Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing cell culture models of tumorigenesis ....................................................... 4 1.4. Key molecules in human cell transformation ............................................................ 6 1.4.1. hTERT .................................................................................................................. 6 1.4.2. H-RasV12 ............................................................................................................. 7 1.4.3. SV40 small t ......................................................................................................... 8 1.4.4. SV40 Large T ....................................................................................................... 8 1.5. Principles of tumor suppression .................................................................................. 9 1.6. p73, a transcription factor of the p53 family ........................................................... 10 1.6.1. Gene and protein organization of p73 ................................................................. 10 1.6.2. p53 family proteins have diverse biological functions ........................................ 11 1.6.3. p73 in cancer development ................................................................................. 12 1.6.4. Regulation of p73 ............................................................................................... 12 1.6.5. Regulation by p73 ............................................................................................... 13 1.7. Scope of the project .................................................................................................... 14 2. MATERIALS AND METHODS .......................................................................... 15 2.1. Materials and Equipment .......................................................................................... 15 2.1.1. Instruments and other technical equipment ......................................................... 15 2.1.2. Glass and plastic ware, consumables .................................................................. 16 2.1.3. Enzymes, PCR reagents and size standards ........................................................ 17 2.1.4. Antibodies........................................................................................................... 17 2.1.5. Oligonucleotides ................................................................................................. 18 2.1.6. Bacterial strains .................................................................................................. 18 2.1.7. Plasmids .............................................................................................................. 19 2.1.8. Eukaryotic cell lines ........................................................................................... 19 2.2. Buffers, media and solutions ..................................................................................... 20 2.2.1. Protein detection ................................................................................................. 20 2.2.2. Flow cytometry ................................................................................................... 21 2.2.3. Cell culture ......................................................................................................... 21 2.2.4. Plasmid isolation from bacteria ........................................................................... 22 2.2.5. Other self-prepared buffers, media and solutions ................................................ 22 I 2.3. Cell culture .................................................................................................................. 23 2.3.1. Cell maintenance ................................................................................................ 23 2.3.2. Tumorigenicity assay .......................................................................................... 23 2.3.3. Growth curves ..................................................................................................... 23 2.3.4. Viability staining ................................................................................................ 24 2.3.5. Transfection ........................................................................................................ 24 2.3.6. Electroporation ................................................................................................... 24 2.3.7. Retroviral transduction ....................................................................................... 24 2.3.8. Adenoviral transduction ...................................................................................... 25 2.3.9. Cell stock freezing .............................................................................................. 25 2.4. DNA work ................................................................................................................... 25 2.4.1. Cloning ............................................................................................................... 25 2.4.2. Plasmid DNA isolation- Miniprep ...................................................................... 26 2.4.3. Plasmid DNA isolation- Midiprep ...................................................................... 27 2.4.4. Plasmid DNA isolation- Maxiprep ...................................................................... 27 2.4.5. DNA purification- spin columns ......................................................................... 27 2.4.6. DNA purification- phenol-chloroform extraction ............................................... 27 2.4.7. DNA quantification ............................................................................................ 28 2.4.8. DNA sequencing ................................................................................................. 28 2.4.9. Mutagenesis ........................................................................................................ 28 2.5. RNA work ................................................................................................................... 28 2.5.1. RNA isolation ..................................................................................................... 28 2.5.2. RNA quantification ............................................................................................. 29 2.5.3. cDNA preparation ............................................................................................... 29 2.5.4. PCR and semi-quantitative RT-PCR ................................................................... 29 2.5.5. PCR primers and amplicon sizes ......................................................................... 30 2.5.6. Real Time PCR ................................................................................................... 30 2.5.7. DNA microarray ................................................................................................. 31 2.6. Protein work ............................................................................................................... 32 2.6.1. Western Blot ....................................................................................................... 32 2.7. Cell-based assays ........................................................................................................ 32 2.7.1. BrdU incorporation assay ................................................................................... 32 3. RESULTS .......................................................................................................... 34 3.1. Validation of the model system ................................................................................. 34 3.1.1. Western Blot ....................................................................................................... 35 3.1.2. Real Time TRAP ................................................................................................ 35 3.1.3. Soft agar