title: Application of RNAi inducible TNFRI knockdown cells to the analysis of TNFalpha-induced cytotoxicity.
abstract: RNA interference (RNAi) has become a popular tool for downregulating in many species including mammalian cells. Therefore, suppression of target genes in mammalian cultured cells using RNAi may represent an ideal alternative to knockout studies for understanding the molecular mechanisms of chemical toxicity. Here, we assessed the potential of RNAi mediated gene knockdown in HeLa and HepG2 cells to cytotoxicity studies. Tumor necrosis factor receptor I (TNFRI) was chosen as a target gene because its signaling has been implicated in xenobiotic-induced toxicity. We optimized the design and performance of a vector-based RNAi experiment and then investigated viability of both HeLa and HepG2 cells exposed to TNFalpha. In addition, we examined gene expression profile of TNFRI knockdown HeLa cells after TNFalpha treatment, and then protein expression levels for several apoptosis-related genes of the cells. In both HeLa and HepG2 cells, TNFalpha exposure resulted in significantly reduced susceptibility of the knockdown cells to the cytotoxicity as compared with those of mock-transfected cells. Furthermore, the gene expression profiling and western blotting revealed that several genes including apoptosis and/or NF-kappaB pathway were downregulated in the knockdown HeLa cells. These results suggest that downregulation of the TNFRI gene in both HeLa and HepG2 cells by RNAi participates in resistance to TNFalpha-induced cytotoxicity. Therefore, this study raises the possibility that RNAi-based gene silencing in mammalian cells may be a valuable tool for elucidating the relationships between phenotypic changes and target gene functions in response to xenobiotic-induced cytotoxicity. Further exposure study using xenobiotics needs to be done to validate the potential utility of RNAi technology.
authors.affiliation: Chemicals Evaluation and Research Institute, Japan, 1600 Shimo-Takano, Sugito, Kitakatsushika, Saitama 345-0043, Japan. saito-fumiyo@ceri.jp
authors.forename: Fumiyo
authors.initials: F
authors.lastname: Saito
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yokota
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Sudo
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Yakabe
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D015153:Blotting, Western; D053181:Caspase 8 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D030781:Organisms, Genetically Modified; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
chemical_list: D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D001761:Bleomycin; C105427:Zeocin; D053181:Caspase 8