Akt constitutive activation correlates with the expression of phosphorylated/inactive form of PTEN. (A) The PI3-K/Akt cascade in MCL cell lines. Granta 519 cells were cultured in serum-free medium for 6 hours with solvent alone (“C”), 50 μM LY294002 (“LY”), 1 μM wortmannin (“Wort”), 20 μM SH-5, or 0.1 μM rapamycin (“R”). Total-cell lysates corresponding to 100 μg of proteins were analyzed by immunoblotting for the indicated proteins. Comparable results were also obtained in SP-53 and Jeko-1 cells (not shown). (B) MCL cell lines express the phosphorylated, inactive form of PTEN. All 3 cell lines investigated were cultured in medium with 10% FCS or medium with 0.5% BSA for 24 hours. Total-cell lysates, corresponding to 100 μg of proteins, were subjected to immunoblotting analysis using specific antibodies to the phosphorylated form of PTEN on Ser380 and Thr382/383. To check the antibody specificity after Western transfer, the membrane (bottom panel) was treated with CIAP for 3 hours at 37°C. Lane 1 was cut before CIAP treatment and used as positive control for the phosphospecific antibody. The samples are Granta 519 (lanes 1 and 2), SP-53 (lane 3), Jeko-1 (lane 4), Jurkat (lane 5), and C6 (lane 6). The Jurkat cell line was used as negative control for PTEN protein expression,10 and the C6 cell line was included as positive control. (C) Immunohistochemistry (magnification, ×400). PTEN protein expression is retained in all MCL cells (top left panel), which also expresses its phosphorylated form (top right panel). Most MCL cells show intense phospho-Akt nuclear signal (bottom left panel), whereas weak nuclear and cytoplasmic staining is evident in reactive mantle and germinal center lymphocytes (top right panel). (D) Original magnification, ×200. Staining with phospho-PTEN (Thr380) of samples treated (top) and untreated (bottom) with calf intestinal alkaline phosphatase for 45 minutes at 37°C. Images were acquired with a Nikon E80i microscope (Nikon, Tokyo, Japan) equipped with a Plan APO 20×/.075 objective and Nikon DS camera. ACT-1 software was used for image processing.
