Molecular pathways controlling DNA damage response

Alhebshi, H 2018, Molecular pathways controlling DNA damage response , PhD thesis, University of Salford.

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Abstract

P53 is inactivated in around 50 % of human neoplasms and more than 26,000 somatic mutations of the p53 have reported in human cancers. P53 network is activated after cells are exposed to the DNA damage and depending on diverse conditions and type of DNA damage, p53 upregulation could lead to repression of the cell cycle progression and induction of apoptosis. Consequently, the p53 protein is considered vital to cancer progression and treatment and has been the focus of intense research. TTC5 (Tetratricopeptide repeat domain 5) or STRAP was identified as p53 cofactor and a key component of the p300/CBP (CREBB binding protein) complex. TTC5 enhances the interaction of JMY (Junction mediating and regulatory protein, p53 co- factor) and P300, leading to increase in HAT (Histone acetyltransferase) activity of P300, increase in p53 acetylation, stability and transcriptional activity.

In this study, we investigated crosstalk between the total p53, p53 phosphorylated at serine 46 (S46), p53 acetylated at lysine 382 (K382) and the TTC5 protein expression in Etoposide treated lung and mesothelioma cancer cell lines (A549, Mero-14, and Mero-25). Our results suggest there is no impact of Etoposide treatment on the TTC5 protein levels in all treated cell lines. Upregulation of p53, K382 and S46 protein expression levels as a response to the DNA damage in A549 and Mero-14 but not in Mero-25 cell lines was observed. Co-immunoprecipitation results showed evidence of the interaction between p53 protein and its co-factor (TTC5). MiRNA-34 family members that are transcriptional p53 targets were also investigated in studied cancer cell lines. Decrease in the miR-34a expression was observed in treated A549, Mero-25, and Mero-14 cancer cells compared to untreated cells, whereas the expression of miR-34b was elevated in treated A549 and Mero-25 but not in Mero-14 mesothelioma cells. We also analysed expression of above mentioned proteins in 220 tissue microarrays (TMA) in human lung cancer tissues using immunohistochemistry (IHC) approach and correlated these data with clinicopathological features of patients. Significant correlation was detected between the p53 expression levels and the grade of lung cancer, which might support the theory that mutant p53 acts as an oncoprotein. In addition, significant correlation was observed between the TTC5 protein expression and age, grade, tumour size and the intensity of Ser-46 protein expression. Interestingly, some cases showed cytoplasmic staining of acetylated p53 at lysine 382 rather than nuclear staining. Immunofluorescence (IF) was used to demonstrate a nuclear staining of the K382 protein in A549 and U2OS cell lines, suggesting that cultured cancer cells might behave differently and/or that in patients tissues there is heterogeneity of K382 subcellular location. Overall, our results indicate that p53 isoforms analysis together with TTC5 expression might have future use as biomarkers, prognostic factors or drug targets in cancer. However, further research is needed to confirm these findings.

Item Type: Thesis (PhD)
Contributors: Krstic- Demonacos, M (Supervisor) and Smyth, LJC (Supervisor)
Schools: Schools > School of Environment and Life Sciences > Biomedical Research Centre
Depositing User: HASEN Alhebshi
Date Deposited: 28 Nov 2018 12:52
Last Modified: 19 Mar 2019 12:03
URI: http://usir.salford.ac.uk/id/eprint/48475

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