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		1. Salivary epidermal growth factor levels decrease in oral cavity cancer patients 1Balicki R., 1Grabowska S.Z., 2Citko A., 2Rogowski F, 1Dziemiańczyk D. 1 Department of Maxillofacial Surgery, Medical University of Bialystok 2 Department of Nuclear Medicine, Medical University of Bialystok Epidermal growth factor (EGF) is a cytokine that contributes to the maintenance of mucosal integrity: an intact oral epithelial barrier and its proper function. It is responsible for epithelial regeneration by means of growth induction and cellular revival. EGF has additional potent and diverse effects on cell migration and matrix synthesis. One of the major sites of EGF synthesis in humans are parotid salivary glands. There is now good evidence that development and progression of epithelial malignancy is associated with the abrogation of normal growth control mechanisms but salivary EGF effect on tumorigenesis and oral cancer biology is unknown. The goal of our study was to investigate the changes of EGF concentration in the whole resting (r) and stimulated (st) saliva in healthy volunteers (Kr, Kst) in comparison with oral cancer patients before (Cr, Cst) and after (C'r, C'st) chirurgical tumour excision. Whole resting and stimulated saliva of 10 patients with oral squamous cell carcinoma was investigated before and two weeks after chirurgical treatment. Volume of saliva was volumetrically determined. Concentration of EGF in saliva was evaluated by sandwich ELISA technique (Quantikinereg; Human EGF Immunoassay kit, RD Systems). Salivary EGF concentration in patients before treatment (Cr=1,7939plusmn;0,57ng/ml, Cst=1,3047plusmn;0,92ng/ml) was lower (p>0,05) than after surgery (C'r=2,0128plusmn;1,66ng/ml, C'st=2,1947plusmn;1,52ng/ml) both in the whole resting and stimulated saliva. As against the control group (Kr=3,3182plusmn;1,83ng/ml, Kst=1,7524plusmn;1,13ng/ml) the values of EGF levels were considerably lower in the whole resting saliva of the patients before surgery (p<0,05). As for the whole stimulated saliva in patients before tumour excision the value of this parameter was inconsiderably lower than in the control (p>0,05). After surgery salivary EGF concentration in the whole resting saliva was still decreased in comparison with the control group (p>0,05). EGF level in the whole stimulated saliva in postreatment cancer patients was higher than in the volunteers group (p>0,05). Our data (especially decreased levels of EGF concentration in saliva before and its contrary tendency after chirurgical treatment) may suggest important role of EGF in oral cancer biology. This problem needs to be more profoundly examined including more numerous groups of patients and longer postreatment time. 2. Treatment planning in prostate cancer based on transrectal USG examination J. Bystrzycka, K.Ślosarek Radiotherapy and Brachytherapy Treatment Planning Department, Centre of Oncology - Institute MSC Gliwice Aim: The aim of the study is to present treatment-planning procedures in brachytherapy of prostate cancer based on USG examination with use to SWIFT system (Nucletron). Introduction: Brachytherapy Department in Institute of Oncology Gliwice is equipped in computed treatment planning system (TPS) SWIFT. The system component is transrectal USG machine, stepper - use to fixed probe and template, and treatment planning system. These elements are integrated and allow to "real time" brachytherapy treatment. Material and method: Before start treatment planning procedures and treatment, patient has to be anaesthetized (spinal anaesthesia). First step is to perform USG examination, to place probe on the depth of prostate base and to send next slices (1mm thickness) until prostate apex. Slices are sending to TPS automatically by network (stepper encoder - connect USG machine and TPS). The next is to prepare pre - plan. First, the prostate base and apex have to be identified. System localized the reference scan automatically (scan between base and apex). Than the doctor contours the prostate - PTV (Planning Treatment Volume) manually or automatically based on interpolation between scans and the organ at risk - urethra. Successively, prescribed dose and catheter placement in prostate is established. The next step is the source "dwell times" optimisation and normalization. Prostate volume covered by reference isodose (100%) and dose in urethra is taken from dose - volume histogram. The doctor inserts needles according to catheter placement. The next is to perform USG examination with implants and to start live planning procedure. Pre - planning slices (contours and catheter location) is to lie on live plan scans. TPS enable to modify needles location (virtual -->live catheter). The next is the source "dwell times" optimisation (according to new, real implant placement) and dose - volume histogram verification. After acceptance, treatment plan is send to treatment control station by network and patient treatment is started. Conclusion: Dose distribution based on USG examination enable to verified implant placement in PTV (prostate) relation to organ at risk (urethra). Integrated treatment planning system enable to modifies catheter location in prostate, directly (pre - planning --> live planning). 3. Cisplatin with aminoflavone wings as an improved apoptosis inducer of cancer cells Ewa Ciesielska1, Kazimierz Studzian1, Elżbieta Zyner2, Justyn Ochocki2, Leszek Szmigiero1 1 Medical University of Łódź, Department of Medicinal Chemistry, Mazowiecka 6/8, 92-215 Łódź 2 Medical University of Łódź, Department of Inorganic Chemistry, Muszyńskiego 1, 90-151 Łódź, Poland In this work we have compared biological properties of cis-diamminedichloroplatinum (cisplatin) and its new analogue cis-[Pt(AF)2Cl2] (AF stands for 3-aminoflavone) containing two bulky aminoflavone wings, as non leaving ligands, instead of ammine groups. Both compounds were tested for their antiproliferative activity against cultured L1210 cells, their DNA damaging properties and ability to induce apoptosis. The new analogue was found to be less more cytotoxic than cisplatin. In terms of IC50 (the drug concentration inhibiting 50% of L1210 cell growth after 72 hours exposure) cisplatin was about 4 times more active. Both complexes reacted with purified calf thymus DNA in a cell-free system producing of DNA interstrand crosslinks. Kinetics of crosslinks formation was very similar for both compounds but maximal level of crosslinks was higher for cisplatin (crosslinked DNA fractions were 0.59 and 0.40 for cisplatin and cis-[Pt(AF)2Cl2], respectively). In cells, however, as assayed by DNA alkaline elution, crosslinks formation was very similar for both compounds. At higher concentrations of drugs, strong degradation of DNA was observed in L1210 cells treated with cis-[Pt(AF)2Cl2] but not in the cells incubated with cis-DDP. This DNA degradation seems to reflect very efficient apoptosis induction by cis-[Pt(AF)2Cl2] as electrophoretic patterns of DNA from cells incubated with this drug showed a ladder typical for apoptotic cells. An additional confirmation of this result was obtained by flow cytometric analysis of drug treated cells. Our results suggest that cis-[Pt(AF)2Cl2] is much more effective apoptosis inducer than its parent compound cisplatin. 4. Identification of nuclear proteins binding to human topoisomerase I Alicja Czubaty Department of Molecular Biology, Institute of Biochemistry, Warsaw University, Poland Human topoisomerase I is a multifunctional protein possessing two distinct enzymatic activities. Its relaxing activity contributes to basal processes like transcription, replication and recombination. Additionally, the enzyme participates in splicing by acting as SR proteins kinase. The purpose of the work was to identify protein partners of human topoisomerase I. To get more detailed information about the sites of binding, the enzyme was divided into four functional domains. Each of these domains was fused with GST and used as a bait in a pull down assay with nuclear protein extract from HeLa cells. Bound proteins were identified by mass spectrometry. The catalytic domain as well as the linker domain did not bind proteins in this assay. On the contrary, the N terminal and core domains bound distinct subsets of proteins. The N terminal domain is expected to be the major site of protein-protein interactions. Within proteins revealed bound to N terminal domain two groups were distinguishable. One group are SR proteins, either playing role in splicing or not. Remaining proteins can be clustered on a base of possessing acidic stretches similar to nucleolin, known protein partner for topoisomerase I. The core domain is the largest part of the enzyme containing almost all catalytic aminoacids. The core domain seemed to bind proteins from two complexes. One is pre-ribosomal complex, the other one pre-spliceosome complex. This result suggests the potential role of topoisomerase I in both pre-RNA splicing or assembly and its contribution to spliceosome complex. 5. Towards identification of features of cis-regulatory regions associated with common patterns of gene expression during hippocampal development Michał Dąbrowski1, Stein Aerts2, Yves Moreau2, Bożena Kamińska1 1Laboratory of Transcription Regulation, Nencki Institute, Warsaw, Poland 2SISTA, ESAT, Katholieke Universiteit Leuven, Leuven, Belgium We employ a combination of computational and experimental approaches to identify features of cis-regulatory regions that underlie common patterns of gene expression during hippocampal development. We analysed the published data from Affymetrix gene profiling of the hippocampal development in mouse (Mody et al., 2001, PNAS 98: 8862-7). Our analysis revealed that most of variations in expression can be explained by the difference in the amplitude of expression, and pattern of up- and down-regulation in the course of development. We sought to identify features of the cis-regulatory regions specifically associated with these two characteristics of expression profiles. Current computational work constitutes of identification of putative regulatory regions and annotating them with motives for consensus binding sites for transcription factors. Then we build higher order features from these motives using purpose-built software. Such features are scored for their statistical association with the amplitude or up/down regulation. The role of the most promising features identified in silico will be tested by perturbation experiments in the hippocampal neuronal culture. This culture system is a good model of the gene regulation in the developing hippocampus (Dabrowski et al., 2003, J. Neurochem. 85: 1279-88). Our strategy will be to interfere with the function of a binding site, or of the transcription factor, and to monitor the effects of such perturbations on the expression of the putative target genes. 6. Expression of recombinant staphylokinase gene in transgenic potato (Solanum tuberosum L.) plants Aneta Gerszberg1, Aneta Wiktorek-Smagur1, Katarzyna Hnatuszko1, Piotr Łuchniak1, Janusz Szemraj2, Andrzej K. Kononowicz1* 1 Department of Cytogenetics and Plant Molecular Biology, University of Lodz, 2 Department of Medical Biochemistry, Medical University of Lodz. * Corresponding author: akononow@biol.uni.lodz.pl Over the past decade, due to progress made in genetic transformation methodology, transgenic plants have become a convenient eukaryotic system for the expression of synthetic genes coding for recombinant proteins of different origin. Moreover, molecular farming that utilize transgenic plants, have become an inexpensive alternative for commercial production of valuable recombinant proteins. This technology has many potential advantages as compared to industrial facilities using fermentation or bioreactor systems for generating recombinant proteins on a large scale. Among the recombinant proteins produced by transgenic plants, those of therapeutic value attract attention of scientists as well as pharmaceutical and biotechnological companies. Here we report on the construction of fusion gene that consists a recombinant staphylokinase (plazminogen activator protein, a promising thrombolytic agent of bacterial origin) and sequences coding for mGFP and ß-glucuronidase markers and its expression in potato (Solanum tuberosum cv. Desiregamma) transgenic plants. The Western blot analysis and biochemical assay were used to confirm the presence of staphylokinase domain in total protein extract from transgenic plants and its amidolytic activity, respectively. This research was supported by the State Committee for Scientific Research (KBN research grant # 6 P04B 003) and, in part, by the University of Lodz (grant 505/0404). 7. DNA double strand break rejoining in M059J and K human glioma cells X-irradiated and treated with signaling pathways inhibitors Iwona Grądzka, Iwona Buraczewska, Irena Szumiel Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw. The aim of this work is verification of the hypothesis that radiosensitization of cancer cells by inhibitors of signalling pathways initiated at growth factor (mitogen) receptors is due to their effect on DNA repair systems. We used two related human glioma cell lines: M059K and M059J, the latter lacking in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) expression. This results in slower DNA double strand break (DSB) rejoining and a substantial increase in M059J cells sensitivity to ionising radiation and bleomycin as compared to M059K cells. Conversely, M059J cells are more resistant to signalling pathways inhibitors: tyrphostine AG 1478 - specific for epidermal growth factor receptor (EGFR) and PD 098059 - acting on MEK 1/2 kinases. In clonogenic growth ability assay, PD 098059 sensitises both glioma cell lines to X-radiation whereas tyrphostine AG 1478 exerts only an additive effect. However, investigation of DSB rejoining efficiency by DNA pulse field gel electrophoresis (PFGE) gives contrasting results. Tyrphostine AG 1478, added before X-irradiation (10 Gy) of the cells, significantly elevates residual DSB levels measured after 30-min (M059K) or 60-min (M059J) repair period, the effect being much more pronounced in M059J cells. Under the same conditions, PD 098059 has no influence on the DSB rejoining rate. The PFGE results may indicate that homologous recombination (HR) system rather than non-homologous end-joining (NHEJ, non-functional in M059J cells) is affected by inhibition of signalling that starts at the plasma membrane. It should be taken into account that cell death processes may accompany DNA repair in a cell culture exposed to growth factor inhibitors and X-radiation and make it difficult for interpretation. Thus, examination of apoptosis/necrosis kinetics may explain inconsistency of cell survival and DNA rejoining rates in M059K and M059J cells treated with X-radiation and signalling pathways inhibitors. 8. Identification of genes important in modulation of radiosensitivity in human melanoma cells Robert Herok1, Krzysztof Fujarewicz2, Maria Wideł1, Joanna Rzeszowska-Wolny1 1Department of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland 2Department of Automatic Control, Institute of Automation, Silesian Technical University, Gliwice, Poland Melanomas are very malignant cancers capable of forming distant metastases. Usually, albeit not always, they are ionising radiation-resistant. During periods between therapy sessions as well as during metastatic spread, selective expansion of neoplastic cell clones occurs. Due to great genetic instability of these neoplasms both genotype and phenotype of arising clones may differ (for example in their radiosensitivity). The goal of our experiments was to check gene expression pattern changes during growth of a new cell population and whether clones obtained differ indeed in their radiosensitivity. Answer to these questions is essential for understanding mechanisms of metastasis. The present study is the first of the planned series. Starting with Me45 melanoma line, three subclones were obtained. Ionising radiation sensitivity of the starting line and that of the subclones was compared. Cell survival tests indicated no statistical differences in radiosensitivity. Total RNA was then isolated from the starting cell line as well as from the subclones and expression levels of some 22 thousand genes were checked using high-density DNA microarrays from Affymetrix. Among genes with significantly altered expression level were those coding for G antigens and other cell surface proteins, transcription factors, protein membrane receptors and other signalling proteins. The study was financed through KBN grants no. PZB-040/PO4/2001 and 4PO5015190. 9. Ionising radiation-induced changes of gene expression patterns in human melanoma cells Robert Herok, Joanna Rzeszowska-Wolny Department of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland Ionising radiation is a factor both increasing the risk of malignancy as well as an important antitumour therapy tool. Damages induced by radiation trigger in cells various signalling pathways, repair mechanisms as well as cause changes in gene expression patterns. Knowledge of processes induced in cells by radiation should help in understanding the basis of different radiosensitivity of various cell types. The goal of this study was to compare gene expression patterns in non-irradiated control cells and cells exposed to ionising radiation as well as to monitor the time course of these changes. Material used in our study was human melanoma Me45 cell line (obtained at the Gliwice Center of Oncology). Cells were irradiated with 4Gy dose and total RNA was then isolated from the cells (a) immediately; (b) after 12 h and (c) after 24 hours following radiation exposure. As a control, non-irradiated cells were used. Expression levels of some 22 thousand genes were then assessed using high density oligonucleotide microarrays, hybridisation equipment and procedures from Affymetrix. Groups of genes were selected for which gene expression pattern differed in a specific manner following various exposure times. Among genes for which expression was substantially increased or decreased in irradiated cells were those coding for: - transcription factors - cell cycle control proteins - proteins participating in metabolic processes - signal transduction proteins - repair proteins The study was financed through KBN grants no. PZB-040/PO4/2001 and 4PO5015190. 10. Analysis of antibody responses induced by peptides mimicking neuroblastoma antigen GD2 ganglioside Irena Horwacik, Dominik Czaplicki, and Hanna Rokita Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland Neuroblastoma is the third most frequent cancer of childhood. Although having the highest spontaneous remission rate among all human malignancies, the disease in advanced stages often cannot be tackled sufficiently. Hence novel therapeutic strategies need to be put forward. One of the new approaches is immunotherapy directed against a glycolipid antigen present on the surface of neuroectodermal cells. The antigen - GD2 ganglioside - is abundant on neuroblastoma cells but not on normal cells. However, due to chemical properties of glycolipid antigens their immunogenicity is low and they are generally difficult to apply in immunotherapy. Yet these antigens can be successfully imitated with mimeotopes - peptide sequences found using phage display technology. The aim of our studies was to investigate the immunogenic potential of peptide sequences mimicking GD2 ganglioside. The peptides of 15 aa in length were isolated by panning of phage displayed peptide libraries with mouse monoclonal antibody specific to GD2 ganglioside (14G2a). We carried out immunizations of BALB/c mice with 2 doses of three different peptide-KLH conjugates in combination with Freund's adjuvant to set the optimal immunization schedule. We showed that the peptide-KLH conjugates posses the ability to elicit humoral response in BALB/c mice and measured the level and reactivity of anti-peptide antibodies with ELISA. Immunotyping of sera samples was also performed to further characterize the immune response induced with our peptide vaccines. In a separate set of experiments we investigated whether sera samples from animals immunized with the peptide mimics recognize the GD2 ganglioside preparations (coated on microtiter plates). We also carried out experiments with human GD2 ganglioside-bearing neuroblastoma cell lines to show the cross-reactivity of the murine sera with the glycolipid expressed on tumour cells. The work was supported by 3P05A 00124 grant from the Polish Ministry of Scientific Research and Information Technology (former State Committee for Scientific Research). 11. Aclarubicin induces the production of reactive oxygen species in human cell lines Katarzyna Kania, Zofia Jóźwiak Department of Thermobiology, University of Łódź, Banacha 12/16 St, Łódź, Poland Aclarubicin (ACL) is one of the anthracycline antibiotics, widely used in chemotherapy of solid tumours and leukaemia. Several mechanisms of anthracycline toxicity have been proposed: intercalation into DNA and inhibition of DNA-topoisomerases, activation of caspases, alteration of the structure and function of mitochondrium and production of the reactive oxygen species (ROS). ROS generated by anthracyclines can induce a number of perturbations in cells, including lipid peroxidation, apoptosis and glutathione depletion. In this report we investigated the ability of aclarubicin to produce ROS in the human cell lines. ROS production was estimated in human fibroblasts derived from the skin of a healthy donor (S-2 cell line), the skin of diabetic patients (C5 cell line) and from the skin of Down's syndrome patients (BB cell line). The level of ROS was studied spectrofluorometrically using the fluorescent probe C2938 - [6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester)] with and without antioxidant PDTC (pyrrolidine dithiocarbamate). This reagent is cleaved by intracellular esterases and releasing product fluoresces in the presence of ROS, in the live cells. Our results have showed an increase of ROS production in aclarubicin-treated cells in comparison to the control cells (drug untreated cells). The addition of antioxidant PDTC inhibits the production of ROS in the fibroblasts treated with drug. 12. The level of chromosomal aberrations in Polish refinery workers Kapka L.(1), Mielżyńska D.(1), Siwińska E.(1), Grzybowska E.(2), Butkiewicz D.(2), Fustinoni S.(3) (1) Institute of Occupational Medicine and Environmental Health, Sosnowiec, Poland. (2) Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Gliwice, Poland. (3) ICP and University of Milan, Milan, Italy. The objective of the project was to evaluate occupational exposure to benzene of workers of two Polish petroleum refineries using selected indicators of individual exposure to benzene (airborne benzene), internal dose (blood and urinary benzene, BB and UB; urinary phenol, UP; trans,trans-muconic acid, t,t-MA; S-phenylmercapturic acid, S-PMA), early effects (chromosomal aberrations, CA) and susceptibility (genetically-based polymorphisms: CYP2E1, NQO1). Determination of urinary cotinine was used to examine an expected confounding effect of tobacco smoke. 101 workers currently exposed to benzene from two Polish refineries: A and B located in different cities were enrolled in the study. 99 controls were selected from two workplaces in the same geographical area and were matched on age, sex and smoking habits. Benzene exposure during work shift was measured using active personal samplers. Biological samples were collected at the end of work shift. Data collected in a standardized questionnaire form included age, sex, work duration, category of job (in refineries), current and lifelong tobacco use, medical history, history of diagnostic, x-ray exposure etc. Significantly higher levels of benzene in air were measured in Refinery A (median 0.61 mg/msup3;) comparing with Refinery B (median 0.11 mg/msup3;). Workers in Refinery A also had detectable BB levels in highest percentage (37% vs 8% in Refinery B and 1% among controls). UB levels were significantly associated with airborne benzene, while UP, t,t-MA and S-PMA concentrations did not reflect the levels of airborne benzene. Polymorphisms investigated were not associated with the levels of biomarkers of dose. Higher levels of CA were found in occupationally exposed groups with the highest frequency of CA in Refinery A where chromosomal aberrations were detected in 33% of workers vs. 14% in controls. Smoking habit had a significant effect on the frequency of CA. 13. Radiation - induced genetic changes in directly exposed and neighbouring cells in vitro M.Konopacka1, J.Rogoliński1, R. Herok1, A.Orlef2, K. Fujarewicz3, J.Polańska3, J.Rzeszowska-Wolny1 1Department of Experimental and Clinical Radiobiology, 2Department of Medical Physics, Center of Oncology, M.Skłodowska-Curie Memorial Institute ul. Armii Krajowej 15, 44-100 Gliwice, Poland, 3 Institute of Automation Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland One of the biological effects of ionising radiation is a phenomenon termed 'bystander effect'. It involves genetic changes occurring in cells that were not directly irradiated but responded to signals transmitted from irradiated cells. It is now well established that irradiated cells secrete cytokines or other soluble factors into the culture medium. The medium from irradiated cells (ICM-irradiation conditioned medium) can initiate genetic changes in non-irradiated cells such as apoptosis, chromosomal aberrations, mutations and modulations of specific proteins` expression. It has been demonstrated that medium from irradiated human epithelial cells induced the bystander response in non-irradiated cells while medium from irradiated human fibroblasts did not. Since this effect depends on the type of cells it seems to be interesting to test the medium-mediated bystander effect in cells in vitro. In the present study the cellular responses to direct irradiation and to treatment of conditioned medium were compared in human leukaemic K562 cells. The cultures were exposed to X-radiation doses between 0 - 8 Gy. After one hour incubation at 37oC the medium (ICM) was removed, filtered and transferred to non-irradiated flasks containing cells from the same line. The cultures were incubated for 36 h prior scoring. The cultures irradiated only, without change of the medium, were incubated in parallel. The genetic changes in cells exposed to radiation or conditioned medium were estimated as a frequency of micronuclei and apoptotic-like bodies. The genetic expression profiles were examined using oligonucleotide arrays that contained probes representing 22 283 human genes. The Affymetrix high density microarrays and equipment were used for hybridisation and read out of the results. The results indicate that X-radiation and conditioned medium induced micronuclei and apoptosis. Both treatments (X-radiation and ICM) induced the change in gene expression patterns. Most of the changes in expression of particular genes were similar; they either increased (for example ORCL5, E2F2, E2F5, MADH6) or decreased (CASP2, MADH1, MADH3, CDK6). Some of genes expressed differentially after X-ray and ICM treatment (BCL2, CDC14A, BUB1). 14. Studies on apoptosis and necrosis in A549 cells caused by cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP Beata Kosmider1, Regina Osiecka1, Elzbieta Zyner2, Justyn Ochocki2, L. Szmigiero3 1Department of Cytogenetics and Plant Molecular Biology, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, Poland (kosbea@biol.uni.lodz.pl) 2Faculty of Pharmacy, Department of Bioinorganic Chemistry, Medical University of Lodz, ul. Muszynskiego 1, 90-151 Lodz, Poland 3Department of Medicinal Chemistry, Medical University of Lodz, ul. Mazowiecka 6, 92-215 Lodz, Poland Non-small cell lung cancer (NSCLC) includes a group of tumours, which respond poorly to drugs. Cis-DDP toxicity still remains a problematic feature, not completely solved by the improvement of supportive care. Therefore, cis-Pt(II) complex of 3-aminoflavone was selected out of cis-DDP analogues as being less toxic towards normal cells and having at least similar or better antitumour activity in comparison with cis-DDP. The aim of this research is to compare cis-Pt(II) complex of 3-aminoflavone and cis-DDP abilities to induce apoptosis and necrosis in human non-small lung cancer cell line A549. Trypan blue dye, fluorescent dyes (acridine orange/ethidium bromide and Hoechst 33258/propidium iodide double staining), MTT and TUNEL assays were used. After cis-Pt(II) complex of 3-aminoflavone application cell viability was only 49% at 4xIC50 after 72-h incubation whereas it was 72.5% at the same time point after cells treatment with cis-DDP. The morphological symptoms of apoptosis were also noticed after cell incubations with fluorescent dyes. The results obtained showed that cis-Pt(II) complex of 3-aminoflavone had higher ability to induce apoptosis in a concentration-dependent manner in cancer cell line A549 despite its lower cytotoxicity (IC50 was 23 micro;M) in comparison with cis-DDP (IC50 was 8.5 micro;M). These observations were confirmed by the results from TUNEL assay, which detects early stages of apoptosis. It suggests beneficial properties of cis-Pt(II) complex of 3-aminoflavone as a possible chemotherapeutic drug. This work was partially supported grants No 502-13-849; 503-316-2 and 502-12-732 from the Medical University of Lodz. 15. Evaluation of the genotoxic properties of cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP in A549 cells Beata Kosmider1, Regina Osiecka1, Elzbieta Zyner2, Justyn Ochocki2, L. Szmigiero3 1Department of Cytogenetics and Plant Molecular Biology, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, Poland (kosbea@biol.uni.lodz.pl) 2Faculty of Pharmacy, Department of Bioinorganic Chemistry, Medical University of Lodz, ul. Muszynskiego 1, 90-151 Lodz, Poland 3Department of Medicinal Chemistry, Medical University of Lodz, ul. Mazowiecka 6, 92-215 Lodz, Poland Lung cancer remains one of the most common causes of cancer-related death worldwide. In view of the central problem of drugs sufficient efficiency in chemotherapy, efforts have focused on the development of alternative platinum-based analogues that can be more effective in cancers treatment. Cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-Pt(II) complex of 3-aminoflavone) represents a novel class of potential antitumour agents. The aim of this research was to assess genotoxicity of cis-Pt(II) complex of 3-aminoflavone and cis-DDP in human non-small lung cancer cell line A549. In order to evaluate genotoxic properties of this chemical compound the comet assay in A549 cells was used. The analysis of DNA damage after 1-h cell incubation with cis-Pt(II) complex of 3-aminoflavone and cis-DDP was carried out including 0.5-h, 1-h and 1.5-h postincubation. The statistically significant increase in tail moments was observed after cis-Pt(II) complex of 3-aminoflavone application in comparison with cis-DDP which can indicate DNA breaks induced by the former compound. On the other hand, the decrease in these values caused by cis-DDP was connected mainly with the presence of DNA-DNA and DNA-protein cross-links. The distribution of tail moments after A549 cells treatment with both compounds was also different. The increase of these values was also noticed after A549 cells postincubation especially with 1 micro;M and 2.5 micro;M of cis-Pt(II) complex of 3-aminoflavone contrary to cis-DDP. Results obtained on the basis of the comet assay could confirm the presence of different mechanisms of action of both tested compounds i.e. the occurrence of DNA breaks (besides cross-links) induced by cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP. This work was partially supported grants No 502-13-849; 503-316-2 and 502-12-732 from the Medical University of Lodz. 16. The examination of XPA protein interaction with selected glycosylases: NTH1, TDG, OGG1-1A and OGG1-2A Małgorzata Krześniak, Marek Rusin Department of Tumor Biology, Center of Oncology - M. Skłodowska-Curie Memorial Institute, Gliwice, Poland Cancer arises as a result of multiple somatic mutations of genes involved in regulation of cellular proliferation, apoptosis, migration, angiogenesis. Attenuation of DNA repair significantly increases cancer risk. Studying the functioning of various DNA repair systems and their interactions and regulation helps to understand cancer formation and response of cancer cells to chemo- and radiotherapy. The goal of this research project was to test the hypothesis that two DNA repair systems: base excision repair and nucleotide excision repair cooperate by physical interaction of their DNA damage recognition elements: glycosylases (BER) and XPA (NER). We selected four glycosylases: NTH1, TDG, OGG1-1A and OGG1-2A. The GST pull-down assay was employed as an experimental procedure. Glutathione-sepharose beads loaded with GST-XPA or GST only (negative control) were incubated with the cell lysates containing the glycosylases. Proteins in the bound fraction were detected using Western blotting. Antibodies used for detection were either polyclonal (recognizing native NTH1 glycosylase) or monoclonal, recognizing the FLAG epitope attached by the genetic engineering to the TDG, OGG1-1A and OGG1-2A glycosylases. These three recombinant proteins were produced in COS-7 cells after the recombinant vectors transfection. None of the glycosylases showed the detectable binding to the GST-XPA fusion protein in the in vitro system that we used. We found however that the recombinant plasmids that we constructed produce easily detectable recombinant TDG, OGG1-1A and OGG1-2A glycosylases linked to the FLAG epitope. They are valuable tools for further biochemical and biological studies on base excision repair. 17. Comparison of DNA damage and repair kinetics in lymphocytes of cancer patients and healthy donors Bożena Lubecka 1, Maria Wideł 1, Zofia Kołosza2, Sylwia Jędruś3, Joanna Rzeszowska-Wolny1 1Department of Experimental and Clinical Radiobiology, 2Department of Cancer Epidemiology, 3Clinic of Oncological Gynecology, Centre of Oncology -Gliwice, Wybrzeże Armii Krajowej 15,44-101 Gliwice , Poland. Correct repair of DNA damage plays a protective role against genotoxic factors, while its impairment leads to mutation and may be a basis of cancerogenesis. Single cell gel electrophoresis (comet assay) is a sensitive method for evaluation of DNA damage and kinetics of its repair and is widely used in environmental mutagenesis and radiobiological studies. In this study the alkaline version of the comet assay was applied for comparison of radiation-induced DNA damage and repair capacity in peripheral blood lymphocytes of 42 patients with cervical carcinoma and 30 healthy women. The results indicate a great variation in response to radiation in both groups under the study. However, the lymphocytes of patients were characterized by significantly higher level of a background (p=0,000005), increased sensitivity to radiation (p=0,002) and reduced DNA repair potential (p<0,001). Furthermore, these features were more pronounced for patients with familial anamnesis. Significant influence of age on background damage was revealed in both groups, but age connection with DNA repair after irradiation was observed in healthy donors only. The smoking status increased the level of background damage (p<0,03) and decreased lymphocyte repair ability (p<0,005) in healthy donors. In the case of patients the inverse relationship was noticed, but significance was not achieved. The observation that tumour regression is connected with lymphocyte repair kinetics suggests that local response depends not only on tumour cell radiosensitivity but may be assisted by host lymphocytes. This work was supported by KBN, Grant No 4P05A.015.19. 18. Atypical cytogenetic aberrations in a patient with CML accelerated phase: a case report Lukyanova A.S., Maslyak Z.V., Loginsky V.E. Institute of Blood Pathology and Transfusion Medicine, Academy of Medical Sciences of Ukraine, Lviv, Ukraine Introduction: Chronic myeloid leukaemia (CML) is a clonal proliferation associated in 90-95% of cases with a specific chromosomal abnormality - Philadelphia (Ph) chromosome and/or bcr-abl fusion gene. The Ph marker is a product of balanced translocation t(9;22)(q34;q11), resulting in the generation of chimeric bcr-abl oncogene. Secondary chromosomal changes in bone marrow of patients with CML may appear before or during progression of the disease. In addition to the Ph chromosome, they include trisomy 8, trisomy 19, monosomy 7, isochromosome 17q, der (22) (so-called extra Ph chromosome), -Y and indicate poor prognosis. The aim: We report a case of Ph-positive CML with multiple typical and atypical secondary aberrations. Materials and methods: A 33-years old patient has been previously treated with hydroxyurea and was in chronic phase of CML during 10 years. Cytogenetic analysis was performed for the first time because the signs of accelerated phase were observed. We initiated 48-hour unstimulated cultures from bone marrow biopsy. Cytogenetic study included karyotyping (GTG) and FISH methods. Results: Karyotyping showed the presence of several clones with multiple numerical and structural aberrations. All the cells were found to express Ph chromosome and an unbalanced translocation t(1;2)(p36;p21). Following the literature data, t(1;2) is not typical secondary abnormality for CML. Besides these changes, we also detected del(6)(q21), del(8q), del(11)(q23), del(18q), der(22) and marker chromosomes as well. The most common clone was 48, XY, t(1;2)(p36;p21), del(6)(q21), del(8q), t(9;22)(q34;q11), del(18q), der(22). Some cells showed loss of 11, 15, 20, 21 chromosomes and absence of der(22), but these changes were not regular. To verify the presence of t(1;2) and to estimate the percentage of cells with an extra Ph chromosome and with an additional copy of chromosome 8 the metaphase FISH was performed. t(1;2) was examined two times. We used the painting probe for the whole chromosome 2 or the painting probe for 2p in combination with the centromere probe for chromosome 1. Both of the studies identified t(1;2)(p36;p21). The detection of Ph and extra Ph markers by dual-colour interphase FISH showed the presence of extra Ph and Ph chromosomes in 95% and 4% of cells, respectively. To score the percentage of cells with trisomy 8 the centromere probe for interphase and metaphase FISH was used. We detected an additional copy of chromosome 8 in 18% of cells. The patient was treated with combined chemotherapy and later with Glivec, but the disease progressed to the blast crisis during 3 months. Conclusions: We observed multiple secondary cytogenetic aberrations in a patient with accelerated phase of CML. One of them - t(1;2)(p36;p21) - is not typical for any of the haematological malignancies; its prognostic significance is to be discussed. The rest are usual for accelerated phase and their presence indicate a poor prognosis. Acknowledgements: I am indebted to dr. B.Pieńkowska-Greła and colleagues from the Cytogenetic Laboratory of the Maria Sklodowska-Curie Memorial Oncology Centre-Institute, Warsaw, Poland for their technical assistance and professional consultations. 19. Microvascular free flaps in reconstructive surgery after extension resections in head and neck region Adam Maciejewski, Janusz Wierzgoń, Cezary Szymczyk Clinic of Oncological Surgery, Center of Oncology - M. Skłodowska-Curie Memorial Institute, Branch in Gliwice The main goal of surgical treatment of head and neck tumours is to achieve macro- and microscopically radical margins and to restore or preserve high quality of life. Based on own experience Reconstructive Surgery Group in Cancer Center in Gliwice introduced free flaps techniques after major resections in head and neck region. Since November 2001 till August 2003 over 80 patients underwent surgical procedures based on microvascular free flaps. In 60% Radial Forearm Free Flap was the technique of choice. In 25 cases were mandibular reconstruction was needed Fibula Free Flap was performed and in two cases iliac crest free flap was harvested. In case of middle face defects reconstruction Rectus Abdominis Free Flap was chosen. In 12 cases were postressective defects were large and composed Anterolateral Thigh Free Flap was harvested and insetted. Twice the complex defects after extended maxillectomy was reconstucted with Subscapular Composite Free Flaps. Overall survival rate based on periodic control and imaging diagnostics was about 90%. 40% of these patients underwent pre- or postoperative radiotherapy and it did not affect flap healing and survival. Based on authors experience the progress on the field of reconstructive surgery will depend on individually chosen free flaps and its goal is to achieve optimal functional and aesthetic outcome. 20. Novel phosphonate derivatives of uracil and thymine - ability to induction apoptosis in human peripheral blood lymphocytes in vitro; comparison to 5-fluorouracil 1Matławska K., 2Kalinowska U., 2Ochocki J., 1Osiecka R. 1Department of Cytogenetics and Plant Molecular Biology, University of Lodz 2Department of Bioinorganic Chemistry, Medical University of Lodz For many years 5-fluorouracil (5-Fu) has been widely used in cancer chemotherapy, especially in the treatment of colorectal, liver, ovarian, head and neck, lung and breast carcinomas. This fluorinated analogue of the uracil can be used alone in monotherapy or in combination with other cytotoxic drugs (e.g., methotrexate, cisplatin) or with agents that are themselves not toxic but that modulate 5-Fu's antitumor activity (e.g. leucovorin, folinic acid). Unfortunately clinical application of this drug is limited by many undesirable effects, such as myelosuppresion, gastrointestinal symptoms, neurotoxicity, cardiotoxicity, hyperpigmentation of skin. Due to that the aim of many studies is to search for new less toxic and more effective compounds. In Department of Bioinorganic Chemistry (Medical University of Lodz) prepared novel phosphonate derivatives of uracil and thymine: 5-uracilmethylphosphonic acid (5-umpa), K /5-umpa adduct and Na /5-umpa adduct. Cytotoxicity and genotoxicity of this compounds were described previously. The aim of this study was to estimate ability of new 5-fluorouracil analogues to induct apoptosis in human lymphocytes. Lymphocytes were isolated from peripheral blood of healthy, non-smoking donors. For detection of apoptotic cells we have used three methods: terminal transferase-mediated dUTP-fluorescensin nick end-labelling assay (TUNEL); double staining with propidium iodide / Hoechst 33258; double staining ethidium bromide / acridine orange. The results indicate that in comparison to 5-fluorouracil this novel analogues poorly support induction of apoptosis in normal human lymphocytes. Project was partly financed by UM grant 50331602. 21. Mitotic instability of human BRCA1 gene cloned in yeast Mariusz Mucha1,2, Jarosław Starzynski1,2, Anna Goc1 and Jan Filipski2 1. Laboratory of Genetics, Copernicus University, Gagarina 9, Toruń, Poland 2. Institut Jacques Monod, 2 Place Jussieu, Tour 43 75251 Paris, France Identification and subsequent mapping and sequencing of human BRCA1 gene has been followed by extensive studies of causes and pathways leading to breast and ovarian cancers. Hundreds of allelic forms of BRCA1 gene have been identified, however, little is known about the molecular mechanisms causing mutations and rearrangements, exceptionally frequent in this chromosomal region. Mutagenesis in the germline cells are mainly caused by incorrect replication and unequal crossing-over between repetitive DNA sequences. The main class of sequences involved in the processes causing chromosomal instability in the human genome are Alu repeats. Experimental data show that Alu-mediated recombination, followed by duplication and/or deletion of large portions of the gene, could be responsible for the dysfunction of BRCA1 transcript and its protein product. We have studied the mitotic stability of the YAC (Yeast Artificial Chromosome) carrying human BRCA1 gene and its flanking regions. S. cerevisiae a good model to study the instability of Alu sequences during mitotic divisions of human germline cells as the yeast chromatin shows some similarity to the chromatin present in the early embryo cells of higher Eucaryota: the cytosine in the CpG dinucleotides is not methylated and the chromatin is less condensed than the one present in somatic cells. Yeast cells containing YAC carrying human BRCA1 gene have been cultivated for 10-15 generations in rich medium. The rare-cutter restriction sites were mapped in the subclones of original YAC by indirect end-labelling, using pulse-field gel electrophoresis and hybridisation with radioactive probe recognising one end of the YAC. Comparison of the autoradiographic pattern of hybridisation bands with the restriction map based on the unmodified sequence extracted from the GenBank shows large deletions and duplications caused probably by unequal crossing-over and incorrect replication of repetitive sequences present in BRCA1 region. 22. Damage to DNA and its repair assessed by the "comet" assay in patients with autonomous thyroid nodules receiving 131-Iodine therapy Jadwiga Nieminuszczy1, Elżbieta Grzesiuk1, Marcin Kruszewski2,3, Maria T. Płazińska4, Wiesław Grzesiuk5 1 Dept of Molecular Biology, Institute of Biochemistry and Biophysics PAS, Warszawa 2 Dept. Radiobiol. and Health Protect ,Institute of Nuclear Chemistry and Technology, Warszawa 3 Dept. Exp. Hematol., Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warszawa 4 Dept. of Nuclear Medicine, University Medical School, Warszawa 5 Dept. of Internal Medicine and Endocrinology, University Medical School, Warszawa The purpose of this study is to evaluate the DNA breakage and base damage with the use of comet assay in radioiodine treated patients with autonomous thyroid nodule. In all the patients thyroid scintigram was performed using 131-I. The thyroid scan showed a single "hot nodule" with suppressed radioactive iodine uptake in remaining thyroid tissue. The dose of administered 131-I was from 14 to 16 mCi. Damage to DNA was estimated in a tissue from "hot nodule" by fine needle aspiratory biopsy and in lymphocytes (PBL). Samples were taken three times: before radioiodine treatment, 12 and 54 days after. Preliminary results (on four patients) indicate a high diversity in the level of DNA damage between individual patients. Generally, in lymphocytes 12 days after 131-I application significant level of DNA breakage and base damage was still observed. However, after 54 days the level of DNA damage in lymphocytes was even lower than in the control. On the contrary, in "hot nodule" cells DNA damage persisted till 54th day after 131-I treatment. Differences in the type of damage between thyroid cells and lymphocytes were also observed. In lymphocytes there was more base damages while in nodule cells single strand DNA breaks prevailed. Our preliminary results indicate that the comet assay can be a valuable tool for monitoring radioiodine treated patients. It can also allowed to estimate proper for each patient 131-I dose. Differences in the type and persistence of DNA damage in lymphocytes and thyroid nodule cells might indicate the different mechanism of DNA damage induction and/or differences in DNA damage repair mechanisms. 23. Recurrent BRCA1/BRCA2 mutations and new aberrations in BRCA1 promoter region in breast and ovarian cancer cases from Upper Silesia in Poland Jolanta Pamuła1, Helena Zientek1, Marzena Siemińska1, Wioletta Pękala1, Ewa Chmielik2, Jadwiga Rogozińska-Szczepka3, Beata Utracka-Hutka4, Marek Rusin1 and Ewa Grzybowska1 1Department of Tumor Biology, 2Department of Pathology, 3I Radiotherapy Clinics, 4Chemotherapy Clinics, Centre of Oncology, Maria Skłodowska- Curie Memorial Institute, Gliwice- Branch, Poland. Introduction. Germline mutations in BRCA1 and BRCA2 tumour suppressor genes cause a hereditary predisposition to breast and ovarian carcinomas. Mutations are distributed through the gene and most frequently lead to truncation of the protein. However, detectable mutations of in those genes have only explained less than half all familial breast and ovarian cancer cases. It has be also estimated that aberrations which affect expression, splicing or stability of transcript maybe responsible for additional 15-20%. Aberrations within BRCA1/2 promoters, which can result in BRCA1/2 protein decrease, could be also associated with an increased risk breast and ovarian cancer. Patients and Methods. One hundred and fifty unrelated probands with strong family history of breast and ovarian cancer, bilateral or early-onset breast cancer were chosen for analysis. We screened by direct sequencing all exons and exon/intron boundaries. Eighty seven breast/ovarian cancer patients without mutations in BRCA1/2 genes were selected for the analysis of BRCA1 promoter region. Results. We found two families with deletions in beta-promoter of BRCA1 (GeneBank NoAc U37574, 2223delAAAAA) one family with mutation, 1827insCdelGGAACA (GeneBank No.Ac U37574) and four polymorphisms in BRCA1 promoter region (GeneBank No.Ac U37574, 2642A>G, 2743T>C, 1983G>C, 1873G>C). We also found five different disease predisposing mutations within BRCA1 gene (185delAG, 300T/G, 4153delA, 5382insC, 5528del1 IV22-6). The results confirms the presence of two strong BRCA1 founder mutations in Polish population-5382insC and 300T>G. The BRCA1 (5528del1 IV22-6) mutation was not reported previously and might be specific to the southern Polish population while the others were recurrent. We also detected two new sequence variants in BRCA1 introns (IVS12-4G-->T and IVS21-31C-->G). Two disease predisposing mutations were detected in BRCA2 (6174delT, 9631delC). In addition, eight new unclassified sequence variants were found within BRCA2 exons (3431T>C, 3446A>G, 3655G>C, 4846G>T, 4988C>T, 6188A>G, 8335A>T, 8341A>T) and 6 new variants in BRCA2 introns (IVS4 67A/C, IVS4 147G/T, IVS12 157A/G, IVS12 183A/G, IVS18 13C/A, IVS24-36C/T). Conclusion. Identified novel aberrations in the BRCA1 promoter suggest that mutation and polymorphisms in this region might be responsible for significant fraction of breast and ovarian cancer cases. Our results lend further support to the need for more detailed functional and epidemiological studies aimed at understanding the role of BRCA1 and BRCA2 promoters in the etiology of breast and cancer. 24. Correlation between gene positioning and gene expression Philimonenko A. A.1, Janacek J.2, Weipoltshammer K.3, Schöfer C.3, and Hozak P.1 1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4 - Krč, Czech Republic; 2Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 3Institute for Histology Embryology, University of Vienna, Vienna, Austria; We have investigated the spatial relationship of transcriptionally active or inactive genes within chromosome territories in the interphase nucleus of human fibroblasts. As a model we used paternally imprinted genes located within the 15q11-q13 region of chromosome 15 (SNRPN locus), deletion of which leads to Prader-Willi syndrome. For the simultaneous detection of genes and chromosome territories, we used in situ hybridisation with commercially available DNA probes to SNRPN locus, and to the q arm of human chromosome 15. A computer program was written which allows the detection of the border of chromosome territory, and the distance between the border and the centre of mass (labelling) of the gene. The analysis demonstrated no significant differences in distances from gene to chromosome territory border between active and inactive genes. Significant portion of genes were found located outside of 15q territory - however, only if the allele were transcriptionally active. In agreement with previously obtained data, we also observed a distinct substructure in interphase chromosome territories. Strongly labelled chromosomal subdomains were surrounded by less intensely labelled areas. Using a thresholding of digital images of human fibroblast nuclei we discriminated these subchromosomal domains and evaluated the distances between their borders and SNRPN loci. The SNRPN gene loci were located preferentially at the borders of chromosomal subdomains. These data support the idea that genes are preferentially located at the outer side of domains with condensed chromatin, but no strict correlation was found between active and inactive genes within the entire chromosomal domain. The only striking difference was the presence of ~ 7% of active genes at relatively large distances from the chromosomal territories. 25. A program for the statistical evaluation of clustering and colocalization patterns in immunogold labelling experiments Philimonenko A. A.1, Janacek J.2, and Hozak P.1 1Department of Cell Ultrastructure Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic (e-mail: hozak@biomed.cas.cz; http://nucleus.biomed.cas.cz/) 2Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the significance of non-random location of gold particles are missing. We have therefore developed a method, which allows the detection of clustering or colocalization of gold particles using the distribution of distances between them (Philimonenko et al., 2000, J. Struct. Biol., Vol. 132, pp. 201-210). The program or plug-ins which are based on this method can be downloaded free of charge on our web-site (http://nucleus.biomed.cas.cz/gold). They allow one to evaluate statistically the observed immunogold labelling patterns for clustering or colocalization. Our program and plug-ins are also a good addition for image analysis software, which accompanies most of the modern CCD cameras for electron microscopes. 26. Oligo(3-hydroxybutyrate)-drug conjugates - a novel drug delivery system. Rapid cellular uptake and multidrug resistance overcoming by OHB-conjugated doxorubicin Valeria Piddubnyak1, Zbigniew Jedliński2, Andrzej Matuszowicz2, Magdalena Głowala1, Piotr Kurcok2, Maria Juzwa2, Mirosław Śnietura3, Zdzisław Krawczyk1. 1Department of Tumor Biology, Centre of Oncology, Gliwice, Poland; 2Centre of Polymer Chemistry, Polish Academy of Science, Zabrze, Poland; 3Department of Tumor Pathology, Centre of Oncology, Gliwice, Poland Extensive search of drug delivery systems that would improve effectiveness of therapy has been performed in recent years. Among the most promising approaches is the application of drugs conjugated to polymeric carrier molecules. It is expected that in conjugated form drugs would have improved pharmacokinetics and biodistribution, and would be released in a controlled manner. In case of cytostatics using in cancer therapy, reduced toxicity to normal cells and the ability to overcome drug resistance is also expected. Aim: The purpose of the present study was: 1) to compare the cytotoxic effect of doxorubicin, either free or conjugated to well-defined tailor made oligo-[R,S]-3-hydroxybutyrates (OHBs); 2) to compare the kinetics of uptake and subcellular localization of both forms of this drug. Methods: The drug cytotoxicity was tested in vitro on the human breast cancer cell lines, doxorubicin-sensitive (MCF7/WT) and doxorubicin-resistant (MCF7/DOX) subclones, using 3-[4,5-dimethylthiazol-2-y]2,5-diphenyltetrazolium bromide (MTT) test and clonogenic-forming assay; the kinetics of drug uptake and localization within the cells were studied by the confocal laser scanning microscopy (CLSM). Results: The OHBs chosen as drug delivery carriers, especially those having MW lower than 1000, did not affect cell viability. Our cytotoxic studies showed that the growth of MCF7/DOX cells was not affected by treatment with 5 µg/ml of free doxorubicin (concentration used for culturing this type of cells), while OHB-conjugated doxorubicin killed approx. 30% of cells after 10 h treatment. The CLSM studies showed that OHB-conjugated doxorubicin localized in the cytoplasm of both cell lines. In contrast, free doxorubicin was localized predominantly in the nuclei of MCF7/WT, whereas in MCF7/DOX the fluorescence of the drug was barely detectable in perinuclear area. Moreover, the uptake of OHB-conjugated doxorubicin by cells was much faster than the uptake of free drug. In fact, the fluorescence within the cytoplasm was clearly visible in both cell lines as soon as 15-30 sec from the addition of the conjugate into the culture medium. Conclusions: These results suggest that OHB-doxorubicin conjugates could overcome multidrug resistance of cancer cells. Our data show that tailor-made biodegradable and biocompatible oligomers of hydroxybutyric acid can be regarded as effective non-toxic vectors for drug delivery in a conjugated form. 27. Proliferation associated gene A (PAG) protein binds to DNA damaged by N-acetoxy-acetylaminofluorene (AAAF) Monika Pietrowska and Piotr Widłak Department of Experimental Clinical Radiobiology, Center of Oncology, M. Skłodowska-Curie Memorial Institute, Gliwice, Poland Proteins that recognize and bind to damaged DNA participate in all repair pathways. Damage recognition is the first step of all repair system. However, several proteins that preferentially bind damaged DNA but do not participate in repair systems have been identified as well. Among such proteins are chromatin proteins HMG-1/2 and histone H1 that bind preferentially DNA damaged by cisplatinum. Non-repair proteins, which specifically recognize DNA damage induced by AAAF or UV irradiation, are poorly characterized. Using an electrophoretic mobility shift assay two different nucleoprotein complexes specific for DNA damaged by AAAF have been detected in nuclear extracts from rat tissues. We attempted to identify proteins that formed such complexes. Proteins binding to DNA damaged by AAAF were purified from nuclear extracts of rat liver using combination of an ion-exchange chromatography and an affinity chromatography on AAAF-damaged-DNA-cellulose. Specific protein binding to DNA damaged by AAAF was then detected in such preparations using Southwestern blot analysis. Major protein that bound AAAF-damaged radioactive probe, that had molecular size of about 24 kDa, was analysed using mass-spectrometry and identified as PAG protein (proliferation associated gene A protein, termed also heme binding-protein 23 kDa, macrophage 23-kD stress protein, peroxyredoxin1). The enhanced binding of PAG protein to AAAF-damaged-DNA-cellulose (and also to cisplatinum-damaged-DNA-cellulose) was further confirmed by Western-blotting. The PAG gene is constitutively expressed in most tissues (the various cell types), but its expression is higher in organs having a higher level of proliferation. The PAG protein has an antioxidant function and protects cells against the oxidative stress, and is localized in either cytoplasm and nucleus. However, the DNA-binding form of the PAG protein has been detected exclusively in nucleus. This work was supported by the Polish State Committee for Scientific Research (KBN) Grant 3PO5A10524. 28. Electronic compensator in combined brachytherapy and teletherapy of gynecological patients Agata Rembielak, Krzysztof Ślosarek, Brygida Białas, Joanna Bystrzycka, Marek Fijałkowski, Krzysztof Nowakowski Brachytherapy Department, Center of Oncology - Institute, Gliwice, Poland Definitive radiation therapy alone combined with teletherapy (TT) and brachytherapy (BT) has been established as an effective form of treatment in patients with advanced cervical cancers. In Center of Oncology - Institute in Gliwice, Poland the following schedule has been used: TT - total dose of 40 - 54 Gy administered in 20 - 27 fx, 2 Gy per fraction, BT - introduced after completing 20 Gy to the entire pelvis from TT. TT is continued on non-BT days with a 4 cm wide midline block to shield critical organs. The use of standard cuboid-in-shape shield in combination with variation in patient shape as well as tissue inhomogeneity can lead to large dose variations in the irradiated volume (risk of hot or cold spots). Dynamic technique of teletherapy and inverse planning option enable to create the individual plan based on BT dose distribution and computed tomography data with no need to use the standard block. The aim of the study is to present the influence of individual compensating filter, instead of standard central shield, on improvement of dose distribution within irradiated area. 29. Isocenter verification in stereotactic radiosurgery using electronic portal imaging device R. Rutkowski1, T. Hauser2, K. Ślosarek1, A. Grządziel1, B. Smolińska1 1Radiotherapy and Brachytherapy Treatment Planning Department, 2Maintenance Department, Center of Oncology - Institute, Gliwice, Poland Purpose: To develop a method of isocenter verification in Stereotactic Radiosurgery (SRS) in order to in precise digital way of isocenter verification by Electronic Portal Imaging Device (EPID). Methods and Materials: Linear accelerator Varian Clinac 2300C/D equipped with EPID and BrainLab stereotactic accessory including micro-Multileaf Collimator (mMLC) and EPID were used. Digital verification method based on Winston-Lutz test, however in digital verification method it is EPID that collects images in order to precise verification of isocenter. During digital verification method mMLC leaves are set to 'H' shaped field (two pairs of leaves in the middle of the field form small square gap). Then first two portal images are taken. Using laser positioners a small metal phantom ball is located in isocenter. To check isocenter invariability several portal images are obtained at various collimator, gantry and couch positions. After each portal field acquisition a quick visual and digital check is done to control if ball is inside square formed by mMLC. The idea of digital analysis is to subtract two portal images: one with phantom ball and second without ball in the same collimator position. Digital check is performed by independent computer program - 'WinIzo'; developed in Treatment Planning Department in MSC Institute in Gliwice, Poland. Digital analyse subtract two portal images (first without ball, second with ball, both with the same collimator position) and shows optical density symmetry distribution. Results: Isocenter verification method thanks to EPID and WinIzo application let to obtain result quickly in easy eye and digital check form. Portal images are collected in database for further analysis. Our takes about 30 minutes. Winston-Lutz test is more time-consuming and takes about 1h. Conclusions: Moreover in presented method images analysis is improved. The correction of ball position can be done after each single portal aqusition and there is no need to wait till the whole test is preformed as in basic winston lutz one. Our method is faster and less expensive then Winston-Lutz test based on portal x-ray film. This method is a part of Quality Assurance (QA) in SRS procedures. 30. Radioprotective and cancer preventive effects of the 1,4-dihydropyridine derivative in human cells Nadzeya I. Ryabokon1, Nataliya V. Nikitchenko1, Joanna Rzeszowska-Wolny2, Gunars J. Duburs3, Rose I. Goncharova1 1Institute of Genetics and Cytology, National Academy of Sciences of Belarus, (antimut@biobel.bas-net.by), 2M.Sklodowska-Curie Memorial Institute, Branch in Gliwice, Centre of Oncology (jwolny@io.gliwice.pl), 3Latvian Institute of Organic Synthesis, (gduburs@osi.lv) Previously it has been reported that some of the derivatives of 1,4-dihydropyridine (1,4-DHP) series are effective antimutagens and reduce the spontaneous, alkylation- and radiation-induced mutagenesis in various test-systems in Drosophila, mouse and fish. Several mechanisms of their actions were studied and/or discussed, among them were antioxidant activity, modulation of DNA repair and apoptosis that can play important role in cancer prevention or therapy [Goncharova et al., 1974, 2002; Kuzhir, 1999; etc.]. The chemical structure of 1,4-DHP derivatives resembles dihydronicotinamide, so it seems probable that they can influence NADH and NAD(P)H metabolism and, in this way, the excision repair and apoptosis [Kuzhir, 1999]. We were interested, if the compounds of this series modulate the levels of DNA damage and apoptosis in control and irradiated human cells. The pilot investigation was carried out using AV-153 1,4-DHP derivative, which showed the highest antimutagen activity in animal experiments [Goncharova et al., 1980; Kuzhir, 1999]. The lymphocytes isolated from peripheral blood of healthy donors were treated with different concentrations of AV-153 dissolved in culture medium. The trypan blue exclusion test did not show any evident cytotoxicity of the compound in a wide range of concentrations (10-11-10-5M) after 24 h of lymphocyte incubation with AV-153. In reality, the antimutagen even decreased the background and radiation-induced cell death frequencies. The alkaline single cell gel electrophoresis (Comet assay) has shown that AV-153 reduces effectively DNA damage in the control and gamma- and X-ray irradiated cells (reduction factor up to 70 %). The analysis of DNA repair kinetics revealed that AV-153 increases the effectiveness of repair process during the first 15-60 min of incubation. The data of micronucleus test in PHA stimulated lymphocytes have demonstrated anticlastogenic effect of AV-153. We demonstrated also that at concentrations which effectively decrease DNA damage AV-153 stimulated apoptosis in human lymphocytes. We conclude that 1,4-DHP derivatives should be further studied as cancer preventive and radioprotective compounds. This work was partially supported by the fellowship grants from the Association for the Support of Cancer Research and the UNESCO (Polish branch) to Dr. N.Ryabokon. 31. Intra S-phase checkpoints. Induction of double strand breaks (DSB) and premature chromosome condensation (PCC) in root meristem cells of Vicia faba Dorota Rybaczek and Janusz Maszewski University of Łódź, Department of Cytophysiology, 90-231 Łódź, ul. Pilarskiego 14, Poland e-mail: doryb@biol.uni.lodz.pl jamasz@biol.uni.lodz.pl The conserved system of cell-cycle checkpoints provides an efficient control mechanism to ensure that DNA synthesis and segregation of chromosomes at mitosis proceed with accuracy high enough to preserve genomic integrity. The checkpoints are signal-transduction pathways specific for either abnormal or incompletely assembled cellular structures. Each of them comprises three essential parts: (1) a way of sensing that a cell cycle event is aberrant or incomplete, (2) means by which this signal is transmitted, and finally, (3) the effectors that delay or block the cell cycle transitions until the problem is resolved. The position of arrest within the cell cycle varies depending on the phase in which the damage is sensed. Since the main role of all these checkpoints is to make a decision, whether or not the cell division cycle has to be continued, particular elements of these checkpoints deserve special attention as promising targets for pharmacological treatment of cancer. Within interphase, the checkpoint-mediated inhibitory pathways give a cell both time and means needed for repair processes to occur before genetic alterations are rendered irreversible and heritable. In yeasts and complex multicellular Eukaryotes, the G1-DNA damage checkpoint prevents from entering the new cell cycle in response to DNA damage. The S-phase damage checkpoint slows down replication when the cell exposed to ionising radiation "senses" breaks, lesions, or modified nucleotide composition of DNA. Another discrete S-M replication checkpoint monitors nuclear DNA structure and delays the onset of mitosis until S phase is complete. The DNA damages preserved or incurred at later stages of the cell cycle are detected by the G2 DNA damage checkpoint, which restrains the onset of mitosis until the mechanisms engaged to repair DNA restore adequate genomic description for the next generation of cells. Studies on root meristems of Vicia faba indicate that a lot of agents may allow the S-phase-arrested cells to override the S-M control system. First of all - caffeine, which is known to induce premature mitosis when cells stay underreplicated. In Vicia, S-phase-blocked cells treated with caffeine start out aberrant mitotic divisions. The full array of aberrations encountered in V. faba includes: chromosomal breaks and gaps representing unreplicated regions of DNA molecules, lost and lagging chromatids and chromosomes, acentric fragments, chromosome bridges and micronuclei. The main conclusions of this work are as follows: 61656; Agents, including caffeine, CDK inhibitors (2-AP), protein kinase inhibitors (sodium vanadate), and activators of protein kinase C (12-meristate-13-acetate - PMA), can override the S-M (replication) checkpoint and induce premature chromosome condensation (PCC). Caffeine and PMA belongs to the most effective stimulators of PCC in S-phase- arrested cells of Vicia faba. Following HU/caffeine treatment, some cells enter "S-PCC". A combined treatment of other chemical agents can override the S-M checkpoint and brings about the "G2-PCC". Structural aspects and regulatory mechanisms of the cell cycle are similar in yeasts, animals, and plants. The checkpoint control systems sense DNA damage or incomplete duplication of the genome, and act to delay the initiation of chromosome segregation to the daughter cells (mitosis). Signalling components, transmitters, and effectors of these pathways are probably highly conserved. These comprise Chk1 kinase and H2AX, phosphorylated specifically in response to HU treatment. 32. Calcium folinate stabilizes neurochemical injuries evoked by methotrexate Anna Shevalye, Vyacheslav Slyshenkov, Igor Zverinsky Institute of Biochemistry NASB, 50 BLK, 230009 Grodno, Belarus, e-mail: covi.lab@km.ru Methotrexate is widely used for the cancer treatment as immunosuppressive drug, its adverse reactions include neurological disorders. Glutathione and glutathione-dependent enzymes play central role in cellular defence against toxic agents and glutathione homeostasis can have effects on the sensitivity of cancer cells to a wide range of drugs. The aim of the present work was to study the state of glutathione system of brain tissue under the administration of methotrexate alone and combined with its antidote calcium folinate. The following neurochemical injuries under the administration of methotrexate in a doze of 2 mg/kg/day (i.p.) to Wistar rats for 4 days were established. The significant decrease of the total glutathione and its reduced form contents in forebrain homogenates was marked. The oxidized glutathione contents did not change. It was shown, that activity of the following enzymes - glutathione reductase and peroxidase, and acetylcholinesterase did not differ from control values, while the glutathione transferase activity increased significantly. The increase of the phospholipids content and antioxidizing capacity of brain tissue was also observed, which most probably might be explained by the activation of adaptation processes of an organism. The combined injections of calcium folinate (17.5 mg/kg) and methotrexate (i.p.) to rats changed the parameters investigated considerably, resulting in the norm values. Thus, the results obtained enable us to conclude that antitoxic effect of calcium folinate can be at least partly mediated by the stabilization of glutathione homeostasis of neuronal cells. 33. Synthesis and characterization of novel hydrophobic porphyrin derivatives suitable for photodynamic therapy Agnieszka Szurko1,2, Gabriela Kramer-Marek1,3, Maria Widel2, Aleksander Sochanik4, Jan Habdas5, Piotr Kus5, Alicja Ratuszna1 1A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland; 2Departament of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland; 3Departament of Tumor Biology, Center of Oncology, Gliwice, Poland; 4Molecular Biology Department, Center of Oncology, Gliwice, Poland; 5Institute of Chemistry, University of Silesia, Katowice, Poland; Photodynamic therapy (PDT) is increasingly applied for treatment of different types of tumours not amenable to surgery, radiotherapy or other conventional treatments. Most widely used in PDT is the first generation of hematoporphyrins, among them Photophrin II. Due to some drawbacks in clinical application of these drugs a number of studies have been undertaken that aim at finding potential novel photosensitizers. In an effort to find such an improved PDT agent we have undertaken syntheses and physico-chemical characterization of some hydrophobic porphyrin derivatives. Their in vitro biological activity was evaluated on human melanoma cell line (Me45) exposed to cationic liposomes that contained novel porphyrins. The dark cytotoxicity and photodynamic efficiency of synthesized porphyrins was determined by tetrazolium chlorimetric reduction assay (MTS) which measures mitochondrial dehydrogenase activity of surviving cells. Alkaline comet assay (single-cell gel electrophoresis) was used to evaluate DNA strand breaks and potential genotoxic effects induced by PDT treatment. Analysis was performed through measuring the tail moment with a comet imaging system. Micronucleus test, apoptosis and assessment of necrosis, expressed as DNA damage, were also done. The results obtained suggest that some of the novel porphyrins may be promising candidates for further use in PDT experiments. 34. Heat shock factor 1 (HSF1) does not activate hsp70i genes but induces caspase-3 dependent apoptosis in spermatogenic cells. Natallia Vydra, Ewa Małusecka, Magdalena Głowala, Katarzyna Lisowska, Michał Jarząb, Konrad Benedyk, Dorota Ścieglińska, Zdzisław Krawczyk, Wiesława Widłak Department of Tumor Biology, Center of Oncology - M. Skłodowska-Curie Memorial Institute, Gliwice, Poland Somatic cells are protected from stress-induced damage by activation of heat shock proteins (HSPs), which posses cytoprotective and anti-apoptotic functions. Heat induced expression of hsp genes is mediated by the heat shock transcription factor 1 (HSF1). Testicular temperature is lower than core body temperature and its elevation causes active elimination of spermatogenic cells. It has been recently shown that expression of the mutated, constitutively active form of the HSF1 leads to degeneration of the seminiferous epithelium in transgenic male mice. To study the mechanism of heat shock response in testes we have constructed transgenic mice in which the expression of constitutively active human HSF1 was driven by the rat spermatocyte-specific Hst70 gene promoter. We obtained three transgenic founders (only females) and established three transgenic lines. Among transgene-positive animals only females were fertile. At autopsy, the testes of transgene-positive males were significantly smaller than those of transgene-negative mice. The transgene expression in testes, detected by RT-PCR and Western blot analysis, was observed from day 15 to 35 of postnatal development. Due to lack of spermatids which should appear at that time, evident degenerative changes in morphology of seminiferous epithelium were observed, from day 22, in transgene-positive males. However, seminiferous tubules of 18-day-old transgenic mice already contained clusters of apoptotic cells with fragmented chromatin. It seems, that appearance of active HSF1 is connected with induction of apoptotic cell death of spermatocytes. Activated caspase-3 was immunohistochemically detected in spermatocytes of 15-day-old males, immediately after appearance of mutant HSF1. Interestingly, mutant heat shock transcription factor did not activate inducible hsp70 genes. To characterize mechanisms involved in disruption of spermatogenesis by HSF1 we employed DNA microarray technique. We compared gene expression profiles in testes of 22-day-old transgenic and wild-type males. We noticed significantly reduced expression of the 63 out of 129 genes (49%) involved in spermatogenesis, 46 out of 114 genes (40%) coding for chaperone proteins, and 92 out of 352 genes (26%) associated with cell cycle. This observation reflected, at the molecular level, the degeneration of seminiferous epithelium. Further microarray experiments are needed to elucidate the role of HSF1 at the earlier developmental stages. 35. Expression of pendrin and sodium-iodide symporter in ret-positive and ret-negative papillary thyroid carcinomas Małgorzata Wiench1, Małgorzata Koligot1, Tomasz Wojdacz1, Jan Włoch2, Agnieszka Czarniecka2, Ewa Chmielik3, Agnieszka Pawlaczek1, Elżbieta Gubała1, Daria Handkiewicz-Junak1, Barbara Jarząb1 1Department of Nuclear Medicine and Endocrine Oncology, 2Clinic of Surgery, 3Department of Tumor Pathology, Center of Oncology - MSC Memorial Institute, Gliwice, Poland. Altered expression of two iodine membrane transporters: NIS (sodium-iodide transporter) and PDS (pendrin) may be an important factor leading to the reduced 131I accumulation in most thyroid cancers. The aim of the study was to investigate the NIS and PDS genes expression in papillary thyroid carcinomas (PTC) by means of real-time Q-PCR and to correlate their expression with RET gene activation. Total RNA was isolated from 64 paired normal and PTC samples using RNeasy Midi and Mini Kits (Qiagen), repurified with RNase-free DNAse I and reverse-transcribed with cDNA Cycle Kit (Invitrogene). The genes' expression was also measured in a residual material left after preparation of cytological smears from fine needle biopsy (FNAB) specimens from 24 patients with lymph node recurrence of PTC. Q-PCR was performed by means of ABI PRISM 7700 Sequence Detection System. Both NIS and PDS mRNA were amplified in multiplex reactions with GUS mRNA as control. A standard concentration curve was prepared from a single sample of hyperfunctioning goiter. In each case the results where normalized to the endogenous control. In cancer samples both NIS and PDS were down-regulated, the expression of NIS was about 10%, of PDS - about 25% of the expression in normal tissue. In tumours NIS expression was significantly correlated with PDS, whereas such relationship was not found in normal samples. Additionally, the expression of NIS and PDS in normal thyroid cells was not predictive of the expression in tumour. In PTC metastases NIS and PDS expression was more significantly decreased than in tumour tissues and was undetectable in 8 and 6 samples for NIS and PDS, respectively. RET/PTC rearrangements were identified in 14/53 (38%) PTC samples. Statistically, we observed a difference in NIS expression in tumour tissues with RET rearrangement compared to those without this alteration. To summarize, NIS and PDS expressions are strongly reduced in papillary thyroid carcinomas; down-regulation is more distinct in lymph node metastases. The expression of NIS and PDS in normal thyrocytes and in tumour cells is differentially regulated. Our results suggest also that NIS expression may be more distinctly reduced in RET-negative tumours. The project was supported by Polish State Committee for Scientific Research, grant no. 305B14622. 36. Gene expression profiling in papillary thyroid carcinoma Małgorzata Wiench1, Jan Włoch2, Krzysztof Fujarewicz3, Krzysztof Simek3, Małgorzata Oczko1, Agnieszka Czarniecka2, Michał Jarząb4, Ewa Chmielik5, Sylwia Szpak1, Elżbieta Gubała1, Andrzej Świerniak3, Barbara Jarząb1 1Department of Nuclear Medicine and Endocrine Oncology, 2Clinic of Surgery, 4Department of Tumor Biology, 5Department of Tumor Pathology, Center of Oncology - MSC Memorial Institute; 3Silesian Technical University, Gliwice, Poland. The aim of the study is to evaluate expression profiles in papillary thyroid carcinomas (PTC) in order to find a set of genes separating tumours and normal tissues and to study the differences in gene expression related to the presence of RET rearrangements. 16 PTC samples and corresponding normal tissues were frozen immediately after excision. About 100-150 mg of tissue was fragmented in liquid nitrogen and homogenized. Total RNA was extracted using RNeasy Midi Kit and repurified with RNeasy Mini Kit (Qiagen), including a digestion step with RNase-free DNase I. 8 µg of RNA was taken for a ds cDNA synthesis reaction (SuperScript II), followed by the synthesis of biotin-labelled cRNA. All samples were hybridised to Human Genome U133A arrays as recommended by Affymetrix. The RET gene rearrangements were found by RT-PCR in six cases. To analyse gene expression pattern in tumour samples the following steps were performed: 1. Preprocessing; 2. Preselection by three alternative procedures (modified Sebestyen criterion - SC, Neighbourhood Analysis - NA and Singular Value Decomposition - SVD); 3. Construction and clustering of set S (434 genes) created as a sum of three gene sets obtained by data preselection; 4. Gene selection by Recurrent Feature Replacement (choice of the best set of genes), by Singular Value Decomposition, by Single Separators and by Data Mining Tool (analysis of paired samples). Difference in gene expression profiles between PTC and normal thyroid tissue is very clear and encompasses a significant number of 434 genes (set S). 258 transcripts were up-regulated and 176 were down-regulated in tumours. Among them there were 55 characterized by the ability to separate tumour and normal samples only by expression signal of this gene (Single Separators). By DMT analysis 49 Single Separators were found to be changed in 16/16 tumours in comparison to paired normal tissues. There were 67 increased (I) and 43 decreased (D) genes in all tumour samples. In addition, 168 genes were changed in 15/16 tumours and further 183 genes in 14/16 PTC. Selection of sets of genes for differentiation of RET-positive and RET-negative PTC tumours was approached by RFR and SVD. RFR selected a set of 14 genes, however further analysis did not unequivocally confirm their biological significance. SVD was unable to find any trend related to the presence of RET rearrangement. Papillary thyroid cancer, investigated in this study, shows a very strong expression profile pattern, which is confirmed also by the unsupervised method of SVD and by the existence of significant number of single separators. Our data widen significantly the list of genes characteristic for the expression profile of papillary thyroid carcinoma proposed previously by Huang et al. The project was supported by Polish State Committee for Scientific Research, grant no. PBZ/KBN/040/P04/2001 37. Differential DNA double strand break fixation dependence on poly(ADP-ribosylation) in L5178Yand CHO cells M. Wojewódzka, B. Sochanowicz, and I. Szumiel Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Poland L5178Y (LY) sublines, LY-R and LY-S, differ in response to combined treatment with poly(ADP-ribose) polymerase inhibitor, aminobenzamide (AB) and ionising radiation: 2mM AB sensitises LY-S but not LY-R cells [Acta Radiol. Oncol. 24, 451]. The high radiation sensitivity of LY-S cells is reasonably explained by deficiency in DNA double strand break (DSB) repair [Radiat. Res., 112, 146]. Since the rejoining of DNA breaks in LY-S cells is not sensitive to DNA-PK inhibitors [Mutat. Res., 409, 31], the high radiation sensitivity is likely due to the impaired function of NHEJ. We investigated the role of poly(ADP-ribosylation) in DSB repair in L5178Y (LY) sublines, LY-R and LY-S, and a pair of CHO lines: wild type (WT) and mutant xrs6 cells. Cells (asynchronous, logarithmic phase) were incubated with 2mM AB at 37oC for 2 h, X-irradiated with 10 Gy and allowed to repair DNA breaks for 15, 60 and 120 min) at 37oC or 25oC. The remaining DSB were estimated by the neutral comet assay. At 37oC no effect of AB treatment on the repair kinetics was observed either in xrs6 or CHO (WT) cells. In contrast, AB inhibited the repair of DSB in LY-S line but not its parental LY-R line, in agreement with the previously observed sensitisation of LY-S cells to X-rays by poly(ADP-ribosylation) inhibition. However, DSB rejoining in the repair competent cell lines, CHO and LY-R, also was affected by AB when the post-irradiation incubation was carried out at 25oC. Analysis of these results together with some earlier data on LY-S cells allowed to interpret these results in terms of Radford's [Int.J. Radiat.Biol., 78, 1081-1093, 2002] model of radiation damage fixation. The impaired repair of DNA breaks in LY-S results in a slow repair of a sector of DSBs (presumably in transcription factories). In the repair competent cell lines, slow down repair is achieved by incubation at 25oC. Then, fixation of DSB enhanced by poly(ADP-ribosylation) inhibition is revealed. The results indicate that poly(ADP-ribosylation) can be an important modulator of the conversion of DNA damage to lethal events. 38. Automated patient positioning ExacTrack system for dynamic techniques in radiotherapy Aleksander Zajusz1, Krzysztof Ślosarek2, Dorota Syguła1, Joanna Bystrzycka2, Roman Rutkowski2 Centre of Oncology - Institute MSC, Gliwice 1 - II Radiotherapy Clinic, 2 - Treatment Planning Department Nowadays, in radiotherapy various dynamic techniques are commonly used. The high precision in patient positioning during whole treatment process using these dynamic techniques is required. In the Centre of Oncology - Gliwice automated patient positioning verification system ExacTrac is used. This system is based on markers localised on patient's skin during all pre-treatment procedures. Afterwards the same markers are read over by digital cameras set on accelerator in the course of treatment sessions. The implementation of ExacTrack system is showed on same example of patient with prostate cancer. Treatment plan for dynamic techniques (IMRT) by means of multi - leaf micro collimator was prepared. ExacTrack system and multi - leaf collimator application in the dynamic technique enable the critical organs protection. The usage of presented patient positioning device assures quite good repeatability but is very time consuming. 39. The androgen receptor (CAG)n and (GGC)n repeats and breast and ovarian cancer risk in BRCA1/2 carriers and non-carriers Helena Zientek1, Jolanta Pamuła1, Michał Jarząb1, Wioletta Pękala1, Marek Rusin1, Ewa Chmielik2, Marta Pacocha1 ,Beata Utracka-Hutka4, Jadwiga Rogozińska-Szczepka3, Katarzyna Lisowska1, Ewa Grzybowska1 1Department of Tumor Biology, 2Department of Pathology, 3I Radiotherapy Clinics, 4Chemotherapy Clinics, Centre of Oncology, Maria Skłodowska- Curie Memorial Institute, Gliwice- Branch, Poland. Introduction. About 5-10% of breast and ovarian cancers occur as a result of highly penetrant germ- line mutations in BRCA1 or BRCA2 tumour suppressor genes. The variation of risk between different families segregating the mutations suggests the existence of important environmental and genetic factors which can modify the cancer risk in mutations carriers and that these modifiers might be also associated with variation in risk in non- carriers. Patients and Methods. We undertook the study of AR polyglutamine and polyglycine repeats length polymorphisms in 3 groups of women: 98 BRCA1/2 mutation carriers, 55 non-carriers with the first degree family history of breast and/or ovarian cancers (BRCAx) and age- matched control group of 109 healthy individuals. The samples chosen for this study had been previously analysed for germ-line mutations in BRCA1 and BRCA2 genes. All participants were assessed for length variation in the (CAG)n and (GGC)n AR repeats using GeneScan analysis with fluorescently labelled primers. As in previous studies, (CAG)n repeat lengths of <22 were classified as short (S), and those of >=22 were classified as long (L). For (GGC)n repeats, those < 17 were classified as short, and those >= 17 were classified as long. The Mann-Whitney test was used to compare the lengths of alleles in cases versus controls. Results. Our data show that long (GGC)n repeat (>=17 repeats) is less common between breast and ovarian cancer cases when compared to general. When the group of mutation carriers (BRCA1/2) was compared to healthy subjects and familial breast cancer cases (BRCAx), there was no observed difference in (CAG)n cumulative length, while a significant decrease in frequency of (GGC)n cumulative >=33 was revealed: 45% in the group of mutation carriers (BRCA1/2) vs. 67.4% in healthy subjects and 71.7% in familial breast cancer patients (BRCAx) (OR 0.4 and 0.32, respectively; p<0.005). Conclusion. These results imply that (CAG )n and especially (GGC)n repeat length can potentially serve as a useful marker to identify a subset of individuals at higher risk of developing breast and ovarian cancer which can also modify the cancer risk in BRCA1/2 mutation carriers. 40. Adaptor protein RukL forms protein-protein complexes with endonuclease activity in HEK293 cells (poster) Kit Yu.Ya.1, Drel' V.R.1, Shuvayeva H.Yu.1, Palyvoda O.Yu.1, Kovalyova V.A.1, Mayevs'ka O.M.1, Fedyshyn Ya.Ya.1, Bobak Ya.P.1, Rzeszowska-Wolny J.4, Gout I.T.2, Buchman V.L.3, Drobot L.B.1 1 Institute of Cell Biology, National Academy of Sciences of Ukraine, Darhomanov Str., 14/16, 79005 Lviv, Ukraine Tel./Fax: (380 322) 72 06 46; e-mail: drobot@biochem.lviv.ua 2 Ludwig Institute for Cancer Research, London, UK 3 The University of Edinburgh, Edinburgh, UK 4Oncology Center, M. Sklodowska-Curie Memorial Institute, Gliwice, Poland RukL protein (regulator for ubiquitous kinase), also known as CIN85 and SETA, belongs to a subfamily of adaptor/scaffold proteins which play an integrating role in the regulation of such fundamental cellular processes and systems as survival and apoptosis, endocytosis mediated by receptor tyrosine protein kinases, and organization of the actin cytoskeleton. RukL contains three typical Src-homology 3 (SH3) domains at the N-terminus, followed by proline-rich, serine-rich regions and carboxy-terminal coiled-coil domain. Multiple modules in the RukL structure involved in protein-protein interactions can provide for formation of ligand clusters with varied properties and subcellular location. To study the nature and biological role of such complexes, the recombinant protein RukL with a Glu-epitope at the C-terminus (RukL Glu-tagged) was purified from transfected HEK293 cells by affinity chromatography on protein G-Sepharose with covalently conjugated anti-Glu-tag antibodies. By SDS polyacrylamide gel electrophoresis with subsequent staining with silver, a set of minor bands in addition to 85 kDa RukL Glu-tagged was detected in the purified preparation of the recombinant protein. Proteins with affinity for nucleic acids were also revealed in the RukL Glu-tagged preparation by retardation of electrophoretic mobility of 32P-labeled oligodeoxyribonucleotides in gel. The RukL Glu-tagged preparation was also shown to hydrolyze both deoxyribonucleotides and plasmid DNA. ZnCl2 and heparin inhibited the DNase activity. These findings suggest the presence of DNases associated with the RukL protein in HEK293 cells. Such complexes were isolated from lysates of HEK293 cells by chromatography on heparin-Sepharose. By elution with 0.5 and 1.0 M NaCl, two fractions with the DNase activity and containing proteins with molecular weights of 83, 80, and 72 kDa were obtained. The reaction was inhibited by ZnCl2 and heparin, and previous precipitation of Ruk-related proteins with anti-Ruk antibodies resulted in the exhaustion of nuclease activity. By immunoblotting with anti-Ruk antibodies, 83 kDa protein immunologically related to the RukL protein was identified in the fractions. It was concluded that the adaptor protein RukL forms complexes with endonucleases in HEK293 cells.
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