1. THE RESULTS OF CLINICOGENEALOGIC AND MOLECULAR GENETIC INVESTIGATIONS OF OVARIAN CANCER PATIENTS

Natalia Antonenkova, Iosif Zalutsky, Galya Porubova, Elena Mokhon

N.N Alexandrov Research Institute of Oncology and Medical Radiology, Minsk, Belarus

          Objectives of the study: Establishing a cohort of probands with ovarian cancer (OC), whose relatives are subject to preventive examination aimed at timely treatment.
          Materials and methods: OC patients aged 24 to 77 years, mean age 51.27±10.31. Clinicogenealogic analysis of the pedigrees of patients treated in the hospital of SI N.N. Alexandrov RIOMR identified 100 families with cancer-burdened familial history. The following mutations were determined using allele-specific PCR technique: BRCA1 (185delAG), BRCA1 (5382 insC), BRCA2 (6174delT).
          Results: The results are presented in Table 1.



          Thus, BRCA1 (185delAG) mutations were found in 29 (29%) patients of 100 BRCA1 (5382 insC) in 28 (28%), BRCA2 (6174delT) in 31 (31%). The relatives of the carriers of germinal mutations predisposing to OC and breast cancer (BC) were invited for preventive examination.
          Conclusions:
1. Activities are underway for early detection of individuals with hereditary predisposition to OC and BC, employing clinicogenealogic and molecular genetic methods of investigation.
2. The adoption of the techniques developed in clinical practice makes it possible to spare the resources needed for preventive examination of persons with high risk of inherited predisposition to OC and BC.

2. ANTITUMOR ACTIVITY OF MANNAN-METHOTREXATE CONJUGATE

Renata Budzynska¹, Dmitry Nevozhay^2,3, Urszula Kanska¹, Monika Jagiello¹, Adam Opolski^2,4, Joanna Wietrzyk², Janusz Boratynski<sup>1,4

1</sup>Laboratory of Biomedical Chemistry,
²Laboratory of Experimental Anticancer Therapy,
²Department of Experimental Oncology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland,
³Kopvillem Institute of Medical Physics, Kirov St, 64, 690068 Vladivostok, Russia; 4J. Dlugosz Academy, Al. Armii Krajowej 13/15, 42-201 Czestochowa, Poland

          Conjugation of anticancer drugs with different carriers has been extensively studied recently as a method of obtaining of improved drug forms. The conjugation often results in increased therapeutic effect, alteration of toxicity profile, and/or selective targeting of therapeutic agent to the tissue of interest. We have synthesized mannan-methotrexate conjugate using methotrexate anhydride and compared its antitumor properties both in vitro and in vivo with free methotrexate. Mannan-methotrexate conjugate showed significantly improved antitumor activity in the model of P388 mouse leukemia disseminated in the peritoneal cavity, following intraperitoneal administration of the chemotherapeutic. Conversely, the antitumor effects of free methotrexate and mannan-methotrexate conjugate were comparable when leukemia was implanted subcutaneously and chemotherapy agents were administered intravenously. These results suggest that mannan-methotrexate conjugate could be further investigated as the potential therapeutic agent for intraperitoneally disseminated tumors.

3. CELL ADHESION PROTEINS EXPRESSION IN PAPILLARY THYROID CANCER

Mykola Chekan¹, Barbara Nikiel², Wojciech Wierzchowski³, Dariusz Lange², Barbara Jarzab¹, Jerzy Stachura<sup>3

1</sup>Dept. of Nuclear Medicine and Endocrine Oncology;
²Dept of Tumor Pathology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland
³Department of Pathomorphology, Jagiellonian University, Krakow, Poland

          Introduction: Many cell adhesion molecules are up-regulated in papillary thyroid cancer (PTC). In our previous microarray studies we have specified several of them (Jarzab et al. 2005), with strong diagnostic accuracy for galectin-3. The aim of the present study was to verify the differences in expression of a panel of cell adhesion proteins by tissue microarray approach.
          Material and methods: Tissue microarray paraffin block with 101 spots (0.6 mm ) of PTC and surrounding normal thyroid tissue (ST) was prepared from 23 patients diagnosed with PTC and operated. EnVision (biotin-free), a highly-sensitive immunochemical technique was applied. 10 spots were not evaluated because of the connective tissue content, the remaining were scored for expression of galectin-3 (LGAL3), MUC1, cadherin P (CDH3), CD44v6 and carbohydrate antigen CA50. Also, the presence of endogenous biotin was visualised by LSAB method. Heterogeneity of reaction intensity was evaluated for all antigens.
          Results. The significant difference between PTC and ST was noted for LGAL3 (100%, intensive reactions in PTC versus 17%, weak in ST ), CA50 (96.4% versus 37.9%), less for CD44v6 (100% versus 69.2%, weak) while CDH3 and MUC1 showed only weak or no difference. High amounts of endogenous biotin were revealed in PTC by labelled streptavidin-biotin without primary antibody.
          Conclusions. Our tissue microarray study does not confirm differences, on the protein level, in expression of MUC1 and cadherin P genes, suggested by clear differences on RNA level, found using DNA microarrays. Simultaneously, our study indicates high expression of carbohydrate CA50 antigen, as well as high content of endogenous biotin in PTC cells, in addition to the well-known overexpression of galectin-3.

Supported by Ministry of Science and Higher Education grant no 2P05A 0 22 30

Mykola Chekan was a fellow of a Fellowship Program totally supported by the National Cancer Institute - Office of International Affairs, NIH, Bethesda, MD, USA.

4. GPR40 & GPR120 RECEPTORS - POTENTIAL TARGETS FOR NEW TREATMENT OPTIONS IN GI TRACT

Adam Cygankiewicz^1,2, Birgitte Holst² Wanda M. Krajewska¹,Thue W. Schwartz<sup>2,3

1</sup>Department of Cytobiochemistry, University of Lodz, Lodz, Poland;
²Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
³ - 7TM Pharma A/S, Hørsholm, Denmark e-mail: adamco@toya.net.pl

          7TM (7 trans-membrane) receptors constitute one of the greatest family in human genome. Many of them create new putative drug targets, hence understanding mechanisms underlying ligand-induced activation, constitutive activity and pharmacological features of these receptors may provide new insights into pathogenesis and novel treatment options for diseases like diabetes, obesity, hypertension and cancer.
          Vast number of 7TM receptors (and among them GPR40 and GPR120) are expressed in the gastrointestinal tract and in the endocrine pancreas and are believed to be involved in the control of the secretion of, for example, insulin and glucagons-like peptide-1.
          Free fatty acids are suggested as natural ligands for both studied receptors. Our investigations have demonstrated that these receptors are also activated in dose-dependent manner by rosiglitazone and other thiazolidinedione drugs as well as other compounds belonging to PPAR activators group.
          In our studies we have also confirmed certain level of constitutive activity of both receptors. More in-depth knowledge about pharmacological features of these receptors may contribute to development of more efficient treatment for hyperglycemias. Alteration in constitutive signaling of 7TM receptors may be involved in other diseases as well.

5. PHOSPHOROTHIOATE ANALOGS OF NUCLEOTIDES ACCELERATE WOUND HEALING

Edyta Gendaszewska-Darmach, Marek Kołodziejczyk, Alina Krystynowicz, Stanisław Bielecki, Maria Koziołkiewicz

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Łódź, Stefanowskiego 4/10, 90-924 Lodz, Poland

          Biomaterials which are carbohydrate polymers (among them bacterial cellulose) have proved in recent years to be promising for many medical applications, in particular for wound healing and tissue regeneration. However, the existing biomaterials are not able to stimulate cell proliferation and migration - processes necessary to achieve formation of new blood vessels (angiogenesis).
          Therefore, there exists the need to evaluate biomaterials with immobilized growth factors that are subsequently released into the wound bed over a period of 2-3 days and stimulate these processes. Our in vitro experiments have shown that phosphorothioate analogs of nucleotides (containing a sulfur atom instead of one of the oxygen atoms at the phosphate group) stimulated the proliferation of human umbilical vein endothelial cells (HUVECs). The highest stimulation of this process was caused by thymidine-5'-thiophosphate (TMPS) (Koziołkiewicz et al., 2001). This effect is associated with higher affinity of the nucleotide to specific P2 receptor and resistance of this compound to the action of nucleolytic enzymes. TMPS was immobilized to microbial cellulose produced at the Institute of Technical Biochemistry and such a wound dressing was tested in preliminary in vivo experiments. These experiments have shown the formation of new blood vessels network after application of the biomaterial and proved its wound-healing potential.

References:
1. Koziolkiewicz M, Gendaszewska E, Maszewska M, Stein CA and Stec WJ (2001) The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5'-nucleotidase. Blood 98:995-1002

6. ROLE OF DAMAGE SPECIFIC DNA POLYMERASES IN MUTAGENICITY OF LIPID PEROXIDATION-INDUCED DNA ADDUCTS

Janowska B., Kurpios D., Komisarski M., Kuśmierek J. T., Tudek B.

Department of Biological Chemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

          Lipid peroxidation leads to the formation of a large family of alkenals, which form cyclic propano- or ethenoadducts to DNA bases bearing side chains of different length. One of the most ubiquitous derivative, trans-4-hydroxy-2-nonenal (HNE) reacts with all four DNA bases with different efficiency (G>C>A>T) forming DNA adducts, as well as DNA-DNA and DNA-protein cross-links. HNE-DNA adducts block DNA synthesis and, when present in bacteria in ssDNA, cause recombination. Removal of these lesions from dsDNA occurs via the Nucleotide Excision Repair (NER) pathway. An equally important mechanism is translesion DNA synthesis. In E.coli, dysfunction of DNA polymerase IV in uvrA mutant dramatically decreased the survival, but had no effect on mutation frequency in comparison to the wild type E.coli. In the strain lacking uvrABC exonuclease and DNA polymerase V, the survival was similar to that of uvrA single mutant, but no HNE-induced mutations were observed. In uvr^- strains which possessed pol II alone or together with pol IV (uvrAdinBumuDC and uvrAumuDC respectively), mutation frequency was lower than in the wild type JM105. In uvrAdinpolB triple mutant, survival decreased dramatically, and mutation frequency increased substantially in comparison to the wild type strain. These results suggest that in E.coli, HNE adducts to DNA bases are processed by damage-specific DNA polymerases, of which DNA polymerase II and IV are able to bypass the adducts in an error-free manner. Lack of DNA pol IV results in the same sensitivity to HNE as the lack of nucleotide excision repair. DNA polymerase V is responsible for mutation induction on HNE-damaged templates. In the strains which possessed pol V, but not II and IV (JM dinBpolB, uvrdinBpolB) half of mutations were 54-nucleotide deletions of the phage polylinker region. Remaining 25% of mutations in uvrdinBpolB strain were double deletions of 54 and 93 (ΔM15) nucleotides resulting from recombination between the sequences of M13 and bacterial F' lacZ genes. These data suggest that HNE-DNA adducts constitute a substantial hindrance for Pol V, which either bypasses these lesions in an error-prone manner or falls off to enable recombination.

7. AN EFFICIENT METHOD FOR THE SYNTHESIS OF ¹⁵N-LABELLED NUCLEOSIDES AND THEIR DERIVATIVES WITH THE USE OF BACTERIAL CULTURE

B.Kalinowski, E.Zarakowska, D.Gackowski

Department of Clinical Biochemistry, Collegium Medicum UMK, Bydgoszcz, Poland

          Isotopically labelled nucleosides and their derivatives that show biological properties are important in many analytical investigations. Accordingly, there is an increasing demand for new and more effective methods of labelled nucleoside synthesis which can be analysed by nuclear magnetic spectroscopy or mass spectrometry.
          In the presented study a method for synthesising ¹⁵N-labelled nucleosides with the help of E.coli cells was used. The bacteria were grown in the ¹⁵N-labelled culture medium (Silantes OD2). Nucleic acids were isolated from bacteria, digested with nuclease P1 and alkaline phosphatase to nucleosides. The hydrolysate was separated by high performance liquid chromatography using a C18 reverse-phase column (Phenomenex luna, size 10 mm x 250 mm). Nucleosides were separated with the help of gradient elution (1.5% acetic acid and methanol). The purified fractions of individual nucleosides were received. The isotopic purity of compounds determined by mass spectrometry was: 99.2% for deoxyadenosine (dA) and deoxythymidine (dT), 97% for deoxycytidine (dC) and deoxyguanosine (dG) and >98% for adenosine (A), uridine (U), guanosine (G) and cytidine (C). Next, labeled dG and G were chemically modified. In the system generating %FFOH radical (¹⁵N₅)-7,8-dihydro-8-oxo-2'-deoxyguanosine ((¹⁵N₅)-8-oxodG) and (¹⁵N₅)-7,8-dihydro-8-oxo-guanosine ((¹⁵N₅)-8-oxoG) were obtained. Also (¹⁵N₄)-deoxyoxanosine ((¹⁵N₄)-dOxo) (¹⁵N₄)-deoxyxanthosine ((¹⁵N₄)-dXao), (¹⁵N₄)-oxanosine ((¹⁵N₄)-Oxo), (¹⁵N₄)-xanthosine ((¹⁵N₄)-Xao) were obtained in the reaction with NO radical.
          The presented method is highly efficient. Collected nucleoside analogues have a high isotopic purity and can have potential use in investigations of processes taking place in living cells.

8. CLINICAL SIGNIFICANCE OF EWS/FLI1 AND EWS/ERG GENE FUSIONS EXPRESSED IN EWING'S FAMILY TUMORS

L. Kisialeu¹, N.V. Lipay<sup>2

1</sup>Department for Elder Children
²Molecular Biology Department, Belarussian Center for Pediatric Oncology and Hematology, pos. Lesnoe, Minsk region, Belarus

          Ewing's sarcoma family of tumors (EFTs) which includes Ewing's sarcoma and peripheral neuroectodermal tumors, has specific chromosomal translocations that result in the fusion of EWS gene and a gene encoding a member of the ETS family of transcription factors, thus forming different chimeric transcription factors, either EWS/FLI1 (90-95%) or EWS/ERG (5-10%). The latter ones may alter in its transactivating capacity. The existence of these alternative fusion genes may provide a basis for a clinical heterogeneity of EFTs.
So we investigated whether the two alternative gene fusion products, EWS/FLI1 and EWS/ERG, define different clinical outcome within EFTs.
50 patients were diagnosed between 1999-2006.
          Nested RT-PCR was performed to detect EWS/FLI1, EWS/ERG transcripts in RNA extracted from samples according to L. Montanaro et al. RNA was isolated by phenol-chloroform extraction described by Chomczynski, Sacchi.
          Molecular genetics analyses were performed for all 50 patients (84 samples). For all patients RT-PCR results were in accordance with histological analysis.
          Event/Relapse-Free-Survival (EFS or RFS) was estimated by Kaplan and Meier and comparison of life-table outcome were performed with log-rank test. The median follow-up duration was calculated from the time of diagnosis for the patients who are event /relapse-free survivors.
          EFT-specific transcripts were found in 40 of 50 patients (80%). For comparison we divided all positive patients into 2 groups according to the fusion gene:
Group 1- patients with EWS/ERG transcripts (10/40 or 25%)
Group 2- patients with EWS/FLI1 transcripts (30/40 or 75%)
          The 5-year EFS in EWS/ERG-group was 37% (median follow-up 22 months), and was not statistically decreased in comparison with EWS/FLI-group (55% with median follow-up 18 months).
          40 patients were next divided in two groups according to high risk (24/40) or standard risk protocol (16/40) and EFS rates were estimated separately for patients in different risk groups with different fusion transcripts.
          High risk: 18/24 patients had EWS/FLI1 transcripts (75%) and 6/24 patients had EWS/ERG transcripts (25%);
          Standard risk: 12/16 patients had EWS/FLI1 transcripts (75%) and 4/16 had EWS/ERG transcripts (25%).
          The 5-year EFS was 33% in EWS/ERG-positive patients with high risk (median follow-up 18 months) in comparison with 55% (median follow-up 18 months) in EWS/FLI-positive patients with high risk. This trend, though, was non-significant.
          Among patients with standard risk 4-year EFS was 38% (median follow-up 51 months) for EWS/ERG group and 67% (median follow-up 17 months) for EWS/FLI1 group. Again, this difference was non-significant.
          There were no significant clinical differences observed between the two groups in event-free survival, or progression-free survival. At the given stage there are no significant proofs of influence of two alternative genes on a clinical outcome of pediatric patients with Ewingxs sarcoma family of tumors. This problem may, however, require further studies.

9. DETECTION OF THE SPECIFIC FUSION GENES WHICH IS CHARACTERIZED EWING'S FAMILY TUMORS FOR PEDIATRIC PATIENTS.

L. Kisialeu¹, N.V. Lipay<sup>2

1</sup>Department for Elder Children;
²Molecular Biology Department, Belarussian Center for Pediatric Oncology and Hematology, pos. Lesnoe, Minsk region, Belarus

          Ewing's sarcoma family of tumors (EFTs) are primitive malignant tumors of bone and soft tissues arising preferentially in children and young adults. EFTs show an extremely aggressive behavior and rapidly disseminates to bones, bone marrow, and lungs. Ewing's tumors are characterized in at least 95% of cases by specific chromosomal rearrangments t(11;22)(q24;q12) and t(21;22)(q22;q12) which corresponding to specific fusion transcripts EWS/FLI1 and EWS/ERG, respectively.
          The aim of our study was to evaluate the clinical significance of the EWS/FLI1, EWS/ERG fusion genes detection by RT-PCR method for diagnostic use in children.
          Nested RT-PCR was performed to detect EWS/FLI1, EWS/ERG transcripts in RNA extracted from samples according to L. Montanaro et al. RNA was isolated by phenol-chloroform extraction, as described by Chomczynski and Sacchi.
          During a 5-year period (2000-2005) we examined 77 samples from 52 patients:
          30 samples- tumor fragments from primary sites; 1 sample-affected lymph node; 28 samples-bone marrow (BM) aspirates; 17 samples-peripheral blood (PB).
          In case of 10 (19%) patients, only BM or PB samples were examined as the materials from primary sites biopsy was not available. All these samples were negative for EFT-specific transcripts.
          For 32 (64%) patients histological analysis was in agreement with RT-PCR results (EFT-specific transcript was detected in at least one sample).
          In case of 10 patients (19%) RT-PCR results influenced primary diagnosis:
          For 1 patient (1.9%) primary diagnosis, osteosarcoma, was substituted for Ewing's tumor (tumor fragments was EWS/FLI1 positive).
          For 1 patient (1.9%) primary diagnosis of EFT has been excluded as EFT-specific transcripts were not detected in primary site biopsy. In this case the rabdomyosarcoma was diagnosed after secondary histological analysis.
          For 2 patients (3.8%) with verified EFT, II stage was replaced by IV stage as the EFT-specific transcripts were detected in BM samples, though morphological analysis did not reveal tumor cells in BM.
          For 6 patients (11.4%), in which histological analysis was insufficient, the presence of the EFT chimeric oncogenes in biological samples allows diagnosing EFT and to start therapy of patients in a grave condition immediately.
          Overall, RT-PCR is a sensitive method for detection of the ET-specific transcripts and it is a relevant tool that allows gathering more accurate data concerning Ewing's family of tumors. Thus, RT-PCR is the essential part of a diagnostic scheme, together with other diagnostic procedures.

10. DAMAGING EFFECT OF SCATTERED RADIATION UPON CELLS IRRADIATED AT DIFFERENT DEPTHS IN A WATER PHANTOM

Maria Konopacka¹, Jacek Rogolinski¹, Aleksander Sochanik², Krzysztof Ślosarek<sup>3

1</sup>Department of Experimental and Clinical Radiobiology
²Department of Molecular Biology
³Radiotherapy and Brachytherapy Treatment Planning Department Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch,Poland

          Radiation generated by linear electron accelerators used in cancer radiotherapy is non-monoenergetic. Energy broadening increases with penetration depth in the irradiated medium such as water. With greater penetration depth the number of interactions with the surroundings also increases. Part of radiation becomes scattered and the fraction of scattered radiation having decreased energy becomes larger at increased depth. A larger portion of scattered radiation, with energy lower than that of the incident beam, should change the biological response of irradiated cells.
          The purpose of this study was to assess the damaging effect of scattered radiation, generated during penetration of medium, upon cultured cells placed inside a water phantom.
          Measurements were performed at different depths (3 - 20 cm) of a water phantom using BEAS-2B (human bronchial epithelial) cell line. Electron radiation (22 MeV) was employed, with radiation dose of 5Gy in build-up (3 cm) depth and two dose rates: 100 or 600 MU/min. Cytogenetic changes in cells irradiated at different depths were assessed and expressed as the frequency of cells with formed micronuclei as well as apoptotic cells. We compared the obtained results with expected data, i.e. those corresponding to doses received at each depth.
          Our results show that, with increasing medium depth, the number of micronucleated as well as apoptotic cells was greater than that which should result from the corresponding dose received. This discrepancy between the observed and expected number of damaged cells becomes greater with increased medium depth and may be caused by the increased fraction of scattered radiation.
          The observed relationship ought to be taken into consideration in both clinical practice and in treatment planning.

This study was supported by MSC Memorial Cancer Center and Institute of Oncology, Branch in Gliwice, Internal Grant No. GW 0307

11. PERFORMANCE OF CYTOGENETIC TECHNIQUES IN IDENTIFYING DNA COPY NUMBER CHANGES

Kostrzewska-Poczekaj M.¹, Giefing M.¹, Jarmuż M.¹, Kujawski M.¹, Martin-Subero J. I.³, Siebert R.³, Grenman R.⁴, Szyfter K.<sup>1,2

1</sup>Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland;
²Department
of Otolaryngology, University of Medical Sciences, Poznan, Poland;
³Institute of Human Genetics, University Hospital Schleswig-Holstein Campus Kiel, Germany;
⁴Department of Otorhinolaryngology - Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, Finland

          The cell line UT-SCC-11, derived from squamous cell carcinoma of the larynx and established at the University of Turku, Finland was analyzed by banding (GTG, QFQ), comparative genomic hybridization (CGH) and the novel, high resolution array-CGH techniques. The aim of the study was to compare the performance of the three applied techniques (banding, CGH and array-CGH) in identifying DNA copy number changes (excluding translocation and inversion).
           Altogether, 21 chromosomal abnormalities have been detected by at least one of the used techniques. Separately, banding detected 12 abnormalities; CGH detected 7 abnormalities and array-CGH detected 18 abnormalities. The highest concordance was observed between banding and array CGH that detected 9/21 (43%) abnormalities. Banding technique presented higher sensitivity than CGH, but identified 2 additional DNA copy number changes that were not confirmed by any of the two other techniques, thus being probably false positives. In contrast, CGH delineated only 6 regions of analogy with array-CGH regions but with only one false positive.
          In conclusion, the best performance was achieved by array-CGH which detected additional 8 aberrations of average size: 25 Mb (2.8-57 Mb) not detected by any of the two other techniques.
          Chosen results from banding, CGH and array-CGH are shown as examples:

1. large 12.6 Mb deletion in 22q was detected by both banding and array-CGH besides classical CGH, but array-CGH detected a small additional 3.5 Mb amplification on chromosome 22,

2. small deletion ca. 4.8 Mb in chromosome 12p is not detectable by binding and classical CGH but is clearly detectable by array-CGH,

Due to low resolution of banding and CGH, array-CGH seems to be the most suitable technique to delineate small deleted and amplified chromosomal regions.

12. VALIDATION OF POTENTIAL MOLECULAR MARKERS OF PAPILLARY THYROID CARCINOMA BY QUANTITATIVE REAL-TIME PCR

Monika Kowal¹, Aleksandra Kukulska¹, Aleksandra Rusin², Małgorzata Kowalska¹, Ewa Chmielik³, Ewa Stobiecka³, Elżbieta Gubała¹, Agnieszka Czarniecka⁴, Jan Włoch⁴, Tomasz Tyszkiewicz¹, Barbara Jarząb<sup>1

1</sup>Department of Nuclear Medicine and Endocrine Oncology;
²Department of Tumor Biology;
³Department of Tumor Pathology;
⁴Oncology Surgery Clinic; Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej ¹⁵, Poland

          Introduction: In our previous DNA microarray profiling studies we specified a multigene classifier of papillary thyroid carcinoma, which included some novel transcripts, not analyzed until now in the context of PTC biology. The aim of this study was to validate them by quantitative real-time PCR on an independent set of PTC samples. We included such new genes as EVA1, CDH3, KCJN2, LRP4, Q-PCT GALE (up-regulated) and HGD, TACSTD2 (down-regulated) and compared them to the well known PTC markers, which were confirmed by our microarray analysis (DPP4, FN1, KRT19, MET, RXRG, ADORA1, up-regulated, and TFF3, down-regulated).
          Material and methods: Total RNA was isolated from 31 paired normal and PTC samples by Chomczynski-Sacchi method. 0.5 µg of RNA was used in the reverse transcription reaction. cDNA was further used in Q-PCR reaction. The expression was measured by Universal Probe Library LNA probes (Roche) and was normalized by the index obtained from 3 reference genes (UBE2D2, HADHA, EIF3S10) by GeNorm software.
          Results: All analyzed genes, except TACSTD2, differentiated tumor and normal samples in a highly significant manner. When the diagnostic efficiency of these genes was assessed by ROC (relative operating characteristic) analysis, we found out that for overexpressed markers, the area under ROC curve (AUC) was higher than 0.9 for all of them and for down-regulated genes this criterion was met for TFF3 and HGD. We trained the multigene classifier on the obtained data and currently we are performing its validation using an independent set of samples.
          Conclusions: Our results are a next step in validating PTC markers before the required verification can be made on routine diagnostic material obtained by fine needle biopsy of benign and malignant thyroid nodules.

Study supported by the Ministry of Science and Higher Education, Poland, Grant No. PBZ-MNiI-2/1/2005

13. INFLAMMATION INCREASES OXIDATIVE DNA DAMAGE REPAIR AND STIMULATES PRENEOPLASTIC CHANGES IN COLONS OF NEWBORN RATS

Paweł Kowalczyk^1,5, Jolanta Jaworek², Michalina Kot², Beata Sokołowska³, Aleksandra Bieleń⁴, Beata Janowska¹, Jarosław M. Cieśla¹, Grzegorz Szparecki¹, Benita Sadoś¹, Barbara Tudek<sup>1,5

1</sup>Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland;
²Department of Physiology, Jagiellonian University College of Medicine, Cracow, Poland; ³Department of Respiratory Research, Medical Research Center, Polish Academy of Sciences, Warsaw, Poland
⁴Cancer Research UK, Clare Hall Laboratories, Blanche Lane, South Mimms, EN6 3PD, United Kingdom; ⁵Institute of Genetics and Biotechnology, Warsaw University, Warsaw, Poland

           Oxidative DNA damage may be a risk factor for development of various pathologies, including malignancy. We studied inflammation triggered modulation of repair activity in the intestines of three-week-old rats injected i.p. with E.coli or S.typhimurium lipoplysacharides (LPS) at doses of 1, 5 or 10 mg/kg. Subsequent formation in these animals of colonic preneoplastic lesions, aberrant crypt foci (ACF) was also investigated. Five days after LPS administration no differences were observed in repair rate of 1,N6-ethenoadenine (εA), 3,N₄-ethenocytosine (εC) and 8-oxoguanine (8-oxoG) in intestines of these rats, as measured by the nicking assay. However, a significant increase in all three repair activities was found within one and two months after S.typhimurium LPS treatment. E.coli LPS significantly increased only the 8-oxoG repair. S.typhimurium LPS stimulated mRNA transcription of pro-inflammatory proteins, lipooxygenase-12 and cyclooxygenase-2, as well as some DNA repair enzymes like AP-endonuclease (Ape1) and εC-glycosylase (Tdg). mRNA level of DNA glycosylases excising εA (Mpg) and 8-oxoG (Ogg1) was also increased by LPS treatment, but only at the highest dose. Transcription of all enzymes increased for up to 30 days after LPS, and subsequently decreased, with the exception of Ape1, which remained elevated even two months after LPS administration. Thus, the repair efficiency of εA, εC and 8-oxoG depends on the availability of Ape1, which increases Ogg1 and Tdg turnover on damaged DNA, as well as presumably stimulates Mpg.
          One and two months after administration of E.coli or S.typhimurium LPS, the number of aberrant crypt foci in rat colons increased in a dose- and time-dependent manner. Thus, inflammation stimulates the repair capacity for εA, εC and 8-oxoG, but simultaneously triggers the appearance of preneoplastic changes in the colon. This may be due to increased oxidative stress and imbalance in DNA repair.

14. MATHEMATICAL DESCRIPTION OF WOUND HEALING ASSAY BY HYPERBOLIC DIFFUSION

Monika Krasowska¹, Krzysztof Małysiak¹, I. Ozerlat², M. Mycielska², Zbigniew J. Grzywna¹, Mustafa B.A. Djamgoz<sup>2

1</sup>Silesian University of Technology Faculty of Chemistry, Gliwice, Poland;
²Division of Cell Molecular Biology, Imperial College, London, UK

          Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of a cell migration is of common interest for biologists as well as for clinicians. The wound healing assay is simple, inexpensive, and one of the earliest- developed methods to study directional cell migration in vitro. The basic steps involve creating a "wound" in a cellular monolayer, capturing the images at the beginning and at regular intervals during cells' migration to close the wound. The above process was modeled in the presented study by symmetric sorption and described by the hyperbolic diffusion equation. We have compared this model with experimental data and show quite a good agreement.

15. SIMULTANEOUS DETECTION OF GENE MUTATIONS IN PEDIATRIC PATIENTS WITH ACUTE MYELOBLASTIC LEUKEMIA

Anatoli Kustanovich, Maya Kryuko, Alexandr Mihas, Tatsyana Savitskaya

BelarusianResearch Center for Pediatric Oncology and Hematology, P/O Lesnoe, Minsk region, 223040, Belarus

           Number of clinically relevant genetic abnormalities detected in acute leukemia increased recently as a result of screening using sequencing, microarrays etc. They include mutations of c-KIT, NPM1, CEBPa, FLT3-ITD, aberrant expression of WT1, BAALC. Modern treatment protocols are taking into account simultaneous detection of mutations, as they can influence treatment outcome. We report here preliminary results of mutational analysis of NPM1, CEBPa, FLT3-ITD and WT1.
          Sequencing of DNA samples for detection of mutations was performed using Genetic Analyzer 3130 (Applied Biosystems, USA).
          Presence of NPM1 gene mutations was evaluated in 15 patients. Mutation was detected in 1 patient (6.6% of observed cases). This frequency corresponds to that found in children (6.5%, Cazzaniga G. et al, 2005) and is lower comparing with one reported for adults (1/3 of AML, Suzuki T et al, 2005). Patient was 8-years-old, his blast cells did not express CD34 and most of them had normal karyotype - features that are typical for patients harboring NPM1 mutation in their blasts. Mutant allele had heterozygous insertion of TCTG nucleotides that corresponded to the most widespread type A mutation described for NPM1. This insertion results in disappearance of tryptophan residues (W) within the nucleolar localization signal domain that leads to retention of protein in cytoplasm (Chen W et al, 2006). Mutational analysis of CEBPa in this patient did not reveal any alterations, while FLT3 gene characterized by homozygous internal tandem duplication. WT1 gene also harbored mutation that included duplication of intron-exon junction before 6^th exon of WT1 gene and tetranucleotide insertion in 6^th exon.
          Analysis of 6 children revealed mutation of WT1 gene in another sample. It was difficult to deduce the right sequence using obtained data. That is why part of WT1 gene was cloned using TOPO TA Cloning kit (Invitrogen) and 10 clones were sequenced. Analysis showed (with help of Jude Fitzgibbon) that this patient was compound heterozygous with frameshift mutations in 7^th exon on both alleles. Mutations in CEBPa and 12 exon of NPM1 gene were not detected in blast cells from this patient. This patient was characterized also by the presence of internal tandem duplication of FLT3 gene. Sequence of allele revealed 77 bp duplication localized in 14th exon of FLT3 gene, corresponding to juxtamembrane domain of FLT3 protein.
          In this study analysis of WT1, NPM1, FLT3 and CEBPa mutations was performed. Frequency of NPM1 mutations in children seems to be lower compared with adults. We did not detect any patient with CEBPa alteration. Mutation of WT1 was found in 33% of the analyzed patients while frequency of FLT3-ITD corresponded to that reported by other authors. Finding of multiple allele variants (>3) of gene can point out to the presence of several subclones of tumor cells. At least 2 patients who were studied for the presence of point mutations had 2-3 simultaneous aberrations that potentially can influence disease outcome and treatment response.

16. THE RESPONSE TO IONIZING RADIATION IN COLON CANCER CELLS DIFFERING IN P53 STATUS

A. Lalik¹, M. Skonieczna², S. Student¹ and J. Rzeszowska-Wolny<sup>1,3

1</sup>Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland;
²Institute of Computer Science, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland;
³Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch in Gliwice, Wybrzeże Armii Krajowej 15, 44-100 Gliwice, Poland

          Colon cancer is the most common type of malignant disease in Europe. In the first decade of the 21^st century 10% of all new cancer cases were cancers of the colon, and in more than half of them the p53 gene coding for a tumour suppressor was mutated. The inactivation of the TP53 protein correlated with aggressive tumor cell phenotypes and with uncontrolled cell proliferation. The protein TP53 is also one of the important factors in the cellular response to DNA damage [1-3].
          The aim of our work was to study the possible influence of the presence of TP53 on the response of colon cells to ionizing radiation. Studies were performed on wild-type and p53 knocked-out HCT 116 cultured cells that originated from colon cancer. The cells were irradiated with 2 or 4 Gy and the DNA damage and repair induced by ionizing radiation were assessed by the micronucleus and comet assays. In the wild-type cells the level of p53 at different time points after irradiation was additionally analyzed by Western blotting.
          The p53 wild type and knocked-out cell lines differed in the nuclear division index and in the level of binucleated cells in the control cultures. The DNA breaks induced by ionizing radiation and kinetics of their rejoining showed also significant differences between cell lines differing in p53 status. The p53 wild-type and knocked-out cells did not however differ in the frequency of micronucleated cells. Our results suggest that TP53 may influence directly the DNA strand break rejoining as well as the proliferation rate of irradiated cells.

References:
1. Didkowska J., Wojciechowska U., Tarkowski W., Zatoński W.A. "Nowotwory złośliwe w Polsce w 2000 roku." Centrum Onkologii, Warszawa 2003
2. Kaeser M. D., Pebernard S., Iggo R. D. (2004) Regulation of p53 Stability and Function in HCT116 Colon Cancer Cells. J. Biol. Chem., 279, 7598-7606
3. Hansen R, Oren M. (1997) p53: From inductive signal to cellular effect. Curr Opin Genet Dev, 7, 46-51.

This study was supported by the Ministry of Science and Higher Education, Poland, Grant No. PBZ-MNiI 2/1/2005

17. RADIATION-INDUCED DNA DAMAGE AND REPAIR IN PERIPHERAL BLOOD LYMPHOCYTES FROM BREAST AND GYNECOLOGICAL CANCER PATIENTS AND THEIR CORRELATION WITH REACTIONS TO RADIOTHERAPY

Lisowska, H.¹, Lankoff, A.¹, Węgierek, A.², Wieczorek, A.², Florek, A.², Kuszewski T.², Góźdź, S.², Wójcik, A.<sup>1,3

1</sup>Department of Radiobiology and Immunology, Institute of Biology, Saint Cross Academy, Kielce, Poland;
²Holy Cross Cancer Center, Kielce, Poland;
³Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, Warsaw, Poland

          Patients treated with identical radiotherapy schedules show a substantial variation in the degree of normal tissue reactions. The identification of radiosensitive patients before therapy would allow optimizing them. In addition, there are data suggesting that the sensitivity to ionising radiation of peripheral blood lymphocytes of cancer patients is higher than that of healthy donors. This effect is especially prominent when chromosomal aberrations induced in G₂ phase of the cell cycle are analysed. The aim of our study was to investigate if the G_2- aberration frequencies in lymphocytes of patients with breast and gynecological cancer are higher than in the case of healthy individuals and whether the in vitro radiosensitivity of lymphocytes derived from a blood sample predicts the effects after radiotherapy. Also, we tested if the kinetics of DNA repair in peripheral blood lymphocytes of breast cancer patients is different from healthy donors
          Peripheral blood of 25 breast and 25 gynecological cancer patients was collected before the onset of radiotherapy, cultured and irradiated with Co-60 after 69 hours of culture time. Chromosome specimens were prepared from cells fixed at 72 hours of culture time. Colcemid was added for 2 hours before harvest. Lymphocytes of 20 healthy donors were cultured and irradiated in the same way like in the case of breast cancer patients. The kinetic of DNA repair was estimated by the alkaline comet assay after 0, 15, 30, 60 and 120 minutes post exposure to radiation.
          The aberration frequencies in lymphocytes of breast cancer patients were on average higher than in the case of healthy donors. There was no difference between aberration frequencies with respect to the gynecological cancer patients. This result suggests, that the radiation sensitivity of lymphocytes of patients might be a marker of breast cancer predisposition, but not gynecological one.
          The capacity of DNA repair in peripheral blood lymphocytes from healthy donors was better then in lymphocytes from breast cancer patients . No significant correlation was observed between the frequency of chromosome aberrations and capacity of DNA repair.
          No marked correlation was observed between the aberration frequency of chromosome aberrations and kinetics of DNA repair as well as degree of normal tissue reaction.

The study was supported by the Ministry of Science and Higher Education, Poland, Grant No. KBN 2 P05D 080 30.

18. BRCA1 MUTATION IN OVARIAN CANCER. MICROARRAY ANALYSIS

Katarzyna Lisowska¹, Magdalena Olbryt¹, Volha Dudaladava^1x, Michał Jarzab¹, Jolanta Kupryjańczyk<sup>2

1</sup>Department of Tumor Biology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland
²Department of Molecular Pathology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland;
^x currently: Department of Medical Biology and Genetics, Grodno State Medical University, Grodno, Belarus

          Rationale: Germline BRCA1 mutation is a major cause of hereditary ovarian cancer. It is not clear whether mutation-linked ovarian cancer has distinct clinical and pathological features. However it is apparent that mutation-bearing women develop ovarian cancer at younger age than non-carriers. It is also anticipated that BRCA1 mutation may influence tumor response to anticancer therapy based on DNA-damaging agents. Our aim was to compare gene expression profile in ovarian cancer from patients with BRCA1 mutation and in sporadic ovarian cancer. Additionally we analyzed gene expression profile in relation to other known molecular and clinical features: TP53 mutation and accumulation, tumor grade, stage, histology, response to chemotherapy, disease free and overall survival.
          Materials and methods: tumor samples were obtained from 2. In total, we analyzed 99 ovarian cancer samples: 26 patients were BRCA1 mutation carriers, 73 were non-carriers. Expression profiling was done using Affymetrix HGU133 PLUS 2.0 chips. Data were analyzed using GeneSpring 7.3.1 software, Bioconductor package (www.Bioconductor.org) and Biocarta algorithm (www.Biocarta.com).
          Results: Tumor samples from ovarian cancer patients with germline BRCA1 mutation have slight but statistically significant changes in gene expression profile as compared to tumors from non-carriers. This was proven in univariate and in multivariate analyses, as well as in the global test. Most significantly affected in tumors from BRCA1 mutation carriers were following signaling pathways (as defined by Biocarta): ATM pathway, "role of BRCA1, BRCA2 and ATR in cancer susceptibility" pathway and cyclin E destruction pathway. Significant changes were observed in gene expression profile also in relation to such features like TP53 mutation, tumor histology, grade, optimal cytoreduction and overall survival but not TP53 protein accumulation, stage and disease free survival.
          Conclusion: Our study shows that ovarian cancer in BRCA1 mutation carriers is characterized by differentially regulated signaling pathways concerned with DNA repair and cell cycle regulation as compared to sporadic ovarian cancer.

This study was partially supported by the Ministry of Science and Higher Education, Poland, Grant No. PBZ-KBN-0491P05/2003/56

19. MOLECULAR SUBTYPES OF OVARIAN CANCER? MICROARRAY ANALYSIS

Katarzyna Lisowska¹, Magdalena Olbryt¹, Michał Jarzab¹, Krzysztof Simek², Jolanta Kupryjańczyk<sup>3

1</sup>Department of Tumor Biology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, Poland
²Department of Automatic Control, Silesian University of Technology, Gliwice, Poland;
³Department of Molecular Pathology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland

          Rationale: Ovarian cancer is difficult to diagnose in early stages due to the lack of evident symptoms, lack of reliable molecular diagnostic markers and imperfect visualization methods. Thus, most cases are diagnosed at an advanced stage. Therapy of choice is than surgery and chemotherapy based on platinum compounds and taksanes. Most cases respond well to the first line chemotherapy but later develop chemoresistance that is the cause of therapeutic failure. Recently several studies have indicated molecular subtypes of different cancers that may differ by clinical course and prognosis. Most significant were studies on breast cancer, one that revealed at least two molecular subtypes with different prognosis: basal-like and luminal-like, and second that identified 70-gene prognostic signature that may help to predict the patients risk of distant metastases. In order to detect possible molecular subtypes of ovarian cancer we analyzed gene expression profile in 99 tumor samples. Unsupervised methods were applied to searched for intrinsic sources of variability in this data set.
          Material and methods: tumor samples were obtained from 3. Expression profiling was done using Affymetrix HGU133 PLUS 2.0 chips. Data were analyzed using Bioconductor package (www.Bioconductor.org) and Singular Value Decomposition algorithm developed by K. Simek.
          Results: Principal Component Analysis did not show any evident subgroups within the analyzed sample set. In SVD analysis we found that first 5 modes (supergenes) were responsible for 28,5% of total variability. First mode (92 genes) alone covered over 8% of variability in the data set. Hierarchical clustering according to the expression of those genes revealed two main clusters. One of them contained mostly clearcell and endometrioid tumors while second cluster contained mostly serous and undifferentiated tumors. Thus, we concluded that first SVD mode represents mostly the differences between histological types of ovarian cancer. Second mode encompassed 216 genes and was not related to any obvious and known feature. Interestingly, when we performed SVD only on a subset of serous and undifferentiated tumors we found that 116 genes of the first mode in this analysis were all included within 216 gene set of second mode selected in the analysis of all tumor samples. Hierarchical clustering on the basis of expression of those 116 genes divided tumors into two distinct clusters that showed strikingly different gene expression profile. This division did not correlate with any known molecular or clinical feature. Analysis of ontology groups revealed that the genes of this mode code mostly for proteins engaged in cell adhesion and communication, response to external stimuli, development, metabolism and immune response.
          Conclusion: Ovarian cancer samples of serous or undifferentiated histology segregate into two separate clusters characterized by distinct gene expression profile. Further studies are necessary to uncover the nature of these two subclasses and to describe their clinical features.

This study was partially supported by the Ministry of Science and Higher Education, Poland, Grant No. 1/0-PBZ-MNiI-2/1/2005

20. ROBUST MULTIDIMENSIONAL QSAR MODELING

Tomasz Magdziarz

Department of Organic Chemistry, Institute of Chemistry, University of Silesia, Katowice, Poland, http://uranos.cto.us.edu.pl/~zchorg

          It has become more evident nowadays that the primary objective of multidimensional methods of Quantitative Structure - Activity Relationships is the illustration of the molecular basis of the investigated activity rather than precise activity prediction. Nonetheless, the latter objective, as well as the former, cannot be gained without reliable mathematical model having high predictive ability. Due to general ambiguity of the interactions of chemical molecules in biological systems, modeling of such systems is prone to provide highly unstable noisy data. Origins of the data noise were classified as: the data, superimposition, molecular similarity, conformational, and molecular recognition noise. We also indicated possible robust solutions that can improve modeling and predictive ability of QSAR modeling. Robust approaches, especially self-organizing mapping (SOM), stochastic model validation (SMV) and modified uninformative variable elimination, coupled with partial least squares (UVE PLS), result in significant improvement of the efficiency of QSAR modeling.

21. MATHEMATICAL DESCRIPTION OF WOUND HEALING ASSAY
BY QUASILINEAR PARABOLIC DIFFUSION.

Krzysztof Małysiak¹, I. Ozerlat², M. Mycielska2, Zbigniew J. Grzywna¹, Mustafa B.A. Djamgoz<sup>2

1</sup>Silesian University of Technology Faculty of Chemistry, Gliwice, Poland
²Division of Cell Molecular Biology, Imperial College, London, UK

          Cell migration is a complex phenomenon that requires the coordination of a numerous cellular processes. It is often investigated by means of the wound healing assay that is based on the observation of scaring up of the scratched cellular monolayer. Wound healing has been given various theoretical descriptions, including the diffusional models with traveling waves. In this study we explored a diffusional model (proposed by Dale, 1995) of a wound healing in the chemoatractor concentration field coupled to the concentration field of cells. We looked for conditions under which the originally used set of coupled, quasilinear partial differential equations could be put in a simpler form that is much more suitable for the analysis and gives a new insight into the mechanism of this model. Finally, we examined the applicability of the model for the wound healing assay done using AT2 cancer cell line.

22. SYNTHESIS AND ANTICANCER ACTIVITY IN VITRO OF 4-SUBSTITUTED-2-BUTYNYLTHIOQUINOLINES

W. Mól¹, M. Matyja¹, K. Szczaurska-Nowak², M. Milczarek², J.Wietrzyk², S.Boryczka<sup>1

1</sup>Medical University of Silesia, Department of Organic Chemistry, Jagiellońska 4, Sosnowiec 41-200, Poland
²Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, Wrocław 53-114, Poland

          Acetylenic derivatives are an important class of compounds that has attracted increasing attention as a source of new anticancer agents. The synthetic methods for their preparation are of interest, especially with regard to the synthesis of enediyne antibiotics and their analogues^1-2. We carried out recently synthesis of novel propargyl thioquinolines which exhibit significant in vitro cytotoxicity^3-4. During the present study we synthesized a series of new 3,4-disubstituted thioquinolines 1 which possess one or two 4-substituted-2-butynyl groups.




          The obtained compounds were tested for their antiproliferative activity in vitro against cancer cell lines either human (SW 707 colrectal carcinoma, CCRF/CEM leukemia, T-47D breast carcinoma) or murine (P388 leukemia, B16 melanoma). The most active compounds have the ID50 values ranging from 0.43 to 4.00 µg/ml which compare to those of the reference substance, cis-platin.

References:
1. G.B.Jones, F.S.Found, Curr. Pharm. Design., 8, 2415 (2002)
2. S. Boryczka, W.Mól, A.Jowsa, Wiad. Chem., 61, 275 (2007)
3. S.Boryczka, J.Wietrzyk, A.Nasulewicz, M.Pełczyńska, A.Opolski, Pharmazie, 57, 733
(2002)
4. W.Mól, A.Naczyński, S.Boryczka, J.Wietrzyk, Pharmazie, 61, 742 (2006)

This study was supported by the Ministry of Science and Higher Education, Poland, Grant No. N405 036 31/2655

23. THE ANALYSIS OF TRANSCRIPTOME OF MEDULLARY THYROID CARCINOMA: SEARCH FOR LINEAGE-SPECIFIC MARKERS

Małgorzata Oczko-Wojciechowska¹, Jan Włoch², Jadwiga Żebracka¹, Małgorzata Kowalska¹, Zbigniew Wygoda¹, Aleksandra Czarniecka², Barbara Jarząb<sup>1

1</sup>Department of Nuclear Medicine and Endocrine Oncology
²Oncology Surgery Clinic; Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland

          Introduction: The analysis of medullary thyroid carcinoma (MTC) transcriptome by gene expression profiling encounters difficulties related to the lack of comparable normal tissue. Para-follicular C cells are embedded in thyroid parenchyma and are not easily accessible to RNA isolation in quantities sufficient for analysis. To omit this difficulty we present other, bioinformatical approach to specify genes characteristic for this histotype. We compare gene expression pattern of MTC to other types of thyroid tumors and normal samples to obtain transcripts specific only for MTC and discard overall cancer-related genes and genes characteristic for normal thyroid contamination. To verify the selected genes we apply quantitative real-time PCR analysis.
          Material and methods: Microarray analysis was carried out by HG-U133A arrays (Affymetrix) in 40 MTC samples: 20 tumors and 20 corresponding normal tissue and 70 different thyroid tumor and normal samples. An independent set of 17 MTC with paired normal tissues and 17 PTC samples was used for QPCR validation.
          Results. Up to now, 8 genes were analyzed: EEF1A2, GRP, NEFL1, SCG2, SCG3, SST, TFF1, TFF3. All analyzed transcripts were overexpressed in MTC samples, when compared to PTC and normal thyroid. The difference between PTC and MTC was statistically significant (p<0.01) for all analyzed transcripts. The significant differences in expression were also seen in comparison between MTC and corresponding normal thyroid tissue for all the genes, with the exception of TFF3.
          Conclusion: We present a gene expression signature of medullary thyroid cancer, selected by the degree of differences in RNA level, not by previous knowledge on its origin, and useful for further analysis of MTC transcriptome.

Supported by the Ministry of Science and Higher Education, Poland, Grant No. 2P05A 159 28

24. HYPOXIA-RESPONSIVE GENES IN B16(F10) MURINE MELANOMA CELLS IN VIVO - VALIDATION OF MICROARRAYS DATA

Magdalena Olbryt¹, Aleksandra Rusin¹, Katarzyna Lisowska¹, Tomasz Cichoń², Tomasz Tyszkiewicz³, Zdzisław Krawczyk<sup>1

1</sup>Department of Tumor Biology
²Department of Molecular Biology; 3Department of Nuclear Medicine and Oncological Endocrynology Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland

          Rationale: Melanoma is the most aggressive skin cancer largely refractory to existing therapies. Lack of effective therapeutic strategies, as well as proper classification systems result in high mortality rate among melanoma patients. One of the most important features of tumor microenvironment is low oxygen tension within tumor mass. Hypoxia induces aggressive phenotype in tumor cells and contributes to cancer progression. It was observed that hypoxia may influence melanocyte transformation and melanoma progression in vivo. Thus, hypoxia-regulated genes seem to be potential therapeutic targets as well as prognostic/predictive markers in melanoma.
          Aim: In our previous study we analyzed in vitro gene expression profile of B16(F10) murine melanoma cells cultured under hypoxic conditions and identified 454 hypoxia-responsive genes (Olbryt et al., 2006). The aim of this study was to investigate whether some genes from selected hypoxia signature are also regulated by hypoxia in experimental murine melanoma tumors.
          Experimental design: Hypoxic areas in B16(F10) tumors were identified immunohistochemically with pimonidazole, an exogenous marker of hypoxia. Using laser-microdissection technique the hypoxic (perinecrotic regions) as well as normoxic ones (in the vicinity of blood vessels) were isolated from frozen, 10 µm thick tumor slices. Expression of the 15 selected genes was analyzed by semiquantitative RT-PCR.
          Results: We confirmed differential expression of 12 genes between hypoxic and normoxic areas in murine melanoma tumors. Among these genes are some linked before to melanoma biology (Lgals3, Vcl, Mxi1, Nme1) as well as those not previously related to melanoma (e.g. Nppb, Selenbp1, Bnip3, Adm). Three genes that in the microarray study showed slightly changed expression (fold change < 2.5) did not reveal differential in vivo expression pattern in the studied material.
          Conclusions: The results obtained herein confirmed differential expression of majority of the analyzed genes between hypoxic and normoxic areas in murine melanoma tumors. Thus, it seems that the molecular response to hypoxia under in vitro conditions may, to some extent, reflect that observed in vivo, at least for the same cell line. To validate genes, the expression of which in microarray study was changed less significantly (fold change < 2.5), more sensitive methods (real-time quantitative RT-PCR) may be required.

The study was supported by the Ministry of Science and Higher Education, Poland, Grant No. 1/0-PBZ-MNiI-2/1/2005

25. STUDY ON THE MECHANISMS OF SELENIUM BIOTRANSFORMATION: SELENOPHOSPHATE SYNTHETASE

Yuliya Preabrazhenskaya, Andriy Moiseenok

Institute of Biochemistry and Pharmacology, Grodno, Belarus

          Selenophosphate synthetase, a key enzyme of selenium metabolic pathway, provides biosynthesis of a new (the 21st) amino acid selenocysteine from selenium and L-serine making an active donor of Se ions, selenophosphate. Products of selenophosphate synthetase (SPS) genes are supposed to be responsible for incorporation of selenium in major selenium-containing proteins. At least two of these, glutathione peroxidase and Sep15, take part in cancerogenesis through support of redox potential balance in the living cell. Selenophosphate synthetase (SPS) activates selenium and transfers it to the other proteins in the form of selenophosphate. The first step catalyzed by SPS is a limiting step in selenium metabolic pathway. Human SPS2 can complement only a mutant product of SelD gene, a SPS from E.coli. Functionally, they differ in selenium binding as SPS2 cannot bind selenium the same as C17S mutant of SPS from E.coli. However, they may be identical in ATP-binding and ATPase activity. We produced 5 truncated mutants (from C-terminus) of SPS from E.coli using pET plasmid as a template and checked them for ATP-binding ability and Mn-ATP-binding. The binding stoichiometry of ATP to SPS and the role of Mn²⁺ in binding were determined using HPLC Agilent system and radioactive-labeled ATP. The role of selenium-containing compounds as an active form of selenium capable of increasing ATPase activity of SPS from E.coli can be established. The interaction of selenium-containing substances with SPS may be one of the important mechanisms of activating enzymatic pathways of antioxidant defense, a way of active release of the microelement from depot or potential alternative (in the case of xenobiotic selenium derivatives) of accomplishing antioxidant effects.

26. GENE EXPRESSION INDUCED BY BRAF ONCOGENE MUTATION IN PAPILLARY THYROID CANCER

Dagmara Rusinek, Małgorzata Wiench, Daria Handkiewicz-Junak, Małgorzata Oczko-Wojciechowska, Małgorzata Kowalska, Grzegorz Gala, Aleksandra Pfeiffer, Barbara Jarząb

Department of Nuclear Medicine and Endocrine Oncology; Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland

          Purpose: Giordano et al. (2005) first indicated distinct differences in gene expression profile of papillary thyroid carcinoma (PTC) related to the presence of BRAF mutations and this observation was confirmed in our own analysis. To validate the differences in gene expression related to the presence of BRAF mutation we used real time QPCR.
          Methods: We performed a meta-analysis of joined sets of our 39 papillary thyroid carcinoma cases and 51 PTC cases analyzed by Giordano et al., based on Bayesian limma method. The verification of the selected genes was carried out on an independent group of PTCs by QPCR normalized to 3 control genes selected by GeNorm.
          Results: So far, 3 genes selected from the microarray analysis were validated, PGF, a placental growth factor related to VEGF; PHLDA1, an evolutionarily conserved proline-histidine rich nuclear protein showing pleckstrin homology-like domain and TM7SF4, a transmembrane molecule preferentially expressed in dendritic cells. PGF and PHLDA1 exhibited a significantly reduced expression in PTCs harbouring BRAF V600E mutation (p<0.01) and TM7SF4 showed higher expression in these PTC cases (p<0.001).
          Conclusions. The obtained results indicate different properties of BRAF-induced papillary thyroid cancers in comparison to other cases. The role of PGF is still unknown, the diminished expression of PHLDA1 may contribute to IGF-1 induced apoptosis while TM7SF4 may take part in antigen presentation by dendritic cells, thus, influence the immune response to PTC.

Supported by the Ministry of Science and Higher Education, Poland, Grant No. N401 101 32/2138

27. BYSTANDER EFFECT IN A MODEL OF COCULTURED IRRADIATED AND NON-IRRADIATED CELLS

A. Szurko^1,2, W. Przybyszewski¹, K. Szołtysek¹, Z. Maniakowski³, M. Wideł<sup>1

1</sup>Department of Experimental and Clinical Radiobiology, Maria-Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15, 44-101 Gliwice
²Department of Solid State Physics, August Chełkowski Memorial Insitute
of Physics, University of Silesia in Katowice, ul. Uniwersytecka 4, 40-007 Katowice; 3Department of Medical Physics, Maria-Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15, 44-101 Gliwice

          There is an increasing body of data corroborating the observation that biological response to ionizing radiation affects, besides directly irradiated cells, also daughter cells (genetic instability) and adjacent non-irradiated cells (bystander effect). Cells present in solid tumors or remaining in close contact under cell culture conditions communicate in various ways. One of the most important is communication via gap-junctions, which makes possible passive transport of ions and small-molecular-weight moieties between cells. Another mechanism involves release of specific mediators into the surrounding milieu by the irradiated cells, which results in inhibited proliferation, increased number of mutations, as well as number of apoptotic cells and micronuclei among non-irradiated cells.
          This report summarizes the results of study on the effect exerted by X-ray-irradiated (2 or 4 Gy, 6 MV, Clinac 600) murine Lewis lung carcinoma cells (LLC) upon non-irradiated cells of the same line. Especially attention-worthy are the results of experiments involving co-cultivated cells of both types, conducted in co-culture vessels with a filter enabling exchange of signal substances among cells but preventing mixing of the latter.
          The ratio of apoptotic cells and cells with cytogenetic damage (micronuclei) was determined microscopically. Alterations of cell-cycle were estimated using flow cytometry. Expression of NF-κB transcription factor was assessed by Western-blot technique.
          A significantly increased ratio of micronuclei was observed not only among directly irradiated cells but also among cells that were co-cultured. Also, the number of cells eliminated via apoptosis was significantly higher in both groups. No major cell cycle-affecting changes were noted following the lower irradiation dose. The observed cyclic fluctuations of NF-κB expression levels require further confirmation.

28. CHLORIN AS POTENTIAL PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY (PDT)

Agnieszka Szurkox ^1,2, Marzena Rams¹, Aleksander Sochanik², Alexeis Mikhailov², Franz-Peter Montforts^#,3, Agnieszka Kozielec³, Maria Widel², Alicja Ratuszna<sup>1

1</sup>A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland, ( xemail: agaspl@o2.pl)
²Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland;
³Institute of Organic Chemistry, University of Bremen, Bremen, Germany, (#email:mont@chemie.uni-bremen.de)

          The crucial role in photodynamic tumor therapy is played by photosensitizers, which are dyes able to accumulate selectively in rapidly growing tumor cells. After activation by exposure to light of a specific wavelength they lead to generation of reactive singlet oxygen and radical species, which trigger a sequence of photochemical and photobiological processes that damage intracellular organelles and lead to cell death. Finding a suitable photosensitizers is important for improving the efficiency of PDT. One of the most promising photosensitizer candidates are chlorins. Chlorins have one pyrrolic double bond reduced, which is concomitant with bathochromic shift of the Q-bands with higher extinction coefficients than those in the corresponding parent porphyrins. Chlorins have been reported to have high singlet oxygen efficiency coupled with 10-fold stronger absorption than HpD or porphyrins in the therapeutic window; hence they are expected to be good photosensitizers.
          Because the mode of transfer strongly influences subsequent localization of photosensitizer in cells and, consequently, determines photodynamic efficiency, we compared cell killing efficiency of chlorin delivered by liposome vehicles or dissolved in DMSO. The study was performed using Lewis lung carcinoma cells (LLC). Cellular distribution of chlorin was studied using confocal microscopy. Dark cytotoxicity and photodynamic efficiency of the explored chlorin were studied by MTS assay. We also studied the possible damaging effect of chlorin on DNA and on cytoskeleton using immunocytochemical staining of actin microfilaments followed by fluorescence microscopy.
          Our preliminary results indicate that conjugation of chlorin with liposomes is an efficient means of transferring the sensitizer into the studied cells, leading to highly efficient photosensitization, whereas non-carrier delivery (DMSO) is rather useless in such experiments. Following PDT with the chlorin photosensitizer we also observed that cytoskeleton microfilaments become shorter and DNA undergoes intense defragmentation processes.

29. THE EFFECT OF COMBINED TREATMENT ON HUMAN PROMYELOCYTIC LEUKEMIA CELL LINE WITH NOVEL ANALOGS AND COMPLEXES OF GENISTEIN AND 1,24 (OH)₂D₃ (PRI-2191)

Marta Świtalska¹, Justyna Zielska¹, Grzegorz Grynkiewicz², Andrzej Kutner², Joanna Wietrzyk<sup>1

1</sup>Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Department of Experimental Oncology, Rudolfa Weigla 12, 53-114 Wroclaw, Poland
²Pharmaceutical Research Institute, L. Rydygiera 8, 01-793 Warsaw, Poland

          Isoflavonoids play regulatory role in the expression of cytochrome P450 enzymes, and also up-regulate vitamin D₃ receptor (VDR) on cancer cells. As a result, increased sensitivity of these cells is noted to 1,25-dihydroxyvitamin D₃, a hormonally active form of vitamin D₃. Isoflavonoids are also able to raise the serum level of active form of vitamin D₃, due to their inhibitory activity on the CYP24, the enzyme involved in the degradation of 1,25-dihydroxyvitamin D₃ and its precursor 25-OH-D₃ to inactive compounds. Another enzyme, CYP27B1, involved in the synthesis of 1,25-dihydroxyvitamin D₃ is stimulated by isoflavonoids. This may result in a similar increase effect of 1,25-dihydroxyvitamin D₃ serum level. Therefore, combined treatment with isoflavonoids and 1,25-dihydroxyvitamin D₃ might be effective in both prevention and in anticancer treatment.
          In order to evaluate the effect of combined application of genistein analogs (IFG-027, IFG-043) and complexes (with schizophylan and xyloglucan) and new vitamin D analog PRI-2191: (24R)-1,24-dihydroxyvitamin D₃), against the cells of human promyelocytic leukemia HL-60, antiproliferative activity and the effects on cell cycle were determined. The synergistic antiproliferative effect was observed for all analogs and complexes used after treatment of HL-60 cell line with PRI-2191. HL-60 cells treated with analogs of genistein and PRI-2191 cumulated in G0/G1 stage. The percentage of cells in G2/M and S phase decreased after combined treatment. This effect was significantly stronger than after treatment with PRI-2191 alone.

This study was supported by the Foundation for Development of Pharmaceutical Sciences, Poland, Grant No. 9/FB/2004

30. COTRASIF: CONSERVATION-AIDED TRANSCRIPTION FACTOR BINDING SITE FINDER

Bogdan Tokovenko Maria Obolenskaya

Institute of Molecular Biology and Genetics, Kyiv, Ukraine; to.bogdan@gmail.com

          Summary: A new tool has been developed for identifying with increased specificity the putative transcription factor binding sites (TFBS) in eukaryotic gene promoters.
          Rationale: Promoter analysis and TFBS identification are essential steps for the identification of gene regulatory networks. Low specificity of the TFBS prediction in eukaryotic gene promoters is a challenging task for modern bioinformatics. Based on our previous research, we observed better specificity of the TFBS search when comparing the promoters of gene orthologs of the evolutionarily close species (e.g. rat and mouse) for the presence of the target TFBS. If the putative TFBS is present in both promoters (with an optional constrain on the similar distance from the transcription start site), then it has higher probability of being biologically meaningful.
          Results: We developed a web-accessible tool (conservation-aided transcription factor binding site finder, COTRASIF) for the conservation-aided TFBS search. It uses Ensembl genome databases, and is currently available for three organisms: human, mouse and rat. Promoters are defined as 800bp upstream from transcription start site, plus the 5' UTR. For the initial TFBS search, classical position-weight matrix approach is used. Initial search results are further analyzed using the gene orthology information available in Ensembl for the selected pair of organisms. Results are presented as a list of genes with the positions of the putative found TFBS in the promoters of both organisms, selected for analysis. Further development plans include the addition of new organisms, and integration of the Gene Ontology functional gene analysis into the results page.

Availability: http://biomed.org.ua/COTRASIF/
Supplementary information:http://biomed.org.ua/COTRASIF/help.html

31. IN VITRO STUDIES OF INDIVIDUAL RADIOSENSITIVITY RELATIONSHIPS IN CERVICAL CANCER PATIENTS BEFORE, DURING AND AFTER RADIOTHERAPY

A.Węgierek³, A. Lankoff¹, H. Lisowska¹, T. Kuszewski³, S.Góźdź³, A. Wójcik<sup>1,2

1</sup>Department of Radiobiology and Immunology, Institute of Biology, Saint Cross Academy, Kielce, Poland
²Radiobiology and Health Protection Department, Institute of Chemistry and Nuclear Technology, Warsaw, Poland
³Holy Cross Cancer Center, Kielce, Poland

          There has been considerable evidence that, even with analogous treatment, individuals differ widely in normal tissue response to radiation injury. It was suggested that this difference might result from a variation in the intrinsic cellular radiosensitivity. However, the mechanisms of such variability are not clear. The aim of the presented investigation was to examine:
a) the correlation between the frequencies of spontaneous and radiation-induced micronuclei (MN) in vitro before radiotherapy
b) the influence of radiotherapy on the frequencies of MN in vivo
c) the correlation between the frequencies of radiation-induced MN in vitro before radiotherapy and the frequencies of radiation-induced MN in vitro during and after radiotherapy.
          The study included 10 patients with cervical cancer. From each donor peripheral blood was collected before radiotherapy (0), 3 weeks after the first radiation fraction (3W) and immediately after completion of radiotherapy (TE). In addition, blood was re-irradiated in vitro with 2Gy. Differences in the individual radiosensitivity of peripheral blood lymphocytes were determined with the micronucleus assay. Lymphocyte cultures were set up by adding 0.5ml of blood to 4.5ml of RPMI 1640 medium supplemented with calf serum, phytohemaglutinin and antibiotics. Cytochalasin B was added 44 hours after the incubation. After subsequent 28 hours the cells were harvested and fixed according to the protocol of Fenech (2000).
          Individual radiosensitivity of lymphocytes before radiotherapy. Part of the blood collected before the beginning radiotherapy was used to assess the intrinsic radiosensitivity of the patients. Our results show a wide variability in the frequencies of both spontaneous and radiation-induced micronuclei in vitro. The yield of spontaneous MN ranged from 9 to 52 MN/1000 BNC. The yield of radiation-induced MN in vitro ranged from 56 to 255 MN/ 1000BNC. Statistical analysis revealed that there was no significant correlation between MN frequencies in the compared groups (p = 0.159).
          Influence of radiotherapy on the MN frequencies in vivo. The mid-treatment samples (3W) from the cervical cancer patients show a marked elevation in the MN frequency. However, a wide inter-individual variability is seen. The yield of radiotherapy-induced MN 3 weeks after the first radiation fraction ranged from 40 to 226 MN/1000 BNC. The samples collected from patients immediately upon completion of radiotherapy (TE) show an enhancement in the MN frequency, which is significantly higher than in spontaneous (0) as well as mid-treatment samples (3W), with the exception of three patients, where the MN frequency was lower as compared to the mid-treatment samples (3W). Statistical analysis revealed that there is no marked correlation between the frequency of spontaneous MN and the frequency of radiotherapy-induced MN neither 3 weeks after the first radiation fraction (p=0.193) nor immediately after completion of radiotherapy (p=0.427).
          Our results show a wide inter-individual variability in the frequency of radiation-induced MN in vitro during and after radiotherapy. The yield of radiation-induced MN in vitro in lymphocytes collected from patients 3 weeks after the first radiation fraction ranged from 123 to 448 MN/1000 BNC. The yield of radiation-induced MN in vitro in lymphocytes collected from patients upon completion of radiotherapy ranged from 64 to 452 MN/1000 BNC. Statistical analysis revealed that there is no significant correlation neither between the frequency of radiation-induced MN in vitro before radiotherapy and the frequencies of radiation-induced MN in vitro during radiotherapy (3W) (p=0.337) nor between the frequency of radiation induced MN in vitro before radiotherapy and the frequencies of radiation-induced MN in vitro upon completion of radiotherapy (p=0.328).

32. CHARACTERIZATION OF GENOMIC INSTABILITY IN EBV-INFECTED B LYMPHOCYTES

Emilia Wiechec^1,2, Sandrine Lacoste², Marie Henriksson³, George Klein3, Sabine Mai<sup>2

1</sup>Institute of Human Genetics, University of Aarhus, 8000 Aarhus C, Denmark
²Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada
³Microbiology and Tumor Biology Centre, Karolinska Institute, Stockholm, Sweden

          Background: Genomic instability is one of the earliest events found in most cancers. Epstein-Barr virus (EBV) infected human B cells display chromosomal aberrations (Kataoka H, 1997; Okubo M, 2001; Kamranvar SA, 2007). These EBV-associated chromosomal aberrations can be linked to telomere dysfunction and chromosomal missegregation.
          Purpose: The overall aim of this study was to characterize the exact post-EBV infection period leading to genomic instability in B-lymphocytes and to determine whether such changes promote tumour development in mice.
          Material and methods: The material for this study included human B-lymphocytes prior to and after EBV infection. Freshly, virally immortalized lymphoblastoid cell lines (LCLs) were followed over a six-month period post-infection, and cells were collected at specific time points. Telomeres were analyzed by fluorescent in situ hybridization (FISH) on both interphase nuclei (3D analysis) and metaphase plates (2D analysis) using the Telomere PNA FISH kit (DAKO). We then characterized the chromosomal abnormalities using spectral karyotyping (SKY).
          Results: Our preliminary results suggest that EBV infected human B cells display telomere-driven genomic instability. The LCL samples display random chromosomal instability including aneuploidy, chromosomal breaks and fusions, translocations and sister chromatid fusions.
          Work in progress: We will continue the analysis of primary human (non-EBV-infected) B cells to confirm or exclude tissue-culture-dependent formation of genomic instability that would have nothing to do with EBV-infection. Then, the EBV infected B-lymphocytes exhibiting a highest rate of genomic changes will be injected into mice to determine whether the observed instability is sufficient to promote tumour progression.

Acknowledgement: This work was supported by grant from CIHR Strategic Training Program. The authors would like to thank Katalin Benedek and Mia Lowbeer for the preparation of cells.

33. COMBINATION OF NEW PRIMER DESIGN AND HIGH RESOLUTION MELTING FOR HIGH THROUGHPUT ANALYSIS OF METHYLATION IN CLINICAL SAMPLES

Tomasz K Wojdacz^1,2, Alexander Dobrovic2^, Lise Lotte Hansen<sup>1

1</sup>Institute of Human Genetics, University of Aarhus, Denmark; ²Department of Molecular Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia

          There is an urgent need for straightforward and reliable methods to examine the extent of methylation changes in tumours. Current methods for single gene methylation determination have various limitations and pitfalls, and contradictory results can be obtained using different protocols [1]. Tumour DNA is a mixture of tumour and normal DNA with a wide range of tumour purity. The sensitivity for detection of methylated sequences should reflect this and should be sensitive to at least 5-10%. We have combined High Resolution Melting and novel primer design for amplification of bisulfite modified DNA regardless of its methylation status in studies of methylation markers. The standard guidelines for primer design to amplify locus of interest for post PCR methylation analyses (MIP, methylation independent primers) advise to avoid CpG dinucleotide in primer sequences or to replace Cs within CpGs by mismatched bases. We have shown that some CpGs in the primer sequence may be necessary; otherwise PCR bias towards unmethylated template may lead to underestimation of the degree of methylation. Moreover, by manipulating the annealing temperature of PCR amplification, we were able to control the efficiency of binding of our MIP primers to the methylated template and therefore reverse PCR bias towards the methylated allele [2]. The combination of the above primer design and a High Resolution Melting platform (HRM) allowed us unambiguously to detect methylated fractions of DNA in the samples containing as little as 0.1% methylated DNA [3]. Furthermore, Methylation Sensitive High Resolution Melting (MS-HRM) can be designed to estimate the methylation levels of screened material when HRM profiles of PCR product of unknown samples are compared with HRM profiles of PCR product derived from the standards with known methylated to unmethylated template ratio.
          MS-HRM analysis can be performed in-tube, which allows for very rapid screening and avoidance of PCR product contamination issues in laboratories. In summary, in-tube methylation analysis with HRM methodology provides a fast and high-throughput tool that has a potential to be introduced into clinical practice.

References:
1. Aggerholm A, Hokland P: DAP-kinase CpG island methylation in acute myeloid leukemia: methodology versus biology? Blood 2000, 95:2997-2999.
2. Wojdacz TK, Hansen LL: Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 2006, 41(3):274, 276, 278.
3. Wojdacz TK, Dobrovic A: Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic acids research 2007, 35(6):e41.

34. MOLECULAR GENETIC DIAGNOSTICS OF THROMBOPHILIA IN CANCER PATIENTS

Iosif Zalutsky, Alexander Mashevsky, Raisa Smolyakova, Elena Mokhon, Dmitri Kovalenko

N.N. Alexandrov Research Institute of Oncology and Medical Radiology, Minsk, Belarus

          The incidence of pulmonary embolism (PE) in cancer patients reaches 25-40%, the highest rates occurring in patients with lung cancer, large intestine cancer and female genital cancer. According to pathomorphology findings, venous thrombosis (VT) is detected in 40-60% of advanced malignant neoplasm cases, being asymptomatic in 70-80% of the patients and with no life-time diagnosis in 50% of them.
          In the past decades, major accomplishments were made in decoding the mechanisms of VT development, which is associated with the introduction of new genetic and immunological methods of investigation and with discovery of hereditary forms of thrombophilia and its molecular markers. Diagnostically significant genetic factors of VT u PE risks are 1691G/A mutations in factor V gene (FV, Leiden) and 20210G/A mutations in factor II gene (FII), in methylenetetrahydrofolate reductase gene (MTHFR C-677-T).
          The objective of the study is molecular genetic detection of mutations in the hemostasis system genes for diagnosis, prevention and treatment of thromboembolic morbidity in cancer patients.
          We used in our studies findings of clinical, instrumental and molecular genetic examinations carried out in stage I-IV cancer patients treated at SI N.N. Alexandrov RIOMR.
          All the patients underwent histological diagnosis (lung cancer, gastric cancer, esophageal cancer, colon cancer, cervical and corpus uteri cancer, bone and soft tissue sarcoma).
          Genomic DNA was extracted from whole blood of cancer patients using Genomic DNA Purification Kit. Molecular genetic examinations of factor V (Leiden) genes, factor II and MTHFR genes were conducted with allele-specific polymerase chain reaction technique using reagent kits of PRONTO, Israel.
          Analysis of the results of examinations performed in 346 cancer patients revealed mutations in the hemostasis system genes in 235 (67.9%) of them. Carrying mutation in factor V gene in the heterozygous state was diagnosed in 49 (14.2%) patients, in the homozygous state - in 21 (6.1%).
          Prothrombin gene mutation resulting in thrombophilia is a genetic defect ranking second in clinical significance. In the course of the examinations the mutation in the heterozygous state was diagnosed in 53 (22.7%) cancer patients, in the homozygous state - in 22 (9.4%). One of the most frequent causes of developing mild and moderate hyperhomocysteinemia is hereditary deficit of 5, 10- MTHFR-folate-dependent enzyme catalyzing the process of remethylation.
          Pleomorphism in MTHFR gene was detected in 174 cancer patients, with dominating heterozygous genotype in 132 (38.1%) patients and the homozygous type in 42 (12.1%) cases.
          The comprehensive molecular genetic analysis of pleomorphisms associated with hemostasis dysfunction may be used for prognostication of VT recurrence and evaluation of PE development risk. G20210A mutation of prothrombin gene was found in combination with FV Leiden mutation in 8 cancer patients, combination of mutations in FV Leiden and MTHFR genes was diagnosed in 25 cases, prothrombin gene mutation associated with MTHFR mutations was noted in 28 patients. The development of severe pulmonary embolism in the postoperative period with unfavourable outcome in 11 cancer patients was associated with concurrent detection of combined mutation in the three hemostasis system genes analysed (factor V Leiden, factor II and MTHFR).
          Thus, genetic defects of FV Leiden gene, G20210A of prothrombin and MTHFR genes were found in 67.9% of patients with malignant neoplasms. The high incidence of detecting genetic disorders in the hemostasis system by means of allele-specific polymerase chain reaction makes it possible to define the degree of PE development risk in cancer patients and to administer pathogenetically-oriented therapy.

35. ANALYSIS OF CHANGES IN GENE EXPRESSION PROFILES INDUCED BY IONIZING RADIATION IN ME45 AND K562 CELLS

²K. Pawełek, ¹R. Herok, ²Z. Grzywna ^1,2J. Rzeszowska-Wolny

¹Department of Experimental and Clinical Radiobiology, M. Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Gliwice, Poland
²Silesian University of Technology, Gliwice, Poland

           The levels of transcripts from all actually active genes (transcriptome, transcription profile) characterize the cell type and its physiological state. Microarray techniques allow determining transcription levels of thousands of genes in one experiment. In our studies transcriptome changes in cells exposed to ionizing radiation were studied by oligonucleotide microarray (Affymetrix) method. Transcription profiles in two human cancer cell lines, Me45 and K562, were determined in control cells and at 4 different time points (20 min, 12h, 24h, 36h) after irradiation with 4Gy. Affymetrix microarrays HU-133 allowed us assessing transcription levels of 22283 genes for each time point. A new approach based on calculating average speed of change of gene expression levels (Vex) was applied to the analysis and Vex distributions were approximated by normal distributions. We observed that in both cell lines the change of transcription level was the highest at first moments after irradiation (SD=225.43 for Me45 cell line and SD=281.9312 for K562 cell line for the 20-min time point). At the next time point (12 h) changes were much slower but they showed a tendency to increase 24 and 36h later. The dynamics of change were different in both cell lines. Analysis of processes that showed the highest change after irradiation revealed that genes coding for proteins of proteasome, oxidative phosforylation and ribosomes were characterized by the highest Vex. The expression levels of some genes belonging to these pathways were studied by Q-RT PCR method.