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		Gliwickie Spotkania Naukowe 2005 Gliwice Scientific Meetings 2005 18-19 November 1. INFLUENCE OF TEMPERATURE ON RADIATION-INDUCED MICRONUCLEI IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES Kinga Brzozowska1 and Andrzej Wojcik1,2 1Institute of Nuclear Chemistry and Technology, Department of Radiation Biology and Health Protection, Warsaw, Poland 2Swietokrzyska Academy, Institute of Biology, Department of Radiobiology and Immunology, Kielce, Poland The level of cytogenetic damage induced by ionizing radiation under in vitro conditions in human peripheral blood lymphocytes is analyzed for the purpose of establishing calibration curves used in biological dosimetry and for assessing the intrinsic radiosensitivity of the blood donor. The irradiation of blood should be performed under strictly controlled physical conditions that allow a high reproducibility of the dose. A factor that is often not regarded is the control of blood temperature during exposure. Available data on the influence of blood temperature on the level of cytogenetic damage is scarce and somewhat contrdictory. We have, therefore, performed experiments to analyze the impact of blood temperature on the level of radiation-induced micronuclei. Blood was exposed to different doses of X-rays (200 kVp, 5 mA, 3 mm Cu filter) at 0, 20 and 370C. Thereafter a standard micronucleus test was performed and micronuclei were analyzed optically on microscopic slides. The results of the experiments will be presented and discussed. 2. MODULATION OF THE ACTIVATION OF NF#922;B AND THE EXPRESSION OF NOS-2 AND COX-2 IN MOUSE EPIDERMIS BY TANNIC ACID - NATURALLY OCCURRING PLANT PHENOL Michał Cichocki, Wanda Baer-Dubowska Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland e-mail: cichocki@amp.edu.pl Tannic acid (TA), the mixture of digallic acid esters of glucose, ubiquitous in edible plants, has been shown to possess antimutagenic and anti-carcinogenic activity in several experimental models, including mouse skin. Mouse skin is one of the best animal models of chemical carcinogenesis which enables to study all stages of this process. Although most human skin cancers are not induced by chemicals, many events in this model could be extrapolated to humans. Moreover the biochemical changes observed in the mouse skin after application of tumor promoter, phorbol ester, 12-O-tertadecanoylphorbol 13-acetate (TPA) are the same as those in humans after UVB radiation. Our earlier studies [1,2] showed the inhibition of the formation of 7,12-dimethylbenz[a]anthracene-diol-epoxides (DMBADE)-dAdo adducts by tannic acid treatment in vitro and in vivo in mouse skin. The aim of the present study was to evaluate weather the inhibition of epidermal DNA adducts formation, which may affect the c-Ha-ras mutation by DMBA, may also influence the activation of NFB and the expression of the enzymes controlled by this transcription factor. We determined the effect of topical application of TA on TPA-induced NF#61547;B subunits IkBα (p35) degradation, and p65 nuclear translocation and DNA binding by Western blot and ELISA assay. The activity and expression of inducible nitric oxide synthase (NOS-2) and cyclooxygenase 2 (COX-2) was measured using the same experimental protocol by specific enzyme activity assays and Western blot. Pretreatment of mice with 16 µmoles of TA 15 minutes before TPA treatment, resulted in significant enhancement of IkBα retention in cytosol (2-fold, in comparison with TPA treated group). Nuclear translocation of p65 and p65-DNA binding caused by TPA was diminished by 60% and 50% respectively. TA significantly reduced the NOS-2 and COX-2 activities and their protein level. NOS-2 activity was reduced by 75% and its protein level by 40%, and COX-2 reduction was by 220% and by 140%, respectively. These results indicate that polyphenol TA affect the NF#61547;B activation which might be related to potential protection against c-Ha-ras mutation. Collectively the results of our present and earlier studies suggest that TA is capable of affecting more than one critical pathway of carcinogenesis and thus will have greater advantage over other single-target potential chemopreventive agents. This work was supported by the State Committee for Scientific Research Poland grant 4P05F049 26 References: 1. Ignatowicz E., Balana B., Vulimiri SV, Szaefer H., Baer-Dubowska W. (2003) Toxicology 189, 199-209. 2. Szaefer H., Cichocki M., Brauze D., Baer-Dubowska W. (2004) Nutrition and Cancer 48, 70-77. 3. SEVERE OXIDATIVE STRESS IN NEWBORN PIGLETS Tomasz Dziaman1, Marek Foksiński1, Daniel Gackowski1, Jolanta Guz1, Rafał Różalski1, Agnieszka Siomek1, Anna Szpila1, Romuald Zabielski2 and Ryszard Oliński1, 1Department of Clinical Biochemistry, The Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University; Karlowicza 24, 85-092 Bydgoszcz, Poland 2Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Science, ul. Instytucka 3, 05-110 Jabłonna, Poland Reactive oxygen species (ROS) can damage different kinds of biomolecules, including proteins, lipids and DNA. Therefore, organisms developed a series of primary antioxidant defenses to protect against these lesions. However, in humans as well as in other mammals during early life the antioxidant systems are poorly developed. Moreover, partial pressure of oxygen in extrauterine environment is much higher than in a womb. Although some studies suggest that increased oxidative stress in newborns may be responsible for many neonatal diseases the specific biochemical markers which can predict the onset of ROS induce damage are largely undefined. The aim of the present study was to evaluate the oxidative status in healthy full-term newborns piglets in first month of their development. We assayed urinary excretion of 8-oxoGua, 8-oxodG and 5HMUra in newborn pigs using HPLC/GS/MS methodology. In addition concentrations of vitamins A, C and E were analyzed in blood serum of newborn piglets (HPLC with UV-absorbance and fluorescent detection). Our experiments demonstrated that the level of all measured oxidatively modified bases/nucleotides excreted with urine increased sharply, shortly after the birth of piglets. Four days after the birth concentration of antioxidant vitamins increased significantly over the level observed in first day. From this moment on, the level of the vitamins dropped to the value characteristic for an adult animal. Simultaneously, excretion rates of the analyzed oxidatively modified bases/nucleosides decreased to the levels characteristic for adult pig. This findings combine indicate that antioxidant vitamins play a major role in the protection against oxidative DNA damage in newborns, scavenging ROS generated shortly after the birth. 4. GENE EXPRESSION SIGNATURE OF HEREDITARY BREAST CANCER Volha Dudaladava1,2,x, Michał Jarząb1, Krzysztof Simek3, Jolanta Pamuła1, Wioletta Pękala1, Tomasz Huzarski4, Jan Lubiński4, Ewa Grzybowska1, Katarzyna Lisowska1 1Department of Tumor Biology, M. Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland 2Permanent address: Institute of Genetics and Cytology, NAS of Belarus, Minsk, Belarus 3Institute of Automatic Control, Silesian University of Technology, Gliwice 4International Hereditary Cancer Center, Pomeranian Medical University, Szczecin, Poland Introduction: Hereditary breast cancer associated with mutations in BRCA1 gene is characterized by some pathological and clinical features that are distinct from sporadic breast cancer. These are e. g. earlier age of manifestation, high tumor grade, high proliferation index, characteristic pushing margins and lymphocyte infiltrate. Despite so bad prognostic indications, it is frequently observed, that patients with BRCA1 mutations have overall survival similar to patients with sporadic breast cancer. The aim of this study was an attempt to elucidate molecular basis underlying described discrepancy by comparing gene expression profiles of BRCA1-associated hereditary breast cancer with sporadic breast cancer cases. Material and methods: We analyzed gene expression in 28 tumor samples obtained from 27 patients: 20 patients with hereditary breast cancer (11 with BRCA1 mutation, 1 with BRCA2 mutation and 8 without proven BRCA mutations) and 8 patients with sporadic breast carcinoma. We also analyzed 6 normal breast tissues. We used HG U133 Plus 2.0 (Affymetrix) oligonucleotyde microarrays. Target preparation, hybrydization and staining were done according to Affymetrix instruction manual. Preprocessing of the microarray data was performed by Robust Multiarray Analysis (RMA). Hierarchical clustering, statistical comparisons analyses and Principal Component Analysis (PCA) and were carried out in GeneSpring 7.2 software (Silicon Genetics). For gene selection, we used both non-parametric Mann-Whitney test and parametric Welch test, False Discovery Rate was estimated by Benjamini-Hochberg algorithm. Singular Value Decomposition was used to obtain orthogonal vectors called characteristic modes or supergenes which represent major independent variability patterns in analyzed data. Quantitative RT-PCR analysis was done using the ABI PRISM 7700 Sequence Detection System instrument and software (Applied Biosystems), and MasterAmp Real-Time RT-PCR Kit (Epicentre). Results: We compared expression profile of over 47 000 transcripts in BRCA1 mutation-linked and BRCA1 mutation negative breast tumor tissues. We were able to select a set of 45 genes differentiating between both types of cancer; however we found that difference rather discrete and connected mostly to the tumor hormonal status. Interestingly, we found, that a difference in gene expression profile between BRCA1( ) and BRCAx tumors is more significant that between BRCA1( ) and sporadic tumors. We found, that several genes connected with selenium metabolism or requiring selenium for it's enzymatic activity, show differential expression between breast cancer and normal breast tissue, as well as between BRCA1( ) and BRCA1(-) tumors. These data were validated by Real-Time RT-PCR. These findings may be of potential practical value in accordance to the attempts of prophylactics of breast cancer in mutation carriers by selenium diet supplementation. The study was supported by the Ministry of Science and Information Society Technologies (grant PBZ-KBN-040/P04/2001). xV.D. is a fellow of a Fellowship Program totally supported by the National Cancer Institute - Office for International Affairs, NIH, Bethesda, MD, USA 5. POLYMORPHISM OF DNA REPAIR GENES, RELATION TO THE REPAIR PROCESS AND CANCER RISK A. Gdowicz, B. Lubecka, R. Oliński, J. Rzeszowska-Wolny Centre of Oncology, M.Sklodowska-Curie Memorial Institute, 44-100 Gliwice The level of DNA damage after exposure to genotoxic factors, the rate of its repair, and the efficiency of repair vary considerably in cells of the same type from different individuals. The genetic background is likely to be one determinant of this diversity of responses, and in particular polymorphism in the coding or regulatory regions of genes which encode enzymes for DNA repair, a factor which also affects the risk of cancer. The aims of the present study were: * To estimate the changes in the risk of cancer that correlate with the presence of polymorphic variants of the genes NBS1 (Gln185Glu), APE1 (Asp145Glu), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), XRCC1 (Arg399Glu). * To study the influence of these polymorphic forms on the process of DNA repair. Polymorphisms were detected by PCR-RFLP using DNA isolated from frozen blood by standard SDS-proteinase K and RNase digestion and phenol-chloroform extraction. Functional tests were performed on lymphocytes isolated from fresh peripheral blood on gradients of Ficoll-Histopaque and exposed in vitro to ionizing radiation. Comet and micronucleus tests were used for assessement of DNA damage and repair. The experimental results for samples obtained from 46 head and neck, 33 lung, 30 cervix and 45 colon cancers patients and 84 healthy donors will be discussed. The work was supported by grant PBZ-KBN-091/PO5/2003/55 6. THE EFFECT OF I.P. ADMINISTRATION OF DIETHYLNITROSAMINE ON THE DNA DAMAGE IN BLOOD LEUKOCYTES IN RATS Ewa Ignatowicz, Wanda Baer-Dubowska Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences, Grunwaldzka 6, 60780 Poznań, Poland e-mail: eignato@amp.edu.pl Oxidative stress related to carcinogen metabolism and activation is important pro-mutagenic and pro-carcinogenic factor as the reactive oxygen species (ROS) play role in all stages of tumorigenesis. Diethylnitrosamine (DEN) is a food-derived carcinogen of gastro-intestinal tract in humans and it is commonly used for the induction of liver cancer in laboratory animals. After metabolic activation by cytochrome P450 (CYP 2E1) DEN becomes an indirect alkylating agent generating the DNA-ethyl adducts and induces oxidative stress due to the contribution to inflammatory reactions. The resulting generation of ROS, induction of NF#954;B and subsequently of iNOS and release of massive amounts of nitric oxide creates the pro-oxidative environment in which oxidative DNA damage occurs. The aim of current study was the assessment of various i.p. doses of DEN (1, 15, 60 and 150 mg/kg body weight) on the DNA damage in peripheral blood leukocytes in male Wistar rats, measured in the single cell alkaline gel electrophoresis (Comet assay). The rate of the DNA damage correlated to the increasing dose of DEN. At the doses of 1 and 15 mg/kg b.wt. no effects in comparison to control were observed (92 and 122% of control value, respectively). DEN administered in the doses of 60 and 150 mg/kg b.wt. caused significant DNA damage (129% and 164% increase in comparison to control, respectively) in the peripheral blood leukocytes. The observed rate of the DNA damage may result from the oxidation of nucleic acid as well as from the formation of adducts. Since animals were sacrificed 24 hours after the DEN administration, a contribution from the repair enzymes should be taken into consideration. The lack of effects from lower doses might be then understood as no direct DNA oxidation and/or more efficient damage repair. Accordingly, in case of higher doses, the overall effect might be the result of increased oxidation and/or insufficient repair. The obtained results indicate that the alkaline comet assay appeared to be a sensitive test for the DNA-damaging effects of carcinogen. The peripheral blood is easily available and the changes observed in leukocytes may reflect the changes appearing in the target organ(s). 7. COMPARISON OF EXPRESSION PROFILE IN BREAST AND OVARIAN CANCER M. Jarząb1, V. Dudaladava1,2, K. Simek3 1Department of Tumor Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland 2Institute of Genetics and Cytology, NAS of Belarus, Minsk, Belarus 3Dept. of Automatic Control, Silesian University of Technology, Gliwice, Poland Background: In majority, microarray studies exploit the differences between cancer and corresponding normal tissues or the molecular differences between tumor histotypes originating from one tissue. However, sound understanding of neoplastic transformation and progression will benefit from comparison of tumors originating from diverse tissues, especially if they share some biological or clinical properties. Such analysis may aid to seek novel therapeutic targets, which are rather tumor-specific than tissue-specific. The aim of our study was to compare the expression profile in breast and ovarian cancer, two female adenocarcinomas with similar genetic background and comparable chemo- and radiosensitivity. Methods: We compared expression profiles of 29 breast carcinomas (BC, analyzed within the project of gene expression profiling of hereditary and sporadic breast cancer, all grade 3 ductal or medullary tumors, 17 ER- ductal carcinomas, 7 ER- medullary ca samples and 5 ER positive tumors) and 16 serous ovarian carcinomas (OC, analyzed within the project of chemosensivity assessment by microarray analysis, all serous ovarian carcinomas, majority grade 3, three samples grade 2). We used GeneChip U133 2.0 Plus microarray and standard amplification procedure. We applied two methods of data pre-processing, GC-RMA and MAS5 algorithm, and compared the obtained results. Results: It seemed clear that two different cancers shall exhibit different expression profiles. We measured the extent of difference between both groups and showed that the number of selected genes depends on the method of preprocessing (14790 genes by MAS5, 12843 by GC-RMA, FDR<5%). Moreover, by various preprocessing approaches different genes are missing. MAS5 algorithm is known to give large variance in low-expressing transcripts, and many low abundance genes were absent in MAS5-obtained data, as compared to GC-RMA. However, GC-RMA (which gives better precision than MAS5), due to quantile normalization which flattens the differences between two significantly distinct subclasses, was loosing many intermediate/high abundance transcripts. In the next step, we performed the unsupervised analysis of BC and OC expression profiles. By Singular Value Decomposition we revealed that the samples were divided into three large clusters, which corresponded to two groups of breast carcinomas (BC1 and BC2) and a separate group of ovarian cancers (OC). These groups were properly separated by expression of two estrogen-related genes, ESR1 and GATA3, which were low in BC1, showed variable and moderate expression in OC and very high expression in BC2. Both these genes were present in the third mode of SVD analysis. We performed the supervised comparison between breast and ovarian tumors. We found out that one of the most upregulated GO classes in breast cancer compared to ovarian ca are the wound-response genes. We compared the expression of genes presented by Chang et al. (signature obtained by serum stimulation of fibroblasts), and we revealed that some of these genes differentiate between breast and ovarian cancer, including AZGP1, PRLR and LTF. To base the comparison of both classes on well-described transcripts, we also used the signature of neoplastic transformation, proposed by Rhodes et al. [2004] in a large meta-analysis of 40 cancer datasets. From 168 probesets that were corresponding to Rhodes genes, majority of genes showed no differences between both classes. 30 transcripts were differentially expressed between BC and OC (the strongest differences were within KDELR2, PLK1, PPP2R5C, ACLY, G3BP, MMP9, TRA1, HSPD1). Conclusions: There are similarities in expression of neoplastic transformation signature genes between breast and ovarian carcinomas. However, such a comparison requires a careful selection of preprocessing method, which may strongly influence the obtained results. The important factor of similarity between the subgroups of breast and ovarian tumors is the estrogen receptor expression. The study was supported by the Ministry of Science and Information Society Technologies(grant number 3 P05A 060 25). 8. FUNCTIONAL INTERACTIONS BETWEEN APOPTOTIC NUCLEASE DFF40/CAD AND HISTONE H1 Magdalena Kalinowska, Xu Lu, Jeffrey C. Hansen, Missag H. Parseghian, William T. Garrard and Piotr Widlak The major apoptotic nuclease, DNA fragmentation factor (DFF40/CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of programmed cell death. Previously we have demonstrated that several chromatin proteins, including HMGB1/2, histone H1 and topoisomerase II, greatly enhances naked DNA cleavage by this nuclease in vitro (histone H1 stimulates DFF40/CAD cleavage of DNA ~20-fold). Here we investigate the mechanism of stimulation of DNA cleavage by histone H1. Addition of histone H1 either during or after caspase-3 treatment of DFF causes the same stimulatory effect on DNA cleavage, indicating that histone H1 affects DFF40/CAD enzyme activity, but not caspase-3-dependent activation of the nuclease. We have found that each of the six somatic cell histone H1 isoforms, which differ in primary sequence, equally activate DFF40/CAD. Using a series of truncation mutants of recombinant mouse histone H1-0, we demonstrate that the H1-0 C-terminal domain (CTD) is responsible for activation of DFF40/CAD. We show further that the intact histone H1-0 CTD and certain synthetic CTD fragments bind to DFF40/CAD. These interactions enhance the ability of DFF40/CAD to bind to DNA. We have concluded that the interactions between the histone H1 CTD and DFF40/CAD target and activate linker DNA cleavage during the terminal stages of apoptosis. 9. HALOGENATED ANESTHETICS: GENOTOXICITY AND INFLUENCE ON HUMAN GENOME T. M. Karpiński1, M. Kostrzewska-Poczekaj1, I. Stachecki2, P. Jałoszyński1, M. Rydzanicz1, K. Szyfter1,4, A. Mikstacki3, R. Szulc2 1Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland 2Clinic of Anesthesiology and Intensive Therapy, Medical University, Poznań, Poland 3Clinic of Intensive Medical Care and Pain Treatment, Regional Hospital, Poznań, Poland 4Clinic of Otolaryngology and Laryngeal Oncology, Medical University, Poznań, Poland Inhalation anesthetics exert some side effects. Health effects for short-term (patients) and long-term occupational exposure differ. An increased hepatotoxicity, nephrotoxicity, migraine and reproduction disorders in operating room personnel were reported. Papers concerning genotoxic and cytotoxic effects of halogenated anesthetics are discordant. The studies aimed for testing genotoxicity of volatile anesthetics (halothane, desflurane, isoflurane and sevoflurane) in human lymphocytes in vitro using comet assay. Because of a high volatility of drugs and to minimize DNA repair, all processes were carried out at 4#186;C. The negative control was water and 1 % DMSO (used as a solvent), when halothane (already proven as genotoxic) served as positive control. Mann-Whitney U-test was employed to estimate statistical significance. An induction of DNA fragmentation by desflurane was as effective at that of halothane. Genotoxicity of isoflurane / sevoflurane did not differ significantly from controls. Genotoxic activity of desflurane was dose-dependent (0.1mM - 10mM). However, when pharmacokinetics is taken into account desflurane appears to be less harmful than halothane for the exposed staff. In studies on inhalation anesthetics concentration (N2O, halothane, sevoflurane, isoflurane) in operating halls of two Poland regions, supported by KBN project No. 6PO5C00521 we found a considerable exceed of highest admissible concentrations. Using comet assay two groups were examined (100 persons each) for DNA damage. The exposed group consisted of surgeons, anesthesiologists and nurses, the control group were the persons not exposed on inhalation anesthetics. The studies did not reveal statistically significant (p<0,05) differences between exposed and group control. Further division in view of work position showed onto anesthesiological nurses as a group of an increased risk. With the aid of genotyping were not affirmed essential differences in distribution of genotypes of DNA repair genes (XRCC1, XRCC3, XPDex6, XPDex23) in the exposed and control group. 10. THE ROLE OF BIOREDUCTIVE ACTIVATION OF 4'-O-TETRAHYDROPYRANYL-DOXORUBICIN IN THE CYTOTOXIC ACTIVITY AGAINST LEUKAEMIA HL60 SENSITIVE CELL LINE AND ITS MULTIDRUG RESISTANT SUBLINES.IN VITRO AND SPECTROFLUOROMETRIC STUDIES Dorota Kostrzewa-Nowak1, Mark J.I. Paine2, Józef Kirkiewicz3, C. Roland Wolf2, Jolanta Tarasiuk1 1Department of Biochemistry, University of Szczecin, Poland 2Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee 3Department of Physics, Maritime University, Szczecin, Poland The clinical usefulness of anthracycline antitumour drugs (e.g. doxorubicin, DOX; daunorubicin, DR) is severely limited by the occurrence of multidrug resistance (MDR) associated with the presence of the membrane transporters (e.g. P-glycoprotein, MRP1), responsible for the active export of drugs out of resistant cells. DOX is a well-known bioreductive drug. In our recent study (Kostrzewa-Nowak et al., Br. J. Cancer, 93, 89-97, 2 005) we have evidenced the important role of bioreductive activation of DOX by exogenously added NADPH-cytochrome P450 reductase (CPR) and NADPH in cytotoxic activity against human promyelocytic leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of multidrug resistance related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). The aim of this study was to examine the reductive activation of 4'-O-tetrahydropyranyl derivative of DOX (THP-DOX, pirarubicin) by human liver CPR and its impact on increasing the cytotoxic activity against sensitive as well as resistant HL60 cell lines. It was found that the presence of tetrahydropyranyl group at the sugar moiety of DOX did not disturb the ability of this derivative to undergo reductive activation by CPR with the formation of reactive metabolites. It was evidenced, similarly to results obtained previously for the parent drug (DOX) that, upon CPR catalysis, THP-DOX underwent only the redox cycling (at low NADPH concentration) or multi-stage chemical transformation (at high NADPH concentration). We have also found, using superoxide dismutase (SOD), that the first stage undergoing according to the mechanism of the redox cycling had the key importance for the metabolic conversion of both compounds examined. Results of in vitro studies confirmed the same behaviour of reductively activated parent drug (DOX) and its tetrahydropyranyl derivative. Our assays showed that the presence of CPR catalysing only the redox cycling of both compounds had no effect in increasing their cytotoxicity against sensitive and MDR tumour cells. In contrast, an important increase in cytotoxic activity of DOX as well as THP-DOX, after their metabolic conversion by CPR, was observed against sensitive HL60 as well as multidrug resistant HL60/VINC and HL60/DOX cells. The interactions of THP-DOX alone (non-activated), THP-DOX acting in the redox cycling as well as reactive metabolites of the drug obtained upon CPR catalysis with naked DNA and intact cells were examined using spectrofluorometric method (Tarasiuk et al. Biochim. Biophys. Acta 1013, 109-117, 1989). Similarly to results obtained for THP-DOX alone, an important quenching of the fluorescence signal for THP-DOX operating in the redox cycling was observed after the addition of naked DNA or during the incubation with sensitive HL60 as well as resistant cells (HL60/VINC and HL60/DOX) due to the intercalation of the drug between base pairs of DNA. In contrast, under the same experimental conditions only a slight decrease in the fluorescence signal was observed for THP-DOX undergoing reductive conversion that indicates another type of interaction of the drug with naked DNA and nuclear DNA of intact cells. The formation of covalent adducts of reactive metabolites of THP-DOX with DNA could be assumed. These studies were supported by the Faculty of Natural Sciences, University of Szczecin, Poland, and Medical Research Council, UK (Grant no. G9203175). 11. BIOLOGICAL ACTIVITY OF PREPARATIONS FROM UNCARIA TOMENTOSA (WILLD.) DC Magdalena Kostrzewska-Poczekaj1, Radosław Pilarski2, Krzysztof Gulewicz2, Krzysztof Szyfter1 1Institute of Human Genetics, Polish Academy of Sciences, Strzeszyńska Str. 32, Poznań 2Institute of Bioorganic Chemistry PAS, Poznań Uncaria tomentosa (Willdenow ex Roemer Schultes) De Candolle also called una de gato, cat's claw and Katzenkralle is a woody wine of South and Central America. Aqueous decoctions of this plant have been used in local folk medicine for at least two thousand years as a remedy on digestive tract, arthritis and rheumatism, immunological and genital diseases. Potential imunostimulating, antinflamatory and anticancer activities have been widely studied. Several phytochemical analyses showed that such activity is associated with pentacyclic and tetracycylic oxindole alkaloids, quinine glycosydes, ursolic acid and a number of phenolic compounds. In this study antiproliferative activities of different extracts obtained form leaves and bark of Uncaria tomentosa originated from Peru and supplied by Andean Medicine Centre were evaluated. The preparations were standardized by HPLC for obtaining their total oxindole alkaloids contents which were from 430 to 50401 mg/100g. Qualitative analyses were also performed indicating different oxidondole alkaloids profiles in the fractionated preparations. The biological assays were performed on HL-60 acute promyelocytic human cell lines, ranging exposed on different extracts for 72 hours. The proliferation rates and cytotoxicity of each preparation were evaluated by applying trypan blue staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay (MTT). The highest antiproliferative activity was showed for enriched alkaloid fraction Bsrt and generally for ethanolic preparations. Total activities of each preparations were determined and compared with particular alkaloid determinations. By calculating Pearson's correlation factors, pteropodine/isomitraphilline and isopteropodine were found to possess the strongest antiproliferative activity (r=0.91 and r=0.74, respectively). 12. MODIFICATION OF THE RATE OF OXIDATIVE DNA DAMAGE REPAIR BY DIETARY FACTORS AND INFLAMMATION IN NEWBORN PIGS Paweł Kowalczyk1, Aleksandra Bieleń1, Beata Sokołowska2, Daniel Laubitz3, Romuald Zabielski3, Barbara Tudek1 1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland 2Institute of Experimental Medicine, Polish Academy of Sciences, Warsaw, Poland 3Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland e-mail: tudek@ibb.waw.pl The diet exerts an important effect on the development and functioning of the digestive tract. We were investigating the effect of supplementation of the diet of sows with grains rich in polyunsaturated fatty acids (PUFA: flax-seed, rape-seed 4 kg/100 kg of feed each), L-carnitine (15 g/100 kg), taurine (100 g/100 kg) and vitamin E (15 g/100 kg) on the rate of oxidative DNA damage excision in colons of their offspring. Diet supplementation was performed from the 80th day of pregnancy till 28th day of lactation. Repair activities in colons of the offspring from one sow were similar in the first, fourth and seventh day after delivery, however they differed between piglets from different mothers. Diet supplementation of mothers had no effect on the repair activity (measured by the nicking assay) of ethenocytosine (#949;C) in the colons of newborn pigs. However the repair activity for 8-oxoG and #949;A increased about twice in pigs, whose mothers were fed with supplemented diet. Thus dietary factors can modulate repair capacity for 8-oxoG, and #949;A, but have no effect on excision rate of #949;C from pigs intestines. 13. PHOTODYNAMIC EFFECT, CELL DEATH PATHWAYS AND SUBCELLULAR LOCALISATION OF TWO LIPOSOME-INCORPORATED SYNTHETIC PORPHYRIN DERIVATIVES Gabriela Krämer-Marek1,2, Carlos Serpa3, Maria Wideł4, Aleksander Sochanik5, Mirosław Śnietura6, Piotr Kuś7, Luis Arnaut3, Alicja Ratuszna1. 1A.Chelkowski Institute of Physics, University of Silesia, Katowice, Poland 2Department of Tumour Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 3Chemistry Department, University of Coimbra, Portugal 4Department of Clinical and Experimental Radiobiology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 5Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 6Department of Tumour Pathology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice, Poland 7Institute of Chemistry, University of Silesia, Katowice, Poland Photodynamic therapy (PDT) of cancer diseases relies on the combined use of photosensitisers (virtually non-cytotoxic by themselves) and harmless visible light. Target cells are photodamaged as a result of singlet oxygen and other reactive oxygen species formation. Photofrin#174; was the first photosensitiser approved for human use. Search for novel drugs with improved efficiency and fewer side effects has been continuing, however, for the past decade. In the present study, two synthetic porphyrin derivatives: 5-(4-hydroxyphenyl)-10,15,20-tritolylporphyrin (C16-TTP) and 5-(4-hexadecyloxyphenyl)-10,15,20-tri-pyridylporphyrin (TPYR-PP) were compared with respect to photodynamic efficiency, cell localization as well as mode of induced cell death. Singlet oxygen yields and lifetimes were determined by directly measuring phosphorescence at 1270 nm. In vitro studies were conducted on three cancer cell lines: human malignant melanoma (Me45), murine melanoma (B16(F10)) and human colon adenocarcinoma (Hct-116). Cationic liposome-plasmid DNA complexes (lipoplexes) were used as vehicles transporting these hydrophobic porphyrins. Cell viability following PDT treatment was monitored by MTS-tetrazolium reduction assay and by a test assessing clonogenic potential of cells. The mode of cell death was investigated with fluorescence microscopy using acridine orange/ethidium bromide (AO/EB) double-staining method. The sensitisers' localisation was analysed using a confocal microscope Both compounds are characterised by high quantum yield of singlet oxygen generation and they displayed high photostability under the conditions used for PDT tests. They revealed negligible dark cytotoxicity but high phototoxicity. The results were both light dose- and cell line-dependent. To reach LD50, only 5 J/cm2 was required. TPYR-PP was slightly more phototoxic than C16-TTP. Analysis of data gathered suggests that the manner of cell death following use of either one of the examined compounds depends on light irradiation dose. In either case, when high dose (15 J/cm2) was used, the dominating effect was necrosis. Almost total inhibition of cellular proliferation was then observed. Confocal laser scanning microscopy showed that both compounds localized in the cytoplasm but not in the nucleus of cells, which explains no DNA damage detection under dark conditions and when using photodynamic doses. Both compounds are photodynamically active, effectively inducing cell death when light activated, presumably due to efficient generation of singlet oxygen. We conclude that porphyrin derivatives of this type seem to be promising agents for PDT treatment of neoplasms. 14. ANALYSIS OF SUCCYNYL DEHYDROGENASE (SDH) SUBUNITS GENE MUTATIONS IN PATIENTS WITH PARAGANGLIOMAS Aleksandra Krawczyk1, Kornelia Hasse-Lazar1, Agnieszka Pawlaczek1, Dagmara Rusinek1, Sylwia Szpak-Ulczok1, Mariola Pęczkowska2, Aleksander Preibisz2, Agata Kubaszek2, Elżbieta Gubała1, Andrzej Januszewicz2, Barbara Jarząb1 1Department of Nuclear Medicine and Endocrine Oncology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland 2Department of Hypertension, National Institute of Cardiology, 04-628 Warszawa, ul. Alpejska 42, Poland Tumors derived from chromaffine tissue include pheochromocytomas (tumors located in adrenal medulla) and paragangliomas (extraadrenal tumors). These tumors are in 20-25% inherited. Paragangliomas are even rarer and are presented either as familial disease or pheochromocytoma-paraganglioma syndrome (PPS). The mutations in SDH genes (SDHB, SDHD) are suspected for causing the syndrome. The aim of present study is to look for germline mutations in SDHB and SDHD genes in patients with pheochromocytomas and/or paragangliomas. DNA was isolated from peripheral blood leukocytes obtained from patients with pheochromocytomas and paragangliomas. The polymerase chain reaction (PCR) was performed for SDHB (exons 2, 3, 4, 6, 7) and SDHD (exons 1, 2, 3). The PCR product was then analyzed with the use of MSSCP (Multiplex Single-Strand Conformation Polymorphism). When the change in the conformation of DNA strand was found, it was then identified by sequencing. We have so far analyzed DNA from 12 patients with diagnosed paragangliomas; we have also examined 72 patients with pheochromocytomas only in order to seek for pheochromocytoma-paraganglioma syndrome. We have found two types of mutations of SDHD in 6/12 (50%) of paraganglioma cases. 33 TGC-TGA substitution was associated with benign tumors, whereas N 721 G-A occurred in the only patient with malignant paraganglioma. Conclusions: 1) Mutations in the SDHD gene appear to be frequent in patients with paragangliomas. 2) Among inherited cases of pheochromocytomas no correlations of the type of SDH mutation with malignancy have been found so far. 15. THE ROLE OF NUCLEOSIDE ANALOGUES AND VITAMINS, ATRA AND D3, IN EPIGENETIC CHANGES (METHYLATION) OF PTEN AND APC GENE PROMOTERS IN MCF-7 BREAST CANCER CELLS Barbara Krawczyk, Krystyna Fabianowska-Majewska Department of Biomedicinal Chemistry, Medical University of Lodz, Mazowiecka Street 6/8,92-215 Lodz, Poland Hypermethylation of promoters of tumour suppressor genes is epigenetic modification of DNA and takes part in combined regulation of gene transcription. We previously documented that the actions of the adenosine analogues, 2-chloro-2'-deoxyadenosine (2CdA, cladribine) and 9-ß-D-arabinosyl-2-fluoro-adenine (F-ara-A, fludarabine) lead to a decrease of genomic DNA methylation. Additionally, results of several studies indicated that natural compounds such as vitamins, which are ligands of nuclear receptors, can be synergists with anticancer drugs. The experiments were aimed at estimation of methylation changes of promoter regions of selected tumour suppressor genes: ERα, BRCA1 (with a specific sequence for CREB), E-cadherin, PTEN and APC in MCF-7 cells (ERα ( ) and non-invasive cells), which grew in the presence of the mentioned above adenosine analogues and natural compounds: all-trans retinoic acid (ATRA) and vitamin D3. MCF-7 cells were cultured (72 hr) in the presence of 2CdA, F-ara-A, 5-aza-deoxycytidine (5-aza-dCyt, a potent inhibitor of DNA methyltransferase), ATRA, and vitamin D3 at their IC50 concentration: 0.17 µM, 15.0 µM and 0.6 µM, 0.34 µM, and 1.5 µM, respectively. The methylation status of gene promoters was estimated using methylation-sensitive restriction analysis (MSRA). We noted that among selected genes only promoters of PTEN, APC, and BRCA1 genes were methylated in MCF-7 cells cultured without drugs. In MCF-7 cells treated with nucleoside analogues or natural compounds for 72 hr we observed that: (i) methylation of PTEN promoter (fragment between #8722;281 and 5 bp) was partially reduced by 2CdA, ATRA, and vitamin D3 and completely eliminated by F-ara-A and 5-aza-dCyt, (ii) methylation of APC promoter (fragment between #8722;113 and 205 bp) was completely eliminated by 2CdA, F-ara-A, 5-aza-dCyt, and ATRA, and partly reduced by vitamin D3, (iii) no effect of any tested drugs and natural compounds on methylation status of BRCA1 promoter (region included sequence for CREB, fragment between #8722;317 and #8722;24 bp) was observed. The findings indicated that promoter methylation of PTEN and APC genes, encoding proteins implicated in regulation of intracellular oncogenic signal transduction (PI3K/Akt and MAP kinase dependent pathways - in the case of PTEN, and Wnt/APC/ß-catenin pathway - in the case of APC) can be modulated by nucleoside analogues and vitamins as well. This fact has dual significance for anticancer strategies. Firstly, it indicates that actions of 2CdA and F-ara-A, antimetabolites of natural nucleosides, are implicated not only in inhibition of DNA synthesis but also in indirect regulation of cell development due to influence on activity of proteins (i.e. PTEN and APC), crucial for cellular signaling pathways. Secondly, the possibility of hypermethylation decrease of tumour suppressor genes (i.e. PTEN and APC) by natural vitamins (i.e. ATRA and D3) seems to be important for carcinogenesis prevention as well as synergistic effects with drug used in chemotherapy. 16. THE REPAIR OF GAMMA-RADIATION-INDUCED DNA DAMAGE IS INHIBITED BY MICROCYSTIN-LR, THE PP1 AND PP2A PHOSPHATASE INHIBITOR A. Lankoff1, J. Bialczyk2 ,D. Dziga2 ,W.W. Carmichael3, I. Gradzka4, H. Lisowska1, T. Kuszewski5, S. Gozdz5, I. Piorun1, A. Wojcik1,4 1Department of Radiobiology Immunology, Institute of Biology, Świętokrzyska Academy, Swietokrzyska 15, 25-406 Kielce, Poland 2Department of Plant Physiology and Development, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland 3Department of Biological Sciences, Wright State University, Dayton, OH, USA 4Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland, 5Department of Medical Physics, Swietokrzyskie Oncology Center, Kielce, Poland, We studied the effect of microcystin-LR, the PP1 and PP2A phosphatase inhibitor, on the repair capacity of radiation-induced DNA damage in human lymphocytes and human glioblastoma cell lines MO59J and MO59K. Human lymphocytes in G0-phase of the cell cycle were pre-treated with MC-LR for 3 hours and irradiated with 2 Gy of gamma radiation. The kinetics of DNA repair was assessed by the comet assay. In addition X-H2AX foci and the frequencies of chromosomal aberrations were analyzed. The pre-treatment with MC-LR inhibited the repair of radiation-induced damage, resulted in reduced numbers of X-H2AX foci and lead to enhanced frequencies of chromosomal aberrations. In order to elucidate the impact of MC-LR on DNA-PK we examined the kinetics of DNA repair in human glioblastoma cells MO59J cells that are deprived of the catalytic subunit of DNA-PK (DNA-PKcs) and in the MO59K cells that have a normal level of DNA-PKcs. Both cell lines were exposed to 10 Gy of X-rays and DNA repair was analyzed by the comet assay. While a strong inhibitory effect was observed in the MO59K, MC-LR also moderately inhibited the repair of damage in the MO59J cells. These results indicate that apart from DNA-PK, other enzymes involved in DNA repair could also be inhibited by MC-LR. Work supported by Ministry of Science and Information Technology, Poland (Project number KBN PO5D 033 26). 17. CHROMOSOMAL RADIOSENSITIVITY IN PERIPHERAL BLOOD LYMPHOCYTES OF LARYNX CANCER PATIENTS H. Lisowska1, A. Lankoff1, A. Padjas2, A. Wieczorek2, A. Florek2, T. Kuszewski2, S. Góźdź2, A. Wójcik1,3 1Department of Radiobiology and Immunology, Institute of Biology, Świętokrzyska Academy, Kielce, Poland 2Świętokrzyskie Cancer Center, Kielce, Poland 3Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland Patients treated with identical radiotherapy schedules show a substantial variation in the degree of early and late normal tissue reactions. The identification of radiosensitive patients before therapy would allow to optimize it. The aim of our study was to investigate whether the in vitro radiosensitivity of lymphocytes derived from a blood sample predicts the effects after radiotherapy in larynx cancer patients. In addition, there is data suggesting that the sensitivity to ionising radiation of peripheral blood lymphocytes of cancer patients is higher than that of healthy donors. This effect is especially prominent when chromosomal aberrations induced in G2 phase of the cell cycle are analysed. The second aim of our study was to investigate if the G2- aberration frequencies in lymphocytes of patients with larynx cancer are higher than in the case of healthy individuals. Peripheral blood of 40 patients was collected before the onset of radiotherapy, cultured and irradiated with Co-60 after 67 hours of culture time. Irradiation was performed in the Swietokrzyskie Oncolgy Center which is located in a different part of Kielce. Therefore, blood cultures were transported to and from the Center and irradiated on ice. Chromosome specimens were prepared from cells fixed at 72 hours of culture time. Colcemid was added for 2 hours before harvest. Lymphocytes of 40 healthy donors were cultured and irradiated in the same way like in the case of larynx cancer patients. No statistically significant correlations were observed between aberration frequencies in lymphocytes and degrees of both early and late normal tissue reactions. The aberration frequencies in lymphocytes of patients were on average higher than in the case of healthy donors. This result suggests, that the radiation sensitivity of lymphocytes of patients with larynx cancer might be a marker of cancer predisposition. 18. SEARCH FOR THE MOLECULAR SIGNATURE RESPONSIBLE FOR DRUG RESISTANCE IN OVARIAN CARCINOMA K. Lisowska, V. Dudaladava, M. Olbryt, M. Jarząb, E. Grzybowska, J. Kupryjańczyk1, Department of Tumor Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; 1) Department of Molecular Pathology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland Ovarian cancer is usually diagnosed at advanced stage. In the majority of cases treatment of the disease encompasses maximal possible cytoreduction and adjuvant chemotherapy. Standard chemotherapy regimen was based on administration of cisplatin and cyclophosphamid. During last few years cisplatin with taxanes became a favored regimen, however this combination is much more expensive. While in general more effective, this regimen not always results in good response of the tumor. About 20 - 30% of patients are not responding to this type of chemotherapy. There are also some evidences, that patients with germline mutation in BRCA1 and 2 genes respond better to standard, cisplatin based chemotherapy. Thus, there is a need for more precise criteria for classification of patients for appropriate chemotherapy. Our aim was to find a set of genes that expression is changed between ovarian cancer cases that are sensitive to chemotherapy and those that are resistant. Among those genes we expect to find potential molecular markers suitable for prediction of individual drug response. We used DNA microarray technology (HG U133 Plus 2.0 oligonucleotide microarrays, Affymetrix) to analyze gene expression profile in ovarian cancer tissues. We analyzed 32 cancer samples from patients that were treated with cisplatin based chemotherapy. We also analyzed 20 samples from patients treated with taxane based chemotherapy. Microarrays from both experiments were normalized together by RMA algorithm, while data analysis was performed for both data sets separately. For genes selection we used Welch test with Benjamini-Hohberg correction for multiple comparison and limma algorithm based on linear models with empirical Bayesian approach. For data analysis patients were divided into several groups according to the level of chemosensitivity/chemoresistance. The poster will show our preliminary results of data analysis. We found, that despite the set of samples from cisplatin treated patients was larger, the difference in gene expression pattern between responders and non-responders in this group was smaller than in the taxane treated cohort. This may be related to the fact, that each chemotherapeutic agent has different mode of action. Probably, in cisplatin treated tumors, p53 status (presence and type of mutation, protein accumulation) is crucial for prediction of response to chemotherapy. The study was supported by the Ministry of Science and Information Society Technologies(grant number 3 P05A 060 25). 19. THE EFFECT OF 1-METHYL PYRIDINIUM SALTS ON THE CYTOTOXIC ACTIVITY OF VINCRISTINE TOWARDS RESISTANT SUBLINES OF HUMAN PROMYELOCYTIC LEUKAEMIA HL60 CELLS, HL60/VINC AND HL60/DOX Agnieszka Maruszewska1, Dorota Kostrzewa-Nowak1, Jan Adamus2, Jerzy Gębicki2, Jolanta Tarasiuk1 1Department of Biochemistry, University of Szczecin, 3c Felczaka St, 71-412 Szczecin, Poland 2 Institute of Applied Radiation Chemistry, Technical University, 90-924 Łódź, Poland Vincristine is among the most effective drugs available for the treatment of leukaemia diseases. However, its antitumour activity is drastically reduced towards tumour cells developing the multidrug resistance (MDR) against a wide range of drugs, structurally dissimilar and having different intracellular targets. MDR phenomenon is associated with the presence of the membrane transporters (e.g. P-glycoprotein, MRP1), responsible for the ATP-dependent export of drugs out of resistant cells. Vincristine exhibits a high cytotoxic activity against human promyelocytic sensitive leukaemia HL60 cell line (IC50 = 1.6 nM). However, an important decrease in its cytotoxic activity is observed against MDR sublines exhibiting two different phenotypes of multidrug resistance related to the overexpression of P-glycoprotein (HL60/VINC, resistance factor IR = 978) or MRP1 (HL60/DOX, IR = 15.8). The aim of this study was to examine the effect of selected 1-methyl pyridinium salts: MNP , (1-methyl-3-nitropyridinium salt) and MDION (3,3,6,6,10-pentamethyl-3,4,6,7-tetrahydro-[1,8(2H,5H)-dion]acridine salt) on the cytotoxic activity of vincristine against HL60/VINC and HL60/DOX cells. In our previous study (Wieczorkowska et al., Free Radic. Res. 37, 1157-62, 2003) it was evidenced that these agents are able to effectively shift an equilibrium between NADH and NAD playing an important role in energy metabolism of cells. MNP and MDION salts were much less cytotoxic themselves against HL60 cells (at micromolar concentration) and conserved an important cytotoxic activity towards resistant HL60/VINC and HL60/DOX cells (IR = 2#61624;4). The high synergistic effect of MNP and MDION salts at 1-2 µM on the cytotoxic activity of vincristine towards both MDR resistant cell lines was observed in the wide range of vincristine concentration (up to 1 µM and 100 nM in the case of HL60/VINC and HL60/DOX, respectively). These studies were supported by the Faculty of Natural Sciences, University of Szczecin, Poland, and by the State Committee for Scientific Research, Warsaw, Poland (Grant no. PBZ-KBN-101/T09/2003). 20. THE ROLE OF LYSOSOMAL IRON IN *NO SIGNALING Sylwia Męczyńskax, Hanna Lewandowska, Marcin Kruszewski Institute of Nuclear Chemistry and Technology, 16 Dorodna Str., 03-195 Warszawa xe-mail: mesyl@ichtj.waw.pl Recently, it has been proved, that a considerable amount of cellular iron remains in the form of complexes with low molecular mass ligands, labile enough to enter the Fenton reaction. This pool has been called the labile iron pool (LIP) [1]. It plays a role, among others, in cellular iron transport, expression of iron regulatory genes, control of the activity of iron containing proteins, and catalysis of the Fenton reactions. It was proposed that a part of the cellular pool of labile iron is confined within the acidic vacuolar compartment (lysosomes). In the present reports the contribution of lysosomal iron to the total amount of cellular labile iron pool is vividly discussed [2-3]. Our previous results show that LIP plays a role in the regulation of nitric oxide-dependent biochemical pathways, forming dinitrosyl iron complexes (DNIC), a group of physiologically important transducers of nitric oxide. Formation of nitrosyl iron complexes, known as 2.03 complexes due to the g value of their characteristic EPR spectra, has been found by several groups of researchers in many kinds of bacteria, plants and animals. It is postulated that DNIC are important factors in nitric oxide-dependent regulation pathways in the cell [4-6]. It has been shown that low molecular-weight DNIC possesses endothelium derived relaxing factor (EDRF) activity [7-8]. In addition, the low molecular-weight DNIC have been shown to modulate redox properties of the cellular interior through the inhibition of glutathione-dependent enzymes, such as reductase, transferase, and peroxidase of glutathione [9-12]. The sources of iron forming DNIC in vivo are still not precisely defined, one of the putative sources being the labile iron pool and another - iron proteins. Neither are defined the cellular compartments, in which DNIC are formed. In the report presented here we show that depletion of lysosomal labile iron pool by either chelation with deferoxamine or lysis inhibition leads to a considerable decrease (down to 50%, depending on the incubation time) of DNIC forming in the cells under the influence of nitric oxide. This would indicate a vital role of lysosomal labile iron in DNIC formation. Taken together, our present and previous results confirm the thesis on the considerable contribution of lysosomal iron to the total labile iron pool in the cell. Bibliography 1) O. Kakhlon, Z.I. Cabantchik, Free Rad. Biol. Med. 2002;33(8):1037-46. 2) Z. Yu, H.L. Persson, J.W. Eaton, U.T Brunk, Free Rad. Biol. Med. 2003;34: 1243-1252. 3) J.W. Persson, Z. Yu, O. Tirosh, J.W.Eaton, U.T. Brunk, Free Rad. Biol. Med.2000; 334:1295-1305. 4) P. A. Rogers, L. Eide, A. Klungland and H. Ding, DNA Repair 2003; 2: 809-817 5) C. Bouton and J.C. Drapier, Sci STKE 2003; 182:17. 6) P. Turella, J. Z. Pedersen, A. M. Caccuri, F. De Maria, P. Mastroberardino, M. L. Bello, G. Federici and G. Ricci, J. Biol. Chem. 2003; 278: 42294-42299. 7) A. F. Vanin, FEBS Lett. 1991; 289:1-3 8) A. Mülsch, P. Mordvintcev, A. F. Vanin and R. Busse, FEBS Lett. 1991: 294: 252-256. 9) M. Boese, M. A. Keese, K. Becker, R. Busse, A. Mulsch, J. Biol. Chem.1997; 272 :21767-21773. 10) M. A. Keese, M. Bose, A. Mulsch, R.H. Schirmer, K. Becker, Biochem. Pharmacol.1997; 54: 1307-1313. 11) K. Becker, S.N. Savvides, M. A. Keese, R.H. Schirmer, P.A. Karplus, Nat Struct Biol. 1998; 5: 267-271. 12) J.S. Stamler, A. Hausladen, Nat Struct Biol. 1998; 5: 247-249. 21. GENE EXPRESSION PROFILE OF B16(F10) MURINE MELANOMA CELLS UNDER HYPOXIC CONDITIONS IN VITRO Magdalena Olbryt1, Michał Jarząb2, Joanna Jazowiecka-Rakus1, Krzysztof Simek3, Aleksander Sochanik1 1Department of Molecular Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch 2Department of Tumor Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch 3Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology in Gliwice Rapidly growing tumors are exposed to hypoxic conditions due to inadequate blood supply. Hypoxia is not only an obstacle for effective radiotherapy and chemotherapy but also strongly contributes to tumor progression. Therefore, better understanding of this phenomenon might improve cancer diagnosis and treatment. We investigated gene expression profile in murine melanoma B16(F10) cultured cells under: (i) hypoxia and (ii) hypoxia-mimicking conditions. Cells were exposed to hypoxic (nominal 1% O2) or hypoxia-mimicking conditions (100µM or 200µM CoCl2), for 24h. Total RNA was isolated and cRNA was hybridized to microarrays (Affymetrix). Data analysis revealed huge changes in gene expression profile when comparing experimental samples and controls: for 1% oxygen experiment 2448 transcripts (app. 19% of total probset number, FDR 5%) and for cobalt chloride 364 transcripts (3.5% of total probset number, FDR 5%) showed differential expression. Genes significantly modulated by cobalt chloride, in comparison to those modulated by hypoxia are related to cytokinesis, mitosis and melanin biosynthesis. Upregulated mRNAs in hypoxia vs. control samples included known hypoxia-induced genes (vegf, p4ha2, hig1), genes previously related to cancer (i.e. ctgf, anxa2, adm, lgals3, nppb, mitf) and genes, to our knowledge, so far not associated with hypoxia (i.e. rras, rnf19). This experiment indicates novel potential hypoxia and/or prognostic markers as well as therapeutic targets for melanoma treatment. It also demonstrates that use of cobalt chloride to induce hypoxia mimicry results in a somewhat different gene expression profile of B16(F10) cells compared to that induced by low oxygen tension. 22. INDIVIDUAL RADIOSENSITIVITY OF PATIENTS WITH BREAST CANCER AND HEALTHY DONORS: ANALYSIS OF DNA DAMAGE AND REPAIR IN PERIPHERAL BLOOD LYMPHOCYTES A. Padjas1, A. Lankoff2, H. Lisowska2, A. Wieczorek1, T. Kuszewski1, S. Góźdź1, A. Wójcik2,3 1Świetokrzyskie Cancer Center, Kielce,Poland 2Department of Radiobiology and Immunology, Institute of Biology, Świętokrzyska Academy, Kielce, Poland 3Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland Individual radiosensitivity of the human population is heterogeneous. In the case of patients undergoing radiotherapy an enhanced curability could be achieved by adjusting the treatment scheme to the individual sensitivity of the patient. There is data suggesting that the radiosensitivity of peripheral blood lymphocytes (PBL) may serve as a model cell system for determining the individual sensitivity to ionising radiation. The analysis of DNA damage and repair in PBL of breast cancer patients undergoing radiotherapy may be important for estmating the susceptibility of the patients side effect of radiotherapy and their risk of developing seconadary cancer. An interesting question is if the kinetics of DNA repair in PBL of cancer patients is different from that of healthy donors and wheather can be correlated with chromosomal aberrations and resistance/sensitivity of the patient to therapy. The aim of our study was to compare the kinetics of DNA repair in PBL of 30 healthy donors and 20 breast cancer patients estimated by the comet assay and to compare the frequency of chromosomal aberrations induced by radiation. Peripheral blood lymphocytes were collected from cancer patients (before radiotherapy) and from healthy donors and irradiated with 2 Gy 60Co. Chromosome slides were prepared from cells fixed at 50 hours after irradiation. The frequency of chromosome aberrations was scored in cells in the first mitotic division. The level of DNA damage was estimated by the alkaline comet assay after 0, 15, 30, 60 and 120 minutes post exposure. The results suggest that the frequency of chromosomal aberrations in cancer patients was higer than in healthy donors and no difference exists between the kinetics of DNA repair and the frequency of chromosomal aberrations. 23. BRAF INITIATING MUTATIONS IN PAPILLARY THYROID CARCINOMA Dagmara Rusinek1, Jadwiga Żebracka1, Małgorzata Oczko-Wojciechowska1, Małgorzata Wiench1, Daria Hankiewicz-Junak1, Grzegorz Gala1, Wojciech Biały, Krzysztof Simek2, Krzysztof Fujarewicz2 1Department of Nuclear Medicine and Endocrine Oncology, MSC Memorial Institute-Center of Oncology, Gliwice 2Institute of Automatic Control, Technical University, Gliwice Introduction: The major molecular pathway leading to papillary thyroid cancer (PTC) includes the activation and alterations in MAPK pathway. Currently, three major points of the initiation have been identified: RET tyrosine kinase (or NTRK) rearrangements cause very early initiation of the whole pathway, while point mutations occurring in the later steps of signaling, i.e. in RAS genes or in BRAF kinase gene constitute the alternative initiating mechanisms. Somatic BRAF mutations have been found in melanomas - in 98% this was a transversion from thymine to adenine at position 1796 (V600E). This mutation activates consitutively the enzymatic activity of the molecule by preventing the hydrophobic interactions between the residues of activation centre and ATP binding residues which are responsible for inactive conformation. BRAFT1796A mutation has been described in 36-39% PTC cases, also in poorly differentiated and anaplastic thyroid ca. Aim: We aimed to study the frequency of somatic BRAF mutations and to relate it to the incidence of RET rearrangements and to the differences in gene expression pattern in papillary thyroid carcinoma. Material and methods: The analysis was carried out in the collection of 45 PTC tumors in which expression profile and RET/PTC were previously analyzed. Total RNA was extracted from postoperative tumor tissue, cDNA synthesis was carried out with gene-specific primers. Exon 15 of BRAF gene was amplified by PCR and analyzed by automated sequencing. . Results: The BRAFT1796A mutation was found in 24 cases of papillary thyroid carcinoma. In one case of PTC we identified a nucleotide substitution, A1799T, coexisting with BRAFT1796A mutation. Similarly only in one patient with papillary thyroid carcinoma we found a deletion of 14 nucleotides. RET/PTC rearrangement were identified in 11/42 cases of PTC, in 20 cases we found no RET rearrangement, in 6 patients we interpret the result without ambiguity. We found BRAF T1796A mutation in two patients with previously detected RET/PTC rearrangement. Now we are verifying this results by repeated sequencing and we carry out the comparison of gene expression profile of papillary thyroid cancer with RET rearrangement vs. BRAF mutation, to reveal whether the difference in initiating event confers the changes in expression profile of these tumors. We are also perform also a comparison to similar study by Giordano et al., where raw microarray data are available. Conclusions: Two molecular events leading to PTC, BRAF mutation and RET/PTC rearrangements, differ in frequency of occurrence in PTC: in our group BRAF mutation prevailed over RET rearrangement and was more than two times frequent. Microarray analysis may allow to delineate changes in gene expression profile associated with these alterations of MAPK pathway. 24. ASSOCIATION BETWEEN PATIENT- AND TUMOR-RELATED FACTORS AND THE GENE EXPRESSION PROFILE OF PAPILLARY THYROID CANCER Sylwia Szpak-Ulczok1, Malgorzata Wiench1, Malgorzata Oczko-Wojciechowska1, Jan Wloch2, Krzysztof Fujarewicz3, Andrzej Swierniak3, Dariusz Lange4, Barbara Jarzab1 1Dept. of Nuclear Medicine and Endocrine Oncology, 2Clinic of Oncological Surgery, 4Dept. of Tumor Pathology; Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland 3Dept. of Automatic Control, Silesian Technical University, Gliwice, Poland We performed the analysis of correlation between gene expression profile of the papillary thyroid cancer PTC tumors and known clinical factors influencing the outcome. Gene expression profile was assessed on Human Genome U133A array (Affymetrix). We examined tumors samples obtained from 49 patients diagnosed with PTC. For further evaluation we included following factors: sex, age, tumor size, capsule invasion, multifocality, vascular invasion, lymph node and distant metastases. We used corrected Welch t-test estimation to analyze how many genes are associated with each factor. We found out that poor differentiation/early recurrence (2 tumors) is connected with a prominent difference in gene expression profile (812 genes changed). For the further analysis we excluded these 2 samples (because of the large scale of difference between two poorly differentiated tumors and the rest of PTCs) and performed the analysis on the remaining 47 well-differentiated PTCs. The strongest factor in this group was sex with 10 sex-related. Large difference appeared for distant metastases: 1486 transcripts showing moderate level of statistical significance. To rank the remaining clinical factors, with low significance, we performed the analysis of uncorrected Welch t-test p-values. In such classification distant metastases are the strongest factor. We compared gene expression profile of 8 patients with mets to 39 samples who did not present metastases within the short follow-up time. We selected 150 genes differentiating both groups of patients. By Support Vector Machine approach this dataset correctly predicted the occurrence of metastases in 45/49 patients. Our conclusions: 1) Some clinically relevant features in PTC (poor differentiation, sex) are strongly and significantly associated with gene expression profile. 2) We revealed that presence of distant metastases is related to gene expression and that it is possible to specify the metastatic signature in PTC. 25. INTRACELLULAR LOCALISATION AND PHOTOTOXIC ACTIVITY OF LIPOSOME ENTRAPPED AMINO ACID PORPHYRIN DERIVATIVES; STUDIES ON HUMAN MALIGNANT MELANOMA Agnieszka Szurko1,2, Aleksander Sochanik3, Maria Widel1, Mirosław Snietura4, Jan Habdas5, and Alicja Ratuszna2 1Department of Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 2A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland 3Department of Molecular Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 4Department of Histopathology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland 5Institute of Chemistry, University of Silesia, Katowice, Poland Numerous clinical trials have demonstrated that PDT is an effective and safe treatment for cancer with comparatively mild side effect and low general toxicity. Much experience has been gained with PDT for malignant disorders of the skin, since skin is readily accessible to treatment by light. PDT plays a substantial part in treatment of non-melanoma skin cancer however poor therapeutic results were reported for pigmented human melanoma and experimental melanoma cell lines. This might be explained by a high content of melanin, which can act as a screen for white light and also as free radical scavenger although the extend of such effect is limited. Melanoma responds poorly also to chemical and radiation therapy and the most effective treatment is surgical excision before the tumour is well advanced. In this contribution we describe the results of experiments with the new potential photosensitisers for PDT, different amino acid porphyrin derivatives. We chose human malignant melanoma (Me45) cell line derived from a lymph node metastasis of skin melanoma in 35-year-old male. It is known that the mode of transfer strongly influence subsequent localization of photosensitiser in cells and consequently modulate cell death pathway therefore cellular distribution and mode of cell death were also explored. Our study with confocal microscopy indicated that use of cationic liposomes as a carrier of porphyrins enabled their efficient transfer into cells. Some of a panel amino acid porphyrin derivatives (glycine-porphyrin) appeared very efficient photosensitiser for melanoma cells with low dark toxicity. The mode of cell death was dependent on applied energy of light and time of post treatment incubation, shifting the apoptosis towards necrotic death at higher doses and longer incubation. 26. PML ISOFORMS I-VI FORM BODIES PARTIALLY CO-LOCALIZING WITH NUCLEAR STRUCTURES BUILT BY WRN PROTEIN AND DIFFERENTIALLY REGULATE THE ACTIVITY OF GENE PROMOTERS Rasa Vaitiekunaite1,2,3, Dorota Butkiewicz1, Małgorzata Krześniak1, Mirosław Śnietura2, Jolanta Pamuła1 and Marek Rusin1 1Department of Tumor Biology 2Department of Pathology, Center of Oncology, Maria Skłodowska-Curie Memorial Institute, 44-101 Gliwice, Poland 3Institute of Oncology, Vilnius University, LT-08660 Vilnius, Lithuania RV is a fellow of the Fellowship Program at Department of Tumor Biology, totally supported by the National Cancer Institute - Office for International Affairs, NIH, Bethesda, MD, USA PML protein may suppress tumor formation by regulating the induction of apoptosis, cellular senescence and by participation in maintenance of genomic stability. These PML functions require its interaction with p53 tumor suppressor protein, and with proteins involved in DNA repair or transcription. The significance of these interactions is only beginning to emerge. Moreover, PML is strongly induced by interferon and is targeted by many viral proteins, indicating that it is a crucial element of cellular, antiviral machinery. To better understand the functioning of PML protein, we began to study the regulation of cellular localization of PML and WRN - the pleiotropic protein involved in recombinational DNA repair, gene transcription and maintenance of correct telomere structure. We created the expression vectors coding for the PML isoforms I to VI fused with the green fluorescent protein (EGFP) and for the WRN fused with the red fluorescent protein (mRFP). We found that mRFP-WRN fusion protein is localized in nucleoli or in numerous, small, nucleoplasmic foci. However, in cancer cell lines of different origin, mRFP-WRN forms distinct, nuclear, toroidal bodies. The WRN point mutation (1184:Leu>Arg), disrupting the native structure of the protein, prevents both: nucleolar localization of WRN and formation of "donut-shaped" bodies, what indicates that the bodies form when structural integrity of WRN is preserved. In spite of the fact that these "donut-shaped" structures morphologically resemble PML bodies, they do not extensively colocalize with any of the studied EGFP-PML isoforms. However, we noticed, that in a subset of cells expressing both fluorescent proteins, the surface of WRN bodies is covered by EGFP-PML protein, what was clearly seen for isoforms I, III, IV. Thus, the isoforms show differences in their ability to interact with WRN bodies. Moreover, we noticed that the co-expression of mRFP-WRN and PML isoforms V or VI significantly increases the frequency of cells with large number of WRN "donuts" and increases the amount of mRFP-WRN, but not the endogenous WRN protein. Using the luciferase reporter assay, we found that the PML V and VI significantly increase the activity of viral promoter (CMV) driving the transcription of the mRFP-WRN sequence from the expression vector. The influence of the PML isoforms on the human gene promoters is conspicuously different. The activity of examined promoters (BRCA1, BRCA2, p16, XPA, WRN) was significantly increased only by isoform IV or by isoforms IV and VI. We conclude that differences in biological activities (e.g. induction of cellular senescence) exerted by PML isoforms, may partially result from differences in sets of genes activated or repressed by these isoforms. Moreover, our data indicate that some PML isoforms may participate in formation of nuclear "compartments" regulating the stability of nuclear proteins. 27. EFFECT OF HEME OXYGENASE-1 ON PROLIFERATION, VIABILITY AND ANGIOGENIC POTENTIAL OF MELANOMA CELLS IN MICE Halina Waś1, Tomasz Cichoń2, Anna Ratajska3, Anna Grochot1, Barbara Węgiel1, Agnieszka Łoboda1, Agnieszka Jaźwa1, Józef Dulak1, Alicja Józkowicz1 1Department of Medical Biotechnology, Faculty of Biotechnology, Jagiellonian University,30-387 Kraków, ul. Gronostajowa 7, Poland 2Department of Molecular Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland 3Department of Pathological Anatomy, Medical University of Warsaw, 02-004 Warsaw, ul. T. Chałubińskiego 5, Poland Background: Heme oxygenase-1 (HO-1), an enzyme degrading heme to biliverdin, carbon monoxide and iron ions, may augment angiogenesis. Additionally, HO-1 can regulate cell cycle and apoptosis. Here we investigated the effect of HO-1 overexpression on the proliferation, viability and angiogenic potential of B16(F10) murine melanoma cell line in vitro and in vivo. Methods and results: Establishing the HO-1 overexpressing cell line (B16(F10)-HO-1) was confirmed by PCR, RT-PCR and western blotting. Overexpression of HO-1 led to a significant increase in melanoma cell proliferation, as measured by BrdU incorporation. Moreover, B16(F10)-HO-1 cells were more resistant to oxidative stress. Four-hour exposure to 200 µM H2O2 resulted in massive death of wild-type melanomas, whereas almost 70% of B16(F10)-HO-1 cells were still viable, as assessed by trypan-blue exclusion. Accordingly, low concentration of H2O2 (0.78-3.12 µM, 18 h) induced apoptosis in B16(F10), but not in B16(F10)-HO-1, as measured by TUNEL assay. Finally, conditioned media harvested from B16(F10)-HO-1 more potently induced endothelial cell proliferation and formation of capillaries by endothelial spheroids than those collected from B16(F10). This effect was not modified by pre-treatment of media with antibodies blocking the vascular endothelial growth factor (VEGF). In accordance, HO-1 overexpression did not influence the generation of VEGF as checked at the promoter, mRNA and protein levels in melanoma. To check the effect of HO-1 overexpression on tumor development 0.2 x 106 B16(F10) or B16(F10)-HO-1 cells were injected intracutaneously into the back of syngenic C57BL/6 mice. Overexpression of HO-1 in melanoma cells shortened survival time of B16(F10)-bearing mice. Surprisingly, there were no differences in tumor size and in tumor necrotic areas between mice bearing B16(F10) and B16(F10)-HO-1 cells. Preliminary analyses suggest, however, that HO-1 overexpression was associated with reduced inflammatory edemas in the tumors and with increased density of melanoma cells. In accordance, the levels of tumor necrosis factor (TNF), in serum and tumor lysates from mice injected with B16(F10)-HO-1 cells were lower and the levels of tumor necrosis factor receptor I (sTNFRI) were higher than from those injected with the wild-type melanoma. Moreover, immunohistochemical staining using anti-CD31 antibodies reveals also the stronger vascularization of HO-1 overexpressing tumors. Interestingly, the levels of vascular endothelial growth factor (VEGF) was significantly increased in tumor lysates from mice injected with B16(F10)-HO-1 cells than from those injected with the wild-type melanoma. Conclusion: HO-1 increases viability, proliferation and angiogenic potential of murine melanoma cell line. Accordingly, overexpression of HO-1 in tumor cells leads to decrease in survival time of melanoma-bearing mice, inhibition of inflammatory reaction, and increase in tumor vascularization. It suggests that reduction of HO-1 activity might be beneficial in therapy of melanoma. Supported by grant PBZ-KBN107/P04/2004 from Polish Ministry for Scientific Research and Information Technology 28. NOVEL GENES IDENTIFIED IN HEPG2 CELLS ACTIVATED BY IL-1 AND IL6 Paulina Węgrzyn, Marta Byrska, Anna Łuchniak, Magdalena Pająk, Anna Pengal, Jolanta Jura, Aleksander Koj Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow Interleukin-1 and interleukin-6 are principal cytokines involved in regulation of expression of acute-phase proteins. Both cytokines are released from activated macrophages, fibrobalsts, keratinocytes and endothelial cells and stimulate liver and other tissues to synthesis of acute phase proteins. To evaluate the changes in the transcript level of hepatoma-derived cell line after stimulation with both cytokines a differential display analysis was carried out. HepG2 cells were treated with IL-1 or IL-6 alone, or with both cytokines for different time periods. Based on the homology data all identified transcripts were classified into 13 groups according to the function of proteins encoded by these transcripts. These proteins are engaged in cellular metabolism, protein synthesis, trafficking, transport, signal transduction, DNA interaction, transcription, posttranscription modification, posttranslation modification, cell proliferation, stress protection, proteases and proteins functionally not yet classified. Forty transcripts (10 - under IL-1 stimulation, 21 - under IL-6 stimulation and 9 - under IL-1/IL-6 stimulation) had no homology with known and functionally classified genes deposited in GenBank entries. Transcript sequences matched BAC and PAC or hypothetical cDNA sequence clones with the identity reachable in more than 85%. Two of them are being now studied in details. Both genes were cloned to analyze their regulation under proinflammatory cytokines treatment and their importance in cell viability. Using Northern and Western blot analysis we studied modulated expression of both transcripts and occurrence of protein products on cellular and tissue level. This work is supported by grants: 2 P05A 011 27 and 2 P04B 022 28 from the State Committee for Scientific Research (Warsaw, Poland). 29. MICRODELETIONS AND CHROMOSOMAL BREAKPOINTS OF RGS AND RGS-LIKE CANCER SUSCEPTIBILITY GENES IN BREAST CANCER Emilia Wiecheć, Lise Lotte Hansen Institute of Human Genetics, The University of Aarhus, The Bartholin Building, Wilhelm Meyers Allé 240, 8000 Aarhus C, Denmark e-mail: emilia@humgen.au.dk,lotte@humgen.au.dk Background: The RGS (regulators of G protein signalling) genes have a very high cancer potency because they encode for proteins which are involved in cellular proliferation, differentiation, response to neurotransmitters, membrane trafficking and embryonic development. These genes are located within a 370 kb region at the chromosome 1q25.3 to which the hereditary prostate cancer (HPC1) locus is tightly linked. The loss of heterozygosity/allelic imbalance results provided information about high level of chromosomal instability reflected in the breakpoints localized between the analyzed microsatellite loci. This study aimed at a detailed mapping of the position of these potential breakpoints in patients with breast cancer. Methods: The breakpoint screening PCR-based analyses were performed in order to determine the size of the deleted parts of the examined RGS genes at the chromosome 1q25.3. The fragile sites flanked by microsatellite markers were divided into small fragments and PCR amplified. The sizes of these fragments varied from 500-1200 bp. A set of 60 primer pairs spaced across chromosome 1q25.3 containing the affected RGS genes were used. Results: PCR analysis revealed intragenic chromosomal breakpoints in the RGSL2, RGS16 and RGS8 in breast tumours. The major common chromosomal breakpoint region was found inside RGS8 and involved intron 3, exon 4 and intron 4. The size of a typical microdeletion localized within the amplified segment (27 kb) ranged from 1 kb (26%), 2 kb (22%), 2.8 kb (11%), 4 kb (4%) to 5 kb (4%). The analyzed region was completely deleted in two breast tumours. Frequent deletions were also found in the potential promoter region of RGS16. Ten of the thirty one patients (32%) shared a common microdeletion of 1.4 kb localized in the close vicinity of exon 1 in RGS16. The third, significant fragile site within the 370 kb region I have identified, was located between the RGSL2 in2 and RGSL2 ex4 microsatellite loci. This chromosomal breakpoint involves exon 3 and 4. Conclusion: These results suggest a possible relationship between microdeletions in the RGS genes and the protein expression. Frequent 1q25.3 chromosomal breakpoints are likely to play a role in breast tumorigenesis as they affect potential tumour suppressor genes. Furthermore, a more extended screening for 1q25.3 chromosomal breakpoints and their implication in breast cancer is required. Key terms: breast cancer, loss of heterozygosity/allelic imbalance, microsatellite markers, RGS genes, microdeletions, chromosomal breakpoint. 30. DNA INTERSTRAND CROSSLINKS ARE INDUCED IN CELLS PRELABELLED WITH 5-BROMO-2'-DEOXYURIDINE AND EXPOSED TO UVC RADIATION A. Wojcik1,2,x, A. Bochenek1, A. Lankoff1, H. Lisowska1, A. Padjas3, C. von Sonntag4 and G. Obe5 1Institute of Biology, Swietokrzyska Academy, Kielce 2Institute of Nuclear Chemistry and Technology, Warszawa 3Swietokrzyskie Oncology Center, Kielce 4Max-Planck-Institut für Bioanorganische Chemie, Mülheim an der Ruhr 5Institute of Genetics, University of Essen, D-451117 Essen, Germany It has been observed previously that 5-bromo-2'-deoxyuridine (BrdU) potentates the effect of UVC radiation on the level of sister chromatid exchanges. It is not known which type of DNA damage is responsible for this enhancing effect and we have proposed this to be the DNA interstrand crosslink (ICL) which, theoretically, may arise in cells that are labeled with BrdU for one round of replication and exposed to UVC radiation. The aim of the present investigation was to verify if ICLs are indeed formed in this irradiation scenario. CHO-K1 cells were prelabelled with BrdU and exposed to UVC. ICLs were detected by the modified version of the comet assay that relies on the reduction of induced DNA migration in the agarose gel. Carboplatin was used as a positive control. We found that BrdU UVC treatment indeed results in a reduction of the damage induced by  -radiation. Furthermore, we observed that CL-V4B cells exposed to BrdU UVC, but not to UVC alone, showed a very high level of chromosomal damage. These cells have a deficient Rad51C paralog that renders them extremely sensitive towards ICLs. Taken together these results clearly show that ICLs are formed in DNA that is prelabeled with BrdU and exposed to UVC radiation.