Browsing by Author "Harries, Sarah"

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    Ataxia telangiectasia mutated protein kinase : a potential master puppeteer of oxidative stress-induced metabolic recycling
    (Hindawi, 2021) Blignaut, Marguerite; Harries, Sarah; Lochner, Amanda; Huisamen, Barbara
    ENGLISH ABSTRACT: Ataxia Telangiectasia Mutated protein kinase (ATM) has recently come to the fore as a regulatory protein fulfilling many roles in the fine balancing act of metabolic homeostasis. Best known for its role as a transducer of DNA damage repair, the activity of ATM in the cytosol is enjoying increasing attention, where it plays a central role in general cellular recycling (macroautophagy) as well as the targeted clearance (selective autophagy) of damaged mitochondria and peroxisomes in response to oxidative stress, independently of the DNA damage response. The importance of ATM activation by oxidative stress has also recently been highlighted in the clearance of protein aggregates, where the expression of a functional ATM construct that cannot be activated by oxidative stress resulted in widespread accumulation of protein aggregates. This review will discuss the role of ATM in general autophagy, mitophagy, and pexophagy as well as aggrephagy and crosstalk between oxidative stress as an activator of ATM and its potential role as a master regulator of these processes.
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    Exploring features of oxidative stress and senescence in the H9c2 cardiomyoblast cell line
    (Stellenbosch : Stellenbosch University, 2023-03) Harries, Sarah; Huisamen, Barbara; Sadie-Van Gijsen, Hanél; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Background: Age-related cardiovascular disease is one of the largest causes of mortality globally. Senescent cells in the heart have emerged as a possible contributor to cardiac disease progression; however, studies elucidating the mechanisms explaining this phenomenon are scarce. The protein kinase Ataxia Telangiectasia Mutated (ATM) has been shown to be involved in DNA damage repair, cellular redox homeostasis, and mitochondrial function, and therefore may play a role in regulating senescence in heart cells. Cardiomyocytes are the most common cell-type in the heart; however, due to logistical and ethical considerations, cardiomyocytes are not always a feasible model for cardiac cell research. Therefore, the aim of this study was to determine if the H9c2 cardiomyoblast cell-line can exhibit features of induced senescence as a foundation to study the role of ATM in cardiac cell senescence. Methods: Early and late passage H9c2 cardiomyoblast cells (EP and LP, respectively) were compared in terms of growth rate, secretome and responsiveness to exogenous hydrogen peroxide and retinoic acid, and were then assessed for markers of senescence and changes in oxidative stress status. Markers measured included intracellular reactive oxygen species (ROS), senescence-associated betagalactosidase (SA-βgal) staining, gene expression levels of p16, p21 and ATM, and protein levels of total and phosphorylated ATM, p53 and H2AX. The anti-senescent properties of the antioxidant polyphenol resveratrol were also explored in EP and LP H9c2 cells. Results: EP and LP cells exhibited little evidence of oxidative stress and senescence and displayed high proliferative capacity, no increase in intracellular ROS and no increase in senescent markers. However, LP cells exhibited upregulation of the DNA damage marker γH2AX and the DNA repair protein ATM. Unexpectedly, a high concentration (25 µM) of resveratrol had a detrimental effect on EP and LP cell culture density and significantly upregulated p21 gene expression in EP cells. Discussion: Despite an increase in DNA damage markers and DNA repair proteins in LP H9c2 cells, other markers of senescence were absent, suggesting that the cells were able to repair the damage without committing to senescence. Intriguingly, a high concentration of resveratrol was detrimental to both EP and LP cultures, and consequently, the use of resveratrol as a cardioprotective antioxidant should be https://scholar.sun.ac.za iv questioned and may be dose-dependent. In conclusion, the H9c2 cells overall showed a resistance to respond to various damaging stimuli. Therefore, this cardiomyoblast cell line may not be suitable for research within the context of senescence owing to its limited ability to develop features of senescence.