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

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Two types of cell death

Apoptosis

Necrosis

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Apoptosis vs. necrosis

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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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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 canmediate 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.

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Shigekazu Nagata (2002)

Lab member: total 21

Cell Death and Diff.Genes to CellApoptosisInt. Immunol.ImmunityExp. Cell Res.Biochem. Biophys. ActaIUBMB/LifeScienceInt. J. CancerCancer 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).

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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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TNF is representative of a still growing family of trimeric surface

proteins, such as lymphotoxin- (LT-), Fas ligand (FasL), receptor-

activator of NF-B ligand (RANKL), CD40 ligand (CD40L), and TNF-

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 TNF 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 TNF to TNFR1 activates

TNFR1-associated death domain protein (TRADD), which interacts

with Fas-associated death domain protein (FADD) to induce

apoptosis; the binding of TNF 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-B activation, but does not result in cell

death. Importantly, the suppression of apoptosis, which is mostly

dependent on NF-B, augments the inflammatory response to TNF.

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.htmlBC Yang

http://www.sghms.ac.uk/depts/immunology/~dash/apoptosis/receptors.html

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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http://www.biotechjournal.com/Journal/jan%202002/janArticle6text.htm

TNF acts through two receptors, TNFR1 & 2. TNFR1 is expressed by all human tissues and is the major signaling receptor for TNF. TNFR2 is mostly expressed in immune cells and mediates limited biological responses. TNFR2 binds both TNF and TNF.

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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

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Caspase 3 Apaf-1Caspase 9Death substrates

Apoptosis

Nature 407, 789 – 795(2000)

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Budd RJ, 2002, J Clin Invest 109:437-441

擋在這裡 !?

Annexin V-positive Loss of the mitochondria

membrane potential Caspase-3, -8, -9 activated Cell size moderately

reduced But, not dead

Sometimes, cells do not die, still intact, low SubGo population

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

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Nature Reviews Immunology 2; 401-409 (2002); doi:10.1038/nri819CYTOTOXIC T LYMPHOCYTES: ALL ROADS LEAD TO DEATH

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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

Cellular stress Cytokiens:IFN-,

TNF-, IL-8 Cytostatic drugs 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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An alternative way to control death signal

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.

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.

Nature. 1998, 396:699-703.

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

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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.

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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

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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)

Death domain: Stereoview of the backbone (N, Calpha, C') of

20 superimposed NMR-derived structures of the Fas death domain.

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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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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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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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A fight between immune cells and cancer

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Fas-L positive tumors with heavily infiltrated lymphocytes

Nasal pharyngeal carcinoma Glioblastoma

Hematoxylin/eosin staining CD3+ cells

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Resistance to killing

Defective Fas pathway Resistance to Granzyme

Fas-L and Neutrophil

Cytotoxic T cells

Innat immunity

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In vivo consequences of Fas-L expression by tumors

(using over-expression system)

Enhanced rejection Delayed rejection

Renca, MH134, L5178Y, B16-BL6, CT26*

Note: *:syngenic, nude, SCID #: allogenic, lpr

Lejeune FJ et al., 1998, Curr. Op. Immunol.

CT26#

B16-F10

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Fas-Lribozyme/EGFP plasmid

Hammerhead ribozyme.

54-mers. Inserted into the

pEGFP-N1. Driven by CMV

promoter.

Br J Cancer, (2001) 85:1185-1192.

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U-373MG cells transfected withFas-Lribozyme/EGFP plasmid

Light microscopy Fluorescent microscopy

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Down-regulation of Fas-L by Fas-Lribozyme

RT-PCR 1. U-373MG 2. EGFP-N1 3. EGFP/Fas-Lribozyme

FACScan

U-373MG

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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

Fas-L on tumorigenesis of glioma in nude mice

Br J Cancer, (2001) 85:1185-1192.

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Fas-LR Control

U-118MG in nude mice FasL associated TIL in particular sites Invasion of tumor cells

Br J Cancer, (2001) 85:1185-1192.

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In vivo consequences of Fas-L expression by tumors

(using over-expression system)

Enhanced rejection Delayed rejection

Renca, MH134, L5178Y, B16-BL6, CT26*

Note: *:syngenic, nude, SCID #: allogenic, lpr

Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol.

CT26#

B16-F10

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Animal model

C57B/6 Melanoma cell line

B6F10 Gene knockdown by

FasL ribozyme Subcutis/lung

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B16-F10 (melanoma) in B6 mice

Control Fas-LRibozyme

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Vector controls Fas-L-ribozyme

Depletion of CD4, CD8 T cells and PMNs affected subcutaneous tumor formation

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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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Depletion of CD4, CD8 T cells and PMNs affected lung metastasis

4410

41 357 95154

Vector controls Fas-Lribozyme

What happened here?

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

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The Third face of Fas signal

Not only “not quite dead enough”!

Stout Rex, 1944

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No death occurred in Jurkat cells during coculture with gliomas

PI-staining

A 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

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Fas Engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1, and the production of interferon- 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.

Rescigno M., et al. 2000

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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.

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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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1 2 3 4 IL-10

-actin

%SubG0 10% 50% 65% 73%

IL-10 expression upon Fas-activation in Jurkat cells by CH-11

1-4: 0, 0.1, 0.5, 1 ng/ml of CH-11 for 24 h

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Fas

Fas-LImmune cells

Tumor

Tissue environment ?

Cytokines ? (IL-10, TGF-)

What will happen when Fas-stimulated immune cells resist to die?

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在動物體內 , 細胞從來不會這樣攤平生長 .

In culture dish

Fas-LR Control

U-118MG in nude mice FasL associated TIL in particular sites Invasion of tumor cells

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Structure of tumor spheroid

Yuhas JM, et al. (1977) Cancer Res 37:3639-3643.

Culture on 0.5% Nobel Agar

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2-D versus 3-D growthOn plate Spheroid

Modification Reduced enhanced references points

IEF

SD

S

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FAK (125)

C-src (60)

Talin (235)

Pyk2 (116)

R V R V R V

Spheroids

Vinculin (116)

?

Proteins associated with extracellular matrix

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Jurkat cells destroy the FasLhigh-spheroids.

U118V~250 m

U118R~260 m

U-118MG (4 weeks) + Jurkat cells (1:5); 3 days coculture

5x105 cells Note: Jurkat cells do not kill tumor cells in dish culture condition

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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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