A role for Bid in etoposide-induced apoptosis in Jurkat T-lymphocytes

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The Bcl-2 family of proteins consists of both anti- and pro-apoptotic members. Bid is a ‘BH3-only’ pro-apoptotic member of this family that is known to be important for the activation of Bax and Bak upon death receptor ligation in type II cells, such as Jurkat T-lymphocytes (clone E6.1). In this pathway, Bid undergoes post-translational modification, most often involving caspase-8-mediated processing, to generate a truncated product (tBid). tBid, in turn, promotes the homo-oligomerization of Bax and Bak, which form protein-permeable pores in the outer mitochondrial membrane to allow the release of intermembrane space proteins, such as cytochrome c,into the cytosol. Upon its release, cytochrome c stimulates the recruitment and activation of caspase-9 within the Apaf-1 apoptosome complex. Because our laboratory has also observed Bid cleavage during etoposide-induced apoptosis, it was of particular interest to investigate the extent to which this event is important for Bax and/or Bak activation in response to this antineoplastic agent. To that end, an RNAi approach was used to stably suppress Bid expression in Jurkat T-cells. Not surprisingly, Bid-deficient cells were found to be highly resistant to apoptosis induced by agonistic anti-Fas antibody, exhibiting reduced cytochrome c release and an impaired activation of all procaspases. Strikingly, these cells were also found to be far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-treated Bid-deficient cells failed to activate Bak, release cytochrome c into the cytosol, or undergo loss of mitochondrial membrane potential when compared to control-transfected cells. It is worth noting that the E6.1 clone was found to be deficient in Bax expression. Furthermore, processing of all procaspases was significantly impaired in Bid-deficient cells as compared to control cells. Combined, these findings strongly suggest that Bid plays an important role in the activation of Bak and the subsequent engagement of the mitochondrial pathway during genotoxic stress-induced apoptosis. (Supported by NIH grants K22 ES011647 (to J.D.R.) and P20 RR016475)