Cellular Signalling 26 (2014) 1567–1575

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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

ER stress signaling in ARPE-19 cells after inhibition of protein kinase CK2 by CX-4945 Johanna Intemann 1,2, Nathaniel Edward Bennett Saidu 1,2, Lisa Schwind 1,2, Mathias Montenarh ⁎,2 Medical Biochemistry and Molecular Biology, University of the Saarland, Building 44, D-66424 Homburg, Germany

a r t i c l e

i n f o

Article history: Received 21 November 2013 Received in revised form 26 February 2014 Accepted 17 March 2014 Available online 29 March 2014 Keywords: Protein kinase ER stress Growth arrest CHOP Apoptosis

a b s t r a c t Protein kinase CK2 is a critical factor for the survival of cells. It is overexpressed in many cancer cells and provides protection against apoptosis in these cells. Inhibition of CK2 kinase activity in various cancer cells leads to apoptosis, which makes CK2 an attractive target for cancer therapy. Little is, however, known about CK2 inhibition in non-cancerous cells. Using the human retinal pigment epithelial cell line ARPE-19, we analyzed the formation of reactive oxygen species (ROS) and the ER stress signaling pathway after CK2 inhibition with CX-4945. Following CK2 inhibition, we did not find any significant generation of ROS in neither ARPE-19 non-cancer cells nor in HCT116 cancer cells. We found an induction of the ER stress pathway including the activation of eIF2α and ATF4 in both cell types. This activation was sufficient for ARPE-19 cells to cope with the ER stress. Furthermore, in contrast to HCT116 cancer cells, there was no induction of the pro-apoptotic transcription factor CHOP and no induction of apoptosis in the ARPE-19 cells. Overexpression of CHOP, however, induced apoptosis in ARPE-19 cells indicating that this step in the ER stress pathway is abrogated in normal cells compared to cancer cell. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The endoplasmic reticulum (ER) is the central organelle within a eukaryotic cell where newly synthesized proteins are processed and properly folded. It is also involved in other processes such as lipid and sterol biosynthesis along with the storing of intracellular Ca2+. This makes the ER absolutely essential to the cell. Disturbances to any of these processes may lead to ER stress and activate signaling pathways collectively termed the unfolded protein response (UPR) [1,2]. The activation of the ER stress signaling pathways occurs in order to re-establish cellular homeostasis. Prolonged or severe ER stress however may cause apoptosis through one or more of the ER stress sensors, namely pancreatic ER kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6). Each one of these stress sensors, when activated, may act proportionately on their respective down-stream targets relative to the stress signal. The ER stress response has been linked to several human and other animal diseases including coronary heart diseases (CHDs) [3,4], chronic ocular hypertension (COH) [5] and cancer [6,7]. Efforts are on the way to develop approaches to exploit ER stress mechanisms for therapy in some of these diseases. In cancer for example, there are ongoing studies aimed at pharmacologically aggravating

⁎ Corresponding author. E-mail address: [email protected] (M. Montenarh). 1 These authors contributed equally to this work. 2 Tel.: +49 6841 1626501; fax: +49 6841 1626027.

http://dx.doi.org/10.1016/j.cellsig.2014.03.014 0898-6568/© 2014 Elsevier Inc. All rights reserved.

chronic ER stress in order to induce apoptotic tumor cell death [6]. Protein kinase CK2, a serine/threonine protein kinase that is composed of two catalytic α- or α′-subunits and two regulatory β-subunits is well known to be implicated in the decision of life and death of the cell [8, 9]. Experimental studies have shown that targeting CK2 induces cell death in vivo [10]. The CK2 inhibitor CX-4945 in particular, has been used to pharmacologically induce cancer cell death. In phase I clinical trials, the inhibition of CK2 using CX-4945 has shown promising clinical benefits in patients [11–13]. Recent studies have also shown that the inhibition of protein kinase CK2 induces apoptosis via ER stress response in specific cancer cells [14]. There is therefore an increasing interest in targeting the ER using CK2 inhibitors in an effort to find treatment or preventive measures for diseases such as cancer. It still, however, remains an open question whether CK2 also plays a role in ER stress signaling in normal cells. In this report, we have examined the activation of the ER stress signaling pathway in human retinal pigment epithelial (ARPE-19) cells upon the inhibition of protein kinase CK2 by CX-4945. The retinal pigment epithelium is a monolayer of cells adjacent to the photoreceptors of the retina. Cells of the retinal epithelium play a critical role in the development and maintenance of those adjacent photoreceptors in the vertebrate retina [15]. It is implicated in the transport of nutrients from the vascular choroid, the formation of the blood–retina barrier and the absorption of scattered light. The ARPE-19 cell line is derived from a primary culture of retinal epithelium cells [16]. Most interestingly, we found that the inhibition of CK2 led to the activation of the eIF2α branch of the ER stress signaling pathway, but in contrast to HCT116

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cancer cells, it failed to induce apoptosis in these normal human epithelial ARPE-19 cells. 2. Materials and methods 2.1. Reagents and antibodies Protease inhibitor cocktail Complete™ was obtained from Roche Diagnostics (Mannheim, Germany). Anti-α-tubulin antibody was obtained from Sigma-Aldrich (Munich, Germany), DMSO from Merck (Darmstadt, Germany). Antibodies against ATF4, CHOP (GADD153), and GAPDH were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Anti-poly(ADP-ribose) polymerase (anti-PARP), anticaspase 3, anti-eIF2α, and anti-phospho-eIF2α (Ser 51) were purchased form Cell Signaling Technology (Frankfurt, Germany). Goat, mouse and rabbit secondary antibodies were all bought from Dianova (Hamburg, Germany). CX-4945 was bought from Selleckchem (Munich, Germany) and tunicamycin from ENZO life sciences (Lörrach, Germany). 2.2. Cell culture ARPE-19 cells (ATCC Number: CRL-2302) were maintained at 37 °C and 5% CO2 in Dulbecco's modified Eagles medium (DMEM) supplemented with 2 mM L-glutamine and 10% fetal calf serum (FCS). In order to obtain positive control lysates for the induction of apoptosis, and for comparison to ARPE-19 cells, HCT116 cancer cells (ATCC Number: CCL-247) were cultured as well. Cells were maintained at 37 °C and 5% CO2 in McCoy's 5A medium (PromoCell, Heidelberg, Germany) containing 10% fetal calf serum (FCS). LNCaP cells are androgen-sensitive prostate cancer cells, which were established from a lymph node metastasis [17]. These cells were cultured to obtain positive control extracts for ER stress induction. Cells were maintained at 37 °C in RPMI 1640, supplemented with 10% FCS in an atmosphere enriched with 5% CO2. The CK2 inhibitor CX-4945 (Selleckchem, Munich, Germany) was dissolved in dimethyl sulfoxide (DMSO) to a 10 mM stock solution. Cells were treated with the CK2 inhibitor, tunicamycin or 5-fluorouracil (5-FU) at the indicated final concentrations for different times or the same volume of vehicle as control.

control) or 10 μM CX-4945 for 24 h to 96 h. The cell proliferation rate was determined by counting the cells in a Neubauer counting chamber (factor of the chamber: 104). Shortly, cells were trypsinized and resuspended in a small amount of cell culture medium. 20 μl of the cellsuspension was mixed with 20 μl of the diazo dye trypan blue to stain dead cells. Only living cells were counted in the Neubauer counting chamber. 2.6. Evaluation of cell viability (MTT assay) In order to determine the effect of CX-4945 on cell viability, cells were seeded at 2 × 104 cells per well to a final volume of 500 μl in a 24-well plate and incubated overnight. Cells were then treated with DMSO (solvent control), 10 μM CX-4945 or left untreated for 24 h to 96 h. Viability of the cells was determined by a colorimetric MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) assay according to the manufacturer's instructions. One hour before the end of treatment, 50 μl MTT (5 mg/ml PBS) was added. The enzymatic reaction took place at 37 °C in a humidified atmosphere. Following 1 h MTT treatment, medium was disposed off and cells were solubilized by adding 500 μl solubilizing solution (10% (w/v) SDS in DMSO and 0.6% (v/v) acetic acid) to each well and allowing the crystals to dissolve completely. The spectrophotometrical absorbance of the purple–blue formazan dye was determined with a Tecan 96 well plate reader (Tecan infinite M200, Crailsheim, Germany) at 595 nm. 2.7. Protein kinase CK2 assay To study in vitro CK2 kinase activity, 30 μg of total protein was mixed with kinase buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT)) to a final volume of 20 μl. 30 μl of CK2 mix (25 mM Tris–HCl, pH 8.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 50 μM ATP, 0.19 mM (final concentration) CK2 specific substrate peptide with the sequence RRRDDDSDDD and 10 μCi/500 μl [32P]γATP) was added and the reaction mix incubated at 37 °C for 10 min. The reaction was stopped on ice and the sample pipetted onto Whatman-P81 cation-exchange paper and washed 3 × 5 min with 85 mM phosphoric acid and 1 × 5 min with ethanol. The filter paper was dried and counted for Čerenkov radiation in a scintillation counter (Liquid Scintillation Analyzer 190S AB/LA; Canberra-Packard GmbH, Dreieich, Germany).

2.3. Plasmids 2.8. Analysis of cell cycle progression The p3xFLAG-CMV-7.1-basic vector was from Sigma-Aldrich (Munich, Germany). To generate the expression plasmid p3xFLAGCMV-7.1-CHOP, the human cDNA of CHOP was cloned into p3xFLAGCMV-7.1-basic vector with EcoRI and BamHI. The sequence of the DNA construct was verified by sequencing. 2.4. Transient transfection Transfection of cells was performed by using the MACSfectin™ reagent (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Briefly, ARPE-19 cells were seeded into a 100 mm dish (1 × 106 cells) in a total volume of 5 ml of cell culture medium. Cells were cultured overnight and were then transfected with MACSfectin™ reagent using a total of 10 μg of plasmid DNA and 20 μl of MACSfectin™ in 500 μl of cell culture medium without FCS. After 24 h of cultivation, the medium was replaced by a fresh one, the cells were cultured for an additional 24 h and then harvested for Western Blot analysis. 2.5. Evaluation of cell proliferation To determine the effect of CX-4945 on cell proliferation rate, 1 × 104 cells per well were seeded into 24-well plates and incubated overnight. Cells were then either left untreated, treated with DMSO (solvent

ARPE-19 cells (5 × 105) were seeded on a 100 mm petri dish and grown overnight. The medium was changed and cells were treated with 0 (control) or 10 μM CX-4945 and incubated for 24 h and 48 h. Media and trypsinized cells were subsequently collected and centrifuged with 200 ×g at 4 °C for 7 min. Cells were washed with cold PBS supplemented with 5 mM EDTA, (PBS/EDTA) then centrifuged under the same conditions as above twice before being re-suspended in PBS/EDTA and fixed with 70% ethanol. Cells were incubated further with 1 mg/ml RNase (Sigma Aldrich) and 400 μM propidium iodide (1 mg/ml, Sigma Aldrich) to label DNA. Cells were then analyzed in a flow cytometer (FACScalibur, 4CS E4021, Becton and Dickinson, Heidelberg, Germany). 10,000 events were counted for each sample. Data were analyzed using Flowjo software (Flowjo, Ashland, USA). 2.9. Analysis of apoptosis using annexin V staining ARPE-19 cells (5 × 105) were seeded and grown on a 100 mm petri dish overnight. The medium was changed and cells were treated with 10 μM CX-4945 and incubated for 24 and 48 h. Cells were trypsinized and collected along with the media from petri dishes, then centrifuged with 200 ×g at 4 °C for 7 min. Cells were washed with binding buffer (BioLegend, Fell, Germany), centrifuged twice and then resuspended in 100 μl binding buffer. They were stained with 5 μl annexin V FITC

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(BioLegend, Fell, Germany) and 10 μl of 0.1 mg/ml propidium iodide (1 mg/ml, Sigma Aldrich, Munich, Germany). Cells were then analyzed in a cytofluorimeter (FACScalibur, 4CS E4021, Becton and Dickinson, Heidelberg, Germany). 10,000 events were counted for each sample. Data were analyzed using Flowjo software (Flowjo, Ashland, USA).

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Mannheim, Germany). The cell lysate was left on ice for 15 min, subjected to sonification (3 × 1 min) at 4 °C and then cell debris was removed by centrifugation at 16,250 ×g at 4 °C for 30 min. The protein content of the supernatant was determined according to the Bradford method using the Bio-Rad protein assay reagent (Bio-Rad, Munich, Germany).

2.10. Determination of O•− 2 in cell culture 2.12. SDS polyacrylamide gel electrophoresis and Western Blot analysis Cells (2 × 104) were seeded in 96 black/transparent wells from Becton and Dickinson overnight. Medium was removed and cells were washed 3 times with phosphate buffer saline (PBS, pH 7.4). For ROS staining, 3.2 mg dihydroethidium (DHE, Sigma Aldrich, Munich, Germany) was dissolved in 100 μl DMSO as stock solution, and diluted with PBS to a 25 μM working solution. 100 μl DHE-working solution was added to each well. After cells were incubated with DHE for 30 min at 37 °C in the dark, they were washed again 3 times with PBS. 100 μl PBS containing 0, 5, 10, 20 or 25 μM CX-4945 was then added directly into each well. For control experiments, 100 μl PBS containing 100 μM H2O2 was added to the wells with either ARPE-19 or HCT116 cells. The fluorescence (Ex/Em: 518 ± 9/606 ± 20 nm) per each well was measured with a plate reader (Tecan infinite M200, Crailsheim, Germany) every 5 min to determine the concentration of O•− 2 inside the cells.

Proteins were separated on a 7.5, 12.5 or 15% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (PVDF) by tank blotting using a transfer buffer containing 20 mM Tris–HCl, pH 8.8 and 150 mM glycine. The membrane was blocked with 5% dry milk in PBS containing 0.1% Tween-20 for 1 h at room temperature and then incubated with the specific antibody which was diluted in PBS with 0.1% Tween-20 containing 1% dry milk powder. The membrane was washed with PBS Tween-20 containing 1% skimmed milk (3 × 10 min), before being incubated with a peroxidase-coupled secondary antibody (anti-rabbit 1:30,000 or anti-mouse 1:10,000) for 1 h at room temperature. The membrane was washed again in PBS Tween-20 (3 × 10 min). Signals were

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Following incubation of ARPE-19 cells, HCT116 cells or LNCaP cells with the test compounds, cells were collected in cold PBS, pH 7.4 and centrifuged together with the cell culture medium at 4 °C and 250 ×g for 7 min. After one washing step with cold PBS, cells were lysed with 100 μl of RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5% sodium desoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS)) supplemented with the protease inhibitor cocktail Complete™ according to the manufacturer's instructions (Roche Diagnostics,

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Fig. 1. CX-4945 efficiently inhibits CK2 kinase activity in ARPE-19 cells. (A) Chemical structure of the protein kinase CK2 inhibitor CX-4945. (B) Endogenous CK2 kinase activity in ARPE-19 cells was determined 24 and 48 h after treatment with DMSO (control) or indicated concentrations of CK2 inhibitor CX-4945. Relative CK2 kinase activities measured in DMSO-treated control cells were set to 100%. The mean of three independent experiments is shown. **p b 0.01 or *p b 0.05.

Fig. 2. The CK2 inhibitor CX-4945 does not induce O•− 2 formation in neither ARPE-19 nor HCT116 cells. Cells were seeded into 96-well plates and incubated overnight. After 30 min of incubation with dihydroethidium (DHE), (A) cells were incubated with DMSO (control) or with 10 μM CX-4945 for various time periods. The fluorescence (Ex/Em: 518 ± 9/606 ± 20 nm) per each well was measured with a plate reader at the indicated times. (B) dose-dependent increase in DHE oxidation in both ARPE-19 and HCT116 cells, indicative of O•− 2 formation following treatment with varying concentrations of CX-4945 for 30 min. H2O2 treatment of HCT116 cells was used as a positive control. Data are expressed as fold increase in DHE oxidation in relation to DMSO (solvent control). The mean of three independent experiments is shown. **p b 0.01 or *p b 0.05.

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developed and visualized by the Lumilight system from Roche Diagnostic (Mannheim, Germany). 2.13. Statistics Microsoft Excel 2007 software was used to analyze the data. Results were expressed as arithmetic mean ± SEM. GraphPad Prism software (GraphPad Inc., USA) was also used for statistical analysis. Results were expressed as arithmetic mean ± SEM. Differences between the experimental groups were analyzed using one-way ANOVA or student's t-test (two-tail, unpaired), statistical significance differences were shown as follows: **p b 0.01 or *p b 0.05. 3. Results 3.1. CX-4945 is a potent inhibitor of protein kinase CK2 activity in ARPE-19 cells Over the last 8 years numerous inhibitors for protein kinase CK2 were reported in literature. It turned out that CX-4945 (Fig. 1A) is a very effective and rather specific inhibitor of CK2 which is currently in phase II clinical trial [11]. Although being very effective in most cancer cells, its optimal concentration has to be tested for every cell line [13]. To find the optimal concentration of CX-4945 for the inhibition of CK2 kinase activity in ARPE-19 cells, these cells were treated with different

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3.2. Protein kinase CK2 inhibition by CX-4945 does not induce O•− 2 production in neither ARPE-19 nor in HCT116 cells By using chemical inhibitors of CK2 such as apigenin or 4,5,6,7tetrabromobenzotriazole (TBB) or anti-sense oligonucleotides or small interfering RNA, Ahmad et al. described the formation of reactive oxygen species (ROS) in various normal and cancer cells [18]. Moreover, Kim et al. showed that deactivation of CK2 enhances the production of ROS in the mouse brain [19]. We therefore tested in the present study whether treatment of ARPE-19 cells and for comparison, HCT116 cells with the CK2 inhibitor CX-4945 may lead to ROS generation. The generation of O•− 2 in cells treated with CX-4945 was analyzed using the dihydroethidium (DHE) assay according to the manufacturer's instructions. As a positive control, we treated cells with H2O2 and as a negative control, cells were treated with DMSO alone (0 μM). Shown in Fig. 2, the treatment of ARPE-19 cells or HCT116 cells with varying

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concentrations (0–20 μM) of CX-4945 for 24 and 48 h. As shown in Fig. 1B, it can clearly be seen that CX-4945 inhibits CK2 kinase activity at a concentration of 5 μM, the CK2 kinase activity was inhibited by about 65 to 70% after 24 and 48 h of CX-4945 treatment. Since there were no gross differences in the CK2 kinase activity between 5 and 20 μM of CX-4945, all subsequent experiments with this inhibitor were performed at 10 μM concentration for up to 24 h and in some cases, up to 48 h.

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Fig. 3. CX-4945 affects the viability of both ARPE-19 and HCT116 cells. (A) ARPE-19 cells were seeded into 24-well plates and were treated with DMSO (solvent control), 10 μM CX-4945 or left untreated for 24 h to 96 h as indicated. Afterwards, cells were counted in a Neubauer counting chamber to determine the cell proliferation rate. (B) HCT116 cells were seeded into 24well plates and were treated with DMSO (solvent control), 10 μM CX-4945 or left untreated for 24 h to 96 h as indicated. Afterwards, cells were counted in a Neubauer counting chamber to determine the cell proliferation rate. (C) ARPE-19 cells and (D) HCT116 cell were seeded in 24-well plates and treated with DMSO as a control, 10 μM CX-4945 or left untreated for 24 h to 96 h. Afterwards, an MTT assay was done to determine the number of viable cells. Data are expressed as a percentage in relation to DMSO (solvent control). The mean of three independent experiments is shown. **p b 0.01 or *p b 0.05. (E) Influence of CX-4945 on cell cycle distribution. ARPE-19 cells were treated for 24 h or 48 h with DMSO (solvent control) or 10 μM CX-4945 and then analyzed by FACS. One representative of at least three similar independent experiments is shown here.

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concentrations of CX-4945 failed to induce an increase in O•− 2 , even after 60 min of treatment (A) and at concentrations of up to 25 μM of CX-4945 (B). Also, the treatment of the ARPE-19 cells with 100 μM of H2O2 failed to induce an increase in O•− 2 formation. On the contrary, applying the same concentration of H2O2 to the cancer cell line HCT116, resulted in a drastic increase in O•− 2 formation. 3.3. Effect of protein kinase CK2 inhibition on cell proliferation Since CX-4945 was able to severely inhibit protein kinase CK2 activity in ARPE-19 cells, we wondered whether this inhibition might have an influence on cell proliferation as well. We counted the cell number of untreated cells, cells treated with the solvent control and with 10 μM CX-4945 for 96 h. As shown in Fig. 3A, the treatment of ARPE-19 cells with CX-4945 led to a slight reduction of cell proliferation, whereas,there was a strong reduction in cell growth in HCT116 cells (B) when treated with CX-4945 in a similar manner. The solvent DMSO had no effect under the same conditions. Furthermore, we performed an MTT assay to analyze cell viability after treatment with CX-4945. Fig. 3C shows that cell viability decreased after 24 and 48 h. After 72 h, however, there was no further decrease in cell viability and after 96 h, the cell viability increased again indicating cell recovery. Thus, there seems to be a transient inhibition of cell proliferation and cell viability which

may help the cells to cope with the ER stress. In contrast to ARPE-19 cells, there was a drastic reduction in cell viability in HCT116 cells after treatment with CX-4945 for 48 h and the viability decreased further after 72 h and 96 h treatment (D). In the next step, we analyzed the cell cycle distribution after the treatment of ARPE-19 cells with CX-4945 using flow cytometry. After the treatment of the cells for 24 and 48 h, we observed a G1-arrest, but no cells in subG1 which would be indicative for apoptosis (Fig. 3E). 3.4. Protein kinase CK2 inhibition by CX-4945 induces the eIF2α branch of ER stress but not CHOP in ARPE-19 cells Experimental data have suggested that CK2 localizes to the ER where it phosphorylates ER resident proteins [20–22]. One of the sensors of ER stress is the ER membrane protein-pancreatic ER kinase (PERK). In cancer cells, it was shown that ER stress leads to the activation of PERK, which phosphorylates the eukaryotic initiation factor 2α (eIF2α). In order to analyze whether this ER stress signaling pathway is activated in normal ARPE-19 cells following CK2 inhibition, we treated ARPE-19 cells with 10 μM CX-4945 for varying time periods as indicated followed by Western Blot analysis. Shown in Fig. 4B, CX-4945 caused a significant transient exacerbated ER stress-induced phosphorylation of eIF2α (especially after 4 h, by over 2 fold), compared to total eIF2α (A),

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Fig. 4. CX-4945 causes ER-stress response over the p-eIF2α branch, but does not induce CHOP in ARPE-19 cells. ARPE-19 cells (A, B, E, F) or HCT116 cells (C, D, G, H) were treated with DMSO or 10 μM CX-4945 for the indicated times. For ATF4 and CHOP positive controls, LNCaP cells were treated with 2 μg/ml tunicamycin (Tun) for 4 h. Cell lysates were prepared and analyzed on a 12.5% SDS-polyacrylamide gel followed by Western Blotting using (A, C) anti-eIF2α, (B, D) anti-p-eIF2α, (E, G) anti-ATF4 or (F, H) anti-CHOP specific antibodies. α-Tubulin was used as a loading control. Band intensity, quantified by Quantity One-4.6.7 imaging system, is reported as a fold change relative to the intensity of the band corresponding to respective control set as 1. One representative of at least 3 Western Blots is shown here.

which remained almost the same. Tunicamycin (Tun), which is known to cause ER stress, was used as a positive control. Shown in the same Fig. 4B, it can be clearly seen that tunicamycin (compared to DMSO solvent control) caused a significant phosphorylation of eIF2α (nearly 4 fold) in ARPE-19 cells after 4 h. A similar result was shown for the HCT116 cells, which also showed a transient increase in p-eIF2α (Fig. 4D), whereas, total eIF2α remained the same over a period of time (Fig. 4C). Activating transcription factor 4 (ATF4) is a down-stream target of eIF2α. Since we observed an increase in the phosphorylation of eIF2α following CK2 inhibition, we wondered whether ATF4 may also be directly targeted by the inhibition of CK2 or indirectly through eIF2α phosphorylation. Fig. 4E shows that CK2 inhibition by CX-4945 also caused a transient increase in the ATF4 level with a maximum between 4 and 24 h (about 1.4 to 2 fold) after treatment with CX-4945. Another transcription factor that is involved in ER stress signaling is the CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), also known as growth arrest- and DNA damaged-inducible gene 153 (GADD153). CHOP is one of the major components of the ER stress-mediated apoptosis pathway. Its activities are regulated by CK2 phosphorylation [23,24], while its transcription is regulated by ATF4 [25,26]. It was therefore interesting to see whether the inhibition of CK2 will in fact have any effect on the endogenous protein levels of CHOP in ARPE-19 cells. We therefore treated ARPE-19 cells with 10 μM

CX-4945 for varying time periods as indicated followed by Western Blot analysis. Shown in Fig. 4F, CK2 inhibition by CX-4945 had no effect on the CHOP protein levels. In the positive control, however, it can be seen that the treatment of LNCaP cancer cells with tunicamycin led to elevated levels of the CHOP protein, which clearly shows that the antibody is able to detect CHOP. Furthermore, in the cancer cell line HCT116, CK2 inhibition by CX-4945 caused an increase in the level of ATF4 (Fig. 4G). In contrast to normal cells however, we observed, an increase in the level of CHOP beginning at 12 h and peaking at 24 h (about 3 fold) after treatment with CX-4945 (Fig. 4H). 3.5. Inhibition of protein kinase CK2 by CX-4945 does not induce apoptosis in ARPE-19 cells Several studies have indicated that CK2 inhibition can lead to apoptotic cell death in a number of cancer cells [27,23,28]. Having shown that CK2 inhibition by CX-4945 does not induce CHOP (which is a marker of ER stress-mediated apoptosis) protein up-regulation in ARPE-19 cells, we further investigated other markers, namely caspase 3 and poly-ADP-ribose polymerase (PARP), of apoptosis following CK2 inhibition. The activation of caspase 3 along with its substrate PARP, is a well known event in the apoptotic process. Here, cells were treated with CX-4945 or with DMSO (solvent control) alone, for 24 h or 48 h. As

J. Intemann et al. / Cellular Signalling 26 (2014) 1567–1575

a positive control, HCT116 cells were treated with 5-fluorouracil (5-FU) for 24 h. Cell extracts were analyzed on a SDS polyacrylamide gel followed by Western Blot with a caspase 3 or PARP specific antibody. As shown in Fig. 5A, CK2 inhibition failed to promote the activation and cleavage of both caspase 3 (A) and PARP (B) in ARPE-19 cells. In

A

contrast to ARPE-19 cells, however, CK2 inhibition by CX-4945 in HCT116 cells led to caspase 3 (C) and PARP cleavage (D), indicating that HCT116 cells enter apoptosis with CK2 inhibition by CX-4945. To further confirm a failed induction of apoptosis in ARPE-19 cells following CK2 inhibition, we analyzed ARPE-19 cells with annexin V

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Fig. 5. CX-4945 induces apoptosis in HCT116 cells (C, D) but not in ARPE-19 cells (A, B). Cells were treated with DMSO, 10 μM CX-4945, or left untreated for 24 and 48 h. As a positive control, HCT116 cells were treated with 5-FU for 24 h. Protein expression and cleavage of caspase 3 and PARP were subsequently analyzed by Western Blot. Proteins were separated on a 15% (A) and (C) or 7.5% (B) and (D) SDS-polyacrylamide gel and blotted on a PVDF membrane. Full-length caspase 3 and its cleavage products (A) and (C) were detected with a caspase 3-specific antibody (8G10), while PARP full-length and its cleavage products (B) and (D) were visualized with the specific antibody #9542. In each case, α-tubulin was used as a loading control. One representative of at least three Western Blots is shown here. (E) Cells were treated for 24 or 48 h with either DMSO (solvent control) or 10 μM CX-4945 and then stained with annexin-V and propidium iodide (PI) before cytofluorimetrically analyzed for apoptotic cell death. Viable cells are presented as both annexin-V and PI negative (Q4). One representative of at least three independent experiments is shown here.

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J. Intemann et al. / Cellular Signalling 26 (2014) 1567–1575

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Fig. 6. Transient transfection of CHOP is able to induce apoptosis in ARPE-19 cells. Seeded cells were cultured overnight and were then transfected with p3xFLAG-CMV-7.1 basic vector or p3xFLAG-CMV-7.1-CHOP using MACSfectin™ transfection reagent. Whole cell extracts were separated through a 12.5% (A), 15% (B) or 7.5% (C) SDS-polyacrylamide gel and transferred onto a PVDF membrane. Overexpression of CHOP (A) was determined with a FLAG-specific antibody. Full-length caspase 3 and its cleavage products (B) were visualized with a caspase 3-specific antibody (8G10), and PARP full-length and cleavage product (C) were detected with the PARP-specific antibody #9542. α-Tubulin was used as a loading control. One representative of at least three Western Blots is shown here.

and propidium iodide staining (Fig. 5E). The quantification of apoptotic cells revealed that 24 and 48 h after treatment with CX-4945, less than 2% of cells were present in the apoptotic fractions, which further confirms that CK2 inhibition using CX-4945 does not induce apoptosis in ARPE-19 cells. 3.6. Overexpression of CHOP induces apoptosis in ARPE-19 cells The data shown so far indicated that the lack in the induction of CHOP might be responsible for the block in apoptosis in ARPE-19 cells. In order to address this issue, we overexpressed CHOP in ARPE-19 cells. The expression of CHOP was analyzed by separating the cell extract in an SDS-polyacrylamide gel followed by a Western Blot with a flagged CHOP specific antibody. Fig. 6A clearly shows the presence of the flagged CHOP in the transfected cells. The cell extract was then further analyzed for apoptosis by Western Blot. The membranes were incubated with either a caspase 3- or a PARP-specific antibody. After the overexpression of CHOP in ARPE-19 cells, there were caspase 3 (Fig. 6B) and PARP cleavages (Fig. 6C), indicating that the cells indeed had gone into apoptosis. 4. Discussion Protein phosphorylation/dephosphorylation is a widely known mechanism which regulates most of the physiological processes in living cells. This process is performed by protein kinases which catalyze phosphate transfer from ATP to substrates. One of these kinases is protein kinase CK2 formerly known as casein kinase 2. There is still a growing body of data that CK2 displays an elevated activity and protein expression in various types of cancer [29]. CK2 supports cancer formation by blocking apoptosis and by stimulation of cell proliferation. Nowadays, CK2 has turned into a promising drug target because the inhibition of CK2 kinase activity leads to the induction of the physiological process of apoptosis which leads to tumor cell death. A number of inhibitors for CK2 were discovered and characterized over the past 8–10 years [10]. One of these inhibitors is CX-4945, which is orally available, highly selective and potent for CK2 and is now in phase II clinical trials. CX-4945 has a broad spectrum of antiproliferative activities in multiple cancer cell lines and in animal tumor models [30,12, 11,13]. So far, however, little is known about CK2 inhibition in normal non-cancerous cells. In cancer cells, it was shown that CK2 inhibition by chemical inhibitors as well as by antisense RNA or siRNA strategies resulted in the formation of reactive oxygen species (ROS) [18,19]. Therefore, it was one of our first experiments to test whether CX-4945

might generate ROS in non-cancerous cells. As shown here, there was no significant indication for the formation of ROS after CX-4945 administration. In cancer cells, there has been an increasing evidence for the generation of ER stress followed by ER stress signaling after CK2 inhibition [28, 14,27,23,31] which finally leads to apoptosis. In both, non-cancerous cells and cancer cells, we show here that CK2 inhibition with CX-4945 leads to a transient increase in the level of phosphorylated eIF2α. Upon ER stress, the α-subunit of the eukaryotic translation initiation factor 2 is phosphorylated by the serine/threonine kinase PERK which is located at the ER membranes. The phosphorylation of eIF2α leads to an attenuation of translation initiation to shut down global protein synthesis and to reduce ER protein folding load. This global reduction in protein synthesis seems to be responsible for the reduction in cell proliferation and cell viability as shown here. In contrast to the cancer cell line HCT116, however, the reduction in cell viability and cell proliferation seems to be transient in ARPE-19 non-cancer cells. This was particularly visible after 96 h of treatment, where there was a recovery of cell viability and an increase in cell proliferation. In addition to the global translational inhibition, phosphorylated eIF2α selectively promotes translation of a number of mRNAs including activating transcription factor 4 (ATF4) which activates gene expression for ER chaperones and transcription factors in order to get rid of the ER stress. From our data presented here, in both cell types, there was an increase in the level of ATF4. In the event of severe ER stress, which cannot be tolerated by the cell, ATF4 transactivates the CCAAT/enhancer-binding protein homologous protein CHOP/ GADD153 which is a bZIP transcription factor that plays a crucial role in ER stress-induced apoptosis [32,1]. Interestingly, and in contrast to most cancer cells, and also to HCT116 cell, which were used here for comparison, in the non-cancerous ARPE-19 cells, we found no induction of CHOP/GADD153. Obviously, normal cells are able to remove ER stress so that the induction of apoptosis is avoided. CHOP/GADD153 seems to be the critical factor for the induction of apoptosis. The overexpression of CHOP in ARPE-19 cells led to the induction of apoptosis, which supports the idea about the critical role of CHOP for the decision about life and death of the cell. Thus, our data presented here show that non-cancerous cells can master ER stress much better than cancer cells which leads to a block in the expression of CHOP and therefore prevents apoptosis. 5. Conclusion In this study, we demonstrated in non-cancer, normal ARPE-19 cells that the inhibition of protein kinase CK2 transiently inhibits cell

J. Intemann et al. / Cellular Signalling 26 (2014) 1567–1575

proliferation through the induction of G1 cell cycle arrest and attenuation of protein synthesis by phosphorylating eIF2α. In contrast to many cancer cells and also to HCT116 cells used here for direct comparison, CK2 inhibition, however, failed to induce apoptosis in normal cells. We further showed that in contrast to cancer cells, the ER pro-apoptotic transcription factor CHOP was not expressed in normal cells indicating that this failure in the expression of CHOP might be responsible for the abrogation of apoptosis. The overexpression of CHOP in normal cells led to apoptosis confirming the key role of CHOP in apoptosis induction. Acknowledgment The authors acknowledge the financial support from the Saarland University and the Landesforschungsförderungsprogramm Saarland (T/1-14.2.1.1.-LFFP 12/23). We thank Claudia Götz for her helpful comments. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

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ER stress signaling in ARPE-19 cells after inhibition of protein kinase CK2 by CX-4945.

Protein kinase CK2 is a critical factor for the survival of cells. It is overexpressed in many cancer cells and provides protection against apoptosis ...
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