title: Characterizing the expression of CYP3A4 and efflux transporters (P-gp, MRP1, and MRP2) in CYP3A4-transfected Caco-2 cells after induction with sodium butyrate and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.
abstract: OBJECTIVE
  To examine the changes in expression levels of CYP3A4 and efflux transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the presence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol-13-acetate (TPA). To characterize the transport of [3H]-digoxin and the metabolism of midazolam in the cells under different inducing conditions.
  
  
  METHODS
  CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were grown for 13-14 days. Transport and metabolism studies were performed on cells induced with NaB and/or TPA for 24 h. The expression and localization of P-gp, MRP1, MRP2, and CYP3A4 were examined by Western blot and confocal microscopy.
  
  
  RESULTS
  In the presence of both inducers, CYP3A4 protein levels were increased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, and P-gp and MRP1 expression were unchanged. Midazolam 1-OH formation exhibited a rank order correlation with increased CYP3A4 protein, whereas [3H]-digoxin transport (a measure of P-gp activity) was unchanged with induction. P-gp and MRP2 were found on the apical membrane, whereas MRP1 was found perinuclear within the cell. CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferential polarization towards the apical side of the cell.
  
  
  CONCLUSIONS
  The present study characterized CYP3A4-Caco-2 cell monolayers when induced for 24 h in the presence of both NaB and TPA. These conditions provide intact cells with significant CYP3A4 and P-gp expression suitable for the concurrent study of transport and metabolism.
authors.affiliation: Department of Biopharmaceutical Sciences, University of California, San Francisco 94143, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Cummins
authors.forename: L M
authors.initials: LM
authors.lastname: Mangravite
authors.forename: L Z
authors.initials: LZ
authors.lastname: Benet
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology*; D018938:Caco-2 Cells; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D003720:Densitometry; D004790:Enzyme Induction / Q000187:drug effects; D018757:GABA Modulators / Q000378:metabolism; D006801:Humans; D007408:Intestinal Absorption / Q000235:genetics; D018613:Microscopy, Confocal; D008874:Midazolam / Q000378:metabolism; D024101:Mitochondrial Proteins; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D012269:Ribosomal Proteins / Q000096:biosynthesis*; D029701:Saccharomyces cerevisiae Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002087:Butyrates; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System; D004268:DNA-Binding Proteins; D018757:GABA Modulators; D008874:Midazolam; D024101:Mitochondrial Proteins; D006899:Mixed Function Oxygenases; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D013755:Tetradecanoylphorbol Acetate; C093947:MRP2 protein, S cerevisiae; C078444:MSH3 protein, human; C104464:CYP3A protein, human; C510163:CYP3A4 protein, human; C421109:multidrug resistance-associated protein 1