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    Comparison of DiOC6(3) uptake and annexin V labeling for quantification of apoptosis in leukemia cells and non-malignant T lymphocytes from children
    (2000) Özgen U.; Savaşan S.; Buck S.; Ravindranath Y.
    Early during apoptosis, there is a reduction in mitochondrial transmembrane potential (MTP) and externalization of phosphatidylserine (PS) in cell membrane prior to eventual cell death. Flow cytometric detection techniques targeting these changes, reduction of DiOC6(3) uptake upon the collapse of MTP and annexin V binding to PS have been successfully used to detect apoptotic cells. These methods have given comparable results when cell lines were used. We compared the two different techniques, DiOC6(3) uptake and Annexin V-propidium iodide co-labeling in the quantification of cytarabine, vincristine and daunorubicin induced apoptosis on three leukemia cell lines (HL-60, CEM, U937), and bone marrow blasts from 26 children with acute myeloid leukemia, 14 with T cell acute lymphoblastic leukemia. Anti- Fas-induced apoptosis in culture-grown peripheral blood T lymphocytes on 18 samples from 9 children with non-malignant conditions were also studied by these techniques. Our results showed that there is a correlation (P < 0.05) between the apoptosis rates measured by these two techniques for drug- induced apoptosis in myeloid and lymphoid blasts, and for anti-Fas mAb- induced apoptosis in T lymphocytes. This data suggests that reduction of the MTP and PS externalization may be common to many apoptotic pathways and techniques targeting either of these changes may be used in quantification of apoptosis in different clinical samples. (C) 2000 Wiley-Liss, Inc.
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    Further elucidation of mechanism of resistance to vincristine in myeloid cells: role of hypochlorous acid in degradation of vincristine by myeloperoxidase
    (Nature Publishing Group, 2000) Özgen Ü.; Savaşan S.; Stout M.; Buck S.; Ravindranath Y.
    Inherent resistance of myeloblasts to vincristine (VCR) has been related to the activity of myeloperoxidase (MPO) which can degrade VCR in the presence of hydrogen peroxide (H2O2). We investigated the relationship between VCR degradation and hypochlorous acid (HOCl) generation from the reaction of H2O2 with chlorine (Cl) as catalyzed by MPO. A cell-free system, three human leukemia cell lines (CEM/CCRF, HL-60, U937) and 15 bone marrow samples from children with acute myeloid leukemia (AML) were studied. VCR cytotoxicity was evaluated by MTT assay and by quantitative measurement of apoptosis. In vitro levels of VCR in cell-free systems were measured by high performance liquid chromatography (HPLC), and intracellular HOCl levels by oxidation of 5-thio-2-nitrobenzoic acid with the accompanying decrease in the absorbency at 412 nm. VCR was degraded by increasing concentrations of HOCl in cell-free systems and this activity was inhibited by taurine, which is known to block HOCl activity. This finding was confirmed by the VCR cytotoxicity studies on cell lines. The HOCl-producing myeloblasts from patients were resistant to VCR. In five samples out of eight HOCl was also detected extracellularly. These results suggest that oxidation by HOCl may be the final step in VCR degradation catalyzed by MPO through its action on intracellular H2O2 and Cl.