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[Cancer Research 64, 8036-8044, November 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Casein Kinase I Attenuates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis by Regulating the Recruitment of Fas-Associated Death Domain and Procaspase-8 to the Death-Inducing Signaling Complex

Kamel Izeradjene, Leslie Douglas, Addison B. Delaney and Janet A. Houghton

Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor–mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKI{alpha} isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKI{alpha} plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytotoxic ligand tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein, belongs to the tumor necrosis factor receptor superfamily of ligands and receptors (1 , 2) . TRAIL induces apoptosis in a wide variety of cultured malignant cell lines but not in nontransformed or normal cells and tissues (1) . TRAIL binds to the apoptosis-inducing receptors DR4 or DR5, which are type I transmembrane receptors, expressed on the surface. TRAIL also binds to non-apoptosis-inducing decoy receptors, which compete with death receptors for the ligand and suppress apoptosis. These include DcR1, DcR2, and osteoprotegerin (3) and may constitute one mechanism by which normal cells can evade the induction of apoptosis by TRAIL. Recombinant trimeric soluble TRAIL also lacks significant toxicity in primate species that possess receptors capable of binding human TRAIL (1 , 2) .

Early biochemical events resulting in apoptosis induction by ligand-induced cross-linking initiate trimerization of the receptor and the formation of a death-inducing signaling complex (3 , 4) . Cross-linking of the TRAIL receptors DR4 or DR5 results in the recruitment of Fas-associated death domain (FADD) and procaspase-8 to the death-inducing signaling complex (3, 4, 5) , similar to the death-inducing signaling complex formed after Fas ligation (5 , 6) . In a homotypic interaction, the death domain of FADD binds to the death domain of the receptor. The death effector domain of FADD in turn interacts with the death effector domain of caspase-8, thereby recruiting this pro-enzyme to the death-inducing signaling complex, where it is activated by dimerization (7 , 8) . Activated caspase-8 subsequently initiates the apoptosis-executing caspase cascade (9) . This cascade is further controlled by "cross-talk" between the intrinsic (mitochondrial) and extrinsic (death receptor) cell death pathways, thereby modulating the outcome of death receptor triggering (10) .

Proapoptotic signaling downstream of TRAIL can be blocked by Bcl-2 or Bcl-xL overexpression in some cell types (11) , whereas others cannot be protected by overexpression of these inhibitory molecules, leading to the concept of two different cell types that use distinct signaling pathways with (type II) or without (type I) the necessity for mitochondrial processing of the cell death signal (6) . Additional complexity is added to the regulatory pathways involved in death receptor sensitivity by proteins that are capable of inhibiting active caspases. These proteins, the inhibitor-of-apoptosis proteins (IAPs), include XIAP, c-IAP1, and c-IAP2, and block apoptosis by directly inhibiting caspases (12) .

In the mitochondrial pathway, a variety of death signals trigger the release of several pro-apoptotic proteins (cytochrome c, Smac/DIABLO, HtrA2/Omi, AIF, and endonuclease G) from the mitochondria to the cytosol or nucleus where they are actively involved in the process of cell death (13) . Once released, cytochrome c forms a complex with Apaf-1 and procaspase-9 in the presence of dATP or ATP, resulting in the activation of this initiator caspase (12) . In contrast, Smac/DIABLO or HtrA2/Omi promotes caspase activation by direct binding to and inhibition of the IAP family of caspase inhibitors, in particular XIAP (12 , 13) . The sensitivity of type II cells to death receptor-mediated apoptosis may be modulated by different mechanisms (6) , including phosphorylation-based signaling pathways (14) . Stimulation of the mitogen-activated protein kinase (MAPK) pathway has antagonized TRAIL-induced apoptosis downstream of Bid cleavage in human breast cancer MCF-7 cells (15) . Furthermore, activation of protein kinase C has inhibited TRAIL-induced caspase activation, mitochondrial events, and apoptosis in a human leukemic T-cell line (16) . Conversely, inhibition of death receptor–mediated ERK1/2 activation was sufficient to sensitize MCF-7 cells to apoptotic signaling induced by TRAIL (15 , 17) . Phosphorylation has also been reported to interfere with the apoptotic cascade downstream of death-inducing signaling complex activation but upstream of full cleavage of caspase-8 or the cleavage of Bid (18 , 19) and can inhibit the cleavage of Bid by caspase-8 (20) .

Casein kinase I (CKI) was one of the first serine/threonine protein kinases to be isolated and characterized (21) . It is a ubiquitous enzyme that can be found in the nucleus and in the cytosol, bound to the cytoskeleton and membranes. The CKI family consists of multiple isoforms encoded by seven different genes (CKI{alpha}, -ß, -{gamma}1, -{gamma}2, -{gamma}3, -{delta}, and -{epsilon}). These isoforms exhibit >50% amino acid homogeneity within the NH2-terminal catalytic domain but contain divergent COOH termini (21) . CKI has been shown to phosphorylate the p75 tumor necrosis factor receptor and to negatively regulate p75-mediated apoptosis (22) and may also play a constitutive and protective role in Fas-mediated apoptosis (20) . Inhibition of CKI has accelerated Fas-mediated apoptosis of HeLa cells, whereas overexpression of CKI delayed the death of these cells exposed to agonistic anti-Fas antibody (20) . In addition, CKI can phosphorylate Bid close to the recognition site for caspase-8 cleavage, thereby preventing cells from undergoing apoptosis (20) . In this study, a mutant of Bid that cannot be phosphorylated was found to be highly sensitive to caspase-8-induced cleavage.

To further investigate mechanisms of resistance to TRAIL in malignant cell lines and how this may be circumvented, we analyzed the role of CKI in the inherent resistance of colon carcinoma and pediatric rhabdomyosarcoma cell lines to TRAIL. We have demonstrated that inhibition of CKI function effectively sensitizes these cell lines to TRAIL at the level of the death-inducing signaling complex by enhancing the recruitment of FADD and procaspase-8 to the receptor complex. Enhancement of death-inducing signaling complex formation is followed by acceleration of TRAIL-induced apoptosis. In isogenic cell lines constructed using HT29, which differ only in the presence or expression level of DN-FADD, Bcl-2, or Bcl-xL, respectively, we demonstrated that sensitization to TRAIL-induced apoptosis in the presence of the specific inhibitor of CKI (CKI-7) involved the enhanced recruitment of FADD and procaspase-8 to the death-inducing signaling complex with enhanced caspase-8 cleavage. Furthermore, activation of mitochondrial signaling pathways necessary for inhibition of the function of XIAP or c-IAP1 were also confirmed. After inhibition of the expression of CKI{alpha} by employing a short hairpin RNA (23) , TRAIL sensitivity was dramatically enhanced in HT29 cells. These findings demonstrate that CKI inhibition augments TRAIL-induced cell death, modulates TRAIL-induced death-inducing signaling complex formation in the absence of elevated TRAIL receptors expression, and enhances the activation of mitochondrial signaling pathways.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
HT29 and HCT8 human colon carcinoma cell lines were obtained from American Type Culture Collection. JR1, derived from a pediatric rhabdomyosarcoma, has been described previously (24) . Human umbilical vascular endothelial cells were obtained from Dr. Michael Kastan (St. Jude Children’s Research Hospital). Human foreskin fibroblasts cells were obtained from Dr. Jeffrey Dome (St. Jude Children’s Research Hospital). Cell lines were maintained in RPMI 1640 (Gibco, Carlsbad, CA) supplemented with 2 mmol/L glutamine and 10% fetal calf serum.

Kinase Assays.
The kinase assay is based on phosphorylation of a CKI-specific peptide substrate using the transfer of the {gamma}-phosphate of [{gamma}-32P]ATP by CKI kinase. Whole-cell extracts from HT29 cells (20 µg) were assayed for CKI kinase activity at 30°C for 15 minutes in kinase buffer [250 mmol/L Tris-HCL (pH 7.5), 250 mmol/L NaCl, 250 mmol/L KCl, 50 mmol/L MgCl2, 250 µmol/L Na3VO4, and 5 µCi of [{gamma}-32P]ATP) containing 1 mmol/L peptide as substrate. An assay for CK2 activity was conducted in the same buffer using 5 µCi of [{gamma}-32P]GTP and 1 mmol/L peptide substrate. The phosphorylated substrate was separated from residual 5-µCi [{gamma}-32P]ATP or 5-µCi [{gamma}-32P]GTP using P81 phosphocellulose paper. Incorporation of [32P] into the substrate was measured using a scintillation counter. The synthetic peptides DDDEESITRR (CKI specific), and RRREEETEEE (CKII specific) were synthesized in the Hartwell Center, St. Jude Children’s Research Hospital. Inhibition of CKII activity was achieved by incubation of cells with 5,6-dichlorobenzimidazole (40 µmol/L; Calbiochem; ref. 25 ) or apigenin (60 µmol/L; Sigma-Aldrich, St. Louis, MO; ref. 25 ).

Production of Recombinant Human Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand.
The cDNA of the extracellular domain of TRAIL corresponding to amino acids 114 to 281 was subcloned into the pET17/b (Novagen, Madison, WI) bacterial expression vector and expressed in the BL21(DE3)pLysE (Novagen) bacterial host. After induction of TRAIL expression using isopropyl-ß-thio-galactosidase (1 mmol/L), bacterial pellets were harvested, and TRAIL was purified after passage through a nickel column (nickel-nitrilo-triacetic acid) followed by a size exclusion column (Amersham Biosciences, Piscataway, NJ) according to published procedures (1 , 6) .

Apoptosis Assays.
Cells were plated at a density of 200,000 cells per well in 12-well plates and after overnight attachment were treated with TRAIL (10–50 ng/mL) in either the absence or presence of the specific CKI inhibitor, CKI-7 (400 µmol/L; Seikagaku Corp., Tokyo, Japan) for up to 24 hours. Cells were subsequently stained with 10 µL of propidium iodide (50 ng/mL) according to the manufacturer’s instructions, incubated for 15 minutes at room temperature in the dark, and immediately analyzed for forward scatter by FACscan (Becton Dickinson, San Jose, CA; ref. 24 ). In some experiments, apoptosis was measured by staining with allophycocyanin (APC)-conjugated annexin V according to the manufacturer’s instructions. Cells were also pretreated with the pancaspase inhibitor z-VAD-fmk (50 µmol/L; Enzyme Systems Products, Livermore, CA) for 1 hour before TRAIL treatment.

Analysis of the Death-Inducing Signaling Complex.
Immunoprecipitation of receptor complexes after treatment of cells for time periods of up to 2 hours with 0.5 µg/mL flag-tagged TRAIL (Upstate, Lake Placid, NY) and analysis of components of the death-inducing signaling complex were conducted as described previously (3) .

Western Blot Analysis.
Western blot analyses were conducted as described previously (6) . Primary antibodies for the detection of caspase-8, Smac/DIABLO, and XIAP were from MBL (Watertown, MA) and for caspase-3, Bid, and poly(ADP-ribose) polymerase (PARP), from BD PharMingen (San Diego, CA). The cytochrome c monoclonal antibody was purchased from Clontech (Palo Alto, CA). Anti-AIF was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-HtrA2/Omi antibody was kindly provided by Dr. Emad Alnemri (Kimmel Cancer Institute, Philadelphia, PA). The c-IAP1 monoclonal antibody was purchased from Alexis Biochemicals (San Diego, CA). Anti-CK1{epsilon} was purchased from BD Transduction Laboratories (San Jose, CA), and anti-CK1{alpha} was purchased from Santa Cruz Biotechnology. Anti-DR5 was from Oncogene (Cambridge, MA). Recognized proteins were detected using horseradish peroxidase–labeled secondary antibodies (Amersham Biosciences).

Effect of Casein Kinase-7 on Expression of DR4 and DR5.
Cells were incubated at 37° for 2 and 24 hours with CKI-7 (400 µmol/L). After washing once with ice-cold PBS, cells were detached by brief trypsinization; incubated with monoclonal antibodies against DR4 (HS101), DR5 (HS201), or control murine immunoglobulin G1; and subsequently incubated with phycoerythrin-conjugated goat antimouse immunoglobulin (Alexis Biochemicals). Monoclonal antibodies were from Alexis Biochemicals. Cells (104) were analyzed by FACScan (Becton Dickinson).

Measurement of Caspase-3 Activation.
Before making the protein extract, floating cells were collected and combined with cells growing on the dish and washed two times with PBS. The cells were lysed in caspase lysis buffer [25 mmol/L HEPES-NaOH (pH 7.4), 0.1% sucrose, 1% CHAPS, 2 mmol/L EDTA, and 10 mmol/L dithiothreitol]. Cell lysates were mixed with caspase assay buffer [25 mmol/L HEPES-NaOH (pH 7.4), 10 mmol/L dithiothreitol, and 50 µmol/L concentration of the fluorogenic substrate Ac-DEVD-AMC (caspase-3)]. After incubation at 37°C for 1 hour, the fluorometric detection of leaved AMC product was performed on a CytoFluor Multi-well plate Reader series 2350 (Millipore, Bedford, MA) using a 400-nm excitation filter and a 530-nm emission filter. Detection of leaved AMC was shown to reflect caspase activity by incubation with 50 µmol/L zVAD-fmk for 30 minutes at 37°C before addition of Ac-DEVD-AMC substrate.

Plasmid Vectors and Transfection.
The retroviral expression vectors pMSCV-I-GFP [expressing green fluorescent protein (GFP)], and pMSCV-DN-FADD (expressing the death domain only of FADD) were kind gifts from Dr. Jill M. Lahti and Dr. Vincent J. Kidd (St. Jude Children’s Research Hospital). The retroviral expression vectors pMSCV-Bcl-xL (expressing human Bcl-xL and GFP) and pMSCV-Bcl-2 (expressing human Bcl-2 protein) were kindly provided by Dr. John Cleveland (St. Jude Children’s Research Hospital). Retroviral supernatants were prepared as described previously (26) . HT29 cells were incubated overnight in a 50% mixture of RPMI 1640 and retroviral supernatants in the presence of Polybrene (8 µg/mL), subsequently washed, and placed in fresh medium; and after 48 hours, cells were sorted for GFP expression by fluorescence-activated cell sorting. The expression of Bcl-2, Bcl-xL, and DN-FADD were confirmed by Western blotting.

Plasmids Expressing Short Hairpin Ribonucleic Acids and Transfection.
The short hairpin RNA sequences were designed using designated software found on the OligoRetriever Database and were prepared by the Hartwell Center, St. Jude Children’s Research Hospital. The sequences encoded inverted repeats of 27 to 29 bp separated by an eight-nucleotide spacer corresponding to nucleotides 193 to 221 (sh193) or 238 to 266 (sh238) of CKI{alpha} cDNA. These were subsequently ligated to the U6 promoter vector (pSHAg-1) compatible with the GATEWAY system (Invitrogen, Carlsbad, CA), used to transport the short hairpin RNA expression cassette into the recipient MSCV-I-GFP retroviral vector. HT29 cells were incubated overnight in a 50% mixture of RPMI 1640 and retroviral supernatants in the presence of Polybrene (8 µg/mL), subsequently washed, and placed in fresh medium; and after 48 hours, cells were tested for GFP expression. The U6 promoter vector pSHAG-1 and the recipient vector MSCV-I-GFP were kindly provided by Dr. Gregory J. Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Design of short hairpin RNA primers from gene accession numbers was conducted from the RNAi OligoRetriever Database.1

Cellular Fractionation.
Cytosolic extracts were prepared using the ApoAlert kit (Clontech) with a Dounce homogenizer and subjected to centrifugation at 700 x g to pellet nuclei. The post-nuclear supernatant was centrifuged at 10,000 x g to pellet the mitochondria-enriched heavy membrane fraction, and the resulting supernatant was further centrifuged at 100,000 x g to obtain the cytosolic fraction. Total proteins (15 µg) were subjected to immunoblot analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Casein Kinase I-7 Inhibits Casein Kinase I and not Casein Kinase 2 Activity.
The specificity of CKI-7 in the inhibition of CKI versus CK2 activity was initially examined using HT29 colon carcinoma cells. Exposure of HT29 cells to the CKI inhibitor, CKI-7 (400 µmol/L), resulted in inhibition of phosphorylation of the CKI-specific peptide substrate (DDDEESITRR; Fig. 1ACitation ). Treatment of cells with the plant flavonoid apigenin, an inhibitor of CK2 activity, had no effect on the activity of CKI. Inhibition of CK2 activity was demonstrated using the classic CK2 inhibitor, 5,6-dichlorobenzimidazole (40 µmol/L), however, the enzyme was not inhibited by CKI-7 (Fig. 1A)Citation .



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  		Fig. 1. A, effect of CKI-7 on CKI and CK2 kinase activity in HT29 cells. Cells were either untreated or treated with the indicated concentrations of each agent for 2 hours and harvested, and the phosphotransferase activity of CKI and CK2 was determined using a CKI- or CK2-specific substrate (DDDEESITRR and RRREEETEEE, respectively). Data represent 32P incorporated (cpm) into the CKI or CK2 substrate peptide. Results are the mean ± SD of two determinations per point. B. CKI-7 sensitizes tumor cells to TRAIL-induced apoptosis. Cells were treated with TRAIL (50 ng/mL) for 24 hours. Caspase-dependent cytotoxicity of TRAIL was only detectable in the presence of the CKI inhibitor, CKI-7 (400 µmol/L), added for 2 hours before and during exposure to TRAIL. Z-VAD-fmk (50 µmol/L) completely abolished TRAIL-induced apoptosis when coincubated with TRAIL. C. z-VAD-fmk abrogates TRAIL-induced apoptosis in the presence of CKI-7. HT29, HCT8, or JR1 cells were treated with CKI-7 (400 µmol/L) for 2 hours before and during exposure to TRAIL (50 ng/mL) for 24 hours. z-VAD-fmk (50 µmol/L) completely abolished TRAIL-induced apoptosis when coincubated with TRAIL. Results are the mean ± SD of two determinations per point.

 
Casein Kinase I Attenuates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis.
To explore the effect of CKI inhibition on the response of HT29 cells to TRAIL, we determined whether CKI-7 affected TRAIL-induced cell death. Treatment with CKI-7 (400 µmol/L) 2 hours before and during incubation with TRAIL (50 ng/mL; 24 hours), resulted in dramatic sensitization of cells to TRAIL-induced apoptosis that could be completely inhibited by the broad-spectrum caspase inhibitor z-VAD-fmk (50 µmol/L; Fig. 1BCitation ), confirming the critical role of caspases in this process. Similarly, in both HCT8 and JR1 cells, inherently resistant to TRAIL (50 ng/mL), apoptosis was induced after pretreatment and coincubation with CKI-7 (400 µmol/L) as determined morphologically and was also inhibited in the presence of z-VAD-fmk (Fig. 1B)Citation . In HT29, HCT8, and JR1 cells, treatment with TRAIL (50 ng/mL) alone for 24 hours induced <10% apoptosis, however, when combined with CKI-7 (400 µmol/L) before and during TRAIL exposure, apoptosis was induced to >90% in all three cell lines (Fig. 1C)Citation . In contrast to tumor cell lines, the combination of TRAIL (50–300 ng/mL) and CKI-7 (400 µmol/L) for 24 hours was not cytotoxic to normal human foreskin fibroblasts cells expressing significant levels of DR5 (Fig. 2)Citation . The same results were observed with human umbilical vascular endothelial cells even at the highest TRAIL concentration.



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  		Fig. 2. A. Human foreskin fibroblasts (HFF) and umbilical vascular endothelial cells (HUVEC) were treated with CKI-7 (400 µmol/L) for 2 hours before and during treatment with increasing concentrations of TRAIL (50–300 ng/mL). Apoptosis was determined 24 hours after TRAIL treatment. B, evaluation of DR4 and DR5 surface expression on human foreskin fibroblasts cells. Results are the mean ± SD of two determinations per point.

 
Recruitment of Fas-Associated Death Domain and Procaspase-8 to the Death-Inducing Signaling Complex Is Regulated by Casein Kinase I.
To determine whether CKI demonstrated a role in regulating TRAIL-induced death-inducing signaling complex formation, HT29 cells were treated with TRAIL (50 ng/mL) in the absence or presence of CKI-7 (400 µmol/L) for up to 2 hours. Immunoprecipitation of receptor complexes followed by Western analysis demonstrated that treatment of HT29 with CKI-7 increased the recruitment of both FADD and procaspase-8 to the death-inducing signaling complex, determined at 1 and 2 hours after the initiation of TRAIL treatment (Fig. 3A)Citation . Similarly, in both HCT8 and JR1 cells (Fig. 3A)Citation , after immunoprecipitation of the TRAIL-induced death-inducing signaling complex, the level of procaspase-8 recruited to the death-inducing signaling complex was significantly enhanced in cells treated with CKI-7 for 2 hours before and during TRAIL exposure. Furthermore, not only was the amount of procaspase-8 recruited to the death-inducing signaling complex increased, but the cleavage of procaspase-8 to p41/43 fragments also increased during CKI inhibition. In CKI-7-treated HCT8 and JR1 cells, the death-inducing signaling complex remained stable for at least 2 hours, whereas in cells treated with TRAIL alone, caspase-8 was no longer recruited and activated at the death-inducing signaling complex. In contrast to DR5, DR4 was not present in the immunoprecipitates. FADD was below the level of detection within the death-inducing signaling complex in both HCT8 and JR1 TRAIL-treated cells, even in the presence of CKI-7. However, retroviral transfection of DN-FADD indicated that in the presence of TRAIL combined with CKI-7, TRAIL-induced apoptosis was inhibited in both cell lines (Fig. 3B)Citation , indicating the importance of FADD in the mechanism of sensitization to TRAIL-induced apoptosis by CKI-7.



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  		Fig. 3. Influence of CKI-7 on formation of the TRAIL receptor complex. A. HT29, HCT8, and JR1 cells were treated for up to 2 hours with Flag-TRAIL precomplexed with anti-Flag antibodies (clone M2; 2 µg/mL) in the presence or absence of CKI-7 (400 µmol/L). The resulting protein complexes were separated by SDS-PAGE and analyzed by Western blot for components of the TRAIL-induced death-inducing signaling complex. B. HCT8 and JR1 cells were stably transfected with plasmids encoding GFP and DN-FADD, and transfected cells were sorted for GFP expression by FACS analysis. Apoptosis was determined 24 hours after TRAIL treatment in the presence or absence of CKI-7. Results are the mean ± SD of two determinations per point.

 
Influence of Casein Kinase I on the Levels of Expression of DR4 or DR5.
Up-regulation of apoptosis-inducing TRAIL receptors could result in enhanced sensitivity for TRAIL-induced apoptosis. We, therefore, examined surface expression of both TRAIL receptors DR4 and DR5 on all three cell lines before and after 2 and 24 hours of treatment with CKI-7. We found marginal expression of DR4 receptor in HCT8; HT29 cells and JR1 cells did not express DR4 receptor. Treatment with CKI-7 resulted in a slight increase in DR4 levels in HCT8 and JR1 cell lines (Fig. 4)Citation . DR5 receptor was expressed at high levels in JR1 cells compared with HT29 and HCT8 cell lines. Upon treatment with CKI-7, DR5 receptor was significantly up-regulated after 24 hours only in HCT8 cell lines. Taken together, CKI inhibition led to the up-regulation of DR5 only in HCT8 cell line and, to a lesser degree, of DR4 in HCT8 and JR1 cells. Thus, increased surface expression of apoptosis-inducing TRAIL receptors could potentially contribute to the increased sensitivity of HCT8 and JR1 cells to TRAIL-induced apoptosis but not for HT29 cells in which the levels of DR4 and DR5 were not modulated upon CKI inhibition.



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  		Fig. 4. Comparison of TRAIL–receptor surface expression in colon and RMS cell lines with or without CKI-7 treatment. FACS analysis of surface expression of DR4 and DR5 without (filled line) or with CKI-7 (400 µmol/L) for 2 hours (solid line) or 24 hours (solid bold line) compared with an isotype-matched control mIgG1 mAb (dashed line). Expression of both receptors was assessed by phycoerythrin fluorescence using flow cytometry.

 
Influence of DN-Fas-Associated Death Domain, Bcl-xL, or Bcl-2 Overexpression.
Using isogenic HT29 cell lines produced by retroviral transfection of specific genes (Fig. 5C)Citation , TRAIL (50 ng/mL) combined with CKI-7 (400 µmol/L) significantly enhanced the cleavage of caspase-8, caspase-3, Bid, and PARP in HT29GFP control cells analyzed at 5 hours after TRAIL treatment (Fig. 5A)Citation . Stable transfection with DN-FADD completely inhibited the induction of apoptosis by TRAIL in the presence of CKI-7 (Fig. 5A and BCitation ; 24 hours). HT29 cells overexpressing Bcl-xL (Fig. 5A and B)Citation or Bcl-2 (Fig. 5B)Citation were examined for their susceptibility to TRAIL-induced apoptosis in the presence of CKI-7. Overexpression of both inhibitory proteins resulted in inhibition of TRAIL-induced cell death. However, whereas Bcl-xL blocked the cleavage of PARP in cells treated with the combination of TRAIL and CKI-7, the cleavage of caspase-8, caspase-3, and Bid was not inhibited.



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  		Fig. 5. CKI-7 enhances TRAIL-induced activation of caspases and release of proapoptotic factors from the mitochondria. HT29 cells were stably transfected with plasmids encoding GFP, DN-FADD, Bcl-2, or Bcl-xL; and transfected cells were sorted for GFP expression by FACS analysis. A. Cells were treated with CKI-7 (400 µmol/L) 2 hours before and during TRAIL stimulation. After 5 hours, lysates were prepared and analyzed by SDS-PAGE using specific antibodies as described in Materials and Methods. B. Cells were treated with TRAIL (50 ng/mL) for 24 hours in the presence or absence of CKI-7. Apoptosis was determined by annexin V staining. C. Expression of DN-FADD, Bcl-2, or Bcl-xL was determined by Western blotting after transfection. D. Inhibition of CKI facilitates TRAIL-induced apoptosis. HT29 cells were treated for up to 3 hours with TRAIL (50 ng/mL) in the presence or absence of CKI-7 (400 µmol/L). Cytoplasmic lysates were subsequently prepared as described in Materials and Methods and analyzed for their content of cytochrome c, Smac/DIABLO, HtrA2/Omi, AIF, XIAP, and c-IAP1 by Western analysis. E. HT29 cells transfected with vector control (GFP), Bcl-2, or Bcl-xL were treated as in D. Equal loading of cytoplasmic proteins was determined by reprobing of blots with ß-actin–specific antibody. F. HT29 cells transfected with vector control (GFP), Bcl-2, or Bcl-xL were treated as in D for up to 6 hours. Equal loading of cytoplasmic proteins was determined by reprobing of blots with ß-actin–specific antibody. G. HT29 cells transfected with vector control (GFP), Bcl-2, or Bcl-xL were treated as in D for up to 6 hours. Caspase-3 activity was defined as relative rates of degradation of Ac-DEVD-AMC over 1 hour at 37°C. Data shown are mean ± SD and representative of three independent experiments.

 
Inhibition of Casein Kinase I Facilitates Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Release of Proapoptotic Factors from the Mitochondria.
The kinetics of release of mitochondrial proteins into the cytosol was examined during CKI-7-induced sensitization of HT29 cells to TRAIL-induced apoptosis. In cells treated with TRAIL alone (50 ng/mL for up to 3 hours), release of cytochrome c, Smac/DIABLO, and HtrA2/Omi into the cytosol could not be detected. In contrast, in cells treated with TRAIL in the presence of CKI-7 (400 µmol/L), cytochrome c, Smac/DIABLO, and HtrA2/Omi were rapidly released from the mitochondria, in the absence of effect of CKI-7 alone (Fig. 5D)Citation . The release of AIF was also enhanced in TRAIL-treated cells in the presence of CKI-7. Inhibition or degradation of XIAP and c-IAP1 was observed only in cells treated with the combination of TRAIL and CKI-7. Data suggest that inhibition of the function of CKI thereby allows TRAIL-induced mitochondrial activation of the cell death signal necessary for the inactivation of XIAP and c-IAP1 and subsequent cell death. Because TRAIL-induced apoptosis was associated with the redistribution of cytochrome c to the cytosol, an event that is inhibited by Bcl-2 (27) , we determined whether Bcl-2 or Bcl-xL was capable of regulating cytochrome c release in HT29 cells treated with TRAIL in the presence of CKI-7. HT29 cells were transfected with vector alone (GFP), Bcl-2, or Bcl-xL and were treated with TRAIL for 3 hours in the presence or absence of CKI-7. As illustrated in Fig. 5ECitation , release of cytochrome c was completely inhibited in HT29 cells overexpressing Bcl-2 or Bcl-xL compared with vector control (GFP) cells treated with the combination TRAIL + CKI-7.

IAPs such as XIAP exert their antiapoptotic function at least in part, downstream of the mitochondrial checkpoint by inhibiting proteolytically processed caspases including caspase-3, caspase-7, and caspase-9. In addition, XIAP has been recently reported to act as a ubiquitin-ligase for activated caspase-3 or caspase-7, thereby promoting the degradation of activated caspases (28) . XIAP expression was therefore examined by Western analysis. After TRAIL treatment in the presence of CKI-7, XIAP was down-regulated in HT29 vector control cells (GFP), whereas cleavage of XIAP was strongly reduced in Bcl-2 and Bcl-xL transfected cells (Fig. 5F)Citation . The same profile was observed for c-IAP1. This indicates that apoptosis was blocked downstream of mitochondria by reduction of the cleavage of XIAP and c-IAP1 proteins in HT29 cells transfected with Bcl-2 or Bcl-xL. A possible explanation for the results in Fig. 5ACitation showing failure of Bcl-xL to reduce caspase-8, Bid, and caspase-3 cleavage after TRAIL treatment in the presence of CKI-7 is that caspase-8 is activated upstream of mitochondria and HT29 cells require down-regulation of XIAP to increase the autocatalytic activity of caspase-3 and breakdown of one of its substrates, PARP. It was recently shown that the conversion of the p20 fragment of cleaved caspase-3 by autocatalysis to the smaller p17 subunit was accelerated by down-regulation of XIAP (29) . To test whether caspase-3 activity was abrogated in cells overexpressing Bcl-2 or Bcl-xL, the proteolytic activity of caspase-3 was measured by testing the cytosolic extracts for their ability to cleave the fluorometric substrate Ac-DEVD-AMC, which is specific for caspase-3. Fig. 5GCitation shows that in HT29 cells transfected with Bcl-2 or Bcl-xL and treated with TRAIL+ CKI-7, caspase-3 activity was dramatically reduced compared with control (GFP) cells. This activity was due to caspases, because zVAD-fmk blocked the activity (Fig. 5G)Citation .

Short Hairpin Ribonucleic Acid Modulates Casein Kinase I{alpha} Expression and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Sensitivity in HT29 Cells.
Recent data has suggested that CKI{alpha} or CKI{epsilon} may play a protective role in Fas-mediated apoptosis (20) . After examination of the levels of expression of both CKI isoforms in HT29, HCT8, and JR1 cells by Western analysis, all cell lines expressed constitutively high levels of CKI{alpha}, in contrast to the expression of CKI{epsilon}, which demonstrated low-level expression in HT29 (Fig. 6A)Citation . Therefore, to examine whether modulation of CKI expression, in particular CKI{alpha}, could influence TRAIL-induced apoptosis, two retroviral vectors containing short hairpin RNA targeting CKI{alpha}, designated sh193 or sh238 were prepared, and their ability to influence CKI{alpha} levels was determined. Transfection of short hairpin RNAs into HT29 cells resulted in suppression of CKI{alpha} expression by sh193, with no effect mediated by sh238 (Fig. 6B)Citation . No effect on the expression of ß-actin was detected. Subsequently, it was determined whether reduction in CKI{alpha} levels with sh193 could sensitize HT29 cells to TRAIL-induced apoptosis. Transfected cells were treated for 24 hours with varying concentrations of TRAIL (10–100 ng/mL). Transfection of HT29 with sh193 resulted in dramatic sensitization of HT29 cells to TRAIL-induced apoptosis to >90%, whereas transfection with sh238 demonstrated no effect (Fig. 6B)Citation .



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  		Fig. 6. Effect of short hairpin RNA on CKI{alpha} expression and on TRAIL sensitivity in HT29. A. Expression of both CKI isoforms was determined by Western analysis. Cell lysates were subsequently prepared as described in Materials and Methods and analyzed for their content of CKI{alpha}, CKI{epsilon}, and ß-actin. B. HT29 cells were stably transfected with empty vector (GFP) or CKI{alpha} short hairpin RNA (sh238 and sh193) and were treated with varied concentrations of TRAIL (10–100 ng/mL) for 24 hours. Results are the mean ± SD of two determinations per point.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Antibody or ligand-mediated cross-linking of death receptors for TRAIL offers an attractive strategy for inducing apoptosis in tumor cells and potentially a therapeutic approach for the treatment of a wide variety of solid tumors. However, cell lines exhibit a wide heterogeneity in their sensitivity to TRAIL-induced apoptosis, and tumor cell lines can remain resistant to TRAIL, even though they express the death receptors DR4 and DR5 (1) . Signal transduction downstream of TRAIL ligation and its inhibition in nontransfected tumor cells still remains to be thoroughly explored, however, understanding the signaling events that control tumor cell death has the likelihood to enhance therapeutic potential for the treatment of human cancers.

Accumulating evidence suggests that CKI may be involved in the regulation of numerous cellular functions (21 , 30) , although there is a paucity of data clearly suggesting a role for CKI as an antiapoptotic factor following ligation of death receptors of the tumor necrosis factor receptor superfamily. One study, however, has demonstrated that CKI can phosphorylate the p75 tumor necrosis factor receptor and negatively regulate p75-mediated apoptosis (22) . In the present study, we have demonstrated that inhibition of CKI leads to sensitization of tumor cells derived from human colon carcinomas and rhabdomyosarcomas to TRAIL-induced apoptosis, in part by controlling the most apical point of intracellular signaling in the TRAIL signaling pathway, the recruitment of FADD and procaspase-8, and activation of caspase-8, at the level of the death-inducing signaling complex. CKI inhibition by a CKI-specific inhibitor (CKI-7; ref. 21 ) resulted in enhanced recruitment of FADD and procaspase-8 to the death-inducing signaling complex in HT29 cells after TRAIL stimulation and also enhanced the extent of caspase-8 cleavage at the death-inducing signaling complex in HCT8 and JR1. These effects occurred in the absence of any effect of inhibition of CKI function on the level of expression of either DR4 or DR5 in the three cell lines and are consistent with inhibition by CKI-7 of the ability of CKI to phosphorylate specific sites on these receptors, thereby enhancing the recruitment of adaptor proteins. The data demonstrate that TRAIL-induced death-inducing signaling complex formation can be modulated by CKI inhibition and that the inhibitory function of CKI on the induction of apoptosis may be more generally applicable to malignant cells derived from different lineages.

After activation of caspase-8 at the death-inducing signaling complex, the cytosolic substrate Bid is rapidly cleaved, leading to the generation of a truncated form of Bid, tBid (31) . Cleaved/truncated Bid translocates to the mitochondria (32) upon its myristoylation (33) , where it triggers initiation of mitochondrial disruption and additional amplification of the death cascade. Phosphorylation of Bid by CKI{alpha} or CKI{epsilon} has been reported to render Bid resistant to caspase-8 cleavage (20) . Recent studies have shown that CKI phosphorylates Bid in the vicinity of the recognition site for caspase-8 and that this phosphorylation renders Bid resistant to cleavage by caspase-8 and protects cells from Fas-induced apoptosis, whereas inhibition of Bid phosphorylation sensitizes cells to Fas-mediated apoptosis (20) . In the current investigation, acceleration of both caspase-8-mediated Bid cleavage and TRAIL-induced apoptosis was demonstrated in cells treated with the CKI inhibitor, CKI-7. Overexpression of DN-FADD in HT29, HCT8, and JR1 cells abrogated TRAIL-induced cell death in the presence of CKI-7, confirming that events upstream of the mitochondria are involved in the mechanism of CKI-7-induced sensitization to TRAIL-induced apoptosis. The current data also suggest that enhanced death-inducing signaling complex formation and activation of caspase-8 also contribute to the enhanced cleavage of Bid when cells are treated with TRAIL in the absence of CKI function.

Signaling events downstream of TRAIL receptor ligation and death-inducing signaling complex formation still remain to be thoroughly explored, in particular the role of mitochondrial signaling pathways for TRAIL-induced apoptosis. In some cell lines, TRAIL-induced apoptosis was shown to be largely independent of mitochondrial pathways (34 , 35) ; however, others have demonstrated that Bcl-xL protected pancreatic carcinoma cells from TRAIL-induced apoptosis (36) ; and inactivation of Bax was recently shown to confer resistance to TRAIL-induced apoptosis in colon cancer cell lines (37) . Taken together, data support the view that cell type–specific differences downstream of active initiator caspases are important in the fate of a cell after ligation of the TRAIL receptors. In HT29 cells overexpressing Bcl-2 or Bcl-xL, TRAIL-induced apoptosis in the presence of CKI-7 was completely inhibited. Even though similar processing of caspase-8, Bid, and caspase-3 was observed in HT29Bcl-xL and HT29GFP, cleavage of PARP was detected only in HT29GFP. These data are consistent with reports demonstrating that in cells overexpressing Bcl-2 or Bcl-xL, Fas-mediated cell death was markedly reduced, despite caspase-8 being processed and active (38) . Thus, the inhibition of caspase-3 activity in Bcl-xL-transfected cells was not limited to the Fas signaling pathway and is also apparent in the TRAIL pathway. The failure of Bcl-xL to reduce caspase-8, Bid, and caspase-3 cleavage after TRAIL treatment in the presence of CKI-7 (Fig. 5A)Citation may be due to caspase-8 being activated upstream of mitochondria, followed by Bid and caspase-3 cleavage. It is conceivable that active caspase-3 (p17 subunit) was inhibited by one of the IAP family members, which was reported by others to bind to and inhibit the activity of caspase-3, as well as caspase-7 and caspase-9 (39) . It was particularly noticeable that XIAP and c-IAP1 were significantly down-regulated in HT29 cells transfected with vector control (GFP) compared with HT29 cells overexpressing Bcl-2 or Bcl-xL. Degradation of XIAP and c-IAP1 correlated with the caspase-3 activity detected in the lysates of HT29 cells transfected with vector control (GFP). This activity was strongly reduced in HT29 cells overexpressing Bcl-2 or Bcl-xL. These data suggest that XIAP and c-IAP1 were mainly responsible for the inhibition of the autocatalytic activity of caspase-3 and breakdown of one of its substrates, PARP. As reported by others (29 , 39 , 40) , conversion of the p20 fragment of cleaved caspase-3 by autocatalysis to the smaller p17 subunit was accelerated by down-regulation of XIAP. The smaller p17 subunit of caspase-3 was not detectable in Western analyses, possibly due to rapid degradation in the cellular lysates.

We have also demonstrated that inhibition of CKI enhanced TRAIL-induced release of proapoptotic factors from mitochondria with concomitant inhibition of the function of members of the IAP family, in the absence of release of proapoptotic factors after TRAIL treatment alone. Both Bcl-2 and Bcl-xL were able to block TRAIL-induced release of cytochrome c and down-regulation of XIAP in the presence of CKI-7. Data demonstrate that activation of the mitochondrial apoptotic pathway is required for the execution of TRAIL-induced apoptosis in both colon carcinoma and rhabdomyosarcoma cells.

In Jurkat cells, CKI{alpha} was determined to be the major isoform of CKI expressed, with CKI{epsilon} as a minor component, although both influenced the phosphorylation of Bid or the induction of Fas-mediated apoptosis (20) . HT29, HCT8, and JR1 cells all expressed CKI{alpha}, whereas HT29 expressed low levels of CKI{epsilon}. Using short hairpin RNA to inhibit the function of CKI{alpha}, TRAIL-induced apoptosis was significantly enhanced to the same level observed when cells were treated with TRAIL in the presence of CKI-7, demonstrating the critical function of CKI in attenuating TRAIL-induced apoptosis.

Based on the current data, we propose a new mechanism promoted by CKI upstream of the function of caspase-8, in that CKI mediates TRAIL resistance at the level of recruitment of adaptor proteins to the receptors. Thus, modulation of CKI is one mechanism by which resistance of human cancer cells to TRAIL may be circumvented, a mechanism that may span cancers derived from different histotypes.


   FOOTNOTES
 
Grant support: NIH awards CA 32613 and CA 87952, Cancer Center Support (CORE) grant CA 21765, and the American Lebanese Syrian Associated Charities.

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.

Requests for reprints: Janet A. Houghton, Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105. Phone: 901-495-3456; Fax: 901-495-3966; E-mail: janet.houghton{at}stjude.org

1 Internet address: http://www.cshl.org/public/science/hannon.html. Back

Received 3/ 2/04. Revised 8/ 9/04. Accepted 8/26/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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