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Charles Eberhart, Jeff S Mumm, Su Chan Lee, Laura Asnaghi; Extracellular DNA promotes retinoblastoma invasion. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1400.
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© ARVO (1962-2015); The Authors (2016-present)
Dissemination of retinoblastoma into the central nervous system or bloodstream represents an extremely serious clinical complication, for which there are no effective therapies. The local environmental cues which promote invasion are poorly understood. DNA precipitates in the form of extracellular DNA (exDNA) have been recognized in retinoblastoma for at least 60 years, but their functional effects have not been investigated. Recently, it has been suggested that exDNA can promote invasion of pancreatic adenocarcinoma. We therefore examined if exDNA might also modulate the pathobiology of retinoblastoma. The possibility that exDNA is released through a unique cell death process called NETosis was also examined.
WERI Rb1 and Y79 retinoblastoma lines were treated with DNase I to target exDNA. Invasion, growth, and proliferation were determined by colorimetric CCK-8, Ki67 and transwell invasion assays, respectively. Activation of apoptosis was determined by cleaved-caspase-3 immunofluorescence assay, or by Western blot, using a cleaved poly (ADP-ribose) polymerase (PARP) at Asp214 antibody. Staining of the exDNA was performed using Sytox Green and an antibody to anti-double strand DNA. Protein components associated with exDNA, such as peptidylarginine deiminase (PADI) and citrullinated histones, were measured by Western blot.
Staining revealed exDNA on and around retinoblastoma cells cultured in vitro in adherent conditions. Treatment with DNase I inhibited invasion by 70 to 80% in retinoblastoma cells at concentrations greater than 0.05 U/µL (p=0.004), but no significant inhibitory effects were observed on growth and proliferation, with a very mild reduction in survival. DNase I treatment also significantly slowed the spread of retinoblastoma cells in a zebrafish orthotopic xenograft model. Citrullinated histones were present in the reninoblastoma cells, and PADI4 mRNA and protein, the main component associated with regulated secretion of exDNA in inflammatory conditions (“NETosis”), was expressed at low levels.
These data indicate that exDNA can play an important role in increasing the invasive properties of retinoblastoma cells, and may represent a new target for therapy. A regulated NETosis-like phenomenon may be important for the release of exDNA in retinoblastoma.
This is a 2020 ARVO Annual Meeting abstract.
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