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[Cancer Research 66, 8662-8671, September 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Calreticulin, a Molecular Chaperone in the Endoplasmic Reticulum, Modulates Radiosensitivity of Human Glioblastoma U251MG Cells

Tomohiro Okunaga1,2, Yoshishige Urata1, Shinji Goto1, Takayuki Matsuo2, Shingo Mizota1,2, Keisuke Tsutsumi2, Izumi Nagata2, Takahito Kondo1 and Yoshito Ihara1,3

1 Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute; 2 Department of Neurosurgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; and 3 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Japan

Requests for reprints: Yoshito Ihara, Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, 852-8523 Nagasaki, Japan. Phone: 81-95-849-7099; Fax: 81-95-849-7100; E-mail: y-ihara{at}net.nagasaki-u.ac.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Radiotherapy is the primary and most important adjuvant therapy for malignant gliomas. Although the mechanism of radiation resistance in gliomas has been studied for decades, it is still not clear how the resistance is related with functions of molecular chaperones in the endoplasmic reticulum. Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. Recently, it was reported that changes in intracellular Ca2+ homeostasis play a role in the modulation of apoptosis. In the present study, we found that the level of CRT was higher in neuroglioma H4 cells than in glioblastoma cells (U251MG and T98G), and was well correlated with the sensitivity to {gamma}-irradiation. To examine the role of CRT in the radiosensitivity of malignant gliomas, the CRT gene was introduced into U251MG cells, which express low levels of CRT, and the effect of overexpression of CRT on the radiosensitivity was examined. The cells transfected with the CRT gene exhibited enhanced radiation-induced apoptosis compared with untransfected control cells. In CRT-overexpressing cells, cell survival signaling via Akt was markedly suppressed. Furthermore, the gene expression of protein phosphatase 2Ac{alpha} (PP2Ac{alpha}), which is responsible for the dephosphorylation and inactivation of Akt, was up-regulated in CRT-overexpressing cells, and the regulation was dependent on Ca2+. Thus, overexpression of CRT modulates radiation-induced apoptosis by suppressing Akt signaling through the up-regulation of PP2Ac{alpha} expression via altered Ca2+ homeostasis. These results show the novel mechanism by which CRT is involved in the regulation of radiosensitivity and radiation-induced apoptosis in malignant glioma cells. (Cancer Res 2006; 66(17): 8662-71)


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The management of patients with glioblastoma multiforme is difficult and poor results have led to a search for novel therapeutic approaches (1). Radiotherapy is the most effective adjuvant modality in the management of glioblastoma multiforme, doubling the median survival rate (2). Radiation-induced cell death is associated with a complex interaction of various factors. The cellular targets of radiation-induced apoptosis are the plasma membrane, cytosol, and nuclear DNA (3). The mechanisms of radiation-induced apoptosis have been studied extensively in terms of p53 status, the Bcl-2 gene family, the Fas-mediated pathway, the ceramide-mediated pathway, the caspase cascade, and the ataxia-telangiectasia-mutated gene (3–5). Nevertheless, it still remains unclear which macroscopic or molecular features determine the response of glioblastoma multiforme to irradiation. For instance, although most studies have shown an association between p53 status and the response to radiotherapy (6), no such association has been convincingly shown in glioblastoma multiforme patients (7).

Recently, it was reported that the Ca2+ of the endoplasmic reticulum and/or cytoplasm plays an important role in radiation-induced apoptosis in association with some of these mechanisms (8–12). In the p53 pathways, Ca2+ and S100B regulated p53-dependent cell growth arrest and apoptosis (8). Bcl-2 and related proteins interfere with intracellular stores and the release of Ca2+ (13). Moreover, ceramide induces an increase in the cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores and activating the capacitative Ca2+ entry pathway, and deletion of Ca2+ from stores is a key to the protective action of Bcl-2 against apoptosis (11). In ataxia-telangiectasia cells, the mobilization of Ca2+ either was absent or increased slowly postirradiation (14). These findings indicate that changes in intracellular Ca2+ homeostasis play a role in the modulation of apoptosis (15).

Calreticulin (CRT) was initially found as a Ca2+-binding protein in the lumen of the endoplasmic reticulum (16). CRT is a multifunctional protein involved in many biological processes that include the regulation of Ca2+ homeostasis (16), intercellular or intracellular signaling, gene expression (17), glycoprotein folding (18), and nuclear transport (19). We found that overexpression of CRT enhanced apoptosis in myocardiac H9c2 cells under conditions inductive to differentiation with retinoic acid (20) or under oxidative stress (21). Moreover, CRT regulates p53 function to induce apoptosis by affecting the rate of degradation and nuclear localization of p53 (12). Furthermore, there have been few reports related to the molecular chaperones and radiation-induced apoptosis of glioma (22). However, it is still not clear whether CRT is involved in the regulatory mechanism for the radiation-induced apoptosis.

In the present study, we investigated the role of CRT in radiosensitivity and radiation-induced apoptosis, using human glioma cell lines. We show here that overexpression of CRT modulates the radiosensitivity of human glioblastoma cells by suppressing Akt/protein kinase B signaling for cell survival via alterations of cellular Ca2+ homeostasis.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and reagents. Antibodies against CRT, calnexin, Grp94, and Erp57 were purchased from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). The anti-Akt and antiphosphorylated Akt (Ser473, Thr309) antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti–protein phosphatase 2A catalytic subunit {alpha} (PP2Ac{alpha}) antibody was from BD Transduction Laboratories (Lexington, KY). The antibodies against PP2A regulatory protein 65 (PP2A-RP65) and PP1a were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Chemicon International (Temecula, CA). The reagents used in the study were all of high grade from Sigma or Wako Pure Chemicals (Osaka, Japan).

Cell culture. Two human glioblastoma cell lines (U251MG and T98G) and a human neuroglioma cell line (H4) were used in this study. The U251MG cells were obtained from Human Science Research Resource Bank (Osaka, Japan). The T98G and H4 cells were obtained from American Type Culture Collection (Manassas, VA). These cells and the CRT gene–transfected U251MG cells were cultured in DMEM supplemented with 10% FCS in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The culture medium was replaced every 2 days.

Gene transfection and selection of cells. A full-length mouse CRT cDNA was cloned from total RNA of mouse RAW264.7 cells by reverse transcription-PCR, and cloned into the mammalian expression plasmid pcDNA3.1 (Invitrogen, Tokyo, Japan) as described before (20). Myr-Akt1 in pUSEamp(+), an expression vector for myristoylated Akt, was obtained from Upstate (Lake Placid, NY). The gene expression vectors were introduced into U251MG cells using LipofectAMINE Plus reagent (Invitrogen) according to the directions from the manufacturer. Stable transfectants were screened by culturing with 500 µg/mL G418. The cloned G418-resistant lines were then screened for expression of CRT. Two independent clonal cell lines (CRT-M5 and CRT-M6) found to express high levels of CRT upon immunoblot analysis were selected and used for the experiments.

Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. Apoptosis was detected by flow cytometry with the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method (23) using an Apop Tag Plus fluorescein in situ apoptosis detection kit (Chemicon International).

Immunoblot analysis. Cultured cells were harvested and lysed in lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 130 mmol/L NaCl, and 1% NP40 including protease inhibitors (20 µmol/L amidinophenyl methanesulfonyl fluoride, 50 µmol/L pepstatin, and 50 µmol/L leupeptin)]. Protein samples were subjected to SDS-PAGE and then transferred to a nitrocellulose membrane as described (21). The membrane was blocked and then incubated with the primary antibody in TBS containing 0.05% Tween 20. The blots were coupled with the peroxidase-conjugated secondary antibodies, washed, and then developed using the ECL chemiluminescence detection kit (GE Healthcare Bioscience, Tokyo, Japan) according to the instructions from the manufacturer.

Cell survival assay and analysis. The cells were trypsinized in a 0.05% trypsin/1 mmol/L EDTA solution and replated in specified numbers into 60-mm dishes for determination of colony-forming ability (24). One day after, these dishes were irradiated with a dose of 0 to 10 Gy. A 60Co source was used for the {gamma}-irradiation of cells. After 14 days of incubation, the contents of the dishes were stained with a Giemsa stain solution (Muto Pure Chemicals, Tokyo, Japan), colonies with >50 cells were counted, and the radiation-surviving fraction (plating efficiency of experimental group/plating efficiency of control group) was determined. Survival curves were generated by combining data from four independent experiments in accordance with linear-quadratic fitting (KaleidaGraph software 4.0).

Protein phosphatase assay. Protein Ser/Thr phosphatase activity was assayed photometrically using Ser/Thr Phosphatase Assay Kit 1 (Upstate), according to the directions from the manufacturer. The activity was assayed in the presence or absence of 10 nmol/L okadaic acid, and the okadaic acid–sensitive activity was estimated as PP2A-specific activity. The phosphopeptide (R-K-pT-I-R-R) was used as a phosphatase substrate. Protein concentrations were determined using a BCA assay kit (Pierce, Rockford, IL).

Northern blot analysis. The full-length rat PP1a catalytic subunit and PP2A catalytic {alpha} cDNAs were generously provided by Dr. Kunimi Kikuchi (Hokkaido University, Hokkaido, Japan; refs. 25, 26). A PstI-SmaI fragment of 600 bp and EcoRI-PvuII fragment of 680 bp were prepared from the cDNAs of PP1ac and PP2Ac{alpha}, respectively, and used as cDNA probes. The probes were labeled with 32P using a Random Primer Labeling kit (Takara Biomedicals, Shiga, Japan). The isolation of cytoplasmic RNA and Northern blotting were essentially done as described before (27).

Assays for release and uptake of Ca2+ in the cell. For the 45Ca2+ release assay, cells were cultured for 48 hours with medium containing 45Ca2+ (1 µCi/mL). After washing with Ca2+-free Earle's balanced salt solution (EBSS; Invitrogen) containing 3 mmol/L EGTA, cells were detached from the culture plates with trypsinization buffer (0.25% trypsin and 0.02% EDTA in EBSS), and the cell suspensions were preincubated in Ca2+-free EBSS at 37°C for 3 minutes and sequentially stimulated with thapsigargin (0.1 µmol/L), ionomycin (2 µmol/L), and monensin (2 µmol/L). The cell suspensions were collected 5 minutes after the addition of each reagent and centrifuged. The radioactivity released from the cells was measured in the supernatant. Cell pellets were lysed and protein amounts were determined using a BCA assay kit (Pierce). 45Ca2+ release was expressed as the cpm subtracted from those recovered in the preceding collection, and normalized to the protein in the corresponding cell pellets. The uptake of Ca2+ was measured radiometrically using the Millipore filtration technique as described previously (21) with a slight modification. The cells were irradiated (5 Gy) for the periods indicated, then washed with EBSS and cultured for 10 minutes in EBSS containing 45Ca2+ (5 µCi/mL). Cells were detached from the culture plates by trypsinization buffer, and the cell suspension was filtered through a 0.45-µm nitrocellulose filter (Bio-Rad, Tokyo, Japan) under vacuum conditions. The filters were rinsed twice with 0.5 mL washing buffer [10 mmol/L HEPES (pH 7.4), 150 mmol/L KCl, 2 mmol/L EGTA, and 2.5 mmol/L MgCl2]. 45Ca2+ uptake was calculated by measuring the radioactivity and standardized using protein concentrations.

Measurement of intracellular free calcium. The cytoplasmic free Ca2+ concentration, [Ca2+]i, was measured with a dual-excitation wavelength spectrofluorophotometer (RF-5500, Shimadzu, Kyoto, Japan) using the fluorescent Ca2+ indicator Fura-2 tetra (acetoxymethyl) ester (Fura-2-AM) essentially as described previously (21).

Luciferase activity assay. Luciferase reporter constructs for the rat PP2Ac{alpha} gene promoter [i.e., pGL3-pro-PP2Ac, pGL3-pro-PP2Ac (C3), and pGL3-pro-PP2Ac (C3-Mut/C)] were prepared using the reporter vector pGL3-Basic (Stratagene, Tokyo, Japan) as described previously (27). Each vector was transfected into cells by using LipofectAMINE 2000 (Invitrogen) according to the instructions from the manufacturer. After 24 hours of transfection, cells were treated with thapsigargin (5 µmol/L) or BAPTA-AM (10 µmol/L) or left untreated for the periods indicated in the text. Then, luciferase activity was assayed with cellular extracts by using a Dual-Luciferase Reporter Assay System (Promega, Tokyo, Japan).

Statistical analysis. Statistical analysis was done using Student's t test or ANOVA (StatView software). Significance was set at P < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Radiosensitivity and expression levels of CRT in human glioma cell lines. To investigate whether the expression level of endoplasmic reticulum chaperones differs in accordance with the radiosensitivity of glia-derived malignant cells, human neuroglioma (H4) and glioblastoma (U251MG and T98G) cells were selected as different categories of glioma. First, to examine the radiosensitivity of each cell line, the colony-forming ability of the cells after {gamma}-irradiation (0-10 Gy) was evaluated, as described in Materials and Methods. As shown in Fig. 1A , H4 exhibited a significant decrease in the surviving fraction after {gamma}-irradiation, compared with U251MG and T98G cells. These results indicate that H4 cells are highly susceptible to irradiation compared with U251MG and T98G cells, suggesting that radiosensitivity differs in different categories of glioma. In Fig. 1B, the expression of endoplasmic reticulum resident chaperones or proteins, such as CRT, calnexin, Grp94, and Erp57, was examined by immunoblot analysis using specific antibodies, and compared between the cell lysate samples. The level of CRT was apparently higher in H4 cells than U251MG or T98G cells. On the other hand, the expression levels of calnexin, Grp94, and Erp57 did not show a significant difference among the cell types.


Figure 1
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  		Figure 1. The expression of CRT is up-regulated in radiosensitive neuroglioma H4 cells. A, radiosensitivity was evaluated in human neuroglioma (H4) and glioblastoma (T98G and U251MG) cells by colony-forming assay after {gamma}-irradiation (5 Gy) as described in Materials and Methods. {blacksquare}, H4; bullet, T98G; {blacktriangleup}, U251MG. Points, mean of four experiments; bars, SD. *, P < 0.01 versus same dose of irradiation for U251MG and T98G. B, expression levels of endoplasmic reticulum chaperones [i.e., CRT, calnexin (CNX), Grp94, and Erp57] were examined in H4, T98G, and U251MG cells by immunoblot analysis using specific antibodies as described in Materials and Methods.

 
Establishment of CRT gene–overexpressing U251MG cells. To investigate the biological significance of the level of CRT to the radiosensitivity of glioma cells, a CRT gene expression vector was constructed and introduced into U251MG cells as described in Materials and Methods. U251MG cells were chosen because they expressed a relatively low level of CRT compared with H4 cells (Fig. 1B). After screening by culturing with G418, the expression level of CRT was characterized immunologically in the G418-resistant transfectants. Two transfectants (CRT-M5 and CRT-M6) expressing high levels of CRT were established and used in subsequent experiments. Figure 2A shows that the expression of CRT increased in CRT-M5 and CRT-M6 cells compared with the parental and mock-transfected (Vector8) U251MG cells. The transfection had no apparent effect on the expression of other endoplasmic reticulum chaperones, such as calnexin and Grp94 (data not shown). Next, the intracellular distribution of CRT was examined by indirect immunofluorescence microscopy, as shown in Fig. 2B. The immunoreactivity for CRT distributed in a perinuclear granular pattern in all cases, including the control and gene-transfected cells, although the signal intensity was increased in the transfectants compared with the control cells (arrows).


Figure 2
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  		Figure 2. Overexpression of CRT enhances radiation-induced cell death in U251MG cells. A, U251MG cells were transfected with the expression vector for the CRT gene, and antibiotic-resistant cell lines were established as described in Materials and Methods. The expression level of CRT was examined by immunoblot analysis in parental cells and cells transfected with a mock vector (Vector8) and the CRT gene expression vector (CRT-M5 and CRT-M6). B, the intracellular distribution of CRT was examined by indirect immunofluorescence (IF) microscopy using a specific antibody in control and gene-transfected cells. C, radiosensitivity was evaluated based on colony-forming ability after {gamma}-irradiation (5 Gy) as described in Materials and Methods in control and CRT gene–transfected cells. {blacktriangleup}, Vector8; bullet, CRT-M5; {blacksquare}, CRT-M6. Points, mean of at least four experiments; bars, SD. *, P < 0.01 versus same dose of irradiation (10 Gy) for Vector8. D, control (Vector8) and gene-transfected (CRT-M5) cells were irradiated (5 Gy) for 10 minutes. Then, at 72 and 96 hours postirradiation, DNA double-strand breaks were detected by the TUNEL method as described in Materials and Methods.

 
Effect of overexpression of CRT on radiosensitivity of U251MG cells. To evaluate the effect of overexpressed CRT on radiosensitivity in U251MG cells, colony-forming ability was examined after 14 days of {gamma}-irradiation in control (Vector8) and CRT-overexpressing (CRT-M5 and CRT-M6) cells (Fig. 2C). The colony-forming ability markedly decreased in CRT-overexpressing cells compared with control cells. These results indicate that overexpression of CRT enhances the radiation-induced cell damage in U251MG cells. To further characterize the enhanced radiation-induced cell damage in CRT-overexpressing cells, apoptotic characteristics were examined by TUNEL assay as described in Materials and Methods (Fig. 2D). Among control cells (Vector8), TUNEL-positive cells bearing DNA-strand breaks appeared after 72 hours of {gamma}-irradiation, and then decreased after 96 hours. In contrast, among CRT-overexpressing cells (CRT-M5), the TUNEL-positive cells appeared after 72 hours of {gamma}-irradiation, and were still detectable 96 hours later. In the assay, some damaged cells were detached from culture plates and removed during the washing step, and the remaining cells attached to the plates were examined, especially after 96-hour exposure to irradiation. As a result, the population of less-damaged surviving clonogens (TUNEL-negative) might be increased in controls than in CRT-overexpressing cells after 96 hours. These results suggest that overexpression of CRT enhances radiation-induced apoptosis, resulting in the enhanced suppression of the ability of U251MG cells to form colonies after {gamma}-irradiation.

Overexpression of CRT suppresses Akt/protein kinase B activity after {gamma}-irradiation. The Akt pathway is known as a pivotal cell survival signal in the cell (28). We previously reported that overexpression of CRT suppressed Akt activity during the cardiac differentiation of H9c2 cells (20). Therefore, we also focused on the Akt pathway in CRT-overexpressing cells treated with irradiation. In Fig. 3A , the phosphorylation status of Akt was examined in control and CRT-overexpressing cells treated with {gamma}-irradiation by immunoblot analysis using the antibodies against phosphorylated Akt. In control cells, the levels of Akt phosphorylated at both Ser473 and Thr309 were increased at 3 hours after the irradiation. On the other hand, in CRT-overexpressing cells, levels of phosphorylated Akt were unchanged after the irradiation. To confirm the functional link between phosphorylation status and activity in Akt, the cells were treated with or without {gamma}-irradiation (5 Gy), and Akt activity was examined after 3 hours, by assessing the phosphorylation of GSK-3{alpha}/ß, a substrate of Akt kinase, as described in Materials and Methods (Fig. 3B). Akt was not activated by {gamma}-irradiation in CRT-overexpressing cells, in spite of the marked activation of Akt in control cells treated with {gamma}-irradiation. These results are consistent with the results for the phosphorylation status of Akt in control and CRT-overexpressing cells treated with {gamma}-irradiation.


Figure 3
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  		Figure 3. Radiation-induced activation of Akt is suppressed in CRT-overexpressing cells. A, the phosphorylation status of Akt was examined in control (Vector8) and CRT-overexpressing (CRT-M5) cells treated with {gamma}-irradiation (5 Gy) by immunoblot analysis using antibodies against Akt phosphorylated at Ser473 and Thr309 as described in Materials and Methods. B, Akt activity was examined in control and CRT-overexpressing cells at 3 hours after the treatment with or without {gamma}-irradiation (5 Gy) by estimating the phosphorylation of GSK-3{alpha}/ß as described in Materials and Methods. C, CRT-overexpressing cells (CRT-M5) were transiently transfected with a mock vector or the expression vector for the myristoylated Akt gene (Myr-Akt1) to overexpress active Akt as described in Materials and Methods. The expression of myristoylated Akt was examined by immunoblot (IB) analysis using the anti-myc antibody in cells transfected with the mock vector (CRT-M5-Mock) and Myr-Akt1 (CRT-M5-Myr-Akt-myc). *, endogenous Akt. D, after 24 hours of transfection with the mock or Myr-Akt1, the cells were irradiated (5 Gy); then, at 72 hours postirradiation, cell damage was estimated by the TUNEL method as described above.

 
Constitutive activation of Akt protects against radiation-induced apoptosis in U251MG cells. To investigate whether the activation of Akt contributes to cellular protection against irradiation in U251MG cells, an expression vector for myristoylated Akt (Myr-Akt1) was introduced into the CRT-overexpressing cells as described in Materials and Methods, to generate cells in which Akt is constitutively activated. After 24 hours of transfection, the expression of Myr-Akt1 was detected by immunoblot analysis using the anti-myc antibody (Fig. 3C). Then, the cells were treated with {gamma}-irradiation (5 Gy), and apoptosis was estimated after 72 hours, by the TUNEL assay as described above. As shown in Fig. 3D, although TUNEL-positive cells were detected among CRT-overexpressing cells, numbers were diminished in the cells transfected with the Myr-Akt expression vector, indicating that activation of Akt plays an important role in cytoprotection against irradiation. This also suggests that a suppressed Akt pathway is a cause of the enhanced susceptibility to radiation-induced apoptosis in CRT-overexpressing cells.

PP2A is up-regulated in CRT-overexpressing U251MG cells. Ionizing radiation is known to trigger the Akt pathway through the activation of epidermal growth factor receptor families and phosphatidylinositol 3-kinase (PI3K; refs. 29, 30). To establish whether overexpression of CRT affects the activity of PI3K, a signaling molecule upstream of Akt, we examined PI3K activity in control and CRT-overexpressing cells treated with {gamma}-irradiation (5 Gy). However, PI3K activity was not suppressed in CRT-overexpressing cells after the irradiation, but rather was slightly increased, compared with that in control cells (data not shown). This suggests that the suppression of the radiation-induced activation of Akt in CRT-overexpressing cells is due to enhanced inactivation of Akt by PP2A (20, 31). In fact, the phosphorylation of Akt was apparently up-regulated in CRT-overexpressing cells treated with okadaic acid (100 nmol/L), a specific inhibitor of PP2A (data not shown). To investigate whether PP2A is influenced by overexpression of CRT, PP2A activity was assayed with cell lysates from control and CRT-overexpressing cells treated with or without {gamma}-irradiation (5 Gy). In Fig. 4A , PP2A activity was always greater in CRT-overexpressing cells rather than controls, although the activity was slightly suppressed by irradiation in both cells. Next, the expression of phosphatases was examined at the level of the protein of transcription by conducting immunoblot and Northern blot analyses, respectively. In Fig. 4B, the protein level of PP2Ac{alpha} increased in CRT-overexpressing cells, compared with controls. However, no significant change was observed in the levels of PP2A regulatory protein 65 (PP2A-RP65), PP1a, and GAPDH between control and CRT-overexpressing cells. Furthermore, the expression of PP2Ac{alpha} was up-regulated at the transcriptional level in CRT-overexpressing cells (Fig. 4C). Together, these results indicate that PP2Ac{alpha} is transcriptionally up-regulated in CRT-overexpressing cells. They also support that PP2A activity is increased by the up-regulated expression of PP2Ac{alpha} in the CRT-overexpressing cells.


Figure 4
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  		Figure 4. The expression of PP2A is up-regulated in CRT-overexpressing cells. A, the activity of PP2A was assayed in control (Vector8) and CRT-overexpressing (CRT-M5) cells at 3 hours after the treatment with or without {gamma}-irradiation (5 Gy). Columns, mean of at least three experiments; bars, SD. *, P < 0.05 versus untreated Vector8 cells. #, P < 0.05 versus Vector8 cells treated with irradiation. B, the protein levels of protein Ser/Thr phosphatases (i.e., PP2Ac{alpha}, PP2A-RP65, and PP1a) and GAPDH were examined by immunoblot analysis using specific antibodies in control and CRT-overexpressing cells at 3 hours after the treatment with or without {gamma}-irradiation (5 Gy). C, transcriptional expression of PP2Ac{alpha}, PP1a, and GAPDH was examined in control and CRT-overexpressing cells by Northern blot analysis as described in Materials and Methods.

 
Ca2+ homeostasis and responses to irradiation are altered in CRT-overexpressing U251MG cells. We previously reported that the gene expression of PP2Ac was controlled by altered Ca2+ homeostasis in myocardiac H9c2 cells (27). To investigate the effect of CRT overexpression on cellular Ca2+ homeostasis, intracellular Ca2+ pools were characterized in control and CRT-overexpressing cells. After 48 hours of loading with 45Ca2+, the cells were washed and resuspended in Ca2+-free buffer. Unidirectional fluxes to the extracellular medium after stimulation with several Ca2+ modulators were then measured as described in Materials and Methods. Thapsigargin, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), ionomycin, a Ca2+ ionophore, and monensin, another ionophore affecting acidic stores, were used to stimulate the cellular Ca2+ pools (Fig. 5A ). The results showed that cellular Ca2+ contents were apparently increased mainly in the thapsigargin-sensitive Ca2+ pools of CRT-overexpressing cells, compared with controls, suggesting that Ca2+ stores in the endoplasmic reticulum were increased in CRT-overexpressing cells. We next examined [Ca2+]i in control and CRT-overexpressing cells after treatment with irradiation (5 Gy). As shown in Fig. 5B, [Ca2+]i was markedly higher in CRT-overexpressing cells than controls at 30 minutes after {gamma}-irradiation (5 Gy), although it increased by {gamma}-irradiation in both cells.


Figure 5
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  		Figure 5. Effect of CRT overexpression on intracellular Ca2+ pools and Ca2+ responses to irradiation in U251MG cells. A, control (U251MG and Vector8) and CRT-overexpressing (CRT-M5 and CRT-M6) cells were cultured with 45Ca2+ (1 µCi/mL) for 48 hours, then detached from the culture dish and resuspended in Ca2+-free EBSS. Cell suspensions were preincubated at 37°C for 3 minutes, and sequentially stimulated with thapsigargin (0.1 µmol/L), ionomycin (2 µmol/L), and monensin (2 µmol/L). The cell suspensions were collected 5 minutes after the addition of each reagent and centrifuged. The radioactivity released from the cells was measured in the supernatant. Cell pellets were lysed and protein amounts were determined using a BCA assay kit (Pierce). Columns, mean of the cpm subtracted from those recovered in the preceding collection, and normalized to the protein in the corresponding cell pellets; bars, SD. *, P < 0.01 versus U251MG or Vector8 cells treated with thapsigargin. B, control and CRT-overexpressing cells were treated with {gamma}-irradiation (5 Gy). [Ca2+]i was measured after the periods indicated using Fura2-AM as described in Materials and Methods. Columns, mean of at least four experiments; bars, SD. *, P < 0.01 versus U251MG or Vector8 cells at 30 minutes after the irradiation. C, control (Vector8) and CRT-overexpressing (CRT-M5) cells were pretreated with or without xestospongin C (5 mmol/L) or Ni2+ (5 mmol/L) for 5 minutes, then treated with {gamma}-irradiation (5 Gy). [Ca2+]i was measured at 30 minutes after the irradiation using Fura2-AM as described above. Columns, mean of at least four experiments; bars, SD. *, P < 0.01 versus Vector8 cells at 30 minutes after the irradiation. **, P < 0.01 versus CRT-M5 cells at 30 minutes after the irradiation. D, control and CRT-overexpressing cells were treated with {gamma}-irradiation (5 Gy). Then, cells were incubated for 10 minutes with 45Ca2+ (5 µCi/mL) at the periods indicated after irradiation. After washing with EBSS, the cells were harvested and 45Ca2+ uptake was measured as described in Materials and Methods. Columns, mean of three independent experiments; bars, SD. *, P < 0.01 versus U251 MG or Vector8 cells at 30 minutes after the irradiation.

 
In U937 cells lacking inositol 1,4,5-triphosphate receptor (IP3R) 1, the irradiation-induced increase of [Ca2+]i is significantly suppressed, suggesting that Ca2+ stores in the endoplasmic reticulum plays an important role in the irradiation-induced increase of [Ca2+]i (32). To investigate whether the IP3R-dependent release of Ca2+ from the endoplasmic reticulum is involved in the mechanism, the effect of xestospongin C (5 µmol/L), an inhibitor of IP3R (21) on the irradiation-induced increase of [Ca2+]i in control and CRT-overexpressing cells was examined. As shown in Fig. 5C, with xestospongin C, the irradiation-induced increase of [Ca2+]i was apparently suppressed in both control and CRT-overexpressing cells; however, the levels were still higher than those of nontreated cells. On the other hand, when the cells were pretreated with Ni2+ (5 mmol/L) to block Ca2+ influx from the extracellular space through Ca2+ channels and Na+/Ca2+ exchangers in the plasma membrane (33), the irradiation-induced increase of [Ca2+]i was significantly suppressed to reach the levels of nontreated cells. To further investigate the effect of CRT overexpression on irradiation-induced Ca2+ influx from the extracellular spaces, 45Ca2+ uptake was examined in control and CRT-overexpressing cells after treatment with irradiation as described in Materials and Methods. As shown in Fig. 5D, the uptake of 45Ca2+ was apparently enhanced in CRT-overexpressing cells after 30-minute exposure to irradiation, compared with controls, although the uptake increased in both control and CRT-overexpressing cells. Together, these results indicate that the IP3R-sensitive release of Ca2+ and Ca2+ influx from the extracellular space both play important roles in the mechanism of the irradiation-induced increase of [Ca2+]i in the cells, and were enhanced by the overexpression of CRT.

The gene promoter activity of PP2Ac{alpha} is up-regulated in CRT-overexpressing cells through cytoplasmic free Ca2+. In the PP2Ac{alpha} gene promoter, cyclic AMP response element (CRE) is a pivotal transcription site, through which the activation is regulated by [Ca2+]i, although both the GC-box and CRE additively contribute to the basal promoter activity (27). To investigate whether the CRE-dependent gene expression of PP2Ac{alpha} is influenced by the overexpression of CRT, the gene promoter activity was examined by assaying the luciferase activity as described in Materials and Methods. Control and CRT-overexpressing cells were transfected with various luciferase vectors [i.e., pGL3-pro-PP2Ac, which contains the entire promoter sequence of PP2Aca (–1,209 to +258); pGL3-pro-PP2Ac (C3), which contains a CRE but no GC-box (–145 to +258); and pGL3-pro-PP2Ac (C3-Mut/C) in which the CRE is disabled by mutation (–145 to +258)]. After 24 hours of transfection, cell lysates were prepared and subjected to an assay for luciferase activity. As shown in Fig. 6A , the level of activity was higher in CRT-overexpressing cells than controls when either pGL3-pro-PP2Ac or pGL3-pro-PP2Ac (C3) was transfected. However, in the case of pGL3-pro-PP2Ac (C3-Mut/C), no activity was detected in the control or CRT-overexpressing cells. These results indicate that the activity of the PP2Ac{alpha} promoter is up-regulated by overexpression of CRT through the CRE. Next, to investigate whether the enhancing effect of overexpressed CRT on the PP2Ac{alpha} promoter is regulated through the change in [Ca2+]i, we examined the promoter activity in control and CRT-overexpressing cells treated with Ca2+ modulators such as thapsigargin and BAPTA-AM. Thapsigargin and BAPTA-AM, a cell-permeable Ca2+ chelator, were used to increase and decrease [Ca2+]i in the cell, respectively (27). In Fig. 6B, control cells transfected with pGL3-pro-PP2Ac (C3) were treated with or without thapsigargin (5 µmol/L for 2 hours), and then the luciferase activity was assayed as described above. The results showed that the activity of the PP2Ac{alpha} promoter was up-regulated in control cells by the increase of [Ca2+]i with thapsigargin treatment. These findings are consistent with those in the case of myocardiac H9c2 cells (27). In Fig. 6C, CRT-overexpressing cells transfected with pGL3-pro-PP2Ac (C3) were treated with or without BAPTA-AM (10 µmol/L for 2 hours), and then the luciferase activity was assayed as described above. The results showed that the activity of the PP2Ac{alpha} promoter was down-regulated in CRT-overexpressing cells by the decrease of [Ca2+]i with BAPTA-AM treatment. Taken together, these results indicate that the activity of the PP2Ac{alpha} promoter was regulated by [Ca2+]i through CRE, and overexpression of CRT up-regulates the promoter activity through the altered homeostasis of Ca2+ in U251MG cell.


Figure 6
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  		Figure 6. The promoter activity of the PP2Ac{alpha} gene is increased in CRT-overexpressing cells through CRE, and is regulated via altered Ca2+ homeostasis. A, left, schematic representation of luciferase vector constructs for the rat PP2Ac{alpha} promoter [i.e., pGL3-pro-PP2Ac, pGL3-pro-PP2Ac (C3), and pGL3-pro-PP2Ac (C3-Mut/C)]. Each luciferase vector construct was generated as described in Materials and Methods. The CRE site (–26) was mutated in pGL3-pro-PP2Ac (C3-Mut/C). Right, luciferase activity of the vector constructs for the PP2Ac{alpha} gene promoter in control and CRT-overexpressing cells. The cells were transiently transfected with the PP2Ac{alpha} promoter-luciferase gene fusion plasmids. After 24 hours of transfection, luciferase activity was assayed with cellular extracts as described in Materials and Methods. Columns, mean of at least three experiments; bars, SD. *, P < 0.01 versus Vector8 cells transfected with pGL3-pro-PP2Ac. #, P < 0.01 versus Vector8 cells transfected with pGL3-pro-PP2Ac (C3). Control and CRT-overexpressing cells were transiently transfected with pGL3-pro-PP2Ac (C3). After 24 hours of transfection, control cells were treated with 5 µmol/L thapsigargin for 2 hours (B) and CRT-overexpressing cells were treated with 10 µmol/L BAPTA-AM for 2 hours (C). Then, luciferase activity was assayed with cell nuclear extracts as described in Materials and Methods. Columns, mean of at least three experiments; bars, SD. *, P < 0.05 versus untreated Vector8 cells. #, P < 0.05 versus untreated CRT-M5 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we focused on functions of endoplasmic reticulum chaperones in malignant gliomas to obtain a new perspective for therapy. To evaluate the association between radiosensitivity and the expression of the molecular chaperones in the endoplasmic reticulum, we used three cultured glia-derived cell lines, i.e., two glioblastoma cell lines (U251MG and T98G) and a neuroglioma cell line (H4). We found that H4 cells were more radiosensitive than U251MG and T98G cells, and the expression level of CRT was specifically higher in H4 than in U251MG or T98G cells (Fig. 1A and B). These findings suggested that the expression level of CRT seems to be related with radiosensitivity in gliomas. Although the expression of CRT is induced by radiation (34, 35), the function of CRT in radiosensitivity is not well understood. To investigate the significance of CRT in radiosensitivity, we established a CRT-overexpressing cell line using U251MG, and examined the effect of the overexpression on the radiosensitivity. It was found that CRT-overexpressing cells showed greater radiosensitivity than control cells, and enhanced radiation-induced apoptosis.

Radiation-induced apoptosis is controlled by various mechanisms, such as the p53 status, the Bcl-2 gene family, the caspase pathways, and so forth (3). Zhao et al. (36) reported that the DNase activation pathway through p53 and Ca2+-mediated DNase {gamma} pathway were involved in the regulation of radiation-induced apoptosis in Molt-4 cells. However, in U251MG cells, the enhancement of radiation-induced apoptosis by CRT may not be due to a p53-dependent mechanism, because of the mutation in the p53 gene (37). On the other hand, apoptosis is regulated by several signaling pathways, including the mitogen-activated protein kinases and Akt pathways (38). The Akt signaling pathway is an important cell survival and antiapoptotic signal in radiation-induced apoptosis (39, 40). In this study, we found that the pathway was significantly suppressed in the CRT-overexpressing cells after {gamma}-irradiation. Moreover, we found that the expression of PP2Ac{alpha} was significantly increased in overexpressing cells compared with control cells. PP2A is known to modulate the activities of several kinases, and is responsible for the dephosphorylation and inactivation of Akt (20, 31). Therefore, these results suggest that Akt signaling was suppressed by the up-regulation of PP2Ac{alpha} expression in CRT-overexpressing cells treated with {gamma}-irradiation.

The Ca2+ concentration of the endoplasmic reticulum or cytoplasm is thought to be a key determinant of radiation-induced apoptosis (9, 11). CRT is a Ca2+-binding molecular chaperone in the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum Ca2+ storage capacity (16). In this study, we found that, in CRT-overexpressing cells, the thapsigargin-sensitive Ca2+ pool was increased, and the levels of [Ca2+]i and Ca2+ influx from the extracellular spaces were both up-regulated, especially after ionizing irradiation. This indicated that CRT overexpression significantly influenced the regulatory mechanism of Ca2+ homeostasis in U251MG cells, although the precise mechanism of CRT action has not yet been fully clarified. In addition, we found that the gene promoter of PP2Ac{alpha} was regulated through the change in [Ca2+]i (Fig. 6B and C). These results strongly suggest a mechanical link between down-regulated Akt signaling and altered Ca2+ homeostasis in CRT-overexpressing cells, and it is consistent with our finding that the antiapoptotic activity of Akt is down-regulated by Ca2+ in myocardiac H9c2 cells (27).

Concerning CRT and apoptosis, Nakamura et al. (10) reported that overexpression of CRT resulted in an increase in the sensitivity of HeLa cells to both thapsigargin- and staurosporine-induced apoptosis. The authors suggested that overexpression of CRT affects the communication between the endoplasmic reticulum and mitochondria to increase the sensitivity to apoptosis via the altered Ca2+ homeostasis, and this has been supported by the study of Arnaudeau et al. (41). We also reported that overexpression of CRT influences the function of SERCA2a under oxidative stress, leading to an alteration of Ca2+ homeostasis (42) and to enhanced susceptibility to apoptosis (20, 21). These findings suggest that the expression level of CRT is well correlated with the susceptibility to apoptosis. In contrast, overexpression of CRT provided resistance to oxidant-induced cell death in renal epithelial LLC-PK1 cells treated with iodoacetamide (43), tert-butylhydroperoxide (44), or hydrogen peroxide (45). In the neuroblastoma x glioma hybrid cell line NG-108-15, suppression of CRT by an antisense oligonucleotide increased sensitivity to ionomycin-induced cytotoxicity (46, 47). The function of CRT in the regulation of apoptosis may differ in specific cell types, and is still controversial. Although almost all studies suggest regulatory functions of CRT in the susceptibility to apoptosis, further investigation is required to clarify the relevance to CRT in cancer biology.

In conclusion, we found that the expression level of CRT was well correlated with radiosensitivity in glioma cell lines, and CRT modulated the radiosensitivity of glioblastoma cell lines by affecting the cell survival pathway of Akt signaling through alterations of Ca2+ homeostasis and responses in the endoplasmic reticulum.


   Acknowledgments
 
Grant support: The 21st Century Center of Excellence program from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (T. Kondo and Y. Ihara).

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 Midori Ikezaki and Akiko Emura for technical assistance.

Received 11/30/05. Revised 5/23/06. Accepted 6/21/06.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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