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.

In this retrospective investigation, the combined analysis of BAX and p16(ink4a/CDKN2) shows subgroups in SCC of the esophagus with favorable (p16(ink4a/CDKN2)/BAX high expressing) or poor prognosis (loss of p16(ink4a/CDKN2)/loss of BAX). We suggest that such a multimarker analysis of apoptosis pathways could be useful for individualization of therapeutic strategies in the future, and suggest prospective studies to confirm these results.