Interferon-γ Sensitizes Resistant Ewing's Sarcoma Cells to Tumor Necrosis Factor Apoptosis-Inducing Ligand-Induced Apoptosis by Up-Regulation of Caspase-8 Without Altering Chemosensitivity
2007; Elsevier BV; Volume: 170; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.060993
ISSN1525-2191
AutoresAndrej Lissat, Thomas Vraetz, Maria Tsokos, Ruth Klein, Matthias Braun, Nino Koutelia, Paul Fisch, María Elena Romero, Lauren Long, Peter Noellke, Crystal L. Mackall, Charlotte M. Niemeyer, Udo Kontny,
Tópico(s)NF-κB Signaling Pathways
ResumoEwing's sarcoma cells are highly susceptible to apoptosis via tumor necrosis factor apoptosis-inducing ligand (TRAIL). Resistance to TRAIL has been linked to deficient expression of caspase-8 in vitro. Here, we report on the status of caspase-8 expression in tumors from patients with Ewing's sarcoma, the effect of interferon-γ on caspase-8 expression and apoptosis, and the role of caspase-8 for TRAIL- and chemotherapy-mediated apoptosis in Ewing's sarcoma. Using immunohistochemistry, we show that low expression of caspase-8 is seen in about 24% of tumors. Interferon-γ induces expression of caspase-8 at concentrations achievable in humans and sensitizes cells to TRAIL. Transfection of wild type but not mutant caspase-8 into caspase-8-deficient Ewing's sarcoma cells restored sensitivity to TRAIL, indicating that up-regulation of caspase-8 is sufficient to restore TRAIL sensitivity. In contrast, no role for caspase-8 in chemotherapy-induced apoptosis was identified, because 1) transfection of caspase-8 or treatment with interferon-γ did not alter the sensitivity of caspase-8-deficient cells to chemotherapeutics, 2) application of chemotherapy did not select for caspase-8-negative tumor cells in vivo, and 3) the caspase-8 status of tumors did not influence survival after chemotherapy-based protocols. In conclusion, our data provide a rationale for the inclusion of interferon-γ in upcoming clinical trials with TRAIL. Ewing's sarcoma cells are highly susceptible to apoptosis via tumor necrosis factor apoptosis-inducing ligand (TRAIL). Resistance to TRAIL has been linked to deficient expression of caspase-8 in vitro. Here, we report on the status of caspase-8 expression in tumors from patients with Ewing's sarcoma, the effect of interferon-γ on caspase-8 expression and apoptosis, and the role of caspase-8 for TRAIL- and chemotherapy-mediated apoptosis in Ewing's sarcoma. Using immunohistochemistry, we show that low expression of caspase-8 is seen in about 24% of tumors. Interferon-γ induces expression of caspase-8 at concentrations achievable in humans and sensitizes cells to TRAIL. Transfection of wild type but not mutant caspase-8 into caspase-8-deficient Ewing's sarcoma cells restored sensitivity to TRAIL, indicating that up-regulation of caspase-8 is sufficient to restore TRAIL sensitivity. In contrast, no role for caspase-8 in chemotherapy-induced apoptosis was identified, because 1) transfection of caspase-8 or treatment with interferon-γ did not alter the sensitivity of caspase-8-deficient cells to chemotherapeutics, 2) application of chemotherapy did not select for caspase-8-negative tumor cells in vivo, and 3) the caspase-8 status of tumors did not influence survival after chemotherapy-based protocols. In conclusion, our data provide a rationale for the inclusion of interferon-γ in upcoming clinical trials with TRAIL. 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A total of 54 formalin-fixed, paraffin-embedded ES tumor specimens were available for immunohistochemical analysis. The samples were obtained during the period from 1986 to 2001 from 47 patients treated at the Pediatric Oncology Branch of the National Cancer Institute (n = 38) and from patients treated at the Children's Hospital of the University of Freiburg, Germany (n = 9). For four patients, samples were available both at initial diagnosis and at relapse; for one patient, at two subsequent relapses; and for another patient, at both initial diagnosis and two subsequent relapses. The majority of samples (n = 52) were derived from diagnostic biopsies at initial presentation or relapse. Two specimens were from tumors removed at surgery as part of multimodal treatment. The morphology of all tumor specimens was homo-geneous and consisted of small round tumor cell aggregates in lobular or sheet-like arrangement. Rosette formation was present only in one case. Immunohistochemical staining (desmin, 12E7, and leukocyte common antigen) and/or detection of EWS/Fli-1 fusion transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) (14 cases) was performed to differentiate the tumor from other round cell tumors. Patients' clinical data were analyzed by retrospective review of medical charts and are summarized in Table 1. Patients were treated after informed consent on Institutional Review Board-approved protocols such as 86-C-169 (n = 25), Euro-E.W.I.N.G. 99 (n = 9), 98-C-0037 (n = 6), and others (n = 7), including 83-C-73, 87-C-10, and 93-C-0125. All protocols contained vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide with the exception of 83-C-73, which only contained vincristine, doxorubicin, and cyclophosphamide. Informed consent for immunohistochemical analysis of archived tissues was waived because the patient identities were anonymized with regard to the investigators on this study.Table 1Characteristics of Patients with Ewing's SarcomaCharacteristicsn = 47%Age at diagnosis (median, range)18.11.0 to 32.8 20 yrs1736Sex Male2757 Female2043Primary tumor site Extremities1634 Chest1021 Pelvis1021 Other1123Primary tumor type Skeletal4085 Extraosseous715Extent of disease Localized3983 Metastatic817Outcome (%, confidence interval) 10-year overall survival6040 to 73 10-year disease-free survival5740 to 69 Open table in a new tab ES cell lines A4573, JR (NCI-EWS 94), SB (NCI-EWS 95), SK-N-MC, TC32, TC71, and 5838 were selected for study based on their different expression of caspase-8 (Table 2).24Kontny HU Hammerle K Klein R Shayan P Mackall CL Niemeyer CM Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis.Cell Death Differ. 2001; 8: 506-514Crossref PubMed Scopus (71) Google Scholar All cell lines have been previously characterized.33Dickman PS Liotta LA Triche TJ Ewing's sarcoma: characterization in established cultures and evidence of its histogenesis.Lab Invest. 1982; 47: 375-382PubMed Google Scholar, 34Giovannini M Biegel JA Serra M Wang JY Wei YH Nycum L Emanuel BS Evans GA EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations.J Clin Invest. 1994; 94: 489-496Crossref PubMed Scopus (138) Google Scholar, 35Liu XF Helman LJ Yeung C Bera TK Lee B Pastan I XAGE-1, a new gene that is frequently expressed in Ewing's sarcoma.Cancer Res. 2000; 60: 4752-4755PubMed Google Scholar, 36Merino ME Navid F Christensen BL Toretsky JA Helman LJ Cheung NK Mackall CL Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.J Clin Oncol. 2001; 19: 3649-3659Crossref PubMed Scopus (20) Google Scholar, 37Toretsky JA Connell Y Neckers L Bhat NK Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides.J Neurooncol. 1997; 31: 9-16Crossref PubMed Google Scholar They were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 100 U/ml penicillin, 100 μg/ml streptomycin, and 4 mmol/L glutamine.Table 2Characteristics of ES Cell Lines UsedCell lineOriginGene rearrangementCaspase-8 protein24Kontny HU Hammerle K Klein R Shayan P Mackall CL Niemeyer CM Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis.Cell Death Differ. 2001; 8: 506-514Crossref PubMed Scopus (71) Google ScholarA457333Dickman PS Liotta LA Triche TJ Ewing's sarcoma: characterization in established cultures and evidence of its histogenesis.Lab Invest. 1982; 47: 375-382PubMed Google ScholarES of boneEWS-FLI1−JR (NCI-EWS 94)36Merino ME Navid F Christensen BL Toretsky JA Helman LJ Cheung NK Mackall CL Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.J Clin Oncol. 2001; 19: 3649-3659Crossref PubMed Scopus (20) Google ScholarES of boneEWS-FLI1−SB (NCI-EWS 95)35Liu XF Helman LJ Yeung C Bera TK Lee B Pastan I XAGE-1, a new gene that is frequently expressed in Ewing's sarcoma.Cancer Res. 2000; 60: 4752-4755PubMed Google ScholarES of boneEWS-FLI1+SK-N-MC34Giovannini M Biegel JA Serra M Wang JY Wei YH Nycum L Emanuel BS Evans GA EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations.J Clin Invest. 1994; 94: 489-496Crossref PubMed Scopus (138) Google ScholarAskin's tumorEWS-FLI1+TC3237Toretsky JA Connell Y Neckers L Bhat NK Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides.J Neurooncol. 1997; 31: 9-16Crossref PubMed Google ScholarPeripheral neuroepitheliomaEWS-FLI1+TC7134Giovannini M Biegel JA Serra M Wang JY Wei YH Nycum L Emanuel BS Evans GA EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations.J Clin Invest. 1994; 94: 489-496Crossref PubMed Scopus (138) Google ScholarES of boneEWS-FLI1+583833Dickman PS Liotta LA Triche TJ Ewing's sarcoma: characterization in established cultures and evidence of its histogenesis.Lab Invest. 1982; 47: 375-382PubMed Google ScholarES of boneEWS-ERG+ Open table in a new tab Recombinant human TRAIL (rh TRAIL) and a crosslinking anti-TRAIL antibody were obtained from Alexis (Grünberg, Germany). Mouse monoclonal anti-caspase-8 antibody was purchased from Biocheck (Muenster, Germany), mouse anti-β-actin antibody was from Sigma (Muenchen, Germany), and goat anti-mouse antibody was from Santa Cruz (Heidelberg, Germany). IFN-γ and caspase inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp(OMe)-fluoromethylketone (Z-IETD-fmk) were obtained from R&D Systems (Wiesbaden, Germany). For immunohistochemical detection of caspase-8, paraffin sections of 5 μm were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase activity was quenched for 10 minutes at room temperature in methanol containing 1.5% hydrogen peroxide. After washing twice with water, sections were subjected to antigen retrieval by incubation in Dako Antigen Retrieval solution, pH 6.2 (Dako Corporation, Carpinteria, CA), for 15 minutes in a microwave oven. The sections were then washed in phosphate-buffered saline (PBS) and incubated for 1 hour with a blocking solution consisting of 10% normal goat serum (Vector Laboratories, Burlingame, CA) and 0.4% Tween 20 (Roche Diagnostics Corporation, Indianapolis, IN) in PBS at room temperature. The mouse monoclonal caspase-8 antibody (Upstate Biotechnology, Lake Placid, NY) was applied overnight at 4°C at a concentration of 1:75. Subsequently, the sections were washed with PBS and incubated with goat anti-mouse immunoglobulins conjugated to peroxidase-labeled dextran polymer (Dako Envision+ Peroxidase) for 30 minutes at room temperature. The peroxidase reaction was developed with 3′,3-diaminobenzidine (Dako), and the slides were counterstained with hematoxylin. The sections were evaluated for intensity of staining and percentage of positive cells. Evaluation of tumor cell percentage was performed on the entire tumor section. Because all samples except two were derived from diagnostic biopsies consisting of small pieces of tissue, evaluation for caspase-8 staining was performed in one representative tumor section. Sections were evaluated for intensity and distribution of staining. The biopsy specimens were evaluated by a pathologist and two other investigators (M.T., M.E.R.) who examined the sections at the same time using a two-head microscope. The latter reviewers were blinded to clinical information. Equivocal cases were discussed, and a consensus score was reached. The intensity was scored as no signal, weak signal, or medium to strong signal. Distribution of staining was evaluated on the entire tumor section using a 20× lens. The percentage of positive cells within various fields was determined, and a mean score was calculated. Total RNA from cell lines was prepared according to the manufacturer's instruction (Peqlab, Erlangen, Germany). A RiboQuant Multi-Probe RNase Protection Assay System (Pharmingen, Hamburg, Germany) was used per manufacturer's instructions. The hAPO5c probe set containing DNA templates for caspases 1 to 10, L32, and glyceraldehyde-3-phosphatase dehydrogenase was used for T7 polymerase direct synthesis of [α32-P]UTP-labeled antisense RNA probes. The probes were hybridized with 5 μg of RNA of the above-described ES cell lines. Samples were then digested with RNase to remove single-stranded (nonhybridized) RNA. Remaining probes were resolved on denaturing 5% polyacrylamide gels. Protein was extracted from cells by detergent lysis with a buffer containing 10% sodium dodecyl sulfate. The lysates were boiled for 10 minutes, and cellular debris was then removed by centrifugation (10 minutes at 14,000 rpm). The protein concentration then was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany). Forty micrograms of protein was boiled for 10 minutes before loading onto a 12% sodium dodecyl sulfate-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and then blocked for 30 minutes in PBS containing 5% dry milk and 0.1% Tween 20 (Sigma). Membranes were incubated with anti-caspase-8 antibody, at a 1:1000 dilution for 4 hours in blocking buffer, washed three times in PBS/Triton X-100, incubated for an additional 30 minutes with a secondary antibody conjugated to horseradish peroxidase at a 1:2000 dilution, washed three times with PBS/Triton X-100, and then developed using enhanced chemiluminescence (Amersham, Braunschweig, Germany). Apoptotic cells were determined by the propidium iodide method.38Nicoletti I Migliorati G Pagliacci MC Grignani F Riccardi C A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunol Methods. 1991; 139: 271-279Crossref PubMed Scopus (4484) Google Scholar In brief, ES cells were plated overnight in a 24-well plate (Falcon Microtest; Becton Dickinson, Heidelberg, Germany) at a concentration of 5 × 104 cells/well in 10% fetal calf serum. The following morning, IFN-γ was added where indicated. After indicated time frames, rh TRAIL was added at a final concentration of 200 ng/ml, together with a crosslinking anti-TRAIL-antibody at a final concentration of 2 μg/ml or chemotherapeutics doxorubicin or etoposide at various concentrations. Twenty-four hours after the addition of TRAIL and 48 hours after the addition of doxorubicin and etoposide, cells in suspension and adherent cells were collected in 12- × 75-mm Falcon Polystyrene tubes and centrifuged at 200 × g. The cell pellet was resuspended in 400 μl of a hypotonic buffer (50 μg/ml propidium iodide and 0.1% sodium citrate plus 0.1% Triton X-100) and placed at 4°C in the dark overnight. Flow cytometric analysis was performed using a FACScan analyzer (Becton Dickinson). The propidium iodide fluorescence of individual nuclei was measured in the red fluorescence, and the data were registered in a logarithmic scale. At least 104 cells of each sample were analyzed. Cells were plated in triplicate in flat-bottom wells at 1 × 105 cells/well in a 24-well microtiter plate. After an overnight incubation, cells were transiently cotransfected with 1 μg of the empty vector pcDNA3, the pcDNA3-wild-type (wt)-caspase-8 vector, or the pcDNA3-mutant (mut)-caspase-8 vector together with 0.8 μg of pEGFP (Clontech Laboratories, Heidelberg, Germany), using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany). The wt caspase-8 vector was kindly provided by V. Castle (University of Michigan, Ann Arbor, MI); and the mut caspase-8 vector, by V. Kidd (St. Jude Children's Research Hospital, Memphis, TN). Twenty-four hours after transfections, cells were treated with rh TRAIL (100 ng/ml) or with various concentrations of doxorubicin. Where indicated, the caspase-8-selective inhibitor Z-IETD-fmk was added at the time of transfection. For immunofluorescence, cells were analyzed with the Axiovert 200 fluorescence microscope (Carl Zeiss, Jena, Germany) 14 hours after the addition of TRAIL. The percentage of transfected cells was determined by flow cytometry, 36 hours after the addition of TRAIL, using a BD FACscan and the CellQuest software. Here, 10,000 cells per sample were analyzed for the fluorescent intensity of enhanced green fluorescent protein (EGFP) in the FL-1 channel. Cells were counted as transfected if their fluorescence intensity was above a cutoff fluorescent intensity level, which was defined as being the 99.9% percentile value of the fluorescent signal of nontransfected cells. Transfection rates for the empty vector plasmid (control) were around 10% for cell line JR and 20% for cell line A4573. Relative transfection rates were calculated as the ratio of the percentage of transfected cells of a given sample to the percentage of transfected cells of untreated controls. Experiments were done in triplicates. For immunoblotting of caspase-8, protein extracts were obtained 24 hours aft
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