Glavni urednik
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- Bu səhifədəki naviqasiya:
- Projekti u sklopu zavoda
- ISTRAŽIVANJE ULOGA PONAVLJAJUĆEG SLIJEDA (HEAT REPEAT) U PROTEINU HUNTINGTIN ASSESSING FUNCTIONS OF THE HEAT REPEAT IN HUNTINGTIN PROTEIN
- Research programme and results
Research programme: During the year 2003. we investigated large number of different new therapeutical approaches to human cancer (gene therapy, antisense therapy, viral anticancer approach, new potential anticancer compounds). The results of the research were published in 53 scientific papers indexed in the Current Content. We proceeded with the experiments in tumor gene therapy by exploring the therapeutic suppressor genes p53 and p21. Human and mouse tumor cell lines were infected by Ad-p53, Ad-p21 and control, dl-312, vector. The strongest inhibitory effect was shown on SW620 and HeLa cell lines. In addition, inhibitory effect on mouse cell lines was weaker than on human cell lines. The wild type p53 gene inserted into the cells with its mutant form, induced the expression of p53 «downstream» genes; in nontreated cells (with mutated p53 or its blocked expression) these genes were not expressed. Potential antitumor effect of newly synthesized small molecules, which were obtained mostly from organic synthesis laboratories of Faculty of Chemical Engineering and Technology, and Laboratory of General and Inorganic Chemistry, Faculty of Science University of Zagreb were tested. More than 100 novel compounds from the following chemical groups were tested: hydroxyurea aminoacid amids, purine and pyrimidine derivatives of L-ascorbic acid, alkylated derivatives of pyrimidines, polyoxometallates, benzo-tieno-quinolones, phenatridines, etc. The antiproliferative activity was tested on five tumor cell lines (derived from five different tumor types) and a normal fibroblast cell line. Pronounced antiproliferative activity of some of the quinolons derivatives, as well as of hydroxyurea amino acid derivatives was shown. Furthermore, new synthesised compounds: benzo(beta)thieno(2,3-c)quinolones; purine substituted 1-aminocyclopropane-1-carboxylic acids and 1-amino-1-hydroxymethylcyclopropane; iodinated and fluorinated 2-(2-hydroxypropyl) and 9-(2-hydroxyethoxy)methyl purine nucleoside; substituted benzimidazolyl-furyl-phenyl-acrylates and naphthol (2,1-beta) furan-carboxylates were evaluated on their antitumor activities. Some of compounds exhibited a strong cytotoxic effect on tumor cell lines. There is an indication that these compounds induced apoptosis. Over the course of this year we have commenced with the research of the indomethacin antitumor activity mechanism in the established colon cancer cell lines. The influence of indomethacin on COX-2 expression as well as of other potential targets (p53, p21, p27, cyclin-E, cyclin-D, APC, beta-catenin, hMSH2) was done immunohistochemically. Iindomethacin raises the expression of p53 and p27 independently of COX-2 expression. We investigated the antitumor effect of Newcastle disease virus LaSote strain. Effect is dose depending cytotoxic against melanoma B16F10, carcinoma SCCVII and fibrosarcoma FsaR but not against normal L929 cells, and significantly suppressed the growth of these transplanted tumors if injected i.p. or into the tumor, but there was no complete regression. We also investigated the therapeutic effects of molecularly targeted approach directed to IGF-2 and/or IGF-1R. Antisense to IGF-1R and IGF-2 or monoclonal antibody to IGF-1R strongly inhibited growth of breast cancer cells. Our scientific program also covered research into the molecular genetics of cancer and some other disease. We have continues our research on laryngeal and gastric cancer tumor samples. In gastric cancer, we have shown that promoters P1 and P4 are not activated and so far, we have shown that only activation of P3 and P4 contributes to LOI in these tumors. The impuls from the clinic encouraged us in our research on gene polymorphism. Some patients with hip implant for unknown reason reject it relatively soon after the surgery, while another group keep it for longer period of time. For that reason, we have started with research on gene polymorphism in TNF-alpha, TGF-beta and interleukin 6. Beta-catenin mutations were analyzed in 120 colon tumors and four different sporadic mutations were confirmed by sequencing. We have analysed loss of heterozygosity (LOH) of nm23-H1, APC, DPC4 and NF1 tumor suppressor genes. The LOH and mutation analysis of the APC gene in sporadic colon cancer showed that LOH was present in 30% of the tumors analyzed. We have found a new yet unpublished sporadic mutation of the APC gene, an AGGT 4 bp insertion in codon 1374. In the LOH analysis of the NF1 gene, 21% of LOH was detected in sporadic colon cancer samples. In the NF1 analysis expression was found to be significantly lower in poorly differentiated tumors as well in tumors classified as Dukes’ C. We elucidate the role of insulin-like growth factor (IGF) family of genes in the development and progression of lung cancer. We found that IGF1 and IGF2 are potent mitogens for human lung cancer cells. IGF-related ligands can act as an autocrine growth factors through action via the type 1 IGF receptor. IGF2 is the predominant growth factor involved in the autocrine growth stimulation of lung epithelial tumor cell lines. Interruption of IGF2 pathways offers the possibility of tumor control with a high therapeutic index. We determined the role and structural changes of FHIT gene in development of tumors. The genomic structure of FHIT overlaps with the FRA3B fragile site and coincides with a genomic region that is known to be frequently involved in allelic loss, genetic rearrangement, and cytogenetic abnormality in solid tumors. Although point mutations within the FHIT gene have rarely been reported, genomic alterations such as homozygous deletions of exons or insertions of intronic sequences and aberrant transcripts of the FHIT gene, as well as the lack of detectable Fhit protein, have all been frequently observed in lung and head and neck cancers. A part of the research is directed to signaling molecules in cancer. Our investigation is aimed to show the effect of transduced proteins into the cells on regulation of the cell cycle. We cloned the vectors TAT-p27, TAT-p27-pt and TAT-p27-N' and after transformation of bacteria, the proteins were expressed in bacteria. Using affinity chromatography and gel filtration, the proteins TAT-p27, TAT-p27-pt and TAT-p27-N' were purified and used for transduction. We have continued with research on IDE in tumor growth. Insulin-degrading enzyme (IDE) is a metalloprotease implicated in insulin degradation and suggested to have a variety of additional functions, including the clearance of amyloid beta peptides of Alzheimer's disease. Little is known about endogenous proteins that may interact with and modulate IDE's activity in the cell. We purified and characterized two proteins from mouse leukemic splenocytes that interact with IDE and inhibit its insulin-degrading activity. A protein of 14 kDa was similar to a competitive IDE inhibitor reported previously. The major inhibitor was identified by amino acid sequencing as ubiquitin, a protein that is post-translationally covalently attached to other intracellular proteins and regulates diverse cellular processes. Our findings suggest a novel role for ubiquitin or perhaps proteins with ubiquitin-like domains in regulating the function of IDE. We also investigated and published the role of the IGF 2 ligand and its receptors, IGF 1 and IGF 2 types, in human gastric cancer. Our findings suggest that members of the IGF family are involved in the pathogenesis of gastric cancer, probably by autocrine/paracrine stimulation of cell growth. Such tumors might be excellent candidates for therapeutic strategies aimed at interference with this pathway. Immunoproliferative tumors were covered by two projects. Defects in lymphocyte development and differentiation lead to disorders which cause different diseases, in most cases lethal for the organism. That’s why mechanisms that strictly control immunocompetent cell maturation have been developed. One of the lymphocyte development control systems is activation of certain transcription factors. They define the place, time and speed of target gene expression. The most important transcription factors in lymphocyte development are Notch proteins, and proteins from the Ikaros gene family, Aiolos, Helios and Eos. During this year we continued the extensive study of human lymphoid transcription factors Aiolos, Helios and Ikaros using various human hematological cell lines as an experimental model. We also screened a number of bone marrow, lymph node and peripheral blood lymphocyte samples from patients with different lymphoproliferative disorders. Using specific PCR primers we designed, different alternatively-spliced isoforms that can be detected. We were investigating the connection between different Helios alternative splicing patterns with immunopathological characteristics of different lymphoma cells, including both patients and cell lines. Flow cytometry with antibodies for both cell surface and cytoplasmic/nuclear proteins was used to determine the developmental stage of malignant cells and to confirm our RT-PCR data. We also studied chronic lymphocytic leukemia (CLL), which is most common type of human leukemia. Thereafter, the incidence increases progressively with the age. CLL is characterized by a progressive expansion of malignant CD5+B1 lymphocytes in the peripheral blood, spleen, lymph nodes and by a myriad of humoral and cellular immunological defects. We are postulating that developmentally and functionally altered T-cell descendants play important role in modulating clinical course of CLL. Namely, imbalance of any of T-cell mediated interactive homeostatic mechanisms in CLL may facilitate deregulation of various protooncogenes, or facilitate certain transduction signals which in turn contribute to defective B-cell activation, their impaired differentiation and/or differentiation arrest accompanied by uncontrolled accumulation in different organ/tissue compartments. Many aspects and the role of various B/T and T/T interactions in modulating the expansion rate of leukemic cells and clinical course of the disease will be modelled by simple nonlinear dynamical models. The project Oxidative Stress and Malignant Disease evaluates pathology and physiology of oxidative stress. The research tends not only to study basic aspects of oxidative stress, in particular lipid peroxidation, but also to develop novel analytical methods and treatments for control of oxidative stress that would attenuate its harmful, pathological consequences. In 2003 the research focused mostly on the pathophysiology of oxidative stress in the immune alterations associated with the bone growth and function. Hence, the interference between lipid peroxidation product 4-hydroxynonenal (HNE) and bioactive peptide BPC 157 were studied in tendocyte growth as well as immunomodulating effects of the ferric sorbitol complex in an experimental model of arthritis. We developed also genuine model of human bone tissue cultures and initiated advanced immuno-electronmicroscopy method for detection of HNE-protein adducts in leukocytes. We investigated the role of acute and chronic application of enkephalins and endorphins on nitric oxide release in human neutrophils and monocyte cell-lines in vitro, and also the involvement of hydrolitic enzymes in these processes. Endomorphin 1 and endomorphine 2 differently regulate relase of nitric oxide from mouse macrophages in cell culture (J774) in vitro and mouse peritoneal macrophages treated with opioide petides in vivo. Also using specific inhibitors for different type of nitric oxide synthase (NOS) we demonstrate type of NOS which is activated with endomorphins. The main goal of the project on modulation of immunological response by bioactive peptides is the development of methods and computer programs for molecular and genetic definition of both bioactive motifs and the secondary structures of proteins in order to design and synthesise peptides responsible for the modulation of immunological responses. The immunomodulation efficacy of newly synthesised peptides was tested experimentally. The Resonant Recognition Model (RRM) of protein bioactivity is applied to the protein secondary structure prediction. The method is based on the physical and mathematical model of the electronic ionic interaction pseudopotential (EIIP). The peptide sequences responsible for infectivity and modulation of immunological responses of field isolates of the infectious bursal disease viruses (IBDV) were determined by the restriction analysis and compared with the data from the date base. In the project Molecular mechanisms of immunosuppression the effects of two herpes viruses of poultry, oncogenic strain of Marek's disease virus (MDV) and vaccinal strain of turkey herpes virus (HVT), on transcription of genes for interferon alpha and gamma was investigated in chickens susceptible (B13/B13) and chicken resistant (B21/B21) to tumor development caused by MDV infection. Oncogenic strain of MDV had negative effect on transcription of genes for interferon alpha and gamma on the first and seventh day post infection in B13/B13 chickens, while it had negative effect only the seventh day in B21/B21 chickens. Vaccinal strain had no influence on transcription of interferon genes the first day post infection, and on the seventh day it had milder negative effect in B21/B21 chickens compared to oncogenic MDV. Our results imply that the ability of oncogenic strain of MDV to block transcription of interferon genes, measured in blood, on the first day post infection of B13/B13 chickens not only causes immunosuppression but also may be related to the oncogenicity of the virus. The aims of the project Regulation of expression of ectopeptidase and opioid receptors were to examine surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. The cells of immature murine T-cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphines. The role of free intracellular calcium in KOR signaling was also examined. T-lymphocyte R1.1 cell line, selectively expressing high density of KOR, and a synthetic KOR-selective ligand, U-69593 served as a model. The data obtained have shown that free intracellular calcium is involved in signalling of KOR expressed on R1.1 cell line. Namely, ligation of KOR with U-69593 altered the level of free intracellular calcium, depending on the state of cell activation: in resting cells it was decreased, whereas in activated cells, it was enhanced. The aims of the project on neurotransmitters in stress and regulation of GABA receptors were a) to elucidate the phenomena of tolerance and physical dependence which appear in animals and humans following prolonged treatment with benzodiazepines and other positive modulators of GABA-A receptors, b) to elucidate the mechanism of the anticonvulsant effect of stress. a) As a model we used human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1 beta 2 gamma2s GABA-A receptors, the most common type of GABA-A receptors found in the brain. Chronic exposure of cells to GABA and muscimol (a GABA receptors agonist) but not to diazepam (s drug that facilitates the function of GABA), enhanced the synthesis of GABA-A receptors, but unlike diazepam, it failed to produce functional uncoupling of GABA and benzodiazepine binding sites. b) We demonstrated that swim stress produced a strong inhibition of 5-HT2A receptors, but this effect does not appear to be directly linked with the effect of stress on the seizure threshold. Peripheral serotonergic markers (platelet serotonin and platelet monoamine oxidase) were studied in the depressed patients and in the patients with posttraumatic stress disorder (PTSD). Our results shown that the determination of platelet serotonin concentration in depressed patients before treatment could contribute to the selection of antidepressent treatment with the best therapeutic results. The relationship between biochemical markers and particular depressive symptoms in was veterans with PTSD shown significant positive correlation among platelet serotonin concentration and different severity of appetite loss that confirm the presumptions on the role of serotonin in the regulation of appetite. We also covered applied research into the molecular-genetic and cellular basis of diseases. Optimal conditions for the cultivation of human skin epithelial cells in selective medium and at a fibroblast feeder layer have been studied and defined. Skin samples were obtained from 25 donors. Keratinocyte cultures have been produced in quantities permitting production of artificial skin transplants for potential clinical use. During the first year of our research we have established a new method of specific gene expression analysis, real-time PCR. This method was used to analyze gene expression of the genes on mRNA level. Projekti u sklopu zavoda:
Oznaka: 0098086 ISTRAŽIVANJE ULOGA PONAVLJAJUĆEG SLIJEDA (HEAT REPEAT) U PROTEINU HUNTINGTIN ASSESSING FUNCTIONS OF THE HEAT REPEAT IN HUNTINGTIN PROTEIN Voditelj/ica projekta: dr. sc. Oliver Vugrek Tel. ++385 1 4560 946 e-mail: ovugrek@irb.hr Program rada i rezultati na projektu: Dokazano je da proteini koji su vezani uz mikrotubule (MAPs) iz TOGp-XMAP215 skupine imaju važnu ulogu u organizaciji mikrotubula (Nature 411: 610-613, 2001). U takvim MAP proteinima se nalazi slijed amino kiselina, tzv. HEAT repeat, koji postoji i u drugim proteinima kao npr. Huntingtin. Mutacija u HEAT-repeatu izaziva dezorganizaciju cijelog mikrotubularnog sustava. Nove spoznaje pokazuju postojeću interakciju Huntingtin proteina s mikrotubularnim sustavom. Nastavak istraživačkog rada ima primarni cilj bolje razumjeti ulogu proteina Huntingtin i važnosti domene HEAT repeata u neuralnim stanicama te razjasniti uloge proteina Huntingtin u mehanizmima dinamičnih procesa vezanih uz citoskelet. Eksperimentalni rad je u početnoj fazi, tj. uspostavljena je stanična kultura (stanice insekta, Sf9) potrebna za korištenje Baculovirus tehnologije, te pripreme za stvaranje knock-out mutanata na staničnom nivou te modelnim organizmima i praćenje fenotipskih promjena uzrokovanih delecijom HEAT repeata. Također, pripremljena je cDNA kompletnog huntingtin gena, koja će se koristiti za ciljanu mutagenezu regije HEAT ponavljajućeg slijeda. Heterologna ekspresija rekombinantnog mutiranog HEAT proteina vršit će se u stanicama insekta. Svojstva rekombinantog HEAT repeata proučavat će se in vitro u eksperimentima praćenja afiniteta za vezanje mikrotubula. Izolacija i pročišćavanje tubulina iz humanih stanica provedena je uspješno.
In previous research, we could demonstrate that proteins associated with microtubules (MAPs) of the TOGp-XMAP215 family play an important role in microtubules organisation (Nature 411: 610-613, 2001). Such MAP proteins contain a characteristic domain, e.g. HEAT repeat, which is also found in Huntingtin protein. Mutations in the HEAT repeat lead to a disorganisation of the cellular microtubules system. New data indicate an existing interaction between Huntingtin and microtubules. This project will investigate a possible involvement of the Huntingtin HEAT repeat with the microtubules system. Therefore, we have established a cell culture model system by using insect cells (Sf9 type) and baculovirus driven heterologous expression of recombinant genes. We have aquired a cDNA clone hosting the complete coding sequence of Huntingtin, which will serve for site directed mutagenesis of the HEAT repeat region. Heterologous expression of mutated HEAT repeat in insect cells and subsequent purification of recombinant protein will allow in vitro binding experiments with microtubules. Tubulin has been succesfully purified from cultured human cells.
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