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[Cancer Research 62, 1815-1821, March 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Atypical Protein Kinase C {zeta} as a Target for Chemosensitization of Tumor Cells1

Rodolphe Filomenko, Florence Poirson-Bichat, Claire Billerey, Jean-Paul Belon, Carmen Garrido, Eric Solary and Ali Bettaieb2

INSERM U517, EPHE, Ecole Pratique des Hautes Etudes, IFR, Institut Féderatif de Recherche 100, Faculties of Medicine & Pharmacy, 21000 Dijon, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of tumor cells to cytotoxic agents simultaneously activates a variety of intracellular signaling pathways. Some of these pathways involve enzymes from the protein kinase C (PKC) family of serine/threonine kinases. This family includes isoenzymes that negatively influence cell death, whereas other demonstrate an opposite effect. The present study analyzes the role of the {zeta} atypical PKC isoform in tumor cell response to cytotoxic agents. Using a histone H1 phosphorylation assay, we showed that both tumor necrosis factor {alpha} and etoposide activate PKC{zeta} in U937 human leukemic cells. Stable transfection of a kinase-dead, dominant-negative PKC{zeta} mutant in U937 cells decreases Bcl-2 expression while increasing the expression of Bax and several procaspases. This transfection also prevents etoposide-induced nuclear factor-{kappa}B nuclear translocation and accumulation of X-linked inhibitor of apoptosis protein. PKC{zeta} inhibition accelerates the occurrence of apoptosis in leukemic cells exposed to etoposide and tumor necrosis factor {alpha}. This sensitization was confirmed in vitro by use of a clonogenic assay. In addition, PKC{zeta} inhibition sensitized tumor cells grown in nude mice to etoposide. These results indicate that PKC{zeta} isoform is a protective signals that is activated in tumor cells exposed to a cytotoxic agent. This inducible resistance factor thus appears an attractive target for chemosensitization of tumor cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxic drugs are commonly used for treating malignant diseases by inducing tumor cell death. These drugs usually enter the cell, are possibly metabolized into a more active derivative, and then interact with a target protein or the DNA. Ideally, the damage that results from this interaction eventually activates the cell death program. In addition to interacting with their intracellular target, anticancer drugs activate both protective and noxious pathways that modulate cellular drug accumulation, drug metabolism, drug-target interaction, and downstream cell death pathways (1) . For example, cytotoxic agents activate lipid-dependent signaling pathways, including generation of ceramide, which contributes to cell death, and diacylglycerol, which demonstrates opposite effects (2 , 3) . Anticancer agents also activate the transcription factor NF-{kappa}B3 by triggering degradation of its inhibitor I{kappa}B, and this response efficiently suppresses apoptotic cell death (4 , 5) . It was shown recently that adenoviral delivery of a modified nondegraded form of I{kappa}B enhances the therapeutic efficacy of the chemotherapeutic compound CPT-11 and the cytokine TNF{alpha}, indicating that inhibition of the protective signal transduction pathways activated by anticancer agents (also called "inducible chemoresistance") is an efficient strategy to improve chemotherapeutic drug efficacy (6) .

Some lipid second messengers, such as diacylglycerol, soluble inositol phosphates, and phosphoinositide 3-kinase products, activate the PKC family of serine/threonine kinases. These kinases mediate a central cytoprotective effect by opposing initiation of cell death pathways by diverse cytotoxic stimuli (7) . Accordingly, inhibitors of PKCs have been shown to enhance the cytotoxic activity of various classes of anticancer drugs and ionizing radiation (8) . The PKC family comprises at least 11 structurally and functionally related isoforms in mammals. Three major subfamilies have been described, based on their differing sensitivities to calcium and lipid activators. These isoenzymes show differential requirements for cofactors, are regulated independently, and are differentially expressed according to individual cell types (9) . All of the PKC isoenzymes do not demonstrate the same influence on cell death pathways. Whereas the PKC{alpha} and -{zeta} isoforms (10 , 11) both demonstrate antiapoptotic activities, the PKC{delta} isoform has been shown to contribute to cell death (12) . Thus, specific inhibition of an antiapoptotic PKC isotype may be more appropriate than nonspecific inhibition of PKCs to sensitize tumor cells to chemotherapeutic drugs.

The present study analyzed the role of PKC{zeta} in tumor cell chemosensitivity. This isoform belongs to the "atypical" subfamily of PKCs, which includes two members, referred to as PKC{lambda}/{iota} and PKC{zeta}. These isoforms differ from other PKCs by their insensitivity to calcium, phorbol esters, and diacylglycerol (13, 14, 15, 16, 17) , whereas their activity is regulated by phosphoinositide 3-kinase, ceramide, and Ras (11 , 18) . To determine whether PKC{zeta} is involved in tumor cell response to chemotherapeutic drugs and cytokines, we stably expressed a kinase-dead, dominant-negative mutant of PKC{zeta} in leukemic and colon cancer cell lines. Our results indicate that PKC{zeta} may be a useful target for tumor cell chemosensitization.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Etoposide (VP-16) and histone H1 were obtained from Sigma Chemical Co.-Aldrich Laboratories (St. Quentin Fallavier, France). Human recombinant TNF{alpha} was obtained from PeproTech-Tebu (Le Perray-en-Yvelines, France) and [{gamma}-32P]ATP (7000 Ci/mmol) from Amersham (Les Ulis, France). All other chemicals were of reagent grade and purchased from local sources. Antibodies included monoclonal antibodies against human procaspase-2 and -3 (PharMingen, San Diego, CA), Bcl-2 and -xL (Dako, Trappes, France), and Bax (Immunotech, Marseille, France) and polyclonal antibodies against human PKC{zeta} (Boehringer-Mannheim, Meylan, France), Mcl-1, I{kappa}B{alpha}, and I{kappa}Bß (Santa Cruz Biotechnology, Santa Cruz, CA). The kinase-defective PKC{zeta} cDNA, generously provided by Jorge Moscat (Molecular Biology Center, Madrid, Spain), was inserted in the pRcCMV plasmid (Invitrogen, Groningen, the Netherlands). The L275T substitution suppresses a critical amino acid for ATP binding in the catalytic domain, thereby inactivating the enzyme, which behaves as a dominant-negative mutant (11 , 19) .

Cell Culture.
The human leukemic U937 and the colon carcinoma HT-29 cell lines were purchased from the American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 and Eagle’s MEM (BioWhittaker, Vervier, Belgium), respectively. These media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 2 mM L-glutamine in an atmosphere of 95% air and 5% CO2 at 37°C. Cell viability was determined by the trypan blue exclusion test. To ensure exponential growth, 24 h before each treatment U937 cells were resuspended at a density of 0.5 x 106 cells/ml in fresh medium and HT-29 cells were detached by trypsinization and reimplanted at 2 x 105 cells/ml. Stable transfection was performed by electroporation at the capacitance of 960 µF and 300 V/0.4 cm for U937 cells and by the use of Superfect (Qiagen, Courtaboeuf, France) for HT-29 cells, followed by selection in the presence of 1 mg/ml G418 (Life Technologies, Inc., Cergy-Pontoise, France) and limiting dilution cloning.

Determination of PKC{zeta} Kinase Activity.
U937 cells (3 x 106) were incubated for 30 min at 4°C in lysis buffer A [20 mM HEPES (pH 7.4), 10 mM EDTA, 125 mM NaCl, 1 mM DTT, 1 mM sodium orthovanadate, 0.5 mg/ml benzamidine, 1% NP40] in the presence of protease inhibitors [0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2.5 µg/ml pepstatin, 5 µg/ml leupeptin]. Samples were centrifuged at 20,000 x g for 25 min, and supernatants were used for immunoprecipitation with 0.5 µg of PKC{zeta} polyclonal antibody and 25 µl of protein A-Sepharose beads. PKC{zeta}-containing immunoprecipitates were washed three times in buffer A, followed by incubation at 32°C for 15 min in a total volume of 40 µl with buffer B [0.5 mM EGTA, 12.5 mM MgCl2, 20 mM HEPES (pH 7.5)] containing 30 µg of histone H1 as substrate and 10 µM [{gamma}-32P]ATP (1.5 µCi/tube). Immunoprecipitates were mixed (v/v) with loading buffer [125 mM Tris-HCl (pH 6.8), 10% ß-mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003% bromphenol blue], boiled for 5 min, and separated by SDS-PAGE. Phosphorylated histone H1 was quantified by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Analysis of Apoptotic Cells.
Apoptosis was assessed by staining the nuclear chromatin with Hoechst 33342 dye. Briefly, untreated and treated cells (1 x 106) were collected, washed with PBS, mounted on glass slides by cytospin centrifugation, fixed for 1 min with methanol, washed with PBS, stained with 1 µg/ml Hoechst 33342 dye for 10 min, and observed under a microscope. The percentage of apoptotic cells (chromatin condensation and nuclear fragmentation) was determined by counting 300 cells.

Clonogenic Assays.
Cells were incubated for 2 h in the presence or absence of various concentrations of cytotoxic agents. U937 cells were then plated at various densities in semisolid medium by the methylcellulose technique (20) , whereas HT-29 cells were layered at various densities on plastic flasks. Cultures were incubated in a humidified 5% CO2 incubator at 37°C, and colonies were scored at day 12.

Western Blot Analyses.
Cells were washed twice with PBS, lysed in RIPA buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 0.5% sodium desoxycholate] in the presence of protease inhibitors [0.1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml pepstatin, 10 µg/ml aprotinin, 2.5 µg/ml trypsin inhibitor, 5 µg/ml leupeptin] for 30 min, then centrifuged for 20 min at 15,000 x g. Fifty µg of supernatant proteins were mixed (v/v) with loading buffer [125 mM Tris-HCl (pH 6.8), 10% ß-mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003% bromphenol blue], boiled for 5 min, separated by SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Ivry sur Seine, France). After nonspecific binding sites were blocked overnight by 5% nonfat milk in PBS containing 0.1% Tween 20, membranes were probed with primary antibodies, and immunoreactive proteins were visualized using peroxidase-conjugated goat antibodies against rabbit or mouse immunoglobulins and chemiluminescent peroxidase substrate (Amersham, Les Ulis, France).

EMSA.
Cell nuclear extracts (4 µg) were prepared as described (21) and incubated with 1 µg of poly(dI-dC) and 100,000 cpm of 32P-end labeled NF-{kappa}B (5'-AGTTGAGGGGCTTTCCCAGGC-3') consensus oligonucleotide in a reaction buffer [2 mM HEPES (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol, 0.5 mM DTT, and 2 µg of BSA]. After 15 min, DNA-protein complexes were separated from free oligonucleotides by electrophoresis in a 4% polyacrylamide gel and detected by a PhosphorImager.

Cell Fractionation.
Mitochondrial and cytosolic (S100) fractions were prepared as described (22) . Briefly, cells were resuspended in ice-cold buffer A [250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 17 µg/ml phenylmethylsulfonyl fluoride, 8 µg/ml aprotinin, 2 µg/ml leupeptin (pH 7.4)], passed through an ice-cold cylinder cell homogenizer, and centrifuged at 750 x g for 10 min to pellet nonlysed cells and nuclei. The supernatant was centrifuged at 10,000 x g for 25 min to pellet the mitochondria. The resulting supernatant was further centrifuged at 100,000 xg for 1 h to generate the S100 or cytosolic fraction (last supernatant) that was frozen at -80°C.

Caspase Activation in a Cell-free System.
The cytosolic fractions from control- and mutant-transfected clones (10 µl; 5–10 mg/ml protein) were incubated for 30 min with 5 mM horse heart cytochrome c (Sigma Chemical Co.-Aldrich) and 1 mM dATP (Pharmacia, Uppsala, Sweden). Caspase-3 activity was then quantified by measuring the cleavage of the fluorometric substrate DEVD-AFC (France Biochem, Meudon, France) using a Berthold fluorometer (Bad Wildbad, Germany) with a 400 nm excitation length and a 505 nm emission length.

In Vivo Studies.
Swiss (nu/nu) mice, 7 weeks of age, were bred in the animal facilities of the Burgundy University (France) and maintained under specified pathogen-free conditions. Animal care and housing were in accordance with institutional guidelines from the French Ethical Committee (Ministère de l’Agriculture, Paris, France) and were under the supervision of authorized investigators. U937 xenografts were established by a s.c. injection of 20 x 106 U937 cells (viability >90%) into the flanks of nude mice randomly assessed to groups of eight animals. As soon as a local tumor was palpable (5 mm diameter), treatment was started with i.p. etoposide (12 mg/kg/day) for 3 consecutive days. Control animals were treated by injection of the vehicle. Tumor volume (V) was determined every 3 days by measuring two perpendicular diameters with a caliper and calculated as: V = a2 x b/2, where a is the width and b is the length of the tumor in mm. Tumor growth inhibition was calculated as the ratio of mean tumor volumes in treated and control groups at a given time, multiplied by 100. Mice were killed by prolonged anesthesia when the tumor volume reached 2000 mm3 in the control group. The statistical significance of differences in tumor volumes between groups was calculated by ANOVA (GraphPad InStat, San Diego, CA) and Student’s t test.

Immunohistochemistry.
Tumor biopsies fixed in 10% formalin were paraffin embedded. Sections (5 µm) were deparaffinized and rehydrated, and slides were soaked in citrate solution for microwave treatment. Endogenous peroxidase was blocked by incubation in 3% H2O2 in water for 10 min at room temperature. After sections were washed in PBS containing 0.1% Triton X-100, nonspecific binding sites were blocked with donkey serum in PBS containing 3% BSA and 0.1% Triton X-100 for 30 min. Sections were incubated overnight at 4°C with a polyclonal antibody directed against the cleaved and active caspase-3 form (Cell Signaling, Beverly, MA), diluted in PBS containing 3% BSA and 0.1% Triton X-100. After washing, sections were incubated with a biotin-conjugated goat antirabbit IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA) diluted in PBS containing 0.1% Triton X-100 and 5% rat serum at 4°C for 2 h. A streptavidin/peroxidase complex was then added (StreptABComplex Duet Reagent Set Kit; Dako). Slides were revealed with DAB (Dako), counterstained with hematoxylin, and mounted with Eukitt.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Both TNF{alpha} and the Cytotoxic Drug Etoposide Activate PKC{zeta} in Human Leukemic Cells.
U937 cells were previously shown to respond to TNF{alpha} treatment with enhanced PKC{zeta} phosphorylation and kinase activity (23) . These results were confirmed by exposing U937 cells to 25 ng/ml TNF{alpha} and then immunoprecipitating PKC{zeta} at various times to measure its ability to phosphorylate histone H1. TNF{alpha} induced a rapid and transient increase in PKC{zeta} activity. This increase reached a maximum (2–3-fold increase) between 10 and 15 min after the beginning of cell treatment, then declined to basal levels after 60 min (Fig. 1A)Citation . Continuous exposure of U937 cells to the cytotoxic drug etoposide, a topoisomerase II inhibitor, at a concentration of 50 µM also enhanced PKC{zeta} kinase activity. This increase became significant after 1 h, reached a maximum (8-fold increase) at 120 min, then declined at 180 min (Fig. 1B)Citation .



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  		Fig. 1. PKC{zeta} activity in U937 cells treated with cytotoxic agents. U937 cells were treated with 25 ng/ml TNF{alpha} (A) or 50 µM etoposide (B) for the indicated times before PKC{zeta} activity was assessed. Histone H1 phosphorylation was visualized by Western blotting (representative experiments shown in bottom panels) and quantified by a PhosphorImager [top panels; means ± SD (bars) of at least three independent experiments]; *, P < 0.05 compared with controls.

 
Expression of a Kinase-defective PKC{zeta} Mutant Prevents PKC{zeta} Activation in U937 Cells.
U937 cells were transfected with either an insertless vector or a kinase-defective PKC{zeta}-containing vector. PKC{zeta} expression in transfected cells was monitored by immunoblotting using a polyclonal antibody raised against the whole protein. Two control- and two PKC{zeta} mutant-transfected clones were selected for further analyses. PKC{zeta} expression was ~3-fold higher in the two clones transfected with the kinase-defective PKC{zeta} mutant (Z4 and Z11) when compared with the two control clones (C1 and C10; Fig. 2ACitation ). We did not detect any decrease in the basal kinase activity in the two PKC{zeta} mutant-containing clones (data not shown). As observed in parental cells, exposure of control cells (transfected with the empty vector) to 50 µM etoposide for 2 h (Fig. 2B)Citation or to 25 ng/ml TNF{alpha} for 10 min (Fig. 2C)Citation increased PKC{zeta} activity. Expression of the kinase-defective PKC{zeta} completely prevented etoposide- (Fig. 2B)Citation and TNF{alpha}-induced (Fig. 2C)Citation activation of endogenous PKC{zeta}.



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  		Fig. 2. Characterization of U937 cells transfected with a kinase-defective PKC{zeta} mutant. U937 cells were transfected with an empty or a PKC{zeta} mutant-containing vector, and four clones were selected for further analyses. A, PKC{zeta} expression was assessed by Western blotting in control (C1 and C10) and PKC{zeta} mutant-containing (Z4 and Z11) clones. Heat shock cognate 70 protein (HSC 70) was used as a loading control, and the PKC{zeta}/HSC 70 ratio was determined by densitometry. B, PKC{zeta} activity induced by exposure to 50 µM etoposide for 2 h was assessed in these clones. Results were related to those obtained in the C1 clone and expressed as means ± SD (bars) of three independent experiments; *, P < 0.05. C, PKC{zeta} activity induced by exposure to 25 ng/ml TNF{alpha} for 10 min in these clones [means ± SD (bars) of three independent experiments]; *, P < 0.05. D, NF-{kappa}B DNA-binding activity was assessed by an EMSA in control-transfected (C1 clone) and PKC{zeta} mutant-transfected (Z11 clone) cells exposed to 50 µM etoposide for indicated times (one representative of three experiments is shown).

 
Because atypical PKCs have been shown to modulate NF-{kappa}B activation (24 , 25) , we analyzed the influence of kinase-defective PKC{zeta} transfection on this molecular target. EMSAs demonstrated that exposure of control-transfected U937 cells to 50 µM etoposide induced a time-dependent increase in NF-{kappa}B DNA binding activity that was not observed in PKC{zeta} mutant-transfected cells (Fig. 2D)Citation .

Etoposide-induced activation of endogenous PKC{zeta} was still observed at a 100-fold lower concentration (0.5 µM), which is closer to clinically relevant concentrations (Fig. 3A)Citation , and this activation was prevented by expression of mutated PKC{zeta} (Fig. 3B)Citation . At this concentration, etoposide induced a time-dependent nuclear accumulation of NF-{kappa}B that correlated with a decrease in I{kappa}Bß expression (Fig. 3C)Citation . In contrast, neither NF-{kappa}B nuclear accumulation nor decreased I{kappa}Bß expression could be detected in PKC{zeta} mutant-expressing cells treated in similar conditions (Fig. 3C)Citation . No change was observed in I{kappa}B{alpha} expression in control and PKC{zeta} mutant cells during etoposide stimulation. Lastly, expression of PKC{zeta} mutant was associated with decreased expression of XIAP, a caspase-inhibitory protein whose gene transcription is activated by NF-{kappa}B (Ref. 26 ; Fig. 3CCitation ).



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  		Fig. 3. Influence of the kinase-defective PKC{zeta} mutant on PKC{zeta} activity and the NF-{kappa}B pathway. U937 cells (A) and PKC{zeta} mutant-transfected cells (B; {blacksquare}, Z4; , Z11) were treated with 0.5 µM etoposide for indicated times before PKC{zeta} activity was measured as in Fig. 1Citation [means ± SD (bars) of three independent experiments]; *, P < 0.05. C, NF-{kappa}B expression (p50) was studied by immunoblotting in nuclear extracts from control-transfected (C1 clone) and PKC{zeta} mutant-transfected (Z11 clone) cells exposed to 0.5 µM etoposide for indicated times. Whole cell extracts were used for immunoblotting analysis of I{kappa}B{alpha}, I{kappa}Bß, and XIAP expression. One representative of three independent experiments is shown.

 
Expression of a Kinase-defective PKC{zeta} Mutant Sensitizes U937 Cells to Cytotoxic Agent-induced Apoptosis.
Immunoblot analyses demonstrated increased expression of several proapoptotic molecules in PKC{zeta} mutant-transfected cells. These molecules included procaspase-2 and -3 and the Bcl-2-related protein Bax (Fig. 4A)Citation . We also identified decreased expression of the antiapoptotic Bcl-2 protein in PKC{zeta} mutant- compared with control-transfected cells. The expression of Bcl-xL and Mcl-1, two other Bcl-2-related antiapoptotic proteins, remained unchanged (Fig. 4A)Citation . In a cell-free system, the combination of cytochrome c (5 µM) and dATP (1 mM) recapitulates some of the postmitochondrial steps of apoptosis, e.g., by activating effector caspases that cleave the DEVD-AFC substrate. Using this assay, we observed that the kinetics of DEVD-AFC substrate cleavage was increased in the cell-free system established from PKC{zeta} mutant-transfected cells compared with the cell-free system established from control-transfected cells (Fig. 4B)Citation . When identified morphologically by staining of nuclear chromatin with the dye Hoechst 33342, apoptosis induced by exposure of whole cells to 0.5 µM etoposide or 25 ng/ml TNF{alpha} for 24 h was 2–3-fold increased in PKC{zeta} mutant- compared with control-transfected cells (Fig. 4C)Citation . Accordingly, a release of cytochrome c from the mitochondria to the cytosol was identified in PKC{zeta} mutant-transfected cells exposed to 0.5 µM etoposide for 24 h, whereas no release was identified in control cells treated in similar conditions (Fig. 4D)Citation . This effect of the PKC{zeta} kinase-dead mutant was not observed at later time points (not shown). Thus, inhibition of PKC{zeta} accelerates the occurrence of apoptosis after treatment with etoposide or TNF{alpha}.



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  		Fig. 4. Influence of PKC{zeta} on cytotoxic agent-induced apoptosis. A, immunoblot analysis of indicated proteins in the four studied clones. One representative of at least three independent experiments is shown. B, effects of cytochrome c and dATP on the caspase substrate DEVD-AFC when added to cell-free extracts from the four studied clones. Dashed lines, C1 and C10 clones; solid lines, Z4 and Z11 clones. Means ± SD (bars) of triplicate measurements in one representative of two experiments. C, apoptotic cells were identified morphologically after nuclear chromatin staining with Hoechst 33342. The four studied clones were treated with etoposide (VP-16, 0.5 µM for 24 h; top panel) or TNF{alpha} (25 ng/ml for 24 h; bottom panel). Mean ± SD (bars) of three independent experiments. D, immunoblot analysis of cytochrome c expression in mitochondria (Mito) and cytosol (Cyto) extracts from control-transfected (clone C10) and PKC{zeta} mutant-transfected (Z4 clone) cells exposed to 0.5 µM etoposide for 24 h.

 
Kinase-defective PKC{zeta} Mutant Sensitizes Tumor Cells to Chemotherapeutic Drug-induced Cytotoxic Activity in Vitro.
To further assess the influence of PKC{zeta} on anticancer drug-induced cytotoxicity, we performed clonogenic assays. Control- and PKC{zeta} mutant-transfected U937 cells were treated for 2 h with various concentrations of etoposide and then cultured in methylcellulose for 12 days before colonies were counted. Stable transfection of PKC{zeta} mutant did not affect the clonogenicity of untreated U937 cells (data not shown). Expression of PKC{zeta} mutant sensitized U937 to the cytotoxic activity of etoposide at concentrations ranging from 0.1 to 5 µM (Fig. 5A)Citation and TNF{alpha} concentrations ranging from 0.1 to 50 ng/ml (Fig. 5B)Citation . To further assess the role of PKC{zeta} in tumor cell sensitivity to cytotoxic drugs, we stably transfected the HT-29 colon cancer cell line with either the empty vector or the kinase-defective PKC{zeta} mutant and exposed the transfected cell populations for 2 h to various concentrations of the chemotherapeutic agent cisplatin. After drug removal, these cells were layered over plastic dishes, and colonies were scored after 12 days of culture. Expression of the kinase-defective PKC{zeta}-sensitized HT-29 cells to cisplatin-induced cytotoxicity, e.g., exposure to 5 µg/ml cisplatin for 2 h, induced a 70% decrease in the colony number in PKC{zeta} mutant-transfected cells compared with a 20% decrease in control-transfected cells (Fig. 5C)Citation .



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  		Fig. 5. Influence of PKC{zeta} on tumor cell sensitivity to cytotoxic agents in vitro. The cytotoxic activities of etoposide (VP-16; A) and TNF{alpha} (B) were measured in control (C1 and C10; solid lines) and kinase-defective PKC{zeta}-transfected (Z4 and Z11; dotted lines) U937 cells by use of a clonogenic assay in methylcellulose. Colonies were scored at day 12 with an inverted microscope. Means ± SD (bars) are representative of three independent experiments carried out in triplicate. C, HT-29 colon cancer cells were stably transfected with the empty vector (solid line) or the kinase-defective PKC{zeta} (dotted line), and then exposed to indicated concentrations of cisplatin before plating at various densities. The colonies were counted at day 12. One representative [mean ± SD (bars)] of triplicates of three independent experiments is shown. *, P < 0.05.

 
Kinase-defective PKC{zeta} Mutant Sensitizes Tumor Cells to Chemotherapeutic Drug-induced Cytotoxic Activity in Vivo.
To further examine the influence of the kinase-defective PKC{zeta} mutant on tumor cell response to cytotoxic agents, we performed in vivo experiments by injecting the parental U937 cells and the transfected clones s.c. into nude mice. As soon as tumors were palpable, animals were treated with either PBS or etoposide (12 mg/kg/day i.p. for 3 consecutive days). Tumor size was then measured every 3 days for 8 days. Etoposide demonstrated limited tumor growth-inhibitory activity in mice bearing parental and control tumors, whereas the drug induced a significant tumor growth inhibition in animals bearing a PKC{zeta} mutant tumor (Fig. 6ACitation and Table 1Citation ; P < 0.0001). Enhanced tumor regression was associated with an increased number of cells labeled with an antiactive caspase-3 when studied at day 4 after the beginning of etoposide treatment (Fig. 6B)Citation . Thus, the kinase-defective PKC{zeta} mutant sensitizes tumor cells to etoposide in vivo.



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  		Fig. 6. Influence of PKC{zeta} on tumor cell sensitivity to cytotoxic agents in vivo. A, parental U937 cells (top panel) and U937 cells transfected with an empty vector (C10; middle panel) or a kinase-defective PKC{zeta} (Z4; bottom panel) were injected sc into nude mice. As soon as tumors were palpable, animals (8/group) were treated with etoposide (VP-16; 12 mg/kg/day i.p. for 3 consecutive days; closed symbols). Controls (A; open symbols) were animals injected with the vehicle alone. Tumor size was measured every 3 days for 8 days. Statistical test was ANOVA. NS, not significant. B, active caspase-3 expression in tumors obtained at day 4 in animals treated with etoposide (right panels) or vehicle alone (left panels).

 

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  		Table 1 Influence of PKC{zeta} on tumor cell sensitivity to cytotoxic agents in vivo

Empty vector-transfected (C10), kinase-defective PKC{zeta}-transfected (Z4) U937 cells, and parental U937 cells were injected s.c. into nude mice. As soon as tumors were palpable, animals (8 per group) were treated with etoposide (VP-16; 12 mg/kg/day i.p. for 3 consecutive days). Controls (vehicle) were animals injected with the vehicle alone. Tumor size was measured every 3 days for 12 days. Doubling times were calculated as described in "Materials and Methods." The median percentage of tumor growth inhibition is shown.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumor cell resistance to chemotherapy can involve a series of mechanisms that prevent drug interaction with its main cellular target, e.g., as a consequence of P-glycoprotein-mediated drug efflux, altered intracellular metabolism, or mutation of a target protein (27) . Abnormal expression of proteins that prevent cell death also accounts for tumor cell resistance (28) . More recently, it was shown that anticancer drugs and cytokines activate protective signaling pathways whose balance with noxious signals determines the fate of the target cell (29) . The present study demonstrates that the atypical PKC{zeta} isoform is one of the protective signals that can be activated by cytotoxic compounds in tumor cells and contribute to this so-called inducible resistance (6) . Using three different experimental conditions, including short- and long-term in vitro assays and in vivo experiments, we show here that the PKC{zeta} kinase mutant behaves as a tumor cell chemosensitizer.

NF-{kappa}B is one of the protective signals that are activated by TNF{alpha} (30, 31, 32) and chemotherapeutic agents (4 , 5 , 33) . This transcription factor has been shown to potently suppress the apoptotic potential of anticancer drugs and cytokines in vitro (26) . Inhibition of NF-{kappa}B through adenoviral delivery of a modified, nonphosphorylatable form of I{kappa}B{alpha} sensitizes chemoresistant tumors to the apoptotic potential of TNF{alpha} and the camptothecin analogue CTP-11, resulting in tumor regression in an animal model (6) . PKC{zeta} takes a central position in the TNF{alpha} signaling pathway that activates NF-{kappa}B. TNF receptor 1 interacts with the death domain kinase RIP through the adaptor molecule TRADD. Although its kinase activity is not required for NF-{kappa}B activation, RIP recruits the atypical PKCs via the recently identified p62/{zeta}-interacting protein (ZIP; Ref. 34 ). In turn, atypical PKCs associate with and directly phosphorylate an I{kappa}B kinase activity, leading to I{kappa}B phosphorylation and degradation and to NF-{kappa}B nuclear translocation (24 , 25 , 35) . Chemosensitization of tumor cells through introduction of a dominant-negative mutant of PKC{zeta} could be mediated partly by the down-regulation of NF-{kappa}B. The present study suggests that etoposide-induced NF-{kappa}B activation involves PKC{zeta} because this activation is prevented by expression of a PKC{zeta} kinase-dead mutant. Etoposide-induced PKC{zeta} activation is associated with the degradation of I{kappa}Bß that permits the nuclear translocation of NF-{kappa}B (36) . These events correlate with an accumulation of the caspase inhibitory protein XIAP, whose expression was shown to be regulated by NF-{kappa}B (37) . All of these changes are prevented by expression of the PKC{zeta} dominant-negative mutant. Thus, targeting PKC{zeta} could be proposed as an alternative strategy for down-regulating NF-{kappa}B-mediated inducible resistance to chemotherapy.

This potential interest for targeting the PKC{zeta} isoform is enforced by its ability to activate additional protective signals that do not depend on NF-{kappa}B activation. For example, PKC{zeta} binds to and is regulated by a widely expressed protein known as Par-4 (38) . This leucine-zipper protein interacts with a zinc finger motif in the regulatory domain of atypical PKCs, which dramatically inhibits their kinase activity. Enforced expression of Par-4 induces apoptosis, which is abrogated by cotransfection of PKC{zeta} but not its kinase-inactive mutant (39) . The level of Par-4 expression has also been demonstrated to determine tumor cell sensitivity to a variety of apoptotic stimuli (40) . Thus, inhibition of PKC{zeta} by transfection of a kinase-dead mutant may increase the ability of Par-4 to promote apoptosis, e.g., by decreasing Bcl-2 expression (41) . In addition, the cytoplasmic and membrane-associated protein p62/ZIP targets the activity of PKC{zeta} to the Kvß2 subunits of potassium channels (42) . Given the importance of these channels for the regulation of some apoptotic pathways (43) , alterations of PKC{zeta} interaction with the protein ZIP could also account for the proapoptotic activity of the PKC{zeta} dominant-negative mutant.

Expression of the PKC{zeta} kinase mutant has been shown here to accelerate the occurrence of apoptosis in U937 cells exposed to either etoposide or TNF{alpha}. Expression of this mutant induces changes in the expression of several proteins involved in apoptotic cell death, including the previously mentioned decrease in Bcl-2 expression as well as an increase in Bax, procaspase-2, and procaspase-3 protein levels. The mechanisms that account for increased procaspase expression could involve the decreased expression of IAPs as a consequence of NF-{kappa}B inhibition (44) . IAPs have been proposed to modulate the expression of procaspases by mediating their proteasome-dependent degradation (45) . These changes in the expression of apoptosis-modulating proteins may explain how cytochrome c-mediated activation of the caspase cascade is more efficient in cell-free extracts from PKC{zeta} dominant-negative mutant-transfected cells than in cell-free extracts from control cells. Thus, PKC{zeta} modulates the apoptotic machinery both at the mitochondrial level, by modifying the Bax/Bcl-2 ratio (46) , and at the postmitochondrial level, by modulating cytochrome c-mediated caspase activation (47) . Although the consequences of PKC{zeta} mutant expression on apoptosis remain limited, the sensitization observed in clonogenic assays and in in vivo experiments and the recent demonstration that Bcl-2 oncoprotein produces multidrug resistance in primary lymphomas developed in vivo indicates that, at least in some tumor types, disruption of apoptosis can have profound effects on tumor treatment outcome (48) .

Broad spectrum inhibitors of PKCs enhance the therapeutic efficacy of numerous antineoplastic agents as well as ionizing radiation in a broad array of cell types (2) . However, PKC isoforms are differentially involved in the regulation of apoptosis (49 , 50) . Thus, compared with nonspecific inhibitors of PKCs, selective inhibitors of antiapoptotic PKC isoenzymes may provide a more efficient strategy for improving the efficacy of chemotherapeutic regimens now in use. Given the structural similarities between PKC isoforms, it has been difficult to identify highly selective chemical inhibitors. Gene therapies using antisense RNA and ribozymes could provide more specific measures to inhibit individual PKC isoforms (51) . We show here that the transfer of a mutated PKC{zeta} gene encoding a kinase-dead mutant protein also efficiently counteracts one of the inducible resistance pathways triggered by cytokines and chemotherapeutic drugs.


   ACKNOWLEDGMENTS
 
We thank Jorge Moscat (Madrid, Spain) for providing the PKC{zeta} cDNA and Guy Laurent (INSERM 9910, Toulouse, France) and Laurent Arnould (U517) for helpful advices.


   FOOTNOTES
 
1 Our group is labeled "La Ligue" by the Ligue Nationale Contre le Cancer and supported by its departmental committees (Saône et Loire, Nièvre, Bourgogne, Haute-Saône). We are also supported by grants from INSERM and ARERS. Back

2 To whom requests for reprints should be addressed, at INSERM U 517, Faculties of Medicine & Pharmacy, 7, boulevard Jeanne d’Arc, 21033 Dijon Cedex, France. Phone: 33 3 80 39 32 56; Fax: 33 3 80 39 34 34; E-mail: ali.bettaieb{at}u-bourgogne.fr Back

3 The abbreviations used are: NF-{kappa}B, nuclear factor-{kappa}B; I{kappa}B, inhibitor of NF-{kappa}B; TNF{alpha}, tumor necrosis factor {alpha}; PKC, protein kinase C; EMSA, electrophoretic mobility shift assay; XIAP, X-linked inhibitory of apoptosis protein; Par-4, prostate apoptosis response-4; RIP, receptor-interacting protein; TRADD, TNF receptor 1-associated death domain; ZIP, {zeta}-interacting protein. Back

Received 5/11/01. Accepted 1/17/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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