title: 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 G1 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 adenovirus-based 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 G1 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.
authors.affiliation: Department of Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan.
authors.forename: Takemi
authors.initials: T
authors.lastname: Kunieda
authors.forename: Naoya
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Masakiyo
authors.initials: M
authors.lastname: Sakaguchi
authors.forename: Teru
authors.initials: T
authors.lastname: Okitsu
authors.forename: Toshinori
authors.initials: T
authors.lastname: Totsugawa
authors.forename: Takamasa
authors.initials: T
authors.lastname: Watanabe
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Matsumura
authors.forename: Masanobu
authors.initials: M
authors.lastname: Maruyama
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Noguchi
authors.forename: Michihiko
authors.initials: M
authors.lastname: Takesue
authors.forename: Norikuni
authors.initials: N
authors.lastname: Shibata
authors.forename: Kenji
authors.initials: K
authors.lastname: Ohmoto
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Noriaki
authors.initials: N
authors.lastname: Tanaka
mesh_terms: D000418:Albumins / Q000032:analysis / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D002453:Cell Cycle; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D010641:Phenotype; D021381:Protein Transport; D014161:Transduction, Genetic
chemical_list: D000418:Albumins; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D015696:Gene Products, tat; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A