Abstract
The rat hepatocytes were immortalized using a temperature-sensitive mutant of SV40 large T antigen (tsT) to develop as a possible substitute for primary hepatocytes. Four rat hepatocyte lines that have been developed and maintained more than passage 50, were characterized for their cellular morphology, T antigen and p53 expression, chromosomes, liver-specific differentiation, telomerase activity and anchorage independent growth. All of four cell lines showed a typical epithelial cell morphology, but the population-doubling time became short with passage: 18 to 60%. T antigen expression was increased with passage about 3 to 65 times at permissive temperature but decreased significantly at non-permissive temperature. The expression level of p53 unchanged during passages was also decreased at non-permissive temperature. The distribution of chromosome number changed somewhat with passage. The production levels of albumin and urea in four cell lines were 2.4 to 13.0% and 7.5 to 19.9% of those produced in primary hepatocytes, respectively and were decreased to an undetectable level with passage. Telomerase activity was increased 10 fold following immortalization of cells, but anchorage independent growth of cells did not develop. These results indicate that conditionally immortalized hepatocytes become dedifferentiated with in vitro passage, which may be caused by marked chromosomal damages that occur with compulsive and continuous replications by the increment of T antigen content with passage and its sequential inhibition of p53 function.
Highlights
Primary hepatocytes have extensively been used to investigate mechanisms of cell growth and differentiation, metabolic processes, biotransformation and genotoxicity of drug, carcinogenesis, and ex vivo gene therapy
Transplantation of hepatocytes has been applied in clinical trials for bioartificial liver
The transcription ability of liver specific genes decreases dramatically when hepatocytes are removed from an animal and cultured in vitro (Clayton et al, 1983; Guguen-Guillouzo et al, 1983a)
Summary
Primary hepatocytes have extensively been used to investigate mechanisms of cell growth and differentiation, metabolic processes, biotransformation and genotoxicity of drug, carcinogenesis, and ex vivo gene therapy. The transcription ability of liver specific genes decreases dramatically when hepatocytes are removed from an animal and cultured in vitro (Clayton et al, 1983; Guguen-Guillouzo et al, 1983a). Such rapid dedifferentiation was partially overcome by culturing the cells in a hormonally defined medium on an extracellular matrix (Rojkind et al, 1980; Dich et al, 1988; Waxman et al, 1990) or coculturing them with non-parenchymal liver cells (Guguen-Guillouzo et al, 1983b; Morin et al, 1986). We report the change of liver-specific differentiation in 4 CIH lines that were established from two rat strains and describe a possible mechanism of differentiation change with in vitro passage
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