| The Glucocorticoid Receptor (GR) activates apoptosis in immature thymocytes through a mechanism that requires transactivation and repression of gene transcription. Several targets of GR regulation have been identified, including Glutathione-S-Transferase M1 (GSTM1) and Nur77 expression, and NFB function, however no specific mechanism for activating GR-regulated apoptosis has been determined. Apoptosis is also induced in immature mouse thymocytes by ceramide analogues, staurosporine, and Fas stimulation, through diverse signal transduction pathways, ultimately resulting in caspase protease activation. Glucocorticoids (GC), ceramide analogues, and staurosporine pathways are inhibited by the Bcl-2-class of apoptosis regulator proteins. In some cell lines, Fas-dependent apoptosis is also blocked by Bcl-2. To identify the involvement of specific genes and components of the apoptosis machinery in this GR-dependent system, a somatic cell genetic approach was taken. A panel of Dexamethasone (Dex)-resistant cell lines (Apt-) isolated in a previous study were characterized with respect to their sensitivity to GR-independent apoptotic signals. While W7.2 cells were sensitive to staurosporine, ceramide, and Fas stimulation, Apt3.8, Apt4.8 and Apt5.8, were found to be resistant to some or all of these treatments. Apt4.8 and Apt5.8, but not Apt3.8, were found to be sensitive to staurosporine-induced apoptosis, whereas, all three Apt- mutants were found to be resistant to ceramide and Fas-mediated apoptosis. Measurement of steady state and Dex-regulated transcripts for Bcl-2-related genes, and Fas-interacting genes, and the caspase gene family, indicated that the observed differential resistance of the Apt- cell lines was not due to altered steady-state levels of any of these gene transcripts. GR-dependent regulation of GSTM1 and Nur77 transcription, and NFkB function was also tested in the Dex-resistant cell lines. GSTM1 expression was not induced in the Apt- cell lines by Dex. TNF a -induced activation of NFkB function was not observed in Apt3.8, and GR-dependent repression of Nur77 expression is defective in Apt4.8. From this genetic analysis of thymocyte apoptosis I conclude that GR-dependent apoptosis occurs via a mitochondria-dependent pathway which overlaps with many other apoptotic pathways including Fas in the WEHI7.2 cell system. |
