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39th Meeting of the Polish Biochemical Society Gdask 1620 September 2003

SESSION 2

Cellular signalling

Organized by J. Baraska, T. Paweczyk

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Lecture

154
Changes of T cell signaling in autoimmune diseases
Ewa Bryl
Zakad Immunopatologii, Akademia Medyczna w Gdasku, ul. Dbinki 7, 80-211 Gdask

The status of T cells is dependent on many factors, including the signals the cells are receiving from the environment, but also the expression of proteins, which are critical for the process of proper activation and signaling. T cells have to integrate many signals, and have the signaling machinery acting appropriately. Changed environment, as well as wrong signaling can result in skewed response of T cells. On the other hand the T cells can cause the pathology by themselves. In many autoimmune diseases T cells do not behave normally, the disturbances can include the well known, major T cell subpopulations: CD4+ and CD8+; however, also some recently described subpopulations of T cells (like CD4+CD28-, CD4+CD28lo or CD3+CD16+56+) are suspected to take part in the pathogenesis of certain autoimmune diseases. Regardless of the recurring question about the reasons (causes) and results (effects), studies of the T cells status can provide a lot of information about the pathomechanisms of autoimmune diseases. Two systemic autoimmune diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are known to have their pathogenesis related to skewed T cells behavior. A characteristic paradox related to T cells behavior exists in the RA. The common thinking about this (and other) autoimmune diseases include expectations that T cells are activated (or even over-activated) causing tissue destruction directly or by

influencing other cells. However, the experimental data about in vitro activation of T cells from peripheral blood of RA patients are quite opposite and indicate their proliferative exhaustion and senescence. The reasons for this paradox is not quite known, however emerging new data bring more insight into it. It is very important to understand that general T cell pool comprises many subsets of cells, and every of them can behave differently, therefore every subpopulation of T cells should be analyzed separately. From the immunologists point of view the most important for the wholesome immune response are CD4+ T cells, with their central role in the immune response. Paradoxically CD4+ T cells isolated from RA patients show the signs of early aging, confirmatory data including lower proliferation capacity measured by populations doubling or DNA incorporation; restricted diversity of T cells receptors; lower number of new thymic emigrants etc. The signal transduction of T cells is also impaired, including defective tyrosine phosphorylation, impaired MAP kinase pathway and increased calcium signal upon interferon g stimulation. Part of the lecture will be devoted to the description of newly discovered relations between the tumor necrosis factor (TNF, a proinflammatory cytokine, the level of which is elevated in RA) and regulation of the expression of CD28 molecule on the T cell surface.

155
NO the universal mediator in cardiovascular system
Stefan Chopicki
Department of Pharmacology, Medical College Jagiellonian University, Krakw

Lecture

Nitric oxide (NO), a molecule hitherto known as environmental pollutant, is now recognized as an ubiquitous mediator in cardiovascular, nervous and immune systems. NO is produced by a family of heme-containing monooxygenases, (nitric oxide synthases) during the oxidative metabolism of L-arginine to L-citrulline. Two of these isoforms are expressed constitutively, i.e. endothelial eNOS (NOS3) and neuronal nNOS (NOS1) whereas the third isoform, an inducible one iNOS (NOS2) is triggered by proinflammatory stimuli such as endotoxin or cytokines (IL-1, IFN-g, TNF-a). Biological responses induced by NO in cardiovascular system are mediated mainly by activation of

intracellular soluble guanylate cyclase, with subsequent elevation of intracellular cGMP, however, cGMP-independent effects are also reported. Importantly, biological action of NO is highly dependent on the cellular environment as it is a highly reactive molecule, which interacts with oxygen, transition metals, and reactive oxygen species (ROS), especially with superoxide anion. The interaction of NO and O2 results in a formation of peroxynitrite, that can further decompose into highly toxic hydroxyl radical (OH). In the presence of thiols peroxynitrite may be transformed to nitrate or S-nitrosothiols. There are numer ous enzymatic sources of O2 in the vascular wall which

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could be involved in decreasing the bioavailability of NO and formation of ONOO . These include NADPH oxidase, xanthine oxidase and mitochondrial respira tory chain. On the other hand, biological activity of O2 produced by these enzymes is under control of numerous antioxidant systems such as intra- and extra-cellular SOD as well as catalase, gluthatione peroxidase or others. Thus, there are multiple determinants of biological activity of endothelial NO and their importance in the physiology and pathology of cardiovascular system is increasingly appreciated. In healthy cardiovascular system endothelial NO released from endothelium by mechanical or chemical stimuli affords vasodilator,

anti-platelet, anti-leukocyte, anti-inflammatory and anti-atherosclerotic activities. It governs the proper functioning of cardiovascular system. On the other hand, the impairment of endothelial NO activity is involved in pathogenesis of a variety of cardiovascular diseases such as atherothrombosis or heart failure. Moreover, dysfunction of endothelial NO has recently gained diagnostic, prognostic and therapeutic significance in cardiovascular diseases. The mechanisms involved in the pathological derangement of endothelial NO activity are still not entirely clear and may involve increased oxidant stress, inadequate endothelial level of tetrahydrobiopterin, increased circulating level of endogenous NOS inhibitor (ADMA) and others. Lecture

156
Capacitative calcium entry mechanism in nonexcitable cells the role of cytoskeleton
Rafa Czajkowski, Berenika Targos, Dorota Supat, Pawe Pomorski, Jolanta Baraska

Pracownia Przekanikw Sygnaw, Instytut Biologii Dowiadczalnej im. M. Nenckiego PAN, ul. Pasteura 3, 02-093 Warszawa

In nonexcitable cells, voltage operated Ca channels 2+ are not expressed and Ca entry from the extracellular 2+ space is regulated by the concentration of Ca ions in the endoplasmic reticulum (ER) pool. The depletion of this pool (the first phase) causes opening of the specific, 2+ voltage-independent Ca channels (SOC channels) in 2+ the plasma membrane and Ca influx (the second 2+ phase), whereas refilling of the ER store blocks Ca entry. This biphasic process has been termed capacitative or store-mediated calcium entry (SMCE). Whereas the mechanism of the first phase is well known, the nature of the signal transmitted from 2+ the ER Ca store to the PM, responsible for the activation of SMCE, is still poorly understood. Two general mechanisms have been proposed to explain how information is transferred from the ER to the PM to produce 2+ store-mediated Ca influx. The first supports the existence of a diffusible messenger and the second the direct conformational coupling between the plasma mem2+ brane Ca channels and proteins resident in the membranes of intracellular stores. In the latter model, the actin cytoskeleton would play a key modulatory role. In this model, a physical interaction between the ER and the PM, involving reversible trafficking of the ER towards the PM has been proposed. We have previously shown that in glioma C6 cells stimulation of P2Y nucleotide receptors by agonists: ATP, UTP and ADP, that results in IP3 generation and depletion of ER stores, 2+ initiates biphasic Ca response consistent with the 2+ typical capacitative model of Ca influx. Also the ac-

2+

tion of thapsigargin, DBHQ and cyclopiazonic acid 2+ (CPA), that block Ca pump in the ER membrane and 2+ promote a leak of Ca from ER stores, induces a similar effect. The aim of the present study was to test the possible role of the actin cytoskeleton in the process of SMCE in glioma C6. To achieve this, two membrane-permeant inhibitors of actin polimerization, cytochalasin D and latrunculin A were used. Treatment of glioma C6 cells with these compounds resulted in the profound changes in the actin cytoskeleton architecture. Regardless of these changes ADP, UTP, DBHQ and CPA were still able to produce cytosolic calcium elevation. However, intracellular calcium store mobiliza2+ 2+ tion and Ca influx through store-operated Ca channels after addition of these compounds were strongly reduced. Cytochalasin D and latrunculin A treatment 2+ also diminished extracellular Ca influx in unstimulat2+ ed glioma C6 cells incubated previously in Ca free buffer. These results favor the secretion-like-coupling model in glioma C6. On the contrary, thapsigargin me2+ diated extracellular Ca influx remains intact after the cytoskeleton disruption. These results indicate that thapsigargin action seems to recruit additional mecha2+ nism for store-mediated Ca entry, perhaps by direct interaction with SOC channels in the plasma membrane.
This study was supported by grants No 6 P05A 012 20, 3 P05A 119 22 and 3 P04A 012 24 from the State Committee for Scientific Research (KBN, Poland).

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Lecture

157
Reactive oxygen species and cell signaling
Andrzej Dbrowski
Gastroenterology and Internal Medicine Department, Medical University of Biaystok, Biaystok

In the last decade it has become increasingly evident that a large variety of extracellular stimuli induce reactive oxygen species (ROS) production which is essential for downstream intracellular signal transduction. Among these stimuli are numerous ligands to tyrosine kinase and G-protein-coupled receptors including: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), angiotensin II, insulin, tumor necrosis factor alpha (TNF-a) and interleukin 1b (IL-1b). The enzymatic source of ligand-stimulated ROS is not completely elucidated, but, cyclooxygenases, lipoxygenases and NADPH oxidase are among the most likely candidates. The downstream targets of ROS have remained largely unexplored. However, it has been found that oxidant bursts are often associated with an increase in tyrosine phosphorylation. Extracellular administration of hydrogen peroxide has been demonstrated to acti-

vate pathways of mitogen-activated protein kinase (MAPK) family members consisiting of extracellular-signal regulated kinase (ERK), p38 MAPK and c-Jun amino-terminal kinase (JNK). It seems likely that ROS-mediated regulation of protein kinase pathways is due to alteration of the tyrosine kinase/phosphatase balance. It has been demonstrated that exogenous oxidants or oxidants generated by peptide growth factor binding can reversibly oxidase and inactivate protein tyrosine phosphatases (PTP). PTPs contain reactive cysteine residues within their active sites. Such reactive cysteine residues are easily modified by ROS and reactive nitrogen species (RNS) providing background for intracellular redox signaling. However, the organization of redox signaling and the use of oxygen to transmit information are proving to be complex and require further research.

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Lecture

Phosphorylation as a signal controlling the entrance of fructose 1,6-bisphosphatase into the nuclei of cardiomyocytes
Andrzej Dugaj, Agnieszka Gizak, Marek Zarzycki, Piotr Mamczur, Dariusz Rakus
Zakad Fizjologii Zwierzt, Uniwersytet Wrocawski, ul. Cybulskiego 30, 50-205 Wrocaw

Although enzymes of carbohydrate metabolism are predominantly located in cytosol (in a free state or partially bound to subcellular structures), several enzymes amongst them glucokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, lactate dehydrogenase and glycogen synthase have also been found in nuclei of different cell types. Recently we have found that fructose 1,6-bisphosphatase (FBPase) is present in nuclei of cardiomyocytes (Gizak and Dzugaj, 2003). Two isozymes of FBPase have been found in vertebrate tissues: liver isozyme is the regulatory enzyme of gluconeogenesis, muscle isozyme participates in glycogen synthesis from noncarbohydrate precursors. Presence of FBPase in nucleus raises a question about its physiological role in this compartment, as well as a question concerning mechanisms involved in FBPase import into the nucleus. A great number of proteins entering the nucleus possess in their primary structure a characteristic nuclear localization sequence (NLS) which consists of three to five basic amino acids separated with non-basic residue. One of such sequence is the KKKGK motif which

is present in primary structure of all known mammalian muscle FBPases. Transport of NLS-containing proteins to the nucleus is mediated by heterodimeric import receptor complex (importin a and b), nuclear pore complex (NPC), and GTPase Ran. The decisive step controlling this precisely regulated process appears to be phosphorylation of a residue near to NLS by Casein Kinase II (CKII) and/or by cyclin-dependent kinases, the kinases which are also directly engaged in the progress of the cell cycle. Despite of earlier reports, liver FBPase isozyme is not phosphorylated in vivo. On the other hand, we have found that rabbit muscle FBPase is phosphorylated (Rakus et al., 2003). Our latest investigation revealed that muscle FBPase is phosphorylated at Ser 210, which is located in close vicinity to FBPase NLS sequence (residues 203207). The analysis of targets of protein kinases strongly suggests that Ser 210 within FBPase structure serves as a substrate for CK2. Thus, one may speculate that subcellular localization of muscle FBPase is controlled by extracellular stimuli or cell cycle dependent phosphorylation/dephosphorylation

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mechanism, and changes in the phosphorylation state of muscle FBPase might be a signal directing the enzyme to the nucleus where it may be involved in the alteration of genes expression or in the mechanisms of cell division or differentiation.

References Gizak A, Dzugaj A (2003) FEBS Lett, 539: 5155. Rakus D, Zarzycki M, Dzugaj A (2003) Acta Biochim Polon, 50: 115121.

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Anna Filipek
Instytut Biologii Dowiadczalnej PAN im. M. Nenckiego, Polska Akademia Nauk, ul. Pasteura 3, 02-093 Warszawa

Lecture

The role of calcium binding proteins from the S100 family in cellular processes

The S100 is a group of low molecular weight (1012 kDa) calcium binding proteins of the EF-hand type. To date 21 different proteins have been assigned to the 2+ S100 family. Binding of Ca to S100 protein induces a conformational change essential for target protein binding. S100 proteins are expressed in different cells and tissues. Within cells most of them exist as dimers and are implicated in the regulation of several processes such as protein phosphorylation, regulation of enzymatic activities, dynamics of cytoskeletal components, cell cycle, proliferation and differentiation and possibly ubiquitinylation. Certain S100 members are released into the extracellular space and stimulate neuronal survival and astrocyte proliferation or cause cell death via apoptosis [1]. Calcyclin (S100A6), a typical member of the S100 family, has been extensively studied in our laboratory. We found that S100A6 interacted in a calcium dependent manner with CacyBP/SIP [2] and Sgt1 [3] proteins. Sgt1 was discovered in yeast as a product of gene required for cell cycle progression and later it appeared that Sgt1 is a component of SCF ubiquitin ligase [4]. cDNA of another protein interacting with S100A6 CacyBP/SIP was originally isolated from mouse

brain library [2] and biochemical properties of recombinant protein were futher investigated [58]. In 2001 Matsuzawa and Reed [9] showed that CacyBP/SIP was a component of a novel complex involved in ubiquitinylation of beta-catenin. Since S100A6 interacts with CacyBP/SIP and Sgt1,we suggest that, at least, some members of the S100 family might regulate ubiquitinylation and protein degradation processes.
References 1. Jastrzbska B, Filipek A (2003) Post Biol Kom, 2: 273283. 2. Filipek J, Kunicki J (1998) J Neurochem, 70: 17931798. 3. Nowotny M, Spiechowicz M, Jastrzebska B, Filipek A, Kitagawa K, Kunicki J (2003) J Biol Chem, in press 4. Kitagawa K, Skowyra D, Elledge SJ, Harper JW, Hieter P (1999) Mol Cell, 1: 2133. 5. Jastrzbska B, Filipek A, Nowicka D, Kaczmarek L, Kunicki J (2000) J Hist Cytochem, 48: 11951202. 6. Nowotny M, Bhattacharya S, Filipek A, Krezel AM, Chazin W, Kunicki J (2000) J Biol Chem, 275: 3117831182. 7. Filipek A, Jastrzebska B, Nowotny M, Kwiatkowska K, Hetman M, Surmacz L, Wyroba E, Kunicki J (2002) J Biol Chem, 277: 2110321109. 8. Filipek A, Jastrzebska B, Nowotny M, Kunicki J (2002) J Biol Chem, 277: 2884828852. 9. Matsuzawa S, Reed JC (2001) Mol Cell, 7: 915926. The work was supported by Grant 3 P04A 043 22 to AF and 6P04A 039 20 to JK from the KBN and by statutory funds provided by the Nencki Institute.

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Signaling by guanylyl cyclases in immune cells
Wojciech Gorczyca, Marcin Kobiaka, Hanna Witwicka, Ewa Kurowska, Jakub Siednienko
Instytut Immunologii i Terapii Dowiadczalnej, Polska Akademia Nauk, ul. Weigla 12, 53-114 Wrocaw

Lecture

Cyclic nucleotides (cAMP and cGMP) are messenger molecules involved in multiple cellular processes including gene expression, differentiation, and maturation. The participation of cAMP in function of immune cells is well documented but although it is well known that cGMP is also present in most of them, mechanisms of its signaling there are still poorly understood. Cyclic

GMP has been suggested to regulate proliferation of lymphocytes, chemotaxis of granulocytes and macrophages, adhesion of granulocytes and macrophages, expression of genes for iNOS and TNFalpha in macrophages and dendritic cells. Intracellular level of cGMP is controlled by opposing action of guanylyl cyclases (GCs), which synthesize the nucleotide from

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GTP, and phosphodiesterases (PDEs) hydrolyzing it to GMP. Guanylyl cyclases exist as cytosolic (soluble, sGC) or membrane-bound (particulate, pGC) enzymes [1]. Both forms differ in structure, mechanisms of activation, localization, and supposedly the cellular effects of their activity are also distinct. Soluble GCs are activated by nitric oxide, the known mediator of diverse immune reactions. Three isoforms (GC-A, GC-B, and GC-C) of particulate cyclases are receptors for peptide hormones and are activated either by natriuretic peptides (GC-A, GC-B) or by guanylin, uroguanylin, lymphoguanylin, and bacterial enterotoxin STa (GC-C). The mRNA for natriuretic peptide A (ANP), an activator of GC-A, has been identified in lymphoid organs of several species. The mRNA for guanylin, uroguanylin, and lymphoguanylin was detected in thymus, lymph nodes, and spleen of opossum. Soluble, particulate or both forms of guanylyl cyclases were reported to be present in different lymphoid organs, T cells, monocytes, macrophages and granulocytes of various species [24]. The profile of GCs expression changes during differentiation and maturation of a given cell type. Once synthesized, cGMP may in turn regulate activities of effector proteins including PDEs and protein kinases (PKG1 and PKG2). The PDE activity is assumed to be the main factor responsible for the duration of the cGMP and/or cAMP signals. Among multiple isoforms of PDEs, enzymes belonging to three families (PDE5, 6 and 9) are cGMP-specific and four fami-

lies (PDE1, 2, 10, 11) may hydrolyze both cGMP and cAMP. Particularly, in peritoneal macrophages the level of cGMP is strictly controlled by the activity of cGMP-regulated PDEs, apparently in a feedback-regulated way [5]. Since some of them are able to hydrolyze also cAMP, they possibly play a dual role: control intracellular concentration of cGMP and affect the level of cAMP, linking these two signaling pathways. It has been well documented that variants of cGMP-regulated kinase 1 (PKG1alfa and PKG1beta) are responsible for cGMP effects in several immune cell types. Antibodies specific to both PKG1 variants detect these enzymes in cells of lymphoid organs, but they are not able to detect them in peritoneal macrophages [2, 3, 5]. All these data clearly indicate that since the expression of enzymes participating in cGMP signaling in immune cells appears as cell-specific and depending on stage of cell development, the role of cGMP in these cells is also variable.
References: 1. Kobiaka M, Gorczyca WA (2000) Acta Biochim Polon, 47: 51728. 2. Kobiaka M, Karwacka-Krupiska K, Zioo E, Fassler R, Strzdaa L, Gorczyca WA (2000). Immunol Lett, 73: 119. 3. Kurowska E, Kobiaka M, Zioo E, Strzdaa L, Gorczyca WA (2002) Arch Immunol Exp Ther, 50: 28994. 4. Kobiaka M, Gorczyca WA (2000) Immunol Lett, 73: 170. 5. Witwicka H, Kobiaka M, Gorczyca WA (2002). Acta Biochim Polon, 49: 8917.

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Lecture

Calcium in excitation-contraction coupling of cardiac myocytes: physiology, hypertrophy, heart failure


Bohdan Lewartowski, Urszula Mackiewicz
Zakad Fizjologii Klinicznej, Centrum Medyczne Ksztacenia Podyplomowego, ul. Marymoncka 99, 01-813 Warszawa

Ca ions are the essential messengers in processes linking cardiac myocyte excitation (action potential) with contractile proteins (excitation-contraction coupling ECC). Depolarization above 40 mV activates sarcolemmal L-type Ca channels and inward Ca current. The current activates Ca channels (RyRs) of sarcoplasmic reticulum (SR). Ca accumulated in the SR due to activity of its Ca-ATPase is released to sarcoplasm, binds to the troponin C and activates contraction. Relaxation is brought about by re-uptake of Ca by the ATPase of the SR and its outward transport by the Na/Ca exchanger. Activity of Ca-ATPase of the SR is inhibited by a protein phospholamban. Inhibition is released by phospholamban phosphorylation by PKA and clamodulin-dependent kinase. A number of proteins bound to RyRs regulate their properties. FKBP12

assambles RyRs in functionally isolated clusters activated by single sarcolemmal Ca channels and stabilises them, which results in complete closure after activation is over. PKA and calmoduline-dependent kinase phosphorylate and phosphatases PP1 and PP2A dephosphorylate RyRs. In heart failure expression of Ca-ATPase of the SR is severily depressed. RyRs are hyperphosphorylated due to uncoupling of PP1 and PP2A and maintained activity of PKA. This results in uncoupling of FKBP12 and destabilization of RyRs which loose SR Ca between the beats. All these events lead to elimination of the SR from ECC and loss of cardiac contractility. All interventions improving function of proteins involved in Ca turnover restore normal funtional and morphological properties of the failing myocytes.

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Lecture

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Excitatory amino acid receptors and calcium fluxes in neurons
Jerzy azarewicz

Instytut Medycyny Dowiadczalnej i Klinicznej im. M. Mossakowskiego, Polska Akademia Nauk, ul. Pawiskeigo 5, 02-106 Warszawa

The glutamate receptors play fundamental role in the excitatory neurotransmission and in regulation of the neuronal plasticity. On the other hand these receptors are known to mediate neurodegeneration in the acute neurological disorders as stroke or brain injury, and in chronic neurodegenration. Calcium ions are involved as second messengers in these physiological and pathogenic processes. The results of in vivo and in vitro studies will be presented, illustrating differential role of extracellular calcium invading neurons via NMDA channels and of the intracellular calcium mobilized from the ryanodine-sensitive pool (in a phenomenon of the calcium-induced calcium release CICR) in the NMDA-mediated responses in different species of laboratory animals. Our data demonstrate that low expression of ryanodine receptors in neurons of the rabbit brain region results in very modest functional expres-

sion of CICR and in low participation of intracellular calcium in generation of the NMDA receptor-mediated intracellular calcium signaling. To the contrary, CICR potentiates the NMDA receptor mediated calcium signals in the rat hippocampal region. Moreover, data will be displayed indicating that group I metabotropic glutamate receptors (mGluRs I) and NMDA receptors are cooperatively mediating the homocysteine-induced neurodegeneration and generation of calcium signaling utilizing mainly the intracellular, IP3 receptor-mediated calcium pool. The possible mechanisms of interactions between mGluRs I, NMDA receptors and IP3 receptors will be discussed. These results point to diversification of calcium signaling in different glutamate receptors mediating physiological and pathological responses in neurons.
Supported by the KBN ordered grant no BPZ-KBN-002/ CD/P05/2000, task no 6.

163
Molecular mechanisms of genomic action of lipophilic hormones
Magorzata Manteuffel-Cymborowska
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa

Lecture

Lipophilic hormones coordinate expression of gene networks in numerous physiological, developmental and metabolic processes. This genomic response, that can be ascribed in large part to nuclear receptor mediation, is the subject of the lecture. However, it should be stressed that the field of nongenomic signalling by hormones is quickly growing, and many important hormone-induced signalling events are membrane-initiated. Nuclear receptors form an evolutionary related superfamily of ligand-inducible transcription factors with conservative functional domains. According to accepted for years classical model, binding of a lipophilic ligand to its nuclear receptor was the sole and absolute requirement for its activation. The activation of nuclear receptor resulted, via a sequence of events including recognizing and binding to specific DNA seqences, in modulation of transcription of a target gene. Now, it has become apparent that nuclear receptors carry both ligand-dependent and ligand-independent transactivation functions and can be alternatively activated in the absence of cognate lipophilic ligands by a variety of extracellular signals. The existence of several mechanisms of nuclear receptor activation within the same

cell leads to the cross-talk between signalling pathways resulting in their synergy or antagonism. Regulation of transcription by nuclear receptors requires also the participation of another class of proteins that are recruited by protein-protein interactions and potentiate (coactivators) or repress (corepressors) transcription. These numerous coregulatory proteins serve several important roles (enzymatic, scaffolding, integration of signalling pathways). They form multicomponent complexes with nuclear receptors and the network of their interactions is variable and still growing (e.g. combinatorial model of transcription initiation). Nuclear receptors, but also their coregulators, are subject to posttranslational covalent modifications (phosphorylation, acetylation, ubiquitylation, sumoylation) that alter their structure and protein-protein interactions. The pattern of coregulatory protein modifications may vary depending on afferent signalling pathwys and determine the functional specificity of coregulator for distinct nuclear receptors and gene promoters. To regulate the expression of the right gene at the right time in a hormone-dependent manner it is important not only to switch on, but also to switch off its transcription.

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Regulation of transcription rate in response to changes in hormone signal intensity is difficult to explain by an old static model according to which the receptor binds to a gene response element and remains associated for as long as the hormone is present in the cellular milieau. Instead, recent reports are consistent with a dynamic hit and run model in which nuclear receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. Thus, a steroid receptor after ligand activation binds to chromatin, recruits

coactivators forming a multicomponent transcriptional complex, activates transcription and is simultanously displaced from the promoter. Such cyclic association and dissociation of steroid receptor-induced complexes permits continous response to hormone signalling. Recent dynamic models of steroid receptor assembly/disassembly mediated by chaperones (glucocorticoid receptor) and by proteasome (human estrogen receptor alpha) will be presented.

164

Lecture

Membrane-initiated steroid signalling (MISS); physiological or pharmacological significance?


Ewa Marcinkowska
Laboratorium Immunologii Rozrodu, Instytut Immunologii i Terapii Dowiadczalnej, ul. Weigla 12, 53-114 Wrocaw

Steroid hormones regulate cellular processes by two distinct modes of action. They bind to intracellular receptors and regulate transcription of target genes. This process takes at least 3060 minutes to occur. Other effects, called membrane-initiated steroid signaling (MISS) are rapid, and occur within seconds after steroid application. The rapid effects are diverse and include changes in intracellular calcium levels, activation of adenyl cyclase, protein kinases, G proteins or nitric oxide synthase. These effects can not be prevented by inhibitors of either DNA transcription or protein synthesis. Less obvious situation concerns steroid antagonists, since they inhibit rapid effects in some cases, while in other they do not. Therefore it is unclear how rapid responses to steroid hormones originate. One hypothesis says that distinct protein receptors, located in plasma membranes exist. Many data obtained with use of vitamin D3 and its analogs point at it. In fact a vitamin D3 binding protein from plasma membranes was isolated and cloning of its cDNA has been recently announced (but not published as yet). This protein is not related to nuclear vitamin D3 receptor. Another possibility is that portion of cellular nuclear steroid receptors or their splicing variants are located close to the plasma membrane, and there interact with exogenously added steroids. This hypothesis is supported by

the observations that some rapid responses to estrogens and progestins are blocked by anti-estrogens and anti-progestins. Moreover classical (nuclear) estrogen receptor coprecipitates with calveolins and with some protein kinases activated in response to estrogens. One more explanation of the membrane-initiated steroid signaling is that allosteric reactions of steroids with membrane proteins that build membrane ion channels affect their functional state. Another open question is physiological relevance of these effects. Rapid responses to steroids are often observed at relatively high concentrations, much higher than blood plasma levels. However physiological significance is questionable, pharmacological meaning seems to be certain. Clinical use of vitamin D3 analogs, which do not cause hypercalcemia in supraphysiological concentrations, but still are able to induce differentiation of leukemia cells or keratinocytes in psoriatic plaques is an example. Another promising case is estren, an analog of estrogen, that could reverse postmenopausal bone loss, without affecting reproductive organs. However the existence of membrane-initiated steroid signaling has been questioned some years ago, at the moment they seem more and more important for pharmacological intervention.

165
Involvement of Ser/Thr kinases in transmitting of insulin signal in the cell
Monika Sakowicz
Zakad Medycyny Molekularnej, Akademia Medyczna w Gdasku, ul. Dbinki 7, 80-211 Gdask

Lecture

Insulin, produced by the pancreas, is the major anabolic hormone whose action is essential for growth, devel-

opment and homeostasis of glucose, fat and protein metabolism. In the mid-1970s, it was observed that insulin

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promoted an increase in Ser/Thr posphorylation of subset of cellular protein. The first of these insulin-activated protein Ser/Thr kinase networks to be fully elucidated was the Ras-Raf-MAPK cascade. At the molecular level, insulin binding to its transmembrane receptor stimulates intrinsic to the intracellular domain of the receptor tyrosine protein kinase activity leads to autophosphorylation of the receptor as well as phosphorylation of a number of intracellular proteins. This result in the activation of Ras and phosphatidylinositol 3-kinase and hence to the activation of a cascade of serine/threonine protein kinases, called the mitogen-activated protein kinases (MAPKs). These include kinase Raf, which phosphorylates MAPK kinase (MEK), which in turn phosphorylates MAPK. Activation of MAPK promotes their nuclear translocation and subsequent phosphorylation of transcription factors leading to insulin-dependent alternations of gene expression. The MAPK cascade is highly conserved amongst

eukaryotic species. There are at least three different MAPKs. These include the extracellular signal-regulated kinases (ERKs), the c-Jun NH2-terminal kinases (JNKs) and the p38 MAPKs. ERKs are stimulated by insulin via phosphorylation of a Thr-Glu-Tyr motif in activation loop and modulate cell growth and differentiation. ERKl and ERK2 were the first MAPKs to be identified and cloned from a vertebrate species. Both ERKs are Ser/ Thr kinase with an impressive portfolio of more than 50 substraters and their activity is most strongly stimulated by activate tyrosine kinase receptors. Protein Ser/Thr kinase cascade involved in the action of insulin is undoubtedly important in the stimulation of the transcription of certain genes and play an important role in regulation of mammalian cells proliferation. Recently the numerous reports indicated the MAPK pathways were involved in many pathological conditions, including cancer, diabetes, inflammation and other diseases.

166
ATP an intracellular metabolic messenger
Adam Szewczyk , Anna Kicinska , Jolanta Skalska
1 2 2

Lecture

1 Pracownia Wewntrzkomrkowych Kanaw Jonowych, Zakad Biofizyki, Instytut Biologii Dowiadczalnej im. M. Nenckiego, PAN, ul. Pasteura 3, 02-093 Warszawa, 2 Instytut Biologii Dowiadczalnej, Polska Akademia Nauk, ul. Pasteura 3, 02-093 Warszawa

It is well establish that adenosine 5-triphosphate (ATP) plays the role of a universal compound being substrate for energy consuming physiological processes. In addition, ATP has a direct intracellular effects on cellular functions. These interactions do not involve ATP hydrolysis. This lecture will present overview on ATP-regulated potassium channels. First, ATP-regulated potassium channels from plasma membrane will be described. These channels present in different cell types such as cardiac, skeletal and smooth muscle cells, pancreatic B-cells or neuronal cells. In pancreatic B-cells the ATP-regulated potassium channels are involved in insulin secretion. They are important receptors for antidiabetic sulfonylureas applied in treatment of diabetes mellitus type II.

Second, ATP-regulated potassium channels are present in inner mitochondrial membrane of liver, heart and brain mitochondria. The primary function of this channel is to allow potasium ions transport into the mitochondrial matrix. This phenomenon could be involved in maintenance of mitochondrial volume homeostasis. Additionally, the cardiac mitochondrial ATP-regulated channel plays an important role in protecting cardiomyocytes during ischemia/reperfusion. Recent data on single channel recordings of mitochondrial ATP-regulated potassium channel will be presented.
The work in authors laboratory was supported in part by the State Committee for Scientific Research (KBN, grants 6P04A02321, 6P04A01019) and in part by NATO Collaborative Grant LST.CLG.979217 and by the grant of the Roche Organ Transplantation Research Foundation (ROTRF 860428181).

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Reactive T cell: from signal to division
Jacek Witkowski
Katedra i Zakad Patomorfologii, Akademia Medyczna, ul. Dbinki 7, 80-211 Gdask

Lecture

The essence of life for a T lymphocyte is to react to a complex gamut of external signals, of which those pro-

vided by an antigen itself are but a major part. The remaining signals and stimuli originate mostly from the

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surface of antigen-presenting cells (APCs), from the long and still elongating list of cytokines, and from a plethora of other sources, including hormones, neuromediators, and molecules embedded in the matrix of connective tissues. All of these diverse signals are integrated at and around the surface of a T lymphocyte and initially transformed into one of the primary responses, including transient rise of the cytoplasmatic concentration of ionized calcium (so called calcium signal), multiple events of thyrosine and/or serine/threonine phosphorylation, and activation of GTP exchange by a group of small GTPases. Further on, three different, but connected and partially overlapping cascades of reactions are activated, and their final outcome culminates in mobilization of a number of transcription factors (including the NFkB Rel family, NF-AT, and AP-1) and activation of a variety of genes. Further fate of such a stimulated T cell is either cytokine production and/or proliferation to form an

effector clone, or activation of a path leading to activation-induced cell death (apoptosis). Eventually most of antigen-responding T cells would undergo apoptosis, leaving behind only a relatively few memory cells. This lecture will concentrate on the following topics: a, early events during signal recognition by a resting, circulating T lymphocyte, including the updated characteristics of molecules involved in the process, especially their participation and function in so called membrane microdomains (rafts); b, updated description of major signal transduction pathways in activated T cells, stressing the roles of phosphorylation, dephosphorylation and proteolysis; c; cellular events that produce an exit of a T cell from resting (G0) phase and entry into the first G1 phase of clonal expansion period; d, characteristics of dynamic proliferative parameters of various subpopulations of human T cells; e, updated information on causes and time course of T cell apoptosis.

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Changes in contractile structures influence on cellular calcium homeostasis
Antoni Wrzosek
Zakad Biochemii Mini, Instytut Biologii Dowiadczalnej im. M. Nenckiego, ul. Pastera 3, 02-093 Warszawa

Lecture

The cytoskeleton forms a network connecting the surface membrane with its array of receptors and channels to the extracellular matrix and intracellular elements such as the nucleus and myofilaments. Mechanical stress induces a variety of hypertrophic responses including an increase in protein synthesis and a reprogramming of gene expression. Calcium signalling has been reported to play an important role in the development of cardiac hypertrophy. Several different ion channels are involved in the sensing of stretch, among them K-selective, Cl-selective, non-selective, and ATP-sensitive potassium channels. Sodium and calcium entering the cells via non-selective ion channels are thought to contribute to the genesis of stretch-induced arrhythmia. Stretch-sensitive channels have been cloned from non-vertebrate and vertebrate species. Chronic stress on the heart activates gene expression in cardiomyocytes and non-myocytes. Step increase in length of the cardiac muscle produced a rapid 2+ potentiation of twitch force but not of the Ca transient. When a quick release was performed during a

contraction, a short-lived increase in the intracellular calcium was observed following the release. These two observations can be explained by increasing in binding constant of troponin C for calcium. After the rapid stretch of the muscle there was a further, slow increase of twitch force which was due entirely to a slow in2+ crease of the Ca transient. Detailed understanding of how mechanical strain on myocardial cells is translated into channel activation will allow identifying new targets for putative antiarrhythmic drugs. The cytochalasin D, a specific F-actin depolymerising agent, 2+ significantly slowed decay of Ca transients and the 2+ rising phase of Ca transients. The effects of cytochalasin D were inhibited by nifedipine, demonstrating that actin cytoskeletal disruption augments 2+ 2+ Ca influx through voltage-sensitive L-type Ca channels. Phalloidin, an F-actin stabilizer, prevented 2+ cytochalasin D-induced alterations of Ca transient kinetics. It was also observed that integrity of F-actin cytoskeleton is an important factor for sarcoplasmic reticulum function.

169
Regulation of calcium signals in animal cell role of mitochondria
Krzysztof Zabocki
Zakad Biochemii Komrki, Instytut Biologii Dowiadczalnej PAN im. M. Nenckiego, ul. Pasteura 3, 02-093 Warszawa

Lecture

Ca plays a pivotal role in the regulation of many intracellular processes such as cell division, gene ex-

2+

pression, exocytosis, fertilization, cell contraction, enzyme activities, control of metabolic pathways and

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intracellular signaling. Therefore, cytosolic Ca con2+ centration ([Ca ]c) must be very precisely controlled. Excitation of the cell by extracellular stimuli triggers a 2+ transient increase in [Ca ]c. Cytosolic calcium may come both from intracellular calcium stores localized in the lumen of endoplasmic reticulum (ER) and from the extracellular milieu. In non-electrically excitable cells calcium influx through plasma membrane (PM) is regulated by the filling state of ER calcium stores. In other words, the depletion of ER is necessary for the activa2+ tion of Ca calcium channels in the PM. ER depletion degree as well as the rate of calcium entry into the cells may depend on the energy status of mitochondria localized in the close vicinity of the calcium channels in the ER and PM, respectively. Energized mitochondria can 2+ take up and transiently buffer Ca . However, because

of low affinity of the mitochondrial Ca uniporter to 2+ Ca , calcium accumulation in the mitochondria may 2+ occur when [Ca ]c exceeded a critical point (ca 500 2+ nM). A local reduction of [Ca ]c due to accumulation of this cation by mitochondria may regulate the activity of calcium channels because of a decrease of the 2+ feed-back effects exerted by [Ca ]c. Moreover, respir2+ ing mitochondria may compete with ER Ca -ATPase 2+ for Ca and therefore protect ER stores against reloading. This may result in a more complete ER depletion of 2+ Ca . So, mitochondria may modify the opening of calcium channels and therefore regulate calcium signals in the cell. These effects are not the same in all types of cells and they depend on the sensitivity of calcium 2+ channels to local changes in [Ca ]c.

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Oral Presentation

12-O-Tetradecanoylphorbol 13-acetate stimulated degradation of IkappaB and expression of cyclooxygenase-2 and inducible nitric oxide synthase in mouse epidermis: The effect of plant phenols
Micha Cichocki, Joanna Piesik, Jarosaw Paluszczak, Wanda Baer-Dubowska
Katedra Biochemii Farmaceutycznej, Akademia Medyczna im. K. Marcinkowskiego, ul. Grunwaldzka 6, 60-780 Pozna

A wide array of phenolic substances present in edible plants, has been reported to possess anticarcinogenic and antimutagenic properties. These include also polyphenol, tannic acid (TA) and phytoalexin 3,5,4-trihydroxystilbene, resveratrol (RES). As one of the possible mechanisms of anticarcinogenic activity of potential chemopreventive agents, modulation of the nuclear transcription factor kappaB (NFkappaB) was postulated. NFkappaB regulates the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), which are involved in inflammation and tumor promotion. Most extracellular signals activate NFkappaB through phosphorylation and proteasome-dependant degradation of its inhibitory protein IkappaB. In this study we assessed the expression of IkappaB alpha, COX-2 and iNOS in mouse epidermis after topical treatment with tumor promoter, 12-O-tetra-

decanoylphorbol-13-acetate (TPA) and its modulation by TA and RES. IkappaB was detected in cytosol by immunoblot analysis. Time course experiments showed that degradation of IkappaB occurred within 12 hours after treatment with TPA. IkappaB recovered to basal levels 6 hours after TPA treatment. Detectable levels of iNOS and COX-2 were found in microsomes 6 hours after TPA treatment. Increased level of iNOS protein resulted in the elevated activity of the enzyme as measured by nitrite formation in cytosol. Pretreatment of mice with TA or RES 15 minutes before topical application of TPA had a moderate effect on the expression of NFkappaB dependent enzymes and degradation of their subunits. However, the modulation of NFkappaB signaling pathway by these two phenolic compounds can not be excluded as the possible mechanism of their antipromotional activity.

171

Oral Presentation

Is cyclosporin A proapoptotic action mediated by inhibition of calcineurin or inhibition of cyclophilins in glioma cells ?
Katarzyna Gawda-Walerych, Magorzata Zawadzka, Beata Pyrzyska, Piotr Kamierczak, Boena Kamiska
Instytut Biologii Dowiadczalnej, Polska Akademia Nauk, ul. Pasteura 3, 02-093 Warszawa

Cyclosporin A a known immunosuppressive drug was shown to trigger apoptosis in glioma C6 cells at 60 mM

concentration. By far it was believed that CsA exerts its action mainly through inhibition of a calcium- and

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calmodulin- dependent phosphatase calcineurin (CaN). To confirm this hypothesis a dominant negative calcineurin mutant (DN-CLN) has been constructed, that was deprived of the major part of the catalytic domain and fused to the green fluorescent protein (GFP). DN-CLN-GFP blocked calcineurin-dependent expression of reporter gene with the equal efficiency as CsA. After transient transfection of glioma C6 cells with a dominant negative calcineurin mutant we observed shrinkage of cell bodies and chromatin condensation, which might suggest that cells undergo programmed cell death. In order to distinguish between inhibition of calcineurin and inhibition of cyclophilins we used an

analog of CsA (NIM 811), which specifically inhibits cyclophilins, but that does not block calcineurin. In fact, after treatment with NIM 811 (60 mM) we observed morphological changes typical for apoptosis. NIM 811 affected viability of C6 glioma cells. The active forms of caspases 7, 9 and 3, which are the hallmarks of apoptotic process were detected as well as cleaved form of PARP after 48 hours treatment with NIM 811. Experiments with NIM 811 indicate that the onset of CsA-induced apoptosis requires not only the inhibition of calcineurin but also the inhibition of cyclophilins.
This work was supported by grant No. PBZ KBN/02/ CD/2000

172
Alicja Jzkowicz , Anneliese Nigisch , Guenter Weigel , Ihor Huk , Jzef Dulak
1 2 2 3 4

Oral Presentation

Opposite effect of prostaglandin-j2 on VEGF synthesis in normoxia and hypoxia


1 Department of Molecular Genetics and Genetic Engineering, Jagiellonian University, ul. Granostajowa 7, 30-387 Krakw, 2 Department of Cardiovascular Surgery, University of Vienna, Vienna, Austria, 3 Department of Vascular Surgery, University of Vienna, Vienna, Austria, 4 Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakw

Vascular endothelial growth factor (VEGF) is one of the major mediators of angiogenesis, produced mostly under hypoxic conditions. In several types of cells 15-deoxy-delta12,14prostaglandin-J2 (15d-PGJ2), a natural ligand of PPARgamma, was shown to upregulate the VEGF synthesis. All those studies were performed, however, in normoxia. Here we compared the effect of 15d-PGJ2 on VEGF production in cells cultured in normoxic and hypoxic conditions. Experiments were carried out on the human microvascular endothelial cells (HMEC-1) incubated for 24 h in normoxia or hypoxia (2% of oxygen). Under normoxic conditions resting HMEC-1 released approximately 20 pg/ml of VEGF protein, whereas in hypoxia the VEGF synthesis was seven-fold higher. In normoxia, treatment of HMEC-1 with 15d-PGJ2 (110 mM) significantly and dose-dependently upregulated the VEGF promoter activity, mRNA expression and VEGF protein release. This aug-

mentation was mediated by induction of heme oxygenase-1 (HO-1) followed by increased synthesis of carbon monoxide and elevated cGMP production. In contrast, under hypoxic conditions the same doses of 15d-PGJ2 decreased the VEGF protein release by 50% and strongly reduced a transcription rate from VEGF promoter. This effect was associated with inhibition of hypoxia inducible factor-1 activity and with decreased stimulation of hypoxia responsive element in promoter of VEGF. Similarly, troglitazone a more specific ligand of PPARgamma increased VEGF synthesis in normoxia and decreased in hypoxia, but its influence was much weaker comparing to 15d-PGJ2. This suggests that some PPARgamma-independent activities contribute to the effects of 15-PGJ2. In summary, we found that 15d-PGJ2 exerts opposite influence on VEGF synthesis in normoxic and hypoxic conditions and that these effects are mediated by distinct signal transduction pathways.

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Oral Presentation

Neuroactive steroids regulation of calcium pump transport activity in stable transfected PC12 cells
Iwona Kawecka, Janusz Szemraj, Ludmia yliska
Instytut Fizjologii i Biochemii, Zakad Biochemii Lekarskiej, Uniwersytet Medyczny w odzi, ul. Mazowiecka 6/8, 92-215 d

De novo steroids synthesis, as well as a steroid metabolic pathway, is present in the brain. Neuroactive steroids have been shown to modulate the excitability of

neuronal membrane. Their concentration in specific brain regions appears to be higher than in the plasma, defining their potential active role in brain cells. In-

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creasing evidence for rapid non-genomic steroid effects has been demonstrated. The signaling pathway for the majority of the steroids is probably transmitted by specific membrane steroid receptors or target proteins (i.e. receptor-mediated binding to ligand-gated ion channels). 2+ The plasma membrane Ca -ATPase (PMCA) is an in2+ tegral part of the Ca regulatory system in eukaryotic 2+ cells playing a prominent role in returning [Ca ]i to basal level. The PMCA diversity results from the alternative splicing, and produces various isoforms composition within the brain. We have previously demonstrated the direct influence of steroids on the activity of the enzyme purified from rat cortical membranes. To elucidate the specific

role of PMCA isoforms the present study was performed on PC12 cells with a stable suppressed expression of PMCA (2 and 3 isoform). These isoforms are characteristic for nervous tissue. From all examined steroids, 17beta-estradiol, pregnenolone, dehydroepiandrosterone and their sulphate derivatives influenced calcium transport and calmodulin stimulation of 2+ Ca -ATPase. Although the precise mechanism of these regulations remains unresolved yet, the CaM-binding 2+ domain of Ca -ATPase is assumed to be a primary site of steroid action. Moreover, the comparison of transport activity in the membranes containing different isoforms composition suggests that specific mechanism of steroid action may depend on PMCA structure.
Supported by the grant No 6P04A 06119 from State Committee for Scientific Research (KBN, Poland).

174

Oral Presentation

Residues of the C-terminal region (T-box) of the Ultraspiracle nuclear receptor DNA-binding domain crucial to specific interaction with the hsp27 response element
Agnieszka Kowalska , Grzegorz Rymarczyk , Iwona Grad , Marek Orowski , Daniel Krowarsch , 1 Andrzej Oyhar
1 Institute of Organic Chemistry, Biochemistry and Biotechnology, Division of Biochemistry, Wrocaw University of Technology, ul. Wybrzee Wyspiaskiego 27, 50-370 Wrocaw, 2 Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wrocaw, ul. Tamka 2, 50-137 Wrocaw
1 1 1 1 2

The steroid hormone 20-hydroxyecdysone (20E) initiates morphogenesis in insects by signaling through a heterodimer of two nuclear receptors encoded by EcR and ultraspiracle (usp) loci. This dimeric receptor modulates transcription via direct interaction with its pseudo-palindromic hormone response elements (HREs) located upstream to the coding sequences of target genes, as in the promoter/enhancer region of the heat-shock protein 27 gene (hsp27). In this study, the functional role of individual residues from the C-terminal fragment (the so called T-box) of the Usp DNA-binding domain (UspDBD) has been addressed using the site-directed mutagenesis. Ten single-residue (K67A through R77A) mutant proteins have been expressed and purified, and their hsp27HRE-binding affinity has been measured by electrophoretic mobility shift assays. We find that mutations at positions E69 or

R77 have no effect on the hsp27HRE-binding, whereas amino acid residues V71, Q72 and R75 are essential for specific DNA-recognition, as observed by a large decrease in affinity for the hsp27 probe. Additionally, interaction of K67 or R68-mutated DBDs with the hsp27HRE is slightly weakened. Interestingly, mutants displaying increased affinity to the HRE studied have also been found (E73A, E74A and Q76A). Since only DBDs mutated at positions V71 or E74 display significant changes in the overall protein structure, as judged by far-UV circular dichroism spectra, the differences in DNA-affinity observed for all other mutants can be attributed to the local perturbation of the protein-DNA contact interface. This is the first report demonstrating the functional role of the UspDBD T-box residues in specific interaction with the hsp27HRE.
This work was supported by the State Committee for Scientific Research (KBN, Poland) grant 3 P04B 009 23.

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1 2 2 2

Oral Presentation

Mechanism of farnesyltransferase inhibitor (FTI) inducted apoptosis of acute myeloid leukaemia cells.
Katarzyna Krzykowska , Marta Szostek , Patrycja Mensah , Aleksander B. Skotnicki , Piotr Laidler
1

1 Instytut Biochemii Lekarskiej, Collegium Medicum Uniwersytetu Jagielloskiego, ul. Kopernika 7, 31-034 Krakw, 2 Katedra i Klinika Hematologii, Collegium Medicum Uniwersytetu Jagielloskiego, ul. Kopernika 17, 31-501 Krakw

Haematopoiesis is a process of proliferation and differentiation of immature pluripotential stem cells

in bone marrow compartment and the release of mature blood cells to the circulation. Genetically based

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abnormalities in the above process could lead to the development of different type of leukaemias. Acute myeloid leukaemia (AML) is characterised by maturation arrest of myeloid lineage and proliferation of leukaemic blasts in the bone marrow space and eventually their release to the peripheral blood. The mutation of Ras oncogen (that is attached to cell membranes by farnesyl anchor) is frequently found in this disease. It seems that its effect on apoptosis is correlated with the RhoB protein dislocation from intercellular membranes to the cytoplasm followed by caspase 9 activation and cytochrom C releasing. Recently the trials of using farnesylation inhibitor (FTI) in the treatment of AML patients were initiated. FTI inhibits the activity of the enzyme responsible for cell proteins farnesylation and blocks intercellular signals transmission. The aim of the study was to evaluate the effect of FTI on localisation of RhoB protein and apoptosis of

leukaemic blast. The bone marrow AML cells were incubated with FTI (incubation time: 24 h, 48, 96 h: concentration of FTI: 30 nM, 150 nM, 300 nM, 600 nM). The early stage of apoptosis was observed in all cases (N=6) by Annexin V. The presence of FTI in cell medium (in every concentration added) induced DNA degradation that was observed mainly in culture ended after 48 and 96 h by DNA ladder method. The identification of RhoB proteins in AML cell extracts was performed by Western blot method. In parallel, the activity of caspase 9 in cultures with and without FTI was assayed. As yet, no correlation between caspase 9 activity, RhoB dislocation from the intercellular membranes and apoptosis of AML cells treated with FTI was found. Our preliminary results confirm that FTI induces apoptosis of AML leukaemic cells in the time and dose dependent manner.

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Oral Presentation

The role of FNIII/10 and FNIII/10 RGD fibronectin fragments in contact inhibition of C3H10T1/2 cells
Joanna Mioszewska, Monika Gos, Halina Trembacz, Magorzata Przybyszewska, Przemysaw Janik
Zakad Biologii Komrki, Centrum Onkologii Instytut im. M. Skodowskiej-Curie, ul. Roentgena 5, 02-781 Warszawa

Introduction: Cell proliferation in vivo and in vitro is a strictly regulated process. One of its control mechanisms is the growth inhibition induced by cellular contact even in the presence of growth factors. Release the cells from this state is associated with cellular transformation and signal transduction disorder. It is generally known that ERK1/ERK2 mitogen activated protein kinases play a critical role in transduction of signal that controls cell proliferation. Our previous experiments confirmed that the signal induced by fibronectin activated FAK and then ERK1/ERK2 had strong influence on contact inhibition in mouse foetal fibroblasts. The objective of the present work was to examine transduction of the proliferative signal in C3H10T1/2 contact inhibited cells transfected with fibronectin fragments. Methods: C3H10T1/2 cells were transfected with genes encoding fibronectin fragments (FNIII/10 and

FNIII/10RGD; confirmed by sequencing analysis). For ERK1/ERK2 phosphorylation Western blotting method was used. Gene expression was assayed using Atlas Gene Expression Array. Results: We found that the phosphorylation pattern of ERK1/ERK2 in FNIII/10 or FNIII/10RGD transfectants during logarithmic growth phase differed as compared to non-transfected C3H10T1/2 cells suggesting changes in signal transduction. The gene expression analysis showed that the cells transfected with fibronectin fragment FNIII/10 or FNIII/10RGD had higher expression of several genes related to proliferation. Conclusion: We suggest that transfection of C3H10T1/2 cells with fibronectin fragments leads to activation of ERK1/ERK2 proteins and induces expression of genes associated with proliferation. It seems that transfected cells can proliferate better than the C3H10T1/2 ones.

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Oral Presentation

177

Mutational analysis of the EcRDBD recognition alpha-helix and T-box region uncovers critical determinants of the functional ecdysteroid receptor interaction with the response element
Marek Orowski , Monika Szyszka , Iwona Grad , Agnieszka Kowalska , Daniel Krowarsch , Andrzej Oyhar
1 1 1 1 2 1

1 Institute of Organic Chemistry, Biochemistry and Biotechnology, Division of Biochemistry, Wrocaw University of Technology, Wybrzee Wyspiaskiego 27, 50-370 Wrocaw, 2 Laboratory of Protein Engineering, Institute of Biochemistry and Molecular Biology, University of Wrocaw, ul. Tamka 2, 50-137 Wrocaw

The steroid hormone 20-hydroxyecdysone controls Drosophila metamorphosis via the heterodimeric receptor composed of two members of the nuclear receptor superfamily ultraspiracle (Usp) and ecdysteroid receptor (EcR). Here, we present new data illustrating how two particular regions of the EcR DNA-binding domain (EcRDBD) contribute to the specificity of the interaction with the natural 20-hydroxyecdysone response element from the hsp27 gene promoter. The functional roles of the individual residues have been probed by alanine scanning. The first region consists of amino acid residues (E19-K31) localized in the EcRDBD region corresponding to the DNA recognition alfa-helix, which accordingly to the structural data obtained for other nuclear receptors, binds the major groove of DNA. As shown previously, the EcRDBD binds hsp27 either as homodimer or heterodimer with UspDBD. The EcRDBD homodimer formation on hsp27 was reduced for mutants E19A, G20A, K22A, R26A, R27A, S28A and K21A. Of these mutants, K22A, R26A, R27A and K31A demonstrated the strongest DNA binding defect. In contrast, mutants G23A and T30A increased the affinity of the EcRDBD towards hsp27. Heterodimerization with UspDBD partially neutralized mutational effects. Mutations at positions K22, R26,

S28 and T30 did not change the EcRDBDs structure as determined by Circular Dichroism (CD) spectroscopy. In contrast, the structure of mutant proteins E19A, G20A, G23A, and R27A was changed compared to the wild-type protein. The second region, localized in the C-terminal extension of the core EcRDBD (R67-E74), corresponds to the so-called T-box which has various functions in different nuclear receptors. The binding of mutants R67A, V71A and P73A to hsp27 was reduced, whereas the greatest DNA binding defect was observed for P73A. In contrast, mutant V72A increased the affinity of the EcRDBD to DNA. In the presence of the UspDBD DNA binding activity of R67A and V71A was impaired moderately, while mutant P73A had the greatest DNA binding defect. Mutant V72A increased the heterodimer affinity to hsp27. The CD spectra of mutants P68A and E74A demonstrated no changes in the EcRDBDs structure, while for E69A, C70A, V71A, V72A and P73A mutation changed the structure, in the case of R67A not significantly. The results define amino acids from recognition alfa-helix and so-called T-box region which are important for hsp27 binding and may play crucial role in protein structure stability or display both functions.
This work was supported by the State Committee for Scientific Research (KBN, Poland) grant 3 P04B 009 23.

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Oral Presentation

The involvement of Abl and Bcr-Abl proteins in Notch signaling pathway in haematpoietic normal and CML CD34+ cells
Barbara Ostrowska , Tomasz Sacha , Marta Szostek , Magdalena Zawada , Aleksander Skotnicki , Piotr 1 Laidler
1 Instytut Biochemii Lekarskiej, Collegium Medicum Uniwersytetu Jagielloskiego, ul. Kopernika 7, 31-034 Krakw, 2 Katedra i Klinika Hematologii, Collegium Medicum Uniwersytetu Jagielloskiego, ul. Kopernika 17, 31-501 Krakw
1 2 2 2 2

Haematopoiesis involves differentiation and proliferation of a small number of multipotent haematopoietic stem cells (HSCs) that give rise to mature blood cells of all lineages. To prevent the exhaustion of stem cell compartment HSCs proliferative activity is tightly restricted. However the molecular mechanisms regulating these processes are not well understood. Even

slight imbalance among them can lead to serious haematological disturbances. Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent HSC where the Philadelphia chromosome appears as a result of t(9,22) reciprocal translocation and the BCR-ABL hybrid oncogene is formed. Bcr-Abl chimeric protein expression leads to myeloid line cells expansion at

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various maturation stages. Exact molecular mechanism of malignant transformation is not well defined. Notch receptor proteins are involved in regulation of various cellular processes like asymmetric cell division, differentiation, proliferation, apoptosis, cell adhesion. The study of Drosophila nervous system development has shown the genetic interaction between Notch, Abl and Disabled proteins that are also found in haematopoietic cell lines. Its known that Notch signaling activates HSCs self-renewal in vivo and is responsible for its immortalisation in vitro and the Notch2 inactivation permits differentiation of murine myeloid line cells. It cannot be excluded that Bcr-Abl chimeric protein could disturb Notch signaling pathway because of an inability to interact with Notch receptor. The aim of this study is to search if interaction between Notch, Abl and/or Bcr-Abl proteins takes place in HSCs. The CD34+ stem and progenitor cells from peripheral blood and bone marrow were isolated by means of mag-

netic cell sorter (Miltenyi Biotec). The immunodetecion of protein-protein interaction in whole cell extracts was performed using custom-made antibody array membranes (Hypromatrix, Santa Cruz). The primary antibodies against protein of interest were spotted on the membranes. The membrane was incubated with CD34+ cell extract, then with primary antibodies against searched proteins and finally with the secondary HRP conjugated antibodies. The enhanced chemiluminescence reaction (ECL Plus, Amersham) was used to detect HRP labelled antibodies. The preliminary results indicate the presence of Notch1-Dab complexes in CML CD34+ cells and probably Notch1-Abl complexes in extracts of both healthy donors and CML patients (n=4) CD34+ cells. However clear-cut conclusion is at present difficult due to observed cross-reactivity of some antibodies and residual presence of anti-CD34 mouse IgG in cell extracts.

179

Oral Presentation

How adhesion, migration and cytoplasmic calcium transients influence interleukin-1beta mRNA stabilization in human monocytes
Pawe Pomorski , Ken Jacobson , Stephen Haskill , Watson Joanna
1 2 2 2

1 Pracownia Przekanikw Sygnaw, Instytut Biologii Dowiadczalnej im. M. Nenckiego PAN, ul. Pasteura 3, 02-093 Warszawa, 2 Lineberger Comprehensive Cancer Center, University of North Carolina, Mason Farm Rd., 27599-7295 Chapel Hill, USA

We investigated the mechanisms by which primary human monocyte migration and the production of important cytokines are co-regulated. Motile monocytes underwent cyclic morphologic and adhesive changes that were associated with intracellular free calcium changes; coordinated with changes of cell adhesion and motility speed. In motile cells, cytokine transcripts were unstable and translationally repressed. Agents that activate monocytes, including lipopolysacharrides (LPS), cytomegalovirus (CMV) and tumor necrosis factor (TNFalfa), have been shown to de-repress translation and these agents stabilize adhesion induced transcripts for IL lbeta and IL 8 and markedly diminished cell migration in the presence of autologous serum.

LPS suppressed Rho A activity and either this agent or C3 transferase elevated intracellular free calcium, stabilized transcripts and, in tandem, inhibited cell migration by preventing tail retraction, a prerequisite for cell translocation. These results therefore suggest that monocyte activating agents inhibit the RhoA pathway and continuously elevate intracellular calcium leading to a concomitant decrease in monocyte migration and stabilization of cytokine transcripts prior to translation. We suspect that downregulation of interleukin production and secretion when cell is still motile can play an important biological role in suppressing cytokine production activity in monocytes before those cells will reach a site of inflammation.

180

Oral Presentation

Expression of membrane antigens in cultured melanoma cell lines in different stages of differentiation
Rafa Sdej, Andrzej Skadanowski
Zakad Enzymologii Molekularnej, Midzyuczelniany Wydzia Biotechnologii UG i AMG, ul. Dbinki 1, 80-211 Gdask

Ecto-5-nucleotidase (eN) is a GPI-anchored enzyme localized in the cell membrane fraction called lipid rafts.

Catalytic function of the enzyme-production of adenosine from AMP is accompanied by antigenic role

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(CD73) and mediating in molecular interaction with extracellular matrix proteins. The main goal of our project is determination of the eN role in differentation, adhesion and motility of melanoma cells. The hypothesis has been put forward that eN also plays a role as an adhesion molecule which promotes cancer cell motility and invasion. Besides, the specific composition of lipid rafts can cause association of eN with neighbouring proteins, engaged in adhesion and signal transduction. Melanoma cell cultures in different stages of development were used as experimental model (human melanoma cells from primary sites in horizontal and vertical phase of growth, cutaneous and noncutaneous metastatic sites, melanotic and amelanotic hamster melanoma). The level of eN expression in the studied lines was determined by Western blotting with specific antibodies. The highest content of this enzyme was found in the cell lines from metastatic sites of noncutaneous

origin, lower in lines derived from primary sites, and cutaneous metastatic sites. Moreover, the level of eN correlates well with Gi alpha2, alpha3- and alpha5integrin subunits expression. As concern, beta1integrin inverse correlation has been observed. Additionally, the expression levels of N-cadherin, uPAR, PKA-I beta, PKA II alpha, Lyn, PI3K and cSrc were established. These signaling proteins were found to be present at high, but slightly diverse level in each type of cells studied. They are proposed to be potential partners interacting with eN. We suppose that eN presence on the surface of cells may represent the initiation process to more aggresive alias metastatic form of the melanoma cells. Our efforts concentrate now on assessment of CD44 and beta3-integrin expression and precise determination of proteins associating with eN by cell extracts fractionation to lipid rafts and cytoskeletal elements in Nycodenz density gradient. Oral Presentation

181

Cyclic GMP affects nuclear factor kappaB activity in human peripheral blood mononuclear cells (PBMC)
Jakub Siednienko, Marcin Kobiaka, Hanna Witwicka, Wojciech Gorczyca
Instytut Immunologii i Terapii Dowiadczalnej, Polska Akademia Nauk, ul. Weigla 12, 53-114 Wrocaw

NF-kappaB plays a key role in the regulation of genes relevant for immune response. This transcription factor may be activated by numerous stimuli and participates in expression of cytokines, chemokines, receptors, adhesion molecules, and enzymes involved in inflammatory reactions. Its constitutive activity prevents spontaneous apoptosis of unstimulated mature immune cells. Several data indicate that among factors that influence NF-kappaB transcriptional activity is also the elevated concentration of intracellular cGMP. The nucleotide has been shown to affect synthesis of TNFalpha, IL-1, IL-2, and NO in different immune cells. In this context the aim of our studies was to determine whether the NF-kappaB activity correlates with intracellular changes of cGMP concentrations in human peripheral blood mononuclear cells (PBMC). First we determined activities of particular (GC-A) and soluble (sGC) guanylyl cyclases by measurements of cGMP

formed in cells in response to specific activators of both enzymes. As complementary to functional measurements, expression of respective genes was determined using RT-PCR. In parallel with these experiments the NF-kappaB activity was determined by means of electro-mobility shift assay (EMSA) using nuclear fractions obtained from respective cells. We found that in freshly isolated human PBMC only soluble GC contributes to the synthesis of cGMP. Stimulation of cells with donors of nitric oxide, the known activator of sGC, correlated with increased activity of NF-kappaB. We also observed enhancement of the NF-kappaB activity after treatment PBMC with membrane-permeable 8Br-cGMP but not with 8Br-cAMP. Taken together, our results indicate that generation of cGMP may influence the NF-kappaB activity in human PBMC. The responsible mechanism remains to be elucidated. Oral Presentation

182

Effect of b1-integrin receptor modulators, echistatin and thrombin on collagen biosynthesis in human dermal fibroblasts
Arkadiusz Surayski, Pawe Sienkiewicz, Jerzy Paka
Zakad Chemii i Analizy Lekw, Akademia Medyczna w Biaymstoku, ul. Kiliskiego 1, 15-230 Biaystok

Collagen biosynthesis in human dermal fibroblasts depends on prolidase, the enzyme which catalyses hydrolysis of imidodipeptides (mainly derivatives of colla-

gen degradation products), releasing proline, which is used for resynthesis of this protein. Prolidase activity is regulated due to the signal induced by activated

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beta1-integrin receptor. Activation of this receptor initiates cascade of signaling pathway, in which several kinases and intracellular proteins are involved, including FAK, Grb-2, SOS, MAPK (ERK1 i ERK2). We studied the influence of echistatin (a well known disintegrin, which blocks beta1-integrin receptor) and thrombin (an agonist of beta1-integrin receptor ) on collagen biosynthesis (measuring 5-[3H]-proline incorporation into proteins, susceptible to action of bacterial collagenase), prolidase activity (colorimetric method) and expression level, beta1-integrin receptor, SOS protein and MAP-kinases (Western blot). All studies were performed using confluent human dermal fibroblasts.

It has been found that echistatin inhibits and thrombin stimulates collagen biosynthesis and prolidase activity and expression level. Analysis of signaling proteins expression levels suggest, that mentioned above beta1-integrin receptor modulators exert their effect on prolidase activity and collagen biosynthesis regulating the expression of SOS protein and MAP-kinases. It has been found that echistatin decreases and thrombin increases expression of these proteins in studied fibroblasts, comparing to control cells. The results suggest that regulation of collagen biosynthesis in human dermal fibroblasts is mediated by signaling cascade from activated beta1-integrin receptor.

183

Oral Presentation

Cyclic GMP-regulated phosphodiesterases are effectors for cGMP in rat peritoneal macrophages
Hanna Witwicka, Marcin Kobiaka, Jakub Siednienko, Ewa Kurowska, Wojciech Gorczyca
Instytut Immunologii i Terapii Dowiadczalnej, Polska Akademia Nauk, ul. Weigla 12, 53-114 Wrocaw

The aim of our studies was to establish which enzymes may participate in cGMP signaling in rat peritoneal macrophages (rPM). We show that synthesis of cGMP in rPM occurs exclusively in response to ANP, the activator of particulate guanylyl cyclase type A (GC-A). Accumulation of the nucleotide is enhanced several-fold in the presence of non-selective inhibitor of phosphodiesterases (PDEs). The PDE activity measured in rPM cellular fractions is independent on 2+ Ca /calmodulin but strongly depends on cGMP concentration. The analysis of the substrate specificity, sensitivity to inhibitors, and cGMP-dependent activity of PDEs present in the fractions obtained after ion exchange chromatography indicates that several isoforms of PDE might be involved in cGMP hydrolysis in rPM. All above findings are supported by RT-PCR which reveals that mRNA specific for GC-A but not for soluble guanylyl cyclase is expressed in rPM. Also expression of PDE2, PDE5 is confirmed by RT-PCR. In-

terestingly, the mRNAs for PDE3A and PDE3B, the enzymes that are cGMP-regulated but specific toward cAMP, are also detected in rPM. It is still unclear whether PDE10 and PDE11 are present in rPM. In summary, our results show that in rPM only GC-A contributes to the synthesis of cGMP and the intracellular concentration of the nucleotide is controlled by the cGMP-dependent PDEs in a feedback-regulated way. Since PKG1 apparently is not expressed in rPM we speculate that in these cells the cGMP-regulated PDEs, specific to both cGMP and cAMP, are effector enzymes for synthesized cGMP. They may play a dual role: by controlling the concentration of cGMP, and also the concentration of cAMP the known mediator of inflammatory response in macrophages. Why PDE2 and PDE3, two oppositely regulated by cGMP enzymes, are expressed in the same cell type remains to be elucidated. Subcellular localization seems to be a critical step in search of their function.

184

Oral Presentation

The participation of MAP kinase pathways in the response of Galleria mellonella to temperature and osmotic challenge
Iwona Wojda, Patryk Kowalski, Teresa Jakubowicz
Department of Invertebrate Immunology, Maria Curie-Sklodowska University, ul. Akademicka 19, 20-033 Lublin

The participation of MAP kinases in the adaptation of yeast and mammalian cells to stress conditions is being studied in many laboratories in the world. So far, rela-

tively little is known about the function of MAP kinases in invertebrate animals. Galleria mellonella (greater wax moth) is an insect whose larvae live in honeybee

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colonies, where feed wax, honey and pollen. We studied the role of MAP kinase pathways in response to stress conditions and immune challenge. Antibodies directed against dually phosphorylated ERK MAP kinases (human p44/p42) recognised two phosphoproteins in Galleria mellonella fat body: ERK-a and ERK-b. We showed that under heat shock conditions (38C) ERK-a MAP kinase was intensively phosphorylated in time-dependent manner while ERK-b-dephosphorylated under the same conditions. The expression/activation of Galleria mellonella ERK MAP kinases was different in hemocytes and in fat body.

We also found that other MAP kinases: p38 and JNK (but not ERK) were activated in isolated fat body and hemocytes upon high external osmolarity. Phosphorylation of p38 was abolished upon the use of specific, membrane permeable inhibitor SB203580. The effect of p38 inhibition by SB203580 on the viability of hemocytes at higher osmolarity depended on salt concentration. We also highlighted the role of carbohydrates such as glucose and trehalose in the adaptation of Galleria mellonella to stress conditions.

185
Induction of the angiogenic phenotype in HUVEC by VEGF
Patrycja Baraska , Hanna Jerczyska , Wiktor Koziokiewicz , Zofia Pawowska , Czesaw Cierniewski
1 1 1 1

Posters
2

1 Zakad Biofizyki Molekularnej i Medycznej, Uniwersytet Medyczny w odzi, ul. Mazowiecka 6/8, 92-215 d, 2 Centrum Mikrobiologii i Wirusologii, Polska Akademia Nauk, ul. Lodowa 106, 92-215 d

Angiogenesis, the formation of new blood capillaries from existing vessels, is a complex process involving endothelial cells under physiological and pathological circumstances. After stimulation with positive regulators of angiogenesis, endothelial cells undergo a complex sequence of events that induces their shift from the quiescent state to angiogenic phenotype. Vascular endothelial growth factor (VEGF) plays the fundamental role as a factor with positive regulatory activity in this process. Since it is an important proangiogenic factor involved in the regulation of various cellular processes, we investigated the effect of VEGF activation in early stages of angiogenesis events. In this study, we characterized the effect of VEGF stimulation of human umbilical vein endothelial cells (HUVEC) on its capability to proliferate, adhere and migrate. The stimulation of quiescent cells with 20 ng/ml VEGF does not signifi-

cantly increase the cell number, but it seems to act as an endothelial cell survival factor under the serum-free medium conditions. Since VEGF has been shown to be involved in the regulation of adhesive response of cells, we investigated the ability of cells cultured in the presence of this factor to adhere to gelatin, fibronectin and collagen. VEGF-stimulated cells appeared to adhere to a greater degree and faster than cells in control cultures. VEGF potently stimulates migration as we determined during the cell migration test (wound healing-like" assay). At the same time we identified the protein pattern of VEGF activated endothelial cells using two-dimensional electrophoresis. Using proteomics we managed to identify the group of proteins which are overexpressed in the angiogenic cells and may modulate signaling pathways involved in the regulation of angiogenesis.

186
Expression of pregnane X receptor gene (PXR) in human polycystic ovaries
1 1 1 2

Posters
1

Natalia Derebecka , Marta Ociepa-Zawal , Marcin Hoysz , Alina Warenik-Szymankiewicz , Wiesaw Trzeciak
1 Katedra i Zakad Biochemii i Biologii Molekularnej, Akademia Medyczna im. K. Marcinkowskiego, ul. wicickiego 6, 60-781 Pozna, 2 Katedra i Klinika Endokrynologii Ginekologicznej, Akademia Medyczna w Poznaniu, ul. Polna 33, 60-535 Pozna

Pregnane X receptor (PXR, NR1I2) is a member of nuclear receptor family and acts as a ligand-dependent transcription factor. It is activated by numerous ligands: xenobiotics and steroids. Its natural ligands are probably 21C steroids. PXR regulates the metabolism of xenobiotics and steroids by the activation of CYP3A4

transcription. The expression of PXR was detected mainly in the liver. One of the PXR isoforms (PXR2) contains a truncated ligand-binding domain and is less effective in induction of CYP3A expression. Since pregnanes act as PXR ligands, it is possible, that the differential expression of this receptor may in-

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fluence the endocrine function of the ovary and play a role in the pathogenesis of polycystic ovary syndrome (PCOS). The main aim of the study was to analyse the expression of PXR gene in polycystic and normal ovaries, focusing on two splice variants PXR1 and PXR2. Total RNA was isolated from 14 polycystic and 6 normal ovaries. PXR mRNAs were reversily transcribed with the use of gene specific primer. Then PXR fragments were amplified by PCR and separated in 2% agarose. Direct sequencing of the fragments was performed in an automated sequencer.

The expression of PXR was detected in 16 (10 polycystic and 6 normal) ovaries. Two fragments (PXR1 and PXR2) were detected in all samples. The expression of PXR2 in polycystic ovaries was higher than in the normal ovaries. No expression was detected in 4 PCO samples The expression of PXR in the ovaries has never been described before. The results of the study suggest that the expression of PXR is important for the endocrine function of the ovary and might play a role in pathogenesis of polycystic ovary syndrome.

187
Prostate cancer and expression of adhesion proteins
Joanna Duliska, Piotr Laidler
Instytut Biochemii Lekarskiej, Collegium Medicum Uniwersytetu Jagielloskiego, ul. Kopernika 7, 31-034 Krakw

Posters

Diets high in fat are associated with an icreased risk of prostate cancer, althought the molecular mechanism is still unknown. In vivo studies have shown that fatty acids can modulate the growth of prostate cancer cells. Long-chain fatty acids, prostaglandins natural ligands and ciglitazone a synthetic factor are bound to peroxisome proliferator-activated receptors (PPAR). PPARs are ligand-activated transcription factors that play important role in regulation of metabolic, developmental and differentiation pathways. Significant changes in cell adhesion are well recognized during cancer progression. Cadherins cell adhesion molecules located in the plasma membrane almost all mediate interaction in vast majority of solid tissues. Some of them, E-cadherins are know as tumor suppressor while the other expression of as N-cadherin is correlated with cancer progression. Therefore we attempted to determine the effect of fatty acids on expression of these important adhesion proteins. Methods: Studies were carried out on human prostate cancer cell lines: PC-3 (androgen-independent) and LNCaP (androgen dependent). The cells were stimu-

lated by linoleic acid, ciglitazone and fenofibrate. Expression of N- and E-cadherins was studied on protein (Western blott) and mRNA levels (RT-PCR, Real-time PCR). Results: Stimulation of PC-3 cells with ciglitazone resulted in appearance of E-cadherin and decreased by about 30% expression of N-cadherin. The restoration expression of E-cadherin in PC-3 cell line and increased expression of this protein in LNCaP cell line after stimulation the cells with linoleic acid was noticed. Conclusion: The expression of E-cadherin and N-cadherins was different in studied cell lines. PC-3 cells expressed N-cadherine and did not or very weakly E-cadherine while LNCaP line showed opposite effect. The obtained results suggest, that the natural and synthetic PPARg ligands increase the expression of E-cadherin tumor suppressor, in PC-3 cell line. The expression of N-cadherin decreased in PC-3 cells stimulated with both ligands and levels of this adhesion protein correlated with invasive phenotype of cancer.
This work was supported by the grant No 6 PO5A 074 21 of the State Committee for Scientific Research (KBN, Poland).

188
Phosducin homologue is present in Blepharisma japonicum
Hanna Fabczak, Katarzyna Sobierajska, Boena Groszyska, Stanisaw Fabczak
Zakad Biologii Komrki, Instytut Biologii Dowiadczalnej im. M. Nenckiego PAN, ul. Pasteura 3, 02-093 Warszawa

Posters

Ciliate Blepharisma japonicum is a photosensitive protozoan, which displays a distinctive motile photophobic response to an increase in light intensity. It has been re-

cently shown that light also elicits transient inositol trisphosphate accumulation in the ciliate. Changes in inositol trisphosphate levels, as well as occurrence of

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photophobic response, are significantly suppressed by agents known to interfere with phosphoinositol turnover in photoreceptor systems of other organisms. These findings, along with the recent localization of an inositol trisphosphate receptor-like protein and GTP-binding protein within the ciliate cortex, may indicate that light transduction mechanism participates in photobehavior of Blepharisma. Our recent immunoblotting studies show that tested ciliates contain a 28-kDa protein, homologous to phosducin. This phosphoprotein is widely distributed in several organisms tissues and it is conjuctured to play a role in regulating the second messenger cascade of vision and G-protein-mediated signaling of other receptor cell systems. Phosducin is phosphorylated in the darkness and

is significantly dephosphorylated upon illumination. The phosphorylation of this protein in vivo occurs via protein kinase type A or G and by Ca2/calmodulin-dependent kinase type II and dephosphorylated by 1 or 2A phosphatases. Immunocytochemical studies show that phosducin is distributed uniformly within cytoplasm of cell adapted to darkness, however upon illumination immediate displacement of phosducin from cytosol to the vacinity of plasma membrane takes place. The presented results are the first identification and morphological localization of phosducin in protozoan ciliate. On the basis of these findings we suggest that the phosducin may be also involved in the cell phototransduction pathway, resulting in the photophobic behavior of Blepharisma.
This study is supported by grant No. 6P04C 057 18 of the Committee for Scientific Research (KBN, Poland).

189
Potassium channel activity from bovine chromaffin granules
Renata Hordejuk , Adam Szewczyk , Krzysztof Doowy
1 2 1

Posters

1 Katedra Fizyki, Zakad Biofizyki, SGGW Warszawa, ul. Rakowiecka 26/30, 02-528 Warszawa, 2 Pracownia Wewntrzkomrkowych Kanaw Jonowych, Zakad Biofizyki, Instytut Biologii Dowiadczalnej im. M. Nenckiego, PAN, ul. Pasteura 3, 02-093 Warszawa

Adrenal chromaffin granules are the intracellular membrane vesicles involved in biogenic amines synthesis and traffic. The uptake of hormones, driven by pH gradient, from the cytosol into chromaffin granules is catalyzed by catecholamine carrier. Secretion of hormones occurs as a result of fusion of chromaffin granule vesicles with plasma membrane. Ion channels with different ion specificity are present in chromaffin granules membranes. Some of them may play an important physiological role by compensating electric charge transfer produced by the vacuolar + type H - ATPase. Properties of a chromaffin granules + K transport was studied by flux measurements using + 86Rb+ (a K analog). They indicate the existence of an + electrogenic, pH-sensitive K transport system in chromaffin granules. The aim of this work was to show the presence of potassium channel with large conduc-

tance in chromaffin granules membranes. In details, potassium channel was characterized, an effect of pH on its kinetic and voltage dependance were shown. These experiments were performed using black lipid membrane method. Granule membranes were reconstituted into artificial membranes made of asolectin. The conductance and selectivity of the channel were examined. At gradient 450 mM/150 mM KCl the current/voltage relation appeared to be linear when examined within membrane potentials of 70 mV with a single channel slope conductance of ~380 pS. Sensi+ tivity of K channel with large conductance on pH changes was examined in 7.0 and 6.4. Results show + that K channel with large conductance may play an important role in acidification of chromaffin granules interior by V-ATPase.
This work is supported by grand 50406020013 and Nencki Institute of Experimental Biology.

190

Posters

Determination in vivo of Bcl-2 and Bax expression in B-CLL cells of patients treated with chemotherapeutic drug(s)
Agnieszka Kobyliska , Jerzy Boski , Tadeusz Robak , Zofia Kiliaska
1 2 2 1

1 Katedra Cytobiochemii, Uniwersytet dzki, ul. Banacha 12/16, 90-237 d, 2 Katedra Hematologii, Uniwersytet Medyczny, ul. Ciokowskiego 2, 93-510 d

B-cell chronic lymphocytic leukemia (B-CLL) is the most common hematological malignancy in Western countries, yet has an unclear etiology. This disease is

characterized by the slow and progressive accumulation in peripheral blood, bone marrow, lymph nodes and other organs of monoclonal, apparently mature,

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CD5+ B lymphocytes. The majority of circulating cells appear to be nondividing and it has been suggested that the clonal excess of B cells results from decreased apoptosis rather than increased proliferation. The development of new chemotherapeutic drugs such as purine nucleoside 2-chlorodeoxyadenosine (cladribine, 2CdA) allows to induce the cell death by apoptosis and it creates new possibilities for B-CLL therapy. We investigated some apoptotic events in mononuclear cells obtained from peripheral blood of patients with B-CLL during and after therapy with 2-CdA (C regimen), and its combination with cyclophosphamide (CC regimen) or 2-CdA with mitoxantrone, and cyclophosphamide (CMC regimen). Western blot technique was performed to estimate expression of antiapoptotic and proapoptotic proteins: Bcl-2 and Bax in leukemic cell homogenates, nuclear and postnuclear fractions originating from blood of untreated (0) and treated B-CLL patients (1st, 5th for C and 1st, 3rd for CC and CMC regimen, respectively). Additionally, blood sam-

ples were taken from patients after 14 days of drug(s) administration (19th and 17th day for C and CC, CMC, respectively). The results obtained revealed that expression of Bcl-2 generally dropped during drug(s) treatment, whereas expression level of Bax protein grew up significantly, resulting in an increase of Bax/Bcl-2 ratio and induction of apoptosis. The lowest level of Bcl-2 expression and the highest increase of Bax/Bcl-2 ratio were observed in cell homogenates isolated from blood patients treated according to CMC regimen (p<0.001, Anova test). The changes in Bcl-2 and Bax expression were accompanied by an appearance of apoptotic morphology and DNA fragmentation in drug-treated leukemia cells, determined by Giemza staining and electrophoresis in agarose gel, respectively. We have also started to analyse the level of Bax/Bcl-2 ratio in mononuclear cells isolated from blood of untreated B-CLL patients cultured in vitro in the presence of 2-CdA or 2-CdA with mafosfamide or 2-CdA with mitoxantrone and mafosfamide. Poster

191
Interleukin-8 serum levels in patients with melanoma
Leszek Kozowski , Irena Zakrzewska , Marek Wojtukiewicz
1 2 1

1 Zakad Onkologii, Akademia Medyczna w Biaymstoku, Biaystok, 2 Zakad Laboratoryjnej Diagnostyki Klinicznej, Akademia Medyczna w Biaymstoku, Biaystok

During the last years, the incidence of malignant melanoma has been rising rapidly in populations of European origin. Malignant melanoma is a potentially fatal skin cancer. Some investigators postulate that IL-8 is involved in the regulation of many processes including proliferation melanoma cells and promotion of angiogenesis. The aim of the study is an attempt to evaluate the usefulness of IL-8 in diagnosis of malignant melanoma. Studies were performed on patients of the age 48-82 with malignant melanoma. Clinical diagnosis was confirmed by histopathologiacl examination. The control group constituted 25 healthy persons. Blood for

analysis was collected under standard conditions prior to treatment. Serum levels of IL-8 were measured using ELISA immunoenzymatic technique. It was decided to treat mean control values 2SD as normal values. Significantly higher IL-8 levels were detected in malignant melanoma compared to control group p<0.01. IL-8 levels were related to stage of disease. The frequency of increased concentration of IL-8 showed a trend to significant increase and a correlation with the clinical stage of disease. The observations allow to conclude that the determination of IL-8 in the serum may by useful in estimation of melanoma progression.

192
Metal ion binding to integrin alphaIIbbeta3, beta3(109352) and its mutants
Kamila Krupska , Lidia Michalec , Szymon Skurzyski , Czesaw Cierniewski
1 1 2 3

Poster

1 Zakad Biofizyki Molekularnej i Medycznej, Uniwersytet Medyczny, ul. Mazowiecka 6/8, 92-215 d, 2 Wydzia Biologii i Ochrony rodowiska, Uniwersytet dzki, d, 3 Centrum Mikrobiologii i Wirusologii, Polska Akademia Nauk, ul. Lodowa 106, 92-215 d

Interaction of Ca and Mn with the purified alphaIIbbeta3, in the presence or absence of ligand peptides (KYGRGDS, LGGAKQAGDV) was tested by equilibrium dialysis. alphaIIbbeta3, either alone or in the complex with peptide ligands, bound the same

2+

2+

amount of divalent cations, i.e. 5-6 mol of Ca or Mn per mol of the receptor. All metal-binding sites in alphaIIbbeta3 have similar 2+ affinity for Mn in the resting state (N = 5.85 1.1

2+

2+

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mol/mol at Kd = 3.65 0.75 mM) and after activation with KYGRGDS (N = 5.61 at Kd = 2.94 mM). The bind2+ ing affinity for Mn was higher than that characteris2+ tic for the medium-affinity Ca -binding sites analyzed o at 21 C. Therefore, if there is a change in the affinity of 2+ Mn -binding sites upon activation of alphaIIbbeta3 by ligand peptides it is not detectable under experimental conditions used. To determine the position of two cation binding sites we used the beta3 (109352) fragment which corresponds to the A domain of beta3 integrin subunit. This region of beta3 is of special importance because it contains the Inhibitory (I), the Ligand Com-

petent (LC) and the RGD binding sites. Then we have focused on studying of the kinetics of folding and unfolding reactions for the recombinant fragment of beta3 subunit. In order to analyze its conformational changes induced upon binding of cations, two tryptofan mutants W129A and W238A were prepared by site directed mutagenesis. We measured spectral and metal-binding properties of two single-point tryptofan mutants of the beta3 (109352) . The purified peptide 2+ fragment beta3 (109352) bound 3 Mn with affinity (N = 2.89 at Kd = 7.88 mM).

193

Poster

The effect of beta-carotene and fatty acids on human cancer cells proliferation and apoptosis
Piotr Laidler , Joanna Duliska , Dorota Gil , Ryszard Nawrocki , Katarzyna Kurpiewska , Aldona 3 Kie-Dembiska
1 Instytut Biochemii Lekarskiej, Collegium Medicum Uniwersytetu Jagielloskiego, ul. Kopernika 7, 31-034 Krakw, 2 Wydzia Chemii, Uniwersytet Jagielloski, Krakw, 3 Biochemia Kliniczna, Collegium Medicum, ul. Kopernika, Krakw
1 1 1 2 2

Despite of extensive knowledge on the role of beta-carotene in major cellular processes there are some controversies in respect to its potential effect on cancer progression. Several functions of retinoids, such as regulators of the cell differentiation, antioxidants and possible anti-carcinogenic factors have been reported. They act through nuclear family receptors retinoid receptor. Their activation is affected by receptor of fatty acid peroxisome proliferator-activated receptor that bound natural ligand (e.g. prostaglandins) as well as synthetic substrate. It has been proposed that beta-carotene and fatty acids may positively regulate the expression of apoptotic genes and in that way affect proapoptotic genes (1). Therefore we studied the effect beta-carotene and fatty acids on proliferation and expression in different human cancer cell lines. Methods: Studies were carried out on human prostate cancer LNCaP, PC-3 and A375, WM 35 melanoma cancer cell lines. The cells were treated by 3 mM and 10 mM beta-carotene in 0.05% THF and by linoleic,

arachidonic (0.5, 2.0, 5.0 mM) and palmitic (1.0, 5.0, 15.0 mM) acids. The proliferation of cells was determineted by ELISA BrdU Proliferation Test and citotoxicity using Cititoxicity Detection Kit (Roche). The apoptosis was studied using DNA ladder method. Expression of apoptotic proteins was determinate at protein (Western blott) and mRNA (RT-PCR) level. Results: The effect of beta-carotene and fatty acids on cancer cells was time-, concentration-, and cell type-dependend. The antiproliferative effect of beta-carotene and fatty acids on melanoma cells was clearly observed. On the contrary, the significant increase in LNCaP cells proliferation in the presence of these compounds was noticed. However, in any case no effect of beta-carotene and fatty acids on the expression of apoptotic proteins Bax/Bcl-2 was found.
References: 1. Sarah J. Roberts-Thomson (2000) Immunology and Cell Biology, 78: 436441. This work was supported by the grant 5RP-DLARFID No QLG1-CT-2000-01185 (EU), Poland

194
Expression of caveolin-1 protein in normal and carcinomatous colorectal tissue
Izabela Masowska , Radzisaw Trzcinski , Adam Dziki , Wanda Krajewska
1 2 2 1

Poster

1 Department of Cytobiochemistry, University of d, ul. Banacha 12/16, 90-237 d, 2 Department of General and Colorectal Surgery, Medical University of d, Plac Hallera 1, 90-647 d

Caveolin-1, a 2124 kDa integral membrane protein is the major structural component of caveolae.

Caveolae are plasma membrane microdomains that can invaginate to form 50100 nm vesicles. Caveolae have

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been reported to contain various recepors, intracellular signaling molecules and proteins. Gene encoding human caveolin-1 is localized to the q31.1 region of human chromosome 7, at the D7S522 locus in fragile site FRA7G. Deletion of the q31 region of human chromosome 7 has been implicated in the pathogenesis of different types of human cancers. The aim of this work was to study protein expression for caveolin-1 in colorectal cancer. Fresh-frozen samples were obtained from surgically ablated colorectal cancers staged according to Dukes classification. Section from normal colonic mucosa at the proximal resection margins were taken at the moment of surgical re-

section. Monoclonal antibody was obtained from Santa Cruz Biotechnology. Expression of caveolin-1 proteins was examined using Western blot technique. Level of caveolin-1 proteins was established by ELISA test. Statistic analysis was done by Mann-Whitney U test. The number of caveolin-positive cases in normal versus carcinomatous tissues was evaluated. In addition the correlation of caveolin-1 expression and the progression of the disease was assessed. Quantitative analysis of caveolin-1 level was done for all positive cases. Obtained data suggest that neoplastic transformation of colon cancer is accompanied by the deregulation of caveolin-1 expression.

195
Study on RAR and RXR receptors in HPV16/18 positive cervical cancer cells
Magdalena Myga , Izabela Parol , Witold Kdzia , Anna Kwaniewska , Anna Godzicka-Jzefiak
1 2 3 2 1

Poster

1 Instytut Biologii Molekularnej i Biotechnologii, Uniwersytet im. Adama Mickiewicza, ul. Midzychodzka 5, 60-371 Pozna, 2 Zakad Ginekologii i Poonictwa, Akademia Medyczna w Lublinie, ul. Staszica 16, Lublin, 3 Zakad Ginekologii Onkologicznej, Akademia Medyczna w Poznaniu, ul. Polna 33, Pozna

Retinoids including vitamin A and its analogs are used as chemopreventive drugs in cervical cancer [1]. Its effect is exerted by activation of retinoic acid receptor (RAR) and retinoic receptor (RXR) heterodimers. These receptors bind to the retinoic acid response element of target gene to regulate gene expression responsible for the inhibition of epithelial cell growth and differentiation [2]. The retinoids also inhibit expression of E6/E7 oncogenic proteins of Human Papillomavirus in HPV transforming HeLa cells. However, their therapeutical effect depends on its specific receptors in target cell. In the present study the coding sequences and expressions of RAR and RXR receptors were analyzed in 70 cervical cancer cells HPV 16/18 positive. The study group of patients had normal or low level of retinoids in blood serum. The RAR and RXR coding sequences were analyzed by PCR/SSCP, and sequenced. The Northern

hybridization was used for the detection of RAR and RXR transcripts in study on cervical cancer and cell culture. The results did not indicate any differences in coding sequences of RAR and RXR in cervical cancer cell. The changes in level of RAR and RXR transcripts were observed in cell culture depending on RA concentration in culture medium. RA at the concentration ratio between 3 6 10 10 pg/ml also inhibits the expression of E6 and E7 HPV16 oncogenic proteins in HeLa cells. Therefore effectiveness of the RA treatment of cervical cancer HPV positive cell will probably depend on factors regulating expression of RAR and RXR receptors as well as concentration of its ligands in blood serum.
References: 1. Eckert RL, Agarwal C, Hembree JR, Choo CK, Sizemore N, Andreatta-van Leyen S, Rorke EA (1995) Adv Exp Med Biol, 375: 3144. 2. Creek KE, Jenkins GR, Khan MA, Hodam JR, Tolleson WH, Pirisi L (1994) Adv Exp Med Biol, 354: 1935.

196
Aleksandra Obrpalska-Stplowska , Andrzej Kdzia , Joanna Pacholska , Anna Godzicka-Jzefiak
1 2 1 1

Poster

Analysis of P1 regulatory region of IGF-1 gene in children with growth retardation


1 Instytut Biologii Molekularnej i Biotechnologii, Uniwersytet im. Adama Mickiewicza, ul. Midzychodzka 5, 60-371 Pozna, 2 Klinika Pediatrii, Instytut Diabetologii i Endokrynologii, Akademia Medyczna w Poznaniu, Pozna

Insulin-like growth factor I (IGF-I) is a 70 amino-acid single-chain peptide which belongs to the family of peptide hormones that includes relaxin and insulin, and

shares a high degree of structural similarity with proinsulin. The human IGF-I gene consists of six exons that are located within a region of over 90 kb on chromo-

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some 12. The gene is transcribed into a large precursor mRNA, which is alternatively spliced to different classes of mRNA: IGF-1A and IGF-1B. In mammals, IGF-1 gene transcription is controlled by two promoters: P1 and P2, that are located 5 to unique leader exon 1 and 2, respectively. IGF-I is produced in most, if not all, tissues. However, liver is the major contributor to the circulating endocrine IGF-I. IGF-I plays a fundamental role in tissue growth and development both as a mediator of many of the growth hormone actions and as a locally acting stimulator. The aim of the present study was analysis of the P1 promoter regulatory region of IGF-1 gene in children with growth disorders. Research was performed on peripheral blood cells from children with growth retardation, normal serum level of growth hormone and low level of IGF1. DNA was isolated from all the samples and PCR/SSCP and sequencing methods were used for analysis of P1 promoter of IGF-1 gene. EMSA studies were used for analysis of binding activity of P1 promoter regulatory re-

gion of IGF-1 gene. Binding reactions were performed with HeLa protein extract and protein extracts from human liver cells. The regulatory region of IGF-1 gene has been identified as a potential binding site for HNF3, HNF1, GATA, C/EBP transcription factors which can participate in transcription regulation of IGF-1 gene expression. Our studies have demonstrated several nucleotide changes within 1000 to 200 segment in children with growth retardation (888 [g], 811[c=>t], 775[a=>g]744 [a=>g], 383 [c=>t] 256 [t=>g] and many others). The gel mobility shift assays indicated that several different DNA-protein complexes could be formed in this region with protein extract from HeLa cells and human hepatocytes. It suggests that nucleotide changes may abolish binding capacity of a particular transcription factor or alter its affinity to the site as well as its ability to interact with other proteins. Using specific competitors in EMSA study we demonstrated that they have no influence on binding activity of HNF3 and HNF1 factors in liver cells. The role of other proteins on activity of P1 promoter is to be analysed.

197
Detection of apoptotic events in B-CLL cells treated in vitro with 2-CdA
Magorzata Rogaliska , Katarzyna Woniak , Agnieszka Kobyliska , Jerzy Boski , Janusz Basiak , 3 1 Tadeusz Robak , Zofia Kiliaska
1 2 1 3 2

Poster

1 Katedra Cytobiochemii, Uniwersytet dzki, ul. Banacha 12/16, 90-237 d, 2 Katedra Genetyki Molekularnej, Uniwersytet dzki, ul. Banacha 12/16, 90-237 d, 3 Katedra Hematologii, Uniwersytet Medyczny, ul. Ciokowskiego 2, 93-510 d

The deoxyadenosine nucleoside analogue 2-chloro-2-deoxyadenosine (2-CdA) alone or with combination with other drugs is currently used treatment of leukemias and lymphomas. 2-CdA is unique compared with traditional antimetabolite drugs in that it is equally active against resting and dividing lymphocytes which may be especially important in B-chronic lymphocytic leukemia (B-CLL), because most cells are in the resting phase. It has been suggested that the clonal excess of malignant B cells arises from still not well known defect(s) in apoptosis. It is accepted that 2-CdA inhibits DNA-repair, hindering RNA-synthesis by the accumulation of strand breaks within DNA, inhibits a number of enzymes involved in nucleic acid metabolism, and causes neoplasm regression by the induction of apoptosis. To asses the apotoxic effects of 2-CdA monotherapy or its combination with other drug(s) we employed several techniques to determine apoptotic events in mononuclear B-CLL cells. B-CLL cells obtained from previously untreated patients were cultured for 48 hours without or with 50 ng/ml 2-CdA; 500 ng/ml mitoxantrone; 1000 ng/ml mafosfamide. Additionally, malignant cells were cul-

tured for the same time with combination of purine nucleoside and mafosfamide or mafosfamide and mitoxantrone. The used drugs, i.e., 2-CdA and mitoxantrone were purchased from Bioton and Jelfa (Poland), respectively, and mafosfamide was donated from Baxter Onc. GmbH (Germany). Cell viability was determined by MTT assay. To evaluate the DNA damage introduced by examined chemotherapeutic agent(s) the alkaline comet assay was performed. The potential of these drugs to induce apoptotic morphological changes in studied B-CLL cultures was estimated by Giemza and Hoechst 33258 propidium iodide staining. The results obtained by comet assay and cell viability test revealed that combined three-drug (2-CdA-mafosfamide-mitoxantrone) treatment of B-CLL cells is generally the most effective in DNA damaging and cell cytotoxity. Strong potential in apoptosis induction of B-CLL cells with this drug combination was confirmed by revealing of apoptotic morphology, i.e., abnormal shape, condensation and nuclear fragmentation, blebbing of the plasma membranes and appearance of apoptotic bodies.

112

Session 2. Cellular signalling

2003
Poster

198

The A/B region of Ultraspiracle protein promotes but does not suffice for effective DNA-independent homodimerization of the receptor
Grzegorz Rymarczyk, Magorzata Nocula, Andrzej Oyhar
Institute of Organic Chemistry, Biochemistry and Biotechnology, Division of Biochemistry, Wrocaw University of Technology, Wybrzee Wyspiaskiego 27, 50-370 Wrocaw

Ultraspiracle protein (Usp) and ecdysteroid receptor protein (EcR) together form a heterodimeric receptor for 20-hydroxyecdysone and therefore constitute an important molecular switch of morphogenetic events during insect metamorphosis. We have previously reported overexpression in E. coli and purification of the full-length His-tagged Usp [Rymarczyk, G., Grad, I., Rusek, A., Owicimska-Rusin, K., Niedziela-Majka, A., Kochman, M., Oyhar, A. (2003) Biol. Chem., 384, 59-69]. The recombinant receptor has been shown to specifically bind to its response elements, whereas gel filtration and crosslinking experiments have demonstrated that the full-length Usp forms homodimers and homotetramers in the absence of DNA. However, the recombinant Usp devoid of its N-terminal A/B region has failed to homodimerize in solution. This suggests

that important determinants responsible for its efficient homodimerization lie in the N-terminal region of the receptor. Here we report expression and purification of two Usp fragments the isolated A/B-region, and the fragment containing both the A/B region and the DBD. Although gel filtration experiments reveal that hydrodynamic properties of both proteins are consistent with existence of homodimers, crosslinking experiments fail to detect any dimeric species in solution. Thus, we conclude, that the A/B region although necessary for DNA-independent homodimerization of Usp is not by itself sufficient for the effective formation of dimers.
This work was supported by grants from the State Committee for Scientific Research (Grant 3 P04B 009 23) and partially from the Wrocaw University of Technology.

199

Poster

Expression of checkpoint kinases Chk1 and Chk2 in human colon carcinoma and normal colorectal tissue
Magdalena Stawiska , Adam Cygankiewicz , Radzisaw Trzciski , Wanda Krajewska
1 1 2 1

1 Department of Cytobiochemistry, University of d, ul. Banacha 12/16, 90-237 d, 2 Department of General and Colorectal Surgery, Medical University of d, Plac Hallera 1, 90-647 d

In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators. Checkpoint kinases 1 and 2 (Chk1 and Chk2) are emerging as the key mediators of diverse cellular responses to genotoxic stress, guarding the integrity of the genome through eukaryotic evolution. Recent studies suggest the fundamental role of Chk1 and Chk2 in the network of genome surveillance pathways which coordinate cell cycle progression with DNA repair and cell survival or death. Defects in these two serine/threonine kinases contribute to the development of both hereditary and sporadic human cancers. The aim of this study was to investigate Chk1 and Chk2 protein expression in colon carcinoma. Fresh-frozen samples were obtained from surgically ab-

lated colorectal cancers staged according to Dukes classification. Section from normal colonic mucosa at the proximal resection margins were taken at the moment of surgical resection. Protein expression was evaluated by Western blot technique with commercial antibodies (Santa Cruz Biotechnology, Cell Signaling Technology) that recognize non-phosphorylated (inactive) and phosphorylated (activated) Chk1 and Chk2. Level of protein expression was estimated by Integrated Optical Density IOD (Gel-Pro Analyzer; Media Cybernetics). Additional analysis was performed using ELISA test. Expression level was correlated with patients age and with conventionally used clinicopathological features of the colon neoplasm. Correlation between levels of inactive an activated forms of Chk1 and Chk2 kinases was analyzed as well.

2003

39th Meeting of the Polish Biochemical Society

113
Poster

200
Diverse effect of PMCA (2 and 3 isoform) blocking in differentiated and non-differentiated PC 12 cells
Janusz Szemraj, Iwona Kawecka, Ludmia yliska

Instytut Fizjologii i Biochemii, Zakad Biochemii Lekarskiej, Uniwersytet Medyczny w odzi, ul. Mazowiecka 6/8, 92-215 d

Plasma membrane Ca -ATPases (PMCAs) are re2+ sponsible for the expulsion of Ca from the cytosol of all eukaryotic cells. In neurons, the high affinity of 2+ PMCAs for Ca and their co-localization with volt2+ age-gated Ca channels make PMCAs an ideal system 2+ for maintaining [Ca ]i at low levels between periods of electrical activity. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. Their expression is regulated in a developmental, tissue- and cell-specific manner. PMCA1 and 4 are found in almost all tissues, whereas PMCA2 and 3 particularly in excitable cells. Alternative splicing affects major regulatory domains of the pumps i.e. calmodulin binding domain and phosphorylation sites. Calmodulin is a protein of primary importance in the regulation of cellular processes de2+ pendent on Ca , serving as an activator of numerous 2+ enzymes. When intracellular Ca level rises, CaM

2+

binds 4 ions of calcium and, next, interacts with site on the fourth cytoplasmic carboxyl terminal and stimu2+ lates Ca pump activity by increasing the affinity and Vmax. In our previous work we have observed an altered kinetic activity of the calcium pump in differentiated PC12 cells with transiently blocked PMCA2 and 3 isoforms. To establish the PMCA2 and 3 roles in proper cell development, using the eukaryotic vector pcDNA3.1, we obtained a stable transfected, non-differentiated PC 12 cell line with impaired expression of two PMCA isoforms. In this model, the activity of calcium pump was two times higher than that of control line, whereas in differentiated PC12 cells without PMCA2 and 3 isoforms the enzyme activity significantly decreased. The presented model system could be useful for studies of calcium signaling in pseudoneuronal cells.
Supported by the grant No 6P04A 06119 from the State Committee for Scientific Research (KBN, Poland)

201
The involvement of Na /H exchanger in the desmopressin-induced platelet procoagulant response
Magdalena Tomasiak , Halina Stelmach , Anna Bodzenta , Marian Tomasiak
1 2 1 2

Poster + +

1 Klinika Alergologii i Chorb Wewntrznych, Akademia Medyczna, ul. M. Curie-Skadowskiej 24, Biaystok, 2 Zakad Chemii Fizycznej, Akademia Medyczna, ul. Mickiewicza 2A, 15-230 Biaystok

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response (PMP formation and procoagulant activity) but the underlying mechanism of this phenomenon is not known. This study was performed to determine, whether the plasma + + membrane Na /H exchanger may be involved in the generation of DDAVP-induced procoagulant activity in human blood platelets. Flow cytometry studies revealed that in vitro platelet response to DDAVP (100200 nM) was associated with the appearance of both degranulated (or fragmented ) and swollen platelets. DDAVP-evoked rise in size heterogeneity was similar to that produced by + + monensin (mimics Na H antiport) and was not ob+ + served in the presence of EIPA (Na /H exchanger inhibitor). Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine we have shown increased and uni-

form binding of this marker to all subsets of DDAVP-treated platelet population. DDAVP-evoked annexin V binding was strongly reduced by EIPA and similar to that exerted by monensin. DDAVP-induced platelet procoagulant response measured as phospholipid-dependent thrombin generation was less pronounced in the presence of EIPA. As judged by sensitive optical swelling assay DDAVP in a dose dependent manner produced rapid rise in the platelet volume. The swelling was inhibited by EIPA and its kinetic was similar to that observed in the presence of monensin. Electronic cell sizing measurements showed increase in mean platelet volume and decrease in platelet count in PRP treated with DDAVP. Altogether the data indicate for a involvement of + + Na /H exchanger to the generation of procoagulant activity in DDAVP-treated platelets.