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IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P-glycoprotein (ABCB1) for Drug
Lasse Saaby1, Hans Christian Cederberg Helms2, Birger Brodin2
1Bioneer-FARMA, Department of Pharmacy, Universitetsparken 2 , DK-2100 Copenhagen, Denmark.
Abstract:
The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell line, IPEC-J2 MDR1, retained its high transepithelilal resistance (>15,000 Ω·cm(2)), which translated into low permeability of the small hydrophilic tracer, mannitol (P < 10(-7) cm·s(-1)). The lipophilic compound, diazepam, displayed high permeability resulting in a dynamic range of 1500 (PDiazepam/Pmannitol) to separate high and low permeability compounds. Human P-gp was expressed predominantly in the apical membrane, as demonstrated by immunocytochemistry, Western blots, and a high efflux ratios (Pbasolateral-apical/Papical-basolateral) of known P-gp substrates. P-gp was demonstrated to be responsible for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages. In conclusion, the IPEC-J2 MDR1 cell line displays high paracellular tightness combined with high expression of human P-gp and low expression of porcine ABC transporters, and it may serve as a useful tool in drug development studies.
Insights
A new cell line, IPEC-J2 MDR1, was developed to screen for P-glycoprotein (P-gp) substrates. This system offers high cell tightness and functional human P-gp expression for drug development.
Area of Science:
- Pharmacology
- Cell Biology
- Drug Discovery
Background:
- P-glycoprotein (P-gp) influences drug distribution and causes cancer drug resistance.
- Developing robust screening systems is crucial for identifying P-gp substrates and understanding drug transport.
Purpose of the Study:
- To create a novel cell line for screening potential P-glycoprotein (P-gp) substrates.
- The cell line requires high paracellular tightness, low non-human ABC transporter expression, and high functional human P-gp (ABCB1) expression.
Main Methods:
- IPEC-J2 cells were transfected with the human MDR1 gene encoding P-gp.
- Cells were selected using geneticin and puromycin.
- Characterization involved measuring transepithelial resistance, permeability of tracers (mannitol, diazepam), immunocytochemistry, Western blots, and substrate profiling with inhibitors.
Main Results:
- The IPEC-J2 MDR1 cell line exhibited high transepithelial resistance (>15,000 Ω·cm(2)) and low mannitol permeability.
- A dynamic range of 1500 was observed for diazepam permeability, distinguishing high/low permeability compounds.
- Human P-gp was apically expressed, functional, stable through passages, and responsible for efflux of known substrates and identified compounds (atenolol, citalopram, mitoxantrone).
Conclusions:
- The IPEC-J2 MDR1 cell line effectively combines high paracellular tightness with high human P-gp expression.
- This cell line serves as a valuable tool for drug development, aiding in the screening of P-gp substrates and overcoming drug resistance.
