Etoposide (VP-16)-related secondary leukemias (t-AML) occur in up to 10 percent of drug-treated patients and are most frequently associated with MLL gene translocations at 11q23. Delineating the mechanism(s) of VP-16-mediated leukemogenesis is an important goal in oncology and is the major focus of this project. Our central hypothesis is that redox cycling of VP-16 initiated by myeloperoxidase (MPO) amplifies the genotoxicity and carcinogenicity of this agent in myeloid precursors which contain high MPO activity. We propose that MPO converts VP-16 to free radical species and oxidized metabolites that induce oxidative DNA damage and initiate recombinogenic events specifically in myeloid precursor stem cells leading to the chromosomal translocations responsible for t-AML. We further hypothesize that antioxidants such as vitamin C or vitamin E homologues will protect against oxidative DNA damage and may prevent t-AML by reducing levels of VP-16 free radical species produced by MPO. Peroxidative activation of the VP-16 E-ring phenol to form phenoxyl and semiquinone radicals, and orthoquinones results in increased DNA strand breakage. It is not known whether enhanced DNA cleavage occurs by interaction of VP-16 oxidation products directly with DNA, by redox cycle-generated reactive oxygen species (ROS), and/or by enhanced inhibition/poisoning of DNA topoisomerase II (topo II). We propose to characterize the precise mechanism(s) by which activation of VP-16 to free radical forms enhances its DNA damaging activity specifically in genomic regions of the MLL gene known to contain breakpoints associated with t-AML.
Specific aims are to: 1. Determine the role of MPO and the bioreductive enzyme NAD(P)H:quinone oxidoreductase (NQO1) in VP-16 metabolic activation, generation of ROS, and oxidative DNA damage in cultured human leukemia cells (HL60, K562) and in normal human CD34+ progenitor cells. 2. Develop an antioxidant recycling system to decrease MPO-generated phenoxyl radicals of VP-16 and attenuate VP-l6-induced oxidative DNA damage. 3. Characterize chemical modifications in topo II upon interaction with oxidatively activated VP-16 that may be responsible for increased DNA strand breakage/recombination events.
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