The overall objective of this project is to isolate and characterize the ATP-dependent glutathione S-conjugate export pump (GS-X pump), clone its encoding cDNA, and examine its expression levels in cisplatin-resistant tumor cells. This proposal is based on evidence that cisplatiN reacts with intracellular glutathione to form a potentially cytotoxic glutathione- platinum chelate complex, and that the GS-X pump plays a role in the elimination of the glutathione-platinum complex from tumor cells. In our preliminary studies, the GS-X pump was found to be functionally overexpressed in a cisplatin-resistant human leukemia HL-60 cell line which we recently developed. In this proposal, we will examine the hypothesis that the GS-X pump in tumor cells is involved in the elimination of the glutathione-platinum complex from tumor cells, and that the overexpression of the GS-X pump confers tumor cell resistance to cisplatin. We will identify the GS-X pump by photoaffinity labeling and purify it by affinity chromatography. Using the polyclonal antibody raised against the purified GS-X pump as well as synthetic oligonucleotide probes deduced from the N-terminal amino acid sequence, human leukocyte cDNA libraries will be screened to isolate the cDNA encoding the GS-X pump. Sequence analysis of the isolated cDNA encoding the GS-X pump will be accomplished. In addition, using the GS-X pump-specific antibody and cDNA probe, we will analyze the differential expression of protein and mRNA levels as well as the cellular localization of the GS-X pump in cisplatin- resistant and sensitive human leukemia cells. This study will for the first time reveal did molecular structure of the GS-X pump and provide new evidence for the molecular mechanisms of glutathione-associated cisplatin resistance in human tumors.
