Cancer Research Meeting Calendar Protein Translation and Cancer
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 Email this article to a friend
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 Mandil, R.
Right arrow Articles by Brodie, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mandil, R.
Right arrow Articles by Brodie, C.
[Cancer Research 61, 4612-4619, June 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Protein Kinase C{alpha} and Protein Kinase C{delta} Play Opposite Roles in the Proliferation and Apoptosis of Glioma Cells1

Revital Mandil, Ely Ashkenazi, Michal Blass, Ilana Kronfeld, Gila Kazimirsky, Guy Rosenthal, Felix Umansky, Patricia S. Lorenzo, Peter M. Blumberg and Chaya Brodie2

Faculty of Life-Sciences, Bar-Ilan University, Ramat-Gan, Israel 52900 [R. M., M. B., I. K., G. K., C. B.]; Department of Neurosurgery, Hadassa Medical Center, Jerusalem, Israel [E. A., G. R., F. U.]; and Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, Maryland 20892 [P. S. L., P. M. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C (PKC) has been implicated in the proliferation and apoptosis of glial tumors, but the role of specific PKC isoforms remains unresolved. Comparing brain tumors differing in degree of malignancy, we found that malignant gliomas expressed higher levels of PKC{alpha} and lower levels of PKC{delta} as compared with low-grade astrocytomas. Consistent with a mechanistic role for these differences, overexpression of PKC{alpha} in the human U87 glioma cell line resulted in enhanced cell proliferation and decreased glial fibrillary acidic protein (GFAP) expression as compared with controls. Reciprocally, overexpression of PKC{delta} inhibited cell proliferation and enhanced GFAP expression. Using PKC chimeras, we found that the regulatory domains of PKC{alpha} and PKC{delta} mediated their effects on cell proliferation and GFAP expression. PKC{alpha} and {delta} have been implicated as potential signaling molecules in apoptosis. Therefore, we examined the role of these isoforms in the resistance of glioma cells to apoptotic stimuli. In U87 cells, manipulation of PKC{alpha} levels had little effect on apoptosis in response to etoposide. In contrast, overexpression of PKC{delta} rendered the U87 cells more sensitive to the apoptotic effect of etoposide, and PKC{delta} was cleaved in these cells by a caspase-dependent process. Furthermore, the glioma cell line U373, which expresses endogenous PKC{delta}, underwent apoptosis in response to etoposide, and the apoptotic response was blocked by the PKC{delta} inhibitor rottlerin. Our results suggest that PKC{alpha} and PKC{delta} play opposite roles in the proliferation and apoptosis of glioma cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PKC3 comprises a family of phospholipid-dependent serine-threonine kinases that play important roles in signal transduction and in the regulation of cell growth, differentiation, and apoptosis (1, 2, 3) . PKC consists of at least 11 isoforms showing diversity in their structures, cellular distributions, and biological functions (4) . The members of the classical PKCs {alpha}, ß1, ß2, and {gamma} bind phorbol esters and are Ca2+dependent. The novel PKCs {delta}, {epsilon}, {eta}, and {theta} do not depend on Ca2+ but bind phorbol esters. The third subfamily includes the atypical PKCs (PKC{zeta} and PKC{iota}/{lambda}), which do not bind either Ca2+ or phorbol esters, and PKCµ and {nu}, which represent a distinct subclass with unique characteristics (5 , 6) . All of the PKC isoforms are composed of an NH2-terminal regulatory domain and a COOH-terminal catalytic domain with serine-threonine kinase activity (7) . Both domains contain conserved (C) regions of extended sequence homology and variable (V) regions (8 , 9) . In the classical PKC isoforms, the regulatory domain contains a Ca2+-binding domain, and in both the classical and novel PKC isoforms, it contains a pseudosubstrate region and a pair of highly conserved zinc fingers (C1 domains) that bind phorbol esters (10) . PKC chimeras have been used to study the role of the regulatory and catalytic domains of different PKC isoforms (11 , 12) .

Malignant gliomas, the most common brain tumor, are refractory to classical chemotherapy and radiotherapy and have a poor prognosis (13) . The molecular mechanisms underlying glial neoplastic transformation have been widely studied, and various signaling pathways including that of PKC have been found to be altered (14, 15, 16, 17) . Thus, PKC activity has been reported to be increased in gliomas and glioma cell lines as compared with astrocytes (18 , 19) , and PKC inhibitors markedly reduced glioma cell proliferation (20 , 21) . Moreover, differential expression of specific PKC isoforms has been reported in gliomas versus normal astrocytes (22 , 23) , and specific targeting of PKC{alpha} by antisense oligonucleotides or by ribozymes blocked growth of glioma cells and increased cell apoptosis (20 , 24) .

In the present study, we examined the expression of specific PKC isoforms in glial tumors with different degrees of malignancy and explored the roles of PKC{alpha} and PKC{delta} in the proliferation and apoptosis of glioma cells. Our results indicate that the expression of PKC{alpha} is increased and the expression of PKC{delta} is decreased in malignant gliomas and that overexpression of PKC{delta} in U87 cells dramatically decreased cell proliferation and rendered the cells more sensitive to apoptosis induced by etoposide.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
An affinity-purified polyclonal anti-PKC{epsilon} antibody against a polypeptide corresponding to amino acids 726–737 of PKC{epsilon} was purchased from Life Technologies, Inc. (Gaithersburg, MD). Monoclonal anti-PKC antibodies were obtained from Transduction Laboratories (Lexington, KY). Polyclonal anti-PKC antibodies were from Santa-Cruz (Santa Cruz, CA). PMA was from Alexis Co. (San Diego, CA). Leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and sodium vanadate were obtained from Sigma Chemical Co. (St. Louis, MO). The human glioma cell lines U373-MG, U87-MG, and T98G were obtained from the American Type Culture Collection, and human primary astrocytes were obtained from Clonetics (Walkersville, MD). [3H]PDBu (10–20 Ci/mmol) was purchased from NEN (Boston, MA).

Tumor Samples.
Tumors from 25 patients were collected during surgical procedures at the Department of Neurosurgery, Hadassah Hospital, Jerusalem, Israel. Tumors were classified as low-grade astrocytomas (Grade II; 18 cases) and GBM (grade IV; 22 cases) according to the WHO classification. Upon removal, tissues were immediately frozen in liquid nitrogen and were kept at -80°C. The mean age of patients with low-grade astrocytomas and GBM was 55 and 62 years of age, respectively. Sample collection and processing were performed according to the regulations of the Committee on Research Involving Human Subjects of the Hadassah Medical Organization Institutional Review Board.

Generation of PKC Chimeras.
The PKC chimeras were generated by exchanging the regulatory and catalytic domains of PKC{alpha}, {delta}, and {epsilon} as described by Acs et al. (25) . PKC{alpha}/{delta} refers to the chimera with the PKC{alpha} regulatory domain and the PKC{delta} catalytic domain, and PKC{delta}/{alpha} refers to the reciprocal chimera. The PKC cDNAs were subcloned into MTH vector. The vector sequence encodes a COOH-terminal PKC-{epsilon}-derived 12 amino acid tag ({epsilon}MTH) that is added to the expressed proteins (26) . The expression of these chimeras and characterization of their activities in C6 cells were described recently (27) .

Construction of PKC-GFP Fusion Proteins.
cDNAs encoding the murine PKC{alpha}, PKC{delta}, and the various PKC chimeras were inserted into the NH2-terminal-enhanced GFP vector pEGFP-N1 (Clontech Laboratories, Palo Alto, CA). The original pEGFP-N1 vector was modified by the addition of an MluI site in the plasmid polylinker. The restriction site was created by ligating a phosphorylated linker containing the Mlul site into pEGFP-N1 digested with SmaI. The construct was verified by sequencing. The clones containing GFP-PKC{delta} or GFP-fused to the different PKC{delta} mutants were constructed by the excision of PKC{delta} or the specific mutants from MTH-PKC plasmids by digestion with XhoI and MluI. The inserts were then ligated into the modified GFP vector using the same restriction sites. DNA sequencing of the GFP-PKC constructs confirmed the intended reading frame.

U87 Cell Cultures and Cell Transfection.
U87 cells (1 x 105 cells/ml) were seeded on tissue culture dishes (10 cm) and were grown in medium consisting of DMEM containing 10% heat-inactivated FCS, 2 mM glutamine, penicillin (50 units/ml), and streptomycin (0.05 mg/ml). The cells were transfected either with the empty vector, PKC{alpha}, PKC{delta}, or the PKC chimeras PKC{alpha}/{delta} and PKC{delta}/{alpha} using LipofectAMINE (Life Technologies, Inc.) as described (28) . Experiments were routinely carried out on a clone of the transfected cells, but all of the results were confirmed on one pool and two additional individual clones.

For overexpression of the GFP-PKC fusion proteins, U87 cells were seeded onto 40-mm round glass coverslips at a density of 5 x 104 cells/coverslip. Twenty-four h later, cells were transfected with the different GFP-PKC constructs using LipofectAMINE Plus reagent, according to the manufacturer’s instructions. All of the experiments were performed 48 h after transfection.

Antisense Treatment.
The following phosphorothioate-modified oligonucleotides were used: PKC{alpha} antisense, CGCATAAACGTCAGCCAT; and PKC{alpha} sense, ATGGCTGACGTTTATGCG. The antisense oligonucleotide sequence was against the first 18 nucleotides downstream from the ATG start site of PKC{alpha} cDNA, and the sense oligonucleotide was used as a control. The oligonucleotides were transfected into the U87 cells at a concentration of 0.5–1 µM using LipofectAMINE as described for the transfection of PKC{alpha} and PKC{delta}.

Preparation of Cell Homogenates.
Cells were washed and resuspended in serum-free medium. The plates were placed on ice, scraped with a rubber policeman, and centrifuged at 1400 rpm for 10 min. The supernatants were aspirated, and the cell pellets were resuspended in 100 µl of lysis buffer [25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5% Na deoxycholate, 2% NP40, 0.2% SDS, 100 µM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50 µM leupeptin; and 0.5 mM Na3VO4] on ice for 15 min. The cell lysates were centrifuged for 15 min at 14,000 rpm in an Eppendorf microcentrifuge. The supernatants were removed, and 2x sample buffer was added to them. Tumors were homogenized in lysis buffer, the lysates were centrifuged, and the supernatants were removed and boiled in 2x sample buffer.

Immunoblot Analysis.
Lysates (20 µg of protein) were subjected to SDS-PAGE (10%) followed by transfer to nitrocellulose membranes. Similar protein contents of the different samples were verified by staining the membranes with 0.1% Ponceau S solution in 5% acetic acid. The nitrocellulose membranes were blocked with 5% dry milk in PBS and subsequently stained with the primary antibody. Specific reactive bands were detected using a goat antirabbit or goat antimouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA), and the immunoreactive bands were visualized by the enhanced chemiluminescence Western blotting detection kit (Amersham, Arlington Heights, IL). Quantification of the bands was done, and the results are presented as relative absorbance.

PKC Kinase Assay.
PKC activity was assayed by measuring the incorporation of 32P from [{gamma}-32P]ATP into substrate in the presence of 100 µg/ml phosphatidylserine and 1 µM PMA, as described previously (27) .

[3H]PDBu Binding.
[3H]PDBu binding was measured using the polyethylene glycol precipitation assay (27) . Briefly, cell lysates (4–60 µg of protein/assay) were incubated with 20 nM [3H]PDBu in the presence of phosphatidylserine. Nonspecific binding, determined in the presence of 30 µM nonradioactive PDBu, was subtracted to give specific binding.

Cell Proliferation Assay.
Cells overexpressing the wild-type PKC{delta} or the PKC{delta} mutants were seeded in triplicate and incubated in the absence or presence of ZnCl2 (30 µM) for 24 h followed by treatment with PMA (30 nM) for an additional 48 h. Cells were pulsed with 0.5 µCi of [3H]thymidine for the last 6 h and then harvested. The incorporation of [3H]thymidine was determined in a Beckman scintillation counter.

Measurement of Apoptosis.
Cell apoptosis was measured using PI staining and analysis by flow cytometry or by ELISA using anti-histone 1 antibodies. Cells (1 x 106/ml) were plated in 6-well plates and treated with the indicated treatments for 24 h. For PI staining, detached cells and trypsinized adherent cells were pooled, fixed in 70% ethanol for 1 h on ice, washed with PBS, and treated for 15 min with RNase (50 µM) at room temperature. Cells were then stained with PI (5 µg/ml) and analyzed on a Becton Dickinson cell sorter. Cells in the sub-G1 population were considered as apoptotic cells. For anti-histone 1 ELISA, fragmented DNA was extracted from the cells and was incubated in 96-well plates coated with anti-histone 1 antibodies for 2 h. Plates were then washed and incubated with anti-DNA antibodies conjugated to peroxidase for an additional 2 h.

Confocal Microscopy.
Confocal fluorescent images were collected with a Bio-Rad MRC 1024 confocal scan head (Bio-Rad) mounted on a Nikon microscope with a 60x planapochromat lens. Excitation at 488 nm was generated by a krypton-argon gas laser with a 522/32 emission filter for green fluorescence. For kinetics of GFP-PKC translocation in living cells, cells plated on a 40-mm round coverslip were enclosed in a Bioptechs Focht Chamber System (Bioptechs, Butler, PA). The chamber was inverted and attached to the microscope stage with a custom stage adapter. A temperature controller set at 37°C was connected, and medium was perfused through the chamber with a Lambda microperfusion pump. Sequential images of the same cell were collected at various time points using LaserSharp Software.

Statistical Analysis.
The results are presented as the mean values ± SE. Data were analyzed using ANOVA and a paired Student’s t test to determine the level of significance between the different groups.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differential Expression of Specific PKC Isoforms in Various Glial Tumors.
We examined the expression of specific PKC isoforms in extracts derived from low-grade astrocytomas and GBM using Western blot analysis. Membranes were probed with antibodies directed against the specific PKC isoforms and with anti-tubulin. The immunoreactive bands were analyzed using densitometry, and the intensities of the specific bands were compared with those of the respective controls.

Fig. 1ACitation shows the mean levels of expression of specific PKC isoforms in 18 tumors of low-grade astrocytomas and 22 tumors of GBM. The level of PKC{alpha} was significantly higher (P < 0.002) and that of PKC{delta} was significantly lower in the GBM tumors compared with the astrocytomas (P < 0.002). In fact, most GBM tumors expressed very low or undetectable levels of PKC{delta}. PKC{epsilon} showed modestly higher expression in GBM, whereas the levels of PKCß, {gamma}, µ, and {zeta} were unchanged. The expression of PKC{eta} and PKC{iota} was low in both tumors (data not shown).



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Expression of specific PKC isoforms in glioma tumors and glioma cell lines. Frozen tumors were homogenized and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with antibodies against the specific PKC isoforms and with anti-tubulin antibody. PKC-specific bands were analyzed by densitometry and were compared with the corresponding tubulin-specific bands. The results represent the mean density values ± SE of each PKC isoform for 18 astrocytoma tumors and for 22 GBM tumors (A). Glioma cell lines and human primary astrocytes were harvested and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with anti-PKC antibodies or with anti-GFAP (B). GFAP- and PKC-specific bands were analyzed by densitometry, and the results represent the mean density values ± SE of three separate experiments (C); *, P < 0.002; **, P < 0.005.

 
We also examined the expression of GFAP and the different PKC isoforms in primary human astrocytes and in the three glioma cell lines, U373, T98G, and U87, for comparison with the glial tumors. Primary human astrocytes expressed high levels of GFAP, whereas lower levels were expressed in the glioma cell lines. The U373 cell line expressed higher levels of GFAP as compared with T987 and U87 cells (Fig. 1B)Citation and a lower proliferation rate (data not shown). The pattern of expression of the different PKC isoforms in the U87 and T98G cells was similar to that in the GBM tumors. In contrast, human astrocytes expressed higher levels of PKC{delta} and ß and lower levels of PKC{alpha} (Fig. 1, B and C)Citation . The expression of PKC{alpha} and PKC{delta} in U373 cells was intermediate between that of the primary astrocytes and that of the U87 and the T987 cells (Fig. 1, B and C)Citation .

Overexpression of PKC{alpha} and PKC{delta} in U87 Cells.
Because major differences were observed in the expression of PKC{alpha} and PKC{delta} in tumors exhibiting different degrees of malignancy and because the U87 glioma cell line showed similar differences, we further examined the role of these isoforms by overexpressing PKC{alpha} and PKC{delta} in the U87 cells. We transfected U87 cells with the control {epsilon}MTH vector and with the corresponding PKC{alpha} and PKC{delta} {epsilon}MTH vectors. The levels of protein expression were analyzed by Western blotting of pooled cultures and of three different overexpressing clones for each of the isoforms as well as of the vector controls. Fig. 2ACitation illustrates a representative Western blot. Using the tagging system described previously (26) , we were able to detect specifically the transfected PKC isoforms with the antibody against PKC{epsilon} (Fig. 2A)Citation . The levels of the overexpressed PKC{alpha} and PKC{delta} in the transfected cells were about 5- and 8-fold higher than the corresponding endogenous PKC, respectively (data not shown). To establish that the overexpressed PKC isoforms were functionally active, we measured specific [3H]PDBu binding on partially purified cell lysates. All of the PKC clones exhibited increased [3H]PDBu binding as compared with the control-transfected cells (data not shown).



View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Morphology, GFAP expression, and proliferation of U87 cells overexpressing the PKC{alpha} and PKC{delta}. Stable transfectants of U87 cells overexpressing PKC{alpha}, PKC{delta}, or the empty vector (CV) were harvested and subjected to SDS-PAGE and Western blot analysis. The membranes were probed with anti-{epsilon} antibody, which recognized the {epsilon}-tag. Top band, the endogenous PKC{epsilon}; bottom band, the PKC{alpha} or PKC{delta} (A). The morphology of the cells was evaluated by phase contrast light microscope (B). GFAP expression was examined in cell lysates using Western blot analysis and anti-GFAP antibody (C). The above results (A–C) are from one representative experiment of four similar experiments. For proliferation assays, cells were plated in 24-well plates and incubated in the absence or presence of PMA (20 nM) for 48 h. [3H]Thymidine was added to the cells for the last 6 h, and the assay was performed as described in "Materials and Methods" (D). The results are expressed as the percentage of the untreated control vector cells and represent the mean ± SE of five separate experiments; *, P < 0.02; **, P = 0.002.

 
The morphology of the PKC-transfected cells differed from that of the control vector cells. Cells overexpressing PKC{alpha} had smaller cell bodies, whereas cells overexpressing PKC{delta} had large cell bodies with long, thick processes. The PKC{delta} transfectants thus resembled differentiated astrocytes (Fig. 2B)Citation . Control vector-transfected cells expressed very low levels of GFAP, similar to those expressed by control-untransfected cells. As shown in Fig. 2CCitation , cells overexpressing PKC{alpha} displayed low levels of GFAP similar to the control cells. In contrast and consistent with their morphological appearance, cells overexpressing PKC{delta} expressed appreciably higher levels of GFAP.

PKC{alpha} and PKC{delta} also affected the rate of proliferation compared with control vector cells. Cells overexpressing PKC{alpha} exhibited a slightly higher rate of proliferation, whereas cells overexpressing PKC{delta} showed a markedly lower rate of proliferation (Fig. 2D)Citation . Incubation of the cells with 20 nM PMA further enhanced the responses observed in the untreated cells.

The Regulatory Domains of PKC{delta} and PKC{alpha} Mediate the Changes in Cell Proliferation and GFAP Expression.
In a recent study (27) , we demonstrated that the regulatory domain of PKC{delta} was responsible for the inhibitory effect of PKC{delta} on the expression of the astrocytic marker, glutamine synthetase. To further characterize the effect of PKC{delta} on the proliferation of U87 cells, we examined the relative contributions of the regulatory and catalytic domains of this isoform. For these studies, we used chimeras between the regulatory and catalytic domains of PKC{alpha} and {delta} combined at the highly conserved hinge region (27) .

U87 cells were transfected with the control vector and with the chimeras PKC{alpha}/{delta} and PKC{delta}/{alpha}. Fig. 3ACitation demonstrates the expression of the PKC chimeras using the tagging system described previously (26) . The PKC chimeras were active as demonstrated by the increased kinase activity of the transfected cells (data not shown).



View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Morphology, GFAP expression, and proliferation of U87 cells overexpressing the PKC chimeras. U87 cells stably overexpressing the control vector (CV) or the PKC chimeras were harvested and subjected to Western blot analysis. The expression of the different chimeras was examined by probing the membrane with the anti- antibody that recognizes the {epsilon}-tag. Top band, the endogenous PKC{epsilon}; bottom bands, the PKC chimeras (A). The morphology of the cells (B), GFAP expression (C), and cell proliferation (D) were determined as described in Fig. 2Citation ; *, P < 0.005; **, P < 0.001.

 
The morphology of the cells overexpressing the chimera PKC{delta}/{alpha} was similar to the morphology of cells overexpressing PKC{delta}. The cells had large cell bodies with multiple processes (Fig. 3B)Citation . In contrast, cells overexpressing the chimera PKC{alpha}/{delta} exhibited a morphology similar to cells overexpressing PKC{alpha}, suggesting that the regulatory domain of PKC{delta} mediates its effect on cell morphology.

The regulatory domain of PKC{delta} also mediated the increased expression of GFAP in the U87 cells. Thus, cells overexpressing the chimera PKC{delta}/{alpha} expressed increased levels of GFAP, similar to cells overexpressing PKC{delta}, whereas cells overexpressing the chimera PKC{alpha}/{delta} exhibited low levels of GFAP, similar to cells overexpressing PKC{alpha} (Fig. 3C)Citation .

Finally, the regulatory domain of PKC{delta} mediated its effects on proliferation in the U87 cells. Similar to cells expressing PKC{delta}, cells overexpressing the chimera PKC{delta}/{alpha} containing the regulatory domain of PKC{delta} also showed a reduced level of cell proliferation, whereas cells expressing the chimera PKC{alpha}/{delta} containing the regulatory domain of PKC{alpha} together with the catalytic domain of PKC{delta} exhibited an increased level of proliferation similar to that observed with cells expressing PKC{alpha} (Fig. 3D)Citation .

Translocation of PKC{alpha}, PKC{delta}, and the PKC Chimeras.
One possible explanation for the differential effects of the chimeras on cell proliferation and GFAP expression is translocation to different cellular compartments after stimulation. Therefore, we examined the translocation of PKC{alpha}, PKC{delta}, and the PKC chimeras in response to PMA. For these experiments, we tagged PKC{alpha}, PKC{delta}, and the PKC chimeras with GFP. PKC-GFP constructs have been shown previously (29) to behave like the unmodified PKCs. Cells were transiently transfected with the specific GFP-PKCs, and the response of the cells to PMA was monitored over a period of 30 min. PMA (100 nM) induced rapid translocation of PKC{alpha} to the plasma membrane. In contrast, PMA induced initial translocation of PKC{delta} to the plasma membrane followed by some translocation of PKC{delta} to the nuclear membrane. The PKC{alpha}/{delta} chimera exhibited a pattern of translocation similar to that of PKC{alpha}, with rapid translocation to the plasma membrane. The PKC{delta}/{alpha} chimera exhibited a pattern of translocation similar to that of PKC{delta}, with translocation to the plasma and nuclear membranes (Fig. 4)Citation . The patterns of translocation, like the biology, thus appear to depend predominantly on the regulatory domain in this system.



View larger version (64K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Cellular localization of PKC, PKC{delta}, and the PKC chimeras in PMA-treated C6 cells. Cells were transiently transfected with GFP-PKC{alpha}, GFP-PKC{delta}, or the different chimeras. After 48 h, cells were treated with PMA (100 nM), and sequential confocal images were taken every 30 s for a period of 30 min. The figures present images taken at time 0 and 30 min after treatment. Cells shown are representative of four independent experiments.

 
PKC{alpha} Does Not Play a Role in the Resistance of Glioma Cells to the Apoptotic Effect of Etoposide.
PKC{alpha} has been suggested to be involved in the resistance of glioma cells to apoptosis (21 , 24) . Therefore, we examined the role of this isoform in the response of U87 cells to etoposide. Treatment of U87 cells with etoposide (50 µM) for 24 h induced apoptosis in only 7% of the cells (Fig. 5B)Citation . To examine the role of PKC{alpha}, we transfected U87 cells with PKC{alpha} oligonucleotide antisense and examined the apoptotic response of the cells. Transfection of the cells with the PKC{alpha} antisense significantly reduced the expression of this isoform as detected by Western blot (Fig. 5A)Citation , whereas it did not affect the expression of PKCß, {gamma}, {delta}, {epsilon}, {zeta}, µ, and {iota} (data not shown). Transfection of the cells with the control PKC{alpha} sense oligonucleotide did not affect the expression of PKC{alpha} (Fig. 5A)Citation or that of the other isoforms (data not shown). Cells transfected with PKC{alpha} antisense exhibited a small enhancement of apoptosis over that observed in control-untreated cells. Treatment of the cells with etoposide induced a small increase in the apoptotic response similar to that observed in control cells and in cells transfected with sense oligonucleotide, suggesting that PKC{alpha} is not involved in the resistance of the cells to etoposide (Fig. 5B)Citation . In contrast, antisense-treated cells exhibited a markedly decreased rate of cell proliferation as compared with control or sense-treated cells (Fig. 5C)Citation .



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Inhibition of PKC{alpha} expression by antisense oligonucleotide does not affect the apoptotic response of U87 cells to etoposide. U87 cells were transfected with PKC{alpha} antisense (AS) or sense (S) oligonucleotides (0.5–1 µM) using LipofectAMINE as described in "Materials and Methods." After 36 h, the cells were harvested and subjected to Western blot analysis for measuring PKC{alpha} expression (A). Cells transfected with either antisense and sense oligonucleotides were incubated with etoposide for 24 h. Cells were then harvested and stained with PI as described in "Materials and Methods." The percentage of the cells present in the sub-G1 domain population of the cells was measured, and the results represent the mean ± SE of four separate experiments (B). Cells treated with either antisense and sense oligonucleotides were pulsed with [3H]thymidine for 24 h, and proliferation was assayed as described in "Materials and Methods" (C). The results are expressed as the percentage of the control-untreated cells and represent the means ± SE of three separate experiments; *, P < 0.005.

 
PKC{delta} Plays a Role in the Apoptosis of Glioma Cells in Response to Etoposide.
Because PKC{delta} has also been associated with apoptotic responses (30, 31, 32) , we examined the effect of this PKC isoform on the apoptosis of glioma cells in response to etoposide. The U87 cells expressing control vector and PKC{alpha}, like the parental U87 cells, showed only a small apoptotic response upon treatment with 50 µM etoposide. In contrast, cells overexpressing PKC{delta} underwent a dramatic apoptotic response to etoposide. Apoptosis was quantitated using PI staining and analysis by flow cytometry. After 24 h of treatment, 40% of the cells underwent apoptosis in response to etoposide (data not shown), whereas 60% of the cells were apoptotic after 48 h (Fig. 6A)Citation . Furthermore, treatment with etoposide of cells overexpressing PKC{delta} resulted in lower cell number and in the appearance of rounded and detached cells, which are characteristic of apoptotic cells, whereas control vector cells and cells overexpressing PKC{alpha} exhibited a morphology similar to untreated cells (Fig. 6B)Citation .



View larger version (62K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Overexpression of PKC{delta} increases the apoptosis of U87 cells in response to etoposide. U87 cells expressing control vector, PKC{alpha}, or PKC{delta} were treated with etoposide (50 µM) for 48 h. Cell apoptosis was determined using PI staining and fluorescence-activated cell sorter analysis (A). The percentage of apoptotic cells was determined, and the results represent the means ± SE of three separate experiments. The morphology of the cells was monitored under phase contrast light microscope (B). The results are representative of four similar experiments.

 
The role of PKC{delta} in the apoptosis of glioma cells was further examined in the cell lines T98G and U373. The T98G cells, which express very small levels of PKC{delta}, exhibited a low level of apoptosis in response to etoposide, similar to that observed in U87 cells. In contrast, U373 cells, which express higher levels of PKC{delta}, underwent marked apoptosis in response to etoposide (Fig. 7A)Citation . Treatment of the U373 cells with the PKC{delta} inhibitor rottlerin inhibited the apoptotic effect of etoposide on the U373 cells, suggesting that the apoptosis observed in these cells was PKC{delta}-dependent (Fig. 7B)Citation .



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Differential apoptosis of various glioma cells lines in response to etoposide. T98G, U87, and U373 cells were treated with etoposide (50 µM) for 48 h, and cell apoptosis was determined using PI staining and fluorescence-activated cell sorter analysis (A). The percentage of apoptotic cells was determined, and the results represent the means ± SE of three separate experiments. U373 cells were treated with etoposide in the presence and absence of rottlerin (5 µM), and cell apoptosis was determined after 48 h (B). The results are the means ± SE of five separate experiments; *, P < 0.005; **, P < 0.002.

 
Etoposide Induces a Caspase-dependent Cleavage of PKC{delta}.
In other systems, cleavage of PKC{delta} has been associated with apoptosis (31 , 32) . We examined whether the glioma system behaves similarly. We treated U87 cells overexpressing PKC{delta} with etoposide (50 µM) for various periods of time and analyzed cell lysates using Western blotting. The level of PKC{delta} decreased after 12 h of etoposide treatment, and a Mr 40,000 cleavage product of PKC{delta} appeared and started to accumulate (Fig. 8A)Citation . No significant changes in the expression of PKC{alpha} were observed in response to etoposide, and no cleavage products were detected (Fig. 8B)Citation . Because PKC{delta} has been reported to be cleaved by caspase-dependent processes (30) , we examined the effects of the cell-permeable caspase inhibitor DEVD.FMK on the cleavage of PKC{delta}. As seen in Fig. 8ACitation , pretreatment of the cells for 1 h with DEVD.FMK reduced significantly the accumulation of the PKC{delta} cleavage product in response to etoposide. Similar results were obtained with another caspase inhibitor, Z-VAD.FMK (data not shown).



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Effect of etoposide on the cleavage of PKC{delta} and PKC{alpha}. U87 cells overexpressing PKC{delta} were treated with etoposide for 12 and 24 h. The cleavage of PKC{delta} (A) and PKC{alpha} (B) was determined using Western blot analysis. The effect of the caspase inhibitor, DEVD.FMK, on the cleavage of PKC{delta} was determined in cells pretreated with this inhibitor for 1 h before the etoposide treatment (A). The results are representative of four similar experiments.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we characterized the expression of specific PKC isoforms in glioma cells and explored the role of PKC{alpha} and {delta} in the proliferation, GFAP expression, and apoptosis of these cells. We found that the glioma tumors expressed PKC{alpha}, ß, {gamma}, {delta}, {epsilon}, µ, and {zeta} and low levels of PKC{eta} and PKC{iota}. Major differences were observed in the expression of PKC{delta} and PKC{alpha} between the GBM and the low-grade astrocytomas. Alterations in the activity of PKC and in the expression of specific PKC isoforms have been reported in gliomas (15 , 18 , 19) . Thus, various studies reported that gliomas and glioma cells exhibited increased PKC activity (18 , 19) and an increased expression of PKC{alpha} and PKC{epsilon} (23 , 33 , 34) . Our results are in agreement with these findings because we found an increased expression of both PKC{alpha} and PKC{epsilon} in the malignant tumors. In contrast, we report for the first time that malignant gliomas expressed lower levels of PKC{delta} as compared with normal astrocytes and with low-grade astrocytomas. In addition to their differential expression in gliomas, specific PKC isoforms play distinct roles in glioma cellular functions. Thus, PKC{alpha} has been implicated in the increased proliferation and decreased apoptosis of glioma cells (20 , 24) , whereas PKC{gamma} and PKC{delta} increased glioma anchorage-independent growth (35) . In a recent report (21) , PKC{eta} was shown to play a role in the PMA-induced proliferation of glioma cells. The results of our study and of others suggest that dysregulation of various PKC isoforms may be involved in the tumorigenesis of gliomas. The differences in the expression of specific PKC isoforms reported in the various studies may result from the use of different experimental systems. Alternatively, these changes can be attributable to the genetic heterogeneity of the tumors that may result in various deletions, amplifications, and point mutations of specific genes.

To further explore the role of PKC{alpha} and PKC{delta} in glioma cell growth, we used glioma cells in which we manipulated PKC{alpha} and PKC{delta} levels by overexpression and, in the case of PKC{alpha}, by antisense. Consistent with the levels of PKC{alpha} and PKC{delta} contributing to the phenotype of the malignant cells, overexpression of PKC{delta} reduced and overexpression of PKC{alpha} increased glioma cell proliferation. PKC{alpha} has been reported previously (20) to be involved in cell proliferation in glioma cells, and its proliferative effect was suggested to be mediated via up-regulation of p21Waf1/Cip1 (34) . In contrast, less is known regarding the role of PKC{delta} in the proliferation of glioma cells. In other systems, PKC{delta} has been associated with the growth control of various cells in a cell type-specific manner; e.g., PKC{delta} inhibited the proliferation of fibroblasts (25) , keratinocytes (36) , glial cells (28) , and Chinese hamster ovary cells (37) . In contrast, in breast cancer cells PKC{delta} has been shown to increase transformation and metastatic progression (38) .

In addition to its inhibitory effect on cell proliferation, PKC{delta} induced marked effects on glioma cell morphology and on the expression of GFAP, whereas small and opposite effects were induced by PKC{alpha}. Thus, U87 cells overexpressing PKC{delta} exhibited larger cell bodies with multiple long processes and increased expression of GFAP as compared with control cells. The role of the intermediate filament GFAP in the function of astrocytes is not fully understood; however, it seems to play a role in the maintenance of the blood-brain barrier and in the ability of astrocytes to form glial scars after injury (39) . GFAP has also been implicated in the tumorigenesis of gliomas, and its expression appeared to correlate inversely with the malignancy of these tumors (39) . In addition, enforced expression of GFAP in glioma cells reduced cell motility, cell invasion, cell morphology, and proliferation (40 , 41) , whereas opposite effects were observed when GFAP-positive glioma cells were transfected with an antisense GFAP plasmid (42) . Thus, the increased expression of GFAP obtained in the PKC{delta} overexpressers may contribute to the marked changes in cell morphology and the decreased cell proliferation observed in these cells.

We found that the regulatory domain of PKC{delta} was responsible for the effects of this isoform on U87 cell morphology, proliferation, and GFAP expression. These results are similar to those we described recently (27 , 29) regarding the regulation of glutamine synthetase expression and cell proliferation by PKC{delta} in C6 glioma cells. Chimeras have been used to delineate the contributions of individual PKC domains to the specific functions of different PKC isoforms in a number of systems. Both the catalytic and the regulatory domains of PKC may determine isoform-specific functions depending on the specific system; e.g., the catalytic domain of PKCß was found to confer isoform-specific function in the differentiation of erythroleukemia cells (11) , and the catalytic domain of PKC{delta} in reciprocal {delta}- and {epsilon}-chimeras mediated PMA-induced macrophage differentiation of mouse promyelocytes (12) . In contrast, the regulatory domain of PKC{epsilon} enhanced cell growth and induced colonies in soft agar in NIH 3T3 cells (25) . Recently, it has been reported (43) that the regulatory domain of PKC{delta} overexpressed by itself inhibited mammary tumor cell metastases.

The differential effects of the chimeras may be attributable to their distinct localization after activation. In response to PMA, the chimera PKC{alpha}/{delta} translocated to the plasma membrane, similar to the translocation observed for PKC{alpha}, whereas the chimera PKC{delta}/{alpha} translocated to the nuclear and plasma membranes, similar to PKC{delta}. Thus, the differential translocation of PKC{alpha} and PKC{delta} may result in the activation of different downstream substrates that may lead to the distinct cellular effects of the chimeras.

We found that U87 cells exhibited a very low degree of apoptosis in response to etoposide and that PKC{alpha} and PKC{delta} played opposite roles in the apoptotic response of glioma cells to this drug. Both PKC{delta} and PKC{alpha} have been implicated in the regulation of cell apoptosis (36 , 44 , 45) . PKC{delta} was reported to be involved in the apoptosis of cells in response to a large number of stimuli including radiation (46) , H2O2 (47) , Fas ligation, and etoposide (32) . The effect of PKC{delta} appeared to be largely dependent on caspases, which results in the cleavage of PKC{delta} (30 , 31 , 44) , although caspase-independent effects of PKC{delta} have also been reported (48) . In contrast, the expression of PKC{alpha} has been mainly associated with resistance to apoptosis. Thus, specific depletion of PKC{alpha} induced apoptosis in glioma (20 , 24) and CHO cells. Similarly, PKC{alpha} has been suggested to increase resistance to chemotherapy by phosphorylating Bcl2 (45) . Although the decreased expression of PKC{alpha} in U87 cells in response to antisense treatment resulted in some spontaneous cell death, the response of the cells to etoposide remained similar, suggesting that the increased expression of PKC{alpha} is not involved in the resistance of U87 cells to the apoptosis induced by etoposide. In contrast, overexpression of PKC{delta} rendered the cells more sensitive to the apoptotic effect of etoposide. The role of PKC{delta} was further supported by the fact that glioma cells that express higher levels of endogenous PKC{delta} exhibited a higher degree of apoptosis in response to etoposide and their apoptotic response was blocked by the PKC{delta} inhibitor rottlerin. Thus, it appears that the low levels of PKC{delta} expressed in the U87 cells confer resistance against the apoptotic effect of etoposide.

The mechanism by which PKC{delta} is involved in the apoptosis induced by etoposide is currently not known. The effect of etoposide in our system appeared to be dependent on cleavage by caspases because the potent caspase inhibitors, Z-VAD.FMK and DEVD.FMK, blocked the cleavage of PKC{delta} in response to etoposide. The results regarding PKC{delta} are similar to the work reported by Reyland et al. (32) in acinar cells. However, in contrast to their results, we did not find an increase in the expression of PKC{alpha} in response to etoposide, suggesting that different cellular systems respond differently to etoposide. Cleavage of PKC{delta} by caspases has been reported in response to various stimuli such as 1-ß-D-arabinofuranosylcytosine (31) , ionizing radiation, and Fas ligation. The cleaved activated product, the PKC{delta} catalytic domain, can then modulate the activity of various important apoptotic-related kinases such as Erk, c-Jun terminal kinase, and the DNA protein kinase (49) . The mechanisms involved in the effects of etoposide on glioma cells are currently being investigated.

In summary, our results demonstrate that malignant gliomas express increased levels of PKC{alpha} and decreased levels of PKC{delta}. The decreased expression of PKC{delta} and the increased expression of PKC{alpha} appeared to play important roles in the proliferative responses of U87 cells. In contrast, only PKC{delta} appeared to be involved in the resistance of U87 cells to the apoptotic effect of etoposide. Our results suggest that imbalance in the expression of PKC{alpha} and PKC{delta} may be involved in the tumorigenesis of gliomas by regulating both proliferation and apoptosis.


   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 Nicol and Andre Bollag Stiftung and by a Research Grant awarded by the Israel Cancer Research Foundation. Back

2 To whom requests for reprints should be addressed, at Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Phone: 972-3-5318266; Fax: 972-3-5350234; E-mail: chaya{at}mail.biu.ac.il Back

3 The abbreviations used are: PKC, protein kinase C; PMA, phorbol myristate acetate; MTH, metallothionein promoter-driven eukaryotic expression vector; GFP, green fluorescent protein; PI, propidium iodide; GBM, glioblastoma multiforme; GFAP, glial fibrillary acidic protein; PDBu, phorbol 12,13 dibutyrate. Back

Received 12/27/00. Accepted 3/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature (Lond.), 308: 693-698, 1984.[Medline]
  2. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature (Lond.), 334: 661-665, 1988.[Medline]
  3. Stabel S., Parker P. J. Protein kinase C. Pharmacol. Ther., 51: 71-95, 1991.[Medline]
  4. Hug H., Sarre T. F. Protein kinase C isoenzymes: divergence in signal transduction?. Biochem. J., 291: 329-343, 1993.
  5. Johannes F. J., Prestle J., Eis S., Oberhagemann P., Pfizenmaier K. PKCµ is a novel, atypical member of the protein kinase C family. J. Biol. Chem., 269: 6140-6148, 1994.[Abstract/Free Full Text]
  6. Hayashi A., Seki N., Hattori A., Kozuma S., Saito T. PKC{nu}, a new member of the protein kinase C family, composes a fourth subfamily with PKCµ. Biochim. Biophys. Acta, 1450: 99-106, 1999.[Medline]
  7. Newton A. C. Protein kinase C. Seeing two domains. Curr. Biol., 5: 973-976, 1995.[Medline]
  8. Burns D. J., Bell R. M. Protein kinase C contains two phorbol ester binding domains. J. Biol. Chem., 266: 18330-18338, 1991.[Abstract/Free Full Text]
  9. Newton A. C. Protein kinase C: structure, function, and regulation. J. Biol. Chem., 270: 28495-28498, 1995.[Free Full Text]
  10. Hurley J. H., Newton A. C., Parker P. J., Blumberg P. M., Nishizuka Y. Taxonomy and function of C1 protein kinase C homology domains. Protein Sci., 6: 477-480, 1997.[Medline]
  11. Walker S. D., Murray N. R., Burns D. J., Fields A. P. Protein kinase C chimeras: catalytic domains of {alpha} and ß II protein kinase C contain determinants for isotype-specific function. Proc. Natl. Acad. Sci. USA, 92: 9156-9160, 1995.[Abstract/Free Full Text]
  12. Wang Q. J., Acs P., Goodnight J., Giese T., Blumberg P. M., Mischak H., Mushinski J. F. The catalytic domain of protein kinase C-{delta} in reciprocal {delta} and {epsilon} chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes. J. Biol. Chem., 272: 76-82, 1997.[Abstract/Free Full Text]
  13. Prados M. D., Levin V. Biology and treatment of malignant glioma. Semin. Oncol., 27: 1-10, 2000.
  14. Baltuch G. H., Yong V. W. Signal transduction for proliferation of glioma cells in vitro occurs predominantly through a protein kinase C-mediated pathway. Brain Res., 710: 143-149, 1996.[Medline]
  15. Bredel M., Pollack I. F. The role of protein kinase C (PKC) in the evolution and proliferation of malignant gliomas, and the application of PKC inhibition as a novel approach to anti-glioma therapy. Acta Neurochir., 139: 1000-1013, 1997.[Medline]
  16. von Deimling A., Louis D. N., Wiestler O. D. Molecular pathways in the formation of gliomas. Glia, 15: 328-338, 1995.[Medline]
  17. Bredel M., Pollack I. F. The p21-Ras signal transduction pathway and growth regulation in human high-grade gliomas. Brain Res. Rev., 29: 232-249, 1999.[Medline]
  18. Couldwell W. T., Antel J. P., Yong V. W. Protein kinase C activity correlates with the growth rate of malignant gliomas: part II. Effects of glioma mitogens and modulators of protein kinase C. Neurosurgery (Baltim.), 31: 717-724, 1992.[Medline]
  19. Couldwell W. T., Uhm J. H., Antel J. P., Yong V. W. Enhanced protein kinase C activity correlates with the growth rate of malignant gliomas in vitro. Neurosurgery (Baltim.), 29: 880-886, 1991.
  20. Ahmad S., Mineta T., Martuza R. L., Glazer R. I. Antisense expression of protein kinase C {alpha} inhibits the growth and tumorigenicity of human glioblastoma cells. Neurosurgery (Baltim.), 35: 904-908, 1994.[Medline]
  21. Hussaini I. M., Karns L. R., Vinton G., Carpenter J. E., Redpath G. T., Sando J. J., VendenBerg S. R. Phorbol-12-myristate-13-acetate induces protein kinase C{eta}-specific proliferative response in astrocytic tumor cells. J. Biol. Chem., 275: 22348-22354, 2000.[Abstract/Free Full Text]
  22. Xiao H., Goldthwait D. A., Mapstone T. The identification of four protein kinase C isoforms in human glioblastoma cell lines: PKC {alpha}, {gamma}, {epsilon}, and {zeta}. J. Neurosurg., 81: 734-740, 1994.[Medline]
  23. Sharif T. R., Sharif M. Overexpression of protein kinase C epsilon in astroglial brain tumor derived cell lines and primary tumor samples. Int. J. Oncol., 15: 237-243, 1999.[Medline]
  24. Sioud M., Sorensen D. R. A nuclease-resistant protein kinase C {alpha} ribozyme blocks glioma cell growth. Nat. Biotechnol., 16: 556-561, 1998.[Medline]
  25. Acs P., Wang Q. J., Bogi K., Marquez A. M., Lorenzo P. S., Biro T., Szallasi Z., Mushinski J. F., Blumberg P. M. Both the catalytic and regulatory domains of protein kinase C chimeras modulate the proliferative properties of NIH 3T3 cells. J. Biol. Chem., 272: 28793-28799, 1997.[Abstract/Free Full Text]
  26. Olah Z., Lehel C., Jakab G., Anderson W. B. A cloning and {epsilon}-epitope-tagging insert for the expression of polymerase chain reaction-generated cDNA fragments in Escherichia coli and mammalian cells. Anal. Biochem., 221: 94-102, 1994.[Medline]
  27. Brodie C., Bogi K., Acs P., Lorenzo P. S., Baskin L., Blumberg P. M. Protein kinase C {delta} (PKC{delta}) inhibits the expression of glutamine synthetase in glial cells via the PKC{delta} regulatory domain and its tyrosine phosphorylation. J. Biol. Chem., 273: 30713-30718, 1998.[Abstract/Free Full Text]
  28. Brodie C., Kuperstein I., Acs P., Blumberg P. M. Differential role of specific PKC isoforms in the proliferation of glial cells and the expression of the astrocytic markers GFAP and glutamine synthetase. Brain Res. Mol. Brain Res., 56: 108-117, 1998.[Medline]
  29. Kronfeld I., Kazimirsky G., Lorenzo P. S., Garfield S. H., Blumberg P. M., Brodie C. Phosphorylation of PKC{delta} on distinct tyrosine residues regulates specific cellular functions. J. Biol. Chem., 275: 35491-35498, 2000.[Abstract/Free Full Text]
  30. Ghayur T., Hugunin M., Talanian R. V., Ratnofsky S., Quinlan C., Emoto Y., Pandey P., Datta R., Huang Y., Kharbanda S., Allen H., Kamen R., Wong W., Kufe D. Proteolytic activation of protein kinase C{delta} by an ICE/CED 3-like protease induces characteristics of apoptosis. J. Exp. Med., 184: 2399-2404, 1996.[Abstract/Free Full Text]
  31. Koriyama H., Kouchi Z., Umeda T., Saido T. C., Momoi T., Ishiura S., Suzuki K. Proteolytic activation of protein kinase C{delta} and {epsilon} by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis. Cell. Signal., 11: 831-838, 1999.[Medline]
  32. Reyland M. E., Anderson S. M., Matassa A. A., Barzen K. A., Quissell D. O. Protein kinase C{delta} is essential for etoposide-induced apoptosis in salivary gland acinar cells. J. Biol. Chem., 274: 19115-19123, 1999.[Abstract/Free Full Text]
  33. Zellner A., Fetell M. R., Bruce J. N., De Vivo D. C., O’Driscoll K. R. Disparity in expression of protein kinase C{alpha} in human glioma versus glioma-derived primary cell lines: therapeutic implications. Clin. Cancer Res., 4: 1797-1802, 1998.[Abstract]
  34. Besson A., Yong V. W. Involvement of p21Waf1/Cip1 in protein kinase C{alpha}-induced cell cycle progression. Mol. Cell. Biol., 20: 4580-4590, 2000.[Abstract/Free Full Text]
  35. Mishima K., Ohno S., Shitara N., Yamaoka K., Suzuki K. Opposite effects of the overexpression of protein kinase C{gamma} and {delta} on the growth properties of human glioma cell line U251 MG. Biochem. Biophys. Res. Commun., 201: 363-372, 1994.[Medline]
  36. Li L., Lorenzo P. S., Bogi K., Blumberg P. M., Yuspa S. H. Protein kinase C{delta} targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector. Mol. Cell. Biol., 19: 8547-8558, 1999.[Abstract/Free Full Text]
  37. Watanabe T., Ono Y., Taniyama Y., Hazama K., Igarashi K., Ogita K., Kikkawa U., Nishizuka Y. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-{delta} subspecies. Proc. Natl. Acad. Sci. USA, 89: 10159-10163, 1992.[Abstract/Free Full Text]
  38. Li W., Jiang Y. X., Zhang J., Soon L., Flechner L., Kapoor V., Pierce J. H., Wang L. H. Protein kinase C-{delta} is an important signaling molecule in insulin-like growth factor I receptor-mediated cell transformation. Mol. Cell. Biol., 18: 5888-5898, 1998.[Abstract/Free Full Text]
  39. Rutka J. T., Murakami M., Dirks P. B., Hubbard S. L., Becker L. E., Fukuyama K., Jung S., Tsugu A., Matsuzawa K. Role of glial filaments in cells and tumors of glial origin: a review. J. Neurosurg., 87: 420-430, 1997.[Medline]
  40. Elobeid A., Bongcam-Rudloff E., Westermark B., Nister M. Effects of inducible glial fibrillary acidic protein on glioma cell motility and proliferation. J. Neurosci. Res., 60: 245-256, 2000.[Medline]
  41. Rutka J. T., Smith S. L. Transfection of human astrocytoma cells with glial fibrillary acidic protein complementary DNA: analysis of expression, proliferation, and tumorigenicity. Cancer Res., 53: 3624-3631, 1993.[Abstract/Free Full Text]
  42. Rutka J. T., Hubbard S. L., Fukuyama K., Matsuzawa K., Dirks P. B., Becker L. E. Effects of antisense glial fibrillary acidic protein complementary DNA on the growth, invasion, and adhesion of human astrocytoma cells. Cancer Res., 54: 3267-3272, 1994.[Abstract/Free Full Text]
  43. Kiley S. C., Clark K. J., Goodnough M., Welch D. R., Jaken S. Protein kinase C{delta} involvement in mammary tumor cell metastasis. Cancer Res., 59: 3230-3238, 1999.[Abstract/Free Full Text]
  44. Emoto Y., Manome Y., Meinhardt G., Kisaki H., Kharbanda S., Robertson M., Ghayur T., Wong W. W., Kamen R., Weichselbaum R. Proteolytic activation of protein kinase C{delta} by an ICE-like protease in apoptotic cells. EMBO J., 14: 6148-6156, 1995.[Medline]
  45. Ruvolo P. P., Deng X., Carr B. K., May W. S. A functional role for mitochondrial protein kinase C{alpha} in Bcl2 phosphorylation and suppression of apoptosis. J. Biol. Chem., 273: 25436-25442, 1998.[Abstract/Free Full Text]
  46. Yuan Z. M., Utsugisawa T., Ishiko T., Nakada S., Huang Y., Kharbanda S., Weichselbaum R., Kufe D. Activation of protein kinase C{delta} by the c-Abl tyrosine kinase in response to ionizing radiation. Oncogene, 16: 1643-1648, 1998.[Medline]
  47. Sun X., Wu F., Datta R., Kharbanda S., Kufe D. Interaction between protein kinase C{delta} and the c-Abl tyrosine kinase in the cellular response to oxidative stress. J. Biol. Chem., 275: 7470-7473, 2000.[Abstract/Free Full Text]
  48. Fujii T., Garcia-Bermejo M. L., Bernabo J. L., Caamano J., Ohba M., Kuroki T., Li L., Yuspa S. H., Kazanietz M. G. Involvement of protein kinase C {delta} (PKC{delta}) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage of PKC{delta}. J. Biol. Chem., 275: 7574-7582, 2000.[Abstract/Free Full Text]
  49. Bharti A., Kraeft S. K., Gounder M., Pandey P., Jin S., Yuan Z. M., Lees-Miller S. P., Weichselbaum R., Weaver D., Chen L. B., Kufe D., Kharbanda S. Inactivation of DNA-dependent protein kinase by protein kinase C{delta}: implications for apoptosis. Mol. Cell. Biol., 18: 6719-6728, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
A. Magnifico, L. Albano, S. Campaner, M. Campiglio, S. Pilotti, S. Menard, and E. Tagliabue
Protein Kinase C{alpha} Determines HER2 Fate in Breast Carcinoma Cells with HER2 Protein Overexpression without Gene Amplification
Cancer Res., June 1, 2007; 67(11): 5308 - 5317.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y.-H. Hsieh, T.-T. Wu, C.-Y. Huang, Y.-S. Hsieh, J.-M. Hwang, and J.-Y. Liu
p38 Mitogen-Activated Protein Kinase Pathway Is Involved in Protein Kinase C{alpha}-Regulated Invasion in Human Hepatocellular Carcinoma Cells
Cancer Res., May 1, 2007; 67(9): 4320 - 4327.
[Abstract] [Full Text] [PDF]

Home page
 Endocr Relat CancerHome page
J. M Haughian, T. A Jackson, D. M Koterwas, and A. P Bradford
Endometrial cancer cell survival and apoptosis is regulated by protein kinase C {alpha} and {delta}
Endocr. Relat. Cancer, December 1, 2006; 13(4): 1251 - 1267.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
V. Aaltonen, J. Koivunen, M. Laato, and J. Peltonen
Heterogeneity of Cellular Proliferation within Transitional Cell Carcinoma: Correlation of Protein Kinase C Alpha/betaI Expression and Activity
J. Histochem. Cytochem., July 1, 2006; 54(7): 795 - 806.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Nakagawa, J. L. Oliva, D. Kothapalli, A. Fournier, R. K. Assoian, and M. G. Kazanietz
Phorbol Ester-induced G1 Phase Arrest Selectively Mediated by Protein Kinase C{delta}-dependent Induction of p21
J. Biol. Chem., October 7, 2005; 280(40): 33926 - 33934.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H. Okhrimenko, W. Lu, C. Xiang, N. Hamburger, G. Kazimirsky, and C. Brodie
Protein Kinase C-{varepsilon} Regulates the Apoptosis and Survival of Glioma Cells
Cancer Res., August 15, 2005; 65(16): 7301 - 7309.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. Hara, Y. Saito, T. Hirai, K. Nakamura, K. Nakao, M. Katsuki, and K. Chida
Deficiency of Protein Kinase C{alpha} in Mice Results in Impairment of Epidermal Hyperplasia and Enhancement of Tumor Formation in Two-Stage Skin Carcinogenesis
Cancer Res., August 15, 2005; 65(16): 7356 - 7362.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
S. Gillespie, X. D. Zhang, and P. Hersey
Variable expression of protein kinase C{varepsilon} in human melanoma cells regulates sensitivity to TRAIL-induced apoptosis
Mol. Cancer Ther., April 1, 2005; 4(4): 668 - 676.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
H.-W. Lo, G. R. Antoun, and F. Ali-Osman
The Human Glutathione S-Transferase P1 Protein Is Phosphorylated and Its Metabolic Function Enhanced by the Ser/Thr Protein Kinases, cAMP-Dependent Protein Kinase and Protein Kinase C, in Glioblastoma Cells
Cancer Res., December 15, 2004; 64(24): 9131 - 9138.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
E. Deutsch, A. Cohen, G. Kazimirsky, S. Dovrat, H. Rubinfeld, C. Brodie, and R. Sarid
Role of Protein Kinase C {delta} in Reactivation of Kaposi's Sarcoma-Associated Herpesvirus
J. Virol., September 15, 2004; 78(18): 10187 - 10192.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Koivunen, V. Aaltonen, S. Koskela, P. Lehenkari, M. Laato, and J. Peltonen
Protein Kinase C {alpha}/{beta} Inhibitor Go6976 Promotes Formation of Cell Junctions and Inhibits Invasion of Urinary Bladder Carcinoma Cells
Cancer Res., August 15, 2004; 64(16): 5693 - 5701.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X.-H. Jiang, S.-P. Tu, J.-T. Cui, M. C. M. Lin, H. H. X. Xia, W. M. Wong, A. O.-O. Chan, M. F. Yuen, S.-H. Jiang, S.-K. Lam, et al.
Antisense Targeting Protein Kinase C {alpha} and {beta}1 Inhibits Gastric Carcinogenesis
Cancer Res., August 15, 2004; 64(16): 5787 - 5794.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Liu, J. Chen, Q. Dai, and R. M. Lee
Phospholipid Scramblase 3 Is the Mitochondrial Target of Protein Kinase C {delta}-induced Apoptosis
Cancer Res., March 15, 2003; 63(6): 1153 - 1156.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. S. Clark, K. A. West, P. M. Blumberg, and P. A. Dennis
Altered Protein Kinase C (PKC) Isoforms in Non-Small Cell Lung Cancer Cells: PKC{delta} Promotes Cellular Survival and Chemotherapeutic Resistance
Cancer Res., February 15, 2003; 63(4): 780 - 786.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. K. Berdiev, J. Xia, B. Jovov, J. M. Markert, T. B. Mapstone, G. Y. Gillespie, C. M. Fuller, J. K. Bubien, and D. J. Benos
Protein Kinase C Isoform Antagonism Controls BNaC2 (ASIC1) Function
J. Biol. Chem., November 22, 2002; 277(48): 45734 - 45740.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
B. C. Morrish and M. G. Rumsby
The 5' Untranslated Region of Protein Kinase C{delta} Directs Translation by an Internal Ribosome Entry Segment That Is Most Active in Densely Growing Cells and during Apoptosis
Mol. Cell. Biol., September 1, 2002; 22(17): 6089 - 6099.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
K. M. Lounsbury, M. Stern, D. Taatjes, S. Jaken, and B. T. Mossman
Increased Localization and Substrate Activation of Protein Kinase C{delta} in Lung Epithelial Cells following Exposure to Asbestos
Am. J. Pathol., June 1, 2002; 160(6): 1991 - 2000.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. T. M. Boudreau, R. Garduno, and T.-J. Lin
Protein Phosphatase 2A and Protein Kinase Calpha Are Physically Associated and Are Involved in Pseudomonas aeruginosa-induced Interleukin 6 Production by Mast Cells
J. Biol. Chem., February 8, 2002; 277(7): 5322 - 5329.
[Abstract] [Full Text] [PDF]

Home page
 The OncologistHome page
A. B. da Rocha, D.R.A. Mans, A. Regner, and G. Schwartsmann
Targeting Protein Kinase C: New Therapeutic Opportunities Against High-Grade Malignant Gliomas?
Oncologist, February 1, 2002; 7(1): 17 - 33.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Blass, I. Kronfeld, G. Kazimirsky, P. M. Blumberg, and C. Brodie
Tyrosine Phosphorylation of Protein Kinase C{delta} Is Essential for Its Apoptotic Effect in Response to Etoposide
Mol. Cell. Biol., January 1, 2002; 22(1): 182 - 195.
[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 Email this article to a friend
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 Mandil, R.
Right arrow Articles by Brodie, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mandil, R.
Right arrow Articles by Brodie, C.

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