The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death.

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16ink4a, PSM-RB is also a potent inhibitor of cell cycle progression in RB-deficient tumor cells. Surprisingly, we identified a tumor cell line that is resistant to the cell cycle inhibitory effects of PSM-RB. This finding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this 'resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the first to show that RB inhibits cellular proliferation through at least two distinct mechanisms - inhibition of cell cycle progression and induction of cell death.