INVESTIGATION OF EXPRESSION AND CHROMOSOMAL LOCALIZATION OF GENE ENCODING TUMOR SPECIFIC PROTEIN WITH MOLECULAR WEIGHT 65 kDa

Ewa Balcerczak¹, Mariusz Panczyk¹, Tadeusz Malewski², Marek Mirowski¹ mirowski@ich.pharm.am.lodz.pl

¹Department of Pharmaceutical Biochemistry, Molecular Biology and Pharmacogenomics Laboratory, Medical University, Muszynskiego 1 Street,90-151 Łódź, Poland; ²Institute of Genetics and Animal Breeding Jastrzębiec, Poland

    P65 gene encodes 65 kDa tumour-associated protein. They seem to be a potential, non-specific tumor marker expressed in many neoplasms. P65 was first time isolated from the MCF-7 human breast cancer cell line. The presence of 65 kDa protein was observed not only in tumor tissue but also in the serum of patients diagnosed with different types of neoplasms. The blood level of P65 in patients suffering from cancer was significantly higher in comparison to healthy men. Also, a 150 bp fragment of P65 gene expression was detected by multiplex semi-quantitative analysis in many different types of tumors. In majority of the investigated carcinomas the presence of this gene expression appeared in advanced clinical stages. Qualitative results were confirmed by real-time PCR analysis. We also established that high levels of P65 are connected with shorter survival time.
    The whole nucleotide sequence of P65 gene is unknown so in the next step, fragment of gene detected in PCR analysis was elongated to 900 bp. Obtained fragment was sequenced and cloned into E. coli by TOPO system where expression of protein was stimulated. The expressed protein was isolated and digested by trypsin. The peptides spectra were compared with protein NCBI data base with the use of MASCOT program. The sequenced P65 gene fragment revealed strong homology to the retrovirus by HERVd FASTA search, other computer analysis by Blat search using the UCSC Genome Browser showed that sequenced fragment is similar to the region of human chromosome 1. These results would be confirmed by hybridization procedures with the RPCI-11 library.

Supported by Grant KBN 2 P05A 098 27
Supported by Grant KBN 2 P05A 098 27

1. ANTITUMOR ACTIVITY OF CARRIER-METHOTREXATE CONJUGATES

Janusz Boratynski¹,², Adam Opolski¹,², Urszula Kańska¹, Monika Jagiełło¹, Renata Budzyńska¹, Dmitry Nevozhay¹, Mohamed Salah Omar¹, Joanna Wietrzyk¹, Tomasz Goszczyński¹

¹Department of Experimental Oncology, Institute of Immunology and Experimental Therapy, PAS, Rudolf Weigl 12, Wrocław, 53-114 Poland; ²J. Dlugosz Academy, Al. Armii Krajowej 13/15 Częstochowa, 42-201 Poland

    Methotrexate (MTX) has been used in chemotherapy of malignancies for several decades. However, low plasma half-life, toxicity to normal proliferating cells and other limitations impel scientists to search for improved forms of MTX. Conjugation of the drug with macromolecular carriers is one of the strategies applied to improve therapeutic properties of anticancer drugs.
    We tested several conjugates of MTX, namely with fibrinogen, albumin, glycated proteins, dextrans and mannan. They were studied both in vitro and in vivo. It allowed us to assess their advantages and disadvantages, and also determined directions for further research. Almost all carrier-MTX conjugates revealed stronger antitumor activity in vivo against i.p. transplanted P388 mouse leukemia as compared to free MTX.

2. DEFECT IN DOUBLE-STRANDED DNA BREAKS REPAIR IN L5178Y-S CELLS IS NOT ASSOCIATED WITH ALTERATIONS IN THE AUTOPHOSPHORYLATION SITES OF DNA-DEPENDENT PROTEIN KINASE

Kamil Brzóska, Marcin Kruszewski, Irena Szumiel

Department of Radiobiology & Health Protection, Institute of Nuclear Chemistry and Technology, Poland

    The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an essential role in the repair of double-stranded DNA breaks through the nonhomologous DNA end joining pathway (NHEJ) by initially recognizing and binding to DNA breaks. It has been shown that DNA-PKcs undergoes autophosphorylation at six sites tightly clustered within 38 residues (ABCDE cluster), and that this autophosphorylation event is critical for its function. Cells with mutated autophosphorylation sites in the ABCDE cluster are defective in the repair of ionising radiation-induced double strand breaks, but show in an in vitro test the same DNA-PK activity as the cells possessing wild type enzyme. This characteristics correspond well with the phenotypic features of the L5178Y-S (LY-S) cell line that is defective in double strand breaks repair, shows a pronounced G1 phase radiosensitivity, but in which the level of DNA-PK activity present in total cell extracts is similar to that of its radioresistant counterpart L5178Y-R (LY-R) cell line.
    In the present study the possible alterations in the sequence encoding the cluster of autophosphorylation sites in the DNA-dependent protein kinase in LY-S cells were examined. Total RNA was extracted from LY-S and LY-R cells and partial cDNA containing ABCDE cluster was synthesized, amplified and sequenced. Obtained sequences were aligned and analysed.
    Despite the presence of phenotypic features indicating the possibility of such alterations, no differences were found between the sequences encoding the autophosphorylation sites in L5178Y-R and L5178Y-S cells. In conclusion, the repair defect in LY-S cells is not related to the structure of the DNA-PKcs autophosphorylation sites in the ABCDE cluster.

This work was supported by the Institute of Nuclear Chemistry and Technology Statutory Grant.

3. WRN EXONUCLEASE ACTIVITY IS BLOCKED BY OXIDATIVE DNA BASE LESIONS

Zuzanna Bukowy¹,⁴, Jeanine A. Harrigan², Dale A. Ramsden³, Vilhelm A. Bohr², Tinna Stevnsner^3*

¹Department of Molecular Biology, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland; ²Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, U.S.A.; ³Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; ³Danish Centre for Molecular Gerontology, Department of Molecular Biology, University of Aarhus, Denmark; ⁴Postgraduate School of Molecular Medicine, Warsaw, Poland

     Werner syndrome (WS) is a premature aging disorder caused by mutations in the WS gene (WRN). Loss of WRN is associated with genomic instability and an elevated incidence of cancer. Although WRN has been suggested to play an important role in DNA metabolic pathways, such as recombination, replication and repair, the precise role of WRN still remains to be determined. WRN is a member of the RecQ family of DNA helicases and possesses ATPase, helicase, and exonuclease activities. Previous studies have shown that the WRN exonuclease is inhibited by certain oxidative lesions, including 8-oxoguanine and 8-oxoadenine, positioned in the digested strand of the substrate. The presence of the Ku heterodimer alleviated WRN exonuclease blockage imposed by these lesions. In the current study, we analyzed several other oxidative lesions, which block WRN exonuclease progression and carefully analyzed their blocking abilities in contrast of two different substrates. We have also shown that Ku stimulates the WRN exonuclease to bypass these lesions. In addition, we also show that the sensitivity towards oxidative lesions is not a general feature of 3. to 5. exonucleases, as the exonuclease activity of AP endonuclease 1 (APE1) was not blocked by oxidative lesions or stimulated by Ku. This study extends the spectrum of lesions which block WRN exonuclease progression and supports a possible function for WRN and Ku in a DNA damage processing pathway.

4. EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS IN THYROID CANCER

Mykola Chekan¹, Michał Jarząb², Barbara Nikel³, Malgorzata Oczko-Wojciechowska¹, Dariusz Lange³, Barbara Jarząb¹.

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

     The problem of sex steroid receptors expression and their influence on development and progression of thyroid tumors is important from the viewpoint of higher thyroid cancer incidence rate among females than in males, especially during puberty (according to different authors male/female ratio ranges from 1:4 to 1:7). This difference is less visible among patients before menarche and after menopause.
     The aim of this study was to analyze estrogen receptor (ER) and progesterone receptor (PR) expression among men and women with different thyroid diseases.
     ER and PR expression was analyzed at transcript level using microarray analysis and at protein level using immunohistochemical staining. 120 probes were used for microarray analysis. Among them: papillary thyroid carcinoma (PTC) - 47 cases, follicular thyroid carcinoma - 12 cases, anaplastic cancer . 6 cases, follicular adenoma . 8 cases and nodular goiter . 36 cases. Immunohistochemical examination of ER and PR expression was conducted on thyroid tissue from 61 patients with different thyroid diseases: PTC . 17 cases, follicular carcinoma . 4 cases, medullary carcinoma . 1 case, nodular goiter . 13 cases, toxic goiter . 10 cases, lymphocytic thyroiditis . 9 cases, follicular adenoma . 8 cases.
     The results of immunohistochemical examination show that majority of ER and PR-positive tissues were from patients with PTC: ER-alpha was positive in 11 of 61 (18%) of examined patients, among them in 8 of 17 (47%) of patients with PTC. PR were positive in 25 of 61 (40%) of examined patients, among them in 15 of 17 patients with PTC. Among all examined cases ER-alpha was observed in thyroid tissue of 18% male and 18% female patients, PR in 45% males and 41% females.
     The results of microarray analysis show that expression of ESR1 was decreased in anaplastic, follicular and medullary carcinomas in comparison with PTC and normal thyroid. ESR1 expression level in PTC and normal thyroid was comparable but range of expression values in PTC was wider. To analyze this difference, hierarchical clustering of PTC and benign thyroid neoplasms/non-neoplastic thyroids (n=91) set was performed. Two subgroups (with higher and lower ESR1 expression level) were not related to the histopathological type of sample. The difference in ESR1 expression was also analyzed in the context of the patients. gender: in men the difference in ESR1 expression level between PTC and benign thyroid tissue is more visible. All tumors with maximal ESR1 expression level were found in women, but the differences between tumors and non-neoplastic thyroid were less visible.
     In conclusion, no difference between ER and PR expression among men and women was found. Increased expression of both ER and PR in PTC was observed.

5. ANTIOXIDATIVE ABILITIES AND LIPID PEROXIDATION IN KIDNEY TUMOR

Marcin Chlabicz¹, Anna Stankiewicz², J. Kudelski¹, Barbara Darewicz¹, Agnieszka Augustyniak², Elżbieta Skrzydlewska²

¹Department of Urology and ²Department of Analytical Chemistry of Medical University of Białystok, 15-230 Białystok, ul. Mickiewicza 2, Poland

     Renal cell carcinoma (RCC) represents 2-3% of all cancers, with the highest incidence in the more developed countries. The worldwide and European increase in incidence is approximately 2%. Renal cell carcinoma is the most frequently occurring solid lesion within the kidney and comprises different RCC types with specific histopathological and genetic characteristics. There is a 1,5:1 predominance of men over women, with peak incidence occurring between 60 and 70 years of age. Aetiological factors include lifestyle factors, such as smoking, obesity and antihypertensive therapy. Common element of above situations that take part in development of kidney cancer is enhanced generation of free radicals. Kidney tissue is particularly susceptible to reactive oxygen species attack and antioxidants play an important role in defence strategy against them.
     The present study aims at examining antioxidant parameters and malondialdehyde . the product of lipid peroxidation as well as the marker of cancer progression . in renal cancer (RCC) patients. The activity of superoxide dismutase, glutathione peroxidase and reductase, thiol compounds, vitamin C and malondialdehyde have been determined in tumors and unchanged tissue of 24 patients with renal cell cancer. The tumor and morphologically unchanged tissue were collected during surgery. The 10% homogenates were used to examinations. Superoxide dismutase activity was determined spectrophotometrically by measuring inhibition of epinephrine oxidation to adenochrome. Glutathione peroxidise and glutathione reductase activities were measured spectrophotometrically by monitoring the oxidation of NADPH to NADP. The level of sulfhydryl groups were measured spectrophotometrically. Lipid peroxidation product malondialdehyde, ascorbic acid were detected by high performance liquid chromatography. Statistical analysis was performed using Wilcoxon Matched-Pairs Signed.Ranks Test and values for p<0.05 were considered significant.
     There were no statistical differences between superoxide dismutase activity in cancer tissue and non-tumor tissues. The present study has indicated a significant increase of glutathione peroxidase activity in tumor tissues as compared with controls in all cases. The glutathione peroxidase activity in kidney tumor was increased up to 400%. There were no statistical differences among kidney tumor and morphologically unchanged tissue in glutathione reductase activity, although was decreased in comparison to control tissue. Changes in the activities of antioxidant enzymes were accompanied by changes in non-enzymatic antioxidant parameters. The present study has evidenced a significant decrease in protein SH groups in tumor tissues as compared with controls. It has been shown that the level of vitamin C insignificantly decreased in tumor kidney tissue. The level of the last of lipid peroxidation product - malondialdehyde was increased on the average by about 150% in kidney cancer tissue.
     In conclusion, the kidney cancer cells are more exposed to oxidative stress than the surrounding noncancerous cells and further understanding of tumour biology from the point of reactive oxygen species may be helpful for establishing a new strategy for cancer therapy.

6. MOLECULAR INTERACTION BETWEEN GD2 GANGLIOSIDE-SPECIFIC ANTIBODY 14G2a AND GD2-MIMICKING PEPTIDES

Dominik Czaplicki¹, I. Horwacik¹, A. Kowalczyk¹, M. Kurciński², A. Koliński² and H. Rokita¹

¹Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 7 Gronostajowa St., ³0-³87 Kraków, Poland; ²Faculty of Chemistry, Warsaw University, 1 Pasteura St., 02-093 Warsaw, Poland

     Molecular mimicry is a phenomenon of structural similarity between two or more different molecules that gives them similar antigenic properties. The molecules which demonstrate mimicry, are related in respect of conformation, although they can differ significantly in terms of chemical nature. Molecular mimicry is naturally observed in a range of pathogens, but is also a useful research tool in experimental immunotherapy. The phenomenon is especially interesting to provide an alternative to non-protein antigens like glycolipids. Those antigens are often aberrant on cancer cells, but due to their chemical properties are poorly immunogenic and therefore difficult to apply in immunotherapy. However, peptide sequences that mimic glycolipid structure are able to elicit immune response which is cross-reactive against the original antigen.
     In our studies we have been searching for active immunotherapy against neuroblastoma, a malignancy of neuroectodermal origin. One of the markers of the disease is GD2 ganglioside, a glycolipid abundantly expressed on the surface of neuroblastoma cells. Using phage-displayed peptide library LX-8/f88-4 and the 14G2a monoclonal antibody specific to GD2 ganglioside, we identified five peptide sequences with high affinity to 14G2a, which was confirmed by ELISA. Further experiments demonstrated that the peptides were able to compete with GD2 for the binding site of the antibody in a dose-dependent manner. Sequence similarity analysis performed with a multiple sequence alignment program revealed two clusters of peptides: (#85, #D, #8) and (#65, #94). Subsequently, a variety of molecular modelling tools was used to further investigate the nature of interaction between peptides and 14G2a binding site.
     Molecular modelling of peptide-14G2a antibody interaction was performed with a reduced lattice representation model CABS (CA, cB and Side groups). The receptor structure (14G2a) was generated from amino acid sequence with a template structure PDB ID: 1SVZ. Then the ligand (peptide) sequence was introduced to the resulting structure and the complex was optimised with Monte Carlo simulation method. 5000 structures of receptor-ligand complex were obtained and subsequently clustered by HCPM method (Hierarchical Clustering of Protein Models). Complex structure from the largest cluster underwent full-atom reconstruction and was further optimised to calculate the ligand binding energy. The same full-atom representation model was used to identify interacting amino acid residues.
     The in silico approach provided valuable information about the structure of the peptide mimics and produced enough data to compare the binding characteristics of the five peptide sequences. Moreover, the results obtained with molecular modelling are in agreement with in vitro assay results. Therefore, the methods used to construct the computer model were correct and could be employed to optimise the peptide sequences for 14G2a binding. Our studies demonstrated that the computational approach and the in vitro methods may successfully complement one another.

The work was supported by 3P05A 00124 grant from the Polish Ministry of Science and Information Technology and N302 034 313063 grant from the Polish Ministry of Science and Higher Education.

7. PARTICIPATION OF THE EGF RECEPTOR IN THE RESPONSE TO X-IRRADIATION IN HUMAN GLIOMA M059 K AND J CELLS

Iwona Grądzka, Barbara Sochanowicz, Iwona Buraczewska and Irena Szumiel

Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, 03-195 Warsaw, Poland

     The role of epidermal growth factor receptor (EGFR) in ionising radiation.induced DNA double-strand break (DSB) rejoining was investigated in two related human glioma M059 K and J cell lines. The latter cells are ionising radiation sensitive due to deficiency in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) . an essential component of DNA-PK-dependent nonhomologous DNA end joining (D-NHEJ).
     DSBs were induced by X-irradiation of the cells (10 Gy) in the presence (or absence) of a specific EGFR kinase inhibitor - tyrphostin AG 1478 (T). DSB rejoining, measured with the use of pulse-field gel electrophoresis, was significantly slowed down by 5 .M T in M059 K but not in M059 J cells. This effect corresponded with a decrease in survival in M059 K but not in M059 J cells subjected to combined (X+T) treatment, as compared with X-irradiation alone. During the repair period, the levels of EGFR in whole cells and nuclei were monitored using enzyme immunosorbent assay (ELISA). The total cellular EGFR level was higher in M059 J than in M059 K cells in agreement with the sensitivity to T treatment. After irradiation, EGFR accumulated in the nuclear fraction of M059 K (but not M059 J) cells and this process could be prevented by T treatment (5 .M, 1 h) preceding irradiation and continued until the end of the experiment. The presented results allow us to suppose that in X-irradiated M059 cells the D-NHEJ system of DSB repair is activated by EGFR. Autophosphorylation of the receptor is essential for this process. X-irradiation-stimulated translocation of EGFR to the nuclei, not observed in M059 J cells, implicates a direct involvement of the receptor in DNA repair through interaction with DNA-PKcs.

IN THE SEARCH OF NOVEL PARTNERS OF THE APOPTOTIC NUCLEASE DFF40/CAD - EXPLOITING OF YEAST TWO-HYBRID SYSTEM

Jakub Hanus¹, Agnieszka Ślusarczyk¹, Magdalena Kalinowska¹, Anna Chełstowska², Piotr Widłak¹

¹Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland; ²Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warszawa, ul. Pawińskiego 5a, Poland

     The major apoptotic nuclease, DNA fragmentation factor (DFF), also termed Caspase-Activated DNase (CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45/ICAD) and a 40 kD latent nuclease subunit DFF40/CAD. Activation of the nuclease depends on caspase-3-mediated cleavage of DFF45/ICAD inhibitor and formation of DFF40/CAD homooligomers, which further may interact with additional activators or inhibitors.
     In order to identify novel proteins that interact with DFF40/CAD we have used yeast .two-hybrid. system. Yeast S. cerevisiae strain AH105 was transformed with human DFF40 (cloned into pGBT9 vector) and then mated with S. cerevisiae strain Y187 carrying mouse brain embryo cDNA library. Several clones have been identified, including DFF45/ICAD ones, that are under further investigation. Currently we are screening human HeLa cell cDNA library with yeast strains either expressing DFF40 alone or co-expressing DFF40 and DFF45 (so called three-hybrid system).

This work was supported by the Ministry of Science Grant N301 058 311763.

8. APOPTOSIS OF LYMPHOCYTES IN H. PYLORI CAG A+ AND CAG A- INDUCED PERIPHERAL BLOOD MONONUCLEAR CELLS OF H. PYLORI INFECTED AND UNINFECTED CHILDREN WITH GASTRITIS.

A. Helmin-Basa¹, L. Gackowska¹, I. Kubiszewska¹, A. Eliaszewicz¹, J. Michałkiewicz¹,³, G. Mierzwa², M. Czerwionka-Szaflarska², D. Dzierżanowski³.

¹Department of Immunology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland; ²Department of Gastroenterology, Pediatry and Alergology, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland; ³Deptartment of Microbiology and Clinical Immunology, Children.s Memorial Hospital Warsaw, Poland

     Background: The aim of this study was to estimate actual H. pylori infection influence on apoptosis of lymphocyte in H. pylori (cagA+ and cagA-) induced peripheral blood mononuclear cells (PBMC) of H. pylori infected (Hp+) and uninfected (Hp-) children with gastritis.
     Materials and Methods: 1) PBMC of Hp+ (n=6) and Hp- (n=5) children and controls (n=6) were used. 2) Quantitative analysis of apoptotic lymphocytes in 72h culture of PBMC with H. pylori cagA+ and cagA- (induced apoptosis) or with medium alone (spontaneous apoptosis) (staining with Annexin V-FITC and Propidium Iodide (PI) and flow cytometry).
     Results:1) Lymphocytes of both Hp+ and Hp- children showed high apoptosis in comparison to control, 2) There were no statistical differences between H. pylori-induced and spontaneous lymphocyte apoptoses, 3) There was higher early spontaneous lymphocyte apoptosis in PBMC of Hp+ children than Hp- ones.
     Conclusions: Gastritis is connected with high sensitivity of peripheral lymphocytes to apoptosis. It is not dependent on H. pylori infection.

9. GHRELIN, A NATURAL LIGAND FOR THE GROWTH HORMONE SECRETAGOGUE RECEPTOR, SENSITIZES BLOOD MONONUCLEAR CELLS TO OXIDATIVE STRESS

M. Kruszewski¹, T. Iwaneńko¹, J. Woliński², R. Zabielski², M. Wojewódzka¹

¹Department of Radiobiology & Health Protection, Institute of Nuclear Chemistry and Technology, Poland; ²Department of Gastrointestinal Physiology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Poland

     Ghrelin, a recently described endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by stomach cells and is a potent regulator of food intake, energy expenditure, adiposity, and growth hormone secretion. However, the functional role of ghrelin in regulation of immune responses remains poorly defined. GHS-R and ghrelin are expressed in human T lymphocytes and monocytes, where ghrelin inhibits the expression of proinflammatory cytokines. Ghrelin exerts potent anti-inflammatory effects and attenuates endotoxin-induced anorexia in a murine endotoxemia model.
     In this work we investigated the effect of ghrelin addition to food on the susceptibility of peripheral blood mononuclear cells to oxidative stressors generated during inflammation. Ghrelin was administered intragastrically to newborn piglets for for 7 days at two doses 7.5 and 15 .g/kg body mass. After treatment, blood was collected by heart puncture and mononuclear cells (MNCs) were isolated by density gradient centrifugation on Histopaque 1137, resuspended in RPMI1640 medium supplemented with 20% of FCS. Isolated cells were exposed to different DNA damaging agents, such as X-radiation (dose 0-3 Gy, 200 kV, 5 mA, dose rate 1.2 Gy/min), H2O2 (0-250 .M) in PBS for 15 min at 4oC, SIN-1 (0-50 .M) in PBS for 15 min at 4oC. The extent of DNA damage was evaluated by alkaline comet assay. To investigate the influence of ghrelin on cells. ability to repair DNA damage, MNCs were irradiated with 2 Gy of X-radiation and disappearance of DNA breaks was monitored 0.5, 1 and 2 h after irradiation.
     We found that ghrelin had a marked effect on the level of X-radiation-, H2O2- and SIN-1-induced DNA damage. In all cases, we observed a significant increase in the level of DNA strand breaks in ghrelin treated animals as compared with untreated controls. However, no effect of ghrelin was observed on cells. capacity to repair DNA damage.
     Our data point to a dual role of ghrelin in inflammation. On the one hand, it is known to inhibit the expression of proinflammatory cytokines and to have a potent anti-inflammatory action; on the other hand, expression of ghrelin potentiates the genotoxicity of oxidative stressors generated during inflammation.

This work was financed from research sources for years 2006-2009 as a scientific grant No. PBZ-KBN-093/P06/2003

10. PRO-APOPTOTIC ROLE OF PI3-K/AKT PATHWAY DURING APOPTOSIS INDUCED BY SELECTED ANTI-CANCER DRUGS

Subbareddy Maddika^1,3, Emilia Wiechec^1,4,5, Lise Lotte Hansen⁴, Marek Los^1,2,3.

¹Manitoba Institute of Cell Biology, Cancer Care Manitoba; ²Manitoba Institute of Child.s Health; ³Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada; ⁴Institute of Human Genetics, University of Aarhus, Denmark; 5Department of Experimental and Clinical Radiobiology, Oncology Center, Maria Sklodowska-Curie Memorial Institute, Gliwice, Poland.

     Introduction: PI3-Kinase/Akt pathway is a well-known cellular pathway, which regulates the cell survival and proliferation. This pathway is frequently activated in various cancers and is responsible for poor response to anti-cancer therapeutics. Intriguingly in this context, Akt-inhibitors have proved to be only moderately successful in experimental cancer therapy. Thus, here we provide a novel approach, where instead of inhibiting the PI3-Kinase/Akt pathway, we redirect this survival pathway towards a death pathway by using selected anti-cancer drugs.. The constitutive activation and nuclear mislocalization of Akt, the central downstream effector molecule of PI3-K plays a positive role in apoptosis induced by selected anticancer drugs.
     Purpose: The overall aim of this study was to investigate the role of PI3-K/Akt signalling pathway in promoting apoptosis induced by anticancer drugs such as Methotrexate and Docetaxel.
     Material and Methods: The material for this study included 293-T, MCF-7 and PC-3 cell lines undergoing chemotherapeutic treatment with selected anticancer drugs like Methotrexate and Docetaxel. The anti-Akt and anti-phospho Akt antibodies are commercially available (Cell Signalling). The level of phospho-Akt and total Akt was detected by immunoblotting with anti-Akt and anti-phospho Akt antibodies respectively. The confocal microscopy was used for visualization of mislocalizated Akt in cell structure in either control, untreated cells or drug treated cells. The percentage of cell death was studied by nicoletti method followed by flowcytometric analysis.
     Results: Our results indicate that activation of Akt is associated with its consequent nuclear translocation during treatment with chemodrugs. The nuclear localization of Akt is dependent on the upstream activation of the PI3-K. The PI3-K inhibition by wortmannin resulted in abolition of nuclear localization of Akt in the presence of anticancer drugs. According to this event, we observed that nuclear Akt enhanced the cell death induced by Methotrexate and Docetaxel in contrast to inhibition of cell death in the presence of drug and PI3-K inhibitor.
     Conclusion: The PI3-K/Akt activation and nuclear location of Akt play an important pro-apoptotic role in cell death induced by Methotrexate and Docetaxel.

11. COCKAYNE SYNDROME GROUP B PROTEIN IS INVOLVED IN REPAIRING OF DNA ADDUCTS INDUCED BY TRANS-4-HYDROXY-2-NONENAL

Leena Maddukuri^1,2, Mette Christiansen⁵, Dominika Dudzinska³, Jolanta Zaim¹, Tomasz Obtulowicz¹, Marek Komisarski¹, Andrzej Wójcik⁴, Jaroslaw Kusmierek¹, Tinna Stevnsner⁵, Vilhelm A. Bohr^5,6, Barbara Tudek^1,3

¹Department of Molecular Biology, IBB PAS, Warsaw, Poland; ²School of Molecular Medicine, Warsaw Medical University, Poland; ³Institute of Genetics and Biotechnology, Warsaw University, Warsaw, Poland; ⁴Institute of Nuclear Chemistry and Technology, Warsaw; 5Danish Center for Molecular Gerontology, Aarhus University, Denmark; 6Laboratory of Molecular Gerontology, National Institute of Aging, NIH, Baltimore, USA

     Cockayne Syndrome (CS) is characterized by progressive multisystem degeneration and is classified as a segmental premature aging syndrome. The CS complementation group B (CSB) protein is engaged in transcription . coupled (TCR) repair, global genome and base excision repair (BER) of some types of oxidative DNA damage and in general transcription. Here we show that cyclic propano- or ethano adducts, which are induced by trans-4-hydroxy-2-nonenal (HNE) may be processed by the TCR pathway. For that reason we studied CSB proficient and deficient human cell lines, as well as several cell lines containing different point mutations in the ATPase domain of the CSB protein. We show for the first time, that CSB cell lines deficient in TCR are hypersensitive to extremely low, physiological concentrations of HNE and exhibit a higher number of sister chromatid exchanges (SCEs) in comparison to their proficient counterpart. We also show that a cell line bearing mutation in motif II of the CSB ATPase is as sensitive to HNE as a CSB-null line. Cell line with a mutation in motif V resembles the wild type, while motif VI renders intermediate sensitivity to HNE. Homology modeling of CSB protein showed that amino acids mutated in motifs II and VI, but not V, were localized in the vicinity of the ATP binding site. Treatment of wild type cells with HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity and activates the TCR pathway. In addition, we observed for the first time that un-repaired HNE-DNA adducts can block transcription in vitro. This data confirms that the TCR pathway is engaged in the processing of HNE-induced DNA lesions. A decreased activity or lack of the TCR pathway may cause inhibition of transcription and may lead to apoptosis.

12. MASS SPECTROMETRY BASED INVESTIGATION OF PROTEIN N-HOMOCYSTEINYLATION IN BLOOD PLASMA

Łukasz Marczak¹, Marta Sikora¹, Joanna Perła-Kaján¹, Hieronim Jakubowski^1,2, T. Twardowski¹, Maciej Stobiecki¹

¹Institute of Bioorganic Chemistry, PAS, Noskowskiego 12/14, 61-704 Poznań; ²Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 185 S Orange Ave, Newark, NJ 07103

     Increased concentration of homocysteine (Hcy) is important factor causing arteriosclerosis, mainspring of early mortality from coronary diseases. There are many mechanisms of toxic action proposed for homocysteine. Among others, the most cited are: induction of oxidative stress, inflammatory factors, responses for non-folded proteins and modification of proteins by homocysteine tiolactone (HTL) - N-homocysteinylation. The latter effect relies on covalent linking of homocysteine to .-amino residue of lysine. It provokes serious physiological consequences. Proteins lose their functions, are toxic and become autoantigens inducing immunological response. Antigens specific to proteins modified by HTL can be used for detection of N-Hcy-proteins and can serve as a diagnostic tool for recognizing diseases caused by increased level of homocysteine in human blood. The alternative method for detection of N-Hcy-proteins could be elaborated using both, classic analytical methods like chromatography and modern techniques like MALDI/TOF mass spectrometry.
     The aim of our project is to work out an efficient medical assay of the level of N-Hcy-proteins in human blood. In this poster the first results on mass spectrometric evaluatrion of N-homocysteinylation degree for chosen proteins are presented. At this stage we relied on elaboration of method for assaying the localization of N-homocysteinylation sites. For this purpose four N-homocysteinylated proteins: cytochrome c, transferrin, mioglobin and albumin were digested with trypsin and analyzed using MALDI/TOF mass spectrometer. Also blood plasma was analyzed in respect of albumin presence in this body fluid. Detailed analysis of results by comparison of peptide maps of control proteins with peptide maps derived from modified samples allowed us to localize N-homocysteinylation sites. Also analysis of control and modified intact model proteins permitted estimation of possible level of N-homocysteinylation of the studied proteins. Elaboration of quantitative approach for so far assayed peptides will provide good and relevant diagnostic technique for evaluating patients with elevated levels of homocysteine in blood.

13. INTERACTION OF DINITROSYL IRON COMPLEXES WITH DNA

Sylwia Męczyńska, Hanna Lewandowska-Siwkiewicz, Marcin Kruszewski

Department of Radiobiology & Health Protection, Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Dorodna Str 16, Poland

     Dinitrosyl iron (II) complexes (DNIC) are an important factor in the NO-dependent regulation of cellular signalling pathways. We examined DNIC interactions with DNA, choosing two ligands: glutathione and histidine, each representing different dinitrosyl iron complex with proteins. Glutathione is a low-molecular thiol compound ubiquitous in all kinds of cells and displaying a wide range of biological activities. Dinitrosyl-dithiol iron (II) complexes are well characterised species, in which iron atom is chelated by two S-2 atoms and two NO molecules. Histidine is an amino acid, which imidazole ring participates in DNIC formation in non-thiol proteins. In the histidine dinintrosyl iron complexes, iron is coordinated to the N7 atom of imidazole ring.
     Complexes of iron with histidine and glutathione were obtained in vitro and the interactions of these complexes with DNA were studied by circular dichroism spectroscopy. Formation of DNIC complexes was monitored by UV/VIS spectroscopy, IR spectroscopy and NMR. We examined the influence of increasing amounts of Fe(II) ions, either in the form of dinitrosyl complexes or in the form of hydrated Fe(II) ions, on DNA in different pH and ionic strength conditions.
     The right band of the DNA spectrum is monotonously decreased by the increase of the FeSO4 concentration. This effect is completely suppressed in pH 7; increasing ionic strength also eliminates this effect gradually. The small decrease in the intensity of the right band of the circular dichroism spectrum of DNA, upon addition of Fe(II) up to 1:1 molar ratio indicates that the interaction between the metal complex and DNA induces only slight modifications to the native conformation of DNA. Disappearance of this effect in pH 7 illustrates that observed effect comes from an external electrostatic binding interaction, between the Fe(II) cation and the negatively charged phosphate groups of DNA. In pH 7 Fe(II) is present in the solution in the form of Fe(OH)2, and therefore, is no longer attracted to DNA. Influence of ionic strength supports this explanation. Similar spectra were found by Silvestri et al. [Wolak M., van Eldik R.: Coord. Chem. Rev., 230, 263-282 (2002)] for Fe (Salen) complexes and also attributed to ionic interaction.

Supported by the Polish State Committee (KBN) statutory grant for the INCT

14. ABCB1 GENE POLYMORPHISM, HAPLOTYPES ANALYSIS AND PROGNOSIS IN COLORECTAL CANCER

Mariusz Panczyk¹, Ewa Balcerczak¹, Krzysztof Jamroziak², Sylwester Piaskowski³, Grażyna Pasz-Walczak⁴, Karolina Całka¹, Aleksandra Sałagacka¹, Marek Mirowski¹

¹Department of Pharmaceutical Biochemistry, Molecular Biology and Pharmacogenomics Laboratory, Medical University of Lodz, ¹ Muszynskiego Street, Poland; ²Department of Haematology, Medical University of Lodz; ³Department of Molecular Pathology and Neuropathology, Medical University of Lodz; ⁴Department of Tumour Pathology, Medical University of Lodz

     The ABCB1 (MDR1) gene encodes P-glycoprotein (P-gp), a 170kD member of adenosine triphosphate.binding cassette (ABC) superfamily of membrane transporters. P-gp is ATP-dependent transporter exporting xenobiotics to extracellular environment. Overexpression of P-gp correlates with high pathological grading of tumours in colon cancer.
     Mutations in ABCB1 gene may change substrates. specificity of P-gp. It was also shown that single nucleotide polymorphisms (SNP) in ABCB1 gene can influence the level of expression of P-gp.
     Relation between C1236T, G2677T/A and C3435T SNPs of ABCB1 gene remains unclear. Maybe this three polymorphisms are linkage disequilibrium (LD), but it is unknown if this genetic variants are located on the same LD-block or haplotype. The implications of genetically determined differences in P-gp function for drug disposition, therapeutic outcome and risk of development of certain diseases are intensively studied.
     The aim of this study was to compare allele frequency of three SNPs in ABCB1 gene namely C1236T, G2677T/A and C3435T, between 100 patients with colorectal cancer and 100 healthy controls of Caucasian origin. The next purpose was to find dependence between allele frequency and histopathological parameters and haplotypes analysis.
     Genotyping of ABCB1C1236T, ABCB1G2677T/A and ABCB1C3435T was performed by automated sequencing and by restriction fragment length polymorphism (RFLP) method, respectively.

Supported by KBN grant: 2 P05F 02628

15. INTERFERON ALPHA IN RAT LIVER AFTER PARTIAL HEPATECTOMY

Maryna Perepelyuk¹, A. Kuklin¹, D. Fedorchenko², M.Yu. Obolenskaya¹

¹Institute of Molecular Biology and Genetics, Str.Zabolotnogo, 150, Kyiv, 03143, Ukraine; ²Institute of Epidemilogy and Infectious Diseases, Str.Amosova, 5, Kyiv, 03038, Ukraine.

     Interferon (IFN)-. treatment is a commom therapy for chronic viral hepatitis, which contributes to hepatocarcinogenesis prevention. Besides, IFN-., combined with chemo- and radiotherapy, is prescribed after surgical removal of tumours. The cancers of different origin vary in their sensitivity to IFN-.. Along with IFN-sensitive cancers (kidney adenocarcinoma, lung sarcoma, malignant melanoma, neuroblastomas, cancers of lymphoid, endocrine and generative organs), there are resistant ones . such as cancers of stomach, liver and colon. Moreover, high and low concentations of IFN-. can cause different response of the organism. The mechanisms of IFN-. action in different situations are still obscure.
     The aim of our study was to investigate the expression of IFN-., some of its targets (PKR and RNAse L) and IFN-receptors during liver transition from quiescence to proliferation in the absense of viral infection. The rats after partial hepatectomy (PHE) and laporotomy (LAP) were used as the corresponding models of G0.S transition and acute phase response. The latter is a compound of postoperative G0.S transition. The expression of investigated genes was assessed by RT-PCR in total liver samples and in isolated hepatocytes (Hep) and Kupffer cells (KC) from intact and operated rats in 1, 3, 6 and 12 hours after surgery. The content of IFN in liver samples was evaluated by antiviral test.
     In the samples from intact liver the expression of all genes manifested itself at RNA level. The isolated Hep and KC differ by the amount of specific RNAs. Kupffer cells were responsible for expression of IFN-. but not hepatocytes. The level of IFN-. receptor- and PKR-specific mRNA was 2 to 3-fold higher in KC than in Hep. It corroborates the fact that Kupfer cells are producers and receivers of cytokines. The amount of RNAse L-specific mRNA was nearly equal in the cells of both types.
     After PHE the content of IFN-. protein and mRNA increases during the first 3 hours after operation with subsequent decrease until 6 .12 hours. After LAP neither IFN-.-specific RNA, nor protein is detected, pointing to the special role of IFN-. in regeneration process. This increase is less than maximum possible response for typical IFN-. inducer . poly(I)-poly(C). IFN-. specific mRNA was shown to be produced by KC and not by hepatocytes. We assume that activation of KC with cell-specific production of IFN-. and responsiveness to it is essential for G0 . G1 transition of hepatocytes and incompatible with the early phases of acute phase response.

16. ABCG2 GENE AND BREAST CANCER RESISTANCE PROTEIN EXPRESSION IN COLORECTAL CANCER

Aleksandra Sałagacka¹, Ewa Balcerczak¹, Grażyna Pasz-Walczak², Karolina Całka¹, Mariusz Panczyk¹, Marek Mirowski¹

¹Molecular Biology and Pharmacogenomics Laboratory, Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Medical University, ¹ Muszynskiego Street, 90-151 Lodz, Poland; ²Department of Tumour Pathology, Medical University of Lodz

     Breast Cancer Resistance Protein (BCRP/ABCG2) is plasma membrane glycoprotein that belongs to the family G of ABC transporters superfamily. It has the ability to pump metabolites as well as xenobiotics out of the cell and plays important protective role against toxic substances. However, overexpression of BCRP is associated with high level of resistance to a wide range of anticancer agents, e.g. mitoxantrone, anthracyclines, camptothecines and could be the reason of chemotherapy failure.
     In the present study, mRNA ABCG2 gene and BCRP protein expression levels were determined in 63 cases of colorectal cancer by real-time PCR and immunohistochemistry, respectively. Both gene and protein expression levels were compared with some histological features like grading and staging to evaluate their potential role as a prognostic marker for colorectal cancer.

17. NEW CHIMERIC PROTEIN ABRAA-VEGF121 IS SELECTIVELY CYTOTOXIC TOWARDS CELLS OVEREXPRESSING VEGFR2 (KDR) RECEPTOR AND INHIBITS GROWTH OF PRIMARY TUMORS.

Andrzej Smagur¹, Mariya Boyko², Nadya Biront², Dorota Szpila¹, Stanisław Szala¹

¹Department of Molecular Biology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland; ²Department of Biochemistry, Ivan Franko National University of Lviv, Hrushevskogo St, 4, Lviv, 79005, Ukraine

     Growth of solid tumors is dependent on successful angiogenesis, in which the key factor is the vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). VEGF, following binding to a specific receptor (KDR, a.k.a. VEGFR2) undergoes cellular internalization. Endothelial cells of tumor vessels, as opposed to those lining up normal blood vesels, are characterized by an increased expression of VEGFR2 receptors.
     The aim of this study was to investigate a novel two-domain protein, the cognitive part of which allows directing the chimeric protein to endothelial cells lining up tumor blood vessels whereas the other, effector domain would entail destruction of targeted cells.
     We constructed a synthetic gene coding for a chimeric protein ABRaA-VEGF121 which combines abrin A chain (ABRaA - a plant toxin that inactivates ribosomes) [Wang et al., 2004]) linked to human VEGF121 via a short aminoacid spacer (G4S). ABRaA-VEGF121 expression was carried out in E. coli BL21(DE3) strain. The protein was isolated from insoluble fraction as inclusion bodies, then it was solubilized and purified (>95%). Additionally, LPS was removed from ABRaA-VEGF121 preparations (<0.0025 EU/ 1.g protein). The obtained protein migrates, under non-reducing conditions, as a ~84kDa homodimer and a ~42kDa monomer; it shows immunoreactivity towards anti-hVEGF121 monoclonal antobodies.
     ABRaA-VEGF121 shows strong cytotoxicity towards PAE/KDR cells overexpressing KDR receptor (LC50 = 0.067 .g/ml), and induces in them apoptotic death, as opposed to PAE cells overexpressing VEGFR1 receptor (PAE/hFlt-1) or wild PAE cell line (LC50 . 27.3 .g/ml).
     ABRaA-VEGF121 inhibits protein biosynthesis in a cell-free protein translation system. Preincubation of ABRaA-VEGF121 with anti-hVEGF121 monoclonal antobodies eliminated cytotoxicity associated with this protein. This indicate that both domains are biologically active.
     Preliminary in vivo studies of anticancer properties of ABRaA-VEGF121 demonstrated inhibition of tumor growth, as observed in the B16(F10) murine melanoma tumor model.

18. CA(1-7)M(2-9) (CAMEL) PEPTIDE IN THE THERAPY OF B16(F10) MURINE MELANOMA TUMORS

Ryszard Smolarczyk¹, Tomasz Cichoń¹, Wojciech Kamysz², Magdalena Głowala-Kosińska³, Rafał Nadolny², Stanisław Szala¹

¹Department of Molecular Biology, ³Department of Tumor Biology; Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland; ²Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdańsk, Al. Hallera 107, 80-416 Gdańsk

     CA(1-7)M(2-9) (CAMEL) is a hybrid peptide combining fragments of Cecropin A (aminoacids 1-7) and Mellitin (aminoacids 2-9). The peptide has been known so far as an effective antibacterial drug. The aim of this study was to check how this drug would affect neoplastic cells and whether it could be useful in anticancer therapy.
     We investigated the interaction of this drug with B16(F10), NIH3T3 and HeCa10 cells in culture. Irrespective of cell type studied the peptide was cytotoxic and its LC50 was ca. 3.M. Following penetration of cells in culture the investigated peptide induced their necrotic death.
     We noted inhibition of primary murine melanoma tumor growth following intratumoral administration of CA(1-7)M(2-9) peptide. However, the observed inhibition of tumor growth lasted only as long as the administration of the peptide. Within 3-4 days of administration cessation, tumor growth was resumed at an increased pace.
     In order to obtain prolonged survival of treated animals we applied a combined therapy consisting of CA(1-7)M(2-9) and IL-12 gene administration. The latter, when administered intratumorally, stimulates the immune system to destroy cancer cells. The applied combination caused inhibition of tumor growth and yielded a statistically significant extension of animal survival.

19. PRELIMINARY STUDIES OF NEW SYNTHETIC CHLORIN-TYPE ANTITUMOUR PHOTOSENSITISER

Agnieszka Szurko*^1,2, Maria Widel², Aleksander Sochanik², Franz-Peter Montforts#³, Agnieszka Kozielec³, Alicja Ratuszna¹

¹A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland, (*email: 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 term photosensitiser is synonymous with any molecule that uses radiant energy, or light, to elicit specific biological responses. In photodynamic therapy (PDT), the photosensitiser takes the role of a drug preferentially localizing in rapidly growing cancer cells and getting activated by cells. exposure to light, generating in the presence of oxygen very reactive cytotoxic species. Each factor, required for PDT, is harmless by itself, but their combination can produce lethal cytotoxic agents that can eradicate tumor cells.
     Chemical reduction of one or two of the peripheral conjugated double bonds in porphyrins. macrocycles gives rise to chlorins, with concomitant bathochromic shift of the Q-bands with higher extinction coefficients than the corresponding parent porphyrins.
     Photostable water-soluble chlorins have been reported that have high singlet oxygen efficiency coupled with 10 times stronger absorption than HpD or porphyrins in the therapeutic window; hence they are expected to be good photosensitisers.
     Since the mode of transfer strongly influences subsequent localization of photosensitiser in cells and, consequently, affects the percentage of killed cells, we compared the killing efficiency of an examined chlorin delivered to the cells in liposomal form 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 determined by MTS assay.
     Our preliminary results indicate that conjugation of chlorin with liposomes is an efficient means of transfering the sensitiser into the cells, leading to highly efficient photosensitization, whereas non-carrier delivery (DMSO) is rather useless in such experiments.

20. LIPOSOME-CONJUGATED NEW PORPHYRIN DERIVATIVES FOR PHOTODYNAMIC THERAPY OF CULTURED TUMOR CELLS

A. Szurko*^1,2, G. Kramer-Marek^1,2, A. Sochanik², M. Widel², P. Kus³, J. Habdas³, J. Szade¹, R. Wrzalik¹, C. Serpa⁴, L. Arnaut⁴ & A. Ratuszna¹

¹A. Chelkowski Institute of Physics, University of Silesia, Katowice, Poland; ²Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; ³Institute of Chemistry, University of Silesia, Katowice, Poland; ⁴Institute of Chemistry, University of Coimbra, Portugal

     In an effort to find a potential agent for PDT we have undertaken a complex studies of synthesis, physico-chemical characterization and evaluation of biological activity of some hydrophobic porphyrin derivatives: amino acid-, cetyl- and pirydyl- porphyrins. We characterised these agents by light absorption and emission studies. Moreover, flash photolysis was used to measure the singlet oxygen quantum yields of porphyrins in aerated toluene. Biological studies were performed on colon adenocarcinoma (Hct116) and human melanoma cells (Me45) in culture. The dark cytotoxicity and photodynamic efficiency of the explored porphyrins was determined by the MTS assay and colony forming ability method. Some of the examined porphyrin derivatives appeared to be very efficient photosensitisers for selected cell lines, with low dark toxicity.
     It has been known that the mode of transfer strongly influences subsequent localization of photosensitiser in cells and, consequently, affects cell death pathway, therefore cellular distribution and mode of cell death were also investigated. Our confocal microscopy studies indicated that use of cationic liposomes as a carrier of porphyrins enabled their efficient transfer into the cells. The mode of cell death was dependent on energy of light applied and time of post-treatment incubation, shifting from apoptosis towards necrotic death at higher light energies and longer incubation periods.
     In vitro PDT experimets using liposome-conjugated novel porphyrins suggest that they might be valuable candidates for further therapeutic studies. Combination of such .second-generation. photosensitisers and targeted nanocarriers would open new routes for PDT.

NF.B SIGNALING CIRCUITS - MODELING OF INTERACTIONS WITH P53 SIGNALING PATHWAY

Katarzyna Szołtysek¹, Joanna Łanuszewska¹, Piotr Widłak¹, Krzysztof Fujarewicz², Jarosław Śmieja², Marek Kimmel²

¹Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch in Gliwice, Poland; ²Institute of Automation, Silesian University of Technology, Gliwice, Poland

     Mathematical models are helpful in describing and understanding of multi-component regulatory modules related to cell signaling and control of gene expression. Mechanisms of cellular responses to stress that depend on NF.B and p53 transcription factors are of particular interest and several mathematical models have been proposed describing each of these two pathways. Both regulatory modules interfere with each other, however details of such interactions are not clear at the moment. Here we aimed to build a mathematical model that will base on experimental data and describe functional interaction between NF.B and p53 signaling pathways. We have used human HCT116 cell line stimulated with TNF. or UV radiation as an experimental model. Two isogenic lines with either functional or deleted p53 gene were compared. The kinetics of changes in levels of different components of the NF.B pathway was measured by Western blot and gel-shift methods in such stimulated cells. The experimental data were fitted to the mathematical model built by our group to study the influence of p53 status on the NF.B regulatory circuits.

This work was supported by the Ministry of Science, Grant KBN 3T11A01929.

21. GENOME-WIDE PREDICTION AND FUNCTIONAL ANALYSIS OF RAT GENES CONTAINING ISRE IN PROMOTER

Bogdan Tokovenko, Maria Obolenskaya

Laboratory of Protein Biosynthesis, Department of Translation Mechanisms of Genetic Information, Institute of Molecular Biology and Genetics, 03143 Kyiv, Zabolotnogo st.150, Ukraine

     Prediction of putative transcription factor binding sites (TFBS) has become an important resource to explore genome organization and predict transcriptional regulation. Computational TFBS prediction provides reliable results in application to prokaryotes and yeast, but in higher eukaryotes accurate and reliable TFBS prediction is an outstanding challenge. In this study, we searched for putative interferon-stimulated response elements (ISREs) in the promoters of protein-coding genes of Rattus norvegicus, as defined and annotated by Ensembl (release 40), and looked at the validity of the TFBS search in terms of biological meaning. The ISRE, a conserved regulatory element of all interferon-stimulated genes (ISGs), is the target for transcriptional activator ISGF3 (IFN-stimulated gene factor-3). Genes containing ISRE can be considered primary interferon-response genes, with the cautionary note on the ability of not only ISGF3, but also of IRFs to bind to ISRE. Finding genes which have biologically meaningful ISRE will allow to better understand the Jak-STAT activated cellular IFN response. A total of 23286 promoters of rat genes were analyzed. Promoter was defined as a sequence extending 1000 nucleotides up the TSS (transcription start site). ISRE position frequency matrix (PFM) was taken from the TRANSFAC 7.0 Public database, accession M00258. To compute matrix-site similarity scores, PFM was converted into position-weight matrix (PWM), with added pseudocounts. TFBS search with 80% threshold produced 5214 TFBS in 4571 promoters. In order to filter away biologically insignificant TFBS, we compared promoters of the orthologues rat and mouse genes. We looked for TFBS occurrence in orthologues genes with an additional constraint for the starts of found TFBSs to be no more than 25bp apart relative to the TSS of each gene. Applying orthology filter produced 850 TFBS in 768 promoters. Set of 768 genes was used for further analysis. Graph of the distribution of found ISREs start sites along the length of the promoter reveals three regions of ISRE localization: 0 to -250, -250 to -550, and above -550 relative to the TSS. It is not yet known whether ISRE TFBS localization has any functional implications. Gene Ontology (GO) categories enrichment analysis was conducted for the 768 gene set against all the rat protein-coding genes, using the GO Tree Machine (GOTM). 49 GO categories (34 in biological_process, 10 in molecular function, 5 in cellular_component) were relatively enriched, using hypergeometric test with p < 0.01 and no multiple-testing correction. Of these, a number of categories were expected based on the generic knowledge about interferon effects (15 categories: cell differentiation, cell cycle, viral life cycle, etc). Relation of other enriched categories to the effects of IFN is not straightforward. However, some expected categories were not significantly enriched, e.g. immune response (enrichment ratio R = 1.38, p = 0.07). Thus, orthology-based filtering of the initial TFBS search does increase the percent of biologically meaningful binding sites, but still is not a sufficient filter by itself. Next step of this research will be to lower the search threshold (to include more putative true-positives), to apply less stringent orthology filtering, apply learning algorithms (trained on known targets), and look for the characteristic TFBS motifs. Genes identified in this research as ISRE-containing will be used to seed the construction of the IFN-.-induced gene regulatory network.

POLYMORPHISM OF XPD312 GENE IN COLORECTAL CANCER PATIENTS INFLUENCES A REPAIR CAPACITY OF OXIDATIVE DNA DAMAGE

M. Wideł¹, A. Gdowicz-Kłosok¹, M. Kłosok², M.S. Wideł³, M. Kryj³, A. Wydmański⁴, R. Oliński⁵, J. Rzeszowska-Wolny¹

¹Department of Experimental and Clinical Radiobiology, Center of Oncology Gliwice; ²Department of Animal Physiology and Ecotoxicology, University of Silesia Katowice; ³Oncological Surgery Clinic; ⁴Department of Radiotherapy, Center of Oncology Gliwice, 5Department of Clinical Biochemistry, Collegium Medicum UMK, Bydgoszcz

     The removal or repair of DNA damage is an important factor for protection of genome integrity. It has been shown that XPD312 (codon 312) polymorphism is connected with lung and other type of cancer, mainly linked with environmental exposure to genotoxic agents. Less is known about XPD polymorphism in colorectal cancer, the malignancy showing permanent increase incidence in west-type countries. Our studies indicate that in this cancer XPD312 polymorphic variant plays a protective role.
     The aim of the presented study was to look for a possible mechanism responsible for increased risk of colorectal cancer in relation to XPD polymorphism. Comparison of the different polymorphic variants capacity to repair of radiation-induced DNA damage was performed on lymphocytes (using comet assay), and simultaneously, the efficiency of removal of oxidatively modified base (8-oxoGua) and nucleotide (8-oxodG) was studied in urine in 46 patients before treatment using HPLC/GS/MS technology.
     Our results indicate that wild type of XPDAsp/Asp312 gene is connected with the less efficient repair capacity of DNA damage, whereas changes of Asp to Asn in one or both alleles significantly increase it. XPD312 protein is ATPase/helicase which plays a role in nucleotide excision repair pathway (NER). The differences in the level of oxidatively damaged base and nucleotide may suggest that individuals differ in using BER or NER pathway for oxidative damage removal from the organism.

22. MODIFICATIONS OF DNA REPAIR ENZYMES

Alicja Winczura^1,2,3, M. Swoboda¹, D. Dudzińska⁴, J Janik.¹, M. Komisarski¹, B. Tudek¹

¹Department of Molecular Biology IBB PAS; ²Recipient of the Fellowship of President of Polish Academy of Sciences; ³Postgraduate School of Molecular Medicine; ⁴Institute of Genetics and Biotechnology, University of Warsaw

     Oxidative stress and lipid peroxidation (LPO) generates plethora of DNA lesions, among which 1,N6-ethenoadenine (.A) and 3,N4-ethenocytosine (.C) have high miscoding potential and may be engaged in carcinogenic process. Two major enzymes involved in excision of .A and .C are: ANPG (alkyl-N-purine DNA glycosylase) and TDG (thymine DNA glycosylase). Our previous studies showed that repair activity toward .C was lower in leukocytes of colon cancer patients than in leukocytes of healthy volunteers.
     We were looking for mutations and polymorphisms in hTDG as well as in hANPG genes of 42 colon cancer patients, in tumor and normal surrounding which did not show histological changes. hTDG and hANPG genes were screened in all exons using MSCCP method. No polymorphism was found in hANPG gene. In case of hTDG gene analysis revealed G/A substitution in exon 5 in normal and tumor tissues from 3 patients. Such substitution changes glycine199 to serine in protein sequence, but it does not change the enzyme activity as it has already been described.
     Activity of BER enzymes may be regulated by protein interactions and modifications. We have examined in vitro influence of oxidative stress products (hydrogen peroxide) and LPO (trans-4-hydroxy-2-nonenal; HNE) on BER proteins- ANPG, HAP1 and Mug.
     HAP1 protein as revealed by mass spectrometry was modified by HNE on residues 93Ser, 52Lys and 151His. Such modification does not have an impact on HAP1 endonuclease activity toward depurinated plasmid. HNE does not influence Mug activity either.
     We observed decrease of ANPG protein activity by HNE. This can be explained by the fact that HNE adduct was found at 136His located in enzyme.s active center. It is possible that such modification may be relevant in living cells.Additionally ANPG was treated with H2O2, which decreased enzyme.s activity only at very high, non-physiological hydrogen peroxide concentrations. For this reason the H2O2 impact on ANPG protein might not be important in vivo.
     These results suggest that changed activity of TDG and ANPG does not depend on mutations or polymorphisms whereas impact of LPO products on ANPG activity may be an important factor.

23. TYPE III HISTONE DEACETYLASES INHIBITION INCREASES THE RATE OF DNA-PK-INDEPENDENT DSB REPAIR

Maria Wojewódzka¹, Marcin Kruszewski¹, Iwona Buraczewska¹, Weizheng Xu², Edmond Massuda², Jie Zhang², Irena Szumiel¹

¹Department of Radiobiology and Health Protection, Institute of Nuclear Chemistry and Technology, 03-195 Warszawa, Dorodna 16, Poland; ²MGI Pharma, Inc., Baltimore, MD 21224, USA

     In mammalian cells sirtuins (type III histone deacetylases, HDAC III), dependent on NAD+ and inhibited by nicotinamide are coded by homologues of the yeast gene SIR2. Histone deacetylases (HDAC) are an important member of a group of enzymes that modify chromatin conformation. In yeast cells, Sir2 participates in repression of transcriptional activity and in DNA double strand break repair It is assumed that certain sirtuins may play a similar role in mammalian cells, by modifying chromatin structure and thus, altering the accessibility of the damaged sites for repair enzymes. It has been postulated by J. Zhang et al. (Bioessays, 2003, 25(8):808-814) that there is a relation between poly(ADP-ribosylation) and sirtuin function in cells with damaged DNA. Interconnections between NAD+ metabolism, poly(ADP-ribosylation), DNA repair and gene expression should allow to modulate the cellular response to agents that damage DNA.
     We investigated the role of sirtuins in DSB repair in a pair of Chinese hamster ovary (CHO) lines: wild type (WT) and radiation sensitive, DSB repair defective mutant line, xrs-6. The latter is defective in DNA-PK.mediated DSB repair pathway due to the deficiency in Ku80 protein. Cells were incubated with sirtuins inhibitor 200 .M GPI 19015/1 (gift from prof. J. Zhang (MIT, USA)) at 37oC for 1 h, X-irradiated with 10 Gy and allowed to repair DNA breaks for 30, 60 and 120 min) at 37oC. The remaining DSB were estimated by the neutral comet assay. We observed the mild effect of GPI treatment on the repair kinetic in both cell lines, but in DSB repair defective mutant cell line, the increase of DNA repair is more pronounced. There, the effect was most marked in G1 phase and practically absent in S and G2 cell cycle phases. The decrease in number of histone .H2AX foci was consistent with repair kinetics measured with the neutral comet assay. The altered repair rate did not affect survival of X-irradiated cells, as estimated at ca 50 % level. Since in G1 xrs6 cells the DNA-PK-dependent non-homologous end-joining, D-NHEJ, does not operate, these results indicate that inhibition of sirtuins modulates DNA-PK-independent (backup) non-homologous end-joining, B-NHEJ. So, B-NHEJ is the DSB repair system affected by sirtuin inhibition to a greater extent than other DSB repair systems.