Two regions of simian virus 40 large T-antigen independently extend the life span of primary C57BL/6 mouse embryo fibroblasts and cooperate in immortalization.

Expression of the SV40 large T-antigen allows primary cells to escape senescence and thereby become immortalized. Immortalization occurs in two steps, extension of life span and acquisition of unlimited cell division potential. By following the increase in expression of a senescence-associated marker with increased cell passage, we show that C57Bl/6 mouse embryo fibroblast (B6MEF) cultures senesce by passage 4. Thus, the development of colonies from cultures transfected with T-antigen expressing constructs indicates extension of life span. Two T-antigen regions independently extended the life span of B6MEF. Expression of either a T-antigen consisting of amino acids 1-147 (T1-147) or a T-antigen consisting of amino acids 251-708 (T251-708) resulted in colony development. However, the colonies expressing these truncated T-antigens could not be expanded into cell lines efficiently. In contrast, coexpression of T1-147 and T251-708 produced colonies that could be expanded into cell lines as efficiently as could colonies expressing full-length T-antigen. Thus, the two regions of T-antigen contain analogous activities that are sufficient to extend cell life span; they cooperate to immortalize primary B6MEF; and they act in trans, indicating that the functions involved are independent.