Prechnical research on gene therapy for oral cancer using a RAS dominant negative mutant

在口腔癌中，RAS癌基因的异常表达是肿瘤生长和转移的关键事件。本研究利用复制缺陷重组腺病毒载体作为基因治疗的基础实验，测试RAS显性阴性突变体N116Y对人舌癌细胞生长的抑制作用。感染N116Y腺病毒（Ad.CMV-N116Y）而不感染由巨细胞病毒启动子驱动表达的LacZ腺病毒（Ad.CMV-LacZ），可显著降低舌癌细胞株（HSC-3、HSC-4、SAS）的体外生长，并诱导其形态异常和凋亡。为了研究N116Y的抑制机制，采用Western blot检测细胞外信号调节激酶（ERR）的激活情况。感染Ad。CMV-N116Y在血清饥饿的HSC-3细胞中抑制EGF刺激后ERK的激活。为了评估N116Y对舌癌细胞生长的肿瘤特异性细胞毒性，我们构建了由人类端粒酶逆转录酶（hTERT）启动子驱动的复制缺陷重组N116Y腺病毒载体（Ad.hTERT-N116Y）或对照病毒载体Ad。hTERT和Ad显示舌癌特异性细胞毒性。hTERT-N116Y而不是Ad。使用MTT法进行hTERT检测。这些发现表明N116Y是人类舌癌基因治疗的潜在候选基因。

In oral cancers, the abnormal expression of RAS oncogene is a critical event in tumor growth and metastasis. This study was performed to test the efficacy of a dominant negative RAS mutant, N116Y, in blocking the growth of human tongue cancer cell lines using a replication-deficient recombinant adenoviral vector as basic experiments for gene therapy. Infection with N116Y adenovirus (Ad.CMV-N116Y) but not with LacZ adenovirus (Ad.CMV-LacZ) in which the expression was driven by the cytomegalovirus promoter, significantly reduced the in vitro growth and induced abnormal morphology and apoptotic picnosis of the tongue cancer cell lines (HSC-3, HSC-4, SAS) studied. To examine the suppressive mechanism of N116Y, the activation of extracellular-signal regulated kinase (ERR) were examined by Western blot analysis. Infection with Ad.CMV-N116Y suppressed the activation of ERK after EGF stimulation in serum-starved HSC-3 cells. To evaluate the tumor specific cytotoxicity of N116Y for suppressing growth of tongue cancer cell lines, we also constructed a replication deficient recombinant N116Y adenovirus vector driven by human telomerase reverse transcriptase (hTERT) promoter (Ad.hTERT-N116Y) or a control virus vector Ad.hTERT and showed tongue cancer-specific cytotoxicity by Ad.hTERT-N116Y but not by Ad.hTERT using the MTT assay. These findings indicate that N116Y is a potential candidate gene for human tongue cancer gene therapy.