Báo cáo khoa học: Signal Transduction pot

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 cel

Candida albicansis 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 LeeSeoul National University, Seoul, REPUBLIC OF KOREARunx proteins are evolutionarily well-conserved transcription fac-tors that are involved in essential aspects of the development ofmetazoan animals ranging from nematodes to humans We foundthat the expression of the nematode RUNX homolog, RNT-1, istightly regulated in that it is expressed only in the intestine andhypodermis at specific developmental stages and that RNT-1 isalmost absent in any tissue at the adult stage Ectopic expression

of RNT-1 resulted in the over-proliferation of the hypodermalcells, indicating that tightly regulated attenuation of the RUNXprotein is required for its proper function in vivo

Recently, it was revealed that the C elegans genome contains onehomolog of the Runx protein partner CBFbeta (BRO-1) bro-1 isexpressed only in the hypodermis at all developmental stages.Knockdown of bro-1 resulted in the up-regulation of RNT-1 in thehypodermis, but not in the intestine, suggesting that bro-1 acts as aco-repressor of RNT-1 We found that the nematode RNT-1 regu-lates its own transcription in the hypodermis, acting on its owncis-acting elements

In situ hybridization experiments showed that the RNT-1 script is still present in the intestine at the adult stage, but not inthe hypodermis, suggesting that RNT-1 might be regulated at theprotein level Consistent with this, RNAi knockdown of ubq-1, apolyubiquitin gene, and uba-1, the E1 gene, resulted in higher sta-bility of RNT-1 in the intestine and the treatment of MG132, aproteasome inhibitor, stabilized RNT-1 in the intestine Takentogether, we conclude that RNT-1 expression is attenuated at theadult stage by two different mechanisms, transcriptional autoregu-lation in the hypodermis and proteasome-mediated degradation inthe intestine

tran-C1-4 Differential cleavage of Dpp precursor modulates morphogen gradient in the Drosophila

J Ku¨nnapuu, I Bjo¨rkgren and O ShimmiUniversity of Helsinki, Helsinki, FINLAND

We have previously shown that two different molecular forms ofDecapentaplegic (Dpp), Drosophila BMP2/4 type ligand, are pro-duced in the embryo (Shimmi et al, Cell 2005) As Dpp precursorcontains two optimal furin recognition sites, we suspect that thesetwo sites can be used for ligand maturation In order to under-stand how Dpp processing is regulated, and why two differentforms of Dpp ligands are used for signaling, we mutated the opti-mal furin recognition sites to study their function The mutation ofthe first furin site drastically dropped the production of mature lig-ands in Drosophila S2 cells In contrast, the mutation of the secondfurin site did not affect the production rates or the signaling inten-sities of ligands, however, mature ligands were produced as a singleform As previous reports indicated that vertebrate BMP4 wassequentially cleaved to help promote the long-range signaling, wefocus 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 howevolutionally conserved furin recognition sites of BMP2/4 type lig-ands contribute to regulate the long- or short-range signaling ofthese ligands during developmental stages

Phenotypic observation of Xenopus laevis MGP

loss of function

B S N Simo˜es, B Simo˜ es1, A C Silva2, M Vitorino2,

N Conceic¸a˜o1, J A Belo2and M L Cancela1

1CCMAR,2IBB, 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 calcificaregula-tion 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¨gi1, C Moll1, L von Lyncker1, N Baumann1,

U Grossniklaus2and R Gross-Hardt1

1ZMBP University of Tu¨bingen, Tu¨bingen, GERMANY,2Institute

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 Sabitov1, K Musabekov1, M K Gilmanov2and

Z S Kudiyarova3

1Chemistry Department, Chair of Colloid Chemistry, Catalysis andOil Chemistry, Al-Faraby’s Kazakh National University, Almaty,KAZAKHSTAN,2M.A Aitkhozhins Institute of Molecular Biologyand Biochemistry, Almaty, KAZAKHSTAN,3Biology Department,Al-Faraby’s Kazakh National University, Almaty, KAZAKHSTAN

It is well known, that regulation of ionic transport the main ture of cell regulation However the mechanisms of regulation ofionic transport in plant cells are not enough investigated For thisreason it is very important to investigate the effect of new powerfulregulator – cytokinine secondary hormone (CSH) on plant cell ionstransport

fea-In laboratory of structure and regulation of enzymes of M.A tkhozhins institute of molecular biology and biochemistry CSHwas discovered and characterized CSH appears its physiologicalaction 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

Ai-It was shown that CSH activates plasmatic membrane H+-ATPasemore than two times The one of the interesting features of studied

H+-ATPase is the next The activity of this enzyme strong dependsfrom Ca2+ and Mg2+ and this enzyme haven’t absolutely anyactivity with sodium and potassium ions We are suggested thatregulatory effects of CSH first of all appear by activation of cal-cium ions transport through plasmatic membrane into the plantcells It is well known that calcium ions are main intracellular mes-senger which regulates many important cell functions CSH by thisproperty is very close to fusicoccine – natural phytotoxin fromFusicoccum 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 KolchanovInstitute of Cytology and Genetics SB RAS, Novosibirsk, RUSSIANFEDERATION

Development of organisms is a very complex process in which alot of gene networks of different cell types are integrated Develop-ment of a cellular automaton that models the morphodynamics ofdifferent cell types is the first step in understanding and analysis ofthe regulatory mechanisms underlying the functioning of develop-mental gene networks A model of a cellular automaton has beendeveloped, which simulates the embryonic development of shootmeristem in Arabidopsis thaliana The model adequately describesthe basic stages in development of this organ in wild and mutanttypes Visualization of the cellular automaton was created thatallows simulating of the process of development The visualization

of cellular automaton model allows estimating distribution of threebiologically meaningful signals, which unambiguously simulate themorphodynamics of the cell tissues The cellular automaton modelintroduced here to investigate the development of primary shootmeristem 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 dynamicsystem and determining the stable state of this biological system byvariation parameters In this visualization of cellular automatonyou can remove some cells at discretion and continue calculationthat allows analyzing and reproducing such experiment as laserexcision

The highly conserved mitochondrial protein

GCP1 is essential for embryo development in

Qiagen AG, Hilden, GERMANY,3Department 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 rara-pamycin 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 ElsonThe Weizmann Institute of Science, Rehovot, ISRAELReversible phosphorylation of tyrosine residues in proteins is acrucial mechanism for regulating cellular and physiological func-tions Protein tyrosine phosphatases (PTPs) are known to be majorregulators 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 thenon-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 thePTB domain of Shc Complex formation is not dependent ontyrosine 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 PTPe leads to reduced association of Shc with Grb2 and to a sub-sequent decrease in ERK activation Interestingly, these effectsoccur readily when signaling is stimulated by Src, but not wheninitiated by Neu or by the EGF receptor This difference is mostlikely the result of Neu’s ability to compete against PTPe for bind-ing the Shc PTB domain: the interaction of Neu with Shc preventsShc’s dephosphorylation and downregulation of the ERK path-way We conclude that PTPe is an important negative regulator ofShc-mediated signaling that acts in a kinase-specific manner

cyt-C1-12 TRKA induction leading to cell death via cell cycle alteration and cH2AX accumulation in cytosol

D Kim and E JungGyeongsang National University School of Medicine, Jinju, REPUB-LIC OF KOREA

In response to NGF, TrkA tyrosine kinase is activated by hosphorylation and plays an important role in neuronal cell survi-val, differentiation, and apoptosis We show here that TrkAoverexpression by the Tet-On system in U2OS cells mimickedNGF-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 incytosol and plasma membrane TrkA overexpression in U2OS cellsinduced 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

autop-by TrkA was inhibited autop-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 largelyincreased in TrkA-overexpressed cells Also, cH2AX and TrkAwere 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 byDNA damage to partly cytosol Here, we first suggest that cH2AXcould be implicated in cell death signaling cascade by TrkA in theabsence or presence of DNA damage

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 interacCoimmunoprecipita-tions 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 Kollarovic1, P Barath1, K Luciakova1and B D Nelson2

1

Cancer Research Institute, Bratislava, SLOVAKIA,2Stockholm

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 (G0-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

G0-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 vitroGST pull downs The results from ChIP assay suggested

changes in protein complex composition on the ANT2 promoter

during G0phase 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 HoferMedical University Vienna, Vienna, AUSTRIAVEGF-A via triggering of VEGFR2 is the major initiator ofangiogenesis Specific signals/genes induced by VEGF-A, but not

by other growth factors and cytokines, have so far not been fullyestablished We have analyzed here the induction of signals andgenes by VEGF-A/VEGFR2 in comparison to EGF and IL-1.HUVEC were induced by VEGF-A, EGF or IL-1, followed byAffymetrix microarray analyses and RT-PCR Specific inhibitors

of NFAT and EGR-1 were used to judge the contribution of thesefactors The data show that VEGF-A/VEGFR2 preferentially trig-gers signals via PLC-gamma, PKC and Ca++, whereas EGF isunable to trigger this pathway and IL-1 is a preferential inducer ofNFkappaB Downstream of PKC and Ca++ the factors EGR-1and 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 alsoregulated by IL-1 This is caused by the presence of overlappingbinding 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 transcriptionfactors Nurr1, Egr3, Hlx1 and Mef2C We propose that bothproperties, 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 asprimary physiological inducer of angiogenesis

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 HoferMedical University Vienna, Vienna, AUSTRIA

The human HLX1 (H2.0-like homeobox 1) gene is a divergedhomeobox gene Microarray data have shown that it is upregulatedselectively 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 theHLX1 gene in endothelial cells and developed tools to study itspotential function in angiogenesis

We show that HLX1 mRNA is upregulated 10-fold by VEGF andanalysed the HLX1 gene by bioinformatics tools This revealed aconserved potential enhancer region 2.9 kb upstream of the startcodon, composed of binding sites for ATF and CREB An analysis

of the protein sequence detected aside the conserved homeodomainthree 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 denoviruses were prepared to achieve overexpression of HLX1 inendothelial cells High expression of HLX1 by the recombinant a-denovirus was confirmed This was used to test the influence ofHLX1 on the induction of certain genes by VEGF MYCN, a geneinvolved in proliferation, was found to be upregulated by HLX1

a-In contrast, the endogenous HLX1 was strongly downregulatedsuggesting a feedback inhibition of the HLX1 gene Further analy-ses of the effects of HLX1 overexpression and inhibition on angio-genesis models are underway

LSHC-CT-2005–518178

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 Bajoghli1, N Aghaallaei1, D Soroldoni1and T Czerny1,2

1University of Veterinary Medicine Vienna, Vienna, AUSTRIA,

2University of Applied Sciences, FH-Campus Wien, Vienna,AUSTRIA

Groucho/Tle proteins constitute a family of highly conserved factors for transcription Interaction of these corepressor proteinswith Tcf/Lef transcription factors leads to repression of targetgenes in absence of canonical Wnt signalling activity Expression

co-of the short family member Aes interferes with this corepressorfunction of Groucho/Tle proteins We misexpressed Aes duringembryonic development of medaka fish and found effects on optic-and otic vesicle outgrowth In addition we observed lateralitydefects during heart development A closer inspection of left-rightasymmetry revealed effects of both Groucho/Tle proteins andcanonical Wnt signalling on asymmetrically expressed TGFbetafamily 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 organformation

When we looked at earlier events during left-right assignment wehowever found different roles for the two pathways BlockingGroucho function strongly affected the formation of the Kupffer¢svesicle (equivalent to the ciliated part of the mouse node) andexpression of the cilia marker Lrd Interestingly at this stagecanonical Wnt signalling neither affected Lrd expression, norKupffer¢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 PLA2 is important in phosphatidic acid-induced Bcl-2 expression through ERK1/2 MAPK

activation

H Choi and J HanDepartment of Biochemistry and Molecular Biology, College ofMedicine, Hanyang University, Seoul, REPUBLIC OF KOREAPhosphatidic acid (PA), the product of PLD-mediated reaction, islipid second messenger that participates in various intracellularsignaling events and regulates a growing list of signaling protein

In this study, we tried to find out the mechanism of Bcl-2 lation by diC8PA treatment in HeLa cell Treatment with diC8PAresulted 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 A2(PLA2), whereas enhancedrather 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 lysophosphatidicacid (LPA), not through arachidonic acid To investigate the rela-tion between ERK1/2 MAPK pathway and Bcl-2 expression, weused MEK1/2 inhibitor, PD98059 Pretreatment with PD98059decreased diC8PA-induced Bcl-2 expression in HeLa cell, indica-

expression In addition, the ERK1/2 MAPK is also associated withLPA-induced Bcl-2 expression When treated with PD98059,ERK1/2 MAPK activation and LPA-induced Bcl-2 expressionwere inhibited Taken together, PLA2 is important in diC8PA-induced Bcl-2 expression by producing LPA which is involved inERK1/2 MAPK activation

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 Athanasiou1, T Stathas2, P Goumas2, S Naxakis2,

E Giannopoulou3and A J Aletras1

1Laboratory of Biochemistry, Department of Chemistry, University

of Patras, Patras, GREECE,2Department of Otolaryngology,

Med-ical School, University of Patras, Patras, GREECE,3Department 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 fibropathogene-sis 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 transducproduc-tion 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 promorepor-ter 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 KimPukyong National University, Busan, REPUBLIC OF KOREAThe growth factors (IGF-I and EGF) are involved in protectingagainst 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 hasbeen the focus of many studies We examined whether two growthfactors, IGF-I and EGF, could protect against AAP-induced celldeath and investigated the protective mechanism involved Based

on the results of MTS assays, Hoechst 33342 cell staining, andDNA fragmentation experiments, AAP induced cell death in adose-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 activatedafter AAP treatment Combined treatment with AAP and IGF-I,

or EGF inhibited caspase-3 activation and PARP cleavage, 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 ofgrowth factors against AAP cytotoxicity was related to MAPKsignaling, which was involved oxidative stress, and Fas signaling,detected growth factors inhibit AAP cytotoxicity through MAPKsignaling Thus, MAPK is involved in the protective effect ofgrowth factors against AAP-induced cell death

consis-C1-24 Role of PKCe in Gelsolin expression by histone deacetylase inhibitor apicidin in human cervix cancer cells

Y Jeon1,2, J You1,3, J Park1, W Choi4, H Lee2and J Han11

Sungkyunkwan University, Suwon, REPUBLIC OF KOREA,2yang University, Daejeon, REPUBLIC OF KOREA,3Konkuk Uni-versity, Chungju, REPUBLIC OF KOREA,4College of Medicineand CBITRC, Konkuk University, Chungju, REPUBLIC OFKOREA

Kon-Down-regulation of gelsolin expression is associated with cellulartransformation and induction of gelsolin exerts antitumorigeniceffects 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 inducesgelsolin mRNA independently of the de novo protein synthesis.Inhibitor study has revealed that the PKC signaling pathway isinvolved in the gelsolin expression Furthermore, inhibition ofPKCe by either siRNA or dominant-negative mutant completelyabrogates the expression of gelsolin by apicidin, indicating thatPKCe is the major isoform for this process In parallel, apicidininduction of gelsolin is antagonized by the inhibition of Sp1 usingdominant-negative Sp1 or specific Sp1 inhibitor mithramycin, andinhibition of PKC leads to suppression of Sp1 promoter activity.Our results provide mechanistic insights into molecular mecha-nisms of gelsolin induction by apicidin

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 typeand 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 ShishkinBakh Institute of Biochemistry, Moscow, RUSSIANFEDERATION

Satellite cells participate in muscle regeneration and hypertrophy.Proliferation and differentiation of satellite cells are regulated by anumber of growth factors, including TGF-beta In this studyhuman myoblasts were cultured in the F-12 media containingsodium pyruvate, gentamicin and fetal calf serum Differentiation

of the myoblasts was induced by incubation in differentiationmedia (containing 2% of horse serum) Using 2D-PAGE it wasshown significantly decreasing of protein fraction with molecularweight 11 kDa and isoelectric point 6.1 By MALDI-TOF mass-spectrometry this protein has been identified as S100A11 calciumbinding protein (calgizzarin).This protein has been described asone 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 ation

differenti-C1-28 Studying the role of tissue transglutaminase in neutrophil granulocyte differentiation

K Csomos, Z Balajthy, G Zahuczky and L FesusUniversity of Debrecen, Debrecen, HUNGARYNeutrophils begin their differentiation in bone marrow and becomematured granulocytes in the circulation system The proliferatingmyeloid cells do not contain tissue transglutaminase (TG2) but dur-ing their differentiation this enzyme is induced and large amount ofthis protein is present in matured cells So far, its exact role in neu-trophil differentiation has remained unclarified All-trans retinoicacid treated NB4 promyelocyte cells provide an appropriate modelsystem to study neutrophil differentiation Lentivirus based anti-TG2 shRNA expression vector was used to establish stabile TG2knockdown NB4 cell line The examination of the normal and TG2knockdown NB4 differentiation revealed that the enzyme isinvolved in the regulation of several genes (i.e gp91 phox) whichare related to neutrophil granulocyte function These results andthe 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 geneexpression analysis was performed using DNA microarray In TG2knockdown cells the expression of 171 genes decreased and 173increased at least 2-fold level Among these identified genes thereare ones involved in neutrophil granulocyte function, transcriptionfactors and apoptosis related genes

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 Higuero1, R M Jean-Mairet1, J Vandekerckhove2and

T Iglesias1

1

Instituto de Investigaciones Biomedicas Alberto Sols, Madrid,

SPAIN,2Department 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

al differentiation Kidins220 (Kinase D interacting substrate of

220 kDa) is a novel trans-membrane protein with unique featuresand 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 thenervous system The localization of Kidins220 at the tips ofextending neurites suggests that it may be participating in proces-ses such as neuritogenesis and/or neurogenesis We have cloned thepromoter and first intron of kidins220 gene and identified severalputative regulatory elements for retinoic acid receptors We havestudied the gene expression pattern of kidins220 in different neur-oblastoma cell lines under several stimuli that modulate changes intheir phenotype, maturation and aggresiveness

in the pyramidal nerve cell of the hippocampus, but the mechanismand functions underlying hippocalcin in the brain remains unclear

To elucidate a role of hippocalcin, we utilized a conditionallyimmortalized hippocampal cell line (H19–7) We show here thatbFGF-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 wasconcurrent with the expression of basic helix-loop-helix (bHLH)transcription factor, NeuroD Hippocalcin suppression blockedbFGF-induced neurite outgrowth and NeuroD expression Stimu-lation of bFGF resulted in the activation of phospholipase C-c(PLC-c) and Ca2+ Hippocalcin expression by bFGF stimulationwas fully blocked by both the PLC-c inhibitor, U73122 and a che-lator of intracellular Ca2+, BAPTA-AM, suggesting that hippocal-cin expression by bFGF stimulation is dependent on PLC-c and

blocked bFGF-induced neurite outgrowth and NeuroD expression.Taken together, these results suggest for the first time that bFGFinduces hippocalcin expression through PLC-c activation and

Ca2+, which leads to neurite outgrowth in H19–7 cells

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 Serine727associated to an absence of

phospho-STAT3 on Tyrosine705is essential for osteoclastogenesis IL6

stimu-lation induced both phosphorystimu-lations, and consequently RAW

264.7 generated macrophages With AG490 a decrease in Serine727

-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

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 a6 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 neugast-rula 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 participmedi-ates 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 Schwarzer1, S Loacker1, M Sayyah1and H Herzog2

1Medical University Innsbruck, Innsbruck, AUSTRIA,2GarvanInstitute for Medical Research, Sydney, AUSTRALIANeuropsychiatric disorders are one of the main challenges ofhuman 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 inepilepsy However, little is known about the mechanism of theendogenous opioid system in epileptogenesis and epilepsy There-fore, we investigated prodynorphin-KO mice (Dyn-/-)in models ofacute seizures, epileptogenesis and epilepsy

Compared with wildtype littermates (Dyn+/+), Dyn-/- miceshowed a significantly reduced seizure threshold as assessed by tail-vein infusion of pentylenetetrazole (PTZ) This phenotype could berescued 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 seizurethreshold in both genotypes Pre-treatment with the kappa selectiveantagonist GNTI completely blocked the rescue effect of U-50488.Consistent with the reduced seizure threshold, Dyn-/- mice showedfaster seizure onset and a prolonged time of seizure activity after icinjection of kainic acid In the PTZ kindling model, Dyn-/- miceshowed a significantly faster kindling progression Three weeksafter 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 stronglysupport 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 OhtsukiKitasato University, Sagamihara, JAPAN

The purpose of this recently, we reported that casein kinase 1(CK1) phophorylates two functional basic proteins [myelin basicprotein (MBP) and tau protein (TP)] in the presence of two sulfat-

ed lipids [sulfatide and cholesterol-3-sulfate (SCS)] in vitro ever, the physiological significance of the CK1-mediated highphosphorylation of these two SCS-BPs at the high level of SCS in

How-an aged brain remains to be elucidated Therefore, the presentstudy has been carried out to characterize the SCS-dependentphosphorylation 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 PKCand CK were detected in the TP fraction; and (iii) p82 and p55was identified as eIF-4B and syndapin 1, respectively These resultssuggest that the accumulated high levels of SCS preferentiallyinduces the CK1-mediated high phosphorylation of TP, eIF-4Band syndapin 1, which are involved in the mechanisms of variousother neuronal diseases including Alzheimer’s disease, and selec-tively suppresses their phosphorylation by PKC and CK2 in thehigh aged rat brain

1Connective Tissue Biochemistry, Caen, FRANCE,2Saint-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 organizaregula-tion and possibly

for other RhoB-dependent responses

C1-38 Neurosteroids protect neural-crest derived cells from apoptosis, tempospatially activating prosurvival kinases

I Charalampopoulos1, C Tsatsanis2, B Vergou1, I Alexaki3,

E Castanas3, A Margioris2and A Gravanis1

1Department of Pharmacology, School of Medicine, University ofCrete, Heraklion, GREECE,2Department 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-derivedPC12 cells, via G protein-associated specific membrane bindingsites 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 ofDHEA 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-apoptoticeffects of DHEA in serum-deprived PC12 cells; (ii) the three kinaseinhibitors completely reversed the induction by DHEA of the anti-apoptotic proteins Bcl-2 and Bcl-xL, as well as the activation ofprosurvival 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, suggestingthe involvement of recently described DHEA specific membranebinding sites These findings suggest a strong neuroprotective rolefor neurosteroids during brain development and ageing

Acknowledgement: Supported by a grant from the PENED03-ED372

GGET-C1-40 Expression and function of arylhydrocarbon receptor in growth plate chondrocytes

M Widerak, K Tahiri, M Dumontier, M Corvol and

J SavouretInserm UMR-S 747 Universite´ Paris 5, Paris, FRANCEArticular (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 expressionand function of AhR in cartilage are not known and this is theaim of the present study Quantitative RT-PCR experiments showthat AhR mRNA is present as non measurable traces in AR and

GR chondrocytes in basal conditions Basal expression of AhRwas robustly induced by Interleukin-1beta (IL-1B, 20h) in GR cellscultured in normoxia (21% oxygen) and to a minor extent in hyp-oxia (0.5% oxygen) IL-1B was inefficient on AR cells We usedthe endogenous gene cytochrome P450–1A1 (CYP1A1) to monitorthe 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 CYP1A1which was further increased after TCDD treatement, in normoxiabut not in hypoxia These observations show that AhR expressionand functionality is restricted to GR chondrocytes in normoxicconditions and cytokine-dependent Our results also suggest thatAhR ligands may exert disruptive effects on growth cartilage, dur-ing development and/or inflammatory processes

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 A2Aadenosine 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 A2A-receptor may serve as a model for the regulation of

A2A-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

A2A-receptor in nervous tissue In addition, coupling of the

recep-tor to its canonical effecrecep-tor 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 A2A-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 Jeyaprakash1, U R Klein2, E A Nigg2and E Conti1,2

1European Molecular Biology Laboratory, Heidelberg, GERMANY,

2Max-Planck-Institute of Biochemistry, Munich, GERMANYThe chromosomal passenger complex (CPC) is an essential regula-tor of mitosis The CPC coordinates multiple chromosomal andcytoskeletal 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, thecomplex moves from the inner centromere to the central spindleduring the metaphase-anaphase transition, and finally translocates

to the midbody during cytokinesis Survivin, Borealin and CENP are the three components of the CPC that regulate theactivity and localization of its enzymatic component, the kinaseAurora-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

IN-We have engineered structure based mutants to dissect the CPCinto different subcomplexes siRNA rescue experiments withmutants reveal that the CPC functions as a single structure unitand the intertwined structural interactions of the core componentslead to a functional interdependence Association of the regulatory

‘passenger’ subunits creates a helical bundle, whose compositemolecular surface presents conserved residues essential for centralspindle and midbody localization

C1-44 Increased stability compensates for lower heparin-binding affinity of FGF-1 mutants

M Zakrzewska1,2, A Wiedlocha2, D Krowarsch1, J Otlewski1and S Olsnes2

1Faculty of Biotechnology, University of Wroclaw, Wroclaw,POLAND,2Department of Biochemistry, Institute for CancerResearch, Oslo, NORWAY

FGF-1 is a powerful signaling molecule with a relatively shorthalf-life in vivo and a denaturation temperature close to physiolo-gical It is widely believed that an essential component of theFGF/FGFR signaling complex is heparin We gradually intro-duced stabilizing mutations into the K132E-FGF-1 variant, whichwas previously shown to be inactive in DNA synthesis stimula-tion, probably due to its lower affinity to heparin We found thatstabilizing mutations of FGF-1 can compensate for the reducedheparin binding in mitogenic activity in NIH3T3 cells Weobserved gradual increase in thymidine incorporation up to thelevel obtained with the wild-type of FGF-1 in the presence ofheparin Neither construct exhibited any difference, compared tothe 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 ity protein against heat and proteolytic degradation and that hep-arin is not crucial in direct FGF/FGFR interaction

Investigation of the phagocytosis signaling in

tissue transglutaminase deficient macrophages

B Toth1, D Aeschlimann2, L Fesu¨s1and Z Szondy1

1University of Debrecen, Debrecen, HUNGARY,2Cardiff

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´tori2, M Ga´csi1and F Erdo}di2

1Department of Experimental Zoology, Balaton Limnological

Research Institute, HAS, Tihany, HUNGARY,2Institute 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 PizzoDuke University Medical Center, Durham, NC, USAPlasminogen (Pg) is the precursor of angiostatins, a group of anti-angiogenic Pg fragments containing lysine- or benzamidine bindingsites inside double looped disulphide structures called kringles Allfive Pg kringles bind lysine, whereas only kringle 5 (K5) bindsbenzamidine In addition, there are two more benzamidine bindingsites in the Pg serine protease domain Both voltage-dependentanion channel (VDAC) and the glucose-regulated protein GRP78are receptors for K5 We found VDAC co-localized with GRP78

on the surface of human prostate tumor 1-LN cells To ate functions of these proteins, either singly or as a complex, weused 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 thethird benzamidine binding site of Pg K5 induces a Ca2+signalingcascade only through VDAC, whereas microplasminogen does itvia GRP78 We demonstrate the existence of a mechanism invol-ving interaction of HK-I and K5 with VDAC, which may function

differenti-to protect cells from apopdifferenti-tosis We also show evidence suggestingthat GRP78 binds to Pg K5 through a region localized in the

NH2-terminus of GRP78, via a mechanism that may keep Pg in anactivation-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 NievesUniversity of Barcelona, Barcelona, SPAIN12-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 growthinduced by serum and that 12-(S)-HETE as well as 5-(S)-HETEand 15-(S)-HETE are mitogenic on 3T6 fibroblast (1) In this work

we study the effect of both enantiomer on cell proliferation Ourresults show that only 12-(S)-HETE was able to induce cell growthand DNA synthesis in 3T6 fibroblast cultures whereas 12-(R)-HETE was inactive Furthermore, we observed that mitogeniceffects of 12-(S)-HETE were correlated with the enhancement ofERK1/2 and AKT phorphorylation whereas 12-(R)-HETE was noteffective on these signal transduction pathways involved in the con-trol of 3T6 fibroblast growth Moreover, we observed that theseeffects can occur through a specific receptor sensitive to pertussistoxin but not identified yet

Acknowledgement: Supported by MEC (BFU2004-04960).Reference

1 Nieves D, Moreno JJ (2006) J Lipid Res 47:2681–2689

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

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 Ca2+

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)2fragments 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 Ca2+ The Ca2+ 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)2from 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 FeltyFlorida International University, Miami, FL, USAEstrogen (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 ously showed that E2-induced DNA synthesis depends on oxidantsignaling Inhibitor of DNA binding protein 3 (Id3) is a redox-sen-sitive gene that mediates vascular lesion formation We propose totest the concept that estrogen-induced vascular lesion formation ismediated by redox sensitive Id3 signaling In this study we exam-ined whether E2-induced endothelial tube formation depends onId3 Endothelial tube formation was significantly inhibited to thelevel of control by overexpression of both MnSOD and catalase aswell 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 showedthat E2-induced oxidants increased Id3 phosphorylation AndRNA interference of Id3 markedly inhibited E2-induced tube for-mation In conclusion, early E2 signaling does not require estrogenreceptor genomic signaling because we can inhibit tube formation

previ-by antioxidants These studies demonstrate that Id3 is an ant signaling molecule in E2 stimulated vascular lesion formationthat may be a useful therapeutic target in the prevention and treat-ment of vasculoproliferative disorders

import-C1-52 Pro- and antioxidant properties of mitochondria-targeted antioxidant mitoQ

D S Izyumov, E V Mostovenko, M V Korotetskaya and

B V ChernyakA.N.Belozersky Institute of Physico-Chemical Biology, MSU,Moscow, RUSSIAN FEDERATION

Mitochondria play a key role in production of reactive oxygen cies (ROS), which take part in signal transduction and cell death

spe-We have studied influence of mitochondria-targeted antioxidantmitoQ, 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 oxidativestress and apoptosis at very low concentration but this processneed a long (about 8 days) preincubation At the same time wehave shown that fluorescent analogue of mitoQ have accumulates

in HeLa during 1–2 h At high concentrations mitoQ caused ficant ROS production and apoptosis Non-toxic concentrations ofmitoQ promoted ROS production and cell death in combinationwith low concentrations of H2O2 and these effects were suppressed

signi-by antioxidant N-acetylcysteine (NAC) indicating that toxicity ofmitoQ was determined by its prooxidant properties Also we haveinduced ROS production using Mitotracker Red, a fluorescent dyewhich is selectively accumulated in mitochondria as a photosensi-tizer Low or high doses of illumination caused secondary ROSproduction and apoptotic or necrotic cell death, simultaneously.MitoQ didn’t affect apoptosis but completely suppressed necrosis.NAC was less effective than mitoQ Mild illumination causedapoptosis, which were insensitive to mitoQ Strong illuminationcaused damage of mitochondria by ROS and necrosis which wasinhibited with mitoQ Thus we showed pro- and antioxidant activ-ity of mitoQ

Calcium-dependent interaction of calmodulin

with synapse-associated proteins of the

MAGUK family

M Konrad1, A Lavie2and I Paarmann3

1Max-Planck-Institute for Biophysical Chemistry, Goettingen,

GER-MANY,2University 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 Bondioni1, G Mantovani1, L Alberti2, C Invitti2, S Corbetta3,

E Peverelli1, A Lania1, P Beck-Peccoz1and A Spada1

1University of Milan, Milan, ITALY,2Istituto Auxologico Italiano

IRCCS, Milano, ITALY,3Policlinico 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

(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 adiponecadiponec-tin 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 SoldatovNational Institute on Aging, Baltimore, MD, USAThe voltage-gated calcium channel is a multi subunit signalingcomplex 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 proteinkinase C activation and cAMP dependent transcription in nuclei isimportant, but not well defined signaling event in excitable cells

By combining FRET microscopy with patch clamp in recombinantexpression system we were able to show the importance of voltage-dependent conformational changes of the pore-forming a1Csubunit

of the Cav1.2 calcium channel in excitation-transcription coupling

We developed a novel 2D wavelet-based image analysis to deciphercalcium channel -dependent signaling pathways The combination

of pixel-by-pixel and 2D wavelet analysis allowed us to reveal amicrodomain organization of the calcium channel plasma mem-brane-activated PKC signaling and intranuclear activation ofcAMP dependent transcription This approach may serve as aframework for intracellular signaling analysis

C1-56 Liposomes as possible carriers for anti-inflammatory and antitumoral compounds

A M Roseanu, F Chelu, M Moisei and M TrifInstitute of Biochemistry, Bucharest, ROMANIALiposomes are efficient carriers for controlled drug delivery andlocal targeting of therapeutic agent to the site of interest

Lactoferrin (Lf) is an iron-binding glycoprotein with potent inflammatory and antitumoral properties The aim of our studieswas to investigate whether the entrapment of Lf in liposomes couldimprove its anti-inflammatory and antitumoral effects The experi-ments were performed in vitro, using human monocytic THP-1cells and murine melanoma B16-F1 cells Previous studies demon-strated that Lf entrapped in liposomes is accumulated by the cellsmore efficiently than the free protein Liposomal formulationincreased the capacity of Lf to affect B16-F1 cell growth and toinduce morphological modifications associated to apoptosis, such

anti-as rim of the cytoplanti-asm, nuclear condensation and fragmentation,appearance of apoptotic bodies The mechanism is suggested toinvolve modulation of the expression of JNK, p-38, ERK 1/2MAPkinase, proteins implicated in cell proliferation and apoptosis

In the case of THP-1 cells, entrapment of Lf in liposomesenhanced the protein ability to reduce pro-inflammatory cytokinesIL-6, TNF-a and IL-8 release mediated by LPS Our resultsrevealed the property of liposomes to enhance the intracellularactivity 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 TECH research program, project CEEX 57/2006(NANOCON-TER)

Investigation of rapid nongenomic effects of

1a,25(OH)2D3 on intracellular calcium in human

peripheral blood mononuclear cells

I Lajdova1, D Chorvat2, V Spustova1and A Chorvatova3

1Slovak Medical University, Bratislava, SLOVAKIA,2International

Laser Centre, Bratislava, SLOVAKIA,3University 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

[Ca2+]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 [Ca2+]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

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 Martynova1, F Eroshkin1, G Ermakova1, A Korotaeva1,

K Smurova2, F Gyoeva3and A Zaraysky1

1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,

Moscow V-437, RUSSIAN FEDERATION,2Lomonosov Moscow

State University, Moscow, RUSSIAN FEDERATION,3Institute 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 movemdiffer-ents

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 vriesThe 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 tothe presence of drug efflux transporters in the blood brain barrier(BBB) restricting the entry of many potentially useful therapeuticdrugs Identifying deranged molecular pathways driving gliomatumor growth resulted in clinical testing of rationally designedmolecular-targeted agents However, many of these agents are sub-strates of the drug transporters P-glycoprotein (P-gp) and/orBreast Cancer Resistance Protein (BCRP) Therefore, appropriatemodels for preclinical in vivo evaluation of such agents shouldcarry the genetic signature of human disease and grow behind anintact BBB, to predict clinical efficacy more accurately than thetraditional used xenograft models We have generated spontaneoushigh-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 mediatingastrocyte-specific expression of Cre Bioluminescence is used tomonitor 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 themajor cell signaling pathways This information can be used toinvestigate the efficacy of (a combination of) agents that targetthese pathways Moreover, by using mice deficient for P-gp andBCRP we can establish whether it will be useful to combine theseagents 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 Lee1, J Lee2and S Kang3

1Laboratory of Biophysics, School of Biological Sciences , SeoulNational University, Seoul, REPUBLIC OF KOREA,2Institute ofMicrobiology, Seoul National University, Seoul, REPUBLIC OFKOREA,3School of Biological Sciences, Institute of Microbiology,Seoul National University, Seoul, REPUBLIC OF KOREACandida 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 geneSKN7 from Candida albicans and its encoding protein Skn7 wasidentified and analyzed To study the roles of SKN7, we knockedout SKN7 gene The skn7/skn7 C albicans mutants are more sensi-tive to oxidative stresses, such as H2O2 and menadione, as like

S cerevisiae skn7mutants Also, In the skn7/skn7 disruptants, theformation of germ tube require shorter time than that in the con-genic wild-type strain, but the mycelium grow slower in variousliquid media Compared with the congenic wild-type strain, skn7/skn7disruptants 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) ofthe SSA1 promoter C albicans Skn7 also contains DNA-bindingdomain and the promoters of those genes have HSEs We showedthat Skn7 can bind to the HSE within the promoters of HWP1gene 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

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 Park1, J Oh2and J Han1

1Dept of Biochemistry and Molecular Biology, College of Medicine,

Hanyang University, Seoul, REPUBLIC OF KOREA,2Department

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

(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¨ rvUniversity of Tartu, Tartu, ESTONIAAsymmetric and synergistic interactions between cAMP-dependentprotein kinase catalytic subunit, its substrates (ATP and kemptide)and inhibitors (H-89, kemptide Ala-analogue LRRAALG-NH2and peptide-nucleoside conjugate inhibitor AdcAhxArg6) werequantified in terms of binding effectiveness of these ligands withthe 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 procedureavoids complications related to the complex catalytic mechanism

of the protein kinase catalyzed reaction It was found that in somecases 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 ‘‘betterbinding - stronger synergism’’ was formulated for cAMP-depend-ent protein kinase catalytic subunit on the basis of this analysisand some linear-free-energy relationships between synergistic effectand ligand affinity were discovered

C1-64 Ghrelin signaling to ERK 1/2: role of G-proteins and beta-arrestins

M Lodeiro1, O Ischenko1, A C Martini2, F F Casanueva1and

J P Camina1

1Laboratory of Molecular Endocrinology, Research Area, ComplejoHospitalario Universitario de Santiago (CHUS) and Department ofMedicine, University of Santiago de Compostela, Santiago de Com-postela, SPAIN,2Physiology Institute, School of Medicine, CordobaNational Institute, Cordoba, ARGENTINA

Ghrelin, an acylated peptidyl gastric hormone, regulates GHrelease, 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 theactivation of MAPK were investigated in HEK 293 cells stablytransfected with the ghrelin receptor GHS-R1a One pathway ismediated 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 Gq/11-dependent and involves

a PKCa/b and Src A third pathway is Gi-dependent and involvesPI3K, PKCe and Src Our study reveals that Gi/o- and Gq/11-pro-teins are crucially involved in the b-arrestin-mediated ERK 1/2activation

Acknowledgements: This work was supported by grants fromthe FIS and the Instituto de Salud Carlos III, Ministerio deSanidad y Consumo and the Secretaria Xeral de Investigacion eDesenvolvemento, Xunta de Galicia (Spain)

Identification of the phosphorylation site of the

histidine kinase of E coli AtoS-AtoC

two-component system

P S Filippou1, L D Kasemian1, C A Panagiotidis2and

D A Kyriakidis1,3

1Laboratory of Biochemistry, Department of Chemistry, Aristotle

University of Thessaloniki, Thessaloniki, GREECE,2Department of

Pharmaceutical Sciences, Aristotle University of Thessaloniki,

Thes-saloniki, GREECE,3The 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

atooperon expression upon acetoacetate induction

C2-1

Deciphering the kinome in basal-like breast

carcinoma for therapeutic usefulness

B Marty1, F Djelti1, I Lebigot1, A Vincent-Salomon1,

F Cruzalegui2, G Tucker2, X Sastre1, J Thiery1, J Hickman2

and T Dubois1

1Institut Curie, Paris, FRANCE,2Institut 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-regulaactiva-ted 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 KikkawaNagoya University Graduate School of Medicine, Nagoya, JAPANHyperglycemia or hyperinsulinemia contributes to poorerendometrial 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 apoor prognosis in endometrial adenocarcinoma patients The aim

of this study was to examine whether the malignant potential ofendometrial cancer enhanced by P-LAP/IRAP is due to increasedglucose uptake via the P-LAP/IRAP-mediated activation of insulinsignaling We transfected P-LAP/IRAP cDNA into A-MEC cells(endometrial adenocarcinoma cell line), and A-MEC-LAP cellsexpressed a remarkably high level of GLUT4 proteins.3H-2-deoxy-glucose uptake which responds to insulin in A-MEC-LAP cells wassignificantly higher than that of A-MEC-pc cells A-MEC-LAPcells exhibited a significant growth-stimulatory effect compared toA-MEC-pc cells A-MEC-LAP cells expressed a remarkably highlevel of p85PI3K protein compared to A-MEC-pc cells, andshowed a higher degree of AKT phosphorylation by insulin stimu-lation In summary, P-LAP/IRAP was involved in the increasingmalignant potential of endometrial cancer mediated by insulin.P-LAP/IRAP was suggested to be a potential new target ofmolecular-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-LaviadaUniversity of Alcala, Alcala de Henares, SPAIN

In the present study, we determined the effects of capsaicin in theintracellular ceramide accumulation, and investigated the roles ofextracellular 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-independenthuman prostate cancer PC-3 cell line Here we report that capsai-cin apoptotic effect was mediated by ceramide generation whichoccurred by sphingomyelin hydrolysis We next confirmed thatcapsaicin could activate ERK and JNK but not p38 Pharmacolo-gical inhibition of JNK kinase, as well as inhibition of ROS by thereducing agent N-acetylcysteine, prevented ceramide accumulationand capsaicin-induced cell death However, inhibition of ceramideaccumulation by the SMase inhibitor D609 did not modify JNKactivation These data reveal JNK as an upstream regulator of cer-amide production Capsaicin-promoted activation of ERK wasprevented with all the inhibitors tested We conclude that capsaicininduces apoptosis in PC-3 cells via ROS generation, JNK activa-tion, ceramide accumulation and secondly, ERK activation

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, transactivainterac-ted

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 Staber1, P Vesely1, C Fuchs1, S Schauer1,

D W Sternberg2and G Hoefler1

1Medical University Graz, Graz, AUSTRIA,2Mount 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 expresexpres-sion, 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 Vesely1, P B Staber1, C Fuchs1, S Schauer1, H Bergler2,

D W Sternberg3and G Hoefler1

1Medical University Graz, Graz, AUSTRIA,2Karl-FranzensUniversity Graz, Graz, AUSTRIA,3Mount Sinai School ofMedicine, New York City, NY, USA

Anaplastic large cell lymphomas (ALCL) are highly proliferatingtumors and commonly express the AP-1 transcription factor JunB

In most cases of ALCL, the fusion tyrosine kinase NPM-ALK ispresent 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 PI3Kinhibition by LY294002 or mTOR inhibition via rapamycin results

in a significant decrease of JUNB protein without affecting itsmRNA level Downregulation of JunB protein leads to inhibition

of proliferation in NPM-ALK positive cells To clarify the lar mechanism of the JunB regulation via PI3K/mTOR we usedpolysomic preparations of ALCL and fibroblast cell lines Thereby

molecu-we found a distinct mechanism regulating JunB translation: JUNBmRNA 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 inthe untranslated region of the JUNB mRNA which is involved inthis regulatory process Our findings reveal that JUNB is a criticaltarget of mTOR and highlight its translational deregulation viaNPM-ALK This is the first study to demonstrate translationalcontrol of a full length AP-1 transcription factor

C2-7 BRAFV600E mutation and oncogenic activation

of MAP kinase by its pseudogene in thyroid tumors

M Zou1, E Y Baitei1, A S Alzahrani1, F A Al-Mohanna1,

N R Farid2, B Meyer1and Y Shi11

King Faisal Specialist Hospital and Research Centre, Riyadh,SAUDI ARABIA,2Osancor Biotech Inc, London, UKActivating BRAF mutation in papillary thyroid carcinoma (PTC)has recently been reported in many studies ranging from 28% to83% 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, 43classic PTCs, six follicular variants of PTC (FVPTC), and threeanaplastic thyroid carcinomas (ATC) BRAF V600Emutation wasdetected in 20 out of 43 PTC (46.51%), and all the three ATC(100%) No mutation was found in 16 multinodular goiters, andsix FVPTCs There is higher frequency of BRAF mutation in clas-sic PTC patients with stages III and IV tumors (12/16, 75%) ascompared to stages I and II tumors (8/27, 29.63%) (P < 0.05,Fisher’s exact test) Interestingly, BRAF pseudogene transcriptswere 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 morefrequently detected in tumors without BRAF mutation (20/27,

(P < 0.01) Furthermore, overexpression of BRAF pseudogene inNIH3T3 cells could activate MAP kinase signaling pathway, trans-form NIH3T3 cells in vitro, and induce tumors in nude mice Thesedata suggest that BRAF mutations are specific to classic PTC andcontribute to the disease progression to poorly differentiated andanaplastic carcinoma BRAF pseudogene activation may also play

an important role in early stage of thyroid tumor development

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 expresoverexpres-sion 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 activmotil-ity 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 Rogalinska1, P Goralski1, J Bednarek1, J Z Blonski2,

J Wesierski-Gadek3, H Piekarski1, M Hanausek4, Z Walaszek4,

T Robak2and Z M Kilianska1

1University of Lodz, Lodz, POLAND,2Medical University of Lodz,

Lodz, POLAND,3Medical University of Vienna, Vienna, AUSTRIA,

4University 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 ± 3C 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 Bednarek1, J Wesierski-Gadek2, M Rogalinska1,

J Z Blonski3, T Robak3and Z M Kilianska1

1University of Lodz, Lodz, POLAND,2Medical University ofVienna, Vienna, AUSTRIA,3Medical University of Lodz, Lodz,POLAND

B-cell chronic lymphocytic leukemia (B-CLL) is a disease terized by an accumulation of long-lived, neoplastic B-lympho-cytes Purine analogs (cladribine, fludarabine) have indicated highactivity against the disease and evidence shows that in B-CLL cellsthese agents exert their cytotoxic effect by induction of apoptosis.Recently, another purine derivative, roscovitine, a selective CDKinhibitor, effective inducer of apoptosis in a large number of can-cers The purpose of this study was to determine chemosensitivity

charac-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 Fwith M are equivalent to that applied in clinical studies, described

as CC and FC program, respectively Exposure of B-CLL cells tothe distinct agents alone, or in combinations strongly affected theirviability Treatment with the CM and FM resulted in the highestreduction of the number of viable cells Interestingly, similar effectwas observed during the incubation of leukemic cells with ROSCalone The tested agents differentially affected expression of apop-tosis-related proteins (caspase-3, caspase-9, Mcl-1 and Bax) andtheir activity status All studied agents, especially ROSC as well asboth used drug combinations strongly reduced the levels of Mcl-1protein, increased Bax/Mcl-1 ratio and cyt c translocation Weconclude that application of ROSC efficiently induces apoptosis ofB-CLL cells, similarly to drug combinations Since ROSC is notgenotoxic, 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 Geiger1, M J Ausserlechner2and P Obexer1

1Tyrolean Cancer Research Institute, Innsbruck, AUSTRIA,

2Molecular 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 forapproximately 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 tothe fact that the majority of aggressive neuroblastoma do notexpress caspase 8 as a result of epigenetic silencing by promoter hy-permethylation, we further investigated the potential function ofFKHRL1 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 8re-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 thatactivation of FKHRL1 and expression of caspase 8 together appear

to be essential to enhance TRAIL-induced apoptosis in stoma The combined data indicate that in neuroblastoma caspase

neurobla-8 is an important regulator in the apoptosis pathway induced byactivated FKHRL1

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, H2O2, 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

Ca2+-dependent calpain-dependent caspase-12 activation

Acknowledgement: Supported by MEC (BFU2004-04960)

M Cha, M Lee and H ParkKyungnam University, Masan, REPUBLIC OF KOREAGleditsiae Semen (GS) has been used in both Korea and China asherbal medicine for the treatment of cephalalgia, catharsis, andother 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 thatGSE induced cytotoxicity in HT-29 cells in a dose-dependent man-ner, and this effect was verified via a lactate dehydrogenase releaseassay and a colony formation assay In particular, HT-29 cellsshowed extensive cell death when treated with 50 lg/ml of GSE;the calculated IC50 value was 20 lg/ml It induced characteristicapoptotic signs in HT-29 cells, including chromatin condensationand DNA fragmentation, occurring within 6–24 h when the cellswere treated at a concentration of 50 lg/ml Interestingly, wedetected the activation of caspase-3 and -9, but not caspase-8, andapoptotic bodies in GSE-treated HT-29 cells Collectively, ourresults indicate that GSE induces apoptosis via a mitochondria-mediated apoptotic pathway, and these findings may be significantwith regard to the development of a new drug for the treatment ofhuman colon carcinoma cells

C2-15 Effect of usnic acid on tissue caspase activity and glutathione level in titanium-implanted subjects

F Odabasoglu1, H Aygun2, O S Yildirim2, Z Halici3,

A Aslan4, A Cakir4, M Halici1and E Cadirci5

1Ataturk Univ, Fac of Pharm, Dept of Biochem, Erzurum,TURKEY,2Ataturk Univ., Fac of Med, Dept of Pediatr andTraumat, Erzurum, TURKEY,3Ataturk Univ, Fac of Med, Dept ofPharm, Erzurum, TURKEY,4Ataturk Univ, Fac of Educ, Dept ofBiol and Chem, Erzurum, TURKEY,5Ataturk Univ, Fac of Pharm,Dept of Pharm, Erzurum, TURKEY

Debris due to the frictions as well as biochemical and magneticreactions following orthopedic implantations may play a role inaseptic loosening through initiating a series of complex cellularreactions between bone and implant Loosening associates withincreased caspase activity (CAS, protease) The present study wasconducted to evaluate the effect of usnic acid (UA) on CAS andglutathione 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 orlocally every 3 day for 21 days or received none Rabbits from theother group served as control Following euthanasia, tissues aroundthe implant were scrapped and then ground within liquid nitrogenfor CAS and GSH There were 3, 3.5, 4.5, and 2-fold increases inactivities of CAS 2, CAS 3, CAS 8 and CAS 9 in Ti-implanted rab-bits compared to control rabbits, which increased further by bothoral and local administrations of UA and OO Olive oil was moreeffective than UA when administered locally, whereas UA was moreeffective than OO when administered orally Surgical interventionwas associated with a 31% reduction in GSH Local and oraladministration of UA and only local administration of OO elevatedthis reduction In conclusion, both UA and OO stimulate apoptosisvia increasing CAS and increase GSH Proapoptotic properties ofthese compounds should be considered in cancer research

Breast cancer tumor suppressor BRCA1

regulates caspase 3 activation

M Ouchi1, S Martin2, J Aglipay2and T Ouchi1

1ENH, Northwestern University, Evanston, IL, USA,2The 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 Lee1, J Jeong1, M Cho1, J Lee1, H Kim2, Y Yun2and

H Lee1

1

Seoul National University, Seoul, REPUBLIC OF KOREA,2Ewha

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 ofDNp63a 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 Benosman1, I Gross2, N Clarke3, A G Jochemsen4,

K Okamoto5, J Loeffler1and C Gaiddon1

1Louis Pasteur University - INSERM U692, Strasbourg, FRANCE,

2INSERM U682, Strasbourg, FRANCE,3School of Pharmacy, versity of Nottingham, Nottingham, UK,4Department of Molecularand Cell Biology, Leiden University Medical Center, Leiden, THENETHERLANDS,5National Cancer Center Research Institute,Radiobiology Division, Tokyo, JAPAN

Uni-During the last decade, extensive data described the mechanisms ofcell death in various cellular contexts Yet, the regulation of neur-onal fate remains poorly explored Among identified players inNeuronal 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-mitoticneurons, we characterized multiple death-inducing conditions thatactivate 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 Potassiumdeprivation, Glutamate or APP-stimulating antibody (a model toAlzheimer’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 bothp53 and E2F-1 transcriptional activity and rescued neurons fromdeath, while shRNA directed against MDMX increased neuronaldeath in the above mentioned conditions Therefore, MDMXappears 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 Pivonˇkova´, P Pecˇinka, O Ticha´ and M FojtaInstitute of Biophysics, Academy of Sciences of Czech Republic,v.v.i., Brno, CZECH REPUBLIC

The tumor suppressor protein p53 belongs to the checkpointproteins 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 interactwith DNA It was found that p53 bound DNA sequence-specific-ally or sequence-nonspecifically to both positively (2) or negativelysupercoiled DNA (3, 4) This work was focused on study of super-coil-selective p53-DNA interactions using immuprecipitation atmagnetic 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 inpresence of higher concentration of CQ, DNA relaxed and p53bound relaxed and linDNA with the same affinity Due to furtherincreasing of CQ concentration, DNA became positively super-coiled and the preference for this DNA was remarkably higheragain Using the same technique we observed that the p53 proteincan 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)

Mutant p53 proteins bind selectively

supercoiled DNA

K Neˇmcova´, M Bra´zdova´, L Cˇincˇa´rova´, P Sˇebest,

H Pivonˇkova´, O Ticha´, M Fojta and E Palecˇek

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

Rapamycin induces p53-dependent apoptosis in

human prostate cancer cells via PI3K pathway

S Numanoglu1, C Biray Avci1, S Yilmaz1, G Saydam2and

C Gunduz1

1Ege University School Of Medicine, Dept of Medical Biology,

Izmir, TURKEY,2Ege 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 IC50 doses of 10, 25,

50 nM, respectively Rapamycin have shown remarkable apoptosis

at 72ndhour 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 diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

K Kim, J Song and Y ParkPusan National University School of Medicine, Busan, REPUBLIC

OF KOREAThe Diphenyleneiodonium (DPI) is widely used as an inhibitor offlavoenzymes, particularly NADPH oxidase In this study, we inves-tigated the effect of DPI on the apoptosis of human RPE cells DPItreatment in ARPE-19 cells evoked a dose- and time-dependentgrowth 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, whichinduces proapoptotic genes in response to DNA damage or irrepar-able cell cycle arrest ROS have been implicated as a key factor inthe 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 levelfrom cells treated for 24 h with DPI, it was not detectable at earlytime points, required to induce p53 expression And DPI-inducedp53 expression was not affected by the ROS scavenger NAC Weconclude 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 Trichet1, C Chipoy1, B Brounais1, P Juin2, F Re´dini1,

D Heymann1and F Blanchard1

1INSERM ERI-7, Nantes, FRANCE,2INSERM U601, Nantes,FRANCE

Oncostatin M (OSM), a cytokine of the IL-6 family, reduces thegrowth and induces differentiation of osteosarcoma cells Giventhe strong interaction between differentiation and apoptosis, weasked here whether OSM could regulate apoptosis of normal ortransformed osteoblasts Alone, OSM did not induce cell death,but OSM-treated osteosarcoma cells or proliferating osteoblastswere particularly more sensitive to apoptosis induced by differentdeath inducers among which staurosporine (STS) Cell deathinduced by OSM+STS was associated with activation of caspase 9and 3 and prevented by the caspase inhibitor Z-VAD-FMK OSMalone 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 isimplicated in reduction of Bcl-2 expression, whereas p53 mediatesenhanced Bax expression By co-immunoprecipitation, we observed

a constitutive interaction between p53 and the C-terminal activation domain of STAT5 Thus we characterize a new signalingpathway (based on p53-STAT5 interaction) used by OSM toincrease Bax and decrease Bcl-2 expression, and thus controllingthe mitochondrial cell death pathway In parallel, strong anti-apoptotic signals are also activated by OSM, via the PKCd andPI3K/Akt pathways Therefore association of OSM with the kinaseinhibitor STS could represent new treatments for wild type p53osteosarcoma

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

trolled by two EWS-FLI1 suppressed growth factor pathways

con-verging on p53 in ESFT cells

FWF grant 18046

C2-26 p53 and rb (retinoblastoma) gene expression in tumoral cells, under the influence of a grape extract

A Niculescu1, L Ghetea1, R Motoc1, G Mihaescu1,

R Huculeci2, C Diaconu3, C Ursaciuc4and G Savi4

1Institute of Genetics-University of Bucharest, Bucharest,ROMANIA,2Multiple Users Research Base – Molecular Biology,University of Bucharest, Bucharest, ROMANIA,3Institute ofVirology ‘‘Stefan S Nicolau’’, Bucharest, ROMANIA,4NationalInstitute for Research & Development in Pathology and BiomedicalSciences ‘‘V Babes’’, Bucharest, ROMANIA

The aim of this study was to establish the in vitro and in vivoeffects of a grape extract (GSE) on Wistar rat Walker sarcoma 256ascitic cells The treatments with GSE were administrated in differ-ent doses and time intervals The analyses were performed by flowcytometry, for the study of apoptosis dynamics, and by Real-TimePCR, for p53 and rb gene level expression detection The resultsobtained revealed that in vitro treatment with GSE increased thenumber of apoptotic cells by increasing the treatment time Themaximum percentage of apoptosis was obtained after 72 h of treat-ment (50,40%), while the control culture presented a percentage of3.45% of apoptotic cells Real-Time PCR analysis showed that theexpression level increased for both genes, at two concentrations ofthe GSE used in the experiments, indicating a increased transcrip-tion activity for p53 and rb The most significant result, for the

in vivoexperiments, was an increase of the life span (by a factor oftwo) 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 resultssuggest that, in vivo, GSE could have a stimulating effect mainly

on the immune system which become more efficient in decreasingthe 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 KimPukyong National University, Busan, REPUBLIC OF KOREAThe purpose of this study is to examine the effect of laurinterolfrom Laurencia okamurai on induction of apoptosis in melanomacells (B16F1) First of all, the effect of laurinterol on cell viabilitywas evaluated by MTT assay Apoptosis induced by laurinterol wasanalyzed using DNA fragmentation, TUNEL assay and caspaseassay in melanoma cells Laurinterol exhibited excellent effect onthe induction of apoptosis compared with etoposide used as posit-ive control in this study It was also observed that transcriptionalactivation of p53, a tumor suppressor gene, by laurinterol wasinvolved 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 therapeuticpotential to develop a novel anti-cancer agent

p53 suppresses cyclooxygenase-2 expression

through down-regulating inducible nitric oxide

synthase in mouse embryonic fibroblast

S Wada1, S Kokura1, Y Naito1, H Ichikawa1, H Ohshima2and

T Yoshikawa1

1Kyoto 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 HanCollege of Medicine Hanyang University, Seoul, REPUBLIC OFKOREA

Phospholipase (PLD) catalyses the hydrolysis of line to generate phosphatidic acid (PA) and choline There are atleast two PLD isozymes, PLD1 and PLD2 Genetic and pharmaco-logical approaches implicate that both PLD isozymes are involved in

phosphatidylcho-a diverse rphosphatidylcho-ange of cellulphosphatidylcho-ar processes, including receptor signphosphatidylcho-aling,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 Inthis study, we examined the role of PLD in taxotere-induced apopto-sis in stomach cell lines; normal stomach cells (NSC) and stomachcancer cells (SNU 484) Taxotere treatment resulted in increase ofboth 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 intaxotere-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 ofchromosomal DNA and increased cell viability Concurrently, bcl-2expression was upregulated, and taxotere-induced activation ofprocaspase-3 was inhibited after PLDs transfection Treatment ofSNU 484 with PA, the product of PLDs, also resulted in upregula-tion of bcl-2 Although, PA-induced bcl-2 expression was blocked bymepacrine, an inhibitor of phospholipase A2 (PLA2), increased bcl-2expression by PA was not abrogated by propranolol, on inhibitor of

PA phospholyhydrolase (PAP) These results indicate that PLA2isclosely related with bcl-2 expression, but not with PAP, induced byPLD 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¨zMiddle East Technical University, Department of BiologicalSciences, 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 thehuman telomerase enzyme PCR-based TRAP is used to determineactivity of telomerase Doxorubicin resistant MCF-7/R were devel-oped from the sensitive breast carcinoma MCF-7 cell line anddevelopment 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 andresistant 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 selectedMCF-7 cells are 107 folds resistant to the drug and these cells overexpress MDR1 and MRP1 genes Doxorubicin application caused

a decrease in Bcl-2 expression level in sensitive cells although nochange was observed in resistant cells Interestingly Bcl-xL levelincreased in sensitive and resistant cells after 72 h incubation indoxorubicin where a slight increase was observed in resistant celllines Bax levels seemed to be unchanged hTERT levels seemed toincrease after 24 h of drug incubations and 72 h doxorubicin incu-bation caused a decrease in telomerase activity in parallel with asmall decrease in hTERT levels in both cells types

PUMA is induced in p16INK4A-expressing

T-ALL cells and increases sensitivity to

glucocorticoid- and Fas-induced apoptosis

M J Ausserlechner1, T Unterkircher1, M Deutsch2,

C Salvador1, M Holzner2, V Porto2and P Obexer2

1Department of Pediatrics, Medical University Innsbruck, Innsbruck,

AUSTRIA,2Tyrolean 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 Ca2+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 Ca2+

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 Ca2+ 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 Seres1, L Gibalova1, Z Sulova1, M Barancik2, J Sedlak3and

A Breier1

1Inst Molec Physiol Genet., SAS, Bratislava, SLOVAKIA,

2Inst Heart Res., SAS, Bratislava, SLOVAKIA,3Inst Exp Oncol,SAS, Bratislava, SLOVAKIA

Expression of drug transporting P-glycoprotein (P-gp) in neoplasticcells is most frequent cause of multidrug resistance (MDR) Wefound that P-gp positive L1210/VCR cells (R) are more sensitive

to high external concentration of Ca2+as parental L1210 cells (S)

We observed the more pronounced calcium uptake and differences

in intracellular localization of Ca2+for R cells Calnexin lular calcium dependent chaperone) was described to ensure matur-ation of P-gp We detected lower levels of calnexin in R as in Scells We detect also downregulation of inositol 1,4,5- triphosphatereceptor channel (IP3R) in R cells cultivated in the presence ofvincristine (VCR) when compared with R cells cultivated in theabsence of VCR However we did not observe any significantchanges in IP3R in S and R cells Resistance to cisplatin (cisPt)associated with downregulation of IP3R was assumed to be con-nected with reduction of apoptosis MDR cells often exhibit aresistance to apoptosis induced by chemotherapeutic agents CisPt

(intracel-is known as un-transportable by P-gp We found that R cells aremore sensitive to cisPt as S cells Interestingly, we detect that Rcells under application of cisPt were entering the apoptosis in alower extent than S cells In contrast, cisPt induced more pro-nounced necrosis in R than in S cells We found predominatingamount 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 toapoptosis 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 RoselovaInstitute of Hematology, Prague 2, CZECH REPUBLIC

To increase the understanding of the mechanism by which cer drugs cause toxicity in normal tissue, we have examined theability of 10 nM actinomycin D (ActD) to induce apoptosis inhuman PBL To specify the primary molecular damage and toascertain which proteins were affected by genotoxic drug action,

antican-we performed in vitro transcription assay for rRNA and RT-PCRand Western blots for thirty apoptosis-related genes and tumoursuppressors We found down-regulation of rRNA synthesis andsubsequent mitochondria-dependent apoptosis While the expres-sion of the majority of examined genes remained indifferentagainst ActD, the cellular level of p53 protein increased, upregulat-ing Puma and p21waf1 mRNA and protein Puma mediated apop-

destabilization likely originating in ARE binding nucleolin vage The stability of the level of Bcl-2 protein, independent of anmRNA decrease, suggests that the protection of anti-apoptoticBcl-2 against proteasomal degradation moderates the apoptoticprocess as well as a temporary increase of caspase-3 inhibitorp21waf1 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

clea-Acknowledgement: This work was supported by grant IGA VZ

00023736 from the Ministry of Health of Czech Republic

N-butyric acid induces p21waf1 controlled

caspase-3 activity in peripheral blood

lymphocytes

I Kalousek, B Brodska, P Otevrelova and P Roselova

Institute of Hematology, Prague 2, CZECH REPUBLIC

To reveal possible mechanism by which a potential

chemo-prevent-ive agent, HDAC inhibitor sodium butyrate (SB), may cause a

normal tissue damage we have examined the ability of 2 mM SB

to induce caspase-3 activity in human PBL The hardly detectable

expression of tumour suppressor p53 in quiescent PBL decreased

after the addition of SB and the expression of pro-apoptotic genes

Bak, Bax, Bid, Puma, Apaf-1 and Smac and anti-apopoptotic

Bcl-2, Bcl-xL, IAP and XIAP was indifferent against SB Cell death

was preceded by increase in expression of BimEL mRNA and by

bell-shaped variation of growth and caspase-3 inhibitor p21waf1

After 24 h of treatment of PBL with SB the profound decrease in

p21waf1 level and the degradation of the PARP was detected

Mit-ochondrial dysfunction was independent of the presence of

pan-ca-spase inhibitor and the cleavage of the PARP wasn’t abrogated by

ROS scavenger The finding that the p21waf1 protein diminution

was independent of the presence of caspase inhibitor and

correla-ted with p21waf1 mRNA decrease prompts speculations about the

significance of p21waf1 mRNA reversible interactions with mRNA

binding proteins for modulated inhibition of executive caspase

activity Conclusions: In PBL, 2 mM SB induced BimEL and

exe-cutive caspase activity independently of expression of p53 and

pro-duction of ROS The course of caspase-3 activation was controlled

by caspase inhibitory effect of p21waf1 likely regulated through

the p21waf1 mRNA transcription or stability

Acknowledgement: This work was supported by grant IGA VZ

00023736 from the Ministry of Health of Czech Republic

C2-37

Overexpression of p21Waf1/Cip1regulates

anticancer drugs-induced apoptosis in HeLa

cells

K Gluhova, Y Trizna, V Shchukin and N Rabaya

Institute of Theoretical and Experimental Biophysics RAS,

Pushchino, RUSSIAN FEDERATION

The experiments on the analysis of HeLa clones with different

lev-els of p21Waf1/Cip1expression were carried out in order to

charac-terize their sensitivity to different cytotoxic compounds commonly

using in anticancer therapy (methyl nitrosoguanidine (MNNG),

cisplatin, taxol, and tumor necrosis factor (TNF)) We found that

clones with elevated p21Waf1/Cip1 expression were more resistant

towards DNA damaging agents At the same time, taxol and

TNF, whose primary targets are cytoskeleton proteins and

mem-brane receptors respectively, markedly stimulated death in cells

with elevated levels of p21Waf1/Cip1 expression We then analyzed

distribution of cells treated with anticancer agents through the cell

cycle In case of DNA-damaging agents, elevated expression of

p21Waf1/Cip1, probably, is a cause of longer periods of check-points,

allowing cells to repair DNA breaks This is accompanied by cell

accumulation on the border between G1/S and G2/M and cell

death inhibition At early time-points of incubation with taxol

accumulation of cells in G2/M fraction occurred Inability of cells

with damaged cytoskeleton to fulfil mitosis causes their death

Since the population of transfected cells in G2/M fraction is larger

that in control, they die more extensively One could conclude that

an application of anti-cancer drugs has to be based on the

prelim-inary estimation of p21Waf1/Cip1expression in transformed cells

C2-38 Caspase-mediated p21Cip/WAF1 cleavage during 3-HK-induced apoptosis of U937 cells

R Yazdanparast and M MoosaviInst Biochem Biophys, Tehran, ISLAMIC REPUBLIC OF IRANWhen human myeloid leukemia cell line (U937) is exposed to thenovel anti-leukemic agent, called 3-hydrogenkwadaphnin (3-HK),both differentiation and apoptosis may occur It remains elusive tounderstand how cells display differential fates in response to a sin-gle stimulus To address this question, here we report the molecu-lar signaling pathways involved in forcing 3-HK-treated U937 cells

to either differentiation or apoptosis After 3-HK (15 nM) ment, a portion of cells adhered to the culture plates while othersremained in suspension The adherent cells showed macrophagescriteria such as NBT reduction, phagocytoses of latex particles andCD14 expression Immunoblotting studies revealed that early acti-vation of ERK1/2 pathway (3 h) along with p21Cip/WAF1(p21) andp27Kip1up-regulation were occurred in adherent cells In contrast,suspension cells showed the characteristics of apoptotic cellsaccompanied with activation of caspase-3, -8 and -9, and also up-regulation of Bax Interestingly, caspase-mediated p21 cleavageaccompanied with activation of JNK1/2 and p38 MAPK, but notERK1/2, were observed during apoptosis in the suspension cells

treat-In addition to getting a deeper understanding of anti-leukemiceffects of 3-HK in U937 cells, our results can have some light onpathways involved in drug-induced differentiation and apoptosis ofleukemia cells

Key words: 3-hydrogenkwadaphnin, Apoptosis, Caspase, entiation, Leukemia, MAP kinase, p21Cip/WAF1

Differ-C2-39 Regulation of human immunoglobulin J-peptide expression in non-small cell lung cancer (NSCLC)

N V Antipova, R I Dmitriev, M I Shakhparonov,

E P Kopancev, L L Zavalova and E D SverdlovShemyakin and Ovchinnikov Institute of Bioorganic Chemistry,Moscow, RUSSIAN FEDERATION

Lung cancer is known to be one of the most intensively gated tumors An increased expression of the immune system pro-teins is observed in the case NSCLC of that can be connected withthe active penetration of B-lymphocytes in the area of malignantneoplasm and presence of great amount of antigen-presenting cellsthere J-chain is an immunoglobulin-like single polymer withmolecular weight of 15 kDa For our experiments there were selec-ted the samples of tumor tissues from central and peripheral areas

investi-of lungs at the I-III stages investi-of carcinoma development which ded high- and low-differentiated tumor forms It was shown thatmajority of investigated patients suffering from NSCLC, withoutany dependence of the area and stage of illness, demonstrate pre-dominantly reduced J-chain expression There were observed adecrease in the J-chain content in the tumor tissues for 42% of theinvestigated samples, an increase for 27% and there were no differ-ence in the content for 31% of the investigated samples In thegroup with central localization of high-differentiated tumor thehalf of the NT sample pares hasn’t shown any changes in theJ-chain, and the rest of samples diverged equally between the regu-lation variants On the basis of these results it can be assumed thatthe low-differentiated tumor form is more regulated, and in thiscase the transformed cells better receive external signals, whetherthe tumor, consisted of high-differentiated cells, is in the isolated,more stable condition Thus, our results suggest that the J-chainexpression in immunocytes depends on the epithelium condition,which affection by NSCLC leads to predominantly decreasedJ-chain expression

1Institute of Molecular Biotechnology of the Austrian Academy of

Science, Vienna, AUSTRIA,2Department of Dermatology and

Inter-disciplinary Center of Clinical Research, University of Mu¨nster,

Mu¨nster, GERMANY,3Department of Immunohematology and

Blood Transfusion, Leiden University Medical Centre, Leiden, THE

NETHERLANDS

The immune system plays an important role in

tumor-immuno-surveillance, but is often compromised due to e.g insufficient

recognition, tolerance induction and immuno-evasion of tumors

Since tolerance induction during tumor growth is one major

prob-lem, identification of a key dominant ‘‘tolerogenic’’ factor in

T cells that directly controls activation of tumor-reactive cytotoxic

T cells in vivo might circumvent these limitations of T cell

immu-notherapy The Casitas B-cell Lymphoma-b protein, Cbl-b, is a

member of the family of Cbl Proteins of this family contain an

tyrosine kinase binding domain, a RING finger, a proline-rich

sequence, and can thus function as both ubiquitin E3 ligases and

molecular adaptors Studies of Cbl-b-deficient mice have revealed

an essential role for this molecule in T cell tolerance induction

Cbl-b-/- T cells show effective activation in the absence of

costim-ulation Thus, Cbl-b functions as a negative regulator of

antigen-specific T cell activation and is a critical mediator of T cell

energy To determine whether Cbl-b contributes to anti-cancer

immunity in vivo we tested the TC-1 cancer cell line in cbl-b

defi-cient mice Intriguingly we found that cbl-b defidefi-cient mice

sponta-neously reject TC-1 tumors As key players in the spontaneous

tumor rejection response CD8+ T cells were identified In

addi-tion in a spontaneous tumor model of UVB-induced skin cancer

cbl-b mice showed dramatically reduced tumor outgrowth

C2-41

Apoptosis of CD4+CD25+ regulatory T cells via

a Bax-dependent pathway regulates CD8+ T cell

immune responses against self antigen

Y Yan1, Z Xiong1, L Lachman2, H Wang1and X Yang1

1Temple University School of Medicine, Philadelphia, PA, USA,

2University of Texas M D Anderson Cancer Center, Houston, TX,

USA

The question of whether T cell responses to more than 2000

serologi-cally-defined tumor antigens are under regulation of naturally

occur-ring CD4+CD25+ regulatory T cells (nTreg cells) has not been

answered To address this issue, we identified a HLA-A2.1-restricted

T cell antigen epitope of self-tumor antigen CML66L, 66Pa

CML66L was identified in chronic myelogenous leukemia (Yang

et al, PNAS USA, 2001) The HLA-A2.1/66Pa peptide complex in vitro

stimulated the in vivo-primed T cells and mediated higher T

cell-medi-ated cytotoxicities of CML66L+ human tumor cells This suggests

that CML66L elicits T cell immune responses On the other hand,

previous reports showed that generation and homeostasis of nTreg

cells require CD28 co-stimulation and interleukin-2 (IL-2) signaling

We demonstrated that anti-apoptotic proteins, such as Bcl-x isoforms

and their interaction protein translationally controlled tumor protein

(TCTP), play a critical role in CD28- and IL-2-signaled T cell

survi-val pathways (Yang et al, Immunity, 1997; Yang et al, Oncogene,

2005) Thus, we wanted to examine our hypothesis that

Bcl-xL-inter-acting pro-apoptotic protein Bax mediates the apoptotic pathway in

nTreg cells, and further regulates T cell immune responses via

modu-lation of survival of nTreg cells We found that nTreg cells regulates

CD8+ T cell responses to 66Pa, and that depletion of nTreg cells via

a pro-apoptotic protein Bax-dependent apoptotic pathway enhances

CD8+ T cell responses to 66Pa These findings provide new direction

in development of novel anti-tumor immunotherapy by modulation

of survival and homeostasis of nTreg cells

C2-42 Role of flavonoids in cancer immunotherapy

D Hotnog, L I Brasoveanu, C M Ionesti and V RomanCenter of Immunology, Institute of Virology, Bucharest, ROMANIAApoptosis is a physiological cell suicide program that is critical forthe development and maintenance of healthy tissues, thereforeaberration of this process can be detrimental Many chronic leuke-mia as well as others neoplasic disorders are characterized bydefective apoptosis The continuing magnitude of cancer problemand the failure of conventional chemotherapy of advance invasivedisease to effect major reductions in the mortality rates for thecommon forms of cancer indicate that new approaches to the con-trol of this disease are critically needed Since apoptosis induction

is the most potent defense against cancer; study of apoptotic cess and different therapeutical agents capable to modulate it couldlead us to an improvement of leukemic patients health Many nat-ural compounds present in the human diet have been studied con-sidering their low level of citotoxicity and potential role asanticancer agents Among them flavonoids (resveratrol,quercitin,currcumin, etc) have been shown to exert multiple pharmacologicaleffects Our data showed the involvement of different flavonoids inapoptotic process, in modulating pro/anti apoptotic proteinsexpression or different signaling pathway by using different tumorcell line The results suggest that flavonoids are important modula-tors of apoptotic pathways, they can open new therapeutically per-spectives for cancer treatment as anticancer agents or assupplementary anticancer agents

pro-C2-43 Activation of non-specific immunity by autologous HSP-peptide complexes from breast adenocarcinoma in human macrophages

M Savvateeva1, E Kuznetsova1, D Zhdanov1, D Kosenkov2,

A Lyashenko3and E S Severin41

Moscow Institute of Medical Ecology, Moscow, RUSSIANFEDERATION,2Moscow Medical Academy, Moscow, RUSSIANFEDERATION,3Center of Medical Biotechnologies, Moscow,RUSSIAN FEDERATION,4Institute of Molecular Diagnostics,Moscow, RUSSIAN FEDERATION

An active immunotherapy generates an adaptive immune response

by introducing an antigene into a patient, often with combinationwith other components that can enhance an immune response tothe antigene Heat-shock proteins form the complexes with a vari-ety of tumor-related antigens via its polypeptide binding domain.Because Hsp is taken up by APCs through the Hsp-receptorsrecognition, its application to antigen delivery systems has beenwell examined We tested the induction of TNF-a in human per-ipheral blood macrophages to investigate activation of non-specificimmunity by autologous Hsp(70, 90, 96)-peptide complexes fromhuman breast adenocarcinoma Hsp-peptide complexes were puri-fied from human breast adenocarcinoma by original protocol TheLPS content was analysed using the LAL assay Peripheral bloodmacrophages were separated from the whole blood by standardprotocol and incubated with different concentrations of autologousHsp-peptide complexes for 2, 24 and 48 h Induction of TNF-a byimmune-modulation drug Polyoxidonium was measured as a pos-itive control Autologous Hsp-peptide complexes induced high lev-els of TNF-a in macrophages in dose-dependent manner after 2 h

of incubation The duration of TNF-a induction was until 48 h ofincubation According to this data we can suggest that autologousHsp(70, 90, 96)-peptide complex from human breast adenocarcino-

ma activate non-specific immune response and it is a possible agentfor the active immunotherapy of breast adenocarcinoma

Overcoming multidrug resistance (MDR) by

targeting protein kinase CK2

G Di Maira1,2, F Brustolon1,2, K Tosoni1,2, J Bertacchini3,

S Marmiroli3, L A Pinna1,2and M Ruzzene1,2

1University of Padova, Padova, ITALY,2VIMM (Venetian Institute

of Molecular Medicine), Padova, ITALY,3University of Modena

and Reggio Emilia, Modena, ITALY

Protein kinase CK2 is a ubiquitous and constitutively active

kin-ase, usually present in the cell as a tetrameric holoenzyme,

com-posed of two catalytic subunits (alpha and/or alpha’) and two

regulatory subunits (beta) It phosphorylates many cellular proteins

and is implicated in the regulation of cell survival, proliferation

and transformation We found that CK2 is overexpressed in some

MDR (multidrug resistant) cell lines, as compared to the parent

cell line, normally sensitive to chemical-induced apoptosis

Interest-ingly, in some cases only the expression of the catalytic subunit is

higher, supporting the existence of CK2 isoforms where alpha is

not combined to beta In all tested apoptosis resistant cell lines,

the blockade of CK2 by means of highly specific inhibitors, or

down-regulation of CK2 expression by the RNA interference

tech-nique, turned out to induce apoptosis, to increase the accumulation

of chemotherapeutic drugs inside the cell, and to potentiate the

pro-apoptotic effect of anticancer agents We therefore suggest that

CK2 can be considered a druggable target to counteract the MDR

phenotype

C2-45

Pleiotrophin as a new target for therapy of

tumors

E Lampropoulou, M Lamprou, A Parthymou,

M Koutsioumpa, C Mikelis and E Papadimitriou

University of Patras, Patras, GREECE

Pleiotrophin (PTN) is an 18 kDa growth factor that has high

affin-ity for heparin PTN interacts with receptor protein-tyrosine

phos-phatase beta/zeta and anaplastic lymphoma kinase, both being

expressed by many types of cancer cells Screening of various

human tumour cell lines and tumour specimens of different origin

revealed that PTN is expressed in many types of cancer, such as

gliomas, melanomas, meningiomas, neuroblastomas,

choriocarcino-mas, leukemias and cancers of pancreas, prostate, stomach, colon,

breast, ovaries and lungs PTN receptors are also up-regulated in a

plethora of tumors and are being tested as targets for anti-cancer

therapy Concerning the biological activity of PTN in cancer, there

is ample evidence that it is a tumor-promoting factor, while it has

also been suggested that it may be implicated in cellular quiescence

rather than an oncogenic phenotype This dual effect of PTN is

also observed in glioblastoma cell lines and may be attributed to

different receptors expressed by different cell lines or different

microenvironment of cells that can affect the form of PTN acting

on cells Clarifying the exact role of PTN in glioblastomas and

other types of tumors could lead to the development of new

thera-peutic tools

Acknowledgements: We thank the European Social Fund

(ESF), Operational Program for Educational and Vocational

Training II (EPEAEK II) and particularly the Program

PYTHAG-ORAS for funding the above work

C2-46 Sulforaphane-induced autophagy protects against apoptosis in human prostate cancer cells

S V SinghUniversity of Pittsburgh, Pittsburgh, PA, USASulforaphane (SFN), a constituent of broccoli, is highly effective inaffording protection against chemically-induced cancer in animalmodels More recent studies have revealed that SFN suppressesgrowth of prostate cancer cells in culture and in vivo by causingapoptosis induction The present study reports a novel response toSFN in PC-3 and LNCaP human prostate cancer cells involvinginduction of autophagy Exposure of PC-3 and LNCaP cells toSFN resulted in several specific features characteristic of autop-hagy including appearance of membranous vacuoles in thecytoplasm and formation of acidic vesicular organelles The SFN-induced autophagy was associated with upregulation, processing,and recruitment to autophagosomes of LC3, which is a mamma-lian homologue of the yeast autophagy regulating protein.Treatment of cells with a specific inhibitor of autophagy (3-methyl-adenine) attenuated localization of LC3 to autophagosomes butexacerbated cytosolic release of cytochrome c as well as apoptoticcell death Even though SFN treatment caused activation of extra-cellular signal-regulated kinases (ERK1/2) as well as a modestincrease in protein level of beclin, both of which are implicated inautophagic response, the SFN-induced autophagy was not affected

by pharmacologic inhibition of ERK1/2 or siRNA-mediatedknockdown of beclin protein While the precise mechanism ofautophagic response in SFN treated cells remains elusive, the pre-sent study indicates that autophagy is a defense mechanism againstSFN-induced apoptosis in human prostate cancer cells This studywas supported in part by NCI grants CA115498 and CA101753

C2-47 Cancer treatment by TAT-mediated protein transduction into the cells

M Grdisa and A MikecinRudjer Boskovic Institute, Zagreb, CROATIATransduction is a biochemical technique for the introduction offull-length proteins into the cells This process occurred in a rapid,concentration dependent fashion that appears to be independent ofreceptors and transporters Transduction of peptides and proteinshas a broad implications in experimental systems for regulatingintracellular processes and has the potential to be use in develop-ment of the new therapeutic strategies for cancer therapy Hyper-proliferation of cancer cells is associated with deregulation of cellcycle progression, which is driven by the activities of CDKs Theyare selective small molecules, which halt G1/S transition andinduce cell death One of them, p27, has significant role in cancerprogression and antitumor drug response, and stems from thereported correlation between low p27 expression and either hightumor grades or poor survival following chemotherapy treatment.Conversely, its overexpression in cancer cells inhibits tumour cellgrowth by enforcing cell cycle arrest and apoptosis, and this hascontributed to p27 being referred to as a tumor suppressor Toexamine a role of p27 in tumor cells apoptosis, transduction ofTAT-p27, TAT-ptp27 and TAT-N’p27, was perform Presence ofTAT fusion proteins was determined in the subcellular fractions aswell as their influence on the expression of cell cycle and apoptosisregulatory proteins Involvment of particular signal transductionpathway was also studied

Sterols and Multi Drug Resistance in cancer

cells

B Rubis and M Rybczynska

University of Medical Sciences, Poznan, POLAND

It has been postulated that phytosterols, compounds studied

previ-ously on their LDL lowering properties, can decrease the cancer

incidence (Tapiero et al., 2002) However, their protective action

has not been elucidated yet (Awad et al., 2002) The analysis of

phy-tosterols contribution to cell viability was studied with MTT test in

a time and dose dependent manner To investigate if the expression

of MDR1 is expression in a RT-PCR reaction was also studied It

was shown that beta-sitosterol significantly decreased the cells

viab-ility The IC50 values for studied cell lines (72 h of incubation time)

were respectively: MCF-7 – 12.55 lM; MCF-7ADR – 13.59 lM

and MDA-MB-231 – 27.22 lM It was shown that the sitosterol

was cytotoxic decreasing the number of MCF-7 and MCF-7ADR

cells by about 60% in a concentration of 2 lM Similar effect was

observed when cells were incubated with cholesterol but in a

con-centration four times higher Beta-epoxy-siosterol did not affect the

viability of any studied cells Moreover, it was also shown that the

cytotoxic effect of beta-sitosterol was observed in both,

estrogen-dependent and estrogen-non-estrogen-dependent cells, however the estrogen

dependent cells were more sensitive It was also shown that the

beta-sitosterol was significantly more cytotoxic in cells with basal

MDR1expression (MCF-7) in the range of concentrations 0.25–

4 lM than in MCF-7/ADR The study of MDR1 expression

regula-tion on the level of specific mRNA showed that it could depend on

the type of the cells and on the basal level of MDR1 expression

Since it was shown that beta-sitisterol is not cytotoxic towards

nor-mal cells (Rahmat et al., 2006), it has been suggested that a diet

reach in phytosterols might be a relevant anticancer factor

Suppor-ted by: 501-01-3302405-08035 PBZ-KBN-094/P06/2003

C2-49

The evaluation of apoptosis in MCF-7 WT and

DOX cells after chemotherapy with multidrug

resistance modifier in vitro

K _Zo´rawski1, J Saczko2, A Chwikowska3, J Kulbacka2,

M.Ługowski2, M Dra˛g-Zalesin´ska4, T Wysocka4and T Banas2

Department of Medical Biochemistry, Poland, Wrocaw,

POLAND,4Department of Histology and Embryology, Wrocaw,

POLAND

The successful treatment of cancer is dependent upon the

effective-ness of cytotoxic anticancer drugs either alone or in combination

with other ways of treatment Unfortunately, most cancers either

are increasingly resistant to any initial treatment or acquire

resist-ance to a board spectrum of anticresist-ancer drugs over time Drugs used

in chemotherapy work in different ways to stop proliferation of

tumor cells or induce cell death Combining more than one drug or

combination with multidrug resistance modifiers may support the

efficiency of applied treatment Cells become simultaneously

resist-ant to wide variety of structurally unrelated compounds Therefore,

understanding the mechanisms of multidrug resistance in human

cancers is crucial for developing novel strategies for treatment The

studies were performed on human breast adenocarcinoma cell lines

doxorubicin sensitive (MCF-7) and resistant and (MCF-7/DX)

The cells were treated with verapamil (10 and 20 lM) during 18 h

and then 24 h with doxorubicin (10 mg/ml) The control cells were

treated separately with verapamil and doxorubicin Neutral comet

assay method associated with DNA fragmentation was used for

apoptosis detection MTT assay was performed to examine the cells

viability as a mitochondria metabolic function after the drugs

uptake Our results confirm that modifier affects on multidrug

resistant cells treated with doxorubicin

C2-50 Treatment of P-gp positive L1210/VCR cells by verapamil and ATRA induced down-regulation

of P-glycoprotein expression/activity

Z Sulova1, D Macejova2, M Seres1, J Sedlak3, L Gibalova1,

J Brtko2and A Breier1

1Institute of Molecular Physiology and Genetics, SAS, Bratislava,SLOVAKIA,2Institute of Experimental Endocrinology, Bratislava,SLOVAKIA,3Institute of Experimental Oncology, SAS,

Bratislava, SLOVAKIAP-glycoprotein (P-gp) is a drug efflux pump of plasma membranewith substrate specificity to drugs with different structure Themultidrug (MDR) phenotype based on overexpression of P-gpoften results more than hundred times higher cells resistance toseveral drugs All-trans retinoic acid (ATRA, ligand of retinoicacid receptors RAR) was described to induce alterations in P-gpexpression and/or activity in different cells L1210/VCR (R) is aP-gp positive cell line, in which P-gp overexpression was achieved

by adaptation of parental L1210 (S) cells to vincristine The topic

of the present paper was the study of relations between regulatorypathways of nuclear receptors for retinoids and P-glycoproteinexpression When compared R and S cells, the levels of mRNAencoding retinoic acid nuclear receptors RARa and c or RARband retinoid X receptors RXRb and RXRc were increased ordecreased, respectively ATRA did not influence the viability of Rcells differently to S cells ATRA alone did not influence the P-gpexpression or the transport activity in R cells In contrast, whenATRA was applied together with verapamil (P-gp inhibitor), a sig-nificant depression of P-gp expression and transport activity wereobserved However, any significant differences in [11, 12-3H]-ATRA uptake were observed neither in sensitive nor in resistantcells Moreover, verapamil did not influence ATRA uptake in anycases Thus, we can conclude that the combined treatment of Rcells with ATRA and verapamil is able to depress P-gp expressionand activity by unknown mechanisms

C2-51 Delphinidin inhibits proliferation and cell cycle progression of FaDu human oral squamous carcinoma cells

S R Noh1, S M Park1, M S Kim1, M K Sung2, J S Kim3and H Yoo1

1Chosun Univ., Gwangju, REPUBLIC OF KOREA,2Department ofFood & Nutrition, College of Human Ecology, SookmyungUniversity, Seoul, REPUBLIC OF KOREA,3Department of AnimalScience and Biotechnology, College of Agriculture, KyungpookNational University, Daegu, REPUBLIC OF KOREAThe growth inhibitory effects of delphinidin on the FaDu humanoral squamous carcinoma cells was evaluated by the MTT assay,and the effects on cell cycle and apoptosis related genes were inves-tigated by flow cytometry, fluorescence microscope and real-timePCR, respectively Delphinidin inhibited the proliferation of FaDuhuman oral squamous carcinoma cells with the IC50s < 60 lg/ml.Flow cytometric investigations demonstrated that delphinidinmainly inhibited cell cycle at the G2/M phase in a dose-dependentmanner with a weak induction of apoptosis, demonstrating thatdelphinidin exert their anti-proliferative effect on FaDu cellsthrough inhibiting cell cycle and inducing apoptosis Delphinidininduced apoptotic cell death was further confirmed by the fluores-cence microscopic analyses of nuclear condensation stained withDAPI Gene expression analysis of anti-apoptotic Bcl2 and pro-apoptotic Bad using real-time PCR showed the decrease of Bcl-2with rapid increase in Bad Our results support delphinidin as acell cycle inhibitor and apoptosis-inducing constituent of anthocya-nidins Further evidence in the modulation of the mitogenic signal-ing in tumor cells is under investigation

Inhibitory effects of pyrrolidine dithiocarbamate

on apoptosis

M Mandl, B Holzer, E Gaudernak, A Triendl and J Seipelt

Max F Perutz Laboratories, Vienna, AUSTRIA

Pyrrolidine dithiocarbamate (PDTC), a substance originally

des-cribed as NFjB inhibitor exhibits pro- as well as anti-apoptotic

properties depending on the model system used Apoptosis or

pro-grammed cell death is a highly conserved and regulated process

which is important for cellular homeostasis In our system PDTC

shows anti-apoptotic effects in puromycin- and embelin-induced

apoptosis The aim of this work is to shed light on the mode of

action of PDTC under these conditions The current data indicate

that PDTC-treated cells are more viable after apoptosis is induced,

independent of the inducer According to this finding, the activity

of executioner caspases 3 and 7 is significant reduced by PDTC

The release of cytochrome c from mitochondria, which is a

hall-mark of the intrinsic pathway of apoptosis seems to be affected by

PDTC only to a minor extent and will be studied in more detail

To investigate the involvement of caspase 3, our experiments were

performed in HeLa cells, in the caspase 3 deficient MCF7 cell line

as well as cells constitutively overexpressing caspase 3 Further

experiments should clarify if PDTC acts on the level of caspases or

if other factors are involved

1Tyrolean Cancer Research Institute, Innsbruck, AUSTRIA,

2Molecular Biology Research Laboratory, Department of Pediatrics,

Medical University Innsbruck, AUSTRIA

Neuroblastoma (NB) is with 10% the most frequent solid tumor in

childhood and emerges from the sympathetic nervous system

Longterm survival rate is still low and drug resistances are more

and more common The proteasome inhibitor Bortezomib (PS-341

Velcade) is an agent, which specifically inhibits the proteasome

pathway We showed that Bortezomib induces apoptosis via a

ca-spase-dependent pathway in neuroblastoma cells Bortezomib

treat-ment was associated with increased mRNA and protein levels of

the pro-apoptotic ‘‘BH3-only’’ protein Noxa, caused loss of the

mitochondrial outer membrane potential and release of cytochrom

c The anti-apoptotic protein Mcl-1 accumulated whereas Bcl-xL

was strongly repressed within 16 h Tetracyclin-regulated Noxa

expression sensitized for Bortezomib induced apoptosis whereas

gene knock-down significantly reduced apoptosis The

anti-apop-totic antagonist of Noxa, Mcl-1, failed to block apoptosis, but the

overexpression of the pro-survival protein Bcl-xL prevented

Bor-tezomib-induced cell death This indicates the involvement of the

intrinsic death pathway We conclude from these data that

Bortez-omib destabilizes Bcl-2 rheostat by induction of Noxa and

repres-sion of Bcl-xL in neuroblastoma cells

C2-54 Cytotoxic activity of the Inula japonica extracts against several human cancer cell lines in vitro

M Lee, M Cha and H ParkKyungnam University, Masan, REPUBLIC OF KOREAThe present study describes the preliminary evaluation of the cyto-toxic activity of the extracts from Inula japonica I japonica wasextracted with methanol, ethanol, acetone, and water, and thencytotoxic activity of these extracts were evaluated The cytotoxicactivity of each extract was assessed by the MTT-dye reductionassay Both ethanol and acetone extracts from I japonica showedthe cytotoxic activity against the HT-29 human colon cancer cells.Furthermore, the ethanol extract was fractionated with n-hexane,diethyl ether, ethyl acetate, and water according to degree of polar-ity The diethyl ether fraction showed the highest cytotoxic activityagainst HT-29 cells, but the other fractions showed low cytotoxicactivity In addition, diethyl ether layer also showed the cytotoxicactivity against various tumor cells, such as human colon carci-noma SW620, human cervix adenocarcinoma HeLa, and humanbreast adenocarcinoma MCF-7 cells as well as HT-29 cells Thesestudies support that extracts of I japonica may be a potential can-didate as possible chemotherapeutic agent against human cancer

C2-55 Glucose-deprived HT-29 cells are sensitive to verrucosidin as a GRP78 down-regulator

M Yoon, K Jo and H ParkKyungnam University, Masan, REPUBLIC OF KOREAGlucose deprivation, a feature of poorly vascularized solid tumors,activates the unfolded protein response (UPR) which is a stress-signaling pathway in tumor cells that is associated with themolecular chaperone GRP78 and induction of GRP78 has beenshown to protect them against programmed cell death Thus, tar-geting glucose-deprived conditions may be a novel strategy in anti-cancer drug development Based on that, we established a novelscreening program for chaperone modulators that preferentiallycytotoxic activity in cancer cells under glucose-deprived conditions.During the course of our screening system, we recently isolated anactive compound, 326-2, from Penicillium verrucosum var cyclopi-

um and identified it as a down-regulator of the grp78 gene Asexpected, 326-2 inhibited the expression of the GRP78 promoterunder glucose-deprived conditions in a dose-dependent mannerwith an IC50value of 50 nM Furthermore, 326-2 was identified asverrucosidin, a pyrone-type polyketide, by ESI-MS and variousNMR spectroscopic methods Interestingly, we found that verru-cosidin prevents UPR-induced expression of protein, such asGRP78, whose expression is induced by glucose-deprived or by 2-deoxyglucose; this effect is not seen under normal growth condi-tions Taken together, the GRP78-inhibitory action of verrucosidinwas dependent on strict hypoglycemic conditions and resulted inselective cell death of glucose-deprived HT-29 cells

Combined anticancer therapy: siRNA and

cytostatics

N Mironova, O Shklyaeva, M Zenkova and V Vlassov

Institute of Chemical Biology and Fundamental Medicine SB RAS,

Novosibirsk, RUSSIAN FEDERATION

One of the obstacles for successful anticancer therapy is acquiring

by tumor cells multiple drug resistance phenotype (MDR), which

causes loss of sensitivity of tumor cells to wide range of

chemo-therapeutics We developed a new animal model of tumor

progres-sion (lymphosarcoma RLS40 in CBA mice) displaying MDR

phenotype, which corresponds to tumor status observed in patients

after one or several courses of chemotherapy The RLS40 tumor

cell line was obtained by cultivation of cell line RLS non-sensitive

to cyclophosphamide on medium with gradually increasing

vinblas-tine concentrations RLS40 was characterized by high levels of

expression of mdra/1b genes and 20-folds decreasing of sensitivity

to cytostatics as compared to parent line

Developed model of lymphosarcoma RLS40 was used to study

effi-ciency of the combined anticancer therapy comprising

cyclo-phosphamide and siRNA targeted MDR1a/1b mRNA In vitro,

cell treatment with siRNA resulted in 3-fold decrease the level of

MDR1a/1b mRNA and restored cell sensitivity towards

cytostat-ics In vivo, simultaneous administration of anti-mdr1a/1b siRNA

and cyclophosphamide caused tumor regression, and in the total

this therapy was at least 2-fold more effective as compare with

conventional therapy

Acknowledgements: This work was supported by RAS

pro-grams ‘‘Molecular and cellular biology’’ and ‘‘Science to

medi-cine’’, and SB RAS Interdisciplinary grant 5.11

C2-57

Curcumin induces apoptosis through

generation of reactive oxygen species: its

suppression by expression of ornithine

decarboxylase

Y F Liao1, H C Hung1, C Y Lin2and G Y Liu2

1Department of Life Sciences, National Chung-Hsing University,

Taichung, TAIWAN,2Institute of Immunology, Chung Shan

Medical University, Taichung, TAIWAN

Curcumin, a well-known dietary pigment derived from food flavor

turmeric (Curcuma longa), exhibits anti-proliferative,

anti-inflam-matory and antioxidant activities Recently, studies have shown

that chemopreventive response of curcumin might be due to the

hyperproduction of reactive oxygen species (ROS) to induce

apop-tosis in tumor cells Ornithine decarboxylase (ODC), the

chemoprevention In our previous studies, ODC overexpression

was shown to prevent TNF-a- and methotrexate-induced apoptosis

via reducing ROS In this study, we found the activity and

expres-sion of ODC were reduced during curcumin treatment

Overexpres-sion of ODC in parental cells could reduce curcumin-induced

apoptosis, which leads to the loss of mitochondrial membrane

potential (Dwm), through diminishing intracellular ROS Moreover,

ODC overexpression prevented the decline the cytochrome c

release and activations of caspase 9 and 3 following curcumin

treatment The results demonstrate that curcumin-induced

apopto-sis is through the down-regulation of ODC protein and occurs

along a ROS-dependent mitochondria mediated pathway

C2-58 Cyclosporin A affects invasiveness and migration of cultured human glioblastoma cells

A Kwiatkowska and B KaminskaThe Nencki Institute of Experimental Biology, Warsaw, POLANDThe invasion of neoplastic cells into brain parenchyma and fastproliferation are hallmarks of glioblastomas Cell migration andproliferation are regulated by common intracellular pathways andseveral studies implicated PI-3K/Akt pathway in those processes

We have demonstrated that exposure of the rat C6 glioma cells tocyclosporin A (CsA) decreases the levels of active Akt concomit-antly with glioma invasiveness Thus, we sought to determine whe-ther CsA inhibits Akt phosphorylation and modulates aninvasiveness of malignant human glioblastoma cells PTEN, a neg-ative regulator of Akt activity, is frequently mutated in glioblasto-mas Therefore, we employed glioblastoma cell lines: LN229 (wildtype PTEN) and T98G (mutated PTEN) We demonstrate that

5 lM CsA decreased the level of phosphorylated Akt in LN229,but not in T98G cells, that correlates with a reduced invasiveness

of LN229 cells through the Matrigel and cell motility in a scratchassay We did not observe any effect of CsA on invasion of T98Gcells CsA at doses used in experiments did not affect viability orproliferation of glioblastoma cells Modulation of Akt activity byoverexpression of a constitutively active Akt partially blocked CsAimpact on invasion in vitro suggesting that CsA action depends oninhibition of PI3K/Akt signaling Our results suggest that CsAaffects an invasiveness of LN229 glioblastoma cells probably byinhibiting of Akt activation, thus the drug has no effect on inva-siveness of T98G cells expressing mutated PTEN

C2-59 Genistein products resulting from free radical action

J Hartmann1, R M Quint2and N Getoff2

1Medical University of Vienna, Waehringer Gu¨rtel 18-20, 1090Vienna, AUSTRIA,2Section of Radiation Biology, Department ofNutrition Sciences, University of Vienna, Althanstr 14, UZA II.,

1090 Vienna, AUSTRIAGenistein (5, 7, 4 ’- trihydroxyisoflavone) is a very active phytoestr-ogene of the isoflavones group It is a natural products, containede.g in soy beans (50%) and red clover Because of its multi-bio-logical properties it has attracted numerous researchers Especiallyits action as an in vitro inhibitor of protein tyrosine kinases, anti-cancer agent etc has been frequently investigated

The present report concerns the genistein products formed by theattack of oxidizing (OH, O2-) as well as reducing (eaq-, H) free rad-icals in aqueous media (pH7.4) The free radicals generated inthe human organism, can also be produced by ionizing radiation

in desired concentration in aqueous solution Therefore, the ments were performed under the influence of c-ray at preciselydetermined conditions Analysis of the solutions were performed

experi-by HPLC-technique It has been established that the yield and thekind of the genistein degradation products depend on the absorbedradiation dose (consecutive action of the free radicals), the appliedsubstrate concentration as well as the kind of the involved primaryradicals The products (aldehydes, carboxylic acids etc.) are of spe-cial interest, since they can strongly influence the biological effect

of genistein, e.g giving rise for undesired biological effects ible variations of reactions mechanisms are also discussed Thisreport is of interest for scientists working in the field of biology,medicine and nutrition

Radiation-induced antitumor action of

genistein Experiments in vitro

J Hartmann1and N Getoff2

1Medical University of Vienna, Waehringer Gu¨rtel 18-20, 1090

Vienna, AUSTRIA,2Section of Radiation Biology, Department of

Nutrition Sciences, University of Vienna, Althanstr 14, UZA II.,

1090 Vienna, AUSTRIA

Isoflavones and related compounds are natural products having

multiple biological properties Genistein (5, 7, 4 / -

trihydroxy-isoflavone) belongs to this group of substances and is a very potent

phytoestrogene Investigations in vitro demonstrated the broad

spectrum of the biological properties of genistein, e.g acting as

anticancer agent, inhibitor of protein tyrosine- and histidine

kinas-es, inhibitor of DNA topoisomerase and many other health

bene-fits The genistein pharmacological activity very likely arises from

its antioxidant properties Experimental results on the radiation

induced antitumor action of genistein were observed in the frame

of experiments in vitro using human cancer cells (MCF-7) as a

model Based on the course of the obtained survival curves, taken

as a function of the absorbed radiation dose (Gy) in oxygenated as

well as airfree media it is concluded that: In oxygenated media the

N/N0ratio (N0= number of cells before -, N = number of cells

after treatment) as a function of absorbed radiation dose shows a

strongly pronounced antitumor effect With increasing genistein

concentrations the antitumor effect is correspondingly enhanced

Combinations of genistein (5 lM or 40 lM) with the cytostatic

agent Mitomycin-C (2.5 lM) show a significant synergism In

air-free media these effects are essentially pronounced Obviously

elec-tron transfer processes from genistein to Mitomycin-C take place

The obtained experimental data demonstrate the antitumor action

of genistein as well as its synergism to MMC The results of these

studies are of special interest in respect to anticancer treatment

C2-61

Comparative effects of boric acid and calcium

fructoborate on breast cancer cells

R N Ciubar1, R I Scorei2, C M Ciofrangeanu1, V Mitran1,

A Cimpean1and D Iordachescu1

1Faculty of Biology, Bucharest University, Bucharest, ROMANIA,

2Craiova University, Craiova, ROMANIA

Recent studies suggested that boron has a chemopreventive role in

prostate cancer We investigated the effects of boric acid (BA), the

naturally occurring form of boron circulating in human plasma,

and calcium fructoborate (CF), a nutritional supplement which has

a chemical structure similar to the natural forms of boron found in

edible plants, on the MDA-MB-231 human breast cancer cell line

Exposure to BA and CF inhibited the proliferation of breast

can-cer cells in a dose-dependent manner To understand the

mechan-ism of cell growth inhibition, Ki67 cell proliferation marker, p53

and apoptosis related proteins expression following BA and CF

treatment were evaluated We found down-regulation of Ki67

expression which may partly explain the chemopreventive effect of

boron compounds Treatment with CF but not BA resulted in a

decrease of p53 and bcl-2 protein levels Furthermore

up-regula-tion of pro-caspase-3 protein, increase in cytosolic cytochrome c

and caspase-3 activity were observed after treatment with CF

indi-cating apoptotic cell deaths Staining with annexin-V and

propi-dium iodide showed that the number of apoptotic cells increased

with higher doses of CF From these results we conclude that while

both BA and CF inhibit the growth of breast cancer cells, only CF

induces apoptosis Further studies will be needed to determine if

BA and CF will be suitable for clinical application in breast cancer

patients

C2-62 Molecular mechanisms of tumour cell growth inhibition by curcumin treatment

C M Ciofrangeanu, A Cimpean, R N Ciubar, V Mitran and

D IordachescuBiology Faculty, Bucharest University, Bucharest, ROMANIANumerous reports suggest that curcumin, a natural polyphenolexerts anti-cancer properties and may be useful for dietary preven-tion of cancer We investigated the effects of curcumin on prolifer-ation and apoptosis-related protein expression of two breast cancercell lines with different in vivo tumorigenicity (MDA-MB-

231 < MDA-MB-435) Curcumin reduced cell growth by 50%after 2 h in 231 cells The response of 435 cells to curcumin is alsorapid, but much weaker (only 10% decrease of viability), althoughthe level of proliferation markers-PCNA and KI67 remainunchanged comparative with 231 cells were their reduction wasobserved after 24 h treatment Following curcumin treatment cellapoptosis occurred after 2 h and the number of annexin V positivecells was much higher in 231 cell line than in 435.We have ana-lyzed the protein expression of p53, Bcl-2, pro-caspase-3 by west-ern blot, cytochrome c release and the caspase-3 activity byELISA The curcumin treatment of 231 cells decreased the level ofBcl-2 and pro-caspase-3 proteins; increased the caspase 3 activitywith 100% after 24 h of treatment and induced a significant release

of cytochrome c into the cytoplasm In both cell lines curcuminhad no effect on the p53 protein expression We conclude that cur-cumin inhibits the growth breast cancer cells with stronger effects

in less tumorigenic cells and induces apoptosis through a pendent pathway

p53-inde-C2-63 Anticancer and cytotoxic activities of olive oil phenolic compounds

F Paiva-Martins1,2, V Rodrigues1,2, J G Coelho1,2,

R Calheiros3and M P M Marques4,5

1Faculdade de Cieˆncias Universidade do Porto, Porto, PORTUGAL,

2CIQ-UP, Porto, PORTUGAL,3Quı´mica-Fı´sica Molecular, sidade de Coimbra, Coimbra, PORTUGAL,4Quı´mica-Fı´sicaMolecular, Coimbra, PORTUGAL,5Departamento de Bioquı´mica,Universidade de Coimbra, Coimbra, PORTUGAL

Univer-Direct evidence for the protective role of olive oil against cancerhas been published Diets containing 15% olive oil were found tosignificantly reduce induced pre-cancerous lesions in rat breast andcolon, and olive oil phenolic extract has shown to improve the bar-rier function of CACO2 cells as well as inhibition of HT115 cellattachment and invasion Oleuropein, the main polyphenol found

in olive fruit, completely regress tumors in 9–12 days, when istered to mice with spontaneous tumors Hydroxytyrosol, one ofthe components of the phenol extract of olive oil, was shown toinhibit the proliferation of both human promyelocytic leukaemiacells HL60 and colon adenocarcinoma cells HT29 and HT20 clone19A, by arresting the cell cycle and inducing apoptosis and to pre-vent an increase in L-isoaspartyl residues, a marker of proteindamage in human melanoma cells (M14) exposed to UV However,studies for the most abundant polyphenols in the oil, are inexist-ent The present work reports the in vitro evaluation of theanti-proliferative and cytotoxic activity of the most important seco-iridoids present in olive oil, the 3,4-DHPEA-EDA and 3,4-DHPEA-

admin-EA, against distinct human cancer cell lines (namely C32 melanomacells) Different growth-inhibition and viability determination meth-ods were used: Trypan Blue exclusion method and Sulphorodamine(SRB) test The toxicity of these compounds towards non-neoplasticcells was also evaluated, through similar experiments

Ornithine decarboxylase prevents cancer

chemotherapeutic drugs-induced apoptosis in

HL-60 cells

C Y Lin1, Y F Liao2, P C Hsu2, H C Hung2and G Y Liu1

1Institute of Immunology, Chung-Shan Medical University,

Taichung, TAIWAN,2Department of Life Sciences, National

Chung-Hsing University, Taichung, TAIWAN

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in

poly-amines (putrescine, spermidine and spermine) synthesis ODC in

several biological functions plays many important roles, including

embryonic development, cell proliferation, and the origin and

pro-gression of neoplastic diseases Elevated ODC activity has been

assumed to be associated with resistance to chemotherapeutic

drugs In our previous studies, overexpression of ODC prevents

TNF-a and methotrexate-induced apoptosis by reducing reactive

oxygen species (ROS), maintaining mitochondrial membrane

potential (Dwm) and inactivating caspases’ activity Herein, we

fur-ther investigated apoptotic mechanisms of ofur-ther chemofur-therapeutic

drugs, including etoposide, paclitaxel and cisplatin and the effects

of ODC on anti-apoptosis and cell cycle These drugs induce

apop-totic cell death, and the mechanisms of apoptosis are through

ROS-dependent, mitochondria-mediated and caspase’s activated

pathways Overexpressed ODC cells are resistant to drugs-induced

apoptosis and keep on the cell cycles without the interference of

G1/S arrest caused by etoposide and G2/M arrest by paclitaxel

The effects on the cell cycles may be resulted from increased

pro-tein expression of cyclin A, cyclin E and CDK4, and

phosphoryla-tion of CDK1 and CDK2

C2-65

Investigating the effect of anticancer

chemotherapeutic drugs on Cdt1 dependent cell

cycle checkpoint

A Stathopoulou1, V Roukos2, C Petropoulou1, C Flordellis1,

Z Lygerou2and S Taraviras1

1Laboratory of Pharmacology, University of Patras, Patra,

GREECE,2Laboratory of General Biology, University of Patras,

Patra, GREECE

The faithful transmission of the genetic material to the daughter

cells requires extreme accuracy in DNA replication, precision in

chromosome distribution and effective repair mechanisms to

min-imize heritable mutations affecting the genetic material To achieve

this fidelity, cells have evolved surveillance mechanisms that

mon-itor the structure of DNA and coordinate repair and cell cycle

pro-gression Recent studies revealed that Cdt1 is a critical and

evolutionarily conserved target of the DNA damage checkpoint,

since exposure to ionizing or UV radiation targets Cdt1 for

degra-dation Cdt1 is an essential component of the cell cycle licensing

machinery that is conserved from yeasts to mammals We have

examined the ability of several anticancer drugs to act through the

Cdt1 dependent cell cycle checkpoint Our findings indicate that

DNA damage induced by MMS, Cisplatin and Doxorubicin lead

to rapid proteolytic destruction of Cdt1, whereas treatment with

Tamoxifen and Etoposide does not affect Cdt1 protein levels

There is no change on Geminin expression levels, the protein

inhibitor of Cdt1 Our results suggest that genotoxic therapies used

against cancer differ in respect to Cdt1 dependent checkpoint

Knowledge of the molecular mechanisms that anticancer drugs

work is important for selecting drugs for combinatorial anticancer

therapies

C2-66 Photodynamic effect of quantum dots coupled with visible light targets mitochondria

dynamics and enhances apoptosis

M Jou1, Y Su1and T Peng1,2

1Chang-Gung University, Tao-Yuan, TAIWAN,2Chang-GungMemorial Hospital, Kee-Lung, TAIWAN

Nano photosensitizers, semiconductor quantum dots (QDs), havegreat potential for cancer treatment due to their potent cytotoxicphotodynamic effect (PDE) upon UV irradiation To improve thelimited biomedical application due to non specific photo toxicityand low tissue penetration of UV, visible light irradiation (490 nmlight or 488 nm laser) was coupled with their use We tested thecapability of using visible light coupled PDE of QDs (525 and 655)

to target mitochondrial dynamics including mitochondrial ment and fission as well as the opening of the mitochondrial per-meability transition pore (MPTP) for an enhanced apoptoticeradication of cells using laser scanning confocal microscopy.Mutant mitochondrial DNA (mtDNA)-induced supra sensitivemitochondria contained cytoplasmic hybrid (cybrid) cells were used

move-to detect any minor alteration in mimove-tochondrial dynamics uponPDE stress Intriguingly, localized mitochondrial area of PDE(mPDE) of QD 525 induced the most significant retardation ofmitochondrial movement, enhancement of mitochondrial fission,branching, swelling, and later fusion and finally the opening ofMPTP Reactive oxygen species generated from the mitochondria(mROS) appeared to be critically involved as a mitochondrial pro-tector (antioxidant), melatonin, but not a MPTP inhibitor, cyclosp-orine A, significantly inhibited visible light coupled PDE of QDsinduced-mROS formation and apoptosis This study demonstratesmitochondria-targeted PDE of QDs coupled with visible light formROS generation may serve as a potential maneuver to manipu-late mitochondria dynamics as well as to potentate mitochondria-mediated apoptotic eradication of cancer cells

(NMe2)2](OMe) (BAM-SiPc) was most potent While non-cytotoxic

in the dark, BAM-SiPc exhibited potent photocytotoxicity with

IC50values around 15–20 nM in HepG2 (human hepatocarcinoma)and HT-29 (human colon adenocarcinoma) cells The photocyto-toxicity correlated well with the level of reactive oxygen specieswhich could increase by up to 5-fold after treatment To study thecellular metabolism, BAM-SiPc was incubated with either HepG2(tumor liver cell) or WRL-68 (normal liver cell) for 24 h before theresidual photosensitizer was extracted and quantified More than40% of BAM-SiPc was metabolized in the normal cell; in contrast,over 95% remained in the tumor cell In the in vivo study, photo-dynamic treatment with BAM-SiPc resulted in a significant reduc-tion in tumor growth in both HepG2 and HT-29 implanted nudemice Histological analyses of the HepG2 tumor after treatmentshowed the presence of apoptotic changes Photodynamic therapywas apparently non-toxic to the mice as shown by serum enzymeassays and histological analyses of the liver sections (Supported by

a grant from the Research Grants Council of the Hong Kong cial Administrative Region, Project no CUHK4569/05M)

Evolution of B16 melanoma following

treatment with several bioactive plant

compounds

L Olariu

Ovidius University, Constanta, ROMANIA

B16 melanoma is an experimental tumor developed on C57B1/6

mices, having a spontaneous metastasis, mainly, in lungs Studies

made on this tumor can be extrapolated to human melanoma

because of the resemblances The experiments started with in vivo

passage of cells from murine melanoma B16F10 line in order to

obtain B16 melanoma tumor The inoculum of cellular suspension

was made subcutaneous on singeneic C57B1/6 line of mice The

inoculated cells kept their selectivity for the host with 100% tumor

development at the place of inoculum but they didn’t maintain

their characteristics as highly metastazic The melanoma was

main-tained through serial subcutaneous passages simultaneously on

sev-eral lots and the treatment with bioactive compounds isolated

from Salvia officinalis and Hedera helix was applied beginning with

the second and the third passage Tumor evolution was greatly

influenced after three days successive treatment with bioactive

compounds, which was initiated at 24 h from melanoma induction;

its occurrence was delayed with 20–44 days, in average, comparing

with control lots Also, tumor attachment was affected by these

treatments as shown by much smaller number of ill mice in treated

lots Regarding dissemination of tumoral cells in lungs there were

no differences between the treated and untreated mice

C2-69

Nitric oxide: a possible mediator of

glibenclamide induced b cell apoptosis

M Ansar1, M Ansari2, A Dehpour3

1Department of Biochemistry & Biophysics, School of Medicine,

Gui-lan University of Medical Sciences, Rasht, ISLAMIC REPUBLIC

OF IRAN,2Department of Clinical Biochemistry, School of

Medi-cine, Tehran University of Medical Sciences, Tehran, ISLAMIC

REPUBLIC OF IRAN,3Department of Pharmacology, School of

Medicine, Tehran University of Medical Sciences, Tehran,

ISLAMIC REPUBLIC OF IRAN

Several recent in vitro studies suggest that glibenclamide induces

apoptosis in b-cell line and human cultured islets In this study we

investigated the role of nitric oxide (NO) and NO synthase (NOS)

blockers, NG-nitro-L-arginine methyl ester (L-NAME) and

Ami-noguanidine (AG), on glibenclamide-induced apoptosis in rat

insu-linoma (RIN-5F) cells Glibenclamide increased NO generation

(measured as nitrite) and decreased cell viability Unlike AG which

is a preferential inhibitor of inducible NOS, L-NAME_ an

inhib-itor more selective for constitutive NOS (cNOS) and D600_ a

blocker of voltage-gated L-type Ca2+ channels inhibited Ca2+

influx into b cells, inhibited the effects of glibenclamide on cell

viability and NO production significantly (P < 0.05) Staining

with Hoechest 33342 and propidium iodide and analysis of DNA

fragmentation by electrophoresis showed that L-NAME and D600,

but not AG, decreased the number of cells with condensed

chro-matin and fragmented nuclei and prevented DNA fragmentation

It means that L-NAME inhibited the effects of glibenclamide on b

cell apoptosis These results suggest that increase of NO

produc-tion following activaproduc-tion of cNOS might be associated with

gliben-clamide-induced apoptosis in b cell line

C2-70 Hypothalamic proline-rich peptide down-modulate doxorubicin-induced bone marrow granulocytes and monocytes apoptosis

G A Kevorkian, T K Davtyan, A A Galoyan,

H L Hayrapetyan, A G Guevorkian, R G Gevorkian and

R M OhanyanH.Buniatian Inst of Biochemistry, Yerevan, ARMENIA

A new family of peptide neurohormones consists of 10–15 aminoacid residues have been discovered and isolated from the neurose-cretory granules of bovine and human neurohypophysis These pep-tides contain high proportion of proline residues and therefore weredesignated as PRPs Bone marrow (BM) was flushed from ratfemurs and red blood cells were lysed with 0, 83% NH4Cl in0.017 M Tris-HCl buffer for 5 min Nucleated cells were washedwith cold RPMI-1640 containing 10% fetal calf serum, 2 mML-glutamine, 1mM sodium pyruvate, 100 U of penicillin and

100 lg of streptomycin per ml and total cell count was determinedusing a hematological analyzer In preliminary experiments wefound that after 24 h incubation of BM cells 64.8 ± 7.9% of gra-nulocytes with high SSF-FCS signal spontaneously undergo toapoptosis, while only 12.8 ± 3.2% of monocytes undergo to apop-tosis spontaneously (Pt= 0.003) However, during the 2–4 h incu-bation of BM cells, we didn’t observed significant differencebetween BM apoptotic granulocytes and monocytes count Exam-ined the effect of PRP-1 and Doxorubicin (Dox) on BM granulo-cytes apoptosis depending from the dose during the 2 and 24 hincubation period PRP-1 during 2 and 24 h cultivation in concen-tration range 250–1000 ng/ml doesn’t influence on BM granulocytesapoptosis In contrast Dox at 1–20 lg/ml concentration induceddose-dependent apoptosis of BM granulocytes during 2 h and 4 hincubation period Dox during 24 h incubation period induced99.8 ± 0.3% of BM granulocytes and 52.8 ± 3.2% monocytesapoptosis However, PRP-1 even at higher concentration signifi-cantly increases neither early and late apoptotic BM granulocytescount nor the intensity of apoptosis

C2-71 Discovery of posttranslationally myristoylated proteins in apoptosis via chemoselective ligation

D D O Martin1, G L Vilas1, J A Prescher2, G Rajaiah3,

J R Falck3, C R Bertozzi2and L G Berthiaume11

University of Alberta, Edmonton, AB, CANADA,2University ofCalifornia, Berkeley, Berkeley, CA, USA,3University of TexasSouthwestern Medical Center, Dallas, TX, USA

In myristoylation the 14-carbon fatty acid myristate is attached tothe N-terminal glycine residue of proteins by N-myristoyltransf-erase Typically a cotranslational process, myristoylation alsooccurs posttranslationally at internal glycine residues revealedupon caspase cleavage during apoptosis Myristoylation is crucialfor proper protein targeting to membranes and function To cir-cumvent the long exposure time required to monitor incorporation

of radioactive myristate into proteins (weeks to months), we oped a non-radioactive methodology The method uses a bioor-thogonal azidomyristate analogue that can be incorporated intoproteins, chemoselectively ligated to tagged triarylphosphines anddetected by western blotting (seconds to minutes) Our methodreadily detected known exogenously and endogenously expressedco- and posttranslationally myristoylated proteins By combiningrational database analysis to recognize putative internal myristoy-lation sites with our new method, we identified five new posttrans-lationally myristoylatable proteins [PKC epsilon, CD-IC2, Bap31,MST3 and the catalytic subunit of glutamate cysteine ligase] Thissuggests that posttranslational myristoylation of caspase-cleavedproteins represents a novel mechanism used to regulate cell death

Antimycin A-induced apoptosis depends on

release of apoptosis-inducing factor from

mitochondria

M Ogita, A Ogita, Y Usuki, K Fujita and T Tanaka

Osaka City University, Osaka, JAPAN

Antimycin A (AMA) blocks mitochondrial electron transport at

complex III thereby inhibiting ATP production Blockade of

elec-tron transport induces the production of reactive oxygen species

(ROS) These cause a necrosis in several mammalian cell lines In

Human promyelocytic leukemia HL-60 cells, AMA was reported to

induce an apoptosis accompanying the fragmentation of nucleus and

chromosomal DNA We newly found that this apoptosis did not

accompany cellular fragmentation AMA decreased intracellular

ATP levels and generated ROS We found that AMA-induced ROS

production was inhibited by ascorbic acid (AA) However, AA did

not restore cell proliferation in AMA-induced apoptosis indicating

no involvement of ROS produced in the cytotoxicity Moreover the

release of cytochrome c from mitochondria and the activation of

ca-spase 3 were not observed On the other hand, the release of Ca2+

into cytosol and the activation of calpains were confirmed These

were characterized as an endoplasmic reticulum (ER)-initiating

apoptosis When potent stresses give to ER, the release of Ca2+

often activates calpains and succeedingly activates caspase 4 In the

AMA-treated cells, apoptosis-inducing factor (AIF), which directly

translocates from mitochondria to nucleus to execute DNA

frag-mentation, was released from mitochondria, instead of the activation

of caspase 4 This release and the cytotoxicity of AMA were

restric-ted by a calpain inhibitor, Z-LL-H We suggest that the apoptotic

induction of AMA in HL-60 cells do not depend on signal

transduc-tions via the release of the cytochrome c, but the release of Ca2+

from ER, the activation of calpains, and then the release of AIF

from mitochondria to cytosol

C2-73

Ca2+-binding sites of transglutaminase 2

revealed by site directed mutagenesis

R Kiraly1,2, E Csosz2, T Kurtan3, Z Keresztessy2and

L Fesus1,2

1Apoptosis and Genomics Research Group of Hungarian Academy of

Sciences, Debrecen, HUNGARY,2Dept of Biochemistry and

Molecular Biology, University of Debrecen, Debrecen, HUNGARY,

3Dept of Organic Chemistry, University of Debrecen, Debrecen,

HUNGARY

Tissue transglutaminase (TG2) is a Ca2+-dependent acyltransferase

which plays important roles in several biological processes such as

apoptosis It has complex structure and enzyme activities including

hydrolyses of GTP The Ca2+-binding sites of TG2 have not been

characterized Our aim was to identify the Ca2+-binding sites using

site directed mutagenesis We have targeted three sites characterised

by negative surface potential and two others selected on the bases

of homology to Ca2+-binding sites of TG3 Multiple or single

mutations were generated at the five potential Ca2+-binding sites

Recombinant TG2 was produced in E coli in GST-fusion and

histi-dine-tagged form The results show that each of the mutants is

defi-cient in transglutaminase activity 45Ca equilibrium dialysis has

demonstrated decreased Ca2+-binding of the mutants Circular

dichroism spectroscopy and GTPase activity measurements

indi-cates that the amino acid substitutions do not cause major

struc-tural alterations GTPase activities of the mutants were not

sensitive for the Ca2+-concentration; some of the mutants showed

highly increased GTPase activity The mutagenised surface patches

may play an important role in the Ca2+-binding and possibly

regu-lation of TG2 function

C2-74 Role of presenilins in signal transduction pathways and cell death in Alzheimer’s disease

B O Popescu1, M Ankarcrona2, A Cedazo-Minguez2,

C A Hansson2, L M Popescu1, R F Cowburn2and

B Winblad2

1’Victor Babes’ National Institute of Pathology, Bucharest,ROMANIA,2Department of Neurobiology, Care Sciences andSociety, Karolinska Institute, Stockholm, SWEDEN

Alzheimer’s disease (AD) is the most frequent cause of age-relatedprogressive dementia, which constantly leads to profound alteration

of cognitive functions and premature death Both cell death andloss of synapses account for the clinical manifestations of the dis-ease Based on genetic and epidemiological data, AD is classified aseither sporadic or familial Mutated presenilins may cause familial

AD by altering neuronal signal transduction pathways, by ing beta-amyloid production or by triggering a number of pro-apoptotic mechanisms Here we present data concerning the local-ization of presenilins in the cell and the complex degradation ofthese proteins by the proteasome and caspase family systems Fur-ther on we explore the mechanisms by which mutated presenilinsrender cells more sensitive to cell death triggers We also showresults obtained in cell culture systems where wild-type presenilin isinvolved in cholinergic muscarinic receptor signal transductionpathways and mutated presenilin species alter phospholipase Cactivity and the resultant Ca2+ responses Presenilin is part ofgamma-secretase complexes and here we also show data supportingthe activity of gamma-secretase during apoptosis In conclusion,these studies provide evidence that presenilin regulates both cho-linergic signal transduction pathways and apoptotic cell death andpresenilin mutations are responsible for both sensitizing neurons todeath and altering neuronal signal transduction

increas-C2-75 Prion protein facilitates hormone-induced differentiation of mouse mammary gland epithelial cells

H Moerwald1,2, S Wurm1, K Crailsheim2and

C Wechselberger1

1Upper Ausrian Research, Linz, AUSTRIA,

2Karl-Franzens-University, Institute of Zoology, Graz, AUSTRIAGPI-linked cellular prion protein (PrP) is highly expressed in neur-onal cells, haematopoietic stem cells, lymphocytes, as well as in sev-eral other tissues like the mammary gland However, thecharacterization of the physiological roles exhibited by this protein isstill in progress and a bewildering number of different biologicalfunctions have been described recently In the present study we haveinvestigated the contribution of PrP to the process of hormone-induced differentiation using the murine mammary gland epithelialcell line EpH4 We generated stable transfected cells exhibitingincreased PrP expression and characterized the behavior of thesecells upon treatment with the lactogenic hormones dexamethasone,insulin and prolactin (DIP) We present evidence that high levels ofPrP facilitate DIP-induced differentiation as judged by increasedexpression of the differentiation marker beta-casein Furthermore,also morphological changes (i.e dome formation) upon hormonetreatment are more pronounced in PrP over-expressing cells Ourdata indicate that PrP is a permissive factor enhancing the differenti-ation-capabilities of mammary gland epithelial cells These resultspoint to a novel physiological aspect of PrP during mammary glanddevelopment and differentiation A role of PrP during cancer devel-opment becomes ever more conceivable

ERK activation by Bax inhibitor-1

overexpression

S Cho, J Kim, E Lee, H Choi, S Kim, H Min, B Kim,

G Kang and H Jeong

Konkuk University, Seoul, REPUBLIC OF KOREA

Multi-cellular organisms eliminate redundant, damaged, or infected

cells by apoptotic cell death Most of the organisms share the basic

apoptosis-regulating genes and the morphorogical and biochemical

features of apoptosis MAPKs are required for the regulation of

most biological processes, including mitosis, proliferation,

differen-tiation and apoptosis Bax inhibitor-1 (BI-1), which was originally

identified as testis enhanced gene transcript (TEGT) in mammals,

can regulate cell death caused by mitochondrial dysfunction or

ele-vated cytosolic Ca2+levels Recent study showed that BI-1 protein

protects cells from oxidative stress, reducing calcium content of

endoplasmic reticulum and ischemia-reperfusion injury BI-1/

TEGT is an evolutionally conserved integral membrane protein

containing multiple membrane-spanning segments and is

predom-inantly localized to intracellular membranes Various studies

revealed that BI-1/TEGT homologues exist in most eukaryotes

including yeasts and plants, and even in prokaryotes In our study,

overexpression of BI-1 was assessed to suppress etoposide-induced

increase of calcium contents in cytoplasm, resulting in increases of

ERK phosphorylation and suppression of ROS production We

confirmed these results using various inhibitors (such as PD98059,

N-acetyl cysteine, BAPTA-AM) or BI-1/TEGT siRNA Taken

together, our results suggest the function of BI-1/TEGT in the

regulation of ERK phosphorylation, ROS production and

apop-totic cell death

C2-77

Phylogenomics of the apoptotic DNA

fragmentation factor

L Eckhart, H Fischer and E Tschachler

Medical University of Vienna, Vienna, AUSTRIA

The degradation of nuclear DNA by DNA fragmentation factor

(DFF) is a key step in apoptosis of mammalian cells Here we have

performed a phylogenomic analysis of the genes encoding the two

DFF subunits, DFFA (also known as ICAD) and DFFB (CAD)

Among the genomes of bilaterian animals, DFF genes were present

in vertebrates and insects, but not in urochordates, echinoderms,

and nematodes Orthologs of DFFA and DFFB were also found in

the genome of Nematostella vectensis, a representative of the

evolu-tionary lineage of the cnidarians, which diverged from the

aforemen-tioned animals more than 600 million years ago A caspase cleavage

site in DFFA, the protein domains mediating the interaction of

DFFA and DFFB, and the amino acid residues critical for

endonuc-lease activity of DFFB were conserved in Nematostella These

find-ings suggest that DFF has been a part of the primordial apoptosis

system of the eumetazoan common ancestor and that the ancient cell

death machinery has degenerated during the evolution of several

bilaterian species, including the prototypical apoptosis model,

Caenorhabditis elegans

C2-78 Galectin-8 induces apoptotic signaling through

a link between phospholipase D, PKA and ERK pathways in Jurkat cells

A Norambuena1, C Metz2,1, L Vicun˜a1, E Pardo1, A Silva1,

C Ca´rcamo3, S Andre´4, H J Gabius4, A Gonza´lez1,2and

A Soza1,2 1Centro de Regulacio´n Celular y Patologı´a, Fac Ciencias Biolo´gicas,Pontificia Universidad Cato´lica de Chile, and MIFAB, Santiago,CHILE,2Depto de Inmunologı´a Clı´nica y Reumatologı´a, Fac Medi-cina, Pontificia Universidad Cato´lica de Chile, Santiago, CHILE,3

Depto Neurologı´a, Fac Medicina, Pontificia Universidad Cato´lica

de Chile, Santiago, CHILE,4Institut fu¨r Physiologische Chemie,Tiera¨rztliche Fakulta¨t, Ludwig-Maximilians-Universita¨t, Mu¨nchen,GERMANY

Galectin-8 (Gal-8) is a mammalian lectin that is secreted via a canonical route and binds beta-galactosides In Jurkat cells, werecently described that Gal-8 interacts with specific b1 integrins lead-ing to sustained ERK activation Here, we studied the functionalconsequences of this effect and explore the participation of phosp-holipase D (PLD) and PKA signaling pathways PLD activationproduces phosphatidic acid (PA) that can act as second messengerfor ERK activation, by recruiting Raf-1 to the plasma membrane,and can also decrease PKA activity, by activating specific cAMPphosphodiesterases (PDE) We found that: 1) Gal-8 induced ERKactivation is sustained for at least 4 h and led to apoptosis, contrast-ing with lower effects of Gal-1 and Gal-3; 2) induction of ERK isinhibited by primary alcohols, which abrogate PLD-production of

non-PA, and by PKA inhibitors; 3) Gal-8 increased PDE activity leading

to decreased basal activity of PKA These results suggest that Gal-8constitutes a novel extracellular stimulus for T cells, which inducesERK activation by involving a nexus between PA and cAMP/PKApathways, with modulating potential upon the immune response(Financed in part by FONDECYT grant# 1050715, FONDAPgrant# 13980001 and MIFAB)

C2-79 Cell distribution of human cell death-inducing DFFa [DNA fragmentation factor]like effector-a, CIDEa

J Santorova1, K Smolkova1, K Janouchova1, M Zackova1,

L Hlavata1, J Vostalova2, M Modriansky2and P Jezek11

Institute of Physiology, Prague, CZECH REPUBLIC,2MedicalFaculty, Palacky´ University, Olomouc, CZECH REPUBLIC

We hypothesize that pro-apoptotic CIDE proteins are sequestered in

a resting position in mitochondria, while apoptosis is initiated byCIDE migration to the nucleus, where it disrupts a complex of a 40-kDa caspase-3-activated nuclease (DFF40 or CAD), & its 45-kDainhibitor (DFF45 or ICAD) [1, 2] Therefore, we monitored mitoch-ondrial, nuclear, and cytosolic localization of recombinant humannative or Lumio- or GFP(RFP)- tagged CIDEa in relation apoptosisinduced by CIDEa overexpression or by CIDEa combined withcamphotecin Studies using confocal microscopy and immunohisto-chemistry were performed with four different cell lines: HeLa, 293Tcells, HepG2, or insulinoma INS-1E cells Higher extent of CIDEawithin the nucleus was observed upon apoptotic initiation CIDEalacking CIDE-C-domain did not localized in mitochondria Thusour data support the above hypothesis that mitochondrial localiza-tion of CIDE proteins [2] serves as sequestering of potentially dangerproteins that put cells into peril of an unavoidable apoptosis Export

of CIDEs from mitochondria then most likely initiates apoptosis.Acknowledgements: Supported by grants GACR 301/05/0221and MSM6198959216

Apoptosis inducing factor contains a PP1

docking motif which is also a penetrating death

sequence

A Godet, V Maire, V Quidville, H Ben Saphir, J Guergnon and

A Garcia, Sr

Institut Pasteur, Paris, FRANCE

The PP1 family of serine/threonine protein phosphatases is

involved in the regulation of many cellular processes including

apoptosis in mammalian cells (1) Apoptosis Inducing Factor

(AIF) is a mitochondrial caspase-independent death regulator In

this study co-immunoprecipitation and western blot experiments

indicated that AIF can interact with the catalytic subunit PP1c

In addition, structural analysis combined with pull down and

survival experiments demonstrate that a short sequence located

in the C terminal portion of AIF is sufficient for PP1 binding,

cell penetration and cell death We recently proposed a new

functional approach called drug phosphatase technology (DPT)

that is based on the design of penetrating sequences that interact

with the two PP1/PP2A families of protein phosphatases (2)

Consistently, our results demonstrate that AIF contains a

PP1-interacting sequence that is also a new target to design

DPT-apoptotic peptides

References

1 Garcia A, Cayla X, Guergnon J, Dessauge F, Hospital V,

Reb-ollo MP, Fleischer A, RebReb-ollo A Serine/threonine protein

phos-phatases PP1 and PP2A are key players in apoptosis Biochimie

(2003) 85:721–726

2 J Guergnon, F Dessauge, V Dominguez, J Viallet, X Cayla,

A Rebollo, V Yuste, S Susin, PE Bost and A Garcia Use of

penetrating peptides interacting with PP1/PP2A proteins as a

basis for a new Drug Phosphatase Technology Mol Pharmacol

(2006) 69:1115–1124

C2-81

An aPKC-Exocyst complex promotes cell

migration: a role for localised JNK1 activation

C Rosse1, E Formstecher2, Y Zhao3, M White4, J Camonis5

and P Parker1

1Cancer research UK, London, UK,2Hybrigenics, Paris, FRANCE,

3Biochemistry, UT Southwestern Medical Center, Dallas, TX, USA,

4

Dept Cell Biology, UT Southwestern Medical Center, Dallas, TX,

USA,5Institut Curie, Paris, FRANCE

Atypical protein kinase C (aPKC) isoforms have been implicated

in cell polarisation and migration through association with Cdc42

and Par6 In distinct migratory models, the Exocyst complex has

been shown to be involved in secretory events and migration Here

we show that the polarised delivery of the Exocyst to the leading

edge of migrating NRK cells is dependent upon aPKCs

Recipro-cally we demonstrate that aPKC localisation at the leading edge is

dependent upon the Exocyst This inter-dependence is shown to

occur through an aPKC-Exocyst interaction mediated by Kibra

Both the aPKCs and the Exocyst are shown to be required for

NRK cell migration and it is further demonstrated that they are

necessary for the localized activation of JNK at the leading edge

The study thus integrates the polarising behaviour of aPKCs, with

the pro-migratory properties of the Exocyst complex, defining a

higher order complex associated with the localised activation of

JNK at the leading edge of migrating cells

C2-82 Bax-induced release of intermembrane space proteins from yeast mitochondria

F N Vo¨gtle1, L K Sanjua´n Szklarz1, V Kozjak-Pavlovic1,

J C Martinou2, C Borner3, N Pfanner1and C Meisinger1

1Institut fu¨r Biochemie und Molekularbiologie, Freiburg,GERMANY,2Departement de Biologie Cellulaire, Geneva,SWITZERLAND,3Institut fu¨r Molekulare Medizin undZellforschung, Freiburg, GERMANY

Programmed cell death plays a role in selective elimination ofdangerous or irreversibly damaged cells in multi-cellular organisms.Although the presence of apoptotic mechanisms in yeast remains acontroversial topic there are many parallels, including several yeastorthologues of crucial mammalian apoptotic proteins and observa-tions pointing to an increased life span of yeast cells by overex-pressing human Bcl-2 These features have established yeast as amodel for apoptosis In S cerevisiae no homologues of the Bcl-2family are present Nevertheless, the release of cytochrome c aftertreatment with human Bax can be stimulated in yeast mitochon-dria in vivo and in vitro and can be suppressed by the addition ofanti-apoptotic Bcl-2 family members How Bax accomplishes thetask of releasing IMS proteins is still not known We tested therole of endogenous mitochondrial proteins in the release of IMSproteins upon Bax treatment of isolated yeast mitochondria from alarge collection of mutants Particularly, we were interested in therole of the protein translocase of the outer membrane (TOM-com-plex) and the sorting and assembly machinery (SAM-complex).Bax induced release of IMS proteins was independent of all TOM,SAM, fusion and fission proteins tested While protein complexesfrom the IMS can be efficiently released up to a size of 230 kDathrough the outer membrane, the inner membrane remains intactand in an import competent state, thus pointing to a selective per-meabilisation of the outer membrane

C2-83 Mutational study of components of the mammalian PI3K-PTEN-Akt in a yeast in vivo cellular system

I Rodriguez, Jr

Universidad Complutense, Spain, SPAINThe mammalian signalling pathway involving class I PI3K (phos-phoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase)and PKB (protein kinase B)/c-Akt has roles in multiple processes,like cell proliferation and apoptosis Its constitutive activation hasbeen implicated in the progression of a wide variety of humantumours To facilitate novel approaches for the molecular analyses

on these proteins, we have reconstituted this pathway by gous expression of PI3K (p110), PTEN and c-Akt in the modelorganism Saccharomyces cerevisiae Expression of hyperactiveforms of mammalian p110 inhibited yeast cell growth, which wascounterbalanced by co-expression of PTEN Moreover, we wereable to reproduce activation of c-Akt in yeast by PI3K Using thisyeast-based system, we have performed i) a functional analysis ofgerm line and somatic human PTEN mutations of clinical rele-vance, ii) a directed mutational analysis of functional domains ofboth PTEN and Akt1, and iii) a random mutagenesis screen forPTEN loss-of-function mutations Our results demonstrate thatyeast is a powerful tool to assess the functionality of differentmutants and isoforms of the components of this pathway, provi-ding a platform for genetic and pharmacological screens

Medical University, Dept Institute for Cancer Research, Clinics of

Internal Med.I, Vienna, AUSTRIA

Activins are members of the TGF-b superfamily, involved in

regu-lation of embryonic development, reproductive processes,

inflam-mation, proliferation and apoptosis Activin signalling occurs via

formation of receptor heterodimers and recruiting Smad proteins

Whereas the signalling pathways for Activin A are well

character-ised, less is known about Activin C signalling and the involved

pathways In this study we compared the biological effects, protein

alteration and transcriptional capacity of Activin C to Activin A,

B and TGF-b in different cell lines In a liver-tumor derived cell

line, Activin C treatment leads to enhanced proliferation in the

presence of serum, whereas a slight induction of apoptosis was

found under serum starvation In both cases, upregulation of

endogenous c-myc, known as a cell cycle regulator and apoptosis

inducer, and strong phosphorylation of the MAP kinase erk1/2,

involved in proliferation and survival, was detected, in contrast to

Activin A and B treatment On the transcriptional level, in a rat

embryonic cell system a smad3/4 consensus element responded

selectively upon Activin C treatment under serum starvation,

whereas under full serum no differences between Activin A, B and

C were detectable Also the promoter of bax, a pro-apoptotic

member of the bcl family, was activated by Activin C, but not by

the other cytokines In conclusion, Activin C signalling differs

from Activin A, B and TGF-b signalling and it appears to

modu-late cell fate between proliferation and apoptosis

Expression of Bcr-Abl, the oncogenic counterpart of the tyrosine

kinase c-Abl is the basis for chronic myelogenous leukemia

Bcr-Abl has acquired paradigmatic status as one of the first successful

cases of modern targeted cancer therapy, but drug resistance and

patient relapse remain problematic Bcr-Abl supposedly serves as a

docking scaffold for a distinct set of proteins linking the protein to

diverse signal transduction pathways that are decisive for

Bcr-Abl-dependent oncogenic transformation Although a lot is known on

individual binary protein-protein interactions of Bcr-Abl,

funda-mental questions on the cellular mechanism of action of Bcr-Abl

are still open, including its critical effectors In particular, there

has been no systematic, comprehensive and comparative study in a

defined cellular setting We are purifying and characterizing

pro-tein-protein complexes of Bcr-Abl and of selected interactors and

identify protein components by LC-MS/MS (liquid

chromatogra-phy coupled to tandem mass spectrometry) Measuring absolute

cellular protein copy numbers and complex stoichiometry enables

the development of pathway models and further strengthens the

approach As a starting point, we have identified a basic set of

Bcr-Abl complex components that interact with Bcr-Abl in a 1:1

or 2:1 stoichiometry constituting the core of the Bcr-Abl molecular

machine and are necessary for Bcr-Abl’s action as an oncogenic

signalling initiator Upon treatment with small-molecule inhibitors,

such as imatinib (Gleevec), the Bcr-Abl complex remodels We

expect that our study will identify components in the pathway that

could be exploited pharmacologically and provide a rationale for

combination therapy

C2-86 ATF-1-mediated hepatocyte growth factor-induced down regulation of TSP-1 expression leads to thyroid cancer cell invasion

C Ghoneim1, M Soula-Rothhut1, C Blanchevoye1,

F Antonicelli2, L Martiny1and B Rothhut1

1CNRS UMR 6198 Laboratory of Biochemistry, Reims, FRANCE,2

CNRS UMR 6198 Laboratory of Dermatology, Reims, FRANCEHepatocyte growth factor (HGF) plays a major role in the patho-genesis of a variety of human epithelial tumors including papillarycarcinoma of the thyroid Previous reports demonstrated thatHGF acting through the Met receptor, repressed thrombospondin-

1 (TSP-1) expression To study the mechanisms by which HGFdown-regulated TSP-1 expression, we transiently transfected apanel of deleted human TSP-1 promoter-reporter plasmids intopapillary thyroid carcinoma cells We identified a region between -

1210 and -1123 bp that is responsive to HGF treatment, and bors a cyclic AMPresponsive element (CRE) at position -1199.Over-expression of various members of the CREB family identifiedactivating transcription factor-1 (ATF-1) as the transcription factorresponsible for HGF induced repression of TSP-1 promoter activ-ity This inhibition was associated with an increase in the level ofnuclear ATF-1 protein Gel shift and antibody super shift studiesindicated that ATF-1 was involved in DNA binding to the TSP-1-CRE site Finally, we utilized shRNA to target ATF-1 and showedthat these shRNA constructs significantly inhibited ATF-1 expres-sion at both the RNA and protein level ATF-1 knockdown pre-vented HGF-induced down-regulation of TSP-1 promoter activityand protein expression and also reduced HGF-dependent tumorcell invasion Taken together, our results indicate that HGF-induced down-regulation of TSP-1 expression is mediated by theinteraction of ATF-1 with the CRE binding site in the TSP-1 pro-moter and that this transcription factor plays a crucial role fortumor invasiveness in papillary carcinoma of the thyroid triggered

har-by HGF

C2-87 Influence of quercetin and cisplatin on cell cycle

of mesothelioma cell lines

A Demiroglu-Zergeroglu1, B Basara1, N Saydan1,

G Yanikkaya-Demirel2and E Kilic3

1Gebze Institute of Technology, Kocaeli, TURKEY,2Centro atories, Istanbul, TURKEY,3Erciyes Universitesi, Clinical Biochem-istry Department, Kayseri, TURKEY

Labor-Malignant Mesothelioma (MM) is a lethal tumour arising from thesurface serosal cells of the pleural, peritoneal and pericardial cavit-ies Although new therapeutic perspectives are being continuouslytried and tested, MM treatment is still remains limited Quercetin(QU) is known as an anti-cancer agent since its anti-proliferativeeffects available on several malignant cells For example, QU treat-ment arrests the cell cycle control points of G0/G1, G1/S andG2/M Cisplatin (CIS) is also widely used chemotherapeutic agents

in treatment of many cancers It forms DNA adducts causing cellcycle arrest in G2/M phase We had previously reported that com-bination of QU and CIS much more effective on cell proliferationthen the single treatment In present study, we examined not onlythe single effect of QU and CIS but also their combination on cellcycle regulation at a dose and time-dependent manner Flow cy-tometry studies of cell cycle distribution showed that single CIStreatment on both SPC111 and SPC212 cells resulted to G2/Mblock at 48 h However, QU induced mainly S phase arrest Bothcell lines gave high percentage of cells in S phase suggesting thatprolongation of S phase when the combination QU and CIS wereadded to the cultures Taking these results together; QU alonemight be a potential agent for the treatment of mesothelioma cellsmoreover when used combination with CIS becomes much moreeffective

Functional characterization of BRCA1/2

unclassified variants observed in the Cypriot

population

C Gurkan, M Loizidou, A Hadjisavvas and K Kyriacou

Cyprus Institute of Neurology & Genetics, Nicosia, CYPRUS

Germ line mutations in BRCA1 and BRCA2 genes are associated

with an increased lifetime risk of breast and/or ovarian cancer

Numerous single nucleotide changes lead to gross truncations in

the BRCA1/2 proteins, and adversely affect their normal function

in key cellular processes Other types of BRCA1/2 variants, such

as missense mutations and in-frame deletions, result in more subtle

changes in the primary amino acid sequence When the functional

significance of such a variant is unknown, it is described as an

unclassified variant (UV) Categorization of these UVs as benign

or pathogenic remains a major challenge and an essential

prere-quisite for improving genetic counselling services This project aims

to establish an in vitro model system that allows functional

charac-terization of any given BRCA1/2 UV Using site-directed

mutagen-esis, we have introduced single nucleotide alterations in the human

BRCA1/2 genes that correspond to selected UVs observed in the

Cypriot population that were previously identified in our

laborat-ory In addition, known pathogenic truncating and missense

muta-tions are used as positive controls We are now setting up

functional assays based on ectopic expression of these BRCA1/2

constructs in selected mammalian cell lines Through these assays,

it will be possible to investigate the functional implications

associ-ated with each UV in comparison with the wild type and known

pathogenic variants

C2-89

Properties and prediction of proto-oncogenes

and tumour suppressor genes

S Furney1, S Madden2, D Higgins2and N Lopez-Bigas1

1

Universitat Pompeu Fabra, Barcelona, SPAIN,2University College

Dublin, Dublin, IRELAND

Cancer results from alterations to DNA that affects either the

expression or the functionality of certain genes, resulting in the

uncontrolled proliferation of a cell Understanding the mechanism

of regulation of cancer genes and the constraints on their coding

sequences is of fundamental importance in understanding the

pro-cess of tumour development Cancer-causing genes have been

tra-ditionally classified as either proto-oncogenes or tumour

suppressor genes Here we test the hypothesis that tumour

suppres-sor genes and proto-oncogenes, due to their involvement in

tu-mourigenesis, have distinct patterns of regulation and coding

selective constraints compared to non-cancer genes We analyse

human cancer gene coding sequence conservation, and examine

factors that may impinge on the appropriate regulation of cancer

genes, such as proximal promoter conservation, presence of

upstream CpG islands, and miRNA targets Indeed, we found

sig-nificantly greater conservation in the promoter regions of

proto-oncogenes, suggesting that these genes are more tightly regulated,

i.e they are more likely to contain a higher density of

cis-regulato-ryelements Furthermore, proto-oncogenes appear to be

preferen-tially targeted by microRNAs and have longer 3’ UTRs In

addition, proto-oncogene evolution appears to be highly

con-strained, compared to tumour suppressor genes and non-cancer

genes Both proto-oncogenes and tumour suppressor genes, possess

more complex gene structures, i.e longer protein and gene

sequences and more exons A number of these trends are

con-firmed in breast and colon cancer gene sets recently identified by

mutational screening

C2-90 The transcription factor ZEB1 (dEF1) promotes tumor cell dedifferentiation by repressing master regulators of epithelial polarity

A SultanMFPL, Vienna, AUSTRIAEpithelial to mesenchymal transition (EMT) is implicated in theprogression of primary tumors towards metastasis and is likelycaused by a pathological activation of transcription factors thatregulate EMT in embryonic development We show that ZEB1 is arepressor of E-cadherin and a major player in EMT during cancerprogression Overexpression of ZEB1 caused downregulation ofE-Cadherin and other epithelial markers in mouse and humanmammary epithelial cells On the other hand, ZEB1 downregula-tion in undifferentiated human cancer cells by RNA interferencewas sufficient to upregulate the expression of epithelial markers,including E-Cadherin and cell polarity genes CRUMBS3 andHUGL2 As shown by chromatin immunoprecipitation ZEB1physically associated with endogenous promoters of these keydeterminants of epithelial differentiation, and strongly repressedtheir promoter activities in reporter assays The upregulation of E-Cadherin expression by ZEB1 knock down was accompanied by adecrease in H3K9 di-, and H3K27 trimethylation and an increase

in H4 acetylation in the E-cadherin promoter, indicating theinvolvement of epigenetic modifications in ZEB1-mediated repres-sion Altogether, our data show that ZEB1 represents a key player

in pathologic EMTs associated with tumor progression

C2-91 LPA2 is necessary for LPA-induced ovarian cancer cell migration and invasiveness

K Jeong, Y Yoo, K Lee, D Yoon and H LeeCollege of Medicine, Konyang University, Daejeon, REPUBLIC OFKOREA

Ovarian cancer represents the fourth leading cause of ted death for women in the Western world, and lysophosphatidicacid (LPA) is a bioactive phospholipid that is involved in variouscellular events, including tumor invasion and metastasis Uponbinding to G protein coupled plasma membrane receptors (LPA1,LPA2, and LPA3), LPA exerts diverse biological effects, includingcell proliferation/survival, induction of neurite retraction, inhibi-tion of gap junctional communication, and stimulation of cellmotility However, detailed role of LPA receptor(s) for LPA-induced cancer cell invasiveness and motility was remain to besolved In the present study, we investigated the role of a LPAreceptor that is critical for invasiveness and motility in ovariancancer cells LPA stimulated ovarian cancer cell invasiveness andmotility in a dose dependent manner that is blocked by pharmaco-logical inhibitors for Gi (pertussis toxin), epidermal growth factor

VPC32183, a LPA1 and LPA3 inhibitor, did not show any effect

on LPA-induced cancer cell invasiveness A siRNA for LPA2abrogated LPA-induced activation of EGFR and p42/p44, as well

as invasiveness Collectively, these finding show the significance ofLPA2 for LPA-induced invasiveness and cell migration that could

be a promising drug target for ovarian cancer cell dissemination

Analysis of TSG101 coding sequence and

expression level in epithelial cells at different

stages of cervical cancer development

J Broniarczyk1, M.Łuczak2, J Zaborowska1, K Pawlak1,

A Jo´zefiak2, A Kwas´niewska3, W Ke˛dzia4and

A Goz´dzicka-Jo´zefiak5

1

Adam Mickiewicz University, Poznan´, POLAND,2University of

Medical Sciences, Poznan´, POLAND,3University of Medical

Sciences, Lublin, POLAND,4University of Medical Sciences,

Poznan, POLAND,5Adam Mickiewicz University, Poznan,

POLAND

Infection with Human Papillomavirus (mainly HPV16, 18 and 33)

has been associated with intraepithelial neoplasia and cervical

can-cer HPV-mediated transformation of epithelial cells has been

recognized as a multi-step process, in which, apart from the

pres-ence of the virus, additional factors are needed It has been

sugges-ted that TSG101 has a very important role in tumor formation

and progression Gene TSG101 was mapped to chromosome 11

p15.1–p15.2 This region of chromosome is known to be associated

with a loss of heterozygosity (LOH) in several tumor types

TSG101 encodes a multidomain protein involved in a range of

bio-logical function: ubiquitination, transcriptional regulation,

endo-somal trafficking, proliferation and cell survival The aim of the

project was to analyze TSG101 coding sequence and expression

level in epithelial cells at different stages of cervical cancer

develop-ment Application of PCR/SSCP methods demonstrated, that there

is no mutation in TSG101 coding sequence Real-Time PCR

analy-sis proved decreased level of TSG101 mRNA in epithelial cells

during different stages of cervical cancer development

C2-93

Evaluation of gene expressions in prognosis of

bladder cancer

S Yilmaz1, C Biray Avci1, Z O Dogan1, S Numanoglu1,

O Nazlı2and C Gunduz1

1Ege Unv School of Medicine, Izmir, TURKEY,2Ege Unv School

of Medicine Dept of Urology, Izmir, TURKEY

Bladder cancer (BCA), the second most common malignancy of

the genitourinary system, is the fourth most common cause of

death from cancer in men Tumor suppressor and oncogenes have

been revealed to play a role in tumorigenesis PIK3Ca, encoding

the p110 a-catalytic subunit phosphatidylinositol 3-kinase, has

been implicated as an oncogene in cancer Overexpression of

epi-thelial growth factor receptor (EGFR) is strongly correlated with

the late-stage, invasive urothelial carcinomas Human telomerase

reverse transcriptase (hTERT) encodes the protein component of

human telomerase In many neoplasms, increased telomerase

activ-ity is associated with poor clinical outcomes p53, a transcription

factor that regulates the cell cycle, is very important for cells in

multicellular organisms to suppress cancer GSTs are a multigene

family of dimeric enzymes that inactivate carcinogens by catalyzing

the conjugation of electrophiles to glutathione Glutathione

S-transferase T1 (GSTT1) is a polymorphic cytosolic enzyme and a

member of the theta class of GSTs and reduced catalytic activity

has been associated with an increased risk of cancer We aimed to

evaluate the expression levels of all these genes that play a role in

tumor progression in BCA Total RNA was isolated from urine

samples of 11 BCA patients Expression of genes was evaluated by

using RT-PCR We found reduced expression of PIK3Ca, EGFR,

GSTT1 and p53 and induced hTERT In conclusion; increased

hTERT expression may be evaluated as a biomarker in cancer

diagnosis The reduced activity of GST enzymes may show

insuffi-cient detoxification of carcinogens in prognosis of BCA

C2-94 Markers of DNA fragmentation and oxidative stress in mononuclear cells of patients with lymphoproliferative diseases

T M Jevtovic Stoimenov, G Kocic, D Pavlovic,

T Cvetkovic, G Bjelakovic and J MiljevicMedical Faculty, Nis, SERBIA AND MONTENEGROLymphoproliferative disorders (LPD) are a subset of immunopro-liferative disorders, and refer to several conditions in whichlymphocytes are over-produced or act abnormally in patients whohave compromised immune systems The most common LPD arechronic lymphatic leukemia and lymphomas Chronic LymphaticLeukemia (CLL) is clonally expansion of mature-appearinglymphocytes involving lymph nodes, a neoplastic disease suscept-ible to antioxidant enzyme alterations and oxidative stress.Although, increased production of ROS and oxidative stress werenon-proteins inducers of mitochondria-dependent apoptosis, inLPD were observed apoptosis disturbance, and also resistantion ofleukemic cells against pro-apoptotic agents We have examined theactivities of catalase (CAT), together with the levels of malondial-dehyde (MDA) and deoxyribonuclease (DNase I) in mononuclearcells of CLL and NHL patients and compared them with those ofnormal subjects of the same age The activity of CAT was signifi-cantly decreased in CLL and NHL mononuclear cells (P < 0.001).The level of MDA was reduced only in group of CLL patients(P < 0.05) compared to the control group, while the activity ofDNase I, as marker of DNA fragmentation was decreased in agroup of nonHodgkin lymphoma, but significantly increased ingroup of CLL patients The aim of DNA fragmentation and oxi-dative stress markers in LPD’s investigation was to contribute tobetter diagnostics and more efficient therapeutic approach to thosediseases On the basis of the results of this paper, we can concludethat investigation of this parameters could be of interests and thatfurther investigation should focused on apoptosis disturbance

C2-95 Sevoflurane induces apoptosis and modulates expression of proapoptotic genes in human colon carcinoma cells

L Glavas-Obrovac, S Kvolik and S MarcziJ.J Strossmayer University of Osijek, School of Medicine Osijek;Clinical Hospital Osijek, Osijek, CROATIA

This study was designed to determine the consequence of singleexposure of carcinoma cells to inhaled anesthetic - sevoflurane,simulating thus the situation occurring in the clinical setting Coloncarcinoma cells (CaCo2) were exposed to anesthetic gas mixtureconsists of O2:N2O:CO2(35:60:5 vol.%) and sevoflurane (3.0 vol.%)for 1 and 2 h Cytotoxicity was analyzed by MTT assay Mode ofcell death, caspase-3 enzyme activity, and expression of genes incontrol and treated cells were tested immediately after exposure tosevoflurane and 24 h later Apoptosis was assessed by FACS analy-sis using annexin and propidium iodide staining Caspase-3 enzymeactivity was measured by EnzChek caspase-3 Assay Kit Expression

of genes (GADPH, p53, caspase-3, and CYP2E1) was examined byRT-PCR Sevoflurane significantly suppressed treated cells’ growth.Percent of apoptotic and necrotic cells increased in comparison withcontrol cells in a time-dependent manner Alteration in caspase-3enzyme activity was also observed Compared to expression in con-trol cells, caspase-3 gene expression changes (>1.5 fold) were found

in treated cells at all time points Expression of p53 gene ately after treatment was unchanged and 24 h later significantlyincreased (for 78%) Expression of CYP2E1 gene also increased (for73%) in the cell exposed to sevoflurane for 2 h, and 24 h later slowdown to 20% In conclusion, sevoflurane in anesthetic dose suppres-ses the colon carcinoma cells’ growth and increases expression ofthe CYP2E1 gene, which could be involved in detoxification proces-ses Does sevoflurane induces apoptosis in CacCo2 cells by p53-dependent or p53-independent pathway remains to be identified