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Hepatocyte‐like mhAT3F cells have been derived from the hepatoma of a transgenic mouse expressing the SV40 large T antigen under the control of the antithrombin III gene regulatory region (Antoine, B., Levrat, F., Vallet, V., Berbar, T., Cartier, N., Dubois, N., Briand, P., and Kahn, A. (1992) Gene expression in hepatocyte‐like lines established by targeted carcinogenesis in transgenic mice. Exp. Cell. Res.200, 175–185; F. Levrat et al., unpublished results). In these cells, the l‐PK gene is transcriptionally activated by glucose, as it is in vivo and in cultured hepatocytes. However, in contrast to the l‐PK gene regulation in the liver and isolated hepatocytes, the glucose responsiveness does not require insulin and is not blocked by cyclic AMP. In mhAT3F cells, the insensitivity to insulin might be due to the replacement of insulin‐dependent glucokinase by insulin‐independent hexokinases able to phosphorylate glucose in the absence of the hormone. The glucose‐dependent activation of the l‐PK gene is delayed, requires ongoing protein synthesis, and is mediated by the same glucose response element as in vivo and in isolated hepatocytes. These results suggest that the glucose‐dependent signaling pathway responsible for the transcriptional activation of glycolytic and lipogenic genes requires glucose phosphorylation, a phenomenon that is insulin‐dependent in the liver but insulin‐independent in cultured hepatoma cells. Nevertheless, the action of glucose 6‐phosphate is most likely indirect.— Lefrançois‐Martinez, A.‐M., Diaz‐Guerra, M.‐J. M., Vallet, V., Kahn, A., Antoine, B. Glucose‐dependent regulation of the L‐pyruvate kinase gene in a hepatoma cell line is independent of insulin and cyclic AMP. FASEB J.8: 89‐96; 1994.

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 ratl-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 ofl-PK and albumin but not α-fetoprotein mRNA transcripts. mhPKT cells mainly metabolized testosterone into androstenedione and 6β-hydroxytestosterone, asin 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 α-naphtoflavone antagonized the TCDD-elicited induction. Phenobarbital slightly increased the CYP2B-mediated activities of pentoxyresorufin-O-depentylase, 2β- and 16β-testosterone hydroxylase. mhPKT cells also had substantial sulfotransferase, UDP-glucuronyltransferase, and glutathioneS-transferase activities. This model may serve as a tool for long-termin vitrostudies of xenobiotic metabolism, potent CYP inducers, and hepatocyte damage due to drugs and other factors.

We have described new well-differentiated mouse hepatocyte-like cell lines (mhAT) derived from transgenic mice expressing simian virus 40 large T antigen under the control of antithrombin III gene promoter (Exp. Cell Res. (1992) 200, 175-185). In an attempt to understand the phenotypic variations of the different cell lines, we analyzed their content in liver-specific transcription factors at the level of both the proteins, by gel shift analysis, and the mRNA, by quantitative reverse-transcription PCR. Moreover, the transactivating ability of endogenous HNF1α and C/EBPα was also evaluated by measuring the activity of transfected synthetic promoters consisting of DNA element homopolymers upstream of a TATA box. High levels of HNF1, HNF3, and HNF4 transcription factors were maintained in mhAT cells. In contrast, C/EBPα was much more variable between the different cell lines and was less abundant than it was in vivo, in the liver. We investigated the influence of HNF1α and C/EBPα on the activity of transfected liver-specific promoters. HNF1α was not limiting for the activity of transfected liver-type pyruvate kinase and albumin promoters. In contrast, the activity of the albumin promoter in the different lines was clearly dependent on the C/EBPα content, which seems, therefore, to be an essential factor modulating the expression of this gene in HNF1α-containing cells. This work shows that the correlations between promoter activities and transacting factor contents in a panel of well-differentiated cultured cells can be used to determine the respective role of transcription factors on the strength of some promoters. Copyright 1993, 1999 Academic Press