Abstract
The Breast Cancer Resistance Protein (BCRP) is a key transporter in drug efflux and drug-drug interactions. However, endogenous expression of Multidrug Resistance Protein 1 (MDR1) confounds the interpretation of BCRP-mediated transport in in vitro models. Here we used a CRISPR-Cas9 edited Madin-Darby canine kidney (MDCK) II cell line (MDCKcMDR1-KO) for stable expression of human BCRP (hBCRP) with no endogenous canine MDR1 (cMDR1) expression (MDCK-hBCRPcMDR1-KO). Targeted quantitative proteomics verified expression of hBCRP, and global analysis of the entire proteome corroborated no or very low background expression of other drug transport proteins or metabolizing enzymes. This new cell line, had similar proteome like MDCKcMDR1-KO and a previously established, corresponding cell line overexpressing human MDR1 (hMDR1), MDCK-hMDR1cMDR1-KO. Functional studies with MDCK-hBCRPcMDR1-KO confirmed high hBCRP activity. The MDCK-hBCRPcMDR1-KO cell line together with the MDCK-hMDR1cMDR1-KO easily and accurately identified shared or specific substrates of the hBCRP and the hMDR1 transporters. These cell lines offer new, improved in vitro tools for the assessment of drug efflux and drug-drug interactions in drug development.
Highlights
The Breast Cancer Resistance Protein (BCRP; gene name ABCG2) discovered in 1998 was originally cloned from a doxorubicin resistant MCF-7 breast cancer cell line.[1]
We present a stable MadinDarby canine kidney (MDCK) cell line overexpressing the human BCRP (hBCRP) transporter which completely lacks endogenous canine MDR1 (cMDR1); it originates from the MDCKcMDR1-KO cell line
The MDCKcMDR1-KO cell line was stably transfected with human BCRP using the BCRP-pcDNA3.1 vector
Summary
The Breast Cancer Resistance Protein (BCRP; gene name ABCG2) discovered in 1998 was originally cloned from a doxorubicin resistant MCF-7 breast cancer cell line.[1] BCRP is expressed in a variety of healthy as well as malignant tissues including membrane barriers and tissues important in drug disposition, e.g., intestinal epithelium, apical membranes of human hepatocytes, kidney proximal tubules, placenta, and the blood-brain barrier.2e4. The BCRP transporter translocates both endogenous and exogenous compounds. Its substrate specificity overlaps with other ABC transporters and the overlap with substrates of Multi Drug Resistance Protein 1. BCRP and MDR1 are concomitantly expressed in many tissues.[6]. In the blood-brain barrier, BCRP and MDR1 cooperate to limit the exposure of the brain to drugs and potentially hazardous compounds.[7]
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