title: In vivo resistance to glucocorticoid-induced apoptosis in rat thymocytes with normal steroid receptor function in vitro.
abstract: Previous studies have shown that although the majority of rat thymic lymphocytes are sensitive to glucocorticoid-induced apoptosis in vivo, a small population of mature thymic lymphocytes remains even after high dose steroid administration. Here, we describe experiments that were performed to understand the molecular basis of the resistance of these cells to glucocorticoid-induced apoptosis. Adrenalectomized rats were treated for 72 h with a bolus dose (5 mg/kg body weight) of dexamethasone to produce a population of thymocytes that survived glucocorticoid administration. Reinjection of these animals with equivalent doses of dexamethasone failed to induce further thymic regression or apoptosis in these cells. Glucocorticoid receptor number and receptor binding affinity for dexamethasone were similar in control and resistant thymocytes. Western blot analysis using epitope-purified antiglucocorticoid receptor antibodies confirmed this observation. To delineate the mechanism of resistance, we evaluated whether cells resistant to dexamethasone in vivo showed any response to this glucocorticoid in vitro. The ability of glucocorticoid to inhibit [3H]lysine incorporation into protein in cells treated with dexamethasone in vitro was equivalent to control cells, indicating that glucocorticoid receptor function was normal in both populations. To evaluate whether in vivo glucocorticoid-resistant thymocytes retain any capacity to undergo apoptosis, in vitro studies were performed on these cells using the calcium ionophore A23187 to induce programmed cell death. Cleavage of chromatin into 30- to 50-kilobase fragments or oligonucleosomal fragments characteristic of apoptosis was observed in both sensitive and resistant thymocytes treated in vitro with A23187. Cells resistant to glucocorticoid in vivo unexpectedly exhibited internucleosomal cleavage of chromatin and apoptosis in response to dexamethasone in vitro. We examined the levels of the apoptosis suppressor Bcl-2 in thymocytes isolated from control and 72 h dexamethasone-treated rats to determine whether increased expression of this protein could explain the resistance to glucocorticoid-induced apoptosis that we observed. Both glucocorticoid-sensitive and -resistant thymocytes expressed similar levels of Bcl-2. Together, these data indicate that resistance to glucocorticoid in vivo is not due to alteration of the glucocorticoid receptor or to expression of Bcl-2, but rather to some endogenous thymic factor and/or cell-to-cell contact that probably alters glucocorticoid receptor signaling.
authors.affiliation: Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: N B
authors.initials: NB
authors.lastname: Oldenburg
authors.forename: R B
authors.initials: RB
authors.lastname: Evans-Storms
authors.forename: J A
authors.initials: JA
authors.lastname: Cidlowski
mesh_terms: D000315:Adrenalectomy; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053938:DNA Fragmentation; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology*; D004351:Drug Resistance; D005938:Glucocorticoids / Q000494:pharmacology*; D008297:Male; D014176:Protein Biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014316:Tritium
chemical_list: D005938:Glucocorticoids; D019253:Proto-Oncogene Proteins c-bcl-2; D014316:Tritium; D003907:Dexamethasone