Transduction of Immortalized Human Hepatocytes with p21 to Enhance Differentiated Phenotypes

Abstract

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G 1 phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirusbased p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immunofluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G 1 checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.

Strategies for immortalization of primary hepatocytes

“…In general, most of the generated hepatocyte-derived cell lines are not tumorigenic, but display reduced or only limited liver-specific functionality [7, 20, 102]. Taking into account that proliferation and differentiation are mutually exclusive in vitro, it has been shown that overexpression of the cdki, p21 and the use of conditional immortalization strategies can stimulate to some extent differentiation of the cells [6, 23, 84, 85, 102, 111, 116-120]. Other anti-dedifferentiation strategies developed to counteract the loss of functionality in primary hepatocyte cultures, including co-culture systems and overexpression of liver-specific genes have also proven usefull [121, 122].…”

Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination

“…Immunofluorescent staining for albumin was conducted with immortalized and reverted 16-T3 cells with a labeled anti-albumin antibody, as previously reported [19]. Staining with Oregon green phalloidin (Molecular Probes, Eugene, OR) and Hoechst dye (Sigma, Saint Louis, MI) was simultaneously performed for specific staining of actin filaments and nuclei, respectively.…”

“…The cells were regularly detached with trypsin and the number of viable cells counted by trypan blue exclusion test. The cell cycle status of both 16-T3 and reverted cells was analyzed by flow cytometry, as previously reported [19].…”

The newly established human hepatocyte cell line: application for the bioartificial liver

“…Chang et al [44] reported that a combination of HDAC and PPAR-gamma ligand therapy for non-small lung cancers led to an increase in p21, cyclin-dependent kinase inhibitor, compared to either therapy alone and was followed by the induction of differentiation. Kunieda et al [45] also reported that p21 was associated with cellular differentiation and senescence in immortalized hepatocytes and the induction of p21 results in the enhancement of albumin and P450 expression. The expression of p21 was up-regulated when terminal differentiation was observed coupled with cell cycle arrest [46][47][48].…”