Noreen M. Robertson

Inhibition of Glucocorticoid-induced Apoptosis by Targeting the Major Splice Variants of BIM mRNA with Small Interfering RNA and Short Hairpin RNA

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirus-mediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells.

Differential Expression of TRAIL and TRAIL Receptors in Allergic Asthmatics Following Segmental Antigen Challenge: Evidence for a Role of TRAIL in Eosinophil Survival

Asthma is a chronic lung disease exhibiting airway obstruction, hyperresponsiveness, and inflammation, characterized by the infiltration of eosinophils into the airways and the underlying tissue. Prolonged eosinophilic inflammation depends on the balance between the cell’s inherent tendency to undergo apoptosis and the local eosinophil-viability enhancing activity. TRAIL, a member of the TNF family, induces apoptosis in most transformed cells; however, its role in health and disease remains unknown. To test the hypothesis that Ag-induced inflammation is associated with TRAIL/TRAIL-R interactions, we used a segmental Ag challenge (SAC) model in ragweed-allergic asthmatics and nonasthmatic patients and analyzed bronchoalveolar lavage (BAL) material for 2 wk. In asthmatic patients, the level of TRAIL in BAL fluid dramatically increased 24 h after SAC, which significantly correlated with BAL eosinophil counts. Immunohistochemical analysis of bronchial biopsies from asthmatic patients demonstrated that TRAIL staining was increased in epithelial, airway smooth muscle, and vascular smooth muscle cells and throughout the interstitial tissue after SAC. This was confirmed by quantitative immunocytochemical image analysis of BAL eosinophils and alveolar macrophages, which demonstrated that expression levels of TRAIL and DcR2 increased, whereas expression levels of the TRAIL-Rs DR4 and DR5 decreased in asthmatic subjects after SAC. We also determined that TRAIL prolongs eosinophil survival ex vivo. These data provide the first in vivo evidence that TRAIL expression is increased in asthmatics following Ag provocation and suggest that modulation of TRAIL and TRAIL-R interactions may play a crucial role in promoting eosinophil survival in asthma.

Regulation of the Human Na/K-ATPase β1 Gene Promoter by Mineralocorticoid and Glucocorticoid Receptors

Expression of the human Na/K-ATPase β1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions −650 to −630, within the β1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the β1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase β1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the β1 promoter effectively down-regulates transcription.

Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils

IL‐5 is a potent eosinophil viability‐enhancing factor that has been strongly implicated in the pathogenesis of IgE‐mediated inflammation in vivo. Recently published data have suggested that IL‐5 (and related cytokines) may act by altering the expression of the anti‐apoptotic regulator Bcl‐2 or its homologues, but this is controversial. The behaviour of the recently described pro‐apoptotic cysteine proteases (caspases) in eosinophils after IL‐5 treatment has not been explored. We examined the effect of IL‐5 on the expression of four major Bcl‐2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti‐apoptotic Mcl‐1 and pro‐apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl‐x, Bcl‐2. IL‐5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl‐2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL‐5 completely blocked caspase processing in spontaneous and dexamethasone‐induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL‐5 acts by altering the balance of anti‐apoptotic and pro‐apoptotic Bcl‐2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.

Inhibition of glucocorticoid-induced apoptosis in 697 Pre-B lymphocytes by the mineralocorticoid receptor N-terminal domain

The glucocorticoid and mineralocorticoid receptors (GR and MR) share considerable structural and functional homology and bind as homodimers to hormone-response elements. We have shown previously that MR and GR can form heterodimers that inhibit transcription from a glucocorticoid (GC)-responsive gene and that this inhibition was mediated by the N-terminal domain (NTD) of MR. In this report, we examined the effect of NTD-MR on GC-induced apoptosis in the GC-sensitive pre-B lymphoma cell line, 697. In GC-treated 697 cells, we demonstrated that stable expression of NTD-MR blocks apoptosis and inhibits proteolytic processing of pro-caspases-3, -8, and -9 and poly(ADP-ribose) polymerase. Importantly, gel shift and immunoprecipitation analyses revealed a direct association between the GR and amino acids 203–603 of NTD-MR. We observed down-regulation of c-Myc and of the anti-apoptotic proteins Bcl-2 and Bfl-1 as well as high levels of the pro-apoptotic proteins Bax and Bid. Conversely, cells stably expressing NTD-MR exhibited increased expression of Bcl-2 and Bfl-1 and diminished levels of Bid and Bax. These data provide a potential mechanism for the observed inhibition of cytochromec and Smac release from the mitochondria of NTD-MR cells and resultant resistance to GC-induced apoptosis. Thus, NTD-MR may mediate GC effects through heterodimerization with GR and ensuing inhibition of GC-regulated gene transcription.

Inhibition of TRAIL-induced apoptosis by IL-8 is mediated by the p38-MAPK pathway in OVCAR3 cells

Introduction: TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of the TNF superfamily of cell death inducing ligands. Interestingly, while malignant cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells do not appear to be sensitive to this ligand, making it a desirable therapeutic compound against many cancers, including many ovarian carcinomas. Interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be at significantly higher level in the ascites from patients with ovarian cancer. We have previously demonstrated a role for IL-8 in blocking TRAILs ability to induce apoptosis in the ovarian cancer cell line, OVCAR3, possibly by repressing the DR4 TRAIL receptor expression and blocking caspase-8 cleavage. In addition, we showed a member of the mitogen-activated protein kinase (MAPK) superfamily, p38γ, is among the genes regulated in OVCAR3 cells by TRAIL and IL-8. The present study further investigates involvement of the p38 MAPK pathway in IL-8s ability to block TRAIL-induced apoptosis in the ovarian surface epithelial cancer cell line, OVCAR3.Results: In this study we demonstrate that p38γ as well as p38α play a significant role in IL-8s ability to block TRAIL-induced apoptosis. Through array analysis, as well as confirmation with other methods, we detected regulation of p38γ and p38α following treatment of the cancer cell line with IL-8 or TRAIL. We also tested two other isoforms of p38 MAPK, p38β and p38δ, but did not find significant regulation by IL-8 or TRAIL. We also examined activation of the p38 MAPK pathway, up-stream as well as down-stream, and noticed activation of the pathway following treatment with TRAIL and decreased activity when IL-8 was introduced. With the use of specific inhibitors, we were able to further confirm the role of this pathway in TRAIL-induced apoptosis, and IL-8s ability to block this apoptosis, in ovarian cancer cell lines.Conclusion: Taken together, these results further solidify the role of IL-8 in blocking the TRAIL-induced apoptosis in these ovarian carcinoma cells and provide new molecular insight into this potentially important therapeutic target.

TRAIL in the airways.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is an important immunomodulatory factor that may play a role in the structural changes observed in the asthmatic airways. In vitro as well as in vivo studies have evidenced a dual role for TRAIL: it can either function as a pro- or anti-inflammatory cytokine on inflammatory cells, participating in the initiation and resolution of inflammatory and immune responses. TRAIL is expressed in the airways by inflammatory cells infiltrated in the bronchial mucosa, as well as by structural cells of the airway wall including fibroblasts, epithelial, endothelial, and smooth muscle cells. By releasing TRAIL, these different cell types may then participate in the increased levels of TRAIL observed in bronchoalveolar lavage fluid from asthmatic patients. Taken together, this suggests that TRAIL may play a role in inflammation in asthma. However, concerning its role is dual in the modulation of inflammation, further studies are needed to elucidate the precise role of TRAIL in the airways.

Evidence for an association of a ribonucleic acid with the purified, unactivated glucocorticoid receptor

The unactivated glucocorticoid-receptor complex (GRC) was purified from rat hepatic cytosol (approximately 4000-fold by specific activity) by a procedure developed in our laboratory. Following elution of unactivated GRC from DEAE-cellulose with a 0.05-0.5 M potassium phosphate gradient, a second gradient of 0.5-1.0 M potassium chloride was started. This gradient eluted material at 0.6 M potassium chloride that incorporated [32P] in vivo and stained with ethidium bromide. A predominant ethidium bromide stained band of 100-110 nucleotides was observed. The presence of this material was dependent on the presence of highly purified GRC since presaturation of cytosol with 50 microM unlabeled triamcinolone acetonide (TA) precluded the appearance of this material. Experiments with partially purified GRC from CEM-C7 cells incubated in vivo with [14C]uridine indicated that the material eluted at 0.6 M potassium chloride incorporated [14C]uridine. Collectively, these data suggest that a RNA is associated with the purified, unactivated form of the GRC.

The N-terminal domain of the mineralocorticoid receptor modulates both mineralocorticoid receptor-and glucocorticoid receptor-mediated transactivation from Na/K ATPase β1 target gene promoter

Mineralocorticoid and glucocorticoid hormones activate the expression of the Na/K ATPase β1 through direct binding of the mineralocorticoid receptor (MR) and glucocorticoid receptors (GR) to a mineralocorticoid-and glucocorticoid-responsive element in the β1 promoter region, but activation of the β1 promoter is inhibited by coexpression of both receptors. Here, using a series of mutated and chimeric receptors, we show that the N-terminal region of MR mediates an inhibitory effect on MR and GR activation from the β1 promoter, in CV-1 cells. Deletion of the N-terminal region of MR (1-603) enhanced MR activation four-fold. Activation by chimeric MR, in which the N-terminus of GR replaces the N-terminal region of MR, was threefold that of wild-type MR. In addition, whereas coexpression of wild-type MR and GR was inhibitory, coexpression of chimeric MR and wild-type GR was nearly equal to that of MR. By contrast, mutated GR lacking its N-terminal region (1–420) was less efficient than the wild type in activating this promoter. These results demonstrate that the N-terminal domains of MR and GR have opposite transactivation properties and that MR region 1–603 is indeed inhibitory for both MR- and GR-mediated regulation of the Na/K ATPase β1 gene promoter.

Evaluation of glucocorticoid sensitivity in 697 pre-B acute lymphoblastic leukemia cells after overexpression or silencing of MAP kinase phosphatase-1

Purpose To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697.Methods Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death.Results MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6±1.9%.Conclusions Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.