The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation

Human Cancer Biology

The Double-Stranded RNA-Activated Protein Kinase Mediates Radiation Resistance in Mouse Embryo Fibroblasts through Nuclear Factor KBandAktActivation Urs von Holzen,1, 6 Abujiang Pataer,2 Uma Raju,3 Dora Bocangel,2 Stephan A.Vorburger,5 Ya nn a L iu ,1Xiaolin Lu,2 Jack A. Roth,2 Bharat B. Aggarwal,4 Glen N. Barber,7 Khandan Keyomarsi,4 Kelly K. Hunt,1and Stephen G. Swisher2

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 2a (eIF-2a).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-nB(NF-nB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined byWestern 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-nBandAkt because bothNF- nB 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-nB 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 phosphatase 2A (13) and the a-subunit of the eukaryotic serine/threonine kinase that is ubiquitously expressed in initiation factor eIF-2. Phosphorylation and activation of eIF- mammalian cells (1). This kinase was first described as a 2a inhibits protein synthesis through the down-regulation of mediator of the antiviral actions of IFN (2, 3). In addition to IFN translation initiation (14, 15). PKR is involved in multiple and dsRNA, many other stimuli can activate PKR, such as cellular pro- and antiapoptotic pathways in normal and cancer cells. stresses (4), tumor necrosis factor-a (TNF-a) (5), the transcrip- Specifically, investigators have shown that PKR interacts tion factor E2F-1(6), lipopolysaccharide (7), and the protein with STAT1 (16, 17), p53 (18), Fas-associated death domain activator of PKR (PACT/RAX; refs. 8–11). Once PKR is activated, (19), TNF receptor–associated factor (20), InB kinase (IKK; it changes its conformation and undergoes autophosphorylation ref. 21), MKK6 (22), and apoptosis signal-regulating kinase-1 (12). This activation of PKR kinase leads to the phosphorylation (23). Furthermore, PKR can regulate the expression of cyclin and activation of several downstream targets, including protein D1(24), c-Myc (25), matrix metalloproteinase-9 (26), and

Authors’ Affiliations: Departments of 1Surgical Oncology, 2Thoracic and Cancer Fund; the Homer Flower GeneTherapy Research Fund; the George Swank Cardiovascular Surgery, 3Experimental Radiation Oncology, and 4Experimental Esophageal Cancer Research Fund and the George Sweeney Esophageal Cancer Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, ResearchFund. Texas; 5Department of Visceral and Transplantation Surgery, Inselspital Bern, Bern, The costs of publication of this article were defrayed in part by the payment of page Switzerland; 6Department of Surgery, University Hospital Basel, Basel, charges. This article must therefore be hereby markedadvertisement in accordance Switzerland; and 7University of Miami School of Medicine, Miami, Florida with18 U.S.C. Section 1734 solely to indicate this fact. Received 12/11/06; revised 2/22/07; accepted 4/24/07. Requests for reprints: Stephen G. Swisher, Unit 445, Department of Thoracic Grant support: National Cancer Institute, NIH grants CA09599, R43 CA97598, and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer and CA16672; The University of Texas M. D. Anderson Esophageal Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-745-4530; Multidisciplinary ResearchProgram; and grants for theCore Laboratory Facility Fax: 713-794-4901;E-mail: [email protected]. fromTenneco and Exxon; The Swiss National Science Foundation Fellowship for F 2007 American Association for Cancer Research. Prospective Researchers [PBZHB-104350 (U. von Holzen)]; the Shooting Down doi:10.1158/1078-0432.CCR-06-2932

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Fig. 1. Radiation sensitivity in PKR+/+ and PKR-/- MEFs. A, clonogenic survival assay of PKR+/+ and PKR-/- MEFs after administration of ionizing radiation therapy at increasing doses. PKR+/+ MEFs show radiation resistance compared withPKR -/- MEFs. B, Clonogenic survival assay of PKR-/- MEFs transfected with either the plasmid containing the PKR+/+ gene or the empty vector plasmid. Partial reconstitution of PKR in the PKR-/- MEFs seemed to induce radioresistance. C and D, clonogenic survival assay of 2-AP ^ treated PKR+/+ (C)andPKR-/- MEFs (D). Cells were treated with1mmol/L 2-AP for 2 hbefore irradiation and cultured in a medium containing 1mmol/L 2-AP. Treatment with2-AP sensitized PKR+/+ MEFs but not PKR-/- MEFs to ionizing radiation. Triplicate experiments were done for eachcell line.

E-selectin (27), and the activation of p38 mitogen-activated the inhibition of PKR with the chemical inhibitor 2-amino- protein kinase (28) and c-Jun NH2-terminal kinase (29) has purine (2-AP), and plasmid transfection of the PKR wild-type been shown to be impaired in PKR-deficient cells. In addition, (wt) gene into PKR-/- MEFs induces radiation resistance. Our researchers have described the activation of nuclear factor-nB results also suggest that PKR exerts this protective effect through (NF-nB) by PKR through the phosphorylation and activation of the activation of NF-nB and Akt because only PKR+/+ cells show IKKh kinase complex, which then phosphorylates and targets up-regulation of these pathways following ionizing radiation. the NF-nB inhibitor InBa for ubiquitination. Removal of InBa Additionally, inhibition of Akt with Deguelin leads to increased allows the active NF-nB p65-p50 complex to translocate into the radiation sensitivity in PKR+/+ cells. This manuscript suggests a nucleus (30). novel role for PKR as a mediator of radiation resistance To date, there have been no reports about the effect of PKR modulated in part through the protective effects of NF-nB and on ionizing radiation therapy. Because many of the above- Akt activation. 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 Materials and Methods fibroblasts (MEF) are much more resistant to ionizing radiation -/- therapy than their PKR knock-out (PKR ) counterparts. Cell lines. PKR+/+ and PKR-/- MEFs were provided by one of the Additionally, we show that this protective effect is reversed by authors (G.N. Barber). Cells were maintained in high-glucose DMEM

www.aacrjournals.org 6033 Clin Cancer Res 2007;13(20) October 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology supplemented with 15% fetal bovine serum, 10 mmol/L glutamine, 100 units/mL penicillin, and 100 Ag/mL streptomycin (Life Technol- ogies Invitrogen) at 37jCin5%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 1mmol/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-nB 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 Biotech- nology), P-PKR pT451and P-eIF-2 a 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 4jC 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, Fig. 2. Induction of apoptosis following radiation therapy. PI staining of PKR+/+ and P-Akt, and NF-nB p65 activation and localization was examined using PKR-/- MEFs 48 h( A)and72h(B) after radiation therapy.There was a marked -/- immunofluorescence. Cells were plated in six-well plates on cover increase in the induction of apoptosis in PKR MEFs, but only a small increase in PKR+/+ cells.Triplicate experiments were done for eachcell line. C, Hoechst staining slides. After treatment, cells were fixed with 2% paraformaldehyde of radiation-treated PKR MEF cells. Apoptotic cell deathwas examined in terms of and permeabilized with 0.3% Triton X-100. Cells were blocked with changes in cell morphology by Hoechst 33342 staining. Radiation-treated PKR-/- 1% normal goat serum for 1 h and then incubated overnight at a MEF cells displayed many apoptotic bodies. dilution of 1:100 with the primary antibody [PKR, NF-nB p65 (Santa Cruz Biotechnology), P-PKR pT451, PeIF-2a pS52, Akt, or P-Akt (Cell Signaling)]. The slides were then washed and incubated with a FITC- or rhodamine-conjugated secondary antibody (Invitrogen) for 1h. radiation, we did clonogenic cell survival assays using MEFs The slides were then mounted with the ProLong Gold antifade that express PKR (PKR+/+) and MEFs that do not express PKR ¶ reagent containing 4 ,6-diamidino-2-phenylindole (DAPI; Invitrogen) (PKR-/-). As shown in Fig. 1A, PKR+/+ MEFs proved to be and analyzed under an Olympus FluoView FV500 laser confocal much more resistant to ionizing radiation than their PKR-/- microscope (Olympus America) after adjustment for background +/+ staining. 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 Results in increased radioresistance by a factor of 2.02, calculated at the surviving fraction of 0.1by dividing radiation dose PKR+/+ MEFs are resistant to ionizing radiation therapy. To of the PKR+/+ MEFs curve with that of the corresponding investigate the role of the PKR in PKR MEF sensitivity to PKR-/- MEFs.

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Plasmid transfection of PKR into PKR-/- MEFs leads to 2-AP, suggesting that the effect of 2-AP was specific for PKR radiation resistance. We then evaluated the effect of restoring (Fig. 1D). PKR expression in PKR-/- MEFs by transfecting PKR-/- MEFs PKR+/+ MEFs show decreased apoptosis after radiation therapy. with a plasmid containing the PKR gene or the empty vector To determine the cellular mechanisms of radioresistance plasmid pcDNA3.1. We used the full length of PKR plasmid induced by PKR, we evaluated the impact of PKR expression for our experiment. We obtained this plasmid from our co- on apoptosis induction after ionizing radiation. Using PI author Dr. G. Barber (University of Miami). PKR-/- MEFs staining and FACS analysis, we found that induction of transfected with the PKR wt plasmid showed increased apoptosis was markedly reduced in the PKR+/+ MEFs, compared radiation resistance after transfection. As shown in Fig. 1B, with the PKR-/- MEFs, suggesting that expression of wt PKR the survival of PKR-/- MEFs after 4 Gy was 0.0689, whereas the blocked radiation-induced apoptosis (Fig. 2A and B). Apoptotic survival of PKR gene transfected MEFs was 0.1217. Restoration cell death was examined in terms of changes in cell morphology of PKR in PKR-/- MEFs resulted in increased radioresistance by Hoechst 33342 staining. Radiation-treated PKR-/- MEF cells by a factor of 1.15 at the surviving fraction of 0.1, suggesting displayed many apoptotic bodies (Fig. 2C). that expression of PKR resulted in enhanced radioresistance PKR is activated by ionizing radiation therapy. We then in MEFs. examined the effect of radiation on PKR expression in PKR+/+ Blocking PKR with 2-AP sensitizes MEFs to radiation and PKR-/- MEFs by Western blot analysis. We found increased therapy. To further examine the role of PKR in the radio- expression of PKR following ionizing radiation in PKR+/+ but resistance of MEFs, we treated PKR MEFs with the PKR inhibitor not PKR-/- cells (Fig. 3A). Radiation therapy also led to PKR 2-AP. As shown in Fig. 1C, PKR+/+ MEFs showed less radiation activation with increased phosphorylated PKR (P-PKR) in resistance after inhibited by 2-AP. The survival fraction of PKR+/+ MEFs as shown by IP experiments (Fig. 3B). Immuno- PKR+/+ MEFs after 4 Gy was reduced to 0.3516, whereas the fluorescence analysis also showed increased PKR expression in survival fraction of PKR+/+ MEFs treated with 2-AP was 0.1508. the cytoplasm around the nucleus (Fig. 3C). These immuno- Inhibition of PKR by 2-AP in PKR+/+ MEFs resulted in decreased fluorescent studies also showed PKR activation by increased radioresistance by a factor of 0.72 (calculated at the surviving P-PKR and P-eIF-2a levels 24 h after irradiation. The expres- fraction of 0.1by dividing radiation dose of the PKR +/+ MEFs sions of P-PKR and P-eIF-2a were inhibited by treatment with treated with 2-AP with that of the corresponding PKR+/+ MEFs). low-dose 2-AP, suggesting functional activity of the inhibitor The PKR-/- MEFs showed no change in radiosensitivity with (Fig. 3C).

Fig. 3. PKR expression after radiation therapy. A, Western blot analysis of the PKR expression in PKR+/+ and PKR-/- MEFs following radiation therapy showing a time-dependent increase in expression of PKR. B, IP Western blot analysis of P-PKR expression in PKR+/+ and PKR-/- MEFs show a dose-dependent increase in expression 48 hafter irradiation in PKR +/+ MEFs. C, immunofluorescent analysis of the effect of radiation therapy on the localization and expression of PKR in PKR+/+ and PKR-/-MEFs. PKR expression increased 4 hafter irradiation. Background staining of nucleoli is seen in PKR -/- cells, which is unspecific.D, immunofluorescent analysis shows increased expression of PKR, P-eIF-2a,andP-PKRinPKR+/+ MEFs 24 h after radiation therapy.The increase in expression was inhibited by treatment with 2-AP. Blue, DAPI staining for DNA in the nucleus. Triplicate experiments were done for each cell line.

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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.

Discussion

The dsRNA-activated protein kinase, PKR, is a critical gene in the cellular antiviral response. Through phosphorylation of the a 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 +/+ Fig. 4. NF-nB is activated in PKR MEFs. A, EMSA showing the activation of increasing PKR activity has a net tumor-suppressive effect NF-nBinPKR+/+ cells, but not in PKR-/- MEFs. B, Immunofluorescent analysis of NF-nB p65 in PKR+/+ and PKR-/- MEFs. The expression of NF- nB p65 in the nucleus through the up-regulation of apoptotic pathways. Despite this increases in PKR+/+ MEFs, but not in PKR-/- MEFs after irradiation. C, Western blot data, the role of PKR as a tumor suppressor is far from clear, analysis of cFLIP in PKR+/+ and PKR-/- MEFs after irradiation. The expression of cFLIP increases in PKR+/+ MEFs, but not in PKR-/- MEFs. h-Actin was used as a with evidence that PKR activation can lead to neoplastic loading control. 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 n Irradiation leads to NF-kB activation in PKR+/+ MEFs. We NF- B. The role of PKR in radiation therapy has not been then evaluated potential mediators of PKR radiation resistance previously reported. In the present study, we show for the first including NF-nB (30). We used EMSA to assess NF-nB–DNA time that in MEFs, PKR is protective to the cytotoxic effects of binding activity in PKR+/+ and PKR-/- MEFs after ionizing radiation therapy. We also present evidence that PKR activation radiation therapy. As Fig. 4A shows, PKR+/+ MEFs show can lead to the up-regulation of the pro-survival Akt pathway, activation of NF-nB after irradiation, which is reduced in and this activation seems dependent on the presence of the -/- n PKR gene. PKR cells. Immunofluorescence also showed NF- B activation +/+ -/- through p65 translocation in PKR+/+ MEFs after irradiation. Our study shows that relative to PKR MEFs, PKR MEFs Translocation of the NF-nB p65 subunit to the nucleus 4 h after are hypersensitive to ionizing radiation therapy. The PKR gene ionizing radiation was shown only in the PKR+/+ cells, but not confers an overall protective cell to MEFs undergoing radiation in PKR-/- cells (Fig. 4B). Western blot analysis also showed therapy. To preclude the possibility that the difference was due increased expression of cFLIP in irradiated PKR+/+ MEFs. cFLIP to differences between the knock-out and wild-type MEFs genetic background, plasmid transduction of PKR was done in is a catalytically inactive variant of caspase-8, the expression -/- n PKR MEFs. Transduction of wild-type PKR partially restored of which is induced by NF- B (Fig. 4C) and may explain the +/+ reduced apoptosis noted following radiation treatment in the radiation-protective effects of PKR cells, suggesting that PKR+/+ cells (Fig. 2). this gene was the critical factor. Inhibition of PKR activity with +/+ the chemical inhibitor 2-AP also reduced the protective effect of Irradiation leads to Akt activation in PKR MEFs. We also +/+ investigated the role of the pro-survival Akt pathway because of PKR only in PKR cells, suggesting that this was the critical a pathway (36). data suggesting that TNF- –induced PKR activation led to Akt -/- +/+ 8 +/+ We also found increased induction of apoptosis in the PKR phosphorylation only in PKR cells. PKR MEFs exhibited +/+ dose- and time-dependent increases in phosphorylation of Akt MEFs when compared with the PKR MEFs. These findings after radiation, whereas PKR-/- MEFs did not show Akt 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 8 Unpublished observation. DNA-damaging agents, such as cisplatin, than their PKR+/+

ClinCancerRes2007;13(20)October15,2007 6036 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Role of PKR in RadiationTherapy counterparts are, supposedly because of impaired DNA damage radiation therapy induced expression of cFLIP, a downstream repair mechanisms. In addition, we found that PKR is up- target of NF-nB. The activation of NF-nB by PKR seems to regulated and activated following ionizing radiation therapy. mediate in part cell survival after ionizing radiation therapy. This activation was shown by the phosphorylation of the PKR Our data concerning NF-nB activation agree with those of a downstream target PeIF-2a. Again, this activation was inhibited previous study demonstrating that PKR may act as a molecular by 2-AP. These observations lead to the proposal that ionizing clock by inducing cell survival through the activation of NF-nB radiation therapy induces PKR activation, which induces pro- before induction of cell death by phosphorylation of eIF-2a survival pathways protective of cellular cytotoxic effects of (39). In our MEFs, this induction of cell survival seemed to radiation therapy. be a more important pathway than did the induction of Consistent with this hypothesis is the observation that PKR apoptosis. activation is involved in proapoptotic as well as antiapoptotic Akt is a serine/threonine kinase that has been shown to be an pathways. In addition, impairment of survival pathways in the important mediator of survival pathways in different normal absence of PKR may play an important role in determining and human tumor cells (40). Akt can be activated by several sensitivity to radiation therapy. We therefore examined some agents. Once Akt is phosphorylated and activated, it promotes of these survival pathways. In particular, we discovered the cell survival by phosphorylating its downstream targets, such as activation of NF-nB, which has been described previously Bad, forkhead transcription factors, IKK, cyclic AMP-responsive (38), 2 to 4 h after ionizing radiation therapy. Our findings element binding protein, glycogen synthase kinase 3, murine confirm that NF-nB is activated following ionizing radiation double minute 2, p21(41),mammalian target of rapamycin therapy as shown by the results of EMSA and increased (42), and caspase-9 (43). This mediates direct, transcriptional, expression of NF-nB p65 in the nucleus. Furthermore, ionizing and metabolic regulation of apoptosis (43). However, the

Fig. 5. Akt expression after radiation therapy. A, Western blot analysis of P-Akt and Akt in PKR-/- and PKR+/+ MEFs following ionizing radiation. The blot shows time- dependent up-regulation of P-Akt after irradiation at 6 Gy in PKR+/+ MEFs but no P-Akt expression in PKR-/- MEFs. Expression of Akt increased in PKR-/- MEFs when compared withthatin PKR +/+ MEFs. h-Actin was used as a loading control. B, Western blot analysis shows dose-dependent up-regulation of P-Akt expression in PKR+/+ MEFs 72 hafter radiation therapy.There was no P-Akt expression in PKR -/- MEFs. Expression of Akt increased in PKR-/- MEFs when compared with that in PKR+/+ MEFs. h-Actin was used as a loading control. C, immunofluorescent staining for P-Akt in PKR-/- and PKR+/+ MEFs following radiation therapy.The expression of P-Akt increased in PKR+/+ MEFs 72 hafter irradiation. There was no P-Akt expression in PKR -/- MEFs. Blue, DAPI staining for DNA in the nucleus; red, P-Akt staining. D, clonogenic survival assay of PKR+/+ and PKR-/- MEFs treated with the Akt inhibitor Deguelin after administration of ionizing radiation therapy at increasing doses. Cells were treated with 100 nmol/L Deguelin for 48 hbefore radiation therapy.Treatment withDeguelin sensitized PKR +/+ MEFs to radiation therapy.Western blot analysis shows decreased phosphorylation of Akt after treatment with Deguelin. h-Actin was used as a loading control.

www.aacrjournals.org 6037 Clin Cancer Res 2007;13(20) October 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology mechanism by which Akt is activated has not been elucidated. In summary, our manuscript shows for the first time that Because our laboratory showed Akt activation following TNF- the IFN-inducible dsRNA-activated protein kinase PKR induced activation of PKR,9 we evaluated the role of this pro- induces radiation resistance in MEFs potentially through the survival pathway in radiation-protective effects of PKR. We up-regulation of the pro-survival NF-nB and Akt pathways. It show dose-dependent phosphorylation of Akt in PKR+/+ MEFs will be important to determine if PKR induces radiation following radiation therapy that is not seen in PKR-/- cells. resistance in human cancer cells as well. Because many These findings suggest a possible interaction between Akt and tumors are radiation resistant, these findings would provide PKR. However, there have been no published findings on this insight into novel therapeutic strategies to overcome radia- potential interaction. Further studies are therefore warranted to tion resistance either through PKR inhibition or down- confirm this interaction. Our finding that Akt inhibition by regulation of the pro-survival NF-nB or Akt pathways. Our Deguelin partially restores radiation sensitivity suggests a results provide evidence that PKR is involved in a novel potential therapeutic approach to increase radiation sensitivity pathway that is protective to the toxic effects of ionizing through the inhibition of the PKR-induced pro-survival radiation therapy. pathway Akt.

Acknowledgments

9 Unpublished observations. We thank Kwang S. Ahn for his assistance with EMSA.

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Urs von Holzen, Abujiang Pataer, Uma Raju, et al.

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