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 numbe r 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 pr oliferate indefinitely; however, such cells are absolutely dependent u pon continued expression of T antigen for maintenance of growth; inact ivation 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)-tsA 58 transgenic mice. We show that these fibroblasts become dependent up on T antigen for maintenance of proliferation only when their normal m itotic life-span has elapsed and that the biological clock that limits the mitotic potential continues to function normally, even in cells e xpressing this immortalizing gene. Our results suggest that random acc umulation of cellular damage is unlikely to be the factor that limits fibroblast division but support the hypothesis that senescence is regu lated via a genetic program.