|
|
|
|
|
|
|
|
||||
|
|
|
Gliwickie Spotkania Naukowe 2008
Mustafa B A Djamgoz A ‘neuroscience’ approach to understanding the pathophysiology of cancer has revealed that acquisition of metastatic potential in carcinomas, including prostate cancer (PCa) and breast cancer (BCa), involves up-regulation of voltage-gated sodium channels (VGSCs) and concomitant down-regulation of potassium channels. Thus, metastatic cancer cells are electrically excitable and, indeed, respond to depolarizing stimuli with regenerative activity. VGSCs are also expressed in vivo and blocking VGSC activity suppresses a range of cellular behaviours integral to the metastatic cascade. Thus, VGSCs are a novel cancer biomarker and therapeutic target. In on-going work, we are investigating the possible underlying cause(s) of the VGSC upregulation in BCa and PCa in a parallel approach since these cancers share many similarities, including hormone sensitivity and metastatic sites (eg bone). Our working hypothesis is that VGSC upregulation occurs at the transition from hormone to growth factor dependency. Most work has been done on epidermal growth factor (EGF) [1,2]. Inhibiting endogenous EGF receptor tyrosine kinase activity with AG1478 suppressed VGSC expression whilst exogenous EGF had the opposite effect. Importantly, the effect of exogenous EGF effect was blocked significantly by co-treatment with the highly specific blocker of VGSCs, tetrodotoxin (TTX), consistent with the following basic scheme: EGF → VGSC upregulation →
enhancement of metastasis A markedly different effect was obtained with insulin/insulin-like growth factor receptor. In the presence of insulin, TTX increased migration of MDA-MB-231 human BCa cells. Thus, VGSC activity would accelerate or decelerate metastatic cell behaviour depending on the biochemical constitution of the extracellular medium. It is concluded (i) that functional VGSC expression is controlled by growth factor, especially EGF receptor signalling and (ii) that VGSC activity can influence metastasis by enhancing cancer cells’ metastatic behaviour as well as homing into metastatic sites. References: 1. Ding
Y, Brackenbury WJ, Onganer PU, Montano X, Porter LM, Bates LF & Djamgoz
MBA (2008). Epidermal growth factor upregulates motility of Mat-LyLu rat
prostate cancer cells partially via voltage-gated Na+ channel
activity. J Cell Physiol. In the
press. 2. Uysal
Onganer P & Djamgoz MBA (2008). Epidermal growth factor potentiates
in vitro metastatic behaviour of human prostate cancer PC-3M cells:
Involvement of voltage-gated sodium channel. Mol Cancer. In the press. Biologically active pyrazines,
the past and the future Martin Doležal Dept. of Pharmaceutical Chemistry and Drug Control, Faculty of
Pharmacy in Hradec Králové, In plants or insects,
pyrazines play the roles of attractants, feromones and signal substances.
Similar substances were found in food and therefore their sensoric properties
were investigated. Pyrazines are also synthesized by a number of fungi, such
as antibiotic aspergillic acid and fungicidal pigment pulcherrimin. Synthetic pyrazines are
used as additives in food manufacture and in tobacco industry. Some of them are
important pharmaceuticals such as sulfonamides sulfapyrazine and sulfalene,
antituberculotic pyrazinamide,
potassium-sparing diuretic amiloride,
drug for smoking addiction treatment varenicline,
hypolipidemid acipimox,
peroral antidiabetic glipizide,
hypnotic eszopiclone,
potential anticancer pyrazine
diazohydroxide and bortezomib,
antiviral 6-fluoro-3-hydroxypyrazine-2-carboxamide. Supporting grant: MSM0021620822 References: 1.
Doležal,
M. Biologicky aktivní pyraziny přírodního a syntetického původu. Chem. Listy 100 (11), 959-966 (2006). 2.
Brown
D.J.: The Pyrazines. J. Wiley & Sons, Inc., THE ROLE OF MITOCHONDRIAL ION CHANNELS IN
ISCHEMIC PRECONDITIONING Krzysztof Dołowy Department Biophysics, Warsaw University
of Life Sciences – SGGW, Nowoursynowska 159, 02-776 Warszawa, Poland. Lack of oxygen (ischemia) kills heart and brain cells after oxygen supply is restored. The repeated episodes of limited oxygen supply to heart and brain cells make them less sensitive to apoptosis (ischemic preconditioning). The molecular mechanism of ischemic preconditioning is not known. The surprising observation that the mitochondrial ATP regulates potassium channel opener protects heart from apoptosis due to ischemia pointed to mitochondria as a key player in the phenomenon. Three mechanisms were suggested. Reactive oxygen species (ROS) production in mitochondria after ischemia is one. The failure of volume regulation of mitochondria during ischemia is the second. And the release of calcium from mitochondria is the third. It is suggested that activation of mitoKATP, mitoBKCa and blocking of mitoCl channels decrease production of ROS after restoration of oxygen supply due to decrease of electric component to proton motive force. This work was supported in part by the Polish Mitochondrial Network
MitoNet.pl and the grant from the President of WULS-SGGW GLYCATION
OF PROTEINS AS THEIR MODIFICATION IN PHYSIOLOGY Gamian Andrzej 1,2, Pietkiewicz Jadwiga 2, Szeja
Wiesław 3, Bartyś Arkadiusz 1 1 Institute of Immunology and Experimental Therapy, Polish
2Department of Medical Biochemistry, 3Department of Chemistry, The advanced glycation end products (AGE) formed during spontaneous reaction of aldehydes and proteins accumulate in tissues and modify several proteins. Glycation is currently considered as a physiological process, whereas the excessive level of accumulating AGEs is correlated with diabetic complications. The group of glycation products is very heterogeneous and only few of them are identified so far, like pentosidine, argpyrimidine, carboxymethyllysine, contributing to only few percent of total products [1]. There are no tests practically that could be in routine use in analytical laboratories for measuring the AGEs in biological samples, while their quantization may serve as a useful marker for monitoring progression of certain pathological processes. Usually, model glycation end products are synthesized from glucose or from such degradation products like glyoxal, methylglyoxal, 3-deoxyglucosone [2]. However, the sera obtained after immunization with a mixture of glycation products possess antibodies in low titer against AGEs [3]. Therefore it is necessary to find the common and specific products of glycation that are formed in human tissues. Synthesis of such products would allow to obtain antibodies for test for glycation [1,2]. In our laboratory a few methods were applied to obtain a panel of model glycation products from several sugars [4]. These procedures involved high pressure glycation (HPG), high temperature (HTG) and also ultrasonic waves (UWG) and microwaves reactor (MWG). The fractionated products were used to prepare rabbit anti AGE sera, which did not recognize products formed from routinely used sugars for glycation and intermediates of Maillard reactions. It appeared in the immunoblotting experiments, that the epitopes on cross-linked glycation products formed in water solutions differ from those originating in dry conditions [3]. The specific immunochemical tests have been elaborated for the determination of protein AGE, anti-AGE antibody and immune complexes. Immunochemical experiments revealed the distinct lower level of circulating serum AGEs in patients with Alzheimer’s disease, in relation to healthy controls [5]. References: 1.
Al-Abed
Y, Kapurniotu A, Bucala R, Meth. Enzymol. 1999, 309, 152-171. 2.
Biemel
KM, Reihl O, Conrad J, Lederer MO, J.
Biol. Chem. 2001, 276,
23405-23412. 3.
Turk
Z, Ljubic S, Turk N, Benko B, Clin.
Chim. Acta 2001, 303, 105-115. 4.
Staniszewska M,
Jarosz S, Jon M, Gamian A, Arch. Immunol. Ther. Exp. 2005, 53, 71-78. 5.
Staniszewska M,
Leszek J, Małyszczak K, Gamian A, Int. J. Geriatr. Psychiatry 2005, 20,
896-897 TOWARDS A GENE EXPRESSION PROFILE TO
CLASSIFY PATIENTS Micheline Giphart-Gassler Department of Toxicogenetics, LUMC, The Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. These late effects limit the dose of radiotherapy. We postulate that variation in the incidence and severity of late complications is at least partly determined by intrinsic genetic differences between individuals. Such variation might be reflected by a difference in genetically regulated responses to radiation such as transcription. Our ultimate goal is to predict the severity of normal tissue damage after radiotherapy by transcription profiling. Assuming that genetic differences between individuals affect the transcriptional response to radiation of all cells, we have chosen T-lymphocytes as a surrogate tissue. We have determined the differences in gene expression after 2Gy in lymphocytes of patients treated for prostate cancer. This difference was used to discriminate between patients with severe late radiation toxicity from patients without complications following radiotherapy. By using a random cross validation strategy, a gene expression profile (classifier) was found that correctly classified 63% of the patients. A better performance was obtained by taking functional gene sets based on gene ontology for classification. Although the results were promising additional studies are needed, also because less correct classification was obtained of patients in an independent validation set. Classical cellular responses to in vitro radiation at a dose of 2Gy are dominated by cell killing and a p53-dependent transcriptional response. The in vitro response to 2Gy in lymphocytes shows large variations between individuals. Interestingly it is not the p53-dependent response that is different between patients groups differing in late toxicity. We propose that late radiation toxicity reflects more sub-lethal effects of radiation in normal tissue. Therefore we hypothesize in the Genepi-lowRT program that in vitro irradiation with low dose ≤100mGy evokes a transcriptional response that relates closer to the late complications to radiotherapy. We intend to identify by micro array analysis, genes or gene sets of which expression after low dose radiation can discriminate between breast cancer patients of the Genepi cohort that differ in normal tissue toxicity. As a first step towards this goal we have analysed gene expression patterns of T-lymphocytes of 2 normal individuals after radiation with a range of low doses to detect possible low-dose specific pathways, dose response relationships and to compare low and high dose transcriptional responses. MOBILE
DNA ELEMENTS THROUGHOUT EVOLUTION Jerzy Jurka Genetic Information Research Institute Eukaryotic genomes contain vast deposits of so-called “repetitive DNA,” derived mostly from active or extinct transposable elements (TEs). Analysis of genomic sequences revealed a wealth of information about the origin, diversity and genomic impact of these elements. DNA sequence analyses revealed new classes of TEs, including Helitrons, Polintons, and numerous superfamilies. TEs are evolutionary precursors of many genes, including the RAG1 protein gene in our immune system. They are the driving force in the evolution of epigenetic regulation and they have long-term genetic impact on genomic stability and evolution. Remnants of ancient TEs appear to be overrepresented in regulatory modules, which raises considerable interest in their impact on gene regulation envisioned decades ago by Britten and Davidson. I will present an overview of recent studies of TEs with emphasis on their evolutionary role in gene regulation and speciation. RESPONSE
OF TUMOUR VASCULATURE TO VASCULAR DISRUPTING AGENTS AND RADIATION Chryso Kanthou, Sara Jane Lunt, Gillan M. Tozer Cancer Research UK Tumour
Microcirculation Group, Academic Unit of Surgical Oncology, School of
Medicine and Biomedical Sciences, University of Sheffield, Beech Hill Road,
Sheffield, S10 2RX, United Kingdom. The tumour vessel network is essential for tumour growth and metastasis and is therefore an important target for therapy. Vascular Disrupting Agents (VDAs) are a relatively new class of low molecular weight drugs that cause necrosis of the tumour mass by inducing selective, rapid and profound disruption of established tumour blood vessels. Combretastatin A-4-Phosphate (CA-4-P) is a lead tubulin-binding VDA, with tumour selectivity currently undergoing advanced clinical development. CA-4-P leads to a rapid rise in vascular permeability within the tumour, which is thought to be central to the mechanism by which blood flow shutdown and vascular collapse occur. Susceptibility to CA-4-P and other VDAs is ascribed to the “immature” nature of blood vessels within the tumour, which have defective pericyte coverage and are highly permeable. Although VDAs induce substantial tumour necrosis, on the whole they are ineffective at halting tumour growth when used as single agents, as tumour re-growth initiates from the peripheral tumour outer rim, which is generally resistant to this type of therapy. Recently, progress has been made when using VDAs in combination with conventional radiotherapy, which targets tumour cells directly. Significant improvements in tumour growth delays have been reported in pre-clinical models and this combination is also undergoing clinical testing with some positive outcomes. The success of this treatment combination is thought to be due to selective targeting of cells within the viable and better-oxygenated tumour outer rim by radiation, while VDAs are more effective within the central hypoxic regions of a tumour. Optimal responses have been shown to be dependent on scheduling. Indeed, radiation is more effective if administered before the VDA, which inevitably results in hypoxia. In addition to targeting tumour cells, radiotherapy also damages blood vessels, and therefore potential interactions between radiation and VDA effects on the tumour vasculature exist. At the cellular level, both CA-4-P and radiation cause rapid remodelling of the endothelial cytoskeleton, and a rise in permeability, and these events occur through similar signaling mechanisms involving activation of RhoA-GTPase signaling pathways. This presentation will evaluate current knowledge of interactions between VDAs and radiotherapy and will address issues of resistance and susceptibility. Understanding the mechanisms by which radiation and VDAs interact to target tumour blood vessels is important in order to maximise the efficacy of this therapeutic strategy. Funded by Cancer Research UK CHARACTERIZING PROTEIN POST TRANSLATIONAL
MODIFICATIONS USING MALDI MASS SPECTROMETRY Rachel Martin Shimadzu Biotech, Post translational modifications (PTMs) are critical to the understanding of proteins in biological systems. They can include, amongst others, the addition of functional groups, the addition of other peptides or proteins, structural changes or substitution/delete/inclusion of amino acids. They are particularly relevant for study as they may alter physical and chemical properties, folding, conformation distribution, stability, activity, and consequently, function of proteins. Examples include phosphorylation for signal transduction, ubiquitination for proteolysis, attachment of fatty acids for membrane anchoring and association, and glycosylation for protein half-life, targeting, and cell:cell and cell:matrix interactions. In this presentation, we investigate the use of MALDI mass spectrometry for the analysis of post translational modifications. We demonstrate not only how the nature of the modification, for example oxidation, phosphorylation or glycosylation, can be determined, but also how the position of the modification can be identified. We will show examples using MALDI TOF, MALDI TOF/TOF and MALDI Ion Trap TOF data on a wide variety of PTMs. THE
TARGETING OF MELANOMA FOR ENDORADIOTHERAPY Walter Mier, Michael Eisenhut, Uwe Haberkorn Universitätsklinikum
Heidelberg, Radiologische Klinik, Abt. The incidence of malignant melanoma continues to
rise. In the Benzamides have been found high and long lasting uptake in melanoid structures of the uveal tract of pigmented C57Bl6 mice. This observation was especially prominent using [125I]BZA (N-(2-diethylaminoethyl)-4-[125I]iodobenzamide) and the B16 melanoma mouse model. It was, therefore, attractive to investigate whether the carrier characteristics of N-(2-(diethylamino)ethyl)benzamide derivatives or compounds combined with the 2-diethylaminoethylamine pharmacophore can be exploited for selective cytostatic drug delivery. Indeed, radiolabeled conjugates of chlorambucil with procainamide (CHL-BZA) and 2-diethylaminoethylamine (CHL-DEAE) revealed high uptake in B16 melanoma cells. The selectivity was confirmed with biodistribution studies in the B16/C57Bl6 mouse melanoma model. The reason for benzamide-melanoma selectivity may be found in the embryonic origin of melanocytes. They are derived from the neural crest, which is a completely different source than that of the surrounding keratinocytes. Melanoma cells resemble, therefore, nerve cells with similar transporters for basic drugs. Owing to our clinical results that radioiodinated BZA can be used as an imaging agent for metastatic melanoma screening experiments with a number of benzamide derivatives have been performed. BYSTANDER EFFECTS AND ADAPTIVE RESPONSES INDUCED BY RADIATION
EXPOSURE This presentation reviews our current knowledge of the mechanisms underlying the induction of bystander effects by low dose low LET ionizing radiation and discusses how they may be related to observed adaptive responses or other protective effects of low doses exposures. Bystander effects appear to be the result of a generalized stress response in tissues or cells. The signals may be produced by all exposed cells but the response appears to require a quoram in order to be expressed. The major response involving low LET radiation exposure discussed in the existing literature is a death response. This has many characteristics of apoptosis but is p53 independent. While a death response might appear to be adverse, the position is argued in this paper, that it is in fact protective and removes damaged cells from the population. Since many cell populations carry damaged cells without being exposed to radiation (so called “background damage”, it is possible that low doses exposures cause removal of cells damaged by agents other than the test dose of radiation. This mechanism would lead to the production of “U-shaped” dose response curves. In this senario, the level of “adaptive” or beneficial response will be related to the background damage carried by the cell population. This model may be important when attempting to predict the consequences of mixed exposures involving radiation and other environmental stressors. IN SILICO STUDY SUGGESTS THAT
RALTEGRAVIR-RESISTANT MUTATIONS MODIFY THE DNA RECOGNITION PROPERTIES OF
HIV-1 INTEGRASE Jean-François Mouscadet
LBPA, CNRS, Ecole Normale
Supérieure de Cachan, France Raltegravir
is the first effective antiretroviral agent belonging to the novel class of
HIV- We
observed that the native and mutant INs show a similarity for general enzyme
folding. We found that the 140-149 catalytic loop is characterised by
a striking conservation of a Ω-shaped hairpin, a structural element involving
144-148 residues, stabilised by multiple H-bonding spanning across the
loop. Folding
of the hairpin is due to the strong conformational preferences of the N, P,
Q, S and G residues. This hairpin which can move from 16 to 4.5 Ǻ
towards the active site as a rigid body in a gate-like manner remains
topologically invariant with respect to the presence of mutations selected under
raltegravir. In contrast, there was a striking difference between the
wild-type and mutant INs in regard to their specific recognition by DNAbases.
The native N155 and Q148 show a clear preference for binding with adenine,
interacting by pair of strong H-bonds whereas the R, K and H mutant strongly
favor pyrimidines. Furthermore, we observed that the secondary mutation G140S
which is readily observed following the selection of the primary Q148(H/R/K)
mutation, modified the mobility of the catalytic loop, thereby probably
reflecting an adaptation to the change of base specificity induced by
Q148R/H/K mutation. This alteration provides a molecular explanation
for Raltegravir inhibition. Indeed, we observed that this compound is an
adenine bioisoster, thus capable to compete with the 5’-AC overhang for the
close contact with Q148 that was experimentally observed. The loss of this
contact is compensated by the H/R/K148 mutant which retains DNA recognition,
while these mutations impairs Raltegravir binding to IN. Finally,
we observed that two Raltegravir fragments act as Adenine thereby suggesting
two possible independent contacts with Q148 and N155, thus providing a
rational for the two independent resistance pathways. CITRATE TRANSPORT IN HUMAN PROSTATE
CANCER CELLS: REGULATION BY FUNCTIONAL VOLTAGE-GATED NA+ CHANNEL
EXPRESSION Maria E. Mycielska and Mustafa B. A. Djamgoz Division of Cell &
Molecular Biology, Neuroscience Solutions to Cancer Research Group, Sir Alexander Fleming
Building, Imperial College London, South Kensington Campus, London SW7 2AZ,
UK Prostate
gland is a unique mammalian organ which produces and releases large amounts
of citrate into prostatic fluid (up to FOLATE, AMINOTHIOLS AND CYSTATHIONINE-Β-SYNTHASE IN HUMAN
PLACENTA Maria Obolenskaya Institute of Molecular Biology and Genetics, Zabolotnogo str. 150, Folate-mediated one-carbon unit metabolism has been a topic of discussion in the obstetrical community for several decades since maternal folate deficiency was related to fetal central nervous system malformations and periconceptual folate supplement was assessed to diminish these manifestations [Ueland, Vollset 2004; van der Put et al., 2001; Finkelstein 2000a]. Folate is present in the organism as a family of structurally related derivates of tetrahydrofolate (THF). In mammalian tissues folate, the generic term, functions as substrate in series of interconnected metabolic cycles involving thymidilate and purine (adenine and guanine) biosynthesis, methionine (Met) cycle, serine and glycine interconversion and metabolism of histidine and formate [van der Put 2001; Suh et al, 2001]. Thus, folate indirectly is involved in essential cell functions, including biosynthesis of nucleic acids and proteins, transmethylation reactions, maintenance of cellular redox status by folate-dependent aminothiols. The mammals cannot synthesize folate de novo and obtain it from the diet and intestinal flora. Methionine cycle is a heart of folate-related metabolism. It is tightly associated with THF cycle. 5-MeTHF after donation of its CH3 group to Hcy remethylation converts into THF that accepts one-carbon unit in result of serine to glycine interconversion catalyzed by serine hydroxyl methylase while synthesis of glycine and 5,10-methylene THF. 5,10-methyleneTHF is reduced to 5-MeTHF by the key enzyme of this cycle - methylenetetrahydrofolate reductase. MTHFR is a polymorphic enzyme. Its alleles with C677 ® T677 or alanine 222 to valine substitutions are highly representative in Caucasian population [Frosst 1995]. This mutation is associated with thermolability and lower catalytic activity of MTHFR, elevated Hcy and lower plasma folate and increased risk of neural tube defects [De Franchis 1995, van der Put 2000, Shields 1999]. Methionine cycle is intimately connected with transsulfuration pathway via homocysteine (Hcy) that may follow either remethylation in methionine cycle and conserve methioine or irreversible catabolism by consequent activities of cystathionine b-synthase (CBS) and cystathionase γ-lyase while synthesis of cysteine used for protein synthesis and/or catabolized to taurine and sulfate. The inability to eliminate Hcy by transsulfuration may make the corresponding tissues especially vulnerable to elevated concentration of Hcy that is considered as a potential marker and cause or contribute to a wide range of obstetrical disorders. The active role of folate related metabolism in human placenta will be discussed on the basis of our own research. MOLECULAR ORGANIZATION IN SELF-ASSEMBLED
MONOLAYERS USED FOR NEURONAL CELL GROWTH Smart Sensors and Integrated Microsystems
(SSIM), Department of Electrical and Computer Engineering, Department of
Biomedical Engineering, The main goal
of the presented project is to develop material surfaces that may be
recognized as biological and can form truly biocompatible interfaces with the
tissue. We are developing two-dimensional (2-D) and three-dimensional (3-D)
scaffoldings to connect neurons over long distances and to investigate neuron
interfaces with microsystems by using biocompatible, self-assembled
monolayers (SAMs) to direct and control the growth of neurons. To accomplish
this goal we need to be able to control the growth of neural tissue as well
as the locations and patterns in which the cells grow. A significant
finding in our preliminary research on the patterned growth of neurons on the
surface of micro electrode arrays has shown that we have the ability to
encourage the selected growth of neural cells into specific micro patterns.
Neuron adhesion efficiency was studied for amino-terminated,
carboxy-terminated and 1:1 mixed alkanethiol SAMs deposited on gold
substrates. By varying both the specific chemistry and pattern size we have
been able to precisely control the growth of the neurons to predetermined 2-D
locations. Using photolithography techniques we have been successful in finding
optimal features for positioning cells. To assess the suitability of SAMs for
neuronal growth, we have investigated correlations of neuron adhesion with
the chemical structure at the SAM surface characterized by Sum Frequency
Generation (SFG) vibrational spectroscopy and with the roughness of gold
substrate and thickness of SAM monolayers studied by X-ray spectroscopy (XPS)
and atomic force microscopy (AFM). We conclude that the neuronal cells
adhesion is not critically affected by the surface roughness within 0.7-2 nm
range or
details of the molecular ordering and orientation of the terminal amino
groups, but only on the chemical functionality displayed at the surface. This
approach can be used to create new methods that help map structure-property
relationships of biohybrid systems and lead to the possibility of utilizing
this method to direct neurons in 3-D scaffoldings to interconnect over
relatively long distances in a controlled and precise manner. This complex
system will be designed to aid in the understanding the neuronal growth and
behavior and may enable precise and localized neuron stimulation and
surveillance for both biological research and medical applications. APOPTIN AND ITS DERIVATIVES AS MOLECULAR TEMPLATES Soumya Panigrahi 1, 2, Emilia Wiechec 3, Subbareddy Maddika 4, Marek Los 5 1 Department of Physiology, Univ. 2 3 Institute of Human Genetics, 4 Department
of Therapeutic Radiology, Yale School of Medicine, New Haven, The BCR-ABL
chimeric oncoproteins are key factors responsible for development and
progress of Imatinib and apoptin had comparable effects on BcrAbl+ CML cells, whereas apoptin was also active on Imatinib-resistant cells. Finally, a computational algorithms for protein modeling to study the 3D structure of apoptin and its docking with Bcr-Abl at the molecular level was applied to gain further insight into mechanism of inhibitory action of apoptin. Our work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents. STATISTICAL PROCESSING OF DNA MICROARRAY
DATA - DETECTING SUBTLE CHANGES OF GENE EXPRESSIONS Joanna Polanska The For some DNA microarray datasets, standard methods of gene expression profile analyses fail to provide statistically significant conclusions, yet there is evidence that these datasets might potentially lead to discovering interesting genetic mechanisms. An example of such datasets are the results of experiments related to low dose irradiation which contain only subtle differential expressions and therefore need special methods of analyses. In the presentation we overview methods of tuning methodologies of DNA data analyses to the problem of detection of subtle (low level) differences and signals in DNA microarray data. The results are based on the analysis of existing DNA microarray datasets related to sensitivity of patients and cells to irradiation.We studied several possible hypotheses concerning developing procedures for detecting low level signals and/or differences in DNA microarray data. These hypotheses concerned different levels of DNA microarray data processing: (i) the level of probes and cells and the level of DNA microarray data quality control, (ii) the level of annotations of genes corresponding to probes, (iii) the level of signal processing and classification algorithms. In the research we have composed and verified algorithms of different structures, based on the analyzed datasets. The methodology for verification of the efficiency of different approaches was based on comparing predictive powers of classifiers with the use of multiple random validation tests. We observed that introducing various changes to constructions of classifiers, such as introducing rules of selections for genes, selecting genes by using quality control procedures or fitting classifiers to hypotheses concerning probability distributions of expression signals, may lead to differences of predictive powers of classifiers of the order of 10-20% of errors in the verification step. Acknowledgement: The work was partially supported by EU grant GENEPI TUMOR DEVELOPMENT MODEL UNDER ANGIOGENIC
SIGNALING WITH DEPENDENCE ON VESSEL IMPAIRMENT Jan Poleszczuk Faculty of Mathematics,
Informatics and Mechanics, Inst. Appl. Math. Mech., In adults the normal physiological role of angiogenesis is restricted to wound healing, menstrual cycle and pregnancy. Unfortunately, it is also essential for the successful growth and development of solid tumours. After reaching avascular dormant state tumour can grow further only by inducing host tissue to sprout capillary tubes which migrate towards and ultimately penetrate the tumour, providing it with a circulating blood supply and, therefore, an additional source of nutrients. Based on the idea that carrying capacity for any solid tumour depends on its vessel density Hahnfeldt, et. al created a mathematical model of tumour growth under angiogenic signalling. Dr. Judith Folkman who discovered process of tumour angiogenis proposed that tumour can be treated by influencing that process. On the basis of the Hahnfeldt, et. al model some protocols of antiangiogenic treatment were proposed. Unfortunately, recent studies show that tumour angiogenesis is highly pathological. Long lasting overexpression of proangiogenic factors (like VEGF) causes impairment and malfunction of newly formed vessels. We propose the model of vessel impairment in the process of tumour angiogenesis, which is based on the idea of the Hahnfeldt, et al. model. In the proposed model carrying capacity depends also on the process of vessel impairment. Simulation of the model solutions shows that tumour vascular dormant state can be reached in two different ways. In addition, in each case efficiency and effects of standard chemotherapy, antiangiogenic treatment and combined treatment are different. NEW INSIGHTS ON BYSTANDER SIGNALLING: THE
ROLE OF DNA DAMAGE SENSING AND REPAIR Kevin M. Prise Centre for Cancer Research & Cell Biology, Queen’s University Cells have evolved complex processes to maintain the stability of their genomes. In response to genotoxic stress, the DNA damage response (DDR) is activated whereby a series of interlinked sensor processes signal to a panel of repair pathways which can attempt to repair the damage. Recent studies have shown compelling evidence for the activation of DDR by ionising radiation even when radiation is not directly deposited in the DNA within the nucleus. Our own studies, using both charged particle and X-ray microbeams, have shown that bystander responses are activated when cells are not directly irradiated through the nucleus. A key modulating role is played by mitochondria under these conditions, probably acting as a source for reactive oxygen species production. In a range of studies we have started to elucidate the relevance of DNA damage and repair pathways in both irradiated and bystander cells. In previous work we have shown formation of γH2AX foci in bystander cells ~ 30 minutes after irradiation and its persistence for up to 48 hours in a range of cell types. This may be due to persistent formation of DNA damage in bystander cells, lack of removal of the γH2AX signal and/or compromised repair. From studies with repair deficient mutants it is clear that dsb repair type processes are involved in bystander cells as cells deficient in components of the non-homologous end-joining pathway show increased bystander responses. In further work, we now have evidence in bystander cells that the initial phosphorylation of H2AX is performed by the ataxia telangiectasia and Rad3-related protein (ATR) rather that ataxia telangiectasia mutated protein (ATM) or DNA dependent protein kinase (DNA-PK). This occurs predominantly in S-phase cells and supports the assertion that damage accumulation in bystander cells leads to stalled replication forks. After this initial ATR-dependent response, ATM dependent signalling plays a role downstream in determining overall biological response. Overall these studies are changing our views of radiation responses in cells and tissues leading to a more comprehensive appreciation of DDR in the maintenance of genomic stability. GENISTEIN
DERIVATIVES AND THEIR BIOLOGICAL ACTIVITY
1Department
of Tumor Biology, 2Pharmaceutical
Research Institute, 3Faculty
of Chemistry, Among health benefits of high genistein intake are
lowered incidence of cardiovascular diseases, prevention of osteoporosis,
attenuation of post-menopausal problems and cancer chemoprevention. Antitumor
activity of this isoflavonoid can manifest itself through a spectrum of
effects including cytotoxic, cytostatic and antiangiogenic action, induction
of tumor cell differentiation, prevention of metastasis, attenuation of
multidrug resistance, reactivation of relevant genes silenced by
methylation and others. Genistein
sensitizes cells to radiotherapy and selected cytostatics, thus it may be
considered as a drug for use in co We report here the
results of our study aimed to determine the molecular mechanism of
cytotoxicity of the highly active synthetic glycosidic derivative of
genistein, termed G21. We show that, in contrast to genistein, G21 exhibits
aneugenic activity and has the ability to alter tubulin dynamics, as well as
to destroy mitotic spindles. Cancer cells exposed to G21 arrest in mitosis at
G2/M phase and are eliminated through apoptosis and mitotic death.
Polymerization of tubulin in an in
vitro assay was found to be inhibited by G21, indicating that this
compound interacted with tubulin directly. To our best knowledge, G21 is the
first derivative of genistein able to efficiently affect microtubule
dynamics. These promising results prompted us to synthesize a novel series of
derivatives in which glycosidic moieties were conjugated with genistein
through 2-5 carbon linkers at C-7. Antiproliferative potential of these new
glycoconjugates of genistein is currently under study. Our results indicate
that some glycosides and glycoconjugates of genistein exhibit higher
cytotoxicity, as compared to genistein, and exert their antiproliferative
action through mechanisms different from those of parent compound, genistein.
TRANSCRIPTION PROFILE CHANGE
AFTER IRRADIATION OR IN BYSTANDER CELLS; DIFFERENCES BETWEEN CELL LINES Joanna Rzeszowska-Wolny1,2, Robert Herok1,2,
Roman Jaksik2, Joanna Polańska2, 1Department of Experimental and Clinical Radiobiology, Maria Skłodowska-Curie Memorial
Cancer Center and Institute of Oncology, 44-100 Gliwice, Poland, 2Institute of Automation Control, Cells exposed to ionizing radiation release factors which can induce DNA damage, chromosomal instability and apoptosis in neighboring untreated cells, phenomena known as bystander effects. We used genome-wide microarrays to compare changes in transcript profiles of human cells grown in culture medium from irradiated cells (irradiation conditioned medium, ICM) with those which occurred in melanoma Me45 and lymphoblastoid K562 cells after IR. Under both conditions, among Me45 transcripts there were more than 10 000 genes present at increased or decreased levels, using the criterion of a >±10% change; and >85% of these were common to growth in ICM and after IR. For K562 cells more than 6 000 genes showed a changed transcript level under bystander as well as irradiated growth conditions, as compared to control cells; the level of more than 3 000 transcripts was higher than that in control cells and in more than 70% of these transcripts the level showed the same response (increase, decrease or no change) and only 0.6% showed an opposite response. Grouping of
transcripts into functional pathways showed that, under both examined
conditions, it was neuroactive ligand- and cytokine-cytokine-receptor
interactions as well as Jak-STAT, MAPK, and other signaling pathways that contained
the largest number of up- or down-regulated transcripts. The similarities
between the responses of the transcriptome in bystander and irradiated cells
and the kinetics of some transcripts’ change were confirmed by quantitative
RT-PCR. The level of transcript groups in some cell-cell communication and
signaling pathways was decreased less during growth in ICM or after
irradiation compared to control cells, an effect of possible importance for
long-term bystander effects such as genome instability. SOMATIC
EXOCYTOSIS OF SEROTONIN MEDIATED BY MOLECULAR MOTORS I. Santamaría-Holek 1Facultad de Ciencias, Universidad Nacional Autónoma de
México, Circuito exterior de Ciudad Universitaria. 04510, D. F., México, 2Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, Circuito exterior de Ciudad Universitaria. 04510, D. F.,
México, 3Facultat de Física, Universitat de Barcelona, Av.Diagonal
647, 08028, Barcelona, We
quantified somatic exocytosis of serotonin in Retzius neurons and explored
the possible contribution of molecular motors and the cytoskeleton on the
mobilization of vesicles induced by stimulation with trains of electrical
impulses. Secretion was quantified
from the increase of fluorescence of FM1- Stimulation of neurons produced a gradual increase in FM1-43 fluorescence for over the next five minutes. The kinetics and latencies of these increases varied from one neuron to another but usually maintaining a sigmoidal shape in one or two steps. Neurons stimulation in the presence of colchicine to uncouple microtubules, failed to evoke fluorescence increases, thus suggesting that vesicle mobilization depended on tubulin-based motor. The kinetics of the fluorescence increase in individual neurons was accounted for by using a model based on a diffusion equation in the presence of external forces, consistent with the contribution of molecular motors to the mobilization of the vesicle clusters towards the membrane in response to electrical activity. Our data show that somatic serotonin secretion in Retzius neurons depends on a motor-based cytoskeletal mobilization of vesicles induced by electrical activity. IDENTIFICATION OF THE
DETERMINANT OF HEPATITIS B VIRUS LIVER TROPISM AND ITS IMPLICATIONS FOR
HEPATOCYTE-SPECIFIC DRUG TARGETING Alexa Schieck1, Andreas Schulze2, Thomas Müller1, Uwe Haberkorn1, Stephan Urban2, Walter Mier1 1Department
of Nuclear Medicine, University Clinics of 2Department
of Molecular Virology, University Clinics of A hallmark of Hepatitis B Virus (HBV) infection are the
extraordinary specificity and efficiency by which virions target hepatocytes
in the liver. This property has been attributed to (a) specific
interaction(s) of (a) viral envelope protein(s) with a yet unknown receptor.
We have recently shown that the subcutaneous administration of low doses of a
myristoylated peptide corresponding to the N-terminal 47 amino acids (a.a.)
of the preS1-domain of the HBV large surfaceprotein blocks HBV infection in
an animal model (Nat. Biotech., 2008). Our observation that this peptide
selectively targets the liver of non HBV-susceptible mice suggests that it
encompasses a species-independent determinant of hepatotropism. We characterized the compulsory a.a. sequence and the role of
the fatty acid modification with respect to their contribution to target the
peptide to hepatocytes and analyzed the kinetics of uptake of fluorescently
labeled derivatives in vivo. Using a series of HBVpreS lipopeptides carrying
deletions, point mutations, D-amino acid exchanges, sequence permutations and
lipid variations we found that (i) N-terminal acylation prevents renal
secretion of the peptide and leads to systemic retention, (ii) a highly
conserved Aside from important implications concerning the species specificity of receptor recognition of HBV, HBVpreS-mediated drug targeting opens a highly selective approach to deliver drugs to hepatocytes or hepatoma cells. Possible applications include the delivery of interferons, inhibitors of HCV or HBV replication, cell cycle inhibitors for HCC treatment, inhibitors of plasmodium falciparum, siRNAs or peptides for MHC-mediated antigen presentation. SYNTHETIC GLYCOMIX FOR MINING NATURAL PRODUCTS AS DRUG LEADS Wiesław Szeja1,
Grzegorz Grynkiewicz2, Jadwiga Zawisza1, Aleksandra Rusin3, 1Silesian 2Pharmaceutical Research Institute Warszawa, 3Institute of Oncology, Majority of proteins are post-translationally modified by glycosylation, which enhances their proper folding and stability, and also influences their functioning. With all due proportion, it can be postulated that low molecular weight ligands, designed as drugs, can also be advantageously modified by chemical glycosylation using natural or synthetic sugars. Suitably selected sugar moieties can render pharmacophoric structures properties desirable in a pro-drug category. In particular, they can serve as: i) an active transport and biodistribution enhancers; ii) protection against phase II metabolism bioconjugation leading to excretion; iii) providers of a lipophilicity element needed for membrane, receptor and binding pockets docking [1]. Several classes of plant polyphenols (flavonols, catechins, isoflavones etc.) are currently studied as antioxidants, detoxicants, chemoprotectants, immunomodulators, antitumor agents, regulators of lipids metabolism and cardiovascular health promoters, but only few lead compounds have reached clinical trials level [2]. Apparently, the main reasons for slow progress in pharmaceutical development of these secondary metabolites in their native state are their sub-optimal physicochemical properties, low bioavailability and unfavorable metabolism. We focused our attention on soy isoflavonoids which are drug candidates in antitumor therapy and started a research program extending from chemical derivatization of genistein to biological activity studies of its new derivatives. From this perspective, of particular interest are regio- and stereoselective syntheses of O-glycosides, and glycoconjugate derivatives of 2,3-unsaturated mono- and disaccharides with genistein. The effect of synthesized compounds on proliferation of selected cancer cell lines will be discussed in some detail. References 1.
G. Grynkiewicz,
W. Szeja, J. Boryski, Synthetic analogs of natural glycosides in drug
discovery 2.
R.A. Dixon,
Isoflavonoids: Biochemistry, Molecular Biology and Biological Functions, MITOCHONDRIAL POTASSIUM CHANNELS Adam Szewczyk Laboratory
of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Mitochondrial potassium channels are believed to contribute to cytoprotection of injured cardiac and neuronal tissues. The following potassium channels have been described in the inner mitochondrial membrane: the ATP-regulated potassium channel, the large conductance Ca2+-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the twin-pore domain TASK-3 potassium channel. The putative functional roles of these channels include changes in mitochondrial matrix volume, mitochondrial respiration, and membrane potential. In addition, the activity of these channels modulates the generation of reactive oxygen species by mitochondria. In this presentation, we discuss recent observations on three fundamental issues concerning mitochondrial potassium channels: (i) their molecular identity, (ii) their interaction with potassium channel openers and inhibitors, and (iii) their functional properties. PROTEOMICS:
AUTOMATED ANALYSIS OF ELECTROPHEROGRAMS
Beata Walczak
Two dimensional gel electrophoresis is a powerful technique of protein separation, based on two independent properties of proteins, i.e., on the differences in their isoelectric points and molecular weights. This method is used as a main tool for protein separation in proteomics, its goal being determination of protein composition in cells and tissues. In order to study differences in protein expression in healthy and diseased tissues, many samples ought to be compared in order to eliminate differences caused by natural diversity of samples. Identification of proteins responsible for a disease studied can be performed based on a comparison of the 2D gel digital images. This, however, requires effective approaches of image enhancement, warping, normalization and significance analysis. In the start-to-end approach proposed by us [1-4], all steps of data analysis are automated. Moreover, data analysis is performed at the pixels level, instead of the spots level [4, 5], thus eliminating main drawbacks of the existing software packages, i.e. with missing elements in the data table (due to the requirement of spots detection). Additionally, multivariate significance analysis is proposed, based on Partial Least Squares discriminant analysis with the randomization tests, which allows minimization of the false discovery rate. References: 1.
K.
Kaczmarek, B. Walczak, S. de Jong and B.G.M Vandeginste, Preprocessing of 2-D
gel electrophoresis images, Proteomics,
4 (2004) 2377-2389 2.
K.
Kaczmarek, B. Walczak, S. de Jong and B.G.M Vandeginste, Feature based fuzzy matching
of 2D gel electrophoresis images, Journal
of Chemical Information and Computer
Sciences, 42 (2002) 1431–1442 3.
K.
Kaczmarek, B. Walczak, S. de Jong and B.G.M Vandeginste, Matching of 2D gel electrophoresis
images, Journal of Chemical Information
and Computer Sciences, 43 (2003)
978–986 4. M. Daszykowski, I. Stanimirova,
A. Bodzon-Kulakowska, J. Silberring, G. Lubec and B. Walczak, 5. E.M. Faergestad, M. Rye, B.
Walczak, L. Gidskehaug, J.P. Wold, H. Grove, X. Jia, K. Hollung, U.G. Indahl,
F. Westad, F. van den Berg and H. Martens, Pixel-based analysis
of multiple images for the identification of changes: A novel approach
applied to unravel proteome patters of 2-D electrophoresis gel images,
Proteomics, 7 (2007) 3450-3461 |
|
|
|
Ostatnia aktualizacja strony
10/01/2008 o godzinie 15:09:02 |
