The objective of this proposal is to study the role of Ich-3, a gene encoding a homolog of mammalian interleukin-1beta converting enzyme (ICE), in apoptosis, sepsis and tumorigenesis. Ich-3-/- mice are resistant to lipopolysaccharide induced lethality, Ich-3-/- thymocytes are partially resistant to Fas-induced apoptosis and older male mice develop tumors. To identify the location of ICH-3 action, immunohistochemistry and Western blot analysis will be used to examine the expression patterns of ICH-3 and its induction by LPS. The results from our Ich-3-/- mice allow us to propose that ICH-3 is an upstream activator of ICE. The enzymatic activity of ICH-3 will be determined to see whether it can cleave pro-IL-1beta and ICE. GraB can cleave ICH-3 in vitro. Immunoprecipitation will be used to determine if ICH-3 is cleaved when cells are induced to die by GraB and whether Ich-3-/- cells are resistant to GraB induced apoptosis. Ich-34- polymorphonuclear leukocytes (PMN) and splenic lymphocytes will be tested to see if they are resistant to apoptosis. Since apoptosis of PMN may contribute to sepsis and Ich-3-/- mice have higher PMN counts, it is possible that Ich-3-/- PMN do not die as readily as wild type. Their longevity will be determined in vivo and in vitro. Since Ich-3-/- thymocytes are partially resistant to Fas- induced- apoptosis, Ich-3-/- splenic lymphocytes will be tested to see if they are also more resistant to Fas. Finally, the identity and origin of the tumors in older Ich-3 mutant male mice will be determined by histological methods and it will be determined whether promotion of tumors in Ich-3 mutants is a result of deficiency in apoptosis. These experiments may reveal that apoptosis genes in the ICE family may function as recessive oncogenes.
