Application of antisense oligonucleotides for EWS-Flil in Ewing's sarcoma.

t（11;22）（q24：q12）易位是尤文氏肉瘤（ES）中检测到的一种特异性染色体异常。易位导致EWS-Flil融合基因。最近的研究评估了融合基因产物作为异常转录因子的转化潜力。然而，EWS-Flil的生物学意义仍然未知。我们发现EWS-Flil融合基因的表达水平与ES细胞的增殖活性之间存在相关性。当EWS-Flil表达被针对融合基因的反义寡核苷酸（AS）抑制时，ES细胞的生长在体外和体内均显著降低。流式细胞仪分析表明，AS对细胞的生长抑制作用是通过细胞周期G1期阻滞介导的。在本研究中，我们的目的是应用针对EWS-Flil的AS抑制ES在体内的生长。为了确定最有效的AS序列，我们使用计算机软件分析EWS-Flil mRNA结构，并确定可能是AS靶点的mRNA环。我们合成了这些AS，并研究了这些AS是否会抑制ES细胞在体外的生长。我们获得了能有效抑制ES细胞生长的AS序列。我们还证实了AS在体内的生长抑制作用。我们接下来研究了AS对ES细胞中三元共聚物诱导的影响。结果表明，AS对ES细胞的作用是抑制细胞生长的，不诱导ES细胞凋亡。因此，我们接下来检查了AS和抗肿瘤药物的联合作用。AS与拓扑异构酶抑制剂共处理可明显诱导ES细胞形成三聚体。这些结果表明，AS和拓扑异构酶抑制剂的联合化疗在临床试验中将是有效的，以最大限度地提高对ES的抗肿瘤作用。

The translocation t(11;22)(q24:q12) is a specific chromosomal abnormality detected in Ewing's sarcoma(ES). The translocation results in an EWS-Flil fusion gene. Recent studies have evaluated transforming potentials of the fusion gene products acting as an aberrant transcription factor. However, the biological significance of EWS-Flil is still unknown. We have found that there is a correlation between the expression levels of the EWS-Flil fusion gene and the proliferative activities of ES cells. When the EWS-Flil expression was inhibited by an antisense oligonucleotide (AS) against the fusion gene, the growth of the ES cells was significantly reduced both in vitro and in vivo. The flow cytometry analysis indicated that the growth inhibition of the cells by AS was mediated by G1 arrest in the cell cycle progression. In the present study, we aimed to apply AS against EWS-Flil to inhibit the growth of ES in vivo. To identify the most effective AS sequence, we analyzed EWS-Flil mRNA structure using computer software, and determined mRNA loops which were possible targets of AS. We synthesized these AS and examined whether these AS would inhibit ES cell growth in vitro. We obtained the AS sequence which could most effectively inhibit the growth of ES cells. We also confirmed the growth inhibitory effects of AS in vivo. We next examined the effects of AS on the induction ofapoptosis in ES cells. We found that the effects of AS on ES cell line was cytostatic and apoptosis was not induced in ES cells by AS. Thus, we next examined the combination effects of AS and anti-tumor drugs. Cotreatment with AS and topoisomerase inhibitors showed strong induction ofapoptosis in ES cells. These results suggest that the combination chemotherapy with AS and topoisomerase inhibitors would be effective to maximize the antitumor effects on ES in clinical trial.