Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor κB Activation in HL60 Cells
2000; Elsevier BV; Volume: 275; Issue: 33 Linguagem: Inglês
10.1074/jbc.275.33.25231
ISSN1083-351X
AutoresMarion P. Boland, Katherine A. Fitzgerald, Luke O'neill,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoTopoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor κB (NFκB) by this drug. Mitoxantrone activated NFκB and stimulated IκBα degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the β isoform of topoisomerase II and express a truncated α isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFκB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFκB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IκB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IκB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFκB activation. Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor κB (NFκB) by this drug. Mitoxantrone activated NFκB and stimulated IκBα degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the β isoform of topoisomerase II and express a truncated α isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFκB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFκB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IκB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IκB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFκB activation. nuclear factor κB tumor necrosis factor fetal bovine serum Imperial Cancer Research Fund 187 Agents that induce stress in cells, such as ionizing radiation, reactive oxygen species, and anti-neoplastic drugs, change the expression of many genes by affecting transcription factors, including AP1, NF-AT, NFκB,1and Egr-1 (1Beiquig L. Chen M. Whisler R.L. J. Immunol. 1996; 157: 160-169PubMed Google Scholar, 2Boland M.P. Foster S.J. O'Neill L.A. J. Biol. Chem. 1997; 272: 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 3Bowie A.G. Moynagh P.N. O'Neill L.A. J. Biol. Chem. 1997; 272: 25941-25950Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 4Brach M.A. Hass R. Sherman M.L. Gunji H. Weichselbaum R. Kufe D. J. Clin. 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A nucleolar localization has, however, been disputed recently, with evidence being presented for the β form being nuclear but not nucleolar (34Cowell I.G. Willmore E. Chalton D. Marsh K.L. Jazrawi E. Fisher L.M. Austin C.A. Exp. Cell. Res. 1998; 243: 232-240Crossref PubMed Scopus (38) Google Scholar). These enzymes may have distinct and overlapping physiological roles, suggested by their differential expression and phosphorylation during mitosis and transformation (31Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar, 32Isaacs R.J. Davies S.L. Sandri M.I. Redwood C. Wells N.J. Hickson I.D. Biochim. Biophys. Acta. 1998; 1400: 121-139Crossref PubMed Scopus (185) Google Scholar). The α form, a substrate for casein kinase II (35Redwood C. Davies S.L. Wells N.J. Fry A.M. Hickson I.D. J. Biol. 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Austin C.A. Mol. Pharmacol. 1999; 56: 1309-1316Crossref PubMed Scopus (87) Google Scholar). In this study, we have investigated the role of topoisomerase II in NFκB activation by mitoxantrone. We have utilized a resistant variant of the promyelocytic cell line HL60, termed HL60/MX2, which, compared with the parental line, is 34-fold more resistant to cell killing by mitoxantrone (45Harker W.G. Slade D.L. Dalton W.S. Meltzer P.S. Trent J.M. Cancer Res. 1989; 49: 4542-4549PubMed Google Scholar, 46Harker W.G. Slade D.L. Drake F.H. Parr R.L. Biochemistry. 1991; 30: 9953-9961Crossref PubMed Scopus (160) Google Scholar). This resistance has been correlated with a complete absence of the expression of the topoisomerase II β isoform. Furthermore, the topoisomerase II α transcript has a deletion in its 3′ end, resulting in a truncated protein with altered subcellular distribution and possibly altered drug sensitivity (47Harker W.G. Slade D.L. Parr R.L. Hoguin M.H. Cancer Res. 1995; 55: 4962-4971PubMed Google Scholar). Our data support a direct relationship between cell killing, NFκB activation, and the targeting of topoisomerase II enzymes by this drug, indicating a novel pathway to NFκB activation involving DNA double strand breaks generated by the poisoning of topoisomerase II. HL60 parental and resistant (HL60/MX2) cells were kindly supplied by Dr. Ian Hickson (ICRF, University of Oxford, Oxford, United Kingdom) (43Son Y.S. Suh J.M. Ahn S.H. Kim J.C. Yi J.Y. Hur K.C. Hong W.-S. Muller M.T. Chung I.K. Cancer Chemother. Pharmacol. 1998; 41: 353-360Crossref PubMed Scopus (35) Google Scholar) and were grown in suspension culture in RPMI 1640 medium supplemented with 20% fetal calf serum, penicillin/streptomycin (100 units/ml and 100 μg/ml, respectively) and l-glutamine (final concentration, 2 mm), all obtained from Life Technologies, Inc. Mitoxantrone and recombinant human TNFα were generous gifts from Wyeth-Ayerst Research (Berks, United Kingdom) and Zeneca Pharmaceuticals Ltd. (Macclesfield, United Kingdom), respectively. ICRF 187 was kindly supplied by Dr. Maxwell Sehested (Department of Pathology, Laboratory Center Rigshospitalet, Copenhagen, Denmark) and Dr. A. M. Creighton (Medicinal Chemistry Laboratory, St. Bartholomew's Hospital Medical College, London, United Kingdom). Merbarone was a gift from Professor William T. Beck (Division of Therapeutics, Cancer Center, College of Medicine, University of Illinois at Chicago, Chicago, IL). Poly(dI-dC) was purchased from Amersham Pharmacia Biotech. T4 polynucleotide kinase and oligonucleotide containing the consensus sequence 5′-GG GAC TTT CC-3′, corresponding to the κ light chain enhancer motif, were purchased from Promega (Southampton, United Kingdom). [γ-32P]ATP (3000 Ci/mmol), was from Amersham Pharmacia Biotech. PhototopeTM horseradish peroxidase Western blot detection kit was from New England Biolabs Ltd. Monoclonal antibodies to the inhibitor protein, IκBα, were a generous gift from Dr. Ron Hay (St. Andrews, United Kingdom). Monoclonal antibodies to human DNA topoisomerase II (α and β) and CPP32 enzymes were purchased from Genosys Biotechnologies Inc. (Cambridge, United Kingdom) and Transduction Laboratories (Lexington, KY), respectively. Rabbit polyclonal antibodies to topoisomerase II α and β were generously provided by Dr. Ian Hickson (ICRF, University of Oxford). All other reagents were purchased from Sigma. For treatments, cells in late log phase of growth were resuspended in complete medium supplemented with 0.5% FBS at a concentration of 1 × 106 cells/ml and incubated at 37 °C in a humidified atmosphere of 5% CO2/95% air (for 16 h prior to stimulation). Following stimulation (4 h unless otherwise stated), incubations were discontinued by the addition of ice-cold phosphate-buffered saline, and either nuclear or whole cell extracts (for IκB and CPP32 determinations) were prepared as described previously (2Boland M.P. Foster S.J. O'Neill L.A. J. Biol. Chem. 1997; 272: 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Treatments using the radiomimetic drug bleomycin were carried out for 2 h only. Where required, cells were preincubated with the topoisomerase II inhibitors ICRF 187 or merbarone for 1 h, prior to the addition of either drug (4 h) or cytokine (1 h). Cell extracts for the analysis of topoisomerase II isoform content were prepared according to the method described by Drake et al. (48Hann C. Evans D.L. Fertala J. Benedetti P. Bjornsti M. Hall D.J. J. Biol. Chem. 1998; 273: 8425-8533Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Protein concentration determinations were made using the Bradford assay with bovine albumin as standard. Nuclear NFκB was assessed by the electrophoretic mobility shift assay using a 22-base pair oligonucleotide containing the human κ light chain enhancer motif, which had previously been end-labeled with [γ-32P]ATP as described (2Boland M.P. Foster S.J. O'Neill L.A. J. Biol. Chem. 1997; 272: 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Typically, 2–4 mg of nuclear extract protein was incubated with radiolabeled oligonucleotide (10,000 cpm) at room temperature for 30 min using the conditions described previously (2Boland M.P. Foster S.J. O'Neill L.A. J. Biol. Chem. 1997; 272: 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Boland M.P. O'Neill L.A. J. Biol. Chem. 1998; 273: 15494-15500Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). NFκB complexes were resolved on 5% acrylamide gels and identified following autoradiography. Equal amounts of whole cell lysate protein (as indicated) were resolved by SDS-polyacrylamide gel electrophoresis (10% running gel) and transferred onto nitrocellulose, and IκBα or CPP32 immunoblot analysis was performed as described previously (2Boland M.P. Foster S.J. O'Neill L.A. J. Biol. Chem. 1997; 272: 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 11Boland M.P. O'Neill L.A. J. Biol. Chem. 1998; 273: 15494-15500Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Samples for topoisomerase II protein detection were resolved on 7% gels and, following transfer, incubated with primary antibody at a dilution of 1:250 (0.4 μg/ml) in 1% dry milk in 1× phosphate buffered saline and 0.5% Tween 20. Secondary antibody was used at a dilution of 1:1000. The blots were developed by chemiluminescent detection (ECL) according to the manufacturer's recommendation. Following treatment, cells were harvested by centrifugation, and nuclear extracts were prepared as described previously (48Hann C. Evans D.L. Fertala J. Benedetti P. Bjornsti M. Hall D.J. J. Biol. Chem. 1998; 273: 8425-8533Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Samples were resolved on 7% gels and, following transfer, incubated with primary antibody to both topoisomerase II isoforms (as described above) or with isoform-specific antibodies (polyclonal) at a dilution of 1:500; samples were resolved in 1% dry milk in 1× phosphate buffered saline and 0.5% Tween 20. Secondary antibody concentrations and detection were also as described above. Previously we have shown that mitoxantrone could activate NFκB in a dose-dependent manner in HL60 promyelocytic leukemia cells (2Boland M.P. Foster S.J. O'Neill L.A. J. Biol. Chem. 1997; 272: 12952-12960Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In this report, we have compared this activation with that in a derived variant of this cell line, HL60/MX2 (42McPherson J.P. Deffie A.M. Jones N.R. Brown G.A. Deuchars K.L. Goldenberg G.J. Anticancer Res. 1997; 17: 4243-4252PubMed Google Scholar, 43Son Y.S. Suh J.M. Ahn S.H. Kim J.C. Yi J.Y. Hur K.C. Hong W.-S. Muller M.T. Chung I.K. Cancer Chemother. Pharmacol. 1998; 41: 353-360Crossref PubMed Scopus (35) Google Scholar, 44Errington 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 (87) Google Scholar), which has reduced levels of both topoisomerase II α and β and is resistant to cell kill by mitoxantrone. NFκB activation was not observed in the HL60/MX2 cell line, compared with a dose-responsive activation in the parental cell line, demonstrated by the detection of protein-DNA complexes in nuclear extracts from drug-treated cells (Fig.1 A). Concentrations of 50–1000 nm mitoxantrone activated NFκB (lanes 2–6) in the parental line, whereas these concentrations were ineffective in the HL60/MX2 cell line (lanes 8–12). However, both cell lines showed comparable NFκB activation following stimulation with the cytokine, TNF (2 ng/ml) (lanes 13 and14, respectively). A dose-dependent degradation of the inhibitor protein, IκBα, a key signal for NFκB activation (9Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2935) Google Scholar), was also observed in the parental but not the HL60/MX2 cell line in response to mitoxantrone (Fig. 1 B). Concentrations from 500 nm clearly induced IκB degradation in the parental line (lanes 3–5) but not in the HL60/MX2 cell line (Fig. 1 B, lanes 9–11), which supported the NFκB activation data. This degradation was comparable in both cell lines in response to TNF (2 ng/ml) (Fig. 1 B, compare lanes 6 and 12). We also tested another DNA damaging agent in both cell lines, bleomycin, the effects of which are not topoisomerase II-dependent (49Sikic B.I. Cancer Surv. 1986; 5: 81-91PubMed Google Scholar). This agent induced IκBα degradation in both HL60 and HL60/MX2 cells (Fig. 1 C, comparelanes 1 and 2 with lanes 3 and4), although its effect was less pronounced than mitoxantrone in parental HL60 cells or TNF in both cell lines (Fig.1 B). Although bleomycin was a weaker activator of NFκB, this may be explained by its ability to cause DNA strand scission resulting in both single and double strand breaks, the former perhaps being a less potent signal for this effect. Densitometric scanning performed on the immunoblot data (Fig. 1 C) showed similar levels of IκBα degradation (47 and 45%) when compared with control values for both parental and variant HL60/MX2 cells in response to bleomycin. Non-topoisomerase II-targeted drugs that damage DNA can therefore activate NFκB and are independent of the topoisomerase II levels in target cells. To confirm the phenotype of both cell lines, Western blot analysis of whole cell extracts was performed with an antibody that recognized both the α and β isoforms of the topoisomerase II enzyme (Fig.2 A). Both isoforms were detected in the parental cell line (Fig. 2 A, lane 1–3), whereas expression of the β isoform was negligible in extracts from the HL60/MX2 cells (lane 4–6). The expression of both isoforms was unaffected by treatment of cells with mitoxantrone (250 and 500 nm, Fig. 2 A, lanes 2–3 and5–6). Formation of transient DNA-topoisomerase II complexes is stabilized by some topoisomerase II poisons, such as mitoxantrone. Employing a band depletion assay on nuclear extracts from treated cells, the functionality of either the drug or the topoisomerase II target can be determined (50Mo Y.-Y. Beck W. Exp. Cell Res. 1999; 252: 50-62Crossref PubMed Scopus (34) Google Scholar). Following treatment of parental HL60 cells with mitoxantrone (Fig. 2 B), the intensity of a band representing both isoforms decreased as drug concentration increased (Fig. 2 B, lanes 1–4). The extraction protocol used in the band depletion assay prevented the resolution of both topoisomerase II isoforms, which migrated as a single band. Importantly, no band depletion was seen following treatment of the resistant variant cells, HL60/MX2 (lanes 5–7). Furthermore, employing an antibody that recognizes the α isoform of topoisomerase II (Fig.2 C), its specific depletion was shown in parental HL60 cells (lanes 1–4) but not in the variant cells, HL60/MX2 (lanes 5–8), confirming the resistance of the α isoform to mitoxantrone in the HL60/MX2 line. In addition, when an antibody that specifically recognizes the β isoform of topoisomerase II was used, mitoxantrone treatment caused a depletion of the β isoform in parental HL60
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