p16INK4a can initiate an autonomous senescence program

The tumor suppressor p16(INK4a) is a potent mediator of cell cycle arrest in transient expression studies, is induced in senescing cells, and can impose morphological features of senescence, Nonetheless, it is unclear whether p16(INK4a) can block cell proliferation irreversibly. We explored this issue using osteogenic sarcoma cell clones with inducible p16(INK4a) expression. Induction of p16(INK4a) for 1 day arrested most cells in G1 phase. If the induction was then interrupted, p16(INK4a) levels returned to baseline and robust growth resumed within 3-5 days. When p16(INK4a) was induced for 6 days DNA synthesis remained strongly inhibited and the cells acquired morphological features of senescence. Moreover, if p16(INK4a) induction was interrupted at this point and the cells were followed for 12 more days, most cells retained these morphologic features and either failed to divide or died. This occurred despite the prompt return of p16(INK4a) expression and retinoblastoma protein phosphorylation toward baseline levels. In fact, some senescing cells appeared to enter S phase. These results demonstrate that a sustained period of p16(INK4a) expression is sufficient in this setting to impose a durable block to cell proliferation and that this state becomes independent of p16(INK4a) expression, hypophosphorylation of pRB, or a strict G1 arrest.