Einfluss der DNA-Bindungskooperativität von p53 auf die Tumorsuppressoraktivität & Beobachtung der Entwicklungsdynamik von Tumorklonen in vitro & in vivo mittels sekretierter Luciferasen

p53 is known as the “guardian of the genome” and plays an important role in the prevention of cancer development. In response to different kinds of cellular stress like DNA damage or oncogene activity p53 binds as a sequence specific transcription factor to the DNA and induces the expression of genes involved in cell cycle arrest, senescence, differentiation and apoptosis. It is known that interaction with cofactors and modifying enzymes is involved in the decision between survival and death by p53. Nevertheless it remains unclear how this decision is made at the level of p53 binding to promoters. p53 forms a tetramer and binds to the DNA in a cooperative manner via the DNA binding domain. For this the H1 helices of two adjacent p53 monomers interact by forming a salt bridge. To determine the role of p53 DNA binding cooperativity for tumour suppression, p53 H1 helix mutants which cover the whole range from low to high cooperativity were generated and analyzed with respect to their genomic binding profiles, transactivation of target genes and response to cellular stress. The results show that the binding spectrum of p53 is extended by DNA binding cooperativity to include degenerated response elements found in proapoptotic genes. Therefore low cooperativity p53 induces cell cycle arrest but prevents the induction of apoptosis. Hence, DNA binding cooperativity modulates the cell fate decision, determines the elimination of damaged cell through apoptosis and contributes to the tumour suppressor activity of p53. Tumours are heterogeneous cell populations that consist of genetically distinct subclones. These subclones arise through the reiterative process of genetic mutation and selection. Most of these cancer clones can be eliminated therapeutically but due to a strong selective pressure during therapy resistant variants can expand. The genetic mutations that contribute to tumour initiation, progression, metastasis and therapy resistance are attractive targets for the development of therapeutic treatments. The contribution of single genes to cancer can be analyzed specifically with RNA interference and the use of short hairpin RNAs (shRNA) by generating a loss-of-function phenotype. In such experiments tumour cells carrying shRNAs can be marked and tracked with fluorescent markers. This works well in in cell culture studies or in leukemia mouse models but not for solid tumours, which comprise 90% of all cancers. Hence a system was generated to mark tumour cells constitutively or in an inducible manner with secreted luciferases. We developed a dual luciferase assay to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. The activities of the secreted Gaussia (GLuc) and Cypridina (CLuc) luciferases can easily and specifically be measured in the supernatant of cultured cells or minimal-invasively in mouse xenograft models in the blood of mice. The luciferase activities also correlate well with the tumour cell number or the tumour size. We show that this dual assay enables the time-resolved monitoring of clonal tumour evolution in a dynamic manner and its suitability for solid tumours as well as for the analysis of genes and their contribution to tumour development, metastasis and therapy. With one of the secreted luciferases as internal control for normalization the variance of the data is reduced and allows a reduction of animal numbers by approximately 75%. Using secreted luciferases and in consideration of the 3R principle we established a methodology to reduce the burden of animals in tumour research.