3. Molecular Basis of Disease 3.1 Oxidative Stress S3.1-1 The role of antioxidants in the cytotoxicity of chemotherapeutic drugs T. Ozben, H. Akbas, I. Akan, S. Akan and M. Timur Department of Biochemistry, Faculty of Medicine, Akdeniz University, Antalya, Turkey. E-mail: ozben@akdeniz.edu.tr A number of drugs used in cancer chemotherapy induce oxidative stress by generation of oxygen free radicals (ROS) which might be an alternative mechanism for their cytotoxic effect via indu- cing apoptosis. In order to clarify the roles of antioxidants in chemotherapy, we investigated Quercetin (3,3’,4’,5,7-pentahyd- roxyflavone) and N-acetylcysteine (NAC) in different cell types treated with anticancer drugs. We studied cytotoxic activity of Topotecan alone and/or in combination with Quercetin in two human breast cancer cell lines, MCF-7 and MDA-MB-231. We also investigated the effect of NAC on MRP1-mediated doxoru- bicin and vincristine cytotoxicity in Human Embryonic Kidney (HEK293) and its MRP1 transfected (293MRP) cells. The viabil- ity of the cells was measured using the colorimetric MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Intracellular ROS was measured using fluorometric 2’,7’- dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Our data indicated increased oxidative status in MCF-7 and MDA- MB-231 cells exposed to Topotecan. Treatment with Quercetin did not inhibit ROS generation, and enhanced cytotoxicity of Topotecan in both cells. In contrast, NAC enhanced resistance against doxorubicine and vincristine in MRP1 overexpressing cells. We conclude that Quercetin and NAC may have diverse effects in the cytotoxicity of chemotheurapeutic drugs depending on their other pharmacological properties which may predomin- ate their antioxidant effects. S3.1-2 Oxidized protein degradation and repair in ageing and oxidative stress B. Friguet Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite ´ Denis Diderot – Paris 7, Paris, France. E-mail: bfriguet@paris7.jussieu.fr Cellular ageing is characterized by the accumulation of oxidatively modified proteins and oxidized protein buildup with age may be due to increased protein damage and/or decreased elimination of oxidized protein. Since the proteasome is in charge of protein turn- over and removal of oxidized protein, its fate during ageing has received special attention, and evidence has been provided for an age-related impairment of proteasome function. In fact, depending on the cellular system investigated, the loss in proteasome activity observed during ageing and upon oxidative stress appears to be due to either or both: (i) decreased proteasome expression and con- tent, (ii) inactivation upon modification of proteasome subunits and (iii) formation of inhibitory proteins. However oxidized pro- teins can be eliminated through degradation but also repair. Oxid- ized protein repair is limited to the reversion of few modifications such as the reduction of methionine sulfoxide by the methionine sulfoxide reductase (Msr) system. We have previously shown that Msr activity is impaired during ageing. To analyse the relationship between oxidative stress, protein oxidative damage and Msr, MsrA has been overexpressed in immortalized WI-38 human fibroblasts. After H202-induced oxidative stress, MsrA-overexpressing cells exhibit lower protein oxidative damage than control cells indica- ting that MsrA may play an important role in cellular defences against oxidative stress by limiting oxidative damage to proteins. S3.1-3 Role of oxidative stress in progressive kidney failure A. Tomasi 1 , S. Uggeri 2 , S. Beergamini 3 , L. Della Casa 3 , A. Albertazzi 2 , L. Lucchi 2 and A. Iannone 1 1 Department Laboratory Medicine, Universita ` di Modena e Reggio Emilia, Modena, Italy, 2 Division of Nephrology, University Hospital, Modena, Italy, 3 Department of Biomedical Sciences, Modena, Italy. E-mail: tomasi@unimore.it The leading cause of morbidity and mortality in patients with end stage renal disease (ESRD), who are maintained on regular dialysis treatment, is cardiovascular disease. Increased free radical production and oxidative stress promote the atherosclerotic pro- cess. The association between haemodialysis and activation of circulating monocytes, both spontaneous and endotoxin-induced release of TNF-alpha are well known. In our research spanning many years, as well as in many other laboratories, there has been a massive attempt to identify reliable biomarkers of oxidative stress. Initially our attention was drawn on direct markers of oxi- dative stress such as malondialdehyde (MDA) and conjugated dienes. More recently, high plasma level of homocysteine, which is considered a putative cardiovascular risk factor by inducing endothelial dysfunction and serum C-reactive protein (CRP), which contributes to monocytes recruitment in the atherosclerotic lesion have been adopted as possible markers of atherosclerotic progression in ESRD patients. It has been postulated that ESRD patients have also a decreased ability to withstand oxidative stress due to a reduced antioxidant capacity, which was also investigated as a putative biomarker. Recently, we have tried to define novel markers of oxidative stress applying proteomic pro- filing technologies, employing both 2-D gel electrophoresis - mass spectrometry and high throughput SELDI technology. Abstracts 17 3.2 DNA Damage Processing S3.2-1 The role of the human mismatch repair system in the processing of modified nucleotides N. Mojas, F. Fischer, M. Lopes, P. Cejka and J. Jiricny Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland. E-mail: jiricny@imcr.unizh.ch The mismatch repair (MMR) system has evolved to correct errors of DNA replication and to prevent illegitimate recombina- tion events. However, MMR is also involved in DNA damage signalling. In MMR-proficient cells, methylating agents such as MNNG activate a cell cycle arrest in the second G2 phase after treatment. The signalling cascade, which is triggered by the ATR and Chk1 kinases, is not activated in MMR-deficient cells. We show that the lesions triggering the cascade are not ‘mismatches’ arising through DNA modification by the methylating agents. Nor are these lesions generated during DNA replication. Our evi- dence suggests that the cell-cycle-arresting lesions are generated through unsuccessful attempts by the MMR system to repair mehylation damage, which leads to the generation of recombino- genic intermediates. DNA recombination enables the cells to cross the mitotic boundary, but the damage that remains kills the cells during the subsequent cell cycle. Thus, in the absence of recombination, the cells become hypersensitive to killing by methylating agents and arrest already in the first G2. We are currently searching for the structures of the recombination inter- mediates that lead to cell death. We are also investigating the role of the MMR system in the cytotoxicity of 5-fluorouracil (FU), which has been reported to kill MMR-proficient cells more efficiently than MMR-deficient ones. In our hands, isogenic MMR-proficient and -deficient cells are equally sensitive to FU. S3.2-2 Base excision repair proteins in cancer prevention and acquired imm une responses H. E. Krokan, M. Akbari, J. Pen ˜ a Diaz, B. Kavli, M. Otterlei, M. Ericsson, H. Aarset, S. Andersen and G. Slupphaug Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway. E-mail: hans.krokan@ntnu.no Base excision repair repairs DNA by removing damaged bases and filling in the gap with correct nucleotides. Uracil-DNA gly- cosylase encoded by the UNG-gene is also required for somatic hypermutation and class switch recombination. Furthermore, Ung-deficient mice have lymphatic hyperplasia and several-fold increased risk of developing B-cell lymphomas. Humans deficient in UNG2 activity display the hyperIgM syndrome (HIGM) phe- notype, including B-cell hyperplasia. We find that lymphomas in mice are of follicular (FL) and diffuse large B-cell type (DLBCL) type. All FLs and 75% of the DLBCLs were monoclonal while 25% were biclonal. Monoclonality was also observed in hyperpl- asia, and could represent an early stage of lymphoma develop- ment. Lymphoid hyperplasia was observed as a significant increase of splenic B-cells. Furthermore, loss of Ung also causes a reduction of T-helper cells, and 50% of the young Ung–/– mice investigated have no detectable NK/NKT-cell population in their spleen. In addition, production of the cytokines interferon c, interleukin-6 and interleukin-2 is clearly different in wild type and in Ung-deficient mice. Thus, Ung-proteins have important functions in the immune system, not only in the process of anti- body maturation, but also for production and functions of immunologically important cell types. The immunological imbal- ances in the Ung-deficient mice may be central in the develop- ment of lymphomas in a background of generalized lymphoid hyperplasia. S3.2-3 New complexities in base excision rep air and relevance to human pathology E. Dogliotti Department of Environment and Primary Prevention, Istituto Superiore di Sanita ` , Rome, Italy. E-mail: dogliott@iss.it Base excision repair (BER) is the major mechanism for repair of oxidative DNA damage. Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA distorting lesions in NER of the genome overall. We show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. We provide the first in vivo evidence that XPC is involved in the repair of 8,5’-cyclopurine 2’-deoxynu- cleosides and of the mutagenic oxidized DNA base 8-hydroxy- guanine (8-OH-Gua). By in vitro reconstitution experiments we uncover a new role of XPC as co-factor in 8-OHGua cleavage by the DNA glycosylase OGG1. XPC complex, by participating to BER of 8-OH-Gua, might contribute to cancer prevention. A provokative hypothesis has recently emerged in which BER enzymes, more than controlling the integrity of the genome, may create unintended consequences. Adaptive increases in BER enzymes have been shown to generate microsatellite instability in chronic inflammation. We detect up-regulation of BER genes in association with microsatellite instability in an inflammation rela- ted cancer (i.e. stomach cancer). Future research should address the mechanistic basis of this phenomenon that might be of great relevance in human pathology. 3.3 DNA Repair in Health, Disease and Aging S3.3-1 DNA repair deficiencies in human premature aging A. Bohr Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland, USA. E-mail: vbohr@nih.gov There are a number of human syndromes where the patients appear much older than their actual chronological age. These conditions are interesting model systems for the study of aging. For many of these conditions, the responsible gene has been cloned, identified, and characterized, thus enabling studies of its molecular function. We are interested in the category of human premature aging conditions that are defective in recQ helicases and associated with genomic instability. These conditions include Werner syndrome, Rothmund–Thompsons syndrome and others. The molecular function of the recQ helicases will be discussed and evidence presented that these proteins participate in an intergral Abstracts 18 manner in the DNA repair process. Another premature aging condition, also associated with DNA repair defects, Cockayne syndrome, will also be discussed. These studies lend credibility to the notion that aging is associated with defects in DNA repair that then lead to genomic instability. S3.3-2 DNA damage repair and the connection with cancer and aging J. Hoeijimakers 1 , J O. Andressoo 1 , L. Niedernhofer 2 , I. van der Pluijm 1 , K. Diderich 1 , A. Lalai 1 , H. de Waard 1 , G. Garinis 1 , J. Mitchell 1 , R.B. Beems 3 , H. van Steeg 3 and G.T.J. van der Horst 1 1 MGC, CBG, Department of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands, 2 University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA, USA, 3 RIVM, Bilthoven, The Netherlands The physico-chemical constitution of our genes does not guaran- tee life-long stability and proper function. A perplexing diversity of lesions is continuously induced in our genes ranging from sin- gle and double strand breaks, to chemical alterations of nucleo- tides including adducts, as well as inter- and intrastrand DNA crosslinks. They arise from ubiquitous, noxious exogenous agents (UV- and X-radiation, chemicals), natural metabolites produced by respiration (notably reactive oxygen species) and from intrin- sic chemical instability of DNA. To protect the vital genetic information from the deleterious effects of DNA injury a net- work of genome care-taking mechanisms has evolved of which a set of DNA repair systems constitute a key component. The clinical impact of the DNA damage problem is still strongly underestimated. Nucleotide excision repair (NER) removes a wide range of helix-distorting lesions in a complex ‘cut and patch’ reaction. There are two sub-pathways: global genome NER critical for preventing mutations and transcription-coupled repair (TCR) counteracting the cytotoxic effects of DNA injury. Inherited NER defects are associated with sun (UV) hypersensi- tive syndromes, including xeroderma pigmentosum (XP, highly cancer-prone), and the severe neuro-developmental conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD). Muta- tions in the multifunctional NER/TCR XPB and XPD helicases are associated with an extreme clinical heterogeneity, ranging from XP to XP combined with CS and TTD. Defects in the NER and crosslink repair endonuclease, ERCC1/XPF, cause XP or XP with multi-system dysfunction. Mouse models have provi- ded important insights into the impact of the NER sub-pathways on human health and the complex genotype-phenotype relation- ship. XPD TTD mice, with a partial defect in both global and TCR are only moderately cancer-prone, but exhibit wide spread premature ageing. XPD XP/CS mutant mice are highly predisposed to cancer, with a milder ageing phenotype. Complete repair defi- ciency in TTDxXPA mice aggravates many premature ageing symptoms, reducing life span to ~3 weeks. Mutations in the ERCC1 gene induce a distinct set of accelerated ageing features, with a rate of onset depending on the severity of the mutation. The correlation between repair defect severity and clinical mani- festation provides strong evidence for the DNA damage theory of ageing. We propose that endogenous oxidative lesions com- promise transcription, inactivate genes, and trigger apoptosis/sen- escence inducing aging. Very cytotoxic interstrand cross-links may also cause cell death, senescence and features of ageing. In contrast, lesions or defects in genetic stability mechanisms caus- ing enhanced levels of DNA damage-induced mutagenesis corre- late with increased carcinogenesis. Various single and double mutant mouse models including conditional mutants have been generated and microarray analysis performed to study these pro- cesses in a controlled, systematic manner. S3.3-3 DNA double strand repair and its relationship to human disease A. Jeggo Genome Damage and Stability Centre, University of Sussex, UK. E-mail: p.a.jeggo@sussex.ac.uk DNA is constantly subject to damage. Cells utilise multiple DNA damage response pathways to maintain genomic stability. A DNA double strand break (DSB) represents a particularly critical DNA lesion that can lead to cell death or carcinogenesis. DSBs are introduced during immune development to generate genetic diver- sity. Curiously, cells exploit the mechanism normally used to maintain genomic stability after exposure to DNA damaging agents to create diversity during immune development. This path- way is DNA non-homologous end-joining (NHEJ), a process of DSB repair. To date, three genetic diseases with defects in NHEJ have been identified. As expected from the role of NHEJ in immune development, such patients display immunodeficiency. Additionally, since a DSB is induced following exposure to ion- izing radiation, such patients and cell lines derived from them dis- play marked radiosensitivity. LIG4 syndrome is a disorder conferred by mutations in DNA ligase IV, the enzyme that effects rejoining during NHEJ. LIG4 patients display pancytopaenia, microcephaly and developmental delay. RS-SCID1 patients have mutations in Artemis, a protein that processes DNA ends prior to rejoining. More recently, a further disorder has been described, which is associated with marked immunodeficiency and mild developmental delay. The protein defective in this disorder is also a NHEJ protein. The distinct clinical features of the patients will be discussed in the context of the protein function. 3.4 Diabetes, Obesity and Metabolic Syndrome S3.4-1 Integration of inflammatory and metabolic signals in obesity and diabetes G. Hotamisligil Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, USA. E-mail: ghotamis@hsph.harvard.edu Obesity is associated with chronic, low grade, inflammatory responses in metabolically active sites. This hightened inflamma- tory status is among the key links between obesity and other associated pathologies, i.e. type 2 diabetes. Recent studies in our group demonstrated the involvement of the inflammatory kinase JNK and endoplasmic reticulum (ER) stress as events involved in both sensing and relaying stress signals and disturb- ing metabolic homeostasis. Obesity generates conditions that increase the demand on the ER and leads to strong activation of JNK in insulin sensitive sites. We have shown that, in both cultured cells and whole animals, experimentally induced ER stress leads to IRE1±-dependent activation of JNK, serine Abstracts 19 phosphorylation of insulin receptor substrate 1 (IRS1), and inhibition of insulin action. Hence, we conclude that JNK acti- vation and ER stress, integrates inflammatory and metabolic responses, plays a crucial role in development of insulin resist- ance and diabetes in obesity and present new targets for the treatment of these disorders. Recently, we developed JNK- or XBP-1 genetic gain- or loss-of function mouse models as well as chemical tools to alter the functional capacity of ER and JNK activity in whole animals to demonstrate the causal link between these pathways and metabolic disease. Recent activities in these areas and identification of cellular and molecular tar- gets and development of new genetic models are underway and will be discussed here. S3.4-2 The metabolic syndrome and adipocytokines Y. Matzuzawa Department of Internal Medicine and Molecular Science, Graduate School of Medicine Osaka University, Osaka, Japan Visceral fat accumulation plays crucial roles in the development of cardiovascular disease as well as diabetes mellitus, hyperlipide- mia and hypertension and so-called metabolic syndrome. Adipo- cyte functions has been intensively investigated in the past 10 years, and adipocytes have been revealed to act as endocrine cells which secrete various bioactive substances.(adipocytokine) Among adipocytokines, tumour necrosis factor-a, plasminogen activator inhibitor type 1, heparin binding epidermal growth fac- tor-like growth factor and visfatin (newly found in visceral fat) are produced in adipocytes as well as other organs, and may con- tribute to the development of vascular diseases. On the contrary to these adipocytokines, adiponectin, an adipose-tissue-specific, collagen-like protein, has been noted as an important antiathero- genic and antidiabetic protein, or as an anti-inflammatory pro- tein. The functions of adipocytokine secretion might be regulated dynamically by nutritional state. Visceral fat accumulation causes dysregulation of adipocyte functions, including oversecretion of TNF-a, PAI-1 and heparin HBEGF and hyposecretion of adipo- nectin, which results in the development of a variety of metabolic and circulatory diseases. In this review, the importance of adipo- cytokines, especially focusing on adiponectin is discussed with respect to cardiovascular diseases. S3.4-3 Nuclear receptors as therapeutic targets to modulate the metabolic synd rome B. Staels UR545, Dpt d’Athe ´ roscle ´ rose, Institut Pasteur de Lille and Faculte ´ de Pharmacie, Universite ´ de Lille II, Lille, France. E-mail: bart.staels@pasteur-lille.fr Cardiovascular disease is significantly increased in patients with the metabolic syndrome and type 2 diabetes. A clustering of risk factors, including dyslipidaemia, insulin resistance, hypertension, inflammation and coagulation disorders result in an increased risk for cardiovascular events in these patients. The Farnesoid X Receptor (FXR) and peroxisome proliferator-activated receptor (PPAR) alpha are members of the nuclear receptor superfamily. Whereas PPARalpha is activated by fatty acids, FXR has recently been identified as a bile acid-activated nuclear receptor. FXR not only controls bile acid synthesis, conjugation and trans- port, but also lipid and glucose metabolism. Activation of PPAR- alpha represents one important pathway that influences vascular function both directly and indirectly. PPARalpha activation induces beneficial effects not only on lipid metabolism, but also influences glucose homeostasis, endothelial function and vessel wall inflammation. PPARalpha agonists in clinical use, such as fibrates, may alter the process of atherosclerosis, especially in subjects with the metabolic syndrome and type 2 diabetes. This presentation will highlight the molecular mechanisms of FXR and PPARalpha action in the prevention and treatment of the metabolic syndrome and atherosclerosis. 3.5 Lipid Related Disorders and Atherosclerosis S3.5-1 Treatment of the metabolic syndrome by targeting hepatocyte nuclear factor-4a by MEDICA R. Hertz and J. Bar-Tana Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem, Israel. E-mail: bartanaj@cc.huji.ac.il Dyslipoproteinemia, NIDDM and essential hypertension often coexist, and synergistically promote ASCVD. The Metabolic Syn- drome that comprises these diseases is driven by nutritional sat- urated fat. Transcriptional modulation of liver genes involved in lipoprotein production and their plasma clearance (apoB, apoC III,MTP), hepatic glucose production (G6Pase, PEPCK), blood coagulation and others is dominated by hepatocyte nuclear fac- tor-4a (HNF4). Activation/suppression of HNF4 transcriptional activity by its acyl-CoA ligands as function of their chain length, unsaturation or extent of substitution, correlates with the repor- ted effects of long chain fatty acid nutrients in the Metabolic Syndrome context. Hence, HNF4 serves as cellular sensor for long chain fatty acids, and targeting HNF4 may offer means for targeting the Syndrome. Long chain methyl-substituted a,x- dicarboxylic acids (MEDICA compounds) are incapable of being esterified into lipids or b-oxidized. MEDICA-CoA specifically binds to HNF4 with K d values in the nM range, and blocks its transcriptional activity. MEDICA compounds induce hypolipide- mia accounted for by a pronounced activation of plasma chylo- microns and VLDL clearance complemented by robust inhibition of liver VLDL production, transcriptional suppression of hepatic glucose production, abrogation of non-alcoholic steatohepatitis, activation of glucose uptake during hyperinsulinemic–euglycemic clamp, and amelioration of ASCVD in animal models of the Metabolic Syndrome. S3.5-2 Why a metabolic syndrome? P. G. Kopelman Institute of Health, University of East Anglia, Norwich, UK. E-mail: p.g.kopelman@qmul.ac.uk The global prevalence of obesity confirms that it has become a major threat to public health. One in five children in Europe are overweight. The rise in obesity in young people is paralleled in adults – obesity rates in men vary from 10% to 27%, in women up to 38%: at least 25% of adults in US have the metabolic syn- drome. The patho-physiological consequence of increased body Abstracts 20 fatness is predictable from an understanding of the relationship between insulin resistance, systemic hyperinsulinaemia and even- tual pancreatic islet cell decompensation. There is a close associ- ation between increasing weight, intra-abdominal fat, impaired glucose tolerance and deleterious alterations in lipid profile that are associated with alterations in pro-inflammatory markers CRP, interleukin-6 and TNF-a. Unsurprisingly these changes are clinically characterised by type 2 diabetes, hypertension and accelerated coronary artery disease. The INTERHEART trial from 52 countries confirms the detrimental impact of abdominal obesity on rates and outcome from myocardial infarction. Of particular worry is that these chronic diseases begin in young people. The great expectation of new therapies to effectively treat obesity has not been fulfilled. Thus a major challenge for 21st century is the prevention of obesity, and the metabolic syn- drome, through populations adopting healthier lifestyles with health-promoting social marketing becoming an important prevention tool. S3.5-3 Transcriptional regulatory networks in the ABCA1/AP3-pathway determine lipid trafficking in macrophages G. Schmitz, M. Grandl, G. Liebisch, A. Boettcher, E. Orso and T. Langmann Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Regensburg, Germany. E-mail: gerd.schmitz@klinik.uni-regensburg.de ABCA1 controls cellular cholesterol and choline-phospholipid release and is required for the generation of pre-b-HDL. Our goal was to characterize gene regulatory networks and ABCA1 associated lipid pathways in human macrophages in cardiovascu- lar disease and under atherogenic and nutritional stimulation. Macrophages from healthy apoE3 donors and patients with monogenetic lipid disorders (ABCA1 deficiency, Niemann–Pick C disease) were incubated with modified lipoproteins, enzymatically modified LDL (E-LDL) and mildly oxidized LDL (Ox-LDL), lipid deloaded with apoA-I and HDL3 and stimulated with reti- noids. We applied Affymetrix DNA-microarrays, TaqMan RT-PCR and computational biology approaches and determined the cellular lipid content and composition using ESI-MS/MS. (i) 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA) induce a nuclear receptor network strongly upregulating apolipoproteins, scavenger receptors, steroid-27-hydroxylase, ABCA1 and ABCG1 resulting in a potent induction of lipid efflux. (ii) E-LDL increases cellular free cholesterol and forma- tion of cholesterol/sphingomyelin-rich rafts in macrophages, whereas Ox-LDL causes accumulation and cell surface expression of ceramide and a stronger binding of apoE. (iii) ABCA1, syntaxin-13 and flotillin-1 operate in both loading conditions with different response rates and downstream signalling involving an inward rectifying K+-channel along the ABCA1/AP-3 secretory pathway. 3.6 Oncogenes and Tumor Suppressors S3.6-1 Molecular pathogenesis of liv er ca ncer M. Ozturk Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey. E-mail: ozturk@fen.bilkent.edu.tr Liver cancer (hepatocellular carcinoma; HCC) is one of the 10 most common cancers worldwide. The major etiology of liver cancer is cirrhosis that is observed mostly in old people with a chronic history of viral (HBV and HCV) and/or non-viral hepa- titis. Liver cancer appears as a heterogeneous disease at the molecular level, probably because of the fact that different etio- logical factors contribute to the development of these cancers, depending on geographical locations. The most shared genetic alterations of liver cancers are mutations affecting p53, b-catenin and Axin1 genes together with the epigenetic or mutational inac- tivation of the p16INK4a gene. Most liver cancers also display hTERT telomerase induction whose mechanisms remain elusive. Gene expression profiling data show deregulated expression of genes involved in proliferation, apoptosis, DNA repair, protein synthesis and liver-specific functions. Taken together these observations provide evidence for critical involvement of senes- cence- and differentiation-regulating events, in addition to well established cell cycle and apoptosis regulatory changes in liver cancer. Among others, ‘p53-retinoblastoma tumor suppressing network’ and ‘canonical Wnt-b-catenin signaling’ appear to play a central role in hepatocellular carcinogenesis. New experimental data incriminating senescence- and differentiation-related molecular changes in the pathogenesis of liver cancer will also be presented. S3.6-2 AP-1 (Fos/Jun) and cancer E. F. Wagner IMP, Research Institute of Molecular Pathology, Vienna, Austria. E-mail: wagner@imp.univie.ac.at AP-1(Fos/Jun) proteins are prototypic oncogenes regulating cell proliferation, differentiation and cell transformation in various organs. We are investigating the specific functions of Fos pro- teins, e.g. Fos, Fra-1 and Fra-2 in development and disease. Transgenic mice expressing Fos develop osteosarcomas (OS), whereas mice lacking Fos are osteopetrotic. It is well established that Fos is phosphorylated in the C-terminus by kinases such as RSK-2 in vitro. We have been studying the role of Fos phos- phorylation in OS development and found that tumors do not progress in the absence of RSK-2. We speculate that the effect on tumor development is due to increased stability of phosphor- ylated Fos protein. When the Fos–/– osteopetrotic mutant was crossed into the p53 background, double mutant mice specifically develop rhabdomyosarcomas (RMS). Re-expressing Fos in Abstracts 21 mutant muscle tumor cell lines induced apoptosis implying a novel function of the oncogene Fos as a potential tumor suppres- sor. Recent data regarding the target genes of Fos in OS and RMS cells obtained by microarray analyses will be presented. The functions of the Jun family members c-Jun and JunB have been analyzed by conditional mutagenesis. Whereas c-Jun was found to function as an oncogene in liver cancer, JunB acts as a tumor suppressor gene in a mouse model of myeloid leukemias. The combined inducible deletion of c-Jun and JunB in the skin of adult mice causes a psoriasis-like phenotype strongly resem- bling the human disease. S3.6-3 Chromatin modifications and their function C. Neilson, H. Santos-Rosa, A. Bannister and T. Kouzarides Gurdon Institute, Tennis Court Road, Cambridge, UK Covalent modifications of histones regulate many biological pro- cesses, the most characterized of which is transcription. We are interested in the molecular mechanisms by which these modifica- tions function. Our focus recently has been the analysis of his- tone methylation at lysines and arginines. We now find that one site of lysine methylation on histone H3 is under the control of proline isomerization. Prolines can exist in a -cis or -trans confor- mation. Enzymes exist (peptidyl prolyl isomerases), which can convert prolines from one conformation to the other. We have found that a yeast protein FPR4 is an enzyme that can isomerize specific prolines in histone H3 and that this isomerization affects lysine methylation of H3. The cross-talk between proline isomeri- zation and lysine methylation has a consequence in the regulation of transcription of certain genes in yeast. These data define a novel non-covalent histone modification that regulates gene expression. 3.7 Intracellular Trafficking in Health and Disease S3.7-1 The yeast vacuolar protein sorting pathway as an experimental model for lysosomal trafficking J. Rytka Department of Genetics, Institute of Biochemistry and Biophysics, Warsaw, Poland. E-mail: rytka@psd.ibb.waw.pl Compartmental organization of eucaryotic cells requires mecha- nisms for precise sorting and distribution of proteins and metab- olites to their target organelles. Due to impressive conservation of intracellular traffic across phyla, a great deal of the current knowledge on the mechanisms of integration of these processes into the cellular interaction network has been acquired from studies on Saccharomyces cerevisiae. A good example is vacuolar/ lysosomal transport, as the yeast vacuole is analogous to the mammalian lysosome, and trafficking to both organelles is remarkably conserved. Defects in vacuolar function lead to pro- tein mislocalization, disturbances in ion homeostasis, affect such complicated processes as osmoregulation and sporulation. Isola- tion and characterization of yeast mutants blocked at various sta- ges of transport pathways are an invaluable method for unravelling the molecular details of vacuolar and lysosomal traf- fic. The activities of lysosomes are critical to many essential cellu- lar processes. Such diseases as Huntington’s, Alzheimer’s, Parkinson’s, tumor invasion and metastasis are all associated with altered lysosomal trafficking. Our research concentrates on Ccz1p, the yeast homolog of mammalian HPS4. The mutated HPS4 protein is responsible for Hermansky–Pudlak syndrome type 4. S3.7-2 Sorting, targeting and delivery in mammalian post-golgi pathways P. Luzio Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK. E-mail: jpl10@cam.ac.uk The targeting and localization of proteins to the correct intracel- lular organelle or cell surface domain is essential for their physio- logical function. In the post-Golgi secretory and endocytic pathways proteins are targeted to many locations, with vesicular traffic being the major mechanism of transport between donor and acceptor compartments. Disruption of membrane traffic events is the molecular basis for many hereditary, autoimmune and other diseases. In addition, several microbial pathogens sub- vert normal membrane traffic and some viruses are able to hijack membrane traffic machinery to avoid recognition by the immune system and/or for the purposes of viral budding. In general, membrane traffic defects in hereditary disease can be subdivided into abnormalities of cargo or of traffic machinery. The former are by far the most common. Mutations in cargo or machinery proteins can result in inappropriate targeting and consequent cel- lular malfunction. This will be illustrated by reference to muta- tions in protein targeting motifs in cargo trafficking to/from the plasma membrane and to lysosomes. An example of traffic machinery in which abnormalities of function can result in dis- ease is that for formation of multivesicular bodies on the endocy- tic pathway. Components of this machinery have been implicated in viral disease, cancer and interacting with spastin, a protein encoded by a hereditary spastic paraplegia gene. S3.7-3 How vesicular stomatitis virus hijacks the endocytic pathway to infect cells P. P. Luyet, I. Le Blanc and J. Gruenberg Biochemistry Department, University of Geneva, Geneva, Switzerland. E-mail: pierre.luyet@biochem.unige.ch Like other enveloped viruses, vesicular stomatitis virus (VSV) infects cells through the endocytic pathway. VSV infection requires transport beyond early endosomes, hence to multivesicu- lar endosomes (MVBs), and then the transcriptionally active nucleocapsid is delivered to the cytoplasm after low pH-mediated fusion of the viral envelope with endosomal membranes. We observed that VSV envelope fusion and nucleocapsid release into the cytosol occur sequentially at two successive steps of the endo- cytic pathway – and not concomitantly as it was thought. Our results indicate that VSV envelope undergoes fusion with the membrane of intra-endosomal vesicles contained within MVBs, thereby releasing the nucleocapsid into the lumen of these vesi- cles. Then, the capsid uses these intra-endosomal vesicles as a Trojan horse to reach late endosomes, from where the nucleocap- sid is delivered to the cytoplasm. This latest step occurs presuma- bly via the back-fusion of the internal vesicles with the endosome limiting membrane, in a process regulated by the endosomal lipid Abstracts 22 lysobisphosphatidic acid under the control of Alix/Vps31p and by the phosphatidylinositol-3-phosphate via Snx16. We propose that proteins and lipids, which are transported from within late endosomes to other cellular destinations, also use this intra-endo- somal trafficking pathway. The functions of other effectors in this pathway will be discussed. 3.8 Neurodegenerative Disorders S3.8-1 Tau and tauopathies: tau phosphorylation and tau assembly J. Avila Centro de Biologı ´ a Molecular ‘Severo Ochoa’, Facultad de Ciencias, Campus de Cantoblanco, Universidad Auto ´ noma de Madrid, Madrid, Spain. E-mail: javila@cbm.uam.es The role of tau protein in different tauopathies, mainly in Alzhei- mer’s disease, will be commented. Tau, a microtubule associated protein, can aberrantly polymerize, in phosphorylated form, yielding the paired helical filaments found in the brain of Alzhei- mer’s disease patients. Tau assembly can be reproduced in vitro by mixing tau protein with polymerization-inducers like heparin or Coenzyme Q0, being the assembly of phosphotau facilitated in the presence of Co.Q0. Polymerization of tau has been also mimified in vivo by using transgenic mouse models. In these models, human tau, bearing some of the mutations found in patients with a tauopathy, FTDP-17, was expressed. Addition- ally, mutations on APP and/or PS-1 proteins will facilitate tau phosphorylation by kinases like GSK3 in mouse models. Thus, a transgenic mouse model overexpressing GSK3 was also charac- terized. In some of these mouse models, a link between tau phos- phorylation and tau assembly has been established. Finally, the possible toxic effect of phosphotau or tau aggregates will be dis- cussed. S3.8-2 Alpha-synucleinopathies M. G. Spillantini Department of Clinical Neurosciences, Brain Repair Centre, University of Cambridge, Cambridge, UK. E-mail: mgs11@cam.ac.uk Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are characterized neuropathologically by the presence of Lewy bodies (LBs) which are intracytoplasmic filamentous inclusions. Alpha-synuclein (alpha-syn) has been implicated in the pathogen- esis of PD based on the presence of missense mutations and abnormal copy number of its gene in familial cases as well as for its presence in LBs. Alpha-syn constitutes also the major compo- nent of the glial cytoplasmic inclusions found in multiple system atrophy. These diseases are therefore grouped under the name of ‘alpha-synucleinopathies’. The mechanism of alpha-syn aggrega- tion in PD and DLB is not yet clear. The presence of ubiquitin in LBs and the association of mutations in the ubiquitin-protea- some pathway with early-onset Parkinsonism, has led to the sug- gestion that abnormalities in ubiquitin-dependent degradation and alpha-synuclein aggregation may be related processes in inclusion formation. However, alpha-syn can be degraded by the proteasome in a ubiquitin independent manner and not all LBs contain ubiquitin. These results suggest that alpha-syn ubiquityla- tion is not a primary event for alpha-syn fibrillization. This is in agreement with the observation that transgenic mouse models of alpha-syn accumulation show very little ubiquitin staining. At present is clear that alpha-synuclein is linked to neurodegenera- tion in PD but the mechanisms and significance of its aggregation remain to be determined. S3.8-3 Friedriech ataxia and other recessive ataxias M. Koenig IGBMC (CNRS-INSERM-ULP), Illkirch, Strasbourg, France Progressive recessive ataxias are severe disabling neurodegenera- tive diseases that affect the cerebellum and/or the spinocerebellar and sensory tracts of the spinal cord. Thirteen genes causing non-metabolic degenerative ataxias have been identified in the recent years. The majority of them encodes for either mitochond- rial or nuclear proteins. The nuclear proteins are ATM, MRE11, tyrosyl-DNA phosphodiesterase 1 (TDP1), aprataxin and sena- taxin, respectively. While the functions of ATM and MRE11 in DNA double strand break repair and of TDP1 immediately upstream of nucleotide excision repair are now well established, the precise function of aprataxin and senataxin remain elusive. The mitochondrial proteins are frataxin, ABC7, twinkle and DNA polymerase gamma (POLGA). Twinkle and POLGA are involved mitochondrial DNA maintenance and frataxin and ABC7 are involved in Fe-S cluster biogenesis. Complete loss of function of only ATM, aprataxin and senataxin results in reces- sive ataxia. Ataxia mutations in the other genes are hypomor- phic, as their complete loss function would not be viable. Friedreich ataxia is caused by intronic trinucleotide expansions which cause severely reduced expression of frataxin and subse- quent mitochondrial and cytosolic Fe-S protein deficiency. We have used conditional and inducible knock-out strategies to artifi- cially recreate the partial frataxin deficiency in mouse models that present with several features of the human disease. Abstracts 23 . searching for the structures of the recombination inter- mediates that lead to cell death. We are also investigating the role of the MMR system in the cytotoxicity. with the membrane of intra-endosomal vesicles contained within MVBs, thereby releasing the nucleocapsid into the lumen of these vesi- cles. Then, the capsid