Molecular Cancer Therapeutics Molecular Diagnostics in Cancer Therapeutic Development: Fulfilling the Promise of Personalized Medicine Targeting the PI3-Kinase Pathway in Cancer
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Mol Cancer Ther. 2005;4:1457-1464
© 2005 American Association for Cancer Research

A role for PKC{zeta} in potentiation of the topoisomerase II activity and etoposide cytotoxicity by wortmannin

Caroline Reis1, Nicole Giocanti1, Christophe Hennequin1,2, Frédérique Mégnin-Chanet1, Marie Fernet1, Rodolphe Filomenko3, Ali Bettaieb3, Eric Solary3 and Vincent Favaudon1

1 Institut National de la Santé et de la Recherche Médicale U612, Institut Curie-Recherche, Centre Universitaire, Orsay, France; 2 Hôpital Saint-Louis, Radiothérapie-Oncologie, Paris, France; 3 Institut National de la Sante et de la Recherche Medicale U517, Faculté de Médecine, Université de Bourgogne, Dijon, France

Requests for reprints: Vincent Favaudon, INSERM, U612, Institut Curie-Recherche, Bât. 110-112, Centre Universitaire, 91405 Orsay Cedex, France. Phone: 33-1-6986-3188; Fax: 33-1-6986-3187. E-mail: vincent.favaudon{at}curie.u-psud.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Enhanced cytotoxicity of etoposide by wortmannin, an inhibitor of enzymes holding a phosphatidylinositol 3-kinase domain, was investigated in eight cell lines proficient or deficient for DNA double-strand break repair. Wortmannin stimulated the decatenating activity of topoisomerase II, promoted etoposide-induced accumulation of DNA double-strand breaks, shifted the specificity for cell killing by etoposide from the S to G1 phase of the cell cycle, and potentiated the cytotoxicity of etoposide through two mechanisms. (a) Sensitization to high, micromolar amounts of etoposide required integrity of the nonhomologous end-joining repair pathway. (b) Wortmannin dramatically increased the susceptibility to low, submicromolar amounts of etoposide in a large fraction of the cell population irrespective of the status of ATM, Ku86, and DNA-PKCS. It is shown that this process correlates depression of phosphatidylinositol 3-kinase–dependent phosphorylation of the atypical, {zeta} isoform of protein kinase C (PKC{zeta}). Stable expression of a dominant-negative, kinase-dead mutant of PKC{zeta} in a tumor cell line reproduced the hypersensitivity pattern induced by wortmannin. The results are consistent with up-regulation of the topoisomerase II activity in relation to inactivation of PKC{zeta} and indicate that PKC{zeta} may be a useful target to improve the efficiency of topoisomerase II poisons at low concentration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Class I phosphatidylinositol 3-kinase (PI3K) plays a pivotal role in the response to growth factor receptors through the synthesis of phosphatidylinositol-3,4,5-triphosphate (PIP3) from the diphosphate and ATP. PIP3 binds to the pleckstrin homology domain of a range of proteins, among others protein kinase B (AktPKB), type I phosphoinositide-dependent kinase (PDK1), and the {zeta} isoform of protein kinase C (PKC{zeta}), and is necessary for PDK1 autophosphorylation. PDK1 phosphorylates AktPKB on Thr308 and Ser473 residues (1–3). Activated AktPKB in turn phosphorylates a range of proteins involved in the control of cell survival and proliferation (4). PDK1 is also involved in the regulatory phosphorylation of several protein kinase C isozymes (5), in particular, the atypical, {zeta} isoform (PKC{zeta}), whose activation requires Thr410 phosphorylation in a PIP3-dependent reaction (6, 7). Consistent with that, and in contrast with conventional PKC isoforms, the phosphorylation of PKC{zeta} is down-regulated by PI3K inhibitors (8).

It has been shown recently that, among other functions, PKC{zeta} interacts with topoisomerase II and regulates its activity. Actually, Plo et al. (9) showed that enforced overexpression of wild-type PKC{zeta} induces resistance to the topoisomerase II poison etoposide correlated with hyperphosphorylation of topoisomerase II serine residues and with a drop in the amount of cleavable complexes [i.e., the reaction intermediates that are liable for the cytotoxicity of topoisomerase II poisons through induction of permanent DNA double-strand breaks (DSB)]. Reciprocally, Filomenko et al. (10) showed that the dominant expression of an inactive PKC{zeta} mutant potentiates the cytotoxicity of etoposide. This prompted us to determine whether wortmannin, a potent PI3K inhibitor would alter the activity of topoisomerase II and switch cells to hypersensitivity to etoposide, a topoisomerase II poison frequently used in antitumor chemotherapy and chemoradiotherapeutic combinations.

Depression of PKC{zeta} phosphorylation has been observed in cells exposed to wortmannin (8). Wortmannin covalently binds to an essential lysine residue in the catalytic phosphate transfer site of PI3K (11), resulting in enzyme inhibition (11, 12). Wortmannin also depresses the activity of other serine/threonine protein kinases whose catalytic site bears close homology to the active site of PI3K. This includes ATM and DNA-PK (13–15), two kinases that are required for the sensing and repair of DSB through nonhomologous end-joining and whose defects impair DSB rejoining (16–19). Wortmannin consistently sensitizes cells to induced killing by ionizing radiation (12, 20–25). Wortmannin-induced sensitization to etoposide has also been reported (22, 26). However, according to Jin et al. (27), DNA-PKCS is not mandatory to the repair of etoposide-induced damage.

To further investigate the mechanisms by which wortmannin sensitizes tumor cells to etoposide, we used nonhomologous end-joining–competent and PKC{zeta}-competent and defective cell lines. Wortmannin unexpectedly increased the susceptibility to low, submicromolar amounts of etoposide in a large fraction of the cell population irrespective of the status of ATM, Ku86, and DNA-PKCS. Using a dominant-negative mutant of PKC{zeta} allowed us to show that PKC{zeta} is the key intermediate in this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Antibodies
Suppliers were as follows: wortmannin, 5-bromo-2'-deoxythymidine, proteinase K, protease inhibitors, phosphatase inhibitors, and mouse monoclonal anti-ß-tubulin antibody (clone 2-28-33) from Sigma-Aldrich Co. (Saint Quentin Fallavier, France); Crithidia fasciculata kinetoplast DNA from Topogen, Inc. (Port Orange, FL); polycarbonate filter membranes (Nuclepore) for DNA elution from Whatman (Banbury, Oxon, United Kingdom); nitrocellulose membrane (0.2-µm pore size) from Schleicher & Schuell (Dassel, Germany); [2-14C]thymidine from Amersham Biosciences (Orsay, France); and products for cell culture from Invitrogen (Cergy Pontoise, France). Sterile etoposide solution (Dakota Pharm, Le Plessis Robinson, France) was stored at liquid nitrogen temperature.

{lambda}-Protein phosphatase and rabbit polyclonal antibodies raised against AktPKB, phospho-Ser473-AktPKB, phospho-Thr308-AktPKB, PKC{zeta}, and phospho-Thr410-PKC{zeta} were from Cell Signaling Technology (Beverly, MA). Goat horseradish peroxidase–conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (Soham, Cambridgeshire, United Kingdom).

Cells
Chinese hamster embryonic lung V79 fibroblasts (clone 79-1/379A) came from Flow Laboratories (Irvine, Scotland). HeLa cells were provided by Dr. J. Coppey (Institut Jacques-Monod, Paris, France). MRC5VI, a SV40-transformed cell line derived from normal human fibroblasts and AT5BIVA, a SV40-transformed cell line derived from fibroblasts of a patient with ataxia telangiectasia (complementation group D), were obtained from Drs. N. Foray and C.F. Arlett (Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom). AT5BIVA cells bear a distinct two-amino-acid deletion on both ATM alleles (28). M059J and M059K cells, kindly provided by Dr. J-P. de Villartay (INSERM, U429, Hôpital Necker, Paris, France) on behalf of Dr. J. Turner (Experimental Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Canada), were originally isolated from a male patient with a glioblastoma (29). M059J cells do not express the DNA-PKCS subunit (17), but ATM is functional in this cell line (30). Expression of the ATM and DNA-PKCS genes is normal in M059K cells (30). Chinese hamster ovary Xrs6 cells were a gift from Dr. D. Averbeck (Institut Curie, Orsay, France) on behalf of Dr. P.A. Jeggo (Genome Damage and Stability Centre, University of Sussex). Xrs6 cells are defective in the Ku86 subunit due to Ku86 gene deletion on one allele and silencing methylation on the other (31, 32).

The human leukemic U937 cell line was purchased from the American Type Culture Collection (Rockville, MD). Cell clones stably transfected with either an empty vector (PKC{zeta}-Co) or a PKC{zeta} kinase-dead, dominant-negative mutant cDNA (PKC{zeta}-DN) were obtained as described (10).

Cell Cultures
U937 cells were cultured in RPMI 1640. All other cell lines were maintained as exponentially growing monolayers in DMEM. For determination of DSB by neutral filter elution, mid-log phase V79 fibroblasts were grown in 25-cm2 flasks seeded at a density of 7 x 105 cells and incubated for 24 hours with 0.1 µCi/mL [2-14C]thymidine. Radioactive thymidine was removed 3 hours before treatment to permit ligation of radiolabeled Okasaki fragments.

Synchronization of HeLa cells at the G1-S junction was achieved using a double thymidine block (33). Cell cycle progression was monitored by dual variable flow cytometry using a FACStarPLUS cytofluorometer (Becton Dickinson, Le Pont de Claix, France) with 5-bromo-2'-deoxythymidine labeling (10 µmol/L, 15 minutes) of S-phase cells as described (34).

Cytotoxicity Determination
The length of exposure to etoposide was 1 hour, unless otherwise stated. Wortmannin (12.5 mmol/L stock solution in DMSO) was usually introduced 30 minutes before etoposide. The DMSO concentration in culture medium was 0.4% throughout. Due to opening of the lactone ring, wortmannin decomposed in the medium within 2 hours (35).

Cell survival was determined in triplicate using a clonogenic assay. For all cell lines except U937 cells, enough cells from mid-log growing subcultures were seeded at constant density in 25-cm2 plastic flasks 4 hours before cytotoxic treatment to obtain ca. 400 colonies in untreated samples. Following treatment, the flasks were washed twice with warm HBSS, supplied with fresh DMEM, and returned to the incubator for 7 to 12 days. Colonies were fixed with methanol, stained with Coomassie blue R-250, and scored by visual examination. Colonies of <50 cells were disregarded. The intra-assay variability was 0.027 ± 0.023.

Exponentially growing U937 cells were incubated for 1 hour in the presence or absence of etoposide at various concentrations, without or with wortmannin (15-minute preincubation and 1-hour cotreatment), rinsed, and plated at various densities in semisolid medium using the methylcellulose technique (36). Colonies were scored at day 12 as described (10). The intra-assay variability was 0.059 ± 0.035.

In experiments using synchronized HeLa cells the cellular multiplicity [i.e., the number of cells (n) per potential colony-forming unit] was measured by digital microscope examination of the culture flasks at the time of treatment. The single-cell surviving fraction (SCSF) was subsequently calculated using the discrete distribution equation (37):

(A)
where Sexp is the experimental cell survival determined from bulk colony scoring and an the fraction of colony-forming units containing n cells.

Assay of Experimental Conditions
Wortmannin did not exert any significant cytotoxic effect when applied alone. Full sensitization to etoposide required 20 or 100 µmol/L wortmannin for cell densities of 103 and 106 per 25-cm2 flask, respectively. At these concentrations, wortmannin inhibits the catalytic activity of all PI3K domain containing enzymes including FRAPmTOR, ATM, and DNA-PKCS, except ATR (12). Higher concentrations of wortmannin did not increase etoposide sensitization, in agreement with other authors (20, 23). Thus, 20 µmol/L (≤103 cells per flask) or 100 µmol/L wortmannin (106 cells per flask) were chosen for subsequent experiments.

DNA Double-Strand Break Determination
Etoposide-induced cleavable complexes and DSB were determined by filter elution at pH 9.6 over polycarbonate filters as described (38). As usual (39), DNA retention (R) was fitted to the time-dependent exponential equation:

(B)
where R0 is the retention at time zero and R{infty} is the retention at completion of repair.

Topoisomerase II Activity
Topoisomerase II activity was measured using kinetoplast DNA decatenation. Samples were assembled in ice in 50 mmol/L Tris-HCl (pH 8.0), 120 mmol/L KCl, 10 mmol/L MgCl2, 0.5 mmol/L DTT, 30 µg/mL bovine serum albumin, and 0.5 mmol/L ATP prepared extemporarily. Incubation was for exactly 12 minutes at 37°C. The reaction was stopped by transfer to ice and addition of 4 µL stop buffer (1% Sarkosyl, 5% glycerol, 0.0005% bromphenol blue, final concentration). Electrophoresis was done in 1% agarose gel in TAE buffer with 0.5 µg/mL ethidium bromide (6 V/cm, 1.5-hour migration). The gels were destained for 30 minutes without agitation and photographed.

Immunoblotting
Cells were washed with ice-cold PBS immediately after treatment and processed in one of two ways. For total extracts, cells were lysed on ice by radioimmunoprecitation assay (RIPA) lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl, 0.1% SDS, 0.5% sodium desoxycholate (pH 8.0)] with 1 mmol/L phenylmethanesulfonyl fluoride plus protease and phosphatase inhibitors and centrifuged at 15,000 x g to remove debris. For preparation of nuclear extracts, cells were submitted to an hypotonic shock [10 mmol/L HEPES, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT buffer (pH 7.9)] for 15 minutes before lysis in ice-cold 20 mmol/L HEPES, 400 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT buffer, 10% glycerol, 0.6% NP40 (pH 7.9), supplemented with 1 mmol/L phenylmethanesulfonyl fluoride plus protease and phosphatase inhibitors. Proteins were titrated using a Bradford colorimetric assay (Bio-Rad, Hercules, CA). After PAGE, the proteins were blotted onto nitrocellulose membrane, incubated with specific primary and secondary antibodies, and revealed using an enhanced chemiluminescence kit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Wortmannin Blocks PI3K-Dependent Phosphorylation in Cells
To determine the effect of wortmannin on the PIP3-dependent pathway, we analyzed the phosphorylation of AktPKB, a known downstream target of PDK1. In agreement with Sonnenburg et al. (8), wortmannin abolished the Ser473/Thr308 phosphorylation of AktPKB in serum-stimulated cells (Fig. 1).



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  		Figure 1. Inhibition of AktPKB and PKC{zeta} phosphorylation by wortmannin. AktPKB, phospho-Ser473-AktPKB, and phospho-Thr308-AktPKB (arrows) were probed by immunoblot analysis (40 µg total protein in each lane) in extracts from asynchronous growing MRC5VI, AT5BIVA, M059J (top), and HeLa cells (middle). Cells were left untreated or treated with wortmannin (WM, 30-min contact), etoposide (VP, 1-h contact), or a combination of both and lysed immediately after treatment. Positive (phosphorylated, Pos) and negative controls (unphosphorylated, Neg) were provided by Cell Signaling Technology. Unphosphorylated AktPKB and ß-tubulin (data not shown) were used as controls. The experiment shows that wortmannin induces rapid and efficient block of AktPKB phosphorylation. In HeLa cells, however, the basal level of AktPKB Ser473 and Thr308 phosphorylation was beyond detection. Parenthetically, this shows that inhibition of AktPKB phosphorylation is not involved in hypersensitivity to submicromolar etoposide. In another experiment (bottom), mid-G1-synchronized HeLa cells (15 h after release from the thymidine block; see Fig. 3) were exposed for 30 min to wortmannin and lysed by RIPA lysis buffer after a rapid wash by ice-cold PBS. The blots (40 µg protein in each lane) were immunoprobed with anti-PKC{zeta}, anti–phospho-Thr410-PKC{zeta}, or anti-tubulin antibodies.

 


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  		Figure 3. Altered cell cycle phase specificity of etoposide (VP) by wortmannin (WM) in HeLa cells. HeLa cells were synchronized (92%) by a double thymidine block before clonogenic assay at the indicated time points. Contact with etoposide was for 40 min. Wortmannin was introduced 10 min before etoposide and was present for up to removal of etoposide. Cell cycle progression was checked in parallel by flow cytometry with 5-bromo-2'-deoxythymidine labeling. Survival values were corrected for the effect of cellular multiplicity as described (34). Bars, SD.

 
Sonnenburg et al. (8) also reported that inhibition of PKC{zeta} phosphorylation occurs in cells exposed to wortmannin. Total extracts from G1-synchronized HeLa cells (see below) were immunoblotted with an anti-phospho-Thr410-PKC{zeta} antibody to confirm this observation. Complete suppression of PKC{zeta} phosphorylation was actually observed in the presence of wortmannin (Fig. 1).

Wortmannin Uncovers Two Subpopulations with Differential Susceptibility to Etoposide in Various Cell Lines
Eight human (HeLa, MRC5VI, AT5BIVA, M059J, M059K, and U937) and rodent (V79 and Xrs6) cell lines were exposed to increasing concentrations of etoposide without or with wortmannin for determination of the drug cytotoxicity. The results are summarized in Fig. 2. Wortmannin alone had no marked lethal effect but increased the slope of the dose-response curves to micromolar etoposide, with the noticeable exception of the DNA-PK-defective M059J and Xrs6 cells. This effect was also weak in U937 cells. Remarkably, wortmannin sensitized a large fraction of the cell population to submicromolar etoposide. This effect occurred irrespective of the status of the main determinants of the nonhomologous end-joining pathway (i.e., DNA-PKCS, Ku86, and ATM). Deciphering the molecular mechanisms involved in this process was the main purpose of this study.



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  		Figure 2. Potentiation of etoposide response by wortmannin (WM) or mutation of PKC{zeta}. The cytotoxicity of etoposide (1-h contact) was determined using a clonogenic assay (see Materials and Methods) in the absence (open symbols) or presence (closed symbols) of wortmannin. Wortmannin was introduced 30 min before etoposide and was removed at the end of treatment. Open arrows indicate survival to wortmannin alone. The curves were fitted to an exponential or biexponential equation. For MRC5VI cells in the absence of wortmannin, a quadratic model was used. Bars, SD. A summary of the fraction of the cell population showing hypersensitivity to submicromolar etoposide (<0.2 µmol/L) in the presence of wortmannin is presented at bottom right. This cell fraction was determined from the ordinate intercept (bold arrows) of the curves drawn for best fit to experimental data in the high range of etoposide concentration. The resistance of MRC5VI cells to etoposide in the absence of wortmannin is presumed to result from reduced topoisomerase II expression (52), whereas the resistance of Xrs6 cells in the high range of drug concentration parallels expression of the wild-type Ku86 allele in a minor fraction of the cell population (31, 32). The relatively low sensitivity of M059J and M059K cells is related to a multidrug resistance phenotype, a common feature in human glioma cell lines (53).

 
Dominant-Negative Mutation of PKC{zeta} Mimics the Effect of Wortmannin at Low Etoposide Concentration
To analyze the role of PKC{zeta} in the response to etoposide, we used U937 clones stably transfected with either an empty vector (PKC{zeta}-Co) or a vector encoding a dominant-negative, kinase-dead mutant of PKC{zeta} (PKC{zeta}-DN). Both clones were assayed for survival to increasing concentrations of etoposide in the absence or presence of wortmannin. The results are shown in Fig. 2. As in other cell lines, wortmannin dramatically enhanced the cytotoxic response to submicromolar etoposide (≤0.2 µmol/L) in a fraction (33%) of the PKC{zeta}-Co cell population. The response of PKC{zeta}-DN cells to etoposide also gave evidence of two subpopulations with differential susceptibility to etoposide, irrespective of whether wortmannin was present or not.

This experiment shows that mutation in the catalytic site of PKC{zeta} mimics the effect of wortmannin in PKC{zeta}-competent cells and sensitizes a large fraction of the cell population to submicromolar etoposide.

Wortmannin Shifts the Peak of Etoposide Cytotoxicity from the S to G1 Phase of the Cell Cycle
To determine whether wortmannin could alter the cell cycle phase specificity of etoposide cytotoxicity, synchronized HeLa cells were exposed briefly (30 minutes) to etoposide at various time points after release from a double thymidine block. Equitoxic doses of etoposide (i.e., 2 µmol/L in the absence of wortmannin versus 0.2 µmol/L in the presence of wortmannin; see Fig. 2) were used in both assays. As expected (40, 41), the toxicity of etoposide alone was maximum in mid-S phase, then decreased rapidly as cells progressed to G1 through G2 and M. Surprisingly, however, maximal susceptibility to 0.2 µmol/L etoposide in the presence of wortmannin was in G1 phase (Fig. 3).

Wortmannin Potentiates the Catalytic Activity of Topoisomerase II
Wortmannin greatly stimulated kinetoplast DNA cleavage by nuclear extracts from G1-synchronized HeLa cells, indicating a large increase in topoisomerase II activity (Fig. 4). Neutral DNA filter elution was subsequently used to measure the kinetics of topoisomerase II–linked cleavable complex reversal and DSB rejoining. Wortmannin induced accumulation of excess DSB, although it did not significantly alter the kinetics of cleavable complex reversal after removal of etoposide (Fig. 5).



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  		Figure 4. Increased topoisomerase II activity by wortmannin (WM) in HeLa cells. G1-synchronized HeLa cells (15 h after thymidine block release; see Fig. 3) were exposed or not to wortmannin (30 min). At the end of incubation, cells were washed twice and nuclear extracts prepared immediately (WM 0-h) or after 1.5 h recovery in drug-free medium (WM 1.5-h). For the kinetoplast assay, each sample (20 µL) contained 0.2 µg kinetoplast DNA (KDNA) and 2 µg total protein. The reaction time (37°C) was exactly 12 min. DOC, decatenated, open circular DNA (product of interest); NDP, nuclease degradation products; DR, decatenated, relaxed DNA.

 


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  		Figure 5. Reversal kinetics of etoposide-induced topoisomerase II cleavable complexes and DSB. [2-14C]Thymidine-labeled V79 fibroblasts were exposed to etoposide (VP, 1-h contact) without (light arrows) or with (bold arrows) preincubation (30 min) with wortmannin (WM). Etoposide was removed at time 0. When present, wortmannin was reintroduced immediately and cells returned to the incubator. Cells were finally washed at the time indicated, collected, and lysed onto polycarbonate filters for neutral elution determination in triplicate. The retention of DNA was measured after 22-mL elution (11 h). Least-squares regression analysis shows that the difference between the rate of cleavable complex reversal (Eq. B; see Materials and Methods) without (k = 0.035 ± 0.011 min–1) and with (kWM = 0.030 ± 0.009 min–1) wortmannin, is not statistically significant. The gap between both curves was the same at time 0 and at the completion of repair and represents excess DSB in the presence of wortmannin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One major problem in assessing the effects of small-molecule inhibitors is that assays with purified enzymes may not be representative of the context in cells or tissues due to uncertainties about drug uptake and catabolism or to the presence of multiple targets with differential affinity or specificity in the various cell compartments. For this reason, results may be weakly correlated with the identity of the intended target. This is the case for wortmannin. Indeed, wortmannin covalently binds to an essential lysine residue in the catalytic phosphate transfer site of PI3K (11), resulting in enzyme inhibition (11, 12). Wortmannin also depresses the activity of other serine/threonine protein kinases whose catalytic site bears close homology to the active site of PI3K. At the concentration used in our experiments, wortmannin inactivates all these kinases except ATR. Our results show that even under these conditions, discrimination between the different pathways involved in potentiation of etoposide cytotoxicity by wortmannin is feasible using a combination of immunoblots (to determine the phosphorylation status of AktPKB and PKC{zeta}, both substrates of PDK1), ATM- and DNA-PK-defective mutants, and a kinase-dead mutant of PKC{zeta}.

The data show that wortmannin sensitizes cells to the lethal effect of etoposide in a dual mode. One mechanism seems to require DNA-PK integrity, results in an increase of the slope of the dose-response curve to etoposide, and predominates at high etoposide concentration (0.5–40 µmol/L). The second mechanism results in a large increase of the cytotoxic response to submicromolar amounts of etoposide (≤0.2 µmol/L). This effect occurs in G1 phase irrespective of the status of ATM, Ku86, and DNA-PKCS.

It is further shown that hypersensitivity to submicromolar etoposide correlates inactivation of PKC{zeta}. Indeed, etoposide elicits a biphasic survival curve in a kinase-dead PKC{zeta}-DN transfectant; this effect mimics the one induced by wortmannin in PKC{zeta}-competent cells (Fig. 2). Such biphasic response indicates that two subpopulations exist in the PKC{zeta}-DN clone and in normal cells in which PKC{zeta} phosphorylation is abolished by wortmannin, with one subpopulation showing hypersensitivity to etoposide at concentrations below 0.2 µmol/L. In consideration of the results from synchronized HeLa cells (Fig. 3), this hypersensitive compartment is likely to match mid-G1 to late-G1 phase, suggesting that PKC{zeta}-mediated phosphorylation of topoisomerase II is required for the control of topoisomerase II activity at this stage. Consistent with this scheme, PKC{zeta} was reported recently to interact with topoisomerase II and down-regulate the catalytic activity of topoisomerase II through serine/threonine phosphorylation (9, 10). Therefore, it may confidently be proposed that wortmannin acts through inhibition of the PI3K-PDK1-PKC{zeta} cascade thus switching topoisomerase II to a hypophosphorylated state with concomitant up-regulation of the topoisomerase II decatenating activity and accumulation of unrejoined DSB in the presence of etoposide.

Topoisomerase II is present as two genetically distinct isoforms [i.e., topoisomerase II{alpha} (p170) and topoisomerase IIß (p180)] that exhibit differential cell cycle regulation. Our study leaves open the question of which isoform is involved in hypersensitivity to submicromolar etoposide. The ß isoform might be a good candidate to this role, because topoisomerase IIß activity is down-regulated by PKC{zeta}-induced phosphorylation (9) and also because its expression is maximum in G1 phase (42). Establishing topoisomerase IIß–defective mutants is necessary to unravel this issue. This is important because authors have recently suggested that topoisomerase IIß may play a part in the cytocidal activity of drugs such as Adriamycin and etoposide (42) and represent an important target for novel drugs (43).

The present study shows that inactivation of PKC{zeta} boosts topoisomerase II activity and potentiates the cytotoxic effect of submicromolar amounts of etoposide. Considering the role played by alteration of PI3K-dependent pathways in the development of cancer (44–46), compounds working to depress PIP3 synthesis (47) or, more specifically, to inhibit the catalytic activity of PDK1, are potentially exciting sensitizers to topoisomerase II poisons. Various inhibitors including n-{alpha}-tosyl-L-phenylalanyl chloromethyl ketone (48), celecoxib (49), 7-hydroxystaurosporine (50), and novel compounds (51) reportedly disrupt PDK1 activity and display antiproliferative potential. Such inhibitors should be assayed in combination with topoisomerase poisons with a view either to reduce the amount of etoposide, or to circumvent quiescence-related resistance.


   Acknowledgments
 
We thank Dr. Jorge Moscat (Universidad Autonoma, Madrid, Spain) for providing the mutant PKC{zeta} cDNA; the Alberta Cancer Board and Dr. Joan Turner (Cross Cancer Institute, University of Alberta, Edmonton, Canada) for the gift of M059J and M059K cell lines; and Drs. P.A. Jeggo, Colin F. Arlett, Dietrich Averbeck, Nicolas Foray, and Jean-Pierre de Villartay for providing part of the cell lines used in this study.


   Footnotes
 
Grant support: Institut National de la Santé et de la Recherche Médicale and the Institut Curie; Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche fellowship (C. Reis); and Ligue Nationale Contre le Cancer (M. Fernet, R. Filomenko, A. Bettaieb, and E. Solary).

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.

Note: C. Reis is currently at the Genome Damage and Stability Centre, University of Sussex, Brighton, East Sussex BN1 9RR, United Kingdom.

M. Fernet is currently at the Institut de Biologie Structurale J.P. Ebel, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

Received 5/17/05; revised 7/18/05; accepted 8/10/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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