Lidija Begović1, Maja Antunovic2, Igor Matic2, Ivana Furcic3, Ana Baricevic4, Valerija Vojvoda Parcina5, Petra Peharec Štefanić2, Biserka Nagy2, Inga Marijanovic2. 1. a Department of Biology , University of J. J. Strossmayer in Osijek , Osijek , Croatia ; 2. b Division of Molecular Biology, Faculty of Science , University of Zagreb , Zagreb , Croatia ; 3. c Institute for Anthropological Research , Zagreb , Croatia ; 4. d Rudjer Boskovic Institute, Center for Marine Research , Rovinj , Croatia ; 5. e Center for Research and Knowledge Transfer in Biotechnology, University of Zagreb , Zagreb , Croatia.
Abstract
PURPOSE: Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell. MATERIALS AND METHODS: Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m(2)) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay. RESULTS: Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells. CONCLUSIONS: Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.
PURPOSE: Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell. MATERIALS AND METHODS: Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m(2)) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay. RESULTS: Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells. CONCLUSIONS: Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.