FADD expression and caspase activation in B-cell lymphomas resistant to Fas-mediated apoptosis

We have previously shown that malignant B cells from non-Hodgkin's lymphoma s (NHL) are resistant to Fas-mediated apoptosis. To determine the mechanism s underlying this resistance, we analysed by Western blotting the expressio n of several apoptotic regulators, caspase 3, caspase 8, FADD and poly(ADP- ribose) polymerase (PARP) in fresh lymphoma cells, isolated from 16 B-NHL b iopsy samples of different histological subtypes, and displaying variable l evels of Fas expression, The profiles of expression of these apoptotic regu lators were monitored in cell lysates at different times following Fas with or without CD40 stimulation, Expression of FADD and of the uncleaved forms of PARP, caspase 3 and caspase 8 were detected in all untreated NHL sample s, Low levels of PARP cleavage were noted in three untreated samples, Fas s timulation alone induced neither significant apoptosis nor significant chan ges in the expression profiles of FADD, caspases 3 and 8 and PARP in the 16 samples, except for variations in FADD and caspase 8 expression levels in a minority of samples. Fas/CD40 costimulation induced apoptosis and cleavag e of caspase 3, caspase 8 and PARP in the ave NHLs tested: expression of FA DD was not modified, Our results showed (1) that induction of apoptosis in B-NHLs bg Fas/CD40 co-stimulation used the same caspase executioner machine ry as the normal Fas pathway and (2) that NHL cells which resisted Fas-medi ated apoptosis displayed no defect in either expression or functionality of caspases 3 and 8, nor in FADD expression, The dysfunction underlying NHL r esistance to apoptosis must therefore lie upstream of caspase 8, or could a lternatively be influenced by anti-apoptotic regulators of the Bcl-2 family .