Effects of zinc on the transcriptome and proteome of human colonic cancer cells (HT-29 cells) [Elektronische Ressource] / Birgit Kindermann

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• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Lehrstuhl für Ernährungsphysiologie


Effects of zinc on the transcriptome and proteome of
human colonic cancer cells (HT-29 cells)


Birgit Kindermann


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. Karl-Heinz Engel
Prüfer der Dissertation:
1. Univ.-Prof. Dr. Hannelore Daniel
2. Univ.-Prof. Dr. Siegfried Scherer


Die Dissertation wurde am 15.11.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 10.02.2005 angenommen.

2






Probleme lassen sich nicht mit den Denkweisen lösen,
die zu ihnen geführt haben
Einstein

3



4 Table of contents
1 Introduction................................................................................. 11
1.1 Physical and chemical properties of zinc ..................................................... 11
1.2 Biological functions of zinc............................................................................ 12
1.2.1 Zinc-dependent enzymes .............................................................................. 14
1.2.2 Role of zinc in DNA replication and transcription .......................................... 15
1.3 Zinc homeostasis............................................................................................ 19
1.4 Pathologies associated with an altered zinc status..................................... 23
1.5 Assessment of the zinc status in humans.................................................... 24
2 The aim of this work ................................................................... 27
3 Materials ...................................................................................... 29
3.1 Equipment........................................................................................................ 29
3.2 Biochemicals and consumables.................................................................... 29
4 Methods ....................................................................................... 31
4.1 Cell culture methods 31
4.1.1 Cell culture of HT-29 cells ............................................................................. 31
4.1.2 Experimental media 31
4.1.3 Transfections................................................................................................. 31
4.1.4 Generation of a double-stable Tet-On/MTF-1 HT-29 cell line ....................... 32
4.2 Fluorescence-optical measurements ............................................................ 32
4.2.1 Proliferation and cell integrity ........................................................................ 32
4.2.2 Free intracellular zinc concentration.............................................................. 33
4.2.3 Determination of lactate levels ...................................................................... 33
4.2.4 Detection of apoptosis................................................................................... 33
4.3 Transcriptome analysis .................................................................................. 34
4.3.1 Isolation of total RNA..................................................................................... 34
4.3.2 DNA arrays.................................................................................................... 34
4.3.3 Northern blot analysis.................................................................................... 35
4.3.4 Real-time RT-PCR ........................................................................................ 36
4.4 Proteome analysis........................................................................................... 37
4.4.1 Western blot analysis 37
4.4.2 2D-PAGE/MALDI-TOF-MS............................................................................ 38
4.5 Molecular biology methods 39
4.5.1 Preparation of DNA fragments for probe synthesis for Northern blot............ 39
4.5.2 Construction of a MTF1/EGFP expression plasmid ...................................... 39
54.5.3 DNA sequencing ........................................................................................... 40
4.6 Other methods applied ................................................................................... 40
4.6.1 Atomic absorption spectrometry (AAS) ......................................................... 40
4.6.2 Confocal laser-scanning microscopy............................................................. 41
4.6.3 Computational search for metal-responsive elements (MREs) ..................... 41
4.7 Statistical analysis .......................................................................................... 41
5 Results......................................................................................... 43
5.1 Transcriptome analysis .................................................................................. 43
5.1.1 Zinc supplementation .................................................................................... 43
5.1.2 Zinc depletion ................................................................................................ 50
5.1.3 Zinc responsiveness of selected zinc-dependent genes............................... 59
5.2 Proteome analysis........................................................................................... 66
5.2.1 Identification of zinc-dependent proteins by 2D-PAGE/MALDI-TOF-MS ...... 66
5.2.2 Assessment of cellular lactate levels............................................................. 70
5.3 Comparison of results from proteome and transcriptome analysis .......... 70
5.3.1 mRNA levels of zinc-dependent proteins ...................................................... 70
5.3.2 Regulation of the Kruppel-like factor 4 on the protein level........................... 71
5.4 Target gene search for MTF-1 ........................................................................ 72
5.4.1 Alterations in transcript levels of zinc-sensitive genes upon
overexpression of MTF-1............................................................................... 72
5.4.2 Computational search for MREs in promoters of the identified
zinc-sensitive genes ...................................................................................... 75
6 Discussion................................................................................... 77
6.1 Transcriptome analysis .................................................................................. 77
6.1.1 Zinc supplementation .................................................................................... 77
6.1.2 Zinc depletion ................................................................................................ 81
6.1.3 Zinc responsiveness of selected zinc-dependent genes............................... 85
6.2 Proteome analysis........................................................................................... 86
6.3 Target gene search for MTF-1 ........................................................................ 90
7 Summary ..................................................................................... 95
8 Zusammenfassung ..................................................................... 97
9 Addendum ................................................................................... 99
9.1 Complete list of genes identified as responsive to zinc depletion............. 99
9.2 Primers........................................................................................................... 106
9.2.1 Zinc supplementation .................................................................................. 106
9.2.2 Zinc depletion .............................................................................................. 107
9.2.3 mRNA levels of zinc-dependent proteins .................................................... 108
6 9.2.4 MTF-1.......................................................................................................... 109
9.3 Vectors ........................................................................................................... 110
9.4 MTF-1 plasmids ............................................................................................. 110
9.4.1 pMTF1 ......................................................................................................... 110
9.4.2 pMTF1/EGFP-TRE...................................................................................... 112
9.5 List of abbreviations ..................................................................................... 116
10 Literature................................................................................ 119

7List of Figures
Fig. 1: Structure of a zinc finger protein and its interaction with DNA............................... 15
Fig. 2: Interrelations between zinc, MTF-1 and metallothionein........................................ 17
Fig. 3: Effects of