Mechanisms Of Glucocorticoid Resistance Research Articles

101. Brain molecular mechanisms of glucocorticoid resistance

101. Brain molecular mechanisms of glucocorticoid resistance

Modulation of 11β-Hydroxysteroid Dehydrogenase Expression by Bombesin: A Possible Mechanism for Glucocorticoid Resistance in Androgen Independent Prostate Cancer

Treatment with glucocorticoids is one of a limited number of options for androgen independent prostate cancer. Neuroendocrine differentiation has been shown to contribute to androgen-independent prostate cancer progression. To study the potential link between neuroendocrine differentiation and the glucocorticoid action, we investigated the effects of the product of neuroendocrine differentiation--bombesin on glucocorticoid metabolizing enzymes--11beta-hydroxysteroid dehydrogenases in PC-3 cells. Our Western analysis, RT-PCR, and activity assays demonstrate that while 18-hour exposure to bombesin reduces 11beta-hydroxy-steroid dehydrogenases-1 profiles (activities 25% less, protein level 29% lower, mRNA levels 45% lower), contrarily it increases 11beta-hydroxysteroid dehydrogenases-2 profiles (activities 34%, protein levels 100%, mRNA levels 120%). Blockade bombesin action with bombesin receptor antagonists and the enzyme degrading bombesin prevented these changes, suggesting the observed modulations were bombesin receptor-specific. In addition, bombesin increased the amounts of interleukin-8 and mRNA of vascular endothelial growth factor receptor 2, which were lowered in the presence of cortisol, suggesting that neuropeptide blockade may extend the benefits of glucocorticoids in treating androgen-independent prostate cancer.

Divergent Mechanisms of Glucocorticoid Resistance in Experimental Models of Pediatric Acute Lymphoblastic Leukemia

Cell line models of glucocorticoid resistance in childhood acute lymphoblastic leukemia (ALL) almost invariably exhibit altered glucocorticoid receptor (GR) function. However, these findings are incongruous with those using specimens derived directly from leukemia patients, in which GR alterations are rarely found. Consequently, mechanisms of glucocorticoid resistance in the clinical setting remain largely unresolved. We present a novel paradigm of glucocorticoid resistance in childhood ALL, in which patient biopsies have been directly established as continuous xenografts in immune-deficient mice, without prior in vitro culture. We show that the GRs from six highly dexamethasone-resistant xenografts (in vitro IC(50) >10 micromol/L) exhibit no defects in ligand-induced nuclear translocation and binding to a consensus glucocorticoid response element (GRE). This finding contrasts with five commonly used leukemia cell lines, all of which exhibited defective GRE binding. Moreover, whereas the GRs of dexamethasone-resistant xenografts were transcriptionally active, as assessed by the ability to induce the glucocorticoid-induced leucine zipper (GILZ) gene, resistance was associated with failure to induce the bim gene, which encodes a proapoptotic BH3-only protein. Furthermore, the receptor tyrosine kinase inhibitor, SU11657, completely reversed dexamethasone resistance in a xenograft expressing functional GR, indicating that pharmacologic reversal of glucocorticoid resistance in childhood ALL is achievable.

Mechanism of glucocorticoid resistance

Many patients have a form of glucocorticoid resistance, yet the mechanism of this serious medical problem has not been established. Previous studies have shown that glucocorticoid resistance is associated with impairment of transcriptional factors, such as activator protein 1 (AP-1), but an integrated view that allows for significant variability in individual patient responses has been unclear. In the current study, the investigators examined murine and human T cells for their responsiveness to glucocorticoids in vitro, aiming to determine whether the degree of signaling during T cell activation, affected responsiveness. Notably, dexamethasone (Dex) did not promote substantial T-cell apoptosis. Rather, Dex suppressed T-cell receptor (TCR)–induced proliferation of naive CD4+ T cells through a mechanism involving downregulation of c-Fos expression and inhibition of AP-1, nuclear factor of activated T cells (NF-AT), and NF-kB transcriptional activity. Importantly, enhancement of TCR signaling by CD28- or IL-2–mediated costimulation blocked the suppressive effect of Dex on c-Fos expression and AP-1 function and restored cellular proliferation. In contrast, NF-kB remained inhibited by Dex. Using specific signaling inhibitors, the investigators demonstrated that costimulatory signaling via MAPK kinase (MEK) and extracellular signal–regulated kinase (ERK) was essential for the development of T-cell resistance to Dex. Selective blockade of MEK/ERK signal transduction abolished the costimulation-induced resistance. Thus, the amount of signaling through the MAPK pathway was critical in determining the effect of Dex on T-cell activation. These results imply that the degree of local costimulation through MEK/ERK, affected by variable processes (including antigenic exposure in individual patients), can modify the effect of glucocorticoids on T cells. These findings raise hope that MAPK inhibitors might have therapeutic utility for glucocorticoid resistance.

Molecular mechanisms of glucocorticoid action and resistance

The actions of glucocorticoid hormones are mediated by an intracellular receptor, the glucocorticoid receptor (GR). The mechanism of action of this ligand-inducible transcription factor is discussed, focusing on mechanisms of glucocorticoid resistance. Three mechanisms are highlighted: ligand-induced down-regulation of the receptor, the dominant-negative inhibition by the β-isoform of the receptor, and repression by the transcription factor NF-κB. It has been shown that these mechanisms can significantly inhibit glucocorticoid signaling, and could therefore seriously decrease the efficacy of glucocorticoids used clinically.

Dexamethasone-resistant human Pre-B leukemia 697 cell line evolving elevation of intracellular glutathione level: an additional resistance mechanism.

Glucocorticoids remain among the most important drugs in the treatment of acute lymphoblastic leukemia (ALL). Although the mechanisms of glucocorticoid resistance have been studied in some T‐cell leukemic cell lines, less work has been done with B‐cell lines. We established a dexamethasone (DEX)‐resistant human pre‐B lineage leukemia cell line (697/DEX) and investigated the mechanism of resistance. 697/DEX was over 430–fold more resistant to DEX compared with the parental cells (697/Neo). Overexpression of Bcl–2 protein was not observed in 697/DEX, different from the mechanism of resistance in Bcl–2–virus‐infected cells (697/Bcl–2). Although the expression of p‐glycoprotein (Pgp) in 697/DEX was positive, its functional activity was not detected. The numbers of glucocorticoid receptors (GR) in 697/DEX and 697/Bcl–2 were significantly lower than those in 697/Neo. In addition, 697/DEX and 697/Bcl–2 had higher levels of glutathione (GSH) than 697/Neo. In the presence of l‐buthionine‐(S, R)‐sulfoximine (BSO), an inhibitor of GSH synthesis, both 697/DEX and 697/Bcl–2 recovered their sensitivity to DEX. Interestingly, cell death by the depletion of GSH did not involve caspase–3/7 activation in 697/Bcl–2 and 697/DEX, different from 697/Neo, suggesting a death mechanism through caspase‐independent programmed cell death or necrosis. In conclusion, DEX‐resistance in 697/DEX was related not only to a GR decrease, but also to an increase in intracellular GSH level in the DEX‐resistant B‐cell leukemia cell line. Circumvention of DEX‐resistance with BSO may offer an approach to overcoming resistance to chemotherapy in B‐cell lineage ALL.

Imbalanced Expression of the Glucocorticoid Receptor Isoforms in Cultured Lymphocytes from a Patient with Systemic Glucocorticoid Resistance and Chronic Lymphocytic Leukemia

The human glucocorticoid receptor (GR) is expressed as two alternatively spliced isoforms, GRα and GRβ. Whereas GRα is a hormone-activated transcription factor, GRβ does not bind glucocorticoids (GCs), is transcriptionally inactive, and is a potential inhibitor of activated GRα. Differential expression of GR isoforms may play a role in generalized or tissue-specific GC resistance. GCs induce apoptosis in neoplastic lymphoid cells; and, defective apoptosis is implicated in the genesis of chronic lymphocytic leukemia (CLL). We studied a patient with generalized GC resistance and CLL. GR number in the patient's transformed lymphocytes was approximately one half that of control cells with a ∼10-fold reduction in binding affinity for dexamethasone.In vitroapoptosis induction in CLL cells was delayed in response to GCs, but not to other apoptosis inducers. Sequencing of the GR cDNA and gene including the 2.3-kb coding region, the intron/exon junctions, the known 5′-regulatory region, and ∼300 bp of the 3′-region revealed no alterations. Western blot with an N-terminal antibody showed normal levels of immunoreactive GR, but quantitative analysis with isoform-specific C-terminal antibodies revealed a markedly reduced GRα expression, and high GRβ expression. These findings indicate that imbalanced expression of the GR isoforms may be a mechanism of GC resistance, and may have implications for tumorigenesis by enhancing cell survival.

Cell cycle-dependent glucocorticoid receptor phosphorylation and activity.

Proliferating cells display striking cell cycle dependence in sensitivity to gene activation by glucocorticoids; they are sensitive in late gap 1/synthesis (G1/S) (late G1 and S phases) but resistant in gap 2/mitotic (G2/M). Here we describe large cell cycle-dependent variations in glucocorticoid receptor (GR) phosphorylation that accompany, and may account for, the changes in sensitivity. GRs are basally phosphorylated and undergo hyperphosphorylation after hormone-induced activation. Identified phosphorylated sites are all in the N-terminal domain. Several lie in a region required for full transactivating activity and reduction of nonspecific binding to DNA. Most are in consensus sequences for cell cycle-associated kinases, suggesting that such kinases phosphorylate GRs. We now show with WCL2 cells (Chinese hamster ovary cells with overexpressed GRs) that: 1) glucocorticoid treatment fails to hyperphosphorylate GRs in G2/M but doubles phosphorylation in S, more than seen with unsynchronized cells; and 2) basal GR phosphorylation is almost three times higher in G2/M than S. These results, along with earlier observations, implicate GR phosphorylation with mechanisms of glucocorticoid resistance in G2/M. Such mechanisms might underlie some forms of glucocorticoid resistance in inflammatory and lymphoproliferative diseases. HPLC phosphopeptide maps of GRs from S and G2/M reveal no significant qualitative differences in phosphorylated sites, consistent with a general increase during G2/M in negative charge of the N-terminal domain. We also show that the previously described increase in GR hormone-binding capacity from G1 to S is accompanied by a parallel increase in GR protein.

Glucocorticoids inhibit cytokine-mediated eosinophil survival.

Glucocorticoids characteristically induce eosinopenia in vivo and are effective for treating allergic and other eosinophilic disorders. We studied the effect of glucocorticoids on cytokine-induced survival of human eosinophils in vitro. Eosinophils were purified from normal or mildly atopic volunteers by Percoll density gradient and incubated for 4 days in the presence of cytokine plus steroid. Cell viabilities were determined by staining cells with fluorescein diacetate and propidium iodide. In the absence of glucocorticoids, human rIL-5 enhanced eosinophil survival in a dose-dependent manner, from 22 fM for a minimal effect to 2200 fM for maximal effect. When eosinophils were cultured with a submaximal concentration of rIL-5 (220 fM), dexamethasone, methylprednisolone, and hydrocortisone inhibited eosinophil survival in a dose-dependent manner. Inhibition was time-dependent and required at least 2 days' exposure of eosinophils to dexamethasone. Dexamethasone, methylprednisolone, and hydrocortisone at 1000 nM inhibited survival by 88 +/- 2, 66 +/- 9 and 37 +/- 7%. In contrast, estradiol and testosterone (1000 nM) had no effect on eosinophil survival. When eosinophils were incubated with varying concentrations of human rIL-5 and 1000 nM dexamethasone, survival inhibition was reduced at higher concentrations of human rIL-5, and completely abolished by human rIL-5 23,000 fM. Human recombinant granulocyte-macrophage CSF, human rIL-3, and human rIFN-gamma also enhanced eosinophil survival in a dose-dependent manner and dexamethasone (1000 nM) strongly inhibited cell survival when submaximal concentrations of these cytokines were used. The effects of dexamethasone were reversed by higher concentrations of granulocyte-macrophage CSF (10 U/ml) and IL-3 (3 ng/ml). However, even 1000 U/ml IFN-gamma did not overcome dexamethasone inhibition, indicating a difference between the mechanism of eosinophil survival induced by IFN-gamma and other cytokines. These results suggest that glucocorticoids exert a direct, inhibitory effect on eosinophil survival, which may be important in the treatment of allergic and other eosinophilic disorders. Antagonism of this effect by higher amounts of cytokine may be a mechanism for glucocorticoid resistance.

The mechanism for glucocorticoid-resistance in a rat hepatoma cell variant that contains functional glucocorticoid receptor

The mechanism of glucocorticoid resistance has been studied in a rat hepatoma cell variant (6.10.2) ,which contains low levels of glucocorticoid receptor (GR). These cells seem to have lost all the glucocorticoid-induced transcriptional responses as measured by the lack of induction of expression of stably integrated mouse mammary tumor virus (MMTV) and the endogenous gene tyrosine aminotransferase (TAT), as well as the transcriptional suppression of GR gene expression. Physico-chemical characterization of the GR in the glucocorticoid resistant 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with regard to size hormone- and DNA-binding. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. Treatment of 6.10.2 cells with 8-bromo-cAMP, which induced the endogenous GR level two-fold, restored responsiveness to glucocorticoids. Secondly, pretreatment of the cells with cycloheximide also led to reacquisition of cellular responsiveness to glucocorticoids. We propose that there exists a “threshold” level of GR, which is required for responsiveness and that under normal culture conditions, the level of GR in 6.10.2 cells is below this threshold. Glucocorticoid responsiveness can be restored by raising the GR level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier (repressor protein) with cycloheximide. Finally, the existence of such a repressor protein for MMTV induction was shown by in vivo titration with an isolated negative cis-element from the MMTV promoter.

A glucocorticoid-resistant rat hepatoma cell variant contains functional glucocorticoid receptor.

The mechanism of glucocorticoid resistance was studied in a rat hepatoma cell variant (6.10.2) which contains low levels of glucocorticoid receptor. These cells seem to have lost glucocorticoid-induced transcriptional responses as measured by the induction of expression of stably integrated mouse mammary tumor virus gene and the endogenous tyrosine aminotransferase gene, as well as the transcriptional suppression of glucocorticoid receptor gene expression. However, characterization of the glucocorticoid resistance in 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with respect to hormone binding and affinity for both nonspecific and specific DNA sequences. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. First, treatment of 6.10.2 cells with 8-bromo-cAMP elevated the endogenous glucocorticoid receptor levels 2-fold and restored responsiveness to glucocorticoids. Second, pretreatment of the cells with cycloheximide also led to acquisition of cellular responsiveness to glucocorticoids. We propose that there exists a "threshold" level of glucocorticoid receptor which is required for responsiveness and that under normal culture conditions, the level of glucocorticoid receptor in 6.10.2 cells is below this threshold. However, glucocorticoid responsiveness can be restored by raising the glucocorticoid receptor level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier with cycloheximide.