Zwischen Zellzyklus-Arrest und Apoptose – p53-Kooperativitätsmutanten in vivo

The p53 protein exerts its tumour suppressive function by determining a cell's fate. The integration of intrinsic and extrinsic cellular stress signals allows p53 as a tetrameric, sequence-specific transcription factor to regulate key target genes that either promote cell cycle arrest, senescence or apoptosis. How this differential expression is regulated is still a subject of intensive research. Re-cently, it has been shown in the Stiewe group that an intrinsic trait of p53 – DNA binding cooperativity – modulates this process. Two charged amino acid residues of the H1-helix (E180 and R181) facilitate cooperative DNA binding. By mutagenesis of these amino acids the ionic interaction between two mon-omers each in the tetramer could be modulated resulting in mutants with dis-tinct cooperative traits. While cooperativity impaired p53 mutants still triggered cell cycle arrest, they failed to induce apoptosis. However, mutations with a higher cooperativity showed an increased rate of apoptosis in comparison to wildtype p53. The objective of this project was to check the effect of these p53 cooperativity mutants on tumour growth and the response to a chemotherapeutic agent in a xenograft tumour mouse model. For this purpose, a p53 deficient clonogenic reporter cell line stably expressing a luciferase was generated and superin-fected with the p53 mutants. After subcutaneous injection of these cells in im-munocompromised mice tumour burden could be quantified repeatedly over time by measuring the activity of the secreted Gaussia luciferase in small blood samples. Concerning the technical aspect of this work it could be shown that i) the reporter cell line is an appropriate tool for studying the p53 response behaving identical to parental HCT 116-cells and ii) that by use of this reporter cell line measurement of Gaussia luciferase activity is an easy, robust and par-ticularly precise and sensitive tool to quantify tumour burden. Utilizing this as-say system, no discernible difference of the spontaneous tumour growth de-pendent on cooperativity could be shown. The issue of this analysis was a rap-id decline in p53’s expression levels in cancer cells. Interestingly, a negative selection dependent on the strength of the cooperativity could be observed that suggests an effect of increasing tumour-suppressive potential with in-creasing DNA binding cooperativity. Despite of this negative selection the re-sponse to a chemotherapeutic agent that facilitates stabilisation of p53 was compromised in a low cooperativity mutant. Genome wide gene expression analysis performed on tumour samples confirm former in vitro data with this model: namely that a low cooperativity is sufficient to induce cell cycle arrest genes, induction of apoptosis, however, is dependent on higher cooperativity that leads to an increased spectrum of transactivated target genes. In further work colleagues were able to show that cooperativity of p53 is nega-tively regulated by phosphorylation of serine 183/185. Phosphorylation of these amino acid residues is potentially conducted by aurora kinase B. Sever-al inhibitors of this kinase, that is overexpressed in various cancers, are partly in advanced stages of clinical trials.