Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 Page 8 of 10 (See figure on previous page.) Figure 3 DIABLO mRNA and protein expression. 3a: Boxplots of DIABLO mRNA expression in 3 neuroblastoma datasets (red), adult tumors (blue) and various normal tissues (green). The boxes represent the 25th to 75th percentile with the median depicted as a horizontal line. Extremes are indicated by the whiskers, and the presence of outliers is indicated by (o). 3b: Array CGH of NGP of the region of chromosome 12q24.31 in which DIABLO is located (arrow). Both the B allele frequency (top) and the Log R ratio (bottom) are shown. The chromosome region is shown underneath the picture. 3c: Immunofluorescence of untreated IMR32 cells. Blue is DAPI, red is mitotracker, green is DIABLO antibody. In the right lower corner the merged pictures are shown. 3d: Cell fractionation of SJNB12 cells 24 h after addition of ABT263. The cytoplasmic fraction (cyto) and organelle fraction (organelle) are shown. Blots were incubated with DIABLO, BCL2 and a-tubulin antibodies. 3e: Co-immunoprecipitation of IMR32 (top) and SKNSH (bottom) with BIRC6 and DIABLO antibodies. Negative control was the immunoprecipitation antibody Flag. Also a protein sample without antibody and for every antibody a sample without protein was used as negative control. Both blots were incubated with DIABLO antibody. IP antibodies are indicated above the blots. (Figure 2c). We then investigated whether BIRC6 silen- cing induced apoptosis. Light microscopy shows a decreased cell number and cell death at 72 h after BIRC6 silencing (Figure 2d). This is confirmed by MTT assays, which shows strongly reduced cell viability after transduction with both BIRC6 shRNAs (Figure 2e, dark grey bars). This phenotype is caused by an apoptotic re- sponse demonstrated by PARP cleavage at 72 h after transduction with both BIRC6 shRNAs on western blot (Figure 2f ). These findings confirm an anti-apoptotic role for BIRC6 in neuroblastoma cells. High BIRC6 levels keep cytoplasmic DIABLO levels low The BIRC6 protein functions both by silencing of DIA- BLO and in the formation of the midbody ring during cell division. Inhibition of each of these functions can result in cell death. We therefore investigated which process causes apoptosis after BIRC6 silencing in neuroblastoma. Apop- tosis induced after inhibiting the midbody-related function of BIRC6 has been found to be mediated by CASP2 [7,8,38]. We inhibited CASP2 by ZVDVAD, a widely used CASP2 inhibitor [39-41] that we have previously used to show that apoptosis induced by silencing of BIRC5 is mediated by CASP2 [26]. ZVDVAD however does not in- hibit apoptosis induced by BIRC6 knockdown in SKNSH and IMR32 cells (Figure 2e). This indicates that this process is not CASP2-mediated, implying that the role of BIRC6 in neuroblastoma is not essential for completion of cell division during midbody ring formation. Alternatively, BIRC6 functions as an IAP that binds DIABLO in the cytoplasm thereby inducing ubiquitina- tion and degradation of DIABLO [1,2]. DIABLO mRNA expression levels in neuroblastoma tumors are surpris- ingly high compared to other types of tumors and com- pared to normal tissue (Figure 3a). Moreover, we confirm an amplification of a region on the chromosome 12q arm in the neuroblastoma cell line NGP, which includes the DIABLO locus (Figure 3b) [42]. We there- fore investigated whether the high BIRC6 expression allows neuroblastoma cells to survive the high levels of the pro-apoptotic protein DIABLO. DIABLO is a mito- chondrial protein, which translocates to the cytoplasm after apoptotic stimuli where it can be degraded by BIRC6. We investigated the cellular localization of DIA- BLO and immunofluorescence indeed shows a clear localization of DIABLO within the mitochondria (Figure 3c), which is confirmed by cell fractionation showing that the majority of DIABLO is localized in the cell organelle fraction (Figure 3d). To test whether DIA- BLO can be functionally activated upon an apoptotic stimulus, we treated neuroblastoma cells with the BCL2 inhibitor ABT263, which results in stimulation of pore formation in the mitochondria [43]. Cell fractionation indicates that the cytosolic DIABLO levels increases 24 h after ABT263 treatment (Figure 3d), demonstrating that DIABLO can be released from the mitochondria and stimulate apoptosis. Untreated cells have low cytoplasmic DIABLO levels (Figure 3d). These levels are probably restricted by BIRC6 activity. To test this hypothesis, we first investi- gated by a co-immunoprecipitation analysis whether BIRC6 and DIABLO physically interact in neuroblast- oma cells. Cell lysates of SKNSH and IMR32 cells were immunoprecipitated with a BIRC6 antibody. Western blot analysis of these precipitates with a DIABLO anti- body shows a strong signal at the correct position for DIABLO. This confirms a physical interaction between both proteins (Figure 3e). Finally, we investigated whether silencing of BIRC6 results in increased DIABLO protein levels. Western blot analysis of SKNSH cells treated with two different shRNAs for BIRC6 shows that the silencing of BIRC6 induced by both hairpins results in a clear increase of DIABLO protein levels (Figure 2f ). These experiments suggest that BIRC6 may effectively inactivate cytoplasmic DIABLO in neuroblastoma cells and can thereby prevent an apoptotic response. Discussion and conclusions In this paper we analyzed aberrations in gene copy num- ber and mRNA expression of genes directly involved in the intrinsic apoptotic pathway in neuroblastoma. BIRC6, known as an inhibitor of the pro-apoptotic pro- tein DIABLO, showed gene copy number gains and increased expression. Silencing of BIRC6 with two shRNAs targeting different parts of the coding sequence resulted in increased cytoplasmic DIABLO levels and