Improvement in the differentiated hepatic phenotype of immortalized human hepatocytes by adenovirus mediated p21 gene transfer.

Abstract

The p21 molecule, a potent cyclin dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle and is involved in terminal cellular differentiation. The overexpression of p21 has been shown to induce differentiation in various cell lines. We have made an effort to establish a reliable human hepatocyte cell line as a source of hepatic function in bioartificial liver (BAL) therapy. In this work, we investigated the effect of p21 on the differential phenotype of simian virus 40 large T antigen (SV40Tag) immortalized human hepatocytic NKNT-3 cells. A recombinant adenoviral vector expressing a p21 gene under control of the cytomegalovirus (CMV) promoter (Ad-p21) was used to efficiently transfer genes into NKNT-3 cells. The morphologic alterations, the cell cycle progression, and the expression of p-450 associated enzymes (CYPs) were carefully examined in NKNT-3 cells that had been infected with Ad-p21. Adenovirus mediated gene delivery of p21 was efficiently achieved in NKNT-3 cells without affecting cellular structure. After Ad-p21 infection, NKNT-3 cells were G0/G1 arrested in cell cycle analysis. NKNT-3 cells that had been infected with Ad-p21 showed differentiated hepatic phenotypes in morphology and improvement in protein expression of CYP 3A4 and CYP 2C9. In the present work, we demonstrate that the exogenous expression of p21 enhances the differential phenotype of immortalized hepatocytic NKNT-3 cells.