Multidrug resistance protein-mediated transport of chlorambucil and melphalan conjugated to glutathione

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Abstract
The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1-mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-alpha-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two- to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.