Oncogene (2002) 21, 2890 2895 2002 Nature Publishing Group All rights reserved 0950 9232/02 $25.

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Dierential regulation of the response to DNA damage in Ewing's sarcoma cells by ETS1 and EWS/FLI-1
Viatcheslav A Soldatenkov1, Irina N Tromova1, Ana Rouzaut1, Frank McDermott1, Anatoly Dritschilo1 and Vicente Notario*,1
1

Department of Radiation Medicine, Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, Washington DC 20007, USA

Ewing's sarcoma (EWS) cells contain levels of poly (ADP-ribose) polymerase (PARP) signicantly higher than other eukaryotic cells. Previously, we cloned the PARP gene promoter region from EWS cells, showed that it contained multiple ETS-binding sites and demonstrated a positive regulation of PARP by ETS1. We now report that, contrary to ETS1, EWS/FLI-1, an aberrant ETS transcription factor present in most EWS cells, is a negative eector of PARP transcription. Because PARP levels have been associated with cellular resistance or sensitivity to genotoxic agents, we studied the eect of modifying PARP levels in EWS cells on their response to DNA damage by modulating the expression of ETS1 or EWS/FLI-1 using antisense methodology. Results show that stable down-regulation of ETS1 increases the resistance of EWS cells to various genotoxic agents, whereas down-regulation of EWS/FLI-1 has pro-apoptotic eects. Because down-regulation EWS/FLI-1 does not dramatically change PARP levels, these results suggest a direct eect for EWS/FLI-1 in the DNA damage response of EWS cells. Since expression of the aberrant fusion proteins by EWS cells is essential for maintaining their neoplastic phenotype, our results suggest that the use of antisense oligonucleotides in combination with chemotherapeutic agents or radiation may be doubly eective by causing both an increase in sensitivity to therapeutic agents and a simultaneous down-regulation, or reversion, of the neoplastic phenotype of EWS cells. Oncogene (2002) 21, 2890 2895. DOI: 10.1038/sj/ onc/1205393 Keywords: antisense; apoptosis; ETS factors; poly(ADP-ribose) polymerase transcription

*Correspondence: V Notario, Experimental Carcinogenesis Laboratory, Department of Radiation Medicine, Georgetown University Medical Center, Research Building, Room E215, 3970 Reservoir Road, NW, Washington DC, 20007 USA; E-mail: notariov@georgetown.edu Received 27 December 2001; revised 31 January 2002; accepted 8 February 2002

Contrary to the majority of mammalian cell types, which express poly(ADP-ribose) polymerase (PARP) at relatively low levels, Ewing's sarcoma (EWS) cells contain high levels of PARP mRNA, protein and polymerase activity (Prasad et al., 1990). To understand the reason for this disparity, we cloned the promoter region of the PARP gene from EWS cells and showed that it encompassed multiple sites matching consensus binding motifs (A/C GGA A/T) for transcription factors of the ETS family (Boulukos et al., 1989; Wasylyk et al., 1993; Sementchenko and Watson, 2000). Further results demonstrated a coordinated regulation of PARP and ETS1 in EWS cells: transiently increased expression of ETS1 in A4573 EWS cells resulted in a strong enhancement (about four-fold) of PARP promoter activity, whereas transfection with antisense Ets1 cDNA constructs mediated a down-regulation of the endogenous levels of both ETS1 and PARP (Soldatenkov et al., 1999a). The Ewing family of tumors is dened by the presence, in nearly 100% of the cases, of characteristic chromosomal translocation events involving the EWS gene (Plougastel et al., 1993), which encodes a RNAbinding protein (Ohno et al., 1994), and genes encoding transcriptions factors of the ETS family such as FLI-1, ERG, ETV1, FEV or E1AF (Rao et al., 1993; Sorensen et al., 1994; Arvand and Denny, 2001). The translocations generate fusion proteins in which the RNA-binding domain at the C-terminus of EWS is replaced by the DNA-binding domain of the ETSfamily proteins (Zucman et al., 1993). The most frequent translocation, t(11;22)(q24;q12), is present in about 95% of EWS cases (Wei et al., 2000; De Alava et al., 2000) and generates a chimeric fusion EWS/ FLI-1 protein with transcription factor activity (Lin et al., 1999). In fact, EWS/FLI-1 fusion genes are heterogeneous: there are at least three breakpoints identied in FLI-1 and two more identied in EWS, resulting in several unique fusion proteins. Using Western analysis and polymerase chain reactions (PCR), with primers specic for the various possible fusion types, we have identied that A4573 EWS cells, our experimental model for high PARP expression, produce a type-3 EWS/FLI-1 fusion protein (data not shown).

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Because EWS/FLI-1 plays an essential role in the malignant phenotype of EWS cells (Ouchida et al., 1995; Kovar et al., 1996; Dohjima et al., 2000), we performed experiments to test whether EWS/FLI-1 had any inuence on the regulation of PARP expression. Transient co-transfection of A4573 cells with a SV40based EWS/FLI-1 expression vector and a PARPpromoter/reporter (cloramphenicol acetyl-transferase, CAT) construct resulted in a marked decrease (about 60% reduction) in PARP promoter activity, which was also reected in a reduction of 17.6+2.8% in the levels of PARP protein in the cells. This result implicated EWS/FLI-1 in the regulation of PARP expression in EWS cells and, interestingly, showed that EWS/FLI-1 and ETS1 had opposite eects on the transcriptional activity of the PARP promoter, suggesting that both transcription factors may be used to modulate the PARP content in EWS cells. It had been shown earlier that treatment of various types of human tumor cells, including EWS, with chemical inhibitors of PARP activity sensitized the cells to ionizing radiation (IR) (Thraves et al., 1986; Leith, 1988; Kelland et al., 1988), suggesting that the regulation of the levels of PARP in the cells played an important role in determining their response to IR and perhaps to other DNA-damaging agents. However, a drawback of the inhibitor studies was the fact that most inhibitors tested were analogs of NAD+, the substrate for PARP but also for a variety of other enzymes, and the observed radiosensitization eects may have resulted from their interaction with a number of enzymatic pathways functionally unrelated to PARP. Therefore, to overcome this limitation, and based on the results mentioned above, our approach to modulate the PARP content in EWS cells and study the eects on the cellular response to IR and other DNA damaging agents involved the use of an RNA antisense (AS) strategy against ETS1, a positive eector of PARP transcription. EWS A4573 cells were transfected with either a recombinant construct expressing AS ETS1 (Soldatenkov et al., 1999a) or the empty vector DNA and initially tested for their response to treatment with etoposide (VP-16). Transfection with AS ETS1 resulted in the isolation of a number of stably-transfected pooled populations with markedly reduced expression levels of ETS1 (AS-ETS-1 and -2, Figure 1a), whereas no dierence was observed in cells transfected with empty vector (A4573 neo, Figure 1a). Transfected ETS-AS-1 cells were used for further characterization. In agreement with our previous observations, the reduction in ETS1 expression in ETS-AS-1 cells correlated with a clear decrease in their PARP content (Figure 1d, UT lanes), suggesting that if results from the inhibitor studies were correct these cells may be more susceptible to DNA damaging agents. However, contrary to these expectations, we observed that, relative to vector-transfected cells (A4573 neo), ETSAS-1 cells were more resistant to the killing action of VP-16 (Figure 1b), as measured by the increase in the percentage of cells with mitochondrial potential break-

down at dierent times of treatment with VP-16 (5 mM). This increased resistance correlated also with decreased levels in ETS-AS-1 cells of caspase-3 activity (Figure 1c) and PARP cleavage (Figure 1d), two later events in VP-16-induced apoptosis (Robertson et al., 2000). Furthermore, this observation could be extended to treatments with several other genotoxic agents, including IR. In all cases tested, ow-cytometric analyses of annexin V-FITC/propidium iodide stained cells performed 24 h after each treatment showed that EWS A4573 cells expressing AS ETS-1 were consistently about 2 3-fold more resistant to apoptosis than vector-transfected, control cells (Table 1). This phenomenon was particularly evident when cells were treated with 5 mM VP-16 (threefold decrease in apoptosis), whereas a rather modest reduction (about 1.6-fold) in the apoptotic response of AS ETS-1 cells was observed after IR (8 Gy) treatment. The dierent degrees of resistance observed after treatment with these genotoxic agents may be a reection of their ability to cause dierent types of DNA damage. Overall, these results on the anti-apoptotic eect of AS ETS1 are in contrast with the pro-apoptotic described in mouse T- and B-lineage cells by inactivation of the Ets1 proto-oncogene (Bories et al., 1995), but they are in agreement with descriptions of a proapoptotic activity for the `sense' p42 isoform of ETS1 in colon cancer cells (Suzuki et al., 1995; Huang et al., 1997). It is possible that the anti- or pro-apoptotic eect of ETS1 or AS ETS1 may be determined by cell type-specic factors such as the endogenous PARP level, the expression of specic target genes or other regulatory mechanisms. On the basis of their opposite eect on PARP transcription, we then tested the eect of AS EWS/ FLI-1 on the cellular response of EWS cells to DNA damage. Our strategy involved the use of AS oligodeoxynucleotides (ODNs) which had been successfully employed previously to down-regulate EWS/FLI-1 expression (Tanaka et al., 1997; Matsumoto et al., 2001). Specically, we used ESAS2, a 25-mer AS ODN spanning the AUG translation-initiation codon of EWS/FLI-1 (Tanaka et al., 1997). Exponentially growing EWS A4573 cells were exposed to either AS (ESAS2) or to sequence-scrambled (SC), control (ESSCE) ODNs. Relative to cells exposed under identical conditions to SC ODN, treatment of EWS A4573 cells with increasing concentrations of AS ODN for 48 h resulted in a decrease (about 50%) in the expression of the EWS/FLI-1 protein (Figure 2a) and a marked inhibition in cell growth (Figure 2b). These results were consistent with data reported previously by other investigators on other EWS cell lines. Treatment of SK-N-MC cells with ESAS2 resulted in a signicant increase in the proportion of cells in G0/G1 (Tanaka et al., 1997) which was paralleled by increased expression of p21 and p27 and the downregulation of the expression of cyclins D1 and E (Matsumoto et al., 2001). However, it is worthwhile mentioning that A4573 cells seemed to be more sensitive to the growth inhibitory action of the AS ESAS2 ODN than

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Figure 1 Down-regulation of ETS1 inhibits apoptosis in Ewing's sarcoma cells. Expression levels of ETS1 protein in untransfected A4573 cells and in cells transfected with vector alone (A4573 neo) or AS ETS1 (AS-ETS-1 and -2) AS cells. (a) Apoptotic response to VP-16 was evaluated by measuring changes in mitochondrial potential (b), caspase-3 activation (c) and PARP cleavage (d) assays on the same cells. Materials and methods: EWS cell were cultured, maintained and transfected as described (Soldatenkov et al., 1999a). Western analysis was performed as described (Soldatenkov et al., 1999a), with an anti-ETS1 monoclonal antibody (Transduction Laboratories, Lexington, KY, USA). Immunodetection of GAPDH with a polyclonal antibody (Trevigen Inc., Gaithersburg, MD, USA) is shown as loading control. A4573 neo and ETS-AS-1 cells were exposed to VP-16 for the indicated times and changes in the mitochondrial potential were evaluated by ow cytometric analysis of cells stained with the potential-sensitive lipophilic cation DePsipher (Trevigen). Cell death was quantied as percentage of cells showing mitochondrial potential breakdown. Caspase-3 activity was assayed by measuring the proteolysis of the specic substrate DEVD-pNA (ApoAlert Caspase-3 assay kit, Clontech, Palo Alto, CA, USA) as recommended by the manufacturers. Enzyme activity is expressed as relative units. Data show mean values+standard error from three independent experiments. PARP cleavage in apoptotic cells was determined by Western analysis of total cell extracts (25 mg protein) with an anti-PARP monoclonal antibody (Enzyme Systems Products, Livermore, CA, USA). The positions of native PARP (116 kDa) and the proteolytic cleavage product (85 kDa) are indicated

Table 1 Eect of antisense ETS on apoptosis of EWS cells by DNA-damaging agents Treatment None Cisplatin (1 mM) VP-16 (5 mM) Camptothecin (0.5 mM) Ionizing radiation (8 Gy) A4573neo 1.63+0.3 32.35+5.2 29.80+4.6 39.60+5.1 17.45+3.2 Cell type ETS-AS 2.54+0.4 14.92+3.0 10.01+3.2 19.62+3.7 10.78+2.8

Results indicate relative percentage of apoptosis in cell cultures 24 h after the treatments shown. Annexin V-FITC staining was used to identify apoptotic cells. Data correspond to mean values+s.d. from three independent tests

previously tested cell lines (RD-ES, SK-N-MC, SK-ES-1, Tanaka et al., 1997). To investigate the eect of AS EWS/FLI-1 ODNs on the response to DNA damage, A4573 cells were exposed to ESAS2 (5 or 10 mM) or SC control (10 mM) for 48 h, followed by treatment with VP-16 (5 mM) for an additional 24 h period. DAPI staining was used to identify cells undergoing apoptosis as exhibiting condensed and fragmented nuclei (Figure 2c, inset). Quantitation of the percentage of apoptotic
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cells showed that AS treatment resulted in a marked sensitization of the EWS cells to apoptotic action of the DNA-damaging agent. These results agree with previous observations (Yi et al., 1997) that AS EWS/ FLI-1 or AS EWS/ERG RNAs were able to sensitize EWS TC135 and 5838 cells, respectively, to etoposide and actinomycin D. Interestingly, AS EWS/FLI-1 RNA was reported to cause a dramatic decrease (about 90%) in the expression of the fusion EWS/ FLI-1 protein which resulted in an apoptosis rate of 40% after 48 h of treatment with VP-16. Our results indicate that similar levels of apoptosis can be attained with a reduction of only about 50% in EWS/FLI-1 expression, in agreement with observations from other AS ODN systems in which it has been shown that a 40 50% inhibition of the target gene product is sucient to elicit a biological response (Gokhale et al., 1999). Our results clearly show that down-regulation of two ETS transcription factors which have opposite eects on the transcriptional regulation of PARP in EWS cells also had reverse eects on the cellular response to DNA damaging agents. Remarkably, down-regulation of PARP by ETS1 AS (Soldatenkov et al., 1999a)

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Figure 2 EWS/FLI-1 antisense oligonucleotides sensitize Ewing's sarcoma cells to apoptosis. (a) Down-regulation of EWS/FLI1 protein expression. (b) Growth inhibition of A4573 cells by EWS/FLI-1 antisense oligonucleotides. (c) Sensitization of EWS A4573 cells to VP-16 induced apoptosis. Inset: DAPI staining of control A4573 cells (UT) and cells exposed to 5 mM VP-16. Materials and methods: For analyses of EWS/FLI-1 protein expression, A4573 cells were incubated with the indicated concentrations of the antisense (ESAS2) or scrambled (ESSC2) oligonucleotides described previously (Tanaka et al., 1997) in medium containing 1% serum. Cells were harvested and lysed 48 h after treatment and total lysates were subjected to Western analysis with an anti-FLI-1 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and conrmed by RT PCR, as described (Zucman et al., 1993). GAPDH detection was used as loading reference. Conditions for Western analyses were as described in Figure 1 legend. For studies of cell growth, cells were treated with antisense (ESAS2) or scrambled (ESSC2) oligomers as above, and the number of viable, trypan blue-excluding cells was assessed 48 h post treatment. Results are expressed as the percentage relative to the cell number in control, untreated samples (cells cultured in complete medium) as the 100%. All experiments were carried out in triplicate and repeated twice. The error bar represents mean and s.d. of the two separate experiments. To study the eect of AS ODNs on VP-16-induced apoptosis, A4575 cells were treated with 10 mM of ESAS2 or ESSC2 ODNs for 48 h, as above, and exposed to 5 mM VP-16 for another 24 h. Following treatment, cells were stained with DAPI (0.5 mg/ml, Sigma Chemical Co., St Louis, MO, USA) and visualized by epiuorescence microscopy using an Olympus Vanox AH-2 microscope (Olympus, Melville, NY, USA). A minimum of 400 500 cells was examined in ve to seven randomly selected elds and apoptotic cells exhibiting condensed and fragmented nuclei (DAPI positive) were scored (Soldatenkov et al., 1999b). Values are expressed as percentage of the total number of nuclei counted. Data represent the mean+standard error from three independent experiments

resulted in increased resistance to DNA damage, whereas the slightly increased PARP levels elicited by EWS/FLI-1 AS ODNs correlated with increased sensitivity to DNA damage in a dose-dependent fashion. These ndings indicate that, in EWS cells, which have unusually high endogenous levels of PARP, changes in PARP per se may not be sucient to cause chemo- or radiosensitization and suggest a possible direct role for ETS1 and EWS/FLI-1 in determining the overall response of EWS cells to DNA damage. The present knowledge of ETS target genes related to apoptosis/DNA damage is quite limited in general, and especially so in the case of EWS cells. A recent

review (Sementchenko and Watson, 2000) included p53, NF-kB, several Bcl-2 family members, Fas and PARP in this category. Other stress-response genes, such as GADD153, have been also described as targets for ETS1 and FLI-1 (Seth et al., 1999). But only PARP has been identied and characterized in EWS cells (Soldatenkov et al., 1999a). More recently, the gene encoding the type II receptor for TGF-b (TGFBR2) has been described as a target for aberrant ETS transcription factors such as EWS/FLI-1 (Hahm et al., 1999) and EWS/ERG or EWS/ETV1 (Im et al., 2000). Therefore, it seemed logical that TGF-b signaling may mediate the sensitizing eect of AS EWS/FLI-1 to
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DNA damaging agents in EWS cells. This possibility appeared to be further strengthened by the observation that landmark molecular events during TGF-bmediated growth inhibition such as down-regulation of cyclin E or up-regulation of p21 and p27 (Massague and Polyak, 1995; Alexandrow and Moses, 1995) are essentially the same triggered by AS EWS/FLI-1 treatment of EWS cells (Tanaka et al., 1997; Matsumoto et al., 2001, as described above. However, the role of TGF-b signaling in DNA-damage response remains somewhat controversial, with certain reports indicating that TGF-b enhanced the lethal eects of DNA damaging agents (Raynal et al., 1997), whereas other studies described that blockade of TGF-b signaling sensitized cells to cytotoxic chemotherapy (Ohmori et al., 1998). It is possible that cell typespecic characteristics determine the quality of the eect of TGF-b on the cellular response to DNA damage. Further studies are necessary to establish a role for TGF-b signaling in our EWS experimental system. Because expression of the fusion proteins in EWS cells is known to be essential for maintaining the oncogenic and tumorigenic properties of the tumor cells (Ouchida et al., 1995; Kovar et al., 1996; Yi et al., 1997; Dohjima et al., 2000), the use of AS ODNs in combination with chemotherapeutic agents or radiation may be doubly eective by causing both an increase in the sensitivity to conventional therapeutic agents and a simultaneous down-regulation of the neoplastic phenotype of the tumor cells. Preliminary experiments have shown that AS EWS/FLI-1 ODNs targeted to the junction sequences of the chromosomal translocation characteristic of EWS were able to enhance their apoptotic response to IR and to prevent the oncogenic activity of the EWS/FLI-1 protein product (data not
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shown). We propose that junction-targeted AS ODNs will more specically render EWS cells sensitive to DNA-damaging agents because they will have a dual eect: enhancing EWS cell killing and down-regulating their neoplastic properties. In addition, the lack of target junction sequences in normal cells may contribute to minimize undesirable secondary eects. Recent advances for the delivery of AS ODNs in vivo (Gokhale et al., 1997, 1999) may help this approach to yield signicantly improved responses in the treatment of EWS, especially in recurrent and/or metastatic cases.
Abbreviations AS, antisense; CAT, chloramphenicol acetyl transferase; EWS, Ewing's sarcoma; IR, ionizing radiation; ODN, oligodeoxynucleotide; PARP, poly(ADP-ribose) polymerase; PCR, polymerase chain reaction; SC, sequencescrambled ODNs

Acknowledgments We are grateful to Dr DK Watson (Medical University of South Carolina, Charleston, SC, USA) for providing ETS1 and EWS/FLI-1 expression plasmids and for discussions and critical suggestions. This work was supported in part by National Institutes of Health grants PO1-CA74175 (to A Dritschilo) and CA64472 (to V Notario) from the National Cancer Institute and by grant DAMD 17-00-10019 (to VA Soldatenkov) from the US Department of Defense. Fluorescence microscopy and FACS analyses were performed using the Microscopy/Imaging and the Flow Cytometry/Cell Sorting Shared Resources of the Lombardi Cancer Center, Georgetown University, Washington, D.C, supported in part by US Public Health Service Grant P30-CA51008.

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