Cells (0.2 105) were suspended in PBS and lysed by repeated freeze-thaw. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34+cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is usually amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In individual studies, etoposide-induced DNA damage andMLLgene rearrangements were demonstrated to be dependent in part on MPO activity in CD34+cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34+cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia. == Introduction == Etoposide [VP-16, 4-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidene–d-gluco-pyranoside)] is usually a DNA topoisomerase II-targeting agent that has been used extensively as an anticancer agent to treat a variety of malignancies in adults and in children (Hande, 1998). However, the use of etoposide has been associated with an increased risk of developing secondary leukemias, especially acute myelogenous leukemia (t-AML), bearing translocations of theMLLgene at human chromosomal band 11q23 (Libura et al., 2005;Felix et al., 2006). Etoposide, which contains a hindered Griseofulvin ring phenol, can be converted to phenoxyl radical forms by the action of peroxidases (Haim et al., 1987;Kagan et al., 1999). Because myeloid progenitor CD34+cells in early stages of maturation contain the enzyme myeloperoxidase (MPO) (Strobl Griseofulvin et al., 1993), we hypothesized that oxidative activation of the etoposide phenolic group by Griseofulvin MPO may lead to MPO-catalyzed oxidative stress, including carcinogenic oxidative modification of DNA (Kagan et al., 2001). Hence, MPO expressed in CD34+cells may make these myeloid progenitors especially sensitive to the leukemogenic action of etoposide. MPO-induced oxidative stress is brought on by this enzyme’s reactive intermediates, which have very high (1.35 V) oxidizing potential (Jantschko et al., 2005;Davies et al., 2008). In the presence of reducing substrates, particularly phenolic compounds such as etoposide, the one-electron oxidation catalyzed by MPO to yield phenoxyl radicals can in turn lead to conversation with a variety of cellular targets including lipids, thiols, ascorbate, proteins, and DNA (Zhang et al., 2002;Borisenko et al., 2004). Depending on the reactivity of the MPO-generated phenoxyl radicals, the oxidation of these cellular constituents may be directly or indirectly involved in MPO-driven oxidations and/or carcinogenesis (Goldman et al., 1999;Kagan et al., 1999). In effect, the reactivity of phenoxyl radicals determines, to a large extent, their overall cytotoxicity and genotoxicity in MPO-expressing CD34+cells, the likely precursors from which t-AML arises. Hence, characterizing the interactions of etoposide phenoxyl radicals with major cellular components is essential for a better understanding of this drug’s effects on cells (Kagan et al., 1999,2001). The most direct way to detect and monitor the free radical MPO-initiated reaction is usually via EPR spectroscopy. We reported previously that EPR detection of a phenoxyl radical of etoposide is usually feasible in MPO-rich human myeloid leukemia HL60 cells (Kagan et al., 2001). EPR detection of the radicals became possible after depletion of GSH and other thiols, suggesting that etoposide radicals (etoposide-O) displayed Rabbit Polyclonal to RAB2B reactivity toward these abundant intracellular reductants (Kagan et al., 1999). Furthermore, possible involvement of secondary reactions of thiol radicals leading to the production of superoxide radicals and other reactive oxygen species were considered as important cytotoxic and genotoxic events (Kagan et al., 1999,2001). To further evaluate whether MPO is usually a cellular determinant of etoposide oxidation, genotoxicity, and leukemogenesis, we evaluated MPO-catalyzed production of etoposide phenoxyl radicals in growth factor-mobilized human CD34+cells, a proximal progenitor model for t-AML. We report for the first time the detection of the EPR signal of etoposide phenoxyl radicals in intact CD34+cells and demonstrate that this process is.