J. Biochem, 1994, Vol. 115, No. 2 213-218
© 1994 Japanese Biochemical Society
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ATP-Dependent Uptake of Anti-Neoplastic Agents by Acidic Organelles1
*Department of Biochemistry and Organic Chemistry, Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567
**Department of Physiology, Kansai Medical University Moriguti, Osaka 570
Daunomycin, an anti-neoplastic agent, is known to be sequestered by acidic organelles in normal and multidrug-resistant cells [Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M., & Pastan, I. (1986) Cancer Res. 46, 5941–5946]. We studied the mechanism of accumulation of daunomycin into acidic organelles using chromaffin granule vesicles and proteoliposomes reconstituted with purified F-type H+-ATPase as model systems. Radiolabeled daunomycin was taken up by chromaffin vesicles upon addition of ATP. Its ATP-dependent uptake was stimulated about 1.4- to 1.8-fold by valinomycin plus K+, but was inhibited by ammonium chloride (10 mM) and nigericin plus K+. Quinidine (5µM), verapamil (5 µM), or vanadate (0.5 mM), inhibitors of P-glycoprotein, had no effect on its uptake. Daunomycin was also taken up by liposomes reconstituted with F-type H+-ATPase. Furthermore, doxorubicin and vinblastine were taken up by these vesicles, whereas colchicine and rhodamine 123 were not. The accumulations of daunomycin and doxorubicin in acidic organelles of cultured cells were decreased by inhibiting vacuolar ATPase by addition of bafilomycin A, or concanamycin A, or by increasing the internal 
pH by addition of nigericin. Melittin and N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide dissipated the dpH and inhibited accumulation of daunomycin in the membrane vesicles and acidic organelles in cultured cells. These results indicate that the pH established by vacuolartype ATPase drives the uptake of daunomycin, doxorubicin or vinblastine into acidic organelles, and that no specific transporters are involved in their uptakes.
1This work was supported by grants for the Human Frontier Science Program, from the Ministry of Education, Science and Culture of Japan
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