Immortalized hepatocytes established with a temperature-sensitive SV40 T antigen could be independent of T antigen

Dong, Seok Ho; Kim, Byung Ho; Nam, Gi Deog; Jang, Jae Young; Kim, Nam Hoon; Lee, Sang Kil; Joo, Kwang Ro; Kim, Hyo Jong; Chang, Young Woon; Lee, Joung Il; Chang, Rin

Abstract

BACKGROUND: Conditionally immortalized hepatocytes (CIH) can be cultured almost indefinitely at permissive temperatures (33 degrees C), but they undergo apoptosis at nonpermissive temperatures (37~39 degrees C) by the release of p53 through inactivation of T antigen, which is called T antigen dependency. This study was aimed at examining if T antigen-independent clones can develop from CIH.
METHODS
CIH established with a temperature-sensitive T antigen (WA1) were cultured continuously at 39 degrees C. Three clones (W39B, W39C, and W39J) survived at this temperature and was subject to following analyses: the morphology, growth, apoptosis, the expression of T antigen and p53, telomerase, and the T antigen gene sequence.
RESULTS
WA1 proliferated at 33 degrees C with the population doubling time of 30.8 +/- 1.7 hours, but they underwent cell death at 39 degrees C. However, T antigen-independent clones (W39B, W39C, and W39C) proliferated at 39 degrees C without undergoing apoptosis, suggesting they lost the temperature-sensitive characteristics. WA1 expressed the T antigen at 33 degrees C, but not at 39 degrees C, and this temperature-sensitive pattern was maintained in T antigen-independent clones. In p53 expression, however, T antigen-independent clones revealed a different pattern. p53 was detected even at 39 degrees C where it normally would not be detected. Telomerase was activated in all the analyzed cell lines. A temperature-sensitive point mutation at nucleotide position 3505 of the WA1 was retained in all T antigen-independent clones.
CONCLUSION
CIH can lost temperature-sensitive characteristics and acquire an ability to proliferate at nonpermissive temperatures. These changes might be related to the change of p53 rather than the change of T antigen itself in these cell lines.