G0 function of BCL2 and BCL-xL requires BAX, BAK, and p27 phosphorylation by Mirk, revealing a novel role of BAX and BAK in quiescence regulation

BCL2 and BCL-x(L) facilitate G(0) quiescence by decreasing RNA content and cell size and up-regulating p27 protein, but the precise mechanism is not understood. We investigated the relationship between cell cycle regulation and the anti-apoptosis function of BCL2 and BCL-x(L). Neither caspase inhibition nor abrogation of mitochondria-dependent apoptosis by BAX and BAK deletion fully recapitulated the G(0) effects of BCL2 or BCL-x(L), suggesting that mechanisms in addition to anti-apoptosis are involved in the cell cycle arrest function of BCL2 or BCL-x(L). We found that BCL2 and BCL-x(L) expression in bax(-/-) bak(-/-) cells did not confer cell cycle effects, consistent with the G(0) function of BCL2 and BCL-x(L) being mediated through BAX or BAK. Stabilization of p27 in G(0) in BCL2 or BCL-x(L) cells was due to phosphorylation of p27 at Ser(10) by the kinase Mirk. In bax(-/-) bak(-/-) cells, total p27 and p27 phosphorylated at Ser(10) were elevated. Re-expression of BAX in bax(-/-) bak(-/-) cells and silencing of BAX and BAK in wild type cells confirmed that endogenous BAX and BAK modulated p27. These data revealed a novel role for BAX and BAK in the regulation of G(0) quiescence.     

Automatic tumor-stroma separation in fluorescence TMAs enables the quantitative high-throughput analysis of multiple cancer biomarkers

The upcoming quantification and automation in biomarker based histological tumor evaluation will require computational methods capable of automatically identifying tumor areas and differentiating them from the stroma. As no single generally applicable tumor biomarker is available, pathology routinely uses morphological criteria as a spatial reference system. We here present and evaluate a method capable of performing the classification in immunofluorescence histological slides solely using a DAPI background stain. Due to the restriction to a single color channel this is inherently challenging. We formed cell graphs based on the topological distribution of the tissue cell nuclei and extracted the corresponding graph features. By using topological, morphological and intensity based features we could systematically quantify and compare the discrimination capability individual features contribute to the overall algorithm. We here show that when classifying fluorescence tissue slides in the DAPI channel, morphological and intensity based features clearly outpace topological ones which have been used exclusively in related previous approaches. We assembled the 15 best features to train a support vector machine based on Keratin stained tumor areas. On a test set of TMAs with 210 cores of triple negative breast cancers our classifier was able to distinguish between tumor and stroma tissue with a total overall accuracy of 88%. Our method yields first results on the discrimination capability of features groups which is essential for an automated tumor diagnostics. Also, it provides an objective spatial reference system for the multiplex analysis of biomarkers in fluorescence immunohistochemistry.     

A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.     

BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

Conformational Changes in BAK,a Pore-forming Proapoptotic Bcl-2 Family Member,upon Membrane Insertion and Direct Evidence for the Existence of BH3-BH3 Contact Interface in BAK Homo-oligomers

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni²⁺-nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the α5-α6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5–10 Å distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.     

Prognostic value of apoptosis in breast cancer (pT1-pT2). A TUNEL, p53, bcl-2, bag-1 and Bax immunohistochemical study

Apoptosis or programmed cell death produces cells breaking into several fragments of nuclei, cytoplasm or both nuclei and cytoplasm, known as apoptotic bodies which can be visualized in haematoxylin-eosin staining. Some genes (promoters and suppressors) control this process and certain mutations may induce the expression of abnormal proteins, which can be detected by immunohistochemical staining. Apoptosis can be detected by the TUNEL method either identifying apoptotic bodies or cells at the initial stages of the fragmentation process. We have studied 186 cases of infiltrating ductal breast carcinoma, stages pT1-pT2, and analysed the prognostic significance of tumour recurrence and overall survival of apoptotic index (AI) through univariate and multivariate analysis. We have also studied the immunohistochemical protein expression of apoptosis promoter and suppressors gene (p53, nuclear expression; bcl-2 and Bax, cytoplasm expression; BAG-1, nuclear and cytoplasm expression). The results indicate prognostic significance of p53 and bcl-2 related to patient death and bcl-2 and tumour size to tumour recurrence, bcl-2 acting as a protector factor (apoptotic suppressor) in both situations. On the other hand, we have not found useful prognostic information of AI either to tumour recurrence or overall survival in univariate or multivariate studies. In this study, Bax expression does not provide a new prognostic role in breast carcinoma, although it contrasts to the bcl-2 action and accelerates death.     

New tissue microarray technology for analyses of gene expression in frozen pathological samples

Tissue microarrays (TMAs) are widely used to analyze gene expression in multiple pathological samples on a single slide. Currently, most TMA slides are made by coring paraffin-embedded tissues and arraying them into a paraffin block, from which TMA sections are cut. However paraffin-based TMA technology may not be compatible with frozen clinical tissue samples, which have a higher quality of RNAs and proteins for preparing TMAs than paraffin-embedded tissue samples. In this study, we developed an alternative TMA technology that is applicable to a broader range of frozen tissue samples. Our method takes advantage of a newly developed array recipient block that can be used to array small tissue cores. After arraying tissue cores, the tissue block can be immediately sectioned on a cryostat microtome to make TMA slides. TMAs made using this method have well-defined array configurations and good tissue/cell morphology. Immunohistochemistry and in situ hybridization study also revealed well-preserved proteins and mRNAs on TMA slides. Our method significantly simplifies TMA preparation and assembly when frozen pathological tissues are used. Our technology provides an alternative tool for creating high-quality TMAs for the general research community to study gene expressions in pathological samples.     

Mesothelioma Symposium 11.30–12.30 Tuesday 16 September 2003. Cytopathology of malignant mesothelioma

The diagnosis of malignant mesothelioma on the cytology of serous effusions is a two‐phase process. First is to determine that the effusion is malignant based on morphological features such as a highly cellular fluid with many large three dimensional cell aggregates, and/or the recognition of minor malignant criteria including prominent cell engulfment, uniformly present very prominent nucleoli, or the finding of very large (giant) cells. In cell block sections, strong positive staining with EMA often with cell membrane accentuation provides compelling support for a cytological diagnosis of malignancy. Second is to recognize that the malignant cells have a mesothelial phenotype and do not represent metastatic malignancy (usually adenocarcinoma). Criteria in support of mesothelioma include the lack of a ‘two cell’ population, that is one native (mesothelial) and one foreign (metastatic), cells with abundant dense staining cytoplasm, the presence of ‘windows’ where mesothelioma cells lie in close apposition and intracytoplasmic glycogen presenting either as small peripheral vacuoles on MGG stained smears or large yellow refractile crescents on Papanicolaou stained smears. In addition, mesothliomas often possess connective tissue stromal cores occurring as either well‐formed collagen within papillary aggregates or lying free as pink (MGG) or light green (Pap) amorphous material in the background of the smear or in loose association with mesothelioma cells. Finally small orange staining squamous‐like cells can occasionally be identified and sometimes this may be a very prominent finding and has resulted in the false impression of a squamous cell carcinoma. Almost certainly these cells represent apoptotic tumour cells. The connective tissue mucin hyaluronic acid may be found as a net‐like pattern in the smear background or as large hard‐edged magenta‐stained vacuoles on MGG‐stained smears. Cell block sections provide architectural information and it is usually possible to separate mesothelioma aggregates with their cuboidal cells, central nuclei and abundant dense cytoplasm arranged in solid, papillary or hollow clusters from those of adenocarcinoma with less dense, often foamy cytoplasm, often composed of columnar cells with elongated nuclei. Aggregate form in adenocarcinoma can be variable but true acini are a rare finding. These cell block sections provide an ideal medium for histochemistry (PAS with and without diastase digestion) and immunocytochemistry. By using a panel of antibodies (Calretinin and CK 5/6, BerEp4, CEA, B72.3) it is almost always possible to distinguish mesothelioma from metastatic adenocarcinoma. Calretinin and CK 5/6 positive staining and absent staining with BerEp4, CEA and B72.3 is considered diagnostic of mesothelioma.     

Color image analysis of cervical neoplasia using RGB computer color specification

OBJECTIVE: To establish quantitative color image analysis for cytology, red, green and blue (RGB) color specification was applied to Papanicolaou-stained cervical smears. STUDY DESIGN: Cell samples used in this study was those from 300 cervical specimens. We analyzed the color tone of nuclei and cytoplasm of the squamous cells in the cervical smear by means of computer image analysis. RESULTS: Papanicolaou stained nuclei displayed basophilic blue to purple. When they were hyperchromatic and deeply stained, B and G values decreased in value. The RGB values of cytoplasm and nuclei decreased significantly (P < .01) as their degree of cellular atypia increased. CONCLUSION: Using RGB color specification to analyze Papanicolaou-stained cervical smears, a significant difference was perceived in the nucleus and cytoplasm between different groups of squamous cells, from normal, dysplastic and squamous cell carcinoma. These findings may help to establish automated cytology.     

Transient over-expression of barley BAX Inhibitor-1 weakens oxidative defence and MLA12-mediated resistance to Blumeria graminis f.sp. hordei

BAX Inhibitor-1 (BI-1) is a conserved cell death suppressor protein. In barley, BI-1 ( HvBI-1 ) expression is induced upon powdery mildew infection and when over-expressed in epidermal cells of barley, HvBI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis . We co-expressed mammalian pro-apoptotic BAX together with HvBI-1, and the mammalian BAX antagonist BCL-X_L in barley epidermal cells. BAX expression led to cessation of cytoplasmic streaming and collapse of the cytoplasm while co-expression of HvBI-1 and BCL-X_L partially or completely, respectively, rescued cells from BAX lethality. When B. graminis was attacking epidermal cells, a green fluorescent protein fusion of HvBI-1 accumulated at the site of attempted penetration and was also present around haustoria. Over-expression of HvBI-1 in epidermal cells weakened a cell-wall-associated local hydrogen peroxide burst in a resistant mlo -mutant genotype and supported haustoria accommodation in race-specifically resistant MLA12 -barley. HvBI-1 is a cell death regulator protein of barley with the potential to suppress host defence reactions.     

Platinum resistant cancer cells conserve sensitivity to BH3 domains and obatoclax induced mitochondrial apoptosis

Resistance to cisplatin chemotherapy remains a major hurdle preventing effective treatment of many solid cancers. BAX and BAK are pivotal regulators of the mitochondrial apoptosis pathway, however little is known regarding their regulation in cisplatin resistant cells. Cisplatin induces DNA damage in both sensitive and resistant cells, however the latter exhibits a failure to initiate N-terminal exposure of mitochondrial BAK or mitochondrial SMAC release. Both phenotypes are highly sensitive to mitochondrial permeabilisation induced by exogenous BH3 domain peptides derived from BID, BIM, NOXA (which targets MCL-1 and A1), and there is no significant change in their prosurvival BCL2 protein expression profiles. Obatoclax, a small molecule inhibitor of pro-survival BCL-2 family proteins including MCL-1, decreases cell viability irrespective of platinum resistance status across a panel of cell lines selected for oxaliplatin resistance. In summary, selection for platinum resistance is associated with a block of mitochondrial death signalling upstream of BAX/BAK activation. Conservation of sensitivity to BH3 domain induced apoptosis can be exploited by agents such as obatoclax, which directly target the mitochondria and BCL-2 family.     

Requirement of BAX for efficient adenovirus-induced apoptosis

Infection of human epithelial cells with adenoviruses induces an apoptosis paradigm that is efficiently suppressed by the expression of viral E1B-19K protein, which is a functional homolog of the cellular antiapoptosis protein BCL-2. The mechanisms of adenovirus (Ad)-induced apoptosis appear to involve the cellular BCL-2 family proapoptotic proteins. Recent genetic studies with fibroblasts derived from mutant mouse embryos indicate that a class of the BCL-2 family proapoptotic proteins (designated BH-123 or multidomain proteins) such as BAX and BAK constitutes an essential component of the core apoptosis machinery in animal cells. We have examined the role of BAX in Ad-induced apoptosis in human epithelial cells using two colon cancer cell lines, HCT116Bax (Bax(+/-)) and HCT116BaxKO (Bax(-/-)) (L. Zhang, J. Yu, B. H. Park, K. W. Kinzler, and B. Vogelstein, Science 290:989-992, 2000). Infection of Bax(+/-) cells with an Ad type 2 mutant (dl250) defective in expression of the E1B-19K protein resulted in enhanced cytopathic effect, large plaques on cell monolayers, fragmentation of cellular DNA, and enhanced cell death. These mutant phenotypes were not efficiently expressed in Bax(-/-) cells, suggesting that BAX is essential for Ad-induced apoptosis. Infection of Bax(+/-) cells with dl250 induced increased levels of an N-terminally processed form of BAX. Cells infected with the 19K mutant also contained enhanced levels of truncated BAX in membrane-inserted form. Our results suggest that at least a part of the mechanism utilized by E1B-19K to suppress apoptosis during Ad infection may involve modulation of the activities of BAX.     

Construction of a tissue microarray with two millimeters cores of endometrioid endometrial cancer: factors affecting the quality of the recipient block

The tissue microarray (TMA) method currently is not used to render a primary diagnosis of cancer, but its scientific value has been proved in studies of various cancer types. TMA technology still is not used often for uterine tumors, however. We investigated the repeatability of histological diagnosis of endometrioid endometrial cancer (EEC) using conventional histology and TMA using 2 mm cores. We examined EEC tissues from 171 patients. Formalin fixed, paraffin embedded tissue donor blocks from EEC specimens were selected and examined histologically. Duplicate 2 mm tissue cores were inserted into a TMA recipient block. EEC tissues were examined as hematoxylin-eosin stained sections from the TMAs. EEC tissue was identified in the TMAs in 158 cases (92.4%) and not found in 13 cases (7.6%). On the TMA slides, both EEC positive cores were identified in 129 cases (75.4%), but only one core in 29 cases (17.0%). Among 342 biopsies of the donor blocks (each case in duplicate), EEC was found in 287 cases (83.9%) using the TMA: 124/146 (84.9%) with superficial infiltration, 153/178 (86.0%) with deep myometrial infiltration, and 10/18 (55.6%) without myometrial infiltration. We concluded that two 2 mm tissue cores from a biopsy of a donor block inserted into a TMA recipient block were sufficient to diagnose EEC in more than 90% of cases. EEC was identified in the TMAs with similar frequency with respect to superficial and deep myometrial infiltration. Cases without myometrial infiltration were identified less often.     

Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

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.     

Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation

Apoptosis during preimplantation development has received much interest because of its potential role in eliminating defective cells. Although development in humans is characterised by a high degree of genetic abnormality, little is known of the regulation of apoptosis in embryos. By PolyA PCR we analysed expression of 11 BCL-2 genes in individual human embryos representative of normal development and in severely fragmented embryos. We demonstrate constitutive expression of BAX in virtually all embryos at all stages of development, and variable expression of BCL2, BCL-XL, BCL-W, MCL-1 BAK, BAD, BOKL, BID, BIK, and BCL-XS. The frequency of expression of pro- and anti-apoptotic BCL-2 members was similar throughout development, except at the two-cell stage where pro-apoptotic genes predominated. Protein expression was confirmed for BCL-2, MCL-1, BCL-X, BAX, BAD, and activated caspase 3. BCL-2 protein was associated with mitochondria but expressed inconsistently in the blastocyst inner cell mass. Consistent differences between morphologically intact and fragmented embryos included the expression of BAK in fragmented but not intact four-cell embryos. Our study addresses the importance of examining single human embryos representative of the viable population for a large number of genes, in order to establish meaningful expression profiles and provide information on overlapping function in a large gene family.     

Malignant lymphoma of follicle centre cells with marked nuclear lobation

Four cases of malignant B-cell lymphoma characterized by a conspicuous component of tumour cells with markedly lobatated nuclei are described. Two exhibited a follicular and two a diffuse growth pattern. The tumour cell population formed a continuous spectrum comprising both cells resembling normal follicle centre cells and multilobated lymphoma cells. Cytomorphological analysis of the multilobated cell group indicated a differentiation series from centroblast-like cells with moderately lobated nuclei to large and medium-sized cells with marked nuclear lobation which revealed features of centrocytes. In three cases (1, 3, and 4) the majority of these multilobated cells showed plasmacytoid differentiation in their cytoplasm in conjunction with the synthesis of monotypical cytoplasmic immunoglobulin. No plasmacytoid features were present in a fourth case (2). In only one case (4) monotypical surface immunoglobulin was detectable on the tumour cells. A close relationship between the multilobated tumour cells and follicle centre cells was further substantiated by the finding of a similar cell variant in the follicle centres of a control group of non-neoplastic lymph nodes. It included cells with plasmacytoid differentiation which synthesized polytypical immunoglobulin. We consider this type of B-cell lymphoma with a conspicuous component of cells with lobated nuclei as a variant of malignant lymphoma, centroblastic/centrocytic.     

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.     

BAK and NOXA Are Critical Determinants of Mitochondrial Apoptosis Induced by Bortezomib in Mesothelioma

Based on promising preclinical efficacy associated with the 20S proteasome inhibitor bortezomib in malignant pleural mesothelioma (MPM), two phase II clinical trials have been initiated (EORTC 08052 and ICORG 05–10). However, the potential mechanisms underlying resistance to this targeted drug in MPM are still unknown. Functional genetic analyses were conducted to determine the key mitochondrial apoptotic regulators required for bortezomib sensitivity and to establish how their dysregulation may confer resistance. The multidomain proapoptotic protein BAK, but not its orthologue BAX, was found to be essential for bortezomib-induced apoptosis in MPM cell lines. Immunohistochemistry was performed on tissues from the ICORG-05 phase II trial and a TMA of archived mesotheliomas. Loss of BAK was found in 39% of specimens and loss of both BAX/BAK in 37% of samples. However, MPM tissues from patients who failed to respond to bortezomib and MPM cell lines selected for resistance to bortezomib conserved BAK expression. In contrast, c-Myc dependent transactivation of NOXA was abrogated in the resistant cell lines. In summary, the block of mitochondrial apoptosis is a limiting factor for achieving efficacy of bortezomib in MPM, and the observed loss of BAK expression or NOXA transactivation may be relevant mechanisms of resistance in the clinic.