Bcl-2: bax and bcl-2: Bcl-x ratios by image cytometric quantitation of immunohistochemical expression in ovarian carcinoma: correlation with prognosis

Bcl-2 is a proto-oncogene which is involved in prolonging cell survival by inhibiting programmed cell death. Bax and bcl-x are members of the bcl-2 family; when overexpressed, they can counteract the ability of bcl-2 to inhibit apoptosis. This suggests a model in which the ratios of bcl-2 to bax and bcl-x can be used to determine response to therapy and prognosis. The expression of bcl-2, bax and bcl-x was studied in 50 ovarian carcinomas. The percentage of positive area immunostained (PPA) in the nucleus and cytoplasm of each ovarian carcinoma was quantitated in 15 high power fields by image cytometry. The ratios were obtained by dividing the PPA of bcl-2 by the PPA of bax and bcl-x. 17 of 50 ovarian carcinomas (34%) stained positively for bcl-2, 39 for bax (78%) and 47 for bcl-x (94%). Although there is no significant statistical correlation between expression of bcl-2, bax or bcl-x and grade (P = 0.15; P = 0. 47; P = 0.56), stage (P = 0.71; P = 0.6; P = 0.42), and overall or disease-free survival (P = 0.26; P = 0.55; P = 0.16), increased bcl-2 expression was demonstrated in patients with shortened overall and disease-free survival. Also, increased expression of bax and bcl-x was associated with increased overall and disease-free survival. Bcl-2:bax and bcl-2:bcl-x ratios less than 1 are associated with survival advantage, although not statistically significant (P = 0.83; P = 0.93). Image cytometric measurement of bcl-2, bax, and bcl-x expression is feasible. There is a tendency for their expression to correlate with prognosis in ovarian carcinomas.     

Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions

The proteins of the bcl-2 family play an important role during apoptosis and may also regulate cell death in response to oxidative stress, which has been implicated in Parkinson's disease. In this study we examined the localization of the pro-apoptotic protein bax, and the anti-apoptotic proteins bcl-2 and bcl-x_L in the substantia nigra (SN) of the adult rat and their response to oxidative stress caused by striatal injections of 6-hydroxydopamine (6-OHDA). Our data show that bcl-2, bcl-x and bax proteins are present in the SN. Bcl-2 and bax are localized primarily in neurons including all those positive for tyrosine hydroxylase (TH). The intraneuronal distribution of bcl-2 and bax were different. Bcl-2 was diffuse throughout the cell while bax was localized in well-defined structures around the nucleus and within processes. Bcl-x staining in neurons was weak, though it was strongly expressed in GFAP-positive astrocytes. 6-OHDA injections, which resulted in loss of dopamine neurons between 7–14 days post-lesion, altered the distribution of bax, bcl-2 and bcl-x proteins in the SN. Bcl-2 and bax were decreased in the TH-positive cells of the SN from 3 to 14 days post-lesion and many TH-positive neurons were bcl-2 negative. Neuronal bcl-x was initially unchanged after lesion, but increased in astrocytes between 3–7 days post-lesion before the increase in GFAP immunoreactivity, which was detectable at days 10–14. While the neuronal distribution of bcl-2 and bcl-x does not change following lesion, bax became evenly distributed thought the soma. Morphological features of apoptosis, including TUNEL labeling and chromatin condensation was not observed. These data suggest that striatal 6-OHDA lesions do not result in classical apoptosis in the SN of the adult rat, even though there are changes in the content and distribution of members of the bcl-2 family of proteins.     

Image cytometric bcl-2:bax and bcl-2:bcl-x ratios in invasive breast carcinoma: correlation with prognosis

BACKGROUND: The bcl-2 family of proteins are important regulators of apoptosis. Some of the members, such as bcl-2 and bcl-x(L), inhibit cell death, whereas others, such as bax and bcl-x(S), promote cell death. We evaluated the ratios of bcl-2:bax and bcl-2:bcl-x expression by image cytometry in invasive breast carcinoma to determine prognostic significance. DESIGN: Five-micron sections of formalin-fixed, paraffin-embedded tissue from 88 invasive breast carcinomas were immunostained using steam antigen retrieval, an avidin biotin-complex technique with automated stainer and primary antibodies against bcl-2 (1/160; Dako, Carpenteria, CA), bax (1/1,500; PharMingen, San Diego, CA), and bcl-x (1/1,500; PharMingen). Positive controls were tonsil (bcl-2) and normal breast (bax and bcl-x) tissue samples. Immunostain was measured in 15 high power fields as percentage positive area (PPA) in nuclei and cytoplasm using the CAS 200 image analyzer (Becton Dickinson, San Jose, CA). RESULTS: Median follow-up was 105 months (range 11-130). Significantly improved disease-free survival was found in patients with a bcl-2:bcl-x ratio > or = 1 by univariate and multivariate analyses. The bcl-2:bax ratio was not predictive of overall or disease-free survival. A significant difference in overall and disease-free survival was found between carcinomas with positive and negative bcl-2 expression by univariate analysis; by multivariate analysis, bcl-2 expression was an independent prognostic factor for disease-free survival. The 5-year survival rates were 77% and 50% in patients with bcl-2-positive and bcl-2-negative carcinomas, respectively. CONCLUSION: A bcl-2:bcl-x ratio > or = 1, assessed by image cytometry, is significantly associated with improved disease-free survival in patients with invasive breast carcinoma. Significantly increased overall and disease-free survival is associated with positive bcl-2 expression.     

Bcl-2 family members and disease

Apoptosis plays an important role during development and in the maintenance of multicellular organisms. Bcl-2 family members affect cell death in either a positive or negative fashion. Although some redundancy exists between family members, expression of certain family members is important during development in an organ-specific manner. The founding family member bcl-2 tends to be highly expressed in the embryo and declines postnatally following differentiation and maturation. Altered expression of bcl-2, as well as other family members, has been observed in disease states potentially affecting treatment modalities. Here we examine the distribution and role death repressors bcl-2, bcl-x(L) and bcl-w as well as death effectors bax and bak play regulating apoptosis in a tissue-specific manner. Understanding the normal role of these proteins during embryogenesis and in the mature organ will give us important insight into what goes awry in various disease states.     

Green fluorescent protein causes mitochondria to aggregate in the presence of the Bcl-2 family proteins

Green fluorescent protein (GFP) has been widely used in a variety of experiments in cell biology. When cells were co-transfected with the GFP gene and the bcl-2 family genes bcl-2, bcl-x(L), and bax, mitochondria appeared to aggregate at the periphery of the nucleus specifically where GFP was expressed. Little aggregation was seen in the presence of other members of the GFP family, EGFP (enhanced GFP), ECFP (enhanced cyan variant), and EYFP (enhanced yellow-green variant). GFP but not EGFP seemed to promote cell death induced by pro-apoptotic Bax. Thus, GFP specifically promotes the aggregation of mitochondria when co-expressed with a member of the Bcl-2 family in association with apoptosis.     

The C-terminus of bax is not a membrane addressing/anchoring signal.

It has been suggested that BCL-2 family members associate with certain organelles through their hydrophobic C-terminus which in the case of bcl-2, appears to play a key role in the regulation of apoptosis. We have investigated the association of bax with microsomal, nuclear and mitochondrial membranes using a cell-free system and found, contrary to bcl-2, that bax binds poorly to these organelles. Deletion of the C-terminal of bax (baxDeltaC) or exchanging the C-terminal ends of bax and bcl-XL suggests that the bax C-terminus is not an addressing/anchoring signal. In agreement with this observation, HL-60 cells transfected with either bax or baxDeltaC show no difference in sensitivity to an apoptotic signal. In the cell-free system, at low pH, bax becomes associated with mitochondria after a change of conformation, a result consistant with its structural homology with certain bacterial toxins. In HL-60 cells, as observed in the cell-free system, bax acquired a protease resistant conformation upon its translocation from the cytosol to the mitochondria after the induction of apoptosis.     

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.     

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.     

Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity,cell survival,and mitochondria number in CHO-DG44 grown in suspension and serum-free media

We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.     

Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies

Although the BCL-2 family constitutes a crucial checkpoint in apoptosis, the intricate interplay between these family members remains elusive. Here, we demonstrate that BIM and PUMA, similar to truncated BID (tBID), directly activate BAX-BAK to release cytochrome c. Conversely, anti-apoptotic BCL-2-BCL-X(L)-MCL-1 sequesters these 'activator' BH3-only molecules into stable complexes, thus preventing the activation of BAX-BAK. Extensive mutagenesis of BAX-BAK indicates that their activity is not kept in check by BCL-2-BCL-X(L)-MCL-1. Anti-apoptotic BCL-2 members are differentially inactivated by the remaining 'inactivator' BH3-only molecules including BAD, NOXA, BMF, BIK/BLK and HRK/DP5. BAD displaces tBID, BIM or PUMA from BCL-2-BCL-X(L) to activate BAX-BAK, whereas NOXA specifically antagonizes MCL-1. Coexpression of BAD and NOXA killed wild-type but not Bax, Bak doubly deficient cells or Puma deficient cells with Bim knockdown, indicating that activator BH3-only molecules function downstream of inactivator BH3-only molecules to activate BAX-BAK. Our data establish a hierarchical regulation of mitochondrion-dependent apoptosis by various BCL-2 subfamilies.     

Direct and selective small-molecule activation of proapoptotic BAX

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.     

Trophic support promotes survival of bcl-x-deficient telencephalic cells in vitro

Survival of immature neurons is regulated by Bcl-xL, as targeted disruption of bcl-x significantly increases cell death in vivo and in vitro. Death of cultured bcl-x-deficient and wild-type telencephalic cells can be prevented by fetal calf serum or chemically-defined medium (ITS), suggesting trophic factors in these media potentiate survival through a pathway independent of Bcl-xL. Addition of trophic factors to basal medium revealed that insulin and insulin-like growth factors (IGFs), but not other trophic factors, reduced apoptosis of wild-type and bcl-x-deficient telencephalic cells. Antibodies raised against IGF-I receptors and wortmannin both attenuated the effects of IGF-I, indicating survival was mediated by IGF-I receptors and phosphatidylinositol 3'-kinase signaling, whereas effects of ITS were only partially reduced by these agents. The survival promoting effects of ITS were reduced in cells lacking both bcl-x and bcl-2, indicating Bcl-2 plays a supportive role to Bcl-xL in maintaining telencephalic cell survival. Furthermore, the ratio of expression of the pro-apoptotic bax gene to the anti-apoptotic bcl-2 gene was reduced in bcl-x-deficient cultures grown in ITS, suggesting that the interaction between these bcl-2 family members may, in part, regulate a Bcl-xL independent survival pathway. Finally, the pro-apoptotic bad gene does not appear to play a role in these interactions as targeted disruption of bad did not alter apoptosis in telencephalic cultures.     

Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.     

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.     

The X-ray structure of a BAK homodimer reveals an inhibitory zinc binding site

BAK/BAX-mediated mitochondrial outer-membrane permeabilization (MOMP) drives cell death during development and tissue homeostasis from zebrafish to humans. In most cancers, this pathway is inhibited by BCL-2 family antiapoptotic members, which bind and block the action of proapoptotic BCL proteins. We report the 1.5 A crystal structure of calpain-proteolysed BAK, cBAK, to reveal a zinc binding site that regulates its activity via homodimerization. cBAK contains an occluded BH3 peptide binding pocket that binds a BID BH3 peptide only weakly . Nonetheless, cBAK requires activation by truncated BID to induce cytochrome c release in mitochondria isolated from bak/bax double-knockout mouse embryonic fibroblasts. The BAK-mediated MOMP is inhibited by low micromolar zinc levels. This inhibition is alleviated by mutation of the zinc-coordination site in BAK. Our results link directly the antiapoptotic effects of zinc to BAK.     

Bcl-2 family gene modulation during spontaneous apoptosis of B-chronic lymphocytic leukemia cells

Malignant cell accumulation in B-cell chronic lymphocytic leukemia (B-CLL) is primarily caused by defective apoptosis rather than increased proliferation. To further understand the role of Bcl-2 family members, known regulators of apoptosis, in the abnormal B-CLL survival, we have measured their mRNA levels in fresh B-CLL cells and in cultures undergoing spontaneous apoptosis. Using RNA protection assays we found constitutive expression of most bcl-2 members with high levels of bcl2, bcl-w, bad, bak, bax, and the bcl-2/bax ratio, compared to normal PBL. Spontaneous apoptosis of B-CLL cells by in vitro culture resulted in decreased bcl-2, bcl-w, bfl-1, mcl-1, bak, bax, and bcl-2/bax expression. The pro-apoptotic member bik was only expressed in 5/19 cases and was not modulated during apoptosis, suggesting that bik is not involved in this process. Thus, several Bcl-2 family genes are regulated during B-CLL spontaneous apoptosis and their relative levels may contribute to in vivo progression of the disease.     

Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.     

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.     

A stapled BID BH3 helix directly binds and activates BAX

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.