UVC-Induced Apoptosis in Drosophila Cells

Abstract

Background
Experiments in mammalian cells suggested that ceramide acts as a signaling molecule in radiation-induced apoptosis, however, it is not well understood how the ceramide synthesis is regulated.
Methods
Quantitative analysis of ceramide: Lipids were extracted from Schneider line 2 (SL2) cells according to Bligh & Dyer method and analyzed by Thin-Layer Chromatography. Caspase 3/7 assay: DEVD-luciferin and luciferase was added to the cell lysate followed by measurement with luminometer.
RNA interference experiment: Double strand (ds) RNA was added to SL2 and incubated for 3 days to allow for turnover of the protein. To induce apoptosis cells were irradiated with UV-C.
Results
Three-fold increase in ceramide was observed 5 hours after irradiation. Cell permeable ceramide, C2 ceramide, can mimic the effect of irradiation and induces Caspase 3/7 activity. Overexpression of ceramidase that hydrolyzes ceramide causes reduction of Caspase 3/7 activity. In RNA interference experiments, Caspase 3/7 activity is decreased only by adding the ds RNA for neutral sphingomyelinase homolog. Other ds RNAs including acid sphingomyelinase homolog, ceramide synthase, and sphingolipid Δ4-desaturase have no significant effect.
Conclusion
In SL2, ceramide have an important role in radiation-induced apoptosis and neutral sphingomyelinase homolog might affect the regulation of ceramide synthesis.