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Conditional immortalization of gunn rat hepatocytes: An ex vivo model for evaluating methods for bilirubin‐UDP‐glucuronosyltransferase gene transfer
Author(s) -
Fox Ira J.,
Chowdhury Namita Roy,
Gupta Sanjeev,
Kondapalli Ravi,
Schilsky Michael L.,
Stockert Richard J.,
Chowdhury Jayanta Roy
Publication year - 1995
Publication title -
hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.488
H-Index - 361
eISSN - 1527-3350
pISSN - 0270-9139
DOI - 10.1002/hep.1840210334
Subject(s) - hepatocyte , microbiology and biotechnology , immortalised cell line , biology , cell culture , chemistry , biochemistry , cell , in vitro , genetics
Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)‐mediated endocytosis are being developed to transfer genes for the correction of bilirubin‐UDP‐glucuronosyltransferase (bilirubin‐UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin‐UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33° C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone‐UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione‐S‐transferase Y p (GST‐Y p ), an oncofetal protein, was expressed in these cells at 33° C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39° C or 37° C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone‐UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST‐Y p expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red‐labeled asialoorosomucoid, and binding and degradation of 125 I‐asialoorosomucoid. After liposome‐mediated transfer of a plasmid containing the coding region of human bilirubin‐UGT 1 , driven by the SV40 large T promoter, active human bilirubin‐UGT 1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful ex vivo evaluation of builirubin‐UGT gene transfer vectors.