Apoptosis 花瓣樹葉的凋零飄落 'Apoptosis' (pronounced as əpo΄tosis ) is of Greek origin, having the meaning "falling off or dropping off", in analogy to leaves falling from trees or petals from flowers, sometimes also called as programmed cell death. 東豐路, 台南 BC Yang

The term 'apoptosis' describes the molecular and morphological processes leading to controlled cellular self-destruction and was first introduced in a publication by Kerr, Wyllie and Currie. Br. J. Cancer, 1972, 26: 239 BC Yang

Two types of cell death Apoptosis Necrosis BC Yang

Apoptosis vs. necrosis BC Yang

Detection of apoptotic cells Hoechest stain DNA ladder Comet assay TUNEL stain; immunohistochmical (terminal transferase) FACS analysis (Annexin V, MC540, propidium iodine for subG0, TUNEL) BC Yang

Identification of death receptors TNF receptor (1985) Kull FC Jr.  Jacobs S.  Cuatrecasas P. (1985) Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity. PNAS 82:5756-5760.  Fas (1989) S Nagata group (1991):   Itoh N.  et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. :233-43, P Krammer group (1989): Trauth BC.  et al.  Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science.  245(4915):301-305. BC Yang

Shigekazu Nagata (2002) Lab member: total 21 Cell Death and Diff. Genes to Cell Apoptosis Int. Immunol. Immunity Exp. Cell Res. Biochem. Biophys. Acta IUBMB/Life Science Int. J. Cancer Cancer Cell Japan Academy Prize, Imperial Prize (Japan) Person of Cultural Merits (Japan)

Peter H. Krammer, 2002 Lab member: total 34

Typically, the cytoplasm begins to shrink following the cleavage of lamins and actin filaments (A). Nuclear condensation, following the breakdown of chromatin and nuclear structural proteins, the nuclei of apoptotic cells may take on a "horse-shoe" like appearance (B). Cells continue to shrink (C). These phagocytic cells are responsible for removing apoptotic cells from tissues in a clean and tidy fashion that avoids many of the problems associated with necrotic cell death. Membrane changes can often be observed morphologically through the appearance of membrane blebs (D) or blisters which often appear towards the end of the apoptotic process. Small vesicles called apoptotic bodies are also sometimes observed (D). BC Yang

The growing subfamily of death receptors is part of the TNF/NGF-receptor superfamily. This superfamily is characterized by a sequence of 2 to 5 cysteine-rich extracellular repeats. The death receptors contain an intracellular death domain (DD), which is essential for transduction of the apoptotic signal. Six members of this subfamily are known so far, namely TNF-R1 (CD120a), CD95 (APO-1/Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2), and DR6.  BC Yang

TNFa is representative of a still growing family of trimeric surface proteins, such as lymphotoxin-a (LT-a), Fas ligand (FasL), receptor-activator of NF-kB ligand (RANKL), CD40 ligand (CD40L), and TNFa-related apoptosis-inducing ligand (TRAIL). Exposure of cells to TNF can result in activation of a caspase cascade leading to apoptosis. However, more commonly, the binding of TNFa to its receptors causes activation of two major transcription factors, AP-1 and NF-kappaB, that in turn induces genes involved in chronic and acute inflammatory responses. The binding of TNFa to TNFR1 activates TNFR1-associated death domain protein (TRADD), which interacts with Fas-associated death domain protein (FADD) to induce apoptosis; the binding of TNFa to TNFR1 can also produce a quite opposite affect if TRADD interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) to activate NF-kappaB, resulting in gene expression and cell survival. TNFR2 triggering can lead to NF-kB activation, but does not result in cell death. Importantly, the suppression of apoptosis, which is mostly dependent on NF-kB, augments the inflammatory response to TNFa. BC Yang

Tumor necrosis factor (TNF) family members play important roles in various physiological and pathological processes, including cell proliferation, differentiation, apoptosis, modulation of immune responses and induction of inflammation. Binding of TNF alpha to TNFR1 results in receptor trimerisation and clustering of intracellular death domains. This allows binding of an intracellular adapter molecule called TRADD (TNFR-associated death domain) via interactions between death domains. TRADD has the ability to recruit a number of different proteins to the activated receptor. Recruitment of TRAF2 (TNF-associated factor 2) leads to activation of NF-kB and the JNK/Ap-1 pathway. http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html BC Yang

TRADD can also associate with FADD, which leads to the induction of apoptosis via the recruitment and cleavage of pro-caspase 8. TNFR1 is also able to mediate apoptosis through the recruitment of an adapter molecule called RAIDD (RIP-associated ICH-1 / CED-3 homologous protein with a death domain). RAIDD associates with RIP through interactions between death domains and can recruit caspase 2 through an interaction with a motif, similar to the death effector domain, known as CARD (caspase recruitment domain). Recruitment of caspase 2 leads to induction of apoptosis. http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html BC Yang

TNF acts through two receptors, TNFR1 & 2 TNF acts through two receptors, TNFR1 & 2. TNFR1 is expressed by all human tissues and is the major signaling receptor for TNFa. TNFR2 is mostly expressed in immune cells and mediates limited biological responses. TNFR2 binds both TNFa and TNFb. http://www.biotechjournal.com/Journal/jan%202002/janArticle6text.htm BC Yang

The ligand for CD95 (CD95L or FasL) is a trimer that on association with the receptor promotes receptor trimerisation that in turns results in intracellular clustering of parts of the receptor called death domains (DD). The death effector domain allows binding of pro-caspase 8 to the CD95-FADD complex. Pro-caspase 8 (also known as FLICE) associates with FADD though its own death effector domain, and upon recruitment by FADD is immediately cleaved to produce caspase 8. This then triggers activation of execution caspases such as caspase 9. The complex of proteins – CD95, FADD and pro-caspase 8 – that trigger apoptosis is also known as DISC or Death Inducing Signaling Complex. http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html BC Yang

Apoptosis Apaf-1 Caspase 3 Caspase 9 Death substrates Nature 407, 789 – 795(2000) Caspase 3 Apaf-1 Caspase 9 Death substrates Apoptosis BC Yang

擋在這裡!? Sometimes, cells do not die, still intact, low SubGo population Annexin V-positive Loss of the mitochondria membrane potential Caspase-3, -8, -9 activated Cell size moderately reduced But, not dead 擋在這裡!? Budd RJ, 2002, J Clin Invest 109:437-441 BC Yang

http://ben-may.bsd.uchicago.edu/bmi/faculty/peter.html BC Yang

Nature Reviews Immunology 2; 401-409 (2002); doi:10 Nature Reviews Immunology 2; 401-409 (2002); doi:10.1038/nri819 CYTOTOXIC T LYMPHOCYTES: ALL ROADS LEAD TO DEATH BC Yang

Activated T-cells stimulated via the CD3/T-cell receptor complex through the recognition of antigen presented by MHC molecules rapidly express Fas-L molecules. In different scenarios, Fas-L molecules cleaved from the T-cell surface by matrix metalloproteases, or Fas-L molecules still anchored in the cell membrane, can induce apoptosis either in the same cell (autocrine suicide), or in a neighbouring T-cell (fratricide), or in a Fas+ target cell (paracrine lysis). http://www.bioscience.org/1997/v2/d/kabelit1/htmls/61-77.htm BC Yang

Factors capable of inducing Fas-L expression in tumor cells An alternative way to control death signal Factors capable of inducing Fas-L expression in tumor cells Cellular stress Cytokiens:IFN-g, TNF-a, IL-8 Cytostatic drugs g irradition GnRH analog UV Oxidative stress Tumor growth inhibitor or promoter Viral products (HIV,Tat, SIV, Nef, Tax, HTLV-1) Ras (Lab Invest 2000) BC Yang

Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was ampliÆed in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL. Expression of DcR3 mRNA. Northern hybridization analysis was done using the DcR3 cDNA as a probe and blots of poly(A)+ RNA (Clontech) from human fetal and adult tissues or cancer cell lines. PBL, peripheral blood lymphocyte. Nature. 1998, 396:699-703.

Inhibition of FasL activity by DcR3 Inhibition of FasL activity by DcR3. a, Human Jurkat T leukaemia cells were incubated with Flag-tagged soluble FasL (sFasL; 5 ng ml-1) oligomerized with anti-Flag antibody (0.1   g ml-1) in the presence of the proposed inhibitors DcR3–Fc, Fas–Fc or human IgG1 and assayed for apoptosis (mean± s.e.m. of triplicates). Nature. 1998, 396:699-703.

Differential Fas signaling efficiency in cell lines. Jagan R Muppidi & Richard M Siegel (2004) Nature Immunology 5, 182 – 189. Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death Differential Fas signaling efficiency in cell lines. BC Yang

TCR restimulation induces redistribution of Fas into lipid rafts before signal complex formation. Nature Immunology 5, 182 – 189. BC Yang

The presence of Fas in lipid rafts controls signaling efficiency. Nature Immunology 5, 182 – 189. BC Yang

Intracellular signaling pathways downstream of tumor necrosis factor (TNF) receptor superfamily receptors. http://arthritis-research.com/content/4/Suppl+3/S243/figure/F1 BC Yang

Death domain structures http://chem.unl.edu/powers/TNFDD/figure/DD-structures.jpg BC Yang

Death domain: Stereoview of the backbone (N, Calpha, C') of 20 superimposed NMR-derived structures of the Fas death domain. Huang, B., Eberstadt, M., Olejniczak, E. T., Meadows, R. P., Fesik, S. W.: NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain. Nature 384 pp. 638 (1996) BC Yang

Apoptosis signaling is regulated and executed by specialized proteins that often carry protein/protein interaction domains. One of these domains is the death effector domain (DED) that is predominantly found in components of the death-inducing signaling complex, which forms at the members of the death receptor family following their ligation. Both proapoptotic- and antiapoptotic-DEDcontaining proteins have been identified, which makes these proteins exquisitely suited to the regulation of apoptosis. Aside from their pivotal role in the control of the apoptotic program, DED-containing proteins have recently been demonstrated to exert their influence on other cellular processes as well, including cell proliferation. BC Yang

The family of the DED-containing proteins True DED proteins are shown above the dividing line. Proteins containing a DED-like domain (or pseudo DED)are shown below the line. DED are shown as yellow boxes, DED-like domains as hatched yellow boxes. The X in c-FLIPL indicates the mutated caspase active site. NLS, nuclear localization signal; NES, nuclear export signal; LZ, leucine zipper; TM, transmembrane; PRR, proline-rich region; SAM, SAM domain. BC Yang

Evidence for active host defense against cancer 80 years of immunotherapy. (Currie GA, 1972, Br. J. Cancer 26: 141) A critique of the evidence for active host defense against cancer, based on personal studies of 27 murine tumors of spontaneous origin. (Hewitt HB, et al. 1976, Br. J. Cancer 33:241) BC Yang

A fight between immune cells and cancer BC Yang

Fas-L positive tumors with heavily infiltrated lymphocytes Nasal pharyngeal carcinoma Glioblastoma Hematoxylin/eosin staining CD3+ cells BC Yang

Resistance to killing Cytotoxic T cells Innat immunity Defective Fas pathway Resistance to Granzyme Innat immunity Fas-L and Neutrophil BC Yang

In vivo consequences of Fas-L expression by tumors (using over-expression system) Enhanced rejection Delayed rejection Renca, MH134, L5178Y, B16-BL6, CT26* CT26# B16-F10 Note: *:syngenic, nude, SCID #: allogenic, lpr Lejeune FJ et al., 1998, Curr. Op. Immunol. BC Yang

Fas-Lribozyme/EGFP plasmid Br J Cancer, (2001) 85:1185-1192. Hammerhead ribozyme. 54-mers. Inserted into the pEGFP-N1. Driven by CMV promoter. BC Yang

U-373MG cells transfected with Fas-Lribozyme/EGFP plasmid Light microscopy Fluorescent microscopy BC Yang

Down-regulation of Fas-L by Fas-Lribozyme U-373MG RT-PCR 1. U-373MG 2. EGFP-N1 3. EGFP/Fas-Lribozyme FACScan BC Yang

Fas-L on tumorigenesis of glioma in nude mice control Fas-Lribozyme 1.5-2 mm3 3-4 mm3 2 in 3 mice 7 in 3 mice ++ + +++ ++ + +(?) + ++ tumor size tumor number infiltrating cells apoptosis liver damage muscle invasion Br J Cancer, (2001) 85:1185-1192. BC Yang

Fas-LR Control Br J Cancer, (2001) 85:1185-1192. U-118MG in nude mice FasL associated TIL in particular sites Invasion of tumor cells BC Yang

In vivo consequences of Fas-L expression by tumors (using over-expression system) Enhanced rejection Delayed rejection Renca, MH134, L5178Y, B16-BL6, CT26* CT26# B16-F10 Note: *:syngenic, nude, SCID #: allogenic, lpr Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol. BC Yang

Animal model C57B/6 Melanoma cell line B6F10 Gene knockdown by FasL ribozyme Subcutis/lung BC Yang

B16-F10 (melanoma) in B6 mice Control Fas-LRibozyme BC Yang

Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation Vector controls Fas-L-ribozyme BC Yang

Granulocytes mediates the Fas-L-associated apoptosis during lung metastasis of melanoma that determines the metastatic behavior (B16F10 in C57BL/6) Br J Cancer, 87: 359 (2002) BC Yang

Depletion of CD4, CD8 T cells and PMNs affected lung metastasis 44 41 357 10 95 154 Vector controls Fas-Lribozyme What happened here? BC Yang

Immune privilege or inflammation. Joe O'Connell et al Immune privilege or inflammation? Joe O'Connell et al. Nature Medicine 7, 271 - 274 (2001) BC Yang

The Third face of Fas signal Not only “not quite dead enough”! Stout Rex, 1944 BC Yang

No death occurred in Jurkat cells during coculture with gliomas PI-staining B C D E A: Jurkat cells alone B: CH-11 (1ng/ml) C: with U-118MG(V) D: with U118MG (R) E: ZB4 (100ng/ml)/CH-11 BC Yang

Rescigno M., et al. 2000 Fas Engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1b, and the production of interferon-g in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses. J Exp Med 192: 1661-1668. BC Yang

Hohlbaum AM, et al. 2001, Fas ligand engagement of resident peritoneal macrophages in vivo induces apoptosis and the production of neutrophil chemotactic factors. J Immunol 167:6217-6224. BC Yang

The IL-10 induction is associated with tumor Fas-L 2: U-373MG(V); 3: U373MG(R); 4: U-118MG(V); 5: U118MG(R) BC Yang

IL-10 expression upon Fas-activation in Jurkat cells by CH-11 1 2 3 4 IL-10 b-actin %SubG0 10% 50% 65% 73% 1-4: 0, 0.1, 0.5, 1 ng/ml of CH-11 for 24 h BC Yang

Tumor What will happen when Fas-stimulated immune cells resist to die? Tissue environment ? Tumor Fas-L Immune cells Fas die Cytokines ? (IL-10, TGF-b) BC Yang

在動物體內, 細胞從來不會這樣攤平生長. Fas-LR Control In culture dish U-118MG in nude mice FasL associated TIL in particular sites Invasion of tumor cells BC Yang

Structure of tumor spheroid Culture on 0.5% Nobel Agar Yuhas JM, et al. (1977) Cancer Res 37:3639-3643. BC Yang

2-D versus 3-D growth On plate Spheroid IEF SDS BC Yang Modification Reduced enhanced references points BC Yang

Proteins associated with extracellular matrix FAK (125) C-src (60) Talin (235) Pyk2 (116) R V R V R V Spheroids Vinculin (116) ? BC Yang

Jurkat cells destroy the FasLhigh-spheroids. U-118MG (4 weeks) + Jurkat cells (1:5); 3 days coculture U118V ~250 mm U118R ~260 mm Note: Jurkat cells do not kill tumor cells in dish culture condition 5x105 cells BC Yang

Jurkat cells inhibit the formation of FasLhigh-tumor spheroids. U118V+ Jurkat (1:4) U118R+ Jurkat (1:4) 48 h 72 h 1x105 cells BC Yang