PMID: 8382211;Abstract:
Based on the finding that glutathione S-transferase Yb1 (GST) gene expression is elevated in the regressing prostate of androgen-ablated rats, we analyzed GST transcript levels during steroid-induced lymphocyte cell death. It was found that GST gene expression was induced in steroid-sensitive cells within 4 hr of dexamethasone treatment, required functional glucocorticoid receptor, and was dose-dependent with regard to hormone. GST expression was not induced in an apoptosis-defective variant that contained normal levels of functional receptor, indicating that GST up-regulation was the result of secondary events that occur during steroid-mediated apoptosis. Using the calcium ionophore A23817 to induce lymphocyte cell death, GST RNA levels were increased in both steroidsensitive and steroid-resistant cell lines, supporting the conclusion that elevated GST expression was the result of cellular processes associated with apoptosis, rather than a direct consequence of steroid-mediated transcriptional control. The cells were also treated with dibutyryl cAMP to cause cell death; however, this mode of killing did not result in GST up-regulation. Taken together, these results suggest that GST induction in dexamethasone-treated T-lymphocytes occurs early in the steroid-regulated apoptotic pathway and that this may be a marker of calcium-stimulated cell death. Based on the known function of GST as an antioxidant defense enzyme and its transcriptional regulation by reactive oxygen intermediates, we propose that the gene product of a primary GR target gene(s) directly or indirectly effects the redox state of the cell. Thus activation of GST gene expression in apoptotic lymphocytes is likely a indicator of oxidative stress, rather than a required step in the pathway. © 1993 Wiley-Liss, Inc.
WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways.
PMID: 6549049;Abstract:
The effects of steroid hormones are mediated by intracellular hormone-specific receptor proteins; the interaction between the hormone and its receptor increases the affinity of the receptor for nuclear binding sites, thereby modulating the expression of specific genes. The glucocorticoid receptor is a soluble protein of relative molecular mass (M(r)) 94,000 (94K), present at a low relative abundance (≤0.01%); it has been purified to near-homogeneity, and specific antisera and monoclonal antibodies have been produced. Purified glucocorticoid receptor binds in vitro with high affinity to defined regions of DNA near regulated promoters, and sequences essential for these interactions are functional in vivo as hormone-dependent transcriptional enhancer elements. We have now cloned complementary DNA (cDNA) for the rat liver glucocorticoid receptor and we describe here a 2.6-kilobase (kb) receptor cDNA isolated following polysome immune-enrichment of receptor messenger RNA with glucocorticoid receptor-specific antibodies. The receptor appears to be encoded by a single-copy gene which specifies a ~6-kb transcript in rat and mouse cells; this mRNA is altered quantitatively and qualitatively in several mutant cell lines with specific defects in receptor function.
PMID: 18992753;PMCID: PMC2646908;Abstract:
Diapause in overwintering adult female Culex pipiens mosquitoes plays an important role in the transmission of West Nile and other encephalitis-inducing flaviviruses. To investigate the dynamic metabolic processes that control Cx. pipiens diapause, we used radioactive tracer techniques with [14C]-glucose to investigate the metabolic fate and flux of glucose in adult mosquitoes reared under diapause (18 °C, short day) and non-diapause (27 °C, long day) conditions. We found that by 72 h post-14C-labeling of 1-day-old mosquitoes, the diapause-destined mosquitoes had converted 46% more 14C-labled glucose into 14C-labled lipid than mosquitoes reared under non-diapausing conditions. When 5-day-old mosquitoes were fed [14C]-glucose, and then switched to water only, the non-diapausing mosquitoes oxidized nearly three times more 14C-labled glycogen and lipid by day 7 than diapausing-mosquitoes. This increased energy expenditure in non-diapausing mosquitoes is most likely due to temperature- and light-dependent increases in the basal metabolic rate. Amongst the diapausing-mosquitoes we analyzed over a subsequent 7-week period, we found that the amount of 14C-labeled glycogen decreased steadily for the first month of diapause, whereas, 14C-labeled-lipid levels were not significantly decreased until after day 35 of diapause, indicating that flux through glycogenolysis is higher than lipolysis during the first month of diapause. Lastly, our analysis revealed that 38% of the initial 14C-labled lipid that was synthesized during the adult pre-diapause phase was still present following the first gonotrophic cycle. About 33% of this remaining 14C-labeled lipid was localized to the newly developed eggs, suggesting that lipid sparing processes during a minimal 7-week long diapause may enhance egg production. © 2008 Elsevier Ltd. All rights reserved.
PMID: 1917967;Abstract:
Glucocorticoid treatment of certain lymphoma cell lines and thymocytes activates a self-destructive pathway of programmed cell death referred to as apoptosis. Calcium and calmodulin (CaM) may be important signals in the apoptotic cascade because an early event is a sustained elevation in cytosolic Ca2+ and CaM inhibitors interfere with the death pathway. In the present study, expression of the CaM gene was examined during glucocorticoid-induced apoptosis in WEHI7.2 lymphocytes. Steady state levels of CaM mRNA were increased up to 10-fold following a 4-6-h exposure of WEHI7.2 cells to 10-6 M dexamethasone. This increase was mediated through the glucocorticoid receptor since the response was not observed in WEHI7.418, a variant line which does not express active glucocorticoid receptor. Induction of CaM mRNA was dose-dependent and highly specific for glucocorticoids, as other steroids were unable to elicit the response. A stringent cell specificity was also observed. Pretreatment of WEHI7.2 lymphocytes with cycloheximide did not interfere with dexamethasone-dependent increases in CaM mRNA levels, and studies with actinomycin D demonstrated that the stability of the transcript was not altered by hormone. Finally, a calmodulin inhibitor elicited a protective effect on WEHI7.2 cells following glucocorticoid exposure. These results indicate that CaM mRNA levels were hormonally controlled in WEHI7.2 lymphocytes and support the putative involvement of CaM in glucocorticoid-induced apoptosis.
