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

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 2α (eIF-2α). 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-κB (NF-κB) 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-κB and Akt because both NF-κB 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-κB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

The double-stranded (ds) RNA-activated protein kinase PKR is a serine/threonine kinase that is ubiquitously expressed in mammalian cells (1). This kinase was first described as a mediator of the antiviral actions of IFN (2, 3). In addition to IFN and dsRNA, many other stimuli can activate PKR, such as cellular stresses (4), tumor necrosis factor-α (TNF-α) (5), the transcription factor E2F-1 (6), lipopolysaccharide (7), and the protein activator of PKR (PACT/RAX; refs. 8–11). Once PKR is activated, it changes its conformation and undergoes autophosphorylation (12). This activation of PKR kinase leads to the phosphorylation and activation of several downstream targets, including protein phosphatase 2A (13) and the α-subunit of the eukaryotic initiation factor eIF-2. Phosphorylation and activation of eIF-2α inhibits protein synthesis through the down-regulation of translation initiation (14, 15). PKR is involved in multiple pro- and antiapoptotic pathways in normal and cancer cells. Specifically, investigators have shown that PKR interacts with STAT1 (16, 17), p53 (18), Fas-associated death domain (19), TNF receptor–associated factor (20), IκB kinase (IKK; ref. 21), MKK6 (22), and apoptosis signal-regulating kinase-1 (23). Furthermore, PKR can regulate the expression of cyclin D1 (24), c-Myc (25), matrix metalloproteinase-9 (26), and E-selectin (27), and the activation of p38 mitogen-activated protein kinase (28) and c-Jun NH₂-terminal kinase (29) has been shown to be impaired in PKR-deficient cells. In addition, researchers have described the activation of nuclear factor-κB (NF-κB) by PKR through the phosphorylation and activation of IKKβ kinase complex, which then phosphorylates and targets the NF-κB inhibitor IκBα for ubiquitination. Removal of IκBα allows the active NF-κB p65-p50 complex to translocate into the nucleus (30).

To date, there have been no reports about the effect of PKR on ionizing radiation therapy. Because many of the above-mentioned pathways are involved in radiation resistance, we evaluated the role of PKR in radiation sensitivity. Our manuscript shows that PKR wild-type (PKR^+/+) mouse embryo fibroblasts (MEF) are much more resistant to ionizing radiation therapy than their PKR knock-out (PKR^−/−) counterparts. Additionally, we show that this protective effect is reversed by the inhibition of PKR with the chemical inhibitor 2-aminopurine (2-AP), and plasmid transfection of the PKR wild-type (wt) gene into PKR^−/− MEFs induces radiation resistance. Our results also suggest that PKR exerts this protective effect through the activation of NF-κB and Akt because only PKR^+/+ cells show up-regulation of these pathways following ionizing radiation. Additionally, inhibition of Akt with Deguelin leads to increased radiation sensitivity in PKR^+/+ cells. This manuscript suggests a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-κB and Akt activation.

Cell lines. PKR^+/+ and PKR^−/− MEFs were provided by one of the authors (G.N. Barber). Cells were maintained in high-glucose DMEM supplemented with 15% fetal bovine serum, 10 mmol/L glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies Invitrogen) at 37°C in 5% CO₂/95% air in a humidified incubator.

Clonogenic survival assay. A clonogenic survival assay was used to investigate the sensitivity of MEFs to radiation therapy as described previously (31). Briefly, increasing doses of radiation (2, 4, or 6 Gy) were given using a ¹³⁷Cs source at a dose rate of 3.7 Gy/min. The MEFs were trypsinized and plated in 100-mm dishes to assay for their colony-forming ability immediately after irradiation. Colonies were counted 10 to 14 days later. Survival curves were plotted using the Sigma Plot software program (Systat Software, Inc.).

PKR and Akt inhibitors. 2-AP, a chemical inhibitor of PKR (Sigma Chemical Co.), was used to inhibit PKR activation in MEFs. PKR^+/+ and PKR^−/− MEFs were pretreated with culture medium containing 1 mmol/L 2-AP for 2 h before irradiation. Deguelin, a natural product extracted from different plants species, was used to inhibit Akt phosphorylation (32). Deguelin was kindly provided by Ho-Young Lee, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center. PKR^+/+ MEFs were pretreated with culture medium containing 100 nmol/L Deguelin for 48 h before irradiation.

Plasmid transfections. To further investigate the role of PKR in radiation therapy, a plasmid containing the wild-type PKR gene or an empty vector plasmid was transfected into MEFs using the Nucleofector system (Amaxa GmbH) according to manufacturer's protocol.

Flow cytometry analysis. To assess the induction of apoptosis following radiation exposure, MEFs were stained using propidium iodide (PI) followed by fluorescence-activated cell sorting (FACS) as described previously (33).

Electrophoretic mobility shift assay. To evaluate the activation of NF-κB following radiation therapy, electrophoretic mobility shift assay (EMSA) was done as described previously (34).

Western blot analysis. Expression of PKR and downstream targets after irradiation was examined using Western blot analysis. This technique has been well described (35). The following primary antibodies were used: the PKR antibody B-10 (Santa Cruz Biotechnology), P-PKR pT451 and P-eIF-2α pS52 (Biosource International), Akt and P-Akt (Cell Signaling Technology), and FLICE-inhibitory protein (cFLIP; Imgenex).

For immunoprecipitation Western blot analysis, beads were incubated at 4°C overnight with one of the following antibodies: PKR antibody B-10 (Santa Cruz Biotechnology) or P-PKR pT451 (Biosource International). Cell lysates were then incubated with these antibody-conjugated beads, and immunoblotting was done as described.

Immunofluorescence staining. The effect of irradiation on PKR, Akt, P-Akt, and NF-κB p65 activation and localization was examined using immunofluorescence. Cells were plated in six-well plates on cover slides. After treatment, cells were fixed with 2% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were blocked with 1% normal goat serum for 1 h and then incubated overnight at a dilution of 1:100 with the primary antibody [PKR, NF-κB p65 (Santa Cruz Biotechnology), P-PKR pT451, PeIF-2α pS52, Akt, or P-Akt (Cell Signaling)]. The slides were then washed and incubated with a FITC- or rhodamine-conjugated secondary antibody (Invitrogen) for 1 h. The slides were then mounted with the ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) and analyzed under an Olympus FluoView FV500 laser confocal microscope (Olympus America) after adjustment for background staining.

PKR^+/+ MEFs are resistant to ionizing radiation therapy. To investigate the role of the PKR in PKR MEF sensitivity to radiation, we did clonogenic cell survival assays using MEFs that express PKR (PKR^+/+) and MEFs that do not express PKR (PKR^−/−). As shown in Fig. 1A, PKR^+/+ MEFs proved to be much more resistant to ionizing radiation than their PKR^−/− counterparts. The survival fraction of PKR^+/+ MEFs after 4 Gy was reduced to 0.2739, whereas the survival fraction of PKR^−/− MEFs was 0.0394. The presence of wt PKR in MEFs resulted in increased radioresistance by a factor of 2.02, calculated at the surviving fraction of 0.1 by dividing radiation dose of the PKR^+/+ MEFs curve with that of the corresponding PKR^−/− MEFs.

Plasmid transfection of PKR into PKR^−/− MEFs leads to radiation resistance. We then evaluated the effect of restoring PKR expression in PKR^−/− MEFs by transfecting PKR^−/− MEFs with a plasmid containing the PKR gene or the empty vector plasmid pcDNA3.1. We used the full length of PKR plasmid for our experiment. We obtained this plasmid from our co-author Dr. G. Barber (University of Miami). PKR^−/− MEFs transfected with the PKR wt plasmid showed increased radiation resistance after transfection. As shown in Fig. 1B, the survival of PKR^−/− MEFs after 4 Gy was 0.0689, whereas the survival of PKR gene transfected MEFs was 0.1217. Restoration of PKR in PKR^−/− MEFs resulted in increased radioresistance by a factor of 1.15 at the surviving fraction of 0.1, suggesting that expression of PKR resulted in enhanced radioresistance in MEFs.

Blocking PKR with 2-AP sensitizes MEFs to radiation therapy. To further examine the role of PKR in the radioresistance of MEFs, we treated PKR MEFs with the PKR inhibitor 2-AP. As shown in Fig. 1C, PKR^+/+ MEFs showed less radiation resistance after inhibited by 2-AP. The survival fraction of PKR^+/+ MEFs after 4 Gy was reduced to 0.3516, whereas the survival fraction of PKR^+/+ MEFs treated with 2-AP was 0.1508. Inhibition of PKR by 2-AP in PKR^+/+ MEFs resulted in decreased radioresistance by a factor of 0.72 (calculated at the surviving fraction of 0.1 by dividing radiation dose of the PKR^+/+ MEFs treated with 2-AP with that of the corresponding PKR^+/+ MEFs). The PKR^−/− MEFs showed no change in radiosensitivity with 2-AP, suggesting that the effect of 2-AP was specific for PKR (Fig. 1D).

PKR^+/+ MEFs show decreased apoptosis after radiation therapy. To determine the cellular mechanisms of radioresistance induced by PKR, we evaluated the impact of PKR expression on apoptosis induction after ionizing radiation. Using PI staining and FACS analysis, we found that induction of apoptosis was markedly reduced in the PKR^+/+ MEFs, compared with the PKR^−/− MEFs, suggesting that expression of wt PKR blocked radiation-induced apoptosis (Fig. 2A and B). Apoptotic cell death was examined in terms of changes in cell morphology by Hoechst 33342 staining. Radiation-treated PKR^−/− MEF cells displayed many apoptotic bodies (Fig. 2C).

PKR is activated by ionizing radiation therapy. We then examined the effect of radiation on PKR expression in PKR^+/+ and PKR^−/− MEFs by Western blot analysis. We found increased expression of PKR following ionizing radiation in PKR^+/+ but not PKR^−/− cells (Fig. 3A). Radiation therapy also led to PKR activation with increased phosphorylated PKR (P-PKR) in PKR^+/+ MEFs as shown by IP experiments (Fig. 3B). Immunofluorescence analysis also showed increased PKR expression in the cytoplasm around the nucleus (Fig. 3C). These immunofluorescent studies also showed PKR activation by increased P-PKR and P-eIF-2α levels 24 h after irradiation. The expressions of P-PKR and P-eIF-2α were inhibited by treatment with low-dose 2-AP, suggesting functional activity of the inhibitor (Fig. 3C).

Irradiation leads to NF-κB activation in PKR^+/+ MEFs. We then evaluated potential mediators of PKR radiation resistance including NF-κB (30). We used EMSA to assess NF-κB–DNA binding activity in PKR^+/+ and PKR^−/− MEFs after ionizing radiation therapy. As Fig. 4A shows, PKR^+/+ MEFs show activation of NF-κB after irradiation, which is reduced in PKR^−/− cells. Immunofluorescence also showed NF-κB activation through p65 translocation in PKR^+/+ MEFs after irradiation. Translocation of the NF-κB p65 subunit to the nucleus 4 h after ionizing radiation was shown only in the PKR^+/+ cells, but not in PKR^−/− cells (Fig. 4B). Western blot analysis also showed increased expression of cFLIP in irradiated PKR^+/+ MEFs. cFLIP is a catalytically inactive variant of caspase-8, the expression of which is induced by NF-κB (Fig. 4C) and may explain the reduced apoptosis noted following radiation treatment in PKR^+/+ cells (Fig. 2).

Irradiation leads to Akt activation in PKR^+/+ MEFs. We also investigated the role of the pro-survival Akt pathway because of data suggesting that TNF-α–induced PKR activation led to Akt phosphorylation only in PKR^+/+ cells.⁸

PKR^+/+ MEFs exhibited dose- and time-dependent increases in phosphorylation of Akt after radiation, whereas PKR^−/− MEFs did not show Akt phosphorylation (Fig. 5A and B). This Akt activation was also shown by immunofluorescent staining where levels of P-Akt were significantly higher in PKR^+/+ MEFs than in PKR^−/− MEFs (Fig. 5C) after radiation.

Inhibition of Akt with Deguelin suppresses the radioprotective effect of PKR. To further examine the role of Akt in the radioresistance of MEFs, we treated MEFs with the Akt inhibitor Deguelin. As shown in Fig. 5D, inhibition of P-Akt expression by 25% as determined by Western blot analysis and densitometry in PKR^+/+ MEFs showed partial restoration of radiation sensitivity following Deguelin therapy; the survival fraction of PKR^+/+ MEFs was 0.3480 after 4 Gy and was reduced to 0.2525 with Deguelin pretreatment. These results suggest that Akt activation may play a role in the radioprotective effects of PKR.

The dsRNA-activated protein kinase, PKR, is a critical gene in the cellular antiviral response. Through phosphorylation of the α subunit of the eukaryotic translation initiation factor 2, protein synthesis is dramatically inhibited, and cellular growth is inhibited. Overexpression of PKR has also been associated with the induction of apoptosis in cancer cells following the transduction of the proapoptotic adenoviral vectors Ad-TNF, Ad-Mda7, and Ad-E2F-1. Increased PKR expression has been associated with increased tumor killing presumably through increased cellular killing. Some authors have postulated that increasing PKR activity has a net tumor-suppressive effect through the up-regulation of apoptotic pathways. Despite this data, the role of PKR as a tumor suppressor is far from clear, with evidence that PKR activation can lead to neoplastic progression in melanoma and colon cancer cells with decreased sensitivity to conventional chemotherapy agents presumable through the up-regulation of pro-survival pathways such as NF-κB. The role of PKR in radiation therapy has not been previously reported. In the present study, we show for the first time that in MEFs, PKR is protective to the cytotoxic effects of radiation therapy. We also present evidence that PKR activation can lead to the up-regulation of the pro-survival Akt pathway, and this activation seems dependent on the presence of the PKR gene.

Our study shows that relative to PKR^+/+ MEFs, PKR^−/− MEFs are hypersensitive to ionizing radiation therapy. The PKR gene confers an overall protective cell to MEFs undergoing radiation therapy. To preclude the possibility that the difference was due to differences between the knock-out and wild-type MEFs genetic background, plasmid transduction of PKR was done in PKR^−/− MEFs. Transduction of wild-type PKR partially restored the radiation-protective effects of PKR^+/+ cells, suggesting that this gene was the critical factor. Inhibition of PKR activity with the chemical inhibitor 2-AP also reduced the protective effect of PKR only in PKR^+/+ cells, suggesting that this was the critical pathway (36).

We also found increased induction of apoptosis in the PKR^−/− MEFs when compared with the PKR^+/+ MEFs. These findings regarding the induction of apoptosis in response to ionizing radiation therapy and increased radiosensitivity in PKR^−/− MEFs are consistent with the findings of a previous study (37) that showed that PKR^−/− MEFs are much more sensitive to DNA-damaging agents, such as cisplatin, than their PKR^+/+ counterparts are, supposedly because of impaired DNA damage repair mechanisms. In addition, we found that PKR is up-regulated and activated following ionizing radiation therapy. This activation was shown by the phosphorylation of the PKR downstream target PeIF-2α. Again, this activation was inhibited by 2-AP. These observations lead to the proposal that ionizing radiation therapy induces PKR activation, which induces pro-survival pathways protective of cellular cytotoxic effects of radiation therapy.

Consistent with this hypothesis is the observation that PKR activation is involved in proapoptotic as well as antiapoptotic pathways. In addition, impairment of survival pathways in the absence of PKR may play an important role in determining sensitivity to radiation therapy. We therefore examined some of these survival pathways. In particular, we discovered the activation of NF-κB, which has been described previously (38), 2 to 4 h after ionizing radiation therapy. Our findings confirm that NF-κB is activated following ionizing radiation therapy as shown by the results of EMSA and increased expression of NF-κB p65 in the nucleus. Furthermore, ionizing radiation therapy induced expression of cFLIP, a downstream target of NF-κB. The activation of NF-κB by PKR seems to mediate in part cell survival after ionizing radiation therapy. Our data concerning NF-κB activation agree with those of a previous study demonstrating that PKR may act as a molecular clock by inducing cell survival through the activation of NF-κB before induction of cell death by phosphorylation of eIF-2α (39). In our MEFs, this induction of cell survival seemed to be a more important pathway than did the induction of apoptosis.

Akt is a serine/threonine kinase that has been shown to be an important mediator of survival pathways in different normal and human tumor cells (40). Akt can be activated by several agents. Once Akt is phosphorylated and activated, it promotes cell survival by phosphorylating its downstream targets, such as Bad, forkhead transcription factors, IKK, cyclic AMP-responsive element binding protein, glycogen synthase kinase 3, murine double minute 2, p21 (41), mammalian target of rapamycin (42), and caspase-9 (43). This mediates direct, transcriptional, and metabolic regulation of apoptosis (43). However, the mechanism by which Akt is activated has not been elucidated. Because our laboratory showed Akt activation following TNF-induced activation of PKR,⁹

we evaluated the role of this pro-survival pathway in radiation-protective effects of PKR. We show dose-dependent phosphorylation of Akt in PKR^+/+ MEFs following radiation therapy that is not seen in PKR^−/− cells. These findings suggest a possible interaction between Akt and PKR. However, there have been no published findings on this potential interaction. Further studies are therefore warranted to confirm this interaction. Our finding that Akt inhibition by Deguelin partially restores radiation sensitivity suggests a potential therapeutic approach to increase radiation sensitivity through the inhibition of the PKR-induced pro-survival pathway Akt.

In summary, our manuscript shows for the first time that the IFN-inducible dsRNA-activated protein kinase PKR induces radiation resistance in MEFs potentially through the up-regulation of the pro-survival NF-κB and Akt pathways. It will be important to determine if PKR induces radiation resistance in human cancer cells as well. Because many tumors are radiation resistant, these findings would provide insight into novel therapeutic strategies to overcome radiation resistance either through PKR inhibition or down-regulation of the pro-survival NF-κB or Akt pathways. Our results provide evidence that PKR is involved in a novel pathway that is protective to the toxic effects of ionizing radiation therapy.

We thank Kwang S. Ahn for his assistance with EMSA.