Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides.
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.
Toretsky
J A
JA
Pediatric Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Connell
Y
Y
Neckers
L
L
Bhat
N K
NK
D001859:Bone Neoplasms / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D018274:Electroporation / Q000592:standards; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D005820:Genetic Testing; D006801:Humans; D008297:Male; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D011471:Prostatic Neoplasms; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012512:Sarcoma, Ewing / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000502:physiology
D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins