Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA
Inhibition of human cancer cell growth by inducible expression of human ribonucleotide reductase antisense cDNA
复制标题
DOI：
10.1089/oli.1.2000.10.111
复制
发表时间：
2000-04-01
期刊：
影响因子：
--
通讯作者：
Yen, Y
中科院分区：
文献类型：
--
作者：
Chen, SY;Zhou, BS;Yen, Y
核糖核苷酸还原酶(RR)是DNA合成和修复中的限速酶。该酶由两个不同的亚基M1和M2组成，M2亚基在肿瘤的发生和转移中起着重要作用。本研究利用诱导型载体系统，将RRM1和M2反义基因导入人口咽癌细胞中。Kb-M1AS、Kb-M2AS和Kb-CAT分别代表含有M1反义、M2反义和CAT报告基因的转基因克隆。在克隆形成实验中，异丙基硫代半乳糖(IPTG)诱导M2反义mRNA表达时，KB-M2AS克隆的集落形成率下降了约50%，而在IPTG诱导下，KB-M1AS克隆的克隆形成率没有明显的抑制。通过(CDP)-C-14还原实验测定RR酶活性，显示IPTG诱导的KB-M2AS克隆在144小时时比未诱导的KB-M2AS克隆降低30%。Northern印迹分析显示，IPTG诱导后48h和144h M2反义mRNA表达达到高峰，144h时M2反义mRNA表达为未诱导对照的33倍，Western印迹分析显示，诱导后72h，KB-M2AS克隆中M2亚基蛋白水平开始下降，144h继续下降至未诱导对照的50%，168h略有恢复。综上所述，通过诱导系统表达M2反义基因可以有效地降低RR M2蛋白的表达，降低酶活性，抑制生长。此外，该方法还可用于未来的反义研究。
Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis and repair. The enzyme consists of two dissimilar subunits, M1 and M2, It is known that the M2 subunit plays a role in tumorgenicity and metastasis, In this study, we transfected human oropharyngeal KB cancer cells with human RR M1 and M2 antisense cDNA expressed by an inducible vector system. The transfectants were double-selected with hygromycin and G418, The clones, designated KB-M1AS, KB-M2AS and KB-CAT, represented transfectant clones that contained M1 antisense cDNA, M2 antisense cDNA, and a CAT reporter gene, respectively. In a colony-forming assay, colony formation for the KB-M2AS clone decreased approximately 50% when M2 antisense mRNA expression was induced by isopropylthiogalactose (IPTG), However, the KB-M1AS clone revealed no significant inhibition under IPTG induction. RR enzyme activity, as measured by (CDP)-C-14 reduction assay, revealed a 30% decrease in the IPTG-induced KB-M2AS clone relative to non-IPTG-induced samples at 144 hours. As shown by Northern blot, expression of the M2 antisense mRNA showed peaks at 48 hours and 144 hours after induction by IPTG, M2 antisense mRNA expression induced by IPTG was 33-fold greater than the uninduced control at 144 hours, Western blot analysis showed that the M2 subunit protein level decreased in the KB-M2AS clone beginning at 72 hours after induction and continued to decrease to 50% of the uninduced control at 144 hours, then showed a slight recovery at 168 hours. In conclusion, M2 antisense mRNA expression by an inducible system can effectively decrease RR M2 protein expression, reduce enzyme activity, and inhibit growth. Furthermore, this approach can be employed in future antisense investigations.