Phosphorylierung der p53 H1-Helix: Rolle für Tumorsuppression und Tumorigenese

Das p53-Tumorsuppressorgen, auch als „Wächter des Genoms“ bekannt, ist das am häufigsten mutierte Gen in humanen Krebszellen. Im physiologischen Zustand wird es durch verschiedene Stimuli wie oxidativer Stress, DNA-Schäden oder Onkogen-Überexpression aktiviert, sodass p53 als Transkriptionsfaktor di...

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Bibliographic Details
Main Author: Niederau, Edmund Constantin
Contributors: Stiewe, Thorsten (Prof. Dr. med.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
Online Access:PDF Full Text
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The p53 tumor suppressor gene, also known as “guardian of the genome”, is the most frequently mutated gene in human cancer cells. In its physiological state, p53 is activated by various stimuli such as oxidative stress, DNA damage, or oncogene overexpression, allowing p53 as a transcription factor to initiate diverse mechanisms with the main goal of tumor suppression. Effects like a transient cell cycle arrest with the purpose of DNA repair, senescence as an irreversible arrest of the cell cycle, and apoptosis, also known as the programmed cell death, play a crucial role. Through these mechanisms, a cell can either be protected from transformation or the proliferation of a damaged cell can be blocked to prevent tumorigenesis. Since cancer cells are not always eliminated by programmed cell death through p53 activation, the necessary question arises under which circumstances p53 triggers the signaling pathway towards senescence or towards apoptosis. To investigate this decision-making, we attempt to use the mechanism of DNA binding cooperativity as a basis. This is because p53 does not act as a monomer but binds to DNA as a tetramer in a sequence-specific manner, allowing a more efficient binding to its promotors and an enhanced transcriptional activity. Interactions within the DNA binding domains, which are mediated by the ionic interactions between the amino acid residues of the so-called H1 helix, are essential for a stable p53 DNA complex. In human p53 this H1 helix contains a glutamate in position 180 and an arginine in position 181 as well as two evolutionarily conserved serine molecules in positions 183 and 185 in the direct vicinity of the H1 helix. Recent studies have shown that mutations in the H1 helix leading to loss of cooperativity can completely abolish p53 transcriptional functions, whereas weakened DNA binding cooperativity leads to a selective loss of apoptosis and directs transcriptional activity towards pro-senescence genes. On the contrary, enhanced DNA binding cooperativity results in a strong transcription of pro-apoptotic genes. In the question of whether the interaction between H1 helices can be influenced, the two serine molecules in positions 183 and 185 may play an interesting role. Phosphorylation of these amino acid residues could result in an enhanced negative charging of the H1 helix region due to the electromagnetic properties of phosphate groups, thus affecting DNA binding cooperativity. Some experimental data suggest that this enzymatic reaction at these serine residues is possibly mediated by Aurora kinases, in particular Aurora B. To further investigate the role of phosphorylation of these serine residues for p53 functions, the cancer cell line HCT116 with phospho-mimic and phospho-deficient mutations at positions 183 and 185 was used in this work. Here, a significantly enhanced cell death of the phospho- deficient mutants could be observed under cytotoxic treatment with doxorubicin. Since the murine genome only contains one conserved serine residue in position 180, performing in-vivo experiments made the S180A mutant mandatory, where the serine residue in position 180 is changed to phospho-deficient alanine. Depending on the experimental set-up, significant differences between wildtype p53 and phospho-deficient p53 in tumor suppression and tumorigenesis were also shown in the in-vivo experiments. Ultimately, taking influence on blocking the mentioned phosphorylation, for example by inhibition of the responsible kinases, could bring crucial benefit to the clinical treatment of cancer. Thus, kinase inhibitors could favorably affect the prognostically important functions of p53 in terms of tumor suppression and prevention of tumorigenesis by more intensively targeting cancer cells to apoptosis. Likewise, a reduced impact of cytotoxic therapy on healthy cells could be achieved by decreased DNA binding cooperativity therapeutically.