Abstract
Ewing's sarcoma (EWS) cells contain levels of poly(ADP-ribose) polymerase (PARP) significantly 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 effector of PARP transcription. Because PARP levels have been associated with cellular resistance or sensitivity to genotoxic agents, we studied the effect 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 effects. Because down-regulation EWS/FLI-1 does not dramatically change PARP levels, these results suggest a direct effect 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 effective by causing both an increase in sensitivity to therapeutic agents and a simultaneous down-regulation, or reversion, of the neoplastic phenotype of EWS cells.
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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:
-
sequence-scrambled ODNs
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Acknowledgements
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 17-00-1-0019 (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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Soldatenkov, V., Trofimova, I., Rouzaut, A. et al. Differential regulation of the response to DNA damage in Ewing's sarcoma cells by ETS1 and EWS/FLI-1. Oncogene 21, 2890–2895 (2002). https://doi.org/10.1038/sj.onc.1205393
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DOI: https://doi.org/10.1038/sj.onc.1205393
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