Detection of NADPH oxidase subunits NOX1 and NOX4 in lung adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1-dependent gene regulation [Elektronische Ressource] / by Malec, Viktor

Detection of NADPH oxidase subunits NOX1 and NOX4 in lung
adenocarcinoma A549 cells, and impact of NOX1 on Nrf2- and HIF-1-
dependent gene regulation





Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen


by
Malec, Viktor
born in
Liberec, Czech Republic




Giessen 2010







From the Department of Internal Medicine II
Director: Prof. Dr. Werner Seeger
of the Faculty of Medicine of the Justus Liebig University Giessen










First Supervisor and Committee Member: PD Dr. Jörg Hänze
Second Supervisor and Committee Member: Prof. Dr. Thomas Kietzmann
Committee Members: Prof. Dr. Ernst Petzinger
Prof. Dr. Andre Menke






thDate of Doctoral Defense: 30 March 2010
Table of content
I. Table of content……………………………………………………………….1
II. List of figures…………………………………………………….……………4
III. Table of abbreviations…………………………………………………………6

I. Table of content
1. Introduction................................................................................................................. 9
1.1 NADPH oxidase........................................................................................................ 9
1.1.1 Functions of NADPH oxidases and NOX subunits ........................................... 9
1.1.2 Subunits of NADPH oxidases.......................................................................... 11
1.1.2.1 NOX1 and NOX4 ..................................................................................... 11
NOX1................................................................................................................ 11
NOX4................................................................................................................ 13
1.1.2.2 p22phox, NOXO, NOXA, p40phox, Rac ................................................. 14
p22phox............................................................................................................. 14
NOXO............................................................................................................... 14
NOXA............................................................................................................... 14
p40phox............................................................................................................. 15
Rac GTPase....................................................................................................... 15
1.1.2.3 Assembling and activation of NADPH oxidase subunits ......................... 15
1.2 Reactive oxygen species ......................................................................................... 18
1.2.1 Types of ROS, oxidative stress and antioxidants............................................. 18
1.2.2 Cellular sources of ROS................................................................................... 19
1.2.3 Mechanisms of ROS impact on cell signaling................................................. 19
1.2.4 ROS, oxygen sensing and hypoxia inducible factor 1 ..................................... 20
1.2.4.1 HIF-1 is a target of oxygen sensors .......................................................... 20
1.2.4.2 Regulation of HIF by the oxygen sensors prolyl 4-hydroxylase and
asparaginyl hydroxylase........................................................................................ 21
1.2.4.3 Impact of reactive oxygen species on HIF regulation .............................. 22
1.3 Nuclear factor (erythroid-derived 2)-like 2............................................................. 24
1.3.1 Regulation of Nrf2 stability and activity ......................................................... 24
1.3.2 Nrf2 target genes.............................................................................................. 25
1.4 Thioredoxin system................................................................................................. 27
1.4.1 Thioredoxin...................................................................................................... 27
1.4.2 Thioredoxin reductase...................................................................................... 28
1.4.3 Function of the thioredoxin system.................................................................. 29
2. Aims of the work ..................................................................................................... 30
Part I.............................................................................................................................. 30
Part II ............................................................................................................................ 30
3. Materials and Methods........................................................................................... 32
3.1 Materials ................................................................................................................. 32
3.1.1 Materials and instruments................................................................................ 32
3.1.2 Chemical agents............................................................................................... 32
3.1.3 Buffers.............................................................................................................. 33
3.1.4 Antibodies........................................................................................................ 34
3.1.5 Oligonucleotide sequences............................................................................... 34
1
3.1.6 siRNAs............................................................................................................. 35
3.1.7 Plasmids ........................................................................................................... 35
3.1.8 Cell culturing ................................................................................................... 36
3.1.8.1 Bacteria, mediums for bacteria culturing and transformation .................. 36
3.1.8.2 Mammalian cells and cell culturing mediums .......................................... 36
3.2 Methods................................................................................................................... 37
3.2.1 Biochemical methods....................................................................................... 37
3.2.1.1 Subcellular fractionation........................................................................... 37
A) Isolation of cytosol, nuclei, membranes and cytoskeleton .......................... 37
B) Separation of Tx-100 soluble and insoluble fraction................................... 38
C) Isolation of plasma membrane..................................................................... 38
D) Isolation of nuclei ........................................................................................ 38
E) Subcellular fraction of samples for HPLC................................................... 38
3.2.1.2 Protein concentration measurement.......................................................... 39
3.2.1.3 Western blot.............................................................................................. 39
3.2.1.4 HPCL protein purification ........................................................................ 40
A) Anion exchange chromatography................................................................ 40
B) Reverse phase chromatography ................................................................... 41
3.2.1.5 2D gel electrophoresis............................................................................... 41
3.2.1.6 MALDI-TOF MS...................................................................................... 42
3.2.1.7 Reactive oxygen species measurement..................................................... 43
3.2.1.8 Luciferase activity..................................................................................... 44
3.2.2 Molecular biological methods.......................................................................... 44
3.2.2.1 Transformation of bacteria and isolation of plasmid ................................ 44
3.2.2.2 RNA isolation, reverse transcription and real-time PCR.......................... 44
3.2.2.3 DNA agarose gel electrophoresis.............................................................. 45
3.2.2.4 RNA interference by synthetic siRNA ..................................................... 46
3.2.2.5 Plasmids .................................................................................................... 46
3.2.3 Cell biological methods ................................................................................... 46
3.2.3.1 Cell culturing under different oxygenation conditions ............................. 46
3.2.3.2 Cell transfection by siRNA and plasmid .................................................. 47
4. Results ........................................................................