THE FOURTH EUROPEAN WORKSHOP
ON CELL DEATH
Colin Adrain
and Seamus J. Martin
Dept. of Genetics, Trinity College,
Dublin, Ireland.
Email:
cadrain@tcd.ie
The CTL/NK protease, granzyme B (GzmB) can provoke apoptosis via activation of the caspase family of proteases, as well as via cleavage of the pro-apoptotic BH-3-only molecule, Bid. To further elucidate the mechanism(s) of GzmB-mediated killing, we have undertaken an extensive proteomic-based screen to seek additional GzmB proteolytic targets. Using this approach, we identified four known GzmB targets, in addition to nine novel substrates of GzmB, including the tumor suppressor protein, PHAPI. Interestingly, PHAPI has recently been identified as a factor that enhances caspase activity within the apoptosome pathway. Over-expression of PHAPI sensitized target cells to NK-mediated apoptosis, suggesting that PHAPI plays a role in modulating apoptosis during NK/CTL-mediated killing. Interestingly, whereas full length PHAPI resides in the nucleus, GzmB-cleaved PHAPI exhibited cytoplasmic staining. We propose that GzmB proteolysis of PHAPI may mobilize the latter from the nucleus to the cytoplasm, enabling PHAPI to enhance caspase activity in the apoptosome pathway.
Hakan
AKÇA1
and Osman Nidai Ozes2
1Pamukkale
University Fakulty of Arts & Sciences Department of Biology, Denizli, Turkey
E-mail:
hakca@pamukkale.edu.tr
The
tumor suppressor protein p53 is most mutated gene in human cancer. The function
of p53 is not restricred to” guarding against oncogenic stress, but also p53
can guard against the presence of DNA damage. One of the principal mechanisms by
which cells achieve this is by regulating the p53 protein level although its
phosphorylation and cellular localization also contribute to the regulation of
its function. Since many tumors secrete growth factor(s) that inhibit apoptosis
and support the growth of cancer cells, we investigated the effects of human
epidermal growth factor (EGF) and Insulin on human TNF-a,
IFN-a, IFN-g
mediated induction of p53 and its transcriptional target, p21 in TNF-a
sensitive human cervical carcinoma cell line, ME180S. We found that cytokines
which we used can increase the cellular levels of p53, p21 and induce apoptosis
in ME180S. However, pretreatment of cells with EGF and insulin can suppress all
these effects of cytokines. To determine which kinase(s) pathway was utilized by
EGF and insulin to show these suppressive effects, cells were pretreated with
inhibitors of MAPK, PI3K and PKC pathways. Among these only PKC inhibitor
reversed all the suppressive effects on EGF and insulin. We also found that
ME180 cells express only z, l, e, i, d, q, b
PKC subtypes and among these EGF and insulin treatment activate only PKC-d
redistribution to the membrane from cytosol. An inhibitor of PKC, GF109203X
inhibited EGF and insulin mediated suppression of cytokines-induced accumulation
of p53, p21 and induction of apoptosis. In summary, we concluded that EGF and
insulin can protect ME180 cells from TNF-a,
IFN-a, IFN-g -
induced apoptosis throug activation of PKC-d.
Early
induction of BH3-only protein Noxa in naïve CD8+ T cells after TCR
triggering.
N.L.
Alves1,3,
I. A. Derks1, R. Gimeno4, A. ten Brinke2, L.A.
Aarden2, F.A. Arosa3, H. Spits4, R.A.W. van
Lier1, E. Eldering1
1Dept
of Experimental Immunology, AMC, Amsterdam. 2 Dept of Immunopathology,
Sanquin, Amsterdam. 3 Lab. of Molecular Immunology, Institute for
Molecular and Cell Biology, Porto, Portugal. 4Dept
of Cell Biology and Histology, AMC, Amsterdam.
E-mail:
n.m.lagesalves@amc.uva.nl
Signalling
through TCR and CD28 drives naïve CD8+ T cells into a
differentiation process which ultimately leads to formation of effector cells.
We evaluated the programme triggered upon CD3/CD28-activation in human naïve
CD8+ T cells, using a novel multiplex assay to analyse the expression
profile of 34 genes involved in apoptosis regulation. Early after activation (24
hrs) we observed a transient induction of anti-apoptotic members (Bcl-xl,
A1/Bfl-1). Notably, the BH3-only member Noxa, although proposed to be
p53-responsive, was rapidly induced and maintained upon activation, in a
PKC-dependent manner. Protein analyses revealed that Noxa induction occurred
specifically upon TCR-engagement. Comparison of Noxa and Puma, another
purported-responsive gene, showed that contrasting with Puma, Noxa was not
induced after irradiation or treatment with cytostatic drugs (e.g. fludarabine).
P53 deficient CD8+ T cells, obtained by RNAi technology, still
displayed TCR-dependent Noxa induction and thus revealed that it is regulated by
other means than p53. This was also confirmed using U2OS cells as a model, where
Puma or Noxa expression was abrogated using RNAi technology. Currently we are
testing the RNAi constructs in primary lymphocytes, analysing the susceptibility
of CD8+ T cells to various apoptotic stimuli when Noxa expression is
impaired. Our data may indicate a new mechanism by which CD8+
T cells after immune activation prepare for death signals in the contraction
phase.
Regulation of heat shock
protein 90 (Hsp90) expression contributes to apoptosis modulation by NF-_B/Rel
transcription factors
Massimo Ammirante°,
Alessandra
Rosati, Antonello Petrella, Arturo Leone and Maria Caterina Turco
Dipartimento di Scienze Farmaceutiche (DIFARMA), Salerno
University, Italy
Email:
mammirante@unisa.it
NF-_B/Rel transcription
factors regulate a number of genes (including iaps, flip, bfl-1/A1,
p53, gadd153, sod-2 and others) involved in apoptosis modulation. The
activity of NF-_B/Rel is stimulated by stress inducers; a major role in
modulating stress responses, including apoptosis, is exerted by heat
shock proteins (HSPs). Among these, HSP90 is known to inhibit apoptotic response
to some antineoplastic drugs; such anti-apoptotic activity relies in part on
HSP90 binding to Akt protein, whose levels are thereby stabilised. Two consensus
sequences for NF-_B/Rel (5'-agcggaggtagttccatcgttt-3' and
5'-atatccgaaaattcccatgtag-3') are found in the Hsp90-_ gene promoter. We
analysed whether the two motifs could bind to NF-_B/Rel complexes in a variety
of cell types. Lymphoid (Jurkat), myeloid (U937), epithelial (HeLa) and
glioblastoma (T98G, U87MG) cells were stimulated with TRAIL, TNF-_ or etoposide
and nuclear extracts were found to specifically bind one or both the NF-_B/Rel
consensus motifs in electrophoretic mobility shift assays (EMSA). Dimers formed
by different NF-_B/Rel subunits appeared to be complexed to the hsp90-
derived motifs in the different cell lines. To investigate whether NF-kB/Rel
activity influenced HSP90 levels, we used decoy phosphorothioate
oligonucleotides or small interfering (si) RNA specific for NF-_B/Rel factors.
In U937 cells, etoposide induced NF-kB/Rel nuclear translocation and raising in
HSP90 protein levels; anti- NF-kB/Rel decoy oligos or siRNA greatly reduced
NF-kB/Rel activation and HSP90 levels detected in Western blot. These findings
indicate that NF-kB/Rel transcription factors regulate HSP90 levels and disclose
a novel mechanism by which NF-kB/Rel activity can modulate apoptosis.
p16INK4A
affects Fas- and Glucocorticoid-induced Apoptosis in Human Leukemia Cells by
Repression of Bcl-2
Ausserlechner
M.J.,1, 2 Obexer P.,3 Kofler R.,1,
3
1Pathophysiology, University of Innsbruck, 2 Department of
Pediatrics, University Hospital of Innsbruck
E-mail: Michael.J.Ausserlechne@uibk.ac.at
INK4A
is the most frequently inactivated gene locus in acute lymphoblastic T-cell
leukemia (T-ALL). T-ALL cells with conditional p16INK4A expression arrested in
the G1 phase of the cell cycle and showed markedly accelerated apoptosis induced
by anti-Fas, glucocorticoid (GC) and a temperature-regulated allel of p53 but
were resistant to various chemotherapeutic agents. We observed accelerated
caspase 3 and caspase 8 cleavage upon Fas treatment in p16INK4A expressing cells.
This phenotype correlated with repression of Bcl-2 after 48 hours of p16INK4A
expression, whereas the cellular levels of Fas,
Flip, FADD, caspase 8 and caspase 3 were not altered. Retrovirally delivered
Bcl-2 reverted the kinetics of Fas- and GC-induced apoptosis to those of
proliferating controls indicating that loss of Bcl-2 was at least in part
responsible for the observed increased sensitivity to Fas- and GC-induced
apoptosis. The combined data suggest that p16INK4A inactivation during T-ALL
tumour development not only prevents cellular senescence but also reduces
sensitivity to Fas and GC by alteration of Bcl-2 rheostat.
Damali
Martin, Deborah Berry and Eric
H. Baehrecke
Center for Biosystems Research, University
of Maryland Biotechnology Institute, College Park, MD 20742, USA.
E-mail:
baehreck@umbi.umd.edu
Apoptosis and autophagic cell death
are the two most prominent morphological forms of programmed cell death that
occur during animal development. While
the mechanisms that regulate apoptosis are well characterized, little is known
about autophagic cell death. We are using Drosophila as a model to study
autophagic cell death, and have combined forward genetic and genomic approaches
to identify genes involved in this process.
The steroid hormone ecdysone triggers autophagic cell death through a
complex signaling hierarchy that includes DNA binding proteins, proteases such
as caspases and matrix metalloproteases, cell remodeling factors, autophagy
proteins, as well as others. The
relationship of these factors to proper regulation of cell death will be
discussed. Significantly, we will
demonstrate a genetic requirement for autophagy genes in programmed cell death.
The association of autophagic cell death with neurodegenerative disorders
and cancer indicate the importance of investigating this understudied form of
programmed cell death.
The counteracting roles of the kinases Akt/PKB and p38 in
c-Myc dependent apoptosis
Kerstin Bellmann,
Julie Martel, Mireille M. Labrie and Jacques Landry
Centre de recherche en cancérologie
de l’Université Laval, CHUQ-HDQ, Québec, Canada
Email:
kerstin.bellmann@crhdq.ulaval.ca
Cells
become extremely sensitive to apoptosis early during the process of cellular
transformation by growth-promoting oncogenes such as c-Myc. Recently, we
provided evidence that chemotherapeutic drugs such as cisplatin lead to c-Myc
dependent apoptosis in Rat1 cells by using the classical mitochondrial sub route
of programmed cell death including release of cytochrome c and activation of Bax.
We have identified the stress-activated MAP kinase p38 as being an essential
element of this pathway. Here, we show that the activation of the pro-survival
kinase Akt by cisplatin is inhibited in cells with deregulated expression of
c-Myc but is activated in cells expressing a non-functional mutant of c-Myc.
Overexpression of active Akt renders the cells resistant to cisplatin and
inhibits all features of apoptosis. Chemical inhibition of Akt together with
activation of p38 led to substantial apoptosis after cisplatin in the cells with
non-functional c-Myc. However, co-transfection studies showed that Akt did not
affect p38 activation. Taken together, our results indicate that Akt and p38 act
independently of each other to generate pro-survival and cell death signals
upstream of the mitochondria in c-Myc dependent apoptosis.
Apoptogenic Effects of Black Tea on Ehrlich’s Ascites
Carcinoma Cells
Animal
Physiology Section, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata-700 054, India
E-mail:
arindam@bic.boseinst.ernet.in
/ arindam19@yahoo.com
Epidemiological studies have
suggested a cancer protective effect of black tea, but the results obtained so
far are not conclusive. In the current study, mechanisms of apoptogenic effect
of black tea extract were delineated in tumor-bearing mice. Black tea
administration to Ehrlich's Ascites Carcinoma (EAC)-bearing Swiss albino mice
caused a significant decrease in the tumor cell count in a dose dependent manner.
Flowcytometric analysis showed an increase in the number of cells in the
sub-G0/G1 phase of cell cycle signifying tumor cell apoptosis by black tea.
These results were further confirmed by nuclear staining that demonstrated
distinct morphological features of apoptosis. Our data also revealed an increase
in the expression of pro-apoptotic protein p53 in EAC cells. It is known that
upon p53 induction, multiple downstream factors contribute to the decision
making between growth arrest and apoptosis. Among those, pro-apoptotic gene Bax
is up-regulated during p53-mediated apoptosis. On the other hand, p53-mediated
growth arrest involves p21 as a major effector.
In our system, increase in p53 expression was followed by moderate
expression of p21/Waf-1 and high expression of Bax at protein levels keeping the
level of anti-apoptotic protein Bcl-2 unchanged. All these observations together
signify that black tea-induced apoptogenic signals overrode the growth-arresting
message of p21, thereby leading the tumor cells towards death.
An amiodarone analogue,
HO3408 protects ischemic hearts by inhibiting the mitochondrial apoptotic
pathway
Zita
Bognar, Gabor Varbiro, Anita Palfi,
Balazs Veres, Antal Tapodi, Balazs Radnai, Balazs Sumegi
Dept. of Biochemistry and Medical
Chemistry, Faculty of Medicine, University of Pécs, Szigeti u. 12, H-7624 Pécs,
Hungary.
E-mail:
zita.bognar@aok.pte.hu
HO 3408 is a modified
Amiodarone analogue. Amiodarone is a widely used and potent antiarrhythmic agent.
Both amiodarone and its metabolite possess antiarrhythmic effect, and both
compounds can contribute to toxic side effects. Here, we compare the effect of HO3408, amiodarone and
desethylamiodarone on mitochondrial energy metabolism in perfused hearts, and
the mitochondrial functions (respiration, membrane potential, and permeability
transition) in isolated mitochondria were also analyzed. Amiodarone and HO 3408,
but not desethylamiodarone protects the mitochondrial energy metabolism in
perfused heart during ischemia re-perfused. Amiodarone, when was present at low
concentrations and predominantly in membrane bound form, protected heart and
mitochondrial energy metabolism from ischemia-reperfusion-induced damages in
Langendorff-perfused heart model. Up to the concentration of 10 mM, the drug
considerably inhibited Ca2+-induced permeability transition, while at higher
concentrations it induced a cyclosporin A independent permeability transition of
its own. At low concentrations, HO 3408 has the same effect as the amiodarone,
by inhibiting the Ca2+-induced mitochondrial swelling, whereas it dissipated the
mitochondrial membrane potential (Dy), and prevented the
ischemia-reperfusion-induced release of apoptosis- inducing factor (AIF). At
higher concentrations, amiodarone but not HO 3408 induced a cyclosporin A (CsA)-independent
mitochondrial swelling. In contrast to these, desethylamiodarone did not inhibit
the Ca2+-induced mitochondrial permeability transition, did not induce the
collapse of Dy in low concentrations, and did not prevent the nuclear
translocation of AIF in perfused rat hearts, but it induced a CsA-independent
mitochondrial swelling at higher concentration, like amiodarone. In conclusion:
amiodarone analogues, like HO3408, can possess cardio-protective effect by
inhibiting the mitochondria apoptotic pathway: the permeability transition, the
collapse of Dy, and the release of the pro-apototic proteins.
Shuttling
Bax from the cytosol to mitochondria: a complex business involving N- and
C-termini
Anna Schinzel1, Thomas
Kaufmann1, Martin Schuler2, David Grubb1 and Christoph Borner1
1Institute
of Molecular Medicine and Cell Research (ZBMZ), Albert-Ludwigs University,
Stefan-Meier-Str. 17, D-79104 Freiburg, Germany; 2Department of
Medicine III, Johannes Gutenberg University, D-55101 Mainz, Germany
Email:
borner@ukl.uni-freiburg.de
The
apoptosis-inducing protein Bax resides inactive in the cytosol of
healthy cells. The C-terminal transmembrane region (TMB) is tucked into
a hydrophobic pocket and an N-terminal epitope is concealed. During
apoptosis Bax undergoes a conformational change involving N-terminal
exposure and translocates to mitochondria to release apoptogenic
factors. How this sequence of events is regulated remains unknown. Both,
the N- and C-termini have been reported to serve as targeting sequence,
and three proteins, Ku70, 14-3-3 and humanin were shown to bi,nd to the
either end of Bax and regulate its mitochondrial translocation. We show
that the C-terminal 23 amino acids of the Bax TMB are both necessary and
sufficient for mitochondrial targeting. Availability of the TMB
targeting sequence depends on Pro168 located within the preceding loop
region. Pro168 mutants of Bax lack apoptotic activity, cannot rescue the
apoptosis-resistant phenotype of Bax/Bak double knock-out cells and are
retained in the cytosol even in response to apoptotic stimuli. In
addition, these cytoplasmic mutants have their N-termini exposed. We
propose that Pro168 links the N- and the C-terminus of Bax and is
required for C-terminal release and mitochondrial targeting once this
link is broken. However, we have not found any evidence that Ku70 or
14-3-3 proteins participate in this process.
The mitochondrial apoptotic pathway in perforin/granzyme-induced
apoptosis mediated by CTL effectors.
Alberto Bosque11,
Julián Pardo1, Javier Naval1, Markus M. Simon2
and Alberto Anel11
1 Departamento de Bioquímica y Biología Molecular y Celular,
Facultad de Ciencias, Universidad de Zaragoza; 2 Max-Planck Institut für
Immunbiologie, Freiburg, Germany
E-mail:
abosque@unizar.es
In order to characterize if the mitochondrial pathway of
apoptosis is implicated in perforin/granzyme-induced death of Fas-resistant
EL4.F15 cells, we performed cytotoxicity assays with the H-2k anti-H-2b CTL
clone BM3.3, and measured in parallel the loss of mitochondrial membrane
potential (∆Ym) in EL4.F15 cells by flow cytometry, separating effector
from targets by CD8 staining. A particular pattern of ∆Ym loss was
observed when analyzing early time points: ∆Ym loss peaked at times as
short as 15 min, concomitant with DNA fragmentation but preceding 51Cr
release from target cells. However, the percentage of cells with low ∆Ym
begun to decrease at 45 min, being almost undetectable by 60 min. By150 min, the
percentage of cells with low ∆Ym increased again. In order to elucidate if
the transient ∆Ym loss was enough to initiate caspase-3 activation through
caspase-9, we performed parallel measurements of ∆Ym, caspase-3 and
caspase-9 activation during perf/gzm-mediated cytotoxicity exerted by CTL on
EL4.F15 cells using fluorescent caspase-9 (FAM-LEHD-fmk) or caspase-3 (SR-DEVD-fmk)
substrates. Caspase-9 and caspase-3 activation were detected concomitantly to
the transient ∆Ym loss, and their activation level increased steadily with
time, paralleling DNA fragmentation, despite the recovery of ∆Ym. Finally,
the general caspase inhibitor Z-VAD-fmk and the specific caspase-9 inhibitor
Z-LEHD-fmk inhibited at the same level DNA fragmentation induced during BM3.3
lysis of EL4.F15 (around 50%) or L1210.3 cells. This reflects our previous
observation on the contribution of both gzmA and gzmB in CTL-induced apoptosis
of EL4.F15 cells, while only the contribution of gzmB could be detected in
L1210.3 cells indicating that in these cell lines, perf/gzmB-induced DNA
fragmentation was entirely dependent on the mitochondrial amplification pathway
and on caspase-9 activation.
Involvement
of serpins in resistance to cell death
Michael
Bots,
Mirjam Rademaker, Guido de Roo and Jan Paul Medema
Department
of Clinical Oncology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA,
Leiden, the Netherlands
E-mail: m.bots@lumc.nl
Tumors
have several mechanisms to escape the immune system. One of these involves
expression of intracellular anti-cytotoxic proteins that modulate the execution
of cell death. Previously, we have
shown that the serine protease inhibitor SPI-6, which inactivates the cytotoxic
protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated
apoptosis, but not cytolysis. Moreover, we find that combined expression of
SPI-6 with a separate serpin, SPI-CI, provides tumor cells with a completely
resistant phenotype and allow escape in vivo. Besides their function as
immune escape molecules, recent data revealed that serpins might play a role in
the inhibition of lysosomal-dependent cell death. As lysosomal-dependent events
play a proximal role in chemotherapeutically induced cell death of non-small
lung cancer cells, we set out to determine whether serpins can convey
chemo-resistance to tumor cells as well.
The
coxsackievirus 2B protein suppresses apoptotic host cell responses by
manipulating intracellular Ca2+ homeostasis.
Michelangelo Campanella,1
Paolo Pinton,1 Frank J.M. van Kuppeveld2 and Rosario
Rizzuto.1
1Department
of Experimental and Diagnostic Medicine, Section of General Pathology and Center
for the study of Inflammatory Disease, Ferrara, Italy. 2Departments
of Medical Microbiology and Biochemistry, University Medical Center Nijmegen,
The Netherlands.
E-mail:
cmm@unife.it
Enteroviruses,
small cytolytic RNA viruses, confer an antiapoptotic state to infected cells in
order to suppress infection-limiting apoptotic host cell responses. This
antiapoptotic state also lends protection against cell death induced by
metabolic inhibitors like actinomycin D and cycloheximide. The identity of the
viral antiapoptotic protein and the underlying mechanism are unknown. Here, we
provide evidence that the coxsackievirus 2B protein modulates apoptosis by
manipulating intracellular Ca2+ homeostasis. Using fluorescent Ca2+
indicators and organelle-targeted aequorins, we demonstrate that ectopic
expression of 2B in HeLa cells decreases the Ca2+ content of both ER
and Golgi, resulting in downregulation of Ca2+ signaling between
these stores and the mitochondria, and increases the influx of extracellular Ca2+.
In our studies towards the physiological importance of the 2B-induced
alterations in Ca2+ signalling, we found that expression of 2B
suppressed cell death induced by various apoptotic stimuli, including
actinomycin D and cycloheximide. 2B mutants that were defective in reducing the
Ca2+ content of the stores failed to protect against apoptosis. These
data implicate a functional role of the perturbation of intracellular Ca2+
compartmentalization in the enteroviral strategy to suppress intrinsic apoptotic
host cell responses. The putative downregulation of an ER-dependent apoptotic pathway is discussed.
The
role of TUCAN in blocking procaspase-9 activation and determining
chemo-sensitivity in non-small-cell-lung cancer cells
Agnieszka
Checinska,
Bas Hoogeland, Carlos Ferreira, Jose A. Rodiguez, Giuseppe Giaccone and Frank A.
E. Kruyt
Department
of Medical Oncology, VU Medical Center, de Boelelaan, 1117,1081 HV Amsterdam,
The Netherlands
Email:
A.Checinska@vumc.nl / Agann@poczta.onet.pl
The
efficacy of chemotherapy of lung
cancer can be dependent on the activation of apoptosis. Chemo-induced apoptosis,
such as by cisplatin (CDDP), is generally activated via the activation of
caspase-9. Alterations in drug-induced apoptosis may represent an important
mechanism of drug resistance. Our results showed that procaspase-9 is not
activated following CDDP treatment in resistant non-small-cell lung cancer cells
(NSCLC). Here, we investigated the mechanism(s) underlying procaspase-9
inhibition in NSCLC cells. First, procaspase-9 was cloned from NSCLC-H460 cells
for determining potential mutations that could lead to defects in procaspase-9
activation. However, sequence analyses revealed no aberrations. The integrity of
procaspase-9 was further confirmed by the observation that FAS/CHX treatment
could overcome the block and trigger processing, as was also observed in
cell-free assays. Next, we monitored the expression of inhibitors of apoptosis (IAPs)
family members in a panel of NSCLC and SCLC cells by Western blotting. At the
protein level, TUCAN and cIAP-2 showed higher levels of expression in NSCLC
cells when compared to SCLC cells. Currently, TUCAN and procaspase-9
interactions are studied in NSCLC cells and RNA interference strategies are used
to silence TUCAN expression and examine the effect on procaspase-9 activation.
A
Role for BAFF in Tumour Immunity.
Paul
Chen,
Katja Simon, Xiaoning Xu, Gavin Screaton
Human
Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe
Hospital, Headington, OX3 9DS
E-mail:
paul.chen@balliol.ox.ac.uk
BAFF is a recent member of the TNF family
and has a unique role in B cell immunity. It is primarily expressed on cells of
myeloid origin and can be cleaved into soluble form like other members of the
TNF family. Elevated serum levels of BAFF seem to correlate with human disease,
particularly in SLE, rheumatoid arthritis, and Sjogren’s syndrome suggesting a
possible critical role for BAFF in several autoimmune diseases. BAFF can
stimulate B cells and act as a survival factor thus attenuating apoptosis, while
it is also known to be involved in B-cell maturation. The elucidation of the
precise mechanism of BAFF however, has been challenging as BAFF binds three
different TNF receptors (BCMA/TACI/BAFF-R) and shares overlapping function with
APRIL, another member of the TNF family. Here, we have found a novel role for
BAFF in which it elicits tumour immunity in a murine melanoma model with weak
immnogenicity and low expression of major histocompatibility complex (MHC) class
I. We further show that tumour immunity is mediated largely by antibodies. This
unique system can potentially allow us to raise antibodies against unknown
tumour antigens which can ultimately aid in determining new tumour antigens and
also raise the possibility in the design of strategies for vaccination against
cancer.
Direct Activation Of Bax By p53 Mediates Mitochondrial
Membrane Permeabilization And Apoptosis
Jerry E. Chipuk1,
Tomomi Kuwana1, Lisa Bouchier-Hayes1, Nathalie M. Droin1,
Donald D. Newmeyer1, Martin Schuler2 and Douglas R. Green1
1Division of Cellular Immunology, La Jolla Institute for
Allergy and Immunology, 10355 Science Center Drive, San Diego, CA, 92121, USA;
2Department of Medicine III, Johannes Gutenberg University, D-55101 Mainz,
Germany.
E-mail:
jerrychipuk@hotmail.com
The tumor suppressor p53 exerts its anti-neoplastic activity
primarily through the induction of apoptosis.
We found that cytosolic localization of endogenous wild type or
transactivation-deficient p53 was necessary and sufficient for apoptosis.
p53 directly activated the pro-apoptotic Bcl-2 protein Bax in the absence
of other proteins to permeabilize mitochondria and engage the apoptotic program.
p53 also released both pro-apoptotic multidomain and BH3-only proteins
sequestered by Bcl-xL. The
transcription-independent activation of Bax by p53 occurred with similar
kinetics and concentrations to those™ produced by activated Bid.
We propose that when p53 accumulates in the cytosol, it can function
analogously to the BH3-only subset of pro-apoptotic Bcl-2 proteins to activate
Bax and trigger apoptosis.
Interleukin
4 production in solid tumors increases cancer cell survival via
upregulation of cflip and Bcl-XL
Concetta Conticello,
Francesca Pedini†, Ann Zeuner†, Mariella Patti‡,
Monica Zerilli‡, Giorgio Stassi‡, Angelo Messina§,
Cesare Peschle†¶, Ruggero De Maria†
Department
of Experimental Oncology, Mediterranean Institute of Oncology, Catania, 95030,
Italy. †Department of Hematology and Oncology, Istituto Superiore
di Sanità, Rome 00161, Italy. ‡Department of Surgical and
Oncological Sciences, University of Palermo, 90127, Italy. §Department
of Biomedical Sciences, General Pathology Section, University of Catania, 95124,
Italy. Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, Pennsylvania 19107, USA
E-mail:
ettaconticello@libero.it
High
levels of IL-4 production in cancer are associated with poor prognosis and often
observed in advanced solid tumors. We found that IL-4 treatment significantly
reduced CD95- and chemotherapeutic drug- induced apoptosis in prostate, breast
and bladder tumor cell lines. Analysis of anti-apoptotic protein expression
revealed that IL-4 stimulation resulted in upregulation of cFlip and Bcl-XL.
Exogenous expression of cFlip inhibited apoptosis induced by CD95 and to a
lesser extent by chemotherapy, while tumor cells transduced with Bcl-XL
were substantially protected both from CD95 and chemotherapeutic drug
stimulation. Moreover, consistent IL-4 production and high expression of
both cFlip and Bcl-XL was observed in prostate, breast and bladder
cancer in vivo. Exposure of normal primary epithelial cells to IL-4
resulted in upregulation of antiapoptotic proteins and increased resistance to
chemotherapy, while primary carcinoma cells, expressing high levels of cFlip and
Bcl-XL, downregulated the antiapoptotic proteins and acquired
sensitivity to apoptosis only if cultured in the absence of IL-4. Thus, IL-4 is
able to protect tumor cells from CD95- and chemotherapy-induced apoptosis by
upregulating anti-apoptotic proteins such as cFlip and Bcl-XL. These
findings may provide useful information for the development of new therapeutic
strategies for cancer.
Cross-talk
between non receptor tyrosine kinases and caspases in the control of apoptosis
and tumor progression.
Silvia Cursi (1,2), Alessandra Rufini (2), Venturina Stagni (1,2), Maria Giovanna di
Bari (1,2), Roberto Testi (2) and
Daniela Barilà (1,2)
1)Dulbecco
Telethon Institute, and 2) Laboratory of Immunology and Signal Transduction
Department of Experimental Medicine and Biochemical Sciences, University of Rome
"Tor Vergata", Via Montpellier,1 00133 Rome, Italy
E-mail:
silviacursi@libero.it
Our work is aimed at the investigation of the
cross-talk between non receptor tyrosine kinases, usually implied in
proliferation signals transduction, and caspases, central players of the
apoptotic signal transduction. The study of the reciprocal control of tyrosine
kinases and caspases can contribute to understand the molecular bases of cancer
development. We
have shown
that c-Abl tyrosine kinase is cleaved by caspases during
death-receptor-induced apoptosis. Upon cleavage the caspase generated fragment
of c-Abl translocates to the nucleus where it contributes to apoptosis
progression. c-Abl and Arg, play a central role in many F-actin-dependent
processes, like cell death, adhesion and migration. Caspase cleavage results in
the loss of the F-actin binding domain suggesting that this event may affect
many F-actin-depending processes. We are currently investigating the role of
caspase cleavage of Abl family kinases in the control and execution of anoikis,
a programmed cell death caused by the loss of adhesion that prevents metastasis
formation. The role of tyrosine phosphorylation in the control and execution of
cell death is still largely obscure. We are currently testing if the activity of
caspases might be regulated by tyrosine phosphorylation. We will present
evidences showing how kinases can directly phosphorylate caspases and how this
modification can modulate apoptosis progression.
Synergy
between TRAIL and DNA damaging tumor therapies depends on Bax but not Bak
Clarissa von Haefen, Jana Wendt,
Dilek Güner, Claus Belka, Peter T. Daniel
Molecular
Hematology and Oncology, University Medical Center Charité, Campus Berlin-Buch,
Humboldt University, Lindenberger Weg 80, 13125 Berlin
Email:
pdaniel@mdc-berlin.de
The
death ligand TRAIL has been suggested as a suitable biological agent for the
selective induction of cell death in cancer cells. Moreover, TRAIL synergizes
with DNA damaging therapies such as chemotherapeutic drugs or ionising
irradiation. Here, we show that synergy of TRAIL and 5-fluorouracil (5-FU), or
ionising irradiation entirely depends on Bax proficiency in human carcinoma
cells. Isobolographic analyses showed a clear synergism of TRAIL and
chemotherapy in Bax-expressing cells. In contrast, the effect was merely
additive in DU145 cells lacking Bax. Notably, DU145 and other cells having lost
Bax such as the frequently employed HCT116 Bax k.o. cell line still express Bak.
Thus, Bak is not sufficient to mediate cross-sensitization and synergism between
5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for
drug-induced apoptosis but failed to confer synergy between TRAIL and 5-FU.
Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU
synergistically trigger oligomerization and clustering of Bax but not Bak. These
data establish distinct roles for Bax and Bak in linking the TRAIL death
receptor pathway to the mitochondrial apoptosis signaling cascade and delineate
a higher degree of specificity in signaling for cell death by multidomain Bcl-2
homologs.
Depletion
of Hsp70-3 leads to MIC-1 dependent growth arrest in tumor cells
Mads
Daugaard JensenQ, Mikkel RohdeQ, Kristian HelinE and Marja JäätteläQ
QApoptosis
Laboratory, Danish Cancer Society, Strandboulevarden 49, 2100-Copenhagen Ø,
Denmark. EDepartment of Experimental Oncology, European Institute
of Oncology, Via Ripamonti 435, 20141-Milan, Italy.
E-mail:
mdj@cancer.dk
Hsp70-3
is a testis specific member of the human heat shock protein (Hsp) 70-gene family
that is necessary for spermatocyte survival. Here, we report that Hsp70-3 is
also necessary for the growth of tumor cells. We found Hsp70-3 expressed in
cancer cell lines of various origins and, remarkably, its expression in tumor
tissue was elevated compared to normal tissue. Depletion of Hsp70-3 by RNA
interference led to programmed cell death in a tumor cell specific manner and
Hsp70-3 depleted tumor cells were arrested in G1 phase of the cell
cycle. An Affymetrix gene expression study of Hsp70-3 depleted HeLa cervix
carcinoma cells revealed a change in expression of several genes involved in
cell cycle progression and apoptosis. MIC-1, a member of the TGF-b
family, was up-regulated 19-fold and was found up-regulated in various cancer
cell lines upon Hsp70-3 depletion. The MIC-1 elevation could be inhibited by the
re-introduction of an Hsp70-3 construct with silent mutations in the RNAi target
site. Furthermore, co-depletion of MIC-1 by RNAi was able to relieve the cells
from the G1-arrest. These results identify Hsp70-3 as an essential
protein for cancer cell growth and survival, thus making it a putative target
for cancer therapy.
Identification
of proteins regulating p73 function
Daniela
Barcaroli2, Eliana Munarriz2, Carine Maisse1,
Seamus Martin3, Anastasis Stephanou4, Richard A. Knight5,
Gerry Melino1,2 and Vincenzo De Laurenzi1,2
1Biochemistry
Lab., IDI-IRCCS, c/o Dept. of Exp. Medicine, Univ. of
"Tor Vergata", Rome,
Italy; 2 MRC, Toxicology Unit,
Leicester University, Leicester, UK; 3Molecular
Cell Biology Lab.,Dept. of Genetics,The Smurfit Institute, Trinity College,Dublin,
Ireland; 4 Medical Molecular Biology
Unit, Institute of Child Health, University College London,
London, UK; 5 Dept. of Cystic
Fibrosis, National Heart and Lung Institute, Imperial College, London UK.
E-mail:
db103@le.ac.uk
p73
belongs to the family of transcription factors that also includes p53 and p63.
The three proteins share a significant degree of sequence homology, these
structural similarities are mirrored by a certain degree of functional overlap
and all members of the family are capable of inducing cell cycle arrest and
apoptosis. p73 activity depends on its steady state protein levels and a number
of evidence suggest that post-transcriptional regulation rather than
transcriptional control plays a major role in p73 response to DNA damage. Upon
DNA damage, p73 becomes activated and stabilized in a pathway that requires a
functional mismatch repair gene MLH1, and the enzymatic activation of c-Abl. In
order to identify proteins that bind and regulate p73 we performed a yeast two
hybrid screening and identified several p73 interacting proteins including
PIAS-1. In addition to their role as inhibitors of activated STATs, PIAS
proteins have been shown to act as SUMO ligases for a number of transcription
factors, and modulate their activities. Our results show that PIAS-1 binds and
sumoylates p73 and is capable of stabilizing its protein levels. PIAS binds,
sumoylates and stabilizes both TA and DN p73 isoforms. PIAS binding alone is
sufficient for p73 stabilization and does not require sumoylation. Sumoylation
of p73 determines its translocation to the nuclear matrix and this results in
loss of activity on p53 responsive promoters and reduction of apoptosis. Finally
PIAS expression also results in p21 down-regulation and and cell proliferation.
In conclusion PIAS is an important regulator of apoptosis and cell cycle,
and its activation counteracts p73 function resulting in cell survival and
proliferation.
Assembling
the p53 network in C. elegans by functional genomics
W.
Brent Derry,
Ruben Bierings*, Martijn van Iersel*, Leukena Cheam*, Thevagi Satkunendran and
Joel Rothman*
Program in Cancer Research, The Hospital
for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8; *Department
of Molecular, Cellular & Developmental Biology, University of California,
Santa Barbara, California, U.S.A. 93106.
Email:
brent.derry@sickkids.ca
To
understand the organization of the p53 signaling network we have taken two
global approaches to identify genes that interact with C. elegans p53, cep-1.
In the first approach, we utilized microarray technology to identify CEP-1
target genes that are activated and repressed by UV radiation. Over 1000 genes
were activated and over 800 repressed in a cep-1-dependent manner.
Over-represented genes upregulated by cep-1 in response to UV included
kinases, phosphatases and collagens, whereas DNA repair genes, transcription
factors, and genes involved in protein degradation were over-represented in the
repressed category. In an effort to sort out direct from indirect targets we
have developed an algorithm to search for putative p53 binding sites in the
genome and correlated these sites with the gene expression profiles. Together
these data reveal a complex and intricate coordination of downstream target
genes by cep-1 that respond to UV radiation. In a complimentary approach
to expression profiling we have recently completed a genome-wide RNAi screen for
cep-1 modifiers in order to uncover negative regulators and parallel
signaling pathways. The most striking class of interactions identified represent
genes that, when knocked down by RNAi, are lethal in a wild type background but
viable in cep-1 loss-of-function mutants. These genes represent a wide
variety of protein classes that include components of degradation machinery and
transcription factors. Other classes of genes that interact with cep-1
identified in our RNAi screen regulate rate of development and body morphology
in a cep-1-dependent manner. Moreover, it appears that cep-1 lies at the
node of an enormously complex genetic network to modulate stress response and
development. Future goals will be aimed at sorting out direct from indirect
interactions.
Messages
from the dead – The influence of apoptotic cell removal on immunity and
peripheral tolerance
Lucie
Dörner,
Heiko Weyd, Dagmar Riess, Diana Macasev, and Peter H. Krammer
Tumor
Immunology Program, German Cancer Research Center, Heidelberg, Germany
E-mail:
l.doerner@dkfz.de
In
a multicellular organism, apoptosis takes place continuously to remove unneeded
cells maintaining tissue homeostasis. The dying cells are taken up by
macrophages and dendritic cells that reside in the tissues as sentinels of the
immune system. Especially dendritic cells play an important role in presenting
scavenged material to T cells thus linking innate and adaptive immunity.
The uptake of apoptotic cells does not lead to an immune response even though
many potential self-antigens are presented. Recent reports suggest that
apoptotic cells play an active part in preventing an immune response by
modulating the dendritic cells that phagocytose them. The aim of this study is
to analyse the phenotypic and functional changes in dendritic cells after uptake
of apoptotic material, possibly leading to a “tolerogenic” dendritic cell or
an activation of regulatory T cells. Possible tolerogenic signals have been
identified in our group. These signals might play an important role in
preventing autoimmunity and maintaining peripheral tolerance.
A serine protease-dependent mechanism of death
receptor-induced apoptosis after caspase arrest.
Georg Dünstl,
T.
Meergans, A. Wendel and Gerard Künstle
Chair of Biochemical Pharmacology, University of Konstanz,
Germany
E-mail:
Georg.Duenstl@uni-konstanz.de
Actinomycin D-sensitized HepG2 cells are highly susceptible
to induction of apoptosis via stimulation of death receptors (TNF-R1, CD95 and
TRAIL receptor). We and others showed that the pan-caspase inhibitor zVADfmk
conferred complete protection of HepG2 cells against apoptosis induced by
TNFalpha or agonistic anti-CD95 antibody. Here we provide evidence that
prevention of apoptosis by zVADfmk is not due to inhibition of caspases but
rather an unspecific effect on other proteases. Incubation of HepG2 cells with
high concentrations of the pan-caspase inhibitor zVADfmk conferred complete
protection against TNF-, TRAIL- and agonistic anti-CD95 antibody-mediated
apoptosis. Interestingly, 200-250-fold lower concentrations of the inhibitor
were sufficient to completely block cytokine-induced activity of caspases. In
contrast, the concentrations of zVADfmk needed to prevent both cytokine-mediated
apoptosis and activation of caspases in primary murine hepatocytes were
virtually identical. Transfection of HepG2 cells with XIAP also completely
failed to prevent TNF-mediated apoptosis, suggesting that the observed
protection by zVADfmk was not due to inhibition of caspases. However, the serine
protease inhibitors TLCK and TPCK conferred protection against TRAIL but only in
presence of zVADfmk. These data suggest a switch to a serine protease-dependent
pathway in HepG2 cells when caspases are inhibited.
CD4+/CD25+
regulatory T cells and their role in immunity
Nadine
Eberhardt, Heiko Weyd, Elisabeth Suri-Payer and Peter H.
Krammer
Tumorimmunology Program, German
Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany
E-mail:
n.eberhardt@dkfz-heidelberg.de
The
immune system has evolved to defeat dangerous pathogens while protecting the own
organism. Therefore, under physiological conditions, T cells can be activated by
foreign proteins but are tolerant to self-proteins. Various mechanisms like
negative selection of self-reactive T cells in the thymus, ignorance of T-cells
for self-proteins sequestered in an organ, induction of T-cell anergy in the
periphery and suppression by regulatory T cells work together to ensure
self-tolerance. CD4+CD25+ regulatory T cells (Treg) are generated in the thymus
and their suppressive activity has been demonstrated in vitro as well as in
vivo. Uptake of apoptotic cells by dendritic cells does not lead to an
immune response, but may lead to tolerance. Our goal is to investigate the
influence of dendritic cells, which have eaten apoptotic cells, on T cell
responses, especially on the generation of Treg.
Lotti
Egger1,3,
Benjamin F. Cravatt2, Donmienne Leung2
and Christoph Borner1
1Institute
of Molecular Medicine and Cell Research (ZBMZ), Albert-Ludwigs University,
Stefan-Meier-Str. 17, D-79104 Freiburg, Germany; 2The
Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037 USA; 3
E-mail:
lotti.egger@mol-med.uni-freiburg.de
Effective execution of apoptosis requires the activation of
caspases. However, in many cases, broad-range caspase inhibitors such as
Z-VAD.fmk do not inhibit cell death because death signaling continues via basal
caspase activities or caspase-independent processes. Although death mediators
acting under caspase-inhibiting conditions have been identified, it remains
unknown whether they trigger a physiologically relevant cell death that shows
typical signs of apoptosis, including phosphatidylserine (PS) exposure and the
removal of apoptotic cells by phagocytosis. Here we show that cells treated with
ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as
cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS
exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the
stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF)
inhibited all these events, indicating that serine proteases mediated the
apoptosis-like cell death and phagocytosis under these conditions. The serine
proteases were found to act upstream of an increase in mitochondrial membrane
permeability. Using an active site directed inhibitor of serine proteases, a
protein of approximately 30 kD was affinity-purified from cell extracts of dying
cells, confirming the activity of serine proteases during the apoptotic process.
Thus, despite caspase inhibition or basal caspase activities, cells can still be
phagocytosed and killed in an apoptosis-like fashion by a serine
protease-mediated mechanism that damages the mitochondrial membrane.
CD40 stimulation increases both the
anti- and pro-apoptotic protein profile of B-CLL cells and renders them
sensitive to autologous CTL-attack
Arnon P. Kater1, Ludo M. Evers1,
Ester B.M. Remmerswaal2, Michiel F. Oosterwijk1,2, René A.W. van Lier2, Marinus
H.J. van Oers2 and Eric Eldering1
Department of Hematology1
and the Laboratory for Experimental Immunology2, Academic Medical Center,
University of Amsterdam, the Netherlands
E-mail:
e.eldering@amc.uva.nl
B cell chronic lymphocytic leukemia (B-CLL)
is a malignancy characterised by dysregulated apoptosis. The extent to which
various apoptotic pathways are still responsive to regulatory signals is
uncertain. B-CLL cells respond to CD40 triggering, leading to T cell dependent
immune responses but how this is achieved is uncertain.This study investigates
the influence of CD40 triggering of B-CLL cells on the expression of virtually
all direct regulators of apoptosis and correlates these findings with the
sensitivity of CD40 triggered B-CLL cells for chemotherapy- and death-receptor
induced apoptosis as well as T cell mediated killing. Upon CD40 stimulation the
constitutive anti-apoptotic profile was further increased by upregulation of
Bcl-xL, Bfl-1/A1 and downregulation of the BH3-only protein Harakiri.
Surprisingly, expression of the BH3-only protein Bid was strongly augmented.
CD40 stimulated B-CLL cells became resistant for drug-induced apoptosis. Despite
upregulation of the CD95 receptor,
CD40 triggering did not lead to enhanced sensitivity for CD95 or TRAIL.
Remarkably, autologous T cell killing, triggered by loading of CLL cells with
viral peptides, was more efficient after CD40 stimulation. This killing was
granzyme B dependent, and CLL targets showed mitochondrial depolarization and
caspase-3 activation. Thus, despite an increased anti-apoptotic profile, CD40
triggered B-CLL cells can be excellent targets for cytotoxic T cells.
TRAIL
resistance of glioblastoma cells by epigenetic silencing of caspase 8
Adriana Eramo1, Fiorenza Lotti1, Giovanni Sette1, Lucia Ricci-Vitiani1,
Roberto Pallini3, Cesare Peschle1 and Ruggero De Maria1,3.
1Department
of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità,
Rome, Italy; 2Department of Neurosurgery, Catholic University, Rome,
Italy; 3Mediterranean Institute of Oncology, Catania, Italy. a.eramo@iss.it
Life
expectancy of patients affected by glioblastoma multiforme is lower than one
year. The therapeutic use of TRAIL has been proposed to treat this disease based
on its ability to kill glioma cell lines in vitro and in vivo. Here, we explored
the possibility to kill primary glioblastoma cells with recombinant TRAIL.
Although glioma cell lines displayed variable levels of caspase 8 expression,
primary glioblastoma cells expressed low to undetectable levels of caspase 8. In
most cases, death receptor-sensitivity strictly correlated with the levels of
caspase 8, with primary cells being completely resistant to TRAIL. Since methyltransferases
mediate the epigenetic silencing of caspase 8 in neuroblastoma cells, we
investigated the ability of the demethylating agent decitabine to sensitize
glioblastoma cells to apoptosis. Decitabine treatment resulted in upregulation
of caspase 8, inhibition of cell growth and sensitization of primary
glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase 8 expression
was able to restore TRAIL-sensitivity in glioblastoma resistant cells,
indicating that decitabine sensitizes primary glioblastoma cells through the
upregulation of caspase 8. Thus, the use of demethylating agents may be required
to prime glioblastoma cells to TRAIL-induced apoptosis. The in vivo
potential of this therapeutic strategy is currently being evaluated in nude mice
xenotransplants.
The evaluation of soluble fas and soluble fas ligand levels
of Bronchoalveolar lavage (bal) in lung cancer patients.
Beril Bahadir Erdogan,
Esra Uzaslan, Ferah Budak, Barbaros Oral, Mehmet Karadag, Dane Ediger, Güher Göral,Ercüment
Ege, Oktay Gözü
Uludag University Medical School, Lung Diseases Department,
16059, Bursa, Turkey
Email:drberilbahadir@hotmail.com
Fas-Fas Ligand (FasL) is one of the major mediator system
that activates programmed cell death. Cleavage of membrane-bound FasL by a
metalloproteinase-like enzyme resulted in the formation of soluble FasL (sFasL).
sFasL as well as the transmembrane form of FasL binds to Fas and transduces
apoptotic signal in Fas-expressing cells. It's suggested that soluble Fas and
soluble FasL has an impact on tumor progress and immune escape of tumor cells
from the host immune system. Since some of Fas antigen expression in the lungs
has been localized to alveolar and bronchial epithelial cells, in this study we
aimed to investigate the soluble Fas (pgr/ml) and soluble FasL levels (pgr/ml)
of BAL fluid in lung cancer patients. Study population was consisted of 27
patients with lung cancer (mean age 63±11 years), 25 control subjects (mean age
48±14 years). BAL was performed before any treatment modality applied to the
lung cancer patients.BAL sFas and sFasL were evaluated by using ELISA method.
The mean levels of sFas was 60.8±56.8 in lung cancer patient and 39.5±25.9 in
control subjects (p>0.05). The mean levels of sFasL
was 51.6±39.2 in cancer patient and 41.2±27.4 in control subjects
(p>0.05). In conclusion, although we did not observe any significant
difference, higher BAL levels of sFas and sFasL levels in lung cancer patients
than control subjects, made us thought that they might have a role in escape of
tumor cells from immun system.
PUMA/bbc3,
a key mediator of p53-induced apoptosis
Miriam
Erlacher1, Andreas
Strasser2 and Andreas Villunger1
1Institute
of Pathophysiology, Innsbruck Medical School, Innsbruck, AUSTRIA
2The
Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia
E-mail:
miriam.erlacher@uibk.ac.at
PUMA/bbc3,
a pro-apoptotic Bcl-2 family member of the BH3-only subgroup, has been
implicated in mediating both, p53-dependent and p53-independent forms of
apoptosis. Previous studies indicate that PUMA/bbc3-deficiency protects primary
lymphocytes, mouse embryonic fibroblasts and colorectal carcinoma cell
lines against DNA damage-induced apoptosis. In our current study, we aimed to
assess the anti-apoptotic potential of PUMA/bbc3 in stress-induced apoptosis in
vivo and to compare the effects of its absence with the effects caused by
absence of the BH3-only protein Bim or overexpression of Bcl-2. Mice lacking
PUMA/bbc3, its relative Bim and animals overexpressing Bcl-2 were therefore
exposed to g-radiation or the glucocorticoid receptor agonist dexamethasone.
Analysis of cell numbers and composition of haematopoietic organs such as bone
marrow, thymus, spleen and lymph nodes revealed that loss of PUMA/bbc3 provides
efficient protection from DNA damage-induced apoptosis but only minor protection
from steroid-induced apoptosis in vivo. Interestingly, both forms of cell
death were inhibited more efficiently in animals expressing a Bcl-2 transgene in
their haematopoietic compartment suggesting that execution of these death
programmes may rely on additional pro-apoptotic Bcl-2 family member. Thus, we
intend to study the consequences of the combined loss of PUMA/bbc3 with other
BH3-only proteins.
Current
studies on the activation of caspase-8 and its role within the nucleus.
Alison
J. Faragher,
Nicholas Harper and Gerald M. Cohen
MRC
Toxicology Unit, Hodgkin Building, PO Box 138, University of Leicester,
Lancaster Road, Leicester, LE1 9HN, UK
Email:
ajf26@le.ac.uk
Previous studies within our lab have
demonstrated that although Fas-associated death domain protein (FADD) and
caspase 8 are obligatory for tumor necrosis factor (TNF) mediated apoptosis they
are not recruited to the tumor necrosis factor receptor 1 (TNF-R1) signaling
complex, suggesting that caspase 8 is activated in a different secondary complex
during TNF mediated apoptosis. It was therefore the aim of this study to
investigate the activation of caspase 8, using antibodies specific to the
catalytically active form of caspase-8, to determine the location and
consequences of caspase 8 activation. We now demonstrate using confocal
microscopy that caspase 8 is activated at an early stage of apoptosis,
coincident with detection of caspase 8 activation with western blotting. Further
investigation demonstrated that the catalytically active form of caspase-8 was
detectable as discrete dots within the nucleoli, suggesting the formation of
secondary complexes. Further work is now underway to determine the substrates of
caspase 8 within the nucleolus.
HTLV-1
Tax protects against CD95-mediated apoptosis by induction of the cellular
FLICE-inhibitory protein (c-FLIP)
Stefanie
C. Fas*,
Andreas Krueger*, Min Li-Weber and Peter H. Krammer
*These
authors contributed equally to this work.
Tumorimmunology Program, German
Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
E-mail:
s.fas@dkfz.de
The
HTLV-1 transactivator protein Tax is essential for malignant transformation of
CD4 T cells, ultimately leading to adult T cell leukemia (ATL). A key feature
that contributes to malignant transformation may be the induction of apoptosis
resistance. In this study we investigated the molecular mechanisms by which
HTLV-1 Tax confers resistance towards CD95-mediated apoptosis. We show that
HTLV-1 transformed Tax expressing T cell lines express elevated levels of c-FLIPL
and c-FLIPS correlating with resistance towards CD95-mediated
apoptosis. Analysis of Bid cleavage revealed the level of apoptosis inhibition
as receptor proximal, indicating a functional relevance of elevated expression
of c-FLIP. Using an inducible system we demonstrate that both resistance towards
CD95-mediated apoptosis and induction of c-FLIP were directly dependent on
activity of the Tax protein. In conclusion, the data provide a potential
mechanism by which expression of HTLV-1 Tax may lead to immune escape of
infected T cells and, thus, to persistent infection and transformation.
Tumorigenesis-associated
sensitization to tumor necrosis factor-mediated cell death depends on cathepsin
B
Nicole
Fehrenbacher*,
Mads Gyrd-Hansen*, Birgit Poulsen*, Ute Felbor†, Tuula
Kallunki*, Marianne Boes‡,
Ekkehard Weber§, Marcel Leist¶ and Marja Jäättelä*
*Apoptosis
Laboratory, Danish Cancer Society, Copenhagen, Denmark,
†Institute of Human Genetics, University of Würzburg, Würzburg, Germany, ‡Department
of Pathology, Harvard Medical School, Boston, MA, §Institute of Physiological
Chemistry, Medical Faculty, Martin-Luther-University, Halle, Germany and ¶Disease
Biology, H. Lundbeck A/S, Valby, Denmark
Email:
Nf@cancer.dk
Tumorigenesis
is associated with several changes that alter the cellular susceptibility to
programmed cell death. Here we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated
lysosomal death pathway. Spontaneous immortalization increased the
susceptibility of wild type murine embryonic fibroblasts (MEFs) to tumor
necrosis factor (TNF)-mediated cytotoxicity over thousand-fold, whereas
immortalized MEFs deficient for lysosomal cysteine protease cathepsin B retained
the resistant phenotype of primary cells. This effect was specific for cysteine
cathepsins, since also lack of Cathepsin L (a lysosomal cysteine protease), but
not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major
executioner protease in classic apoptosis) inhibited the
immortalization-associated sensitization of MEFs to TNF. Akin to spontaneous
immortalization, oncogene-driven transformation with v-Ha-ras or c-src
sensitized cells to the cysteine cathepsin-mediated death pathway. Importantly,
exogenous expression of cathepsin B partially reversed the resistant phenotype
of immortalized cathepsin B deficient MEFs, and the inhibition of cathepsin B
activity by pharmacological inhibitors or RNA interference attenuated
TNF-induced cytotoxicity in immortalized and transformed wild type cells. Thus,
cysteine cathepsins, which are commonly over-expressed in human cancer, may
counteract tumorigenesis and enhance therapeutic responses by sensitizing cells
to programmed cell death.
Structural
analysis of Fas and FADD death domain Interactions
Brian J. Ferguson1
and Paul C. Driscoll1,2
1Department of Biochemistry and Molecular Biology, University
College London, Gower Street, London WC1E 6BT 2Ludwig institute of Cancer
Research, 91 Riding House Street, London W1W 7BS.
E-mail:
ferguson@biochem.ucl.ac.uk
In order to study in vitro the interaction between the death
domains of Fas (Fas-DD) and FADD (FADD-DD), a Fas-DD fusion protein was
engineered that is suitable for structural studies. A titration of soluble
Fas-DD into 15N-labelled FADD-DD was monitored by heteronuclear NMR experiments
and resulted in monotonic loss of FADD-DD signals from all but the flexible N-
and C-terminal regions, consistent with the formation of a large, soluble
complex. This result has been repeated using a non-fusion Fas-DD with a single
point mutation that improves the WT domain's solubility characteristics.
Mutational analysis shows the interaction between Fas-DD and FADD-DD can be
disrupted by substituting residues on any surface of Fas-DD. Our data is
consistent with the formation of a hetero-oligomeric Fas-DD/FADD-DD complex in
which multiple surfaces of both death domains take part, extending previous data
which suggested a primary role for one specific region on each of the two
domains (namely the a2/a3 surface). Current work is focussing on further
characterisation of the physicochemical and hydrodynamic properties of the
complex formed by the two species in solution and attempts are being made to
study the affects of the Fas-DD mutants in a cellular context.
Nele
Festjens, Michael Kalai, Ann Meeus, Tom
Vanden Berghe, Joel Smet*, Rudy Van Coster*, Xavier Saelens and Peter
Vandenabeele
Molecular
Signaling and Cell Death Unit, Department of Molecular Biomedical Research, VIB,
Ghent University, Technologiepark 927, 9052 Zwijnaarde, Belgium. *Department
of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University
Hospital, De Pintelaan, 185, 9000 Ghent, Belgium
TNF
and dsRNA both lead to necrosis in L929 fibrosarcoma cells. Mitochondria-
produced reactive oxygen species (ROS) were suggested to mediate both necrotic
signaling pathways. Therefore, we compared the effects of two chemical
antioxidants butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT) on
TNF- and dsRNA-induced necrosis. Although both chemicals equally reduced the
levels of ROS detected in TNF treated cells, BHA was much more anti-necrotic
than BHT. In dsRNA-induced cell death both compounds reduced the levels of ROS.
Nevertheless, BHT did not affect the necrotic outcome, while BHA shifted the
response to apoptosis. These results suggest that inhibition of necrotic cell
death does not depend exclusively on ROS scavenging and imply that BHA has
additional effects lacking for BHT. Therefore, we investigated the effect of BHA
and BHT on electron transport chain complex activities. We show that BHA and BHT
are both able to inhibit NADH-dehydrogenase (complex I) activity in a
concentration dependent manner but BHA is more effective than BHT. Support for
the involvement of mitochondrial respiration in TNF- and dsRNA-induced necrotic
cell death was also provided by studies with electron transport chain inhibitors.
These results demonstrate that the mitochondrion is a major crossroad in the
pathways leading to apoptosis and necrosis.
Comparison of substrate cleavage by
initiator caspase-8 and -10 in apoptotic pathways
Ute Fischer,
Ajoy
Kumar Samraj, Reiner U. Jänicke and Klaus Schulze Osthoff
Institute of Molecular Medicine,
University of Düsseldorf, Germany
e-mail:
Ute.Fischer@uni-duesseldorf.de
While the key role of
caspase-8 in death receptor-mediated apoptosis is well established, the function
of its closest homologue, caspase-10, is largely unknown. There is no doubt that
caspase-10 can be recruited to death receptor signaling complexes, but it
remains controversial whether caspase-10 can substitute for caspase-8 and
whether it exerts a specific or redundant role. The different phenotype of
patients with caspase-10 mutations suffering from autoimmune lymphoproliferative
syndrome-type II and patients with caspase-8 mutations, might suggest that both
initiator caspases serve a specific function. A key to the understanding of
caspase function is the analysis of its downstream targets. For instance,
cleavage of RIP kinase-1 by caspase-8 shuts down NF-kB controlled cell survival
pathways. Furthermore, caspase-8 initiates the mitochondrial apoptotic pathway
by cleaving Bid to its proapoptotic form tBid. However, specific substrates of
caspase-10 are as yet completely unknown. Therefore we have compared caspase-8
and -10 regarding their substrate cleavage. We found several proteins that are
differentially cleaved by caspase-10 and caspase-8. The functional consequences
of these differences in substrate cleavage for the propagation of the apoptotic
signal and the interference with the mitochondrial pathway will be discussed.
Tumor-associated
E-cadherin mutations – their role in apoptosis signalling and cisplatin
treatment
Margit
Fuchs, Elena Fricke, Christine Hermannstädter, Gisela
Keller, Heinz Hoefler, Birgit Luber
Institute
of Pathology, Klinikum rechts der Isar (Technical University Munich), Trogerstr.
18, 81675 Munich, Germany
E-mail:
giti.fuchs@gmx.net
The
cell adhesion molecule E-cadherin acts as a tumor and invasion suppressor and is
frequently downregulated or mutated in tumors. Recent studies of MDA-MB-435S
cells transfected with tumor-associated mutant E-cadherin variants with
alterations in exons 8 or 9 showed that these mutations affect cell adhesion,
proliferation and tumorigenicity. In the present study, we investigated the
effect of wild-type (wt) and mutant E-cadherin in MDA-MB-435S
transfectants on important apoptosis-regulators (Akt/PKB, p38 and p53) and
responsiveness to the chemotherapeutic agent cisplatin. While Akt/PKB expression
was similar in all cell lines, the kinase activity was 2-fold increased in wt-E-cadherin
expressing cells compared to mutant E-cadherin expressing or parental E-cadherin
negative MDA-MB-435S cells. The activity and expression of p38 were induced by
E-cadherin expression independently of the mutation status. Further, wt-
and mutant E-cadherin transfectants showed reduced cisplatin sensitivity
compared to parental cells. Since p53 influences the sensitivity of cells to
chemotherapeutic agents, we investigated its role dependent on E-cadherin
mutations. Transfection of MDA-MB-435S cells with wt- or mutant
E-cadherin neither altered the genetic p53 background nor the p53 expression
level. In conclusion, our results suggest that the expression level and/or
mutation status of E-cadherin influence apoptosis signalling and cisplatin
sensitivity of tumor cells.
Apoptosis
induced by farnesyltransferase inhibition. Initiation by conformational changes
of Bax and Bak and Mcl-1 elimination and execution by caspases and AIF.
María Gómez-Benito,
Isabel Marzo, Alberto Anel and Javier Naval.
Departamento de Bioquímica y
Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza,
Spain.
E-mail:
Marigomb@unizar.es
We
have studied the mechanism of apoptosis elicited by the farnesyltransferase
inhibitor BMS-214662 in human myeloma cell lines (RPMI-8226, U266 and
NCI-H929). BMS-214662 at low,
inhibitory doses, induced proapoptotic conformational changes in Bax and Bak,
reduction of Mcl-1 levels, mitochondrial membrane potencial loss,
cytochrome c release, caspase activation, AIF nuclear translocation,
phosphatidylserine exposure and development of apoptotic morphology.
Western blot analysis of cell extracts revealed the activation of at
least caspases 2, 3, 8 and 9 in all
cells. Cotreatment with the general caspase inhibitor Z-VAD-fmk
significantly prevented BMS-214662-induced death in U266 and RPMI 8226
cells for the first 30 and 48 h
respectively, but not at longer times. It was unable to
prevent cell death in NCI-H929 cells. Z-VAD-fmk prevented nor the
conformational changes of Bax and
Bak neither the rapid decrease of Mcl-1 levels. Selective
caspase inhibitors Ac-DEVD-CHO, Ac-LEHD-CHO, Z-IETD-fmk or Z-VDVAD-fmk
prevented some morphological and
biochemical features of apoptosis but not cell death.
These results suggest that apoptosis is initiated by a Bax/Bak/Mcl-1-dependent
mechanism. However, in some cases, is not sufficient per se to induce
mitochondrial failure and release of apoptogenic proteins and an
unidentified caspase must be
activated to initiate these processes.
Apoptosis
in the eye: a model of photoreceptor degeneration.
Violeta
Gomez-Vicente,
Maryanne Donovan and Thomas G. Cotter
Tumour Biology Laboratory, Biochemistry
Department, Biosciences Institute, University College Cork, Cork, Ireland
E-mail:
vgvicente@hotmail.com
Retinitis pigmentosa (RP) is the name of a
heterogeneous group of genetic diseases leading to progressive photoreceptor
loss. Though a large number of mutations, involving at least 32 different genes,
have been identified so far, photoreceptors share a common final: death by
apoptosis. The study of the apoptotic mechanisms in this and other retinal
degenerations has represented a difficult task due to the complexity and variety
of cell types in the retina, which are not easy to isolate in high numbers.
Therefore, the employ of a transformed mouse photoreceptor cell line (661W)
pretends to be a practical approach to the problem. In a few examples of RP
(PDE6B, GCAP1, KIF3A) an increase in the cytoplasmic calcium levels occurs as a
result of the genetic defect. Since calcium has been previously related to the
induction of programmed cell death, this metabolic overload might be the
triggering factor. Concerning this, the use of two apoptotic-inducing agents,
which mimic the conditions of the disease in vivo, has allowed us to
describe the occurrence of different apoptosis pathways in 661W cells, including
the participation of the endoplasmic reticulum-associated protease, caspase-12,
not linked to photoreceptor degeneration to date.
Douglas R. Green, Edith Janssen, Nathalie
Droin, Stephen Schoenberger
Division
of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San
Diego, CA, USA. La Jolla Institute for Allergy and Immunology
E-mail: DGreen5240@aol.com
TRAIL, a member of the TNF ligand family,
can induce apoptosis upon its engagement of TRAIL receptors, a finding that has
led to its experimental use as a cancer chemotherapeutic.
Its normal physiological roles, however, have remained elusive.
Studies with TRAIL knockout mice have suggested that TRAIL may be
required for the regulation of autoimmunity and the process of negative
selection in T cell development. The
latter finding, however, remains highly controversial.
We have identified a fundamental biological process that is regulated by
expression of TRAIL. This has been
shown by identification of differentiation states in the relevant cells that
lead to expression, or not of TRAIL, and inhibition of TRAIL (by antibodies or
decoy receptor) or use of TRAIL KO mice, results in a conversion of one
differentiation state to the other. We
will discuss our latest findings and the implications for TRAIL-based therapies.
Deregulated
survivin expression is relevant for disease prognosis in esophageal tumors with
loss of p16INK4a
Dilek
Güner,
Philipp G. Hemmati, Patricia Grabowski, Peter T. Daniel
Molecular Hematology and Oncology, University Medical Center Charité,
Campus Berlin-Buch, Humboldt University, Lindenberger Weg 80, 13125 Berlin
E-mail:
dguener@gmx.de
Deregulation
of the Rb-pathway and G1-restriction point control is a frequent event in cancer.
We previously observed that intact p16ink4/CDCN2 gene expression is
associated with good outcome in patients with esophageal squamous cell
carcinoma. Here, we addressed the role of additional regulators. A cohort of 53
patients was investigated who underwent resection of squamous cell carcinoma of
the esophagus with a curative intent to treat. Here, we show that deregulation
of G1 cell cycle restriction point control due to overexpression of cyclin D1/E
or loss of p16/p21/p27/Rb is associated with poor prognosis. Moreover, we
establish that elevated expression of the IAP protein survivin is associated
with a poor disease prognosis. The novel finding of this study is, however, that
survivin expression was related to poor prognosis only in patients with intact
p16INK4a expression or lack of cyclin D1 expression. This delineates
a clinically relevant link between cell cycle regulatory properties by survivin
and G1 restriction point control. This is in accordance with recent findings
that assign a preferentially cell cycle regulatory role to survivin.
Crosstalk
between Caspases and cysteine cathepsins in TNF-induced cell death.
M.
Gyrd-Hansen, J. Nylandsted, M. Hoyer Hansen &
M.Jaattela
Apoptosis
Laboratory, Danish Cancer Society, Copenhagen, Denmark
E-mail: mgh@cancer.dk
Caspases
have long been considered as key executioners of all apoptosis. However, recent
data suggest that also lysosomal cysteine proteases, when released into the
cytosol, can mediate and/or execute tumour necrosis factor (TNF)-induced
programmed cell death(PCD). Here, we studied the interplay between caspases and
cathepsins in TNF-induced PCD in MCF7 cells. We show, while TNF-induced PCD in
MCF7 cells is dependent on caspase activity, cysteine cathepsins are released
into the cytosol and participate in the death process. Lysosomal membrane
permeabilisation (LMP) and the ensuing release of cysteine cathepsins after
TNF-treatment was blocked by pre-treatment with caspase inhibitor zVAD-fmk or
overexpression of Bcl2, but was unaffected by depletion of Apaf-1. Also, ectopic
expression of caspase-3 accelerated release of lysosomal enzymes, but did not
alter the extend of release. Inconcordance, inhibition of cysteine cathepsins
had no effect on TNF-induced cytochrome c release. However, cysteine cathepsins
were needed for full activation of caspase-3 upon TNF-treatment in
MCF7-caspase-3 cells. Together, these data suggest that TNF-induced LMP occurs
downstream of Bcl2, but upstream apoptosome formation and effector caspase
activation, that cytosolic cysteine cathepsins are needed for activation of
effector caspases and that caspase-3 can mediate a positive feedback loop to
accelerate LMP.
Enhanced
Caspase-8 Recruitment to and activation at the DISC is Critical for
Sensitisation of Human Hepatocellular Carcinoma cells to TRAIL-induced Apoptosis
by Chemotherapeutic Drugs
Tobias
L. Haas1, Tom M. Ganten1,2, Jaromir Sykora1,2, Heiko Stahl1,
Martin R.
Sprick1, Stefanie C. Fas3,
Andreas Krueger3, Markus A. Weigand1, Anne Grosse- Wilde1, Wolfgang Stremmel2,
Peter H. Krammer3 and Henning Walczak1
1Div.
of Apoptosis Regulation, German Cancer Research Center (DKFZ), Heidelberg,
Germany. 2Dept. of Internal
Medicine, University of Heidelberg, Heidelberg, Germany.
3Div. of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg,
Germany.
E-mail: T.Haas@dkfz-Heidelberg.de
TRAIL
is discussed as a promising anticancer therapeutic agent. However, about 60% of
tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of
tumour resistance to TRAIL we investigated hepatocellular carcinoma cell lines
that exhibit differential sensitivity to TRAIL. Pre-treatment with
chemotherapeutic drugs, e.g. 5-fluorouracil (5-FU), rendered the TRAIL-resistant
hepatocellular carcinoma cell lines sensitive to TRAIL-induced apoptosis.
Analysis of the TRAIL death-inducing signalling complex (DISC) revealed
upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the
DISC was substantially increased after 5-FU sensitisation while FADD recruitment
remained essentially unchanged. 5-FU pre-treatment downregulated cFLIP and
specific cFLIP downregulation by siRNA was sufficient to sensitise
TRAIL-resistant hepatocellular carcinoma cell lines for TRAIL-induced apoptosis.
Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased
effectiveness of caspase-8 recruitment to and activation at the DISC facilitated
by the downregulation of cFLIP and the consequent shift in the ratio of
caspase-8 to cFLIP at the DISC. In addition, other cellular mechanisms of TRAIL
sensitisation will be presented.
Fernando
Abaitua, L. Planelles,
D. Rodríguez, C. Martinez-A., Michel Hahne
Institut de Génétique Moléculaire
de Montpellier (IGMM),CNRS UMR5535
Email:
hahne@igm.cnrs-mop.fr
The TNF-like ligands APRIL and BLyS are close relatives and
share the capacity to bind the receptors TACI and BCMA.
BLyS has been shown to play an important role in B cell homeostasis and
autoimmunity, but the biological role of APRIL remains less well defined.
We used APRIL transgenic (Tg) mice to further analyse the effect of APRIL
in humoral responses. Following
immunisation with a T cell-independent type 1 antigen, APRIL Tg mice showed
increased IgM and IgG titres throughout the immune response.
Induction of a T cell-dependent humoral response by administration of
vaccinia virus resulted in elevated IgM titres in the transgenic mice up to 100
days after immunisation; in contrast, IgG responses were downregulated more
rapidly in APRIL Tg than in control mice. Induction
of distinct signals by TNF ligand family members is well established for example,
Fas ligand stimulates T cells during the early activation phase, whereas it
mediates apoptosis at later phases of T cell activation. We are presently
testing whether the observed distinct signalling of APRIL can be also observed
during T cell-dependent humoral responses against non-viral antigens.
Exogenous
H2O2: A new anti-apoptotic effect in wheat roots.
Email:
amjad46pk@yahoo.com
Early
stages of ontogenesis in plants are accompanied with programmed death of
individual cells and tissues. We studied the effect of exogenous H2O2
on apoptosis and antioxidant systems during early ontogenesis in different
organs of etiolated wheat seedlings. Here we report very first time, a new
anti-apoptotic effect of exogenous H2O2 in plant system.
Internucleosomal nDNA fragmentation analysis revealed anti-apoptotic and
growth enhancing effect of exogenous H2O2 treatment in
wheat roots. Exogenous H2O2 (100mM) treatment completely
abolished the nDNA fragmentation, observed during normal ontogenesis in roots of
control seedlings. Significant (P<0.01) increase in root mass, number,
ascorbate and catalase level in treated roots (compared with control), further
supported the growth enhancing/anti-apoptotic effect of exogenous H2O2.
Contrary to roots, H2O2 treatment induced apoptosis in coleoptile and initial leaf and was
accompanied with lower ascorbate and catalase levels while increased level of
proteases overlapping with course of apoptosis
induction. In coleoptile, after 48hours of
treatment H2O2 induced
apoptosis, which was 24 hours earlier as compared with control. Analysis
of ascorbate and catalase dynamics with special
reference to apoptosis showed that both are key molecules in the
signaling pathway for modulating the H2O2 induced
apoptosis. Collectively a dual, organ specific
anti/pro-apoptotic effect of H2O2 was observed.
Pathologically elevated cyclic hydrostatic pressure induces
CD95-mediated apoptotic cell death in vascular endothelial cells
C Hasel*, S Dürr*, A Bauer, S Brüderlein, T Tambi, U Bhanot
and P Möller
Institute of Pathology, University of Ulm, D-89081 Ulm,
Germany
E-mail: cornelia.hasel@medizin.uni-ulm.de
We describe cyclic hydrostatic pressure of 200/100 mmHg with
a frequency of 85/min as a hemodynamically relevant pathologic condition
enforcing apoptosis on endothelial cells. After 24 hrs apoptotic cell death was
significantly increased, with only a slightly higher value at 48 hrs compared to
untreated cells and cells subjected to physiological hydrostatic pressure 120/80
mmHg. This went along with an increase of CD95 and CD95L surface expression,
cleavage of caspases 3 and 8, elevated JNK expression and induction of
phosphorylated c-jun. Furthermore, an induction in DNA-binding at the regulatory
element of the CD95L promoter was observed. These findings were most prominent
at 24 hrs, >the CD95/CD95L expression
pattern switching back to basic levels after 48 hrs and thus implicating a
vulnerable window for endothelial cell apoptosis triggered by pathological
hydrostatic pressure after which the constitutive CD95-/CD95L+ status is
restored. Caspase inhibitors and a CD95L neutralizing antibody almost completely
prevented endothelial cell apoptosis
E-mail: thomas.hofmann@hpi.uni-hamburg.de
The CD95 (Fas/APO-1) death receptor
triggers apoptosis by activation of caspase-8. Promyelocytic leukemia-nuclear
bodies (PML-NBs) are important nuclear tumour suppressor domains which regulate
CD95-induced apoptosis through an yet unknown mechanism. Here we identify
FLICE-associated huge protein (FLASH), a protein previously described to
regulate procaspase-8 activation, as a molecular link between PML-NBs and the
CD95 death pathway. FLASH interacts with the constitutive PML-NB component Sp100
and predominantly localises to the cell nucleus and PML-NBs under physiological
conditions. Following CD95 ligation FLASH translocates in a Crm1-dependent
manner from PML-NBs and the cell nucleus into the cytoplasm, which unmasks
FLASHs apoptogenic activity. In the cytoplasm, FLASH accumulates at the
mitochondria where it associates with endogenous caspase-8. Interestingly,
depletion of endogenous Sp100 by RNA interference significantly increases
cytoplasmic localisation of FLASH and potentiates CD95-induced apoptosis. Taken
together, our data identify FLASH as a molecular link between the CD95 death
receptor and PML-NBs, and uncover a novel unexpected nuclear apoptosis pathway
targeting caspase-8 activation. This pathway is initiated by release of
Sp100-bound FLASH from PML-NBs, its export into the cytoplasm and translocation
to mitochondria and procaspase-8, which results in proteolytic activation of
caspase-8.
Bcl-2
inhibits autophagy via its localization to the ER
Maria
Høyer-Hansen1, Ida Stenfeldt Mathiasen1, David Andrews2,
Brian Leber2, Lone Bastholm3, Folmer Elling3,
Michelangelo Campanella4 and Marja Jäättelä1.
1Apoptosis
Laboratory, Danish Cancer Society, Copenhagen, Denmark, 2McMaster
University, Hamilton, Ontario, Canada, 3Institute of Molecular
Pathology, Faculty of Health Sciences, University of Copenhagen, Denmark, 4Department
of Experimental and Diagnostic Medicine, Section of General Pathology,
University of Ferrara, Italy.
E-mail: Mhh@cancer.dk
The
vitamin D analogue, EB1089, is a promising anti-cancer agent. In MCF-7 breast
cancer cells it triggers a calcium-dependent programmed cell death (PCD) that
can be inhibited by Bcl-2. Here we show that EB1089-induced PCD can be
classified as autophagic degeneration. EB1089 triggered a massive increase in
autophagic vesicles and the inhibition of autophagy by 3-methyladenine rescued
cells from EB1089-induced death. Interestingly, a Bcl-2 variant localized to the
ER prevented EB1089-induced autophagy and DNA fragmentation, whereas it failed
to abolish the mitochondrial apoptosis pathways induced by tumor necrosis factor
or thapsigargin. Vice versa, Bcl-2 variants localized to mitochondria or cytosol
inhibited the mitochondrial death pathways, but failed to inhibit autophagy.
Interestingly, the ER-localized Bcl-2 reduced the steady state Ca2+
amount in the ER. Moreover, the EB1089-induced reduction in ER calcium level was
not as pronounced in cells expressing ER-located Bcl-2 as in cells expressing
mitochondrial or cytosolic Bcl-2. These results suggest that Bcl-2-mediated
protection against EB1089-induced autophagy requires the localization to the ER
and occurs via the reduction in ER calcium stores.
The
regulation of neuronal cell fate and protection from apoptosis by the
interaction of the Brn-3 and p73 family of proteins
Chantelle
D Hudson, David s Latchman, Vishanie Budrum-Mahadeo
Institute
of Child Health, University College London, UK
E-mail: c.hudson@ich.ucl.ac.uk
The
Brn-3a and Brn-3b POU transcription factors are expressed in the developing
nervous system where Brn-3a is associated with neuronal differentiation and
survival, whereas Brn-3b is associated with proliferation.
It has been shown that Brn-3a directly interacts with the
p53 protein and this association resulted in differential regulation of
gene targets which effect cell fate i.e. death or differentiation. Here we show
using affinity chromatography, that the p73 isoforms also physically interacted
with Brn-3a and Brn-3b and this interaction occurs via the Brn-3 POU domain and
amino acids 316-423 of p73 which includes the oligomerization domain which is
critical for this interaction. The transactivation potential of the Brn-3a/p73
complex was tested on p53 target genes where Brn-3a was shown to potentiate the
transcriptional effect of p73 on the p21 promoter. In agreement with a
functional effect, co-expression of Brn-3a and p73 increased cell cycle arrest,
with a more significant increase observed with p73β. Also the p73 mediated
activation of the pro-apoptotic genes Bax and Noxa was repressed upon
co-expression of Brn-3a. Therefore, it appears that association of the Brn-3a
transcription factors with the p53 family of proteins including the p73 family
can regulate the genes transcribed and hence affect survival and fate of
neuronal cells that co-express these proteins.
Histone
deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced
apoptosis in lymphoid malignancies.
Satoshi
Inoue,
Marion MacFarlane, Nicholas Harper, Luise Wheat, Martin Dyer, and Gerald Cohen
MRC
Toxicology Unit, University of Leicester, UK
E-mail: IS47@le.ac.uk
Chronic
lymphocytic leukemia (CLL) is characterized by the accumulation of mature
lymphocytes that fail to undergo appropriate apoptosis. CLL is currently
incurable and new therapies are required. Apoptosis may be induced by activation
of cell surface death receptors (extrinsic pathway) or by perturbation of
mitochondria (intrinsic pathway). As most chemotherapeutic agents activate the
intrinsic pathway, novel agents are required which activate the extrinsic
pathway thereby circumventing resistance mechanisms against the intrinsic
pathway. Activation of the extrinsic apoptotic pathway via TRAIL receptor
ligation has been suggested to be a novel therapeutic target for various
malignancies. However, for reasons that remain unclear, CLL and other lymphoid
malignancies are resistant to TRAIL and other apoptosis- inducing ligands. We
report that low concentrations of histone deacetylase (HDAC) inhibitors,
including depsipeptide and trichostatin A, which alone failed to induce
apoptosis, markedly sensitize CLL cells and other primary lymphoid malignancies
to TRAIL-induced apoptosis. These combinations were not toxic to normal
lymphocytes. HDAC inhibitors sensitized to TRAIL-induced apoptosis by
facilitating formation of an active death inducing signaling complex (DISC),
leading to the rapid activation of caspase-8. Thus, the combination of HDAC
inhibitors and TRAIL may be valuable in the targeted treatment of CLL and other
hemopoietic malignancies.
Functional
characterization of ASAP complexes in Apoptosis and RNA processing.
A.
Joselin, K. Schulze-Osthoff and C. Schwerk
Institute
of Molecular Medicine, University of Düsseldorf (Medical School), Universitätsstr.
1, D-40225 Düsseldorf, Germany
Email:
Alvin.Joselin@uni-duesseldorf.de
Different
isoforms of a novel protein complex termed the Apoptosis and Splicing Associated
Protein (ASAP) were purified from HeLa cells. ASAP complexes are composed of the
polypeptides SAP18, RNPS1 and distinct isoforms of the Acinus protein. Acinus is
cleaved during apoptosis by caspases releasing a 23 kDa fragment which has been
implicated in mediating apoptotic chromatin condensation. RNPS1, a spliceosome
associated RNA-binding protein with an SR-domain, has been described as general
activator of pre-mRNA splicing and component
of the exon-exon junction complex involved in export and surveillance of mRNA.
Functions of ASAP in apoptosis and mRNA processing have been confirmed employing
microinjection and in vitro splicing analyses.
To further characterize the function of ASAP in vitro, recombinant
wild-type and mutant complexes were produced in the baculovirus system.
Destruction of the caspase-3 cleavage site of Acinus does not seem to affect the
progression of apoptosis in microinjection experiments.
For a detailed investigation of ASAP function in vivo, we have
generated HeLa cells allowing inducible knockdown of complex subunits by RNAi.
Analysis of these cell lines should enable determination of the contribution of
the individual subunits during apoptosis and distinct steps of mRNA metabolism.
Data obtained during these experiments will be presented.
Possible
function of HPV E7 in HDAC inhibitor induced apoptosis of cervical carcinoma
cells
Handan
Karaduman, F. Rösl and P. Finzer
German Cancer Research Center Heidelberg, Research
Program Applied Tumor Virology, Division Viral Transformation Mechanism (F030)
INF 280D-69120 Heidelberg,Germany
Email:
H.Karaduman@dkfz.de
Particular
types of “high risk” of human papilloma virus (HPV) are causally involved in
the development of the cancer of the cervix. In recent studies we have shown
that in HPV-positive cervical carcinoma cell lines, inhibitors of histone
deacetylase (HDAC) cause cell cycle arrest and apoptosis. Cell death was
preceded by degradation of the pocket proteins pRB and p107 in a
HPV-E7-dependent manner, while the E2F expression remained uneffected. Free E2F,
under G1, conditions creates a
conflict situation whereby the cells undergo apoptosis by induction of
proaoptotic isoforms of p73. Viral
oncoprotein such as HPV-E7 can bind directly via a LXCXE sequence motif to the
pocket domain of pRB and abrogate
the association of RB with HDAC to allow tumor cells to overcome the repressive
effect of pRB. To gain insight into the role of
E7 on pRB degradation during the HDAC inhibition, several experimental
approaches were taken. These included the attenuation of E7 expression by siRNA
in cervical carcinoma cells, stable overexpression
of E7 in HPV-negative / RB-positive rhabdomyosarcoma cells and expression
of E7 and pRB in RB/HPV double negative Saos-2 cells under transient
transfection conditions. Our preliminary results indicate that, E7 is involved
in the selective degradation of pocket protein family members after HDAC
inhibition. The role of E7 mediated pRB degradation by HDAC inhibitors
for further therapeutical perspectives in the treatment
of cervical cancer will be discussed.
Study
of the structure and Function of TNFR Superfamily Members
Fiona
Kimberley,
Gavin Screaton
Human
Immunology unit, Institute of Molecular Medicine, University of Oxford
E-mail:
Fiona.Kimberley@chch.ox.ac.uk
The
Tumour Necrosis Factor Receptor Superfamily encompasses type-I transmembrane
proteins, with highly conserved cysteine bonds in the extracellular domain that
define a common structural framework. Crystallographic
studies of several receptor-ligand complexes have shown that a trimeric ligand
recruits three receptor molecules, this juxtaposition of intracellular domains
initiating intracellular signalling. However,
work on patients suffering from autoimmune lymphoproliferative syndrome (ALPS),
linked the disease to heterozygous mutations in Fas that disrupted the
ligand-binding domain of the molecule but dominantly interfered with signalling.
Consequently, a pre-ligand association domain (PLAD) was defined, and
this was found to be necessary for subsequent ligand binding, defining a new
signalling mechanism. In this study,
I have investigated the presence of a PLAD in the TRAIL receptors: the death
domain (DD) containing molecules, DR4/5, and the decoy receptors DcR1/2, which
lack a DD. I have also studied a
disease caused by mutations in the TNFR1 gene, tumour necrosis factor receptor
associated periodic syndrome (TRAPS), to search for clues as to a
structure-function relationship in these molecules.
German
Cancer Research Center, Heidelberg, Germany
E-mail: s.kleber@dkfz.de
The
CD95ligand/receptor (CD95L/R) system is best characterized as a trigger of
apoptosis. This is also the case in the adult brain following stroke, where
engagement of CD95 induces death through caspase activation. However, whether
additional pathways are also involved in CD95-induced damage is unclear yet. To
further address this issue, we examined the pathway downstream of the CD95L/R
system, after transient middle cerebral artery occlusion (MCAo). In wt and gld
(with a non functional CD95L) mice subjected to 90 minutes MCAo and
different reperfusion times, proteins from the ischemic and non-ischemic
hemisphere were extracted and analysed by immunoprecipitation and Western blot.
Downstream of the CD95L/R system a pathway parallel to the caspase pathway was
detected. This pathway involves the Ras, PKB/AKT and GSK3b molecules. In wt mice, Ras was inactivated early post-reperfusion and
18h afterwards reactivated, whereas no change in Ras activity was detectable in gld
mice. PKB/AKT was activated and GSK3b
was inactivated upon ischemia/reperfusion in wt animals, and remained unchanged
in gld mice. The Ras/AKT/GSK3b pathway signals survival in other
models, whether this is also the case in stroke should be addressed in further
studies.
Therapeutic
Neutralization of CD95 Ligand Prevents Apoptosis and Promotes Axonal
Regeneration and Functional Recovery after Spinal Cord Injury
Stefan
Klussmann1, Deana Demjen1, Susanne Kleber1,
Bram Stieltjes2, Cecilia Zuliani1, Ulrich A. Hirt4,
Henning Walczak4, Werner Falk5, Marco Essig2,
Lutz Edler3, Peter H. Krammer1, Ana Martin-Villalba1
1Tumorimmunology
Program, 2Department of Radiology, and 3Biostatistics Unit,
German Cancer Research Center, Heidelberg, Germany; 4Apogenix
Biotechnology AG, Heidelberg, Germany; 5Department
of Internal Medicine I, Regensburg, Germany.
Email:
s.klussmann@dkfz.de
Apoptosis
contributes to the secondary damage after spinal cord injury (SCI). In order to
examine the involvement of the CD95- and TNF-ligand/receptor (L/R) systems in
SCI-induced damage, we acutely inhibited the activities of these ligands in mice
subjected to SCI. Therapeutic neutralization of CD95L strongly decreased
apoptotic cell death, whereas blocking TNF did not exhibit any significant
effect. Strikingly, anti-CD95L-treated mice regained the ability to initiate
active hindlimb movements, while control mice remained almost completely
paraplegic. Moreover, this improvement in locomotor function was accompanied by
an increase in regenerating nerve fibers at the lesion site and upregulation of
the growth-associated protein-43 (GAP-43). In summary, we have shown that
neutralization of CD95L promotes axonal regeneration and functional recovery in
adult spinal cord-injured mice. Thus, this strategy may constitute a potent
approach to treat human spinal injuries.
Aspirin prevents apoptosis and NF-kB
activation induced by H2O2 in HeLa cells
Ozgur Kutuk
and Huveyda Basaga
aBiological Sciences and
Bioengineering Program, Sabanci
University, 34956 , Orhanli-Tuzla, Istanbul, Turkey
Email: ozgurkutuk@su.sabanciuniv.edu
The classical pathway of NF-kB activation by several inducers
mainly involves the phosphorylation of IkBa by a signalsome complex composed of IkBa kinases (IKKa and IKKb).
However, in some cell types hydrogen peroxide (H2O2) has
been shown to activate an alternative pathway that does not involve the
classical signalsome activation process. In this study we demonstrate that H2O2
induced NF-kB activation in HeLa cells through phosphorylation and degradation of IkB proteins as shown by immunblot analysis. Our studies reveal that a
commonly used non-steroid anti-inflammatory drug, acetylsalicylic acid (aspirin)
prevents H2O2-induced NF-kB
activation in a dose-dependent manner through inhibition of phosphorylation and
degradation of IkBa
and IkBb. Differential staining and DNA fragmentation analysis also show that
aspirin preloading of HeLa cells also prevents H2O2-induced
apoptosis in a dose-dependent manner with maximum efficiency at a concentration
of 10 mM. Additionally, aspirin effectively prevents caspase-3 and caspase-9
activation by H2O2. These results suggest that NF-kB activation is involved in H2O2-induced apoptosis
and aspirin may inhibit both processes simultaneously.
Verena
Labi1, Franziska
Müllauer1, David Bernhard1, Leigh Coultas2,
Lorraine O´Reilly2, Andreas Strasser2 and Andreas
Villunger1
1Institute
of Pathophysiology, Innsbruck Medical School, Innsbruck, AUSTRIA
2The
Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia
E-mail: verena.labi@uibk.ac.at
The
pro-apoptotic function of the BH3-only protein Bmf is regulated, in part, at the
post-translational level. Sequestration of Bmf to the cytoskeleton is mediated
by its interaction with dynein light chain (DLC) 2, a component of the actin-
based Myosin V motor complex. Apoptosis caused by detachment of adherent cells (anoikis),
UV-irradiation or treatment with actin-depolymerizing agents, have been reported
to free Bmf, together with DLC 2, allowing it to inactivate Bcl-2. To assess its
physiological role in vivo, we generated mice lacking bmf by
homologous recombination. Bmf-/- animals derived from
heterozygous intercrosses displayed no obvious abnormalities and were born at
the expected frequency. In wt animals Bmf was found to be strongly expressed in
haematopoietic tissues but an initial analysis of Bmf-/- mice
revealed normal cell numbers in and composition of all haematopoietic organs
analyzed. Furthermore, when Bmf-deficient lymphocytes where compared with
wt cells for their in vitro response to various apoptosis inducing agents
no significant differences were observed.
In order to evaluate its proposed role in anoikis we investigated
apoptosis induction in mouse
embryonic fibroblasts and endothelial cells derived from wt and Bmf-deficient
animals. Preliminary results indicate that Bmf may be crucial mediator of
endothelial cell apoptosis.
Heat
shock-induced non-apoptotic cell death mediated by protein aggregation.
Philippe S. Nadeau,
Kerstin Bellmann and Jacques Landry
Centre de recherche en cancérologie
de l’Université Laval, CHUQ-HDQ, Québec, Canada
E-mail:
jacques.landry@med.ulaval.ca
Heat shock (HS) induced in several cell lines a cell death process that
was non-apoptotic based on a number of morphological and biochemical
characteristics. HS-induced nuclear deformation was clearly distinguishable from
the nuclear alterations associated with apoptosis and was not accompanied by the
activation of the mitochondrial pathway of apoptosis.
There was no Bax activation, cytochrome c release or caspase activation,
and cell death was insensitive to Bcl2 overexpression. Furthermore HS-induced
cell death was highly inhibited in cells that overexpressed heat shock proteins
(Hsp), whereas classical apoptosis as induced by cisplatin was not. HS caused an
accelerated denaturation of proteins whose aggregation and subsequent toxicity
can be prevented by Hsp that function as molecular chaperones helping in the
refolding or targeting for proteasomal degradation. We found morphological
evidence that a non-apoptotic cell death process similar to that induced by HS
was activated upon inhibiting the proteasome or expressing proteins with poly-Q
extension that aggregate in the cells. We suggest that a gain of toxic activity
by the aggregated misfolded proteins is responsible for cell death during HS
following a process that is similar to the toxicity associated with protein
aggregation in conformational diseases.
CD95
signaling by the combination of bioinformatical and experimental approaches.
Inna
Lavrik, Martin Bentele, Roland Eils and Peter H. Krammer.
Division of Immunogenetics,
Tumorimmunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer
Feld 280, 69120 Heidelberg, Germany.
E-mail:i.lavrik@dkfz.de
We
established a data-based approach for a generalized model of CD95-induced
apoptosis with parameters estimated on the basis of quantitative experimental
data. The resulting model of CD95-induced apoptosis consists of 41 molecules, 36
reactions, and 54 parameters. For the generation of experimental data, we chose
the human B-lymphoblastoid cell line SKW 6.4, previously classified as type I
cells by their high amount of DISC formation. Cells were stimulated with
different concentrations of agonistic anti-APO-1 antibody for various periods of
time (from 5 minutes to 4 days). Each sample was evaluated by three independent
approaches. Cell death was determined by flow cytometry, caspase activity was
measured by fluorometric activity, and the change of concentration of major
apoptotic molecules was evaluated by western blot. The obtained experimental
data in combination with mathematical modelling provide new insights into
CD95-mediated apoptosis and allow to predict the threshold of life and death.
Our model suggests that the threshold of CD95-induced apoptosis is determined
upstream in the DISC. The ratio
between active receptors and c-FLIP as well as the ratio between binding rates
of c-FLIP to DISC and of procaspase-8 to DISC are highly relevant parameters for
this threshold. We are now able to simulate the mechanism of CD95 induced
apoptosis under different conditions (e.g. for different expressions of c-FLIPL,
c-FLIPS or FADD), thereby predicting a higher or lower resistance to apoptosis.
The
cardioprotective effect of urocortin during ischaemia/reperfusion involves the
prevention of apoptosis and mitochondrial damage.
1Medical
Molecular Biology Unit, Institute of Child Health, University College London,30
Guilford Street, London WC1N 1EH. 2
National Heart and Lung Institute, Royal Brompton Hospital, London.
E-mail:
K.Lawrence@ich.ucl.ac.uk.
Urocortin
(Ucn), is a small endogenous peptide found in the heart and
has potent anti apoptotic effects caused by simulated ischaemia/reperfusion
(I/R) injury, in primary rat cardiac myocytes. The mechanism of this
cardioprotection is unclear but at least part of this protection involves de-novo
protein synthesis. Therefore we have used Affymetrix gene chips to look for
global gene changes caused by Ucn treatment to rat neonatal primary cardiac
myocytes. From the gene chip data,
several molecules of interest were altered by Ucn treatment. We have
concentrated on three so far; a Katp channel, a phosholipase A2
enzyme and protein kinase C epsilon. We have found that these three molecules
are involved in the cardioprotective mechanism of action of Ucn and
pharmacological manipulation of these end effectors protects cardiac myocytes
from apoptotic cell death. Significantly, we have also found that Ucn protects
cardiac myocyte mitochondria from membrane damage produced by I/R and that all
three proteins involved in Ucns cardioprotective effect are localised to cardiac
myocyte mitochondria. Furthermore, as well as protecting myocytes from apoptotic
cell death, these proteins, when modulated, prevent damage caused by I/R to
mitochondria. It seems plausible therefore that this mitochondrial protection
may be responsible for enhanced cell survival and this mechanism may be via
proteins regulated by Ucn.
cFLIPL inhibits TRAIL-mediated NF-kB activation
and apoptosis induction in human keratinocytes.
Wachter T.1, Hausmann
D.1, Kerstan A.1, Sprick, MR.2, Mc Pherson K.1,
Stassi G.3, Walczak, H.
2, Bröcker E.-B.1, Leverkus M.1
1University of Würzburg Medical School, Department
of Dermatology, Würzburg, Germany
E-mail: leverkus_m@klinik.uni-wuerzburg.de
Human keratinocytes express death receptors like CD95,
TRAIL-R1/-R2 and undergo apoptosis following treatment with TNF-related
apoptosis-inducing ligand (TRAIL) or CD95 ligand (CD95L). The intracellular
inhibitor cFLIPL inhibits death receptor-mediated apoptosis and is
highly expressed in primary human keratinocytes when compared to transformed
HaCaT keratinocytes. In order to study the role of cFLIPL in more
detail, we established a panel of stable monoclonal cFLIPL-overexpressing
HaCaT keratinocytes by viral transduction using a retroviral bicistronic vector
system. Functional analysis revealed that relative cFLIPL levels
correlated with resistance to TRAIL in these lines, while CD95, TRAIL-R1 - R4
and initiator caspase expression were unchanged compared to control lines.
Biochemical characterization of caspase activation following TRAIL treatment
showed that caspase 8 activation was inhibited at the death inducing signalling
complex (DISC) in cFLIPL-overexpressing lines, while DISC recruitment
and partial cleavage of caspase 8 was unaffected. We next asked if nonapoptotic
signals like activation of the transcription factor NF-kB are modulated by cFLIPL.
Surprisingly cFLIPL specifically blocked TRAIL-induced NF-kB
activation and TRAIL-dependent induction of the NF-kB target gene IL-8. Taken together our data demonstrate that cFLIPL
is not only a central anti-apoptotic modulator of death receptor signals, but
also an inhibitor of TRAIL-induced NF-kB activation and proinflammatory target
gene expression. cFLIPL may thus represent a “silencer” of
proapoptotic and proinflammatory responses to death receptor ligation in
keratinocytes and its modulation may not only influence apoptosis sensitivity
but may also lead to altered death receptor-dependent inflammation of the skin.
Identification
and relevance of the cd95-binding domain in the n-terminal region of ezrin
F. Lozupone, L.
Lugini, P. Matarrese., F. Luciani, C. Federici, E. Iessi, G. Stassi, W. Malorni.
and S. Fais.
Istituto Superiore di Sanità
Viale Regina Elena, 299 00161 - ROME - ITALY
E-mail: lozupone@iss.it
The
CD95 (Fas/APO-1) linkage to the actin cytoskeleton through ezrin is an essential
requirement for susceptibility to the CD95-mediated apoptosis in CD4+ T cells.
We have previously shown that moesin was not involved in the binding to CD95.
Here we further support the specificity of the ezrin/CD95 binding, showing that
radixin did not bind CD95. The ezrin region specifically and directly involved
in the binding to CD95 was located in the middle lobe of the ezrin FERM domain,
between aminoacids 149 and 168. In this region ERM show 60-65% identity, as
compared to the 86% identity in the whole FERM domain. Transfection of two
different human cell lines with a GFP-tagged ezrin mutated in the CD95-binding
epitope, induced a marked inhibition of CD95-mediated apoptosis. In these cells
the mutated ezrin did not co-localize nor co-immunoprecipitate with CD95.
Further analysis showed that the mutated ezrin, while unable to bind CD95, was
fully able to bind actin, thus preventing the actin linkage to CD95. Altogether,
our results support the specificity of ezrin in the association to CD95 and the
importance of the ezrin-to-CD95 linkage in CD95-mediated apoptosis. Moreover,
this study suggests that a major role of ezrin is to connect CD95 to actin, thus
allowing the CD95 polarization on the cells and the occurrence of the following
multiple cascades of the CD95 pathway.
Caspase
activation impedes proteasome function during apoptosis thus inhibiting
degradation of the pro-apoptotic molecules Smac and Omi:
siRNA targeting of XIAP to assess its potential in promoting Smac/Omi
degradation.
1MRC
Toxicology Unit, Hodgkin Building, Leicester, LE1 9HN, UK & 2Department of Biochemistry, Technion-Israel
Institute of Technology, Haifa, Israel
E-mail: mm21@le.ac.uk
The
ubiquitin/proteasome system regulates protein turnover by degrading
polyubiquitinated proteins. To date almost all studies on the relationship of
apoptosis and the proteasome have emphasised the key role of the proteasome in
the regulation of apoptosis, by virtue of the ability of the proteasome to
degrade regulatory molecules involved in apoptosis. We now report that induction
of apoptosis may also regulate the activity of the proteasome. We demonstrate
that caspase activation during apoptosis results in the caspase-dependent
cleavage of three specific subunits of the 19S regulatory complex of the
proteasome, S6′ (Rpt5), and S5a (Rpn10), whose role is to recognize
polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a
and S2 (Rpn1), holds together the lid and base of the 19S regulatory complex.
This caspase-mediated cleavage inhibits the proteasomal degradation of cellular
substrates, including the pro-apoptotic molecules Smac and Omi, so facilitating
the execution of the apoptotic programme by providing a feed-forward
amplification loop. Using siRNA targeting we have further examined the role of
XIAP as the potential E3 ligase in the degradation of mitochondrial-released
Smac and Omi. The effects of silencing XIAP expression on the sensitivity of
tumour cells to TRAIL-induced apoptosis will also be discussed.
Developmental
down-regulation of apoptotic signalling proteins in post-mitotic tissue.
S.D.
Madden,
M. Donavan and T.G. Cotter
Cell
Development and Disease Laboratory, Biosciences Institute, University College
Cork, Ireland.
E-mail: maddensd@yahoo.co.uk
An
investigation into the expression of key developmental apoptotic proteins in the
mouse retina revealed a down-regulation of caspase 3, 9 and APAF-1 expression in
the adult compared to the pup. This coincided with a loss of apoptosome function
in the adult retina. We therefore investigated whether both the reduced
expression of these apoptotic regulators and the subsequent loss of apoptosome
function occurred in other adult murine post-mitotic tissues such as the brain,
heart and skeletal muscle. Caspase 3 and APAF-1 protein and mRNA levels
decreased dramatically from pup to adult. Caspase 9 protein levels decreased
less dramatically from pup to adult and were still easily detectable in the
adult. Interestingly, caspase 9 mRNA levels remained unchanged between the pup
and adult. We then investigated apoptosome function through the ability of
cytochrome C to induce caspase 9 cleavage in cell free extracts (CFE). We found
that we could not induce caspase 9 cleavage in pup or adult tissue, despite the
fact that there are high levels of APAF-1 and caspase 9 in pup tissue. We
therefore hypothesise that there may be an inhibitor present in the pup tissue
that prevents induction of caspase 9 cleavage.
Understanding the demolition phase of
apoptosis
Dept. of Genetics, Trinity College,
Dublin, Ireland.
Email: martinsj@tcd.ie
Apoptosis is coordinated
by a family of cysteine proteases-the caspases-that dismantle the cell by
targeting numerous proteins for limited proteolysis. The mammalian caspase
family contains 13 members, a subset of which participate in apoptosis, with the
remainder likely to be involved in the processing of pro-inflammatory cytokines.
Diverse cellular stresses provoke apoptosis by damaging mitochondria and
result in the release of factors (such as cytochrome c) which trigger
caspase activation and cell death as a consequence. Here, we discuss the
hierarchical nature of the caspase activation cascade that is triggered by
efflux of cytochrome c and the role of specific caspases within this
cascade in targeting cellular proteins for degradation.
Data will also be presented relating to global proteomic analyses of
apoptotic cells. These data suggest
that several hundred proteins are targeted for limited proteolysis during the
terminal phase of apoptosis.
Binding
specificity and regulation of the serine protease and PDZ domains of HtrA2/Omi
L.
Miguel Martins(1,3),
Benjamin E. Turk(2), Lewis Cantley(2) and Julian Downward
(1)Signal Transduction Lab, Cancer
Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK; (2)Division of
Signal Transduction, Beth Israel Deaconess Medical Center and Department of Cell
Biology, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA,
(3)Present address: Cell Death Regulation Lab, MRC Toxicology Unit, Leicester,
UK
E-mail: lmm24@le.ac.uk
Inhibitor
of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of
caspases. The serine protease HtrA2/Omi has an amino terminal IAP interaction
motif like that found in Reaper, which displaces IAPs from caspases leading to
enhanced caspase activity. The cell death promoting properties of HtrA2/Omi are
not only exerted through its capacity to oppose IAP inhibition of caspases, but
also through its integral serine protease activity. We have used peptide
libraries to determine the optimal substrate sequence for cleavage by HtrA2 and
also the preferred binding sequence for its PDZ domain. Using these peptides, we
show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless
it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment
also increases its protease activity. Unexpectedly, binding of XIAP to the
Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity,
suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria
following an apoptotic stimulus, binding to IAPs may switch their function from
caspase inhibition to serine protease activation. Thus while IAP over-expression
can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent
cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may
limit the overall cellular protection that can be provided by these proteins.
NF-kB
activation inhibits bax gene expression induced by p73 tumour suppressor
protein.
Valeria Marzano1,
Roberta Cianfrocca1, Barbara Marinari1, Antonio Costanzo2,
Massimo Levrero3, Enza Piccolella1 and Loretta Tuosto1
1Dept.
Cell. and Develop. Biol. Univ. of Rome "La Sapienza"; 2Dept.
Dermatol. Univ. of Rome "Tor Vergata"; 3Fondazione Andrea
Cesalpino, Univ. of Rome "La Sapeinza", Rome Italy.
E-mail: tuosto@uniroma1.it
Identification
of a novel post-translational modification of Apaf-1 after apoptosome activation
Gavin
P. McStay
and Douglas R. Green
Division
of Cellular Immunology, La Jolla Institute for Allergy & Immunology, 10355
Science Center Drive, San Diego, CA 92121, USA.
E-mail: gavin_mcstay@hotmail.com
After
the release of cytochrome c from the mitochondrial inter-membrane space, a large
multi-protein complex, called the apoptosome, is formed.
This complex is comprised of Apaf-1, caspase-9, cytochrome c and
formation is dependent on the presence of dATP. Upon addition of exogenous cytochrome c to HEK293T S100
cytosolic extracts a shift in the molecular weight of Apaf-1 was observed.
This modified Apaf-1 appeared rapidly after cytochrome c addition, before
degradation of Apaf-1 in a caspase-dependent manner was observed.
This modification does not appear to be dependent on caspase-3, but
dependent on caspase-9 activity, determined using the broad-spectrum caspase
inhibitors qVD and zVAD. Progress
is underway to determine the identity of this modification and so far does not
appear to be due to characterized post-translational modifications, such as,
ubiquitination, sumoylation, NEDDylation or ISGylation of Apaf-1. Strategies to identify this novel modification of Apaf-1 will
be described and the role it has in apoptosome formation.
Developmentally Regulated
Changes in Apoptotic Stress-induced c-Jun N-Terminal Kinase Activation in the
Oligodendrocyte Lineage
Grisha Pirianov,
Anthony Jesurasa and Huseyin Mehmet
Institute of Reproductive and
Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology,
Faculty of Medicine, Imperial College London, Hammersmith Hospital, London W12
0NN, U.K.
E-mail: g.pirianov@imperial.ac.uk
Oligodendrocytes (OL) are the major myelin
forming cells in the central nervous system. Oligodendrocyte precursor cells (OPC)
are particularly vulnerable to stress-induced death in the developing brain. In
the present study, we examined JNK signalling in three distinct paradigms of
oligodendrocyte apoptosis: exposure to ultraviolet (UV) irradiation, or
treatment with staurosporine (SSP) or with C2-ceramide (C2). We found that in
OPC, JNK activation and phosphorylation of the transcription factor c-Jun was
strongly upregulated in response to all three apoptotic stimuli and this was
also associated with a dramatic reduction in cell viability. In contrast, mature
OL were more resistant to stress-induced apoptosis and this was reflected in
lower levels of JNK activation and c-Jun phosphorylation. This was not a
universal response mediated by changes in JNK expression since JNK protein
levels were not altered by OL maturation. Indeed, mature OL remained very
sensitive to C2 in the absence of JNK activation, suggesting that ceramide can
utilise alternative apoptotic signalling pathways. Using specific
immunodepletion to remove JNKs 1 and 2, we found that active JNK3 formed a
substantial part of stress stimuli-induced JNK activity in OPC.
Finally, we observed that the JNK peptide inhibitor, D-JNKI (which is
based on the JNK binding domain of JNK interacting protein) blocked JNK activity
and partially protected against UV-induced OL cell death in OPC. These results
indicate that JNK is differentially activated in OL depending on the stage of
differentiation.
DIAP1
differentially binds to caspases and IAP antagonists
Anna
Zachariou1, Tencho Tenev1, Lakshmi Goyal2,
Julie Agapite2, Hermann Steller2 and Pascal Meier1
1)The Breakthrough Toby Robins Breast Cancer Research
Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester
Beatty Laboratories, Fulham Road, London SW3 6JB, UK.
2) Howard Hughes Medical Institute, Rockefeller University, New York, NY
10021
E-mail: pascal.meier@icr.ac.uk
The
Drosophila inhibitor of apoptosis protein DIAP1 ensures cell viability by
directly inhibiting caspases. In cells destined to die this IAP-mediated
inhibition of caspases is overcome by IAP-antagonists. Genetic evidence indicate
that IAP-antagonists are non-equivalent and function synergistically to promote
apoptosis. We will provide
biochemical evidence for the non-equivalent mode of action of Reaper, Grim, Hid
and Jafrac2. We find that these IAP-antagonists display differential and
selective binding to specific DIAP1 BIR domains. In particular, we find that Rpr
and Grim associate with the BIR1 and BIR2 regions of DIAP1 with equal efficiency.
Hid interacts preferentially with the BIR2 domain and only weakly with the BIR1
while Jafrac2 binds exclusively to the BIR2 domain. Consistently, we show that
each DIAP1 BIR region associates with distinct caspases.
The differential DIAP1 BIR interaction seen both between initiator and
effector caspases and within IAP-antagonist family members suggests that
different IAP-antagonists inhibit distinct caspases from interacting with DIAP1.
Consistently, we find that Rpr but not Hid blocks the binding of drICE to DIAP1.
We also provide evidence that Rpr, Grim and Hid induce cell death predominantly,
if not exclusively, in an IAP-binding dependent manner.
Heat stress down-regulates c-FLIP and sensitises cells to Fas
receptor-mediated apoptosis
A Meinander1,2, SEF Tran1,3, L Sistonen1,3,
and JE Eriksson1,4
(1) Turku Centre for Biotechnology, Turku, Finland, (2)
Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland,
(3) Department of Biology, Åbo Akademi University, Turku, Finland, (4)
Department of Biology, University of Turku, Finland
E-mail: annika.meinander@btk.utu.fi
Cell stress and programmed cell death are two important
signaling pathways regulating survival and death of the cells of an organism and
thereby maintaining tissue homeostasis. We are investigating common denominators
between these two signaling responses, particularly when activated
simultaneously, to help understand the regulation of death receptor signaling
during stress, fever, or inflammation. We found that exposure to a mild heat
stress rapidly sensitized Jurkat, HeLa and H9 cells to Fas-mediated apoptosis.
Neither inducible expression of Hsp70 nor the MAPK pathways seems to be involved
in this sensitization. The sensitizing effect of stress was observed both in
Type II (Jurkat) and Type I (H9) cells, indicating that apoptotic signaling via
the mitochondria is not essential for the heat-increased apoptosis. Instead,
heat stress on its own induced down-regulation of FLIP in all tested cell lines.
The increase of caspase-8 cleavage upon heat treatment combined with
Fas-stimulation was dependent on the stress-mediated down-regulation of FLIP. We
are currently studying the mechanism of FLIP down-regulation and subsequent
sensitization. In support of the FLIP down-regulation as determinant of
sensitization, the restoration of FLIP levels during recovery after heat
treatment correlates with a decrease of the sensitivity.
Mitochondrial intermembrane space proteins are released in a
single step during apoptosis.
Cristina
Muñoz-Pinedo,
Joshua Goldstein, Ana Guío-Carrión, Jean-Ehrland Ricci, Roger Tsien, and
Douglas R. Green.
La Jolla Institute for Allergy
and Immunology. 10355 Science Center Drive, San
Diego, CA 92121, USA.
E-mail: cmunoz@liai.org
The
permeabilization of the mitochondrial outer membrane to release proteins from
the intermembrane space into the cytosol is likely to be the pivotal event in
the process of apoptosis. Studies in single cells suggest that the release of
Cytochrome c is sudden, rapid, and complete, and that it can proceed before
detectable changes in mitochondrial membrane potential.
Other studies indicate that Smac/DIABLO release coincides with cytochrome
c release; however, many studies have shown a differential release of
intermembrane space proteins. We employed real-time single cell analysis to
analyze the translocation of several molecules. A recently described technology
permitting in situ fluorescent labeling of proteins tagged with a short
tetracysteine-containing sequence was used to label Smac, Adenylate Kinase and
Cytochrome c. Apoptotic inducers triggered the release of all these proteins in
individual cells prior to mitochondrial depolarization. The duration of release
was short, and proceeded in the presence of caspase inhibitors. Furthermore,
Smac and Adenylate Kinase were released with the same kinetics of Green
Fluorescent Protein tagged-Cytochrome c from individual cells coexpressing
combinations of these proteins. The kinetics of release of several proteins from
the intermembrane space, and its temporal relation to Bax translocation will be
discussed.
Ueli
Nachbur,
Nadia Corazza and Thomas Brunner
Division
of Immunpathology; Institute of Pathology; University of Berne; Murtenstrasse
31; CH-3010 Berne, Switzerland
E-mail: ueli.nachbur@pathology.unibe.ch
Lipid
rafts are small cholesterol- and sphingolipid-rich domains of the cell membrane,
and are known to be crucial in diverse signaling pathways, such as T cell
receptor signaling or induction of cell death via TNF receptor 1. Upon now,
nothing is known about the role of membrane lipid rafts in the regulation of Fas
(CD95/APO1) ligand (FasL)-mediated cytotoxicity. Since coordinated expression of
FasL protein on the cell surface of cytotoxic T cells is important for its
apoptosis-inducing activity, we have investigated the possibility whether cell
surface FasL associates with lipid rafts and whether raft association is
important for FasL activity. We will show evidence, that FasL is partially
localized in lipid rafts and that this association is crucial for its cytotoxic
activity. Disruption of lipid rafts in cytotoxic T cells leads to a reduced
induction of cell death in the target cells. Furthermore we are interested in
the factors that recruit FasL to the lipid raft compartments of the cell
membrane. For this purpose we are employing different FasL mutants, which are
analyzed for raft-association and cytotoxicity
Hsp70
promotes tumour cell survival by inhibiting lysosomal membrane permeabilization
1,3Jesper
Nylandsted, 1,3Mads Gyrd Hansen, 1Agnieszka
Danielewich, 1Nicole Fehrenbacher, 1Ulrik Lademann, 1Maria
Høyer-Hansen, 2Gabriele Multhoff, 1Mikkel Rohde and 1Marja
Jäättelä
1Apoptosis Laboratory, Danish Cancer Society,
Strandboulevarden 49, DK-2100 Copenhagen, Denmark and 2Department of
Hematology, University Hospital Regensburg, Franz-Josef Strauss Allee 11,
D-93053 Regensburg, Germany.
E-mail: jnl@cancer.dk
Lysosomes function as signal integrators
in many models of apoptosis- and necrosislike programmed cell death. Here we
show that heat shock protein 70 (Hsp70) is localized to the lysosomal membranes
of cancer cells and that Hsp70 positive lysosomes display increased size and
resistance against chemical and physical membrane destabilization. Furthermore,
Hsp70 expression in diverse cell types effectively inhibited cytokine-,
anti-cancer drug- and transformation-induced cell death at the level of the
leakage of cathepsins from lysosomes to the cytosol. These data identify Hsp70
as the first survival protein that functions by inhibiting the death-associated
permeabilization of lysosomes.
Obexer
P.,1 Geiger K.,1 Ausserlechner M.J.,2,
3 Meister B.1, 2
1
Tyrolean Cancer Research Institute, Innsbruck, 2 Department of
Pediatrics, University Hospital of Innsbruck, and 3 Institute of
Pathophysiology, University of Innsbruck
E-mail: Petra.Obexer@uibk.ac.at
The neuroblastoma
(NB) is the most frequent extracranial solid tumor in childhood.
Phosphatidylinositol 3`-kinase (PI3K) and PKB/Akt and its target, the forkhead
transcription factor FKHRL1 have been suggested as possible downstream
regulators of neurotrophin mediated cell survival in neuroblastoma cells. To
analyze, whether this pathway is critical for survival and cell cycle
progression we generated a retrovirus expressing a regulated FKHR-L1-(A3)-ER*
fusion protein and infected SH-EP and LAN-1 neuroblastoma cells and the primary
tumor cell line STA-NB15. Activation of FKHR-L1-(A3) by 4-OH-tamoxifen induced
apoptotic cell death in STA-NB15 and in SH-EP cells after 48 hours and reduced
proliferation in LAN-1 as measured by propidium iodide and MTT-assay. This was
associated with the induction of the cell cycle inhibitor p27Kip1
whereas expression of the proapoptotic FKHR-L1 target bim was not detected. Ectopic expression of crmA inhibited FKHR-L1-(A3) induced apoptosis up to
48 hours whereas overexpression of bcl2 prevented apoptosis up to 72 hours in
SH-EP cells. 4-OH-tamoxifen treated SH-EP-FKHR-L1 showed an increased
chemosensitivity to cisplatin, doxorubicin, etoposide and vinblastine as
measured by FACS-analysis and MTT assay. The data suggest that the transcription
factor FKHR-L1 critically regulates cell death and cell cycle progression in
neuroblastoma cells.
Activation of the mitogen-activated protein kinase (MAPK)
pathway attenuates TRAIL-induced apoptosis by preventing tBid-mediated release
of cytochrome c from mitochondria in breast tumor cells
Gustavo Ortiz-Ferrón*,
Stephen W. Tait#, Gema Robledo*, Evert de Vries#, Jannie Borst#, and Abelardo López-Rivas*.
*Instituto de Parasicología y Biomedicina, CSIC, Granada,
Spain. #Division of Cellular Biochemistry, The Netherlands. Cancer Institute,
Amsterdam, The Netherlands.
E-mail:gustavo@ipb.csic.es
A high percentage of breast cancers show increased activity
of the MAPK pathway. We have studied the impact of an activated MAPK/Erk1/2
pathway on the regulation of tumour necrosis factor-related (TRAIL)-induced
apoptosis in breast tumour MCF-7 cells. When we activated the MAPK pathway,
TRAIL-induced apoptosis was inhibited in a protein synthesis-independent manner.
The release of cytochrome c was blocked but caspase-8 activation and BID
cleavage still occurred. We studied tBID translocation to the mitochondria with
a GFP-tBid fusion protein and found that translocation was inhibited at least
partially when the MAPK pathway was activated. Translocation of a GFP-tBid
protein with a truncated BH3 domain was also inhibited. Furthermore, in
digitonin-permeabilised cells, tBid-induced cytochrome c release from
mitochondria was also reduced by MAPK activation. Altogether, our data indicate
that the MAPK pathway inhibits TRAIL-induced apoptosis in MCF-7 cells at the
level of tBID translocation to the mitochondria.
Caspase-14 expression, regulation and processing
during epidermal differentiation
Petra Ovaere,
S. Lippens, C. Van Den Broecke, G. Denecker, E. Van Damme, E. Tschachler, H.
Demol, M. Puype, J. Vandekerckhove, P. Vandenabeele and W. Declercq
Molecular Signalling and Cell Death Unit, Department
for Molecular Biomedical Research, Technologiepark 927, B-9052 Ghent-Zwijnaarde,
VIB, University of Ghent
E-mail: petra.ovaere@dmbr.ugent.be
Caspase-14 is expressed in a
limited number of epithelia, including the epidermis. This is in contrast to the
other caspase family members who are ubiquitously expressed. Until now the
keratinocyte terminal differentiation pathway leading to programmed cell death
has not been unravelled yet. In the epidermis pro-apoptotic caspases-3, -6, -7
and -8 are only present as precursor forms, suggesting that these caspases do
not participate in programmed cell death during normal epidermal differentiation.
Caspase-14 is not present in the proliferating cells of the basal layer, but in
the differentiating suprabasal layers procaspase-14 is expressed. Caspase-14
activation by proteolytic processing is correlated with epidermal cornification.
Addition of caspase inhibitors to in vitro skin equivalent cultures does
not block caspase-14 activation. We identified a caspase-14 processing activity
present in epidermal extracts giving rise to identical p20 and p10 caspase-14
fragments as observed in skin. In general, caspases are activated by cleavage at
Asp residues. Surprisingly, caspase-14 is not processed at an Asp residue during
activation. Caspase-14 expression can be regulated by vitamin D and retinoic
acids, similar to other keratinocyte differentiation-associated proteins. Taken
together, our observations strongly suggest that caspase-14 may be involved in
the keratinocyte terminal differentiation program.
Sphingosine kinase as a 'sensor'
during chemotherapy-induced apoptosis in prostate cancer cells.
Dimitri Pchejetski, V. Garcia, T. Levade and O. Cuvillier
Inserm U466, CHU Rangueil, 31059
Toulouse, France
E-mail: dimitri.pchejetski@toulouse.inserm.fr
Chemotherapy is a major treatment used on late stages of prostate cancer;
however the efficacy of anticancer agents is not equal in different cell lines.
Regulation of growth and apoptosis, important factors in cancer development, has
been linked to sphingolipids such as ceramide and sphingosine 1-phosphate and
importantly to sphingosine kinase. Our objective is to determine whether
sphingosine kinase is implicated in chemoresistance of prostate cancer cells.
Camptothecine, a known apoptosis inducer in LNCaP prostate cancer cells, is much
less effective in PC-3 cell line. On the contrary docetaxel
treatment caused massive loss of cell viability in PC-3, but had much lesser
effect in LNCaP. Docetaxel and camptothecine induced inhibition of sphingosine
kinase only in cell lines sensitive to the drugs, but not in the resistant ones.
Enforced expression of sphingosine kinase in
PC-3 and LNCaP cells restored their resistance to chemotherapy, which is
reversed by sphingosine kinase inhibition. Cell death, induced by docetaxel and
camptothecine in sensitive cell lines had distinct apoptotic features, as
confirmed by fluorescent microscopy and flow cytometry. In addition, sphingosine
kinase inhibition was associated with processing of XIAP, caspases and PARP.
These results show that apoptosis induction by docetaxel and camptothecine in
prostate cancer cells is correlated with sphingosine kinase inhibition. Ability
of sphingosine kinase to determine the resistance of cancer cells to
chemotherapy might propose inhibition of this enzyme for potential application
in cancer treatment.
Multiple
members of the TNF superfamily contribute to IFNg-mediated inhibition of
erythropoiesis.
Francesca Pedini1,
Nadia Felli1, Ann Zeuner1,2, Eleonora Petrucci1,
Ugo Testa1, Jeffrey A. Winkles3, Cesare Peschle1,4,
Ruggero De Maria1,2
1Department
of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità,
Rome, Italy; 2Mediterranean Institute of Oncology, Catania, Italy; 3Department
of Vascular Biology, American Red Cross, Rockville, MD; 4Kimmel
Cancer Institute, Thomas Jefferson University, Philadelphia, PA
E-mail: fpedini@iss.it
TRAIL and CD95 ligand are two members of
the tumor necrosis factor family that have been proposed to contribute to the
homeostasis of the erythroid compartment. TWEAK is a recently identified member
of this family, predominantly produced by activated monocytes, that exerts
pleiotropic effects on different types of hematopoietic and non-hematopoietic
cells. We observed that primary human erythroid precursors express low levels of
TWEAK and its receptor Fn14, and respond to TWEAK stimulation by reducing their
expansion and maturation, while the monocytic lineage is unaffected by exogenous
TWEAK exposure. IFN-g treatment of differentiating hematopoietic progenitor
cells results in suppression of growth and differentiation, pointing to a
possible involvement of this cytokine in hematopoietic failure syndromes. We
found that IFN-g upregulates the expression of TWEAK, Fn14 and TRAIL on
differentiating erythroid cells, and that the simultaneous neutralization of
TWEAK, TRAIL and CD95 ligand on isolated erythroblasts completely restored
erythroid growth and differentiation in the presence of IFN-g. Our data indicate
that TWEAK has anti-erythropoietic properties and that multiple interactions
between TWEAK, TRAIL, CD95 ligand and their corresponding receptors mediate the
impairment of erythropoiesis triggered by IFN-g.
A
serine protease is involved in the initiation of DNA damage-induced apoptosis
Lucy
T.C. Peltenburg1, Daphne B. Pontier1,
Elza C. de Bruin1, Dorothea Meersma1, Peter A. van Veelen2
and Jan Paul Medema1
1Department
of Clinical Oncology and 2Department of Immunohematology and Blood
Transfusion, Leiden University Medical Center, Leiden, The Netherlands
E-mail: l.t.c.peltenburg@lumc.nl
Caspases
are considered to be the key effector proteases of apoptosis. We studied the
role of caspases in apoptotic cell death of a human melanoma cell line.
Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of
poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did
prevent DEVDase activity. We excluded a role for caspase‑2, which is a
relatively zVAD-fmk-resistant caspase, in mediating etoposide-induced cell death
of these cells. In contrast, caspase activation and PARP degradation were
blocked by pretreatment of the cells with the serine protease inhibitor AEBSF (Pefabloc).
More importantly, while zVAD-fmk could not rescue melanoma cells from
etoposide-induced death, the combination with AEBSF resulted in substantial
protection. In etoposide-treated rat fibroblasts, this serine protease inhibitor
could also block caspase activity. We therefore conclude that a serine protease
regulates an alternative initiation mechanism that leads to caspase activation
and PARP cleavage. The involvement of several known apoptotic serine proteases,
such as Granzyme B and HtrA2/Omi, could be excluded. We are currently trying to
identify the novel apoptotic protease by isolating active serine proteases from
cytosolic fractions of dying melanoma cells and rat fibroblasts using
immobilized AEBSF.
Development of hyperplasia in aged
APRIL transgenic mice
L. Planelles, G. Hardenberg, C.E. Carvalho-Pinto, S. Smaniotto, W.Savin,, E. Eldering,
J. de Jong, C. Martínez-A, J.P. Medema and M. Hahne
Institut de Génétique Moléculaire de Montpellier
(IGMM),CNRS UMR5535
Email: planelles@igm.cnrs-mop.fr
The TNF-like ligand APRIL
(a proliferation-inducing ligand) is a secreted protein, named for its capacity
to stimulate tumour cell proliferation in vitro. APRIL transcript levels
are reported to be low in normal tissues, among which the highest levels are
found in peripheral blood leucocytes (PBLS). In contrast, higher mRNA levels
have been detected in tumour cell lines and in a variety of primary tumour
tissues including lymphoma and colon carcinoma. In addition, endogenous APRIL
protein has been detected in human myeloid leukaemia cell lines, such as U937
and Mono Mac 1. We have previously described the generation of APRIL transgenic
(Tg) mice displaying detectable protein levels of the transgene. APRIL Tg mice
at age of 6-12 weeks revealed no signs of T or B cell hyperplasia. In contrast,
when mice are 9-12 months of age we observed in about 30% of them hyperplasia in
mesenteric lymph nodes and Peyer's Patches, which is accompanied by a
disorganisation of affected lymphoid tissues. This appears to be related to an
increased activity of B1 cells.
Double
level of protection from death receptors and inflammatory cytokines in
neural stem cells
Lucia Ricci-Vitiani1,
Francesca Pedini1, Cristiana Mollinari2, Fabrizio
Condorelli3, Gerolama Condorelli4, Désirée Bonci1,
Alessandra Bez5, Eugenio Parati5, Cesare Peschle1,
Ruggero De Maria1,6
1Department
of Ematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità,
Rome, Italy; 2Cattedra
Oftalmologia, Campus Bio-Medico, Rome, Italy (Fondazione G.B. Bietti);
3DISCAFF,
University of Piemonte Orientale, Novara, Italy; 4Department of
Biology and Molecular and Cellular Pathology and Institute of Endocrinology and
Experimental Oncology of C.N.R., Federico II University of Naples, Italy; 5Laboratory
of Neurobiology, Department of Neurobiology and Neurorestorative Therapies,
National Neurological Institute "C. Besta", Italy; 6Mediterranean
Institute of Oncology, Italy
E-mail: lriccivitiani@yahoo.it
Neural stem cells (NSCs) are self-renewing
populations present in both the developing and adult nervous system of all
mammals, which proliferate and differentiate into neurons and glia. In several
pathological conditions, death receptor (DR) ligands exert a deleterious effect
on neurons, whilst primitive neural cells migrate and survive in the site of
lesion despite the presence of inflammatory cytokines. We show that, even in the
presence of pro-apoptotic cytokines, DRs are unable to generate death signals in
NSCs. Accordingly, differently from their neuronal progeny, human embryonic and
adult NSCs did not express caspase-8, whose presence is required for initiating
the caspase cascade. However, exogenous or cytokine-mediated expression of
caspase-8 was not sufficient to restore their DR sensitivity. Searching for
molecules potentially able to block DR death-inducing signaling complex (DISC)
showed that primitive neural cells expressed high levels of PED/PEA-15, a death
effector domain-containing protein that localized in the DISC of NSCs and
prevented caspase-8 recruitment and activation. Moreover, lentiviral-mediated
delivery of PED/PEA-15 antisense resulted in dramatic down regulation of the
endogenous gene and sensitization of NSCs to apoptosis mediated by inflammatory
cytokines and DRs. Thus, absence of caspase-8 and high expression of PED/PEA-15
constitute two levels of NSC protection from apoptosis induced by DRs.
xA novel role for Survivin-DEx3: regulation of Aurora B sub-cellular distribution during
interphase.
Jose A. Rodriguez1, Simone W. Span1, Susanne M.A. Lens2, Rene H. Medema2, Frank A.E. Kruyt1
and Giuseppe Giaccone1.
1Department of Medical Oncology, VU
University Medical Center and 2Division of Molecular Biology, Netherlands Cancer
Institute, Amsterdam, The Netherlands
Email:ja.rodriguez@vumc.nl
Survivin is a member of
the inhibitor of apoptosis protein (IAP) family involved in the regulation of
apoptosis and mitotic progression. The mitotic function of Survivin is reflected
by its dynamic co-localization with the Aurora B kinase and INCENP throughout
mitosis. Survivin is essential for the localization of Aurora B to centromeres
and the spindle midzone. Disfunction of Aurora B and Survivin may contribute to
tumorigenesis. Alternative splicing produces two additional Survivin isoforms
with yet unknown function: Survivin-2B and Survivin-DEx3. We found that, as
previously shown for Survivin, the localization of Aurora B during interphase
reflects a dynamic equilibrium between nuclear import and export. However, the
transport of both proteins appears to be regulated in a largely independent
manner. Co-expression with Survivin-DEx3 relocates Aurora B to the nucleus, but
does not alter the cytoplasmic localization of Survivin. Survivin-DEx3 interacts
in vivo and co-localizes in the nucleus with Aurora B, but not with Survivin.
Interestingly, Aurora B/Survivin-DEx3 co-localization is disrupted during
mitosis, when Aurora B co-localizes with Survivin. Our data suggest that
different Survivin isoforms may regulate the subcellular localization of Aurora
B through the cell cycle, Survivin playing a major role during mitosis and
Survivin-DEx3 contributing to Aurora B nuclear import during interphase.
Rapamycin enhances doxorubicin-induced apoptosis in human
melanoma and hematopoietic cells independently of PI3K/Akt inhibition.
Raffaella Avellino*, Simona Romano*,
Antonello Petrella#, Annalisa Lamberti*, Rita Bisogni*, Salvatore Venuta° and Maria
Fiammetta Romano*
*Dipartimento di Biochimica e
Biotecnologie Mediche (DBBM), Università Federico II, Napoli, Italy.
#Dipartimento di Scienze Farmaceutiche (DIFARMA) University of Salerno. °Department of Experimental Medicine (DMSC), University of
Catanzaro, Italy.
E-mail: romano@dbbm.unina.it
NF-kB/Rel-regulated genes are involved in
oncogenic processes and promote cell survival following stimuli which lead to
cell death. The macrolide antibiotic rapamycin has recently been shown to exert
an anticancer effect mainly ascribed to inhibition of the PI3K/Akt pathway. Our
study was aimed at investigating if the pro-apoptotic activity exerted by the
macrolide antibiotic could be ascribed to a downmodulation of NF-kB/Rel nuclear
activity. To this end we analysed the effect of rapamycin on apoptosis induced
by the anthracyclin drug doxorubicin. We found that rapamycin greatly enhanced
apoptosis in a human melanoma cell line and also induced IkBa degradation
and NF-kB/Rel
nuclear translocation. IkBa degradation was detected after 3
hrs of incubation and was complete after 5 hrs. Addition of rapamycin to the
cultures inhibited IkBa phosphorylation and degradation
and, at the same time, NF-kB/Rel
nuclear levels appeared reduced. To verify if rapamycin was still able to
further reduce cell survival in condition of PTEN hyperexpression, we
transiently transfected PTEN cDNA in melanoma cells and analysed the effect of
rapamycin on doxorubicin-induced apoptosis. Our data shows that cells
hyperexpressing PTEN remain sensitive to the apoptosis-enhancing effect of
rapamycin when apoptosis was induced by doxorubicin and not by cisplatin that in
our system did not induce NF-kB.
We next investigated the effect of the macrolide agent in the leukemic cell line
Jurkat and verified that rapamycin also inhibited doxorubicin-induced NF-kB/Rel nuclear translocation these
cells. Our results suggest that the downmodulation of the protective function of
NF-kB/Rel
transcription factors may represent a novel mechanism by which rapamycin
enhances apoptosis in cancer cells, envisioning promising results of the
macrolid agent also in the treatment of PTEN-positive tumors.
Biochemical
Features of CD47-Mediated Caspase-Independent Cell Death in B Chronic
Lymphocytic Leukemia
Gaël
Roué, Marlène Bras, Victor J. Yuste, Cécile Delettre, Rana
Moubarak and Santos A. Susin
Apoptose et Système Immunitaire,
CNRS-URA 1961, Institut Pasteur, 25 rue du Dr. Roux. 75015 Paris, France
Email:
roue@pasteur.fr
Ligation
of CD47 by its natural ligand thrombospondin, or cross-linking by CD47
antibodies, triggers caspase-independent cell death in normal and leukemic B
cells. This kind of cell death is characterized by the cytoplasmic events of
apoptosis. After B cell stimulation via CD47, a rapid mitochondrial
transmembrane potential disruption is accompanied by the production of reactive
oxygen species and phosphatidylserine exposure. Surprisingly, mitochondrial
dysfunction does not induce the release of the pro-apoptotic proteins cytochrome
c or AIF, and consequently neither oligonucleosomal nor large scale DNA
fragmentation could be observed. All the features characterizing this type of
apoptosis could not be inhibited by specific caspase inhibitors and did not
involve the Bcl-2 family of proteins. On another hand, CD47-cell death
signalling involves a complete reorganization of the cytoskeleton and could be
regulated by inhibitors of actin polymerization. Since mitochondria seem
re-distributed after CD47 stimulation, the study of potential links between CD47
receptor, cytoskeleton, and mitochondria is currently under investigation. Taken
together, our results may help to elucidate a new cell death pathway. Our
findings could also contribute to the identification of novel targets that might
overcome some forms of tumour-associated mechanisms of B cell drug-resistance.
Translational
control in apoptosis versus necrosis
Xavier
Saelens, Michael Kalai, Ann Meeus and Peter Vandenabeele.
Molecular
Signalling and Cell Death Unit, Department for Molecular Biomedical Research,
VIB and Ghent University, Fiers-Shell-Van Montagu building, B9052 Ghent, Belgium.
E-mail: Xavier.Saelens@dmbr.UGent.be.
Apoptosis
is associated with a rapid decline in protein synthesis, consistent with the
phosphorylation and/or the cleavage of some translation initiation factors (eIFs).
Since, little is known about the biochemistry of necrosis, we compared de
novo protein synthesis, ribosomal RNA integrity, eIF2-alpha phosphorylation
and PKR phosphorylation and cleavage in apoptosis and necrosis. In necrosis
induced by dsRNA in Jurkat cells de novo protein synthesis persisted. In
contrast to apoptosis, 28S ribosomal RNA fragments, eIF2-alpha phosphorylation,
and phosphorylation and cleavage of PKR were absent in necrosis induced by dsRNA
or TNF. Phosphorylation of full length PKR and eIF2-alpha in apoptosis
correlated in time with caspase activity and the appearance of caspase-mediated
PKR fragments. We demonstrate that both the N-terminal (ND) and the Kinase
domain (KD) fragments of PKR, as generated by caspase cleavage, interact with
full length PKR and that KD leads to PKR and eIF2-alpha phosphorylation.
Remarkably, ND enhances the capacity of KD to phosphorylate eIF2-alpha and to
suppress the expression of a reporter. We propose that cells exposed to a
necrotic stimulus, retain the capacity to synthesize proteins while in apoptosis
both caspase-mediated PKR fragments cooperate to activate full length PKR and
contribute to the shut down of translation.
Cytotoxicity
of Cerebrospinal Fluid Samples from Human Infants with Cerebral
Hypoxic-Ischaemic Injury Implicates CD95/Fas as a Major Component of the
Inflammatory Response
Katerina Sakoulas, Kristin Leifsdottir and
Huseyin Mehmet
*Weston
Laboratory, Division of Paediatrics, Obstetrics, and Gynaecology, faculty of
Medicine, Imperial College London, Hammersmith Hospital, London W12 0NN,
United Kingdom.
E-mail: ks399@imperial.ac.uk
Cerebral
hypoxic-ischaemic injury (HII) is a major cause of neurological impairment in
human infants. Although inflammatory cytokines are thought to contribute to
neural cell loss following HII, the progression of the inflammatory response or
any correlation between brain cytokine levels, cytotoxicty and clinical outcome
are not known. As part of an ethically approved pilot study, we obtained 40
cerebrospinal fluid (CSF) samples from 20 new-born human infants (two serial
lumbar punctures from each) approximately half of were diagnosed with HII.
First, we measured the cytokine levels in the CSF samples, specifically
interleukins -1beta, -6, -10 and -12, tumour necrosis factor alpha, soluble
CD95/Fas (sFas), interferon gamma and CD95/Fas ligand (FasL). Next, we utilised
the B-cell lymphoma cell lines, A20 (FasL-sensitive) and A20R (FasL-resistant),
as a model system to determine the cytotoxicity of the CSF samples. In analysing
the cytokine profiles of CSF samples from healthy infants and those with HII, we
observed a degree of correlation between cytokine levels and cytotoxicity. For
example, the levels of FasL in CSF samples were directly related to the extent
of apoptosis induced by the CSFs in A20 cells. On the other hand, CSF samples
with high levels of sFas were protective against FasL-induced cell death in A20
cells. These results suggest that Fas and FasL are major contributors to the
inflammatory response following cerebral HII.
B-MYB
regulates the stress response though direct transactivation of key survival
genes.
Giorgia Santilli, Rebekka Schwab
and Arturo Sala
Molecular
Haematology and Cancer Biology Unit, Institute of Child Health, 30 Guilford
street, London WC1N 1EH, U.K.
E-mail: a.sala@ich.ucl.ac.uk
B-MYB
is a ubiquitous protein that belongs to the MYB family of transcription factors.
B-MYB gene expression is growth regulated and it has an essential role in
mammalian development. B-MYB knockout mice arrest their growth at the bastocyst
stage. This has lent credence to the hypothesis that B-MYB may play a key role
in cell proliferation through transactivation of target genes involved in cell
cycle control. Recent experiments conducted in our laboratory with a
dominant-negative inhibitor suggest that B-MYB’s transcriptional activity is
not required for cell proliferation. Instead, we have identified B-MYB’s
target genes that are involved in the control of apoptosis. Ectopic B-MYB’s
overexpression can confer an antiapoptotic phenotype suggesting that B-MYB’s
transcriptional activity could be required for cell survival. We go on to show
that B-MYB’s transactivating potential is enhanced by stress and that this
results in modification of B-MYB protein and modulation of target genes.
Interestingly, retroviral infection of murine fibroblasts with a transcriptional
dominant-negative B-MYB molecule can ablate stress-dependent gene expression and
increase cell death. We conclude that B-MYB is a stress-activated protein that
promotes cell survival through direct regulation of antiapoptotic genes. These
results could explain why B-MYB is critically required during mammalian
development.
Differential modulation of endotoxin responsiveness by
human caspase-12 polymorphisms.
Maya Saleh1, John P. Vaillancourt1, Rona K.
Graham2, Emad S. Alnemri4, Richard S. Hotchkiss5, Timothy G. Buchman5, Barbara
A. Zehnbauer6, Michael R. Hayden2, Lindsay A. Farrer3, Sophie Roy1 and Donald W.
Nicholson1
Biochemistry and Molecular Biology, Merck
Frosst Centre for Therapeutic Research, New Jersey, USA.
E-mail:
maya_saleh@merck.com
Caspases play an essential role in
mediating key proteolytic events in inflammatory cascades and in the apoptotic
cell death pathway. They functionally segregate into two distinct sub-families:
those involved in cytokine maturation (human caspases 1, 4 and 5) and those
involved in cellular apoptosis (human caspases 2, 3, 6, 7, 8, 9 and 10).
Although caspase-12 is phylogenetically-related to the cytokine processing
caspases, its role in mice has been proposed to be a mediator of ER
stress-induced apoptosis including amyloid-É¿ cytotoxicity, therefore
suggesting a contributory role of this protease to Alzheimer disease. Here
we show that in humans, a polymorphism in caspase-12 results in the synthesis of
either a truncated CARD-containing prodomain-only protein (Csp12-S) or a
full-length tripartite caspase proenzyme (Csp12-L). The readthrough SNP
which encodes Csp12-L was confined entirely to populations of African descent
and conferred hypo-responsiveness to LPS-stimulated cytokine production in ex
vivo whole blood, but had no significant effect on apoptotic sensitivity. The
frequency of the Csp12-L allele was elevated in African American patients with
severe sepsis. Thus Csp12-L attenuates the inflammatory and innate immune
response to endotoxins and in doing so may constitute a risk factor for
developing sepsis
The tyrosine kinase Lck is a positive regulator of the mitochondrial
apoptosis pathway by regulating Bak expression
E-mail: ajoy@uni-duesseldorf.de
Tyrosine kinases of the Src family have
been implicated in key biological processes. Here we report that p56Lck,
a lymphoid-specific Src kinase, plays a crucial role for the activation of the
mitochondrial apoptosis pathway. Lck-deficient Jurkat T-cells were completey
resistant to anticancer drugs. In contrast, apoptosis sensitivity to death
receptors such as CD95 was not altered, indicating that a specific interference
of Lck with the mitochondrial pathway. Reexpression of Lck restored sensitivity
to drug-induced apoptosis and triggered mitochondrial cytochrome c release and
subsequent caspase activation. Further analysis identified that sensitization by
Lck was independent of stress-activated protein kinases and classical mediators
of T cell receptor signaling, but essentially involved the Bcl-2 member Bak.
Expression of Bak was completely absent in Lck-deficient cells, while,
importantly, reexpression of Lck transcriptionally triggered Bak expression and
conferred sensitivity to drug-induced apoptosis, which was associated with a
proapoptotic conformational change of Bak. Furthermore, in vitro the truncated
fragment of Bid specifically activated Bak and cytochrome c release only from
mitochondria of Lck expressing cells. These results do not only demonstrate a
sentinal role of Lck in drug resistance, but also delineate a hitherto unknown
pathway of Src kinases in regulation of expression of Bcl-2 proteins.
Emma Sanzari,
Fiammetta Romano, Federico Chiurazzi, Michela Garofalo, Ciro Zanca and Gerolama
Condorelli.
Università degli Studi di Napoli
Federico II, Naples, ITALY
Email:
gecondor@unina.it
Chronic
lymphocytic leukaemia (CLL) is characterized by the accumulation of long-lived B
cells that results from a failure of cells to undergo apoptosis. The mechanisms
underlying apoptosis resistance of B-CLL cells are presently unknown. B-CLL are
also resistant to TRAIL-induced apoptosis. The mechanism underlying
TRAIL-resistance could be related to the function of inhibitory molecules. PED/PEA-15
is a DED-containing protein that interferes with signalling from the DISC. This
induced us to explore the possibility that PED expression levels correlate with
TRAIL-resistance. Our analysis was conducted on peripheral blood lymphocytes
from 35 B-CLL-affected patients. To down-modulate PED expression levels we
treated the cells with phosphorothioate oligodeoxynucleotides antisense or with
cycloheximide. PED expression, analyzed by western blot and immunofluorescence,
was markedly reduced by the treatment of both the antisense and
the cycloheximide. The percentage of apoptosis, measured as hypodiploidy
in flow cytometry, was 15% in the cells incubated only in medium o with control
scrambled oligonucleotide and 35% in cultures kept with PED antisense. PED
antisense was also able to overcome to TRAIL resistance. Finally, we observed
that cycloheximide sensitizes B-CLL cells to TRAIL-induced apoptosis and this
effect correlated with down-regulation of PED expression. In conclusion, PED
down-modulation determined a significant increase of both basal and
TRAIL-induced apoptosis.
DNA
damage induces rapid degradation of the anti-apoptotic p73 isoform, Dnp73
A. Emre Sayan1, Carine Maisse2, Eliana Munarriz1,
Daniela Barcaroli1, Gerry Melino1,2 and Vincenzo De
Laurenzi1,2
1
Medical
Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Lancaster
Road, P.O. Box 138, Leicester LE1
9HN, UK. 2Biochemistry Laboratory,
IDI-IRCCS, C/O Department of Experimental Medicine and Biochemical Sciences,
University of Rome "Tor Vergata", 00133 Rome, Italy
E-mail: aes16@le.ac.uk
p73
belongs to the family of transcription factors that also includes p53 and p63. The three
proteins share a significant degree of sequence homology, these structural
similarities are mirrored by a certain degree of functional overlap and all
members of the family are capable of inducing cell cycle arrest and apoptosis. The TP73
gene has two promoters that independently control the expression of a full
length TAp73 protein and a ∆Np73 isoform that lacks the amino terminal TA
domain. DNA damage can
induce phosphorylation of TAp73 by c-Abl, leading to stabilization of the TAp73
protein, while deregulated expression of E2F1 can transcriptionally activate
TAp73 expression. The resultant
elevation of TAp73 results in the induction of apoptosis. Antithetically,
∆Np73 inhibits both TAp73- and p53-induced apoptosis. The TP73 gene
maps to a region (1p36.33) that is frequently deleted in neuroblastoma,
suggesting that loss of p73 function may play a role in the development of this
tumor. However, in keeping with an anti-apoptotic and potentially oncogenic role
for the N-terminally deleted versions of the p73 protein, expression of
∆Np73 is an adverse prognostic marker for neuroblastoma patients. It would
appear that fine tuning of TAp73 and ∆Np73
ratios within the individual cellular context dictates the functional outcome,
and it therefore comes as no surprise that enhanced expression of the
∆Np73 forms, rather than inactivating mutations within p73, is associated
with cancer development. We show that DNA damage determines a rapid degradation
of ∆Np73 therefore releasing the block exerted on p53 and p73 allowing
cell cycle arrest and apoptosis to occur. Degradation is proteosome dependent
and specific for the ∆Np73 while p53 and TAp73 are not affected.
The
role of B-MYB in neuroblastoma cell survival and resistance to chemotherapy
Rebekka Schwab, Giorgia Santilli, Arturo Sala
Molecular
Haematology and Cancer Biology, Institute of Child Health, University College
London
E-mail:
rschwab@ich.ucl.ac.uk
Neuroblastoma is a malignant childhood
cancer. The tumour originate from neural crest cells and affected sites are the
neural crest-derived organs adrenal medulla and ganglia.
Several lines of evidence suggest that the
transcription factor B-Myb plays an important role in neuroblastoma. Not only
expression of B-Myb in neuroblastoma patients correlates with poor survival but,
also, B-Myb expression in neuroblastoma cell lines has been linked to enhanced
cell survival and reduced differentiation potential. B-Myb has been shown to
regulate apoptosis, to play an important role in embryonal development, and to
be required for progression through S-phase, being expressed at the G1-S
boundary. We are currently testing the efficacy of synthetic B-Myb siRNAs in
neuroblastoma cell lines. In preliminary experiments we have observed that
siRNA-mediated downregulation of B-Myb results in increased spontaneous
apoptosis and sensitisation of neuroblastoma cells to chemotherapeutic drugs.
B-Myb expression is greatly increased in tranformed cells compared to
non-transformed counterparts. This might suggest that B-Myb acts as a oncogene,
perverting normal progression through the cell cycle and/or increasing cell
survival. Thus, our strategy of downregulating B-Myb with siRNAs might be a
powerful tool to specifically kill cancer cells, since we have observed that
normal cells do not depend on B-Myb expression to the same extend as transformed
cells. To further investigate the role of B-myb in neuroblastoma we are
developing a transgenic mouse model in which the B-myb cDNA will be
overexpressed in organs/tissues derived from neural crest cells, under the
control of the tyrosine hydroxylase promoter (PTH).
Effect
of D-Galactosamine on oxidative stress and apoptosis in the liver of rats
Sule
Seçkin
, Makbule Aydın , Sueyda Alsancak , Müjdat Uysal
Istanbul
University , Istanbul Faculty of Medicine , Department of Biochemistry,Çapa ,
34093 , Istanbul , Turkey
E-mail: sseckin@istanbul.edu.tr
This
study was done to clarify the effect of high dose of D-Galactosamine (GalN) on
oxidative stress and apoptosis in experimental rats. Malondialdehyde (MDA) level
which is used as an indicator of lipid peroxidation, increased significantly,
24hr after intraperitoneal administration of D-GalN (1g/kg body weight) to rats.
Hepatic TNF-a level decreased significantly compared with that in control group.
No DNA fragmentation was observed by gel electrophoresis. These results suggest
that high dose of D-Galn induces oxidative stress but not apoptosis in the liver
of experimental rats.
Homeostatic
regulation of IAP protein levels
John
Silke,
Tobias Kratina, Diep Chu, David L.Vaux
The
Walter & Eliza Hall Institute, Melbourne, Australia
E-mail:
silke@wehi.edu.au
Most
inhibitor of apoptosis (IAP) proteins bear RING domains that enable them to
promote ubiquitination of themselves and other interacting proteins. Here we
show that the RING domains also allow IAPs to homeostatically control the levels
of other IAPs. A previously undefined region extending C terminal to the RING
allows IAPs to directly interact with each other, leading to their
ubiquitination and degradation. Expression of a construct containing the RING of
cIAP1 was able to deplete melanoma cells of endogenous XIAP, markedly reducing
their clonogenicity when treated with cisplatin. Other RING containing proteins
that interact, such as MDM2 and MDMX, bear RING domains with flanking regions
closely resembling those of the IAPs, suggesting that homeostatic control of
proteins levels by RING domains is a common occurrence, and may enable their
levels to be manipulated for therapeutic ends. Our findings explain several
previously puzzling observations; why cIAP1 levels are elevated in XIAP
knock-out mice, why cIAP1 RING and DIAP1 are cytotoxoic and why it is difficult
to establish stable cell lines over-expressing IAPs. Our findings suggest models
for how IAP levels are regulated, and how IAP antagonists such as Grim promote
IAP degradation.
Fas
Ligand (FasL): an adjuvant to generate monoclonal antibodies that reject tumour.
Katharina
Simon,
Paul Chen, Matthew Frayne and Gavin Screaton
MRC
Human Immunology Unit, Weatherall Institute of Molecular, Medicine, John
Radcliffe Hospital, Oxford OX3 9DS, UK
E-mail: katja.simon@ndm.ox.ac.uk
FasL
does not only induce apoptosis but also possesses pro-inflammatory properties.
Indeed, in a murine model, FasL expressed on melanoma elicits tumour immunity
mediated by antibodies. We generated 4 monoclonal antibodies (mAb) from mice
that were protected from wild type melanoma after treatment with irradiated FasL
expressing melanoma. All antibodies recognise specifically the melanoma cell
line against which they were raised, as well as an allogeneic murine melanoma
cell line. They do not react with non-transformed syngeneic cells nor any other
transformed cell line tested. Three mAb are of the IgM isotype and are specific
for gangliosides. Gangliosides are well conserved glycosylated lipids, known to
be upregulated upon transformation of melanocytes. Anti-ganglioside mAb are to
date one of the most promising antibodies used in anti-tumour clinical trials.
More importantly, the fourth mAb of IgG2a isotype protects mice from tumour
growth in vivo. It recognises a glycosylated residue on a protein back bone.
These results indicate how FasL can be used as an adjuvant to generate novel
anti-tumour antibodies able to reject tumours in vivo.
Distinct
but overlapping substrate specificities of caspase-10 and caspase-8.
Sprick,
M.R.,
Buffy, V.; Pfeiffer, D; Haas, T.L. and Walczak, H.
Division of Apoptosis
Regulation, Tumour Immunology Program, German Cancer Research Center (DKFZ), Im
Neuenheimer Feld 580, 69120Heidelberg, Germany
E-mail:
m.sprick@dkfz.de
It
was previously shown that caspase-10, like caspase-8, is recruited to and
activated at the CD95 and TRAIL-DISC in a stimulation- and FADD-dependent manner.
A controversy remains whether both caspases are functionally redundant.
Additionally, it remained unclear whether both catalytically active caspase-10
isoforms, caspase-10a and caspase-10d, share the same functions and
specificities. To compare the activities of the different caspases and their
isoforms, we devised a method to generate highly active recombinant caspase-10
and -8. Using these proteins in assays utilizing tetrapeptide substrates as well
as recombinant proteins, we find that both proteins have largely overlapping
substrate specificities. However, caspase-10 has a marked preference for some
substrates. This result suggests a function of caspase-10 which is possibly
different from that of caspase-8.
A novel sigma2-receptor ligand as an inducer of
caspase-independent death of cancer cells in vitro and in vivo.
Marie
Stampe Andersen, Ingrid Fossar-Larsen and Marja Jäättelä.
Apoptosis
Laboratory, Danish Cancer Society, Copenhagen, Denmark.
E-mail: msa@cancer.dk
Tumor
cells have often acquired resistance towards classic caspase-mediated apoptosis.
The understanding of caspase-independent cell death mechanisms is therefore
becoming more important in the development of novel cancer therapies. Here we
show that a novel sigma2-receptor ligand (S2RL) effectively induces
caspase-independent programmed cell death in tumor cells of various origins.
S2RL induced an initial hyperpolarization of the plasma membrane potential
followed by a continuous depolarization and a rapid rise in the lysosomal pH.
Lipid antioxidants alpha- and gamma-tocopherol, but not other antioxidants such
as butylated hydroxyanisol and N-acetyl cysteine, completely blocked the plasma
membrane effects, the rise of lysosomal pH and the cell death. Furthermore, the
cell death was partially inhibited by a cathepsin B inhibitor CA-074-Me
suggesting a possible involvement of lysosomal proteases. Also tumor cells
protected against most other anti-cancer drugs by ectopic expression of Hsp70 or
Bcl-2 were effectively killed by S2RL. Importantly, the S2RL was well tolerated in
vivo and showed an anti-tumorigenic effect in a syngenic tumor xenograft
model in BALB/c mice. These results hold promise for S2RL as a novel anti-cancer
drug alone or in combination with conventional chemotherapeutics.
Regulation of anti-apoptotic programmes in
activated T cells by costimulatory signals through CD28 and 4-1BB/CD137
Lilian
Stärck, Christian Scholz, Bernd Gillissen, Bernd Dörken,
Peter T. Daniel
Molecular Hematology and
Oncology, University Medical Center Charité, Campus Berlin-Buch, Humboldt
University, Lindenberger Weg 80, 13125 Berlin
E-mail: staerck@rrk-berlin.de
T cell costimulatory
molecules are essential for T lymphocyte proliferation and inhibition of
activation induced cell death. While signaling pathways activated following the
ligation of the costimulatory molecule CD28 are well defined, less is known
about the molecular events induced by alternative costimulators. CD137/4-1BB, a
costimulatory receptor which belongs to the tumor necrosis factor receptor
family, plays an important role during late primary T cell stimulation. Here, we
demonstrate that CD137/4-1BB-mediated inhibition of activation induced cell
death involves up-regulation of the anti-apoptotic proteins FLIPS and Bcl-xL.
Inhibition of cell death and up-regulation of FLIPS and Bcl-xL were abolished by
blocking the phosphatidyl-inositol 3-kinase or the AKT/protein kinase B, two
kinases which so far had only been implied in CD28-mediated inhibition of
activation induced cell death. Our findings demonstrate that costimulatory
molecules, although belonging to different protein families and participating in
distinct upstream signaling pathways, employ common downstream signaling
pathways.
STAT1:
a novel regulator of the apoptotic programme
Anastasis
Stephanou
Medical Molecular Biology Unit, Institute of Child
Health, University College London, London, UK
E-mail: a.stephanou@ich.ucl.ac.uk
The STAT-1 transcription factor has been
implicated as a tumour suppressor by virtue of its ability to inhibit cell
growth and promoting apoptosis. However, the mechanisms by which STAT-1 mediates
these effects remain unclear. Using human and mouse STAT-1-deficient cells, we
show here that STAT-1 is required for optimal DNA damage-induced apoptosis. The
basal level of the p53 inhibitor Mdm2 is increased in STAT-1 -/- cells,
suggesting that STAT-1 is a negative regulator of Mdm2 expression.
Correspondingly, both basal p53 levels, and those induced by DNA damage were
lower in STAT-1 -/- cells. Therefore, p53 is less stable in STAT-1 deficient
cells. In agreement with this lower p53 response to DNA damage, the induction of
p53 responsive genes, such as Bax, Noxa and Fas, was reduced in STAT-1 deficient
cells. Conversely, STAT-1 overexpression enhances transcription of these genes,
an effect that is abolished if the p53 response element in their promoters is
mutated. Moreover, STAT-1 interacts directly with p53, an association which is
enhanced following DNA damage. Therefore, in addition to negatively regulating
Mdm2, STAT-1 also acts as a coactivator for p53. Hence STAT-1 is another member
of a growing family of protein partners able to modulate the p53-activated
apoptotic pathway.
Functional
Extrinsic and Intrinsic Apoptotic Pathways in Human Fetal Mesenchymal Stem Cells
Nigel
Kennea, Christina Stratou, Andreas Neparus and Huseyin Mehmet.
Division of Paediatrics, Obstetrics and
Gynaecology, Imperial College London, Hammersmith Hospital, London, United
Kingdom.
E-mail: christina.stratou@imperial.ac.uk
Although stem cells offer considerable
promise for the correction of neurodegenerative diseases and acute brain injury;
a major obstacle is the poor survival of grafted cells. We have identified a
population of human fetal mesenchymal stem cells (FMSCs) that can be
differentiated into neural cells. To determine whether FMSCs have functional
mitochondrial and death receptor pathways and to investigate whether survival
can be prolonged by inhibition of death signalling, FMSCs were subjected to
three death stimuli: serum withdrawal, Fas ligation or treatment with
staurosporine (SSP). Apoptosis was defined using morphological and biochemical
methods. Cytochrome c localisation was determined by immunofluorescence, and
activation of caspases by cleavage-specific antibodies. DNA fragmentation was
confirmed by TUNEL. Activation of the mitochondrial pathway by SSP was
demonstrated by the release of cytochrome c from mitochondria and cleavage of
caspases 9 and 3 followed by DNA fragmentation indicated by TUNEL labelling.
Serum withdrawal resulted in a similar pattern of molecular events, although the
time course was longer. Caspase 9 inhibitors, and pan-caspase inhibitor
z-VAD.fmk attenuated apoptosis triggered by SSP treatment. Functional death
receptor machinery in FMSCs was demonstrated by rapid induction of apoptotic
death by Fas ligand, which was partially inhibited by the selective caspase-8
inhibitor IETD.fmk. These results provide an insight into the mechanisms of
human FMSC apoptosis and may be useful in the design of pharmacological agents
to prolong the survival of stem cell grafts in vivo.
Caspase-independent
cleavage of Bid at aspartate is essential for DNA damage-induced apoptosis
Stephen
Tait,
Arlette Werner, Evert de Vries, Eric Eldering* and Jannie Borst
Immunology
Department, Netherlands Cancer Institute, Amsterdam, Netherlands.*Dept. of
Experimental Immunology, Academic Medical Centre, Amsterdam, Netherlands.
E-mail: s.tait@nki.nl
We
have selected Jurkat clones on the basis of resistance to CD95/Fas induced
apoptosis. These clones proved cross-resistant to apoptosis induced by DNA
damaging anti-cancer drugs and É¡-radiation. Using an in vitro cytochrome C
release assay, we have mapped CD95 resistance downstream of Bid cleavage but
upstream of mitochondrial activation and cytochrome C release. Interestingly,
Trail receptor stimulation was able to bypass this resistance, causing
cytochrome C release and apoptosis in these cells. In vivo expression of a
non-cleavable Bid mutant (D59E D75E) effectively blocked CD95 and Trail receptor
but also, surprisingly, DNA damage induced apoptosis. Secondly, immunodepletion
of Bid completely abrogated the ability of cytosolic extracts from DNA damage
stimulated Jurkat cells to cause cytochrome C release in vitro. DNA-damage
induced cytochrome C release in Jurkat cells was insensitive to zVAD addition or
RNAi knockdown of caspase-2. Collectively, these data demonstrate an essential
role for caspase independent Bid cleavage at aspartate in response to DNA damage
in these cells. Secondly, these data show that a novel regulatory point exists
downstream of Bid cleavage but upstream of cytochrome C release that can
differentially regulate mitochondrial activation in response to distinct
apoptotic stimuli.
Inhibitory
effect of Turkish black tea on azoxymethane-induced aberrant crypt foci in colon
carcinogenesis of Sprague Dawley rats
Sehime
Temel, E Evke, T Gulten, Z Kahveci
Medical
Genetic Dept., Uni. Of Uludag, Faculty of Medicine, Bursa, Turkey
E-mail: eulukaya@uludag.edu.tr
Colon
cancer is the second most common cause of cancer death in many industrialized
countries. A number of reviews have adressed the health-promoting effects of tea
extracts in experimental models of different types of neoplasia, including lung,
stomach, mammary gland and colon. Although the effect of tea in
anticarcinogenesis is not well defined, it is supposed that carcinogens are
detoksified by tea polyphenols therefore cell proliferation is inhibited and
apoptosis is increased.
If
DNA is damaged, some genes are activated and apoptosis is induced. P53 is one of
the most important of these activated genes. P53 is mutated or deleted in more
than 50% of human tumors and is known as guardian of genome. If the DNA damage
can’t be repaired pre-apoptotic proteins are activated (bax) through the
mitochondria and cell death occurs
by apoptosis. The role of Cox (cyclooxygenase) has been studied in colorectal
tumorigenesis by man y investigators. There are two known isoforms of Cox; Cox-1
and Cox-2. The latter is not detectable in the majority of normal tissues,
including the colonic epithelium, but is induced in vitro by various agents
including tumour promoters, growth factors, and cytokines. In view of this
knowledge we aimed to evaluate the interaction between tea, Cox-2, p53 and Bcl-2
in azoxymethane-induced colon carcinogenesis of rats. The occurence and the
multiplicity of ACF (aberrant crypt formation) was assesed by
immunohistochemical staining . The data showed that the incidence of aberrant
crypt formation was significantly
increased compared to the control group. Moreover, immunohistochemical analyses
showed that the expression extent of cox-2, p53 and bcl-2 was decreased in
experimental group.
The Drosophila IAP, DIAP1, interacts with caspases through
an evolutionarily conserved mechanism that is distinct from XIAP
Tencho
Tenev,
Anna Zachariou and Pascal Meier
The Breakthrough Toby Robins Breast Cancer Research
Centre, Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester
Beatty Laboratories, Fulham Road, London SW3 6JB, UK.
E-mail: pmeier@icr.ac.uk
Direct
interaction of XIAP1 with the catalytic pocket of caspase-3 and -7 results in
the steric occlusion of these caspases blocking their access to substrates.
Residues within a small segment N-terminal to XIAP’s BIR2 domain are essential
for the binding of XIAP to activated caspase-3 and –7. Our biochemical data on
the interaction between the Drosophila IAP DIAP1 and caspases expose significant
differences between DIAP1 and XIAP. Intriguingly, DIAP1 does not contain
sequence homology to the caspase-binding residues of XIAP predicted to be
strictly conserved in IAPs capable of binding caspases; yet, DIAP1 specifically
interacts with caspases. Our studies indicate that IAP:caspase association may
occur in distinct modes depending on the IAPs and caspases involved. We have
investigated the mechanism through which DIAP1 associates with effector caspases
and have found that DIAP1 and caspases interact through a bimodular binding
mechanism. Importantly, the residues on drIce and Dcp-1 that are crucial for
DIAP1-binding are evolutionarily conserved in caspases ranging from insects to
humans. Our observations suggest that certain mammalian IAPs, other than XIAP,
may bind to caspases in a DIAP1-like manner.
Identification
of a caspase-2 activating complex
Antoine
Tinel
and Jurg Tschopp
Department
of Biochemistry, University of Lausanne, Chemin de Boveresses, 155 CH-1066
Epalinges, Switzerland
Email:
Antoine.Tinel@ib.unil.ch
Caspase-2
is one of the most conserved caspases through evolution. It is CARD-containing
caspase and was shown to be processed early during many apoptotic processes.
However its function and its mechanism of activation remain largely unknown.
Recently caspase-2 was shown to be activated in an apoptosome-like complex of
unknown composition. Here we show that this complex involved RAIDD, a known
adaptor for caspase-2, and a recently cloned DD-containing protein PIDD. Pidd is
a new p53-responsive gene but is also expressed in a constitutive manner in
several tissues. PIDD interacts with RAIDD and is able to induce caspase-2
processing and activation upon overexpression in a RAIDD-dependent manner.
PIDD-induced caspase-2 activation was caspase-3 independent. Moreover both PIDD
and RAIDD shift to the same high molecular weight fractions as caspase-2 upon
incubation at 37oC and we show that these three proteins are in the
same complex. HeLa cells stably-expressing PIDD showed constitutive caspase-2
activation. Pidd is the first protein that is able to induce caspase-2
auto-activation in a RAIDD-dependent manner. Upstream stimuli specific for PIDD
“activation” and subsequent caspase-2 activation remain to be found.
Calnexin suppresses ceramide- and GD3 synthase (ST8)-induced
apoptosis.
Barbara Tomassini*, Florence Malisan*, Luigi
Franchi*, Chiara Nicolo'*, Gloria Brea Calvo, Takashi Saito and Roberto Testi*
*University of Rome Tor Vergata, Laboratory of Immunology and
Signal Transduction, Department of Experimental Medicine and Biochemical
Sciences, Via Montpellier 1, 00133 Rome.
E-mail: tomassini@med.uniroma2.it
An accelerated activity of the GD3 synthase (ST8), with
consequent GD3 accumulation, is part of the response to environmental stressors
in different cell types. Depending on specific, yet largely undefined, cellular
settings, this can be followed by cellular adaptation or apoptosis, the latter
mostly due to GD3-induced mitochondrial damage. Here we show that the
subcellular localization of the ST8 could significantly affect the biological
outcome of GD3 accumulation. We found that binding to the molecular chaperone
calnexin causes the retention of the ST8 within the endoplasmic reticulum (ER)
and prevents its relocalization to the Golgi. Calnexin-dependent ER retention
does not affect the activity of the ST8, yet the de novo synthesized GD3 largely
fails to reach the mitochondria. Accordingly, overexpression of calnexin
suppresses the pro-apoptotic activity of the ST8, while the loss of calnexin
sensitizes the cells to stress-induced apoptosis. Reconstitution of calnexin
confers protection to calnexin-deficient cells. Thus, by affecting the
subcellular distribution of the ST8, calnexin controls the biological outcome of
ceramide and GD3 accumulation, revealing itself as a novel regulator of the
stress response.
BAG-1
proteins protect cardiac myocytes from simulated ischemia/reperfusion-induced
apoptosis via an alternate mechanism of cell survival independent of the
proteasome
Paul A Townsend1,
Tiziano Scarabelli2, Christopher Carroll1, Kevin M
Lawrence1, Ramsey I Cutress3, Graham Packham3,
Anastasis Stephanou1, and David S Latchman1.
1Medical
Molecular Biology Unit, Institute of Child Health, University College London,
London, UK
E-mail:
p.townsend@ich.ucl.ac.uk
BAG-1
proteins interact with the HSC70 and HSP70 heat shock proteins and have been
proposed to promote cell survival by coordinating the function of these
chaperones with the proteasome, to facilitate protein degradation.
Analyses in cancer cells have demonstrated BAG-1 is required for cell
protection following various stresses. Since ischemia (I) and reperfusion (R)
are important stress signals in acute and chronic heart disease, we have
examined the role of BAG-1 proteins in cardiac myocytes (CM) and the intact
heart. BAG-1S and BAG-1L, were
rapidly induced following ischemia in CM and this was maintained during
reperfusion. In control hearts, BAG-1S and BAG-1L were readily detectable in
both the nucleus and cytoplasm, however, BAG-1S did not relocate to the nucleus
following simulated-I/R. BAG-1
interacted with both RAF-1 and HSC70 and binding to HSC70 was increased
following I/R. Overexpression of
human BAG-1S and BAG-1M isoforms significantly reduced CM apoptosis following
simulated-I/R. Whereas, BAG-1L or
BAG-1S fused to a heterologous nuclear localization sequence failed to protect
CM. Finally, overexpression of
BAG-1 deletion and point mutants unable to bind HSC70/HSP70 failed to offer
cardioprotection. A deletion mutant
lacking the N-terminal ubiquitin-like domain which mediates interaction with the
proteasome still promoted cardioprotection. Therefore, BAG-1 has a novel
cardioprotective role, mediated via association with HSC70/HSP70, which is
critical upon cytoplasmic localization but independent of the BAG-1
ubiquitin-like domain. Therefore,
BAG-1 can influence cellular response to stress by multiple mechanisms,
potentially influenced by the cell type and nature of the stress signal and is
thus a key player in apoptosis modulation.
Response of lung cancer patients to cisplatin-based
anti-cancer treatments: Effects on the levels of cleaved cytokeratin 18 and sFas
Engin
Ulukaya1,
Meryem Yilmaz1, Arzu Yilmaztepe1, Berrin Zik2,
Mehmet Karadag3
1
Uludag
University Medical School, Biochemistry Department, 16059, Bursa, Turkey; 2
Uludag University Veterinary School, Histology Department, 16059, Bursa,
Turkey; 3 Uludag University Medical School, Lung Diseases Department,
16059, Bursa, Turkey
Email:
eulukaya@uludag.edu.tr
Lung
cancer is one of the major causes of death from malignancy, with poor rates of
long-term survival. The main reason is resistance of lung cancers to treatment
either initially or when secondary tumours arise. The detection of the cleaved
cytokeratin 18, also named M30, in patient sera after the application of
chemotherapeutic drugs may be a predictor of the response to cancer therapy. M30
is a feature of epithelia-originated cells undergoing apoptosis and can be
detected in human serum using ELISA. In the study presented here, we have
measured M30 and sFas levels in 18 patients with lung cancer before and 24 or 48
hour after cisplatin-based treatments. We found that the M30 levels increased
significantly increased at both time points. Similarly, sFas levels increased at
24 hours, and remained high at 48 hours after treatment. These results are
consistent with the induction of lung cancer apoptosis in patients undergoing
cisplatin-based therapy. Future
studies will be aimed at relating these apoptosis markers with patient survival
parameters (e.g. patient’s response to treatment, progression free survival,
overall survival). Any significant correlations will indicate the benefit of
using M30 and sFas responses to predict treatment outcome.
Phosphatidic acid induces
permeability transition and the collapse of the membrane potential in isolated
rat liver mitochondria.
Gabor Varbiro,
Zita Bognar, Balazs Veres, Antal Tapodi, Balazs Radnai, Balazs Sumegi
Dept. of Biochemistry and
Medical Chemistry, Faculty of Medicine, University of Pécs, Szigeti u. 12,
H-7624 Pécs, Hungary.
E-mail: gabor.varbiro@aok.pte.hu
The role of phospholipids
in the apoptotic process has recently been implicated. Evidence show that
cardiolipin and phophogycerol affect the mitochondria. In our study, we analyzed
the effect of phosphatidic acid on the mitochondrial permeability transition and
membrane potential. Rat liver mitochondria was isolated and was treated by
different concentrations of phosphatidic acid. The mitochondrial permeability
was detected by the accompanying large amplitude swelling, indicated by the
alteration of light scattering. The changes in the membrane potential was
detected by the release of the mitochondrial dye, Rhodamine 1,2,3. Our results
show that phosphatidic acid induces permeability transition and the collapse of
the membrane potential in a concentration dependent manner. This effect was
inhibited by Cyclosporin A, a widely used inhibitor of permeability transition.
Our results were further supported by detecting the release of cytochrome c from
the mitochondria to the supernatant following phosphatidic acid administration.
Similarly Endonuclease-G was also released from the mitochondria following the
administration of phophatidic acid. In the presence of Cyclosporin A,
phosphatidic acid did not induce the release of mitochondrial pro-apoptotic
proteins. Other glycerophospholipids like phosphatidylcholine, or cardiolipin
did not mimic the effect of phosphatidic acid. When Jurkat cells were loaded
with Rhodamine 1,2,3; phosphatidic acid induced its release from the
mitochondria. The activation of caspase-3, an effector in the apoptotic process
was also observed in Jurkat cell line following the administration of
phosphatidic acid. These data indicate, that phophatidic acid contributes to the
apoptotic process, however further studies are necessary to elucidate its role
in in vivo models as well.
An extramitochondrial
role for frataxin in apoptosis suppression
Natascia Ventura*, Ivano Condo* and Roberto Testi
Laboratory
of Immunology and Signal Transduction, Department of Experimental Medicine,
University of Rome "Tor Vergata"
E-mail:
natascia.ventura@med.uniroma2.it
Defective
frataxin expression in humans causes Friedreich's Ataxia (FA), an autosomal
recessive neurodegenerative syndrome characterized by progressive ataxia,
dysarthria, skeletal abnormalities, glucose intolerance and cardiomyopathy.
Frataxin is a highly conserved mitochondrial protein involved in the biogenesis
of iron/sulfur clusters (ISC) and iron homeostasis. Low frataxin levels affect
mitochondrial energy metabolism as a consequence of altered biogenesys of ISC
enzymes, which include several components of the mitochondrial electron
transport chain. Degeneration of frataxin-deficient cells has therefore been
mostly attributed to inefficient oxidative phosphorylation leading to low ATP
and/or high reactive oxygen species (ROS) production. FA cells are more sensitive to oxidative stress, yet
the involvement of frataxin in cell survival is still obscure. With this work we
show for the first time that an extramitochondrial pool of frataxin exists in
different cell types. We demonstrate that a cytosolic frataxin (_1-frtx)
construct, lacking the mitochondrial signal peptide, appears largely functional.
Indeed, by transient overexpression, we show that _1-frtx prevents, as well as
wild type (wt-frtx), ceramide-induced ROS accumulation and apoptosis in
transformed cells with the same extent of Bcl-2. Moreover, differently from
Bcl-2, both wt- and _1-frtx protect against anti-Fas induced apoptosis
suggesting a possible role for frataxin besides its mitochondrial localization.
An essential role for the
BH3-only protein Bim in the establishment of central tolerance
Andreas
Villunger1,
Vanessa Marsden2, Miriam Erlacher1, Philippe Bouillet2,
William R Heath2 and Andreas Strasser2
1Institute
of Pathophysiology, Innsbruck Medical School, Innsbruck, Austria. 2The
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
E-mail: Andreas.Villunger@uibk.ac.at
TCR/CD3-ligation
induces apoptosis in semi-mature CD4+8-HSA+
thymocytes and this helps establish immunological tolerance and constitutes one
of the safeguards against autoimmune disease. We have analyzed several knock-out
and transgenic mouse lines and found that TCR/CD3-ligation induced killing of
semi-mature thymocytes occurred independently of Fas and ‘death receptor’-signaling
in general but required the pro-apoptotic BH3-only protein Bim and could be
inhibited by Bcl-2. Loss of Apaf-1 or caspase-9, which act downstream of the
Bcl-2 family protein family, provided only minor protection, indicating that the
‘apoptosome’ functions as an amplifier rather than as an essential initiator
of this death program. These results reveal the mechanisms of apoptosis in
negative selection of semi-mature thymocytes and have implications for
immunological tolerance and autoimmunity.
Cell
death activation by the proteasome inhibitor PS-341 (Velcade(tm)) in Non-Small
Cell Lung Cancer.
Jens
Voortman,
Giuseppe Giaccone and Frank A.E. Kruyt.
Department
of Medical Oncology, VU Medical Center, de Boelelaan 1117,
1081
HV Amsterdam, The Netherlands.
E-mail: j.voortman@vumc.nl
The
small molecule PS-341 (Velcade(tm)), a specific and reversible inhibitor of the
proteasome, is a promising new agent for the treatment of cancer. It has shown
to be effective in chemotherapy resistant tumor cells suggesting a unique
mechanism of action. Our aim is to study the mechanism underlying PS-341-induced
cell death in non-small cell lung cancer (NSCLC) cells in order to identify
factors that facilitate or suppress its activity. For this, cytotoxicity of
PS-341 in H460 NSCLC cells was measured using MTT - and propidium-iodide
staining assays. PS-341 induced a time and concentration dependent increase in
toxicity that was accompanied by an increase in the subG1 population and G2-M
phase arrest. SubG1 accumulation could be prevented by co-treatment with the
caspase-inhibitor zVAD-fmk illustrating caspase-dependent cell death.
Overexpression of Bcl-2 partially suppressed PS-341 cytotoxicity, suggesting a
role for mitochondria in PS-341 induced apoptosis. To have a more realistic
model, we examined cytotoxicity of PS-341 under hypoxia, which often occurs in
tumors. Preliminary studies
indicate a caspase-dependent enhancement of PS-341 toxicity under hypoxic
conditions. Further mechanistic studies are in progress, including to determine
the involvement of the NF-kB pathway that is regulated by the
proteasome-dependent degradation of its inhibitor, IkB.
Lentiviral
expression of TRAIL in human tumor cells.
Till
Wenger1, Klaus-Michael Debatin2, Ingrid Herr1
2Department of Pediatric Oncology/Therapy, German Cancer Research Center (DKFZ),
INF 280, 69120 Heidelberg, Germany; 2University Children’s
Hospital, Pritzwitzstr. 43, 89075 Ulm, Germany
E-mail:
t.wenger@dkfz.de
TRAIL
induces apoptosis in transformed
cells but not in most normal cells in vitro. The ability of soluble TRAIL
to induce apoptosis in normal human hepatocytes in vitro, as well as the
rapid clearance and large amounts of protein needed made us seek alternative
ways for the use of recombinant TRAIL in tumor therapy. Lentiviruses,
such as HIV, are members of the retrovirus family. Unlike classical retroviral
vectors, VSV-G pseudotyped lentiviral vectors have an extremely broad tropism
and can be concentrated to very high titers. We generated HIV-1 based second
generation lentiviral vectors at high titers (>109 IU/ml) capable
of efficiently infecting a variety of human cancer cell lines and primary cells
at moderate MOI (0,5-2). A vector containing human TRAIL was capable of
expressing TRAIL in different cell lines and of inducing apoptosis in the
B-cell-line BJAB and the NSCLC line P693. Apoptosis occured rapidly after
infection and was cell-cell-contact dependent, suggesting bystander killing of
surrounding cells by infected cells. The infected cells were also able to kill
co-cultured target cells. Soluble TRAIL appeared to be absent from the
supernatant of infected cells. These findings suggest a strictly
cell-cell-contact mediated killing by TRAIL expressed on the surface of infected
cells. We however found that, after prolonged culture of the transduced cells,
only TRAIL-transduced cells
survived, in contrast to a GFP control. We examined the cell surface expression
of TRAIL and the TRAIL receptors 1 and 2 and found complete absence of receptor
surface expression in the TRAIL- transduced cells, rendering the cells resistant
to TRAIL-induced apoptosis, but not to apoptosis induced by other stimuli. The
down-regulation of the receptors was not due to decreased transcription, as
demonstrated by RT-PCR, and not solely due to selection of receptor negative
cells since it also occurred in FADD-dominant-negative expressing BJAB cells. Since intracellular expression of
TRAIL receptors could be detected, we suggest intracellular retention of the
receptors by the ligand as a mechanism for this phenomenon.
Partial cleavage of RasGAP by caspases is required for
survival in mild stress conditions.
Jiang-Yan Yang*, David Michod*, Joël Walicki*, Brona M. Murphy^,
Shailaja Kasibhatla#, Seamus J. Martin^, and Christian Widmann*
* Institut de biologie cellulaire et de morphologie (IBCM),
Biology and Medicine Faculty, Lausanne University, Switzerland; ^ Molecular Cell
Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity
College, Dublin, Ireland; # Maxim Pharmaceuticals, San Diego, California, USA.
E-mail: jiang-yan.yang@ibcm.unil.ch
Ectopic expression of the N-terminal fragment resulting from
the partial cleavage of RasGAP by caspases results in the activation of the
anti-apoptotic Ras-PI3K-Akt pathway. Here we demonstrate that this fragment is
generated in a caspase-3-dependent manner in stressed cells that do not undergo
apoptosis. Subjected to the same low stress conditions, cells, in which the
wild-type RasGAP protein was replaced with an uncleavable mutant, could not
activate Akt, could not prevent an amplification of the caspase-3 activity, and
eventually underwent apoptosis. Formation
of fragment N in response to low caspase-3 activation is therefore critical for
survival of stressed cells. Executioner caspases control therefore the extent of
their own activation by a feedback regulatory mechanism initiated by the partial
cleavage of RasGAP.
Th2
cytokines induce chemotherapy resistance in epithelial tumors via PI3K/AKT
pathway
M. Zerilli, M. Patti, M. Bini, M. Perez Alea, A. Gorgone, R. Vitale, M. Todaro and G.
Stassi.
Molecular
and Cellular Pathophysiology Lab. Dpt of Surgical and
Oncological Science, Università degli Studi di Palermo, Italy
E-mail: monicazerilli@hotmail.com
Several studies designate apoptosis as the
predominant mechanism by which cancer cells die in response to chemotherapeutic
drugs. Moreover, it was suggested that anti-apoptotic molecules activation play
a pivotal role in the chemotherapeutic drugs resistance. We have recently
demonstrated that the presence of Th2 cytokines in thyroid cancer
microenvironment increase FLIP, Bcl-2 and Bcl-xl expression levels and protect
cancer cells from chemotherapeutic drugs induced apoptosis. Particularly, in
this study we demonstrate that IL-4 and IL-10 promote proliferation and
chemotherapy resistance activating the PI3K/AKT signal pathway. Therefore, we
extensively studied cell survival-related substrates and their modulation by Th2
cytokines in epithelial tumors. These substrates include members of intrinsic
cell death machinery such as BAD and caspase 9, and forkhead family members and
the transcriptional factor NF-kB, which seems to exert a key role in promoting
anti-apoptotic genes expression. These findings elucidate a key molecular
mechanism by which tumor cells escape conventional chemotherapy.
Ineffective
death receptor signaling in polycythemia vera erythroid progenitor cells
Ann Zeuner
*°, Signore M.*, Pedini F.*, Messina C.*, Peschle C.*, De Maria R*°.
* Department of
Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome,
Italy
° Istituto
Oncologico del Mediterraneo, Viagrande (Catania), Italy
Email:
a.zeuner@iss.it
Polycythemia
vera (PV) is a clonal hematopoietic disorder characterized by dysregulated
erythrocyte production. TNF family members act as negative regulators of
erythropoiesis by inducing erythroblast growth arrest or apoptosis. In the
hypothesis that defective death receptor (DR) signaling may contribute to
unnecessary erythroid cell expansion in PV, we analyzed purified erythroblast
populations obtained from PV patients for sensitivity to DR stimulation. Whereas
normal erythroblasts undergo growth and differentiation arrest, and ultimately
cell death, in response to CD95L and TRAIL, PV erythroblasts from the majority
of patients show partial or total resistance to the growth-inhibitory effect of
CD95 and TRAIL receptors, which was still correlated with a differentiative
delay. Caspase activation following CD95 stimulation was lower in the majority
of PV samples, indicating a possible alteration in death receptor signalling
pathways. As a possible consequence of lower caspase activation, CD95-stimulated
PV erythroblasts maintain higher levels of the erythroid transcription factor
GATA-1 compared to normal erythroblasts. The majority of erythroblasts from
CD95-resistant PV patients displayed higher levels of FLIP than controls,
indicating a possible mechanism for the inefficient transduction of death
receptor-mediated antiproliferative signals. These results indicate in defective
death receptor signaling a potential mechanism of unrestrained hematopoietic
cell production in polycythemia vera.
BMP5- a new player in the control of interdigital
cell death acting hrough Smad and MAPK signalling patwhays.
Vanessa Zuzarte-Luís
and Juan Hurlé
Dpto. Anatomy and Cell Biology, University of Cantabria,
Spain.
E-mail: uc24658@correo.alumnos.unican.es
The process of apoptosis plays a key role in the sculpturing
of the shape of the limbs. The formation of free digits in all amniote
vertebrates is preceded by the occurrence of an area of programmed cell death in
the interdigital mesoderm. The molecular machinery responsible for signalling
and executing apoptosis is highly conserved however its regulation has still
some links missed. The Bone Morphonenetic Proteins (BMP-2, -4 and -7) have been
identified as the triggering signals for limb programmed cell death. These
secreted factors are expressed in the preapoptotic limb mesoderm, and apoptosis
is induced and inhibited respectively in gain and loss of function experiments.
We cloned a new member of this family, Bmp5, which has not been previously
implicated in the process of apoptosis during limb development. Bmp5 exhibited a
regulated pattern of expression in the interdigital tissue, in agreement with a
possible role in the control of cell death. Gain-of-function experiments using
the human recombinant protein revealed that BMP5, like the other BMP proteins,
is able to induce apoptosis in the undifferentiated mesoderm and growth of the
prechondrogenic mesenchyme. Furthermore we characterized the intracellular
effectors of BMPs in the developing limb autopode. Both Smad and MAPK signalling
pathways are activated. Activation of Smad signalling implies both the
upregulation of gene transcription and protein phosphorilation of Smads 1, -6
and -8. The MAPK p38 is also phosphorilated by BMPs in the limb mesoderm and the
treatment with the its inhibitor, revealed that this kinase has a major function
in the BMP-mediated chondrogenic effect but is also the responsible for the
activation of several genes associated with the onset of interdigital apoptosis.
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