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。本研究采用复制缺陷型重组腺病毒载体作为基因治疗的基础实验，检测显性负性RAS突变体N116 Y对人舌癌细胞系生长的抑制作用。N116 Y腺病毒（Ad. CMV-N116 Y）感染舌癌细胞株（HSC-3、HSC-4、SAS）后，细胞生长明显下降，细胞形态发生异常，并出现凋亡。为了研究N116 Y的抑制机制，通过Western印迹分析来检测细胞外信号调节激酶（ERR）的激活。在血清饥饿的HSC-3细胞中，用Ad. CMV-N116 Y感染抑制EGF刺激后ERK的活化。为了评价N116 Y对舌癌细胞株的特异性杀伤作用，我们构建了人端粒酶逆转录酶（hTERT）启动子驱动的复制缺陷型重组N116 Y腺病毒载体（Ad. hTERT-N116 Y）和对照病毒载体Ad.hTERT，并通过MTT法检测了Ad. hTERT-N116 Y对舌癌细胞株的特异性杀伤作用。这些结果表明，N116 Y是一个潜在的候选基因，为人类舌癌的基因治疗。

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.