We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH3 demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH3 into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH3–mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH3–mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH3 and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.

The ether phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) is a potent inducer of apoptosis in human tumor cells. We show that ET-18-OCH3-induced apoptosis is associated with activation of the c-Jun NH2-terminal kinase (JNK) signaling. The addition of ET-18-OCH3 to distinct human leukemic cells (HL-60, U937, and Jurkat), which undergo rapid apoptosis on treatment with ET-18-OCH3, induced a dramatic and sustained increase in the of c-jun mRNA level that was associated with activation of activator protein-1 transcription factor. We found that ET-18-OCH3 induced a persistent activation of JNK in HL-60 cells that was detected before the onset of apoptosis, the latter being assessed by DNA fragmentation and by the appearance of phosphatidylserine on the external leaflet of the plasma membrane. The inductions of JNK after HL-60 monocyte/macrophage differentiation and ET-18-OCH3-mediated apoptosis were distinguished by the different activation patterns, transient versus persistent, respectively. ET-18-OCH3 analogues unable to induce apoptosis failed to activate JNK. ET-18-OCH3-dependent JNK activation was not detected in K562 cells, which did not undergo apoptosis on treatment with ET-18-OCH3. Phorbol myristate acetate inhibited both ET-18-OCH3-induced apoptosis and sustained JNK activation; thus, persistent JNK activation by ET-18-OCH3 is associated with the capacity of this ether phospholipid to induce apoptosis. Furthermore, antisense oligonucleotides directed against c-jun blocked ET-18-OCH3-induced apoptosis, indicating a role for c-Jun in this apoptotic response. These data indicate that JNK activation and c-Jun are involved in the induction of apoptosis by ET-18-OCH3.