c-FL/P overexpression in ALCL. (A-C) Immunohistochemical detection of extrinsic apoptotic pathway proteins in reactive lymph node and ALK⁺ anaplastic large-cell lymphoma (ALCL) tumors. (A) CD95/FAS was weakly expressed in a subset of centroblasts within the germinal centers (GCs) of reactive lymph nodes (left). Strong cytoplasmic and membranous CD95/FAS expression was observed in most ALK⁺ ALCL tumors (right). (B) CD95L/FASL was strongly positive in the mantle and marginal zones of reactive lymph nodes (left). CD95L/FASL was not expressed in most ALK⁺ ALCL tumors (right). Infiltrating small reactive lymphocytes strongly expressed CD95L/FASL and served as internal positive controls in all tumor specimens. (C) c-FLIP expression was restricted to a subset of GC cells (left). Strong cytoplasmic expression for c-FLIP was observed in most ALK⁺ ALCL tumors (right). (D-E) c-FLIP up-regulation may result from activation of PI3K/Akt and JAK3/ALK oncogenic pathways. Immunohistochemistry: × 200 original magnification (20 ×/0.75 NA objective; left panels) or × 400 (40 ×/0.75 NA objective; right panels). Images were obtained with a BX51 Olympus microscope, a DP12 Olympus camera, and a universal semi-apochromat UPlan FI lens. (D, left panel) Karpas 299 and SU-DHL1 cells were treated with a PI3K inhibitor, LY294002, at concentrations of 0, 5, or 20 μg/μL. Whole-cell lysates were then prepared at 48 hours. Immunoblots showed that serine 473 phosphorylation of Akt is almost completely inhibited at a concentration of 20 μg/μL that is associated with a substantial decrease of c-FLIP levels. (D, right panel) Karpas 299 and SU-DHL1 cells were infected with the constitutively active, myrAkt adenovirus²¹  at an MOI of 20, which significantly increases ^Ser473pAKT levels in these cells. Expression of myrAkt was confirmed by Western blot analysis using a monoclonal antibody specific for the hemagglutinin (HA) tag. An adeno-β-Gal adenovirus construct expressing β-Gal served as a control in this experiment. Whole-cell lysates were prepared from control and infected cells 48 hours after infection. Immunoblots showed a dramatic increase of Akt phosphorylation, which was associated with an increase of c-FLIP_S splice variant. (E) ALK⁺ ALCL cells were treated with 2 known JAK3 inhibitors, WHI-P131 and WHI-P154, at concentrations previously shown to inhibit JAK3 and ALK enzymatic activity. ²⁵ Whole-cell lysates were prepared 24 hours following treatment. Immunoblots demonstrate that c-FLIP levels are decreased after treatment with WHI-P131 or WHI-P154 in a dose-dependent manner.