Characterization of the Interleukin-1β-Converting Enzyme/Ced-3-Family Protease, Caspase-3/CPP32, in Hodgkin's Disease
1999; Elsevier BV; Volume: 154; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65398-9
ISSN1525-2191
AutoresKeith F. Izban, Tamara Wrone-Smith, Eric D. Hsi, Bertram Schnitzer, Maria Eugenia Quevedo, Serhan Alkan,
Tópico(s)Hepatitis B Virus Studies
ResumoApoptosis (programmed cell death) serves an important role in the normal morphogenesis, immunoregulation, and homeostatic mechanisms in both normal and neoplastic cells. Caspase-3/CPP32, a member of the ICE/Ced-3-family of cysteine proteases, is an important downstream mediator of several complex proteolytic cascades that result in apoptosis in both hematopoietic and nonhematopoietic cells. Previous studies have demonstrated that caspase-3 is commonly expressed in classical Hodgkin's disease (CHD); however, the biological significance of its expression in Hodgkin's disease is unknown. In this report, the expression of caspase-3 in nodular lymphocyte predominance Hodgkin's disease (NLPHD) was evaluated by immunohistochemistry; in addition, we investigated the role of caspase-3 in CD95 (Fas)-mediated apoptosis in three CHD cell lines. Formalin-fixed, paraffin-embedded tissue sections from 11 cases of NLPHD were immunostained for caspase-3 using a polyclonal rabbit antibody that detects both the 32-kd zymogen and the 20-kd active subunit of the caspase-3 protease. Only 1/11 cases of NLPHD demonstrated caspase-3 immunopositivity in lymphocytic/histiocytic cells. Caspase-3 expression was also evaluated in three CHD cell lines, HS445, L428, and KMH2. Whereas caspase-3 expression was detected in HS445 and L428 cell lines, no expression was found in KMH2 cells by immunohistochemical staining. Treatment of HS445 and L428 cell lines for 72 hours with agonistic CD95 monoclonal antibody induced marked apoptosis that was significantly inhibited by pretreatment with the caspase-3 inhibitor, DEVD-FMK, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and flow cytometric analysis of 7-amino-actinomycin D staining. In addition, a significant increase in caspase-3 activity as determined by an enzyme colorimetric assay was detected in HS445 and L428 cells after 48 hours of CD95 stimulation. In marked contrast, treatment of caspase-3-deficient KMH2 cells with anti-CD95 mAb did not demonstrate an increase in caspase-3 activity or induce apoptosis. These data demonstrate caspase-3 is important for CD95-mediated apoptosis in CHD cell lines. In addition, the majority of NLPHD cases examined in this study failed to express detectable levels of caspase-3, suggesting these tumor cells may be resistant to apoptotic stimuli dependent on caspase-3 activity. Furthermore, these data suggest the differential expression of caspase-3 noted between NLPHD and CHD may provide additional evidence that each is a unique disease entity. Apoptosis (programmed cell death) serves an important role in the normal morphogenesis, immunoregulation, and homeostatic mechanisms in both normal and neoplastic cells. Caspase-3/CPP32, a member of the ICE/Ced-3-family of cysteine proteases, is an important downstream mediator of several complex proteolytic cascades that result in apoptosis in both hematopoietic and nonhematopoietic cells. Previous studies have demonstrated that caspase-3 is commonly expressed in classical Hodgkin's disease (CHD); however, the biological significance of its expression in Hodgkin's disease is unknown. In this report, the expression of caspase-3 in nodular lymphocyte predominance Hodgkin's disease (NLPHD) was evaluated by immunohistochemistry; in addition, we investigated the role of caspase-3 in CD95 (Fas)-mediated apoptosis in three CHD cell lines. Formalin-fixed, paraffin-embedded tissue sections from 11 cases of NLPHD were immunostained for caspase-3 using a polyclonal rabbit antibody that detects both the 32-kd zymogen and the 20-kd active subunit of the caspase-3 protease. Only 1/11 cases of NLPHD demonstrated caspase-3 immunopositivity in lymphocytic/histiocytic cells. Caspase-3 expression was also evaluated in three CHD cell lines, HS445, L428, and KMH2. Whereas caspase-3 expression was detected in HS445 and L428 cell lines, no expression was found in KMH2 cells by immunohistochemical staining. Treatment of HS445 and L428 cell lines for 72 hours with agonistic CD95 monoclonal antibody induced marked apoptosis that was significantly inhibited by pretreatment with the caspase-3 inhibitor, DEVD-FMK, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and flow cytometric analysis of 7-amino-actinomycin D staining. In addition, a significant increase in caspase-3 activity as determined by an enzyme colorimetric assay was detected in HS445 and L428 cells after 48 hours of CD95 stimulation. In marked contrast, treatment of caspase-3-deficient KMH2 cells with anti-CD95 mAb did not demonstrate an increase in caspase-3 activity or induce apoptosis. These data demonstrate caspase-3 is important for CD95-mediated apoptosis in CHD cell lines. In addition, the majority of NLPHD cases examined in this study failed to express detectable levels of caspase-3, suggesting these tumor cells may be resistant to apoptotic stimuli dependent on caspase-3 activity. Furthermore, these data suggest the differential expression of caspase-3 noted between NLPHD and CHD may provide additional evidence that each is a unique disease entity. Increased understanding of the physiological and pathological processes of programmed cell death, or apoptosis, at the molecular level will provide insights into carcinogenesis and potentially create new opportunities for development of novel prognostic markers and therapeutic tools for the treatment of various neoplasms. One of the earliest cell death-regulating genes to be identified was the proto-oncogene Bcl-2, an apoptosis inhibitor that appears to block a step in an evolutionarily conserved pathway involved in apoptosis.1Tsujimoto Y Croce CM Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma.Proc Natl Acad Sci USA. 1986; 83: 5214-5218Crossref PubMed Scopus (1068) Google Scholar, 2Vaul DL Cory S Adams JM Bcl-2 gene promotes haematopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.Nature. 1988; 335: 440-442Crossref PubMed Scopus (2716) Google Scholar Subsequent investigations led to the isolation of a homologue of Bcl-2 in the nematode Caenorhabitis elegans. 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In this report, we demonstrate caspase-3 plays an important role in CD95-mediated apoptosis in classical Hodgkin's disease (CHD) cell lines. Furthermore, we demonstrate that nodular lymphocyte predominance Hodgkin's disease (NLPHD) lacks caspase-3 expression by immunophenotypic analysis. The lack of caspase-3 expression in NLPHD may contribute to the development and pathogenesis of this disease by imbuing tumor cells with resistance to caspase-3-dependent apoptotic pathways. Formalin-fixed, paraffin-embedded tissue sections from 11 cases of NLPHD were selected from the surgical pathology files of Loyola University Medical Center and the University of Michigan Medical School for immunohistochemical determination of caspase-3. Diagnosis of NLPHD was performed using established criteria on lymph node biopsy histology and immunohistochemistry.24von Wasielewski R Werner M Fischer R Hansmann ML Hubner K Hasenclever D Franklin J Sextro M Diehl V Georgii A Lymphocyte-predominant Hodgkin's disease: An immunohistochemical analysis of 208 reviewed Hodgkin's disease cases from the German Hodgkin's Study Group.Am J Pathol. 1997; 150: 793-803PubMed Google Scholar, 25Harris NL Jaffe ES Stein H Banks P Chan JK Cleary ML Delsol G De Wolf-Peeters C Falini B Gatter KC Isaacson PG Knowles DM Mason DY Muller-Hermelink H-K Pileri SA Piris MA Ralfkiaer E Warnke RA A revised European-American classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group.Blood. 1994; 84: 1361-1392PubMed Google Scholar NLPHD was diagnosed by the finding of typical nodular architecture and lymphocytic/histiocytic (L&H) cells with the appropriate CD20- and CD45RB-positive immunophenotype. Morphology assessment of NLPHD cases was performed on 4-μm tissue sections with hematoxylin-eosin. Immunoperoxidase staining of lymph node sections with the antibodies listed in Table 1 was performed using a Ventana 320 automated stainer (Ventana Medical Systems, Tucson, AZ) and a streptavidin/horseradish peroxidase detection kit (Ventana), with microwave antigen retrieval and trypsin pretreatment used as necessary. The chromogen was 3,3′-diaminobenzidine tetrahydrochloride (DAB).Table 1Antibodies Used in Immunohistochemical Staining of NLPHDAntibodySourceDilutionLCA (PD7/26/16 and 2B11)DAKO (Carpinteria, CA)1:50CD30 (Ber-H2)DAKO1:40CD20 (L26)DAKO1:100EMA (E29)DAKO1:100CD15 (Leu-M1)Becton Dickinson, (San Jose, CA)1:50CD45RO (A6)Zymed Laboratories (San Francisco, CA)1:50 Open table in a new tab The CHD cell lines KMH2, L428, and HS445 were used in this study. KMH2 and L428 cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). HS445 and Jurkat cell lines were obtained from the American Type Culture Collection (Manassas, VA). Cell lines were cultured in RPMI 1640 (Gibco-BRL, Grand Island, NY) supplemented with 20% (v/v) heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 2 mmol/L L-glutamine (Gibco-BRL), 25 mmol/L Hepes (Sigma), and antibiotic-antimycotic solution (Sigma). All cell lines were maintained at 37°C in a humidified incubator at 5% CO2. Four-micron-thick formalin-fixed, paraffin-embedded tissue sections from each case of NLPHD were deparaffinized in xylene, hydrated in graded alcohol, and pretreated for antigen retrieval in 10 mmol/L citrate buffer, pH 6.0, for 10 minutes. Cytospins from CHD cell lines were fixed in a 1:1 mixture of acetone and methanol for 10 minutes. Staining was performed using polyclonal rabbit anti-human CPP32 (1:200 titer, DAKO Corp., Carpinteria, CA) and a Vectastain ABC peroxidase, rabbit IgG detection kit (Vector Laboratories, Burlingame, CA) with 3-amino 9-ethyl carbazole (AEC) as the chromogen. The chromogen DAB was used for paraffin-embedded specimens. Formalin-fixed, paraffin-embedded tissue sections from three cases of caspase-3-positive nodular sclerosis Hodgkin's disease and a reactive tonsil were used as positive controls for caspase-3 staining. For apoptosis assays, 1 × 106 cells from each cell line were cultured in 24-well tissue culture plates (Falcon, Lincoln Park, NJ) and incubated with 500 ng/ml of agonistic anti-CD95 monoclonal antibody (mAb) (clone CH11, mouse IgM, Upstate Biotechnology, Lake Placid, NY) for indicated time periods, with or without 1 hour preincubation with 10 μmol/L caspase-3 peptide inhibitor Ac-Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD-FMK, Clontech, Palo Alto, CA). Detection of apoptosis in CHD cell lines by terminal deoxynucleotidyl tranferase-mediated dUTP nick end-labeling (TUNEL) was quantitated using the ApopTag in situ apoptosis peroxidase detection kit (Oncor, Gaithersburg, MD). Cytospin preparations of cells were fixed in 1% formaldehyde for 15 minutes followed by 1 hour fixation in 70% ethanol at −20°C. After a brief wash in FA buffer (Difco Laboratories, Detroit, MI), each slide was incubated at room temperature (RT) for 10 minutes with equilibration buffer followed by 1 hour incubation at 37°C with TdT enzyme (or deionized water (dH2O) for negative controls) diluted with the reaction buffer. The TdT reaction was stopped with stop/wash buffer and each specimen was briefly washed with FA buffer before 30 minute incubation with anti-digoxigenin-peroxidase at RT. After a series of washes with FA buffer, each slide was developed with DAB/hydrogen peroxide (Sigma) color substrate for 6 minutes at RT. All slides were counterstained with hematoxylin. A CD95-sensitive Jurkat T cell line was used as a positive control for apoptosis. A positive reaction for apoptosis was characterized by brown/black coloration of the nuclear or perinuclear region of the cell. Apoptotic cells were quantitated by 1000-cell count at 400× magnification. The 7-Amino Actinomycin D (7-AAD) staining method to measure cell viability was performed per manufacturer's protocol using Via-Probe 7-AAD (PharMingen, San Diego, CA). Briefly, anti-CD95 mAb-treated and untreated cells (1 × 106 cell/ml) were washed twice in cold PBS and resuspended in 1× binding buffer (10 mmol/L Hepes/NaOH (pH 7.4), 140 mmol/L NaCL, and 2.5 mmol/L CaCl2). Resuspended cells were then incubated for 20 minutes at 20–25°C in the dark with 5 μl of 7-AAD. Samples (30,000 events per sample) were then quantitated on an Epics XL-MCL flow cytometer (Coulter, Miami Lakes, FL), recorded in LIST mode, and registered on logarithmic scales. 7-AAD emission was detected in the FL-3 channel (>650 nm). Analysis was performed using Coulter System II software. Caspase-3 activity was determined using the ApoAlert CPP32/Caspase-3 colorimetric assay kit (Clontech). After a 48-hour incubation with anti-CD95 mAb, duplicate samples of untreated and treated cells (2 × 106 cells) were washed in cold PBS, resuspended in 50 μl cell lysis buffer, and incubated on ice for 10 minutes. Cell lysates were pelleted, followed by transfer of the supernatants to microcentrifuge tubes. Fifty microliters of 2× reaction buffer with 5 mmol/L DTT and 5 μl of 1 mmol/L DEVD-p-nitroanilide (pNA)-conjugated CPP32 substrate were added to each tube, followed by 1 hour incubation in a water bath at 37°C. A control reaction of treated cells without DEVD-pNA was included. Optical density (OD) for each specimen was determined at 405 nm using the EL 312e microplate reader (Bio-Tek Instruments, Winooski, VT). For quantification of protease activity, sample values were plotted on a calibration curve derived from the OD values obtained from each of five standards (range: 0–20 nmole pNA). For each sample, units of CPP32 activity were determined by the following formula: Units of CPP32 activity=(ΔOD) × (calib. Curve slope)-1 × (nmole pNA/OD)where ΔOD is the change in optical density from the control reaction without conjugated substrate. All 11 specimens demonstrated architectural changes consistent with NLPHD and were composed predominantly of large nodules with focal areas of diffuse effacement. Typical nodules contained characteristic L&H cells intermixed in a background of small lymphocytes and occasional epithelioid histiocytes separated by compressed intervening paracortical areas composed of small lymphocytes and scattered plasma cells. In all specimens, L&H cells demonstrated positive staining for CD20 and LCA. In seven specimens, L&H cells expressed EMA, with one case also expressing CD30. In all cases, L&H cells were negative for CD15 and CD45RO. In 10 of 11 cases of NLPHD, including the case which expressed CD30, L&H cells were negative for caspase-3 expression by immunohistochemical staining as represented in Figure 1A. In one case, caspase-3 immunopositivity was detected in the cytoplasm in a few scattered L&H cells (Figure 1B). In contrast, caspase-3 expression was demonstrated both in Hodgkin Reed-Sternberg (HRS) cells and in background lymphocytes in three cases of nodular sclerosis Hodgkin's disease (Figure 1C). In addition, tonsil tissue positive controls demonstrated caspase-3 immunopositivity concentrated predominantly in germinal center cells of secondary follicles (Figure 1D). Three CHD cell lines (HS445, L428, and KMH2) were analyzed for caspase-3 expression by immunohistochemistry. HS445 and L428 consistently demonstrated substantial cytoplasmic immunostaining for caspase-3 (Figure 2 and data not shown). However, in contrast, repeated immunohistochemistry assays failed to detect expression of caspase-3 in the KMH2 cell line (Figure 2). Activation of the CD95 receptor by ligand or agonistic mAb is known to induce apoptosis with concomitant proteolytic cleavage and activation of caspases, including caspase-3, in CD95-positive neoplasms.19Trauth BC Klas C Peters AM Matzku S Moller P Falk W Debatin KM Krammer PH Monoclonal antibody-mediated tumor regression by induction of apoptosis.Science. 1989; 245: 301-305Crossref PubMed Scopus (1662) Google Scholar, 20Yonehara S Ishii A Yonehara M A cell killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor.J Exp Med. 1989; 169: 1747-1756Crossref PubMed Scopus (1427) Google Scholar, 21Suda T Nagata S Purification and characterization of the Fas ligand that induces apoptosis.J Exp Med. 1994; 179: 873-878Crossref PubMed Scopus (501) Google Scholar, 22Suda T Takahashi T Golstein P Nagata S Molecular cloning and expression of the Fas ligand, a novel member of the TNF family.Cell. 1993; 75: 1169-1178Abstract Full Text PDF PubMed Scopus (2438) Google Scholar, 23Takahashi T Tanaka M Inazawa J Abe T Suda T Nagata S Human Fas ligand: gene structure, chromosomal location and species specificity.Int Immunol. 1994; 6: 1567-1574Crossref PubMed Scopus (416) Google Scholar To investigate the effect of CD95 stimulation with potential activation of caspase-3 in Hodgkin's disease, we examined the effect of agonistic CD95 mAb on CHD cell lines. The HS445 and L428 cell lines displayed a significant increase in apoptosis after 72 hours' treatment with anti-CD95 mAb as quantitated by both flow cytometric analysis with 7-AAD (Figure 3)staining and the TUNEL assay (Figure 4, Table 2). CD95-induced apoptosis was significantly inhibited in these cells by the caspase-3 peptide inhibitor, DEVD-FMK, as demonstrated by the decrease in number of apoptotic cells to nearly background levels (Figure 3, Figure 4, Table 2). In contrast, the KMH2 cell line demonstrated consistent resistance to anti-CD95 mAb treatment with no effect by the addition of DEVD-FMK (Figure 3 and Table 2). Resistance of KMH2 cells to CD95-mediated apoptosis was not due to lack of CD95 expression, as all three cell lines expressed similarly high levels of CD95 as determined by flow cytometric staining.26Alkan S Hsi ED Wrone-Smith T CD95 (Fas antigen: APO-1) mediated apoptosis on Hodgkin's disease cell lines.Mod Pathol. 1998; 11 (abstr. 950): 162AGoogle ScholarFigure 4TUNEL assay for apoptosis. Compared to untreated cells (A), the L428 cell line displayed a considerable increase in apoptosis (dark brown/black cells) after 72 hour incubation with 500 ng/ml agonistic CD95 (Fas) mAb (B). Pretreatment of L428 cells with 10 μM DEVD-FMK decreased the number of apoptotic cells to near baseline levels (C). Stimulation of KMH2 cells with anti-CD95 mAb with or without DEVD-FMK pretreatment showed no increase in apoptosis compared to untreated cells (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2Apoptosis Rates Induced in Anti-CD95 mAb Treated or Untreated CHD Cell Lines as Determined by TUNEL AssayApoptosis*1 × 106 cells were untreated or treated with 500 ng/ml anti-CD95 mAb (CH11) for 72 hours with or without pretreatment with 10 μM caspase-3 peptide inhibitor, DEVD-FMK. Apoptosis was quantitated by TUNEL staining as described in Materials and Methods.CHD cell lineUntreatedAnti-CD95Anti-CD95+ DEVD-FMKHS4457.5%20.5%9.9%L4287.8%30.2%10.8%KMH21.0%1.3%1.0%* 1 × 106 cells were untreated or treated with 500 ng/ml anti-CD95 mAb (CH11) for 72 hours with or without pretreatment with 10 μM caspase-3 peptide inhibitor, DEVD-FMK. Apoptosis was quantitated by TUNEL staining as described in Materials and Methods. Open table in a new tab The significant inhibition of apoptosis by a caspase-3 inhibitor in HS445 and L428 cells, and the lack of apoptosis induced by caspase-3-deficient KMH2 cells suggests caspase-3 is important for CD95-mediated apoptosis in CHD cell lines. To further substantiate caspase-3 cleavage and activation in CD95-mediated apoptosis in CHD, each cell line was evaluated for changes in caspase-3 activity before and after treatment with anti-CD95 mAb using an enzyme colorimetric assay (Figure 5). Forty-eight-hour treatment revealed approximately tenfold increases in caspase-3 activity in HS445 and L428 cells in contrast to no difference detected in treated KMH2 cells. Positive control CD95-sensitive Jurkat T cells displayed a fivefold increase in caspase-3 activity after 24 hours of treatment with anti-CD95 mAb. Among the caspases identified in humans thus far, caspase-3 is probably one of the most relevant and best studied as regards to apoptosis in hematopoietic cells. Caspase-3 (CPP32, Yama, apopain) has been shown to be a key effector molecule in the downstream execution of various apoptotic stimuli.9Schlegel J Peters I Orrenius S Miller DK Thornberry NA Yamin TT Nicholson DW CPP32/apopain is a key interleukin 1β converting enzyme-like protease involved in Fas- mediated apoptosis.J Biol Chem. 1996; : 1841-1844PubMed Google Scholar, 10Enari M Hug H Nagata S Involvement of an ICE-like protease in Fas-mediated apoptosis.Nature. 1995; 375: 78-81Crossref PubMed Scopus (797) Google Scholar, 11Enari M Talanian RV Wong WW Nagata S Sequential activation of ICE-like and CPP32- li
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