RT Journal Article
SR Electronic
T1 Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 180
OP 188
DO 10.1124/dmd.115.066506
VO 44
IS 2
A1 King Leung Fung
A1 Khyati Kapoor
A1 Jessica N. Pixley
A1 Darrell J. Talbert
A1 Alexandra D.T. Kwit
A1 Suresh V. Ambudkar
A1 Michael M. Gottesman
YR 2016
UL http://dmd.aspetjournals.org/content/44/2/180.abstract
AB The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacologic evaluation of these proteins in vitro remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the BacMam baculovirus gene transfer system. The purpose of the study was to evaluate the efficacy of BacMam baculovirus to transduce cells grown in monolayers. In a porcine kidney cell line, LLC-PK1 cells, baculoviral transduction is successful only via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and preincubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [3H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, coexpression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp– or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system