Sequence and helicity requirements for the proapoptotic activity of Bax BH3 peptides

Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl-2 and Bcl-XL inhibit apoptosis by binding to proapoptotic proteins such as Bax, thereby preventing chemotherapy-induced or radiation-induced release of cytochrome c from mitochondria and subsequent activation of the caspase protease cascade. Efforts to inhibit Bcl-2 or Bcl-XL function in tumor cells have focused on developing agents to inhibit the interactions of these proteins with proapoptotic proteins. Peptides derived from the BH3 domains of proapoptotic proteins have been shown to disrupt the interactions of Bcl-2 and Bcl-XL with key binding partners in cell-free reactions and to promote cellular apoptosis. However, less is known about the targets of BH3 peptides in intact cells as well as the sequence, length, and conformational requirements for peptide biological activity. In this report, we show that cell-permeable Bax BH3 peptides physically disrupt Bax/Bcl-2 heterodimerization in intact cells and that this disruption correlates with peptide-induced cell death. A point-mutant, control peptide that failed to disrupt intracellular Bax/Bcl-2 interactions also failed to promote apoptosis. To determine important sequence, length, and structural requirements for peptide activity, we generated and systematically analyzed the biological activities of 17 Bax BH3 peptide variants. Peptides were quantitatively examined for their ability to inhibit Bax/Bcl-2 and Bax/Bcl-XL heterodimerization in vitro and to promote cytochrome c release from mitochondria isolated from Jurkat, HL-60, U937, and PC-3 cells. Our results define 15 amino acids as the minimal length required for Bax BH3 peptide biological activity and show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH2 terminal to the core. In addition, circular dichroism spectroscopy revealed that high alpha-helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimization of Bax BH3 peptide as a therapeutic anticancer agent.     

Natural BH3 mimetic (-)-gossypol chemosensitizes human prostate cancer via Bcl-xL inhibition accompanied by increase of Puma and Noxa

Antiapoptotic members of the Bcl-2 family proteins are overexpressed in prostate cancer and are promising molecular targets for modulating chemoresistance of prostate cancer. (-)-Gossypol, a natural BH3 mimetic, is a small-molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 currently in phase II clinical trials as an adjuvant therapy for human prostate cancer. Our objective is to examine the chemosensitization potential of (-)-gossypol in prostate cancer and its molecular mechanisms of action. (-)-Gossypol inhibited cell growth and induced apoptosis through mitochondria pathway in human prostate cancer PC-3 cells and synergistically enhanced the antitumor activity of docetaxel both in vitro and in vivo in PC-3 xenograft model in nude mouse. (-)-Gossypol blocked the interactions of Bcl-xL with Bax or Bad in cancer cells by fluorescence resonance energy transfer assay and overcame the Bcl-xL protection of FL5.12 model cells on interleukin-3 withdrawal. Western blot and real-time PCR studies showed that a dose-dependent increase of the proapoptotic BH3-only proteins Noxa and Puma contributed to the cell death induced by (-)-gossypol and to the synergistic effects of (-)-gossypol and docetaxel. The small interfering RNA knockdown studies showed that Noxa and Puma are required in the (-)-gossypol-induced cell death. Taken together, these data suggest that (-)-gossypol exerts its antitumor activity through inhibition of the antiapoptotic protein Bcl-xL accompanied by an increase of proapoptotic Noxa and Puma. (-)-Gossypol significantly enhances the antitumor activity of chemotherapy in vitro and in vivo, representing a promising new regime for the treatment of human hormone-refractory prostate cancer with Bcl-2/Bcl-xL/Mcl-1 overexpression.     

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.     

The proteasome inhibitor bortezomib sensitizes cells to killing by death receptor ligand TRAIL via BH3-only proteins Bik and Bim

Previously, we showed that the proteasome inhibitor bortezomib/Velcade (formerly PS-341) synergizes with the protein tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL), a ligand for certain death receptors, to induce apoptosis in cell lines derived from prostate and colon cancers. Because apoptosis is often triggered by BH3-only proteins of the Bcl-2 family, we have explored the hypothesis that bortezomib contributes to the apoptosis by up-regulating their levels. Indeed, bortezomib induced increases of Bik and/or Bim in multiple cell lines but not notably of two other BH3-only proteins (Puma and Bid) nor other family members (Bax, Bak, Bcl-2, and Bcl-xL). The increase in Bik levels seems to reflect inhibition by bortezomib of its proteasome-mediated degradation. Importantly, both Bik and Bim seem central to the proapoptotic function of bortezomib, because mouse embryo fibroblasts in which the genes for both Bik and Bim had been disrupted were refractory to its cytotoxic action. Similarly, the synergy between bortezomib and TRAIL in killing human prostate cancer cells was impaired in cells in which both Bik and Bim were down-regulated by RNA interference. Further evidence that bortezomib acts through the mitochondrial pathway regulated by the Bcl-2 family is that deficiency for APAF-1, which acts downstream of Bcl-2, also blocked its apoptotic effect. These results implicate BH3-only proteins, in particular both Bik and Bim, as important mediators of the antitumor action of bortezomib and establish their role in its enhancement of TRAIL-induced apoptosis.     

Chronic lymphocytic leukemia requires BCL2 to sequester prodeath BIM, explaining sensitivity to BCL2 antagonist ABT-737

Antiapoptotic B cell leukemia/lymphoma 2 (BCL2) family proteins are expressed in many cancers, but the circumstances under which these proteins are necessary for tumor maintenance are poorly understood. We exploited a novel functional assay that uses BCL2 homology domain 3 (BH3) peptides to predict dependence on antiapoptotic proteins, a strategy we call BH3 profiling. BH3 profiling accurately predicts sensitivity to BCL2 antagonist ABT-737 in primary chronic lymphocytic leukemia (CLL) cells. BH3 profiling also accurately distinguishes myeloid cell leukemia sequence 1 (MCL1) from BCL2 dependence in myeloma cell lines. We show that the special sensitivity of CLL cells to BCL2 antagonism arises from the requirement that BCL2 tonically sequester proapoptotic BIM in CLL. ABT-737 displaced BIM from BCL2's BH3-binding pocket, allowing BIM to activate BAX, induce mitochondrial permeabilization, and rapidly commit the CLL cell to death. Our experiments demonstrate that BCL2 expression alone does not dictate sensitivity to ABT-737. Instead, BCL2 complexed to BIM is the critical target for ABT-737 in CLL. An important implication is that in cancer, BCL2 may not effectively buffer chemotherapy death signals if it is already sequestering proapoptotic BH3-only proteins. Indeed, activator BH3-only occupation of BCL2 may prime cancer cells for death, offering a potential explanation for the marked chemosensitivity of certain cancers that express abundant BCL2, such as CLL and follicular lymphoma.     

The pro-apoptotic protein,Bik, exhibits potent antitumor activity that is dependent on its BH3 domain

The Bcl-2 homology 3 (BH3) domain is present in most members of the Bcl-2 protein family and is required to confer the death-inducing properties of pro-apoptotic members, including Bax, Bak, Bad, and Bik, in cell-based assay systems. To determine whether the BH3 domain possesses a similar role in tumor tissues in vivo, we overexpressed the wild-type Bik protein and its BH3-deleted counterpart, using adenoviral technology, in chemoresistant human tumor prostate (PC-3) and colon (HT-29) cell lines growing in vitro and in vivo. Bik caused apoptosis in both PC-3 and HT-29 cells in vitro by inducing the release of cytochrome c from mitochondria to cytoplasm, resulting in the catalytic activation of caspases 9, 7, and 3 and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. When the BH3 domain was deleted from the Bik protein, no effect on mitochondrial activity or cell morphology could be observed. Furthermore, intratumoral injection of an adenovirus vector expressing the Bik gene, but not the deleted BH3 Bik gene, suppressed the growth of PC-3 and HT-29 xenografts established in nude mice. Histological examination of tumors from mice treated with the wild-type Bik adenoviral construct demonstrated cellular debris, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive staining, and morphological changes associated with apoptosis. In contrast, tissue sections obtained from tumors treated with the BH3-deleted Bik adenoviral construct showed no evidence of apoptosis. Thus, our results suggest that the BH3 domain is required for the antitumor activity of the Bik protein and provides a novel therapeutic approach for cancer therapy.     

Treatment of B-RAF mutant human tumor cells with a MEK inhibitor requires Bim and is enhanced by a BH3 mimetic

B-RAF is frequently mutated in solid tumors, resulting in activation of the MEK/ERK signaling pathway and ultimately tumor cell growth and survival. MEK inhibition in these cells results in cell cycle arrest and cytostasis. Here, we have shown that MEK inhibition also triggers limited apoptosis of human tumor cell lines with B-RAF mutations and that this effect was dependent on upregulation and dephosphorylation of the proapoptotic, Bcl-2 homology 3–only (BH3-only) Bcl-2 family member Bim. However, upregulation of Bim was insufficient for extensive apoptosis and was countered by overexpression of Bcl-2. To overcome apoptotic resistance, we treated the B-RAF mutant cells both with MEK inhibitors and with the BH3 mimetic ABT-737, resulting in profound synergism and extensive tumor cell death. This treatment was successful because of both efficient antagonism of the prosurvival Bcl-2 family member Mcl-1 by Bim and inhibition of Bcl-2 and Bcl-x_L by ABT-737. Critically, addition of ABT-737 converted the predominantly cytostatic effect of MEK inhibition to a cytotoxic effect, causing long-term tumor regression in mice xenografted with human tumor cell lines. Thus, the therapeutic efficacy of MEK inhibition requires concurrent unleashing of apoptosis by a BH3 mimetic and represents a potent combination treatment for tumors harboring B-RAF mutations.     

The histone deacetylase inhibitor suberic bishydroxamate regulates the expression of multiple apoptotic mediators and induces mitochondria-dependent apoptosis of melanoma cells

Histone deacetylase (HDAC) inhibitors have attracted much interest because of their ability to arrest cell growth, induce cell differentiation, and in some cases, induce apoptosis of cancer cells. In the present study, we have examined a new HDAC inhibitor, suberic bishydroxamate (SBHA), for its effect on a panel of human melanoma cell lines. We report that it induces varying degrees of apoptosis in the melanoma lines but not in melanocytes and fibroblasts. Induction of apoptosis was caspase dependent and was associated with induction of changes in mitochondrial membrane permeability, which could be inhibited by overexpression of Bcl-2. The changes in mitochondria were independent of caspase activation and were associated with changes in conformation of Bax. SBHA down-regulated several key antiapoptotic proteins including X-linked inhibitor of apoptosis and the Bcl-2 family proteins, Bcl-XL and Mcl-1. In contrast, it induced up-regulation of the Bcl-2 family proapoptotic proteins, Bim, Bax, and Bak. In addition, SBHA induced relocation of the protein Bim to mitochondria and its association with Bcl-2. De novo protein synthesis was required for initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide, inhibited SBHA-induced conformational changes in Bax as well as changes in mitochondrial membrane permeability and activation of caspase-3. These results suggest that SBHA induces apoptosis by changing the balance between proapoptotic and antiapoptotic proteins in melanoma cells. The protein Bim may be a key initiator of apoptosis in cells treated with SBHA.     

Apoptosis induction by 1alpha,25-dihydroxyvitamin D3 in prostate cancer

Calcitriol [1alpha,25-dihydroxyvitamin D3] is the natural ligand of the vitamin D receptor (VDR). Using cultured prostate cancer (PC) cell lines, LN-CaP and ALVA-31, we studied the effects of 1alpha,25(OH)2-Vitamin D3 (VD3) on expression of several apoptosis-regulating proteins including: (a) Bcl-2 family proteins (Bcl-2, Bcl-X(L), Mcl-1, Bax, and Bak); (b) the heat shock protein 70-binding protein BAG1L; and (c) IAP family proteins (XIAP, cIAP1, and cIAP2). VD3 induced decreases in levels of antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1, BAG1L, XIAP, cIAP1, and cIAP2 (without altering proapoptotic Bax and Bak) in association with increases in apoptosis. In contrast to VDR-expressing LN-CaP and ALVA-31 cells, VDR-deficient prostate cancer line Du-145 demonstrated no changes in apoptosis protein expression after treatment with VD3. In sensitive PC cell lines, VD3 activates downstream effector protease, caspase-3, and upstream initiator protease caspase-9, the apical protease in the mitochondrial ("intrinsic") pathway for apoptosis, but not caspase-8, an initiator caspase linked to an alternative ("extrinsic") apoptosis pathway triggered by cytokine receptors. VD3 induced declines in antiapoptotic proteins and also stimulated cytochrome c release from mitochondria by a caspase-independent mechanism. Moreover, apoptosis induction by VD3 was suppressed by overexpressing Bcl-2, a known blocker of cytochrome c release, whereas the caspase-8 suppressor CrmA afforded little protection. Thus, VD3 is capable of inhibiting expression of multiple antiapoptotic proteins in VDR-expressing prostate cancer cells, leading to activation of the mitochondrial pathway for apoptosis.     

BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/BAK

BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-X_L with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK.     

Destruction of tumor vasculature and abated tumor growth upon VEGF blockade is driven by proapoptotic protein Bim in endothelial cells

For malignant growth, solid cancers must stimulate the formation of new blood vessels by producing vascular endothelial growth factor (VEGF-A), which is required for the survival of tumor-associated vessels. Novel anticancer agents that block VEGF-A signaling trigger endothelial cell (EC) apoptosis and vascular regression preferentially within tumors, but how the ECs die is not understood. In this study, we demonstrate that VEGF-A deprivation, provoked either by drug-induced tumor shrinkage or direct VEGF-A blockade, up-regulates the proapoptotic BH3 (Bcl-2 homology 3)-only Bcl-2 family member Bim in ECs. Importantly, the tumor growth inhibitory activity of a VEGF-A antagonist required Bim-induced apoptosis of ECs. These findings thus reveal the mechanism by which VEGF-A blockade induces EC apoptosis and impairs tumor growth. They also indicate that drugs mimicking BH3-only proteins may be exploited to kill tumor cells not only directly but also indirectly by ablating the tumor vasculature.     

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway sensitizes MDA-MB468 human breast cancer cells to cerulenin-induced apoptosis

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.     

BH3 domains as BCL-2 inhibitors: prototype cancer therapeutics

It is likely that most, if not all, cancer cells possess a mechanism for opposing programmed cell death, or apoptosis. One mechanism often used by cancer cells to escape apoptosis is the overexpression of B cell leukaemia/lymphoma-2 protein (BCL-2) or related antiapoptotic proteins. Inhibiting BCL-2, therefore, is an anticancer strategy worthy of attention. BCL-2 homology 3 (BH3) domains are α-helical segments found in BCL-2 family member proteins. In pro-apoptotic members, the BH3 domain is necessary for pro-apoptotic function. It has been shown that short peptides derived from the BH3 region possess intrinsic pro-death activity. Furthermore, certain peptides of this group exert their pro-death function by specifically binding BCL-2 and opposing BCL-2’s antideath function. Therefore, BH3 domains are prototype BCL-2 inhibitors. Mimetics of BH3 domains, whether derived from peptides or small molecules, have promise as cancer therapeutics.     

BH3 domains as BCL-2 inhibitors: prototype cancer therapeutics

It is likely that most, if not all, cancer cells possess a mechanism for opposing programmed cell death, or apoptosis. One mechanism often used by cancer cells to escape apoptosis is the overexpression of B cell leukaemia/lymphoma-2 protein (BCL-2) or related antiapoptotic proteins. Inhibiting BCL-2, therefore, is an anticancer strategy worthy of attention. BCL-2 homology 3 (BH3) domains are alpha-helical segments found in BCL-2 family member proteins. In pro-apoptotic members, the BH3 domain is necessary for pro-apoptotic function. It has been shown that short peptides derived from the BH3 region possess intrinsic pro-death activity. Furthermore, certain peptides of this group exert their pro-death function by specifically binding BCL-2 and opposing BCL-2's antideath function. Therefore, BH3 domains are prototype BCL-2 inhibitors. Mimetics of BH3 domains, whether derived from peptides or small molecules, have promise as cancer therapeutics.     

Loss of the BH3-only protein Bmf impairs B cell homeostasis and accelerates gamma irradiation-induced thymic lymphoma development

Members of the Bcl-2 protein family play crucial roles in the maintenance of tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Proapoptotic BH3-only members of the Bcl-2 family are essential for initiation of cell death, and they function by activating the proapoptotic Bcl-2 family members Bax and/or Bak, either directly or indirectly through binding to prosurvival Bcl-2 family members. Bax and Bak then elicit the downstream events in apoptosis signaling. Mammals have at least eight BH3-only proteins and they are activated in a stimulus-specific, as well as a cell type-specific, manner. We have generated mice lacking the BH3-only protein Bcl-2-modifying factor (Bmf) to investigate its role in cell death signaling. Our studies reveal that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV irradiation. Remarkably, loss of Bmf protected lymphocytes against apoptosis induced by glucocorticoids or histone deacetylase inhibition. Moreover, bmf(-/-) mice develop a B cell-restricted lymphadenopathy caused by the abnormal resistance of these cells to a range of apoptotic stimuli. Finally, Bmf-deficiency accelerated the development of gamma irradiation-induced thymic lymphomas. Our results demonstrate that Bmf plays a critical role in apoptosis signaling and can function as a tumor suppressor.     

Molecular targeting of BCL2 and BCLXL proteins by synthetic BCL2 homology 3 domain peptide enhances the efficacy of chemotherapy

Chemotherapeutic agents are known to induce programmed cell death or apoptosis. The activation of cellular antiapoptotic defense that prevents the translation of drug-induced damage into cell death is the key factor in cellular antiapoptotic resistance that decreases the chemotherapeutic effectiveness of a broad spectrum of anticancer drugs. A novel proapoptotic anticancer drug delivery system (DDS) was designed to simultaneously induce apoptosis and suppress antiapoptotic cellular defense. The system includes three main components: 1) anticancer drug camptothecin (CPT) as an apoptosis inducer, 2) synthetic BCL2 homology 3 domain (BH3) peptide as a suppressor of cellular antiapoptotic defense, and 3) poly(ethylene glycol) (PEG) polymer as a carrier. The above DDS was studied in vitro using A2780 human ovarian carcinoma cells and in vivo on nude mice bearing xenografts of human ovarian tumor. The results obtained in both series of experiments corroborate each other. They show that the designed DDS provided intracellular delivery of active components and suppressed cellular antiapoptotic defense, leading to the more pronounced induction of caspase-dependent signaling pathway of apoptosis compared with CPT alone and simple CPT-PEG conjugate. Including BH3 peptide in complex DDS decreased apoptotic cellular defense, substantially increased toxicity of the whole complex, and provided high antitumor activity. Therefore, the proposed novel multicomponent proapoptotic anticancer drug delivery system has high potential to enhance the efficacy of chemotherapy.     

Therapeutic potential of a peptide targeting BCL-2 cell guardians in cancer

A promising approach to cancer therapy is to elicit apoptosis with "BH3 mimetic" drugs, which target proteins of the BCL-2 family. As of yet, however, such drugs can target only certain BCL-2 family proteins. Hence, in this issue of the JCI, LaBelle et al. assess instead the therapeutic potential of a "stapled" BH3 peptide from the BIM protein, which inactivates all its prosurvival relatives. The peptide killed cultured hematologic tumor cells and abated growth of a leukemia xenograft, without perturbing the hematopoietic compartment. Hence, such peptides might eventually provide a new way to treat refractory leukemias.     

Adenoviral natural born killer gene therapy for malignant glioma

Glioblastoma is a lethal neoplasm resistant to conventional radiotherapy and chemotherapy. Natural born killer (NBK), also known as Bcl-2-interacting killer (BIK), is a death-promoting Bcl-2 family protein sharing with Bcl-2 only the Bcl homology 3 (BH3) domain. We here report that an adenoviral vector encoding NBK (Ad-NBK) uniformly induces cell death in 12 human malignant glioma cell lines. Ad-NBK-induced cell death involves neither quantitative mitochondrial cytochrome c release nor caspase 8, 9, 7, or 3 processing and is unaffected by the viral caspase inhibitor, cytokine response modifier A (CRM-A), or selective caspase 8 or 9 inhibitors. In contrast, Ad-NBK-induced cell death is inhibited by the broad-range caspase inhibitor, zVAD-fmk, or by adenoviral gene transfer of the X-linked inhibitor of apoptosis protein (XIAP). Further, Ad-NBK-induced cell death is inhibited by Bcl-2 or Bcl-xL gene transfer. Interestingly, Bcl-2- and Bcl-xL-transfected glioma cells, which are partially protected from Ad-NBK-induced cell death, accumulate much higher levels of NBK than are ever observed in control-infected cells. This indicates that complex formation with Bcl-2 or Bcl-xL sequesters NBK in an inactive form and that free NBK, rather than an NBK-mediated depletion of free antiapoptotic Bcl-2 family proteins, is the proximate mediator of Ad-NBK-induced cell death. Conversely, proteasome inhibition-mediated accumulation of NBK strongly enhances Ad-NBK-induced cell death. Finally, Ad-NBK-infected LN-229 glioma cells are not tumorigenic in nude mice. Thus Ad-NBK triggers an XIAP- and zVAD-fmk-sensitive cell death pathway in glioma cells with potential therapeutic value, provided that NBK expression can be selectively targeted to cancer cells.