The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor B and Akt Activation

Authors:
von Holzen U, Pataer A, Raju U, Bocangel D, Vorburger SA, Liu Y, Lu X, Roth JA, Aggarwal BB, Barber GN, Keyomarsi K, Hunt KK, Swisher SG
In:
Source: Clin Cancer Res
Publication Date: (2007)
Issue: 13(20): 6032-9
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic fibroblast, mouse (MEF) immort
Species: mouse
Tissue Origin: embryo
Platform:
Nucleofectorâ„¢ I/II/2b
Abstract
PURPOSE: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. EXPERIMENTAL DESIGN: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. RESULTS: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR(+/+) but not PKR(-/-) cells. CONCLUSIONS: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.