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Chair(s): Hauer-Jensen, Martin
(SY052) Reorganization of membrane rafts into large signaling platforms in endothelium initiates apoptosis.
Kolesnick, Richard1, 1 Molecular Pharmacology and Chemistry, New York, New York, USA
ABSTRACT- Recent evidence suggests clustering of plasma membrane rafts into ceramide-enriched platforms serves as a transmembrane signaling mechanism for a subset of cell surface receptors and environmental stresses. These cell membrane domains transmit signals for stimuli as diverse as CD95, CD40, TCR, chemotherapy, UV-C as well as infection with pathogenic bacteria and viruses. Upon stimulation, a secreted form of acid sphingomyelinase (ASM) translocates into and generates ceramide within rafts by sphingomyelin hydrolysis. Ceramide, which possesses the unique capability to self-associate, provides the driving force for coalescence of these sub-microscopic rafts into the large signaling platforms. Whereas ionizing radiation activates ASMase to generate ceramide and initiate apoptosis, we studied the possible role of ceramide-enriched platforms in radiation-induced death. Our results show that exposure of Jurkat cells to 10Gy induced ceramide elevation and platform formation within seconds. Confocal microscopy showed that platforms contained ASMase, ceramide and Fas. Disrupting rafts by the cholesterol depleting reagents -cyclodextrin and nystatin inhibited radiation-induced ceramide increase and platform formation. Cholesterol depletion also inhibited radiation-induced apoptosis by 50-80% in a time- and dose-dependent manner. Furthermore, clonogenic assays showed disruption of rafts by cholesterol depletion protected against reproductive cell death, increasing the Dq of the dose-survival curve from 0.0±0.78Gy to 3.97±0.49Gy without significant change in the D0 (1.04±0.12Gy and 1.11±0.12Gy, respectively). The dose modification factor at D10 was 2.6, associated with an average 2 logs increase in survival for any dose within the range of 5-10Gy. Endothelial cells are 20-fold enriched in secretory ASMase compared with any other cell in the body and are particularly sensitive to radiation-induced apoptosis in vitro and in vivo via the ASMase pathway. Our recent data show that ionizing radiation also uses ceramide-rich platforms to signal apoptosis in endothelium, an event that regulates microvascular dysfunction in the GI tract and in experimental tumors in mice. Potentially, modulating this pathway might lead to development of ceramide-based therapies in management of some tumor types.
Key words: ceramide, apoptosis, rafts, clonogenic
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