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[Cancer Research 63, 172-178, January 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

Role of Apical Caspases and Glucocorticoid-regulated Genes in Glucocorticoid-induced Apoptosis of Pre-B Leukemic Cells1

Sonia L. Planey2, Marc T. Abrams2, Noreen M. Robertson and Gerald Litwack3

Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glucocorticoid (GC) sensitivity in hematopoietic cells requires the activation and nuclear translocation of the glucocorticoid receptor (GR) and the subsequent activation of caspases. To gain insight into the caspase cascade responsible for the execution phase of GC-induced apoptosis, 697 pre-B leukemic cells were stably transfected with dominant negative forms of caspase-8, caspase-9, or caspase-10 and the caspase-8 inhibitor CrmA. We observed that inhibition of caspase-9 or caspase-10 activity, but not caspase-8, caused partial resistance of 697 cells to GC-induced apoptosis. Inhibition of multiple caspases through the use of specific peptide inhibitors had an additive effect and caused complete resistance. To identify GR-regulated genes upstream of caspase activation in 697 cells, we performed DNA microarray analysis. 113 genes were identified, which were induced or repressed at least 3-fold by GC. Surprisingly, mitogen-activated protein kinase phosphatase-1 (MKP-1), a GR-induced gene in other cell types, was repressed 3-fold and correlated with an induction of JNK activity. These results suggest the involvement of mitogen activated protein kinases and apical caspase-9 and caspase-10 in the GC-induced apoptosis of pre-B lymphocytes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GCs4 are steroid hormones that have diverse tissue-specific effects involved in homeostasis and response to stress. Almost all of the biological consequences of exposure to GCs are mediated through the GR, a ligand-activated transcription factor and member of the nuclear receptor superfamily (1) . In its unactivated state, the GR is complexed with heat shock proteins and immunophilins (2) . Upon binding hormone, the GR becomes activated by the ATP-dependent release of these associated proteins, inducing a conformational change in the receptor and resulting in its translocation into the nucleus. There, it regulates target gene expression by (a) activating or repressing transcription by direct binding to GREs in the promoters of specific genes or (b) repressing transcription by binding to and inhibiting the function of other transcription factors such as AP-1 and nuclear factor {kappa}B (reviewed in Ref. 3 ).

GCs induce apoptosis in numerous cell types, including immature lymphocytes and various malignancies of lymphoid origin and thus have become one of the most common therapies for corresponding leukemias and lymphomas (4) . Universally, apoptosis is mediated through the activation of caspases. These aspartate-specific cysteine proteases cleave specific substrates within a cell, resulting in a conserved series of biochemical and morphological changes (5) . At least two major pathways of caspase activation have been described: the receptor-mediated extrinsic pathway and the mitochondrial-mediated intrinsic pathway. Caspase-8 and caspase-10 are initiators of the extrinsic pathway where they are activated in response to death receptor engagement by ligands belonging to the TNF superfamily (6) . The intrinsic pathway involves mitochondrial disruption by proapoptotic Bcl-2 family members and consequent release of factors such as cytochrome c that promote caspase-9 activation (7) . Both pathways culminate in the activation of downstream effector caspase-3, caspase-6, and caspase-7 and can cooperate to enhance apoptosis through caspase-8-mediated cleavage of Bid (8) .

We and others have observed that GC-induced apoptosis is associated with release of cytochrome c from the mitochondria5 (9) and that Bcl-2 overexpression can delay (10) or inhibit this process (11) , confirming a primary role for the intrinsic pathway. To this end, experiments using thymocytes from knockout mice have shown an absolute requirement for caspase-9 and Apaf-1 (12) in GC-induced apoptosis but not for caspase-3 (13) , suggesting redundancy among the effector caspases. We examined the involvement of apical caspases and GR-regulated genes in the GC-induced cell death of human 697 pre-B acute lymphoblastic leukemia cells. Using a combination of specific caspase inhibitors and stably transfected DN caspase genes, we demonstrate a requirement for caspase-9 and caspase-10, but not caspase-8, in GC-induced apoptosis.

GC-induced apoptosis can be modulated by the transfection of genes that affect the activity of the GR and related cofactors (e.g., RAP46/Bag-1; Ref. 14 ) as well as by genes that affect functions of the universal apoptotic machinery (e.g., Bcl-2). However, it is still unclear which specific GR-regulated genes are involved in the decision stage of GC-induced apoptosis, downstream of the GR but upstream of caspase activation. Using microarray technology, we have found that MKP-1, a GR-induced gene in other tissues, was strongly repressed in 697 cells and correlated with an increase in JNK/stress-activated protein kinase activity. Thus, GC-mediated cell death in 697 cells may involve GR-dependent repression of MKP-1 and subsequent activation of the proapoptotic JNK pathway (reviewed in Ref. 15 ).


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Restriction enzymes and other molecular biology reagents were obtained from Promega (Madison, WI), Roche (Indianapolis, IN), or New England Biolabs (Beverly, MA). Horseradish peroxidase-conjugated antibodies and enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). TA and RU486 were purchased from Sigma Chemical Company (St. Louis, MO). All tissue culture media and supplements were from Invitrogen (Carlsbad, CA). The caspase inhibitors, Z-LEHD-FMK and Z-DQMD-FMK, and the negative control, Z-FA-FMK, were purchased from Enzyme Systems Products (Livermore, CA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).

Cell Culture and Stable Transfections.
Cells (697) are a cloned human pre-B leukemic cell line derived from childhood acute lymphoblastic leukemia, which carries the t(1;19) translocation. All cell lines were cultured with RPMI media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, (100 units/ml), and streptomycin (100 µg/ml) at 37°C under 5% CO2. Exponentially growing cells were used throughout all experiments at a concentration of 5 x 105 -1 x 106 cells/ml.

Mammalian expression vectors were constructed as described previously (16 , 17) . The cell lines 697-Neo and 697-CrmA, 697-Bcl-2, 697-DN-caspase-8, 697-DN-caspase-9, and 697-DN-caspase-10 were generated by resuspending 1 x 107 cells in serum-free RPMI containing 10 µg of the expression plasmids: pcDNA3.0/Neo; pcDNA3.0/CrmA; pcDNA3.0/Bcl-2; pcDNA3.0/DN-caspase-8; pcDNA3.0/DN-caspase-9; or pcDNA3.0/DN-caspase-10. The cells were electroporated using a 4-mm gap cuvette at 0.22kV, 500 microfarads, and 27–31 ms. Cells were cultured for 18–48 h before the addition of G418 at 1 mg/ml. After selection for 4 weeks, cells were subcloned by limiting dilution and screened by immunoblot analysis for protein expression using an {alpha}-T7 mouse mAb (Novagen, Madison, WI) against the T7 epitope tag within the expression plasmid.

Determination of Cell Number, Viability, and Caspase Activity.
Cells (1 x 106 cells/ml) were seeded in 24-well plates and incubated at 37°C for 2 days in the presence or absence of 1 µM TA. Throughout the 48-h time course, cell viability was determined by trypan blue exclusion using a hemocytometer. Caspase-3 activity was detected after a 20-h treatment with 100 nM TA, using ApoAlert (BD Biosciences, Palo Alto, CA) according to the manufacturer’s protocol.

Gel Electrophoresis and Western Blotting.
Cells were washed with PBS and lysed with radioimmunoprecipitation assay buffer containing 1x Complete protease inhibitors (Roche). Total protein was quantitated using the BCA assay (Pierce, Rockford, IL). A total of 20–40 µg of whole cell extracts was electrophoresed in SDS-polyacrylamide gels and transferred to nitrocellulose for Western blotting. Membranes were blocked overnight with 10% nonfat milk/1x PBS/0.1% Tween 20 and incubated with the {alpha}-T7 Tag mAb (1:5000; Novagen), {alpha}-CrmA mAb (1:1000; BD Biosciences), {alpha}-Bcl-2 mAb (1:800; BD Biosciences), MKP-1 rabbit polyclonal antibody (1:100; Santa Cruz Biotechnology), or HDJ-2 (Lab Vision, Fremont, CA) in 1x PBS-5% nonfat milk for 1 h at room temperature, followed by a 1-h incubation with horseradish peroxidase-labeled donkey antirabbit or sheep antimouse antibodies diluted 1:2500. Proteins were detected using enhanced chemiluminescence (Amersham) or SuperSignal (Pierce) reagents. The total protein stain in Fig. 6BCitation was performed using Gelcode Silver (Pierce).



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  		Fig. 6. MKP-1 protein levels are reduced, and JNK activity is increased by GC in 697 cells. A, anti-MKP-1 immunoblot of total protein extracts from 697 cells treated with 100 nM TA or TA plus 2 µM RU486 (RU) for 1–24 h. The nonspecific immunoreactive species (bottom band) serves as a loading control. B, total protein extracts from cells treated with 100 nM TA, with or without RU486, were subject to a JNK activity assay using GST-cJun (1–79) as described in the "Materials and Methods." A 1-h treatment with anisomycin (Ani, 20 µg/ml) was performed as a positive control for induction of JNK activity, and a 24-h treatment with vehicle alone (V) was performed as a negative control. A total protein stain and an immunoblot against HDJ-2 are shown as loading controls for the c-Jun substrate and the cell extracts, respectively. Fold induction (FI) of JNK activity is indicated.

 
DNA Microarray Chip Analysis.
The Affymetrix GeneChip system was used to determine the expression profiles of 697 cells treated with 100 nM TA or vehicle (ethanol) for 4 h. Total RNA from each sample was extracted using Qiagen’s RNeasy kit (Valencia, CA). Preparation of biotinylated cRNA and hybridization to Affymetrix U95A DNA Chips was performed at the University of Pennsylvania Microarray Core Facility. Analyses of the resulting images and data files were performed using Affymetrix Microarray Suite 5.0 and the NetAffx website. Although it has been demonstrated that expression changes > 2-fold are significant in Affymetrix microarrays (18) , our more stringent cutoff is likely to significantly decrease the occurrence of false positives (19) . ESTs with no putative function were excluded from the list of 113 regulated genes shown in Table 1Citation .


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  		Table 1 Genes regulated by TA in 697 cells

The 113 genes induced or repressed 3-fold or greater by GC were loosely categorized based on protein function, although many of these genes likely fit into more than one category.

 
RT-PCR.
Cells (697) were treated for 4 h with 100 nM TA, harvested by centrifugation, and frozen immediately on dry ice. Total cellular RNA was isolated from frozen samples using Qiagen’s RNeasy kit. RNA concentrations were determined by measuring the absorbance of each sample at 260 and 280 nm. RT-PCR was performed on 1 µg of each RNA sample using Clontech’s Titanium One-Step RT-PCR Kit and the following gene-specific primer pairs (GAPDH: 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGG TCAGGTCCACC-3'; Bcl2: 5'-ATGGCGCACGCTGGGAGAACGGGGTA CGAC-3' and 5'-TCACTTGTGGCTCAGATAGGCACCCAGGGT-3'; CK:5'-ATGGTTCTGGAGAGCGTTATGTTTGCCATTT-3' and 5'-TCACACCC CAAGCTTCCTCTTCTGGTGGAA-3'; FKBP54: 5'-GAACAATGAAGAA AGCCCCACAGCCACTGT-3' and 5'-TCATACGTGGCCCTCAGGTTTC TCTTCTTC-3'; GPCR18: 5'-GCTCATCTCTCACACAGAC-3' and 5'-CTG TGAGAGCTCCAAGAATC-3'; and MKP-1: 5'-AATCCTGCCCTTTCTGT ACCTG-3' and 5'-ATGGTGGCTGACCGGGAAATG-3'. The amplification was performed as recommended by the manufacturer. PCR products were electrophoresed in a 1.0% agarose gel and visualized by ethidium bromide staining.

Taqman Quantitative RT-PCR.
Total RNA was prepared using the Qiagen RNeasy-mini method. Taqman reactions were performed using Taqman One-Step RT-PCR Master Mix reagents (Applied Biosystems, Foster City, CA). The 18S rRNA control primer/probe set was purchased from Applied Biosystems, and the MKP-1 primer/probe set was purchased form Midland Certified Reagents (Midland, TX). The sequence of the 6-carboxyfluorescein-labeled MKP-1 probe is 5'-6FAM-CCCGGTCAGCCACCATCTGCC-TAMRA. The MKP-1 primer sequences are 5'-CACTGCCAGCAGCATT-3' and 5'-CTCGATTAGTCCTCATAAGGTAAGCA-3'. Reactions were performed in duplicate in an ABI Prism 7900 Sequence Detection System using the recommended cycling conditions. Data were analyzed using ABI SDS software. Threshold cycle (Ct) values were determined by the software as described previously (20) . Gene expression of treated cells relative to control cells was quantitated using 2Ct of treated cells - Ct of control cells. In Fig. 5CCitation , the expression of MKP-1 in vehicle-treated cells is normalized to a value of 1.



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  		Fig. 5. MKP-1 mRNA is repressed by GC in 697 cells. A, RT-PCR analysis of cells treated with vehicle, 100 nM TA, or TA plus 2 µM RU486 for 4 h. Each treatment group was normalized to contain the same vehicle concentration, 0.2% ethanol. GAPDH is included as a control. B, Taqman real-time quantitative RT-PCR on samples treated as above for 4 or 8 h. 18S rRNA is included as a loading control, and each condition was tested in duplicate. The amplification plots show the accumulation of MKP-1 PCR products at a lower cycle number in the absence of TA. C, the relative expression of MKP-1 was quantitated as described in the "Materials and Methods" using the threshold cycle (Ct) values, i.e., the earliest cycle at which a PCR product is detectable. Average relative MKP-1 mRNA levels are compared for cells treated with vehicle (), TA (), and TA plus RU486 ( ).

 
JNK Activity Assay.
To determine JNK activity, cells were lysed in buffer containing 20 mM HEPES (pH 7.5), 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 1x Complete protease inhibitors (Roche), 1 µM microcystin, 25 µM glycerol phosphate, 1 mM sodium orthovanadate, and 10 mM sodium fluoride. One mg of cell extract from each condition was incubated with 5 µg of a GST fusion protein containing 79 NH2-terminal amino acids of c-Jun (21) . Samples were then incubated with glutathione-agarose (Sigma Chemical Company) for 3 h. Agarose beads were pelleted, washed three times with lysis buffer, and resuspended in 30 µl of 20 mM HEPES (pH 7.5), 20 mM MgCl2, 2 mM DTT, 10 µM ATP, 20 µCi 33P-ATP, and the phosphatase inhibitors listed above. Kinase reactions were incubated at room temperature for 90 min, resuspended in 12 µl of 4x SDS-PAGE sample buffer and boiled. Samples were electrophoresed on a 4–20% gradient gel, which was fixed in 40% methanol/10% acetic acid, dried, and exposed to film at -70°C for 48 h. Band intensities were quantitated using Molecular Dynamics ImageQuant Software (Amersham Pharmacia Biotech), and fold induction of JNK activity was normalized to vehicle.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To study the involvement of specific apical caspases in GC-induced cell death, we stably transfected DN forms of caspase-8, caspase-9, or caspase-10, containing active-site mutations (16 , 17) , into the human pre-B leukemic cell line, 697. Fig. 1ACitation shows the expression of DN caspase-8, caspase-9, and caspase-10 in six stably transfected clonal cell lines. To characterize the effect of these DN caspases on GC-induced apoptosis, cells were treated with the GC, TA, for 48 h. Fig. 1BCitation demonstrates the effects of GC on the viability of a representative clone for each of the stably transfected cell lines. DN-caspase-9 and DN-caspase-10 partially inhibited GC-induced cell death by 50 and 40%, respectively, whereas DN-caspase-8 and vector alone had no effect on viability. This suggests that the apical caspase-9 and caspase-10 are essential for mediating GC-triggered cell death in 697 cells, whereas caspase-8, a key mediator in Fas- and TNF-induced apoptosis, is dispensable.



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  		Fig. 1. Establishment and characterization of 697 cells expressing DN apical caspases. A, samples (1–6) containing whole cell extracts prepared from cells (1 x 106/ml) transfected with pcDNA3.0/DN-caspase-8, pcDNA3.0/DN-caspase-9, or pcDNA3.0/DN-caspase-10 were subjected to SDS-PAGE followed by immunoblotting with a {alpha}-T7 mAb. B, 697 cells (1 x 106 cells/ml) stably transfected with vector alone or with DN-caspase-8, DN-caspase-9, or DN-caspase-10 were cultured in the presence () or absence () of TA (1 µM) for 48 h, and viability was determined by trypan blue exclusion. C, whole cell lysates from 697 cells stably transfected with DN caspase-8, caspase-9, or caspase-10 and treated with 100 nM TA () or 100 nM TA + RU486 for 20 h ( ) or untreated () were incubated with DEVD-AFC at 37°C. Each treatment group was normalized to contain the same vehicle concentration, 0.2% ethanol. After 30 min, the release of AFC was monitored by a fluorimeter. D, 697 cells (1 x 106 cells/ml) stably transfected with vector alone or with DN-caspase-8, DN-caspase-9, or DN-caspase-10 were cultured in the presence () or absence () of TRAIL (1 µg/µl) for 18 h, and viability was determined by trypan blue exclusion. Data shown are means and SDs for two experiments performed in triplicate.

 
To measure the proteolytic activity of the downstream effector caspase-3 in response to GC in these DN cell lines, lysates from cells treated with TA for 20 h were incubated with the fluorogenic peptide substrate DEVD-AFC. Fig. 1CCitation shows that DEVD-specific caspase-3 activity was repressed in the DN-caspase-9 cell line but not in cells expressing DN-caspase-8, DN-caspase-10, or vector alone (data not shown). To determine whether induction of caspase-3 activity was dependent on the GR, we treated the cells with TA in the presence of a 20-fold excess of the receptor antagonist RU486. The GR antagonist completely inhibited caspase-3 induction in response to GC. Thus, inhibition of endogenous caspase-9 but not caspase-8 or caspase-10 activity in 697 cells effectively blocks GC-induced activation of the downstream effector caspase-3.

To further characterize the expression of DN apical caspases in 697 cells, we examined the effect of TRAIL on cell viability. Fig. 1DCitation shows that 697 cells expressing vector alone, DN-caspase-9, or DN-caspase-10 exhibit decreased viability after 18 h of treatment with TRAIL. In contrast, cells expressing DN-caspase-8 maintain their viability. Because TRAIL-induced apoptosis is known to be initiated through caspase-8-mediated signaling, these data suggest that stable expression of DN-caspase-8 in 697 cells effectively blocks endogenous caspase-8 activity. The result that inactivation of the death receptor-associated caspase-10 did not block TRAIL-induced cell death is consistent with observations in some cell types (22) but not others (23) .

To examine the effect of multiple caspase inhibition on GC-induced apoptosis in 697 cells, wild-type- and DN-transfected cells were treated with specific peptide-based caspase inhibitors. These included Z-LEHD-FMK, a caspase-9 inhibitor; Z-DQMD-FMK, a caspase-3/-6 inhibitor; and Z-FA-FMK, a negative control. As demonstrated in Fig. 2Citation , treatment with Z-FA-FMK had no effect on GC-induced cell death in any of the DN-caspase expressing cell lines (second series, compare to Fig. 1BCitation ). Inhibition of caspase-9 with Z-LEHD-FMK failed to protect vector or DN-caspase-8-transfected cells from apoptosis after 48 h but conferred 75–80% resistance when combined with DN-caspase-9 or caspase-10 (third series). This trend was also observed using the caspase-3/-6 inhibitor, Z-DQMD-FMK, in which apoptotic resistance increased from ~25 to ~65% in combination with DN-caspase-9 or caspase-10 (fourth series). Simultaneous treatment of empty vector or DN-caspase-transfected cells with both Z-LEHD-FMK and Z-DQMD-FMK provided near-complete protection from cell death, demonstrating the additive effects of multiple caspase inhibition (fifth series).



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  		Fig. 2. DN-caspase-9 and DN-caspase-10 partially protect against GC-induced apoptosis in 697 cells. 697 cells stably transfected with DN-caspase-8 (8), DN-caspase-9 (9), DN-caspase-10 (10) or vector alone (V) were pretreated for 1 h with the cell permeable inhibitors Z-LEHD-FMK (caspase-9 inhibitor), Z-DQMD-FMK (caspase-3/-6 inhibitor), or Z-FA-FMK (negative control inhibitor) at a final concentration of 20 µM and incubated at 37°C in the presence or absence of 1 µM TA for 48 h. Viability was determined by trypan blue exclusion. Data shown are mean and SD for two experiments performed in triplicate.

 
Because of their known ability to modulate caspase activity, we stably transfected 697 cells with CrmA or Bcl-2 (Fig. 3A)Citation . Cowpox virus CrmA is a member of the serpin family that is a potent inhibitor of initiator caspases, including caspase-8 and caspase-1 (24) . Stable expression of CrmA in 697 cells had no effect on cell viability after 48 h of treatment with TA (Fig. 3B)Citation , suggesting that caspase-8 activation is dispensable. Bcl-2 regulates the release of cytochrome c from the mitochondria and is a key upstream mediator of caspase-9 activation (16) . Cells containing stably transfected Bcl-2 were partially resistant to TA at a level similar to DN-caspase-9, confirming the role of the mitochondria in caspase-9 activation after treatment with GC.



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  		Fig. 3. Effect of CrmA and Bcl-2 expression on GC-induced apoptosis in 697 cells. A, samples containing whole cell extracts prepared from 1 x 106 cells/ml transfected with vector alone (pcDNA3.0), CrmA (pcDNA3.0/CrmA), or Bcl-2 (pcDNA3.0/Bcl-2) were subjected to SDS-PAGE followed by immunoblotting with {alpha}-CrmA or {alpha}-Bcl-2 mAbs. B, at time 0, 697 cells stably transfected with either vector alone, CrmA, or Bcl-2 were cultured in the presence () or absence () of 1 µM TA, and survival was analyzed over time by trypan blue exclusion. Data shown are mean and SD for two experiments performed in triplicate.

 
GCs induce caspase activity by signaling through the GR, but the key GR-regulated genes in this pathway remain elusive. To identify genes that are induced or repressed by GR, 697 cells were treated with vehicle or 100 nM TA for 4 h. The rationale for choosing a relatively early time point was to identify genes that are primary transcriptional targets of GR and are upstream of caspase activation (caspase-3 cleavage can be detected after a 12-h exposure to TA; Ref. 6 ). Microarray analysis was performed using Affymetrix U95A oligonucleotide chips containing >12,000 genes. A scatterplot of the transcriptional profile (Fig. 4A)Citation reveals relatively few changes in gene expression. Excluding several ESTs with no known or putative function, 52 genes were induced and 61 genes were repressed at least 3-fold by GC. These genes are listed and categorized in Table 1Citation .



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  		Fig. 4. Gene profiling of GC-treated 697 cells. A, total RNA isolated from 697 cells treated for 4 h with vehicle or 100 nM TA was subject to Affymetrix microarray analysis. Of the 12,583 genes tested on the U95A DNA Chip, 5121 genes were clearly present at detectable levels in either or both samples. Genes in which the detected signal showed a >=3-fold difference between samples fall outside of the two lines near the center of the scatterplot. B, total RNA from 697 cells treated with 100 nM TA for 4 h was analyzed by RT-PCR for the presence of selected genes. GAPDH was included as a control.

 
Many known GR-regulated genes that are induced by GC were identified, providing quality control for the microarray experiment. These included GILZ (25) , inhibitor of nuclear factor-{kappa}B (26) , choline kinase (27) , gp130 (28) , granzyme A (29) , and osteopontin (30) . Reported GC-repressed genes that were confirmed by this experiment included Bcl-2 (31) , the low-density lipoprotein receptor (32) , the VLDL receptor (33) , insulin-like growth factor binding protein 4 (34) , and sialyltransferase (35) . In addition, genes not previously identified as being affected by GC were induced or repressed more than three fold in the TA-treated 697 cells. These included factors involved in apoptosis (Bim), survival pathways (FKHRL1, phosphatidylinositol 3'-kinase) MAPK signaling (ERG, Elk-1, MKP-1, MKP-5), and G-protein coupled receptor signaling (transducin {alpha}, GPCR-18, Rgs2). RT-PCR was performed on selected genes with GAPDH included as a control (Fig. 4B)Citation .

One of the more unexpected findings from the microarray data is that MKP-1 is repressed by GC in 697 cells. In contrast, it has been observed that the MKP-1 gene, which contains three consensus GREs (36) , is induced by GC in RBL-2H3 mast cells and NIH3T3 fibroblasts (36) . MKP-1 expression exerts antiapoptotic effects that are mediated by Thr- and Tyr-dephosphorylation of JNK and p38 mitogen-activated protein kinase (37) .

Repression of MKP-1 in 697 cells after 4 h was confirmed by RT-PCR (Fig. 5A)Citation . Hormone binding to the GR was essential for MKP-1 repression because the addition of a 20-fold excess of RU486 returned MKP-1 expression levels to that of untreated cells. To obtain quantitative data on MKP-1 steady-state mRNA levels, we performed real-time Taqman RT-PCR on RNA isolated from 697 cells after 4 or 8 h of treatment with TA or TA + RU486. MKP-1 expression was repressed 3-fold at both time points, an effect that was blocked by the addition of RU486 (Fig. 5, B and C)Citation . Immunoblot analysis of whole cell extracts from 697 cells treated with TA demonstrates that MKP-1 protein levels steadily decrease over the course of 24 h but remain unchanged in the presence of RU486 (Fig. 6A)Citation .

Because MKP-1 has been shown to inhibit JNK activation (38) , we reasoned that loss of MKP-1 protein expression in 697 cells might correlate with an increase in JNK activity. Thus, we performed a JNK activity assay by monitoring the ability of whole-cell extracts to phosphorylate a fragment of the JNK substrate c-Jun. The protein synthesis inhibitor, anisomycin, was used as a positive control. In Fig. 6BCitation , JNK activity in 697 cells is increased at 4, 8, and 24 h after treatment with TA but blocked by RU486, demonstrating GR dependence. Equal loading of GST-c-Jun was confirmed using a total protein stain (Fig. 6BCitation , middle panel). As an additional control, whole cell extracts used in the JNK assay were immunoblotted against the chaperone HDJ-2, which was present at higher levels in cells treated with RU486 at 8 and 24 h.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using dominant negative caspase constructs together with specific caspase inhibitors, we investigated the role of apical caspases in the execution of GR-mediated apoptotic cell death in 697 cells. We have shown that among the death receptor-associated caspases, caspase-10, but not caspase-8, is essential for a full apoptotic response to GC. DN-caspase-10 showed significant inhibition of GC-induced apoptosis, whereas expression of DN-caspase-8 and CrmA had no effect. These data offer an important functional distinction between caspase-8 and caspase-10, which are homologous but have different substrate profiles (23) . The importance of caspase-10 in physiologically relevant lymphocyte apoptosis is underscored by its high occurrence of mutation in hereditary Autoimmune Lymphoproliferative Syndrome (39) .

DN-caspase-10 was unable to inhibit caspase-3 activity in 697 cells, suggesting that the ability of caspase-10 to mediate GC-induced apoptosis is caspase-3 independent. This implies that caspase-3 deficiency does not completely prevent GC-induced apoptosis in 697 cells and that other downstream effector caspases such as caspase-6 and caspase-7 may be involved. Indeed, studies have shown that caspase-6 and caspase-7 are activated in lymphocytes in response to GC (40, 41, 42) . Thus, GC-induced apoptosis in 697 cells is caspase-9 and caspase-10 dependent (Fig. 1B)Citation and partially caspase-3 independent (Fig. 1C)Citation .

We have used oligonucleotide microarray technology to identify GR-regulated genes in 697 cells, expanding the data set previously generated using other GC-sensitive hematopoietic cell lines (43 , 44) . Although there is some overlap between GR-regulated genes in 697 pre-B and CEM T ALL cells, we did not observe the large transcriptional changes in genes involved in ATP generation, protein synthesis, and RNA synthesis reported for the latter. Conversely, the CEM study did not identify many of the GR-regulated genes discovered in 697 cells, suggesting the possibility of tissue-specific, mechanistic differences in GC-induced apoptosis. While this manuscript was in preparation, microarray data were reported for 697 cells treated with dexamethasone (45) . The striking overlap in the data sets serves as an effective quality control check for the technology, whereas the differences may reflect subtle technical variations or ligand-binding effects of TA versus dexamethasone. Among these differences, the most notable are the presence of the Bcl-2 family member, Bim, and the MAPK phosphatase, MKP-1, in our data set.

We have found that MKP-1 mRNA is down-regulated 3-fold after a 4 or 8 h treatment with TA in 697 cells. Conversely, MKP-1 is induced by GC in at least three other cell lines and contains GREs within its promoter. Interestingly, there are several examples of GRE-containing promoters having the ability to respond both positively and negatively to GC under different conditions. The relative concentrations of GR cofactors that modify chromatin structure, CREB-binding protein and p300, have been shown to determine whether the GRE-containing mouse mammary tumor virus promoter responds positively or negatively to dexamethasone in HeLa cells (46) . It is tempting to speculate that differences in GR cofactor expression between cell lines may determine whether MKP-1 GREs are positively or negatively regulated by GR. Alternatively, it has been suggested that the relative abundance of the non-ligand binding GR-ß isoform may be responsible for such tissue-specific effects (47) .

Repression of MKP-1 is an attractive mechanism for the regulation of GC-induced apoptosis. MKP-1 is an antiapoptotic protein because its presence or overexpression protects various tissues from cell death induced by UV (48) , TNF (49) , cisplatin (50) , and Fas ligand (51) . MKP-1 is overexpressed in a variety of human cancers (52 , 53) and may contribute to unregulated proliferation through inhibition of MAPK activity.

MKP-1 dephosphorylates and inhibits the activity of the extracellular signal-regulated kinase, p38, and JNK MAPKs in vitro, but JNK is likely to be the most physiologically relevant substrate (38 , 50) . Therefore, our observation that JNK activity is induced between 4 and 24 h after TA treatment in 697 cells is likely to be caused, in part, by the loss of MKP-1 activity. The important role of JNK in promoting apoptosis is well characterized (54) . For example, T cells from jnk2 (-/-) mice show various apoptotic defects (55) and inhibiting JNK activation prevents drug-induced apoptosis in chronic myelogenous leukemia cells (56) . Furthermore, JNK signaling through c-Jun has been shown to induce expression of the proapoptotic Bcl-2 related protein, Bim (57) , which was induced by TA in 697 cells in this study (Table 1)Citation . It remains to be determined if c-Jun activation is a critical event in GC-induced apoptosis of leukemic cells.

GC-induced apoptosis was one of the first recognized forms of programmed cell death but remains one of the least understood. Our results indicate a novel, caspase-3 independent role for caspase-10 in this pathway. Moreover, microarray technology has provided new mechanistic insight into how GC exposure leads to caspase activation. Rather than a linear series of biochemical events, it is likely that multiple GR-induced genes activate a network of pathways that contribute to apoptosis.


   ACKNOWLEDGMENTS
 
We thank Drs. Emad Alnemri and Srinivasa Srinivasula for the contribution of the dominant negative caspase plasmid constructs, Dr. Don Baldwin for his technical expertise in DNA microarray technology, and members of the Litwack Lab for their helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Research Grant AI/HL40976 (to G. L.), ALA Grant RG-034N (to N. M. R.), and NIH Training Grant 5T32 DK07705 (to S. L. P.). Back

2 These two authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Thomas Jefferson University, 233 South 10th Street, BLSB #350, Philadelphia, PA 19107. Phone: (215) 503-4634; Fax: (215) 503-5393; E-mail: Gerry.Litwack{at}mail.tju.edu Back

4 The abbreviations used are: GC, glucocorticoid; GR, glucocorticoid receptor; TA, triamcinolone acetonide; DN, dominant negative; MAPK, mitogen-activated protein kinase; MKP-1, MAPK phosphatase-1; GRE, glucocorticoid response element; TNF, tumor necrosis factor; JNK, c-Jun N-terminal kinase; mAb, monoclonal antibody; RT-PCR, reverse transcription-PCR; GST, glutathione S-transferase; TRAIL, TNF-related apoptosis-inducing ligand; GAPDH, glyceraldehydes-3-phosphate dehydrogenase. Back

5 S. L. Planey, A. Derfoul, A. Steplewski, N. M. Robertson, and G. Litwack. Inhibition of glucocorticoid-induced apoptosis in 697 pre-B lymphocytes by the mineralocorticoid receptor N-terminal domain, submitted for publication. Back

Received 8/ 1/02. Accepted 10/31/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kumar R., Thompson E. B. The structure of the nuclear hormone receptors. Steroids, 64: 310-319, 1999.[Medline]
  2. Pratt W. B. The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J. Biol. Chem., 268: 21455-21458, 1993.[Free Full Text]
  3. McEwan I. J., Wright A. P., Gustafsson J. A. Mechanism of gene expression by the glucocorticoid receptor: role of protein-protein interactions. Bioessays, 19: 153-160, 1997.[Medline]
  4. Gaynon P. S., Carrel A. L. Glucocorticosteroid therapy in childhood acute lymphoblastic leukemia. Adv. Exp. Med. Biol., 457: 593-605, 1999.[Medline]
  5. Thornberry N. A., Lazebnik Y. Caspases: enemies within. Science (Wash. DC), 281: 1312-1316, 1998.[Abstract/Free Full Text]
  6. Ashkenazi A., Dixit V. M. Death receptors: signaling and modulation. Science (Wash. DC), 281: 1305-1308, 1998.[Abstract/Free Full Text]
  7. Saleh A., Srinivasula S. M., Acharya S., Fishel R., Alnemri E. S. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J. Biol. Chem., 274: 17941-17945, 1999.[Abstract/Free Full Text]
  8. Li H., Zhu H., Xu C. J., Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell, 94: 491-501, 1998.[Medline]
  9. Chandra J., Niemer I., Gilbreath J., Kliche K. O., Andreeff M., Freireich E. J., Keating M., McConkey D. J. Proteasome inhibitors induce apoptosis in glucocorticoid-resistant chronic lymphocytic leukemic lymphocytes. Blood, 92: 4220-4229, 1998.[Abstract/Free Full Text]
  10. Haldar S., Jena N., DuBois G. C., Takayama S., Reed J. C., Fu S. S., Croce C. M. Purification and characterization of the bcl-2 protein. Arch. Biochem. Biophys., 315: 483-488, 1994.[Medline]
  11. Alnemri E. S., Fernandes T. F., Haldar S., Croce C. M., Litwack G. Involvement of BCL-2 in glucocorticoid-induced apoptosis of human pre-B leukemias. Cancer Res., 52: 491-495, 1992.[Abstract/Free Full Text]
  12. Yoshida H., Kong Y. Y., Yoshida R., Elia A. J., Hakem A., Hakem R., Penninger J. M., Mak T. W. Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell, 94: 739-750, 1998.[Medline]
  13. Kuida K., Zheng T. S., Na S., Kuan C., Yang D., Karasuyama H., Rakic P., Flavell R. A. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature (Lond.), 384: 368-372, 1996.[Medline]
  14. Kullmann M., Schneikert J., Moll J., Heck S., Zeiner M., Gehring U., Cato A. C. RAP46 is a negative regulator of glucocorticoid receptor action and hormone-induced apoptosis. J. Biol. Chem., 273: 14620-14625, 1998.[Abstract/Free Full Text]
  15. Davis R. J. Signal transduction by the JNK group of MAP kinases. Cell, 103: 239-252, 2000.[Medline]
  16. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Ahmad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell, 91: 479-489, 1997.[Medline]
  17. MacFarlane M., Ahmad M., Srinivasula S. M., Fernandes-Alnemri T., Cohen G. M., Alnemri E. S. Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J. Biol. Chem., 272: 25417-25420, 1997.[Abstract/Free Full Text]
  18. Wodicka L., Dong H., Mittmann M., Ho M. H., Lockhart D. J. Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat. Biotechnol., 15: 1359-1367, 1997.[Medline]
  19. Wan Y., Nordeen S. K. Overlapping but distinct gene regulation profiles by glucocorticoids and progestins in human breast cancer cells. Mol. Endocrinol., 16: 1204-1214, 2002.[Abstract/Free Full Text]
  20. Martell M., Gomez J., Esteban J. I., Sauleda S., Quer J., Cabot B., Esteban R., Guardia J. High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA. J. Clin. Microbiol., 37: 327-332, 1999.[Abstract/Free Full Text]
  21. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev., 7: 2135-2148, 1993.[Abstract/Free Full Text]
  22. Kischkel F. C., Lawrence D. A., Tinel A., LeBlanc H., Virmani A., Schow P., Gazdar A., Blenis J., Arnott D., Ashkenazi A. Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8. J. Biol. Chem., 276: 46639-46646, 2001.[Abstract/Free Full Text]
  23. Wang J., Chun H. J., Wong W., Spencer D. M., Lenardo M. J. Caspase-10 is an initiator caspase in death receptor signaling. Proc. Natl. Acad. Sci. USA, 98: 13884-13888, 2001.[Abstract/Free Full Text]
  24. Ray C. A., Black R. A., Kronheim S. R., Greenstreet T. A., Sleath P. R., Salvesen G. S., Pickup D. J. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1ß converting enzyme. Cell, 69: 597-604, 1992.[Medline]
  25. D’Adamio F., Zollo O., Moraca R., Ayroldi E., Bruscoli S., Bartoli A., Cannarile L., Migliorati G., Riccardi C. A new dexamethasone-induced gene of the leucine zipper family protects T lymphocytes from TCR/CD3-activated cell death. Immunity, 7: 803-812, 1997.[Medline]
  26. Aljada A., Ghanim H., Assian E., Mohanty P., Hamouda W., Garg R., Dandona P. Increased I{kappa}B expression and diminished nuclear NF-{kappa}B in human mononuclear cells following hydrocortisone injection. J. Clin. Endocrinol. Metab., 84: 3386-3389, 1999.[Abstract/Free Full Text]
  27. Oka T., Perry J. W. Glucocorticoid stimulation of choline kinase activity during the development of mouse mammary gland. Dev. Biol., 68: 311-318, 1979.[Medline]
  28. Schooltink H., Schmitz-Van de Leur H., Heinrich P. C., Rose-John S. Up-regulation of the interleukin-6-signal transducing protein (gp130) by interleukin-6 and dexamethasone in HepG2 cells. FEBS Lett., 297: 263-265, 1992.[Medline]
  29. Aebischer F., Schlegel-Haueter S. E. Glucocorticoids modulate the induction of BLTE/granzyme A activity in the murine T-cell hybridoma PC60. Immunopharmacology, 23: 181-190, 1992.[Medline]
  30. Singh K., Balligand J. L., Fischer T. A., Smith T. W., Kelly R. A. Glucocorticoids increase osteopontin expression in cardiac myocytes and microvascular endothelial cells. Role in regulation of inducible nitric oxide synthase. J. Biol. Chem., 270: 28471-28478, 1995.[Abstract/Free Full Text]
  31. McConkey D. J., Chandra J., Wright S., Plunkett W., McDonnell T. J., Reed J. C., Keating M. Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX. J. Immunol., 156: 2624-2630, 1996.[Abstract]
  32. Al Rayyes O., Wallmark A., Floren C. H. Additive inhibitory effect of hydrocortisone and cyclosporine on low-density lipoprotein receptor activity in cultured HepG2 cells. Hepatology, 26: 967-971, 1997.[Medline]
  33. Ensler K., Mohammadieh M., Broijersen A., Angelin B., Gafvels M. Dexamethasone stimulates very low density lipoprotein (VLDL) receptor gene expression in differentiating 3T3–L1 cells. Biochim. Biophys. Acta, 1581: 36-48, 2002.[Medline]
  34. Cheung P. T., Wu J., Banach W., Chernausek S. D. Glucocorticoid regulation of an insulin-like growth factor-binding protein-4 protease produced by a rat neuronal cell line. Endocrinology, 135: 1328-1335, 1994.[Abstract]
  35. Wang X. C., Smith T. J., Lau J. T. Transcriptional regulation of the liver ß-galactoside {alpha} 2, 6-sialyltransferase by glucocorticoids. J. Biol. Chem., 265: 17849-17853, 1990.[Abstract/Free Full Text]
  36. Kassel O., Sancono A., Kratzschmar J., Kreft B., Stassen M., Cato A. C. Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of MKP-1. EMBO J., 20: 7108-7116, 2001.[Medline]
  37. Keyse S. M. Protein phosphatases and the regulation of MAP kinase activity. Semin. Cell Dev. Biol., 9: 143-152, 1998.[Medline]
  38. Slack D. N., Seternes O. M., Gabrielsen M., Keyse S. M. Distinct binding determinants for ERK2/p38{alpha} and JNK map kinases mediate catalytic activation and substrate selectivity of map kinase phosphatase-1. J. Biol. Chem., 276: 16491-16500, 2001.[Abstract/Free Full Text]
  39. Wang J., Zheng L., Lobito A., Chan F. K., Dale J., Sneller M., Yao X., Puck J. M., Straus S. E., Lenardo M. J. Inherited human caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II. Cell, 98: 47-58, 1999.[Medline]
  40. Miyashita T., Nagao K., Krajewski S., Salvesen G. S., Reed J. C., Inoue T., Yamada M. Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3. Cell Death Differ., 5: 1034-1041, 1998.[Medline]
  41. Hakem R., Hakem A., Duncan G. S., Henderson J. T., Woo M., Soengas M. S., Elia A., de la Pompa J. L., Kagi D., Khoo W., Potter J., Yoshida R., Kaufman S. A., Lowe S. W., Penninger J. M., Mak T. W. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell, 94: 339-352, 1998.[Medline]
  42. Komoriya A., Packard B. Z., Brown M. J., Wu M. L., Henkart P. A. Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med., 191: 1819-1828, 2000.[Abstract/Free Full Text]
  43. Obexer P., Certa U., Kofler R., Helmberg A. Expression profiling of glucocorticoid-treated T-ALL cell lines: rapid repression of multiple genes involved in RNA-, protein- and nucleotide synthesis. Oncogene, 20: 4324-4336, 2001.[Medline]
  44. Tonko M., Ausserlechner M. J., Bernhard D., Helmberg A., Kofler R. Gene expression profiles of proliferating versus G1-G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis. FASEB J., 15: 693-699, 2001.[Abstract/Free Full Text]
  45. Yoshida N. L., Miyashita T. U. M., Yamada M., Reed J. C., Sugita Y., Oshida T. Analysis of gene expression patterns during glucocorticoid-induced apoptosis using oligonucleotide arrays. Biochem. Biophys. Res. Commun., 293: 1254-1261, 2002.[Medline]
  46. Kino T., Nordeen S. K., Chrousos G. P. Conditional modulation of glucocorticoid receptor activities by CREB-binding protein (CBP) and p300. J. Steroid Biochem. Mol. Biol., 70: 15-25, 1999.[Medline]
  47. Vottero A., Chrousos G. P. Glucocorticoid Receptor ß: view I. Trends Endocrinol. Metab., 10: 333-338, 1999.[Medline]
  48. Franklin C. C., Srikanth S., Kraft A. S. Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis. Proc. Natl. Acad. Sci. USA, 95: 3014-3019, 1998.[Abstract/Free Full Text]
  49. Guo Y. L., Kang B., Williamson J. R. Inhibition of the expression of mitogen-activated protein phosphatase-1 potentiates apoptosis induced by tumor necrosis factor {alpha} in rat mesangial cells. J. Biol. Chem., 273: 10362-10366, 1998.[Abstract/Free Full Text]
  50. Sanchez-Perez I., Martinez-Gomariz M., Williams D., Keyse S. M., Perona R. CL100/MKP-1 modulates JNK activation and apoptosis in response to cisplatin. Oncogene, 19: 5142-5152, 2000.[Medline]
  51. Srikanth S., Franklin C. C., Duke R. C., Kraft R. S. Human DU145 prostate cancer cells overexpressing mitogen-activated protein kinase phosphatase-1 are resistant to Fas ligand-induced mitochondrial perturbations and cellular apoptosis. Mol. Cell. Biochem., 199: 169-178, 1999.[Medline]
  52. Bang Y. J., Kwon J. H., Kang S. H., Kim J. W., Yang Y. C. Increased MAPK activity and MKP-1 overexpression in human gastric adenocarcinoma. Biochem. Biophys. Res. Commun., 250: 43-47, 1998.[Medline]
  53. Kurt R. A., Urba W. J., Smith J. W., Schoof D. D. Peripheral T lymphocytes from women with breast cancer exhibit abnormal protein expression of several signaling molecules. Int. J. Cancer, 78: 16-20, 1998.[Medline]
  54. Leppa S., Bohmann D. Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene, 18: 6158-6162, 1999.[Medline]
  55. Behrens A., Sabapathy K., Graef I., Cleary M., Crabtree G. R., Wagner E. F. Jun N-terminal kinase 2 modulates thymocyte apoptosis and T-cell activation through c-Jun and nuclear factor of activated T cell (NF-AT). Proc. Natl. Acad. Sci. USA, 98: 1769-1774, 2001.[Abstract/Free Full Text]
  56. Mc Gee M. M., Campiani G., Ramunno A., Nacci V., Lawler M., Williams D. C., Zisterer D. M. Activation of the c-Jun N-terminal kinase (JNK) signaling pathway is essential during PBOX-6-induced apoptosis in chronic myelogenous leukemia (CML) cells. J. Biol. Chem., 277: 18383-18389, 2002.[Abstract/Free Full Text]
  57. Harris C. A., Johnson E. M., Jr. BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons. J. Biol. Chem., 276: 37754-37760, 2001.[Abstract/Free Full Text]



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