Toretsky J A, Connell Y, Neckers L, Bhat N K
Pediatric Branch, National Cancer Institute, Bethesda, MD 20892, USA.
J Neurooncol. 1997 Jan;31(1-2):9-16. doi: 10.1023/a:1005716926800.
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.
尤因肉瘤家族性肿瘤（EFT）存在相互易位，其中约90%发生在11号和22号染色体长臂之间，即t(11;22)(q24;q12)，导致由EWS和FLI-1基因融合产生嵌合蛋白。为了确定EWS-FLI-1蛋白是否导致尤因肉瘤表型，我们使用序列特异性反义寡脱氧核苷酸（ODN）来阻断其表达。我们评估了一系列针对断点区域的反义ODN，以阻止融合信使核糖核酸的翻译。ODN首先在无细胞体外翻译系统中进行评估。发现外源性添加核糖核酸酶H是抑制翻译所必需的。将显示完全抑制翻译的ODN电穿孔导入EFT细胞系TC-32细胞中。通过蛋白质免疫印迹分析评估融合蛋白和EWS蛋白水平。在导入针对断点区域的反义ODN的TC-32细胞中，观察到融合蛋白减少了40 - 60%。反义序列降低了TC-32细胞的活力，但对前列腺癌细胞系无此作用。由于对t(11:22)基因产物的抑制与对细胞活力的影响相关，融合蛋白的减少可能因此为深入了解EFT的生物学特性提供线索。
