Analysis of a novel cytotoxic drug induced TRAIL-R2 signalling complex using a new FACS-based Smac/DIABLO release assay

The induction of programmed cell death (apoptosis) in tumour cells is an essential cellular and molecular process in response to cancer treatment and important for its success. Classically two forms of apoptosis have been distinguished, the extrinsic and the intrinsic apoptosis. Recently novel mechanisms have been proposed that blur the lines between the two apoptosis pathways. One of these novel mechanisms has been proposed by the Mohr/Zwacka laboratory. It involves a new death receptor-independent caspase-8 inducing complex and modulation by the TRAIL-R2 (DR5). The DR5 modulation appears to be mediated by JNK activation, up regulation of a phosphatase and subsequent dephosphorylation of Bid. Dephosphorylated Bid can be cleaved by active caspase-8 and can then convey the apoptotic signal onto mitochondria, which respond with the release of proapoptotic factors such as cytochrome c and Smac/DIABLO into the cytosol. As the release of Smac/DIABLO is important in apoptosis and possibly impaired in DR5-silenced cells, we developed a FACS based Smac/DIABLO release assay in 5-Flurouracil (5-FU) stimulated HCT116 cancer cells. Using this assay we found that DR5 knock-down cells show a markedly reduced Smac/DIABLO release, which corresponds to decreased mitochondrialmembrane depolarisation in these cells. These mechanistic differences can explain the significantly reduced apoptosis in 5-FU treated DR5 silenced HCT116 cells. Moreover, this FACS method is a useful tool to understand apoptosis signalling in cancer cells