Transcriptomic and proteomic analyses of gene expression in fibroblasts
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1 Research Collection Doctoral Thesis Transcriptomic and proteomic analyses of gene expression in fibroblasts Author(s): Bumke, Maja Alexandra Publication Date: 2002 Permanent Link: Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library
2 Diss. ETH No Transcriptomicand proteomicanalyses of gene expressionin fibroblasts A dissertation submitted to the SWISS FEDERALINSTITUTEOF TECHNOLOGYZÜRICH for the degree of DOCTOROF NATURALSCIENCES Presentedby MajaAlexandraBumke Diplome d'etudes Approfondies Biochimie - UniversiteParis VII Born September1,1974 from Germany accepted on the recommendationof Prof. Dr. Dario Neri, examiner Prof. Dr. Leonardo Scapozza, co-examiner Dr. Giuliano Elia, co-examiner Zürich, September2002
3 Summary In this work, gene expression in fibroblasts has been studied by proteomic and transcriptomic techniques, with particularattention to the modificationsinduced by extracellularph changes. The ph of tissues varies during development, in physiological processes and in disease (e.g. cancer). However, the influence of extracellularph on gene expressionhas been studied only for a limited numberof genes. For this work, primary fibroblastswere chosen as a model system, since these cells adapt their intracellularph on the basis of the ph of the culture medium, and due to the fact, that fibroblasts are responsible for the synthesis of a large portion of extracellular matrix (ECM) components in health and disease. Normal human dermal fibroblasts were cultured at two different ph values (ph 6.7 and 7.5). The expression of secreted proteins, as well as the expression of mrna transcripts were studied. The aim was to identify components of the ECM, which are differentially modulated by extracellular ph and might be aberrantly expressed in a tumoral environment, and to get a deeper insight in the regulation of the transcriptionalprogramas a response to a ph change. Additionally, endothelial cells cultured in vitro have been studied in a differentmodel system ofgrowing and resting cells. For the proteomic analysis of the fibroblast cell culture model, two-dimensional gel electrophoresis (2D PAGE) was performed, and for protein visualization several staining and imaging techniques (silver staining methods and fiuorescent dye) were compared. For the identification of protein spots from 2D gels, different analyses were performed: N-terminal Edman sequencing, Peptide Mass Fingerprinting using MALDI-TOF mass spectrometry, as well as collision induced dissociation spectra analysis of peptides separated by microcapillary liquid tandem mass chromatography- spectrometry(ulc-ms/ms). The transcriptionalprogram of fibroblasts at the two different ph values, and in the presence and absence of serum, was studied by quantitative RT-PCR and Northern blotting, and a genome-wide analysis was performed using Affymetrix GeneChip technology.
4 mass Summary Using 2D PAGE and mass spectrometry, the measurementsof about 600 protein spots led to the identification of at least 170 different spots; many proteins being identified in various isoforms, this corresponds to the identificationof 40 differentproteins. Several proteins were found to be differentiallyexpressed by fibroblastsat the two different ph values. Using the Affymetrix GeneChip system, the expression of 2068 genes (out of the represented on the chip) was found to be modulated by more than two-fold after the changeof the culture medium ph to a more acidic value. Genes displaying a modulated pattern of expression included, among others, cell cycle regulators (consistent with the Observationthat acidic ph abolishes the growth of fibroblasts in culture) and relevant extracellular matrix (ECM) components. Among the proteins found to be differentially expressed by fibroblasts at the two ph conditions, the ones with the strongest differences were agrin and tetranectin (identifiedby at ph 6.7 and 7.5 respectively), and e.g. extracellularmatrix - 2D PAGE spectrometry, protein ECM-2 and stanniocalcin STC-1 (identifiedby Affymetrix GeneChip analysis). These and other upregulatedproteins might be interestingcandidates for further studies. The relevance to physiological and pathological processes of these Undings will have to be confirmed, e.g. performing antibody selection followed by immunohistochemicalanalysis of healthyand diseasedtissue samples, to evaluate their eventualdifferential expression. u
5 Zusammenfassung Der ph-wertvon Geweben variiert während der Embryonalentwicklung, in physiologischen Prozessen, sowie im Rahmen gewisser Krankheitsbilder wie zum Beispiel Krebs. Der Einfluss des extrazellulären ph-wertes auf die Transkription von Genen wurde bisher jedoch nur bei einer sehr begrenzten Anzahl von Genen untersucht. In der vorliegenden Arbeit wurde der Einfluss des extrazellulären ph-wertes auf die Genexpression mit Hilfe eines breit gefächerten erforscht. Proteomics- und Transcriptomics-Ansatzes Als Zellkulturmodellwurde eine Fibroblasten-Primärkulturgewählt, da für diese Zellen zum einen bereits bekannt war, dass sie ihren intrazellulären ph-wert an den ph-wert des umgebendennährmediums anpassen und sie zum anderen einen Grossteil der Komponenten der extrazellulären Matrix synthetisieren. Normale humane Haut-Fibroblastenwurden bei zwei verschiedenen ph-werten (ph 6.7 und ph 7.5) kultiviert. Anschliessend wurden die Expressionsmuster sowohl auf Protein- als auch auf mrna-ebene bei den beiden ph-werten verglichen. Ziel war es, auf diesem Wege Komponenten der extrazellulären Matrix zu identifizieren, deren Expression in Abhängigkeit vom extrazellulären ph-wert moduliert wird und die daher in einer tumoralen Umgebung eine Rolle spielen könnten. Weiterhin sollte mit diesem experimentellenansatz ein tieferes Verständnis der Transkriptionsregulation in Antwort auf Veränderungen des ph-wertes gewonnen werden. Die proteomische Analyse des Fibroblasten-Zellkulturmodells wurde mittels 2D-Gelelektrophorese durchgeführt. Für den Proteinnachweis in den Gelen wurden unterschiedliche Färbemethodenund bildgebende Verfahren (verschiedene Silberfärbungen und Fluoreszenzfarbstoffe) herangezogen und miteinanderverglichen. Zur Identifizierung gefärbter Proteine von den 2D-Gelen wurden unterschiedliche Analyseverfahren verwendet: N-terminale Edman-Sequenzierung, Peptidmassen Fingerprinting unter Verwendung von MALDI-TOF Massenspektrometrie, sowie Milo-okapillarflüssigkeitschromatographie-Tandem-Massenspektrometrie(^LC-MS/MS). in
6 Zusammenfassung Die Transkriptionsmustervon Fibroblasten bei den zwei untersuchten ph-werten, sowie zudem jeweils in Gegenwart und Abwesenheitvon Serum, wurden mit Hilfe von RT-PCR und Northern Blot Analyse untersucht. Eine weitere, genomumgreifendeanalyse wurde mit der Affymetrix GeneChip Technologie durchgeführt. 2D-Gelelektrophorese und massenspektrometrische Messungen von ungefähr 600 Proteinspots führten zur Identifizierung von mehr als 170 Proteinspots, mehreredavon waren unterschiedliche Isoformen, so dass insgesamt 40 gänzlichverschiedene Proteine identifiziert wurden. Mehrere Fibroblasten-Proteine mit unterschiedlichen Expressionsmustem konnten bei den zwei untersuchtenph-werten identifiziert werden. Die Affymetrix GeneChip Technologie zeigte, dass die Expression von 2068 Genen (von insgesamt auf dem Chip vorhandenengenen) nach der Änderung des extrazellulären ph-wertes um mehr als das Expressionsmuster zeigten, gehörten unter anderem die Gene von Zell-Zyklus-Regulatoren Zweifache moduliert wurde. Zu den Genen, die ein moduliertes (in Übereinstimmung mit der Beobachtung, dass ein saurer ph-wert das Wachstum von Fibroblastenin Kultur verhindert) und von einigen Komponentender extrazellulären Matrix. Zu den Fibroblasten-Proteinen, die bei den untersuchten ph-werten die grössten Expressionunterschiedeaufweisen,zählten unter anderem Agrin und Tetranectin, die mittels 2D-Gelelektrophorese und Massenspektrometrie identifiziert werden konnten, sowie ECM2, ein Protein der extrazellulären Matrix, und Stanniocalcin STC-1, die mittels Affymetrix GeneChip Analyse ermitteltwurden. Diese und weitere Proteine, die eine verstärkte Expression bei einer Veränderung des ph-wertes erfahren, könnten interessante Kandidaten für weitere Studien sein. Zum Beispiel könnten Antikörper-Selektionierungund anschliessendeimmunohistochemischeanalyse von gesundenund tumoralen Geweben verwendet werden, um zu untersuchen,ob diese Proteine in vivo unterschiedliche Expressionmuster aufweisen. IV
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