AL-Jumaily, Rakad Mohammed Khamas (2018) Molecular cytology of hypoxic cancer and stem cells: an epigenetic approach. Doctoral thesis, Keele University.

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Abstract

Oxygen is an essential factor for life in many organisms. Oxygen concentrations vary widely across different human tissues, but in general, are much lower than the 21% oxygen in atmospheric air. In spite of this, most research continues to be based on the culture of cells in an air oxygen (21% O2) environment. This ignores the mounting evidence of the role of physiological oxygen levels in the maintenance of survival, proliferation, stemness, genetic and epigenetic changes. The positive benefits of low oxygen tension on the maintenance of properties of several cells including embryonic and mesenchymal stem cells are well established while data describing negative impacts via genomic stability are few and conflicting. The role of cytosine modifications in cancer cells in response to hypoxia is poorly understood either in vitro or in vivo. In this study, we aimed to determine the role of low oxygen in the regulation of DNA methylation marks (5mC and 5hmC) and their associated genes including DNMT1/3A/3B/3L and TET1/2/3 in different cell lines including human embryonic stem cells, human mesenchymal stem cells and cancer cells. In addition to above, we also sought to corroborate upon and extend previous studies describing the effect of reduced oxygen on a range of cellular aspects including proliferation, metabolic activity, stemness and differentiation. To achieve these aims we cultured cells in either air oxygen, a fully defined 2% O2 environment (Hypoxycool media, tri-gas workstation), or in a multiuser tri-gas incubator with handling in a standard biological safety cabinet.

Routine culture of stem cells in physiological normoxia enhanced the functional profile of stem cell populations including proliferation, metabolic activity and stemness. In contrast, culture of cancer cells in reduced oxygen caused significant decreases in growth profiles (vs. air oxygen). Quantitative RT-PCR and Western blots results of cells cultured in reduced oxygen revealed significant transcriptional translational downregulation of DNMT3B and TET1 vs. air oxygen (except for TET1 in cancer cell lines cultured in 2% O2 where it increased significantly), accompanied by significantly reduced in levels of 5mC and 5hmC (again except for 5hmC in cancer cell lines cultured in 2% O2 where significant increases were noted). Noteworthy was that the downregulation in gene expression of DNMT3B was associated with an increase in its CpG promoter methylation. Importantly, these changes observation was associated with increases HIF2A, at protein levels in most cell types investigated. The role of physiological oxygen in these changes was confirmed by transitioning cancer cell lines between 2% O2 and air oxygen and detailing the reversibility of both DNMT3B and DNMT3L expression at mRNA level and promoters CpG island methylation. Together these data suggest that the level of 5mC is reduced by HIF2A, via DNMT3B- mediated methylation.

In conclusions, our cells display oxygen-sensitive methylation patterns where de novo methylation is linked to oxygen culture and correlates directly with transcriptional and translational regulation of the de novo methylase DNMT3B. Delineating cancer and stem cell biology under niche-like culture conditions will ultimately enhance our understanding of mechanisms of action promoting improvements to both tumour-targeting medicines and regenerative medicine applications.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Medicine and Health Sciences > Institute for Science and Technology in Medicine
Depositing User: Lisa Bailey
Date Deposited: 12 Dec 2018 11:09
Last Modified: 12 Dec 2018 11:09
URI: http://eprints.keele.ac.uk/id/eprint/5582

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