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• W2045461964 abstract "In this study, we evaluated the influence of protein kinase Cζ (PKCζ) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-ζJ and U937-ζB cells, enforced PKCζ expression, conferred by stable transfection of PKCζ cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKCζ-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKCζ overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKCζ-overexpressing cells, we found that PKCζ interacted with both α and β isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKCζ (rH-PKCζ) was incubated with purified topoisomerase II isoforms, rH-PKCζ interacted with topoisomerase IIβ but not with topoisomerase IIα. PKCζ/topoisomerase IIβ interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase IIβ is a substrate for PKCζ, and that PKCζ may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase IIβ activity through its kinase function. In this study, we evaluated the influence of protein kinase Cζ (PKCζ) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-ζJ and U937-ζB cells, enforced PKCζ expression, conferred by stable transfection of PKCζ cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKCζ-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKCζ overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKCζ-overexpressing cells, we found that PKCζ interacted with both α and β isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKCζ (rH-PKCζ) was incubated with purified topoisomerase II isoforms, rH-PKCζ interacted with topoisomerase IIβ but not with topoisomerase IIα. PKCζ/topoisomerase IIβ interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase IIβ is a substrate for PKCζ, and that PKCζ may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase IIβ activity through its kinase function. double-strand breaks 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide atypical multidrug resistant phenotype DNA protein cross-links etoposide recombinant human PKCζ myelin basic protein protein kinase Cζ phenylmethylsulfonyl fluoride dithiothreitol mitogen-activated protein kinase DNA topoisomerases II are nuclear enzymes that modify DNA topology by their ability to break and reseal both strands in concert. Topoisomerases II have important functions in DNA replication and can serve as a cancer chemotherapy target. Indeed, drugs such as etoposide (VP-16) or mitoxantrone, form drug-topoisomerase II-DNA ternary complexes referred to as “cleavable complex.” The primary cytotoxic effect of these so-called “topoisomerase II inhibitors” is not by inhibition of topoisomerase II activity but rather by stabilizing topoisomerase II cleavable complexes. This interaction prevents the DNA-resealing step normally catalyzed by topoisomerase II. The ternary complex constitutes a latent DNA-damaging state, which is ultimately converted to an irreversible DNA double-strand break (DSB).1 Although the mechanism by which complex formation mediates cell death is still poorly understood, it has been largely documented with few exceptions that the amount of cleavable complexes and the subsequent number of DNA breaks correlates with cytotoxicity (1Davies S.M. Robson C.N. Davies S.L. Hickson I.D. J. Biol. Chem. 1988; 263: 17724-17729Abstract Full Text PDF PubMed Google Scholar). These observations suggest that abnormal intracellular distribution or a decrease in expression level, activity, and sensitivity of the inhibited topoisomerase may have major impacts on topoisomerase inhibitor clinical efficacy. This has been confirmed by the molecular characterization of the so-called atypical multidrug resistant phenotype (at-MDR) resulting from selection by topoisomerase II inhibitors. Indeed, at-MDR cells display cross-resistance to other topoisomerase II inhibitors and have been associated with a number of functional and/or structural topoisomerase II alterations, including decreased catalytic activity, abnormal interaction between topoisomerase II and nuclear matrix, reduced expression, point mutation and, finally, altered phosphorylation (2Beck W.T. Danks M.K. Wolverton J.S. Chen M. Granzen B. Kim R. Suttle D.P. Adv. Pharmacol. 1994; 29B: 145-169Crossref PubMed Scopus (56) Google Scholar).The role of phosphorylation on topoisomerase II function has been debated and remains controversial. Indeed, previous studies have shown that topoisomerase II contains potential serine phosphorylation sites and that this enzyme is a substrate for various serine kinases, including casein kinase II, p34cdc2 kinase, and classic protein kinase C (PKC). In a cell-free system, PKC-induced phosphorylation of topoisomerase II results in an increase in its catalytic activity by enhancing ATP hydrolysis (3Corbett A.H. DeVore R.F. Osheroff N. J. Biol. Chem. 1992; 267: 20513-20518Abstract Full Text PDF PubMed Google Scholar, 4Corbett A.H. Fernald A.W. Osheroff N. Biochemistry. 1993; 32: 2090-2097Crossref PubMed Scopus (56) Google Scholar). In the absence of antineoplastic drugs, phosphorylation has a negligible effect on other steps of topoisomerase II catalytic cycle, including DNA binding or DNA cleavage/religation equilibrium. However, in the presence of VP-16 or amsacrine, phosphorylation decreases the ability of drugs to stabilize DNA-topoisomerase II complexes, apparently by increasing the rates of religation of DNA by the enzyme (5DeVore R.F. Corbett A.H. Osheroff N. Cancer Res. 1992; 52: 2156-2161PubMed Google Scholar). Other studies have provided indirect evidences that PKC might also influence topoisomerase II function in vivo. For example, PKC inhibitors, such as suramin or staurosporine, decrease topoisomerase II phosphorylation and catalytic activity in intact cells as well as drug-induced topoisomerase II-mediated cleavage (6Zwelling L.A. Altschuler E. Mayes J. Hinds M. Chan D. Cancer Chemother. Pharmacol. 1991; 29: 48-52Crossref PubMed Scopus (7) Google Scholar, 7Funayama Y. Nishio K. Takeda Y. Kubota N. Ohira T. Ohmori T. Ohta S. Ogasawara H. Hasegawa S. Saijo N. Anticancer Res. 1993; 13: 1981-1988PubMed Google Scholar). However, the role of topoisomerase II phosphorylation in drug resistance has been minimized on the basis of independent studies that have shown that, in at-MDR cells, topoisomerase II could be either hyperphosphorylated or hypophosphorylated (8Takano H. Kohno K. Ono M. Uchida Y. Kuwano M. Cancer Res. 1991; 51: 3951-3957PubMed Google Scholar, 9Chen M. Wolverton J.S. Beck W.T. Proc. Am. Assoc. Cancer Res. 1992; 33: 2707Google Scholar, 10Ritke M.K. Murray N.R. Allan W.P. Fields A.P. Yalowich J.C. Mol. Pharmacol. 1995; 48: 798-805PubMed Google Scholar).At least 12 different isoforms of PKC have been characterized so far and have been separated into three categories based on the Ca2+ requirement for activation and phorbol ester binding activity. Conventional PKCs (α, βI, βII, and γ) are Ca2+-dependent phorbol ester receptor kinases; novel PKCs (δ, ε, θ, and η) are Ca2+-independent phorbol ester receptor kinases; and atypical PKCs (ζ, ι, λ, and υ) are independent of both Ca2+ and phorbol ester. Previous studies have shown that topoisomerase II is phosphorylatedin vitro by each of the conventional PKC isoforms (11Wells N.J. Fry A.M. Guano F. Norbury C. Hickson I.D. J. Biol. Chem. 1995; 270: 28357-28363Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the influence of these PKC isozymes on cellular topoisomerase function in vivo is still largely unknown. Moreover, to the best of our knowledge, the influence of atypical PKC isozymes on topoisomerase II phosphorylation and function has not been investigated.PKC ζ is an atypical PKC isoform, which is activated directly or indirectly by a variety of important signaling molecules, including ceramide (12Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar, 13Muller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (469) Google Scholar), phosphatidic acid (14Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar), and diacylglycerol generated from phosphatidylcholine hydrolysis (15Bjorkoy G. Overvatn A. Diaz-Meco M.T. Moscat J. Johansen T. J. Biol. Chem. 1995; 270: 21299-21306Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), phosphoinositide 3-kinase lipid products (16Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar), and p21Ras (17Berra E. Diaz-Meco M.T. Dominguez I. Municio M.M. Sanz L. Lozano J. Chapkin R.S. Moscat J. Cell. 1993; 74: 555-563Abstract Full Text PDF PubMed Scopus (343) Google Scholar). PKC ζ has emerged as a critical regulator of a number of cellular functions, including proliferation, differentiation, and apoptosis inhibition (18Moscat J. Sanz L. Sanchez P. Diaz-Meco M.T. Adv. Enzyme Reg. 2001; 41: 99-120Crossref PubMed Scopus (10) Google Scholar). Despite the critical role of this enzyme in cellular signaling, its implication in the regulation of topoisomerase II function has never been examined. This study was aimed to evaluate the effect of PKC ζ overexpression on the formation of cleavable complexes and cytotoxicity induced by VP-16 in the human leukemic U937 cells.DISCUSSIONThis study shows that PKCζ overexpression in U937 cells resulted in inhibition of apoptosis and increased survival of U937 cells treated with VP-16 and mitoxantrone, two topoisomerase II inhibitors. Enforced PKCζ expression resulted in a marked decrease in VP-16-induced DPC and DNA DSB, whereas the level of topoisomerase IIα and topoisomerase IIβ expression was unchanged compared with control cells. These results suggest that PKCζ can interfere with topoisomerase II function. In fact, we found that PKCζ-overexpressing cells exhibited reduced topoisomerase II catalytic function as measured by the decatenation assay. Altered topoisomerase II catalytic cycle may explain reduced drug-induced DNA damage and cytotoxicity. Thus, this study shows for the first time that a specific PKC isozyme may inhibit topoisomerase II catalytic activity and VP-16-induced apoptosis and cytotoxicity by interfering with drug-induced DNA damage.Based on the kinase function of PKCζ, we hypothesized that PKCζ overexpression might result in abnormal topoisomerase II phosphorylation. In fact, we found that, in PKCζ-overexpressing cells, PKCζ was not only found to interact with topoisomerase IIα and topoisomerase IIβ but also that these two topoisomerase II isoforms were heavily phosphorylated on serine residues. These results suggest that, in PKCζ-overexpressing cells, PKCζ not only directly or indirectly interacts with the two topoisomerase II isoforms but also phosphorylates these enzymes. However, using a cell-free system, we described that only topoisomerase IIβ is a substrate for PKCζ and that PKCζ inhibits topoisomerase IIβ activity. This result suggests that, in PKCζ-overexpressing cells, PKCζ interacts directly with topoisomerase IIβ and inhibits topoisomerase IIβ catalytic activity. This hypothesis is consistent with the role of this topoisomerase IIβ form in the cytotoxicity of topoisomerase II inhibitors (23Brown G.A. McPherson J.P., Gu, L. Hedley D.W. Toso R. Deuchars K.L. Freedman M.H. Goldenberg G.J. Cancer Res. 1995; 55: 78-82PubMed Google Scholar, 24Errington F. Willmore E. Tilby M.J., Li, L., Li, G., Li, W. Baguley B.C. Austin C.A. Mol. Pharmacol. 1999; 56: 1309-1316Crossref PubMed Scopus (86) Google Scholar).With regard to topoisomerase IIα, the fact that this enzyme was found to interact in vivo, but not in vitro, with PKCζ, suggests that, in PKCζ-overexpressing cells, PKCζ/topoisomerase IIα interaction involves one or several other proteins required for the constitution of this complex. Moreover, the fact that, in PKCζ-overexpressing cells, topoisomerase IIα was found to be constitutively phosphorylated whereas, in vitro, PKCζ was unable to phosphorylate this enzyme, suggests that topoisomerase IIα is phosphorylated by another PKCζ-regulated kinase. In this perspective, it is interesting to note that in a recent study topoisomerase IIα was found to be phosphorylated in intact cells by ERK2, the effector serine kinase of the classic MAPK module (25Shapiro P.S. Whalen A.M. Tolwinski N.S. Wilsbacher J. Froelich-Ammon S.J. Garcia M. Osheroff N. Ahn N.G. Mol. Cell. Biol. 1999; 19: 3551-3560Crossref PubMed Google Scholar). Based on previous studies, which have documented that PKCζ is a downstream target of MAPK (26Berra E. Diaz-Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar, 27van Dijk M.C. Hilkmann H. van Blitterswijk W.J. Biochem. J. 1997; 325: 303-307Crossref PubMed Scopus (63) Google Scholar), topoisomerase IIα phosphorylation could result from PKCζ-mediated ERK2 activation in PKCζ-overexpressing cells. The fact that, in these cells, ERK2 was found to be constitutively activated and accumulated in the nucleus (data not shown) supports this hypothesis.The role of atypical PKC isoforms, including PKCζ, in cell survival has been previously documented. Indeed, it has been described that the blockade of PKCζ or PKCλ/ι with dominant-negative mutants or antisense oligonucleotides is sufficient to promote apoptosis (28Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 29Murray N.R. Fields A.P. J. Biol. Chem. 1997; 272: 27521-27524Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The inactivation of PKCζ by caspase-dependent proteolysis during apoptosis induced by UV (30Frutos S. Moscat J. Diaz-Meco M.T. J. Biol. Chem. 1999; 274: 10765-10770Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) or by cisplatin (31Basu A. Akkaraju G.R. Biochemistry. 1999; 38: 4245-4251Crossref PubMed Scopus (84) Google Scholar) strengthens the role of PKCζ in the cellular protection against genotoxic stress. The mechanism by which atypical PKC isoforms exert their anti-apoptotic effect has received a great deal of attention. These studies strongly suggested that NF-κB signaling pathways could play an important role in PKCζ-induced inhibition of apoptosis (32Lallena M.J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). Indeed, NF-κB is a negative regulator of apoptosis induced by genotoxic agents, including topoisomerase II inhibitors (33Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2926) Google Scholar, 34Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2500) Google Scholar). Therefore, we cannot rule out that PKCζ overexpression may result in the activation of anti-apoptotic signals that interfere with the post-damage apoptotic response and, therefore, contribute to drug resistance.To conclude, we propose a model in which, upon PKCζ accumulation in the nucleus, this enzyme interacts with and phosphorylates nuclear topoisomerase IIβ. Topoisomerase IIβ hyperphosphorylation reduces catalytic function and decreases formation of ternary complexes and drug-induced cytotoxicity. If so, nuclear PKCζ accumulation might function to regulate topoisomerase II function. Although very little is known about expression and subcellular localization of PKCζ in tumor cells, PKCζ may translocate to the nucleus upon stimulation by differentiating agents (35Bertolaso L. Gibellini D. Secchiero P. Previati M. Falgione D. Visani G. Rizzoli R. Capitani S. Zauli G. Br. J. Haematol. 1998; 100: 541-549Crossref PubMed Scopus (36) Google Scholar), growth factors (36Wooten M.W. Zhou G. Wooten M.C. Seibenhener M.L. J. Neurosci. Res. 1997; 49: 393-403Crossref PubMed Scopus (50) Google Scholar, 37Neri L.M. Martelli A.M. Borgatti P. Colamussi M.L. Marchisio M. Capitani S. FASEB J. 1999; 13: 2299-2310Crossref PubMed Scopus (103) Google Scholar), cytokines (38Marchisio M. Bertagnolo V. Celeghini C. Vitale M. Capitani S. Zauli G. Anat. Rec. 1999; 255: 7-14Crossref PubMed Scopus (12) Google Scholar), or hypoxia (39Mizukami Y. Hirata T. Yoshida K. FEBS Lett. 1997; 401: 247-251Crossref PubMed Scopus (62) Google Scholar). Whether PKCζ alters topoisomerase II function in these conditions will be the subject of further investigations. DNA topoisomerases II are nuclear enzymes that modify DNA topology by their ability to break and reseal both strands in concert. Topoisomerases II have important functions in DNA replication and can serve as a cancer chemotherapy target. Indeed, drugs such as etoposide (VP-16) or mitoxantrone, form drug-topoisomerase II-DNA ternary complexes referred to as “cleavable complex.” The primary cytotoxic effect of these so-called “topoisomerase II inhibitors” is not by inhibition of topoisomerase II activity but rather by stabilizing topoisomerase II cleavable complexes. This interaction prevents the DNA-resealing step normally catalyzed by topoisomerase II. The ternary complex constitutes a latent DNA-damaging state, which is ultimately converted to an irreversible DNA double-strand break (DSB).1 Although the mechanism by which complex formation mediates cell death is still poorly understood, it has been largely documented with few exceptions that the amount of cleavable complexes and the subsequent number of DNA breaks correlates with cytotoxicity (1Davies S.M. Robson C.N. Davies S.L. Hickson I.D. J. Biol. Chem. 1988; 263: 17724-17729Abstract Full Text PDF PubMed Google Scholar). These observations suggest that abnormal intracellular distribution or a decrease in expression level, activity, and sensitivity of the inhibited topoisomerase may have major impacts on topoisomerase inhibitor clinical efficacy. This has been confirmed by the molecular characterization of the so-called atypical multidrug resistant phenotype (at-MDR) resulting from selection by topoisomerase II inhibitors. Indeed, at-MDR cells display cross-resistance to other topoisomerase II inhibitors and have been associated with a number of functional and/or structural topoisomerase II alterations, including decreased catalytic activity, abnormal interaction between topoisomerase II and nuclear matrix, reduced expression, point mutation and, finally, altered phosphorylation (2Beck W.T. Danks M.K. Wolverton J.S. Chen M. Granzen B. Kim R. Suttle D.P. Adv. Pharmacol. 1994; 29B: 145-169Crossref PubMed Scopus (56) Google Scholar). The role of phosphorylation on topoisomerase II function has been debated and remains controversial. Indeed, previous studies have shown that topoisomerase II contains potential serine phosphorylation sites and that this enzyme is a substrate for various serine kinases, including casein kinase II, p34cdc2 kinase, and classic protein kinase C (PKC). In a cell-free system, PKC-induced phosphorylation of topoisomerase II results in an increase in its catalytic activity by enhancing ATP hydrolysis (3Corbett A.H. DeVore R.F. Osheroff N. J. Biol. Chem. 1992; 267: 20513-20518Abstract Full Text PDF PubMed Google Scholar, 4Corbett A.H. Fernald A.W. Osheroff N. Biochemistry. 1993; 32: 2090-2097Crossref PubMed Scopus (56) Google Scholar). In the absence of antineoplastic drugs, phosphorylation has a negligible effect on other steps of topoisomerase II catalytic cycle, including DNA binding or DNA cleavage/religation equilibrium. However, in the presence of VP-16 or amsacrine, phosphorylation decreases the ability of drugs to stabilize DNA-topoisomerase II complexes, apparently by increasing the rates of religation of DNA by the enzyme (5DeVore R.F. Corbett A.H. Osheroff N. Cancer Res. 1992; 52: 2156-2161PubMed Google Scholar). Other studies have provided indirect evidences that PKC might also influence topoisomerase II function in vivo. For example, PKC inhibitors, such as suramin or staurosporine, decrease topoisomerase II phosphorylation and catalytic activity in intact cells as well as drug-induced topoisomerase II-mediated cleavage (6Zwelling L.A. Altschuler E. Mayes J. Hinds M. Chan D. Cancer Chemother. Pharmacol. 1991; 29: 48-52Crossref PubMed Scopus (7) Google Scholar, 7Funayama Y. Nishio K. Takeda Y. Kubota N. Ohira T. Ohmori T. Ohta S. Ogasawara H. Hasegawa S. Saijo N. Anticancer Res. 1993; 13: 1981-1988PubMed Google Scholar). However, the role of topoisomerase II phosphorylation in drug resistance has been minimized on the basis of independent studies that have shown that, in at-MDR cells, topoisomerase II could be either hyperphosphorylated or hypophosphorylated (8Takano H. Kohno K. Ono M. Uchida Y. Kuwano M. Cancer Res. 1991; 51: 3951-3957PubMed Google Scholar, 9Chen M. Wolverton J.S. Beck W.T. Proc. Am. Assoc. Cancer Res. 1992; 33: 2707Google Scholar, 10Ritke M.K. Murray N.R. Allan W.P. Fields A.P. Yalowich J.C. Mol. Pharmacol. 1995; 48: 798-805PubMed Google Scholar). At least 12 different isoforms of PKC have been characterized so far and have been separated into three categories based on the Ca2+ requirement for activation and phorbol ester binding activity. Conventional PKCs (α, βI, βII, and γ) are Ca2+-dependent phorbol ester receptor kinases; novel PKCs (δ, ε, θ, and η) are Ca2+-independent phorbol ester receptor kinases; and atypical PKCs (ζ, ι, λ, and υ) are independent of both Ca2+ and phorbol ester. Previous studies have shown that topoisomerase II is phosphorylatedin vitro by each of the conventional PKC isoforms (11Wells N.J. Fry A.M. Guano F. Norbury C. Hickson I.D. J. Biol. Chem. 1995; 270: 28357-28363Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, the influence of these PKC isozymes on cellular topoisomerase function in vivo is still largely unknown. Moreover, to the best of our knowledge, the influence of atypical PKC isozymes on topoisomerase II phosphorylation and function has not been investigated. PKC ζ is an atypical PKC isoform, which is activated directly or indirectly by a variety of important signaling molecules, including ceramide (12Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar, 13Muller G. Ayoub M. Storz P. Rennecke J. Fabbro D. Pfizenmaier K. EMBO J. 1995; 14: 1961-1969Crossref PubMed Scopus (469) Google Scholar), phosphatidic acid (14Nakanishi H. Exton J.H. J. Biol. Chem. 1992; 267: 16347-16354Abstract Full Text PDF PubMed Google Scholar), and diacylglycerol generated from phosphatidylcholine hydrolysis (15Bjorkoy G. Overvatn A. Diaz-Meco M.T. Moscat J. Johansen T. J. Biol. Chem. 1995; 270: 21299-21306Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), phosphoinositide 3-kinase lipid products (16Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar), and p21Ras (17Berra E. Diaz-Meco M.T. Dominguez I. Municio M.M. Sanz L. Lozano J. Chapkin R.S. Moscat J. Cell. 1993; 74: 555-563Abstract Full Text PDF PubMed Scopus (343) Google Scholar). PKC ζ has emerged as a critical regulator of a number of cellular functions, including proliferation, differentiation, and apoptosis inhibition (18Moscat J. Sanz L. Sanchez P. Diaz-Meco M.T. Adv. Enzyme Reg. 2001; 41: 99-120Crossref PubMed Scopus (10) Google Scholar). Despite the critical role of this enzyme in cellular signaling, its implication in the regulation of topoisomerase II function has never been examined. This study was aimed to evaluate the effect of PKC ζ overexpression on the formation of cleavable complexes and cytotoxicity induced by VP-16 in the human leukemic U937 cells. DISCUSSIONThis study shows that PKCζ overexpression in U937 cells resulted in inhibition of apoptosis and increased survival of U937 cells treated with VP-16 and mitoxantrone, two topoisomerase II inhibitors. Enforced PKCζ expression resulted in a marked decrease in VP-16-induced DPC and DNA DSB, whereas the level of topoisomerase IIα and topoisomerase IIβ expression was unchanged compared with control cells. These results suggest that PKCζ can interfere with topoisomerase II function. In fact, we found that PKCζ-overexpressing cells exhibited reduced topoisomerase II catalytic function as measured by the decatenation assay. Altered topoisomerase II catalytic cycle may explain reduced drug-induced DNA damage and cytotoxicity. Thus, this study shows for the first time that a specific PKC isozyme may inhibit topoisomerase II catalytic activity and VP-16-induced apoptosis and cytotoxicity by interfering with drug-induced DNA damage.Based on the kinase function of PKCζ, we hypothesized that PKCζ overexpression might result in abnormal topoisomerase II phosphorylation. In fact, we found that, in PKCζ-overexpressing cells, PKCζ was not only found to interact with topoisomerase IIα and topoisomerase IIβ but also that these two topoisomerase II isoforms were heavily phosphorylated on serine residues. These results suggest that, in PKCζ-overexpressing cells, PKCζ not only directly or indirectly interacts with the two topoisomerase II isoforms but also phosphorylates these enzymes. However, using a cell-free system, we described that only topoisomerase IIβ is a substrate for PKCζ and that PKCζ inhibits topoisomerase IIβ activity. This result suggests that, in PKCζ-overexpressing cells, PKCζ interacts directly with topoisomerase IIβ and inhibits topoisomerase IIβ catalytic activity. This hypothesis is consistent with the role of this topoisomerase IIβ form in the cytotoxicity of topoisomerase II inhibitors (23Brown G.A. McPherson J.P., Gu, L. Hedley D.W. Toso R. Deuchars K.L. Freedman M.H. Goldenberg G.J. Cancer Res. 1995; 55: 78-82PubMed Google Scholar, 24Errington F. Willmore E. Tilby M.J., Li, L., Li, G., Li, W. Baguley B.C. Austin C.A. Mol. Pharmacol. 1999; 56: 1309-1316Crossref PubMed Scopus (86) Google Scholar).With regard to topoisomerase IIα, the fact that this enzyme was found to interact in vivo, but not in vitro, with PKCζ, suggests that, in PKCζ-overexpressing cells, PKCζ/topoisomerase IIα interaction involves one or several other proteins required for the constitution of this complex. Moreover, the fact that, in PKCζ-overexpressing cells, topoisomerase IIα was found to be constitutively phosphorylated whereas, in vitro, PKCζ was unable to phosphorylate this enzyme, suggests that topoisomerase IIα is phosphorylated by another PKCζ-regulated kinase. In this perspective, it is interesting to note that in a recent study topoisomerase IIα was found to be phosphorylated in intact cells by ERK2, the effector serine kinase of the classic MAPK module (25Shapiro P.S. Whalen A.M. Tolwinski N.S. Wilsbacher J. Froelich-Ammon S.J. Garcia M. Osheroff N. Ahn N.G. Mol. Cell. Biol. 1999; 19: 3551-3560Crossref PubMed Google Scholar). Based on previous studies, which have documented that PKCζ is a downstream target of MAPK (26Berra E. Diaz-Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar, 27van Dijk M.C. Hilkmann H. van Blitterswijk W.J. Biochem. J. 1997; 325: 303-307Crossref PubMed Scopus (63) Google Scholar), topoisomerase IIα phosphorylation could result from PKCζ-mediated ERK2 activation in PKCζ-overexpressing cells. The fact that, in these cells, ERK2 was found to be constitutively activated and accumulated in the nucleus (data not shown) supports this hypothesis.The role of atypical PKC isoforms, including PKCζ, in cell survival has been previously documented. Indeed, it has been described that the blockade of PKCζ or PKCλ/ι with dominant-negative mutants or antisense oligonucleotides is sufficient to promote apoptosis (28Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 29Murray N.R. Fields A.P. J. Biol. Chem. 1997; 272: 27521-27524Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The inactivation of PKCζ by caspase-dependent proteolysis during apoptosis induced by UV (30Frutos S. Moscat J. Diaz-Meco M.T. J. Biol. Chem. 1999; 274: 10765-10770Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) or by cisplatin (31Basu A. Akkaraju G.R. Biochemistry. 1999; 38: 4245-4251Crossref PubMed Scopus (84) Google Scholar) strengthens the role of PKCζ in the cellular protection against genotoxic stress. The mechanism by which atypical PKC isoforms exert their anti-apoptotic effect has received a great deal of attention. These studies strongly suggested that NF-κB signaling pathways could play an important role in PKCζ-induced inhibition of apoptosis (32Lallena M.J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). Indeed, NF-κB is a negative regulator of apoptosis induced by genotoxic agents, including topoisomerase II inhibitors (33Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2926) Google Scholar, 34Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2500) Google Scholar). Therefore, we cannot rule out that PKCζ overexpression may result in the activation of anti-apoptotic signals that interfere with the post-damage apoptotic response and, therefore, contribute to drug resistance.To conclude, we propose a model in which, upon PKCζ accumulation in the nucleus, this enzyme interacts with and phosphorylates nuclear topoisomerase IIβ. Topoisomerase IIβ hyperphosphorylation reduces catalytic function and decreases formation of ternary complexes and drug-induced cytotoxicity. If so, nuclear PKCζ accumulation might function to regulate topoisomerase II function. Although very little is known about expression and subcellular localization of PKCζ in tumor cells, PKCζ may translocate to the nucleus upon stimulation by differentiating agents (35Bertolaso L. Gibellini D. Secchiero P. Previati M. Falgione D. Visani G. Rizzoli R. Capitani S. Zauli G. Br. J. Haematol. 1998; 100: 541-549Crossref PubMed Scopus (36) Google Scholar), growth factors (36Wooten M.W. Zhou G. Wooten M.C. Seibenhener M.L. J. Neurosci. Res. 1997; 49: 393-403Crossref PubMed Scopus (50) Google Scholar, 37Neri L.M. Martelli A.M. Borgatti P. Colamussi M.L. Marchisio M. Capitani S. FASEB J. 1999; 13: 2299-2310Crossref PubMed Scopus (103) Google Scholar), cytokines (38Marchisio M. Bertagnolo V. Celeghini C. Vitale M. Capitani S. Zauli G. Anat. Rec. 1999; 255: 7-14Crossref PubMed Scopus (12) Google Scholar), or hypoxia (39Mizukami Y. Hirata T. Yoshida K. FEBS Lett. 1997; 401: 247-251Crossref PubMed Scopus (62) Google Scholar). Whether PKCζ alters topoisomerase II function in these conditions will be the subject of further investigations. This study shows that PKCζ overexpression in U937 cells resulted in inhibition of apoptosis and increased survival of U937 cells treated with VP-16 and mitoxantrone, two topoisomerase II inhibitors. Enforced PKCζ expression resulted in a marked decrease in VP-16-induced DPC and DNA DSB, whereas the level of topoisomerase IIα and topoisomerase IIβ expression was unchanged compared with control cells. These results suggest that PKCζ can interfere with topoisomerase II function. In fact, we found that PKCζ-overexpressing cells exhibited reduced topoisomerase II catalytic function as measured by the decatenation assay. Altered topoisomerase II catalytic cycle may explain reduced drug-induced DNA damage and cytotoxicity. Thus, this study shows for the first time that a specific PKC isozyme may inhibit topoisomerase II catalytic activity and VP-16-induced apoptosis and cytotoxicity by interfering with drug-induced DNA damage. Based on the kinase function of PKCζ, we hypothesized that PKCζ overexpression might result in abnormal topoisomerase II phosphorylation. In fact, we found that, in PKCζ-overexpressing cells, PKCζ was not only found to interact with topoisomerase IIα and topoisomerase IIβ but also that these two topoisomerase II isoforms were heavily phosphorylated on serine residues. These results suggest that, in PKCζ-overexpressing cells, PKCζ not only directly or indirectly interacts with the two topoisomerase II isoforms but also phosphorylates these enzymes. However, using a cell-free system, we described that only topoisomerase IIβ is a substrate for PKCζ and that PKCζ inhibits topoisomerase IIβ activity. This result suggests that, in PKCζ-overexpressing cells, PKCζ interacts directly with topoisomerase IIβ and inhibits topoisomerase IIβ catalytic activity. This hypothesis is consistent with the role of this topoisomerase IIβ form in the cytotoxicity of topoisomerase II inhibitors (23Brown G.A. McPherson J.P., Gu, L. Hedley D.W. Toso R. Deuchars K.L. Freedman M.H. Goldenberg G.J. Cancer Res. 1995; 55: 78-82PubMed Google Scholar, 24Errington F. Willmore E. Tilby M.J., Li, L., Li, G., Li, W. Baguley B.C. Austin C.A. Mol. Pharmacol. 1999; 56: 1309-1316Crossref PubMed Scopus (86) Google Scholar). With regard to topoisomerase IIα, the fact that this enzyme was found to interact in vivo, but not in vitro, with PKCζ, suggests that, in PKCζ-overexpressing cells, PKCζ/topoisomerase IIα interaction involves one or several other proteins required for the constitution of this complex. Moreover, the fact that, in PKCζ-overexpressing cells, topoisomerase IIα was found to be constitutively phosphorylated whereas, in vitro, PKCζ was unable to phosphorylate this enzyme, suggests that topoisomerase IIα is phosphorylated by another PKCζ-regulated kinase. In this perspective, it is interesting to note that in a recent study topoisomerase IIα was found to be phosphorylated in intact cells by ERK2, the effector serine kinase of the classic MAPK module (25Shapiro P.S. Whalen A.M. Tolwinski N.S. Wilsbacher J. Froelich-Ammon S.J. Garcia M. Osheroff N. Ahn N.G. Mol. Cell. Biol. 1999; 19: 3551-3560Crossref PubMed Google Scholar). Based on previous studies, which have documented that PKCζ is a downstream target of MAPK (26Berra E. Diaz-Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (252) Google Scholar, 27van Dijk M.C. Hilkmann H. van Blitterswijk W.J. Biochem. J. 1997; 325: 303-307Crossref PubMed Scopus (63) Google Scholar), topoisomerase IIα phosphorylation could result from PKCζ-mediated ERK2 activation in PKCζ-overexpressing cells. The fact that, in these cells, ERK2 was found to be constitutively activated and accumulated in the nucleus (data not shown) supports this hypothesis. The role of atypical PKC isoforms, including PKCζ, in cell survival has been previously documented. Indeed, it has been described that the blockade of PKCζ or PKCλ/ι with dominant-negative mutants or antisense oligonucleotides is sufficient to promote apoptosis (28Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 29Murray N.R. Fields A.P. J. Biol. Chem. 1997; 272: 27521-27524Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The inactivation of PKCζ by caspase-dependent proteolysis during apoptosis induced by UV (30Frutos S. Moscat J. Diaz-Meco M.T. J. Biol. Chem. 1999; 274: 10765-10770Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) or by cisplatin (31Basu A. Akkaraju G.R. Biochemistry. 1999; 38: 4245-4251Crossref PubMed Scopus (84) Google Scholar) strengthens the role of PKCζ in the cellular protection against genotoxic stress. The mechanism by which atypical PKC isoforms exert their anti-apoptotic effect has received a great deal of attention. These studies strongly suggested that NF-κB signaling pathways could play an important role in PKCζ-induced inhibition of apoptosis (32Lallena M.J. Diaz-Meco M.T. Bren G. Paya C.V. Moscat J. Mol. Cell. Biol. 1999; 19: 2180-2188Crossref PubMed Google Scholar). Indeed, NF-κB is a negative regulator of apoptosis induced by genotoxic agents, including topoisomerase II inhibitors (33Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2926) Google Scholar, 34Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2500) Google Scholar). Therefore, we cannot rule out that PKCζ overexpression may result in the activation of anti-apoptotic signals that interfere with the post-damage apoptotic response and, therefore, contribute to drug resistance. To conclude, we propose a model in which, upon PKCζ accumulation in the nucleus, this enzyme interacts with and phosphorylates nuclear topoisomerase IIβ. Topoisomerase IIβ hyperphosphorylation reduces catalytic function and decreases formation of ternary complexes and drug-induced cytotoxicity. If so, nuclear PKCζ accumulation might function to regulate topoisomerase II function. Although very little is known about expression and subcellular localization of PKCζ in tumor cells, PKCζ may translocate to the nucleus upon stimulation by differentiating agents (35Bertolaso L. Gibellini D. Secchiero P. Previati M. Falgione D. Visani G. Rizzoli R. Capitani S. Zauli G. Br. J. Haematol. 1998; 100: 541-549Crossref PubMed Scopus (36) Google Scholar), growth factors (36Wooten M.W. Zhou G. Wooten M.C. Seibenhener M.L. J. Neurosci. Res. 1997; 49: 393-403Crossref PubMed Scopus (50) Google Scholar, 37Neri L.M. Martelli A.M. Borgatti P. Colamussi M.L. Marchisio M. Capitani S. FASEB J. 1999; 13: 2299-2310Crossref PubMed Scopus (103) Google Scholar), cytokines (38Marchisio M. Bertagnolo V. Celeghini C. Vitale M. Capitani S. Zauli G. Anat. Rec. 1999; 255: 7-14Crossref PubMed Scopus (12) Google Scholar), or hypoxia (39Mizukami Y. Hirata T. Yoshida K. FEBS Lett. 1997; 401: 247-251Crossref PubMed Scopus (62) Google Scholar). Whether PKCζ alters topoisomerase II function in these conditions will be the subject of further investigations. We thank Dr. Ways (Lilly Corporate Center, USA) who kindly provided the PKCζ-overexpressing U937 cell lines." @default.
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• W2045461964 title "Overexpression of the Atypical Protein Kinase C ζ Reduces Topoisomerase II Catalytic Activity, Cleavable Complexes Formation, and Drug-induced Cytotoxicity in Monocytic U937 Leukemia Cells" @default.
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