Disruption of WT1 Gene Expression

Molecular Cancer Therapeutics 1467

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

Jane Renshaw,1 Rosanne M. Orr,2 cell death. In addition, novel potential WT1 target genes Michael I. Walton,2 Robert te Poele,2 were identified in each cell line. These studies highlight a Richard D. Williams,1 Edward V. Wancewicz,3 new layer of complexity in the regulation and function of Brett P. Monia,3 Paul Workman,2 and the WT1 gene product and suggest that antisense directed Kathryn Pritchard-Jones1 to WT1 exon 5 might have therapeutic potential. [Mol Cancer Ther 2004;3(11):1467–83] 1Section of Paediatrics, and 2Cancer Research UK Centre of Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, United Kingdom and 3Isis Pharmaceuticals, Inc., Introduction Carlsbad, California The Wilms’ tumor gene (WT1) is located at the human chromosome region 11p13 and encodes a developmentally regulated transcription factor that is essential for normal Abstract genitourinary development (reviewed in refs. 1, 2). In the Deregulated expression of the Wilms’ tumor gene (WT1) adult, WT1 expression is restricted to specific cell types in has been implicated in the maintenance of a malignant kidney, gonads, hematopoietic and nervous system, and phenotype in leukemias and a wide range of solid tumors mesothelium (1). However, inappropriate and/or over- through interference with normal signaling in differentia- expression of WT1 has been reported in leukemias and a tion and apoptotic pathways. Expression of high levels of wide range of solid tumors including prostate, breast, and WT1 is associated with poor prognosis in leukemias and lung as well as thyroid, testicular and ovarian carcinomas, breast cancer. Using real-time (Taqman) reverse transcrip- melanoma, and mesothelioma (2). Although an oncogenic role tion-PCR and RNase protection assay, we have shown in these tumors has not been proven, experimental evi- up-regulation of WT1 expression following cytotoxic treat- dence suggests that expression of WT1 may contribute to ment of cells exhibiting drug resistance, a phenomenon the maintenance of a malignant phenotype through a va- not seen in sensitive cells. WT1 is subject to alternative riety of mechanisms including inhibition of differentiation splicing involving exon 5 and three amino acids (KTS) at and apoptosis and increased proliferation (reviewed in ref. 2). the end of exon 9, producing four major isoforms. Exon 5 The WT1 protein consists of 10 exons with an activator/ splicing was disrupted in all cell lines studied following a repressor domain near the NH2 terminus and four zinc cytotoxic insult probably due to increased exon 5 skipping. fingers of the Cys2-His2 type at the COOH terminus. Disruption of exon 5 splicing may be a proapoptotic signal Alternative translation initiation site usage (3, 4) and because specific targeting of WT1 exon 5–containing alternative splicing of WT1 mRNA (5) produces multiple transcripts using a nuclease-resistant antisense oligonu- WT1 protein isoforms. The two alternative splice regions cleotide (ASO) killed HL60 leukemia cells, which were correspond to the 17 amino acids of exon 5 (present only in resistant to an ASO targeting all four alternatively spliced mammals) and the last three amino acids (KTS) of exon 9 transcripts simultaneously. K562 cells were sensitive to (conserved in all vertebrates; ref. 6). Insertion of KTS alters both target-specific ASOs. Gene expression profiling the spacing between zinc fingers 3 and 4, disrupting following treatment with WT1 exon 5–targeted antisense sequence-specific DNA binding and, in transient cotrans- showed up-regulation of the known WT1 target gene, fection assays, transcriptional regulation (reviewed in thrombospondin 1, in HL60 cells, which correlated with ref. 2). In addition, +KTS isoforms have been shown to colocalize with splicing factors in the nucleus, suggesting a role in RNA processing (7). In these assays, the presence or absence of exon 5 has little impact. Received 3/10/04; revised 8/6/04; accepted 9/15/04. The four major isoforms of WT1, designated WT1 (+/+), Grant support: Children’s Cancer Unit Fund, Royal Marsden Hospital NHS WT1 (+/À), WT1 (À/+), and WT1 (À/À) to indicate the Trust, Sutton, UK (J. Renshaw, R.D. Williams) and Cancer Research UK presence or absence of exon 5/KTS, respectively (see Fig. 1), (R.M. Orr, M.I. Walton, R. te Poele, P. Workman, and K. Pritchard-Jones). are in general quoted as being coexpressed in a fixed ratio The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked in normal tissues (5) and this is true for the presence or advertisement in accordance with 18 U.S.C. Section 1734 solely to absence of KTS (the WT1 KTS ratio). Studies of transgenic indicate this fact. mice have shown that WT1 isoforms with and without Requests for reprints: Jane Renshaw, Section of Paediatrics, Institute of the KTS insert have separate and vital functions in sex Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, United Kingdom. Phone: 44-208-7224327; Fax: 44-208-7224321. determination and gonadal development, consistent with a E-mail: [email protected] fixed expression ratio (8, 9). However, we and others have Copyright C 2004 American Association for Cancer Research. shown that WT1 exon 5 ratios differ according to tissue and

Mol Cancer Ther 2004;3(11). November 2004

in gonadal tumors they were nearer equivalence or marginally underrepresented. These findings were in- triguing because the individual isoforms of WT1 seem to mediate different downstream cell type–specific biological effects that may involve isoform-specific interactions with coregulatory binding proteins such as Par4 and cyclic AMP response element binding protein binding protein (reviewed in ref. 2). For example, Par4 interacts Figure 1. Schematic representation of WT1 alternatively spliced variants. Four main splice variants are represented schematically to show with both exon 5 and the zinc fingers of WT1 resulting in the presence or absence of exon 5 and KTS along with their designations: opposing effects on transcription and, in exon 5 interaction, WT1 (+/+), (+/À), (À/+), and (À/À) and WT1 + and À exon 5. the rescue of 293 cells from lethal UV light treatment (25, 26). It is therefore probable that Par4 may mediate both differentiation stage as well as between species (6, 10). positive and negative interactions with WT1 dictated, at Differential splicing of exon 5 has been confirmed in least in part, by the relative ratio of WT1 + to À exon 5 human kidney and Wilms’ tumor samples through analysis isoforms. of native WT1 protein (11). Mice lacking the WT1 exon 5 Numerous reports have described cell type–specific insert develop normally and are not compromised with regulation of WT1 expression following induction of respect to fertility, embryo viability, or the capacity to differentiation in leukemic, embryonal stem cell, and lactate, suggesting that exon 5 is redundant in genitouri- carcinoma cell lines accompanied by growth arrest and nary development and function at least in mice (12). apoptosis in some cell types (27–30). We initiated the Nevertheless, the presence of the exon 5 insert and the present study to determine whether similar alterations in maintenance of the correct balance between WT1 + exon the regulation of WT1 mRNA levels and/or alternative 5 and WT1 À exon 5 isoforms has been suggested to be splicing were induced following an apoptotic stimulus essential for the regulation of critical cellular functions such such as cytotoxic drug treatment. The possibility of as proliferation, differentiation, and resistance to chemo- differential regulation of WT1 expression in cell lines therapeutic drugs (13–16). Furthermore, disruption of exon sensitive to or with acquired resistance to a cytotoxic agent 5 splicing has been suggested to affect the regulation of was investigated using two paired cell lines: the paired genes downstream in the WT1 pathway, as has been shown ovarian papillary cystadenocarcinoma cell lines, CH1-S in Wilms’ tumors that express reduced levels of WT1 exon and CH1-R (sensitive to and with acquired resistance to 5 variants relative to those lacking exon 5 (17). cisplatin, respectively), and the corresponding testicular In leukemias, expression of very high levels of WT1 has germ cell tumor lines, GCT27-S and GCT27-R. The levels been correlated with poor response to treatment (18–21), of total WT1 and individual alternatively spliced WT1 whereas high expression of WT1 mRNA has been shown variants were measured in these cell lines following to predict poor prognosis in breast cancer patients (22). cisplatin treatment and, in the erythroleukemia cell line Numerous studies using ectopic overexpression of indi- K562, following treatment with doxorubicin. A combina- vidual WT1 isoforms have indicated that WT1 may in- tion of radioactive reverse transcription-PCR (RT-PCR), terfere with apoptotic pathways, but the results are diverse, quantitative real-time (Taqman) RT-PCR, and a RNase often conflicting, and cell type specific. For example, protection assay (RPA) was used. regulation of the antiapoptotic gene Bcl-2 by exon 5– We report here the novel observation that both WT1 exon containing isoforms has been suggested to mediate induced 5 splicing and total WT1 mRNA transcript expression were resistance to apoptosis, because cells expressing the WT1 dynamically regulated following cytotoxic treatment. Dis- (+/À) isoform and Bcl-2 are resistant to staurosporine-, ruption of exon 5 splicing resulted from the down- vincristine-, and doxorubicin-induced apoptosis (16). How- regulation of WT1 exon 5–containing transcripts relative ever, K562 cells stably expressing the various WT1 isoforms to those lacking exon 5, probably by a mechanism of did not exhibit increased resistance to doxorubicin or induced exon 5 skipping. Induction of total WT1 expression cisplatin, although induction of differentiation by 12-O- was seen only in those cell lines displaying a resistant tetradecanoylphorbol 13-acetate was partially inhibited phenotype. The downstream sequelae of disrupted exon 5 (23). By contrast, high levels of WT1 (+/À) expression splicing were investigated further using the leukemic cell have been shown to suppress epidermal growth factor lines K562 and HL60 and a combination of antisense and receptor (EGFR) and induce late-onset, p53-independent gene expression profiling technology. Both cell lines apoptosis in some cell types (24). It should be noted that express high levels of WT1, and HL60 cells have been enforced expression of individual isoforms simultaneously shown previously to be resistant to WT1-directed antisense alters both exon 5 and KTS ratios, making interpretation of oligonucleotides (ASOs), which kill K562 cells (31). These their individual functions difficult. Definitive evidence that results provide novel insights into the role of WT1 exon 5 WT1 overexpression contributes to a resistant phenotype is splicing and total WT1 expression in the regulation of cell still lacking in studies using this approach. survival signaling pathways. Antisense targeted to WT1 In our previous study, exon 5–containing WT1 tran- exon 5 may have broader therapeutic potential than scripts were shown to be in excess in leukemias, whereas previously described WT1-directed ASOs.

Mol Cancer Ther 2004;3(11). November 2004

Materials and Methods FCS and 0.2% Agar Noble (Difco Laboratories, Detroit, Oligonucleotides MI) and incubated at 37jC and colonies were counted after The 20-mer, 2V-O-methoxyethyl chimeric oligonucleoti- 14 days. In several separate experiments, plating efficien- des consisting of a central window of eight 2V-deoxy cies ranged from 22% to 38%, with 800 to 1,600 cells initially unmodified sugar residues with flanking 2V-O-methox- plated. yethyl regions and a fully thioated backbone were RNA Extraction and Radioactive RT-PCR synthesized by Isis Pharmaceuticals Inc. (Carlsbad, CA) At the appropriate times following drug treatment, cells as described previously (32). The two active compounds were harvested and RNA was extracted using Trizol (Life selected for this study were ISIS 16609, sequence Technologies Ltd., Paisley, United Kingdom). RNA (1 Ag) 5V-GCCCTTCTGTCCATTTCACT-3V, targeting exon 5 (ASW- was reverse transcribed with SuperScript II and random T1exon 5) and ISIS 16601, sequence 5V-CACATACA- hexamer primers (100 pmol, Invitrogen Ltd., Paisley, CATGCCCTGGCC-3V, targeting the 3V untranslated region United Kingdom) in a final volume of 20 AL according to (UTR) of WT1 (ASWT13VUTR). Two oligonucleotides of the the manufacturer’s instructions. RT-PCR of all four WT1 same chemistry were used as controls: ISIS 15770, sequence alternatively spliced mRNA transcripts was carried out 5V-ATGCATTCTGCCCCCAAGGA-3V, a 5-10-5 gapmer tar- using 1 AL cDNA, [a-32P]dCTP, reduced cold dCTP, and geting murine c-raf kinase (ASmc-raf) and lacking homol- primer pair 297 and 298 spanning WT1 exons 4 to 10. RT- ogy to any known human sequence, and ISIS 105730, PCR methodology and method validation have been sequence 5V-CCATCGACCTGCACCGATCA-3,V a scrambled described in detail previously (10). Four individual reaction sequence of ASWT13VUTR (ASWT1scram). mixes for each sample were set up in parallel and amplified Cell Culture and Drug Treatment for 26, 29, 32, and 35 cycles, respectively, and PCR products K562 cl.6 cells, a subclone of the parent erythroleukemia 482 (À/À), 491 (À/+), 533 (+/À), and 542 (+/+) bp long (33), were kindly provided by Prof. Adrian Newland and were separated on standard denaturing polyacrylamide Dr. Xu-Rong Jiang (London Hospital Medical College, gels. Levels of [32P]dCTP incorporated into all four London, United Kingdom). HL60 promyelocytic leukemia transcripts were visualized and analyzed using a Storm cells were obtained from the American Type Culture 860 PhosphorImager and ImageQuant software (Amer- Collection (Manassas, VA). K562 cl.6 and HL60 cells were sham Pharmacia Biotech UK Ltd., Little Chalfont, Buck- grown in RPMI 1640 (HEPES buffered) supplemented with inghamshire, United Kingdom) followed by adjustment for 10% FCS (Biowest, Ringmer, East Sussex, United Kingdom) the number of possible sites of incorporation of 32P in the and penicillin (100 units/mL). The in-house paired ovarian alternatively spliced PCR products. cell lines CH1-S and CH1-R (34) and testicular germ cell RNase Protection Assay tumor cell lines GCT27-S and GCT27-R (35) were cultured A cDNA sequence spanning the whole of WT1 exon 5 in DMEM (bicarbonate buffered) supplemented with 10% and 70 bp of exon 4 was amplified by RT-PCR from a FCS. Doxorubicin (Sigma-Aldrich Co. Ltd., Gillingham, sample of normal human testis. PCR products were Dorset, United Kingdom) and cisplatin (Johnson Matthey subcloned by standard methods and sequenced (automat- Technology Centre, Reading, Berkshire, United Kingdom) ed fluorescent sequencing) using an ABI PRISM 310 were dissolved in sterile saline and cells were treated Genetic Analyzer (Applied Biosystems, Applera United (100 AL drug in saline to 10 mL cultures) either continu- Kingdom, Warrington, Cheshire, United Kingdom). High ously (doxorubicin) or for 2 hours (cisplatin) at a specific activity WT1 antisense riboprobes were generated 32 concentration determined previously as the IC99 in clono- using 1 Ag of purified linearized vector, [a- P]UTP, and a genic assays: K562, 0.1 Amol/L doxorubicin; CH1-S, Maxiscript labeling kit (Ambion Europe Ltd., Huntindon, 3.8 Amol/L; CH1-R, 18.5 Amol/L; GCT27-S, 9.3 Amol/L; Cambridgeshire, United Kingdom) according to the and GCT27-R, 40 Amol/L cisplatin. Antisense or control manufacturer’s instructions but with the addition of oligonucleotides were dissolved in PBS and introduced into single-stranded binding protein (1 AL, Amersham Phar- K562 or HL60 cells by low-voltage electroporation. Briefly, macia Biotech UK). RPA analyses were carried out appropriately diluted ASOs (40 AL) were combined with according to the manufacturer’s instructions using total cell suspension (360 AL) at 2 Â 107 cells/mL and cells were RNA (f10 Ag) and the RPA III kit (Ambion Europe). WT1 electroporated (Bio-Rad Gene Pulser II electroporation RNase protected fragments, 121 (+ exon 5) and 70 (À exon system with Pulse Controller Plus capacitance extender 5) bp, along with control actin (Ambion Europe) protected accessory module, Bio-Rad Laboratories Ltd., Hemel fragments, were separated in 1 hour using 8% standard Hempstead, Hertfordshire, United Kingdom) using 300 V denaturing polyacrylamide gels. Levels of [32P]UTP in- and a capacitor value of 1,000 AF, diluted to 10 mL, and corporated into the protected fragments were visualized incubated at 37jC for various times. and analyzed using a Storm 860 PhosphorImager as Clonogenic Cell Survival Assays above. Following drug treatment, appropriate aliquots of cells Real-time (Taqman) PCR were serially diluted in complete medium. Aliquots (2 mL) The primer pairs and probes for the quantitation of WT1, of diluted cells were added to polystyrene tubes (Elkay thrombospondin 1 (THBS1), and glypican 5 (GPC5) levels Products Ltd., Basingstoke, Hampshire, United Kingdom) were designed using the Primer Express program (Applied containing medium (3 mL) supplemented with 20% Biosystems) according to the recommended guidelines:

Mol Cancer Ther 2004;3(11). November 2004

WT1 forward primer 5V-TACCCAGGCTGCAATAAGAG- West Sussex, United Kingdom.) and concentrated using ATATTTTAAG-3V, reverse primer 5V-CCTTTGGTGTCTT- Microcon YM-30 centrifugal filter devices (Millipore, TTGAGCTGGTC-3V, and probe 5V-CACTGGTGAGAAAC- Watford, Hertfordshire, United Kingdom). cDNA micro- CATACCAGTGTGACTTCAAGGACT-3V; THBS1 forward array analyses of each sample were done in duplicate. The primer 5V-GACAGCATCCGCAAAGTGACT-3V,reverse preparation of the cDNA microarray slides, and fluorescent primer 5V-GACAGTGACACTCAGTGCAGCTATC-3V, and labeling of polyA+ mRNA samples using Superscript II probe 5V-TGAGCTGAGGCGGCCTCCCCTA-3V; and GPC5 and Cy5-labeled or Cy3-labeled dCTP, was carried out as forward primer 5V-GGGCTGCCGGATTCG-3V, reverse prim- described previously (36). The microarray hybridization er 5V-CTGGTGCAACATGTAGGCTTTT-3V, and probe 5V- was carried out according to a published protocol (37). The CGCGGGCAGGACCTGATCTTCA-3V. The primers were so-called Institute of Cancer Research gene set, consisting designed to amplify across exon/exon boundaries and of 5,603 IMAGE cDNA clones, is also described in detail in were confirmed to be lacking significant homology with this latter publication along with the image collection and any known DNA sequences by a search of the HGMP data analysis procedures. Briefly, array images were database. All other genes were analyzed using Assays-on- acquired with an Axon GenePix 4000 scanner (Axon Demand Gene Expression Products (Applied Biosystems). Instruments, Foster City, CA) and analyzed with the Axon Taqman analysis was carried out according to the GenePix Pro 3 software package (Axon Instruments). Data manufacturer’s instructions using an Applied Biosystems were filtered for quality by automated spot flagging and 7700 Sequence Detector. Each assay sample was analyzed manual inspection. Fluorescence intensity ratios (I = Cy5/ in triplicate and multiplexed to facilitate the measurement Cy3) were calculated after background subtraction and of gene expression levels relative to 18s rRNA expression normalized to the median expression ratio of all high (rRNA control reagents, Applied Biosystems) using the quality spots. Intensity ratios were transferred to a Micro- standard curve method. soft Access database for IMAGE clone to gene assignments, Protein Extraction and Estimation Using Western data filtering, and group queries. Gene assignments were Immunoblotting checked and updated using National Center for Biotech- At the appropriate times following drug treatment, f5 Â nology Information UniGene, formatted for local database 106 cells were harvested, washed in PBS, and lysed using import by a Perl script.4 2% SDS containing 10% v/v protease inhibitor cocktail (Sigma-Aldrich). Sample protein concentrations were esti- Results mated using the Bio-Rad detergent-compatible microtiter Dynamic Alteration of WT1 exon 5 Alternative Splic- plate protein assay according to the manufacturer’s ing and Total WT1 Expression following Treatment of instructions. Prior to electrophoresis, an aliquot of each Gonadal Cell Lines with Cisplatin sample was treated with RNase-free DNase (2 units, 2+ The paired human ovarian cell lines CH1-S and CH1-R as Ambion Europe) in the presence of Mg (5 mmol/L), total well as the corresponding testicular germ cell tumor cell A j j volume 20 L, at 37 C for 30 minutes followed by 75 C for lines GCT27-S and GCT27-R have been shown previously 5 minutes to minimize protein band distortion. Electro- to undergo apoptosis following equitoxic concentrations of phoretic separation of the + and À exon 5 WT1 isoforms cisplatin (38, 39). Following treatment with cisplatin at IC A 99 (20 g protein loaded) was achieved using NuPAGE 10% concentrations (concentration of drug reducing cell surviv- Bis-Tris precast gels (Invitrogen, Groningen, Netherlands) al by 99% of control levels in clonogenic assays), the run with NuPAGE MOPS and SDS denaturing running relative levels of all four WT1 alternatively spliced tran- buffer. WT1 proteins were analyzed using a 1:1,000 dilution scripts were analyzed using radioactive RT-PCR (Fig. 2). of the rabbit polyclonal WT(C-19) (Santa Cruz Biotechnol- WT1 exon 5 ratios were rapidly reduced in all four cell ogy, Santa Cruz, CA). Horseradish peroxidase–conjugated lines, reaching a nadir at 8 hours post-treatment, whereas V anti-rabbit F(ab )2 fragment secondary antibodies (Amer- the WT1 KTS ratios remained relatively constant through- sham Bioscience UK Ltd.) were used at 1:2,000 dilution, out (data not shown). Control 0-hour exon 5 ratios and detected using the Enhanced Chemiluminescence Plus ratios in samples processed at various times during the system (Amersham Bioscience UK Ltd.), and visualized 24-hour period were not significantly different to those of and analyzed using a STORM PhosphorImager 860 and untreated, logarithmically growing cells. In both paired cell ImageQuant software. For WT1 estimation, a nonspecific lines, the extent of disruption of WT1 exon 5 ratios was band, running slightly faster than WT1 and shown greater in the resistant line than in the parent sensitive line, previously to be minimally affected by antisense treatment, the mean F range of WT1 exon 5 ratios when expressed as was used to adjust for differences in loading and sample a percentage of control ratios being 65.7 F 10% versus 48.5 protein concentration. F 11.5% for the CH1-S and CH1-R cell lines, respectively, PolyA + mRNA Isolation and cDNA MicroarrayAnalysis and 76.0 F 14.6% versus 32.4 F 8% for the GCT27-S and Â Multiple (10 ) aliquots of K562 and HL60 cells were GCT27-R cell lines, respectively, at 8 hours post-treatment. treated with ASO (10 Amol/L) as described above. After 24 hours, total RNA was extracted and the samples were pooled. PolyA+ mRNA was isolated and purified from total RNA using an Oligotex kit (Qiagen Ltd., Crawley, 4 http://www.hgmp.mrc.ac.uk/~rdwillia/unigene.html.

Mol Cancer Ther 2004;3(11). November 2004

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

Figure 2. Dynamic regulation of WT1 exon 5 transcript ratios following treatment with cisplatin. WT1 exon 5 ratios (see Fig. 1) were determined using radioactive RT-PCR (as illustrated in Fig. 3) at various time points over 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 5 transcript ratios are expressed as a percentage of ratios at 0 hour.

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 of absolute alterations in the levels of the + and À exon 5 transcripts. We therefore examined the levels of WT1 amplification products with and without exon 5 generated in these samples during the logarithmic phase of polymer- ase amplification (26 cycles; see Fig. 3 for ovarian cell lines and Fig. 4 for germ cell tumor cell lines). In the CH1-S and GCT27-S parent sensitive cell lines, exon 5–containing transcripts were reduced to 63% and 69% of control levels, respectively, at 8 hours after cisplatin treatment, whereas the levels of transcripts lacking exon 5 remained essentially unchanged 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 spliced Figure 3. Radioactive RT-PCR analysis of WT1 + and À exon 5 transcripts had recovered to near control levels, although transcript levels following IC99 cisplatin treatment of CH1-S and CH1-R overall levels were slightly reduced. By contrast, total WT1 cells. Levels of WT1 amplification products with and without exon 5 transcript levels were up-regulated in the resistant CH1-R generated in (A) CH1-S and (B) CH1-R cells were determined using radioactive RT-PCR following 26 cycles (logarithmic phase) of amplifica- and GCT27-R lines following treatment, although the tion. Aa and Ba, typical gel images (inset) of all four alternatively spliced induction kinetics differed (Figs. 3Ba and Bb and 4Ba and RT-PCR products from selected time points along with superimposed line Bb). In CH1-R cells, disruption of exon 5 splicing and up- graphs generated from these images. Ab and Bb, normalized (adjusted regulation of alternatively spliced transcript levels occurred for length) levels of + exon 5 (open columns) and À exon 5 (closed columns) WT1 transcripts over the 24-hour time course. C and D, simultaneously, and the excess production of WT1 À exon confirmation of total WT1 transcript levels in CH1-S (C) and CH1-R (D) 5 transcripts (459%) as compared with WT1 + exon 5 cells using real-time (Taqman) analysis.

Mol Cancer Ther 2004;3(11). November 2004

Dynamic Alteration ofWT1exon 5 Alternative Splicing and Total WT1 Expression following Treatment of a Leukemic Cell Line with Doxorubicin 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 have been shown to be relatively resistant to induction of apoptosis, which does not occur until 48 to 72 hours following a variety of apoptotic stimuli including treatment with the clinically used anthracycline, doxorubicin (40). To determine whether the response to a cytotoxic insult was different in a leukemic cell line as opposed to a gonadal cell line, WT1 exon 5 ratios and total WT1 levels were determined in K562 cells following an IC99 treatment with doxorubicin using RPA analysis (Fig. 5). As seen in the ovarian and germ cell tumor lines following cisplatin treatment, there was a rapid reduction of WT1 exon 5 ratios reaching a nadir of 57% at 8 hours and partially recovering 24 hours following doxorubicin treatment (Fig. 5A). Total WT1 levels are shown in Fig. 5B. After an initial reduction, both WT1 + and À exon 5 transcripts were up-regulated in concert in a manner similar to that seen in CH1-R cells but achieving maximum levels 16 hours following treatment

Figure 4. Radioactive RT-PCR analysis of WT1 + and À exon 5 transcript levels following IC99 cisplatin treatment of GCT27-S and GCT27-R cells. Levels of WT1 amplification products with and without exon 5 generated in (A) GCT27-S and (B) GCT27-R cells were determined using radioactive RT-PCR following 26 cycles (logarithmic phase) of amplification. Aa and Ba, typical gel images (inset) of all four alternatively spliced RT-PCR products from selected time points along with superimposed line graphs generated from these images. Ab and Bb, normalized (adjusted for length) levels of + exon 5 (open columns) and À exon 5 (closed columns) WT1 transcripts over the 24-hour time course. C and D, 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 À exon 5 transcript levels and levels) at 8 hours post-treatment. Up-regulation of total ratios following IC99 doxorubicin treatment of K562 cells. Levels of WT1 WT1 expression was apparent by 16 hours and maximal by + and À exon 5 transcripts in K562 cells were determined using RPA analysis at various time points over a 24-hour time course following 24 hours post-treatment in this cell line, accompanied by a treatment with IC99 doxorubicin. A, WT1 exon 5 transcript ratios in K562 recovery and an overshoot of the WT1 exon 5 ratios. cells following doxorubicin treatment expressed as a percentage of ratios Confirmation of the differential regulation of total WT1 at 0 hour. B, total WT1 mRNA levels normalized to actin. C, typical gel expression in the resistant as compared with the sensitive images of RNase protected fragments (inset) along with line graphs generated from these images. D, normalized (adjusted for length) levels of cell lines was achieved using real-time (Taqman) RT-PCR + exon 5 (open columns) and À exon 5 (closed columns) WT1 analysis of the same samples (Figs. 3C and D and 4C and D). transcripts over 24 hours following doxorubicin treatment.

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(Fig. 5C and D). Again, there was an excess production of WT1 À exon 5 transcripts (217%) as compared with WT1 + exon 5 transcripts (125%), consistent with increased exon 5 skipping during post-transcriptional splicing. Down-Regulation of WT1exon 5 ^ Containing Tran- scripts Using a Nuclease-Resistant ASO To further analyze the impact that disruption of exon 5 splicing might have on cell viability and downstream signaling, we used an antisense approach. WT1 exon 5 transcripts were targeted specifically in an attempt to mimic the drug-induced down-regulation of exon 5 transcript expression seen in this study. An ASO targeting all four WT1 alternatively spliced transcripts simultaneously was deemed an essential comparator to control for those effects due solely to a balanced down-regulation of WT1 expression. From a primary screen of 25 2V-O-methoxyethyl ASOs targeted through the 5V UTR to 3V UTR sequences of WT1 (data not shown), two active oligonucleotides were selected for further evaluation: ISIS 16609, targeting exon 5 (ASW- T1exon 5), and ISIS 16601, targeting the 3V UTR region of WT1 (ASWT13VUTR). The control ASO used was ISIS 15770 (ASmc-raf), directed to mouse c-raf and with no sequence homology to human DNA. K562 cells were used as this cell line had been shown previously to be sensitive to an ASO directed to the translational start site of WT1 (31). Validation of Antisense Activity Initial experiments using RPA analysis examined total WT1 transcript levels 5 hours following electroporation of K562 cells with 10 Amol/L ASWT13VUTR and ASWT1exon 5 as compared with electroporation in the presence of PBS alone or the control ASO, ASmc-raf (Fig. 6A). Total WT1 transcript levels were equivalent in PBS-treated and ASmc- raf-treated cells and reduced to 55% and 30% of PBS control levels following treatment with ASWT13VUTR and ASW- T1exon 5, respectively. The WT1 exon 5 ratios did not alter as total WT1 levels were reduced following treatment with ASWT13VUTR. Following treatment with ASWT1exon 5, WT1 exon 5–containing transcripts were reduced to <10% of control levels, whereas those lacking exon 5 were only minimally affected (Fig. 6B). These data show that ASWT1exon 5 targets WT1 exon 5–containing transcripts selectively at early time points and also confirm postspliced exon 5–containing transcripts as the primary target. At 24 Figure 6. Efficient targeting WT1 mRNA transcripts by ASWT13VUTR hours following treatment, WT1 levels were reduced to and ASWT1exon 5. RPA analysis of total WT1 levels in K562 cells 5 (A) 26% and 22% of control levels by ASWT13VUTR and and 24 (C) hours following treatment with PBS, ASmc-raf,ASWT13VUTR ASWT1exon 5, respectively (Fig. 6C). At this time point, (AS-3 VUTR), and ASWT1exon 5 (AS-exon 5) expressed as a percentage of PBS levels. B and D, same samples expressed as counts (PhosphorImager both WT1 + and À exon 5 transcripts were reduced by units) and plotted individually as WT1 + exon 5 transcript levels (open ASWT1exon 5. However, selective targeting of WT1 + exon columns) and WT1 À exon 5 transcript levels (closed columns). E, typical 5 transcripts was still apparent, the WT1 exon 5 ratios being gel images of WT1 fragments generated by radioactive RT-PCR analysis V (top) and RPA analysis (bottom) of ASO (10 Amol/L) – treated K562 cells. 0.7 as compared with 1.8 following ASWT13 UTR (Fig. 6D). F, radioactive RT-PCR analysis of WT1 exon 5 ratios in K562 cells 4 hours WT1ASO Activity Is BothTime and Dose Dependent following treatment with 0.5 – 10 Amol/L ASWT13VUTR (open columns) The concentration-dependent and time-dependent na- 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 in ture of WT1 transcript reduction induced by both active K562 cells over the first 4 hours following treatment with 10 Amol/L ASOs during the first 4 hours of treatment was confirmed ASWT13VUTR (open columns) and ASWT1exon 5 (closed columns) using radioactive RT-PCR, RPA, and real-time PCR. expressed as a percentage of ASmc-raf (10 Amol/L) – treated levels and Examples of typical images of WT1 splice variants determined by radioactive RT-PCR. H, RPA analysis of total WT1 transcript levels 4 hours following treatment with 0.5 – 10 Amol/L ASWT13VUTR following treatment are shown in Fig. 6E (top) using RT- (open columns) and ASWT1exon 5 (closed columns) expressed as a PCR and Fig. 6E (bottom) using RPA. Using RT-PCR, the percentage of ASmc-raf (10 Amol/L) – treated levels.

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Antisense activity was subsequently confirmed in the HL60 cells used for cell survival studies using real-time (Taqman) RT-PCR (see below). Basal WT1 transcript levels in K562 cells were shown to be f2-fold higher than in HL60 cells. Efficient Targeting of WT1 Protein Expression in Both K562 and HL60 Cells by ASWT1 exon 5 and ASWT13VUTR A reduction in WT1 isoform levels was shown at 24 hours following WT1-directed ASO treatment of both K562 and HL60 cells lines but not at 4 hours (data not shown). Although adequate separation of the WT1 + and À exon 5 isoforms was achieved using NuPAGE gels, the splitting of the WT1 signal reduced the sensitivity of the Western blots, making visual interpretation of the protein bands difficult. Therefore, for clarity, typical Western blot images are showninFig.7(insets) along with the line graphs generated from these bands using the ImageQuant software. ASWT1scram was used as control following confirmation that both WT1 isoform levels and exon 5 ratios were equivalent following PBS, ASWT1mc-raf, and ASWT1- scram treatment at 10 Amol/L concentration (data not shown). WT1 exon 5–containing isoforms were overrepre- sented in both leukemia cell lines, suggesting a correlation between alternatively spliced mRNA transcript levels and WT1 protein isoform expression. In these experiments, there was a slight reduction of nonspecific band signal in antisense-treated samples compared with control samples but no dose response effect between 5 and 10 Amol/L antisense concentrations. Concentration-dependent reduction of total WT1 isoform levels with maintenance of isoform ratios was seen in both K562 and HL60 cell lines following ASWT13VUTR treatment. Differential targeting of WT1 + exon 5 isoforms by ASWT1exon 5 was also seen in both cell lines as shown by the concentration-dependent Figure 7. Efficient targeting of WT1 protein expression in both K562 and HL60 cells by ASWT13VUTR and ASWT1exon 5. Western immuno- reduction in the WT1 exon 5 isoform ratios (see Table 1). blot analysis of WT1 protein 24 hours following ASWT13VUTR (5 and However, targeting of exon 5 isoforms was associated with 10 Amol/L) and ASWT1scram (10 Amol/L; Con) treatment of K562 (A) and HL60 (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 of Table 1. WT1 exon 5 ratios and total WT1 protein levels in K562 WT1 protein bands (inset) are shown along with the superimposed and HL60 cells following ASWT1 exon 5 and ASWT13VUTR ImageQuant line graphs generated from these bands using the ImageQuant F software. treatment (mean range of two experiments)

Treatment K562 HL60 (24 h) WT1 + exon 5 transcripts were shown to be selectively and WT1 exon % Total WT1 exon % Total linearly reduced by ASWT1exon 5 over a concentration 5 ratios WT1 5 ratios WT1 range of 0.5 to 10 Amol/L at 4 hours (Fig. 6F) and selective activity could be seen as early as 30 minutes following ASWT1scram 3.25 F 0.75 100.00 3.39 F 0.39 100.00 treatment at 10 Amol/L concentration (Fig. 6G). By contrast, (10 Amol/L) the WT1 exon 5 ratios were not reduced by treatment ASWT13VUTR 4.45 F 0.40 37.2 F 7.6 3.70 F 0.70 81.8 F 8.2 with ASWT13VUTR. Total WT1 transcripts were reduced (5 Amol/L) F F F F at 4 hours following treatment with both ASOs in a ASWT13VUTR 4.75 0.15 20.7 7.6 3.90 0.40 45.3 12.6 A concentration-dependent manner (Fig. 6H using RPA). (10 mol/L) WT1 F F Because we had clearly shown efficient targeting of WT1 AS scram 3.24 0.27 100.00 4.28 0.18 100.00 (10 Amol/L) expression in K562 cells, we used a simple nonradioactive ASWT1exon 5 2.20 F 0.27 40.2 F 16.6 2.41 F 0.12 69.6 F 5.0 A RT-PCR screen to compare the activity of 10 mol/L (5 Amol/L) ASWT1exon 5 and ASWT13VUTR in K562 and HL60 cells 24 ASWT1exon 5 1.59 F 0.11 23.7 F 12.7 1.82 F 0.03 27.5 F 3.1 hours following treatment. Equivalent activity was shown (10 Amol/L) in both cell lines using both ASOs (data not shown).

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reduced overall levels of WT1 protein as was seen at the mRNA level at 24 hours following treatment (cf. Fig. 6D). A recent report showed that WT1 exon 5 isoforms mediate specifically the transactivation of an antisense WT1 promoter in the first intron of the WT1 gene probably by interaction with an accessory protein (41). The regulatory antisense WT1 mRNA product has been shown previously to positively regulate WT1 protein levels (42), providing a potential mechanism by which selective targeting of WT1 exon 5–containing variants results in the subsequent down-regulation of WT1 protein expression. Cell Survival Studies Two publications have shown growth inhibition and induction of apoptosis using ASOs targeted to the translation initiation site of WT1 in K562 cells, MM6 myelomonocytic cells, and samples of fresh leukemic cells, whereas granulocyte-macrophage colony-forming units and HL60 cells were unaffected (31, 43). To determine whether this cell type–specific cytotoxicity was WT1ASO target sequence specific, the survival of K562 and HL60 cells following treatment with both WT1-targeted ASOs was determined using a soft agar clonogenic assay. In K562 cells (Fig. 8A), the control ASO used in the validation experiments, ASmc-raf, was nontoxic at concentrations up to 10 Amol/L, whereas the control ASO, ASWT1scram, was nontoxic at 5 Amol/L but showed slight nonspecific WT1 unrelated toxicity (f20% loss of cell survival at 10 Amol/L; data not shown). The IC50 concentrations of both WT1 ASOs were comparable at 7 and 8 Amol/L, respectively, confirming the ability of WT1-targeted ASOs to reduce cell survival in K562 cells regardless of the targeted sequence. In HL60 cells (Fig. 8B), however, ASWT13VUTR and the control ASO, ASWT1scram, did not reduce clonogenic survival, in keeping with the previous report (31). Analysis of the residual HL60 cells from the clonogenic assays using real-time PCR showed that the absence of cytotoxicity was not due to the failure of ASWT13VUTR to target WT1 mRNA in these experiments (Fig. 8C). By contrast, ASWT1exon 5 reduced cell survival to 41% of control levels at 10 Amol/L. Thus, in HL60 cells, down-regulation of WT1 expression in itself was not sufficient to reduce cell survival, but when down-regulation of WT1 was associated Figure 8. Inhibition of cell survival by ASWT13VUTR and ASWT1 exon 5 in K562 and HL60 cells. Soft agar clonogenic assays of cell survival with disruption of WT1 exon 5 ratios, significant cell kill following ASO treatment of K562 (A) and HL60 (B) cells. Control ASO was achieved. (K562 cells: ASmc-raf and HL60 cells: ASWT1scram; n); ASWT13VUTR cDNA Microarray Screen of WT1 Target Gene Expres- (E); ASWT1exon 5 (.). Points, mean of quadruplicate determinations; bars, SD. C, confirmation of efficient targeting of WT1 transcripts at sion in HL60 and K562 CellsTreated with ASWT13VUTR 24 hours posttreatment in the residual HL60 cells from the experiment in and ASWT1exon 5 B using real-time PCR analysis. cDNA microarray analysis was used to examine which, if any, of the known WT1 target genes were differentially by more than one clone. Gene expression profiles compared regulated by ASWT13VUTR and ASWT1exon 5 in the first-strand cDNA populations from cells treated with 10 leukemic cell lines. The differential cytotoxicity profiles of Amol/L ASWT13VUTR or ASWT1exon 5 and cells treated ASWT13VUTR and ASWT1exon 5 in HL60 and K562 cells with 10 Amol/L ASWT1scram as the reference. In K562 also provided an opportunity to investigate whether any of cells, no alteration in the expression of any of the putative these genes were regulated in a manner that correlated WT1 target genes was detected following treatment with with disruption of isoform ratios and also with toxicity. either active ASO (data not shown). These genes seem, Of 42 known putative WT1 target genes, 36 (listed in therefore, not to contribute to the cytotoxic activity of either Table 2) were represented in our in-house gene expression ASO in this cell line. In HL60 cells, which were resistant to array of 5,603 cDNA clones. Eleven genes were represented ASWT13VUTR treatment, again no alteration in the expression

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Table 2. Microarray analysis of the expression of putative WT1 target genes 24 hours following treatment of HL60 cells with 10 Amol/L ASWT1exon 5 or ASWT13VUTR using 10 Amol/L ASWT1scram as the reference

Symbol Gene name IMAGE clone ASWT13VUTR ASWT1exon 5 vs. ASWT1scram vs. 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 243549 1.27 F 0.10 0.96 F 0.02 (AMV) oncogene homologue 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 F VDR Vitamin D (1,25-dihydroxyvitamin D3) receptor 815816 0.89 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).

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of any of these genes by ASWT13VUTR was noted. How- ever, treatment with ASWT1exon 5, which decreased cell survival by 60%, resulted in the differential expression of MYC, ornithine decarboxylase (ODC1), and THBS1 (Table 2). The proto-oncogene MYC plays a key role in cell proliferation, differentiation, and apoptosis and is reported to be amplified in HL60 cells (44), whereas THBS1 exhibits numerous biological activities including effects on cell adhesion, migration, proliferation, and angiogenesis (45). Using real-time Taqman RT-PCR, the differential regulation of both MYC and THBS1 were confirmed in the same samples used for the microarray analysis (data not shown) and also in an independent experiment that included an additional 48-hour time point to assess the duration of altered gene expression. This latter experiment confirmed down-regulation of WT1 expression in HL60 cells by both ASOs over 48 hours (Fig. 9A). Down-regulation of MYC following ASWT1exon 5 was seen at 24 hours (Fig. 9B) but not to the degree seen in the microarray samples (Table 2). In addition, at 48 hours, MYC expression was reduced by both ASOs revealing a lack of correlation between down- regulation of MYC and cell survival. Basal levels of MYC were 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 ASWT1exon 5 but not ASWT13VUTR was confirmed at both 24 and 48 hours following treatment (Fig. 9C) and was shown to be ASWT1exon 5 dose dependent (Fig. 9D). These data implicate the THBS1 gene as a bona fide downstream target of WT1. In these studies, THBS1 was only responsive in HL60 cells when down-regulation of WT1 was accompanied by disruption of exon 5 isoform ratios. THBS1 expression levels were f10 fold lower in K562 cells than in HL60 cells and were not disrupted by WT1ASO treatment. Figure 9. Confirmation of disruption of gene expression following WT1 Identification of Novel Putative WT1-Responsive ASO treatment of HL60 and K562 cells using real-time (Taqman) RT-PCR Genes analysis. WT1 (A), MYC (B), and THBS1 (C) transcript levels in HL60 cells In addition to the known WT1 regulated genes, we 24 and 48 hours following treatment with 10 Amol/L ASWT1scram (gray columns), ASWT13VUTR (open columns), and ASWT1exon 5 (black examined the expression profiles of the remaining genes in columns), expressed as a percentage of ASWT1scram levels. D, dose- the expression array. A further 10 genes were found to be dependent increase of THBS1 in HL60 cells 24 hours following modestly up-regulated (1.6- to 2.6-fold) specifically in HL60 ASWT1exon 5 treatment (closed columns) and confirmation of lack of WT1 disruption of THBS1 following ASWT13VUTR (open columns). TM4SF9 (E) cells following AS exon 5 treatment, mirroring the (E) and GPC5 (F) transcript levels in K562 cells following WT1 ASO pattern of differential regulation displayed by THBS1 treatment as in HL60 cells above. (Table 3A). Similarly, a further five genes showed a pattern of down-regulation concordant with MYC and ODC1. Our gene profile database was therefore searched for ASW- cells and genes commonly disrupted in this cell line by both T1exon 5–induced disruption of gene expression specifi- ASOs (Table 4A and B, respectively). From this latter cally in K562 cells. Eight genes were found to be modestly category, the down-regulation of the genes encoding up-regulated in this category, whereas only two were tetraspan 5 (TM4SF9)andGPC5 by both ASOs was found to be down-regulated (Table 3B). Significantly, these confirmed using real-time PCR (Fig. 9E and F, respective- represent a different set of genes to those disrupted in ly). Both genes may potentially contribute to WT1-directed HL60 cells by ASWT1exon 5. A further search for genes, antisense activity in this cell line. Transmembrane proteins common to both cell lines and disrupted by ASWT1exon 5, of the tetraspanin superfamily are implicated in a diverse revealed seven that were commonly down-regulated but range of biological phenomena, including cell motility, none that were differentially up-regulated convincingly in metastasis, cell proliferation, and differentiation (reviewed both cell lines (Table 3C). in refs. 46, 47). TM4SF9 was not expressed in HL60 cells. This exercise was repeated to identify genes specifically GPC5 is one of the membrane-bound heparan sulfate up-regulated or down-regulated by ASWT13VUTR in K562 proteoglycan family of genes, members of which have been

Mol Cancer Ther 2004;3(11). November 2004

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

Symbol Gene name IMAGE HL60 K562 clone ASWT13VUTR ASWT1exon 5 ASWT13VUTR ASWT1exon 5 vs. ASWT1scram vs. ASWT1scram vs. ASWT1scram vs. 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 0.09 IL-1h Interleukin-1h 491763 0.63* 2.14 F 0.04 f 0.78 F 0.02 BTG2 BTG family, member 2 213136 1.04 F 0.13 2.49 F 0.62 0.99 F 0.01 0.96 F 0.02 NR4A2 Nuclear receptor subfamily 4, A, member 2 898221 0.91* 2.23 F 0.29 0.91* 1.13 F 0.17 NDRG1 N-myc downstream regulated 842863 1.28 F 0.77 2.11 F 0.04 0.97 F 0.04 1.25 F 0.03 SLC2A3 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 0.06 DTR Diphtheria toxin receptor 35828 1.09 F 0.19 1.94 F 0.12 f 1.02* TYROBP TYRO protein tyrosine kinase 148469 0.84 F 0.06 1.82 F 0.01 0.65* 0.84 F 0.11 binding protein 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 0.01 PHLDA1 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 0.03 MYC v-myc AMV oncogene homologue 417226 0.85* 0.49 F 0.02 0.87 F 0.16 0.75 F 0.01 ODC1 Ornithine decarboxylase 1 796646 0.87 F 0.07 0.58 F 0.03 1.22 F 0.11 0.82 F 0.04 ICSBP1 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 0.13 NME1 Nonmetastatic cells 1, protein(NM23A) 845363 0.92 F 0.08 0.57 F 0.07 1.20 F 0.31 0.81 F 0.12 TRAP1 Heat shock protein 75 897570 1.07 F 0.15 0.66 F 0.06 1.45 F 0.14 1.20 F 0.02 RPL19 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, 71672 0.95 F 0.12 1.07 F 0.09 0.91* 1.88 F 0.23 a polypeptide PDE8A Phosphodiesterase 8A 289972 f 1.02* 0.74 F 0.03 1.76 F 0.07 PIR Pirin 234237 0.96* 0.96 F 0.06 1.20 F 0.19 1.71 F 0.06 MAPK8 Mitogen-activated protein kinase 8 119133 f 1.16 F 0.04 1.13* 1.70 F 0.01 PPIB Peptidylprolyl isomerase B (cyclophilin B) 756600 1.03 F 0.34 1.14 F 0.05 1.08 F 0.36 1.69 F 0.07 APRT Adenine phosphoribosyltransferase 897774 0.98 F 0.32 1.08 F 0.03 1.09 F 0.21 1.66 F 0.24 NQO2 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 0.05 MPP1 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 0.03 BTK Bruton agammaglobulinemia 2014424 1.56 F 0.30 0.95 F 0.04 1.43 F 0.10 0.53* tyrosine kinase

C. HL60 and K562 Cells NSEP1 Nuclease-sensitive element binding 949932 1.17 F 0.03 0.45 F 0.05 1.49 F 0.23 0.37 F 0.06 protein 1 AHCY S-adenosylhomocysteine hydrolase 840364 0.86 F 0.07 0.50* 0.86 F 0.11 0.44 F 0.01 CDC45L CDC45 (Saccharomyces cerevisiae, 453107 0.95 F 0.24 0.51 F 0.06 1.61 F 0.33 0.41 F 0.04 homologue) like ANXA11 Annexin A11 810117 0.91* 0.55 F 0.04 1.25 F 0.22 0.48 F 0.08 APLP2 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 0.04 UMPK UMP kinase 344243 0.88* 0.64 F 0.08 1.19 F 0.24 0.56 F 0.07 CUL4A Cullin 4A 2310644 1.16 F 0.05 0.66 F 0.02 1.70 F 0.14 0.61*

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

proposed to function in cellular growth control and region (51), raising the possibility that GPC5 might be morphogenesis (48). GPC5 has been mapped to chromo- overexpressed in K562 cells. Real-time PCR analysis some 13q31-2 (49) and 13q32 (50). Interestingly, K562 cells confirmed that expression of GPC5 was in excess of 40- have been shown to harbor an amplicon spanning this fold higher in K562 cells compared with HL60 cells.

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In addition to the genes listed in Table 4B, several genes all four lines was a slowdown in S-phase transit, whereas were found to be differentially regulated by both WT1- transient G2 arrest or G2-M block at later time points was directed ASOs in both cell lines. For example, the gene seen dependent on cell line and dose of cisplatin. In the encoding adaptor-related protein complex 2, beta1 (AP2B1) present studies, no temporal correlation between induc- was up-regulated 2- and 3-fold, glucose phosphate isom- tion of WT1 in CH1-R or GCT27-R cells and the previ- erase (GPI) increased 1.6- and 3-fold, and KIAA0220 pro- ously reported cell cycle effects was apparent. Lack of G1 tein was increased f2-fold by both ASOs in HL60 and arrest is not due to a p53-null phenotype because all K562 cells, respectively. Other genes were differentially four cell lines show partial G1-S arrest, associated with regulated by ASWT13VUTR, specifically in HL60 cells or induced p53 and p21, following g-irradiation (data not commonly in both HL60 and K562 cells. Because shown). ASWT13VUTR was not cytotoxic to HL60 cells, these genes Drug-Induced Disruption of WT1exon 5 Splicing May have not been listed in this report. However, some may be Constitute a Cell Stress Response genuine WT1-responsive genes. Alternative pre-mRNA splicing is a fundamental mechanism of gene expression that can be regulated dependent on sex, development stage, or tissue, and in response to extracellular stimuli such as growth factors, hormones, and Discussion cytokines (59–61). Recent studies have identified a com- Does Drug-Induced Induction of WT1 Contribute to posite exonic splice control unit, which combines an exon Chemoresistance Mechanisms? recognition element with splice silencer elements. The As mentioned previously, expression of WT1 is dynam- splice control unit seems to govern alternative splicing in a ically regulated following induction of differentiation in a cell type–specific manner and in response to activation of variety of cell lines (27–30). However, to our knowledge, protein kinase C or Ras signaling pathways (61). The dis- this is the first report of induction of WT1 expression covery of signal-responsive splice elements provides a link following cytotoxic drug treatment of cell lines displaying a between extracellular signals and regulation of exon var- resistant phenotype, a phenomenon absent in the sensitive iant transcription. cell lines studied. These findings support the notion that In these studies, we have provided the first evidence that increased WT1 expression may contribute to chemoresist- the alternative splicing of exon 5 of WT1 mRNA is subject ance mechanisms, allowing cells to survive following drug to dynamic regulation in response to drug treatment, therapy. However, at this stage, we can only speculate on whereas KTS splicing is not disrupted. Much interest has the mechanism(s) of induction and the downstream effects focused recently on the activation of stress-activated pro- of induced WT1 expression. tein kinase/c-Jun-NH2-kinase and p38 mitogen-activated Little information exists regarding the regulatory factors protein kinase cascades following cisplatin treatment controlling WT1 expression. Several ubiquitous and tissue- (62, 63). Whether WT1 possesses signal-responsive ele- specific transcription factors have been shown to activate ments that respond to activation of these or other protein or repress the WT1 promoter in transient transfection kinases is unknown. Nevertheless, in the cell lines studied, assays including Sp1, WT1, GATA-1, Pax-2, Pax-8, and the disruption of WT1 exon 5 splicing seemed to be an nuclear factor-nB (NF-nB; refs. 52–56). Regulation by early stress response to a toxic stimulus independent of NF-nB may be relevant here, because treatment of cells the tissue of origin of the cell lines, the relative chemo- with DNA-damaging agents or various cytokines and sensitivity, or the drug used. We propose that increased mitogens can result in its translocation from the cyto- exon 5 skipping is the most likely mechanism. plasm to the nucleus and the modulation of the appro- Does Disruption of WT1exon 5 Ratios Provide a Pro- priate target genes. Ectopic expression of NF-nB has been apoptotic or Cell Survival Signal? shown to increase the transcription of endogenous WT1, Inthesestudies,wehaveattemptedtomimicthe indicating that members of the NF-nB/Rel family may disruption of WT1 exon 5 splicing seen following drug be involved in a regulatory cascade leading to WT1 treatment using an ASO targeted specifically to exon 5– activation (57). Further studies are clearly required to containing WT1 transcripts. ASWT1exon 5 induced both determine the mechanisms responsible for, and the signif- disruption of exon 5 splicing and down-regulation of WT1 icance of, the differential regulation of WT1 expression in levels. The latter effect may be due to down-regulation of sensitive and resistant cell lines following cytotoxic drug the exon 5 transactivated regulatory antisense WT1 mRNA treatment. product, which has been shown previously to positively Because G1 arrest associated with p53-independent regulate WT1 protein levels (42). Cell survival studies regulation of endogenous p21CIP1 by WT1 has been shown, showed loss of cell viability in both K562 and HL60 cell it is tempting to hypothesize that induction of WT1 con- lines by ASWT1exon 5, despite HL60 cells being resistant to tributes to chemoresistance by inducing cell cycle arrest, the effects of a balanced down-regulation of WT1 isoforms. thereby allowing DNA repair and avoidance of replica- These data support the view that the drug-induced tion using a damaged template. However, in previous disruption of exon 5 splicing constitutes a proapoptotic studies using both paired cell lines, G1 arrest was not signal. It may be that the up-regulation of WT1expression observed following cisplatin treatment (38, 58). Common to shown here in resistant cell lines following drug treatment

Mol Cancer Ther 2004;3(11). November 2004

Table 4. Microarray expression profiling of genes disrupted specifically in K562 cells by ASWT13VUTR (A) and ASWT13VUTR and ASWT1exon 5 (B)

Symbol Gene name IMAGE HL60 K562 clone ASWT13VUTR ASWT1exon 5 ASWT13VUTR ASWT1exon 5 vs. ASWT1scram vs. ASWT1scram vs. ASWT1scram vs. ASWT1scram

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 269292 f 1.12* 2.03 F 0.52 1.11 F 0.05 region OY11.1 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, 770901 0.75 F 0.05 0.97 F 0.04 0.45* 0.85 F 0.12 nonreceptor type 9 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) 839888 1.26 F 0.01 1.01 F 0.05 0.48* 1.00 F 0.05 intracellular binding protein 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 897006 0.93 F 0.15 1.16* 0.57 F 0.09 1.13 F 0.06 reg kinase 1A 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, 950689 1.24 F 0.03 1.14 F 0.18 1.82 F 0.10 1.66 F 0.10 RP/EB family 2 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*

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

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temporarily overrides this proapoptotic signal, providing Identification of Previously Unreported WT1-Respon- the cells with a greater opportunity for damage repair and sive Genes escape from apoptosis. Interference with proapoptotic Of the remaining genes on our expression array, f2% signaling following cell damage can be expected to provide were significantly altered (directly or indirectly) by WT1- a survival advantage, although additional resistance directed ASO treatment, generally in a cell type–specific mechanisms are likely to contribute to the resistant and WT1 antisense target-specific manner. Our initial aim phenotype of these cell lines. was to search for evidence of differential regulation of Differential Regulation of Putative WT1 Target Genes downstream genes following disruption of WT1 exon 5 following Disruption of WT1exon 5 Ratios ratios; clearly, this occurs in both HL60 and K562 cells. Some 42 putative WT1 target genes have been identified However, of the 31 genes affected by ASWT1exon 5 mainly as a result of transient transfection studies with treatment and listed in Table 3, only 7 were common to reporter constructs (for a recent list, see ref. 2). However, both cell lines, highlighting yet again the cell type with a few exceptions such as p21CIP1 and EGFR (13, 23), specificity of WT1 activity. Overall, the changes in little convincing evidence of WT1-directed regulation of the expression were modest at 24 hours. It is possible that endogenous expression of these putative target genes has more extensive alterations in expression may be evident at been forthcoming. Cell context specificity further compli- later time points, or differential regulation may be lost, as in cates the search for bona fide WT1 target genes, because the case of MYC. interactions between WT1 and coregulatory proteins may These studies fall short of confirming which, if any, of influence both the transcriptional regulatory properties these genes contribute to the cytotoxic activity of ASW- of WT1 and target recognition. Consequently, there are T1exon 5 but can be used as the basis for further studies. few clues as to how the various isoforms of WT1 interact Worthy of comment is the up-regulation of interleukin-1h intracellularly to exert their biological effects or indeed (IL-1h) in HL60 cells. IL-1h, like tumor necrosis factor, how disruption of WT1 isoform ratios might induce altered initiates the activation of signal cascades by the recruitment biological function. of adapter proteins to its receptor. In many cancer cell lines, In this study, we have combined the use of antisense both IL-1h and tumor necrosis factor induce apoptosis, and cDNA microarray technologies in an attempt to whereas untransformed cell types are not sensitive unless identify which WT1-responsive genes are differentially mRNA translation or protein synthesis is blocked (65). regulated following disruption of WT1 isoform ratios in The differential regulation of the genes listed in Table 4A a manner correlating with inhibition of cell survival. by ASWT13VUTR, specifically in K562 cells, shows that a As found previously, few changes in the expression levels different set of genes respond to the balanced down- of the identified WT1 putative target genes were noted regulation of WT1 isoforms as compared with when down- and none were seen in K562 cells. Differential regulation regulation of WT1 is accompanied by disruption of exon 5 of MYC and its reported downstream target ODC1 was ratios (listed in Table 3B). It is possible that networks of observed at 24 hours following ASWT1exon 5 in HL60 genes and gene cascades may be controlled not only by the cells, but subsequent and equivalent down-regulation of regulation of the overall levels of WT1 but also by MYC by ASWT13VUTR at 48 hours revealed a lack of cor- regulation of exon 5 ratios. Interaction with the various relation between MYC expression and cytotoxicity. stress/apoptotic/differentiation/growth factor signaling Unlike MYC expression, the 5-fold up-regulation pathways is a possibility that requires further investigation. of THBS1 seemed to correlate with the cytotoxic activity Finally, there are some genes that respond to down- of ASWT1exon 5 in HL60 cells, although the mechanism of regulation of WT1 regardless of whether isoform ratios are such activity in vitro is not obvious. Thrombospondin is a disrupted. One of these, GPC5, may represent a bona fide 450-kDa extracellular matrix–bound trimeric glycoprotein WT1 target gene as predicted by the presence of two WT1 that is expressed and secreted by platelets and a wide consensus binding sequences in its promoter region. GPC5 variety of cell types. It has been suggested to play an constitutes one of the membrane-bound heparan sulfate important albeit complex role in controlling cancer cell proteoglycan family of genes, members of which have been growth and metastasis in vivo and variously implicated in proposed to function in cellular growth control and cancer cell adhesion, migration, invasion, proliferation, and morphogenesis (48). Overexpressed in K562 cells, GPC5 apoptosis-dependent inhibition of angiogenesis (reviewed was of particular interest because it is expressed in cells of in ref. 45). Endogenous repression of THSB1 by WT1 has mesenchymal origin during development and shows a been shown previously in response to overexpression of similar pattern of expression to WT1 in the developing c-Jun (64). Repression involved a factor secreted by c-Jun- kidney and gonads (49, 66, 67). In addition, GPC5 has been activated cells, which triggered a signal transduction suggested to be a candidate gene for at least some of the pathway culminating in the binding of WT1 to the THBS1 phenotypic features of 13q syndrome, a developmental promoter. By contrast, our present studies suggest a clear disorder with a pattern of defects that shows overlap with correlation between disruption of WT1 exon 5 ratios and both WAGR and DDS and also with WT1 knockout mice endogenous regulation of THBS1, a novel observation. (49, 68, 69). Whether this activity is direct or involves a coregulatory In summary, the results of this study support a role for protein remains to be determined. WT1 in the maintenance of viability and proliferative

Mol Cancer Ther 2004;3(11). November 2004

capacity in cancer cells and as a mediator of survival 15. Kudoh T, Ishidate T, Moriyama M, Toyoshima K, Akiyama T. G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK signals following cytotoxic drug treatment. Downstream complexes. Proc Natl Acad Sci U S A 1995;92:4517 – 21. signaling seems to involve the orchestrated regulation of 16. Mayo MW, Wang CY, Drouin SS, et al. WT1 modulates apoptosis by WT1 exon 5 splicing and total WT1 expression. Using ASOs transcriptionally upregulating the Bcl-2 proto-oncogene. EMBO J directed to both exon 5 and the 3V UTR of WT1, we have 1999;18:3990 – 4003. 17. Baudry D, Faussillon M, Cabanis MO, et al. Changes in WT1 splicing are shown cell type–specific and antisense target-specific associated with a specific gene expression profile in Wilms’ tumor. regulation of genes, some of which may prove to be novel Oncogene 2002;21:5566 – 73. WT1 target genes. Disruption of WT1 exon 5 ratios by 18. Bergmann L, Miething C, Maurer U, et al. High levels of Wilms’ tumor ASWT1exon 5 was shown to reduce the cell survival of gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood 1997;90:1217 – 25. HL60 cells that are resistant to other WT1-targeted ASOs. 19. Inoue K, Sugiyama H, Ogawa H, et al. WT1 as a new prognostic factor ASWT1exon 5 may therefore have a broader therapeutic and a new marker for the detection of minimal residual disease in acute potential than previously described WT1 ASOs. In prelim- leukemia. Blood 1994;84:3071 – 9. inary studies, we have shown significant antitumor activity 20. King-Underwood L, Renshaw J, Pritchard-Jones K. Mutations in the against K562 cells grown in vivo using both WT1-directed Wilms’ tumor gene WT1 in leukemias. Blood 1996;87:2171 – 9. ASOs (70). We are extending these studies to explore the 21. Trka J, Kalinova M, Hrusak O, et al. Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic therapeutic potential of these compounds in other WT1- significance, correlation with disease status and residual disease detection expressing tumors such as prostate cancer, breast cancer, by flow cytometry. Leukemia 2002;16:1381 – 9. and childhood embryonal cancers. 22. Miyoshi Y, Ando A, Egawa C, et al. High expression of Wilms’ tumor suppressor gene predicts poor prognosis in breast cancer patients. Clin Cancer Res 2002;8:1167 – 71. 23. Carrington D, Algar E. Overexpression of murine WT1 +/+ and À/À isoforms has no effect on chemoresistance but delays differentiation in the Acknowledgments K562 leukemia cell line. Leuk Res 2000;24:927 – 36. The authors thank Dr. Dan Williamson (Section of Molecular Carcinogen- 24. Englert C, Hou X, Maheswaran S, et al. WT1 suppresses synthesis of esis, ICR, Sutton, UK) for the design of the GPC5 primers and probe and the epidermal growth factor receptor and induces apoptosis. EMBO J Kathryn R. Taylor for excellent technical assistance. 1995;14:4662 – 75. 25. Richard DJ, Schumacher V, Royer-Pokora B, Roberts SG. Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1. Genes Dev 2001;15:328 – 39. References 26. Johnstone RW, See RH, Sells SF, et al. A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms’ 1. Reddy JC, Licht JD. The WT1 Wilms’ tumor suppressor gene: how much tumor suppressor WT1. Mol Cell Biol 1996;16:6945 – 56. do we really know? Biochim Biophys Acta 1996;1287:1 – 28. 27. Phelan SA, Lindberg C, Call KM. Wilms’ tumor gene, WT1, mRNA is 2. Scharnhorst V, van der Eb AJ, Jochemsen AG. WT1 proteins: functions down-regulated during induction of erythroid and megakaryocytic differen- in growth and differentiation. Gene 2001;273:141 – 61. tiation of K562 cells. Cell Growth Differ 1994;5:677 – 86. 3. Bruening W, Pelletier J. A non-AUG translational initiation event 28. Sekiya M, Adachi M, Hinoda Y, Imai K, Yachi A. Downregulation of generates novel WT1 isoforms. J Biol Chem 1996;271:8646 – 54. Wilms’ tumor gene (wt1) during myelomonocytic differentiation in HL60 4. Scharnhorst V, Dekker P, van der Eb AJ, Jochemsen AG. Internal cells. Blood 1994;83:1876 – 82. translation initiation generates novel WT1 protein isoforms with distinct 29. Kudoh T, Ishidate T, Nakamura T, Akiyama T. Constitutive expression biological properties. J Biol Chem 1999;274:23456 – 62. of the Wilms tumor suppressor gene WT1 in F9 embryonal carcinoma cells 5. Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE. induces apototic cell death in response to retinoic acid. Oncogene Alternative splicing and genomic structure of the Wilms tumor gene WT1. 1996;13:1431 – 9. Proc Natl Acad Sci U S A 1991;88:9618 – 22. 30. Scharnhorst V, Kranenburg O, van der Eb AJ, Jochemsen AG. 6. Kent J, Coriat AM, Sharpe PT, Hastie ND, van Heyningen V. The Differential regulation of the Wilms’ tumor gene, WT1, during differentiation evolution of WT1 sequence and expression pattern in the vertebrates. of embryonal carcinoma and embryonic stem cells. Cell Growth Differ Oncogene 1995;11:1781 – 92. 1997;8:133 – 43. 7. Larsson SH, Charlieu JP, Miyagawa K, et al. Subnuclear localization of 31. Algar EM, Khromykh T, Smith SI, Blackburn DM, Bryson GJ, Smith PJ. WT1 in splicing or transcription factor domains is regulated by alternative A WT1 antisense oligonucleotide inhibits proliferation and induces splicing. Cell 1995;81:391 – 401. apoptosis in myeloid leukaemia cell lines. Oncogene 1996;12:1005 – 14. 8. Hammes A, Guo JK, Lutsch G, et al. Two splice variants of the Wilms’ 32. Baker BF, Lot SS, Condon TP, et al. 2V-O-(2-methoxy)ethyl-modified tumor 1 gene have distinct functions during sex determination and nephron anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively formation. Cell 2001;106:319 – 29. increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 9. Hastie ND. Life, sex, and WT1 isoforms—three amino acids can make all translation initiation complex in human umbilical vein endothelial cells. J Biol the difference. Cell 2001;106:391 – 4. Chem 1997;272:11994 – 2000. 10. Renshaw J, King-Underwood L, Pritchard-Jones K. Differential splicing 33. Alder S, Ciampi A, McCulloch EA. A kinetic and clonal analysis of of exon 5 of the Wilms tumor (WTI) gene. Genes Chromosomes Cancer heterogeneity in K562 cells. J Cell Physiol 1984;118:186 – 92. 1997;19:256 – 66. 34. Hills CA, Kelland LR, Abel G, Siracky J, Wilson AP, Harrap KR. 11. Iben S, Royer-Pokora B. Analysis of native WT1 protein from frozen Biological properties of ten human ovarian carcinoma cell lines: calibration human kidney and Wilms’ tumors. Oncogene 1999;18:2533 – 6. in vitro against four platinum complexes. Br J Cancer 1989;59:527 – 34. 12. Natoli TA, McDonald A, Alberta JA, Taglienti ME, Housman DE, 35. Kelland LR, Mistry P, Abel G, et al. Establishment and characterization Kreidberg JA. A mammal-specific exon of WT1 is not required for of an in vitro model of acquired resistance to cisplatin in a human testicular development or fertility. Mol Cell Biol 2002;22:4433 – 8. nonseminomatous germ cell line. Cancer Res 1992;52:1710 – 6. 13. Englert C, Maheswaran S, Garvin AJ, Kreidberg J, Haber DA. Induction 36. Lee YF, John M, Edwards S, et al. Molecular classification of synovial of p21 by the Wilms’ tumor suppressor gene WT1. Cancer Res 1997; sarcomas, leiomyosarcomas and malignant fibrous histiocytomas by gene 57:1429 – 34. expression profiling. Br J Cancer 2003;88:510 – 5. 14. Hewitt SM, Saunders GF. Differentially spliced exon 5 of the Wilms’ 37. Clark J, Edwards S, John M, et al. Identification of amplified and tumor gene WT1 modifies gene function. Anticancer Res 1996;16:621 – 6. expressed genes in breast cancer by comparative hybridization onto

Mol Cancer Ther 2004;3(11). November 2004

microarrays of randomly selected cDNA clones. Genes Chromosomes The Wilms’ tumor suppressor gene WT1 is negatively autoregulated. J Biol Cancer 2002;34:104 – 14. Chem 1994;269:6198 – 206. 38. Ormerod MG, O’Neill C, Robertson D, Kelland LR, Harrap KR. cis- 54. Wu Y, Fraizer GC, Saunders GF. GATA-1 transactivates the WT1 Diamminedichloroplatinum(II)-induced cell death through apoptosis in hematopoietic specific enhancer. J Biol Chem 1995;270:5944 – 9. sensitive and resistant human ovarian carcinoma cell lines. Cancer Chemo- 55. Dehbi M, Pelletier J. PAX8-mediated activation of the wt1 tumor ther Pharmacol 1996;37:463 – 71. suppressor gene. EMBO J 1996;15:4297 – 306. 39. Huddart RA, Titley J, Robertson D, Williams GT, Horwich A, Cooper 56. Dehbi M, Ghahremani M, Lechner M, Dressler G, Pelletier J. The paired- CS. Programmed cell death in response to chemotherapeutic agents in box transcription factor, PAX2, positively modulates expression of the human germ cell tumor lines. Eur J Cancer 1995;5:739 – 46. Wilms’ tumor suppressor gene (WT1). Oncogene 1996;13:447 – 53. 40. Benito A, Grillot D, Nunez G, Fernandez-Luna JL. Regulation and 57. Dehbi M, Hiscott J, Pelletier J. Activation of the wt1 Wilms’ tumor function of Bcl-2 during differentiation-induced cell death in HL-60 suppressor gene by NF-B. Oncogene 1998;16:2033 – 9. promyelocytic cells. Am J Pathol 1995;146:481 – 90. 58. Walton MI, Koshy, P, Kelland LR. Characterization of a human germ cell 41. Moorwood K, Salpekar A, Ivins SM, et al. Transactivation of the WT1 tumor GCT27 and its cisplatinum resistant counterpart—p53 status and antisense promoter is unique to the WT1[+/À] isoform. FEBS Lett 1999; mechanism of cell death. Proc AACR 1995;316. 456:131 – 6. 42. Moorwood K, Charles AK, Salpekar A, Wallace JI, Brown KW, Malik K. 59. Lopez AJ. Developmental role of transcription factor isoforms Antisense WT1 transcription parallels sense mRNA and protein expression generated by alternative splicing. Dev Biol 1995;172:396 – 411. in fetal kidney and can elevate protein levels in vitro. J Pathol 1998;185: 60. Lopez AJ. Alternative splicing of pre-mRNA: developmental conse- 352 – 9. quences and mechanisms of regulation. Annu Rev Genet 1998;32:279 – 305. 43. Yamagami T, Sugiyama H, Inoue K, et al. Growth inhibition of human 61. Konig H, Ponta H, Herrlich P. Coupling of signal transduction to leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleo- alternative pre-mRNA splicing by a composite splice regulator. EMBO J tides: implications for the involvement of WT1 in leukemogenesis. Blood 1998;17:2904 – 13. 1996;87:2878 – 84. 62. Sanchez-Perez I, Martinez-Gomariz M, Williams D, Keyse SM, Perona 44. Collins SJ. The HL-60 promyelocytic leukemia cell line: proliferation, R. CL100/MKP-1 modulates JNK activation and apoptosis in response to differentiation, and cellular oncogene expression. Blood 1987;70:1233–44. cisplatin. Oncogene 2000;19:5142 – 52. 45. Sargiannidou I, Zhou J, Tuszynski GP. The role of thrombospondin-1 63. Kyriakis JM, Banerjee P, Nikolakaki E, et al. The stress-activated in tumor progression. Exp Biol Med (Maywood) 2001;226:726 – 33. protein kinase subfamily of c-Jun kinases. Nature 1994;369:156 – 60. 46. Maecker HT, Todd SC, Levy S. The tetraspanin superfamily: molecular 64. Dejong V, Degeorges A, Filleur S, et al. The Wilms’ tumor gene product facilitators. FASEB J 1997;11:428 – 42. represses the transcription of thrombospondin 1 in response to over- 47. Berditchevski F. Complexes of tetraspanins with integrins: more than expression of c-Jun. Oncogene 1999;18:3143 – 51. meets the eye. J Cell Sci 2001;114:4143 – 51. 65. Madge LA, Li JH, Choi J, Pober JS. Inhibition of phosphatidylinositol 3- 48. Filmus J, Selleck SB. Glypicans: proteoglycans with a surprise. J Clin kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin- Invest 2001;108:497 – 501. dependent apoptosis. J Biol Chem 2003;278:21295 – 306. 49. Saunders S, Paine-Saunders S, Lander AD. Expression of the cell 66. Pritchard-Jones K, Fleming S, Davidson D, et al. The candidate Wilms’ tu- surface proteoglycan glypican-5 is developmentally regulated in kidney, mor gene is involved in genitourinary development. Nature 1990; 346:194 – 7. limb, and brain. Dev Biol 1997;190:78 – 93. 67. Rackley RR, Flenniken AM, Kuriyan NP, Kessler PM, Stoler MH, 50. Veugelers M, Vermeesch J, Reekmans G, Steinfeld R, Marynen P, Williams BR. Expression of the Wilms’ tumor suppressor gene WT1 during David G. Characterization of glypican-5 and chromosomal localization of mouse embryogenesis. Cell Growth Differ 1993;4:1023 – 31. human GPC5, a new member of the glypican gene family. Genomics 68. Kreidberg JA, Sariola H, Loring JM, et al. WT-1 is required for early 1997;40:24 – 30. kidney development. Cell 1993;74:679 – 91. 51. Rodley P, McDonald M, Price B, Fright R, Morris C. Comparative 69. Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A. YAC genomic hybridization reveals previously undescribed amplifications and complementation shows a requirement for Wt1 in the development of deletions in the chronic myeloid leukemia-derived K-562 cell line. Genes epicardium, adrenal gland and throughout nephrogenesis. Development Chromosomes Cancer 1997;19:36 – 42. 1999;126:1845 – 57. 52. Cohen HT, Bossone SA, Zhu G, McDonald GA, Sukhatme VP. Sp1 is a 70. Orr RM, Renshaw J, Valenti MR, et al. In vivo studies of antisense critical regulator of the Wilms’ tumor-1 gene. J Biol Chem 1997;272: 2901 – 13. oligonucleotides targeted to the mRNA of Wilms’ tumor gene in tumor- 53. Rupprecht HD, Drummond IA, Madden SL, Rauscher Fr, Sukhatme VP. bearing mice. Proc AACR 2001;42:849.

Mol Cancer Ther 2004;3(11). November 2004

Downloaded from mct.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. Disruption of WT1 gene expression and exon 5 splicing following cytotoxic drug treatment: Antisense down-regulation of exon 5 alters target gene expression and inhibits cell survival

Jane Renshaw, Rosanne M. Orr, Michael I. Walton, et al.

Mol Cancer Ther 2004;3:1467-1484.

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