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Investigating the role of ATRX in telomeric stability and escape from crisis

Geiller, Helene E. B. 2020. Investigating the role of ATRX in telomeric stability and escape from crisis. PhD Thesis, Cardiff University.
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Cancer cells gain replicative immortality through the upregulation of telomere maintenance mechanisms, and 10-15% of tumours activate the alternative lengthening of telomeres (ALT). The ATRX gene has been strongly linked to ALT and is often mutated in cancers that upregulate this pathway. The aim of this project was to establish the effect the loss of the ATRX gene and telomere dysfunction have on the initiation of the ALT phenotype. The ATRX gene was knocked out in two human cell types: epithelial cancer cells and primary fibroblasts. Upon loss of ATRX and the induction of telomere stress, cells successfully escaped a telomere-driven crisis in both models. In the fibroblast cultures, clones exclusively immortalised by the upregulation and maintenance of ALT. The loss of ATRX led to telomere length heterogeneity pre-crisis, providing evidence that it plays a role in the maintenance of telomere chromatin. In contrast, in epithelial cells, upon the loss of telomerase and the induction of a telomere-driven crisis, ALT-like elongation was observed at 5 chromosome ends in 4.7% of escapees for which the insertion length appeared to be chromosome and allele specific. A further 18% of clones that died during crisis, displayed initiation of an ALTlike process, providing evidence that epithelial cells can transiently switch to ALT, but ultimately require telomerase activity for long-term survival. Finally, telomeres from ALT clones were sequenced and showed an increase in non-canonical variant repeats suggesting the use of telomeric DNA as template for elongation. In conclusion, the loss of ATRX combined with telomere stress during crisis, is sufficient to initiate the ALT mechanism, and this study provides an insight into the transition from normal cells to malignancy upon the activation of the ALT mechanism, thus providing a tractable model to gain a better understanding of the ALT pathway.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Last Modified: 04 May 2020 08:54

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