Functional analyses of multidrug resistance protein 3 (MRP3) and characterization of a retinoic acid resistant human leukemia cell line (HL60-ATRA)

Drug resistance is a major obstacle to successful cancer chemotherapy. MRP1 and MRP2 are ATP-binding cassette transporters that confer resistance to natural product cytotoxic drugs. We recently isolated a new MRP subfamily member, MRP3, which was found to be most closely related to MRP1, and have analyzed its drug resistance activity and substrate selectivity profile. HEK293 cells transfected with an MRP3 expression vector displayed ∼4-fold resistance to etoposide, ∼2-fold resistance to vincristine, and ∼1.7-fold resistance to methotrexate, compared to control transfected cells. Reduced accumulation of radiolabeled etoposide was observed in the MRP3-transfected cells, consistent with the operation of a plasma membrane efflux pump. Using enriched membrane vesicles prepared from MRP3-transfected HEK293 cells, we have demonstrated that expression of MRP3 markedly stimulates the ATP-dependent transport of glucuronide E₂17βG, glutathione conjugates LTC ₄ and DNP-SG, antimetabolite methotrexate and bile acid glycocholate. This transporter is thus concluded, like MRP1 and MRP2, to be competent in the transport of glucuronides, glutathione S-conjugates, and methotrexate, albeit at low-moderate affinity. Unlike all the other characterized mammalian ABC transporters, human MRP3 has the additional feature of transporting the monoanionic human bile constituent glycocholate.; In unraveling mechanisms of drug resistance, resistant cell lines have proved to be helpful in providing visions at cellular level. Retinoic acid (RA) is a differentiation agent widely utilized in both cancer treatment and prevention regimens, but the rapid resistance to this agent has limited its clinical effectiveness. To explore ATRA resistance mechanisms, we have developed and characterized a human leukemia cell line highly resistant to all-trans retinoic acid, HL60-ATRA. Cytotoxicity studies revealed no increased cross-resistant of HL60-ATRA to a wide variety of chemotherapeutic drugs, including anthracyclines and alkylating agents. By contrast with other reported ATRA resistant HL-60 sublines, alterations of the RAR-α receptor were not detected in HL60-ATRA cells. Furthermore, no obvious decrease was observed in HL60-ATRA cells regarding the expression of RAR-α protein and its retinoic acid binding activities. Comparative genomic hybridization (CGH) analysis uncovered significant amplifications at 6q23-24 and 18q22-23 in HL60-ATRA cells. Taken together, our results suggest that ATRA resistance does not contribute increased resistance to cytotoxic anticancer drugs and heavy metals, that it does not rely upon mutations and/or diminished activities of RAR-α receptor, and that stable genomic alterations associated with ATRA resistance can be identified using the CGH approach.