Publications

The signal transduction pathways activated by the proinflammatory cytokine interleukin-1 (IL-1) have been the focus of much attention because of the important role that IL-1 plays in inflammatory diseases. A number of proteins have been described that participate in the post-receptor activation of the transcription factor nuclear factor kappaB (NF-kappaB), and stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK). It has also emerged that the type I IL-1 receptor (IL-1RI) is a member of an expanding receptor superfamily. These related receptors all have sequence similarity in their cytosolic regions. The family includes the Drosophila melanogaster protein Toll, the IL-18 receptor (IL-18R), and 10 Toll-like receptors (TLRs), TLR-1 to TLR-10, which bind to microbial products, activating host defense responses. Because of the similarity of IL-1RI to Toll, the conserved sequence in the cytosolic region of these proteins has been termed the Toll-IL-1 receptor (TIR) domain. The same proteins activated during signaling by IL-1RI also participate in signaling by other receptors with TIR domains. The receptor superfamily is evolutionarily conserved; members also occur in plants and insects, where they also function in host defense. The signaling proteins that are activated are also conserved across species. Differences are, however, starting to emerge in signaling pathways activated by different receptors. This receptor superfamily, therefore, represents an ancient signaling system that is a critical determinant of the innate immune and inflammatory responses.

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-κB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-κB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-κB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-κB and stimulation of IκBα degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive toN-acetyl-l-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-κB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-κB activation and IκBα degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6,6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-κB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-κB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-κB activation by tumor necrosis factor but not interleukin-1.Taken together, the results argue against a role for H2O2 in NF-κB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-κB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation. It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-κB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-κB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-κB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-κB and stimulation of IκBα degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive toN-acetyl-l-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-κB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-κB activation and IκBα degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6,6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-κB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-κB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-κB activation by tumor necrosis factor but not interleukin-1. Taken together, the results argue against a role for H2O2 in NF-κB activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-κB through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation. The inducible, higher eukaryotic transcription factor NF-κB has an important role in the regulation of a number of genes involved in immune and inflammatory responses. It is activated in many cell types by a wide range of stimuli including the proinflammatory cytokines interleukin-1 (IL-1) 1The abbreviations used are: IL-1, interleukin-1; BHA, butylated hydroxyanisole; t-BHP, tert-butyl hydroperoxide; DDTC, diethyldithiocarbamate; DFO, desferrioxamine; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; MDA, malondialdehyde; NAC, N-acetyl-l-cysteine; PDTC, pyrrolidine dithiocarbamate; PGA, pyroglutamic acid; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; TNF, tumor necrosis factor; FBS, fetal bovine serum; PBS, phosphate-buffered saline. and tumor necrosis factor (TNF) and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (reviewed in Ref. 1Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4599) Google Scholar). In endothelial cells (ECs), activation of NF-κB is central to the regulation of many genes by IL-1 and TNF such as the cell adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (2Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1572) Google Scholar) and tissue factor (3Bierhaus A. Zhang Y. Deng Y. Mackman N. Quehenberger P. Haase M. Luther T. Muller M. Bohrer H. Greten J. Martin E. Baeuerle P.A. Waldherr R. Kisiel W. Ziegler R. Stern D.M. Nawroth P.P. J. Biol. Chem. 1995; 270: 26419-26432Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Recently, NF-κB has been identified in an activated form in the ECs of atherosclerotic plaques (4Brand K. Page S. Rogler G. Bartsch A. Brandl R. Knuechel R. Page M. Kaltschmidt C. Baeuerle P.A. Neumeier D. J. Clin. Invest. 1996; 97: 1715-1722Crossref PubMed Scopus (714) Google Scholar), and it has been suggested that NF-κB may play a central role in the initiation of atherosclerosis (5Collins T. Lab. Invest. 1993; 68: 499-508PubMed Google Scholar). NF-κB exists in a latent form in the cytoplasm of unstimulated cells comprising a transcriptionally active dimer bound to an inhibitor protein, IκB. The currently known subunit members of the NF-κB family in mammals are p50, RelA (p65), c-Rel, p52, and RelB, while multiple forms of IκB also exist, namely IκBα, β, γ, and Bcl-3 (reviewed in Ref. 6Liou H.C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (517) Google Scholar). The predominant form of NF-κB activated in cells is a p50/RelA heterodimer, which is associated with IκBα in resting cells. Upon stimulation with agents such as IL-1 and TNF, IκBα is rapidly phosphorylated on two serine residues (Ser32 and Ser36), which targets the inhibitor protein for ubiquitination and subsequent degradation by the 26 S proteasome (reviewed in Ref. 7Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). This allows NF-κB to translocate to the nucleus and activate target genes by binding with high affinity to κB elements in their promoters. The phosphorylation and degradation of IκBα are tightly coupled events (7Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). Thus, it is likely that agents that activate NF-κB do so through the activation of a specific IκBα kinase or, alternatively, by inactivating a particular phosphatase. A high molecular mass kinase complex that phosphorylates IκBα on Ser32 and Ser36 has been identified (8Chen Z.J. Parent L. Maniatis T. Cell. 1996; 84: 853-862Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). The upstream events that lead to phosphorylation of IκBα are unclear. A model has been proposed whereby diverse agents all activate NF-κB by causing oxidative stress (an increase in intracellular reactive oxygen intermediates (ROS)) (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar). In particular, H2O2 has been implicated as a common second messenger in the various pathways leading to NF-κB activation (10Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar). This hypothesis is based on several of lines of evidence. First, in some cell types H2O2 has been shown to be released in response to agents that also activate NF-κB (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 10Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 11Los M. Schenk H. Hexel K. Baeuerle P.A. Droge W. Schulze-Osthoff K. EMBO J. 1995; 14: 3731-3740Crossref PubMed Scopus (297) Google Scholar). Second, direct addition of H2O2 to culture medium has been shown to activate NF-κB in some cell lines (12Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3420) Google Scholar, 13Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1269) Google Scholar). Third, overexpression of the H2O2-metabolizing enzyme catalase in a mouse epidermal cell line was shown to attenuate the activation of NF-κB by TNF and okadaic acid, while overexpression of the H2O2-producing enzyme superoxide dismutase potentiated the activation, suggesting a role for H2O2 in these pathways to NF-κB (10Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (430) Google Scholar). Other work has suggested that some of these observations are cell-specific. H2O2 had no stimulatory effect on NF-κB in a number of other cell types (14Israel N. Gougerot-Pocidalo M.A. Aillet F. Virelizier J.C. J. Immunol. 1992; 149: 3386-3393PubMed Google Scholar, 15Bradley J.R. Johnson D.R. Pober J.S. Am. J. Pathol. 1993; 142: 1598-1609PubMed Google Scholar, 16Brennan P. O'Neill L.A.J. Biochim. Biophys. Acta. 1995; 1260: 167-175Crossref PubMed Scopus (209) Google Scholar, 17Moynagh P.N. Williams D.C. O'Neill L.A.J. J. Immunol. 1994; 153: 2681-2690PubMed Google Scholar), while Suzuki et al.(18Suzuki Y.J. Mizuno M. Packer L. Biochem. Biophys. Res. Commun. 1995; 210: 537-541Crossref PubMed Scopus (42) Google Scholar) showed that in COS-1 cells, overexpression of catalase did not block activation of NF-κB by either TNF or PMA. It has also been suggested that oxidative stress facilitates but does not mediate NF-κB activation (14Israel N. Gougerot-Pocidalo M.A. Aillet F. Virelizier J.C. J. Immunol. 1992; 149: 3386-3393PubMed Google Scholar). Another line of evidence implicating oxidative stress and H2O2 as central to NF-κB activation has been the effect of antioxidants in inhibiting NF-κB activation in response to diverse stimuli. Two compounds in particular have been extensively used, the glutathione precursor and radical scavengerN-acetyl-l-cysteine (NAC) and the putative antioxidant pyrrolidine dithiocarbamate (PDTC). However, the effect of NAC on NF-κB is also somewhat cell-specific, in that although it has proved inhibitory in some cells (12Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3420) Google Scholar, 13Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Crossref PubMed Scopus (1269) Google Scholar), we and others have reported NAC-insensitive pathways to NF-κB (16Brennan P. O'Neill L.A.J. Biochim. Biophys. Acta. 1995; 1260: 167-175Crossref PubMed Scopus (209) Google Scholar, 17Moynagh P.N. Williams D.C. O'Neill L.A.J. J. Immunol. 1994; 153: 2681-2690PubMed Google Scholar, 19Suzuki Y.I. Mizuno M. Packer L. J. Immunol. 1994; 153: 5008-5015PubMed Google Scholar). PDTC seems to be a better general inhibitor of NF-κB and, in fact, has been proposed as a specific universal inhibitor of NF-κB that acts independently of the activating agent and cell type used (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar). However, in addition to its radical scavenging and metal-chelating properties (9Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1448) Google Scholar, 20Sunderman F.W. Ann. Clin. Lab. Sci. 1991; 21: 70-81PubMed Google Scholar), PDTC can also exert a pro-oxidant effect in some cells by increasing oxidized glutathione levels (21Nobel C.S.I. Kimland M. Lind B. Orrenius S. Slater A.F.G. J. Biol. Chem. 1995; 270: 26202-26208Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 22Brennan P. O'Neill L.A.J. Biochem. J. 1996; 320: 975-981Crossref PubMed Scopus (123) Google Scholar), which also leads to an inhibition of NF-κB (22Brennan P. O'Neill L.A.J. Biochem. J. 1996; 320: 975-981Crossref PubMed Scopus (123) Google Scholar, 23Mihm S. Galter D. Droge W. FASEB J. 1995; 9: 246-252Crossref PubMed Scopus (153) Google Scholar). Information on the role of oxidative stress and H2O2 in cytokine stimulation of NF-κB in ECs remains limited, mainly coming from studies using PDTC or NAC to perturb particular genes downstream of NF-κB activation (24Weber C. Erl W. Pietsch A. Strobel M. Ziegler-Heitbrock H.W.L. Weber P.C. Arterioscler. Thromb. 1994; 14: 1665-1673Crossref PubMed Google Scholar, 25Orthner C. Rodgers G.M. Fitzgerald L.A. Blood. 1995; 86: 436-443Crossref PubMed Google Scholar, 26Marui N. Offermann M.K. Swerlick R. Kunsch C. Rosen C.A. Ahmad M. Alexander R.W. Medford R.M. J. Clin. Invest. 1993; 92: 1866-1874Crossref PubMed Scopus (978) Google Scholar). Given the importance of NF-κB in ECs together with the often cell-specific nature of the effect of H2O2 and antioxidants on NF-κB activation, we decided to investigate the role of H2O2 and oxidative stress in NF-κB activation in ECs using both primary and transformed ECs. Our results show that although H2O2 activates NF-κB in transformed ECs, it is unlikely to have a role in the cytokine-mediated pathways to NF-κB in transformed or primary ECs. In transformed cells, TNF but not IL-1 was sensitive to PDTC, while in primary cells neither stimulus was inhibited. Further, we show that the ability of PDTC to inhibit NF-κB activation by TNF in transformed ECs involves inhibition of iron-catalyzed lipid peroxidation that is not important for activation of NF-κB by IL-1. The immortalized human endothelial cell line ECV304 (27Takahashi K. Sawasaki Y. Hata J.-I. Mukai K. Goto T In Vitro Cell. Dev. Biol. 1990; 25: 265-274Crossref Scopus (418) Google Scholar) and human Jurkat T cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, United Kingdom). Pooled human umbilical vein endothelial cells (HUVECs) were obtained at first passage from Clonetics Corporation (San Diego, CA). RPMI 1640 medium, heat-inactivated fetal bovine serum (FBS), trypsin-EDTA, and penicillin-streptomycin-glutamine were from Life Technologies, Inc. (Paisley, Scotland). Human recombinant IL-1α was a gift from NCI, National Institutes of Health (Frederick, MD), while human recombinant TNFα was a gift from Dr. Steve Foster (Zeneca Pharmaceuticals, Macclesfield, UK). The 22-base pair oligonucleotide, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, containing the NF-κB consensus sequence (underlined), T4 polynucleotide kinase, and the Cytotox 96TM nonradioactive cytotoxicity assay were from Promega Corp. (Madison, WI). The 22-base pair oligonucleotide, 5′-AGT TGA GGC GAC TTT CCC AGG C-3′, containing the mutated NF-κB consensus sequence (underlined), the rabbit polyclonal antibody to human IκBα, and the antisera to the NF-κB subunits p50 and c-Rel were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antiserum to the NF-κB subunit RelA (p65) was a gift from Dr. Jean Imbert (INSERM, Marseille, France). [γ-32P]ATP (3000 Ci/mmol) and enhanced chemiluminescence (ECL) reagent were from Amersham International (Aylesbury, UK). Poly(dI·dC) was from Pharmacia Biosystems (Milton Keynes, UK). All other reagents, including heparin (sodium salt), human recombinant acidic fibroblast growth factor, medium 199 (HEPES modification), PDTC (ammonium salt), diethyldithiocarbamate (DDTC), pyrrolidine, pyroglutamic acid, NAC, desferrioxamine, allopurinol, the spin traps 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), FeCl3, H2O2, PMA, butylated hydroxyanisole (BHA),tert-butylhydroperoxide (t-BHP), thiobarbituric acid, anti-mouse IgG peroxidase conjugate, scopoletin, horseradish peroxidase (type II), and catalase were from Sigma (Poole, UK). ECV304 cells were grown in medium 199 (HEPES modification) containing 10% (v/v) FBS, and passaged when confluent using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA. HUVECs were grown in medium 199 containing 20% FBS, 10 ng/ml acidic fibroblast growth factor, and 90 μg/ml heparin. The medium was changed every 48 h, and cells were passaged when 80–90% confluent using trypsin-EDTA. Jurkat T cells were grown in RPMI 1640 medium containing 10% FBS. All media were supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine, and cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Cells were pretreated with the test compounds or left untreated before the addition of IL-1, TNF, PMA, or H2O2 as described in the figure legends. All experiments were carried out in complete medium at 37 °C. PDTC, DDTC, and pyrrolidine were dissolved in PBS; pyroglutamic acid (PGA), desferrioxamine (DFO), and FeCl3were dissolved in H2O; NAC was dissolved in 25 mm Tris-HCl, pH 7.5; BHA was dissolved in ethanol; and PMA, allopurinol, TEMPO, and DMPO were dissolved in Me2SO. NAC and PGA were adjusted to pH 7.4 with 1 n NaOH. None of the vehicles had any effect on NF-κB alone at the concentrations used. No metal spatulas were used. The effect of the compounds on cell viability was assessed using the Cytotox 96TM nonradioactive cytotoxicity assay, as described by the manufacturers. This system uses lactate dehydrogenase release as an index of cell toxicity. At the concentrations used here, NAC interfered with this assay, and thus the magnitude of release of lactate dehydrogenase activity from NAC-treated cells was determined directly by monitoring spectrophotometrically the decrease in absorbance at 340 nm in the presence of 75 mmTris-HCl, pH 7.2, containing 150 mm KCl, 0.2 mmNADH, and 4.8 mm sodium pyruvate. Measurement of LDH release from intact cells and examination of monolayer morphology revealed that none of the compounds used were toxic to the cells at the exposure times and concentrations used here. Nuclear extracts were prepared using a modified version of the method of Osborn et al. (28Osborn L. Kunkel S. Nable G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2336-2340Crossref PubMed Scopus (1370) Google Scholar). Confluent ECV304 cells or HUVECs in six-well plates (3-ml volume) were treated as described in the figure legends. Stimulation was terminated by removal of medium followed by washing twice with 3 ml of ice-cold PBS (0.145m NaCl, 0.027 m phosphate buffer, pH 7.6). Washed cells were then scraped into 1 ml of hypotonic buffer (10 mm Hepes buffer, pH 7.9, containing 1.5 mmMgCl2, 10 mm KCl, 0.5 mmdithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride). Stimulation of Jurkat T cells (5 × 106 in 1 ml of medium) was terminated by the removal of cells into 5 ml of ice-cold PBS, followed by centrifugation (170 g, 10 min). Cell pellets were resuspended in 1 ml of hypotonic buffer. Nuclear extracts were subsequently prepared as described previously (17Moynagh P.N. Williams D.C. O'Neill L.A.J. J. Immunol. 1994; 153: 2681-2690PubMed Google Scholar). Protein concentrations were determined using the method of Bradford (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). Extracts were then stored at −20 °C and assayed for NF-κB activity the next day. Nuclear extracts (2 μg of protein) were incubated with 10,000 cpm of a 22-base pair oligonucleotide containing the NF-κB consensus sequence that had previously been labeled with [γ-32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. Incubations were performed for 30 min at room temperature, in the presence of 2 μg of poly(dI·dC) as nonspecific competitor and 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 4% glycerol, and 100 μg/ml nuclease-free bovine serum albumin. For competition studies, unlabeled wild type or mutant NF-κB oligonucleotides were added to the binding reaction 30 min before the addition of the radiolabeled probe. In experiments involving antisera to NF-κB subunits, 0.5 μl of a specific antiserum to p50, RelA, or c-Rel was incubated with nuclear extracts for 20 min on ice prior to the binding reaction. All incubation mixtures were subjected to electrophoresis on native 5% (w/v) polyacrylamide gels, which were subsequently dried and autoradiographed. Confluent ECV304 cells in six-well plates (3-ml volume) were treated as described in the figure legends. Treatment was terminated by washing monolayers twice with ice-cold PBS. Cells were then scraped into 1 ml of ice-cold radioimmune precipitation buffer (1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS in PBS) containing 10 μg/ml phenylmethylsulfonyl fluoride, 7 μg/ml aprotinin, and 1 mm Na3VO4. Following further disruption of cells by passage through a 21-gauge needle (5 strokes), an additional 0.1 mg/ml phenylmethylsulfonyl fluoride was added to samples, which were then incubated on ice for 45 min. Samples were then centrifuged at 14,000 × g for 20 min at 4 °C, and the supernatant was removed as cell lysate. Supernatants were assayed for protein (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar). Equal amounts of protein (2–4 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose, and IκBα immunoblot analysis was performed as described previously (30Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). H2O2released from ECV304 cells was measured by the horseradish peroxidase-mediated oxidation of the fluorescent probe scopoletin to its nonfluorescent oxidized form (31Harpe J.D.L. Nathan C.F. J. Immunol. Methods. 1985; 78: 323-336Crossref PubMed Scopus (137) Google Scholar). Since hydrogen peroxide freely diffuses across the cell membrane, measuring extracellular release is an indication of intracellular levels. Confluent monolayers of ECV304 cells in six-well plates (3-ml volume) were washed twice with Hanks' balanced salt solution to remove phenol red and serum. Monolayers were incubated with 3 ml of assay solution containing stimulant (IL-1 (10 ng/ml), TNF (10 ng/ml), or PMA (100 ng/ml)), sodium azide (1 mm), scopoletin (1 μm), and horseradish peroxidase (0.2 units/ml) in Hanks' balanced salt solution, for 30 min at 37 °C. The supernatant was then transferred to a test tube and cooled to room temperature, and the fluorescence was measured on a Perkin-Elmer LS 50B fluorimeter using an excitation wavelength of 390 nm and an emission wavelength of 460 nm. Controls included cell-free plates with full assay mix and cells incubated in the absence of peroxidase. The specific decrease in fluorescence due to H2O2 was assessed by measuring scopoletin oxidation for each sample in the presence and absence of catalase (50 units/ml). H2O2 concentration was then determined against a standard curve, using concentrations of 0.05–0.5 μm H2O2, as determined spectrophotometrically (ε240 = 43.6m−1 cm−1). Protein concentrations of samples were determined (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar) following alkali digestion of monolayers. H2O2 release was then expressed as pmol/min/mg of protein. Lipid peroxidation was assessed by the TBARS assay, which detects mainly malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters. TBARS were measured by a modification of the method of Ohkawa et al. (32Ohkawa H. Ohishi N. Yagi K. Anal. Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23298) Google Scholar). Confluent monolayers of ECV304 cells in 100-mm dishes were treated as described in the figure legends and washed in PBS before undergoing three cycles of freeze-thawing in 200 μl of water. A 20-μl aliquot was subsequently removed for Bradford protein determination (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216428) Google Scholar), and 800 μl of assay mix (0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, 9.4% (v/v) acetic acid, pH 3.5) was added to the remaining sample. Samples were incubated for 60 min at 95 °C, cooled to room temperature, and centrifuged at 14,000 ×g for 10 min, and the absorbance of the supernatants was read at 532 nm against a standard curve prepared using the MDA standard (10 mm 1,1,3,3-tetramethoxypropane in 20 mmTris-HCl, pH 7.4). Results were calculated as nmol of MDA equivalents/mg of protein and expressed as a percentage of matched control values. Significance was evaluated by Student's t test. Fig.1 A shows that nuclear extracts from untreated ECV304 cells contained trace amounts of NF-κB, the levels of which varied slightly between experiments (lanes 1, 9, 17, and 25). Following treatment with 10 ng/ml IL-1 (lanes 2–8) or 10 ng/ml TNF (lanes 10–16), NF-κB was activated, as evidenced by the increased retardation of the DNA probe containing the κB motif. This activation was evident from 5 min (lanes 2 and 10) and peaked at 30 min (lanes 4 and 12). NF-κB activity was strong for at least 4 h (lanes 6 and 14) and still detectable at 24 h (lanes 8 and 16). Treatment of cells with 100 ng/ml PMA (lanes 18–24) gave a somewhat different time course of activation in that active NF-κB only became detectable at 30 min (lane 20) and was greatly reduced by 24 h (lane 24). Prolonged treatment of ECV304 cells with H2O2 led to activation of NF-κB. When cells were treated with 0.2 mm H2O2 for 4 h, a strong activation was observed (compare lanes 28 and 25). In contrast to the rapid response seen for IL-1 and TNF, the H2O2-stimulated activation was not detectable after 1 h (compare lanes 26 and 25) and did not peak until 4 h (lane 29). H2O2-mediated activation was also more transient than that seen for IL-1 and TNF, with activity greatly decreased by 8 h (compare lanes 29 and 28) and identical to control levels at 24 h (compare lanes 30 and 25). A concentration of 0.2 mmH2O2 was optimal in this effect. Fig.1 B confirms that the protein-DNA complexes activated by IL-1, TNF, PMA, and H2O2 were all specific for NF-κB, since 18 or 180 fmol of unlabeled NF-κB wild type consensus sequence effectively competed with each binding activity, while the same concentrations of a mutant NF-κB oligonucleotide containing a single base pair change in the consensus sequence failed to compete with binding. Since this is the first study reporting NF-κB activation in ECV304 cells, we also characterized the NF-κB subunits present in the complexes activated by the four stimuli. Fig. 1 Cdemonstrates that IL-1, TNF, PMA, and H2O2activated similar NF-κB complexes. Using specific antisera to p50, RelA, and c-Rel, the same pattern of supershifting was seen for IL-1-, TNF-, PMA-, and H2O2-activated NF-κB (lanes 1–4, 5–8, 9–12, and 13–16, respectively). This revealed the presence of two main NF-κB complexes. Antiserum to p50 affected both the lower and upper complex (lanes 2, 6, 10, and 14), while RelA antiserum only reacted with the upper complex (lanes 3, 7, 11, and 15). There was no detectable reaction with c-Rel antiserum (lanes 4, 8, 12, and 16). Hence, it was likely that IL-1, TNF, PMA, and H2O2 were activating two NF-κB complexes, tentatively identified as p50/p50 homodimers and p50/RelA heterodimers. Fig. 1 D shows that IL-1, TNF, and PMA also activated NF-κB in HUVECs as has been well characterized by others (2Collins T. Read M.A. Neish A.S. Whitley M.Z. Thanos D. Maniatis T. FASEB J. 1995; 9: 899-909Crossref PubMed Scopus (1572) Google Scholar). IL-1 (10 ng/ml) gave a similar time course of activation of NF-κB to that seen in ECV304 cells, with activity detectable after 5 min (compare lanes 2 and 1), maximal at 1 h (lane 5) and still apparent at 24 h (lane 8). Activation of NF-κB by TNF (10 ng/ml) and PMA (100 ng/ml) at a single time point (1 h) is also shown (compare lanes 10 and 11, respectively, with lane 9). In contrast to ECV304 cells, H2O2 failed to activate NF-κB in HUVECs. No activation was apparent upon a 2- or 4-h incubation of HUVECs with either 0.2 or 0.4 mm H2O2 (comparelanes 12–15 with lane 9). Higher doses of H2O2 also failed to activate NF-κB (not shown). These results highlight an important dif

No abstract is provided for this article.

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. 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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Wells N.J. Hickson I.D. Biochim. Biophys. Acta. 1998; 1400: 121-139Crossref PubMed Scopus (185) Google Scholar). There are two isoforms of topoisomerase II enzymes in mammalian cells, an α and a β form, which differ in their molecular weight and are the products of two separate genes. Their amino-terminal sequence is highly conserved, but they only maintain 35% homology in their carboxyl termini, which has been proposed to account for differences in their cellular distribution (31Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar, 33Drake F.H. Hofmann G.A. Bartus H.F. Mattern M.R. Crooke S.T. Mirabelli C.K. Biochemistry. 1989; 28: 8154-8160Crossref PubMed Scopus (423) Google Scholar). The α form is localized to the nuclear matrix, whereas the β form is non-matrix-associated and localizes preferentially in the nucleoli. 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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In effect, anthracyclines and anthracenediones form ternary complexes with DNA and the enzyme and stimulate DNA cleavage in a sequence-specific manner (37Capranico G. Binaschi M. Biochim. Biophys. Acta. 1998; 1400: 185-195Crossref PubMed Scopus (113) Google Scholar). Correlations between the level of target enzyme, drug induced strand breaks, and sensitivity toward drug-induced cell killing have been shown (38Davies S.M. Robson C.N. Davies S.L. Hickson I.D. J. Biol. Chem. 1988; 263: 17724-17729Abstract Full Text PDF PubMed Google Scholar), although there remains some controversy regarding the preferential targeting of different isoforms by different categories of topoisomerase II-targeted drugs (39Brown G.A. McPherson J.P. Gu L. Hedley D.W. Toso R. Deuchars K.L. 40 Freedman M.H. Goldenberg G.J. Cancer Res. 1995; 55: 78-82PubMed Google Scholar, 40Fry A.M. Chresta C.M. Davies S.M. Walker M.C. Harris A.L. Hartley J.A. Masters J.R.W. Hickson I.D. 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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

No abstract is provided for this article.

No abstract is provided for this article.

The NLRP3 inflammasome regulates the maturation of the pro-inflammatory cytokines IL-1 β and IL-18, which are important mediators in inflammatory disorders such as atherosclerosis, Type 2 diabetes and Alzheimer’s disease. Given its role as an inflammatory gatekeeper, NLRP3 expression is tightly controlled. It is transcriptionally regulated by NF- κ B as well as post-translationally by S-nitrosylation and ubiquitination. We decided to test whether NLRP3 is also regulated at the post-transcriptional level. We found that the NLRP3 3’ untranslated region (UTR) contains an evolutionarily conserved target site for miRNA-223 and we confirmed miR-223 as a direct negative regulator of NLRP3 using a luciferase reporter assay. Furthermore, overexpressing the miRNA reduces NLRP3 levels and IL-1 β production following activation with NLRP3 agonists. Since miR-223 is differentially expressed during myeloid differentiation, it could alter the threshold for inflammasome activation in different cell types. We have now generated a transgenic mouse deficient in the miR-223 target site in the NLRP3 3’UTR, which will allow us for the first time to study the importance of miRNA regulation of NLRP3 in vivo and in the context of inflammatory disease models. Interestingly, the human NLRP3 3’UTR can be alternatively polyadenlyated, giving rise to a truncated form of the transcript that lacks regulatory elements. While mRNA stability was not affected, a luciferase reporter assay showed that LPS stimulation could enhance expression of both 3’UTR isoforms. Using an RNA pulldown approach we identified potential RNA binding proteins that could mediate this effect. Ultimately, understanding the post-transcriptional control of NLRP3 expression will help to explain cell type-specific expression of NLRP3 and could potentially provide new opportunities to interfere with inflammasome activation in diseases such as Type 2 diabetes.

The field of immunometabolism has demonstrated that metabolites can lead double lives as immunomodulators. Itaconate is perhaps the best example of such a moonlighting molecule, and has been shown to have multiple anti-inflammatory effects in macrophages. Itaconate is significantly upregulated under inflammatory conditions, and can promote an anti-inflammatory phenotype by reducing oxidative stress and blocking transcriptional responses to lipopolysaccharide (LPS) in murine macrophages. Antibacterial and protumor effects have also been described for itaconate and, most recently, reports have surfaced of its possible modulatory roles during Zika virus infection in murine neurons. We posit here that itaconate is a crucial determinant of innate immune responses, and may potentially be harnessed therapeutically to treat inflammatory diseases.

No abstract is provided for this article.

Conference Abstract| November 01 1996 DAUNORUBICIN ACTIVATES NFkB AND POTENTIATES TNF-INDUCED GENE EXPRESSION Marion P. Boland; Marion P. Boland 1Inflammation Group, Department of Biochemistry, Trinity College, Dublin 2 Search for other works by this author on: This Site PubMed Google Scholar Luke A.J. O'Neill Luke A.J. O'Neill 1Inflammation Group, Department of Biochemistry, Trinity College, Dublin 2 Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (4): 601S. https://doi.org/10.1042/bst024601sb Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation Marion P. Boland, Luke A.J. O'Neill; DAUNORUBICIN ACTIVATES NFkB AND POTENTIATES TNF-INDUCED GENE EXPRESSION. Biochem Soc Trans 1 November 1996; 24 (4): 601S. doi: https://doi.org/10.1042/bst024601sb Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

A molecular basis for asbestos-induced inflammation may lie in a particular component of the inflammasome protein complex.

The discovery of the Toll-like receptors (TLRs) has revolutionised the field of innate immunity. One unresolved question regarding LPS signalling is whether there is a role for tyrosine kinases downstream of the LPS receptor. Studies in mice deficient in Bruton’s tyrosine kinase have previously shown that they are defective in their responses to LPS. Further investigation into the role of Btk in LPS signalling has directly implicated Btk downstream of TLR4, both with respect to p38 MAPK activation and activation of the transcription factor NFκB. In fact Btk is activated by LPS and has been shown to directly bind TLR4 and the key proximal signalling proteins involved in LPS-induced NFκB activation, MyD88, Mal and IRAK-1. These recent findings point to a direct role for Btk in LPS signal transduction and raise interesting questions regarding the mode of activation of Btk following LPS stimulation and the precise nature of the pathways activated downstream of Btk. A better understanding of how Btk functions in LPS signalling will have important implications for inflammatory and autoimmune disorders and therapies thereof.