Conditional immortalization of gunn rat hepatocytes

An ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer

Ira J. Fox, Namita Roy Chowdhury, Sanjeev Gupta, Ravi Kondapalli, Michael L. Schilsky, Richard J. Stockert, Jayanta Roy-Chowdhury

Research output: Contribution to journalArticle

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Abstract

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 Yp(GST-Yp), 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-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 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 for ex vivo evaluation of bilirubin-UGT gene transfer vectors.

Original languageEnglish (US)
Pages (from-to)837-846
Number of pages10
JournalHepatology
Volume21
Issue number3
DOIs
StatePublished - 1995

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bilirubin glucuronoside glucuronosyltransferase
Gunn Rats
Asialoglycoprotein Receptor
Hepatocytes
Bilirubin
Albumin Receptors
Viral Tumor Antigens
Genes
Androsterone
Polyomavirus Transforming Antigens
Glutathione Transferase
Moloney murine leukemia virus
Severe Combined Immunodeficiency
DNA
Viral Proteins
Endocytosis
Liposomes
Cultured Cells
Plasmids

ASJC Scopus subject areas

  • Hepatology

Cite this

Conditional immortalization of gunn rat hepatocytes : An ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer. / Fox, Ira J.; Roy Chowdhury, Namita; Gupta, Sanjeev; Kondapalli, Ravi; Schilsky, Michael L.; Stockert, Richard J.; Roy-Chowdhury, Jayanta.

In: Hepatology, Vol. 21, No. 3, 1995, p. 837-846.

Research output: Contribution to journalArticle

Fox, Ira J. ; Roy Chowdhury, Namita ; Gupta, Sanjeev ; Kondapalli, Ravi ; Schilsky, Michael L. ; Stockert, Richard J. ; Roy-Chowdhury, Jayanta. / Conditional immortalization of gunn rat hepatocytes : An ex vivo model for evaluating methods for bilirubin-UDP-glucuronosyltransferase gene transfer. In: Hepatology. 1995 ; Vol. 21, No. 3. pp. 837-846.
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abstract = "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 Yp(GST-Yp), 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-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 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 for ex vivo evaluation of bilirubin-UGT gene transfer vectors.",
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