Abstract
BackgroundWe investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively).MethodBy an ultra-sensitive quantitative PCR-based assay, McfDNA and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions.ResultsOur data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia.ConclusionThis study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.
Highlights
We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively)
NcfDNA and McfDNA stability in supernatants from DLD1 and SW620 cell cultures As shown in Fig. 1a (DLD1 cell line) and Fig. 1b (SW620 cell line), the concentration of NcfDNA, measured via the detection of the long nuclear amplicon, dropped rapidly at D1: only 4% (DLD1 cells, Fig. 1a) and 7% (SW620 cells, Fig. 1b) of the concentration measured at D0 could be recovered
We show the high stability of short extracellular DNA amplicons of nuclear and mitochondrial origins in cell culture medium in the absence of cells: 60% of NcfDNA and McfDNA were recovered after 24 h of incubation, and 40% of this amount is recovered after 48 h of incubation
Summary
We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively). Hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia. The presence of hypoxic cells in solid tumours is closely associated with a poor prognosis and survival in patients with many types of cancer.[17,18,19] Epidemiological studies have associated higher mortality and increased cancer aggressivity in patients with intermittent hypoxia (IH) due to obstructive sleep apnoea.[20,21]
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