Fas Ligand Is Present in Tumors of the Ewing's Sarcoma Family and Is Cleaved into a Soluble Form by a Metalloproteinase
1998; Elsevier BV; Volume: 153; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65708-2
ISSN1525-2191
AutoresNicholas Mitsiades, Vassiliki Poulaki, Vassiliki Kotoula, Alvaro Leone, Maria Tsokos,
Tópico(s)Signaling Pathways in Disease
ResumoFas ligand (FasL) exists in transmembrane and soluble forms and induces apoptosis on cross-linking with the Fas receptor. We evaluated the biological significance of FasL and Fas in 61 tumor tissues and 9 cell lines of the Ewing's sarcoma family of tumors (ESFT). FasL was present in 62.5% and Fas in 79.4% of primary ESFT. Metastatic tumors had higher expression of FasL (95%), suggesting association with a metastatic phenotype. FasL was detected in the cytoplasm and membrane of ESFT cells by immunofluorescence. Western blotting revealed transmembrane and soluble FasL in cytosolic extracts and soluble FasL in conditioned media. Both transmembrane and soluble FasL induced apoptosis of Fas-sensitive Jurkat cells in co-culture experiments with ESFT cells or their media. Treatment with phenanthroline and the synthetic metalloproteinase inhibitor BB-3103 reduced the levels of soluble FasL in the media, suggesting that in ESFT, FasL is processed by a metalloproteinase and released in the extracellular milieu. The released soluble FasL may serve to attack cells of the immune system and/or interfere with the binding of transmembrane FasL with Fas, and results in down-regulation of transmembrane FasL. Synthetic metalloproteinase inhibitors may modify the ratio of transmembrane to soluble FasL. Fas ligand (FasL) exists in transmembrane and soluble forms and induces apoptosis on cross-linking with the Fas receptor. We evaluated the biological significance of FasL and Fas in 61 tumor tissues and 9 cell lines of the Ewing's sarcoma family of tumors (ESFT). FasL was present in 62.5% and Fas in 79.4% of primary ESFT. Metastatic tumors had higher expression of FasL (95%), suggesting association with a metastatic phenotype. FasL was detected in the cytoplasm and membrane of ESFT cells by immunofluorescence. Western blotting revealed transmembrane and soluble FasL in cytosolic extracts and soluble FasL in conditioned media. Both transmembrane and soluble FasL induced apoptosis of Fas-sensitive Jurkat cells in co-culture experiments with ESFT cells or their media. Treatment with phenanthroline and the synthetic metalloproteinase inhibitor BB-3103 reduced the levels of soluble FasL in the media, suggesting that in ESFT, FasL is processed by a metalloproteinase and released in the extracellular milieu. The released soluble FasL may serve to attack cells of the immune system and/or interfere with the binding of transmembrane FasL with Fas, and results in down-regulation of transmembrane FasL. Synthetic metalloproteinase inhibitors may modify the ratio of transmembrane to soluble FasL. FasL is a transmembrane protein of the tumor necrosis factor (TNF) family, with an amino-terminal cytoplasmic and a carboxy-terminal extracellular region (type II protein). Its molecular weight ranges from 37 to 42 kd,1Suda T Takahashi T Golstein P Nagata S Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family.Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2429) Google Scholar probably reflecting differences in levels of glycosylation. In addition to the transmembrane FasL (tm-FasL), a soluble form has been described as well. The human soluble FasL (s-FasL) has a molecular weight of 23–26 kd and consists of the extracellular domain of the FasL molecule.2Tanaka M Suda T Takahashi T Nagata S Expression of the functional soluble form of human fas ligand in activated lymphocytes.EMBO J. 1995; 14: 1129-1135Crossref PubMed Scopus (603) Google Scholar Recent studies have shown that s-FasL originates from cleavage of tm-FasL by an unidentified metalloproteinase-like enzyme.3Tanaka M Suda T Haze K Nakamura N Sato K Kimura F Motoyoshi K Mizuki M Tagawa S Ohga S Hatake K Drummond AH Nagata S Fas ligand in human serum.Nat Med. 1996; 2: 317-322Crossref PubMed Scopus (649) Google Scholar, 4Kayagaki N Kawasaki A Ebata T Ohmoto H Ikeda S Inoue S Yoshino K Okumura K Yagita H Metalloproteinase-mediated release of human Fas ligand.J Exp Med. 1995; 182: 1777-1783Crossref PubMed Scopus (772) Google Scholar, 5Mariani SM Matiba B Baumler C Krammer PH Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases.Eur J Immunol. 1995; 25: 2303-2307Crossref PubMed Scopus (213) Google Scholar Both tm- and s-FasL bind to the Fas (APO-1/CD95) receptor and induce apoptosis.1Suda T Takahashi T Golstein P Nagata S Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family.Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2429) Google Scholar, 2Tanaka M Suda T Takahashi T Nagata S Expression of the functional soluble form of human fas ligand in activated lymphocytes.EMBO J. 1995; 14: 1129-1135Crossref PubMed Scopus (603) Google Scholar, 3Tanaka M Suda T Haze K Nakamura N Sato K Kimura F Motoyoshi K Mizuki M Tagawa S Ohga S Hatake K Drummond AH Nagata S Fas ligand in human serum.Nat Med. 1996; 2: 317-322Crossref PubMed Scopus (649) Google Scholar, 6Kiener PA Davis PM Rankin BM Klebanoff SJ Ledbetter JA Starling GC Liles WC Human monocytic cells contain high levels of intracellular Fas ligand. Rapid release following cellular activation.J Immunology. 1997; 159: 1594-1598PubMed Google Scholar, 7Martinez-Lorenzo MJ Alava MA Anel A Pineiro A Naval J Release of preformed Fas ligand in soluble form is the major factor for activation-induced death of Jurkat T cells.Immunology. 1996; 89: 511-517Crossref PubMed Scopus (90) Google Scholar Fas is a type I transmembrane protein of the TNF/nerve growth factor receptor superfamily.8Oehm A Berhmann I Falk W Pawlita M Maier G Klas C Li-Weber M Richards S Dhein J Trauth BC Ponstingl H Krammer PH Purification and molecular cloning of the APO-1 cell surface antigen, a member of the TNF/NGF receptor superfamily.J Biol Chem. 1992; 267: 10709-10715Abstract Full Text PDF PubMed Google Scholar The Fas/FasL system plays an important role in immune homeostasis9Brunner T Mogil RJ LaFace D Yoo NJ Mahboubi A Echeverri F Martin SJ Force WR Lynch DH Ware CF Green DR Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas.Nature. 1995; 373: 441-444Crossref PubMed Scopus (1266) Google Scholar, 10Ju ST Panka DJ Cui H Ettinger R el-Khatib M Sherr DH Stanger BZ Marshak-Rothstein A Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation.Nature. 1995; 373: 444-448Crossref PubMed Scopus (1450) Google Scholar and participates in T cell-mediated cytotoxicity.11Kägi D Vignaux F Ledermann B Bürki K Depraetere V Nagata S Hentgartner H Golstein P Fas and perforin pathways as major mechanisms of T-cell mediated cytotoxicity.Science. 1994; 265: 528-530Crossref PubMed Scopus (1461) Google Scholar, 12Lowin B Hahne M Mattmann C Tschopp J Cytolytic T-cell cytotoxicity is mediated through perforin, and Fas lytic pathways.Nature. 1994; 370: 650-652Crossref PubMed Scopus (983) Google Scholar, 13Rensing-Ehl A Frei K Flury R Matiba B Mariani S Weller M Aebischer P Krammer P Fontana A Local Fas/Apo-1 (CD95) ligand-mediated tumor cell killing in vivo.Eur J Immunol. 1995; 25: 2253-2258Crossref PubMed Scopus (201) Google Scholar According to a recently proposed model, FasL-expressing tumor cells use FasL as a cytolytic effector molecule to kill Fas-expressing activated lymphocytes ("counterattack" model).14O'Connell J O'Sullivan GC Collins JK Shanahan F The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand.J Exp Med. 1996; 184: 1075-1082Crossref PubMed Scopus (866) Google Scholar, 15Hahne M Rimoldi D Schroter M Romero P Schreier M French LE Schneider P Bornand T Fontana A Lienard D Cerottini J Tschopp J Melanoma cell expression of Fas(Apo-1/CD95) ligand: Implications for tumor immune escape.Science. 1996; 274: 1363-1366Crossref PubMed Scopus (1191) Google Scholar The group of small round cell sarcomas in children and adolescents that is collectively referred to as the Ewing's sarcoma family of tumors (ESFT) includes morphological variants of Ewing's sarcoma and peripheral primitive neuroectodermal tumor (PNET). All ESFTs are characterized by specific chromosomal translocations. The most commonly encountered translocation, t(11;22) (q24;q12), results in the fusion of the EWS and Fli-1 genes.16Horowitz ME DeLaney TF Malawar MM Tsokos MG Ewing's sarcoma family of tumors: Ewing's sarcoma of bone and soft tissue and the peripheral primitive neuroectodermal tumors.in: Pizzo PA Poplack DG In Principles and Practice of Pediatric Oncology. JB Lippincott, Philadelphia1993: 795-821Google Scholar Clinical studies have shown that despite a significant initial response to conventional treatment, a high percentage of patients with ESFT suffer a recurrence at metastatic sites.16Horowitz ME DeLaney TF Malawar MM Tsokos MG Ewing's sarcoma family of tumors: Ewing's sarcoma of bone and soft tissue and the peripheral primitive neuroectodermal tumors.in: Pizzo PA Poplack DG In Principles and Practice of Pediatric Oncology. JB Lippincott, Philadelphia1993: 795-821Google Scholar Since the "counterattack" model suggests that the FasL may offer a survival advantage to tumors, we investigated the role of FasL in the biology of ESFT. In this study, we show that ESFTs frequently express Fas and FasL. A significantly higher level of FasL expression in metastatic than primary ESFTs supports the theory that FasL-expressing clones survive and undergo expansion in metastatic sites. Our in vitro data demonstrate that ESFT express not only tm-FasL, but also s-FasL and release s-FasL in the media. Both tm- and s-FasL induce apoptosisin vitro. The levels of s-FasL in the media are reduced by metalloproteinase inhibitors, suggesting cleavage by a metalloproteinase. Collectively, these data support the hypothesis that there is a dynamic equilibrium between tm- and s-FasL that can be manipulated by agents such as metalloproteinase inhibitors. Seven previously described and one unpublished ESFT cell lines were used in this study. Specifically, the TC-71, TC-32, A4573, 5838, SK-N-MC, CHP100 (clone-S and -L), and TC-268 cell lines were shown to have the characteristic translocation and/or EWS/Fli-1 fusion gene product of the ESFT.17Cavazzana AO Navarro S Noguera R Reynolds PC Triche TJ Olfactory neuroblastoma is not a neuroblastoma but is related to primitive neuroectodermal tumor (PNET).Prog Clin Biol Res. 1988; 271: 463-473PubMed Google Scholar, 18Sorensen PHB Liu XF Delattre O Rowland JM Biggs CA Thomas G Triche TJ Reverse transcriptase PCR amplification of EWS/FLI-1 fusion transcripts as a diagnostic test for peripheral primitive neuroectodermal tumors of childhood.Diagn Mol Pathol. 1993; 2: 147-157Crossref PubMed Scopus (140) Google Scholar, 19Sorensen PHB Wu JK Berean KW Lim JF Donn W Frierson HF Reynolds CP L-pez-Terrada D Triche TJ Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing's sarcoma.Proc Natl Acad Sci USA. 1996; 93: 1038-1043Crossref PubMed Scopus (83) Google Scholar, 20Whang-Peng J Triche TJ Miser J Kao-Shan S Tsai S Israel M Cytogenetic characterization of selected small round cell tumors of childhood.Cancer Genet Cytogenet. 1986; 21: 185-208Abstract Full Text PDF PubMed Scopus (274) Google Scholar The unpublished cell line, TC-248, was established in our laboratory from an ESFT that exhibited the typical EWS/Fli-1 fusion product as well (data not shown). The T-cell leukemia cell line Jurkat (American Type Culture Collection, Manassas, VA) was used in functional experiments. All cells except Jurkat were grown in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Walkersville, MD) with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal calf serum (FCS) (GIBCO/BRL, Gaithersburg, MD), unless stated otherwise. Jurkat cells were grown in RPMI (GIBCO/BRL) with 10% FCS and antibiotics as above. Sections from 61 formalin-fixed paraffin-embedded tumor tissue specimens obtained from 49 patients with ESFT and stored in the files of the Laboratory of Pathology at the National Cancer Institute (NCI) were stained for FasL. Forty of these tumors were primary and 21 were metastatic. In 12 cases, primary and metastatic tumor tissue from the same patient was available for comparison. In 34 primary and 19 metastatic tumors from which additional sections were available, immunocytochemical staining for Fas receptor was performed. For immunofluorescence and immunoperoxidase staining, the anti-FasL rabbit polyclonal antibodies C-20 (against the extracellular C-terminus) and Q-20 (against the intracellular N-terminus) (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Both antibodies were used in the presence or absence of the corresponding blocking peptide (amino acid residues 260–279 for C-20 and residues 2–19 for Q-20, Santa Cruz) to confirm specificity of staining. The monoclonal anti-FasL antibodies clone 33 (0.25 μg/ml) (Transduction Laboratories, Lexington, KY) and G247–4 (1:500 dilution) (Pharmingen, San Diego, CA), as well as the polyclonal antibodies Ab-3 (1:100 dilution) (Oncogene Research, Cambridge, MA) and C-20 (1:500 dilution) and Q-20 (1:500 dilution) were used in the immunoblotting experiments. Both monoclonal antibodies are directed against the extracellular portion of the FasL molecule. For detection of the Fas receptor on paraffin sections, the anti-Fas rabbit antibody Ab-1 was used (Oncogene Research) in the presence or absence of its blocking peptide (amino acid residues 321–335) (Oncogene Research). The neutralizing anti-FasL antibody NOK-2 (Pharmingen) and the cytotoxic anti-Fas monoclonal CH-11 antibody (Panvera, Madison, WI) were used in the cytotoxic cell assays. Cells (1 × 106) from all 9 ESFT cell lines were scraped, centrifuged briefly, and lysed for 30 minutes on ice in a lysis buffer (50 mmol/L Tris-HCl, pH 8.0, containing 120 mmol/L NaCl and 1% Igepal), supplemented with the Complete-TM mixture of proteinase inhibitors (Boehringer Mannheim, Indianapolis, IN). The samples were cleared by centrifugation (14,000 rpm for 30 minutes at 4°C) and assessed for protein concentration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) was performed (30 μg of protein per lane) and the proteins were electroblotted onto nitrocellulose membranes. After 1 hour incubation in blocking solution (20% IgG-free normal horse serum in PBS, GIBCO/BRL), the membranes were exposed to the primary antibody overnight at 4°C. After washing in PBS, the secondary peroxidase-labeled secondary antibody (Amersham, Arlington Heights, IL) was added at 1:10,000 dilution for 40 minutes at room temperature. The proteins were visualized with the enhanced chemiluminescence (ECL) technique (Amersham). An endothelial cell lysate, provided by Transduction Laboratories as a positive control for the anti-FasL antibody clone 33, was used along with the ESFT cell lysates in our immunoblotting experiments. Conditioned media were generated from 3 of the 9 ESFT cell lines (TC-248, TC-268 and TC-71). These lines expressed the highest levels of FasL in the cell pellets and were selected for further analysis. Following brief washing in Hanks' balanced salt solution (HBSS) (GIBCO/BRL) and a 4-hour washing in serum-free DMEM, ESFT cells were then incubated with fresh serum-free DMEM medium overnight. The overnight-conditioned media were collected and centrifuged for 5 minutes at 2,000 rpm. The supernatants were supplemented with a proteinase inhibitor cocktail of 10 μg/ml aprotinin (Sigma, St. Louis, MO), 1 mmol/L phenylmethylsulfonyl fluoride (Sigma) and 25 μmol/L leupeptin (Sigma). To increase the sensitivity of detection of s-FasL by Western blotting, the conditioned media were subjected to 50-fold concentration with Centricon-10 filters (Amicon, Beverly, MA). The concentrates were mixed with 5× Laemmli buffer, electrophoresed on 12% SDS-PAGE gels and immunoblotted with the anti-FasL monoclonal antibody G247–4 (Pharmingen) at 1:500 dilution (1 μg/ml). Air-dried cytospins from cultured cells were fixed in −20°C acetone for 10 minutes. Subsequently, the cytospins were washed and blocked for 1 hour with 20% normal goat serum and 3% bovine serum albumin (Sigma) solution in PBS. The cytospins were then washed in PBS and incubated overnight at 4°C with the C-20 or Q-20 rabbit polyclonal anti-FasL antibodies at 1:100 dilution (1μg/ml), in the presence or absence of a 10-fold excess of the corresponding blocking peptide. Subsequently, the cytospins were washed in PBS and incubated with fluorescein isothiocyanate (FITC)-labeled anti-rabbit IgG (Boehringer Mannheim, Indianapolis, IN) (1:75 dilution) for 1 hour at room temperature. Fluorescent signals were visualized with a Zeiss standard fluorescence microscope equipped with an epifluorescence illuminator and FITC narrow-band filter. Immunohistochemical detection of Fas and FasL was performed as previously described.21Mitsiades N Poulaki V Kotoula V Mastorakos G Tseleni-Balafouta S Koutras DA Tsokos M Fas/Fas ligand up-regulation, and Bcl-2 down-regulation may be significant in the pathogenesis of Hashimoto's thyroiditis.J Clin Endocrinol Metab. 1998; 83: 2199-2203Crossref PubMed Scopus (93) Google Scholar Briefly, 5-micron paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval by incubation in 10 mmol/L citrate buffer for 15 minutes in a microwave oven. Endogenous peroxidase activity was quenched for 30 minutes in methanol containing 0.5% H2O2. The sections were washed in PBS and blocked for 1 hour in 20% normal goat serum in PBS. The primary antibodies, anti-FasL Q-20 (0.7 μg/ml) and anti-Fas Ab-1 (2.5 μg/ml) respectively, were applied overnight in the presence or absence of a 10-fold excess of the corresponding blocking peptides. Subsequently, the sections were washed in PBS and incubated with a biotinylated anti-rabbit antibody (1:500 dilution) for 1 hour at room temperature. After washing with PBS, the sections were covered with the Vectastain Elite Avidin Biotin Complex Reagent (Vector Laboratories, CA) for 30 minutes. The peroxidase reaction was developed with 3,3′-diaminobenzidine and the slides were counterstained with methyl green. Positive staining was evaluated subjectively by two independent observers for both percentage of positive cells and intensity of staining on a scale of 1 to 3 (interobserver agreement in 95% of cases). The product of the intensity of staining and percentage of positive cells was used for final classification into grade 0 (no staining), grade 1 (0.1–0.3), grade 2 (0.4–0.6), grade 3 (0.7–1), or grade 4 (>1). Following treatment as indicated below, the cells were incubated with 1 mg/ml MTT (Sigma) in fresh media for 4 hours at 37°C. Subsequently, a mixture of isopropanol and 1N HCl (24:1, v/v) was added under vigorous pipetting to dissolve the formazan crystals. Dye absorbance (A) in viable cells was measured at 570 nm, with 630 nm as a reference wavelength. Cell death was estimated with the formula: % specific death=|A(untreated cells) - A(treated cells)A(untreated cells) This method, a non-radioactive analogue of the [3H]-Thymidine-DNA fragmentation assay, was used to detect apoptosis in the co-culture experiments described below. The DNA fragmentation ELISA kit (Boehringer Mannheim) was used. Target cells were labeled overnight with 5′-bromo-2′-deoxy-uridine (BrdU) according to the manufacturer's instructions and subsequently were co-cultured with effector cells. The amount of fragmented DNA in the target cells was quantified according to the manufacturer's instructions. The results were expressed as percentages of the value in control cells. Air-dried cytospins were labeled with the in situ cell death kit-Fluorescence (Boehringer Mannheim) following the instructions of the manufacturer and were viewed with a Zeiss standard fluorescence microscope equipped with an epifluorescence illuminator and FITC narrow-band filter. The ability of the TC-248 and TC-32 ESFT effector cells to kill target lymphocytes in a Fas-dependent manner was evaluated as previously described22Shiraki K Tsuji N Shioda T Isselbacher KJ Takahashi H Expression of fas ligand in liver metastases of human colonic adenocarcinomas.Proc Natl Acad Sci USA. 1997; 94: 6420-6425Crossref PubMed Scopus (267) Google Scholar with minor modifications. Briefly, ESFT cells (5 × 105 cells/well grown up to 90% confluency) were fixed lightly (0.6% paraformaldehyde in PBS for 15 minutes) and, after adequate washing in HBSS, incubated with a Jurkat cell suspension (105 cells in 400 ml DMEM supplemented with 1% calf serum, GIBCO/BRL), at an effector to target ratio 10:1 and in the presence or absence of the FasL neutralizing NOK-2 antibody (10 μg/ml). After a 48-hour incubation, Jurkat cell death was evaluated with the MTT assay, as described above. The percentage of Jurkat cell death in each well was calculated with the equation: %cell death= A(target along)-[A(target with effector)-A(effector alone)]A(target alone) ESFT cell-induced Jurkat cell death was also evaluated with a DNA fragmentation ELISA that measured DNA fragmentation of BrdU-labeled Jurkat cells co-cultured with viable TC-248 cell monolayers for 48 hours in the presence or absence of NOK-2 antibody (10 μg/ml). The Jurkat cell suspension was collected at the end of the experiment with vigorous pipetting. Media conditioned with TC-248 and TC-71 cells for 48 hours were concentrated 30-fold with Centriprep-10 filters (Amicon) and mixed with a Jurkat cell suspension at a 1:1 v:v ratio. Thus the final concentration of media was 15-fold, much less than the 50-fold concentration used to detect s-FasL by Western blotting. The Jurkat cell concentration in the final sample was 1.25 × 105 cells/ml in DMEM supplemented with 1% calf serum. Media conditioned with TC-248 and TC-71 were added in the presence or absence of NOK-2 FasL neutralizing antibody (10 μg/ml). Control wells consisted of Jurkat cells in fresh medium with 1% calf serum. Percentage of Jurkat cell death in each well was evaluated with the MTT assay and estimated with the equation: %cell death= (Jurkat cells in control medium)-(Jurkat cells in ESFT-cell conditioned medium)(Jurket cells in control medium) In addition, cells from duplicate wells were centrifuged onto positively charged slides and stained for apoptosis with the TUNEL method. The percent of apoptotic cells was evaluated subjectively. Cells from the TC-248, TC-268, and TC-71 cell lines were grown to 70–80% confluence. Subsequently, the cells were washed in HBSS and incubated for 18 hours with the CH-11 anti-Fas antibody (500 ng/ml, in DMEM medium with 10% calf serum) at 37°C. Cells grown in the absence of CH-11 antibody were used as negative controls. The Fas-sensitive Jurkat cell line was used as a positive control. Cell survival was evaluated with the MTT assay. TC-248 cells were grown in serum-containing medium for 2 days, then washed twice in HBSS and incubated for 4 hours in serum-free DMEM with the general zinc-chelating agent 1,10-phenanthroline (0.1 mmol/L, Sigma) or with one of the following metalloproteinase inhibitors: BB-3103 (10 μM, generous gift of British Biotech, Oxford, UK), TIMP-1 (1 μg/ml), and TIMP-2 (1 μg/ml). To eliminate any possible traces of pre-existing s-FasL, this medium was discarded and replaced with fresh DMEM containing the same agents as before, at the same concentrations. The final incubation was 18 hours for BB-3103, TIMP-1, and TIMP-2 and 8 hours for 1,10-phenanthroline. At the end of the incubation times, the media were collected, subjected to 50-fold concentration, and tested for s-FasL by immunoblotting, as discussed previously The χ2Tanaka M Suda T Takahashi T Nagata S Expression of the functional soluble form of human fas ligand in activated lymphocytes.EMBO J. 1995; 14: 1129-1135Crossref PubMed Scopus (603) Google Scholar test was used to compare numbers of positive and negative primary versus metastatic tumors. Intensity of staining in total number of primaries versustotal number of metastatic tumors was evaluated with a two-sided unpaired t-test. In cases with primary and metastatic tumors from the same patient available for comparison, the two-sided pairedt-test was used. All other comparisons were examined with the one-factor analysis of variance repeated measures method. We investigated the presence of the tm- and s-FasL in ESFT cells and media by immunoblotting. All antibodies against FasL (G247–4, clone 33, Ab-3, C-20, and Q-20) identified a 37–40 kd band in the cell lysates of all 9 ESFT cell lines. This band co-migrated with the FasL band of the control endothelial cells and corresponds to the tm-FasL (Figure 1A and inset). A band of approximately 24 kd that corresponds to the s-FasL was detected with the G247–4 antibody in concentrated conditioned media and cell lysates from ESFT cell lines (TC-248, TC-268, TC-71) (Figure 1B). Tm-FasL was not detected in any of the media with either clone 33 or G247–4, thus ruling out cell contamination. Although both FasL-specific monoclonal antibodies used in the immunoblotting experiments were directed against the extracellular domain of FasL, clone 33 stained preferentially for tm-FasL and G247–4 exhibited higher affinity for s-FasL. Furthermore, the G247–4 antibody also detected an additional doublet of approximately 31 kd (Figure 1B) that corresponds to nonglycosylated or partially glycosylated tm-FasL.2Tanaka M Suda T Takahashi T Nagata S Expression of the functional soluble form of human fas ligand in activated lymphocytes.EMBO J. 1995; 14: 1129-1135Crossref PubMed Scopus (603) Google Scholar, 5Mariani SM Matiba B Baumler C Krammer PH Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases.Eur J Immunol. 1995; 25: 2303-2307Crossref PubMed Scopus (213) Google Scholar Having detected the presence of tm- and s-FasL in ESFT cells by immunoblotting, we assessed the cellular localization of these proteins by immunofluorescence staining of acetone-fixed cytospins from the TC-248, TC-268, and TC-71 cell lines. Both antibodies showed diffuse cytoplasmic and paranuclear dot-like staining that was more prominent with the C-20 (Figure 2A) than the Q-20 antibody (Figure 2B). A granular pattern at the periphery of the cells, consistent with membranous staining, was additionally observed with the Q-20 antibody (Figure 2B). These data show that FasL is expressed both on the surface and in the cytoplasm of ESFT cells. All staining was totally abolished in the presence of the corresponding blocking peptide that confirmed specificity (data not shown). To exclude the possibility that FasL expression in ESFT is a random trait acquired from prolonged propagation of tumor cellsin vitro, we studied the expression of FasL by immunohistochemistry in sections of 61 primary and metastatic tumor tissues from 49 patients. Thirty-nine of 49 patients (79.6%) were found positive for FasL in at least one tumor specimen. Metastatic tumors exhibited a higher incidence of staining than primary tumors. Specifically, among the 61 tumor specimens examined, 25 of 40 (62.5%) primary and 20 of 21 (95%) metastatic were FasL-positive. This difference was statistically significant (P < 0.006). Metastatic tumors also exhibited a statistically significant higher grade of staining than primary tumors (P= 0.000467), as shown in tissue sections in Figure 3, A and B). Positive tumor cells demonstrated both diffuse cytoplasmic and peripheral membrane staining in a rim-like pattern. The staining for FasL disappeared when the antibody was applied simultaneously with the corresponding blocking peptide. To verify the significance of the observed higher FasL staining in metastatic ESFT, we also evaluated paired primary and metastatic tumor tissues from the same patients. A total of 12 pairs of tumor tissues were analyzed and in 8 of them a higher grade of tumor cell staining was observed in the metastasis (Figures 3C and D). The remaining 4 showed no change. The difference in the grade of staining in the paired primary and metastatic tumor tissues was statistically significant (P = 0.0086). Positive endothelial cells within tumor tissues served as internal positive controls. Because the apoptotic signal of FasL is transduced via the Fas receptor, we also investigated the presence of Fas in 53 ESFT tissues, 34 of which were primary and 19 metastatic. Fas was expressed in 40 (75.47%) ESFT in toto. Specifically, 27 of the primary (79.4%) and 13 of the metastatic (68%) tumors were found positive for the Fas receptor (Figure 3E). However, the difference in the frequency and grade of staining for the Fas receptor in primary and metastatic ESFT was not statistically significant (P = 0.36 and P = 0.6425, respectively). Staining for Fas disappeared when the antibody was applied simultaneously with the corresponding blocking peptide. To evaluate the biological activity of the tm-FasL on the surface of ESFT cells, we used TC-248 or TC-32 cells as cytotoxic effectors in co-culture experiments with target Fas-expressing Jurkat cells. Two different assays were used for data confirmation: the MTT assay and a DNA fragmentation ELISA. Because the MTT assay is a cell viability test, the ESFT effector cells were lightly fixed to allow assessment of viability of target Jurkat cells. On the contrary, viable ESFT cells were used in the DNA fragmentation ELISA, in which quantification of specific DNA fragmentation of target cells was based on previous labeling of Jurkat cells with BrdU. With the MTT assay, Jurkat cells co-cultured with TC-248 cells exhibited 76 ± 2% cell death (mean ± SD). The Jurkat cell death was Fas/FasL-specific, because it was inhibited to 30.4 ± 0.9% in the presence of the neutralizing anti-FasL NOK-2 antibody (P < 0.000001). With the DNA fragmentation ELISA, Jurkat cells grown in the pre
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