Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.
Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 μM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.
Sigma-Aldrich Corporation, St. Louis, Missouri.
Kathleen E
KE
Sampson
Sigma-Aldrich Corporation, St. Louis, Missouri.
Amanda
A
Brinker
Sigma-Aldrich Corporation, St. Louis, Missouri.
Jennifer
J
Pratt
Sigma-Aldrich Corporation, St. Louis, Missouri.
Neetu
N
Venkatraman
Sigma-Aldrich Corporation, St. Louis, Missouri.
Yongling
Y
Xiao
Sigma-Aldrich Corporation, St. Louis, Missouri.
Jim
J
Blasberg
Sigma-Aldrich Corporation, St. Louis, Missouri.
Toni
T
Steiner
Sigma-Aldrich Corporation, St. Louis, Missouri.
Maureen
M
Bourner
Sigma-Aldrich Corporation, St. Louis, Missouri david.thompson@sial.com.
David C
DC
Thompson
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000187:drug effects; D002999:Clone Cells; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004347:Drug Interactions; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006801:Humans; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015262:Xenobiotics / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology
D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D015262:Xenobiotics; C513055:ABCB1 protein, human; C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human