Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556002
Title: BCRP (ABCG2) expression, function and regulation in model and intestinal epithelial cells
Author: Wright, Jamie Andrew
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2011
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Abstract:
The breast cancer resistance protein (BCRP, ABCG2) is a member of the ATP binding cassette transporters (ABC transporters) that functions as an efflux transporter alongside other family members such as MDR1 and MRP2 to remove foreign entities from the cell. These efflux transporters often have overlapping substrate specificity that together comprise a formidable barrier in preventing cell exposure to foreign compounds, but in doing so inevitably influence drug absorption, elimination and distribution of a significant number of prescribed drugs. This thesis has used established in vitro epithelial cell models such as MDCKII and Caco-2 cells to examine the function and regulation of the less characterised of the three main efflux transporters, BCRP, in the context of epithelial expression with particular focus on human intestinal cells. Several complementary techniques have been used including cellular accumulation of Hoechst 33342, a known MDR1/BCRP bi-substrate in the presence of selective pharmacological inhibitors, together with immunocytochemistry, immunoblotting and PCR techniques. Of most importance were functional assays of transepithelial substrate flux measured across reconstituted epithelial layers. BCRP substrates were identified by comparing bi-directional transport directly across native MDCKII monolayers to hBCRP/mBcrp and MDR1 transfected MDCKII cell layers in a high throughput assay. This technique identified several BCRP substrates including the selective BCRP agent nitrofurantoin. The human intestinal Caco-2 cell system was used as a model for BCRP mediated transport and induction of ABC transport activity. BCRP is expressed at differing levels in 2 cell-strains; saturable nitrofurantoin transport was evident in the high-expressing strain. Ko143, a specific BCRP inhibitor, inhibited nitrofurantoin secretion by this Caco-2 cell strain and allowed partition of BCRP mediated secretory flux from MDR1 for bi-substrates. The aryl hydrocarbon receptor (AhR) agonist β-naphthoflavone (BNF) and the peroxisome proliferator receptor gamma (PPARγ) agonist rosiglitazone increased the BCRP mediated net flux of the selective substrate, nitrofurantoin and this correlated with an increase in both BCRP mRNA and protein expression. BCRP regulation in Caco-2 cells is likely to be controlled by more than one pathway and it appears that in the human gut a complex network of distinct nuclear receptors regulate the expression of BCRP. The present work suggests that the Caco-2 cell model together with nitrofurantoin as a substrate could serve as a screen for potential inducers of BCRP.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.556002  DOI: Not available
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