Durg Resistance Mediated by Overexpression of MRPI in an Inducible System

Abstract

The superfamily of A TP-binding cassette transport proteins known as multidrug resistance proteins (MRPs) are important to cancer therapy due to their function in conferring tumor cells with resistance to multiple drugs. MRP 1 is one of six members in this family that has been identified previously and has been strongly implicated in the reduction of tumor cell sensitivity to chemotherapeutic drugs. Because of its location in the membranes of many tumor cell lines, the MRPI transport protein plays a crucial role in the uptake and efflux of chemotherapuetic drugs. In this study, we have utilized K562 acute myeloid leukemia (AML) cells transfected with MRP 1 cDNA in an inducible expression system. By introducing a plasmid containing MRPI under control of a tetracycline-response promotor into a K562 , cell line, it is possible to induce the expression of MRP 1 through the addition of doxycycline (Dox), a derivative of tetracycline. The response protein functions as a control switch for MRP 1 expression by "turning on" transcription of the protein when Dox is added to the cell media. Drug resistance observed in the transfected cell line can then be attributed solely to MRP 1 overexpression due to the fact that the presence of Dox is necessary for MRPI expression to occur. Following demonstration of MRP 1 overexpression in transfected cells induced with Dox, both induced and uninduced cells were treated with several chemotherapeutic agent. Using cytotoxicity assays, we determined that transfectants overexpressing MRPI displayed a reduction in sensitivity to vincristine and daunorubicin during long-term exposure (72 hours) and to methotrexate during short-term (4 hour) exposure. Increased resistance to the antifolate drug, ZD1694, was not observed during either long- or shortterm exposure. As expected, net uptake of radio labelled methotrexate (eH]MTX ) in the transfected K562 cell line was greater for uninduced cells while the efflux rate for eH]MTX was slightly faster in induced cells (i.e. cells overexpressing the MRPI transporter) due to the fact that MRPI has the ability to pump drug out of the cells. Thus, using a tightly controlled system of expression, we have strong evidence to support the role of MRP I-mediated drug resistance in tumor cell lines. This may be important in regard to providing effective treatments for future cancer patients.