2, beta1 (AP2B1)was up-regulated 2- and 3-fold, glucose phosphate isom-erase (GPI) increased 1.6- and 3-fold, and KIAA0220 pro-tein was increased f2-fold by both ASOs in HL60 andK562 cells, respectively. Other genes were differentiallyregulated by ASWT13VUTR, specifically in HL60 cells orcommonly in both HL60 and K562 cells. BecauseASWT13VUTR was not cytotoxic to HL60 cells, these geneshave not been listed in this report. However, some may begenuine WT1-responsive genes.

DiscussionDoes Drug-Induced Induction of WT1Contribute to

ChemoresistanceMechanisms?As mentioned previously, expression of WT1 is dynam-

ically regulated following induction of differentiation in avariety of cell lines (27–30). However, to our knowledge,this is the first report of induction of WT1 expressionfollowing cytotoxic drug treatment of cell lines displaying aresistant phenotype, a phenomenon absent in the sensitivecell lines studied. These findings support the notion thatincreased WT1 expression may contribute to chemoresist-ance mechanisms, allowing cells to survive following drugtherapy. However, at this stage, we can only speculate onthe mechanism(s) of induction and the downstream effectsof induced WT1 expression.

Little information exists regarding the regulatory factorscontrolling WT1 expression. Several ubiquitous and tissue-specific transcription factors have been shown to activateor repress the WT1 promoter in transient transfectionassays including Sp1, WT1, GATA-1, Pax-2, Pax-8, andnuclear factor-nB (NF-nB; refs. 52–56). Regulation byNF-nB may be relevant here, because treatment of cellswith DNA-damaging agents or various cytokines andmitogens can result in its translocation from the cyto-plasm to the nucleus and the modulation of the appro-priate target genes. Ectopic expression of NF-nB has beenshown to increase the transcription of endogenous WT1 ,indicating that members of the NF-nB/Rel family maybe involved in a regulatory cascade leading to WT1activation (57). Further studies are clearly required todetermine the mechanisms responsible for, and the signif-icance of, the differential regulation of WT1 expression insensitive and resistant cell lines following cytotoxic drugtreatment.

Because G1 arrest associated with p53-independentregulation of endogenous p21CIP1 by WT1 has been shown,it is tempting to hypothesize that induction of WT1 con-tributes to chemoresistance by inducing cell cycle arrest,thereby allowing DNA repair and avoidance of replica-tion using a damaged template. However, in previousstudies using both paired cell lines, G1 arrest was notobserved following cisplatin treatment (38, 58). Common to

all four lines was a slowdown in S-phase transit, whereastransient G2 arrest or G2-M block at later time points wasseen dependent on cell line and dose of cisplatin. In thepresent studies, no temporal correlation between induc-tion of WT1 in CH1-R or GCT27-R cells and the previ-ously reported cell cycle effects was apparent. Lack of G1

arrest is not due to a p53-null phenotype because allfour cell lines show partial G1-S arrest, associated withinduced p53 and p21, following g-irradiation (data notshown).

Drug-Induced Disruption ofWT1exon 5 SplicingMayConstitute a Cell Stress Response

Alternative pre-mRNA splicing is a fundamental mech-anism of gene expression that can be regulated dependenton sex, development stage, or tissue, and in response toextracellular stimuli such as growth factors, hormones, andcytokines (59–61). Recent studies have identified a com-posite exonic splice control unit, which combines an exonrecognition element with splice silencer elements. Thesplice control unit seems to govern alternative splicing in acell type–specific manner and in response to activation ofprotein kinase C or Ras signaling pathways (61). The dis-covery of signal-responsive splice elements provides a linkbetween extracellular signals and regulation of exon var-iant transcription.

In these studies, we have provided the first evidence thatthe alternative splicing of exon 5 of WT1 mRNA is subjectto dynamic regulation in response to drug treatment,whereas KTS splicing is not disrupted. Much interest hasfocused recently on the activation of stress-activated pro-tein kinase/c-Jun-NH2-kinase and p38 mitogen-activatedprotein kinase cascades following cisplatin treatment(62, 63). Whether WT1 possesses signal-responsive ele-ments that respond to activation of these or other proteinkinases is unknown. Nevertheless, in the cell lines studied,the disruption of WT1 exon 5 splicing seemed to be anearly stress response to a toxic stimulus independent ofthe tissue of origin of the cell lines, the relative chemo-sensitivity, or the drug used. We propose that increasedexon 5 skipping is the most likely mechanism.

Does Disruption ofWT1exon 5 Ratios Provide a Pro-apoptotic or Cell Survival Signal?

In these studies, we have attempted to mimic thedisruption of WT1 exon 5 splicing seen following drugtreatment using an ASO targeted specifically to exon 5–containing WT1 transcripts. ASWT1exon 5 induced bothdisruption of exon 5 splicing and down-regulation of WT1levels. The latter effect may be due to down-regulation ofthe exon 5 transactivated regulatory antisense WT1 mRNAproduct, which has been shown previously to positivelyregulate WT1 protein levels (42). Cell survival studiesshowed loss of cell viability in both K562 and HL60 celllines by ASWT1exon 5, despite HL60 cells being resistant tothe effects of a balanced down-regulation of WT1 isoforms.These data support the view that the drug-induceddisruption of exon 5 splicing constitutes a proapoptoticsignal. It may be that the up-regulation of WT1expressionshown here in resistant cell lines following drug treatment





Table 4. Microarray expression profiling of genes disrupted specifically in K562 cells by ASWT13VUTR (A) and ASWT13VUTR andASWT1exon 5 (B)

Symbol Gene name IMAGEclone

HL60 K562





A. ASWT13VUTR

TCOF1 Treacher Collins-Franceschetti syndrome 1 815535 1.13 F 0.14 0.82 F 0.08 2.06 F 0.09 0.86 F 0.08

5VOY11.1 Sim to Ovis Aries Y chromosome repeat

region OY11.1

269292 f 1.12* 2.03 F 0.52 1.11 F 0.05

ZDHHC9 Zinc finger, DHHC domain containing 9 767419 1.12* 1.16 F 0.14 2.02 F 0.23 1.16 F 0.16

OS-9 Amplified in osteosarcoma 767419 1.12* 1.16 F 0.14 2.02 F 0.23 1.16 F 0.16

CBS Cystathionine h synthase 769857 f 1.03* 1.85 F 0.19 1.08 F 0.02

T54 T54 protein 26910 0.93* 1.13* 1.79 F 0.23 1.30 F 0.06

FLJ11184 Hypothetical protein FLJ11184 502891 1.02 F 0.06 0.80 F 0.07 1.77 F 0.28 0.72 F 0.01

FLJ10439 Hypothetical protein FLJ10439 773324 0.90* 0.78 F 0.03 1.75 F 0.30 1.07 F 0.05

CHAF1B Chromatin assembly factor 1B (p60) 756769 1.13 F 0.01 0.90 F 0.07 1.74 F 0.03 1.07 F 0.02

ELK4 ELK4, ETS-dom protein (SRF access prot 1) 236155 0.99* 1.03* 1.70 F 0.06 0.97*

DEFA1 Defensin a1, myeloid-related sequence 247483 0.91* 0.78* 0.27* 1.11 F 0.18

SSA1 Sjogren syndrome antigen A1 282956 f 1.01 F 0.18 0.35* 1.19*

MGST1 Microsomal glutathione S-transferase 1 768443 0.72 F 0.13 1.06 F 0.08 0.40 F 0.07 1.38 F 0.01

ROCK1 Rho-associated, coiled-coil cont prot kinase 1 80649 1.13 F 0.11 1.42* 0.43* 1.09*

FGFR1 Fibroblast growth factor receptor 1 154472 f 1.20* 0.45* 1.01 F 0.01

PTPN9 Protein tyrosine phosphatase,

nonreceptor type 9

770901 0.75 F 0.05 0.97 F 0.04 0.45* 0.85 F 0.12

KBF2 H-2K binding factor-2 731339 0.80 F 0.19 1.15 F 0.04 0.47 F 0.11 1.11 F 0.08

FIBP Fibroblast growth factor (acidic)

intracellular binding protein

839888 1.26 F 0.01 1.01 F 0.05 0.48* 1.00 F 0.05

NCSTN Nicastrin 199645 0.77 F 0.02 1.09 F 0.01 0.52 F 0.01 1.01 F 0.04

PLAUR Plasminogen activator, urokinase receptor 810017 0.94 F 0.17 1.39 F 0.47 0.52* 1.04 F 0.05

DYRK1A Dual-specificity Tyr-(Y)-phosphorylation

reg kinase 1A

897006 0.93 F 0.15 1.16* 0.57 F 0.09 1.13 F 0.06

JUND Jun D proto-oncogene 767784 0.71 F 0.04 0.81 F 0.14 0.58 F 0.05 0.91 F 0.12

B. ASWT13VUTR and ASWT1exon5

TARDBP TAR DNA binding protein 293576 1.23 F 0.33 1.49 F 0.05 3.77 F 1.16 2.63 F 0.48

TARDBP TAR DNA binding protein 417855 f 0.86* 1.70 F 0.09 1.72 F 0.01

SMA5 SMA5 470261 1.15* 1.24 F 0.24 2.50 F 0.66 2.30 F 0.36

ARL2 ADP-ribosylation factor-like 2 452780 1.26 F 0.06 1.24 F 0.08 2.21 F 0.21 1.69 F 0.20

MTIF2 Mitochondrial translational initiation factor 2 50754 1.19 F 0.35 1.09 F 0.15 2.21 F 0.08 1.65 F 0.19

TRA1 Tumor rejection antigen (gp96) 1 897690 1.13 F 0.07 0.97 F 0.03 2.13 F 0.12 1.67*

PCK2 Phosphoenolpyruvate carboxykinase 2 625923 1.16* 1.09 F 0.03 1.95* 1.82 F 0.16

MT1L/1X Metallothionein 1L/1X 297392 1.15 F 0.09 1.03 F 0.05 1.99 F 0.32 1.66 F 0.12

CIRBP Cold inducible RNA binding protein 1493383 0.95 F 0.02 0.92 F 0.14 1.93 F 0.10 1.53 F 0.10

MAPRE2 Microtubule-associated protein,

RP/EB family 2

950689 1.24 F 0.03 1.14 F 0.18 1.82 F 0.10 1.66 F 0.10

WARS Tryptophanyl-tRNA synthetase 855786 0.98 F 0.08 0.99 F 0.14 1.81 F 0.44 1.55 F 0.12

ILF1 Interleukin enhancer binding factor 1 40781 1.05 F 0.19 0.90 F 0.06 1.74 F 0.45 2.01 F 0.10

SEC13L1 SEC13 (S. cerevisiae)-like 1 897636 1.05 F 0.31 1.05 F 0.02 1.63 F 0.35 1.72 F 0.01

ASNS Asparagine synthetase 1493527 0.87* 0.87 F 0.02 1.57 F 0.34 1.93 F 0.20

ATP5I ATP synthase 782439 1.10* 1.07 F 0.15 1.55 F 0.10 2.08 F 0.09

TM4SF9 Tetraspan 5 812967 f 0.91* 0.43 F 0.10 0.58 F 0.09

GPC5 Glypican 5 1416502 0.55* 0.90* 0.41* 0.78*

CUL4B Cullin 4B 2244482 f 1.06* 0.62 F 0.15 0.62 F 0.05

NRAP Nebulin-related anchoring protein 611407 f 0.91* 0.45* 0.66 F 0.01

TSC22 TGFh-stimulated protein TSC-22 868630 f f 0.46* 0.68*

EFNA1 Ephrin-A1 1743833 f f 0.45* 0.67*






temporarily overrides this proapoptotic signal, providingthe cells with a greater opportunity for damage repair andescape from apoptosis. Interference with proapoptoticsignaling following cell damage can be expected to providea survival advantage, although additional resistancemechanisms are likely to contribute to the resistantphenotype of these cell lines.

Differential Regulation of PutativeWT1Target Genesfollowing Disruption ofWT1exon 5 Ratios

Some 42 putative WT1 target genes have been identifiedmainly as a result of transient transfection studies withreporter constructs (for a recent list, see ref. 2). However,with a few exceptions such as p21CIP1 and EGFR (13, 23),little convincing evidence of WT1-directed regulation of theendogenous expression of these putative target genes hasbeen forthcoming. Cell context specificity further compli-cates the search for bona fide WT1 target genes, becauseinteractions between WT1 and coregulatory proteins mayinfluence both the transcriptional regulatory propertiesof WT1 and target recognition. Consequently, there arefew clues as to how the various isoforms of WT1 interactintracellularly to exert their biological effects or indeedhow disruption of WT1 isoform ratios might induce alteredbiological function.

In this study, we have combined the use of antisenseand cDNA microarray technologies in an attempt toidentify which WT1-responsive genes are differentiallyregulated following disruption of WT1 isoform ratios ina manner correlating with inhibition of cell survival.As found previously, few changes in the expression levelsof the identified WT1 putative target genes were notedand none were seen in K562 cells. Differential regulationof MYC and its reported downstream target ODC1 wasobserved at 24 hours following ASWT1exon 5 in HL60cells, but subsequent and equivalent down-regulation ofMYC by ASWT13VUTR at 48 hours revealed a lack of cor-relation between MYC expression and cytotoxicity.

Unlike MYC expression, the 5-fold up-regulationof THBS1 seemed to correlate with the cytotoxic activityof ASWT1exon 5 in HL60 cells, although the mechanism ofsuch activity in vitro is not obvious. Thrombospondin is a450-kDa extracellular matrix–bound trimeric glycoproteinthat is expressed and secreted by platelets and a widevariety of cell types. It has been suggested to play animportant albeit complex role in controlling cancer cellgrowth and metastasis in vivo and variously implicated incancer cell adhesion, migration, invasion, proliferation, andapoptosis-dependent inhibition of angiogenesis (reviewedin ref. 45). Endogenous repression of THSB1 by WT1 hasbeen shown previously in response to overexpression ofc-Jun (64). Repression involved a factor secreted by c-Jun-activated cells, which triggered a signal transductionpathway culminating in the binding of WT1 to the THBS1promoter. By contrast, our present studies suggest a clearcorrelation between disruption of WT1 exon 5 ratios andendogenous regulation of THBS1 , a novel observation.Whether this activity is direct or involves a coregulatoryprotein remains to be determined.

Identification of Previously UnreportedWT1-Respon-sive Genes

Of the remaining genes on our expression array, f2%were significantly altered (directly or indirectly) by WT1-directed ASO treatment, generally in a cell type–specificand WT1 antisense target-specific manner. Our initial aimwas to search for evidence of differential regulation ofdownstream genes following disruption of WT1 exon 5ratios; clearly, this occurs in both HL60 and K562 cells.However, of the 31 genes affected by ASWT1exon 5treatment and listed in Table 3, only 7 were common toboth cell lines, highlighting yet again the cell typespecificity of WT1 activity. Overall, the changes inexpression were modest at 24 hours. It is possible thatmore extensive alterations in expression may be evident atlater time points, or differential regulation may be lost, as inthe case of MYC .

These studies fall short of confirming which, if any, ofthese genes contribute to the cytotoxic activity of ASW-T1exon 5 but can be used as the basis for further studies.Worthy of comment is the up-regulation of interleukin-1h(IL-1h) in HL60 cells. IL-1h, like tumor necrosis factor,initiates the activation of signal cascades by the recruitmentof adapter proteins to its receptor. In many cancer cell lines,both IL-1h and tumor necrosis factor induce apoptosis,whereas untransformed cell types are not sensitive unlessmRNA translation or protein synthesis is blocked (65).

The differential regulation of the genes listed in Table 4Aby ASWT13VUTR, specifically in K562 cells, shows that adifferent set of genes respond to the balanced down-regulation of WT1 isoforms as compared with when down-regulation of WT1 is accompanied by disruption of exon 5ratios (listed in Table 3B). It is possible that networks ofgenes and gene cascades may be controlled not only by theregulation of the overall levels of WT1 but also byregulation of exon 5 ratios. Interaction with the variousstress/apoptotic/differentiation/growth factor signalingpathways is a possibility that requires further investigation.

Finally, there are some genes that respond to down-regulation of WT1 regardless of whether isoform ratios aredisrupted. One of these, GPC5 , may represent a bona fideWT1 target gene as predicted by the presence of two WT1consensus binding sequences in its promoter region. GPC5constitutes one of the membrane-bound heparan sulfateproteoglycan family of genes, members of which have beenproposed to function in cellular growth control andmorphogenesis (48). Overexpressed in K562 cells, GPC5was of particular interest because it is expressed in cells ofmesenchymal origin during development and shows asimilar pattern of expression to WT1 in the developingkidney and gonads (49, 66, 67). In addition, GPC5 has beensuggested to be a candidate gene for at least some of thephenotypic features of 13q syndrome, a developmentaldisorder with a pattern of defects that shows overlap withboth WAGR and DDS and also with WT1 knockout mice(49, 68, 69).

In summary, the results of this study support a role forWT1 in the maintenance of viability and proliferative





capacity in cancer cells and as a mediator of survivalsignals following cytotoxic drug treatment. Downstreamsignaling seems to involve the orchestrated regulation ofWT1 exon 5 splicing and total WT1 expression. Using ASOsdirected to both exon 5 and the 3V UTR of WT1 , we haveshown cell type– specific and antisense target-specificregulation of genes, some of which may prove to be novelWT1 target genes. Disruption of WT1 exon 5 ratios byASWT1exon 5 was shown to reduce the cell survival ofHL60 cells that are resistant to other WT1-targeted ASOs.ASWT1exon 5 may therefore have a broader therapeuticpotential than previously described WT1 ASOs. In prelim-inary studies, we have shown significant antitumor activityagainst K562 cells grown in vivo using both WT1-directedASOs (70). We are extending these studies to explore thetherapeutic potential of these compounds in other WT1-expressing tumors such as prostate cancer, breast cancer,and childhood embryonal cancers.

Acknowledgments

The authors thank Dr. Dan Williamson (Section of Molecular Carcinogen-esis, ICR, Sutton, UK) for the design of the GPC5 primers and probe andKathryn R. Taylor for excellent technical assistance.

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2004;3:1467-1484. Mol Cancer Ther Jane Renshaw, Rosanne M. Orr, Michael I. Walton, et al. inhibits cell survivaldown-regulation of exon 5 alters target gene expression andfollowing cytotoxic drug treatment: Antisense

gene expression and exon 5 splicingWT1Disruption of

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