Session
I: Caspases
Sunday April 2nd
BCR-INDUCED APOPTOSIS IS
INITIATED BY A zVAD-INSENSITIVE PROTEASE WHICH IS UNDER DIRECT CONTROL OF CD40
Eric ElderingH, Susanne M.A. Lens*H, Bianca F.A. den Drijver*, Ingrid A.M. Derks, Jannie BorstI, Marinus H.J van Oers' and René A.W. van Lier*H
Depts.
of *Immunobiology, CLB, HClinical
Immunology Laboratory, AMC, ICellular
Biochemistry, NCI, and 'Hematology,
AMC, Amsterdam, The Netherlands
B-cell
antigen receptor (BCR)-induced apoptosis is accompanied by cleavage of caspase (casp)
3. We have shown that in the B-cell line Ramos it takes place independently from
the adaptor protein FADD and is not inhibited by overexpression of FLIP. Thus,
the initial stages of BCR-mediated apoptosis differ strikingly from the
CD95-pathway. In addition, after BCR cross-linking, conversion of casp-3 from
p32 to p20 can not be blocked by the broad spectrum caspase inhibitor z-VAD-fmk,
while processing of the related executioner casp-7 can be blocked completely. In
contrast to z-VAD-fmk, ligation of CD40 prevents BCR-induced casp-3 processing
and apoptosis completely, even when this signal is delivered 12 hours after
cross-linking of the BCR. Finally, triggering of the BCR induces the release of
cytochrome C from mitochondria in a z-VAD-fmk insensitive but CD40-regulated
fashion. We conclude that the BCR can couple to the classical caspase cascade
via a specific proteolytic pathway which is under direct control of T-helper
signals. Calpain (Ruiz-Vela etal., EMBO 18, 4988, ’99) and caspase-2 (Chen
etal., J Imm 163, 2483, ’99) have recently been proposed as mediators
of BCR-induced apoptosis, but no direct comparison of BCR- vs. CD95-triggering
involving these proteases has yet been made. Our current efforts include
expression of caspase-2 constructs in Ramos with which we will be able to
further dissect the BCR- and CD95-pathways.
A regulatory role for Apaf 1 in Caspase 9
function
Srinivasa M. Srinivasula,
Ayman saleh, Manzoor Ahmad, Teresa Fernandes-Alnemri, Emad S. Alnemri.
Center
for Apoptosis Research and Department of Microbiology and Immunology, Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA
Caspases play a fundamental role in the initiation and execution of apoptosis. Though Apaf 1 is required for oligomerization and autocatalytic activation of caspase 9, its role in the caspase cascade after processing of caspase 9 is not yet clear. Using pure recombinant Apaf 1, completely processed and unprocessed caspase 9 and procaspase 3 we provide evidence that Apaf 1 is the only factor required for the activation of caspase 9 and caspase 3. Apaf 1 also positively regulates the enzymatic activity of caspase 9 by forming a holoenzyme complex with fully processed or unprocessed caspase 9. At physiological concentrations both completely and unprocessed caspase 9 have no enzymatic activity , but express equivalent enzymatic activity towards procaspase 3 when incubated with Apaf 1, cytochrome c and dATP. In contrast completely processed prodomain mutant (R56A) of caspase 9, which can not bind to CARD domain of Apaf 1, remains enzymatically inactive towards procaspase 3 even inpresence of Apaf 1, cytochrome c and dATP. These data suggests that fully processed caspase 9 requires binding to Apaf 1 for expression of its optimal activity. We believe that such a regulation of processed caspase 9 activity by Apaf 1 or any other isoforms of Apaf 1 that may exist in cell plays an important role in cell differentiation and death.
Session
I: Caspases
Sunday April 2nd
DRADD
is a nuclear-localized death domain adaptor protein that binds caspase-9 and
promotes apoptosis
Colin Adrain, Mary T. Harte,
Helen Egan, Liam Carlin, Elizabeth A. Slee and Seamus J. Martin
Molecular Cell Biology
Laboratory, Department of Genetics, The Smurfit Institute, Trinity College,
Dublin 2, Ireland
Apoptosis
is coordinated by a family of cysteine proteases, the caspases, that dismantle
the cell by cleaving a subset of cellular proteins after aspartic acid residues.
Caspases are frequently activated at the onset of apoptosis by recruitment to
adaptor proteins that promote aggregation of particular caspases and facilitate
their processing. Caspase-9 is activated in the cytosol through recruitment to
Apaf-1, a human homologue of the C elegans
CED-4 protein. To identify other caspase-9-binding proteins, we performed a
two-hybrid screen with the N-terminal caspase recruitment domain (CARD) region
of human caspase-9. This screen yielded multiple copies of a protein which we
have called DRADD. DRADD possesses a C-terminal death domain as well as a
caspase-recruitment domain (CARD) that is specific for caspase-9. Whereas
transient overexpression of full-length DRADD did not promote apoptosis,
truncations of this protein containing the CARD domain did so. Interestingly, in
contrast to most caspase adaptor proteins described to date, DRADD expression is
strongly localized to the nucleus. DRADD may be involved in initiating caspase
activation in response to stimuli that provoke nuclear damage.
Apoptin
induces apoptosis specifically in human tumor cells: studies on its mechanism
Astrid A.A.M. Danen-van
Oorschot1,2, Arend van Zon1, Maud C.M.J. Seelen2
and Mathieu H.M. Noteborn1,2
1
Molecular Cell B
2
Leadd BV, PO Box 9503, 2300 RA, Leiden, The Netherlands
Apoptin,
a protein derived from chicken anemia virus (CAV), induces apoptosis in various
human transformed or tumor cell lines, even those lacking p53 or overexpressing
Bcl-2. Interestingly, apoptin does not induce apoptosis in any of the human
normal diploid cells tested to date. Elucidation of the molecular mechanism of
apoptin-induced apoptosis is important for its potential use as an anti-tumor
agent. Moreover, it may shed light on the characteristics underlying the
transformed state of a cell, which possibly provides new drug targets for cancer
therapy. Experiments with inhibitors of specific caspases indicate that
activation of upstream caspases is not essential, but downstream caspases do
seem to be required for apoptin-induced apoptosis. In immunofluorescence assays,
active caspase-3 could be detected in cells undergoing apoptin-induced apoptosis.
In transformed or tumor cells apoptin is mainly present in the nucleus, whereas
in normal diploid cells apoptin is predominantly located in the cytoplasm.
Mutations preventing nuclear localization of apoptin also reduce its apoptotic
activity. However, a fusion protein of apoptin and a strong NLS results in
nuclear localization in normal diploid cells, but not in induction of apoptosis.
These observations indicate that nuclear localization is important, but not
sufficient for apoptin-induced apoptosis.
Session
I: Caspases
Sunday April 2nd
Two
Cell Death Pathways leading to Necrosis and Apoptosis : Differential Role
of Caspases and Mitochondria
Geert
van Loo, Geertrui Denecker, Margino Steemans, Dominique Vercammen, Wim Declercq,
Walter Fiers, Johan Grootten & Peter Vandenabeele
Molecular
Signalling and Cell Death Unit, Department of Molecular Biology, Flemish
Interuniversity Institute for Biotechnology (VIB)
University of Gent (RUG),
Ledeganckstraat 35, B-9000 Gent (Belgium)
Apoptosis
and necrosis are often described as two distinct ways of cell death. However,
most comparative studies are based on the use of different cell lines or
unrelated stimuli. In order to compare the signalling pathways of both types of
cell death in the same cells initiated by death domain receptors, murine L929sA
fibrosarcoma cells were transfected with the human Fas receptor (CD95). The
cells treated with agonistic anti-Fas exhibited a clear apoptotic phenotype,
while TNF treatment led to necrosis. We studied the release of mitochondrial
factors in both types of cell death and the influence of Bcl-2. We will also
provide evidence for a protective role of caspase-8 in the necrotic process. The
presence of caspase-inhibitors blocks Fas-induced apoptosis, but later on the
cells die by necrosis. The necrotic process is not accompanied with activation
of caspases and is prevented by the addition of oxygen radical scavengers and
serine protease inhibitors. This indicates that TNF-R1- and Fas-initiated
necrosis occur independent of the FADD-procaspase-8 effector axis. It also
implies that in the absence of caspase activation another cell death pathway
than apoptosis may be initiated from the death domain receptors or death domain
containing associated proteins, in which serine proteases and generation of
mitochondrial reactive oxygen intermediates are implicated.
The
Viral nucleocapsid protein of the transmissible gastroenteritis coronavirus,
TGEV, is a target for caspase -6 and -7
J.-F. Eléouët1,
E. Slee2,4, F. Saurini1,
N. Castagné1, D. Poncet1, C.
Garrido3, E. Solary3,
and S. J. Martin2,4.
1VIM,
INRA, 78352 Jouy-en-Josas, France; 2Molecular
Cell Biology Laboratory, National University of Ireland, Maynooth, Co. Kildare, Ireland; 3U.F.R.
Médecine et Pharmacie, INSERM U517, 7 Boulevard Jeanne d'Arc 21000 Dijon,
France. 4Present address: Division of Molecular and Cell Biology,
The Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland
Transmissible
gastroenteritis coronavirus (TGEV), which causes acute and fatal diarrhea in
newborn pigglets, exerts much of its cytotopathic effects through induction of
apoptosis of its host cell. The present study shows that infection of the human
rectal tumor cell line HRT118, modified to express the porcine aminopeptidase N
which is the receptor for TGEV, results in the activation of caspases -3, -6,
-7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin and a-fodrin in host cells. Interestingly, apoptosis of
TGEV-infected HRT18 cells was associated with proteolysis of the TGEV
nucleocapsid (N) protein. N protein cleavage, that was inhibited by cell
permeable caspase inhibitors, was reproduced
in vitro by incubating the protein with either caspase-6 or caspase-7. The
site of N protein cleavage by caspases was mapped to VVPD359Ø.
These results indicate that cells undergoing cell death upon viral infection can
alter viral structural proteins through caspase-mediated proteolysis.
Session
II: Mitochondria and cytotoxic drugs
Sunday April 2nd
Regulation of
caspase-dependent but not caspase-indipendent cell death by the multidrug
resistance protein, P-glycoprotein
Ricky W. Johnstone, Astrid
A. Ruefli, Kelly M. Tainton and Mark J. Smyth.
The Austin Research
Institute, Heidelberg, Victoria, Australia
Multidrug
resistance (MDR) is characterized by the expression of P-glycoprotein (P-gp), a
170kD ATP-dependent drug efflux protein. As well as effluxing xenotoxins,
functional P-gp can confer resistance to caspase-dependent apoptosis induced by
a range of different stimuli including Fas ligand, TNF, UV irradiation and serum
starvation. However, P-gp positive cells remain sensitive to caspase-indipendent
death induced by cytotoxic T cell granule proteins, perforin and granzyme B. It
is therefore possible that agents that induce cell death in a
caspase-indipendent manner might circumvent P-gp-mediated MDR. We now evidence
that Hexamethylene bisacetamide (HMBA) can induce equivalent caspase-indipendent
cell death in both P-gp positive and negative cell lines. The HMBA-induced death
pathway is marked by release of cytochrome c from the mitochondria and reduction
of Bcl-2 protein levels. In addition, functional P-gp can specifically inhibit
the activation of caspases-8 and –3, caspase-9 remains unaffected. These
studies greatly enhance our understanding of the molecular cell death events
that can be regulated by functional P-gp and highlight the potential clinical
use of drugs that function via a caspase-indipendent pathway for the treatment
of MDR tumors.
Oxidation
of a Critical Thiol Residue of the Adenine Nucleotide Translocator Enforces
Bcl-2-Independent Permeability Transition Pore Opening and Apoptosis
Paola Costantini1,
Anne-Sophie Belzacq2, Helena L.A.
Vieira1, Nathanael Larochette1,
Manuel de Pablo1, Naoufal Zamzami1,
Santos A. Susin1, Catherine Brenner2,
and Guido Kroemer1
1 Centre
National de la Recherche Scientifique, ERS 1984, 19 rue Guy Môquet, F-94801
Villejuif, France; 2 Centre National
de la Recherche Scientifique, UPRES-A6022, Université Technologique de Compiègne,
F-60205 Compiègne, France
Mitochondrial
membrane permeabilization is a critical event in the process leading to
physiological or chemotherapy-induced apoptosis. This permeabilization event is
at least in part under the control of the permeability transition pore complex (PTPC),
which interacts with oncoproteins from the Bcl-2 family as well as with tumor
suppressor proteins from the Bax family, which inhibit or facilitate membrane
permeabilization, respectively. Here we show that thiol crosslinking agents
including diamide, dithiodipyridine (DTDP), or bis-maleimido-hexane (BMH) can
act on the adenine nucleotide translocator (ANT), one of the proteins within the
PTPC. ANT alone reconstituted into artificial lipid bilayers suffices to confer
a membrane permeabilization response to thiol crosslinking agents. Diamide, DTDP,
and BMH but not tert-butylhydroperoxide
or arsenite cause the oxidation of a critical cysteine residue (Cys 56) of ANT.
Thiol modification within ANT is observed in intact cells, isolated mitochondria,
and purified ANT. Recombinant Bcl-2 fails to prevent thiol modification of ANT.
Concomitantly, a series of different thiol crosslinking agents (diamide, DTDP,
and BMH, phenylarsine oxide) but not tert-butylhydroperoxide or arsenite induce mitochondrial membrane
permeabilization and cell death irrespective of the expression level of Bcl-2.
These data indicate that thiol crosslinkers cause a covalent modification of ANT
which, beyond any control by Bcl-2, leads to mitochondrial membrane
permeabilization and cell death.
Session
II: Mitochondria and cytotoxic drugs
Sunday April 2nd
Biochemical
differences in apoptosis induced by CD95 ligation or thymidilate-synthase
inhibition
Instituto de
Parasitología y Biomedicina, CSIC, Granada, Spain
Drugs
that inhibit dTTP synthesis induce apoptosis in a wide range of cell types, and
are currently used in chemotherapy. Despite of this, the mechanism of induction
of cell death is not fully understood. Previous results of our lab indicate that,
in hematopoietic cells, p53 accumulation or CD95‑CD95L are not implicated
in death response to the thymidilate‑syntase inhibitor 5'fluor, 2'deoxyuridine
(FUdR, Floxuridine). In this report, we have examined the mechanism of apoptosis
induced either by FUdR or CD95‑triggering in hematopoietic U937 cells.
Caspase-8, a classically death receptor‑associated caspase, was activated
by both types of death inducers. Neverthless, inhibition of caspases‑8
with a specific peptide caused only a slight delay in FUdR‑induced death,
while completely inhibited CD95‑induced apoptosis. Furthermore, inhibition
of caspases‑8 inhibited cytochrome C release only when celIs were treated
with anti‑CD95, but not with FUdR. Requirement of ATP was also addressed.
While apoptotic death induced by FUdR was reduced by depletion of ATP, anti‑CD95‑induced
apoptosis was not inhibited. Studies of mitochondrial depolarization and
cytochrome C release showed that ATP was required in FUdR‑induced
apoptosis at a pre‑mitochondrial or mitochondrial level.
GRANZYME B INDUCED
APOPTOSIS IS REGULATED BY PRO- AND ANTI-APOPTOTIC MEMBERS OF THE BCL-2 FAMILY
Vivien R.Sutton, David
Huang*, Michael Cancilla*, Ricky Johnstone, Karin Sedelies, Astrid Ruefli,
Joanne Davis and Joseph Trapani.
The Austin Research Institute, Heidelberg, Vic., 3084
and * The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne
Hospital, Vic., 3050
We
have previously shown that Bcl-2 overexpression in mouse FDC-P1 or human Jurkat
cells blocks the apoptotic changes induced by GrB, including mitochondrial
depolarisation, cytochrome c release, DNA fragmentation, and plasma membrane
damage. We now report that sensitivity to GrB was restored by co-expression of
the pro-apoptotic bcl-2-related protein BID, whereas the same cells remained
protected from growth factor deprivation or treatment with staurosporine. BID
cleavage was observed only in response to GrB, but not to other apoptotic
stimuli. Time-course studies demonstrated that cleavage of either overexpressed
or endogenous BID was extremely rapid (within 2 minutes) and was not slowed by
over-expression of Bcl-2. We next examined the effect of introducing mutations
in the cleavage sites of BID. BID in which the GrB cleavage site (D75) was
mutated to Ala was unable to restore GrB-mediated apoptosis to Bcl-2 expressing
cells. However, mutation of the caspase 8 site (D59) did not prevent GrB-induced
BID cleavage or apoptosis. In untransfected Jurkat cells, inhibition of caspases
with z-VAD-fmk abrogated GrB -induced nuclear damage, whereas BID cleavage and
cytochrome c release were still observed, and the cells failed to survive in
clonogenic growth assays. Collectively, our findings indicate that Bcl-2
controls a key upstream event in GrB-mediated cell death, and that GrB-induced
cell death (both caspase-dependent and -independent) is initiated by direct BID
cleavage by GrB. Consequently, GrB-induced mitochondrial events do not require
active caspases.
Session
II: Mitochondria and cytotoxic drugs
Sunday April 2nd
GD3 GANGLIOSIDE INDUCES
APOPTOSIS VIA DIRECT MITOCHONDRIAL DAMAGE IN A BCL-2 CONTROLLED FASHION
M.R.
Rippo*, F. Malisan*, L. Ravagnan#, B. Tomassini*, I. Condo'*, P. Costantini#,
S.A. Susin#, A. Rufini*, G. Kroemer# and R. Testi*
*Laboratory of Signal Transduction, Department of
Experimental Medicine and Biochemical Sciences, University of Rome "Tor
Vergata" and # Centre National de la Recherche Scientifique, UPR 420,
Villejuif, France
GD3
ganglioside is an intracellular effector of Fas-induced apoptotic signal
mediated by ceramide. GD3, indeed, accumulates in hematopoietic cells upon Fas
and ceramide stimulation and blocking the GD3 neosynthesis strongly delays the
Fas- or ceramide- induced apoptotic program. Moreover, exogenous GD3 can
directly induce DNA fragmentation and apoptosis. In hematopoietic cells, GD3
induces a massive mitochondrial damage. Here we show that on isolated
mitochondria GD3, but not other gangliosides, induces mitochondrial swelling,
external mitochondrial membrane disruption, and release of apoptogenic factors
such as cytochrome c and AIF (Apoptosis Inducing Factor). All these events are
prevented by treatment with cyclosporine A, suggesting that GD3 induces
permeability transition (PT) pore opening. Supernatants of GD3 treated
mitochondria, but not GD3 alone, induce DNA fragmentation of isolated nuclei,
indicating that the target for GD3 is on mitochondria. Accordingly, suppression
of GD3 synthase (ST8) expression in intact cells substantially prevents
ceramide-induced DYm dissipation, indicating that endogenously synthesized GD3
induces mitochondrial changes in vivo. Moreover enforced expression of Bcl-2
prevents GD3-induced mitochondrial changes and apoptosis. All these results show
that mitochondria are the key destination for apoptogenic GD3 along the lipid
pathway to programmed cell death.
Luigi Ravagnan 1, Santos A. Susin 1,
Eric Daugas 1, 2, Kumiko Samejima 3,
Naoufal Zamzami 1, Markus Loeffler 1,
Paola Costantini 1, Karine F. Ferri 1,
Theano Irinopoulo 4, Marie-Christine
Prévost 5, William C. Earnshaw 3,
and Guido Kroemer1
1 Centre
National de la Recherche Scientifique, ERS 1984, 19 rue Guy Môquet, F-94801
Villejuif, France; 2 Assistance
Publique - Hôpitaux de Paris, Service de Néphrologie B, Hôpital Tenon, 20 rue
de la Chine, F-75020, France; 3 University
of Edinburgh, Institute for Cellular and Molecular Biology, Edinburgh EH93JR,
Midlothian, Scotland; 4 INSERM U430, Hôpital
Broussais, 14 rue Didot; F-75014 Paris, France; 5 Unité d'Oncologie Virale, Institut Pasteur, 28 rue du Dr. Roux,
F-75724 Paris cedex 15, France
Apaf-1-/-
or caspase-3-/- cells treated with staurosporine (STS) manifest
apoptosis-associated alterations including the translocation of AIF from
mitochondria to nuclei, large scale DNA fragmentation and initial chromatin
condensation (stage 1). However, when compared to normal control cells, Apaf-1-/-
or caspase-3-/- cells fail to exhibit oligonucleosomal chromatin digestion and
a more advanced pattern of chromatin condensation (stage 2). Microinjection of
such cells with recombinant AIF only causes peripheral chromatin condensation,
whereas microinjection with activated caspase-3 or its down-stream target CAD
causes a more pronounced type of chromatin condensation. This observation has
been confirmed in a cell free system. When added to purified HeLa nuclei, AIF
causes stage-1 chromatin condensation and large-scale DNA fragmentation, whereas
CAD induces stage-2 chromatin condensation and oligonucleosomal DNA degradation.
To suppress the chromatin condensation-inducing activity of cytoplasmic extracts
from cells undergoing apoptosis, both CAD and AIF must be neutralized
concomitantly. As a result, at least two redundant parallel pathways may lead to
chromatin processing during apoptosis. One of these pathways involves caspases,
ICAD, as well as CAD, and leads to oligonucleosomal DNA fragmentation and
advanced chromatin condensation. The other pathway, caspase-independent,
involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin
condensation.
Session
II: Mitochondria and cytotoxic drugs
Sunday April 2nd
Differential
role of caspase 8 and Bid cleavage during CD95- and anticancer drug induced
caspase activation
Division of Immunology and
Cell Bilogy, University of Muenster, Germany
* Department of Radiation
Oncology, University of Tuebingen, Germany
Multiple
interconnected pathways are involved in CD95- and anticancer drug-induced
apoptosis. One common feature for the receptor-dependent and -independent
pathways to apoptosis is the activation of the executioner caspase 3. Both
pathways can be differentiated based on caspase 8 or caspase 9 requirement for
caspase 3 activation. To analyze the relative position of caspases 8, 9, 6, and
3 in the caspase cascade of CD95- and anticancer drug-induced apoptosis we
utilized caspase 8 deficient, and BclXl or caspase 9 dominant negative (9DN)
Jurkat cell line. Although caspase 8 has been previously reported to be
dispensable for death receptor-independent apoptosis, we observed the activation
of caspase 8 in response to etoposide or staurosporine. In addition, we found
that Bid, known as caspase 8 substrate, is also activated by anticancer drugs in
the presence or absence of caspase 8. Caspases 9, 6, and 3 activation were
inhibited in Jurkat-BclXL and ñ9DN cell lines stimulated with anticancer drugs.
Interestingly, caspase 8 and Bid activation were also inhibited in these cell
lines indicating that caspase 8 and Bid activation occur downstream of
mitochondria in anticancer drug mediated apoptosis. We conclude that caspase 8
plays a role of an executioner caspase in anticancer drug induced apoptosis in
contrast to CD95- induced apoptosis. We propose that Bid not only constitutes a
link between receptor dependent and independent pathways to apoptosis but also
enables an amplification loop for the execution of the anticancer drug induced
apoptosis.
Session
III: Signaling
Sunday April 2nd
Activation
of the Caspase Cascade and the Effect of Ras in Detachment Induced Apoptosis
M. Rytömaa and J.
Downward
Signal
Transduction Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields,
London WC2A 3PX, U.K
Epithelial
and endothelial cells undergo apoptosis upon detachment from the matrix, a
phenomenon called anoikis. It is known that the activated Ras is able to protect
cells from anoikis through its downstream effectors PI 3-kinase and Akt/PKB. The
pathways involved in anoikis and the way Ras effectors work are not yet clear
and this work gives some new insight to this area. We have shown that detachment
of MDCK cells from the matrix leads to the activation of caspase-8 and
downstream effector caspases. Interestingly, in MDCK cells stably expressing V12
Ras, v-Akt, or dominant negative FADD, no detachment-induced caspase-8
activation or DNA fragmentation were detected. The activation of caspases can be
blocked by inhibitor peptides, such as z-VAD, which also protects cells from
anoikis. Detachment of MDCK cells from matrix also leads to release of
cytochrome c form mitochondria to cytoplasm and this is blocked by V12 Ras as
well as v-Akt. Importantly, the release of cytochrome c is caspase dependent
since z-VAD is able to prevent it. However, an other downstream effector of Ras,
Raf, is not able to block cytochrome c release even through it is blocking
detachment induced apoptosis.
Regulation of Cell Survival by
the MAPK-activated kinase, RSK1 and the Phosphoinositide-Dependent Kinase, PDK1
1 Department of Cell
Biology, Harvard Medical School, Boston, Massachusetts, 02115, USA
2 Department of Pediatric
Hematology/Oncology, Dana Farber Cancer Institute, Boston, Massachusetts 02115,
USA
Aberrant
cell death or inappropriate cell survival are primary causes in diseases as
diverse as neoplastic transformation, autoimmunity, and neurodegeneration. Over
the last few years a handful of signaling molecules have been identified as
transducers of growth-factor-regulated cell survival signals. Both the
PI3-kinase/Akt and the MEK/MAPK signaling casettes constitute key regulatory
pathways promoting cell survival. Whereas the molecular basis of Akt’s
pro-survival signal is multifactorial and a number of downstream effectors have
been identified, the downstream effectors of the mammalian MEK/MAPK cell
survival signal have not been previously described. These studies identify a
pro-survival role for a downstream target of the MEK/MAPK signaling pathway,
RSK1. Experiments in vitro and in vivo demonstrate that RSK1 directly
phosphorylates BAD at the serine residues critical for abrogation of BAD’s
pro-apoptotic function. A novel constitutively active RSK1 mutant confers
constitutive BAD phosphorylation and protection from IL-3 withdrawal-induced
cell death. Conversely, kinase-inactive RSK1 mutants antagonize both BAD
phosphorylation and the MEK survival signal. BAD mutations that prevent
phosphorylation by RSK1 also inhibit RSK1-mediated cell survival. Additionally,
we have identified a novel inhibitor of Phosphoinositide-Dependent Kinase-1
(PDK-1). PDK1 activates a growing number of kinases in the AGC kinase
superfamily including PKA, PKC, Akt, RSK, SGK, and S6 kinases-1 and -2. PDK-1 is
thus positioned at the apex of numerous kinase cascades governing multiple
aspects of normal and aberrant cellular functions including cell survival.
Consistent with the requirement for PDK1 in activating the pro-survival, BAD
kinases Akt, RSK and PKA, pharmacological inhibition of PDK1 eliminated IL-3
dependent BAD phosphorylation and led to cell death in IL-3-dependent 32D cells.
Session
III: Signaling
Sunday April 2nd
A
nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of
c-Abl and JNK
Daniela
Barilá1, Raffaella Mangano, Stefania
Gonfloni, Marina Moro, Jana Kretzschmar, Dirk Bohmann, Roberto Testi2
and Giulio Superti-Furga
European Molecular Biology
Laboratory Meyerhofstrasse 1 69117 Heidelberg, Germany. 1present
address: University of Rome “Tor Vergata” Department of Experimental
Medicine and Biochemical Sciences” Via di Tor Vergata 135 00133 Rome, Italy. 2University
of Rome “Tor Vergata” Department of Experimental Medicine and Biochemical
Sciences” Via di Tor Vergata 135 00133 Rome, Italy
The
nuclear function of the c-Abl tyrosine kinase is not well understood. In order
to identify nuclear substrates of Abl, we constructed a constitutively active
and nuclear form of the protein. The expression of a constitutively active form
of Abl in the nucleus is highly toxic for the cell. We found that active nuclear
Abl efficiently phosphorylates c-Jun, a transcription factor not previously
known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on
tyrosine 170, both proteins interacted via the SH2 domain of Abl. Surprisingly,
elevated levels of c-Jun activated nuclear Abl, resulting in activation of the
JNK serine/threonine kinase. This phosphorylation circuit generates nuclear
tyrosine phosphorylation and represents a reversal of previously known
signalling models. We are currently investigating the structural requirements of
Abl and the possible role of Jun phosphorylation in the induction of cell death.
Departamento
de Imunologìa y Oncologìa. Centro Nacional de Biotecnologìa
The
role of dimerization in G protein-coupled receptor signaling has recently been
demonstrated. Two different families of these receptors appear to follow
distinct strategies during the formation of this supramolecular complex. The
GABA receptor is composed of two subunits that require close association to
transmit the GABA signal within the neuron, allowing activation of potassium
channels. The chemokine receptor undergoes a ligand-mediated homodimerization
process that is required for Ca2+ flux
and chemotaxis. Here we show that, in the chemokine response, heterodimerization
is also permitted between given receptor pairs, specifically between the CCR2
and the CCR5 receptors, but not between the CCR2 and the CXCR4 receptors. This
heterodimerization results in recruitment of the signaling complex associated to
both receptors, as measured by association of JAK tyrosine kinases and STAT
transcription factors to the receptor complex. This has functional consequences,
since calcium responses are triggered in the presence of MCP-1 and RANTES at
concentrations 10-100 fold lower than the threshold for each chemokine when
added individually. In addition, expression of a CCR2 death receptor with the
Tyr139Phe mutation, which impairs CCR2 responses, blocks responses triggered
through the CCR5 receptor. These results may be relevant for in
vivo chemotaxis under limiting chemokine concentrations. They also show the
structural constraints on chemokine receptors for homo- and heterodimer
formation in the presence of the appropriate ligands, and the implications of
dimer formation for increasing the sensitivity and dynamic range of the
chemokine response. The role of dimerization in G protein-coupled receptor
signaling has recently been demonstrated. Two different families of these
receptors appear to follow distinct strategies during the formation of this
supramolecular complex.
Session
III: Signaling
Sunday April 2nd
The
GABA receptor is composed of two subunits that require close association to
transmit the GABA signal within the neuron, allowing activation of potassium
channels. The chemokine receptor undergoes a ligand-mediated homodimerization
process that is required for Ca2+ flux
and chemotaxis. Here we show that, in the chemokine response, heterodimerization
is also permitted between given receptor pairs, specifically between the CCR2
and the CCR5 receptors, but not between the CCR2 and the CXCR4 receptors. This
heterodimerization results in recruitment of the signaling complex associated to
both receptors, as measured by association of JAK tyrosine kinases and STAT
transcription factors to the receptor complex. This has functional consequences,
since calcium responses are triggered in the presence of MCP-1 and RANTES at
concentrations 10-100 fold lower than the threshold for each chemokine when
added individually. In addition, expression of a CCR2 death receptor with the
Tyr139Phe mutation, which impairs CCR2 responses, blocks responses triggered
through the CCR5 receptor. These results may be relevant for in
vivo chemotaxis under limiting chemokine concentrations. They also show the
structural constraints on chemokine receptors for homo- and heterodimer
formation in the presence of the appropriate ligands, and the implications of
dimer formation for increasing the sensitivity and dynamic range of the
chemokine response.
Session
IV: Neurology
Sunday April 2nd
Fas/CD95/APO-1
can Function as a DeathReceptor for Neuronal Cells in Vitro and in Vivoand is
Upregulated Following Cerebral Hypoxic-Ischemic Injury to theDeveloping Rat
Brain
Ursula Felderhoff-Mueser1,2, Deanna L. Taylor1,
KirstyGreenwood1, Mary Kozma1,
DietgerStibenz3, Umesh C. Joashi1, A.
David Edwards1 and Huseyin Mehmet1
1Weston
Laboratory,Division of Paediatrics, Obstetrics and Gynaecology, Imperial College
ofScience, Technology and Medicine, Hammersmith Hospital, Du Cane Road,London
W12 0NN, UK and 2Clinic for
Neonatology and 3Institutfür
Diagnostikforschung (IDF) der FU Charité Children´s Hospital,Virchow Campus,
Humboldt University 13353 Berlin, Germany
Fas/CD95/Apo-1
is a cell surface receptor that transduces death signalsfollowing activation and
has been implicated in triggering apoptosis ininfected or damaged cells in
disease states. Apoptosis is a major mechanismof neuronal loss following
hypoxic-ischaemic injury to the developing brain, although the role of Fas in
thisprocess has not been studied in detail. In the present study, we
haveinvestigated the expression and function of Fas in neuronal cells invitro
and in vivo. Fas was found to be expressed in the 14 day old rat brain, with
strongestexpression in the cortex, hippocampus and cerebellum. Cross-linking of
Fasinduced neuronal apoptosis both in neuronal PC12 cells in culture and
following intracerebral injection in vivo, indicating that neuronal Fas was
functional as a death receptor. Fasinduced death was caspase dependent in
primary neuronal cultures and wasblocked by the selective caspase 8 inhibitor
IETD. Finally, cerebral hypoxia ischaemia resulted in a strong lateralised
upregulation of Fas inthe hippocampus, that peaked six to twelve hours after the
insult and wasgreater on the side of injury. These results suggest that Fas may
beinvolved in neuronal apoptosis following hypoxic-ischaemic injury to the
developing brain.
Differential Effects of
Bcl-2, Caspase Inhibition and PARP Deletion on Survival of Intracerebral
Neuronal Transplants
1Molecular Toxicology,
Faculty of Biology, Konstanz University, D-78457 Konstanz, Germany. 2Section for
Neuronal Survival, Wallenberg Neuroscience Center, Lund University, S-22362 Lund,
Sweden
The
causes of death of transplanted neurons are not known in detail, but apoptotic
mechanisms are likely to play a role. We examined whether overexpression of
Bcl-2, inhibition of caspases or deletion of poly-(ADP-ribose) polymerase (PARP)
may enhance the survival of grafted neurons. Initially, cells were prepared from
mice overexpressing human Bcl-2 or lacking PARP, or from their wild-type
littermates. The bcl-2 transgene resulted in protection of neurons from
staurosporine exposure or growth factor withdrawal in vitro. To model
pretransplantation stress more closely in vitro, we stored dissociated cells for
eight hours in the same type of medium as used for intracerebral transplantation.
This resulted in massive cell death as quantified by lactate
dehydrogenase-release, and increased DNA-fragmentation. This cell loss was
strongly reduced by different caspase inhibitors, but neither Bcl-2 nor PARP had
any significant protective effect. Finally, cells from rats, mice or human
embryos were grafted into the striatum of hemiparkinsonian rats. Caspase
inhibition significantly increased neuronal survival, functional recovery and
fibre outgrowth after several weeks. Bcl-2 or PARP had no effect in murine
grafted neurons. Cell death in neuronal transplants seems to involve
PARP-independent apoptotic mechanisms that involve the activation of caspases,
but that bypass negative regulation by Bcl-2.
Session
IV: Neurology
Sunday April 2nd
APOPTOSIS
CONTRIBUTES TO NEURONAL LOST FOLLOWING SPINAL CORD TRAUMA
Deana Demjen, Ana
Martin-Villalba, Susanne Kleber, Manfred Zimmermann, Johannes Schenkel
Institute of Physiology and
Pathophysiology, University of Heidelberg, Germany
The
spinal cord is made of the motor and sensory pathways between brain and body.
Injury of the spinal cord results in a disruption of these pathways and in
permanent dissability of a typically young patient. There is growing evidence
that neuronal death following spinal cord trauma may be in part an apoptotic
event. We investigated the presence of apoptotic cells in an animal model of
spinal cord trauma. Numerous TUNEL-positive cells appeared both adjacent to and
distant from the injured site 24 hours after transection of the spinal cord.
TUNEL-positive cells were still present but to a lesser extent three days after
the injury. Apoptosis was accompanied by increased expression of the
transcription factor c-Jun and of the death-ligands TNF and CD95-Ligand. Further
examination of gld mice (expressing a mutated CD95-Ligand protein) and
TNF-knockout mice are currently being undertaken. Our finding helps unraveling
the mechanism underlying neuronal lost following spinal cord injury.
SYNERGISTIC INHIBITION OF
CD95 LIGAND AND TNF ATTENUATES BRAIN DAMAGE IN STROKE
Ana Martin-Villalba1,2,
Johannes Schenkel1, Susanne Kleber1,
Johannes Vogel1, Henning Walczak2,
Heinz Krestel3, Michael Hahne4,
Peter H. Krammer2
1
Department of Physiology, University of Heidelberg, Heidelberg 2Tumorimmunology
Programm, German Cancer Research Center, Heidelberg
3 Departments of Molecular Neuroscience and Cell Physiology, Max Planck
Institute for medical research, Heidelberg, 4Department
of immunology, National Center of Biotechnology, Madrid
Stroke
is the most common cause of death in the western world and still its
therapeutical possibilities remain low. There is evidence that the
death-inducing ligands TNF and CD95-Ligand are involved in triggering ischemic
neuronal death. We examined the contribution to brain damage of these ligands in
a rodent model of stroke. In the present study we show that mice deficient in
TNF (tnf -/-) and in functional
CD95L (gld) are protected against
brain ischemia. This protection is greatly enhanced in mice lacking both death
ligands (gld/tnf
-/-). In wild-type mice
therapeutic application of antibodies against TNF and CD95L diminished infarct
volumes and significantly improved survival of the animals. Most importantly,
intact functionality of rescued neurons in vivo was demonstrated by the almost
normal locomotor performance exhibited by treated animals. Thus, neutralization
of CD95L and TNF may support the treatment of stroke.
Session
IV: Neurology
Sunday April 2nd
C. Volbracht, M. Leist, P. Nicotera
Department
of Molecular Toxicology, University of Konstanz, D-78457 Konstanz, Germany
Early
loss of neurites followed by delayed damage of neuronal somata is a feature of
several neurodegenerative diseases. Microtubule depolymerizing agents such as
colchicine or nocodazol were used in murine cerebellar granule cells (CGC) to
mimic these pathological changes. Colchicine (1 µM) or nocodazol (1 µM)
triggered fragmentation of microtubuli resulting in axo-dendritic degeneration
followed by apoptosis. The microtubule stabilizing agent taxol prevented both
fragmentation of microtubules and cell death. Apoptosis was accompanied by
activation of caspases and phosphatidylserine (PS) translocation to the outer
surface of the plasma membrane. Caspase inhibitiors prevented the initial
apoptotic changes, although the axo-dendritic net was lost. Depletion of
intracellular ATP by the mitochondrial poisons NO and MPP+ or by deoxyglucose
prevented activation of caspases, exposure of the phagocytosis marker PS and
apoptotic death, while the primary effect of microtubule disruption still
occured. Conversely, repletion of cellular ATP by enhanced glycolysis restored
all apoptotic features. These findings indicate that the intracellular ATP level
may determine whether degenerating neurons are rapidly removed by apoptosis or
whether they persist as severely damaged cells.
V. De
Laurenzi, D. Barcaroli, A. Costanzo*, M. Ranalli, M. Levrero* and G. Melino
IDI-IRCCS, Biochemistry Lab, c/o Department of
Experimental Medicine, University of Rome Tor Vergata; 00133 Rome, Italy. *Gene
Expression Laboratory, Fondazione A. Cesalpino, University La Sapienza, 00161
Rome, Italy
The
p53-homologue p73, has been mapped to a region (1p36.33) which is frequently
deleted in neuroblastomas, suggesting that its alterations may play a role in
the development of tumours of the nervous system. However, unlike p53, mutant
p73 has rarely been found in human tumours.
Few studies have directly investigated functions of p73, and their
activities have been largely assumed based on their structural similarities with
the p53 homologue. Here we show
that:
(1)
p73 is expressed in human cells as several alternatively spliced forms, a,
b, g, d, e
and z which show different abilities to homo/hetero-dimerize
with each other and with p53. (2) Induction of apoptosis by p73 in response to cisplatin is
regulated by c-Abl. DNA damaging agents, through MLH1 and c-Abl, stabilize the
protein level of p73 by increasing its half-life. (3) p73 functions as trigger of differentiation in neuroblastoma cell
lines. Indeed, p73 expression is upregulated during neuroblastoma cell
differentiation induced by dimethylsulphoxide and retinoic acid. Our results
indicate that the overexpression of all p73 isoforms is sufficient to trigger
the neuronal differentiation of neuroblastoma cells in vitro, while the
transcriptionally inactive Ala156Val mutants (or dominat negative p73D84) of all isoforms failed to do so. p73-induced
differentiation was accompanied by MYCN down regulation, NCAM transactivation,
pRB down regulation. The ability of p73 to act as a determinant of neuronal
differentiation is specific and it is not shared by their homologue p53, which
is indeed capable to induce cell death under the same conditions. In conclusion,
p73 seems involved both in tumour suppression and development.
Session
IV: Neurology
Sunday April 2nd
Cleavage
of Alzheimer's b-amyloid precursor protein by g-secretase liberates an
intracellular peptide that promotes apoptosis
T-cell apoptosis Unit,
Laboratory of Cellular and Molecular Immunology, NIAID, National Institutes of
Health, Bethesda, Maryland 20892
It
is believed that Alzheimer's Disease (AD) is a result of extracellular
accumulation of the amyloidogenic form of Ab peptide (Ab42). Ab is produced by
the sequential proteolysis of the b-amyloid precursor protein (APP) by b- and g-
secretases1. Some Alzheimer's cases are familial (FAD) and are due to mutations
in APP itself, presenilin-1 (PS1) or presenilin-2 (PS2) (ref. 2-5). Presenilins
are highly homologous proteins that are required for g-secretase activity6. Of
more importance, mutations in presenilins and APP are theorized to cause FAD by
augmenting the formation of Ab42 (ref. 7,8). Aside from its pathological
relevance, the physiological consequences of APP proteolysis remains unknown.
The discovery that the three FAD genes enhance programmed cell death (PCD) has
hinted at a potential link between the dysregulation of apoptosis and
neurodegeneration observed in AD9-11. The parallelism between apoptotic and
amyloidogenic effects suggests that the FAD proteins could modulate apoptosis by
directing APP cleavage. Here we report that the APP intracellular domain (AID)
fragment liberated after g-secretase cleavage acts as a positive regulator of
apoptosis. Thus, overproduction of AID, as in the case of FAD, could add to the
toxic effect of Ab42 aggregates in Alzheimer's patients further accelerating
neurodegeneration.
Session
V: TRAIL Signaling
Monday
April 3rd
TRAIL-induced
apoptosis is not inhibited by overexpression of Bcl-2 or Bcl-xL
Department
of Immunogenetics, German Cancer Research Center, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany
Tumor
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a novel
member of the TNF family and has recently been shown to exert tumoricidal
activity in vivo in the absence of any
observable toxicity. However, the signaling pathways triggered by TRAIL
stimulation and the mechanisms involved in resistance against TRAIL-mediated
apoptosis are still poorly defined. Therefore, we addressed the question to
which extent the mitochondrial pro-apoptotic potential contributes to
TRAIL-induced apoptosis and whether protection of mitochondria interferes with
apoptosis mediated by TRAIL. We show that TRAIL-induced apoptosis involves late
dissipation of mitochondrial membrane potential (ΔΨm) and cytochrome c release that follows activation of caspase-8 and
caspase-3 and induction of DNA fragmentation. In addition, inhibition of
caspase-8 but not caspase-9 or ñ3 prevents mitochondrial permeability
transition (PT) and apoptosis. Various cell lines overexpressing the
anti-apoptotic proteins Bcl-2 or Bcl-xL
are not or only marginally protected against TRAIL-induced apoptosis. In
contrast, apoptosis induced by the chemotherapeutic drug etoposide was severely
impaired in these cells. Thus, TRAIL induces apoptosis via a caspase signaling
cascade that executes apoptosis independently of the pro-apoptotic machinery of
mitochondria. Since most chemotherapeutic drugs used in the treatment of
malignancies lead to apoptosis by engagement of the mitochondrial pro-apoptotic
machinery our data suggest a novel alternative anti-tumor strategy by using
TRAIL against Bcl-2- or Bcl-xL-overexpressing
tumors that have acquired resistance to conventional anti-tumor chemotherapy.
Structure
of the apoptosis inducing TRAIL‑DR5 complex
Juthathip Mongkolsapaya1, Jonathan M. Grimes2,
Nan Chen1, Xiao‑Ning Xu1, David I Stuart2,
E. Yvonne Jones2 and Gavin R Screaton1
1M3C
Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital.
Oxford OX3 9DS, UK; 2 Structural Biology, Wellcome
trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford, OX3
7BN, UK
Apoptosis
plays a significant role in the immune system in processes such as T cell
development, cytotoxicity and T cell homeostasis. It can be initiated by the
interaction of cell surface receptors with their cognate ligands. A paradigm for
this interaction is TNF and TNF‑R1. These two proteins define two large
families of type II and type I membrane proteins respectively. DR4 and DR5,
which are the members of the TNF‑R family, both interact with the ligand
TRAIL. TRAIL also interacts with two other receptors, DcR1, and DcR2, which have
no or a truncated death domain and act as decoys preventing apoptotic signaling
through DR4 and DR5. We have solved the crystal structure of a complex between
TRAIL and DR5 at 2.2 A. Three receptor molecules are arranged around a central
TRAIL trimer. An additional 15 amino acid loop and variability in the D and E
strands of the TRAIL together with a change in the alignment of the two receptor
domains are among a number of unique surface features which confer specificity
of TRAIL for its receptors.
Session
V: Trail Signaling
Monday April 3rd
Caspase-8 and FADD/Mort1
are necessary components of the TRAIL-R2 Death-Inducing Signalling Complex
(DISC)
Martin R. Sprick1,
Markus A. Weigand1, Anne Grosse-Wilde1,
Melanie Logemann1, Heiko Stahl1, Eva
Rieser1, Peter Juo2,
John Blenis2, Peter H. Krammer1,
and Henning Walczak1
1Tumorimmunology
Program, German Cancer Research Center, Heidelberg, Germany
2Department
of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA
The
TNF-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis
in a number of tumor cell lines while being non-toxic to normal tissues.
Currently, five receptors have been shown to bind TRAIL, namely TRAIL-Receptor†(TRAIL-R)†1
to TRAIL-R4 and osteoprotegerin. Two of these receptors, TRAIL-R1 and TRAIL-R2,
have been shown to induce apoptosis in a caspase-dependent manner. Recently, a
number of studies have addressed the composition of the death-inducing
signalling complex (DISC) associated with TRAIL-R1 and TRAIL-R2 by
overexpressing at least one putative component of the TRAIL-DISC. Yet, it
remained unclear which molecules are recruited to the DISC upon TRAIL
stimulation under native conditions. We thus compared the protein complex
induced by stimulation with TRAIL and
CD95L under non-overexpression conditions in various cell lines. We could
demonstrate that both FADD and Caspase-8 are necessary for the formation of the
TRAIL-R2-DISC.
Active
effector caspases and cleaved cytokeratins are sequestered into cytoplasmic
inclusion following TRAIL-induced apoptosis
MRC Toxicology Unit,
Hodgkin Building, University of Leicester, P.O. box 138, Lancaster Road,
Leicester LEI 9HN, UK
Tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, in
transformed human breast epithelial MCF-7 cells, resulted in a time-dependent
activation of the initiator caspases-8 and -9 and the effector caspase-7.
Cleavage of caspase-8 and its preferred substrate, Bid, preceded processing of
caspases-7 and -9, indicating that caspase-8 is the apical initiator caspase in
TRAIL-induced apoptosis. Using transient transfection of C-terminal tagged green
fluorescent protein fusion constructs, caspases-3, -7 and -8 were localized
throughout the cytoplasm of MCF-7 cells. TRAIL-induced apoptosis resulted in
activation of caspases-3 and –7 and the redistribution of most of their
detectable catalytically active small subunits into large spheroidal cytoplasmic
inclusions, which lacked a limiting membrane. These inclusions, which were also
induced in untransfected cells, conteined cytokeratins 8, 18 and 19, together
with both a phosphorylated form and a caspase-cleavage fragment(s) of
cytokeratin 18. Similar inclusions were found follwing TRAIL treatment of lung,
cervical and non-transformed breast epithelial cells. We propose that effector
caspase-mediated cleavage of cytokeratins, resulting in disassembly of the
cytoskeleton and formation of cytoplasmic inclusions, may be a characteristic
feature of epithelial cell apoptosis and that sequestration of active caspases
within these inclusions may limit their potential damage to neighbouring cells.
Session
VI: Trail biology
Monday April 3rd
Markus A. Weigand, Martin
Sprick, Anne Grosse-Wilde, Silke Maier, Dorothee Suess, Eva Rieser, Peter H.
Krammer, and Henning Walczak
Department of
Immunogenetics, German Cancer Research Center, Im Neuenheimer Feld 280, 69120
Heidelberg, Germany
Peripheral
deletion of T cells is essential for the homeostasis of the immune system. This
mechanism involves the CD95 and TNF system. Recently, also the TRAIL system has
been postulated to contribute to peripheral deletion of T cells. However, the
regulation and function of TRAIL and its receptors on peripheral T cells and
intracellular apoptotic signaling pathways are largely unknown. Using monoclonal
antibodies specific for the individual TRAIL-receptors we demonstrate that on
freshly isolated human T†cells TRAIL-R1 and TRAIL-R2 are significantly
expressed, while there is only marginal expression of TRAIL-R3 and TRAIL-R4.
Whereas the CD95 receptor is upregulated upon stimulation of T cells with PHA/IL-2,
TRAIL-receptor surface expression is not increased. Increased CD95 receptor
expression is associated with increased sensitivity to CD95-mediated apoptosis.
In contrast, peripheral T†cells are insensitive to TRAIL-induced apoptosis.
This phenotype does not change during culture with various physiological stimuli.
T cells can, however, be sensitized for TRAIL with cycloheximide or actinomycin
D. Apoptosis induction by plate bound agonistic antibodies revealed that
TRAIL-induced apoptosis in sensitized T†cells is mainly mediated by TRAIL-R2,
while there is no significant role of TRAIL-R3 and TRAIL-R4 in determining the
resistance of peripheral T†cells to TRAIL. TRAIL-induced apoptosis in T†cells
is characterized by cleavage of caspase-8, caspase-3, PARP and DNA†fragmentation.
These results suggest differential roles for the CD95 and TRAIL systems in the
homeostasis of the immune system.
INVOLVEMENT OF TRAIL RECEPTOR 2 IN THYMIC
NEGATIVE SELECTION
A
Katharina Simon & Gavin R Screaton
Developing
T lymphocytes bearing self reactive antigen-receptors undergo apoptosis. This
process called negative selection requires T cell mediated recognition of
antigenic peptides+MHC as well as caspases. It is not known whether surface
molecules other than the TCR such as death receptors are involved in negative
selection. We have investigated the role of Trail and and its receptors 1 and 2
in the human thymus where negative selection can be mimicked by anti-CD3 or
superantigens. Thymocytes are not susceptible to an anti-Trail receptor 2
(Trail-r-2) polyclonal antibody inducing death in the T cell line Jurkat.
However, in the presence of a sublethal dose of anti-CD3 thymocytes die in
response to the anti-Trail-r-2 antibody as well as in response to crosslinked
soluble Trail. Furthermore in human thymus organ cultures lethal doses of
anti-CD3 treatment can be inhibited by soluble Trail. Finally the depletion of
superantigen-specific V beta families in thymus organ culture is inhibited by a
soluble Trail-r-2 but not by soluble Fas or TNFr. These results indicate that
Trail and Trail-r-2 play a role in anti-CD3 and superantigen induced deletion of
thymocytes. It remains to be shown whether this is true in antigen-induced
deletion and whether other death receptors play a role in negative selection.
Session
VI: Trail biology
Monday April 3rd
Regulation
of TRAIL sensitivity in primary and transformed human keratinocytes
M. Leverkus1,
M. Neumann2, T. Mengling1,
E.- B. Bröcker1, P. H. Krammer3 and
H.Walczak3
Department of 1Dermatology
and 2Pathology, University of Würzburg,
Germany, 3Tumor Immunology Program,German
Cancer Research Center, Heidelberg, Germany
Tumor
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been shown
to exert potent cytotoxic activity against many tumor cell lines but not against
normal cells. It has been hypothesized that this difference in TRAIL sensitivity
between normal and transformed cells might be due to the expression of the non
death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and TRAIL-R4, presumably by
competition for limited amounts of TRAIL. In order to assess the regulation of
resistance versus sensitivity to TRAIL in primary as well as transformed
keratinocytes, we examined TRAIL sensitivity, TRAIL receptor expression and
intracellular signalling events induced by TRAIL. Although TRAIL induced
apoptosis in primary as well as transformed keratinocytes, a marked difference
in sensitivity could be observed with primary keratinocytes (PK) being five-fold
less sensitive to TRAIL than transformed keratinocytes (TK). Yet, both cell
types exhibited similar TRAIL receptor surface expression, suggesting that
expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of sensitivity
to TRAIL. Biochemical analysis of the signalling events induced by TRAIL
revealed that PK could be sensitized for TRAIL and, similarly, for TRAIL-R1-,
and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide
(CHX). This sensitization concomitantly resulted in processing of caspase-8,
which did not occur in TRAIL-resistant PK. These data indicate that an early
block of TRAIL-induced apoptosis was present in PK as compared to TK or PK
treated with CHX. Interestingly, cFLIP levels, high in PK and low in TK and
several other squamous cell carcinoma cell lines, decreased rapidly after
treatment of PK with CHX correlating with the increase in TRAIL sensitivity and
caspase-8 processing. Furthermore, ectopic expression of cFLIPL
in TK by transfection with a cFLIPL
expression vector resulted in resistance to TRAIL-mediated apoptosis of these
cells. Thus, our results demonstrate that TRAIL sensitivity in PK is primarily
regulated at the intracellular level rather than at the receptor level.
Session
VII: Clinical aspects of apoptosis
Monday
April 3rd
FLIP
(FLAME-1/I-FLICE) and Bcl-xL protect thyrocytes from Fas-mediated destruction in
Graves’ disease
Giorgio
Stassi1, Diana Di Liberto1, Ann Zeuner2,
Matilde Todaro1, Felicia Farina1, Antonella
Stoppacciaro3, Luigi Ruco3, Francesco Grignani4
and Ruggero De Maria2.
1Istituto
di Anatomia Umana, University of Palermo; 2Istituto di Patologia
Generale, University of Catania; 3Dipartimento di Medicina
Sperimentale e Patologia, University of “La Sapienza”, Roma; 4Istituto
di Clinica Medica, University of Perugia
We
previously demonstrated that following autoimmune inflammation, autocrine or
paracrine interactions between Fas and its ligand (FasL) mediate thyrocyte
destruction in Hashimoto’s thyroiditis (HT). Differently from HT, thyroid
autoimmune processes leading to Graves’ disease (GD) result in
autoantibody-mediated TSH receptor stimulation rather than in thyrocyte
destruction. Given that thyrocytes constitutively express FasL, we have
investigated why autoimmune inflammation in GD does not seem to proceed to
Fas-mediated thyrocyte destruction. Immunohistochemical and immunoblot analyses
of thyroid samples showed that all GD thyrocytes constitutively express FasL,
while Fas was mostly detected in thyrocytes in proximity to infiltrating
lymphocytes. However, differently from HT, we could not detect neither apoptosis
nor caspase activation in vivo in Fas-positive GD thyrocytes. Importantly,
extensive searching for pro-apoptotic and anti-apoptotic factors in thyrocytes
showed that HT thyrocytes overexpressed caspase-8 and caspase-3, the dominant
mediators of Fas-induced apoptotic cascade. By contrast, GD thyrocytes
overexpressed FLIP and Bcl-xL, two major anti-apoptotic proteins. Importantly,
analysis of freshly isolated primary thyrocytes transduced with either FLIP or
Bcl-xL showed that both proteins were able to completely block FasL-induced
apoptosis in thyrocytes, suggesting a major role for FLIP or Bcl-xL in
protection from thyrocyte depletion in GD. Taken together, our results provide
molecular evidence explaining the presence or the absence of thyrocyte
destruction in thyroid autoimmune diseases.
G.
Condorelli, M.Latronico, R. Roncarati, J.K. Aycock, F. Farina, John Ross, Jr.,
G. Stassi
II Faculty of medicine,
“La Sapienza” University, Rome; Kimmel Cancer Center, Thomas Jefferson
University; UCSD, La Jolla, CA; Human Anatomy Section, University of Palermo
Heart
failure (HF) is a final pathological condition in cardiovascular diseases of
various etiology. Cardiomyocyte cell loss characterizes HF and accounts for the
decreased cardiac function. We have investigated whether cardiomyocyte apoptosis
plays a causal role in HF. As model systems, we used isolated neonatal
cardiomyocytes, transgenic animals, a rat model of pressure-overload HF and
human specimens from HF patients. We determined the contribution of the pro- and
anti-apoptotic pathways generated from the TNF-a
receptors in cardiomyocyte survival. Moreover, we are determining the relative
contribution of pathways generated from gp130 in rescuing cardiomyocytes from
oxidative stress. We have also associated cardiomyocyte apoptosis with
decompensation of cardiac function during pressure overload. To determine
whether apoptotic genes play a causal role in HF, transgenic mice expressing
selectively in the cardiomyocyte compartment apoptotic genes were generated.
Pro-caspase 3 overexpression induced a mild phenotype in unstressed animals; on
the contrary, mice were prone to death after ischemia-reperfusion injury. Bcl-xl
overexpression rescues the HF phenotype of genetically induced HF. All together,
our data strongly suggest that by modulating the activity of apoptotic genes, it
is possible to alter the course of HF.
Session
VII: Clinical aspects of apoptosis
Monday
April 3rd
Cyclosporin
A-insensitive CD95 (Fas) ligand expression in the pathogenesis of acute
intestinal Graft-versus-Host Disease
Christoph Wasem*, Corina
Frutschi*, Diana Arnold*, Tesu Liná, Douglas R. Greenß, Christoph Mueller*,
and Thomas Brunner*
*Division of
Immunopathology, Institute of Pathology, University of Berne, Switzerland, áGI
Division, Department of Medicine, Northwestern Medical School, Chicago, IL, USA,
and ßLa Jolla Institute for Allergy and Immunology, San Diego, CA, USA
Graft-versus-host
disease (GvHD) represents a serious complication after bone marrow
transplantation. Anti-host immune responses are often treated with the
immunosuppressant cyclosporin A (CsA), however, with limited success. We have
previously shown in an experimental model for acute intestinal GvHD that
expression of CD95 (Fas/APO-1) ligand by donor-derived T cells and resulting
cytotoxicity is a crucial disease-promoting factor. We have thus investigated
whether CsA can block activation-induced CD95 ligand expression by donor-derived
T cells and whether CD95 ligand expression is impaired during GvHD. Whereas CsA
was found to efficiently inhibit activation-induced CD95 ligand expression in
control splenic and intestinal intraepithelial lymphocytes, it only marginally
affected CD95 ligand expression by activated donor-derived T cells during GvHD.
This was found to be the result of extensive in vivo accumulation and storage of intracellular CD95 ligand
protein which is released in a protein synthesis-independent and CsA-insensitive
manner. In addition, in vivo treatment
of animals with CsA completely blocked T cell responses to tetanus toxoid but
could only partially reverse the development of GvHD and CsA-insensitive CD95
ligand expression, suggesting that alternative, calcineurin-independent,
signaling pathways may be involved in CD95 ligand expression and cytotoxicity
during GvHD.
Proapoptotic
FasL Is present in Normal Kidney Tubules and Inflammed Glomeruli
C.
Lorz, A. Ortiz, P. Justo, J. Egido.
Fundacion Jimenez Diaz,
Universidad Autonoma de Madrid, Madrid, Spain
Fas
expression is increased during renal injury and Fas activation promotes
glomerular injury. However, there is little information on FasL expression in
the kidney. We now report that FasL is present in normal mouse and rat kidney.
In situ hybridization and immunohistochemistry showed that renal tubular
epithelium is the main site of Fasl expression in the normal kidney. De novo
FasL expression by glomerular cells was present in two models of glomerular
injury: rat immune‑complex
proliferative glomerulonephritis and murine lupus nephritis. FasL was also
detected in murine proximal tubular epithelial cells in culture. The biological
significance of tubular epithelial cell FasL was further investigated. Tubular
epithelium‑derived FasL Induced apoptosis in Fas sensitive lymphoid cell
lines (A20) but not in Fas resistant lymphoid cell lines (A2OR). Neutralizing
anti‑FasL antibodies prevented this effect. By contrast, non‑stimulated
tubular epithelial cells well relatively resistant to apoptosis induced by high
concentrations of FasL and FasL did not induce apoptosis in an autocrine
fashion. However, tubular epithelial cells stimulated with inflammatory
mediators that increased cell surface Fas expression were sensitized to
apoptosis induced by FasL.
Session
VII: Clinical aspects of apoptosis
Monday April 3rd
Marcello
Pinti, Jessica Pedrazzi, Milena Nasi, and Andrea Cossarizza
Department of Biomedical Sciences, University of Modena
and Reggio Emilia, Italy
We
developed a quantitative-competitive RT-PCR assay for the analysis of the 2 main
forms of CD95 mRNA and for CD95L mRNA, and studied their levels in chronic and
acute HIV infection. A301 and its infected clone ACH-2 mounted the same amount
of plasmamembrane CD95, but have different ratios between total and membrane
form of CD95 mRNA. In comparison with U937 cells, the infected clone U1 had a
consistent decrease in CD95 expression, mirrored by a decrease in membrane CD95
mRNA. In respect to A301, ACH-2 expressed about 400 (basal) or 10 (induced) fold
less CD95L mRNA, which was undetectable in U937 and U1. Almost all lymphocytes
from patients with primary HIV infection were CD95+; anti-CD95 mAbs or binding
to CD95L+ cells induced rapid apoptosis, but CD95L mRNA was not expressed, and
poorly inducible. Our data indicate that a complex balance exists between
proapoptotic events, likely triggered by the host to limit viral production, and
anti-apoptotic events likely triggered by the virus to increase its production
and survival.
Session
VIII: Transcriptional regulation of apoptosis
Tuesday April 4th
Steroid
regulation of programmed cell death during Drosophila development.
Eric H. Baehrecke.
Center
for Agricultural Biotechnology, University of Maryland Biotechnology Institute,
College Park, MD 20742
The
steroid hormone ecdysone activates caspase-dependent cell death of salivary
glands. Salivary gland cell death is preceded by increased transcription of rpr
and hid, the CED4 homologue dark, the capase dronc, and several
ecdysone-regulated transcription factors. The ecdysone-regulated EcR, BR-C and
E74 genes regulate cell death, but their function in other processes indicate
that more specific mediators of cell death exist. E93 is a steroid-regulated
gene that is expressed in dying cells, and encodes a novel nuclear protein. BR-C,
E74 and E93 function are required for programmed cell death of salivary glands,
and E93 mutant salivary glands are arrested prior to changes in the cytosol and
plasma membrane that accompany this cell death. Expression of E93 results in
programmed cell death that reflects the pattern of ectopic expression. E93
requires the function of the H99 genetic interval that contains the rpr, hid,
and grim cell death genes to induce nuclear changes diagnostic of apoptosis. In
contrast, expression of E93 is sufficient to induce the removal of cells and
engulfment by phagocytes in the absence of the H99 genes. These studies indicate
that E93 regulates a novel cell death program involving the coordination of
distinct caspase-mediated subcellular events, and specifies steroid-activated
destruction of cells.
Natural
retinoic acids inhibit the activation induced T-cell death by modifying the
function of Nur77
Eva Szegezdi1,
Uwe Reichert2, Laszlo Fesus1
and Zsuzsa Szondy1
1Medical
School of Debrecen, Department of Biochemistry and Molecular Biology
2Centre
International de Recherches Dermatologiques Galderma, Sophia Antipolis, France
Crosslinking
of the T-cell receptor (TCR) induces cell death in T-cells that is based on
Fas/FasL interaction. The TCR activation can initiate the translocation of FasL
in the membrane or it can induce FasL expression. The activation of FasL
requires the expression of the Nur77 orphan nuclear receptor family members, in
case of the lack of this step no apoptosis happens. Natural retinoic acids
inhibit TCR mediated cell death without inhibiting the expression of Nur77. The
goal of our studies was to identify the step where retinoic acids interfere with
the TCR signalling pathway. In our experiments the effect of various retinoic
acid analogues on the activation-induced apoptosis of Jurkat T-cells was studied.
It was proven that the TCR activation initiates de novo FasL synthesis, while
natural retinoids prevented it. To determine which signalling pathway is
modified by retinoids Jurkat cells were transfected with a vector carrying a
Nur77 response element (NBRE) connected to luciferase reporter gene. Our results
show that all trans retinoic acid and the RARa
analogue prevented the increase of Nur77 transactivation initiated by T-cell
receptor activation. The results show that retinoids act through the RARa
receptor in the Nur77 signalling pathway. By decreasing the DNA binding activity
of Nur77 they regulate activation induced cell death.
Session
VIII: Transcriptional regulation of apoptosis
Tuesday April 4th
Apolipoprotein
J (Clusterin), a protein required for neuroblastoma cell survival, is directly
regulated by the B-myb oncoprotein and expressed in advanced disease stages
Maria
Cervellera 1, Giuseppe Raschella 2, Giorgia Santilli 1, Barbara Tanno 2, Andrea
Ventura 1, Camillo Mancini 2, Bruno Calabretta 3, and Arturo Sala 1*.
1Laboratory of Molecular
Pharmacology and Pathology, Consorzio Mario Negri Sud, 66030 S. Maria Imbaro,
Italy; 2ENEA Section of Toxicology and Biomedical Sciences, 00060 Rome, Italy;
3Department of Microbiology/Immunology, Kimmel Cancer Institute, Thomas
Jefferson University, Philadelphia, PA 19107
B-Myb
is a ubiquitiously expressed transcription factor involved in the regulation of
cell survival, proliferation, and differentiation. B-myb expression in
neuroblastoma correlates with advanced stages of the disease and poor patients1
survival. In an attempt to isolate B-Myb-regulated genes which may explain the
role of B-Myb in tumorigenesis, RDA (Representational Difference Analysis) was
performed in neuroblastoma cell lines with different levels of B-Myb expression.
One of the genes whose mRNA levels were enhanced in B-Myb expressing cells was
ApoJ/Clusterin SGP-2/TRMP-2 (ApoJ/Clusterin, thereof), previously implicated in
cell survival. Here we show that the human ApoJ/Clusterin gene contains in its
51 flanking region a Myb-binding site which interacts with bacterially
synthesized B-Myb protein and mediates B-Myb-dependent transactivation of the
ApoJ/Clusterin promoter in transient transfection assays. Moreover, ApoJ/Clusterin
expression was detected in most tumor samples of neuroblastoma patients in
advanced disease stages. Neuroblastoma cell lines transfected with a ApoJ/Clusterin
antisense expression vector showed reduced resistance to apoptosis induced by
the chemiotherapic drug doxorubicin. These results suggest that aberrant B-Myb
expression may promote tumor progression in neuroblastoma patients by induction
of ApoJ/Clusterin expression and increased resistance to apoptotic cell death.
INTERFERON
REGULATORY FACTOR-1 AND -2 regulate constitutive and INTERFERON-GAMMA-induced
expression of multiple caspases in monocytic cells
Ann
Zeuner1, Adriana Eramo3, Giovanni Rizzo2, Livia
Manzella1, Angelo Messina1, Cesare Peschle3,
Francesco Grignani2 and Ruggero De Maria2
Istituto
di Patologia Generale, University of Catania1; Istituto di Clinica
Medica, University of Perugia2; Laboratorio di Ematologia e
Oncologia, ISS, Rome3
Caspases
are central components of the apoptotic machinery, responsible for proteolytic
cleavage of key substrates and regulated cell disassembly. Interferon-gamma (IFN-g)
has
been shown to increase sensitivity to apoptosis in a variety of cells, but the
molecular mechanisms underlying this phenomenon have not been identified. We
found that exposure of monocytic cell lines to IFN-g
induces upregulation of several caspases, including caspase-1, caspase-2,
caspase-3, caspase-7 and caspase-8 in, resulting in enhanced susceptibility to
cell death following a variety of apoptotic stimuli. In order to identify IFN-g-induced transcriptional activators of caspases, the
monocytic cell line U937 was transfected with interferon regulatory factor-2
(IRF-2), a negative regulator of interferon transcription. Unexpectedly, IRF-2
expression completely abolished both constitutive and IFN-g-induced expression of multiple caspases, suggesting
that IRF-2 inhibits several pro-apoptotic genes by acting as dominant negative
of interferon consensus sequence binding proteins. Importantly, U937 cells
stably transfected with an antisense for interferon regulatory factor-1 (IRF-1)
displayed the same phenotype of IRF-2 overexpressing cells, demonstrating that
IRF-2 acts by targeting endogenous IRF-1, which is required for constitutive and
IFN-g-induced
expression of multiple caspases. These findings may explain the anti-oncogenic
and oncogenic potentials of IRF-1 and IRF-2.
Session
VIII: Transcriptional regulation of apoptosis
Tuesday April 4th
Adriana
Eramo1,2, Ann Zeuner1,3, Nadia Felli1,
Srinivasa M. Srinivasula2, Emad S. Alnemri2, Ugo Testa1,
Gianluigi Condorelli2, Cesare Peschle1,2, Ruggero De Maria2,3.
Dept. of
Hematology-Oncology Istituto Superiore di Sanita’, Rome, Italy1;
Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA2;
Institute of General Pathology, University of Catania, Italy3
We
previously demonstrated that caspase-mediated cleavage of GATA-1 results in
growth and differentiation arrest of immature erythroid cells, as a major
mechanism responsible for negative regulation of erythropoiesis. TAL-1 is a
helix-loop-helix trascription factor required for early blood cell development
and terminal erythropoiesis. Immunofluorescence and immunoblot analysis showed
that prolonged death receptor (DR) stimulation in erythroid cells induced TAL-1
and Bcl-xL downregulation prevented by caspase inhibitors. Direct demonstration
that TAL-1 is a target of caspases was obtained by testing wild type and
putative caspase-resistant TAL-1 mutants for in vitro and in vivo cleavage.
Recombinant caspase-3, -7 and -8 were able to cleave TAL-1 in vitro at Asp 180
and Asp 293. A similar cleavage pattern followed by proteolytic TAL-1
degradation was found in vivo by analyzing CD95-stimulated erythroblasts and
lymphoid cell lines expressing TAL-1. Importantly, expression of a
caspase-resistant TAL-1 double mutant (D180E and D293E) in erythroid progenitors
was able to prevent amplification of caspase activation, GATA-1 degradation,
Bcl-xL downmodulation, and impaired erythropoiesis induced by erythropoietin
deprivation or DR triggering. The antiapoptotic activity of uncleavable TAL-1
was particularly evident in the presence of low levels of erythropoietin and
saturating amount of CD95L, a culture condition that did not allowed survival of
erythroid cells expressing caspase-resistant GATA-1. Immunoblot and
semiquantitative RT-PCR analysis in erythroid cells showed that TAL-1 induced a
strong upregulation of Bcl-xL, the major erythroid antiapoptotic factor. In
conclusion, we provide evidence that TAL-1 acts as a survival factor in
erythroid cells by sustaining Bcl-xL expression and preventing massive caspase
activation
Novel approach for
enhancing the susceptibility of cancer cells to chemotherapeutic drug induced
apoptosis: Inhibition of NF‑kB through intracellular
delivery of IkB by HSV-1 structural protein VP22
Christopher Stroh, Juergen
Held, Wolfgang Wybranietz, and Klaus Schulze-Osthoff
Institute of Experimental
Dermatology, University of Muenster, Germany
One
major problem in chemotherapy of cancer is the activation of the transcription
factor NF‑kB
by many of the used chemotherapeutic compounds thereby rendering the cells more
resistant to apoptosis. A promising strategy for increasing the effectiveness of
chemotherapeutic drugs is the inhibition of NF‑kB
by overexpression of its inhibitory protein IkBa.
However, an unsolved problem of these genetherapeutic approaches is the low
transfection efficiency. Here we report the intracellular delivery of IkBa
by VP22, a structural protein of herpes simplex virus type 1 (HSV‑1). VP22
has the unique feature that upon transfection the expressed protein migrates out
of transfected cells and is taken up by virtually all surrounding cells where it
accumulates in the nucleus. This intercellular transport was also shown to be
possible with VP22 fusion proteins. To take
advantage of the possibility to deliver IkBa
to nearly 100% of target cells several VP22‑IkBa
constructs were made and tested. The expressed VP22‑IkBa
fusion proteins proved to be highly effective in inhibiting the activation of NF‑kB
and enhancing the susceptibility to apoptosis of different cell cell lines after
TNF and etoposide treatment. When cell‑free extracts of cells expressing
either VP22, IkB,
or VP22‑IkBa
proteins were incubated with HeLa cells VP22 and
VP22‑IkBa
but not IkBa
were taken up into the cells within min. Only VP22‑IkBa
proteins were able to block NF‑kB
activation in this import assay. At present we are performing large scale
purification of VP22‑IkBa
constructs to examine their effect in in
vivo models.
Session
IX: Anti- and pro-apoptotic mechanism
Tuesday April 4th
Follicular
dendritic cells provide GC B cells with two anti-apoptotic signals
Cell Biology and Histology,
Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ,
Amsterdam
Follicular
dendritic cells (FDCs) select B lymphocytes that have undergone affinity
maturation of their B cell receptors (BCR) during germinal center (GC) reactions.
Recently, we demonstrated that GC B cells apoptosis requires cathepsin activity
downstream of caspase-3. Moreover, we showed that FDCs keep the caspase route
inactive and additionally switch off endonuclease. Interestingly, FDCs contain
cytoplasmic cystatin A, a natural inhibitor of cathepsins. Therefore we
investigated this protein and its anti-apoptotic properties towards GC B cells.
We showed that cystatin A was present in and was secreted by FDCs. Moreover, we
demonstrated that recombinant cystatin A could block endonuclease activity of GC
B cells and that anti-cystatin A antibodies reverted endonuclease inhibition.
Furthermore, we showed that the cystatin A protein is found back in the GC B
cells after contact with the FDCs. Finally, we demonstrated that B cells present
in FDC-B cells clusters upregulate FLIP. From these data we conclude that FDCs
provide binding GC B cells with two anti-apoptotic signals: cystatin A blocks
endonuclease activity in GC B cell nuclei and FLIP expression blocks Fas
signalling.
Engagement
of CD4 before TCR triggering regulates both Bax- and Fas-mediated apoptosis
L.
Tuosto*, F. Somma*, M.M. Di Somma*, M.S. Gilardini Montani°, E. Cundari§
and E. Piccolella*.
*Dept. Cell. and Develop.
Biol. University of Rome "La Sapienza", Italy; °Dept. Environmental
Sciences, University "Della Tuscia", Viterbo, Italy; §Center
of Evolutionary Genetics, CNR, Rome, Italy
In
this work we wanted to clarify the CD4-dependent molecular mechanisms which
regulate the susceptibility to both Fas- and bcl-2-dependent apoptotic pathway
of antigen-activated human memory T cells.To address this issue, we used an
experimental system of viral and alloantigen-specific T cell lines and clones
and two ligands of CD4 molecules, Leu3a mAb and HIV gp120. We demonstrate that
CD4 engagement before TCR triggering suppresses the TCR-mediated transcription
of Flice-inhibitory protein (FLIP) and transforms memory T cells from an
AICD-resistant phenotype to an AICD-susceptible phenotype. Moreover, the
evidence that FasL mRNA expression was inhibited but the apoptotic programs were
executed prompted us to analyze bcl-2 dependent pathways. The data demonstrate
that while Bax and Bcl-2 expressions are coordinately modulated by
antigen-activation, the engagement of CD4 separately from TCR influences the
expression of the proapoptotic protein Bax independently of the anti-apoptotic
protein Bcl-2. The increased expression of Bax was associated to the dissipation
of the mitochondrial transmembrane potential (DYm) suggesting a novel
immunoregulatory function of CD4 and demonstrating that both PCD and AICD are
operative in CD4+ memory T cells. Furthermore, the analysis of the mechanisms by
which IL-2 and IL-4 cytokines exert their protective function on anti-CD4
treated T cells show that they were able to revert the susceptibility to
Bax-mediated but not to Fas-dependent apoptotic pathways.
Session
IX: Anti- and pro-apoptotic mechanism
Tuesday April 4th
REQUIREMENT FOR
TRANSFORMING GROWTH FACTOR-beta-1 (TGF-b1)
IN CONTROLLING T CELL APOPTOSIS
Cellular Immunology Section, OIIB, NIDCR, NIH, USA
TGF-b1, a potent immunoregulatory molecule, was found to
control the life and death decisions of T lymphocytes. Both thymic and
peripheral T cell apoptosis was increased in mice lacking TGF-b1 compared to wildtype littermates. Engagement of the T
cell receptor enhanced this aberrant T cell apoptosis as did signaling through
either the death receptor Fas or the TNFa receptor. Strikingly, the absence of TGF-b1 resulted in disruption of mitochondrial membrane potential (Dm) in these T cells, which marks the point of no-return in a cell
condemned to die. Thus, TGF-b1
may protect T cells at multiple sites in the death pathway, particularly by
maintaining the integrity of mitochondria. These findings may have broad
implications not only for T cell selection and death in immune responses and in
the generation of tolerance, but also for defining the mechanisms of programmed
cell death in general
Map
kinase‑mediated regulation of Fas, TNF and TRAIL receptor‑induced
apoptosis in HeLa cells
Turku Centre for
Biotechnology, P.O.Box 123, FIN‑20500 Turku, Finland
TNF,
Fas, and TRAIL receptors are highly specific physiological mediators of
apoptosis. Previously we have shown that some FasR‑insensitive cell lines
are able to redirect the signal from apoptosis into survival by induction of
MAPK/ERK. HeLa cells are insensitive both to FasR and TNF‑R1 and TRAIL‑Rs‑induced
apoptosis. Therefore we wanted to
determine whether MAPK could have a similar effect on other death receptor
signaling. Inhibition of MAPK was sufficient to sensitize the cells to FasR and
TRAIL‑Rs‑mediated apoptosis but not to signaling by TNF‑R1
which can be explained by the fact that TNF‑R1 but not FasR nor TRAIL‑Rs
stimulation could strongly activate NF‑kB,
a known survival mechanism. However, when
the cells were sensitized with cycloheximide, overexpression of constitutively
active MKK1 could rescue the cells from apoptosis induced by all three
respective receptors, indicating that MAPK has a dominant protecting effect over
death receptor stimulation. The protection occurs at the level or upstream of
caspase‑8 and is protein synthesis independent. Current studies aim at
characterizing the molecular mechanisms behind this effect.
Session
IX: Anti- and pro-apoptotic mechanism
Tuesday April 4th
Bernassola
F., Corazzari M., Barcaroli D., Melino G. and De Laurenzi V
Dept.
of Experimental Medicine, University of Rome Tor Vergata, Via di Tor Vergata
135, 00133 Rome, Italy
Tissue-Transglutaminase
(tTG or TGase 2) is a Ca++-dependent enzyme that catalyses either e-(g-glutamyl)lysine
or N1, N8 (g-glutamyl) spermidine isopeptide bonds. At
least in some models, TGase 2 expression has been associated with apoptosis and
it has been proposed that its activation leads to the irreversible assembly of a
cross-linked protein scaffold in cells undergoing apoptosis. This results in a
strong structure which is resistant to mechanical and chemical attacks. Thus,
TGase 2-catalyzed protein polymerisation contributes to the ultrastructural
changes typical of dying apoptotic cells; in addition, it stabilise the
integrity of the apoptotic cells preventing the release of harmful intracellular
components into the extracellular space and, consequently, inflammation and scar
formation. The construct used for the targeting deleted part of exon 5, exon 6
containing the active site and intron 5. One in 1,500 clones was found
recombined, allowing the production of hetero/homo-zygous mice. Complete absence
of TGase 2 was demonstrated RT-PCR and western blot analysis. Transglutaminase
activity measured on liver and thymus extracts showed however a minimal residual
activity in TGase 2 -/- mouse. PCR analysis of RNA extracted from the same
tissues demonstrated that at least TGase 1 (normally present in the skin) is
also expressed in this tissues and could be responsible for the residual minimal
activity. TGase 2 -/- mice showed no major developmental abnormalities, and
histological examination of the major organs appeared normal. Induction of
apoptosis ex vivo in TGase 2 -/- thymocites and in vitro on TGase 2 -/- Mouse
Embryonal Fibroblasts showed only minor differences.
FLIP
activates NF-kB and ERK signaling pathways
and can divert Fas ligand-induced death signals to survival
Nils Holler#,
Takao Kataoka#, Ralph C. Budd*#,
Fabio Martinon#, Kim Burns#, Michael
Hahne#, Margot Thome#,
Norman Kennedy* and Jürg Tschopp#
#Institute
of Biochemistry, University of Lausanne, BIL Biomedical Research Center, Chemin
des Boveresses 155, CH-1066 Epalinges, Switzerland
*Immunobiology Program,
Department of Medicine, The University of Vermont
Activation
of Fas (CD95) by its ligand (FasL) rapidly induces cell death through
recruitment and activation of caspase-8 via the adaptor protein FADD. However,
Fas signals do not always result in apoptosis, but can also trigger a pathway
that leads to proliferation. We investigated at what level the two conflicting
Fas signals bifurcate and what protein(s) might be inplicated in switching the
response. We found that under conditions where proliferation of CD3-activated
human T lymphocytes is increased by recombinant FasL, there is recruitment of
the caspase-8 inhibitor and FADD-interacting protein FLIP occurs. Fas-recruited
FLIP can interact with TRAF1, TRAF2 as well as with the kinases RIP and Raf-1,
resulting in the activation of the NF-kB and ERK signaling pathways. In T cells
these two signal pathways are critical for IL-2 production and indeed, increased
expression of FLIP in T cells results in enhanced IL-2 production. We provide
evidence that FLIP is not simply an inhibitor of death-receptor induced
apoptosis but that FLIP also mediates the activation of NF-kB and ERK signaling
pathways by virtue of its capacity to recruit the respective adaptor proteins.
Thus, FLIP can switch the pro-apoptotic activity of Fas to differentiation
and/or proliferation
Session
IX: Anti- and pro-apoptotic mechanism
Tuesday April 4th
The
pro- and anti- apoptotic properties of cFLIPshort depend upon its intracellular
compartmentalisation
Imperial Cancer Research
Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. *Present address: UCSF
Cancer Center, 2340 Sutter Street, Room S-233, San Francisco, California
94143-0218,USA
The
death effector domain containing E8 gene product from equine herpesvirus-2,
designated FLICE inhibitory protein (vFLIP), and its cellular homologue,
cFLIPshort, have been reported to inhibit the activation of caspase-8 by death
receptors. The cFLIPshort in contrast to its viral counterpart has also been
described to act as a potent inducer of apoptosis. We report here that the
pro-apoptotic effect of cFLIPshort when expressed above a certain threshold is
mediated through intracellular filaments similar to the recently described death
effector filaments (DEF). The vFLIP is incapable of forming DEF-like structures
and acts thereby independent of its expression level exclusively as an
anti-apoptotic protein. The cFLIPshort protein, however, only exhibits its
protective site against CD95 induced death when present in a disperse cellular
localisation. The coexpression of the vFLIP E8 protein interferes with DEF
formation thus allowing the accumulation of cFLIPshort as a protective protein.
The expression of members of the Bcl-2 family also prevents cFLIPshort to form
DEF-like structures. Endogenous DEF-like structures by cFLIP can be detected
after treatment with the anti-tumor ether lipid ET-18-0CH3 that is thought to
act intracellulary upon the CD95 receptor. We conclude that the pro- or
anti-apoptotic property of cFLIPshort depends upon its intracellular
compartmentalisation.
Session
X: Heat Shock Proteins
Tuesday April 4th
Hsp70
protects tumour cells from cell death by at least two distinct mechanisms
Marja Jäättelä and
Jesper Nylandsted.
Apoptosis Laboratory,
Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark
The
major heat shock protein Hsp70 protects cells from cell death induced by tumour
necrosis factor, heat and several chemotherapeutic drugs. Hsp70 is a chaperone
protein containing an ATP binding domain capable of hydrolysing ATP and a
peptide binding domain binding to the substrates. To study its mechanism of
action, we expressed the wild type Hsp70 or its deletion mutants lacking these
domains in apoptosis sensitive WEHI-S tumour cells. Whereas wild type Hsp70
conferred an effective protection against apoptosis induced by heat and TNF, the
deletion mutant lacking the ATP binding domain protected only against heat and
the deletion mutant lacking the peptide binding domain protected only against
TNF. The protection from heat occurred prior to the activation of caspases,
whereas protection from TNF took place downstream of the activation of
caspase-3-like proteases. Thus, Hsp70 can inhibit apoptosis at two different
steps of the apoptotic pathway by two clearly distinct mechanisms utilising
different domains of the protein.
Interaction
of SODD with Hsp70/Hsc70 via the BAG domain
Institute of Biochemistry, University of Lausanne,
Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland
Spontaneous
signalling events through death domain (DD) containing receptors have to be
prevented as they can result in inappropriate cell death. A novel protein has
been charachterized (SODD, for silencer of DD) that binds to the DD of non
activated TNF-receptor 1 thereby negatively regulating its signaling capacities.
We identified an Hsp70/Hsc70 binding domain in SODD: the BAG domain. We found
that the BAG domain of SODD interacts with the ATPase domain of Hsp70/Hsc70.
Hsp70 interacts with the death receptor TRAMP/DR3 in the presence of SODD. We
propose that SODD may act as an adaptor protein that targets Hsp70/Hsc70 to the
cytoplasmic domain of death receptors, thereby locking them in a silent state.
Session
X: Heat Shock Proteins
Tuesday April 4th
Inhibition
of Hsp70 synthesis leads to apoptotic death of breast cancer cells
Jesper Nylandsted, Karsten
Brand, Michael Strauss and Marja Jäättelä.
Apoptosis Laboratory and
Department of Cell Cycle and Cancer, Danish Cancer Society, Copenhagen, Denmark
and Max-Planck- Gesellschaft, Max Delbrück Centre for Molecular Medicine,
Berlin-Buch, Germany
Hsp70
is an anti-apoptotic chaperone protein preferentially expressed in human tumors
and tumor cell lines. Here, we demonstrate that the expression of Hsp70 is
required for the survival and growth of human breast cancer cells. Inhibition of
the synthesis of Hsp70 by adenoviral transfer of antisense Hsp70 cDNA
(Ad.asHsp70) resulted in massive apoptosis of all breast cancer cell lines
tested, whereas the survival of non-transformed breast-derived cells were not
affected. Anti-apoptotic proteins Bcl-2 and Bcl-x as well as caspase-inhibitors
CrmA, ZVAD-FMK and DEVD-CHO failed to rescue tumor cells from Ad.asHsp70-induced
apoptosis. Furthermore, sensitivity of breast cancer cells to Ad.asHsp70-induced
apoptosis was independent of the expression of functional p53 tumor suppressor
protein and estrogen receptor. Thus, the inhibition of the synthesis or the
function of Hsp70 may form basis for the development of novel highly specific
strategies for treatment of otherwise therapy resistant breast tumors.
Session
XI: Apoptosis and tumors
Tuesday April 4th
Jan Paul Medema1,
Joan de Jong1, Sandra Bres1, Ton Schumacher2,
Mireille Toebes2, Kees Melief1 and Rienk Offringa1.
1Dept.
of Immunohematology and Blood transfusion, Leiden University Medical Center,
Leiden, The Netherlands
2Dept.
of Tumorimmunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
The
murine protease inhibitor SPI-6 belongs to the large family of Serpin Protease
Inhibitors . SPI-6 has a high specificity for the cytotoxic granule-assoaciated
protease granzymeB (GrB). Expression of SPI-6 is mainly found in cytotoxic T
cells (CTLs). Since CTL can efficiently lyse target cells with the use of
perforin and GrB, it is thought to protect itself against its own cytotoxic
potential by the expression of SPI-6. Since several tumor cells have been shown
to be resistant to CTL-induced killing both in vitro and in vivo we set out to
determine the underlying mechanisms that control this resistance. Previously we
showed that certain tumor cells that express the apoptosis inhibitor c-FLIP
become resistant to the CD95-pathway and can thereby escape from CTL-induced
apoptosis in vivo. Here we show that expression of the GrB-inhibitor SPI-6
completely inhibits CTL-induced apoptosis through the perforin/GrB pathway .
More importantly, northern analysis revealed that a SPI-6 homologue is expressed
at very high levels in tumor cells which are resistant to CTL-induced apoptosis
in vitro and in vivo. Currently we are cloning this factor and will try to
characterise its function in tumor growth.
Jennifer L. Rohn1,
Astrid Danen-van Oorshot1, 2, Patrick
Voskamp1, Martine Miltenburg1
and Mathieu H. M. Noteborn1,2
1Leadd
BV, and 2Leiden University Medical
Center, Leiden, The Netherlands
Apoptin
(VP3) is a chicken anemia virus-encoded protein that is able to induce apoptosis
in cells in which it is expressed. The ability of Apoptin to kill exhibits a
remarkable specificity; namely, transformed or tumor cells are susceptible,
whereas normal cells are completely resistant. Moreover, the localization of
Apoptin correlates with this tumor-specific apoptotic activity. Specifically,
while Apoptin is found in the cytoplasm of normal cells, it is situated in the
nucleus of tumor cells. We have hypothesized that the differential killing
ability of Apoptin could result from the protein responding differently to a
tumor-specific state. The nature of this state could in principle consist of a
state-specific modification of Apoptin, the binding of Apoptin to a
state-specific co-factor, or a combination of both. To investigate the first
possibility, we examined whether Apoptin is differentially modified in tumor
versus normal cells; preliminary evidence suggests that Apoptin is indeed
phosphorylated in a state-dependent manner. To study the second possibility, we
performed a yeast two-hybrid screen of a tumor library using full-length Apoptin
as “bait”, and isolated a number of bona fide associating proteins, some of
which possess clear tumor-specific phenotypes. These ongoing studies support the
hypothesis that the tumor-specific apoptotic activity of Apoptin may respond to
an underlying, and possibly important, fundamental difference in state between
the normal and the transformed cell.
Session
XI: Apoptosis and tumors
Tuesday April 4th
Engagement
of the Apoptotic Machinery in Prostate Cancer
Marco
Marcelli1, Michela Marani1,
Lydia Sturgis2, Joe Haidacher2,
Roberta Mannucci3, Ildo Nicoletti3,
and Larry Denner2.
1VA
Medical Center and Baylor College Of Medicine, Houston, TX; 2Texas
Biotechnology Corp., Houston,TX; 3Perugia
Univ. Medical School, Perugia, Italy
Metastatic
prostate cancer occurs when cells escape hormone ablation-induced apoptosis.
Cell lines derived from different metastatic sites have varied genotypes and
exhibit cell-specific and inducer-specific differences in sensitivity to
engagement of the apoptotic machinery. These differences are evident based on
analyses of dissipation of the mitochondrial membrane potential, regulation and
localization of the Bcl-2 family members, release of cytochrome c from the
mitochondria to the cytoplasm, proteolytic processing and catalytic activation
of caspases, cleavage of the death substrates DFF and PARP, and expression of
morphological features seen in TUNEL staining. Thus, while the LNCaP line is
very sensitive to engagement of the apparatus, PC-3 cells are relatively inert
to many inducers. Further, inducing agents such as staurosporin elicit different
responses than viral-mediated overexpression of caspase-3, caspase–7, or Bax.
In some but not all prostate cancer cell lines, the Bcl-2/Bax family mediates
the apoptotic response. Regulation of engagement of these apoptotic signalling
intermediates in therapeutic intervention will be discussed.
Molecular
analysis of radiation-induced apoptosis in paraffin-embedded tumor and normal
tissue of rectal cancer patients
Lucy T.C. Peltenburg1,
Corrie A.M. Marijnen1, Iris D.
Nagtegaal2, Adri A. Mulder-Stapel2,
J. Han J.M. van Krieken3 and Peter I.
Schrier1.
Departments of Clinical Oncology1
and Pathology2,
Leiden University Medical Center, Leiden, Department of Pathology3, Nijmegen University Hospital, The Netherlands
Numerous
studies of molecular factors that influence apoptosis have been done in in
vitro tissue culture systems. In contrast, we have investigated the
occurrence and modulation of apoptosis in human tumors that were treated with
radiotherapy. We analyzed a large collection of tumor tissue samples, derived
from a randomized trial on preoperative radiotherapy of rectal cancer patients.
This trial included over 1400 patients, of which pre- and postoperative tumor
samples are available, thus providing a unique system to investigate the role of
apoptosis in vivo. Proteins were
extracted from paraffin-embedded pre-treatment biopsies, tumors and normal
mucosa of 50 irradiated and 50 non-irradiated patients. As a measure of
apoptosis, we determined the ratio of full-length and cleaved gelsolin by
Western blotting. To evaluate the reliability of this method, the numbers of
apoptotic cells were also determined by immunohistochemistry using an antibody
specific for the apoptosis-induced cleavage product of cytokeratin 18. By using
these assays, expression of oncogenes, tumor suppressor genes and the Bcl-2
family members can as well be quantified. The methods of these analyses, as well
as the differences in the level of apoptosis and correlations with apoptosis
determinants in irradiated and non-irradiated tissues will be discussed.
Session
XI: Apoptosis and tumors
Tuesday April 4th
Modulation
of Survivin expression by CD40L in B‑chronic lymphocytic leukemia
(B‑CLL)
Luisa
Granziero, Paola Circosta, Giuliana Strola, Massimo Geuna, Daniela Gottardi,
Pier Carlo Marchisio, Dario Altieri, Marco Chilosi, Federico Caligaris‑Capplo
and Paolo Ghia.
Istituto
per la Ricerca e la Cura del Cancro, Candiolo (TO); Divisione di Immunologia
Clinica, Ospedale Mauriziano Umberto 1, Dipartimento Scienze Biomediche ed
Oncologia Umana, Università di Torino
B‑CLL
is a human malignancy that primarily involves defects in apoptosis. Although the
malignant cells progressively accumulate in
vivo, they rapidly undergo apoptosis when cultured in vitro; supporting a role for the microenvironment. A new family
of inhibitors of apoptosis proteins (IAP) has been cloned in humans and so far
six different members have been recognized (cIAP1, cIAP2, NAIP, XIAP, Survivin
and Apollon) that suppress apoptosis by caspase and pro‑caspase inhibition.
We analyzed the expression of these genes in CD19+/CD5+ cells from peripheral
blood of 10 B‑CLL patients at diagnosis and after 7 days of culture with
and without the soluble form of the CD40 ligand (CD40L). The in vitro apoptosis of cells exposed to CD40L was significantly
reduced. RT‑PCR analysis showed that the expression of IAPs was not
modified after stimulation, with the notable exception of survivin, which was
strongly up‑regulated after CD40 activation. The results were confirmed at
protein level, by FACS and western blot analyses. In immunohistochemistry,
malignant cells in bone marrow and lymph nodes from 6 B‑CLL patients, were
highly positive for survivin while they were negative in the blood. Taken
together, these observations indicate that a signal through CD40 might be
responsible for the induction of an apoptosis-resistant phenotype in B‑CLL
cells, mainly in secondary lymphoid tissues, suggesting a possible mechanism for
the accumulation of malignant cells.
Protection
against an non‑immanogenic melanoma by vaccination with apoptotic tumor
cells coupled with TNF‑a
V.S.
Zimmerman, Patrizia Rovere, A. Bondanza, F. Curnis, A. Corti, C.Rugarli, A.A.
Manfredi.
Hospital San Raffaele,
Laboratory of Tumor Immunology, Milano
Apoptotic
cells are usually cleared by phagocytosis without causing inflammation. In
vivo, tumors cells, which contain relevant tumor associated antigens, die by
apoptosis in an environment devoid of inflammatory signals and do not induce an
efficace immune response. We have shown previously that vaccination with
apoptotic tumor cells induces a functional and long lasting immune response
against tumor. However, this response is limited by the production of
immunosoppressive cytokines including IL-10. In order to augment the
efficiency of the procedure we vaccinated C57BL/6 with apoptotic B16 melanoma
cells coupled covalently with the pro‑inflammatory cytokine TNF‑a. This coupling was achieved by the way of a biotin‑avidin bridge.
We have verified in vitro that the TNF‑a
coupled to the apoptotic tumor cells is bioactive since it induced cell death
and dendritic cell maturation. In vivo, the
vaccination with apoptotic B16 melanoma coupled with TNF‑a
induced the priming of tumor specific CTL and protection (rejection or delay in
the growth) against a further challenge with living B16.These results confirm
that the cytokine environment at the site of apoptotic cell clearance determines
the establishment of immune response towards intracellular antigens and provide
a tool for clinical intervention.