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Title: RECQL4 : linking DNA replication and bone tumourigenesis
Author: Wiltshire, Tammy
ISNI:       0000 0004 8506 9751
Awarding Body: University of Lincoln
Current Institution: University of Lincoln
Date of Award: 2019
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As part of cell division, the initiation of DNA replication is an important regulatory mechanism that maintains genomic stability over generations. RECQL4 belongs to the RecQ DNA helicase family holding an important role in the initiation of DNA replication. RECQL4 mutations can lead to disorders including type II RothmundThomson (RTS) syndrome. These patients demonstrate predisposition to osteosarcoma (OS) development. OS is a primary bone tumour showing extensive chromosomal instability. We hypothesise mesenchymal stem cells (MSC) differentiating to osteoblasts are particularly sensitive to RECQL4 mutations, which may lead to impaired differentiation and OS. Our aim is to establish a direct link between the impairment of replication initiation, consequent chromosomal instabilities and deregulation in osteoblast differentiation, and OS development. A model that phenocopies the effect of RECQL4 mutations was established in ASC52telo cells to apply acute and chronic pressure on replication initiation using PHA-767491, which inhibits DDK that acts upstream of RECQL4 in replication initiation (PHA cells). We monitored cell viability and chromosomal instability characteristics in these cells, and RTS cell lines. To establish if PHA cells sustained differentiation capability, the cells were cultured using supplemented media for osteoblast and adipocyte differentiation, and were analysed by histochemical staining and immunofluorescence. Presence of DNA damage was quantified using γH2AX, and activation of the DNA damage response was assayed by western blotting. The cells were cultured in ultra-low attachment plates to test for anchorageindependent growth, and further analysed by cell count, MTS and luminescence assays. To identify protein-protein interactions of RECQL4, GFP-tagged RECQL4 HeLa and U2OS cells were treated with SAHA, or hydroxyurea, pulled down with GFP-nanotrap, and analysed by mass spectrometry and western blot. We confirmed reduced proliferation rate while maintaining viability in PHA cells. Assaying mitochondrial membrane potential revealed no significant effect on mitochondrial function. Successful differentiation of PHA treated MSCs into osteoblasts and adipocytes was confirmed. Expression of osteoblast differentiation markers: calcium, and RUNX2 was influenced by PHA. An increase was also observed in chronic PHA cells under normal medium, indicating malignant transformation. Sustained DNA damage was shown in chronic PHA-767491 treated ASC52telo cells, with a higher degree of CHK1 phosphorylation, anchorage-independent growth and reduced contact inhibition. We found that the RECQL4 mutated cell line AG05013tert was more sensitive to inhibition of replication initiation. Increase of DNA damage markers was observed in AG05013tert cells, but not in AG18375 and AG03587. Presence of MCM10 and PP2A in RECQL4 complexes was confirmed, and novel interactions with HDX and EN-2 were found. Overall, we demonstrated that chronic interference with DNA replication initiation leads to sustained DNA damage with characteristics of genomic instability, activated DNA damage response that may become impaired over time, and may induce transformation. To further these studies, RECQL4 knockdown using lentiviral transduction in osteoblasts would verify the cellular changes we propose, which lead to chromosomal instabilities and OS development. Novel protein interactions with RECQL4 could highlight new pathways with a direct and/or indirect role in tumourgenesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available