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mRNA expression profiling of the ATP-binding cassette transporter family

University of British Columbia

Multidrug resistance (MDR) in cancer is characterized by cross-resistance to a variety of structurally unrelated chemotherapeutic compounds. Altered expression levels of P-glycoprotein (Pgp) and multidrug resistance-associated protein-1 (MRP1) are strongly associated with MDR. Pgp and MRP1 are members of the ATP-binding cassette (ABC) superfamily of membrane proteins. Members of this family transport a wide spectrum of substrates (e.g. peptides, metals, ions, toxins and chemotherapeutic drugs) across cell membranes. Completion of the human genome-sequencing project revealed there are close to 50 ABC genes (Dean et al., 2001); however, function or substrate specificity is known for only a few. It is anticipated that several of these proteins may also contribute to MDR and mutations or altered expression levels of a number of ABC proteins are causal of diseases such as cystic fibrosis and Tangier disease. Despite these associations, there has been no systematic attempt to profile expression of ABC transcripts. We have developed a competitive-RT-PCR assay to examine RNA expression profiles of 35 human ABC transcripts. This assay uses RNA competitors for each transcript, and fluorescently labeled PCR products are resolved using capillary electrophoresis. The data is highly reproducible and accurate. We have examined ABC transcript expression profiles in sensitive and vincristineresistant cell lines, as well as cells exposed short-term (24 hours) to various chemotherapeutic drugs. Vincristine-selection demonstrated multiple, possibly sequential, expression changes may collectively contribute to MDR, whereas short-term drug exposure revealed few expression changes, suggesting that altered mRNA expression is not a common response to xenobiotic exposure. ABC expression profiles were also generated for 12 normal human tissues. Analysis of individual transcripts and the collective expression profiles provided insights into the relative contribution of ABC proteins to MDR, defense against xenobiotic toxins and phospholipid and cholesterol homeostasis. Another study examined the effect o f enforced expression o f the proapoptotic BAX cDNA on the collective ABC expression profile and relative drug-resistance of a cell line. Our results suggest Pgp-mediated MDR may be regulated by BAX protein in some cell lines. The continued systematic evaluation of ABC transcript profiles under various experimental conditions should provide further insights into the normal physiological roles of ABC proteins.

Thesis/Dissertation

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2009-09-15

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http://hdl.handle.net/2429/12788

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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