SERINE-PROTEASE INHIBITORS BLOCK NEUTRAL SPHINGOMYELINASE ACTIVATION,CERAMIDE GENERATION, AND APOPTOSIS TRIGGERED BY DAUNORUBICIN

To address the role of a plausible protease cascade in daunorubicin-tr iggered apoptosis, we evaluated the effect of cell-permeant protease i nhibitors on its signal transduction pathway. Treatment of U937 and HL -60 cells with 0.5-1. mu M of the chemotherapeutic drug daunorubicin i nduced a greater than 30% activation of neutral sphingomyelinase activ ity within 4-10 min with concomitant sphingomyelin hydrolysis and cera mide generation. DNA fragmentation and the classical morphological fea tures of apoptosis were observed within 4-6 h. Pretreatment of cells w ith the serine protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone (20 mu M) or dichloroisocoumarin (20 mu M) for 30 min inhibite d daunorubicin-induced neutral sphingomyelinase activation, sphingomye lin hydrolysis, ceramide generation, and apoptosis. Other cell-permean t protease inhibitors such as pepstatin, leupeptin, and antipain had n o such effect. The apoptotic response could be restored by the additio n of 25 mu M cell-permeant C6-ceramide. Daunorubicin-induced NF-kappa B activation was inhibited by dichloroisocoumarin but not by N-tosyl-L -phenylalanyl chloromethyl ketone, suggesting that this transcription factor can be activated independently of ceramide and is not directly implicated in the apoptotic pathway. These results suggest that inhibi tors of serine proteases can act upstream of ceramide in drug-triggere d apoptosis and that neutral sphingomyelinase activation is either dir ectly or indirectly serine protease dependent.