Myeloperoxidase Increases Chemosensitivity of Leukemia Cells through the Generation of Reactive Oxigen and Nitrogen Species.

Myeloperoxidase (MPO) is a specific enzyme whose expression is restricted to the late stage of myeloid differentiation such as promyelocytes and more matured cells. In a pathological situation, however, morphologically immature blasts of acute myeloid leukemia (AML) express MPO at variety of percentages that has been utilized for the diagnosis of AML. On the other hand, the expression of MPO has been shown to have prognostic value for AML cases. Several groups including ours reported that the high percentage of MPO positive blasts at diagnosis was related to the favorable prognosis. Recently, we also found that the expression of the MPO gene in CD133 positive cells also related to the better prognosis of AML cases, suggesting the MPO in the very immature fraction, that is thought to contain leukemia stem cells, is related to the chemosensitivity of AML cells. These results made us hypothesize that MPO directly changes chemosensitivity of leukemia cells through its enzymatic activity. To test this hypothesis, we transfected wild type and enzymaticaly defective MPO (dMPO) cDNA into MPO-negative leukemia cells line, K562, generating K562-MPO and K562-dMPO. Control cells were K562-vector that were transfected with empty vector. The expression of MPO protein was shown in K562-MPO cells, but only precursor of MPO protein, apo-pro-MPO was expressed in K562-dMPO cells as expected. The enzymatic activity was shown only in K562-MPO cells. When these cells were treated with cytosine arabinoside (AraC), K562-MPO cells showed decreased proliferation by WST1 assay compared with wild type K562, K562-vector and K562-dMPO. The expression of Annexin V was increased in AraC-treated K562-MPO suggesting these cells died through apoptosis. Using flowcytometer or confocal microscopy with aminophenyl fluorescein (APF), reactive oxygen species (ROS) were detected in wild type K562 and K562-MPO cells after treatment with AraC. The amount of ROS shown by the intensity of APF-fluorescence increased more in K562-MPO compared with wild type K562 or K562-vector. The difference in the APF-fluorescence observed AraC-treated K562-vector and K562-MPO cells was strongly enhanced by the addition of hydrogen peroxide as a donor of ROS. However, this difference completely disappeared with the addition of N-acetyl cystein (NAC), an inhibitor of ROS, suggesting the involvement of MPO to generate ROS in K562-MPO cells. Although flowcytometer with DAF-2DA failed to detect the significant differences in the amount of reactive nitrogen species (RNS) among AraC-treated wild type K562, K562-vector and K562-MPO cells, immunoblotting against the nitrated tyrosine residue demonstrated the increased nitration of protein in K562-MPO cells compared with wild type or K562-vector. It was suggested that RNS was generated in K562-MPO treated with AraC. These results supported our working hypothesis that MPO in leukemia cells enhanced the sensitivity against chemotherapeutic agents through the generation of ROS and RNS.