Effect of Long-term Passage on Liver-specific Functions in Immortalized Hepatocytes with a Temperature-sensitive Mutant of SV40 T Antigen

Kim, Byung Ho; Seong, Se Ra; Lee, Woo In; Dong, Seok Ho; Kim, Hyo Jong; Chang, Young Woon; Lee, Joung Il; Chang, Rin

Abstract

BACKGROUND/AIMS: The transcription of liver specific genes decreases dramatically in primary hepatocyte culture. In this study, we evaluated the long-term differentiation of immortalized hepatocytes.
METHODS
Immortalized rat hepatocytes were established with a temperature-sensitive mutant of SV40 T antigen and were cultured until they divided more than 200 doublings. We measured the change of various differentiations and investigated its mechanism.
RESULTS
The epithelial cell morphology was not changed, but their size were decreased about 20% in the later passage, in which chromosomal changes were marked. In the early passage, albumin secretion and urea synthesis were reduced to 1/6 and 1/5 level of primary hepatocytes, respectively, and they were decreased to the undetectable level in the later passage. The expression of T antigen was increased about 29 times in the later passage. Telomerase activity was progressively increased according to the passage. In immortalized hepatocytes the mean telomere length was not changed, but the length variation increased.
CONCLUSIONS
The differentiation of immortalized hepatocytes disappears after long-term passage possibly due to marked chromosomal changes that occur with compulsive and continuous replications by the increased expression of T antigen which inhibits sequentially the function of p53.