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 SWIFT system for 3D real time brachytherapy planning in prostate cancer B. Bialas, J. Bystrzycka, M. Fijalkowski, K. ¦losarek Aim: The aim of the study is to present treatment-planning procedures in brachytherapy of prostate cancer based on US examination with use SWIFT system (Nucletron). INTRODUCTION: Brachytherapy Department in Institute of Oncology Gliwice is equipped with computed treatment planning system (TPS) SWIFT. The SWIFT system consists of transrectal US machine, stopper - use to fix probe and template, treatment planning system. All these elements are integrated and allow planning brachytherapy dose distribution in "real time". Material and method: Patient has to be anaesthetized (spinal anaesthesia) before planning procedures and treatment start. First step of procedure is to perform US examination, to place probe on the depth of prostate base and to send next slices (1mm thickness) to prostate apex (through the whole prostate) Slices are sending to TPS automatically by network (stepper encoder, US machine and TPS are connected). The next step is to prepare pre - plan:
According to the pre - plan doctor inserts needles and transrectal US examination with implant in place is performed. The live planning procedure starts:
After acceptance by staff, treatment plan is send to treatment control station by network and treatment is started. Conclusion: dose distribution based on US examination make possible verification of implant placement in PTV (prostate) in relation to organ of risk (urethra). Integrated treatment planning system enables modification of catheter location in prostate, directly in real time of procedure (pre - planning; live planning). DNA double-strand break repair and its implication in cancer M. Chovanec Laboratory of Molecular Genetics, Cancer Research Institute, Vlárska 7, 833 91 Bratislava 37, Slovak Republic DNA double-strand breaks (DSBs) are considered the most lethal form of DNA damage. They can be induced either exogenously by such agents as ionising radiation (IR) and a wide range of chemical compounds, or endogenously by such agents as reactive oxygen species. Moreover, DSBs arise in DNA as intermediates during several cellular processes. Unrepaired or misrepaired DSBs can lead to cell death, genomic instability, and hence cancer. To combat these detrimental effects, two main pathways have evolved for the repair of DSBs: homologous recombination (HR) and non-homologous end-joining (NHEJ). In vertebrates, NHEJ pathway is represented by the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunit (DNA-PKcs) and the DNA targeting subunit (Ku70/80 heterodimer). Another NHEJ factor is DNA ligase IV, which functions in a tight complex with the Xrcc4 protein. The two main candidates for nucleolytic processing stage of NHEJ are the Mre11-Rad50-Nbs1 complex and the Artemis protein. The Mre11-Rad50-Nbs1 complex presumably functions also in early event in vertebrate HR. Moreover, vertebrate HR involves Rad51 protein along with its five paralogues (Rad51, Rad51C, Rad51D, Xrcc2 and Xrcc3), as well as Rad52, Rad54, Rad54B proteins, and the breast cancer susceptibility proteins, Brca1 and Brca2. Vertebrate cells deficient in components of DSB repair display sensitivity to IR and cross-linking agents, spontaneous and IR-induced chromosomal instability, centromere abnormalities and altered kinetics of DSB rejoining. Moreover, many HR and NHEJ components are essential for viability in vertebrates. The essential role of the HR or NHEJ in vertebrates is further strengthened by the discovery that mutations in some of the HR or NHEJ genes are associated with cancer-prone syndromes, e.g. Nijmegen breakage syndrome, Ataxia telangiectasia-like disorder, LIG4 syndrome, severe combined immune deficiency, and radiosensitive severe combined immune deficiency. Molecular basis of these syndromes and their relation to cancer predisposition will be discussed in more detail. Assessment of biomarkers of exposure, effect and individual susceptibility in workers exposed to mineral fibres M. Dusinská, M. Barancoková, A. Kazimirová, A. Horská, Z. Dzupinková, K. Volkovová, M. Staruchová, A. Kocan, L. Wsolová, A. Collins2, S. Kyrtopoulos3 1Institute of Preventive and Clinical Medicine, Bratislava, Slovakia, 2 Institute for Nutrition Research University of Oslo, Norway, 3 National Hellenic Research Foundation, Athens, Greece An occupational biomonitoring was conducted in 3 factories producing asbestos, glass fibres and rockwool. Personal as well as workplace exposure for PAHs and fibres was measured together with personal dosimetry. The levels of asbestos in asbestos factory 3 - 5 times overstepped the Slovak occupational limit. Just presence of basalt glass fibres was confirmed in both rockwool and glass fibre factories. Individual PAH congener levels in occupational atmosphere range from tenths to several hundreds mg/m3. Altogether 239 exposed, and 148 controls were investigated. Subjects were clinically examined; questionaired for life style and diet, blood and 24h urine were sampled. DNA damage (strand breaks [SBs], formamidopyrimidine glycosylase [FPG] -, endonuclease III [EndoIII] -, and 3-Methyladenine DNA glycosylase [AlkA]- sensitive sites), using the comet assay in peripheral blood lymphocytes were assessed. Micronuclei and chromosome aberrations; genetic polymorphisms of xenobiotic-metabolising and DNA repair enzymes; individual DNA repair capacity in lymphocyte extracts; intrinsic antioxidants, antioxidant enzymes; proinflammatory mediators and immune markers, were also measured. Exposed asbestos workers, especially men, had higher level of oxidative DNA damage (EndoIII sites, P=0.005). Similarly, in glass fibre factory exposed men had more oxidised pyrimidines than non-exposed. The difference between exposed and controls in SBs was significant in glass fibre (P=0.03) as well as in rockwool factory (P=0.05); this was again more pronounced in men, and in group of non-smokers (P=0.004). DNA damage correlated with age in exposed groups combined (R2=0.16, P=0.000, n=239), which may reflect the length of exposure to the fibres, rather than age itself as there was no such correlation found in controls (R2=0.004, P=0.000, n=100). The only biomarker of genotoxicity that has been shown to predict cancer risk is chromosome aberrations. Chromosome aberrations were elevated in the asbestos-exposed workers, and especially in those who smoked (P<0.05) but not in glass fibre or rockwool factories. Level of micronuclei was not affected by exposure to any fibres. However, in the three populations combined (n=388), micronucleus frequency correlated positively with EndoIII- (r=0.21, P<0.001), FPG- (r=0.29, P<0.001), and AlkA-sensitive sites (r=0.22, P<0.001). Similar correlation was seen in all subgroups: controls; exposed; men; women. It is unexpected that markers of DNA damage correlate so strongly with the 'downstream' marker of chromosome stability. There was also an interesting inverse correlation between DNA repair rate and micronucleus frequency especially in the rockwool factory. Associations between DNA damage, exposure, smoking, sex and genotype were found in all three populations. Funded by the EC (contract no. QLK4-CT-1999-01629). Transgenic plants as a tool to improve sulphur amino acid content in plants Holger Hesse, Oliver Kreft, Stefanie Maimann, Michaela Zeh, Rainer Höfgen Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany Essential amino acids and cysteine represent an indispensable component in the diet of all mammals including man. Plants represent the only source widely available to meet this demand. Major crops, such as cereals and legumes, are low in Met and an attempt to manipulate the biosynthetic pathway is a major interest of molecular plant breeding. According to this it can be assumed that Met synthesis, accumulation and consumption are under high regulatory control. Amongst the amino acids underrepresented sulphur containing amino acids such as cysteine and methionine are by far the most important with respect to world nutrition. It is therefore of utmost importance to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality. This article is intended to serve two purposes. The first is to provide a brief review on the physiology of cysteine and methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. Recent studies suggest that Met synthesis in plants has to be controlled at the level of competition between CgS and TS for their common substrate OPHS. This information can be used to develop strategies to improve methionine content of plants with a higher nutritional value. In this paper, a summary of our current understanding of the regulatory network with the focus on efforts to understand and manipulate the carbon flux into Met is given. Labile iron pool, oxidative DNA damage and cancerogenesis Marcin Kruszewski Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, 03-195 Warsaw, Poland Department of Experimental Haematology and Cord Blood Bank, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, 02-781 Warsaw, Poland The unique abilities of iron to change its oxidation state and redox potential in response to the changes of liganding environment makes this metal essential for almost all living organisms. Iron-containing enzymes are the key components of many essential biological reactions, such as energy metabolism, oxygen transport, DNA synthesis and repair, detoxification of reactive oxygen species (ROS) and its reaction products and numerous other reactions catalysed by oxygenases, peroxygenases, etc. However, the same biochemical properties that make iron beneficial in many biological processes might be a drawback in some particular conditions, namely, when improperly shielded iron can catalyse one-electron reductions of oxygen species that lead to production of very reactive free radicals. Trace amounts of "free" iron can catalyse production of a highly toxic hydroxyl radical via Fenton/Haber-Weiss reaction cycle. Iron-driven generation of oxygen-derived free radicals is known to induce oxidation of proteins, lipids and lipoproteins, nucleic acids, carbohydrates and other cellular components. An oxidative damage to the vital cellular components might have in turn a deleterious effects at cellular and tissue levels, leading to the cell death, tissue necrosis and degenerative diseases or cell phenotype changes and cancer formation. The critical factor appears to be the availability and abundance of cellular labile iron pool (LIP) that constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need of iron, its uptake and storage. To avoid an excess of harmful "free" iron, the LIP is kept at the lowest sufficient level by transcriptional and posttranscriptional control of the expression of principal proteins involved in iron homeostasis. The putative sources of cellular LIP, its homeostasis and its role in the cellular response to oxidative stress and cancerogenesis are discussed. Functional analysis of gluB gene in Solanum tuberosum plants Magdalena Krzymowska, Anna Barabasz, Agnieszka Mac, Kamil Witek1, Beata Bieniak2, Maria Charzyńska2, Jacek Hennig1 1Institute of Biochemistry and Biophysics, PAS, Pawińskiego 5A, 02-106 Warsaw, Poland, 2Institute of Experimental Plant Biology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland There is considerable evidence that 1,3-E-glucanases (glucan endo-1,3-E-glucosidases; EC 3.2.1.39) are part of plant defence systems. Although the major interest in 1,3-E-glucanases stems from their possible role in the response of plants to microbial pathogens, there are some reports that these enzymes are also involved in diverse developmental processes of healthy plants. We isolated the gluB gene coding for a novel 1,3-E-glucanase from potato (Solanum tuberosum cv. Désirée). cDNA and corresponding genomic sequence were characterized. Northern blot hybridization showed the presence of gluB mRNA at a very low level in mature leaves of uninfected plants. A considerable increase of expression was observed after infection with potato virus Y (PVY) or Phytophtora infestans and after treatment with salicylic acid or BTH. We also found that transcript accumulation of gluB increases during flower development, reaching a maximum in stigmatic cells during anthesis. To study the function of the GluB protein we have generated transgenic potato plants expressing gluB under 35S promoter in sense and antisense orientation. The susceptibility of the plants, with elevated levels of glucanase, was reduced upon powdery mildew and P. infestans challenge. Interestingly, the changes in the gluB gene product level correlated with some changes in the appearances of the plants. The plants overproducing GluB were more compact and stunted in comparison with nontransgenic control. The cells of internodes of gluB plants were characterized by reduced elongation (mainly in pith parenchyma) in direction of the main shoot axis and simultaneously were radially extended. We did not observe any changes in tuber size and production. Our data suggest that the gluB gene product may play an important role both in plant development and in the defence response against pathogen infection. Wrong assumptions and misinterpretations in molecular biology, biochemistry and bioinformatics Jacek Leluk Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University, Pawińskiego 5a, 02-106 Warsaw, Poland The interdisciplinary character of research work became typical in many research studies of present century. The disciplines such as bioinformatics, computational biology, genomics, proteomics or molecular modelling assemble researchers representing various fields of science and specialization. The dialogue between biologist, computer scientist, physician, mathematician, chemist and physicist is now an ordinary feature that occurs in performing the common projects of their interest. As a consequence there appears important problem of proper mutual understanding within the interdisciplinary group of researchers. It is especially important while interpreting the results of theoretical analysis, modelling and simulation performance, and correct further application of the results. The problem of effective dialogue and understanding between collaborators starts at very early stage of cooperation. It concerns diversity and imperfection at terminology and nomenclature level. The terminology and definitions used in biological sciences are often unclear, misused, mistaken or misinterpreted. The same problem appears with respect to the tools elaborated by computer scientists and applied to accomplish the molecular biology and bioinformatics projects. The algorithms and software are often constructed in a perfect way from the programist's or mathematician's point of view, they have perfect logical architecture, but they do not comprise all significant parameters that describe a biological process. It may happen, that some algorithm that is free of any internal inconsistency becomes useless because of purpose and subject of its application. It may happen that the examined processes do not correspond to the rules of the theoretical approach. As a consequence there is a risk of making wrong or at least incomplete hypotheses and theories, which are far from actual processes that are to be described by the theoretical models. The presented examples concern the errors in fundamental assumptions of many theoretical approaches and wrong way of their application. The examples refer to the statistical analysis of protein evolutionary variability based on stochastic matrices, which ignore the genetic background of mutational protein variability, Markovian interpretation of amino acid replacement within homologous protein sequences, hypothesis of correlated mutations, Monte-Carlo methods for computing tertiary structure of the protein, interpretation of X-ray structural data and theoretical protein folding methods. The special attention was focused on the most popular methods, commonly assumed as reliable and trustworthy. The antioxidant potential of transgenic plants Marcin Łukaszewicza and Jan Szopab aInstitute of Genetics and Microbiology, bInstitute of Biochemistry and Molecular Biology, Wrocław University, Przybyszewskiego 63-77, 51-148 Wrocław, Poland There is a growing interest in the probiotic properties of various natural antioxidants having beneficial effects on human health. Oxidation products such as hydroperoxides or superoxide radicals formed during free radical reactions are the causative agents of many diseases. Data gathered so far strongly indicate further need to conduct a research on impact, mechanisms of function and degradation pathways of various antioxidants. Plants are rich source of such compounds. Specific only for plants are flavonoids which are supposed to play an important role in protection against UV irradiation and osmotic, oxidative or heat shock stresses. 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. As 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, 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 by expression modulation of three different groups of enzymes: regulatory protein (14-3-3), biosynthetic pathway genes (chalcone synthase, chalcone isomerase, dihydroflavanone reductase) and end-product modifying enzyme (glucosyltransferase). 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 for each group of expressed enzymes. In potato plants the most effective was overexpression of DFR, and have been shown to produce mainly one anthocyanin compound. Flax plants with greatly increased antioxidant capacity have been obtained by simultaneous introduction of three flavonoid biosynthetic pathway genes. Fibres of these plants are potentially very interesting from pharmaceutical point of view. Free flaps in reconstructive surgery in head and neck region Adam Maciejewski, Janusz Wierzgoń, Cezary Szymczyk, Bogusław M±ka Center of Oncology, Maria Skłodowska-Curie Memorial Institute, 44-101 Gliwice, Poland The main goal of surgical treatment of head and neck tumours is to achieve macro- and microscopically radical margins and to restore or preserve high quality of life. Based on own experience Reconstructive Surgery Group in Cancer Center in Gliwice introduced free flaps techniques after major resections in head and neck region. Since November 2001 till August 2003 over 80 patients underwent surgical procedures based on microvascular free flaps. In 60% Radial Forearm Free Flap was the technique of choice. In 25 cases were mandibular reconstruction was needed Fibula Free Flap was performed and in two cases iliac crest free flap was harvested. In case of middle face defects reconstruction Rectus Abdominis Free Flap was chosen. In 12 cases were postressective defects were large and composed Anterolateral Thigh Free Flap was harvested and insetted. Twice the complex defects after extended maxillectomy was reconstructed with Subscapular Composite Free Flaps. Overall survival rate based on periodic control and imaging diagnostics was about 90%. 40% of these patients underwent pre- or postoperative radiotherapy and it did not affect flap healing and survival. Based on authors experience the progress on the field of reconstructive surgery will depend on individually chosen free flaps and its goal is to achieve optimal functional and aesthetic outcome. Poly (ADP-ribose) reactivates stalled DNA topoisomerase I: relevance to genomic stability and cancer therapy Maria Malanga and Felix R. Althaus Institute of Pharmacology and Toxicology - University of Zurich - Tierspital, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland DNA topoisomerase I (topo I) plays an essential role in controlling the level of DNA supercoiling and releasing the torsional stress that is generated during DNA transactions. In the course of the topo I catalytic cycle, a DNA single strand break is produced that, under normal conditions, is very short lived and escapes damage surveillance systems. However, when acting on damaged DNA, topo I may get trapped in the vicinity of DNA lesions leading to an accumulation of enzyme-linked nicked DNA (stalled topo I). If unrepaired this may cause genomic instability or cell death. In fact, the potency of the anticancer drug camptothecin and its analogues (commonly known as topo I poisons) is directly related to their ability to stabilize such topo I-DNA complexes. We have found that poly (ADP-ribose), the catalytic product of poly (ADP-ribose) polymerases (PARPs), targets specific domains of topo I and reprograms the enzyme to remove itself from DNA and close the resulting gap. In particular, two nuclear members of the PARP family, PARP-1 and PARP-2, act as poly (ADP-ribose) carriers to stalled topo I sites and induce repair of enzyme associated-DNA strand breaks. In our studies, camptothecin was used to stabilize topo I cleavage complexes, mimicking topo I stalling in the vicinity of DNA lesions. In addition, the DNA nicking activity of topo I was completely blocked by poly (ADP-ribosyl)ated PARPs, both in the presence and absence of camptothecin. Thus, by counteracting topo I-induced DNA damage, PARP-1 and PARP-2 act as positive regulators of genomic stability. Moreover, the observation that poly (ADP-ribose) antagonizes camptothecin action may explain the cytotoxicity potentiation effect of PARP inhibitors used in combination with topo I poisons. Bicistronic strategy in angiogenic therapy Maciej Malecki, Przemyslaw Janik Department of Cell Biology, Center of Oncology, Warsaw, Poland Angiogenic gene therapy is a very promising procedure but requires large amounts of pharmaceutical-grade plasmid DNA. Overexpression of angiogenic genes like VEGF, FGF causes new vessel formation and improves the clinical state of patients. In this regard a bicistronic plasmid DNA vector encoding two proangiogenic factors, VEGF165 and FGF-2 have been constructed. The construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) which permits both genes to be translated from a single bicistronic mRNA. The IRES sequence allows for a high efficiency of gene expression in vivo. The pVIF vector was characterized in vitro and in vivo. In vivo angiogenesis studies showed that the bicistronic vector encoding two proangiogenic factors induces the formation of new vessels significantly more than pVEGF165 or pFGF-2 alone. It is worth noticing that the combined proangiogenic approach with VEGF165 and FGF-2 is more powerful and efficient than single gene therapy. Can current biomarkers be applied in the monitoring of low environmental exposures? Results from an investigation on traffic wardens of Rome city. Francesca Marcon and Riccardo Crebelli Istituto Superiore di Sanita', Rome, Italy. Air pollution from vehicle exhaust is a relevant health problem in urban areas. The composition of the pollution is complex, including many chemicals known to have high potential genotoxic effects, such as benzene, 1,3-butadiene, benzo[a]pirene, in addition to particulates and traditional air pollutants (i.e. carbon monoxide and nitrogen monoxide). In recent years, in Italy, there was a progressive reduction of the levels of exposure and the doses encountered today are in most cases quite low. However, the risk for human health associated to this low exposure is not yet clear, mainly because there is a lack of information on the biological effects produced by the low doses. In this respect, the biomonitoring of human population occupationally exposed to low levels of urban air pollutants could be useful to evaluate the adverse effects resulting from the low dose exposure. With this aim, our laboratory was involved in a study on a group of traffic policemen showing a profile of exposure comparable to the one of other outdoor workers and the general population of Rome city. The sample included 206 subjects, consisting of 143 exposed individuals working in the urban traffic and 63 control policemen employed in the offices, matched by age, gender and smoking habits. The study included the analysis of markers of external and internal exposure, of genotoxic effects and susceptibility. The assessment of baseline chromosomal damage was performed using classical cytogenetic endpoints, sister chromatid exchanges (SCE), micronuclei (Mn), and the Comet assay in subjects genotyped for CYP1A1, CYP2E1, GSTM1, GSTT1, and DT-diaphorase polymorphisms. The average benzene exposure detected during the workshift was 9.5 and 3.8 µg/m3 in exposed individuals and controls, respectively. The frequency of SCE was significantly influenced by smoking habits, but no differences were observed between exposed and control individuals. Similarly, the Mn frequency was unaffected by the occupational exposure to traffic fumes, whereas it was mainly modulated by the age and gender of the study subjects. In order to improve the sensitivity of the method to excision-repairable lesions, a modified protocol with the exposure of the cells to the DNA polymerase inhibitor cytosine arabinoside (Ara-C), was applied to 78 subjects, but the results failed to demonstrate any significant effect of chemical exposure (occupational or related to smoking habits). Moreover, the analysis of DNA damage by comet assay did not highlight any statistical significant difference between exposed and control workers. A mutagen sensitivity assay was also performed on a subgroup of 31subjects with the aim to investigate the relative role of genetic and environmental factors on individual susceptibility to genotoxic agents. The analysis of chromosomal aberrations and the assessment of the kinetics of DNA repair by comet assay revealed a modulator effect of smoking habits and GSTM1-null genotype on DNA sensitivity to X-radiation, likely to be due to the higher expression of enzymes involved in the repair of oxidative DNA damage in heavy smokers and GSTM1-null subjects. On the whole, the results obtained in this work are in agreement with those of previous investigations on populations with low or moderate exposure to atmospheric pollutants, which indicate that the contribution of environmental pollution to background levels of genetic damage may be barely detectable, while important non-occupational factors, such as smoking habits, may have a dominant role in determining the results of biomonitoring studies. However, in the near future, genomic based new technologies such as microarray will probably be useful to identify early, sensitive biomarkers of xenobiotic exposure by examining changes in gene expression profile, thus improving the efficiency of risk assessment. This work was partially supported by the Italian Ministry of Environment (project PR-22IS). PCR performed at low denaturation temperatures - PCR melting profiles Masny A., Płucienniczak A. Institute of Biotechnology and Antibiotics, Staroscinska 5, 02-516 Warsaw, Poland; masnya@iba.waw.pl; apl@iba.waw.pl We show that using low denaturation temperatures (80-88 degrees C) during ligation mediated PCR (LM PCR) of bacterial DNA leads to the amplification of limited sets of less stable DNA fragments. A set of electrophoretic patterns of such fragments obtained at different denaturation temperatures forms the PCR melting profile (PCR MP). A single pattern obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very specific for the given bacterial genome and may be used for strain characterisation and differentiation. The method may also be used for amplification and isolation of the less stable DNA fragments in a genome. Application of cytogenetic markers in the polluted region of Upper Silesia Grażyna Motykiewicz Upper Silesia is a heavy industrialized region of southern Poland. It encompasses over 12 thousand square kilometres and is inhabited by almost 5 million people. Environmental pollution mainly originates from coal-based heavy industry, gasoline exhaust from automobile traffic, and combustion of coal for domestic heating. As a result of coal burning and/or processing, among the most prevalent mutagenic and carcinogenic air pollutants in Silesia are the polycyclic aromatic hydrocarbons (PAH) and heavy metals. At the second half of last century Silesia was one of the most polluted regions in the world. It has been postulated that increased incidence of cancer and other genetic diseases including infertility and congenital malformations, observed in residences of Silesia, is the result of significant damage to genetic material from long-term exposure to environmental pollution. Over last ten years, cytogenetic methods were successfully used in Silesia to reveal DNA damage in human samples. Studies were performed on populations exposed to airborne pollutants at low-dose environmental and extremely high occupational levels. The control populations from mostly rural north-eastern Poland were also included using the same criteria for subject enrolment. Both male and female populations were studied. A preliminary study on children from Silesia was also performed but was lacking control population. The employed methods included sister chromatid exchanges (SCE), chromosomal aberrations (CA) and micronuclei formations (MN). All the methods were performed on cultured white blood cells. The data obtained with cytogenetic methods were compared with markers of exposure performed on urine samples including the Ames test, the level of 1-hydroxypyrene, and cotinine. As a part of the study, we also tested the level of aromatic adducts in situ and in isolated DNA samples. The blood lead and cadmium levels were also controlled but only among children. In three independent projects we were able to show statistically significant increase of the levels of both SCE and CA in Silesian populations as compared to controls. The data were analysed by conventional statistics, ANOVA method and a multiple regression model. A dose-response, seasonal variation in marker level, and additive effects of smoking and exposure were found using the SCE method. The highest levels of SCE were seen among coke oven workers and the lowest in control samples. There was statistically significant influence of the season on SCE level. The highest values were found in samples collected in winter, when air pollution originates from both industrial and domestic heating sources. Using a multiple regression model, smoking was a major factor influencing the level of SCE. There was a clear additive effect of exposure and smoking in the group of coke oven workers. Although the CA method has also significantly revealed the damage caused by environmental pollution there was no correlation between those two markers. A borderline correlations were found between the level of aromatic adducts, tested by 32P-postlabelling method, and CA as well as SCE, p=0.05 and p=0.03, respectively. A positive correlation (p=0.003) was found between blood lead level and formation of MN in children population. Based on our data performed on Silesian populations exposed to airborne pollutants, cytogenetic markers are adequately sensitive, providing valuable information on potential risk of genetic-based diseases including cancer. Mapping of the hot spots of recombination in human DNA cloned in S. cerevisiae Mariusz Mucha, Jaroslaw Starzynski, Anna Goc and Jan Filipski Institut Jacques Monod, 2 Place Jussieu, Tour 43 75251 Paris, France Pracownia Genetyki, Uniwersytet M. Kopernika, ul. Gagarina 9, Torun, Poland The crossing over and gene conversion events occurring during meiosis in eucaryotic organisms are clustered in the short segments of chromosomes called hotspots of recombination. It has been demonstrated, that in yeast and mammals, these clusters are located in the vicinity of initiation sites. Initiation of meiotic recombination is associated with the formation of DNA double strand breaks (DSB) followed by DNA strand exchange, repair of heteroduplexes and resolution of Holliday junctions. The location and activity of the hotspots of recombination depend on several factors such as: local DNA sequence features, local chromatin characteristics, distance from the chromosomal loop attachment sites or distance from the centromere and telomere. It has been shown that the DNA segments corresponding to the cold spots of recombination in mammalian cells scarcely recombine in yeast cells if cloned as Yeast Artificial Chromosomes (YAC). The segments frequently recombining in mammalian cells conserve this property when cloned in artificial chromosomes or plasmids. The aim of our project was to identify the DNA features, which make a mammalian DNA segment recombination-prone in yeast. We studied several YACs, most of them quite stable in yeast. The YAC 745D12 carrying the HLA class I antigen gene cluster contained six CpG islands and two long minisatellite sequences, the YAC 225A3 contained a single CpG island located 166 kb from telomere C and a region rich in GC nucleotides, YAC XY206 contained also a single CpG island located 8 kb from the telomere C, YAC A85D10 contain a long minisatellite sequence and no CpG island. The CpG islands, with one exception, were hypersensitive to nuclease. The island accompanying the human G6PD gene has also been subcloned from YAC XY206 into a yeast plasmid in front of the cDNA segment coding ADE2HI gene. This construct complemented the auxotrophic mutation ade2 of the yeast. Apparently the island was acting as a transcription promoter. Its function was dependent upon the integrity of the sequences present in the island. We interpret this finding as suggesting that the sequences present in the island are recognised by yeast transcription factors. Three classes of DNA sequences were associated with meiotic DSB. First class contained some, but not all of the CpG islands. The second class contained the GC-rich region in the YAC 225A3. The DSB formed in this region showed spacing corresponding to the length of the chromatin loops in yeast. The third class was associated with minisatellite sequences. We suggest that meiotic DSB are formed at least in some in of these sites during meiosis in human germ-line cells. Transcription factor function search: combining genomics and single-gene approaches to discover new regulatory networks in plant biology Bernd Müller-Röber University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Str. 24-25, Haus 20, D-14476 Golm, Germany; e-mail: bmr@rz.uni-potsdam.de Transcription factors (TFs) regulate the expression of downstream target genes and thereby contribute to the establishment of complex traits in higher plants, including developmental features, cell differentiation, biosynthetic pathways and adaptation to environmental stresses. The evolution of higher plants is tightly coupled to TF evolution and the high TF number in plants places them on the same level of complexity with e.g. Drosophila. Several examples demonstrate a large biotechnological potential of plant TFs for the modification of traits. We use genomics and single-gene approaches to investigate the function of currently under-explored plant-specific TF genes, using Arabidopsis thaliana as a model. More than 150 TF genes were over-expressed in transgenic plants, including those of the DOF and NAC families. TF function is deduced by microscopic and macroscopic phenotype analysis, the identification of potential target genes and interacting protein factors. RNAi lines are generated for a selected number of TF genes. Functional analysis is enhanced through in-house development of suitable bio-informatics tools. Glutathione transferase activity and genotoxic damage in human placentas from environmentally exposed pregnancies Obolenskaya Maria Institute of Molecular Biology and Genetics National Academy of Sciences of the Ukraine, Zabolotnogo str. 150, Kiev 03143, Ukraine; Introduction: The early protection of children from environmental hazards has become of particular importance in the Ukraine in view of the industrial pollution and the accident at Chernobyl. Also the increasing Ukrainian cancer incidences support the notion that the lifetime environmental exposures may contribute to cancer risk in exposed individuals. Extracorporally the foetus is protected from environmental factors by maternal (mainly hepatic) detoxification and by placental detoxification. However, genotoxic exposures that occur transplacentally are evidenced in the form of macromolecular damage measurable in the placenta. Damage to placental DNA cannot only document individual exposure and the efficiency of detoxification but can serve as a surrogate for DNA damage occurring in the foetal tissues. The underlying hypothesis of this study was that placental detoxification efficiency reflects the individual genotype of detoxifying enzymes and the environmental exposure and influences on the foetus development. The objectives of this study were to compare the biomarkers of metabolism with: individual genotype of polymorphic detoxifying enzymes in placenta - cytochrome P4501A1 (CYP1A1) and glutathione transferase P1 (GSTP1); polycyclic aromatic hydrocarbon (PAH) adducts in DNA; clinical status of mother and newborn. Material and methods: The placentas (186 samples) were obtained during the period 1991 - 2002 from several areas of the Ukraine and neighbouring region of Byelorussia, some exposed to high levels of radioactivity, some exposed to high levels of PAH and others exposed to low levels of both. They were classified into 8 groups according to areas of origin: the radioactively contaminated areas with higher (Group 1) and lower (Groups 2, 3) Summary Effective Equivalent Annual Exposure Dose (in mSv), where the samples were obtained in 1991 - 1993 (Groups 1, 2) and 1999 (Group 3); the chemically polluted areas monitored for ambient levels of benzo(a)pyrene (BP, ng/m3) and arranged in the order of decreasing pollution (Groups 4 - 6) where the samples were obtained in 1992 (Group 4), 1992 - 1993 (Group 5) and 2002 (Group 6); the area judged as clean (Group 7, 1992 - 1993); the group with variable pathologies of pregnancy (Group 8, 1995) from area with combined radioactive and chemical pollution at intermediate level. The parameters that were studied: GST1 and glutathione reductase (GSSG-R) 2 activities in placental cytosol; thiobarbituric acid reactive substances (TBARS) 3 and reduced low molecular weight (rLMW) thiols4; ethoxycoumarine de-O-ethylase (ECOD) activity in microsomes5; relative amount of GSTP1-specific mRNA in total placental RNA by Northern hybridisation; immunohistochemical detection of GSTP1 specific antigen; CYP1A1 Ile462Val6 and GSTP1 Ile104Val7 polymorphism; PAH-DNA adducts by competitive chemiluminescence immunoassay8. Questionnaires and clinical data concerning mother and newborn are used in analysis. Results and discussion: The comparison of metabolism biomarkers at the carriers of different genotypes has revealed the stronger impact of CYP1A1 genotype than GSTP1 one. GST and GSSG-R activities, TBARS concentration are statistically significantly higher and rLMW thiols are lower at the carriers of CYP1A1 Ile/Val genotype than at carriers of Ile/Ile genotype. GST activity and rLMW thiols decrease and GSSG-R activity increases in the row of GSTP1 Ile/Ile, Ile/Val and Val/Val isoforms. The higher is the radioactive contamination and chemical pollution the lower is GST activity and higher is the concentration of PAH-DNA adducts. But the difference between both types of exposure is evidenced in other parameters. In radioactively contaminated area down-regulation of ECOD activity, GSTP1 specific mRNA concentration in total RNA and GSTP1 specific antigen is combined with two-fold increase of TBARS concentration as an indirect index of radioactive exposition. In chemically polluted area on the contrary up-regulation of ECOD activity and GSTP1 specific mRNA is observed as an indicator of CYP1A1 and GSTP1 induction. Reducing treatment of cytosol with dithiotreitol nonsignificantly increases GST activity in the samples from radioactively contaminated areas while nearly 2.5 times increases it in the samples from chemically polluted area. We suggest that down-regulation of GSTP1 expression plays the primary role in the radioactively contaminated areas and serves as a prerequisite of PAH-DNA adducts accumulation. Up-regulation of GSTP1 expression combined with posttranslational inhibition of GSTP1 activity manifests itself in PAH-polluted areas. Despite the challenge of less number of individuals of different genotypes in each of classified group the data demonstrate contributions by both genotype and exposure in the detoxification. Lower GST activity in placental cytosol statistically significantly correlates with elevated frequencies of hypoxia, anaemia and risk of premature pregnancy interruption at mothers and with worse status of new-borns according to Apgar coefficient. Thus PAH-DNA adducts and GST activity in placental cytosol may be used as more and less specific biomarkers of environmental exposure while together with genotype of detoxifying enzymes they may serve as prognostic factors for new-borns. References: 1. Habig W. et al. (1974) J. Biol. Chem. 249, 7130-7139. 2. Carlberg J., Mannervik B. (1975) J. Biol. Chem. 250, 5475-5480. 3. Gavrilov V. et al. (1987) Voprosu Med. Chem. 33, 118-122. 4. Sedlak J., Lindsay R. (1968) Analyt. Biochem. 25, 192-195. 5. Greenlee W., Polalnd A. (1978) J.Pharmac. Exp. Ther. 205, 596-605. 6. Shields P. et al. (1993) Cancer Res. 53, 3486-3492. 7. Harries L. et al. (1997) Carcinogenesis 18, 641-644. 8. Divi R. et al. (1999) Cancer Res. 59, 4829-4833. The molecular links between oxidative damage to DNA and cancer Ryszard Olinski, Daniel Gackowski, Rafal Rozalski, Marek Foksinski, Karol Bialkowski Department of Clinical Biochemistry, The L. Rydygier Medical University in Bydgoszcz Karlowicza 24, 85-092 Bydgoszcz, Poland, e-mail: ryszardo@amb.bydgoszcz.pl A wide variety of oxidative DNA lesions are present in living cells. One of the best known lesions of this type is 8-oxoguanine (8-oxoGua), which has been shown to have mutagenic properties. Our works demonstrated an influence of antioxidative vitamins and labile iron pool on the background level of 8-oxoGua in cellular DNA (1-4). Our recently published results of analysis of urinary excretion of the oxidatively modified base/nucleoside combined with the determination of background level of 8-OH-dGuo in leukocytes DNA and measurement of 8-OH-Gua repair activity appears to predict an individual's susceptibility to tobacco-related lung cancer (5). An involvement of 8-oxoGua in the origin and/or progression of cancer will be reviewed. It is concluded that a severe oxidative stress manifested as a high level of 8-oxoGua in cellular DNA as well as in urine of cancer patients is a consequence of development of many types of cancer. Although at present it is impossible to answer directly the question concerning involvement of oxidative DNA damage in cancer etiology it is likely that oxidative DNA base modifications may serve as a source of mutations that initiate carcinogenesis (i.e. they may be causal factors responsible for the process). 1/. Gackowski D., Kruszewski M., Bartlomiejczyk T., Jawien A., Ciecierski M., Olinski R., J. Biol. Inorg. Chem. 7, 548-550, 2002 2/. Oliński R., Gackowski D., Foksiński M., Różalski R., Roszkowski K., Jaruga P., Free Rad. Biol. Med. 33, 192-200, 2002 3/. Rozalski R., Gackowski D., Roszkowski K., Foksinski M. and Olinski R. Cancer Epidemiology, Biomarkers Prevention. 11(10), 1072-5, 2002 4/. Gackowski D., Banaszkiewicz Z., Rozalski R., Jawien A. and Olinski R. Intern. J Cancer, 101, 395-397, 2002 5/. Gackowski D., Speina E., Zielinska M., Kowalewski J., Rozalski R., Siomek, A., Paciorek T., Tudek B., Olinski R., Cancer Res. 63, 4899-02, 2003 Analysis of differences in protein substitution patterns based on statistical tests Marcin Pacholczyk Silesian Technical University, Gliwice Protein substitution matrices are widely used in protein alignment algorithms. Protein similarities may reveal degree of evolutionary relatedness among different organisms. Comparison of substitution matrices derived using data from two groups of bacterial genomes with different GC content, makes possible analysis of differences in protein substitution patterns being possibly the result of dissimilar environmental conditions. The analysis was carried out using standard nonparametric tests with G statistic. Nuclear myosin I and actin are required for RNA polymerase I transcription 1Philimonenko V.V., 2Iben S., 1Dingová H., 1Kyselá K., 2Grummt I., 3de Lanerolle P., and 1Hozák P. 1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4-Krč, Czech Republic 2German Cancer Research Center, Im Neuenheimer Feld 280, D69120 Heidelberg, Germany 3University of Illinois at Chicago, Chicago, IL 60612, USA Recently, nuclear myosin I (NMI) was shown to be involved in RNA polymerase II transcription. We report for the first time that actin and NMI are required for rRNA synthesis. Electron microscopy reveals that both actin and NMI are localized within the nucleoli, and their distribution is highly specific. NMI colocalizes with nascent rRNA in the dense fibrillar component and its presence here is transcription-dependent. When nuclear extracts are fractionated, significant amounts of actin and NMI are detected in fractions containing partially purified Pol I and its transcription factors. Co-immunoprecipitation experiments demonstrate association of actin with murine initiation-competent Pol I holoenzyme complexes. The rate of Pol I transcription in vitro is dramatically reduced by antibodies directed against NMI, while addition of purified NMI stimulates transcription from mouse rDNA promoter in a dose-dependent manner. Anti-actin antibodies specifically inhibit formation of full-length transcripts, but not ACU trimers in abortive initiation assay, indicating that actin plays a role in promoter clearance or transcript elongation. Microinjections of anti-actin and anti-NMI antibodies significantly reduce the level of nucleolar transcription in cultured HeLa cells, demonstrating the requirement for these proteins for transcription in vivo. We conclude that the acto-myosin molecular motor can be generally required for the movement of DNA relative to transcription complexes. Covalent binding of anthracycline antibiotics to DNA M. Piestrzeniewicz, D. Wilmańska, M. W±sowska, K. Studzian, I. Oszczapowicz, M. Gniazdowski Department of Medicinal Chemistry, Institute of Physiology and Biochemistry Medical University of ŁódĽ; Mazowiecka 6/8, 92-215 ŁódĽ, Poland; e-mail: magn@csk.am.lodz.pl Department of Antibiotics, Institute of Biotechnology and Antibiotics; Staro¶cińska 5, 02-516 Warszawa; e-mail: oszczapowiczi@iba.waw.pl It has been largely documented that doxorubicin (DOX), daunorubicin (DRB) and their analogues with 3'NH2 group in daunosamine moiety form with the exocyclic 2NH2 group of guanine a covalent bond via a methylene group from formaldehyde (CH2O). It is postulated that a Shiff base type intermediate is formed between CH2O and NH2 group in the course of the reaction. This reaction is supposed to occur in the cell. The analogues of anthracycline antibiotics with formamidine functionality bonded to daunosamine moiety and containing bulky morpholine (DRBM and DOXM) or hexamethylene (DRBH and DOXH) rings attached are studied in our laboratory. These substituents structurally hinder formation of Schiff base-intermediates with CH2O. A decrease of DNA template transcriptional activity upon preincubation of the derivatives and DNA in the presence of CH2O and spectrophotometric estimation upon washing out non-covalently bound drugs indicate on covalent binding of morpholine analogues of DRB and DOX. Covalent binding of hexamethylene derivatives to DNA is hardly detectable under these conditions. Electrophoretic analysis of drug-DNA complexes formed in the presence of CH2O indicate that the analogues as their parent compounds induce labile "virtual" cross-links in DNA. Since HPLC analyses indicate that the drugs molecules remain integral in the condition of CH2O - dependent complex formation with DNA these experiments suggest another, yet unidentified route leading to covalent binding of amidine derivatives to DNA. Comparison of the results obtained at the subcelular level with cytotoxicity estimations indicates that there a high correlation between cytotoxicity of anthracyclines and transcriptional template activity of drug-DNA complexes formed in the presence of CH2O (r=0.792; n=9) while no correlation was found between cytotoxicity and their non-covalent interactions with DNA measured by binding constants. These data confirm a notion that covalent attachment of anthracyclines to DNA is an essential event leading to cytotoxicity. DNA repair genes: polymorphisms and risk of cancer Marek Rusin Department of Tumor Biology, Center of Oncology, Maria Skłodowska-Curie Memorial Institute, 44-101 Gliwice, Poland Infrequent, mutant versions of many genes, including tumor suppressor and DNA repair genes, are associated with significant increase of cancer risk (even 1000 fold). However, the human gene pool contains probably hundreds, or even more, frequent gene alleles modulating cancer risk only slightly, what makes their identification very difficult. In order to identify the alleles, researchers use a candidate gene approach by selecting genes plausibly involved in cancer formation. One of the selected groups of genes code for the proteins of DNA repair. An allele may be regarded as the bona fide marker of cancer risk if its influence on cancer formation was shown in a large, well controlled case-control analysis, preferably performed on different populations and by different research teams. Equally important are the functional, mechanistic studies on the link between the change of DNA or protein sequence and alteration of protein activity as well as the influence of altered protein functioning on cell and tissue physiology. Judging by those standards, there are only a few alleles of DNA repair genes, whose influence on cancer risk have been relatively well documented. The strong candidates for the true cancer risk markers are: hOGG1 Ser326Cys, XRCC1 Arg194Trp, BRCA2 Asn372His polymorphisms. The major findings published by others on these polymorphisms will be presented together with the epidemiological data and results of functional experiments performed in our laboratory on polymorphic sequences of MGMT - DNA repair gene and HSC70 - stress-response gene. Nucleotide excision repair - new role in oxidative damage repair? Joanna Rzeszowska-Wolny Department of Experimental and Clinical Radiobiology Maria Sklodowska-Curie Center of Oncology, 44-100 Gliwice, Poland Individuals differ in their reaction to the same dose of genotoxic agents and in DNA repair kinetics and efficiency (Palyvoda et al. 2002). As has been shown for aromatic DNA adducts and UV-induced DNA damage, these differences in response to genotoxic factors may depend on the genetic background and particularly on the existence of polymorphic variants of genes coding for DNA repair enzymes (Matullo et al. 2001, Qiao et al. 2002, Spitz et al. 2001). To examine this hypothesis in respect to ionizing radiation, we assessed the effect of polymorphisms of repair genes coding proteins which take part in different repair mechanisms on induction and repair of single strand breaks and on the frequency of micronuclei and of apoptosis in lymphocytes from different donors exposed to X-radiation in vitro. The results concerning polymorphism of XRCC1, XPD, XPA and MGMT will be presented. Ionizing radiation induces single and double strand breaks and a spectrum of oxidative damage in DNA molecules. While the breaks are repaired by homologous recombination or nonhomologous end joining mechanisms, oxidative damage is mainly removed by base excision repair (BER) although recently interconnections between BER and nucleotide excision repair (NER) have been shown in yeast and bacteria and mutations in genes coding for NER reduce the level and rate of global removal of X-irradiation-induced DNA damage (Gellon et al. 2001, Doetsch et al. 2001, Weinfeld et al. 2001). Comparison of the results of DNA repair in irradiated lymphocytes obtained from donors carrying different polymorphic variants of repair genes showed that polymorphism in genes coding for XPD and XPA proteins correlates with the change in kinetics of DNA repair and influences the induction of apoptosis. XPD gene codes for a helicase, which is a component of TFIIH. This multiprotein and multifunctional factor is required for both NER and transcription initiation by RNA polymerases I and II (Weber et al. 1990, Reardon et al. 1996, Hoogstraten et al. 2002). XPA protein is also the component of NER mechanism. Polymorphism of XRCC1 protein engaged in BER did not influence the lymphocyte answer to irradiation. Our results suggest that NER takes part in repair of ionizing radiation-induced DNA damage and also that polymorphism in genes coding for proteins of this pathway can influence an individual's response to ionizing radiation. Supported by Grant 4 P05A 015 19 from the State Committee for Scientific Research Chromosomal aberrations and cancer risk Maria M. S±siadek Department of Genetics, Wroclaw Medical University, Wroclaw, Poland Process of carcinogenesis is closely related to the accumulation of genetic alterations in a single cell. This accumulation results from an imbalance between the induction of DNA-damage and its repair, which is one of the most important factors modulating individual susceptibility to mutagens and thus the individual cancer risk. Alterations in DNA repair machinery results in genomic instability, which manifests itself as chromosomal or molecular instability. Chromosomal instability is one of characteristic features of cancers. In solid tumours, as well as in haematological malignancies, chromosomal instability is expressed by accumulation of structural and numerical aberrations. Chromosomal aberrations can play a key role in cancer initiation and progression, or can be a feature of genetic instability that cells acquire during tumour development. The increased chromosomal instability (both, spontaneous and induced) has been observed not only in tumour cells but also in the normal tissues of cancer patients. The in vitro susceptibility to clastogenic effect of bleomycin has been accepted as a predictor of cancer risk. However, it can be used only for a group not for an individual prognosis. Therefore, there is still an unanswered question how to employ the cytogenetic methods in the evaluation of individual cancer risk. Manipulation of flax for improved fibre extractability Urte Schlüter Risoslash; National Laboratory, Denmark Flax (Linum usitatissimum L.) is a multipurpose crop of great antiquity. The flax fibres are long, thin and lustrous and can produce textiles and linen cloth of great quality. More recently flax also provoked interest as a source for novel industrial purposes including fibre reinforced plastics and pulp and paper applications. Unfortunately, the processing of flax still has to rely on the initial microbial decomposition of the fibre cell walls (retting) - a method that is time- and area-consuming and often rather unreliable. During the retting process hemicelluloses, pectins and lignins are loosened from the cellulose fibres and manipulation of the fibre cell wall composition could help to improve extractability of the fibres. To test this, two different approaches have been chosen. Firstly, fungal pectinases will be introduced into the flax plant and expressed in the stem in order to weaken the pectin bondage between the fibres. Secondly, the fibre cell wall composition will be manipulated by downregulation of hemicellulose and lignin related enzymes. The obtained transformants will be tested for their retting characteristics and fibre quality. Green tea in cancer prevention Elżbieta Skrzydlewska Department of Analytical Chemistry, Medical Academy of Białystok, Poland The critical action during carcinogenesis is transformation of normal cells by genotoxic carcinogens [chemicals, radiation or viruses], which affect specific codons in DNA and represent a somatic mutation of oncogenes or tumor suppressor genes. Growth and development of cells with such a modified DNA and additional alterations of the genetic elements, leads to typical neoplastic cells with the characteristic gene structure and phenotypic expression. Some epidemiological studies have found an inverse association between green tea consumption and the risk of cancer. It has been shown that green tea can block the formation of mutagens and carcinogens from precursor. Besides that green tea and their polyphenols inhibit the biochemical activation of genotoxic carcinogens. Moreover green tea increases their detoxification through the induction of metabolic enzymes - related phase I enzymes and phase II enzymes. Tea polyphenols also influence molecular events at the level of the gene. EGCG effects an action of tumor promoters on transcription factors such as AP-1 or NF-kB, what lead to control of the activity of transforming growth factors TGF-alfa and TGF-ß. Genotoxic carcinogens and also oxidative processes enhanced during carcinogenesis in cells lead to formation of reactive oxygen species that alter DNA. Green tea, as a nonoxidized-nonfermented product receiving from tealeaves, contains several polyphenolic components, mainly flavonoids such as epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate, which possess antioxidant properties. The possible mechanism of green tea as a typical natural antioxidant is connected with inhibition of activity of enzymes participating in free radicals generation, such as xanthine oxidase, lipooxygenases and cyclooxygenases and connected with induction of antioxidant enzymes such as superoxide dismutase. Subsequent steps in the development of neoplasia involve the growth control of the early neoplastic cells. The active ingredients of tea decrease effectively these sequences. Last results suggest that the gallate structure of catechins is important for growth inhibition of tumor cell lines by these compounds. The effect of promoters involves blockage of cellular growth control messages through gap junctions and tea polyphenols restore effective gap junction communication and hence inhibit the action of promoters. Despite of proved antioxidant and cancer chemoprotective properties of main components of green tea i.e. catechins, studies of last years on several human cohort and case-control have indicated significant positive relationships between green tea consumption and cancers of various organs. Thus the problem of participation of green tea in cancer prevention is still open. Targeted chemotherapy. Principles, new targets, challenges for the future Beata Utracka-Hutka Center of Oncology, Gliwice, Poland 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. Many of them are already used in the clinic improving patients outcome and giving new hope for the future. Biological implications of failure in solid tumors treatment Małgorzata Wierzbicka Institute of Human Genetics PAS, Department of Otolaryngology, Head Neck Oncological Surgery, Poznan University of Medical Sciences, Poznan, Poland Objective: Loco-regional relapses, distant metastases and second tumour development constitute majority of head and neck cancer (HNSCC) treatment failures. Occurrence of second primary tumours after curative treatment is current problem in contemporary oncology. The mutagen sensitivity is well known marker to predict patient proneness to develop the second tumour. Another aspect is influence of genes on individual susceptibility. GST, CYP and CCND1 genotypes have been associated with HNSCC outcome. In this report a prospective, case-control study in which we determined the induced mutagen sensitivity i.e. frequency of mutagen-induced chromatid breaks (breaks per cell; b/c) in peripheral blood lymphocytes (PBLs) in patients with multiple primary tumours (MPT) compared with patients with single HNSCC and healthy controls is presented. The relationship between spontaneous and induced mutagen sensitivity and age, smoking, alcohol consumption, index and second primary tumour site and interval between both tumors occurrence was estimated for MPT group. GSTM1, GSTT1, GSTM3, CYP1A1, CYP2E1XPD, XRCC1 and XRCC3 genotypes were analysed. Material and method: 36 patients with MPT and two control groups: 52 patients with one malignancy and 47 healthy individuals were analysed. The differences between examined patients and control groups were estimated and differences among the patients with MPT spontaneous and induced b/c level were compared using U Mann-Whitney and Spaerman Rank Correlation. Results: The b/c level in PBLs of patients with MPT ranged from 0,26 to 4,12 (mean 1,53) and was significantly higher (p<0,000006) both compared with patients with one malignancy (b/c ranged from 0,02 to 3,08; mean 0,74) and healthy controls (b/c ranged from 0,04 to 1,14; mean 0,41). The spontaneous b/c level in PBLs of patients with MPT ranges from 0 to 0,22 with mean score b/c=0,84. Induced b/c scores between tumors deriving from one tissue, i.e. HNSCC and tumours of different sites and histologicaly divergent were not statistically significant (p=0,051) whereas spontaneous mutagen sensitivity in patients with SCCHN (b/c=0,07) have been statistically lower then in patients with MPT of different sites and histology (b/c=0,13; p=0,012). Spontaneous mutagen sensitivity in patients with smoking-related tumours (b/c=0,07) has been lower than in patients with not smoking-related MPT (b/c=0,11; p=0,05). An increase of b/c index was observed in almost all chromosomal arms. The majority of chromosomal locations with the increased proportion of breaks in the group of patients with multiple tumours were identified as regions where loci involved in DNA repair, cell cycle regulation suppressor genes and oncogenes were found. GSTM1 , CYP1A1 1/4 and combinations of: GST T1 /GST M1 , GST M null/CYP 1A1 and GST M null/CYP 1A1 were also associated with MPT group. Conclusions: Statistically higher induced individual susceptibility in MPT patients compared with single tumour and healthy controls were confirmed. Comparable induced mean b/c was found in patients with two smoking-related cancers as well as with not smoking related tumours. In opposite, baseline spontaneous b/c in MPT localization distinguished according to exogenous factors compliance were found; more sensitive occurred to be individuals who develop not smoking related tumour or second primary different then HNSCC. Flax engineering for fibre improvement Magdalena Wróbel1, Jacek Żebrowski2 and Jan Szopa1 1Institute of Biochemistry and Molecular Biology, University of Wrocław, Przybyszewskiego 63, 51-148 Wrocław, Poland 2Plant Biotechnology and Cytogenetics Dept., Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland Flax (Linum usitatissimum L.) is an annual plant cultivated in temperate climate. Although the whole genus Linum has about 230 species, the only one, Linum usitatissimum, remains a very important plant of commercial use, serving as a fibre donor for the textile industry and linseed oil production. In order to improve the properties of flax fibres, which are of lower quality than those of cotton origin, transgenic plants synthesizing polyhydroxybutyrate (PHB) in stem tissue were generated and analysed. Poly-ß-hydroxybutyrate (PHB) is a hydrophobic, thermoplastic polymer produced by numerous bacteria as a source of carbon and energy. PHB can be called 'green' plastic, because it is completely degraded by microorganisms in the different environmental conditions, like soil or seawater. In this study bacterial pathway of PHB synthesis was transferred into flax by Agrobacterium mediated transformation. Three genes, used throughout this study, encoding enzymes necessary for PHB production derived from Ralstonia eutropha cells. We have prepared two constructs, a multigene construct that contains three genes for PHB production (phb A, phb B, phb C) and a single construct bearing only phb A gene encoding E-ketothiolase, the first enzyme of PHB biosynthetic route. E-ketothiolase was suggested to be rate-limiting enzyme for PHB production and it was placed under the control of recently isolated and characterized 14-3-3 promoter. Acetyl-CoA is the substrate needed in the PHB synthesis. Since there is a high amount of acetyl-CoA in chloroplasts, the PHB enzymes were directed into flax plastids. Transgenic plants obtained after transformation with single construct mainly served as a transformation and regeneration control. The level of PHB in selected transgenic lines was analysed using the GC-MS method. Transgenic plants produced over 70-fold higher level of polymer than untransformed, wild type plants. The PHB accumulation in plastids caused changes in their shape and size, which were confirmed using transmission electron microscopy. Biomechanical parameters of obtained transgenic plants were characterized. Young's modulus (E) was measured and compared to control plants. The E parameter ranged from 24.1 to 54.4 MPa in plants transformed with the triple construct (26.9 MPa for control plants). Thus the increase in PHB level affects the mechanical properties of flax stem. Since acetyl-CoA is a key compound in many metabolic processes occurred in cells, the metabolite profile of transgenic plants was determined. Polymer production caused the alterations in the metabolite composition, for example: some amino acids (e.g. lysine), fatty acids (e.g. 18:0, 18:2, 16:0) and citrate cycle intermediates (e.g. citrate, isocitrate) were decreased in transgenic plants. However the PHB accumulation did not have any negative effect on fertility or growth of transgenic lines, which were observed in other species and when 35S CaMV promoter was used for the expression of PHB genes. The obtained transgenic plants with higher PHB content and improved mechanical properties can serve as a source of industrially important flax cultivars. Transgenic crops in the world Janusz Zimny Plant Breeding and Acclimatization Institute, Radzików, Poland World population, now 6.0 billion, has doubled during last 40 years and is expected to grow to 10 billion, by 2050. Some 2 billion people already lack food security and about 800 millions are suffering hunger. There is a hope that genetically modified crops could help to solve this problem. Genetically modified crops were introduced in agriculture in 1994. According to Clive James between 1996 and 2002 the global area of GM crops cultivation, has increased from 1.7 million hectares to 58.7 million hectares. The use of GM crops was in 2002 limited mainly to four countries: USA (66%), Argentina (23%), Canada (6%) and China (4%). Only two European Union countries Spain and Germany were growing GM crops in 2001 in very small amount. However other European countries (Rumania and Bulgaria) started to grow GM crops also. The number of countries growing GM crops was 16 in 2002, with new users like India, Colombia and Honduras. Since 1996 adoption of transgenic plants in agriculture has been very crop-specific. Mainly herbicide tolerant crops such as soybean and cotton and insect resistant crops such as Bt cotton and Bt maize have been cultivated. Although it seems that the use of GM crops is expanding rapidly there is still a very controversial discussion around potential environmental and health risks and unclear benefits. With respect to the above, it is a need to create a biological safety system that among others should result from the Convention on Biological Diversity and the Protocol on Biological Safety signed in Cartageny. The biological safety system is a global issue and its goal is to ensure a safe use of present and future GMO's. Using Gaussian mixtures to infer structure in microarray data Joanna Polańska Institute of Automatic Control, Silesian University of Technology, ul.Akademicka 16, 44-100 Gliwice, e-mail: jpolanska@ia.polsl.gliwice.pl The aim of the talk is to present application of the Gaussian mixture model to the analysis of DNA microarray data. The most characterisic property of DNA microarray data on genes expressions profiles is the existence of very large number of measurements with only few related or strongly related to the result of the conducted experiment. Therefore it is necessary to develop methods to select genes that take part in the studied process. One obvious method is repeating experiments and selecting genes by using the hypothesis that expressions of genes urelated to the experiment results will take random values in the subsequent measurements, while those involved in the process will always be either over or underexpressed. However, repeating experiments many times can be very expensive. Also selecting genes by oversimplified criteria will obviously lead to loss of some information. One of the alternative approaches, which can be used when the number of repetitions is low or even when there is only one experiment, is the method of Gaussian mixtures. Gaussian mixtures method allows approximating any probability density function with arbitrary precision, by a sum of normal pdfs. The use of Gaussian mixtures method has a twofold effect: (1) gives predictions of probabilities of results of experiments, (2) provides information about the structure of the analyzed data by estimating numbers and parameters (means, standard deviations) of normal components. We give arguments that the Gaussian mixture method can be very useful in the analysis of DNA microarray data. The hypothesis, which can be used to select genes in experiments, is that normal components or some compositions of normal components in the observed data, correspond to the processes that take part in the studies sample. We present numerical methodology necessary to estimate normal components by maximizing likelihood function. Two main approaches to solve likelihood maximization are Expectation Maximization (EM) method and Metropolis Hastings sampling. We illustrate the method by showing the use of the Gaussian mixtures for the analysis of the expression profiles data on the Bystander effect comprising genetic changes induced in unirradiated cells by signals emitted from irradiated cells. Expression profiling by DNA microarray in papillary thyroid cancer Barbara Jarz±b*, Jan Włoch1, Dariusz Lange2, Małgorzata Wiench*, Krzysztof Fujarewicz3, Krzysztof Simek3, Małgorzata Oczko*, Agnieszka Czarniecka1, Ewa Chmielik2, Elżbieta Gubała*, Michał Jarz±b4, Joanna Hucz*, Monika Gałęza-Kulig*, Andrzej ¦wierniak3. *Dept. of Nuclear Medicine and Endocrine Oncology, 1Clinic of Oncological Surgery, 2Dept. of Tumor Pathology 4Dept. of Tumor Biology, Maria Sklodowska-Curie Memorial Institute, Gliwice Branch, Gliwice, Poland 3Silesian Technical University, Gliwice, Poland Functional genomics offers new possibilities of looking for genes participating in malignant transformation and allows to improve cancer classification. However, only a few papers apply this technique to thyroid cancer. This seems important not only from the diagnostic point of view. The molecular mechanisms leading to different types of thyroid tumors are not completely understood. RET protooncogene activation, as a consequence of chromosomal rearrangement, is regarded at present as the most important initiating event in the development of papillary carcinomas. However, in those papillary thyroid tumors which are RET-negative the molecular mechanisms of carcinogenesis are unknown. In the present study the expression profiling by high density oligonucleotide microarray was applied to analyze following problems: 1. Does expression profiling give a diagnostically useful set of genes for differential diagnosis between papillary thyroid carcinoma and normal/benign thyroid tissue? 2. Are there any differences in gene expression related to the presence of RET rearrangements which may be detected by DNA microarray analysis? For this analysis, 23 papillary thyroid ca samples were obtained during thyroid surgery of 17 females and 6 males aged 5-71 years. 16 patients were younger than 45 years at surgery. Seven of them exhibited the presence of RET rearrangement, as judged by RT-PCR. All were euthyroid during thyroid surgery with TSH range from 0,88 to 3,06 mU/l. PTC samples and corresponding normal thyroid tissues were frozen immediately after thyroid surgery. All samples were hybridized to GeneChip U133A arrays. 16 paired tumour/ normal tissue samples were included into training set. Different approaches were used to analyze gene expression data. Affymetrix Data Mining Tool software was used for paired analysis, an own method of unpaired analysis for the selection of non-overlapping genes was also applied. Some of the selected genes were subsequently checked by real-time Q-PCR and a good correlation was shown (correlation coefficients exceeded 0.9 for 7 of 8 analysed genes). However, none of the selected genes proved to be an ideal marker of papillary thyroid cancer at univariate analysis. Thus, a neural-network based analysis was applied. We used three different methods of gene preselection (among them two supervised methods and Singular Value Decomposition as an unsupervised method) followed by Support Vector Machine technique with a linear kernel for classification. All the methods gave a very clear separation of gene expression profiles in tumours and normal samples, however, the sets of genes obtained showed some differences which need further analysis to choose the optimal method for diagnostic purposes. The set of 31 genes chosen by Recursive Feature Replacement was able to categorize tumour tissue properly, when the tumour contained at least 30% of cancer cells. The subdivision of analyzed PTCs into RET-positive and RET-negative tumours has not revealed a clearly distinct expression pattern. Our results corroborate the previously postulated rather stable molecular profile of PTC and provide new clues for diagnostic purposes as well as specify new genes to be analysed for their significance in malignant transformation of thyroid cells. Simultaneously, the data obtained until now do not indicate any clear difference in expression profile dependent on the presence of RET rearrangements. Supported by grant PBZ/KBN/040/P04/2001 |
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