1. 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
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2. 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
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3. Two types of cell death
Apoptosis
Necrosis
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4. Apoptosis vs. necrosis
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5. 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)
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6. 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:  
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):
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7. 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)

8. Peter H. Krammer, 2002
Lab member: total 34

9. 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).
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10. 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. 
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11. 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.
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12. 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.
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13. 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.
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14. 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.
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15. 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.
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16. Apoptosis Apaf-1 Caspase 3 Caspase 9 Death substrates
Nature 407, 789 – 795(2000)
Caspase 3
Apaf-1
Caspase 9
Death substrates
Apoptosis
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17. 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:
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18.
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19. Nature Reviews Immunology 2; 401-409 (2002); doi:10
Nature Reviews Immunology 2; (2002); doi: /nri819 CYTOTOXIC T LYMPHOCYTES: ALL ROADS LEAD TO DEATH
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20. 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).
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21. 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)
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22. 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:

23. 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:

24. 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.
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25. TCR restimulation induces redistribution of Fas into lipid rafts before signal complex formation.
Nature Immunology 5, 182 – 189.
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26. The presence of Fas in lipid rafts controls signaling efficiency.
Nature Immunology 5, 182 – 189.
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27. Intracellular signaling pathways downstream of tumor necrosis factor (TNF) receptor superfamily receptors.
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28. Death domain structures
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29. 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)
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30. 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.
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31. 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.
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32. 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)
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33. A fight between immune cells and cancer
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34. Fas-L positive tumors with heavily infiltrated lymphocytes
Nasal pharyngeal carcinoma
Glioblastoma
Hematoxylin/eosin staining
CD3+ cells
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35. Resistance to killing Cytotoxic T cells Innat immunity
Defective Fas pathway
Resistance to Granzyme
Innat immunity
Fas-L and Neutrophil
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36. 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.
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37. Fas-Lribozyme/EGFP plasmid
Br J Cancer, (2001) 85:
Hammerhead ribozyme.
54-mers.
Inserted into the pEGFP-N1.
Driven by CMV promoter.
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38. U-373MG cells transfected with Fas-Lribozyme/EGFP plasmid
Light microscopy
Fluorescent microscopy
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39. Down-regulation of Fas-L by Fas-Lribozyme
U-373MG
RT-PCR
1. U-373MG
2. EGFP-N1
3. EGFP/Fas-Lribozyme
FACScan
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40. Fas-L on tumorigenesis of glioma in nude mice
control Fas-Lribozyme
1.5-2 mm mm3
2 in 3 mice in 3 mice
(?)
tumor size
tumor number
infiltrating cells
apoptosis
liver damage
muscle invasion
Br J Cancer, (2001) 85:
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41. 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
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42. 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.
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43. Animal model C57B/6 Melanoma cell line B6F10
Gene knockdown by FasL ribozyme
Subcutis/lung
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44. B16-F10 (melanoma) in B6 mice
Control Fas-LRibozyme
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45. Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation
Vector controls
Fas-L-ribozyme
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46. 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)
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47. Depletion of CD4, CD8 T cells and PMNs affected lung metastasis44 41
357 10 95
154
Vector controls
Fas-Lribozyme
What happened here?
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48. Immune privilege or inflammation. Joe O'Connell et al
Immune privilege or inflammation? Joe O'Connell et al. Nature Medicine 7, 271 - 274 (2001)
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49. The Third face of Fas signal
Not only
“not quite dead enough”!
Stout Rex, 1944
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50. No death occurred in Jurkat cells during coculture with gliomas PI-stainingB 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
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51. 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:
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52. 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:
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53. The IL-10 induction is associated with tumor Fas-L
2: U-373MG(V); 3: U373MG(R); 4: U-118MG(V); 5: U118MG(R)
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54. IL-10 expression upon Fas-activation in Jurkat cells by CH-11
IL-10
b-actin
%SubG % 50% 65% 73%
1-4: 0, 0.1, 0.5, 1 ng/ml of CH-11 for 24 h
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55. 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)
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56. 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
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57. Structure of tumor spheroid
Culture on 0.5% Nobel Agar
Yuhas JM, et al. (1977) Cancer Res 37:
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58. 2-D versus 3-D growth On plate Spheroid IEF SDS BC Yang
Modification Reduced enhanced references points
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59. Proteins associated with extracellular matrix
FAK (125)
C-src (60)
Talin (235)
Pyk2 (116)
R V R V R V
Spheroids
Vinculin (116) ?
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60. 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
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61. Jurkat cells inhibit the formation of FasLhigh-tumor spheroids.
U118V+ Jurkat (1:4)
U118R+ Jurkat (1:4)
48 h
72 h
1x105 cells
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