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		LECTURES ROLE OF CRABP I AND II IN REGULATING SENSITIVITY OF TUMOR CELLS TO RETINOIDS AND IONIZING RADIATION Blaese Marcel A., Santo-Hoeltje Lan, Rodemann H. Peter Section of Radiobiology and Molecular Environmental Research, Clinic of Radiooncology, Eberhard-Karls University, 72076 Tuebingen, Germany Aim: Retinoids like all-trans retinoic acid (ATRA) and 13-cis retinoic acid (13c-RA) inhibit proliferation, induce differentiation and enhance radiosensitivity of many tumor cells in vivo and in vitro. The effects of retinoids are mediated by two cytoplasmic retinoic acid binding proteins (CRABPs) as well as nuclear retinoic acid receptors RARs and RXRs, which regulate the expression of several target genes.Because of its minor side-effects retinoids could be an alternative to standard tumor therapy protocols for patients with high risk of normal tissue reaction. Therefore it is necessary to identify prognostic factor(s) which enable us to predict a possible successful retinoid/irradiation therapy. Methods: To elucidate the relation of RA- plusmn; IR-treatment and clonogenic inactivation we measured the basic and modulated mRNA and protein level of all RA-receptors as well as cytoplasmic RA-binding proteins (CRABP I, II) by RT-PCR and Western blotting and the clonogenic survival by using colony formation assay on 7 tumor cells and two normal human fibroblasts. To prove whether CRABPI is involved in determination retinoid sensitivity and radiosensitization in tumor cells we transfected the high RA-sensitive cervix carcinoma cell line HTB35 with CRABP I by using the regulable Tet-on over-expression vector system. Results: No correlation could be observed between clonogenic inactivation and basic or modulated RA-receptor expression in all cell lines used. Furthermore, also the RA-sensitive cell lines showed no induction of tumor-suppressorgene RARb2 as it was described in the literature. In contrast we could demonstrate a strong correlation between clonogenic survival and the basic expression of CRABP I (RNA and protein) under RA- plusmn; IR-treatment. Moreover, retinoid sensitive HTB35 cells could be switched to retinoid insensitive cells by transfection with CRABPI gene. These cells where insensitive against both retinoid treatment alone or in combination with irradiation. Conclusion: The response of tumor cells to RA- plusmn; IR-treatment seems to be directly dependent on basic CRABP I level. Therefore, CRABP I could be a possible important prognostic factor in alternative tumor therapy for clinical use especially in patients with high risk of normal tissue reaction. INDIVIDUAL RADIOSENSITIVITY: MOLECULAR MECHANISM AND CLINICAL IMPACT Brammer Ingo, Borgmann Kerstin, Kasten Ulla, Dikomey Ekkehard Institute of Biophysics and Radiobiology, University Hospital Hamburg-Eppendorf, Germany The individual radiosensitivity as measured with lymphocytes or fibroblasts show a broad variation. This variation might play a role in the individual risk of normal tissue complications after radiotherapy. The reason for the variation in the individual radiosensitivity are only partly understood. For fibroblasts, there is a clear correlation between the cellular sensitivity as measured by colony assay and the repair capacity for DNA double-strand breaks. The number of non-repaired double strand breaks is directly correlated with the number of lethal chromosomal aberrations. The differences in repair appear to be rather small; the repair capacity varied only between 95 and 98 %. The differences appear not to be regulated by different basic levels in the expression or the activity of DNA-repair genes as studied for the key proteins of the DNA-PK complex. There are also no differences in the localisation of these proteins or the induction by ionising irradiation. The repair capacity is also not affected by differences in the differentiation status of the fibroblast strains as determined by the fraction of terminally differentiated fibrocytes. The repair capacity, however, was found to be related to the extent of the acute radiation-induced permanent G1-arrest, which makes up a considerable part of the inactivation of fibroblasts. The data indicate that both processes, double-strand break repair and G1-arrest, are influenced by a common upstream signal protein, probably p53. This is presently under investigation. PREDICTIVE ASSAY COMPARISON BETWEEN VARIOUS BIOLOGICAL END-POINTS AND VARIOUS DOSES Cebulska-Wasilewska Antonina 1, 2 1 Department of Radiation and Environmental Biology, The H. Niewodniczański Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Kraków 2 Department of Epidemiology, CM UJ, Kraków, Poland The challenge for molecular epidemiology and environmental studies is to improve the process for assessing risk to human health from exposure to genotoxic agents. Human biomonitoring, as a tool to identify and potentially quantify the risk of environmental exposures, has gained increasing interest especially in the area of cancer risk assessment and diseases treatment. There are several major reasons for which biomarkers may be used in epidemiological research: to improve the assessment of health risk associated to exposure; to identify subgroups of persons of different susceptibility altering the effects of the exposure or treatments; to measure early health outcome with some predictive significance; and to differentiate disease subtypes with potentially different etiologies and strategies of treatment. In order to define genotoxic effectiveness of any agent or action and to realize genetic or carcinogenic hazards, it is very important to search for correlation between evidence of the induced genotoxic damage in exposed living organisms or exposed humans with accurate measures of treatments or exposures.The desirable features of biological markers of genotoxic action are that biomarker measure should indicate a quantitative reaction to the action and should be associated with a health risk related to the genotoxic outcome. That is why, characterization of the dose response process is often done by the use of biomarkers detected in assays predictive of potential toxic outcomes and pathological changes. This creates necessity to extrapolate from high doses (experimental, occupational or accidental) to low dose region. There may be many confounding factors affecting the simple extrapolation. On the other side, an individual's genetic constitution and lifestyle, e.g., diet and levels of physical activity, can also affect the body's response to exogenous agents. In this paper are discussed influence of the shape of dose/exposure effect relationship, polymorphism and competence of DNA damage repair on the decency of the searched association between a genotoxic outcome and health risk. Chromosome aberrations as structural or numerical chromosome changes, resulting from direct DNA breakage or from inhibition of DNA repair or synthesis, measured in peripheral blood lymphocytes have been used in occupational health surveillance programs in order to assess genotoxic risks. The concept for this biomarker assumes that the extent of genetic damage in peripheral lymphocytes reflects similar events in the precursor cells for carcinogenic processes. Many results of cytogenetic damage detected in our human monitoring studies have shown both an association with adverse health outcome on one side and the influence of confounding factors related to the life style on the other. The full potential of the molecular techniques, that have had a dramatic effect on the many insights in the clinical and research applications, lies in deriving quantitative outcomes of cytogenetic investigations. In our studies, for the purpose of retrospective biological dosimetry, in the first cellular division are applied both, unstable chromosome aberration frequencies (dicentrics, rings, fragments) and translocations (analyzed with FISH techinque), and in the second cellular division are studied sister chromatid exchanges and high frequency cells. Susceptibility to the environmental agent actions is evaluated in studies on the variation between responses to the challenging dose of UV or X-rays followed by the evaluation of the repair capacity of the DNA damage induced by a challenging dose. The induced and residual DNA damage is analyzed with the use of SCGE assay (also known as a Comet) assay. Susceptibility and repair capacities of healthy donors and cancer patients are also compared. Studies have shown a good correlation between various measures of the induced in vivo or in vitro DNA and cytogenetic damage levels. In the paper are also discussed results from studies on susceptibilities and effectiveness of the induced damage repair performed in groups of occupationally exposed and unexposed healthy donors (475 samples investigated) on one hand, and patients with diagnosed cancer on the other. The possible effects of occupational exposures, and influence of the diet and other confounding factors is shown. Prospective use of a challenging dose of radiation combined with the comet assay as a predictive assay is suggested and limitation discussed. Acknowledgments: Research was partially supported by grant: EC EXPAH QLK4-CT-2000-00091. GENETICALLY MODIFIED ORGANISMS - INTRODUCTION Chorąży Mieczysław Department of Tumor Biology, Center of Oncology - Maria Skłodowska-Curie Memorial Institute, Branch in Gliwice Modifying the genotype by introduction of a particular gene or gene sets into the genome of several animal and plant species opened new research possibilities in basic biological research, biomedicine, microbiology, agriculture, forestry and animal breeding. For dozens of years transgenic bacteria have been widely used for making bioproducts for use in medicine, agriculture, households, animal breeding, etc. Genetically modified organisms (GMOs) have caused a worldwide debate about benefits and risks, especially with reference to plants. Large areas have been planted with genetically modified (GM) soybeans, corn, wheat, barley, rice, canola and cotton. Proponents of GM plants claim that bioengineering of plants is the only way to feed hungry people from the "Third world" and make life in less developed countries easier. GM plants are made resistant to disease and herbicides, they can resist extreme weather conditions, can grow on a high salt-containing soil, etc. Other GM plants can thrive on heavy metal-rich soil (copper, cadmium, mercury, etc.) owing to genes coding for phytochelatins - peptides that immobilize metals in nonsoluble complexes deposited in plant cells. More evident benefits include: GM plants synthetizing higher level of proteins, vitamins (A, E), monounsaturated fatty acids, fruits and vegetables suitable for long-term storage, fast growing trees with modified lignin to yield better paper, orange trees that initiate flowering and fruit production in the first year of growth, etc. The humanitarian and charitable perspectives seem a bit cynical and are shadowed anyway by intense and fruitful research, animated and supported by big corporations. These aim at developing miracle GM plants with a "technology protection system", as a way to protect their investments and future interests. These efforts resulted in development of "terminator technology" that make feasible at will a GM plant bearing sterile seeds, useless for the successive sowing. GM plants can contaminate with pollen wild species, thus posing a real danger of eliminating wild type species. Field trials with GM plants bearing Bt transgene harboring toxin and therefore resistant to insects appear to render soil sterile and less valuable for agriculture. Besides, several species of insects and larvae feed on Bt plants became resistant to Bt toxin. Use of herbicide Rundup by farmers cultivating GM beets resistant to this herbicide caused after a few years deep disturbance in the fields: some insects living on weeds disappeared and together with them birds which fed on weed's seeds. There is more convincing data indicating that genetically engineered plants pose a real risk for ecology. There is a risk for invasiveness of GM plants into native plant ecosystems, the effect upon soil micro-ecosystems, and fauna. There is a risk of developing weeds tolerant to herbicides by transfer of herbicide-tolerant traits from GM plants. Eating GM food constituting risk for human health cannot be excluded: allergic response to new types of protein (e.g. cryo protein in frost-resistant GM maize), transfer of DNA fragments stemming from virus vectors used for gene transfer into plant cells. No major objections have been imposed on experiments aiming at GM plants' use for manufacture of pharmaceuticals. The mighty mouse produced by transfer of rat growth hormone gene opened the possibility of applying this technology to growing livestock faster and on a lesser food supply. But it appeared soon those animals (pigs, cows, sheep) breeded with this technology develop several pathologies: nephritis, pneumonia, gastrointestinal disorders, ulcers, synovitis, etc. Obviously, no single growth factor (such as the growth hormone) is sufficient to the growth of animals. Breeding of "Belgian blue" monster cows by crossing animals lacking (natural knock-out) myostatin gene prompted studies on mice with experimentally knocked-out myostain gene. The encouraging results obtained with mouse create temptation to do the same with cow. Will it work, or again, some unexpected pathology would arise? Using GM animals as bioreactors for producing biofarmaceuticals (blood clotting factors, peptide hormones, enzymes immunoglobulins, vaccines, etc.) in general does not cause serious debates. Ethically acceptable are also trials to modify histocompatibility systems and tissue antigens in animals, could-be organ donors. No obstacles are posed for using transgenic laboratory animals in basic research. Knock-out and transgenic mice contributed priceless information to elucidate mechanisms of various pathways involved in induction and pathogenesis of a great number of diseases, including cancer. Yet, in my opinion, dreams and experiments leading to transgenic goats or cows that will produce silk in their milk (owing to introduction of spider silk gene into their genome) are not acceptable. Similarly, any interest in producing more virulent species of viruses and bacteria, carrying e.g. toxins, and aimed to be used as biological weapons, is not acceptable. DISCOVERY OF RNAi PHENOMENON - TURNING-POINT IN FUNCTIONAL GENOMICS Figlerowicz Marek, Alejska Magdalena, Malinowska Nelli Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland e-mail: marekf@ibch.poznan.pl The sequencing of several complete genomes and the elaboration of a DNA microarray technology are the most important achievements, which have created proper ground for the development of modern functional genomics. The latter can be defined as the studies of how the entire genome works and how the expression of the individual gene influences the activity of other genes. However, there is one additional condition which needs to be satisfied if one wishes to study how the genome is functioning: an effective and simple method of selective inducing and silencing of the expression of each individual gene must be available. Unfortunately, for a long time such a technique was not at our disposal. The recent discovery of RNA interference (RNAi) totally changed this situation, providing us with a new powerful tool for genome studies. RNAi is a post-transcriptional process of gene silencing induced by double-stranded RNA (dsRNA) homologous to the dsDNA fragment within which the silenced gene is encoded. The foundations for the discovery of RNAi phenomenon were laid by scientists working on gene co-suppression and antisense RNA. In the first case it was observed that the introduction of an additional gene copy into the plant genome often does not lead to an increase of its expression, but on the contrary, to its inhibition. Equally puzzling results were obtained while conducting antisense RNA research. It was observed that gene expression can be inhibited by introducing into the cell an oligoribonucleotide complementary to the mRNA fragment. It was believed that the oligoribonucleotide binds to mRNA or directly to a gene and thus prevents translation or transcription processes. Contrary to scientists' expectations, the same result was brought about when RNA with a sequence identical to the mRNA fragment was applied. Interestingly, the phenomenon of gene silencing was intensified when both molecules, i.e. the complementary one and that identical to a mRNA sequence were used simultaneously as dsRNA. The first attempts to explain the above phenomena appeared towards the end of the 1990s. Research on plant gene co-suppression and antisense RNA showed that in both cases we are dealing with the same phenomenon of RNAi. It was determined that the transcription of silenced genes takes place uninterruptedly. However, the selected transcription products are identified in some way and degraded. It was also shown that, induced in one cell, RNAi can spread over the whole organism. The discovery of RNAi provides completely new possibilities for the research on the gene functioning, without the necessity of altering the genome structure. Thus, at any moment of organism development one can inhibit the expression of a specific gene (or several genes) even if it is important for the life of the organism under study. Literature: 1. G.J. Hannon, RNA interference. Nature 2002, 418, 244-251 2. RNA silencing and noncoding RNA- series of articles, Science 2002, 296, 1259-1273 DECIPHERING MENIN'S INTERACTION NETWORKS AS A TOOL TO TACKLE ITS BIOLOGICAL ROLE Gaudray Patrick CNRS - UNSA UMR 6549, Instabilite et Alterations des Genomes, Faculte de Medecine, Nice, France Multiple Endocrine Neoplasia type 1 (MEN1, OMIM 131100) is a cancer predisposition syndrome inherited as a dominant trait. It affects a variety of endocrine tissues, in particular parathyroids, endocrine pancreas, anterior pituitary, foregut-derived neuroendocrine tissues and adrenal cortex. Other tissues are affected in MEN1 patients, although less frequently: cutaneous proliferations such as angiofibroma, collagenoma, lipoma or melanoma, and peripheral or central nervous system. The MEN1 gene, which is localized onto chromosome 11q13, was identified in 1997. It consists of 10 exons, spanning 9 kb of genomic sequence, and encoding a protein of 610 aminoacids (Menin). Menin does not reveal homologies to any other known proteins. The only motifs which have been recognized in the Menin sequence are two leucine zippers, and two nuclear localization sequences (NLS) in the carboxyterminal part of the protein. Although a mouse knock-out model is available, the function of Menin is still elusive. Beside what can be learnt from animal models, Menin's function can be anticipated neither from its protein sequence nor from its mutation profile in MEN1 patients. Whatever its function could be, Menin is supposed to play a role in defined regulation pathways leading to the control of cell growth (MEN1 is primarily an hyperplastic syndrome) and/or the maintenance of genomic integrity. It is thus expected that deciphering the interaction network(s) in which Menin is implicated will enable us to tackle its biological role. Proteins of known function have been shown to interact with Menin : JunD, NF-KappaB, Smad3, Pem, Nm23H1, GFAP, vimentin, and probably P53. Their partnership with Menin may correspond to a regulation of their activity, but their relevance to the various traits of MEN1 pathogenicity is not established. Consistent with the fact that MEN1 represents a complex syndrome with -at the same time- a strict tissue specificity and a relatively broad spectrum of both associated tumors and phenotype expressivity, the presently known protein partners of Menin seem to drive it through various cellular compartments to act in different regulation pathways. In this respect, they have provided informations that will turn out to be essential in the understanding of the pathogenicity of MEN1. GENETIC EFFICIENCY OF LOW-DOSE ionizing RADIATION IN small MAMMALS under chronic irradiation Goncharova R, Rabokon N., Smolich I. Institute of Genetics and Cytology of National Academy of Sciences of Belarus Earlier we have established the genetic effects of low dose chronic irradiation in bank vole (somatic and germ cells, embryos), in pond carp (fertilized eggs, embryos, fry) and in laboratory mice (somatic and germ cells) in the range of doses from near-background to 10 cGy. These low dose effects observed in mammals and fish are not expected from extrapolation of high dose experiments. For understanding the reasons of this discrepancy the comparative analysis of genetic efficiency of low doses chronic irradiation and higher doses acute irradiation was carried out in natural populations of bank vole which inhabited two sites differing in ground radionuclide deposition. For comparing efficiency the linear regression model of dose-effect curve was used. Dose-effect equations were obtained for animals from two chronically irradiated bank vole populations. The mean population absorbed doses were in the range of 0.04-0.68 cGy, the main part of the absorbed doses consisted of external radiation by 137Cs ?-rays. Dose-effect equations for acute irradiation to 137Cs ?-rays (10-100 cGy) were determined for the same populations. Comparison of genetic efficiency was made by extrapolation, sing the regression coefficient ß and doubling dose estimation. For chronic exposure doubling doses calculated from low-dose experiments are 0.1-2 cGy and the doubling doses determined from high-dose experiments are in the range of 5-20 cGy. Our hypothesis that the doubling dose estimate is calculated in higher-dose ionizing radiation experiments should be much higher than the deduced from the low dose line regression equation was verified. The doubling dose estimates for somatic cells of bank vole and those for germ cells of laboratory mice are in close agreement. Radiosensitivity of bank vole chromosomes was shown to be practically the same as that for human lymphocytes since doubling dose estimates for acute irradiation close to each other. For low LET radiation higher genetic efficiency of chronic low doses in comparison with the higher doses of acute gamma-irradiation (137Cs source) was proved by three methods. EXPRESSION PROFILING BY DNA MICROARRAY IN ENDOCRINE - RELATED CANCER Jarząb Barbara Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Institute, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland Application of powerful, high - through - put genomics technologies creates new possibilities of cancer classification and therapy prediction. Oligonucleotide based DNA microarrays and cDNA microarrays constitute two possibilities of investigating tumor expression profiles. In the paper, the capabilities of high density oligonucleotide microarrays will be analyzed for expression profiling in endocrine - related cancer with the focus on breast cancer as an example of hormone - dependent cancer and thyroid cancer as an example of hormone - producing and - dependent cancer. At present, high density oligonucleotide microarrays, synthetised in situ by photolithography and supplied by AffymetrixR, enable the analysis of expression of 22 000 human genes on one chip and are based on short, 25 mer - oligos, with 11 pairs used per gene analyzed. A similar number of genes (25 000) has been reported to be used in breast cancer expression profiling on AgilentR - produced chips, where 60 - mer oligos synthetised by ink - jet like technology were applied. The ability to measure the expression level of thousands of genes in one tumor sample allows its characterization and classification. The analysis of tumor expression profiles may be performed in a sample chosen by macroscopic criteria, be supported by microscopic selection of frozen sections or by laser - directed microdissection. This last approach supplies the best material for investigation, requires, however, additional amplification of RNA and is difficult for clinical applications. An important issue is then, whether the expression profile of stromal and infiltrating cells will be essential for the prognostic and predictive purposes in examined tumors. In breast cancer, the clinical predictors fail to clarify accurately breast tumors according to their metastatic potential, while a supervised classification of oligonucleotide microarrays presented recently by van't Veer (2002) and colleagues enabled to identify a metastatic signature in low risk breast cancer patients. The 'prognosis classifier' used by them consisted of 70 genes and was obtained by analysis of correlation of the expression of circa 5000 differently expressed genes with the 5 years outcome. Most of the genes included were known to participate in cell cycle regulation, invasion, metastasis and angiogenesis. However, genes expressed in non-cancer cells present in tumor contributed also to the prognosis classifier and the expression pattern of the whole set of genes, not their particular role, was crucial for the prediction of outcome. Papillary thyroid cancer constitutes another example of endocrine - related cancer, where the prediction of the outcome based on molecular features is expected to support clinical decisions, as only 15% of those cancers exhibit poor prognosis and require radical therapeutic measures, which are today supplied to a much wider group of patients. Recent analysis of Huang et al. (2002) revealed highly consistent expression profile in papillary thyroid carcinoma, despite its clinical heterogeneity. Our own experiences are still scarce. We observed also a distinct decrease of expression of thyroid specific genes, with the highest fall in TPO expression level. However, the pattern of genes with increased expression did not parallel the changes reported by Huang et al. Our attention was focused on SSX genes, which belong to cancer testis genes. In physiologic conditions their expression is limited to testis and normal thyroid and has not been described in thyroid ca, while a clear overexpression was noted in both tumors analyzed by us until now. Microarray-based approaches for classification of endocrine - related cancer require further intensive basic and translational research to obtain new diagnostic and predictive criteria, useful for clinical purposes. TRANSPOSABLE ELEMENTS: HORIZONTAL TRANSFER AND MECHANISMS OF INTEGRATION Jurka Jerzy Genetic Information Research Institute 2081 Landings Drive, Mountain View, CA 94024, USA Transposable elements (TEs) are specialized DNA or RNA fragments capable of surviving in intragenomic niches. They are commonly and perhaps unjustifiably referred to as "selfish" or "parasitic" elements. TEs can be divided into two major classes: retroelements and DNA transposons. The former include non-LTR retrotransposons and retrovirus-like elements, using reverse transriptase for their reproduction prior to integration into host DNA. The latter depend mostly on host DNA replication, with the possible exception of rolling-circle transposons recently discovered by our team. TEs can be transferred horizontally between different species. This applies primarily to cut-and-paste DNA transposons and LTR-retroelements. Most evidence for horizontal transfer of TEs is based on phylogenetic analysis, but more direct evidence also exists. To be transferred between different species, TEs need to overcome several barriers and become successfully integrated into host DNA. The barriers include: immune systems, cell membranes, elimination by recombination, and intracellular silencing by DNA methylation and RNA degradation. TEs are active mostly in germline cells. Based on our recent work, I will show how transposition in paternal or maternal germlines can lead to different distribution of TEs for different chromosomes. This may be of significance for evolution of eukaryotic systems. Far from being selfish parasites, TEs should perhaps be viewed as relatively harmless elements continuously probing intragenomic responses to foreign DNA/RNA and contributing to eukaryotic evolution. MECHANISMS OF ACTION AS A BASIS FOR THE DEVELOPMENT OF NEW ANTITUMOR COMPOUNDS Konopa Jerzy Gdańsk University of Technology, Department of Pharmaceutical Technology and Biochemistry, Gdańsk Two examples will be presented to illustrate in what way the studies on the mechanism of action of antitumor drugs and compounds may augment the development of new antitumor drugs. Our earlier studies on the mode of action of mitoxantrone have demonstrated that after metabolic activation this drug induces covalent interstrand crosslinks in DNA of tumor cells. In the formation of crosslinking bonds, diaminoalkylo groups present in the side chains of mitoxantrone are responsible, while anthraquinone chromophore probably facilitates the initial docking of the drug molecule within DNA. The above observations made us put forward a hypothesis that the linkage of diaminoalkylo moiety to another polycyclic system capable of DNA intercalation might result in obtaining new antitumor compounds. This hypothesis was verified by the development of several new groups of antitumor compounds, i.e. imidazoacridinones, triazoloacridinones and 4-methyl-1-aminoacridinones. The derivatives of imidazoacridinone have been most extensively studied. These compounds display antitumour activity towards a number of experimental tumours, especially towards colon carcinomas, including human colon tumour xenografts in nude mice. Imidazoacridinones intercalate into DNA, induce covalent interstrand DNA crosslinking after metabolic activation, and are topoisomerase II inhibitors. The most active imidazoacridinone derivative, C-1311, is currently in the process of preparation for the Ist phase of clinical studies. In the 60ties/70ties, in our Department, a group of 1-nitro-9-aminoacridines was developed exhibiting high cytotoxic and antitumor activity towards some experimental tumours. One of these derivatives was registered in Poland as an antitumour drug under the name of Ledakrin (Nitracrine). The broader interest in 1-nitroacridines was discouraged by their considerable toxicity. The studies on the mechanism of action of Ledakrin and analogues showed that, after metabolic activation, these derivatives induce covalent DNA crosslinks and that the nitro group in position 1 of acridine played the major role in this mechanism of action as well as antitumor activity. Modulation of the properties of 1-nitro group, especially its susceptibility to reduction, by introducing appropriate substituents into acridine ring system, resulted in the development of new derivatives with decreased toxicity, but still displaying strong activity towards colon and prostate tumours, including human tumour xenografts in nude mice. One of these derivatives, C-1748, is in the process of preparation for the Ist phase of clinical studies. TRANSLATIONAL REGULATION OF THE BRCA1 GENE Krzyżosiak Włodzimierz Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznań, Poland With the rapid progress of research on transcripts of eukaryotic genes and the entire transcriptomes it has become clear that mRNAs are regulated at many levels by a variety of trans-acting factors and cis-elements hidden in their sequences and structures. A good example is the BRCA1 mRNA, which is involved in sporadic breast and ovarian cancer mainly through reduced expression. Two BRCA1 mRNAs containing different leader sequences show different patterns of expression. In a normal mammary gland mRNA with a shorter leader sequence, 5'-UTRa is expressed only, whereas in breast cancer tissue mRNA with a longer leader, 5'-UTRb is expressed also. We show that translation efficiency of transcripts containing 5'-UTRb is 10 times lower than those containing 5'-UTRa. The structures of 5'-UTRa and 5'-UTRb were determined by chemical and enzymatic probing aided by a new method developed for monitoring the number of co-existing stable conformers. Specificfactors responsible for reduced translation of mRNA containing 5'-UTRb were determined using a variety of transcripts with mutations in the leader sequence. These factors include a stable secondary structure formed by truncated Alu element and upstream AUG codons. The novel mechanism by which BRCA1 may be involved in sporadic breast and ovarian cancer is proposed. It is based on the expression patterns of BRCA1 mRNAs and differences in their translatability. According to this mechanism the deregulation of BRCA1 transcription in cancer, resulting in a higher proportion of translationally inhibited transcripts containing 5'-UTRb, contributes to the decrease in the BRCA1 protein observed in sporadic breast and ovarian cancer. POSITIONAL CLONING OF TUMOR SUPPRESSOR GENES IN PARATHYROID TUMORIGENESIS Larsson Catharina Pharmacology Biovitrum, Stockholm Abstract not received TRANSGENIC PLANTS OVEREXPRESSING BIOLOGICALLY ACTIVE COMPOUNDS Łukaszewicz Marcin, Szopa Jan Institute of Microbiology, Institute of Biochemistry and Molecular Biology, Wrocław University, Przybyszewskiego 63-77, 51-148 Wrocław, Poland Flavonoids are a large group (about 7 000) of secondary metabolites ubiquitous in all vascular plants. New compounds are being identified in nature and chemically synthesized. The core structure of flavonoids (C6-C3-C6) is two aromatic rings (A and B) joined by 3 carbon bridge, which most often forms the third C-ring. Flavonoids are classified into various groups according to the modification (saturation, hydroxylation) of the C ring. The main structure (aglycone) very often carries one or more sugar residues. The glycosides are stored mainly in vacuoles. The multitude of structures implicates variable functions. In plants, flavonoid compounds are supposed to play an important role in protection against UV irradiation, and osmotic, oxidative or heat shock stresses. As pigments determining the colour of flowers and fruits flavonoids may be either attractants or repellents. They are also active in the plant-micro organism interactions (promotion of symbiosis and protection against pathogens). Biological activity of flavonoids is not limited to plants. As ingredients of animal (human) diet, flavonoids have been shown to have great impact on human health. They show antimicrobial, antiviral, antiphlogistic, antioxidant, antisclerosis, analgesic and anticancer activity. Positive impact on cardiovascular, digestive and respiratory systems has been also well documented. Thus, flavonoids are used as pharmaceuticals in the form of purified compounds or as components of plant tissue mixtures. There is a growing interest in the impact of flavonoid doses and their quality present in the food on human health, as well as in the possibility of modification of the food composition to promote human health. In this context the flavonoids biosynthesis pathway in potato and flax plants have been modified. Constructs coding regulatory gene (14-3-3 protein) and structural genes of flavonoids biosynthesis pathways, i.e. chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavanone reductase (DFR); have been introduced in sense and antisense orientation under strong, constitutive promoter from Cauliflower Mosaic Virus (CaMV35S). CHS, CHI and DFR genes have been introduced either separately, or two or all the three genes simultaneously. For this purpose, a special construct enabling introduction of up to five genes has been prepared. Plants with overexpression and repression of flavonoids have been obtained. Plants with higher amounts of flavonoids show higher resistance to pathogens attack. Potato plants with overexpression of DFR have been shown to produce mainly one anthocyanin compound. DNA REPAIR CAPACITY IN LYMPHOBLASTS FROM SISTERS DISCORDANT FOR BREAST CANCER Motykiewicz Grazyna, Faraglia Beatrice, Wang Lillian, Terry Mary Beth, Senie Ruby T., Santella Regina M. Departments of Environmental Health Sciences and Epidemiology, Mailman School of Public Health, Columbia University, 701 West 168th St., New York, NY 10032 The mutagen sensitivity assay is one of the approaches used to investigate individual DNA repair capacity. This method is based on the premise that after in vitro treatment with a test mutagen, DNA from subjects with defective repair will be more damaged than DNA from those with an efficient repair system. However, very little is known about unmeasured processes that occur between cell treatment and final assessment of DNA damage. To develop a more precise assay, we modified the traditional mutagen sensitivity assay to also include measurement of DNA damage after culturing cells in the absence of mutagen. First we treated apparently normal and Xeroderma Pigmentosum lymphoblastoid cell lines with various doses of benzo(a)pyrene diol epoxide (BPDE), and harvested cells at different time points. A polyclonal antiserum against BPDE-DNA was used to quantitate levels of adducts by immunoslot-blot and immunohistochemistry. Selected conditions included treatment with 10?M BPDE, a 4h culture in mutagen-free medium, and immunohistochemical measurement of BPDE-DNA adducts. The method was then applied in a pilot study to 50 lymphoblastoid lines from sisters discordant for breast cancer. There was no significant difference between cases and controls in the level of BPDE-DNA adducts in lymphoblasts harvested immediately after BPDE treatment. However, after a 4h culture in mutagen-free medium, the level of adducts was significantly higher (p=0.006) among cases than in controls. There was a two-fold increase in mean adduct removal in lines from non-affected as compared to affected sisters (44% and 22% decrease, respectively). DNA repair capacity was predictive of case status (p=0.04) in logistic regression analysis. This method, which can be easily applied to large numbers of samples should be useful in studies to investigate the role of DNA repair in cancer risk. OXIDATIVE DNA DAMAGE AND REPAIR; INSIGHT FROM DETERMINATION OF 8-OXOGUANINE (8-oxoGua) AND 8-OXO-2' DEOXOGUANOSINE (8oxodGuo) IN EXTRACELLULAR FLUIDS Olinski Ryszard, Rozalski Rafał, Gackowski Daniel, Foksinski Marek Department of Clinical Biochemistry, The Ludwik Rydygier Medical University in Bydgoszcz, Karlowicza 24, 85-092 Bydgoszcz 8-oxoguanine has been recognized as a biomarker of oxidative DNA damage by endogenously generated oxygen radicals. Following excision from DNA the modified base (8-oxoGua) or nucleoside (8-oxodGuo) is excreted into urine (or another extracellular fluid) where their presence has been acknowledged to be reflective of overall oxidative stress. The presence of these compounds can also mirror an involvement of different DNA repair pathways, namely base excision repair and nucleotide excision repair towards removal of these two lesions. These aspects will be discussed during the presentation and our published results (1,2,3) as well as the recently obtained results concerning experiments with mOGG1 knock out mice will be described. 1. Gackowski D., Różalski R., Roszkowski K., Jawień A., Foksiński M., Oliński R., 8-oxo-7,8-dihyrdroguanine and 8-0xo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet. Free Radical Res. 35: 825-832, 2001. 2. Oliński R., Gackowski D., Foksiński M., Różalski R., Roszkowski K., Jaruga P., Oxidative DNA damage: assessment of the role in carcinogenesis, atherosclerosis and acquired immunodeficiency syndrome. Free Rad.Biol.Med. 33: 192- 200, 2002. 3. Rozalski R., Gackowski D., Roszkowski K., Foksinski M. and Olinski R. The level of 8-oxoguanine - possible repair product of oxidative DNA damage - is higher in urine of cancer patients than in control subjects. Cancer Epidemiology, Biomarkers Prevention. 2002 Oct;11(10):1072-5. EFFECT OF ANTIOXIDANT VITAMINS ON RADIATION INDUCED APOPTOSIS Ortmann Elisabeth1, Mayerhofer Thomas1, Getoff Nikola2, Kodym Reinhard1 1Department of Radiobiology, Clinic for Radiotherapy and Radiobiology, General Hospital Vienna, 2Ludwig Botzmann Institute for Radiation Chemistry and Radiation Biology The University of Vienna, Austria Among the various forms of radiation-induced cell death, apoptosis was found to be important in the cellular radio-sensitivity of tumors and normal tissues alike. Radiation-induced reactive oxygen species (ROS) as well as ROS produced during signal transduction events play a role in the process of apoptosis.. Anti-oxidant vitamins have been used in clinical settings to suppress normal tissue toxicities due to their radical scavenger capacity. Therefore we investigated how a-tocopherol, ascorbic acid, and b-carotene influence radiation-induced apoptosis. We studied the human lymphoblastic leukemia cell line MOLT-3 which undergoes apoptosis within 8 hours after exposure to ionizing radiation. MOLT-3 cells were incubated with 0.01mM, 1mM or 100mM of the vitamins mentioned above as well as with all combinations of vitamins. Cells were treated with the vitamins prior or after irradiation with 3 Gy. Eight hours after irradiation apoptosis was scored morphologically. Vitamin A, C, and E as well as all combinations thereof were found not to influence apoptosis in unirradiated cells when given at concentrations mentioned. When given prior to irradiation vitamins A and E showed a significant radio-protection at concentrations of 0.01mM as well as 1mM. Combinations of vitamins did not influence this behavior significantly. When given immediately after irradiation we observed no alteration of the cellular radio-sensitivity at concentrations of 0.01mM and 1mM while we observed a radio-sensitization at 100mM. Anti-oxidant vitamins apart from vitamin C show a significant radio-protective effect in the low dose range while high concentrations of vitamins tend to work as radio-sensitizers. Therefore clinical administration of anti-oxidant vitamins should consider the time and dose effect found to obtain the desired effect. CHICKEN DOMAIN OF ALPHA-GLOBIN GENES: ORGANIZATION AND REGULATION OF GLOBIN GENES EXPRESSION Razin Sergey V., Iarovaia Olga, Ioudinkova Elena Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia Chicken domain of alpha-globin genes represents a classical example of "weak" domains with poor-defined boundaries. It is located in permanently open chromatin area and overlaps a house-keeping gene expressed in cells of different lineages. Nevertheless, the globin genes are expressed only in erythroid cells. In non-erythroid cells the whole domain is repressed by mechanisms which, apparently, have nothing to do with a change in the mode of chromatin packaging. We have identified two blocks of regulatory elements that might contribute to the repression of alpha-globin genes in non-erythroid cells. The first one includes matrix attachment region, CTCF-dependent insulator and elements blocking the continuous transcription of the whole domain. The second one is a differently methylated region located upstream to the whole cluster of alpha-globin genes. These CpG-rich region is extensively methylated in non-erythroid cells and remains non-methylated in erythroid cells. In a model experiment we have demonstrated that, being methylated, this region suppress activity of promoters of alpha-globin genes. In erythroid cells, productive expression of alpha-globin genes seems to be controlled at the post-transcriptional level. We studied, by in situ hybridisation and confocal microscopy, the distribution of the transcripts of the chicken alpha A gene in the course of induced terminal differentiation of the erythroleucemic AEV cells. Furthermore, we analyzed the size distribution and overall quantity of transcripts of this gene present in nuclei and cytoplasm using Northern-blot hybridization. The results obtained suggest that globin genes are actively transcribed at both early and late stages of erythroid cell differentiation. Most surprisingly, we found that, prior to induction, the globin RNA accumulates mainly in the peri-nucleolar areas. Transport of globin mRNA from nuclei to cytoplasm seems, thus, to occur only in terminally differentiated cells, whereas in the dividing erythroleukemic cells (pre)mRNA is retained in nuclei. CELL-CYCLE-DEPENDENT CONTRIBUTION OF NON-HOMOLOGOUS END JOINING AND HOMOLOGOUS RECOMBINATION TO THE REPAIR OF RADIATION-INDUCED DNA DOUBLE-STRAND BREAKS IN MAMMALIAN CELLS Rothkamm Kai, Krüger Ines, Löbrich Markus Universität des Saarlandes, Fachrichtung Biophysik, Germany Two enzymatically distinct pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR) contribute to the repair of DNA double-strand breaks (DSBs) in eukaryotic cells. Although most of the enzymes involved are surprisingly conserved from yeast to man, the relative importance of NHEJ and HR shows considerable variation. Yeast cells primarily rely on HR for the repair of radiation-induced DSBs, while NHEJ is believed to be the major repair pathway in mammalian cells. The objective of the present work was to assess the relative contribution of NHEJ and HR to the repair of radiation-induced DSBs in synchronized populations of Chinese hamster ovary cells. To that end, we employed an immunological approach based on microscopic detection of fluorescent foci of the phosphorylated histone g-H2AX that was recently shown to detect individual DSBs induced by ionizing radiation (Rogakou et al., JCB 146:905-916, 1999) and provides a quantitative measurement of the repair of individual breaks in single cells. We show that V3 cells compromised in NHEJ exhibit strongly impaired DSB repair after irradiation with 1 Gy in all phases of the cell cycle. irs1SF cells deficient in homologous recombination, in contrast, show no repair defect in the G1 phase, a partial impairment in S but a substantial defect in G2. Furthermore, the radiosensitivity of irs1SF cells is marginal in G1 but dramatically increases in G2, while V3 cells are highly sensitive throughout the cell cycle. This shows that NHEJ is important in all cell cycle phases while HR is restricted to late S/G2 where both pathways contribute equally to DSB repair and radioresistance. In contrast, breaks introduced by the replication inhibitor aphidicolin are repaired by homologous recombination, and irs1SF but not V3 cells show hypersensitivity to aphidicolin treatment. PREMATURE MITOSIS (PCC) - A PRESERVED ABILITY OF CHROMATIN TO REPLICATE THE DNA Rybaczek Dorota University of Łódź, Department of Cytophysiology, 90-231 Łódź, ul. Pilarskiego 14, Poland To preserve genomic stability, nuclear DNA must be replicated once and the resulting sister chromatids partitioned equally between each of the daughter cells. Temporal ordering of these events is controlled tightly by the so-called 'cell cycle checkpoints', which are signal-transduction pathways specific for either abnormal or incompletely assembled cellular structures. The checkpoint-mediated inhibitory pathways block or slow down cell cycle progression until the abnormalities are repaired and the molecular components assembled properly, thus providing means to assure a correct and adequate genetic inheritance to each of the cells produced by mitosis. One of such responses, the S-M checkpoint delays the onset of mitosis (M) while DNA synthesis (S) is underway. Our studies on root meristems of Vicia indicate that, following the hydroxyurea (HU)-mediated arrest of DNA replication, molecular signals generated by caffine, 2-aminopurine, or sodium vanadate may invoke mechanisms allowing the cells to override the S-M dependency control system. The ensuing events result in a variety of aberrant mitotic divisions including chromosomal breaks and gaps, lost and lagging chromatids and chromosomes, acentric fragments, chromosome bridges and micronuclei. Electron microscopic studies indicate that, depending on the chemical agent applied to induce premature condensation of incompletely replicated chromosomes (PCC), interphase and mitotic cells reveal some characteristic ultrastructural features, comprising both chromatin and cytoplasmic structures. Long-term experiments show thay root cells displaying PCC become transformed into apoptotic cells. Furthermore, using Feulgen cytophotometry and 3H-thymidine autoradiography, we have evidenced that the frequency and appearance of PCC vary significantly depending on the stage at which hydroxyurea-blocked S-phase cells were enforced by caffeine to enter mitosis. Despite the condensed state, pulverized chromosomes in HU/caffeine-treated S-phase cells stimulated to PCC still preserve the ability to incorporate 3H-thymidine. However, the validity of acquired licence for DNA replication is limited merely to anaphase and telophase cells. This result suggests that resumption of S-phase activities in cells displaying symptoms of PCC is strictly dependent on the Anaphase Promoting Complex/Cyclosome (APC/C)-mediated decline in the amount of B-type cyclins and, consequently, on the degradation of protein kinases indispensable for the metaphase-anaphase transition. INHIBITOROTHERAPY OF CANCER Siewiński Maciej, Saleh Yousif, Gryboś Marian Department of Obstetrics and Gynaecology, Medical University of Wrocław, Chałubińskiego 3, PL-50-368 Wrocław, tel. 4871-3209741 The hypothesis that cysteine endopeptidases play a fundamental role in tumor malignancy becomes well established. Activity of these enzymes can be regulated on the level of the enzyme synthesis and by inhibitors. Specific endogenous cysteine peptidase inhibitors (CPI) serve as an ultimate control mechanism for tumor cysteine endopeptidases activity in vivo. In the light of the hypothesis the aim of therapeutically procedures should be an attempting to reduce activity of these enzymes. 1. Correlation between tumor invasiveness, metastasis and neoplastic transformation and activity of cysteine endopeptidases particularly cathepsins B and L in "cascades of proteases and caspases enzymes. 2. The activity of cysteine proteinases, their activators (marker of tumor aggresivity) and their autogenously inhibitors - total, active and in complex forms (marker of organism defense again cancer aggressivity) are investigated in body fluids and homogenates of tissues 3. Inhibition in vivo and in vitro cysteine peptidases using inhibitors from human urine, placenta and white egg. 4. Administration of vitamin E on stimulation on macrophages, interleukin 1, lymphocytes, interleukin 6 levels and increased level of T-kininogen (cysteine peptidases' inhibitor). THE POTENTIAL OF TRANSGENIC PLANTS FOR SUBUNIT VACCINE PRODUCTION Sirko Agnieszka, Kazimierczuk Kacper, Wawrzyński Adam Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawinskiego 5A, 02-106 Warsaw, Poland Subunit vaccines are defined as those containing one ore more pure or semipurified antigens. Availability of complete sequences of many pathogens allows now for production of the recombinant antigens in heterologous production systems. The traditional systems used for the production of subunit vaccines include bacterial, yeast and mammalian cells cultures. Recent advances in plant transformation techniques made it possible to employ plant cells and plants as vaccines production systems. Using plants for these purposes is extremely attractive for a number of reasons, including economics of production, purification and delivery - especially if edible plant parts will be used for oral vaccination, possibility to extend the shelf life - if the vaccine will be stored in plant organs (e.g. seeds) and purified only when required, as well as safety for vaccinated individuals since plant pathogens generally do not infect humans and animals. The plant systems have also some disadvantages, including low expression levels and a lack of correct (animal-specific) posttranslational glycosylation. However, several strategies successfully adopted to increase protein expression level in plants have been reported. During the last decade many bacterial and viral antigens have been produced in plants. The immune responses to plant-derived antigens and protection against pathogens observed in many cases confirmed efficacy of this system for subunit vaccine production. The aim of experiments conducted in our laboratory is to produce the selected antigens of Helicobacter pylori (UreB, HspB and HspA) and of equine herpes virus 1, EHV-1, (gC and gD) in transgenic plants. The respective plant expression cassettes have been prepared and their functionality confirmed in a transient in planta expression system. A novelty of our current approach is: (i) production of antigens as fusion proteins with mammalian cytokines that are supposed to act as immunological adjuvants and (ii) targeting the products into endoplasmic reticulum in order to increase their yields. Selection of transgenic tobacco plants with the sufficient level of antigens expression is in progress. The next planned steps are transformation of other plant species (carrot or lettuce) and verification of the efficacy of the potential vaccines in experimental models. A short review of the experimental data from various laboratories will be shown and discussed along with our results. GENOMICS AND PROTEOMICS AS A TOOL FOR ANALYSIS OF PHOSPHOLIPID HYDROPEROXIDE GLUTATHIONE PEROXIDASE EXPRESSION AND TRANSFORMATION DURING SPERMATOGENESIS Sztajer Helena Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, Braunschweig, Germany It was found that selenoprotein phospholipid glutathione peroxidase (PHGPx) is abundantly formed in spermatids as an enzymatically active and soluble protein and is transformed into inactive form in mature spermatozoa. As such it accounts for at least 50% of the keratin-like material that embeds the mitochondrial helix in the midpiece of sperematozoa. Preliminary clinical data reveal that the PHGPx contents of human sperm samples correlate with fertility related parameters such as morphological integrity, motility and, less evidently, with viability. Additionally it was observed that glutathione (GSH), the normal PHGPx reducing substrate, drops in late spermatogenesis to undetectable level. The mechanism leading to the burst of PHGPx synthesis and transformation during spermatogenesis are being investigated by RNA expression analysis and proteome analysis of defined stages of spermatogenesis in rats. It is anticipated that the data will ultimately provide a key to understand and possibly intervene with male fertility problems. PHENOTYPIC AND GENOTYPIC INDICATIONS FOR AN IMPAIRED DNA REPAIR CAPACITY IN LARYNGEAL CANCER SUBJECTS Szyfter Krzysztof 1,2, Gajęcka Marzena1, Rydzanicz Małgorzata 1, Wierzbicka Małgorzata 2 1. Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland 2. Clinic of Otolaryngology, K.Marcinkowski University of Medical Sciences, Poznań, Poland The presented studies are a part of long-lasted interest in biology and genetics of tobacco smoke-associated laryngel cancer. Interindividual differences in susceptibility to pathogens and drugs have been recently extensively studied because of an association with an (i) estimation of an individual risk to develop cancer, (ii) a variable progression of cancer and (iii) a clinician's claim to individualise cancer therapy. Concerning laryngeal cancer, a slight significance of genetic risk factor was shown in numerous studies on genes/enzymes involved in the processes of metabolic activation and detoxication of tobacco smoke carcinogens. Following the same line we have focused our attention an impact of DNA repair process. First, DNA repair damage induced by bleomycin or S9-activated benzo(a)pyrene was determined in peripheral blood leukocytes using alkaline comet-assay. Laryngeal cancer subjects (n=52) were shown to have higher levels of spontaneous and mutagen-induced DNA damage as compared to healthy controls (n=56). A level of spontaneous DNA damage tended to increase with tumour aggressiveness. A percentage of individuals with the arbitrary chosen efficient DNA repair was higher for controls for the both used mutagens. Then, a genotyping in the group of laryngeal cancer subjects (males, n=293) and in the matched controls (n=322) was performed for the genes coding one activating enzyme, 3 detoxifying enzyme and 3 DNA repair enzymes was performed. The latter group included the genes XPD, XRCC1 and XRCC3 representing, respectively, NER, BER and non-homologous end rejoining. All three mechanisms are involved in removal of DNA lesions induced by tobacco smoke. The following polymorphisms were studied by PCR-RFLP: transversion A-C at 35931 for XPD, transition C-A at 28152 for XRCC1 and transition C-T at 18067 for XRCC3. There were found only two XPD alleles significantly over-represented in laryngeal cancer that could be interpreted as an increase of genetic risk. Besides, there was discovered an accumulation of gene defects in laryngeal cancer that substantially contributed to the genetic risk. Recently, a group 46 subjects with the second primary tumour was included to the study. Preliminary results seem to indict for a higher frequency of defects of DNA repair genes in case of second primary tumour. HOW MOLECULAR BIOLOGY MAY IMPROVE OUTCOME OF RADIATION ONCOLOGY INTERACTIONS Tarnawski Rafał Department of Experimental and Clinical Radiobiology Center of Oncology - M. Skłodowska-Curie Memorial Institute, Branch in Gliwice Recent progress in molecular biology have broadened our knowledge about DNA repair and cell cycle control. Nowadays we better understand the mechanisms of interactions between inonizing radiation and cell survival or death. Radiation therapy had been developed as an empirical skill and its effectiveness has not been changed by the advances basic biological sciences. Although the major improvement in radiotherapy outcome has been driven by technology and physics, the combination of promising new biological modifying drugs with radiation therapy has significant potential for improving anti-tumor responses over radiation treatment alone. In general these modifiers may be classified according to mechanims of their action: I Modifiers of tumor response: 1) Proliferation a. Growth factor receptors (EGFR. Her2Neu) 2) Radiosensitivity a. Modification of intrinsic radiosensitivity i Increase apoptotic threshold ii. Decrease DNA repair b. Complementary cell kill i. S-phase specific cytotoxins c. Additive cell kill 3) Hypoxia a. Hypoxic radiosensitizers (nimorazole, etc) b. Oxygenators (EPO, oxygen carriers, etc) c. Decrease oxygen consumption hyperglycemia, etc) d. Hypoxia directed therapy i. cytotoxins, bioreductive drugs (tirapazamine, etc} ii. hyperthermia iii bacteria gene therapy vectors (clostridia, salmonella, etc) e. Hypoxia response modifiers (Glycolysis, VEGF, HIF, etc...) f. Vascular targeting drugs, inhibitors of angiogenesis (combretastatin, etc) II Modifiers of normal tissue 1) Modifiers of survival or the number of clonogenic cells a. Increase protiferation (KGF, etc) b. Block apoptosis (pifithrin, etc) 2) Modifiers of the function-differentiation of normal cells a. Stem cell therapy b. Cytokine therapy long-chain adducts of fatty acids derivatives to dna bases, mutations and repair Tudek B., Kowalczyk P., Cieśla J.M., Komisarski M., Kuśmierek J.T. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland Oxidative stress enhances lipid peroxidation (LPO), implicated in the promotion and progression stages of carcinogenesis, particularly under conditions of chronic inflammation and infections. One of the most abundant products of LPO, trans-4-hydroxy-2-nonenal (HNE), was shown to be mutagenic and carcinogenic, however, its oxidised derivative, 2,3-epoxy-4-hydroxynonanal (EH), is much more potent mutagen and carcinogen. Our studies on M13 phage have revealed that all four DNA bases, A, C, G and T, are targets for HNE. DNA synthesis by T7 DNA polymerase is stopped at A, C, G and T sites in M13 DNA pretreated with HNE, albeit the reactivity of bases is different and follows the order G3C>A3T. Mutagenicity studies in M13 phage system showed increased mutation rates, among which mainly recombination events were observed, followed by base substitutions and frameshifts, the latter occurring in runs of A, C or G. Over 50% of base substitutions were C(r)T transitions, followed by G(r)C and A(r)C transversions. In E.coli strain recA deficient the frequency of recombination events decreased significantly, suggesting that recombination repair system is involved in removing HNE/EH adducts to DNA bases. Since at present, only reaction of HNE with dG was described in literature, we have undertaken studies on all four deoxynucleosides. The HPLC analysis of reaction mixtures showed formation of several products in each case, and reactivity follows the order dG>dC>dTraquo;dA. MS of the reaction mixtures showed peaks corresponding to HNE-dN 1:1 adducts, and in lower abundance, to 2:1 and 3:1 adducts. In dA, dC and dG reactions peaks corresponding to heptyl-substituted e-adducts were detected, what indicates that during reaction HNE is oxidized to EH, probably by oxygen from air. The four most abundant products of the HNE-dC reaction were temporarily characterised on the basis of MS, UV and pKa evaluation: A is N3-substituted HNE-dC (cyclic or linear), B and C are N4-substituted HNE-dC, whereas D is dehydrated heptyl-substituted etheno-dC adduct. Thus, these long chain adducts to DNA bases arrest DNA synthesis, initiate recombination, base substitutions and frameshift mutations. TOWARDS DNA BASED COMPUTER - THE INITIAL STATE Unold Olgierd Institute of Engineering Cybernetics, Wroclaw University of Technology Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland phone: (48 71) 320 20 15, fax: (48 71) 321 26 77 e-mail: unold@ci.pwr.wroc.pl This lecture presents the possibility to build DNA based computer using the available equipment of the laboratory in Medical University of Wroclaw. Our approach is based on the experiment conducted by Profesor Ehud Shapiro of the Weizmann Institute of Science [reported in the November 22, 2001 issue of Nature]. Shapiro has found a way to use DNA as a truly general-purpose computer, suitable for solving any kind of problem. The computation method proposed by a group of scientists headed by Shapiro uses two types of DNA molecules: software, or computation molecules, which are about 40 base pairs long and contain the instructions for the computation, and input molecules, which contain strings of six bases that represent the problem to be solved. A computation happens when these two types of molecules interact. Each has a sticky end, meaning one strand of the double helix is longer than the other, exposing a sequence of bases that are not paired. When the sticky end of an input molecule bumps into a software molecule that has a sticky end that fits, the bases of the two sticky ends join together, and an enzyme present in the solution seals them together. The molecule is then cut in a different place by a second enzyme, exposing another sticky end so that a second step can take place. The number of steps depends on the number of computations coded into the software DNA strand. Up to now a group of scientists from Wroclaw University of Technology and Medical University of Wroclaw has worked out the program which simulates the experiment performed by Shapiro in a test tube. During the simulation virtual DNA molecules are hybridized and divided like in the normal, biological experiment. Experiments with DNA-manipulating enzymes different then used by Shapiro are currently in progress. CHEMOTHERAPY - WHERE ARE YOU GOING? PRINCIPLES, NEW TARGETS, CHALLENGES FOR FUTURE Utracka-Hutka Beata Department of Chemotherapy, Center of Oncology - Maria Skłodowska-Curie Memorial Institute, Branch in Gliwice The effective use of cancer therapy requires an understanding of the principles of tumor biology, cellular kinetics, pharmacology and drug resistance. Thanks to the development of the new effective chemotherapeutic agents coupled with our expanding knowledge about the administration and combination of these agents we are now able to cure almost 20% of all new cases by chemotherapy along. Combination of chemotherapy with other modalities of treatment like radiotherapy and surgery improves greatly the chance of curing. The lecture focuses on the principles responsible for the development of modern combination regimens. This is followed by description of new chemotherapeutic drugs, and description of the new exciting agents as angiogenesis, COX-2 and epidermal growth factor receptor inhibitors. THERAPY FOR LYMPHOMA: CURRENT CONCEPTS AND NEW APPROACHES Walewski Jan Maria Skłodowska-Curie Memory Cancer Center - Institute of Oncology, Warszawa There is a common belief that we are approaching a new era in diagnosis of lymphoma. Impressive advances in molecular techniques provided new tools for investigating multiple genetic abnormalities in distinct lymphoma subtypes and hold promise of generating data that will make possible establishing a classification based on disease pathogenesis. Current WHO classification based on combined analysis of all diagnostic data available, i.e. morphology, immunophenotype, cytogenetics, and clinical information is successful in defining disease entities that are clinically relevant. Chemotherapy, although declared as having reached plateau of efficacy some time ago, continues to be a mainstay of treatment. Intensified regimens developed over the last decade offer cures for majority of patients with highly aggressive disease types like Burkitt's and T-cell lymphoblastic lymphoma. High-dose chemotherapy with autologous stem cell support evolved as a routine and safe treatment for recurrent aggressive lymphoma that is two-fold as effective as conventional chemotherapy in terms of survival. Allogeneic stem cell transplantation is being widely investigated with ultimate goal of reducing morbidity and mortality due to graft versus host disease and enhancing graft versus lymphoma effect. Monoclonal antibodies are entering clinical practice as powerful new agents and they are new hope for patients with indolent lymphoma. Evolving science of cytokines, receptors and signal transduction pathways has already reached the bedside with new options of targeted therapy. Accumulating results of pharmacogenomic studies open new insights in mechanisms of treatment successes and failures. Variety of emerging options implies that most of lymphoma patients should be considered as possible candidates for prospective clinical trials. WHY SPERMATOGENIC CELLS ARE SENSITIVE TO ELEVATED TEMPERATURE? THE ROLE OF HEAT SHOCK PROTEINS Widłak Wiesława Department of Tumor Biology, Centre of Oncology, M. Skłodowska-Curie Memorial Institute Gliwice, Poland Somatic cells are protected from thermal insult and other stress conditions by inducing a set of heat shock proteins (HSPs). Major function of HSPs is to maintain other proteins in their native folded state, facilitate proper folding of nascent polypeptides and repair or promote degradation of unfolded proteins, thus HSPs are called "molecular chaperones". In contrast to somatic cells, mechanisms of the response to stress conditions in germ cells are not clear at the moment. In majority of mammals testicular temperature is lower than the core body one, and its elevation disrupts spermatogenesis leading to infertility. Pachytene spermatocytes of the first meiotic division are the most thermo-sensitive cells, and mild hyperthermia will induce gross apoptosis within one day. Spermatogenic cells specifically express two members of the mammalian multigene hsp70 family. One of them, named hst70 in rat and hsp70.2 in mouse, is activated in pachytene spermatocytes during prophase of meiosis I. Second one, called hsc70t in mouse, is expressed in spermatids. Both genes are developmentally regulated. Both are not stress-inducible and do not protect spermatogenic cells against heat shock. The hsp70.2 gene codes for protein involved in desynapsis of synaptonemal complexes as well as chaperoning of CDC2 kinase required for completion of the meiotic division. Homozygotic removal of the hsp70.2 gene leads to male infertility (due to spermatocytes apoptosis), whereas females remain fertile. The function of spermatid-specific HSC70t protein remains unknown. The expression pattern and function of the stress/heat inducible hsp70i (i - inducible) genes in the testis is more elusive. According to our observation heat inducibility of the hsp70i genes in the rat testes significantly decreases during postnatal development. One can postulate that inducible hsp70 genes are not activated in spermatogenic cells and an observed expression originate from somatic cells, which are in minority in mature testes. However, it has been shown by others that HSP70i can be synthesized in spermatocytes, which is correlated with induction of apoptosis in such cells. Hsp genes are activated during thermal stress by heat shock transcription factor 1 (HSF1). In unstressed cells of higher eukaryotes HSF1 exists as an inactive monomer, whose basal activity is negatively regulated by heat shock proteins. In response to stress, HSF monomers aggregate into homotrimers that bind avidly at conserved nGAAn repeats (HSE - heat shock element) and activate target hsp genes. A previous study suggested, that activation of HSF1 would be a major trigger for the induction of apoptosis in germ cells. Our preliminary data shows that in transgenic mice which constitutively express in spermatocytes active trimeric form of HSF1 (human HSF1 gene is under control of the hst70 gene promoter) the germinal epithelium of seminiferous tubules is severely affected. Sperm quality is directly linked to embryo quality and injury caused in spermatogenic cells by thermal stress is passed on the next generation. The mechanism that actively eliminates germ cells exposed to thermal stress might be an important factor involved in maintaining the quality of the progeny.
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