Abstract

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.