Investigations from our laboratory and others have shown that overexpression of wild-type pRb inhibits apoptosis induced or mediated by TGF-β1, ionizing radiation and p53 in HUH-7, Saos-2, and HeLa cells (Table 2). Suppression of RB expression and inactivation of pRb by TGF-β1 and okadaic acid are coincident with induction of apoptosis. Apoptosis induced by inhibition of pRb expression with antisense RB S-oligonucleotides and with TGF-β1 links apoptosis to cell cycle exit at the G1-phase. Furthermore, increased E2F-1 activity promotes both S-phase entry and apoptosis. Thus, apoptosis initiated by the inhibition of pRb expression or suppression of the ability of pRh to bind its effector proteins may be due to increased E2F-1 activity. Is E2F-1 promotion of premature S-phase entry a ticket to apoptosis? We think this is unlikely since greater than 90% of preapoptotic HUH-7 cells were arrested in the G1-phase during TGF-β1 induced apoptosis. Generally, E2F-1 is bound and sequestered by un- or hypophosphorylated pRb during the G1-phase. Subsequently, pRb phosphorylation results in the release of E2F-1 and allows entry into S-phase. Late in the cell cycle during M-phase, E2F-1 is sequestered again to facilitate teentry into the next round of replication. Therefore, we hypothesize that the timing of E2F-1 expression and activity is important for both cell proliferative and apoptotic pathways. Overexpression or untimely increases in E2F-1 activity may signal initiation of apoptosis (see Fig. 7). In addition, an increase in E2F-1 levels below the apoptotic threshold may facilitate induction of apoptosis through a p53- dependent pathway. This is in agreement with the observation that expression of p53 can arrest cells in G1, but E2F-1 expression at non-apoptotic levels induces apoptosis in cooperation with p53 (41). In conclusion, pRb appears to function in the negative regulation of both cell proliferation and apoptosis. Dependent upon the signals received by the cell, abrogation of pRb suppressor activity through decreased expression levels, phosphorylation or binding of inhibitor proteins allows either cell proliferation or apoptosis to occur.