The long-term objectives of this project focus on the human glucocorticoid receptor (GR), its gene, and glucocorticoid action in human leukemias. DNA sequences encoding a large part of the human GR have just been obtained, in what may be the first isolation of a gene encoding a human transcription regulatory protein, one whose occupancy by steroids leads to death of leukemic lymphoblasts. This DNA, along with clones of sensitive and resistant cells from the well-studied acute lymphoblastic leukemic line (CEM) will be used in a combination of methods, involving molecular cloning, protein purification and microsequencing, steroid binding, and somatic cell genetics to study human GR and its mechanism of action.
Specific aims will include comparisons of human, rat and mouse GR genes, measurement of GR mRNA levels and comparison of GR genes in mutant and wild-type CEM cells as well as in genetically glucocorticoid-resistant humans and monkeys. The human GR will be further purified so that sequence data and monoclonal antibodies may be obtained. The binding of phenylpyrazolo glucocorticoids will be studied to see whether they interact solely with the classic GR. Cell ploidy, exposure time to and concentration of glucocorticoids will be evaluated for their bearing on effective use of the steroids to produce cell kill. Additional r+ly- (lysis defective) cell lines of several classes of leukemia will be treated with azadeoxycytidine to see if they will shift to ly+. Somatic cell hybrids between r+ly- cells and resistant cells with defective receptors will be made to test the function of the receptor from the ly- cell. Steroid sensitive CEM cells will be examined for oncogene/transforming gene expression and phosphatidyl inositol turnover, in the presence and absence of glucocorticoids.
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