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		Gliwickie Spotkania Naukowe 2006 Gliwice Scientific Meetings 2006 17-18 November NUCLEOTIDE RECEPTORS AND THEIR ROLE IN CELLULAR SIGNALLING IN GLIOMA C6 CELLS Jolanta Barańska Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warszawa, Poland Extracellular nucleotides (ATP, ADP, UTP, UDP, and ATP metabolite, adenosine) induce a variety of responses and regulate a variety of functions in many cells of different origin. They act through two large families of receptors: P1, sensitive to adenosine and P2, sensitive to ATP, ADP, UTP and UDP. The P2 receptors include the intrinsic ion channel P2X receptors and G protein-coupled P2Y receptors. Among P2Y receptors, P2Y2 responds to ATP and UTP, whereas P2Y1 and P2Y12, both respond to ADP. P2Y1 and P2Y2 receptors are coupled to PLC and are responsible for Ca2 release, while P2Y12 is negatively coupled to adenylate cyclase. P2Y1, P2Y2 and P2Y12 receptors are all expressed in rat glioma C6 cells. They are not only positively coupled to PLC and negatively to adenylate cyclase but are also able to modulate activity of ERK1/ERK2 and phosphatidylinositol 3-kinase (PI3-K). These effects depend on physiological conditions of the cell. When the cells are incubated in a medium devoid of serum, nucleotides cause transient activation of ERK1/2. In non-starved cells, ADP markedly decreases the PI3-K activity, whereas in serum starved it causes an increase of the enzyme activity. Blocking of the P2Y1 receptor by MRS2179 additionally increases this ADP response, suggesting that P2Y1 contributes to glioma signalling by negative regulation of P2Y12 action, whereas P2Y12 has an opposite effect. During long-term (up to 96h) serum starvation, the cells change fibroblast-like flat morphology to a rounded one. This process is not a differentiation toward astrocytes since glial fibrillary acidic protein (GFAP) expression, standard astrocyte marker, decreases during serum deprivation. Moreover, in the cells starved for 72 and 96h, the P2Y1 receptor is low or almost undetectable and under such conditions P2Y12 receptor is the main player, responsible for ADP-evoked signal transduction. The P2Y12 receptor activates ERK1/2 kinase phosphorylation, known cell proliferation regulator, and stimulates Akt activity. These effects were reduced by AR-C69931MX, specific antagonist of the P2Y12 receptor. The shift in expression of nucleotide receptors from P2Y1 to P2Y12 seems to be a new and important self-regulating nechanism acting not in favor of differentiation but promoting cell growth and adapting for survival under inhospitable conditions. PERSPECTIVES OF CANCER IMMUNOTHERAPY Marek Jakóbisiak Department of Immunology, Medical University of Warsaw, Banacha 1a, building 1F, 02-097 Warsaw, Poland Tumor immunotherapy is based on application of selected cytokines demonstrating antitumor activity, monoclonal antibodies directed against tumor antigens, cytotoxic T lymphocytes, and active immunization using tumor vaccines. As efficacy of most of these therapies remains unsatisfactory alternative therapeutic approaches are being developed. Among cytokines, only interleukin 2, interferon α, and tumor necrosis factor have found limited application in cancer patients. Monoclonal antibodies, including radioimmunoconjugates, are gaining a place in tumor therapy. T cytotoxic lymphocytes engineered to express chimeric T cell receptors are able to destroy tumor cells including those which have lost the expression of major histocompatibility complex molecules. Promising immunotherapeutic strategies are based on application of cancer vaccines consisting of gene-modified tumor cells. Tumor cells engineered to express major histocompatibility molecules, costimulatory proteins, and/or various cytokines induce effective immune response against tumor antigens. CANCER TRANSCRIPTOME: FACTORS DETERMINING GENE EXPRESSION PROFILES Barbara Jarząb Nuclear Medicine and Endocrine Oncology Department, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland The clinical applications of microarray-based analyses in oncology raise much interest among both biologists and clinicians and require many specific questions to be solved. There are some important issues which should be considered for realization of this goal, among them the question of quality assurance of microarray analysis, which includes validation of the biological quality of the supplied samples (percentage of cancer cells in tumor samples) as well as analytical points (quality and quantity of RNA available, quality control of microarray analyses) . For the validity of final conclusions, the problems related to the biological heterogeneity of tumors and other clinical and biological factors influencing gene expression need to be understood. Also, data mining and statistical analysis require special tools, designed for microarray-derived data. Their understanding by their end-users - biologists and clinicians -is still far from being perfect. These aspects have to be taken into consideration at evaluation of the results. In the presentation examples of analysis will be shown in laryngeal cancer, thyroid cancer, lung cancer, pancreatic cancer and breast cancer. HIJACKING CELL SURVIVAL PATHWAYS TO SELECTIVELY KILL CANCER CELLS Subbareddy Maddika, Tadeusz J. Kroczak, Srilekha Maddika, Emilia Wiechec, Anne Zuse, Nina Poric, Iran Rashedi, Mehdi Eshraghi, Soumya Panigrahi, and Marek Los. Dept. Biochemistry and Medical Genetics, Dept. Human Anatomy and Cell Science, Manitoba Institute of Cell Biology, CancerCare Manitoba, 675 McDermot Ave. Rm. ON6010,Winnipeg, MB R3E 0V9 We have recently identified a novel role for the PI3-K/Akt pathway during programmed cell death induced by apoptin, as well as selected anticancer drugs. We show for the first time that apoptin interacts with the p85 regulatory subunit, through the proline-rich region of apoptin and the SH3 domain of PI3-K, leading to the constitutive activation of PI3-K. Downstream of PI3-K, Akt is activated and translocated to the nucleus together with apoptin, most likely through a piggy-back mechanism. Nuclear Akt phosphorylates p27kip1 at the Thr-157 site, which in turn mediates p27kip1 downregulation by proteosome-dependent degradation. Cyclin A-associated CDK2 is activated aberrantly upon p27kip1 downregulation and activated CDK2 translocates to the cytoplasm. CDK2 directly phosphorylates Bcl2, thus mediating its degradation and further activation of mitochondrial death pathway. Apoptin-facilitated nuclear Akt, in contrast to its cytoplasmic pool, appears to be a positive regulator, rather than the repressor of apoptosis. This also holds true for apoptosis induced by anticancer drugs, such as methotrexate, taxol, doxorubicin and cisplatin. Our observations indicate that PI3-K/Akt pathways have a dual role in both survival and cell death processes depending on the stimulus. The implicated link between the survival and cell death pathways during apoptosis opens new pharmacologic opportunities to modulate apoptosis in cancer. STRUCTURE-FUNCTION RELATIONSHIPS IN DNA-FRAGMENTATION FACTOR Gregor Meiss Institute of Biochemistry Justus-Liebig-University Gießen Heinrich-Buff-Ring 58 35392 Gießen Germany Fragmentation of chromosomal DNA is a biochemical hallmark of apoptotic cell death. Among candidate proteins involved in this process, the DNA-fragmentation factor (DFF) plays a major role. It is a heterodimeric complex of the nuclease DFF40/CAD and its inhibitor and chaperone DFF45/ICAD-L. Using sequence- and structure-based mutational analyses in combination with biochemical and biophysical characterization of protein variants, we have identified functionally important regions in the nuclease subunit DFF40/CAD. Determining the catalytic centre and the DNA binding region allowed us to assign specific roles for particular amino acid residues involved in DNA binding and cleavage by this enzyme. Here, I present structure-function data relating to the regulation of DFF40/CAD at the level of protein folding, nuclear transport as well as DNA binding and cleavage. BIOACTIVE SMALL MOLECULES CAN MODULATE RADIATION-INDUCED BYSTANDER EFFECTS IN HUMAN CELLS AND TISSUE EXPLANTS Carmel Mothersill, Nalini Agnihotri and Colin Seymour Juravinski Cancer Centre, and the Dept of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada L8S 4K1 Bystander effects including apoptosis and chromosomal instability in unirradiated cell populations receiving signals from irradiated cells are now accepted. Neither the nature of the signals nor the pathways involved in signal propagation or transduction are fully understood. Previously we have shown that very low concentrations of the monoamine oxidase inhibitors (MAOI's), l-deprenyl and clorgyline can change bystander responses although probably not by inhibiting MAO. We tested the hypothesis that other small signaling or bioactive molecules such as serotonin, L-DOPA, glycine or nicotine can also modulate or simulate bystander signal production by irradiated cells. We now have data which suggest that nano to micromolar concentrations of these agents can change bystander induced cell death. Zofran and Kitryl, inhibitors of 5HT type 3 receptors, and reserpine, also blocked the bystander effect although it is not clear that the target for the bioactivity of the drugs tested is the cell membrane. Evidence from measurements apoptosis frequencies and of bcl 2 expression in surviving colonies, could suggest mitochondrial targets have a role to play. The results may be important in the search for novel drugs for optimizing radiotherapy treatment or in the prevention of second cancers following therapy. OXIDATIVE DNA DAMAGE; CAUSE OR CONSEQUENCE OF CANCER? Ryszard Olinski, Rafal Rozalski, Daniel Gackowski, Marek Foksinski, Jolanta Guz, Tomasz Dziaman, Anna Szpila, Agnieszka Siomek Department of Clinical Biochemistry, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun Reactive oxygen species (ROS) can cause oxidative damage to DNA resulting in potentially enhanced cancer risk. Commonly used biomarkers of oxidative DNA damage include measure of 8-oxo 7,8-dihydro-2'-deoxyguanosine (8-oxodGua) and its corresponding base, 8-oxoGua. These modifications can be determined either in cellular DNA or in urine where their excretion represents the average rate of oxidative DNA damage in the total body. Using recently developed methodology ((LC-GC-MS) we have found that urinary excretion of 8-oxoGua and 8-oxodG does not depend on diet in the case of humans and mice. Our study with cancer patients undergoing chemotherapy strongly suggest that cell death does not contribute to urinary excretion rate of the modifications. These data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair. In order to assess the role of oxidative DNA damage in cancer development we decided, for the first time, to analyse the broad spectrum of oxidative DNA damage biomarkers; urinary excretion of the base/nucleoside modification as well as the level of oxidative DNA damage and repair. In our recently published works have been found that the levels of oxidative DNA damage in leukocytes were significantly higher while the concentrations of the antioxidant vitamins were significantly lower in colon and lung cancer patients than in control group. Moreover, the same direction of the changes has been found in patients with adenoma. This, in turn, suggests that the changes in aforementioned biomarkers of oxidative stress are characteristic for cancer development. Although there is a little room for doubt on the basis of available experiments that oxidative DNA damage has some role to play in the pathogenesis of cancer, the quantitative relationship between the measured DNA damage and the rate of mutation and cancer is still lacking. Age is a single the most important factor which may contribute to cancer development. There is a possibility that a common link of these pathological conditions is oxidative damage to DNA. We have demonstrated that the level of 8-oxodG in leukocytes' DNA showed statistically significant correlation with the age of the examined subjects (n = 256), and the level of urinary 8-oxoGua and 8-oxodG followed the same pattern. On the basis of the presented correlative association between oxidative DNA damage parameters and age it seems reasonable to state that the damage may be one of the substantial factors in human ageing. IMMUNOTHERAPY OF NEUROBLASTOMA WITH PEPTIDE VACCINES BASED ON MOLECULAR MIMICRY OF GD2 GANGLIOSIDES Hanna Rokita Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Gronostajowa St., 7; 30-387 Kraków, Poland Neuroblastoma remains the third most frequent cancer of childhood. Despite of the application of intensive treatment regiments, the majority of high-risk patients are eventually relapsing. Therefore new therapeutic approaches are needed to eradicate residual tumour cells that might improve the survival of the high-risk group neuroblastoma patients. Immunotherapy of neuroblastoma is one of the currently developed strategies. They include the passive and active therapies targeting a neuroblastoma antigen - GD2 ganglioside. Gangliosides expressed on neuroectodermally derived tumours, including neuroblastoma and melanoma, remain weakly immunogenic in tumour-bearing organisms and induce predominantly immunoglobulin M antibody responses in the immunised host. However, the gangliosides GM2, GD2 and GD3 make the cells susceptible to immune attack by antibodies. Therefore, approaches are made to convert the GD2 ganglioside into a peptide mimetic to induce GD2 cross-reactive IgG antibody responses in mice. Peptide mimics of GD2 ganglioside constitute surrogate antigens of the glycolipid, able to elicit immunity to the self-antigen, although the original one may not. We are working on application of a peptide vaccine to GD2 ganglioside that is T-cell dependent, highly immunogenic, and specifically directs the antibody response to a protective epitope. First, we searched through LX-8/f88-4 phage displayed peptide library with the 14G2a monoclonal antibody specific to GD2 ganglioside. A number of phage clones binding to 14G2a was identified. Within those clones five families bearing distinct peptide sequences were found. The peptides were synthesised and used in further experiments. Thus, the immunogenicity of the peptides and the ability to elicit cross-reactive humoral response against GD2 were tested in vivo. Additionally, effector functions of the sera from the peptide immunised mice were investigated in the CDC assay on the IMR-32 cells. Concomitantly, sera of 16 patients who were diagnosed with neuroblastoma at the Jagiellonian University Children's Hospital, were collected and tested in ELISA for reactivity with GD2 ganglioside and selected peptides. The work was supported by the grants 3P05A 00124 and 2P05A 03429 from the Ministry of Education and Science. INTERCELLULAR COMMUNICATION IN X-IRRADIATED CELLS AND ITS INFLUENCE ON GENE EXPRESSION PROFILES Joanna Rzeszowska-Wolny M. Skłodowska-Curie Cancer Center and Institute of Oncology, Gliwice, Poland Cells exposed to ionising radiation (IR) transmit signals which induce DNA and chromosome damage, mutation, and apoptosis in non-irradiated cells, termed bystander effects, which are also induced by growth in medium from irradiated cells (irradiation conditioned medium, ICM). To examine possible changes in transcript profiles following transfer of cells into ICM we used oligonucleotide microarrays, sampling after 36 h to detect persistant effects which could be relevant to radiotherapy. The levels of <200 transcripts in Me45 (melanoma) changed >2-fold after ICM and IR, but using the criterion of a >1.1-fold change 6037 and 5616 showed increased or decreased levels, respectively after growth in ICM and the corresponding figures after IR were 6867 and 4785; more than half of those whose level changed were common to both ICM and IR. Grouping of transcripts into functional pathways (Kyoto Encyclopedia of Genes and Genomes) revealed significant differences (p<0.01) in the numbers which were up- or downregulated by both ICR and IR in certain groups, most markedly in the neuroactive ligand-receptor interactions (up), oxidative phosphorylation (down), and proteasome component (down) groups. These differences were confirmed by real time RT-PCR. Signaling factors in the medium of irradiated cells therefore cause reprogramming of transcript levels, revealing a new facet of the bystander effect. ZINC-DEPENDENT METALLOPROTEASES AND THEIR INHIBITORS AXIS IN BIOLOGY AND DIAGNOSTIC OF CANCERS Aleksander L. Sieron1, Aleksandra Auguściak-Duma1, Marta Lesiak1,Ksymena Urbanek1, Anna Szydło1, M. Kajor2 1Department of General and Molecular Biology and Genetics, Medical University of Silesia in Katowice; 2Department of Pathomorphology, Medical University of Silesia in Katowice Matrix metalloproteases are believed to play an important role in carcinogenesis via the degradation and remodelling of tumour surrounding extra cellular matrix, which could explain their association with survival. Although, many in vitro studies, animal models, and clinical research clearly showed involvement of MMPs in a number of critical steps during tumour growth and invasion, most synthetic MMP inhibitors, designed as anticancer agents, failed to improve patients outcome in clinical trials. The problems show that our understanding of the working mechanisms of MMPs in tumour biology is still poor. The most studied MMPs in tumourigenesis are MMP-2 and -9. A decade ago, for example it was reported that the levels of MMP-2 and MMP-9 in human gastric carcinoma tissues were enhanced and related to the survival of the patients. Since then the prognostic value of MMPs for carcinoma patients has been confirmed, however with variable success, in an ample of different studies and clinical trials testing the effect of MMP inhibitors for patients with various types of cancer. The major obstacle to develop valuable tests is that most of proteases is produced and secreted as inactive pro-enzymes, activated by proteolytic cleavage, and controlled in their activity by interaction with tissue specific inhibitors of MMPs (TIMPs). Disturbances in these processes are of eminent importance in tumour invasion and metastasis. Therefore, for diagnostic approaches it is critical to determine if the MMPs are at latent or activated state and what is the ratio of MMPs to TIMPs. Degradation of extra cellular matrix is always linked to its remodelling required synthesis of new matrix involving action of numerous molecules including zinc-dependent procollagen C- and N-endopeptidases. Their activity is essential for providing self assembling collagen monomers by proteolytic removal of globular propeptides flanking the triple-helix. Also, at least for procollagen C-endopeptidase, it has been shown that this enzyme has broad spectrum of substrates including, up regulated in tumours, laminin V gamma 2 chain, prolysyl oxidase, and related to angiogenesis perlecan. The role of procollagen converting enzymes in tumourigenesis is still poorly understood and their natural inhibitors yet have to be identified. In this presentation I will discuss the current knowledge on structure and function of metzincins, TIMPs, and other inhibitors, their expression in cancers and already available, as well as potential diagnostic clinical tests for detection of metzincins including MMPs/TIMPs status in cancer patients. WHAT IF NOT APOPTOSIS? Ewa Sikora Nencki Institute of Experimental Biology, Warsaw, Poland Induction of apoptotic cell death with anticancer drugs or irradiation is correlated with tumor response. In addition, failure in anticancer treatment is due to drug resistance in cancer cells, and the phenomenon of drug resistance is considered to be almost equal to resistance to apoptosis. However the strong correlation between induction of apoptosis and drug sensitivity is not necessarily correlated with overall tumor sensitivity. Even if apoptotic cell death is blocked, non-apoptotic cell death could be achieved, leading to cell dead. Anticancer drugs can induce other cell death mode, such as authophagy, mitotic catastrophe or proliferative cell death (cellular senescence), which have different characteristic from apoptosis. Moreover, the cell death mode strongly depends on cancer cell type and drug concentration. The main hallmark of apoptosis is activation of executor caspases followed by DNA fragmentation and apoptoti bodies formation. We showed that curcumin, a natural dye, induced caspase-3 activity and subsequent oligonucleosomal DNA fragmentation in human HL-60 cells, HL-60 cells with multidrug resistance phenotype (MDR), and resistant to undergo apoptosis upon etoposide treatment differentiated HL-60 cells. However, in human Jurkat cells curcumin used at the same concentration failed to cause DNA fragmentation despite the significant reduction of cell survival and activation of caspase-3 sufficient to cleave DFF45 factor which is the DFF40 endonuclease inhibitor. Inhibitory effect of curcumin on DFF40 activity resulted from curcumin binding to the active center of DFF endonuclease. In other resistant to apoptosis cells, namely human HCW-2 derived from HL-60 cells and Bcr/Abl-transfected mouse 32 progenitor cells, which are a model of chronic myeloid leukemia, curcumin strongly inhibited cell proliferation and affected cell viability, induced caspase-3 activation and DNA fragmentation. However, both caspase-3 activation and DNA fragmentation followed G2/M cell cycle arrest, together with increased mitotic index and cellular and molecular morphology resembling not apoptotic ones, but those described for mitotic catastrophe. Relatively low concentration of curcumin did not induce cell death in human colon cancer HCT116 cells. Instead, the symptoms of cellular senescence were observed in these cells. Upon curcumin- or doxorubicin-treatment HCT116 cells became giant/polyploid and expressed active ß-SAgalactosidase, a common marker of cellular senescence. Altogether, our results show that the same drug can induce many different cellular responses and apoptosis blockade can be overcome in cancer cells by inducing other types of cell demise. TOO MANY WAYS TO DIE: CELL DEATH-CELL SURVIVAL DILEMMA IN CANCER CHEMOTHERAPY Andrzej M. Skladanowski Laboratory of Molecular and Cellular Pharmacology, Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland Cell death by apoptosis induced by chemoterapeutic treatment attracted enormous attention during the last 15 years. It has been long believed that apoptosis is the major, if not the only, pathway leading to death of tumor cells as a result of antitumor therapy. However, recent studies provide cumulative evidence that other cell death types and pathways are activated by antitumor agents such as paraptosis, mitotic catastrophe, autophagy, and even programmed forms of necrosis. To add more confusion, some cell death pathways seem not to be mutually exclusive e.g. apoptosis and autophagy or mitotic catastrophe and apoptosis may partially overlap or integrate, providing in this way a variety of different cellular responses to drug action. The best described examples are insufficient caspase activation and ATP levels which may function as molecular switches between apoptotic and necrotic death. The molecular mechanisms of apoptosis have been studied for many years and are currently known in great details. In contrast, molecular and biochemical phenomena which are associated with other death pathways have only started to be characterized. Survival of tumor cells after drug treatment depends not only on the ability of these cells to activate different cell death programs but also on survival signaling mediated by e.g. PI3K/Akt/PKB, MAP kinases and inhibitors of apoptosis or IAPs (e.g. survivin). Both cell death and survival pathways can be defective or abnormally active in tumor cells which may lead to higher drug sensitivity in one case or drug resistance in the other. Modern therapeutic approaches are based on the modulation of these pathways by chemical inhibitors which could be combined with other antitumor agents to restore the ability of cells to die and improve drug efficacy. In this presentation, the different cell death and survival signaling pathways induced in tumor cells after treatment with antitumor agents will be reviewed and their role in the cytotoxic and antitumor action of chemotherapeutic agents will be discussed. In particular, the issue will be raised whether induction of a particular cell death pathway could be predictive for the final therapeutic effect of drug treatment. WHAT MATHEMATICAL MODELLING CAN HELP WITH IN ANALYSIS OF SIGNALLING PATHWAYS? Jarosław Śmieja Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland Following rapid developments in new experimental techniques, mathematical modeling of signaling pathways that control intracellular biological and chemical processes is gaining increasing interest in biomedical research. Though the models are unavoidably much simplified, they can significantly contribute to the biological field. Knowledge about dynamics of the processes involved in a given pathway facilitates better planning of experiments. Mathematical models can help to formulate or reject new hypotheses about unknown processes underlying results observed in experimental work. As a result, directions to be taken in experimental work may be suggested by mathematical models. Moreover, modeling can be used to analyze perturbed behavior even before experiments are undertaken, and answer the question if the desired effects are possible. Finally, analysis of dynamics can indicate time points, at which measurements should be taken to gain maximum information from experiments. This work presents how mathematical analysis influenced investigation of Interferon-beta stimulated signaling pathway. The model includes several feedback loops, and comprises both early and late responses of a cell to IFN-b treatment. The early response involves phosphorylation of STAT proteins, their subsequent dimerization and nuclear import of newly formed complexes. Once in the nucleus, those complexes act as transcription factors for early genes. Also in nucleus they undergo dephosphorylation followed by nuclear export of products of this process. Among the early genes is IRF1, and IRF1 protein is a member of complexes activating late gene expression. Here, experimental work yielded surprising results that could not be explained by already known mechanisms regulating the pathway. Two main questions arose from these results: 1) Why, despite unperturbed phosphorylation of STAT1, STAT1 homodimer levels decrease very rapidly, and 2) What causes cytoplasmic accumulation of IRF1 protein, known to be an active transcription factor for late genes activated in the investigated pathway. Careful analysis of the mathematical model made it possible to state hypotheses about molecular mechanisms that could be the basis for the observed behavior and was subsequently used to plan a series of experiments, currently in progress, that should expand our knowledge about interferon-induced pathways. This work is partially supported by BW-409-RAu1/2006. MODERN-RADIOONCOLOGY Rafał Tarnawski M. Skłodowska-Curie Cancer Center and Institute of Oncology, Gliwice, Poland Intensity modulated radiotherapy (IMRT) is an emerging concept of high-precision radiotherapy, a modality characterized by adaptation to patient and tumor characteristics. Modern radiotherapy has unprecedented technical capabilities to create conformal dose distributions allowing for avoidance of critical structures Precision of IMRT is based on precise immobilization, advanced imaging, computer optimized treatment planning. Functional imaging may potentially help in delineation of target volume, tumor staging, visualization of possible microscopic tumor infiltration, estimation of radioresistance (Biological Target Volume). Modification of radiosensitivity nowadays is based on common use of radio-chemotherapy, but many molecular targeted drugs are used in clinical trials. Better understanding of tumor biology may potentially introduce molecular biology techniques to combined treatment (radio-chemotherapy) as prognostic/predictive factors or response modifications. MODULATION OF OXIDATIVE DNA DAMAGE REPAIR BY OXIDATIVE STRESS AND NEOPLASTIC TRANSFORMATION Barbara Tudek1,2, T. Obtułowicz1, M. Swoboda1, J. Janik1, P. Kowalczyk1, J.M. Cieśla1, B. Janowska1, R. Oliński3, D. Laubitz4, R. Zabielski4 1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, 2Institute of Genetics and Biotechnology, Warsaw University, Poland, 3Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland, 4Department of Physiological Sciences, Warsaw Agricultural University, Warszawa, Poland Activity of DNA repair enzymes depends on many factors, such as gene polymorphisms, mRNA and protein level, as well as enzymes activation and inhibition. We report here on the modulation of repair activities of base excision repair enzymes eliminating from DNA oxidatively formed lesions by inflammation and neoplastic transformation. In newborn piglets, which on the third day after birth were supplemented with iron (75 or 200 mg in a single sc injection) increased repair activity for 8-oxoguanine (8-oxoG), and two lipid peroxidation-derived DNA damages, 1,N6-ethenoadenine (eA) and 3,N4-ethenocytosine (eC) was transiently observed. This was probably due to de novo enzyme synthesis, since 8-oxoG glycosylase (OGG1) quantity in the intestine epithelium increased on the 7th day after supplementation, and subsequently gradually decreased. Inflammation induced in newborn rats by i.p. injection of E. coli lipopolysacharide (LPS) increased the repair activity for 8-oxoG in their intestines and the effect was observed even two months after LPS administration. This coincided with the stimulation of mRNA synthesis of abasic sites endonuclease, APE1, which can activate OGG1 glycosylase, in vitro up to 400-fold. LPS induced also preneoplastic changes - aberrant crypts foci (ACF) in colons of rats. Inflammatory processes probably change also repair capacity in humans. Both repair activity for 8-oxoG and mRNA level of APE1, but not OGG1, was higher in blood leukocytes of colon cancer patients in comparison to healthy controls. Cancer tissues also differ from unaffected surrounding in repair capacity of oxidative DNA damage. Lung tumors reveal lower 8-oxoG repair activity than normal lung tissue, however this is neither correlated to OGG1 and APE1 mRNA level, nor OGG1 protein level, but may depend on interaction with other Base Excision Repair regulating proteins, like XRCC1, TP53 or tuberine or direct oxidation of repair proteins. Modulation of repair enzymes activities may be a cell response to oxidative stress on its way to neoplastic transformation. QUALITY CONTROL IN THE CELL NUCLEUS BY THE UBIQUITIN-PROTEASOME SYSTEM (UPS) Anna von Mikecz, Min Chen, Thomas Rockel, Andrea Scharf Institut für Umweltmedizinische Forschung (IUF) at Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany Compartmentalized proteolysis provides an efficient tool for controlling degradation of specific proteins by regulating their subcellular localization. Considering the central role of the ubiquitin-proteasome system (UPS) in cellular processes, detailed knowledge of the time and place of substrate ubiquitination and proteolysis proves to be essential to our understanding of the molecular mechanisms that regulate cell structure, function, development and disease. Consistent with this idea we and others have shown that the UPS is an active player in the cell nucleus. Accumulating evidence suggests involvement of the protein ubiquitination machinery in both, epigenetic regulation of gene expression and nuclear quality control. We analyse nucleoplasmic protein clusters that contain components of the UPS with respect to their (i) protein composition, (ii) ubiquitination capacity, and (iii) proteasomal activity in order to define functional versus pathological protein aggregates / clusters. A tight balance of ubiquitination and proteolysis within or near such nucleoplasmic clusters may decide whether the cell nucleus sustains gene expression or the development of diseases, in particular those of neurodegenerative and autoimmune nature. DOES ACTIVE DFF NEED REGULATION? (HOW MANY STEPS FOR REGULATION OF APOPTOTIC NUCLEASES?) Piotr Widłak M. Skłodowska-Curie Cancer Center and Institute of Oncology, Gliwice, Poland One of the biochemical hallmarks of programmed cell death is DNA breakdown. The major apoptotic nuclease, DNA fragmentation factor (DFF), also termed Caspase-activated DNase (CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45/ICAD) and a 40 kD latent nuclease subunit DFF40. Activation of caspase-3 results in DFF45 cleavage and release of active DFF40 that forms homo-oligomers. Binding of additional factors to such DFF40 homo-oligomers further regulates the nuclease activity. To date, histone H1, HMGB1/2 and TOPO2 have been reported to stimulate the nuclease activity, while nucleophosmin and CIIA have been identified as the nuclease inhibitors. It has been recently reported that DFF40/CAD is involved in maintaining genomic stability apparently playing a role of tumor suppressor (Yan et al, 2006, PNAS 103: 1504-9). This unexpected yet fascinating finding boosted efforts aimed to fully decipher mechanism involved in regulation of this nuclease. Currently, we have set up appropriate yeast two- and three-hybrid system in aim to identify novel partners of DFF40. In conclusion, DFF is regulated by multiple pre- and post-activation fail-safe steps, which include requirements during translation for DFF45 (and HSPs) to mediate appropriate folding to generate a potentially activatable nuclease, the synthesis in stoichiometric excess of the inhibitor, and the presence of additional activators/inhibitors that regulate the nuclease after removal of its inhibitor. COORDINATION OF APOPTOTIC DNA DEGRADATION AND CELL CORPSE ENGULFMENT IN C. ELEGANS Ding Xue MCDB, Univ. of Colorado, Boulder, CO 80309 Programmed cell death or apoptosis is a fundamental aspect of animal development and tissue homeostasis. Abnormal apoptosis may underlie many human diseases including cancers, autoimmune disorders and neurodegenerative disorders. We use the nematode Caenorhabditis elegans as a model system and a combination of genetic, biochemical, molecular biological, and functional genomic approaches to study how apoptosis is regulated, activated and executed. Using a functional genomic approach, we have identified nine cell-death related nucleases (CRN nucleases) that act in two independent pathways and in both dying cells and phagocytes to promote chromosome fragmentation during apoptosis and complete DNA degradation after dying cells are engulfed. Interestingly, defects in either DNA degradation pathway in the dying cell can compromise engulfment of apoptotic cell corpses in sensitized genetic backgrounds, suggesting that the apoptotic DNA degradation process and the cell corpse engulfment process are intrinsically connected. Further molecular genetic and biochemical analyses of these new cell-death nucleases and their interactions with other cell death factors in C. elegans will help elucidate how the apoptotic DNA degradation process and the cell corpse engulfment process are coordinated, regulated, and executed. ROLE OF MOLECULAR CHAPERONE IN CELL TRANSFORMATION Maciej Żylicz International Institute of Molecular and Cell Biology in Warsaw The p53 tumour suppressor gene encodes a sequence specific transcription factor, which is mutated in the vast majority of human cancer. One of the foremost characterized target genes of p53 is the mdm2 gene. MDM2 protein possesses E3 ubiquitin ligase activity towards p53. Through its ability to ubiquitinate p53 and target it for proteasomal degradation, MDM2 plays key role in retaining p53 at very low levels in non stress conditions. MDM2 oncoprotein also possesses numerous p53-independent activities, which contribute to the development of tumours where mdm2 is overexpressed, mostly by gene amplification. It should be stressed that not all MDM2 client proteins are targeted by it for proteasome degradation; hence not all involvements of MDM2 can be explained by its E3 ligase activity. Recently we have shown that Hsp90, in an ATP-dependent reaction, retains wt p53 in the conformation, which allows binding to the specific promoter sequence (Walerych et al., 2004). Hsp90 binds also to the mutant p53, but this interaction is indirect. With the use of highly purified proteins, we identified intermediate reactions that lead to the assembly of the multi chaperone complex (mutp53-Hsp40-Hsc70-Hop-Hsp90) (King et al., 2001). Given that MDM2 protein can bind ATP, interact with the HSP90 chaperone, and play a role in nascent p53 protein biosynthesis, we have evaluated and have found that MDM2 protein possesses an intrinsic molecular chaperone activity. MDM2 can function like the Hsp90 chaperone in the protection of citrate synthase and firefly luciferase from aggregation. MDM2 can also promote ATP-dependent in vitro p53 DNA-binding activity to the p21 derived promoter sequence. Although the E3-ligase inactive MDM2 mutant still maintains the chaperone-like activity, the ATP-binding mutant MDM2 protein (K454A) lacks the chaperone activity. The mdm2 cotransfected with wild-type p53 stimulates efficient p53 protein folding in vivo and this effect is abrogated when using the ATP-binding defective form of MDM2. This is the first demonstration that MDM2 possesses an intrinsic molecular chaperone activity and indicates that the ATP-binding function of MDM2 can mediate its chaperone function towards p53 (Wawrzynow et al., 2006).