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		1. THE EFFECT OF INHIBITION OF PARP ON THE FREQUENCY OF HOMOLOGOUS RECOMBINATION IN CHO-K1 WILD TYPE AND XRS-6 MUTANT CELL LINE T. Bartłomiejczyk, M. Wojewódzka, M. Kruszewski Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195, Warszawa, Poland The aim of this project was to examine the role of PARP-1 (poly(ADP-ribose) polymerase-1) in repair of DNA double strand breaks. In mammalian cells DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmid carrying two nonfunctional copies of the galactosidase (lacZ) gene in a tandem array. As a result of recombination they give rise to a functional copy of beta-galactosidase. Isolated transfected clones were used to examine the effect of ADP-ribosylation inhibition on the frequencies of spontaneous and X-ray- (2 Gy) induced recombination. The cells were incubated with the PARP-1 inhibitor (AB) and recombination frequency was determined with histochemical or flow cytometry methods. Cells were cultured in the medium containing G418 or left to grow without G418 (5 days before and 2 days after treatment). This treatment allows to distinguish between reciprocal gene exchange/HR (loss of neo gene) and gene conversion (neo gene is still present and cells are resistant to G418). The level of -galactosidase activity (reflecting the frequency of spontaneous recombination) in transfected CHO-K1 cells was 2-3 times lower than in xrs-6 cells, whereas frequency of recombination per generation measured by flow cytometry was one order of magnitude lower. However, significant differences between individual clones have been observed. Irradiation of the cells with 2 Gy of X-rays insignificantly elevated the level of the enzyme in both cell lines. As expected, in non-irradiated cells, release of the selective pressure (7 days without G418) significantly elevated the level of beta-galactosidase activity as compared with that in cells cultured with G418. These results suggest that the defect in NHEJ-mediated DSB repair pathway results in elevated frequency of HR. No effect of inhibition of poly(ADP)rybosylation was observed in this experimental model. 2. ETOPOSIDE-INDUCED APOPTOSIS OF HL-60 CELLS MAY BE ENHANCED BY PYRROLIDINE DITHIOCARBAMATE (PDTC). Ilona Bednarek Department of Biotechnology and Genetic Engineering, Medical University of Silesia; Narcyzow 1 Street, Sosnowiec, Poland. Chemotherapy is one of the main strategies used for medical intervention in cancer treatment. Most of antineoplastic drugs induce programmed cell death - apoptosis. Agents that interfere with the ability of an antineoplastic drug to induce apoptosis may limit drug efficacy. Reactive oxygen species activate cell death but mostly by non-apoptotic mechanism causing damage to the surrounding tissue. According to the published data cells killed in vivo in the presence of hydrogen peroxide are not phagocytosed until after they have begun to leak their contents into the extracellular space, what may induce a cycle of chronic inflammation and further interference with chemotherapy action. The overall effectiveness of chemotherapy might by improved by co-administering antioxidants with chemotherapeutic agent. The aim of the presented study was to determine, whether the antioxidant: pyrrolidine dithiocarbamate (PDTC) enhances etoposide-induced apoptosis of cancer cells. Using HL-60 cells, human acute promyelocytic leukemia cells, we show that PDTC used at 12.5 - 25 µM concentration administrated with etoposide increases the apoptotic cell death. Proliferation of HL-60 cells treated with etoposide dropped down with increasing dose of drug, the values of cytotoxic index (IC50) was estimated as 17 µM (24 hrs incubation). The highest apoptotic cell death induction was observed in drugs combination: 12.5 µM of PDTC with 4 µM of etoposide. Quantitative analysis of bcl-2 and bax mRNA showed changes in bcl2/bax ratio: from 0.525 for etoposide-treated HL-60 cells to 0.334 in HL-60 treated with PDTC and etoposide. On the basis of presented results a conclusion can be made that administration of antioxidant pyrrolidine dithiocarbamate increases etoposide antineoplastic efficiency regarding to human HL-60 leukemia cells apoptosis. 3. PLASMID DNA COMBINED WITH CYCLOPHOSPHAMIDE INHIBITS B16(F10) MELANOMA METASTASES SPREAD INTO MOUSE LUNGS. Tomasz Cichoń, Ryszard Smolarczyk, Stanisław Szala Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch. The aim of the presented study was to examine if gene therapy using plasmid DNA constructs encoding genes of anti-angiogenic proteins can be effectively applied to inhibit experimental B16 (F10) melanoma metastases in mouse lungs. Two different therapy approaches were used. The first was based on transferring plasmid DNA into muscle tissues via electroporation; second involved systemic transfer of polyplexes formed by the plasmid construct and PEI 750 kDa or PEI 25 kDa. In experiments involving constructs' transfer via electroporation the number of lung metastases was decreased two-fold only in mice treated with endostatin and sFlt-1 genes. No prolonged mice survival was observed as compared to controls. Both experimental and control animals died at approximately the same time. In therapy mediated by polyplexes two-fold decrease in the number of metastases was seen in both mice treated with constructs encoding antiangiogenic proteins as in mice treated with constructs containing empty plasmid. Neither this solution resulted in prolonged animal survival. The decreased number of metastases resulting from use of polyplexes was a result of CpG motifs present in plasmid backbone and not of therapeutic genes' presence in the tested constructs. Survival rates were improved by treatment of mice with a combination of cyclophospamide and plasmid DNA. Our data indicate for the first time that only specific combinations of plasmid DNA with cyclophosphamide can inhibit metastatic spread and significantly prolong survival rates. 4. DNA ADDUCTS FORMATION AND DNA CROSSLINKING BY C-1748, A POTENT ANTITUMOR 4-METHYL-1-NITROACRIDINE IN HUMAN COLON CANCER CELLS A. Dyrcz, J. Lewandowska, A. Bartoszek, J. Konopa Gdańsk University of Technology, Dept. of Pharmaceutical Technology and Biochemistry, Gdańsk, Poland 4-Substituted 1-nitroacridines represent a new group of acridine derivatives synthesized at Gdansk University of Technology. Compared to parent 1-nitroacridines, these compounds exhibit lower toxicity and enhanced antitumor efficacy especially against colon and prostate cancers. Initiation of the first phase of clinical evaluation is proposed for the leading derivative, 4-methyl-1-nitroacridine, denoted C-1748. The introduction of an electron donating methyl group into position 4 (para to 1-nitro) decreased the susceptibility of 1-nitro group to reduction. This 1-nitro group is crucial for biological activity and also for the ability of 1-nitroacridines to form DNA adducts and induce interstrand DNA crosslinking. In the present study, we compare DNA covalent binding properties of C-1748 and its parent 4-unsubstituted analogue - C-857. Two radioactive methods were used: 32P-post-labelling technique for detection of covalent DNA binding and modified Parsons method for detection of interstrand DNA crosslink formation. The maps of 32P-labelled adducts formed by C-1748 displayed more chromatographic spots (adducts) than those obtained for C-857 under experimental conditions in human colon carcinoma HT29 cells. The chromatographic pattern of DNA adducts observed in cell-free system resembled the ones observed in cell system. Using Parson's method it was shown that C-1748 and C-857 were able to form DNA crosslinks in human colon cancer HCT-8 cells in a dose-dependent manner. The DNA crosslinking bonds induced by 1-nitroacridinones were stable in alkaline conditions but they were sensitive to high temperature. In conclusion, obtained results suggest that this new generation of 1-nitroacidines is able to bind covalently to DNA and crosslink DNA. Thus DNA seems to be their major molecular target for covalent modification. 5. SECOND PRIMARY TUMORS OF HEAD AND NECK - LOH PATTERN ANALYSIS AND ASSESSMENT OF THEIR CLONALITY STATUS. Maciej Giefing1 *, Małgorzata Rydzanicz1 *, Katarzyna Szukała1, Aldona Woźniak3, Małgorzata Wierzbicka2, Krzysztof Szyfter1,2 and Maciej Kujawski1 1Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland 2Department of Otolaryngology, Medical University of Poznań, Poland 3Department of Clinical Pathomorphology, Medical University of Poznań, Poland *These authors contributed equally to this work The reason of treatment failures in head and neck tumors is often linked to the appearance of second primary tumors (SPT). There are three mechanisms of SPT development of clonal or non clonal secondary tumors: 1o via micrometastases (clonal); 2o from a common carcinogenic field (SFT) (partially clonal); 3o via independent events (from different carcinogenic fields - "true" SPT) (not clonal). In this study a set of 12 microsatellite markers was used to find similarities and/or differences in allelic imbalance patterns between 22 pairs of tumors (index and SPT) obtained from the Department of Otolaryngology, K. Marcinkowski Medical University, Poznań, Poland and to confirm if two tumors originated and progressed from a single cell indicating clonal progression or independently in different locations as a result of unrelated initiations of carcinogenesis. The results allowed to distinguish unrelated second primary tumors in 9 (41%) cases and in 4 (18%) cases particular tumors carried clonal genetic changes. In 9 (41%) cases the results were insufficient or ambiguous to state the clonality status. Assessing the clonality of diagnosed tumors is not only a matter of theoretical dispute but carries important clinical implications including chemoprevention, radiotherapy and general patient management and thus should become a routine molecular diagnostic tool. 6. ENERGY CHARGE AND CELL PROLIFERATION ACTIVITY AS MARKERS OF THE MALIGNANCY IN GLIOMAS. J. Głogowska-Ligus, A. Gruchlik, A. Wilczok, A. Zajdel, U. Mazurek Department of Molecular Biology and Medical Genetics, Medical University of Silesia, Katowice, Poland. Molecular analysis is currently being used in clinical diagnostics of tumors. One of the methods for assessment of tumor malignancy is determination of transcriptional activity of histone H3 genes. Expression of H3 encoding genes correlate with DNA synthesis both in normal and malignant cells while H3 transcripts are absent in cells that do not divide. Tumor cells are characterized by high proliferation, inhibition of differentiation and changes in metabolism. ATP concentration, which reflects balance between synthesis and hydrolysis in reactions requiring energy is directly associated with cell metabolism. The depletion of ATP synthesis influences adenylate energy charge of the cell. The aim of our study was to investigate proliferation activity in gliomas of different malignancy and to compare expression level of histone H3 genes with energy metabolism in gliomas. The studied material consisted of brain tissue obtained form stereotactic biopsy. Biopsy specimens were divided into two parts: one part was used for RNA extraction and another one for extraction of phosphorylated nucleotides. Total RNA extracted form the material served as a template in Real-Time RT-PCR (TaqMan) for determination of the transcriptional activity of histone H3 genes using ABIPRISM 7700 Sequence Detector System and ß-actin as an internal control. Each result was normalized on the basis of ß-actin transcript content. HPLC analyses of ATP, ADP and AMP were carried out using a Hewlett Packard 1100 system with a diode array UV-VIS detector. The extracts kept at -70oC were melted just before analysis, immediately filtered (0.22 µm, Millipore) and injected onto the C18 Eurospher column (4 x 250 mm, 5 µm, Knauer). A gradient mixture of methanol, ammonium acetate (0.2 M) and water was used for elution. Separations were performed at 30oC using 1 ml/ min flow rate. Determination of ATP, ADP and AMP was based on retention times and spectral characteristics comparison with the corresponding standards. A library search function of the HPLC ChemStation software was used in all experiments. Amounts of the adenylate nucleosides were calculated using appropriate calibration curves. Our study indicates that the histone H3 genes were expressed in all examined samples. The highest level of mRNA H3 was found in glioblastoma. This corresponded to the low energy charge. On the contrary, astrocytoma II and III showed lower levels of mRNA H3 and higher energy charge. 7. THE ROLE OF ERCC3/XPB IN GLOBAL AND TRANSCRIPTION-COUPLED DNA REPAIR Ján Gurský1, Ivana Rybanská1, Edmund Paul Salazar2, Erika Kimlíčková1, Lawrence Hadley Thompson2 and Miroslav Piršel1 1Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovak Republic 2Lawrence Livermore National Laboratory, Livermore, California, U.S.A. Mutation of the ERRC3/XPB gene in humans gives rise to the distinct autosomal recessive disorders with a striking clinical heterogeneity: xeroderma pigmentosum associated with Cockayne's syndrome (XP/CS) and trichothiodystrophy (TTD). XPB is a subunit of a multifuctional RNA polymerase II general initiation factor TFIIH and codes for 3'-5' DNA helicase essential for both nucleotide excision repair (NER) and transcription. In NER, it unwinds DNA around the damage by about 25-30 base pairs forming an open complex. In transcription, XPB functions at multiple steps to promote efficient initiation and promoter escape by RNA polymerase II. There have been only five XP-B patients with three naturally occuring mutations identified so far. The XPB gene was shown to be a homologue of the hamster ERCC3 gene. Hamster cell lines belonging to the third complementation group are a unique source for the structure - function analysis of the ERCC3/XPB protein. To study the role of the ERCC3/XPB protein in the removal of variuos DNA damages, we focused on the role of NER and TCR in the removal of DNA damage caused by ultraviolet (UV-C)-irradiation and hydrogen peroxide (H2O2) in the wild type and 9 UV-sensitive ERCC3 hamster mutant cell lines. All the tested mutant cell lines are very sensitive to killing by UV-C and are unable to recover RNA synthesis after 10 Jm-2 UV-C, suggesting a defect in TCR. They also have limited global NER capacity measured by the single cell gel electrophoresis assay (0.25 Jm-2). Their sensitivity to H2O2 is the same as in the wild type cell lines suggesting they possess functional global BER. This work was supported by Science and Technology Assistance Agency under the contract no. APVT-51-003202 and by the U.S.-Slovak Science and Technology Joint Fund under the project no. 031/2001. 8. HUMORAL IMMUNE RESPONSES INDUCED IN MICE WITH 12-AA CONSTRAINED PEPTIDES WHICH MIMIC GD2 GANGLIOSIDE Irena Horwacik, Dominik Czaplicki, Hanna Rokita Faculty of Biotechnology, Jagiellonian University, 7 Gronostajowa St., 30-387, Kraków Neuroblastoma is the most common extracranial tumour in childhood. Despite applying standard treatment including intensive induction, surgery, radiotherapy, myeloablative therapy with stem cell transplantation the majority of the high risk patients eventually die of relapsing disease. This stresses the need for new therapeutical approaches to treat minimal residual disease, which might improve the survival of high risk group neuroblastoma patients. Immunotherapy of neuroblastoma is one of the currently developed strategies. It aims to mobilise innate and adoptive immunity of patients for recognising and eradicating tumor cells. Our goal was to develop active specific immunotherapy of neuroblastoma, which targets neuroblastoma associated carbohydrate antigen, namely GD2 ganglioside. Gangliosides have been shown to be weakly immunogenic, since they belong to a group of T cell independent antigens. For this reason, we are working on application of peptide vaccines, which could structurally and functionally mimic GD2 ganglioside. We have panned phage display peptide library LX-8, which encodes for 12 aa peptides constrained with disulphide bridge, using mouse monoclonal anti-GD2 ganglioside antibody 14G2a. Five peptides sequences have been identified. The synthesised peptides have been shown to bind to 14G2a antibody in competition assay using GD2 ganglioside-positive human neuroblastoma cell line IMR-32. Using BALB/c mice model we tested the peptides for their ability to induce GD2 ganglioside specific immune responses. Animals were immunised with 3 doses of peptides conjugated to carrier protein KLH (keyhole limpet hemocyanin) in combination with adjuvants: Freund's adjuvant and GM-CSF. GD2 ganglioside boosting has been applied for the final immunisation step. Three weeks after the last dose of our vaccine sera samples were collected and analysed for anti-GD2 ganglioside humoral responses. In order to find the best epitope that mimics functionally GD2 ganglioside we have analysed the sera for the level and isotype of the antibodies that bind to the immobilised GD2 ganglioside with ELISA. In addition, flow cytometry technique has been utilised to show the cross-reactivity of the murine sera with the glycolipid expressed on neuroblastoma cells. The work was supported by 3P05A 00124 grant from the Polish Ministry of Scientific Research and Information Technology. 9. THERAPEUTIC EFFECT OF VASOSTATIN IN THE TREATMENT OF B16(F10) MURINE MELANOMA Joanna Jazowiecka-Rakus, Magdalena Jarosz, Stanisław Szala Department of Molecular Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch. It has become almost indisputable that angiogenesis i.e. formation of new blood vessels out of pre-existing capillaries, is essential to development of the tumor vasculature. The discovery of specific antiangiogenic inhibitors would have important therapeutic applications in the treatment of cancer. Vasostatin, the N-terminal domain of calreticulin, is a potent endogenous inhibitor of angiogenesis and tumor growth. In this study we compared vasostatin gene therapy strategy with administration of purified recombinant vasostatin in B16(F10) murine melanoma model. We applied the combination of intramuscular gene transfer of vasostatin by electroporation and cyclophosphamide using an antiangiogenic schedule of drug dosing. In such combined therapy we observed synergistic antitumor as well as extended survival of treated mice. However, when melanoma-bearing mice received i.t. recombinant vasostatinwe observed a more significant suppression of tumor growth and prolonged survival. These initial results suggest that vasostatin protein applied in cancer therapy is more effective than combination of vasostatin gene and cyclophosphamide and that recombinant vasostatin might find potential therapeutic use in the future. Key words: vasostatin; gene therapy; angiogenesis; cyclophosphamide This study was supported by a grant (No.007/2002) from Polpharma Foundation. 10. EXPRESSION OF EXTRACELLULAR MATRIX METALLOPROTEINASES : MMP-1, MMP-3, MT1-MMP (MMP-14) AND MT2-MMP (MMP-15) IN LIVER BIOPSIES OF PATIENTS WITH CHRONIC HEPATITIS C M. Jurzak1, U. Mazurek1, C. Kruszniewska1, W. Mazur2, Z. Gonciarz2, T. Wilczok1 1Department of Molecular Biology an Medical Genetics, 2Department of Internal Medicine, Medical University of Silesia, Katowice, Poland Prolonged activation of hepatic stellate cells caused by HCV infection together with accumulation of extracellular matrix components may result in liver fibrosis developing into cirrhosis. Removal of factor which caused liver injury may lead to regression to the initial stage of liver fibrosis as a result of activating extracellular matrix metalloproteinases, which may degrade components produced by activated hepatic stellate cells. Liver biopsies and blood of sixty patients with chronic hepatitis C were examined in this study. The aim of this study was to determine the transcriptive activity of genes encoding extracellular matrix metalloproteinases in liver biopsy in patients with chronic HCV infection and to determine the transcription activity according to presence or absence of RNA HCV in liver biopsy, genotype of HCV, stage and grade in histopathological picture QRT-PCR was performed using sequence detector ABI PRISMTM 7700. SSCP and reversed hybridization (test INNOLiPA HCV II) were used. It was shown that the presence of RNA HCV in liver biopsies of patients with chronic hepatitis C irrespective of genotype HCV: 1b or 3 are accompanied by overexpression of genes encoding MMP-3 and MMP-15 but without influence on the expression of genes encoding MMP-1 and MMP-14. No changes of the expression of genes encoding extracellular matrix metalloproteinases: MMP-1, MMP-3, MMP-14 and MMP-15 in liver biopsies of patients with chronic hepatitis C with stage: F1, F2 and grade: G1, G2 in histological pictures of liver biopsies were shown. Level of HCV viraemia in blood was directly proportional to the level of HCV viraemia in liver biopsies and may allow determining the level of HCV viraemia in liver. On the basis of RNA HCV in blood the level of viraemia in the liver can be estimated. 11. REGULATION OF THE HUMAN APOPTOTIC NUCLEASE ENDONUCLEASE G Magdalena Kalinowska, Wojciech Garncarz, Monika Pietrowska and Piotr Widłak Department of Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland Endonuclease G (EndoG) is a mitochondrial enzyme that turns to be an apoptotic nuclease when released from mitochondrial intermembrane space. EndoG is a DNA/RNA non-specific nuclease. However, at physiological ionic strength, RNA is a much more favorable substrate of EndoG, as compared to chromatin. This indicates that (i) EndoG could be an apoptotic RNase and/or (ii) for efficient cleavage of double-stranded DNA in vivo EndoG requires additional co-activators. In the present study we have searched for factors that affected activity of human EndoG. EndoG forms in vitro complexes with AIF and FEN-1 but not with PCNA. Interestingly, heat shock proteins 70 interact with EndoG and are involved in regulation of its activity. Purified Hsp70 prevented stimulation of EndoG activity by other nuclear factors in the ATP-dependent manner. 12. DNA DAMAGE IN CHILDREN EXPOSED TO LEAD Lucyna Kapka1, Diana Anderson2, Marcin Kruszewski3, Ewa Siwińska1, Tomasz Ołdak4, Danuta Mielżyńska1 1Institute of Occupational Medicine and Environmental Health, Dept. of Genetics Toxicology, Sosnowiec, Poland 2 University of Bradford, Dept. of Biomedical Sciences, Bradford, United Kingdom 3Institute of Nuclear Chemistry and Technology, Dept. Radiobiology and Health Protection, Warsaw, Poland 4M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Dept. Experimental Hematology and Cord Blood Bank, Warsaw, Poland Environmental exposure to lead in childhood remains a serious environmental health problem in the Silesia region. Lead is known to be a toxin mostly affecting nervous and hematopoietic systems, however its genotoxic potential has also been shown. The objective of the study was to confirm contribution of lead to the development of cytogenetic damage and to assess selected genotoxic effects of environmental exposure to lead in children. Additional aim was to examine if the micronuclei formation results from a break of chromosome or damage of karyokinetic spindle. Examined population was composed of 67 nine year old children, living in the region where non-ferrous ores are extracted and processed and 15 control children of the same age. Exposure to lead was assessed by lead in blood (PbB) determination (AAS). The following biomarkers of DNA damage were included: (1) frequency of micronuclei (MN) - to confirm the relationships between PbB and MN levels observed in our previous study, (2) MN-FISH with specific pan-centromeric probe - to distinguish between MN caused by chromosome breakage and MN cause by chromosome malsegregation, (3) single strand breaks (SSB) by comet assay (alkaline version) expressed as tail moment-TM, (4) FPG sensitive sites by comet assay modified by use of DNA glycosylase expressed as TM - to estimate DNA bases oxidation, as well as (5) mutation frequency in (a) locus TCR in lymphocytes (number of CD4 /CD3- cells) and (b) locus GPA in erythrocytes (number of N0 and NN cells) in MN heterozygotes. Environmental exposure to lead resulted in significantly increased levels of PbB, although average level was much below the value of biological exposure limit = 10 mg/dl. Median value in exposed children was 4.8 mg/dl compared to the controls: 2.5 mg/dl. The results showed significant difference in SSB level between exposed and control groups: median value was 1.81 vs. 0.92, respectively. Environmental exposure caused significant induction of MN (2.8 vs. 1.0). The level of micronuclei with centromeric signals was also significantly increased in exposed children. Examined children had higher level of FPG sensitive sites and mutation frequency in GPA locus compared to control children while no effect of exposure was found in relation to the frequency of mutations in locus TCR. In conclusion, our results indicate that although environmental exposure to lead was not high, it resulted in measurable biological effects in examined children (SSB, MN, MN-FISH, FPG, GPA). Higher level of micronuclei with centromeric signals suggests that the formation of micronuclei probably results from a damage to karyokinetic spindle. 13. THE ROLE OF rrp1 IN THE REPAIR OF DNA DAMAGE IN SCHIZOSACCHAROMYCES POMBE. Paweł Karpiński and Dorota Dziadkowiec Institute of Genetics and Microbiology, Wrocław University, ul. Przybyszewskiego 63/77, 51-148 Wrocław, e-mail: dorota@microb.uni.wroc.pl Homologous recombination is an important process assisting in the repair of DNA double-strand breaks. In eukaryotic cells, proteins belonging to the Rad51 (Rhp51 in fission yeast) group, functionally homologous to the Escherichia coli RecA protein, take part in homologous pairing and strand exchange, essential to the DNA repair process. It was previously shown that sfr1 gene in Schizosaccharomyces pombe participates in the Rhp51-dependent recombination repair pathway that does not require the Rhp57 protein. Here we report that another protein, Rrp1, possibly involved in chromatin remodelling and DNA repair, may also take part in this pathway. The strain devoid of the rrp1 gene is viable and does not show any apparent growth defect upon exposure to UV or MMS. It is also proficient in mating-type switching. The double mutant Drrp1Drhp57 but not Drrp1Dsfr1 or Drrp1Drhp51 is more sensitive to UV and MMS than respective single mutants, suggesting that, as sfr1, rrp1 is epistatic to rhp51, but not rhp57. Further studies will be needed to determine the exact role of these genes in different aspects of recombination DNA repair in fission yeast. 14. CHARACTERIZATION OF A SILENCER LOCATED WITHIN CpG ISLAND UPSTREAM TO THE CHICKEN ALPHA-GLOBIN GENE CLUSTER Denis Klochkov*, Elena S. Ioudinkova and Sergey V. Razin Laboratory of Structural-Functional Organization of Chromosomes,Institute of Gene Biology of the Russian Academy of Sciences, 34/5 Vavilov Street, 117334 Moscow, Russia *Correspondence to D. Klochkov (e-mail: dklochkov@mail.ru) It was shown that the DNA fragment of 200 base pairs size located within the CpG island at 2.5 - 4.5 kbp distance upstream of the chicken alpha-globin gene cluster contains binding site for multifunctional protein factor CTCF and possesses a silencer activity, but not an insulator or promoter activity. The formation of complex between CTCF and the DNA fragment under study was demonstrated using electrophoretic mobility shift assays. The data are discussed with underlying hypothesis suggesting that the discovered silencer plays role in regulation of the expression of ggPRX gene (chicken analogue of human housekeeping gene "-14"), transcribed in the direction opposite to the alpha-globin genes transcription. 15. THAMINE PREVENTS X-RAY INDUCTION OF GENETIC CHANGES IN HUMAN LYMPHOCYTES IN VITRO Maria Konopacka1, Jacek Rogoliński1, Andrzej Orlef2 1Department of Experimental and Clinical Radiobiology, 2Department of Medical Physics, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland Oxidative stress influences DNA and other biomolecules damage via oxidative changes to their chemical structure. These changes are believed to increase the risk of cancer, heart disease and aging processes. It has been demonstrated that antioxidants such as ascorbic acid, tocopherols and flavonoids give protection against oxidative damage and several degenerative diseases, including cancer. Little is known about whether B group vitamins give protection against DNA damage in human cells. The water-soluble vitamin B1 (thiamine) is commonly included in multivitamin preparations and it is nontoxic for humans. In the present study the effects of thiamine on the extent of spontaneous as well as irradiation-induced DNA damage was measured in cultured human lymphocytes. Cultures were exposed to increasing concentrations of thiamine (0-500 µg/ml) and irradiated with X-rays. The DNA damage was estimated as the frequency of micronuclei and apoptotic or necrotic morphological changes in fixed cells. The results show that thiamine alone did not induce genetic changes. A significant decrease in the fraction of apoptotic and necrotic cells was observed in lymphocytes irradiated in the presence of vitamin B1 at concentrations between 1-100 µg/ml compared to those irradiated in the absence of thiamine. Vitamin B1 at 1 and 10 µg/ml decreased also the extent of radiation-induced formation of micronuclei. Vitamin B1 had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The results indicate that vitamin B1 protects human cells from radiation-induced genetic changes. 16. THE FUNCTIONAL IMPACT OF POLYMORPHISMS AND MUTATIONS IN SELECTED DNA REPAIR GENES: XPA, XPB AND RECQ1 ON THE PROTEIN LEVEL, LOCALIZATION AND ACTIVITY. Małgorzata Krześniak1, Dorota Butkiewicz1, Małgorzata Mrzygłodzik1, Rasa Vaitiekunaite1, Jolanta Pamuła1, Curtis C. Harris2, Marek Rusin1 1Department of Tumor Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 2National Cancer Institute, NIH, Bethesda, United States of America The inter-individual variation in DNA repair efficiency and cancer susceptibility are associated with polymorphisms of DNA repair genes. Previously, we and others have found sequence alterations within the coding and non-coding regions of genes involved in DNA metabolism. It is not known whether and how these particular sequence alterations change the functioning of the encoded proteins. We focused on two genes involved in nucleotide excision repair system - XPA, XPB and on the RECQ1 gene encoding one of the human helicases from the RecQ family. This gene family contains at least three genes (WRN, BLM and RTS), whose mutations are associated with cancer-prone genetic diseases (e.g. Werner syndrome, Bloom syndrome, Rothmund-Thomson syndrome). For our functional analyses we have selected the common polymorphism of XPA (-4G>A) located four residues upstream the start codon, which in previous molecular epidemiological analyses showed significant association with lung cancer risk. Two relatively infrequent XPB polymorphisms (117:Lys>Arg; 402:Gly>Cys) were selected due to their localization within the evolutionary conserved regions of the protein. Finally, we studied the functional impact of three sequence alterations of RECQ1 helicase gene: 248:Ala>Pro, 487:Lys>Thr (polymorphic allele), 566:Thr>Ala (sequence alteration found in HeLa cells) arranged as 8 different sequence combinations. RECQ1 helicase is not well studied protein and its cellular function remains unknown. Our luciferase reporter assay showed that co-transfection of RECQ1 expression vector with the reporter vector causes fourfold increase of the reporter gene activity when compared with the co-transfection of the LacZ control expression vector, indicating that RECQ1 facilitates gene expression. The high RECQ1 expression in the lung suggests that its sequence alterations may influence lung cancer risk. Using the luciferase reporter assay, host cell reactivation assay, in vivo protein labeling by the fluorescent tags and Western blotting we assessed the influence of the sequence alterations on the expression level, activity and cellular localization of the studied DNA repair proteins. The detailed results of the analyses will be presented. 17. INDUCTION OF UNIQUE STRUCTURAL CHANGES IN GUANINE-RICH DNA REGIONS BY THE TRIAZOLOACRIDONE C-1305 RESULTS IN ISOFORM-SPECIFIC STABILIZATION OF TOPOISOMERASE II-DNA CLEAVABLE COMPLEXES Krzysztof Lemke1,3, Marcin Wojciechowski1, William Laine2, Christian Bailly2, Annette K. Larsen3, Vibe H. Oestergaard4, Anni H. Andersen4 and Andrzej M. Skladanowski1 1Laboratory of Molecular and Cellular Pharmacology, Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland. 2INSERM U-524 and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Lille, France 3Group of Biology and Pharmacogenetics of Human Tumors, CNRS UMR 8113, Ecole Normale Supérieure, Cachan and Institut Gustave-Roussy, Villejuif, France. 4Department of Molecular Biology, University of Aarhus, C. F. Mollers Alle, Building 130, 8000 Aarhus C, Denmark. C-1305 is a triazoloacridone with potent activity in lung and colon cancer models. We have recently reported that C-1305 is a topoisomerase II poison which is able to induce topoisomerase II-mediated cleavable complexes in vitro as well as in living cells [Lemke et al., 2004]. An unusual feature of C-1305 is the induction of low levels of very toxic cleavable complexes in tumor cells. Topoisomerase II is a nuclear enzyme which can catalyze catenation/decatenation reaction as well as knotting/unknotting of DNA. This protein exists as two different isoforms: the alpha isoform which is restricted to proliferating tissues and which frequently is deregulated in human tumors and the beta isoform which constitutively is expressed in all tissues. Unexpectedly, only the alpha isoform of topoisomerase II was found to be covalently associated with DNA following exposure of living cells to C-1305 in marked contrast to amsacrine, a classic topoisomerase II inhibitor, which stimulated cleavable complexes with both isoforms. To explore the molecular mechanisms underlying this phenomenon, we have investigated the sequence specificity of DNA binding for C-1305 and other triazoloacridones in comparison with classic topoisomerase II inhibitors and other DNA interacting compounds. These studies have revealed that C-1305 is able to induce structural distortions in DNA regions containing guanine tracts. Studies with other triazoloacridone derivatives as well as topoisomerase inhibitors did not give similar results. The experiments carried out with isolated enzymes (topoisomerase II α and ß) and DNA have shown strong DNA cleavage site within the guanine-rich region as well as differences between topoII isoforms in terms of enzyme binding to DNA in presence of C-1305. One of the explanations of these findings might be the interaction of the drug with specific regions/sequences of DNA or distortion of DNA structure in the way that only one of the isoforms of DNA topoisomerase II would be able to bind to its substrate. Based on our results, we propose a model of the complex between C-1305 and a short oligonucleotide containing 5'-AGGGT-3' sequence, where drug-DNA complex is stabilized by hydrogen bonds between triazoloacridone and neighbouring bases. The whole structure could also be stabilized by the p-p interactions between triazoloacridone chromophore and guanine as well as interactions of positively charged side chain within the minor groove of DNA. Together, we here show that the topoisomerase II inhibitor triazoloacridone C-1305 binds strongly to DNA at guanine-rich regions resulting in unique conformational alterations. This effect leads to specific DNA cleavage by only the a isoform of topoisomerase II. Our results suggest that C-1305 might specifically influence the expression of genes that are regulated by guanine-rich elements in the promoter regions. Lemke K, Poindessous V, Larsen AK, Skladanowski A. The antitumor triazoloacridone C-1305 is a topoisomerase II poison with unusual properties. Mol Pharmacol. 66 (4): 1-9 (2004) 18. NEW ANTITUMOUR DERIVATIVE OF CIS-PLATINUM: STRUCTURE AND BIOLOGICAL ACTIVITY OF PT(II) COMPLEX WITH METHYL 3,4-DIAMINO-2,3,4,6-TETRADEOXY-a-L-LYXO-HEXOPYRANOSIDE H. Lewandowska1, K.Samochocka2, M. Kruszewski1, L. Fuks1 1University of Warsaw, Department of Chemistry, Zwirki i Wigury 101, 02-089 Warsaw, Poland. 2Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland. Cationic platinum-based antitumor drugs containing the DMSO molecules appear to be of special interest, since as a rule their therapeutic activity is of the same magnitude as that of cis-platinum and they are believed to be only marginally nephrotoxic [1-4]. Herein, we report our structural, spectroscopic and biological studies of a novel cis-platinum-like, DMSO containing complex with expected antitumor properties. This cationic complex consists of one chloride anion, methyl-3,4-diamino-2,3,4,6-tetradeoxy-#61537;-L-lyxopyranoside (C7H16N2O2) and dimethylsulfoxide (DMSO) molecules forming a square-plane. The sugar moiety is a modified carbohydrate part of the commonly used in therapy antineoplastic anthracycline antibiotics, daunorubicin and doxorubicin, where it acts as a minor groove binder contributing close to 40% to free energy of DNA-drug binding [5-7]. It has been obtained by courtesy from prof. Priebe (Texas University, Houston, USA). Biological tests performed using leukemia L1210 cells for the investigated platinum(II) complex or the free lyxo-hexopyranoside, respectively, have shown that toxicity of the title complex is similar to that of cis-platin. No significant toxicity of the aminosugar was found, up to the highest concentration tested. Although the ID50 values for the title complex and the cis-platin (studied as the reference drug) appeared to be similar within the experimental system applied, the ID90 values show that cis-platin is about two times more toxic than the investigated complex. Bibliography 1. T.A. al Allafi, L.J. Rashan, Bull. Chim. Farm., 2001, 140, 205. 2. G. Sava, T. Giraldi, G. Mestroni, G. Zassinovich, Chem. Biol. Interact., 1983, 45, 1. 3. M. Tonew, E. Tonew, W. Gutsche, K. Wohlrabe, A. Stelzner, H.P. Schröder, B. Hein, Zentralbl. Bakteriol. Mikrobiol. Hyg [A], 1984, 257, 108. 4. Y.S. Kim, K.M. Kim, R. Song, M.J. Jun, Y.S. Sohn, J. Inorg. Biochem., 2001, 87, 157. 5. Pasini, Gazz. Chim. Ital., 1987, 117, 763. 6. W. Priebe, Curr. Pharmacol. Des., 1995, 1, 51. 7. K. Samochocka et al., J. Chem. Soc., Dalton Trans., 2003, 11, 2177. 19. PROGNOSTIC VALUE OF LYMPHOCYTES IN CERVICAL CARCINOMA B. Lubecka1, Z. Kołosza2, M.Wideł1, S. Jędruś3, E. Wojciechowska4, A. Czuba4, J. Rzeszowska-Wolny1 1Department of Experimental and Clinical Radiobiology2, Department of Cancer Epidemiology, 3Clinic of Oncological Gynaecology, 4Department of Tumour Pathology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch. Objective: The aim of our study was to test whether DNA damage in peripheral blood lymphocytes may be a factor predicting development of cancer and results of local treatment. Methods: The tested group consisted of 56 patients with cervical cancer in clinical stage IIB- IIIB and the control group of 38 healthy women. Lymphocytes were isolated from fresh blood, taken from patients before treatment (radiotherapy or radiochemotherapy). DNA damage and its repair were measured after irradiation with X rays (2Gy) using alkaline comet test. Lymphocyte subpopulations were detected by monoclonal antibodies and flow cytometry. Results: Lymphocytes of patients with cancer presented a higher level of background DNA damage (p<0.00001), increased radiation induced damage (p<0.00005) and reduced repair capacity (p<0.00001) in comparison with healthy donors. In both groups broad interindividual differences in DNA repair kinetics were observed. Blood of patients and normal controls contained comparable numbers of T, B, CD4 , CD8 and NK lymphocytes. Testing the relationship between the distribution of subpopulations and DNA repair kinetics we found that patients who were characterized by more efficient repair in lymphocytes (lower level of residual DNA damage after 180 min of repair) had a higher number of T, CD4 lymphocytes and a lower number of NK cells than patients with less efficient DNA repair (p=0.021, 0.021, 0.005 respectively) The data obtained using the comet test were compared with clinical results of treatment. A higher efficiency of DNA repair in lymphocytes was characteristic for patients with faster regression of tumour. About 93% individuals whose lymphocytes repaired DNA damage better, exhibited complete or almost complete reduction of tumour mass after termination of radiotherapy. A faster regression of tumour was characteristic for patients with a higher number of T lymphocytes (p=0.013) and a lower number of NK cells (p=0.017). This suggests that lymphocytes which repair DNA damage more efficiently probably survive better genotoxic effects of radiotherapy and participate in tumour elimination. Conclusions: DNA repair capacity of lymphocytes may be an important factor in determining susceptibility to cancer and response to cancer therapy. Differences in response of lymphocytes to genotoxic factors may result from different sensitivity of their subpopulations. 20. EXPRESSION OF PORCINE ENDOGENOUS RETROVIRUSES SUBTYPES (PERV-A, PERV-B AND PERV-C) IN IMMUNOSUPPRESSED PIGS G.Machnik1, D. Sypniewski1, U. Mazurek2, J. Stojko3, I. Bednarek1 1 Department of Biotechnology and Genetic Engineering, Medical University of Silesia 2 Department of Molecular Biology and Medical Genetics, Medical University of Silesia 3 Department of Bioanalysis and Environmental Studies, Medical University of Silesia Possibility of breaking the cross-species barrier by some infectious particles is the main obstacle for developing xenotransplants. Expression of porcine endogenous retroviruses (PERVs) does not pose any threat for their host (pig) in a physiological state. However, immunosuppressed recipient is vulnerable to numerous infectious agents including endogenous viruses or latent viruses that normally do not manifest their presence. This may play an important role in post-transplantational complications. The aim of this study was to assess if immunosuppression influences PERV-A, PERV-B and PERV-C expression in domestic pig (Sus scrofa domestica). The studied material consisted of peripheral blood samples collected after transplantation from pigs treated with cyclosporin A (Sandimmum Neoral; 4mg/kg body weight) and Solu-Medrol (500mg/kg). We used genotype-specific PCR with primer sets described earlier by Bösch et al (2000). The results indicate that immunosuppression with the drug set used in this study (immunosuppressant steride) causes a significantly elevated PERV-A expression (p=0.028) compared with a control group, while it does not affect expression of PERV-B (p=0.203) or PERV-C (p=0.817). These results are in concordance with literature data showing that PERV-A has the strongest capability to express and propagate (its full-length-cDNA is most frequently found in the pig genome). Since it is difficult to eliminate the hazard of PERVs expression in xenotransplantation by conventional methods (e.g. protease inhibitors), PERVs silencing by means of antisense oligonucleotide strategy should be developed. The study was supported by the grant from the State Committee for Scientific Research (No. KBN 048/PO5/2001/10). 21. INDUCTION OF APOPTOSIS BY 1-NITROACRIDINE DERIVATIVE - C-1748 (4-METHYL-1-NITROACRIDINE) IN HUMAN COLON CANCER HCT-8 Anna Moś-Rompa, Jerzy Konopa Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Poland The derivatives of 4-methyl-1-nitroacridine are a group of antitumor compounds developed in our Department. The most active derivative of 4-methyl-1-nitroacridines, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine denoted C-1748, displays very high antitumor activity against a number of human prostate cancer xenografts in nude mice: LnCaP, JCA, PC3 and TSU, as well as towards human colon cancer HCT-8. This compound shows a decreased general toxicity compared to other derivatives of 1-nitroacridine and exhibits high cytotoxic activity in vitro towards cells derived from solid tumors. The 4-methyl-1-nitroacridine C-1748 has been selected for the I phase clinical trials. We studied in HCT-8 human colon cancer cells the mechanism of apoptosis induced by C-1748 in biologically relevant concentration, corresponding to EC90 value. The fluorescence microscopy studies of HCT-8 cells incubated with C-1748 showed morphological changes such as chromatin condensation and formation of apoptotic bodies, after 12 hr of treatment. The induction of internucleosomal DNA fragmentation, characteristic for apoptosis, was observed in HCT-8 cells after 12 hr of exposure to C-1748 by agarose gel electrophoresis. The ability of 4-methyl-1-nitroacridine to induce apoptosis in HCT-8 cells was also demonstrated by flow cytometry methods. The disruption of mitochondrial membrane potential (∆Ψm) appeared after 12 hr of incubation HCT-8 cells with C-1748. The loss of phospholipid asymmetry of the plasma membrane in HCT-8 cells was evident starting from 24 hr of treatment. Percentage of HCT-8 cells with active caspase-3 amounted to 6% after 12 hr of incubation of cells with the drug and after 72 hr it reached about 15%. The TUNEL assay confirmed the occurrence of DNA fragmentation in HCT-8 cells. However, this DNA fragmentation was not specific to any phase of the cell cycle. The 4-methyl-1-nitroacridine studied induced apoptosis in HCT-8 cells in a time dependent manner. Nuclear DNA fragmentation and morphological changes induced by C-1748 in HCT-8 cells occurred in the same time as the decrease of mitochondrial membrane potential and appearance of activated caspase-3. 22. MITOGENIC SIGNALLING BY BRADYKININ RECEPTORS IN CERVICAL CANCER Joanna Orchel1, Urszula Mazurek1, Bogdan Michalski2, Anna Belowska2, Tomasz Zieliński2 1Department of Molecular Biology and Medical Genetics, Medical University of Silesia. Narcyzów street 1, 41-200 Sosnowiec 2Department and Clinic of Obstetrics and Gynecology , Medical University of Silesia. Tychy In a large survey of cancers, receptors for bradykinin were most commonly found on tumor cells and on established cancer lines. Bradykinin plays an important role in enhanced vascular permeability in tumor tissue, improves transportation of nutrients to tumor cells, facilitates metastasis and sustains tumor growth. The two characterized bradykinin receptors are designated B1 and B2. The B1 receptor shows greater preference for the bradykinin metabolite [des-Arg9]bradykinin than for bradykinin itself. It has been suggested that mitogenic signalling may display a high degree of cellular specificity. The precise role of each bradykinin receptor in controlling of both normal and cancer cell division needs to be clarified. Moreover, the importance of various intracellular pathways in transduction of signals generated by bradykinin receptors has not been properly elucidated. The purpose of this study was to characterize the cellular mechanisms underlying mitogenic activity exerted by bradykinin in cervical cancer. The analyzed caterial was biopsy samples of cervical cancer. The genes related to mitogenic pathways were selected. The relationship between studied genes was established and illustrated as a gene map. The map will be used in analyses of transcripts of the studied genes based on oligonucleotide microarray HU 133 A. 23. INCREASE OF ER-BETA5 mRNA COPY NUMBER CORRELATES WITH HIGH PROLIFERATIVE ACTIVITY IN HUMAN COLON CANCER Monika Paul-Samojedny1, Danuta Kokocińska2, Zbigniew Lorentz2, Urszula Mazurek1, Tadeusz Wilczok1 1Department of Molecular Biology and Medical Genetic, 2Department of Surgery and Transplantology, Medical University of Silesia, Katowice, Poland Introduction: Research conducted on colon cancer cell lines show that growth of tumour cells is influenced by the action of estrogen receptors. The working efficiency of "wild-type" estrogen receptors depends not only on the transcriptional activity of estrogen receptors (ERs)-encoding genes but also on the part of posttranscriptional ERs mRNA modification in total pool of ERs RNA. The tumour ability for metastasis depends on proliferation potential. Therefore, proliferative activity of tumour centre cells of the copy number of posttranscriptional mRNA ER-beta variants was analyzed in accordance with the standards of the American Joint Committee on Cancer (AJCC). Material and Method: Colon cancer tissues were obtained from 41 patients after surgery. The patients were divided into groups taking into account the classification of colon adenocarcinoma according to the AJCC. The RNA from 164 segments of colon tissues was extracted using the modified phenol-chloroform method. The tissue sample of each patient consists of the margin, centre of tumour as well as normal tissue. QRT-PCR reaction was performed for all RNA extracts. RT-QPCR was also performed using the Quanti Tect SYBR Green Kit and sequence detector ABI PRISM7000. Results: Positive correlation among mRNA H3j copy number and mRNA ER-beta5 copy number showed stages I and IV of colon adenocarcinoma clinical classification (the rang correlation R Spearman test). Conclusion: Existence of positive correlation among transcriptional activity of H3 gene and ER-beta5 mRNA copy number indicated the unquestionable role of this variant in the control of the proliferative potential in colon cancer tissue. 24. RADIATION THERAPY GEL DOSIMETRY Loukas Petrokokkinos1, Loukas Sakelliou1, Tadeusz Biegański2, Marek Kozicki3, Angelos Angelopoulos1 and Janusz M. Rosiak4 1Nuclear and Particle Physics Section, Physics Department, University of Athens, Panepistimioupolis, Ilisia, 157 71 Athens, Greece 2Department of Radiology, Polish Mother's Memorial Research Institute, Rzgowska 281/289, 93-388, Lodz, Poland 3Department of Textile Finishing, Faculty of Engineering and Marketing of Textiles, Technical University of Lodz, Zeromskiego 116, 90-543 Lodz, Poland 4Institute of Applied Radiation Chemistry, Technical University of Lodz, Wroblewskiego 15, 93-590 Lodz, Poland The main aim of radiation therapy is to deliver a high radiation dose to the tumour while on the same time keeping the dose at surrounding healthy tissue minimal. Recent advanced techniques of conformal radiotherapy, as Stereotactic Radiosurgery, Intestity Modulated Radiation Therapy and Brachytherapy, manage to deliver dose using high dose-gradient radiation fields so that the dose distribution closely fits the shape of the target. Due to these complex shapes and high dose gradients used, new precise three-dimensional dose verification methods are necessary. The only such unconventional method nowadays, capable of measuring three-dimensional dose distributions directly, is the polymer gel dosimetry. The polymer gel dosimetry was established to overcome the limitations of up-to-date techniques and to obtain a full three-dimensional dose distribution from a single measurement with high spatial resolution of dose. For this method polymer gel dosimeters have been developed that consist of a gelatin matrix in which monomers are embedded. Since the major ingredient of these systems is water, their exposition to radiation causes water radiolysis products formation. The radical products initiate polymerisation of monomers, the degree of which directly relates to the absorbed dose. The process results in the change of chemical and physical properties of the irradiated part of the gel. The appropriate imaging techniques like optical, ultrasound, and in particular three-dimensional nuclear magnetic resonance gives a precise three-dimensional image of the dose distribution and allows the reconstruction of dose distribution in any preferred plane of the gel. Nowadays in the clinical environment the new irradiation techniques urge for such new methods of dosimetry capable of precise evaluation of the calculated dose distributions. Therefore, gel dosimetry paired with MRI scanning appears as a promising tool. In this study we provide basic characteristic of selected gel compositions and the results of applying the method in conformal radiotherapy techniques. Acknowledgements: Loukas Petrokokkinos gratefully acknowledges the financial support from the European Commission, Marie Curie fellowship no HPMT-GH-01-00228-07. This work was partly supported by the Greece-Poland bilateral Research Agreement. 25. THE 1,4-DIHYDROPYRIDINE DERIVATIVE PROMOTES DNA REPAIR THROUGH STIMULATION OF POLY(ADP-RIBOSYLATION) Nadzeya I. Ryabokon1,2, Rose I. Goncharova1, Gunars J. Duburs3, Joanna Rzeszowska-Wolny2 1Institute of Genetics and Cytology, NAS of Belarus, (rgoncharova@igc.bas-net.by), 2M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch ( jwolny@io.gliwice.pl),3Latvian Institute of Organic Synthesis, ( gduburs@osi.lv) Among 1,4-dihydropyridine derivatives (1,4-DHPs), the analogs of nicotinamide and remote analogs of NAD and NADP attract a special attention because of a crucial role of the mentioned coenzymes in cells. It seems probable that such type analogs can influence NAD and NADP metabolism and, in this way, the excision repair through poly(ADP-ribosylation) [Kuzhir, 1999] in which NAD is used as a substrate. In our studies, the data of alkaline single cell gel electrophoresis (Comet Assay) had shown that one of such 1,4-DHPs (AV-153) reduced significantly (up to 70%) the endogenous, radiation- (X- and gamma-rays) and chemically-induced (oxidized and alkylated) DNA damage in lymphocytes of healthy donors and lymphoblastoid cell lines HL-60 and Raji. This effect of AV-153 was observed in a wide range of concentrations, from 10-9 to 10-6 M, it had reverse dose-effect relationship and it appeared from first minutes of repair. The fact that the most effective concentrations of AV-153 (10-9 and 10-8 M) correspond to the intracellular concentrations of NAD , ATP and nicotinamide that take part in poly(ADP-ribosylation), supports the hypothesis that 1,4-DHPs could modulate the synthesis of poly(ADP-ribose) (PAR). The reverse relationship observed between the AV-153 concentration and DNA damage reduction suggests that AV-153 may stimulate or inhibit poly(ADP-ribosylation) depending on its concentration. We compared the level of PAR induced by H2O2 treatment in presence or absence of AV-153. The level of PAR was measured by immunocytochemical test with anti-PAR polyclonal antibody and computer quantification of PAR as pixel intensity per cell square unit. We found that AV-153 at concentration of 10-9 M stimulated synthesis of PAR up to 120% during the first minute of repair process after H2O2 treatment, the concentrations of 10-8-10-6 M were less effective and 10-5 M had slight inhibitory effect. The level of PAR in presence of AV-153 inversely correlated with AV-153 concentration (R2 = 0.99, P < 0.05). The efficiency of DNA repair in the presence of AV-153 showed positive correlation with the inducible level of PAR at the first minute of repair process (r > 0.9, P < 0.05). The AV-153 stimulated increase of PAR level correlated with increase of DNA repair rate. The data obtained demonstrate one of the mechanisms of 1,4-DHPs action on DNA repair through promotion of PAR synthesis. This is the first evidence of poly(ADP-ribose) polymerase-1 (PARP-1) stimulation by compound in non-genotoxic concentrations. It opens the further possibilities for modulation of PARP-1 activity and study of the mechanisms of PARP action. This work has been carried out at the Department of Experimental and Clinical Radiobiology, M. Skłodowska-Curie Memorial Cancer Center and Institute of Oncology (Gliwice Branch) and partially supported by the fellowship grants from the Association for the Support of Cancer Research, the UNESCO (Polish Committee) and the National Cancer Institute (Bethesda, USA). 26. DNA DAMAGE INDUCED BY 1-NITROACRIDINE DERIVATIVES IS REPAIRED BY HOMOLOGOUS RECOMBINATION Michał Sabisz, Magdalena Hyży, Krzysztof Lemke and Andrzej Składanowski Laboratory of Molecular and Cellular Pharmacology, Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Gdansk, Poland 1-nitroacridines are potent antitumor compounds with high activity toward ovary, breast and prostate cancer. Previous studies have shown that 1-nitroacridines are activated by cellular enzymes and bind covalently to DNA, and that covalent binding and crosslinking of DNA correlate well with the cytotoxic and antitumor activities of biologically active 1-nitroacridines. Our studies have shown that, covalent modification of DNA by Ledakrin but not C-857 leads to topoisomerase I-associated DNA cleavage in vitro. We have also demonstrated in LNCaP human prostate carcinoma cells incubated with 1-nitroacridines that the level of DNA-protein complexes greatly increased for C-857 and Ledakrin but to much lesser extent for C-1748 with increasing concentrations of studied compounds. The kinetics of appearance and removal of these complexes differed from those induced by camptothecin and m-AMSA, classical inhibitorsof DNA topoisomerases. Studies using Immuno-Complex of Enzyme (ICE) assay where one can determine the existence of the covalent DNA-topoisomerase complexes have shownthat topoisomerase I and topoisomerase II are differently involved in DNA-protein complexes induced by 1-nitroacridines. All studied 1-nitroacridines induced high levels of double-strand breaks (DSB) in LNCaP cells. Interestingly, simultaneous incubation of LNCaP cellswith aphidicolin, a well known inhibitor of DNA replication, and 1-nitroacridines led to reduced levels of DSBs. This suggests that DSBs are generated by collisions of the replication fork with DNA-topoisomerase I complex stabilized by DNA adducts produced by 1-nitroacridines. We have also observed high number of sister chromatid exchanges (SCE) in CHO cells treated with C-1748 and to much lesser extent with Ledakrin and C-857 or DNA topoisomerase II inhibitor,m-AMSA. It is known that generation of SCE in drug-treated cells is associated with the ongoing DNA replication and/or inhibition of DNA topoisomerases. DSBs are very toxic cellular lesions and, if left unrepaired, they lead to impaired cell function or direct cell killing. One of the DNA repair mechanisms which could repair DBSs which originate from replication fork-stalling or during attempted replication over a single strand break is homologous recombination (HR). To clarify the potential role of HR in DNA repair of DNA lesions produced by 1-nitroacridines, we exposed CHO cells which have deficient BRCA2 gene and have only partially functional HR pathway to studied 1-nitroacridines. We observed that cells deficient in HR DNA repair are about 5-10-fold more sensitive to 1-nitroacridines compared to parental cells which are HR-proficient. Interestingly, CHO xrs-6 cells, in which Ku80 gene is mutated and non-homologous end joining pathway is inactive, are equally sensitive to 1-nitroacridines as parental cells. Finally, we compared gene expression profiles by DNA microarray technology in LNCaP/A40 cells which are about 40-fold resistant to C-1748 and parental LNCaP cells. Interestingly, C-1748-resistant cells exhibit about 3 times lower expression levels of BRCA1 and BRCA2 than parental LNCaP cells. Taken together, all available data suggest that DBSs induced by 1-nitroacridines are repaired by homologous recombination.< br> 27. TRANSCRIPTION ACTIVITY ANALYSIS OF GENES REGULATING APOPTOSIS ASSOCIATED WITH TNF SUPERFAMILY RECEPTORS IN CD34- CELLS WITH DIFFERENT ADHESION CAPABILITIES USING OLIGONUCLEOTIDE MICROARRAY TECHNIQUE Justyna Samelska1, Urszula Mazurek1, Joanna Głogowska-Ligus1, Monika Paul-Samojedny1, Rafał Stojko2, Andrzej Witek2 1Department of Molecular Biology and Medical Genetics, Medical University of Silesia, ul. Narcyzów 1, 41-200 Sosnowiec; 2Department and Clinic of Obstetrics and Gynecology, Medical University of Silesia, ul. Medyków 14, 40-752 Katowice Ligota The cells presenting inherent adhesion capability have stable metabolism. Reduction of cell adhesion capability may be associated either with cell cycle stage that precedes the cell division or with cell damage leading to changes in transcription activity profile of proapoptotic and antyapoptotic genes. The aim of the study was to estimate the transcription activity of genes associated with death receptors apoptotic pathways, as well as the analysis of genes expression profiles in cells with different adhesion capability. The material used in this study were cell cultures of human CD34- progenitor cells with different adhesion capabilities, isolated from cord blood. The variant I were CD34-NP cells (with reduced adhesion capability) and variant II were CD34-P cells (with inherent adhesion capability, controle). Absolute and comparative analyses were performed using HG_U133A oligonucleotide DNA microarrays, instrumentation and software tools from Affymetrix. 139 genes transcripts associated with death receptors apoptotic pathways were analyzed usingthe HG_U133A DNA microarray. The transcripts were divided into three groups: GROUP I - proapoptotic genes (111 transcripts), GROUP II - antiapoptotic genes (22 transcripts), GROUP III - genes that, depending on their expression regulation, can promote as well as inhibit apoptosis (6 transcripts). Absolute analysis allowed to determine which among 139 examined transcripts are present, marginally present or absent in CD34-NP and CD34-P cells. Comparative analysis yielded information how transcription activity of analyzed groups of genes changed in CD34-NP cells compared to CD34-P cells. The obtained preliminary results permit to assume that in CD34- cells changes of the adhesion capabilities can be associated with the changes of transcription activity of both proapoptotic and antiapoptotic genes. 28. THE LEVEL OF 8-OXO-2`-DEOXYGUANOSINE IN LEUKOCYTE DNA OF CANCER PATIENTS UNDERGOING CHEMOTHERAPY. Agnieszka Siomek1, Marek Foksiński1, Jerzy Tujakowski2, Daniel Gackowski1, Rafał Różalski1, Karol Białkowski1, Tomasz Dziaman1, Jolanta Guz1, Marek Jurgowiak1 and Ryszard Oliński1 1Department of Clinical Biochemistry, The Ludwik Rydygier Medical University in Bydgoszcz, Karlowicza 24, 85-092 Bydgoszcz, Poland, 2Department of Clinical Oncology, Center of Oncology in Bydgoszcz, Romanowskiej 2, 85-793 Bydgoszcz, Poland The anticancer therapy involves a wide spectrum of drugs. Some of them are attributed to intracellular production of ROS which can be generated by both enzymatic and nonenzymatic mechanisms. ROS may cause damage to biological molecules, including proteins, lipids, and DNA. Production of different kinds of DNA lesions, particularly free radical- modified DNA bases are responsible for mutations. 8-Oxo-2`-deoxyguanosine (8-oxo-dG) is one of the typical biomarkers of oxidative stress. Materials and methods: The study comprised a group of 34 cancer patients undergoing chemotherapeutic treatment with different drugs like Cis-platine, vepesid, bleomycine, adriblastine. Patients were suffering from different cancer types like ovarian cancer, breast cancer and adenocarcinoma. Blood samples were collected before chemotherapy and one day after the treatment. Leukocytes were isolated on Histopaque 1119 solution (Sigma), according to the procedure laid down by the manufacturer. HPLC with electrochemical and UV-absorbance detection was used to determine the level of 8-oxo-dG in leukocytes DNA. Results: The mean levels of 8-oxo-dG in leukocytes DNA before chemotherapy and one day after the treatment were 10.46 plusmn;3.96 /10 6 dG molecules and 11.79plusmn; 5.03 /10 6 dG, respectively. This difference was statistically significant (p<0.02). Conclusion: In the present study we observed that the level of 8-oxo-dG in leukocytes DNA was higher one day after the treatment than before chemotherapy. Some reports have shown that adriamycin derivatives and some other drugs used in chemotherapy can produce ROS, what in turn may be responsible for oxidative stress at the level of the whole organism. In our study the level of 8-oxo-dG in leukocytes DNA remains elevated up to 24-th hour after the treatment. This suggests that in human leukocytes in vivo some of DNA lesions may avoid the repair processes. Some nucleobase modifications lead to mutagenesis. Since 8-oxo-dG posses mutagenic properties it may be responsible for secondary cancers after chemotherapy. 29. INDUCTION OF CELL DEATH BY IMIDAZOACRIDINONE DERIVATIVE C-1311 IN T LYMPHOBLASTOID LEUKEMIA MOLT4 CELLS. Anna Skwarska, Ewa Augustin, Jerzy Konopa Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Poland The imidazoacridinone derivative C-1311 is a highly potent antitumor agent with a broad spectrum of activity against different experimental tumors. C-1311 has been synthesized in Gdansk University of Technology and is currently undergoing phase I clinical trials. This compound has previously been shown to display DNA-binding properties and to inhibit activity of topoisomerase II. One of the early biological effects induced by pharmacologically relevant doses of C-1311 (EC90 concentrations) was arrest in the G2M phase of the cell cycle. C-1311 induced apoptosis of L1210 murine leukemia cells and to much lesser extent in ostegogenic sarcoma cells as well as abortive mitosis followed by cell death of HT29 human colon adenocarcinoma cells. The aim of this work was to investigate the ability of C-1311 to induce apoptosis of T lymphoblastoid leukemia MOLT4 cells. Cell death studies were carried out over continuous exposure times varying from 3 to 72 h at EC90 concentration of the drug. DAPI staining of cytospin preparations was performed to analyze the cellular morphology. Mitochondrial membrane potential was measured by flow cytometry using JC-1 fluorochrome. Caspase-3 activity and phosphatydilserine externalization was evaluated using commercially available assay kit on flow cytometry. DNA fragmentation was analyzed by 1,8% agarose gel electrophoresis. Microscopic examination of MOLT4 cells showed alterations of the nuclear morphology after 24 h but cells with more condensed chromatin and apoptotic body-like structures appeared after 48 and 72 h. Percentage of Annexin-V positive cells increased gradually upon treatment starting from 12 h and after 72 h it reached about 70%. However, by this latter time cells were also stained red (due to PI) which indicated late stages of apoptosis and/or necrosis. Disruption of mitochondrial transmembrane potential was observed in the cells after 30 h of incubation with C-1311. Induction of caspase-3 activity was detectable in 30% of cell population after 39 h of treatment and after 72 h this percentage increased to 80%. Characteristic internucleosomal DNA fragmentation was observed only after 48 and 72 h. These results indicate that C-1311 induced apoptosis of MOLT4 cells in a time-dependent manner and both mitochondria and caspase-3 are involved in this process. 30. ASSESSMENT OF HBV DNA AND TGF-ß1 AND ITS RECEPTORS TßRI, TßRII, TßRIII mRNA LEVELS IN THE QUALIFICATION OF PATIENTS WITH THE CHRONIC HBV INFECTION FOR THE INTERFERON α TREATMENT Barbara Strzałka1, Urszula Mazurek1, Tadeusz Wilczok1, Bogdan Marek2 1Department of Molecular Biology and Medical Genetics, Medical University of Silesia, Sosnowiec, Poland 2Division of Pathophysiology Department of Pathophysiology and Endocrinology, Medical University of Silesia, Zabrze, Poland The frequency of HBV infection as well as the serious consequences for patient's health and life resulting from the insufficiency of liver and the probability of the development of cancer, force to constant searching for new techniques to complete the virological diagnosis. Such procedure permits putting the patients through an appropriate form of treating and individualizing methods of the therapy used. The aim of the study was the estimation of the applicability of the designed QPCR for HBV DNA and QRT-PCR for TGF-ß1 and its receptors: TßRI, TßRII and TßRIII mRNA in the qualification of patients with the chronic HBV infection for the interferon α treatment. The first stage of the research embraced designing, empirical optimization and the assessment of the specificity QPCR for HBV DNA and QRT-PCR for TGF-ß1and receptor's mRNA. The second stage of the study deals with analysis of HBV DNA isolates for the purpose of indication of the HBV polymorphic types the designed oligonucleotydes, specifity and the number of HBV DNA copies in patients with chronic virus hepatitis B treated with interferon α. The last stage deals with the indication of the dynamics of changes in the expression of TGF-ß1 genes and the receptors TßRI, TßRII and TßRIII in peripheral blood in patients put through treating and in healthy people, and then the indication of reciprocal relations between parameters marked in blood as well as the inflammatory activity and the degree of fibrosis in the liver. The number of DNA HBV copies in plasma conditioning the set-back of HBV replicative activity and a good prognosis of the therapy with interferon α in patients put through the treatment, was fixed on the level of 4.52 x 108 of the virus copies/ml of plasma. The transcription activity of the TGF-ß1 gene in peripheral blood in patients suffering from chronic virus hepatitis B correlated with the expression of TßRI, TßRII receptors genes while it was not influencing the expression of the gene of the TßRIII receptor. Indicated by means of the QRT-PCR, the number of mRNA TGF-ß1 copies in 1 µg of the total RNA in peripheral blood in patients suffering from chronic virus hepatitis B was significantly higher comparing to healthy people, and was used as a marker, differentiating patients treated with interferon α with the degree of inflammatory activity in liver < or ≥ 3 points in 4-point Scheuer's scale. 31. PREVALENCE OF PORCINE CYTOMEGALOVIRUS (PCMV) IN PIG HERDS INBRED FOR XENOTRANSPLANTATION D. Sypniewski1, G. Machnik1, U. Mazurek2, B. Gajda3, Z. Smorąg3, I. Bednarek1 1Department of Biotechnology and Genetic Engineering, Medical University of Silesia 2Department of Molecular Biology and Medical Genetics, Medical University of Silesia 3Department of Physiology of Animal Breeding, National Research Institute of Animal Production, Balice/Kraków Xenotransplatation opens new perspectives for medicine, however the problem linked with these procedures is the threat of pathogen transmission. Infections with porcine cytomegalovirus (PCMV), a member of Herpesviridae, are latent in most cases and do not manifest any clinical symptoms. Nevertheless, when activated, PCMV causes severe systemic injury resulting, in many cases, with death. Immunosuppression used in transplantation procedures is a state that enables many latent viruses to express. What is more, porcine CMV is capable of activating recipient's latent viruses of the Herpesviridae family. Thus, it is crucial to exclude PCMV-carriers from herds inbred for xenotransplantation. The purpose of the study was to monitor different herds of domestic pig (Sus scrofa domestica) for PCMV frequency. The analyzed material was samples of peripheral blood collected from pigs from three different herds of the National Research Institute of Animal Production, Balice/Kraków. For detection of PCMV DNA with nested-PCR primer sets described earlier by Fryer et al (2000) were used. We found significant differences in the prevalence of PCMV genome among the herds: herd SZ 35.2% (6 positive individuals per 17 individuals in total), herd BS 84.2% (59/70), and herd NZ 71.4% (25/35). The results indicate that herds with minimal risk of PCMV spread can be established. The study was supported by the grant of the State Committee for Scientific Research (No. KBN 048/PO5/2001/10). 32. BIOLOGICAL ACTIVITY AND SINGLET MOLECULAR OXYGEN PRODUCTION OF SYNTHETIC PORPHYRIN DERIVATIVES; POTENTIAL NEW PHOTODYNAMIC THERAPY AGENTS. Agnieszka Szurko1,2, Gabriela Kramer-Marek2, Aleksander Sochanik3, Mirosław Snietura4, Piotr Kus5, Jan Habdas5, Alicja Ratuszna2, Maria Widel1 1Departament of Experimental and Clinical Radiobiology, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland; 2A. Chekowski Institute of Physics, University of Silesia, Katowice, Poland; 3Departament of Molecular Biology, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland; 4Department of Histopathology, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland; 5Institute of Chemistry, University of Silesia, Katowice, Poland; Background and purpose: Photodynamic therapy (PDT) is a treatment method which uses the combination of photosensitiser and visible light to induce a cytotoxic effect in cancerous tissue. The therapeutic effect is mediated through two types of reactions: the first generating free radicals and other reactive oxygen species, such as superoxide radical anion and lipid peroxidation products and the second occurring solely in the presence of oxygen and generating highly reactive singlet oxygen, O2 (1Dg). These species are cytotoxic and disruptive to cellular structures (DNA, lipid membranes, mitochondria and lysosomes). The high quantum yield of singlet oxygen is a critical feature for effective photosensitisation. Other chemical prerequisites for good photosensitisers are: stability, purity and high absorption coefficient in the phototherapeutic window (600-800 nm). The desired biological features of photosensitisers are: low dark toxicity, selective accumulation and prolonged retention in tumour cells. The porphyrins, as agents with good photosensitising properties are very promising for PDT. Materials and methods: We have undertaken the complex studies involving synthesis, physico-chemical characterisation and evaluation of biological activity of new synthetic aminoacid-, cholesteryl-, pyridyl- and cetyl- porphyrin derivatives. On the basis of physico-chemical properties some of them were selected for biological studies including cellular accumulation, dark cytotoxicity and photodynamic efficiency in different lines of human tumour cells. Cationic liposomes were used as carriers for transfer of porphyrins into the cells. Cellular accumulation of drugs was assessed on the basis of luciferase activity measured in lysates of cell cultures previously transfected with liposomes complexed to plasmid DNA carrying luciferase reporter gene. The confocal microscopy was also used for visualization of drugs in cell structures. Cytotoxicity and photodynamic activity was determined by the tetrazolium colorimetric reduction assay (MTS assay). The modes of cell death after PDT treatment (apoptosis or necrosis) were studied by differential staining with acridine orange/ethidium bromide. Results: Our results indicate that at least two probed synthetic porphyrin derivatives are relatively nontoxic without light treatment and are highly effective with regard to phototoxicity in human malignant melanoma and colon carcinoma cell lines and may be promising candidates for further use in PDT experiments. Selected data will be presented. 33. EXPRESSION OF CELL CYCLE REGULATORY GENES IN CD34- CELLS ISOLATED FROM CORD BLOOD Bogdan Waksmański1, Rafał Stojko3, Joanna Głogowska-Ligus2, Monika Paul-Samojedny2, Andrzej Witek3, Urszula Mazurek2 1Department of Perinatology and Gynecology, 2Department of Molecular Biology and Medical Genetics, 3Department of Gynecology and Obstetrics, Medical University of Silesia, Katowice Introduction: Cell cycle is responsible for most important events in the cell life. Accordingly, from cellular context signaling pathways can recruit components linked with DNA repair, activation of apoptosis, breaking of transcription, activation of arrangements checking cell walking by definite points of cell cycle, cell proliferation and differentiation. Expression profile of cell cycle regulatory genes can determine the ultimate fate of cells. Mechanism regulating self-renewal and cell fate decisions in human cord CD34- cells are poorly understood. Purpose: The aim of the study was to determine (by oligonucleotide microarray assay) the expression profile of cell cycle regulatory genes in cord CD34- cells with different ability for adhesion. Material and Method: Material for the study included cord CD34- cell lines with ent ability for adhesion. The CD34 cells were isolated from cord blood in Ficoll and uroproline gradient (F/U). CD34 cells were divided into CD34- and CD34 populations using Progenitor Cell Isolation Kit and MiniMidiMACS Starting Kit. Gene expression was evaluated using HG_U133A microarrays (Affymetrix). The absolute and comparative analyses was carried out by means of Affymetrix GeneChip Analysis Suite 5.0 software. The results obtained after comparative analysis were sorted to single out genes with changed expression, using the software Affymetrix Data Mining Tool. Results: The HG_U133A microarray contains 607 transcripts for cell cycle regulatory genes. Increase of CCND3, CDKN1A, YWHAQ genes expression and decrease of CDC2, CCNB1 and BUB3 genes expression were observed in CD34- cells with weak ability for adhesion (CD34-NP) in comparison with CD34-cells with normal ability for adhesion (CD34-P). Conclusion: In case of CD34- cells with weak ability for adhesion take place inhibition of crossing G2/M cell cycle check point. 34. LOSS OF HETEROZYGOSITY/ALLELIC IMBALANCE OF THE RGS AND RGS-LIKE CANCER SUSCEPTIBILITY GENES AT THE CHROMOSOME 1Q25.3 REGION IN PRIMARY SPORADIC BREAST CANCER. Emilia Wiecheć1,2 1 Department of Human Genetics, The University of Aarhus, The Bartholin Building, Wilhelm Meyers Allé 240, 8000 Aarhus C, Denmark; e-mail: emilia@humgen.au.dk 2 Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch., ul.Armii Krajowej 15, 44-100 Gliwice, Poland. Background: Loss of heterozygosity / allelic imbalance in tumors plays an important role in the identification of new cancer susceptibility genes with a prognostic significance. Allelic imbalance describes either a deletion or an amplification of an allele. Recent studies have led to the identification of four genes from the regulators of G protein signalling family within a 370 kb region at chromosome1q25.3. This chromosomal region is rearranged in breast tumors and is tightly linked to the familial prostate cancer (HPC1). Aims: The overall aim of this study was to investigate loss of heterozygosity in the RGSL2, RGSL1, RGS16 and RGS8 genes in 226 patients with primary sporadic female breast cancer and to localize the potential chromosomal breakpoints on the strength of the observed rearrangements. Methods: Polymorphic microsatellite loci are the most important marker type used to detect loss of heterozygosity in this study. Each microsatellite locus was scored by PCR amplification with locus-specific, fluorescent-labelled primers. The amplification products were separated by capillary electrophoresis using ABI 310 Genetic Analyzer. Results: Microsatellite markers RGS16 A and RGSL1 ex5 exhibited the highest frequency (65% and 64%, respectively) of loss of heterozygosity among all the markers analysed. The 1q25.3 region showed a high genomic instability in primary sporadic breast cancer. LOH analysis points to the potential chromosomal breakpoint between the markers. Conclusion: The high rate of chromosomal rearrangements at 1q25.3 region suggests the implication of RGS genes in tumor development, particularly breast cancer. Key terms: breast cancer, loss of heterozygosity, allelic imbalance, microsatellite markers, RGS genes, chromosomal breakpoint. 35. THE EFFECT OF MISTLETOE EXTRACT (ISCADOR P) ON THE LYSOSOMAL PHYSIOLOGY Anna Wieczorek, Teodora Król, Małgorzata Łysek-Gładysińska, Klaudia Staszczyk Department of Cell Biology, Institute of Biology, The Świętokrzyska Academy, Kielce, Poland. The lysosomes are organelles responsible for a diverse spectrum of cellular functions. In physiolological conditions the lysosomal system is stable. The activity of pharmacological substances may induce a disturbance of lysosomal physiology, which is manifested by a change of activity of lysosomal enzymes. It results in cellular dysfunction and may eventually lead to a variety of diseases. In the present study we investigated the effect of iscador P on the ultrastructure and activity of some hydrolases of the lysosomal compartment of liver cells. Iscador is the trade name of extracts of Viscum album, which are widely used as complementary cancer therapies in Europa. Preclinical data suggests immunostimulatory and cytotoxic effects of mistletoe extracts, although its mechanism of action is largely unknown. Mistletoe extracts contain a number of components considered biologically active, including lectins and viscotoxins. The extracts are biologically and biochemically standardized.The biologic effects of mistletoe lectin have been studied most extensively. Their mechanism of action is probably 2-fold. On the one hand, mistletoe lectins can stimulate immunological relevant effector cells, on the other hand, mistletoe lectins have shown direct growth inhibitory effects on tumor cells. Depending on the concentration, treatment with mistletoe lectins results in death via apoptosis or necrosis.While the clinical efficacy of iscador in cancer is being investigated, toxicity and potential interactions with standard chemoterapeutic agents are unknown. The experiment was carried out on 5-month-old Swiss male mice. The mice serving as a control (the first group) were injected intraperitoneally with 0.9% NaCl, while the mice from the experimental groups were injected with iscador P (iscador P is extracted from mistletoe plants growing on pine) in doses 0.1mg/kg b.w., 1mg/kg b.w. and 2mg/kg b.w. After due time (24h) the animals were decapitated and segments of liver were immediately taken for biochemical and morphological studies. In the lysosomal fraction of the hepatocytes the activity of the following enzymes was estimated: cathepsin D and L (Cath D and Cath L, EC 3.4.23.5, EC 3.4.22.15), acid phosphatase (AcP, EC 3.1.3.2) and beta-glucuronidase (BGRD, EC 3.2.1.31). The activity of the enzymes were expressed in micro;moles/mg of protein/hour. Data were analyzed statistically using Student's t-test. Administration of iscador to male mice caused a dose-dependent reduction of the acivity of all examined hydrolases and protein breakdown in liver cells compared to control group. Electron micrographs showed reduction of number and sizes of primary and secondary lysosomes and decrease of autophagic vacuole formation. From our experiment we may conclude that iscador P causes lysosomal disorders. 36. METABOLIC ACTIVATION OF ANTITUMOR 9-AMINO-1-NITROACRIDINE DERIVATIVES C-857, C-1748 AS A PRELIMINARY STEP OF THE COVALENT BINDING OF THIS DRUG TO DNA A. Wiśniewska, Z. Mazerska J. Konopa Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdańsk University of Technology, 80-952 Gdańsk The aim of the presented study was to explain the role of reducing transformations of 9-amino-1-nitroacridine derivatives in their biological activity and toxicity and to create a background for prediction of metabolic pathways that would be possible in the future patients. Compounds C-857 and C-1748 belong to the new set of antitumor 9-amino-1-nitroacridine derivatives developed in our laboratory headed by Prof. Konopa. These compounds are structural analogs of the Polish antitumor drug, ledakrin. One of them, C-1748, which caused lower toxicity in animals than ledakrin was selected to phase I of clinical trials. It was shown earlier that metabolic activation of 1-nitroacridines is necessary for activity of these compounds towards tumor cells. Futhermore, the formation of the adducts between DNA and activated form of 9-amino-1-nitroacridine derivatives was demonstrated by 32P-postlabeling analyses in tumor cell as well as with activation systems in vitro. In the presented work the studies on the reductive transformations of these agents in the presence of dithiothreitol and various fractions of rat liver microsomes have been described. The studied processes were carried out under identical conditions, in which the covalent binding of these compounds with DNA was shown earlier. The transformations were observed spectrophotometrically and monitored by HPLC RP with MS spectra analysis. We demonstrated that C-857 was less reactive than ledakrin under the studied conditions. Nevertheless, its metabolic transformations of C-857 have led not only to the reduction of nitro group but also to the activation of the acridine ring for nucleophilic substitution in position 2 and 4 and the activation of 9-amino group for electrophilic attack. Therefore, we postulated that three reactive places in acridine ring are possible to form covalent binding of acridine to two strands of DNA. On the other hand, a new less toxic derivative C-1748 turned out to be less reactive with DTT and with microsomal enzymes than C-857. Thus, we hypothesized that high susceptibility of 9-amino-1-nitroacridines to reducing metabolism would be responsible for high toxicity of these compounds. In our investigation we also considered that metabolic transformations of the studied compounds were postulated to be the preliminary step in the covalent binding of these agents to DNA, which is crucial to their antitumor activity. In this respect, the knowledge on the metabolite pathways of the potent medicine, C-1748 will be also helpful in the design of the optimal dose schedule in the future human therapy. 37. ANALYSIS OF GENE EXPRESSION ASSOCIATED WITH IMMORTALIZATION PERFORMED IN STEM CELLS CHARACTERIZED BY DIFFERENT ABILITY TO ADHESION ISOLETED FROM UMBILICAL BLOOD Agnieszka Witkowska1, Rafał Stojko2, Joanna Głogowska Ligus1, Urszula Mazurek1, Monika Paul-Samojedny1, Andrzej Witek2, Tadeusz Wilczok1 1Departments of Molecular Biology and Medical Genetics; 2Gynecological Endocrinology; Medical University of Silesia, ul. Narcyzów 1, 41-200 Sosnowiec 2 Telomerase, a ribonucleoprotein polymerase which takes part in the maintenance of telomeric DNA length, is one of the most important and probably conditional factor in cells immortalization processes. Deregulation of telomerase activity in somatic cells is thought to be involved in oncogenesis.The aim of our study was to determine a model gene expression profile connected with telomerase - dependent telomere maintenance and immoralization in cells that are not tissue specialized, as a reference to following trials performed in neoplasm tissues. It is assumed that stem cells characterized by different adhesion ability can vary in degrees of readiness to immortalization on the transcriptional stage. Results: Transcriptional activity of selected group of genes associated with telomerase were compared: transcripts composed the telomerase complex (9 genes), potentially activators and inhibitors of hTERT transcription - a catalytic protein component with reverse transcritase activity - (10 genes), hTERT stabilization factors in cell (4 genes), and also transcripts having an effect on telomerase complex activity in general (23 genes) The analysis was performed by oligonucleotide DNA microarray technique using Affimetrix GeneChip U133A. This method enables parallel analysis of selected groups of genes at the same time and estimate a tendency to changes in expression, depending on examined probes. Among 25 marked activators of immortalization 11 factors revealed high and middle-high transcriptional activity, from 15 inhibitors of this process only 2 factors with high transcriptional activity were detected. The changes in transcriptional activity depending on ability to adhesion in different cells was observed in 7 cases. Results have been shown in tables and schemes considering semiquantitative basis of method. Metabolic dependences among particular factors have been shown in diagrams.We paid attention to some interpretation problems when comparing outcomes with quantitative analysis of the same single transcripts performed by polymerase chain reaction method based on fluorescent TaqMan methodology. Because of this ambiguity the results seem to be insufficient for making conclusion regarding a degree of readiness to immortalization in stem cells which differ in the ability to adhere. In spite of this, the study proved valuable in assessing a group of factors that may be useful in pathologic prognosing or diagnosing and in determination of genes important in some particular aspect of metabolism. It also forms a good basis for checking up a gene expression profile by conventional quantitive methods performed in parallel. Our results are a preliminary research concerned with establishing gene expression profile in neoplastic tissues. 38. VEGF ISOFORMS AND ITS RECEPTORS OF TYPE 1 AND 2 EXPRESSION IN PATIENTS WITH NON-HODGKIN LYMPHOMAS Dariusz Woszczyk, Joanna Gola, Urszula Mazurek, Bogusław Michalski, Tadeusz Wilczok Vascular endothelial growth factor (VEGF) is the most important new vessel formation regulator in the process of physiological and neoplastic angiogenesis. The mRNA VEGF level and VEGF protein expression positively correlate with a tendency to tumor progression in human malignancies. Hypoxia is the most potent stimulator of VEGF synthesis. There have been four receptors for VEGF identified ; VEGFR-1, (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt-4) and Neuropilin-1. It is believed that the most powerful angiogenetic stimulation is mediated by the KDR activation, with recognition of a comparatively poor role of Flt-1 in this process. It seems that it could be of considerable importance in the process of angiogenesis inhibition and inactivation of circulating VEGF protein. In the presented study mRNA -VEGF expression and their receptors of the 1 and 2 type have been determined. in the group of 14 subjects with diagnosed malignant lymphomas of high and low malignancy grade The VEGF expression has been assessed for the whole panel of six isoforms, moreover, apart from Flt and KDR receptors, soluble receptor Flt-1 expression was also performed. Significantly, higher levels of KDR receptor in high grade malignancy lymphomas were observed, compared to lymphoproliferative disorders of lower grade malignancy. This can confirm its crucial role in the process of angiogenesis stimulation. Flt-1 expression was comparable in both subgroups of patients. Analyzing expression of six isoforms, a higher activity of VEGF165 and VEGF189 was found in the individuals with high grade malignancy lymphomas. Expression of other isoforms did not show any significant differences when analyzed in both subgroups of patients. Signal pathways engaged in VEGF receptors activation are schematically presented. 39. SECONDARY LIPID PEROXIDATION PRODUCTS IN CULTURED HUMAN MELANOMA CELLS Alicja Zajdel1, Adam Wilczok1, Małgorzata Latocha2 1Departament of Biopharmacy, Medical University of Silesia 2Departament of Cell Biology, Medical University of Silesia The peroxidation of unsaturated fatty acids gives rise to short-chain carbonyl compounds, which contribute to peroxidative cell damage by inhibiting DNA, RNA, and protein synthesis, inhibiting respiration and depleting glutathione. Aldehydic lipid peroxidation products, especially the 4-hydroxylkenals (HAK), due to their high reactivity, display marked biological effects in a concentration-dependent manner. These aldehydes can initiate the processes of spontaneous mutagenesis and carcinogenesis. The two most toxic 4-hydroxylkenals are 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). These aldehydic compounds can affect and modulate, at very low non toxic concentration, several cell functions, including signal transduction, gene expression, cell proliferation and differentiation, cellular growth inhibition, apoptosis induction, and more generally, response of the target cell. Data pertaining to the amounts of different aldehydes in cancer cells after photodynamic therapy, are limited. This prompted us to determine aldehyde levels in laser irradiated and non-irradiated human melanoma cells (SK-MEL) in the presence or absence of delta-aminolevulinic acid (ALA). Caco-2 cells, which do not contain melanin, were used for comparison. Cells were cultured under standardized conditions using Petri dishes and Cellstar 93 incubator (370 C, 5% CO2). ALA(100 µg/ml) was added to the cultivation medium 1 hour before irradiation. Aldehydic products of lipid peroxidation, after extraction with acetonitrile, were detected as their 2,4-dinitrophenylhydrazone (DNP) derivates using HPLC-MS-MS. A gradient mixture of water and methanol (acidified with acetic acid) and C-18 column were used for HPLC separations. Diode-array (250 - 500 nm) detector was used to collect spectra of the separated compounds. Negative ionization was applied in TIC, SIM, product of, and MRM modes during MS analysis. The following secondary lipid peroxidation products were present in the analyzed SK-MEL cultures: hydroxybutanal, hydroxyheptanal, hydoxyoctanal, hydroxyhexenal, hydroxynonenal, hydroxynonanal, pentanal, hexenal, and hexanal. The addition of ALA and laser irradiation, and even the simultaneous application of both factors, did not influence concentrations of the analyzed aldehydes. In SK-MEL cells, while in Caco-2 a marked increase (3-7 fold) of hydroxyhexenal, hydroxynonenal, hydroxybutanal, hydoxyoctanal, and hydroxynonanal was observed. Is it a result of protective action of melanin? 40. GENE EXPRESSION PROFILE IN B16(F10) MURINE MELANOMA IN VITRO UNDER HYPOHIC CONDITIONS. Aleksander Sochanik, Joanna Jazowiecka-Rakus, Magdalena Olbryt Department of Molecular Biology, Center of Oncology, Maria Skłodowska-Curie Memorial Institute, Gliwice Branch Weak oxygenation of tumors results from disturbed blood flow or limited oxygen diffusion. Hypoxia is prevalent in tumors exceeding 1mm3. It bears impact upon proliferation, genetic stability, angiogenesis and apoptosis. Poor oxygenation is a serious obstacle to radio- and chemotherapy. In this study we compared expression profile of genes in cells of B16(F10) murine melanoma cultured in a standard incubator with cobalt chloride (hypoxia mimicry) added to medium or in either a static Billups-Rothenberg chamber (nominal 1% oxygen) or hypoxic incubator with flow of a gas mixture containing 1% oxygen. Total RNA was isolated from 24-hour cultures. cDNA and cRNA was synthesized and the expression profiles were assessed using murine microarrays from Affymetrix. Numerical fluorescence data processed by Affymetrix software were analyzed using singular value decomposition (SVD) method. Clusterization of genes chosen by SVD method shows distinct differences between experimental samples and controls. Cobalt chloride (chemical mimicry of hypoxia) induces also a set of genes distinct from those obtained using two methods of lowering oxygen concentration in cell culture. The conditions of the experiment abruptly change the expression profile of genes when oxygen cocncentration is lowered. Among differentiating genes dominate those with increased expression (cobalt chloride - 80/109, Billups chamber - 86/97, hypoxic incubator - 60/85). Using the three approaches described a very strongly increased expression was obtained for two genes: interferon stimulated protein and galectin-3. When only lowering of oxygen concentration is considered there is also a sharp increase in the expression of vinculin, connective tissue growth factor, VEGF and Cyr61. EXPRESSION OF TNFα, TGFß1 GENES AND THEIR RECEPTORS IN LOW AND HIGH GRADE NON-HODGKIN LYMPHOMAS Joanna Gola, Dariusz Woszczyk1, Magdalena Jurzak, Urszula Mazurek Department of Molecular Biology and Medical Genetics, Faculty of Pharmacy 1Department and Clinic of Internal Diseases and Oncological Chemotherapy Medical University of Silesia in Katowice, Poland Tumor necrosis factor-α (TNFα) is pleiotropic cytokine mainly produced by stimulated monocytes, macrofages and T lymphocytes subsets. It plays a role in inflammation, infection, endotoxin-induced septic shock, cahexia and cancer and modulates cell growth, differentiation and metabolism in many cell types. TNFα activities are mediated via two receptors: tumor necrosis factor receptor type I (TNFR1 - 55kDa) and tumor necrosis factor receptor type II (TNFR2 - 75kDa). Both receptors are expressed by majority of cell types. TNFα signaling pathways include activation of transcription factors such as NFĸB and AP-1. Those factors have been implicated in many regulatory effects on gene expression. TGFß1 is a multifunctional cytokine playing a role in different physiological reactions. TGFß1 can inhibit or stimulate the growth of the same type of cells. Transforming growth factor beta may also induce apoptosis in various cell types. There are three types of TGFß1 receptors: TßRI, TßRII and TßRIII. The initiation of signal transduction into the inside of a cell is only possible through TßRI stimulation. TGFß1 is first bound with TßRIII and only then does this complex join with TßRII or directly with TßRI. The attachment of a TGFß molecule to TßRII causes the activation of TßRI and phosphorylation of the intracellular Smad2 or Smad3 proteins. The new complex including Smad4 is carried to the cell nucleus, thus carrying the signal from the cell surface and influencing the transcriptive activity of TGFß1-dependent genes. TNFα is known to antagonize most of TGFß regulatory effects on gene expression. It has been shown that NFĸB activation inhibits or induces Smad7 expression depending on cell types. Smad7 interferes with Smad phosphorylation by TßRI and subsequent translocation into cell nucleus. Others have demonstrated that in human dermal fibroblasts TNFα prevents TGFß-induced Smad-specific gene transactivation without inducing detectable levels of Smad7. The molecular mechanism of such interaction in lymphomas is still unclear. We examined samples from patient with high and low-grade non-Hodgkin lymphomas. The quantitative analysis of mRNA TNFα, TGFß and their receptors was carried out with the use of Sequence Detector ABI PRISM 7000 (Applied Biosystems). It was shown that the mRNA level of TNFα, TNFR1 and TNFR2 as well as TGFß and TßRI, TßRII, TßRIII reflects divided the low and high-grade non-Hodgkin lymphomas.
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