Inhibition of Akt increases total p27 levels and induces cell-cycle arrest in ALCL cells. (A) Akt-II inhibitor induced gradual decrease of pAkt (serine 473) levels. At a concentration of 10 μM, Akt-II induced almost complete absence of pAkt at 12 hours. Total Akt was also probed using the same membrane. No substantial changes were noticed in Akt levels. Top panel, SU-DHL1; bottom panel, Karpas 299. (B) Immunoprecipitation studies revealed a decrease in threonine phosphorylation of p27 (top panel) and an increase in total p27 levels in Karpas 299 cells treated with Akt-II inhibitor at 12 hours. WB indicates Western blot; and IP, immunoprecipitation. Densitometry of the immunoblot bands showed a substantial decrease in the threonine-phosphorylated p27/immunoglobulin G (IgG) ratio that was associated with increased total p27/IgG ratio. (C) Cell cycle analysis using BrdU uptake and flow cytometry in Karpas 299 cells 24 hours after treatment with Akt-II inhibitor. The S-phase fraction was 9% in cells treated with 5 μM of the Akt-II inhibitor compared with 39% in untreated (control) cells. Similar results were obtained for SU-DHL1 cells. (D) Total p27 levels after proteasome inhibition in ALCL cells. Treatment of Karpas 299 cells with LLnL and MG132 proteasome inhibitors for 16 hours resulted in a significant increase of total p27 levels (lanes 2 and 4 compared with lane 1), due to decreased p27 degradation through the ubiquitin-proteasome system. LLnL and MG132 were used at a concentration of 35 μM each and were previously shown to adequately block proteasome activity (data not shown). Pretreatment of ALCL cells with proteasome inhibitors for 4 hours followed by treatment of cells with both proteasome inhibitors and Akt-II for 12 hours resulted in no additional increase of total p27 levels (lanes 3 and 5), which demonstrates complete blockade of proteasome-mediated degradation.