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Cell Growth & Differentiation Vol. 13, 237-246, May 2002
© 2002 American Association for Cancer Research

Protein Kinase C{delta} Overexpression Enhances Radiation Sensitivity via Extracellular Regulated Protein Kinase 1/2 Activation, Abolishing the Radiation-induced G2-M Arrest1

Yoon-Jin Lee, Jae-Won Soh, Nicholas M. Dean, Chul-Koo Cho, Tae-Hwan Kim, Su-Jae Lee2 and Yun-Sil Lee2,,3

Laboratory of Radiation Effect, Korea Cancer Center Hospital, Seoul 139-706, Korea [Y-J. L., C-K. C., T-H. K, S-J. L., Y-S. L.]; Division of Molecular Life Sciences, Ewha Womans University, Seoul 120-750, Korea [Y-J. L.]; Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008 [N. M. D]; and Department of Biochemistry and Molecular Biophysics and Herbert Comprehensive Cancer Center, Columbia University, New York, New York 10032 [J-W. S]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Protein kinase C (PKC) has been widely implicated in regulation ofcell growth/cell cycle progression and apoptosis. However,the role of PKC{delta} in radiosensitivity and cell cycle regulation remains unclear. Overexpression of PKC{delta} increased Ca2+-independent PKC activity without altering other PKC isoforms (PKC{alpha}, -ß1, -{epsilon}, and -{zeta}), and extracellular regulated protein kinase (ERK) 1/2 activity was also increased in PKC{delta}-specific manner. A clonogenic survival assay showed that PKC{delta}-overexpressed cells had more radiosensitivity and pronounced induction of apoptosis than control cells. Flow cytometric analysis revealed that PKC{delta} made the cells escape from radiation-induced G2-M arrest. Moreover, p53 and p21Waf induction by radiation were higher in PKC{delta}-overexpressed cells than control cells, and PKC{delta}-mediated apoptosis was reduced, when radiation-induced ERK1/2 activity was inhibited by PD98059. Furthermore, PKC{delta} antisense and rottlerin, PKC inhibitor-abrogated PKC{delta}-mediated radiosensitivity and reduced ERK1/2 activity to the control vector level. These results demonstrated that PKC{delta} overexpression enhanced radiation-induced apoptosis and radiosensitivity via ERK1/2 activation, thereby abolishing the radiation-induced G2-M arrest and finally apoptosis.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
PKC4 is a multigene family of serine/threonine kinases that are central to many signal transduction pathways, involving cellular growth, transformation, and differentiation. PKC-family genes encode at least 12 distinct isoforms of lipid-regulated serine/threonine kinases, and PKC isoforms can be classified into three groups, based on their structure and cofactor requirement: (a) classical PKCs (-{alpha}, -ß1, -ßII, -{gamma}), which can be activated by DAG or calcium; (b) novel PKCs (-{delta}, -{epsilon}, -{eta}, -{theta}, -µ), which can be activated by DAG but not by calcium; and (c) atypical PKCs (-{zeta}, -{iota}), which are not responsive to either DAG or calcium (1, 2, 3) .

The complexity in the regulation of PKC{delta} activity, which includes not only regulation by DAG and tyrosine phosphorylation but also association to tyrosine kinases, suggests an important role of this PKC isozyme in proliferation and cell death (4 , 5) . Proteolytic cleavage and generation of an active CAT fragment of PKC{delta} has been visualized after treatment of DNA-damaging agent (6) , and the cleaved CAT fragment of PKC{delta} is constitutively activated and is sufficient to induce apoptosis when overexpressed (7) . The inhibition of apoptosis by overexpression of Bcl-2 or Bcl-XL is associated with a block of PKC{delta} cleavage (8 , 9) . Therefore, it is likely that PKC{delta}-mediated apoptosis may proceed by two distinct mechanisms, namely proteolytic cleavage (after DNA damage) and allosteric activation (after direct activation).

Phorbol esters such as TPA and phorbol 12,13-dibutyrate activates the Raf-MEK-ERK pathway. Treatment of cells with TPA results in the activation of c-Raf and MAPK within minutes (10) , and DN mutant PKC decreases the total protein level of MAPK (11) , which suggests the involvement of PKC in the signaling pathway leading to MAPK activation. Whereas it is generally accepted that the activation of the ERK cascade leads to mitogenic effect (12 , 13) , MAPK signaling cascades are also involved in many cellular responses, including apoptosis. For example, MAPK/ERK inhibitor PD98059 blocks asbestos-induced apoptosis (14) , and a requirement for ERK in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells and ovarian cell lines (15 , 16) has also been demonstrated. Moreover, persistent activation of ERK1/2 contributes to glutamate-induced oxidative toxicity (17) .

Ionizing radiation activates growth-responsive MAPK cascade (18 , 19) leading to different cell cycle checkpoint control, and this cascade is an important response of the cells to stress. Radiation induces double-strand breaks (dsbs), and the repair of dsbs follows two pathways; nonhomologous recombinational repair (occurring in G1) and homologous recombination repair (20 , 21) . Homologous recombinational repair occurs in G2, when homologous undamaged double-strand DNA is present to serve as a template for repair and leads to fewer mutations than does nonhomologous repair (21) . Therefore, the G2-M checkpoint is essential for the proper repair of DNA damage by ionizing radiation.

In this article, we describe how PKC{delta} overexpression enhanced radiosensitivity and radiation-induced apoptosis, and this occurred via ERK1/2 activation, abolishing the radiation-induced G2-M arrest and finally apoptosis.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Overexpression of PKC{delta} Increased Ca2+-independent PKC Activity and Phospho-EKR1/2 Expression.
To elucidate whether PKC{delta} transfection affected other PKC isoforms, PKC activity and PKC protein expression levels were assessed. As shown in Fig. 1ACitation , Ca2+-independent PKC activity was much higher in PKC{delta}-overexpressing cells, although Ca2+-dependent PKC activity was slightly activated. Western blot analysis also revealed that PKC{delta} protein expression was significantly increased without altering other PKC isoforms (PKC{alpha}, -ß1, -{epsilon}, and -{zeta}) and that PKC{delta} overexpression spontaneously induced its translocation to membrane fraction even without stimuli (data not shown). Phospho-ERK1/2 expression was also increased, whereas phospho-p38 MAPK was down-regulated and phospho-JNK expression was not changed (Figs. 1, B and C)Citation . When transient transfection of PKC-CAT domain PKC{delta}-CAT, the constructs of which contained only the CAT domains with the inhibitory NH2-terminal domains deleted, a similar effect was observed (Fig. 1C)Citation .



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Fig. 1. PKC activity and phospho-EKR1/2 expression were increased in a PKC{delta}-specific manner. A, Ca2+-dependent and -independent PKC activity was determined in Triton X-100 lysates of control vector (pLTR) and PKC{delta}-overexpressing NIH3T3 cells, as described under "Materials and Methods." Assays were performed with extracts from three separate dishes. Error bar, mean ± SE, using a modified PKC{alpha} pseudosubstrate peptide as a phosphate acceptor. Similar results were obtained in two separate independent experiments. B, protein extracts of vector control and PKC{delta}-overexpressed NIH3T3 cells were prepared, separated by SDS-PAGE, and analyzed by Western blot analysis. C, left, NIH3T3 cells were transfected with PKC{delta}-CAT vector and the control vector pcDNA3, and cellular proteins were extracted by cell lysis in PKC extraction buffer. HA-tagged PKC proteins were immunoprecipitated from 300 µg of cell extracts by using 3 µg of an anti-HA antibody and 30 µl of protein G-Sepharose after a 3-h incubation of 4°C. Immune complex kinase reactions were performed at 30°C for 30 min in the presence of 10 µg of the GST-MARCKS substrate and 5 µCi of [{gamma}-32P]ATP. The reaction products were then analyzed by SDS-PAGE and autoradiography. Right, NIH3T3 cells were transfected with PKC{delta}-CAT vector and the control vector pcDNA3, and cellular proteins were extracted by cell lysis, separated by SDS-PAGE, and analyzed by Western blot analysis.

 
Overexpression of PKC{delta} Induced Decreased Clonogenic Survival and Increased Apoptosis after Radiation.
Clonogenic survival assay revealed that PKC{delta}-overexpressed cells were more radiosensitive than nonoverexpressing cells. To confirm these phenomena, transient transfection was used. PKC{delta}-DN mutant transfection induced radioresistance, and PKC{delta}-CAT transfection showed radiosensitive (Fig. 2IA)Citation . To elucidate whether the clonogenic cell death was associated with the induction of apoptosis, nuclear morphological changes were examined using Heochst 33568 staining. A dose-dependent increase of apoptosis after radiation was observed in both PKC{delta}-overexpressing and non-PKC{delta}-overexpressing cells, however, the induction of apoptosis was more pronounced in PKC{delta}-overexpressing cells (Fig. 2B)Citation .



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Fig. 2. Overexpression of PKC{delta} decreased clonogenic survival and increased apoptosis after radiation. A, surviving fraction of vector control and PKC{delta}-overexpressed NIH3T3 cells (top), PKC{delta}-DN [(K->R) point mutation at the ATP binding site] mutant cells (middle), and PKC{delta}-CAT mutant cells (bottom), which constructs contained only the CAT domains, were obtained by colony-forming assay after irradiation. B, DNA fragmentations of PKC{delta}-overexpressing (top) or PKC{delta}-KR mutant (bottom) NIH3T3 cells were measured by Hoechst 33258 staining 48 h after 4- and 8-Gy radiation as described under "Materials and Methods." Error bar, means ± SD from three independent experiments. *, significantly different from control vector cells at P < 0.05.

 
Escape of PKC{delta} Overexpression from Radiation-induced G2-M-Phase Cell Cycle Arrest.
To determine to what extent the cell cycle was affected by radiation, as well as the role of PKC{delta}, the cell cycle progression was examined by propidium iodide staining. In the control, clear G2-M block was observed from 6 h, and the block lasted through 24 h after irradiation. However, in the case of PKC{delta}-overexpressing cells, G2-M block was seen from 12 h after radiation, and it already recovered at 24 h, which suggested that PKC{delta} overexpression shortened the length of radiation-induced G2-M arrest induced by radiation (Fig. 3)Citation .



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Fig. 3. Escape from radiation-induced G2-M phase cell cycle arrest by PKC{delta} overexpression. NIH3T3 cells from control vector and PKC{delta}-overexpressed cells were harvested after incubation for the periods indicated after 5-Gy radiation, and cell cycle distribution was analyzed by a flow cytometer after staining with propidium iodide.

 
Radiation-induced p21Waf Induction Was Less in PKC{delta}-overexpressed Cells.
To determine whether p21Waf induction was involved by inhibiting cyclin/CDK complex, the association of p21Waf with CDK2 or CDK4 was examined. As seen in Fig. 4ACitation , as the amount of p21Waf increased from 6 h after radiation. more p21Waf association with CDK2 and CDK4 was found in the control cells. On the other hand, less association was found in the PKC{delta}-overexpressing cells. Coimmunoprecipitation also showed decreased binding of p21Waf and cyclin A or cyclin B1 in the PKC{delta}-overexpressing cells (Fig. 4B)Citation . These data suggested that PKC{delta} overexpression made the cells escape from the radiation-induced G2-M phase cell cycle arrest.



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Fig. 4. Radiation-induced p21Waf induction was less in PKC{delta}-overexpressing cells. A, indicated time intervals after 5 Gy irradiation, cell lysates (500 mg) of vector control and PKC{delta}-overexpressed NIH3T3 cells were immunoprecipitated (IP) with anti-CDK2 or anti-CDK4 antibodies, and anti-cyclin A or -B antibodies, separated on SDS-polyacrylamide gels, tranferred to nitrocellulose, and incubated with anti-p21WAF1 antibody, followed by enhanced chemiluminescence detection. WB, Western blotting. B, the density of specific band was scanned and quantified. Values are expressed as fold-induction compared with control. A representative data from three independent experiments is shown.

 
G2-M Transition after Radiation Was Increased in the PKC{delta}-overexpressing Cells.
To examine whether PKC{delta}-overexpression also affected radiation-induced G2-M arrest by altering cyclin/CDK activity, Western blotting analyses of cyclin A and -B1 were performed. Fig. 5Citation shows that cyclin A and -B1 expression were increased from 3 h of irradiation in the control cells; however, an even stronger expression was seen in PKC{delta}-overexpressing cells. In addition, CDK2 and CDC2 activities were increased significantly more in the PKC{delta}-overexpressing cells than in the control cells. Coimmunoprecipitation of cyclin A or -B1 with CDC2 also revealed a similar pattern, i.e., a stronger radiation effect in the PKC{delta}-overexpressing cells.



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Fig. 5. G2-M transition after radiation was increased in PKC{delta}-overexpressing cells. At indicated time intervals after 5-Gy irradiation, protein extracts (60 mg) of growing vector control and PKC{delta}-overexpressing NIH3T3 cells were prepared, separated by SDS-PAGE, and analyzed by Western blotting for cyclin A, B, and CDC2. Cell lysates (200 µg) were immunoprecipitated (IP) with anti-CDK2 or CDC2 antibodies, and kinase activity was assayed using histone H1 as a substrate. Or cell lysates (500 µg) were immunoprecipitated (IP) with anti-cyclin A antibody, separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with anti-CDK2 or anti-CDC2 antibodies followed by enhanced chemiluminescence detection. WB, Western blotting.

 
Expressions of Apoptosis-related Proteins in the PKC{delta}-overexpressing Cells after Radiation.
Because p53 protein is known to be an inducer of G1 or G2-M phase arrest and p21Waf expression level was different between the PKC{delta}-overexpressing and control cells (Fig. 4)Citation , p53 protein expression after radiation was examined. Induction of p53 protein was increased from 3 h of irradiation in both of the cell lines; however, there was a more pronounced effect in the PKC{delta}-overexpressed cells. The induction of p21Waf was dependent on p53 protein expression, however, more in the PKC{delta}-overexpressed cells. The above results indicated a good correlation between p53 and p21Waf protein expressions and induction of apoptosis (Fig. 6)Citation .



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Fig. 6. Expressions of apoptosis-related proteins in PKC{delta}-overexpressing cells after radiation. At indicated time intervals after 5-Gy radiation, protein extracts (60 µg) of growing vector control and PKC{delta}-overexpressing NIH3T3 cells were prepared, separated by SDS-PAGE, and analyzed for p21Waf and p53 by Western blot.

 
Radiation-induced ERK1/2 Activation Was Responsible for PKC{delta}-mediated Radiosensitivity.
Because increased expression of phospho-ERK1/2 was detected in the PKC{delta}-overexpressed cells, ERK1/2 activation after irradiation was examined. As shown in Fig. 7ACitation , radiation-induced ERK1/2 activation was more pronounced in the PKC{delta}-overexpressed cells, whereas p38 MAPK activation was decreased. When treated with PD98059, MEK inhibitor, PKC{delta}-mediated apoptosis was returned to the level of the control cells (Fig. 7B)Citation . When treated with SB202190, selective inhibitor of p38 MAPK, induction of apoptosis was not inhibited, thus demonstrating that ERK1/2 activation by PKC{delta} overexpression was responsible for the increased induction of apoptosis.



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Fig. 7. Radiation-induced ERK1/2 activation was responsible for PKC{delta}-mediated radiosensitivity. A, at indicated time intervals after 5-Gy radiation, protein extracts (60 µg) of growing vector control and PKC{delta}-overexpressed NIH3T3 cells were prepared, separated by SDS-PAGE, and analyzed for phospho-ERK1/2, phospho-JNK and phospho-p38 by Western blot. B, DNA fragmentation measured by Hoechst 33258 staining 48 h after 5-Gy radiation with or without pretreatment with 50 µM PD98059 as described under "Materials and Methods." Error bar, mean ± SD from three independent experiments.

 
Treatment with PKC{delta} Antisense or Rottlerin, PKC Inhibitor, Abrogated PKC{delta}-mediated Radiosensitivity.
To examine whether PKC{delta} was directly involved in radiosensitivity, the PKC{delta}-overexpressing cells were treated with PKC{delta} antisense oligonucleotides and PKC inhibitor, rottlerin. When treated with two antisenses (ISIS 11514 and ISIS 11515), a strong inhibition of PKC{delta} was detected in the PKC{delta}-overexpressing cells without altering other PKC isozymes (PKC{alpha}, -ß, -{epsilon}, and -{zeta}). In the case of rottlerin, there was no change in PKC{delta} expression, because it acts only to prevent cytosol-to-membrane translocation of PKC{delta} (22) . Phospho-ERK1/2 expression was inhibited by PKC{delta} antisense and rottlerin, whereas p38 MAPK was not affected by PKC{delta} antisense treatment. Down-regulated p38 by PKC{delta} overexpression did not recover to the control level, which suggested that down-regulation of p38 might be an indirect effect of PKC{delta}. The harvesting of the cells at 24 h of AS or PKC inhibitor treatment may not be enough to recover p38 phosphorylation. In the case of phospho-JNK expression, it was slightly inhibited by rottlerin (Fig. 8A)Citation . When radiation-induced cell cycle distribution was examined after PKC{delta} antisense or rottlerin treatments, radiation-induced G2-M phase arrest was found to be restored to the control vector level (Fig. 8B)Citation . Moreover, increased radiation sensitivity of the PKC{delta}-overexpressing cells was reduced by PKC{delta} antisense or rottlerin treatments, evidenced by clonogenic survival assay and induction of apoptosis (Fig. 8, C and D)Citation .



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Fig. 8. Treatment with PKC{delta} antisense-abrogated PKC{delta}-mediated radiosensitivity. A, protein extracts (60 µg) of growing vector control and PKC{delta}-overexpressed NIH3T3 cells with or without PKC{delta} antisense (ISIS 11514, 11515), sense oligonucleotides (ISIS 22083, ISIS 22086), or rottlerin (5 µM) were prepared, separated by SDS-PAGE, and analyzed by Western blotting. B, control vector and PKC{delta}-overexpressing NIH3T3 cells with or without PKC{delta} antisense (ISIS 11514, 11515), sense oligonucleotides (ISIS 22083, ISIS 22086), or rottlerin (5 µM) were harvested after incubation for the periods indicated after 5 Gy radiation, and cell cycle distribution was analyzed after by a flow cytometer staining with propidium iodide. C, surviving fraction of vector control and PKC{delta}-overexpressing NIH3T3 cells with or without PKC{delta} antisense (ISIS 11514, 11515), sense oligonucleotides (ISIS 22083, ISIS 22086), or rottlerin (5 µM) was obtained by colony-forming assay after irradiation. D, DNA fragmentation measured by Hoechst 33258 staining 48 h after 5-Gy radiation as described under "Materials and Methods." Error bar, mean ± SD from three independent experiments.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Although all of the members of the PKC family share many similarities, PKC isoforms are also likely to have different biological functions. In the present study, we established NIH3T3 cells that overexpressed the Ca2+-independent novel PKC and PKC{delta}, and found that expression of phospho-ERK1/2, PKC downstream, was increased in these cells, whereas expressions of p38 MAPK and JNK were slightly reduced, which suggested that PKC{delta} directly regulated ERK1/2 pathway. Because it is generally accepted that the activation of the ERK cascade leads to cell proliferation (13 , 14) , we compared growth rates: the PKC{delta}-overexpressing cells showed increased growth rate compared with control vector cells (data not shown), suggesting that PKC{delta}-mediated ERK1/2 overexpression accelerated cell growth.

Recently it has been demonstrated that PKC{delta} was cleaved and a CAT fragment was produced during apoptosis induced by ionizing radiation (7 , 8) or DNA damaging agent (23) . The proteolytic cleavage of PKC{delta} was inhibited by the expression of antiapoptotic proteins Bcl-2 and Bcl-XL and also by pretreatment with the interluekin-1ß-converting enzyme (ICE)/caspase-1 inhibitor peptide (9 , 10) . These results suggest that the activation of PKC{delta} may play a role in apoptosis. However, in the present study, although a large amount of PKC{delta} translocation to membrane fraction was observed, we could not detect any proteolytic degradation in the PKC{delta}-overexpressing cells.

We observed in the present study that the PKC{delta}-overexpressing cells induced radiosensitivity, and this was correlated with increased induction of apoptosis (Fig. 2)Citation , increased radiation-induced ERK1/2 activation, and increased basal level of phospho-ERK1/2 expression in the PKC{delta}-overexpressing cells (Fig. 7A)Citation . In our data, phospho-p38 expression levels were more inhibited in PKC{delta}-overexpressed cells than in control cells. We do not know exact reasons; however, there are some studies about cross-talk among the MAPKs (24 , 25) , and ERK1/2 phosphorylation by PKC{delta} overexpression may affect the protein phosphorylation.

Because MAPK cascade has been shown to be involved in radiosensitivity (26) , we treated the cells with PD98059. Radiation-induced apoptosis was blocked by PD98059 in PKC{delta}-overexpressing cells (Fig. 7B)Citation , which suggested that PKC{delta}-mediated ERK1/2 activation was important for radiosensitivity. These observations were somewhat surprising, because ERK pathway is known to be critical in the control of cellular growth and cell survival responses in many different cell systems, which includes those received by tyrosine kinase, G protein-coupled and cytokine receptors (27 , 28) : All of the studies support a general view that activation of the ERK pathway delivers a survival signal which counteracts proapoptotic effects associated with JNK and p38 activation (29) . On the other hand, a requirement of ERK activation in mediating cisplatin-induced apoptosis of HeLa cells and human ovarian cell lines (16 , 17) and UVB-induced apoptosis (12) have also been demonstrated. Moreover, persistent activation of ERK1/2 contributes to glutamate-induced oxidative toxicity (18) .

Flow cytometric analysis revealed that PKC{delta}-overexpressing cells escaped from the G2-M phase arrest induced by radiation (Fig. 3)Citation and molecular biological study also revealed decreased radiation effect on G2-M phase cell cycle arrest in the PKC{delta}-overexpressing cells; binding activity of p21Waf with cyclin A or –B1 was decreased (Fig. 4)Citation , even although p21Waf induction by radiation, which was p53-independent pathway, was higher in PKC{delta}-overexpressing cells. We do not know how this discrepancy happened. However, the induction of p21Waf does not always mean increased binding to CDKs. Other possibility may be that p21Waf may bind other cyclins such as cyclin D and E (30) .

Numerous studies demonstrated that cell cycle delay induced radioresistance and altered apoptosis. The enhanced apoptosis of the PKC{delta}-overexpressing cells after radiation was most likely caused by acceleration of cell cycle progression after radiation. It has been proposed that S-phase perturbation by DNA-damaging agents occurs in conjunction with the G2-M block, and the cells enter mitosis prematurely, before completion of DNA synthesis (31) . In support of the above contention, cyclin B1 expression and its associated kinase activity was found to be increased by radiation (Fig. 5)Citation , which might be resulting in accelerated exit from the G2-M arrest with subsequent formation of micronuclei (32) . These phenomena were also promoted by PKC{delta} overexpression, which suggests that PKC{delta} helped the cells escape from radiation-induced G2-M arrest, which resulted in increased apoptosis. Enhanced exit from G2-M phase after radiation by PKC{delta} overexpression may result in entry into the next cell cycle, whereas not all of the radiation-induced DNA damage is completely repaired, thereby possibly leading to reinduction of apoptosis and formation of micronuclei. These findings are consistent with our observed second waves of p53 and p21Waf upon radiation in the PKC{delta}-overexpressing cells (Fig. 6)Citation .

Pardo et al. (33) suggested that increased sensitivity toward DNA-damaging drugs was caused by wild-type p53, irrespective of whether the cells were normal or tumor-derived; enhanced level of p53 in all of the cell lines examined led the cells to apoptosis and finally cell death. This was in accordance with our findings in which the response to ionizing radiation was dependent on the initial level of cyclin D1, and cyclin D1 and p53 expression levels were higher in PKC{delta}-overexpressed cells (data not shown). The higher level of cyclin D1 led to higher p53 induction after radiation and apoptosis and ultimately resulted in clonogenic cell death. We could not offer any suggestion on the mechanism of how PKC{delta} was related to cell cycle-related proteins. However, treatment with PKC{delta} antisense or rottlerin reduced ERK1/2 activation and abrogated PKC{delta}-mediated radiosensitivity, and PKC inhibition restored the escaped radiation-induced cell cycle arrest (Fig. 8)Citation ; therefore, regulation of cell cycle most likely occurred downstream of PKC{delta}-ERK pathways.

In conclusion, PKC{delta} overexpression enhanced radiation-induced apoptosis and radiosensitivity. This occurred via ERK1/2 activation, thereby abolishing the radiation induced G2-M arrest and finally apoptosis. Although the PKC-driven signaling systems affected by radiation have not been clearly characterized, our data indicate that PKC activity is certainly associated with the MAPK response and G2-M phase arrest in radiation sensitivity. In preclinical model systems, modulation of PKC was found to potentiate the activity of cytotoxic agents as well as ionizing radiation (3) . Accumulating evidence together with the above consideration indicates selective targeting of PKC to be useful in improving the therapeutic efficacy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
Rottlerin, and PD98059 were purchased from Calbiochem (La Jolla, CA), and bisbenzimide trihydrochloride (Heochst 33258) was from Sigma Chemical Co (St. Louis, MO). Anti-PKC{alpha}, -ß1, -{delta}, -{epsilon}, and -{zeta} antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-MAPK, anti-phospho MAPK (P202/Y204), anti-p38 MAPK, anti-phospho-p38 MAPK, anti-phospho-JNK and -MEK1/2 (Ser217/221) polyclonal antibodies were from New England BioLabs (Beverly, MA). Anti-p53 antibody was from Calbiochem (Oncogene Research Products, Cambridge, MA); and anti-cyclin B1, anti-CDC2 antibodies, and rabbit polyclonal anti-CDK2, -CDK4, and -p21Waf antibodies were from Santa Cruz Biotechnology.

Generation of Overexpressing Cell Line.
NIH3T3 cells were grown in DMEM supplemented with 4 mM glutamine and 10% fetal bovine serum. The NIH3T3 cells were transfected either with empty vector as a control (pLTR) or with PKC{delta} (cloned into the EcorR1 site of pLTR to create PKC{delta}; Ref. 21 ) expression vectors using LipofectAMINE (Life Technologies, Inc., Gaithersburg, MD) by following the procedure recommended by the manufacturer. The transfected cells were subsequently grown in selection medium. pLTR-based constructs, had the selectable marker xanthine-guanine phosphoribosyltransferase, and cells that were transfected with these constructs were grown in DMEM supplemented with 0.2 mM hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine, and 80 µM mycophenolic acid. After 10–16 days in selection medium, single colonies were picked and subsequently examined for the presence of PKC protein by Western blot analysis. PKC{delta}-DN and PKC-CAT mutant expression vector were kindly supplied by Dr. J. W. Soh (Columbia University, New York City; Ref. 34 ). Briefly, pHANE is a mammalian expression vector that contains a cytomegalovirus promoter, Kozak translation initiation sequence, ATG start codon. NH2-terminal HA epitope tag, EcoRI cloning site, and stop codon. It was generated into pcDNA3 (Invitrogen) after digestion with BamH1 and EcoRI. pHANE was used to generate PKC mutants with an NH2-terminal HA tag. pHACE is a mammalian expression vector that contains a cytomegalovirus promoter, Kozak translation initiation sequence, ATG start codon, EcoRI cloning site, COOH-terminal HA epitope tag, and stop codon. It was generated into pcDNA3 after digestion BamH1 and EcoRI pHACE was used to generate PKC mutants with an COOH-terminal HA tag. pHACE-PKC-WT expression plasmids were generated by ligating full-length open reading frames of different PKC isoforms into pHACE, digested with EcoRI. pHACE-PKC-KR expression plasmids were generated by ligating full-length open reading frames of PKC isoforms with a K->R point mutation at the ATP binding site into pHACE-digested with EcoRI. pHANE-PKC-CAT expression plasmids were generated by ligating cDNA fragments encoding only the CAT domains of PKC isoforms into pHANE digested with EcoRI. All of the cDNA fragments of PKC mutants were generated by PCR and were analyzed to confirm their sequences with an automated DNA sequencer.

Antisense Oligonucleotides Treatment.
Chimeric oligonucleotides of PKC{delta} antisense oligonucleotides (ISIS 11514 and ISIS 11515) and scrambled control antisense (ISIS 22083 and ISIS 22086) were provided by ISIS Pharmaceuticals (Carlsbad, CA). PKC{delta}-overexpressed cells were transfected with Lipofection for introducing PKC{delta} or scrambled antisense, as described previously (35) . Scrambled and antisense oligonucleotide sequences were treated for overnight, and serum-added medium was changed.

Irradiation.
Cells were plated in 3.5-, 6-, or 10-cm dishes and incubated at 37°C under humidified 5% CO2-95% air in culture medium until 70–80% confluent. Cells were then exposed to {gamma}-rays with 137Cs {gamma}-ray source (Atomic Energy of Canada, Ltd., Ontario, Canada) with dose rate of 3.81 Gy/min.

Colony-forming Assay.
Clonogenicity was examined by colony-forming assay, as described previously (36 , 37) . Cells were seeded into 6-cm Petri dishes at densities to produce ~500 colonies per dish in the control and were incubated for 7–14 days. Colonies were fixed with a mixture of 75% methanol and 25% acetic acid, and stained with 0.4% trypan blue. The number of colonies consisting of 50 or more cells was scored.

Detection of Apoptosis.
Cells were plated on glass slides and irradiated. After 24 or 48 h, cells were fixed in 70% ethanol, were washed with PBS, and were incubated with 1 µg/ml bisbenzimide trihydrochloride in PBS (Heochst 33258) for 30 min in the dark. Specimens were viewed by fluorescence microscopy using Olympus BX-40 microscope. At least 200 cells for each determination were scored. Apoptosis was characterized by chromatin condensation and fragmentation.

Assay of PKC Activity.
PKC activity in cell lysates was determined as described previously (36) , and protein content was estimated with a commercial assay kit (Bio-Rad, Hercules, CA). Protein fractions were incubated at 30°C for 10 min in reaction mixture containing phospholipid micelles (phosphatidylcholine and phosphatidylserine; Sigma Chemical Co.), [{gamma}-32P]ATP (3000 Ci/mmol; NEN Life Science Products, Boston, MA), and serine-substituted PKC{alpha} pseudosubstrate peptide (Life Technologies, Inc.). The samples were dried on P81 filter paper and washed with 0.45% phosphoric acid. After a final rinse with acetone, the amount of incorporated 32P was determined. Because modified PKC pseudosubstrate peptide can be phosphorylated by purified PKC{alpha}, -ß, -{gamma}, -{delta}, -{epsilon}, -{zeta}, and -{eta}, this assay detects the total activity of both Ca2+-dependent and Ca2+-independent isoforms in cells. Thus, Ca2+-dependent PKC activity was calculated by subtracting Ca2+-independent activity from the total PKC activity.

Cell Cycle Analysis.
For cell cycle analysis, cells were fixed in 80% ethanol at 4°C for at least 18 h. The fixed cells were then washed once with PBS-EDTA and resuspended in 1 ml of PBS. After the addition of 10 µl each of propidium iodide (5 mg/ml) and RNase (10 mg/ml), the samples were incubated for 30 min at 37°C and analyzed with a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

PAGE and Western Blot.
Cells were solubilized with lysis buffer [120 mM NaCl, 40 mM Tris (pH 8.0),and 0.1% NP40] and boiled for 5 min, and an equal amount of protein (40 µg/well) was analyzed on 7.5–10% SDS-PAGE. After electrophoresis, proteins were transferred onto a nitrocellulose membrane and processed for immunoblotting. When antibodies against phospho-specific peptides were used, blots were stripped by washing 3 times with TBS-T [10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween 20 (0.1%)] for 5 min each at room temperature, 30 min at 55°C with stripping buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol], and, finally, 3 times with TBS-T at room temperature for 5 min each. The stripped blots were then reprobed with corresponding non-phospho-specific antibodies to ensure equal protein loading.

Immunoprecipitation.
Cell lysates were incubated with anti-CDK2, -CDC2, -cyclin, A or -cyclin B1 polyclonal antibodies (Santa Cruz Biotechnology) or normal rabbit serum. The immunocomplex was collected on protein A-Sepharose (Sigma) and analyzed by SDS-PAGE using enhanced chemiluminescence detection (ECL; Amersham International).

Immunoprecipitation and Immune Complex Kinase Assay.
Cell lysates were incubated with primary antibody, and immunocomplexes were collected on protein A-Sepharose beads and resuspended in kinase assay mixture containing [{gamma}-32P]ATP (NEN Life Science, Boston, MA) and histone H1 (Life Technologies, Inc.) as substrates. Proteins were separated on SDS-polyacrylamide gels, and bands were detected by autoradiography.


    Footnotes
 
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.

1 Supported by the Nuclear Research and Development Program from the Ministry of Science and Technology of Korea. Back

2 S-J. L. and Y-S. L. contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Laboratory of Radiation Effect, Korea Cancer Center Hospital, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, Korea. Phone: 82-2-970-1325; Fax: 82-2-977-0381; E-mail: yslee{at}kcchsun.kcch.re.kr Back

4 The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent kinase, DAG, diacylglycerol; TPA, 12-O-tetradecanoylphorbol 13-acetate; ERK, extracellular regulated protein kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAP/ERK kinase; JNK, c-Jun NH2-terminal kinase; DN, dominant-negative; CAT, catalytic. Back

Received for publication 9/11/01. Revision received 3/19/02. Accepted for publication 4/ 1/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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