2 REGIONS OF SIMIAN-VIRUS-40 LARGE T-ANTIGEN INDEPENDENTLY EXTEND THELIFE-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 di vision potential. By following the increase in expression of a senesce nce-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 e ither a T-antigen consisting of amino acids 1-147 (T1-147) or a T-anti gen consisting of amino acids 251-708 (T251-708) resulted in colony de velopment. However, the colonies expressing these truncated T-antigens could not be expanded into cell lines efficiently In contrast, coexpr ession of T1-147 and T251-708 produced colonies that could be expanded into cell lines as efficiently as could colonies expressing full-leng th T-antigen. Thus, the two regions of T-antigen contain analogous act ivities that are sufficient to extend cell life span; they cooperate t o immortalize primary B6MEF; and they act in trans, indicating that th e functions involved are independent. (C) 1998 Academic Press.