Inhibition of the PI3K/Akt pathway leads to up-regulation of p27^kip1 and loss of cyclin D1 expression. PI3K/Akt pathway inhibition studies were performed with the 4 MCL cell lines and the non-Akt-activated control cell line RAJI using 3 different inhibitors: LY294002, wortmannin, and Akt inhibitor (Calbiochem). Comparable results were obtained for each MCL cell line, and results with Granta 519 using LY294002 or Akt inhibitor are shown as representative experiments. (A,C) Proliferation/viability as assessed by MTT test. Results are averages of 3 individual experiments and are displayed as percent absorbance of control cells (untreated) with Granta 519 (□), Z138C (), and RAJI (▪). A significant reduction in absorbance to 50% of control values after 48 hours is seen in the MCL cell lines, whereas the non-Akt-activated control RAJI shows no significant reduction. (B,D) Western blot analysis with Granta 519 demonstrates Akt inactivation at 8 hours, followed by abrogation of phosphorylation of FRKHL-1, p27^kip1, and GSK-3β by 24 hours. The cell-cycle inhibitor p27^kip1 shows a gradual increase in expression levels over time, and cyclin D1 is dramatically down-regulated. In contrast, cdk4 and cyclin E remain constant. α-tubulin expression is shown as loading control.