Role of Apical Caspases and Glucocorticoid-Regulated Genes in Glucocorticoid- Induced Apoptosis of Pre-B Leukemic Cells1

[CANCER RESEARCH 63, 172–178, January 1, 2003] Role of Apical Caspases and Glucocorticoid-regulated Genes in Glucocorticoid- induced Apoptosis of Pre-B Leukemic Cells1

Sonia L. Planey,2 Marc T. Abrams,2 Noreen M. Robertson, and Gerald Litwack3 Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT two major pathways of caspase activation have been described: the receptor-mediated extrinsic pathway and the mitochondrial-mediated Glucocorticoid (GC) sensitivity in hematopoietic cells requires the ac- intrinsic pathway. Caspase-8 and caspase-10 are initiators of the tivation and nuclear translocation of the glucocorticoid receptor (GR) and extrinsic pathway where they are activated in response to death the subsequent activation of caspases. To gain insight into the caspase cascade responsible for the execution phase of GC-induced apoptosis, 697 receptor engagement by ligands belonging to the TNF superfamily pre-B leukemic cells were stably transfected with dominant negative (6). The intrinsic pathway involves mitochondrial disruption by pro- forms of caspase-8, caspase-9, or caspase-10 and the caspase-8 inhibitor apoptotic Bcl-2 family members and consequent release of factors CrmA. We observed that inhibition of caspase-9 or caspase-10 activity, such as cytochrome c that promote caspase-9 activation (7). Both but not caspase-8, caused partial resistance of 697 cells to GC-induced pathways culminate in the activation of downstream effector apoptosis. Inhibition of multiple caspases through the use of specific caspase-3, caspase-6, and caspase-7 and can cooperate to enhance peptide inhibitors had an additive effect and caused complete resistance. apoptosis through caspase-8-mediated cleavage of Bid (8). To identify GR-regulated genes upstream of caspase activation in 697 We and others have observed that GC-induced apoptosis is asso- cells, we performed DNA microarray analysis. 113 genes were identified, ciated with release of cytochrome c from the mitochondria5 (9) and which were induced or repressed at least 3-fold by GC. Surprisingly, that Bcl-2 overexpression can delay (10) or inhibit this process (11), mitogen-activated protein kinase phosphatase-1 (MKP-1), a GR-induced gene in other cell types, was repressed 3-fold and correlated with an confirming a primary role for the intrinsic pathway. To this end, induction of JNK activity. These results suggest the involvement of mito- experiments using thymocytes from knockout mice have shown an gen activated protein kinases and apical caspase-9 and caspase-10 in the absolute requirement for caspase-9 and Apaf-1 (12) in GC-induced GC-induced apoptosis of pre-B lymphocytes. 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 INTRODUCTION human 697 pre-B acute lymphoblastic leukemia cells. Using a combination of specific caspase inhibitors and stably transfected DN GCs4 are steroid hormones that have diverse tissue-specific effects caspase genes, we demonstrate a requirement for caspase-9 and involved in homeostasis and response to stress. Almost all of the caspase-10, but not caspase-8, in GC-induced apoptosis. biological consequences of exposure to GCs are mediated through the GC-induced apoptosis can be modulated by the transfection of GR, a ligand-activated transcription factor and member of the nuclear genes that affect the activity of the GR and related cofactors (e.g., receptor superfamily (1). In its unactivated state, the GR is complexed RAP46/Bag-1; Ref. 14) as well as by genes that affect functions of the with heat shock proteins and immunophilins (2). Upon binding hor- universal apoptotic machinery (e.g., Bcl-2). However, it is still unclear mone, the GR becomes activated by the ATP-dependent release of which specific GR-regulated genes are involved in the decision stage these associated proteins, inducing a conformational change in the of GC-induced apoptosis, downstream of the GR but upstream of receptor and resulting in its translocation into the nucleus. There, it caspase activation. Using microarray technology, we have found that regulates target gene expression by (a) activating or repressing tran- MKP-1, a GR-induced gene in other tissues, was strongly repressed in scription by direct binding to GREs in the promoters of specific genes 697 cells and correlated with an increase in JNK/stress-activated or (b) repressing transcription by binding to and inhibiting the func- protein kinase activity. Thus, GC-mediated cell death in 697 cells may tion of other transcription factors such as AP-1 and nuclear factor ␬B involve GR-dependent repression of MKP-1 and subsequent activa- (reviewed in Ref. 3). tion of the proapoptotic JNK pathway (reviewed in Ref. 15). 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 MATERIALS AND METHODS leukemias and lymphomas (4). Universally, apoptosis is mediated through the activation of caspases. These aspartate-specific cysteine Materials. Restriction enzymes and other molecular biology reagents were obtained from Promega (Madison, WI), Roche (Indianapolis, IN), or New proteases cleave specific substrates within a cell, resulting in a con- England Biolabs (Beverly, MA). Horseradish peroxidase-conjugated antibod- served series of biochemical and morphological changes (5). At least ies and enhanced chemiluminescence reagents were purchased from Amer- sham Pharmacia Biotech (Piscataway, NJ). TA and RU486 were purchased Received 8/1/02; accepted 10/31/02. from Sigma Chemical Company (St. Louis, MO). All tissue culture media and The costs of publication of this article were defrayed in part by the payment of page supplements were from Invitrogen (Carlsbad, CA). The caspase inhibitors, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Z-LEHD-FMK and Z-DQMD-FMK, and the negative control, Z-FA-FMK, 1 Supported by NIH Research Grant AI/HL40976 (to G. L.), ALA Grant RG-034N (to were purchased from Enzyme Systems Products (Livermore, CA). All other N. M. R.), and NIH Training Grant 5T32 DK07705 (to S. L. P.). chemicals were purchased from Fisher Scientific (Pittsburgh, PA). 2 These two authors contributed equally to this work. Cell Culture and Stable Transfections. Cells (697) are a cloned human 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: pre-B leukemic cell line derived from childhood acute lymphoblastic leukemia, (215) 503-5393; E-mail: [email protected]. which carries the t(1;19) translocation. All cell lines were cultured with RPMI 4 The abbreviations used are: GC, glucocorticoid; GR, glucocorticoid receptor; TA, media supplemented with 10% fetal bovine serum, 2 mML-glutamine, (100 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, 5 S. L. Planey, A. Derfoul, A. Steplewski, N. M. Robertson, and G. Litwack. Inhibition reverse transcription-PCR; GST, glutathione S-transferase; TRAIL, TNF-related apopto- of glucocorticoid-induced apoptosis in 697 pre-B lymphocytes by the mineralocorticoid sis-inducing ligand; GAPDH, glyceraldehydes-3-phosphate dehydrogenase. receptor N-terminal domain, submitted for publication. 172

␮ units/ml), and streptomycin (100 g/ml) at 37°C under 5% CO2. Exponentially One-Step RT-PCR Master Mix reagents (Applied Biosystems, Foster City, growing cells were used throughout all experiments at a concentration of CA). The 18S rRNA control primer/probe set was purchased from Applied 5 ϫ 105-1 ϫ 106 cells/ml. Biosystems, and the MKP-1 primer/probe set was purchased form Midland Mammalian expression vectors were constructed as described previously Certified Reagents (Midland, TX). The sequence of the 6-carboxyfluorescein- (16, 17). The cell lines 697-Neo and 697-CrmA, 697-Bcl-2, 697-DN- labeled MKP-1 probe is 5Ј-6FAM-CCCGGTCAGCCACCATCTGCC- caspase-8, 697-DN-caspase-9, and 697-DN-caspase-10 were generated by TAMRA. The MKP-1 primer sequences are 5Ј-CACTGCCAGCAGCATT-3Ј resuspending 1 ϫ 107 cells in serum-free RPMI containing 10 ␮gofthe and 5Ј-CTCGATTAGTCCTCATAAGGTAAGCA-3Ј. Reactions were per- expression plasmids: pcDNA3.0/Neo; pcDNA3.0/CrmA; pcDNA3.0/Bcl-2; formed in duplicate in an ABI Prism 7900 Sequence Detection System using pcDNA3.0/DN-caspase-8; pcDNA3.0/DN-caspase-9; or pcDNA3.0/DN- the recommended cycling conditions. Data were analyzed using ABI SDS caspase-10. The cells were electroporated using a 4-mm gap cuvette at 0.22kV, software. Threshold cycle (Ct) values were determined by the software as 500 microfarads, and 27–31 ms. Cells were cultured for 18–48 h before the described previously (20). Gene expression of treated cells relative to control addition of G418 at 1 mg/ml. After selection for 4 weeks, cells were subcloned cells was quantitated using 2Ct of treated cells Ϫ Ct of control cells. In Fig. 5C, the by limiting dilution and screened by immunoblot analysis for protein expres- expression of MKP-1 in vehicle-treated cells is normalized to a value of 1. sion using an ␣-T7 mouse mAb (Novagen, Madison, WI) against the T7 JNK Activity Assay. To determine JNK activity, cells were lysed in buffer

epitope tag within the expression plasmid. containing 20 mM HEPES (pH 7.5), 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM Determination of Cell Number, Viability, and Caspase Activity. Cells EDTA, 0.05% Triton X-100, 1ϫ Complete protease inhibitors (Roche), 1 ␮M (1 ϫ 106 cells/ml) were seeded in 24-well plates and incubated at 37°C for 2 microcystin, 25 ␮M glycerol phosphate, 1 mM sodium orthovanadate, and 10 days in the presence or absence of 1 ␮M TA. Throughout the 48-h time course, mM sodium fluoride. One mg of cell extract from each condition was incubated ␮ cell viability was determined by trypan blue exclusion using a hemocytometer. with 5 g of a GST fusion protein containing 79 NH2-terminal amino acids of Caspase-3 activity was detected after a 20-h treatment with 100 nM TA, using c-Jun (21). Samples were then incubated with glutathione-agarose (Sigma ApoAlert (BD Biosciences, Palo Alto, CA) according to the manufacturer’s Chemical Company) for 3 h. Agarose beads were pelleted, washed three times protocol. with lysis buffer, and resuspended in 30 ␮lof20mM HEPES (pH 7.5), 20 mM ␮ ␮ Gel Electrophoresis and Western Blotting. Cells were washed with PBS MgCl2,2mM DTT, 10 M ATP, 20 Ci 33P-ATP, and the phosphatase and lysed with radioimmunoprecipitation assay buffer containing 1ϫ Com- inhibitors listed above. Kinase reactions were incubated at room temperature plete protease inhibitors (Roche). Total protein was quantitated using the BCA for 90 min, resuspended in 12 ␮lof4ϫ SDS-PAGE sample buffer and boiled. assay (Pierce, Rockford, IL). A total of 20–40 ␮g of whole cell extracts was Samples were electrophoresed on a 4–20% gradient gel, which was fixed in electrophoresed in SDS-polyacrylamide gels and transferred to nitrocellulose 40% methanol/10% acetic acid, dried, and exposed to film at Ϫ70°C for 48 h. for Western blotting. Membranes were blocked overnight with 10% nonfat Band intensities were quantitated using Molecular Dynamics ImageQuant milk/1ϫ PBS/0.1% Tween 20 and incubated with the ␣-T7 Tag mAb (1:5000; Software (Amersham Pharmacia Biotech), and fold induction of JNK activity Novagen), ␣-CrmA mAb (1:1000; BD Biosciences), ␣-Bcl-2 mAb (1:800; BD was normalized to vehicle. Biosciences), MKP-1 rabbit polyclonal antibody (1:100; Santa Cruz Biotech- nology), or HDJ-2 (Lab Vision, Fremont, CA) in 1ϫ PBS-5% nonfat milk for RESULTS 1 h at room temperature, followed by a 1-h incubation with horseradish peroxidase-labeled donkey antirabbit or sheep antimouse antibodies diluted To study the involvement of specific apical caspases in GC-induced 1:2500. Proteins were detected using enhanced chemiluminescence (Amer- cell death, we stably transfected DN forms of caspase-8, caspase-9, or sham) or SuperSignal (Pierce) reagents. The total protein stain in Fig. 6B was caspase-10, containing active-site mutations (16, 17), into the human performed using Gelcode Silver (Pierce). pre-B leukemic cell line, 697. Fig. 1A shows the expression of DN DNA Microarray Chip Analysis. The Affymetrix GeneChip system was caspase-8, caspase-9, and caspase-10 in six stably transfected clonal used to determine the expression profiles of 697 cells treated with 100 nM TA cell lines. To characterize the effect of these DN caspases on GC- or vehicle (ethanol) for 4 h. Total RNA from each sample was extracted using induced apoptosis, cells were treated with the GC, TA, for 48 h. Fig. Qiagen’s RNeasy kit (Valencia, CA). Preparation of biotinylated cRNA and 1B demonstrates the effects of GC on the viability of a representative hybridization to Affymetrix U95A DNA Chips was performed at the Univer- sity of Pennsylvania Microarray Core Facility. Analyses of the resulting clone for each of the stably transfected cell lines. DN-caspase-9 and images and data files were performed using Affymetrix Microarray Suite 5.0 DN-caspase-10 partially inhibited GC-induced cell death by 50 and and the NetAffx website. Although it has been demonstrated that expression 40%, respectively, whereas DN-caspase-8 and vector alone had no changes Ͼ2-fold are significant in Affymetrix microarrays (18), our more effect on viability. This suggests that the apical caspase-9 and stringent cutoff is likely to significantly decrease the occurrence of false caspase-10 are essential for mediating GC-triggered cell death in 697 positives (19). ESTs with no putative function were excluded from the list of cells, whereas caspase-8, a key mediator in Fas- and TNF-induced 113 regulated genes shown in Table 1. apoptosis, is dispensable. RT-PCR. Cells (697) were treated for 4 h with 100 nM TA, harvested by To measure the proteolytic activity of the downstream effector centrifugation, and frozen immediately on dry ice. Total cellular RNA was isolated caspase-3 in response to GC in these DN cell lines, lysates from cells from frozen samples using Qiagen’s RNeasy kit. RNA concentrations were de- treated with TA for 20 h were incubated with the fluorogenic peptide termined by measuring the absorbance of each sample at 260 and 280 nm. RT-PCR was performed on 1 ␮g of each RNA sample using Clontech’s Titanium substrate DEVD-AFC. Fig. 1C shows that DEVD-specific caspase-3 One-Step RT-PCR Kit and the following gene-specific primer pairs (GAPDH: activity was repressed in the DN-caspase-9 cell line but not in cells 5Ј-CCACCCATGGCAAATTCCATGGCA-3Ј and 5Ј-TCTAGACGGCAGG expressing DN-caspase-8, DN-caspase-10, or vector alone (data not TCAGGTCCACC-3Ј; Bcl2: 5Ј-ATGGCGCACGCTGGGAGAACGGGGTA shown). To determine whether induction of caspase-3 activity was CGAC-3Ј and 5Ј-TCACTTGTGGCTCAGATAGGCACCCAGGGT-3Ј; CK: dependent on the GR, we treated the cells with TA in the presence of 5Ј-ATGGTTCTGGAGAGCGTTATGTTTGCCATTT-3Ј and 5Ј-TCACACCC a 20-fold excess of the receptor antagonist RU486. The GR antagonist CAAGCTTCCTCTTCTGGTGGAA-3Ј; FKBP54: 5Ј-GAACAATGAAGAA completely inhibited caspase-3 induction in response to GC. Thus, Ј Ј AGCCCCACAGCCACTGT-3 and 5 -TCATACGTGGCCCTCAGGTTTC inhibition of endogenous caspase-9 but not caspase-8 or caspase-10 Ј Ј Ј Ј TCTTCTTC-3 ; GPCR18: 5 -GCTCATCTCTCACACAGAC-3 and 5 -CTG activity in 697 cells effectively blocks GC-induced activation of the TGAGAGCTCCAAGAATC-3Ј; and MKP-1: 5Ј-AATCCTGCCCTTTCTGT downstream effector caspase-3. ACCTG-3Ј and 5Ј-ATGGTGGCTGACCGGGAAATG-3Ј. The amplification was performed as recommended by the manufacturer. PCR products were To further characterize the expression of DN apical caspases in 697 electrophoresed in a 1.0% agarose gel and visualized by ethidium bromide cells, we examined the effect of TRAIL on cell viability. Fig. 1D staining. shows that 697 cells expressing vector alone, DN-caspase-9, or DN- Taqman Quantitative RT-PCR. Total RNA was prepared using the Qia- caspase-10 exhibit decreased viability after 18 h of treatment with gen RNeasy-mini method. Taqman reactions were performed using Taqman TRAIL. In contrast, cells expressing DN-caspase-8 maintain their 173

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. Induced Ͼ3x Repressed Ͼ3x Transcription factors and cofactors Forkhead protein (FKHRL1) ATF3: activating transcription factor 3 (CREB family) BTEB1: basic transcription element binding protein 1 DNA-damage-inducible transcript 3, C/EBP family MAD-4 (regulates c-myc) Runt-related transcription factor 1 (aml1 oncogene) Zinc finger protein 189 Zinc finger protein 36, EGF and TPA inducible Zinc finger protein 216 E2F family transcription factor 2 ERG transcription factor (Ets family) TIEG: TGFB inducible transcription factor ELK-1 oncogene (Ets family) SRY-box 4 (HMG-box family) Meis1-related protein 2 (MRG2) CBX4: chromobox homolog 4, Pc2 Signal transduction GILZ protein, inhibits NFKB Human transforming growth factor-␤-2 Rgs2 regulator of G-protein signaling 2 Insulin-like growth factor binding protein 4 STAT-induced STAT inhibitor-2 (SOCS-2) TNFSF4: tumor necrosis factor family, member 4 IKB ␣ (NF-␬-B inhibitor) EGF-like-domain, multiple 5 (MEGF9) Interleukin 6 signal transducer (gp130) Regulator of G-protein signalling 16 EFS2: signal transduction protein (SH3 containing) G protein ␣ subunit z (transducin ␣) Antagonist of FGF signaling (sprouty-1) G protein-coupled receptor 18 PDZ-GEF: guanine nucleotide exchange factor Ephrin-B2; ligand of Eph-related receptors Anchor protein 13; regulates localization of type II PKA FGFR2: fibroblast growth factor receptor 2 Adenylate cyclase 7 Cell cycle and apoptosis Bim-EL, BCL2-like 11 (apoptosis facilitator) Bcl-2 (B-cell lymphoma protein 2) Cdc5 (cell cycle regulator important for G2/M transition) SPHAR: S-phase response (cyclin-related) BTG2, may function in cell cycle control GG2-1 TNF-induced with death effector domain Kinases Diacylglycerol kinase epsilon (DGK) DYRK3 tyrosine-phosphorylation regulated kinase Choline kinase Phosphoinositide 3-kinase (PI3k) Urokinase plasminogen activator STK10: serine/threonine kinase 10 Coagulation factor X C8FW protein kinase HCK: hemopoietic cell kinase GS3955 protein, Region highly similar to C8FW Phosphatases PSPHL: phosphoserine phosphatase-like, CO9 CL-3 phosphoserine phosphatase Dual specificity phosphatase MKP-1 Dual specificity phosphatase MKP-5 PTPN7: tyrosine phosphatase, nonreceptor type 7 Other enzymes Granzyme A UDP-galactose ceramide galactosyltransferase Fatty-acid-Coenzyme A ligase, long-chain 2 PDIR: protein disulfide isomerase-related ATPase, Class VI, type 11B Cystathionase (cystathionine gamma-lyase) Fucosyltransferase 4 Sialyltransferase 9 (GM3 synthase) Transketolase 1 Polysialyltransferase-1 AU RNA binding protein/enoyl-Coenzyme A hydratase X-prolyl aminopeptidase Superoxide dismutase 2, mitochondrial Phosphoribosylformylglycinamidine synthase Ion transport Sodium bicarbonate cotransporter 2 Solute carrier family 16, member 1 Copper transporter 2 Potassium inwardly-rectifying channel J12 Polycystin-2, similar to voltage-activated Ca2ϩ channels Stanniocalcin 2; metal ion homeostasis Cytoskeleton and adhesion Osteopontin (Secreted phosphoprotein-1) Cdc42 effector; binds the Rho GTPase Cdc42 AIM1, ␤␥-crystallin superfamily CHL1: cell adhesion molecule, homology to L1CAM S72869: putative cytoskeletal protein CAPG: capping protein (actin filament), gelsolin-like Coronin, actin binding protein Lymphocyte-associated proteins SE20-4: T-cell lymphoma-associated tumor antigen NFATC3: nuclear factor of activated T cells SLAM: signaling lymphocytic activation molecule T-cell receptor, ␣ MHC class I CD79B antigen (immunoglobulin-associated ␤) Cytohesin binding protein HE ETR101 protein, TPA induced in leukemic cells CD22 Antigen, expressed in B lymphocytes RAG1: recombination activating gene 1 Other/unknown KIAA0878 protein, ras superfamily member Solute carrier family 9, isoform 3 regulatory factor 1 RIGUI (Per1 circadian rhythm protein) Low density lipoprotein receptor Immunophilin FKBP54 Very low density lipoprotein receptor Thioredoxin interacting protein ADP-ribosylation factor-like 7 TBC1D1: TBC1 domain family, member 1 Human putative transmembrane protein Peroxin 13; docking target for the PTS1 receptor MyoD family inhibitor, represses myogenesis DiGeorge syndrome critical region protein 6 Fusion RNA binding protein PRG1: proteoglycan 1, secretory granule Hypoxia induced protein DEC1 TACC2: transforming acidic coiled-coil protein 2 Insulin induced gene 1, CL-6 Hereditary hemochromatosis protein Adaptor-related protein complex 3, sigma 1 subunit viability. Because TRAIL-induced apoptosis is known to be initiated Z-LEHD-FMK, a caspase-9 inhibitor; Z-DQMD-FMK, a caspase-3/-6 through caspase-8-mediated signaling, these data suggest that stable inhibitor; and Z-FA-FMK, a negative control. As demonstrated in Fig. expression of DN-caspase-8 in 697 cells effectively blocks endoge- 2, treatment with Z-FA-FMK had no effect on GC-induced cell death nous caspase-8 activity. The result that inactivation of the death in any of the DN-caspase expressing cell lines (second series, compare receptor-associated caspase-10 did not block TRAIL-induced cell to Fig. 1B). Inhibition of caspase-9 with Z-LEHD-FMK failed to death is consistent with observations in some cell types (22) but not protect vector or DN-caspase-8-transfected cells from apoptosis after others (23). 48 h but conferred 75–80% resistance when combined with DN- To examine the effect of multiple caspase inhibition on GC-induced caspase-9 or caspase-10 (third series). This trend was also observed apoptosis in 697 cells, wild-type- and DN-transfected cells were using the caspase-3/-6 inhibitor, Z-DQMD-FMK, in which apoptotic treated with specific peptide-based caspase inhibitors. These included resistance increased from ϳ25 to ϳ65% in combination with DN- 174

Fig. 1. Establishment and characterization of 697 cells expressing DN apical caspases. A, samples (1–6) containing whole cell extracts prepared from cells (1 ϫ 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 ␣-T7 mAb. B, 697 cells (1 ϫ 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 (u) or absence (p)ofTA (1 ␮M) 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 (u) or 100 nM TA ϩ RU486 for 20 h ( )or untreated (o) 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 moni- tored by a fluorimeter. D, 697 cells (1 ϫ 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 (u)or absence (o) of TRAIL (1 ␮g/␮l) for 18 h, and viability was determined by trypan blue exclusion. Data shown are means and SDs for two experiments performed in triplicate. 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). Because of their known ability to modulate caspase activity, we stably transfected 697 cells with CrmA or Bcl-2 (Fig. 3A). 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), 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 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 ϫ 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 ␣-CrmA or ␣-Bcl-2 mAbs. B, at time 0, 697 cells stably transfected with either vector alone, CrmA, or Bcl-2 were cultured in the presence (u) or absence (o)of1␮M TA, and survival was analyzed over time by trypan blue exclusion. Data shown are mean and SD for two experiments performed in triplicate.

the role of the mitochondria in caspase-9 activation after treatment with GC. 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 Ͼ12,000 genes. A scatterplot of the transcriptional profile (Fig. 4A) reveals relatively few changes in gene Fig. 2. DN-caspase-9 and DN-caspase-10 partially protect against GC-induced apo- expression. Excluding several ESTs with no known or putative func- ptosis in 697 cells. 697 cells stably transfected with DN-caspase-8 (8), DN-caspase-9 (9), tion, 52 genes were induced and 61 genes were repressed at least 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), 3-fold by GC. These genes are listed and categorized in Table 1. or Z-FA-FMK (negative control inhibitor) at a final concentration of 20 ␮M and incubated Many known GR-regulated genes that are induced by GC were at 37°C in the presence or absence of 1 ␮M TA for 48 h. Viability was determined by trypan blue exclusion. Data shown are mean and SD for two experiments performed in identified, providing quality control for the microarray experiment. triplicate. These included GILZ (25), inhibitor of nuclear factor-␬B (26), choline 175

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 Ն3-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.

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 3Ј-kinase) MAPK signaling (ERG, Elk-1, MKP-1, MKP-5), and G- protein coupled receptor signaling (transducin ␣, GPCR-18, Rgs2). RT-PCR was performed on selected genes with GAPDH included as a control (Fig. 4B). 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 fibro- blasts (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). 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 ϩ 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). 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). 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. 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 ␮M RU486 for 4 h. Each treatment group 6B, JNK activity in 697 cells is increased at 4, 8, and 24 h after was normalized to contain the same vehicle concentration, 0.2% ethanol. GAPDH is treatment with TA but blocked by RU486, demonstrating GR depend- included as a control. B, Taqman real-time quantitative RT-PCR on samples treated as ence. Equal loading of GST-c-Jun was confirmed using a total protein 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 stain (Fig. 6B, middle panel). As an additional control, whole cell products at a lower cycle number in the absence of TA. C, the relative expression of extracts used in the JNK assay were immunoblotted against the 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 chaperone HDJ-2, which was present at higher levels in cells treated relative MKP-1 mRNA levels are compared for cells treated with vehicle (o), TA (u), and with RU486 at 8 and 24 h. TA plus RU486 ( ). 176

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 Fig. 6. MKP-1 protein levels are reduced, and JNK activity is increased by GC in 697 lines may determine whether MKP-1 GREs are positively or nega- cells. A, anti-MKP-1 immunoblot of total protein extracts from 697 cells treated with 100 tively regulated by GR. Alternatively, it has been suggested that the nM TA or TA plus 2 ␮M RU486 (RU) for 1–24 h. The nonspecific immunoreactive species relative abundance of the non-ligand binding GR-␤ isoform may be (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 responsible for such tissue-specific effects (47). (1–79) as described in the “Materials and Methods.” A 1-h treatment with anisomycin Repression of MKP-1 is an attractive mechanism for the regulation ␮ (Ani, 20 g/ml) was performed as a positive control for induction of JNK activity, and a of GC-induced apoptosis. MKP-1 is an antiapoptotic protein because 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 its presence or overexpression protects various tissues from cell death substrate and the cell extracts, respectively. Fold induction (FI) of JNK activity is induced by UV (48), TNF (49), cisplatin (50), and Fas ligand (51). indicated. MKP-1 is overexpressed in a variety of human cancers (52, 53) and may contribute to unregulated proliferation through inhibition of DISCUSSION MAPK activity. MKP-1 dephosphorylates and inhibits the activity of the extracel- Using dominant negative caspase constructs together with specific lular signal-regulated kinase, p38, and JNK MAPKs in vitro, but JNK caspase inhibitors, we investigated the role of apical caspases in the is likely to be the most physiologically relevant substrate (38, 50). execution of GR-mediated apoptotic cell death in 697 cells. We have Therefore, our observation that JNK activity is induced between 4 and shown that among the death receptor-associated caspases, caspase-10, 24 h after TA treatment in 697 cells is likely to be caused, in part, by but not caspase-8, is essential for a full apoptotic response to GC. the loss of MKP-1 activity. The important role of JNK in promoting DN-caspase-10 showed significant inhibition of GC-induced apopto- apoptosis is well characterized (54). For example, T cells from jnk2 sis, whereas expression of DN-caspase-8 and CrmA had no effect. (Ϫ/Ϫ) mice show various apoptotic defects (55) and inhibiting JNK These data offer an important functional distinction between activation prevents drug-induced apoptosis in chronic myelogenous caspase-8 and caspase-10, which are homologous but have different leukemia cells (56). Furthermore, JNK signaling through c-Jun has substrate profiles (23). The importance of caspase-10 in physiologi- been shown to induce expression of the proapoptotic Bcl-2 related cally relevant lymphocyte apoptosis is underscored by its high occur- protein, Bim (57), which was induced by TA in 697 cells in this study rence of mutation in hereditary Autoimmune Lymphoproliferative (Table 1). It remains to be determined if c-Jun activation is a critical Syndrome (39). event in GC-induced apoptosis of leukemic cells. DN-caspase-10 was unable to inhibit caspase-3 activity in 697 cells, GC-induced apoptosis was one of the first recognized forms of suggesting that the ability of caspase-10 to mediate GC-induced programmed cell death but remains one of the least understood. Our apoptosis is caspase-3 independent. This implies that caspase-3 defi- results indicate a novel, caspase-3 independent role for caspase-10 in ciency does not completely prevent GC-induced apoptosis in 697 cells this pathway. Moreover, microarray technology has provided new and that other downstream effector caspases such as caspase-6 and mechanistic insight into how GC exposure leads to caspase activation. caspase-7 may be involved. Indeed, studies have shown that caspase-6 Rather than a linear series of biochemical events, it is likely that and caspase-7 are activated in lymphocytes in response to GC (40– multiple GR-induced genes activate a network of pathways that con- 42). Thus, GC-induced apoptosis in 697 cells is caspase-9 and tribute to apoptosis. caspase-10 dependent (Fig. 1B) and partially caspase-3 independent (Fig. 1C). ACKNOWLEDGMENTS We have used oligonucleotide microarray technology to identify GR-regulated genes in 697 cells, expanding the data set previously We thank Drs. Emad Alnemri and Srinivasa Srinivasula for the contribution generated using other GC-sensitive hematopoietic cell lines (43, 44). of the dominant negative caspase plasmid constructs, Dr. Don Baldwin for his technical expertise in DNA microarray technology, and members of the Although there is some overlap between GR-regulated genes in 697 Litwack Lab for their helpful discussions. pre-B and CEM T ALL cells, we did not observe the large transcriptional changes in genes involved in ATP generation, protein synthesis, REFERENCES and RNA synthesis reported for the latter. Conversely, the CEM study did not identify many of the GR-regulated genes discovered in 697 1. 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