Basic Study for The Gene-Targeted Therapy in Lung cancer

研究了L-myc反义核酸对高表达L-myc基因的人小细胞肺癌细胞株NCI-H209的增殖抑制作用。合成的15聚体寡脱氧核苷硫代磷酸(OPT)主要定位于细胞核，少量存在于细胞质中，并呈浓度依赖关系抑制细胞增殖。此外，将翻译起始点为L-myc的反义DNA作用于该细胞系，发现L-myc蛋白呈浓度依赖性地抑制细胞的增殖。反义OPT对小细胞肺癌细胞株的生长抑制作用与L-myc基因的表达水平相关。与反义OPT具有相同核苷酸长度但随机顺序的混合序列对照OPT对细胞增殖没有影响。正义OPT处理后对细胞增殖有一定的抑制作用，这种抑制可能是OPT非序列特异性抑制，而不是常规的反义基因抑制。在反义处理的NCI-H209细胞中，L-MYCmRNA表达增强，L-MYC蛋白表达降低。结合L-myc基因的独特特点，包括1)在小细胞肺癌中频繁扩增和过度表达，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基因是基于分子生物学诊断的小细胞肺癌反义基因治疗的很好的候选靶基因。

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.