Constitutive activation of P13-K/Akt and mTOR pathways promotes MCL cells proliferation. (A) Inhibition of PI3-K/Akt and mTOR pathways strongly reduces the proliferation of MCL cell lines. Granta 519 and SP-53 cells were cultured at 10⁵/mL in DMEM and RPMI, respectively, supplemented with 10% FCS for 24 hours with either solvent alone (“C”), LY294002 (“LY”) at 5 or 10 μM, wortmannin (“Wort”) at 1 or 5 μM, SH-5 at 10 or 20 μM, or rapamycin (“R”) at 0.1 or 1 μM. DNA synthesis was assessed by ³H-thymidine incorporation after 6 hours. Results were expressed as mean (± SD) counts per minute of quadruplicate wells. (B) Antiproliferative effects of PI3-K and TORC1 inhibition in primary MCL cultures. Effects of PI3-K or TORC-1 inhibition on the division index of 5 primary MCL cultures were evaluated by CFDA-SE labeling coupled with cyclin D1 staining. The results are relative to a 3-day culture period. Globally, cultures treated with LY294002 (10 μM) or rapamycin (0.1 μM) showed a significantly lower division index compared with controls. Effects of LY294002 (10 μM) and rapamycin (0.1 μM) were also investigated in the same primary MCL cultures stimulated by rhsCD40-L alone or in combination with IL-4, showing that PI3-K or TORC-1 inhibition also significantly decreased the division index in the presence of these stimuli. (C) The PI3K/Akt and mTOR/p70^S6K pathways are constitutively activated in MCL cell lines. Granta 519, SP-53, and Jeko-1 cells were cultured in medium with 10% FCS or medium with 0.5% bovine serum albumin (BSA) for 24 hours. Total-cell lysates, corresponding to 100 μg of proteins, were subjected to immunoblotting analysis using phosphospecific antibodies.