Apoptosis causes the death of about half of the mitotic neuronal or glial cells during early periods of nervous system development. Sphingolipids, in particular ceramide, are known to participate critically in the induction of apoptosis. Ceramide is mainly generated by membrane-bound sphingomyelinase upon receptor-mediated stimulation by extracellular cytokines and growth factors. Mitogenic factors and sphingolipids occasionally share the intracellular signal transduction by common protein cinase cascades. This may lead to apoptosis specifically in rapidly dividing cells. The molecular mechanism and regulation of apoptosis in mitotic cells, however, is poorly understood. In this project investigation of this mechanism will be approached by use of sphingolipid analogues, in particular ceramide mimics, for induction of apoptosis in neuroblastoma and glioma cells. New compounds will be synthesized and used for the analysis of apoptotic signal cascades. The analysis of these signal cascades in mitotic cells will be utilized for the rationale design of an anti-tumor treatment by induction of apoptosis in cancer of the nervous system. The use of ceramide mimics in place of ceramide itself will enable a specific targeting of proteins in different ceramide dependent signaling pathways. We synthesized a novel ceramide analogue, 2-palmitoylamino-1,3-propanediol (C16-serinol) which was derived from the beta-hydroxy amide motif present in ceramide. Preliminary results with this compound revealed a highly apoptotic potential specifically in rapidly dividing neuroblastoma cells. C16-serinol was used as affinity ligand for preparation of a protein fraction from rat brain containing three serine/threonine specific protein kinases with molecular masses of 50, 70, and 95 kDa. The 70 kDa protein was identified as protein kinase Czeta which is known to be regulated by binding of ceramide and is involved in apoptosis. PKCzeta could be directly activated by C16-serinol in an in vitro phosphorylation assay. C 16-serinol and additional analogues may thus be useful for isolation of sphingolipid-binding proteins participating in apoptotic signal cascades.
Our specific aims i nclude: i) the purification and characterization of these proteins by use of sphingolipid-analogues as affinity ligands; ii) the generation of experimental tumors in nude mice and the use of the new analogues For treatment by induction of apoptosis. Eventually, we will study the regulation of apoptosis in mitotic cells during nervous system development.
