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The Escape of Temperature-Sensitive T Antigen Immortalized Rat Hepatocytes From Conditional Immortalization

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Abstract:

Conditionally immortalized hepatocytes (CIH) established with a gene for the temperature-sensitive mutant of the T antigen (tsT) have characteristics to stop proliferating and to differentiate at nonpermissive temperatures (37–39°C) due to inactivation of the T antigen. Therefore, they may be a good alternative to primary hepatocytes for experimental investigations or clinical applications. Deinduction of the T antigen results in a transient increase of p53 in these cells, leading to reexpression of normal senescence because of the telomere attrition occurring during the early stages of immortalization. To determine this T antigen dependency for the maintenance of immortality, a type of rat CIH was cultured continuously at 39°C. The frequency of occurrence of T-antigen-independent clones ranged from 0.053% to 0.093%. These clones maintained the temperature-sensitive property of the T antigen; nevertheless, they were able to progress to the S phase and proliferate without undergoing apoptosis at 39°C as at 33°C, a permissive temperature. The temperature-sensitive point mutation of tsT was not affected in these clones and the T antigen was functioning properly. The integrity of the p53 pathway was also maintained from the point of Western blot analysis of p21. Although the telomerase continued to be expressed and the telomere length was maintained, marked chromosomal damage could not be avoided in these cells. It is a plausible explanation that this escape phenomenon from conditional immortalization may be related to the change of other genes involved in cell cycles, which have yet to be elucidated. In conclusion, CIH could lose their temperature-sensitive characteristics without the change of tsT, itself, and the T antigen is not always necessary to maintain their immortality. Therefore, the results obtained from experimental investigations using these cells should be interpreted carefully, and unpredictable phenotypic changes should also be taken into consideration when using them in clinical applications.

Keywords: Cell growth; Hepatocytes; Immortalization; T antigen independence; Temperature-sensitive SV 40 large T antigen

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000005783982864

Affiliations: 1: Department of Internal Medicine, Kyung Hee University College of Medicine, Seoul 130-702, Korea 2: Preventive Medicine, Kyung Hee University College of Medicine, Seoul 130-702, Korea 3: Department of Biochemistry, Ajou Universtiy School of Medicine, Suwon 447-749, Korea

Publication date: July 1, 2005

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.

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