p27 deficiency cooperates with Bcl-2 but not Bax to promote T-cell lymphoma

The effect of Bcl-2 on oncogenesis is complex and expression may either delay or accelerate oncogenesis. The pro-oncogenic activity is attributed to its well characterized anti-apoptotic function while the anti-oncogenic function has been attributed to its inhibition of cellular proliferation. Recent studies demonstrate that p27 may mediate the effects of Bcl-2 on cellular proliferation. We hypothesized that p27 may suppress tumor formation by Bcl-2 family members. To test this hypothesis, cell cycle inhibition and lymphoma development were examined in Lck-Bcl-2 and Lck-Bax38/1 transgenic mice deficient in p27. Strikingly, p27 deficiency synergistically cooperates with Bcl-2 to increase T cell hyperplasia and development of spontaneous T cell lymphomas. Within 1 year, >90% of these mice had developed thymic T cell lymphomas. This high penetrance contrasts with a one year incidence of <5% of thymic lymphoma in Lck-Bcl-2 or p27 -/- mice alone. In contrast, p27 deficiency had no effect on tumor formation in Lck-Bax38/1 transgenic mice, another model of T cell lymphoma. Histologically the lymphomas in p27 -/- Lck-Bcl-2 mice are lymphoblastic and frequently involve multiple organs suggesting an aggressive phenotype. Interestingly, in mature splenic T cells, Bcl-2 largely retains its anti-proliferative function even in the absence of p27. T cells from p27 -/- Lck-Bcl-2 mice show delayed kinetics of CDK2 Thr-160 phosphorylation. This delay is associated with a delay in the up regulation of both Cyclin D2 and D3. These data demonstrate a complex relationship between the Bcl-2 family, cellular proliferation, and oncogenesis and demonstrate that p27 up-regulation is not singularly important in the proliferative delay observed in T cells expressing Bcl-2 family members. Nonetheless, the results indicate that p27 is a critical tumor suppressor in the context of Bcl-2 expression.     

The presence of 19-kDa Bcl-2 in dividing cells

The 26-kDa bcl-2 gene product inhibits apoptosis and cell proliferation. Cleavage of Bcl-2 into a 22-kDa fragment inactivates its anti-apoptotic activity and is a key event in apoptosis. Here, and in recent work, we describe massive 19-kDa Bcl-2 immunoreactivity in non-apoptotic cells, suggesting a link with viability rather than cell death. Loss of 19 kDa Bcl-2 in adriamycin-induced apoptotic cells underlines this. G2/M-phase accumulation of cells by nocodazole-treatment also results in loss of 19 kDa Bcl-2. Next to its well-documented cytoplasmic localization, a substantial pool of Bcl-2 resides in nuclei. Hampered nuclear localization of Bcl-2 leads to a loss of cell cycle repression. This has led us to point at a pivotal role for nuclear Bcl-2 in cellular proliferation. In this report, cellular fractionation of bcl-2 transfected cells in various phases of the cell cycle reveals a constitutive cytoplasmic pool of 19 kDa Bcl-2. Nuclear 19-kDa Bcl-2 immunoreactivity is far more pronounced in rapidly dividing nuclei compared with more quiescent nuclear fractions. This implicates that ongoing cell proliferation involves cleavage of nuclear Bcl-2 with a 19-kDa fragment.     

Effects of differential overexpression of Bcl-2 on apoptosis, proliferation, and telomerase activity in Jurkat T cells.

The effects of Bcl-2 overexpression on several of its multifunctional characteristics, which include anti-apoptotic properties, impeding of cell proliferation, and telomerase activity, were examined in four Jurkat T cell clones overexpressing different levels of Bcl-2. When treated with anti-Fas or staurosporine, only three of the four clones showed resistance to apoptosis that correlated with the level of Bcl-2 expression. Surprisingly, the clone having no anti-apoptotic characteristic expressed the highest level of Bcl-2. When all the clones were treated with anti-Fas the processing of caspase-2, -3, and -7 but not -8 was inhibited in the resistant clones to a similar extent by the differential overexpression of Bcl-2. However, with staurosporine treatment the processing of all the caspases examined was inhibited to a similar degree by the different levels of Bcl-2 expression in the resistant clones. These results suggest that Bcl-2 blocked Fas-mediated cell death by acting downstream of caspase-8, which is in contrast to staurosporine-induced apoptosis where Bcl-2 is acting upstream of caspase-8. When the anti-proliferative effect of Bcl-2 was examined, a direct correlation between a decrease in cell proliferation and the level of Bcl-2 overexpressed in the clones was observed. The clone overexpressing the greatest amount of Bcl-2 protein, which had no resistance to apoptosis, had the slowest proliferative rate. This suggests that the anti-apoptotic effect of Bcl-2 can be separated from its anti-proliferative effect. The possible effect of overexpression of Bcl-2 on telomerase activity, which is known to control the proliferative capacity of normal cells and cellular senescence, was also determined. Our results suggest that Bcl-2 had no effect on telomerase activity or telomere length in the clones. In summary, our results further suggest that some properties of Bcl-2, such as anti-apoptotic and inhibition of cell proliferation, are individual features of a multifaceted protein.     

Overexpression of Bcl-2 prevents neomycin-induced hair cell death and caspase-9 activation in the adult mouse utricle in vitro

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside-induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside-induced hair cell death can be prevented by broad-spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside-induced hair cell death requires activation of caspase-9. Caspase-9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside-induced hair cell death is mediated by the mitochondrial (or "intrinsic") cell death pathway. The Bcl-2 family of pro-apoptotic and anti-apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl-2 is an anti-apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl-2 to examine the role of Bcl-2 in neomycin-induced hair cell death. Overexpression of Bcl-2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl-2 overexpression prevented neomycin-induced activation of caspase-9 in hair cells. These results suggest that the expression level of Bcl-2 has important effects on the pathway(s) important for the regulation of aminoglycoside-induced hair cell death.     

Human hepatitis B virus X protein induces apoptosis in HepG2 cells: role of BH3 domain

The smallest protein of hepatitis B virus, HBX, has been implicated in the development of liver diseases by interfering with normal cellular processes. Its role in cell proliferation has been unclear as both pro-apoptotic and anti-apoptotic activities have been reported. We showed molecular evidence that HBX induced apoptosis in HepG2 cells. A Bcl-2 Homology Domain 3 was identified in HBX, which interacted with anti-apoptotic but not pro-apoptotic members of the Bcl-2 family of proteins. HBX induced apoptosis when transfected into HepG2 cells, as demonstrated by both flow cytometry and caspase-3 activity. However, HBX protein may not be stable in apoptotic cells triggered by its own expression as only its mRNA or the fusion protein with the glutathione-S-transferase was detected in transfected cells. Our results suggested that HBX behaved as a pro-apoptotic protein and was able to induce apoptosis.     

Serine-70 phosphorylated Bcl-2 prevents oxidative stress-induced DNA damage by modulating the mitochondrial redox metabolism

Bcl-2 phosphorylation at serine-70 (S70pBcl2) confers resistance against drug-induced apoptosis. Nevertheless, its specific mechanism in driving drug-resistance remains unclear. We present evidence that S70pBcl2 promotes cancer cell survival by acting as a redox sensor and modulator to prevent oxidative stress-induced DNA damage and execution. Increased S70pBcl2 levels are inversely correlated with DNA damage in chronic lymphocytic leukemia (CLL) and lymphoma patient-derived primary cells as well as in reactive oxygen species (ROS)- or chemotherapeutic drug-treated cell lines. Bioinformatic analyses suggest that S70pBcl2 is associated with lower median overall survival in lymphoma patients. Empirically, sustained expression of the redox-sensitive S70pBcl2 prevents oxidative stress-induced DNA damage and cell death by suppressing mitochondrial ROS production. Using cell lines and lymphoma primary cells, we further demonstrate that S70pBcl2 reduces the interaction of Bcl-2 with the mitochondrial complex-IV subunit-5A, thereby reducing mitochondrial complex-IV activity, respiration and ROS production. Notably, targeting S70pBcl2 with the phosphatase activator, FTY720, is accompanied by an enhanced drug-induced DNA damage and cell death in CLL primary cells. Collectively, we provide a novel facet of the anti-apoptotic Bcl-2 by demonstrating that its phosphorylation at serine-70 functions as a redox sensor to prevent drug-induced oxidative stress-mediated DNA damage and execution with potential therapeutic implications.     

Bcl-2 inhibits apoptosis by increasing the time-to-death and intrinsic cell-to-cell variations in the mitochondrial pathway of cell death

BH3 mimetics have been proposed as new anticancer therapeutics. They target anti-apoptotic Bcl-2 proteins, up-regulation of which has been implicated in the resistance of many cancer cells, particularly leukemia and lymphoma cells, to apoptosis. Using probabilistic computational modeling of the mitochondrial pathway of apoptosis, verified by single-cell experimental observations, we develop a model of Bcl-2 inhibition of apoptosis. Our results clarify how Bcl-2 imparts its anti-apoptotic role by increasing the time-to-death and cell-to-cell variability. We also show that although the commitment to death is highly impacted by differences in protein levels at the time of stimulation, inherent stochastic fluctuations in apoptotic signaling are sufficient to induce cell-to-cell variability and to allow single cells to escape death. This study suggests that intrinsic cell-to-cell stochastic variability in apoptotic signaling is sufficient to cause fractional killing of cancer cells after exposure to BH3 mimetics. This is an unanticipated facet of cancer chemoresistance.     

Characterization of peptide aptamers targeting Bfl-1 anti-apoptotic protein

Bfl-1, an anti-apoptotic protein of the Bcl-2 family, has been identified as a potential therapeutic target for B-cell malignancies. We describe herein the first characterization of peptide aptamers selected against Bfl-1. We show that most of the Bfl-1 peptide aptamers do not interact with Bcl-2, Bcl-xL, or Mcl-1 in yeast and that some of them restore the pro-apoptotic activity of Bax in yeast in which Bax and Bfl-1 proteins are coexpressed. When expressed in mammalian cells, peptide aptamers interact with Bfl-1 and sensitize B-cell lines to apoptosis induced by chemotherapeutic agents. We further demonstrate that a nonconstrained peptide derived from one aptamer variable region reverses Bfl-1 anti-apoptotic activity in HeLa cells through disruption of Bax-Bfl-1 interaction. This peptide also promotes cell death in lymphoma B-cell lines expressing a high level of Bfl-1 and sensitizes these cells to drug-induced apoptosis. Taken together, these results further validate Bfl-1 as a therapeutic target for malignant B-cells and suggest that peptide aptamers may be a useful tool for guiding the identification of small compounds that target the anti-apoptotic Bfl-1 protein.     

Molecular dynamics simulations of the Bcl-2 protein to predict the structure of its unordered flexible loop domain

B-cell lymphoma (Bcl-2) protein is an anti-apoptotic member of the Bcl-2 family. It is functionally demarcated into four Bcl-2 homology (BH) domains: BH1, BH2, BH3, BH4, one flexible loop domain (FLD), a transmembrane domain (TM), and an X domain. Bcl-2’s BH domains have clearly been elucidated from a structural perspective, whereas the conformation of FLD has not yet been predicted, despite its important role in regulating apoptosis through its interactions with JNK-1, PKC, PP2A phosphatase, caspase 3, MAP kinase, ubiquitin, PS1, and FKBP38. Many important residues that regulate Bcl-2 anti-apoptotic activity are present in this domain, for example Asp34, Thr56, Thr69, Ser70, Thr74, and Ser87. The structural elucidation of the FLD would likely help in attempts to accurately predict the effect of mutating these residues on the overall structure of the protein and the interactions of other proteins in this domain. Therefore, we have generated an increased quality model of the Bcl-2 protein including the FLD through modeling. Further, molecular dynamics (MD) simulations were used for FLD optimization, to predict the flexibility, and to determine the stability of the folded FLD. In addition, essential dynamics (ED) was used to predict the collective motions and the essential subspace relevant to Bcl-2 protein function. The predicted average structure and ensemble of MD-simulated structures were submitted to the Protein Model Database (PMDB), and the Bcl-2 structures obtained exhibited enhanced quality. This study should help to elucidate the structural basis for Bcl-2 anti-apoptotic activity regulation through its binding to other proteins via the FLD.     

Nonlinear regulation of commitment to apoptosis by simultaneous inhibition of Bcl-2 and XIAP in leukemia and lymphoma cells

Apoptosis is a complex pathway regulated by the concerted action of multiple pro- and anti-apoptotic molecules. The intrinsic (mitochondrial) pathway of apoptosis is governed up-stream of mitochondria, by the family of Bcl-2 proteins, and down-stream of mitochondria, by low-probability events, such as apoptosome formation, and by feedback circuits involving caspases and inhibitor of apoptosis proteins (IAPs), such as XIAP. All these regulatory mechanisms ensure that cells only commit to death once a threshold of damage has been reached and the anti-apoptotic reserve of the cell is overcome. As cancer cells are invariably exposed to strong intracellular and extracellular stress stimuli, they are particularly reliant on the expression of anti-apoptotic proteins. Hence, many cancer cells undergo apoptosis when exposed to agents that inhibit anti-apoptotic Bcl-2 molecules, such as BH3 mimetics, while normal cells remain relatively insensitive to single agent treatments with the same class of molecules. Targeting different proteins within the apoptotic network with combinatorial treatment approaches often achieves even greater specificity. This led us to investigate the sensitivity of leukemia and lymphoma cells to a pro-apoptotic action of a BH3 mimetic combined with a small molecule inhibitor of XIAP. Using the computational probabilistic model of the apoptotic pathway, verified by experimental results from human leukemia and lymphoma cell lines, we show that inhibition of XIAP has a non-linear effect on sensitization towards apoptosis induced by the BH3 mimetic HA14-1. This study justifies further ex vivo and animal studies on the potential of the treatment of leukemia and lymphoma with a combination of BH3 mimetics and XIAP inhibitors.     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.     

Unknotting the roles of Bcl-2 and Bcl-xL in cell death

The antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL play important roles in inhibiting mitochondria-dependent extrinsic and intrinsic cell death pathways. It seems that these two proteins have distinct functions for inhibiting extrinsic and intrinsic cell death pathways. The overexpression of Bcl-2 is able to inhibit not only apoptotic cell death but also in part nonapoptotic cell death, which has the role of cell cycle arrest in the G1 phase, which may promote cellular senescence. The overexpression of Bcl-2 may also have the ability to enhance cell death in the interaction of Bcl-xL with other factors. The overexpression of Bcl-xL enhances autophagic cell death when apoptotic cell death is inhibited in Bax(-/-)/Bak(-/-) double knockout cells. This review discusses the previously unexplained aspects of Bcl-2 and Bcl-xL functions associated with cell death, for better understanding of their functions in the regulation.     

Bcl-2 expression in synovial fibroblasts is essential for maintaining mitochondrial homeostasis and cell viability

The regulation of proliferation and cell death is vital for homeostasis, but the mechanism that coordinately balances these events in rheumatoid arthritis (RA) remains largely unknown. In RA, the synovial lining thickens in part through increased proliferation and/or decreased synovial fibroblast cell death. Here we demonstrate that the anti-apoptotic protein, Bcl-2, is highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68-negative, fibroblast-like synoviocyte population. To determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA and osteoarthritis synovial tissues. In addition, Ad-Rbz-Bcl-2-induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. The general caspase inhibitor zVAD.fmk blocked caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation, but not loss of transmembrane potential or viability, indicating that cell death was independent of caspase activation. Ectopically expressed Bcl-xL inhibited Ad-Rbz-Bcl-2-induced mitochondrial permeability transition and apoptosis in Ad-Rbz-Bcl-2-transduced cells. Thus, forced down-regulation of Bcl-2 does not induce a compensatory mechanism to prevent loss of mitochondrial integrity and cell death in human fibroblasts.     

Cyclin-dependent kinase inhibitors,roscovitine and purvalanol,induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.     

The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death

Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-X(L) bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.     

Cellular neuroprotective mechanisms in cerebral ischemia: Bcl-2 family proteins and protection of mitochondrial function

Mitochondria are central to brain cell response to ischemia, with critical roles in generation of ATP, production of free radicals, and regulation of apoptotic cell death. Changes in the permeability of the outer mitochondrial membrane to regulators of apoptosis can control ischemic cell death and this permeability is directly controlled by the Bcl-2 family of proteins. The Bcl-2 family regulate apoptosis by several mechanisms including affecting the formation of apoptotic protein-conducting pores in the outer mitochondrial membrane. The anti-apoptotic protein Bcl-2 improves neuron survival following various insults, and is protective even when administered after stroke onset in a rat model of focal ischemia. Despite intense study, the precise molecular mechanisms underlying protection by the anti-apoptotic members of the Bcl-2 family are not completely understood. This review focuses on the mechanisms by which Bcl-2 family members control the permeability of the mitochondrial membrane and influence other aspects of mitochondrial function after brain ischemia, concluding with discussion of the potential use of Bcl-2 for the treatment of cerebral ischemia.