Chemoresistance in b cell lymphoma role of apoptosome activation

CHEMORESISTANCE IN B CELL LYMPHOMA: ROLE OF APOPTOSOME ACTIVATION Sun Yu (MBBS, China/ Licentiate, Sweden) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2005 ACKNOWLEDGEMENTS This study was initiated as collaboration between the Department of Physiology, National University of Singapore, and the Institute of Environmental Medicine, Karolinska Institutet I would like to express my gratitude to the followings Shazib Pervaiz, my supervisor, Associate Professor in the Department of Physiology, National University of Singapore, thanks for bringing me into this interesting scientific area Your knowledgeable guidance and smart idea from the beginning really built the basis of this study I appreciate the flexible environment you provided and constant support Bengt Fadeel, my supervisor, Associate Professor in the Division of Molecular Toxicology of Karolinska Institutet, thanks for your scientific guidance, knowledgeable discussion, constant encouragement, and the fully support I also thank you for the opportunities you gave me to attend international conferences from where I opened my eyes to the scientific world and really indulge in it Sten Orrenius, my supervisor, Professor in the Division of Toxicology of Karolinska Institutet, thanks for the effort from you to initiate this collaboration And also thanks for every wonderful discussion with you on this study Even you were very busy, but you really put much energy into the study and make it going smoothly I am grateful for all the scientific discussions and technique supports from the following people in Karolinska Institutet, Stockholm: Bertrand Joseph, Ping Huang, Leta Nutt, Barbett Bartling, Mihails Baryshev, Souren Mkrtchian, Yvonne Hofmann and Jue Wang Thanks for your selfless sharing your experience and idea with me My dear colleagues from Apoptosis and Tumour Biology Lab in Physiology, I really cherish the friendship among us I am proud of to be the one of you and to be working with you Christopher, thanks for your patience to teach me the western blot and others Jayshree and Tini, thanks for the general guidance and instructions of the lab, which provide me an easier and comfort working Tze Wei, specially thanks to you for your always help and scientific discussion even when I was far away in Sweden And thanks all the other members from the lab for your entertainment with me and friendly environment you created I specially want to thank and give this thesis to my family, my husband, my parents and my brother During my PhD study, you are always with me and support me far away from China Thanks for all the love you give me This work was supported, in part, by grants from the National Medical Research Council of Singapore, Swedish Children’s Cancer Foundation, the Swedish Research Council, and the joint graduate student program of Karolinska Institutet and the National University of Singapore i TABLE OF CONTENTS Acknowledgements i Table of Contents ii Summery vi List of Tables vii List of Figures viii Abbreviations xi List of Publications xiv PART I INTRODUCTION 1 B CELL LYMPHOMA 1.1 Genetic background of Burkitt lymphoma 1.2 Sensitivity of BL to chemotherapy APOPTOTIC SIGNALING PATHWAY 2.1 Genetic control of apoptosis 2.2 Apoptotic signaling pathways in mammals THE APOPTOTIC MACHINERY 3.1 Extrinsic (death receptor-mediated) apoptosis signaling: DISC formation and regulation 3.2 Intrinsic (mitochondria-dependent) apoptosis signaling: apoptosome formation and activation 10 CHEMORESISTANCE: DEFECTIVE OF APOPTOSOME ACTIVATION IN APOPTOTIC SIGNALING PATHWAY 16 4.1 Regulation of Apoptosis by anti-apoptotic members of Bcl-2 family 17 4.2 Defective expression of Apaf-1 with chemoresistance 20 4.3 Heat Shock Proteins as inhibitors of apoptosome formation 21 4.4 Other factors regulating apoptosome formation 22 4.5 Inhibition of apoptosome function by targeting caspases, the role of IAPs in chemoresistance 23 ii OTHER KEY PLAYERS IN CHEMORESISTANCE, FROM BL GENETIC BACKGROUND 26 5.1 EBV related proteins 26 5.2 c-MYC and P53 27 PART II AIM OF THE STUDY 31 PART III MATERIAL AND METHODS 32 Cell lines 32 Apoptosis-inducing agents and induction of apoptosis 32 Generation of stable Apaf-1 over-expressing cell lines 34 Apoptosis assays 35 4.1 Morphological assessment of nuclear condensation and fragmentation by Hoechst 33342 35 4.2 Analysis of DNA fragmentation assessment by PI incorporation 35 4.3 MTT Reduction assay 36 4.4 Determination of caspase activities 36 4.5 Detection of membrane Phosphatidylserine (PS) exposure 37 4.6 Analysis of mitochondrial transmembrane potential 37 4.7 Detection of apoptotic related proteins by western blot analysis 40 In vitro cell-free systems 40 5.1 Generation of S-100 cytosols and cell-free apoptosis assays 41 5.2 Assessment of apoptosome formation 41 5.3 Immunoprecipitation 42 5.4 Cytochrome c and Smac release 42 Molecular approaches 43 6.1 Apaf-1 sequencing 43 6.2 AsHsp27 (antisense) pCDNA3.1 (+) construct 44 6.3 Transient transfection of AsHsp27 44 Appendix: Buffers used in the study 45 PART IV RESULTS 48 iii Human BL-derived cell lines are resistant to apoptosis triggered by the chemotherapeutic drug, etoposide: evidence for a post-mitochondrial defect 48 1.1 Raji B cells are resistant to etoposide-induced apoptosis 48 1.2 BL cell lines are resistant to etoposide-induced caspase activation both in vivo system and in cell free system 51 1.3 Post-mitochondrial apoptotic defects in the Raji BL cell line 55 1.4 Subthreshold levels of Apaf-1 in the cytosol of human BL cells 60 1.5 Defective apoptosome activation in the human Raji BL cell line 60 1.6 Overexpression of Apaf-1 restores sensitivity to apoptosis in the Raji BL cell line 67 Plasma membrane sequestration of Apaf-1 contributes to BL cells resistance to etoposide and staurosporine-induced apoptosis 71 2.1 Plasma membrane sequestration of Apaf-1 in chemoresistance BL cells 71 2.2 Reversal of chemoresistance in human BL cells upon liberation of Apaf-1 78 The role of IAPs and Smac in chemoresistant BL cells 78 3.1 IAPs are highly expressed in BL-derived cell lines, and serve to modulate apoptosis in these cells in an apoptosome-dependent manner 80 3.2 Smac release is defective in BL-derived cell lines: introduction of cell-permeable Smac N7 peptides potentiate Raji cells to apoptosis in an Apaf-1-dependent manner 85 Other apoptotic inhibitors in BL cell lines 88 Alternative apoptotic pathway in BL cells 92 PART V DISCUSSION 103 The role of the apoptosome in controlling the sensitivity of cancer cells to chemotherapeutic agents 103 Intracellular requirements for post-mitochondrial molecules, such as the IAPs, to control apoptosis in BL-derived cell lines 107 Cytochrome c and Smac release in BL cells 111 Alternative apoptosis window opened in chemoresistant cells 113 The role of other inhibitors of apoptosis in the modulation of apoptosis in BL cells 114 iv Proteasome in apoptosis and cancer therapy 115 PART VI CONCLUSIONS 117 PART VII REFERENCES 119 v Summary Chemoresistance is a major clinical problem in the treatment of cancer, and is thought to be related to an underlying resistance to apoptosis (programmed cell death) in cancer cells In the present study, Burkitt’s lymphoma (BL)-derived B cell lines were used to investigate the intracellular signaling pathways involved in apoptosis induction by chemotherapeutic agents; in particular, we focused on post-mitochondrial signaling and its implications for chemoresistance Apaf-1 (apoptotic protease-activating factor-1) is the main component of a protein complex termed the apoptosome, which is required for caspase activation downstream of mitochondria Upon release from these organelles, cytochrome c binds to Apaf-1 thus driving its oligomerization; this results in the activation of caspase-9 and caspase3, and the rapid dismantling of the cell In the B cell lines utilized herein, cytochrome c failed to stimulate apoptosome formation and caspase activation, and this was shown to be due to insufficient levels of Apaf-1 in the cytosolic compartment However, the level of Apaf-1 in total cellular extracts was normal Enforced expression of Apaf-1 increased its concentration in the cytosol, restored cytochrome c-dependent caspase activation, and rendered the prototypic BL cell line Raji sensitive to etoposide- and staurosporine-induced apoptosis Subsequent investigations revealed that, in Raji BL cells, the bulk of Apaf-1 was sequestered in the plasma membrane Moreover, liberation of Apaf-1 using lipid raft-disrupting agents served to sensitize BL cells to chemotherapeutic drugs Our studies also show that IAPs (inhibitor of apoptosis proteins) are highly expressed in BL cell lines The combination of Smac peptides, which serve to antagonize IAPs, thereby alleviating caspase inhibition, and staurosporine triggered apoptosis in Apaf-1-overexpressing BL cells, but failed to so in mock transfectants Similar results were obtained when Smac peptides were added together with the proteasome inhibitor, lactacytsin Hence, Smac-mediated potentiation of apoptosis was shown to be Apaf-1-dependent, and IAPs were concluded to modulate the sensitivity to apoptosis in this model In sum, these studies are the first to show that plasma membrane sequestration of Apaf1, a key component of the apoptosome, may render cancer cells resistant to treatment From the point of view of clinical utilization of apoptosis-targeted therapies, our results suggest that IAP modulation using, for instance, Smac peptides or Smac mimetics, may be beneficial only if sufficient amounts of Apaf-1 are present Our results also suggest that proteasome inhibition-triggered apoptosis is dependent on apoptosome formation The latter findings may be of potential importance since clinical trials using proteasome inhibitors are currently ongoing in several hematological malignancies vi LIST OF TABLES Table EBV status in BL derived cell lines used in the study 28 Table P53 status in BL derived cell lines used in the study 30 vii LIST OF FIGURES INTRODUCTION Figure Apoptotic signaling pathway, the key role of apoptotic complex formation Figure Apaf-1 isoforms and schematic model for apoptosome formation 12 Figure Overall structure of the WD40-deleted Apaf-1 bound to ADP 15 Figure IAPs family members and schematic model for functional domains 25 MATERIALS AND METHODS Figure Methodology flow chart 33 Figure Apoptosis analysis by Hoechst 33342 staining and PI incorporation 38 Figure Time-dependent AMC release curves 39 RESULTS Figure Raji cells were resistant to etoposide treatment 49 Figure Raji cells failed to expose phophatidyl serine upon etoposide treatment 50 Figure 10 Raji cells were defective in caspases activation as indicated 52 Figure 11 Quantification of CD95 and Bcl-2 expression in Raji and Jurkat cells 53 Figure 12 BL cells were resistant to etoposide 54 Figure 13 Defective caspases activation in BL cells 56 Figure 14 Etoposide induced mitochondria changes in Raji cells 58 Figure 15 Cytochrome c release upon triggering apoptosis in Raji cells 59 Figure 16 Exogenous cytochrome c failed to induce caspase-3 activation in S100 cytosols from BL cells 61 Figure 17 Wide type caspase-3 could be cleaved independent of apoptosome in BL cell lines 62 Figure 18 Subthreshold level of cytosolic Apaf-1 in BL cell lines 63 Figure 19 Apoptosome formation in Jurkat cells 65 Figure 20 Defective apoptosome formation in Raji cells 66 Figure 21 Stable overexpression of Apaf-1 in Raji B cells 68 viii Figure 22 Overexpression Apaf-1 sensitized Raji cells to etoposide and staurosporine 69 Figure 23 Overexpression Apaf-1 sensitize S100 cytosol of Raji cells to dATP and cytochrome c apoptosis 70 Figure 24 Apaf-1 cytosolic distribution in Raji cells by immunofluorescent staining 72 Figure 25 Plasma membrane sequestration of Apaf-1 in BL cells 73 Figure 26 Methyl-beta-cyclodextran (MCD) or cytochalasin B (CB) mobilize Apaf-1 from plasma membrane to cytosol in Raji cells 75 Figure 27 Defective Apaf-1 redistribution by MCD in Jurkat cells 76 Figure 28 Mobilization of plasma membrane associated Apaf-1 sensitized Raji cells S100 cytosol to apoptosis 77 Figure 29 MCD sensitized BL cells to etoposide induced apoptosis 79 Figure 30 IAPs especially cIAP2 highly expressed in Raji BL cells 81 Figure 31 Immunodepletion of cIAP2 could not sensitize Raji S100 cytosol to cytochrome c and dATP induced caspase activation 82 Figure 32 Immunodepletion of cIAP2 sensitized S100 cytosol to dATP and cytochrome c in RajiApaf-1 cells 83 Figure 33 Immunodepletion of cIAP2 sensitized Raji S100 cytosol to dATP and cytochrome c in RajiApaf-1 cells 84 Figure 34 Defective Smac release in Raji cells upon staurosporine treatment 86 Figure 35 Exogenous Smac N7 peptide sensitized cell death was Apaf-1 dependent 87 Figure 36 Bcl-2 and Hsp27 expression in BL cells 90 Figure 37 IAPs were highly expressed in BL cell lines 91 Figure 38 Transient transfection of antisense Hsp27 silenced Hsp27 expression to 50% in DG75 cells 94 Figure 39 BL derived cell lines showed different sensitivities to staurosporine and lactacystin induced cell death 95 Figure 40 Cytochrome c and Smac release were cell type dependent in BL derived cell lines upon 1uM staurosporine treatment 96 Figure 41 Cytochrome c release was not caspases dependent, however Smac release was caspases dependent in MutuI and Ramos cells 97 Figure 42 Lactacystin and staurosporine induced caspases independent cell death in Ramos 98 ix REFERENCES Acehan, D., Jiang, X., Morgan, D G., Heuser, J E., Wang, X., and Akey, C W (2002) Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation Mol Cell 9, 423-432 Altieri, D C (2003) Survivin, versatile modulation of cell division and apoptosis in cancer Oncogene 22, 8581-8589 Arnoult, D., Gaume, B., Karbowski, M., Sharpe, J C., Cecconi, F., and Youle, R J (2003) Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization Embo J 22, 4385-4399 Arnt, C R., Chiorean, M V., Heldebrant, M P., Gores, G J., and Kaufmann, S H (2002) Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP1 in situ J Biol Chem 277, 44236-44243 Askew, D S., Ashmun, R A., Simmons, B C., and Cleveland, J L (1991) Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis Oncogene 6, 1915-1922 Bairey, O., Zimra, Y., Shaklai, M., Okon, E., and Rabizadeh, E (1999) Bcl-2, Bcl-X, Bax, and Bak expression in short- and long-lived patients with diffuse large B-cell lymphomas Clin Cancer Res 5, 2860-2866 Bartke, T., Pohl, C., Pyrowolakis, G., and Jentsch, S (2004) Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase Mol Cell 14, 801-811 Beere, H M., and Green, D R (2001) Stress management - heat shock protein-70 and the regulation of apoptosis Trends Cell Biol 11, 6-10 Beere, H M., Wolf, B B., Cain, K., Mosser, D D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R I., Cohen, G M., and Green, D R (2000) Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome Nat Cell Biol 2, 469-475 Benedict, M A., Hu, Y., Inohara, N., and Nunez, G (2000) Expression and functional analysis of Apaf-1 isoforms Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9 J Biol Chem 275, 8461-8468 Blum, K A., Lozanski, G., and Byrd, J C (2004) Adult Burkitt leukemia and lymphoma Blood 104, 3009-3020 119 Boatright, K M., Renatus, M., Scott, F L., Sperandio, S., Shin, H., Pedersen, I M., Ricci, J E., Edris, W A., Sutherlin, D P., Green, D R., and Salvesen, G S (2003) A unified model for apical caspase activation Mol Cell 11, 529-541 Brieger, A., Boehrer, S., Schaaf, S., Nowak, D., Ruthardt, M., Kim, S Z., Atadja, P., Hoelzer, D., Mitrou, P S., Weidmann, E., and Chow, K U (2004) In bcr-ablpositive myeloid cells resistant to conventional chemotherapeutic agents, expression of Par-4 increases sensitivity to imatinib (STI571) and histone deacetylase-inhibitors Biochem Pharmacol 68, 85-93 Bruey, J M., Ducasse, C., Bonniaud, P., Ravagnan, L., Susin, S A., Diaz-Latoud, C., Gurbuxani, S., Arrigo, A P., Kroemer, G., Solary, E., and Garrido, C (2000) Hsp27 negatively regulates cell death by interacting with cytochrome c Nat Cell Biol 2, 645652 Burgess, D H., Svensson, M., Dandrea, T., Gronlund, K., Hammarquist, F., Orrenius, S., and Cotgreave, I A (1999) Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1 Cell Death Differ 6, 256-261 Cain, K., Bratton, S B., Langlais, C., Walker, G., Brown, D G., Sun, X M., and Cohen, G M (2000) Apaf-1 oligomerizes into biologically active approximately 700-kDa and inactive approximately 1.4-MDa apoptosome complexes J Biol Chem 275, 6067-6070 Cain, K., Brown, D G., Langlais, C., and Cohen, G M (1999) Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspaseactivating complex J Biol Chem 274, 22686-22692 Cain, K., Langlais, C., Sun, X M., Brown, D G., and Cohen, G M (2001) Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome J Biol Chem 276, 41985-41990 Cande, C., Cecconi, F., Dessen, P., and Kroemer, G (2002) Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? J Cell Sci 115, 4727-4734 Cao, G., Xiao, M., Sun, F., Xiao, X., Pei, W., Li, J., Graham, S H., Simon, R P., and Chen, J (2004) Cloning of a novel Apaf-1-interacting protein: a potent suppressor of apoptosis and ischemic neuronal cell death J Neurosci 24, 6189-6201 Chauhan, D., Li, G., Hideshima, T., Podar, K., Mitsiades, C., Mitsiades, N., Catley, L., Tai, Y T., Hayashi, T., Shringarpure, R., et al (2003) Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance Blood 102, 3379-3386 120 Cheson, B D (2004) What is new in lymphoma? CA Cancer J Clin 54, 260-272 Chipuk, J E., and Green, D R (2005) Do inducers of apoptosis trigger caspaseindependent cell death? Nat Rev Mol Cell Biol 6, 268-275 Concannon, C G., Orrenius, S., and Samali, A (2001) Hsp27 inhibits cytochrome cmediated caspase activation by sequestering both pro-caspase-3 and cytochrome c Gene Expr 9, 195-201 Conradt, B., and Horvitz, H R (1998) The C elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9 Cell 93, 519529 Danial, N N., and Korsmeyer, S J (2004) Cell death: critical control points Cell 116, 205-219 Dierlamm, J., Baens, M., Wlodarska, I., Stefanova-Ouzounova, M., Hernandez, J M., Hossfeld, D K., De Wolf-Peeters, C., Hagemeijer, A., Van den Berghe, H., and Marynen, P (1999) The apoptosis inhibitor gene API2 and a novel 18q gene, MLT, are recurrently rearranged in the t(11;18)(q21;q21)p6ssociated with mucosaassociated lymphoid tissue lymphomas Blood 93, 3601-3609 Du, C., Fang, M., Li, Y., Li, L., and Wang, X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition Cell 102, 33-42 Fadeel, B., Orrenius, S., and Pervaiz, S (2004) Buried alive: a novel approach to cancer treatment Faseb J 18, 1-4 Fanidi, A., Harrington, E A., and Evan, G I (1992) Cooperative interaction between c-myc and bcl-2 proto-oncogenes Nature 359, 554-556 Farrell, P J., Allan, G J., Shanahan, F., Vousden, K H., and Crook, T (1991) p53 is frequently mutated in Burkitt's lymphoma cell lines Embo J 10, 2879-2887 Fong, W G., Liston, P., Rajcan-Separovic, E., St Jean, M., Craig, C., and Korneluk, R G (2000) Expression and genetic analysis of XIAP-associated factor (XAF1) in cancer cell lines Genomics 70, 113-122 Fu, W N., Bertoni, F., Kelsey, S M., McElwaine, S M., Cotter, F E., Newland, A C., and Jia, L (2003) Role of DNA methylation in the suppression of Apaf-1 protein in human leukaemia Oncogene 22, 451-455 Fu, W N., Kelsey, S M., Newland, A C., and Jia, L (2001) Apaf-1XL is an inactive isoform compared with Apaf-1L Biochem Biophys Res Commun 282, 268-272 121 Fulda, S., Wick, W., Weller, M., and Debatin, K M (2002) Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo Nat Med 8, 808-815 Gaidano, G., Ballerini, P., Gong, J Z., Inghirami, G., Neri, A., Newcomb, E W., Magrath, I T., Knowles, D M., and Dalla-Favera, R (1991) p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia Proc Natl Acad Sci U S A 88, 5413-5417 Green, D R., and Kroemer, G (2004) The pathophysiology of mitochondrial cell death Science 305, 626-629 Gross, A., Yin, X M., Wang, K., Wei, M C., Jockel, J., Milliman, C., ErdjumentBromage, H., Tempst, P., and Korsmeyer, S J (1999) Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death J Biol Chem 274, 1156-1163 Guo, F., Nimmanapalli, R., Paranawithana, S., Wittman, S., Griffin, D., Bali, P., O'Bryan, E., Fumero, C., Wang, H G., and Bhalla, K (2002) Ectopic overexpression of second mitochondria-derived activator of caspases (Smac/DIABLO) or cotreatment with N-terminus of Smac/DIABLO peptide potentiates epothilone B derivative-(BMS 247550) and Apo-2L/TRAIL-induced apoptosis Blood 99, 3419-3426 Hanahan, D., and Weinberg, R A (2000) The hallmarks of cancer Cell 100, 57-70 Hao, Y., Sekine, K., Kawabata, A., Nakamura, H., Ishioka, T., Ohata, H., Katayama, R., Hashimoto, C., Zhang, X., Noda, T., et al (2004) Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function Nat Cell Biol 6, 849-860 Harlin, H., Reffey, S B., Duckett, C S., Lindsten, T., and Thompson, C B (2001) Characterization of XIAP-deficient mice Mol Cell Biol 21, 3604-3608 Hausmann, G., O'Reilly, L A., van Driel, R., Beaumont, J G., Strasser, A., Adams, J M., and Huang, D C (2000) Pro-apoptotic apoptosis protease-activating factor (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L) J Cell Biol 149, 623-634 Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E., and Rickinson, A (1991) Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein protects infected B cells from programmed cell death Cell 65, 1107-1115 Hengartner, M O (2000) The biochemistry of apoptosis Nature 407, 770-776 Higuchi, M., Izumi, K M., and Kieff, E (2001) Epstein-Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: protein binds to the 122 cytoskeleton through TNF receptor cytoplasmic factors Proc Natl Acad Sci U S A 98, 4675-4680 Hill, M M., Adrain, C., Duriez, P J., Creagh, E M., and Martin, S J (2004) Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes Embo J 23, 2134-2145 Horvitz, H R (1999) Genetic control of programmed cell death in the nematode Caenorhabditis elegans Cancer Res 59, 1701s-1706s Hu, S., Vincenz, C., Ni, J., Gentz, R., and Dixit, V M (1997) I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis J Biol Chem 272, 17255-17257 Hu, S., and Yang, X (2003) Cellular inhibitor of apoptosis and are ubiquitin ligases for the apoptosis inducer Smac/DIABLO J Biol Chem 278, 10055-10060 Hu, Y., Benedict, M A., Ding, L., and Nunez, G (1999) Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis Embo J 18, 3586-3595 Hu, Y., Benedict, M A., Wu, D., Inohara, N., and Nunez, G (1998) Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation Proc Natl Acad Sci U S A 95, 4386-4391 Imoto, I., Yang, Z Q., Pimkhaokham, A., Tsuda, H., Shimada, Y., Imamura, M., Ohki, M., and Inazawa, J (2001) Identification of cIAP1 as a candidate target gene within an amplicon at 11q22 in esophageal squamous cell carcinomas Cancer Res 61, 66296634 Ishitsuka, K., Hideshima, T., Hamasaki, M., Raje, N., Kumar, S., Hideshima, H., Shiraishi, N., Yasui, H., Roccaro, A M., Richardson, P., et al (2005) Honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and independent apoptosis Blood Jacobs, T W., Prioleau, J E., Stillman, I E., and Schnitt, S J (1996) Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer J Natl Cancer Inst 88, 1054-1059 Jacobson, M D., Weil, M., and Raff, M C (1997) Programmed cell death in animal development Cell 88, 347-354 Jain, M., Arvanitis, C., Chu, K., Dewey, W., Leonhardt, E., Trinh, M., Sundberg, C D., Bishop, J M., and Felsher, D W (2002) Sustained loss of a neoplastic phenotype by brief inactivation of MYC Science 297, 102-104 123 Jia, L., Patwari, Y., Kelsey, S M., Srinivasula, S M., Agrawal, S G., Alnemri, E S., and Newland, A C (2003) Role of Smac in human leukaemic cell apoptosis and proliferation Oncogene 22, 1589-1599 Jia, L., Srinivasula, S M., Liu, F T., Newland, A C., Fernandes-Alnemri, T., Alnemri, E S., and Kelsey, S M (2001) Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells Blood 98, 414-421 Jiang, X., Kim, H E., Shu, H., Zhao, Y., Zhang, H., Kofron, J., Donnelly, J., Burns, D., Ng, S C., Rosenberg, S., and Wang, X (2003) Distinctive roles of PHAP proteins and prothymosin-alpha in a death regulatory pathway Science 299, 223-226 Johansson, A C., Steen, H., Ollinger, K., and Roberg, K (2003) Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine Cell Death Differ 10, 1253-1259 Kagan, V E., Gleiss, B., Tyurina, Y Y., Tyurin, V A., Elenstrom-Magnusson, C., Liu, S X., Serinkan, F B., Arroyo, A., Chandra, J., Orrenius, S., and Fadeel, B (2002) A role for oxidative stress in apoptosis: oxidation and externalization of phosphatidylserine is required for macrophage clearance of cells undergoing Fasmediated apoptosis J Immunol 169, 487-499 Karpova, M B., Schoumans, J., Ernberg, I., Henter, J I., Nordenskjold, M., and Fadeel, B (2005) Raji revisited: cytogenetics of the original Burkitt's lymphoma cell line Leukemia 19, 159-161 Kashkar, H., Haefs, C., Shin, H., Hamilton-Dutoit, S J., Salvesen, G S., Kronke, M., and Jurgensmeier, J M (2003) XIAP-mediated caspase inhibition in Hodgkin's lymphoma-derived B cells J Exp Med 198, 341-347 Kelliher, M A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B Z., and Leder, P (1998) The death domain kinase RIP mediates the TNF-induced NF-kappaB signal Immunity 8, 297-303 Kerr, J F., Wyllie, A H., and Currie, A R (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics Br J Cancer 26, 239257 Kischkel, F C., Lawrence, D A., Tinel, A., LeBlanc, H., Virmani, A., Schow, P., Gazdar, A., Blenis, J., Arnott, D., and Ashkenazi, A (2001) Death receptor recruitment of endogenous caspase-10 and apoptosis initiation in the absence of caspase-8 J Biol Chem 276, 46639-46646 Kluck, R M., Bossy-Wetzel, E., Green, D R., and Newmeyer, D D (1997a) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis Science 275, 1132-1136 124 Kluck, R M., Martin, S J., Hoffman, B M., Zhou, J S., Green, D R., and Newmeyer, D D (1997b) Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system Embo J 16, 4639-4649 Krammer, P H (2000) CD95's deadly mission in the immune system Nature 407, 789-795 Kurland, J F., Kodym, R., Story, M D., Spurgers, K B., McDonnell, T J., and Meyn, R E (2001) NF-kappaB1 (p50) homodimers contribute to transcription of the bcl-2 oncogene J Biol Chem 276, 45380-45386 Lee, H H., Dempsey, P W., Parks, T P., Zhu, X., Baltimore, D., and Cheng, G (1999) Specificities of CD40 signaling: involvement of TRAF2 in CD40-induced NF-kappaB activation and intercellular adhesion molecule-1 up-regulation Proc Natl Acad Sci U S A 96, 1421-1426 Li, F., Srinivasan, A., Wang, Y., Armstrong, R C., Tomaselli, K J., and Fritz, L C (1997a) Cell-specific induction of apoptosis by microinjection of cytochrome c BclxL has activity independent of cytochrome c release J Biol Chem 272, 30299-30305 Li, L., Thomas, R M., Suzuki, H., De Brabander, J K., Wang, X., and Harran, P G (2004) A small molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell death Science 305, 1471-1474 Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S M., Ahmad, M., Alnemri, E S., and Wang, X (1997b) Cytochrome c and dATP-dependent formation of Apaf1/caspase-9 complex initiates an apoptotic protease cascade Cell 91, 479-489 Liston, P., Fong, W G., Kelly, N L., Toji, S., Miyazaki, T., Conte, D., Tamai, K., Craig, C G., McBurney, M W., and Korneluk, R G (2001) Identification of XAF1 as an antagonist of XIAP anti-Caspase activity Nat Cell Biol 3, 128-133 Liston, P., Young, S S., Mackenzie, A E., and Korneluk, R G (1997) Life and death decisions: the role of the IAPs in modulating programmed cell death Apoptosis 2, 423-441 Liu, J R., Opipari, A W., Tan, L., Jiang, Y., Zhang, Y., Tang, H., and Nunez, G (2002) Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance Cancer Res 62, 924-931 Liu, X., Kim, C N., Yang, J., Jemmerson, R., and Wang, X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c Cell 86, 147-157 125 MacFarlane, M., Merrison, W., Bratton, S B., and Cohen, G M (2002) Proteasomemediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro J Biol Chem 277, 36611-36616 MacKenzie, A., and LaCasse, E (2000) Inhibition of IAP's protection by Diablo/Smac: new therapeutic opportunities? Cell Death Differ 7, 866-867 Madesh, M., Antonsson, B., Srinivasula, S M., Alnemri, E S., and Hajnoczky, G (2002) Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization J Biol Chem 277, 5651-5659 Magdalena, C., Dominguez, F., Loidi, L., and Puente, J L (2000) Tumour prothymosin alpha content, a potential prognostic marker for primary breast cancer Br J Cancer 82, 584-590 Marcucci, G., Byrd, J C., Dai, G., Klisovic, M I., Kourlas, P J., Young, D C., Cataland, S R., Fisher, D B., Lucas, D., Chan, K K., et al (2003) Phase and pharmacodynamic studies of G3139, a Bcl-2 antisense oligonucleotide, in combination with chemotherapy in refractory or relapsed acute leukemia Blood 101, 425-432 Martin, S J (2002) Destabilizing influences in apoptosis: sowing the seeds of IAP destruction Cell 109, 793-796 Martinon, F., Burns, K., and Tschopp, J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta Mol Cell 10, 417-426 Medema, J P., Scaffidi, C., Kischkel, F C., Shevchenko, A., Mann, M., Krammer, P H., and Peter, M E (1997) FLICE is activated by association with the CD95 deathinducing signaling complex (DISC) Embo J 16, 2794-2804 Micheau O., and Tschopp J (2003) Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes Cell 114, 181-190 Miyashita, T., Krajewski, S., Krajewska, M., Wang, H G., Lin, H K., Liebermann, D A., Hoffman, B., and Reed, J C (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo Oncogene 9, 1799-1805 Moriishi, K., Huang, D C., Cory, S., and Adams, J M (1999) Bcl-2 family members not inhibit apoptosis by binding the caspase activator Apaf-1 Proc Natl Acad Sci U S A 96, 9683-9688 Moroni, M C., Hickman, E S., Denchi, E L., Caprara, G., Colli, E., Cecconi, F., Muller, H., and Helin, K (2001) Apaf-1 is a transcriptional target for E2F and p53 Nat Cell Biol 3, 552-558 126 Munro, S (2003) Lipid rafts: elusive or illusive? Cell 115, 377-388 Nakano, K., and Vousden, K H (2001) PUMA, a novel proapoptotic gene, is induced by p53 Mol Cell 7, 683-694 Ng, P W., Porter, A G., and Janicke, R U (1999) Molecular cloning and characterization of two novel pro-apoptotic isoforms of caspase-10 J Biol Chem 274, 10301-10308 Nicholson, D W., Ali, A., Thornberry, N A., Vaillancourt, J P., Ding, C K., Gallant, M., Gareau, Y., Griffin, P R., Labelle, M., Lazebnik, Y A., and et al (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis Nature 376, 37-43 Nicoletti, I., Migliorati, G., Pagliacci, M C., Grignani, F., and Riccardi, C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry J Immunol Methods 139, 271-279 Nowak, D., Boehrer, S., Brieger, A., Kim, S Z., Schaaf, S., Hoelzer, D., Mitrou, P S., Weidmann, E., and Chow, K U (2004) Upon drug-induced apoptosis in lymphoma cells X-linked inhibitor of apoptosis (XIAP) translocates from the cytosol to the nucleus Leuk Lymphoma 45, 1429-1436 Oda, E., Ohki, R., Murasawa, H., Nemoto, J., Shibue, T., Yamashita, T., Tokino, T., Taniguchi, T., and Tanaka, N (2000) Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis Science 288, 1053-1058 Okada, H., Suh, W K., Jin, J., Woo, M., Du, C., Elia, A., Duncan, G S., Wakeham, A., Itie, A., Lowe, S W., et al (2002) Generation and characterization of Smac/DIABLO-deficient mice Mol Cell Biol 22, 3509-3517 Oliferenko, S., Paiha, K., Harder, T., Gerke, V., Schwarzler, C., Schwarz, H., Beug, H., Gunthert, U., and Huber, L A (1999) Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton J Cell Biol 146, 843-854 Orre, R S., Cotter, M A., 2nd, Subramanian, C., and Robertson, E S (2001) Prothymosin alpha functions as a cellular oncoprotein by inducing transformation of rodent fibroblasts in vitro J Biol Chem 276, 1794-1799 Pandey, P., Saleh, A., Nakazawa, A., Kumar, S., Srinivasula, S M., Kumar, V., Weichselbaum, R., Nalin, C., Alnemri, E S., Kufe, D., and Kharbanda, S (2000) Negative regulation of cytochrome c-mediated oligomerization of Apaf-1 and activation of procaspase-9 by heat shock protein 90 Embo J 19, 4310-4322 127 Parat, M O., Stachowicz, R Z., and Fox, P L (2002) Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells Biochem J 361, 681-688 Pasquini, L A., Besio Moreno, M., Adamo, A M., Pasquini, J M., and Soto, E F (2000) Lactacystin, a specific inhibitor of the proteasome, induces apoptosis and activates caspase-3 in cultured cerebellar granule cells J Neurosci Res 59, 601-611 Paul, C., Manero, F., Gonin, S., Kretz-Remy, C., Virot, S., and Arrigo, A P (2002) Hsp27 as a negative regulator of cytochrome C release Mol Cell Biol 22, 816-834 Phan, R T., and Dalla-Favera, R (2004) The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells Nature 432, 635-639 Piva, R., Gianferretti, P., Ciucci, A., Taulli, R., Belardo, G., and Santoro, M G (2005) 15-Deoxy-delta 12,14-prostaglandin J2 induces apoptosis in human malignant B cells: an effect associated with inhibition of NF-kappa B activity and downregulation of antiapoptotic proteins Blood 105, 1750-1758 Pozo-Guisado, E., Merino, J M., Mulero-Navarro, S., Lorenzo-Benayas, M J., Centeno, F., Alvarez-Barrientos, A., and Salguero, P M (2005) Resveratrol-induced apoptosis in MCF-7 human breast cancer cells involves a caspase-independent mechanism with downregulation of Bcl-2 and NF-kappaB Int J Cancer 115, 74-84 Rajkumar, S V., Richardson, P G., Hideshima, T., and Anderson, K C (2005) Proteasome inhibition as a novel therapeutic target in human cancer J Clin Oncol 23, 630-639 Ravagnan, L., Gurbuxani, S., Susin, S A., Maisse, C., Daugas, E., Zamzami, N., Mak, T., Jaattela, M., Penninger, J M., Garrido, C., and Kroemer, G (2001) Heat-shock protein 70 antagonizes apoptosis-inducing factor Nat Cell Biol 3, 839-843 Reed, J C (2003) Apoptosis-targeted therapies for cancer Cancer Cell 3, 17-22 Rehm, M., Dussmann, H., and Prehn, J H (2003) Real-time single cell analysis of Smac/DIABLO release during apoptosis J Cell Biol 162, 1031-1043 Renatus, M., Stennicke, H R., Scott, F L., Liddington, R C., and Salvesen, G S (2001) Dimer formation drives the activation of the cell death protease caspase Proc Natl Acad Sci U S A 98, 14250-14255 Riedl, S J., Li, W., Chao, Y., Schwarzenbacher, R., and Shi, Y (2005) Structure of the apoptotic protease-activating factor bound to ADP Nature 434, 926-933 128 Robertson, J D., Gogvadze, V., Zhivotovsky, B., and Orrenius, S (2000) Distinct pathways for stimulation of cytochrome c release by etoposide J Biol Chem 275, 32438-32443 Rodriguez, J., and Lazebnik, Y (1999) Caspase-9 and APAF-1 form an active holoenzyme Genes Dev 13, 3179-3184 Ruf, I K., Rhyne, P W., Yang, H., Borza, C M., Hutt-Fletcher, L M., Cleveland, J L., and Sample, J T (1999) Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma Mol Cell Biol 19, 1651-1660 Ruiz-Vela, A., Albar, J P., and Martinez, C A (2001) Apaf-1 localization is modulated indirectly by Bcl-2 expression FEBS Lett 501, 79-83 Ruiz-Vela, A., Gonzalez de Buitrago, G., and Martinez, A C (2002) Nuclear Apaf-1 and cytochrome c redistribution following stress-induced apoptosis FEBS Lett 517, 133-138 Saleh, A., Srinivasula, S M., Balkir, L., Robbins, P D., and Alnemri, E S (2000) Negative regulation of the Apaf-1 apoptosome by Hsp70 Nat Cell Biol 2, 476-483 Salvesen, G S., and Dixit, V M (1999) Caspase activation: the induced-proximity model Proc Natl Acad Sci U S A 96, 10964-10967 Salvesen, G S., and Duckett, C S (2002) IAP proteins: blocking the road to death's door Nat Rev Mol Cell Biol 3, 401-410 Samali, A., Cai, J., Zhivotovsky, B., Jones, D P., and Orrenius, S (1999) Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells Embo J 18, 2040-2048 Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K J., Debatin, K M., Krammer, P H., and Peter, M E (1998) Two CD95 (APO-1/Fas) signaling pathways Embo J 17, 1675-1687 Schattner, E J (2002) Apoptosis in lymphocytic leukemias and lymphomas Cancer Invest 20, 737-748 Schimmer, A D., Welsh, K., Pinilla, C., Wang, Z., Krajewska, M., Bonneau, M J., Pedersen, I M., Kitada, S., Scott, F L., Bailly-Maitre, B., et al (2004) Smallmolecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity Cancer Cell 5, 25-35 Schmitt, C A., and Lowe, S W (1999) Apoptosis and therapy J Pathol 187, 127137 129 Schmitt, C A., and Lowe, S W (2002) Apoptosis and chemoresistance in transgenic cancer models J Mol Med 80, 137-146 Seiffert, B M., Vier, J., and Hacker, G (2002) Subcellular localization, oligomerization, and ATP-binding of Caenorhabditis elegans CED-4 Biochem Biophys Res Commun 290, 359-365 Sekine, K., Hao, Y., Suzuki, Y., Takahashi, R., Tsuruo, T., and Naito, M (2005) HtrA2 cleaves Apollon and induces cell death by IAP-binding motif in Apollondeficient cells Biochem Biophys Res Commun 330, 279-285 Selvakumaran, M., Lin, H K., Miyashita, T., Wang, H G., Krajewski, S., Reed, J C., Hoffman, B., and Liebermann, D (1994) Immediate early up-regulation of bax expression by p53 but not TGF beta 1: a paradigm for distinct apoptotic pathways Oncogene 9, 1791-1798 Simons, K., and Toomre, D (2000) Lipid rafts and signal transduction Nat Rev Mol Cell Biol 1, 31-39 Smith, T F., Gaitatzes, C., Saxena, K., and Neer, E J (1999) The WD repeat: a common architecture for diverse functions Trends Biochem Sci 24, 181-185 Soengas, M S., Alarcon, R M., Yoshida, H., Giaccia, A J., Hakem, R., Mak, T W., and Lowe, S W (1999) Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition Science 284, 156-159 Soengas, M S., Capodieci, P., Polsky, D., Mora, J., Esteller, M., Opitz-Araya, X., McCombie, R., Herman, J G., Gerald, W L., Lazebnik, Y A., et al (2001) Inactivation of the apoptosis effector Apaf-1 in malignant melanoma Nature 409, 207-211 Soengas, M S., and Lowe, S W (2003) Apoptosis and melanoma chemoresistance Oncogene 22, 3138-3151 Sprick, M R., Rieser, E., Stahl, H., Grosse-Wilde, A., Weigand, M A., and Walczak, H (2002) Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8 Embo J 21, 4520-4530 Srinivasula, S M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E S (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization Mol Cell 1, 949957 Srinivasula, S M., Datta, P., Fan, X J., Fernandes-Alnemri, T., Huang, Z., and Alnemri, E S (2000) Molecular determinants of the caspase-promoting activity of 130 Smac/DIABLO and its role in the death receptor pathway J Biol Chem 275, 3615236157 Stennicke, H R., Jurgensmeier, J M., Shin, H., Deveraux, Q., Wolf, B B., Yang, X., Zhou, Q., Ellerby, H M., Ellerby, L M., Bredesen, D., et al (1998) Pro-caspase-3 is a major physiologic target of caspase-8 J Biol Chem 273, 27084-27090 Strasser, A., Harris, A W., Bath, M L., and Cory, S (1990) Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2 Nature 348, 331-333 Strasser, A., Harris, A W., Huang, D C., Krammer, P H., and Cory, S (1995) Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis Embo J 14, 61366147 Sun, C., Cai, M., Gunasekera, A H., Meadows, R P., Wang, H., Chen, J., Zhang, H., Wu, W., Xu, N., Ng, S C., and Fesik, S W (1999) NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP Nature 401, 818-822 Sun, X M., Butterworth, M., MacFarlane, M., Dubiel, W., Ciechanover, A., and Cohen, G M (2004) Caspase activation inhibits proteasome function during apoptosis Mol Cell 14, 81-93 Susin, S A., Lorenzo, H K., Zamzami, N., Marzo, I., Snow, B E., Brothers, G M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., et al (1999) Molecular characterization of mitochondrial apoptosis-inducing factor Nature 397, 441-446 Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., and Takahashi, R (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death Mol Cell 8, 613-621 Takasawa, R., and Tanuma, S (2003) Sustained release of Smac/DIABLO from mitochondria commits to undergo UVB-induced apoptosis Apoptosis 8, 291-299 Tamm, I., Kornblau, S M., Segall, H., Krajewski, S., Welsh, K., Kitada, S., Scudiero, D A., Tudor, G., Qui, Y H., Monks, A., et al (2000) Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias Clin Cancer Res 6, 1796-1803 Thome, M., and Tschopp, J (2001) Regulation of lymphocyte proliferation and death by FLIP Nat Rev Immunol 1, 50-58 Tinel, A., and Tschopp, J (2004) The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress Science 304, 843-846 131 Verhagen, A M., Ekert, P G., Pakusch, M., Silke, J., Connolly, L M., Reid, G E., Moritz, R L., Simpson, R J., and Vaux, D L (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins Cell 102, 43-53 Vogelstein, B., and Kinzler, K W (2004) Cancer genes and the pathways they control Nat Med 10, 789-799 Vousden, K H., and Lu, X (2002) Live or let die: the cell's response to p53 Nat Rev Cancer 2, 594-604 Vucic, D., Stennicke, H R., Pisabarro, M T., Salvesen, G S., and Dixit, V M (2000) ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas Curr Biol 10, 1359-1366 Wang, J., Chun, H J., Wong, W., Spencer, D M., and Lenardo, M J (2001) Caspase-10 is an initiator caspase in death receptor signaling Proc Natl Acad Sci U S A 98, 13884-13888 Wang, J., Zheng, L., Lobito, A., Chan, F K., Dale, J., Sneller, M., Yao, X., Puck, J M., Straus, S E., and Lenardo, M J (1999) Inherited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II Cell 98, 47-58 Weis, M., Schlegel, J., Kass, G E., Holmstrom, T H., Peters, I., Eriksson, J., Orrenius, S., and Chow, S C (1995) Cellular events in Fas/APO-1-mediated apoptosis in JURKAT T lymphocytes Exp Cell Res 219, 699-708 Wilson, J B., Bell, J L., and Levine, A J (1996) Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice Embo J 15, 3117-3126 Wiman, K G., Magnusson, K P., Ramqvist, T., and Klein, G (1991) Mutant p53 detected in a majority of Burkitt lymphoma cell lines by monoclonal antibody PAb240 Oncogene 6, 1633-1639 Wolf, B B., Schuler, M., Li, W., Eggers-Sedlet, B., Lee, W., Tailor, P., Fitzgerald, P., Mills, G B., and Green, D R (2001) Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity J Biol Chem 276, 34244-34251 Wright, K M., Linhoff, M W., Potts, P R., and Deshmukh, M (2004) Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis J Cell Biol 167, 303-313 132 Yan, N., Gu, L., Kokel, D., Chai, J., Li, W., Han, A., Chen, L., Xue, D., and Shi, Y (2004) Structural, biochemical, and functional analyses of CED-9 recognition by the proapoptotic proteins EGL-1 and CED-4 Mol Cell 15, 999-1006 Yang, J., Liu, X., Bhalla, K., Kim, C N., Ibrado, A M., Cai, J., Peng, T I., Jones, D P., and Wang, X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked Science 275, 1129-1132 Yang, L., Mashima, T., Sato, S., Mochizuki, M., Sakamoto, H., Yamori, T., Oh-Hara, T., and Tsuruo, T (2003a) Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide Cancer Res 63, 831-837 Yang, Q H., Church-Hajduk, R., Ren, J., Newton, M L., and Du, C (2003b) Omi/HtrA2 catalytic cleavage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates caspase activity in apoptosis Genes Dev 17, 1487-1496 Yoshida, H., Kong, Y Y., Yoshida, R., Elia, A J., Hakem, A., Hakem, R., Penninger, J M., and Mak, T W (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development Cell 94, 739-750 Youn, H J., Kim, H S., Jeon, M H., Lee, J H., Seo, Y J., Lee, Y J., and Lee, J H (2005) Induction of caspase-independent apoptosis in H9c2 cardiomyocytes by adriamycin treatment Mol Cell Biochem 270, 13-19 Zhang, X., Uthaisang, W., Hu, L., Ernberg, I T., and Fadeel, B (2005) Epstein-Barr virus-encoded latent membrane protein promotes stress-induced apoptosis upstream of caspase-2-dependent mitochondrial perturbation Int J Cancer 113, 397-405 Zhang, X D., Gillespie, S K., and Hersey, P (2004) Staurosporine induces apoptosis of melanoma by both caspase-dependent and -independent apoptotic pathways Mol Cancer Ther 3, 187-197 Zou, H., Henzel, W J., Liu, X., Lutschg, A., and Wang, X (1997) Apaf-1, a human protein homologous to C elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 Cell 90, 405-413 Zou, H., Yang, R., Hao, J., Wang, J., Sun, C., Fesik, S W., Wu, J C., Tomaselli, K J., and Armstrong, R C (2003) Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP J Biol Chem 278, 8091-8098 133 ... repeating-containing EPR-1/BIRC5 effector cell protease receptor-1/ baculoviral IAP repeating-containing BRUCE/BIRC6 BIR repeat-containing ubiquitin-conjugating enzyme/ baculoviral IAP repeating-containing (also... TABLES Table EBV status in BL derived cell lines used in the study 28 Table P53 status in BL derived cell lines used in the study 30 vii LIST OF FIGURES INTRODUCTION Figure Apoptotic signaling... BCL2-antagonist/killer Bcl-2-associated X protein B- cell lymphoma protein Bcl-2 homology BH3 interacting domain death agonist truncated Bid baculoviral-IAP-repeat Burkitt’s lymphoma bromphenol blue caspase