Untersuchung zur Korrelation der RNF20 abhängigen Ubiquitinylierung des Histons H2B mit der Expression des Tumorsuppressorgens p53 in Inflammationsprozessen und Inflammation assoziierter Kanzerogenese
Zellbiologie
Zusammenfassung der Projektergebnisse
The monoubiquitiylation of core histone H2B to H2Bub1 is a RNF20 ligase driven post-translational modification, which participates in numerous chromatin biological processes, which lead to changes in transcription, replication and repair of DNA. Multiple studies in diverse solid tumor entities showed that a downregulation of RNF20 and thus reduced H2Bub1 levels ultimately lead to a susceptibility to tumorigenesis. A further look into the molecular processes behind these observations lead to the discovery of interactions between the RNF20/H2Bub1 axis and a positive regulation of core tumor suppressor p53. In recent experiments, the Professor Moshe Oren lab of the Weizmann Institute of Science in Rehovot, Israel, showed an unexpected upregulation of H2Bub1 upon p53 knockdown in a mammary epithelial cell line, which could be interpreted as a compensatory mechanism for the loss of a crucial tumor suppressor gene. In order to further examine this new observation, a colorectal cancer cell line was challenged with p53 knockdown in order to investigate its influence on H2Bub1 and RNF20 expression shown on protein and RNA level. Findings were not consistent in three different experiments, therefore another approach was launched by creating a RNF20 knockdown in the same cell line. On protein level in Western blotting, RNF20 knockdown and consequently H2Bub1 downregulation was successful, but no significant reduction of p53 was shown. The investigation of these experiments on a RNA level by real time PCR showed unfortunately an insufficient knockdown of RNF20, thus observations in Western blotting are also not reliably applicable. When challenged by DNA damage treatment in the shape of chemotherapeutic agents such as Doxorubicin and Cisplatin, p53 and p21, a p53-dependent cell cycle arrest mediator protein, were significantly upregulated as a cell damage response system both shown in Western blot an rtPCR. However, results of H2Bub1 expression on protein level as well as RNF20 expression on RNA level were inconsistent, which in the case of H2Bub1 most likely originates from an impaired binding force of the H2Bub1 specific antibody. Over and above that the short termed project touched interesting aspects in an area of molecular processes in the inflammation-cancer continuum worth being more investigated in the future, but further experiments, such as the repetition of all above described experiments in order to see beyond technical inconsistencies, a repetition of the same experiments in the original cell line the discovery has originally been made in order to highlight potentially existing cell specific circumstances as well as embark on the analysis of proliferation of cells following p53 and RNF20 knockdown until conclusions can be drawn that could lead to results worth publishing and being of economic value in the future.
