THE BIOLOGICAL CLOCK THAT MEASURES THE MITOTIC LIFE-SPAN OF MOUSE EMBRYO FIBROBLASTS CONTINUES TO FUNCTION IN THE PRESENCE OF SIMIAN-VIRUS-40 LARGE TUMOR-ANTIGEN

Normal mammalian fibroblasts cultured in vitro undergo a limited number of divisions before entering a senescent phase In which they can be maintained for long periods but cannot be induced to divide. In rodent fibroblasts senescence can be prevented by expression of sinian virus 40 large tumor antigen (T antigen). Cells expressing T antigen can proliferate indefinitely; however, such cells are absolutely dependent upon continued expression of T antigen for maintenance of growth; inactivation of T antigen results in a rapid and irreversible entry into a postmitotic state. To determine when, after the initial expression of T antigen, fibroblasts become dependent upon it for continued growth, we serially cultivated embryonic fibroblasts prepared from H-2K(b)-tsA58 transgenic mice. We show that these fibroblasts become dependent upon T antigen for maintenance of proliferation only when their normal mitotic life-span has elapsed and that the biological clock that limits the mitotic potential continues to function normally, even in cells expressing this immortalizing gene. Our results suggest that random accumulation of cellular damage is unlikely to be the factor that limits fibroblast division but support the hypothesis that senescence is regulated via a genetic program.