Identification of the tumour associated gene S100A14 and analysis of its regulation [Elektronische Ressource] / von Agnieszka Pietas

Agnieszka Pietas

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• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2004
• Nombre de lectures: 41
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Identification of the tumour-associated gene
S100A14 and analysis of its regulation

DISSERTATION

zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Diplom-Biotechnologin Agnieszka Pietas
geb. am 14. April 1975 in Lublin, Polen
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD

Gutachter/innen: 1. Prof. Dr. Harald Saumweber
2. PD Dr. Christine Sers
3. Prof. Dr. Iver Petersen


Datum der Promotion: 22.11.2004
I

Table of Contents
List of Figures...................................................................................................... IV
List of Tables VII
List of Abbreviations......................................................................................... VIII
Zusammenfassung............................................................................................... X
Abstract............................................................................................................... XII
1 Introduction...................................................................................................1
1.1 Multi-Step Progression of Tumours ............................................................1
1.2 The S100 Protein Family ............................................................................3
1.2.1 Genomic Organization and Chromosomal Localization of
S100 Genes .......................................................................................4
1.2.2 Biological Functions ...........................................................................5
1.2.3 Association with Human Diseases .....................................................7
1.3 Aim of This Work ......................................................................................10
2 Materials and Methods ...............................................................................11
2.1 Materials ...................................................................................................11
2.1.1 Chemicals ........................................................................................11
2.1.2 Kits11
2.1.3 Materials...........................................................................................12
2.1.4 Enzymes ..........................................................................................12
2.1.5 Antibodies12
2.1.6 Mammalian Cell Lines......................................................................13
2.1.7 E. coli Strain .....................................................................................15
2.1.8 RNA Samples from Mammalian Cell Lines ......................................15
2.1.9 Tissue Specimens............................................................................16
2.1.10 Plasmids and Expression Constructs...............................................16
2.1.11 Oligonucleotides...............................................................................18
II

2.2 Methods....................................................................................................19
2.2.1 Bacterial Culture...............................................................................19
2.2.2 Culturing of Mammalian Cells ..........................................................21
2.2.3 Preparation, Enzymatic Manipulation and Analysis of DNA .............26
2.2.4 Dual-Luciferase Reporter Assay ......................................................33
2.2.5 Preparation and Analysis of RNA.....................................................34
2.2.6 Analysis of Proteins..........................................................................38
2.2.7 S100A14 Antibody Generation.........................................................44
2.2.8 Immunofluorescence Analysis..........................................................45
2.2.9 Immunohistochemistry .....................................................................46
2.2.10 Tissue Microarrays (TMA) Generation .............................................48
2.2.11 Statistical Analysis............................................................................48
2.2.12 Bioinformatics...................................................................................48
3 Results.........................................................................................................50
3.1 Identification of the Human S100A14 cDNA .............................................50
3.1.1 Screening of SSH cDNA Libraries....................................................50
3.1.2 Sequence Analysis of S100A14 cDNA51
3.2 Expression Profile in Tumour Cell Lines, Normal, and
Neoplastic Tissues....................................................................................53
3.2.1 S100A14 mRNA Level in Tumour Cell Lines....................................53
3.2.2 Expression Profile in Normal Human Tissues ..................................56
3.2.3 S100A14 mRNA Level in Tumour Tissues.......................................58
3.2.4 S100A14 Protein Expression in Lung Tumours and
Association with Clinicopathological Factors....................................60
3.2.5 S100A14 Protein Expression in Breast Tumours and opathological Factors62
3.2.6 S100A14 is not Re-Expressed Following Growth of
Human Cancer Cell Lines Transplanted into Mice ...........................65
3.3 Subcellular Localization of the S100A14 Protein ......................................66
3.4 Genomic Organization and Chromosomal Localization of the
S100A14 Gene .........................................................................................69
III

3.5 Identification and Characterization of the Promoter for the
S100A14 Gene .........................................................................................75
3.6 ERBB Ligands Induce S100A14 Expression at the
Transcriptional Level in 9442 Cells...........................................................79
3.6.1 Effects of Signalling Pathways Inhibition on Activation of
S100A14 by the EGF .......................................................................82
3.6.2 EGF-Induced S100A14 Gene Expression is Dependent on
de novo Protein Synthesis................................................................86
3.7 Transcriptional Induction by Protein Kinase C ..........................................87
4 Discussion...................................................................................................89
4.1 Identification of the S100A14 cDNA..........................................................89
4.2 S100A14 is Differentially Expressed in Human Tumours .........................90
4.3 Identification and Characterization of the Genomic Locus of
S100A1496
4.4 Oncogenic Signalling Pathways Mediate S100A14
Transcriptional Induction.........................................................................100
5 References ................................................................................................110
Danksagung.......................................................................................................127
Curriculum Vitae129
Publications130
Eidesstattliche Erklärung .................................................................................132

IV


List of Figures
Fig. 1 The S100 gene cluster on human chromosome 1q21............................5
Fig. 2 The scoring system used for immunohistochemical analysis
of S100A14...........................................................................................47
Fig. 3 Nucleotide and deduced amino acid sequence of the human
S100A14 gene......................................................................................52
Fig. 4 Alignment of the predicted amino acid sequences of the
human S100A14 and the mouse orthologue 1110013O05RIK
with the most homologous S100 family members ................................53
Fig. 5 Expression of the S100A14 mRNA in normal human tissues...............57
Fig. 6 Expression profile of S100A14 in human tumours59
Fig. 7 S100A14 protein is overexpressed in primary lung tumour
tissue in comparison to normal lung tissue...........................................60
Fig. 8 S100A14 localizes to plasma membrane in lung tumour
tissue....................................................................................................61
Fig. 9 S100A14 protein is up-regulated in primary breast carcinoma
relative to normal breast tissue.............................................................63
Fig. 10 S100A14 overexpression correlates with ERBB2
overexpression in primary breast tumours............................................63
Fig. 11 S100A14 localizes to plasma membrane in breast cancer
tissue64
Fig. 12 S100A14 is not re-expressed in lung cancer cell line
xenografts from nude mice ...................................................................65
Fig. 13 Cellular localization of S100A14-V5 protein in human lung