Molecular genetic analysis of glucocorticoid-induced thymocyte apoptosis.

I have used a molecular genetic approach to study early events in the gene network that precede apoptotic commitment in glucocorticoid-induced thymocyte apoptosis. A panel of recessive, apoptotic-deficient (Apt⁻) mutants were isolated that are cross resistant to several diverse apoptotic treatments. These results indicated that the signal pathways initiated by glucocorticoids, gamma radiation, and c-AMP analog treatment converge to a common apoptotic pathway. Complementation analysis of Apt⁻ cell lines has defined five independent complementation groups that appear to represent mutations in genes that are required for apoptotic commitment. In addition, I have characterized induced gene expression patterns characteristic of dexamethasone (dex)-induced apoptosis and have found that glutathione-s-transferase (GST), Dag8 (a gene of unknown function) and calmodulin (Cam) transcript levels are elevated following dex treatment. Dex-treatment of Apt⁻ cell lines does not change GST or Cam transcript levels which suggests that these cell lines are blocked in early steps of the apoptotic pathway. In contrast, the dominant oncogene, Bcl-2, blocks apoptosis and appears to affect a relatively late event in the apoptotic pathway since the pattern of dex-induced gene expression is normal in cells that express this protein. Since the Apt⁻ cells contain wild type levels of functional glucocorticoid receptor (GR), GST and Cam do not appear to be primary GR target genes, but seem to respond to cellular events that occur prior to apoptotic commitment. In support of this conclusion, it was found that GST transcript levels increase in calcium ionophore-induced apoptotic cells. In contrast, Dag8, transcript levels increased in dex-treated Apt⁻ cells indicating that Dag8 is most likely a primary GR target gene. Furthermore, Dag8 expression was found to be restricted to thymocyte containing tissues and its locus was mapped to the H2 complex of chromosome 17, a region that is known to contain many immunologically important genes. Finally, a model is presented to describe a common apoptotic pathway in murine thymocytes and proposes that an increase in oxidative stress precedes calcium mobilization in response to glucocorticoid treatment