A New Determinant of Glucocorticoid in Lymphoid Cell Lines Sensitivity

The SAK cell line, derived from a spontaneous thymic lymphoma in an AKR mouse, is resistant to lysis by glucocorticoids in spite of the presence of functional glucocorticoicl receptor. Receptor function was determined by hormone binding analyses, as well as characterization of hormonal effects on cell growth and on the accumulation of murine leukemia virus and metallothionein mRNAs. SAK cells were fused with a receptor-defective (and therefore resistant) variant of a well-characterized murine thymoma line, W7. The resulting hybrids are glucocorticoid sensitive, demonstrating complementation of the receptor defect in W7 cells by the functional glucocorticoid receptor of SAK. This fusion shows that SAK cells are resistant to the hormone clue to the absence of another function designated 'T ' for lysis. SAK cells were also fused with glucocorticoid-sensitive W7 cells (containing wild-type receptor), generating glucocorticoid-sensitive hybrids, which demonstrate that the dexamethasone-resistant phenotype of the SAK cells is recessive. Resistant derivatives of this hybrid were found which still contain the full amount of receptor. Chromosome analysis revealed that, on the average, the resistant derivatives had lost two chromosomes, suggesting segregation of chromosomes carrying genetic material necessary for the "lysis" function. The drug 5-azacytidine (a known inhibitor of DNA methylation) has been shown to cause heritable changes in gene expression. Treatment of SAK cells with 5-azacytidine generated glucocorticoid-sensitive clones at high frequency, suggesting that the gene(s) involved in the "lysis" function are intact and have been inactivated through a process such as differentiation. It has been known for over thirty years that T-lymphocytes, during early stages of differentiation, are lysed by glucocorticoid hormones. In addition, a number of neoplastic T-cell lines derived from both human (1) and mouse (2, 3) are sensitive to lysis by glucocorticoids (1--4). A variety of mutagenic treatments have been used to generate dexamethasone(dex, a synthetic glucocorticoid) resistant derivatives ofglucocorticoidsensitive lymphoid cell lines in order to study the biology of the lytic response, as well as the mechanism of action of glucocorticoids. Numerous studies have demonstrated that glucocorticoid receptor binding and translocation of the steroid hormone into the nucleus are a necessary step for the response; further steps in the cytolytic pathway remain obscure (reviewed in references 5-7). In spite of considerable effort, no resistant variant resulting from a defect in a function other than the receptor has ever been found (8, 9). Moreover, complementation between receptor-defective variants has never been observed (8, 10), indicating that all variants obtained so far result from defects in the same genetic locus. SAK is a cell line derived from a spontaneous thymic lymphoma in an AKR/J mouse. SAK cells do not lyse in response to dex; instead, the cells continue to grow and also form aggregates. This report provides both biological and biochemical evidence that the glucocorticoid receptor in SAK cells is functional. The presence of functional glucocorticoid receptor in this dex-resistant T-ceU line makes it possible to use a genetic approach to begin to analyze other steps in the cytolytic pathway. Genetic experiments are described which show that at least one other function, in addition to hormone binding to the receptor, is required for the cytolytic response to glucocorticoids. Treatment of SAK cells with 5-azacytidine (an inhibitor of DNA methylation) causes this "lysis" function to be activated and results in a cytolytic response to glucocorticoids. Our results support the hypothesis that this "lysis" function is inactivated in SAK cells possibly as a result of differentiation. Thus, SAK cells may provide a model for a THE JOURNAL OF CELL BIOLOGY VOLUME 96 FEBRUARY 1983 409-415 © The Rockefeller University Press • 0021-9525/83/02/0409/07 $1.00 4 0 9 on Jne 7, 2017 D ow nladed fom Published February 1, 1983