): The long-term goals of this proposal are to define the role of the sphingomyelin cycle in antiproliferative pathways of cells. The sponsor's laboratory has characterized the sphingomyelin cycle by which vitamin D3 and TNF-alpha activated a neutral sphingomyelinase, which hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide inhibits cell proliferation, induces differentiation, and stimulates apoptosis. Recent work by the applicant shows that dexamethasone (dex), a classic inducer of apoptosis and component of ALL induction regimens stimulates ceramide generation by an acid sphingomyelinase (A-SMase) and may commit leukemic lymphoblasts to apoptosis. Specific goals of this project are: (Phase 1) 1. Define acid SMase's role in dex-induced ceramide generation and apoptosis. Planned objectives are: a) determine if A-SMase is sufficient (or participates) in dex-induced apoptosis by showing elevated A-SMase activity generates ceramide, affects cell viability, and sensitizes leukemic cells to dex-induced apoptosis by overexpression of recombinant A-SMase; b) establish the effects of decreased A-SMase activity on inhibition of dex-induced ceramide generation and cell death by blocking protein translation using antisense oligonucleotides; by inhibiting A-SMase maturation through blocking transport to lysosomes; and stimulating proteolytic degradation of A-SMase with desipramine. 2. Define the mechanism of dexamethasone regulation of A-SMase. Since dex binding to cytosolic glucocorticoid receptors and nuclear translocation initiates transcriptional events, objectives are: a) determine the effects of dex on A-SMase transcription and mRNA stability by northern blot analyses; b) evaluate protein regulation by demonstrating quantitative changes (increased translation, posttranslational processing, or decreased degradation) by western blot analyses and enzyme activity. (Phase II) 3. Characterize the dex-induced ceramide generation. The form of dex-induced A-SMase (70kDa endolysosomal A-SMase, 57kDa A-SMase of unknown function or a unique enzyme) will be determined. These studies will define the role of A-SMase in dex-induced apoptosis of leukemic cells and may disclose therapeutic targets for inducing cell death in leukemia and other cancers.
