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Role of Peroxiredoxin 6 in Human Melanoma

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Role of Peroxiredoxin 6 in Human Melanoma Role of Peroxiredoxin 6 in human melanoma Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Alexandra Schmitt aus Würzburg Würzburg 2015 Eingereicht am:__________________________ Mitglieder der Promotionskommission: Vorsitzender:____________________________ Gutachter:______________________________ Gutachter:______________________________ Tag des Promotionskolloquiums:___________________ Doktorurkunde ausgehändigt am:___________________ Eidesstattliche Erklärung Gemäß §4, Abs. 3, Ziff. 3, 5 und 8 der Promotionsordnung der Fakultät für Biologie der Bayerischen Julius-Maximilians-Universität Würzburg Hiermit erkläre ich ehrenwörtlich, dass ich die vorliegende Dissertation selbständig angefertigt und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet habe. Ich erkläre weiterhin, dass die vorliegende Dissertation weder in gleicher, noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat. Weiterhin erkläre ich, dass ich außer den mit dem Zulassungsantrag urkundlich vorgelegten Graden keine weiteren akademischen Grade erworben oder zu erwerben versucht habe. Würzburg, Januar 2015 ______________________________________ Alexandra Schmitt Table of contents 1. Abstract ................................................................................................................. 1 2. Zusammenfassung ............................................................................................... 3 3. Introduction ........................................................................................................... 5 3.1 Melanoma ......................................................................................................... 5 3.1.1 Common melanoma signaling pathways ............................................................... 7 3.1.2 Oxidative stress in melanoma ...............................................................................10 3.2 The Xmrk and HERmrk melanoma models ..................................................... 11 3.3 Peroxiredoxin 6 ............................................................................................... 12 3.3.1 Peroxidase activity of PRDX6 ...............................................................................12 3.3.2 iPLA2 activity of PRDX6 ........................................................................................13 3.3.3 PRDX6 in cancer ..................................................................................................15 3.4 Aim of the thesis.............................................................................................. 16 4. Material and methods ......................................................................................... 17 4.1 Material ........................................................................................................... 17 4.1.1 Cell lines ...............................................................................................................17 4.1.2 Plasmids ...............................................................................................................17 4.1.3 Oligonucleotides for cloning and real-time-PCR ...................................................18 4.1.4 siRNAs .................................................................................................................20 4.1.5 Antibodies.............................................................................................................20 4.1.6 Inhibitors, drugs and compounds ..........................................................................21 4.1.7 Enzymes ..............................................................................................................22 4.1.8 Transfection reagents ...........................................................................................23 4.1.9 Kits .......................................................................................................................23 4.1.10 Technical equipment ..........................................................................................24 4.1.11 Buffer and media ................................................................................................24 4.1.11.1 Standard buffer ............................................................................................24 4.1.11.2 Cell culture media ........................................................................................25 4.1.11.3 Cell culture buffer .........................................................................................25 4.1.11.4 Bacterial culture media .................................................................................26 4.2 Methods .......................................................................................................... 27 4.2.1 Cell culture methods .............................................................................................27 4.2.1.1 Maintenance of cell lines ................................................................................27 4.2.1.2 Cryopreservation of cell lines .........................................................................27 4.2.1.3 Thawing of cell lines .......................................................................................27 4.2.1.4 Lentiviral infection and establishment of stable transgenic cell lines ...............27 4.2.1.5 siRNA Transfection ........................................................................................28 4.2.1.6 Proliferation and cell growth assays ...............................................................29 4.2.1.6.1 Cell growth assay performed by manual cell counting .............................29 4.2.1.6.2 Analysis of BrdU incorporation .................................................................29 4.2.1.6.3 xCELLigence assay .................................................................................29 4.2.1.7 Luciferase assay ............................................................................................30 4.2.1.8 Immunofluorescence ......................................................................................30 4.2.1.9 Gas chromatography analysis ........................................................................31 4.2.1.10 PGE2-ELISA .................................................................................................32 4.2.2 Protein methods ...................................................................................................32 4.2.2.1 Cell lysate preparation ...................................................................................32 4.2.2.2 SDS-PAGE and western blot .........................................................................32 4.2.2.3 Co-immunoprecipitation .................................................................................33 4.2.3 DNA methods .......................................................................................................33 4.2.3.1 Restriction enzyme digestion and ligation ......................................................33 4.2.3.2 Plasmid construction and site-directed mutagenesis ......................................34 4.2.3.3 Transformation, colony screen and plasmid preparation ................................36 4.2.4 RNA and cDNA methods ......................................................................................37 4.2.4.1 RNA extraction and cDNA synthesis ..............................................................37 4.2.4.2 Real-time-PCR ...............................................................................................37 4.2.4.3 Microarray ......................................................................................................38 5. Results ................................................................................................................. 39 5.1 Regulation of PRDX6 ...................................................................................... 39 5.1.1 PRDX6 is a direct target of Xmrk and the human EGFR .......................................39 5.1.2 PRDX6 is regulated by the PI3K pathway .............................................................42 5.1.3 Reactive oxygen species have no influence on PRDX6 level in melanoma cells and after HERmrk stimulation ........................................................................................43 5.2 Localisation of PRDX6 and its interaction with PCNA ..................................... 44 5.3 Function of PRDX6 in human melanoma cells ................................................ 47 5.3.1 Knockdown of PRDX6 reduces proliferation .........................................................47 5.3.2 Knockdown of PRDX6 decreases P-Rb protein and P-SRC family kinase levels ..51 5.3.3 PRDX6 influences proliferation via its iPLA2 activity..............................................52 5.3.4 Knockdown of PRDX6 lowers the amount of arachidonic acid and decreases the level of PGE2 in UACC-62 cells .....................................................................................57 5.3.5 The proliferative function of the iPLA2 activity of PRDX6 is mediated via AA ........59 6. Discussion .......................................................................................................... 62 6.1 PRDX6 in other tumor entities ......................................................................... 62 6.2 Analysis of PRDX6 levels and its regulation in melanocytes and melanoma
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