ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter.
MRP is a recently isolated ATP-binding cassette family transporter. We previously reported transfection studies that established that MRP confers multidrug resistance [Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T. & Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652] and that expression of MRP is associated with enhanced cellular efflux of lipophilic cytotoxic agents [Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A. & Kruh, G. D. (1995) Cancer Res. 55, 5342-5347]. To examine the biochemical mechanism by which MRP confers multidrug resistance, drug uptake experiments were performed using inside-out membrane vesicles prepared from NIH 3T3 cells transfected with an MRP expression vector. ATP-dependent transport was observed for several lipophilic cytotoxic agents including daunorubicin, etoposide, and vincristine, as well as for the glutathione conjugate leukotriene C4 (LTC4). However, only marginally increased uptake was observed for vinblastine and Taxol. Drug uptake was osmotically sensitive and saturable with regard to substrate concentration, with Km values of 6.3 microM, 4.4 microM, 4.2 microM, 35 nM, and 38 microM, for daunorubicin, etoposide, vincristine, LTC4, and ATP, respectively. The broad substrate specificity of MRP was confirmed by the observation that daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, the glutathione conjugates S-(p-azidophenacyl)-glutathione (APA-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), arsenate, and the LTD4 antagonist MK571. This study establishes that MRP pumps unaltered lipophilic cytotoxic drugs, and suggests that this activity is an important mechanism by which the transporter confers multidrug resistance. The present study also indicates that the substrate specificity of MRP is overlapping but distinct from that of P-glycoprotein, and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression of MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that the transporter represents a widespread and remarkably versatile cellular defense mechanism.
Paul
S
S
Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Breuninger
L M
LM
Tew
K D
KD
Shen
H
H
Kruh
G D
GD
D016475:3T3 Cells; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents / Q000378:metabolism*; D001149:Arsenates / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003630:Daunorubicin / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016133:Polymerase Chain Reaction; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D001149:Arsenates; D001386:Azides; D003630:Daunorubicin; D005978:Glutathione; D019803:Glutathione Disulfide; D017997:Leukotriene C4; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011422:Propionates; D011804:Quinolines; D011994:Recombinant Proteins; C030190:S-(2,4-dinitrophenyl)glutathione; C059141:verlukast; C024939:S-(4-azidophenacyl)glutathione; C025657:arsenic acid