Engagement of CD99 Induces Apoptosis Through a Calcineurin-Independent Pathway in Ewing's Sarcoma Cells
1998; Elsevier BV; Volume: 153; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65707-0
ISSN1525-2191
AutoresHae Won Sohn, Eun Young Choi, Soon Ha Kim, Im‐Soon Lee, Doo Hyun Chung, Uhna Sung, Dae Yeon Hwang, Sa Sun Cho, Bokkyoo Jun, Ja June Jang, Je G., Seong Hoe Park,
Tópico(s)NF-κB Signaling Pathways
ResumoProgrammed cell death (PCD) is a prominent feature of the development of the immune and nervous systems. In both systems, widespread PCD occurs in primitive progenitor cells during development. In this study, we demonstrated that Ewing's sarcoma (ES) cells, undifferentiated neural precursors, underwent apoptosis upon engagement of CD99 with anti-CD99 monoclonal antibody. Apoptosis via CD99 occurred only in the undifferentiated state of ES cells, but not in differentiated ES cells. CD99-induced apoptosis in ES cells appeared to require de novo synthesis of RNA and protein as well as caspase activation. Cyclosporin A, known to be a potent inhibitor of both calcineurin activation and mitochondrial permeability transition pore opening, inhibited CD99-mediated apoptosis, whereas FK-506, a specific calcineurin inhibitor, did not, indicating the induction of CD99-mediated apoptosis through a calcineurin-independent pathway. Furthermore, the dying cells displayed the reduction of mitochondrial transmembrane potential (ΔΨm). These results suggest that CD99 engagement induce CsA-inhibitable mitochondrial permeability transition pore opening, followed by a reduction of ΔΨm and caspase activation, thereby leading to apoptosis. Based on these results, we suggest the possible involvement of CD99 in the apoptotic processes that occur during nervous system development and also its application in immunotherapeutic trials for ES cases. Programmed cell death (PCD) is a prominent feature of the development of the immune and nervous systems. In both systems, widespread PCD occurs in primitive progenitor cells during development. In this study, we demonstrated that Ewing's sarcoma (ES) cells, undifferentiated neural precursors, underwent apoptosis upon engagement of CD99 with anti-CD99 monoclonal antibody. Apoptosis via CD99 occurred only in the undifferentiated state of ES cells, but not in differentiated ES cells. CD99-induced apoptosis in ES cells appeared to require de novo synthesis of RNA and protein as well as caspase activation. Cyclosporin A, known to be a potent inhibitor of both calcineurin activation and mitochondrial permeability transition pore opening, inhibited CD99-mediated apoptosis, whereas FK-506, a specific calcineurin inhibitor, did not, indicating the induction of CD99-mediated apoptosis through a calcineurin-independent pathway. Furthermore, the dying cells displayed the reduction of mitochondrial transmembrane potential (ΔΨm). These results suggest that CD99 engagement induce CsA-inhibitable mitochondrial permeability transition pore opening, followed by a reduction of ΔΨm and caspase activation, thereby leading to apoptosis. Based on these results, we suggest the possible involvement of CD99 in the apoptotic processes that occur during nervous system development and also its application in immunotherapeutic trials for ES cases. Apoptosis, or programmed cell death (PCD), is the mechanism by which cells die through the activation of their intrinsic suicide program in response to changes in external milieu or irreparable cell damages. Apoptosis, in which the cell actively participates in its demise, has been characterized by morphological changes such as chromatin condensation, nuclear fragmentation, internucleosomal DNA fragmentation, and cytoplasmic blebbing.1Kerr JFR Shrinkage necrosis: a distinct mode of cellular death.J Pathol. 1971; 105: 13-20Crossref PubMed Scopus (708) Google Scholar Apoptosis requires that the dying cell be metabolically active, and is often dependent on RNA and protein synthesis.2Yazdanbakhsh K Choi JW Li Y Lau LF Choi Y Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcriptional factor Nur 77.Proc Natl Acad Sci USA. 1995; 92: 437-441Crossref PubMed Scopus (95) Google Scholar, 3Ferrer I Olive M Ribera J Planas AM Naturally occurring (programmed) and radiation-induced apoptosis are associated with selective c-Jun expression in the developing rat brain.Eur J Neurosci. 1996; 8: 1286-1298Crossref PubMed Scopus (75) Google Scholar Recently, various triggering factors and processes of apoptosis have been widely investigated in vitro and in vivo.4Kroemer G The proto-oncogene Bcl-2 and its role in regulating apoptosis.Nature Med. 1997; 3: 614-620Crossref PubMed Scopus (1722) Google Scholar, 5Hengartner MO Death cycle and swiss army knives.Nature. 1998; 391: 441-442Crossref PubMed Scopus (111) Google Scholar One of the common initial manifestations of the apoptotic process, irrespective of cell types and inducing stimuli, is a disruption of mitochondrial membrane function, including a dissipation of the mitochondrial transmembrane potential (ΔΨm) due to the opening of the mitochondrial permeability transition (PT) pores.4Kroemer G The proto-oncogene Bcl-2 and its role in regulating apoptosis.Nature Med. 1997; 3: 614-620Crossref PubMed Scopus (1722) Google Scholar In many systems, mitochondrial PT pore opening is inhibited by cyclosporin A (CsA)6Petronilli V Nicolli A Costantini P Colonna R Bernardi P Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.Biochem Biophys Acta. 1994; 1187: 255-259PubMed Google Scholar, 7Petronilli V Cola C Massari S Colonna R Bernardi P Physiological effectors modify voltage sensing by the cyclosporin A-sensitive permeability transition pore of mitochondria.J Biol Chem. 1993; 268: 21939-21945Abstract Full Text PDF PubMed Google Scholar, 8Crompton M Ellinger H Costi A Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.Biochem J. 1988; 255: 357-360PubMed Google Scholar via a mechanism involving a mitochondrial cyclophilin, but not by calcineurin.6Petronilli V Nicolli A Costantini P Colonna R Bernardi P Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.Biochem Biophys Acta. 1994; 1187: 255-259PubMed Google Scholar Despite the extensive studies on PCD during development, little is known about the major cell surface proteins controlling PCD and its intracellular signaling pathway at the early stage of neural ontogeny. Ewing's sarcoma (ES) is a rare, small-round-cell undifferentiated tumor of bone and soft tissues. ES has been described to represent the stage of either very early pluripotential cells or primitive neuroectodermal cells9Moll R Lee I Gould VE Berndt R Roessner A Franke WW Immunocytochemical analysis of Ewing's tumors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeniety and pluripotential differentiation.Am J Pathol. 1987; 127: 288-304PubMed Google Scholar, 10Cavazzana AO Miser JS Jefferson J Triche TJ Experimental evidence for a neural origin of Ewing's sarcoma of bone.Am J Pathol. 1987; 127: 507-518PubMed Google Scholar that can differentiate along a neuronal, glial, Schwannian, melanocytic, neuroendocrine, or even ectomesenchymal pathway. ES is considered to be closely related to primitive neuroectodermal tumor (PNET), because they share a common chromosomal abnormality and the high expression of CD99 molecules on their cell surfaces. However, they have some differences in neuronal differentiation potential. PNET has neuronal features such as dense core granules, whereas ES lacks any trace of neuronal differentiation.11Hara S Adachi Y Kaneko Y Fujimoto J Hata J Evidence for heterogeneous group of neuronal differentiation of Ewing's sarcoma.Br J Cancer. 1991; 64: 1025-1030Crossref PubMed Scopus (17) Google Scholar, 12Tetsuro S Akihiro U Jun-ichi H Neurogenic potential of Ewing's sarcoma cells.Virchows Arch. 1997; 430: 41-46Crossref PubMed Scopus (19) Google Scholar Recently, in vitro culture studies have described that ES cell lines possess the ability to differentiate along neuronal pathways in response to various stimuli of differentiating agents.12Tetsuro S Akihiro U Jun-ichi H Neurogenic potential of Ewing's sarcoma cells.Virchows Arch. 1997; 430: 41-46Crossref PubMed Scopus (19) Google Scholar, 13Noguera R Triche TJ Navarro S Tsokos M Llombart-Bosch A Dynamic model of differentiation in Ewing's sarcoma cell lines.Lab Invest. 1992; 62: 143-151Google Scholar, 14Kodama K Doi O Higashiyama M Yokouchi H Tateishi R Mori Y Differentiation of a Ewing's sarcoma cell line towards neuronal and mesenchymal cell lineages.Jpn J Cancer Res. 1994; 85: 335-338Crossref PubMed Scopus (18) Google Scholar One report has shown that the mRNA expression patterns of a neural differentiation marker, NF-L, in ES cell lines were different in PNET, but similar in undifferentiated neural tissues.12Tetsuro S Akihiro U Jun-ichi H Neurogenic potential of Ewing's sarcoma cells.Virchows Arch. 1997; 430: 41-46Crossref PubMed Scopus (19) Google Scholar CD99 is a ubiquitous 32-kd transmembrane protein encoded by themic2 gene,15Gelin CF Aurit F Phalipon A Raynal B Cole S Kaczorek M Bernard A The E2 antigen, a 32 kD glycoprotein involved in T-cell adhesion process, is the MIC2 gene product.EMBO J. 1989; 8: 3253-3259Crossref PubMed Scopus (151) Google Scholar in particular, highly expressed in human cortical thymocytes, Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) cells, pancreatic islet cells, and Leydig and Sertoli cells.16Dworzak MN Fritsch G Buchinger P Fleischer C Printz D Zellner A Schöllhammer A Steiner G Ambros PF Gadner H Flow cytometric assessment of human MIC2 expression in bone marrow, thymus, and peripheral blood.Blood. 1994; 83: 415-425PubMed Google Scholar, 17Fellinger EJ Garin-Chesa P Triche TJ Huvos AG Rettig WJ Immunohistochemical analysis of Ewing's sarcoma cell surface antigen p30/32MIC2.Am J Pathol. 1991; 139: 317-325PubMed Google Scholar Recently, it has been reported that engagement of CD99 induces homotypic cell aggregation,18Bernard G Zoccola D Deckert M Breittmayer J-P Aussel C Bernard A The E2 molecule (CD99) specifically triggers homotypic aggregation of CD4+CD8+ thymocytes.J Immunol. 1995; 154: 26-32PubMed Google Scholar, 19Hahn J-H Kim MK Choi EY Kim SH Sohn HW Ham DI Chung DH Kim TJ Lee WJ Park CK Ree HJ Park SH CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.J Immunol. 1997; 159: 2250-2258PubMed Google Scholar up-regulation of T cell receptor and major histocompatibility complex molecules,20Choi EY Park WS Jung KC Kim SH Park SH Engagement of CD99 induces upregulation of TCR and MHC I and II molecules on the surface of human thymocytes.J Immunol. 1998; 161: 749-754PubMed Google Scholar and apoptosis in immature thymocytes.21Bernard G Breittmayer J-P Matteis M Trampont P Hofman P Senik A Bernard A Apoptosis of immature thymocytes mediated by E2/CD99.J Immunol. 1997; 158: 2543-2550PubMed Google Scholar In the present study, we demonstrate that CD99-induced apoptosis occurs only in undifferentiated ES cells, not in differentiated ES cells, as it does in immature cortical thymocytes. We suggest that CD99 might trigger apoptosis in a certain developmental stage during neural ontogeny and also that CD99 might be used as a target for immunotherapy of ES patients. The monoclonal antibody (MAb) DN16 (IgG1) used for CD99 activation was produced in our laboratory and described previously.22Jung KC Kim TJ Chung DH Lee GK Hahn J-H Park SH An antibody against E2/MIC2 antigen (CD99): identification and characterization.J Kor Cancer Assoc. 1996; 28: 350-358Google Scholar We purchased rabbit anti-mouse immunoglobulin Ab and FITC-conjugated goat anti-mouse immunoglobulin Ab from Sigma Chemical Co. (St. Louis, MO) and mouse anti-NF-H MAb (NCL-NF200) from Novocastra Laboratories (New Castle upon Tyne, UK). N6-O2-Dibutyryladenosine-3,5′-cyclic monophosphate (db-cAMP) and 3,3′dihexyloxacarbocyanine iodide (DiOC6(3) were purchased from Sigma Chemical Co. Calcium ionophore A23187 was from Boehringer Mannheim Biochemicals (Mannheim, Germany). Apoptosis inhibitors, such as actinomycin D (Act D), cycloheximide (CHX), cyclosporin A (CsA), and EGTA, were also from Sigma Chemical Co. FK-506 was kindly provided by Dr. J. K. Shin (Dana-Farber Cancer Institute). Z-VAD-fmk and Z-FA-fmk were obtained from Enzyme System Products (Livermore, CA). RD-ES (human Ewing's sarcoma), SK-N-MC (human PNET), and SK-N-SH (human neuroblastoma) cells were obtained from the American Type Culture Collection (Rockville, MD), and CADO-ES1 cells (human Ewing's sarcoma) were obtained from the German Collection of Microorganism and Cell Cultures (Braunschweig, Germany). Two human neuroblastoma cell lines, SK-N-AS and SH-SY5Y, were generous gifts of Dr. Chong-Jae Kim (Seoul National University College of Medicine, Seoul, Korea). RD-ES and CADO-ES1 cells were maintained in RPMI 1640 supplemented with 15% and 10% fetal bovine serum (FBS; Life Technology, Gaithersburg, MD), respectively. SK-N-MC, SK-N-SH, SK-N-AS, and SH-SY5Y cells were cultured in Dulbecco's minimal essential medium supplemented with 10% FBS. For differentiation induction of RD-ES and CADO-ES1 cells, the cells were cultured in Dulbecco's minimal essential medium supplemented with 10% FBS in the presence of 2.5 mmol/L and 0.25 mmol/L db-cAMP, respectively. The medium was changed every 3rd day and maintained for up to 12 days.12Tetsuro S Akihiro U Jun-ichi H Neurogenic potential of Ewing's sarcoma cells.Virchows Arch. 1997; 430: 41-46Crossref PubMed Scopus (19) Google Scholar For immunofluorescence staining, db-cAMP-treated and untreated ES cells were fixed in 95% methanol for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS, and washed three times with PBS containing 1% FBS. The cells were then blocked in 10% FBS for 30 minutes and stained with mouse anti-NF-H MAb overnight at 4°C. After washing three times in PBS containing 1% FBS for 15 minutes, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin Ab and mounted on the glass with mounting media (GEL/MOUNT, Biomeda Corp., Foster City, CA). Confocal analysis was performed with a 600MRC equipped with an argon/krypton laser (BioRad Labs, Hercules, CA). Green fluorescence was detected at λ > 515 nm after excitation at 488 nm. For the MAb-induced cell death assay, cells (5 × 105 cells/well) were placed in 24-well plates and incubated with 10 μg/ml anti-CD99 MAb and the secondary Ab for cross-linking of CD99 or the secondary Ab alone for the indicated period. In the inhibition experiment of cell death induced by CD99 or calcium ionophore A23187, RD-ES cells were treated with 10 μg/ml anti-CD99 MAb or 10 μmol/L A23187, respectively, at 37°C in the presence or absence of 0.1 μg/ml Act D, 1.0 μg/ml CHX, 0.1 to 200 μmol/L CsA, 1.5 mmol/L EGTA containing 1.5 mmol/L MgCl2, and various concentrations of FK-506 for the indicated period. To examine the requirement of caspases in CD99-induced apoptosis, RD-ES cells were pretreated with 20 μmol/L Z-VAD-fmk (a general caspase inhibitor) or Z-FA-fmk (a control cystein protease inhibitor) for 2 hours. The cells were then incubated for 6 hours in the presence or absence of DN16 MAb. Cell death was quantified by trypan blue dye exclusion. All experiments were performed at least three times, and a representative result of the experiments is shown in figures. Cells were fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, for 24 hours at 4°C. Then they were post-fixed in 0.1% osmium tetroxide in the same solution for 1 hour, dehydrated, transferred to propylene oxide, and embedded in epoxy resin (Polyscience Co., Warrington, PA). Ultrathin sections were stained with saturated aqueous uranyl acetate and lead citrate and examined under an electron microscope (Hitachi, H-600) at 75 kV. Cells (1 × 106) were incubated with anti-CD99 MAb (1 μg/100 μl) in PBS containing 1% bovine serum albumin and 0.1% sodium azide for 30 minutes at 4°C, washed with PBS, and stained with FITC-conjugated goat anti-mouse immunoglobulin Ab. After three washes, cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA). Apoptosis was assessed by TdT-mediated dUTP nick end-labeling (TUNEL) assay23Traganos F Melamed MR Darzynkiewicz Z Single step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation.Cytometry. 1995; 20: 172-180Crossref PubMed Scopus (155) Google Scholar using the APO-DIRECT apoptosis detection kit (Pharmingen, San Diego, CA). Control or anti-CD99 Ab-treated cells (1 × 106 to 2 × 106) were fixed with 4% paraformaldehyde, washed in PBS, resuspended in 70% cold ethanol, and stored overnight. After washing the cells with PBS, the TUNEL assay was performed according to the manufacturer's protocol. After the induction of apoptosis with anti-CD99 MAb (2 μg/ml) for 2 hours, cells (1 × 106) were incubated for 15 minutes in 1 ml of 20 nmol/L DiOC6(3) at 37°C, followed by analysis on a FACScan (excitation, 488 nm; emission, 525 nm).24Zamzami N Marchetti P Castedo M Zanin C Vayssière J-L Petit PX Kroemer G Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocytes death in vivo.J Exp Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1101) Google Scholar The control experiment was performed in the presence of 50 μmol/L carbamoyl cyanide m-chlorophenylhydrazone (mClCCP), an uncoupling agent that abolishes ΔΨm, for 15 minutes at 37°C. Recently, it has been reported that CD99 mediates apoptosis in immature thymocytes and Jurkat cells.21Bernard G Breittmayer J-P Matteis M Trampont P Hofman P Senik A Bernard A Apoptosis of immature thymocytes mediated by E2/CD99.J Immunol. 1997; 158: 2543-2550PubMed Google Scholar The findings that ES/PNET is a neoplastic counterpart of primitive neuroectodermal cells9Moll R Lee I Gould VE Berndt R Roessner A Franke WW Immunocytochemical analysis of Ewing's tumors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeniety and pluripotential differentiation.Am J Pathol. 1987; 127: 288-304PubMed Google Scholar, 10Cavazzana AO Miser JS Jefferson J Triche TJ Experimental evidence for a neural origin of Ewing's sarcoma of bone.Am J Pathol. 1987; 127: 507-518PubMed Google Scholar, 11Hara S Adachi Y Kaneko Y Fujimoto J Hata J Evidence for heterogeneous group of neuronal differentiation of Ewing's sarcoma.Br J Cancer. 1991; 64: 1025-1030Crossref PubMed Scopus (17) Google Scholar and that these cells express a large amount of CD99 on their surfaces17Fellinger EJ Garin-Chesa P Triche TJ Huvos AG Rettig WJ Immunohistochemical analysis of Ewing's sarcoma cell surface antigen p30/32MIC2.Am J Pathol. 1991; 139: 317-325PubMed Google Scholar, 25Kover H Dworzak M Strehl S Kovar H Gadner H Sazler-Kuntschik M Overexpression of the pseudoautosomal gene MIC2 in Ewing's sarcoma and pheripheral primitive neuroectodermal tumor.Oncogene. 1990; 5: 1067-1070PubMed Google Scholar led us to ask whether the ligation of CD99 with anti-CD99 MAb DN16 could induce cell death in ES/PNET cell lines. In this experiment, cells from two ES cell lines (RD-ES and CADO-ES1) and one PNET cell line (SK-N-MC) were incubated with DN16 in the presence of cross-linking Ab. Within 2 hours after the DN16 treatment, cell death was clearly observed in the ES cells. Maximal cell death was evident within 6 hours (Figure 1A). However, in SK-N-MC, which has recently turned out to be PNET,26Ida K Kobayashi S Taki T Hanada R Bessho F Yamamori S Sugimoto T Ohki M Hayashi Y EWS-FLI-1 and EWS-ERG chimeric mRNA in Ewing's sarcoma and primitive neuroectodermal tumor.Int J Cancer. 1995; 63: 500-504Crossref PubMed Scopus (46) Google Scholar CD99-induced cell death did not occur within 6 hours (Figure 1B) or even after 24 hours (data not shown), indicating that CD99 molecules were able to deliver a death signal only in ES cells, but not in SK-N-MC cells. In addition, neuroblastoma cells, such as SK-N-SH, SK-N-AS, and SH-SY5Y, were also resistant to CD99-mediated death after a 6-hour engagement (Figure 1B). Among the three types of tumors tested, ie, PNET, neuroblastoma, and ES cells, which differ in their neurogenic potentials and the levels of CD99 expression, ES cells are considered to be the tumor of the most undifferentiated stage.12Tetsuro S Akihiro U Jun-ichi H Neurogenic potential of Ewing's sarcoma cells.Virchows Arch. 1997; 430: 41-46Crossref PubMed Scopus (19) Google Scholar, 27Rettig WJ Garin-Chesa P Huvos AG Ewing's sarcoma: new approaches to histogenesis and molecular plasticity.Lab Invest. 1992; 66: 133-137PubMed Google Scholar As it was necessary to prepare cells in various stages of differentiation, we performed the in vitro induction of neural differentiation of RD-ES and CADO-ES1 cells by db-cAMP treatment. After 12 days of the induction of ES cells with db-cAMP, morphological changes such as elongated cytoplasmic process and varicosity formation were observed under phase contrast microscopic examination (Figure 2, B and D). Furthermore, in the indirect immunofluorescence analysis, the expression of NF-H, a neural differentiation marker, was clearly visualized in differentiated CADO-ES1 (Figure 2E) and RD-ES cells (data not shown). As shown in Figure 2E, NF-H was mainly localized in the perinuclear cytoplasm and in the elongated neuritic process of the differentiated CADO-ES1 cells. Subsequent experiments were done on both the undifferentiated and the differentiated ES cells to investigate whether the induction of CD99-mediated apoptosis is dependent on the degree of differentiation. Interestingly, apoptosis occurred only in the undifferentiated form of ES cells (Figure 3A). The expression level of CD99 was dramatically decreased in the differentiated ES cells (Figure 3B). All these data suggest that CD99 can deliver a death signal into undifferentiated ES cells that express CD99 on their surfaces at a high level.Figure 2The morphological changes and neurofilament expression after the differentiation induction of ES cells with db-cAMP. A–D: Cell culture morphology of RD-ES (A andB) and CADO-ES1 (C andD) cells under phase contrast microscopy. RD-ES and CADO-ES1 cells were cultured in the absence (A and C) or presence (B and D) of db-cAMP for 12 days. The differentiated cells show long cytoplasmic processes, and some neurites terminated in growth cones (B andD). E: Differentiated CADO-ES1 cells were stained with anti-NF-H MAb and analyzed by confocal microscopy. Note the neuritic process and perinuclear localization of NF-H.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Resistance to CD99-induced cell death and the down-regulation of CD99 in the differentiated ES cells. A: Resistance to CD99-induced cell death in db-cAMP-treated ES cells. ES cells were cultured in the presence or absence of db-cAMP for 12 days. In 24-well plates, the db-cAMP-treated or untreated cells (5 × 105 cells) were plated and incubated with DN16 (anti-CD99 MAb) and the secondary Ab (rabbit anti-mouse immunoglobulin Ab) for cross-linking of CD99 (filled bars) or with the secondary Ab alone (control,open bars) for 6 hours. All values are the averages of the experiments done in duplicates. The percentage of dead cells was assessed by trypan blue dye exclusion. B: Effect of db-cAMP on the expression of CD99. Live cells were gated and analyzed on a FACScan flow cytometer. The expression level of CD99 was markedly reduced in the db-cAMP-treated ES cells (filled areas) as compared with the untreated ES cells (open areas). Thedotted line indicates the staining pattern with a negative control Ab.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Apoptosis has been characterized by morphological changes such as chromatin condensation, nuclear fragmentation, internucleosomal DNA fragmentation, and cytoplasmic blebbing.1Kerr JFR Shrinkage necrosis: a distinct mode of cellular death.J Pathol. 1971; 105: 13-20Crossref PubMed Scopus (708) Google Scholar The electron microscopic analysis of RD-ES cells treated with DN16 MAb clearly showed several ultrastructural changes comparable to those of apoptotic cells (Figure 4, A–C), as compared with the control Ab-treated cells (Figure 4D). CD99-induced apoptosis in RD-ES cells was further confirmed by the TUNEL method.23Traganos F Melamed MR Darzynkiewicz Z Single step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation.Cytometry. 1995; 20: 172-180Crossref PubMed Scopus (155) Google Scholar Although a typical ladder pattern of DNA fragmentation was not clear on agarose gel electrophoresis (data not shown), TUNEL assay revealed that the percentage of apoptotic cells in the DN16 MAb-treated RD-ES cells was nearly five times higher than that in the control Ab-treated cells. Approximately 20% of the cells treated with DN16 MAb contained incorporated FITC-dUTP (Figure 5, right), whereas only 4.5% of the control Ab-treated cells were positive for FITC-dUTP (Figure 5, left). It is likely that CD99 induces extremely limited endonuclease activities, as a DNA ladder pattern was not seen on gel electrophoresis and the labeling intensity of FITC-dUTP was weak. As many apoptotic processes require de novo synthesis of RNA and/or protein,2Yazdanbakhsh K Choi JW Li Y Lau LF Choi Y Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcriptional factor Nur 77.Proc Natl Acad Sci USA. 1995; 92: 437-441Crossref PubMed Scopus (95) Google Scholar, 3Ferrer I Olive M Ribera J Planas AM Naturally occurring (programmed) and radiation-induced apoptosis are associated with selective c-Jun expression in the developing rat brain.Eur J Neurosci. 1996; 8: 1286-1298Crossref PubMed Scopus (75) Google Scholar we tested whether this is also the case in CD99-induced apoptosis in RD-ES cells. As shown in Figure 6, the CD99-mediated apoptosis in RD-ES cells was completely blocked after treatment with a RNA synthesis inhibitor, Act D (Figure 6B), or a protein synthesis inhibitor, CsA (Figure 6C), at the concentration of 0.1 μg/ml or 1.0 μg/ml, respectively. These results indicate that de novo synthesis of both RNA(s) and protein(s) are necessary for the process of apoptosis via CD99 in RD-ES cells. An increasing amount of evidence has shown that the alteration of mitochondrial membrane function plays a crucial role in the induction of apoptosis.28Zamzami N Marchetti P Castedo M Decaudin D Macho A Hirsch T Susin SA Petit PX Mignotte B Kroemer G Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.J Exp Med. 1995; 182: 367-377Crossref PubMed Scopus (1443) Google Scholar, 29Kroemer G Petit PX Zamzami N Vayssière J-L Mignotte B The biochemistry of apoptosis.FASEB J. 1995; 9: 1277-1287Crossref PubMed Scopus (969) Google Scholar, 30Ankarcrona M Dypbukt JM Bonfoco E Zhivotovsky B Orrenius D Lipton SA Nicotera P Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function.Neuron. 1995; 15: 961-973Abstract Full Text PDF PubMed Scopus (1702) Google Scholar As CsA is a potent inhibitor of mitochondrial PT pore opening,6Petronilli V Nicolli A Costantini P Colonna R Bernardi P Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.Biochem Biophys Acta. 1994; 1187: 255-259PubMed Google Scholar, 8Crompton M Ellinger H Costi A Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.Biochem J. 1988; 255: 357-360PubMed Google Scholar we examined the effect of CsA in CD99-induced apoptosis. When RD-ES cells were pretreated with CsA at the various concentrations ranging from 0.1 to 200 μmol/L, CD99-induced apoptosis was completely inhibited at the concentration above 100 μmol/L CsA (Figure 7). It has been well known that CsA can inhibit apoptosis not only through the modification of mitochondrial PT pores but also through inactivation of calcineurin by complex formation with cyclophilin A.31Fruman DA Klee CB Bierer BE Burakoff SJ Calcineurin phosphatase activity in T lymphocytes is inhibited by FK506 and cyclosporin A.Proc Natl Acad Sci USA. 1992; 89: 3686-3690Crossref PubMed Scopus (765) Google Scholar Therefore, to rule out the possible involvement of calcineurin in CD99-induced apoptosis, we examined the effect of FK-506, a more selective calcineurin inhibitor, in RD-ES cells. The apoptosis in RD-ES cells was not protected by the treatment of FK-506 (ranging from 10 to 100 nmol/L; Figure 8A), indicating that apoptosis induced by CD99 cannot be blocked by inhibition of calcineurin. Interestingly, calcium ionophore, which induces calcium signaling and activates calcineurin in a calcium-dependent manner, was also able to induce the death of RD-ES cells. Figure 8B shows that both CsA and FK-506 protect RD-ES cells from the calcium-ionophore-induced apoptosis. These data strongly suggest that CD99-induced apoptosis is mediated by mitochondrial PT pore opening, regardless of Ca2+/calmodulin-dependent calcineurin activity.Figure 8Effects of CsA, FK-506, and EGTA on apoptosis induced by anti-CD99 MAb or calcium ionophore. RD-ES cells were preincubated with CsA or FK-506 for 2 hours before the induction of apoptosis by anti-CD99 MAb at 10 μg/ml (A) or calcium ionophore A23187 at 10 μmol/L (B). In the case of EGTA tr
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