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J. Biol. Chem., Vol. 275, Issue 29, 22461-22469, July 21, 2000
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From the Departments of We examined the mechanism of
H2O2-induced cytotoxicity and its
relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to
H2O2, and at concentrations up to about 20-25
µM, the killing was mediated by apoptosis. There was
limited evidence of lipid peroxidation, suggesting that the effects of
H2O2 do not involve hydroxyl radical. When
HL-60 cells were exposed to H2O2 in the
presence of the spin trap
Myeloperoxidase Is Involved in
H2O2-induced Apoptosis of HL-60 Human Leukemia
Cells*
,¶
Medicine and
§ Radiology (Free Radical and Radiation Biology Graduate
Program), The University of Iowa College of Medicine and The University
of Iowa Cancer Center, Iowa City, Iowa 52242
-(4-pyridyl-1-oxide)-N-tert-butylnitrone
(POBN), we detected a 12-line electron paramagnetic resonance spectrum
assigned to the POBN/POBN· N-centered spin adduct previously
described in peroxidase-containing cell-free systems. Generation of
this radical by HL-60 cells had the same H2O2
concentration dependence as initiation of apoptosis. In contrast,
studies with the K562 human erythroleukemia cell line, which is often
used for comparison with the HL-60, and with high passaged HL-60 cells
(spent HL-60) studied under the same conditions failed to generate
POBN·. Cellular levels of antioxidant enzymes superoxide
dismutase, glutathione peroxidase, and catalase did not explain the
differences between these cell lines. Interestingly, the K562 and spent
HL-60 cells, which did not generate the radical, also failed to undergo H2O2-induced apoptosis. Based on this we
reasoned that the difference in H2O2-induced
apoptosis might be due to the enzyme myeloperoxidase. Only the
apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme.
When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid
hydrazide, which are inhibitors of myeloperoxidase, they no longer
underwent H2O2-induced apoptosis. Hypochlorous
acid stimulated apoptosis in both HL-60 and spent HL-60 cells,
indicating that another oxidant generated by myeloperoxidase induces
apoptosis and that it may be the direct mediator of
H2O2-induced apoptosis. Taken together these
observations indicate that H2O2-induced
apoptosis in the HL-60 human leukemia cell is mediated by
myeloperoxidase and is linked to a non-Fenton oxidative event marked by
POBN·.
*
This investigation was supported by Grant P01 CA66081
awarded by the National Cancer Institute, Department of Health and
Human Services; The Iowa Leukemia and Cancer Research Fund; The Dr. Richard O. Emmons Memorial Fund; and The Mamie C. Hopkins Fund.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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