Daunorubicin Activates NFkB and Induces kB-dependent Gene Expression in HL-60 Promyelocytic and Jurkat T Lymphoma Cells*

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, NFkB, 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 NFkB. 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 NFkB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IkB degradation and increased the expression of an NFkB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFkB-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 NFkB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFkB 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 NFkB 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 NFkB 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 (1). Although its mechanism of anti-tumor action is uncertain, DNA is believed to be a primary target (2). 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 (2). Irrespective of the initial insult, anthracyclines, along with a variety of agonists, ultimately activate the event of programmed cell death or apoptosis in cells (3). 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 (4). 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 (5), and the activation of effector molecules such as interleukin-1 converting enzyme-like proteases (6). Recent studies suggest that the neutral lipid ceramide may also play a role in mediating druginduced apoptosis (7, 8). 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-a, interleukin-1b, g-interferon, nerve growth factor, Fas ligand, and 1,25-dihydroxyvitamin D3 (9). A general role in growth arrest and suppression is suggested by its ability to induce cell differentiation, cellcycle arrest, apoptosis, or cell senescence (9), although a mitogenic role has been demonstrated in certain cell types (10). In a recent study, daunorubicin was shown to increase ceramide levels in cells following induction of the enzyme ceramide synthase (7). 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 TNFinduced apoptosis, but also with the activation of NFkB (11). 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 (12). The * This work was supported by funding from the CAM Research Department, Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kindgom, the National Pharmaceutical Biotechnology Centre, Trinity College, Dublin, and a grant from the Cancer Research Advancement Board, Ireland (to M. P. B. and L. O. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed. Tel.: 353-1-6082449; Fax: 353-1-677-2400; E-mail: [email protected]. 1 The abbreviations used are: TNF, tumor necrosis factor; PDTC, pyrrolidine dithiocarbamate; ROS, reactive oxygen species; NFkB, nuclear factor kB; CAT, chloramphenicol acetyltransferase. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 20, Issue of May 16, pp. 12952–12960, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.