Abstract 1144 Poster Session II, Sunday, 5/2 (poster 70)

The caspases are key enzymes in the apoptotic pathway, cleaving and activating other caspases as well as substrates essential for normal cellular function. Blocking apoptosis by blocking caspase activation or cleavage of target substrates is a potential therapeutic approach to conditions such as hypoxic-ischaemic encephalopathy (HIE) where apoptosis contributes significantly to the pathophysiology. For caspase inhibition to be an effective strategy, cells must be rescued from death, and the surviving cells must be functional. Several mammalian and viral genes are known to function as inhibitors of caspases, such as MIHA (Mammalian IAP Homologue A), crmA (Cytokine Response Modifier A) and a novel gene where the active site of crmA has been replaced by the active site of another viral caspase inhibitor, p35. We have addressed these issues by using these genes to determine whether human neuronal cells exposed to apoptotic stresses mimicking hypoxic-ischaemic conditions are protected from apoptosis, and in another cell line; to determine the function of cells protected from apoptosis by caspase inhibition. Methods: Transient and stable transfection of NT2 and WEHI7 cells. Transfected cells were subjected to a variety of death stimuli including autoactivating caspases, and potential mediators of cell death in HIE such as hydroxy nonenal and free radicals, and survival determined by several methods including uptake of propidium iodide, staining with Annexin V and morphology. Western blots determined the expression or degradation of the caspase inhibitors. Clonal assay was done in soft agar culture. Results: Caspase inhibitors can protect human neuronal cells from caspase mediated cell death and from cell death induced by potential mediators of cell death in HIE. WEHI7 cells exposed to death stimuli can be rescued by caspase inhibition, and are able to proliferate in a clonal assay. Conclusions: Cells may be rescued from death by caspase inhibition and are functional. This validates the therapeutic approach of blocking caspases in conditions such as HIE.