Characterization of Nek9 as a transcriptional regulator of p53-mediated networks involved in cellular arrest and apoptosis
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Date
2021
Authors
Crisostomo, Leandro Gabriel Villota
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Abstract
Oncogenesis is the consequence of the transformation of normal cells into cancerous cells. Cellular kinases are vital enzymes that facilitate the transfer of phosphate groups between two molecules. The disruption of these messengers may lead to genomic instability, resulting in death, or oncogenesis. NEK9, a human kinase, is responsible for mitotic progression and spindle formation. This thesis describes how the dysregulation of NEK9 in human cells alters the expression of tumour-suppressing pathways, disrupting mitotic progression, leading to transformation, cell cycle arrest, and the induction of apoptosis. The overexpression of NEK9 in adenovirus-infected MEF cells was shown to enhance the transformative properties of the virus, suggesting it may possess oncogenic properties. NEK9 was shown to suppress p53 activity independent of adenovirus, suggesting an inherent ability to regulate p53-promoters. The depletion of NEK9 in p53-mutant cells led to the upregulation of tumour-suppressor genes such as PIG3, PUMA, and GADD45α. The expression of proapoptotic and antiapoptotic markers in NEK9-depleted cells were mainly cell-dependent, despite their p53 wild-type status. A kinase-inactive variant (D194N) and RCC1-deletion variant (ΔRCC1) of NEK9 were shown to cause a delay in the S-phase of cells. Cells with prolonged expression of these variants also exhibited signs of apoptosis. Altogether, these findings support the hypothesis that NEK9 acts as a transcriptional regulator to p53-regulated genes responsible for the induction of cell cycle arrest and apoptosis.
A high throughput in vivo screen was developed to identify NEK9 substrates. Co-localization between an mCherry-NEK9-LacI fusion protein with a GFP-tagged NEK6 consensus site indicated a positive interaction. However, co-localization was also detected between an mCherry-LacI fusion protein that lacked NEK9 and the GFP-tagged NEK6 consensus sequence, suggesting further optimization of the assay is required. Establishment of NEK9’s binding partners would aid in characterizing it’s involvement in various cellular pathways.
Additionally, this thesis set out to map the temporal expression of adenoviral genes during infection of normal human cells. The boundaries of early, intermediate, and late viral gene activation were defined and discussed. Overall, these results detail the transcriptional cycle of adenovirus, by carefully examining the chronological events that occur during infection.
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Keywords
Kinase, Cancer, Apoptosis, Cellular Arrest, Senescence, Nek9, Cell cycle, Adenovirus, Droplet Digital PCR
Citation
Crisostomo, L., Soriano, A. M., Mendez, M., Graves, D. & Pelka, P. Temporal dynamics of adenovirus 5 gene expression in normal human cells. PLoS One 14, (2019).