Donald J. Gruol

Isolation of new types of dexamethasone-resistant variants from a cAMP-resistant lymphoma

We have developed a sequential selection procedure for the isolation of novel steroid-resistant variants of the murine thymoma WEHI-7. The first step involves the isolation of cell lines with an altered cAMP-dependent protein kinase (cAPK) activity by selection for resistance to dibutyryl cAMP (dbcAMP). The second step involves the selection for resistance to dexamethasone (dex) which results in the isolation of variants with decreased receptor function and a cAMPrdexr phenotype. The initial selection, to cAMPr, serves as a permissive step since isolation of spontaneous glucocorticoid resistance from wild-type WEHI-7 does not occur at a measurable frequency. The results demonstrate a potential role for cAPK in regulating the functional levels of glucocorticoid receptor and suggest that mutations in other cellular functions that affect receptor activity could lead to steroid resistance in lymphoid cells.

Nuclear interactions of wild type and variant glucocorticoid receptors

Nuclease digestion of nuclei from glucocorticoid sensitive and resistant lymphoma cell lines was used to study the nuclear compartmentalization of wild type and variant glucocorticoid receptors. In comparison with wild type, the variant line (S49 143r) had an increased capacity to translocate to the nucleus (nti), but was more completely released from nuclei by nuclease digestion. Approximately 20% of the receptor in wild type nuclei was resistant to release by DNase I digestion, while only less than 5% of the receptor from nti nuclei was retained under the same conditions. Studies with wild type nuclei show that the nuclease resistant portion of receptors was also more resistant to release by increased ionic strength.

Analysis of glucocorticoid receptor subspecies binding to DNA-cellulose and isolated nuclei☆

Conversion of the glucocorticoid receptor into a DNA-binding protein results in the generation of several distinct receptor subspecies (peaks A-E) which can be resolved by anion exchange chromatography. In vitro, the fraction of the receptor population (approx. 40%) which gains a capacity to bind DNA-cellulose is preferentially transformed into the peak A species by a process that was enhanced by the presence of KCl. At 0.4 M KCl, virtually all of the DNA-binding receptor was in the peak A form. Isolated nuclei also exhibit a receptor binding profile similar to that observed with DNA-cellulose.

Modulation of Glucocorticoid-Induced Responses by Cyclic AMP in Lymphoid Cell Lines

Glucocorticoid hormones affect immune function, in part, through their capacity to elicit a cytolytic response in thymocytes (Claman, 1972; Thompson and Lippman, 1974). The lytic process is dependent upon continued RNA and protein synthesis, and may represent induced transcription of one or more specific genes whose expression leads to programmed cell death (Munck and Crabtree, 1981; Young et al., 1980). Evidence for the existence of “lysis gene(s)” has been obtained by cell fusion experiments (Gasson and Bourgeois 1983a; Gasson and Bourgeois, 1983b) and by work demonstrating reactivation of cytolysis in the steroid-resistant SAK-8 cell line (Gassen et al., 1983; Bourgeois and Gasson, 1985). While cell cycle arrest (Harmon et al., 1979) and DNA degradation (Wyllie, 1980) have been found to be part of the autolytic process, the exact nature of the lysis-mediating function(s) remains to be discovered. It is important to keep in mind, however, that in the intact animal, thymocyte killing resulting from release of adrenal steroids is often part of an overall reaction to stress (Baxter and Rousseau, 1979). This raises the possibility that other stress-related signals, such as catecholamines, might also be integrated into the lymphoid cytolytic program. Thus, in T-cells, second messenger systems such as cAMP, calcium and phospholipid metabolites may play a contributing role in modulating the action of glucocorticoids.