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

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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.