During the last year we have continued to employ both biochemical and genetic approaches to the study of the acquisition of steroid resistance in human leukemic cells. In particular we have concentrated on the analysis of the normal glucocorticoid receptors of the human lymphoid cell line IM-9 in order to provide a basis for the ultimate analysis of glucocorticoid receptor mutants in the clonal glucocorticoid-resistant human leukemic cell lines we have previously isolated. Using high-resolution 2-dimensional gel electrophoresis of affinity labeled, immunopurified IM-9 receptors we have identified two isoforms of the 92 kilodalton human glucocorticoid receptor. Analysis of tryptic and chymotryptic digests of these isoforms has demonstrated that the charge heterogeneity resides in the 24 kilodalton tryptic fragment of the receptor which contains the steroid binding site. In addition, we have determined: (1) that there is no covalent charge modification of the 92 kilodalton steroid binding protein during activation of the steroid-receptor complex to its DNA binding form; and (2) that only the more basic of the two 92 kilodalton isoforms can bind to DNA. These results suggest that activation of the steroid-receptor complex does not involve dephosphorylation of the steroid binding protein. Our anti-human glucocorticoid receptor antibodies have also been used to obtain a partial cDNA clone of IM-9 glucocorticoid receptor mRNA. This clone was obtained after immunological screening of a lambda gt11 cDNA expression library. The availability of this clone will allow direct analysis of mutant receptor mRNA's and genes as well as provide a powerful reagent for the study of glucocorticoid receptor gene organization and regulation. Thus, we are in an advantageous position to study both the protein chemistry of mutant glucocorticoid receptors and the mutated genes which encode them. (D)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA032226-07
Application #
3170193
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1988-08-01
Project End
1993-06-30
Budget Start
1988-08-01
Budget End
1989-06-30
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Henry M. Jackson Fdn for the Adv Mil/Med
Department
Type
DUNS #
City
Rockville
State
MD
Country
United States
Zip Code
20852
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Ramdas, J; Harmon, J M (1998) Glucocorticoid-induced apoptosis and regulation of NF-kappaB activity in human leukemic T cells. Endocrinology 139:3813-21
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Miyashita, Y; Miller, M; Yen, P M et al. (1993) Glucocorticoid receptor binding to rat liver nuclei occurs without nuclear transport. J Steroid Biochem Mol Biol 46:309-20
Powers, J H; Hillmann, A G; Tang, D C et al. (1993) Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells. Cancer Res 53:4059-65
Scherrer, L C; Picard, D; Massa, E et al. (1993) Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90. Biochemistry 32:5381-6
Palmer, L A; Hukku, B; Harmon, J M (1992) Human glucocorticoid receptor gene deletion following exposure to cancer chemotherapeutic drugs and chemical mutagens. Cancer Res 52:6612-8

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