Anthracyclines modulate multidrug resistance protein (MRP) mediated organic anion transport.
We studied the ATP-dependent uptake of dinitrophenyl-glutathione (GS-DNP) into plasma membrane vesicles derived from parental GLC4 cells and from multidrug resistant GLC4/ADR cells. The latter have a high expression of the multidrug resistance protein (MRP). Uptake of GS-DNP into membrane vesicles from GLC4/ADR cells was highly stimulated by the addition of ATP, compared to the uptake into membrane vesicles from GLC4 cells. This ATP-dependent uptake into membrane vesicles from GLC4/ADR cells was saturable with a Km of 1.2 +/- 0.2 microM and a Vmax of 560 +/- 80 pmol/mg prot./min. ATP stimulated GS-DNP uptake with a Km of 187 +/- 4 microM. This uptake was specifically inhibited by a polyclonal serum raised against a fusion protein containing a segment of MRP. The ATP-dependent uptake of GS-DNP was not only inhibited by organic anions, such as oxidized glutathione (GSSG), methotrexate (MTX) and some bile acids, but also by non-anionic natural product drugs, such as anthracyclines, vinca alkaloids and etoposide (VP-16). Uptake of GSSG and MTX into membrane vesicles from GLC4/ADR cells could be stimulated by ATP. The ATP-dependent uptake of GSSG had a Km of 43 +/- 3 microM and a Vmax of 900 +/- 200 nmol/mg protein/min. The ATP-dependent uptake of GS-DNP seemed to be non-competitively inhibited by the anthracycline daunorubicin (DNR), whereas the ATP-dependent GSSG uptake seemed to be competitively inhibited by DNR. A substrate binding site on MRP is proposed that comprises a pocket in which both DNR and GS-DNP or GSSG bind in random order to different, only partly overlapping sites. In this pocket binding of a second compound is influenced by the compound which was bound first.
Heijn
M
M
Academic Hospital Vrije Universiteit, Department of Medical Oncology, Amsterdam, The Netherlands.
Hooijberg
J H
JH
Scheffer
G L
GL
Szabó
G
G
Westerhoff
H V
HV
Lankelma
J
J
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D018943:Anthracyclines / Q000494:pharmacology*; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003630:Daunorubicin / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014407:Tumor Cells, Cultured
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D018943:Anthracyclines; D003630:Daunorubicin; D005978:Glutathione; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C030190:S-(2,4-dinitrophenyl)glutathione