Tài liệu Báo cáo khoa học: Oral Presentations Integration of Metabolism and Survival pdf

Sabitov” Laboratory of Enzyme Structure and Regulation, Aithozhin’s Institute of Molecular Biology and Biochemistry,’ Department of Chemistry, Al-Faraby’s Kazakn National University.. "

Oral Presentations

Integration of Metabolism and Survival

OP-1

The ankyrin repeat and SOCS box-containing

protein Asb-9 targets creatine kinase B for

degradation

M A Debrincat', J G Zhang’, T A Willson’, B T Kile’,

S L Masters’, L M Connolly’, R J Simpson’, H M Martin’,

N A Nicola’ and D J Hilton?

"Cancer and Haematology/Molecular Medicine, The Walter

and Eliza Hall Institute of Medical Research, Melbourne,

Australia,’Cancer and Haematology, The Walter and Eliza Hall

Institute of Medical Research, Melbourne, Australia,’ Molecular

Medicine, The Walter and Eliza Hall Institute of Medical

Research, Melbourne, Australia,‘The Joint Proteomics Laboratory

of the Walter and Eliza Hall Institute of Medical Research and

Ludwig Institute for Cancer Research, Melbourne, Australia

E-mail: debrincat@wehi.edu.au

The suppressors of cytokine signalling (SOCS) proteins inhibit

cytokine signalling by direct interaction with Janus kinases

(JAKs) or activated cytokine receptors In addition to the N-ter-

minal and SH2 domains that mediate these interactions, SOCS

proteins contain a C-terminal SOCS box Evidence suggests

SOCS box-containing proteins act as part of an elongin-cullin-

SOCS (ECS) E3 ubiquitin ligase complex, marking target pro-

teins for degradation The specificity of the complex is deter-

mined by the protein interaction motif located upstream from the

SOCS box A number of other protein families that possess a

SOCS box have been identified, the largest of which are the ank-

yrin repeat and SOCS box-containing Asbs While it is known

that the SOCS proteins are involved in the negative regulation of

cytokine signalling, the biological and biochemical functions of

the Asbs are undefined To understand the functional role of Asb

proteins, a proteomic approach was implemented and creatine

kinase B (CKB) was identified as a specific binding partner of

Asb-9 Transfection of increasing concentrations of a tagged

Asb-9 construct into 293T cells increased the polyubiquitination

of CKB and resulted in a concomitant decrease in total CKB

levels within the cell The targeting of CKB for degradation by

Asb-9 was entirely SOCS box-dependent The interaction has

been confirmed iv vivo and suggests that Asb-9 may act as a

specific ubiquitin ligase-regulating CKB abundance

OP-2

Signal transduction of cytokinine

M Gilmanov', S Ibragimova', Sv Sadykova', Zh Basygaraev

and A Sabitov”

Laboratory of Enzyme Structure and Regulation, Aithozhin’s

Institute of Molecular Biology and Biochemistry,’ Department of

Chemistry, Al-Faraby’s Kazakn National University

E-mail: baltakay@mail.ru

1

The cytokinine the most important phytohormone, which is con-

trolling the division of the plant, cells We carried out the investi-

gation of cytokinine signal transduction We were discovered the

most important participant of cytokinine signal transduction

This participant was purified from germinated wheat grains by hydrophobic chromathography on octyl-sepharose 4B CL and by reversed-phase chromatography on RP-18 type column Thus we were named the isolated substance as secondary cytokinine hor- mone or shorter mediator of cytokinine (MC) The MC is very powerful phytohormone as it shows the physiological activity at concentration 1000 times less than cytokinine In contrast of cytokinine the MC appears own biochemical activities For exam- ple, MC is activated NADP-glutamate dehydrogenase and H+ATP-ase, while the cytokinine not be able to activate this enzymes The purified MC is competitive repressed the binding

of tritium-labeled fusicoccine with fusicoccine receptors on plas- matic membrane winch were isolated from roots of Zea mays sprouts It was shown that the binding of MC with fusicoccine receptors led to increasing the level of cytoplasmic calcium ions Then calcium ions by participation of inositides is activated the proteinkinase C Proteinkinase C was isolated by gel chromatog- raphy on sephacryle S-300 column and by ion-exchange chroma- tography on DE-52 column This enzyme is the last participant

of the signal transduction of cytokinine

Very little is known about the effect of exercise on the molecular regulation of polypeptide synthesis in skeletal muscle Here, the effect of contractions on skeletal muscle eukaryotic elongation factor 2 (eEF2) phosphorylation and eEF2 kinase activity was investigated In response to contractions im situ, there was a rapid (i.e 15s) fivefold increase in eEF2 phosphorylation at Thr56 in the contracting gastrocnemius muscle of rats that was maintained

at this level during 30 min of contractions, with no change in the non-stimulated contralateral muscle Furthermore, eEF2 phos- phorylation was higher in both soleus and extensor digitorum longus muscles of mice when contracted ex vivo, indicating that the mechanism behind this increase is related to local factors No change in im vitro eEF2 kinase activity was observed in the con- tracted rat muscles at any time-point or when measured at pH 6.8 versus 7.2 Furthermore, the increase in eEF2 phosphoryla- tion occurred at a time before any change in AMPK activity was observed and was normal in contracting muscles of mice expres- sing non-functional AMPK However, Ca’ *-calmodulin potently increased the activity of skeletal muscle eEF2 kinase when meas- ured in vitro Taken together, these data indicate that the inhibi- tion of protein synthesis in contracting muscle may arise from phosphorylation of eEF2 via a Ca**-calmodulin-eEF2 kinase cascade

Integration of Defence and Survival

OP-4

Investigation of multidrug resistance in

docetaxel and doxorubicin-resistant MCF-7 cell

lines

O Darcansoy Iseri!, M Demirel Kars', U Giindiiz' and

F Arpacf

Department of Biological Sciences, Middle East Technical

University, Ankara, Turkey,’ Department of Oncology, Guilhane

Military Academy School of Medicine, Ankara, Turkey

E-mail: dozlem@metu.edu.tr

Ineffectiveness of anticancer drugs during chemotherapy or recur-

rence of malignancy after therapy is a frequently observed situation

in cancer chemotherapy Multidrug resistance (MDR) phenomenon

is defined as the resistance of tumor cells to various cytotoxic drugs

It is a major impediment to successful treatment of breast cancer

using chemotherapy Cancer cells either strengthen the already pre-

sent systems necessary for the removal of toxins from cells or acquire

resistance to cytotoxic drugs Members of the ATP-binding cassette

(ABC) transporter superfamily have an important role in MDR

Among these, proteins coded by the ABCB1 (MDRI1), ABCC1

(MRPI1), and ABCG2 (BCRP) genes are the most important trans-

porters related to MDR phenotype In this study, effects of expres-

sion levels of the MDR1I, MRP1, BCRP genes on the development

of docetaxel and doxorubicin resistance using a model MCF-7

breast carcinoma cell line is evaluated Docetaxel and doxorubicin

were applied to cell culture in dose increaments and resistant sub-

lines were developed Cytotoxicity analysis of drugs was performed

in wild type and developed resistant sublines to test development

of resistance Total RNA was isolated from cells, converted to

cDNA and amplified by using gene (MDR1, MRPI, and BCRP)-

specific primers by RT-PCR Western blot analysis and immuno-

staining were performed to determine the related protein levels

OP-5 GSK3: identification of a novel mechanism

controlling inflammation in the brain

E Beurel!, S Michalek” and R Jope!

‘Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Birmingham, AL, USA,’Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA E-mail: beurel@uab.edu Controlling inflammation is a major challenge in the brain where inflammation has devastating consequences because, since dam- aged neurons cannot be replaced, neuroinflammation contributes

to neurodegenerative diseases In response to lipopolysaccharide (LPS), brain microglia, and astrocytes activate cytokines produc- tion, such as IL-6 Glycogen synthase kinase-3 (GSK3) regulating transcription factors, was studied as a regulator of neuroinflam- mation In mouse primary astrocytes, we examined if GSK3- regulated IL6 production stimulated by LPS and its amplified production caused by co-administered interferon-y Both LPS- induced IL-6 production and also its potentiation by interferon-y are highly dependent on GSK3 With both IL-6 production was abolished by GSK3 inhibitors, demonstrating the key role of GSK3 in neuroinflammation The mechanism of LPS-induced IL-6 production potentiated by interferon-y was due to GSK3 activation and nuclear depletion of GSK3 This suggests that interferon-y plus LPS potentiates IL-6 production by activating cytosolic GSK3 which can control the activation of transcription factors that activate IL-6 expression Chromatin immunoprecipi- tation is being used to identify the transcriptional targets of GSK3, such as NF-«B that regulate IL-6 production This study identified GSK3 as a key regulator of neuroinflammation, estab- lishing that GSK3 inhibitors provide a new strategy to counteract the devastating effects of neuroinflammation

Rhythmic Signals: the Setting of Biological Time

OP-6

Computational search of the interaction

between melanopsin and cryptochrome-2

proteins

E B Unal', B Erman? and I H KavakliF

"Computational Sciences and Engineering, Koc University, Istan-

bul, Turkey,°Chemical and Biological Engineering, Koc University,

Istanbul, Turkey E-mail: evunal@ku.edu.tr

Circadian rhythms are the biological processes that oscillate in the

biochemical, physiological and behavioral functions of organisms

with a periodicity of approximately 24 h without any external cues

In mammals, circadian rhythm is generated by molecular clock,

which is located at suprachiasmatic nuclei (SCN) part of brain

Circadian rhythm is reset by external factors such as light The

cryptochromes,which was first discovered in Arabidopsis,are the

blue-light photoreceptors They absorb light and transmit the elec-

tromagnetic signal to blue-light dependent of signal transduction

In mammals, the cryptochromes and melanopsin have been pro-

posed as circadian photoreceptor pigments that exist in the inner

retina to transmit signal to the SCN to tell the time of day Both

humans and mice have two cryptochrome proteins; CRY1 and

CRY2 CRY2 is mostly expressed in retinal cells Based on current

evidence we propose that CRY2 may interact with melanopsin to

mediate the light dependent signal-transduction in mammals.We have taken both computational and experimental approaches in order to show possible interaction between them during the circa- dian photoreception First, we have predicted 3-D structure of cryptochrome using EsyPred, Robetta programs.Then, we have shown that both cryptochrome and melanopsin may interact in silico using various computational softwares, such as AUTO- DOCK, HEX.We are currently verifying our computational data taking experimental approach, specifically the FRET

OP-7

Mitochondrial electron transport chain reactivity in the brain and eye tissues under circadian rhythm alterations

M B Yerer'’, M P Alcolea Delgado”, S Aydogan',

F J Garcia-Palmer” and P R Salom?

"Faculty of Medicine, Department of Physiology, University of Erciyes, 38039 Kayseri, Spain,’ Department of Biochemistry and Molecular Biology, University of Balearic Islands, Mallorca, Spain E-mail: eczbetul@yahoo.com

Mitochondria plays a central role in energy-generating processes within the cell thorough the electron transport chain (ETC), the primary function which is ATP synthesis via oxidative

phosphorylation (OXPHOS) which is shown to be related to age-

ing and apoptosis when this balance is destroyed under different

circumstances This study is performed to investigate the effects

of alterations in the physiological melatonin levels via the circa-

dian rhythm changes, on the mitochondiral ETC in brain and

eye and how these changes are correlated to the pineal gland

melatonin receptor expressions Fifty Sprague-Dawley male rats

weighing 200-250 ¢ were used in five groups of different circa-

dian rhythms The control group was 12/12 h of light/dark (L/D)

cycle Different circadian rhythms of 24/0 h L/D, 0/24h L/D,

16/8 h L/D and 8/16 h L/D cycles were applied to the groups for

1 week, respectively, in special cages where the duration of the

light and the climate can be adjusted The melatonin receptors,

MELI1 and MEL2 expressions were determined by real-time PCR

in the pineal gland The mitochondria of the brain and eye tis-

sues were isolated from the homogenates and the activation of

the mitochondrial OXPHOS complexes were determined by spec-

trophotometric micro-methods described before Plasma melato-

nin levels were also determined by ELISA kit (IBL, Turkey)

Related to circadian rhythms, the plasma melatonin levels were

the highest in the 0/24 L/D group compared to the other groups

(p < 0.05) and the MEL1 and MEL2 receptor expressions were

also altered significantly by the circadian rhythms (p < 0.05) The Complex I activity is found to be decreased significantly in the 24/0 L/D group compared to the control and the 0/24 h L/D group (p < 0.05) in the brain mitochondria whereas it was sig- nificantly higher in the eye mitochondria compared to the control (p < 0.05) Complex III activities were slightly lower in the 24/ 0h L/D group, whereas there was a significant increase in the eye mitochondria in all the groups compared to the control (p < 0.05) Furthermore, there was a significant increase in the Complex IV and V activities in the brain mitochondria were sig- nificantly higher 24/0 L/D group compared to its control (p < 0.05) whereas they were found to be unaffected in the eye mitochondria As a consequence, this is the known first report to show the MELI and MEL2 receptor expressions by real-time PCR under different circadian rhythms These alterations both in the receptors and the plasma melatonin levels are found to be correlated with the mitochondrial respiratory chain complexes which are directly related to the energy metabolism in the cells during the ageing process and the apoptosis This study was granted by NATO Science Fellowships A2 Programme of TUBI-

TAK

Signaling and Cancer: Nuclear Receptor Connection

OP-8

Dysregulated Msx and DIx gene expression in

epithelial odontogenic tumors

S Ghoul-Mazgar', B Ruhin’, D Hotton? and A Berdal?

‘Laboratoire de Biologie Oro-Faciale et Pathologie, INSERM

U714-IF R-58, Universités Paris 7 and Paris 6 Laboratoire

d’Histologie-Embryologie, Faculté de Médecine Dentaire de

Monastir, Tunisia,’Stomatology and Maxillofacial Surgery

Department, Pitié Salpétriére University Hospital, Paris Cedex 13,

France,’ Laboratoire de Biologie Oro-Faciale et Pathologie

INSERM U714-IFR-58, Universités Paris 7 and Paris 6, France

E-mail: ghoulsonia@yahoo.fr

Odontogenic tumors are rare pathologies, mostly benign, located

in maxillary area The most frequently observed benign epithelial

odontogenic tumors is called ameloblastoma and may although

give rise to the extremely rare malignant epithelial odontogenic

tumors, usually named odontogenic carcinomas.The differential

diagnosis between these tumors is so difficult regarding the

diverse clinical prognosis and therefore management Homeodo-

main proteins comprise transcription factors that are essential in

many developmental processes Homeodomain is encoded by a

highly conserved 60 amino acid sequence called homeobox that is

responsible for specific interactions with DNA Non-clustered ho-

meobox genes are called non-HOX and include the Msx and Dlx

gene family In this study, we examined the Dlx and Msx gene

expression by RT-PCR and in situ hybridization in recurrent 13

benign ameloblastomas and one malignant clear cell odontogenic

carcinoma (CCOC) Our data show specific expression pattern of

Msx and Dix gene in the CCOC compared with benign amelobl-

astomas Furthermore, exploring the expression pattern of signal

molecules by RT-PCR, Bmp2 was shown to be inactivated in the

carcinoma, but not Bmp4 Malignancy of epithelial odontogenic

carcinoma seems to be a multistep and highly heterogeneous pro-

cess requiring activation and deactivation of multiple and specific

genes suggesting exploration of homeogene expression to discrim-

inate benign ameloblastomas and odontogenic carcinomas

OP-9 Ligand-specific dynamics of the androgen

receptor on its target promoter in living cells

T L Klokk!, P Kurys!, C Elbi*, A K Nagaich?,

A Hendarwanto’, T Slagsvold', C Y Chang®, G L Hager? and F Saatcioglu!

"Department of Molecular Biosciences, University of Oslo, Oslo, Norway,’Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, USA, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, MD, USA E-mail: t.i.klokk@imbvy.uio.no Androgen receptor (AR) mediates the action of androgens, which are important in the development and maintenance of the male reproductive system and in pathologic conditions such as pros- tate cancer This is the basis for the routine use of antiandrogens

to block AR function in disease states, but little is known on the mechanisms involved We studied ligand-dependent AR interac- tion with a target promoter in vivo, using photobleaching micros- copy, kinetic modeling, FRET analysis, and in vitro chromatin remodeling The interaction of agonist-bound AR with the MMTV promoter was rapid and transient In the presence of antagonist, and with a transcriptionally impaired AR mutant, an even faster interaction was seen due to decreased residence time

on the promoter The short residence times seen for AR in response to all ligands support the ‘hit-and-run’ model and three- dimensional genome-scanning hypothesis of transcription factor action Furthermore, agonist and partial antagonists, but not pure antagonists, induced the recruitment of a chromatin-re- modeling complex to the HRE Finally, FRET analysis in vive demonstrated both intermolecular and intramolecular interac- tions between the N- and C- termini of AR at the HRE Thus, three-dimensional scanning of the genome space, ligand-depend- ent modulation of AR kinetic properties, recruitment of chroma- tin remodeling complexes and proper intermolecular and intramolecular interactions are all critical for the im vive function

of AR on its target promoter

Cell Surface Receptors and Downstream Targets

OP-10

Mutations of the growth hormone receptor

gene in Turkish patients with Laron-type

dwarfism

A Arman', N Yordam’, A Ozon’, P Isguvenỷ, A Coker* and

I Peker!

'The Faculty of Engineering, Marmara University, Istanbul,

Turkey,’ The Department of Pediatrics, Division of Endocrinology,

Hacettepe University, Ankara, Turkey,?The Department of

Endocrinology, Goztepe Government Hospital, Istanbul,

Turkey,’ The Department of Biology, Art and Sciences, Marmara

University, Istanbul, Turkey E-mail: aarman@eng.marmara.edu.tr

Growth hormone (GH) mediates its growth, fat and carbohy-

drate metabolism through insulin-like growth factor-I (IGF-I)

Interaction of GH with the GH receptor (GHR) is necessary for

systemic and local production of IGF-I that mediates GH

actions Mutations in the GHR cause severe postnatal growth

failure and the disorder is an autosomal recessive genetic disease

called Laron-type dwarfism, characterized by elevated serum GH

associated with low levels of IGF-I In this research, our purpose

was to analyse the GHR gene for mutations in eight patients

with Laron-type dwarfism Eight patients were selected based on

their phenotypic characteristics and their genomic DNAs were

isolated from bloods of eight laron children Exon 2-9 specific

polymerase chain reactions (PCRs) and their flanking splice sites

were amplified PCR The PCR products were purified and se-

quenced We defined three missense mutations (S40L, V125A,

I506L), one nonsense mutation (W157X), one sense mutation

(G168G), one exon 3 deletion (exon 3 deletion) and one frame-

shift mutation (G insertion in exon 2) located in the extracellular

domain of GHR in eight patients Acknowledgment: This project

was supported by Turkish Republic State Planning Organization

OP-11

Human XLas signals more efficiently than the

a-subunit of the stimulatory G protein (GSz)

in vitro

A Linglart!, M J Mahon’, T Dean’, T J Gardella’,

H Jueppner7 and M Bastepe”

’ Pediatric Endocrimology and INSERM U561, Saim Vincent de

Paul Hospital, Paris, France,’ Endocrine Unit, Department of

Medicine, Massachusetts General Hospital and Harvard Medical

School, Boston, MA, USA E-mail: bastepe@helix.mgh

harvard.edu

XLas is partly identical to the a-subunit of the stimulatory G pro-

tein (Gs) with an extended N-terminus Using adenoviral expres-

sion and cells that endogenously lack Gsa and XLas (GnasE2—/—

cells), we investigated human XLas (hXLas) Immunofluorescence

microscopy showed membrane and punctate perinuclear staining

for hXLas On metal affinity chromatography, hXLas co-purified

with a histidine-tagged Gal Furthermore, a PTH(1-15) analog,

which normally prefers binding to a Gsa-coupled form of

PTHRI, bound to membranes from GnasE2—/— cells co-expres-

sing PTHRI1 and hXLas Also, hXLas was able to mediate basal

and agonist-induced cAMP accumulation However, while hXLas

was expressed at lower levels than Gsa in transduced cells, basal

cAMP level in cells expressing hXLas was “twofold higher than

in cells expressing Gs Similarly, basal ERK 1/2 phosphorylation

in GnasE2-/- cells transiently expressing hXLas was markedly

enhanced Isoproterenol treatment also resulted in significantly higher levels of cAMP accumulation in hXLas-expressing cells than Gso-expressing cells, whereas PTH-induced cAMP accumu- lation appeared similar in cells co-expressing PTHR1 and either hXLas or Gsa These findings indicate that hXLas can act as a more potent signaling protein than Gsa, which may have implica- tions in responses mediated typically by Gsz

OP-12

Growth inhibition of c6 glioma cells in monolayer and spheroid cultures by the

combination of carvedilol and imatinib

M Erguven!, A Bilir?, S Tuna’ and N Akev!

‘Department of Biochemistry, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey," Department of Histology and Embryology, Istanbul Faculty of Medicine, Istanbul, Turkey E-mail: merguven@istanbul.edu.tr

Rat C6 glioma is a chemoresistant experimental brain tumour, which is difficult to treat with a combination of drugs A new tyrosine kinase inhibitor, imatinib (Gleevec), has recently been found efficacious in the pre-clinical trials for glioblastoma (GBM) Carvedilol (Dilatrend), antihypertensive drug, has been demon- strated to reverse multidrug resistance (MDR) to anticancer drugs

in several tumor cell lines in vitro However, any possible modula- tory effect of carvedilol on brain tumours and on the efficacy of imatinib has not yet been evaluated experimentally In the present study, we have investigated whether carvedilol provides synergistic

or antagonistic effect on imatinib-induced cytotoxicity in mono- layer and spheroid cultures of malignant C6 glioma cells C6 glioma cells in monolayer and spheroid cultures were treated with the combination of carvedilol and imatinib in concentration of

10 pM The cell proliferation, morphology, spheroid volumes, bromodeoxyuridine-labelling index (BrdU-LI) were evaluated The expression levels of caspase-3, caspase-9, hypoxia inducible factor-1 (HIF-1), Bcl-2 and platelet-derived growth factor receptor alpha (PDGFR«) were examined by Western blot analysis The statistical significance was analysed by using the Student’s ứ-test The results demonstrated that carvedilol and imatinib in combina- tion display enhanced antitumour activity in vitro against experi- mental rat C6 glioma in monolayer and spheroid cultures

OP-13 Non-traditional ways of B lymphocyte regulation: receptors for acetylcholine and thrombin

S Komisarenko', R Grailhe*, Y Petrova’, J P Changeux’,

W Bahou’ and M Skok!

'Palladin Institute of Biochemistry, Kiev, Ukraine,’ Pasteur Institute, Paris, France,’ State University of New York, Stony Brook, NY, USA E-mail: svk@ biochem.kiev.ua

Immune system is highly specialized to recognize and destroy the invading pathogens and transformed self-cells For this purpose,

it is equipped with unique antigen-specific receptors and employs specific ways of cell-to-cell communication However, both the development and functioning of immune cells are regulated by various external factors including those of nerve and blood coagu- lation systems Correspondingly, lymphocytes express receptors for non-immune mediators, such as acetylcholine and throm- bin We demonstrated the presence of nicotinic acetylcholine

receptors (nAChRs) and protease-activated receptors type 3

(PAR3) on mouse B lymphocytes using specific antibodies gener-

ated to functionally important parts of these receptors: compo-

nents of acetylcholine-binding site or thrombin-cleavage site It

was shown that signaling through nicotinic receptors stimulated

proliferation of B lymphocyte-derived cell lines, supported B-lym-

phocyte survival in the bone marrow, but limited its activation in

mature state In contrast, PAR3 activation inhibited B-cell line

proliferation, but enhanced CD40-mediated B-lymphocyte prolif-

eration It is concluded that signaling through nAChR and PAR3

affects basic vital functions of B lymphocytes, such as prolifer-

ation and survival, and interferes with their activation pathways

These data show the ways, by which acetylcholine, nicotine or

thrombin can regulate the humoral branch of immunity

OP-14

Identification and characterization of a novel

transcriptionally active domain in the linker

region of the TGFf-regulated Smad3 protein

M Siderakis, V Prokova, S Mavridou, P Papakosta and

D Kardassis

Department of Medicine, University of Crete and IMBB-FORTH,

Heraklion, Crete, Greece E-mail: kardasis@imbb.forth.gr

Our structure-function analysis of human Smad3 protein, a key

mediator of TGF6 signaling in mammalian cells revealed that the

Signaling Through lon-channels

OP-15

Sigma’ receptor/beta1 integrin complex: a

novel target site for breast cancer cell

adhesion

R Mahen, C Palmer, C Edwards, M Djamgoz and E Aydar

Imperial College London, Divisions of Biology, Cell & Molecular

Biology and Molecular Biosciences, Sir Alexander Fleming

Building, South Kensington Campus, London, UK

E-mail: e.aydar @imperial.ac.uk

The sigma receptor is a novel protein that is highly expressed

in cancer cells and tissues, and has been shown to modulate

proliferation and adhesion of breast cancer cells The mechan-

ism of action of the sigmal receptor in these processes; how-

ever, has not been elucidated Using the Single Cell Adhesion

Measuring Apparatus (SCAMA; confocal microscopy, sigmal

receptor RNAi, immunoprecipitation, surface biotinylation and

lipid raft fractionation, the function of sigmal receptor in adhe-

sion of MDA-MB-231 breast cancer cells was investigated

Functional studies using SCAMA revealed that disruption of

lipid rafts eliminated sigma receptor modulation of adhesion

Moreover,immunoprecipitation experiments in MDA-MB-231

cells,demonstrated that sigmal receptor and betal integrin are

associated Furthermore, both confocal microscopy experiments

and surface biotinylation experiments indicated that both appli-

cation of sigma receptor drugs and knock-down of the sigmal

receptor increased the betal integrin expression in the mem-

brane Lipid raft fractionation experiments in MDA-MB-231

cells demonstrated that both application of the sigma receptor

drugs and the knock-down the sigmal receptor levels, betal in-

tegrin protein in lipid rafts fraction of MDA-MB-231 cells were

altered All these data suggest that sigmal receptor is associated

with betal integrin and is likely modulate betal integrin levels

middle, non-conserved, linker domain has an autonomous and potent transactivation function The region with the maximal transactivation capacity was the 143-248 that consist of almost the entire linker domain and the first 18 amino acids of the MH2 domain The corresponding regions in Smad4 as well as in Smad1, which is a key mediator in Bone Morphogenetic Protein (BMP) signaling, are also transcriptionally active in mammalian cells further supporting the important role of this domain for Smad function Smad3 mutants bearing an internal deletion of the 200-230 region or single amino acid substitutions in two highly conserved residues of this region (Q222A and P229A) had severe defects in oligomerization and transcriptional activation of target promoters In contrast, mutagenesis of a non-conserved amino acid residue in the same region (N218A) did not affect any Smad function examined Using a protein-protein interaction assay based on biotinylation in vivo, we were able to show that the Smad3 mutant with the internal deletion of amino acids 200—

230 is unable to interact physically and functionally with the his- tone acetyltransferase p/CAF Our data support an essential role

of the previously uncharacterized middle region of Smad3 for nuclear functions, such as transcriptional activation and interac- tion with Smad coactivators

Therefore, sigmal receptor is likely to be a novel target for breast cancer metastasis

OP-16

HERG potassium channels and heart disease

S Kuyucak School of Physics, University of Sydney, NSW, Sydney, Australia E-mail: serdar@ physics.usyd.edu.au

The heartbeat is controlled by electrical signals mediated by the flow of ions through specialized ion channels Of the channels that contribute to cardiac electrical activity, potassium channels encoded by the Human ether-a-go-go-related gene (HERG) have been of particular interest for many reasons First, mutations in HERG are the cause of one-third of cases of congenital long QT syndrome, an inherited cause of sudden cardiac death Secondly, HERG is the molecular target for the vast majority of drugs that cause drug-induced long QT syndrome, the commonest cause of drug-induced arrhythmias and cardiac death Thirdly, HERG channels have very unusual biophysical properties, which suggest that they may act as an endogenous antiarrhythmic agent There- fore, understanding the operation of HERG channels has become

an important goal-post in medicine, physiology and pharmacol- ogy While there is no crystal structure for the HERG protein yet, homology models based on the crystal structure of bacterial potassium channels provide a promising avenue for progress In this talk, a survey of advances made in the field using a com- bined experimental/simulation approach will be given This involves using homology models of HERG to find the important residues on the protein that are involved in channel dynamics (e.g toxin binding, fast inactivation) and then test these hypothe- ses via mutagenesis experiments This information is then used to refine the model, followed by further tests

Signaling and Apoptosis

"Department of Pharmacology, National University of Singapore,

Yong Loo Lin School of Medicine, Singapore,’ Department of

Biochemistry, National University of Singapore, Yong Loo Lin

School of Medicine, Singapore,’ Department of Pathobiology,

University of Illinois at Urbana Champaign, Urbana, IL, USA

E-mail: sharmila@nus.edu.sg

We investigated the molecular mechanisms that regulate the

apoptosis of acinar cells induced by crambene (1-cyano-2-hydrox-

y-3-butene-CHB), a plant nitrile As evidenced by annexin

V-FITC staining, crambene treatment for 3 h induced the apop-

tosis but not necrosis of pancreatic acini Caspase 3, 8 and 9

activity in acini treated with crambene were significantly higher

than in untreated acini Treatment with caspase 3, 8 and 9 inhibi-

tors inhibited annexin V staining, as well as caspase 3 activity,

pointing to an important role of these caspases in crambene-

induced acinar cell apoptosis The mitochondria membrane

potential was collapsed in crambene-treated acini than in

untreated that displayed polarized mitochondria Also, the treat-

ment of acini with crambene induced the release of cytochrome

C by mitochondria than in untreated acini Neither TNF-a nor

Fas ligand levels were changed in pancreatic acinar cells after

crambene treatment These results provide evidence for the induc-

tion of pancreatic acinar cell apoptosis in vitro by crambene and

suggest the involvement of mitochondrial pathway in pancreatic

acinar cell apoptosis

OP-18

Androgen inhibition of apoptosis in prostate

cancer cells is due to downregulation of JNK

activation

P I Lorenzo and F Saatcioglu

Department of Molecular Biosciences, University of Oslo, Oslo,

Norway E-mail: petri@imby.uio.no

Androgens have a significant role in normal prostate physiology,

as well as in prostate cancer Removal of androgens during the

initial stages of prostate cancer causes tumor regression via a

combination of reduced cellular proliferation and increased apop-

tosis We have studied the molecular mechanisms of apoptosis in

prostate cancer cells, focusing on the inhibition of apoptosis by

androgens We show that androgen treatment protects LNCaP

prostate cancer cells from thapsigargin (TG)- and 12-0-tetradeca-

noyl-13-phorbol-acetate (TPA)-induced apoptosis through a

mechanism that involves the inhibition of c-Jun N-terminal kin-

ase (JNK) The inhibitory effect of androgens on JNK activation

is dependent on the androgen receptor (AR) since it is blocked

by the androgen antagonist bicalutamide The inhibition of JNK

by androgens is independent of the stimulus that activates JNK,

since it occurs readily when the JNK pathway is activated in

response to TG, TPA or ultraviolet irradiation (UV) The inhibi-

tory effect of androgens on JNK _ activation and apoptosis

requires new gene expression, which is consistent with the time

required to observe this effect ATP depletion experiments indi-

cate that inhibition of JNK activation by androgens is mediated,

at least in part, through an increase in phosphatase activity This

crosstalk between AR and JNK signaling pathways may have

important implications for both normal prostate physiology, as well as prostate cancer progression

OP-19

Enhancement of peptidylarginine deiminase 4 enzymatic activity assists apoptosis

C.-Y Lin', Y.-F Liao”, P.-C Hsu’, H.-C Hung? and G.-Y Liu!

"Institute of Immunology, Chung Shan Medical University, Tai- chung, Taiwan,’Department of Life Sciences, National Chung- Hsing University, Taichung, Taiwan,’ Department of Medicine, Da Chien General Hospital, Taiwan E-mail: mt229.tw@yahoo.com.tw PAD4 post-translationally converts peptidylarginine to citrulline

It plays an essential role in immune cell differentiation and apop- tosis A haplotype of single nucleotide polymorphism (SNP) in PAD4 is functionally relevant as a rheumatoid arthritis (RA) gene It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody Inducible PAD4 causes haematopoietic cell death (Liu et al 2006) apopto- sis Herein, we further investigate whether the increase of PAD4 enzymatic activity induces apoptosis In Tet-On Jurkat T cells, ionomycin (Ion) only treatment did not induce apoptosis; how- ever; it promoted inducible PAD4-decreased cell viability and enhanced apoptosis Zn vitro PAD enzyme activity assay, we dem- onstrated PAD4 enzyme activity of SNP relative to RA was higher than wild type (WT) relative to non-RA following Ca+ + treatment The effect of PAD4 SNP-induced apoptosis was superior to PAD4 WT In addition, both Ion and PAD4 SNP sy- nergistically provoked apoptosis compared with both Ion and PAD4 WT Western blotting data showed apoptosome activation during the programming cell death Concurrently, the expression

of Bcl-xL was downregulated remarkably and Bax upregulated in Ion treatment cells These data demonstrated that increasing PAD4 enzyme activity could enhance apoptosis through mitoch- ondrial pathway and provide a conceivable explanation in the pathogenesis of RA following the upregulation of PAD4 activity

OP-20

Glucose-induced impairment of the insulin-signaling pathway in mouse pancreatic beta cells

E Tsilibary', P Venieratos', A Charonis” and P Kitsiou!

"Institute of Biology, National Center for Scientific Research

‘Demokritos’, Greece,’ Foundation for Biomedical Research of the Academy of Athens, Athens, Greece

E-mail: effie@bio.demokritos.gr Type 2 diabetes is characterized by progressive pancreatic B-cell dysfunction and apoptosis Recent reports provided evidence for

an autocrine role of insulin on the signalling cascade in B-cell growth, function and survival are concerned We examined the effects of chronic glucose stress on insulin signalling Exposure of BTC-6 cells to high glucose resulted in impairment of insulin-sti- mulated phosphorylation of IRS-2 These changes were accom- panied by significant impairment of IRS-2-associated PI3-kinase activation, and substantially decreased activation of Akt We also examined mTOR kinase, a downstream effector of Akt, which stimulates protein synthesis in response to Akt phosphorylation High glucose abolished insulin-induced activation of mTOR without affecting mTOR expression, thus protein synthesis should also be impaired A mechanism by which Akt promotes cell survival includes phosphorylation of the pro-apoptotic

protein BAD, which keeps the pro-apoptotic protein BAX

engaged to Bcl-XL Exposure of cells to high glucose also resul-

ted in suppression of insulin-stimulated phosphorylation of BAD

without affecting BAD expression In conclusion, we demonstra-

ted that chronic exposure of pancreatic B-cells to increased glu-

cose concentration resulted in impaired activation of the IRS-2/

PI3-kinase/Akt signalling pathway in response to insulin The

observed defects in insulin signalling may eventually have a neg-

ative effect on B-cell function and survival

OP-21

Caspase-dependent and geldanamycin-

enhanced cleavage of co-chaperone p23 in

leukemic apoptosis

G Gausdal', B T Gjertsen’, K Fladmark!, H Demol’,

J Vandekerckhove*® and S O Deskeland!

"Department of Biomedicine, University of Bergen, Bergen,

Norway,’ Department of Medicine, Haukeland University Hospital,

Bergen, Norway, Department of Biochemistry, University of Ghent,

Ghent, Belgium E-mail: gro.gausdal@biomed.uib.no

The co-chaperone p23 is a component of the Hsp90 multiprotein

complex and is an important modulator of Hsp90 activity

Hsp90 client proteins involved in oncogenic survival signaling are

often found to be mutated in leukemia, and the integrity of the

Hsp90 complex could therefore be important for leukemic cell

survival We demonstrate here that the Hsp90 co-chaperone p23

is cleaved in leukemic cell lines treated with commonly used che-

motherapeutic drugs The cleavage of p23 paralleled the activa-

tion of procaspase 3 and was suppressed by z-DEVD-FMK

Interestingly, p23 cleavage was also observed in caspase 3-defici-

ent MCF-7 cells, and in vitro translated 35S-p23 was cleaved by

both caspase 3 and 7 Two caspase target sites were identified in

the C-terminal sequence EVD142GAD145, and only Asp to Ala

mutagenesis of both sites (D142/145A) completely blocked p23

cleavage The Hsp90 inhibitor geldanamycin, which inhibits p23

binding to Hsp90, did not induce cell death or p23 cleavage on

its own, but enhanced anthracycline-induced caspase activation,

p23 cleavage and apoptosis This implies that Hsp90 inhibition

amplifies caspase activation Geldanamycin also enhanced

Embryonic Stem Cells

OP-23

Expression of the cholinergic system

components during mouse embryonic stem

cell differentiation

L Paraoanu, G Steinert and P Layer

Developmental Biology, Darmstadt University of Technology,

Darmstadt, Germany E-mail: paraoanu@ bio.tu-darmstadt.de

Expression of cholinesterases during phases of embryonic devel-

opment is a general phenomenon However, no precise function

for cholinesterases could be described during early developmental

stages We have examined the expression of cholinesterases and

other cholinergic components during in vitro differentiation of

CGR8& cells, an embryonic stem cell line Undifferentiated mono-

layers of these cells can be maintained in culture, or embryoid

bodies can be generated in vitro The embryoid bodies were

caspase cleavage of 35S-p23 in vitro, indicating that the associ- ation of p23 to Hsp90 protects against cleavage These findings underscore the importance of the Hsp90-complex in antileukemic treatment, and suggest that p23 may have a role in survival signaling

OP-22 Overview how colorectal cancer cells avoid cell elimination; mechanisms of immune- and chemo-resistance

B Pajak', H Engi', J Molnar! and A Orzechowski?

"Institute of Medical Microbiology and Immunology, Faculty of Medicine, University of Szeged, Szeged, Hungary

E-mail: bepaj@wp.pl,’ Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland

The resistance of cancer cells to deletion allows tumors to grow and develop In the past, several tactics were shown how colon a- denocarcinomas avoid cell deletion and maintain cell viability In particular, colorectal cancer cells resist death ligands-induced apoptosis by expressing antiapoptotic proteins, including FLIP

By direct interaction with FADD, FLIP inhibits the signal des- cending from death receptors in COLO 205 cells Colorectal can- cer cells also stimulate own survival by the cytoplasmic retention

of proapoptotic protein clusterin In contrast, in normal cells clus- terin translocates to the nucleus and induces cell death We found that apoptotic activity of clusterin is dependent on calcium ions, and depletion of intracellular calcium caused extensive death of COLO 205 cells Other type of strategy of chemotherapy-induced cell death is the activity of multidrug resistance proteins (MDR) These cell membrane efflux pumps actively expel the drugs from the cell interior to prevent their action on intracellular targets Upon phenothiazine derivatives rhodamine 123 accumulated within cells interior, which indicates the reversal of Pgp efflux pump in chemoresistant COLO 320 cell line The variety of antia- poptotic mechanisms found in colorectal cancer cells and the knowledge how complex they are renders the anticancer therapy a challenge but the more we know how to sensitize cancer cells to death signals the more likely promise to eliminate them

allowed to differentiate in the absence of further additives or treated with retinoic acid, and collected at various times for fur- ther analysis The cholinesterase expression was analyzed by reverse transcriptase polymerase chain reaction, histochemistry, and activity measurement Low levels of cholinesterase activity and transcripts could be detected already in undifferentiated cells The phases of differentiation are accompanied by increased ace- tylcholinesterase transcripts Butyrylcholinesterase expression was initially constant, decreasing at later differentiation stages All transcripts of muscarinic cholinergic receptors were present; how- ever, their expression did not vary throughout the differentiation steps Choline-acetyltransferase was also detected, indicating that the cells are possibly able to produce acetylcholine With these results we could show that mouse embryonic stem cells express a primitive cholinergic system Its function remains to be deci- phered

Differentiation of Stem Cells

OP-24

Human umbilical cord stroma cells

differentiate into neurons in vitro

S Karahuseyinoglu!, O Cinar*, F Kara* and A Can!

"Department of Histology-Embryology, Ankara University School

of Medicine, Ankara, Turkey,’ Department of Infertility, Etlik

Maternity and Women’s Health Research Training Hospital,

Ankara, Turkey,’ Department of Obstetrics, Zubeyde Hanim

Maternity Hospital, Ankara, Turkey E-mail: alpcan@medicine

ankara.edu.tr

A recently isolated source for stem cells is the mesenchymal cells

of human umbilical cord stroma Although neuronal differenti-

ation has been demonstrated in human umbilical cord stroma cells

(HUSCSs), there is a still discordance among studies about the

potential of these cells to differentiate into neurons The aim of

this study was to differentiate HUSCSs into neurons and investi-

Gene Therapy

OP-25

Cationic nanoparticle synthesis and their use

in gene transfer

G Guven!, M Turkoglu! and E Piskin?

' Hacettepe University Chemical Eng Dept,’ Hacettepe University

Chemical Eng & TUBITAK-BIOMEDTEK E-mail: gguven@

hacettepe.edu.tr

Polymer microspheres would be synthesized by use of an emulsi-

fier-free emulsion polymerization, which gives more pure, and

monodisperse samples In this method the particle formation

occurs when the growing radicals reach to a certain critical chain

length depending upon the solvency of the continous phase In

this study, monosized non-functional poly(styrene/N-[3-dimethyl-

amino )propyl|methacrylamide/poly(ethyleneglycoljethylethermetha-

crylate) [poly(St/(PEG-EEM/DMAPM)] and functional group

carrying poly(styrene/N-[3-dimethylamino)propyl|methacrylamide/

poly(ethyleneglycol) methacrylate) [poly(St/PEG-MA/DMAPM)|

nanoparticles were synthesized by emulsifier-free emulsion copo-

lymerization in the presence of a cationic initiator 2,2’-azobis(2-

methyl propionamidine) dihydrochloride (V-50) The spherical

particles with diameters in the nanometer range and cationic surface

charge were prepared Generally, the polymeric nanoparticle dimen-

sions are below 100 nm The smallest particle size (71 nm) with very

low polydispersity index and very high surface charge was obtained

by increasing water content This particle size was achieved by using

both of the functional (PEG-MA) and non-functional (PEG-EEM)

Nanoparticles with such a high amount of surface charge make

these materials useful for the gene transfer especially

OP-26

Peripheral pool of CD8+ lymphocytes contains

T cells that recognize syngeneic MHC class Il

molecules

E S Zvezdova, T S Grinenko, L A Pobezinsky,

E L Pobezinskaya and D B Kazansky

Russian N.N Blokhin Cancer Research Center

E-mail: katyazvezdova@yahoo.com

A number of publications appeared, which assume that autoim-

mune cells exist in the pool of CD8+ T lymphocytes with the

gate the temporal development potency of newly differentiated neuronal cells The spatio-temporal distribution and the quantifi- cation of various proteins during differentiation, immunofluores- cent single and multi-immunolabeling techniques were performed using a series of antibodies raised against major cellular markers for neurons, such as MAP1, MAP2, GFAP, tau, NSE, NFM, B- III tubulin, NeuN, and thyrosine hydroxylase MAP1, a specific structural protein in the cytoplasm, MAP2, specific for dendrites and NFM were all positive during the course of the entire differ- ention period whereas tau and B-III tubulin were detected at only certain time-points of differentiation While NeuN stained faintly, GFAP and thyrosine hydroxylase were both negative since no dif- ferentiation toward astrocytes and dopaminergic neurons was accomplished Conclusively, it appears that throughout the differ- entiation of HUCSCs into neurons, expression of neuronal mark- ers present a diversity which follows a similar progress as seen in embryologic development of neurons of the body

potential to recognize both MHC class I and MHC class I mole- cules To test this, we generated autoreactive T-cell hybridomas with the ability to recognize both allogeneic MHC class I mole- cule and MHC class II molecules: the LN CD8+ T cells from C57BL/6 mice were activated in the presence of allogeneic MHC class I molecules Kbm3 and fused with BW5147 thymoma cells, expressing CD4 coreceptor The percentage of autoreactive hy- bridomas in CD8+ T cells was 9.3% One of these hybridomas expressed two a-chains One of these a-chains help to recognize syngeneic MHC class If molecules, while the other allogeneic MHC class I molecule Kbm3 To elucidate would the same situ- ation be observed in TCRa‘'° mice capable to express single a-chain of TCR, we obtained hybridomas from their CD8+ T cells The frequency of self MHC class II reactive hybridomas was lower in contrast with that of wild type 1.3% We have shown that in the peripheral pool of wild-type CD8+ T cells the autoreactive T cells exist that recognize foreign MHC class I and self MHC class II molecules One part of these cells expresses the TCR with dual specificity to MHC class I and MHC class I molecules, while the other expresses two a-chains, one defines the specificity to MHC class I molecule and the other to syngeneic MHC class II molecule

OP-27

Salmonella typhimurium aroB-encoding murine IL-18 or CD40L: evaluation for gene therapy

D Aydin‘, A Gunel-Ozcan”, N Menager’, G Esendagli',

M O Guc*, M Hayran’, E Kansu! and D Guc!

"Institute of Oncology, Department of Basic Oncology, Hacettepe University, Ankara, Turkey,’ Faculty of Medicine, Department of Medical Biology and Genetics, Kirikkale University, Kirikkale, Turkey,’ Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK,‘ Faculty of Medicine,

Department of Pharmacology, Hacettepe University, Ankara, Turkey, Institute of Oncology, Department of Preventive Oncology, Hacettepe University, Ankara, Turkey

E-mail: didemaydin@ gmail.com Attenuated Salmonella typhimurium (ST) strains have being investigated as promising vectors for gene therapy In our study,

STaroB strain was evaluated as a gene delivery vehicle for the

first time Eukaryotic expression vectors, pTARGET and

peDNA3, carrying murine IL-18 or CD40L genes were construc-

ted to enhance immune responses of the host for further cancer

therapy studies mRNAs used to synthesize cDNAs to amplify

IL-18 and CD40L DNAs were obtained from lipopolysaccharide-

induced adherent peripheral blood mononuclear cells, and Phor-

bol 12-Myristate 13-Acetate and Ionomycin-induced splenocytes

respectively A bioactive IL-18 molecule consisting of IFN-B sig-

nal and mature IL-18 sequences was constructed by overlap

extension method The plasmids were transformed into STaroB

by electroporation While in vitro stability of pTARGET con-

structs was very low, pcDNA3 constructs were highly stable

HEK293 cells were transfected with pcDNA3 constructs by a cat-

ionic liposome and mRNA expressions of the molecules were

shown by RT-PCR STaroB carrying pcDNA3 constructs were

injected to mice intravenously Plasmids carrying insert DNAs

were found statistically more stable than vector only control in

vivo; however, in vivo stability of all was below 1% and mRNA

expressions in liver and spleen could not be detected by RT-

PCR Our preliminary results showed that STaroB carrying

pIARGET or pcDNA3 may not be suitable to investigate the

effects of a therapeutic gene in a disease model

OP-28

Kanamicin-induced adaptive mutation in E coli

cells harbouring plasmids with direct repeats

S C Ribeiro, P H Oliveira, D M F Prazeres and

G A Monteiro

Departamento de Engenharia Quimica e Bioldgica, Instituto

Superior Técnico, Lisbon, Portugal

E-mail: pcoliveira@mail ist.utl pt

This study describes a kanamycin stress-induced adaptive

mutation in Escherichia coli cells harbouring either a candidate

Therapeutic Enzymes

OP-29

Engineering of nucleoside kinases to improve

the activation of nucleoside analog prodrugs

M Konrad!, S Ort!, C Monnerjahn! and A Lavie7

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

Germany,’ Department of Biochemistry and Molecular Genetics,

University of Ilinois, Chicago, IL, USA E-mail: mkonrad@

gwdg.de

The objective of our study is to improve therapeutic enzyme-pro-

drug systems Compounds, such as AZT for the treatment of HIV

infections, ACV and GCV used against Herpes virus, or the antican-

cer compounds AraC and gemcitabine, enter cells only in the un-

phosphorylated form (prodrug) and need to be transformed by

different kinases to their pharmacologically active triphosphate

state that interferes with DNA replication Based on crystal struc-

ture analyses of various enzyme—nucleotide complexes we first

designed mutants of the human TMP kinase (hTMPK) that phos-

phorylate AZTMP up to 200-fold faster than wild type Expression

of this enzyme in human cells leads to 10-fold higher intracellular

concentrations of AZTTP and to enhanced HIV inhibition Sec-

ondly, the prodrugs ACV and GCV are not phosphorylated by

human kinases, but are converted to their monophosphates by

HSV1-TK, which has been used in enzyme/prodrug-dependent can-

cer suicide gene therapy trials With the aim of improving this thera-

peutic system we generated enzyme variants, which show selective

plasmid DNA vaccine (pGPV-PV) or its mammalian expression vector backbone (pCI-neo) [1] The mutation, which was shown

to occur due to the presence of a 28 bp direct repeat flanking 1.6 (pClI-neo) and 3.2 kb (pGPV-PV) intervening sequences, was responsible for the acquisition of a kanamycin resistance pheno- type in different FE coli strains (DH5a, Top 10F’, HBI0I and JM109) This could be explained as follows: as a result of slip- page events, the Neo’ gene falls under the control of a promoter- like sequence located at the origin of replication, thus enabling cells to strive in kanamycin The Poisson distribution of mutants determined by Luria-Delbriick fluctuations tests and reconstruc- tion experiments surprisingly, but unequivocally, indicates that this plasmid slippage event behaves adaptively This study high- lights the need to carefully design plasmid vectors avoiding, for example, repetitive sequences that are genetically unstable and may give rise to unforeseen problems during plasmid DNA pro- duction and subsequent utilization

Reference:

1 Bahloul et al Vaccine 1998; 16: 417-425

and efficient phosphorylation of GCV Thirdly, an engineered human dCK variant catalyzes more efficiently the activation of the prodrugs AraC and gemcitabine Thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intra- cellular protein transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy

OP-30 Effect of N2A connectin/titin on disassembly

of p94/capn3 caused by autolysis in IS2

Y Ono!, F Torii’, K Ojima’, N Doi’, K Yoshioka’, D Labeit*,

S Labeit*, K Suzuki®, K Abe”, T Maeda® and H Sorimachi!

"Department of Enzymatic Regulation for Cell Functions, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo, Japan,’Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan, CREST, Japan Science and Technology (JST), Saitama, Japan,*University of Mannheim, Mannheim, Germany,’ New Frontiers Research Laboratories, Toray Industries Inc., Kanagawa, Japan,’ Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan E-mail: yakoono@rinshoken.or jp

p94/calpain3 is the skeletal muscle-specific member of calpains, Ca** -regulated cytosolic cysteine protease family Defective p94 protease activity originated from gene mutations causes muscular

dystrophy called calpainopathy, indicating p94s’ indispensability

for muscle survival One of unique properties of p94 is its very

rapid and exhaustive autolytic activity, which is proposed to be

regulated by its binding protein connectin/titin Zn vitro analysis

of p94 autolysis revealed that autolytic cleavage in each p94-spe-

cific insertion sequence, NS, ISI, and IS2, causes different impact

on molecular integrity of p94 as a protease; autolysis in IS2, but

not in ISI, causes disassembly of autolyzed fragments Using

proteinase trapping system to semiquantitatively assay p94 auto-

lysis, where p94 was expressed in yeast as a hybrid protein

between DNA binding and activation domains of the yeast tran-

scriptional activator Gal4, N2A connectin was shown to suppress

p%4 autolytic disassembly Proximity of IS2 autolytic and connec-

tin-binding sites in p94 suggested that N2A connectin interferes

with IS2 autolysis It was also shown that N2A connectin with

mdm mutation that is causative of dystrophy in mice and abol-

ishes connectin—p94 interaction was incapable of suppressing p94

autolytic disassembly These data indicate that p94-connectin

interaction plays an important role in the control of p94 func-

tions by regulating autolytic decay of p94

OP-31

Prevention of thermal aggregation of yeast

alcohol dehydrogenase by arginine and

a-cyclodextrin

A Barzegar and A A Moosavi-Movahedi

Institute of Biochemistry and Biophysics, University of Tehran,

Tehran, Iran E-mail: barzegar@ibb.ut.ac.ir

Yeast alcohol dehydrogenase (YADH) is a tetrameric enzyme

and is a widely studied as a metaloenzyme for its well-known

RNA Interference

OP-32

RNA interference shows critical involvement

for NF-IkappaB p65 in the production of Wnt-1

protein

S Tsai!, C Cheng’, C Sun”, C Tzeng* and A H Wang*

"Liver Research Unit, Department of Medical Research, Chi Mei

Medical Center, Tainan, Taiwan,’ Division of Hepatogastroentero-

logy, Department of Internal Medicine, Chi Mei Medical Center,

Tainan, Taiwan,’ Department of Pathology, Chi Mei Medical

Center, Tainan, Taiwan,‘Core Facilities for Proteomics Research

and Institute of Biological Chemistry, Academia Sinica, Taipei,

Taiwan E-mail: sltsai@mail.chimei.org.tw

The link of proto-oncogenic protein Wnt-I with NF-kappaB

activity has been reported in PC12 cells, a rat pheochromocytom-

a cell line of neural crest lineage, showing that the Wnt-1-medi-

ated survival of these cells is dependent on NF-lkappaB

activation, and that stable expression of Wnt-l increases NF-

IkappaB activity In Wnt-1-mediated breast tumorigenesis, Wnt-1

is reported to be a fundamental signaling event in metastatic pro-

gression of human cancers We have shown that enhanced NF-

IkappaB-associated Wnt-1 protein expression is detected in the

majority of hepatoma samples from both hepatitis B and C

patients Insight into how NF-lkappaB regulates Wnt-1 protein

production may help design highly effective therapeutic agents in

treating Wnt-l-producing cancers and prevent their metastatic

progression RNA interference (RNAi) was used in this study to

assess the role of p50 and p65 of NF-lkappaB in the regulation

of Wnt-1 protein production by Het-1A cells, a human esopha-

biotechnological importance Arginine (Arg) and «cyclodextrin (a-CD) are the universal reagents that are effective in assisting re- folding of recombinant proteins from inclusion bodies The results showed that YADH (0.5 mg/ml) initiate to aggregation after 15 min at 45 °C and 5 min at 50 °C in sodium phosphate buffer (pH 7.5) In the presence of Arg (100 mM) aggregation phenomenon completely suppressed at 50 °C until 2 h and a-CD suppressed aggregation until 40 min (aggregation initiates after

40 min) in the same condition The fluorescence and UV-Vis data show that enzyme structure does not change in the presence

of these additives The residual activity of YADH at 50 °C after

50 min was very low and negligible (<5%) and in the presence of

100 mM Arg its activity completely inhibit whereas maintained

at =<30% in the presence of 100 mM a-CD We propose Arg has

a major factor for diminishing the electrostatic forces and a-CD has a role for controlling of the hydrophobicity due to decreasing the aggregation of YADH

geal cancer cell line with Wnt-1 protein production Cultured Het-1A cells were transfected with sequence-specific double-stran- ded small interfering RNAs (siRNAs) of P50, P65, and Wnt-1 respectively All these siRNAs did not inhibit NF-kappaB acti- vation in Het-1A cells The production of Wnt-1l protein was totally abolished in cells transfected with P65 siRNAs and parti- ally reduced in cells transfected with P50 siRNAs, indicating a pivotal requirement for P65 in Het-1A production of Wnt-l protein

OP-33 Molecular mechanism of paclitaxel-induced apoptosis in ER+/- breast cancer cell lines: MCF-7 and MDA-MB-231

E D Arisan, O Kutuk, A Verim and H Basaga Biological Sciences and Bioengineering Program, Faculty of Science and Engineering, Sabanci University, Istanbul, Turkey E-mail: ebuyuktuncer@su.sabanciuniv.edu

Taxanes such as paclitaxel (Pac) and docetaxel are cytotoxic agents, which act by promoting deformation of stable microtu- bules, inhibiting the normal dynamic reorganization of microtu- bule networks required for mitosis However, the development intrinsic/acquired of resistance against Pac is a major obstacle to successful cancer chemotherapy Bcl2 is believed to have role in drug resistance since Bcl2 overexpression is very frequent especi- ally in ER+ breast cancer tumors through ER-responsive ele- ment in its promoter Bcl2 also causes prevention of Pac-induced

expression of apoptosis-related proteins It was our aim to study

the role of Bcl2 in induced resistance to apoptosis in breast can-

cer cell lines treated with taxanes MCF-7 (ER+) and MDA-

MB-231 (ER-) breast cancer cell lines were treated with 20 and

50 nM Pac Cytotoxicity was determined with MTT assay after

cells were treated with the drug for 24-72 h 20 nM Pac treat-

ment for 24h inhibited growth of MCF-7 by 30% and MDA-

MB-231 by 44% Cytotoxic effect was dose-dependent as 50 nM

Oxidative Stress

OP-34

ANKRD1 gene shows opposite atrial versus

ventricular expression kinetics at heart failure

M Torrado!, B Nespereira!, Y Bouzamayor!, A Centeno’,

E Lopez” and A Mikhailov!

"Institute of Health Sciences, University of La Corufa, La Coruiia,

Spain,” Experimental Surgery Unit, University Hospital ‘Juan

Canalejo’, La Coruria, Spain E-mail: margot@udc.es

Background: It has been suggested that the cardiac ankyrin

repeat domain 1 (ANKRD1) gene is differentially regulated in

cardiac chambers both during development and at heart failure

Aim: To determine a ANKRDI1 gene expression fingerprint

throughout the heart, accounting for region- and disease-specific

aspects Methods: Heart failure in neonatal piglets was produced

by intravenous administration of cardio-toxic antibiotic, Doxoru-

bicin (2 mg/kg) ANKRD1 mRNA and protein levels were deter-

mined in both atrial (A) and ventricular (V) chambers of control

and failing hearts, using quantitative PCR, Northern blot hybrid-

ization, and Western blot Results: In the normal heart, AN-

KRD1 showed a fairly left-right (L-R) asymmetric distribution

with a twofold higher protein level in the LA when compared to

the RA, and an eightfold ANKRDI1 protein enrichment in the

LV over the RV In failing hearts, ANKRDI1 transcript and pro-

tein contents were significantly augmented in each ventricle (two-

to threefold change in mRNA/protein levels), but reduced in each

atrium (twofold change in mRNA/protein levels) Owing to these

opposite expression kinetics, the normal atrio-ventricular pattern

of ANKRD1 distribution was abolished in failing hearts Conclu-

sions: (a} ANKRDI expression is distinctly regulated in cardiac

chambers in the postnatal heart; (b) the inverse gene expression

response in atria when compared to ventricles at advanced heart

failure may be considered a molecular sigh of severity of cardiac

disease

OP-35

Peroxynitrite induces red blood cell membrane

reorganization

M N Starodubtseva!, T G Kuznetsoval, J.C Ellory’,

T A Kuznetsova? and S O Abetkovskaya*

'Gomel State Medical University, Gomel, Belarus,’ Department of

Physiology, Anatomy and Genetics, University of Oxford, Oxford,

UK,°A V Lykov Heat and Mass Transfer Institute of NASB,

Minsk, Belarus E-mail: marysta@mail.ru

Peroxynitrite is formed in many pathological human processes

This study aims at studying peroxynitrite-induced rearrangements

in red blood cells (RBC) by using atomic force microscopy

(AFM; both taping and contact mode) in air Peroxynitrite

(2.5 mM) increases RBC membrane rigidity Young’s modulus of

RBC membrane fixed by 1% glutaraldehyde increases from

Pac caused growth inhibition by 52% and 64% for 24h in both cell line DNA fragmentation experiments showed that 20 and

50 nM Pac induced apoptosis To understand the role of Bcl2 in apoptotic response Bcl2 mRNA levels was checked by qPCR Pac at both concentrations decreased Bcl2 expression by 1.8- and 2.1-fold in ER+ and 1.2- and 1.7-fold in ER- cells compared to control.To further clarify the role of Bcl2 we have prepared Bcl2 siRNA cells and the experiments are in progress

18.7 + 7.5 P (vn = 8) for control cells to 29.2 + 2.6 P (n = 10,

P = 0.001) for peroxynitrite-treated RBCs Peroxynitrite treat- ment of RBCs leads to formation of macroareas with different mechanical properties and composition in the RBC membrane The peak of the spatial distribution of torsion angle for peroxyni- trite-treated RBC membranes is shifted and broadened in com- parison with control RBC membranes Peroxynitrite at a lower concentration (300 1M) also causes the lipid macrodomain for- mation The spatial distribution density of AFM cantilever oscil- lation phase change is bimodal with the modes at 49.30 + 7.27° and 333.79 + 4.76° (n = 28) The membrane of control samples

is characterized by a unimodal distribution with mode at 46.90 + 1.78° (P = 0.001, n = 20) The findings are evidence of increasing heterogeneity of the mechanical properties of RBC membrane under peroxynitrite action that seems to be one from the most likely mechanisms of peroxynitrite-induced enhancement

of ion permeability of the RBC membrane Acknowledgment:

We thank The Physiological Society for financial support

OP-36

Oxidative stress fails to induce protein

misfolding in replicating cells

G Kiss, P Csermely and C Séti Department of Medical Chemistry, Semmelweis University, Budapest, Hungary E-mail: csaba@ puskin.sote.hu Molecular chaperones or stress proteins guard the cellular con- formational homeostasis of proteins, protecting against a num- ber of stresses The strongest inducer of stress proteins is the accumulation of misfolded proteins Proper function of chaper- ones as well as a robust mounting of the stress response is required for longevity Indeed, a hallmark of ageing is the increase of aggregated and oxidized proteins However, little is known about the causal relationship between protein misfold- ing, oxidative stress and chaperone induction In our present studies using different cell lines we examined the relationship between protein misfolding, aggregation and chaperone induc- tion As expected, heat shock, the archetype of stress as well

as geldanamycin induced the uniform elevation of the major chaperones, Hsp70 and Hsp90 In contrast, the oxidative stres- sor hydrogen peroxide induced only Hsp90 Investigating the hydrophobic surface exposure to detect protein misfolding in live cells, the fluorescence of ANS, an apolar probe was applied In contrast to heat shock, hydrogen peroxide was unable to induce the ANS signal of purified model proteins, cytosolic extracts and live cells Moreover, did not result in significant protein aggregation These results suggest that short- term oxidative stress does not induce bulk protein denaturation and stress response, raising the question what is the depend- ence of protein oxidation and misfolding during ageing and in degenerative diseases

DNA Damage Processing

OP-37

Chromosome remodelling and DNA replication

in B subtilis

P Soultanas

Centre for Biomolecular Sciences, University of Nottingham,

Nottingham, UK E-mail: panos.soultanas@nottingham.ac.uk

In Bacillus subtilis three essential proteins known as Dnal, DnaD

and DnaB have been implicated in a “primosomal cascade’ that

initiates DNA replication We have recently discovered that both

proteins also exhibit global DNA remodelling activities Our ini-

tial studies on DnaD and DnaB have revealed oligomeric pro-

teins that remodel DNA [1, 2] We have suggested that the DNA

remodelling activities of DnaD and DnaB are similar to the

Escherichia coli H-NS and HU DNA remodelling activities but

whether their biological significance is analogous to these pro-

teins still remains an open question Interestingly, there are no

H-NS and HU homologues in B subtilis and other Gram-posit-

ive bacteria Recently, we have proposed a model where DnaD

and DnaB act as global or local (at the vicinity of oriC) DNA-

remodelling proteins to render the chromosomal DNA replica-

tion proficient This is compatible with previous suggestions that

DnaD recruitment to the bacterial membrane by DnaB is the

main regulatory event in the initiation of B subtilis DNA replica-

tion I will present our latest data on the function/structure rela-

tionships of DnaI, DnaD and DnaB proteins in relation to

initiation of DNA replication and chromosomal remodelling

References

1 Turner IJ, Scott DJ, Allen S5, Roberts CJ, Soultanas P FEBS

Lett 2004; 57T: 460-464

2 Zhang W, Carneiro MJVM, Turner IJ, Allen S, Roberts CJ,

Soultanas P J Mol Biol 2005; 351: 66-75

OP-38

Coordination of DNA base excision repair

studied by photoreactive DNA probes and

functional assay

O I Lavrik

Institute of Chemical Biology and Fundamental Medicine,

Novosibirsk, Russia E-mail: lavrik@niboch.nsc.ru

Photoaffinity-labeling technique has been developed to study

coordination of base excision repair (BER) proteins Photoreac-

tive DNA intermediates of BER pathways were created in sys-

tems reconstituted of purified proteins and in cellular/nuclear

extracts to identify proteins interacting with damaged DNA The

main target proteins interacting with branch point BER interme-

diate were identified as Poly(ADP-ribose)polymerasel (PARP1),

flap endonucleasel (FEN1), DNA polymerase beta (Pol beta)

and apurinic/apyrimidinic endonucleasel (APE1) The results

indicate that APEI and PARPI interact preferentially with

nicked BER intermediate carrying 3’-photoreactive dNMP resi-

due and the 5’-sugarphosphate moiety, whereas intermediate with

5’-phosphate is less favourable interaction partner Thus, PARP1

and APEI1 can discriminate intermediates of SP and LP BER

pathways to regulate BER DNA repair synthesis catalysed by

Pol beta is modulated by the interplay between Pol beta, APE,

PARPI and XRCC1 APEI1 can perform stimulation of strand-

displacement synthesis catalysed with Pol beta and proofreading

function while PARPI inhibits these reactions PARP1 inhibits

activity of FENI preventing formation of intact DNA structure

The inhibition by PARPI1 of these activities was not completely

reversed by poly(ADP-ribosyl)ation Our study further estab- lished that photoaffinity labelling combined to functional assay is

a powerful tool to explore proteomic ensembles of DNA repair Acknowledgement: RFBR grant 05-04-48319

OP-39

A new family of nucleases: the uracil-DNA- specific nuclease of pupating insects

A Bekesi!, F Felfoldi*, M Pukanesik!, V Muha!, I Zagyva',

I Leveles', E Hunyadi-Gulyas*, K Medzihradszky*, A Erdei* and B Vertessy!

"Institute of Enzymology, Budapest, Hungary,’MolCat Bt, Buda- pest, Hungary, Proteomics Laboratory, Biol.Res.Ctr., Szeged, Hungary,*Immunology Department, Eotvos University, Budapest, Hungary E-mail: vertessy@enzim.hu

Uracil in DNA may arise by cytosine deamination or thymine replacement and is removed by members of the uracil-DNA gly- cosylase (UDG) superfamily The surprising lack of the major UDG-superfamily member UNG from the fruitfly genome may allow the fly to tolerate thymine-replacing incorporation of uracil

in its DNA Such incorporation is usually prevented by the enzyme dUTPase that removes dUTP from the DNA polymeriza- tion pathway However, fruitfly larval tissues do not contain any detectable dUTPase (the enzyme being confined to the imaginal

disks, progenitors of adult fly organs; Békési et al J Biol Chem

2004; 279: 22362) Here, we asked if the putative presence of ura- cil-DNA in larval tissues, potentially accumulated due to lack of both UNG and dUTPase, might trigger any physiological response Such response would require macromolecular (e.g pro- tein) factors specifically recognizing uracil-DNA; we therefore set out to identify such proteins from Drosophila extracts We show that the most intensive hit of uracil-DNA pull-down experiments

is a uracil-DNA specific nuclease (UDE) with no detectable homology to other proteins except a group of sequences of other pupating insects UDE is upregulated right before pupation by

an ecdysone-controlled mechanism potentially involving Write BFTZ-Fl Data imply the existence of a novel DNA nuclease family specific for uracil-DNA with a possible role in cell death during metamorphosis

OP-40

Zooming on metals in DNA degradation

M Fuxreiter, L Mones and I Simon Institute of Enzymology, Budapest, Hungary E-mail: monika@ enzim.hu

Sequence-specific degradation plays pivotal role in defense against foreign DNA Most repair enzymes requisite bivalent metal ions for hydrolyzing the DNA backbone, the number of which is a long-standing puzzle in molecular biology Despite the wealth of structural data, the role, and contribution of the metal ions to the different steps of the catalytic choreography remains elusive Recently, the mismatch repair initiating MutH enzyme revealed similarity to PDD/ExK endonucleases, especially to the type II restriction enzyme BamHI concerning the metal ion arrangement To probe whether these enzymes could employ the same mechanism for their action that might be even further gen- eralized for other repair enzymes we computed the activation barrier of the potential pathways in BamHI We found that the general base mechanism is less likely than recruiting the attacking OH- nucleophile from the bulk phase Based on their contri- bution to the total catalytic effect, Metal ion A ligating the

nucleophile was concluded to be of primary importance for the

reaction, while the removal of metal ion B was less dramatic

Thus, we propose a unified catalytic scheme involving only one

obligatory metal ion at the active site that serves to stabilize the

attacking nucleophile, which is coming from the bulk phase and not assisted by a general base or substrate A second, variable group is required to facilitate the nucleophilic attack via interac- tions with the pentavalent transition state

DNA Repair in Health, Disease and Aging

OP-41

Base excision repair pathway at telomeric

DNA

M Muftuoglu, P Opresko and V Bohr

Laboratory of Molecular Gerontology, National Institute on Aging,

National Institutes of Health, Baltimore, MD, USA

Telomeres consist of protein-DNA complexes that protect the

ends of linear chromosomes from inappropriate chromosome

fusions by DNA repair pathways Telomere-repeat binding fac-

tors 1 and 2 (TRF1 and TRE2) bind specifically to duplex telo-

meric DNA and regulate telomere length When bound to

telomeres, TRF2 allows the cell to recognize the telomeres as

chromosome ends rather than double strand breaks in DNA

Defects in TRF2 induce telomere dysfunction that causes telo-

mere end fusions, replicative senescence, apoptosis, and genomic

instability Oxidative damage causes loss of telomeric DNA, and

we found previously that oxidative DNA base damage disrupts

TRFI and TRF2 binding to telomeric DNA, emphasizing the

importance of DNA repair at telomeres We are investigating the

function of base excision repair (BER) in preserving telomeres,

and specifically roles for TRF2 in cooperation with BER proteins

for repair pathways in preventing genomic instability We dem-

onstrated that TRF2 physically interacts with DNA polymerase

beta (pol beta), a critical enzyme in BER, and stimulates pol beta

DNA synthesis on both telomeric and nontelomeric substrates,

and in reconstituted BER reactions Our data suggest that TRF2

can participate in BER together with pol beta during repair of

oxidative lesions at the telomeres, and/or in genomic regions out-

side of the telomeres

OP-42 Sequence variations of fanca gene in Turkish

patients with fanconi anemia

G Balta, F Gumruk, H Okur, A Gurgey and C Altay Department of Pediatrics, Section of Pediatric Hematology, Faculty of Medicine, Hacettepe University, Ankara, Turkey E-mail: gbalta@hacettepe.edu.tr

Fanconi anemia (FA) is a DNA repair deficiency disorder charac- terized by bone marrow failure, developmental defects, cancer pre- disposition, and sensitivity to DNA cross-linking damage To date,

12 different complementation groups have been described FA pro- teins function in a DNA damage-response network including BRCAI, BRCA2, and RADS1 The molecular defects in FANCA gene account for 65-70% of all cases In this study, a total of 26 patients from a panel of 50 unrelated Turkish families were assigned to group A by using linkage analysis Genomic DNA or cDNA of these patients were screened for the sequence variations

in the complete coding (43 exons) and flanking sequences of the FANCA gene by SSCP/HD analysis Ironically while sequencing

of the variant bands led to the identification of many homozygous, non-pathogenic mutations including missense/silent mutations, base substitutions, and deletions/insertions in introns, only a few pathologic mutations could be detected Despite much effort, the molecular defects in the most of Turkish group A patients go undetected Given that all of the exons could be amplified by PCR

in almost all of the patients, it is unlikely that large deletions could

be cause of the disease either This study proved that mutation analysis of FANCA gene is quite difficult and inefficient, which makes pre-natal diagnosis and detection of carriers in Turkish population almost impossible Acknowledgment: This study was supported by Hacettepe University Research Fund (02G116)

Diabetes, Obesity and Metabolic Syndrome

OP-43

The role of TGFB1 in glycosaminoglycan

metabolism in diabetes mellitus

N Yu Yevdokimova

Molecular Immunology, Institute of Biochemistry, Kyiv, Ukraine

E-mail: berezan@mathber.carrier.kiev.ua

The dysregulation of glycosaminoglycan (GAG) metabolism is a

typical feature of renal diabetic complications TSP-1-dependent

TGFBI activation is known to mediate numerous ECM disor-

ders in diabetic kidney However, its role in the metabolism of

GAGs is studied insufficiently We observed that high glucose

enriched the mesangial ECM with hyaluronic acid (HA) of

high-molecular weight (> 2000 kDa) We detected the upregula-

tion of hyaluronan synthase 2 (HAS2) mRNA without altera-

tions of HAS3 expression High glucose also stimulated the

production and TSP-1-dependent activation of TGFBI The blockage of TGFBI action by neutralizing antibodies or by stopping TGFBI activation abolished the effect of high glucose

on HAS2 mRNA expression and normalized HA synthesis Exogenous TGFBI revealed the same effect on HAS2 expres- sion, as high glucose did We consider that TSP-1/TGF61 axis

is involved in high glucose influence on HA metabolism In contrast, high glucose decreased the level of heparan sulphate (HS) in mesangial ECM and led to the undersulphation of HS molecules The blockage of TGFBI intensified the pathological changes of HS, while exogenous TGF61 increased HS synthesis Therefore, the pathological alterations of HS under the action

of high glucose take place despite the upregulation and activa- tion of TGFB1 Hence, our data underlined the ambiguity of TGFBI involvement in the abnormalities of GAGs metabolism

in diabetes mellitus

OP-44

The effect of -675 4G/5G polymorphism on

PAI-1 gene expression and adipocyte

differentiation

D Ozel!, M Berberoglu’, H Aktas* and N Akar!

"Pediatric Molecular Genetics Department, Ankara University

School of Medicine, Ankara, Turkey,’ Pediatric Endocrinology

Department, Ankara University School of Medicine, Ankara,

Turkey,’ The Laboratory for Translational Research, Harvard

University School of Medicine, Boston, MA, USA

E-mail: duygu_ozel@hotmail.com

Plasminogen Activator Inhibitor I (PAI-1), a member of the ser-

ine proteinase inhibitor family is not only inhibits fibrinolysis but

also has complex interactions with cellular matrix, further inhib-

its proteolysis and important in cardiovascular diseases Obesity

is a metabolic disorder, associated with increased PAI-1 concen-

tration in the circulation This increase is also related to insulin

resistance, dyslipidemia and cardiovascular diseases In our study

we investigated the frequency of -675 4G/5G PAI-1 polymorph-

ism located at -675 in promoter of the gene in Turkish pediatric

obese patients and the effect of this polymorphism on PAI-1 gene

expression and on the adipocyte differentiation The frequency of

PAI-1 4G/5G genotype was determined as previously described

We constructed a dual-glo luciferase effect reporter assay to

investigate of this deletion on the PAI-1 promoter activity in pre-

adipocytes, adipocytes, and HUVEC cells We also investigated

the effects of troglitazone and ciglitazone treatments on PAI-1

promoter activity 4G/4G genotype was significantly higher than

the 5G/ 5G genotype in Turkish pediatric obese patients In the

present study, we demonstrated that 4G/4G genotype increases

the PAI-1 promoter activity PAI-1 increases the differentiation

of pre-adipocytes to adipocytes Troglitazone and the ciglitazone

had similar effects on PAI-1 expression and decrease the level of

PAI-1 in HUVEC, undifferentiated and differentiated 3T3 Ll cells

OP-45 Effect of modifications of Grb14 protein expression on insulin signaling and sensitivity

N Carré, J Girard and A Burnol

Institut Cochin, INSERMU567 CNRS UMR8104 Endocrinology, Metabolism, Cancer Department, Paris, France,

E-mail: capitaine@cochin.inserm.fr Obesity and type 2 diabetes are rapidly expanding in most coun- tries and become a worldwide health issue These physiopatho- logical states are characterized by peripheral insulin resistance, which leads to an increase in glucose and fatty acids plasmatic concentrations Grbl4, a member of the Grb7 adaptor protein family, directly binds to insulin receptors and inhibits their tyro- sine kinase activity We demonstrated that Grb14 was specifically expressed in insulin target tissues, and that its level of expression was inversely correlated with insulin sensitivity in insulin-resistant rodent models and type 2 diabetics To investigate the physiologi- cal role of Grb14, we modified its level of expression by overex- pressing the protein with a recombinant adenovirus or by decreasing its expression using a siRNA approach Grb14 overex- pression in primary cultures of mouse hepatocytes only induced a slight decrease in ERK1/2 activation by insulin On the other hand, a 70% decrease in Grbl4 expression following Grbl4 siRNA transfection was accompanied by a significant increase in insulin-induced Akt and ERK1/2 phosphorylation These results show that modifications in Grbl4 expression level can rapidly modulate insulin signalling and action, suggesting that Grb14 can

be considered as a new target for the development of insulin-sen- sitizing agents

Lipid-related Disorders and Atherosclerosis

OP-46

Associations of TSH levels with blood lipids,

metabolic syndrome, coronary risk factors,

coronary heart disease in Turkish adult

G Hergenc!, A Onat, S Albayrak?, A Karabulut*,

S Turkmen’, I Sari* and G Can®

"Yildiz Tech University, Department of Biology, Istanbul,

Turkey,°Turkish Society Cardiology, Istanbul, Turkey,*Izzet

Baysal Univ Duzce Medical Fac, Department of Cardiology,

Duzce, Turkey,“Sipami Ersek Cardiovascular Surgery Center,

Cardiology Department, Istanbul, Turkey, Gaziantep Med Fac,

Department of Cardiology, Gaziantep, Istanbul, Turkey,°Istanbul

Univ Cerrahpasa Med Fac, Cardiology Institute, Istanbul, Turkey

E-mail: ghergenc@superonline.com

Epidemiological data related to the role of thyroid hormones in

coronary heart disease (CHD) and metabolic syndrome (MS) in

Turkish adults are lacking Thyroid-stimulating hormone (TSH)

was measured in the 2004 follow up of the Turkish Adult Risk

Factor Study, with the aim of investigating thyroid hormone sta-

tus as a risk factor in CHD and MS in the population sample

A subgroup of the cohort (nm = 512, mean age 52 years)

was screened for TSH Thyroid status was classified according to

cut-offs of 4.2 and 0.3 wU/ml for TSH No distinction was made

between overt and SC thyroid states T and LDL-Chol was low-

est, waist circumference unexpectedly highest in the hyperthyroid

group Women (1.3 wU/ml) had significantly higher TSH levels

than men (0.95 pU/ml) Prevalence of hypo- and hyper-thyoidism

in men and women were 1.7/7% and 4.4/5% respectively TSH values were not significantly different among groups diagnosed

as having or not CHD, MS and DM Multivariate linear regres- sion analysis revealed TChol as the sole independent covariate of TSH levels in men and both sexes combined To conclude, TSH was found not to contribute to CHD, hypercholesterolaemia, MS and DM risk in logistic regression analyses Prospective follow ups may give a better understanding concerning the thyroid sta- tus and CHD risk in Turkish adults

OP-47

The carboxy-terminal region of apoA-l is

important for the biogenesis of HDL in vivo

A Chroni', A Duka”, G Koukos” and V Zannis”

‘Institute of Biology, National Center for Scientific Research Demokritos, Athens, Greece,’ Boston University School of Medicine, Boston, MA, USA E-mail: achroni@ bio.demokritos.gr Apolipoprotein A-I (apoA-I) promotes ABCAI1-mediated lipid efflux that results in the initial lipidation of apoA-I This leads to the formation of discoidal HDL and, after cholesterol esterifica- tion by the enzyme LCAT, of spherical a-HDL particles Follow- ing adenovirus-mediated gene transfer in apoA-I-deficient mice the plasma HDL levels were greatly reduced in mice expressing the C-terminus deletion mutants apoA-I[del(185—243)] and apoA- I[del(220—243}] and were normal in mice expressing the WT

apoA-I, the apoA-I[del(232—243)] deletion mutant or the apoA-

I[E191A/H193A/K195A] point mutant Electron microscopy and

two-dimensional gel electrophoresis showed that the apoA-

I[del(1 85—243}] and apoA-I[del(220—243)] mutants formed mainly

prebl HDL particles and few spherical particles enriched in

apoE, while WT apoA-I, apoA-I[del(232-243)] or apoA-I[E191A/

H193A/K195A] formed spherical a-HDL particles Consistently

with these in vivo data, previous in vitro data showed that the

apoA-I[del(185—243)] and apoA-I[del(220—-243)] mutants had

diminished ABCA1-mediated cholesterol efflux, while the apoA-

I[del(232—243)] mutant had normal cholesterol efflux The apoA-

I[E191A/H193A/K195A] mutant that is normal in HDL biogen-

esis has normal ABCA1-mediated cholesterol efflux The findings

indicate that (a) the C-terminal region 220-231 of apoA-I is

important for formation of HDL in vivo and (b) mutations in

apoA-I that diminish its functional interactions with ABCAI

diminish the formation of HDL in vivo

OP-48

Genetic risk factors and early ischemic stroke

S Stankovic!, N Majkic-Singh', Z Jovanovic” and D Alavantic?

"Institute for Medical Biochemistry, University School of

Pharmacy & Clinical Center of Serbia, Belgrade, Serbia &

Montenegro,’ Institute of Neurology , Clinical Center of Serbia,

Belgrade, Serbia & Montenegro,’ Laboratory for Radiobiology and

Molecular Genetics, VINCA Institute of Nuclear Sciences,

Belgrade, Serbia & Montenegro E-mail: sanjast@EUnet.yu

The aim of this study was to determine whether the DNA poly-

morphism in apolipoprotein (apo)B, E and angiotensin-converting

enzyme (ACE) is associated with occurrence of ischemic stroke

(IS) in young adults and to find out the relationship between IS,

lipids, apolipoproteins, blood pressure (BP) levels and those poly-

morphisms The possible association of apoB (Xbal, EcoRI,

MspI, I/D, 4311 A/G), apoE (hal) and ACE (I/D) polymorph-

isms were analyzed in 65 IS survivors younger than 65 and 591

age-matched healthy subjects The occurrence of stroke was pro-

ven by computed tomography or magnetic resonance of the brain

The results showed that patients with IS presented statistically sig-

nificant higher frequencies of X (XbaI), I (I/D), M1 (MspI), A

(4311 A/G), E4 allele, and lower E2 allele than control subjects

(P < 0.01) These results are discussed in the sense of finding

risk/protective haplotype for IS, lighting the role of circulating

lipids, BP levels in the pathogenesis of IS subtypes in different genotypes in this study ApoB, triglycerides and MAP were the best predictors of IS ApoE34 genotype/apoEF4 allele and cigarette smoking/hypertension act synergistically, increasing an indivi- dual’s propensity to have a cerebral ischemic event Additional knowledge of the role of genes in IS may improve our under- standing of the cause of IS, provide new insights in prevention and factors that influence the outcome of IS, as new therapeutic targets when preventive strategies have failed

OP-49 Plasma fibrinogen level: an independent risk factor for long-term survival in Chinese patients with peripheral artery disease?

up was 6 years All variables were first correlated with survival rates using Kaplan—Meier analysis and compared by means of the log-rank test Significant risk factors were identified, and mul- tivariate Cox regression analysis was used to evaluate the inde- pendent contribution of the fibrinogen level to the risk of mortality During follow up, 95 patients (68.3%) died The over- all survival rate was 77.7% at 3 years, 56.8% at 5 years, and 31.2% at 10 years (SE: 0.05, 0.06, and 0.07, respectively) All- cause mortality rate increased with an elevated fibrinogen level Eighty percentage of patients with a fibrinogen level > 3.4 g/l had a survival time of <3 years (P = 0.002) This relation was also demonstrated within patients with critical ischemia The plasma fibrinogen level was thus identified as an independent risk factor for mortality in PAD patients after adjusting for con- founding factors

Oncogenes and Tumor Suppressors

OP-50

An approach for deciphering molecular

patterns involved in genistein effects in

human prostate

R Laslo', H Klocker’, I Rowland’, R L Hancok’,

R S Pardini* and A I Baba?

"Nutrition and Toxicology, University of Agricultural Sciences and

Veterinary Medicine of Cluj, Cluj-Napoca, Romania,’ UROLAB-

IBK, Innsbruck Medical University, Innsbruck, Austria,’ NICHE,

CMB, University of Ulster, Coleraine, UK,* Cancer Research

Laboratory, CABR, University of Reno, NV, USA, Comparative

Oncology Department, University of Agricultural Sciences and

Veterinary Medicine of Cluj, Cluj-Napoca, Romania

E-mail: laslora@yahoo.com,; rlaslo@usamvycluj.ro

cDNA microarrays experiments revealed that Genistein caused a

differential expression of several genes Our further question was

if deciphering gene expression data we can predict alternatively spliced variants and protein-encoded sequences of these genes, which could be implicated in prostate cancer prevention by set- ting-up preventive gene sets, and transcriptionally silent genes

To collect accurate information about alternative splicing and protein-encoded sequences predicted associated with genes of interest we investigated known data in the databases using two different computational methods/software (ENSEMBL and JIG- SAW) Three prediction criteria were measured: accuracy on genes, on exons and on nucleotides The goal was to provide consistent, reproducible predictions based on statistical principles, even n case of conflicting evidence about gene structure Then,

we interpreted deciphering those data to find the relationship between genes of interest, alternative splice forms and protein- encoded sequences function and structure We found some inter- esting features that are presented here We discuss the results of genes of interest in order to temp establishing of preventive gene

set They concern the coverage quality, in terms of consistency

and reproducible, of the protein-encoded sequences of genes of

interest, an EST analysis, the validation of splice types, and

finally the link between gene expression analysis/alternative spli-

cing and prostate cancer prevention

OP-51

Ornithine decarboxylase interferes with tumor

necrosis factor alpha-mediated matrix

metalloproteinase-9 productions on

macrophage-like differentiation

Y Liao!, H Hung! and G Liv

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

Taichung, Taiwan,’ Institute of Immunology, Chung-Shan Medical

University, Taichung, Taiwan E-mail: yafanliao@yahoo.com.tw

Proteolytic activity of matrix metalloproteinase-9 (MMP-9) is

necessary for a variety of macrophage functions such as extrava-

sation, migration, and tissue remodeling Ornithine decarboxylase

(ODC) is a rate-limiting enzyme of polyamine biosynthesis Poly-

amines restrain immune response in activated macrophage The

data reported here show the critical role of ODC in the inhibition

of MMP-9 By using 12-0-tetradecanoylphorbol-13-acetate

(TPA)-differentiated human promyelocytic HL-60 and promono-

cytic U-937 cell lines, we found that polyamines blocked the

expression, secretion, and activation of MMP-9 Furthermore,

overexpression of ODC interferes with MMP-9 enzyme activity,

tumor necrosis factor alpha (TNF-«) expression and nuclear fac-

tor-kappaB (NF-kB) activation The secretion of MMP-9 was

induced by stimulating recombinant TNF- « (rTNF- «) in ODC

overexpressed cells Pyrrolidinedithiocarbamate (PDTC), an

inhibitor of NF-kB, suppressed TPA-induced MMP-9 enzyme

activity Difluoromethylornithine (DFMO), an irreversible inhib-

itor of ODC, and rTNF- œ could not rescue MMP-9 secretion

after PDTC treatment Therefore, these data indicated that ODC

inhibits TNF- & expression and then outcome the NF-kB activa-

tion and that regulatory mechanism may provide the molecular

differentiation basis for ODC’s ability to suppress MMP-9

expression and phagocytosis

OP-52

Phosphorylation by Neu directs tyrosine

phosphatase epsilon to activate Src in

mammary tumorigenesis

A Elson, D Berman and S Granot-Attas

Department of Molecular Genetics, The Weizmann Institute of

Science, Rehovot, Israel E-mail: ari.elson@ weizmann.ac.il

Aberrant tyrosine phosphorylation is a well-established cause of

cellular transformation Protein tyrosine phosphatases (PTPs)

help regulate this process; few PTPs support, rather than inhibit,

survival of tumor cells We present genetic and biochemical evi-

dence that the receptor-type PTP epsilon (RPTPe) supports

transformation by linking Neu with downstream events Cells

from mammary epithelial tumors induced in vivo by Neu in mice

genetically lacking RPTPe are less transformed and proliferate

slowly At the molecular level RPTPe activates Src, a known col-

laborator of Neu in mammary tumorigenesis, as well as the Src-

related kinases Yes and Fyn Accordingly, absence of RPTPe

reduces Src activity and alters Src phosphorylation in tumor cells,

RPTPe dephosphorylates and activates Src in heterologous sys-

tems, and Src binds a substrate-trapping mutant of RPTPe The

role of Neu is central, since Neu phosphorylates RPTPe specific-

ally at Y695 at its C-terminus, thereby directing RPTPe to acti-

vate Sre Finally, the altered morphology of Neu-induced

mammary tumor cells lacking RPTPe is corrected by exogenous Src or RPTPe We conclude that a Neu-RPTPe-Sre pathway exists in mammary tumor cells, and that phosphorylation of RPTPe by Neu directs the phosphatase to activate Src in a man- ner required for complete transformation Inhibition of RPTPe may be useful to augment direct pharmacologic inhibition of Src

in fighting mammary tumors

OP-53

Both C and N-terminal regions of p53 are required for its optimal Mdm2-mediated degradation

R Hjerpe!, F Aillet!, T Hay” and S Rodriguez!

Proteomics Unit, CIC BioGUNE, Derio, Spain,’ University of Dundee, Dundee, UK E-mal: mrodriguez@ cicbiogune.es Multiple mechanisms have been reported to regulate the activity

of the tumor-suppressor p53 Different cellular ubiquitin ligases (E3s), such as Mdm2, COP-1, and Pirh2, have been proposed to recognize and modify p53 These E3s are upregulated by p53 as well as by genotoxic stress and each has been found to directly promote p53 ubiquitination and degradation via the ubiquitin- proteasome pathway To face the challenge of elucidating the molecular mechanisms providing specificity for the recognition of p53 and the physiologic significance of various E3s, we used a

‘transfer of signals strategy’ using both B-galactosidase and GFP fusions This strategy allowed us to isolate the minimal p53 N- and C-terminal regions involved in its Mdm2-mediated ubiquity- lation and degradation These minimal sequences are able to con- fer the capacity of being degraded to unrelated proteins, resulting

in the degradation of the fusion protein to the same extent as the wild-type p53 molecule We found that, independently of the type

or size of the fusion protein, both GFP and B-galactosidase show Mdm2-mediated degradability after having been fused to identi- cal p53 regions As the chimeric proteins do not contain the DNA-binding domain of p53, we effectively dissect regions involved in proteolysis, avoiding those problems associated with the transcriptional capacity of p53 This strategy is ready to be used to identify p53 regions involved on its degradation mediated

by other cellular and viral E3s

OP-54

In vivo dynamics of MN1TEL suggest that its immobility may be crucial in leukemias caused

by this fusion protein

M Ter Haar, M Meester-Smoor, M Janssen, A Houtsmuller and E Zwarthoff

Department of Pathology, Erasmus MC, Rotterdam, the Netherlands E-mail: e.zwarthoff@erasmusmc.nl The MNITEL fusion gene is created by a leukemia-associated translocation and combines the MNI1 gene from chromosome 22 with the TEL (ETV6) gene from chromosome 12 TEL is a mem- ber of the ETS family of transcription factors and contains a DNA-binding domain (DBD) TEL represses transcription from ETS-responsive elements MN1 contains transcription-activating domains MNI can stimulate transcription from several promot- ers by interaction with coactivators such as p300 and members of the p160 family and can enhance expression driven by the reti- noic acid receptor In the MNITEL protein the transactivating domains from MNI are combined with the TEL DBD MNITEL stimulates transcription but is unable to synergize with the retinoic acid receptor and is not stimulated by p300 or p160 coactivators To get an insight in the physical properties of the proteins, we decided to examine their behaviour in vive For this

we generated inducible cell lines expressing GFP-tagged proteins

Protein mobility was examined using the fluorescence recovery

after photobleaching (FRAP) technique It was found that MN1

is completely mobile and that a small, but significant fraction of

TEL was immobile We presume that this is due to DNA bind-

ing Interestingly, a much larger fraction of MNITEL was immo-

bile Our current hypothesis is that MNITEL may promote

leukemogenesis by occupying TEL responsive elements, resulting

in transcription from genes that are normally repressed by TEL

OP-55

Ewing sarcoma oncoprotein EWS-FLI1 activity

is enhanced by RNA helicase A

J A Toretsky, H V Erkizan, O D Abaan and A Uren

Georgetown University E-mail: au26@georgetown.edu

RNA helicase A (RHA), a member of the DEXH box helicase

family of proteins, is an integral component of protein complexes

that regulate transcription and splicing EWS-FLI1 oncoprotein

is expressed as a result of a chromosomal translocation that

occurs in patients with Ewing’s Sarcoma Family of Tumors

(ESFT) Although more than 95% of the tumors carry EWS-

FLI1, therapeutic applications using this target have not been

developed Using phage display library screening, we identified

an EWS-FLIl-binding peptide containing homology to RHA

and characterized human RHA protein as a potential EWS-FLI1

interacting protein We observed endogenous RHA and EWS-

FLI1 in the same protein complex in ESFT cell lines GST pull-

down and ELISA assays with recombinant proteins showed that

EWS-FLI1 directly bound to RHA Chromatin immunoprecipita-

tion experiments demonstrated both proteins bound to EWS-

FLII target gene promoters RHA stimulated the transcriptional

activity of EWS-FLI1-regulated promoters, including Id2, in

ESFT cells RHA expression in mouse embryonic fibroblasts cells

stably transfected with EWS-FLI] enhanced the anchorage-inde-

pendent phenotype of EWS-FLI1 alone Reduction of RHA pro-

tein levels by siRNA in ESFT cell lines decreased their growth

rate Our results provide strong evidence for EWS-FLI1 and

RHA interaction and its oncogenic consequences This finding

may lead to development of better therapeutic agents that may

target EWS-FLI1 and RHA interaction

OP-56

Role of p38 MAP kinases in oncogene-induced

malignant transformation

A Nebreda, I Dolado, A Cuadrado, V Lafarga and A Swat

CNIO, Melchor Fernandez Almagro, Madrid, Spain

E-mail: anebreda@cnio.es

We are investigating the role of p38 MAPKs in the processes of

cell proliferation, apoptosis and malignant transformation

induced by oncogenes Oncogenic H-RasV12 induces a more dra-

matic transformed phenotype in p38a-deficient mouse fibroblasts

than in their wild type counterparts The inhibitory effect of p38

MAPKs on H-RasV12-driven transformation was confirmed in

NIH3T3 fibroblasts overexpressing the p38 MAPK activator

MKK6 Interestingly, p38 MAPKs do not inhibit transformation

induced by all oncogenes but seem to rather specifically modulate

signalling pathways activated by oncogenic Ras We have also

observed that, in fibroblasts expressing H-Ras V12, there is an

inverse correlation between p38a activity levels and the ability of

the cells to lose contact inhibition and grow in multiple layers

Consistent with these results, p38a—/— fibroblasts grow to a

higher saturation density than wild-type fibroblasts Moreover,

p38a-deficient cells proliferate better than wild-type cells in the

presence of low serum concentrations and are also more resistant

to apoptosis induced by oncogenes Finally, we have found that p38a—/— cells have impaired motility, which correlates with a less robust actin cytoskeleton These results indicate that p38 MAPKs may modulate cancer cell-associated traits at multiple levels We have performed proteomic and microarray experiments to iden- tify new proteins that potentially function downstream of p38 MAPKs in tumorigenesis regulation

OP-57

Abnormal expression of gamma catenin

correlates with poor prognosis and liver

metastases of breast carcinoma

N Erin‘, J Weisz’, D Shearer” and G Clawson?

‘Internal Medicine and Human Gene Therapy Unit, Akdeniz University School of Medicine, Antalya, Turkey

E-mail: nerin@akdeniz.edu.tr ,° Obstetrics and Gynecology, Penn State University, Hershey, PA, USA; Gittlen Cancer Foundation, Penn State University, Hershey, PA, USA

We previously developed a cell line from a heart metastases of 4T1 murine breast carcinoma This cell line named as 4THMpc behaved more aggressively than the parental cell line (4T1) and formed multiple macroscopic liver metastases Expressions of over 12 000 genes were determined in liver metastases, 4T1 and 4THMpc primary tumors Comparison of gene array results demonstrated that expression of adherence junctions proteins were markedly decreased in liver metastases compared to primary tumors formed by 4Tl and 4THMpc Among these proteins gamma catenin is examined at protein level Immunoblots from tumor lysates demonstrated the presence of gamma catenin in both 4T1 and 4THMpce primary tumors 4T1 primary tumors dis- played more gamma catenin than 4THMpc Immunohistochemi- cally, patches of cells were stained in both groups Interestingly, cytoplasmic staining was observed in both groups but membrane staining was detectable only in 4T1 tumor cells Instead, nuclear and perinuclear staining was observed in 4THMpc tumor Gamma catenin expression was undetectable in liver metastases Similarly, hepatocytes were mostly unstained, except around metastases and the central vein These findings demonstrate that abnormal expression of gamma-catenin correlates with the more aggressive tumor and an indicator of liver metastatic breast can- cer We here also demonstrated that liver metastases of breast

carcinoma do not express gamma catenin

OP-58 Molecular and cellular characterization of GPCR mas-induced tumour formation

W Z Lin, S Y Tsang, S S T Lee and W T Cheung Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China E-mail: wtcheung@cukh.edu.hk Mas was originally isolated from a human epidermoid carcinoma and predicted to be an orphan GPCR Recently, we have identi- fied a surrogate agonist for mas using phage-displayed peptide library [1] With dihydrofolate reductase gene as a selection mar- ker, several stable mas-transformed CHO cell clones were isola- ted, and mas transgene expression was further amplified by stepwise addition of increasing doses of methotrexate Mas-trans- formed CHO cells induced solid tumour formation in nude mice Genetic analyses indicated that tumorigenicity of mas-transfected CHO cells depends on the sites of chromosomal integration and the levels of mas expression, suggesting that mas overexpression

is not sufficient to induce tumour formation Histochemical ana- lyses revealed that tumour was composed of mas-transfected cells