#Department of Experimental Medicine, University of L'Aquila, 67100 L'Aquila, $Department of Experimental Medicine and Pathology, University of Rome "La Sapienza, 00161 Rome, @Department of Immunobiology, Institute of Cell Biology, CNR, 00137 Rome, *Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", 00133 Rome.

Corresponding author: Roberto Testi

The early signals generated following crosslinking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 crosslinking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected and it appeared required for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 crosslinking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently from the PC-PLC / aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signaling in a dominant negative fashion. Fas/APO-1 crosslinking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activities are induced. These results strongly suggest that a PC-PLC / aSMase pathway directly contributes to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.