Exp Mol Med.  2000 Mar;32(1):29-37.

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

Affiliations
  • 1Department of Internal Medicine, Kyung Hee University College of Medicine, Seoul, Korea. kimbh@khmc.or.kr

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.

Keyword

chromosome; conditional immortalization; liverspecific differentiation; Rat hepatocytes; temperature-sensitive SV40 large T antigen; telomerase

MeSH Terms

Animal
Antigens, Polyomavirus Transforming/biosynthesis
Cell Adhesion
Cell Differentiation
Cell Division
Cell Line, Transformed
Cell Transformation, Viral*
Chromosome Aberrations
Liver/cytology*
Protein p53/metabolism
Rats
Telomerase/metabolism
Time Factors
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