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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA041407-14S1
Application #
6134716
Study Section
Endocrinology Study Section (END)
Program Officer
Mccarthy, Susan A
Project Start
1985-12-01
Project End
2000-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Webb, M S; Miller, A L; Howard, T L et al. (2018) Sequential gene regulatory events leading to glucocorticoid-evoked apoptosis of CEM human leukemic cells:interactions of MAPK, MYC and glucocorticoid pathways. Mol Cell Endocrinol 471:118-130
Aiyar, Sarah E; Park, Hoyong; Aldo, Paulomi B et al. (2010) TMS, a chemically modified herbal derivative of resveratrol, induces cell death by targeting Bax. Breast Cancer Res Treat 124:265-77
Garza, Anna S; Miller, Aaron L; Johnson, Betty H et al. (2009) Converting cell lines representing hematological malignancies from glucocorticoid-resistant to glucocorticoid-sensitive: signaling pathway interactions. Leuk Res 33:717-27
Ji, Zhenyu; Mei, Fang C; Miller, Aaron L et al. (2008) Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells. J Biol Chem 283:21920-5
Webb, M S; Miller, A L; Thompson, E Brad (2007) In CEM cells the autosomal deafness gene dfna5 is regulated by glucocorticoids and forskolin. J Steroid Biochem Mol Biol 107:15-21
Miller, Aaron L; Webb, M Scott; Thompson, E Brad (2007) Comparison of two structurally diverse glucocorticoid receptor agonists: cortivazol selectively regulates a distinct set of genes separate from dexamethasone in CEM cells. Steroids 72:673-81
Ji, Zhenyu; Mei, Fang C; Johnson, Betty H et al. (2007) Protein kinase A, not Epac, suppresses hedgehog activity and regulates glucocorticoid sensitivity in acute lymphoblastic leukemia cells. J Biol Chem 282:37370-7
Ilies, Marc Antoniu; Seitz, William A; Johnson, Betty H et al. (2006) Lipophilic pyrylium salts in the synthesis of efficient pyridinium-based cationic lipids, gemini surfactants, and lipophilic oligomers for gene delivery. J Med Chem 49:3872-87
Copik, Alicja J; Webb, M Scott; Miller, Aaron L et al. (2006) Activation function 1 of glucocorticoid receptor binds TATA-binding protein in vitro and in vivo. Mol Endocrinol 20:1218-30
Miller, Aaron L; Webb, M Scott; Copik, Alicja J et al. (2005) p38 Mitogen-activated protein kinase (MAPK) is a key mediator in glucocorticoid-induced apoptosis of lymphoid cells: correlation between p38 MAPK activation and site-specific phosphorylation of the human glucocorticoid receptor at serine 211. Mol Endocrinol 19:1569-83

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