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FADD Deficiency Causes Changes in Apoptosis and Necroptosis of Mouse Embryonic Fibroblasts

Introduction: Necrosis, like apoptosis, can be strictly regulated and that form of necrosisis called necroptosis. FADD (Fas-associateddeathdomain)protein is a key molecule of extrinsic apoptotic pathway that transduces signal from the membrane death receptors to caspase 8, but it also plays pivotal role in activation of necroptosis. UV irradiation triggers intrinsic apoptotic pathway via DNA damage and caspase 9 and extrinsic pathway via death receptor trimerization and caspase 8. Material and Methods: The effect of FADD deficiency on cell survival and activation of cell death was investigated using knock-out mouse embryonic fibroblasts (FADD-/-) irradiated with different doses of UVB radiation. Cell viability was estimated using MTT test, caspase activity using commercial assays and detection of necroptosis using MTT test with necrostatin-1. Results and Discussion: Results showed that FADD-/- fibroblasts have lower proliferation rate than wild-type cells as their viability was reduced in comparison to wild-type cells, following the exposure to UVB radiation at intensity range 100–600 J/m2. Increased activation of caspases 3/7 and caspase 9 was detected in the irradiated FADD-/- fibroblasts and these cells did not have an increased viability in the presence of necrostatin-1 in comparison to the wild type. Caspase 8 activation was not detected in either cell type after the exposure to 300J/m2 of UVB. Conclusion: From results we can conclude that UVB radiation in FADD-/- fibroblasts causes stronger induction of apoptosis due to intrinsic pathway activation. Necroptosis can be excluded as the cause of increased UV sensitivity of FADD-/- cells. However, cell death of wild type fibroblasts seems to be partly due to apoptosis and partly due to necroptosis. Lack of caspase 8 activation indicates that the extrinsic pathway of apoptosis is not involved in UVB-induced apoptosis of mouse embryonic fibroblasts.

UVB; necroptosis; apoptosis

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