Glucocorticoid-mediated co-regulation of RCAN1-1, E4BP4 and BIM in human leukemia cells susceptible to apoptosis.

Glucocorticoids (GCs) are known to induce apoptosis of leukemia cells via gene regulatory changes affecting key pro-and anti-apoptotic genes. Three genes previously implicated in GC-evoked apoptosis in the CEM human T-cell leukemia model, RCAN1, E4BP4 and BIM, were studied in a panel of human lymphoid and myeloid leukemia cell lines. Of the two RCAN1 transcripts, the synthetic GC Dexamethasone (Dex) selectively upregulates RCAN1-1, but not RCAN1-4, in GC-susceptible Sup-B15, RS4;11, Kasumi-1 cells but not in GC-resistant Sup T1 and Loucy cells.

Correlation of glucocorticoid-mediated E4BP4 upregulation with altered expression of pro- and anti-apoptotic genes in CEM human lymphoblastic leukemia cells.

In Caenorhabditiselegans, motorneuron apoptosis is regulated via a ces-2-ces-1-egl-1 pathway. We tested whether human CEM lymphoblastic leukemia cells undergo apoptosis via an analogous pathway. We have previously shown that E4BP4, a ces-2 ortholog, mediates glucocorticoid (GC)-dependent upregulation of BIM, an egl-1 ortholog, in GC-sensitive CEM C7-14 cells and in CEM C1-15mE#3 cells, which are sensitized to GCs by ectopic expression of E4BP4.

E4BP4 facilitates glucocorticoid-evoked apoptosis of human leukemic CEM cells via upregulation of Bim.

Background: Synthetic GCs serve as therapeutic agents for some lymphoid leukemias because of their ability to induce transcriptional changes via the GC receptor (GR) and trigger apoptosis. Upregulation of the BH3-only member of Bcl-2 family proteins, Bim, has been shown to be essential for GC-evoked apoptosis of leukemic lymphoblasts. Using human T cell leukemic sister clones CEM-C7-14 and CEM-C1-15, we have previously shown that the bZIP transcriptional repressor, E4BP4, is preferentially upregulated by GCs in CEM-C7-14 cells that are susceptible to GC-evoked apoptosis, but not in refractory CEM-C1-15 cells.

Quantitative polymerase chain reaction analysis by deconvolution of internal standard.

Background: Quantitative Polymerase Chain Reaction (qPCR) is a collection of methods for estimating the number of copies of a specific DNA template in a sample, but one that is not universally accepted because it can lead to highly inaccurate (albeit precise) results. The fundamental problem is that qPCR methods use mathematical models that explicitly or implicitly apply an estimate of amplification efficiency, the error of which is compounded in the analysis to unacceptable levels.

Results: We present a new method of qPCR analysis that is efficiency-independent and yields accurate and precise results in controlled experiments.

Glucocorticoid evoked upregulation of RCAN1-1 in human leukemic CEM cells susceptible to apoptosis.

Background: Glucocorticoid hormones (GCs) induce apoptosis of leukemic T-cells by transcriptional regulation via the GC receptor, GR. In the human leukemic CEM cell culture model, RCAN1 has been identified as one of the genes that is specifically upregulated only in the GC-sensitive CEM C7-14 cells, but not in the GC-resistant CEM-C1-15 sister cells in correlation with GC-evoked apoptosis. RCAN1 gene encodes two major protein isoforms of the regulator of calcineurin (RCAN1), RCAN1-1 and RCAN1-4 via alternative splicing of exons 1 and 4 respectively, to exons 5-7.

Calcium-dependent upregulation of E4BP4 expression correlates with glucocorticoid-evoked apoptosis of human leukemic CEM cells.

Glucocorticoid (GC)-evoked apoptosis of T-lymphoid cells is preceded by increases in the intracellular Ca2+ concentration ([Ca2+]i), which may contribute to apoptosis. This report demonstrates that GC-mediated upregulation of the bZIP transcriptional repressor gene, E4BP4, is dependent on [Ca2+]i levels, and correlates with GC-evoked apoptosis of GC-sensitive CEM-C7-14 cells. Calcium chelators EGTA and BAPTA reduced [Ca2+]i levels and protected CEM-C7-14 cells from Dex-evoked E4BP4 upregulation as well as apoptosis.

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis.

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out.

Microarray-based expression profiling of normal and malignant immune cells.

Recent advances in gene microarray technology have facilitated global analyses of gene expression profiles in normal and malignant immune cells. Great strides have been made in our understanding of molecular differences among various types of immune cells, the process of T and B cell activation, and the genomic changes that convert normal cells to malignant ones. Genomic analysis has become a crucial aspect of cancer classification, diagnosis, therapy, and prognosis.

Glucocorticoids and polyamine inhibitors synergize to kill human leukemic CEM cells.

Glucocorticoids are well-known apoptotic agents in certain classes of lymphoid cell malignancies. Reduction of intracellular polyamine levels by use of inhibitors that block polyamine synthesis slows or inhibits growth of many cells in vitro. Several such inhibitors have shown efficacy in clinical trials, though the toxicity of some compounds has limited their usefulness.

Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells.

Sensitivity to glucocorticoid (GC)-evoked apoptosis in lymphoid cell lines correlates closely with GC-mediated suppression of c-Myc expression. To establish a functional role for c-Myc in GC-mediated apoptosis, we have stably expressed MycER(TM), the human c-Myc protein fused to the modified ligand-binding domain of the murine estrogen receptor alpha, in GC-sensitive CEM-C7-14 cells. In CEM-C7-14 cells, MycER(TM) constitutively imparts c-Myc functions.

The delayed induction of c-jun in apoptotic human leukemic lymphoblasts is primarily transcriptional.

Because of their ability to induce lymphoid cell apoptosis, glucocorticoids have been used for decades to treat certain human leukemias and lymphomas. Studies presented in this paper complement our previous work demonstrating that sustained induction of the proto-oncogene c-jun plays a crucial role in the glucocorticoid-induced apoptotic pathway in CEM cells, human leukemic lymphoblasts. Results from measurements of c-jun mRNA half-life with RNase protection assays and of transcription by nuclear run-on assays indicate that, in the dexamethasone-sensitive cloned CEM-C7 cells, c-jun is induced at the transcriptional level.

Glucocorticoid mediated transcriptional repression of c-myc in apoptotic human leukemic CEM cells.

Suppression of c-myc has been implicated as a critical event in some glucocorticoid-evoked apoptotic systems. It is therefore of interest to understand the mechanism of glucocorticoid-regulation of the c-myc gene. In the present study, a detailed analysis of dexamethasone (Dex)-evoked regulation of the human c-myc gene in human leukemic CEM-C7 cells has been performed.

Hormonal regulation of physiological cell turnover and apoptosis.

Physiological cell turnover plays an important role in maintaining normal tissue function and architecture. This is achieved by the dynamic balance of cellular regeneration and elimination, occurring periodically in tissues such as the uterus and mammary gland, or at constant rates in tissues such as the gastrointestinal tract and adipose tissue. Apoptosis has been identified as the prevalent mode of physiological cell loss in most tissues.

Structure-apoptotic potency evaluations of novel sterols using human leukemic cells.

Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM-C7-14 human leukemic cells. In addition to testing 15-ketocholestenol (K15), 15-ketocholestenol hydroxyethyl ether (CK15), and 7-ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25-hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC > K15 > CK15 > CK7.

Glucocorticoids, oxysterols, and cAMP with glucocorticoids each cause apoptosis of CEM cells and suppress c-myc.

In clones of the CEM human acute lymphoblastic leukemic cell line, glucocorticoids, oxysterols and activators of the cAMP pathway acting synergistically with glucocorticoids, each can cause apoptotic cell death. Morphologically and kinetically, these deaths resemble one another. The kinetics are striking: in each case, after addition of the lethal compound(s), an interval of approximately 24 h follows, during which cell growth continues unabated.

Resistance of human leukemic CEM-C1 cells is overcome by synergism between glucocorticoid and protein kinase A pathways: correlation with c-Myc suppression.

Glucocorticoids (GCs) induce apoptosis in lymphoid cells that contain functional GC receptors (GRs). However, GC resistance often is seen in cells with demonstrable GRs; one such line is CEM-C1. We have tested the hypothesis that positive interactions between GC and cyclic AMP (cAMP) regulate GC actions in CEM clones.

Agonist-specific modulation of glucocorticoid receptor-mediated transcription by immunosuppressants.

Although the immunosuppressive drugs FK506, rapamycin and cyclosporin A have been reported to potentiate transcriptional activation mediated by a non-saturating concentration of the glucocorticoid receptor agonist dexamethasone, the precise mechanism(s) underlying these responses remains unclear. The murine L-929-derived LMCAT cell line stably transfected with the mouse mammary tumor virus promoter-chloramphenicol acetyl transferase reporter gene construct was utilized in the present study to further investigate the mechanism(s) underlying this dexamethasone potentiation as well as the possible agonist specificity of this potentiation. The present data demonstrate that pretreatment (2 h) of LMCAT cells with 10 microM FK506, rapamycin or cyclosporin A results in the potentiation of reporter gene transcription mediated not only by dexamethasone (approximately 12-fold), but also by hydrocortisone (approximately 6-fold) and triamcinolone acetonide (approximately 2.

Trans-retinoic acid and glucocorticoids synergistically induce transcription from the mouse mammary tumor virus promoter in human embryonic kidney cells.

Human embryonic kidney (K293) cells transfected with a mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc) were utilized to investigate the potential effects of trans-retinoic acid (tRA), either by itself or in combination with glucocorticoid (GC) hormones, on a well-characterized, GC-sensitive transcriptional response. tRA or the synthetic GC hormone dexamethasone induced transcription from the MMTV promoter in a dose-dependent manner, with 1 micromol tRA and 1 micromol dexamethasone alone causing a four- to six-fold and a 40-fold induction of basal transcription, respectively. Simultaneous treatment with 1 micromol dexamethasone and 1 micromol tRA resulted in a synergistic transcriptional response that was 120-fold higher than basal level and 2.

Stimulation of tissue plasminogen activator production by retinoic acid: synergistic effect on protein kinase C-mediated activation.

Trans retinoic acid (t-RA) stimulated the production of tissue plasminogen activator (tPA) in HeLa-S3 and human umbilical vein endothelial cells (huvecs) in a dose-dependent manner with maximal release (four to five times control) at 40 nmol/L and 40 mumol/L, respectively. In endothelial cells, the stimulation of tPA production by phorbol 12-myristate 13-acetate (PMA) was potentiated 1.9-fold by 10 mumol/L t-RA, or 1.

Characterization of a novel glutathione S-transferase isoenzyme from mouse lung and liver having structural similarity to rat glutathione S-transferase 8-8.

In mouse lung, glutathione S-transferase (GST, EC 2.5.1.

Purification and characterization of dinitrophenylglutathione ATPase of human erythrocytes and its expression in other tissues.

S-(2,4-dinitrophenyl)glutathione (Dnp-SG) ATPase of human erythrocytes has been purified to apparent homogeneity by affinity chromatography. In reduced denaturing gels, the subunit Mr value of Dnp-SG ATPase was found to be 38,000. Dinitrophenyl glutathione (Dnp-SG) stimulated the hydrolysis of ATP by the purified enzyme whereas oxidized glutathione (GSSG) did not, indicating that Dnp-SG and GSSG are transported from the erythrocytes by different transporters.

Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues.

The suitability of mouse as an animal model for studying the glutathione S-transferase (GST)-mediated detoxification mechanisms has been studied by analyzing the expression of the alpha, mu, and pi classes of glutathione S-transferase isoenzymes in mouse brain, heart, kidney, spleen, liver, and muscle. Individual isoenzymes from each of these tissues have been purified, characterized, and classified into the three known classes of GST. These studies demonstrate that GST isoenzymes are variably expressed in different mouse tissues, suggesting that their expression is tissue specific.

Glutathione S-transferase and P-glycoprotein in multidrug resistant Chinese hamster cells.

Glutathione S-transferases (GSTs) have been reported to be elevated in some forms of hepatic carcinogenesis, in multidrug resistant (MDR) cells exhibiting elevated P-glycoprotein, and in cells resistant to alkylating agents independent of the MDR phenotype. The reported elevation of GST in association with the MDR phenotype and the overexpression of P-glycoprotein along with induction of GST in hepatic carcinogenesis suggest a correlation in the two mechanisms of cellular detoxification. To evaluate this hypothesis we examined the expression of GSTs in an MDR Chinese hamster fibroblast cell line overexpressing P-glycoprotein.