Dedifferentiation of conditionally immortalized hepatocytes with long-term in vitro passage

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, liverspecific 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.

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

“…A maximum of 178 ng cm -2 h -1 was achieved corresponding to a cell specific productivity of 17 pg d -1 calculated on the basis that 5 · 10 6 cells formed a confluent monolayer in culture dishes of 20 cm 2 surface. Hence, the cellspecific albumin secretion rate of porcine hepatocytes cultured in dishes was lower than reported for the respective cells cultivated in an in vivo-like microenvironment supported by a flat membrane bioreactor and the 30 pg d -1 secreted by primary rat hepatocytes (Kim et al 2000). The maximum productivity of the hepatocytes grown under DG conditions was up to 2.6-fold more compared to those cells grown under single gel mode where the secretion potential could be maintained at a nearly constant level of 70 ng cm -2 h -1 for 16 days.…”

Tumor-initiating stem cells in liver cancer

“…Hepatocytes were immortalized and dedifferentiated using a temperature-sensitive mutant of SV40 large T antigen. 79 Moreover, cell matrix interaction may determine and maintain the differentiated phenotype of hepatocytes by regulating hepatic genes and liver transcription factors. 55 Hepatocyte cultures dedifferentiate rapidly in vitro, resulting not only in decreased liver-specific functions, but also in dedifferentiated morphology: the cells flatten, depolarize and lose many of the surface characteristics of normal hepatocytes in vivo.…”

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells

“…To determine the T antigen content, the cells were cultured at a permissive (33 o C) and a non-permissive temperature (39 o C) for 2 days and Western blot analysis was performed (Kim et al, 2000). COS-7 cells and normal RPE cells were used as positive and negative controls, respectively.…”