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RNA expression profiles of the human ABC transporter family in patient-derived leukemia cell lines and in HL60 cells exposed to chemotherapeutic drugs

University of British Columbia

Poor outcome in Acute Myeloid Leukemia (AML) is associated with the elevated expression of P-glycoprotein (Pgp) and the Multidrug-Associated Resistance Protein (MRP1), members of the ATP-Binding Cassette (ABC) protein superfamily. However, a proportion of A M L patient blasts demonstrates drug-efflux that is not correlated with Pgp or MRP1 expression. The AML1, 2 and 3 cell lines, derived from AML patient blasts at relapse, exhibit ATP-dependent daunorubicin accumulation with no Pgp and low MRP1 expression. This evidence strongly suggests the presence of energy-dependent processes distinct from Pgp that contribute to daunorubicin efflux. The large number of human ABC proteins (>50) and the complex nature of drug efflux in Multiple Drug Resistance (MDR) deems it reasonable to expect that currently uncharacterized ABC proteins may contribute to drug efflux in AML. Our laboratory has developed a Competitive-Reverse Transcription-Polymerase Chain Reaction (C-RT-PCR) assay to examine global mRNA expression of 40 human A BC transcripts to evaluate their contribution to MDR in cancer cell lines. This assay uses RNA competitors for each transcript and the fluorescently labeled PCR products are detected using an automated DNA sequencer. Surprisingly, C-RT-PCR expression profiles of the AML cell lines revealed that the majority of ABC transcripts examined were detectable (75%) and that considerable patient heterogeneity was evident, providing no clear indication of which ABCs might be relevant to MDR. To address this issue, we examined whether chemotherapeutic drugs could induce expression of ABC genes. The human HL60 leukemic cell line was exposed for 24 hours to individual chemotherapeutic drugs at low drug concentrations. Using C-RT-PCR, significant expression changes were detected with only 2% of the transcripts examined. Among these, a 6.2 ± 1.1-fold increase in MRP7 (p<0.01), a novel member of the Multidrug Resistance-Associated Protein subfamily of ABC transporters, was observed with daunorubicin treatment (1/2 IC ₅₀). Efforts then focused on generating a MRP7 cDNA construct to be used in drug resistance assays of transfected cells. The absence of significant expression changes in response to drug exposure might suggest that induction of ABC gene expression may not play a significant role in the development of drug resistance in HL60 cells.

Thesis/Dissertation

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2009-08-04

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http://hdl.handle.net/2429/11582

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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