X Ma1, H E Bazan. 1. Department of Ophthalmology and Neuroscience Center, LSU Health Sciences Center, New Orleans, Louisiana 70112, USA.
Abstract
PURPOSE: To examine the role of platelet-activating factor (PAF) on apoptosis of corneal epithelial cells exposed to radiation. METHODS: Rabbit corneal epithelial (RCE) and human corneal epithelial (HCE) cells were exposed to UVC radiation and then to carbamyl PAF (cPAF) for different increments of time. The PAF antagonist BN50739 was added 30 min before cPAF. The caspase inhibitors Ac-DEVD-CHO and Ac-YVAD-CHO were added 1 h before, and the phospholipase A(2) (PLA(2)) inhibitor MAFP was added 3 h before UVC irradiation. FITC-dUTP TUNEL and DAPI staining were performed to assess the percentage of apoptotic cells. DNA ladder analysis was used to investigate apoptosis induced by different intensities of UVC (50-600 J/m(2)) with or without cPAF. Caspase activation and release of cytochrome c from mitochondria to cytosol were determined by Western blot. RESULTS: While only 2.7% of RCE cells were DAPI positive in controls incubated for 12 h, 44% of cells were stained positive 4 h after irradiation; these values increased to 63% in the presence of cPAF. Cells incubated with cPAF alone were similar to controls. TUNEL staining and DNA laddering showed also increased in apoptosis after PAF treatment of UV-irradiated cells and BN50739 blocked the effect of cPAF. cPAF increased caspase-3 activation induced by UV irradiation in HCE cells. Cytochrome c release from mitochondria to cytosol was observed 30 min after irradiation. cPAF almost doubled the release of cytochrome c at 30 min and 1 h. Here, too, BN50739 blocked the PAF effect. No release of cytochrome c by PAF was seen in non-irradiated cells, even at higher concentrations. MAFP caused a decrease in cytochrome c release from UV-treated cells, and caused an even greater inhibition of cytochrome c release in cells stimulated with PAF. CONCLUSIONS: PAF increases RCE and HCE apoptosis caused by UV irradiation by stimulating PLA(2), producing an early release of cytocrome c from mitochondria and activating caspase-3 by a receptor-mediated mechanism. This accelerating effect of PAF on the apoptotic cascade only occurred when corneal epithelial cells had been previously damaged by UV radiation.
PURPOSE: To examine the role of platelet-activating factor (PAF) on apoptosis of corneal epithelial cells exposed to radiation. METHODS:Rabbit corneal epithelial (RCE) and human corneal epithelial (HCE) cells were exposed to UVC radiation and then to carbamyl PAF (cPAF) for different increments of time. The PAF antagonist BN50739 was added 30 min before cPAF. The caspase inhibitors Ac-DEVD-CHO and Ac-YVAD-CHO were added 1 h before, and the phospholipase A(2) (PLA(2)) inhibitor MAFP was added 3 h before UVC irradiation. FITC-dUTP TUNEL and DAPI staining were performed to assess the percentage of apoptotic cells. DNA ladder analysis was used to investigate apoptosis induced by different intensities of UVC (50-600 J/m(2)) with or without cPAF. Caspase activation and release of cytochrome c from mitochondria to cytosol were determined by Western blot. RESULTS: While only 2.7% of RCE cells were DAPI positive in controls incubated for 12 h, 44% of cells were stained positive 4 h after irradiation; these values increased to 63% in the presence of cPAF. Cells incubated with cPAF alone were similar to controls. TUNEL staining and DNA laddering showed also increased in apoptosis after PAF treatment of UV-irradiated cells and BN50739 blocked the effect of cPAF. cPAF increased caspase-3 activation induced by UV irradiation in HCE cells. Cytochrome c release from mitochondria to cytosol was observed 30 min after irradiation. cPAF almost doubled the release of cytochrome c at 30 min and 1 h. Here, too, BN50739 blocked the PAF effect. No release of cytochrome c by PAF was seen in non-irradiated cells, even at higher concentrations. MAFP caused a decrease in cytochrome c release from UV-treated cells, and caused an even greater inhibition of cytochrome c release in cells stimulated with PAF. CONCLUSIONS:PAF increases RCE and HCE apoptosis caused by UV irradiation by stimulating PLA(2), producing an early release of cytocrome c from mitochondria and activating caspase-3 by a receptor-mediated mechanism. This accelerating effect of PAF on the apoptotic cascade only occurred when corneal epithelial cells had been previously damaged by UV radiation.
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