title: A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function.
abstract: Stably transfected MDCK/hPepT1-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein were established to quantify the relationship between transgene hPepT1 expression levels and its functional kinetics in facilitating peptide and peptide-like drug uptake and transport in vitro. The hPepT1 sequence was amplified from Caco-2 cell mRNA, inserted into the pcDNA3.1 -V5&His TOPO plasmid, and transfected into MDCK cells. Transgene protein levels were quantified by Western Blot analysis utilizing a standard curve generated with a positive control protein containing a V5&His epitope. Three clones expressing different levels of the hPepT1 fusion protein (low, medium, and high) were selected for the functional characterization with [14C]Gly-Sar and [3H]carnosine. The MDCK/hPepT1 cells expressed a novel hPepT1/epitope tag protein with an apparent molecular mass of 110 kDa. The [14C]Gly-Sar uptake in the transfected cells was sodium-independent and pH-dependent, demonstrating enhanced uptake, the rate of which increased significantly from the weakly to strongly expressing hPepT1 MDCK/hPepT1 -V5&His clones as compared to the mock cell line at pH 6.0. The uptake and permeability of [14C]Gly-Sar and [3H]carnosine demonstrated a direct correlation between the hPepT1 level of expression, uptake, and transport capabilities. Molecular and functional characterization of the MDCK/hPepT1-V5&His cell line confirmed a directly proportional relationship between Vmax and Papp versus the molar levels of hPepT1 transgene expression. This stably transfected hPepT1 cell line may serve as a useful in vitro model for screening and quantifying peptide and peptide-like drug transport as a function of hPepT1 expression in drug discovery.
authors.affiliation: Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854-8022, USA.
authors.forename: Dea
authors.initials: D
authors.lastname: Herrera-Ruiz
authors.forename: Teresa N
authors.initials: TN
authors.lastname: Faria
authors.forename: Rajinder K
authors.initials: RK
authors.lastname: Bhardwaj
authors.forename: Julita
authors.initials: J
authors.lastname: Timoszyk
authors.forename: Olafur S
authors.initials: OS
authors.lastname: Gudmundsson
authors.forename: Paul
authors.initials: P
authors.lastname: Moench
authors.forename: Doris A
authors.initials: DA
authors.lastname: Wall
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Smith
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Knipp
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D045744:Cell Line, Tumor; D017931:DNA Primers; D004151:Dipeptides / Q000378:metabolism; D004285:Dogs; D004337:Drug Carriers; D006801:Humans; D007668:Kidney; D008565:Membrane Proteins / Q000378:metabolism; D000074011:Peptide Transporter 1; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D027981:Symporters / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D017931:DNA Primers; D004151:Dipeptides; D004337:Drug Carriers; D008565:Membrane Proteins; D000074011:Peptide Transporter 1; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; D027981:Symporters