title: Selective downregulation of the MDR1 gene product in Caco-2 cells by stable transfection to prove its relevance in secretory drug transport.
abstract: Considerable interest is focused on overcoming multidrug resistance (MDR) in cancer chemotherapy. The in vitro experiments to characterize P-glycoprotein's (P-gp) function and to decrease its effects have led to a variety of strategies such as addition of competitors or supplementation of the medium with oligonucleotides complementary to the 5'-end of the MDR1-mRNA. For the Caco-2 cell line, an in vitro model for absorption screening, expressing multiple transporters including P-gp, which pumps substances back into the apical solution, P-gp activity might mask other relevant transport proteins' activity. The objective of the present study was to construct a Caco-2 subline with reduced P-gp expression level. Caco-2 cells were transfected by electroporation with two different mammalian expression vectors, and the obtained subclones were investigated at RNA (Northern blotting, RT-PCR), protein (FACS analysis), and functional (transport studies) levels for reduction in P-gp expression. Northern blotting showed that the levels of transcription of the inserted gene were different among the several clones, but those results did not completely correlate with the FACS analysis for P-gp expression. The clones with the strongest reduction in P-gp expression detected by the FACS analysis also showed the lowest secretory fluxes of the P-gp substrate talinolol in transport studies. Repetition of FACS analysis after 7 and 24 months on 20 to 30 passage older subclones still showed reduction in P-gp expression and indicated that they are stably transfected. The new cell lines constructed in the present study provide the possibility to perform in vitro absorption studies in a cell system composed of differentiated enterocytes growing as a monolayer like the normal Caco-2 cell line but with a lower down to almost lacking expression of P-gp.
authors.affiliation: School of Pharmacy, Martin Luther University, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle/Saale, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Hilgendorf
authors.forename: Hilde
authors.initials: H
authors.lastname: Spahn-Langguth
authors.forename: Magdalena
authors.initials: M
authors.lastname: Rhedin
authors.forename: Carl-Gunnar
authors.initials: CG
authors.lastname: Regårdh
authors.forename: Björn
authors.initials: B
authors.lastname: Löwenadler
authors.forename: Peter
authors.initials: P
authors.lastname: Langguth
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism* / Q000493:pharmacokinetics*; D001692:Biological Transport; D018938:Caco-2 Cells; D002999:Clone Cells; D015536:Down-Regulation; D005434:Flow Cytometry; D019450:Genes, MDR; D006801:Humans; D016254:Mutagenesis, Insertional; D010539:Permeability / Q000187:drug effects; D011412:Propanolamines / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D011412:Propanolamines; C011550:talinolol; D014700:Verapamil