Basic Study for The Gene-Targeted Therapy in Lung cancer

我们研究了L-myc反义DNA对过表达L-myc基因的人小细胞肺癌（SCLC）细胞株NCI-H209的抗增殖作用。本研究使用的合成DNA为15mer oligodeoxynucleoside phosp硫代酸寡聚核苷（OPT），该DNA能快速融入NCI-H209细胞，且主要定位于细胞核，细胞质中定位较弱。将该细胞系暴露于转化翻译起始位点L-myc蛋白的5个L-myc反义DNA中，在1.6 ~ 10muM浓度范围内以浓度依赖性的方式抑制细胞增殖。此外，在包括NCI-H209在内的三种不同SCLC细胞系中，该反义DNA的生长抑制作用与L-myc表达水平相关。与反义OPT具有相同核苷酸长度但顺序随机的混合序列控制OPT对细胞增殖没有任何影响。感官OPT处理诱导了一定程度的细胞增殖抑制，这可能是一种非序列特异性的OPT抑制，独立于常规的反义基因抑制，这在其他系统中也有报道。在反义处理的NCI-H209细胞中观察到L-myc mRNA的丰富表达和L-myc蛋白的表达减少，这表明覆盖翻译起始位点的反义OPT通过抑制靶mRNA的核糖体翻译而不是通过抑制基因的转录起作用。结合L-myc基因的独特特征，包括1)在SCLC中经常扩增和过表达状态，以及2)在成人组织中表达非常有限和低水平，目前的数据表明，L-myc是基于分子生物学诊断的SCLC反义DNA治疗的良好候选靶基因。

We evaluated the antiproliferative effect of L-myc antisense DNA in NCI-H209, a human small cell lung cancer (SCLC) cell line overexpressing the L-myc gene.The synthetic DNA used in the present study was 15mer oligodeoxynucleoside phosphorothioate (OPT), which showed rapid incorporation into NCI-H209 cells, and which localized mainly in the cell nucleus and weakly in the cytoplasm.The exposure of this cell line to s L-myc antisense DNA convering the translational initiation site L-myc proteins inhibited the cell proliferation in a concentration-dependent manner through the concentrations of 1.6muM to 10muM.Furthermore, the growth inhibition by this antisense DNA was correlated with the level of L-myc expression in three different SCLC cell lines including NCI-H209.A mixed sequence control OPT having the same nucleotide length as the antisense OPT but in random, order, did not show any effect on cell proliferation.The sense OPT treatment induced the some inhibition of cell proliferation, which migh be non-sequence-specific inhibition by OPT independent of conventional antisense gene inhibition, which was reported in other systems.Abundant expression of L-myc mRNA and decreased expression of L-myc protein were observed in the antisense-treated NCI-H209 cells, suggesting that the antisense OPT covering the translational initiation site acted by inhibiting ribosomal translation of the target mRNA,rather than by inhibiting transcription from the gene.Together with unique characteristics of the L-myc gene, including 1) a frequently amplified and overexpressed state in SCLC,and 2) very restricted and low level expression in human adult tissues, the present data indicate that L-myc is a good candidate for the target gene for antisense DNA therapy in SCLC based on molecular biological diagonosis.