INHIBITION OF EWS-FLI-1 FUSION PROTEIN WITH ANTISENSE OLIGODEOXYNUCLEOTIDES

Ewing's sarcoma family of tumors (EFT) contain reciprocal translocatio ns, of which approximately 90% occur between the long arm of chromosom es 11 and 22, t(11;22)(q24;q12), resulting in the formation of chimeri c proteins generated by a fusion of the EWS and FLI-1 genes. To determ ine if EWS-FLI-1 protein is responsible for the Ewing sarcoma phenotyp e 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 translat ion of the fusion messenger RNA. ODN were first evaluated in a cell-fr ee in vitro translation system. Exogenously added RNase I-I was found to be required for translation inhibition. bi 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 We stern blot analysis. A 40-60% decrease in the fusion protein was obser ved in TC-32 cells with antisense ODN directed toward the breakpoint r egion. Cell viability was reduced with antisense sequences in TC-32 ce lls but not in a prostate cancer cell line. Since inhibition of t(11;2 2) gene product is correlated to effects on cell viability, reduction of the fusion protein may thus offer insight into the biology of EFT.