title: siRNA targeting NBS1 or XIAP increases radiation sensitivity of human cancer cells independent of TP53 status.
abstract: NBS1 is essential for the repair of radiation-induced DNA double-strand breaks (DSBs) in yeast and higher vertebrate cells. In this study, we examined whether suppressed NBS1 expression by small interference RNA (siRNA) could enhance radiation sensitivity in cancer cells with different TP53 status. We used human non-small cell lung cancer cells differing in TP53 gene status (H1299/wtp53 cells bearing wild-type TP53 or H1299/mp53 cells bearing mutant TP53). A DNA cassette expressing siRNA targeted for the NBS1 gene was transfected into those cell lines, and radiation sensitivity was examined with a colony-forming assay. Cellular levels of NBS1 and other proteins were analyzed using Western blotting. We found that the radiation sensitivity of H1299/wtp53 and H1299/mp53 cells was enhanced by transfection of the DNA cassette. In the NBS1-siRNA-transfected cells, we observed decreased constitutive expression of NBS1 protein and decreased radiation-induced accumulation of phosphorylated NBS1 protein. In addition, radiation-induced expression of the transcription factor NF-kappaB (NFKB) and XIAP (X-chromosome-linked inhibitor of apoptosis protein) was suppressed by NBS1-siRNA. Enhanced X-ray sensitivity after NBS1-siRNA transfection was achieved in TP53 wild-type cells and sensitivity was even more pronounced in TP53 mutant cells. The transfection of siRNA targeted for XIAP also enhanced X-ray sensitivity even more for TP53 mutant cells compared to TP53 wild-type cells. Our data suggest that the sensitization to radiation results from NBS1-siRNA-mediated suppression of DNA repair and/ or X-ray-induced cell survival signaling pathways through NFKB and XIAP. siRNA targeting appears to be a novel radiation-sensitizing agent, particularly in human TP53 mutant cancer cells.
authors.affiliation: Department of Biology, Nara Medical University School of Medicine, Kashihara, Nara, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Ohnishi
authors.forename: Zorica
authors.initials: Z
authors.lastname: Scuric
authors.forename: Robert H
authors.initials: RH
authors.lastname: Schiestl
authors.forename: Noritomo
authors.initials: N
authors.lastname: Okamoto
authors.forename: Akihisa
authors.initials: A
authors.lastname: Takahashi
authors.forename: Takeo
authors.initials: T
authors.lastname: Ohnishi
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism* / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D020868:Gene Silencing / Q000528:radiation effects; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000008:administration & dosage; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents; D016159:Tumor Suppressor Protein p53; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human