Generation of reporter cell lines by genome editing to probe p53 activity
Tolay, Nazife (2018) Generation of reporter cell lines by genome editing to probe p53 activity. [Thesis]
The p53 protein is defined as a sequence-specific transcription factor and functions as a tumor suppressor protein. The p53 protein is involved in diverse cellular processes which are important for controlling cell cycle arrest and apoptosis. The regulation of p53 is governed by an autoregulatory negative feedback loop between p53 and MDM2. Mutations in the TP53 gene and defects in the regulation of p53 are mostly associated with tumor initiation, invasion, and metastasis. Therefore, restoration of p53 functions to target cancer cell viability has been used for cancer therapy. Genome editing techniques have been used as an effective method to correct mutations, to integrate a gene of interest, and to knock out genes. We generated reporter cell lines by using genome editing methods to probe the transcriptional activation of p53. We showed that TALEN induced genome editing is an effective method to integrate a reporter gene into a targeted safe harbor site. We investigated the effects of various compounds on the transcriptional activity of p53 by using these reporter cell lines and found that some of these compounds increased the transcriptional activity of p53. We also analyzed the effects of the compounds on cell viability in either the presence or absence of p53 and we showed that these compounds caused cell death independent of p53. Additionally, we identified that all compounds stabilized the p53 protein in HCT 116 p53 WT cells. We showed that this stabilization was because of damage-induced post-translational modification of p53. Lastly, we showed that these compounds did not block the interaction between MDM2 and p53. In summary, we developed and tested screening tools to identify modifiers of p53.
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