EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma ... | Nature

Oncogene (2001) 20, 6653 ± 6659 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

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EWS-ATF-1 chimeric protein in soft tissue clear cell sarcoma associates with CREB-binding protein and interferes with p53-mediated trans-activation function Yasuo Fujimura1, Habibur Siddique1, Leo Lee2, Veena N Rao1 and E Shyam P Reddy*,1 1

Program of Cancer Genetics, Cancer center, MS #481, Department of Biochemistry, School of Medicine, MCP Hahnemann University, Broad and Vine, Philadelphia, Pennsylvania, PA 19102, USA; 2SAIC, Frederick, Maryland, MD 21702, USA

The recurrent t(12;22) (q13;q12) chromosomal translocation associated with soft tissue clear cell sarcoma results in a chimeric protein EWS-ATF-1 that acts as a constitutive transcriptional activator. The CBP/p300 transcriptional coactivator, which links various transcriptional factors to basal transcription apparatus, participates in transcriptional activation, growth and cell cycle control and dierentiation. In this study, we show that EWS-ATF-1 associates constitutively with CBP both in vitro and in vivo. Both EWS and ATF-1 fusion domains are needed for this interaction. Here, we demonstrate that EWS-ATF-1 represses p53/CBP-mediated transactivation function. Overexpression of CBP can counteract this repressive eect of EWS-ATF-1. Taken together, these ®ndings suggest that one of the mechanisms by which EWS-ATF-1 may cause tumors is through targeting CBP/p300 resulting in the loss of function of p53. This novel mechanism may be responsible for the development of these and other related solid tumors. Oncogene (2001) 20, 6653 ± 6659. Keywords: EWS-ATF-1; CBP; p53; Ewing's sarcoma; clear cell sarcoma Introduction Soft tissue clear cell sarcoma or malignant melanoma of soft parts is rare and aggressive neuroectodermal tumor that occurs in the deep soft tissue usually near tendons and aponeuroses (Epstein et al., 1984). Recent molecular characterization of this tumor reveals recurrent chromosomal translocation t(12;22) (q13;q12) resulting in the fusion of EWS gene to Activating Transcription Factor 1 (ATF-1) gene (Zucman et al., 1993). The EWS gene on chromosome 22 is a member of RNA binding protein family which includes TLS/FUS, hTAFII68, SARFH/Cabeza (Bertolotti et al., 1996; Immanuel et al., 1995; Stolow and Haynes, 1995). Chromosomal translocations associated with EWS gene are found in Ewing family tumors and other solid tumors. In these tumors, the EWS gene is

*Correspondence: ESP Reddy Received 23 January 2001; revised 23 May 2001; accepted 31 May 2001

fused to various transcriptional factors like ETS family members Fli-1 (Ben-David et al., 1991; Prasad et al., 1992; Delattre et al., 1992), erg (Rao et al., 1987; Reddy et al., 1987; Sorensen et al., 1994), ETV1 (Jeon et al., 1995), E1A-F (Kaneko et al., 1996; Urano et al., 1996), FEV (Peter et al., 1997), Tumor suppressor gene WT-1 (Gerald et al., 1995), orphan nuclear receptor TEC (Labelle et al., 1995), and C/EBP family CHOP (Panagopoulos et al., 1996). In addition, TLS/FUS, a gene closely related to EWS, is also shown to be involved in chromosomal translocations with erg in human myeloid leukemia (Ichikawa et al., 1994; Panagopoulos et al., 1994), and with CHOP in myxoid liposarcoma (Crozat et al., 1993; Rabbitts et al., 1993). EWS has several conserved RNA binding motifs called RNP-CS and RGG boxes (Burd and Dreyfuss, 1994). We have shown that EWS preferentially binds to poly G and poly U RNA (Ohno et al., 1994). The RNA binding activity of EWS is located in the RGG box (Ohno et al., 1994) which is present in the carboxy terminal region. In these aberrant chimeric proteins, this carboxyl terminal RNA binding domain of EWS is replaced by DNA-binding domain of several transcriptional factors. EWS-Fli-1, EWS-erg and TLS/FUS-erg have been shown to function as sequence speci®c transcriptional activators (Ohno et al., 1993, 1994; May et al., 1993; Bailly et al., 1994; Prasad et al., 1994). Antagonizing EWS-fusion gene expression in the tumors showed reduced tumorigenicity and clonogenicity, suggesting that a certain threshold level of expression of these chimeric products is needed for maintaining oncogenicity (Ouchida et al., 1995). We have found that over expression of aberrant fusion proteins (EWS-erg, EWS-Fli-1 and TLS/FUS-erg) and its normal counterpart (Fli-1 and erg) in mouse ®broblasts inhibit apoptosis induced by either serum deprivation or calcium ionophore treatment, indicating the anti-apoptotic role of these fusion proteins in these tumors (Yi et al., 1997). In the EWS-ATF-1 protein, the amino-terminal domain of EWS is juxtaposed with DNA binding domain of ATF-1 (Figure 1). ATF-1 is a member of CREB/ATF basic leucine-zipper type of transcriptional factor family, which includes CREB and CREM. CREB and ATF-1 share signi®cant homology in Kinase inducible domain (KID), basic and leucine-

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Figure 1 Schematic representation of the functional domains of EWS-ATF-1 and CBP. (a) Normal EWS, ATF-1, and their chimeric product EWS-ATF-1 are shown. Arrowhead indicates breakpoint of the fusion protein. Carboxy-terminal region of EWS containing RGG box, which functions as an RNA binding domain, is shown. RNP-CS represents the domain conserved in a variety of RNA binding proteins. Basic and leucine zipper domain of ATF-1 responsible for DNA binding and dimerization are shown. The amino-terminal region of ATF-1 containing the Protein Kinase A (PKA) site is replaced by amino-terminal half of EWS in EWS-ATF-1. (b) Schematic representation of co-activator CBP is shown. Three cysteine histidine rich region and Bromodoamin are shown. The region possess Histone Acetyl-Transferase (HAT) is indicated. KIX represents CREB binding site (aa446 ± 684)

zipper domain. Increasing concentrations of cAMP or calcium induce phosphorylation of KID domain of CREB resulting in augmented transactivation properties of CREB. The interaction between CBP and CREB is dependent on the phosphorylation status of Ser 133 in the KID domain, which represents a protein kinase A phosphoacceptor site (Chrivia et al., 1993; Arias et al., 1994). Similarly, when phosphorylated at Ser 63 (corresponding to Ser 133 of CREB), ATF-1 can bind to CBP and show augmented transcriptional activation to certain target promoters (Shimomura et al., 1996). In EWS-ATF-1, the N-terminal region of KID domain including the PKA phosphoacceptor site at Ser 63 was replaced by the N-terminal region of EWS gene (Figure 1), resulting in the loss of the phosphoacceptor site. We and others have demonstrated that EWS-ATF-1 functions as a constitutive transcriptional activator on certain target promoters (Fujimura et al., 1996; Brown et al., 1995). CBP/p300 are large nuclear phospho-proteins which function as cofactors to various transcriptional factors and seem to have a key role in growth and cell cycle control, cellular dierentiation and development. The growing numbers of factors that are found to be Oncogene

regulated by CBP/p300 include CREB, ATF-1, c-Myb, c-fos, c-Jun, STATs, GATA-1, Ets, MyoD1 and nuclear receptors (reviewed in Giles et al., 1998). Recent studies show that p53 also requires this cofactor for its transcriptional activation function (Lill et al., 1997; Avantaggiati et al., 1997; Gu et al., 1997; Somasundaram and El-Deiry, 1997). In these pathways, CBP/p300 is thought to work as a signal integrator between multiple transcriptional factors. CBP/p300, which interact with other cofactors such as NcoA-1, pCIP-1 and TFH110, has histone acetyl transferase activity that makes the holoenzyme complex to transmit the activating signals. CBP/p300 and other cofactors are thought to participate in chromatin remodeling through its histone acetylation activity to open chromatin structure. Both histones and other transcriptional factors including p53 are acetylated by CBP/p300 resulting in increased ability to bind target DNA sequences suggesting a broad function for CBP/ p300 (Gu and Roeder, 1997). The translocation of CBP has been found in therapy-related myelodysplasia and acute myeloid leukemia. In these translocations CBP is fused to MLL/ALL-1 and MOZ respectively, although the functional study of these fusion genes remains to be


determined (Satake et al., 1997; Borrow et al., 1996; Gu et al., 1992). Several DNA viral oncoproteins were shown to target transcriptional cofactor CBP/p300 suggesting that this phenomenon may contribute to neoplasia. We speculated whether a similar mechanism for cellular transformation is used by EWS-ATF-1 fusion protein. To test this hypothesis, we studied the interaction of EWS-ATF-1 and CBP. In this study, we show that EWS-ATF-1 associates constitutively with CBP and represses the p53-mediated transcriptional activation function through regulation of CBP/ p300. To our knowledge, this is the ®rst report showing that aberrant chimeric proteins inhibit p53 signaling pathways by squelching transcriptional coactivator CBP.

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Results EWS-ATF-1 interacts with CBP both in vitro and in vivo Both CREB and ATF-1 proteins were shown earlier to bind to KIX domain of CBP. To test whether EWSATF-1 binds to CBP/p300, we performed co-immunoprecipitation assays with in vitro translation products of EWS-ATF-1 and CBP (KIX domain) (Figure 2a). Both EWS-ATF-1 and KIX domain of CBP (aa446 ± 684) were co-translated and labeled with 35S-methionine (Figure 2a, lane 1), then subjected to immunoprecipitation with anti-ATF-1 monoclonal antibody. KIX domain of CBP was co-immunoprecipitated along with EWS-ATF-1 (Figure 2a, lanes 5, 6). Similar results were observed when EWS-ATF-1 and KIX domain of

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Figure 2 EWS-ATF-1 interacts with CBP in vitro and in vivo. (a) Co-immunoprecipitation studies of in vitro translated CBP and EWS-ATF-1. EWS-ATF-1 or CBP RNAs were translated in vitro either alone or together with CBP RNA. These [35S]methioninelabeled translated products were immunoprecipitated with monoclonal antibody raised against carboxy-terminal region of ATF-1. Lane 1, In vitro co-translation of EWS-ATF-1 and CBP (aa446 ± 684); Lane 2, In vitro translation of EWS-ATF-1; Lane 3, In vitro translation of CBP (aa 446 ± 684) (KIX domain of CBP); Lane 4, EWS-ATF-1 and CBP proteins were synthesized separately and then mixed and subjected to immunoprecipitation with monoclonal antibody raised against carboxy-terminal region of ATF-1; Lanes 5 and 6, EWS-ATF-1 and CBP proteins were synthesized together by co-translation and then this reaction mixture was immunoprecipitated with the above mentioned ATF-1 monoclonal antibody. Lane 7, represents in vitro translation carried out in the absence of RNA (negative control); Lane 8, in vitro translated EWS-ATF-1 was immunoprecipitated with anti-ATF-1 antibody; Lane 9, in vitro translated CBP (aa446 ± 684) was immunoprecipitated with anti-ATF-1 antibody. (b) GST pull down assay. Schematic representation of EWS-ATF-1 deletion mutants used in the in vitro binding studies are shown. GST or GST-CBP (aa 446 ± 684) was incubated with in vitro translated [35S]methionine-labeled proteins and the bound complexes were characterized by polyacrylamide gel electrophoresis and visualized by autoradiography. Lanes 1 ± 5 represents input of in vitro translation products. Lanes 6 ± 15, represent [35S]methionine-labeled products bound to GST and GST-CBP. (c) Puri®ed GST and GST-CBP (aa 446 ± 684) proteins used for GST pull down assays are shown by Coomassie staining. (d) Immunoblot analysis of SU-CCS-1 whole cell extracts with anti-ATF-1 monoclonal antibody after immunoprecipitation with CBP (lane 3), normal rabbit IgG (lane 2). Lane 1, detection of EWS-ATF-1 in whole cell extract without immunoprecipitation Oncogene


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CBP were translated separately (Figure 2a, lanes 2 and 3), then mixed and subjected to the immunoprecipitation analysis (Figure 2a, lane 4). Since a kinase regulatory domain of EWS-ATF-1 is replaced by the EWS regulatory domain (Fujimura et al., 1996), we speculated that phosphorylation of ATF1 fusion domain by PKA may not be necessary to bind to CBP. To investigate the nature of the interaction in vitro, CBP-KIX domain was expressed as a GST-fusion protein, and then tested its ability to bind to in vitro translated EWS-ATF-1 using GST pull-down assay (Figure 2b,c). Consistent with our previous results, EWS-ATF-1 binds to GST ± CBP (446 ± 684) whereas unphosphorylated ATF-1 does not bind under these experimental conditions. In order to further understand mechanisms of interaction, we in vitro translated EWS N-terminal fusion domain and ATF-1 C-terminal fusion domain separately (Figure 2a) and subjected them to GST pulldown assay (Figure 2b). Neither EWS N-terminal half nor ATF-1 C-terminal half showed detectable interaction with KIX domain of CBP under these experimental conditions, indicating that fusion partners by themselves are not sucient to bind to CBP (Figure 2b; lanes 13, 15). It appears that the aberrant fusion of EWS to ATF-1 may cause conformational change to the fusion protein that facilitates binding to CBP. Next, we asked whether this interaction occurs in vivo in malignant melanoma of soft part (MMSP). SUCCS-1 is one of MMSP cell lines, which expressed EWS-ATF-1 (Brown et al., 1995). We tested whether immunoprecipitation of CBP brings down EWS-ATF-1 in co-immunoprecipitation assays (Figure 2d). Using anti-CBP antibody, we obtained a band with a molecular mass around 60 kDa (Figure 2d, lane 3). This band co-migrates with the EWS-ATF-1 band obtained from Western analysis (Figure 2d, lane 1). We conclude from these results that EWS-ATF-1 associates with CBP both in vitro and in vivo. EWS-ATF-1 represses p53-mediated trans-activation Recent studies revealed that p53 requires CBP/p300 for its transcriptional activation function (Lill et al., 1997; Avantaggiati et al., 1997; Gu et al., 1997). Since EWSATF-1 interacts with CBP, we hypothesized that EWSATF-1 may sequester CBP/p300 (which is needed for p53 transactivation function) leading to the inhibition of p53-mediated trans-activation function. To test this hypothesis, we studied the eect of EWS-ATF-1 on p53-mediated transcriptional activation function. U2OS cells, which express wild type p53 (Kastan et al., 1992), were transfected with various amounts of EWSATF-1 expression plasmid along with p53 reporter and CAT assays were performed (Figure 3a). EWS-ATF-1 repressed p53-mediated trans-activation in a dose dependent manner (Figure 3a). Nearly half of the activity (49%) compared to control was observed when 1 mg of EWS-ATF-1 expression plasmid was added to the cells. To eliminate the possibility that EWS-ATF-1 aects endogenous p53 expression levels, we also

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checked these eects in Saos-2 cells which do not express p53 (Figure 3b). We co-transfected p53 expression plasmid along with EWS-ATF-1 expression plasmid into Saos-2 cells. Again, EWS-ATF-1 revealed similar extent of repression (55%) of p53-mediated transcriptional activation (Figure 3b). We wanted to know whether sequestration of CBP is involved in this repressive eect. To test this hypothesis, we transfected increasing concentration of CBP along with EWSATF-1 (Figure 3c). Interestingly, over expression of CBP relieved EWS-ATF-1 mediated repression in a dose dependent manner (Figure 3c). These results suggest that EWS-ATF-1 interfere with p53-mediated transactivation by sequestration of CBP/p300 proteins. Discussion CBP/p300 plays a pivotal role in transcriptional control of broad aspects of cellular regulation including dierentiation, homeostasis, and growth control. In this study, we have shown that EWS-ATF-1 associates with CBP both in vitro and in vivo through the KIX domain of CBP (Figure 2a ± c). We also demonstrate that EWS-ATF-1 fusion protein interferes with the p53-mediated transcriptional activation through competition between p53 and EWS-ATF-1 in binding to CBP (Figure 3). The tumor suppressor gene p53, which is mutated in several human cancers, plays an important role in the regulation of cell cycle and DNA repair (Levine, 1997). The transcriptional activity of p53 is regulated through the binding with CBP/p300 cofactor. Viral oncoproteins such as adenovirus E1a, HTLV-I Tax, and human papilloma virus E6 are known to interfere with p53 mediated trans-activation function through the binding with CBP and was suggested that this may be in part responsible for transformation by these viruses. Inhibition of histone acetylase activity of CBP by E1a, which is involved in remodeling of chromatin structure, might explain one of the consequences of inhibition. In the case of human T-cell leukemia virus type-I (HTLV-I) viral protein Tax, it's competition with p53 in binding to CBP might account for repression of the p53mediated transcriptional activity (Ariumi et al., 2000). HTLV-1 TAX binds to KIX domain of CBP. Recent studies reveal that p53 also binds to KIX domain of CBP besides the carboxy terminal and C/H1 region (Van Orden et al., 1999). Both p53 and Tax were shown to bind to KIX domain in a mutually exclusive fashion. It is possible to assume such squelching mechanism is operative in EWS-ATF-1 since EWSATF-1 also binds to KIX domain. There is an increasing body of evidence indicating that the gene dosage of CBP/p300 is critical to keep proper cellular function. The patients of hereditary disease Rubinstein-Taybi syndrome are heterozygous at CBP locus resulting in developmental abnormality. In some of these cases, one allele is inactivated by large deletion suggesting haplo-insuciency model of this disease. These patients are prone to develop neural and


developmental tumors (Miller and Rubinstein, 1995). Truncated mutations of p300 were found in epithelial cancers which was accompanied inactivation of the second allele (Gayther et al., 2000). In a mouse model, CBP nullizygous mutants die in the mid-gestation. The hemizygous mutants of CBP develop leukemia supporting the suggestion that certain level of expression of CBP is required to prevent tumor development (Kung et al., 2000). Taken together, these ®ndings support the view that CBP/p300 has a tumor suppressor function. It appears from our results that aberrant fusion proteins and viral proteins follow similar mechanism in cellular transformation. This includes targeting transcriptional cofactors (such as CBP/p300 etc.) that play an essential role to transmit and combine several signal transduction cascades. Therefore, this novel molecular mechanism may be responsible for the initiation and/or progression of solid tumors involving EWS-ATF-1. It is possible other EWS/TLS chimeric proteins may use similar pathway in transformation.

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Materials and methods Plasmids PG13CAT and MG15CAT were kindly provided by B Vogelstein (Kern et al., 1992). pRc/CMV mCBP-HA-1 was a gift from R Goodman. Construction of EWS-ATF-1 and deletion mutants were described elsewhere (Fujimura et al., 1996). Wild type p53 was cloned into pSG5 vector. CREB interaction domain (aa446 ± 684) of CBP was generated by PCR using pcDNA3/CBP as a template and cloned into pGex2TK (Amersham Pharmacia Biotech). Tissue culture and transfection Saos-2 cells and U-2 OS cells were purchased from the American Type Culture Collection (Rockville, MD, USA). SU-CCS-1 cells were obtained from AL Epstein. Saos-2 cells were grown in McCoy's 5A medium supplemented with 15% c

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Figure 3 EWS-ATF-1 represses p53-mediated trans-activation in a dose-dependent manner. (a) U-2 OS human osteosarcoma cells

were transiently transfected with 2 mg of p53 reporter plasmid PG13-CAT, 2 mg of pCMVb (b-galactosidase) along with various amounts of expression vector encoding EWS-ATF-1. All CAT values were normalized to each other based on the respective bgalactosidase activity. Percentage of the control was calculated based on CAT value of the equimolar empty vector transfected cells. Error bars are standard deviations of three independent experiments. (b) Saos-2 cells were co-transfected with 2 mg of PG13CAT, 10 mg of pCH110 (b-galactosidase as internal control), 0.1 mg of p53 expression plasmid along with various concentrations (1, 3 or 10 mg) of EWS-ATF-1 expression plasmid or pcDNA3 empty vector. Percentage of the control was calculated based upon fold activation of CAT value of the empty vector transfected cells. Error bars are standard deviations of three independent experiments. (c) Overexpression of CBP relieves the repressive eect of EWS-ATF-1 on p53-mediated trans-activation. U-2 OS cells were transfected with 1 mg of EWS-ATF-1 expression vector, 2 mg of PG13-CAT, 2 mg of pCMVb (as an internal control) along with indicated amount of full-length CBP expression vector. The value obtained with the reporter alone was arbitrarily set as 100. Results shown are average of three independent experiments with standard deviations Oncogene


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fetal bovine serum, 50 units/ml penicillin, 50 mg/ml streptomycin. Both U-2 OS cells and SU-CCS-1 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 mg/ml streptomycin. The cells were transfected by calcium phosphate precipitation method on a 10-cm plate. Forty-eight hours after transfection, the cells were harvested and chloramphenicol acetyl transferase (CAT) activity and b-galactosidase activity were measured. CAT activity was measured using Fuji Bioimage analyser and transfection eciency was normalized using bgalactosidase activity as previously described (Fujimura et al., 1996). Co-immunoprecipitation and Western blot assay Con¯uent 10-cm plates of SU-CCS-1 cells were washed twice with cold phosphate-buered saline and lysed in lysis buer with freshly added protease inhibitors (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, 0.5% IGEPAL CA630 (SIGMA), 0.1 mM PMSF and complete protease inhibitor cocktail (Boehringer Mannheim)). After incubation on ice for 20 min, the lysate was centrifuged at 20 000 g for 10 min at 48C. Supernatants were diluted with one volume of lysis buer without IGEPAL CA-630 and subjected to immunoprecipitation using 1 mg anti-CBP antibody (A-22) or control sera (Santa Cruz). Immunocomplex were crosslinked to 15 ml protein A/G PLUS Agarose (Santa Cruz) for overnight at 48C. The beads were washed three times with lysis buer containing 0.25% of IGEPAL CA-630. Proteins were solubilized with SDS sample buer and separated on

10% SDS ± PAGE gels. After the transfer, membrane was incubated in blocking solution (5% blocking materials (BioRad) in 16TBS) for overnight. Western blot assay were performed using anti-ATF-1 mouse monoclonal antibody (25C10G) (Santa Cruz) as primary antibody. Immunocomplex were detected using chemiluminescence-based system (Amersham) as described by the manufacturer. GST Pull-down assay GST-CBP (KIX) and GST were expressed and puri®ed as described previously. For in vitro binding assay, 10 ml of 35Smethionine labeled in vitro translated proteins was diluted with binding buer (20 mM Tris-HCl [pH 7.8], 150 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol) and incubated with 2 ml of GST ± CBP KIX beads or GST beads for 1 h at 48C. The beads were washed ®ve times with binding buer, boiled in SDS sample buer and loaded on 12% SDS-polyacrylamide gel. The gel was stained with Coomassie brilliant blue, dried and scanned using a Fuji BioImaging analyser.

Acknowledgments We thank other colleagues of Reddy and Rao's laboratories for their kind cooperation. This work is supported in part by NIH grants RO1 CA 85343, RO1 CA 58642 and US Army medical research and command grant DAMD17-99-1-9060 to ESP Reddy and CA 57322 to VN Rao.

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