Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-speci®c BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a singlevector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-de®cient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wildtype HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x L and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not suf®cient to confer sensitivity to Nbk.

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramidemediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct e ect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramideinduced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This in¯uence of Bax on ceramide-induced apoptosis was con®rmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C 2 -ceramide, sensitivity of Bax knock-out HCT116 cells was signi®cantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.

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