We propose that the multidrug resistance-associated protein (MRP) and glutathione S-transferases (GSTs) cooperate synergistically to confer high level multidrug resistance (MDR) to many anticancer drugs. By synergy we mean that the total effect on drug resistance of increases in both MRP and GST is greater than the sum of the effects on drug resistance of MRP and GST taken independently. We suggest that the mechanism of this synergy involves the conjugation of drugs, or their toxic metabolites, to glutathione (GSH) by GSTs. It is believed that these GSH-toxin conjugates are better substrates for the MRP-associated drug efflux pump than are the unconjugated drugs or metabolites. This hypothesis is appealing because, while GSTs can conjugate a number of anticancer drugs with GSH, the effect of increased GST on drug resistance has been inconsistent. Consequently, it has been suggested that a second gene product may be necessary for GSTs to fully confer drug resistance. Recent reports have strongly indicated that MRP is an ATP-dependent, GSH- conjugate exporter. Therefore, MRP is the prime candidate for the second gene product involved in GST-mediated drug resistance. To test this hypothesis, we will use model parental cell lines that express low basal levels of MRP and GST. By stable transfection of these cells, derivative cell lines will be established that express increased levels of MRP with or without concomitantly increased levels of the isozymes of GST. These cell lines will be examined for relative resistance to the cytotoxic effects of a panel of drugs and xenobiotic toxins. Comparison of cytotoxicity dose-response data for cells that express only increased MRP versus cells that express increased levels of MRP-GST isozyme combinations will allow the identification and quantitation of pharmacodynamic interactions between MRP and GSTs. Additionally, the relationship between resistance and maintenance of intracellular GSH pools will be explored. With these derivative cell lines, the relative contributions to resistance of drug conjugating enzymes, such as GSTs, and MRP can be evaluated. These cells will be used to test our hypothesis that GST and MRP can cooperate synergistically to confer high level resistance to select antineoplastic drugs. In order to examine the mechanism of any observed synergy between MRP and the isozymes of GST, the kinetics of drug accumulation and efflux will be determined in the derivative cell lines. These pharmacokinetic studies will establish whether or not increased GST levels facilitate drug export, by the formation GSH-drug conjugates, in cells having increased MRP.
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