Cancer Research Versailles No Abst Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamane, K.
Right arrow Articles by Kinsella, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamane, K.
Right arrow Articles by Kinsella, T. J.
[Cancer Research 65, 4362-4367, May 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

CK2 Inhibits Apoptosis and Changes Its Cellular Localization Following Ionizing Radiation

Kazuhiko Yamane1 and Timothy J. Kinsella1,2

1 Department of Radiation Oncology, Case Western Reserve University and 2 Case Comprehensive Cancer Center/University Hospitals of Cleveland, Cleveland, Ohio

Requests for reprints: Timothy J. Kinsella, Department of Radiation Oncology, LTR6068, University Hospitals of Cleveland/Case Comprehensive Cancer Center, 11100 Euclid Avenue, Cleveland, OH 44106-6068. Phone: 216-844-2530; Fax: 216-844-4799; E-mail: timothy.kinsella{at}uhhs.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we show that CK2 (casein kinase II, CKII) participates in apoptotic responses following ionizing radiation (IR). Using HeLa human cervical carcinoma cells, we find that transfection of small interfering RNA against the CK2 {alpha} and/or {alpha}' catalytic subunits results in enhanced apoptosis following IR damage as measured by flow cytometry techniques, compared with a control small interfering RNA. Within 2 to 6 hours of IR, CK2 {alpha} partially localizes to perinuclear structures, whereas a marked nuclear localization of {alpha}' occurs. Treatment with a pan-caspase inhibitor or transfection of ARC (apoptosis repressor with caspase recruitment domain) suppresses the apoptotic response to IR in the CK2-reduced cells, indicating involvement of caspases. Additionally, we find that CK2 {alpha} and/or {alpha}' reduction affects cell cycle progression independent of IR damage in this human cell line. However, the G2-M checkpoint following IR is not affected in CK2 {alpha}- and/or {alpha}'-reduced cells. Thus, our data suggest that CK2 participates in inhibition of apoptosis and negatively regulates caspase activity following IR damage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CK2 (formally, casein kinase II) is a protein Ser/Thr kinase complex that forms a heterotetramer mainly consisting of {alpha}2ß2, {alpha}{alpha}'ß2, or {alpha}2'ß2, where {alpha} and {alpha}' are the catalytic subunits and ß is the noncatalytic subunit (for reviews, see refs. 1, 2). The highly conserved amino acid sequences of CK2 from yeast to humans suggest the importance of CK2 in cellular functions, although its major function(s) are not clearly understood. The {alpha} and {alpha}' subunits have significant homology in NH2-terminal kinase regions but have diverse COOH-terminal tail regions, which are highly conserved between species. CK2 is essential for basic cell viability because a double knockout of the two catalytic subunits is lethal in yeast (3). However, a single knockout of either {alpha} or {alpha}' remains viable, indicating complementary functions of the catalytic subunits, at least with respect to viability in yeast. In male mice, a knockout of CK2 {alpha}' is reported to be sterile and defective in spermatogenesis (4), whereas a knockout of CK2 ß shows embryonic lethality in mice (5). CK2 also plays an antiapoptotic role through different pathways, including protection of Bid from cleavage by caspase 8 (6–9). Recently, antitumor activity was found using a peptide that inhibits CK2 activity (10) and through treatment using an antisense CK2 {alpha} oligonucleotide (11).

CK2 phosphorylates many substrates that regulate the cell cycle, including p53 (12), Cdc2 (13), and BRCA1 (14). Following UV light damage, CK2 phosphorylates p53 and I{kappa}B (15, 16). A role for CK2 has not been determined following ionizing radiation (IR) damage, which principally causes DNA double-strand breaks. Therefore, in this study, we assess the role of CK2 in response to IR damage using human HeLa cervical carcinoma cells. Because a double knockout of {alpha} and {alpha}' subunits is lethal and because the functions of {alpha} can compensate the functions of {alpha}', we used three different small interfering RNAs (siRNA) against the {alpha} and/or {alpha}' subunits. We found that a reduction of CK2 catalytic subunits by siRNA significantly enhanced apoptosis and reduced the G2-M population following IR. However, a G2-M checkpoint arrest following IR damage was not affected by CK2 reduction. We also found that the CK2 {alpha}' subunit translocates into the nucleus within 6 hours of IR treatment, supporting a role for CK2 in response to IR damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture, reagents, and transfection of small interfering RNA. HeLa cells were grown in RPMI 1640 supplemented with 10% FCS in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The caspase inhibitor z-VAD was obtained from Biomol (Plymouth Meeting, PA).

The three CK2 siRNAs used were 5'-GAUGACUACCAGCUGGUUCdTdT ({alpha} siRNA), 5'-UCAAGAUGACUACCAGCUGdTdT ({alpha}10 siRNA, against {alpha} with 100% homology and against {alpha}' with 90% homology), and 5'-CAGUCUGAGGAGCCGCGAGdTdT ({alpha}' siRNA). The negative control siRNA was 5'-GCUCAGAUCAAUACGGAGAdTdT. Additionally, a Chk1 siRNA (5'-GCGTGCCGTAGACTGTCCAdTdT) was used as a positive control (17) to assess apoptosis and cell cycle changes following IR. All siRNAs were obtained from Dharmacon (Lafayette, CO). All were annealed with the complementary strand with dTdT overhangs. Unless stated, the {alpha} siRNA (but not {alpha}10 siRNA) was used for reduction of the {alpha} subunit. Transfection was done with Oligofectamine as recommended by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). Transfection was done for 30 hours, and HeLa cells were then washed and trypsinized. Next, the cells from one well of a 24-well plate were divided into two to three wells, and RPMI 1640 was added to the culture before irradiation. The efficiencies of transfection were ~90% according to the maximum protein level reduction. Transfection with plasmid DNA was done using Fugene 6, as recommended by the manufacturer (Roche, Indianapolis, IN), using the above culture medium.

Ionizing radiation treatment. Following the transfection procedures (30 hours) and plating for 20 hours in complete medium, cells were irradiated using a model S109 137Cs radiation source at a dose rate of 4.1 Gy/min (J.L. Shepherd and Associates, San Fernando, CA). The cells were then returned to the incubator under the same culture conditions and maintained for the indicated times.

Western blotting. Total cell extracts were prepared by trichloroacetic acid precipitation to detect CK2 {alpha} and {alpha}' using antibodies (N-18 and C-20, respectively, Santa Cruz Biotechnology, Santa Cruz, CA) and to detect Myc using a specific antibody (9E10, Babco, Berkeley, CA). After extraction of trichloroacetic acid with ether, the DNA was sheared by sonication before loading onto gels. An actin antibody (Sigma, St. Louis, MO) was also used for Western blotting as a control.

Flow cytometry. HeLa cells were fixed with 90% ethanol at –20°C for 60 minutes to a few days, incubated with RNase and stained with propidium iodide, and then subjected to flow cytometry (Coulter, Epics XLMCL, Miami, FL). To measure the G2-M checkpoint response, cells were fixed using 0.25% formaldehyde in the medium before the ethanol treatment and then double-stained with mitotic protein monoclonal-2 antibody (Upstate, Waltham, MA) and propidium iodide. Nocodazole (0.3 mg/mL) was added to the medium and cells were cultured for 24 hours as a general method (18). To perform terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) staining, cells were also fixed with 0.25% formaldehyde in the medium before the ethanol treatment and then stained with the Apo-Direct kit (eBioscience, San Diego, CA).

Confocal microscopy. HeLa cells were irradiated and cultured as described above. At various time points following IR, cells were then fixed with methanol and acetone (1:1) and stained with anti-{alpha} or anti-{alpha}' antibodies (N-18 and C-20, respectively; Santa Cruz Biotechnology). Samples were washed, stained with secondary antibodies (Alexa Fluor 633 anti-goat IgG, A21082, Molecular Probes, Eugene, OR) and 4',6-diamidino-2-phenylindole (DAPI), and then subjected to confocal microscopy. Mitotracker (M7514, Molecular Probes) was used for mitochondria staining (30 µmol/L, 1 hour). Transfection of endoplasmic reticulum–targeted green fluorescent protein (GFP; ref. 19) was used for staining of the endoplasmic reticulum. The confocal images were obtained with a Zeiss LSM510 inverted confocal microscope system (Carl Zeiss, Oberkochen, Germany) equipped with a tunable T-sapphire laser (Mira-F-V5-XW-220) with a diode pump (Verdi 5 W) to obtain the images with the different dyes. Image analysis was carried out with Adobe Photoshop Software (Adobe Systems, San Jose, CA).

Gene isolation and site-specific mutagenesis. Genes encoding ARC (apoptosis repressor with caspase recruit domain) and Cdc2 were amplified by a PCR using human placenta total cDNA, and cloned into a pcDNA3-based vector (Invitrogen Life Technologies). The mutations were introduced using Quikchange (Stratagene, La Jolla, CA). All amplified and mutated sequences were confirmed by DNA sequencing in all coding regions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A sub-G1 population is enhanced in CK2-reduced cell following ionizing radiation. We used a RNA interference technique (20) for reduction of the protein levels of the CK2 catalytic subunits, using transfection of specific siRNAs against the two catalytic subunits {alpha} and/or {alpha}'. The two antibodies against {alpha} or {alpha}' specifically recognized each subunit by Western blotting (Fig. 1A). After siRNA transfection, the protein levels of the CK2 {alpha} and {alpha}' subunits were markedly reduced, whereas a control siRNA transfection did not result in changes in protein levels of the CK2 subunits nor actin (control; Fig. 1B). The {alpha}10 siRNA was designed to target both {alpha} and {alpha}' with 100% and 90% homologies, respectively, and seems to reduce {alpha} to a greater extent than {alpha}' (Fig. 1B). The strong inhibition by siRNA continued for at least 48 hours, following siRNA transfection (30 hours) and then 20 hours in complete media (Fig. 1C). The effects of IR treatment in CK2-reduced HeLa cells were assessed for up to 48 hours, based on this continued inhibition of CK2 catalytic unit protein expression (Fig. 1C).



View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. CK2 siRNA transfection reduces protein expression of the specific catalytic units and results in an enhanced sub-G1 population following IR. A, total cell lysates of HeLa cells were analyzed by Western blotting using antibodies against CK2 {alpha} or {alpha}', or both. B, HeLa cells were transfected with the indicated single siRNAs for 30 hours. The medium was then changed and cells were cultured for 20 hours. Total cell lysates were analyzed by Western blotting using antibodies against {alpha}, {alpha}', and actin (control), respectively. C, time course of inhibition of CK2 {alpha} and {alpha}' subunits following initial siRNA transfection for 30 hours and incubation for 20 hours (time 0 hour). Cells were harvested at time 0, 24, and 48 hours. Total cell lysates were analyzed by Western blotting using antibodies to {alpha}, {alpha}', and actin (control). D, HeLa cells were transfected with indicated siRNAs for 30 hours and cultured for 20 hours, then irradiated (time 0) to 0, 10, or 30 Gy. Cells were further cultured following IR for the indicated times and then analyzed with flow cytometry.

 
HeLa cells were transfected with siRNA against {alpha} and {alpha}' for 30 hours, cultured for 20 hours, and then irradiated to 0, 10, or 30 Gy (time 0). CK2 reduction using siRNA against both {alpha} and {alpha}' seemed to result in an increased sub-G1 population at 24 and 48 hours following IR compared with control-transfected cells (Fig. 1D). The control siRNA-transfected HeLa cells showed an IR dose- and time-dependent enhancement in the G2-M population, consistent with a recent report using HeLa cells (21). A single siRNA transfection against either CK2 {alpha} or {alpha}' also resulted in an increased sub-G1 population following IR (Fig. 1D), suggesting that each CK2 catalytic subunit is important for inhibition of apoptosis following IR. Additionally, CK2-reduced HeLa cells using both {alpha} + {alpha}' siRNA or {alpha}' siRNA alone showed a significant G1 cell cycle delay at 24 hours after 30 Gy IR. These results on cell cycle progression in CK2-reduced HeLa cells are further analyzed (see Fig. 4).



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. CK2-reduced cells have a normal G2-M checkpoint response after IR but show delayed cell cycle progression even without IR damage. A, HeLa cells were transfected with indicated siRNAs (CK2 {alpha} + {alpha}' or Chk1) for 30 hours and cultured for 20 hours before IR (30 Gy). Next, nocodazole was added to the medium and cells were cultured for an additional 24 hours. The cells were then fixed with formaldehyde and stained with mitotic protein monoclonal-2 antibody conjugated with FITC and with propidium iodide and analyzed by flow cytometry. G0-G1 (G1) and G2 (G2) populations are indicated (arrowheads). The gated regions are M phase populations (M), which are quantified as the percent M phase cells per total cell population. B, to evaluate the effect of CK2 reduction without IR, HeLa cells were transfected with indicated siRNAs for 30 hours and then cultured for 20 hours. Nocodazole was then added to the medium for 24 hours and cells were harvested at 0, 8, and 24 hours following nocodazole treatment for flow cytometry. Each cell cycle population is calculated by gating of M phase and by Modfit software. The sub-G1 population is not shown.

 
Enhanced apoptosis following ionizing radiation is confirmed by a TUNEL assay in CK2-reduced cells. To further confirm and characterize the apoptotic response following IR treatment in the CK2-reduced HeLa cells, we used a TUNEL and propidium iodide double-staining technique with formaldehyde fixation to reduce the sub-G1 population. We found that CK2 reduction in HeLa cells results in a significant TUNEL-positive population with the use of a double ({alpha} + {alpha}') siRNA transfection even without IR treatment (Fig. 2, top). However, following IR treatment (10 Gy), we also find a time-dependent increase in the apoptotic population in the CK2-reduced cells (24 and 48 hours following IR). In contrast, a very low TUNEL-positive population is found in the (negative) control siRNA-transfected cells with or without treatment with IR. It should be noted that the TUNEL-positive populations do not directly correspond to the sub-G1 populations in Fig. 1D because the TUNEL labeling is an enzymatic treatment to label each cell, whereas a sub-G1 population represents total fragmented DNA detected by flow cytometry.



View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. A TUNEL-positive population is enhanced in CK2- and Chk1-reduced cells following IR. HeLa cells were transfected with the indicated siRNA for 30 hours and cultured for 20 hours. Cells were then irradiated to 10 Gy and cultured for 24 or 48 hours. Harvested cells were then cross-linked with formaldehyde, fixed, and then labeled according to the TUNEL method using fluorescein dUTP. Samples were stained with propidium iodide and analyzed by flow cytometry. G0-G1 (G1) and G2-M (G2) populations are indicated (arrowheads). The gated regions are TUNEL-positive populations that are quantified as the percentage of TUNEL-positive cells per total cell population. Note that cross-linking can reduce the sub-G1 population because it fixes cellular structures and prevents fragmentation of DNA during ethanol fixation.

 
Use of a single siRNA ({alpha}, {alpha}', or {alpha}10) transfection also shows an increased TUNEL-positive fraction at 48 hours following 10 Gy (Fig. 2, bottom), indicating that the {alpha} and {alpha}' subunits are independently important for the inhibition of apoptosis following IR damage. We also used a Chk1 siRNA transfection into HeLa cells as a positive control because Chk1 is an essential protein for cell survival and DNA damage response (22, 23). An increased TUNEL-positive fraction is also found in Chk1-reduced HeLa cells (15%), but this fraction is further increased to 55% at 48 hours following 10 Gy (Fig. 2, bottom), similar to the data in CK2-reduced cells.

CK2 {alpha} and {alpha}' show differential patterns of cellular localization following ionizing radiation. To examine the localization of the CK2 {alpha} or {alpha}' catalytic subunits, we stained nontransfected HeLa cells with anti-CK2 antibodies and analyzed their localization by confocal microscopy. Both CK2 {alpha} and {alpha}' localized mainly to the cytoplasm in the absence of IR treatment (0 Gy, 24 hours; Fig. 3A). Following IR treatment to 10 Gy and a 24-hour period in complete medium, CK2 {alpha} became partially localized to perinuclear regions. The staining patterns of mitochondria (using Mitotracker) did not exclusively correspond to the CK2 {alpha} pattern (CK2 {alpha} at 10 Gy, a merged panel with yellow color when colocalized; Fig. 3A). Using a higher IR dose (30 Gy) and a longer observation time (48 hours following IR), nuclear fragmentation was seen and CK2 {alpha} remained partially localized to perinucleic structures. Further confocal microscopy studies using HeLa cells transfected with endoplasmic reticulum-targeted GFP show that CK2 {alpha} is partially localized to the endoplasmic reticulum (data not shown). On the other hand, CK2 {alpha}' markedly changed its localization to the nucleus following IR treatment at 10 Gy and culture for 24 hours, whereas a higher dose (30 Gy) and longer observation (48 hours following IR) showed persistent nuclear localization with evidence of nuclear fragmentation (Fig. 3A). The preferential localization of CK2 {alpha} and {alpha}' in cytoplasm without DNA damage is due to incubation with Mitotracker (compare with Fig. 3B).



View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. The CK2 catalytic subunits show a differential pattern of cellular localization following IR. A, nontransfected HeLa cells were treated with IR (10 Gy) and cultured for 24 hours or treated to a higher IR dose (30 Gy) and cultured for 48 hours. Cells were stained with Mitotracker, fixed, and then stained with anti-{alpha} or {alpha}' antibody. Next, samples were stained with secondary antibodies and DAPI and subjected to confocal microscopy. B, nontransfected HeLa cells were irradiated to 10 Gy and cultured for indicated times. Samples were fixed without Mitotracker staining, immunostained and stained with DAPI, and, finally, subjected to confocal microscopy.

 
We next determined the time course of changes in the localization of CK2 {alpha} and {alpha}' in nontransfected HeLa cells over the initial 24 hours following 10 Gy IR treatment (Fig. 3B). The perinuclear localization of CK2 {alpha} began at 2 hours and continued to increase to 24 hours, whereas the nuclear localization of CK2 {alpha}' was not evident until ~6 hours following IR. These data suggest that CK2 is a relatively late-response protein to IR damage compared with early IR–response proteins, such as 53BP1, which can form nuclear foci within 10 minutes of IR treatment (24). Whereas IR causes multiple types of IR damage, including DNA double-strand breaks (which are considered the primary lesion leading to cytotoxicity), DNA single-strand breaks, and DNA-protein cross-links, these lesions are typically repaired (or misrepaired) within the first 1 to 2 hours following IR. The observed time course of localization changes of CK2 {alpha} and {alpha}' of 2 to 6 hours following IR suggests that the participation of CK2 {alpha} and {alpha}' in IR damage response are temporally related downstream events and that the CK2 catalytic subunits seem to have different cellular targets following IR damage (see Discussion).

CK2-reduced cells have a normal G2-M checkpoint response after ionizing radiation. To examine whether CK2 participates in the G2-M arrest following IR, the G2-M checkpoint response in CK2 knockdown HeLa cells was measured by double staining with propidium iodide and mitotic protein monoclonal-2 antibody after nocodazole treatment (Fig. 4). Nocodazole is generally used for a G2-M checkpoint assay (18, 25). IR treatment (30 Gy) strongly inhibits entry into mitosis in control cells and CK2-reduced cells (Fig. 4A, 60% to 4.2% and 38% to 0.2%, respectively), indicating that CK2 is not essential for the G2-M checkpoint. Again, we used Chk1-reduced HeLa cells as a positive control. No inhibition of entry into mitosis following IR treatment was found in Chk1 siRNA-treated cells with nocodazole (8.3% and 11%; Fig. 4A). These results indicate that CK2 and Chk1 have different effects on the G2-M checkpoint response in HeLa cells following IR damage.

We previously found a significant delay in cell cycle progression in CK2-reduced cells (Fig. 1D). To determine if this delay was related to IR damage, we used nocodazole to stop the cell cycle at M phase in the absence of IR (Fig. 4B). Cell cycle progression was delayed in both CK2 {alpha}– and CK2 {alpha}'–reduced cell populations compared with the control. These results suggest that CK2 also participates in cell cycle progression from G1 to M phase, being consistent with a previous report in yeast (3). When CK2 {alpha}' is reduced, the G1 population was significantly higher (19% at 24 hours) than in the control cells (2.8% at 24 hours), suggesting preferential involvement of {alpha}' in G1-S progression. These data are consistent with the results in Fig. 1D. When CK2 {alpha} is reduced, the G2 population was higher (26% at 24 hours) than in {alpha}'-reduced cells (13% at 24 hours), suggesting a possible role of CK2 {alpha} in G2-M progression.

A caspase inhibitor inhibits apoptosis following DNA damage. The enhanced apoptotic response in CK2-reduced cells (Figs. 1 and 2) suggests that CK2 controls apoptosis in the presence of IR damage. Therefore, we examined the effect of a general caspase inhibitor, z-VAD. When z-VAD was added to either CK2 {alpha}- and {alpha}'-reduced cells or Chk1-reduced cells, apoptosis was strongly inhibited after IR treatment, indicating the involvement of caspases (Fig. 5A). The G2-M population decreased in CK2-reduced cells after IR treatment, suggesting again that CK2 also regulates cell cycle.



View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. z-VAD and ARC partially inhibit apoptosis in CK2-reduced cells after IR treatment. A, HeLa cells were transfected with the indicated siRNAs for 30 hours and then cultured for 20 hours. The caspase inhibitor z-VAD (Biomol) or the solvent, DMSO, was added to the medium. Next, cells were irradiated to 30 Gy, cultured for 48 hours, and then analyzed by flow cytometry. B, Myc epitope–tagged ARC, its mutants, and the vector were transfected into HeLa cells. Proteins were analyzed by Western blotting using anti-Myc antibody. C, protocol for initial siRNA transfection (CK2 {alpha} + {alpha}' or Chk1 siRNA) followed by plasmid transfection of ARCs or vector for 68 hours and IR (0 or 30 Gy) after 20 hours. Cells were then harvested at 48 hours following IR. D, flow cytometry results of cell populations from the above protocol at 48 hours following IR (0 or 30 Gy).

 
Recently, it was reported that ARC could be phosphorylated by CK2 at Thr149, which regulates the apoptosis inhibitory function of ARC (26). Therefore, we expressed ARC and its mutants, 149A (Thr to Ala, an unphosphorylatable form) and 149E (Thr to Glu, a phosphorylation-mimicking form), in HeLa cells (Fig. 5B). After treatment with CK2 siRNA or Chk1 siRNA (Fig. 5C), the wild-type ARC reduces the extent of apoptosis in both IR-treated cells and nontreated cells (Fig. 5D). However, the mutant ARC transfectants did not show significant differences in the reduction of apoptosis compared with the wild-type ARC transfectants. These results suggest that Thr149 is not critical for the inhibition of apoptosis induced by IR damage, which differs from the apoptosis mechanism found in a previous study (26).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that apoptosis is enhanced following IR by a reduction of the CK2 {alpha} and/or {alpha}' subunits through siRNA transfection in HeLa cells (Figs. 1 and 2). A small subset of CK2 {alpha} changes localization to perinuclear structures, whereas a marked nuclear accumulation of {alpha}' occurs over 24 hours following IR (Fig. 3). This is the first report indicating dynamic localization changes of the CK2 catalytic subunits after IR. Taken together with our observed apoptosis induction using {alpha} and {alpha}' siRNAs (Fig. 2), our results suggest that CK2 {alpha} and {alpha}' may have multiple preferential targets involved in inhibition of apoptosis following IR. Additionally, a pan-caspase inhibitor, z-VAD, reduces the apoptotic responses to IR in CK2-reduced cells (Fig. 5A), suggesting that CK2 regulates caspase activity.

We questioned whether ARC could be one of the downstream targets because ARC has a CK2 phosphorylation site that regulates receptor-mediated apoptosis (26). However, both wild-type and mutant ARCs have similar antiapoptotic effects, indicating that point mutations at the phosphorylation site did not change the apoptotic response to IR (Fig. 5D). We conclude that ARC has a nonspecific inhibitory function in IR-mediated apoptosis, which is very different from receptor-mediated apoptosis previously reported (26). Therefore, it is possible that there are other critical targets downstream of CK2 in the IR damage response. We are currently attempting to define these targets.

Because Chk1 is an essential protein involved in both cell survival and in an IR damage response (22, 23), we used a Chk1 siRNA as a positive control. In the G2-M checkpoint response after IR treatment, the ATR-Chk1 pathway is recognized to be the central pathway, whereas the ATM-Chk2 pathway is a supporting pathway (27, 28). Consistent with previous reports (22, 23), we find that Chk1 siRNA transfection into HeLa cells induces apoptosis even in the absence of IR damage, but also enhances apoptosis following IR treatment similar to the response to CK2 siRNA-transfected HeLa cells (Fig. 2). On the other hand, our data suggest that Chk1, but not CK2, is involved in the G2-M checkpoint response following IR (Fig. 4A). The caspase inhibitor z-VAD reduces the extent of apoptosis following IR treatment in both CK2- and Chk1-reduced cells, suggesting involvement of caspases in both pathways (Fig. 5).

CK2 affects the progression of the cell cycle from G1 to M phase even without IR damage (Figs. 1 and 4B). The G2-M population in CK2-reduced cells is also reduced in the presence of z-VAD and IR (Fig. 5A). These data are consistent with a prior study suggesting that CK2 is required for cell cycle progression during G1 and G2 phases in yeast (3). It is also reported that overexpression of CK2 {alpha} is associated with increased cell proliferation in transformed fibroblasts (29). Our data suggest that {alpha}' contributes to G1-S progression, and that {alpha} contributes to G2-M progression at least in part in HeLa cells (Fig. 4B). Because Cdc2 kinase is the central cell cycle regulator whose Ser39 is phosphorylated by CK2 (13), we examined whether a 39A mutation of Cdc2 affects the cell cycle progression in CK2reduced cells. However, we found no clear differences in the cell cycle distribution between CK2-reduced cells highly expressing wild-type Cdc2 and Cdc2 39A mutants after IR treatment (data not shown).

To examine the initial induction of DNA double-strand breaks by IR and subsequent repair at 30 minutes, we used pulse field gel electrophoresis of chromosomal DNA following 0, 20, and 50 Gy. There was no enhancement in DNA breaks on chromosomes in CK2-reduced cells compared with controls (data not shown). These results suggest that CK2 is not involved in initial double-strand break repair at least under these conditions. This may be reasonable because CK2 has no typical regions related to DNA repair enzymes. We speculate that CK2 can control apoptosis downstream of IR-related DNA repair and its cell cycle effects.

In summary, we show that the CK2 catalytic subunits, {alpha} and {alpha}', independently contribute to inhibition of apoptosis after IR damage. This is the first report indicating involvement of CK2 in IR-induced apoptosis and demonstrating a relocalization of the CK2 catalytic subunits within 2 to 6 hours of IR damage.


   Acknowledgments
 
Grant support: NIH grant CA84578 (T.J. Kinsella), Flow Cytometry Core Facility, Confocal Microscopy Core Facility, and Radiation Resource Core Facility of the Comprehensive Cancer Center of Case Western Reserve University Hospitals of Cleveland grant P30 CA43703-12.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Song-mao Chiu of Case Western Reserve University for discussion, Michael Sramkoski for flow cytometry techniques, Dr. Minh Lam for confocal microscopy techniques, and Dr. Clark Distelhorst and Michael Malone for the endoplasmic reticulum–targeted GFP plasmid.

Received 11/ 2/04. Revised 3/ 1/05. Accepted 3/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Litchfield DW. Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J 2003;369:1–15.[CrossRef][Medline]
  2. Meggio F, Pinna LA. One-thousand-and-one substrates of protein kinase CK2? FASEB J 2003;17:349–68.[Abstract/Free Full Text]
  3. Hanna DE, Rethinaswamy A, Glover CV. Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae. J Biol Chem 1995;270:25905–14.[Abstract/Free Full Text]
  4. Xu X, Toselli PA, Russell LD, Seldin DC. Globozoospermia in mice lacking the casein kinase II {alpha}' catalytic subunit. Nat Genet 1999;23:118–21.[CrossRef][Medline]
  5. Buchou T, Vernet M, Blond O, et al. Disruption of the regulatory ß subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Mol Cell Biol 2003;23:908–15.[Abstract/Free Full Text]
  6. Desagher S, Osen-Sand A, Montessuit S, et al. Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8. Mol Cell 2001;8:601–11.[CrossRef][Medline]
  7. Wang H, Davis A, Yu S, Ahmed K. Response of cancer cells to molecular interruption of the CK2 signal. Mol Cell Biochem 2001;227:167–74.[CrossRef][Medline]
  8. Guo C, Yu S, Davis AT, et al. A potential role of nuclear matrix-associated protein kinase CK2 in protection against drug-induced apoptosis in cancer cells. J Biol Chem 2001;276:5992–9.[Abstract/Free Full Text]
  9. Ahmed K, Gerber DA, Cochet C. Joining the cell survival squad: an emerging role for protein kinase CK2. Trends Cell Biol 2002;12:226–30.[CrossRef][Medline]
  10. Perea SE, Reyes O, Puchades Y, et al. Antitumor effect of a novel proapoptotic peptide that impairs the phosphorylation by the protein kinase 2 (casein kinase 2). Cancer Res 2004;64:7127–9.[Abstract/Free Full Text]
  11. Slaton JW, Unger GM, Sloper DT, Davis AT, Ahmed K. Induction of apoptosis by antisense CK2 in human prostate cancer xenograft model. Mol Cancer Res 2004;2:712–21.[Abstract/Free Full Text]
  12. Meek DW, Simon S, Kikkawa U, Eckhart W. The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II. EMBO J 1990;9:3253–60.[Medline]
  13. Russo GL, Vandenberg MT, Yu IJ, et al. Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle. J Biol Chem 1992;267:20317–25.[Abstract/Free Full Text]
  14. O'Brien KA, Lemke SJ, Cocke KS, Rao RN, Beckmann RP. Casein kinase 2 binds to and phosphorylates BRCA1. Biochem Biophys Res Commun 1999;260:658–64.[CrossRef][Medline]
  15. Keller DM, Zeng X, Wang Y, et al. A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1. Mol Cell 2001;7:283–92.[CrossRef][Medline]
  16. Kato T Jr, Delhase M, Hoffmann A, Karin M. CK2 is a C-terminal I{kappa}B kinase responsible for NF-{kappa}B activation during the UV response. Mol Cell 2003;12:829–39.[CrossRef][Medline]
  17. Zhao H, Watkins JL, Piwnica-Worms H. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Sci U S A 2002;99:14795–800.[Abstract/Free Full Text]
  18. Cortez D, Guntuku S, Qin J, Elledge SJ. ATR and ATRIP: partners in checkpoint signaling. Science 2001;294:1713–6.[Abstract/Free Full Text]
  19. Thomenius MJ, Wang NS, Reineks EZ, Wang Z, Distelhorst CW. Bcl-2 on the endoplasmic reticulum regulates Bax activity by binding to BH3-only proteins. J Biol Chem 2003;278:6243–50.[Abstract/Free Full Text]
  20. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002;3:737–47.[CrossRef][Medline]
  21. Al-Mohanna MA, Al-Khodairy FM, Krezolek Z, et al. p53 is dispensable for UV-induced cell cycle arrest at late G(1) in mammalian cells. Carcinogenesis 2001;22:573–8.[Abstract/Free Full Text]
  22. Takai H, Tominaga K, Motoyama N, et al. Aberrant cell cycle checkpoint function and early embryonic death in Chk1(–/–) mice. Genes Dev 2000;14:1439–47.[Abstract/Free Full Text]
  23. Liu Q, Guntuku S, Cui XS, et al. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 2000;14:1448–59.[Abstract/Free Full Text]
  24. Rappold I, Iwabuchi K, Date T, Chen J. Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways. J Cell Biol 2001;153:613–20.[Abstract/Free Full Text]
  25. Yamane K, Taylor K, Kinsella TJ. Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway. Biochem Biophys Res Commun 2004;318:297–302.[CrossRef][Medline]
  26. Li PF, Li J, Muller EC, et al. Phosphorylation by protein kinase CK2: a signaling switch for the caspase-inhibiting protein ARC. Mol Cell 2002;10:247–58.[CrossRef][Medline]
  27. Cliby WA, Roberts CJ, Cimprich KA, et al. Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO J 1998;17:159–69.[CrossRef][Medline]
  28. Brown EJ, Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev 2000;14:397–402.[Abstract/Free Full Text]
  29. Orlandini M, Semplici F, Ferruzzi R, et al. Protein kinase CK2{alpha}' is induced by serum as a delayed early gene and cooperates with Ha-ras in fibroblast transformation. J Biol Chem 1998;273:21291–7.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
K. Yamane, J. E. Schupp, and T. J. Kinsella
BRCA1 Activates a G2-M Cell Cycle Checkpoint following 6-Thioguanine-Induced DNA Mismatch Damage
Cancer Res., July 1, 2007; 67(13): 6286 - 6292.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
J. S. Kim, J. I. Eom, J.-W. Cheong, A. J. Choi, J. K. Lee, W. I. Yang, and Y. H. Min
Protein Kinase CK2{alpha} as an Unfavorable Prognostic Marker and Novel Therapeutic Target in Acute Myeloid Leukemia
Clin. Cancer Res., February 1, 2007; 13(3): 1019 - 1028.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. C. Tapia, V. A. Torres, D. A. Rodriguez, L. Leyton, and A. F. G. Quest
Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription
PNAS, October 10, 2006; 103(41): 15079 - 15084.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
P. Salinas, D. Fuentes, E. Vidal, X. Jordana, M. Echeverria, and L. Holuigue
An Extensive Survey of CK2 {alpha} and {beta} Subunits in Arabidopsis: Multiple Isoforms Exhibit Differential Subcellular Localization
Plant Cell Physiol., September 1, 2006; 47(9): 1295 - 1308.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
X. Li, B. Guan, S. Maghami, and C. J. Bieberich
NKX3.1 Is Regulated by Protein Kinase CK2 in Prostate Tumor Cells
Mol. Cell. Biol., April 15, 2006; 26(8): 3008 - 3017.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamane, K.
Right arrow Articles by Kinsella, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamane, K.
Right arrow Articles by Kinsella, T. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online