Qutob, Haitham
(2014)
DNA damage response and anti-apoptotic proteins expression in NPMc+ mutated cells in acute myeloid leukaemia.
PhD thesis, University of Nottingham.
Abstract
Nucleophosmin 1 (NPM1) is a multi-functional phosphoprotein, which shuttles between the nucleolus and the cytoplasm. It participates in many cellular processes including ribosome biogenesis and transport, centrosome duplication, and also contributes to the control of genomic stability. NPM1 interacts with many proteins including those that participate in DNA damage repair processes. C-terminal mutations in NPM1 occur in 35% of patients with acute myeloid leukaemia and are associated with a good prognosis. They are characterised by delocalisation of NPM1 into the cytoplasm. This may have a role in the sensitivity of mutant cells to chemotherapy via inactivation of the DNA damage response processes.
Firstly, we aimed to identify potential differences in DNA repair in response to double-strand breaks (DSB) and alkylating inducers and then assess them through the comet assay in the wild-type and mutant NPM1 cell lines. The percentage and kinetics of DNA damage in both cell lines was identical, indicating that DNA lesions were repaired efficiently in both the wild-type and mutant cell lines. The γ-H2AX foci were also evaluated which increased to similar levels in mutant and wild-type cells lines after exposure to DNA damaging agents and decreased with similar rates when the cells were allowed 24 hours to repair the damage. Interestingly, following DNA damage, the amount of NPM1 increased significantly in NPMc+ cells, both in the nucleus and the cytoplasm, which was not seen in NPM1 wild-type cells.
Next, we determined the subcellular localisation of APE1, which is a DNA repair enzyme in the base excision repair pathway and a transcriptional co-activator. APE1 has previously been shown to associate with NPM1. Using confocal microscopy, we found that the APE1 in both wild-type and NPMc+ mutant cell lines is predominately localised in the cytoplasm, while it is translocated into the nucleus after the cells were exposed to MMS, presumably to play a role in the DNA damage repair mechanism.
As we could find no apparent difference in DNA repair between NPM1 mutated and wild type cells we went on to look at proteins involved in cell survival - BCL-2 and MCL-1. The NPMc+ mutant cell line expressed the highest level of mRNA BCL-2 and MCL-1 when compared to the NPM1 wild-type cell lines. The NPMc+ mutated cells have previously been shown to be sensitive to the effect of all-trans retinoic acid (ATRA). We looked to see whether ATRA has an effect on the anti-apoptotic proteins. The results demonstrate that the BCL-2 and MCL-1 levels were down-regulated to a greater level in the NPM1 mutant cell line than in the NPM1 wild-type cells. In patient samples, the BCL-2 and MCL-1 mRNA down-regulation was seen in 3/5 and 5/5 NPMc+ samples, respectively. Thus, these finding indicate that BCL-2 and MCL-1 mRNA expression is down-regulated following ATRA in NPMc+ mutant cells.
Finally, siRNA was used to decrease levels of either total NPM1 or mutant NPM1 alone. Results showed that in the NPM1 mutant cells there was a down-regulation of BCL-2 mRNA, while in the NPM1 wild-type cells, no effect on the BCL-2 mRNA level was found. Furthermore, levels of P53 were up-regulated in the mutant NPM1 cells after knocking-down the total NPM1, whereas the wild-type cell line showed no change in the P53 level. These results provide evidence suggesting that down-regulation of NPM1 in the NPMc+ cell line increases total P53, possibly by interaction of ARF and HDM2, resulting in down-regulation of the BCL-2 in the mutated cells. In conclusion, perturbation of NPM1 subcellular localisation in NPMc+AML has no effect on the DNA repair mechanisms but we found differences in the anti-apoptotic proteins expression compared to NPM1 wild-type cells.
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