Etablierung eines NSCLC-Modells zur Untersuchung der Rolle der p53 DNA Bindungskooperativität für Zellschicksalsentscheidungen in vitro und in vivo

The tumor suppressor p53 acts as a cellular hub which processes a multitude of stress signals. By ensuring a tight control of the cell cycle and the state of the genome it acts as a safeguard of genomic integrity of a cell and - as an answer to cellular stresses - decides the cell’s fate mainly through its function as a transcription factor. Depending on the type and extent of the damage, the answer can reach from transient cell cycle arrest with concurrent start of DNA-repair programs to an irreversible cell cycle arrest by senescence or differentiation up to – ultimately – the removal of the cell from the organism via p53-mediated apoptosis. p53 DNA binding cooperativity plays an important role for target gene selection and thus for p53-dependent cell fate. p53 binds DNA as a tetramer in a cooperative manner. The structural basis for cooperativity is constituted by intermolecular interactions between the two oppositely charged amino acid residues glutamate 180 and arginine 181 in the helix H1 of the DNA binding domains of two p53 monomers. Mutations of these residues can lower or raise cooperativity and therefore DNA binding affinity of p53. This influences target gene selection, because high cooperativity expands the target gene spectrum to genes with p53 response elements deviating from the p53 consensus sequence. While target genes involved in cell cycle arrest mostly display canonical response elements and can therefore be transactivated by both low- and high-cooperative p53, promoters of apoptosis-promoting target genes often show imperfect, low-affinity p53 binding sites which can only be bound by high-cooperative p53. The influence of binding cooperativity on long-term proliferation behavior of tumor cells in vitro and in vivo has not been assessed comparatively yet. A primary objective of this work was therefore to establish cell lines with stable expression of p53-cooperativity mutants by means of which the entire spectrum of H1-Helix interactions strength and the associated impact on cell fate can be investigated. For this purpose an inducible p53 expression system with the different cooperativity mutants, which spanned the entire spectrum of cooperativity – from abrogated up to higher than physiological – was cloned. Furthermore, the system was labeled with a reporter, in order to monitor p53 expression. The created fusion proteins were expressed correctly and exerted p53-specific effects only after activation. For an optimal comparability of the influence of cooperativity on cell fate, it was attempted to integrate the p53 construct into a given locus in p53-null H1299 cells by means of gene targeting; this means endonuclease-mediated targeted integration into the Rag1 locus via homologous recombination to generate isogenic cell lines. The recombination rate was very low; furthermore the positively tested clones exhibited different p53-protein levels and did not show inducible and effective activity of p53. Thus, they were not suitable for further investigation of binding cooperativity. It can be presumed that because of a variety of reasons – e.g. the epigenetic status of the Rag1 locus or mutations in the DNA repair system – Meganuclease-mediated targeted integration in H1299 cells is only possible with very low efficiency. Thereupon additional cell lines with a stable expression of p53 fusion constructs were generated through conventional transfection and selection. They exhibited good expression of the constructs and strong p53- and cooperativity dependent effects on the cell fate upon p53 activation. For instance, cells showed reduced proliferation kinetics with increasing cooperativity. However, due to the fact that cells were not strictly isogenic, it could be observed that in some of the used clones different protein levels showed an effect on the cellular outcome upon p53 activation which in part masked influence of binding cooperativity. On the other hand, it could also be clarified that by means of careful selection of clones this model can display the influence of p53 DNA binding cooperativity very well. Hence, it is very suitable for additional experiments. Understanding the mechanisms of p53-mediated cell fate decisions is of great importance, thus further research on binding cooperativity as one modulator of these decisions should be conducted. There is a variety of possibilities for further investigations with this model, of which I suggest xenograft studies.