): The objective of this project is to study the role of radiation-induced ceramide generation via sphingomyelinase (ASMase) in the induction of apoptosis in CNS glial cells, with emphasis on oligodendrocytes (OLs). A novel signal transduction system has recently been character in which the lipid second messenger, ceramide, is linked to the stress activated kinase (SAPK/JNK) cascade, resulting in cell death by apoptosis. Each of the CNS glial cell types, the myelin-forming OLs and astrocytes, varies in its sensitivity to ionizing radiation and to exogenous ceramide in inducing apoptosis. These differences create distinct, opposing clinical relevancies. OLs are radiosensitive and undergo apoptosis following radiation-induced ceramide generation via ASMase. The CNS is a dose limiting tissue for therapeutic irradiation, partly because of radiation-induced cell death of OLs, and as hypothesized in this project, partly because of loss of clonogenic viability of OL progenitors. Thus the issue is ultimately to protect OLs. In contrast astrocytes are radioresistant, show no radiation-induced ceramide generation, and show no ceramide-dependent apoptosis. The majority of gliomas are astrocytic in character, and are known for a high degree of radioresistance. Thus the issue is ultimately to sensitize astrocytes. To investigate the role of radiation-induced ceramide generation via ASMase in contributing to the mechanisms of radioresistance/sensitivity, glial cell lines derived from an ASMase-deficient transgenic mouse (ASM-KO) will be generated. Primary glial cultures will be immortalized by transduction with SV40 large T antigen. In the ASM-KO vs. wild type mouse in vivo, and in these new ASMasc(+/-) cell lines and in existing glial cell lines in vitro, the kinetics and magnitude of ASMase activation, ceramide generation, SAPK/JNK activation, and apoptosis will be characterized by specific biochemical assays, in response to irradiation. Agents which inhibit specific points in the ceramide-SAPK/JNK pathway will be tested in vitro for OL lines, and stimulatory agents for astrocyte lines. To validate these concepts for OLs in vivo, ASMase(+/-) OL lines will be transfected with reporter genes and transplanted intracranially in the nude rat. After irradiation, the level of apoptosis and clonogenic survival of transplants will be assessed by histochemical techniques. Useful agents identified by in vitro screening will be tested in this in vivo model for protection of OLs from radiation-induced cell death.
