Antioxidant defense and redox responses to telomere homolog oligonucleotides in human dermal fibroblasts: a model for investigating redox signaling responses to DNA damage

• Main Author: Lee-Bellantoni, Margaret S.
• Language: en_US
• Published: Boston University 2019
• Subjects: Medicine; DNA damage
• Online Access: https://hdl.handle.net/2144/37162

Thesis (Ph.D.)--Boston University === PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [e-mail]. Thank you. === It has been demonstrated that oligonucleotides homologous to the 3' telomere repeat sequence TTAGGG (T-oligos) stimulate DNA damage responses that are also induced by disruption of the telomere loop structure. Adaptive defense against oxidative stress and UV or ionizing radiation has been reported, but adaptive antioxidant defense as a response to mimicking telomere loop exposure has not been described. The T-oligos pTT and pGTTAGGGTTAG were added to human dermal fibroblast cultures to investigate whether mimicking telomere loop disruption stimulates antioxidant defense. pTT stimulated mitochondrial superoxide dismutase protein levels within 72 hours. Cell yields were higher after H202 exposure in fibroblasts pretreated with pTT for 72 hours compared to diluent pretreated cells. Intracellular reactive oxygen species (ROS) levels, measured by flow cytometry and the dichlorofluorescein diacetate probe, increased during T-oligo treatment as compared to diluent and oligonucleotide controls. The time course and degree of ROS stimulation corresponded to the time course for activation and/or induction of p53 and p21/Cip1/Waf1. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated this increase and fibroblasts retrovirally transduced to produce dominant negative p53 failed to display increased ROS, implicating that the T-oligos induced ROS through p53-responsive NADPH oxidases. A horseradish peroxidase assay for extracellular H20 2 showed no H20 2 release with pTT treatment. To determine whether there was induction of senescence, an endpoint response to increased ROS and prolonged T-oligo treatment in fibroblasts, the senescence-associated β-galactosidase assay was conducted in parallel with the DCF assay. Only the 11mer T-oligo treatment modestly increased the number of β-galactosidase positive cells by 72 hours (<30% of cells). This is the first report suggesting that antioxidant defense and ROS signaling are part of the broad adaptive response in mammalian cells presumably initiated by telomere loop disruption and mimicked by T-oligos. T-oligo treatment thus offers a new model for studies of ROS signaling in human dermal fibroblasts, allowing exploration of the relationships between DNA damage, ROS, oxidative stress, and the evolution of cellular defense mechanisms. === 2031-01-01