The three transforming regions of SV40 T antigen are required for immortalization of primary mouse embryo fibroblasts.
Simian virus 40 (SV40) is a small DNA tumor virus whose early region gene product, large T antigen, is sufficient to immortalize primary rodent cells and transform established rodent cell lines. Three functional domains of large T antigen are required for transformation of the rat embryo fibroblast REF 52 cell line: the extreme amino-terminal region, a domain which binds p105Rb family members, and the bipartite p53-binding region. Many studies have attempted to define the activities and regions of SV40 large T antigen required for immortalization of mouse embryo fibroblasts (MEFs). In most of these studies, investigators have used survival of T antigen-expressing primary MEF colonies at the time when controls MEFs undergo senescence as a measurement of 'immortalization' and concluded that immortalization of MEFs is correlated with large T antigen's ability to sequester the human tumor suppressor gene product p53 and separable from its p105Rb-binding or N terminal functions. In order to more rigorously define the regions of SV40 large T antigen required for escape from senescence, individual T antigen-expressing primary MEF colonies were systematically subcultured for > 60 population doublings beyond the time of control MEF senescence under conditions known to limit the number of spontaneously immortalized cells. We found that although interaction of T antigen with p53 was sufficient to substantially extend the lifespan of MEFs, all three SV40 large T antigen domains required for REF 52 transformation were necessary to immortalize primary MEFs. These results indicate that p53 inactivation alone is insufficient to immortalize primary MEFs; rather, immortalization requires multiple activities of T antigen which are also required for efficient transformation.
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Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
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D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D016922:Cellular Senescence / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D016159:Tumor Suppressor Protein p53