C L Cummins1, L M Mangravite, L Z Benet. 1. Department of Biopharmaceutical Sciences, University of California, San Francisco 94143, USA.
Abstract
PURPOSE: 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.
PURPOSE: 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. Midazolam1-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: P Anderle; E Niederer; W Rubas; C Hilgendorf; H Spahn-Langguth; H Wunderli-Allenspach; H P Merkle; P Langguth Journal: J Pharm Sci Date: 1998-06 Impact factor: 3.534
Authors: M J Flens; G J Zaman; P van der Valk; M A Izquierdo; A B Schroeijers; G L Scheffer; P van der Groep; M de Haas; C J Meijer; R J Scheper Journal: Am J Pathol Date: 1996-04 Impact factor: 4.307
Authors: M J Flens; M A Izquierdo; G L Scheffer; J M Fritz; C J Meijer; R J Scheper; G J Zaman Journal: Cancer Res Date: 1994-09-01 Impact factor: 12.701