Defects in apoptosis underpin both tumorigenesis and drug resistance, and because of these defects chemotherapy often fails. Understanding the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death, provides a paradigm to explain the relationship between cancer genetics and treatment sensitivity and should enable a more rational approach to anticancer drug design and therapy.

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.

The essential upstream steps in granzyme B–mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B–resistant Bcl-2–overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B–treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.