ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells.
To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.
Naito
M
M
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo.
Hamada
H
H
Tsuruo
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001665:Binding Sites; D001692:Biological Transport; D002166:Camptothecin / Q000494:pharmacology; D002462:Cell Membrane; D003609:Dactinomycin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D005472:Fluorouracil / Q000494:pharmacology; D007700:Kinetics; D007951:Leukemia, Myeloid / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology; D014750:Vincristine / Q000378:metabolism* / Q000494:pharmacology
D003609:Dactinomycin; D014750:Vincristine; D014747:Vinblastine; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D014700:Verapamil; D008274:Magnesium; D005472:Fluorouracil; D002166:Camptothecin