The development of drug resistance in leukemia cells constitutes the major reason for treatment failure in acute myeloid leukemia (AML). Although a number of prognostic factors such as cellular morphology and cytogenetic abnormalities have been identified, the mechanisms by which cellular resistance to chemotherapy occurs are poorly understood. We propose to build upon observations derived from our physiologic or """"""""functional"""""""" assays for a multidrug resistance (MDR) phenotype which reveal that daunorubicin accumulation, retention and cytotoxicity can be enhanced by cyclosporin A (CsA) or verapamil in blast cells from more than half of newly diagnosed AML patients. The major thrust will be the correlation of our """"""""functional"""""""" evidence for MDR with expression of P-glycoprotein (Pgp) or mdr1 transcripts. In addition, we will also study multidrug resistance-associated protein (MRP) a newly discovered and cloned membrane protein associated with drug resistance in human neoplastic cell lines. MRP has been found to be overexpressed in the human leukemia cell line HL- 60/Adr, which displays a classical MDR phenotype and impaired accumulation and retention of drug, but does not overexpress Pgp. Flow cytometry and reverse transcriptase-PCR (RT-PCR) will be utilized to identify Pgp expression in leukemic cells, using different approaches than in our previous proposal. MRP expression will be determined by RT-PCR. To validate these assays, these data will be correlated with our """"""""functional"""""""" assays for an MDR phenotype, and also with outcome after treatment and clinical parameters, in a consecutive cohort of newly diagnosed patients treated on clinical trials, as well as with data and samples acquired during the previous granting period. Serial evaluations will be done at the time of treatment failure or relapse. Finally, we will also initiate a phase I trial of CsA, a modulator of Pgp-associated MDR, in combination with daunorubicin, etoposide and cytarabine, which is a regimen that could be used as initial induction therapy for AML. The UMCC has a large population of leukemia patients, extensive preliminary data on the proposed projects, a history of productive clinical-laboratory interactions and represents an excellent environment in which to study these important questions.
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