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, NFkappaB, 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 NFkappaB. 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 NFkappaB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IkappaB degradation and increased the expression of an NFkappaB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFkappaB-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 NFkappaB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFkappaB 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 NFkappaB 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 NFkappaB may be involved in apoptosis, this effect may be an important aspect of the cellular responses to this agent.