The fundamental objective of this grant is to understand how the activated glucocorticoid receptor (GR) exerts one of its major biological actions, apoptosis of malignant lymphoid cells. Significant progress has been made on each of the previous specific aims. In lymphoid cells, the abrupt down-regulation of the protooncogene c-myc has been identified as a key step in glucocorticoid-evoked apoptosis. New results in CEM C7 cells show that glucocorticoids also down-regulate other important regulatory genes, but induce the GR and c-jun. From these facts, a model for the mechanism of glucocorticoid-evoked apoptosis in CEM cells has been formed. Four of the six new specific aims will test aspects of this model. 1) The transcriptional/post-transcriptional nature of the glucocorticoidal regulation over c-myc will be determined by nuclear run-on and mRNA stability experiments. Regulatory sequence analysis in promoter:reporter transfections, plus DNA or RNA binding experiments will be used to determine the mechanisms of the control. 2) The hypothesis that in the apoptotic pathway loss of Myc leads to reduced expression of other regulatory genes will be tested. Enzyme activities, protein, and mRNA levels will be followed after reducing Myc with glucocorticoid or anti sense oligonucleotides. 3) Differential display to find other genes regulated by both glucocorticoids and Myc will be carried out, these genes cloned and characterized. 4) Cellular Jun and Fos levels will be varied while following the effects on Myc and apoptosis. Domain mapping of the GR for lethal function has shown the DNA Binding Domain (DBD) to be critical, with the kinetics of death different depending on the DBD:GR context. When associated with a Steroid Binding Domain (SDB), the DBD mediates glucocorticoid-dependent apoptosis after a long lag. Transfected alone, the DBD or the DBD mutant 465* cause cell death more quickly.
Two specific aims pursue the basic and practical consequences of these findings. Systematic structure/function analyses of the DBD will be carried out: 1. In context with the SDB; and 2. As a unique lethal fragment. Effective ways to deliver the latter, in development of a new gene-based therapeutic method will be sought. Recombinant DBD fragments will be studied for structure and function. Protein partners will be sought and DNA binding sites will be defined.
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