Apoptosis by anthracyclines at therapeutic concentrations in MDR1+ human leukemic cells.
Induction of apoptosis by daunorubicin (DNR) and idarubicin (IDA) was evaluated cytofluorometrically in CEM and CEM-MDR1+ leukemic cells exposed to drug concentrations similar to peak plasma levels obtainable in vivo (DNR 200-400 ng/ml, IDA 50-100 ng/ml, 30' incubation), and differentiating apoptosis from necrosis (FITC-annexin V+/propidium iodide- and + cells, respectively). Firstly, to set experimental conditions, apoptosis was evaluated in CEM cells at 3, 6, 12, 18, 24, 48, 72, and 96 hours from end of drug incubation, the maximal increase being noted at 24-48 hours. Net apoptosis rates were determined after subtraction of the spontaneous activity observed in untreated cells. The apoptotic effect from varying drug type and concentration was compared at 24 hours in CEM-MDR1+ cells, with and without co-incubation with MDR1 functional downregulator cyclosporin A (CSA) used at therapeutic concentration (1500 ng/ml). The results indicated that, at drug concentrations likely to be approached in vivo as a short-lasting peak level (IDA 100-200 ng/ml) with increased-dose IDA (> 12-15 mg/m2), pro-apoptotic effects by IDA+CSA in CEM-MDR1+ cells were significantly greater than by DNR+CSA, and corresponded to the levels observed with IDA 50 ng/ml without CSA in control CEM cells. This in vitro study demonstrates that it is possible to determine in the same sample cell fluorescence related to anthracyclines, apoptotic cells (FITC-annexin V positive), and necrotic cells (propidium iodide positive), and confirms that cytofluorimetric evaluation of apoptosis can reliably predict the effects of anthracycines in function of drug type, concentration and, in MDR1+ cells, concurrent MDR1 inhibition. Extension of this assay to the clinical ground may be warranted.
Chiodini
B
B
Division of Hematology, Ospedali Riuniti, Bergamo, Italy.
Bassan
R
R
Barbui
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D017304:Annexin A5 / Q000502:physiology; D000903:Antibiotics, Antineoplastic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D002462:Cell Membrane / Q000502:physiology; D003630:Daunorubicin / Q000031:analogs & derivatives / Q000633:toxicity*; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D019450:Genes, MDR; D006801:Humans; D015255:Idarubicin / Q000633:toxicity*; D015459:Leukemia-Lymphoma, Adult T-Cell; D008563:Membrane Lipids / Q000378:metabolism; D009336:Necrosis; D010718:Phosphatidylserines / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017304:Annexin A5; D000903:Antibiotics, Antineoplastic; D008563:Membrane Lipids; D010718:Phosphatidylserines; C054440:idarubicinol; D015255:Idarubicin; D003630:Daunorubicin