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.

SUMMARY1. The effects of chronic conduction block with tetrodotoxin (TTX) of the cervical sympathetic trunk on synapses in the superior and stellate ganglia were examined in vitro with intracellular recording techniques.2. The mean maximum amplitude of excitatory post-synaptic potentials (e.p.s.p.s) evoked in superior cervical ganglion neurones by stimulation of the cervical sympathetic trunk was increased significantly after 2 or 4 days of block. The electrical properties of the ganglion cells were not appreciably changed by the period of inactivity.3. Chronic conduction block for 4 days also resulted in a significant increase in the amplitude of the e.p.s.p.s. evoked in stellate ganglion cells by active collaterals of the blocked fibres.4. The number of steps in the synaptic response elicited in individual stellate ganglion neurones by graded stimulation of the ansa subclavia and the subelavian trunk (which join to form the cervical sympathetic trunk) was increased after 4 days of conduction block.5. These results show that elimination of impulse activity in the cervical sympathetic trunk increased synaptic efficacy at the disused synapses in the superior cervical ganglion and at the active synapses in the stellate ganglion. In the latter case, collaterals of the blocked fibres apparently formed new synaptic connexions on stellate ganglion cells.

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Intercellular communication is considered to have a role during pattern specification processes in early embryonic development. This report analyzes the changing gap junctional communication properties of chick neuroepithelial cells depending on their position relative to the segmental partitions of the rhombencephalon. Intercellular electrical coupling and dye transfer were studied with microelectrode techniques. Neuroepithelial cells were electrically coupled irrespective of their location relative to interneuromeric boundaries. Iontophoretic injection of biocytin or Lucifer Yellow into single cells inside the rhombomeres was followed by transjunctional diffusion to the surrounding cells. In contrast, dye transfer was strictly limited when the diffusion zone contacted the cells forming the interneuromeric limits. Label injected into the boundary cells did not spread to other cells at all. Avian interrhombomeric boundaries are thus sites of reduced junctional permeability during early morphogenesis.

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