Protein kinase CK2 regulates redox homeostasis through NF ...

University of Southern Denmark

Protein kinase CK2 regulates redox homeostasis through NF-κB and Bcl-xL incardiomyoblasts

Schaefer, Susanne; Guerra, Barbara

Published in:Molecular and Cellular Biochemistry

DOI:10.1007/s11010-017-3085-y

Publication date:2017

Document version:Final published version

Citation for pulished version (APA):Schaefer, S., & Guerra, B. (2017). Protein kinase CK2 regulates redox homeostasis through NF-κB and Bcl-xL incardiomyoblasts. Molecular and Cellular Biochemistry, 436(1-2), 137–150. https://doi.org/10.1007/s11010-017-3085-y

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Protein kinase CK2 regulates redox homeostasis through NF-jBand Bcl-xL in cardiomyoblasts

Susanne Schaefer1 • Barbara Guerra1

Received: 14 March 2017 / Accepted: 30 May 2017

� Springer Science+Business Media New York 2017

Abstract Oxygen consumption is particularly elevated in

cardiac cells as they are equipped with a large number of

mitochondria and high levels of respiratory chain compo-

nents. Consequently, production of reactive oxygen species

(ROS) is tightly controlled as an imbalance in redox

reactions can lead to irreversible cellular damage. siRNA-

mediated down-regulation of protein kinase CK2 has been

implicated in the accumulation of ROS in cells. The pre-

sent study was undertaken in order to investigate the role of

CK2 in redox homeostasis in cardiomyoblasts. We found

that inhibition or silencing of CK2 causes elevated levels of

ROS, notably superoxide radical, and this is accompanied

by suppression of NF-jB transcriptional activity and

mitochondrial dysfunction. We show that CK2 regulates

the expression of manganese superoxide dismutase, the

enzyme catalyzing the dismutation of superoxide, in cancer

cells but not in cardiomyoblasts. Furthermore, we report

evidence that impaired expression of CK2 results in

destabilization of the Bcl-2 mammalian homolog Bcl-xL,

which is known to stabilize the mitochondrial membrane

potential, through a mechanism involving disruption of the

chaperone function of heat shock protein 90. Analysis of

differential mRNA expression related to oxidative stress

revealed that CK2 silencing caused a statistically signifi-

cant deregulation of four genes associated with the oxida-

tive damage, i.e., Fmo2, Ptgs1, Dhcr24, and Ptgs2.

Overall, the results reported here are consistent with the

notion that CK2 plays a role in conferring protection

against oxidative stress by positively regulating pro-sur-

vival signaling molecules and the protein folding machin-

ery in cardiomyoblasts.

Keywords CK2 � ROS � NF-jB/RelA � Bcl-xL � HSP-90 �Cardiomyoblasts

Introduction

Reactive oxygen species (ROS) comprise a group of highly

reactive molecules that are derived from molecular oxygen

and formed as a by-product of aerobic metabolism. Mito-

chondria are considered the major source of ROS, being the

site for ATP generation through oxidative phosphorylation

[1]. The primary ROS generated in these organelles is

superoxide anion (O2�-) that is the precursor of other

biologically relevant ROS such as hydroxyl radical (OH�)and hydrogen peroxide (H2O2, reviewed in [2]). Superox-

ide is produced both enzymatically by NADPH oxidase,

cytochrome P450-dependent oxygenase, xanthine oxidase,

and nitric oxide synthase, and non-enzymatically by direct

transfer of an electron to O2 [3]. Scavenger enzymes (e.g.,

thioredoxin peroxidases, glutathione peroxidases, catalase,

and superoxide dismutase) control the production of ROS

by keeping their concentration in the picomolar range that

is necessary for preserving cellular homeostasis. When

ROS are excessively produced or antioxidants are not

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11010-017-3085-y) contains supplementarymaterial, which is available to authorized users.

& Barbara Guerra

[email protected]

1 Department of Biochemistry and Molecular Biology,

University of Southern Denmark, Campusvej 55,

5230 Odense M, Denmark

123

Mol Cell Biochem

DOI 10.1007/s11010-017-3085-y

adequately expressed, cells undergo life-threatening

oxidative stress, which results in protein oxidation, lipid

peroxidation, and/or DNA damage (reviewed in [4]).

Compelling evidence supports the notion that ROS also

play an important role in cellular signaling and commu-

nication by acting as secondary messengers [5]. In this

respect, protein kinases and phosphatases are affected by

direct or indirect redox modifications. Oxidative stress has

been shown to inhibit phosphotyrosine phosphatases (i.e.,

PTP1A, PTP1B, and PTEN) and activate protein kinases

(e.g., Src, Lyn, and ATM); however, there are several cases

where especially high levels of H2O2 have resulted in the

inactivation of various receptor as well as intracellular

protein kinases [5, 6].

The transcription factor nuclear factor-jB (NF-jB)-mediated signaling pathway contributes to redox home-

ostasis generally by targeting genes that would attenuate

ROS to promote cell survival. Inhibitory jB kinase (IKK),

a Ser/Thr kinase primarily responsible for the activation of

NF-jB, becomes inactive by endogenously produced nitric

oxide or H2O2. Furthermore, NF-jB positively modulates

the expression of scavenger enzymes such as manganese

superoxide dismutase (MnSOD), copper-zinc superoxide

dismutase, and glutathione S-transferase (for a review see

[7]). In turn, it has been shown that enhanced ROS interfere

with the NF-jB pathway at different levels, sometimes

causing opposite effects (i.e., stimulation or inhibition)

mostly attributed to cell-specific differences and/or

methodologies and stimulation of different upstream

pathways (for a review, see [7]).

Recent evidence has shown that chemical inhibition or

RNA interference-mediated down-regulation of protein

kinase CK2 leads to accumulation of ROS in cancer

cells, suggesting that CK2 may provide protection

against oxidative stress [8–11]. Protein kinase CK2 is a

constitutively active Ser/Thr kinase composed of two

catalytic a and/or a0 subunits and two regulatory bsubunits (reviewed in [12–15]). CK2 expression and

activity are deregulated in many human diseases, and

while much work has been undertaken regarding its role

in cancer development, its function in non-malignant

cells has not been extensively investigated particularly

with respect to redox homeostasis.

In the present study, we have employed cardiomyoblasts

to investigate whether cellular depletion or chemical inhi-

bition of CK2 results in ROS accumulation and, if so, the

mode by which CK2 regulates proteins that contribute to

redox homeostasis. We provide evidence that CK2 exerts

protection against oxidative stress by positively regulating

NF-jB/RelA transcription activity and preserving the HSP-

90-mediated molecular chaperone machinery.

Materials and methods

Cell culture and treatment

The rat cardiomyoblast cell line H9c-2 was purchased from

the American Type Culture Collection (ATCC, Rockville,

MD, USA) and cultivated at 37 �C under a 5% CO2

atmosphere in Dulbecco’s modified Eagle’s medium

(DMEM, Invitrogen, Taastrup, Denmark) supplemented

with 10% fetal bovine serum (FBS, Biochrom AG, Berlin,

Germany). Cells were treated with 1,3-Dichloro-6-[(E)-((4-

methoxyphenyl)imino)methyl] diben- zo(b,d) furan-2,7-

diol—hereafter referred to as D11 (DTP, NIH/NCI,

Rockville, MD, USA)—hydrogen peroxide (H2O2, Sigma-

Aldrich, Schnelldorf, Germany), vitamin C (Sigma-

Aldrich), carbonyl cyanide 4-(trifluoromethoxy)phenylhy-

drazone (FCCP, Santa Cruz Biotechnology, Heidelberg,

Germany), and TNFa (Sigma-Aldrich) as indicated in the

figure legends. Down-regulation of protein expression was

carried out by RNA interference as previously described

[16]. Here, sets of four small interfering RNA duplexes

(ON-TARGET plus SMART pools, Dharmacon, Lafayette,

CO, USA) directed against NF-jB, CK2a, and CK2a0,respectively, were used. Where indicated, cells have been

serum-starved for 12 h prior stimulation with TNFa.

Retrovirus production and preparation of a cell line

stably overexpressing Bcl-xL

Viral particles carrying a pBABE vector containing the

human sequence of Bcl-xL were produced in the Phoenix-

ECO packaging cell line as previously described [17]. For

the stable expression of Bcl-xL, the H9c-2 cell line at 50%

confluence was transduced with Bcl-xL-encoding viral

particles that were diluted in a fresh growth medium in the

ratio of 1:1 in the presence of 8 lg/ml polybrene for 24 h.

Cells successfully transduced were selected with 0.3 lg/ml

puromycin (Thermo Fisher Scientific, Rockford, IL, USA)

for 3 days.

ROS measurement

Reactive oxygen species were detected by incubating cells

with 5 lM CellROX� Green reagent and 5 lM dihy-

droethidium (DHE), respectively, (both from Molecular

Probes, Paisley, UK) for 30 min at 37 �C after washing the

cells in PBS and prior to trypsinization. Pellets obtained by

centrifugation of the cells at 3009g for 5 min were re-

suspended in PBS containing 0.1% BSA and immediately

analyzed on a FACSCalibur (BD Biosciences, San Jose,

CA, USA). For each analysis, 10,000 events were recorded.

Mol Cell Biochem

123

Acquired data were processed by Cell Quest Pro Analysis

software (BD Biosciences).

Mitochondrial membrane potential measurement

JC-1 dye (Molecular Probes) was employed for determin-

ing changes in the mitochondrial membrane potential. JC-1

was added to the treated cells, the treatment of which is

described in the figure legends, at a concentration of 5 lg/ml for 10 min prior to harvest by trypsinization. Cell pel-

lets were re-suspended in PBS containing 0.1% BSA and

directly analyzed by flow cytometry. Cells incubated with

70 lM FCCP for 2 h served as positive control.

Preparation of whole cell lysate, Western blot

analysis, and antibodies

Cells were harvested and further processed for Western

blot analysis as previously described [18, 19]. The fol-

lowing primary antibodies were employed in the study:

rabbit monoclonal anti-NF-jB/RelA and rabbit mono-

clonal anti-Bcl-xL (both from Cell Signaling Technol-

ogy, Beverly, MA, USA); mouse monoclonal anti-b-actin (Sigma-Aldrich); rabbit polyclonal anti-phospho-

NF-jB/p65 (S529) and rabbit monoclonal anti-phospho-

CDC37 (S13, both from Abcam, Cambridge, MA, USA);

mouse monoclonal anti-CDC37, mouse monoclonal anti-

MnSOD and rabbit polyclonal anti-HSP-90 (all from

Santa Cruz Biotechnology, Heidelberg, Germany);

mouse monoclonal anti-Bcl-xL and mouse monoclonal

anti-HSP-90 (both from Thermo Fisher Scientific).

Rabbit polyclonal anti-CK2a0 was obtained by immu-

nizing rabbits with a specific peptide sequence of human

CK2a0 (SQPCADNAVLSSGTAAR). Rabbit polyclonalanti-CK2a was obtained by immunizing rabbits against

the human full-length protein sequence. Mouse mono-

clonal anti-CK2a/a0 was obtained from KinaseDetect

Aps, Odense, Denmark.

NF-jB transcription factor assay

Determination of the transcription activity of NF-jB was

carried out essentially as reported in [20] employing the

NF-jB transcription factor assay kit (TransAM, Active

Motif, Rixensart, Belgium). In brief, whole cell lysate

(20 lg) was incubated with a specific DNA oligonucleotide

containing the NF-jB consensus site immobilized on a

96-well plate. NF-jB bound to its target DNA was detected

by a specific primary antibody directed against NF-jB(p65). A secondary antibody conjugated to HRP was added

to provide a colorimetric readout at 450 nm quantified by

spectrophotometry (Versamax, ELISA, Molecular Devices,

Sunnyvale, CA, USA).

Immunostaining and in situ proximity ligation assay

Immunostaining and in situ proximity ligation assay were

carried out essentially as described in [20] employing

rabbit monoclonal anti-Bcl-xL (Cell Signaling Technol-

ogy) and mouse monoclonal anti-HSP-90 (Thermo Fisher

Scientific) antibodies.

Oxidative stress gene analysis by quantitative

RT-PCR array

Preparation of total RNA samples from cells treated as

described in the figure legends was carried out by phenol–

chloroform extraction and subsequent silica-membrane-

based purification combined with on-column DNase

digestion according to the miRNeasy Mini handbook from

Qiagen (Hilden, Germany). RNA integrity was assessed by

Agilent 2100 bioanalyzer (Agilent Technologies, Wald-

bronn, Germany). Purified RNA was used as template for

cDNA preparation using the RT2 First strand kit (Qiagen).

Expression analysis of 84 genes associated with oxidative

stress and reactive oxygen species was carried out with the

Qiagen RT2 Profiler PCR array according to the manu-

facturer’s instructions in 96-well plates with a StepOne-

PlusTM real-time cycler (Applied Biosystems, Nærum,

Denmark). Five house-keeping genes served for normal-

ization of the data. Normalized data were analyzed using

the DDCT method: DDCT = DCT (experimental sample

group) -DCT (control group), and the fold-change was

calculated based on DDCT with 2-DDCT for positive

changes or with -1/2-DDCT for negative changes [21, 22].

Statistical analysis

Statistical significance of differences between means of

two groups was determined by the two-tailed t test (stu-

dent’s t test). The levels of significance are indicated in the

figure legends.

Results

Chemical inhibition or down-regulation of protein

kinase CK2 results in the generation of ROS

in cardiomyoblasts

We have previously reported evidence that knockdown of

CK2 is accompanied by the generation of superoxide in

human glioblastoma cells [10]. The largely unexplored role

of CK2 in redox homeostasis in non-cancerous cells

prompted us to investigate whether CK2 down-regulation

had an effect on the intracellular levels of ROS in car-

diomyoblasts. As shown in Fig. 1a (left panel), siRNA-

Mol Cell Biochem

123

Mol Cell Biochem

123

mediated silencing of the individual catalytic subunits of

CK2 resulted in the accumulation of intracellular ROS

(CellROX-positive cells) as measured by the CellROX�

oxidative stress reagent, a fluorogenic probe employed for

the detection of ROS in live cells. Similar results were

obtained at 24 h following treatment of cells with

increasing concentrations of D11, a recently identified

highly specific small-molecule inhibitor of CK2 (Fig. 1a,

right panel, [23]). Mitochondrial oxidative phosphorylation

primarily produces superoxide anion that is the precursor

of most ROS [24]. Hence, we measured superoxide levels

by flow cytometry (DHE-positive cells) following cell

labeling with dihydroethidium (DHE), a fluorescence

marker employed for superoxide detection [25]. Results in

Fig. 1b show a significant accumulation of superoxide in

cells either depleted of CK2a and -a0, respectively, ortreated with increasing concentrations of D11 for 24 h with

respect to control experiments. It is worth noting that

changes in ROS levels appeared to be more dramatic in the

case of CK2 inhibition. Pre-treatment of cells with the ROS

scavenger vitamin C significantly reduced accumulation of

superoxide induced by depletion or inhibition of CK2

(Fig. 1c). Experiments carried out in the presence of H2O2

served as a positive control. In all experiments involving

siRNA transfection, silencing of the individual catalytic

subunits of CK2 was at least 70% as compared to control

experiments (data not shown).

Increase in cellular ROS is indicative of loss of mito-

chondrial membrane potential (DWm, [26]). Hence, in

order to verify whether accumulation of ROS was

accompanied by mitochondrial depolarization, we moni-

tored changes in DWm by flow cytometry using the JC-1

dye, a cell permeable reagent fluorescing orange-red (J-

aggregates form) in the case of intact DWm and emitting

green fluorescence (monomeric form) in the case of

membrane potential loss [27]. Results shown in Fig. 1d

clearly indicate that the membrane potential was lost in a

time and concentration-dependent fashion in cells treated

with D11. Cells were treated with carbonyl cyanide-4-

(trifluoromethoxy)phenylhydrazone (FCCP), a mitochon-

drial uncoupler, served as a positive control [28]. Addi-

tionally, incubation with vitamin C resulted in partial

restoration of DWm and, concomitantly, reduction of ROS

accumulation (Fig. 1e). We employed siRNA transfection

to down-regulate the individual CK2 catalytic subunits.

Loss of DWm at 72 h post-transfection correlated with CK2

silencing; however, the increase in green JC-1 fluorescence

signal was not as significant as in the case of cells treated

with D11 (data not shown). Overall, data reported above

demonstrate that silencing or inhibition of CK2 results in

ROS accumulation and loss of DWm in cardiomyoblasts.

CK2 protects cardiomyoblasts from oxidative stress

through the NF-jB-mediated pathway

It has been reported that murine embryonic fibroblasts

deficient in TNF-associated factor (TRAF) 2 and -5 or

NF-jB/RelA show high levels of ROS when stimulated

with TNFa, indicating that one of the pro-survival func-

tions of NF-jB is to counteract TNFa-mediated formation

of ROS [29]. Interestingly, CK2 has been shown to stim-

ulate the transcription activity of NF-jB at multiple levels

by phosphorylating NF-jB at Ser529, activating IKK2 and

promoting IjBa degradation (reviewed in [30, 31]). Given

the fact that CK2 modulates the activity of NF-jB in

cancer cells, we investigated whether similar regulation

occurred in cardiomyoblasts. Down-regulation of the

individual catalytic subunits of CK2 resulted in decreased

phosphorylation of NF-jB/RelA at Ser529 (Fig. 2b, left

panel) in cells left untreated or stimulated with 10 ng/ml

TNFa for 8 h as determined by the analysis of NF-jBphosphorylation levels at the activating Ser536 (Fig. 2a).

Correspondingly, CK2 depletion was followed by a sig-

nificant decrease of NF-jB/RelA transcription activity in

the presence of TNFa as compared to si-scr-transfected

cells (Fig. 2c, left panel). Similar results were obtained

when cells were treated with D11 in the presence and

absence of TNFa, respectively (Fig. 2b, c, right panels),

demonstrating that the kinase activity of CK2 is required

for NF-jB/RelA transcription activity in cardiomyoblasts.

Next, we investigated whether silencing of CK2 affected

TNFa-mediated induction of ROS accumulation by stain-

ing cells with DHE reagent and analyzing fluorescence

bFig. 1 siRNA-mediated silencing and inhibition of CK2 leads to

accumulation of ROS in cardiomyoblasts, respectively. a Cells were

transfected with control (si-scr), CK2a (si-CK2a), and CK2a0 (si-

CK2a’) siRNA for 72 h (left), respectively, or treated with vehicle

(DMSO) and increasing concentrations of D11 for 24 h (right),

respectively. Accumulation of ROS (CellROX-positive cells) was

quantified by flow cytometry by staining cells with 5 lM CellROX�

Green for 30 min. b Detection of superoxide anion (DHE-positive

cells) was carried out by staining cells with 5 lM DHE for 30 min.

Cells were analyzed by Flow cytometry. c To the cells that were

treated as described in (a) was added 100 lM vitamin C (Vit C) 6 h

prior analysis as indicated in the figure. Detection of superoxide anion

was performed as in b. d Cells treated with vehicle or increasing

concentrations of D11 for 24 h and 48 h, respectively, were subjected

to FACS analysis following staining with 5 lg/ml JC-1 for 10 min for

measurement of mitochondrial membrane potential changes. JC-1

monomer-positive cells indicative of loss of DWm are expressed as

percentage of total cells. e Control experiments where cells were pre-

treated with 100 lM vitamin C prior incubation with DMSO or

60 lM D11 for 24 h as indicated in the figure. Analysis was carried

out as described in (d). In all experiments, treatment with 100 lMH2O2 for 5 h or 70 lM FCCP for 2 h served as positive control. Data

are shown as the mean ± standard deviation of three experiments

performed independently. *P B 0.05, **P B 0.005, ***P B 0.0001

denote statistically significant differences with respect to control

experiments. NC unstained cells

Mol Cell Biochem

123

Fig. 2 Down-regulation of CK2 impairs the transcriptional activity

of NF-jB resulting in ROS accumulation. a Cells were transfected

with control (si-scr) or siRNA against NF-jB/RelA (si-NF-jB/RelA)for 72 h. Where indicated, cells were treated with 10 ng/ml TNFaduring the last 5 h of incubation time. Whole cell lysate was subjected

to Western blot analysis of NF-jB expression and phosphorylation

levels. b-actin detection was used as a control for equal loading.

b Cells were transfected with scramble, CK2a and CK2a0-siRNA for

72 h (left), respectively, or treated with 0.1% DMSO (C) and D11 at

the indicated concentrations for 24 h, respectively, (right). Stimula-

tion of NF-jB/RelA was induced with 10 ng/ml TNFa in the last

15 min of incubation time. Whole cell lysates were analyzed by

Western blot for the expression and phosphorylation levels of the

indicated proteins. b-actin detection was used as a control for equal

loading. c Whole lysates from cells transfected as above (left) or

treated with 60 lM D11 for 24 h (right) were subjected to NF-jB/RelA transcription activity assay as described in Materials and

Methods. Where indicated, cells were additionally stimulated with

10 ng/ml TNFa in the last 15 min of incubation time. d Flow

cytometry analysis of DHE-stained cells following cell transfection

with scramble, CK2a, and -a0 siRNA, respectively. Cells were

stimulated with 10 ng/ml TNFa in the last 5 h of incubation time.

Experiments were repeated at least three times, obtaining similar

results. Data are shown as the mean ± standard deviation. *P B 0.05,

**P B 0.001, ***P B 0.0001 denote statistically significant differ-

ences to control

Mol Cell Biochem

123

signals by flow cytometry (DHE-positive cells, Fig. 2d).

Cellular depletion of the individual subunits of CK2

induced increased ROS levels with respect to control cells.

A further increase in ROS formation was observed when

cells were incubated with TNFa. These data indicate that

NF-jB-mediated regulation of ROS levels in cardiomy-

oblasts is dependent, at least in part, on the cellular

expression of protein kinase CK2.

Much evidence indicates that NF-jB regulates redox

homeostasis by up-regulating MnSOD, the enzyme con-

verting O2-� into H2O2 [32–34]. Hence, we investigated

whether enhanced levels of superoxide resulted from

decreased expression or impaired function of MnSOD

following inhibition or silencing of CK2. We titrated car-

diomyoblasts with increasing concentrations of TNFa for

8 h or a fixed concentration of the cytokine (i.e., 10 ng/ml)

for up to 24 h for determining optimal treatment conditions

(Fig. 3a, b). Subsequently, we analyzed the expression of

endogenous MnSOD before and after cell incubation with

30 ng/ml TNFa for 24 h, respectively. As shown in

Fig. 3c, stimulation of cells with TNFa resulted in a slight

increase in the expression of MnSOD. However, neither

down-regulation (Fig. 3c) nor inhibition (Fig. 3d) of CK2

resulted in a significant decrease in the expression of the

scavenger enzyme and only a slight effect was observed

following down-regulation of NF-jB (Fig. 3c). To confirm

observations reported above, an in vitro colorimetric assay

(OxiSelectTM superoxide dismutase activity assay) was

performed for the measurement of MnSOD activity in

whole cell lysate following cell treatment as reported in

Fig. 3c, left panel. However, we could not measure any

significant change in the activity of MnSOD supporting the

findings reported above (data not shown). The obtained

data suggest that the expression of MnSOD may not be

subjected to regulation by CK2 and, alternatively, that its

stability makes it difficult to reveal any change in the

expression levels under the given experimental conditions.

Numerous studies carried out mostly employing cancer

cell lines have shown that treatment of cells with TNFamodulates the expression of scavenger enzymes in an NF-

jB-dependent fashion and that suppression of NF-jB-me-

diated signaling leads to increased TNFa-mediated ROS

formation [33]. As we observed no significant changes in

MnSOD expression levels following CK2 silencing in

cardiomyoblasts, we investigated the effect of deregulated

expression of CK2 in the human osteosarcoma-derived

RS3.22 cell line which constitutively expresses CK2a-HAand Myc-CK2b under a tetracycline-regulated transcrip-

tional activator [35]. The expression of MnSOD in whole

lysate from cells that were treated as described in Fig. 4a

was investigated by Western blot analysis. The expression

levels of MnSOD in cells transfected with si-scr and treated

with 30 ng/ml TNFa for 8 h increased about 2-fold as

compared to control cells (i.e., si-scr-transfected cells).

Fig. 3 Effect of CK2 inhibition or down-regulation on the expression

of MnSOD in cardiomyoblasts. a Determination of MnSOD expres-

sion levels by Western blot analysis in untreated or TNFa-treatedcells for the indicated times. b Analysis of MnSOD expression levels

from cells treated with increasing concentrations of TNFa for 8 h.

c Western blot analysis of whole lysates from cells transfected with

siRNA against the individual catalytic subunits of CK2 or with

scramble siRNA (si-scr) before and after incubation with 30 ng/ml

TNFa for 8 h. d Analysis of MnSOD expression levels from cells

incubated with 60 lM D11 for 24 h. Where indicated, 30 ng/ml

TNFa was added in the last 8 h of incubation time. Experiments were

repeated three times, obtaining similar results

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123

MnSOD signal intensity was roughly 3.5-fold higher in

cells expressing CK2a-HA and stimulated with the cyto-

kine as compared to control experiment. Conversely, in the

presence of TNFa down-regulation of NF-jB/RelA and

CK2a, respectively, resulted in decreased expression of

MnSOD, which was close to basal levels in the case of NF-

jB/RelA silencing. Similar results were obtained in cells

incubated with 60 lM D11 for 24 h and stimulated with

30 ng/ml TNFa for 8 h (Fig. 4b).

Overall, results reported above support the notion that

multiple mechanisms regulate redox homeostasis. In cancer

cells, changes in the expression levels of MnSOD are

dependent on an intact NF-jB-mediated signaling and

CK2. The expression of MnSOD is not regulated in a

similar fashion in cardiomyoblasts, and it seems to be

independent of functional CK2, suggesting that alternative

mechanisms may play a major role in protecting cells from

oxidative stress.

Down-regulation of CK2 destabilizes B-cell

lymphoma-extra large (Bcl-xL) in cardiomyoblasts

The anti-apoptotic protein Bcl-xL, a mammalian homolog

of Bcl-2 whose expression is positively regulated by NF-

jB, has been shown to stabilize DWm to maintain mito-

chondrial membrane homeostasis through a mechanism

involving TNF complex II-mediated binding to ROS

modulator 1 (Romo1) [36]. Under these conditions, Romo1

recruits Bcl-xL to reduce DWm resulting in ROS produc-

tion [37–40]. As we verified that CK2 positively modulates

NF-jB in cardiomyoblasts, we investigated whether CK2

silencing resulted in changes in Bcl-xL expression levels

before and after stimulation with TNFa. As shown in

Fig. 5a, siRNA-mediated down-regulation of NF-jB,CK2a, or CK2a0 resulted in lower expression levels of Bcl-

xL as compared to control cells. Similar results were

obtained following treatment with D11 to investigate the

effect of CK2 inhibition on Bcl-xL expression (Fig. 5b)

and by immunostaining of cells that were subjected to

simultaneous knockdown of CK2a and a0 with anti-Bcl-xL

antibody (Fig. 5c, d).

An early event in Romo1-mediated ROS production is

the binding of Romo1 to Bcl-xL, which occurs under basal

conditions or in cells stimulated with TNFa resulting in

decrease of DWm [37]. We postulated that CK2-mediated

down-regulation of Bcl-xL expression could represent an

alternative way to suppress the protective function of Bcl-

xL on DWm to maintain mitochondrial homeostasis. We,

therefore, examined whether overexpression of Bcl-xL

prevented accumulation of ROS in cells depleted of CK2aand a0, respectively. Indeed, constitutive overexpression of

Bcl-xL in cardiomyoblasts prevented ROS formation in

cells expressing low levels of CK2 in the absence and

presence of TNFa, respectively, indicating that CK2

Fig. 4 CK2-mediated regulation of MnSOD expression levels in

RS3.22 cell line. a Cells were transfected with siRNA against CK2aor NF-jB/RelA as shown in the figure (left). Where indicated, the

expression of CK2a-HA was induced in the absence of tetracycline.

Where indicated, cells were stimulated with 30 ng/ml TNFa in the

last 8 h of incubation time. Whole cell lysates were analyzed by

Western blot employing antibodies against the indicated proteins.

Detection of b-actin served as loading control. b Cells were treated

with 60 lM D11 for 24 h. Where indicated, 30 ng/ml TNFa were

added during the last 8 h of incubation time. MnSOD expression

levels were analyzed by Western blot. Detection of CDC37

phosphorylated at Ser13 (S13) served as control for successful

inhibition of CK2 kinase activity. Data shown are representative of

three independent experiments

bFig. 5 Down-regulation of CK2 attenuates the expression of Bcl-xL

in cardiomyoblasts. a Cells were treated essentially as described in

Fig. 4a. Whole cell lysates were analyzed by Western blot using

antibodies against the indicated proteins. b Analysis of Bcl-xL

expression levels by Western blot from cells that were treated as

described in Fig. 4b. c Cells transfected with scramble siRNA or

siRNA against CK2a and a0 were stained with rabbit monoclonal

anti-Bcl-xL antibody. Negative control (NC) refers to scramble

siRNA-transfected cells incubated with biotinylated secondary anti-

body and, subsequently, Alexa Fluor 488-conjugated streptavidin.

Cell nuclei were visualized by DAPI staining. d Western blot analysis

of HSP-90, Bcl-xL, and CK2 protein expression levels. e Control andBcl-xL-overexpressing cells were transfected with scramble siRNA or

siRNA for the silencing of CK2a/a0, respectively, as indicated in the

figure. Analysis of superoxide formation was carried out by Flow

cytometry following staining of cells with DHE reagent. Where

indicated, TNFa (20 ng/ml) was added in the last 5 h of incubation

time. *P B 0.05

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antagonizes ROS accumulation by preserving intact Bcl-xL

expression (Fig. 5e and Suppl. Fig. S1).

Previous studies have shown that CK2-mediated phos-

phorylation of the co-chaperone CDC37 at Ser13 is a

necessary event for the stabilization of heat shock protein

90 (HSP-90)–CDC37 heterocomplex with client proteins

including ErbB2, EGFR, Raf, MEK, PTEN, AKT, and

mTOR [41–45]. We postulated that CK2 down-regulation

impaired Bcl-xL expression as a result of indirect disrup-

tion of HSP-90 chaperone function as HSP-90 has been

shown to interact with and stabilize Bcl-xL [46]. To show

this, we analyzed the association between HSP-90 and Bcl-

xL by in situ proximity ligation assay employing antibodies

directed against the aforementioned proteins. As shown in

Fig. 6, cells lacking CK2 resulted in a significant decrease

in signal intensity as compared to si-scr-transfected cells,

suggesting that the observed decrease in the expression

levels of Bcl-xL may result from destabilization of HSP-

90–CDC37 heterocomplex.

Differential gene expression profiling

of cardiomyoblasts in CK2-mediated oxidative stress

induction

Oxidative stress that results from ROS accumulation can

modulate multiple signaling pathways and thus a wide

variety of biological processes by linking cell surface sig-

nals to changes in gene expression. To clarify differential

mRNA expression in cardiomyoblasts resulting from ROS

accumulation induced by CK2 down-regulation, we

examined the expression of 84 genes related to oxidative

stress and oxidative defense using the oxidative stress RT2

profiler PCR array. The differential expression of genes

was plotted on a volcano plot as shown in Fig. 7a. Genes

up-regulated with fold-change C1.5 and P\ 0.05

consisted of flavin-containing monooxygenase 2 (Fmo2)

and prostaglandin-endoperoxide synthase 2 (Ptgs2), while

genes down-regulated with fold-change B-1.5 and

P\ 0.05 included 24-dehydrocholesterol reductase

(Dhcr24) and prostaglandin-endoperoxide synthase 1 (Pt-

gs1). The analysis also identified a number of genes whose

up-regulation was C1.5 although changes were not defined

as statistically significant (Fig. 7a; Suppl. Table S1). Based

on scatter plot analysis where the log of the fold-change in

gene expression in control cells was plotted against log of

the fold-change in CK2-knockdown cells, we could deter-

mine that the aforementioned group of genes had a low

expression profile making any change in their expression

among different datasets not statistically significant

(Fig. 7b). Overall, the observed differences in gene

expression suggest that CK2 modulates a subset of genes

linked to oxidative stress and ROS metabolism.

Discussion

The role of protein kinase CK2 in the regulation of a wide

range of intracellular processes has been mainly studied in

cancer cells (for reviews see [13, 14, 47, 48]). In this

respect, compelling evidence has indicated that CK2 is

markedly elevated both at the protein and mRNA levels

conferring protection from induction of cell death and

supporting cell survival and proliferation. Although

CK2 has been linked to various human diseases including

cancer, its role in non-cancerous cells particularly with

respect to mitochondrial redox homeostasis has remained

largely unexplored. The mitochondrial respiratory chain is

one of the major sources of ROS whose production is

related to oxygen consumption. The latter process is

especially prominent in cardiomyocytes that are equipped

Fig. 6 Analysis of the association between HSP-90 and Bcl-xL. Cells

that were treated as described in Fig. 5c were stained with antibodies

against HSP-90 and Bcl-xL, respectively. Detection of complexes was

revealed by in situ PLA (left). NC: scramble siRNA-transfected cells

subjected to in situ PLA where incubation with one of the primary

antibodies was omitted (negative control). Bar graph refers to the

determination of number of signals per cell as distinct red fluores-

cence spots performed by computer-assisted image analysis (right).

Data are expressed as mean ± standard deviation. Experiments were

repeated three times, obtaining similar results. In all cases, cell

pictures were taken at 409 magnification. *P B 0.001

Mol Cell Biochem

123

with scavenger enzymes and non-enzymatic systems to

prevent oxidative damage. In this study, we have reported

evidence that down-regulation of CK2 expression or its

inhibition, though not affecting the cell viability (data not

shown) significantly, results in the accumulation of ROS

and this effect is accompanied by loss of mitochondrial

membrane potential. Similar to what was previously

reported, changes in redox homeostasis appear to be more

significant when CK2 is inhibited than when its expression

levels are down-regulated, an effect most likely associated

to the long incubation time necessary to induce CK2

silencing [11].

Two main NF-jB-regulated pathways exist in cells: the

canonical pathway mostly induced by NF-jB stimuli and

the non-canonical signaling pathway induced by certain

TNF family cytokines. However, NF-jB can be directly

modulated by a variety of post-translational modifications

including phosphorylation, acetylation, and methylation

[31]. We show here that CK2 modulates the activity of NF-

jB/RelA in cardiomyoblasts and that its down-regulation

results in ROS production in cells left untreated or exposed

to TNFa, suggesting that CK2 modulates redox home-

ostasis through NF-jB/RelA.An important observation that emerged from this study

is that cancer cells promptly responded to TNFa treatment

by inducing MnSOD synthesis. Correspondingly, altered

expression of CK2 resulted in modulation of MnSOD

expression levels most likely through the NF-jB signaling

pathway. Conversely, similar results were not obtained

with cardiomyoblasts (in this respect, down-regulation of

NF-jB/RelA resulted only in a marginal decrease in

MnSOD expression), suggesting that alternative detoxifi-

cation mechanisms regulated by CK2 through or indepen-

dent from NF-jB might be taking place in

cardiomyoblasts. In this respect, NADPH oxidase is a

multi-component complex producing ROS after ischemic

injury. Deactivation of CK2 in mouse brain has been

shown to induce accumulation of ROS and neural cell

death as a result of increased NADPH oxidase activity,

demonstrating that CK2 is a negative modulator of this

enzyme [9].

Consistent with the notion that NF-jB regulates the

expression of genes antagonizing oxidative stress, it has

been shown that c-Rel and RelA directly activate the

expression of the apoptosis inhibitor Bcl-xL [36]. As

Romo1-mediated sequestration of Bcl-xL has been shown

to promote DWm destabilization and subsequent ROS

generation, we examined whether CK2 down-regulation

had an effect on Bcl-xL [37]. We show for the first time

that chemical inhibition or silencing of CK2 results in

decreased expression levels of Bcl-xL in cardiomyoblasts

identifying an alternative strategy to block Bcl-xL function

and induce ROS formation beside Romo1-mediated

sequestration. We failed to observe up-regulation of Bcl-xL

expression levels following treatment with TNFa of cells

transfected with si-scr as compared to control experiment

(Fig. 5a). This may indicate that a longer incubation time

with TNFa is necessary to induce the expression of Bcl-xL.

Results shown in Fig. 6 suggest that impaired expression of

Bcl-xL may result from CK2-mediated disruption of HSP-

90 chaperone function; however, one cannot exclude that

Fig. 7 Down-regulation of CK2 determines the changes in the

expression levels of genes related to oxidative stress. a Total RNA of

cells transfected with scramble siRNA (control) or siRNA against

CK2a and CK2a0 was extracted and purified as described in the

Materials and Methods and subsequently subjected to quantitative

PCR array for the analysis of 84 genes related to oxidative stress.

Log2 fold-changes (x axis) are plotted against -log10 of P-values (y

axis) in the volcano plot. The dashed black line indicates P = 0.05

with values above the line having P\ 0.05 and values below the line

having P[ 0.05. Points outside the dashed red lines indicate values

having a log2 fold-change B-0.58 or C0.58 corresponding to fold-

change B-1.5 and C1.5, respectively. b Scatter plot showing genes

differentially transcribed in cells transfected with siRNA against

CK2a and a0. The fold regulation cut-off was set as 1.5. Results

shown are derived from three independent data sets. (Color

figure online)

Mol Cell Biochem

123

impaired NF-jB transcription activity following CK2

down-regulation may additionally be responsible for the

observed decrease in Bcl-xL expression levels as previ-

ously reported in the case of tumor cells treated with CK2

inhibitors [49].

An additional finding that emerged from our study is the

elevated expression of two ROS-related genes, namely,

Fmo2 and Ptgs2, following simultaneous down-regulation

of CK2a and a0. Flavin-containing monooxygenases

(FMOs) are a family of NADPH-dependent proteins spe-

cialized in the oxidation of xeno-substrates in the presence

of flavin adenine dinucleotide (FAD). FMO2 is the most

abundant of the FMOs and it is particularly expressed in

lung although appreciable amounts are also found in nasal

tissue, heart, and brain (reviewed in [50, 51]). Studies

carried out with yeast FMO showed that its expression is

significantly enhanced as a result of accumulation of mis-

folded proteins in the endoplasmic reticulum as part of an

unfolded protein response (UPR) which triggers up-regu-

lation of endoplasmic reticulum-resident proteins involved

in protein folding [52]. Bearing in mind that CK2-depen-

dent phosphorylation of CDC37 is essential for the stability

of HSP-90–CDC37-containing complex, it is conceivable

that stimulation of FMO gene expression in cardiomy-

oblasts may result from the presence of misfolded proteins

as a result of impaired HSP-90 chaperone function.

Stimulation of ptgs-2-gene expression was also observed

in cells lacking CK2. This is in line with previous studies

showing that the expression of ptgs-2 is induced by

oxidative stress in various cancer cell lines and in cardiac

tissue [53, 54]. The reason why ptgs-2-transcript levels are

increased following ROS accumulation remains to be

determined; however, it has been postulated that this might

be a mechanism to abort cell death as ptgs-2 is known to

reduce apoptotic susceptibility [55].

The analysis of genes related to oxidative stress also

revealed a significant down-regulation of the expression of

Dhcr24 and Ptgs1. Dhcr24 encodes for 24-dehydrocholes-

terol reductase (DHCR24), a key enzyme in cholesterol

biosynthesis. DHCR24 has been recently shown to act as

anti-oxidant through twomechanisms: the first dependent on

cholesterol and the second one through a direct scavenging

of hydrogen peroxide [56, 57]. Interestingly, compelling

evidence has pointed out to a dual role of DHCR24 in pro-

tecting cells against oxidative stress. Its up-regulation during

an acute response is associated with elevated cholesterol

levels. In contrast, DHCR24 levels decline upon prolonged

exposure to oxidative stress and its down-regulation seems to

be part of an alternative pro-survival strategy associatedwith

destabilization of the tumor suppressor protein p53 [57]. We

have obtained results in line with these findings. However, it

remains to be determined whether down-regulation of

DHCR24 correlates with decreased expression levels of the

corresponding protein and whether this event results in

destabilization of p53 as part of a protective mechanism

against cell death.

Taken together, results reported here demonstrate that

CK2 provides protection against oxidative stress in car-

diomyoblasts through cellular mechanisms involving the

NF-jB-mediated signaling pathway and the Bcl-2 mam-

malian homolog Bcl-xL. It has been previously shown that

Romo1 controls redox homeostasis by recruiting Bcl-xL to

reduce DWm. We propose a new mechanism by which

silencing of CK2 correlates with attenuation of HSP-90-

molecular chaperone function and reduced Bcl-xL levels

resulting in oxidative stress. Although changes in the

expression of a gene may not necessarily lead to alteration

in the abundance of the corresponding protein, analysis of a

PCR array for profiling the expression of genes related to

oxidative stress further supports the notion that CK2 exerts

a pro-survival role also in non-cancerous cells. This may

occur by positively regulating the activity of proteins with

chaperone activity and those with a pro-survival function

during a prolonged exposure to oxidative stress.

Acknowledgements The authors would like to thank Dr. Olaf-Georg

Issinger (University of Southern Denmark) for critically reading the

manuscript; Dr. David W. Litchfield (University of Western Ontario)

for the generous gift of the RS3.22 cell line; Dr. Tuula Kallunki

(Danish Cancer Society) for providing the Bcl-xL viral vector; Dr.

Phillip Hallenborg (University of Southern Denmark) for expertise in

establishing Bcl-xL-overexpressing cardiomyoblasts; and Tina H.

Svenstrup for technical assistance. We thank the Drug Synthesis and

Chemistry Branch, Developmental Therapeutics Program, NCI, USA,

for providing us with viable samples. This work was supported by the

Danish Council for Independent Research-Natural Sciences (Grant

1323-00212A to B. Guerra).

References

1. Brown GC (1992) Control of respiration and ATP synthesis in

mammalian mitochondria and cells. Biochem J 284:1–13. doi:10.

1042/bj2840001

2. Taverne YJHJ, Bogers AJJC, Duncker DJ, Merkus D (2013)

Reactive oxygen species and the cardiovascular system. Oxid

Med Cell Longev. doi:10.1155/2013/862423

3. Turrens JF (2003) Mitochondrial formation of reactive oxygen

species. J Physiol 552:335–344. doi:10.1113/jphysiol.2003.

049478

4. Castaldo SA, Freitas JR, Conchinha NV, Madureira PA (2016)

The tumorigenic roles of the cellular REDOX regulatory systems.

Oxid Med Cell Longev 2016:1–17. doi:10.1155/2016/8413032

5. Truong TH, Carroll KS (2013) Redox regulation of protein

kinases. Crit Rev Biochem Mol Biol 48:332–356. doi:10.3109/

10409238.2013.790873

6. Knock GA, Ward JPT (2011) Redox regulation of protein kinases

as a modulator of vascular function. Antioxid Redox Signal

15:1531–1547. doi:10.1089/ars.2010.3614

7. Morgan MJ, Liu Z-G (2010) Crosstalk of reactive oxygen species

and NF-jB signaling. Cell Res 21:103–115. doi:10.1038/cr.2010.

178

Mol Cell Biochem

123

8. Montenarh M (2009) DMAT, an inhibitor of protein kinase CK2

induces reactive oxygen species and DNA double strand breaks.

Oncol Rep 21:1593–1597. doi:10.3892/or_00000392

9. Kim GS, Jung JE, Niizuma K, Chan PH (2009) CK2 is a novel

negative regulator of NADPH oxidase and a neuroprotectant in

mice after cerebral ischemia. J Neurosci 29:14779–14789. doi:10.

1523/JNEUROSCI.4161-09.2009

10. Guerra B (2012) Downregulation of protein kinase CK2 induces

autophagic cell death through modulation of the mTOR and

MAPK signaling pathways in human glioblastoma cells. Int J

Oncol 41:1967–1976. doi:10.3892/ijo.2012.1635

11. Qaiser F, Trembley JH, Kren BT et al (2014) Protein Kinase CK2

Inhibition Induces Cell Death via Early Impact on Mitochondrial

Function. J Cell Biochem 115:2103–2115. doi:10.1002/jcb.24887

12. Guerra B, Issinger O-G (1999) Protein kinase CK2 and its role in

cellular proliferation, development and pathology. Electrophore-

sis 20:391–408. doi:10.1002/(SICI)1522-2683(19990201)20:

2\391:AID-ELPS391[3.0.CO;2-N

13. Guerra B, Issinger O-G (2008) Protein kinase CK2 in human

diseases. Curr Med Chem 15:1870–1886

14. St-Denis NA, Litchfield DW (2009) Protein kinase CK2 in health

and disease. Cell Mol Life Sci 66:1817–1829. doi:10.1007/

s00018-009-9150-2

15. Trembley JH, Chen Z, Unger G et al (2010) Emergence of protein

kinase CK2 as a key target in cancer therapy. BioFactors

36:187–195. doi:10.1002/biof.96

16. Guerra B, Iwabuchi K, Issinger O-G (2014) Protein kinase CK2 is

required for the recruitment of 53BP1 to sites of DNA double-

strand break induced by radiomimetic drugs. Cancer Lett

345:115–123. doi:10.1016/j.canlet.2013.11.008

17. Hallenborg P, Feddersen S, Francoz S et al (2012) Mdm2 controls

CREB-dependent transactivation and initiation of adipocyte dif-

ferentiation. Cell Death Differ 19:1381–1389. doi:10.1038/cdd.

2012.15

18. Olsen BB, Issinger OG, Guerra B (2010) Regulation of DNA-

dependent protein kinase by protein kinase CK2 in human

glioblastoma cells. Oncogene 29:6016–6026. doi:10.1038/onc.

2010.337

19. Olsen BB, Wang S-Y, Svenstrup TH et al (2012) Protein kinase

CK2 localizes to sites of DNA double-strand break regulating the

cellular response to DNA damage. BMC Mol Biol 13:7. doi:10.

1186/1471-2199-13-7

20. Guerra B, Fischer M, Schaefer S, Issinger O-G (2015) The kinase

inhibitor D11 induces caspase-mediated cell death in cancer cells

resistant to chemotherapeutic treatment. J Exp Clin Cancer Res

34:125. doi:10.1186/s13046-015-0234-6

21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene

expression data using real-time quantitative PCR and the 2(-Delta

Delta C(T)) Method. Methods 25:402–408. doi:10.1006/meth.

2001.1262

22. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data

by the comparative C(T) method. Nat Protoc 3:1101–1108

23. Guerra B, Hochscherf J, Jensen NB, Issinger O-G (2015) Iden-

tification of a novel potent, selective and cell permeable inhibitor

of protein kinase CK2 from the NIH/NCI Diversity Set Library.

Mol Cell Biochem 406:151–161. doi:10.1007/s11010-015-2433-z

24. Marchi S, Giorgi C, Suski JM et al (2012) Mitochondria-ros

crosstalk in the control of cell death and aging. J Signal Trans-

duct. doi:10.1155/2012/329635

25. Peshavariya HM, Dusting GJ, Selemidis S (2007) Analysis of

dihydroethidium fluorescence for the detection of intracellular

and extracellular superoxide produced by NADPH oxidase. Free

Radic Res 41:699–712. doi:10.1080/10715760701297354

26. Kim I, Rodriguez-Enriquez S, Lemasters JJ (2007) Selective

degradation of mitochondria by mitophagy. Arch Biochem Bio-

phys 462:245–253. doi:10.1016/j.abb.2007.03.034

27. Cottet-Rousselle C, Ronot X, Leverve X, Mayol J-F (2011)

Cytometric assessment of mitochondria using fluorescent probes.

Cytom A 79:405–425. doi:10.1002/cyto.a.21061

28. To M-S, Aromataris EC, Castro J et al (2010) Mitochondrial

uncoupler FCCP activates proton conductance but does not block

store-operated Ca(2?) current in liver cells. Arch Biochem

Biophys 495:152–158. doi:10.1016/j.abb.2010.01.004

29. Sakon S, Xue X, Takekawa M et al (2003) NF-kappaB inhibits

TNF-induced accumulation of ROS that mediate prolonged

MAPK activation and necrotic cell death. EMBO J

22:3898–3909. doi:10.1093/emboj/cdg379

30. Viatour P, Merville M-P, Bours V, Chariot A (2005) Phospho-

rylation of NF-kappaB and IkappaB proteins: implications in

cancer and inflammation. Trends Biochem Sci 30:43–52. doi:10.

1016/j.tibs.2004.11.009

31. Oeckinghaus A, Hayden MS, Ghosh S (2011) Crosstalk in NF-jBsignaling pathways. Nat Immunol 12:695–708. doi:10.1038/ni.

2065

32. Jones PL, Ping D, Boss JM (1997) Tumor necrosis factor alpha

and interleukin-1beta regulate the murine manganese superoxide

dismutase gene through a complex intronic enhancer involving

C/EBP-beta and NF-kappaB. Mol Cell Biol 17:6970–6981

33. Djavaheri-Mergny M, Javelaud D, Wietzerbin J, Besancon F

(2004) NF-kappaB activation prevents apoptotic oxidative stress

via an increase of both thioredoxin and MnSOD levels in

TNFalpha-treated Ewing sarcoma cells. FEBS Lett 578:111–115.

doi:10.1016/j.febslet.2004.10.082

34. Kairisalo M, Korhonen L, Blomgren K, Lindholm D (2007)

X-linked inhibitor of apoptosis protein increases mitochon-

drial antioxidants through NF-kappaB activation. Biochem

Biophys Res Commun 364:138–144. doi:10.1016/j.bbrc.2007.

09.115

35. Vilk G, Saulnier RB, Pierre RS, Litchfield DW (1999) Inducible

Expression of Protein Kinase CK2 in Mammalian Cells: EVI-

DENCE FOR FUNCTIONAL SPECIALIZATION OF CK2

ISOFORMS. J Biol Chem 274:14406–14414. doi:10.1074/jbc.

274.20.14406

36. Chen C, Edelstein LC, Gelinas C (2000) The Rel/NF-kappaB

family directly activates expression of the apoptosis inhibitor

Bcl-x(L). Mol Cell Biol 20:2687–2695

37. Kim JJ, Lee SB, Park JK, Yoo YD (2010) TNF-alpha-induced

ROS production triggering apoptosis is directly linked to Romo1

and Bcl-X(L). Cell Death Differ 17:1420–1434. doi:10.1038/cdd.

2010.19

38. Genestier L, Bonnefoy-Berard N, Rouault JP et al (1995) Tumor

necrosis factor-alpha up-regulates Bcl-2 expression and decreases

calcium-dependent apoptosis in human B cell lines. Int Immunol

7:533–540

39. Vander Heiden MG, Chandel NS, Williamson EK et al (1997)

Bcl-xL regulates the membrane potential and volume home-

ostasis of mitochondria. Cell 91:627–637

40. Shirakata Y, Koike K (2003) Hepatitis B virus X protein induces

cell death by causing loss of mitochondrial membrane potential.

J Biol Chem 278:22071–22078. doi:10.1074/jbc.M301606200

41. Miyata Y (2009) Protein kinase CK2 in health and disease: CK2:

the kinase controlling the Hsp90 chaperone machinery. Cell Mol

Life Sci 66:1840–1849. doi:10.1007/s00018-009-9152-0

42. Pratt WB, Toft DO (2003) Regulation of signaling protein

function and trafficking by the hsp90/hsp70-based chaperone

machinery. Exp Biol Med (Maywood) 228:111–133

43. Echeverrıa PC, Bernthaler A, Dupuis P et al (2011) An interac-

tion network predicted from public data as a discovery tool:

application to the Hsp90 molecular chaperone machine. PLoS

ONE 6:e26044. doi:10.1371/journal.pone.0026044

44. Taipale M, Krykbaeva I, Koeva M et al (2012) Quantitative

analysis of HSP90-client interactions reveals principles of

Mol Cell Biochem

123

substrate recognition. Cell 150:987–1001. doi:10.1016/j.cell.

2012.06.047

45. Fu J, Koul D, Yao J et al (2013) Novel HSP90 inhibitor NVP-

HSP990 targets cell-cycle regulators to ablate Olig2-positive

glioma tumor-initiating cells. Cancer Res 73:3062–3074. doi:10.

1158/0008-5472.CAN-12-2033

46. Caldas-Lopes E, Cerchietti L, Ahn JH et al (2009) Hsp90 inhi-

bitor PU-H71, a multimodal inhibitor of malignancy, induces

complete responses in triple-negative breast cancer models. Proc

Natl Acad Sci USA 106:8368–8373. doi:10.1073/pnas.

0903392106

47. Duncan JS, Litchfield DW (2008) Too much of a good thing: the

role of protein kinase CK2 in tumorigenesis and prospects for

therapeutic inhibition of CK2. Biochim Biophys Acta

1784:33–47. doi:10.1016/j.bbapap.2007.08.017

48. Trembley JH, Wang G, Unger G et al (2009) Protein kinase CK2

in health and disease: CK2: a key player in cancer biology. Cell

Mol Life Sci 66:1858–1867. doi:10.1007/s00018-009-9154-y

49. Ravi R, Bedi A (2002) Sensitization of tumor cells to Apo2

ligand/TRAIL-induced apoptosis by inhibition of casein kinase

II. Cancer Res 62:4180–4185

50. Krueger SK, Williams DE (2005) Mammalian flavin-containing

monooxygenases: structure/function, genetic polymorphisms and

role in drug metabolism. Pharmacol Ther 106:357–387. doi:10.

1016/j.pharmthera.2005.01.001

51. Cashman JR, Zhang J (2006) Human flavin-containing

monooxygenases. Annu Rev Pharmacol Toxicol 46:65–100.

doi:10.1146/annurev.pharmtox.46.120604.141043

52. Suh JK, Robertus JD (2000) Yeast flavin-containing monooxy-

genase is induced by the unfolded protein response. Proc Natl

Acad Sci USA 97:121–126

53. Gilroy DW, Colville-Nash PR, Willis D et al (1999) Inducible

cyclooxygenase may have anti-inflammatory properties. Nat Med

5:698–701. doi:10.1038/9550

54. Sun Y, Chen J, Rigas B (2009) Chemopreventive agents induce

oxidative stress in cancer cells leading to COX-2 overexpression

and COX-2-independent cell death. Carcinogenesis 30:93–100.

doi:10.1093/carcin/bgn242

55. Sun Y, Tang XM, Half E et al (2002) Cyclooxygenase-2 over-

expression reduces apoptotic susceptibility by inhibiting the

cytochrome c-dependent apoptotic pathway in human colon

cancer cells. Cancer Res 62:6323–6328

56. Lu X, Kambe F, Cao X et al (2008) 3beta-Hydroxysteroid-

delta24 reductase is a hydrogen peroxide scavenger, protecting

cells from oxidative stress-induced apoptosis. Endocrinology

149:3267–3273. doi:10.1210/en.2008-0024

57. Kuehnle K, Crameri A, Kalin RE et al (2008) Prosurvival effect

of DHCR24/Seladin-1 in acute and chronic responses to oxidative

stress. Mol Cell Biol 28:539–550. doi:10.1128/MCB.00584-07

Mol Cell Biochem

123