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Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides
J A Toretsky1, Y Connell, L Neckers
1Pediatric Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Abstract:
Ewing's sarcoma family of tumors (EFT) contain reciprocal translocations, of which approximately 90% occur between the long arm of chromosomes 11 and 22,t(11;22)(q24;q12) resulting in the formation of chimeric proteins generated by a fusion of the EWS and FLI-1 genes. To determine if EWS-FLI-1 protein is responsible for the Ewing sarcoma phenotype we have used sequence-specific antisense oligodeoxynucleotides (ODN) to block its expression. We have evaluated a series of antisense ODN directed toward the breakpoint region in an effort to prevent translation of the fusion messenger RNA. ODN were first evaluated in a cell-free in vitro translation system. Exogenously added RNase H was found to be required for translation inhibition. ODN that showed complete inhibition of translation were electroporated into TC-32 cells, a EFT cell line. Fusion protein and EWS protein levels were evaluated by Western blot analysis. A 40-60% decrease in the fusion protein was observed in TC-32 cells with antisense ODN directed toward the breakpoint region. Cell viability was reduced with antisense sequences in TC-32 cells but not in a prostate cancer cell line. Since inhibition of t(11:22) gene product is correlated to effects on cell viability reduction of the fusion protein may thus offer insight into the biology of EFT.
Insights
Antisense oligodeoxynucleotides targeting the EWS-FLI-1 fusion gene reduced its expression in Ewing sarcoma cells. This inhibition of the EWS-FLI-1 fusion protein correlated with decreased cell viability, offering insights into Ewing sarcoma family of tumors biology.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Ewing sarcoma family of tumors (EFT) are characterized by specific chromosomal translocations.
- The most common translocation, t(11;22)(q24;q12), results in the EWS-FLI-1 fusion gene and its chimeric protein.
- The role of the EWS-FLI-1 fusion protein in the EFT phenotype requires further elucidation.
Purpose of the Study:
- To investigate the functional role of the EWS-FLI-1 fusion protein in EFT.
- To assess the efficacy of antisense oligodeoxynucleotides (ODN) in inhibiting EWS-FLI-1 expression.
- To correlate EWS-FLI-1 inhibition with cellular effects in EFT.
Main Methods:
- Sequence-specific antisense ODN were designed to target the breakpoint region of the EWS-FLI-1 fusion messenger RNA.
- ODN efficacy was tested in a cell-free in vitro translation system with RNase H.
- Antisense ODN were electroporated into TC-32 EFT cells, and protein levels were analyzed via Western blot.
Main Results:
- Antisense ODN targeting the EWS-FLI-1 breakpoint effectively inhibited translation in vitro when supplemented with RNase H.
- Electroporation of antisense ODN into TC-32 cells led to a 40-60% reduction in EWS-FLI-1 fusion protein levels.
- A significant decrease in TC-32 cell viability was observed with antisense ODN treatment, while a prostate cancer cell line remained unaffected.
Conclusions:
- Antisense ODN can effectively reduce EWS-FLI-1 fusion protein expression in EFT cells.
- Inhibition of the EWS-FLI-1 fusion protein is associated with reduced cell viability in EFT.
- Targeting the EWS-FLI-1 fusion product holds potential for understanding and treating Ewing sarcoma family of tumors.