title: Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-kappaB activation in mouse embryonic stem cells.
abstract: SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5-1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IkappaBalpha degradation leading to inhibition of NF-kappaB activation. Further detailed analysis revealed that IkappaBalpha is a direct substrate of SAG-SCF(beta-TrCP) E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-kappaB.
authors.affiliation: Division of Radiation and Cancer Biology, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Mingjia
authors.initials: M
authors.lastname: Tan
authors.forename: Yueming
authors.initials: Y
authors.lastname: Zhu
authors.forename: Jordan
authors.initials: J
authors.lastname: Kovacev
authors.forename: Yongchao
authors.initials: Y
authors.lastname: Zhao
authors.forename: Zhen-Qiang
authors.initials: ZQ
authors.lastname: Pan
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Spitz
authors.forename: Yi
authors.initials: Y
authors.lastname: Sun
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects; D020672:I-kappa B Proteins / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics; D011839:Radiation, Ionizing; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000528:radiation effects; D015533:Transcriptional Activation / Q000235:genetics / Q000528:radiation effects; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D020672:I-kappa B Proteins; D016328:NF-kappa B; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D044767:Ubiquitin-Protein Ligases; C104998:1-stearoyl-2-arachidonoylglycerol kinase; D017853:Phosphotransferases (Alcohol Group Acceptor)