Transcript A malicious kiss of Fas and FasL:

New insight in death signaling:
on Fas and FasL
Bei-Chang Yang, Ph.D. (楊倍昌)
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
Department of Microbiology and Immunology, College of Medicine, National
Cheng Kung University (NCKU), Tainan, Taiwan.

Research center for Society, Technology and Medicine (STM center); NCKU
Tainan, Taiwan.
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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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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.
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東豐路, 台南
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Some types of death: apoptosis, autophage, and necrosis
AUTOPHAGE
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Detection of apoptotic cells
•
•
•
•
Hoechst stain
DNA ladder
Comet assay
TUNEL stain; immunohistochmical (terminal
transferase)
• FACS analysis (Annexin V, MC540, propidium
iodine for subG0, TUNEL)
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• 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)
– P Krammer group (1989): Trauth BC. et al. Monoclonal
antibody-mediated tumor regression by induction of
apoptosis. Science 245(4915):301-305.
– S Nagata group (1991): Itoh N. et al. The polypeptide
encoded by the cDNA for human cell surface antigen Fas
can mediate apoptosis. Cell 66:233-243.
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Shigekazu Nagata
Peter H. Krammer, 2002
For a man who spends most of his time thinking
about death, Shigekazu Nagata is remarkably
upbeat. For over a decade, he has been making a
name for himself with research on apoptosis, the
mechanism of programmed cell death, and
during that time he has watched the field come
alive.
Nature Medicine 7, 759 (2001)
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Nature 407, 789 – 795(2000)
 Nagata, S. (Fas);
Krammer, P. (Apo1)
 CD95/CD95-L
 Transducing death
signal
 Involving in
cytotoxicity, immune
homeostasis, tumor
formation, etc.
Caspase 3
Death substrates
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Apoptosis
Apaf-1
Caspase 9
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Fas counterattack mechanism
 Hahne, M., D. Rimoldi, M. Schroter, P. Romero, M. Schreier, L. E. French, P.
Schneider, T. Bornand, A. Fontana, D. Lienard, J. C. Cerottini, and J. Tschopp.
1996. Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for
tumor immune escape. Science 274:1363.
 Strand, S., W. J. Hofmann, H. Hung, M. Muller, G. Otto, D. Strand, S. M.
Martani, W. Stremmel, P. H. Krammer, and P. R. Galle. 1996. Lymphocyte
apoptosis induced by CD95(APO-1/Fas) ligand-expressing tumor cells-a
mechanism of immune evasion ? Nature Med. 2:1361.
 O’Connell, J., G. C. O’Sullivan, J. K. Collins, and F. Shanahan. 1996. The Fas
counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas
ligand. J. Exp. Med. 184:1075.
 Seino, K., N. Kayagaki, K. Okumura, and H. Yagita. 1997. Antitumor effect of
locally produced CD95 ligand. Nature Med. 3:165.
 O’Connell, J., M. W. Bennett, G. G. O’sullivan, D. Roche, J. Kelly, J. K. Collins,
and F. Shanahan. 1999. Fas counter-attack the best form of tumor defense?
Nature Med. 5:267.
 Seino, K. I., N. Kayagaki, N. Tsukada, K. Fukao, H. Yagita, and K. Okumura.
1997. Transplantation of CD95 ligand-expressing grafts. Influence of
transplanation site and difficulty in protecting allo- and xenigrafts.
Transplantation 64:1050.
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Many tumor cells express FasL
Nat Med 5:267 (1999); Nat Med 7:271 (2001)
Joe O'Connell, et al. Fas counter-attack- the best form of tumor defense?
Tumor
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Immune privilege or inflammation?
Joe O'Connell et al. Nature Medicine 7, 271 - 274 (2001)
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In vivo consequences of FasL
expression by tumors
(using over-expression system)
 Enhanced rejection
Renca, MH134,
L5178Y,
B16/BL6,
CT26*
Note: *:syngenic, nude, SCID
 Delayed rejection
CT26#
B16-F10
#:
allogenic, lpr
Ref: Lejeune FJ et al., 1998, Curr. Op. Immunol.
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Animal model

C57B/6
 Melanoma cell line
B6F10
 Gene knockdown by
FasL ribozyme
 Subcutis/lung
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FasLribozyme/EGFP plasmid
 Hammerhead
ribozyme.
 54-mers.
 Inserted into the
pEGFP-N1.
 Driven by CMV
promoter.
Chio CC, Wang YS, Chen YL, Lin SJ and Yang BC (2001) Down-regulation of
Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumor infiltrating
cells, and liver damage but accelerates tumor formation in nude mice.
Br J Cancer 85:1185-1192
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Effect of FasL on growth and
death of B16F10 cells
Cell growth rate
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Apoptosis in 3-day culture
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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
44
10
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Vector controls
What happened here?
357
95
154
Fas-Lribozyme
Br J Cancer, 87: 359 (2002)
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In subcutaneous environment: FasL on tumor cells
mediates inactivation of neutrophils
5x105cells subcutaneous injection
Control
Fas-Lribozyme
FasL on tumor cells mediates inactivation of
neutrophils. J. Immunol. (2003) 171:1183-1192
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Depletion of CD4, CD8 T cells and PMNs
affected subcutaneous tumor formation
Vector controls
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Fas-L-ribozyme
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Cell death
 Tumor FasLhigh impairs
neutrophil activation:
gelatinase B, ROS, CD-11b,
and FasL (In coculture for
24 h).
Gelatinase B
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ROS
J. Immunol. (2003) 171:1183-1192
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The action of FasL:
It is organ dependent!
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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)
i.v. 5 x 105 cells /mouse; Observed at day 14; (bar = 1 cm)
Voice against Fas counter attack mechanism:
Kang, S. M. et al. 1997. Fas ligand expression in islets of
Langerhans does not confer immune privilege and instead
targets them for rapid destruction. Nature Med. 3:738.
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Ryan AE,1 Shanahan F, O’Connell J, and Houston AM (2005)
Addressing the ‘‘Fas Counterattack’’ Controversy: Blocking Fas
Ligand Expression Suppresses Tumor Immune Evasion of Colon Cancer In vivo.
Cancer Res 65: 9817-9823 (困獸之鬥?)
• Figure 1. FasL antisense RNA suppresses FasL expression by
CMT93 cells.
• Figure 2. Effect of reduced FasL expression by CMT93 cells on
tumor development.
• Figure 3. Analysis of tumors derived from CMT93/AS, CMT93/C
and parental cells inoculated s.c. in C57BL/6 mice.
• Figure 4. Down-regulation of FasL expression leads to increased
infiltration of TILs.
• Figure 5. Constitutively expressed FasL does not trigger
neutrophil recruitment in vivo.
No Fas-mediated apoptosis at all!
What Joe O’Connell was thinking then?
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Increase in Th17 cells
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FasL reverse signaling
 Suzuki I. and Fink PJ.. Maximal proliferation
of cytotoxic T lymphocytes requires reverse
signaling through Fas ligand. J. Exp. Med.,
187:123, 1998.
 Boursalian TE, Fink PJ, Mutation in Fas
Ligand Impairs Maturation of Thymocytes
Bearing Moderate Affinity T Cell Receptors,
J. Exp. Med., 198, 349, 2003.
Pamela J. Fink, h.D.
 Sun M, Ames KT, Suzuki I,. Fink PJ, The
Cytoplasmic Domain of Fas Ligand
Costimulates TCR Signals, J. Immunol., 177,
1481, 2006.
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Department of Immunology
University of Washington
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The structure of FasL
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The cytoplasmic domain of FasL is
sufficient to mediate costimulation.
A, Murine FasL and z (CD3)
fusion constructs encode an Nterminal myristylation motif (M)
that tethers the molecules to the
membrane, the relevant
cytoplasmic domains, FKBP3,
and a C-terminal HA epitope.
The addition of FK1012 or
AP1510 induces heterooligomers of these membranebound proteins. B, Jurkat cells
cotransfected with combinations
of CID constructs, firefly
luciferase reporter (NFAT or AP1) and Renilla luciferase reporter
(pRL-TK) were subjected to CID
analysis.
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J. Immunol., 177, 1481, 2006
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Ectopic expression of FasL in NIH3T3
(A). Full-length and truncated FasL. Adapted from Orlinick JR, et al. J Biol Chem
1997, 272:32221-29. Jodo S, et al. J Immunol 2005, 174:4470-4474. FasL-△70:
deleting the N-terminal 2-70 aa; FasL-△33: deleting the N-terminal 2-33 aa.
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Full-length FasL suppresses lung metastasis, while
truncated FasL promotes tumor formation.
Nude mice were injected with
5x105 cells of NIH3T3 cells
carrying FasL as indicated via
tail vein. After 4 weeks,
spontaneous transformed
NIH3T3 tumor nodules formed
in the lung. Ling sections were
stained with H/E. Tumor
nodules show dark purple
stain.
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Truncated FasL promote cell transformation
(enhances anchorage-indecent growth)
FasL△33 and FasL△70 enhanced anchorage-independent growth. 1.5 ×104 cells
were cultured in 0.3% noble agar for 2-3 weeks. Foci were observed under a
microscope. Representative shape of colony (x100) was shown (A). Number of foci
was counted (B). Values was shown the mean ± SEM. (* p<0.05 compared with N1).
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FasL enhances cell migration and invasion.
In the single cell motility assay, cell migration distance in 6 h was recorded every 5
min by time lapse video under a microscope. Total distance of cell migration was
taken to represent cell mobility. (C) In invasion assay, cells were seeded on matrigel
(1 mg/ml) coated transwell plates in 16 h. Invading cells were calculated for
quantification. Values was shown the mean ± SEM. (* p<0.05, ** p<0.001 compared
with N1, # p<0.05 compared with FasL)
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Phospho-STAT3 and STAT3 were analyzed by Western blot (B). Cells were incubated in 1% FBS in
16 h and detected the expressions of cyclin D1 by Western blotting (C) and RT-PCR (D).
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FasL reverse signal, A speculation
FasL-△70
Full-length
FasL
Cell membrane
?
PRD
ERK, PI3K
CK1
Stat3, cyclinD1
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Inhibitory signal
regulating immune
responses
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A proposed FasL-Met signal pathway.
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Do you still believe in Fas counterattack mechanism?
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Nature is working in a way that is
not always as been told.
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