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Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity

Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases... [Cancer Biology & Therapy 8:3, 245-252; 1 February 2009]; ©2009 Landes Bioscience Research Paper Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity 1, 1 1 2 2 Abujiang Pataer, * Stephen G. Swisher, Jack A. Roth, Christopher J. Logothetis and Paul G. Corn 1 2 Departments of Thoracic and Cardiovascular Surgery, and Genitourinary Medical Oncology; The University of Texas MD Anderson Cancer Center; Houston, TX USA Key words: PKR, gene therapy, adenovirus 1-6 RNA-dependent protein kinase is an interferon-induced, signal transduction and differentiation. While PKR controls the double-stranded (ds), RNA-activated serine/threonine protein expression of multiple genes and signaling pathways, its function kinase involved in the eukaryotic response to viral infection. While in suppressing or promoting mammalian cell growth is somewhat PKR also functions in cellular differentiation, growth control and controversial. apoptosis, its role in human cancer remains poorly understood. To In support of its role in growth suppression, for example, treat- explore a role for PKR in human cancer, we evaluated PKR expres- ment of murine cell lines with dsRNA, TNFα or lipopolycacharide sion and function in a series of cancer cell lines from different leads to a PKR-dependent apoptosis, which may be due to phospho- tumor types. We observed that PKR protein expression is high in rylation of eIF-2α, but also to the expression of other pro-apoptotic 7,8 various cancer cells and low in normal cells. Knockdown of PKR factors such as Fas. In addition, PKR induces apoptosis in epithelial protein expression by PKR siRNA induced cell death, indicating a cancer cell lines in response to adenoviral overexpression of TNFα, PKR-dependent survival pathway under normal growth conditions. E2F-1 and the melanoma differentiation-associated gene 7 (mda7), a 9-12 Inhibition of PKR signaling using a dominant negative adenoviral novel tumor suppressor gene. Lastly, in studies of primary human PKR mutant (Ad-Δ6PKR) also induced cancer cell apoptosis via a cancers from the head and neck, thyroid and colon, increasing PKR mechanism that blocks activation of AKT-mediated survival while expression has been correlated with more well-differentiated tumors simultaneously inducing ER stress. ER stress-mediated apop- and diminished proliferative activity, suggesting that PKR regulates 13-15 tosis was evidenced by unregulated expression of phosphorylated tumor suppression. JNK (p-JNK), phosphorylated cJun (p-cJun), and caspase-4 and Other studies, however, do not support a tumor suppressor role was significantly reduced in cancer cells treated with JNK and for PKR and raise the alternative hypothesis that PKR functions in caspase-4 inhibitors. We further demonstrated that inhibition of growth promotion. For example, PKR knockout mice do not show 16,17 PKR signaling via either siRNA or Ad-Δ6PKR sensitizes cancer increased spontaneous rates of tumor development. In addition, cells to etoposide or cisplatin-mediated cell death. Our results increased expression and activity of PKR has been reported in some 18,19 suggest a rationale to develop therapeutic strategies that target human tumor types and correlates with neoplastic progression. PKR signaling in human cancer cells. Biochemical studies have shown that PKR can phosphorylate IKK, leading to activation of the NFκB anti-apoptotic signaling Introduction 20 pathway. PKR can also induce the expression of several key survival genes known to be dependent on NFκB, such as c-IAP1, c-IAP2 and RNA-dependent protein kinase (PKR) is an interferon-induced, A20. PKR also “crosstalks” with multiple signaling pathways that double-stranded (ds), RNA-activated serine/threonine protein kinase 1-3 activate and engage a number of transcription factors implicated in that has a well-established role in anti-viral defense mechanisms. oncogenesis. In response to dsRNAs and interferons, activated PKR inhibits Several recent studies from our group further support the hypoth- cellular protein translation by phosphorylation of eIF-2α, an event esis that PKR promotes cellular survival. First, using PKR wild-type that induces cellular apoptosis. In the absence of viral infections, (+/+) and knockout (-/-) MEFs to deduce PKR function, treatment however, PKR also plays a role in other important cellular functions with tumor necrosis factor (TNF) activated AKT and NFκB in including growth control, apoptosis regulation, cell proliferation, a PKR-dependent manner. In conjunction with this, PKR was required for TNF-induced NFκB-dependent anti-apoptotic protein *Correspondence to: Abujiang Pataer; Departments of Thoracic and Cardiovascular expression. Secondly, PKR was shown to positively regulate an Surgery; Unit 23 or Department of Genitourinary Medical Oncology; Unit 1374; The University of Texas MD Anderson Cancer Center; 1515 Holcombe Blvd.; Houston, TX AKT-dependent survival pathway in human lung cancer cells. 77030 USA; Tel.: 713.792.8905; Fax: 713.794.4901; Email: apataer@mdander- Thirdly, PKR mediates resistance to radiation therapy in mouse son.org embryo fibroblasts through the activation of AKT and NFκB- Submitted: 10/09/08; Revised: 11/17/08; Accepted: 11/08/08 dependent signaling pathways. Collectively, these results suggest that PKR can function to enhance cell survival, although this phenotype Previously published online as a Cancer Biology & Therapy E-publication: may depend on the stress signal, cell-type and physiologic context. http://www.landesbioscience.com/journals/cbt/article/7386 www.landesbioscience.com Cancer Biology & Therapy 245 Inhibition of PKR leads to cancer cell death Figure 1. Knockdown of PKR by siRNA induces cell death in cancer cell lines. (A) Western blot analysis of PKR protein expression in human lung (A549 and H460), prostate (PC3 and LNcaP), breast (MCF-7 and T47D), and colon (SW620, HT-29 and KM12L4) cancer cell lines and normal cells (NHBEs and HMECs). The expression of actin was used as a loading control. (B) Immunofluorescence microscopy with antibodies against PKR (green) demonstrated cytosolic expression of PKR in A549 and PC3 cancer cells and diffuse expression of PKR in normal cells (NHBEs and HMECs). (C) Western blot and flow cytometric analyses of PKR protein expression and cell death in human lung (A549), prostate (PC3), breast (T47D) cancer cell lines and normal cells (NHBEs) cells after 72 hrs of treatment with PKR siRNA and luc SiRNA. Indicated at the bottom is the percent cell death induced by PKR siRNA and Luc siRNA treat- ment in each of these cell lines. Actin expression was analyzed as a loading control. In the present study, we explored the role for PKR in promoting contrast, we observed a diffuse expression pattern of PKR in NHBEs resistance to chemotherapy. We show that inhibition of PKR and HMECs normal cells (Fig. 1B). signaling using a dominant negative PKR mutant (Ad-Δ6PKR) acts We next investigated the effect of silencing PKR protein expres- synergistically with chemotherapy to induce cell death in human sion on cell viability. As revealed by immunoblotting and FACS cancer cell lines. The induction of cell death was due in part to the analysis, treatment of A549, PC3 and T47 cancer cells with PKR activation of an endoplasmic reticulum (ER) stress pathway. Our siRNA effectively knocked down expression of PKR protein and results suggest a therapeutic strategy to target PKR signaling in the induced apoptosis (Fig. 1C). Conversely, treatment with luc siRNA treatment of human cancer. did not knock down expression of PKR protein nor induce apop- tosis (Fig. 1C). Normal NHBEs cells treated with PKR siRNA also Results underwent apoptosis, but to a much lesser extent than the cancer cells (12% versus ~45–50% in normal and cancer cells, respectively) Knock down of PKR by siRNA in cancer cells induces cell (Fig. 1C). Taken together, these results suggest differential function death. We first investigated the expression and cellular localization of of PKR between normal and cancer cells. PKR in human cancer cells as well as normal cells. Figure 1A shows Inhibition of PKR by adenoviral mutant PKR (Ad-Δ6PKR) that PKR protein expression is high in multiple cell lines derived induces cell death in cancer cells. We have previously shown that from human lung (A549, H460), prostate (PC3, LNCaP), breast a deletion mutant of PKR (Δ6PKR) can act as a dominant negative (MCF7, T47D) and colon (SW620, HT-29, KM12L4) cancers. inhibitor of PKR function. As shown by flow cytometric analysis In contrast, PKR expression was low in normal human bronchial of human lung (A549 and H460), prostate (PC3 and LNCaP) and epithelial cells (NHBEs) and normal human mammary epithelial breast (MCF-7 and T47D) cancer cells, adenoviral infection with cells (HMECs). Δ6PKR (Ad-Δ6PKR) induced cell death within 72 hrs of infection We next investigated the pattern of PKR expression in two cancer cell lines (A549 and PC3) and the two normal cell lines (NHBEs and (Fig. 2A). In contrast, Ad-Δ6PKR was not toxic to NHBE or HMEC HMECs) by confocal immunofluorescence analysis. We observed normal cells (Fig. 2A). +/+ both cytosolic and nuclear localization of PKR in A549 and PC3 Our previous studies in PKR MEFs indicated that PKR activates cancer cells with the predominant signal in the cytosol (Fig. 1B). In an AKT-dependent anti-apoptotic survival pathway by upregulating 246 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 Inhibition of PKR leads to cancer cell death different environmental and metabolic 26,27 stimuli. Under conditions of stress (for example with hypoxia and/or nutrient deprivation) misfolded/unfolded proteins accumulate in the ER and stimulate a signaling pathway known as the “unfolded protein response” (UPR). The UPR permits cells to either adapt to the stress until the stress resolves, or alternatively, promotes apoptosis when the stress related damage becomes irre- versible. Previous results suggested that PKR plays an important role in ER stress-induced retinal neuron damage in mice. We next tested whether Ad-Δ6PKR- mediated apoptosis of cancer cell lines resulted from activation of ER-stress. To do this, we measured downstream target proteins that are activated via phosphory- lation in response to ER-stress, including JNK and c-Jun. A549 and PC3 cancer cell lines were treated for 72 hours with either PBS, Ad-Luc, Ad-Δ6PKR or endoplasmic reticulum targeting melanoma differen- tiation-associated gene 7 (Ad-ER-mda7). Ad-ER-mda7 served as a positive control. As shown in Figure 3A, expression of phospho- JNK and phospho-cJun was upregulated after Ad-Δ6PKR and Ad-ER-mda7 transduction but not after Ad-Luc transduction (Fig. 3A), suggesting that activation of JNK or cJun was the mechanism by which Ad-Δ6PKR medi- ated cell death. Figure 2. Inhibition of PKR by Adenoviral mutant PKR (Ad-Δ6PKR) vector induces cell death in Human caspase-4 is localized to the ER cancer cells. (A) Flow cytometric analysis of cell death in A549, H460, PC3, LNCaP, MCF-7, T47D, membrane and is specifically activated by and NHBEs and HMECs cells 72 hours after treatment with PBS, Ad-luc (3000 vp) or Ad-Δ6PKR (3000 required for ER stress-induced apoptosis. vp). Experiments were performed in triplicate; data are presented as the means (error bars, SDs). (B) We next investigated whether caspase-4 Western blot analysis of phosphorylated PKR (p-PKR), PKR, phosphorylated AKT (p-AKT), and AKT1/2 was involved in Ad-Δ6PKR-induced cell protein expression in A549 and PC3 cell lysates 72 hours after treatment with PBS, Ad-luc (3000 vp) or Ad-Δ6PKR (3000 vp). β-Actin expression was analyzed as a loading control. death. Caspase-4 was cleaved to its active form in A549 and PC3 cancer cells treated with Ad-Δ6PKR or Ad-ER-mda7 (positive 473 23,25 levels of phospho-AKT (Ser ). Conversely, Ad-Δ6PKR inhib- control) but not in those treated with PBS or Ad-Luc (Fig. 3A). 473 +/+ 25 ited phospho-AKT (Ser ) in PKR MEFs. To test whether To more directly determine the importance of JNK and caspase-4 Ad-Δ6PKR would also inhibit AKT phosphorylation in cancer cells, activation in Ad-Δ6PKR-mediated cell death, A549 and PC3 cancer we infected A549 and PC3 cells with Ad-Δ6PKR. As revealed by cells were infected with Ad-Δ6PKR in the presence or absence of a immunoblotting, a high level of PKR expression was observed in chemical JNK inhibitor (SP600125, 10 μmol/L), a peptide inhibitor A549 and PC3 cancer cells after being infected with Ad-Δ6PKR of caspase-4 (Ac-LEVD-CHO, 10 μmol/L) or combination of (Fig. 2B), whereas low levels of endogenous PKR protein were the two inhibitors. In both cancer cell lines, inhibitory treatment observed in cells infected with the control contructs. Ad-Δ6PKR reduced the amount of AdΔ6PKR-induced cell death as measured infected cancer cells demonstrated decreased expression of phospho- by FACS. In A549 cells, apoptosis was reduced from 28.5% to 15% 451 473 PKR (Thr ) and phospho-AKT (Ser ). We could not detect after JNK inhibition, 16.8% after caspase 4 inhibition, and 7% decreased expression of phosphorylated PKR and AKT1/2 in Ad-Luc after treatment with both inhibitors (p < 0.05, significantly different infected cancer cells (Fig. 2B). These results further confirmed that from untreated). Similarly in PC3 cells, apoptosis was reduced from Ad-Δ6PKR can inhibit phospho-AKT (Ser ) in cancer cells. 29.9% to 17% after JNK inhibition, 16.4% after caspase 4 inhibi- Activation of the endoplasmic reticulum (ER) stress response tion, and 6.7% after treatment with both inhibitors (Fig. 3B) (p contributes to mutant PKR (Ad-Δ6PKR)-induced apop- < 0.05, significantly different from untreated). Conversely, neither tosis. The endoplasmic reticulum functions in normal cellular inhibitor reduced the amount of cell death induced by overexpres- homeostasis by ensuring proper protein folding in response to sion of Ad-Bak, a Bcl-2 family member that induces apoptosis in a www.landesbioscience.com Cancer Biology & Therapy 247 Inhibition of PKR leads to cancer cell death Figure 3. Adenoviral mutant PKR (Ad-Δ6PKR) induces the endoplasmic reticulum (ER) stress-mediated cell death pathway. (A) Western blot analysis of phos- phorylated JNK (p-JNK), JNK, phosphorylated cJun (p-cJun), cJun and caspase-4 protein expression in A549 and PC3 cell lysates 72 hrs after treatment with Ad-luc (3000 vp), Ad-ER-mda7 (3000 vp) or Ad-Δ6PKR (3000 vp). β-Actin expression was analyzed as a loading control. (B) Flow cytometric analysis of cell death in A549 and PC3 cancer cells 72 hrs after treatment with PBS, Ad-Luc (3000 vp) or Ad-Δ6PKR (3000 vp) with or without caspase-4 (Ac-LEVD-CHO, 10 μmol/L) or JNK chemical inhibitors (10 μmol/L). Experiments were performed in triplicate; data are presented as the means (error bars, SDs) *p < 0.05 vs. untreated control, Wilcoxon rank-sum test. (C) Immunofluorescent confocal microscopic analysis of PKR protein expression. Analysis of antibody against PKR stained green, ER stained red with ER-Tracker dyes, and nuclei stained blue with DAPI, demonstrated colocalization (orange) of PKR protein and the ER marker in Ad-Δ6PKR-treated A549 and PC3 cancer cells 72 hrs after transfection. +/+ PKR-independent manner (data not shown). Together, these results (PKR ) MEFs to bulky adduct DNA damage caused by UV suggest that Ad-Δ6PKR cell death is mediated in part through radiation or alkylating chemotherapeutic agents such as cisplatin activation of ER stress and downstream effectors such as JNK and and melphalan. To confirm and expand on these results, we first +/+ -/- caspase-4. compared the viability of PKR and PKR MEFs following treat- We next analyzed the pattern of PKR expression in A549 lung ment with DNA damaging agents (etoposide, cisplatin, mitomycin), cancer and PC3 prostate cancer cells after treatment with PBS, an anti-metabolite (5-fluouracil [5FU]) and an anti-tubulin (Taxol). -/- Ad-Luc or Ad-Δ6PKR. Confocal immunofluorescence analysis In agreement with Bergeron et al. we found that PKR MEFs cells +/+ revealed that PKR levels were markedly higher in the cytosol of are much more susceptible than PKR MEFs to cell death induced A549 and PC3 cells after treatment with Ad-Δ6PKR (Fig. 3C). by cisplatin or melphalan (Fig. 4A) (p < 0.05). In contrast, there was The precise subcellular localization of the PKR (green) was further no difference following treatment with Mitomycin C. The enhanced confirmed by comparing its expression pattern with an ER molec- sensitivity to DNA-damage induced apoptosis was not restricted to -/- ular marker (red) known to reside within the ER compartment akylating agents, as PKR MEFs were also more sensitive to DNA (Fig. 3C). Treatment with Ad-Δ6PKR led to an increase in PKR damage by etoposide, a Topoisomerase II poison. Interestingly, -/- +/+ within the ER, as evidenced by enhanced colocalization fluorescence PKR MEFs were also more susceptible than PKR MEFs to cell (yellow-orange) in the overlay images. These results further support death induced by 5FU but not Taxol. -/- a role for the endoplasmic reticulum in Ad-Δ6PKR induced cellular To control for the possibility that PKR MEFs acquired genetic apoptosis. change(s) outside the PKR locus that could account for these results, +/+ Adenoviral mutant PKR (Ad-Δ6PKR) sensitizes cancer cells we next transfected PKR MEFs with PKR siRNA and then +/+ to some chemotherapeutic agents. In a single study previously treated them with cisplatin or etoposide. PKR MEFs transfected -/- published by Bergeron et al. PKR-deficient (PKR ) mouse-embry- with PKR siRNA underwent chemotherapy-induced apoptosis to a -/- +/+ onic fibroblasts (MEFs) were more sensitive than PKR-proficient similar degree as PKR MEFs (Fig. 4B). In contrast, PKR MEFs 248 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 Inhibition of PKR leads to cancer cell death (55% with etoposide and 50% with cisplatin) and PC3 (57% with etoposide and 48% with cisplatin) cancer cells (Fig. 5) (p < 0.05, significantly different from untreated or Ad-luc treated). In contrast, Ad-luc did not have any significant effects on cell viability, either alone or in combination with chemotherapy. Discussion PKR is a stress-activated kinase induced in response to multiple different stimuli including viral infections, interferons, interleukins, TNFα, lipopolysaccharide and 1-6,22 ionizing radiation. In the present study, we tested whether knockdown of PKR by PKR siRNA or inhibi- tion of PKR by adenoviral mutant PKR (Ad-Δ6PKR) alters cell viability or chemotherapy-mediated apop- tosis in human cancer cells. We demonstrated that PKR protein expression is high in various cancer cell lines and low in normal cells (Fig. 1A). We have inves- tigated the pattern of PKR expression in two cancer cell lines (A549 and PC-3) and two normal cell lines (NHBE and HMECs) by confocal immunofluores- cence analysis. We observed both the cytosol (most) and nuclei (less) localization of PKR in A549 and PC-3 cancer cells. We observed a diffuse expression pattern of PKR in NHBE and HMECs normal cells. Once activated, PKR translocates to the nucleus. Unlike the known cytoplasmic function of PKR, the role of PKR in the nucleus has yet to be defined. Dr. Barber’s laboratory has demonstrated that both NFAR-1 and -2 (nuclear factors associated with dsRNA) are substrates for PKR. Dr. Barber’s results indicate that the NFARs may facilitate double-stranded RNA-regulated -/- Figure 4. PKR-deficient mouse-embryonic fibroblasts (PKR ) are susceptible to chemo- gene expression at the level of post-transcription and +/+ -/- therapeutic agents. (A) Flow cytometric analysis of apoptosis in PKR and PKR MEF cells possibly contribute to host defense-related mechanisms after 72 hrs of treatment with etoposide, cisplatin, 5-FU, melphalan, Taxol or mitomycin. in the cell. Knockdown of PKR by PKR siRNA Experiments were performed in triplicate; data are presented as the means (error bars, SDs) induced cell death in these cancer cells under normal *p < 0.05 vs. corresponding control, Wilcoxon rank-sum test. (B) Flow cytometric analysis +/+ -/- growth conditions, underscoring the dependence of of apoptosis in PKR and PKR MEF cells after 72 hrs of treatment with PKR siRNA and luc SiRNA with or without etoposide (ET) or cisplatin (CDDP) treatment. Experiments were these cells on PKR signaling even in the absence of performed in triplicate; data are presented as the means (error bars, SDs) *p < 0.05 vs. exogenous stress stimuli (Fig. 1C). Inactivation of corresponding control, Wilcoxon rank-sum test. PKR by dominant negative adenoviral mutant PKR (Ad-Δ6PKR) selectively and effectively induces cell transfected with a control (Luciferase) siRNA retained a relative death may partly due to reduction of phospho-AKT (Ser ) protein -/- resistance to etoposide or cisplatin when compared to PKR MEFs. (Fig. 2A and B). Collectively, these results demonstrate that a PKR-dependent survival We have also determined the long-term effect of PKR siRNA pathway mediates resistance to some chemotherapeutic agents. or Ad-Δ6PKR treatment on cell growth using a clonogenic assay. We next wanted to test whether inhibition of PKR signaling Both PKR siRNA and Ad-Δ6PKR treatments caused significant would confer a similar chemosensitization effect in human cancer reduction in clonogenic potential of A549 and PC3 cells (data not cells. To do this, we examined the apoptotic effects of Ad-Δ6PKR shown). Despite similar phenotypes, however, we postulate that alone or in combination with etoposide (10 μmol/L), cisplatin (10 inactivation of PKR signaling using siRNA may not be functionally μmol/L), or both drugs in A549 and PC3 cancer cells after 72 hrs equivalent to inactivation using dominant negative Ad-Δ6PKR. treatment. By flow cytometric analysis, Ad-Δ6PKR alone resulted Downregulation of PKR protein by siRNA would be predicted in cell death rates of 25% in A549 cells and 24% in PC3 cells (Fig. to inhibit both apoptotic and survival pathways equally. In this 5). Treatment with etoposide or cisplatin as single agents resulted case, cell fate (survival versus death) would reflect the balance in cell death rates of 10% and 8.6% respectively in A549 cells of pro-apoptotic versus anti-apoptotic pathways and depend on and 11% and 9% respectively in PC3 cells (Fig. 5). Importantly, which pathway was dominant in a particular cancer cell (cell-type the combination of Ad-Δ6PKR with either etoposide or cisplatin dependent). In contrast, inactivation of PKR by dominant negative resulted in a synergistic enhancement of apoptosis in both A549 PKR may result predominantly in cell death because Ad-Δ6PKR www.landesbioscience.com Cancer Biology & Therapy 249 Inhibition of PKR leads to cancer cell death preferentially induces ER-stress mediated apoptosis in cancer cells (cell-type independent). Future studies aim to elucidate the precise mechanism(s) of PKR siRNA and Δ6PKR mediated apoptosis in cancer cells versus normal cells. PKR associates with ribosomes, 1-3 mainly to the 40S subunits. In a recent study by Onuki et al. PKR was shown to be involved in ER stress-mediated apoptosis induced by tunicamycin treatment of SK-N-SH human neuroblastoma cells. In that study, tunicamycin treatment increased expression levels of phos- phoylated PKR in the nucleus. Another recent study showed a role for PKR in endoplasmic reticulum (ER) stress-induced neuronal cell death in cases of Alzheimer’s disease and Huntington’s disease. In our study, we demonstrated that activation of ER stress occurs as a consequence of deregulating PKR signaling with Figure 5. Adenoviral mutant PKR (Ad-Δ6PKR) enhanced etoposide (ET)- or cisplatin (CDDP)-mediated cell death in cancer cells. Flow cytometric analysis of apoptosis in Ad-Δ6PKR-transfected A549 and PC3 cancer cells, mutant PKR (Δ6PKR). This was with or without etoposide or cisplatin treatment after 72 hrs. Experiments were performed in triplicate; data are evidenced by increased expression presented as the means (error bars, SDs) *p < 0.05 by Wilcoxon rank-sum test compared with corresponding of phospho-JNK and phosphor-Jun, control (ET, CDDP, Ad-Δ6PKR, Ad-Luc + ET or Ad-Luc + CDDP). and increased cleavage of caspase-4. Taken together, these results suggest -/- significant “crosstalk” between the PKR and ER-stress signaling susceptibility of PKR MEFs to cytotoxic chemotherapy was not pathways. due increased proliferation (and hence potentially increased exposure -/- The ER stress-mediated cell death pathway involves recruitment to the drug), since PKR MEFs actually grow ~50% more slowly +/+ of the cytosolic adaptor TRAF2 to the ER membrane, where TRAF2 than PKR MEFs (data not shown). Secondly, inhibition of PKR activates the apoptosis-signaling kinase 1 (ASK1). Activation of signaling using a dominant-negative mutant sensitized cancer cells ASK1 leads in turn to activation of JNK and mitochondria-depen- to etoposide and cisplatin-mediated cell death. As with MEFs, the dent caspases. In this study, we demonstrated that activation of increased chemosensitivity of Ad-Δ6PKR infected cells was not due JNK and caspase-4 is essential for Ad-Δ6PKR-mediated cell death. increased proliferation since Ad-Δ6PKR caused growth inhibition We hypothesize two possible mechanisms for this. First, inhibition (data not shown). Our working hypothesis is that PKR regulates an of PKR by Ad-Δ6PKR reduces expression of survival factors such AKT and NFκB-dependent survival pathway in response to chemo- as phospho-AKT. Secondly, inactivation of PKR by Ad-Δ6PKR therapy treatment. Since this mechanism mediates PKR-dependent disrupts normal PKR dimerization such that inactive PKR dimers radioresistance, future studies will address if AKT1/2 and NFκB play or monomers translocate to the ER and induce stress. PKR exists in a similar role in promoting chemoresistance. the cell in at least three forms: an inactive monomer associated with Our data add to a growing body of evidence that support a role ribosomes, an inactive dimer in the cytoplasm, and an active dimer. for PKR in promoting tumor cell survival. It has been shown that PKR can also switch from an inactive dimer to an active dimer by a PKR activation contributes to neoplastic progression and decreased 1-6 mechanism involving dsRBM interactions. sensitivity to conventional chemotherapy agents in melanoma and We previously demonstrated that PKR plays a critical role in colon cancer cells, presumably through upregulation of pro-survival 25 - 34 radio-resistance. In that study, we found that PKR-deficient (PKR pathways such as NFκB. Nussbaum et al. have proposed that high /- ) mouse-embryonic fibroblasts (MEFs) were more susceptible to PKR activity in breast cancer cells was responsible for activation of +/+ 25 radiation-induced cell death than PKR-proficient (PKR ) MEFs. protooncogenes such as the platelet-derived growth factor (PDGF) This resistance was mediated in part through PKR-dependent and c-fos that stimulate cell proliferation. We have previously upregulation of AKT1/2. In the present study, we provide demonstrated that PKR can indirectly regulate AKT1/2 and NFκB- 23,25 evidence that PKR also contributes to chemotherapy resistance. dependent survival pathways. This regulation is likely critical -/- First, in agreement with Bergeron et al. PKR MEFs were more for the tumor promoting properties of PKR, since AKT is known +/+ sensitive than PKR MEFs to apoptosis due to bulky adduct to promote cellular survival and NFκB blocks apoptosis induced 30 36-38 DNA damage caused by cisplatin and melphalan. The increased by chemotherapeutic agents. It is possible; therefore, that PKR 250 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 Inhibition of PKR leads to cancer cell death promotes cancer cell growth through activation of survival factors medium overnight. Solution A was prepared by adding 3.6 μL of the such as AKT1/2, NFκB or activation of protooncogenes such as c-fos desired 10 μM siRNA to 60 μL of the siRNA transfection medium, in human cancers. We are now examining whether the Ad-Δ6PKR and solution B, by adding 3.6 μL of the transfection reagent to inhibits activation of NFκB or c-fos. 14.5 μL of the siRNA transfection medium. Both solutions were Lastly, our results suggest a therapeutic strategy to target PKR mixed gently and kept at room temperature for five min, after which signaling in the treatment of human cancer. As proof of principal they were combined to form an siRNA-siRNA transfection reagent for this concept, injection of melanoma tumor cells transfected with complex. This complex was added to cells for five hrs. Luciferase a PKR-specific short hairpin RNA (shRNA) expressing plasmid siRNA was used as a control. resulted in inhibition of pulmonary metastatic nodules in mice. In Immunofluorescent cellular localization studies. A549 lung primary human thyroid carcinomas, Terada et al. reported significant cancer cells and PC3 prostate cancer cells were grown on chamber correlations between high PKR expression, vascular invasion, and the slides to 70% confluence (5 x 10 cells/well) and then infected with presence of satellite tumor nodules. These data suggest that PKR Ad-luc or Ad-Δ6PKR or treated with PBS as a negative control. may play a role in cancer cell metastases. Further investigation is Seventy-two hours later, cells were washed with PBS and fixed with required to determine the effect of PKR inhibition on the develop- 4% paraformaldehyde/PBS for confocal microscopic analysis as ment of metastases. previously described. In brief, cells were blocked with 1% normal goat serum for one hr and then incubated overnight at a dilution Materials and Methods of 1:100 with the primary PKR antibody. Next, the slides were washed to remove primary antibody, rinsed with PBS, and placed Cell lines and reagents. Human lung (A549 and H460), pros- tate (PC3 and LNcaP), breast (MCF-7 and T47D), colon (SW620, in a prewarmed staining solution containing ER-Tracker red dyes HT-29 and KM12L4) cancer cell lines and normal cell lines (Molecular Probes) for 15–20 min at 37°C. Then the slides were (normal human bronchial epithelial cells [NHBEs] and normal washed and incubated with a fluorescein isothiocyanate-conjugated human mammary epithelial cell [HMECs]) were obtained from secondary antibody (Invitrogen, Eugene, OR) for one hr. Next, the +/+ the American Type Culture Collection (Manassas, VA). PKR slides were mounted with ProLong Gold antifade reagent containing -/- and PKR MEFs were obtained from Dr. Glen Barber (University 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) and analyzed with 7-9 of Miami School of Medicine, Miami, FL. Caspase-4 and JNK an Olympus FluoView FV500 laser confocal microscope (Olympus inhibitors were obtained from Calbiochem (San Diego, CA). Final America, Melville, NY) after adjustment for background staining. working solutions were diluted in medium to contain <0.01% of Statistical analysis. Data reported in the figures are the means dimethyl sulfoxide. All experiments using these compounds were and standard deviations (SD) of three independent experiments. performed under subdued lighting conditions. Differences from untreated controls were considered statistically Adenovirus production and plasmid constructs. We have devel- significant in all experiments at p < 0.05. oped an adenoviral vector expressing a mutant form (Δ6PKR) of PKR. Acknowledgements The Δ6PKR protein has a deletion of six amino acids (AAs 361–366, We thank Bingbing Wang for her technical assistance and Debbie LFIQME) between catalytic domains IV and V, cannot autophospho- Smith for her assistance in preparing the manuscript. rylate or activate substrates, and acts as a dominant negative inhibitor This work was supported by grants from the National Cancer of PKR function. Construction of the adenoviral luciferase (Ad-luc) Institute, National Institutes of Health: P01 CA78778-01A1 (J.A.R., 23,25 and Ad-Δ6PKR vectors has been reported previously. The trans- S.G.S.), SPORE 2P50-CA70970-04, SBIR S/C 1R43 CA86587-1 duction efficiencies of adenoviral vectors in cancer cell lines were (S.G.S.), and The University of Texas M.D. Anderson Cancer Center determined by infecting cells with Ad-LacZ and then quantifying the Support Core Grant (CA 16672); by gifts to the Division of Surgery titers needed to transduce at least 70% of the cells. from Tenneco and Exxon for the Core Laboratory Facility; by the Flow cytometric analysis. We measured apoptotic cells by W.M. Keck Foundation; by a sponsored research agreement with propidium iodide staining and fluorescence-activated cell sorting Introgen Therapeutics, Inc., (SR93-004-1); and by support from (FACS) analysis. Cells were harvested; pelleted by centrifugation; the Homer Flower Gene Therapy Fund, the Charles Rogers Gene resuspended in phosphate-buffered saline (PBS) containing 50 μg/ Therapy Fund, and the George P. Sweeney Esophageal Research mL propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate; Fund. and subjected to vortex mixing prior to FACS analysis (FL-3 channel, Acknowledgements FACScan; Becton-Dickinson, Mountain View, CA). This work was supported by grants from the National Cancer Immunoblot analysis. At 72 hrs after transfection, the cell extracts Institute of Health: SPORE 5P50 CA70907 and The University were prepared and immunoblot assays performed as previously of Texas M.D. Anderson Cancer Center Support Core Grant: described. Antibodies to JNK, phosphorylated JNK (phospho- CA16672. Additional support was provided from the Homer Flower JNK), cJun, phosphorylated cJun (phospho-cJun), caspase 4, PKR Gene Therapy Fund, Charles Rogers Gene Therapy Fund, and the (K-17), phosphorylated PKR (Thr ), AKT1/2, phosphorylated George P. Sweeney Esophageal Research Fund. AKT (Ser ), and β-actin (control) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). References 1. Barber GN. Host defense, viruses and apoptosis. Cell Death Differ 2001; 8:113-26. Small interfering RNA transfections. PKR and luc siRNA 2. Williams BRG. Signal integration via PKR. Sci STKE 2001; 89:2. were obtained from Santa Cruz Biotechnology. Transfections were 3. Katze MG. The war against the interferon-induced dsRNA-activated protein kinase: Can performed according to the manufacturer’s instructions. Briefly, viruses win? J Interferon Res 1992; 12:241-8. 1 x 10 cells were seeded onto 12 well plates with antibiotic-free www.landesbioscience.com Cancer Biology & Therapy 251 Inhibition of PKR leads to cancer cell death 4. Jagus R, Joshi B, Barber GN. PKR, apoptosis and cancer. Int J Biochem Cell Biol 1999; 31. Onuki R, Bando Y, Suyama E, Katayama T, Kawasaki H, Baba T, et al. An RNA-dependent 31:123-38. protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer’s disease. EMBO J 2004; 23:959-68. 5. Gil J, Esteban M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 2000; 5:107-14. 32. Bando Y, Onuki R, Katayama T, Manabe T, Kudo T, Taira K, et al. Double-strand RNA dependent protein kinase (PKR) is involved in the extrastriatal degeneration in Parkinson’s 6. Gil J, Alcamí J, Esteban M. Induction of apoptosis by double-stranded-RNA-dependent disease and Huntington’s disease. Neurochem Int 2005; 46:11-8. protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NFkappaB. Mol Cell Biol 1999; 9:4653-63. 33. Wu J, Kaufman RJ. From acute ER stress to physiological roles of the unfolded protein 7. Der SD, Yang YL, Weissmann C, Williams BR. A double-stranded RNA-activated protein response. Cell Death Differ 2006; 13:374-84. kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci USA 34. Kim SH, Gunnery S, Choe JK, Mathews MB. Neoplastic progression in melanoma and 1997; 94:3279-83. colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR. Oncogene 2002; 21:8741-8. 8. Balachandran S, Kim CN, Yeh WC, Mak TW, Bhalla K, Barber GN. Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death 35. Nussbaum JM, Major M, Gunnery S. Transcriptional upregulation of interferon-induced signaling. EMBO J 1998; 17:6888-902. protein kinase, PKR, in breast cancer. Cancer Lett 2003; 196:207-16. 9. Pataer A, Vorburger SA, Barber GN, Chada S, Mhashilkar AM, Zou-Yang H, et al. 36. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt promotes cell survival Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apop- by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999; 96:857-68. tosis of lung cancer cells via upregulation of the double-stranded RNA-dependent protein 37. Nakanishi C, Toi M. Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs. Nat kinase (PKR). Cancer Res 2002; 62:2239-43. Rev Cancer 2005; 5:297-309. 10. Vorburger SA, Pataer A, Yoshida K, Barber GN, Xia W, Chiao P, et al. Role for the double- 38. Pommier Y, Sordet O, Antony S, Hayward RL, Kohn KW. Apoptosis defects and chemo- stranded RNA activated protein kinase PKR in E2F-1-induced apoptosis. Oncogene 2002; therapy resistance: molecular interaction maps and networks. Oncogene 2004; 23:2934-49. 21:6278-88. 39. Delgado André N, De Lucca FL. Knockdown of PKR expression by RNAi reduces pulmo- 11. Holzen UV, Bocangel D, Pataer A, Vorburger SA, Liu Y, Lu X, et al. Role for the double- nary metastatic potential of B16-F10 melanoma cells in mice: Possible role of NFkappaB. stranded RNA-activated protein kinase PKR in Ad-TNFalpha gene therapy in esophageal Cancer Lett 2007; 258:118-25. cancer. Surgery 2005; 138:261-8. 40. Terada T, Maeta H, Endo K, Ohta T. Protein expression of double-stranded RNA-activated 12. Pataer A, Vorburger SA, Chada S, Balachandran S, Barber GN, Roth JA, et al. Melanoma protein kinase in thyroid carcinomas: correlations with histologic types, pathologic param- differentiation-associated gene-7 protein physically associates with the double-stranded eters and Ki-67 labeling. Hum Pathol 2000; 31:817-21. RNA-activated protein kinase PKR. Mol Ther 2005; 11:717-23. 13. Haines GK, 3rd, Becker S, Ghadge G, Kies M, Pelzer H, Radosevich JA. Expression of the double-stranded RNA-dependent protein kinase (p68) in squamous cell carcinoma of the head and neck region. Arch Otolaryngol Head Neck Surg 1993; 119:1142-7. 14. Haines GK, 3rd, Panos RJ, Bak PM, Brown T, Zielinski M, Leyland J, et al. Interferon- responsive protein kinase (p68) and proliferating cell nuclear antigen are inversely distrib- uted in head and neck squamous cell carcinoma. Tumour Biol 1998; 19:52-9. 15. Terada T, Maeta H, Endo K, Ohta T. Protein expression of double-stranded RNA activated protein kinase in thyroid carcinomas: correlations with histologic types, pathologic param- eters and Ki-67 labeling. Hum Pathol 2000; 31:817-21. 16. Yang YL, Reis YF, Pavlovic J, Aguzzi A, Schäfer R, Kumar A, et al. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J 1995; 14:6095- 17. Abraham N, Stojdl DF, Duncan PI, Méthot N, Ishii T, Dubé M, et al. Characterization of transgenic mice with targeted disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR. J Biol Chem 1999; 274:5953-62. 18. Kim SH, Gunnery S, Choe JK, Mathews MB. Neoplastic progression in melanoma and colon cancer is associated with increased expression and activity of the interferon-inducible protein kinase, PKR. Oncogene 2002; 21:8741-8. 19. Roh MS, Kwak JY, Kim SJ, Lee HW, Kwon HC, Hwang TH, et al. Expression of double- stranded RNA-activated protein kinase in small-size peripheral adenocarcinoma of the lung. Pathol Int 2005; 55:688-93. 20. Li M, Shillinglaw W, Henzel WJ, Beg AA. The Rela(p65) subunit of NFkappaB is essential for inhibiting double-stranded RNA-induced cytotoxicity. J Biol Chem 2001; 276:1185-94. 21. Donze O, Deng J, Curran J, Sladek R, Picard D, Sonenberg N. The protein kinase PKR: A molecular clock that sequentially activates survival and death programs. EMBO J 2004; 23:564-71. 22. Barber GN. The dsRNA-dependent protein kinase, PKR and cell death. Cell Death Differ 2005; 12:563-70. 23. Takada Y, Ichikawa H, Pataer A, Swisher SG, Aggarwal BB. Genetic deletion of PKR abrogates TNF-induced activation of IκBα kinase, JNK, Akt and cell proliferation but potentiates p44/p42 MAPK and p38 MAPK activation. Oncogene 2006; 21:1-12. 24. Pataer A, Bocangel D, Chada S, Roth JA, Hunt KK, Swisher SG. Enhancement of adenoviral mda-7 mediated cell killing in human lung cancer cells by geldanamycin and its 17-allylamino-17-demethoxy analogue. Cancer Gene Ther 2006; 10:1-7. 25. von Holzen U, Pataer A, Raju U, Bocangel D, Vorburger SA, Liu Y, et al. The double- stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation. Clin Cancer Res 2007; 13:6032-9. 26. Moenner M, Pluquet O, Bouchecareilh M, Chevet E. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res 2007; 67:10631-4. 27. Malhotra JD, Kaufman RJ. Endoplasmic reticulum stress and oxidative stress: A vicious cycle or a double-edged sword? Antioxid Redox Signal 2007; 12:2277-93. 28. Shimazawa M, Ito Y, Inokuchi Y, Hara H. Involvement of double-stranded RNA-dependent protein kinase in ER stress-induced retinal neuron damage. Invest Ophthalmol Vis Sci 2007; 48:3729-36. 29. Kim R, Emi M, Tanabe K, Murakami S. Role of the unfolded protein response in cell death. Apoptosis 2006; 11:5-13. 30. Bergeron J, Benlimame N, Zeng-Rong N, Xiao D, Scrivens PJ, Koromilas AE, et al. Identification of the interferon-inducible double-stranded RNA-dependent protein kinase as a regulator of cellular response to bulky adducts. Cancer Res 2000; 60:6800-4. 252 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity

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Copyright © 2009 Landes Bioscience
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[Cancer Biology & Therapy 8:3, 245-252; 1 February 2009]; ©2009 Landes Bioscience Research Paper Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity 1, 1 1 2 2 Abujiang Pataer, * Stephen G. Swisher, Jack A. Roth, Christopher J. Logothetis and Paul G. Corn 1 2 Departments of Thoracic and Cardiovascular Surgery, and Genitourinary Medical Oncology; The University of Texas MD Anderson Cancer Center; Houston, TX USA Key words: PKR, gene therapy, adenovirus 1-6 RNA-dependent protein kinase is an interferon-induced, signal transduction and differentiation. While PKR controls the double-stranded (ds), RNA-activated serine/threonine protein expression of multiple genes and signaling pathways, its function kinase involved in the eukaryotic response to viral infection. While in suppressing or promoting mammalian cell growth is somewhat PKR also functions in cellular differentiation, growth control and controversial. apoptosis, its role in human cancer remains poorly understood. To In support of its role in growth suppression, for example, treat- explore a role for PKR in human cancer, we evaluated PKR expres- ment of murine cell lines with dsRNA, TNFα or lipopolycacharide sion and function in a series of cancer cell lines from different leads to a PKR-dependent apoptosis, which may be due to phospho- tumor types. We observed that PKR protein expression is high in rylation of eIF-2α, but also to the expression of other pro-apoptotic 7,8 various cancer cells and low in normal cells. Knockdown of PKR factors such as Fas. In addition, PKR induces apoptosis in epithelial protein expression by PKR siRNA induced cell death, indicating a cancer cell lines in response to adenoviral overexpression of TNFα, PKR-dependent survival pathway under normal growth conditions. E2F-1 and the melanoma differentiation-associated gene 7 (mda7), a 9-12 Inhibition of PKR signaling using a dominant negative adenoviral novel tumor suppressor gene. Lastly, in studies of primary human PKR mutant (Ad-Δ6PKR) also induced cancer cell apoptosis via a cancers from the head and neck, thyroid and colon, increasing PKR mechanism that blocks activation of AKT-mediated survival while expression has been correlated with more well-differentiated tumors simultaneously inducing ER stress. ER stress-mediated apop- and diminished proliferative activity, suggesting that PKR regulates 13-15 tosis was evidenced by unregulated expression of phosphorylated tumor suppression. JNK (p-JNK), phosphorylated cJun (p-cJun), and caspase-4 and Other studies, however, do not support a tumor suppressor role was significantly reduced in cancer cells treated with JNK and for PKR and raise the alternative hypothesis that PKR functions in caspase-4 inhibitors. We further demonstrated that inhibition of growth promotion. For example, PKR knockout mice do not show 16,17 PKR signaling via either siRNA or Ad-Δ6PKR sensitizes cancer increased spontaneous rates of tumor development. In addition, cells to etoposide or cisplatin-mediated cell death. Our results increased expression and activity of PKR has been reported in some 18,19 suggest a rationale to develop therapeutic strategies that target human tumor types and correlates with neoplastic progression. PKR signaling in human cancer cells. Biochemical studies have shown that PKR can phosphorylate IKK, leading to activation of the NFκB anti-apoptotic signaling Introduction 20 pathway. PKR can also induce the expression of several key survival genes known to be dependent on NFκB, such as c-IAP1, c-IAP2 and RNA-dependent protein kinase (PKR) is an interferon-induced, A20. PKR also “crosstalks” with multiple signaling pathways that double-stranded (ds), RNA-activated serine/threonine protein kinase 1-3 activate and engage a number of transcription factors implicated in that has a well-established role in anti-viral defense mechanisms. oncogenesis. In response to dsRNAs and interferons, activated PKR inhibits Several recent studies from our group further support the hypoth- cellular protein translation by phosphorylation of eIF-2α, an event esis that PKR promotes cellular survival. First, using PKR wild-type that induces cellular apoptosis. In the absence of viral infections, (+/+) and knockout (-/-) MEFs to deduce PKR function, treatment however, PKR also plays a role in other important cellular functions with tumor necrosis factor (TNF) activated AKT and NFκB in including growth control, apoptosis regulation, cell proliferation, a PKR-dependent manner. In conjunction with this, PKR was required for TNF-induced NFκB-dependent anti-apoptotic protein *Correspondence to: Abujiang Pataer; Departments of Thoracic and Cardiovascular expression. Secondly, PKR was shown to positively regulate an Surgery; Unit 23 or Department of Genitourinary Medical Oncology; Unit 1374; The University of Texas MD Anderson Cancer Center; 1515 Holcombe Blvd.; Houston, TX AKT-dependent survival pathway in human lung cancer cells. 77030 USA; Tel.: 713.792.8905; Fax: 713.794.4901; Email: apataer@mdander- Thirdly, PKR mediates resistance to radiation therapy in mouse son.org embryo fibroblasts through the activation of AKT and NFκB- Submitted: 10/09/08; Revised: 11/17/08; Accepted: 11/08/08 dependent signaling pathways. Collectively, these results suggest that PKR can function to enhance cell survival, although this phenotype Previously published online as a Cancer Biology & Therapy E-publication: may depend on the stress signal, cell-type and physiologic context. http://www.landesbioscience.com/journals/cbt/article/7386 www.landesbioscience.com Cancer Biology & Therapy 245 Inhibition of PKR leads to cancer cell death Figure 1. Knockdown of PKR by siRNA induces cell death in cancer cell lines. (A) Western blot analysis of PKR protein expression in human lung (A549 and H460), prostate (PC3 and LNcaP), breast (MCF-7 and T47D), and colon (SW620, HT-29 and KM12L4) cancer cell lines and normal cells (NHBEs and HMECs). The expression of actin was used as a loading control. (B) Immunofluorescence microscopy with antibodies against PKR (green) demonstrated cytosolic expression of PKR in A549 and PC3 cancer cells and diffuse expression of PKR in normal cells (NHBEs and HMECs). (C) Western blot and flow cytometric analyses of PKR protein expression and cell death in human lung (A549), prostate (PC3), breast (T47D) cancer cell lines and normal cells (NHBEs) cells after 72 hrs of treatment with PKR siRNA and luc SiRNA. Indicated at the bottom is the percent cell death induced by PKR siRNA and Luc siRNA treat- ment in each of these cell lines. Actin expression was analyzed as a loading control. In the present study, we explored the role for PKR in promoting contrast, we observed a diffuse expression pattern of PKR in NHBEs resistance to chemotherapy. We show that inhibition of PKR and HMECs normal cells (Fig. 1B). signaling using a dominant negative PKR mutant (Ad-Δ6PKR) acts We next investigated the effect of silencing PKR protein expres- synergistically with chemotherapy to induce cell death in human sion on cell viability. As revealed by immunoblotting and FACS cancer cell lines. The induction of cell death was due in part to the analysis, treatment of A549, PC3 and T47 cancer cells with PKR activation of an endoplasmic reticulum (ER) stress pathway. Our siRNA effectively knocked down expression of PKR protein and results suggest a therapeutic strategy to target PKR signaling in the induced apoptosis (Fig. 1C). Conversely, treatment with luc siRNA treatment of human cancer. did not knock down expression of PKR protein nor induce apop- tosis (Fig. 1C). Normal NHBEs cells treated with PKR siRNA also Results underwent apoptosis, but to a much lesser extent than the cancer cells (12% versus ~45–50% in normal and cancer cells, respectively) Knock down of PKR by siRNA in cancer cells induces cell (Fig. 1C). Taken together, these results suggest differential function death. We first investigated the expression and cellular localization of of PKR between normal and cancer cells. PKR in human cancer cells as well as normal cells. Figure 1A shows Inhibition of PKR by adenoviral mutant PKR (Ad-Δ6PKR) that PKR protein expression is high in multiple cell lines derived induces cell death in cancer cells. We have previously shown that from human lung (A549, H460), prostate (PC3, LNCaP), breast a deletion mutant of PKR (Δ6PKR) can act as a dominant negative (MCF7, T47D) and colon (SW620, HT-29, KM12L4) cancers. inhibitor of PKR function. As shown by flow cytometric analysis In contrast, PKR expression was low in normal human bronchial of human lung (A549 and H460), prostate (PC3 and LNCaP) and epithelial cells (NHBEs) and normal human mammary epithelial breast (MCF-7 and T47D) cancer cells, adenoviral infection with cells (HMECs). Δ6PKR (Ad-Δ6PKR) induced cell death within 72 hrs of infection We next investigated the pattern of PKR expression in two cancer cell lines (A549 and PC3) and the two normal cell lines (NHBEs and (Fig. 2A). In contrast, Ad-Δ6PKR was not toxic to NHBE or HMEC HMECs) by confocal immunofluorescence analysis. We observed normal cells (Fig. 2A). +/+ both cytosolic and nuclear localization of PKR in A549 and PC3 Our previous studies in PKR MEFs indicated that PKR activates cancer cells with the predominant signal in the cytosol (Fig. 1B). In an AKT-dependent anti-apoptotic survival pathway by upregulating 246 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 Inhibition of PKR leads to cancer cell death different environmental and metabolic 26,27 stimuli. Under conditions of stress (for example with hypoxia and/or nutrient deprivation) misfolded/unfolded proteins accumulate in the ER and stimulate a signaling pathway known as the “unfolded protein response” (UPR). The UPR permits cells to either adapt to the stress until the stress resolves, or alternatively, promotes apoptosis when the stress related damage becomes irre- versible. Previous results suggested that PKR plays an important role in ER stress-induced retinal neuron damage in mice. We next tested whether Ad-Δ6PKR- mediated apoptosis of cancer cell lines resulted from activation of ER-stress. To do this, we measured downstream target proteins that are activated via phosphory- lation in response to ER-stress, including JNK and c-Jun. A549 and PC3 cancer cell lines were treated for 72 hours with either PBS, Ad-Luc, Ad-Δ6PKR or endoplasmic reticulum targeting melanoma differen- tiation-associated gene 7 (Ad-ER-mda7). Ad-ER-mda7 served as a positive control. As shown in Figure 3A, expression of phospho- JNK and phospho-cJun was upregulated after Ad-Δ6PKR and Ad-ER-mda7 transduction but not after Ad-Luc transduction (Fig. 3A), suggesting that activation of JNK or cJun was the mechanism by which Ad-Δ6PKR medi- ated cell death. Figure 2. Inhibition of PKR by Adenoviral mutant PKR (Ad-Δ6PKR) vector induces cell death in Human caspase-4 is localized to the ER cancer cells. (A) Flow cytometric analysis of cell death in A549, H460, PC3, LNCaP, MCF-7, T47D, membrane and is specifically activated by and NHBEs and HMECs cells 72 hours after treatment with PBS, Ad-luc (3000 vp) or Ad-Δ6PKR (3000 required for ER stress-induced apoptosis. vp). Experiments were performed in triplicate; data are presented as the means (error bars, SDs). (B) We next investigated whether caspase-4 Western blot analysis of phosphorylated PKR (p-PKR), PKR, phosphorylated AKT (p-AKT), and AKT1/2 was involved in Ad-Δ6PKR-induced cell protein expression in A549 and PC3 cell lysates 72 hours after treatment with PBS, Ad-luc (3000 vp) or Ad-Δ6PKR (3000 vp). β-Actin expression was analyzed as a loading control. death. Caspase-4 was cleaved to its active form in A549 and PC3 cancer cells treated with Ad-Δ6PKR or Ad-ER-mda7 (positive 473 23,25 levels of phospho-AKT (Ser ). Conversely, Ad-Δ6PKR inhib- control) but not in those treated with PBS or Ad-Luc (Fig. 3A). 473 +/+ 25 ited phospho-AKT (Ser ) in PKR MEFs. To test whether To more directly determine the importance of JNK and caspase-4 Ad-Δ6PKR would also inhibit AKT phosphorylation in cancer cells, activation in Ad-Δ6PKR-mediated cell death, A549 and PC3 cancer we infected A549 and PC3 cells with Ad-Δ6PKR. As revealed by cells were infected with Ad-Δ6PKR in the presence or absence of a immunoblotting, a high level of PKR expression was observed in chemical JNK inhibitor (SP600125, 10 μmol/L), a peptide inhibitor A549 and PC3 cancer cells after being infected with Ad-Δ6PKR of caspase-4 (Ac-LEVD-CHO, 10 μmol/L) or combination of (Fig. 2B), whereas low levels of endogenous PKR protein were the two inhibitors. In both cancer cell lines, inhibitory treatment observed in cells infected with the control contructs. Ad-Δ6PKR reduced the amount of AdΔ6PKR-induced cell death as measured infected cancer cells demonstrated decreased expression of phospho- by FACS. In A549 cells, apoptosis was reduced from 28.5% to 15% 451 473 PKR (Thr ) and phospho-AKT (Ser ). We could not detect after JNK inhibition, 16.8% after caspase 4 inhibition, and 7% decreased expression of phosphorylated PKR and AKT1/2 in Ad-Luc after treatment with both inhibitors (p < 0.05, significantly different infected cancer cells (Fig. 2B). These results further confirmed that from untreated). Similarly in PC3 cells, apoptosis was reduced from Ad-Δ6PKR can inhibit phospho-AKT (Ser ) in cancer cells. 29.9% to 17% after JNK inhibition, 16.4% after caspase 4 inhibi- Activation of the endoplasmic reticulum (ER) stress response tion, and 6.7% after treatment with both inhibitors (Fig. 3B) (p contributes to mutant PKR (Ad-Δ6PKR)-induced apop- < 0.05, significantly different from untreated). Conversely, neither tosis. The endoplasmic reticulum functions in normal cellular inhibitor reduced the amount of cell death induced by overexpres- homeostasis by ensuring proper protein folding in response to sion of Ad-Bak, a Bcl-2 family member that induces apoptosis in a www.landesbioscience.com Cancer Biology & Therapy 247 Inhibition of PKR leads to cancer cell death Figure 3. Adenoviral mutant PKR (Ad-Δ6PKR) induces the endoplasmic reticulum (ER) stress-mediated cell death pathway. (A) Western blot analysis of phos- phorylated JNK (p-JNK), JNK, phosphorylated cJun (p-cJun), cJun and caspase-4 protein expression in A549 and PC3 cell lysates 72 hrs after treatment with Ad-luc (3000 vp), Ad-ER-mda7 (3000 vp) or Ad-Δ6PKR (3000 vp). β-Actin expression was analyzed as a loading control. (B) Flow cytometric analysis of cell death in A549 and PC3 cancer cells 72 hrs after treatment with PBS, Ad-Luc (3000 vp) or Ad-Δ6PKR (3000 vp) with or without caspase-4 (Ac-LEVD-CHO, 10 μmol/L) or JNK chemical inhibitors (10 μmol/L). Experiments were performed in triplicate; data are presented as the means (error bars, SDs) *p < 0.05 vs. untreated control, Wilcoxon rank-sum test. (C) Immunofluorescent confocal microscopic analysis of PKR protein expression. Analysis of antibody against PKR stained green, ER stained red with ER-Tracker dyes, and nuclei stained blue with DAPI, demonstrated colocalization (orange) of PKR protein and the ER marker in Ad-Δ6PKR-treated A549 and PC3 cancer cells 72 hrs after transfection. +/+ PKR-independent manner (data not shown). Together, these results (PKR ) MEFs to bulky adduct DNA damage caused by UV suggest that Ad-Δ6PKR cell death is mediated in part through radiation or alkylating chemotherapeutic agents such as cisplatin activation of ER stress and downstream effectors such as JNK and and melphalan. To confirm and expand on these results, we first +/+ -/- caspase-4. compared the viability of PKR and PKR MEFs following treat- We next analyzed the pattern of PKR expression in A549 lung ment with DNA damaging agents (etoposide, cisplatin, mitomycin), cancer and PC3 prostate cancer cells after treatment with PBS, an anti-metabolite (5-fluouracil [5FU]) and an anti-tubulin (Taxol). -/- Ad-Luc or Ad-Δ6PKR. Confocal immunofluorescence analysis In agreement with Bergeron et al. we found that PKR MEFs cells +/+ revealed that PKR levels were markedly higher in the cytosol of are much more susceptible than PKR MEFs to cell death induced A549 and PC3 cells after treatment with Ad-Δ6PKR (Fig. 3C). by cisplatin or melphalan (Fig. 4A) (p < 0.05). In contrast, there was The precise subcellular localization of the PKR (green) was further no difference following treatment with Mitomycin C. The enhanced confirmed by comparing its expression pattern with an ER molec- sensitivity to DNA-damage induced apoptosis was not restricted to -/- ular marker (red) known to reside within the ER compartment akylating agents, as PKR MEFs were also more sensitive to DNA (Fig. 3C). Treatment with Ad-Δ6PKR led to an increase in PKR damage by etoposide, a Topoisomerase II poison. Interestingly, -/- +/+ within the ER, as evidenced by enhanced colocalization fluorescence PKR MEFs were also more susceptible than PKR MEFs to cell (yellow-orange) in the overlay images. These results further support death induced by 5FU but not Taxol. -/- a role for the endoplasmic reticulum in Ad-Δ6PKR induced cellular To control for the possibility that PKR MEFs acquired genetic apoptosis. change(s) outside the PKR locus that could account for these results, +/+ Adenoviral mutant PKR (Ad-Δ6PKR) sensitizes cancer cells we next transfected PKR MEFs with PKR siRNA and then +/+ to some chemotherapeutic agents. In a single study previously treated them with cisplatin or etoposide. PKR MEFs transfected -/- published by Bergeron et al. PKR-deficient (PKR ) mouse-embry- with PKR siRNA underwent chemotherapy-induced apoptosis to a -/- +/+ onic fibroblasts (MEFs) were more sensitive than PKR-proficient similar degree as PKR MEFs (Fig. 4B). In contrast, PKR MEFs 248 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 Inhibition of PKR leads to cancer cell death (55% with etoposide and 50% with cisplatin) and PC3 (57% with etoposide and 48% with cisplatin) cancer cells (Fig. 5) (p < 0.05, significantly different from untreated or Ad-luc treated). In contrast, Ad-luc did not have any significant effects on cell viability, either alone or in combination with chemotherapy. Discussion PKR is a stress-activated kinase induced in response to multiple different stimuli including viral infections, interferons, interleukins, TNFα, lipopolysaccharide and 1-6,22 ionizing radiation. In the present study, we tested whether knockdown of PKR by PKR siRNA or inhibi- tion of PKR by adenoviral mutant PKR (Ad-Δ6PKR) alters cell viability or chemotherapy-mediated apop- tosis in human cancer cells. We demonstrated that PKR protein expression is high in various cancer cell lines and low in normal cells (Fig. 1A). We have inves- tigated the pattern of PKR expression in two cancer cell lines (A549 and PC-3) and two normal cell lines (NHBE and HMECs) by confocal immunofluores- cence analysis. We observed both the cytosol (most) and nuclei (less) localization of PKR in A549 and PC-3 cancer cells. We observed a diffuse expression pattern of PKR in NHBE and HMECs normal cells. Once activated, PKR translocates to the nucleus. Unlike the known cytoplasmic function of PKR, the role of PKR in the nucleus has yet to be defined. Dr. Barber’s laboratory has demonstrated that both NFAR-1 and -2 (nuclear factors associated with dsRNA) are substrates for PKR. Dr. Barber’s results indicate that the NFARs may facilitate double-stranded RNA-regulated -/- Figure 4. PKR-deficient mouse-embryonic fibroblasts (PKR ) are susceptible to chemo- gene expression at the level of post-transcription and +/+ -/- therapeutic agents. (A) Flow cytometric analysis of apoptosis in PKR and PKR MEF cells possibly contribute to host defense-related mechanisms after 72 hrs of treatment with etoposide, cisplatin, 5-FU, melphalan, Taxol or mitomycin. in the cell. Knockdown of PKR by PKR siRNA Experiments were performed in triplicate; data are presented as the means (error bars, SDs) induced cell death in these cancer cells under normal *p < 0.05 vs. corresponding control, Wilcoxon rank-sum test. (B) Flow cytometric analysis +/+ -/- growth conditions, underscoring the dependence of of apoptosis in PKR and PKR MEF cells after 72 hrs of treatment with PKR siRNA and luc SiRNA with or without etoposide (ET) or cisplatin (CDDP) treatment. Experiments were these cells on PKR signaling even in the absence of performed in triplicate; data are presented as the means (error bars, SDs) *p < 0.05 vs. exogenous stress stimuli (Fig. 1C). Inactivation of corresponding control, Wilcoxon rank-sum test. PKR by dominant negative adenoviral mutant PKR (Ad-Δ6PKR) selectively and effectively induces cell transfected with a control (Luciferase) siRNA retained a relative death may partly due to reduction of phospho-AKT (Ser ) protein -/- resistance to etoposide or cisplatin when compared to PKR MEFs. (Fig. 2A and B). Collectively, these results demonstrate that a PKR-dependent survival We have also determined the long-term effect of PKR siRNA pathway mediates resistance to some chemotherapeutic agents. or Ad-Δ6PKR treatment on cell growth using a clonogenic assay. We next wanted to test whether inhibition of PKR signaling Both PKR siRNA and Ad-Δ6PKR treatments caused significant would confer a similar chemosensitization effect in human cancer reduction in clonogenic potential of A549 and PC3 cells (data not cells. To do this, we examined the apoptotic effects of Ad-Δ6PKR shown). Despite similar phenotypes, however, we postulate that alone or in combination with etoposide (10 μmol/L), cisplatin (10 inactivation of PKR signaling using siRNA may not be functionally μmol/L), or both drugs in A549 and PC3 cancer cells after 72 hrs equivalent to inactivation using dominant negative Ad-Δ6PKR. treatment. By flow cytometric analysis, Ad-Δ6PKR alone resulted Downregulation of PKR protein by siRNA would be predicted in cell death rates of 25% in A549 cells and 24% in PC3 cells (Fig. to inhibit both apoptotic and survival pathways equally. In this 5). Treatment with etoposide or cisplatin as single agents resulted case, cell fate (survival versus death) would reflect the balance in cell death rates of 10% and 8.6% respectively in A549 cells of pro-apoptotic versus anti-apoptotic pathways and depend on and 11% and 9% respectively in PC3 cells (Fig. 5). Importantly, which pathway was dominant in a particular cancer cell (cell-type the combination of Ad-Δ6PKR with either etoposide or cisplatin dependent). In contrast, inactivation of PKR by dominant negative resulted in a synergistic enhancement of apoptosis in both A549 PKR may result predominantly in cell death because Ad-Δ6PKR www.landesbioscience.com Cancer Biology & Therapy 249 Inhibition of PKR leads to cancer cell death preferentially induces ER-stress mediated apoptosis in cancer cells (cell-type independent). Future studies aim to elucidate the precise mechanism(s) of PKR siRNA and Δ6PKR mediated apoptosis in cancer cells versus normal cells. PKR associates with ribosomes, 1-3 mainly to the 40S subunits. In a recent study by Onuki et al. PKR was shown to be involved in ER stress-mediated apoptosis induced by tunicamycin treatment of SK-N-SH human neuroblastoma cells. In that study, tunicamycin treatment increased expression levels of phos- phoylated PKR in the nucleus. Another recent study showed a role for PKR in endoplasmic reticulum (ER) stress-induced neuronal cell death in cases of Alzheimer’s disease and Huntington’s disease. In our study, we demonstrated that activation of ER stress occurs as a consequence of deregulating PKR signaling with Figure 5. Adenoviral mutant PKR (Ad-Δ6PKR) enhanced etoposide (ET)- or cisplatin (CDDP)-mediated cell death in cancer cells. Flow cytometric analysis of apoptosis in Ad-Δ6PKR-transfected A549 and PC3 cancer cells, mutant PKR (Δ6PKR). This was with or without etoposide or cisplatin treatment after 72 hrs. Experiments were performed in triplicate; data are evidenced by increased expression presented as the means (error bars, SDs) *p < 0.05 by Wilcoxon rank-sum test compared with corresponding of phospho-JNK and phosphor-Jun, control (ET, CDDP, Ad-Δ6PKR, Ad-Luc + ET or Ad-Luc + CDDP). and increased cleavage of caspase-4. Taken together, these results suggest -/- significant “crosstalk” between the PKR and ER-stress signaling susceptibility of PKR MEFs to cytotoxic chemotherapy was not pathways. due increased proliferation (and hence potentially increased exposure -/- The ER stress-mediated cell death pathway involves recruitment to the drug), since PKR MEFs actually grow ~50% more slowly +/+ of the cytosolic adaptor TRAF2 to the ER membrane, where TRAF2 than PKR MEFs (data not shown). Secondly, inhibition of PKR activates the apoptosis-signaling kinase 1 (ASK1). Activation of signaling using a dominant-negative mutant sensitized cancer cells ASK1 leads in turn to activation of JNK and mitochondria-depen- to etoposide and cisplatin-mediated cell death. As with MEFs, the dent caspases. In this study, we demonstrated that activation of increased chemosensitivity of Ad-Δ6PKR infected cells was not due JNK and caspase-4 is essential for Ad-Δ6PKR-mediated cell death. increased proliferation since Ad-Δ6PKR caused growth inhibition We hypothesize two possible mechanisms for this. First, inhibition (data not shown). Our working hypothesis is that PKR regulates an of PKR by Ad-Δ6PKR reduces expression of survival factors such AKT and NFκB-dependent survival pathway in response to chemo- as phospho-AKT. Secondly, inactivation of PKR by Ad-Δ6PKR therapy treatment. Since this mechanism mediates PKR-dependent disrupts normal PKR dimerization such that inactive PKR dimers radioresistance, future studies will address if AKT1/2 and NFκB play or monomers translocate to the ER and induce stress. PKR exists in a similar role in promoting chemoresistance. the cell in at least three forms: an inactive monomer associated with Our data add to a growing body of evidence that support a role ribosomes, an inactive dimer in the cytoplasm, and an active dimer. for PKR in promoting tumor cell survival. It has been shown that PKR can also switch from an inactive dimer to an active dimer by a PKR activation contributes to neoplastic progression and decreased 1-6 mechanism involving dsRBM interactions. sensitivity to conventional chemotherapy agents in melanoma and We previously demonstrated that PKR plays a critical role in colon cancer cells, presumably through upregulation of pro-survival 25 - 34 radio-resistance. In that study, we found that PKR-deficient (PKR pathways such as NFκB. Nussbaum et al. have proposed that high /- ) mouse-embryonic fibroblasts (MEFs) were more susceptible to PKR activity in breast cancer cells was responsible for activation of +/+ 25 radiation-induced cell death than PKR-proficient (PKR ) MEFs. protooncogenes such as the platelet-derived growth factor (PDGF) This resistance was mediated in part through PKR-dependent and c-fos that stimulate cell proliferation. We have previously upregulation of AKT1/2. In the present study, we provide demonstrated that PKR can indirectly regulate AKT1/2 and NFκB- 23,25 evidence that PKR also contributes to chemotherapy resistance. dependent survival pathways. This regulation is likely critical -/- First, in agreement with Bergeron et al. PKR MEFs were more for the tumor promoting properties of PKR, since AKT is known +/+ sensitive than PKR MEFs to apoptosis due to bulky adduct to promote cellular survival and NFκB blocks apoptosis induced 30 36-38 DNA damage caused by cisplatin and melphalan. The increased by chemotherapeutic agents. It is possible; therefore, that PKR 250 Cancer Biology & Therapy 2009; Vol. 8 Issue 3 Inhibition of PKR leads to cancer cell death promotes cancer cell growth through activation of survival factors medium overnight. Solution A was prepared by adding 3.6 μL of the such as AKT1/2, NFκB or activation of protooncogenes such as c-fos desired 10 μM siRNA to 60 μL of the siRNA transfection medium, in human cancers. We are now examining whether the Ad-Δ6PKR and solution B, by adding 3.6 μL of the transfection reagent to inhibits activation of NFκB or c-fos. 14.5 μL of the siRNA transfection medium. Both solutions were Lastly, our results suggest a therapeutic strategy to target PKR mixed gently and kept at room temperature for five min, after which signaling in the treatment of human cancer. As proof of principal they were combined to form an siRNA-siRNA transfection reagent for this concept, injection of melanoma tumor cells transfected with complex. This complex was added to cells for five hrs. Luciferase a PKR-specific short hairpin RNA (shRNA) expressing plasmid siRNA was used as a control. resulted in inhibition of pulmonary metastatic nodules in mice. In Immunofluorescent cellular localization studies. A549 lung primary human thyroid carcinomas, Terada et al. reported significant cancer cells and PC3 prostate cancer cells were grown on chamber correlations between high PKR expression, vascular invasion, and the slides to 70% confluence (5 x 10 cells/well) and then infected with presence of satellite tumor nodules. These data suggest that PKR Ad-luc or Ad-Δ6PKR or treated with PBS as a negative control. may play a role in cancer cell metastases. Further investigation is Seventy-two hours later, cells were washed with PBS and fixed with required to determine the effect of PKR inhibition on the develop- 4% paraformaldehyde/PBS for confocal microscopic analysis as ment of metastases. previously described. In brief, cells were blocked with 1% normal goat serum for one hr and then incubated overnight at a dilution Materials and Methods of 1:100 with the primary PKR antibody. Next, the slides were washed to remove primary antibody, rinsed with PBS, and placed Cell lines and reagents. Human lung (A549 and H460), pros- tate (PC3 and LNcaP), breast (MCF-7 and T47D), colon (SW620, in a prewarmed staining solution containing ER-Tracker red dyes HT-29 and KM12L4) cancer cell lines and normal cell lines (Molecular Probes) for 15–20 min at 37°C. Then the slides were (normal human bronchial epithelial cells [NHBEs] and normal washed and incubated with a fluorescein isothiocyanate-conjugated human mammary epithelial cell [HMECs]) were obtained from secondary antibody (Invitrogen, Eugene, OR) for one hr. Next, the +/+ the American Type Culture Collection (Manassas, VA). PKR slides were mounted with ProLong Gold antifade reagent containing -/- and PKR MEFs were obtained from Dr. Glen Barber (University 4',6-diamidino-2-phenylindole (DAPI; Invitrogen) and analyzed with 7-9 of Miami School of Medicine, Miami, FL. Caspase-4 and JNK an Olympus FluoView FV500 laser confocal microscope (Olympus inhibitors were obtained from Calbiochem (San Diego, CA). Final America, Melville, NY) after adjustment for background staining. working solutions were diluted in medium to contain <0.01% of Statistical analysis. Data reported in the figures are the means dimethyl sulfoxide. All experiments using these compounds were and standard deviations (SD) of three independent experiments. performed under subdued lighting conditions. Differences from untreated controls were considered statistically Adenovirus production and plasmid constructs. We have devel- significant in all experiments at p < 0.05. oped an adenoviral vector expressing a mutant form (Δ6PKR) of PKR. Acknowledgements The Δ6PKR protein has a deletion of six amino acids (AAs 361–366, We thank Bingbing Wang for her technical assistance and Debbie LFIQME) between catalytic domains IV and V, cannot autophospho- Smith for her assistance in preparing the manuscript. rylate or activate substrates, and acts as a dominant negative inhibitor This work was supported by grants from the National Cancer of PKR function. Construction of the adenoviral luciferase (Ad-luc) Institute, National Institutes of Health: P01 CA78778-01A1 (J.A.R., 23,25 and Ad-Δ6PKR vectors has been reported previously. The trans- S.G.S.), SPORE 2P50-CA70970-04, SBIR S/C 1R43 CA86587-1 duction efficiencies of adenoviral vectors in cancer cell lines were (S.G.S.), and The University of Texas M.D. Anderson Cancer Center determined by infecting cells with Ad-LacZ and then quantifying the Support Core Grant (CA 16672); by gifts to the Division of Surgery titers needed to transduce at least 70% of the cells. from Tenneco and Exxon for the Core Laboratory Facility; by the Flow cytometric analysis. We measured apoptotic cells by W.M. Keck Foundation; by a sponsored research agreement with propidium iodide staining and fluorescence-activated cell sorting Introgen Therapeutics, Inc., (SR93-004-1); and by support from (FACS) analysis. Cells were harvested; pelleted by centrifugation; the Homer Flower Gene Therapy Fund, the Charles Rogers Gene resuspended in phosphate-buffered saline (PBS) containing 50 μg/ Therapy Fund, and the George P. Sweeney Esophageal Research mL propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate; Fund. and subjected to vortex mixing prior to FACS analysis (FL-3 channel, Acknowledgements FACScan; Becton-Dickinson, Mountain View, CA). This work was supported by grants from the National Cancer Immunoblot analysis. At 72 hrs after transfection, the cell extracts Institute of Health: SPORE 5P50 CA70907 and The University were prepared and immunoblot assays performed as previously of Texas M.D. Anderson Cancer Center Support Core Grant: described. Antibodies to JNK, phosphorylated JNK (phospho- CA16672. Additional support was provided from the Homer Flower JNK), cJun, phosphorylated cJun (phospho-cJun), caspase 4, PKR Gene Therapy Fund, Charles Rogers Gene Therapy Fund, and the (K-17), phosphorylated PKR (Thr ), AKT1/2, phosphorylated George P. Sweeney Esophageal Research Fund. AKT (Ser ), and β-actin (control) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). References 1. 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Cancer Biology & Therapy – Taylor & Francis

Published: Feb 1, 2009

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