Systematische Analyse der Fitness-Effekte von TP53-Mutationen in Tumorzellen unter Nährstoffmangel und Aktivierung von p73
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Philipps-Universität Marburg
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Abstract
TP53 is the most commonly mutated gene in human tumors. Mutations of the tumor suppressor p53 are found in almost all tumor entities, on average in 50 % of all patients. The majority of mutations are single missense mutations which are located in the DNA binding domain and lead to the expression of a mutated full length protein. Besides a loss of function (LOF) or inhibition of the remaining wildtype-allele, known as dominant-negative effect (DN), a mutation can lead to a gain of function (GOF), so that tumor cells can proliferate more rapidly, metastasize or become resistant to cytostatic drugs. Mechanistically, GOF manifests itself in metabolic changes, also under nutrient deficiency, or inhibition of other tumor suppressors such as p73. So far, this has only been shown for a few p53 mutations but many different ones are present in patients which differ in their consequences. The aim of this project was to investigate different mutations regarding their fitness effects under nutrient deprivation conditions such as glucose or glutamine starvation as well as a possible inhibition of p73. The human colon carcinoma cell line HCT116 was used, which was modified using CRISPR/Cas9, so that a library of all possible mutations for hotspot codon 175 could be introduced by recombination. Drug treatment or nutrient deprivation was followed by extraction of genomic DNA, which was amplified by PCR and subsequently sequenced, revealing enrichment or depletion of cells with specific mutations. This depletion or enrichment led to the characterization of p53 mutations as WT-like, LOF, or pLOF, but no GOF was detected for any of the TP53R175 mutations. To analyze the GOF effect in regard to the inhibition of p73, the isoforms TAp73α or TAp73β were overexpressed in a doxycyclin-inducible manner. Here, for none of the TP53R175 R175 mutations a GOF with respect to inhibition of p73 was observed. This work confirms the different impacts of various amino acid changes in the p53 protein concerning viability in nutrient deficient conditions.
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Created: 2023Issued: 2024-07-22Updated: 2024-07-22
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
GOFnutrient deprivationhomology directed repair
DFG-subjects
TP53CRISPR/Cas9Nährstoffentzugp73TP53-Mutationen
DDC-Numbers
610
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Borowek, Anna: Systematische Analyse der Fitness-Effekte von TP53-Mutationen in Tumorzellen unter Nährstoffmangel und Aktivierung von p73. : Philipps-Universität Marburg 2024-07-22. DOI: https://doi.org/10.17192/z2023.0616.