The objective of this investigation remains the elucidation of the basis for the acquisition of glucocorticoid resistance in the human leukemic cell line CEM-C7. Specific mutations responsible for loss of glucocorticoid receptor (GR) function in spontaneously occurring glucocorticoid-resistant (dex-r) mutants will be cloned and sequenced to determine whether they are randomly distributed throughout the human GR (hGR) gene, or are localized to specific regions. Transient transfection assays will be used to ascertain whether any of these mutants act as dominant negative repressors of receptor function. Restriction fragment length polymorphisms in the 5' and 3' regions of the hGR gene will be used to assess the frequency, extent, and distribution of deletions in the hGR gene in dex-r mutants induced by cytotoxic cancer chemotherapeutic drugs with different mechanisms of action to determine if specific regions of the hGR gene are particularly susceptible to breakage, and whether individual drugs have specific mutagenic """"""""signatures."""""""" The pathway of steroid-induced growth arrest will be investigated. The basis for tissue-specific autoregulation of hGR expression will be determined by isolation, and functional characterization of hGR promoter regions responsible for hGR regulation in T and B cells. The role of positive autoregulation of hGR expression in steroid-induced cell death will be evaluated by stable transfection of an hGR gene under the control of the metal-inducible mouse metallothionein I gene promoter into cells lacking endogenous functional hGR. hGR concentration will be correlated with growth arrest to determine whether positive autoregulation is a necessary component in growth arrest or simply a pleiotypic response. In addition, construction of subtractive cDNA libraries will be used to attempt to isolate other genes involved in steroid-induced growth arrest. Identification of the mutations responsible for loss of hGR function in spontaneously occurring and drug-induced dex-r mutants will provide new information about the function of this trans-acting regulator of gene expression. In addition, an understanding of mechanisms responsible for the growth arrest of glucocorticoid-sensitive human leukemic cells in culture, and the mechanisms responsible for the acquisition of steroid resistance could provide the foundation for similar studies in steroid resistant leukemias, thus permitting the determination of when steroid treatment would be beneficial, and when such treatment and its associated side effects would best be withheld.
Showing the most recent 10 out of 25 publications
