RAT EMBRYO FIBROBLASTS IMMORTALIZED WITH SIMIAN-VIRUS-40 LARGE T-ANTIGEN UNDERGO SENESCENCE UPON ITS INACTIVATION

Introduction of simian virus 40 T antigen into rodent fibroblasts give s rise to cells that can proliferate indefinitely but are dependent up on it for maintenance of their growth once the normal mitotic life spa n has elapsed. Inactivation of T antigen in these immortalized cells c auses rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the fo llowing parallels between the tsa cells after inactivation of T antige n and senescent rat embryo fibroblasts: (i) growth arrest is irreversi ble; (ii) it occurs in G(1) as well as G(2); (iii) the G(1) block can be partially overcome by stimulation with 20% fetal calf serum, but th e G(2) block cannot be overcome; (iv) 20% fetal calf serum induces c-f os, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1 ) are upregulated upon growth arrest. These results suggest that T-ant igen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells . Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.