Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts.
Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.
Department of Medicine, New England Medical Center, Boston, Massachusetts.
C A
CA
Slapak
J M
JM
Lecerf
J C
JC
Daniel
S B
SB
Levy
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides / Q000494:pharmacology; D002451:Cell Compartmentation; D002738:Chloroquine / Q000494:pharmacology; D003630:Daunorubicin / Q000378:metabolism* / Q000494:pharmacology; D003847:Deoxyglucose / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D004347:Drug Interactions; D004351:Drug Resistance; D004734:Energy Metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008985:Monensin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D019810:Sodium Azide; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D004140:Dinitrophenols; D008562:Membrane Glycoproteins; D008744:Methylamines; D014638:Vanadates; D010042:Ouabain; D002738:Chloroquine; D008985:Monensin; D019810:Sodium Azide; D003847:Deoxyglucose; C027451:methylamine; D014700:Verapamil; D005033:Ethylmaleimide; D003630:Daunorubicin