Activity and inducibility of drug-metabolizing enzymes in immortalized hepatocyte-like cells (mhPKT) derived from a L-PK/Tag1 transgenic mouse.
This report describes the establishment and characterization of the mhPKT cell line derived from the liver of a transgenic mouse harboring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence of the rat L-type pyruvate kinase (L-PK) gene. mhPKT cells had a prolonged life span, expressed the SV40-encoded nuclear large T antigen when grown in glucose-enriched medium, and induced tumors when injected subcutaneously into athymic (nu-nu) mice. Growth on petri dishes or filters yielded multiple layers of cuboid cells, with numerous spaces between adjacent cells that were closed by junctional complexes. These bile canaliculi-like structures exhibited numerous microvilli in which villin, an actin-binding brush-border protein, colocalized with actin. These bile canaliculi-like structures appeared to be functional as they accumulated fluorescein. mhPKT cells conserved the expression of the liver-specific transcription factors HNF1, HNF3, HNF4, and DBP together with substantial levels of L-PK and albumin but not alpha-fetoprotein mRNA transcripts. mhPKT cells mainly metabolized testosterone into androstenedione and 6beta-hydroxytestosterone, as in vivo. 3-Methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) markedly increased ethoxyresorufin-O-deethylase activity and the related cytochrome P450 (CYP) 1A1/2 protein, whereas alpha-naphtoflavone antagonized the TCDD-elicited induction. Phenobarbital slightly increased the CYP2B-mediated activities of pentoxyresorufin-O-depentylase, 2beta- and 16beta-testosterone hydroxylase. mhPKT cells also had substantial sulfotransferase, UDP-glucuronyltransferase, and glutathione S-transferase activities. This model may serve as a tool for long-term in vitro studies of xenobiotic metabolism, potent CYP inducers, and hepatocyte damage due to drugs and other factors.
Courjault-Gautier
F
F
Département Sécurité du Médicament, Centre de Recherche de Vitry-Alfortville, Rhône-Poulenc Rorer SA, Vitry-sur Seine, France.
Antoine
B
B
Bens
M
M
Vallet
V
V
Cluzeaud
F
F
Pringault
E
E
Kahn
A
A
Toutain
H
H
Vandewalle
A
A
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D001648:Bile Canaliculi; D002241:Carbohydrates; D002461:Cell Line, Transformed / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004032:Diet; D015870:Gene Expression; D005978:Glutathione / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013539:Simian virus 40; D013739:Testosterone / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D002241:Carbohydrates; D004364:Pharmaceutical Preparations; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D014157:Transcription Factors; D013739:Testosterone; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D011770:Pyruvate Kinase; D005978:Glutathione