Title: Daunorubicin Activates NFκB and Induces κB-dependent Gene Expression in HL-60 Promyelocytic and Jurkat T Lymphoma Cells
Abstract: The anthracycline antibiotic, daunorubicin, can induce programmed cell death (apoptosis) in cells. Recent work suggests that this event is mediated by ceramide via enhanced ceramide synthase activity. Since the generation of ceramide has been directly linked with the activation of the transcription factor, NFκB, this was investigated as a novel target for the action of daunorubicin. Here we describe how treatment of HL-60 promyelocytes and Jurkat T lymphoma cells with daunorubicin results in the activation of the transcription factor NFκB. The effect of daunorubicin was evident following 1–2 h treatment, which was in contrast to the time course of activation obtained with the cytokine, tumor necrosis factor, where NFκB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IκB degradation and increased the expression of an NFκB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFκB-linked reporter gene, suggesting a synergy between these two agents in this response. These events were sensitive to the iron chelator, deferoxamine mesylate (desferal), and the anti-oxidant and metal chelator pyrrolidine dithiocarbamate. A structurally related compound, mitoxantrone, which, unlike daunorubicin, is unable to undergo redox cycling in cells, also activated NFκB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFκB activation at a range of concentrations previously reported to block apoptosis induced by this drug. However, this agent could inhibit increases in ceramide induced by daunorubicin, in addition to blocking ceramide synthase activity from HL-60 cells which was activated in response to daunorubicin treatment. These data therefore suggest that the effect of daunorubicin on NFκB is unlikely to involve ceramide, but may involve reactive oxygen species generated as a result of endogenous cellular processes rather than reductive metabolism of the drug. As NFκB may be involved in apoptosis, this effect may be an important aspect of the cellular responses to this agent. The anthracycline antibiotic, daunorubicin, can induce programmed cell death (apoptosis) in cells. Recent work suggests that this event is mediated by ceramide via enhanced ceramide synthase activity. Since the generation of ceramide has been directly linked with the activation of the transcription factor, NFκB, this was investigated as a novel target for the action of daunorubicin. Here we describe how treatment of HL-60 promyelocytes and Jurkat T lymphoma cells with daunorubicin results in the activation of the transcription factor NFκB. The effect of daunorubicin was evident following 1–2 h treatment, which was in contrast to the time course of activation obtained with the cytokine, tumor necrosis factor, where NFκB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IκB degradation and increased the expression of an NFκB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFκB-linked reporter gene, suggesting a synergy between these two agents in this response. These events were sensitive to the iron chelator, deferoxamine mesylate (desferal), and the anti-oxidant and metal chelator pyrrolidine dithiocarbamate. A structurally related compound, mitoxantrone, which, unlike daunorubicin, is unable to undergo redox cycling in cells, also activated NFκB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFκB activation at a range of concentrations previously reported to block apoptosis induced by this drug. However, this agent could inhibit increases in ceramide induced by daunorubicin, in addition to blocking ceramide synthase activity from HL-60 cells which was activated in response to daunorubicin treatment. These data therefore suggest that the effect of daunorubicin on NFκB is unlikely to involve ceramide, but may involve reactive oxygen species generated as a result of endogenous cellular processes rather than reductive metabolism of the drug. As NFκB may be involved in apoptosis, this effect may be an important aspect of the cellular responses to this agent. The anthracycline antibiotic, daunorubicin, is widely used in cancer chemotherapy with proven therapeutic benefit in the treatment of a variety of neoplasia (1Calabresi P. Chabner B.A. Gilman A.G. Rall T.W. Nies A.S. Taylor P. Pharmacological Basis of Therapeutics. 8th Ed. Pergamon Press, New York1990: 1202-1263Google Scholar). Although its mechanism of anti-tumor action is uncertain, DNA is believed to be a primary target (2Powis G. Powis G. Prough R.A. Metabolism and Action of Anti-cancer Drugs. Taylor and Francis Ltd., London1987: 211-246Google Scholar). Its ability to cause strand scission may be mediated by stabilizing a cleavable complex between DNA and the enzyme, topoisomerase II, and/or oxygen radicals arising from redox cycling following its bioreduction. Additionally, bioreduction products and reactive oxygen species have been associated with anthracycline induced alkylation of cellular macromolecules, DNA intercalation and cross-linking, lipid peroxidation, and cell membrane damage (2Powis G. Powis G. Prough R.A. Metabolism and Action of Anti-cancer Drugs. Taylor and Francis Ltd., London1987: 211-246Google Scholar). Irrespective of the initial insult, anthracyclines, along with a variety of agonists, ultimately activate the event of programmed cell death or apoptosis in cells (3Barry M.A. Behnke C.A. Eastman A. Biochem. Pharmacol. 1990; 40: 2353-2362Crossref PubMed Scopus (914) Google Scholar). Their ability to induce this pathway may be a mechanism underlying their therapeutic efficacy in certain tumor types. The development of the apoptotic morphology is well defined; however, signaling pathways that may act as primary mediators of apoptosis and growth suppression are poorly characterized (4Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2429) Google Scholar). Some of those relevant to the cytotoxic action of chemotherapeutic drugs include the triggering of CD95/CD95-L interaction resulting in a type of autocrine suicide (5Friesen C. Herr I. Krammer P.H. Debatin K-M. Nature Med. 1996; 2: 574-577Crossref PubMed Scopus (952) Google Scholar), and the activation of effector molecules such as interleukin-1 converting enzyme-like proteases (6Kaufmann S.H. Desnoyers S. Ottaviano Y. Davidson N.E. Poirier G.G. Cancer Res. 1993; 53: 3976-3985PubMed Google Scholar). Recent studies suggest that the neutral lipid ceramide may also play a role in mediating drug-induced apoptosis (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar, 8Jaffrezou J-P. Levade T. Bettaieb A. Andrieu N. Bezombes C. Maestre N. Vermeersch S. Rousse A. Laurent G. EMBO J. 1996; 15: 2417-2424Crossref PubMed Scopus (351) Google Scholar). Ceramide is a putative second messenger which can also be generated following the activation of distinct sphingomyelinase activities in response to a range of extracellular agents including TNF-α, 1The abbreviations used are: TNF, tumor necrosis factor; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; NFκB, nuclear factor κB; CAT, chloramphenicol acetyltransferase. 1The abbreviations used are: TNF, tumor necrosis factor; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; NFκB, nuclear factor κB; CAT, chloramphenicol acetyltransferase.interleukin-1β, γ-interferon, nerve growth factor, Fas ligand, and 1,25-dihydroxyvitamin D3 (9Hannun Y.A. Obeid L.M. Trends Biol. Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (573) Google Scholar). A general role in growth arrest and suppression is suggested by its ability to induce cell differentiation, cell-cycle arrest, apoptosis, or cell senescence (9Hannun Y.A. Obeid L.M. Trends Biol. Sci. 1995; 20: 73-77Abstract Full Text PDF PubMed Scopus (573) Google Scholar), although a mitogenic role has been demonstrated in certain cell types (10Boucher L.-M. Wiegmann K. Futterer A Pfeffer K. Mak T.W. Kronke M. J. Exp. Med. 1995; 181: 2059-2068Crossref PubMed Scopus (190) Google Scholar). In a recent study, daunorubicin was shown to increase ceramide levels in cells following induction of the enzyme ceramide synthase (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). Furthermore, inhibition of this enzyme by the mycotoxin, fumonisin B1, blocked apoptosis induced by daunorubicin. The regulated biosynthesis of ceramide may represent a signaling mechanism by which apoptotic events are induced by this drug. The generation of ceramide following activation of lysosomal acid sphingomyelinase has not only been linked with TNF-induced apoptosis, but also with the activation of NFκB (11Yang Z. Costanzo M. Golde D.W. Kolesnick R.N. J. Biol. Chem. 1993; 268: 20520-20523Abstract Full Text PDF PubMed Google Scholar). This inducible transcription factor has been implicated in the regulation of many genes which code for mediators of the immune, acute phase, and inflammatory responses (12Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar). The DNA-binding protein complex recognizes a discrete nucleotide sequence (5′-GGGACTTTCC-3′) in the upstream regions of a variety of responsive genes. Subunits belonging to the NFκB family comprise five members in mammals: p50, p65 (RelA), c-Rel, p52, and RelB. These proteins share a conserved 300-amino acid sequence in the N-terminal portion, termed the Rel homology domain, which mediates DNA binding, protein dimerization, nuclear localization, and binding of the inhibitor protein IκB (either α or β) (12Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar). Various dimer combinations of these proteins have distinct DNA binding specificities and may serve to activate specific sets of genes (13Lin R. Gewert D. Hiscott J. J. Biol. Chem. 1995; 270: 3123-3131Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In resting cells, the NFκB dimer is sequestered in the cytosol by associating with IκB, and can be “liberated” from this complex by a variety of inducers. A simple model for NFκB activation is as follows. Phosphorylation of IκB by specific activated protein kinase(s) tags it for proteolytic degradation (14Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). This facilitates the nuclear translocation of activated NFκB complexes, whereupon binding to cognate sequences, gene expression is activated. The signaling pathways linking receptor stimulation to NFκB activation are poorly defined. A number of kinases have been implicated in the phosphorylation of IκB, the most notable being a recently identified ubiquitination-dependent multisubunit protein kinase (14Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). Phosphorylation at two specific residues, serine 32 and 36, on IκBα, is thought to lead to its ubiquitination and subsequent degradation by the proteosome, facilitating NFκB release and translocation into the nucleus (15Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1314) Google Scholar). Mutants lacking these residues cannot undergo phosphorylation (and subsequent proteolytic degradation) in response to a variety of stimuli (16Traenckner E.B-M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (931) Google Scholar) suggesting that many signal transduction pathways converge on the putative IκB kinase(s). In addition to ceramide being implicated as an upstream regulator of IκBα phosphorylation, a model has been proposed whereby reactive oxygen species (ROS) act as second messengers in this event (17Schulze-Osthoff K. Los M. Baeuerle P.A. Biochem. Pharmacol. 1995; 50: 735-741Crossref PubMed Scopus (256) Google Scholar). Evidence to support this model is based on the ability of H2O2 to activate NFκB (18Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3415) Google Scholar) and the inhibitory effects of antioxidants such as N-acetylcysteine (a thiol antioxidant and glutathione precursor) and pyrrolidine dithiocarbamate (PDTC), which is also a metal chelator, on NFκB activation (18Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3415) Google Scholar, 19Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 20Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 21Brennan P. O'Neill L.A.J. Biochim. Biophys. Acta. 1995; 1260: 167-175Crossref PubMed Scopus (208) Google Scholar). The link between ceramide and ROS in signaling either transcriptional activation or apoptotic events is unclear (22Buttke T.M. Sandstrom P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2099) Google Scholar,23Jacobson M.D. Trends Biol. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (729) Google Scholar). In a model for NFκB proposed by Baeuerle and Henkel (12Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar), agonist-stimulated ceramide generation lies upstream of an event which triggers H2O2 production, leading to the activation of this transcription factor. Because previous work demonstrated the production of ROS and ceramide in response to daunorubicin (2Powis G. Powis G. Prough R.A. Metabolism and Action of Anti-cancer Drugs. Taylor and Francis Ltd., London1987: 211-246Google Scholar, 7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar), we investigated the effect of this agent on NFκB activation. We have found that daunorubicin signals NFκB activation in HL-60 promyelocytes and Jurkat T cells by a PDTC-sensitive mechanism, suggesting the involvement of ROS, and not activated ceramide synthase. The importance of this signal for daunorubicin-mediated apoptosis is discussed. HL-60 and Jurkat T cells (both obtained from the European Collection of Animal Cell Culture (ECACC, Salisbury, United Kingdom) were grown in suspension culture in RPMI 1640 supplemented with 10% fetal calf serum, penicillin/streptomycin (100 units/ml and 100 mg/ml, respectively), and l-glutamate (2 mmfinal concentration), all obtained from Life Technologies, Inc. (Paisley, United Kingdom). Recombinant human TNFα was a gift from Zeneca Pharmaceuticals Ltd., Macclesfield, United Kingdom. Mitoxantrone was also a generous gift from Wyeth-Ayerst Research (United Kingdom). Poly(dI·dC) was from Pharmacia Biosystems (Milton Keynes, United Kingdom), T4 polynucleotide kinase and oligonucleotide containing the consensus sequence (5′-GGGACTTTCC-3′), corresponding to the κ-light chain enhancer motif, were purchased from Promega (Southampton, United Kingdom). [γ-32P]ATP (3000 Ci/mmol), [14C]chloramphenicol (56 mCi/mmol), [1-14C]palmitoyl-coenzyme A (55 mCi/mmol), and ECL reagent were from Amersham (Aylesbury, United Kingdom). Diacylglycerol kinase was from Calbiochem (United Kingdom). Rabbit polyclonal antibody preparations to the DNA-binding subunits of NFκB (c-Rel and RelA) and the inhibitor protein IκBα were from Santa Cruz Biotechnology Inc. Mutant NFκB oligonucleotide was also from Santa Cruz. An antiserum to the p50 subunit of NFκB was a generous gift from Dr. Jean Imbert (INSERM, Marseille). All other reagents were purchased from Sigma (Poole, Dorset, United Kingdom) unless otherwise stated. For treatments, cells in late log phase of growth were resuspended in fresh medium at a concentration of 1 × 106/ml and incubated at 37 °C in a humidified atmosphere of 5% CO2, 95% air. Where required, cells were preincubated with inhibitors (fumonisin B1 and PDTC for 60 min, and desferal for 16 h) prior to the addition of drug (4 h). Following stimulation, incubations were discontinued by the addition of ice-cold phosphate-buffered saline, and either nuclear or whole cell extracts were prepared as described previously (24Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Protein determinations were made using the Bradford assay with bovine albumin as standard. The transactivating potential of activated NFκB complexes was assessed following transfection of cells (25Queen C. Baltimore D. Cell. 1983; 33: 741-748Abstract Full Text PDF PubMed Scopus (389) Google Scholar) with a plasmid containing five NFκB consensus sequences upstream of a chloramphenicol acetyltransferase reporter gene (pCATTM-Promoter plasmid, a gift from Dr. Tim Bird, Immunex Corp., Seattle, WA). Following treatment (indicated in legends), extracts prepared from harvested cells were assayed for CAT activity as described previously (26Moynagh P.N. Williams D.C. O'Neill L.A.J. Biochem. J. 1993; 294: 343-347Crossref PubMed Scopus (81) Google Scholar). Statistical significance was evaluated by employing Student's t test for unpaired data. 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 (24Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Typically, 4 μg of nuclear extract protein was incubated with radiolabeled oligonucleotide (10,000 cpm) at room temperature for 30 min using conditions as described previously (24Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). NFκB complexes were resolved on 5% acrylamide gels and identified following autoradiography. To identify the subunit components of activated NFκB complexes, supershift analysis was carried out where extracts from treated cells were preincubated with antibody preparations to p50, RelA (p65), and c-Rel subunit components on ice for 30 min prior to the addition of labeled probe. A similar protocol was employed in competition studies (incubations were at room temperature), where mutant and wild type NFκB consensus sequence were assessed for their ability to block binding of activated complexes to labeled wild type NFκB probe. Equal amounts of whole cell lysate protein (as indicated) were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and IκBα immunoblot analysis was performed as described previously (24Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Secondary antibody was used at a dilution of 1:400. The blots were developed by ECL according to the manufacturers recommendations. Ceramide was quantified by the diacylglycerol kinase assay as described (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar), with some modifications. In brief, following stimulation, cell pellets were extracted with 600 μl of chloroform, methanol, 1 n HCl (100:100:1, v/v/v). Following alkaline hydrolysis (1 h at 37 °C), re-extracted samples were dried down and redissolved in 50 μl of reaction buffer (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). The reaction was started by the addition of 40 μg/ml (4 milliunits/ml)Escherichia coli diacylglycerol kinase followed closely by 10 μCi of [γ-32P]ATP. Reaction termination was as described following incubation at room temperature for 90 min. The level of ceramide was determined by comparison with a standard curve generated with known amounts of ceramide (ceramide type III; Sigma). This activity was measured in HL-60 microsomal membranes as described previously (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). In general, 50 × 106 cells were pelleted following drug treatment and disrupted in 300 μl of homogenization buffer by repeatedly passing through a 26-gauge needle. Microsomal membrane protein (37.5 μg) was incubated in a 250-μl reaction volume/mixture as described (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar) with dihydrosphingosine as substrate. The reaction was started by the addition of 3.6 μm (0.2 μCi) [1-14C]palmitoyl-coenzyme A, the incubation was allowed to proceed for 1 h at 37 °C, and stopped by extraction with an equal volume of chloroform/methanol (2:1, v/v). The substrate concentrations chosen were based on those reported to allow maximal enzyme activity to be monitored (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar), with 100 μmdihydrosphingosine being optimal. Following TLC as described (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar), radioactivity corresponding to synthesized dihydroceramide was determined using an InstantImagerTM (Packard Instrument Co., Meriden, CT). Treatment of both HL-60 and Jurkat T cells with the anthracycline antibiotic, daunorubicin, resulted in the activation of NFκB, which was dose-dependent and time-responsive (Fig. 1 A, C, andD). Fig. 1 A illustrates data obtained with HL-60 cells, where activation of NFκB is demonstrated by the appearance of DNA-protein complexes. The activation was time-dependent occurring from 1 to 4 h and was sustained up to 24 h (Fig.1 A). This was in contrast to that seen with TNF, where the activation of NFκB was rapid occurring within minutes of cellular stimulation (Fig. 1 B). Concentrations of daunorubicin employed were similar to those previously reported to induce apoptosis in this cell line (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar), with activation being apparent at 0.05 μm, and peaking at 0.5 μm (Fig.1 C). These concentrations also paralleled that reported for ceramide elevation induced by this drug (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). Data for TNF (0.6 and 2.5 ng/ml) are shown in Fig. 1 C for comparison purposes. A less potent induction was observed in Jurkat T cells (Fig. 1 D) where weak activation could be detected at 0.125 μm and a strong signal observed at 2.5 μm daunorubicin. The binding specificity of activated complexes was demonstrated by competition studies in which unlabeled oligonucleotide containing NFκB consensus sequence inhibited the appearance of retarded complexes, whereas a mutant oligonucleotide had no effect at equivalent concentrations (Fig. 2 A). Analysis of specific subunit components in activated NFκB complexes revealed the presence of p50 and to a lesser extent p65/RelA as indicated by enhanced retardation of labeled complexes following gel electrophoresis (Fig. 2 B). Although supershifted complexes were not seen with anti-c-Rel antibodies, a weaker signal when compared with control lanes suggested the presence of this subunit component in the complex (Fig. 2 B, lane 2). HL-60 cells treated with daunorubicin at doses which resulted in NFκB activation were examined for degradation of the inhibitor protein, IκBα, a critical event in the activation of this transcription factor (12Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4591) Google Scholar). A marked degradation of this inhibitor protein was observed which was dose-responsive (Fig. 3). In support of the proposed model for IκBα degradation, a doublet was observed prior to degradation (open arrow), most probably corresponding to the phosphorylated form of this protein, which is a signal for its degradation (15Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1314) Google Scholar). At the highest concentrations of daunorubicin employed (2.5 μm), IκBα degradation was complete (Fig. 3, lane 6). Following transfection of Jurkat T cells with a CAT reporter gene construct containing five NFκB sites upstream of a chloramphenicol acetyltransferase (CAT) reporter gene, the effects of daunorubicin on κB-dependent gene expression were investigated. Daunorubicin induced expression of CAT activity in a dose dependent fashion (Fig. 4 A). At 0.25 μmdaunorubicin, a concentration previously shown to activate NFκB, CAT activity was increased 4-fold over control values (unstimulated cells), indicating that induced complexes were transcriptionally active. In addition, daunorubicin and TNF were found to synergize in this response (Fig. 4 B). Combining a concentration of TNF which was marginally inducing (1.2-fold over control) with a concentration of daunorubicin inducing a 6-fold increase in CAT activity, resulted in a 14-fold induction. This synergy suggests that TNF and daunorubicin activated NFκB by different pathways, as was previously suggested from the different time courses of activation observed for these two agonists (Fig. 1, A and B). We next investigated the mechanism by which daunorubicin activates NFκB. In cells pretreated with the metal chelators desferal and PDTC (which also has anti-oxidant properties), activation was inhibited (Fig. 5 A), as indicated by a diminished signal corresponding to activated complexes. Desferal (1 mm) inhibited the response by 38% with no further inhibition being observed at higher doses (lanes 3–5). PDTC was the more potent inhibitor of the two at equivalent concentrations employed, inhibiting the response by 63% at 1 mm(lane 8). PDTC and desferal were also found to inhibit the daunorubicin-mediated increase in κB-dependent CAT expression (Fig. 5 B). We next determined whether the mechanism of NFκB activation involved redox cycling of daunorubicin. For this purpose, we used a closely related anthraquinone, mitoxantrone, which does not undergo redox cycling (27Fisher G.R. Patterson L.H. Cancer Chemother. Pharmacol. 1992; 30: 451-458Crossref PubMed Scopus (32) Google Scholar, 28Vile G.F. Winterbourne C.C. Cancer Chemother. Pharmacol. 1989; 24: 105-108Crossref PubMed Scopus (29) Google Scholar). Mitoxantrone was found to be as potent an activator of NFκB in HL-60 as daunorubicin (Fig.6 A), with an effect being evident from 0.05 μm. In addition, PDTC was found to inhibit this activation, with 1 mm completely blocking the response induced by 1.0 and 0.2 μm mitoxantrone (Fig.6 B). These results suggested that activation of NFκB by daunorubicin involved the generation of ROS via endogenous cellular processes rather than through redox cycling of the drug per se. Finally, we tested the effect of a specific ceramide synthase inhibitor, fumonisin B1, for its ability to block daunorubicin-induced NFκB activation at a range of concentrations previously reported to block apoptosis induced by this drug (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). Fumonisin B1 failed to inhibit NFκB activation at all concentrations tested (Fig. 7 A) and, furthermore, no inhibition of daunorubicin-mediated CAT activation was observed (data not shown). However, treatment of HL-60 cells with daunorubicin (0.5 and 10 μm) for 4 h increased ceramide levels, as shown in Fig. 7 B, with 10 μm causing a 2.7-fold increase over controls. This effect was inhibited by fumonisin B1 (300 μm), with ceramide levels being reduced to that in unstimulated cells. Furthermore, treatment of HL-60 cells with daunorubicin (10 μm) for 4 h was found to increase microsomal ceramide synthase activity more than 2-fold over controls. 300 μm fumonisin B1 inhibited this activity, reducing it to below control levels, which was consistent with its ability to act as a competitive inhibitor toward dihydrosphingosine (Fig. 7 B). This confirmed a previous report where induction of ceramide synthase was found to mediate increases in ceramide levels in cells in response to daunorubicin (7Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). However, our data indicated that this process was not involved in NFκB activation here. We therefore concluded that increases in ROS, generated by endogenous cellular processes, but not ceramide synthase induction, were mediating the effect of daunorubicin on NFκB activation and κB-driven gene expression. In this study, we present the report that daunorubici