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Alexander Ogilvy, De Fen Shen, Yujuan Wang, Chi-Chao Chan, Mones Abu-Asab; Extranuclear DNA: a Normal Phenomenon or an Indication of Pathology?. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1782.
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© ARVO (1962-2015); The Authors (2016-present)
Extranuclear DNA (enDNA) occurs in the cytoplasm outside of nuclear membrane. The appearance of enDNA in cells is an understudied and enigmatic phenomenon. Mechanism of enDNA generation, conditions, and functions remain mostly unknown. The answer to whether or not enDNA is merely a normal phenomenon or an indication of a pathological state within the cell could have major implications in the realms of diagnostic and preventative medicine. Through our experiment we sought to investigate whether or not the appearance of enDNA within the RPE layer of the mouse eye was due to a pathological state, or just a normal cellular event.
Four mouse strains were studied using transmission electron microscopy (TEM) and immunolabeling EM. The study collection encompassed wild-type negative control C57BL/6J, and C57BL/6N (on rd8 background) strain as a positive control; as well as two mutant mice strains of Ccl2-/- (rd8 background) and double knockout Ccl2-/-/Cx3cr1-/- (rd8 background). Mice ages were 1.5-6 months old, the eyes were fixed in 2.5% glutaraldehyde for 24 hours, embedded in epoxy resin, and stained with uranyl acetate and lead citrate. Mouse eyes studied for immunolabeling were fixed in 4% formalin, embedded in LR white resin, labeled with rabbit-anti-mouse histone antibody and gold-conjugated protein A, and stained with aqueous uranyl acetate.
Extranuclear DNA was observed to be extensive in the neuroretina with rd8-like lesions (retinal dystrophy), and only occurred in the control mouse eyes when the RPE cell appeared unhealthy and with abnormal nuclear membrane. Observed enDNA was confirmed by immunolabeling. Cells containing enDNA often contained abnormal mitochondria and/or deteriorating nuclei. Cells with enDNA lacked apoptotic signs, suggesting that such enDNA leakage was not part of the apoptotic programmed cell death.
Our results suggest that enDNA is due to chromatin leakage that is unrelated to the apoptotic process, and that enDNA could be associated with a pathological state, senescence, or yet an undescribed death mechanism. The lack of apoptotic signs in cells with enDNA, as well as their unhealthy appearance, support our hypothesis that enDNA is not a normal event in healthy cells or during apoptosis, but is instead a sign of a pathological condition, senescence, or a new death mechanism.
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