Local irradiation (IR) is a standard cancer treatment. Aside from direct cytotoxic effect via lethal DNA damage, IR also impacts the phenotype of tumor cells, alters the tumor microenvironment and consequently enhances activation of the immune system, which could lead to bystander and abscopal effects of IR. Activation of cytosolic pattern recognition receptors, i.e. DNA sensors and subsequent induction of cytokines could be one of the mechanisms causing these effects. The main producers of cytokines after IR were reported to be immune cells, however they can also be produced by tumor cells. Therefore, the aim of this study was to investigate whether IR causes upregulation of cytosolic DNA sensors in B16F10 melanoma cells and whether this leads to cytokine release. B16F10 mouse melanoma cells were irradiated with graded doses (0.5, 2, 4, 6, 8 Gy) and cell survival was determined by clonogenic assay. The presence of DNA in the cytosol after IR was determined by immunofluorescence labelling. RNA was purified 5, 24, 48 or 72 hours after IR of cells and thereafter q-PCR was performed to determine the expression of some cytosolic DNA sensors. The expression of IFNß1 and TNF-? was measured at the mRNA level by q-PCR and at the protein level by immunofluorescence labelling. The survival of melanoma cells was already reduced at IR with 0.5 Gy and only 4% of the cells survived at IR with 8 Gy. The presence of cytosolic DNA after IR was confirmed. IR of the tumor cells increased the expression of cytosolic DNA sensors DDX60, DAI/ZBP1 and p204 in a time- and dose-dependent manner. Namely, the expression of all three cytosolic DNA sensors was significantly elevated in B16F10 melanoma cells at 24 h after IR and expression reached peak at 48 h after IR with? doses higher than 4 Gy. At 72 h post-IR, the expression of cytosolic DNA sensors decreased to the levels of control-unirradiated cells. The expression of cytokines, IFNß1 and TNF-? was upregulated on mRNA and protein levels at doses higher than 4 Gy. The results of our study demonstrate that IR can trigger the production of IFNß1 and TNF-? via the sensing of DNA released into the cytosol after irradiation in B16F10 melanoma cells, which could be involved in the bystander and abscopal effects of radiation.
COBISS.SI-ID: 3013755