Hormone-Dependent Tumor Regression in Vivo by an Inducible Transcriptional Repressor Directed at the PAX3-FKHR Oncogene1

[CANCER RESEARCH 60, 5803–5814, October 15, 2000] Hormone-dependent Tumor Regression in Vivo by an Inducible Transcriptional Repressor Directed at the PAX3-FKHR Oncogene1

Kasirajan Ayyanathan, William J. Fredericks, Carola Berking, Meenhard Herlyn, Christopher Balakrishnan, Edward Gunther, and Frank J. Rauscher, III 2 The Wistar Institute, Philadelphia, Pennsylvania 19104

ABSTRACT domain (5). These studies suggest that these PAX proteins have sustained a gain of function that leads to ARMS tumorigenesis (2). In alveolar rhabdomyosarcomas (ARMSs), a specific chromosomal However, the PAX3-FKHR-activated target genes responsible for translocation creates a fusion transcription factor, PAX3-FKHR, that is ARMS have not been defined. oncogenic due to transcriptional activation. As a strategy for down- regulation of PAX3-FKHR target genes, we created conditional PAX3 The PAX gene family consists of nine members that are unified by repressors by fusing the PAX3 DNA-binding motifs to the hormone the presence of the paired box DNA-binding domain and are subclas- binding domain (HBD) of the estrogen receptor and to the KRAB repres- sified based on their genomic organization. PAX proteins play regu- sion domain. We validated proper expression, specific DNA binding, latory roles in pattern formation during organogenesis (6). Ectopic corepressor interaction, and nuclear localization for the KRAB-PAX3- expression of several PAX genes in NIH/3T3 cells induces cellular HBD protein and showed it to be a 4-hydroxytamoxifen-dependent tran- transformation and tumor formation in nude mice, suggesting that scriptional repressor of transiently transfected and integrated PAX3 re- deregulated expression of PAX proteins could play a role in human porters in ARMS cells. We established ARMS cell lines that exhibited tumorigenesis (6, 7). Furthermore, suppression of apoptosis by PAX stable expression of the conditional PAX3 repressor proteins and used proteins is crucial for their complex developmental role and could them to down-regulate the malignant growth under low serum or anchorage-independent conditions in a hormone-dependent manner. Terminal account for their tumorigenic potential (8, 9). In accordance with this, deoxynucleotidyl transferase-mediated nick end labeling assays revealed antisense inhibition of PAX genes results in growth arrest and apo- that hormonal activation of the PAX3 repressors induced extensive apoptosis in tumor cell lines (10–12). Although evidence supports a role ptosis that correlated with down-regulation of BCL-XL expression. SCID for PAX3 in protection of cells from apoptosis during development, mice that were engrafted with the KRAB-PAX3-HBD ARMS cell lines the mechanism has not been determined (11). and were implanted with 4-hydroxytamoxifen timed-release pellets exhib- The developmental abnormalities and alterations in gene expression ited suppression of tumor growth and an altered vascularity that was not observed in the splotch mouse model, which contains mutations in the observed in the control mice. These observations strongly suggest that we PAX3 DNA-binding domains, has suggested several downstream have directly repressed the PAX3 target genes that are deregulated by the target genes regulated by PAX3 (13, 14). Candidate targets include mi PAX3-FKHR oncogene in ARMS. (15), myoD and myogenin (16), pax7 (11) and others. Similarly, transfection of the PAX3-FKHR fusion protein present in ARMS into INTRODUCTION heterologous cells has been shown to up-regulate the expression of pdgfr-␣ and c-met, the receptor for hepatocyte scatter factor (17). Chromosomal translocations that result in the creation of chimeric Whether any of these candidate genes play a role in ARMS tumori- transcription factors that deregulate specific target genes are a type of genesis remains to be clarified. genetic alteration frequently associated with oncogenesis (1). Differ- It is generally hypothesized that the enhanced transcriptional acti- ent modes of deregulation include gain or loss of transcriptional vation potential of PAX3-FKHR is responsible for ARMS. We have 3 activation or repression function. The model system, ARMS , chosen previously engineered synthetic PAX3 repressors using the KRAB in this study provides a clear example for this phenomenon for human repression domain and demonstrated that expression of a PAX3- pediatric solid tumors (2). KRAB repressor in the ARMS Rh30 cell line could inhibit malignant ARMSs occur due to a highly specific chromosomal translocation growth (18). The KRAB domain functions as a potent DNA binding- event [t(2;13) (q35;q14)] that juxtaposes the DNA-binding domains of dependent transcriptional repression module by recruiting the KAP-1 PAX3 with the transcriptional activation domain of FKHR (3). In corepressor (19, 20). Other repression domains such as the SNAG transfection assays, PAX3-FKHR, the causative oncogene in ARMS, domain from the GFI-1 proto-oncogene (21) and the WT-1 repression functions as a more potent activator of transcription than the wild-type domain derived from the Wilms’ tumor gene (22) do not use the PAX3 (4). Less frequently, another translocation [t(1;13) (p36;q14)] KAP-1 corepressor mechanism. The KRAB and the SNAG domains fuses the PAX7 DNA-binding domain to the same FKHR activation are well suited for the creation of engineered repressors due to their small size and strong repression potentials when fused to heterologous Received 5/5/00; accepted 8/17/00. DNA-binding domains. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with We were interested in developing conditional PAX3 repressors to 18 U.S.C. Section 1734 solely to indicate this fact. examine the immediate consequences of repressing PAX3 target genes 1 K. A. was supported by the American Cancer Society NP-954 Research Training Grant. W. J. F. was supported by Wistar Basic Cancer Research Training Grant CA09171. in ARMS cell clones. The use of conditional repressors avoids sec- F. J. R. is supported in part by NIH Grants CA52009, Core Grant CA10815, DK49210, ondary changes in cells that might be selected by constitutive expres- GM54220, DAMD17-96-1-6141, ACS NP-954, the Irving A. Hansen Memorial Founda- sion of transcriptional repressors. Several conditional eukaryotic ex- tion, the Mary A. Rumsey Memorial Foundation, and the Pew Scholars Program in the Biomedical Sciences. pression systems based on either inducible transcription or conditional 2 To whom requests for reprints should be addressed, at The Wistar Institute, 3601 activity due to fusion to the HBD of steroid receptors have been Spruce Street, Philadelphia, PA 19104. Phone: (215) 898-0995; Fax: (215) 898-3929; developed (23). Of these, the HBD fusion confers rapid temporal E-mail: [email protected]. 3 The abbreviations used are: ARMS, alveolar rhabdomyosarcoma; 4-OHT, 4- regulation to the functionality of heterologous proteins. Furthermore, hydroxytamoxifen; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse specific mutations in the HBDs make these receptors very selective to transcription-PCR; DD-PCR, differential display RT-PCR; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; HBD, hormone-binding domain; FBS, fetal synthetic ligands without being influenced by endogenous hormones bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. (24). HBD fusions with transcription factors including PAX5 (25–27) 5803

Fig. 1. A, chimeric repressor plasmids. The KRAB-PAX3-HBD plasmid combines the KRAB repression domain with the ERTM HBD of the estrogen receptor by fusion to the PAX3 paired box (PB) and homeodomain (HD) DNA-binding motifs. The KRAB(DV)-PAX3-HBD plasmid contains a mutant KRAB domain, which lacks repression potential. The SNAG-PAX3-HBD plasmid harbors the SNAG domain; a repression module derived from the GFI-1 gene. B, the CD19–2(A-ins)-TK-LUC-ZeoR reporter plasmid is described in “Materials and Methods.” C, the pcDNA3-KRAB-MNP-HBD plasmid is a novel expression vector for the construction of conditional transcriptional repressor fusion proteins using the KRAB repression domain and the ERTM HBD. The resulting conditional repressors would be myc epitope-tagged (M) and nuclear-localized (N) and would repress a set of endogenous target genes specified by the DNA-binding domain of choice fused in-frame into the polylinker (P). and enzymes such as STAT6 (28) have been successfully made to growth as tumors in SCID mice, were studied using ARMS Rh30 cell generate hormone-dependent conditional alleles. clones expressing the KRAB-PAX3-HBD repressors. The results of In this study, we generated hormone-inducible, conditional alleles these experiments suggest that we have successfully used the induc- of a KRAB-PAX3 protein by fusing it to the HBD of the murine ible repressor strategy to down-regulate the set of PAX3 target genes estrogen receptor ERTM, which exhibits selectivity to 4-OHT (24). that are activated by the PAX3-FKHR oncoprotein. Furthermore, we Hormone-dependent changes in biological properties such as growth have used the conditional PAX3 repressors in ARMS cells to explore in low-serum medium, apoptosis, anchorage-independent growth, and whether the cellular survival factor BCL-XL might be a PAX3 target 5804

Fig. 2. A, characterization of KRAB-PAX3 and KRAB-PAX3-HBD proteins expressed in COS-1 cells that were transiently transfected with the pcDNA3, pcKRAB-PAX3, and pcKRAB-PAX3- HBD expression plasmids and metabolically labeled with 35S-methionine. Whole cell lysates prepared in radioimmunoprecipitation assay buffer were immunoprecipitated using preimmune-, or ␣-PAX3-, and ␣-KRAB-IgGs and analyzed by SDS-PAGE and fluorography. KRAB-PAX3 (Mr 55,000) and KRAB-PAX3-HBD (Mr 88,000) proteins are indicated by the half-arrows. B, DNA- binding properties of KRAB-PAX3 and KRAB- PAX3-HBD proteins. Recombinant KRAB-PAX3 and KRAB-PAX3-HBD proteins were synthesized in vitro and used in gel shift assays using 32P- labeled e5 probe. For KRAB: PAX3: KAP-1 complex analysis, the binding reactions contained 1 ␮l (ϳ3 ␮g) of COS-1 nuclear extract as a source of the KAP-1 corepressor. Binary complexes (KRAB- PAX3:e5DNA and KRAB-PAX3-HBD:e5DNA) and ternary complexes (KRAB-PAX3: KAP-1: e5DNA and KRAB-PAX3-HBD:KAP-1:e5DNA) are indicated on the autoradiogram. FP, free probe. C, expression of the KRAB-PAX3-HBD transcript in Rh30-KPHBD-cl22 cells. Northern blot analysis of 20 ␮g of total RNA from un-induced and 4-OHT-induced Rh30-KPHBD-cl22 cells. The po- sitions of the 28S and 18S rRNA species are indicated (top). The ethidium bromide-stained gel is shown to control for loading (bottom).

gene and be involved in ARMS tumorigenesis. These studies repre- derived as HindIII and BamHI fragments encoding amino acids 1–90 of the sent a first step in the identification of important oncogenic targets in KOX1 cDNA. The 5Ј oligonucleotide primer incorporated a HindIII site and a ARMS by creation of a biological system well suited for analysis Kozak consensus immediately before the KOX-1 initiator methionine using differential gene expression array technologies. (5Ј primer, 5Ј-TTTTAAGCTTCCACCATGGATGCTAAGTCAC-3Ј). The 3Ј oligonucleotide primer incorporated a BamHI site after amino acid 90 of KOX-1 (3Ј primer, 5Ј-TTTTGGATCCAGTCTCTGAATCAGGATG-3Ј). The MATERIALS AND METHODS resulting pcDNA3-KRAB-PAX3-STOP plasmid contains the KRAB domain, Cell Lines. The NIH/3T3 cell line was maintained in DMEM supple- followed by a small linker encoding amino acids GSGVP, followed by amino acids 11–381 of the PAX3 DNA-binding domain. After amino acid 381, the mented with 10% calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin at 37°C in 5% CO under sterile conditions. The COS-1 PAX3-STOP protein is terminated by a vector-derived stop codon (18). The 2 TM cells were grown in Iscove’s modified Dulbecco’s medium containing 10% pcKRAB-PAX3-HBD plasmid was constructed by fusing the HBD, ER of TM FBS and other components, as above. The parental Rh30 cell line and its clonal the murine estrogen receptor, to pcKRAB-PAX3-STOP. The ER DNA was ϩ TM derivatives were maintained in RPMI 1640 containing 10% calf serum sup- generated by PCR amplification from the pBS ER plasmid (24) template plemented as above. using a pair of oligonucleotide primers designed to incorporate flanking EcoRI Construction of Expression Plasmids. The previously described sites (5Ј primer, 5Ј-GCATGAATTCTATGGGTGCTTCAGGAG-3Ј;3Ј T3 pcDNA3-PAX3-STOP plasmid, which expresses a hybrid mouse-human promoter primer, 5Ј-AATTAACCCTCACTAAAGGG-3Ј). The PCR product PAX3 protein, was used as a base to construct the KRAB-PAX3 wild-type and was digested with EcoRI and ligated to the unique EcoRI site just 5Ј of the mutant fusion genes (4, 18). This plasmid was digested with HindIII and vector-derived stop codon in the pcKRAB-PAX3-STOP plasmid to create an

BamHI and ligated to the wild-type or the mutant (D18V19 changed to A18A19) in-frame fusion. The pcSNAG-PAX3-HBD plasmid was constructed by fusing KRAB repression domains (19). The KRAB domain-encoding fragments were the HBD to the pcDNA3-SNAG-PAX3 plasmid, as described above. The generated by PCR amplification from the pM1-KOX-1 template (29) and were fragment encoding the SNAG domain was generated by overlapping PCR 5805

HBD proteins used for EMSA were synthesized using the Promega TnT T7 transcription and translation system. In parallel reactions, 35S-labeled proteins were prepared and analyzed by 10% SDS-PAGE and fluorography to confirm and normalize specific in vitro synthesis. For the corepressor supershift studies, COS-1 nuclear extract was used as a source of the KAP-1 corepressor (ϳ3 ␮g/␮l, prepared as described previously; Refs. 4 and 31). For the antibody supershift studies, ␣-KRAB or ␣-PAX3 IgG (2 ␮g/␮l) was included in the EMSA-binding reactions. Northern Analysis for KRAB-PAX3-HBD Transcript. Total RNA was isolated from Rh30-KPHBD-cl22 cells using the TRIzol reagent (Life Tech- nologies, Inc., Rockville, MD) and electrophoresed on 1% formaldehyde- agarose gels. Prior to RNA isolation, Rh30-KPHBD-cl22 cells were either un-induced (treated with 0.1% ethanol as a solvent control) or induced with 500 nM 4-OHT. The gel was stained with ethidium bromide to assess the integrity and equal loading of the samples and then transferred to a nylon membrane (Hybond). The membrane was prehybridized, hybridized with radiolabeled PAX3-KRAB probe, and washed to a final stringency of 0.2ϫ SSC, 0.2% SDS, at 65°C, before autoradiography. Indirect Immunofluorescence. Subcellular localization of KRAB-PAX3 and KRAB-PAX3-HBD proteins in Rh30, Rh30-pcDNA-cl5, and Rh30- KPHBD-cl22 cells grown on cover glasses was conducted using previously described procedures for immunofluorescence (31). After fixation with 1% Fig. 3. Transcriptional repression of the PAX reporter plasmid by the pcKRAB-PAX3- paraformaldehyde and permeabilization with 0.2% Triton X-100 (Sigma HBD is hormone dependent. NIH/3T3 cells were transiently transfected with indicated Chemical Co.), the antigens were localized using ␣-PAX3 or ␣-HBD primary expression plasmids along with a constant amount of the CD19–2(A-ins)-TK-LUC rabbit antibodies, followed by detection with a secondary biotinylated ␣-rabbit ␤ reporter and CMV- -D-galactosidase plasmids. Transfected cultures were induced with IgG and avidin-FITC (Vector Laboratories, Inc.). The nuclei were counter- 500 nM 4-OHT or treated with 0.1% ethanol as a solvent control for 24 h. The luciferase ␮ activities were normalized using ␤-D-galactosidase activity. stained for DNA with 0.5 g/ml Hoechst 33258 (Sigma Chemical Co.) and the cells were visualized using a Leica confocal laser-scanning microscope. The HC-20 antibody to the HBD of the murine estrogen receptor was obtained from amplification. The 5Ј primer was designed to incorporate an EcoRI and a Santa Cruz Biotechnology Inc. BamHI site, a Kozak consensus sequence, and the SNAG domain sequences Transient Transfections and Reporter Assays. The transcription assays (encoding amino acids 1–15; 5Ј primer, 5Ј-GAATTCGGATCC ACCATGC- were performed on NIH/3T3 cells that were transiently transfected with a CACGTTCTTTCCTGGTTAAATCTAAAAAAGCGCACTCTTACC-3Ј). LipofectAMINE mixture containing 1 or 2.5 ␮g of the expression plasmids The3Ј primer contained the remaining portion of the SNAG domain (amino (pcDNA3, pcKRAB-PAX3, and pcKRAB-PAX3-HBD), 0.5 ␮g of CD19– acids 16–20 in an antisense orientation), followed by BglII and SalI sites 2(A-ins)-TK-LUC, and 0.25 ␮g of CMV-␤-D-galactosidase plasmids, as de- (3Ј-primer, 5Ј-GTCGACAGATCTGGAGTAGTCCGGACCCGGAG- scribed previously (18). Transfected cells were treated with 0.1% ethanol as a AACGCGGCTGGTGGTAAGAGTGCGCTTTTTTAG-3Ј). These two oligo- solvent control for un-induced dishes or were induced with 500 nM 4-OHT nucleotides were annealed and amplified to yield a 105-bp fragment that was (Research Biochemicals International, Natick, MA). After 24 h, the cells were used as a template in the PCR reaction with a pair of flanking primers: washed twice with Tris-buffered saline, and the cell extracts were prepared in (5Ј-GTCAGAATTCGGATCCACC-3Ј; and 3Ј primer, 5Ј-CCAAGTCGACA- reporter lysis buffer and assayed for luciferase and ␤-galactosidase activities as GATCTGGAG-3Ј). The resulting PCR product was digested with BamHI and described (31). BglII and cloned into the BamHI site in the pcDNA3-PAX3-STOP plasmid. Generation of Stable Rh30 Cell Clones. Rh30 cell transfectants contain- The general-purpose vector pcDNA3-KRAB-MNP-HBD was constructed in ing the conditional PAX3 repressor plasmids depicted in Fig. 1A or the two steps. First, the KRAB domain was amplified using the 5Ј HindIII primer pcDNA3 vector were isolated as individual colonies using cloning rings and described above and a 3Ј primer that incorporated a myc-epitope tag (EQKLI- were expanded into cell lines after selection for stable resistance to 500 ␮g/ml SEEDL) and a nuclear localization signal of the E1A gene (KRPRP) imme- G418 (Mediatech, Inc., Herndon, VA). Twenty-four independent cell lines diately after amino acid 89 of the KRAB domain, followed by a BamHI site. were tested for expression of the PAX3-HBD repressor proteins by immuno- Next, the ERTM DNA was generated by PCR amplification using a 5Ј primer precipitation with ␣-PAX3 IgG. Dual-stable inducible PAX3 repressor/PAX3 designed to incorporate a polylinker composed of BamHI, EcoRI, EcoRV, and reporter cell clones were generated in the Rh30-KPHBD-cl22 cell line after ClaI sites, and the 3Ј T3 primer. This fragment was cleaved with BamHI and transfection with the CD19–2(A-ins)-TK-LUC-ZeoR plasmid and selection NotI, and then these two fragments were cloned into the HindIII and NotI sites with 500 ␮g/ml G418 and 100 ␮g/ml Zeocin. The PAX3 repressor/PAX3 of the pcDNA3 vector to generate the final construct. The nucleotide sequences reporter cell lines, designated as HBDLUC clones, were screened for repres- of all PCR-derived constructs were confirmed by sequencing both strands. The sion of luciferase after induction with 4-OHT. The luciferase activities of the previously described PAX reporter plasmid, CD19–2(A-ins)-TK-LUC (30), HBDLUC clones were normalized to a protein content of 1.0 A595 unit in the was modified by incorporation of a ZeocinR cassette derived as a PvuII BioRad protein assay. fragment from the pcDNA3.1Zeo plasmid (Invitrogen) to create the DD-PCR. Gene expression profiles of un-induced and 4-OHT-induced CD19–2(A-ins)-TK- Rh30-KPHBD-cl22 cells were analyzed using DD-PCR. Total RNA was LUC-ZeoR plasmid. isolated using the TRIzol reagent and poly(A)ϩ mRNA was purified using the

COS-1 Transfection, Extract Preparation, and Immunoprecipitation. oligo-(dT)25 Dynabeads (Dynal, Inc., Lake Success, NY). First-strand cDNA The expression plasmids depicted in Fig. 1A were transfected into COS-1 cells was synthesized using the Life Technologies, Inc. cDNA synthesis system and for 6 h with a mixture of DNA:lipofectAMINE in the ratio 1:6 in optiMEM, quantitated by spectrophotometry. Differential subtraction display PCR was followed by growth for 48 h in DMEM containing 10% FBS. Transfected cells conducted as described (32), and the samples were electrophoresed on a were metabolically labeled with 35S-methionine, and the cell extracts prepared sequencing gel and autoradiographed. in radioimmunoprecipitation assay buffer were subjected to immunoprecipita- Low-Serum and Poly-HEMA-MTT Assays. The growth assays of Rh30, tion analysis with ␣-KRAB, ␣-PAX3, and ␣-HBD antibodies, as described Rh30-KPHBD-cl22, Rh30-SPHBD-cl8, and Rh30-K(DV)PHBD-cl24 cell previously (4, 18). lines in low-serum (0.1% FBS) medium was conducted in 24-well tissue EMSA. The DNA-binding potentials of KRAB-PAX3 and KRAB-PAX3- culture plates and was repeated twice with at least 10 replicates. The anchor- HBD proteins were assessed by EMSA performed using 32P-labeled e5 DNA age-independent growth of parental Rh30 and Rh30-KPHBD-cl22 cells was probe, as described previously (4, 31). The KRAB-PAX3 and KRAB-PAX3- evaluated using poly-HEMA-coated plates. In both assays, the proportion of 5806

Fig. 4. A, C, and E, immunoprecipitation analysis of PAX3 repressor protein expression in stable Rh30 cell clones using ␣-PAX3 antibody. The endogenous PAX3- FKHR fusion protein and the chimeric PAX3 repressor proteins are indicated. A, KRAB-PAX3-HBD protein expression in clones cl11, cl13, cl19, and cl22. C, aKRAB(DV)-PAX3-HBD protein expression in clones cl3, cl13, and cl24. E, SNAG-PAX3-HBD protein expression in clones cl4, cl7, cl8, and cl24. B, D, and F, hormone- dependent transcriptional repression of luciferase activity in conditional PAX3 repressor Rh30 cell clones transiently transfected with the CD19–2(A-ins)-TK-LUC reporter plasmid. B, Rh30-KPHBD clones, cl11, cl13, cl19, and cl22. D, Rh30-K(DV)PHBD clones, cl3, cl13, and cl24. F, Rh30-SPHBD cell clones, cl4, cl7, cl8, and cl24. IVT, in vitro translated protein.

viable cells at each time point was determined by MTT assay, as described (5Ј-primer, 5Ј-GCGACGGGCCCATGGGTGCTTCAG G-3Ј; and 3Ј-primer, previously (33). 5ЈGGTGGGCCCCTGATATCACAAGTCCTCTTCAGAAATGAGCTTTTG- Apoptosis Assays. Apoptosis assays were performed using the ApoAlert CTCGATCGTGTTGGGGAAGCC-3Ј) was used to amplify a 1010-bp product DNA Fragmentation and the ApoAlert Annexin V Assay kits according to the from the SCID mouse tumor samples that were derived from Rh30-KPHBD- manufacturer’s instructions (Clontech Laboratories, Inc.). Assays were con- cl22 cells. ducted on SCID mouse tumor sections or on Rh30-KPHBD-cl22 cells that Tumor Growth Inhibition Assays in SCID Mice. The tumorigenic po- were grown on coverslips in low-serum medium under either un-induced tentials of Rh30-pcDNA-cl5 and Rh30-KPHBD-cl22 cell lines were evaluated (0.1% ethanol) or 4-OHT-induced conditions (500 nM, 48 h). The polyclonal after s.c. injection into female CB17-SCID mice, 6 weeks of age. Evidence of rabbit antibody for immunoblot detection of BCL-XL (bcl-x, Ab-1) and the tumor growth became apparent after 10 days, at which time the mice were antibody for detection of human ␣-tubulin as a loading control were obtained divided into two groups of five mice each (un-induced and 4-OHT induced). from Oncogene Research Products (Cambridge, MA). The mice of the 4-OHT-induced group were implanted with 35-mg timed- Semiquantitative RT-PCR Analysis. For RT-PCR analysis, RNA was release pellets specified to maintain a 200-nM circulating concentration of isolated from the un-induced or the 4-OHT-induced Rh30-KPHBD-cl22 and 4-OHT for 21 days (Innovative Research of America, Sarasota, FL). Improved Rh30-SPHBD-cl8 cells grown in low-serum medium using the TRIzol reagent. implant success was ensured by application of DERMABOND topical skin The reverse transcription reactions were performed on 5 ␮g of total RNA using adhesive (Ethicon, Inc., Somerville, NJ) over the wounds. Alternate day oligo-dT primers with the Ready-To-Go You-Prime First-Strand Synthesis measurements of tumor volumes were made using a tumorimeter (Cancer Beads (Pharmacia Biotech). The PCR reactions were carried out with 2.5 ␮lof Technologies, Inc., Tucson, AZ). After 3 weeks, the mice were sacrificed and reverse transcription reactions as templates. A pair of primers specific for the the wet weights of the tumors were recorded. A portion of each tumor was Ј Ј Ј human BCL-XL transcript (5 primer, 5 -CAGCAGCAGTTTGGATGC-3 ; fixed in formalin for H&E staining and histopathological evaluation. 3Ј-primer, 5Ј-CCACAGTCATGC CCG TC-3Ј) was used to amplify the 448-bp product. A specific primer pair (5Ј-primer, 5Ј-TCAGCGCAGGGG- RESULTS CGCCCGGTTCTT T-3Ј; and 3Ј-primer, 5Ј-ATCGACAAGACCGGCTTC- CATCCGA-3Ј) was used to amplify the 345-bp product from the NeoR gene. Conditional PAX3 Repressors and Integrated Reporter. We A pair of primers specific for the HBD of the murine estrogen receptor have previously demonstrated that we could engineer a PAX3 tran- 5807

repressors is not limited to the set of target genes selected by the PAX3 DNA-binding domains as in this study. The general purpose KRAB-MNP-HBD vector (Fig. 1C) was designed for use with other DNA-binding domains of interest that could be inserted into the poly-linker between the KRAB repression domain and the ERTM domain. This plasmid features incorporation of a nuclear localization signal and a c-myc epitope tag to aid in detection. The resulting conditional chimeric repressors can be applied for repression of the set of endogenous target genes of choice determined by the inserted DNA binding motif. Characteristics of Engineered Repressor Proteins. Immunopre- cipitation analysis of transfected COS-1 cell extracts using both ␣-KRAB and ␣-PAX3 IgGs (Fig. 2A) demonstrates that the pcKRAB- PAX3 (55 kDa) and pcKRAB-PAX3-HBD (88 kDa) proteins are expressed in vivo at the size predicted by their cDNAs and are recognized by the appropriate antibodies. The EMSA analysis (Fig. 2B) indicated the presence of clear binary complexes of both KRAB- PAX3:e5DNA and KRAB-PAX3-HBD:e5DNA, which confirmed that both KRAB-PAX3 and KRAB-PAX3-HBD proteins exhibited the anticipated DNA-binding properties. We have previously shown,

Fig. 5. A, hormone-dependent repression of an integrated CD19–2(A-ins)-TK-LUC- reporter gene. The dual-stable HBDLUC Rh30 cell lines that contain the inducible PAX3 repressor/PAX3 reporter alleles were assayed for repression of luciferase activities after induction with 4-OHT. B, the fold-repression, defined as the ratio between the un-induced and 4-OHT-induced luciferase values, is depicted for several independent HBDLUC clones. scriptional repressor by fusing the KRAB domain to the PAX3 DNA- binding domains. We found that constitutive expression of a PAX3- KRAB protein could inhibit the malignant growth of ARMS cells (18). In the present study, a KRAB-PAX3 plasmid was converted to a conditional repressor, KRAB-PAX3-HBD, by fusion of the ERTM domain in-frame to the COOH terminus (Fig. 1A). The ERTM domain is a well-established, tamoxifen-selective, mutant version of the HBD of the murine estrogen receptor that confers hormone-dependent functionality to heterologous fusion proteins (24). The KRAB(DV)- PAX3-HBD protein features a mutant KRAB repression domain that lacks transcriptional repression potential and is a useful control to confirm that the elicited biological responses depend on a functional repression module (19). Furthermore, by stable integration of the 6ϫ CD19–2(A-ins)-TK-LUC luciferase reporter plasmid (Fig. 1B), we have created inducible PAX3 repressor/reporter (HBDLUC) cell lines that permit convenient monitoring of the repression function in a chromatin-mediated state. The modular nature of the engineered repressor is shown by the use of another conditional repressor plasmid, Fig. 6. Hormone-dependent inhibition of malignant growth. The growth rate of Rh30 stable cell lines in low-serum medium in the presence or absence of 4-OHT was monitored SNAG-PAX3-HBD, which was created using the SNAG repression over a 10-day period by MTT assay and plotted as A550 versus Time (in days). A, Rh30 module of the GFI-1 gene (21).4 Clearly, the use of engineered cells; B, Rh30-KPHBD-cl22 cells; C, Rh30-SPHBD-cl8 cells; D, Rh30-K(DV)PHBD- cl24 cells. MTT assay of anchorage-independent growth in poly-HEMA-coated dishes under un-induced or 4-OHT-induced conditions. E, Rh30 cells; F, Rh30-KPHBD-cl22 4 Unpublished results. cells. 5808

Fig. 7. A, BCL-XL expression in Rh30-SPHBD- cl8 (Lanes 1 and 2) and Rh30-KPHBD-cl22 cell lysates (Lanes 3 and 4) obtained after growth in low-serum medium (0.1%) under un-induced (Lanes 1 and 3) and 4-OHT-induced (Lanes 2 and 4) conditions. Depicted are immunoblot analyses of ␮ ␣ 100 g of whole cell lysates using -BCL-XL- specific antibody (top). Expression of ␣-tubulin in the same lysates was assessed as a control for equal loading (bottom). Semiquantitative RT-PCR analy- R sis for BCL-XL (top) and Neo (bottom) transcripts under un-induced and 4-OHT-induced conditions in the absence of cycloheximide (B) and in the presence of 50 ␮g/ml cycloheximide (C).

by competition with unlabeled homologous oligonucleotide, that KRAB-PAX3-HBD repressor indicated proper expression, DNA- binding of PAX3 protein to the e5 binding site probe is specific (4). binding activity, and a significant degree of hormone-dependent tran- Furthermore, these binary complexes were confirmed to contain the scriptional repression activity in NIH/3T3 cells. NIH/3T3 cells do not predicted proteins because they were efficiently super-shifted when express PAX3, hence, lack any endogenous PAX3-dependent tran- ␣-KRAB or ␣-PAX3 IgG was included during the DNA-binding scriptional activation function (data not shown; Ref. 34). reactions (data not shown). It was also evident that the KRAB domain Conditional PAX3 Repressor in ARMS Stable Cell Lines. Our present in the binary complex was amenable for protein-protein in- primary interest, however, was to evaluate the conditional PAX3 teractions because ternary complexes of KRAB-PAX3:KAP-1: repressor in Rh30 ARMS cell lines that express the endogenous e5DNA or KRAB-PAX3-HBD:KAP-1:e5DNA were observed when PAX3-FKHR oncogenic transcriptional activator. Therefore, we gen- COS-1 nuclear extract was included in the gel-shift reaction as a erated stable Rh30 cell clones, which expressed the conditional PAX3 source of the KAP-1 corepressor (20). We have also found that the repressors and then evaluated their repression potentials after transient SNAG-PAX3-HBD protein efficiently binds the e5 DNA probe but transfection of the CD19–2(A-ins)-TK-LUC reporter plasmid. In con- does not form a ternary complex with KAP-1 (data not shown). As trast to the 10-fold repression observed in transiently transfected would be expected with the ERTM posttranslational regulation system, NIH/3T3 cells (Fig. 3), in Rh30 cells the repression achieved ranged we have shown that similar levels of KRAB-PAX3-HBD transcript from 2–4-fold, consistent with the presence of a competing endoge- are expressed in Rh30-KPHBD-cl22 cells grown under both un- nous activation due to PAX3-FKHR. Hormone-dependent repression induced and 4-OHT-induced conditions (Fig. 2C). Immunofluores- of the reporter plasmid was observed in all of the Rh30-KPHBD cence microscopy indicated that in the absence of hormone the clones (e.g., Ϫcl11, cl13, cl19, and cl22; Fig. 4B) that correlated well KRAB-PAX3-HBD protein was predominantly cytoplasmic (Fig. 10A, top), but after induction with 4-OHT it was located in the nucleus with the expression levels of the KRAB-PAX3-HBD fusion protein (Fig. 10A, bottom). (Fig. 4A). The observed conditional transcriptional repression ap- Hormone-dependent Luciferase Repression. Transcriptional re- peared to be dominant over transcriptional activation elicited by the pression assays that were carried out in NIH/3T3 cells transiently endogenous PAX3-FKHR oncogene. Similarly, all of the SNAG- Ϫ transfected with the KRAB-PAX3 plasmid showed constitutive re- PAX3-HBD-expressing Rh30 cell clones (e.g., cl4, cl7, cl8, and pression that depended on the dose of the input expression plasmid but cl24; Fig. 4F) showed a good correlation between hormone-dependent not on the presence of 4-OHT. In contrast, cells transfected with 1 ␮g repression of luciferase activities and expression levels of the SNAG- of the KRAB-PAX3-HBD plasmid exhibited a significant dependence PAX3-HBD protein (Fig. 4E). As expected, the Rh30 cell clones (e.g., on the presence of 4-OHT to repress the luciferase reporter and Ϫcl3, cl13, and cl24; Fig. 4C) that expressed the mutant KRAB(DV)- typically manifested a 10-fold repression (Fig. 3). A hormone-inde- PAX3-HBD repressor protein (Fig. 4C) showed no repression (Fig. pendent repression was also observed at a high input dose (2.5 ␮g) of 4D). Thus, repression was found to completely depend on the pres- KRAB-PAX3-HBD plasmid. As expected, the vector-transfected cells ence of a functional repression domain (KRAB or SNAG) but was not showed no repression (Fig. 3). Overall, our characterization of the restricted to a particular type of repression mechanism. 5809

Fig. 8. Hormone-dependent suppression of tumorigenic growth of Rh30 cell clones in SCID mice by activation of the KRAB-PAX3-HBD repressor protein. Fifteen CB17 SCID mice received s.c. injections of Rh30-pcDNA-cl5 or Rh30- KPHBD-cl22 clonal cell lines. Tumor-bearing mice (five each) either were induced by implantation of slow release 4-OHT pellets or were un-induced. Progression of tumor volumes for Rh30-pcDNA- cl5 (pcDNA; A) and Rh30-KPHBD-cl22 (KPHBD; B) mice. Solid symbols represent 4-OHT-induced mice, whereas open symbols indicate un-induced mice. Volumes (C) and wet weights (D)ofthe dissected tumors.

Repression of the Integrated PAX3 Reporter and Endogenous acterization of these differentially expressed mRNA species is cur- Genes. Our intended goal was to apply the PAX3 conditional repres- rently under investigation, and candidate genes are being evaluated for sor strategy to specifically down-regulate endogenous target genes functional relevance to the malignant phenotype of ARMS. that are activated by the PAX3-FKHR oncogene. Hence, we produced Growth Properties of Conditional PAX3 Repressor ARMS Cell stable cell clones containing both the KRAB-PAX3-HBD plasmid Lines. To further substantiate the inducible PAX3 repressor Rh30 (NeoR) and the CD19–2(A-ins)-TK-LUC reporter plasmid (ZeoR), cell lines as pertinent model systems for the study of PAX-3-FKHR designated as HBDLUC cell lines. This allowed us to correlate re- target genes in ARMS, we used the Rh30-KPHBD-cl22 cell line to pression of an integrated artificial reporter gene target with reversion examine whether KRAB-PAX3-HBD would function as a conditional of ARMS malignant growth characteristics and changes in expression repressor of ARMS malignant growth. Growth properties of Rh30 and of endogenous PAX3 target genes. Several independent clonal cell Rh30-KPHBD-cl22 cells were studied in the presence or absence of lines were tested for hormone-dependent repression of the chromatin- 4-OHT under full-serum (10%) and reduced-serum (0.1%) conditions. integrated luciferase gene, and the results obtained for a selected The growth in full serum was not significantly changed relative to number of clones are depicted in Fig. 5A, which shows that the growth of a parallel un-induced culture on activation of KRAB- normalized luciferase activities varied from 103–106 luciferase units. PAX3-HBD protein by 4-OHT (data not shown). However, under The fold repression ranged from Ͼ1.5–5 for different clonal cell lines low-serum conditions, a significant reduction in the number of viable (Fig. 5B). The variation observed between clones most likely reflects Rh30-KPHBD-cl22 cells over a 10-day period was observed when the differences in reporter copy number and integration site. The range of KRAB-PAX3-HBD repressor was activated by 4-OHT and growth repression potentials obtained with the chromatin-integrated reporter was evaluated by MTT assay (Fig. 6B). Rh30-SPHBD-cl8 cells ex- clones are consistent with those observed in transient assays in Rh30 pressing the SNAG-PAX3-HBD protein manifested similar hormone- cells (Fig. 4), further supporting the contention that the PAX3 repres- dependent inhibition of cell growth (Fig. 6C). As expected, Rh30- sors could dominantly repress endogenous genes despite the presence K(DV)PHBD-cl24 cells did not show any growth retardation under of the PAX3-FKHR protein. either conditions (Fig. 6D), similar to parental Rh30 cells (Fig. 6A). Our studies have indicated that repression of PAX3-FKHR target Growth of un-induced Rh30-KPHBD-cl22 cells (Fig. 6B) was also genes is a strategy for reversion of malignant growth of ARMS cells. similar to that of Rh30 cell line (Fig. 6A). These results clearly Thus, we have established a relevant system to model the conditional indicate that conversion of the inactive repressor protein to an active repression of endogenous PAX3-FKHR target genes. To demonstrate form by 4-OHT is responsible for this growth inhibition in Rh30- that hormone-dependent repression of endogenous genes was occur- KPHBD-cl22 and Rh30-SPHBD-cl8 cells. The malignant phenotype ring in this ARMS cell system, DD-PCR analysis was performed of the Rh30-KPHBD-cl22 cells was further examined in poly-HEMA- using RNA made from un-induced and 4-OHT-induced Rh30- coated tissue culture plates to evaluate anchorage-independent growth KPHBD-cl22 cells. When different combinations of anchor and arbi- potential. As expected, the parental Rh30 cell line exhibited abundant trary primers were used, the DD-PCR products displayed significant growth under the conditions of the poly-HEMA assay, and the growth expression pattern differences (data not shown). Isolation and char- was equivalent under un-induced and 4-OHT-induced conditions (Fig. 5810

Fig. 9. A, un-induced and 4-OHT-induced tumors in SCID mice bearing Rh30-pcDNA-cl5 or Rh30-KPHBD-cl22 cell tumors. The site of implantation of 4-OHT pellets is indicated by the arrows. B, RT-PCR analysis of tumors derived from Rh30-KPHBD-cl22 SCID mice (un- induced: Lanes 1, 2, and 3; 4-OHT-induced: Lanes 4, 5, and 6) and of tumors derived from Rh30-pcDNA-cl5 control mice (un-induced: Lanes 7, 8, and 9; 4-OHT-induced: Lanes 10, 11, and 12). The arrow indicates the specific product from the pcKRAB-PAX3-HBD plasmid (KPHBD).

6E). In marked contrast, the Rh30-KPHBD-cl22 cell line showed a To test whether the KRAB-PAX3-HBD protein is a direct repressor dramatic suppression of growth potential only on 4-OHT treatment of BCL-XL, Rh30-KPHBD-cl22 cells were induced with 4-OHT for (Fig. 6F). 10 h in the presence of cycloheximide. The RT-PCR analyses (Fig.

Our data indicate that activation of the KRAB-PAX3-HBD repres- 7C) indicated that amplification of the BCL-XL product continued to sor renders the Rh30-KPHBD-cl22 cells very sensitive to inhibition of be diminished compared with the un-induced controls. However, the growth in low-serum medium as evidenced by a loss of cell viability BCL-XL product generated from the cycloheximide-treated sample by day 8 (Fig. 6B). The PAX3 repressor also inhibited growth under was not repressed as fully as the 4-OHT-induced, noncycloheximide- anchorage-independent conditions as seen above, however, there was treated sample. This diminution may be attributed to a reduced level no evidence of drastic cell loss. We were interested to know whether of the KRAB-PAX3-HBD protein after the cycloheximide treatment the difference in growth kinetics observed between the two different (data not shown). Overall, these results suggest that repression of the assays could be due to apoptosis. Hence, we carried out two inde- BCL-XL product does not require de novo protein synthesis and pendent apoptosis assays (TUNEL and Annexin V) using Rh30- BCL-XL is a probable candidate for a direct target gene of PAX3- KPHBD-cl22 cells that were grown in low serum under un-induced or KRAB-HBD. 4-OHT-induced conditions. On 4-OHT induction, the TUNEL assays Hormone-dependent Suppression of Tumorigenesis in SCID showed a significant increase in the proportion of cells stained with Mice. Our previous studies had demonstrated that PAX3-KRAB ex- streptavidin-FITC (Fig. 10B). This enhanced staining reflects incor- pression could suppress Rh30 cell tumorigenesis in SCID mice (18). poration of biotin-16-dUTP at DNA strand breaks and is a sensitive Thus, we were interested in whether conditional suppression of tu- indication of apoptotic cell death. The level of nuclear staining by morigenesis in vivo could be demonstrated using the newly estab- propidium iodide was the same in the presence or absence of 4-OHT lished conditional PAX3 repressor cell line Rh30-KPHBD-cl22. We induction, ruling out nonspecific cytotoxicity (data not shown). En- generated tumors by injecting SCID mice with Rh30-pcDNA-cl5 or hanced immunodetection of Annexin V was also observed after Rh30-KPHBD-cl22 cells. After a 10-day period to establish tumor 4-OHT induction (data not shown), supporting the correlation be- growth, half of the mice having comparably sized tumors were im- tween KRAB-PAX3-HBD activity and apoptosis in low-serum cul- planted with slow-release pellets containing the 4-OHT inducer. Sub- ture. sequently, tumor measurements were made at regular intervals for 3 These findings suggested that the KRAB-PAX3-HBD protein weeks, and these results over time are shown in Fig. 8A for Rh30- might have repressed a PAX3 target gene linked to protection from pcDNA-cl5 mice and in Fig. 8B for Rh30-KPHBD-cl22 mice. Data apoptosis, consistent with the role described for PAX proteins during for the measured tumor volumes are presented in Fig. 8C. After development (10, 11). It has previously been reported that PAX8 can sacrifice, tumors were isolated by dissection, and their wet weights are promote cell survival by activating antiapoptotic factors of the BCL plotted in Fig. 8D. These results agree with the tumor volume esti- family (8). We evaluated expression of the BCL-XL survival factor in mates and clearly indicate that in the presence of 4-OHT only the Rh30-KPHBD-cl22 cells that were grown in low serum under un- Rh30-KPHBD-cl22 mice showed a reduction in tumor growth, induced or 4-OHT-induced conditions. Immunoblot analysis of whole whereas the Rh30-pcDNA-cl5 mice did not. The mice bearing the cell lysates indicated that the BCL-XL protein levels from 4-OHT- Rh30-pcDNA-cl5 and the Rh30-KPHBD-cl22 tumors were photo- induced cells were decreased compared with un-induced cells (Fig. graphed before sacrifice (Fig. 9A). RT-PCR analysis of tumor RNA Ј Ј 7A). RT-PCR analysis using BCL-XL-specific primers indicated a using the HBD 5 and 3 primers indicated that only Rh30-KPHBD- ϩ reduced amplification of the BCL-XL product from reactions from cl22 (-/ 4-OHT)-injected mice expressed the KRAB-PAX3-HBD 4-OHT-induced samples compared with the un-induced samples. transcript (Fig. 9B). Furthermore, the tumors from the un-induced or These data indicate that activation of KRAB-PAX3-HBD protein 4-OHT-induced pcDNA-cl5 mice and the un-induced KPHBD-cl22 results in repression of BCL-XL transcription (Fig. 7B). mice contained many blood vessels in the H&E-stained tumor tissue 5811

Fig. 10. A, immunofluorescent localization of the KRAB-PAX3-HBD protein with ␣-HBD antibody in Rh30-KPHBD-cl22 cells under un-induced (top) or 4-OHT-induced conditions (bottom). B, TUNEL assays of Rh30-KPHBD-cl22 cells that were grown in low-serum medium under un-induced (top) or 4-OHT-induced conditions (bottom). C, H&E-stained tumor sections from SCID mice with Rh30-pcDNA-cl5 tumors (left) and Rh30-KPHBD-cl22 tumors (right) under un-induced (top) or 4-OHT-induced (bottom) conditions. Note the diminished vascularity in Rh30-KPHBD-cl22 mice that were implanted with the 4-OHT time-release pellets. D, TUNEL assays of tumor tissue sections from SCID mice with Rh30-pcDNA-cl5 tumors (left) and Rh30-KPHBD-cl22 tumors (right) under un-induced (top) or 4-OHT-induced (bottom) conditions. sections (Fig. 10C). On the contrary, in the 4-OHT-induced KPHBD- pcDNA-cl5 mice and the un-induced KPHBD-cl22 mice) showed a cl22 mice significantly fewer blood vessels were evident. It is pres- similar low level of staining, indicating very little apoptosis in vivo ently unknown whether the reduced angiogenesis is directly related to (Fig. 10D). However, an enhanced streptavidin-FITC staining was the KRAB-PAX3-HBD protein or a secondary consequence of the observed in the tumor sections from the 4-OHT-induced KPHBD-cl22 reduced tumor burden. TUNEL assays performed on the tumor sec- mice, indicative of KRAB-PAX3-HBD-induced apoptotic cell death tions from the negative control mice (un-induced or 4-OHT-induced in vivo. 5812

DISCUSSION PAX3 repressor Rh30 cell lines under low-serum conditions is en- tirely consistent with previous studies in which an antisense approach In this study, we have generated ARMS cell lines with conditional was used to down-regulate PAX3-FKHR in Rh30 ARMS cells (10). PAX3 repressors to establish a system to understand the biological Furthermore, it has been clearly demonstrated that down-regulation of role and identify endogenous target genes of the PAX3-FKHR onco- PAX3 function, either by mutation in splotch mice (14), or by reduced protein. The conditional PAX3 repressors were generated by fusing expression in diabetic mice (36), leads to extensive apoptosis. It is the KRAB or SNAG repression domains and PAX3 DNA-binding well established that many of the PAX family proteins function during TM motifs to the tamoxifen-selective ER HBD of the estrogen receptor. organogenesis to protect cells from apoptosis (9, 11, 14). Previous We believe that application of our conditional repressors to the ARMS studies have linked the antiapoptotic function of PAX8 to activation model system is especially relevant for evaluating the cellular path- of the BCL family protein, BCL-2 (8). Our studies have demonstrated ways aberrantly regulated by the PAX3-FKHR protein. One important that the KRAB-PAX3-HBD protein can down-regulate expression of advantage of using the ERTM system is that rapid activation of the the BCL-XL cellular survival factor and induce apoptosis in Rh30 cell transcription factor-ER fusion protein can be achieved by a simple lines in vitro and in SCID mice in vivo. These observations suggest ligand-mediated on/off mechanism (24, 35). Thus, the immediate that PAX3-FKHR may contribute to oncogenesis in ARMS by acti- consequences of the KRAB-PAX3-HBD repressor on biological prop- vating the antiapoptotic BCL-XL survival factor. One advantage of the erties contributing to malignant growth and tumorigenesis can be ER system for studying transcription factors like KRAB-PAX3-HBD studied without selection for secondary mechanisms. is that activation of the ER-fusion protein can occur in the absence of Our confidence in the conditional repressors applied in this study is de novo protein synthesis. Thus, in the presence of protein synthesis supported by the following principal results that support their effec- inhibitors such as cycloheximide, a distinction can be made between tive dominant negative function. First, the KRAB-PAX3-HBD and directly regulated target genes and those secondary targets whose SNAG-PAX3-HBD proteins were fully competent in binding to the e5 regulation depends on transcription and translation of a primary target target DNA in EMSA analysis. The super-shift experiments showed gene. Our data supports the hypothesis that BCL-XL is a direct PAX3 that the KRAB-PAX3-HBD protein was also fully capable of binding target gene, because the repression by KRAB-PAX3-HBD was the KAP-1 corepressor, which is a prerequisite for KRAB-mediated largely insensitive to inhibition by cycloheximide. Recent studies transcriptional repression (20). Conditional repressors using the have confirmed that PAX3 and PAX3-FKHR can directly regulate the SNAG domain do not rely on KAP-1 association, thus, may have an BCL-XL via direct binding to the promoter (37). extended applicability in biological systems in which KAP-1 is not It is likely that regulation of apoptosis by PAX3 may involve present, such as in insect cells, or in certain differentiated cell types several other mechanisms in addition to BCL-XL. Recent expression (31). Second, the ability of the KRAB-PAX3-HBD and SNAG- profiling has shown that PAX3-FKHR can activate expression of the PAX3-HBD conditional repressors to induce transcriptional repres- SLUG protein (34). Other studies have shown that SLUG plays an sion was tightly regulated by the ligand, 4-OHT. Moreover, the degree antiapoptotic role in E2A-HLF-induced tumorigenesis (38). In addi- of transcriptional repression achieved by the conditional KRAB- tion, PAX3 is known to regulate c-MET, the receptor for the hepato- PAX3-HBD repressor was as potent as the repression observed with cyte growth factor/scatter factor, which has also been implicated in the constitutive KRAB-PAX3 repressor, implying that fusion of the apoptosis (39, 40). It is interesting that apoptosis was only observed ER HBD did not interfere with the expected function. Third, in Rh30 when the KRAB-PAX3-HBD protein was activated under low-serum stable clones, the KRAB-PAX3-HBD and the SNAG-PAX3-HBD conditions, implying that the Rh30 cells may only depend on PAX3- protein expression levels were higher than that of the endogenous FKHR for protection against apoptosis when deprived of certain PAX3-FKHR protein; hence, the repressors would be expected to factors present in serum. Substantial evidence supports a role for have a competitive advantage for PAX3-FKHR target genes. Fourth, growth factor signaling in ARMS. PAX3-FKHR has been shown to the conditional PAX3 repressor proteins exhibited hormone-depen- activate expression of insulin-like growth factor II, which has a dent nuclear localization, which may account for the hormone-depen- well-established role in ARMS malignant growth (34, 41). Other dent biological effects in Rh30 cells. Fifth, the KRAB-PAX3-HBD studies have defined a role for the wild-type FKHR protein in linking and SNAG-PAX3-HBD proteins exhibited potent repression of either insulin signaling to a cellular proapoptotic response (41, 42). It is not transiently transfected or integrated reporter genes in Rh30 ARMS known whether PAX3-FKHR exerts any dominant-negative effect on cells. The conditional repressors were as effective in repression of the wild-type FKHR protein derived from the nontranslocated allele in integrated reporter plasmids as transiently transfected reporter plas- ARMS. Protection from apoptosis by PAX3-FKHR is likely to be a mid. Furthermore, the repression of endogenous genes by the condi- key oncogenic mechanism in ARMS that will require further identi- tional repressors was confirmed by the DD-PCR analysis. In all cases, fication and validation of the essential target genes. the conditional repression was dominant over any activation mediated Our demonstration that KRAB-PAX3-HBD Rh30 cell tumors show by the endogenous PAX3-FKHR. In addition, there was a striking pronounced hormone-dependent inhibition of growth confirms our correlation between the expression levels of the PAX3 repressors and previous findings and emphasizes that in ARMS the tumorigenic the degree of transcriptional repression. Sixth, the conditional PAX3 potential is dependent on PAX3-FKHR. In SCID mice experiments, repressors exhibited hormone-dependent suppression of characteristic use of the timed-release pellets allowed maintenance of the blood malignant properties such as: (a) growth in low serum; (b) anchorage- levels of 4-OHT and activation of the conditional repressor through- independent growth; and (c) tumorigenicity in SCID mice. Further- out the course of the experiment. The tumors that grew from control more, both the KRAB-based as well as the SNAG-based conditional cells without the repressor or from Rh30-KPHBD-cl22 cells without PAX3 repressors exhibited similar inhibitory properties, demonstrat- the 4-OHT inducer were heavily vascularized. It is interesting that ing that the growth inhibition did not depend on a particular type of only the Rh30-KPHBD-cl22 tumors derived from the 4-OHT-treated repression mechanism. Finally, the temporal control afforded by the mice showed very little evidence of vascularization in the H&E- conditional PAX3 repressor allowed observation of immediate stained tumor sections. We are currently investigating whether the changes in growth under low-serum conditions that led us to demon- KRAB-PAX3-HBD protein may have repressed angiogenic factors. strate that the KRAB-PAX3-HBD protein induces apoptosis. We believe that our conditional PAX3 repressor strategies have The hormone-dependent apoptosis exhibited in the conditional targeted a major oncogenic mechanism in ARMS, protection from 5813

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and inhibits G1 arrest induced by interleukin-2 withdrawal. Mol. Cell. Biol., 16: ACKNOWLEDGMENTS 6263–6272, 1996. 22. Madden, S. L., Cook, D. M., Morris, J. F., Gashler, A., Sukhatme, V. P., and We are grateful to Dr. Satish Parimoo (Skin Biology, Johnson & Johnson, Rauscher, F. J., III. Transcriptional repression mediated by the WT1 Wilms tumor Skillman, NJ) for providing reagents and guidance with DD-PCR protocols. gene product. Science (Washington DC), 253: 1550–1553, 1991. We thank Dr. Beat Schafer for the 6xCD19–2 (A-ins)-TK-LUC reporter 23. Wang, Y., Xu, J., Pierson, T., O’Malley, B. W., and Tsai, S. Y. Positive and negative plasmid. We thank Trevor Littlewood (ICRF, London) for the HBD-ER™ regulation of gene expression in eukaryotic cells with an inducible transcriptional regulator. Gene Ther., 4: 432–441, 1997. plasmid. Richelle Takemoto and Rachel Beurmann of Dr. Meenhard Herlyn’s 24. Littlewood, T. D., Hancock, D. C., Danielian, P. S., Parker, M. G., and Evan, G. I. 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Kasirajan Ayyanathan, William J. Fredericks, Carola Berking, et al.

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