A non-death function of the mitochondrial apoptosis apparatus in immunity.

Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.

Increased Ripk1-mediated bone marrow necroptosis leads to myelodysplasia and bone marrow failure in mice.

Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate Vav CreBaxBakBid triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis.

Bcl-2 family proteins regulate apoptosis by controlling the release of mitochondrial cytochrome c via the Bax/Bak channel. However, recent studies have also implicated several members of this family in the regulation of mitochondrial fission/fusion dynamics. It has been debated whether the role of Bcl-2 proteins in mitochondrial morphogenesis is functionally distinct from their role in apoptosis, with some arguing that Bax/Bak-induced mitochondrial fission promotes apoptosis-associated cytochrome c release, while others suggest that these functions are separable. Here we review this emerging area and argue for a role for the Bcl-2 family as novel regulators of mitochondrial morphogenesis.

Proapoptotic activities of protein disulfide isomerase (PDI) and PDIA3 protein, a role of the Bcl-2 protein Bak.

Protein disulfide isomerase (PDI) family proteins are classified as enzymatic chaperones for reconstructing misfolded proteins. Previous studies have shown that several PDI members possess potential proapoptotic functions. However, the detailed molecular mechanisms of PDI-mediated apoptosis are not completely known. In this study, we investigated how two members of PDI family, PDI and PDIA3, modulate apoptotic signaling. Inhibiting PDI and PDIA3 activities pharmacologically alleviates apoptosis induced by various apoptotic stimuli. Although a decrease of PDIA3 expression alleviates apoptotic responses, overexpression of PDIA3 exacerbates apoptotic signaling. Importantly, Bak, but not Bax, is essential for PDIA3-induced proapoptotic signaling. Furthermore, both purified PDI and PDIA3 proteins induce Bak-dependent, but not Bax-dependent, mitochondrial outer membrane permeabilization in vitro, probably through triggering Bak oligomerization on mitochondria. Our results suggest that both of PDI and PDIA3 possess Bak-dependent proapoptotic function through inducing mitochondrial outer membrane permeabilization, which provides a new mechanism linking ER chaperone proteins and apoptotic signaling.

Role of Bax/Bcl-2 family members in green tea polyphenol induced necroptosis of p53-deficient Hep3B cells.

Green tea polyphenol (GTP) is one of the most promising chemopreventive agent for cancer; it can inhibit cancer cell proliferation and induce apoptosis through p53-dependent cell signaling pathways. Unfortunately, many tumor cells lack the functional p53, and little is known about the effect of GTP on the p53-deficient/mutant cancer cells. To understand the p53-independent mechanisms in GTP-treated p53-dificient/mutant cancer cells, we have now examined GTP-induced cytotoxicity in human hepatoma Hep3B cells (p53-deficient). The results showed that GTP could induce Bax and Bak activation, cytochrome c release, caspase activation, and necroptosis of Hep3B cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore (MPTP), were interdependently activated by GTP, with translocation and homo-oligomerization on the mitochondria. Bax and Bak induce cytochrome c release. Importantly, cytochrome c release and necroptosis were diminished in Hep3B cells (Bax(-/-)) and Hep3B cells (Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate GTP-induced cytochrome c release and necroptosis. Together, the findings suggested that GTP-induced necroptosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

BH3-only activator proteins Bid and Bim are dispensable for Bak/Bax-dependent thrombocyte apoptosis induced by Bcl-xL deficiency: molecular requisites for the mitochondrial pathway to apoptosis in platelets.

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.

Oxaliplatin sensitizes human colon cancer cells to TRAIL through JNK-dependent phosphorylation of Bcl-xL.

BACKGROUND & AIMS: Oxaliplatin sensitizes drug-resistant colon cancer cell lines to tumor necrosis factor-related apoptosis inducing ligand (TRAIL), a death receptor ligand that is selective for cancer cells. We investigated the molecular mechanisms by which oxaliplatin sensitizes cancer cells to TRAIL-induced apoptosis. METHODS: We incubated the colon cancer cell lines HT29 and V9P, which are resistant to TRAIL, with TRAIL or with oxaliplatin for 2 hours, followed by TRAIL. Annexin V staining was used to measure apoptosis; RNA silencing and immunoblot experiments were used to study the roles of apoptosis-related proteins. Site-directed mutagenesis experiments were used to determine requirements for phosphorylation of Bcl-xL; co-immunoprecipitation experiments were used to analyze the interactions among Bcl-xL, Bax, and Bak, and activation of Bax. RESULTS: Oxaliplatin-induced sensitivity to TRAIL required activation of the mitochondrial apoptotic pathway; reduced expression of Bax, Bak, and caspase-9, and stable overexpression of Bcl-xL, reduced TRAIL-induced death of cells incubated with oxaliplatin. Mitochondrial priming was induced in cells that were sensitized by oxaliplatin and required signaling via c-Jun N-terminal kinase and phosphorylation of Bcl-xL. Mimicking constitutive phosphorylation of Bcl-xL by site-directed mutagenesis at serine 62 restored sensitivity of cells to TRAIL. Co-immunoprecipitation experiments showed that oxaliplatin-induced phosphorylation of Bcl-xL disrupted its ability to sequestrate Bax, allowing Bax to interact with Bak to induce TRAIL-mediated apoptosis. CONCLUSIONS: Oxaliplatin facilitates TRAIL-induced apoptosis in colon cancer cells by activating c-Jun N-terminal kinase signaling and phosphorylation of Bcl-xL. Oxaliplatin-induced sensitivity to TRAIL might be developed as an approach to cancer therapy.

A role for proapoptotic Bax and Bak in T-cell differentiation and transformation.

Proapoptotic Bax and Bak are the key B-cell lymphoma-2 family members mediating apoptosis through the intrinsic pathway. Cells doubly deficient for Bax and Bak are profoundly resistant to apoptotic stimuli originating from multiple stimuli. Here we describe mice in which Bax and Bak have been deleted specifically in T-cells using Lck-Cre. In these T cell-specific BaxBak-deficient mice, early T-cell progenitors accumulate in the thymus, with relative depletion of more mature T cells. In addition, bone marrow progenitor cells fail to progress to the double positive stage when cultured on OP9 stromal cells expressing the Notch ligand Delta-like 1, consistent with a critical role for Bax and Bak in early T-cell development. Over time, T cell-specific BaxBak-deficient mice progress to an aggressive T-cell lymphoblastic leukemia/lymphoma. Interestingly, quantitative real-time polymerase chain reaction analysis of BaxBak-deficient T-cell lymphomas does not display amplification of the Notch signal transduction pathway, commonly activated in T-cell leukemia in both mouse and man. Bax and Bak, key regulators of the intrinsic pathway of apoptosis, are thus required to prevent T-cell malignancy, and for normal T-cell differentiation, regulating early T-cell development at the stage of early T-lineage progenitor cells.

The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer.

The development of selective inhibitors for discrete anti-apoptotic BCL-2 family proteins implicated in pathologic cell survival remains a formidable but pressing challenge. Such precisely tailored compounds would serve as molecular probes and targeted therapies to study and treat human diseases driven by specific anti-apoptotic blockades. In particular, MCL-1 has emerged as a major resistance factor in human cancer. By screening a library of stabilized alpha-helix of BCL-2 domains (SAHBs), we determined that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. X-ray crystallography and mutagenesis studies defined key binding and specificity determinants, including the capacity to harness the hydrocarbon staple to optimize affinity while preserving selectivity. MCL-1 SAHB directly targets MCL-1, neutralizes its inhibitory interaction with pro-apoptotic BAK and sensitizes cancer cells to caspase-dependent apoptosis. By leveraging nature's solution to ligand selectivity, we generated an MCL-1-specific agent that defines the structural and functional features of targeted MCL-1 inhibition.

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer.

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours.

Inhibition of Bak activation by VDAC2 is dependent on the Bak transmembrane anchor.

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.

Selective roles for antiapoptotic MCL-1 during granulocyte development and macrophage effector function.

During hematopoiesis, myeloid cell leukemia-1 (MCL-1) mediates the survival of bone marrow progenitors and lymphocytes. However, its requirement during myeloid cell differentiation, development, and effector function is less clear. Lineage-specific deletion of MCL-1 in myeloid precursors results in neutropenia due to death during differentiation. The loss of mature neutrophils induced by Mcl-1 deletion was not rescued by genetic deletion of proapoptotic Bim and Puma or by exogenous cytokine treatment. However, blockade of intrinsic apoptosis by lineage-specific deletion of both multidomain proapoptotics Bax and Bak was capable of rescuing the neutropenia associated with Mcl-1 deletion. In the monocytic lineage, despite efficient Mcl-1 deletion, monocytes and macrophages undergo normal development. During the phagocytosis of extracellular bacteria, macrophages concomitantly increase the expression of both MCL-1 and BIM. However, Mcl-1-deficient macrophages exhibit increased sensitivity to death during bacterial phagocytosis that can be abolished by codeletion of Bim. These data suggest that MCL-1 may be necessary to antagonize BIM during macrophage effector responses. Thus, MCL-1 plays selective roles in myeloid development, being required for neutrophil development and setting the threshold for apoptosis during a macrophage effector response.

Vesicular stomatitis virus induces apoptosis primarily through Bak rather than Bax by inactivating Mcl-1 and Bcl-XL.

Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-X(L), which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-X(L). However, inactivation of both Mcl-1 and Bcl-X(L) was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-X(L) indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.

BH3-only protein bid participates in the Bcl-2 network in healthy liver cells.

UNLABELLED: Bcl-2 homology domain 3 (BH3)-only protein Bid is posttranslationally cleaved by caspase-8 into its truncated form (tBid) and couples with stress signals to the mitochondrial cell death pathway. However, the physiological relevance of Bid is not clearly understood. Hepatocyte-specific knockout (KO) of Bcl-xL leads to naturally-occurring apoptosis despite co-expression of Mcl-1, which shares a similar anti-apoptotic function. We generated Bcl-xL KO, Bcl-xL/Bid double KO, Bcl-xL/Bak double KO, Bcl-xL/Bax double KO, and Bcl-xL/Bak/Bax triple KO mice and found that hepatocyte apoptosis caused by Bcl-xL deficiency was completely dependent on Bak and Bax, and surprisingly on Bid. This indicated that, in the absence of Bid, Bcl-xL is not required for the integrity of differentiated hepatocytes, suggesting a complicated interaction between core Bcl-2 family proteins and BH3-only proteins even in a physiological setting. Indeed, a small but significant level of tBid was present in wild-type liver under physiological conditions. tBid was capable of binding to Bcl-xL and displacing Bak and Bax from Bcl-xL, leading to release of cytochrome c from wild-type mitochondria. Bcl-xL-deficient mitochondria were more susceptible to tBid-induced cytochrome c release. Finally, administration of ABT-737, a pharmacological inhibitor of Bcl-2/Bcl-xL, caused Bak/Bax-dependent liver injury, but this was clearly ameliorated with a Bid KO background. CONCLUSION: Bid, originally considered to be a sensor for apoptotic stimuli, is constitutively active in healthy liver cells and is involved in the Bak/Bax-dependent mitochondrial cell death pathway. Healthy liver cells are addicted to a single Bcl-2-like molecule because of BH3 stresses, and therefore special caution may be required for the use of the Bcl-2 inhibitor for cancer therapy.

Apogossypol derivatives as antagonists of antiapoptotic Bcl-2 family proteins.

Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5' substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax-/-bak-/- mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer.

The third model of Bax/Bak activation: a Bcl-2 family feud finally resolved?

Bax and Bak, two functionally similar, pro-apoptotic proteins of the Bcl-2 family, are known as the gateway to apoptosis because of their requisite roles as effectors of mitochondrial outer membrane permeabilization (MOMP), a major step during mitochondria-dependent apoptosis. The mechanism of how cells turn Bax/Bak from inert molecules into fully active and lethal effectors had long been the focal point of a major debate centered around two competing, but not mutually exclusive, models: direct activation and indirect activation. After intensive research efforts for over two decades, it is now widely accepted that to initiate apoptosis, some of the BH3-only proteins, a subclass of the Bcl-2 family, directly engage Bax/Bak to trigger their conformational transformation and activation. However, a series of recent discoveries, using previously unavailable CRISPR-engineered cell systems, challenge the basic premise that undergirds the consensus and provide evidence for a novel and surprisingly simple model of Bax/Bak activation: the membrane (lipids)-mediated spontaneous model. This review will discuss the evidence, rationale, significance, and implications of this new model.

Structural insight into tanapoxvirus-mediated inhibition of apoptosis.

Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double-stranded DNA virus belonging to the poxviridae that causes mild monkeypox-like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl-2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl-2 fold comprising 7 α-helices and utilizes the canonical Bcl-2 binding groove to engage proapoptotic host cell Bcl-2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain-swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain-swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus-mediated inhibition of host cell apoptosis and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. DATABASE: Structural data are available in the Protein Data Bank (PDB) under the accession numbers 6TPQ, 6TQQ and 6TRR.

Cisplatin overcomes Bcl-2-mediated resistance to apoptosis via preferential engagement of Bak: critical role of Noxa-mediated lipid peroxidation.

Increased expression of antiapoptotic Bcl-2 proteins confers therapeutic resistance in various cancer types. Targeting Bcl-2 proteins by small molecules or activating alternative pathways to bypass Bcl-2-mediated protection to promote apoptosis are two approaches to overcoming therapeutic resistance. Here, we show that cisplatin triggers a Bak-dependent pathway to induce apoptosis in Bcl-2-overexpressing MCF-7 cells. p53-mediated induction of Noxa expression, generation of lipid peroxidation end products and induction of Noxa-Mcl-1 interaction are necessary for this pathway to function. Although Puma is also induced by cisplatin treatment, it is not required for apoptosis. Similarly, reactive oxygen species production by cisplatin did not have any effect on cisplatin-induced apoptosis in MCF-7 Bcl-2 cells. Furthermore, p53 promotes cisplatin-induced apoptosis by directly binding and counteracting Bcl-x(L) antiapoptotic function. In conclusion, our findings suggest a novel mode of action for cisplatin to overcome Bcl-2-mediated protection against apoptosis, which requires preferential activation of Bak and p53-mediated upregulation of Noxa protein levels and lipid peroxidation.

BAX and BAK proteins are required for cyclin-dependent kinase inhibitory drugs to cause apoptosis.

In previous reports, we have shown in SH-SY5 cells that olomoucine and roscovitine, two inhibitory drugs of cyclin-dependent kinases, caused apoptosis independent of the extrinsic pathway. In this experimental paradigm, apoptosis was refractory to the protective effects of either Bcl-2 or Bcl-XL overexpression. We are now reporting that the failure of Bcl-XL to prevent dell death was consistent with no effect on the kinetics of caspase activation and cytochrome c release. To further characterize this issue, we have discarded a direct effect of either olomoucine or roscovitine on mitochondrial permeability transition. Moreover, we have evidence that an intrinsic pathway took place in SH-SY5Y cells by showing the mitochondrial translocation of a GFP-Bax construct on transfection and treatment with cyclin-dependent kinase inhibitory drugs. Finally, we tested the effect of olomoucine and roscovitine on wild-type, bax-/-, bak-/-, and double bax-/-bak-/- mouse embryonic fibroblasts (MEF). In wild-type MEFs, both drugs induced cell death by apoptosis in a dose-dependent manner. In bax-/-, bak-/-, and, particularly, double bax-/-bak-/- MEFs, we observed the inhibition of apoptosis. In conclusion, olomoucine and roscovitine caused apoptosis through an intrinsic pathway, with Bax and Bak proteins being involved.