Kurt C. Almquist

Multidrug Resistance Protein (MRP)-mediated Transport of Leukotriene C and Chemotherapeutic Agents in Membrane Vesicles DEMONSTRATION OF GLUTATHIONE-DEPENDENT VINCRISTINE TRANSPORT

The 190-kDa multidrug resistance protein (MRP) has recently been associated with the transport of cysteinyl leukotrienes and several glutathione (GSH) S-conjugates. In the present study, we have examined the transport of leukotriene C (LTC) in membrane vesicles from MRP-transfected HeLa cells (T14), as well as drug-selected H69AR lung cancer cells which express high levels of MRP. V and K values for LTC transport by membrane vesicles from T14 cells were 529 ± 176 pmol mg min and 105 ± 31 nM, respectively. At 50 nM LTC, the K (ATP) was 70 μM. Transport in T14 vesicles was osmotically-sensitive and was supported by various nucleoside triphosphates but not by non- or slowly-hydrolyzable ATP analogs. LTC transport rates in membrane vesicles derived from H69AR cells and their parental and revertant variants were consistent with their relative levels of MRP expression. A 190-kDa protein in T14 membrane vesicles was photolabeled by [3H]LTC and immunoprecipitation with MRP-specific monoclonal antibodies (mAbs) confirmed that this protein was MRP. LTC transport was inhibited by an MRP-specific mAb (QCRL-3) directed against an intracellular conformational epitope of MRP, but not by a mAb (QCRL-1) which recognizes a linear epitope. Photolabeling with [3H]LTC was also inhibitable by mAb QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC transport. However, the ability of alkylated GSH derivatives to inhibit transport increased markedly with the length of the alkyl group. S-Decylglutathione was a potent competitive inhibitor of [3H]LTC transport (K 116 nM), suggesting that the two compounds bind to the same, or closely related, site(s) on MRP. Chemotherapeutic agents including colchicine, doxorubicin, and daunorubicin were poor inhibitors of [3H]LTC transport. Taxol, VP-16, vincristine, and vinblastine were also poor inhibitors of LTC transport but inhibition by these compounds was enhanced by GSH. Uptake of [3H]vincristine into T14 membrane vesicles in the absence of GSH was low and not dependent on ATP. However, in the presence of GSH, ATP-dependent vincristine transport was observed. Levels of transport increased with concentrations of GSH up to 5 mM. The identification of an MRP-specific mAb that inhibits LTC transport and prevents photolabeling of MRP by LTC, provides conclusive evidence of the ability of MRP to transport cysteinyl leukotrienes. Our studies also demonstrate that MRP is capable of mediating ATP-dependent transport of vincristine and that transport is GSH-dependent.

Non-P-glycoprotein-mediated multidrug-resistant human KB cells selected in medium containing adriamycin, cepharanthine, and mezerein

Human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500, and C-A1000, were isolated in selection medium containing increasing concentrations of adriamycin, 1 μg/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100 nM of mezerein, a protein kinase C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D, VP-16, and colchicine. The accumulation of adriamycin and vincristine was decreased in C-A500 cells and the efflux of adriamycin from C-A500 was enhanced compared with parental KB-3-1 cells. These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpressMDR1. Multidrug-resistance-associated protein (MRP) andMRP mRNA were expressed in the adriamycin-resistant KB cells, C-A120, C-A500, and C-A1000, but not in parental KB-3-1 and revertant C-AR cells. TheMRP gene was amplified in all the MDR cells that overexpressed MRP mRNA. DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincristine and that both the increased expression of MRP and decreased levels of topoisomerase II underlie the drug resistance in C-A120, C-A500, and C-A1000 cell lines.