Maria Grazia Cifone*, Ruggero De Maria#, Paola Roncaioli*, Maria Rita Rippo#, Miyuki Azuma$, Lewis L. Lanier$, Angela Santoni# and Roberto Testi@#.

Corresponding author: Roberto Testi

@Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", $DNAX Research Institute, Palo Alto, CA, USA, *Department of Experimental Medicine, University of L'Aquila and #Department of Experimental Medicine, University of Rome "La Sapienza".

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after crosslinking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 mAb was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled sphingomyelin vesicles as substrates, showed strong sphingomyelinase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell permeant C2-ceramide. These data indicate that CD95 crosslinking induces sphingomyelin breakdown and ceramide production through an acidic sphingomyelinase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.