Development and characterization of LLC-PK1 cells containing Sprague-Dawley rat Abcb1a (Mdr1a): comparison of rat P-glycoprotein transport to human and mouse.
BACKGROUND
P-glycoprotein is localized in numerous tissues throughout the body and plays an important role in the disposition of many xenobiotics. The contribution of P-glycoprotein-mediated drug transport is being evaluated in early drug discovery stages, particularly for compounds targeted to the central nervous system, using in vitro tools including cell lines expressing P-glycoprotein. Previous work in our laboratory suggests there are species differences in P-glycoprotein transport activity between humans and animals. The rat Abcb1a form of P-glycoprotein (formerly known as Mdr1a), the predominate isoform in the brain, has not been described in a functional cell system. Here, we describe the development and characterization of LLC-PK1 cells expressing rat Abcb1.


METHODS
We cloned rat Abcb1a and generated a stable LLC-PK1 cell line. Expression and function of the cells were evaluated by immunoblot analysis, cytotoxicity analysis, cellular accumulation assays, and transcellular transport of probe substrates. The transport ratios of structurally diverse compounds obtained from parental cells or cells stably transfected with human ABCB1, mouse Abcb1a or rat Abcb1a were compared.


RESULTS
Two forms of rat Abcb1a were cloned from Sprague-Dawley cDNA that differ by six amino acids and a base pair deletion. The intact form was stably transfected in LLC-PK1 cells. Immunoblot analysis demonstrated expression of the protein. The cells demonstrated P-glycoprotein-mediated function by directional transport of dexamethasone, ritonavir, and vinblastine in a transwell assay that was inhibited in the presence of cyclosporin A, verapamil, or quinidine. Likewise, the cells showed reduced cellular accumulation of Rh123 by FACS analysis that was reversed in the presence of cyclosporin A. These cells showed >or=350-fold resistance to colchicine, doxorubicin, vinblastine, and taxol and were sensitized in the presence of verapamil or cyclosporin A. Of 179 chemically diverse compounds evaluated, approximately 20% of the compounds evaluated were predicted to be substrates in one species but not in other species.


CONCLUSIONS
Taken together, these data suggest these cells will be useful for evaluation of rat Abcb1a-mediated transport and for evaluation of species-specific P-glycoprotein-mediated transport.
Booth-Genthe
Catherine L
CL
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA. boothgenthe@verizon.net
Louie
Steven W
SW
Carlini
Edward J
EJ
Li
Bing
B
Leake
Brenda F
BF
Eisenhandler
Roy
R
Hochman
Jerome H
JH
Mei
Qin
Q
Kim
Richard B
RB
Rushmore
Thomas H
TH
Yamazaki
Masayo
M
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics* / Q000378:metabolism*; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006801:Humans; D018374:LLC-PK1 Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; C095122:multidrug resistance protein 3