Differential induction and activation of NF-kappaB transcription complexes in radiation-induced chromosomally unstable cell lines.
Radiation-induced genomic instability is a delayed effect of ionizing radiation that may contribute to radiation carcinogenesis. Prior microarray studies investigating gene expression changes in genomically unstable cell lines isolated after radiation exposure uncovered the differential expression of the NF-kappaB p105 mRNA. In this study, the functionality of the NF-kappaB pathway was examined to determine its role in regulating gene expression changes after oxidative stress in chromosomally stable and unstable human-hamster hybrid clones. Basal DNA-binding activity assays showed no significant differences between the clones; however, further experiments established differences in NF-kappaB induction in three chromosomally unstable clones after acute hydrogen peroxide treatment. A second assay was used to confirm this differential activity in the chromosomally unstable clones by studying reporter gene activation after treatment with hydrogen peroxide. Yet an initial upstream analysis of the pathway revealed no significant increase in the level of IkappaBalpha inhibitor protein in the unstable clones. Downstream tests analyzing the induction of the antiapoptotic target protein Bcl-2 found variable induction among the stable and unstable clones. These differences did not translate to a reduction in clonogenic survival after acute exposure to oxidative stress, as the irradiated but chromosomally stable clone displayed the most sensitivity. Due to its role in regulating a diverse set of cellular functions, including responses to oxidative stress, alterations in the NF-kappaB pathway in chromosomally unstable clones may regulate the differential physiology of a subset of chromosomally unstable clones and could contribute to the perpetuation of the phenotype. However, a specific role for defective induction and activation of this pathway remains unidentified.
Snyder
Andrew R
AR
Molecular and Cell Biology Graduate Program, University of Maryland, Baltimore, Maryland, USA. asnyder@wistar.upenn.edu
Morgan
William F
WF
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D042822:Genomic Instability / Q000235:genetics / Q000528:radiation effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D051550:I-kappa B Kinase; D008156:Luciferases; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011839:Radiation, Ionizing; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation
D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D006861:Hydrogen Peroxide; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human