The involvement of PI3′K/PKB signaling in p53-dependent apoptosis and in EPO-mediated cell survival.

(A) Constitutive activation of PKB in DP16.1/p53ts cells. DP16.1/p53ts cells were left untreated, treated with EPO alone (15 minutes), wortmannin (WMN, 100 nM for 60 minutes) alone, or with both (WMN for 60 minutes followed by EPO for 15 minutes). Cell extracts were prepared and analyzed by SDS-PAGE and immunoblotting with an antibody specific for phosphorylated PKB (Ser473). (B) Activated PI3′K limits p53-dependent apoptosis. Cells were cultured at 37°C or 32°C for 12 hours in the presence or absence of 100 nM wortmannin (WMN) or 5 μM LY294002 (LY). The percentage of cells that showed evidence of apoptosis was determined on the basis of morphology as described in “Materials and methods” and is presented as the mean ± SEM (n = 3). DP16.1, parental p53⁻ cells; DP16.1/p53ts(S), sensitive subclone that undergoes p53-dependent apoptosis at 32°C; DP16.1/p53ts(R), resistant subclone that undergoes p53-dependent growth arrest but not apoptosis when cultured at 32°C. (C) EPO does not prevent reduction in the level of phosphorylated (Ser473) PKB. DP16.1/p53ts cells or parental DP16.1 cells were harvested at 0 to 9 hours (as indicated) after shifting to 32°C. Then, 50 μg whole cell lysate (in 2 × SDS-loading buffer) was applied to a polyacrylamide gel, separated by electrophoresis, and blotted onto a PDVF membrane. The membrane was immunoblotted sequentially with antiphosphoserine 473 PKB, total PKB (Cell Signaling), and β-actin (Sigma) antibodies. (D) EPO-mediated cell survival in the presence of LY294002. DP16.1/p53ts cells were cultured at 32°C for 12 hours. EPO and LY294002 were added to the cultures at the time of the temperature shift. The percentage of cells undergoing apoptosis was measured by the TUNEL assay.