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B and Induces
B-dependent Gene Expression in HL-60 Promyelocytic and
Jurkat T Lymphoma Cells
(Received for publication, October 1, 1996, and in revised form, February 17, 1997)
From the Department of Biochemistry, Trinity College, Dublin,
Ireland and § CAM Research Department, ZENECA
Pharmaceuticals, Mereside, Alderley Park, Macclesfield,
Cheshire SK10 4TG, United Kingdom
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, NF
B, 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 NFB. 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 NFB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IB
degradation and increased the expression of an NFB-linked reporter
gene. In addition, the drug was found to strongly potentiate the
ability of tumor necrosis factor to induce an NFB-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 NFB in a pyrrolidine dithiocarbamate-sensitive manner. A
specific inhibitor of ceramide synthase, fumonisin B1, had no effect on
daunorubicin induced NFB 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 NFB 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 NFB may be involved in apoptosis, this
effect may be an important aspect of the cellular responses to this
agent.
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