High expression of human CYP2C in immortalized human liver epithelial cells

Cell lints stably expressing high levels of single isozymes of human CYP2C genes (CYP2C8, CYP2C9. CYP2C18 and CYP2C19) have been successfully generated by transfecting liver epithelial human cells (THLE) with an appropriate expression vector. To this aim, cDNAs encoding for each CYP2C gene;were inserted by blunt-ended cloning into the unique insertion site of the singular expression vector pCMVneo. The recombinant pCMV2C8, pCMV2C9. pCMV2C18 and pCMV2C19 vectors were liposome-mediated transfected into THLE cells. The resulting transgenic cells, designated as T5-2C8. T5-2C9, T5-2C18 and T5-2C19, were cloned and the expression of the ectopic gene, mRNA and protein, was investigated by RT-PCR and Western blot analysis. The functionality of each expressed CYP2C was assessed by determining specific catalytic activities in these cells. that is, taxol-6-hydroxylation for CYP2C8; diclofenac-4'-hydroxylation for CYP2C9; S-mephenytoin-4'-hydroxylation for CYP2C18; S-mephenytoin-4'-hydroxylation for CYP2C19. As a result of the combined strategies used here, the transfected cells showed activities four to seven times higher than those of 24-hour cultured hepatocytes. (C) 1999 Elsevier Science Ltd. All rights reserved.