1 The present study was performed to investigate the ability of the multidrug resistance protein (MRP1) to transport dierent cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC 4 /Adr) and a MRP1-transfected cell line (S1(MRP)). 2 As substrates we used 3 H-labelled derivatives of the hydrophilic monoquaternary cation N-(4',4'-azo-n-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. 3 MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC 4 /Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. 4 ATP-and GSH-dependent transport of daunorubicin and vincristine into GLC 4 /Adr membrane vesicles was inhibited by the MRP1-speci®c monoclonal antibody QCRL-3. 5 MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations 510 mM. The apparent K M value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC 50 of 0.4 mM. 6 In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP-and GSH-dependent manner. APDA is not a substrate for MRP1.

Summary Sublines of the human small-cell lung carcinoma (SCLC) cell line GLC4 with acquired resistance to teniposide, amsacrine and mitoxantrone (GLC4/VM20 ,, GLC4/AM3, and GLC4/MIT60 ,, respectively) were derived to study the contribution of DNA topoisomerase Ila and -,B (Topolla and -,B) to resistance for Topolltargeting drugs. The cell lines did not overexpress P-glycoprotein or the multidrug resistance-associated protein but were cross-resistant to other TopoIl drugs. GLC4/VM20x showed a major decrease in Topolla protein (54%; for all assays presented in this paper the GLC4 level was defined to be 100%) without reduction in TopoII,B protein; GLC4/AM3X showed only a major decrease in TopoII,B protein (to 18%) and not in Topolla.In GLC4/MIT60 ,, the Topolla and -,B protein levels were both decreased (Topolla to 31%; TopoII,B protein was undetectable). The decrease in Topolla protein in GLC4/VM20 and GLC4/MIT60,,, was mediated by decreased Topollcx mRNA levels. Loss of Topolla gene copies contributed to the mRNA decrease in these cell lines. Only in the GLC4/MIT60x cell line was an accumulation defect observed for the drug against which the cell line was made resistant. In conclusion, Topollcx and -/3 levels were decreased differentially in the resistant cell lines, suggesting that resistance to these drugs may be mediated by a decrease in a specific isozyme.

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Intracellular glutathione-conjugate transport was evaluated in the human small cell lung carcinoma cell line GLC 4 with low multidrug resistance protein (MRP 1 ) expression and its 300؋ doxorubicin-resistant, MRP 1 -over-expressing, GLC 4 -Adr subline. Transport of non-toxic concentrations of monochlorobimane and 5-chloro-methyl fluorescein diacetate was evaluated using fluorescence microscopy. After exposure to these compounds, fluorescence was observed especially in intracellular vesicles in GLC 4 -Adr. Immunotransmission electron microscopy showed that MRP 1 was present in the vesicle membranes and plasma membrane, while inside the vesicles the glutathione conjugate of 1-chloro-2,4-dinitrobenzene could be detected. Experiments with brefeldin A, which induces arrest in vesicle release from the Golgi complex, indicated that these vesicles may originate from the trans-Golgi network. In GLC 4 -Adr cells, doxorubicin also was transported in vesicles, with an arrest in vesicle release from the Golgi complex. Our results indicate that MRP 1 functions as a glutathione-conjugate transporter not only at the plasma membrane but also in intracellular secretory vesicles. Int.