title: Using the BacMam Baculovirus System to Study Expression and Function of Recombinant Efflux Drug Transporters in Polarized Epithelial Cell Monolayers.
abstract: 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 [(3)H]-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.
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland.
authors.forename: King Leung
authors.initials: KL
authors.lastname: Fung
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland.
authors.forename: Khyati
authors.initials: K
authors.lastname: Kapoor
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland.
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Pixley
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland.
authors.forename: Darrell J
authors.initials: DJ
authors.lastname: Talbert
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland.
authors.forename: Alexandra D T
authors.initials: AD
authors.lastname: Kwit
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.affiliation: Laboratory of Cell Biology, National Institutes of Health, National Cancer Institute, Bethesda, Maryland mgottesman@nih.gov.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Gottesman
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D018938:Caco-2 Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D018374:LLC-PK1 Cells; D061985:Madin Darby Canine Kidney Cells; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008942:Mitoxantrone / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism*; D013552:Swine
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D026901:Membrane Transport Proteins; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D008942:Mitoxantrone