Báo cáo khoa học: Signal Transduction pot

Signal Transduction
C1-1
Histone modifications regulate morphogenesis
in C. albicans
D. Hnisz and K. Kuchler
MFPL, Vienna, AUSTRIA
Candida albicans is an opportunistic human pathogen with remark-
able phenotypic plasticity. Its diploid cells can undergo an epi-
genetic phase-transition termed the white-opaque switching.
Whereas white cells have a round shape and are unable to mate,
opaque cells have an oval shape and are mating-competent.
While the contribution of transcription factors to phase changes is
widely investigated, little is known about epigenetic mechanisms
underlying switching. During the white-opaque transition, phase-
commitment is dependent on a single master locus WOR1, but it
includes changes in the expression levels of about 400 genes. The
major goal of our studies was to identify genes regulating white-opa-
que switching and analyze their regulatory mechanisms.
With bioinformatic tools we identified all histone-modifying genes
of C. albicans. We created homozygous deletion mutants and
assayed the influence of the deletions on the phase transitions, thus
establishing functional categories for switching modulators. Quanti-
tative mating assays show that all the mutants maintain wild-type
mating efficiencies arguing that the modulators have a effect speci-
fic rather than global effect. We demonstrate with real-time PCR
analyses that the expression of the modulators is independent of
WOR1. Our results suggest a new model of the regulation of white-
opaque switching that includes at least another so far unidentified
second master regulator that has a mutual transcriptional depend-
ence on WOR1 to determine the phase outcome and the transcrip-
tional feed-backs are mediated by histone-modifying cofactors.

C1-2
Glutathione regulates the transition from
growth to development in Dictyostelium
discoideum
J. Kim, C. Choi, S. Jeong, J. Seo and S. Kang
Seoul National University, Seoul, REPUBLIC OF KOREA
Glutathione is a ubiquitous tripeptide, found in most plants,
microorganisms, and all mammalian tissues and most prevalent
reducing thiol-containing compound in eukaryotic cells. Glutathi-
one serves as cellular thiol redox buffer to maintain a thiol/disul-
fide redox potential, and also known to participate in many
cellular processes. Disruption of GCS encoding c-glutamylcysteine
resulted in glutathione auxotrophy and developmental defect in
Dictyostelium discoideum. And GCS-null cells showed different
developmental progress, depending on the level of GSH. To under-
stand defective development, we investigated the development in
suspension. GCS-null cells depleted GSH did not aggregate and
were still single cells in suspension with addition of cAMP. Inter-
estingly, the addition of 1mM GSH induced them to aggregate.
However, the defect in aggregation of them was not rescued by
dithiothreitol, N-acetylcysteine and ascorbic acid. GCS-null cells
fail to decrease the expression of the growth-stage gene cprD, and
do not induce the expression of cAR1 (cAMP receptor), acaA (a-
denylyl cyclase A) and lagC (aggregation marker) that required for
the earliest stages of development. Dictyostelium cells that enter
the development stage use G protein-coupled receptor signaling to
direct chemotactic migration to a source of cAMP. We overex-
pressed cAR1, the most important receptor for cAMP signal cas-
cade, in GCS-null cells. cAR1 overexpressing GCS-null cells still
fail to aggregate in suspension. These results suggest GCS-null cells

are defective in production of the extracellular cAMP that serves
as the extracellular chemoattractant and in cAMP signal cascade
in D. discoideum development.
C1-3
Tissue-specific regulation of RNT-1 function in
C. elegans
J. Shim, K. Lee and J. Lee
Seoul National University, Seoul, REPUBLIC OF KOREA
Runx proteins are evolutionarily well-conserved transcription fac-
tors that are involved in essential aspects of the development of
metazoan animals ranging from nematodes to humans. We found
that the expression of the nematode RUNX homolog, RNT-1, is
tightly regulated in that it is expressed only in the intestine and
hypodermis at specific developmental stages and that RNT-1 is
almost absent in any tissue at the adult stage. Ectopic expression
of RNT-1 resulted in the over-proliferation of the hypodermal
cells, indicating that tightly regulated attenuation of the RUNX
protein is required for its proper function in vivo.
Recently, it was revealed that the C. elegans genome contains one
homolog of the Runx protein partner CBFbeta (BRO-1). bro-1 is
expressed only in the hypodermis at all developmental stages.
Knockdown of bro-1 resulted in the up-regulation of RNT-1 in the
hypodermis, but not in the intestine, suggesting that bro-1 acts as a
co-repressor of RNT-1. We found that the nematode RNT-1 regu-
lates its own transcription in the hypodermis, acting on its own
cis-acting elements.
In situ hybridization experiments showed that the RNT-1 tran-
script is still present in the intestine at the adult stage, but not in
the hypodermis, suggesting that RNT-1 might be regulated at the
protein level. Consistent with this, RNAi knockdown of ubq-1,a

polyubiquitin gene, and uba-1, the E1 gene, resulted in higher sta-
bility of RNT-1 in the intestine and the treatment of MG132, a
proteasome inhibitor, stabilized RNT-1 in the intestine. Taken
together, we conclude that RNT-1 expression is attenuated at the
adult stage by two different mechanisms, transcriptional autoregu-
lation in the hypodermis and proteasome-mediated degradation in
the intestine.
C1-4
Differential cleavage of Dpp precursor
modulates morphogen gradient in the
Drosophila
J. Ku
¨
nnapuu, I. Bjo
¨
rkgren and O. Shimmi
University of Helsinki, Helsinki, FINLAND
We have previously shown that two different molecular forms of
Decapentaplegic (Dpp), Drosophila BMP2/4 type ligand, are pro-
duced in the embryo (Shimmi et al, Cell 2005). As Dpp precursor
contains two optimal furin recognition sites, we suspect that these
two sites can be used for ligand maturation. In order to under-
stand how Dpp processing is regulated, and why two different
forms of Dpp ligands are used for signaling, we mutated the opti-
mal furin recognition sites to study their function. The mutation of
the first furin site drastically dropped the production of mature lig-
ands in Drosophila S2 cells. In contrast, the mutation of the second
furin site did not affect the production rates or the signaling inten-
sities of ligands, however, mature ligands were produced as a single
form. As previous reports indicated that vertebrate BMP4 was

sequentially cleaved to help promote the long-range signaling, we
focus on two different stages, the early embryo and the wing ima-
ginal disc, in which Dpp works as a morphogen, to investigate the
in vivo function of different forms of Dpp. We will discuss how
evolutionally conserved furin recognition sites of BMP2/4 type lig-
ands contribute to regulate the long- or short-range signaling of
these ligands during developmental stages.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 133
C1-5
Phenotypic observation of Xenopus laevis MGP
loss of function
B. S. N. Simo
˜
es, B. Simo
˜
es
1
, A. C. Silva
2
, M. Vitorino
2
,
N. Conceic¸ a
˜
o
1
, J. A. Belo
2
and M. L. Cancela

1
1
CCMAR,
2
IBB, CBME – University of Algarve, Faro, PORTUGAL
Matrix Gla protein (MGP) belongs to the family of vitamin
K-dependent Gla proteins and is known to be involved in regula-
tion of extracellular matrix calcification and maintenance of carti-
lage and soft tissue integrity during growth and development. We
have previously determined the spatial pattern of XlMGP expres-
sion during embryogenesis and showed that XlMGP transcripts are
expressed during gastrulation in the dorsal mesoderm along Bra-
chet’s cleft, as well as in the ventral mesoderm. To understand the
role of MGP in vertebrate development, zygotic knockdown of
MGP messages was performed using morpholino oligos against
XlMGP. Specific and control morpholino oligos were injected into
the blastomeres of Xenopus embryos at 4–8 cell stage and resulting
phenotypes were analysed by the expression pattern of develop-
mental marker genes. Our data suggests that MGP knockdown
induces changes in localization of different gene markers, thus
affecting subsequent events in Xenopus early development.
Acknowledgements: NC is supported by a post-doctoral grant
(SFRH/BPD/9451/2002), AS and MV by PhD grants (SFRH/BD/
10035/2002 and SFRH/BD/24765/2005, respectively) and BS was a
recipient of a technical fellowship. This work is supported by pro-
ject POCI /BIA-BCM/58677/2004.
C1-6
Lachesis restricts gametic cell fate in the female
gametophyte of Arabidopsis
C. Ka

¨
gi
1
, C. Moll
1
, L. von Lyncker
1
, N. Baumann
1
,
U. Grossniklaus
2
and R. Gross-Hardt
1
1
ZMBP University of Tu
¨
bingen, Tu
¨
bingen, GERMANY,
2
Institute
of Plant Biology, Zu
¨
rich, SWITZERLAND
In flowering plants, the egg and sperm cells form within haploid
gametophytes. The female gametophyte of Arabidopsis consists of
two gametic cells, egg cell and central cell, that are flanked by five
accessory cells. We asked why some cells differentiate gametic cell
fate whereas others become accessory cells.

In a screen for regulators of egg cell fate we isolated the lachesis
(lis) mutant which forms supernumerary egg cells. In lis mutants
accessory cells differentiate gametic cell fate, indicating that Lis is
involved in a mechanism that prevents accessory cells from adopt-
ing gametic cell fate. The expression pattern of Lis suggests that
this mechanism is generated in gametic cells, implying that lateral
inhibition patterns the female gametophyte. Lis is homologous to
the yeast splicing factor PRP4. The puzzling link between the regu-
lation of gametic cell fate and the splicing apparatus is corrobor-
ated by the finding that defects in a second splicing factor,
CLOTHO, also result in the formation of supernumerary egg cells.
Possible implications will be discussed.
C1-7
Cytokinine secondary hormone activates
plasmatic membrane H
+
-ATPase which is
important regulatory machine of the plant cell
A. N. Sabitov
1
, K. Musabekov
1
, M. K. Gilmanov
2
and
Z. S. Kudiyarova
3
1
Chemistry Department, Chair of Colloid Chemistry, Catalysis and
Oil Chemistry, Al-Faraby’s Kazakh National University, Almaty,

KAZAKHSTAN,
2
M.A. Aitkhozhins Institute of Molecular Biology
and Biochemistry, Almaty, KAZAKHSTAN,
3
Biology Department,
Al-Faraby’s Kazakh National University, Almaty, KAZAKHSTAN
It is well known, that regulation of ionic transport the main fea-
ture of cell regulation. However the mechanisms of regulation of
ionic transport in plant cells are not enough investigated. For this
reason it is very important to investigate the effect of new powerful
regulator – cytokinine secondary hormone (CSH) on plant cell ions
transport.
In laboratory of structure and regulation of enzymes of M.A. Ai-
tkhozhins institute of molecular biology and biochemistry CSH
was discovered and characterized. CSH appears its physiological
action at the concentration hundred times less than other phyto-
hormones. We have studied the effect of CSH on main ion trans-
port enzyme - plasmatic membrane H
+
-ATPase from wheat grain.
It was shown that CSH activates plasmatic membrane H
+
-ATPase
more than two times. The one of the interesting features of studied
H
+
-ATPase is the next. The activity of this enzyme strong depends
from Ca
2+

and Mg2
+
and this enzyme haven’t absolutely any
activity with sodium and potassium ions. We are suggested that
regulatory effects of CSH first of all appear by activation of cal-
cium ions transport through plasmatic membrane into the plant
cells. It is well known that calcium ions are main intracellular mes-
senger which regulates many important cell functions. CSH by this
property is very close to fusicoccine – natural phytotoxin from
Fusicoccum amygdali.
C1-8
Mathematical model of the Arabidopsis thaliana
morphogenesis in a cellular automaton terms
I. R. Akberdin, E. A. Ozonov, V. V. Mironova,
D. N. Gorpinchenko, N. A. Omelyanchuk, V. A. Likhoshvai and
N. A. Kolchanov
Institute of Cytology and Genetics SB RAS, Novosibirsk, RUSSIAN
FEDERATION
Development of organisms is a very complex process in which a
lot of gene networks of different cell types are integrated. Develop-
ment of a cellular automaton that models the morphodynamics of
different cell types is the first step in understanding and analysis of
the regulatory mechanisms underlying the functioning of develop-
mental gene networks. A model of a cellular automaton has been
developed, which simulates the embryonic development of shoot
meristem in Arabidopsis thaliana. The model adequately describes
the basic stages in development of this organ in wild and mutant
types. Visualization of the cellular automaton was created that
allows simulating of the process of development. The visualization
of cellular automaton model allows estimating distribution of three

biologically meaningful signals, which unambiguously simulate the
morphodynamics of the cell tissues. The cellular automaton model
introduced here to investigate the development of primary shoot
meristem of the Arabidopsis thaliana in embryogenesis under differ-
ent initial parameters of the model. It allows recognizing of signifi-
cant parameters, which greatly influence on behaviour of dynamic
system and determining the stable state of this biological system by
variation parameters. In this visualization of cellular automaton
you can remove some cells at discretion and continue calculation
that allows analyzing and reproducing such experiment as laser
excision.
Abstracts Signal Transduction
134 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-9
The highly conserved mitochondrial protein
GCP1 is essential for embryo development in
Arabidopsis thaliana
P. F. Huesgen
1
, K. Haußu
¨
hl
2
, P. Dessi
3
, J. Adamski
4
, E. Glaser
3
and I. Adamska

1
1
Fachbereich Biologie, Universita
¨
t Konstanz, Konstanz, GERMANY,
2
Qiagen AG, Hilden, GERMANY,
3
Department of Biochemistry and
Biophysics, Stockholm University, Stockholm, SWEDEN,
4
GSF-
Forschungszentrum fu
¨
r Umwelt und Gesundheit, Neuherberg,
GERMANY
Glycoproteases (GCP) are a family of putative Zn-metalloendopep-
tidases that are found highly conserved in taxonomically diverse
species. Our phylogenetic analysis revealed that all prokaryotes
contain only one GCP, either of the gcp1-type (Bacteria) or the
gcp2-type (Archaea). Eukaryotic organisms contain two gcp genes,
one of each type. GCP1 is a predicted to target to the mitochon-
dria, while has no recognizable signal sequence and is probably
located in the nucleus and/or cytoplasm.
We cloned the gcp1 gene from Arabidopsis thaliana and raised a
polyclonal antibody against the purified recombinant protein. With
this antiserum, we demonstrated that GCP1 is located in the inner
membrane of plant mitochondria. The antiserum also detected spe-
cific bands in murine and human protein extracts. In Arabidopsis,
we detected a high GCP1 level in developing leaves, roots, flowers

and pods of mature plants, as compared with fully developed
organs or mature seeds, where only traces of GCP1 were present.
Using immunocytochemistry we investigated the tissue specific
expression of GCP1 and demonstrated that this protein is strongly
expressed in axial meristems. We demonstrate that homozygous
GCP1 knock-out mutants are not viable. Embryos of these mutants
were arrested at the globular stage and failed to undergo the trans-
ition to heart stage. Based on our data we propose that the mitoch-
ondrial GCP1 is essential for cell division and/or differentiation in
plants, and suggest that GCP1 plays a similar role in other species.
C1-10
Phospholipase D1 regulates neurogenin1
expression via mTOR during bFGF-induced
neurite outgrowth in H19–7cells
J. Koo, M. Yoon and J. Han
Department of Biochemistry and Molecular Biology, Hanyang
University, Seoul, REPUBLIC OF KOREA
Phospholipase D (PLD) catalyzes the hydrolysis of phospholipids,
mainly phosphatidylcoline (PC), resulting in formation of phosphati-
dic acid (PA). PA has been associated with many aspects of mamma-
lian physiology, which include cell proliferation, survival,
transformation, progression and differentiation. Recently, PA has
been identified as an activator of mTOR signaling pathway. PA binds
to mTOR to promote various signals. We previously showed that
PLD1 activity is upregulated during neurogenesis induced by basic
fibroblast growth factor (bFGF) treatment in H19–7 cells. Subse-
quently, we investigated the role of PLD1 in neurogenesis, specifically
expression of neurogenin1 (Ngn1), bHLH family which plays critical
role in regulation of neural stem cell differentiation. To figure out the
effect of PLD1 on Ngn1 expression through mTOR signal, we treated

permeable PA to H19–7 cell under differentiation condition. Per-
meable PA treatment upregulated mTOR and Ngn1 expressions. Fur-
thermore, inhibition of PLD1 activity by PLD1 siRNA showed that
expressions of mTOR and Ngn1 were completely inhibited. However,
mTOR and Ngn1 expression levels were upregulated when PLD1
activity was increased by bFGF in H19–7 cells. To further confirm
the role of PLD1 on Ngn1 expression through mTOR, we treated ra-
pamycin to inhibit mTOR signal. Treatment of rapamycin showed
completely inhibited Ngn1 expression and neurogenic morphological
change. These results suggest that PLD1 regulates Ngn1 expression
through mTOR signaling pathway during neurogenesis in H19–7 cell.
C1-11
Tyrosine phosphatase epsilon is a negative
regulator of the signal transducer Shc
J. Kraut-Cohen and A. Elson
The Weizmann Institute of Science, Rehovot, ISRAEL
Reversible phosphorylation of tyrosine residues in proteins is a
crucial mechanism for regulating cellular and physiological func-
tions. Protein tyrosine phosphatases (PTPs) are known to be major
regulators of this process in vivo. This study examines the relation-
ship between PTP epsilon (PTPe) and the adaptor and signal trans-
ducer molecule Shc. Basal interactions were observed between the
non-receptor form of PTPe (cyt-PTPe) and p52 or p66 Shc in Jur-
kat T-cells and in Ras transformed NIH3T3 fibroblasts. This inter-
action is dependent on the N-terminal part of cyt-PTPe and on the
PTB domain of Shc. Complex formation is not dependent on
tyrosine phosphorylation of Shc in signaling processes as non-
phosphorylatable Y-to-F mutants of Shc can still bind cytPTPe.
Cyt-PTPe can down-regulate phosphorylation of endogenous Shc
at tyrosines 239/240 and 317. In correlation, expression of cyt-

PTPe leads to reduced association of Shc with Grb2 and to a sub-
sequent decrease in ERK activation. Interestingly, these effects
occur readily when signaling is stimulated by Src, but not when
initiated by Neu or by the EGF receptor. This difference is most
likely the result of Neu’s ability to compete against PTPe for bind-
ing the Shc PTB domain: the interaction of Neu with Shc prevents
Shc’s dephosphorylation and downregulation of the ERK path-
way. We conclude that PTPe is an important negative regulator of
Shc-mediated signaling that acts in a kinase-specific manner.
C1-12
TRKA induction leading to cell death via cell
cycle alteration and
c
H2AX accumulation in
cytosol
D. Kim and E. Jung
Gyeongsang National University School of Medicine, Jinju, REPUB-
LIC OF KOREA
In response to NGF, TrkA tyrosine kinase is activated by autop-
hosphorylation and plays an important role in neuronal cell survi-
val, differentiation, and apoptosis. We show here that TrkA
overexpression by the Tet-On system in U2OS cells mimicked
NGF-mediated activation pathway, leading to tyrosine phosphory-
lation of cellular proteins, even in the absence of ligand engage-
ment. Overexpressed TrkA appeared to be mainly accumulated in
cytosol and plasma membrane. TrkA overexpression in U2OS cells
induced the morphological change to neuron-like cells and inter-
rupted cell cycle progression, especially on a G1-S transition,
which led to cell death at a time-dependent manner. The cell death
by TrkA was inhibited by its specific tyrosine kinase inhibitor,

GW441756. p53 upregulation upon DNA damage was decreased
by TrkA overexpression, whereas p21 was upregulated by TrkA in
a p53-independent manner. Interestingly, cH2AX was largely
increased in TrkA-overexpressed cells. Also, cH2AX and TrkA
were colocalized in cytosol in the absence of DNA damage, indica-
ting that two proteins might have a functional relation. Moreover,
TrkA overexpression altered nuclear localization of cH2AX by
DNA damage to partly cytosol. Here, we first suggest that cH2AX
could be implicated in cell death signaling cascade by TrkA in the
absence or presence of DNA damage.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 135
C1-13
Identification of SIVA and PDCD2 as novel XIAP
interaction partners
U. Resch and R. deMartin
Department of Vascular Biology and Thrombosis Research, Medical
University, Vienna, AUSTRIA
The X-linked inhibitor of apoptosis (XIAP) is a specific inhibitor of
proapoptotic caspases 3, 7 and 9. In addition, XIAP plays a role in
intracellular signaling cascades involved in cellular response to stress
or inflammation. Recently we have shown that XIAP activates the
NF-jB pathway, which is dependent on the ubiquitin-E3-ligase
activity within the RING-domain of XIAP. However, the underlying
molecular mechanism for this activation is unclear.
Therefore, a yeast-two-hybrid screen with full-length as well as
truncated versions of XIAP was performed. We identified two
novel XIAP-interacting proteins, CD-27 binding protein (SIVA)
and programmed cell death-2 (PDCD2). The three BIR domains
of XIAP were dispensable for this interaction. Coimmunoprecipita-

tion experiments in HEK293 cells verified these interactions with
both wild type and its E3-ligase mutant of XIAP. Furthermore,
SIVA was found to be ubiquitinated when overexpressed along
with XIAP but not with its E3-ligase mutant. To test if these pro-
teins influence the XIAP-induced NF-jB activity, reporter gene
assays were performed. We found that SIVA had a inhibitory
effect on the XIAP-induced NF-jB activity, whereas PDCD2 had
no effect. This suggests that XIAP mediated ubiquitination of
SIVA inhibits its inhibitory effect on NF-jB activation, whereas
the functional consequences of the interaction with PDCD2 remain
to be elucidated.
C1-14
NF1 and Smad proteins, are they partners?
G. Kollarovic
1
, P. Barath
1
, K. Luciakova
1
and B. D. Nelson
2
1
Cancer Research Institute, Bratislava, SLOVAKIA,
2
Stockholm
University, Stockholm, SWEDEN
The linkage between mitochondrial oxidative phosporylation and
cytosolic ATP utilization needs co-operation between molecular
machine called ATPase and adenine nucleotide translocator (ANT).
ANTs are antiporters of inner mitochondrial membrane that

exchange cytosolic ADP for mitochondrial ATP synthesized by
ATPase. There are four ANT isoforms in human cells. Expression of
the ANT2 isoform is unique because it is growth dependent.
The promoter region of human ANT2 gene is composed of five main
regulatory elements which bind two transcription factors. Three bind
Sp1 protein, two of which activate ANT2 expression and one of
them represses ANT2 transcription. The other two regions bind
transcription factor NF1 and repress ANT2 transcription. One of
them (G
0
-R element) has been shown to be responsible for growth
dependent regulation of ANT2 transcription.
The aim of our work is to describe the mechanism of ANT2 repres-
sion in quiescent cells mediated by NF1 and other proteins. First we
found that there are additional binding sites for Smad proteins near
G
0
-R element. Therefore, we performed co-immunoprecipitation
experiments and showed presence of Smad and NF1 in one protein
complex. Interaction of Smad and NF1 was also confirmed by
in vitro GST pull downs. The results from ChIP assay suggested
changes in protein complex composition on the ANT2 promoter
during G
0
phase of cell cycle. Finally, the proteins of Smad family
are involved in TGFb signalling pathway. And indeed, TGFb
decrease the level of ANT2 mRNA as demonstrated by RT-PCR.
We believe that our data strongly suggest novel role for NF1 tran-
scription factor that cooperate with Smad proteins in growth
dependent transcriptional repression.

C1-15
VEGF induces a specific gene repertoire in
endothelial cells
J. Schultes, B. Schweighofer, J. Pomyje, M. Bilban, H. Mayer and
E. Hofer
Medical University Vienna, Vienna, AUSTRIA
VEGF-A via triggering of VEGFR2 is the major initiator of
angiogenesis. Specific signals/genes induced by VEGF-A, but not
by other growth factors and cytokines, have so far not been fully
established. We have analyzed here the induction of signals and
genes by VEGF-A/VEGFR2 in comparison to EGF and IL-1.
HUVEC were induced by VEGF-A, EGF or IL-1, followed by
Affymetrix microarray analyses and RT-PCR. Specific inhibitors
of NFAT and EGR-1 were used to judge the contribution of these
factors. The data show that VEGF-A/VEGFR2 preferentially trig-
gers signals via PLC-gamma, PKC and Ca++, whereas EGF is
unable to trigger this pathway and IL-1 is a preferential inducer of
NFkappaB. Downstream of PKC and Ca++ the factors EGR-1
and NFAT are important regulators. Gene activation via PLC-
gamma provides VEGF with the potency to induce a wide spec-
trum of genes, which includes a large proportion of genes also
regulated by IL-1. This is caused by the presence of overlapping
binding sites for NFAT and NFkappaB in these genes. In addi-
tion, a smaller number of genes was found to be strongly induced
by VEGF, but not by EGF or IL1. These include the transcription
factors Nurr1, Egr3, Hlx1 and Mef2C. We propose that both
properties, the ability to induce a large number of genes in com-
mon with inflammatory mediators, and a small group of exclu-
sively regulated genes, is important for the role of VEGF as
primary physiological inducer of angiogenesis.

Acknowledgement: Supported by FWF-NFN94–3 and EC
LSHC-CT-2005–518178.
C1-16
Functional analysis of a homeobox gene
upregulated by VEGF in endothelial cells
J. Schultes, B. Schweighofer, J. Pomyje, C. Sturtzel and E. Hofer
Medical University Vienna, Vienna, AUSTRIA
The human HLX1 (H2.0-like homeobox 1) gene is a diverged
homeobox gene. Microarray data have shown that it is upregulated
selectively by VEGF, the main trigger and key regulator of angio-
genesis, and not by the general growth factor EGF or the inflam-
matory cytokine IL-1. We investigated here the regulation of the
HLX1 gene in endothelial cells and developed tools to study its
potential function in angiogenesis.
We show that HLX1 mRNA is upregulated 10-fold by VEGF and
analysed the HLX1 gene by bioinformatics tools. This revealed a
conserved potential enhancer region 2.9 kb upstream of the start
codon, composed of binding sites for ATF and CREB. An analysis
of the protein sequence detected aside the conserved homeodomain
three different potential functional motifs, an SH3 binding site, ser-
ine/threonine phosphorylation sites, and a kinase binding site.
To analyse the function of HLX1 in angiogenesis recombinant a-
denoviruses were prepared to achieve overexpression of HLX1 in
endothelial cells. High expression of HLX1 by the recombinant a-
denovirus was confirmed. This was used to test the influence of
HLX1 on the induction of certain genes by VEGF. MYCN, a gene
involved in proliferation, was found to be upregulated by HLX1.
In contrast, the endogenous HLX1 was strongly downregulated
suggesting a feedback inhibition of the HLX1 gene. Further analy-
ses of the effects of HLX1 overexpression and inhibition on angio-

genesis models are underway.
Acknowledgement: Supported by FWF-NFN94–3 and EC
LSHC-CT-2005–518178.
Abstracts Signal Transduction
136 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-17
Signaling events downstream of MuSK and
their role during neuromuscular synapse
formation
V. Nizhynska, R. Neumueller and R. Herbst
Medical University Vienna, Vienna, AUSTRIA
The formation of the neuromuscular synapse (NMS) is regulated
by the nerve-derived heparan sulphate proteoglycan agrin and the
muscle-specific kinase MuSK. Agrin induces a signal transduction
pathway via MuSK, which induces pre- as well as postsynaptic dif-
ferentiation. Most importantly, activation of MuSK leads to the
phosphorylation and redistribution of acetylcholine receptors (AC-
hRs) and other postsynaptic proteins to synaptic sites. The accu-
mulation of high densities of AChRs at postsynaptic regions
represents a hallmark of NMS formation and is required for
proper NMS function. The steps that follow MuSK activation and
that lead to AChR clustering are however largely unknown.
Using MuSK mutant muscle cells, we have shown that MuSK car-
rying mutations in a juxtamembrane tyrosine or in the activation
loop tyrosines is unable to induce AChR clustering. In particular, a
13 aa juxtamembrane region of MuSK is necessary and sufficient
to regulate NMS development in vitro and in vivo. Further, we have
shown that phosphoinositide 3-kinase (PI3-K) represents a compo-
nent of the agrin/MuSK signaling pathway. Muscle cells treated
with specific PI3-K inhibitors are unable to form full-size AChR

clusters in response to agrin and AChR phosphorylation is
reduced. Moreover, agrin-induced activation of Rac and Cdc42 is
abolished in the presence of PI3-K inhibitors. These results put
PI3-K downstream of MuSK as regulator of AChR phosphoryla-
tion and clustering. Its role during agrin-stimulated Rac and Cdc42
activation suggests a critical function during cytoskeletal reorgani-
zations, which lead to the redistribution of actin-anchored AChRs.
C1-18
Cell-specific control of Wnt target genes: role of
epigenetic modifications and differential
promoter binding by TCFs
A. Hecht, S. Woehrle and B. Wallmen
University of Freiburg, Freiburg, GERMANY
The recurrent use of a limited number of signaling systems in
embryonic development poses the fundamental question of how
the same effector molecules can generate distinct tissue-specific
responses. Canonical Wnt signaling provides a highly attractive
model system to address this problem. It performs important and
widespread functions during ontogenesis, yet, it engages only beta-
catenin and members of the TCF family of DNA-binding proteins
to control a large variety of ubiquitous and tissue-specific target
genes. TCF proteins are considered to act as bimodal switches in
both the activation and the repression of their target genes.
Accordingly, they are believed to remain constantly bound to tar-
get promoters. However, constant promoter occupancy by TCF
proteins does not readily explain how distinct groups of Wnt target
genes can be differentially regulated in a cell-type specific and
developmentally controlled manner. Here, we report a systematic
comparison of Wnt-responsiveness, TCF promoter occupancy and
epigenetic status of known Wnt/beta-catenin targets in different

cell types. Analysis of DNA methylation patterns and histone
modifications at promoter regions revealed that Wnt-inducibility
correlates with DNA hypomethylation and active histone marks.
In contrast, non-responsive promoters showed hypermethylation
and repressive histone modifications. Moreover, Wnt-responsive-
ness correlates with differential promoter occupation by TCFs.
Notably, in contrast to current models, TCF factors are not pre-
sent on promoter regions of non-responding genes. We hypothesize
that distinct promoter occupancy by TCF proteins and epigenetic
control mechanisms form a multi-layered control system to achieve
differential regulation of Wnt target gene expression.
C1-19
Canonical Wnt signalling and Groucho proteins
affect left-right asymmetry
B. Bajoghli
1
, N. Aghaallaei
1
, D. Soroldoni
1
and T. Czerny
1,2
1
University of Veterinary Medicine Vienna, Vienna, AUSTRIA,
2
University of Applied Sciences, FH-Campus Wien, Vienna,
AUSTRIA
Groucho/Tle proteins constitute a family of highly conserved co-
factors for transcription. Interaction of these corepressor proteins
with Tcf/Lef transcription factors leads to repression of target

genes in absence of canonical Wnt signalling activity. Expression
of the short family member Aes interferes with this corepressor
function of Groucho/Tle proteins. We misexpressed Aes during
embryonic development of medaka fish and found effects on optic-
and otic vesicle outgrowth. In addition we observed laterality
defects during heart development. A closer inspection of left-right
asymmetry revealed effects of both Groucho/Tle proteins and
canonical Wnt signalling on asymmetrically expressed TGFbeta
family members in the axial mesoderm. Both Aes and Wnt1 ectopi-
cally activate Lefty and Spaw genes, resulting in bilateral expres-
sion and consequently leading to laterality defects in organ
formation.
When we looked at earlier events during left-right assignment we
however found different roles for the two pathways. Blocking
Groucho function strongly affected the formation of the Kupffer¢s
vesicle (equivalent to the ciliated part of the mouse node) and
expression of the cilia marker Lrd. Interestingly at this stage
canonical Wnt signalling neither affected Lrd expression, nor
Kupffer¢s vesicle formation. Therefore Groucho/Tle proteins regu-
late left-right patterning at two different levels of the pathway,
whereas canonical Wnt signalling specifically acts during later sta-
ges of left-right development.
C1-20
PLA
2
is important in phosphatidic acid-induced
Bcl-2 expression through ERK1/2 MAPK
activation
H. Choi and J. Han
Department of Biochemistry and Molecular Biology, College of

Medicine, Hanyang University, Seoul, REPUBLIC OF KOREA
Phosphatidic acid (PA), the product of PLD-mediated reaction, is
lipid second messenger that participates in various intracellular
signaling events and regulates a growing list of signaling protein.
In this study, we tried to find out the mechanism of Bcl-2 up-regu-
lation by diC8PA treatment in HeLa cell. Treatment with diC8PA
resulted in significantly increased expression of Bcl-2 in HeLa cell.
Moreover diC8PA-induced Bcl-2 expression was blocked by mepa-
crine, an inhibitor of phospholipase A
2
(PLA
2
), whereas enhanced
rather by propranolol, an inhibitor of diC8PA phospholydrolase
(PAP). Treatment of 1,2-Dipalmitoryl-sn-Glycero-3-phosphate
(DPPA) instead of diC8PA also increased Bcl-2 expression indica-
ting that Bcl-2 expression is mediated through lysophosphatidic
acid (LPA), not through arachidonic acid. To investigate the rela-
tion between ERK1/2 MAPK pathway and Bcl-2 expression, we
used MEK1/2 inhibitor, PD98059. Pretreatment with PD98059
decreased diC8PA-induced Bcl-2 expression in HeLa cell, indica-
ting that ERK1/2 MAPK was closely related with Bcl-2
expression. In addition, the ERK1/2 MAPK is also associated with
LPA-induced Bcl-2 expression. When treated with PD98059,
ERK1/2 MAPK activation and LPA-induced Bcl-2 expression
were inhibited. Taken together, PLA
2
is important in diC8PA-
induced Bcl-2 expression by producing LPA which is involved in
ERK1/2 MAPK activation.

Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 137
C1-21
Inhibitors of protein kinase A, protein kinase C
and MAP kinases enhance the IL-13-induced
expression of IL-6 by nasal polyp fibroblasts
S. D. Athanasiou
1
, T. Stathas
2
, P. Goumas
2
, S. Naxakis
2
,
E. Giannopoulou
3
and A. J. Aletras
1
1
Laboratory of Biochemistry, Department of Chemistry, University
of Patras, Patras, GREECE,
2
Department of Otolaryngology, Med-
ical School, University of Patras, Patras, GREECE,
3
Department of
Pharmacology, Medical School, University of Patras, Patras,
GREECE
Nasal polyposis is a chronic inflammatory disease of the nasal mu-

cosa that is characterized by inflammatory cell infiltration, modifi-
cations of epithelial differentiation, basement membrane
thickening, extracellular matrix accumulation, and oedema. IL-13
is a cytokine, generated by Th2 cells, implicating in the pathogene-
sis of various diseases characterized by fibrosis. It modulates the
collagen homeostasis, enhances the TIMP-1 and inhibits the IL-1b-
induced MMP-1 and -3 production by skin fibroblasts, and up-
regulates the expression of b1 integrin, VCAM-1, IL-6 and MCP-1
in lung fibroblasts.
In this study the effect of IL-13 on the fibrotic factor IL-6 produc-
tion by nasal polyp fibroblasts and he signal transduction pathway
mediating this effect, are investigated. Polyp fibroblasts in culture
expressed IL-13 receptors. ELISA and RT-PCR showed that IL-13
up-regulated the IL-6 expression in a dose and time-dependent
manner and this effect was not mediated by TGF-b1. RT-PCR
showed that IL-13 did not affect the expression of TGF-b1 and its
receptors. Using specific inhibitors it was found that the inhibitors
of protein kinases A and C, and of ERKs and JNKs enhanced the
stimulatory effect of IL-13, while the inhibitors of cyclooxygenases,
tyrosin kinases and NF-jB activation strongly suppressed this
effect.
C1-22
Endothelin-1 activates Glut1 transcription via
both PKC’s and MAPK signaling pathways
Y. Kao and J. C. Fong
National Yang-Ming University, Taipei, TAIWAN
We have demonstrated previously that endothelin-1 (ET-1) may sti-
mulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes
via both protein kinase C (PKC) - and p42/p44 mitogen-activated
protein kinase (MAPK)-dependent pathways. In the present study,

we further explored the molecular mechanism involved. Our results
indicate that both novel PKCe- and MAPK-dependent pathways
are involved in ET-1 activation of Glut1 transcription and there is
no interaction between PKCe and MAPK at the kinase activity
level. By using deletion and mutation constructs of luciferase repor-
ter driven by Glut1 promoter with enhancers 1 and 2, we were able
to identify NF-jB and Sp1 binding sites on enhancer 2 as the ET-1
response elements. In concord, chromatin immunoprecipitation and
co-immunoprecipitation experiments demonstrated that in cells
treated with ET-1, NF-jB and Sp1 may form a binding complex
bound to enhancer 2. While nuclear contents of both NF-jB and
Sp1 were increased by ET-1, only the increase in Sp1 required de
novo protein synthesis. In addition, we provide evidence that ET-1-
induced Sp1 expression requires both PKCe- and MAPK/CREB-
dependent pathways, whereas activation of NF-jB by ET-1 is
mediated by a PKCe/reactive oxygen species (ROS) cascade. Taken
together, our results strongly suggest that by activating NF-jB via
PKCe/ROS cascade and increasing Sp1 expression through both
PKCe- and MAPK/CREB-dependent pathways, ET-1 may activate
Glut1 transcription by inducing interaction between nuclear NF-jB
and Sp1 as well as their binding to enhancer 2.
C1-23
Effect of growth factors on
acetaminophen-induced liver injury
T. Nam, H. Hwang and I. Kim
Pukyong National University, Busan, REPUBLIC OF KOREA
The growth factors (IGF-I and EGF) are involved in protecting
against chemotherapeutic drug-induced cell death in human hepa-
toma cells. Acetaminophen (AAP) hepatotoxicity is a leading cause
of liver failure and the prevention of AAP-induced cell death has

been the focus of many studies. We examined whether two growth
factors, IGF-I and EGF, could protect against AAP-induced cell
death and investigated the protective mechanism involved. Based
on the results of MTS assays, Hoechst 33342 cell staining, and
DNA fragmentation experiments, AAP induced cell death in a
dose-dependent manner. According to Western blot analysis, treat-
ment with AAP increased the level of poly (ADP-ribose) polym-
erase (PARP) fragments in cells compared to that in control cells,
and caspase-3, a key signaling molecule in apoptosis, was activated
after AAP treatment. Combined treatment with AAP and IGF-I,
or EGF inhibited caspase-3 activation and PARP cleavage, consis-
tent with the ability of growth factors to restore the level of gluta-
thione (GSH) and cell viability in GSH and MTS assays,
respectively. We investigated whether the protective effect of
growth factors against AAP cytotoxicity was related to MAPK
signaling, which was involved oxidative stress, and Fas signaling,
detected growth factors inhibit AAP cytotoxicity through MAPK
signaling. Thus, MAPK is involved in the protective effect of
growth factors against AAP-induced cell death.
C1-24
Role of PKC
e
in Gelsolin expression by histone
deacetylase inhibitor apicidin in human cervix
cancer cells
Y. Jeon
1,2
, J. You
1,3
, J. Park

1
, W. Choi
4
, H. Lee
2
and J. Han
1
1
Sungkyunkwan University, Suwon, REPUBLIC OF KOREA,
2
Kon-
yang University, Daejeon, REPUBLIC OF KOREA,
3
Konkuk Uni-
versity, Chungju, REPUBLIC OF KOREA,
4
College of Medicine
and CBITRC, Konkuk University, Chungju, REPUBLIC OF
KOREA
Down-regulation of gelsolin expression is associated with cellular
transformation and induction of gelsolin exerts antitumorigenic
effects. In this study, we show that protein kinase C (PKC) signa-
ling pathway is required for the induction of gelsolin by the his-
tone deacetylase inhibitor apicidin in HeLa cells. Apicidin induces
gelsolin mRNA independently of the de novo protein synthesis.
Inhibitor study has revealed that the PKC signaling pathway is
involved in the gelsolin expression. Furthermore, inhibition of
PKCe by either siRNA or dominant-negative mutant completely
abrogates the expression of gelsolin by apicidin, indicating that
PKCe is the major isoform for this process. In parallel, apicidin

induction of gelsolin is antagonized by the inhibition of Sp1 using
dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and
inhibition of PKC leads to suppression of Sp1 promoter activity.
Our results provide mechanistic insights into molecular mecha-
nisms of gelsolin induction by apicidin.
Abstracts Signal Transduction
138 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-25
Prion protein at the interface of MAPK and TGF-
beta signaling
S. Wurm and C. Wechselberger
Upper Austrian Research GmbH, Linz, AUSTRIA
Members of the transforming growth factor-beta (TGF-beta)
superfamily control a multitude of cellular processes, including cell
growth, differentiation and apoptosis. On the other hand, TGF-
beta’s have also been shown to act as tumor-promoting cytokines,
underscoring the complexity of this signaling network. Conven-
tional TGF-beta signaling involves a heteromeric transmembrane
receptor complex which leads to the phosphorylation of intracellu-
lar Smad proteins and finally to the transcriptional regulation of
target genes. Auxiliary, MAPK cascades have been described to be
activated by TGF-beta and furthermore to modulate the conven-
tional Smad response.
Previous studies have shown that also GPI-anchored proteins can
influence TGF-beta signaling. Now we were able to reveal for the
first time by co-immunoprecipitation in HEK293 cells that also pri-
on protein (PrP), a GPI-linked protein whose physiological roles
are still poorly defined, interacts directly with members of the
TGF-beta receptor complex. We have already shown that PrP
modulates both, the conventional as well as the MAPK-pathways

in TGF-beta activated mouse mammary epithelial cells. Recent
findings corroborate the assumption that PrP can block the p42/44
MAPK pathway, e.g. stimulated by EGF, another key regulator of
cell growth and differentiation. In response, endogenous TGF-
beta1 production is enhanced in cells over-expressing PrP. As
TGF-beta as well as EGF signaling is often disturbed in tumori-
genesis, PrP could fulfill an unexpected task during cancer develop-
ment.
C1-26
Supportive evidence of desmin’s role in TGF-ß
signaling and early cardiomyogenesis
C. Fuchs, M. Stary and G. Weitzer
Max F. Perutz Laboratories/Medical University of Vienna, Vienna,
AUSTRIA
Desmin is a type III intermediate filament protein and contributes
to the stability of the myocardium. Desmin specifically supports
fusion of myoblasts and the commitment and differentiation of
cardiomyocytes in myogenesis and cardiomyogenesis. Expression
of brachyuri and nkx2.5 is modulated by desmin. For our study,
we used murine embryonic stem cell derived embryoid bodies
(EBs) where cardiomyogenesis is faithfully recapitulated.
Constitutive expression of desmin in EBs results in an enhanced
expression level of the TGF-ß family member nodal, and shows up-
regulation of islet-1, sparc and nkx2.5. Vice versa, des
-/-
EBs show
a decreased expression level of nodal, islet-1, sparc and nkx2.5
compared to wild type. We tested the influence of desmin in TGF-
ß signaling by using an inhibitor of ALK4 receptor, SB 431542. In
wild type and des

-/-
EBs, expression of brachyuri, islet-1 and nkx2.5
were downregulated by SB 431542 whereas desmin overexpression
in SB 431542 treated EBs rescued expression of brachyuri, islet-1
and nkx2.5.
These results strongly suggest a role of desmin in TGF-ß signaling
and early cardiomyogenesis.
C1-27
Dynamics of S100A11 protein in human
myoblasts after stimulation of differentiation
A. Makarov, L. Kovalyov, K. Lisitskaya and S. Shishkin
Bakh Institute of Biochemistry, Moscow, RUSSIAN
FEDERATION
Satellite cells participate in muscle regeneration and hypertrophy.
Proliferation and differentiation of satellite cells are regulated by a
number of growth factors, including TGF-beta. In this study
human myoblasts were cultured in the F-12 media containing
sodium pyruvate, gentamicin and fetal calf serum. Differentiation
of the myoblasts was induced by incubation in differentiation
media (containing 2% of horse serum). Using 2D-PAGE it was
shown significantly decreasing of protein fraction with molecular
weight 11 kDa and isoelectric point 6.1. By MALDI-TOF mass-
spectrometry this protein has been identified as S100A11 calcium
binding protein (calgizzarin).This protein has been described as
one of the messengers involved in signal transduction from TGF-
beta receptor (Sakaguchi et al, 2004). We believe down-regulation
of this protein can be one of the factors dependable for decreasing
of myoblast sensitivity to TGF-beta during myogenic differenti-
ation.
C1-28

Studying the role of tissue transglutaminase in
neutrophil granulocyte differentiation
K. Csomos, Z. Balajthy, G. Zahuczky and L. Fesus
University of Debrecen, Debrecen, HUNGARY
Neutrophils begin their differentiation in bone marrow and become
matured granulocytes in the circulation system. The proliferating
myeloid cells do not contain tissue transglutaminase (TG2) but dur-
ing their differentiation this enzyme is induced and large amount of
this protein is present in matured cells. So far, its exact role in neu-
trophil differentiation has remained unclarified. All-trans retinoic
acid treated NB4 promyelocyte cells provide an appropriate model
system to study neutrophil differentiation. Lentivirus based anti-
TG2 shRNA expression vector was used to establish stabile TG2
knockdown NB4 cell line. The examination of the normal and TG2
knockdown NB4 differentiation revealed that the enzyme is
involved in the regulation of several genes (i.e. gp91 phox) which
are related to neutrophil granulocyte function. These results and
the phenomenon that the enzyme translocates to the nucleus in sug-
gest that TG2 modulates gene expression.
In order to find the genes which are modulated by TG2 total gene
expression analysis was performed using DNA microarray. In TG2
knockdown cells the expression of 171 genes decreased and 173
increased at least 2-fold level. Among these identified genes there
are ones involved in neutrophil granulocyte function, transcription
factors and apoptosis related genes.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 139
C1-29
Role of protein L-isoaspartyl
o-methyltransferase in neuronal differentiation

of P19 embryonal carcinoma
S. Hong, S. Lee and S. Hong
Department of Genetic Engineering, Sungkyunkwan University,
Suwon, REPUBLIC OF KOREA
Protein L-isoaspartyl-( D-aspartyl) o-methyltransferase (PIMT, EC
2.1.1.77) is a cytosolic enzyme that methylates the side chain carb-
oxyl group of racemized L-aspartyl or L-isoaspartyl residues in pro-
teinaceous substrates with S-adenosylmethionine (AdoMet) as a
methyl donor. This enzyme is expressed highly in the brain but the
functions of PIMT in it are poorly understood. P19 embryonic carci-
noma cells are pluripotent cells that can undergo irreversible differ-
entiation into derivatives of three germ layers. And P19 cells can be
induced to differentiate into neuron-like cells by treating all-trans
retinoic acid (at-RA). The purpose of this study to investigate the
relationship of PIMT to neurogenesis. After treatment of P19 cells
with atRA, PIMT mRNA level and activity were measured during
neuronal differentiation. After 2 days of t-RA treatment, the PIMT
mRNA levels increased and reached the highest level at day 4 and
maintained the level during the further differentiation. The activity
of PIMT increased by 20% after 2 days of differentiation and
showed similar level during further differentiation. But at day 8 of
neuronal differentiation when P19 neurons become mature, PIMT
activity increased by 79% compared to that of control. These results
suggest that PIMT might be related to neurogenesis and influenced
by the retinoic acid receptor pathway.
C1-30
Interaction between the neurotrophin
receptor target kidins220 and the ERM
membrane-cytoskeleton linker protein Moesin
A. M. Higuero

1
, R. M. Jean-Mairet
1
, J. Vandekerckhove
2
and
T. Iglesias
1
1
Instituto de Investigaciones Biomedicas Alberto Sols, Madrid,
SPAIN,
2
Department of Medical Protein Research, VIB, Ghent,
BELGIUM
Kinase-D interacting substrate of 220 kDa (Kidins220), also
known as ARMS, is a protein predominantly expressed in develop-
ing brain that was originally identified as a protein kinase D
(PKD) substrate and as a downstream target of the neurotrophin
receptors. PKD is a serine/threonine kinase related to the PKC su-
perfamily that serves as a diacylglycerol receptor. One of its best-
characterized functions is its role in regulating the fission of trans-
port carriers from the golgi to the plasma membrane. On the other
hand, neurotrophins are fundamental factors in the development
of the nervous system influencing processes such as neuronal survi-
val, differentiation, synaptic plasticity and axonal and dendritic
ramifications. In order gain insight into Kidins220’s function, we
decided to identify its physiological binding partners. Using a pro-
teomics approach, we identified the cytolinker protein Moesin,
which belongs to the Ezrin/Radixin/Moesin (ERM) family, as a
Kidins220 interacting protein. We confirmed this interaction by co-

immunoprecipitation from hippocampal neurons and PC12 cell ly-
sates, and by colocalization studies. The ERM proteins are key
factors in cytoskeletal processes underlying diverse cellular func-
tions such as cell division, adhesion, migration, morphology and
intracellular signal transduction. In neurons, ERM proteins have
been implicated in developmental growth, morphology and migra-
tion. These results suggest that the ERMs link the plasma mem-
brane protein Kidins220 to the neuronal cytoskeleton and that this
interaction might be relevant for the neurotrophin-induced cyto-
skeletal remodeling.
C1-31
Kidins220: A novel neuronal protein that is
up-regulated during neuroblastoma
differentiation
C. Lo
´
pez-Mene
´
ndez
1
,T.Lo
¨
fstedt
2
, M. Ovenberger
2
, S. Pahlman
2
and T. Iglesias
1

1
Instituto de Investigaciones Biome
´
dicas, Madrid, SPAIN,
2
University Hospital MAS, Malmo
¨
, SWEDEN
Neuroblastoma is a childhood tumor derived from the developing
synpathetic nervous system that retains many characteristics of
immature neural cells. Despite their tumoral origin, neuroblastoma
cell lines can be induced to differentiate in vitro by several agents,
including retinoic acid and phorbol esters. In vitro differentiated
neuroblastoma cells have a neuronal phenotype as judged by their
morphology, extension of neurites and expression of biochemical
and functional neuronal markers. Therefore, neuroblastoma cells
are considered a useful model to study the initial phases of neuron-
al differentiation. Kidins220 (Kinase D interacting substrate of
220 kDa) is a novel trans-membrane protein with unique features
and unknown function. It was first cloned as a physiological sub-
strate for protein kinase D1 and later as a substrate of neurotro-
phin and ephrin receptor. Kidins220 is abundantly expressed in the
nervous system. The localization of Kidins220 at the tips of
extending neurites suggests that it may be participating in proces-
ses such as neuritogenesis and/or neurogenesis. We have cloned the
promoter and first intron of kidins220 gene and identified several
putative regulatory elements for retinoic acid receptors. We have
studied the gene expression pattern of kidins220 in different neur-
oblastoma cell lines under several stimuli that modulate changes in
their phenotype, maturation and aggresiveness.

C1-32
A novel role for hippocalcin in bFGF-induced
neurite outgrowth of H19–7 cells
D. Oh, J. Cho, S. Park and J. Han
Department of Biochemistry and Molecular Biology, College of
Medicine, Hanyang University, Seoul, REPUBLIC OF KOREA
Hippocalcin is a Ca
2+
binding protein which is expressed mainly
in the pyramidal nerve cell of the hippocampus, but the mechanism
and functions underlying hippocalcin in the brain remains unclear.
To elucidate a role of hippocalcin, we utilized a conditionally
immortalized hippocampal cell line (H19–7). We show here that
bFGF-induced hippocalcin expression is involved in neurite out-
growth of H19–7 cells. Increased expression of hippocalcin dramat-
ically elongated neurites induced by bFGF stimulation and was
concurrent with the expression of basic helix-loop-helix (bHLH)
transcription factor, NeuroD. Hippocalcin suppression blocked
bFGF-induced neurite outgrowth and NeuroD expression. Stimu-
lation of bFGF resulted in the activation of phospholipase C-c
(PLC-c) and Ca
2+
. Hippocalcin expression by bFGF stimulation
was fully blocked by both the PLC-c inhibitor, U73122 and a che-
lator of intracellular Ca
2+
, BAPTA-AM, suggesting that hippocal-
cin expression by bFGF stimulation is dependent on PLC-c and
Ca
2+

. Moreover, both U73122 and BAPTA-AM completely
blocked bFGF-induced neurite outgrowth and NeuroD expression.
Taken together, these results suggest for the first time that bFGF
induces hippocalcin expression through PLC-c activation and
Ca
2+
, which leads to neurite outgrowth in H19–7 cells.
Abstracts Signal Transduction
140 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-33
IL6 inhibits RANKL-induced osteoclastogenesis
by diverting cells into the macrophage lineage:
implication of STAT3
V. Trichet, L. Duplomb, M. Baud’Huin, C. Charrier,
F. Blanchard and D. Heymann
INSERM ERI-7, Nantes, FRANCE
Osteoclasts are bone-resorptive cells differentiated from hematop-
oı
¨
etic precursors upon RANKL activation. Some studies demon-
strated that IL6 indirectly up-modulates osteoclastogenesis through
the production of RANKL by osteoblasts. To investigate the direct
effect of IL6 on osteoclast, we used the monocyte cell line RAW
264.7 which differentiates into osteoclast in presence of RANKL.
The addition of IL6 irreversibly inhibited RANKL-induced osteo-
clastogenesis in a dose-dependant manner. Furthermore, IL6
decreased the expression of osteoclast markers but up modulated
macrophage markers. To understand this phenomenon, we focused
on STAT3, the main signaling molecule activated by IL6. Any of
two STAT3 inhibitors used affected the IL-6 effect. However, these

experiments revealed that STAT3 is mandatory for osteoclastogene-
sis. Indeed both inhibitors completely abolished RANKL-induced
osteoclastogenesis of RAW 264.7. We showed that a basal level of
phospho-STAT3 on Serine
727
associated to an absence of phospho-
STAT3 on Tyrosine
705
is essential for osteoclastogenesis. IL6 stimu-
lation induced both phosphorylations, and consequently RAW
264.7 generated macrophages. With AG490 a decrease in Serine
727
-
phosphorylation leaded to an inhibition of osteoclastogenesis.
Finally, we showed that IL6 inhibits osteoclasts differentiation of
mouse bone marrow precursors and human PBMCs. In conclusion,
IL6 inhibits RANKL-induced osteoclastogenesis by diverting cells
into the macrophage lineage, and the activated-STAT3 level and its
form of phosphorylation control osteoclastogenesis.
C1-34
Expression and role of a6 integrin in the early
embryo
A. Dimiropoulos, K. Konstantopoulos, M. Christopoulos and
N. Zagris
Department of Biology, University of Patras, Patras, GREECE
The integrin family of transmembrane proteins composed of het-
erodimers of a and b subunits transfer information from the extra-
cellular environment to the interior of the cell and vice versa. We
studied the expression pattern of a 6 subunit in the early chick
embryo by RT-PCR and immunofluorescence. a6 integrin mRNA

presence was first detectable at the blastula stage (XIII). It was
intriguing to detect the a6A and a6B mRNA splice variants during
the gastrula stage (HH2). a6 immunoreactivity was first detectable
in the epiblast and the hypoblast at the blastula stage, was intense
in the cells ingressing through the primitive streak during the gast-
rula stage (HH3–4) and was weak during the early neurula stage
(HH5). Later in development, immuno- reactivity was prominent in
the brain and the neural tube. The neural crest cells migrating to
the pharyngeal arches and to the eye expressed a6 integrin strongly.
The expression of a6 was strong in lens, was strong in the myotome
in the somites, intense in the myocardium and endocardium in the
heart and in the walls of dorsal aorta and gut. Inhibition of func-
tion of a6 integrin by blocking antibodies indicated that a6 medi-
ates the guided migration of cells and participates in brain and
heart morphogenesis in the early embryo.
Acknowledgements: Supported by grants from the European
Social Fund (ESF), Operational Program for Educational and
Vocational Training II (EPEAEK II) particularly the Program
‘PYTHAGORAS II’ and from the University of Patras (‘K. Kar-
atheodoris’ grant B. 397).
C1-35
Opioids in epilepsy: lessons from prodynorphin
KO mice
C. Schwarzer
1
, S. Loacker
1
, M. Sayyah
1
and H. Herzog

2
1
Medical University Innsbruck, Innsbruck, AUSTRIA,
2
Garvan
Institute for Medical Research, Sydney, AUSTRALIA
Neuropsychiatric disorders are one of the main challenges of
human medicine with epilepsy being one of the most common seri-
ous disorders of the brain. Increasing evidence suggests that neuro-
peptides, particularly the opioids, play an important role in
epilepsy. However, little is known about the mechanism of the
endogenous opioid system in epileptogenesis and epilepsy. There-
fore, we investigated prodynorphin-KO mice (Dyn-/-)in models of
acute seizures, epileptogenesis and epilepsy.
Compared with wildtype littermates (Dyn+/+), Dyn-/- mice
showed a significantly reduced seizure threshold as assessed by tail-
vein infusion of pentylenetetrazole (PTZ). This phenotype could be
rescued entirely by the kappa opioid receptor specific agonist U-
50488, but not the mu opioid receptor specific agonist DAMGO.
The delta opioid receptor specific agonist SNC80 decreased seizure
threshold in both genotypes. Pre-treatment with the kappa selective
antagonist GNTI completely blocked the rescue effect of U-50488.
Consistent with the reduced seizure threshold, Dyn-/- mice showed
faster seizure onset and a prolonged time of seizure activity after ic
injection of kainic acid. In the PTZ kindling model, Dyn-/- mice
showed a significantly faster kindling progression. Three weeks
after local injection of kainic acid into the dorsal hippocampus,
Dyn-/- mice displayed an increased extent of granule cell layer dis-
persion and neuronal loss along the rostro-caudal axis of the ipsi-
and partially the contralateral hippocampus. Our data strongly

support a critical role for dynorphin in the regulation of hippo-
campal excitability, indicating an anticonvulsant role of kappa opi-
oid recepors.
C1-36
The biological significance of novel
CK1-mediated phosphorylation of tau protein
and its associated proteins in rat brain
K. Suzuki, H. Sasaki, F. Kawakami and K. Ohtsuki
Kitasato University, Sagamihara, JAPAN
The purpose of this recently, we reported that casein kinase 1
(CK1) phophorylates two functional basic proteins [myelin basic
protein (MBP) and tau protein (TP)] in the presence of two sulfat-
ed lipids [sulfatide and cholesterol-3-sulfate (SCS)] in vitro. How-
ever, the physiological significance of the CK1-mediated high
phosphorylation of these two SCS-BPs at the high level of SCS in
an aged brain remains to be elucidated. Therefore, the present
study has been carried out to characterize the SCS-dependent
phosphorylation of TP and its associated proteins by CK1 in the
TP fraction from rat brain.
By the obtained result it was found that (i) in the presence of SCS,
CK1 phosphorylated TP and its associated proteins (p82 and p55)
in the partially purified TP fraction from rat brain; (ii) both PKC
and CK were detected in the TP fraction; and (iii) p82 and p55
was identified as eIF-4B and syndapin 1, respectively. These results
suggest that the accumulated high levels of SCS preferentially
induces the CK1-mediated high phosphorylation of TP, eIF-4B
and syndapin 1, which are involved in the mechanisms of various
other neuronal diseases including Alzheimer’s disease, and selec-
tively suppresses their phosphorylation by PKC and CK2 in the
high aged rat brain.

Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 141
C1-37
IL-1beta upregulates Smad7 via NFjB activation
in human chondrocytes
C. Bauge
´
1
, J. Attia
1
, S. Leclercq
2
, P. Galera
1
and
K. Boumediene
1
1
Connective Tissue Biochemistry, Caen, FRANCE,
2
Saint-Martin
Private Clinic, Caen, FRANCE
We have previously shown that Interleukin-1b (IL-1b), a key-cytoki-
ne in osteoarthritis (OA) pathology, impairs TGFb signaling,
through TbRII down-regulation and Smad7 up-regulation. This
mechanism could account for the reduced responsiveness of OA
chondrocytes to TGFb and the cartilage breakdown associated with
this disease. The aim of this present study was to investigate the
molecular mechanism underlying the IL-1ß-induced stimulation of
Smad7 in human articular chondrocytes (HAC). HAC were treated

with IL-1ß in the presence of TGFß1, PDTC (a repressor of NFjB
pathway) or cycloheximide (a translation inhibitor). Then, mRNA
steady-state and protein levels were estimated by real-time RT-PCR
and immunocytology. Furthermore, transient transfections of p65
expression vector or siRNA targeted p65 were achieved to define its
effect of this transcription factor on Smad7 expression. We showed
that TßRII overexpression restores TGFß response of HAC. How-
ever, this effect was total only for short time incubation, suggesting
the implication of a subsequent mechanism. Moreover, IL-1b causes
a late induction of the inhibitory Smads, Smad7. This effect is direct
as it does not require de novo synthesis. In addition, we established,
by experiments of gain/loss function, that the up-regulation of
Smad7 by IL-1ß is mediated through NFjB pathway and especially
p65 subunit. These findings enlighten the regulatory process of IL-1b
on Smad7 expression. Understanding the molecular basis for IL-1ß
induction of Smad7 and reduction of chondrocytes-responsiveness to
TGFß provides news insight into molecular mechanisms of OA and
may facilitate identification of novel approaches for its treatment.
C1-39
Transcriptional regulation of the small GTPase
RhoB gene by the transforming growth factor b
signaling pathway
E. Vasilaki, E. Papadimitriou, C. Stournaras and D. Kardassis
University of Crete, Heraklion, GREECE
Small GTPases of the rho family control key biological processes
such as cell growth, apoptosis and actin cytoskeleton organization.
We have shown previously that transforming growth factor b
(TGFb) induced a rapid, non-genomic, activation of RhoA and
RhoB in Swiss 3T3 fibroblasts and that this activation was associ-
ated with actin cytoskeleton reorganization. We now show that

TGFb increases the steady state mRNA levels of RhoB but not of
RhoA gene in HaCaT keratinocytes. This activation was observed
as early as 1 h post-TGFb addition and remained for 24 h. The early
transcriptional activation of RhoB gene by TGFb was abolished
using a specific MEK1 kinase inhibitor suggesting the involvement
of the MEK/ERK pathway in this process. Using adenovirus-medi-
ated gene transfer, we observed RhoB gene induction by Smad2 and
Smad3. TGF-b induced transcriptional up-regulation of the RhoB
gene and actin polymerization were not observed in Smad3
-/-
cells
but both phenotypes were rescued by adenoviral mediated exogen-
ous expression of Smad3. Both the TGFb/Smad and the MEK/
ERK pathways activated the human RhoB promoter via distinct
promoter regions but there was no evidence for functional coopera-
tivity between these two pathways on RhoB gene transcription. Over
expression of RhoB was associated with a decrease in RhoB promo-
ter activity suggesting a mechanism of auto inhibition operating in
RhoB gene regulation. In summary, our findings indicate that TGFb
regulates the function and the expression of the small GTPase RhoB
via non-genomic and genomic pathways and that this dual regula-
tion is important for actin cytoskeleton organization and possibly
for other RhoB-dependent responses.
C1-38
Neurosteroids protect neural-crest derived cells
from apoptosis, tempospatially activating
prosurvival kinases
I. Charalampopoulos
1
, C. Tsatsanis

2
, B. Vergou
1
, I. Alexaki
3
,
E. Castanas
3
, A. Margioris
2
and A. Gravanis
1
1
Department of Pharmacology, School of Medicine, University of
Crete, Heraklion, GREECE,
2
Department of Clinical Chemistry,
School of Medicine, University of Crete, Heraklion, GREECE,
3
Department of Exp Endocrinology, School of Medicine, University
of Crete, Heraklion, GREECE
We have recently shown that neurosteroid dehydroepiandrosterone
(DHEA) at 1 nM protects from apoptosis neural crest-derived
PC12 cells, via G protein-associated specific membrane binding
sites and subsequent activation within minutes of prosurvival tran-
scription factors CREB and NFjB, upstream effectors of the anti-
apoptotic Bcl-2 proteins (Charalampopoulos et al, PNAS 2004;
FASEB J 2006). We now describe the signalling pathways,
involved in the transduction of the neuroprotective effects of
DHEA. Specifically, we have the following data: (i) Wortmannin,

PD98059 and PP2, inhibitors of prosurvival kinases PI3K, MEK1/
2 and Src respectively, completely blocked the anti-apoptotic
effects of DHEA in serum-deprived PC12 cells; (ii) the three kinase
inhibitors completely reversed the induction by DHEA of the anti-
apoptotic proteins Bcl-2 and Bcl-xL, as well as the activation of
prosurvival transcription factors CREB and NFjB; (iii) DHEA at
10 nM induced within minutes the phosphorylation of ERK1/2/
MEK1/2, PI3K/Akt and Src kinases in serum-deprived PC12 cells;
(iv) the effect of DHEA on prosurvival kinase activation was mim-
icked by non permeable DHEA-BSA conjugate and was reversed
by Pertussis toxin and glucocorticoids and androgens, suggesting
the involvement of recently described DHEA specific membrane
binding sites. These findings suggest a strong neuroprotective role
for neurosteroids during brain development and ageing.
Acknowledgement: Supported by a grant from the GGET-
PENED03-ED372.
C1-40
Expression and function of arylhydrocarbon
receptor in growth plate chondrocytes
M. Widerak, K. Tahiri, M. Dumontier, M. Corvol and
J. Savouret
Inserm UMR-S 747 Universite
´
Paris 5, Paris, FRANCE
Articular (AR) and growth plate (GR) cartilage is in a physiologi-
cal state of variable hypoxia, which may be altered by inflamma-
tion or trauma. The Aryl hydrocarbon receptor (AhR) is activated
by xenobiotic ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) or benzo(a)pyrene (tobacco tar fraction). The expression
and function of AhR in cartilage are not known and this is the

aim of the present study. Quantitative RT-PCR experiments show
that AhR mRNA is present as non measurable traces in AR and
GR chondrocytes in basal conditions. Basal expression of AhR
was robustly induced by Interleukin-1beta (IL-1B, 20h) in GR cells
cultured in normoxia (21% oxygen) and to a minor extent in hyp-
oxia (0.5% oxygen). IL-1B was inefficient on AR cells. We used
the endogenous gene cytochrome P450–1A1 (CYP1A1) to monitor
the functionality of AhR in chondrocytes. AR and GR chondro-
cytes did not respond to TCDD treatement (20 h) in basal condi-
tions regardless of oxygen pressure. In GR cells only, the increase
in AhR expression by IL-1B stimulated the expression of CYP1A1
which was further increased after TCDD treatement, in normoxia
but not in hypoxia. These observations show that AhR expression
and functionality is restricted to GR chondrocytes in normoxic
conditions and cytokine-dependent. Our results also suggest that
AhR ligands may exert disruptive effects on growth cartilage, dur-
ing development and/or inflammatory processes.
Abstracts Signal Transduction
142 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-41
TEGT suppresses stress-induced apoptosis and
shows differential mRNA expression during
early development
S. Cho, J. Kim, J. Kim, H. Choi, B. Kim, S. Kim and E. Lee
Konkuk University, Seoul, REPUBLIC OF KOREA
Successful embryonic development is dependent on the time and
stage-specific expression of proper genes, but information on speci-
fic gene expression during early stages before zygotic gene activa-
tion is limited, especially in pigs. In this study, we compared the
transcript levels among porcine immature, in vitro-matured porcine

oocytes and 2–4 cell stage embryos cleaved after in vitro fertiliza-
tion. Using annealing control primer (ACP)-based Gene Fishing
PCR, we detected 56 different bands showing differential transcript
level and identified nine genes such as KCRF, CAMSAP1, SMP1,
FLJ20647, LOC132321, NADH1, NADH6, HERC3, and TEGT.
Different from other 8 genes, TEGT was highly expressed at the
immature-stage, and its expression was decreased, while transcript
levels of other eight genes were increased after oocyte maturation.
We originally cloned and sequenced porcine TEGT (ptestis
enhanced gene transcript) gene and found that expression of
pTEGT could suppress the etoposide- or staurosporine-induced
apoptosis by inhibiting caspase activation. Interestingly, ERK
phosphorylation was induced by pTEGT expression. The anti-
apoptotic function of pTEGT was disappeared by treatment of
PD98059, a specific MEK1 inhibitor. The transgenic mouse over-
expressing TEGT also showed higher ERK phosphorylation.
Taken together, TEGT-induced ERK activation seems to be
important for its anti-apototic effect.
C1-42
An autocrine mechanism leads to productive
effector-coupling of the A
2A
adenosine receptor
in SH-SY5Y neuroblastoma cells
E. Ibrisimovic, C. Nanoff and H. Drobny
Medical University of Vienna, Vienna, AUSTRIA
We have investigated if SH-SY5Y cells which endogenously
express an A
2A
-receptor may serve as a model for the regulation of

A
2A
-receptor signalling in nerve cells. We found that receptor acti-
vation facilitated the release of noradrenalin and that the receptor
molecule was targeted to cell extensions; both findings positively
reproduced previous evidence from brain slices on the role of the
A
2A
-receptor in nervous tissue. In addition, coupling of the recep-
tor to its canonical effector adenylyl cyclase (AC) was contingent
on the exposure of cells to retinoic acid (RA) followed by incuba-
tion with serum-free medium. This treatment led to cell differenti-
ation and to coupling of the A
2A
-receptor to neurotransmitter
release and cAMP formation. We found that the increased cAMP
formation was the consequence of an altered regulation of the cat-
alyst: upon cell differentiation (but not in the proliferative pheno-
type), the regulatory pattern was consistent with the presence of
type I (III) and type V (VI) AC isoforms. Our current hypothesis
is that RA induced AC-sensitization via an autocrine mechanism
whereby secreted soluble factors affect the responsiveness of aden-
ylyl cyclase. Despite a heightened activation of AC, the receptor
dependent facilitation of noradrenalin was not mediated by cAMP
but by a G-protein independent pathway that employs the ARF6
guanine nucleotide exchange factor, ARNO.
C1-43
Structure of a survivin-borealin-INCENP core
complex reveals how chromosomal passengers
travel together

A. A. Jeyaprakash
1
, U. R. Klein
2
, E. A. Nigg
2
and E. Conti
1,2
1
European Molecular Biology Laboratory, Heidelberg, GERMANY,
2
Max-Planck-Institute of Biochemistry, Munich, GERMANY
The chromosomal passenger complex (CPC) is an essential regula-
tor of mitosis. The CPC coordinates multiple chromosomal and
cytoskeletal events, such as the correction of centromere-microtu-
bules attachment, the stabilization of the spindle and the comple-
tion of cell division. In performing these diverse functions, the
complex moves from the inner centromere to the central spindle
during the metaphase-anaphase transition, and finally translocates
to the midbody during cytokinesis. Survivin, Borealin and IN-
CENP are the three components of the CPC that regulate the
activity and localization of its enzymatic component, the kinase
Aurora-B. We have determined the 1.4 A
˚
resolution crystal struc-
ture of the regulatory core of the CPC and explored the require-
ments for targeting the CPC to the central spindle and midbody.
We have engineered structure based mutants to dissect the CPC
into different subcomplexes. siRNA rescue experiments with
mutants reveal that the CPC functions as a single structure unit

and the intertwined structural interactions of the core components
lead to a functional interdependence. Association of the regulatory
‘passenger’ subunits creates a helical bundle, whose composite
molecular surface presents conserved residues essential for central
spindle and midbody localization.
C1-44
Increased stability compensates for lower
heparin-binding affinity of FGF-1 mutants
M. Zakrzewska
1,2
, A. Wiedlocha
2
, D. Krowarsch
1
, J. Otlewski
1
and S. Olsnes
2
1
Faculty of Biotechnology, University of Wroclaw, Wroclaw,
POLAND,
2
Department of Biochemistry, Institute for Cancer
Research, Oslo, NORWAY
FGF-1 is a powerful signaling molecule with a relatively short
half-life in vivo and a denaturation temperature close to physiolo-
gical. It is widely believed that an essential component of the
FGF/FGFR signaling complex is heparin. We gradually intro-
duced stabilizing mutations into the K132E-FGF-1 variant, which
was previously shown to be inactive in DNA synthesis stimula-

tion, probably due to its lower affinity to heparin. We found that
stabilizing mutations of FGF-1 can compensate for the reduced
heparin binding in mitogenic activity in NIH3T3 cells. We
observed gradual increase in thymidine incorporation up to the
level obtained with the wild-type of FGF-1 in the presence of
heparin. Neither construct exhibited any difference, compared to
the wild-type, in binding to FGFR and downstream signaling.
They all exhibited increased half-life in the absence of heparin.
Interestingly, stable mutants with reduced affinity to heparin were,
in contrast to the wild-type, effectively translocated into the cell
in the absence of heparin. Our results suggest that the main role
of heparin in FGF-signaling is to protect this naturally low-stabil-
ity protein against heat and proteolytic degradation and that hep-
arin is not crucial in direct FGF/FGFR interaction.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 143
C1-45
Investigation of the phagocytosis signaling in
tissue transglutaminase deficient macrophages
B. Toth
1
, D. Aeschlimann
2
, L. Fesu
¨
s
1
and Z. Szondy
1
1

University of Debrecen, Debrecen, HUNGARY,
2
Cardiff
University, Cardiff, UK
The rapid and efficient phagocytosis of apoptotic cells plays a cru-
cial role in preventing secondary necrosis and inflammation. Macr-
ophages play a central role in this clearance process. Engulfment
of dying cells initiates cytoskeletal reorganization in macrophages
regulated by small GTP-ases. Tissue transglutaminase is a GTP
binding protein, and also has transamidation function. Our labor-
atory previously has shown that TG2-/- mice has a defect in pha-
gocytosis of apoptotic cells and on long-term autoimmunity is
developed. Here we show, that phagocytosis of TG-/- macrophages
is also defective under in vitro condition and this is related to
altered cytoskeletal reorganization and have a defect in the signa-
ling pathway that lead to rac activation. We have focused on the
signaling events upstream rac and identification of what function
of TG2 plays role in the clearace of apoptotic cells. A series adeno-
viruses have been generated to transduce mouse TG2, crosslinking
deficient TG2, GTP-binding deficient TG2, fibronectin-binding
deficient or secretion deficient enzyme. Using adenoviral gene
transfer, rescue experiments with TG2-/- cells were carried out to
identify whether TG2 is required in intracellular cell signalling
directly controlling cytoskeletal rearrangements or in cell-cell com-
munication.
C1-46
Nitric oxide synthase expression in
synchronized and asynchronous cell cultures
Z. Serfo} zo}
1

,R.Ba
´
tori
2
,M.Ga
´
csi
1
and F. Erdo} di
2
1
Department of Experimental Zoology, Balaton Limnological
Research Institute, HAS, Tihany, HUNGARY,
2
Institute of Medical
Chemistry, University of Debrecen, Debrecen, HUNGARY
Nitric oxide (NO) modulates various cellular events including meta-
bolism, motility, cell survival and apoptosis. We investigated the
expression of NO synthase (NOS) isoforms during the progression
of the cell cycle in synchronized and asynchronous cultures of HU-
VEC, CHO, or HaCaT cells. The NOS immunofluorescence
increased in mitotic state in all the three investigated cell types
exhibiting the most intensive labeling around centromers and mito-
tic spindles. Increased NOS level was detected in cells arrested at
metaphase, compared to that of asynchronous cells by immuno-
blots. In cells released from cell cycle inhibition the NOS level
showed cyclic changes with peak intensities of 7–8 h periods after
the release. In HUVEC, 56 kDa and 112 kDa S-nitrosylated pro-
teins were detected and their nitrosylation level showed similar peri-
odic changes to the NOS level during cell cycle. Upon treatment of

CHO cells with L-NAME, a NOS inhibitor, the number of cells in
prophase increased and distortion of the microtubular structure was
apparent in cells in both interphase and methaphase. L-NAME
induced also apoptotic cell death. The above results suggest that the
NOS/NO pathway play an important role in the regulation of mito-
sis and its influence might be exerted via S-nitrosylation of proteins.
This study was supported by an ETT grant 244/2006 to F.E.
C1-47
Plasminogen structural domains exhibit
different functions when associated with cell
surface GRP78 or VDAC
M. Gonzalez-Gronow, S. Kaczowka, S. Payne, F. Wang,
G. Gawdi and S. Pizzo
Duke University Medical Center, Durham, NC, USA
Plasminogen (Pg) is the precursor of angiostatins, a group of anti-
angiogenic Pg fragments containing lysine- or benzamidine binding
sites inside double looped disulphide structures called kringles. All
five Pg kringles bind lysine, whereas only kringle 5 (K5) binds
benzamidine. In addition, there are two more benzamidine binding
sites in the Pg serine protease domain. Both voltage-dependent
anion channel (VDAC) and the glucose-regulated protein GRP78
are receptors for K5. We found VDAC co-localized with GRP78
on the surface of human prostate tumor 1-LN cells. To differenti-
ate functions of these proteins, either singly or as a complex, we
used human hexokinase I (HK-I) as a specific ligand for VDAC,
and microplasminogen as an specific ligand for GRP78. We identi-
fied a putative sequence in microplasminogen responsible for bind-
ing to the COOH-terminus of GRP78, which appears to be the
third benzamidine binding site of Pg. K5 induces a Ca
2+

signaling
cascade only through VDAC, whereas microplasminogen does it
via GRP78. We demonstrate the existence of a mechanism invol-
ving interaction of HK-I and K5 with VDAC, which may function
to protect cells from apoptosis. We also show evidence suggesting
that GRP78 binds to Pg K5 through a region localized in the
NH
2
-terminus of GRP78, via a mechanism that may keep Pg in an
activation-resistant configuration when it binds to the cell surface.
C1-48
Enantioselective effect of 12(S)-HETE on 3T6
fibroblast growth
J. J. Moreno and D. Nieves
University of Barcelona, Barcelona, SPAIN
12-Lipoxygenase and cytochrome P-450 pathways lead to the for-
mation of two enantiomers of 12-hydroxyeicosatetraenoic acids
(12-S-HETE and 12-R-HETE). Recently, we suggested that 12(S)-
HETE produced by CYP is involved in the 3T6 fibroblast growth
induced by serum and that 12-(S)-HETE as well as 5-(S)-HETE
and 15-(S)-HETE are mitogenic on 3T6 fibroblast (1). In this work
we study the effect of both enantiomer on cell proliferation. Our
results show that only 12-(S)-HETE was able to induce cell growth
and DNA synthesis in 3T6 fibroblast cultures whereas 12-(R)-
HETE was inactive. Furthermore, we observed that mitogenic
effects of 12-(S)-HETE were correlated with the enhancement of
ERK1/2 and AKT phorphorylation whereas 12-(R)-HETE was not
effective on these signal transduction pathways involved in the con-
trol of 3T6 fibroblast growth. Moreover, we observed that these
effects can occur through a specific receptor sensitive to pertussis

toxin but not identified yet.
Acknowledgement: Supported by MEC (BFU2004-04960).
Reference
1. Nieves D, Moreno JJ (2006) J. Lipid Res. 47:2681–2689.
Abstracts Signal Transduction
144 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-49
The 3rd intracellular loop of somatostatin
receptor 5 is crucial for arrestin binding and
receptor internalization
E. Peverelli, G. Mantovani, S. Bondioni, A. Lania,
P. Beck-Peccoz and A. Spada
University of Milan, Milan, ITALY
Somatostatin exerts inhibitory effects on hormone secretion and
cell proliferation by interacting with five different receptors (SST1-
SST5). b-arrestins have been implicated in regulating SST internal-
ization but the structural domains mediating this effect are largely
unknown. The aim of this study was to characterize the intracellu-
lar mechanisms responsible for internalization of human SST5 in
the rat pituitary cell line GH3 and to identify the SST5 structural
domains involved in this process. To this purpose we evaluated by
fluorescence microscopy the ability of wt and progressive C-ter-
minal truncated and 3rd cytoplasmatic loop mutants SST5 to asso-
ciate with barrestin and to internalize under SS28 stimulation. The
truncated mutants were comparable to the wt receptor with respect
to recruitment of barrestin2 and internalization, whereas the third
loop mutants R240W, S242A and T247A showed the abolishment
of arrestin translocation and a significant reduction of receptor
internalization upon SS28 stimulation. Moreover, we evaluated the
ability of simultaneous mutation of these three residues (RST) and

C-terminal truncated receptors to internalize. The progressive trun-
cation of C-terminal tail resulted in a progressive increased inter-
nalization with respect to full-length RST mutant. Our results
indicate the SST5 3rd intracellular loop as an important mediator
of barrestin/receptor interaction and receptor internalization, while
the role of the C-terminal tail would be to sterically prevent beta-
arrestin/receptor interaction in basal conditions.
C1-50
Activation of protease-activated receptors 2
(PAR-2) of HT29 cells by abzymes from breast
milk
G. Barrera, R. Portillo, A. Mijares, M. Rocafull, J. del Castillo
and L. E. Thomas
Instituto Venezolano de Investigaciones Cientificas, Caracas,
VENEZUELA
PAR-2 (Proteinase-activated receptor type2) is highly expressed at
plasma membrane of small intestinal epithelial cells. These
receptors are couple to G-proteins and are activated by proteolytic
cleavage. In addition, breast milk contains catalytic antibodies (ab-
zymes) with proteolytic activity. Secretory immunoglobulin A
(sIgA) from human milk may regulate signal transduction in intes-
tinal cells by cleaving and activating PAR-2, resulting in Ca
2+
mobilization. sIgA was prepared from human milk by ammonium
sulfate precipitation, jacaline affinity chromatography and DEAE-
sepharose chromatography. Purity was evaluated by SDS-PAGE
and immunoblot. Bands of 150, 300 and 450 KDa were detected,
corresponding to single, dimeric and trimeric sIgA conformation,
respectively. F(ab)
2

fragments were obtained from sIgA by pepsine
digestion. The proteolytic activities, for both sIgA and its F(ab)
2
fragments, were evaluated by zymography using casein-bovine
serum albumin as substrate. The association of proteolytic activa-
tion of PAR-2 by F(ab)
2
with intracellular calcium concentration
was evaluated in HT-29 fluorimetric single cell assay. F(ab)
2
increased intracellular Ca
2+
. The Ca
2+
response was inhibited by
pertussi toxin (1 lg/ml, for 4 h), indicating that F(ab)
2
-induced
activation of PAR-2 is mediate through Gi protein. The observa-
tion that IgA-F(ab)
2
from breast milk cleaves and activates intesti-
nal PAR-2 suggests a novel regulation mechanism for this receptor
in neonate intestine.
C1-51
Estrogen-induced vascular lesions formation is
mediated by redox sensitive Id3 signaling
Q. Felty
Florida International University, Miami, FL, USA
Estrogen (E2) is a risk factor for cardiovascular disease presuma-

bly by promoting abnormal proliferative vascular lesions and sub-
sequent thickening of the vasculature. The mechanism by which E2
is involved in the development of this lesion is not clear. We previ-
ously showed that E2-induced DNA synthesis depends on oxidant
signaling. Inhibitor of DNA binding protein 3 (Id3) is a redox-sen-
sitive gene that mediates vascular lesion formation. We propose to
test the concept that estrogen-induced vascular lesion formation is
mediated by redox sensitive Id3 signaling. In this study we exam-
ined whether E2-induced endothelial tube formation depends on
Id3. Endothelial tube formation was significantly inhibited to the
level of control by overexpression of both MnSOD and catalase as
well as co-treatments with ebselen and N-acetylcysteine as deter-
mined by 3-D Matrigel Assay and HUVECs co-cultured with fi-
broblasts. Western Blot analysis and confocal microscopy showed
that E2-induced oxidants increased Id3 phosphorylation. And
RNA interference of Id3 markedly inhibited E2-induced tube for-
mation. In conclusion, early E2 signaling does not require estrogen
receptor genomic signaling because we can inhibit tube formation
by antioxidants. These studies demonstrate that Id3 is an import-
ant signaling molecule in E2 stimulated vascular lesion formation
that may be a useful therapeutic target in the prevention and treat-
ment of vasculoproliferative disorders.
C1-52
Pro- and antioxidant properties of
mitochondria-targeted antioxidant mitoQ
D. S. Izyumov, E. V. Mostovenko, M. V. Korotetskaya and
B. V. Chernyak
A.N.Belozersky Institute of Physico-Chemical Biology, MSU,
Moscow, RUSSIAN FEDERATION
Mitochondria play a key role in production of reactive oxygen spe-

cies (ROS), which take part in signal transduction and cell death.
We have studied influence of mitochondria-targeted antioxidant
mitoQ, which can be accumulated in mitochondria due to its posit-
ive charge. Treatment of HeLa cells with H2O2 induced ROS pro-
duction and cell death. MitoQ greatly suppressed this oxidative
stress and apoptosis at very low concentration but this process
need a long (about 8 days) preincubation. At the same time we
have shown that fluorescent analogue of mitoQ have accumulates
in HeLa during 1–2 h. At high concentrations mitoQ caused signi-
ficant ROS production and apoptosis. Non-toxic concentrations of
mitoQ promoted ROS production and cell death in combination
with low concentrations of H2O2 and these effects were suppressed
by antioxidant N-acetylcysteine (NAC) indicating that toxicity of
mitoQ was determined by its prooxidant properties. Also we have
induced ROS production using Mitotracker Red, a fluorescent dye
which is selectively accumulated in mitochondria as a photosensi-
tizer. Low or high doses of illumination caused secondary ROS
production and apoptotic or necrotic cell death, simultaneously.
MitoQ didn’t affect apoptosis but completely suppressed necrosis.
NAC was less effective than mitoQ. Mild illumination caused
apoptosis, which were insensitive to mitoQ. Strong illumination
caused damage of mitochondria by ROS and necrosis which was
inhibited with mitoQ. Thus we showed pro- and antioxidant activ-
ity of mitoQ.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 145
C1-53
Calcium-dependent interaction of calmodulin
with synapse-associated proteins of the
MAGUK family

M. Konrad
1
, A. Lavie
2
and I. Paarmann
3
1
Max-Planck-Institute for Biophysical Chemistry, Goettingen, GER-
MANY,
2
University of Illinois, Chicago, IL, USA,
3
Max-Planck-
Institute for Brain Research, Frankfurt, GERMANY
Membrane-associated guanylate kinase (MAGUK) homologs have
been identified at cell-cell contact sites in organisms from Dro-
sophila to man. They are multidomain proteins, encompassing at
least one PDZ domain, an SH3 domain, and a guanylate kinase
(GK)-like domain. The subfamily comprising the synapse-associated
proteins (SAPs) SAP90/PSD-95, SAP97/hDlg, SAP102/NE-Dlg,
and PSD-93/Chapsyn-110 contain three amino-terminal PDZ
domains; CASK and its homologs have a calmodulin-dependent
protein kinase (CaMKII)-like domain at the N-terminus; the zonula
occludens proteins ZO-1, ZO-2, and ZO-3 have an extended C-ter-
minal region; p55 and other members of the fourth subfamily con-
sist mainly of the three core domains. Different modes of inter- and
intramolecular interactions are proposed to occur between the SH3
and GK domains and the so-called HOOK region located between
these two domains. The GK domain lacks critical residues in the
ATP binding site and is devoid of enzymatic activity; it appears to

have evolved as a protein-protein interaction module that associates
with a novel class of proteins designated GKAP Comparison of the
1.3 A
˚
structure of the GK domain of human CASK with the struc-
tures of GMP kinases shows important differences in the GMP
binding site. By using surface plasmon resonance spectroscopy we
characterized the high affinity (Kd of 50–200 nM) interaction of cal-
modulin with various MAGUKs, the HOOK region being of critical
importance for complexation. Our findings suggest that calmodulin
could act as a trigger molecule to switch MAGUKs from a closed
to an open conformation where protein binding sites are unmasked.
C1-54
BMI inversely correlates with PKA expression
and activity in adipocytes from lean and obese
subjects
S. Bondioni
1
, G. Mantovani
1
, L. Alberti
2
, C. Invitti
2
, S. Corbetta
3
,
E. Peverelli
1
, A. Lania

1
, P. Beck-Peccoz
1
and A. Spada
1
1
University of Milan, Milan, ITALY,
2
Istituto Auxologico Italiano
IRCCS, Milano, ITALY,
3
Policlinico San Donato IRCCS, Milan,
ITALY
In human adipocytes cAMP-dependent pathway mediates signals
originating from the activation of badrenergic receptors, regulating
important metabolic processes. cAMP effects are mainly mediated
by PKA, that is composed by two catalytic and two regulatory R
subunits. There are four different R subunit genes
(R1A,R1B,R2A,R2B) expressed with a tissue-specific pattern and
with distinct roles. Studies indicate R2B isoform as the most
expressed in mouse adipose tissue while its presence is limited else-
where. In our study, the expression of PKA R subunits was evalu-
ated in human subcutaneous and visceral adipose tissue from 10
lean subjects (BMI < 25) and 55 obese patients (BMI > 30). Real-
time PCR showed that, as in mice, R2B is the most abundant tran-
script, both in obese and normal subjects. A significant negative
correlation was observed between R2B expression levels and BMI,
insulin levels, HOMA-IR, with a positive correlation with adiponec-
tin and adiponectin receptors 1&2 mRNA levels. Both PKA activity
and glycerol release were significantly higher in adipocytes from

lean subjects when compared with those measured in primary cul-
tures obtained from obese patients. This is the first study evaluating
the relative expression of the different PKA isoforms in human adi-
pose tissue. Our results indicating BMI-related differences in R2B
expression suggest that similar differences in PKA activity may
modulate the lipolytic response to badrenergic activation.
C1-55
Voltage-gated calcium channel dependent
intracellular signaling
E. Kobrinsky, S. Thomas and N. Soldatov
National Institute on Aging, Baltimore, MD, USA
The voltage-gated calcium channel is a multi subunit signaling
complex. It is the major voltage-dependent regulator of intracellu-
lar calcium signaling. Coupling of plasma membrane voltage chan-
ges to intracellular signaling such as plasma membrane protein
kinase C activation and cAMP dependent transcription in nuclei is
important, but not well defined signaling event in excitable cells.
By combining FRET microscopy with patch clamp in recombinant
expression system we were able to show the importance of voltage-
dependent conformational changes of the pore-forming a
1C
subunit
of the Ca
v
1.2 calcium channel in excitation-transcription coupling.
We developed a novel 2D wavelet-based image analysis to decipher
calcium channel -dependent signaling pathways. The combination
of pixel-by-pixel and 2D wavelet analysis allowed us to reveal a
microdomain organization of the calcium channel plasma mem-
brane-activated PKC signaling and intranuclear activation of

cAMP dependent transcription. This approach may serve as a
framework for intracellular signaling analysis.
C1-56
Liposomes as possible carriers for
anti-inflammatory and antitumo ral compounds
A. M. Roseanu, F. Chelu, M. Moisei and M. Trif
Institute of Biochemistry, Bucharest, ROMANIA
Liposomes are efficient carriers for controlled drug delivery and
local targeting of therapeutic agent to the site of interest.
Lactoferrin (Lf) is an iron-binding glycoprotein with potent anti-
inflammatory and antitumoral properties. The aim of our studies
was to investigate whether the entrapment of Lf in liposomes could
improve its anti-inflammatory and antitumoral effects. The experi-
ments were performed in vitro, using human monocytic THP-1
cells and murine melanoma B16-F1 cells. Previous studies demon-
strated that Lf entrapped in liposomes is accumulated by the cells
more efficiently than the free protein. Liposomal formulation
increased the capacity of Lf to affect B16-F1 cell growth and to
induce morphological modifications associated to apoptosis, such
as rim of the cytoplasm, nuclear condensation and fragmentation,
appearance of apoptotic bodies. The mechanism is suggested to
involve modulation of the expression of JNK, p-38, ERK 1/2
MAPkinase, proteins implicated in cell proliferation and apoptosis.
In the case of THP-1 cells, entrapment of Lf in liposomes
enhanced the protein ability to reduce pro-inflammatory cytokines
IL-6, TNF-a and IL-8 release mediated by LPS. Our results
revealed the property of liposomes to enhance the intracellular
activity of Lf and suggest that liposomal protein may have poten-
tial therapeutic use in the prevention and/or treatment of inflam-
matory and cancer diseases.

Acknowledgements: This work was supported by MATNAN-
TECH research program, project CEEX 57/2006(NANOCON-
TER).
Abstracts Signal Transduction
146 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-57
Investigation of rapid nongenomic effects of
1a,25(OH)2D3 on intracellular calcium in human
peripheral blood mononuclear cells
I. Lajdova
1
, D. Chorvat
2
, V. Spustova
1
and A. Chorvatova
3
1
Slovak Medical University, Bratislava, SLOVAKIA,
2
International
Laser Centre, Bratislava, SLOVAKIA,
3
University of Montreal,
Montreal, PQ, CANADA
Steroid hormone 1a,25(OH)2D3 (D3) acts via both slow, genomic
and rapid, nongenomic mechanisms, yet we still lack knowledge
about pathways implicated in rapid actions of the hormone. Here,
we examined nonenomic effects of D3 on intracellular calcium
mobilization and entry in resting human PBMC from healthy vol-

unteers. D3 induced biphasic increase in intracellular calcium con-
centration, determined using Fluo-3 fluorescent probe. Initial
D3-stimulated calcium rise was sensitive to thapsigargin, indicating
its originates in calcium release from intracellular stores. 2APB, an
inhibitor of capacitative calcium entry, significant decreased
[Ca
2+
]i in PBMC treated with D3 and abolished the biphasic
response, while nifedipine had no effect on the D3-induced calcium
entry. These findings suggest that D3 promotes two-step calcium
response through calcium release from internal stores, followed by
store refilling via capacitative, but not L-type calcium channels.
Besides, D3 prevented calcium entry induced by BzATP, specific
agonist of P2X7 receptors and reduced 4AP-stimulated [Ca
2+
]i
increase. D3 also reduced BzATP and 4AP-stimulated ethidium
bromide fluorescence, confirming inhibitory effect of the hormone
on calcium influx through P2X7 channel. Presented results demon-
strate for the first time that, in healthy human PBMC, D3 induces
rapid biphasic effect on intracellular calcium, while inhibiting per-
meability of P2X7 channel.
Acknowledgements: This work was supported by Slovak
Research and Development Agency under the contract No APVT-
21-033002 and No APVT-21-019702.
C1-58
The homeodomain factor Xanf can bind with
the LIM-domain protein Zyxin in early
development of the neural plate of Xenopus
laevis

N. Martynova
1
, F. Eroshkin
1
, G. Ermakova
1
, A. Korotaeva
1
,
K. Smurova
2
, F. Gyoeva
3
and A. Zaraysky
1
1
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Moscow V-437, RUSSIAN FEDERATION,
2
Lomonosov Moscow
State University, Moscow, RUSSIAN FEDERATION,
3
Institute of
Protein Research, Pushchino, RUSSIAN FEDERATION
One of the crucial questions of modern developmental biology is
how patterning of an embryo onto cells territories acquiring differ-
ent fates is coordinated with cells morphogenetic movements sha-
ping the embryonic body. By using yeast two-hybrid system, we
have established that a key transcriptional regulator of the anterior
neural plate patterning, the homeodomain factor Xanf1, can

directly bind to Zyxin, which is known to be involved in regulation
of the actin cytoskeleton dynamics. This interaction was confirmed
by the coimmunoprecipitation of Xanf1 and Zyxin from Xenopus
embryos and the co-localisation of proteins expressed in cultured
cells. By using a set of deletion mutants, we have determined pro-
tein domain, responsible for this interaction. In accordance with
these data, we have found that within the anterior neurectoderm,
Zyxin transcription notably enhances at the late gastrula stage, i.e.
just at the place and time where and when Xanf1 is being
expressed. We have investigated also some effects exerted by differ-
ent dominant-negative and dominant-positive mutants of Zyxin on
the early development of the neural anlage. Taken together, these
results indicate that the cytoskeletal protein Zyxin can be involved
in regulation of genes expression in cells of the anterior neural
plate through the interaction with the transcription factor Xanf1.
C1-59
Creation/implementation of clinically relevant
high-grade glioma mouse models for
optimizing chemotherapeutic treatment
N. A. de vries
The Netherlands Cancer Institute, Amsterdam,
THE NETHERLANDS
High-grade gliomas are highly infiltrative and among the deadliest
of human cancers. Chemotherapy failure is at least partly due to
the presence of drug efflux transporters in the blood brain barrier
(BBB) restricting the entry of many potentially useful therapeutic
drugs. Identifying deranged molecular pathways driving glioma
tumor growth resulted in clinical testing of rationally designed
molecular-targeted agents. However, many of these agents are sub-
strates of the drug transporters P-glycoprotein (P-gp) and/or

Breast Cancer Resistance Protein (BCRP). Therefore, appropriate
models for preclinical in vivo evaluation of such agents should
carry the genetic signature of human disease and grow behind an
intact BBB, to predict clinical efficacy more accurately than the
traditional used xenograft models. We have generated spontaneous
high-grade gliomas in mice by using Cre/loxP conditional kRas-
V12;Ink4a/Arf and kRasV12;Ink4a/Arf;Pten mice following stereo-
tactic intracranial injection of a self-deleting lentivirus mediating
astrocyte-specific expression of Cre. Bioluminescence is used to
monitor tumor growth non-invasively. Furthermore cell lines will
be isolated from spontaneous primary tumors. Using these more
‘‘patient-like’’ mouse models we will characterize the status of the
major cell signaling pathways. This information can be used to
investigate the efficacy of (a combination of) agents that target
these pathways. Moreover, by using mice deficient for P-gp and
BCRP we can establish whether it will be useful to combine these
agents with drug-transport inhibitors.
C1-60
Skn7 regulates the formation of germ tube by
binding the promoter of some hypha-specific
genes in Candida albicans
S. Lee
1
, J. Lee
2
and S. Kang
3
1
Laboratory of Biophysics, School of Biological Sciences , Seoul
National University, Seoul, REPUBLIC OF KOREA,

2
Institute of
Microbiology, Seoul National University, Seoul, REPUBLIC OF
KOREA,
3
School of Biological Sciences, Institute of Microbiology,
Seoul National University, Seoul, REPUBLIC OF KOREA
Candida albicans, one of the most frequently isolated fungal patho-
gens of humans, can grow with a variety of morphologies from ye-
asts to hyphae. A putative two-component response regulator gene
SKN7 from Candida albicans and its encoding protein Skn7 was
identified and analyzed. To study the roles of SKN7, we knocked
out SKN7 gene. The skn7/skn7 C. albicans mutants are more sensi-
tive to oxidative stresses, such as H
2
O
2
and menadione, as like
S. cerevisiae skn7 mutants. Also, In the skn7/skn7 disruptants, the
formation of germ tube require shorter time than that in the con-
genic wild-type strain, but the mycelium grow slower in various
liquid media. Compared with the congenic wild-type strain, skn7/
skn7 disruptants show increased transcriptional level of hypha-spe-
cific genes such as HYR1, ECE1, HWP1, and ALS1. Skn7 in
S. cerevisiae was found to bind the heat shock element (HSE) of
the SSA1 promoter. C. albicans Skn7 also contains DNA-binding
domain and the promoters of those genes have HSEs. We showed
that Skn7 can bind to the HSE within the promoters of HWP1
gene. Therefore these results suggested that Skn7 bind the promot-
ers of some hypha-specific and virulence genes to regulate the mor-

phological changes of C. albicans.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 147
C1-61
Anthocyanins inhibit airway inflammation and
hyperresponsiveness in a murine asthma model
S. Park, W. Shin, J. Seo and E. Kim
Korea Institute of Toxicology, Daejeon, REPUBLIC OF KOREA
Asthma is a common chronic inflammatory disease regulated by
coordination of T-helper cell type 2 (Th2) cytokines and inflamma-
tory signal molecules. Additionally, oxidative stress may play an
important role in airway inflammation such as eosinophilia, mucus
hypersecretion, and airway hyperresponsiveness (AHR). In the pre-
sent report, we investigated whether anthocyanins would reduce
airway inflammation in a mouse asthma model immunized and
challenged with ovalbumin (OVA). OVA inhalation elicited inflam-
matory responses characterized by eosinophilia and increased lipid
hydroperoxide (LPO) in bronchoalveolar lavage (BAL) fluid,
enhanced pause (Penh), increased glycoprotein and proliferating
cell nuclear antigen (PCNA) expressions in mucus hypersecretion,
and an increased expression of various cytokines and cyclooxyge-
nase (COX) 2 in lung tissues. All parameters were attenuated in a
dose-dependant manner by the administration of anthocyanins.
These results suggest that anthocyanins may attenuate the develop-
ment of asthma by downregulating Th2 cytokines, proinflammato-
ry cytokines, and COX-2. Our findings suggest that anthocyanins
have positive contributions as a dietary supplement for the preven-
tion of asthma.
C1-62
Phospholipase D is important in Der f 2 induced

expression of IL-8 and IL-13 in human bronchial
epithelial cells
S. Park
1
,J.Oh
2
and J. Han
1
1
Dept of Biochemistry and Molecular Biology, College of Medicine,
Hanyang University, Seoul, REPUBLIC OF KOREA,
2
Department
of Pediatrics, College of Medicine, Hanyang University, Seoul,
REPUBLIC OF KOREA
The purpose of this study was to identify the role of PLD in Der f
2 induced IL-8 and IL-13 expression. The major house dust mite
allergen, Der f 2, stimulates the PLD in human bronchial epithelial
cell line (BEAS-2B). PLD activity was increased within 5 min after
exposure of Der f 2. The well-known PLD activator PKC-a was
found to be translocated to membrane from cytosol in Der f 2
treated BEAS-2B cells. To determine whether the effects of Der f 2
on PLD occurred as a consequence of PKC activation, BEAS-2B
cells were pretreated for 30 min with PKC inhibitor (RO320432).
RO320432 reduced the effects of Der f 2 induced PLD activation
suggesting that PKC-a acts as upstream activator of PLD in Der f
2 treated BEAS-2B cells. Also, the p38 MAPK inhibitor
(SB203580) prevented PLD activation. Der f 2 enhanced IL-8 and
IL-13 expressions in BEAS-2B cells. We found that the expressions
of IL-8 and IL-13 were increased when PLDs were activated with

Der f 2 in BEAS-2B cells. To confirm the role of PLD in IL-8 and
IL-13 expression, we transfected the PLD1 and PLD2, and their
dominant negative forms. Interestingly, we found that only PLD1,
not PLD2, overexpressed IL-8 and IL-13. These results indicate
that Der f 2 might activate PLD through PKC-a activation and
p38 MAPK phosphorylation which induces IL-8 and IL-13 expres-
sion in BEAS-2B cells.
C1-63
Synergistic effects and reversible inhibition of
cAMP-dependent protein kinase catalytic
subunit
A. Kuznetsov and J. Ja
¨
rv
University of Tartu, Tartu, ESTONIA
Asymmetric and synergistic interactions between cAMP-dependent
protein kinase catalytic subunit, its substrates (ATP and kemptide)
and inhibitors (H-89, kemptide Ala-analogue LRRAALG-NH
2
and peptide-nucleoside conjugate inhibitor AdcAhxArg
6
) were
quantified in terms of binding effectiveness of these ligands with
the free enzyme, the enzyme-ATP and enzyme-kemptide com-
plexes. A simple kinetic procedure was proposed for characteriza-
tion of these interactions, by using the second-order rate constants,
calculated from the steady-state reaction kinetics. This procedure
avoids complications related to the complex catalytic mechanism
of the protein kinase catalyzed reaction. It was found that in some
cases synergistic enhancement of ligand binding occurs in the pres-

ence of substrates. This phenomenon is typical for synergistic inter-
action between ligands and the enzyme. The principle ‘‘better
binding - stronger synergism’’ was formulated for cAMP-depend-
ent protein kinase catalytic subunit on the basis of this analysis
and some linear-free-energy relationships between synergistic effect
and ligand affinity were discovered.
C1-64
Ghrelin signaling to ERK 1/2: role of
G-proteins and beta-arrestins
M. Lodeiro
1
, O. Ischenko
1
, A. C. Martini
2
, F. F. Casanueva
1
and
J. P. Camina
1
1
Laboratory of Molecular Endocrinology, Research Area, Complejo
Hospitalario Universitario de Santiago (CHUS) and Department of
Medicine, University of Santiago de Compostela, Santiago de Com-
postela, SPAIN,
2
Physiology Institute, School of Medicine, Cordoba
National Institute, Cordoba, ARGENTINA
Ghrelin, an acylated peptidyl gastric hormone, regulates GH
release, food intake and energy homeostasis and exerts others func-

tions including effects on cell proliferation through the activation
of the MAPK cascade. The signaling pathways associated to the
activation of MAPK were investigated in HEK 293 cells stably
transfected with the ghrelin receptor GHS-R1a. One pathway is
mediated by the barrestins 1 and 2, and requires entry of the recep-
tor into a multiprotein complex with the barrestins, Src, Raf-1,
and ERK 1/2. A second pathway is G
q/11
-dependent and involves
a PKCa/b and Src. A third pathway is G
i
-dependent and involves
PI3K, PKCe and Src. Our study reveals that G
i/o
- and G
q/11
-pro-
teins are crucially involved in the b-arrestin-mediated ERK 1/2
activation.
Acknowledgements: This work was supported by grants from
the FIS and the Instituto de Salud Carlos III, Ministerio de
Sanidad y Consumo and the Secretaria Xeral de Investigacion e
Desenvolvemento, Xunta de Galicia (Spain).
Abstracts Signal Transduction
148 ª 2007 The Authors Journal compilation ª 2007 FEBS
C1-65
Identification of the phosphorylation site of the
histidine kinase of E. coli AtoS-AtoC
two-component system
P. S. Filippou

1
, L. D. Kasemian
1
, C. A. Panagiotidis
2
and
D. A. Kyriakidis
1,3
1
Laboratory of Biochemistry, Department of Chemistry, Aristotle
University of Thessaloniki, Thessaloniki, GREECE,
2
Department of
Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thes-
saloniki, GREECE,
3
The National Hellenic Research Foundation 48,
Vas. Constantinou Ave 11635, Athens, GREECE
The sensor histidine kinase AtoS together with AtoC/Az constitute
a two-component signal transduction system (TCS) in E. coli,
involved in the regulation of the atoDAEB operon. Upon activa-
tion by acetoacetate, AtoS autophosphorylates and subsequently
phosphorylates AtoC which is essential for the transcriptional
regulation of the atoDAEB operon, the products of which are
involved in the catabolism of short-chain fatty acids. AtoS, has the
structural characteristics of an integral membrane protein and
structurally comprises three putative transmembrane domains, a
HAMP, a PAS, a PAC and the catalytic domain of the histidine
kinase. Sequence comparisons with other histidine kinases revealed
the presence of a characteristic ‘‘H-box’’ in AtoS with histidine-398

as a possible phosphorylation site. In the present study, chemical
stability tests of phosphorylated cytosolic form of AtoS, and sub-
stitution of histidine-398 to leucine through site directed mutagen-
esis, pointed towards the direction that histidine is indeed the
phosphorylated residue in AtoS. The alteration of this putative
phosphorylation site has also been demonstrated to affect the bio-
logical activity of AtoS, i.e. its ability to activate AtoC and induce
ato operon expression upon acetoacetate induction.
C2-1
Deciphering the kinome in basal-like breast
carcinoma for therapeutic usefulness
B. Marty
1
, F. Djelti
1
, I. Lebigot
1
, A. Vincent-Salomon
1
,
F. Cruzalegui
2
, G. Tucker
2
, X. Sastre
1
, J. Thiery
1
, J. Hickman
2

and T. Dubois
1
1
Institut Curie, Paris, FRANCE,
2
Institut de Recherches Servier,
Croissy sur Seine, FRANCE
Our objective is to identify alterations in intracellular signaling
pathways to reveal key kinases involved in the progression of
basal-like breast cancers. We investigated the phosphoproteome of
these poor prognostic carcinomas with no targeted therapy using
Western blot (WB) and a technology of reverse phase protein
(RPP) microarray. Data indicated that Akt and mTOR are activa-
ted in the basal-like population. This up-regulated PI3K signaling
pathway could be the result of less PTEN expression that we
observed in these biopsies. In parallel, the signaling pathway pro-
files of basal-like human cell lines (BT20, HCC38 and HCC1937)
was compared to that found in basal-like biopsies. WB analysis
showed high levels of Akt phosphorylation indicating that PI3K
(mutated in 25% breast cancers) pathway is up-regulated in the
three basal-like cell lines. In contrast to BT20, known to express
an active PI3K mutant, the activation of Akt in HCC38 and
HCC1937 resulted from a low/lack PTEN expression. As reported,
EGFR and Met may be over-expressed in basal-like subtype.
Therefore we aim to establish the changes of phosphoproteome in
basal-like cell lines upon stimulation of these receptors. We showed
that EGFR is expressed at higher levels in BT20 compared to
HCC38 and HCC1937. In contrast, Met is expressed at similar lev-
els in the three basal-like cell lines. EGF or HGF induced the
phosphorylation of EGFR and Met, respectively, and other signa-

ling molecules such as ERK, Akt, Src and FAK. Our study may
suggest potential therapeutic targets (proteins) and strategies for
this sub-pathology of breast cancers.
C2-2
P-LAP/IRAP-induced cell proliferation and
glucose uptake in endometrial carcinoma cells
via insulin receptor signaling
K. Shibata, H. Kajiyama, M. Terauchi and F. Kikkawa
Nagoya University Graduate School of Medicine, Nagoya, JAPAN
Hyperglycemia or hyperinsulinemia contributes to poorer
endometrial cancer survival. It was shown that P-LAP/IRAP trans-
locates to the plasma membrane in response to insulin stimulation.
Recently, we demonstrated that P-LAP/IRAP is associated with a
poor prognosis in endometrial adenocarcinoma patients. The aim
of this study was to examine whether the malignant potential of
endometrial cancer enhanced by P-LAP/IRAP is due to increased
glucose uptake via the P-LAP/IRAP-mediated activation of insulin
signaling. We transfected P-LAP/IRAP cDNA into A-MEC cells
(endometrial adenocarcinoma cell line), and A-MEC-LAP cells
expressed a remarkably high level of GLUT4 proteins.
3
H-2-deoxy-
glucose uptake which responds to insulin in A-MEC-LAP cells was
significantly higher than that of A-MEC-pc cells. A-MEC-LAP
cells exhibited a significant growth-stimulatory effect compared to
A-MEC-pc cells. A-MEC-LAP cells expressed a remarkably high
level of p85PI3K protein compared to A-MEC-pc cells, and
showed a higher degree of AKT phosphorylation by insulin stimu-
lation. In summary, P-LAP/IRAP was involved in the increasing
malignant potential of endometrial cancer mediated by insulin.

P-LAP/IRAP was suggested to be a potential new target of
molecular-targeted therapy for endometrial cancer.
C2-3
Ceramide production is involved in
capsaicin-induced apoptosis in the
androgen-independent prostate cancer PC-3
cells
A. M. Sanchez, S. Malagarie-Cazenave, N. Olea, D. Vara and
I. Diaz-Laviada
University of Alcala, Alcala de Henares, SPAIN
In the present study, we determined the effects of capsaicin in the
intracellular ceramide accumulation, and investigated the roles of
extracellular signal-regulated protein kinase (ERK), c-Jun N-ter-
minal kinase (JNK) and p38 signaling pathways in the antiprolifer-
ative effect of capsaicin exerted in the androgen-independent
human prostate cancer PC-3 cell line. Here we report that capsai-
cin apoptotic effect was mediated by ceramide generation which
occurred by sphingomyelin hydrolysis. We next confirmed that
capsaicin could activate ERK and JNK but not p38. Pharmacolo-
gical inhibition of JNK kinase, as well as inhibition of ROS by the
reducing agent N-acetylcysteine, prevented ceramide accumulation
and capsaicin-induced cell death. However, inhibition of ceramide
accumulation by the SMase inhibitor D609 did not modify JNK
activation. These data reveal JNK as an upstream regulator of cer-
amide production. Capsaicin-promoted activation of ERK was
prevented with all the inhibitors tested. We conclude that capsaicin
induces apoptosis in PC-3 cells via ROS generation, JNK activa-
tion, ceramide accumulation and secondly, ERK activation.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 149

C2-4
TCTP induced signaling pathways
M. Kim, J. Jung and K. Lee
Ewha Womans Univ., Seoul, REPUBLIC OF KOREA
Inhibition of Na, K-ATPase has been implicated in the pathogene-
sis of hypertension via its effect on smooth muscle reactivity and
myocardial contractility. In our previous studies, we demonstrated
that translationally controlled tumor protein (TCTP) acts as a
cytoplasmic repressor of Na,K-ATPase and that transgenic mice
over-expressing TCTP developed systemic arterial hypertension.
Thus, TCTP seems to play a key role in maintaining the cells’ ion
homeostasis and dysregulation of its expression may lead to
disease progression, such as hypertension, via repression of
Na,K-ATPase activity. In the present study, we demonstrated
TCTP induced signaling pathways that might be related to the
development of hypertension. TCTP overexpression by adenoviral
system inhibited the Na,K-ATPase activity by less than 50% and
induced Src kinase phosphorylation. Activated Src kinase interac-
ted with both Na,K-ATPase and EGF receptor, transactivated
EGFR, and activated Ras/Raf/MEK/ERK, which were inhibited
by genistein, PP2 and PD98059. In addition, TCTP overexpression
activated EGFR-independent PI3K/Akt, suggesting anti-apoptotic
function for the protein in HeLa cells. Our results suggest that
Na,K-ATPase inhibition by TCTP overexpression activated
EGFR-dependent signaling pathways which might be related
to the pathogenesis of hypertension, and also activated EGFR-
independent pathways which might be related to the anti-apoptosis
of the cell.
C2-5
NPM-ALK induces JUNB and converts its role

from a tumor suppressor to an oncogene
P. B. Staber
1
, P. Vesely
1
, C. Fuchs
1
, S. Schauer
1
,
D. W. Sternberg
2
and G. Hoefler
1
1
Medical University Graz, Graz, AUSTRIA,
2
Mount Sinai School of
Medicine, New York City, NY, USA
The balanced chromosomal rearrangement t(2;5) leading to
nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is associ-
ated with certain human lymphomas and sarcomas. High expres-
sion of CD30 and JunB are a hallmark of NPM-ALK expressing
neoplasm. In contrast to the prototypic AP-1 transcription factor
c-Jun, JunB exerts an antioncogenic function in most cell types. Its
functional role in the context of NPM-ALK remains uncertain.
We found that: 1) Aberrant NPM-ALK expression leads to IL-3
independent outgrowth of Ba/F3 cells. 2) NPM-ALK induces acti-
vation of MEK-ERK MAP kinase pathway. 3) NPM-ALK expres-
sion induces JunB and CD30 expression, which is undetectable in

the corresponding wild type cells and can be reversed by MEK-
inhibition. 4) Interruption of the NPM-ALK kinase domain
impedes JunB and CD30 expression. 5) Specific down-modulation
of JUNB mRNA using small hairpin (sh) RNA avoids CD30
expression and arrests the cell cycle in G1 phase of NPM-ALK
expressing cells. 6) Ectopic JunB expression in NPM-ALK trans-
genic Ba/F3 cells leads to enhanced proliferation in the absence of
IL3, whereas ectopic JunB expression in WT Ba/F3 is not suffi-
cient to provoke IL3 independence and even leads to reduced pro-
liferation in the presence of IL3. Thus, both, NPM-ALK and JunB
are essential to induce CD30 expression and malignant transforma-
tion. The presence of NPM-ALK establishes the oncogenic role of
JunB.
C2-6
Translational control of JUNB via the fusion
tyrosine kinase NPM-ALK in ALC(L) lymphoma
P. Vesely
1
, P. B. Staber
1
, C. Fuchs
1
, S. Schauer
1
, H. Bergler
2
,
D. W. Sternberg
3
and G. Hoefler

1
1
Medical University Graz, Graz, AUSTRIA,
2
Karl-Franzens
University Graz, Graz, AUSTRIA,
3
Mount Sinai School of
Medicine, New York City, NY, USA
Anaplastic large cell lymphomas (ALCL) are highly proliferating
tumors and commonly express the AP-1 transcription factor JunB.
In most cases of ALCL, the fusion tyrosine kinase NPM-ALK is
present and leads to activation of the PI3Kinase/mTOR pathway.
Using EMSA supershift analysis of ALCL cell-lines and NPM-
ALK transduced BA/F3 cells we demonstrate pronounced activa-
tion of JUNB via NPM-ALK. Moreover we reveal that PI3K
inhibition by LY294002 or mTOR inhibition via rapamycin results
in a significant decrease of JUNB protein without affecting its
mRNA level. Downregulation of JunB protein leads to inhibition
of proliferation in NPM-ALK positive cells. To clarify the molecu-
lar mechanism of the JunB regulation via PI3K/mTOR we used
polysomic preparations of ALCL and fibroblast cell lines. Thereby
we found a distinct mechanism regulating JunB translation: JUNB
mRNA is shifted from the polysomic to monsomic and RNP frac-
tions upon serum withdrawal and/or rapamycin triggered inhibi-
tion of mTOR. Moreover, we present a highly conserved motive in
the untranslated region of the JUNB mRNA which is involved in
this regulatory process. Our findings reveal that JUNB is a critical
target of mTOR and highlight its translational deregulation via
NPM-ALK. This is the first study to demonstrate translational

control of a full length AP-1 transcription factor.
C2-7
BRAF
V600E
mutation and oncogenic activation
of MAP kinase by its pseudogene in thyroid
tumors
M. Zou
1
, E. Y. Baitei
1
, A. S. Alzahrani
1
, F. A. Al-Mohanna
1
,
N. R. Farid
2
, B. Meyer
1
and Y. Shi
1
1
King Faisal Specialist Hospital and Research Centre, Riyadh,
SAUDI ARABIA,
2
Osancor Biotech Inc, London, UK
Activating BRAF mutation in papillary thyroid carcinoma (PTC)
has recently been reported in many studies ranging from 28% to
83%. The BRAF mutation has not been studied in the Arab popu-

lation. In the present study, we investigated BRAF mutation from
68 thyroid tumors from Saudi Arabia: 16 multinodular goiters, 43
classic PTCs, six follicular variants of PTC (FVPTC), and three
anaplastic thyroid carcinomas (ATC). BRAF
V600E
mutation was
detected in 20 out of 43 PTC (46.51%), and all the three ATC
(100%). No mutation was found in 16 multinodular goiters, and
six FVPTCs. There is higher frequency of BRAF mutation in clas-
sic PTC patients with stages III and IV tumors (12/16, 75%) as
compared to stages I and II tumors (8/27, 29.63%) (P < 0.05,
Fisher’s exact test). Interestingly, BRAF pseudogene transcripts
were detected in seven of 16 (43.75%) multinodular goiters, 18 of
43 (41.86 %) classic PTCs, and one of six (16.67%) FVPTCs.
There is an inverse correlation between BRAF pseudogene activa-
tion and BRAF mutation. The pseudogene transcripts were more
frequently detected in tumors without BRAF mutation (20/27,
74.37%) than those with BRAF mutation (7/27, 25.93%)
(P < 0.01). Furthermore, overexpression of BRAF pseudogene in
NIH3T3 cells could activate MAP kinase signaling pathway, trans-
form NIH3T3 cells in vitro, and induce tumors in nude mice. These
data suggest that BRAF mutations are specific to classic PTC and
contribute to the disease progression to poorly differentiated and
anaplastic carcinoma. BRAF pseudogene activation may also play
an important role in early stage of thyroid tumor development.
Abstracts Signal Transduction
150 ª 2007 The Authors Journal compilation ª 2007 FEBS
C2-8
Valproic acid modulates cell motility and
MAP-kinase activity in a cell type-specific

manner
K. Gotfryd, G. Skladchikova, E. Lepekhin, V. Berezin, E. Bock
and P. Walmod
University of Copenhagen, Copenhagen, DENMARK
Valproic acid (VPA) is an anticonvulsant, which might be used for
the treatment of cancer. Furthermore, the drug is a known terato-
gen, and both the teratogenic potential and the anti-cancer proper-
ties of VPA may be caused by effects on the proliferation, motility,
survival, and differentiation of cells. We here demonstrate that
VPA caused a reduction in the motility of L929 cells in a manner
correlating with the activity of the extracellular signal-regulated
kinases (ERK) 1 and 2 in the mitogen activated protein (MAP)
kinase pathway. Inhibition of motility could in L929 cells be mim-
icked by the mitogen-activated kinase kinase (MEK) inhibitor
PD98059. Furthermore, the effect could be rescued by overexpres-
sion of constitutively active MEK2 but not by expression of consti-
tutively active Ras, suggesting that VPA affects signalling
downstream of Ras, but upstream of MEK1/2, probably at the
level of Raf. An investigation of a total of ten cell lines revealed
that the expression and activity of Raf proteins, as well as effects
of VPA on cell motility and ERK1/2 activity, were highly cell
type-specific. These data suggest that effects of VPA on cell motil-
ity and ERK1/2 activity may be modulated by cell type-specific
differences in the expression or activity of Raf-A, -B, or -C. This
observation is important for the potential use of VPA as an anti-
cancer drug.
C2-9
In vitro studies of nuclear fraction of leukemic
cells treated with anticancer drug(s) by thermal
technique

M. Rogalinska
1
, P. Goralski
1
, J. Bednarek
1
, J. Z. Blonski
2
,
J. Wesierski-Gadek
3
, H. Piekarski
1
, M. Hanausek
4
, Z. Walaszek
4
,
T. Robak
2
and Z. M. Kilianska
1
1
University of Lodz, Lodz, POLAND,
2
Medical University of Lodz,
Lodz, POLAND,
3
Medical University of Vienna, Vienna, AUSTRIA,
4

University of Texas, San Antonio, TX, USA
Using differential scanning calorimetry (DSC), the in vitro effect of
purine analogs, i.e., cladribine or fludarabine combined with ma-
fosfamide (the active form of cyclophosphamide in vitro)-CM
and FM, and also Campath-1H on B-cell chronic lymphocytic le-
ukemia (B-CLL) cell nuclei was examined. Above agents are
known as potent inducers of apoptosis - the process which is inhib-
ited during development of B-CLL. DSC produces plots of excess
heat capacity as a function of temperature, which resolves the
components of nuclei on the basis of their different thermal trans-
ition of chemically-induced changes in stabilization of nuclear pro-
teins and DNA. For comparison, DSC, cytometric and Western
blot analyses were performed on mononuclear cells isolated from
blood of B-CLL patients. The obtained results revealed the
decrease (or even loss) of endotherm at 95 ± 3 °C in nuclear prep-
arations isolated from leukemia cells. These changes correlated
with reduction of the number of viable cells. The diversities in
expression of some apoptosis-related proteins (members of Bcl-2
family, cytochrome c) were observed. Our results indicate that the
changes in DSC profiles reflect susceptibility of individual patients
and seems to predict the most effective drug-treatment for B-CLL
patients.
C2-10
Pro-apoptotic activity of different purine
derivatives in B-CLL cells
J. D. Bednarek
1
, J. Wesierski-Gadek
2
, M. Rogalinska

1
,
J. Z. Blonski
3
, T. Robak
3
and Z. M. Kilianska
1
1
University of Lodz, Lodz, POLAND,
2
Medical University of
Vienna, Vienna, AUSTRIA,
3
Medical University of Lodz, Lodz,
POLAND
B-cell chronic lymphocytic leukemia (B-CLL) is a disease charac-
terized by an accumulation of long-lived, neoplastic B-lympho-
cytes. Purine analogs (cladribine, fludarabine) have indicated high
activity against the disease and evidence shows that in B-CLL cells
these agents exert their cytotoxic effect by induction of apoptosis.
Recently, another purine derivative, roscovitine, a selective CDK
inhibitor, effective inducer of apoptosis in a large number of can-
cers. The purpose of this study was to determine chemosensitivity
of B-CLL cells to cladribine (C), fludarabine (F), mafosfamide (M)
and roscovitine (ROSC) alone and additionally, to the combina-
tions of C and F with M. The in vitro combinations of C and F
with M are equivalent to that applied in clinical studies, described
as CC and FC program, respectively. Exposure of B-CLL cells to
the distinct agents alone, or in combinations strongly affected their

viability. Treatment with the CM and FM resulted in the highest
reduction of the number of viable cells. Interestingly, similar effect
was observed during the incubation of leukemic cells with ROSC
alone. The tested agents differentially affected expression of apop-
tosis-related proteins (caspase-3, caspase-9, Mcl-1 and Bax) and
their activity status. All studied agents, especially ROSC as well as
both used drug combinations strongly reduced the levels of Mcl-1
protein, increased Bax/Mcl-1 ratio and cyt c translocation. We
conclude that application of ROSC efficiently induces apoptosis of
B-CLL cells, similarly to drug combinations. Since ROSC is not
genotoxic, its application for first line therapy would be of advant-
age.
C2-11
FKHRL1 leads to re-expression of caspase 8 in
neuroblastoma cells
K. Geiger
1
, M. J. Ausserlechner
2
and P. Obexer
1
1
Tyrolean Cancer Research Institute, Innsbruck, AUSTRIA,
2
Molecular Biology Research Institute, Department of Pediatrics,
Medical University Innsbruck, AUSTRIA
Neuroblastoma (NB), a pediatric malignancy of neural crest origin
is the most common solid tumor in children and accounts for
approximately 10% of all childhood cancers. We previously repor-
ted that FKHRL1 (FoxO3a) triggers apoptosis via the mitochon-

dria in human SH-EP and STA-NB15 neuroblastoma cells. Due to
the fact that the majority of aggressive neuroblastoma do not
express caspase 8 as a result of epigenetic silencing by promoter hy-
permethylation, we further investigated the potential function of
FKHRL1 on the extrinsic death pathway in neuroblastoma cells.
For this purpose we retrovirally transduced a 4OH-tamoxifen
(4OHT) inducible FKHRL1(A3)ERtm transgene into neuroblasto-
ma cells. We observed that activated FKHRL1 induces caspase 8
re-expression in caspase 8 deficient neuroblastoma cells. Further-
more we demonstrate that expression of transgenic caspase 8 sensi-
tized for FKHRL1-induced apoptosis. In addition we showed that
activation of FKHRL1 and expression of caspase 8 together appear
to be essential to enhance TRAIL-induced apoptosis in neurobla-
stoma. The combined data indicate that in neuroblastoma caspase
8 is an important regulator in the apoptosis pathway induced by
activated FKHRL1.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 151
C2-12
Equol induces apoptosis through cytochrome
c-mediated caspase cascade in breast cancer
MDA-MB-453 cells
E. Choi and G. Kim
Plant Resources Research Institute, Seoul, REPUBLIC OF KOREA
This study investigated the role of the caspase activation cascade
in extrinsic and intrinsic apoptosis induced by equol in human
breast cancer MDA-MB cells. First, the antiproliferative effect of
equol was determined in cells treated with 1–100 lM equol for 24,
48, and 72 h. Equol significantly inhibited cell proliferation in a
dose- and time-dependent manner (P < 0.05). Exposure to 50 or

100 lM equol for 72 h strongly promoted apoptosis. Under the
same conditions, remarkable cytochrome c release was observed.
Subsequently, caspase-9, which acts in mitochondria-mediated
apoptosis, was cleaved by equol at high concentrations, but cas-
pase-8 activation of receptor-mediated apoptosis was not observed.
At both equol concentrations, the caspase-8 and -9 activity assays
showed similar patterns. In addition, equol treatment activated
caspase-3, which is downstream from caspase-9, and this was
accompanied by the cleavage of capase-6 and -7. Activation of
these caspases leads to increased activation of PARP, lamin, and
ICAD. This study suggests that equol induces the intrinsic path-
way of apoptosis via caspase-9 and cytochrome c, independent of
caspase-8, in human breast cancer MDA-MB-453 cells.
C2-14
Epoxyeicosatrienoic acids induce apoptosis in
3T6 fibroblast cultures through calpain/caspase
cascades
J. J. Moreno and D. Nieves
University of Barcelona, Barcelona, SPAIN
Arachidonic acid (AA) can be metabolized by the epoxygenase
activity of cytochromes P-450 (CYP) producing epoxyeicosatrie-
noic acids (EETs). Finally, the cytosolic epoxide hydrolase catalyze
the hydratation of the EETs to dihydroxyeicosatetraenoic acids
(DHETEs). Besides mitogenic effect, EETs have also been des-
cribed as survival factors. Thus, 14,15-EET inhibited apoptosis
induced by serum withdrawal, H
2
O
2
, etoposide or excess free AA

on renal epithelial cells (1). However, we observed that EETs and
DHETEs inhibit 3T6 fibroblast proliferation induced by PDGF
and induce apoptosis. In this work, we proposed to study the
mechanism by means of induction of apoptosis by EETs/DHETEs.
Our results show that EETs (5,6-EET, 8,9-EET, 11,12-EET, 14,15-
EET) or DHETEs (5,6-DHETE, 11,12-DHETE) (0.1–1 lM) in the
presence of PDGF induce phosphatidylserine externalization
(measured by annexin V-binding), caspase and calpain activities
and DNA fragmentation (quantified using a TUNEL assay). Our
results show that caspase-12 and caspase-3 but not caspase-8 and
caspase-9 are involved in these events. Moreover, calpeptin, a cal-
pain inhibitor, decreases the enhancement of caspase-12 induced
by EETs. Considering that Martı
´
nez and Moreno (2) reported that
EETs were able to induced a marked calcium influx in 3T6 fibro-
blast, we propose that EETs/DHETEs cause apoptosis through
Ca
2+
-dependent calpain-dependent caspase-12 activation.
Acknowledgement: Supported by MEC (BFU2004-04960).
References
1. Chen JK, Capdevila J, Harris RC (2001) Mol. Cell Biol.
21:6322–6331.
2. Martinez J, Moreno JJ (2005) Biochem. Pharmacol. 70:733–39.
C2-13
Induction of cytotoxicity and apoptosis in
HT-29 cells by a gleditsiae semen extract
M. Cha, M. Lee and H. Park
Kyungnam University, Masan, REPUBLIC OF KOREA

Gleditsiae Semen (GS) has been used in both Korea and China as
herbal medicine for the treatment of cephalalgia, catharsis, and
other diseases. However, the apoptosis of GS against human can-
cer cells has not previously been investigated. The primary objec-
tive of this study was to determine the mechanisms inherent in GS-
induced cytotoxicity and apoptosis, using methanolic extract of GS
(GSE) in HT-29 human colon carcinoma cells. We found that
GSE induced cytotoxicity in HT-29 cells in a dose-dependent man-
ner, and this effect was verified via a lactate dehydrogenase release
assay and a colony formation assay. In particular, HT-29 cells
showed extensive cell death when treated with 50 lg/ml of GSE;
the calculated IC
50
value was 20 lg/ml. It induced characteristic
apoptotic signs in HT-29 cells, including chromatin condensation
and DNA fragmentation, occurring within 6–24 h when the cells
were treated at a concentration of 50 lg/ml. Interestingly, we
detected the activation of caspase-3 and -9, but not caspase-8, and
apoptotic bodies in GSE-treated HT-29 cells. Collectively, our
results indicate that GSE induces apoptosis via a mitochondria-
mediated apoptotic pathway, and these findings may be significant
with regard to the development of a new drug for the treatment of
human colon carcinoma cells.
C2-15
Effect of usnic acid on tissue caspase activity
and glutathione level in titanium-implanted
subjects
F. Odabasoglu
1
, H. Aygun

2
, O. S. Yildirim
2
, Z. Halici
3
,
A. Aslan
4
, A. Cakir
4
, M. Halici
1
and E. Cadirci
5
1
Ataturk Univ, Fac of Pharm, Dept of Biochem, Erzurum,
TURKEY,
2
Ataturk Univ., Fac of Med, Dept of Pediatr and
Traumat, Erzurum, TURKEY,
3
Ataturk Univ, Fac of Med, Dept of
Pharm, Erzurum, TURKEY,
4
Ataturk Univ, Fac of Educ, Dept of
Biol and Chem, Erzurum, TURKEY,
5
Ataturk Univ, Fac of Pharm,
Dept of Pharm, Erzurum, TURKEY
Debris due to the frictions as well as biochemical and magnetic

reactions following orthopedic implantations may play a role in
aseptic loosening through initiating a series of complex cellular
reactions between bone and implant. Loosening associates with
increased caspase activity (CAS, protease). The present study was
conducted to evaluate the effect of usnic acid (UA) on CAS and
glutathione level (GSH) in Ti-implanted tissues. Femurs of rabbits
in five groups of total six groups were subperiostally implanted with
Ti. Then, they received UA (30 mg/kg) and olive oil (OO) orally or
locally every 3 day for 21 days or received none. Rabbits from the
other group served as control. Following euthanasia, tissues around
the implant were scrapped and then ground within liquid nitrogen
for CAS and GSH. There were 3, 3.5, 4.5, and 2-fold increases in
activities of CAS 2, CAS 3, CAS 8 and CAS 9 in Ti-implanted rab-
bits compared to control rabbits, which increased further by both
oral and local administrations of UA and OO. Olive oil was more
effective than UA when administered locally, whereas UA was more
effective than OO when administered orally. Surgical intervention
was associated with a 31% reduction in GSH. Local and oral
administration of UA and only local administration of OO elevated
this reduction. In conclusion, both UA and OO stimulate apoptosis
via increasing CAS and increase GSH. Proapoptotic properties of
these compounds should be considered in cancer research.
Abstracts Signal Transduction
152 ª 2007 The Authors Journal compilation ª 2007 FEBS
C2-16
Breast cancer tumor suppressor BRCA1
regulates caspase 3 activation
M. Ouchi
1
, S. Martin

2
, J. Aglipay
2
and T. Ouchi
1
1
ENH, Northwestern University, Evanston, IL, USA,
2
The Mount
Sinai School of Medicine, New York, NY, USA
Deregulation of apoptosis or programmed cell death, can lead to
many human pathologies including cancer. Caspases are the major
regulators of the apoptoic response. Therefore it is reasonable to
suggest the inactivation of the caspase response is a crucial factor
in cancer development. BRCA1, the breast cancer tumor suppres-
sor has previously been shown to be involved in many functional
pathways including apoptosis. However, the precise mechanism of
its tumor suppression remains to be elucidated. Our analysis to
date, suggests that the abrogation of caspase 3 activation following
UV, in the presence of mutant BRCA1 is clinically relevant to the
functional role of BRCA1 as a tumor suppressor. We observe that
inactivation of caspase 3 by unphosphorylated BRCA1 involves
XIAP. Increased activity of the IAP family members has been sug-
gested to provide a survival advantage to cells, in particular cancer
cells therefore indicating their potential as anticancer targets. Inter-
estingly, a number of reports have indicated that caspase 3 defici-
ency is significant in the resistance of breast cancer cells to
chemotherapeutic drugs. Therefore our analysis of caspase 3 acti-
vation status induced by chemotherapeutic agents, and the effect
of BRCA1 on this activation status will provide insight into the

chemoresistance of breast cancer cells. Thus indicating the possibil-
ity of this present study to specifically elucidate the role of caspase
3 in breast cancer and the possibility of inhibitory caspase 3 pro-
teins including XIAP and unphosphorylated BRCA1 as anti-cancer
drug targets.
C2-17
Dynamic sumoylation and desumoylation
controls the repressor activity of DNp63a by
regulating the subcellular localization
H. Lee
1
, J. Jeong
1
, M. Cho
1
, J. Lee
1
, H. Kim
2
, Y. Yun
2
and
H. Lee
1
1
Seoul National University, Seoul, REPUBLIC OF KOREA,
2
Ewha
University, Seoul, REPUBLIC OF KOREA
DNp63a, a homologue of p53, is exclusively expressed in stem cells

and progenitor cells of the stratified epithelia. It promotes cell sur-
vival by acting as a transcription repressor towards p53 and related
TAp63/TAp73, consistent with its expression in cancer cells. Here
we report that the repressor activity of DNp63a is regulated by
SUMO-1 modification. Disruption of the sumoylation site compro-
mised the repressor activity of DNp63a towards TAp63c, but less
towards p53. Furthermore, expression of SUMO protease SuPr-1
reduced the repressor activity of DNp63a towards TAp63c and
TAp73b, whereas p53-mediated trans-activation was not affected.
In zebrafish embryogenesis, sumoylation-defective mutant failed to
rescue the epidermal defects caused by the scheduled over-expres-
sion of TAp63c, while the wild-type DNp63a did, signifying the
importance of DNp63a sumoylation in vivo. In search for the
molecular mechanism, we found that sumoylated DNp63a co-
localized with PML, which was increased upon c-irradiation and
disturbed by enzymatically active SuPr-1 expression. In contrast,
binding to HDACs or counterpart transactivators, nucleoplasmic
shuttling, or ubiquitination were largely unaffected. These results
indicate that sumoylation of DNp63a confers selective repressor
activity towards TAp63, which is counterbalanced by SuPr-1-medi-
ated desumoylation at PML nuclear body. Collectively, the sumoy-
lation-desumoylation switch regulates the subcellular localization
of DNp63a and provides a fine-tuning mechanism for DNp63a-
mediated transcription repression.
C2-18
Regulation of neuronal survival by MDMX, p53
and E2F-1
S. Benosman
1
, I. Gross

2
, N. Clarke
3
, A. G. Jochemsen
4
,
K. Okamoto
5
, J. Loeffler
1
and C. Gaiddon
1
1
Louis Pasteur University - INSERM U692, Strasbourg, FRANCE,
2
INSERM U682, Strasbourg, FRANCE,
3
School of Pharmacy, Uni-
versity of Nottingham, Nottingham, UK,
4
Department of Molecular
and Cell Biology, Leiden University Medical Center, Leiden, THE
NETHERLANDS,
5
National Cancer Center Research Institute,
Radiobiology Division, Tokyo, JAPAN
During the last decade, extensive data described the mechanisms of
cell death in various cellular contexts. Yet, the regulation of neur-
onal fate remains poorly explored. Among identified players in
Neuronal death, p53 and E2F-1 are prominent transcription fac-

tors that induce apoptosis in neurons under specific stresses.
MDMX is an analogue of the p53 inhibitor MDM2 whose impli-
cation in neuronal death is still uncertain. In cultured post-mitotic
neurons, we characterized multiple death-inducing conditions that
activate simultaneously or selectively p53 or E2F-1. Those condi-
tions include DNA damaging agents Neocarzinostatin and Cisplat-
in, and more neuron-specific stresses such as Potassium
deprivation, Glutamate or APP-stimulating antibody (a model to
Alzheimer’s disease’s Amyloid protein stimulation). Interestingly,
MDMX protein was phosphorylated at S367 before being lost dur-
ing the apoptotic process. Loss of MDMX was restored using sev-
eral protease inhibitors, suggesting a post-translational regulation
of MDMX. Moreover, overexpression of MDMX inhibited both
p53 and E2F-1 transcriptional activity and rescued neurons from
death, while shRNA directed against MDMX increased neuronal
death in the above mentioned conditions. Therefore, MDMX
appears to be a new key player in adult Neuronal survival that lev-
erages not only p53, but E2F-1 activities as well.
C2-19
Recognition of negatively or positively
supercoiled DNA topoisomers by the p53
protein
H. Pivon
ˇ
kova
´
, P. Pec
ˇ
inka, O. Ticha
´

and M. Fojta
Institute of Biophysics, Academy of Sciences of Czech Republic,
v.v.i., Brno, CZECH REPUBLIC
The tumor suppressor protein p53 belongs to the checkpoint
proteins. Its function is closely related to maintaining genetic integ-
rity of the cells and to defence against malignant transformation
(1). Biological activity of p53 is dependent on its ability to interact
with DNA. It was found that p53 bound DNA sequence-specific-
ally or sequence-nonspecifically to both positively (2) or negatively
supercoiled DNA (3, 4). This work was focused on study of super-
coil-selective p53-DNA interactions using immuprecipitation at
magnetic beads. We tested influence of intercalative drug chloroqu-
ine (CQ) on p53 binding. We found that in the absence of CQ,
p53 bound scDNA with higher preference then linDNA, but in
presence of higher concentration of CQ, DNA relaxed and p53
bound relaxed and linDNA with the same affinity. Due to further
increasing of CQ concentration, DNA became positively super-
coiled and the preference for this DNA was remarkably higher
again. Using the same technique we observed that the p53 protein
can differentiate among DNA topoisomers differing in a few su-
perhelix turns, both in the presence or absence of CQ.
Acknowledgements: This work was supported by GACR (301/
05/0416, 204/07/P476), GAAS CR (IAA500040701) and MEYS
CR (LC06035).
References
1. Levine, A.J., et al. 2005, editor G.P. Zambetti.
2. Mazur, S.J., et al. J Mol Biol, 1999, 292(2).
3. Palecek, E., et al. Oncogene, 1997, 15(18).
4. Fojta, M., Pivonkova, H., et al. Eur J Biochem, 2004, 271(19)
Signal Transduction Abstracts

ª 2007 The Authors Journal compilation ª 2007 FEBS 153
C2-20
Mutant p53 proteins bind selectively
supercoiled DNA
K. Ne
ˇ
mcova
´
, M. Bra
´
zdova
´
,L.C
ˇ
inc
ˇ
a
´
rova
´
,P.S
ˇ
ebest,
H. Pivon
ˇ
kova
´
, O. Ticha
´
, M. Fojta and E. Palec

ˇ
ek
Institute of Biophysics, Academy of Sciences of the Czech Republic,
v.v.i., Brno, CZECH REPUBLIC
It has been shown previously that DNA superhelicity influences
significantly DNA binding by the wild type (wt) p53 protein (1, 2).
Full length wtp53 binds selectively to scDNA (supercoil-selective,
SCS binding) regardless of the presence or absence of the p53 con-
sensus sequence (p53CON). In addition, the p53 sequence-specific
binding to certain target sites can be enhanced by negative super-
coiling (2). In this work we studied DNA interactions of full-length
mutant p53 proteins (mutp53) (R175H, G245S, R248W, R249Q,
R273C, R273H). Some of the mutp53s exhibited significant bind-
ing to p53CON in $500 bp DNA fragments at 0 °C. All of the
mutants retained the SCS DNA binding (in the absence of
p53CON). Competition assays and selective manipulation of the
central and/or C-terminal DNA binding domains of the mutp53
proteins revealed critical role of the C-terminal domain in the SCS
binding [as established previously for the wt p53 (1)]. The structure
selective DNA binding of mutp53 has been proposed to play
important roles in its active role in tumorgenesis (gain of func-
tion).
Acknowledgements: This work was supported by MEYS CR
(1K04119, LC06035), GACR (204/06/P369) and GAAS CR
(IAA500040701).
References
1. Brazdova M, Palecek J, Cherny DI, et al. (2002) Nucleic Acids
Res. 30(22), 4966–74.
2. E. Palecek, D. Vlk, V. Stankova, et al. (1997) Oncogene, 15,
2201–2209.

C2-21
Rapamycin induces p53-dependent apoptosis in
human prostate cancer cells via PI3K pathway
S. Numanoglu
1
, C. Biray Avci
1
, S. Yilmaz
1
, G. Saydam
2
and
C. Gunduz
1
1
Ege University School Of Medicine, Dept. of Medical Biology,
Izmir, TURKEY,
2
Ege University School Of Medicine, Dept. of
Haematology, Izmir, TURKEY
Prostate cancer (PCA) is one of the most common cancers among
men. Because of treatment limitations, there has been intense pub-
lic and scientific interest in the use of other approaches to control
the growth of PCA for its treatment. Rapamycin has been reported
to inhibit metastatic prostate tumor growth and angiogenesis in
in-vivo mouse models. We aimed to investigate the role of gene
expressions (PIK3CA, RB1 and p53) in rapamycin-induced apop-
tosis of PCA cells; PC3, DU145 and LNCAP we performed cyto-
toxicity, apoptosis and expression analysis. Rapamycin was used in
treatments of 1 nM, 10 nM, 25 nM, 50 nM and 100 nM. Cyto-

toxic assays were performed by using Trypan blue dye exclusion
and XTT assay and apoptosis with Acridine orange/Ethidium bro-
mide. Gene expressions were examined by RT-PCR. Cytotoxic
effects of rapamycin in DU145, PC3 and LNCAP were detected in
dose and time dependent manner with the IC
50
doses of 10, 25,
50 nM, respectively. Rapamycin have shown remarkable apoptosis
at 72
nd
hour in treated cells. Gene expressions analysis showed up
regulation of PIK3CA (20.98%), RB1 (352.00%) and p53
(121.22%) genes in PC3 cell line. DU145 cell line exhibited up regu-
lation of RB1 (33.88%) and p53 (24.26%) and reduced gene expres-
sion of PIK3CA (16.75%). PIK3CA (50.30%) and RB1 (14.49%)
gene expressions were down regulated and expression of p53
(78.96%) was increased in LNCAP cell line. In conclusion; rapamy-
cin-induced regulation of these genes may be further exploited for
devising chemopreventive and/or therapeutic strategies for PCA.
C2-22
NADPH oxidase inhibitor diphenyleneiod onium
induces ROS-independent p53 expression and
apoptosis in human RPE cells
K. Kim, J. Song and Y. Park
Pusan National University School of Medicine, Busan, REPUBLIC
OF KOREA
The Diphenyleneiodonium (DPI) is widely used as an inhibitor of
flavoenzymes, particularly NADPH oxidase. In this study, we inves-
tigated the effect of DPI on the apoptosis of human RPE cells. DPI
treatment in ARPE-19 cells evoked a dose- and time-dependent

growth inhibition, and also induced DNA fragmentation and pro-
tein content of the proapoptotic factor Bax. In addition, DPI signifi-
cantly induced the expression and phosphorylation of p53, which
induces proapoptotic genes in response to DNA damage or irrepar-
able cell cycle arrest. ROS have been implicated as a key factor in
the activation of p53 by many chemotherapeutic drugs. Recent data
on the regulation of intracellular ROS by DPI are controversial.
Therefore, we analyzed whether DPI could contribute to the genera-
tion of intracellular ROS. Although there was increase in ROS level
from cells treated for 24 h with DPI, it was not detectable at early
time points, required to induce p53 expression. And DPI-induced
p53 expression was not affected by the ROS scavenger NAC. We
conclude that DPI induces the expression of p53 by ROS-independ-
ent mechanism in ARPE-19 cells, and renders cells sensitive to drug-
induced apoptosis by induction of p53 expression.
C2-23
Sensitization of osteosarcoma cells to
apoptosis by Oncostatin M depends on STAT5
and p53
V. Trichet
1
, C. Chipoy
1
, B. Brounais
1
, P. Juin
2
,F.Re
´
dini

1
,
D. Heymann
1
and F. Blanchard
1
1
INSERM ERI-7, Nantes, FRANCE,
2
INSERM U601, Nantes,
FRANCE
Oncostatin M (OSM), a cytokine of the IL-6 family, reduces the
growth and induces differentiation of osteosarcoma cells. Given
the strong interaction between differentiation and apoptosis, we
asked here whether OSM could regulate apoptosis of normal or
transformed osteoblasts. Alone, OSM did not induce cell death,
but OSM-treated osteosarcoma cells or proliferating osteoblasts
were particularly more sensitive to apoptosis induced by different
death inducers among which staurosporine (STS). Cell death
induced by OSM+STS was associated with activation of caspase 9
and 3 and prevented by the caspase inhibitor Z-VAD-FMK. OSM
alone induced activation of p53, STAT1, 3 and 5 transcription fac-
tors. By using chemical inhibitors, dominant negative (dn) STATs,
shRNA and knock out cells, we demonstrated that STAT5 is
implicated in reduction of Bcl-2 expression, whereas p53 mediates
enhanced Bax expression. By co-immunoprecipitation, we observed
a constitutive interaction between p53 and the C-terminal trans-
activation domain of STAT5. Thus we characterize a new signaling
pathway (based on p53-STAT5 interaction) used by OSM to
increase Bax and decrease Bcl-2 expression, and thus controlling

the mitochondrial cell death pathway. In parallel, strong anti-
apoptotic signals are also activated by OSM, via the PKCd and
PI3K/Akt pathways. Therefore association of OSM with the kinase
inhibitor STS could represent new treatments for wild type p53
osteosarcoma.
Abstracts Signal Transduction
154 ª 2007 The Authors Journal compilation ª 2007 FEBS
C2-24
Human Vaccinia-Related Kinase 1 (VRK1)
regulation and its implication in cell proliferation
A. Valbuena and P. A. Lazo
Instituto de Biologı
´
a Molecular y Celular del Ca
´
ncer, Salamanca,
SPAIN
VRK1 is a new human Ser-Thr protein kinase with a possible role
in cell proliferation. Tumor suppressor p53 induces responses
aimed to protect the cell allowing damage repair by stopping the
cell cycle or inducing apoptosis. VRK1 phosphorylates p53 in
Thr18 leading to p53 accumulation and transcriptional activation.
A persistent accumulation of p53 would induce permanent cell
arrest or apoptosis, so p53 accumulation must be transient and
thus it is probable the existence of an autoregulatory mechanism
between VRK1 and p53. We have identified an autoregulatory
loop between p53 and its activator VRK1. There is an inverse cor-
relation between VRK1 and p53 levels in cell lines. Induction of
p53 by UV light and its overexpression promotes a VRK1 down-
regulation which is dependent on a transcriptionally active p53,

which activates the targeting of VRK1 protein for proteolytic deg-
radation in the lisosomal pathway. There is a disruption of p53-
VRK1 autoregulatory loop in some human lung carcinomas. In
human biopsies VRK1 is expressed where cellular proliferation
takes place, and it is lost as cells stop dividing and differentiate.
Levels of VRK1 mRNA are reduced 1 day after serum deprivation
in normal fibroblasts, and levels of protein, which is very stable,
diminish after 3–6 days, when the cell cycle is stopped and cells dif-
ferentiate. Readdition of serum leads to a recovery of VRK1
mRNA levels at the same time as early response genes as c-Fos and
c-Myc and VRK1 protein levels are recovered parallel to PCNA
and inversely to p27, a cell cycle inhibitor. VRK1 deficiency induced
by siRNA leads to cell proliferation abnormalities. VRK1 is a new
element in the p53 regulation pathway and a key protein in progres-
sion of the cell cycle that might be deregulated in human tumors.
C2-25
EWS-FLI1 controls cell cycle and death by
suppressing distinct signalling pathways
converging on p53
J. Ban, D. N. T. Aryee, I. Bennani and H. Kovar
Children’s Cancer Research Institute, Vienna, AUSTRIA
P53 plays a central role in regulating cellular fate in response to
various stresses. In Ewing
´
s sarcoma (ESFT), p53 alterations are
rare and it remains to be established, how the tumor cells escape
the checkpoint function of p53. Silencing of the ESFT oncogene
EWS-FLI1 by RNAi results in cell cycle arrest and death. Using
genetic and chemical compounds targeting components of candi-
date EWS-FLI1 regulated growth factor pathways, and forcing the

expression of selected signalling molecules, we report a role for
EWS-FLI1 in keeping p53 in check. Suppression of EWS-FLI1
leads to nuclear accumulation of phospho-p53 and induction of
the cell cycle inhibitor CDKN1A. We identified two independent
signalling pathways contributing to this observation: i) NOTCH,
activated via re-expression of the ligand JAG1, which stimulated
accumulation of unphosphorylated p53 via induction of the tran-
scription factor HEY1, and ii) the TGFb pathway, activated via
re-expression of the type II receptor and responsible for p53 phos-
phorylation via stimulation of ATM kinase. HEY1-mediated p53
accumulation sufficed to induce CDKN1A and cell cycle arrest but
not cell death. TGFb inhibitors interfered with p53 phosphoryla-
tion and protected ESFT cells from EWS-FLI1 silencing-induced
death but not from p53 and CDKN1A accumulation. These results
suggest that cell cycle arrest and death are independently con-
trolled by two EWS-FLI1 suppressed growth factor pathways con-
verging on p53 in ESFT cells.
Acknowledgements: Supported by GEN-AU-Ch.I.L.D. and
FWF grant 18046.
C2-26
p53 and rb (retinoblastoma) gene expression in
tumoral cells, under the influence of a grape
extract
A. Niculescu
1
, L. Ghetea
1
, R. Motoc
1
, G. Mihaescu

1
,
R. Huculeci
2
, C. Diaconu
3
, C. Ursaciuc
4
and G. Savi
4
1
Institute of Genetics-University of Bucharest, Bucharest,
ROMANIA,
2
Multiple Users Research Base – Molecular Biology,
University of Bucharest, Bucharest, ROMANIA,
3
Institute of
Virology ‘‘Stefan S. Nicolau’’, Bucharest, ROMANIA,
4
National
Institute for Research & Development in Pathology and Biomedical
Sciences ‘‘V. Babes’’, Bucharest, ROMANIA
The aim of this study was to establish the in vitro and in vivo
effects of a grape extract (GSE) on Wistar rat Walker sarcoma 256
ascitic cells. The treatments with GSE were administrated in differ-
ent doses and time intervals. The analyses were performed by flow
cytometry, for the study of apoptosis dynamics, and by Real-Time
PCR, for p53 and rb gene level expression detection. The results
obtained revealed that in vitro treatment with GSE increased the

number of apoptotic cells by increasing the treatment time. The
maximum percentage of apoptosis was obtained after 72 h of treat-
ment (50,40%), while the control culture presented a percentage of
3.45% of apoptotic cells. Real-Time PCR analysis showed that the
expression level increased for both genes, at two concentrations of
the GSE used in the experiments, indicating a increased transcrip-
tion activity for p53 and rb. The most significant result, for the
in vivo experiments, was an increase of the life span (by a factor of
two) of the rats treated with GSE (150 lg/kg), but, in this case,
none of the two genes (p53 and rb) level was modified. This results
suggest that, in vivo, GSE could have a stimulating effect mainly
on the immune system which become more efficient in decreasing
the tumor progression rate.
C2-27
Laurinterol from Laurencia okamurai induces
apoptosis via p53 activation
M. M. Kim, N. Rajapakse, E. Mendis, S. H. Lee and S. K. Kim
Pukyong National University, Busan, REPUBLIC OF KOREA
The purpose of this study is to examine the effect of laurinterol
from Laurencia okamurai on induction of apoptosis in melanoma
cells (B16F1). First of all, the effect of laurinterol on cell viability
was evaluated by MTT assay. Apoptosis induced by laurinterol was
analyzed using DNA fragmentation, TUNEL assay and caspase
assay in melanoma cells. Laurinterol exhibited excellent effect on
the induction of apoptosis compared with etoposide used as posit-
ive control in this study. It was also observed that transcriptional
activation of p53, a tumor suppressor gene, by laurinterol was
involved in induction of the apoptosis using reporter gene assay.
Western blot analysis showed that laurinterol increased the expres-
sion level of phosphorylated p53. These results suggest that laurin-

terol the from Laurencia okamurai may provide a therapeutic
potential to develop a novel anti-cancer agent.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 155
C2-28
p53 suppresses cyclooxygenase-2 expression
through down-regulating inducible nitric oxide
synthase in mouse embryonic fibroblast
S. Wada
1
, S. Kokura
1
, Y. Naito
1
, H. Ichikawa
1
, H. Ohshima
2
and
T. Yoshikawa
1
1
Kyoto Prefectural University of Medicine, Kyoto, JAPAN,
2
University of Shizuoka, Shizuoka, JAPAN
Inducible nitric oxide synthase (iNOS), tumor suppressor p53 and
cyclooxygenase-2 (COX-2) may be co-regulated with each other.
Elevated expression of COX-2 has been reported in stromal cells in
the early stage of carcinogenesis. However, the roles of iNOS and
p53 on expression of COX-2 have not been clearly established. We

have investigated the effects of iNOS and p53 gene deficiencies on
COX-2 expression in mouse embryonic fibroblasts. The following
mice were used for embryonic fibroblasts: (1) Wild-type (WT,
iNOS+/+, p53+/+), (2) iNOS-deficient (iNOS-/-, p53+/+), (3)
p53-deficient ( iNOS+/+, p53-/-) and (4) iNOS-p53 double knock-
out (iNOS-/-, p53-/-). They were treated with 1 lg/ml of LPS and
100 IU/ml of IFN-gamma for 3, 6, 9, 16, 24, 48 and 72 h. Expres-
sions of COX-2 were analyzed by Western-blotting. In fibroblasts
from WT mice, expression of iNOS was detected after 48 h,
whereas one in p53-deficient was earlier (16 h). Cox-2 protein
expression of p53-deficient sustained longer than one of WT. In
iNOS-deficient fibroblasts, protein expression of COX-2 was
reduced earlier than one of iNOS-expressed cells. Exogenous NO
donor (0.8 mM of S-nitrosoglutathione) in the activated iNOS-defi-
cient cells augmented COX-2 protein expression. In conclusion,
loss of functions of p53 genes enhances COX-2 expression by indu-
cing iNOS expression. There results indicates one of the potential
mechanisms for the tumor progression of p53-deficient.
C2-29
The impact of CD43 expression on cell growth
K. Ja
¨
a
¨
ger, J. Viil, L. Kadaja-Saarepuu and T. Maimets
Institute of Molecular and Cell Biology, University of Tartu, Tartu,
ESTONIA
CD43 is a transmembrane protein expressed in hematopoietic cells
but also in cancer cells of non-hematopoietic origin. Elevated levels
of CD43 induce the activation of ARF and p53 protein which

results in cell death. In cells lacking either ARF or p53, CD43 sti-
mulates cell growth which refers to its potential role in cancer for-
mation. The next step would be to further investigate the signaling
pathway between CD43 and p53 and to study the ways CD43
influences cell division. Fas protein, belonging to the tumor necro-
sis factor receptor family, mediates apoptotic signaling upon stimu-
lation by its ligand FasL. In colon cancer cells, the reduction in
the expression of Fas receptor has been reported. Current results
show that CD43 lowers the expression of Fas receptor in cancer
cell lines providing them with higher resistance against Fas depend-
ent apoptosis. As CD43 is abnormally expressed in colon cancer
cells and promotes cell growth without affecting cell cycle, the inhi-
bition of apoptosis could explain the ability of CD43 to support
cell proliferation. Improper degradation of beta-catenin protein is
one of the main causes for colon cancer development. Overex-
pressed beta-catenin translocates to the nucleus and regulates the
expression of genes stimulating cell proliferation. It has been
shown that in colon cancer cells CD43 interacts with beta-catenin
and stimulates its ability to promote cell growth. The results of
our study exploiting RNAi technique support the idea that both
beta-catenin and CD43 are necessary to stimulate cell growth:
silencing the expression of either gene causes the reduction in the
number of colonies and inhibits the activation of beta-catenin
dependent promoter.
C2-30
Phospholipase D suppresses taxotere-induced
cell death through bcl-2 retaining in stomach
cancer cells
J. Cho, S. Hong, E. Kim, S. Park, S. Kwon, G. Lee, C. Park and
J. Han

College of Medicine Hanyang University, Seoul, REPUBLIC OF
KOREA
Phospholipase (PLD) catalyses the hydrolysis of phosphatidylcho-
line to generate phosphatidic acid (PA) and choline. There are at
least two PLD isozymes, PLD1 and PLD2. Genetic and pharmaco-
logical approaches implicate that both PLD isozymes are involved in
a diverse range of cellular processes, including receptor signaling,
membrane transport control, and actin cytoskeleton reorganization.
Several recent studies reported that PLD has a role in signaling path-
ways that oppose apoptosis and promote cell survival in cancer. In
this study, we examined the role of PLD in taxotere-induced apopto-
sis in stomach cell lines; normal stomach cells (NSC) and stomach
cancer cells (SNU 484). Taxotere treatment resulted in increase of
both PLDs expression and activity. When PLD was selectively inhib-
ited by 1-butanol treatment, taxotere-induced apoptosis was exacer-
bated in both of NSC and SNU484. To confirm the role of PLD in
taxotere-induced apoptosis, PLDs were transfected into SNU 484.
Overexpression of PLD isozymes resulted in inhibition of taxotere-
induced apoptotic cell death, evidenced by decreased degradation of
chromosomal DNA and increased cell viability. Concurrently, bcl-2
expression was upregulated, and taxotere-induced activation of
procaspase-3 was inhibited after PLDs transfection. Treatment of
SNU 484 with PA, the product of PLDs, also resulted in upregula-
tion of bcl-2. Although, PA-induced bcl-2 expression was blocked by
mepacrine, an inhibitor of phospholipase A2 (PLA
2
), increased bcl-2
expression by PA was not abrogated by propranolol, on inhibitor of
PA phospholyhydrolase (PAP). These results indicate that PLA
2

is
closely related with bcl-2 expression, but not with PAP, induced by
PLD activation.
C2-31
Effects of doxorubicin on telomerase and
apoptosis in doxorubicin resistant and sensitive
MCF-7 cells
U. Eskiocak,O
¨
. Darcansoy
_
Is¸ eri, M. Demirel Kars, A. Bic¸ er and
U. Gu
¨
ndu
¨
z
Middle East Technical University, Department of Biological
Sciences, Ankara, TURKEY
Bcl-2 and Bcl-xL are two of the anti-apoptotic members, and Bax
is a pro-apoptotic member of the Bcl-2 related protein family.
hTERT gene encodes the catalytic protein component of the
human telomerase enzyme. PCR-based TRAP is used to determine
activity of telomerase. Doxorubicin resistant MCF-7/R were devel-
oped from the sensitive breast carcinoma MCF-7 cell line and
development of resistance was demonstrated by XTT and analysis
of resistance related MDR1 and MRP1 mRNA levels by RT-PCR.
Differential dose and time dependent response of sensitive and
resistant cells to doxorubicin were evaluated by viable cell counts,
expression analysis of Bcl-2, Bcl-xL, Bax and hTERT mRNA lev-

els and telomerase activity determination. Doxorubicin selected
MCF-7 cells are 107 folds resistant to the drug and these cells over
express MDR1 and MRP1 genes. Doxorubicin application caused
a decrease in Bcl-2 expression level in sensitive cells although no
change was observed in resistant cells. Interestingly Bcl-xL level
increased in sensitive and resistant cells after 72 h incubation in
doxorubicin where a slight increase was observed in resistant cell
lines. Bax levels seemed to be unchanged. hTERT levels seemed to
increase after 24 h of drug incubations and 72 h doxorubicin incu-
bation caused a decrease in telomerase activity in parallel with a
small decrease in hTERT levels in both cells types.
Abstracts Signal Transduction
156 ª 2007 The Authors Journal compilation ª 2007 FEBS
C2-32
PUMA is induced in p16INK4A-expressing
T-ALL cells and increases sensitivity to
glucocorticoid- and Fas-induced apoptosis
M. J. Ausserlechner
1
, T. Unterkircher
1
, M. Deutsch
2
,
C. Salvador
1
, M. Holzner
2
, V. Porto
2

and P. Obexer
2
1
Department of Pediatrics, Medical University Innsbruck, Innsbruck,
AUSTRIA,
2
Tyrolean Cancer Research Institute, Innsbruck,
AUSTRIA
The cell cycle inhibitor p16INK4A is frequently deleted in acute
lymphoblastic T-cell leukemia. Leukemia cells designed to condi-
tionally express p16INK4A arrest in the G0/G1 phase of the cell
cycle and show increased sensitivity to glucocorticoids (GC) and
accelerated death receptor induced apoptosis. Upon tetracycline-
regulated expression of p16INK4A Bcl-2 and Survivin were
repressed whereas the BH3-only protein PUMA was strongly
induced. To functionally validate these regulations we retrovirally
expressed Bcl-2 and Survivin and knocked-down endogenous
PUMA in p16INK4A-expressing cells. Expression of Bcl-2 and
PUMA-shRNA significantly reduced cell death, whereas transgenic
Survivin did not alter sensitivity. These results indicate that the
deletion of p16INK4A during leukemia development not only
deregulates cell cycle but also alters the balance of pro- and anti-
apoptotic Bcl-2 proteins, thereby causing apoptosis resistance to
certain therapeutic agents.
C2-33
Life/death decisions in growth factor signaling:
critical role for RAF, AKT and BCL-2 family
proteins
J. Smigelskaite, S. Scheidl, A. Kuznetsov, M. Schneider and
J. Troppmair

Daniel Swarovski Research Laboratory, Innsbruck, AUSTRIA
Lack of sufficient growth factors is a frequent stimulus for the
induction of apoptosis that can be delayed through the expression
of RAF, AKT or BCL-2 proteins. It is currently unclear, which
events translate the lack of survival signal into a death stimulus
and how they are controlled by survival proteins. Interleukin-3
(IL-3)-dependent parental 32D cells or 32D cells expressing activa-
ted versions of RAF or AKT or overexpressing BCL-2 were used
in growth factor abrogation experiments. Alteration in mitochond-
rial ROS and Ca
2+
levels were monitored by confocal imaging fol-
lowing loading of cells with MitoSOXTM Red or Rhod-2. Protein
expression was verified by immunoblotting following SDS-PAGE.
Factor dependent myeloid (FDM) cells are derived from wild type
or bax-/- bak-/- double knockout mice and are grown in the pres-
ence of IL-3. We show that following growth factor IL-3 with-
drawal reactive oxygen species (ROS) induced mitochondrial Ca
2+
overload functions as an RAF-suppressible apoptosis trigger, while
others demonstrated the requirement to inactivate MCL-1 via an
AKT-dependent pathway. We thus have begun to systematically
analyze a possible link between these two control mechanisms. Our
results obtained so far suggest that MCL-1 stability is not affected
by RAF-signaling, or anti- (N-acetyl-L-cysteine) or pro-oxidants
(t-BHP). However, activated AKT, which we established as an
effector of RAF in these cells before, also suppressed mitochond-
rial ROS production and Ca
2+
overload. Similar results were

obtained with BCL-2.
Life/death decisions following growth factors signaling may hinge
on cooperative decision making by RAF and BCL-2 family pro-
teins dependent signals.
C2-34
Changes of calcium homeostasis and apoptosis
in P-gp positive L1210/VCR cells
M. Seres
1
, L. Gibalova
1
, Z. Sulova
1
, M. Barancik
2
, J. Sedlak
3
and
A. Breier
1
1
Inst. Molec. Physiol. Genet., SAS, Bratislava, SLOVAKIA,
2
Inst. Heart Res., SAS, Bratislava, SLOVAKIA,
3
Inst. Exp. Oncol,
SAS, Bratislava, SLOVAKIA
Expression of drug transporting P-glycoprotein (P-gp) in neoplastic
cells is most frequent cause of multidrug resistance (MDR). We
found that P-gp positive L1210/VCR cells (R) are more sensitive

to high external concentration of Ca
2+
as parental L1210 cells (S).
We observed the more pronounced calcium uptake and differences
in intracellular localization of Ca
2+
for R cells. Calnexin (intracel-
lular calcium dependent chaperone) was described to ensure matur-
ation of P-gp. We detected lower levels of calnexin in R as in S
cells. We detect also downregulation of inositol 1,4,5- triphosphate
receptor channel (IP
3
R) in R cells cultivated in the presence of
vincristine (VCR) when compared with R cells cultivated in the
absence of VCR. However we did not observe any significant
changes in IP
3
R in S and R cells. Resistance to cisplatin (cisPt)
associated with downregulation of IP
3
R was assumed to be con-
nected with reduction of apoptosis. MDR cells often exhibit a
resistance to apoptosis induced by chemotherapeutic agents. CisPt
is known as un-transportable by P-gp. We found that R cells are
more sensitive to cisPt as S cells. Interestingly, we detect that R
cells under application of cisPt were entering the apoptosis in a
lower extent than S cells. In contrast, cisPt induced more pro-
nounced necrosis in R than in S cells. We found predominating
amount of antiapoptotic Bcl-2 protein and proapoptotic Bax pro-
tein in Bcl-2:Bax complexes obtained by immunoprecipitation from

R and S cells, respectively. Thus, R cells are more resistant to
apoptosis than S cells that may be linked with alteration in intra-
cellular calcium homeostasis.
C2-35
Apoptosis in normal peripheral blood
lymphocytes treated with low concentration of
actinomycin D
I. Kalousek, B. Brodska, P. Otevrelova and P. Roselova
Institute of Hematology, Prague 2, CZECH REPUBLIC
To increase the understanding of the mechanism by which antican-
cer drugs cause toxicity in normal tissue, we have examined the
ability of 10 nM actinomycin D (ActD) to induce apoptosis in
human PBL. To specify the primary molecular damage and to
ascertain which proteins were affected by genotoxic drug action,
we performed in vitro transcription assay for rRNA and RT-PCR
and Western blots for thirty apoptosis-related genes and tumour
suppressors. We found down-regulation of rRNA synthesis and
subsequent mitochondria-dependent apoptosis. While the expres-
sion of the majority of examined genes remained indifferent
against ActD, the cellular level of p53 protein increased, upregulat-
ing Puma and p21waf1 mRNA and protein. Puma mediated apop-
tosis was accompanied by p21waf1 and Bcl-2 mRNA
destabilization likely originating in ARE binding nucleolin clea-
vage. The stability of the level of Bcl-2 protein, independent of an
mRNA decrease, suggests that the protection of anti-apoptotic
Bcl-2 against proteasomal degradation moderates the apoptotic
process as well as a temporary increase of caspase-3 inhibitor
p21waf1. Conclusions: In PBL cultured in vitro, a 10 nM concen-
tration of ActD induced the p53 and Puma dependent mitochond-
rial way of apoptosis moderated by Bcl-2 and p21waf1.

Acknowledgement: This work was supported by grant IGA VZ
00023736 from the Ministry of Health of Czech Republic.
Signal Transduction Abstracts
ª 2007 The Authors Journal compilation ª 2007 FEBS 157