Comparison of Function and Relative Transporter Protein Concentrations in Caco-2 Cells with Single and Double Knockouts of the ABCB1, ABCG2, and ABCC2 Genes

By: Emile G. Plise, Jonathan Cheong, and Laurent Salphati, Drug Metabolism and Pharmacokinetics, Genentech, Inc. South San Francisco, CA


Compare the MDR1, BCRP, and MRP2 transporter function and their relative protein expression levels in Caco-2 wild-type versus single and double knockout cells.


Caco-2 wild-type (wt) cells and cells with single (KO) and/or double (DKO) knockouts for the ABCB1 (MDR1), ABCG2 (BCRP), and ABCC2 (MRP2) genes were purchased from Sigma-Aldrich (St. Louis, MO) in pre-cultured 24-well plates. Cells were dosed with 2.5 µM of prototypical probe substrates [digoxin (MDR1), topotecan (MDR1/BCRP) and vinblastine (MDR1/MRP2)] in HBSS containing 100 µM lucifer yellow. Samples were taken at 45, 90, and 135 minutes and were analyzed on an AB Sciex Qtrap 5500. A ProteoExtract kit was used to extract plasma membrane proteins from cells with the same passage. The proteins were reduced (DTT), alkylated (iodoacytamide) and digested with trypsin (24 h at 37°C). Signature peptide fragments and heavy peptides were used for calibration standards and internal standards, respectively. Peptide samples were analyzed on an Agilent 6460A MS in MRM mode.


The endogenous transporter expression levels of MDR1, BCRP, and MRP2 in the Caco-2 wt cells were 0.48, 0.91, and 0.83 fmol/µg of plasma membrane protein, respectively.  All three substrates exhibited B to A / A to B ratios >14 in the wild-type cells. In the MDR1 KO cells the ratios for the substrates markedly decreased [digoxin (1.3), topotecan (3.8), and vinblastine (2.9)] with ratios >1 suggesting BCRP (topotecan) or MRP2 (vinblastine) involvement. The transporter protein levels for all deleted genes were BLOQ and it appears the knockouts did not cause compensatory changes in the endogenous expression of the transporters examined.


The endogenous levels of MDR1, BCRP, and MRP2 in the Caco-2 wt cells are sufficient to transport substrates. The function, protein levels, and the specific gene deletion of these transporters have been confirmed in both the KO and DKO cells. These Caco-2 cell lines could prove to be a valuable tool to study the transport of compounds without the use of non-specific or multiple MDR1, BCRP, and MRP2 inhibitors.

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