Reexpression of the retinoblastoma protein in tumor cells induces senescence and telomerase inhibition

Normal human diploid cells senesce in vitro and in vivo after a limited number of cell divisions, This process known as cellular senescence is an underlying cause of aging and a critical barrier for development of human cancers, We demonstrate here that reexpression of functional pRB alone in RB/p53-defective tumor cells via a modified tetracycline-regulated gene expression system resulted in a stable growth arrest at the G0/G1 phase of the cell cycle, preventing tumor cells from entering S phase in response to a variety of mitogenic stimuli, These cells displayed multiple morphological changes consistent with cellular senescence and expressed a senescence-associated beta-galactosidase biomarker, Further studies indicated that telomerase activity, which was assumably essential for an extended proliferative life-span of neoplastic cells, was abrogated or repressed in the tumor cell lines after induction of pRB (but not p53) expression, Strikingly, when returned to an nonpermissive medium for pRB expression, the pRB-induced senescent tumor cells resumed DNA synthesis, attempted to divide but most died in the process, a phenomenon similar to postsenescent crisis of SV40 T-antigen-transformed human diploid fibroblasts in late passage, These observations provide direct evidence that overexpression of pRB alone in RB/p53-defective tumor cells is sufficient to reverse their immortality and cause a phenotype that is, by all generally accepted criteria, indistinguishable from replicative senescence, The results suggest that pRB may play a causal role in the intrinsic cellular senescence program.