TUMOR-CELL RESISTANCE TO APOPTOSIS DUE TO A DEFECT IN THE ACTIVATION OF SPHINGOMYELINASE AND THE 24 KDA APOPTOTIC PROTEASE (AP24)

Signal transduction pathways involved in apoptotic cell death are poor ly understood, although recent studies have implicated sphingomyelin h ydrolysis and generation of the second messenger, ceramide, Previous w ork in this laboratory demonstrated that a serine protease termed AP24 was activated by TNF or UV light and induced DNA fragmentation in iso lated nuclei, This study extended these findings to examine the role o f these enzymes in apoptosis of the U937 cell line and the mechanism o f resistance of its variant, U9-TR, Although this subclone was selecte d by growth in TNF, it was unexpectedly found to resist apoptosis indu ced by UV light, but was still sensitive to anti-Fas-induced DNA fragm entation, Here we show that in contrast to normal U937 cells, UV light and TNF both failed to activate neutral or acidic sphingomyelinase or AP24 in the U9-TR variant, However, anti-Fas activated both neutral a nd acidic sphingomyelinase in the variant comparable to that seen in p arental U937, The U9-TR variant could be sensitized to TNF or UV light activation of both sphingomyelinase and DNA fragmentation by the prot ein phosphatase inhibitors okadaic acid and calyculin A, Furthermore, exogenous bacterial-derived sphingomyelinase caused U9-TR activation o f AP24 and DNA fragmentation comparable to that in the parental U937, Exposure of permeabilized U937 cells to ceramide caused internucleosom al DNA cleavage that was blocked by an inhibitor of AP24, Taken altoge ther, these findings demonstrate that TNF or UV light activate sphingo myelinase that leads to generation of ceramide resulting in activation of AP24 and DNA fragmentation in sensitive cells, A selective defect in signals leading to sphingomyelinase activation can confer resistanc e to apoptosis even though the variant is still sensitive to downstrea m apoptotic signals such as nuclear DNA fragmentation by activated exo genous AP24.