1533

Previous publications have demonstrated MPO-catalyzed oxidative activation of etoposide (VP-16) to phenoxyl radical and quinone metabolites in human myeloid leukemia HL-60 cells (Chem. Res. Toxicol.19: 937-43, 2006; Cancer Res. 61: 7777-84, 2001). Metabolic activation of VP-16 results in enhanced DNA damaging activity mediated through poisoning of DNA topoisomerase II (topo II). We hypothesized that MPO-catalyzed activation of VP-16 in myeloid progenitor CD34+ cells enhances DNA damage and recombination events that are responsible for VP-16-induced secondary myeloid leukemias (t-AML). We now provide experimental evidence to indicate that MPO found in human CD34+ cells catalyzes production of VP-16 phenoxyl radicals and VP-16 ortho-quinone, resulting in greater VP-16-induced DNA damage. Using mobilized CD34+ cells isolated from umbilical cord blood, mature (heavy subunit) MPO was detected by immunoblotting and enzymatic activity was demonstrated by oxidation of tetramethylbenzidine. MPO levels and activities were reduced (to <20% of control levels) by incubation with the heme synthesis inhibitor, succinylacetone (SA). Formation of VP-16 phenoxyl radicals and of a VP-16 ortho-quinone-glutathione adduct were demonstrated in intact CD34+ cells by electron spin resonance (EPR) and in cell lysates by LC-ESI-ion trap mass spectrometry (MS) and MS/MS, respectively. For MS analyses, a stable isotope-labeled form of the glutathione adduct was synthesized and employed for quantitative analysis. After SA pretreatment to reduce MPO levels, the EPR signal was eliminated, indicating dependence on MPO for formation of VP-16 phenoxyl radicals. Similarly, SA pretreatment reduced the level of VP-16 ortho-quinone glutathione adduct from 69 to 29 pmol/million CD34+ cells. When CD34+ cells were pretreated with SA, VP-16 (5 µM)-induced DNA damage as assessed by the Comet assay (after 30 min) was significantly reduced compared to CD34+ cells with replete MPO. Results indicate an MPO-dependent component of VP-16-induced DNA damage in CD34+ cells consistent with the central idea that MPO-catalyzed oxidative activation of VP-16 contributes to leukemogenesis in CD34+ progenitors. Together, these results set the stage for investigation of MLL gene rearrangements (known to be associated with t-AML) dependent on MPO-catalyzed activation of VP-16. Support: R01 CA90787; TATRC/DoD (USAMRAA Prime Award W81XWH-05-2-0066)

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA