Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.     

Robust lanthanide-based assays for the detection of anti-apoptotic Bcl-2-family protein antagonists

Anti-apoptotic Bcl-2-family proteins (Bcl-2, Bcl-x(L), Bfl-1, Mcl-1, Bcl-W and Bcl-B) have been recently validated as drug discovery targets for cancer, owed to their ability to confer tumor resistance to chemotherapy or radiation. The anti-apoptotic activity of Bcl-2 proteins is due to their ability to heterodimerize with their pro-apoptotic counterparts (proteins such as Bad, Bim or Bid) via a conserved peptide region termed BH3. Thus, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Here, we report on the development and evaluation of two novel Lanthanide-based assays that are formatted for high-throughput screening of small molecules capable of antagonizing BH3-Bcl-2 interactions. The assay conditions, robustness and reproducibility (Z' factors) are described. These assays represent useful tools to enable further studies in the search for novel, safe and effective anti-cancer agents targeting Bcl-2-family proteins.     

Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.     

Characterization of the signal that directs Tom20 to the mitochondrial outer membrane

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.     

Apoptosis modulatory activities of transiently expressed Bcl-2: Roles in cytochrome c release and Bax regulation

Bcl-2 and Bcl-X_L are pro-survival members of the Bcl-2 family. These proteins have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival through blocking Bax translocation from the cytosol to mitochondria and by preventing the release of cytochrome c. However, it has been recently reported that transiently expressed Bcl-2 unexpectedly leads to significant cell toxicity. To study this intriguing phenomenon, we have carried out further analyses into the properties of transiently expressed Bcl-2. We found that various isoforms of human and different species of Bcl-2 were equally capable of inducing apoptosis. In addition, we discovered that transient expression of Bcl-2, unlike its pro-survival homolog Bcl-X_L, can lead to the release of cytochrome c from mitochondria and that the resulting cell death can be inhibited by caspase and calpain inhibitors. Moreover, we have shown that unlike the pro-apoptotic protein Bid, the toxicity associated with the transient expression of Bcl-2 occurs independent of the activity of the endogenous Bax. Finally, we found that in spite of its intrinsic toxicity, transiently expressed Bcl-2 is fully capable of blocking the ectopically expressed Bax from localizing to mitochondria. Taken together, these studies demonstrate that transiently expressed Bcl-2 displays opposing functional properties.     

Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane

The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane.     

Bcl-x(L) overexpression delays the onset of autophagy and apoptosis in hyperosmotic recombinant Chinese hamster ovary cell cultures

Hyperosmolality in recombinant Chinese hamster ovary (rCHO) cell cultures induces autophagy and apoptosis. To investigate the effect of Bcl-x_L overexpression on autophagy and apoptosis in hyperosmotic rCHO cell cultures, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x_L overexpression was subjected to hyperosmolality resulting from NaCl addition in a batch culture and nutrient supplementation in a fed-batch culture. In the batch culture, Bcl-x_L overexpression suppressed apoptosis, as evidenced by a decreased amount of cleaved caspase-7 and PARP. Concurrently, Bcl-x_L overexpression also delayed autophagy, as indicated by reduced LC3 conversion, from LC3-I to LC3-II. As a result, the cell viability and EPO production were improved by Bcl-x_L overexpression. In the fed-batch culture, the simultaneous application of Bcl-x_L overexpression and nutrient feeding increased the culture longevity and maximum EPO concentration. Taken together, Bcl-x_L overexpression delayed autophagy and apoptosis in hyperosmotic rCHO cell cultures, resulting in increased EPO production.     

Bcl-2 inhibits apoptosis induced by mitochondrial uncoupling but does not prevent mitochondrial transmembrane depolarization

Bcl-2 overexpression protects cells from apoptosis induced by many cytotoxic agents. In this study, we investigated the effects of uncoupling mitochondrial electron transport in both HL60 wild-type and Bcl-2-overexpressing cells using the protonophore carbonyl cyanide m-chlorophenylhydrazone. We found that uncoupling mitochondrial electron transport induced apoptosis in wild-type, but not in Bcl-2-overexpressing cells. To investigate the mechanism of action of Bcl-2-mediated inhibition of cyanide m-chlorophenylhydrazone-induced apoptosis, we measured the mitochondrial transmembrane potential (DeltaPsi(m)) after uncoupling mitochondrial electron transport and found that both HL-60 wild-type and Bcl-2-overexpressing cells similarly depolarize following cyanide m-chlorophenylhydrazone exposure. Western blot analysis demonstrated that Bcl-2 overexpression did not completely block cytochrome c release from mitochondria after uncoupling mitochondrial electron transport. Since Bcl-2 may act as an antioxidant, we studied the effect of altering the cellular redox state prior to uncoupling mitochondrial electron transport in Bcl-2-overexpressing cells. Depletion of mitochondrial (but not cytosolic) glutathione induced apoptosis in Bcl-2-overexpressing cells and negated the protective effect of Bcl-2. Furthermore, following glutathione depletion, Bcl-2-overexpressing cells were sensitized to undergo cyanide m-chlorophenylhydrazone-induced apoptosis. These data suggest that the action of Bcl-2 is dependent, in part, on the cellular and mitochondrial redox state.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制

Bcl-2家族蛋白在调控线粒体功能和细胞色素C释放中起重要作用。最近发现Bcl-2分子通过与其他促凋亡分子相互作用调控线粒体外膜通透性,其具体分子机制尚不完全清楚。本课题组采用化学生物学方法,在研究Bax/Bak非依赖的细胞凋亡途径中,发现了一些小分子化合物能够诱导Bim表达量急剧升高,Bim能转位到线粒体上,与Bcl-2相互作用增强,并直接促进Bcl-2构象变化。有意义的是,Bim可以诱导Bcl-2功能发生转换并能够形成大的复合体通道来介导细胞色素C释放。研究结果提示Bcl-2分子可变成促凋亡分子,参与Bax/Bak非依赖的细胞色素C释放和细胞凋亡。     

Bcl-x(S) induces an NGF-inhibitable cytochrome c release

Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L)

How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.     

Regulation of Bcl-2 proteins and of the permeability of the outer mitochondrial membrane

In many apoptotic responses, pro-apoptotic members of the Bcl-2 family trigger the permeabilization of the outer mitochondrial membrane, thereby allowing the release of mitochondrial apoptogenic factors that contribute to caspase activation in the cytosol. The mechanisms that lead to the activation of pro-apoptotic Bcl-2 family members and to the permeabilization of the outer mitochondrial membrane are not yet completely understood. Here, we attempt to summarize our current view of the mechanisms that lead to these events, regarding both additional proteins that were recently suggested to be involved, and the roles of lipids.     

Adenovirus-mediated transfer of Bcl-X(L) protects neuronal cells from Bax-induced apoptosis

Bax-mediated apoptosis in neurons is involved in many pathologic conditions affecting the central nervous system, including degenerative diseases, stroke, and trauma. Two molecules belonging to the Bcl-2 family, Bcl-2 and Bcl-X(L), protect cells from Bax-induced apoptosis and show distinct expression patterns in adult neurons, with downregulated Bcl-2 and highly upregulated Bcl-X(L) expression. To investigate the biological functions of these two molecules in Bax-mediated apoptosis in neurons, we transduced various levels of Bcl-X(L) or Bcl-2 via adenoviral vectors into nerve growth factor (NGF)-treated PC12 cells. Overexpression of Bax induced drastic apoptosis in NGF-treated PC12 cells. Bcl-X(L) expressed at a wide range of levels conferred a high level of protection against Bax-mediated apoptosis. In contrast, Bcl-2 at various levels conferred far less protection against apoptosis. Moreover, Bcl-X(L) protected PC12 cells from apoptosis induced by NGF withdrawal. These data indicate that Bcl-X(L)-mediated protection is the major pathway that suppresses apoptosis in NGF-treated PC12 cells and that Bcl-X(L) would be a more relevant target of manipulation in future treatment strategies, including gene therapies.     

Structure-based discovery of a new class of Bcl-xL antagonists

Apoptosis, or programmed cell death, plays a key role in normal tissue homeostasis ensuring a proper balance between cell production and cell loss. Anti-apoptotic Bcl-2-family proteins are central regulators of the apoptotic pathway and due to their ability to confer tumor resistance to chemotherapy or radiation, have been recently validated as targets for cancer drug discovery. Since the crucial interaction between pro- and anti-apoptotic members occurs via a conserved region located on the surface of the protein, a viable way to inhibit the anti-death activity of Bcl-2 proteins is to design small molecule inhibitors that occupy this cavity. Here, we describe a structure-based approach that led to the identification of four small molecule inhibitors directed at the hydrophobic groove on the surface of the Bcl-2 family protein Bcl-xL. The compounds were characterized in a number of assays including in vitro binding using 15N-labeled protein, a displacement DELFIA assay, and a cell-based viability assay with human cancer cells.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

A New Assay Based on Fluorescence Resonance Energy Transfer to Determine the Binding Affinity of Bcl-xL Inhibitors

We developed a new assay of Bcl-x_L inhibitors based on fluorescence resonance energy transfer that occurs between an AEDANS-labeled Bak-BH3 peptide and three tryptophans in the BH1 and BH2 domains of Bcl-x_L. The method can tolerate up to 5% DMSO, and it was validated with several Bcl-x_L inhibitors. It can be adapted to screen for compounds targeting other Bcl-2 family proteins.     

TAT-mediated endocytotic delivery of the loop deletion Bcl-2 protein protects neurons against cell death

Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Deltaloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Deltaloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Deltaloop occurs at 37 degrees C but not at 4 degrees C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Deltaloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.     

Deficiency of pro-apoptotic Hrk attenuates programmed cell death in the developing murine nervous system but does not affect Bcl-x deficiency-induced neuron apoptosis

The BCL-2 family includes both pro- and anti-apoptotic proteins, which regulate programmed cell death during development and in response to various apoptotic stimuli. The BH3-only subgroup of pro-apoptotic BCL-2 family members is critical for the induction of apoptotic signaling, by binding to and neutralizing anti-apoptotic BCL-2 family members. During embryonic development, the anti-apoptotic protein BCL-X(L) plays a critical role in the survival of neuronal populations by regulating the multi-BH domain protein BAX. In this study, the authors investigated the role of Harakiri (HRK), a relatively recently characterized BH3-only molecule in disrupting the BAX-BCL-X(L) interaction during nervous system development. Results indicate that HRK deficiency significantly reduces programmed cell death in the nervous system. However, HRK deficiency does not significantly attenuate the widespread apoptosis seen in the Bcl-x (-/-) embryonic nervous system, indicating that other BH3-only molecules, alone or in combination, may regulate BAX activation in immature neurons.