One of the limiting factors of successful chemotherapy of cancer is the emergence of multidrug resistance (MDR), which is often associated with a reduction in intracellular drug accumulation. While overexpression of transport proteins, such as P-glycoprotein (Ppg) and the multidrug resistance associated protein (MRP), have been shown to cause MDR in some cells, not all multidrug resistant cells that exhibit reduced drug accumulation overexpress Pgp and/or MRP. For instance, a Pgp-and MRP-negative variant of the human breast cancer cell line MCF7, MCF7/MX, that has been selected for mitoxantrone resistance, exhibits unusual cross-resistance to several camptothecin analogs such as topotecan and irinotecan. Intracellular accumulation of both mitoxantrone and topotecan is reduced in these cells, whereas no alterations in the targets for these drugs, topoisomerases II and I, respectively, are detected. These observations led to the hypothesis that MCF.MX cells express a novel drug transport system, tentatively name MTPR for mitoxantrone topotecan resistance. It is the goal of this grant application to identify and characterize the molecular basis for MTPR. A cDNA expression library from MCF7/MX cells was constructed in the episomal mammalian expression vector pREP3 and transfected into parental MCF7/WT cells. 80 individual mitoxantrone and topotecan resistant clones were isolated from the transfected cells and pREP3 plasmids were rescued by ampicillin selection of bacteria transformed with total cellular DNA. The rescued plasmids will be analyzed for the presence of a MCF7/MX derived cDNA and its overexpression in MCF7/MX cells, followed by sequencing. This CDNA will then be used as a probe to study MTPR gene expression in cell lines and normal tissues as well as leukemia samples from patients treated with topotecan. To confirm that the MTPR gene can cause MDR, parental MCF7/WT cells will be transfected with an MTPR expression construct and the Transfected cells assayed for mitoxantrone and topotecan resistance. To establish whether the MTPR protein has drug transport activity as predicted, drug uptake and efflux studies will be performed with transfected cells. Furthermore, studies to determine whether the MTPR protein binds ATP and drugs will also be performed. The characterization of the MTPR gene/protein and the study of its role in drug resistance are expected to help better understand MDR and result in improved therapy. In addition, since bone marrow Toxicity appears to be a major dose limiting factor in chemotherapy with camptothecin analogs, the MTPR gene may also provide a new gene for bone marrow stem cell protective gene therapy.
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