Hypoxia, a common feature of solid tumours, is associated with poor prognosis, a more aggressive tumour phenotype and therapeutic resistance. Tumour hypoxia induces changes in the chromatin structure, with induction of the heterochromatin mark H3K9me3 frequently observed. The upregulation of H3K9me3 was observed with co-incidental upregulation of the methyltransferase Suv39H1 in three (FTC133, U87 and HCT116) different cancer cell lines exposed to hypoxic conditions (0.1% O2). Thus, it was hypothesised that Suv39H1 was a candidate for the tri- methylation of H3K9 and the first part of this study was focused on investigating the potential regulatory mechanisms. RT-qPCR analysis showed no changes in the levels of Suv39H1 mRNA in hypoxia suggesting a post transcriptional regulation mechanism. In mild hypoxic conditions (O2 â¥1%) the cells failed to induce H3K9me3 and Suv39H1. However, treatment with proteasome inhibitor MG132 in mild hypoxia induced Suv39H1 upregulation, suggesting the involvement of the proteasomal pathway in the regulation of Suv39H1 stability in hypoxia. Under normal oxygen conditions Suv39H1 is degraded via MDM2 dependent ubiquitination, which is blocked by the protein Sirt1. However, the levels of Sirt1 were highly downregulated and MDM2 protein levels were maintained following hypoxic exposure. It has been previously reported that ATM is activated in response to hypoxia and negatively regulates MDM2 activity in response to DNA damage. To test the involvement of ATM and MDM2 in regulating Suv39H1 levels in hypoxia, the ATM specific inhibitor (Ku55933) was used together with MDM2 knockdown under normal oxygen conditions and hypoxia. As predicted, highest levels of Suv39H1 were observed when ATM is catalytically active (hypoxia) and MDM2 inactive (knockdown). The results of this study indicate an important role that ATM has in regulating chromatin structure in response to stress conditions, such as hypoxia, through regulation of MDM2. As ATM plays a crucial role in the hypoxia induced DNA damage response, the second part of this study was focused on uncovering ATM activation in hypoxia. It has been previously reported that the acetyltransferase Tip60 is involved in ATM activation in response to DNA damage. However, if Tip60 plays a role in the hypoxia induced activation of ATM hasnât been tested to date. In order to do this a Tip60 specific inhibitor, TH1834, was used. FTC133 and HCT116 cells were treated with TH1834 under hypoxic conditions and the levels of the catalytically active form of ATM were assessed by Western blot and IF. The obtained results showed a clear involvement of Tip60 in the hypoxia induced activation of ATM. Finally, as the activation of ATM in hypoxia has been related to S-phase arrest, the cell cycle distribution of FTC133 and HCT116 cells were analysed by FACS. The result showed that ATM activation in FTC133 cells seems independent of S-phase arrest. Heterochromatin is suggested to be important for cancer cell survival. This study proposes that ATM is involved in maintaining the heterochromatic state under hypoxia. ATM is an extensively researched target for anticancer therapy. Thus, uncovering this mechanism enables new insight into targeting hypoxic tumour cells.
|Date of Award
|1 Aug 2019
- The University of Manchester
|Kaye Williams (Supervisor), Costas Demonacos (Supervisor) & Roben Gieling (Supervisor)