Bude studována exprese pomocí DNA microarrays u leukémií, vhodných kontrolních buněk a pro in vitro populace. Změny budou korelovány s epigenetickými charakteristikami chromatinu na úrovni jeho modifikací (metylace, acetylace histonů, metylace DNA). Strukturální charakteristiky chromatinu budou zkoumány v místech se změnami exprese pomocí fluorescenční in situ hybridizace, spektrální mikroskopie a imunoFISH. Budou testovány protinádorové látky s úzkým spektrem účinku na úrovni exprese. Využitím pokročilých metod vizualizace jaderných stuktur a moderní konfokální mikroskopie bude studován mechanismus vzniku a progrese leukémií. Cílem bude navrhnout vhodné markry pro diagnostiku. Domníváme se, že studium genů měnících expresi a epigenetických mechanismůstojících za těmito změnami nám umožní navrhnout cílové molekuly pro léčbu.; Using DNA microarrays, gene expression will be studied in leukemia, in control cell populations and in cell cultures. Changes will be correlated to epigenetic characteristics of chromatin at the level of its modifications (methylation, acetylation). Structural characteristics of chromatin at sites with altered expression will be studied using fluorescence in situ hybridization (FISH), spectral microscopy and immunoFISH. Drugs with narrow spectrum of genes with altered expression wil be studied. Using advanced techniques of gene or protein visualization and top level confocal microscopy, the mechanisms of the induction and progression of leukemia will be investigated with the goal to suggest appropriate markers for diagnostics. We hope that our study ofgene expression in parallel to epigenetics will allow us to propose target molecules of effective therapy.

• MeSH
• čipová analýza proteinů metody   využití   MeSH
• histondeacetylasy analýza   MeSH
• hybridizace in situ fluorescenční metody   využití   MeSH
• inhibitory histondeacetylas MeSH
• intracelulární signální peptidy a proteiny analýza   MeSH
• leukemie diagnóza   MeSH
• restrukturace chromatinu MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• hematologie a transfuzní lékařství
• onkologie
• radiologie, nukleární medicína a zobrazovací metody
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

To understand how genes are distributed on chromosomes we bring new insights into gene positional clustering and its properties. We have made a large-scale analysis of three types of differentiation and we observed that genes that subsequently enter into different cell processes are positionally clustered on chromosomes. Genes from the clusters are transcribed subsequently with respect to time kinetics and also to position. This means that the genes related to a cellular process are clustered together, independent of the period of time during which they are active and important for the process. Our results also demonstrate not only that there are general regions of increased or decreased levels of gene expression, but also that, in fact, in some chromosome regions we can find clustering of genes related to specific cell processes. The results provided in this paper also support the theory of "transcription factories" and show that transcription of genes from the clusters is managed by softer epigenetic mechanisms.

• MeSH
• buněčná diferenciace genetika   MeSH
• buňky K562 MeSH
• DNA primery genetika   MeSH
• financování organizované MeSH
• genetická transkripce MeSH
• granulocyty cytologie   metabolismus   MeSH
• HL-60 buňky MeSH
• lidé MeSH
• megakaryocyty cytologie   metabolismus   MeSH
• monocyty cytologie   metabolismus   MeSH
• multigenová rodina MeSH
• myelopoéza genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• trombopoéza genetika   MeSH
• Check Tag
• lidé MeSH

V této publikaci přinášíme přehled vědeckých prací, ve kterých nám technologie cDNA microarrays umožnila nové náhledy do procesů maligní transformace. Díky této technologii dnes můžeme sledovat v jeden okamžik expresní aktivitu tisíců genů. S pomocí vhodných nástrojů můžeme naměřené hodnoty dávat do souvislostí, které byly ještě nedávno prakticky nemyslitelné. V naší laboratoři jsme vyvinuli nové nástroje a postupy pro analýzu microarrays a pro zpracování výsledků do podoby použitelné v onkologickém výzkumu. Pro sledování některých charakteristik je nutné použít vizualizační nástroje. Například pro sledování aktivity určitých oblastí chromosomů jsou velmi vhodné transkripční mapy (dále TM). Implementovali jsme vlastní software pro generování TM a díky kombinaci jeho výstupů s dosavadními znalostmi jsme formulovali závěry našich experimentů. Intuitivním rozšířením TM jsme dosáhli mnoha dalších vizualizací např. hustoty aktivních genů, kumulativní exprese oblastí atp. Tyto nástroje by měly v budoucnosti pomáhat klinikům při diagnostice zhoubných malignit a určování následné léčby.

In this paper we bring a scientific review of studies in which cDNA microarrays technology allowed us to create new insights into malignant transformation processes. We can monitor parallel expression activity of thousands of genes using this technology. Using appropriate tools we can evaluate biological correlations that were practically impossible few years ago. We have developed new tools and techniques transforming the data into form suitable for oncological research. Special visualizations are necessary for some types of studies. E.g. transcription maps (TMs) are a proper tool for studying activity of chromosomal regions. We have implemented our own software for generating TMs. On the basis of combination of its outputs and knowledge gained so far we have formulated our conclusions. Using intuitive TM extensions we have obtained many useful visualizations, e.g. density of active genes, cumulative expression etc. These tools are supposed to help clinicians while forming diagnoses of malignant diseases and while treatment planning.

• MeSH
• biomedicínský výzkum metody   trendy   MeSH
• C-DNA diagnostické užití   MeSH
• financování organizované MeSH
• lékařská onkologie metody   trendy   MeSH
• leukemie diagnóza   genetika   imunologie   MeSH
• lidé MeSH
• mikročipové analytické postupy metody   využití   MeSH
• nádory tračníku diagnóza   genetika   imunologie   MeSH
• stanovení celkové genové exprese metody   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Recurring chromosomal abnormalities are associated with specific tumour types. The EWSR1 and FLI1 genes are involved in balanced translocation t(11;22)(q24;q12), which is present in more than 85% of Ewing sarcomas. In our previous study, we have found that the fusion genes pertaining to both derivative chromosomes 11 and 22 in Ewing sarcoma cell nuclei are shifted to the midway nuclear position between the native EWSR1 and FLI1 genes. In this contribution we focused our attention at nuclear positioning of other genetic elements of chromosomes 11 and 22 in order to find if the whole derivative chromosomes or only their translocated parts change their nuclear positions in comparison with the native chromosomes. Using repeated fluorescence in situ hybridization and high-resolution cytometry, 2D radial positions of EWSR1, BCR, FLI1, BCL1 genes and fluorescence weight centres of chromosome territories were compared for intact and derivative chromosomes 11 and 22 in nuclei of three Ewing sarcoma samples. Significant radial shift was obtained for the derivative EWSR1, FLI1 and BCL1 genes and for the derivative chromosome 11 compared with the intact ones and not very significant for chromosome 22 and the BCR gene. Our results also suggest that the mean nuclear positions of fusion genes are determined by the final structure of the derivative chromosomes and do not depend on the location of the translocation event.

• MeSH
• buněčné jádro genetika   MeSH
• chromozomální aberace MeSH
• Ewingův sarkom genetika   patologie   MeSH
• financování organizované MeSH
• genová dávka MeSH
• hybridizace in situ fluorescenční MeSH
• lidé MeSH
• lidské chromozomy, pár 11 MeSH
• lidské chromozomy, pár 22 MeSH
• lymfocyty cytologie   MeSH
• nádory kostní tkáně genetika   patologie   MeSH
• pravděpodobnost MeSH
• proteiny vázající kalmodulin fyziologie   genetika   MeSH
• proteiny vázající RNA fyziologie   genetika   MeSH
• protoonkogenní protein c-fli-1 fyziologie   genetika   MeSH
• sekvenční homologie nukleových kyselin MeSH
• translokace genetická MeSH
• velikost buňky MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• srovnávací studie MeSH

OBJECTIVE: Hematopoietic stem cells (enriched in fraction of CD34+ cells) have the ability to regenerate hematopoiesis in all of its lineages, and this potential is clinically used in transplanting bone marrow or peripheral blood stem cells. Our objective was to assemble a suitable method for evaluating gene expression in enriched populations of hematopoietic stem cells. We compared biologic properties of cells cultured ex vivo obtained using two different ways of immunomagnetic separation (positive selection of CD34+ cells and negative selection of Lin- cells) by means of a cDNA microarray technique. METHODS: CD34+ and Lin- cells were enriched from peripheral blood stem cell (PBSCs) grafts of patients with non-Hodgkin's lymphoma. Isolated cells were in the presence of cytokine PBSCs, Flt-3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. At days 0, 4, 6, 8, 10, 12, and 14 cells were harvested and analyzed by cDNA microarrays. Total cell expansion, CD34+, colony-forming unit for granulocyte-macrophage and megakaryocytes expansion, vitality, and phenotype of cells were also analyzed. RESULTS: cDNA microarray analysis of cultured hematopoietic cells proved equivalence of the two enrichment methods for PBSC samples and helped us characterize differentiating cells cultured ex vivo. CONCLUSION: Our methodologic approach is helpful in characterizing cultured hematopoietic cells cultured ex vivo, but it is also suitable for more general purposes. Equivalence of CD34+ and Lin- selection methods from PBSC samples proved by cDNA microarray may have an implication for graft manipulation in an experimental setting of hematopoietic transplantation. Total cell expansion and colony formation and phenotype from CD34+ selected and from Lin- samples were comparable.

• MeSH
• antigeny CD34 imunologie   MeSH
• financování organizované MeSH
• imunofenotypizace MeSH
• imunomagnetická separace MeSH
• komplementární DNA MeSH
• lidé MeSH
• lymfom genetika   imunologie   terapie   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transplantace kmenových buněk MeSH
• Check Tag
• lidé MeSH

The progression of colorectal cancer involves accumulation of various genetic and epigenetic events that dramatically change gene expression. The aim of this study was to investigate a possible new approach to the diagnosis of colorectal carcinoma patients, based on their gene expression profiles. Human 19K cDNA microarrays were used to analyze the gene expression profiles of 18 colorectal carcinoma patients. Transcriptome maps (TMs) were analyzed to detect chromosomal regions that could serve as potential diagnostic markers for colon cancer. A comparison of TMs showed chromosome regions with conserved changes of gene expression typical of colorectal cancer in general, and also patient-specific variable regions. We identified 195 genes with significantly altered expression in colon cancer. Functional analysis of the regulated genes distinguished three main categories: biological processes, cellular components, and molecular functions. We found that different patients had chromosome regions characterized by very similar changes of gene expression, probably linked to the most fundamental events in carcinogenesis. On the other hand, variable chromosome regions can be patient-specific. The variable regions may provide further information on the individual pathogenesis and prognosis of the patient. Comparison of TMs is proposed as a tool to facilitate diagnosis and treatment planning for individual patients.

• MeSH
• DNA nádorová genetika   MeSH
• financování organizované MeSH
• genetická transkripce MeSH
• kolorektální nádory diagnóza   genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mapování chromozomů MeSH
• nádorové biomarkery genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

• MeSH
• apoptóza účinky záření   MeSH
• buněčné jádro enzymologie   genetika   metabolismus   účinky léků   MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   MeSH
• buňky HT-29 MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové antagonisté a inhibitory   metabolismus   MeSH
• financování organizované MeSH
• histondeacetylasy metabolismus   MeSH
• histony metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory histondeacetylas MeSH
• interfáze účinky léků   MeSH
• kyselina máselná farmakologie   MeSH
• kyseliny hydroxamové farmakologie   MeSH
• lidé MeSH
• malobuněčný karcinom enzymologie   metabolismus   patologie   MeSH
• nádorové buněčné linie MeSH
• nádory plic enzymologie   metabolismus   patologie   MeSH
• nádory tračníku enzymologie   metabolismus   patologie   MeSH
• plod MeSH
• Check Tag
• lidé MeSH